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CLINICAL STUDIES ON THE FOLLOWING INGREDIENTS:

Zinc (as chelate)

The antioxidant properties of a novel zinc-carnosine chelate compound

Abstract

A zinc-carnosine chelate compound, Z-103, attenuates gastric mucosal injuries and inhibits the increase of lipid peroxide in the gastric mucosa induced by burn shock or ischemia-reperfusion. However, the exact mechanism of the antioxidative effect of Z-103 is not clear. The antioxidant properties of a novel anti-peptic ulcer agent Z-103 in vitro were compared with those of zinc ion and l-carnosine. Z-103 scavenged superoxide anion radicals. Z-103 and ZnSO4, but not l-carnosine, inhibited the superoxide generation from polymorphonuclear leukocytes stimulated by opsonized zymosan, and also inhibited the generation of hydroxyl radicals by the Fenton reaction. The increase of lipid peroxides produced by rat brain homogenates and liver microsomes was also inhibited by Z-103 and ZnSO4. These findings indicate that the strong anti-ulcer and antioxidative actions of Z-103 in vivo are due to a combination of these antioxidant actions in vitro.

Reference:

  1. Yoshikawa, Kamigyo-ku, Kyoto. The antioxidant properties of a novel zinc-carnosine chelate compound. Biochimica et Biophysica Acta (BBA) – General Subjects. 14 Nov 1991:V1115, Issue1; P15-22. doi:10.1016/0304-4165(91)90005-2

http://www.sciencedirect.com/science/article/pii/0304416591900052

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Zinc extraction from chloride solutions with mixtures of solvating and chelating reagents

Abstract
An equimolar mixture of weakly basic and chelating reagent was used for extracting zinc(II) from acid chloride solutions at various concentrations of chloride and hydrogen ions. N,N-dihexylpyridine-3-carboxamide was used as a weak base and 1-phenyldecane-1,3-dione (β-diketone) as a chelating extractant. In a three-step extraction–scrubbing–stripping process, zinc ions were transferred from the chloride into the sulfate solutions. In the first step, Zn(II) was extracted by N,N-dihexylpyridine-3-carboxamide. The composition of the extracted complexes (solvate or ion pairs) depends on HCl concentration. After zinc(II) extraction from acid chloride solutions, Cl ions were scrubbed out from the organic phases with ammonia solutions. In this step, the extractants transfer zinc ions according to one of two possible mechanisms: from the solvate into the chelate, or from the ion pair into the chelate. The stripping of zinc ions from the chelate complex with β-diketone was carried out with sulfuric acid solutions. For both systems under investigation (weakly and strongly acidic solutions), the use of the reagent mixture enables the effective extraction of zinc(II) and removal of chloride ions from the organic phase. Owing to the possibility of using the extractant mixture for the recovery of Zn(II) from the chloride solutions of varied acidity, a partial protonation of the weakly basic extractant in the stripping step has no significant effect on the efficiency of a subsequent extraction–scrubbing–stripping process.

Reference:
Marcin Stasiak, Magdalena Regel-Rosocka, Aleksandra Borowiak-Resterna. Zinc extraction from chloride solutions with mixtures of solvating and chelating reagents. Hydrometallurgy. June 2016: V162,P57—62. doi:10.1016/j.hydromet.2016.02.017

Chelation behavior of various flavonols and transfer of flavonol-chelated zinc(II) to alanylaspartic dipeptide: A PCM/DFT investigation

Abstract
Alanylaspartic dipeptide (AlaAsp) and zinc(II)-flavonol complex could represent a metal-binding site in proteins and a metal-ion releasing agent, respectively. Chelation of zinc(II) by either AlaAsp or flavonol ligands in aqueous solution has been examined using DFT methods with polarizable continuum model (PCM/DFT). Coordination geometry, complexation stoichiometry, coordination bond strength, preferable metal-binding site on ligands and effect of water coordination on the stability of complexes have been addressed. In several cases, the long-range corrected density functional CAM-B3LYP allows the most accurate prediction of both structural and spectroscopic data. The preferential transfer of flavonol-chelated zinc(II) to AlaAsp under solvation is attainable through the ligand-exchange reaction. The energy barrier of such reaction is significantly dependent on the degree of hydrogen bonding within the transition state. In summary, either hydroxylation or methoxylation at particular positions on the 3-hydroxyflavone backbone significantly affects the reactivity of flavonol chelates in the metal-ion transfer.

Reference:
Nuttawisit Yasarawana, Khajadpai Thipyaponga, Vithaya Ruangpornvisutib. Chelation behavior of various flavonols and transfer of flavonol-chelated zinc(II) to alanylaspartic dipeptide: A -PCM/DFT investigation. Journal of Molecular Structure. March 2016: V1107, P278-290. doi:10.1016/j.molstruc.2015.11.059

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Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: Possible contribution of characteristic amino acid residues

Abstract
Zinc is an essential trace element for human growth and development. In this work, zinc-chelating peptides from rapeseed protein hydrolysates produced with alcalase were investigated by affinity chromatography with immobilized zinc and Sephadex G-25 gel filtration. Four small peptides, namely, Ala-Arg, Asn-Ser-Met (NSM), Gly-Lys-Arg, and Glu-Pro-Ser-His, were obtained and identified by reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The zinc-chelating ability of the four peptides was further validated by inductively coupled plasma atomic emission spectrometry (ICP-AES). NSM was found to exhibit the highest zinc-chelating rate, which was better than that of reduced glutathione. We speculated that the Asn residue at the amino-terminus might facilitate this zinc-chelating ability. Therefore, utilizing small peptides from rapeseed protein as novel carriers for zinc supplement was feasible.

Reference:
Ningning Xie et. al., Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: Possible contribution of characteristic amino acid residues. Food Chemistry. April 15, 2015: V173, P210-217. doi:10.1016/j.foodchem.2014.10.030

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New alicyclic thiosemicarbazone chelated zinc(II) antitumor complexes: Interactions with DNA/protein, nuclease activity and inhibition of topoisomerase-I

Abstract
Two new zinc(II) complexes, Zn(chtsc-N-Me)2 and Zn(chtsc-N-Ph)2 where chtsc = cyclohexanone thiosemicarbazone; chtsc-N-Ph = cyclohexanone N(4)-phenyl thiosemicarbazone, were isolated and characterized by X-ray crystallography. The interaction of these complexes with DNA and protein were studied using calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) as the respective models, and marked activity was observed. The complexes exhibited efficient DNA cleavage activity via the oxidative pathway involving singlet oxygen as the reactive oxygen species. The topoisomerase inhibition assay showed that, even at low concentrations, both Zn(chtsc-N-Me)2 and Zn(chtsc-N-Ph)2 are capable of impairing enzymatic occupation of human topoisomerase-I, a significant feature of anticancer drugs. The results of in-vitro anti-proliferation tests carried out against five different human tumor cells lines gave GI50 values lower than 5 μg/mL, which indicates that these complexes are potentially advantageous as anticancer agents.

Reference:

  1. Vikneswarana, Naser Eltaher Eltayebb, S. Ramesha,R. Yahya. New alicyclic thiosemicarbazone chelated zinc(II) antitumor complexes: Interactions with DNA/protein, nuclease activity and inhibition of topoisomerase-I. Polyhedron. February 17, 2016: V105, P89-95. doi:10.1016/j.poly.2015.12.012

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Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD)

Abstract
To elucidate the role of Zn2+-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia–ischemia, PC12 cells were exposed to Oxygen–Glucose Deprivation (OGD) solution mimicking the hypoxic–ischemic condition in neuron, and the effect of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn2+ chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3 h, 6 h and 24 h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1 h, but decrease at 3 h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3 h and 6 h, P < 0.01 at 24 h respectively compared to control) and GluR2 expression (P < 0.05 at 3 h, 6 h and 24 h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic–ischemic condition.

Reference:
Zhao Liu et. al., Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD). Journal of Trace Elements in Medicine and Biology. July 2015: V31, P45-52. doi:10.1016/j.jtemb.2015.03.003

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Synthesis and initial in vitro biological evaluation of two new zinc-chelating compounds: Comparison with TPEN and PAC-1

Abstract
The lipophilic, cell-penetrating zinc chelator N,N,N′,N′,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce apoptosis in various cell types. The relationship between apoptosis-inducing ability and zinc affinity (Kd), have been investigated with two new model compounds, ZnA-DPA (3) and ZnA-Pyr (4), and compared to that of TPEN and PAC-1. The zinc-chelating o-hydroxybenzylidene moiety in PAC-1 was replaced with a 2,2′-dipicoylamine (DPA) unit (ZnA-DPA, 3) and a 4-pyridoxyl unit (ZnA-Pyr, 4), rendering an order of zinc affinity TPEN > ZnA-Pyr > ZnA-DPA > PAC-1. The compounds were incubated with the rat pheochromocytoma cell line PC12 and cell death was measured in combination with ZnSO4, a caspase-3 inhibitor, or a ROS scavenger. The model compounds ZnA-DPA (3) and ZnA-Pyr (4) induced cell death at higher concentrations as compared to PAC-1 and TPEN, reflecting differences in lipophilicity and thereby cell-penetrating ability. Addition of ZnSO4 reduced cell death induced by ZnA-Pyr (4) more than for ZnA-DPA (3). The ability to induce cell death could be reversed for all compounds using a caspase-3-inhibitor, and most so for TPEN (1) and ZnA-Pyr (4). Reactive oxygen species (ROS), as monitored using dihydro-rhodamine (DHR), were involved in cell death induced by all compounds. These results indicate that the Zn-chelators ZnA-DPA (3) and ZnA-Pyr (4) exercise their apoptosis-inducing effect by mechanisms similar to TPEN (1) and PAC-1 (2), by chelation of zinc, caspase-3 activation, and ROS production.

Reference:

  1. Alexander H. Åstranda et. al., Synthesis and initial in vitro biological evaluation of two new zinc-chelating compounds: Comparison with TPEN and PAC-1. Bioorganic & Medicinal Chemistry. September 1,2013: V21, Issue 17; P5175-5181. doi:10.1016/j.bmc.2013.06.037

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Inhibition of the lymphoid tyrosine phosphatase: The effect of zinc(II) ions and chelating ligand fragments on enzymatic activity

Abstract
A 96-member chelator fragment library (CFL-1.1) was screened to identify inhibitors of the lymphoid tyrosine phosphatase in the absence and presence of zinc acetate. Fragments that inhibit LYP activity more potently in the presence of zinc, fragments that rescue LYP activity in the presence of inhibitory concentrations of zinc, and fragments that inhibit LYP activity independent of zinc concentration were identified. Of these, 1,2-dihydroxynaphthalene was the most potent inhibitor with an IC50 value of 2.52 ± 0.06 μM after 2 h of incubation. LYP inhibition by 1,2-dihydroxynaphthalene was very similar to inhibition by 1,2-naphthoquinone (IC50 = 1.10 ± 0.03 µM), indicating that the oxidized quinone species is likely the active inhibitor. The inhibition was time-dependent, consistent with covalent modification of the enzyme.

Reference:
Megan K. Thorsona, David T. Puertab, Seth M. Cohenb, Amy M. Barriosa. Inhibition of the lymphoid tyrosine phosphatase: The effect of zinc(II) ions and chelating ligand fragments on enzymatic activity. Bioorganic & Medicinal Chemistry Letters. August 14, 2014: V24, Issue 16; P 4019-4022. doi:10.1016/j.bmcl.2014.06.016

Vitamin C

Combined treatment with minodronate and vitamin C increases bone mineral density and strength in vitamin C-deficient rats

Abstract

Objectives

Reduced bone quality caused by vitamin C deficiency in older persons may lead to incidental fragility fractures during bisphosphonate treatment, although bisphosphonate increases bone mineral density (BMD). This study aimed to evaluate the effects of minodronate and ascorbic acid (Aa) on BMD, bone quality, and bone strength in Aa-deficient osteogenic disorder Shionogi (ODS) rats.

Methods

Six-month-old ODS rats were divided into four groups (n = 20 per group): (1) Aa supplementation (Aa+); (2) Aa-deficient (Aa); (3) Aa supplementation and minodronate administration (Aa+ + Mino); and (4) Aa-deficient and minodronate administration (Aa + Mino). BMD, bone strength, bone histomorphometry, and bone quality determined using Fourier transform infrared spectroscopy imaging (FTIRI) were evaluated after 4 and 8 weeks.

Results

BMD was significantly higher in the Aa+ + Mino group than in the Aa group (p < 0.05). Bone strength was significantly higher in the Aa+ and Aa+ + Mino groups than in the Aagroup (p < 0.05). Furthermore, bone strength was significantly higher in the Aa+ + Mino group than in the Aa + Mino group (p < 0.05). Minodronate treatment irrespective of Aa supplementation significantly decreased bone resorption compared with the Aa+ and Aagroups (p < 0.05). No significant differences in the parameters evaluated by FTIRI were observed between the groups.

Conclusions
Aa supplementation improved bone strength in ODS rats. Combined treatment with minodronate and Aa, but not minodronate alone, improved bone strength and increased BMD. Aa is required for bone health because it is essential for osteoblast differentiation.

Reference:
Toyohito Segawa et. al., Combined treatment with minodronate and vitamin C increases bone mineral density and strength in vitamin C-deficient rats. Osteoporosis and Sarcopenia. March 2016: V2, Issue 1; P30-37. doi:10.1016/j.afos.2016.01.002

http://www.sciencedirect.com/science/article/pii/S2405525515300224

The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis

Abstract
Vitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP-LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs.

Reference:
Rajnikumar Sangani. The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis. Stem Cell Research. September 2, 2015: V15, Issue 2; P312-321. doi:10.1016/j.scr.2015.06.002

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In vitro effects of vitamins C and E, n-3 and n-6 PUFA and n-9 MUFA on placental cell function and redox status in type 1 diabetic pregnant women

Abstract
The aim of this investigation was to determine the in vitro effects of vitamin C and E, n-3 and n-6 PUFA and n-9 MUFA on placental cell proliferation and function in type 1 diabetes.

Placenta tissues were collected from 30 control healthy and 30 type 1 diabetic women at delivery. Placental cells were isolated and were cultured in RPMI medium supplemented with vitamin C (50 μM), vitamin E (50 μM), n-3 PUFA (100 μM), n-6 PUFA (100 μM) or n-9 MUFA (100 μM). Cell proliferation, cell glucose uptake and intracellular oxidative status were investigated.

Our results showed that basal placental cell proliferation, glucose uptake, malondialdehyde (MDA) and carbonyl proteins were higher while intracellular reduced glutathione (GSH) levels and catalase activities were lower in placentas from diabetic women as compared to controls. Vitamins C and E induced a modulation of placental cell proliferation and glucose consumption without affecting intracellular redox status in both diabetic and control groups. N-3 and n-6 PUFA diminished placental cell proliferation and enhanced intracellular oxidative stress while n-9 MUFA had no effects in the two groups. Co-administration of n-3 or n-6 PUFA and vitamin C or E were capable of reversing back the PUFA-decreased cell proliferation and normalizing placental cell function and redox status especially in diabetes.

In conclusion, PUFA and antioxidant vitamin combinations may be beneficial in improving placenta function and in reducing placental oxidative stress in type 1 diabetic pregnancy.

Reference:
Djamila Mezouara et. al., In vitro effects of vitamins C and E, n-3 and n-6 PUFA and n-9 MUFA on placental cell function and redox status in type 1 diabetic pregnant women. Placenta. Available online 15 April 2016: In Press, Accepted Manuscript — Note to user. doi:10.1016/j.placenta.2016.04.013

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Chapter 43 – Vitamin C, Antioxidant Status, and Cardiovascular Aging

Abstract

The aging process is characterized by a progressive decline of cellular integrity and function resulting from the structural modification of macromolecules including formation of oxidized lipid species, advanced glycated products, nitrosylated proteins, and DNA mutations and epimutations. Lifestyle factors, including nutrition, which modulate the aging trajectory must do so because they modulate the accumulation of such macromolecular damage. Cardiovascular diseases are the most common cause of death among older people in the Western World and their burden is increasing globally. Therefore, the development of effective strategies to attenuate aging of the vascular system could make a substantial contribution to lowering CVD risk and improving the quality of life of older people. Vitamin C plays an important role in vascular health but the benefits of supplementation with vitamin C remain controversial. Current evidence suggests that there are unlikely to be population-wide benefits on cardiovascular health of vitamin C supplementation in older people. It seems more likely that those population groups with low vitamin C status, because of inadequate vitamin C intake or for genotypic reasons, or with high levels of oxidative damage may benefit from extra vitamin C intake. This opens the way for more effective public health interventions to enhance vitamin C status which use a personalized (or stratified) approach.

Reference:
Ammar W. Ashor,Mario Siervo, John C. Mathers. Chapter 43 – Vitamin C, Antioxidant Status, and Cardiovascular Aging. Molecular Basis of Nutrition and Aging. 2016: A Volume in the Molecular Nutrition Series; P 609-619. doi:10.1016/B978-0-12-801816-3.00043-1

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High-dose vitamin C and cancer

Abstract

Vitamin C (ascorbic acid, ascorbate) is a basic compound that is of great importance with its role in various enzymatic reactions including the synthesis of collagen, as well as with its redox functions. Vitamin C has become the center of interest in cancer studies, in consequence of the facts that connective tissue changes and vitamin C deficiency were first alleged to be associated with cancer in the 1950s; and that high doses of vitamin C was asserted to be cytotoxic for cancer cells, later on. The results of the first study carried out in the 1970s were promising; but afterwards, the studies were ascertained to be faulty. Despite the positive results achieved from some laboratory and animal experiments, randomized clinical trials did not verify those findings, and no clear benefit of vitamin C for cancer treatment could be demonstrated. As for studies, where its use in combination with other cancer treatment regimens was assessed, conflicting results were obtained. Although intake of high doses of vitamin C is alleged to be harmless, based on that it is in the group of water soluble vitamins and is not stored in the body, there are many side effects and drug interactions reported in the literature. For now, it is better to abstain from this treatment, until the benefit of the treatment (if any) is clearly demonstrated, considering the potential side effects and interactions.

Reference:

Ahmet Unlu, M.D.a, Onder Kirca, M.D.b, Mustafa Ozdogan, M.D.b, , , Erdinç Nayır, M.D., High-dose vitamin C and cancer. Journal of Oncological Science. January 2016: V1; P 10-12. doi:10.1016/j.jons.2015.11.010

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Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes

Abstract

Background

Intra-articular injections of local anesthetics are commonly used to enhance post-operative analgesia following orthopedic surgery as arthroscopic surgeries. Nevertheless, recent reports of severe complications due to the use of intra-articular local anesthetic have raised concerns.

Reference:

Jun Tian, , Yan Li. Comparative effects of vitamin C on the effects of local anesthetics ropivacaine, bupivacaine, and lidocaine on human chondrocytes. Brazilian Journal of Anesthesiology (English Edition). January-February 2016: V66, Issue 1; P 29-36. doi:10.1016/j.bjane.2015.01.006

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Effect of vitamin C on collagen structure of cardinal and uterosacral ligaments during pregnancy

Abstract

Objective

This study aimed to investigate changes in collagen structure in the cardinal and uterosacral ligaments of rats that were administered vitamin C during pregnancy.

Study design

Eighteen female rats were divided into three groups: six pregnant rats administered 1.25 mg/ml/day of vitamin C during pregnancy (Group A); six non-pregnant rats that were not administered vitamin C (Group B); and six pregnant rats that were not administered vitamin C during pregnancy (Group C). Fifteen days after delivery, the uteruses of all rats were removed. The intensity of staining (mild, moderate or severe) and the extent of positive staining areas (%) of type I and type III collagen H scores for types I and III collagen, and intensity of elastin fibres in the cardinal and uterosacral ligaments were investigated immunohistochemically. Differences between groups were analysed using Kruskal–Wallis and independent samples tests.

Results

The intensity and extent of type I and type III collagen, the H scores for type I and type III collagen, and the ratio of type III collagen H score: type I collagen H score differed significantly between groups. Pregnant rats administered vitamin C (Group A) had significantly higher values compared with non-pregnant rats (Group B): intensity of type I collagen (p = 0.001), extent of type I collagen (p ≤ 0.001), H score for type I collagen (p ≤ 0.001), intensity for type III collagen (p = 0.002), extent of type IV collagen (p = 0.007), H score for type III collagen (p = 0.017), typeIII collagen H score: type I collagen H score (p = 0.039) and intensity of elastin fibres (p = 0.097). A significant difference in the ratio of type III collagen H score: type I collagen H score was found between pregnant rats administered vitamin C (Group A) and pregnant rats not administered vitamin C (Group C) (p = 0.002).

