Research On Quality Control Of The Seeds Of Herpetospermum Pedunculosum(Sex.)

Herpetospermum pedunculosum(Sex.)(Cucurbitaceae) is distributed in southwest China, Nepal, and northeast India. The dried ripe seeds of H. pedunculosum have been used for the treatment of liver diseases as a Tibetan medicinal herb in China. The quality control of H. pedunculosum had ever been recorded by Chinese pharmacopoeia of edition1977version. Tibetan medicine health pharmaceutical standards Volume One (1995version) is the current quality standard. However, there are only morphological identification and microscopic identification; Qualitative identification just includes the identification of alkaloid. In order to control inherent quality of medicinal materials of H. pedunculosum accurately and comprehensively, it is necessary to study quality control of the seeds of H. pedunculosum systematically,This study has significance for the further development and use of the drug.In this paper, the quality standard on the H. pedunculosum was established from three parts, namely, medicinal materials examination, medicinal materials identification and determination of the active ingredients’content in by multi-components assay of single marker. The contents are as follows:1. The examination of medicinal materialsThe content of moisture, total ash, ethanol-soluble extractives and heavy metal of medicinal materials were detected. According to the results, the content limitation of all indexes was defied. The moisture content of H. pedunculosum on the range of4.66%-10.97%, so that the moisture content should not exceed12%tentatively; The total ash content of H. pedunculosum on the range of3.89%-7.31%, so that the total ash content should not exceed8%tentatively; The ethanol-soluble extractives content of H. pedunculosum on the range of12.55%-17.21%, so that ethanol-soluble extractives content of H, pedunculosum should not less than12%tentatively. The range of5kinds of heavy metal content as follows, the content of Pb, Cb, As, Hg and Cu range from0.00to2.60mg/kg,0.00to0.23mg/kg,0.01to0.13mg/kg,0.00to0.14mg/kg, and2.85to9.11mg/kg, respectively. 2. The identification of medicinal materials(1) Taking herpetolide A and herpetotriol as control substances, the medicinal materials of H. pedunculosum is indentified by TCL. Compared with the control, twelve batches of medicinal materials of H, pedunculosum which come from different producing areas showed the same color spots at the corresponding position on the same condition. In addition, these spots are separated excellently and distinctly, so TLC can be acted as qualitative identification method for the medicinal materials of H. pedunculosum(2) The study of HPLC fingerprinting of medicinal materials of H. pedunculosum. Taking herpetolide A as control substance, HPLC fingerprinting of twelve batches of samples was detected. As a result, the fingerprinting of18common peaks were acquired, and the similarities of fingerprinting between every beach of samples and reference substance are more than0.9. It was found that the chromatography conforme to Chinese herbal fingerprinting because of high chiomatogram response value, smooth baseline, and good separating degree. It is significantly to discern the false from the genuine of medicinal materials of H. pedunculosum.3. The quantitative analysis of two compounds by Multi-components Assay of single marker simultaneouslyTaking herpetolide A as control substances, the content of herpetotriol is assayed by establishing relative correction factor between herpetolide A and herpetotriol. The comparison of external standard method and multi-components quantitive of one marker method shows that the relative error was in the range of2%. So, the above results further indicated that the above two methods not exist significant error and can be used for the study of quality criterion on the H. pedunculosum.4. Based on the above reseach, proposed the quality standard of H. Pedunculosum is as follows:Herpetospermum pedunculosum(Sex.)(Cucurbitaceae) is distributed in southwest China, Nepal, and northeast India. The dried ripe seeds of H. pedunculosum have been used for the treatment of liver diseases as a Tibetan medicinal herb in China.[Identification](1) To an accurately weighed quantity equivalent to1.0g of medicinal materials of H. Pedunculosum in40mL of methanol with ultrasonic treatment for30minutes, cool, dilute with methanol to weight, shake well and filter, withdraw1mL of the successive filtrate, test solution is got. Dissolve a quantity of herpetolide A with methanol to produce a solution of1mg per mL, use the methanol solution as the reference solution. Carry out the method for thin-layer chromatography(Appendix VI B), imbibe3μL of test solution and2μL of reference solution, apply to the gel GF254plate, using a mixture of chloroform-ethyl acetate-formic acid (5:1:0.2) as the mobile phase, after developing and removal of the plate, dry in air, view at365nm. The colour and position of the principal spot in the chromatogram obtained with the test solution correspond to the principal spot obained with reference solution.(2)To an accurately weighed quantity equivalent to1.0g of medicinal materials of H. Pedunculosum in40mL of methanol with ultrasonic treatment for30minutes, cool, dilute with methanol to weight, shake well and filter, withdraw1mL of the successive filtrate, test solution is got. Dissolve a quantity of herpetotriol with methanol to produce a solution containing lmg of herpetolide A per mL, use the methanol solution as the reference solution. Carry out the method for thin-layer chromatography (Appendix VI B), imbibe3μL of test solution and2μL of reference solution, apply to the gel GF254plate, using a mixture of chloroform-acetone-formic acid (8:4.3:0.7) as the mobile phase, after developing and removal of the plate, dry in air,view at254nm. The colour and position of the principal spot in the chromatogram obtained with the test solution correspond to the principal spot obained with reference solution,examination] Water Not more12%, using oven drying method.Total ash Not more8%(Appendix IX K, ash determination method)Alcohol solublility extractum Not more12%(Appendix X A)[Assay] Complies with Chinese pharmacopoeia of edition2010version high performance liquid chromatography (Appendix VI D)Reference solution Dissove accurately weighted quantity of herpetolide A and herpetotriol respectively to produce solutions of6.8μg and5.76μg per mL separately.Test solution To an accurately weighed quantity equivalent to about1.0g of----in a conical flask, add critically20mL of methanol, close, weigh, ultrasound, cool, weigh, dilute with methanol to weight, shake well, filter, tranfer the successive filtrate, store in4℃, use the successive filtrate as the reference solution.[proceduce] Inject10μL of reference solution and test solution into the column and record the chromatogram.The content of herpetolide A and herpetotriol is not less than0.7%、3.6%.[Storage] Preserve in a cool and dry place, protected from moth.