HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines

Ang, Lee Fung,Yam, Mun Fei,Fung, Yvonne Tan Tze,Kiang, Peh Kok,Darwin, Yusrida KOREAN PHARMACOPUNCTURE INSTITUTE 2014 Journal of pharmacopuncture Vol.17 No.4

Objectives: Quercetin and curcuminoids are important bioactive compounds found in many herbs. Previously reported high performance liquid chromatography ultraviolet (HPLC-UV) methods for the detection of quercetin and curcuminoids have several disadvantages, including unsatisfactory separation times and lack of validation according the standard guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Methods: A rapid, specific, reversed phase, HPLC-UV method with an isocratic elution of acetonitrile and 2% v/v acetic acid (40% : 60% v/v) (pH 2.6) at a flow rate of 1.3 mL/minutes, a column temperature of $35^{\circ}C$, and ultraviolet (UV) detection at 370 nm was developed. The method was validated and applied to the quantification of different types of market available Chinese medicine extracts, pills and tablets. Results: The method allowed simultaneous determination of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the concentration ranges of $0.00488-200{\mu}g/mL$, $0.625-320{\mu}g/mL$, $0.07813-320{\mu}g/mL$ and $0.03906-320{\mu}g/mL$, respectively. The limits of detection and quantification, respectively, were 0.00488 and $0.03906{\mu}g/mL$ for quercetin, 0.62500 and $2.50000{\mu}g/mL$ for bisdemethoxycurcumin, 0.07813 and $0.31250{\mu}g/mL$ for demethoxycurcumin, and 0.03906 and $0.07813{\mu}g/mL$ for curcumin. The percent relative intra day standard deviation (% RSD) values were $0.432-0.806{\mu}g/mL$, $0.576-0.723{\mu}g/mL$, $0.635-0.752{\mu}g/mL$ and $0.655-0.732{\mu}g/mL$ for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and those for intra day precision were $0.323-0.968{\mu}g/mL$, $0.805-0.854{\mu}g/mL$, $0.078-0.844{\mu}g/mL$ and $0.275-0.829{\mu}g/mL$, respectively. The intra day accuracies were 99.589%-100.821%, 98.588%-101.084%, 9.289%-100.88%, and 98.292%-101.022% for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and the inter day accuracy were 99.665%-103.06%, 97.669%-103.513%, 99.569%-103.617%, and 97.929%-103.606%, respectively. Conclusion: The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of commercial Chinese medicine products containing the flour flavonoids as their principle components in the extracts.

HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines

Lee Fung Ang,Mun Fei Yam,Yvonne Tan Tze Fung,Peh Kok Kiang,Yusrida Darwin 대한약침학회 2014 Journal of pharmacopuncture Vol.17 No.4

Objectives: Quercetin and curcuminoids are importantbioactive compounds found in many herbs. Previouslyreported high performance liquid chromatographyultraviolet (HPLC-UV) methods for the detection ofquercetin and curcuminoids have several disadvantages,including unsatisfactory separation times and lack ofvalidation according the standard guidelines of the InternationalConference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals forHuman Use. Methods: A rapid, specific, reversed phase, HPLC-UVmethod with an isocratic elution of acetonitrile and 2%v/v acetic acid (40% : 60% v/v) (pH 2.6) at a flow rate of1.3 mL/minutes, a column temperature of 35°C, and ultraviolet(UV) detection at 370 nm was developed. Themethod was validated and applied to the quantificationof different types of market available Chinese medicineextracts, pills and tablets. Results: The method allowed simultaneous determinationof quercetin, bisdemethoxycurcumin, demethoxycurcuminand curcumin in the concentration ranges of 0.00488 ─ 200 μg/mL, 0.625 ─ 320 μg/mL, 0.07813─ 320 μg/mL and 0.03906 ─ 320 μg/mL, respectively. The limits of detection and quantification, respectively,were 0.00488 and 0.03906 μg/mL for quercetin,0.62500 and 2.50000 μg/mL for bisdemethoxycurcumin,0.07813 and 0.31250 μg/mL for demethoxycurcumin,and 0.03906 and 0.07813 μg/mL for curcumin. Thepercent relative intra day standard deviation (% RSD)values were 0.432 ─ 0.806 μg/mL, 0.576 ─ 0.723 μg/mL, 0.635 ─ 0.752 μg/mL and 0.655 ─ 0.732 μg/mLfor quercetin, bisdemethoxycurcumin, demethoxycurcuminand curcumin, respectively, and those forintra day precision were 0.323 ─ 0.968 μg/mL, 0.805 ─0.854 μg/mL, 0.078 ─ 0.844 μg/mL and 0.275 ─ 0.829μg/mL, respectively. The intra day accuracies were99.589% ─ 100.821%, 98.588% ─ 101.084%, 9.289% ─100.88%, and 98.292% ─ 101.022% for quercetin, bisdemethoxycurcumin,demethoxycurcumin and curcumin,respectively, and the inter day accuracy were99.665% ─ 103.06%, 97.669% ─ 103.513%, 99.569% ─103.617%, and 97.929% ─ 103.606%, respectively. Conclusion: The method was found to be simple, accurateand precise and is recommended for routinequality control analysis of commercial Chinese medicineproducts containing the flour flavonoids as theirprinciple components in the extracts.