Conclusions

The administration of vitamin C to rats during pregnancy had a favourable impact on collagen structure in the cardinal and uterosacral ligaments, suggesting that vitamin C supplementation during pregnancy may help to prevent pelvic organ prolapse and stress urinary incontinence.

Reference:

R.B. Findik et. al., Effect of vitamin C on collagen structure of cardinal and uterosacral ligaments during pregnancy. European Journal of Obstetrics & Gynecology and Reproductive Biology. June 2016: V201; P31-35. doi:10.1016/j.ejogrb.2016.03.022

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Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow

Abstract
A number of studies have revealed that Type I diabetes (T1D) is associated with bone loss and an increased risk of fractures. T1D induces oxidative stress in various tissues and organs. Vitamin C plays an important role in the attenuation of oxidative stress; however, little is known about the effect of T1D induced oxidative stress on the regulation of vitamin C transporter in bone and bone marrow cells. To investigate this, T1D was induced in mice by multiple low dose injections of streptozotocin. We have demonstrated that endogenous antioxidants, glutathione peroxidase (GPx) and superoxide dismutase (SOD) are down-regulated in the bone and bone marrow of T1D. The vitamin C transporter isoform SVCT2, the only known transporter expressed in bone and bone marrow stromal cells (BMSCs), is negatively regulated in the bone and bone marrow of T1D. The μCT imaging of the bone showed significantly lower bone quality in the 8 week T1D mouse. The in-vitro study in BMSCS showed that the knockdown of SVCT2 transporter decreases ascorbic acid (AA) uptake, and increases oxidative stress. The significant reversing effect of antioxidant vitamin C is only possible in control cells, not in knockdown cells. This study suggested that T1D induces oxidative stress and decreases SVCT2 expression in the bone and bone marrow environment. Furthermore, this study confirms that T1D increases bone resorption, decreases bone formation and changes the microstructure of bones. This study has provided evidence that the regulation of the SVCT2 transporter plays an important role not only in T1D osteoporosis but also in other oxidative stress-related musculoskeletal complications.

Reference:

Rajnikumar Sangani. Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow. Experimental and Molecular Pathology. December 2013: V95, Issue 3; P298-306. doi:10.1016/j.yexmp.2013.08.007

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Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8+ memory T cell production capacity of these cells in vivo

Abstract

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4+ T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8+ T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ+ CD8+ T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44high CD62Llow CD8+ effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors.

Reference:

Young-Joo Jeong. Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8+ memory T cell production capacity of these cells in vivo. Immunobiology. July 2014: V219, Issue 7; P554-564. doi:10.1016/j.imbio.2014.03.006

Calcium (as citrate)

Calcium citrate: a new biomaterial that can enhance bone formation in situ

Abstract

Objective

To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone regeneration in a bone defect rabbit model.

Methods

Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citrate-rhBMP-2 (CC-rhBMP-2) group and rhBMP-2 only group. Two 10 mm-long and 5 mm-deep bone defects were respectively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate (10 mg) combined with rhBMP-2 (2 mg) or rhBMP-2 alone were implanted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD /-test and a subsequent /-test were adopted for statistical analysis.

Results

Histomorphometric analysis revealed newly formed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±L26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks (P<0.05 for all). At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group.

Conclusion

From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly enhances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions.

Reference:

Wang Li-ming et. Al., Calcium citrate: a new biomaterial that can enhance bone formation in situ. Chinese Journal of Traumatology. V15, Issue 5; P291-296. doi:10.3760/cma.j.issn.1008-1275.2012.05.007

http://www.sciencedirect.com/science/article/pii/S1008127515303333

Persisting Hypocalcemia after Surgical Parathyroidectomy: The Differential Effectiveness of Calcium-Citrate vs. Calcium-Carbonate with Acid Suppression

Abstract

The effectiveness of oral calcium (Ca) may be contingent on patient’s factors beyond compliance, such as proton-pump inhibitor (PPI) use and the choice of calcium supplements. A 32 year-old Hispanic male with end-stage renal disease on peritoneal dialysis (PD) underwent successful surgical parathyroidectomy (intact parathyroid hormone level: 2,328 pg/mL; post-surgical: 287-69 pg/mL [normal: 8.5-72.5]). His post-operative course was complicated by severe and recurrent hypocalcemia as outpatient and he needed repeated admissions for IV Ca-gluconate. Initially, severe hypocalcemia (corrected Ca: 4.8-5.6 mg/dL; nadir ionized Ca: 0.57-0.69 mmol/L) was attributed solely to medical non-compliance with oral Ca-carbonate (3750 mg, x3/day between meals) and calcitriol (2-4 mcg/day). Recognizing co-existing treatment with PPI, oral Ca-supplement was changed to calcium-citrate (2,850 mg x3/day), with prompt resolution of hypocalcemia (corrected Ca 8.1-8.3 mg/dL). This current case and the included literature review emphasizes the disproportionate effectiveness of Ca-citrate in subjects with achlorhydria.

Reference:

Sabahat Afshan, M.D et. al., Persisting Hypocalcemia after Surgical Parathyroidectomy: The Differential Effectiveness of Calcium-Citrate vs. Calcium-Carbonate with Acid Suppression.
The American Journal of the Medical Sciences. April 22,2016: Available online ; In Press, Accepted Manuscript — Note to users. doi:10.1016/j.amjms.2016.04.007

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Aqueous solubility of calcium citrate and interconversion between the tetrahydrate and the hexahydrate as a balance between endothermic dissolution and exothermic complex formation

Abstract

Aqueous solubility of calcium citrate tetrahydrate was found to decrease with increasing temperature, while solubility of hexahydrate increased with a transition temperature at 51.6 °C. Excess citrate increased calcium citrate solubility but decreased the calcium ion activity of the saturated solution with an initial solubility overshooting to form supersaturated solutions indicating binding of calcium to citrate with an association constant of 3.6 ± 0.1 × 104, ΔHº = −5.07 ± 0.04 kJ mol−1, ΔSº = 70.3 ± 0.3 J mol−1 K−1 at 25 °C. Dissolution of the tetrahydrate and hexahydrate was found to have ΔHº = 27 ± 9 kJ mol−1, ΔSº = −218 ± 30 J mol−1 K−1 and ΔHº = 57 ± 7 kJ mol−1, ΔSº = −126 ± 24 J mol−1 K−1, respectively, as determined from the temperature dependence of solubility corrected for complex formation. The exothermic complex formation results in inverse solubility only for the tetrahydrate with its moderate endothermic dissolution, which also precipitates at ambient temperature rather than the less soluble hexahydrate.

Reference:Martina Vavrusova, Leif H. Skibsted. Aqueous solubility of calcium citrate and interconversion between the tetrahydrate and the hexahydrate as a balance between endothermic dissolution and exothermic complex formation. International Dairy Journal. June 2016: V57, P20-28. doi:10.1016/j.idairyj.2016.02.033

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Anti-inflammatory effects of calcium citrate in RAW 264.7 cells via suppression of NF-κB activation

Abstract

Here we aimed to investigate the anti-inflammatory effects of calcium citrate in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The anti-inflammatory effects of calcium citrate were investigated by assessing pro-inflammatory factors (NO, ROS, NF-κB, iNOS, and COX-2) and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). Treatment of cells with calcium citrate (10–100 μM) significantly reduced the generation of intracellular reactive oxygen species and increased the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase in LPS-stimulated macrophages. Calcium citrate was further shown to inhibit NO production in LPS-stimulated RAW 264.7 cells. The expression levels of iNOS, COX-2, and NF-κB were also suppressed by treatment with calcium citrate. Calcium citrate was furthermore found to significantly inhibit the production of IL-1β, IL-6, and TNF-α in response to LPS-stimulation. These findings demonstrate that calcium citrate may be an effective anti-inflammatory agent.

Reference: Eun-Young Choia, Hak-Ju Kimb, Ji-Sook Han. Anti-inflammatory effects of calcium citrate in RAW 264.7 cells via suppression of NF-κB activation. Environmental Toxicology and Pharmacology. January 2015: V39, Issue 1; P27-34. doi:10.1016/j.etap.2014.11.002

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Efficacy of Mixtures of Magnesium, Citrate and Phytate as Calcium Oxalate Crystallization Inhibitors in Urine

Purpose

The main aim of the current study was to evaluate the effectiveness of mixtures of magnesium, citrate and phytate as calcium oxalate crystallization inhibitors.

Materials and Methods

A turbidimetric assay in synthetic urine was performed to obtain induction times for calcium oxalate crystallization in the absence and presence of different mixtures of inhibitors. The morphology of calcium oxalate crystals in the absence or presence of inhibitors and mixtures of the inhibitors was evaluated in 2 crystallization experiments at low and high calcium oxalate supersaturation. The crystals formed were examined using scanning electron microscopy.

Results

Examination of crystallization induction times revealed clear inhibitory effects of magnesium, citrate and phytate on calcium oxalate crystallization, supporting usefulness in the treatment and prevention of calcium oxalate nephrolithiasis. Significant synergistic effects between magnesium and phytate were observed. Scanning electron microscopy images revealed that phytate is a powerful crystal growth inhibitor of calcium oxalate, totally preventing the formation of trihydrate and monohydrate. In addition to crystallization inhibition capacity, citrate and magnesium avoided calcium oxalate crystallization by decreasing its supersaturation.

Conclusions

The synergistic effect between magnesium and phytate on calcium oxalate crystallization suggests that a combination of these 2 compounds may be highly useful as antilithiasis therapy.

Reference:Felix Grases, , Adrian Rodriguez, Antonia Costa-Bauza. Efficacy of Mixtures of Magnesium, Citrate and Phytate as Calcium Oxalate Crystallization Inhibitors in Urine. The Journal of Urology. September 2015: V194, Issue 3; P812-849. doi:10.1016/j.juro.2015.03.099

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Superior calcium bioavailability of effervescent potassium calcium citrate over tablet formulation of calcium citrate after Roux-en-Y gastric bypass

Abstract

Background

Calcium supplementation is commonly recommended for patients after Roux-en-Y gastric bypass to avert bone loss. To test the hypothesis that effervescent (liquid) potassium-calcium-citrate (PCC) might be more bioavailable than a tablet formulation of calcium citrate (Citracal Petite), the present study compared a single dose response of the 2 compounds. The present study was conducted at the University of Texas Southwestern Medical School at Dallas.

Methods

A total of 15 patients who had undergone Roux-en-Y gastric bypass were included in a 2-phase, crossover, randomized study comparing the single-dose bioavailability of PCC versus Citracal Petite. After following a restricted diet for 1 week, the participants ingested either a single dose of 400 mg elemental calcium as PCC or Citracal Petite. Sequential serum and urine samples were collected for a 6-hour period after the dose and analyzed for calcium, parathyroid hormone, and acid-base parameters.

Results

Compared with citracal petite, PCC significantly increased the serum calcium concentrations at 2, 3, and 4 hours after the oral load. The peak to baseline variation and increment in serum calcium (area under the curve) were significantly greater after PCC (P = .015 and P = .002, respectively). Concurrently, the baseline to nadir variation and decrement in serum parathyroid hormone (area over the curve) were significantly greater after PCC (P = .004 and P = .005, respectively). Moreover, compared with Citracal Petite, PCC caused a significantly greater increment in urinary citrate (P < .0001) and potassium (P = .0004) and a significantly lower increase in urinary ammonium (P = .045).

Conclusion

In patients who have undergone Roux-en-Y gastric bypass, PCC was superior to Citracal Petite in conferring bioavailable calcium and suppressing parathyroid hormone secretion. PCC also provided an alkali load.

Reference:Khashayar Sakhaee, M.D., Charles Pak, M.D. Superior calcium bioavailability of effervescent potassium calcium citrate over tablet formulation of calcium citrate after Roux-en-Y gastric bypass. Surgery for Obesity and Related Diseases. September–October 2013: V9, Issue 5; P 743-748. doi:10.1016/j.soard.2011.11.011

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Effect of calcium citrate on bone integration in a rabbit femur defect model

Abstract

Objective

To explore effect of calcium citrate on bone integration in a rabbit femur defect model, and to compare the bone formation with different sizes by radiological and histological study.

Methods

Twenty-four male Japanese white rabbits were randomly divided into three groups (Group A, B, C) in this study. Under anesthesia, defects of four sizes (1.2, 1.5, 2.0 and 2.5 mm) were created in each of the rabbits. Commercially pure calcium citrate powder was placed inside the medullary compartment of the femur (Experimental), while in the contralateral femur (Control) nothing was implanted. The defects were analyzed using radiography and histological analysis by using Imagepro-Plus 6.0 software after animal was sacrificed at 4th(Group A), 6th(Group B) and 8th(Group C) weeks postoperatively. Four samples were analyzed for each size of defect and each healing period.

Results

The histological and the radiologic evaluation were performed after sacrification of all rabbits on postoperative 4th and 6th weeks, It showed significant difference between the experimental group and the control group when these defects were less than or equal to 2.0 mm. No statistical difference was observed when these defects were larger than 2.0 mm at all healing periods except at the 4th week.

Conclusions

Calcium citrate affects the early periods of bone defects healing mechanism in Japanese white rabbits positively, especially when the defect is not too large. We suggest further studies on calcium citrate to determine the effects of various dosages, administration ways and the experimental time on the bone defects.

Reference:Wei Zhang et. al.. Effect of calcium citrate on bone integration in a Asian Pacific Journal of Tropical Medicine rabbit femur defect model. April 2012: V5, Issue 4; P310-314. doi:10.1016/S1995-7645(12)60045-5

Biochemical control of bone loss and stone-forming propensity by potassium-calcium citrate after bariatric surgery

Abstract

Background

Patients undergoing Roux-en-Y gastric bypass (RYGB) surgery are prone to developing bone loss and kidney stones. The goal of the present study was to test the hypothesis that an effervescent formulation of potassium calcium citrate (PCC) would avert metabolic complications by providing bioavailable calcium and alkali.

Methods

A total of 24 patients with RYGB underwent a 2-phase crossover randomized trial comparing PCC and placebo. During the last 2 days of each 2-week phase, the serum and 24-hour urine samples were analyzed for calcium and bone turnover markers, acid base status, and urinary stone risk factors.

Results

Compared with placebo, PCC marginally reduced the serum parathyroid hormone level and significantly decreased urinary deoxypyridinoline by 12% (P <.001) and serum type 1 collagen C-telopeptide by 22% (P <.01). PCC significantly increased the net gastrointestinal alkali absorption, citrate, and pH and significantly lowered the urinary net acid excretion (P <.001). The urinary saturation of uric acid decreased significantly (P<.001). The supersaturation of calcium oxalate and brushite did not change despite an increase in calcium and pH. In untreated urine samples with citrate concentrations altered to mimic those of placebo and PCC, calcium oxalate agglomeration was significantly inhibited by PCC.

Conclusion

In RYGB patients, PCC supplementation inhibited bone resorption by providing bioavailable calcium, reduced the urinary saturation of uric acid, and increased the inhibitor activity against calcium oxalate agglomeration by providing alkali that increased urinary pH and citrate.

Reference:Khashayar Sakhaee, M.D., , Carolyn Griffith, R.N., Charles Y.C. Pak, M.D. Biochemical control of bone loss and stone-forming propensity by potassium-calcium citrate after bariatric surgery. Surgery for Obesity and Related Diseases. January–February 2012: V8, Issue 1; P 67-72. doi:10.1016/j.soard.2011.05.001

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Calcium supplementation attenuates citrate-related changes in bone metabolism: A placebo-controlled crossover study in healthy volunteers

Abstract

Background

Citrate is the anticoagulation of choice in apheresis procedures. Citrate anticoagulation results in a short-term increase in serological markers of bone turnover, with uncertain clinical significance.

Aim

To understand the effect of calcium supplementation on serological bone turnover markers during an acute citrate load as a mimic of citrate anticoagulation during apheresis procedures.

Methods

A placebo-controlled, crossover study was conducted in 22 healthy volunteers. Volunteers received a standardized citrate load at a fixed dose of 1.5 mg/kg of body weight/min for 80 min for three times and a single placebo infusion as a control. Each intervention was separated by a wash-out interval of 2 to 3 weeks. During two citrate infusions, volunteers received an additional calcium supplementation, consisting of either oral administration of calcium carbonate or an i.v. bypass infusion of calcium gluconate. Serial blood samples were collected for the determination of ionized calcium (iCa), intact parathyroid hormone (iPTH) and markers of bone remodeling, C-telopeptide of type 1 collagen (CTX) and osteocalcin (OC).

Results

The infusion of citrate without calcium supplementation resulted in an increase in the bone formation marker OC and the bone resorption marker CTX, in addition to the changes in iPTH and iCa. The administration of calcium by either oral administration or as an i.v. bypass infusion attenuated the observed changes in CTX, but showed no effects on the elevation of the bone formation marker OC. There was no difference in the attenuation of CTX between the two calcium formulations. However, the i.v. application of calcium gluconate had a superior effect in reducing the change of serum iPTH and iCa as compared to the oral administration of calcium carbonate.

Conclusions

Calcium supplementation is an effective method in damping the citrate-related transient increase of the serological bone resorption marker CTX. As a mimic for the citrate-based apheresis procedure, our data may enforce the prophylactic application of calcium supplementation to attenuate the short-term elevation of bone resorption related to an acute citrate load.

Reference:

Ying Chen et. al.. Calcium supplementation attenuates citrate-related changes in bone metabolism: A placebo-controlled crossover study in healthy volunteers. Bone. September 2011: V49, Issue 3; P506-512. doi:10.1016/j.bone.2011.05.014

Manganese (as sulfate)

Solubility of Manganese Sulfate Monohydrate in the Presence of Trace Quantities of Magnesium Sulfate Heptahydrate in Water

Abstract

The solubility of aqueous manganese sulfate monohydrate has been measured in the presence of magnesium sulfate as the second salt. Over the temperature range from 273 to 353 K, aqueous manganese sulfate on its own exhibited an unusual behavior in that its solubility reached a maximum at an intermediate temperature of 293K. The solubilities of manganese sulphate under and over the maximum solubility temperature were first predicted individually by the electrolyte NRTL model. The binary interaction parameters and solubility parameters were universal parameters that were applied to any salts consisting of manganese, magnesium and sulfate ions, and they were newly obtained from the binary solubility data. The predicted solubility curves could represent the experimental data quantitatively, and adequately described the effect of co-salt of magnesium. The experimental solubility data were also satisfactorily correlated with the electrolyte NRTL model.

Reference:Kouji Maeda ET.AL., Solubility of Manganese Sulfate Monohydrate in the Presence of Trace Quantities of Magnesium Sulfate Heptahydrate in Water. AsiPacific Journal of Chemical engineering. March 15 2008: V11, Issue 5-6; P423-435,2003. DOI: 10.1002/apj.5500110602

http://onlinelibrary.wiley.com/doi/10.1002/apj.5500110602/abstract?systemMessage=Subscribe+and+renew+is+currently+unavailable+online.+Please+contact+customer+care+to+place+an+order%3A++http%3A%2F%2Folabout.wiley.com%2FWileyCDA%2FSection%2Fid-397203.html++.Apologies+for+the+inconvenience.

Redox spectrophotometric method involving electrolytically generated manganese(III) sulphate with diphenylamine for the determination of ascorbic acid present in the samples of various fruits, commercial juices and sprouted food grains

Abstract

A spectrophotometric method was developed for ascorbic acid present in various fruits, commercial fruit juices and sprouted food grains. The method involves the oxidation of ascorbic acid with excess manganese(III) following reduction of unreacted manganese(III) with diphenylamine or barium diphenylamine sulphonate forming a product λmax 570 nm the system 1 or 540 nm the system 2 and decrease in the colour intensity is proportional to the concentration of vitamin C with quantification range 0.3–3.0 μg ml−1. The molar absorptivity and Sandell’s sensitivity values of the system 1 and the system 2 were 1.829 × 104 and 1.813 × 104mol−1 cm−1 and 0.0096 and 0.0097 μg cm−2 respectively. The stoichiometry was 4:1 between manganese(III) and diphenylamine. The ascorbic acid contents of the same samples were determined separately following the procedures of the developed method as well as the reference method and the results were comparable.

