| Objective:Codonopsis Radix is a traditional Chinese medicine commonly used for tonifying qi,strengthening spleen and lungs,and mostly used in the formula of prescriptions and clinical treatment in the form of decoction pieces.Dividing specifications and grades of traditional Chinese medicine decoction pieces is an important means to facilitate market transactions.At present,the standard classification of Codonopsis Radix decoction pieces in the market is not uniform,which results in the problems of chaotic market circulation and uneven quality of Codonopsis Radix decoction pieces.In this paper,the grade study on Codonopsis Radix decoction pieces is performed to establish the grade evaluation standards for Codonopsis Radix decoction pieces.Through the quality comparison of Codonopsis Radix decoction pieces of different grades,the grading quality standards of Codonopsis Radix decoction pieces were established.The effects of processing methods on the content of effective ingredients in Codonopsis Radix decoction pieces were determined by measuring the contents of various ingredients in Codonopsis Radix products and processed products.Methods:1.According to the 2015 Chinese Pharmacopoeia and the national and local processing standards,Codonopsis Radix decoction pieces include Dangshen tablets and rice-fried Dangshen.Basing on the "Specification Standards for 76 Kinds of Medicinal Materials" and " Standards of Specification Grades for Electronic Transaction of 200 Kinds of Chinese Medicinal Materials" and "Traditional Chinese Medicine Commodity",the main production areas of Dangshen such as Shanxi and Gansu,and the Chinese medicinal materials markets including Hebei Anguo,Anhui Bozhou and Haerbin Sankeshu et al.are investigated,and the samples of Codonopsis Radix decoction pieces from different origins and grades are collected to be used for experiments.Quantitative indicators such as diameter,thickness,quality,impurity content,and mildew of Codonopsis Radix decoction pieces were measured,and various statistical methods such as variability analysis,correlation analysis,and one-way analysis of variance were used to analyze the grade of Codonopsis Radix decoction pieces.The correlation between the appearance traits index and the commodity grade were established.2.According to the 2015 Chinese Pharmacopoeia,the collected Codonopsis Radix decoction pieces(Dangshen tablets and rice fried Dangshen)were identified by microscopy and thin layer chromatography.The routine inspections of Codonopsis slices including moisture,total ash,and alcohol-soluble extracts were performed according to the 2015 Chinese Pharmacopoeia(Part One)appendix and the fourth chapter.The correlation analysis and one-way analysis of variance were used to compare the water,ash,and extracts of Codonopsis tablets of different grades,and the differences in water content,total ash,and extracts.Then the standards of water limit,total ash limit,and limits of extracts of Dangshen tablets and rice fried Dangshen were drawn up.Based on the previous researches,UV-visible spectrophotometry was used to determine the polysaccharide content of Codonopsis Radix decoction pieces at 490 nm.Correlation analysis and one-way analysis of variance were used to compare the polysaccharide content of deferent grades of Dangshen tablets,Dangshen tablets and rice fried Dangshen.The polysaccharide content limits of Dangshen tablets and rice fried Dangshen were drawn up respectively,and the quantitative research method of Codonopsis Radix decoction pieces was established.Based on the previous researches HPLC method was used to study the characteristic map of Codonopsis Radix decoction pieces,and methodological investigations were performed,including stability,precision,and reproducibility.The HPLC characteristics of Codonopsis tablets and rice fried Dangshen were established and characterized.The HPLC characteristics of Codonopsis Radix decoction pieces of different grades,different origins and different processed products were compared,and the quality standards of Codonopsis Radix decoction pieces were established.3.According to HPLC characterization and literature research of Codonopsis Radix decoction pieces,effective ingredients including syringin,tangshenoside I,lobetyolin and atractylenolide III were selected and their contents were determined by HPLC.Correlation analysis and one-way analysis of variance were used to statistically analyze the measurement results,and the effects of the processing on the content of effective ingredients in Codonopsis Radix were studied.Results:1.Basing on the data from literatures and market researches,Codonopsis Radix decoction pieces grades were as follows:(1)Divided into multiple specifications and grades by taking diameter,thickness,and whethersulfured or not as indicators: including the Codonopsis festival(4 grades)and Codonopsis sulphur free Inch section(4 grades),Party election films(4 grades),Party sulphur-free selection films(4 