Anti-inflammatory and Analgesic Effects of Elephantopus tomentosus Ethanolic Extract

Mun Fei Yam,Lee Fung Ang,Omar Ziad Ameer,Ibrahim Muhammad Salman,Hesham Abdul Aziz,Mohd. Zaini Asmawi 사단법인약침학회 2009 Journal of Acupuncture & Meridian Studies Vol.2 No.4

Elephantopus tomentosus is widely used in Asia, especially in Malaysia, for the treatment of pain and inflammation. In the present study, the analgesic and antiinflammatory effects of a 95% ethanol extract of E. tomentosus were investigated in different experimental models. In the anti-inflammation study, 1000 mg/kg of extract significantly reduced carrageenan-induced hind paw edema (p < 0.05) and inhibited abdominal permeability compared with control (p < 0.01). The analgesic activity was assayed in several experimental models in mice: (1) hot plate, (2) tail flick, (3) writhing test; and rats: carrageenan-induced hyperalgesia pain threshold test. However, at the doses tested, no significant activity was found in the hot plate test and the tail flick test. E. tomentosus ethanol extract at 1000 mg/kg significantly (p < 0.05) increased hyperalgesia pain threshold and inhibited writhing activity. The results suggest that E. tomentosus ethanol extract at 1000 mg/kg dose is effective in anti-inflammatory and non-steroidal anti-inflammatory drug type anti-nociception activities. Elephantopus tomentosus is widely used in Asia, especially in Malaysia, for the treatment of pain and inflammation. In the present study, the analgesic and antiinflammatory effects of a 95% ethanol extract of E. tomentosus were investigated in different experimental models. In the anti-inflammation study, 1000 mg/kg of extract significantly reduced carrageenan-induced hind paw edema (p < 0.05) and inhibited abdominal permeability compared with control (p < 0.01). The analgesic activity was assayed in several experimental models in mice: (1) hot plate, (2) tail flick, (3) writhing test; and rats: carrageenan-induced hyperalgesia pain threshold test. However, at the doses tested, no significant activity was found in the hot plate test and the tail flick test. E. tomentosus ethanol extract at 1000 mg/kg significantly (p < 0.05) increased hyperalgesia pain threshold and inhibited writhing activity. The results suggest that E. tomentosus ethanol extract at 1000 mg/kg dose is effective in anti-inflammatory and non-steroidal anti-inflammatory drug type anti-nociception activities.

Antioxidant and Hepatoprotective Effects of Murdannia bracteata Methanol Extract

Mun Fei Yam,Lee Fung Ang,Chung Pin Lim,Omar Ziad Ameer,Ibrahim Muhammad Salman,Mahfoudh Al-musli Mohammed,Mohd. Zaini Asmawi,Muthanna Fawzy Abdulkarim,Ghassan Zuhair Abdullah,Mariam Ahmad 사단법인약침학회 2010 Journal of Acupuncture & Meridian Studies Vol.3 No.3

Murdannia bracteata (C. B. Clarke) is a local plant that is widely used in Malaysia as a traditional remedy for various diseases of the kidney and liver, including inflammation and cancer. In the present study, we investigated the antioxidant and hepatoprotective activities of M. bracteata methanol extract (MB). 2,2’-diphenyl-1-picrylhydrazyl radical scavenging activity, lipid peroxidation inhibition and trolox equivalent antioxidant capacity of MB were determined. The hepatoprotective activity of MB was studied using a CCl4-induced liver toxicity model in rats. The hepatoprotective effect was assessed by monitoring the plasma malondialdehyde level and serum alanine transaminase and aspartate transaminase activities. Histopathological changes of hepatic tissue were also investigated. The results indicated that MB possessed potential antioxidant, lipid peroxidation inhibition and free radical scavenging activities. Pretreatment of rats with MB (500 mg/kg and 1000 mg/kg per os) before induction of CCl4-induced hepatotoxicity showed a dosedependent reduction in the necrotic changes in hepatic tissue. The increases in plasma malondialdehyde level, serum alanine transaminase and aspartate transaminase activities were also significantly inhibited by MB. The total phenolic content of MB determined using Folin-Ciocalteu assay was found to be 10%. The results of the present study indicated that the hepatoprotective effect of MB is most likely due to its antioxidant and free radical scavenging properties.

