Capillary Electrophoresis Separation of Artepillin C: Determination in Brazilian Green Propolis.

Propolis is important in complementary and alternative medicine having well-known therapeutic applications. Artepillin C, a main component of Brazilian (green) propolis, has attracted great attention for its anticancer action. Consequently, the synthesis of artepillin C has been reported but, due to the limited yield and elevated costs, this biomolecule is largely produced from Brazilian propolis. We report the capillary electrophoresis (CE) separation of artepillin C in Brazilian propolis also comparing the results with those of HPLC-UV-MS. Optimal separation was obtained with a simple buffer constituted of sodium tetraborate 30 mM pH 9.2 and detection at 210 nm. Artepillin C and the polyphenols of propolis were fully separated with a voltage gradient of 30 to 8 kV and a current of 300 µA for a total run of 50 min. The sensitivity of CE-UV was 22 times greater than HPLC-UV and 100 times more than HPLC-MS with also a stronger reduction in the run time and a greater robustness and reproducibility. The development of CE as an effective and reliable method for the analysis of artepillin C is desired as the standardized quality controls are essential before propolis or its biomolecules can be adopted routinely in nutraceuticals, food ingredients and therapeutic applications.

Structural definition of terrestrial chondroitin sulfate of various origin and repeatability of the production process.

We report results on the structure, physicochemical characteristics and purity of chondroitin sulfate (CS) samples derived from three largely available and common biological sources such as bovine and porcine trachea and chicken keel bones with the aim to define their structural signatures. Many lots of CS produced by a manufacturer at industrial scale were characterized with a view to assess the reproducibility of the process as not controlled extractive procedures may produce final products with variable structure and biological contaminants as well as not constant clinical efficacy and safety. By using standardized source animal tissues and manufacturing procedure, highly pure CS (∼92 %) products with constant structure and characteristics were obtained. Bovine CS showed a lower molecular weight (MWw of ∼21,500 Da) than porcine (MWw of ∼26,000 Da) and chicken (MWw of ∼35,900 Da) products with a CV% of ∼2.0-7.5 and a polydispersity variability of 0.7-2.7 %. The ratio between the sulfate groups main located in position 4 and 6 of N-acetyl-galactosamine (4/6 ratio) was ∼1.70 for bovine CS versus a value of 3.60 for porcine and ∼2.70 for chicken samples with a overall charge density of 0.92-0.93 and a CV% of 2.1-2.5. The final products also showed the presence of a very low and constant content of other co-purified bio(macro)molecules (hyaluronic acid, keratan sulfate, dermatan sulfate, heparan sulfate, nucleic acids and proteins), calcium and sodium, and the absence of versican. Finally, a high reproducibility of molecular weight values, disaccharide composition, specific optical rotation and particle dimension was observed. The observed parameters are structural signatures useful to specifically identify the origin of CS and obtained by a standardized and highly reproducible manufacturing process. The compositional profile determined from this study provides a measure of the norm and range of variation in CS samples of terrestrial origin produced under standardized production protocol to which future pharmaceutical/nutraceutical final products can be compared. Moreover, the physicochemical properties including molecular weight, disaccharide composition, presence of natural contaminants and particle dimension were characterized to provide the basis of CS of high quality for application as pharmaceutical/nutraceutical active agents.

Short- and Long-Term Effectiveness of Supplementation with Non-Animal Chondroitin Sulphate on Inflammation, Oxidative Stress and Functional Status in Obese Subjects with Moderate Knee Osteoarthritis before and after Physical Stress: A Randomized, Double-Blind, Placebo-Controlled Trial.

