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Objective:To establish a method of measuring the contents of gallic acid, brevifolin, corilagin, geraniin, ellagic acid and rutin in Phyllanthus urinaria L. simultaneously with fingerprint study for analysis. Methods:Phyllanthus urinaria L. was extracted by ultrasound with 50% methanol. Chromatographic separation was performed on a Phenonmenex Luna C18 (4.6 mm×250 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B) with gradient elution. The flow rate was 1.0 ml/min. The column temperature was 25 ℃, and the injection volume was 10 μl. The detection wavelength was 270 nm. HPLC fingerprints of Phyllanthus urinaria L. from different habitats was established. PCA and OPLS-DA were used to analyze the differences in chemical components of different habitats. Results:Gallic acid, brevifolin, corilagin, geraniin, ellagic acid and rutin showed good linearity at 0.042 8-0.641 6, 0.033 4-0.501 4, 0.142 2-2.133 1, 0.383 1-5.746 5, 0.063 1-0.946 2 and 0.019 2-0.287 8 μg, respectively. The average recovery rate of them was 103.65%, 96.39%, 101.85%, 95.04%, 98.79% and 98.33%, respectively. The HPLC fingerprints of different habitats contained 14 characteristic common peaks, and six compounds characteristic peaks were identified. PCA analysis showed that the chemical components of Phyllanthus urinaria L. from different habitats were different. Geraniin, ellagic acid and corilagin were screened by OPLS-DA. Conclusions:The method is efficient, accurate and sensitive, which can be used to measure the six components in Phyllanthus urinaria L.. The established HPLC fingerprint of different habitats combined with the measrurement method of six components can be used for the quality control and evaluation of Phyllanthus urinaria L..
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Objective: To study the chemical constituents of Phyllanthus urinaria. Methods: Compounds were isolated and purified by the normal phase silica gel, Sephadex LH-20, MCI gel, RP-18, and semi-manufactured preparation HPLC method. Their structures were identified by the methods of 1H-NMR and 13C-NMR combined with physicochemical property. Results: Sixteen compounds were isolated from the petroleum ether and ethyl acetate fraction in 95% ethanol extract of P. urinaria and their structures were gallic acid (1), caffeic acid (2), ethyl gallate (3), methyl gallate (4), 4-ethoxybenzoic acid (5), diisobutyl phthalate (6), dibutyl phthalate (7), (4R,6R)-2,3-dihydromenisdaurilide (8), (4R,6S)-2,3-dihydromenisdaurilide (9), aquilegiolide (10), menisdaurilide (11), cassipourol (12), (9Z,12Z)-nonadeca-9,12-dienoic acid (13), methyl linoleate (14), stigmasterol (15), and (8R,8'S,7S)-4'-(3″- methoxyrhamnopyranosyl) oxy-8'-hydroxy-3,3',4-trimethoxy-8-hydroxymethyl-lign-7-9'-lactone (16). Conclusion: Compounds 5-7, 10, 11, 13, and 14 are isolated from this plant for the first time, and compounds 8, 9, 12, and 16 are first isolated from the plants in genus Phyllanthus L. for the first time.
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Objective: To establish the HPLC fingerprint and its evaluation system for the whole plant of Phyllanthus urinaria. Methods: Diamonsil C18 column (250 mm × 4.6 mm, 5 μm)was used with acetonitrile and 0.2% acetic acid solution in gradient elution mode. The detective wavelength was 270 nm and the flow rate was 1.0 mL/min. The fingerprints for the whole plant of P. urinaria and phenolic acids part from nine different sources were set up, the similarity assay for the whole plant of P. urinaria from nine different sources was carried out to evaluate their quality by Chinese material medica (CMM) fingerprint similarity evaluation system ( 2004A edition), and the peaks were identified by comparing the reference substance with LC-MS/MS. Results: The common mode of HPLC fingerprints for the whole plant of P. urinaria were set up. There were 16 common peaks in the fingerprints and eight peaks were identified. The cluster analysis and principal component analysis were applied to studying the HPLC fingerprint and chemical pattern recognition. Conclusion: The combination of fingerprint and chemical pattern recognition is an effective method for the quality control of the whole plant of P. urinaria. The study lays the foundation for the full quality evaluation for the whole plant of P. urinaria.
