Traditional Chinese Medicine Of SSP Quality And The Material Basis Research Of The Anti Osteoporosis

Objective: Previous studies have demonstrated that the SSP has obviousanti osteoporosis, and confirm the total flavonoids from semen sojaepraeparatum for effective part of osteoporosis, but it is not clear the specificrole of the effective components. This study adopts ultraviolet-visiblespectrophotometry （UV） determination of semen sojae praeparatum totalflavonoids content and characters of observation and comparison, contentdetermination of total flavonoids in semen sojae praeparatum, guarantee thequality of SSP; at the same time, in our own laboratory extraction of effectiveisolation of semen sojae praeparatum points, were identified by using thetechnique of HPLC-ESI-MS/MS its, make its independent effects on ratosteoblasts in vitro, from the cellular level to observe its effect on proliferationof osteoblast function and interaction, to further clarify the material basisMethod:1the quality of semen sojae praeparatum1.1The TLC identification conditionUsing chloroform-methanol-water （9:2:1）below10℃placed lowersolution as the agent, on silica gel GF254thin layer plate, spread out to dry,under the UV lamp （254nm） review.1.1.1Identification of semen sojae Preparatum accessories, the mulberryleaves of Artemisia annua.1.1.1.1Tin conditionsOn silica gel G thin layer plate, toluene-ethyl formate and formic acid（5:4:1） as developing agent, expansion, removed, dried, the UV lamp （365nm）under review.1.2Dfferent areas （containing home-made SSP） observation and UV methodfor the determination of total flavonoid content in the. 1.2.1Dfferent of origin of fermented soybean traits were observed, includingthe surface color, Pili color and so on.1.2.2UV method for the determination of conditions of total flavonoidscontentUsing genistein as control, sample blank solution with0.01mol/L NaOHaqueous solution, at261nm measured absorbance, and draw standard curve.1.3Determination of semen sojae praeparatum daidzin, daidzein, glycitin,glycitein, genistein and genistin content by HPLC method1.3.1Cromatographic conditionHPLC gradient elution gradient:0-10min,13%A,87%B;10-40min,25%A,75%B:40-55min,30%A,70%B;55-65min,80%A,20%B. Flow rate is1mL/min; the detection wavelength was261nm, column temperature was atroom temperature, the injection volume was25μL.1.4Use Liquid chromatography-mass spectrometry technology HPLC-ESI-MS and identification of flavonoids in semen sojae Preparatum1.4.1Cromatographic conditionHPLC linear elution gradient for:0-10min,13%A,87%B;10-40min,25%A,75%B:40-55min,30%A,70%B;55-80min,80%A,20%B. Flow of150μL/min; column temperature was at room temperature. the injectionvolume was0.3μL.1.4.2Mass spectrometry conditionsThe positive,negative ion detection mode.Spray voltage4.0-5KV;capillary voltage of+15or-20; capillary temperature300℃; sheath gas flowrate:40ARB; the auxiliary gas flow rate:50ARB; tube lens compensationvoltage:+18or-40; a mass scan range of m/z300-1500; collision energy:30%-40%.1.5Study on the chemical ingredients in SSP1.5.1The preparation method of SSP20kg black beanswashed and dried. Artemisia （10kg） and annua leaves（10kg）, boiled three times. And place the soft bean into the oven at37℃for7days. 1.5.2Extraction of active componentsSSP （20kg） degreased by petroleum, extracted by ethanol reflux （75%）,supernatant was collected, concentrated.1.5.3Separation of active components of SSP1.5.3.1Crude parts of active ingredients in SSPThe extract obtained after extraction with ethyl acetate extraction,"theethyl acetate layer", n-butanol, supernatant was collected, concentrated,extract markers obtained concrete.1.5.3.1.1Crude parts of ethyl acetateThe ethyl acetate layer by dry weight, with petroleum ether and ethyl acetatedissolved and silica gel with samples, respectively with petroleum ether,petroleum ether and ethyl acetate in different proportions, ethyl acetate, ethylacetate and methanol, methanol elution ratio, but with the filtrate,concentrating, too extract, and mark.1.5.3.1.2Crude parts of n-butanolThe n-butanol fraction drying and weighing, dissolving, filtering, andslag, stay clear filtrate, macroporous resin column, and washed with ethanol ofdifferent concentrations, respectively, with effluent, concentration, theremaining slag.1.5.3.2Ethyl acetate layer segmentationThe above is the ethyl acetate part, respectively, by silica gel, MCI, andreversed phase C-18column, and were separated with thin layer technology.1.5.4Identification of active components structure in SSPAll kinds of monomers and flavonoids obtained above controlproducts,application NMR technology,to identify the monomer structure.2Effects of activate components in SSP on osteoblast proliferation2.1Culture and identification of rat calvarial osteoblasts （OB） primary cellAccording to the modified method of tissue block, isolated from neonatal（24h） SD rat calvarial osteoblasts.Identification of osteoblasts by alkalinephosphatase staining, cells were observed under inverted microscope.2.2Effects of activate components on OB cell proliferation in vitro by MTT methodSeparation will have to determine the different monomers of sampleconcentration gradient is set to （10-5,10-6,10-7,10-8,10-9mol/L）, reaction timewas24h; survival rate was determined by