Reference: B. Shyla, G. Nagendrappa. Redox spectrophotometric method involving electrolytically generated manganese(III) sulphate with diphenylamine for the determination of ascorbic acid present in the samples of various fruits, commercial juices and sprouted food grains. Food Chemistry. 1 June 2013,: V138, Issue 2-3; P2036-2042. doi:10.1016/j.foodchem.2012.11.076

Bioaccumulation and locomotor effects of manganese sulfate in Sprague–Dawley rats following subchronic (90 days) inhalation exposure

Abstract

Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic compound that was introduced as an antiknock additive to replace lead in unleaded fuel. The combustion of MMT results in the emission of fine Mn particulates mainly in the form of manganese sulfate and manganese phosphate. The objective of this study is to determine the effects of subchronic exposure to Mn sulfate in different tissues, on locomotor activity, on neuropathology, and on blood serum biochemical parameters. A control group and three groups of 30 male Sprague–Dawley rats were exposed 6-h/day, 5 days/week for 13 consecutive weeks at 30, 300, or 3000 μg/m3 Mn sulfate. Locomotor activity was measured during 36 h using an Auto-Track System. Blood and the following tissues were collected and analyzed for manganese content by neutron activation analysis: olfactory bulb, globus pallidus, caudate/putamen, cerebellum, frontal cortex, liver, lung, testis, and kidney. Neuronal cell counts were obtained for the caudate/putamen and the globus pallidus and clinical biochemistry was assessed. Manganese concentrations were increased in blood, kidney, lung, and testis and in all brain regions in the 3000 μg/m3exposure group. Significant differences were also noted in the 300 μg/m3 exposure group. Neuronal cell counts for the globus pallidus were significantly different between the two highest exposed groups and the controls. Locomotor activity for all exposure concentrations and resting time for the middle and highest concentrations for the two night resting periods were significantly increased. Total ambulatory count was decreased significantly for all exposure concentrations. Biochemical profiles also presented significant differences. No body weight loss was observed between all groups. These results suggest that neurotoxicity could occur at low exposure levels of Mn sulfate, one of the main combustion products of MMT.

Reference:

Danielle Tapina, Greg Kennedyb, Jean Lambertc, Joseph Zayed.Bioaccumulation and locomotor effects of manganese sulfate in Sprague–Dawley rats following subchronic (90 days) inhalation exposure. March 1,2006: V211, Issue 2; P166-174. doi:10.1016/j.taap.2005.07.007

Maternal–fetal Distribution of Manganese in the Rat Following Inhalation Exposure to Manganese Sulfate

Abstract

Studies examining the pharmacokinetics of manganese during pregnancy have largely focused on the oral route of exposure and have shown that the amount of manganese that crosses the rodent placenta is low. However, limited information exists regarding the distribution of manganese in fetal tissues following inhalation. The objective of this study was to determine manganese body burden in CD rats and fetuses following inhalation of a MnSO4 aerosol during pregnancy. Animals were evaluated following pre-breeding (2 weeks), mating (up to 14 days) and gestational (from gestation day (GD) 0 though 20) exposure to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 h/day, 7 days/week. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. Fetal tissues were examined on GD 20 and included whole blood, lung, liver, brain, and skull cap. Maternal lung manganese concentrations were increased following exposure to MnSO4 at ≥0.05 mg Mn/m3. Maternal brain and placenta manganese concentrations were increased following exposure of pregnant rats to MnSO4 at ≥0.5 mg Mn/m3. Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at ≥0.5 mg Mn/m3. Manganese concentrations within all other fetal tissues were not different from air-exposed controls. The results of this study demonstrate that the placenta partially sequesters inhaled manganese, thereby limiting exposure to the fetus.

Reference:David C. Dorman et. al.. Maternal–fetal Distribution of Manganese in the Rat Following Inhalation Exposure to Manganese Sulfate. NeuroToxicology. August 2005: V26, ssue 4; P625-632. doi:10.1016/j.neuro.2004.08.004

Copper (as gluconate)

Copper inclusion in cellulose using sodium d-gluconate complexes

Abstract

Copper containing cellulose material is of growing interest, e.g. offering alternative in the field of antimicrobials. Solutions of copper d-gluconate complexes (Cu2+–DGL) were used to introduce copper ions into a swollen cellulosic matrix. A ligand exchange mechanism forms the chemical basis of the sorption process. Copper sorption in cellulose was studied in the range between pH 6 and 13. An estimate for the complex stabilities of the Cu-cellulose system could be derived from the calculated species distribution of the different Cu2+–DGL complexes present. Spectrophotometry and cyclic voltammetry of Cu2+–DGL complex solution were used to confirm the presence of different species participating in the ligand exchange reaction. The pH dependent uptake of Cu2+ ions in the cellulose matrix can be explained on the basis of the relative stabilities of Cu2+–DGL complex vs. Cu2+–cellulose complexes. In comparison to pH 10, higher copper content was observed at pH 6 and 13. Copper content was limited by carboxyl content of cellulosic materials, thus in analogy to the structure of Cu2+–DGL complexes participation of the carboxyl group as complex forming site is proposed. At high Cu2+-concentration and longer time of immersion in the copper complex solutions formation of solid deposits was observed on the surface of the treated fibres.

Reference:Hossam E. Emam, Avinash P. Manian, Barbora Široká, Thomas Bechtold, Copper inclusion in cellulose using sodium d-gluconate complexes. Carbohydrate Polymers. October 15, 2012: V90, Issue 3, P1345-1352. doi:10.1016/j.carbpol.2012.07.003

Role of copper gluconate/triethanolamine in irinotecan encapsulation inside the liposomes

Abstract

A novel method for encapsulating irinotecan into liposomes containing copper gluconate buffered to pH 7.0 with triethanolamine (TEA) has recently been developed. In the present study, the mechanism dictating drug encapsulation and retention inside those liposomes was investigated. Spectroscopic analyses revealed that irinotecan interacted with copper gluconate/TEA in solution. Fourier transformed infrared (FT-IR) spectroscopy indicated a strengthening of the hydrogen bonds involving the hydroxyl groups when solutions of irinotecan and copper gluconate/TEA are mixed at a 1:1 molar ratio. The intensity of the circular dichroism (CD) signal of copper gluconate/TEA increased in the presence of equimolar amounts of irinotecan. The addition of irinotecan to liposomes containing copper gluconate/TEA at 50 °C induced a shift of the absorption bands from 370 nm to 378 nm as well as a 60% quenching of the drug fluorescence at 440 nm suggesting the occurrence of irinotecan self association. Irinotecan encapsulation was found to be kinetically and stoichiometrically correlated with the release of TEA from the liposomes. The results suggested that the encapsulation of irinotecan was mediated by TEA in association with copper gluconate, leading to a final drug complex that is retained inside the liposomes. A neutral antiport exchange loading mechanism between irinotecan and TEA is proposed.

Reference:Awa Dicko, Paul Tardi, Xiaowei Xie, Lawrence Mayer. Role of copper gluconate/triethanolamine in irinotecan encapsulation inside the liposomes. International Journal of Pharmaceutics. June 7, 2007: V337, Issue 1-2; P219-228. doi:10.1016/j.ijpharm.2007.01.004

Disulfiram and Copper gluconate in cancer chemotherapy;

a review of the literature

Abstract:

Repurposing non- cancer related drugs with possible antitumoral activities is a promising strategy for identifying prospective new anticancer drugs in a cost efficient and time saving way. Repurposing disulfiram has recently become of interest because of its pre-clinically described anticancer effects against various human cancers, which include breast, cervical, colorectal, lung, melanoma, prostate as well as myeloma and leukaemia. Epidemiological studies reveal a trend to reduced cancer risks in cancer patients using disulfiram for chronic alcoholism treatment while already reported literature point to the efficacy of disulfiram on cancer cell lines. Disulfiram has been shown to be effective either as a stand alone or in combination with other drugs against metastatic liver cancer, lung cancer, prostate cancer, glioblastoma and melanoma. Preclinical studies indicate that disulfiram when combined with copper ions acts as a proteasome inhibitor, to induce oxidative stress, reduce NFƙB (Nuclear factor ƙappa Binding) activity and enhance the sensitivity of cancer cells to chemotherapeutic agents. This study analyzed existing literature and found that disulfiram in combination with copper gluconate is a promising therapeutic agent for use in cancer chemotherapy.

Reference:Georgewill Udeme Owunari et. al..Disulfiram and Copper gluconate in cancer chemotherapy; a review of the literature. Cancer Research Journal. September30, 2014: V2, Issue 5; P88-92. doi: 10.11648/j.crj.20140205.12

CMO (cetyl myristolate)

Synthesis of cetyl myristoleate and evaluation of its therapeutic efficacy in a murine model of collagen-induced arthritis

Abstract

Cetyl myristoleate (CM) was reported by Diehl and May [J Pharm Sci 83 (1994) 296] to block inflammation and prevent adjuvant-induced arthritis in rats. To verify this earlier work, we have synthesized pure CM and tested its anti-arthritic properties in a collagen-induced arthritis model in DBA/1LacJ mice. Multiple intraperitoneal injections of CM in 450 and 900 mg kg−1 doses resulted in a significantly lower incidence of disease and caused a modest but significant diminution in clinical signs in those mice that developed arthritis. CM administered in daily oral doses of 20 mg kg−1 also reduced the incidence of arthritis and caused a small reduction in the clinical signs in mice that developed arthritis. Although the protective effect of CM in collagen-induced arthritis observed in the present study was less dramatic than that reported earlier, our results confirm the anti-arthritic properties of pure CM.

Reference:

Kenneth W Hunter Jr.a, , , Ruth A Gaulta, Jeffrey S Stehouwerb, Suk-Wah Tam-Chang, Synthesis of cetyl myristoleate and evaluation of its therapeutic efficacy in a murine model of collagen-induced arthritis. Pharmacological Research. January 2003: V47, Issue 1, P43-47. doi:10.1016/S1043-6618(02)00239-6

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Cetyl myristoleate isolated from swiss albino mice: An apparent protective agent against adjuvant arthritis in rats

Abstract

Cetyl myristoleate was isolated from National Institutes of Health, general purpose, Swiss albino mice that were immune to the polyarthritis induced in rats with Freund’s adjuvant. This substance, or material synthesized from cetyl alcohol and myristoleic acid, afforded good protection against adjuvant-induced arthritic states in rats. In limited comparisons, cetyl oleate, also found in Swiss albino mice, gave lesser protection, whereas cetyl myristate and cetyl elaidate, the trans-isomer of cetyl oleate, appeared to be virtually ineffective. Dosage of the protective compound as well as the site of injection of Freund’s adjuvant was important.

Reference:Harry W. Diehl†* andEverette L. May. Cetyl myristoleate isolated from swiss albino mice: An apparent protective agent against adjuvant arthritis in rats. Journal of Pharmaceutical Sciences. March 1994: V83, Issue 3; P296-299. DOI: 10.1002/jps.2600830307

Statement on the safety of ‘Cetyl Myristoleate Complex’ as an ingredient in food supplements

ABSTRACT

Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies was asked to update its opinion on the safety of ‗Cetyl Myristoleate Complex‘ (CMC) as a novel food ingredient in the light of additional information submitted by the applicant. In its previous opinion of 2010, the Panel concluded that the safety of CMC as an ingredient in food supplements at an intake of 3.3 g per day has not been established. This conclusion was based on the considerations that in the absence of appropriate data on absorption, distribution, metabolism and excretion, the provided toxicological data were insufficient. In 2012, the Commission requested EFSA to review and update its opinion by taking into account a new subchronic 90- day oral toxicity study conducted with ―Cetylated Fatty Acid Esters Powder 50 %‖ in mice. In its opinion of 2013, the Panel considered that a new 90-day study cannot serve as a reliable source of information supporting the absence of adverse effects of CMC. The dossier of this new mandate contains three new references which were not submitted and hence not considered in the previous assessments. The Panel notes that two references do not address the concerns expressed by the Panel in its previous assessments. The third reference provided is a report on an in vitro hydrolysis study demonstrating a low rate of hydrolysis of cetyl myristoleate and cetyl myristate. The Panel notes the low rate of hydrolysis of the two esters found in this in vitro hydrolysis study and therefore reiterates the need for adequate safety information on the unhydrolysed esters contained in CMC as expressed in its opinions of 2010 and 2013. The Panel concludes that, even after considering the newly submitted information, the safety of ‗Cetyl Myristoleate Complex‘ has not been established.

Reference: Panel on Dietetic Products, Nutrition and Allergies. European Food Safety Authority. Statement on the safety of ‘Cetyl Myristoleate Complex’ as an ingredient in

food supplements1. EFSA Journal. 2014: 13; (6):3261, P9. DOI: 10.2903/j.efsa.2013.3261

http://www.efsa.europa.eu/en/efsajournal/pub/3261

CMO (Cerasomol- cis -9-Cetyl Myristoleate) in the Treatment of Fibromyalgia: An Open Pilot Study

Abstract

Purpose: To investigate the efficacy and tolerability of CMO (cerasomol- cis -9-cetyl myristoleate) in the treatment of fibromyalgia. Design: Open study: 21 days’ treatment following a 7-day baseline without treatment. Materials and Methods: Thirteen adult patients with fibromyalgia. Following a 7-day pre-treatment baseline, patients were treated with a mixture of CMO + sea cucumber and shark cartilage extracts. All treatments were administered orally. Patients kept daily diary cards recording the severity of generalized pain, fatigue and sleep disturbance (scale 0-4). A symptom profile was made using an 84-point questionnaire. Adverse events were recorded. Results: Ten patients completed the study. One subject did not record baseline symptoms. Two patients had to discontinue treatment because of adverse effects. One had severe indigestion and the other developed muscle spasms and a skin rash. The mean (SD) scores for the severity of pain, fatigue, and sleep disturbance during the baseline were 2.63 (0.55), 2.50 (0.54) and 2.33 (0.480). These improved to 1.70 (0.82), 1.83 (0.82) and 1.73 (1.11) during the last 7 days of treatment. There were individual differences in response with five patients showing good response and five showing little or no response. From the questionnaires those showing the best response had more severe pain and cognitive dysfunction than the non-responders. In addition to the two withdrawals, one further patient reported gastrointestinal symptoms. Conclusions: This open study provides evidence of a beneficial effect of CMO in the treatment of fibromyalgia. A double-blind, placebo-controlled study is now required to confirm this. This should also include a symptom profile questionnaire, which may help to distinguish responders and non-responders.

Reference: A. M. Edwards. CMO (Cerasomol- cis -9-Cetyl Myristoleate) in the Treatment of Fibromyalgia: An Open Pilot Study. Journal of Nutritional & Environmental Medicine. July 13, 2009: V11, Issue 2; P105-111. DOI:10.1080/13590840120060849

Glucosamine Sulfate

Kinetics of cathodic processes in Cu(II) gluconate solutions containing an excess of sulfate

Abstract

Chronopotentiometry and linear potential sweep (LPS) voltammetry were applied to study cathodic processes occurring at the Cu electrode in Cu(II) solutions containing gluconate (L) and excess of sulfate at 2 < pH < 5. The data obtained are indicative of sufficiently high lability of both CuL+ and CuSO4 complexes. The effective diffusion coefficients obtained from transition times (τ) are in the range from 4.4 × 10−6 to 3.7 × 10−6 cm2 s−1 and decrease with the molar fraction of CuL+. No adsorbed electrochemically active complexes (EAC) were detected. Additional τ′ observed at pH 5 results from the reduction of surface Cu2O that is formed at the open-circuit potential.

LPS voltammograms contain two current maxima conditioned by Cu(II) and H3O+reduction, respectively. The first of them contains irregularities that disappear when perchlorate is substituted for sulfate. Cu2+ aqua-ions take part in the consecutive transfer of two electrons whereas CuL+ complexes seem to be electrochemically inert. Cathodic charge transfercoefficients (α = 0.43 ± 0.03), determined by different methods, correlate well. A role of adsorption processes has also been discussed.

Highlights

► Kinetics of Cu(II) reduction in gluconate solutions, containing an excess of sulfate, is studied. ► Effective diffusion coefficients decrease with the molar part of Cu(II)-gluconate complexes. ► LPS voltammograms contain two current maxima conditioned by Cu(II) and H3O+ reduction. ► Reduction of surface Cu2O is observed at pH 5.

Reference:

  1. Survila, S. Kanapeckaitė., Kinetics of cathodic processes in Cu(II) gluconate solutions containing an excess of sulfate. Electrochimica Acta. September 1, 2012: V78, P359-364. doi:10.1016/j.electacta.2012.06.010

http://www.sciencedirect.com/science/article/pii/S0013468612009164

The reverse glucosamine sulfate pathway: application in knee osteoarthritis

Abstract

Glucosamine is a natural amino sugar and a normal constituent of glycosaminoglycans in the cartilage matrix and synovial fluid of joints. Crystalline glucosamine sulfate salt has been approved as a medicinal product for the treatment of osteoarthritis in several European countries. Nevertheless, although it has been prescribed for more than 10 years, it is only dueto the research in the last 5 years that the scientific basis underlying its beneficial effects are starting to be clarified. In randomised, double-blind, placebo-controlled trials, this compound clinically controls pain and produces beneficial effects in patients with knee osteoarthritis, possibly delaying the appearance of long-term structural changes in the joint (i.e., it has a structure-modifying effect). Furthermore, it has an excellent toxicity profile. Despite the different lines of investigation that have been followed, the mechanism of action of glucosamine sulfate still remains to be clearly defined. However, the activity of glucosamine sulfate has recently been related to its capacity to downregulate the catabolic effects of pro-inflammatory molecules, such as IL-1, which are present in osteoarthritic cartilage.

Reference:Gabriel Herrero-Beaumont et. al.. The reverse glucosamine sulfate pathway: application in knee osteoarthritis. Expert Opinion on Pharmacotherapy. January 26, 2007: V8, Isssue 2; P215-225. DOI:10.1517/14656566.8.2.215

Evaluation of the Physicochemical Stability and Skin Permeation of Glucosamine Sulfate

Abstract

Glucosamine sulfate (GS) is known to stop the degenerative process of osteoarthritis. Because most of the GS formulation on the market is in the oral form, an alternative formulation such as a transdermal delivery system (TDS) is necessary in order to increase patient compliance. As the initial step to develop a TDS of GS, the physicochemical stability and permeation study in rat skin were examined. Evaluation of the stability of GS at different pHs showed the compound to be most stable at pH 5.0. The degradation rate constant at 25°C was estimated to be 5.93 ×10− 6 hr− 1 (t90~ 2.03 years) in a pH 5 buffer solution. Due to its hydrophilic characteristic, low skin permeability was expected of GS. However, the skin permeation rate was determined to be 13.27 µg/cm2/hr at 5% concentration. Results of this study suggest the possibility of developing GS into a transdermal delivery system.

Reference: Marion Kanwischera.et. al.. Evaluation of the Physicochemical Stability and Skin Permeation of Glucosamine Sulfate. Drug Development and Industrial Pharmacy. September 26,2008: V31, Issue 1; P 91-97. DOI:10.1081/DDC-44181

A review of glucosamine for knee osteoarthritis: why patented crystalline glucosamine sulfate should be differentiated from other glucosamines to maximize clinical outcomes

Abstract

The European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) treatment algorithm for knee osteoarthritis (OA) recommends symptomatic slow-acting drugs for osteoarthritis (SYSADOAs) first line for the medium to long term management of OA, due to their ability to control pain, improve function, and delay joint structural changes. Among SYSADOAs, glucosamine is probably the most widely used intervention. In the present review of glucosamine for knee OA, we have investigated whether the evidence is greater for the patented crystalline glucosamine sulfate (pCGS) preparation (Rottapharm/Meda) than for other glucosamine formulations. Glucosamine is actually widely available in many forms, as the prescription-grade pCGS preparation, generic and over-the-counter formulations of glucosamine sulfate (GS) and food supplements containing glucosamine hydrochloride (GH), which vary substantially in molecular form, pharmaceutical formulation and dose regimens. Only pCGS is given as a highly bioavailable once daily dose (1500 mg) with a proven pharmacological effect. pCGS consistently reaches the plasma levels of around 10 μM required to inhibit interleukin-1 induced expression of genes involved in the pathophysiology of joint inflammation and tissue destruction, compared with sub-therapeutic levels achieved with GH. It is evident, from careful consideration of the evidence base, that only the pCGS formulation of glucosamine reliably provides an effect size on pain that is higher than that of paracetamol and equivalent to that provided by non-steroidal anti-inflammatory drugs. In comparison, the effect size on pain of non-crystalline GS preparations and GH from randomized controlled trials is repeatedly demonstrated to be zero. In addition, there is evidence that chronic administration of pCGS has disease-modifying effects, with a reduction in the need for total joint replacement surgery lasting for at least 5 years after treatment cessation. Consequently, the pCGS preparation (Rottapharm/Meda) is the logical choice, with demonstrated medium-term control of pain and lasting impact on disease progression.

