| Folk herbal medicine is the treasure of Chinese nation and the mutual wealth of human beings.However,the precious experience of herbal medicine and affluent medical resources are in the danger of disappearance,due to the economic development of the modern society,the destruction of ecological environment,and the deficiency of herbal medicine records—limited studies of chemical constituents and restriction of clinical application.So,it is urgent to protect and utilize them.By applying modern technical method,we can use the treasury to establish the bridge between the folk medicine and modern technology sufficiently.It has an actual sense of not only spreading the culture of traditional medicine,but also promoting the investigation and development,serving the human beings and transforming it into commodity's advantages,which consequently advance economy development rapidly in local region.According to the aforementioned,we selected the effective substance of Lemonfragrant Angelica Root and the critical process of Huxin extract as the studying topic,combined the fundamental research and application to inherit and develop the culture of Chinese medicine.1.Research on chemical constituents and quality control of Lemonfragrant Angelica RootLemonfragrant Angelica is a species endemic to China.Its root and the whole plant are used as crude drag recorded in Iconographia Plantarum Chinese Materia Medica Chinese Materia Medica dictionary and many local medical books,such as Guangxi herbal medicine Jiangxi herbal medicin Zhejing common folk herbal medicine and common herbal medicine manual from Guangzhou troops.The main components were always wroten as starch,flavonoid glycoside,essential oil,organic acid and carbohydrate,common herbal medicine manual from Guangzhou troops introduced its actions:root,circulating the blood and eliminating stasis,promoting the flow of qi to relieve pain,stoping coughing and eliminating phlegm,applied to angina pectoris,gastralgia,chronic cough and also venomous snake biting.The effect of Lemonfragrant Angelica Root on cardiovascular diseases is reliable and clear that it is used as a monarch drug in the well-known Chinese medical preparation "HUXIN Capsule".But the lack of researches on chemical components of Lemonfragrant Angelica Root make the investigation just focus on isodillapiol,isodillapiolglycol andβ-sitosterol isolated in petroleum ether part from the root,there was no more records about the chemical constituents in the literature at home and abroad.(1).GC-MS analysis of essential oil from Lemonfragrant Angelica RootOur study initially used GC-MS method to analyse and identify essential oil from Lemonfragrant Angelica Root,47 compounds were identified including apiol, panaxynol,myristicin,isoelemicine,caryophyllene,hexadecanoic acid,hexadecanoic acid, linoleic,etc.The pharmacologic action of partial components was related to the action of Lemonfragrant Angelica Roots.(2).Chemical constituents from Roots of Lemonfragrant AngelicaLemonfragrant Angelica roots(10kg)was extracted with 95%ethanol and filtered. The filtrate was concentrated by rotary evaporation,the resulting residue was partitioned between H2O and petroleum ether,then by EtOAc,n-butanol.The petroleum ether fraction and EtOAc fraction were subjected to silica gel column,then gradiently eluted with Petroleum eher-EtOAc respectively.Similar fractions were combined with TLC plates screening,then repeated to the silica gel column and recrystallized to gain purified compounds.12 compounds were isolated and 8 compounds were identified by the the IR,MS,1H and 13C-NMR spectrograph.One of them is a new compound and the rest 7 compounds are obtained from this plant from the first time.(3).Founded a new TLC method for identifying two types of components from Lemonfragrant Angelica Rootâ… .TLC method for the essential oil from Lemonfragrant Angelica RootThe coarse powder of plant material(20g)was extracted by distillating for 4h to give the volatile oil,and then the oil was dissolved in 10ml ether to prepare the sample solution, when 1,3-Benzodioxole,4,7-dimethoxy-5-(1E)-1-propenyl were served as standard. According to the thin layer chromatography ruled by Chinese Pharmacopoeia,took 10μl the sample solution and 10ul standard solution to have small spot on the same thin layer plate;used petroleum ether-acetone(9:0.5)as the developer;took out and volatilized the solvent when the developer moved up to the leading edge,and finally sprayed Phospho-molybdic acid,heating for visualizing the spots.There was the same spot on the same position of the sample comparing standard solution.