Study On The Hemostatic Activity Part Of Tibetan Medicinal Herb Lamiophlomis Rotata

The plant Lamiophlomis rotata(Benth.)Kudo(LR)is of genus Lamiophlomis and once of genus Phlomis from the Labiatae family,which is called as DABA or DABUBA in Tibetan.It distributes wild at an altitude of 3000 m in the Qinghai Xizang plateau in China,especially in the south of Gansu,northwest of Sichuan,west of Yunnan,and southeast of Qinghai and Xizang.It has been used as a traditional drug by Chinese minorities such as Tibetans Mongolians and Na Xi for thousands of years.In Sichuan Herbals and Yunnan Flora,it was recorded to cure incised and gunshot wounds on humans and animals with miraculous effects.Since 1982,Pro.Liang had studied the pharmacology,phytochemical and clinical application of LR,and DUYIWEI pellet, which was composed of the aqueous extract of LR(LRAE),had been licensed as a new drug in 1992.Since 1995,DUYIWEI pellet,DUYIWEI capsule,and DUYIWEI medical material have been recorded in Pharmacopoeia of P.R.China with the effects of hemostasis,alleviating pain,detumescence,promoting blood circulation to remove blood stasis and reinforcing marrow.Recently,LR is used to stop bleeding and alleviate pain after operations;meanwhile it can be used to cure some diseases in dentistry,gynecology and arthrophlogosis.Iridoid glycosides and flavanoids are two main components of this plant.8-O-acetylshanzhiside methylester and luteolin-7-O-glycoside were the main compounds of these two kinds' components respectively.Contents of total flavonoid and luteolin have been used as the quality specification of this plant and its preparation in the Pharmacopoeia of P.R.China.But the analgesia and haemostasis effective composition of LR hasn't been elucidated.Iridoid glycosides,flavanoids and phenethanolside have been reported as the potential analgesia effective composition of LR.At the same time, there is no report about the study on the haemostasis effective composition of LR. Recently,LR has been generally used in clinic and the secondly exploitation of LR attracted more and more attention.Un-identification of the effective composition of LR has holden back the advancing study on new preparation and quality specification of LR. In order to solve the insufficiency about LR,improve the quality of preparation and medical material of LR,and implement the secondly exploitation of LR,some innovative works are carried out in this dissertation as followings: (1)Phytochemitry study of the hemostatic activity part of LR resulted five iridoid glycosides been isolated and identified.At the same time,the absolute structure of a new compound was elucidated by X-ray,and HPLC-MS technology was used to analysis iridoid glycosides in LR and its preparation for the first time.Content of total iridoid glycosides in LR and its extractions was firstly determined by the first derivative spectrophotometry with 8-O-acetyl shanzhiside methylester as standard. This method may be beneficial for the Pharmacopoeia of P.R.China.(2)The macroporous adsorptive resins chromatographic column combining with polyamide chromatographic column was originally used to separate the flavonoids ingredients(P₁),iridoid glycosides ingredients(P₂)and maximus polarity components ingredients(P₃.mainly of carbohydrate)from LRAE.The enriching technical process of the hemostatic activity part of LR was vested the invention patent,which may improve the secondly exploitation of LR.(3)The hemostatic activities of LRAE and P₁,P₂ and P₃ were estimated in mice or rats. As a result,LRAE and P₂ showed satisfactory hemostatic effect with dose-effect relationship and P₃ and P₄ showed no hemostatic effect.(4)LRAE and P₂ showed some effects of improving liver function and reducing blood lipids.These results confirm the hemostatic and thromboplastic effects of LR can be implemented by improving the synthetic function of liver.No produce mortality was found in the acute toxicity test of P₂ in mice.This dissertation consists of four chapters:Chapter 1:Review of studies on Lamiophlomis rotata and genus Phlomis were reviewed(1)Review on Lamiophlomis rotata:After reviewing the study of LR from eighty directions,such as herb record,plant taxonomy,medical material appearance and resource,phytochemistry,pharmacology,clinical application,preparation exploitation and quality specification,some insufficiency was drew off.