| ObjectiveDalbergia sissoo Roxb.is belonged to family Leguminosae and genus Dalbergia.,which is a subtropical evergreen broad leaved tree species.Its heartwood was used as Chinese medicine lignum dalbergiae odoriferae.Indian rosewood wide spreading,grow fast,but so far we didn't have seen relevant research reports of India Ro se-wood leaf chemical components and the comprehensive utilization.To explore the medicinal value of India rosewood leaves,explore and utilize the resources comp rehensively,several parts as pharmacognosy identification,screening of effective p arts for pharmacology,chemical separation and identification,extraction and purific ation process of total flavonoids,content determination and fingerprint were studiedMethodsBased on the morphological features to make morphological description of the original plants of sissoo leaf of,to make identification according to the identificat ion method by the shape and properties,to make paraffin section with the relati ve method,to make identification of paraffin section?skin production and powder characteristics by microscopic identification method.Take KM mice as experimental models,anticoagulant,anti-inflammatory and analgesic as experiment index,to make pharmacodynamics screening of different polar parts of sissoo leaves.Using column chromatography and solvent method,recrystallization etc.for sis soo leaf alcohol extracts chemical separation and purification,and analysis throuh the constant,mass spectrometry?1H-NMR and 13C-NMR spectral analysis,structu ral identification of the separation of the components.According to the 2010 edition of Chinese pharmacopoeia appendix B ? thin-layer chromatography test of the TL C method,to take identification of sissoo leaf by TLC method.Take Biochanin A as reference substance,using uv spectrophotometry method to detect the total isoflavones content in sissoo leaf,and according to the orthogonal design method to optimize the extraction process the total isoflavone of sissoo leaf.UPLC method is used to simultaneously determine sissoo heartwood,leaf,flower,fruit,bark and other parts of Biochanin A content.Using HPLC method and different regions of sissoo leaves in different fingerprint comparison study,the same msthod is used to construct sissoo fingerprint.Results1 The shape,section and microscopic characteristics of Dalbergia sissoo.leafIt's shape characteristics:Leaf blade ovate or elliptic,apex acute,obtuse head,base rounded or cuneate,4?7 cm long,2?3 cm wide,1.5?3 cm petiole;Raw product surface dark green,on the surface of the green;Bask in one with a pale green;Leaf blade nearly leathery,papery or thinly leathery,relatively smooth surface;the smell is slightly sweet,slightly bitter taste.Microscopic identification points summarized as:(1)the transverse section view:libriform outside vascular bundle,ray cell,phloem cells and lateral veins vascular bundle area column contains more calcium oxalate crystal or Fang Jing;the two leaf sides are of different planes,palisade tissue and spongy tissue differentiation is obvious;(2)Surface view:the leaf surface is smooth,the porosity distribution in the leaf lower epidermis,the main is flat shaft type and infinitive;There are many yellow non glandular hairs;the lower epidermis stomatal index is 15.2%?17.6%,pulse island is 7?10/mm2(3)powder:visible many fibers and fiber,1ome branch shaped crystal fibers;Calcium oxalate column crystal and Fang Jing exists in parenchyma cells,the more visible isolated in bast fibers and wood fibers;Non-glandular hair is composed of two cells,short cells near the head of the larger cell color is deeper,wall wart is obvious;Catheter main screw line.2 different polar parts pharmacodynamic study of Dalbergia sissoo leafThe Dalbergia sissoo leaves was extracted by 70%ethanol,extraction was pro cessed by petroleum ether,chloroform,ethyl acetate,n-butyl alcohol,therefore get four parts extractions.On anticoagulant,anti-inflammatory and analgesic three i ndicators,jacaranda leaves different polar parts of India for pharmacodynamics rese arch.Anticoagulant test dose group compared with normal saline control group befo re and after dosing coagulation time for t test,n-butyl alcohol and petroleum ether group of P values were less than 0.05,has a significant meaning.Anti-inflammator y experiments,dosage group compared with normal saline control group before and after dosing t test mice ear swelling inhibition rate,which the P value is less tha n 0.05,chloroform group has a significant meaning;Ethyl acetate group P value is less than 0.01,there is a very significant meaning.Analgesic experiments,dosag e group compared with normal saline control group before and after the dosage in mice body torsion times t test,P values each dosage group were less than 0.05,significant meaning;Ethyl acetate group P value is less than 0.01,there is a very significant meaning.3 quality evaluation on Dalbergia sissoo.leaf3.1 chemical constutents research of sissoo.leafDalbergia Sissoo Leaves contains amino acids,proteins,organic acids,phenols,sugars,triterpene,flavonoids,volatile compounds,etc.Six compounds were isolated,identified from Sissoo.leaves,they are:Bioch anin A?1,3-Dihydroxy-6-methoxy-9H-xanthen-9-one,Biochenin A 7-0-[?