Orthosiphon stamineus Leaf Extract Protects Against Ethanol-Induced Gastropathy in Rats

Mun Fei Yam,Lee Fung Ang,Ibrahim Muhammad Salman,Omar Ziad Ameer,Vuanghao Lim,Lai Man Ong,Mariam Ahmad,Mohd. Zaini Asmawil,Rusliza Basir 한국식품영양과학회 2009 Journal of medicinal food Vol.12 No.5

Orthosiphon stamineus Benth., which is used as a gastroprotective herbal remedy in Malaysia, was assessed for its anti-ulcerogenic activity against ethanol-induced ulcers in rats. Fifty percent methanol was used to extract the oven-dried O. stamineus leaves. The extract was then lyophilized with a rotary evaporator and freeze-dried. Oral administration of O. stamineus methanolic extract (OSME) (125, 250, 500, and 1,000mg/kg) was found to significantly decrease the ulcer index (P<.01, P<.001, P<.001, and P<.001, respectively). Histological study of a section of the rat stomach also showed a marked improvement in the gastric mucosal damage in groups receiving OSME. In order to further investigate the gastroprotective mechanism of OSME, mucus secretion and lipid peroxidation level were estimated in vitro and ex vivo. OSME exhibited dose-dependent stimulation of mucus secretion (r=0.718, P<.001) and inhibition of lipid peroxidation in rat gastric mucosal homogenates (both in vitro [r=0.819, P<.05] and ex vivo [r=0.981, P<.05]). It was concluded that the gastroprotective mechanism of OSME was partly due to its ability to inhibit lipid peroxidation and stimulate gastric mucus secretion.

A Simple Isocratic HPLC Method for the Simultaneous Determination of Sinensetin, Eupatorin, and 30-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus Extracts

Mun Fei Yam,Elsnoussi Ali Hussin Mohamed,Lee Fung Ang,Li Pei,Yusrida Darwis,Roziahanim Mahmud,Mohd. Zaini Asmawi,Rusliza Basir,Mariam Ahmad,유혜현,김동현,정혜진 사단법인약침학회 2012 Journal of Acupuncture & Meridian Studies Vol.5 No.4

Orthosiphon stamineus extracts contain three flavonoids (3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20 mM phosphate buffer (NaH2-PO4) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1 ml/minute, a column temperature of 25_C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3’-hydroxy-5,6,7,4’- tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052 e250 mg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 mg/ml for 3’-hydroxy-5,6,7,4’-tetramethoxyflavone, 0.0153 and 0.122 mg/ml for sinensetin and 0.0305 and 0.122 mg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048e0.368, 0.025e0.135, and 0.05e0.476 for 3’-hydroxy-5,6,7,4’-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333e1.688, 0.722e1.055, and 0.548e1.819, respectively. The accuracy for intraday were 91.25%e103.38%, 94.32%e109.56%, and 92.85%e109.70% for 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%e103.92%, 103.58% e104.57%, and 103.9%e105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.

Antidiabetic Properties and Mechanism of Action of Orthosiphon stamineus Benth Bioactive Sub-fraction in Streptozotocin-induced Diabetic Rats

Elsnoussi Ali Hussin Mohamed,Mun Fei Yam,Lee Fung Ang,Ali Jimale Mohamed,Mohd. Zaini Asmawil 사단법인약침학회 2013 Journal of Acupuncture & Meridian Studies Vol.6 No.1

Orthosiphon stamineus is a popular folk medicine widely used to treat many diseases including diabetes. Previous studies have shown that the sub-fraction of chloroform extract was able to inhibit the rise of blood glucose levels in a glucose tolerance test. This study was carried out to evaluate the chronic effect and possible mechanism of action of the bioactive chloroform sub-fraction of O. stamineus using streptozotocin-induced diabetic rats and in vitro methods. Administration of the chloroform extract sub-fraction 2 (Cƒ2-b) at a dose of 1 g/kg twice daily on diabetic rats for 14 days showed a significant lowering (p < 0.05) of the final blood glucose level compared to the pretreatment level. However, there were no significant differences in the plasma insulin levels post-treatment compared to the pretreatment levels for all doses of Cƒ2-b. Conversely, Cƒ2-b at a concentration of 2 mg/mL significantly increased (p < 0.001) the glucose uptake by the rat diaphragm muscle. The increase in glucose uptake was also shown when the muscle was incubated in a solution containing 1 IU/mL of insulin or 1 mg/mL of metformin. Furthermore, the effect of this sub-fraction on glucose absorption in the everted rat jejunum showed that Cƒ2-b at concentrations of 0.5 mg/mL, 1 mg/mL and, 2 mg/mL significantly reduced the glucose absorption of the jejunum (p < 0.05–0.001). Similarly, the absorption of glucose was also inhibited by 1 mg/mL and 2 mg/mL of metformin (p < 0.001). These results suggest that the effect of Cƒ2-b may be due to extra-pancreatic mechanisms. There was no evidence that the plant extract stimulated the release of insulin in order to lower the blood glucose level.