Chondroitin Sulfate in USA Dietary Supplements in Comparison to Pharma Grade Products: Analytical Fingerprint and Potential Anti-Inflammatory Effect on Human Osteoartritic Chondrocytes and Synoviocytes.

The biological activity of chondroitin sulfate (CS) and glucosamine (GlcN) food supplements (FS), sold in USA against osteoarthritis, might depend on the effective CS and GlcN contents and on the CS structural characteristics. In this paper three USA FS were compared to two pharmaceutical products (Ph). Analyses performed by HPAE-PAD, by HPCE and by SEC-TDA revealed that the CS and GlcN titers were up to -68.8% lower than the contents declared on the labels and that CS of mixed animal origin and variable molecular weights was present together with undesired keratan sulfate. Simulated gastric and intestinal digestions were performed in vitro to evaluate the real CS amount that may reach the gut as biopolymer. Chondrocytes and synoviocytes primary cells derived from human pathological joints were used to assess: cell viability, modulation of the NF-κB, quantification of cartilage oligomeric matrix protein (COMP-2), hyaluronate synthase enzyme (HAS-1), pentraxin (PTX-3) and the secreted IL-6 and IL-8 to assess inflammation. Of the three FS tested only one (US FS1) enhanced chondrocytes viability, while all of them supported synoviocytes growth. Although US FS1 proved to be less effective than Ph as it reduced NF-kB, it could not down-regulate COMP-2; HAS-1 was up-regulated but with a lower efficacy. Inflammatory cytokines were markedly reduced by Ph while a slight decrease was only found for US-FS1.

First small-molecule PROTACs for G protein-coupled receptors: inducing α 1A-adrenergic receptor degradation.

Proteolysis targeting chimeras (PROTACs) are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitin-proteasome system (UPS), thereby selectively reducing the target protein level by the ubiquitin-proteasome pathway. Nowadays, small-molecule PROTACs are gaining popularity as tools to degrade pathogenic protein. Herein, we present the first small-molecule PROTACs that can induce the α 1A-adrenergic receptor (α 1A-AR) degradation, which is also the first small-molecule PROTACs for G protein-coupled receptors (GPCRs) to our knowledge. These degradation inducers were developed through conjugation of known α 1-adrenergic receptors (α 1-ARs) inhibitor prazosin and cereblon (CRBN) ligand pomalidomide through the different linkers. The representative compound 9c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression, which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Compositional Monosaccharide Analysis of Morus nigra Linn by HPLC and HPCE Quantitative Determination and Comparison of Polysaccharide from Morus nigra Linn by HPCE and HPLC.

BACKGROUND: Morus nigra Linn is not only treated as health food but also medicine in Chinese history. OBJECTIVE: Here, we have extracted and separated the heteroglycan to monosaccharides of the dry fruits. METHOD: After heteroglycan being hydrolysised, then were we derivatived the monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP), and then subsequently HPCE and HPLC were used to separate. HPCE, which held an uncoated capillary (d=75µm) and detected by PDA at 245 nm with borate buffer, the voltage was set at 15 kV and capillary temperature was 25°C. There is Yilite column (Hypersil BDS C18 5µm, 4.6mm×250mm) with HPLC and detected by UV at 245 nm and capillary temperature 25°C.The High Performance capillary electrophoresis method and High Performance Liquid Chromatog-raphy method were compared on monosaccharide composition and quantitative determination of polysac-charide from the black mulberry. RESULTS: Through several indicators including standard curve, precision, reproducibility, stability and recovery rate, the result of the two methods is basically consistent while they have complementary ad-vantages. CONCLUSION: Both methods are suitable for the determination of the black mulberry polysaccharide, but each has its own merits.

Simultaneous determination of seven nucleosides in inonotus obliquus by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneous determination of seven nucleosides and nucleobases, including adenine, uracil, adenosine, hypoxanthine, uridine, guanosine, and inosine in Inonotus obliquus samples of different batches. METHODS: Based on the mode of HPCE, uncoated fused silica capillary (75 μm × 64.5 cm, 56 cm of effective length) was used with separation voltage of 20 kV. The 55 mmol·L-1 sodium borate solution was selected as the running buffer solution (pH 9.5). The detection wavelength was set at 260 nm. The sample was injected at 5 kPa × 6 s and column temperature was maintained at 30℃. RESULTS: Good linear relationship was obtained between the response peak areas and the concentrations of all the seven nucleosides (r > 0.998 8). The average recoveries of the method were between 96.46%-102.12%. The contents of the seven nucleosides in the Inonotus obliquus samples of different origins were different. CONCLUSION: The established method is reliable, accurate, and can be used for the quality control of Inonotus obliquus.

