阅读说明：本技术 一种从小叶莲中提取黄酮苷类化合物的方法及其应用 (Method for extracting flavonoid glycoside compounds from fructus Podophylli and application thereof ) 是由 孙彦君 冯卫生 陈辉 韩红艳 王俊敏 郝志友 张艳丽 陈豪杰 赵晨 韩瑞杰 于 2019-09-03 设计创作，主要内容包括：本发明涉及从小叶莲中提取黄酮苷类化合物的方法及其应用,可有效解决从小叶莲中提取黄酮苷类化合物,实现在制备抗氧化剂和抗宫颈癌药物中的应用问题,所述的黄酮苷类化合物为从小叶莲中提取的桃儿七酮苷A(Sinoflavonoidgs A)和桃儿七酮苷B(Sinoflavonoidgs B),具有抗氧化作用,对人宫颈癌细胞HeLa具有细胞毒活性,有效用于制备抗氧化剂和抗人宫颈癌细胞HeLa的药物。本发明原料丰富,制备方法易操作,有效用于从小叶莲中提取黄酮苷类化合物桃儿七酮苷A(Sinoflavonoidgs A)、桃儿七酮苷B(Sinoflavonoidgs B),开拓了小叶莲的药用价值和商业价值,并为制备抗氧化药物和抗人宫颈癌细胞HeLa药物提供了技术支持,有显著的经济和社会效益。(The invention relates to a method for extracting flavonoid glycoside compounds from fructus podophylli and application thereof, which can effectively solve the application problem of extracting the flavonoid glycoside compounds from the fructus podophylli and realizing the application in preparing antioxidant and anti-cervical cancer drugs. The method has rich raw materials and easy operation of the preparation method, is effectively used for extracting the flavonoid glycoside compounds of the sinopodophyllin A (Sinoflavoidodgs A) and the sinopodophyllin B (Sinoflavoidodgs B) from the lobular nelumbinis, develops the medicinal value and the commercial value of the lobular nelumbinis, provides technical support for preparing antioxidant drugs and anti-human cervical carcinoma cell HeLa drugs, and has remarkable economic and social benefits.)

1. A method for extracting flavonoid glycoside compounds from fructus Podophylli is characterized in that the flavonoid glycoside compounds are sinopodophyllin A and sinopodophyllin B, and the molecular structural formula of the flavonoid glycoside compounds is as follows:

The extraction method comprises the following steps:

Taking 6-9kg of A PodophyllA drug as A raw material, heating and refluxing with 2-5 times of 75-95% ethanol at 90-95 ℃ for 1.5-2 hours, recovering ethanol under reduced pressure to obtain an extract-like ethanol extract, suspending the extract-like ethanol extract in 2-3.2L of distilled water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 3 times at 2-3.2L each for 1.5-2 hours, separating the ethyl acetate extraction part by silicA gel column chromatography, sequentially performing gradient elution with petroleum ether-acetone mixed solvent systems with volume ratios of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:100, 100:200 and 0:100, each gradient elution with 9.1-13L of eluent, 10-15 mGF in ^-1, 500mL of each, collecting 260: 70, 100:200, 0:70, 100: 70: 1.1-70: 10: 70, 70: 100: 70: 1: 10: 1.

^-12. The method for extracting flavonoid glycoside compounds from podophyllum parviflorA according to claim 1, characterized in that 9kg of podophyllum parviflorA is taken as A raw material, 18L of 95% ethanol with A volume ratio is heated and refluxed for 3 times, the extraction temperature is 95 ℃, the extraction time is 1.5 hours each time, ethanol is recovered under reduced pressure to obtain an extract-like ethanol extract, the extract-like ethanol extract is suspended in 3.2L of distilled water, 3 times of 3.2L of petroleum ether, dichloromethane, ethyl acetate and n-butanol with A volume ratio of 1.5 hours each time, the ethyl acetate extraction part is subjected to primary separation by silicA gel column chromatography, the primary separation is sequentially carried out by A petroleum ether-acetone mixed solvent system with A volume ratio of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:200, 0: 100:10, 100: 10: 70, 100:70, 100: 200: 10: 70: 10: 70: 10: 70: 1.7: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 35: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 35: 1: 70, the gradient of acetone, the gradient chromatography, 10: 35-35: 10: 35: 10: 35: 10: 35: 10.

