A Multilevel Study of Eupatorin and Scutellarein as Anti-Amyloid Agents in Alzheimer's Disease.

Today, Alzheimer's disease (AD)-the most common neurodegenerative disorder, which affects 50 million people-remains incurable. Several studies suggest that one of the main pathological hallmarks of AD is the accumulation of abnormal amyloid beta (Aß) aggregates; therefore, many therapeutic approaches focus on anti-Aß aggregation inhibitors. Taking into consideration that plant-derived secondary metabolites seem to have neuroprotective effects, we attempted to assess the effects of two flavones-eupatorin and scutellarein-on the amyloidogenesis of Aß peptides. Biophysical experimental methods were employed to inspect the aggregation process of Aß after its incubation with each natural product, while we monitored their interactions with the oligomerized Aß through molecular dynamics simulations. More importantly, we validated our in vitro and in silico results in a multicellular organismal model-namely, Caenorhabditis elegans-and we concluded that eupatorin is indeed able to delay the amyloidogenesis of Aß peptides in a concentration-dependent manner. Finally, we propose that further investigation could lead to the exploitation of eupatorin or its analogues as potential drug candidates.

Eupatorin 3'-O-glucopyranoside, a trimethoxyflavonoid glucoside from the aerial parts of Salvia mellifera.

Salvia mellifera, native to California, Baja California, and Mexico, is a medicinal herb traditionally used to relieve pain, body aches, including chronic pain. A detailed phytochemical investigation of aerial parts of S. mellifera was accomplished to find species-specific markers and to differentiate the closely related, often (un)intentionally substituted with S. apiana. A total of 22 metabolites, including flavonoids (1-14), triterpenoids (15-18), diterpenoids (19-21), and phenylpropanoid (22), were isolated and characterized thoroughly. Among the isolates, eupatorin 3'-O-glucopyranoside (1) was identified as undescribed phytochemical and detailed structure elucidation was achieved through extensive NMR and mass spectral data analysis.

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines.

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer. Eupatorin, a flavonoid with a wide range of phytomedical activities, suppresses proliferation of and induces apoptosis of multiple cancer cell lines. However, low solubility, poor bioavailability, and rapid degradation limit its efficacy. The aim of our study was to evaluate whether the use of mPEG-b-poly (lactic-co-glycolic) acid (PLGA) coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines. Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology, surface charge, particle size, drug loading, encapsulation efficiency and in vitro drug-release profile. The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test. Apoptosis was then determined by Hoechest staining, cell cycle analysis, NO production, annexin/propidium iodide (PI) assay, and Western blotting. The results indicated that eupatorin was successfully entrapped in Fe3O4@mPEG-b-PLGA nanoparticles with an efficacy of (90.99 ± 2.1)%. The nanoparticle's size was around (58.5 ± 4) nm with a negative surface charge [(-34.16 ± 1.3) mV]. In vitro release investigation showed a 30% initial burst release of eupatorin in 24 h, followed by sustained release over 200 h. The MTT assay indicated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 µM and 75 µM, respectively. Next, apoptosis was confirmed by nuclear condensation, enhancement of cell population in the sub-G1 phase and increased NO level. Annexin/PI analysis demonstrated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency. Finally, Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles. Encapsulation of eupatorin in Fe3O4@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin. Based on these results, this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose, making it a suitable candidate for pharmacological uses.

