Study On The Quality Of The Introducted Dalbergia Sissoo Roxb. In China

ObjectiveDalbergia sissoo Roxb.is the plant belongs to Leguminosae and Dalbergia.,which is the evergreen broad-leaved speciesan that native in subtropical and tropical country such as Indian.The heartwood was used as Chinese herbal medicines,it was named fanjiang.It was introduced to china dozens of years ago and the amount has increased rapidly recent years.But the study of its medicinal properties is not enough.The effective part and effective ingredient are not clear,the study on the pharmacological of the main ingredient is few and the quality standard is blank.So,it is hard to evaluat the quality of the drug scientifically.Therefore,it is necessary to study the chemical constituents,pharmacological and fingerprint of Dalbergia Roxb systematically,so as to provide a scientific basis for the modernization and standardization quality standards of Dalbergia sissoo Roxb.Methods1.By heating and refluning Dalbergia sissoo Roxb.powder with 85%ethanol to get the ethanol extractive.Get the concentration.The petroleum ether part,chloroform part,ethyl acetate part and n-butanol part can be obtained through extraction by different solvents with different polarity.These different extractions were purified by the methods of chromatography,extraction,solvent,precipitation,crystallization and so on.So as to get the monomeric compounds.Identify compound structures by the method of physical and chemical and spectral(including IR,MS,UV,NMR,etc.).Analysis the chemical composition of ether extract by GC-MS.2.We got 13 samples from Guangdong Zhanjiang,Guangdong Fengshun,Guangdong Taishan,Guangdong Guangzhou,Guangdong Jiangmen and Yunnan province,etc.Refer to the character,microscopic and chromogenic method of lignum dalbergiae odoriferae in Chinese Pharmacopoeia(2015 Edition,general rule 2001 and general rule 0502)to test the thin layer chromatography(TLC)behaver of Dalbergia sissoo Roxb.to identify it.Refer to the method of general rule 2201 in Chinese Pharmacopoeia(2015 Edition)to test the content of alcohol extract of Dalbergia sissoo Roxb.Test the content of ether extract.Set dalbergin and other five monomeric compounds as reference to test the quality concentration of monomeric compounds in Dalbergia sissoo Roxb.3.Build the HPLC fingerprint of Dalbergia sissoo Roxb samples by HPLC;Analysis the ether extract by GC-MS to find out the main components and build the GC-MS fingerprint of Dalbergia sissoo Roxb.4.Measure the content of Pb,Cr,Cd,Hg,As,Cu,Fe,Zn,Mn inorganic elements in the 13 samples of Dalbergia sissoo Roxb by atomic absorption spectrometry.5.Test the DPPH radical scavenging ability of the seven compounds so as to find out the antioxidant activity;Test the inhibition to cell proliferation of U-87 MG to test the antitumor activity of seven compounds in Dalbergia sissoo Roxb.Results1.The compound I was extracte from the petroleum ether part.It was identified as Dalbergiphenol.The compounds ?,? and ? were separated from the chloroform part,they were identified as 4-Methoxydalbergione,0-Methyldalbergin and Dalbergin.The compounds?,? and ? were separated from the ethyl acetate part,they were identified as 7-0-Methylplathymenin,Plathymenin and Biochanin A.Twenty three components of Dalbergia sissoo Roxb were identified by GC-MS.The main contents are DIBP,2(1H)-Naphthalenone,octahydro-4a-phenyl-,trans-,Dibenz[a,c]cycloheptane,1,2,9-trimethoxy-,Dalbergiphenol,1H-Pyrrolo[2,3-f]quinolin-9-ol,1,2,3,5,7-pentamethyl-,Coumarine 6-methoxy-3-(4-methoxyphenyl)2H-1-benzopyran-2-one,1,7,7-Trimethyl-3-phenethylidenebicyclo[2.2.1]heptan-2-one,7-hydroxy-3-(4-methoxyphenyl)-.Dalbergiphenol is the highest among them.Except sample 