Studies On The Analyses And Detection Of Stilbene And Anthraquinone Compounds In Crude Drug And Cultures Of Rhizoma Polygoni Cuspidati

Polygonum Cuspidatum Sieb.et Zucc, a kind of perennial herb of Polygonaceae, widely distributes in the south areas from Qinling mountain of China. Its radix and rhizome are commonly used traditional Chinese medicine. Rhizoma Polygoni Cuspidati contains many constituents such as stilbene, anthraquinone, flavone and phenol. In these constituents, resveratrol and piceid are the main stilbene, while emodin and physcione are main anthraquinone. Because of difference of geography and growth environment, the components in Rhizoma Polygoni Cuspidati from various origins are diverse. Variety and complexity of chemical components in traditional Chinese medicine are the physical foundation to develop curative effects. However, at present the quality control on crude drug of Rhizoma Polygoni Cuspidati always only use one component, such as emodin,which can't reflect its internal quality comprehensively. In order to control quality of Rhizoma Polygoni Cuspidati roundly, it is necessary to develope the chromatography methods to simultaneously detecte more components such as stilbene and anthraquinone. Resveratrol and piceid, which exist in Rhizoma Polygoni Cuspidati, have been proved to have significant bioactivites. So the need of crude drug is increased. Tissue culture technique is one effective measure to accelerate medicinal plant propagatine speed and solve the shortage of resource. The tissue culture technique is paied more attention. Because of different culture conditions, the sample processing is often large. Sometimes the content of target component is low. Therefore, it is necessary to establish a sensitive, accurate and rapid analytical method which is suitable for tissue culture. In the paper, the methods of polyamide thin-layer chromatography with fluorescence detection and HPLC-MS are suitable for culture, due to their sensitivity and accuracy.The modern analytical technique is applied to study fingerprints by using figures to describe the internal chemical information of traditional Chinese medicine. In this case, fingerprints method is a new method to reflect the differences of chemical information. It can be used to control the quality of the traditional Chinese medicine and drugs preparation through integrative information. It's a better method to control the quality of tradition Chinese medicine. So it is necessary to develop the fingerprints of Rhizoma Polygoni Cuspidati.Objective: To establish thin-layer chromatography (TLC) and HPLC methods for qualitative and qualitative determiation of stilbene and anthraquinone in the crude drug of Rhizoma Polygoni Cuspidati. To establish a sensitive and specific HPLC fingerprints method and get the control chromatogram to evaluate the quality of Rhizoma Polygoni Cuspidati. Methods: 1. Identification of crude drug of Rhizoma Polygoni Cuspidati by TLC. (1) The separation of resveratrol, piceid and emodin was obtained on polyamide film with microemulsion as mobile phase, combing methyl acid and acetone as the modifier. Fluorescence detection was employed. (2) Identification of (Z) - and (E) - resveratrol and piceid by TLC. The separation of (Z) - and (E) - resveratrol and piceid was obtained on polyamide film with microemulsion as mobile phase, combing methyl acid and methanol as the modifier. Fluorescence detection was employed. (3) Identification of 5 anthraquinone by TLC. To separation and identify 5 anthraquinone on silica G with petroleum, acetoacetate ether and methyl acid as mobile phase and fluorescence detection. 2. Establishment of fingerprint: (1) Extraction: Extract conditions were optimized by comparing extract method, solvent and time. (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 resolution and theoretical plate of piceid peak. (4) In this chromatographic condition, to investigation the precision, reproducibility and stability of this experiment. (5) Prepare test solutions of samples from different locality, inject to HPLC, get fingerprint chromatograms and compared. 3. Assay: Adjust proportion of mobile phase to reduce analysis time, determinate the content of piceid, resveratrol, emodin and physcion. 4. Study on separation and identification of the (Z) - and (E) - resveratrol and piceid in Rhizoma Polygoni Cuspidati by HPLC-MS. Establish Simultaneous determinate of (Z) - and (E) - resveratrol and piceid by HPLC–MS with acetonitrile and 0.1% methyl acid as mobile phase gradient elution. The detection mode of multiple reaction monitoring was utilized. 