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines / 药物分析学报

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer.Eupatorin,a flavonoid with a wide range of phytomedical activities,suppresses proliferation of and in-duces apoptosis of multiple cancer cell lines.However,low solubility,poor bioavailability,and rapid degradation limit its efficacy.The aim of our study was to evaluate whether the use of mPEG-b-poly(lactic-co-glycolic)acid(PLGA)coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines.Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology,surface charge,particle size,drug loading,encapsulation efficiency and in vitro drug-release profile.The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test.Apoptosis was then determined by Hoechest staining,cell cycle analysis,NO production,annexin/propidium iodide(PI)assay,and Western blotting.The results indicated that eupatorin was successfully entrapped in Fe3O4@mPEG-b-PLGA nanoparticles with an efficacy of(90.99 ± 2.1)％.The nanoparticle's size was around(58.5 ± 4)nm with a negative surface charge[(-34.16 ± 1.3)mV].In vitro release investigation showed a 30％initial burst release of eupatorin in 24 h,followed by sustained release over 200 h.The MTT assay indicated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 μM and 75 μM,respectively.Next,apoptosis was confirmed by nuclear condensation,enhancement of cell population in the sub-G1 phase and increased NO level.Annexin/PI analysis demonstrated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency.Finally,Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles.Encapsulation of eupatorin in Fe3O4@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin.Based on these results,this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose,making it a suitable candidate for pharmacological uses.

Eupatorin Suppressed Tumor Progression and Enhanced Immunity in a 4T1 Murine Breast Cancer Model.

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti-breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti-breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1ß. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model.

Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS.

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Apoptotic and antimetastatic activities of betulin isolated from Quercus incana against non-small cell lung cancer cells.

BACKGROUND: Globally, the prevalence and mortality rates of lung cancer have been escalated with the increasing trend of tobacco smoking. The toxicity and irresponsive nature of the available drugs for lung cancer treatment demands an alternative approach. METHODS: In this study, four known compounds namely, cirsimaritin (4',5, -dihydroxy-6,7-di-methoxyflavone) (1), eupatorin (5,3'-dihydroxy-6,7,4'-trimethoxyflavone) (2), betulin (Lup-20 (29)-ene-3, 28-diol) (3), and ß-amyrin acetate (12-Oleanen-3yl acetate) (4) have been isolated from the leaves extract of Quercus incana. Preliminary screening of these natural compounds (1-4) was performed against non-small cell lung carcinoma (NCI-H460) and normal mouse fibroblast (NIH-3T3) cell lines. RESULTS: The compounds were found to be antiproliferative against cancer cells with wide therapeutic index in comparison to the normal cells. Effects of betulin (3) on cell migration, invasion, apoptosis, and expression of important apoptosis- and metastasis-related markers were observed at different concentrations. The results showed significant dose-dependent induction of apoptosis after the treatment with betulin (3) followed by increased expression of the caspases family (ie, caspase-3, -6, and -9), proapoptotic genes (BAX and BAK), and inhibiting anti-apoptotic genes (BCL-2L1 and p53). Furthermore, wound healing and transwell invasion assays suggested that betulin (3) could also regulate metastasis by inhibiting MMP-2/-9. Osteopontin, a central regulator of apoptosis and metastasis was also inhibited in a dose-dependent manner. CONCLUSION: The present findings suggest that betulin (3) can be an attractive chemotherapeutic target for treating resistant lung cancers.

Changes and degradation kinetics of some bioactive compounds in dried Orthosiphon aristatus (Java tea) leaves during elevated temperature storage.

BACKGROUND: Orthosiphon aristatus is a traditional medicinal herb mostly used in Southeast Asia and which has many health benefits. Packaging types and storage temperatures were investigated in order to select the best conditions for producing high bioactive compounds (BC) from two kinds of dried O. aristatus leaves. RESULTS: Blanched leaves were vacuum packed in polypropylene (PP) and aluminum foil laminated with polyethylene terephthalate and polyethylene (PET/Al/PE) and dried in a freeze dryer (B_FD) or heat pump-assisted dehumidified dryer (B_HPD60) at 60 °C prior to storage at 15, 25 and 35 °C for 6 months. Leaves in PET/Al/PE bags had higher total phenolic content (TPC), antioxidant activity (AOA) and BC than in PP bags (P ≤ 0.05). Storage at 15 °C retained the highest TPC and AOA in PET/Al/PE bags (P ≤ 0.05). The degradation kinetics for BC, sinensetin and eupatorin followed first-order kinetics. Half-lives (t1/2 ) for BC in PET/Al/PE were higher than in PP and were the highest at 15 °C for both packaging types. CONCLUSIONS: Low temperature and PET/Al/PE bags provided the highest bioactive compound retention. The dried leaves from B_HPD60 and packed in PET/Al/PE bags had higher resistance to degradation of sinensetin than B_FD in PP bags. © 2018 Society of Chemical Industry.