grades);(2)Classification by diameter: first-class(0.8cm-1.6cm in diameter),second-class(Diameter 0.6cm-1.4cm),third-class(diameter 0.4cm-1.2cm),and divided into Codonopsis thick slices or Codonopsis short segments by thickness,or sieved with sieves with different millimeter holes;market research results are: Dangshen tablets Based on the diameter,it is divided into: selected(0.8cm or more in diameter),general election(0.6cm-0.8cm),middle(0.4cm-0.6cm),small(0.3cm-0.4cm),and general goods(not screened);rice fried Dangshen mostly for unified goods.The research group collected a total of 98 batches of Codonopsis Radix decoction pieces(67 batches of Dangshen tablets and 31 batches of rice fried Dangshen).According to "Classification Standards for Electronic Trading Specifications of 200 Kinds of Chinese Medicinal Materials",the various local Chinese medicine processing specifications and the related literatures,as well as the actual situation of the markets,two quantifiable property indicators including diameter and thickness(observing the mildew and oil spill of each batch of samples)were selected to be determined as indicators of their appearance by statistical analysis methods such as variability analysis,correlation analysis,one-way analysis of variance,histograms,and statistical descriptions.According to the mean value calculation,the grade standard of Codonopsis Radix decoction pieces is formulated as follows: Dangshen tablets or segments: Dangshen thick slice: thickness 2-4 mm;Dangshen short section: thickness 5-10 mm.First grade(general selection): no less than 0.6cm in diameter,no worms,mildew;Second grade(medium selection): no less than 0.4cm in diameter,no worms,mildew;Third grade(small selection): no less than 0.3cm in diameter,no worms,mildew;General goods: all diameters,no worms,mildew.Rice Fried Dangshen: Rice fried Dangshen thick slices: thickness 2-4 mm,no worms,mildew;Rice Fried Dangshen short section: 5-10 mm thick,no insects,mildew.2.The 98 batches of Codonopsis Radix decoction pieces collected by our group are the dried roots of Codonopsis pilosula(Franch.)Nannf.Dangshen tablets or segments and rice fried Dangshen cross-sections: wooden plug cells from tens to ten,with single or clustered stone cells on the outside.The inner layer of the plug is narrow and the cells are slightly spindle-shaped.The phloem is broad,and the milk tube group is often arranged radially along with the sieve tube group.The outer side often forms a cluster distribution with the surrounding thin-walled cells,accounting for about 1/2 of the phloem;the formation layer is obvious and looped.Xylem ducts are arranged radially,accounting for about 1/3 to 2/3 of the root.The parenchyma cells contain inulin,which are fan-shaped,round or irregular,with radial texture visible on the surface,and starch granules.Dangshen tablets or segments and rice fried Dangshen sibiricum were identified by inulin,stone cells,milk tubes,cork cells,trapezoidal or reticulated ducts.Codonopsis glycoside was used as a reference substance,and was developed on a silica gel G thin plate with n-butanol-glacial acetic acid-water(7:1:0.5)as a developing agent,and a 10% sulfuric acid ethanol solution was used as a developer at 90℃.Under the conditions,the color is heated and clear,and they are examined under sunlight and ultraviolet light(365nm).Microscopy identification items and thin layer identification items were established for the quality standard of Codonopsis Radix decoction pieces.The moisture,total ash,and alcohol-soluble extractives of Codonopsis Radix decoction pieces(67 batches of Dangshen tablets or segments,31 batches of rice fried Dangshen)were determined according to the general provisions of the 2015 edition of the Chinese Pharmacopoeia.Based on our previous researches,the Codonopsis pilosula polysaccharide content was determined.The moisture of Dangshen tablets or segments is 8.56% ~ 15.81%,the total ash content is 2.65% ~ 5.81%,the alcohol-soluble extracts are 63.08%~80.40%,the polysaccharide content is 10.02% ~ 25.78%;the water content of rice fried Dangshen pilosula is 5.05% ~ 12.40%,the total ash 2.75% to 5.61%,alcohol-soluble extractives from 51.30% to 75.38%,polysaccharide content of 4.47% to 30.13%.And according to the principle of average ± 20%,and the qualified rate of samples above 80%,it is proposed that the moisture content of Dangshen tablets or sections should not exceed 13.0%,the total ash content should not exceed 5.0%,the extract content should not be less than 55.0%,and the polysaccharide content should not be less than 12.0%;the moisture content of rice fried ginseng must not exceed 10.0%,the total ash content must not exceed 5.0%,the extract content must not be less than 55.0%,and the polysaccharide content must not be less than 12.0%.The inspection items and content determination items for the quality standard of Codonopsis Radix decoction pieces were established.Characteristic chromatogram of Codonopsis Radix decoction pieces by HPLC was