Studies On Chemical Constituents From Solanum Lyratum Thunb. And Pharmacokinetics Of Relative Active Ingredients

Solanum lyratum Thunb. (Solanaceae) called "Baimaoteng" in traditional Chinese medicine had antitumor actiity. The research object was Solanum lyratum Thunb. using antitumor activity as guideline. The compounds of alkaloid were isolated and purified by various chromatographic techniques and identified by their physicochemical properties and spectral data. MTT method was used to determined the antitumor activity of the compounds obtianed from Solanum lyratum Thunb. using Hela cells. The present study provided a method for the determination of chlorogenic acid and caffeic acid in Solanum lyratum Thunb. simultaneously by high performance liquid chromatography (HPLC). Besides, HPLC-UV and HPLC-ELSD were established to determine the concentration of strychnine and (3-O-β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl-(1â†'4)-β-D-galactopyranoside-(25ξ)-solanidan-3β,23β-diol) in Solanum lyratum Thunb., respectively. An HPLC-UV method was set up for comparative study on pharmacokinetics and bioavailability of chlorogenic acid after oral administration chlorogenic acid and extract of Solanum Lyratum Thunb. in rats.1. Studies on chemical constituents from Solanum lyratum Thunb.8 compounds from the ethanol extraction of Solanum lyratum Thunb. were isolated by silica gel, macroporous resin, Sephadex LH-20, thin layer chromatography, semipreparative HPLC and recrystallization. The structures were elucidated by NMR and measuring physicochemical property as N-trans-feruloyl-3-methyldopamine (1), strychnine (2), N-trans-feruloyloctopamine (3), linarin (4), dihydroleptinidine (5), Saladulcidine (6), (3-O-β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl-(1â†'4)-β-D-galactopyranoside-(25ξ-solanidan-3β,23β-diol) (7), daucosterol (8). Compounds (1)-(6) were obtained from the plant for the first time.2. Studies on antitumor activity of compoundsMTT assay was applied to screen the anti-tumor effect of the extracts and compounds of Solanum lyratum Thunb.. The result showed that most extracts of Solanum lyratum Thunb. had different degrees of antineoplastic activity, and had found antineoplastic activity against Hela cells of compound 4 for the first time.3. To establish an HPLC-UV method for simultaneous determination of chlorogenic acid and caffeic acid in Solanum lyratum Thunb.To establish a RP-HPLC-UV method for simultaneous determination the contents of chlorogenic acid and caffeic acid in Solanum lyratum Thunb., the operation was carried out on a Diamonsil-ODS C18 column (250 mm×4.6 mm,5μm) with the mobile phase consisting of a mixture of methanol-0.05%phosphoric acid (25:75, v/v), and UV detection wavelength was 327 nm. The calibration curve of mass concentration of chlorogenic acid was linear in the range of 0.5-32.0μg·mL-1 (r=0.9999). The mean recovery of chlorogenic acid was 99.9%(RSD=1.5%). The calibration curve of mass concentration of caffeic acid was linear in the range of 10.0-640.0 ng·mL-1 (r=0.9999). The mean recovery of caffeic acid was 99.9%(RSD=1.4%).20 batches of Solanum lyratum Thunb. were determined under the conditions. The HPLC method is found simple, accurate, and reproducible. It can be used for the determination of the contents of chlorogenic acid and caffeic acid in Solanum lyratum Thunb..4. To establish an HPLC-UV method for determination of strychnine in Solanum lyratum Thunb.A RP-HPLC-UV method was found to determine strychnine in Solanum lyratum Thunb. using a Diamonsil-C18 column (250 mm×4.6 mm,5μm), eluted with acetonitrile-10 mmoL·L-1 phosphate (8:92, v/v, adjusted to pH 2.5 by phosphoric acid) as mobile phase and UV detection wavelength was 254 nm. Mass concentration of