Eupatorin and Salvigenin Potentiate Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in HT-29 and SW948 Human Colon Cancer Cells

Background: Cancer persists as one of the world's most pressing maladies. Notable points about chemotherapy are drug side effects which are almost universally encountered. Emerging knowledge focusing on mechanisms of toxicity due to chemotherapy has led to characterization of novel methods, including the exploitation of natural compounds, in combination therapies. Flavonoids are natural polyphenolic compounds that play protective roles against tumor cell development. The focus of this study was apoptotic effects of two flavonoids, eupatorin and salvigenin, in combination with doxorubicin on a cellular model of colon cancer. Method: Upon establishing a non-effective dose of doxorubicin, and effective doses of eupatorin (100µM) and salvigenin (150µM) via MTT, morphological features of apoptosis were distinguished using DAPI staining and cell cycle blockage in the sub-G1 phase. Apoptosis was determined by annexin/ PI and western blotting. ROS levels and MMP were measured to show any role of mitochondria in apoptosis. Results: Co-administration of flavonoids with doxorubicin induced apoptosis via the mitochondrial pathway as mitochondrial membrane potential and ROS production were changed. Annexin/PI analysis demonstrated that apoptosis frequency was increased with the combination treatments in colon cancer cells. Finally, the combination of these flavonoids with doxorubicin increased the Bax/Bcl-2 ratio, caspase-3 expression and PARP cleavage. Conclusion: Combination of flavonoids with doxorubicin induces apoptosis and enhances effect on cancer cells which might allow amelioration of side effects by dose lowering.

Anti-inflammatory activity of standardized dichloromethane extract of Salvia connivens on macrophages stimulated by LPS.

CONTEXT: A previous study demonstrated that the chloroform extract of Salvia connivens Epling (Lamiaceae) has anti-inflammatory activity. OBJECTIVE: Identification of the active components in the dicholorometane extract (DESC), and, standardization of the extract based in ursolic acid. MATERIAL AND METHODS: DESC was prepared by percolation with dichlromethane and after washed with hot hexane, its composition was determined by CG-MS and NMR, and standardized by HPLC. The anti-inflammatory activity was tested on acute TPA-induced mouse ear oedema at doses of 2.0 mg/ear. The cell viability of macrophages was evaluated by MTT method, and pro- and anti-inflammatory interleukin levels were measured using an ELISA kit. RESULTS: Ursolic acid, oleanolic acid, dihydroursolic acid and eupatorin were identified in DESC, which was standardized based on the ursolic acid concentration (126 mg/g). The anti-inflammatory activities of DESC, the acid mixture, and eupatorin (2 mg/ear) were 60.55, 57.20 and 56.40% inhibition, respectively, on TPA-induced ear oedema. The IC50 of DESC on macrophages was 149.4 µg/mL. DESC (25 µg/mL) significantly reduced TNF-α (2.0-fold), IL-1ß (2.2-fold) and IL-6 (2.0-fold) in macrophages stimulated with LPS and increased the production of IL-10 (1.9-fold). DISCUSSION: Inflammation is a basic response to injuries, and macrophages are involved in triggering inflammation. Macrophage cells exhibit a response to LPS, inducing inflammatory mediators, and DESC inhibits the biosynthesis of the pro-inflammatory and promote anti-inflammatory cytokines. CONCLUSION: DESC has an anti-inflammatory effect; reduced the levels of IL-1ß, Il-6 and TNF-α; and increases IL-10 in macrophages stimulated with LPS. Ursolic acid is a good phytochemical marker.

High-performance Thin-layer Chromatography Method Development, Validation, and Simultaneous Quantification of Four Compounds Identified in Standardized Extracts of Orthosiphon stamineus.