3. The method for extracting flavonoid glycoside compounds from fructus podophylli as claimed in claim 1, wherein the method comprises heating and refluxing fructus podophylli 6kg as raw material with 30L of 75% ethanol at 90 deg.C for 3 times, recovering ethanol under reduced pressure for 2 hours at 90 deg.C to obtain extract-like ethanol extract, suspending in 2L of distilled water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 3 times, each for 2L for 2 hours, subjecting the ethyl acetate extraction portion to silicA gel column chromatography, sequentially subjecting the ethyl acetate extraction portion to gradient elution with petroleum ether-acetone mixed solvent system with volume ratio of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:100, 100:200 and 0:100, subjecting each gradient to gradient elution with 9.1L of petroleum ether-acetone mixed solvent system with flow rate of 10mLm in ^-1, subjecting each gradient to GF, collecting 260 GF, subjecting each GF, subjecting each fluid fraction to silicA gel thin layer chromatography analysis with 254-gel chromatography, subjecting to silicA gel chromatography to 1 min, subjecting to TLC to silicA gel chromatography, subjecting to gel chromatography to preparation, subjecting to TLC to silicA gel chromatography to chromatography, subjecting to silicA gel chromatography to obtain crude oil chromatography with 10-acetone chromatography, subjecting crude oil chromatography with 10, subjecting crude oil chromatography to crude oil chromatography with crude oil extraction rate of 100:0, crude oil chromatography with crude oil chromatography, crude oil chromatography with crude oil extraction, crude oil extraction rate of 100:0, crude oil extraction, crude.

4. The method for extracting flavonoid glycoside compounds from podophyllum parvifolium as claimed in claim 1, wherein 8kg of podophyllum parvifolium is used as a raw material, 24L of 85% ethanol with a volume ratio is heated and refluxed for 3 times, the extraction temperature is 92 ℃, the extraction time is 1.5 hours each time, ethanol is recovered under reduced pressure to obtain an extract-like ethanol extract, the extract-like ethanol extract is suspended in 2.8L of distilled water, the extract-like ethanol extract is sequentially extracted for 3 times with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 2.8L each time for 1.5 hours, the ethyl acetate extraction part is subjected to silica gel column chromatography for primary separation, the extract-like ethanol extract is sequentially subjected to gradient elution with petroleum ether-acetone mixed solvent systems with volume ratios of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:200, 0: 100: 10: 70, the eluent is subjected to gradient elution with eluent composed of 11.7L of 13 mGF ^-1, the eluent with a volume ratio of 1: 70: 10: 1.70: 10: 1: 10: 1.7: 10: 1: 10: 1.7: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 10: 1: 1.7: 1, the eluent is used as a silica gel-96, the eluent is used as a silica gel flow chromatography, the eluent is used as a silica gel chromatography, the eluent.

5. The method for extracting flavonoid glycoside compounds from podophyllum emodi, as claimed in claim 1, is characterized in that 7kg of podophyllum emodi medicinal material is taken as A raw material, 28L of 75% ethanol in volume ratio is heated and refluxed for 3 times, the extraction temperature is 90 ℃, the extraction time is 2 hours each time, ethanol is recovered under reduced pressure to obtain an extractum-like ethanol extract, the extractum-like ethanol extract is suspended in 2.4L of distilled water, 2.4L of petroleum ether, dichloromethane, ethyl acetate and n-butanol are sequentially extracted for 3 times, 2.4L of 2 hours each time, the ethyl acetate extraction part is subjected to silicA gel column chromatography primary separation, the gradient elution is sequentially performed by A petroleum ether-acetone mixed solvent system with the volume ratio of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:100, 100:200 and 0:100, each gradient is performed by 10.4L of eluent, the flow rate is 12 Lm ^-1, each gradient is performed by A silicA gel thin-gel thin layer chromatography, the gradient chromatography, the eluent is obtained by collecting 260 portions of 260: 35 portions, the eluent is performed by the thin layer chromatography, the silicA gel chromatography, the thin layer chromatography, the eluent is performed by the thin layer chromatography, the eluent is performed by the thin layer chromatography, the gradient chromatography, the eluent is performed by the thin layer chromatography, the gradient chromatography, the eluent is performed by the eluent, the eluent.

6. Use of the sinopodophyllin A and sinopodophyllin B extracted from fructus Podophylli by the method of claims 1-5 in preparing antioxidant.

7. The use of the sinopodophyllin A and sinopodophyllin B extracted from fructus Podophylli by the method of claims 1-5 in preparing anti-human cervical cancer cell HeLa medicine.

Technical Field

The invention relates to medicine, in particular to a method for extracting flavonoid glycoside compounds from fructus podophylli and application thereof.

Background

Oxidative stress means that active oxygen free radicals generated in organism tissues or cells exceed the using capacity or the removing capacity of original oxygen free radicals, and the redox balance is damaged, so that excessive accumulation of the active oxygen free radicals and related metabolites thereof are generated in vivo to cause certain damage to the organism, and therefore, a certain pathological state is presented. Oxidative stress is thought to be responsible for many degenerative diseases, such as cancer, chronic inflammation, cardiovascular diseases, neurological diseases, diabetes, alzheimer's disease, parkinson's disease, and the like. Therefore, inhibition of oxidative stress-like damage is one of the important ways to treat diseases associated with oxidative stress.

