阅读说明：本技术 一种从荷叶中联产多种活性物质的方法 (Method for co-producing multiple active substances from lotus leaves ) 是由 李响 于 2019-07-17 设计创作，主要内容包括：本发明公开了一种从荷叶中联产多种活性物质的方法,该包括以下步骤：提取、萃取得各活性物质部分,再分别对各部分进行分离纯化,最终得到了8种生物碱单体、3种黄酮单体以及4种多酚单体,且本申请制备得到的荷叶总生物碱、荷叶总黄酮以及荷叶总多酚均可直接应用于不同的领域。本发明提供的方法操作简便,生产周期短,分离效率高,工艺稳定,成本低廉,可实现工业化从荷叶中联产多种活性物质,得到的单体的纯度达95％以上,有效成分回收率大于70%,极大地利用了荷叶资源。(The invention discloses a method for co-producing multiple active substances from lotus leaves, which comprises the following steps: extracting and extracting to obtain active substance parts, and then respectively separating and purifying the active substance parts to finally obtain 8 alkaloid monomers, 3 flavone monomers and 4 polyphenol monomers, wherein the lotus leaf total alkaloids, the lotus leaf total flavones and the lotus leaf total polyphenols prepared by the method can be directly applied to different fields. The method provided by the invention has the advantages of simple operation, short production period, high separation efficiency, stable process and low cost, can realize industrial co-production of various active substances from lotus leaves, ensures that the purity of the obtained monomer reaches more than 95 percent, ensures that the recovery rate of active ingredients is more than 70 percent, and greatly utilizes lotus leaf resources.)

1. A method for co-producing a plurality of bioactive substances from lotus leaves is characterized by comprising the following steps:

1) cleaning and drying folium Nelumbinis, pulverizing, sieving with 80 mesh sieve, extracting with 65-95% methanol or ethanol under ultrasonic-assisted heating for 2-3 times at 60-85 deg.C for 0.5-2 hr, mixing extractive solutions, vacuum filtering to remove residue, and concentrating under reduced pressure to obtain extract;

2) dispersing the extract obtained in the step 1) with water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and further separating the chloroform part, the ethyl acetate part and the n-butanol part;

3) processing the chloroform part obtained in the step 2) by ultrafiltration membrane equipment, processing the obtained filtrate by nanofiltration membrane equipment, concentrating the obtained filtrate under reduced pressure, and freeze-drying or spray-drying to obtain total alkaloids of lotus leaves;

4) separating the alkaloids in folium Nelumbinis by medium-low pressure preparative liquid chromatography, sequentially performing gradient elution with 0.1-0.2% triethylamine water solution and acetonitrile as eluent, bottling, collecting eluate, standing for crystallization, filtering, drying, and identifying to obtain the following specific 8 alkaloids: nuciferine, dehydronuciferine, liensinine, dehydronuciferine, amacrine, baperine, O-demethylnuciferine, N-demethylnuciferine;

5) concentrating the ethyl acetate part obtained in the step 2) under reduced pressure to dryness, dissolving with distilled water, passing the obtained water solution through macroporous adsorbent resin, eluting with 10 times of column volume of deionized water, sequentially eluting with water and 50-75% ethanol, washing respectively to colorless, collecting ethanol eluate, concentrating, and drying to obtain folium Nelumbinis total flavone;

6) loading the lotus leaf total flavonoids obtained in the step 5) on a Sephadex LH-20 column chromatography, performing gradient elution by using dichloromethane-methanol, detecting by TCL, combining the same components, performing semi-preparative HPLC on the components, and finally separating by using acetonitrile-water as an eluent to obtain the following 3 flavonoid compounds: quercetin, luteolin, and 3-methoxyl quercetin, wherein the 3-methoxyl quercetin is obtained by separating folium Nelumbinis for the first time;

7) concentrating the n-butanol part obtained in the step 2) under reduced pressure to dryness, dissolving with distilled water, subjecting the obtained water solution to polyamide column chromatography, performing gradient elution with ethanol-water, each gradient elution being 3-5 times the column volume, collecting 30-50% ethanol eluate, concentrating and drying to obtain total polyphenol of lotus leaves;

8) the semi-preparative HPLC for the total polyphenol in the lotus leaves obtained in the step 7) takes methanol-water as a mobile phase, the flow rate is 1ml/min, effluents in different times are collected, and the polyphenol substances in the following 4 are obtained by final separation: catechin, epicatechin, Nympholide a2, and Nympholide B3.

2. The method for co-producing multiple bioactive substances from lotus leaves as claimed in claim 1, wherein: the dosage of the extraction solvent in the step 1) is the mass of the medicinal materials: the volume of the solution is 1Kg and 10-40L; the ultrasonic power was 350W.