Reference: Eugene J. Kucharza et. al..A review of glucosamine for knee osteoarthritis: why patented crystalline glucosamine sulfate should be differentiated from other glucosamines to maximize clinical outcomes. CrossMark. February 26,2016: DOI:10.1185/03007995.2016.1154521

Glucosamine sulphate for the management of arthrosis: A controlled clinical investigation

Summary

A new preparation of pure glucosamine sulphate, in injectable and oral form, was investigated in a controlled clinical trial in patients with osteoarthrosis. Two groups of 15 in-patients received either 400 mg glucosamine sulphate daily (12by the intramuscular and 3 by the intra-articular route) for 7 days, followed by 2 weeks at 1.5 g daily of oral glucosamine sulphate in 3 divided doses, or an intramuscular injection daily of a piperazine/chlorbutanol combination for 7 days, followed by oral placebo during the following 2 weeks. Semi-quantitative scoring of pain at rest and during active and passive movements, of restricted function and, where possible, of walking time over 20 metres, were taken as therapeutic activity indices, and tested before and after 1 and 3 weeks of treatment. Patients were positively questioned daily for possible intolerance symptoms. Laboratory tests were recorded before and after treatment. With both initial parenteral treatments, each symptom significantly improved, with a trend for faster and greater recovery with glucosamine, mainly in restricted function. During the maintenance period, a further significant improvement was recorded in the group receiving glucosamine, whereas with placebo the symptom scores rose to almost the pre-treatment levels. A similar pattern was shown in the measurement of walking speed. Clinical and biological tolerance were excellent with both treatments. No drug-related complaints were recorded, nor signs of interference in other illnesses or interactions with other drug treatments. It is suggested that injectable and/or oral treatment with pure glucosamine sulphate should be considered for the basic therapy of primary or secondary osteoarthrosis, mainly because it restores articular function to a certain extent.

Reference: G. Crollea & E. D’estea. Glucosamine sulphate for the management of arthrosis: A controlled clinical investigation. Current Medical Research and Opinion. August 11, 2008: V7, Issue 2, P104-109. DOI:10.1185/03007998009112035

A Double-Blind Randomized Placebo Controlled Parallel Group Study Evaluating the Effects of Ibuprofen and Glucosamine Sulfate on Exercise Induced Muscle Soreness

Abstract

Objective: The primary objective of this study is to compare the effects of ibuprofen with placebo on post-exercise induced muscle soreness. The secondary objective is to explore the effects of glucosamine sulphate versus placebo on post-exercise induced muscle soreness. Ibuprofen is a nonsteroidal anti-inflammatory drug which relieves pain and reduces inflammation. Glucosamine sulphate is a naturally occurring amino monosaccharide in the human body. It is biosynthesized from glucose, and is used to form glycosamineglycan, a constituent of proteoglycans, and an important component of the extracellular matrix of articular cartilage.

Methods: Ibuprofen 1,200 mg/day, 1,500 mg/day glucosamine sulphate, or placebo was given orally daily for 22 days to three groups, each consisting of 20 healthy men [aged 24.3 ± 3.1 years] in a double-blind, randomized controlled parallel group study. Subjects carried out an intensive eccentric exercise of the first dorsal interosseous muscle of the left hand on a standardized hand exerciser on day 14. Muscle tenderness was assessed by the pressure pain stimulation on days 0, 14 [before exercise], 14 [immediately after exercise], 15, 16 [the days of maximal tenderness], and 22.

Results: Muscle tenderness on days 0 and 14 [before exercise] was not different in the three groups. Muscle tenderness was significantly increased in the group consuming 1,500 mg/day glucosamine sulphate as compared with the placebo group day 15 and day 16 after the exercise There was no significant difference between ibuprofen versus placebo. None of the participants reported any serious adverse effects.Conclusion: Glucosamine sulphate facilitates muscle tenderness whereas systemic administration of ibuprofen is not capable of inhibiting experimentally induced muscle tenderness/soreness.

Reference:Lars Arendt-Nielsena et. al.. A Double-Blind Randomized Placebo Controlled Parallel Group Study Evaluating the Effects of Ibuprofen and Glucosamine Sulfate on Exercise Induced Muscle Soreness. Journal of Musculoskeletal Pain. January 16,2010: V15, Issue 1; P21-28. DOI:10.1300/J094v15n01_04.

A SIMPLE AND VALIDATED LC METHOD FOR THE SIMULTANEOUS ANALYSIS OF GLUCOSAMINE AND CHONDROITIN SULFATE EQUIVALENT IN DIETARY PRODUCTS

Abstract

A simple and reliable reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine simultaneously a glucosamine and chondroitin sulfate equivalent in dietary products. The procedure is based upon the reaction of o-phthaldialdehyde with glucosamine and galactosamine coming from the galactosaminoglycan hydrolysis. The hydrolysis reaction was carried out with hydrochloric acid (7.5 N) at 80°C for 8 hr, whereas, the pre-column derivatization reaction was carried out in alkaline media for 1 min at ambient temperature. The chromatographic separations were performed on a Phenomenex Synergi 4 μ fusion-RP 80 A (250 mm × 3.0 mm i.d.) using a mobile phase consisting of a mixture of sodium acetate buffer (pH 5.9; 0.05 M) and methanol (85:15, v/v). UV-DAD detection at λ = 340 nm was used. Linear responses were observed and the limit of quantitation for both aminosaccharides was about 60 pmol. The intra-day precision (RSD) was ≤1.8% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results (99.3–101.0%) with RSD ranging from 1.1 to 2.1%.

Reference: Rita Gattia, Paolo Andreattab, Maria G. Gioiac & Silvia Boschettib. A SIMPLE AND VALIDATED LC METHOD FOR THE SIMULTANEOUS ANALYSIS OF GLUCOSAMINE AND CHONDROITIN SULFATE EQUIVALENT IN DIETARY PRODUCTS. Journal of Liquid Chromatography & Related. December 1, 2010: V33, Issue 19; P 1760-1775. DOI:10.1080/10826076.2010.526829

Effects of Glucosamine in the Treatment of Osteoarthritis

Abstract

This paper describes the effects and efficacy of glucosamine sulfate for the treatment of osteoarthritis. In vitro evidence is presented that supports the beneficial effects of glucosamine sulphate in treating osteoarthritis in both human and animal models. The mechanisms of glucosamine action are described including stimulation of the production of glycoaminoglycans, which are important building blocks of the structure of articular cartilage, inhibition of interleukin-1, and enhancement of the components of synovial fluid, such as increased quality and quantity of hylunonic acid. Glucosamine sulfate has demonstrated in clinical trials to be a relatively safe, effective treatment for the pain associated with mild to moderate osteoarthritis of the human knee joint. However, many of the clinical trials have had short-term follow-up and relatively small sample sizes. No research has been reported regarding the prevention of osteoarthritis in healthy joints with the use of glucosamine, and further research is needed to determine if joints other than the knee can be successfully treated with glucosamine sulfate.

Reference: Kenneth A. Olson. Effects of Glucosamine in the Treatment of Osteoarthritis. Journal of Manual & Manipulative Therapy. July 18, 2013: V12, Issue 2; P 100-106. DOI:10.1179/106698104790825356.

MSM methysulfonylmethane

Efficacy of methylsulfonylmethane (MSM) in osteoarthritis pain of the knee: a pilot clinical trial

Summary

 

Objective

Osteoarthritis (OA) is the most common form of arthritis and the second most common cause of long-term disability among middle-aged and older adults in the United States. Methylsulfonylmethane (MSM) is a popular dietary supplement used as a single agent and in combination with other nutrients, and purported to be beneficial for arthritis. However, there is paucity of evidence to support the use of MSM.

Methods

A randomized, double-blind, placebo-controlled trial was conducted. Fifty men and women, 40–76 years of age with knee OA pain were enrolled in an outpatient medical center. Intervention was MSM 3 g or placebo twice a day for 12 weeks (6 g/day total). Outcomes included the Western Ontario and McMaster University Osteoarthritis Index visual analogue scale (WOMAC), patient and physician global assessments (disease status, response to therapy), and SF-36 (overall health-related quality of life).

Results

Compared to placebo, MSM produced significant decreases in WOMAC pain and physical function impairment (P < 0.05). No notable changes were found in WOMAC stiffness and aggregated total symptoms scores. MSM also produced improvement in performing activities of daily living when compared to placebo on the SF-36 evaluation (P < 0.05).

Conclusion

MSM (3 g twice a day) improved symptoms of pain and physical function during the short intervention without major adverse events. The benefits and safety of MSM in managing OA and long-term use cannot be confirmed from this pilot trial, but its potential clinical application is examined. Underlying mechanisms of action and need for further investigation of MSM are discussed.

Reference: Dr L.S. Kim, N.D. et. al., Efficacy of methylsulfonylmethane (MSM) in osteoarthritis pain of the knee: a pilot clinical trial 1 2. Osteoarthritis and Cartilage. March 2006: V14, Issue 3; P286-294. doi:10.1016/j.joca.2005.10.003

Systematic review of the nutritional supplements dimethyl sulfoxide (DMSO) and methylsulfonylmethane (MSM) in the treatment of osteoarthritis

Summary

Objective

Conventional treatment of osteoarthritis (OA) with non-steroidal anti-inflammatory drugs is associated with serious gastrointestinal side effects and in view of the recent withdrawal of some cyclo-oxygenase-2 inhibitors, identifying safer alternative treatment options is needed. The objective of this systematic review is to evaluate the existing evidence from randomised controlled trials of two chemically related nutritional supplements, dimethyl sulfoxide (DMSO) and methylsulfonylmethane (MSM) in the treatment of OA to determine their efficacy and safety profile.

Methods

The electronic databases [Cochrane Library, Medline, Embase, Amed, Cinahl and NeLH (1950 to November 2007)] were searched. The search strategy combined terms: osteoarthritis, degenerative joint disorder, dimethyl sulfoxide, DMSO, methylsulfonylmethane, MSM, clinical trial; double-blind, single blind, RCT, placebo, randomized, comparative study, evaluation study, control. Inclusion and exclusion criteria were applied. Data were extracted and quality was assessed using the JADAD scale.

Results

Six studies were included [evaluating a total of 681 patients with OA of the knee for DMSO (N = 297 on active treatment); 168 patients for MSM (N = 52 on active treatment)]. Two of the four DMSO trials, and both MSM trials reported significant improvement in pain outcomes in the treatment group compared to comparator treatments, however, methodological issues and concerns over optimal dosage and treatment period, were highlighted.

Conclusion

No definitive conclusion can currently be drawn for either supplement. The findings from all the DMSO studies need to be viewed with caution because of poor methodology including; possible unblinding, and questionable treatment duration and dose. The data from the more rigorous MSM trials provide positive but not definitive evidence that MSM is superior to placebo in the treatment of mild to moderate OA of the knee. Further studies are now required to identify both the optimum dosage and longer-term safety of MSM and DMSO, and definitive efficacy trials.

Reference: S. Brien, B.Sc., M.Sc., Ph.D. et. al.. Systematic review of the nutritional supplements dimethyl sulfoxide (DMSO) and methylsulfonylmethane (MSM) in the treatment of osteoarthritis. Osteoarthritis and Cartilage. November 2008: V16, Issue 11; P 1277-1288. doi:10.1016/j.joca.2008.03.002

The Anti-inflammatory Effects of Methylsulfonylmethane on Lipopolysaccharide-Induced Inflammatory Responses in Murine Macrophages

Abstracts

Methylsulfonylmethane (MSM), also known as dimethyl sulfone and methyl sulfone, is an organic sulfur-containing compound that occurs naturally in a variety of fruits, vegetables, grains, and animals, including humans. In the present study, we demonstrated the anti-inflammatory effects of MSM in lipopolysaccharide (LPS)-stimulated murine macrophages, RAW264.7 cells. MSM significantly inhibited the release of nitric oxide and prostaglandin E2 by alleviating the expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-stimulated RAW264.7 cells. Furthermore, the levels of interleukin-6 and tumor necrosis factor-α were decreased by MSM treatment in cell culture supernatants. Further study indicated that the translocation of the p65 subunit of nuclear factor (NF)-κB to the nucleus was inhibited by MSM treatment in LPS-stimulated RAW264.7 cells, in which it helped block degradation of inhibitor of NF-κB. In addition, in vivo studies demonstrated that topical administration of MSM at 500—1250 μg/ear resulted in similar inhibitory activities in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema. Collectively, theses results indicate that MSM inhibits LPS-induced release of pro-inflammatory mediators in murine macrophages through downregulation of NF-κB signaling.

Reference: Yoon Hee Kim et. al.. The Anti-inflammatory Effects of Methylsulfonylmethane on Lipopolysaccharide-Induced Inflammatory Responses in Murine Macrophages. Biological and Pharmaceutical Bulletin. April 1, 2009: V32, Issue 4; P651-656. doi.org/10.1248/bpb.32.651

Randomised, Double-Blind, Parallel, Placebo-Controlled Study of Oral Glucosamine, Methylsulfonylmethane and their Combination in Osteoarthritis

Abstract

Objective: Glucosamine, classified as a slow-acting drug in osteoarthritis (SADOA), is an efficacious chondroprotective agent. Methylsulfonylmethane (MSM), the isoxidised form of dimethyl-sulfoxide (DSMO), is an effective natural analgesic and anti-inflammatory agent. The aim of this study was to compare the efficacy and safety of oral glucosamine (Glu), methylsulfonylmethane (MSM), their combination and placebo in osteoarthritis of the knee.

Patients and design: A total of 118 patients of either sex with mild to moderate osteoarthritis were included in the study and randomised to receive either Glu 500mg, MSM 500mg, Glu and MSM or placebo capsules three times daily for 12 weeks. Patients were evaluated at 0 (before drug administration), 2, 4, 8 and 12 weeks post-treatment for efficacy and safety. The efficacy parameters studied were the pain index, the swelling index, visual analogue scale pain intensity, 15m walking time, the Lequesne index, and consumption of rescue medicine.

Results: Glu, MSM and their combination significantly improved signs and symptoms of osteoarthritis compared with placebo. There was a statistically significant decrease in mean (± SD) pain index from 1.74 ± 0.47 at baseline to 0.65 ± 0.71 at week 12 with Glu (p < 0.001). MSM significantly decreased the mean pain index from 1.53 ± 0.51 to 0.74 ± 0.65, and combination treatment resulted in a more significant decrease in the mean pain index (1.7 ± 0.47 to 0.36 ± 0.33; p < 0.001). After 12 weeks, the mean swelling index significantly decreased with Glu and MSM, while the decrease in swelling index with combination therapy was greater (1.43 ± 0.63 to 0.14 ± 0.35; p < 0.05) after 12 weeks. The combination produced a statistically significant decrease in the Lequesne index. All treatments were well tolerated.

Conclusion: Glu, MSM and their combination produced an analgesic and anti-inflammatory effect in osteoarthritis. Combination therapy showed better efficacy in reducing pain and swelling and in improving the functional ability of joints than the individual agents. All the treatments were well tolerated. The onset of analgesic and anti-inflammatory activity was found to be more rapid with the combination than with Glu. It can be concluded that the combination of MSM with Glu provides better and more rapid improvement in patients with osteoarthritis.

Reference: P. R. Usha , M. U. R. Naidu. Randomised, Double-Blind, Parallel, Placebo-Controlled Study of Oral Glucosamine, Methylsulfonylmethane and their Combination in Osteoarthritis. Clinical Drug Investigation. June 2004: V24,Issue 6; P353-363. DOI: 10.2165/00044011-200424060-00005

A Multicentered, Open-Label Trial on the Safety and Efficacy of Methylsulfonylmethane in the Treatment of Seasonal Allergic Rhinitis

ABSTRACT

Background: Seasonal allergic rhinitis (SAR) affects more than 23 million Americans annually, and current epidemiologic studies indicate that its prevalence within the United States is increasing. Numerous clinical observations and case studies have led researchers to hypothesize that methylsulfonylmethane (MSM) may help ameliorate the symptoms associated with SAR.

Objective: The primary goal of this study was to evaluate the efficacy of MSM in the reduction of SAR-associated symptoms. This study also examined possible adverse reactions associated with methylsulfonylmethane supplementation. Finally, this study attempted to elucidate the method of action by which MSM elicits its effect on allergy symptoms.

Design: Fifty-five (55) subjects were recruited for the study. All met the criteria for participation in the study. 50 subjects completed the study. Those subjects completing the study consumed 2600 mg of MSM orally per day for 30 days. Clinical respiratory symptoms and energy levels were evaluated by a Seasonal Allergy Symptom Questionnaire (SASQ) at baseline and on days 7, 14, 21, and 30. Immune and inflammatory reactions were measured by plasma immunoglobulin E (IgE) and C-reactive protein at baseline and on day 30. An additional inflammatory biomarker, plasma histamine, was measured in a subset of subjects (n = 5).

Results: Day 7 upper and total respiratory symptoms were reduced significantly from baseline (p < 0.01 and p < 0.005, respectively). Lower respiratory symptoms were significantly improved from baseline by week 3 (p < 0.001). All respiratory improvements were maintained through the 30-day visit. Energy levels increased significantly by day 14 (p < 0.0001); this increase continued through day 30. No significant changes were observed in plasma IgE or histamine levels. The results of this study are promising. It would be worthwhile to conduct a larger, randomized, double-blind, placebo-controlled study to establish further if MSM would be a useful agent in the treatment of symptoms associated with SAR.

Conclusion: The results of this study suggest that MSM supplementation of 2600 mg/day for 30 days may be efficacious in the reduction of symptoms associated with SAR. Furthermore, few side effects are associated with the use of this compound. Recent acute and subacute chronic toxicologic data on the same source of MSM as used in this study, further validate the safety of this product.

Reference: Eleanor Barrager et. al..A Multicentered, Open-Label Trial on the Safety and Efficacy of Methylsulfonylmethane in the Treatment of Seasonal Allergic Rhinitis. The Journal of Alternative and Complementary Medicine. July 2004: V8, Issue 2; P167-173. doi:10.1089/107555302317371451.

Oleanolic Acid

Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes

ABSTRACT

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with a combination of synovium joint inflammation, synovium hyperplasia, and destruction of cartilage and bone. Oleanolic acid acetate (OAA), a compound isolated from Vigna angularis, has been known to possess pharmacological activities, including anti-inflammation and anti-bone destruction. In this study, we investigated the effects of OAA on RA and the underlying mechanisms of action by using a type-II collagen-induced arthritis (CIA) mouse model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. Oral administration of OAA decreased the clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum total and anti-type II collagen IgG, IgG1, and IgG2a levels. OAA administration reduced Th1/Th17 phenotype CD4+ T lymphocyte expansions and inflammatory cytokine productions in T cell activated draining lymph nodes and spleen. OAA reduced the expression and production of inflammatory mediators, such as cytokines and matrix metalloproteinase (MMP)-1/3, in the ankle joint tissue and RA synovial fibroblasts by down-regulating Akt, mitogen-activated protein kinases, and nuclear factor-κB. Our results clearly support that OAA plays a therapeutic role in RA pathogenesis by modulating helper T cell immune responses and matrix-degrading enzymes. The immunosuppressive effects of OAA were comparable to dexamethasone and ketoprofen. We provide evidences that OAA could be a potential therapeutic candidate for RA.

Reference:

Jin Kyeong Choi et. al., Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes. Toxicology and Applied Pharmacology. January 1, 2016: V290, P1-9. doi:10.1016/j.taap.2015.11.005

Pharmacology of oleanolic acid and ursolic acid

Abstract

Oleanolic acid and ursolic acid are triterpenoid compounds that exist widely in food, medicinal herbs and other plants. This review summarizes the pharmacological studies on these two triterpenoids. Both oleanolic acid and ursolic acid are effective in protecting against chemically induced liver injury in laboratory animals. Oleanolic acid has been marketed in China as an oral drug for human liver disorders. The mechanism of hepatoprotection by these two compounds may involve the inhibition of toxicant activation and the enhancement of the body defense systems. Oleanolic acid and ursolic acid have also been long-recognized to have antiinflammatory and antihyperlipidemic properties in laboratory animals, and more research is warranted to develop a therapy for patients. Recently, both compounds have been noted for their antitumor-promotion effects, which are stimulating additional research in this field. Oleanolic acid and ursolic acid are relatively non-toxic, and have been used in cosmetics and health products. The possible mechanisms for the pharmacological effects and the prospects for these two compounds are discussed.