Contents Determination of Luteoloside,Quercetin and Hyperoside in the Extract of Lonicera japonica by HPCE / 中国药房

OBJECTIVE:To establish a method for contents determination of luteoloside,quercetin and hyperoside in Lonicera japonica. METHODS:HPCE was performed silica capillary column with detection wavelength of 360 nm and separation voltage of 20 kV,electrokinetic sampling,sampling voltage of 15 kV,sampling time of 5 s,operation temperature of 25 ℃.The buffer was consisted of 60 mmol/L sodium tetraborate-50 mmol/L natrium carbonicum-50 mmol/L hydroxypropyl-β-cyclodextrin(pH 9.2). RE-SULTS:The linear ranges of luteoloside,quercetin and hyperoside were 0.06-0.56mg/mL (r=0.9881),0.08-0.56 mg/mL (r=0.9892),0.06-0.49 mg/mL(r=0.9796),respectively. RSDs of precision,stability and reproducibility tests were all lower than 2.0%. The recoveries were 96.12%-99.77%(RSD=1.29%,n=6),95.90%-98.35%(RSD=0.89%,n=6),94.07%-97.45%(RSD=1.33%,n=6),respectively. CONCLUSIONS:The method is simple,accurate,stable and reproducible,and can be used for simultaneous determination of luteoloside,quercetin and hyperoside in L. japonica.

Identification of velvet antler by random amplified polymorphism DNA combined with non-gel sieving capillary electrophoresis.

Mitochondrial DNA of velvet antler was amplified with random amplified polymorphic DNA (RAPD) technique and the PCR products were detected with non-gel sieving capillary electrophoresis to establish a RAPD-HPCE method used for identifying the authenticity of velvet antler or it counterfeits. Factors that could affect the PCR amplification and capillary electrophoresis were optimized. Under the optimized conditions, namely, 20 mmol L(-1) NaH2PO4-Na2HPO4-2 mmol L(-1) EDTA buffer solution [0.8% (W/V) HPMC, 15 mmol L(-1) TBAP and pH 7.3], -10 kV injection voltage and -8 kV separation voltage, Cervus nippon Temminck antler, Cervus elaphus Linnaeus antler, Rangifer tarandus antler, Cervus canadensis antler and Elaphurus davidianus antler were analyzed. The analysis on the similarity of obtained elctrophoretograms showed that there were significant differences in similarities of different velvet antlers, which could be used for the quick identification of the authenticity of velvet antler samples. It can be found that the technique of RAPD combined with HPCE is advantageous in rich polymorphism, high detection rate, simple and convenient performance, high efficiency, rapidness and sensitivity, indicating that it should be suitable for the quick identification of the authenticity of velvet antler samples.

Determination of gallic acid in Quanshen pieces of two different colors by HPCE / 中国药学杂志

OBJECTIVE: To establish a high-performance capillary electrophoresis (CE) method for determinating gallic acid in commercial Quanshen pieces of two different colors, amaranth and brownish red. METHODS: CE-UV method was used in the following condition: BGE 20 mmol·L-1 sodium borate solution, voltage 20 kV, detection wavelength 290 nm, sample injection 10 s at 5 kPa, capillary temperature room temperature. RESULTS: A quantitative method was successfully developed to determine gallic acid in Quanshen pieces within 6 min. The contents of gallic acid in 7 batches of amaranth colored Quanshen pieces were 2.70% (Hubei province), 12.08% and 7.79% (Shandong province), 2.01%, 5.78%, 6.97%, and 4.22% (Hebei province), respectively. The contents of gallic acid in brownish red colored Quanshen pieces were 1.35% (Hubei province), 1.81% and 3.42% (Shandong province), 1.87%, 3.42%, 0.44%, and 1.52% (Hebei province), respectively. CONCLUSION: The method is rapid, economical and simple and can provide a powerful quality control measure for Quanshen pieces. The contents of gallic acid in the amaranth colored Quanshen pieces are higher than those in the brownish red colored pieces.