CONTEXT: Orthosiphon stamineus is a medicinal herb widely grown in Southeast Asia and tropical countries. It has been used traditionally as a diuretic, abdominal pain, kidney and bladder inflammation, gout, and hypertension. AIMS: This study aims to develop and validate the high-performance thin layer chromatography (HPTLC) method for quantification of rosmarinic acid (RA), 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF), sinensitin (SIN) and eupatorin (EUP) found in ethanol, 50% ethanol and water extract of O. stamineus leaves. MATERIALS AND METHODS: HPTLC method was conducted using an HPTLC system with a developed mobile phase system of toluene: ethyl acetate: formic acid (3:7:0.1) performed on precoated silica gel 60 F254 TLC plates. The method was validated based on linearity, accuracy, precision, limit of detection, limit of quantification (LOQ), and specificity, respectively. The detection of spots was observed at ultraviolet 254 nm and 366 nm. RESULTS: The linearity of RA, TMF, SIN, and EUP were obtained between 10 and 100 ng/spot with high correlation coefficient value (R2) of more than 0.986. The limit of detection was found to be 122.47 ± 3.95 (RA), 43.38 ± 0.79 (SIN), 17.26 ± 1.16 (TMF), and 46.80 ± 1.33 ng/spot (EUP), respectively. Whereas the LOQ was found to be 376.44 ± 6.70 (RA), 131.45 ± 2.39 (SIN), 52.30 ± 2.01 (TMF), and 141.82 ± 1.58 ng/spot (EUP), respectively. CONCLUSION: The proposed method showed good linearity, precision, accuracy, and high sensitivity. Hence, it may be applied in a routine quantification of RA, SIN, TMF, and EUP found in ethanol, 50% of ethanol and water extract of O. stamineus leaves. SUMMARY: HPTLC method provides rapid estimation of the marker compound for routine quality control analysis.The established HPTLC method is rapid for qualitative and quantitative fingerprinting of Orthosiphon stamineus extract used for commercial product.Four identified markers (RA, SIN, EUP and TMF) found in three a different type of O. stamineus extracts specifically ethanol, 50% ethanol and water extract were successfully quantified using HPTLC method. Abbreviations Used: HPTLC: High-performance thin layer chromatography; RA: Rosmarinic acid; TMF: 3'-hydroxy-5,6,7,4'-tetramethoxyflavone; SIN: Sinensitin; EUP: Eupatorin; E: Ethanol; EW: 50% ethanol; W: Water; BK: Batu Kurau; KB: Kepala Batas; S: Sik; CJ: Changkat Jering; SB: Sungai Buloh.

Mechanism of vasorelaxation induced by eupatorin in the rats aortic ring.

Previous studies demonstrated that eupatorin content in Orthosiphon stamineus fractions correlated with their vasorelaxation activity. Even with previous studies, there is still very little information on the vasorelaxation effect of eupatorin, and not many scientific studies had been carried out. Therefore, the present study was designed to investigate the vasorelaxation activity and mechanism of action of eupatorin. The vasorelaxation activity and the underlying mechanisms of eupatorin was evaluated on thoracic aortic rings isolated from Sprague Dawley rats. Eupatorin caused the relaxation of aortic rings pre-contracted with phenylephrine with and without endothelium (pD2=6.66±0.13, EMAX=99.72±6.39%; pD2=6.10±0.22, EMAX=65.78±8.01%), and also the relaxation of endothelium-intact aortic rings pre-contracted with potassium chloride (pD2=6.20±0.30, EMAX=71.89±12.25%). In the presence of Nω-nitro-l-arginine methyl ester (pD2<4.60, EMAX=24.91±6.39%), methylene blue (pD2=6.05±0.38, EMAX=66.79±9.69%), ODQ (pD25.84±0.32, EMAX=60.47±9.6%), indomethacin (pD2=6.27±0.21, EMAX=76.03±9.45%), tetraethylammonium (pD2=6.09±0.35, EMAX=69.35±11.31%), 4-aminopyridine (pD2=6.34±0.12, EMAX=76±6.1%), barium chloride (pD2=6.47±0.14, EMAX=79.61±10.02%), atropine (pD2=6.36±0.29, EMAX=86.47±12.95%) and propranolol (pD2=6.49±0.26, EMAX=83.2±12.01%), relaxation stimulated by eupatorin was significantly reduced. Eupatorin was also found to be active in reducing Ca(2+) release from sarcoplasmic reticulum and in blocking calcium channels. The present study demonstrates the vasorelaxation effect of eupatorin involving NO/sGC/cGMP and indomethacin pathways, calcium and potassium channels, and muscarinic and beta-adrenergic receptors.