 

Reference:

Jie Liu. Pharmacology of oleanolic acid and ursolic acid. Journal of Ethnopharmacology. December 1, 1995: V49, Issue 2; P57-68. doi:10.1016/0378-8741(95)90032-2

Cardiovascular, antihyperlipidemic and antioxidant effects of oleanolic and ursolic acids in experimental hypertension

Summary

Cardiovascular (systolic and diastolic blood pressure, heart rate), antihyperlipidemic (tryglycerides, total cholesterol and lipoprotein fractions), antioxidant (glutathione peroxidase – GPx, and superoxide dismutase – SOD), diuretic/saluretic and hypoglycemic activity of 98% pure oleanolic (OA) and ursolic (UA) acid were studied in Dahl salt-sensitive (DSS), insulin resistant rat model of genetic hypertension. Both OAand UA displayed low toxicity, with LC50 0.10 and 0.95 mg/ml, respectively. Although both triterpenoids did not have direct hypotensive effect, after 6-week application in a daily dose 60 mg/kg b.w., i.p., they prevented the development of severe hypertension. The antihypertensive effect was attributed to their potent diuretic-natriuretic-saluretic activity; direct cardiac effect (heart rate decrease by 34% and 32%, respectively); antihyperlipidemic (more than two times decrease of LDLand triglycerides); antioxidant (GPx increase by 12% and 10%, respectively;SOD increase by 12% and 22%, respectively), and hypoglycemic (blood glucose decrease by 20% and 50%, respectively) effects on the DSSrats. Except for the antihyperlipidemic effects, the other described above in vivo antihypertensive effects of OAand UAare reported for the first time and the underlying mechanisms are currently under investigation.

Reference:

L.O. Somovaa, A. Nadara, P. Rammanana, F.O. Shodeb. Cardiovascular, antihyperlipidemic and antioxidant effects of oleanolic and ursolic acids in experimental hypertension. Phytomedicine. 2003: V10, Issue 2-3; P 115-121. doi:10.1078/094471103321659807

Inhibition of cytochrome P450 2E1 expression by oleanolic acid: hepatoprotective effects against carbon tetrachloride-induced hepatic injury

Abstract

The protective effects of oleanolic acid on carbon tetrachloride-induced hepatotoxicities and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with oleanolic acid prior to the administration of carbon tetrachloride significantly prevented the increase in serum alanine aminotransferase and lactate dehydrogenase activity and liver lipid peroxidation in a dose-dependent manner. Hepatic glutathione levels and glutathione-S-transferase activities were not affected by treatment with oleanolic acid alone but pretreatment with oleanolic acid protects carbon tetrachloride-induced depletion of hepatic glutathione levels. The effects of oleanolic acid on the cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation were investigated. Treatment of mice with oleanolic acid resulted in a significant decrease of P450 2E1-dependent p-nitrophenol and aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. These results show that the protective effects of oleanolic acid against the carbon tetrachloride-induced hepatotoxicity may, at least in part, be due to its ability to block bioactivation of carbon tetrachloride mainly by the inhibition of expression and activities of P450 2E1.

Reference: Hye Gwang Jeong. Inhibition of cytochrome P450 2E1 expression by oleanolic acid: hepatoprotective effects against carbon tetrachloride-induced hepatic injury. Toxicology Letters. April 12, 1999: V105, Issue 3; P215-222. doi:10.1016/S0378-4274(99)00004-1

Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system

Summary

The effect of naturally occurring triterpenoid compounds such as glycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin on the immune system was studied using Balb/c mice. Intraperitoneal treatments with 5 doses of these terpenoid compounds were found to enhance the total white blood cells (WBC) count. In ursolic acid, oleanolic acid and nomilin treated animals the maximum total WBC count was observed on the 6th day, while in glycyrrhizic acid treated animals it was observed only on the 9th day after the drug treatment. In ursolic acid, oleanolic acid and nomilin treated animals the percentage of increase in the total WBC count was to 91.48 ± 4.6%, 135.75 ± 6.4% and 117.33 ± 17.9% respectively. In the glycyrrhizic acid treated animals the total WBC count was increased to 114.9 ± 18%. Bone marrow cellularity and α-esterase positive cells were also enhanced by the treatment with these terpenoids. Treatment with various triterpenoids along with antigen produced an enhancement in the specific antibody titre and the number of plaque forming cells (PFC) in the spleen. Triterpenoids remarkably inhibited delayed type hypersensitivity reaction (DTH). These results indicate the immunomodulatory activity of naturally occurring triterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin.

Reference:

T.J. Raphaela, G. Kuttana. Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system. Phytomedicine. 2003: V10, Issue 6-7; P483-489. doi:10.1078/094471103322331421

Anti-AIDS Agents. 30. Anti-HIV Activity of Oleanolic Acid, Pomolic Acid, and Structurally Related Triterpenoids1

Abstract

Oleanolic acid (1) was identified as an anti-HIV principle from several plants, including Rosa woodsii (leaves), Prosopis glandulosa (leaves and twigs), Phoradendron juniperinum (whole plant), Syzygium claviflorum (leaves), Hyptis capitata (whole plant), and Ternstromiagymnanthera (aerial part). It inhibited HIV-1 replication in acutely infected H9 cells with an EC50value of 1.7 μg/mL, and inhibited H9 cell growth with an IC50 value of 21.8 μg/mL [therapeutic index (T. I.) 12.8]. Pomolic acid, isolated from R. woodsii and H. capitata, was also identified as an anti-HIV agent (EC50 1.4 μg/mL, T. I. 16.6). Although ursolic acid did show anti-HIV activity (EC50 2.0 μg/mL), it was slightly toxic (IC50 6.5 μg/mL, T. I. 3.3). A new triterpene (11) was also isolated from the CHCl3-soluble fraction of R. woodsii, though it showed no anti-HIV activity. The structure of 11 was determined to be 1β-hydroxy-2-oxopomolic acid by spectral examination. Based on these results, we examined the anti-HIV activity of oleanolic acid- or pomolic acid-related triterpenes isolated from several plants. In addition, we previously demonstrated that derivatives of betulinic acid, isolated from the leaves of S. claviflorum as an anti-HIV principle, exhibited extremely potent anti-HIV activity. Accordingly, we prepared derivatives of oleanolic acid and evaluated their anti-HIV activity. Among the oleanolic acid derivatives, 18 demonstrated most potent anti-HIV activity, with an EC50 value of 0.0005 μg/mL and a T. I. value of 22 400.

Reference:

Yoshiki Kashiwada et. al..Anti-AIDS Agents. 30. Anti-HIV Activity of Oleanolic Acid, Pomolic Acid, and Structurally Related Triterpenoids1J. Nat. Prod. July 31,1998: V6; P1090-1095. DOI: 10.1021/np9800710

Boswellia Serrata

Boswellia serrata extract attenuates inflammatory mediators and oxidative stress in collagen induced arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease which leads to destruction of joints. Current treatment modalities for RA either produce symptomatic relief (NSAIDs) or modify the disease process (DMARDs). Though effective, their use is also limited by their side effects. As a result, the interest in alternative, well tolerated anti-inflammatory remedies has re-emerged. Our aim was to evaluate the antioxidant and antiarthritic activity of Boswellia serrata gum resin extract (BSE) in collagen induced arthritis. Arthritis was induced in male Wistar rats by collagen induced arthritis (CIA) method. BSE was administered at doses of 100 and 200 mg/kg body weight once daily for 21 days. The effects of treatment in the rats were assessed by biochemical (articular elastase, MPO, LPO, GSH, catalase, SOD and NO), inflammatory mediators (IL-1β, IL-6, TNF-α, IL-10, IFN-γ and PGE2), and histological studies in joints. BSE was effective in bringing significant changes on all the parameters (articular elastase, MPO, LPO, GSH, catalase, SOD and NO) studied. Oral administration of BSE resulted in significantly reduced levels of inflammatory mediators (IL-1β, IL-6, TNF-α, IFN-γ and PGE2), and increased level of IL-10. The protective effects of BSE against RA were also evident from the decrease in arthritis scoring and bone histology. The abilities to inhibit proinflammatory cytokines and modulation of antioxidant status suggest that the protective effect of Boswellia serrata extract on arthritis in rats might be mediated via the modulation of immune system.

Reference: Sadiq Umar et.al., Boswellia serrata extract attenuates inflammatory mediators and oxidative stress in collagen induced arthritis. Phytomedicine. May 15,2014: V21, Issue 6; P847-856. doi:10.1016/j.phymed.2014.02.001

Efficacy and tolerability of Boswellia serrata extract in treatment of osteoarthritis of knee – A randomized double blind placebo controlled trial

Summary

Osteoarthritis is a common, chronic, progressive, skeletal, degenerative disorder, which commonly affects the knee joint. Boswellia serrata tree is commonly found in India. The therapeutic value of its gum (guggulu) has been known. It posses good anti-inflammatory, anti-arthritic and analgesic activity.

A randomized double blind placebo controlled crossover study was conducted to assess the efficacy, safety and tolerability of Boswellia serrata Extract (BSE) in 30 patients of osteoarthritis of knee, 15 each receiving active drug or placebo for eight weeks. After the first intervention, washout was given and then the groups were crossed over to receive the opposite intervention for eight weeks. All patients receiving drug treatment reported decrease in knee pain, increased knee flexion and increased walking distance. The frequency of swelling in the knee joint was decreased. Radiologically there was no change. The observed differences between drug treated and placebo being statistically significant, are clinically relevant. BSE was well tolerated by the subjects except for minor gastrointestinal ADRs. BSE is recommended in the patients of osteoarthritisosteoarthritis of the knee with possible therapeutic use in other arthritis.

Reference:N. Kimmatkara, V. Thawanib, , , L. Hingoranic, R. Khiyanid. Efficacy and tolerability of Boswellia serrata extract in treatment of osteoarthritis of knee – A randomized double blind placebo controlled trial. Phytomedicine. 2003: V10, Issue 1; P3-7. doi:10.1078/094471103321648593

Effects of gum resin of Boswellia serrata in patients with chronic colitis.

Abstract

Patients studied here suffered from chronic colitis characterized by vague lower abdominal pain, bleeding per rectum with diarrhoea and palpable tender descending and sigmoid colon. The inflammatory process in colitis is associated with increased formation of leukotrienes causing chemotaxis, chemokinesis, synthesis of superoxide radicals and release of lysosomal enzymes by phagocytes. The key enzyme for leukotriene biosynthesis is 5-lipoxygenase. Boswellic acids were found to be non-redox, non-competitive specific inhibitors of the enzyme 5-lipoxygenase. We studied the gum resin of Boswellia serrata for the treatment of this disease. Thirty patients, 17 males and 13 females in the age range of 18 to 48 years with chronic colitis were included in this study. Twenty patients were given a preparation of the gum resin of Boswellia serrata (900 mg daily divided in three doses for 6 weeks) and ten patients were given sulfasalazine (3 gm daily divided in three doses for 6 weeks) and served as controls. Out of 20 patients treated with Boswellia gum resin 18 patients showed an improvement in one or more of the parameters: including stool properties, histopathology as well as scanning electron microscopy, besides haemoglobin, serum iron, calcium, phosphorus, proteins, total leukocytes and eosinophils. In the control group 6 out of 10 patients showed similar results with the same parameters. Out of 20 patients treated with Boswellia gum resin 14 went into remission while in case of sulfasalazine remission rate was 4 out of 10. In conclusion, this study shows that a gum resin preparation from Boswellia serrata could be effective in the treatment of chronic colitis with minimal side effects.

Reference:Gupta I et. al..Effects of gum resin of Boswellia serrata in patients with chronic colitis.Planta Medica. 2001: V67, Issue 5; P391-395. DOI: 10.1055/s-2001-15802

Pharmacology of an extract of salai guggal ex-Boswellia serrata, a new non-steroidal anti-inflammatory agent

Abstract

Pharmacological evaluation of alcoholic extract of salai guggal (AESG) has been carried out in experimental animals. AESG displayed marked anti-inflammatory activity in carrageenan induced oedema in rats and mice and dextran oedema in rats. It was equally effective in adrenalectomised rats. In formaldehyde and adjuvant arthritis, AESG produced prominent anti-arthritic activity but no significant effect was observed in cotton pellet-induced granuloma test. It inhibited inflammation induced increase in serum transaminase levels and leucocyte counts but lacked any analgesic or anti-pyretic effects. The gestation period or parturition time in pregnant rats or onset time of castor oil-induced diarrhoea was unaffected by AESG and no significant effect was seen on cardiovascular, respiratory and central nervous system functions. No ulcerogenic effects were found in the rat stomach. The oral and intraperitoneal LD50 was greater than 2 g/Kg in mice and rats.

Reference:G. B. Singh, C. K. Atal. Pharmacology of an extract of salai guggal ex-Boswellia serrata, a new non-steroidal anti-inflammatory agent. Agents and Actions. June 1986: V18, Issue3; PP407-412. DOI 10.1007/BF01965005

Boswellia Serrata, A Potential Antiinflammatory Agent: An Overview

Abstract

The resin of Boswellia species has been used as incense in religious and cultural ceremonies and in medicines since time immemorial. Boswellia serrata (Salai/Salai guggul), is a moderate to large sized branching tree of family Burseraceae (Genus Boswellia), grows in dry mountainous regions of India, Northern Africa and Middle East. Oleo gum-resin is tapped from the incision made on the trunk of the tree and is then stored in specially made bamboo basket for removal of oil content and getting the resin solidified. After processing, the gum-resin is then graded according to its flavour, colour, shape and size. In India, the States of Andhra Pradesh, Gujarat, Madhya Pradesh, Jharkhand and Chhattisgarh are the main source of Boswellia serrata. Regionally, it is also known by different names. The oleo gum-resins contain 30-60% resin, 5-10% essential oils, which are soluble in the organic solvents, and the rest is made up of polysaccharides. Gum-resin extracts of Boswellia serrata have been traditionally used in folk medicine for centuries to treat various chronic inflammatory diseases. The resinous part of Boswellia serrata possesses monoterpenes, diterpenes, triterpenes, tetracyclic triterpenic acids and four major pentacyclic triterpenic acids i.e. β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid, responsible for inhibition of pro-inflammatory enzymes. Out of these four boswellic acids, acetyl-11-keto-β-boswellic acid is the most potent inhibitor of 5-lipoxygenase, an enzyme responsible for inflammation.

Reference:M. Z. Siddiqui. Boswellia Serrata, A Potential Antiinflammatory Agent: An Overview. Indian J Pharm Sci. May-Jun 2011: V73, Issue 3; P255-261. doi: 10.4103/0250-474X.93507

Effect of Boswellia serrata on intestinal motility in rodents: inhibition of diarrhoea without constipation

 

Abstract

Clinical studies suggest that the Ayurvedic plant Boswellia serrata may be effective in reducing diarrhoea in patients with inflammatory bowel disease. In the present study, we evaluated the effect of a Boswellia serrata gum resin extract (BSE) on intestinal motility and diarrhoea in rodents.

BSE depressed electrically-, acetylcholine-, and barium chloride-induced contractions in the isolated guinea-pig ileum, being more potent in inhibiting the contractions induced by acetylcholine and barium chloride.

The inhibitory effect of BSE on acetylcholine-induced contractions was reduced by the L-type Ca2+ channel blockers verapamil and nifedipine, but not by the sarcoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid, by the phosphodiesterase type IV inhibitor rolipram or by the lipoxygenase inhibitor zileuton.

3-acetyl-11-keto-β-boswellic acid, one of the main active ingredients of B. serrata, inhibited acetylcholine-induced contractions.

BSE inhibited upper gastrointestinal transit in croton oil-treated mice as well as castor oil-induced diarrhoea. However, BSE did not affect intestinal motility in control mice, both in the small and in the large intestine.

It is concluded that BSE directly inhibits intestinal motility with a mechanism involving L-type Ca2+ channels. BSE prevents diarrhoea and normalizes intestinal motility in pathophysiological states without slowing the rate of transit in control animals. These results could explain, at least in part, the clinical efficacy of this Ayurvedic remedy in reducing diarrhoea in patients with inflammatory bowel disease.

Reference:Francesca Borrelli et. al..Effect of Boswellia serrata on intestinal motility in rodents: inhibition of diarrhoea without constipation. British Journal of Pharmacology. June 2006: V148, Issue 4; P553-560. DOI: 10.1038/sj.bjp.0706740

 

Hyaluronic Acid

Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues

Abstract

Osteoarthritis is a critical disease that comes from degeneration of cartilage tissue. In severe cases surgery is generally required. Tissue engineering using scaffolds with stem cell transplantation is an attractive approach and a challenge for orthopedic surgery. For sample preparation, silk fibroin (SF)/hyaluronic acid (HA) scaffolds in different ratios of SF/HA (w/w) (i.e., 100:0, 90:10, 80:20, and 70:30) were formed by freeze-drying. The morphological, mechanical, and physical clues were considered in this research. The morphological structure of the scaffolds was observed by scanning electron microscope. The mechanical and physical properties of the scaffolds were analyzed by compressive and swelling ratio testing, respectively. For the cell experiments, scaffolds were seeded and cultured with human umbilical cord-derived mesenchymal stem cells (HUMSCs). The cultured scaffolds were tested for cell viability, histochemistry, immunohistochemistry, and gene expression. The SF with HA scaffolds showed regular porous structures. Those scaffolds had a soft and elastic characteristic with a high swelling ratio and water uptake. The SF/HA scaffolds showed a spheroid structure of the cells in the porous structure particularly in the SF80 and SF70 scaffolds. Cells could express Col2a, Agg, and Sox9 which are markers for chondrogenesis. It could be deduced that SF/HA scaffolds showed significant clues for suitability in cartilage tissue engineering and in surgery for osteoarthritis.

Reference:

Jirayut Jaipaew et. al., Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues. Materials Science and Engineering: C. July 1, 2016: V64, P173-182. doi:10.1016/j.msec.2016.03.063

Copper Sulfate

ANTIMICROBIAL EVALUATION OF COPPER SULFATE (II) ON STRAINS OF ENTEROCOCCUS FAECALIS. IN VITRO STUDY

Abstract:

Abstract: Introduction: Controlling Enterococcus faecalis is of vital importance in endodontics, as this pathogen is associated with endodontic failure. Experimental evidence has shown that copper has antibacterial activity against other pathogens with similar characteristics. The objective of this study was to determine the antimicrobial activity of copper (II) or cupric (SC-II) sulfate on strains of Enterococcus faecalis and to compare it with the most commonly used antimicrobials. Methodology: We used 33 strains of Enterococcus faecalis isolated from different clinical pictures in different Chilean hospitals. The minimum inhibitory concentration (MIC) of SC-II, chlorhexidine and calcium hydroxide was determined by the broth microdilution technique, following the recommendations given by the Clinical and Laboratory Standards Institute. Results: The MIC for CHX varied in the range of 5-10 µg/ml; SC-II from 1.5 to 12 µg/ml, and HC was >32 mg/ml. The geometric mean of SC-II was 6 µg/ml, lower than that of CHX, which was 7.29 µg/ml. Conclusions: SC-II showed antimicrobial activity at lower concentrations than CHX. HC (which could have been affected by the buffer effect of the broth microdilution technique) showed high values, not comparable to other compounds. We suggest carrying out further studies on the properties of SC-II, such assessing its biocompatibility and reaction with other materials to be used clinically in endodontic therapy.

Reference: Marisol Sierra, Aldo Sanhueza, Rául Alcántara, Gabriela Sánchez., ANTIMICROBIAL EVALUATION OF COPPER SULFATE (II) ON STRAINS OF ENTEROCOCCUS FAECALIS. IN VITRO STUDY. Journal of Oral research. Vol 2, No 3. DOI: 10.17126/JORALRES.2013.026

Copper Sulfate-Induced Hemolytic AnemiaInhibition of Glucose-6-Phosphate Dehydrogenase and Other Possible Etiologic Mechanisms

ABSTRACT

Many substances have been found to cause hemolytic anemia in persons whose erythrocytes are deficient in glucose-6-phosphate dehydrogenase (G6PD). However, the basis for the hemolysis observed with heavy metals has not been elucidated. In 1965, the author treated a patient who experienced severe acute hemolytic anemia after accidental ingestion of a saturated solution of copper sulfate. At that time, only one previous report1 of copper sulfate-induced hemolytic anemia in man could be found. Subsequently, two papers2,3 have documented several cases of copper sulfateinduced hemolytic anemia. It also has become apparent that this disorder has been repeatedly recorded in India. The case observed by the author is briefly recorded and investigations of possible mechanisms are described.

Reference:Virgil F. Fairbanks, MD. Copper Sulfate-Induced Hemolytic AnemiaInhibition of Glucose-6-Phosphate Dehydrogenase and Other Possible Etiologic Mechanisms. Jama International Medecine. October 1,1967: V12, Issue 4; P428432. doi:10.1001/archinte.1967.04410010042005.