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<p><b>OBJECTIVE</b>To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus (HBV) replication and expression in HBV transient transfection model in vitro.</p><p><b>METHODS</b>The eukaryotic expression plasmid pHBV1.1, which contains 1.1-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells.</p><p><b>RESULTS</b>After the transfection of plasmid pHBV1.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells (P<0.05), and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group (P<0.05); Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group (P<0.01); Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group (P<0.01).</p><p><b>CONCLUSIONS</b>The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.</p>
Subject(s)
Humans , Hep G2 Cells , Hepatitis B , Drug Therapy , Hepatitis B virus , Physiology , Phyllanthus , Plant Extracts , Pharmacology , Transfection , Virus ReplicationABSTRACT
This study was aimed to establish the HPLC fingerprint ofPhyllanthus urinaria Linn and Phyllanthusamarus Linn, in order to provide evidences for the study on material basis. Analysis was performed on an INDUSTRIES Epic C18 120A (5μm, 250 mm × 4.6 mm) column eluted with the acetonitrile (A) - water (0.1% phosphoric acid, V/V) gradient system as mobile phase. The wavelength was 254 nm and the flow rate was 1mL·min-1. The column temperature was 30℃. The injection volume was 10 μL. The results showed that the HPLC fingerprint ofPhyllanthus urinaria L. andPhyllanthus amarus L. were established. It was concluded that the method was simple, accurate and reproducible. This study provids experimental data for rapid quality identification and comprehensive evaluation ofPhyllanthus urinaria L. andPhyllanthus amarus L..
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<p><b>OBJECTIVE</b>To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma (HCC) using early treatment by Compound Phyllanthus Urinaria L. (CPUL) on patients with preneoplastic hepatitis B virus (HBV)-associated HCC.</p><p><b>METHODS</b>A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins (five up-regulated genes URG4, URG7, URG11, URG12 and URG19, and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software. Fifty-two patients were in the treatment group and 50 patients were in the control group. CPUL was used in the treatment group for 3 years, while the control group did not receive any treatment. The changes in HBV-DNA level, number of antibodies, and hepatocarcinogenesis occurred were observed. Patients who did not develop HCC were followed up for another 2 years.</p><p><b>RESULTS</b>HBV-DNA levels decreased ⩾2log in 22.2% (10/45) of patients in the treatment group in contrast to only 5.0% (2/40) of patients in the control group (P=0.0228). The number of antibodies that were tested positive in the treatment group (1.08±1.01) was significantly lower compared with the control group (2.11±1.12) after 24 months of drug treatment (P<0.01). Both the positive rates of anti-URG11 (33/52) and anti-URG19 (31/52) were over 60% at baseline in the two groups, and were decreased to 48.1% (25/52) and 46.2% (24/52) respectively at 36 months of drug treatment, while the rates increased to 68.0% (34/50) and 66.0% (33/50) respectively (P=0.0417, P=0.0436) in the control group. The positive rate of anti-DRG2 was increased to 55.8% (29/52) at 36 months of drug treatment, while in the control group was decreased to 36.0% (18/50, P=0.0452). Among the 102 patients who developed HCC, 2 were in the treatment group and 9 were in the control group, meaning that a significant difference between the two groups (P=0.0212). In 11 patients who developed HCC, anti-URG11 and anti-URG19 were always positive, while anti-DRG2 was negative. Patients newly developing HCC were 6 (20.0%) in the control group, and only one (2.5%) in the treatment group (P=0.0441) during 2-year follow-up after the end of the treatment.</p><p><b>CONCLUSIONS</b>Anti-URG11, anti-URG19 and anti-DRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC. CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.</p>
Subject(s)
Humans , Antibodies, Viral , Blood , Carcinoma, Hepatocellular , Therapeutics , Virology , DNA, Viral , Hep G2 Cells , Hepatitis B virus , Genetics , Allergy and Immunology , Virulence , Liver Neoplasms , Therapeutics , Virology , Phyllanthus , Chemistry , Plant Extracts , Therapeutic Uses , Precancerous Conditions , VirologyABSTRACT
Objective To establish culture method for Phyllanthus urinaria. Methods To study the possible effective factors of culture condition by comparing with different explants,sucrose,plant growth substance,and its ratio. Results The inductivity of stem was the highest about 55.56%,but callus of leaves could not be induced.6-BA was the most important factor on callus induction,followed by 2,4-D NAA and sucrose. Conclusion The optimal medium to induce and subculture is MS+2,4-D 0.5 mg/L+6-BA 1.0 mg/L+NAA 0.1 mg/L+sucrose 10 g/L.
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Objective To investgate the inhibitory effect of Phyllanthus Urinaria L compound on proliferation of hepatoma cell HePG2 in vitro and to explore its mechanism.Methods The influence of different concentrations of Phyllanthus Urinaria L compound at different time on HePG2 proliferation was compared by MTT colorimetric assay and cell growth curve assay.The cell apoptotic rate and morphological changes of HePG2 were observed by flow cytometry,fluorescence microscope and electron microscope.Results Phyllanthus Urinaria L compound can inhibit the proliferation of human hepatoma cell line HePG2.Within a certain limit of concentrations,the higher the concentration and the longer the acting time,the stronger the inhibition.Co-cultured with 500 ? g/mL Phyllanthus Urinaria L compound for 72 h,the inhibitory rate reached 93.58 % and IC50 was 240 ? g/mL.Phyllanthus Urinaria L compound at different concentrations had an certain effect on inducing cell apoptosis.Conclusion Phyllanthus Urinaria L compound can inhibit the proliferation of hepatoma cell,and its mechanism may be related with the induction of hepatoma cell HePG2 apoptosis.