Malignant tumor is a common disease and frequently encountered disease which seriously threatens human health and life. Today, cancer has become the leading cause of death worldwide. Cervical cancer is a malignant tumor that occurs in the female reproductive system, is the most common malignant tumor in gynecology, and is common in women aged 50-55 years. According to statistics, the incidence and mortality of cervical cancer accounts for about 50% of the world in Asia range, and the incidence and mortality of cervical cancer accounts for over 1/3 of the world in China. In recent years, the age of onset of cervical cancer has gradually become younger, and the average age of onset of cervical cancer has been reduced from 60 years to 51.7 years worldwide. At present, no ideal medicine for treating cervical cancer exists in the market, and the medicine has the defects of poor curative effect, large side effect and the like. The Chinese herbal medicine has a long history of application in the aspect of resisting tumors, can improve the immunity of the organism while killing tumor cells, and has small side effect. Therefore, the first problem to be solved by pharmaceutical researchers is to search high-efficiency and low-toxicity anti-cervical cancer active substances from Chinese herbal medicines and develop novel anti-cervical cancer medicines with strong selectivity and low toxic and side effects.

The herba Podophylli is dried mature fruit of Podophyllum hexandrum of Podophyllum of berberidaceae. The podophyllum hexandrum is a medicinal plant with a long history, and the ancient 'Shennong herbal Jing' has records: kill virulent pathogen, treat cough and throat diseases, and relieve restlessness due to pathogenic wind. It should not be added into soup. The herbs of the later generations are also recorded, and are mainly used for activating blood circulation to dissipate stagnation, dispelling wind and eliminating dampness, insect and snake bite, traumatic injuries, cardialgia and stomachache, cough due to wind-cold evil, irregular menstruation, aconitum pendulum poisoning, rheumatism, muscle and bone pain, tracheitis and other symptoms. The sinopodophyllum hexandrum is widely distributed, and China is mainly distributed in Sichuan, Qinghai, Tibet, Gansu and Shaanxi. Lobelia chinensis is originally recorded in the Yue Wang dynasty as a traditional Tibetan medicine and has a long medicinal history. The chemical composition research shows that the compound mainly contains lignans and flavonoids, and the flavonoids have important and wide biological activities, such as oxidation resistance, virus resistance, tumor resistance, free radical removal and the like. The B-ring glycosidation flavonoid glycoside compound related by the invention is found in the plant of the genus Podocarpus for the first time, and the bioactivity of the compound is not reported in patents or documents so far.

Disclosure of Invention

In view of the above situation, in order to overcome the defects of the prior art, the invention aims to provide a method for extracting flavonoid glycoside compounds from fructus podophylli and application thereof, which can effectively solve the application problem of extracting flavonoid glycoside compounds from fructus podophylli and realizing the application in preparing antioxidants and anti-cervical cancer drugs.

The technical scheme of the invention is that the method for extracting flavonoid glycoside compounds from the small leaf lotus comprises the following steps:

Taking 6-9kg of A PodophyllA drug as A raw material, heating and refluxing the raw material with 2-5 times of weight of the raw material and 75-95% of ethanol for 3 times at 90-95 ℃ for 1.5-2 hours, recovering ethanol under reduced pressure to obtain an extractum-like ethanol extract, suspending the extractum-like ethanol extract in 2-3.2L of distilled water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 3 times at 2-3.2L for 1.5-2 hours, separating the ethyl acetate extraction part by silicA gel column chromatography, sequentially performing gradient elution with petroleum ether-acetone mixed solvent systems with volume ratios of 100:0, 100:5, 100:7, 100:10, 100:30, 100:50, 100:70, 100:100, 100:200 and 0:100, each gradient with 9.1-13L of eluent, 10-15 mSfynin ^-1, 500mL of each gradient with volume of 350: one flow rate, collecting 260: 254, 254: 1 min, 70: 70, 100: 70: 10: 70: 10: 70: 1.20: 1: 10: 70: 1: 70: 1: 10: 1.

The compounds of sinopodophyllin A (Sinoflavoidougs AI) and sinopodophyllin B (Sinoflavoidougs BII) extracted from herba Potentillae Discoloris have antioxidant effect, cytotoxic activity to human cervical cancer cell HeLa, and can be effectively used for preparing antioxidant and medicine for resisting human cervical cancer cell HeLa.

The method has rich raw materials and easy operation of the preparation method, is effectively used for extracting flavonoid glycoside compounds of the sinoflavonoine glycoside A (Sinoflavoidodgs A I) and the sinoflavonoine glycoside B (Sinoflavoidodgs B II) from the lobular lotus, develops the medicinal value and the commercial value of the lobular lotus, provides technical support for preparing antioxidant drugs and anti-human cervical cancer cell HeLa drugs, and has obvious economic and social benefits.

Drawings

FIG. 1 shows the NMR spectrum of compound I of the present invention.

FIG. 2 shows the NMR spectrum of Compound I of the present invention.

FIG. 3 is a HMBC spectrum of compound I of the present invention.

FIG. 4 is an HSQC spectrum of Compound I of the present invention.

FIG. 5 shows the NMR spectrum of Compound II of the present invention.

FIG. 6 shows the NMR spectrum of Compound II of the present invention.

FIG. 7 is a HMBC spectrum of compound II of the present invention.

FIG. 8 is an HSQC spectrum of compound II of the present invention.

Detailed Description

The following examples are provided to explain the present invention in detail.

The invention may be embodied in the form of the following examples.