Acute copper sulfate poisoning

Abstract

Acute copper sulfate poisoning constituted 33.6 per cent of all cases of poisoning admitted to the Irwin Hospital, New Delhi, in 1961. The highest incidence was found in the hospitals of Delhi and Uttar Pradesh.

Forty-eight cases of acute copper sulfate poisoning were studied, with special reference to clinical features and for evidence of biochemical and histopathologic injury. An autopsy was performed in nine fatal cases. The levels of serum total copper, serum ionic copper and whole blood copper were found to be much higher in these patients than in normal subjects. A significant correlation was found between the levels of whole blood copper and the severity of manifestations.

There were superficial or deep ulcerations of gastric and intestinal mucosa. Histologic examination of the liver revealed dilatation of central veins, varying degrees of liver cell necrosis and bile thrombi. In the kidneys there was congestion of glomeruli, swelling or necrosis of tubular cells, and in some cases haemoglobin casts were observed.

Reference:H.K. Chuttani, M.D., D.T.M et. al..Acute copper sulfate poisoning. The American Journal of Medicine. November 1965: V39, Issue 5; P849-854.doi:10.1016/0002-9343(65)90105-1

Ion association and solvation behavior of copper sulfate in binary aqueous–methanol mixtures at different temperatures

Abstract

The specific conductance (κ) of copper sulfate in binary mixtures (methanol–water) with the alcohol mass fractions of 0%, 20% and 40% at different temperatures of (298.15, 303.15, 308.15 and 313.15) K was measured experimentally. The conductivity data have been analyzed using the Fuoss–Shedlovsky conductivity equation. The molar conductance (ΛM), the limiting molar conductance (Λ0), the association constants (KA), the Walden product (Λ0η0), and the standard thermodynamic parameters of association (ΔG°A, ΔH°A and ΔS°A) were calculated and discussed. The results show that, the molar conductance and the limiting molar conductance values were decreased as the relative permittivity of the solvent decreased while, the association constant increased. Also the results show that the values of the molar conductance, the limiting molar conductance and the association constant were increased as the temperature increased indicating that the association process is an endothermic one.

Reference:Esam A. Gomaa, Mohamed A. Tahoon,. Ion association and solvation behavior of copper sulfate in binary aqueous–methanol mixtures at different temperatures. Journal of Molecular Liquids. February 2016: V214, P19-23. doi:10.1016/j.molliq.2015.11.046

Histopathological evaluation of supportive effects of Rosa damascene on mice testes, following long term administration of copper sulfate

Abstract

Objective

To evaluate the supportive effects of Rosa damascene (R. damascene) essential oil on epididymal sperm quality and histopathology of testes following long term administration of copper sulfate in mice.

Materials and methods

The study comprised of four different groups of six mice as follows: group Cu, which received 0.1 mL copper sulfate solution at dose of 100 mg/kg, group R which received 0.1 mL R. damascene essential oil at dose of 1 mg/kg, treatment group (T) which received copper sulfate solution (100 mg/kg) and treated by R. damascene essential oil (1 mg/kg), and control group (C) which received the same volume of normal saline. The supplements were gavaged in all animals every other day, during experimental period. All animals of each experimental group were sacrificed 42 days after the beginning of experiment.

Results

Results showed that sperm concentration, motility and viability in group Cu were significantly decreased after 6 weeks, and severe degenerative changes were observed in testicular tissues in comparison with the control group. In treatment group, significant improve in the sperm count, motility and viability, and normal architecture in most seminiferous tubules with organized epithelium was observed compared to the group Cu.

Conclusions

Administration of the essential oil of R. damascene owning to its antioxidant properties is able to protect the testis and epididymal sperm from the adverse effects of copper poisoning in mice.

Reference: Ehsanollah Sakhaee1, Ladan Emadi2, Hamidreza Siahkouhi. Histopathological evaluation of supportive effects of Rosa damascene on mice testes, following long term administration of copper sulfate. Asian Pacific Journal of Reproduction. March 1, 2016: V5, Issue 1; P46-50. doi:10.1016/j.apjr.2015.12.008

A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage

Abstract

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.—Makris, E. A., MacBarb, R. F., Responte, D. J., Hu, J. C., Athanasiou, K. A. A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage.

Reference: Eleftherios A. Makris, et al..A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage. FASEB Journal. June 2013: V27; Issue 6; P2421-2430. doi: 10.1096/fj.12-224030.

Copper ability to induce premature senescence in human fibroblasts

Abstract

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Reference: Liliana Matos, et. al..Copper ability to induce premature senescence in human fibroblasts. Springer Age. June 22,2011: V34, Issue 4; P783-794. doi: 10.1007/s11357-011-9276-7

Chondroitin Sulfate

Determining equilibrium osmolarity in poly(ethylene glycol)/chondrotin sulfate gels mimicking articular cartilage

Abstract

We present an experimentally guided, multi-phase, multi-species polyelectrolyte gel model to make qualitative predictions on the equilibrium electro-chemical properties of articular cartilage. The mixture theory consists of two different types of polymers: poly(ethylene gylcol) (PEG), chondrotin sulfate (ChS), water (acting as solvent) and several different ions: H+, Na+, Cl−. The polymer chains have covalent cross-links whose effect on the swelling kinetics is modeled via Doi rubber elasticity theory. Numerical studies on equilibrium polymer volume fraction and net osmolarity (difference in the solute concentration across the gel) show a complex interplay between ionic bath concentrations, pH, cross-link fraction and the average charge per monomer. Generally speaking, swelling is aided due to a higher average charge per monomer (or a higher particle fraction of ChS, the charged component of the polymer), low solute concentration in the bath, a high pH or a low cross-link fraction. A peculiar case arises at higher values of cross-link fraction, where it is observed that increasing the average charge per monomer leads to gel deswelling.

Reference:

  1. Sircara, , , E. Aisenbreyb, S.J. Bryantb, c, d, D.M. Bortze. Determining equilibrium osmolarity in poly(ethylene glycol)/chondrotin sulfate gels mimicking articular cartilage. Journal of Theoretical Biology. January 7,2015: V364, P397-406. doi:10.1016/j.jtbi.2014.09.037

Synthesis and characterization of some cellulose/chondroitin sulphate hydrogels and their evaluation as carriers for drug delivery

 

Abstract

Mixed hydrogels based on natural, biodegradable and biocompatible polysaccharides, such as cellulose (C) and chondroitin sulphate (CS) in various mixing ratios were prepared by a crosslinking technique and characterized by swelling behaviour, FTIR spectroscopy, scanning electron microscopy, toxicity and biocompatibility tests.

The mixed cellulose/chondroitin sulphate hydrogels have been loaded with 7-[2-nitroxiacetyl-oxy-3-(4-acetyl-amino-phenoxy)-propyl]-8-morpholino-1,3-dimethyl-xanthine, a novel nitric oxide donor compound with a lower toxicity and a higher anti-inflammatory activity than its parent molecules, paracetamol and theophylline. Swelling and release kinetics have been also studied. It has been established that an increase of CS content in hydrogels composition leads to a higher swelling ratio for all formulations and to a decreased released amount of nitric oxide donor compound. It has been found that the swelling occurs by an anomalous swelling mechanism, while the release of nitric oxide donor compound follows a diffusion controlled mechanism.

Reference:Ana-Maria Oprea et.al..Synthesis and characterization of some cellulose/chondroitin sulphate hydrogels and their evaluation as carriers for drug delivery. Carbohydrate Polymers. January 4, 2012: V87, Issue 1; P721-729. doi:10.1016/j.carbpol.2011.08.052

In vitro and preliminary in vivo toxicity screening of high-surface-area TiO2–chondroitin-4-sulfate nanocomposites for bone regeneration application

 

Abstract

The goal of this study was to prepare nontoxic, biomimetic TiO2/chondroitin-4-sulfate nanocomposites with osteointegration ability for biomedical applications. Nanocomposites with higher surface area were subjected to bioactivity study and obtained bone-like layer with stoichiometric Ca/P ratio of 1.64 and 1.66. The susceptibility of nanocomposites against Staphylococcus aureus (∼16 mm) and Escherichia coli (∼12 mm) is favorable in preventing the risk of bone diseases and postoperative infections. Adequate swelling and degradations properties were favorably achieved to reduce the risk of nanoparticle accumulation in cell organelles. Moreover, the toxicity in AGS cell line and biocompatibility in osteoblast-like MG-63 cell line showed no significant mitochondrial damage. In addition, the in vitro expression of osteoblast inducing genes (OCN, OPN, ALP and COL 1) and their up-regulation, and 20% of increased hatching rate in preliminary in vivo (zebrafish) analysis were favorable for the nanocomposite at the ratio of 2:0.50 than pure TiO2. Hence, it can be concluded that among the prepared nanocomposites TCs.5 is a promising biomimetic biomaterial that can be used for advanced orthopedic research and other applications.

Reference:Kavitha Kandiah et. al..In vitro and preliminary in vivo toxicity screening of high-surface-area TiO2–chondroitin-4-sulfate nanocomposites for bone regeneration application. Colloids and Surfaces B: Biointerface. April 1,2015: V128, P347-356. doi:10.1016/j.colsurfb.2015.02.027

Oral chondroprotection with nutraceuticals made of chondroitin sulphate plus glucosamine sulphate in osteoarthritis

Abstract

Oral supplementation of chondroitin sulphate plus glucosamine helps repair the articular surface in osteoarthritis. Chondroitin-S reduces the concentration of the pro-inflammatory cytokines and transcription factor involved in inflammation. GlcN.S enhances cartilage specific matrix components and prevents collagen degeneration in chondrocytes by inhibiting hydrolytic enzymes, and preventing the oxidation of lipids and proteins. Chondroitin-S plus GlcN.S are slow-acting drugs that alleviate pain and partly restore joint function in OA patients. Orally administered pharmaceutical-grade chondroitin-S plus GlcN.S stabilize the joint space narrowing and significantly decrease the number of patients with new erosive OA. They are safe and no adverse events have ever been reported; they are recommended by EULAR and OARSI. The cost/effectiveness of the oral chondroitin-S plus GlcN.S therapy derives from the reduction of costs for physiotherapy, and for gastroprotective and non-steroidal drugs. The synergistic association of these two world-widely preferred nutraceuticals is a step forward in the management of OA.

Reference: Carlo Bottegoni et. al..Oral chondroprotection with nutraceuticals made of chondroitin sulphate plus glucosamine sulphate in osteoarthritis. Carbohydrate Polymers. August 30,2014: V109, P126-138. doi:10.1016/j.carbpol.2014.03.033

Curcumin

Curcumin directly inhibits the transport activity of GLUT1

Abstract

Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin’s inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin.

Reference:

Leesha K. Gunnink et. al., Curcumin directly inhibits the transport activity of GLUT1. Biochimie. June 2016: V125, P179-185. doi:10.1016/j.biochi.2016.03.014

Curcumin loaded gum arabic aldehyde-gelatin nanogels for breast cancer therapy

Abstract

Curcumin, a widely studied hydrophobic polyphenol with anticancer potential is loaded in gum arabic aldehyde-gelatin (GA Ald-Gel) nanogels to improve its bioavailability and therapeutic efficacy towards cancer cells. Physicochemical properties of the curcumin loaded GA Ald-Gel nanogels are investigated by different techniques including dynamic light scattering (DLS), NMR spectroscopy and scanning electron microscopy (SEM). These nanogels exhibit hydrodynamic diameter of 452 ± 8 nm with a zeta potential of − 27 mV. The nanogels possess an encapsulation efficiency of 65 ± 3%. Potential of the nanogels for controlled release of curcumin is illustrated by in vitro drug release studies. Hemocompatibility and cytocompatibility of the drug loaded nanogels are evaluated. In vitro cytotoxicity of the bare and curcumin loaded nanogels are analyzed by MTT assay towards MCF-7 cells. The results manifest that curcumin loaded nanogels induce toxicity in MCF-7 cells. Confocal laser scanning microscopy (CLSM) studies indicate in vitrocellular uptake of the nanogels in MCF-7 cells. All these results prove the suitability of the curcumin loaded GA Ald-Gel nanogels for cancer therapy.

Reference:P.R. Sarika , Rachel James Nirmala, Curcumin loaded gum arabic aldehyde-gelatin nanogels for breast cancer therapy. Materials Science and Engineering: C. August 1,2016: V65, P331-337. doi:10.1016/j.msec.2016.04.044

Analysis of anti-depressant potential of curcumin against depression induced male albino wistar rats

Abstract

The present study investigated the antidepressant potential of curcumin in olfactory bulbectomy and forced swimming test models of depression in male albino rats under chronic treatment. The experimental animals were divided into four groups, and curcumin was administered for 45 days. Our results showed that the curcumin significantly reduced olfactory bulbectomy-induced behavioral abnormalities including deficits in step-down passive avoidance, increased activity in the open area and immobility time. Chronic administration of curcumin significantly reversed levels of 3, 4-dihydroxyphenylacetic acid, noradrenaline, serotonin and 5-hydroxyindoleacetic acid in the hippocampus region of male albino rats. Also, curcumin normalizes the levels of dopamine, noradrenaline, and 5-hydroxyindoleacetic acid in the frontal cortex of rats. Taking all these results together, it may suggest that curcumin is potent compound acting against the depression in the male albino rats.

Reference:Xue-run Changa, 1, Li Wangb, , 1, Jing Lic, Dian-shui Wu. Analysis of anti-depressant potential of curcumin against depression induced male albino wistar rats. Brain Research. July 1, 2016: V1642, P219-225. doi:10.1016/j.brainres.2016.03.010

Interference with Akt signaling pathway contributes curcumin-induced adipocyte insulin resistance

Abstract

Previous study has shown that curcumin directly or indirectly suppresses insulin signaling in 3T3-L1 adipocytes. However, the underlying mechanism remains unclear. Here we experimentally demonstrate that curcumin inhibited the ubiquitin-proteasome system (UPS) function, activated autophagy, and reduced protein levels of protein kinase B (Akt) in a dose- and time-dependent manner in 3T3-L1 adipocytes, accompanied with attenuation of insulin-stimulated Akt phosphorylation, plasma membrane translocation of glucose transporter type 4 (GLUT4), and glucose uptake. These in vitro inhibitory effects of curcumin on Akt protein expression and insulin action were reversed by pharmacological and genetic inhibition of autophagy but not by inhibition of the UPS and caspases. In addition, Akt reduction in adipose tissues of mice treated with curcumin could be recovered by administration of autophagy inhibitor bafilomycin A1 (BFA). This new finding provides a novel mechanism by which curcumin induces insulin resistance in adipocytes.

Reference: Deling Zhang et. al. Interference with Akt signaling pathway contributes curcumin-induced adipocyte insulin resistance. Molecular and Cellular Endocrinology. July 5,2016: V429, P1-9. doi:10.1016/j.mce.2016.04.013

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype

Abstract

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.

Reference:Grażyna Mosieniak et. al. Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype. The International Journal of Biochemistry & Cell Biology. May 2016: V74, P33-43. doi:10.1016/j.biocel.2016.02.014

Curcumin protects cardiac myocyte against hypoxia-induced apoptosis through upregulating miR-7a/b expression

Abstract

Objective

Curcumin has properties of anti-inflammation, anti-oxidation, anti-infection and anti-tumor, benefiting for the treatment of many diseases. The present study was aimed to investigate the role of curcumin in myocardial infarction (MI) and its potential mechanism involving transcription factor specific protein 1 (SP1).

Methods

After receiving curcumin, C57BL/6 mice subjected to left anterior descending (LAD) coronary artery occlusion to induce MI model. Infarct size was measured by triphenyl tetrazolium chloride staining. In vitro experiments, mouse cardiac myocytes (MCM) subjected to hypoxia after the incubation of curcumin, miR-7a/b and SP1 expression levels were detected by real-time PCR and western blot. Caspase-3 activity and TUNEL assay were performed to assess the cell apoptosis.

Results

In animal experiments, curcumin significantly reduced the infarct size compared with the control. It also up-regulated miR-7a/b expression and down-regulated SP1 expression. In hypoxia-induced MCM, curcumin led to the decrease of cell apoptosis. Transfected MCM with miR-7a/b inhibitor, curcumin induced the decrease of cell apoptosis and SP1 expression was reversed. Transfected with pcDNA-SP1, the decrease of cardiac myocytes apoptosis after the treatment of curcumin was also reversed.

Conclusion

Curcumin pre-treatment protected against hypoxia-induced cardiac myocytes apoptosis through the up-regulation of miR-7a/b and the down-regulation of SP1 expression.

Reference:Hai-Hua Geng et. al..Curcumin protects cardiac myocyte against hypoxia-induced apoptosis through upregulating miR-7a/b expression. Biomedicine & Pharmacotherapy. July 2016: V81, P258-264. doi:10.1016/j.biopha.2016.04.020

Boron

Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1)

Abstract

The aim of this study was to determine the effects of boron (B) on the cell-survival, proliferation, mineralization and mRNA expression of mineralized tissue-associated proteins. Additionally, determination of the effects of B on the BMP-4, -6 and -7 protein levels of pre-osteoblastic cells (MC3T3-E1) was also intended. The effects of B (pH 7.0) concentrations (0, 0.1, 1, 10, 100, 1000, 2000, 4000, 8000 and 10,000 ng/ml) on the survival of the cells were evaluated at 24 and 96 hrs with MTT assay. To evaluate the proliferation in long term, MC3T3-E1 cells were treated with different concentrations of B (0, 0.1, 1, 10, 100 and 1000 ng/ml) and were counted on days 2, 5, and 14. While in short term, decreased cell survival rate was observed at 1000 ng/ml and above, at long term no statistically significant difference was detected in different B concentrations applied. Slight decreases at the proliferation of the B-treated groups were determined on days 5 and 14 but one-way analysis of variance revealed that the difference was statistically insignificant. In mineralization assay, increased mineralized nodules were apparently observed in B treatment (1 and 10 ng/ml concentrations) groups. Based on quantitative RT-PCR results, remarkable regulation in favor of osteoblastic function for Collagen type I (COL I), Osteopontin (OPN), Bone Sialoprotein (BSP), Osteocalcin (OCN) and RunX2 mRNA expressions were observed in B treatment groups in comparison with untreated control groups. Increased BMP-4, -6 and -7 protein levels were detected at 0.1, 1, 10 and 100 ng/ml B concentrations. Results of the study suggest that at the molecular level B displays important roles on bone metabolism and may find novel usages at the regenerative medicine.

Reference:

Sema S. Hakkia, Buket S. Bozkurtb, Erdogan E. Hakkic. Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1). Journal of Trace Elements in Medicine and Biology. October 2010: V24, Issue 4; P243-250. doi:10.1016/j.jtemb.2010.03.003

Encapsulated boron as an osteoinductive agent for bone scaffolds

Abstract

The aim of this study was to develop boron (B)-releasing polymeric scaffold to promote regeneration of bone tissue. Boric acid-doped chitosan nanoparticles with a diameter of approx. 175 nm were produced by tripolyphosphate (TPP)-initiated ionic gelation process. The nanoparticles strongly attached via electrostatic interactions into chitosan scaffolds produced by freeze-drying with approx. 100 μm pore diameter. According to the ICP-OES results, following first 5 h initial burst release, fast release of B from scaffolds was observed for 24 h incubation period in conditioned medium. Then, slow release of B was performed over 120 h. The results of the cell culture studies proved that the encapsulated boron within the scaffolds can be used as an osteoinductive agent by showing its positive effects on the proliferation and differentiation of MC3T3-E1 preosteoblastic cells.

