Multiflorum, Caulis Medicine Quality Control

Radix Polygoni Multiflori is the dry root tuber of Polygonum Multiflorum Thunb, which belongs to the family of Polygonaceae. It is reported that the dry root tuber has the quality of bitter and astringent, which can loosening bowel to relieve constipation. Caulis Ploygoni Multiflori is the dry vine of Radix Polygoni Multiflori. It is reported that the dry vine has the quality of sweet and little bitter, which can be mainly used to treat insomnia, impairment caused by overstrain, colliquative sweating and blood deficiency. Radix Polygoni Multiflori and Caulis Ploygoni Multiflori are the different part of the same plant, but they have different efficiency.Utility control variety and complexity of chemistry components of Traditional Chinese Medicine(TCM) are the physical foundation to produce curative action. So it is necessary to determine the content of active principle in TCM.1. Determination of Stilbene Glycoside in Radix Polygoni Multiflori and Caulis Ploygoni Multiflori by RP-HPLC Stilbene Glycoside is one of the main reconstituent in Radix Polygoni Multiflori. It has showed in modern pharmacology that Stilbene Glycoside is bioactive components in Radix Polygoni Multiflori.Chp (2005) Vol I has recorded the assaying method of Stilbene Glycoside in Radix Polygoni Multiflori. We want to establish a method for determine the content of Stilbene Glycoside in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori by RP-HPLC.Objective: To establish a method for determine the content of Stilbene Glycoside in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori by RP-HPLC and compare the content of Stilbene Glycoside in three medical material.Methods: (1)Extraction: An optimal extracting condition was chosen by comparing different experimental results. (2) Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better chromatogram. (3)Preparation of standard curve: Prepare a series of the reference solutions,determine peak areas,then the regression equation was obtained with the content of Stilbene Glycoside as abscissa and the relative peak area as ordinate.(4)System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of Stilbene Glycoside.(5) Specificity test: 20μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system, then every peak within 20 min was recorded. (6)Precision test: The same test solutions were taken and injected to the instrument for five times; determine the peak areas of Stilbene Glycoside.(7)Reproducibility test: Six shares of the same concentration test solution were prepared and the peak area was determined, respectively(.8)Stability test: Transfer the sample solution from Radix Polygoni Multiflori to determine the peak area at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hour, respectively.(9)Recovery test: Transfer 6 shares of Radix Polygoni Multiflori and add the reference solutions, respectively. Detection of the content of Stilbene Glycoside each.(10)Determination of the detection limit:Dilute the reference solution until the value of S/N is more than 3. The relevant concentration is the detection limit. ( 11 ) Assay: Under above-mentioned conditions, determine the content of Stilbene Glycoside in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori.Results: ( 1 ) Extraction: The method of supersonic wave-extraction with 50% ethanol for 30 mins was simple, quick and stable.(2)The HPLC separation was performed on a C18 analytical column, Using a mixture of methanol- acetonitrile-1% formic acid (15: 10: 75) as a mobile phase, at a flow rate of 1.0mL/min, with UV detection at 320nm. The temperature of column was set at 30?C. Injection volume was 20μl.(3)The regression equation: Y =7.17×107X-1.00×105, r=0.9996 (n=6), the linear range of Stilbene Glycoside was from 20.0μg to 160.0μg.(4)System suitability test: Under the above conditions, the peak of Stilbene Glycoside was separated well with the resolution of more than 1.5 and about 2500 of theoretical plate(.5)Specificity test: It showed that there weren't any disturbance in blank solvent and the peak of Stilbene Glycoside appeared at the same time in the test solution.(6)Precision test: The precision of sample was good and the RSD was 1.19%. ( 7 ) The reproducibility of sample: The reproducibility of sample was good and the RSD was 1.30%(.8)The test solution was stable in 48 hours and the RSD was 1.26%.(9)Recovery test: The average recovery was 99.13% and the RSD was 0.78%.(10)The detection limit of Stilbene Glycoside was 1.6ng.