It has recently been demonstrated that chronic supplementation with nonanimal chondroitin sulfate (nonanimal CS) in overweight subjects with knee osteoarthritis (OA) improves the function, pain and inflammation, but there are no studies of its effectiveness in an acute setting. In 48 obese subjects with moderate knee OA, we investigated the effectiveness of nonanimal CS supplementation for eight weeks on the inflammation, functional status, oxidative stress, cartilage catabolism markers, metabolic profile and body composition, by Dual-Energy X-ray Absorptiometry (DXA) at the baseline, after 15 days and at the end of the eight-week study. To evaluate the acute effectiveness on inflammation, 15-min cycle training sessions were done 15 days after the start of the study and at the end. C-reactive protein (CRP) was assayed in blood samples collected before and after the two cycling exercises. The 48 obese subjects (M and F, 20-50 years, body mass index (BMI) 30-35 kg/m2) were randomly assigned to an experimental group (N = 24, 600-mg tablet of nonanimal CS/day) or the control group (N = 24, placebo). The between-groups analysis of covariance showed a significant effect on the Western Ontario and McMaster Universities Arthritis index (WOMAC) scale (p = 0.000) and CRP (p = 0.022). For intra-group differences, the result was significant in the CS group for BMI, WOMAC, CRP, total cholesterol and Homeostasis Model Assessment (HOMA). In these obese adults with OA, nonanimal CS improved the inflammation, knee function, metabolic profile and body composition.

Chondroitin/dermatan sulfate purified from corb (Sciaena umbra) skin and bone: In vivo assessment of anticoagulant activity.

The present work deals with the extraction and purification of chondroitin sulfate/dermatan sulfate from skin (CSG) and bone (CBG) of corb (Sciaena umbra). Electrophoresis of these polymers in barium acetate buffer on cellulose acetate revealed two fractions similar to dermatan sulfate and chondroitin sulfate. The in vivo anticoagulant activity of both chondroitin sulfate/dermatan sulfate (CS/DS) were evaluated, at 25 and 75 mg kg-1 of body weight (b.w), using activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests. Results showed that aPTT of CSG and CBG at 75 mg kg-1 of b.w were prolonged by 1.59 and 1.48-fold respectively, compared with the control. Further, toxicity studies on liver performed by the catalytic activity of transaminases in plasma, oxidative stress markers and hepatic morphological changes demonstrated that CSG and CBG at both doses are not toxics. In summary, the higher activity and lower toxicity of both CS/DS, especially at 25 mg kg-1 of b.w, recommended these compounds as a better drug candidate.

Anise Hyssop Agastache foeniculum Increases Lifespan, Stress Resistance, and Metabolism by Affecting Free Radical Processes in Drosophila.

Anise hyssop, Agastache foeniculum, is a widely used medicinal herb with known antioxidant properties. We studied how dietary supplementation with dried A. foeniculum leaf powder affected physiological and metabolic traits as well as activities of antioxidant enzymes and markers of oxidative stress in Drosophila melanogaster. Dietary hyssop extended the lifespan in a sex and genotype independent manner over a broad range of concentrations up to 30 mg/ml. Dietary supplementation with the herb significantly increased fecundity, resistance to oxidative stress and starvation. Higher transcript levels of Drosophila insulin-like peptide (dilp2) and decreased dilp3 and dilp6 transcripts together with increased levels of glycogen and triacylglycerols support an alteration of insulin signaling by the plant extract. Increased enzymatic activities of superoxide dismutase and aconitase as well as elevated protein and low molecular mass thiols also supported an alteration of free radical process in flies treated with dietary A. foeniculum leaf powder. Thus, physiological and metabolic traits as well as free radical processed may be affected by active compounds detected in extracts of anise hyssop leaves and contribute to the increased lifespan and reproductive (egg-laying) activity observed.

Antiviral activity of different extracts of standardized propolis preparations against HSV.

BACKGROUND: Viral infections are among the most common problems in health-care practice. Natural products offer great promise as potentially effective antiviral drugs. Propolis is a honeybee product with biological properties and therapeutic applications. We aimed to investigate the antiviral activity of different extracts of Standardized Propolis Preparations (M.E.D.®) with glycol, ethanol, glycerol and soya oil, against herpes simplex type-1 (HSV-1) and type 2 (HSV-2) viruses. METHODS: Chemical composition and antiviral activity of each extract were determined. The selective index (SI=CC50/EC50) was determined as a parameter to indicate the in vitro antiviral activity of the extracts compared with acyclovir as the control. RESULTS: SI values of glycol, ethanol, glycerol, soya oil extracts and acyclovir were determined as 6.8, 4.1, 2.2, 3.3 and 6.3 against HSV-1, and as 6.4, 7.7, 1.9, 4.2 and 2.9 against HSV-2, respectively. Glycolic propolis extract was found to possess a greater antiviral activity than acyclovir for both HSV-1 and 2, while glycolic, ethanolic and soya oil preparations were found to have more significant activity than acyclovir for HSV-2. CONCLUSIONS: It was determined that standardized propolis preparations have antiviral bioactivity against HSV.