â…¡.Identified method for the water-solubility part from Lemonfragrant Angelica RootThe coarse powder of plant material(2.5g)was extracted with CH3OH(25ml)by reflux for 1h.After filtered and concentrated the filtrate,the residule was dissolved with water(20ml)to subject to AB-8 type of macroporous resin column(1.5cm×8cm).The followed process was:firstly eluted by water,then by 95%EtOH,collected the 95%EtOH eluted solution and recovered EtOH,the residue was dissolved with 2ml MeOH and using citriodorumin as standard.According to the thin layer chromatography ruled by Chinese Pharmacopoeia took 10μl sample solution and 10ul standard solution to have small spot on the same thin layer plate;used Methyl benzene-Ethyl formate-MeOH-Formic acid((7:2:1:0.3))as the developer and pre-saturated for 15min;took out and volatilized the solvent when the developer moved up to the leading edge,and finally sprayed aluminium trichloride solution,inspecting in the UV lamp(365nm).There was the same spot on the same position of the sample compared with standard solution.(4).Initially developed fingerprint HPLC spctrum for Lemonfragrant Angelica Rootâ… .Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18(250mm×4.6mm,5μm)column as stationary phase,when acetonitrile(A)and 0.3%hydrogenphosphate(B)were served as gradient elution in the flow rate of 1.0ml/min.Detective wavelength was set at 320nm, simultaneously,the column temperature was maintained at 20℃.â…¡.Preparation of standard solutionUsing 5.0mg Bdp as standard,weighed accurately,then dissolved in 25ml MeOH and mixed for injecting.â…¢.Preparation of sample solutionWeighed 1.0g coarse powder of plant material accurately;dissolved with 50ml 50%EtOH;weighed and extracted by reflux for 1h,weighed again to make up the lose weight with 50%EtOH.The extracted solution was filtered through 0.45μm syringe filter for HPLC injecting. â…£.Determination methodTook 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.(5)Founded firstly HPLC quantity determination method of two Phenylpropanoid componens from Lemonfragrant Angelica Rootâ… .Quantity determination method of Bdp from Lemonfragrant Angelica Roota.Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18(250mm×4.6mm,5μm)column as stationary phase,when MeOH(A)and H2O(B)were served as elution in the proportion of 70:30.Detective wavelength was set at 280nm.b.Preparation of standard solutionAn accurately weighed standard Bdp was dissolved in MeOH to prepare the standard solution with a concentration of 200μg/ml.c.Preparation of sample solutionWeighed 0.5g coarse powder of plant material accurately;dissolved with 50ml MeOH and then extracted by ultrasound for 30min;weighed again to make up the lose weight with 50%MeOH after descending to room temperature;finally filtered and took the filtrate as sample solution.d.determination methodTook 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.â…¡.Quantity determination method of citriodorumina.Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18(250mm×4.6mm,5μm)column as stationary phase,when acetonitrile(A)and H2O(B)were served as elution in the proportion of 45:55.Detective wavelength was set at 320nm.b.Preparation of standard solutionAn accurately weighed citriodorumin standard was dissolved in MeOH to prepare the standard solution with a concentration of 10μg/ml.c.Preparation of sample solutionWeighed 0.5g coarse powder of plant material accurately;dissolved with 50ml MeOH and then extracted by ultrasound for 30min;weighed again to make up the lose weight with 50%MeOH after descending to room temperature;finally filtered and took the filtrate as sample solution.d.determination method Took 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.(6).Process of Lemonfragrant Angelica root extract with maeroporous resinHPLC fingerprint of Lemonfragrant Angelica Root was adopted as index to investigate the purified process of Lemonfragrant Angelica Root extract with AB-8 type of macroporous resin.The results indicated that the HPLC fingerprints of the extract before and after absorption by macroporous resin were similar.2.Research on purification process of Huxin Extract with macroporous resinThe prescription of"Huxin" which use Lemonfragrant Angelica Root as monarch drug has vivid "Lingnan" feature and practically curative effect.Respecting that most drugs of the prescription such as(Ostericum citriodorum,Adenosma glutinosum,Acori Gramineus and Evodiae)contain a large amount of essential oil(equal to more than 0.5%of crude drug in prescription)which is similar in activity to the whole prescription,we chose drop pill praeparatum which have the advantages of stabilizing the essential oil by solidification and dissolving quickly.The refining processes of the extracted solution was an important part of the drop pill preparation.Based on the research of macroreticular resin processes for single drug in the prescription,we selected macroreticular resin to purify the compound extraction.By the content of total flavonoids(marked by icariin),the Retention ratio of main Components in HPLC fingerprints and the Phenols compounds inspected by color reaction,kinetic adsorption curve,the type and dosage of elution solvent were investigated. The results indicated that this refining process could reduce the yield of extract effectively and retain the main components of the HPLC fingerprints.