7 contains 42.53%,the other samples contains over 53.10%.2.The plant characteristics of Dalbergia sissoo Roxb as follows:Arbor,evergreen,the high is 10?20 m;Often have root nodules;Stems are erect,bark gray,rough,thick and deep cleft.Leaves bipinnate,obovate,3-5 cm long;Stipules lanceolate.Racemes;Flowers are papilionaceous,bisexual and zygomorphous.The corollas have long stalk and the color is pale white or yellow.Calyx canister shape.Keel valve and ala is oblanceolate,base attenuate.10 Stamens are diadelphous.Carpels are monocarpellary with ovary superior and single locule,three ovules to six ovules contains in it.The pods are linear or oblong to bandlike,it has seeds 12(-3),the shape is flat,Kidney-shaped and exalbuminous.Florescence is March to May.The fruiting season is October to December.The heartwood of Dalbergia sissoo Roxb is cylindrical or irregular lumps.The color of surface is yellow-brown or reddish-brown.There are brown and yellow stripes,texture is hard with less oily.It smells micro-incense and taste slightly sour.Heavy or suspended in water.Black oil will escape when it is burned,and it will be more fragrant.The ash is white.The microscopic characteristics of Dalbergia sissoo Roxb as follows:There are more parenchyma cells,irregular polygon,slight lignified cell wall,some contains calcium oxalate prismatic crystal.The wide of wood-ray cells is 1?2 lines.The diameter of the catheter is 180 ?m to 200 ?m,it often contains brown lumps,and they are bordered pits.Libriform fiber pit is not obvious.The powder of Dalbergia sissoo Roxb is brown,fiber bundles,its colorless or yellowish brown,the diameter is 10 to 24 ?m,the cell wall is thick;Fragments of bordered pit have different size,the complete with diameter of about 180 ?m and its often contains brown substance;The parenchyma cell is irregular polygon,often contains brown substance,some contains calcium oxalate prismatic crystal.The main differences of microscopic characteristics between Dalbergia sissoo Roxb and Dalbergia odorifera T.Chen as follows:Crystal fibers in Dalbergia odorifera T.Chen and the fibers often contains brown material.The fibers of Dalbergia sissoo Roxb have no crystal fibers and the fibers have no brown material.The TLC of alcohol extract of Dalbergia sissoo Roxb shows 2 spots when observed under UV lamp(365nm),which is a distinctive identification.The content of alcohol extraction of the 13 samples is betweenl.28%to 11.122%.The alcohol content are more than 10%are samples S1,S3,S4,S7,S8,S9.The essential oil is between 0.78%to 3.44%.The mass fraction of Dalbergiphenol in the wood of Dalbergia sissoo Roxb samples is between 0.56%to 2.63%,the mass fraction of 4-Methoxydalbergione is between 0.05%to 1.24%,the mass fraction of 0-Methyldalbergin is between 0.06%to 0.35%,the mass fraction of Dalbergin is between 0.76%to 2.35%,the mass fraction of 7-0-Methylplathymenin is between 0.07%to 0.18%,the mass fraction of Plathymenin is between 0.04%to 0.91%.The similarity of 10 samples of HPLC fingerprint is greater than 0.8 among the 13 samples,the similarity of the rest three samples between 0.136 and 0.776.The fingerprint of thirteen samples peaks appear that the relative standard deviation of all relative retention time is less than 3%,the relative standard deviation of relative peak area of fingerprint are different.Chromatographic system established in HPLC can separate chromatographic peaks very well in Dalbergia sissoo Roxb and the selected samples common peaks are stable,common peaks area in the total area of chromatographic peaks is about 90%.GC-MS fingerprint signed eight common peaks as Dalbergia sissoo Roxb fingerprint characteristic peaks,with NIST1998 standard MS spectrum library matching