ABSTRACT
Rotundic acid(RA),an ursane-type pentacyclic triterpene acid isolated from the dried barks of Ilex rotunda Thunb.(Aquifoliaceae),possesses diverse bioactivities.To further study its pharmacokinetics,a simple and sensitive liquid chromatography with triple quadrupole mass spectrometry(LC-QqQ-MS/MS)method was developed and validated to quantify RA concentration in rat plasma and tissue using etofesalamide as an internal standard(IS).Plasma and tissue samples were subjected to one-step protein precipitation.Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 col-umn(4.6 mm×50 mm,5 μm)under gradient conditions with eluents of methanol:acetonitrile(1∶1,V/V)and 5mM ammonium formate:methanol(9∶1,V/V)at 0.5mL/min.Multiple reaction monitoring transitions were performed at m/z 487.30 → 437.30 for RA and m/z 256.10 → 227.10 for IS in the negative mode.The developed LC-QqQ-MS/MS method exhibited good linearity(2-500 ng/mL)and was fully validated in accordance with U.S.Food and Drug Administration bioanalytical guidelines.Dose proportionality and bioavailability in rats were determined by comparing pharmacokinetic data after single oral(10,20,and 40 mg/kg)and intravenous(10 mg/kg)administration of RA.Tissue distribution was studied following oral administration at 20 mg/kg.The results showed that the absolute bioavailability of RA after administration at different doses ranged from 16.1％to 19.4％.RA showed good dose proportionality over a dose range of 10-40 mg/kg.RA was rapidly absorbed in a dose-dependent manner and highly distributed in the liver.In conclusion,this study is the first to systematically elucidate the absorption and distribution characteristics of RA in rats,which can provide additional information for further development and evaluation of RA in drug metabolism and pharmacokinetic studies.
ABSTRACT
OBJECTIVE： To synthesize and identify rotundic acid esters derivatives， and to evaluate in vitro antitumor activity. METHODS： Using rotundic acid as raw material， rotundic acid esters derivatives were synthesized by 28-esterification， 3β and 23-position hydroxyl group combined with acid anhydride. The structure of rotundic acid esters derivatives were identified by HRMS， 1H-NMR， 13C-NMR and physicochemical properties. The in vitro antitumor activities were evaluated by MTT assay against paclitaxel （positive control）， rotundic acid and rotundic acid esters derivatives with human cervical cancer cells HeLa， human malignant melanoma cells A375， human lung adenocarcinoma cells SPC-A1 and human liver cancer cells HepG2 （take IC50 as an indicator）. RESULTS： Totally 6 kinds of rotundic acid esters derivatives were synthesized， i.e. rotundic acid methyl ester（compound 1）， 3， 23-O-di（acetyl） rotundic acid methyl ester（compound 2）， 3， 23-O-di（propionyl） rotundic acid methyl ester（compound 3）， 3， 23-O-di（butyryl） rotundic acid methyl ester（compound 4）， 3， 23-O-di（phthaloyl） rotundic acid methyl ester（compound 5） and 3， 23-O-di（succinyl） rotundic acid methyl ester（compound 6）. Compared with paclitaxel， the antitumor activity of compound 5 and compound 6 to Hela， A375， HepG2 and SPC-A1 was similar to it， and there was no significant difference between them （P＞0.05）； the IC50 of rotuntic acid and other compounds decreased with statistical significance （P＜0.05）. CONCLUSIONS： In the study， 6 kinds of rotundic acid esters derivatives are synthesized and compound 5 and compound 6 have significant antitumor activity in vitro， which can provide reference for further study.
ABSTRACT
Objective:To design and synthesize series of rotundic acid derivatives by introducing aromatic ester groups with rotundic acid as the parent nucleus, test their anti-tumor activity in vitro,investigate the structure-activity relationship of rotundic acid derivatives in inhibiting tumor cell proliferation, and obtain the novel rotundic acid derivatives with high anti-tumor activity. Method:Compounds 1-8 were synthesized with rotundic acid as the initial raw material through the 28-etherification,3β and 23di-aromatic esterification eaction. The anti-tumor activities in vitro were evaluated by MTT assay against A375 (human malignant melanoma cells),HeLa (human cervical cancer cells),SPC-A1 (human lung adenocarcinoma cells),and HepG2 (human liver cancer cells). Result:Compounds 2-8 were new compounds. Their structures were identified by melting point (MP),high resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS),1H nuclear magnetic resonance (1H-NMR) and 13 C nuclear magnetic resonance (13 C-NMR). MTT results showed that compounds 3,5 and 8 exhibited significant anti-tumor activity, especially compound 5 was found to have the best inhibition activity on HeLa,A375, HepG2 and SPC-A1 with IC50 values of (5.25±1.08),(5.99±0.88),(3.31±1.89),(5.74±1.78) μmol·L-1, 1.92,3.22,3.79, 3.72 times of that of rotundic acid,respectively. Conclusion:Compound 5 has significant anti-tumor activity with great significance for further research and development of new anti-tumor medicines.