MTT method in the hole, the ODrepresentation.Result:1Thin layer identification resultsThrough TLC identification can be seen, in the chromatography of the test,and reference substance chromatography corresponding position, show thesame color of the main spots, spots were clear, the Rf values conform to therequirements. Through TLC identification, with mulberry leaf and artemisinincontrast medicinal materials as reference, using the same piece of thin layerplate the same developing solvent system examining method for identificationat the same time, in the chromatogram of the test, the chromatographiccorresponding to the position, show the same color of the main spots, spotswere clear, separation effect is ideal.2UV determination flavonoids and SSPOne of the highest levels of Sndong JuanCheng （2011） samples was5.14%,the lowest for yangjiang in Gangdong province （2011）,0.911%,4times.3HPLC of daidzin, daidzein, glycitin, glycitein, genistein, genistin sixcompounds content determination results3.1Survey of methodology3.1.1Linear relationshipWith reference substance concentration （including g/ml）（X） as theabscissa, integral value of peak area （Y） as the ordinate, draw standard curve.3.1.2Precision of test resultsDaidzin, daidzein, glycitin, glycitein, genistein, genistin six referencesubstance content of RSD value was0.572%,0.829%,0.498%，0.572%,0.773%,0.557%showed that instrument precision is good.3.1.3The stability test resultsIn the detection of48hours daidzin, daidzein, glycitin, glycitein, genistein, genistin content of the reference substance solution stability, RSD value was0.562%,1.002%,0.636%,0.562%,1.194%,0.551%, the stability is good.3.1.4Repeatability test resultsBy6times the determination results of daidzin, daidzein, glycitin, glycitein,genistein, genistin content determination, the RSD were1.553%,2.198%,2.158%,1.838%,1.731%,1.759%, that method with good reproducibility.3.1.5Recovery test resultsAdd sample recovery experiment showed that daidzin, daidzein, glycitin,glycitein, genistein, genistin the average recovery of genistein glycosides were100.620%,100.210%,100.510%,100.210%,98.599%,98.953%, RSD were0.727%,2.243%,0.944%,0.727%,1.376%,2.287%（n=9）, the method isaccurate and reliable.4From HPLC/ESI/MS/MS total ion flow chart and HPLC showed that mostof the components from the raw black beans, by positive and negative ionsfragment ion information, identify one from flavonoids Atalantoflavone ofmulberry leaves, and2from Qinghao of artemisinin and celery flavonoid.Other flavonoids from black beans, respectively daidzein, genistein, glycitein3kinds of flavonoids and its composition of glycosides.5chemical composition research of SSP5.1Identification of components in SSP11compounds from the extraction and separation of SSP of the CommunistParty of China, under the joint action of NMR and LC-MS technology,and toidentify the structure of, respectively, syringic acid （I）, daidzin（II）, daidzein（Ⅲ）, glycitin（IV）, glycitein（V）, genistein（VI）, genistin（VII）, apigenin（VIII）,β-sitosterol（IX）, campesterol （x）, stigmasterol （XI）.5.2Effects of chemical composition in SSP on bone cell proliferation5.2.1Morphological observation and identification of osteoblastPrimary cultures from24h to36h, inverted phase contrast microscope,visible from the skull fragment climb out of osteoblast, adherent growth,radially short fusiform or triangle, cells arranged no direction.Passage cellinitial inoculation was spherical, suspended in culture medium,24h observed in most cells attached to the wall, stretch; after3d incubation, OB shapebasically stable; culture about5d cells were cobblestone-like.Alkaline phosphatase staining for alkaline phosphatase staining: after theexperiment, observation of the cytoplasm in purple blue precipitate under amicroscope, a typical ALP positive reaction.Cells staining positive cells ofdifferent depths, were long fusiform and scaly, into bone cells alkalinephosphatase staining positive rate of up to93%.5.2.2Effect of SSP chemical composition on the proliferation ofosteoblasts5.2.2.1daidzein,daidzin,genistein,genistin,glycitein on10^-6,10^-7,10^-8,10^-9mol/L MTT showed showed a positive correlation between the drug groupand the concentration, at10^-5mol/L,showed the cell toxicity.Conclusion:1UV method was applied to from14different producing area and theflavonoids content determination of SSP, the result shows that differentregions main composition content of SSP difference is very big.2Extraction and separation in the SSP11compounds, after structureidentification and fluid composition analysis, can determine its respectivelyDaidzin, daidzein, glycitin, daidzein, genistein and genistin clove acid, celery,and plant sterols, and of the Daidzin, daidzein, glycitin, daidzein, genisteinand genistin six compounds using HPLC and UV methods for contentdetermination.3Daidzein, daidzin, genistin, genistein, glycitein in10^-5、10^-6、10^-7、10^-8、10^-9mol/L, proliferation activity and the concentration of daidzein, daidzin,genistin, genistein, glycitein each drug group,10^-9,10^-8,10^-7,10^-6, mol/Lwith the increase of concentration, but the cell proliferation in10^-5mol/Lfound cell toxicity, some tips: the flavonoids extraction in SSP, has obviouscell proliferation, semen sojae praeparatum has strong anti osteoporosisactivity.