5. Study on tissue culture of Rhizoma Polygoni Cuspidati. Using TLC and HPLC methods, which have been established in this experiment, to choose the culture strain with shorter culture cycle and high content of resveratrol or piceid. The selected strain will be used to enlarge culture and further study.Results: 1. Identification of crude drug of Rhizoma Polygoni Cuspidati by TLC. (1) The optimal mobile phase to separate and identify the resveratrol, piceid and emodin is the microemulsions containing 80% water-methyl acid-acetone (1.0:1.0:0.5). Compared with general mobile phase, the resolution and detection were improved markedly. (2) The best mobile phase to separate and identify the (Z) - and (E) - resveratrol and piceid was the microemulsions containing 4%SDS, 80% water methyl acid methanol (1.0:2.0:1.0). Compared with general TLC, resolutions were improved markedly. (3) Using petroleum benzin, acetic ether and methyl acid (15:5:2) as mobile phase can separate and identify 5 anthraquinones. 2. Establishment of fingerprint (1) Extraction: The method of supersonic wave-extraction with methanol for 30mins was simple, quick and stable. (2) The HPLC system was performed on C18 analytical column gradient eluted with a mixture consisting of acetonitrile, 0.1% H₃PO₄ at a flow rate of 1mL·min^-1. The temperature of column was 30℃.The UV detection wavelength was set at 230nm. Injection volume was 10μL. (3) Under the above condition, the peak corresponding to piceid of the test solution was separated well with the resolution of more than 1.5 and about 30000 of theoretical plate. The retention time of piceid was about 12 minute. (4) Same piceid peak as reference peak, the precision of sample was good and the RSD values of relative retention time and relative area were between 0.10%⁰.16% and 0.87%².4%, respectively. The reproducibility of sample was good and the RSD values of relative retention time and relative area were between 0.13%⁰.19% and 2.0%³.0%, respectively. The test solution was stable in 12 hours, The RSD values of relative retention time and relative area were between 0.13%⁰.30% and 1.0%³.0%, respectively. (5) Get relevant fingerprint chromatograms. Same piceid peak as reference peak, compared with 10 different producing areas'fingerprints, get 14 common peaks, similarity is more than 0.90. 3. Assay: (1) piceid, resveratrol, emodin and physcion of the regression were: y=1.32×10³x-85.8,r=0.9997;y=709x+19.5,r=0.9999;y=409x+4.05,r=0.9999;y=134x+4.46,r=0.9985,respectively. (2) The precision of sample was good and the RSD values of areas were between 1.4%².0%, respectively. (3) The reproducibility of sample was good and the RSD values of contents were between 2.2%³.0%. (4) The test solution was stable in 12 hours, The RSD values of areas were between 1.6%².3%, respectively. (5) The average recoveries of piceid, resveratrol, emodin and physcion were 94.4%⁹8.4%,and their RSD values were 1.3%².2%, respectively. The results of the content of piceid, resveratrol, emodin and physcion were difference from different producing areas for producing areas and collection period were different. 4. Study on separation and identification of the (Z) - and (E) - resveratrol and piceid in Rhizoma Polygoni Cuspidati by HPLC-MS. Acetonitrile and 0.1% methyl acid as mobile phase gradient elution, mass spectrometric conditions were in the negative mode. The mass transition ion-pairs of (Z) - and (E) -resveratrol were m /z 227/185,143. The retention times were 23.73 minute and 20.95minute, respectively. The ion-pairs of (Z) - and (E) - piceid were m /z 389/227. The retention time was 15.15minute and 10.18 minute. The lowest detection limitation of resveratrol and piceid were 1.9×10^-2and 5.4×10^-2μg·mL^-1, respectively. 5. Study on tissue culture of Rhizoma Polygoni Cuspidati. The best mobile phase to separate and identify the tissue culture was the microemulsions containing 4%SDS, 80% water methyl acid methanol (1.0:2.0:1.0). The result of TLC showed that the content of resveratrol and piceid were increased in the root of cultivation Rhizoma Polygoni Cuspidati when the root was radiated by ultraviolet. However, the content was still lower than that in crude drug from Si Chuan province. The contents of resveratrol and piceid in the root of cultivation Rhizoma Polygoni Cuspidati were 2.15mg·g^-1and 4.69mg·g^-1, determined by HPLC. But, the content of resveratrol and piceid in callus and hairy roots were lower. Conclusion: To establish polyamide thin-layer chromatography with fluorescence detection and HPLC determination for stilbenes and anthraquinones in crude drug and cultures of Rhizoma Polygoni Cuspidati,simultaneously. The method is simple, quick, sensitive, and useful to identification and detection of Rhizoma Polygoni Cuspidati. It is also a new method to screen culture conditions. The fingerprint chromatogram is useful to control the quality of Rhizoma Polygoni Cuspidati and offer a more precise method to identification Rhizoma Polygoni Cuspidati from different sources. It is a good method to confirm commons and differences in cultures and crud drugs of Rhizoma Polygoni Cuspidati by fingerprint chromatogram technology.