Reference: Menemşe Gümüşderelioğlu et. al. Encapsulated boron as an osteoinductive agent for bone scaffolds. Journal of Trace Elements in Medicine and Biology. July 2015: V31, P120128. doi:10.1016/j.jtemb.2015.03.008

Silicon and boron differ in their localization and loading in bone

Abstract

Silicon and boron share many similarities, both chemically and biochemically, including having similar effects on bone, although their mechanisms of action are not known. Here we compared the loading of silicon and boron into bone, their localization and how they are influenced by age (growth & development), to obtain further clues as to the biological effects of these elements and, especially, to see if they behave the same or not. Bone samples were obtained from two different studies where female Sprague Dawley rats had been maintained on a normal maintenance diet for up to 43 weeks. Total bone elemental levels were determined by ICP-OES following microwave assisted acid digestion. Silicon and boron levels in the decalcified bones (i.e. the collagen fraction) were also investigated. Silicon and boron showed marked differences in loading and in their localization in bone. Highest silicon and lowest boron concentrations were found in the under-mineralized bone of younger rats and lowest silicon and highest boron concentrations were found in the fully mineralized bone of the adult rat. Overall, however total bone silicon content increased with age, as did boron content, the latter mirroring the increase in calcium (mineral) content of bone. However, whereas silicon showed equal distribution in the collagen and mineral fractions of bone, boron was exclusively localized in the mineral fraction. These findings confirm the reported association between silicon and collagen, especially at the early stages of bone mineralization, and show that boron is associated with the bone mineral but not connective tissues. These data suggest that silicon and boron have different biological roles and that one is unlikely, therefore, to substitute for the other, or at least boron would not substitute for Si in the connective tissues. Finally, we noted that silicon levels in the mineral fraction varied greatly between the two studies, suggesting that one or more nutritional factor(s) may influence the loading of Si into the mineral fraction of bone. This and the nature of the interaction between Si and collagen deserve further attention.

Reference:Ravin Jugdaohsingha,Liliana D. Pedroa, Abigail Watsona, b, Jonathan J. Powella. Silicon and boron differ in their localization and loading in bone. Bone Reports. January 2015: V1, P9-15. doi:10.1016/j.bonr.2014.10.002

Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering

Abstract

Scaffold-based bone defect reconstructions still face many challenges due to their inadequate osteoinductive and osteoconductive properties. Various biocompatible and biodegradable scaffolds, combined with proper cell type and biochemical signal molecules, have attracted significant interest in hard tissue engineering approaches. In the present study, we have evaluated the effects of boron incorporation into poly-(lactide-co-glycolide-acid) (PLGA) scaffolds, with or without rat adipose-derived stem cells (rADSCs), on bone healing in vitro and in vivo. The results revealed that boron containing scaffolds increased in vitro proliferation, attachment and calcium mineralization of rADSCs. In addition, boron containing scaffold application resulted in increased bone regeneration by enhancing osteocalcin, VEGF and collagen type I protein levels in a femur defect model. Bone mineralization density (BMD) and computed tomography (CT) analysis proved that boron incorporated scaffold administration increased the healing rate of bone defects. Transplanting stem cells into boron containing scaffolds was found to further improve bone-related outcomes compared to control groups. Additional studies are highly warranted for the investigation of the mechanical properties of these scaffolds in order to address their potential use in clinics. The study proposes that boron serves as a promising innovative approach in manufacturing scaffold systems for functional bone tissue engineering.

Reference: Ayşegül Doğan et. al.. Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering. Materials Science and Engineering: C. November 1, 2014: V44, P246-253. doi:10.1016/j.msec.2014.08.035

Dietary boron does not affect tooth strength, micro-hardness, and density, but affects tooth mineral composition and alveolar bone mineral density in rabbits fed a high-energy diet

Abstract

The objective of this study was to determine whether dietary boron (B) affects the strength, density and mineral composition of teeth and mineral density of alveolar bone in rabbits with apparent obesity induced by a high-energy diet. Sixty female, 8-month-old, New Zealand rabbits were randomly assigned for 7 months into five groups as follows: (1) control 1, fed alfalfa hay only (5.91 MJ/kg and 57.5 mg B/kg); (2) control 2, high energy diet (11.76 MJ and 3.88 mg B/kg); (3) B10, high energy diet + 10 mg B gavage/kg body weight/96 h; (4) B30, high energy diet + 30 mg B gavage/kg body weight/96 h; (5) B50, high energy diet + 50 mg B gavage/kg body weight/96 h. Maxillary incisor teeth of the rabbits were evaluated for compression strength, mineral composition, and micro-hardness. Enamel, dentin, cementum and pulp tissue were examined histologically. Mineral densities of the incisor teeth and surrounding alveolar bone were determined by using micro-CT. When compared to controls, the different boron treatments did not significantly affect compression strength, and micro-hardness of the teeth, although the B content of teeth increased in a dose-dependent manner. Compared to control 1, B50 teeth had decreased phosphorus (P) concentrations. Histological examination revealed that teeth structure (shape and thickness of the enamel, dentin, cementum and pulp) was similar in the B-treated and control rabbits. Micro CT evaluation revealed greater alveolar bone mineral density in B10 and B30 groups than in controls. Alveolar bone density of the B50 group was not different than the controls. Although the B treatments did not affect teeth structure, strength, mineral density and micro-hardness, increasing B intake altered the mineral composition of teeth, and, in moderate amounts, had beneficial effects on surrounding alveolar bone.

Reference: Sema S. Hakki et. al.. Dietary boron does not affect tooth strength, micro-hardness, and density, but affects tooth mineral composition and alveolar bone mineral density in rabbits fed a high-energy diet. Journal of Trace Elements in Medicine and Biology. January 2015: V29, P208-215. doi:10.1016/j.jtemb.2014.10.007

Boron influences immune and antioxidant responses by modulating hepatic superoxide dismutase activity under calcium deficit abiotic stress in Wistar rats

Abstract

The influence of Boron (B) supplementation on immune and antioxidant status of rats with or without abiotic stress induced by dietary calcium (Ca) restriction was studied in a feeding trial of 90 days. Wistar strain rats (3–4 wk age, n = 84) were divided into 7 dietary groups (4 replicates of 3 each) viz., normal-calcium (100%) basal diet alone (NC, control) or supplemented with B at 5 (NCB-5), 10 (NCB-10), 20 (NCB-20) and 40 ppm (NCB-40) levels; low-calcium (50%) basal diet alone (LC) or supplemented with 40 ppm B (LCB-40). After 75 days of experimental feeding, rats were challenged with intraperitoneal injection of sheep RBCs to assess their humoral immunity. At the end of the trial, cell-mediated immunity was assessed as foot pad reaction to sheep RBCs injected into the hind leg paws. Eight rats from each group were sacrificed to collect blood for estimation of minerals and total antioxidant activity, and liver for superoxide dismutase gene expression analysis. Supplementation of graded levels of B (5, 10, 20 and 40 ppm) as borax in NC diets significantly increased (P < 0.01) the footpad thickness and serum total antioxidant activity, hepatic expression levels of both Cu-Zn SOD (SOD1) and Mn-SOD (SOD2) mRNAs. The erythrocytic SOD activity and humoral response did not differ significantly among the dietary groups. In Ca restricted groups, humoral immune response was significantly decreased (P < 0.01) compared to control but increased (P < 0.05) with 40 ppm B supplementation. Serum levels of copper (Cu) and zinc (Zn) remained similar among the dietary groups, while the manganese (Mn) content was significantly decreased (P < 0.01) with increased levels of dietary B. In conclusion, B supplementation increased the hepatic mRNA expression levels of both SOD isoenzymes, thereby improving the immune and antioxidant status.

Reference: T. Vijay Bhasker et. al.. Boron influences immune and antioxidant responses by modulating hepatic superoxide dismutase activity under calcium deficit abiotic stress in Wistar rats. Journal of Trace Elements in Medicine and Biology. July 2016: V36, P73-79. doi:10.1016/j.jtemb.2016.04.007

Avocado Soy Unsaponifiables (Asu)

Symptomatic efficacy of avocado–soybean unsaponifiables (ASU) in osteoarthritis (OA) patients: a meta-analysis of randomized controlled trials 1

Summary

Objective

To evaluate the efficacy of preparations with avocado–soybean unsaponifiables (ASUs) in osteoarthritis (OA) patients using meta-analysis on randomized controlled trials (RCTs).

Method

RCTs from systematic searches were included if they explicitly stated that hip and/or knee OA patients were randomized to either ASU or placebo. The co-primary outcome was reduction in pain and Lequesne index, leading to effect size (ES), calculated as the standardized mean difference. As secondary analysis, the number of responders to therapy was analyzed as odds ratios (ORs). Restricted maximum likelihood methods were applied for the meta-analyses, using mixed effects models.

Results

Four trials – all supported by the manufacturer – were included, with 664 OA patients with either hip (41.4%) or knee (58.6%) OA allocated to either 300 mg ASU (336) or placebo (328). Average trial duration was 6 months (range: 3–12 months). Though based on heterogeneous results, the combined pain reduction favored ASU (I2 = 83.5%, ES = 0.39 [95% confidence intervals: 0.01–0.76], P = 0.04). Applying the Lequesne index also favored ASU (I2 = 61.0%, ES = 0.45 [0.21–0.70], P = 0.0003). Secondarily, the number of responders following ASU compared to placebo (OR = 2.19, P = 0.007) corresponded to a number needed to treat of six (4–21) patients.

Conclusions

Based on the available evidence, patients may be recommended to give ASU a chance for e.g., 3 months. Meta-analysis data support better chances of success in patients with knee OA than in those with hip OA.

Reference:

  1. Christensen, M.Sc., E.M. Bartels, D.Sc., A. Astrup, M.D., Ph.D., H. Bliddal, M.D., Ph.D. Symptomatic efficacy of avocado–soybean unsaponifiables (ASU) in osteoarthritis (OA) patients: a meta-analysis of randomized controlled trials 1. Osteoarthritis and Cartilage. April 2008: V16, Issue4; P399-408. doi:10.1016/j.joca.2007.10.003

Management of Osteoarthritis with Avocado/Soybean Unsaponifiables

Abstract

Osteoarthritis (OA) is a painful and life-altering disease that severely limits the daily activities of millions of Americans, and it is one of the most common causes of disability in the world. With obesity on the rise and the world’s population living longer, the prevalence of OA is expected to increase dramatically in the coming decades, generating burdensome socioeconomic costs. This review summarizes current pharmaceutical, nonpharmaceutical, and prospective new treatments for OA, with primary focus on the dietary supplement avocado/soybean unsaponifiables (ASU). ASU modulates OA pathogenesis by inhibiting a number of molecules and pathways implicated in OA. Anticatabolic properties prevent cartilage degradation by inhibiting the release and activity of matrix metalloproteinases and increasing tissue inhibitors of these catabolic enzymes. ASU also inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor. Anabolic properties promote cartilage repair by stimulating collagen and aggrecan synthesis via inhibition of inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor, ERK, and prostaglandin E2. Chondroprotective effects are mediated by correcting growth factor abnormalities, increasing TGF-β, and decreasing vascular endothelial growth factor (VEGF) in synovial fluid. ASU also inhibits cholesterol absorption and endogenous cholesterol biosynthesis, which mediate reactive oxygen species pathology in chondrocytes. At the clinical level, ASU reduces pain and stiffness while improving joint function, resulting in decreased dependence on analgesics.

Reference: Blaine A. Christiansen,1 Simrit Bhatti,2 Ramin Goudarzi,3 and Shahin Emam. Management of Osteoarthritis with Avocado/Soybean Unsaponifiables. Cartilage. January 2015: V6, Issue 1; P30-44. doi: 10.1177/1947603514554992

Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes

Abstract

Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.

Reference:Louis Lippiello, Joseph V. Nardo, Robert Harlan, and Tiffany Chiou. Metabolic Effects of Avocado/Soy Unsaponifiables on Articular Chondrocytes. Evid Based Complement Alternat Med. June 2008: V5, Issue 2; P191-197. doi: 10.1093/ecam/nem132

Protective effects of total fraction of avocado/soybean unsaponifiables on the structural changes in experimental dog osteoarthritis: inhibition of nitric oxide synthase and matrix metalloproteinase-13

Abstract

Introduction

The aims of this study were, first, to investigate the in vivo effects of treatment with avocado/soybean unsaponifiables on the development of osteoarthritic structural changes in the anterior cruciate ligament dog model and, second, to explore their mode of action.

Methods

Osteoarthritis was induced by anterior cruciate ligament transection of the right knee in crossbred dogs. There were two treatment groups (n = 8 dogs/group), in which the animals received either placebo or avocado/soybean unsaponifiables (10 mg/kg per day), which were given orally for the entire duration of the study (8 weeks). We conducted macroscopic and histomorphological analyses of cartilage and subchondral bone of the femoral condyles and/or tibial plateaus. We also conducted immunohistochemical analyses in cartilage for the following antigens: inducible nitric oxide synthase, matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS)4 and ADAMTS5.

Results

The size of macroscopic lesions on the tibial plateaus was decreased (P = 0.04) in dogs treated with the avocado/soybean unsaponifiables. Histologically, in these animals the severity of cartilage lesions on both tibial plateaus and femoral condyles, and the cellular infiltration in synovium were significantly decreased (P = 0.0002 and P = 0.04, respectively). Treatment with avocado/soybean unsaponifiables also reduced loss of subchondral bone volume (P < 0.05) and calcified cartilage thickness (P = 0.01) compared with placebo. Immunohistochemical analysis of cartilage revealed that avocado/soybean unsaponifiables significantly reduced the level of inducible nitric oxide synthase (P < 0.05) and MMP-13 (P = 0.01) in cartilage.

Conclusions

This study demonstrates that treatment with avocado/soybean unsaponifiables can reduce the development of early osteoarthritic cartilage and subchondral bone lesions in the anterior cruciate ligament dog model of osteoarthritis. This effect appears to be mediated through the inhibition ofinducible nitric oxide synthase and MMP-13, which are key mediators of the structural changes that take place in osteoarthritis.

Reference: Christelle Boileau et. al..Protective effects of total fraction of avocado/soybean unsaponifiables on the structural changes in experimental dog osteoarthritis: inhibition of nitric oxide synthase and matrix metalloproteinase-13. Arthritis Res Therapy. March 16, 2009: V11, Issue 2; P41. doi: 10.1186/ar2649

Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination

Abstract

Background

Osteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression.

Findings

Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 μg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1β, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1β, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytesWestern blot and immunostaining confirmed NF-κB translocation inhibition.

Conclusions

We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.

Reference: Stacy L Ownby et. al..Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination. Journal of Inflamation. March 28, 2014: V11, Issue 8; doi: 10.1186/1476-9255-11-8.

Collagen Type II

The effect of type II collagen on MSC osteogenic differentiation and bone defect repair

Abstract

The function of type II collagen in cartilage is well documented and its importance for long bone development has been implicated. However, the involvement of type II collagen in bone marrow derived mesenchymal stem cell (BMSC) osteogenesis has not been well investigated. This study elucidated the pivotal role of type II collagen in BMSC osteogenesis and its potential application to bone healing. Type II collagen-coated surface was found to accelerate calcium deposition, and the interaction of osteogenic medium-induced BMSCs with type II collagen-coated surface was mainly mediated through integrin α2β1. Exogenous type II collagen directly activated FAK-JNK signaling and resulted in the phosphorylation of RUNX2. In a segmental defect model in rats, type II collagen-HA/TCP-implanted rats showed significant callus formation at the reunion site, and a higher SFI (sciatic function index) scoring as comparing to other groups were also observed at 7, 14, and 21 day post-surgery. Collectively, type II collagen serves as a better modulator during early osteogenic differentiation of BMSCs by facilitating RUNX2 activation through integrin α2β1-FAK-JNK signaling axis, and enhance bone defect repair through an endochondral ossification-like process. These results advance our understanding about the cartilaginous ECM-BMSC interaction, and provide perspective for bone defect repair strategies.

Reference:

Li-Hsuan Chiu et. al., The effect of type II collagen on MSC osteogenic differentiation and bone defect repair. Biomaterials. March 2014: V35, Issue 9; P2680-2691. doi:10.1016/j.biomaterials.2013.12.005

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

Abstract

Introduction

Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.

Methods

Col-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.

Results

Col-Treg clones are characterized by their specific cytokine profile (IL-10highIL-4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.

Conclusions

Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.

Reference:Hélène Asnagli et. al..Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis. Arthritis Res Therapy. May 22, 2014: V16, Issue 3; R115. doi: 10.1186/ar4567

Influence of collagen type II and nucleus pulposus cells on aggregation and differentiation of adipose tissue-derived stem cells

Abstract

Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem cells (ASCs) in a 3D environment. ASCs were cultured in collagen type I or type II hydrogels alone, or co-cultured in transwells with micromass NP cells for 4 and 14 days. ASCs seeded in collagen type II gels acquired dentritic cell shapes, and orchestrated cell density-dependent gel contraction rates. Up-regulation of collagen type X, but not of other chondrogenic markers was observed at day 4, irrespective of the hydrogel type. Strikingly, in co-cultures with NP cells, more pronounced differentiation of ASCs along the cartilaginous lineage was observed (up-regulation of collagen IIA, IIB and aggrecan gene expression, as well as stronger alcian blue staining), when ASCs were embedded in collagen type II in comparison with type I hydrogels. Interestingly, strong cellular condensations/aggregations were observed in ASC-seeded type II, but not type I gels, and this aggregation was markedly delayed when the same gels were co-cultured with NP cells. The NP cell-mediated inhibition of ASC aggregation in collagen type II gels coincided with down-regulation of integrin subunit α2 gene expression. We conclude that soluble factors released by NP cells can direct chondrogenic differentiation of ASCs in collagen hydrogels, and that combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilage/NP lineage relative to collagen type I hydrogels.

Reference:Z F Lu et. al..Influence of collagen type II and nucleus pulposus cells on aggregation and differentiation of adipose tissue-derived stem cells. J Cell Mol Med. December 2008: V12, Issue 6B; P2812-2822. doi: 10.1111/j.1582-4934.2008.00278.x

Hydroxyl Radical Modification of Collagen Type II Increases Its Arthritogenicity and Immunogenicity

Abstract

Background

The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA.

Methodology/Principal Findings

Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by •OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatorymarkers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group.

Conclusions/Significance

Neo-antigenic epitopes were generated on •OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.

Reference:Uzma Shahab et. al..Hydroxyl Radical Modification of Collagen Type II Increases Its Arthritogenicity and Immunogenicity. PLoS One. February 3, 2012: V7, Issue 2, Pe31199. doi: 10.1371/journal.pone.0031199

Bromelain

Bromelain: A natural proteolytic for intra-abdominal adhesion prevention

Abstract

Introduction

Peritoneal adhesions are pathological fibrous connections between peritoneal surfaces resulting from incomplete peritoneal repair. Adhesions cause various health problems ranging from pelvic pain and bowel obstruction to infertility. To date, no effective agent exists for intra-abdominal adhesion prevention. Bromelain is the crude extract of the pineapple and it has fibrinolytic, antithrombotic, and anti-inflammatory properties. Bromelain has been shown to be effective for removing necrotic tissues and has been found to be effective for treating various wounds, inflammatory conditions, and thrombotic pathologies. In the present study, we evaluated bromelain as a novel agent for preventing intra-abdominal adhesions.

Methods

Group 1 (control group): Adhesions were produced by cecal abrasion method, and no treatment was applied. Group 2 (i.p. bromelain-treated group): After adhesion formation, 10 mg/kg/BW of bromelain dissolved in 1 mL saline solution was applied intraperitoneally for 10 days. Group 3 (i.p. saline-treated group): After adhesion formation, 1 mL saline solution was applied intraperitoneally for 10 days. On postoperative day 10, all animals were sacrificed.

Results

All 30 rats survived surgery. Throughout the follow-up period, no complications were observed. Statistically significant differences were found between the groups with regards to macroscopic adhesion scores, inflammation, fibrosis and neo-vascularization (p < 0.001, <0.001, p = 0.001, p = 0.002, respectively). Macroscopic and histopathologic (inflammation, fibrosis, neo-vascularization) adhesion scores were lowest in the bromelain-treated group.

Conclusion

Bromelain, acting through its barrier, anti-inflammatory, antioxidant, and proteolytic effects and without increasing bleeding tendency or having any adverse effects on wound healing, may be a suitable agent for intra-abdominal adhesion prevention.

Reference:

Ahmet Sahbaz et. al., Bromelain: A natural proteolytic for intra-abdominal adhesion prevention. International Journal of Surgery. February 2015: V14, P 7-11. doi:10.1016/j.ijsu.2014.12.024

Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy

Abstract

Background

Bromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored.

Methods

The growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay.

Results

Bromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy.

Conclusion

We report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in particular in combination, on a panel of gastrointestinal cancer cell lines with different phenotypes and characteristics. These effects apparently resulted from cell cycle arrest, apoptosis and autophagy. Towards the development of novel strategies for the enhancement of microscopic cytoreduction, our results lay the basis for further evaluation of this formulation in locoregional approaches to peritoneal surface malignancies and carcinomatosis.