Simultaneous determination of seven phenolic acids from Xanthii Herba and Xanthii Fructus by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneousn determination of nchlorogenic acid, caffeic acid, ferulic acid, isochlorogenic acid A, isochlorogenic acid C, neochlorogenic acid, and cynarin in the herbs of Xanthii Herba and Xanthii Fructus. METHODS: 50 mmol·L-1 borax-water was used as buffer solution (pH 9.54). The separation was performed on an uncoated fused silica capillary (64.5 cm×75 μm, 56 cm of effective length) maintained at 25℃ at voltage of 25 kV. The detection wavelength was 326 nm, and the sample was injected at 25 kPa×6 s. RESULTS: The calibration curves of the seven phenolic acid showed good linearity (r>0.9994) in the ranges of the tested concentrations, and the average recoveries of the method were between 99.85%-102.70%. CONCLUSION: The method is simple, accurate and reproducible, and can be used for the quality evaluation and control of Xanthii Herba and Xanthii Fructus.

Determination of Atractylenolide Ⅰ in Atractylodes Macrocephala by High Performance Capillary Electro-phoresis / 中国药师

Objective: To establish a method for the determination of atractylenolide Ⅰ in Atractylodes macrocephala by HPCE. Methods:The separation conditions of HPCE were as follows:50 mmol·L-1 borate buffer was used as the running buffer, the UV de-tection was set at 210 nm, the separation voltage was 20 kV, the column temperature was 25℃ and the pressure injection was 50 mbar × 5 sec. Results:The lower detection limit was 0. 5 μg·ml-1 . The concentration of atractylenolide Ⅰ showed a linear plot within the range of 2-100 μg·ml-1(r=0. 996 0). The average recovery was 97. 1%, and the intra-and inter-day RSD was 1. 3% and 2. 5%, respectively. Conclusion:The established method is simple, sensitive and economic. The method is suitable for the quality control of atractylodes macrocephala.

Content Determination of Calceolarioside A in the Leaves of Fraxinus mandschurica by HPCE / 中国药房

OBJECTIVE:To establish a method for the content determination of calceolarioside A in the Leaves of Fraxi-nus mandschurica. METHODS:HPCE was performed on the capillary of uncoated fused silica capillary,buffer solution of borate buffered saline(20 mmol/L,pH 9.3),separation voltage of 20 KV by pressure injection(injection pressure was 50 bar with 3 s), detection wavelength was 350 nm,and column temperature was 30 ℃. RESULTS:The linear range of calceolarioside A was 50-250μg/ml(r=0.999 7);RSDs of precision,stability and reproducibility tests were less than 3%,recovery was 97.48%-102.08%(RSD=1.12,n=6). CONCLUSIONS:The method is simple,good reproducibility,and can be used for the content determination of calceolarioside A in the Leaves of F. mandschurica.

Simultaneous determination of six alkaloids in Aconitum plants by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneous determination of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine and benzoylmesaconine in the herbs from Aconitum plants.

Simultaneous determination of seven nucleosides in Isatidis Radix by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis( HPCE) method for simultaneous determination of seven nucleosides, including epigoitrin, uracil, thymine, adenosine, hypoxanthine, guanosine and uridine in Isatidis Radix. METHODS: The chromatographic separation was performed on an uncoated fused silica capillary(75 μm × 64.5 cm, 56 cm of effective length) with separation voltage of 20 kV. 60 mmol · L-1 Na2B4O7-15 mmol · L-1 β-CD was selected as the running buffer solution(pH 9.6). The detection wavelength was set at 254 nm. The sample was injected at 5 kPa × 5 s and column temperature was maintained at 25°C. RESULTS: The calibration curves of the seven nucleosides showed good linearity (r > 0.9994). The average recoveries of the method were between 97.15%-100.40%. The contents of the seven nucleosides in Isatidis Radix samples of different batches were different. CONCLUSION: The established method is simple, accurate and can be used for the quality control of Isatidis Radix.

