| Toddalia asiatica ( L.) Lam. (Rutaceae) is widely used as a folk medicine in many Asian and Africa countries. The root and its bark have been used for the treatment of traumatic injury, rheumatism, gall etc. The hemostatic activity, chemical constituents,quality control methods of Toddalia asiatica ( L.) Lam. and pharmacokinetics of active components were investigated with the modern pharmacological methods and analytical technologies in this study.The hemostatic activities of Toddalia asiatica ( L.) Lam. extracts with ethanol of various concentrations were investigated by determining the bleeding time (BT) and coagulant time (CT) of mice. The experimental data were analyzed using one-way ANOVA, which indicated that the 70% ethanol extract had excellent hemostatic activity. The extract of 70% ethanol was fractionated with chloroform, ethyl acetate and n-butanol successively. The chloroform layer was found to be the most active fraction with hemostatic activity. Blood coagulation parameters of rats, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time(TT), fibrinogen level (FIB) and platelet morphology, were tested after 3 weeks treatment with the chloroform fraction of Toddalia asiatica ( L.) Lam. extract. The thrombin times (TT) was significantly shortened and the fibrinogen level (FIB)significantly increased (p<0.05). Meanwhile, the platelet number was increased and the morphology was changed by extension and pseudopodium formation. Increasing fibrinogen levels, promoting the endogenous coagulation pathway and increasing the platelet number may be the main mechanism of hemostatic activity of Toddalia asiatica ( L.) Lam. extract.To define the hemostatic components of Toddalia asiatica ( L.) Lam., the chemical constituents in chloroform layer of 70 % ethanol extract of Toddalia asiatica ( L.)Lam. were isolated by using multiple column and thin layer chromatographic techniques,and 21 compounds were obtained. The structures of 16 compounds were fully elucidated by chemical and spectroscopic methods (NMR, MS). The identified compounds include: Zanthocadinanine A (1), pimpinellin (2),isopimpinellin (3),phellopterin (4), amottianamide (5) chelerythrine (6), nitidine (7), chlorogenic acid (8),toddalolactone (9), protopine (10), skimmianine (11), dictamine (12), toddalenone(13), ?-sitosterol (14), bergapten (15) and 8-Hydroxy-6-methoxycoumarin (16).Among them, compound 1 and 16 were isolated from Toddalia for the first time. The hemostatic activities of main chemical constituents obtained from Toddalia asiatica(L.) Lam. were investigated by determining the bleeding time and coagulant time of mice. The results of experiment were analyzed using one-way ANOVA, pimpinellin showed excellent hemostatic activity. Blood coagulation parameters of rats, including PT, APTT, TT, FIB and platelet morphology were tested after 3 weeks treatment with pimpinellin, the results indicated that thrombin times (TT) was significantly shortened and the fibrinogen level (FIB) significantly increased (p<0.05). Meanwhile, the platelet number was increased and the morphology was changed by extension and pseudopodium formation. Increasing fibrinogen levels, promoting the endogenous coagulation pathway and changing the platelet morphology may be the main mechanism of hemostatic activity of pimpinellin.The chromatographic fingerprint (CFP) of Toddalia asiatica ( L.) Lam. from different resources was established by HPLC with UV detection (HPLC-UV). The separation was conducted on a C1g column with 0.75% acetic acid in water and acetonitrile as the mobile phase. There are 20 "common peaks" in the fingerprints,among them, 9 peaks were identified by comparing with the reference substances. The similarities of Toddalia asiatica ( L.) Lam. samples were evaluated with angle cosine and correlation coefficient as the measure. The similarity results indicate the HPLC-FPs of Toddalia asiatica ( L.) Lam. from different resources varied slightly.From the fingerprints it was found that the content of coumarins and alkaloids in root bark is much higher than in root xylem of Toddalia asiatica ( L.) Lam. Toddalia asiatica ( L.) Lam., Zanthoxylum nitidum(Roxb.)DC. and Zanthoxylum simullans Hance can be accurately identified by using the established HPLC-FPs.The gas chromatographic fingerprints of essential oils in Toddalia asiatica ( L.)Lam. was established. The fingerprint analysis was performed on HP-5 column with programmed column temperature and FID as the detector. The GC-FPs of samples from different resources was analyzed. Twenty one common peaks were identified by GC/MS with NIST database. The relative content of common peaks was more than 92% of the total content of the essential oils in of Toddalia asiatica ( L.) Lam. by area normalization. The constituents in essential oils of Toddalia asiatica ( L.) Lam. leaves and root were compared based on their GC-FPs, the results showed that their constituents are quite different.A high performance liquid chromatography (HPLC) method was developed for simultaneous quantitative determination of toddalolactone, chelerythrine,8-hydroxy-6-methoxycoumarin, isopimpinellin, pimpinellin and phellopterin in Toddalia asiatica ( L.) Lam. The analysis was performed on a C18 column using gradient elution with the mobile phase consisting of 0.75% acetic acid in water and acetonitrile. The method provided good accuracy and good precision and has been successfully applied to quantitative determination of six active components in Toddalia asiatica ( L.) Lam. obtained from 10 different resources.A rapid and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous determination of three bioactive coumarins of Toddalia asiatica extract, pimpinellin, isopimpinellin and phellopterin, in rat plasma for the first time. Phenacetin was used as the internal standard (IS). Plasma samples were extracted by liquid-liquid extraction with methyl tert-butyl ether. The chromatographic separation was carried out on an ACQUITY UPLCTM BEH C18 column with an isocratic mobile phase consisting of methanol-5 mmol/L ammonium acetate (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive ionization mode. This validated method was successfully applied to a pharmacokinetic study in rats after oral administrations of the three bioactive coumarins and the extract of Toddalia asiatica(L.) Lam., respectively. The Tmax and Cmax in rats receiving Toddalia asiatica ( L.)Lam. extract were 1.83±0.35 h and 1.62×103±521 ng/mL for pimpinellin, 1.33 ±0.31 h and 882±234 ng/mL for isopimpinellin,0.83±0.25 h and 63.9±25.7 ng/mL for phellopterin. While Tmx, and Cmax in rats having the coumarin monomer were 0.68±0.28 h and 1.56×103 ± 990 ng/mL for pimpinellin,2.12±0.81 h and 788 ± 243 ng/mL for isopimpinellin,0.64 ± 0.24 h and 59.9±15.4 ng/mL for phellopterin. The AUC0-t of Toddalia asiatica ( L.) Lam. extract was significantly increased. It was indicated that pharmacokinetic of coumarins affected by other constituents in Toddalia asiatica ( L.) Lam.extract.In conclusion, by utilizing various professional knowledge and analytical techniques, the hemostatic activity of Toddalia asiatica ( L.) Lam. was determined,the constituents in Toddalia asiatica ( L.) Lam. were investigated, quality control methods based on HPLC and GC were established and the pharmacokinetic study on three active coumarins in Toddalia asiatica ( L.) Lam.were conducted. This study would be useful for new drug development from the Toddalia asiatica ( L.) Lam. |
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