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OBJECTIVE: To prepare high-purity rotundic acid, and to determine the contents of syringoside, peduncloside and rotundic acid in Ilicis rotundae and its refined products. METHODS: I. rotundae saponins was prepared by ethanol extraction method and hydrolyzed with methanol and sodium hydroxide; rotundic acid monomer was obtained after recrystallization. The contents of syringoside, peduncloside and rotundic acid in I. rotundae, its refined product 1 and its refined product 2 were determined by RP-HPLC. The determination was performed on an Agilent XDB C18 column with mobile phase consisted of water-acetonitrile (gradient elution) at the flow rate of 1. 2 mL/min. The column temperature was 30 ℃, and the detection wavelength was set at 210 nm. The sample size was 10 μL. RESULTS: The purity of rotundic acid exceeded 99％. The linear ranges of syringing, peduncloside and rotundic acid were 0. 18-3. 22, 0. 59-10. 36 and 0. 20-3. 53 μg, respectively (r=0. 999 9). RSDs of precision, stability (24 h) and reproducibility tests were all lower than 2. 0％ (n=6-7). The average recoveries were 96. 08％-100. 81 ％ (RSD≤1. 98％, n=6). The contents of syringoside,peduncloside and rotundic acid in I. rotundae were 1. 61％,7. 26％, 1. 29％ (RSD≤1. 08％,n=3); those of refined product 1 were 0,82. 59％,4. 18％ (RSD≤1. 67％,n=3); those of refined product 2 were 0, 73. 29％, 7. 41％ (RSD≤1. 15％,n=3),respectively. CONCLUSIONS: Prepared rotundic acid has high purity and is simple in preparation, safe and environmentally-friendly. RP-HPLC method that established is simple and reliable, and can meet the requirements for rapid analysis of I. rotundae and its refined products.
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Objective:To study the metabolism of pedunculoside treated with rat intestinal bacteria in vitro. Methods:Pedunculo-side and rat intestinal bacteria were incubated in vitro for 0, 4, 8, 24 and 48 hours under anaerobic condition. After extracted repeat-edly by ethyl acetate, the metabolites in the incubation media were qualitatively and quantitatively analyzed by HPLC. Results:Totally 90. 8% of pedunculoside was transformed to M2 after incubated with rat intestinal bacteria in vitro for 48 hours, and a detailed compari-son of HPLC profiles between M2 and rotundic acid showed M2 was rotundic acid. Conclusion: Pedunculoside can be metabolized to rotundic acid by rat intestinal bacteria in vitro.
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Objective: To investigate the chemical constituents from the aerial part of Rabdosia flexicaulis. Methods: Various column chromatography techniques were used to isolate and purify the compounds and their structures were identified by the spectral data. Results: Fifteen compounds were isolated and identified as rotundic acid (1), tormentic acid (2), corsolic acid (3), 23-hydroxyursolic acid (4), maslinic acid (5), teuclatriol (6), 3,6-dihydroxy-1-menthen (7), 1- hydroxypinoresinol (8), epipinoresinol (9), lariciresinol (10), caffeic acid (11), rosmarinic acid (12), 3'-O-methyl-rosmarinic acid (13), methyl 4'-O-methyl-rosmarinate (14), and methyl 3-dehydroxyl-rosmarinate (15). Conclusion: All the compounds are isolated from the species for the first time, among which compounds 1, 6, 7, 9, 13, and 14 are isolated from the plants in genus Rabdosia (Bl.) Hassk. for the first time.
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Objective: To study the chemical constituents from Millettia speciosa. Methods: The compounds were isolated and purified by silica gel, Sephadex LH-20, ODS column chromatography, and recrystallization. The structures were identified using physicochemical and spectral data. Results: Thirteen compounds were isolated from M. speciosa and identified as docosanoic acid (1), tetracosane (2), octadecane (3), hexacosanoic acid (4), β-sitosterol acetate (5), β-sitosterol (6), syringin (7), maackiain (8), formononetin (9), ψ-baptigenin (10), rotundic acid (11), pedunculoside (12), and daucosterol (13). Conclusion: Compounds 5, 7, and 10-12 are obtained from this plant for the first time.