| ObjectiveTo establish a quality standard of Herba Gendrussae,which can be a scientific critera for identifing the authenticity of Herba Gendrussae slices and its merits of the quality,even provided a safe,effective,stable,uniform and controlled clinical medicine.The common style of Infrared identification method and infrared spectrum was building,which will facilitate foundation of fingerprint chromatogram database and bring a novel method for further investigating the HPLC fingerprint chromatogram of Herba Gendrussae in quality control and evaluation.Methods1 Accordance to National drug quality control methods,identifying the appearance,powder microstructure and TLC of Herba Gendrussae slice.2 Impurity,Water,Total Ash and Acid-insoluble Ash.(See ChP appendixIX H,? K,2010 edition)3 Extractives(See ChP appendix X A,X D,2010 edition).Ultraviolet spectrum(See ChP appendix V A,2010 edition)was used to measure the total content of flavonoids in the decoction pieces,standard solution is prepared by rutin CRS.4 Applying the infrared spectrum to make scanning in the range of 4000?400cm-1 of Herba Gendrussae,using SPSS17.0 to make clustering analysis for all samples which aim to build an infrared spectrum of identifying methods of Herba Gendrussae slice.5 Fingerprint chromatograph of Herba Gendrussae slice was established by HPLC.Result1 The quality of Herba Gendrussae1.1 AppearanceHerba Gendrussae was short cylindrical irregular segments.Some of them were single seeds,which were between 40cm to 90cm in height and about 3mm in diameter.They appeared green-yellow,light green or yellowish-brown on surface with yellow holes.The branches of them were many in number,resembling four ridges and touched with purple.The shorter branch was 2cm to 6cm in height and the irregular leaves were some 2cm in thickness,fragile,broke easily and their segments looked yellow-white and fibroid and had pith in the holes.The leaves looked to each other with small stems.They were as thick as a piece of paper and fragile.When stretched thoroughly,they displayed the shape of Lanceolate or Strip lanceolate with the length of 4cm to 14cm and the width of 1cm to 2cm.Their original point was pointed and the basins were wedged-shaped.Their leaves were light green or green-brown on surface while the back looked sage green with leaf veins were touched with purple.Give off wild aroma,a bit bitter and spicy.1.2 Microscopic identification of decoction powderYellowish-green to yellowish-brown.Numerous stone cells singly scattered or several connected regularly,brownish-yellow,bright yellow or greenish-yellow,sub-rounded,sub-triangular,rectangular or sub-square,20-80 ?m in diameter,20-160 ?m long,2-10 ?m thick,striation well-marked,some containing yellow substance in lumen;some sub-rounded or sub-square with relatively thin-walled,60 ?m in diameter,pits extremely dense and relatively large.Glandular singly scattered,scales with a 4-celled spherical head,60-90 ?m in diameter,stalk unicellular,short,7-16 ?m in diameter,some containing yellow contents.Stomata anisocytic,subsidiary cells 2-3.Parenchymatous cells containing prisms of calcium oxalate.Non-glandular hairs 2-4 celled,straight or curved at the apex,up to about 300 ?m long.Vessels mainly bordered pitted,with some reticulate,scalar form and spiral ones,20-50 ?m in diameter,bordered pits elliptical,arranged tightly.1.3 TLC identificationTake 2 g of the powder,add 50 mL of ethanol to it,ultra-sonicate the solution for 30 minutes,filter,evaporate the filtrate to dryness,dissolve the residue in 1 mL of ethanol as the test solution.The same method was used to make contrasting medicinal solution.Carry out the method for thin layer chromatography(Appendix VI B),using silica gel G as the coating substance and a mixture of petroleum ether(60-90?)and ethyl acetate(8:2)as the mobile phase.Apply separately to the plate 5 u L of each of the above two solutions.After developing and removal of the plate,dry in air,spray with a 10%solution of sulfuric acid in ethanol and heat at 105? to the extent that the spots appeared.The spots in the chromatogram obtained with the test solution,which corresponding display the same colors as that of the spots in the chromatogram obtained with the reference drug solution.1.4 Water,total ash,acid-insoluble ashWater(10 test samples)was as high as 11.96%,the lowest 9.04%,the average 10.76%,and the difference between the highest and lowest water content was 2.92%;Total ash was up to 16.70%,the lowest 4.72%,the average 9.23%,the highest and lowest total ash is 11.98%.Acid insoluble ash was as high as 1.31%,the lowest 0,32%,the average 0.57%.The difference of the highest and lowest acid insoluble ash component was 0.99%.1.5 Water-soluble extract results10 test samples of water-soluble extract(hot dip method),the highest content was up to 27.41%,the lowest 0.64%,the average 17.90%,the highest and lowest amount of water-soluble extract was 16.77%.1.6 Flavonoids content determinationThe highest total content of flavonoids(12 samples)in the test was 7.60 mg/g,the minimum was 2.72 mg/g,with a mean of 4.67 mg/g,the maximum and the minimum amount of water-soluble extract was from 4.88 mg/g.2 Infrared spectrumTo gain infrared spectrum of 12 batches samples,we prepared sample tablets,which scanned in the range of 4000?400cm-1,KBr tablet as a background,deducted interference of H2O and CO2.By analysing infrared spectru,we preliminary build a database of Herba Gendrussae,confirming and classifying 10 shared peaks,3400 cm-1?2924 cm-1?1642 cm-1?1426 cm-1?1378 cm-1?1324 cm-?1242 cm-1?1152 cm-1?1051 cm-1?769 cm-1,separately.Meanwhile,10 batches of infrared spectrum were under clustering analysis after they were divided into 5 categories,sample 3,8,1,7,4,5,9,Sample 11,12,Sample2,Sample 10 and Sample 6,separately.3 HPLC fingerprint studyVolume was ODS-18 packed,Mobile phase was methanol?0.3%glacial acetic acid,gradient elution,detecting wavelength was 245nm.Assay(See ChP Appendix VI D),internal standard was Quercetin.12 batches samples of Herba Gendrussae fingerprint chromatogram were matched with Chromatographic peak,getting 11 common peaks and 12 batches sample slices of HPLC fingerprint peak appearance similarity being high.The appearance of the data analysis revealed 11 common peaks in chromatographic peak area,accounting for more than 90%of total peak area;samples show stability in 24 hours.Initially established HPLC fingerprint spectrum method of Herba Gendrussae slice,12 samples have different similarity,0.915?0.913?0.984?0.988?0.981?0.521?0.789?0.738?0.937?0.945?0.957?0.926,separately.Except sample of 6,7,8 were less than 80%in similarity,other samples were more than 90%in similarity.We have already confirmed 11 shared peak,highly similarity in the shape of peak.Conclusion1 Establishing a quality control method for Appearance,Microscopic identification of decoction powder and TLC identification of Herba Gendrussae slice.2 According to experimental results,a preliminary quality standards of Herba Gendrussae slice was:water should not more than 13.0%;total ash should not more than 11.0%;Acid-insoluble ash should not more than 2.0%;Water-soluble extract should not less than 8.0%;Calculated on dry product,total flavonoids content rutin(C27H30O18)should not less than 0.25%.3 A database of infrared spectrum of Herba Gendrussae has been originally built up,which consists of 10 shared peaks.The approach to identifying Herba Gendrussae proved to be practical.Through infrared spectrum,Herba Gendrussae can be classified into five categories and most of the circulating products in the market belonging to the first category.4 A database of HPLC fingerprint has been originally build up:the measured samples similarity is generally greater than 0.90,there were 11 common peaks,benefitting the producing of Herba Gendrussae to develop in the direction of modernization,standardization and internationalization. |
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