established.The relative retention time of seven characteristic peaks was determined by taking peak 5(Lobetyolin)as reference peak,which were 0.26(peak 1),0.39(peak 2),0.51(peak 3),0.66(peak 4),1.00(peak 5),1.02(peak 6)and 1.30(peak 7),respectively.The characteristic peaks were identified by reference addition method,and the results were as follows: Peak 1-codonopyrrolidium B,peak 3-geniposide,peak 4-tangshenoside I,peak 5-lobetyolin and peak 7-atractylenolide III.Comparing the ranks of Dangshen tablets or segments,there is a significant difference between the relative peak area of peak 1 in the general goods and first grade,and a significant difference in the relative peak area of peak 3 between the first grade and the third grade.There is a certain difference in the relative peak area of No.1,No.2,No.3,and No.7 peaks of different places of origin of Dangshen tablets or segments.Compared with the rice fried Dangshen from different origins,the relative peak areas of the No.1,No.2,No.6,and No.7 peaks of Guizhou rice fried Dangshen are significantly different from those of Shanxi and Gansu.Compared with Dangshen tablets or sections and rice fried Dangshen,the relative peak areas of No.1 and No.6 peaks are different.A grade standard for the medicinal materials of Codonopsis Radix decoction pieces was established and submitted.3.HPLC method for simultaneous determination of syringin,tangshenoside I,lobetyolin and atractylenolide III was established.Methodological investigation results: syringin exhibits a good linear relationship in the concentration range of 0.25 μg/mL ~ 6.67 μg/mL.The regression equation is Y = 119120X-10886(r = 0.9995,n = 6),and the average sample recovery is 98.55%;tangshenoside I has a good linear relationship in the concentration range of 20.00 μg/mL ~ 400.00 μg/mL.The regression equation is Y = 20288X-98945(r = 0.9998,n = 6),and the average sample recovery rate is 102.56%;lobetyolin shows a good linear relationship in the concentration range of 15.00 μg/mL ~ 300.00 μg/mL.The equation is Y = 24690X+249436(r = 0.9996,n = 6),and the average sample recovery rate is 101.81%;atractylenolide III has a good linear relationship in the concentration range of 0.39 μg/mL ~ 23.50 μg/mL,regression The equation is Y = 44256X-10755(r = 0.9998,n = 6),and the average sample recovery is 97.32%.Results of 7 batches of Dangshen tablets or segments: syringin content: 0.0112 mg/g ~ 0.0222 mg/g,tangshenoside I content: 0.4887 mg/g ~1.1379 mg/g,lobetyolin content: 0.2281 mg/g ~ 0.4304 mg/g The content of atractylenolide III was 0.0257 mg/g ~ 0.0877 mg/g;the determination results of 7 batches of rice fried Dangshen: syringin content: 0.0149 mg/g ~ 0.0350 mg/g,tangshenoside I content: 0.4825 mg/g ~ 1.13743 mg/g,lobetyolin content: 0.2262 mg/g ~ 0.5177 mg/g,atractylenolide III content: 0.0222 mg/g ~ 0.1072 mg /g.The results showed that the contents of syringin,tangshenoside I and lobetyolin were increased,while the content of atractylenolide III was decreased after rice-fried.Conclusion:1.The product grade standards for Codonopsis Radix decoction pieces are initially established basing on the literature research,origin investigation and analysis of experimental data results,as well as referring to the "Classification Standards for Electronic Trading Specifications of 200 Kinds of Chinese Medicinal Materials" and local standards and standards,and combination with market conditions,and using SPSS statistical analysis to screen the diameter and thickness of Codonopsis Radix decoction pieces as indicators of appearance characteristics.The commodity grade standards formulated in this study are closely combined with the actual market of Codonopsis Radix decoction pieces,and have certain practicability and scientificity,which will provide guidance for the evaluation of Codonopsis Radix decoction pieces in the current market.2.According to the results of Microscopic identification,thin layer identification,routine inspection(moisture,total ash,extracts),determination of polysaccharide content and characteristic maps of 98 batches of Codonopsis Radix decoction pieces and SPSS statistical analysis,the quality standards of Codonopsis Radix decoction pieces was established.On the basis of the 2015 edition of the Chinese Pharmacopoeia,this standard adds the items of Codonopsis pilosula polysaccharide content determination and the HPLC characteristic map and provides a method to control the quality of Codonopsis Radix decoction pieces more scientifically and comprehensively.3.HPLC chromatography method was established for the determination of active ingredients(syringin,tangshenoside I,lobetyolin,and atractylenolide III)in 14 batches of Codonopsis Radix decoction pieces.The method exhibits good accuracy,specificity and feasibility.The determination results and comparative studies provide a theoretical basis for the effects of processing on the content of active ingredients in Codonopsis. |
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