(2)Review on genus Phlomis:The literature data on the phytochemical and biological investigations on the genus of Phlomis are summarized with 92 references.More than 150 compounds isolated from 35 plants of Phlomis species were exhibited with various bioactivities including antinociceptive,anti-inflammatory,antidiabetic, cytotoxic and cytostatic,antioxidant activity,antiulcerogenic,antiprotozoal, antimutagenic,antimicrobial and antifungal activities.From genus Phlomis,which has very genetic relationship with genus Lamiophlomis,some replacing resources of LR may be found.Chapter 2:Study on the hemostatic effects and mechanism of LR and iridoid glycosides ingredients(P₂).The hemostatic activity of LRAE and P₁,P₂ and P₃,which were isolated from LRAE,was estimated in mice or rats.As a result,LRAE and P₂ showed satisfactory hemostatic effect with dose-effect relationship and P₃ and P₄ showed no hemostatic effect. At the same time,LRAE and P₂ showed some effects of improving liver function and reducing blood lipids by decreasing the levels of AST,ALT,Chol and LDL-C et al.All these changes had a significant dose-effect and time-effect relationship.These results confirm the hemostatic and thromboplastic effects of L.rotata might be implemented by improving the synthetic function of liver.Result of acute toxicity test of P₂ showed, orally administered P₂ at the doses of 0.1-20 g/kg to mice did not produce mortality within 24 h.Chapter 3:Phytochemical study on the hemostatic activity part of LR(1)Separation of compositions in LR:According to the different of physico- chemical property between compositions in LR,macroporous adsorptive resins chromatographic column combining with polyamide chromatographic column was originally used to separate them from LRAE.All compositions were successfully separated as follows:flavonoids ingredients(P₁)with yield of 10.25%and content of 64.97%, iridoid glycosides ingredients(P₂)with yield of 19.77%and content of 66.92%, maximus polarity components ingredients(P₃)with yield of 44.30%and mainly carbohydrate.These parts were valid for the pharmacology study.(2)Isolation and identification iridoid glycosides:Using silica gel column chromatography and polyamide column chromatography,five iridoid glycosides with higher content have been isolated and identified as 8-O-acetylshanzhiside methylester (â… ),loganin(â…¡),shanzhiside methylester(â…¢),phloyosideâ…¡(â…£),and 8-dehydroxy shanzhiside(â…¤).Among them,â…¤was a new compound.(3)X-ray analysis the structure of new compound:With the extensive NMR spectroscopy techniques and X-ray investigation on the new compound 5,the absolute configurations of C-1,C-5,C-6,C-8 and C-9 was(S),(S),(R),(R),and(R) respectively.The Glc moiety was found to adopt a chair-like conformation,the absolute configurations at its five stereogenic C-atoms being(R),(R),(S),(R),and(R) from C-1' to C-5',respectively.(4)Qualitative and quantitative analysis of iridoid glycosides in LR with HPLC-PAD-APCI/MS:LR was studied by HPLC-PAD-APCI/MS for the first time.Under the optimized condition,the analysis could be finished in 45 min on the symmetry reversed-phase C₁₈column combined with negative ion detection.Five iridoid glycosides were identified as 8-O-acetylshanzhiside methylester,loganin, 8-dehydroxy shanzhiside,phloyosideâ…¡,and shanzhiside methylester with markers and four unknown iridoid glycosides were presumed as penstemoside,a pair of isomeride of schismoside and phloyosideâ… .Furthermore the outstanding and concomitant appearance of[M-H]^-,[M-H+33]^-,and[M-H+60]^- could be regarded as the characteristic ion to distinguish the iridoid glycosides,which has an ester group at the C-4 position in acetas acid buffer as eluent in negative MS mode,and the iridoid glycosides with a carboxyl group at C-4 only produced the molecular ion[M-H]^-.At the same time,the quality control method for LR was firstly established.Calibration linearity was good(R=0.9991).Chapter 4:Determination and enrichment of iridoid glycosides ingredients(P₂).(1)The first derivative spectrophotometry with polyamide chromatographic column as pre-column to eliminate flavonoids was established firstly to determine the content of total iridoid glycosides in LR and its extractions.8-O-acetyl shanzhiside methylester was used as standard and the determining wavelength was 250 nm.(2)After comparing the abilities of absorption and antiabsorption of ten different type's macroreticular resin,XDA-1 macroreticular resin was used to enrich the iridoid glycosides from LRAE.The quiet and dynamic state absorptions were 16.95 mg/ml and 15.18 mg/ml respectively.50%Ethanol was the best solvent of antiabsorption and the content of total iridoid glycosides in eluate was 67.53%,eluting recovery rate is 90.06%.The macroreticular resin could be reused more than 8 times.