strychnine showed the good linear relationship at the range of 1.0-16.0μg·mL-1(r=0.9999). The average recovery of strychnine was 99.1%and RSD was 3.2%.10 batches of Solanum lyratum Thunb. were studied. The method is simple, accurate and can be used for the determination of strychnine in Solanum lyratum Thunb..5. To establish an HPLC-ELSD method for determination of (3-O-β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl-(1â†'4)-β-D-galactopyranoside-(25ξ)-solanidan-3β,23β-diol) in Solanum lyratum Thunb.An HPLC-ELSD method was established for the determination of (3-O-β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl-(1â†'4)-β-D-galactopyranoside-(25ξ)-solanidan-3β, 23β-diol) in Solanum lyratum Thunb. using Elite-ODS C18 (200 mm×4.6 mm,5μm) with the mobile phase consisting of a mixture of acetonitrile-0.02%triethylamine (56:44, v/v). The parameters of drift tube and gas flow rate of the detector were set at 95℃and 2.3 L-min-1, respectively. The linear range was 0.40-4.0μg and the correlation coefficient was 0.9996. The average recovery was 99.7%. The HPLC-ELSD method is found accurate, sensitive and reproducible. The method can be used for the determination of (3-O-β-D-glucopyranosyl-(1â†'2)-β-D-glucopyranosyl-(1â†'4)-β-D-galactopyranoside-(25ξ)-sola-nidan-3β,23β-diol) in Solanum lyratum Thunb..6. Comparative study on pharmacokinetics and bioavailability of chlorogenic acid and extract of Solanum lyratum Thunb. in ratsAn high-performance liquid chromatographic method has been developed to determine the concentration of chlorogenic acid in rat plasma and to compare the pharmacokinetic parameters and bioavailability of chlorogenic acid after oral administration of chlorogenic acid and extract of Solanum lyratum Thunb.. Liquid-liquid extraction was applied to process plasma samples using isopropanol as extractant. Supernatant was dried by N2 at 40℃, and residue was redissolved in 100μL mobile phase. Puerarin was used as internal standard. The HPLC method was used with a Diamonsil-C18 column (250 mm×4.6 mm,5μm), eluted with methanol-0.05% phosphoric acid (23:77, v/v) as mobile phase with UV detection wavelength 327 nm. The calibration curve of chlorogenic acid was linear in the range of 0.0500-15.0 mg·L-1 (r=0.9973) with mean recovery of 70.50%(RSD=4.9%) for chlorogenic acid. LLOQ of the method was 0.0500 mg·L-1. The method was reproducible and reliable with intra-day precision better than 5.1%, inter-day precision better than 9.5%, accuracy within-7.1%. Three groups of rats were administrated orally chlorogenic acid, extract of Solanum lyratum Thunb., intravenous injection chlorogenic acid, respectively. The pharmacokinetic parameters were evaluated using non-compartmental methods by the DAS 2.0 practical pharmacokinetics program. The results of pharmacokinetics analysis were as follows:Tmax(0.49±0.32), (0.81±1.08) h; T1/2(2.66±0.48), (2.63±1.12), (0.91±0.10) h; Cmax(0.52±0.20),(0.35±0.08), (9.99±0.73)mg·L-1; AUC(0-t) (1.87±0.91), (0.93±0.46), (4.33±0.39) mg·L-1·h-1; AUC(0-∞) (2.09±0.92), (1.09±0.44), (4.42±0.38) mg·L-1·h-1; Ke(0.27±0.05), (0.32±0.17), (0.77±0.09) h-1. The absolute bioavailability of chlorogenic acid after oral administration chlorogenic acid was 4.800%, and the relative bioavailability of chlorogenic acid after oral administration extract of Solanum lyratum Thunb. was 52.20%in rats. The results indicate that the HPLC method is found to be accurate and reliable. It can be used for the pharmacokinetics of chlorogenic acid in plasma of rats. There were significant pharmacokinetic differenceses between oral administration of chlorogenic acid and the extract of Solanum lyratum Thunb.. Absorption of chlorogenic acid in rats could be restrained by other components of Solanum lyratum Thunb..