| Polygonum capitatum,also named as sungrass,capitate knotweed herb and hydrangea,was the dry whole grass or above-ground part from Polygonum capitatum Buch.-Ham.ex D.Don.Whole grass was used as medicine,tasted bittee pungent and cool.It mainly contained flavonoids,phenolic acids,lignans and other compounds.Modern pharmacological research indicated that polygonum capitatum had pharmacological effects such as antibacterial,anti-inflammatory and analgesic,antioxygentation and hypoglyceic.Previous studies showed that polyphenol represented by Davidiin had a good hypoglycemic effcet,and the content was higer than gallic acid,quercetin and other substances,which had good development and reserch value.Therefore,Davidiin was the study object in this article,and its preparation process and metabolite in diabetes model were investigated.Firstly,a method was established to detect the content determination of Polygonum capitatum.At present,Polygonum capitatum was included in the “Quality Standards of Traditional Chinese Medicine and Ethnic Medicinal Materials of Guizhou Province(2003 Edition)” and “Standards of Traditional Chinese Medicinal Mterials of Hunan Province(2009 Edition)”.The sample solution was prepared using the hydrochloric acid hydrolysis method.Quercetin-3-rhamnoside and other components can also be hydrolyzed to produce quercetin,and the content determination item used quercetin as the quality control indicator.The content determination item of Relinqing granule made from Polygonum capitatum used gallic acid as the quality control indicator(collected in the 2015 edition of “ Chinese Pharmacopoeia ”).In order to improve quality standards,this study established a HPLC content determination method using gallic acid,Davidiin and quercetin-3-rhamnoside as indicators.Waters Atlantis T3 column(4.6?250 mm,5 ?m)was adoped.The mobile phase was acetonitrile(A)-0.2% formic acid(B)with the gradient elution(0-10 min,7%A-15%A;10-30 min,15%A-20%A;30-45,20%A-25%A;45-50 min,25%A-7%A)at the flow rate of1.2 m L/min.The injection volume was 10 ?L,the detection wavelength was 280 nm and the column temperature was room temperature.The results showed that galiic acid,Davidiin and quercetin-3-rhamnoside had good linear relationships within the ranges of 0.0380?0.2280 ?g,0.3560?2.1360 ?g and 0.0684?0.4140 ?g,the correlation coefficients were between 0.9996?0.9998 and the average recoveries(n=6)were 101.96%,98.77% and 106.26%,respectively.By comparing different extraction methods,the heating reflux method extraction was determined.The extraction time and methanol concention were optimized to determine the method of sample preparation.A total of 13 batches of Polygonum capitatum were tested.The contents of gallic acid,Davidiin and quercetin-3-rhamnoside are 0.2715?1.1167 mg/g,1.6910?10.9266 mg/g and 0.7236?3.2265 mg/g.The developed method is simple and feasible,which is suitable for the content determination and quality control of Polygonum capitatum.Secondly,studied on the extraction process of polyphenol Davidiin from Polygonum capitatum.By examining different extraction methods,determined the heating reflux method to extract Davidiin.Examined the influence of various factors on the extraction rate,useing Davidiin content as indicators and taking different ethanol concentraton,solventmaterial ratio and extraction time as single factors.Response surface methodology(RSM)was used to optimize the extraction process of Davidiin on the base of single-factor experiment to determine the optimal extraction conditions.The optimal process condition was as follow: extraction solvent was 65% ethanol,solvent-material ratio was 24(m L/g),extraction time was 60 min,extraction times was one.Under the above conditions,the extraction rate of Davidiin can reach 95.46%.After scale-up experiment based on the optimal precess condition,the extraction rate of Davidiin was 85.62%.Then,studied on the purfication process of polyphenol Davidiin from Polygonum capitatum by macroporous resin.The loading solution was the concentrated solution after extraction based on the optimal precess condition.HPD-750 was selected as the suitable macroporous resin by static adsorption and desorption experiment.With the combination of transfer rate and purity of Davidiin as comprehensive evaluation indicators,several factors include loading concentration,loading rate,diameter-height ratio,loading volume were evaluated by dynamic adsorption experiment.The optimal purification conditions were: loading concentration 2.37 mg/m L,loading rate 3 BV/h,diameter-height ratio 1:1.5,loading volume 3 BV,water elution 12 BV,15% ethanol elution 12 BV,25% ethanol elution 7BV,90% ethanol elution 10 BV,flow rate 3 BV/h.The 25% ethanol elution fraction was collected and concentrated to dryness.In this process,the transfer rate of Davidiin was 80.61% and the purity was 74.23%.Acording to the best purification process,the final transfer rate of Davidiin was 60.99% and the purity was 72.47% during the entire preparation process after scale-up the experiment.Finally,studied on the identification of metabolites of polyphenol Davidiin from Polygonum capitatum in SD diabetes-model.A SD diabetes-model was established combined a high-fat diet with streptozotocin,which were kept in metabolic cages and administered orally at 8 am every day for 4 weeks.The Davidiin group was administered at a dose of 90 mg/kg,and the model group was given the same volum of saline.Urine,feces and blood sample were collected and detected by UHPLC-Q-TOF-MS.Identified its major metabolites and inferred the transfermation pathway in the diabetes model.The results showed that Davidiin and related matabolites were not found in blood,and a total of 6 metabolites were found in urine.After oral administration,a one-phase metabolic hydrolysis reaction mainly occured to generate small polar molecules,which were then combined with glucuronic acid and sulfonic acid to undergo two-phase metabolic reactions to form large polar substances that were excreted by the urine.In summary,the article mainly sdudied the extrsction and purification of polyphenol Davidiin,which provided a basis for subsequent large-scale preparation.At the same time,a metabolite identification study of Davidiin in a diabetes model was conducted to provide a theoretical foundation for the study of Davidiin's hypoglycemic mechanism. |
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