Chondroitin Sulfate Safety and Quality.

The industrial production of chondroitin sulfate (CS) uses animal tissue sources as raw material derived from different terrestrial or marine species of animals. CS possesses a heterogeneous structure and physical-chemical profile in different species and tissues, responsible for the various and more specialized functions of these macromolecules. Moreover, mixes of different animal tissues and sources are possible, producing a CS final product having varied characteristics and not well identified profile, influencing oral absorption and activity. Finally, different extraction and purification processes may introduce further modifications of the CS structural characteristics and properties and may lead to extracts having a variable grade of purity, limited biological effects, presence of contaminants causing problems of safety and reproducibility along with not surely identified origin. These aspects pose a serious problem for the final consumers of the pharmaceutical or nutraceutical products mainly related to the traceability of CS and to the declaration of the real origin of the active ingredient and its content. In this review, specific, sensitive and validated analytical quality controls such as electrophoresis, eHPLC (enzymatic HPLC) and HPSEC (high-performance size-exclusion chromatography) able to assure CS quality and origin are illustrated and discussed.

Oral Bioavailability and Pharmacokinetics of Nonanimal Chondroitin Sulfate and Its Constituents in Healthy Male Volunteers.

The pharmacokinetic profile of a new 800-mg tablet of nonanimal chondroitin sulfate (CS) (Mythocondro®, 800-mg tablets, Gnosis S.p.A., Italy) was investigated vs an animal CS in healthy volunteers for a total period of 48 hours. After a single 2400-mg dose of the test and the reference formulation, total CS, the compositional disaccharides (ΔDi6S, ΔDi4S and ΔDi0S), and the overall charge density were quantified in plasma. The safety and tolerability profile after a single dose of this new nonanimal CS tablets was excellent. After baseline-corrected concentrations, an overall greater plasma concentration was observed after 24 hours of ∼44% and after 48 hours of ∼45% from administration of nonanimal when compared to animal-derived CS. Moreover, nonanimal CS increases the specific sulfation in the 6-position of N-acetyl-galactosamine in human plasma CS and, as a consequence, the overall charge density, reaching double values (0.91), after 48 hours compared to bovine CS and to endogenous CS. In conclusion, nonanimal CS, possessing a lower molecular weight than an animal-derived sample, produces a greater CS concentration for a more prolonged period of time in plasma and an increase in charge density and specific 6-sulfation of endogenous plasma CS.

Selective treatment to reduce contamination of propolis by polycyclic aromatic hydrocarbons (PAHs) still preserving its active polyphenol component and antioxidant activity.

The adverse effects on health and environment caused by polycyclic aromatic hydrocarbons (PAHs) are critical problems. EFSA has defined 16 priority PAHs that are both genotoxic and carcinogenic, and identified eight (PAH8) priority PAHs as good indicators of the toxicity and occurrence in food. Food supplements containing propolis were also found to contain relatively high quantities of PAHs. We report about an extractive procedure which is able to purify propolis from a high content of PAHs using a balanced mixture of ethanol and water solvents. Extracts were characterised for total content of polyphenols, for in vitro antioxidant activity, and single classes of polyphenols evaluated by HPLC-ESI-MS. Obtained propolis extracts were found to have PAH8 and specific benzo[a]pyrene content below limits recommended by EFSA. The reported extractive procedure is easily applicable for possible industrial productions and may also be adopted to the purification of polyphenols from other plant extracts and natural sources.

Safety assessment of non-animal chondroitin sulfate sodium: Subchronic study in rats, genotoxicity tests and human bioavailability.