and combined with relevant literature,we determined the chemical composition of 8 common peaks.With the peaks area normalization method to measure the relative contents of chemical composition combined accounted from 68.106%to 89.919%of the total ether extract.Use the similarity evaluation software;The 13 samples of Dalbergia sissoo Roxb ether extract fingerprint have higher similarity.The similarity is above 0.9000 except the S13 sample.3.Atomic absorption spectrometry is used for testing the content of lead(Pb),mercury(Hg),copper(Cu)and chromium(Cr),iron(Fe)and manganese(Mn),cadmium(Cd),arsenic(As),zinc(zinc)in thirteen samples.The test results show that the content of lead is from 0 to 0.02 mg/kg,cadmium content is from 0.01 to 0.09 mg/kg,arsenic content is from 0.00 to 0.15 mg/kg,mercury content is from 0.00 to 0.08 mg/kg,copper content is from 6.68 to 25.72 mg/kg,chromium content is from 0.49 to 13.96 mg/kg,iron content is from 30.90 to 177.29 mg/kg,the content of manganese is from 0.00 to 16.43 mg/kg,the content of zinc t is from 0.00 to 22.03 mg/kg.Among the nine elements,the ranking of average content is:iron,zinc,the lowest average level is lead.As time by the elements and heavy metals content increased in tree.But the heavy metals are not more than the Chinese pharmacopoeia restrictions for lead,cadmium,arsenic and mercury content.4.The 7 components were obtained from Dalbergia sissoo Roxb all show some effectivity on removal of DPPH free radical and the effectivity increased when the concentration increased.7-O-Methylplathymenin,Plathymenin and Biochanin A effectivity most and the IC50 value of them are concentrate upon 20 ?g/ml;Dalbergiphenol,4-Methoxydalbergione,0-Methyldalbergin and Dalbergin effectivity weakest and the IC50 alue concentrate upon 38 to 58 ?g/ml.The seven monomer compounds show inhibitory effectt to the cells of U-87 MG.4-Methoxydalbergione effectivity most,even with low concentration(1.15 jig/ml),it also shows tremendous inhibitory effect.The inhibition enhanced gradually when concentration increased from low(6.25 ?g/ml)to high(100 ?g/ml),IC50 alue concentration between 1.15 and 64.81 ?g/ml.Conclusion1.All of the seven compounds that separated from the wood of Dalbergia sissoo Roxb are flavonoid.There are two neoflavonoid,two chalcone,two flavanones and one isoflavone.The main Five compounds Dalbergiphenol,4-Methoxydalbergione,0-Methyldalbergin,Dalbergin ang Plathymenin contains about 90%of total composition of Dalbergia sissoo Roxb in HPLC.The main constituent of ether extract in Dalbergia sissoo Roxb is Dalbergiphenol,it contains over 50%in Dalbergia sissoo Roxb.It can be the index to control the quality of Dalbergia sissoo Roxb.2.Dalbergia sissoo Roxb has the obvious traits on the plants.The main traits of the plant,microscopic traits and the Thin-Layer Chromatography behavior can be used to identify Dalbergia sissoo Roxb.The main Five compounds Dalbergiphenol,4-Methoxydalbergione,0-Methyldalbergin,Dalbergin ang Plathymenin contains about 90%of totalcomposition of Dalbergia sissoo Roxb in HPLC.The method is reliable.The method of HPLC finger-print and GC-MS finger-print are easy and stable.They can be used as the index to control the quality of Dalbergia sissoo Roxb.The elements and heavy metals content won't increased more then the restrictions in Dalbergia sissoo Roxb.It's safe as drugs.3.The 7 flavonoid compounds show some effectivity on removal of DPPH free radical and the effectivity increased when the concentration increased.7-0-Methylplathymenin,Plathymenin and Biochanin A effectivity stronger.4-Methoxydalbergione shows the most powerful inhibitory effectt to the cells of U-87 MG.effectivity even with low concentration(1.15 ?g/ml).