3. The method for co-producing multiple bioactive substances from lotus leaves as claimed in claim 1, wherein: the temperature of ultrafiltration and nanofiltration in the step 3) are controlled to be 40-60 ℃, and the pressure is controlled to be 0.6-2.2 MPa.

4. The method for co-producing multiple bioactive substances from lotus leaves as claimed in claim 1, wherein: the macroporous adsorption resin in the step 5) is AB-8 macroporous resin, the sample loading concentration is 20 mg/mL, the sample loading volume is 20mL, the diameter-height ratio is 1:30, and the ethanol concentration is 70%.

5. The use of the total alkaloids from lotus leaves obtained in step 3) of claim 1 in the preparation of lipid-lowering and weight-reducing drugs, foods or cosmetics.

6. Use of the lotus leaf total flavonoids obtained in step 5) of claim 1 in the preparation of cosmetics.

7. Use of the lotus leaf total polyphenols obtained in step 7) of claim 1 for the preparation of cosmetics.

Technical Field

The invention belongs to the technical field of separation and purification of active ingredients of traditional Chinese medicines, and particularly relates to a method for co-producing multiple active substances from lotus leaves.

Background

The lotus leaves are dry leaves of Nelumbo nucifera Gaertn, a plant of Nelumbo nucifera of Nymphaeaceae, are distributed in all countries of the world, are distributed in most areas of China, are mainly produced in Hubei, Hunan, Zhejiang and Jiangsu fields and the like, and have the planting area of about 100 mu of a thousand. The Hubei Honghu of the company is famous aquatic product, and is rich in lotus roots, which is one of the main special products of the Honghu. The lotus leaves are bitter and neutral, enter liver, spleen and stomach channels, and have the effects of relieving summer heat, clearing heat, growing hair, clearing yang, dissipating blood stasis, stopping bleeding and the like. In document 45 of the ministry of health (1991) in 11 months 1991, lotus leaves are listed in the list of "food and medicine" in batch 2. Lotus leaf has the functions of losing weight and reducing fat, and the record of the 'being taken by lotus leaf and being thin and bad' is recorded in the compendium of materia Medica. The lotus leaf contains protein, vitamin C, fat, carbohydrate, carotene, thiamine, nicotinic acid and other substances, and also contains various microelements such as iron, calcium, phosphorus and the like, and flavonoid compounds and various alkaloids including nuciferine, amacrine, protophylline, N-demethylnuciferine and the like. The flavonoids and alkaloids in the lotus leaves have obvious biological activity and physiological function, and the lotus leaf extract has the effects of reducing fat and losing weight, resisting oxidation, inhibiting hypercholesterolemia, arteriosclerosis, inhibiting bacteria and the like, so the research on the lotus leaf extract becomes a focus of attention in the fields of medicine, beauty treatment and food.

The lotus leaves are rich in active substances, including polyphenols, flavonoids, alkaloids, saponins, steroids, organic acids, polysaccharides, amino acids, volatile oil and other substances, and can play remarkable roles in resisting oxidation, resisting wrinkles, replenishing water, whitening, fading color spots, brightening the skin and the like when being used in a cosmetic formula.

Because the planting amount of Honghu lotus roots is large, a series of products derived from Honghu lotus roots are very many, but the Honghu lotus roots are mostly processed and manufactured by lotus roots, lotus seeds and the like, the lotus leaves are not fully utilized at present, and the resource waste is greatly caused. The extraction yield of the traditional method for extracting the effective substances of the lotus leaf powder is not high at present, and a large amount of organic solvent is used in the extraction process, so that the method is not beneficial to practical production and application.

The invention fully utilizes lotus leaf resources, expands the application field of the lotus leaf resources, reduces the pollution problem to the environment caused by the rotten lotus leaves, extracts and purifies the active ingredients in the lotus leaf resources, has simple process and low cost, can co-produce various bioactive substances, and is further applied to different fields.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for co-producing various bioactive substances from lotus leaves. The method has the advantages of simple operation, short production period, high separation efficiency, stable process, low cost, and can realize industrialized co-production of multiple bioactive substances in folium Nelumbinis, the obtained active substances have purity of above 95%, and the recovery rate of effective components is more than 70%.