Simultaneous determination of six index components in ligustri lucidi fructus by MEKC-DAD / 中国药学杂志

OBJECTIVE: To establish a micellar electrokinetic chromatography(MEKC) method for simultaneous determination of salidroside, tyrosol, ligustroflavone, specnuezhenide, oleanolic acid, and ursolic acid in Ligustri Lucidi Fructus. METHODS: 60 mmol·L-1 borax-10 mmol·L-1 SDS-30 mmol·L-1 hydroxypropyl-beta-cyclodextrin-10% methyl alcohol was used as the buffer solution (pH 9.03). Uncoated fused silica capillary (75 μm×64.5 cm, 56 cm of effective length) was used with separation voltage of 20 kV. The detection wavelength was set at 210 nm. The column temperature was maintained at 25°C, and the sample was injected at 5 kPa×6 s. RESULTS: The calibration curves of the six index components showed good linearity (r>0.95) in the range of the tested concentrations, and the average recoveries of the method were between 94.57% and 102.07% (RSD<5%). CONCLUSION: The method is simple, accurate and reproducible, and can be used for the quality control of Ligustri Lucidi Fructus.

Simultaneous Determination of Seven Anions and Organic Acids in Huo-Xue Tong-Luo Injection by HPCE / 世界科学技术-中医药现代化

This study was aimed to establish a method for simultaneous determination of seven anions and organic acids in Huo-Xue Tong-Luo (HXTL) injection by HPCE. With tartaric acid as the internal standard, separation was performed on an uncoated fused silica capillary (50 μm × 64. 5 cm, 56 cm of effective length). The 14 mmol·L-1 potassium acid phthalate and 0.1 mmol·L-1 hexadecyl trimethyl ammonium chloride were selected for the running buffer solution (pH 5.6). The separation voltage was -16 kV. The detection wavelength was set at 210 nm. The column temperature was maintained at 25 ℃ . The sample was injected at 50 mbar×4 s. The results showed that calibration curves of chloride ion, sulfuric acid root ion, formate ions, malic acid, succinic acid, iodate ion and acetic acid ions showed good linear relationship 41.4-248.2 μg·mL-1 (r = 0.999 3), 12.5-74.8 μg·mL-1 (r = 0.999 8), 18.2-109.1 μg·mL-1 (r = 0.999 8), 20.3-121.6 μg·mL-1 (r = 0.999 5), 17.2-103.1 μg·mL-1 (r=0.999 1), 17.6-105.6μg·mL-1 (r=0.999 6), 51.6-309.6μg·mL-1 (r=0.999 7), respectively. The average recoveries were 102.6%, 97.3%, 102.2%, 99.0%, 99.2%, 97.8%, and 103.4%, respectively. The RSD were 1.7%, 2.0%, 1.6%, 2.6%, 2.1%, 2.9%, and 1.0%, respectively (n = 6). It was concluded that the method was accurate and reproducible. It was suitable for the determination of anions and organic acids in HXTL injection.

Simultaneous Content Determination of Hesperidin and Phillyrin in Bao-He-Wan by HPCE / 世界科学技术-中医药现代化

This study was aimed to establish a HPCE method for the content determination of hesperidin and phillyrin in Bao-He-W an (BHW). Fused silica capillary (75 cm í 50 μm) was employed and 30 mmol·L-1 borax so-lution (8% acetonitrile, pH9.64) was served as the running buffer. Other conditions were as follows: electrokinetic injection was 50 kPa í 20 s; analytical voltage was 20 kV; temperature was 20℃; and detection wavelength was 254 nm. The silica capillary was flushed with 0.1 mol·L-1 sodium hydrate and the running buffer for 10 min before each injection, respectively. The results showed that the linearity of hesperidin was in the range of 0.10~2.40 mg·mL-1 (r=0.999 4), the average recovery was 99.85% and RSD=2.34%. The phillyrin was in the range of 0.07~0.84 mg·mL-1 (r=0.999 2), the average recovery was 99.16% and RSD=2.78%. It was concluded that the method was rapid and sensitive. It can be used for the quality control of content determination of hesperidin and phillyrin in BHW.

Establishment of a quantitative method for tetramethylpyrazine hydrochloride in rat plasma using pyramide as internal standard / 中国药学杂志

OBJECTIVE: To develop an HPCE method of tetramethylpyrazine hydrochloride in rat plasma using pyramide as the internal standard. METHODS: The separation was performed on a fused-silica uncoated capillary with an inner diameter (ID) of 75 μm, an effective length of 50 cm and a total length of 60 cm. The optimum HPCE conditions were as follows: 100 mmol·L-1 SDS-30 mmol·L-1 sodium tetraborate solution (40:60) was used as the background electrolyt, the samples were injected at the anodic end at 0.5 psi for 6 s, separation voltage was 20 kV at positive power, capillary temperature was 20°C, and detection wavelength was 295 nm. RESULTS: The linear range was 1.12-286.72 mg·L-1(r=0.9996), and the inter-day and intra-day precisions expressed as RSDs were less than 4.21% and 4.74%, respectively. The average recoveries were 91.22%-97.48%, and the limit of detection was 0.16 mg·L-1. CONCLUSION: This method is rapid and accurate to quantify tetramethylpyrazine hydrochloride in rat plasma.