(11)The result showed the content of Stilbene Glycoside in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori was 1.956%~3.364%, 1.891%~3.504%, 0.011%~0.252% ,respectively.Conclusion: It is the first time to establish the RP-HPLC method to determine the content of Stilbene Glycoside in Caulis Ploygoni Multiflori.The method is sample and feasible, high accurate and precise. It can be used for content determination of Stilbene Glycoside in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori. 2. Determination of anthraquinone in Radix Polygoni Multiflori and Caulis Ploygoni Multiflori by RP-HPLC Anthraquinone is the other active component in Radix Polygoni Multiflori, which contains Emodin, Rhein, Chrysophanol, and Physcion.Caulis Ploygoni Multiflori is the dry vine of Radix Polygoni Multiflori. Chp(2005)Vol I hasn't recorded the assaying method of anthraquinone in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori. We want to establish a method for determine the content of anthraquinone in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori by RP-HPLC.Objective: To establish a method for determine the content of anthraquinone in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori by RP-HPLC and compare the content of anthraquinone in three medical material.Methods:(1)Extraction: An optimal extracting condition was chosen by comparing different experimental results.(2)Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better chromatogram.(3)Preparation of standard curve: Prepare a series of the reference solutions,determine peak areas,then the regression equation was obtained with the content of anthraquinone as abscissa and the relative peak area as ordinate.(4)System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of anthraquinone(.5)Specificity test: 20μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system, and then every peak within 20 minutes was recorded.(6)Precision test: The same test solutions were taken and injected to the instrument for five times; determine the peak areas of anthraquinone.(7)Reproducibility test: Six shares of the same concentration test solution were prepared and the peak area was determined, respectively.(8)Stability test: Transfer the sample solution from Radix Polygoni Multiflori to determine the peak areas at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hour, respectively.(9)Recovery test: To transfer 6 shares of Radix Polygoni Multiflori and add the reference solutions, respectively. Detection of the content of anthraquinone each. ( 10 )Determination of the detection limit:To dilute the reference solutions until the value of S/N are more than 3. The relevant concentrations are the detection limits.(11)Assay: Under above-mentioned conditions, determine the content of anthraquinone in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori. Results:(1)Extraction: The powder was extracted by supersonic wave with 70% methanol for 30 min and the solution was extracted with trichlormethane. The supernatant layer was extracted under reflux with trichlormethane.(2)The HPLC separation was performed on a C18 analytical column, using a mixture of methanol-acetonitrile-1% phosphoric acid (75:10:15) as a mobile phase, at a flow rate of 1.0mL/min, with UV detection at 254nm.The temperature of column was set at 30?C. Injection volume was 20μl.(3)The regression equation:YEmodin = 53470X– 36480,r=0.9991(n=6);YRhein = 86420X–150360,r=0.9993(n=6);YChrysophanol = 148295X–70207,r=0.9997(n=6);YPhyscion = 35061X–38742,r=0.9996(n=6).The linear range of Emodin, Rhein, Chrysophanol, and Physcion were 20.0~160.0μg/mL, 0.5~100.0μg/mL, 0.5~100.0μg/mL and 0.5~100.0μg/mL, respectively(.4)System suitability test: Under the above conditions, the peak of Emodin was separated well with the resolution of more than 1.5 and about 2500 of theoretical plate(.5)Specificity test: It showed that there weren't any disturbance in blank solvent and the peaks of Emodin, Rhein, Chrysophanol, and Physcion was good(.6)Precision test: The precision of sample was good and the RSD of Rhein, Emodin, Chrysophanol, and Physcion were 0.591%, 1.035 %, 0.771 % and 1.352 %, respectively.(7)The reproducibility of sample: The reproducibility of sample was good and the RSD of Emodin and Physcion were 0.153% and 2.976%.(8)The test solution was stable in 48 hours and the RSD of Emodin and Physcion were 2.656% and 1.487 %.(9)Recovery test: The average recoveries of Emodin and Physcion were 99.75% and 99.02% and the RSD of Emodin and Physcion were 0.489% and 0.969 %. ( 10 ) The detection limits of Emodin, Rhein, Chrysophanol, and Physcion were 1.5ng, 2.5ng, 1.6ng and 4.0ng, respectively.