(1).Study on the kinetic adsorption process with AB-8 macroreticular resin for the purification of Huxin extractUsing icariin as index and combining HPLC fingerprints to study the refinement process with AB-8 macroreticular resin.The results showed that the maximum dosage(the leak point was 5%)was 164.61mg total flavonoids/g dry resin,and the main components in the HPLC fingerprints were retained.(2).Study on the kind of elution solvent during the purification process with AB-8 macroreticular resin of Huxin extractAccording to the elution ratio,HPLC fingerprint and the literature on refinement process of single drug,95%EtOH was used as solvent.(3).Study on the dosage of elution solvent during the purification process with AB-8 macroreticular resin of Huxin extractBased on the elution ratio,HPLC fingerprint and the literature on refinement process of single drug,we finally concluded that 2 folds of column volume of 95%EtOH should be used to elute.(4).Verification to the refinement process parameters by the color reaction of the Phenols compoundsThe processing parameters were confirmed respecting to the color reactions of the Phenols compounds marked by the chloride ferric(2%)-ferricyanatum Kalium(1%)solution. The results suggested that the Phenols compounds leak far less than 5%,compared with that of initial solution;after eluting 2 folds of column volume of 95%EtOH,the concentration of phenols compounds in the subsequent eluted solution was far less than 5%, compared with that of initial solution.As the case stands,we can retain phenols compounds during the refinement process.3.Evaluation on Huxin extract with macroporous resin using "Bioactivity and FingerPrint"as an indexThis experiment kept the product in the retention time in the main fingerprints and retained its bioactivity when compared with the initial extracts:the dia-column liquor, water eluant and the ethanol eluant respectively,which have a representative action of resisting myocardial ischemia injury.When using "Bioactivity and FingerPrint" as an index, this method also establishes a reasonable refined process of Huxin extract with macroporous resin.The retention time of the main peaks in HPLC chromatogram after the treatment of resin remained more than 90%without apparent leakage of the main peaks in water eluent, moreover,the HPLC chromatograms of the initial physic liquor and the ethanol eluant were similar,both indicating that the quantity of Lemonfragrant Angelica Root,Ilex pubescem, Adenosma glutinosum and Epimedii in the "Huxin" compound extract was reserved, including icariin and Bdp.The biological results of protecting myocardial ischemia after the experimental rats intragastically administrated with initial extract,the dia-column liquor,water eluant and the ethanol eluant,show as follows:the initial extract and the ethanol eluant can significantly reduce myocardial ischemia induced injury,which inhibit the upgrade of the ST segment in ECG and the myocardial enzyme in serum,and diminish the infarction area.However, dia-column liquor and water eluant has a weak action to attenuate myocardial ischemia injury;neither effect on volume percentage of infarction area occupied the risk area,nor decrease myocardial enzyme AST and LDH.The ability of different eluants to protect against myocardial ischemia injury was:the ethanol eluant(0.4g/kg/d)＞the initial extract (0.4g/kg/d)＞the water eluant(0.4g/kg/d).It demonstrated that the active compounds were principally enriched in the ethanol eluant,and the process of refinement with macroporous resin remained the bioactivity of the initial extract.This project not only discovers a new compound,but also identifies seven compounds from Lemonfragrant Angelica roots.We establish a system to standardize the quality of the plant material which includes the identification of TLC,the quantitive determination and chromatogram of HPLC.The outcomes can be applied in the "Secondary Development" of "Huxin Capsule".Without application of new technologies,there will be no innovation of traditional chinese medicine products.There always exists a doubt that if the functions of the drug remains while the amount of formula seems less after the treatment with new technology.This topic puts forward and carries out evaluation index of "Bioactivity and Fingerprint".In other words,according to the retention time of main components represented by main peak of "HPLC fingerprints" and the bioactivity represented by the key pharmacodynamics experiment,it is possible and applicable to make evaluations on new technology of medicament preparation. |
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