Reference:Afshin Amini et. al. Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy. J Exp Clin Cancer Res. November 12, 2014: V33, Issue 1; P92. doi: 10.1186/s13046-014-0092-7

Bromelain Surface Modification Increases the Diffusion of Silica Nanoparticles in the Tumor Extracellular Matrix

Abstract

Tumor extracellular matrix (ECM) represents a major obstacle to the diffusion of therapeutics and drug delivery systems in cancer parenchyma. This biological barrier limits the efficacy of promising therapeutic approaches including the delivery of siRNA or agents intended for thermoablation. After extravasation due to the enhanced penetration and retention effect of tumor vasculature, typical nanotherapeutics are unable to reach the nonvascularized and anoxic regions deep within cancer parenchyma. Here, we developed a simple method to provide mesoporous silica nanoparticles (MSN) with a proteolytic surface. To this extent, we chose to conjugate MSN to Bromelain (Br–MSN), a crude enzymatic complex, purified from pineapple stems, that belongs to the peptidase papain family. This surface modification increased particle uptake in endothelial, macrophage, and cancer cell lines with minimal impact on cellular viability. Most importantly Br–MSN showed an increased ability to digest and diffuse in tumor ECM in vitro and in vivo.

Reference: Alessandro Parod et. al.. Bromelain Surface Modification Increases the Diffusion of Silica Nanoparticles in the Tumor Extracellular Matrix. ACS Nano. August 13, 2014: V8, Issue 10; P9874-9883. doi: 10.1021/nn502807n

Lipase

Orientating lipase molecules through surface chemical control for enhanced activity: A QCM-D and ToF-SIMS investigation

Abstract

Bio-active materials consisting of lipase encapsulated within porous silica particles were engineered to control the adsorption kinetics and molecular orientation of lipase, which play critical roles in the digestion kinetics of triglycerides. The adsorption kinetics of Candida antartica lipase A (CalA) was monitored using quartz crystal microbalance with dissipation (QCM-D) and controlled by altering the hydrophobicity of a silica binding support. The extent of adsorption was 2-fold greater when CalA was adsorbed onto hydrophobic silica compared to hydrophilic silica. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) fragmentation patterns, in conjunction with multivariate statistics, demonstrated enhanced exposure of the lipase’s catalytic domain, specifically the histidine group responsible for activity, when CalA was adsorbed on hydrophilic silica. Consequently, lipid digestion kinetics were enhanced when CalA was loaded in hydrophilic porous silica particles, i.e., a 2-fold increase in the pseudo-first-order rate constant for digestion when compared to free lipase. In contrast, digestion kinetics were inhibited when CalA was hosted in hydrophobic porous silica, i.e., a 5-fold decrease in pseudo-first-order rate constant for digestion when compared to free lipase. These findings provide valuable insights into the mechanism of lipase action which can be exploited to develop smarter food and drug delivery systems consisting of porous lipid-based materials.

Reference:

Paul Joycea, Ivan Kempsonb, Clive A. Prestidge,. Orientating lipase molecules through surface chemical control for enhanced activity: A QCM-D and ToF-SIMS investigation. Colloids and Surfaces B: Biointerfaces. June 1, 2016: V142, P173-181. doi:10.1016/j.colsurfb.2016.02.059

Significant elevations of serum lipase not caused by pancreatitis: a systematic review

Abstract

Background

Many authors advocate lipase as the preferred serological test for the diagnosis of pancreatitis and a cut-off level of three or more times the upper limit of normal (ULN) is often quoted. The literature contains no systematic review that explores alternative causes of a lipase level over three times as high as the ULN. Such a review was therefore the objective of this study.

Methods

The EMBASE and MEDLINE databases (1985 to August 2013) were searched for all eligible articles. Predetermined data were extracted and independently analysed by two reviewers.

Results

In total, data from 58 studies were included in the final analysis. The following causes other than pancreatitis of lipase levels exceeding three times the ULN were found: reduced clearance of lipase caused by renal impairment or macrolipase formation; other hepatobiliary, gastroduodenal, intestinal and neoplastic causes; critical illness, including neurosurgical pathology; alternative pancreatic diagnoses, such as non-pathological pancreatic hyperenzymaemia, and miscellaneous causes such as diabetes, drugs and infections.

Conclusions

A series of differential diagnoses for significant serum lipase elevations (i.e. exceeding three times the ULN) has been provided by this study. Clinicians should utilize this knowledge in the interpretation and management of patients who have lipase levels over three times as high as the ULN, remaining vigilant for an alternative diagnosis to pancreatitis. The medical officer should be aware of the possibility of incorrect diagnosis in the asymptomatic patient.

Reference: Ahmer M Hameed,1 Vincent W T Lam,1,2 and Henry C Pleass. Significant elevations of serum lipase not caused by pancreatitis: a systematic review. HPB (Oxford). February 2015: V17, Issue 2; P9-112. doi: 10.1111/hpb.12277

Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males

Abstract

Background

In addition to their general role in the hydrolysis of storage lipids, bumblebee lipases can participate in the biosynthesis of fatty acids that serve as precursors of pheromones used for sexual communication.

Results

We studied the temporal dynamics of lipolytic activity in crude extracts from the cephalic part of Bombus terrestris labial glands. Extracts from 3-day-old males displayed the highest lipolytic activity. The highest lipase gene expression level was observed in freshly emerged bumblebees, and both gene expression and lipase activity were lower in bumblebees older than 3 days. Lipase was purified from labial glands, further characterized and named as BT-1. The B. terrestris orthologue shares 88% sequence identity with B. impatiens lipase HA. The molecular weight of B. terrestris lipase BT-1 wasapproximately 30 kDa, the pH optimum was 8.3, and the temperature optimum was 50°C. Lipase BT-1 showed a notable preference for C8-C10 p-nitrophenyl esters, with the highest activity toward p-nitrophenyl caprylate (C8). The Michaelis constant (Km) and maximum reaction rate (Vmax) for p-nitrophenyl laurate hydrolysis were Km = 0.0011 mM and Vmax = 0.15 U/mg.

Conclusion

This is the first report describing neutral lipase from the labial gland of B. terrestris. Our findings help increase understanding of its possible function in the labial gland.

Reference: Jana Brabcová et. al..Characterization of Neutral Lipase BT-1 Isolated from the Labial Gland of Bombus terrestris Males. PLoS One. November 8, 2013: V8, Issue 11; P e80066. doi: 10.1371/journal.pone.0080066

Lipoprotein composition in patients with type 1 diabetes mellitus: Impact of lipases and adipokines

Abstract

Objective

High cardiovascular mortality in patients with type 1 diabetes (T1DM) is widely recognized. Paradoxically, these patients have been shown to have elevated HDL-C and reduced apoB-containing lipoproteins. The purpose of this investigation was to further characterize the lipoprotein composition in T1DM and to assess the role that lipases and adipokines may play in these differences.

Methods

T1DM patients (89) attending the Diabetes Clinic at the University of Miami and 42 healthy controls were recruited. Clinical characteristics, lipoprotein composition (by ultracentrifugation and HPLC), leptin, and adiponectin were measured in the full cohort, while a subgroup had LPL and hepatic lipase measured.

Results

Subjects were predominately Caucasian and Hispanic. HgbA1c’s were above goal while their mean duration of diabetes was > 20 years. LPL was 2-fold elevated in diabetic women versus controls (+ 107%{p = 0.001}) with no difference in men. Hepatic lipase was reduced 50% {p < 0.001} in women but increased 50% {p = 0.079} in men. Leptin was similar to controls in women but reduced in men (− 60%{p < 0.001}). Adiponectin was elevated in both genders (men: + 55%{p = 0.018}; women: + 46%{p = 0.007}).

LDL-C was reduced in both diabetic men (− 33%{p < 0.001}) and women (− 24%{p < 0.001}) while HDL-C trended higher only in men (+ 13%{p = 0.064}). Both total apoB (men: − 31%{p < 0.001}; women: − 17%{p = 0.016}) and triglycerides (men: − 49%{p < 0.001}; women: − 31%{p = 0.011}) were reduced in both genders while total apoA-I was increased in both (men: + 31%{p < 0.001}; women: + 19%{p = 0.008}). Both men and women had increases in LpA-I (+ 66%{p < 0.001}; + 40%{p = 0.001}) which accounted for essentially the entire increase in HDL mass. VLDL lipids (men: − 53 → 70%; women: − 31 → 57%) were lower as was apoB (particle number) in men (− 51{p < 0.001}) with a similar trend in women (− 35%{p = 0.066}). Cholesterol esters in the particle core were depleted in both genders relative to both apoB (men: − 41%; women: − 37%) and triglycerides (men: − 38%; women: − 34%) (all{p < 0.009}). There were similar differences in IDL.

HDL-L lipids (except triglycerides) (men: + 45 → 74%; women: + 49 → 77%{p < 0.006}), apoA-1 (men: + 162%; women: + 117%{p < 0.001}), and apoA-II (men: + 64%{p = 0.008}; women: + 55%{p = 0.014}) were higher in T1DM patients. These differences produced dramatic increases in LpA-I (men: + 221%; women + 139%{p < 0.001}) and total HDL-L mass (men: + 85%; women: + 78%{p < 0.001}). ApoM (men: + 190%; women: + 149%{p < 0.001}) was also dramatically increased. Conversely, HDL-D lipids were lower in both genders (− 20% → 50%) while apoA-I was not different in either. ApoA-II was lower only in the diabetic women (− 25%{p = 0.015}).

LPL activity correlated primarily with IDL(−), LDL(−), HDL-L(+), and HDL-D(−) only in the women. HL correlated weakly with VLDL(+), LDL(+), HDL-L(−), and HDL-D(+) in women but had much stronger correlations with VLDL(−), IDL(−), and HDL-L(+). Adiponectin correlated with VLDL(−), IDL(−), LDL(−), HDL-L(+), and HDL-D(−) in women but only HDL-L(+) and HDL-D(−) in men. Leptin correlated with very few parameters in women but did correlate weakly with several HDL-L(−) and HDL-M(−) parameters.

Conclusion

Lipoprotein composition and adipokine concentrations in both genders as well as lipase activities in the women would be expected to reduce the atherosclerotic risk in these patients with T1DM. These data suggest that there are functional lipoprotein abnormalities responsible for their CV risk that are not reflected in their plasma concentrations.

Reference: Thomas A. Hughes et. al..Lipoprotein composition in patients with type 1 diabetes mellitus: Impact of lipases and adipokines. Journal of Diabetes and its Complications. May 4, 2016: V 30, Issue 4; P657-668. doi:10.1016/j.jdiacomp.2016.01.018

G0/G1 Switch Gene 2 controls adipose triglyceride lipase activity and lipid metabolism in skeletal muscle

Abstract

Objective

Recent data suggest that adipose triglyceride lipase (ATGL) plays a key role in providing energy substrate from triglyceride pools and that alterations of its expression/activity relate to metabolic disturbances in skeletal muscle. Yet little is known about its regulation. We here investigated the role of the protein G0/G1 Switch Gene 2 (G0S2), recently described as an inhibitor of ATGL in white adipose tissue, in the regulation of lipolysis and oxidative metabolism in skeletal muscle.

Methods

We first examined G0S2 protein expression in relation to metabolic status and muscle characteristics in humans. We next overexpressed and knocked down G0S2 in human primary myotubes to assess its impact on ATGL activity, lipid turnover and oxidative metabolism, and further knocked down G0S2 in vivo in mouse skeletal muscle.

Results

G0S2 protein is increased in skeletal muscle of endurance-trained individuals and correlates with markers of oxidative capacity and lipid content. Recombinant G0S2 protein inhibits ATGL activity by about 40% in lysates of mouse and human skeletal muscle. G0S2 overexpression augments (+49%, p < 0.05) while G0S2 knockdown strongly reduces (−68%, p < 0.001) triglyceride content in human primary myotubes and mouse skeletal muscle. We further show that G0S2 controls lipolysis and fatty acid oxidation in a strictly ATGL-dependent manner. These metabolic adaptations mediated by G0S2 are paralleled by concomitant changes in glucose metabolism through the modulation of Pyruvate Dehydrogenase Kinase 4 (PDK4) expression (5.4 fold, p < 0.001). Importantly, downregulation of G0S2 in vivo in mouse skeletal muscle recapitulates changes in lipid metabolism observed in vitro.

Conclusion

Collectively, these data indicate that G0S2 plays a key role in the regulation of skeletal muscle ATGL activity, lipid content and oxidative metabolism.

Reference: Claire Laurens1 et. al..G0/G1 Switch Gene 2 controls adipose triglyceride lipase activity and lipid metabolism in skeletal muscle. Molecular Metabolism. April 22, 2016: In Press, Uncorrected Proof — Note to users. doi:10.1016/j.molmet.2016.04.004

Protease

Protease expression in giant cell tumour of bone: A comparative study on feline and human samples

Abstract

Human giant cell tumour of bone (GCTB) is a rare low grade of malignancy tumour with tendency to recur. During tumourigenesis the bone remodeling balance is subverted by the tumour cellular components that interacting with bone matrix induce release of growth factors and cytokines, promoting cell proliferation and bone resorption. The master regulators of this positive feed-back are acid and neutral proteases that destroying extracellular matrix increase osteolysis. In contrast, in cats, very few data are reported on GCTB biological activity. In this study, histological features and metalloproteinase (MMPs) and urokinase plasminogen activator system (uPA) expression were compared in human and feline GCTB and differences in distribution and intensity related to histological pattern and clinical behaviour were determined. In both species, the overexpression of these molecules suggested a strong and complex cross-talk between tumour and microenvironment.

Reference:

Leonardo Leonardia, Irene Quattrinib, Franco Ropertoc, Maria Serena Benassib. Protease expression in giant cell tumour of bone: A comparative study on feline and human samples. Research in Veterinary Science. October2, 2013: V95, Issue 2; P310-315. doi:10.1016/j.rvsc.2013.04.011

Design of protease-resistant peptide ligands for the purification of antibodies from human plasma

Abstract

A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations.

Reference:Stefano Menegatti et. al..Design of protease-resistant peptide ligands for the purification of antibodies from human plasma. Journal of Chromatography A. May 6, 2016: V1445, P93-104. doi:10.1016/j.chroma.2016.03.087

Structure and Mechanism of Rhomboid Protease*

Abstract

Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

Reference:Ya Ha,Yoshinori Akiyama, and Yi Xue. Structure and Mechanism of Rhomboid Protease. J Biol Chem. April 12, 2013: V288, Issue 22; P15430-15436. doi: 10.1074/jbc.R112.422378

Protease inhibitor monotherapy is effective in controlling human immunodeficiency virus 1 shedding in the male genital tract

Abstract

Cross-sectional study comparing seminal human immunodeficiency virus type 1 (HIV-1) shedding in patients receiving boosted protease inhibitor monotherapy (mtPI/rtv) (n = 66) versus triple therapy (TT) (n = 61). Seminal HIV-1 shedding rates in patients with undetectable plasma HIV-RNA were 16.0% on mtPI/rtv compared with 28.6% on TT (p 0.173). Aviraemic status and time on viral suppression were independently associated with lack of seminal HIV-1 shedding. During TT, non PI/rtv-based regimens were associated with a better control of HIV infection in semen despite similar time on viral suppression. The use of mtPI/rtv in well-controlled patients is not associated with increased seminal HIV excretion compared with TT.

Reference:A. Torres-Cornejo et. al. Protease inhibitor monotherapy is effective in controlling human immunodeficiency virus 1 shedding in the male genital tract. Clinical Microbiology and Infection. January 2016: V22, Issue 1; P 98.e7-98.e10. doi:10.1016/j.cmi.2015.09.028

Amylase

Low serum amylase in association with metabolic syndrome and diabetes: A community-based study

Abstract

Background

Low serum amylase levels may reflect impaired exocrine-endocrine relationship in the pancreas. However, few clinical studies have addressed this issue. Therefore, in this epidemiological study, we investigated whether low serum amylase was associated with the pathogenesis of impaired insulin action: metabolic syndrome (MetS) and diabetes.

Research Design and Methods

Serum amylase, cardiometabolic risk factors, MetS (Adult Treatment Panel III criteria), and diabetes were examined in 2,425 asymptomatic subjects aged 30-80 years who underwent medical checkups recently (April 2009-March 2010) and 5 years ago.

Results

Clinical variables, except for age and estimated glomerular filtration rate (eGFR), shifted favorably with increasing serum amylase levels. Plasma glucose levels at 1- and 2-hr during OGTT increased significantly with decreasing serum amylase levels. Multiple logistic analyses showed that, compared with highest quartile of serum amylase, lowest quartile was associated with increased risk for MetS and diabetes after adjustment for confounding factors [odds ratio (95% CI), 2.07 (1.39-3.07) and 2.76 (1.49-5.11), respectively]. In subjects who underwent checkups 5 years ago (n = 571), lower amylase at the previous checkup were associated with larger numbers of metabolic abnormalities at the recent checkup. The fluctuation over time in serum amylase levels in subjects with low serum amylase at the previous checkup was slight and was unaffected by kidney dysfunction.

Conclusions

Our results indicate that low serum amylase is associated with increased risk of metabolic abnormalities, MetS and diabetes. These results suggest a pancreatic exocrine-endocrine relationship in certain clinical conditions.

Reference:

Kei Nakajima et. al., Low serum amylase in association with metabolic syndrome and diabetes: A community-based study. Cardiovascular Diabetology. April 17,2011: DOI: 10.1186/1475-2840-10-34

http://cardiab.biomedcentral.com/articles/10.1186/1475-2840-10-34

Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

ABSTRACT

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment

Reference: Anna E. Nikitkova et. al . Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii. Appl Environ Microbiol. March 2012: V78, Issue 6; P1865-1875. doi: 10.1128/AEM.07071-11

Analysis on evolutionary relationship of amylases from archaea, bacteria and eukaryota

Abstract

Amylase is one of the earliest characterized enzymes and has many applications in clinical and industrial settings. In biotechnological industries, the amylase activity is enhanced through modifying amylase structure and through cloning and expressing targeted amylases in different species. It is important to understand how engineered amylases can survive from generation to generation. This study used phylogenetic and statistical approaches to explore general patterns of amylases evolution, including 3118 α-amylases and 280 β-amylases from archaea, eukaryota and bacteria with fully documented taxonomic lineage. First, the phylogenetic tree was created to analyze the evolution of amylases with focus on individual amylases used in biofuel industry. Second, the average pairwise p-distance was computed for each kingdom, phylum, class, order, family and genus, and its diversity implies multi-time and multi-clan evolution. Finally, the variance was further partitioned into inter-clan variance and intra-clan variance for each taxonomic group, and they represent horizontal and vertical gene transfer. Theoretically, the results show a full picture on the evolution of amylases in manners of vertical and horizontal gene transfer, and multi-time and multi-clan evolution as well. Practically, this study provides the information on the surviving chance of desired amylase in a given taxonomic group, which may potentially enhance the successful rate of cloning and expression of amylase gene in different species.

Reference: Shaomin Yan and Guang Wu. Analysis on evolutionary relationship of amylases from archaea, bacteria and eukaryota. World J Microbiol Biotechnol. January 8, 2016: V32, P24. doi: 10.1007/s11274-015-1979-y

The Gastric/Pancreatic Amylase Ratio Predicts Postoperative Pancreatic Fistula With High Sensitivity and Specificity

Abstract

This article aims to identify risk factors for postoperative pancreatic fistula (POPF) and evaluate the gastric/pancreatic amylase ratio (GPAR) on postoperative day (POD) 3 as a POPF predictor in patients who undergo pancreaticoduodenectomy (PD).

POPF significantly contributes to mortality and morbidity in patients who undergo PD. Previously identified predictors for POPF often have low predictive accuracy. Therefore, accurate POPF predictors are needed.

In this prospective cohort study, we measured the clinical and biochemical factors of 61 patients who underwent PD and diagnosed POPF according to the definition of the International Study Group of Pancreatic Fistula. We analyzed the association between POPF and various factors, identified POPF risk factors, and evaluated the predictive power of the GPAR on POD3 and the levels of serum and ascites amylase.

Of the 61 patients, 21 developed POPF. The color of the pancreatic drain fluid, POD1 serum, POD1 median output of pancreatic drain fluid volume, and GPAR were significantly associated with POPF. The color of the pancreatic drain fluid and high GPAR were independent risk factors. Although serum and ascites amylase did not predict POPF accurately, the cutoff value was 1.24, and GPAR predicted POPF with high sensitivity and specificity.

This is the first report demonstrating that high GPAR on POD3 is a risk factor for POPF and showing that GPAR is a more accurate predictor of POPF than the previously reported amylase markers.

Reference: Shuo Jin, MD, et. al. The Gastric/Pancreatic Amylase Ratio Predicts Postoperative Pancreatic Fistula With High Sensitivity and Specificity. Medicine (Baltimore). January 26, 2015: V94, Issue 3; P e339. doi: 10.1097/MD.0000000000000339