(11)The result showed the contents of the Free emodin and Free Physcion in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori were 0.08%~0.041% and 0.013%~0.101%, 0.036%~0.067% and 0.012%~0.068%, 0.005%~0.013% and 0.005%~0.015%, respectively. The Glycoside emodin and Glycoside Physcion in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori were 0.009%~0.019% and 0.006%~0.049%, 0.011%~0.025% and 0.002%~0.020%, 0.002%~0.006% and 0.004%~0.011%, respectively.Conclusion: It is the first time to establish the RP-HPLC method to determine the content of anthraquinone in Caulis Ploygoni Multiflori.The method is sample and feasible, high accurate and precise. It can be used for content determination of anthraquinone in Radix Polygoni Multiflori, Radix Polygoni Multiflori Preparata and Caulis Ploygoni Multiflori. Variety and complexity of chemistry components of TCM are the physical foundation to produe curative action. However, one or several components always can't reflect its internal quality comprehensively. The modern analytical technique is applied to study fingerprints by using figures to describe the internal chemical information of TCM. In this case, fingerprints just are a new method to reflect the differences of description and quantity of chemical information. It can be used to control the quality of TCM and drugs preparation through integrative information.1. Studies on fingerprints of Radix Polygoni Multiflori, Radix Polygoni Multiflori PreparataRadix Polygoni Multiflori is one of the frequently used drugs. Its quality control is based on the contents of Glycoside and Anthraquinone. Obviously, it isn't enough. Radix Polygoni Multiflori Preparata as the processing drugs of Radix Polygoni Multiflori, there are lots of differents between them. The research indicated that fingerprints can give a full-scale evaluation of Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata.Objective: To establish HPLC fingerprints of Radix Polygoni Multiflori and get control chromatogram; to compare the fingerprints of Radix Polygoni Multiflor collected from different producing areas. It provided a new method as a comprehension quality control item for Radix Polygoni Multiflori.Methods:(1)Extraction: An optimal extracting condition was chosen by comparing different experimental results.(2)Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. ( 3 ) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of Emodin.(4)Specificity test : 20μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system, then every peak within 80 min was recorded.(5)Precision test: The same test solutions were taken and injected to the instrument for six times and the relative retention time and peak areas were determined, respectively. Meanwhile, similarity of the fingerprints was compared.(6)Reproducibility test: Six shares of the same test solution of Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata were prepared. In addition, the relative retention time and peak area was determined, respectively. Meanwhile, similarity of the fingerprints was compared.(7)Stability test: Transfer the sample solution from Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata to determine the relative retention time and peak area at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hour, respectively. Meanwhile, similarity of these fingerprints was compared(.8)Development of fingerprints: The test solution of Radix Polygoni Multiflori was prepared from 19 batches of different producing areas and a batch of reference sample and then their relevant fingerprint chromatograms were obtained; the test solution of Radix Polygoni Multiflori Preparata was prepared from 19 batches of different producing areas and a batch of reference sample and then their relevant fingerprint chromatograms were obtained.Results: ( 1 ) Extraction: The method of supersonic wave-extraction with methanol for 30 min was simple, quick and stable.(2)The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and 0.1%H3PO4 at the flow rate of 1mL/min with UV detector. The temperature of column was set at 30?C. Injection volume was 20μl(.3)System suitability test: Under the above conditions, the peak of Emodin was separated well with the resolution of more than 1.5 and about 2500 of theoretical plate.(4)Specificity test: It showed that there weren't any disturbance in blank solvent and the peak corresponding to Stilbene Glycoside, Emodin and Physcion appeared at the same time that named 7, 16 and 19 in the test solution.(5)Precision test: The similarity of all of the fingerprints is more than 0.90, coinciding with the demand of fingerprints. ( 6 ) The reproducibility of sample: The similarity of all of the fingerprints is more than 0.90, coinciding with the requirements of fingerprints. The reproducibility is acceptable.(7)The test solution was stable in 48 hours. The similarity of all of the fingerprints is more than 0.90, coinciding with the requirements of fingerprints. ( 8 ) Development of relevant fingerprint chromatograms: the fingerprint chromatograms of reference sample and different producing areas were obtained.