Chondroitin sulfate, an amino sugar polymer made of glucuronic acid and N-acetyl-galactosamine, is used in dietary supplements to promote joint health. Commonly used chondroitin sulfate is of animal origin and can pose potential safety problems including bovine spongiform encephalopathy (BSE). The objective of the present study was to investigate potential adverse effects, if any, of microbial derived chondroitin sulfate sodium (CSS) in subchronic toxicity, genotoxicity and bioavailability studies. In the toxicity study, Sprague Dawley rats (10/sex/group) were gavaged with CSS at dose levels of 0, 250, 500 and 1000 mg/kg body weight (bw)/day for 90-days. No mortality or significant changes in clinical signs, body weights, body weight gain or feed consumption were noted. Similarly, no toxicologically relevant treatment-related changes in hematological, clinical chemistry, urinalysis and organ weights were noted. Macroscopic and microscopic examinations did not reveal treatment-related abnormalities. In vitro mutagenic and clastogenic potentials as evaluated by Ames assay, chromosomal aberration test and micronucleus assay did not reveal genotoxicity of CSS. In pharmacokinetic study in human, CSS showed higher absorption as compared to chondroitin sulfate of animal origin. The results of subchronic toxicity study supports the no-observed-adverse-effect level (NOAEL) for CSS as 1000 mg/kg bw/day, the highest dose tested.

Effect of nonanimal high- and low-molecular-mass chondroitin sulfates produced by a biotechnological process in an animal model of polyarthritis.

BACKGROUND/AIMS: We planned to report on the effect of two nonanimal chondroitin sulfates (CSs) with different molecular masses produced using an innovative biotechnological process in an adjuvant arthritis animal model. METHODS: The experiments included healthy animals, untreated arthritic animals and arthritic animals having been administered 900 mg/kg of either of the two CS samples daily. Arthritic score, γ-glutamyltransferase (GGT) activity in hind paw joint tissue homogenates, plasmatic C-reactive protein (CRP) and pro-inflammatory cytokines IL-1ß and IL-6 were assayed. RESULTS AND CONCLUSIONS: Low-molecular-mass (LMM) CS significantly reduced the arthritic score by up to about 30% from 14 to 28 days. In contrast, no significant differences were observed for high-molecular-mass (HMM) CS, even if a trend in its capacity to decrease the arthritic score by up to about 11% was observed. Additionally, LMM CS was able to significantly decrease GGT activity by approximately 31% and plasmatic CRP levels by about 9%. Both nonanimal CS samples were effective in reducing plasmatic levels of proinflammatory cytokines. A greater efficacy was also observed for LMM CS compared with a pharmaceutical-grade CS of extractive origin, while the efficacy of the HMM CS sample was found to be rather similar. The greater effect of LMM CS in reducing arthritic parameters may be related to its lower molecular mass with respect to HMM CS and natural CS.

Human milk glycosaminoglycans: the state of the art and future perspectives.

Recently, a complete characterization and detailed evaluation of the glycosaminoglycans of human milk were performed. The total glycosaminoglycans content in milk from healthy mothers having delivered term or preterm newborns showed a constant pattern which was essentially composed of two main polysaccharides: chondroitin sulfate (60-70%) and heparin (30-40%). Moreover, considerable variations of glycosaminoglycans concentration were found during the first month of lactation, the highest values being present in colostrum compared to mature milk. Metabolism and potential biological functions of human milk glycosaminoglycans are hypothesized and future studies are encouraged.

Anti-inflammatory activity of chondroitin sulphate: new functions from an old natural macromolecule.

Chondroitin sulphate (CS) is recommended by the European League Against Rheumatism as a symptomatic slow-acting drug for the treatment of osteoarthritis on the basis of numerous clinical trials and meta-analyses. Furthermore, recent clinical trials have also demonstrated the possible structure-modifying effects of CS. This review focuses on recent experimental results and data available in the scientific literature regarding the anti-inflammatory properties of CS with a view to understanding the molecular basis responsible for its activity. Several animal studies have demonstrated that orally administered CS significantly inhibited hind paw oedema, synovitis and destruction of the articular cartilage in a dose-dependent manner. Furthermore, CS proved to have a beneficial effect in slowing down the development of adjuvant arthritis and in reducing disease markers, findings which support its beneficial activity in humans as a chondroprotective drug. Finally, several in vitro studies have focused on the hypothesis that CS may reduce inflammatory processes by acting on the nuclear translocation of NF-κB, which is closely associated with the blood biomarkers of inflammation, primarily IL-1, IL-6 and C-reactive protein.

Quality of different chondroitin sulfate preparations in relation to their therapeutic activity.