-D-apiofurano syl-(1?6)-p-D-glucopyranoside]?Biochenin A 7-0-[?-Dapiofuranosyl-(1?5)-?-D-apiof uranosyl-(1?6)-?-D-glucopyranoside]?Quercetin 3-O-?-D-glucopyranoside?Quercetin-3-O-rutinoside.3.2.To set up Dalbergia Sissoo Leaves total isoflavones content determination method and optimize the extraction processDalbergia Sissoo leaf flavonoids was optimized by using orthogonal design meth od extraction process,determine the best process as follows:70%ethanol extraction,extraction time for 30 min,solid-liquid ratio 1:200,extraction times as 2 times;Us ing the ultraviolet spectrophotometric method,obtained the best flavonoids extraction process under India cocobolo total isoflavones content in leaf was 30.041 ±0.28mg/g,RSD was 0.93%.3.3 Dalbergia Sissoo Leaf thin layer identificationWith element of Biochanin A etc.as the reference substance,under 365nm UV lamp,in the chromatogram of the test,on the reference substance chromatography c orresponding position,it has the same color spots.3.4 Content determination of Biochanin A in different parts of Dalbergia Sissoo.Biochanin A respectively in the concentration of 0.017?502?g had good linear relationship within A(r = 0.9998),the average sample recovery rate was 99.02%.The content of Biochanin A comparison in each part of Dalbergia Sissoo.:Sissoo.flower>heartwood>leaf>fruit?sapwood>bark,content of Biochanin A in bark is low,sometimes hard to check out.Biochanin A content in flower is much higher than in leaf,and the comprehensive exploitation and utilization of the leaf,also can consider to undertake the development and utilization,strengthen Dalbergia Sissoo.resources sustainable utilization.3.5 Dalbergia Sissoo.Leave fingerprintAfter similarity software analysis determine the characteristics of 15 common peak peak,form Dalbergia Sissoo.leaves medicinal materials HPLC fingerprint,there are two characteristic peaks were confirmed,13 peaks is 6-OH-Biochanin A,15 peaks for Biochanin A.The similarity of 9 batches sample out of 19 of Sissoo.leaf fingerprint and contrast fingerprint above 0.90,five batches in 0.80 above,the similarity of the rest of the samples are under 0.80.3.6 Quality of Dalbergia Sissoo.leaves in different regions,different monthIn different habitats and different leaf,six of the main ingredients in the discovery of guangzhou university of Chinese medicine YaoWangShan Sissoo.leaf quality is better than that of NanYao test center in zhanjiang and HuaZhou;In different leaves of several main ingredients change has certain regularity.Dalbergia Sissoo.fingerprint similarity is low,at different positions of the heart wood components than other parts of the difference is bigger,heartwood of chroma tographic peak basic is concentrated in the 15?22 min,and leaves,twigs,flowers,fruit and other parts of the chromatographic peak distribution is relatively homogene.ous,and from its uv absorption spectra of fingerprint chromatogram peak can be se en that differences in composition with other parts of the heartwood.When choosin g common peak also found heartwood and other parts of the common peak is less,only Biochanin A relatively obvious.After eliminating heartwood,compared to othe r parts,the results found that fruit similarity is low,other parts of the similarity degree is higher.ConclusionThe morphological features and epidermis,transverse section and powder microscopic characteristics,can effectively to make pharmacognosy identification of Dalbergia Sissoo.leaf.The four parts of Dalbergia Sissoo.leaf:petroleum ether,chloroform,ethyl acetate,n-butyl alcohol all have anticoagulant,anti-inflammatory and analgesic effect,the main parts of the anticoagulant effect is a place of n-butyl alcohol and petroleum ether part,anti-inflammatory efficacy of ethyl acetate and chloroform part is the main part,the main parts of the analgesic efficacy is ethyl acetate,n-butyl alcohol and chloroform,the results are statistically significant,especially ethyl acetate and n-butyl alcohol has the extremely significant significance,which is the key parts of leaf in the follow-up development.The ethyl acetate and n-butanol part of Dalbergia Sissoo.leaf are mainly contain flavonoids ingredients,such as Biochanin A isoflavone class is given priority to,has been separated in the six compounds,there are three classes for isoflavones,two flavonoids,one anthracene ketone derivative;The total flavonoids under the best extraction technology of in the leaf measured Sissoo.leaf total isoflavones content in leaf was 30.041±0.28mg/g,suggest Sissoo.leaf total content of flavonoids in the leaves is high level,the extraction technology is simple,stable,in favour of the comprehensive exploitation and utilization of Sissoo.leaves.the determination of Biochanin A from different parts of the Sissoo.by UPLC method which has the characteristics of rapid,stable,the results show that Biochanin A,takes up the highest content of flower,the content in the leaves is close to the heartwood,so the development potential is bigger.Combined with the shape and microscopic identification,thin layer identification,content determination and fingerprint of modern analysis methods,the Sissoo.leaf quality evaluation systemcan be well established. |
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