(9)Data analysis: The similarity of whole data was analyzed with software, including 19 batches of different producing areas and a batch of reference sample .The similarity of Radix Polygoni Multiflori was 0.874~0.992 and the similarity of Radix Polygoni Multiflori Preparata was 0.667~0.995.Conclusion: We have established the HPLC fingerprints of Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata from different producing areas, got reference fingerprint chromatogram, compared the fingerprints from various sources, and studied their difference with similarity analysis, then generally evaluated the quality of Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata. The precision and reproducibility of the method is well, which is useful to actualize standardization planting and can be used for the quality control for Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata.2. Studies on fingerprints of Caulis Ploygoni MultifloriCaulis Ploygoni Multiflori as a medicine to calm down and decline fat, there is no unified standard to control the quality.We established the fingerprint chromatogram of Caulis Ploygoni Multiflori with HPLC, get reference fingerprint chromatogram and set up the common pattern of HPLC fingerprint.Objective: To establish HPLC fingerprints of Caulis Ploygoni Multiflori and get control chromatogram; to compare the fingerprints of Caulis Ploygoni Multiflori collected from different producing areas. It provides a new method as a comprehension quality control item for Caulis Ploygoni Multiflori.Methods:(1)Extraction: An optimal extracting condition was chosen by comparing different experimental results.(2)Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. ( 3 ) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of Emodin.(4)Specificity test : 20μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system, then every peak within 110 min was recorded.(5)Precision test: The same test solution was taken and injected to the instrument for six times and the relative retention time and peak area were determined, respectively. Meanwhile, similarity of the fingerprints was compared.(6)Reproducibility test: Six shares of the same test solution of Caulis Ploygoni Multiflori were prepared. In addition, the relative retention time and peak area was determined, respectively. Meanwhile, similarity of the fingerprints was compared(.7)Stability test: Transfer the sample solution from Caulis Ploygoni Multiflori to determine the relative retention time and peak area at 0, 2, 4, 6, 8, 10, 12, 24 and 48 hour, respectively. Meanwhile, similarity of these fingerprints was compared(.8)Development of fingerprints: The test solution of Caulis Ploygoni Multiflori was prepared from 19 batches of different producing areas and then their relevant fingerprints were obtained.Results: ( 1 ) Extraction: The method of supersonic wave-extraction with ethyl acetate for 30 min was simple, quick and stable.(2)The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and 0.1%H3PO4 at the flow rate of 1mL/min with UV detector. The temperature of column was set at 30?C. Injection volume was 20μl(.3)System suitability test: Under the above conditions, the peak of Emodin was separated well with the resolution of more than 1.5 and about 2500 of theoretical plate.(4)Specificity test: It showed that there weren't any disturbance in blank solvent and the peak corresponding to Stilbene Glycoside, Emodin and Physcion appeared at the same time that named 4, 13 and 15 in the test solution.(5)Precision test: The similarity of all of the fingerprints is more than 0.90, coinciding with the demand of fingerprints. ( 6 ) The reproducibility of sample: The similarity of all of the fingerprints is more than 0.90, coinciding with the requirements of fingerprints. The reproducibility is acceptable.(7)The test solution was stable in 48 hours. The similarity of all of the fingerprints is more than 0.90, coinciding with the requirements of fingerprints(.8)Development of fingerprints: The fingerprint chromatograms of reference sample and different producing areas were obtained.(9)Data analysis: The similarity of whole data was analyzed with software, including 19 batches of different producing areas whose similarity was 0.673~0.99.Conclusion: We have established the HPLC fingerprints of Caulis Polygoni Multiflori from different producing areas, got reference fingerprint, compare the fingerprints from various sources, and study their difference with similarity analysis, then generally evaluate the quality of Caulis Polygoni Multiflori. The precision and reproducibility of the method is well, which is useful to actualize standardization planting and can be used for the quality control for Caulis Polygoni Multiflori.