| Objective:At present,the research on the fruits of Alpinia zerumbet from Sichuan is relatively blank and its application is relatively limited.This article aims to analyze the chemical components of essential oil in Alpinia zerumbet fruits from Sichuan,to clarify the types and contents of essential oil,to reveal the material basis of effective components in essential oil in Alpinia zerumbet fruits.Study the flavonoids in Alpinia zerumbet fruits,to optimize the suitable method for determination of total flavonoids.Study the extraction and purification process of total flavonoids,to preliminarily identify and determine the content of flavonoids in Alpinia zerumbet fruits,to reveal the material basis of flavonoids effective components in Alpinia zerumbet fruits,and to provide some reference for quality control of Alpinia zerumbet fruits.Study the analgesic effect of Alpinia zerumbet fruits produced from Sichuan,provide scientific basis for the research and development of new drugs for treating epigastric pain,sciatica and rheumatic arthralgia,and provide research basis for the further development and utilization of Alpinia zerumbet fruits resources.Methods:(1)The essential oil was extracted by steam distillation,the GC-MS was used to separate and analyze the essential oil,and the NIST08.LIB library was searched and the chemical components were determined by comparing CAS numbers and references,the percentage of each chemical component was calculated by chromatographic peak area normalization method.(2)Using rutin as reference substance,the content of total flavonoids in Alpinia zerumbet fruits was estimated by UV-vis method.Using reflux extraction method.Firstly,a single factor experiment was carried out to investigate the influence of ethanol concentration,material-to-liquid ratio,extraction temperature and extraction time on the content of total flavonoids in Alpinia zerumbet fruits and three levels were optimized.Then,the best extraction process of total flavonoids in Alpinia zerumbet fruits was determined by Box-Behnken method.(3)The macroporous adsorption resin was used to purify the crude extract of total flavonoids in Alpinia zerumbet fruits.It was investigated that the adsorption rate and desorption rate of total flavonoids were affected by sample concentration,ethanol concentration,sample loading amount,water washing amount,ethanol dosage and other factors,in order to determine the best purification technology of total flavonoids in Alpinia zerumbet fruits.(4)The UPLC-QTOF-MS/MS method was used to separate and identify the flavonoids in Alpinia zerumbet fruits.A rapid and accurate method for simultaneous determination of hyperoside and isoquercitrin,(two flavonoid monomer components)in the Alpinia zerumbet fruits,was established by HPLC.The DPPH method and ABTS method were used to study the in vitro antioxidant activity of Alpinia zerumbet fruits.(5)The hot plate method was used to determine the licking time of mice after the last dose of 30 min,60min,90 min,and 120 min after continuous gavage with water extract of Alpinia zerumbet fruits,42% alcohol extract,water and aspirin for 7 days.The writhing method was used to observe the mice’s writhing reaction times within 15 minutes after the last dose of water extract,42% alcohol extract,water and aspirin for 7 days.Results:(1)Ninety-one chemicals were identified of the essential oil in the of Alpinia zerumbet fruits from Sichuan,accounting for 93.61% of the total essential oil,and mainly containing terpenoids and aromatic compounds.It mainly contains β-pinene,limonene,α-terpineol and α-pinene,accounting for53.4% of the total essential oil,It was also found for the first time that the flavor components of Alpinia zerumbet accounted for 60.85% of the total essential oil.(2)The UV-vis determination method and the best extraction technology of total flavonoids in Alpinia zerumbet fruits from Sichuan was established:the ethanol concentration was 42%,the ratio of solid to liquid was 1:17(g/ m L),and the extraction time was 2.5h at 80℃,the total flavonoids content of Alpinia zerumbet fruits was about 8.4 mg/g.(3)The most suitable macroporous adsorption resin for the purification of total flavonoids in the Alpinia zerumbet fruits produced from Sichuan is polar NAK-9 resin,the best purification process of total flavonoids in Alpinia zerumbet fruits has been established as follows: 6BV0.5mg/m Lsample concentration,1m L/min adsorption,31 BV water consumption,8BV 40% ethanol eluted at 1m L/min volume flow rate,the purity of total flavonoids increased from 8.6% to 36.5%,and the purity increased 4.2times.(4)The UPLC-QTOF-MS/MS method was used to quickly and accurately identify two flavonoids mainly containing hyperoside and isoquercitrin in the fruits of Alpinia zerumbet from Sichuan.The established HPLC method is simple and accurate,and can be used for the simultaneous determination of hyperoside and isoquercitrin in the fruits of Alpinia zerumbet from Sichuan.,in which the contents of hyperoside and isoquercitrin were 21.34μg/g and40.67μg/g respectively.Moreover,the 42% alcohol extract of Alpinia zerumbet fruits has antioxidant activity in vitro before and after purification,and the antioxidant activity after purification is obviously higher than that before purification,and the antioxidant activity after purification is equivalent to the positive drug VC.(5)The high and middle doses of water extraction and alcohol extraction of Alpinia zerumbet fruits can prolong the licking time of mice stimulated by hot plate.The effect of alcohol extract of Alpinia zerumbet fruits is better than that water extract,and it is dose-dependent.The high dose of water extract of Alpinia zerumbet fruits can reduce the times of writhing reaction in mice.Conclusion:This experiment made clear the types and contents of essential components in Alpinia zerumbet fruits from Sichuan,and revealed the material basis of effective components in essential oil.The determination method of total flavonoids in Alpinia zerumbet fruits is established,which is simple and quick to operate.In this study,the extraction process and purification process of total flavonoidsin the fruits of Alpinia zerumbet from Sichuan were optimized,and the operations were simple and feasible.The material basis of flavonoids in the fruits of Alpinia zerumbet from Sichuan is preliminarily identified.The established HPLC method is simple and accurate,and can be used for the simultaneous determination of hyperoside and isoquercitrin in the fruits of Alpinia zerumbet from Sichuan.The purified total flavonoids from Alpinia zerumbet fruits has good antioxidant activity in vitro.The water extract and 42% alcohol extract of Alpinia zerumbet fruits have central analgesic effect;while the water extract of Alpinia zerumbet fruits also has peripheral analgesic effect.This study can provide a certain reference value for establishing the quality standard for content determination of Alpinia zerumbet fruits,and is beneficial to the further development and utilization of medicinal plant resources of Alpinia zerumbet fruits. |
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