Study on chemical constituents of Artemisia integrifolia / 中草药

Objective: To study the chemical constituents from the aerial parts of Artemisia integrifolia. Methods: The chemical constituents were separated and purified by column chromatographies and HPLC. Their structures were determined on the basis of spectroscopic analyses (1H-NMR, 13C-NMR, 2D-NMR, and MS). Results: Fifteen compounds were isolated from the methanol extract in the aerial parts of A. integrifolia with the structures identified as α-curcumene (1), β-sitosterol-3-O-β-D-glucoside (2), zingiberone (3), 3-(2-hydroxyphenyl) propanoic acid methyl ester (4), (E)-o-hydroxycinnamic acid (5), eupatorin (6), cirsimaritin (7), artemetine (8), loliolide (9), luteolin-7-O-β-D-glucopyranoside (10), (+)-pinoresinol (11), α-spinasterol (12), reynosin (13), 3α-hydroxy-1(10),4,11(13)-diager-12,6α-olide (14), and scopoletin (15). Conclusion: Compounds 1 and 3 are isolated from the plants of Artemisia Linn for the first time and compounds 4-9, 11, 13, and 14 are isolated from this plant for the first time.

Flavonoids Affect the Light Reaction of Photosynthesis in Vitro and in Vivo as Well as the Growth of Plants.

Flavonoids retusin (5-hydroxy-3,7,3',4'-tetramethoxyflavone) (1) and pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) (2) were isolated from Croton ciliatoglanduliferus Ort. Pachypodol acts as a Hill reaction inhibitor with its target on the water splitting enzyme located in PSII. In the search for new herbicides from natural compounds, flavonoids 1 and 2 and flavonoid analogues quercetin (3), apigenin (4), genistein (5), and eupatorin (6) were assessed for their effect in vitro on the photosynthetic electron transport chain and in vivo on the germination and growth of the plants Physalis ixocarpa, Trifolium alexandrinum and Lolium perenne. Flavonoid 3 was the most active inhibitor of the photosynthetic uncoupled electron flow (I50 = 114 µM) with a lower log P value (1.37). Results in vivo suggest that 1, 2, 3, and 5 behave as pre- and postemergent herbicides, with 3 and 5 being more active.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts.

BACKGROUND: Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3'-hydroxy-5, 6, 7, 4'-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. OBJECTIVE: The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. MATERIALS AND METHODS: The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 µm, 3 × 150 mm) with 18 min separation time. RESULTS: The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. CONCLUSION: The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products.

Determination of Three Active Flavones in Orthosiphon stamineus by HPLC / 医药导报

Objective To develop a method for content determination of sinensetin,eupatorin,and 3′-hydroxy-5,6,7, 4′-tetramethoxyflavone in Orthosiphon stamineus. Methods The determination was carried out on a Symmetry C18 column (4.6 mm×250 mm,5 μm) by HPLC.The mobile consisted of acetonitrile and water containing 0.05% H3 PO4 in gradient elution. The flow rate was 1 mL?min-1 ,the column temperature was 30 ℃ ,the detected wavelength was set at 365 nm, and the injection volume was 10 μL. Results The peak areas and the sample quantity of the three components presented good linear relationship in the range of 0. 50 - 5. 00 μg for sinensetin,0. 50 - 5. 00 μg for eupatorin, and 0. 05 - 0. 50 μg for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone.The average recoveries were 101.26%,100.28% and 99.66%,respectively. RSD were 1.73%, 0.82% and 1.67%, respectively. Conclusion The method is proved to be simple,accurate and can be used for the quality evaluation of Orthosiphon stamineus.