OBJECTIVES: Chondroitin sulfate is currently recommended by the European League Against Rheumatism (EULAR) as a SYSADOA (symptomatic slow acting drug for osteoarthritis) in Europe in the treatment of knee and hand osteoarthritis based on research evidence and meta-analysis of numerous clinical studies. Furthermore, recent clinical trials demonstrated its possible structure-modifying effects. Chondroitin sulfate, alone or in combination with glucosamine or other ingredients, is also utilized as a nutraceutical in dietary supplements in Europe and the USA. However, it is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants and many other factors contribute to the overall biological and pharmacological actions of these agents. We aim to review the quality control of chondroitin sulfate in pharmaceutical-grade preparations and nutraceuticals. KEY FINDINGS: Pharmaceutical-grade formulations of chondroitin sulfate are of high and standardized quality, purity and properties, due to the stricter regulations to which this drug is subjected by local national health institutes as regards production and characteristics. On the contrary, as several published studies available in literature indicate, the chondroitin sulfate quality of several nutraceuticals is poor. Additionally, there are no definite regulations governing the origin of the ingredients in these nutraceuticals and the origin of the ingredients in natural products is the most important factor ensuring quality, and thus safety and efficacy, in particular for chondroitin sulfate, due to its extraction from different sources. CONCLUSIONS: Due to the poor chondroitin sulfate quality of some nutraceuticals, we conclude that stricter regulations regarding their quality control should be introduced to guarantee the manufacture of high quality products for nutraceutical utilization and to protect customers from low-quality, ineffective and potentially dangerous products. There is a need for specific and accurate analytical procedures, which should be enforced to confirm purity and label claims both for raw materials and finished chondroitin sulfate products, and also to govern the origin of ingredients. Until these stricter regulations are in place, then it is strongly recommended that pharmaceutical-grade chondroitin sulfate is used rather than food supplements.

Capillary electrophoresis determination of glucosamine in nutraceutical formulations after labeling with anthranilic acid and UV detection.

A new robust CE method for the determination of the glucosamine (GlcN) content in nutraceutical formulations is described after its derivatization with anthranilic acid (2-aminobenzoic acid, AA). The CE separation of derivatized GlcN with AA was performed on an uncoated fused-silica capillary tube (50 microm I.D.) using an operating pH 7.0 buffer of 150 mM boric acid/50 mM NaH2PO4 and UV detection at 214 nm. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for GlcN was linear over the selected concentration range from 240 to 2400 pg (40-400 microg/mL) with a correlation coefficient greater than 0.980. The intra- and inter-day variations (CV%) were between 0.5 and 0.9 for migration time, and between 2.8 and 4.3 for peak area, respectively. The LOD and the LOQ of the method were approximately 200 and 500 pg, respectively. The intra- and inter-day accuracy was estimated to range from 2.8% to 5.1%, while the percent recoveries of GlcN in formulations were calculated to be about 100% after simple centrifugation for 10 min, lyophilization and derivatization with AA. The CE method was applied to the determination of GlcN content, in the form of GlcN-hydrochloride or GlcN-sulfate, of several nutraceutical preparations in the presence of other ingredients, i.e. chondroitin sulfate, vitamin C and/or methylsulfonylmethane (MSM) as well as salts and other agents. The quantitative results obtained were in total conformity with the label claims.

Analytical aspects of pharmaceutical grade chondroitin sulfates.

Chondroitin sulfate is a very heterogeneous polysaccharide in terms of relative molecular mass, charge density, chemical properties, biological and pharmacological activities. It is actually recommended by EULAR as a symptomatic slow acting drug (SYSADOA) in Europe in the treatment of knee osteoarthritis based on meta-analysis of numerous clinical studies. Chondroitin sulfate is also utilized as a nutraceutical in dietary supplements mainly in the United States. On the other hand, chondroitin sulfate is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants, and many other factors contribute to the overall biological and pharmacological actions of these agents. The aim of this review is to evaluate new possible more specific analytical approaches to the determination of the origin and purity of chondroitin sulfate preparations for pharmaceutical application and in nutraceuticals, such as the evaluation of the molecular mass values, the constituent disaccharides, and the specific and sensitive agarose-gel electrophoresis technique. Furthermore, a critical evaluation is presented, together with a discussion of the limits of these analytical approaches. Finally, the necessity for reference standards having high specificity, purity and well-known physico-chemical properties useful for accurate and reproducible quantitative analyses will be discussed.