In order to realize the invention, the technical scheme adopted by the invention is as follows: the method for co-producing various bioactive substances from lotus leaves comprises the following steps:

1) cleaning and drying folium Nelumbinis, pulverizing, sieving with 80 mesh sieve, extracting with 65-95% methanol or ethanol under ultrasonic-assisted heating for 2-3 times at 60-85 deg.C for 0.5-2 hr, mixing extractive solutions, vacuum filtering to remove residue, and concentrating under reduced pressure to obtain extract;

2) dispersing the extract obtained in the step 1) with water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and further separating the chloroform part, the ethyl acetate part and the n-butanol part;

3) processing the chloroform part obtained in the step 2) by ultrafiltration membrane equipment, processing the obtained filtrate by nanofiltration membrane equipment, concentrating the obtained filtrate under reduced pressure, and freeze-drying or spray-drying to obtain total alkaloids of lotus leaves;

4) separating the alkaloids in folium Nelumbinis by medium-low pressure preparative liquid chromatography, sequentially performing gradient elution with 0.1-0.2% triethylamine water solution and acetonitrile as eluent, bottling, collecting eluate, standing for crystallization, filtering, drying, and identifying to obtain the following specific 8 alkaloids: nuciferine, dehydronuciferine, liensinine, dehydronuciferine, amacrine, baperine, O-demethylnuciferine, N-demethylnuciferine;

5) concentrating the ethyl acetate part obtained in the step 2) under reduced pressure to dryness, dissolving with distilled water, passing the obtained water solution through macroporous adsorbent resin, eluting with 10 times of column volume of deionized water, sequentially eluting with water and 50-75% ethanol, washing respectively to colorless, collecting ethanol eluate, concentrating, and drying to obtain folium Nelumbinis total flavone;

6) loading the lotus leaf total flavonoids obtained in the step 5) on a Sephadex LH-20 column chromatography, performing gradient elution by using dichloromethane-methanol, detecting by TCL, combining the same components, performing semi-preparative HPLC on the components, and finally separating by using acetonitrile-water as an eluent to obtain the following 3 flavonoid compounds: quercetin, luteolin, and 3-methoxyl quercetin, wherein the 3-methoxyl quercetin is obtained by separating folium Nelumbinis for the first time;

7) concentrating the n-butanol part obtained in the step 2) under reduced pressure to dryness, dissolving with distilled water, subjecting the obtained water solution to polyamide column chromatography, performing gradient elution with ethanol-water, each gradient elution being 3-5 times the column volume, collecting 30-50% ethanol eluate, concentrating and drying to obtain total polyphenol of lotus leaves;

8) the semi-preparative HPLC for the total polyphenol in the lotus leaves obtained in the step 7) takes methanol-water as a mobile phase, the flow rate is 1ml/min, effluents in different times are collected, and the polyphenol substances in the following 4 are obtained by final separation: catechin, epicatechin, Nympholide a2, and Nympholide B3.

The dosage of the extraction solvent in the step 1) is the mass of the medicinal materials: the volume of the solution is 1Kg and 10-40L; the ultrasonic power is 350W;

in the step 3), the temperature of ultrafiltration and nanofiltration are controlled to be 40-60 ℃, and the pressure is controlled to be 0.6-2.2 MPa;

in the step 5), the macroporous adsorption resin is AB-8 macroporous resin, the sample loading concentration is 20 mg/mL, the sample loading volume is 20mL, the diameter-height ratio is 1:30, and the ethanol concentration is 70%.

The invention also provides application of the lotus leaf total alkaloids obtained in the step 3) in preparing lipid-lowering and weight-losing medicines, foods or cosmetics.

The invention also provides the application of the lotus leaf total flavonoids obtained in the step 5) in the preparation of cosmetics.

The invention also provides application of the lotus leaf total polyphenol obtained in the step 7) in preparing cosmetics.

Compared with the prior art, the invention has the advantages that:

the invention provides a method for co-producing a plurality of active substances from lotus leaves, which comprises the following steps: extracting and extracting to obtain active substance parts, and then respectively separating and purifying the active substance parts to finally obtain 8 alkaloid monomers, 3 flavone monomers and 4 polyphenol monomers, wherein the lotus leaf total alkaloids, the lotus leaf total flavones and the lotus leaf total polyphenols prepared by the method can be directly applied to different fields. In the method, for the separation and extraction of the total alkaloids in the lotus leaves, a membrane separation technology is adopted and combined with medium-low pressure preparative liquid chromatography for separation, so that various alkaloid monomers are quickly and efficiently obtained; for the separation and purification of lotus leaf total flavonoids, the invention combines macroporous adsorption resin, Sephadex LH-20 column chromatography and semi-preparative HPLC for use, and obtains 3 flavonoid monomers by high-efficiency separation, wherein 3-methoxyl quercetin is separated from lotus leaves for the first time; for separation and purification of lotus leaf total polyphenol, polyamide column chromatography is combined with semi-preparative HPLC, and 4 polyphenol monomers are separated efficiently. The monomer compound provides important guarantee for further developing the available value of the lotus leaves.

The method for co-producing multiple active substances from lotus leaves optimizes the extraction and separation steps and reduces the production cost, the content of the active monomers obtained according to the method is more than 95 percent, and the recovery rate of the active ingredients is more than 70 percent.

Detailed Description