Simultaneous determination of five nucleosides in Antrodia camphorata by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneous determination of five nucleosides and nucleobases, including adenine, adenosine, uridine, guanosine and inosine in antrodia camphorate of different batches. METHODS: Based on the mode of HPCE, uncoated fused silica capillary (75 μm×64.5 cm, 56 cm of effective length) was used with separation voltage of 22 kV. 60 mmol·L-1 sodium borate was selected for the running buffer solution (pH 9.3). The detection wavelength was set at 260 nm. The sample was injected at 50 mbar×6 s and column temperature was maintained at 28°C. RESULTS: The calibration curves of the five nucleosides showed good linearity (r>0.9995). The average recoveries of the method were between 98.83%-101.08%. The contents of the five nucleosides in Antrodia camphorate samples of different batches were different. CONCLUSION: The established method is reliable, accurate and can be used for the quality control of Antrodia camphorata.

Determination of 10 phenolic compounds in extract from Bidens bipinnata by HPCE / 中草药

Objective: To develop a novel method using high-performance capillary electrophoresis coupled with DAD (HPCE-DAD) for the simultaneous determination of 10 phenolic compounds (luteolin-7-O-glucoside, rutin, quercetin-3-glucoside, quercetin-3- rhamnoside, salicylic acid, luteolin, kaemferol, quercetin, gallic acid, and protocatechuic acid) in Bidens bipinnata. The extraction efficiency of above mentioned compounds was compared using six different ethanol/water solutions. Methods: The effects of pH value and concentration of buffer, applied voltage, and temperature on the separation were researched. The 10 compounds were baseline separated within 16 min in a 60 cm length capillary at a separation voltage of 25 kV with a running buffer consisting of 25 mmol/L borate (pH 9.5) at wavelength of 214 nm. Results: Excellent linearities of luteolin-7-O-glucoside, rutin, quercetin-3-glucoside, quercetin-3-rhamnoside, salicylic acid, luteolin, kaemferol, quercetin, gallic acid, and protocatechuic acid were observed at 5.0-120, 5.0-120, 5.0-120, 5.0-120, 1.0-120, 2.5-120, 5.0-120, 2.5-120, 2.5-120, and 2.5-120 mg/L, respectively. The detection limits were at 2.5, 2.5, 2.5, 2.5, 0.5, 1.0, 2.5, 1.0, 1.0, and 1.0 mg/L, respectively. The relative standard deviations (RSD) of precision were below 5.17% (n = 6). The mean recoveries for 10 phenolic compounds in B. bipinnata ranged from 94.4% to 105.8%, with RSD values of 0.32%-4.33%. Conclusion: The contents of luteolin-7-O-glucoside, rutin, quercetin-3- glucoside, quercetin-3-rhamnoside, salicylic acid, luteolin, kaemferol, quercetin, gallic acid, and protocatechuic acid in B. bipinnata are 95.04-105.84, 78.03-110.45, 96.45-177.96, 178.78-300.00, 62.06-66.54, 71.08-72.85, 77.39-95.30, 68.27-77.43, 68.47- 88.47, and 107.24-142.43 μg/g, respectively.

Determination of mangiferin in Baihe Zhimu decoction by HPCE / 国际中医中药杂志

Objective To establish HPCE method for determining the mangiferin in Baihe Zhimu decoction.Methods Rutin was used as internal standard.An uncoated fused silica capillary (77 cm×75 μm,effective length of 70 cm) was served as separation gate.The electrolyte buffer was composed of 12 mmol/L borax-MeOH (90 ∶ 10).20 kV voltages were applied and detection wavelength was set at 318nm.Results Mangiferin was successfully separated within 20 rin,the linear response range was 0.06～1.14 mg/ml.The average recovery was 99.0％.Conclusion The method was simple,rapid and well reproducible.It could be used as a reliable tool for the quantity control of Baihe Zhimu decoction and descriptions containing mangiferin.