WO2022247097A1

WO2022247097A1 - Method for separating active ingredients from siraitia grosvenorii flowers

Info

Publication number: WO2022247097A1
Application number: PCT/CN2021/122932
Authority: WO
Inventors: 张宝堂; 谭家忠; 郑思佳; 蒋乐文; 范斌
Original assignee: 湖南绿蔓生物科技股份有限公司
Priority date: 2021-05-28
Filing date: 2021-10-09
Publication date: 2022-12-01
Also published as: CN113041279A; CN113041279B

Abstract

A method for separating active ingredients from Siraitia grosvenorii flowers, comprising: S1, extracting Siraitia grosvenorii flowers with water vapor to obtain wet flowers and a first extracting solution; S2, extracting the wet flowers with ethanol as a solvent at 25-65°C to obtain a second extracting solution and a first filter residue, concentrating the second extracting solution, and performing solid-liquid separation to obtain a third extracting solution and a wet residue; S3, concentrating the third extracting solution, and standing to obtain a crystal and a crystallization mother liquor; and S4, eluting a macroporous adsorption resin chromatographic column on the crystallization mother liquor sequentially with an acid solution, an alkali solution, and an alcohol solution to respectively obtain an acid eluent, an alkali eluent, and an alcohol eluent. According to the method, essential oil, flower dew, polysaccharide, Grosvenorine, alkaloid and triterpenoid saponin of Siraitia grosvenorii flowers can be obtained at the same time, and efficient comprehensive separation of Siraitia grosvenorii flowers is achieved.

Description

A method for isolating effective components from Luo Han Guo flowers

technical field

The invention relates to the field of extraction, separation and purification of biological functional components, in particular to a method for isolating active components from Luo Han Guo flowers.

Background technique

Siraitia grosvenorii (formerly known as Momordica grosvenorii), also known as Siraitia grosvenorii, is the fruit of the genus Siraitia grosvenorii in the vine family Cucurbitaceae. Mainly produced in Guilin, Guangxi, China, Yongzhou, Shaoyang, Huaihua, Hunan, Guangdong and other places.

Luo Han Guo flowers are unisexual, dioecious, inflorescence peduncle, pedicel, sepals, and petals are covered with pilose and glandular hairs, male flowers are axillary, and 5-10 flowers are arranged in racemes; bracts 1, oblong; calyx 5 lobed , lobes with linear pointed tail; petals 5, light yellow, slightly reddish, ovate, about 2 cm long, with pointed tail at apex; stamens 3, anthers separated. 1 piece with 1 room, and the other 2 pieces with 2 rooms. Female flowers solitary in leaf axils or 2-5 clustered at the top of the total pedicel, calyx tube apex 5-lobed, petals 5, obovate, apex short and pointed, ovary inferior, connate with calyx tube, styles 3, stigmas 2 divergent, with staminodes 3. The gourd is round, oblong or obovate, dark brown-red when young, blue when mature, and covered with hairs. The flowering period of Luo Han Guo is from June to August.

Luo Han Guo flowers contain polyphenols, phenolic acids, flavonols, flavanols, polysaccharides, volatile oils, alkaloids and other components. Among them, there are many studies on its flavonoids, but it is very important to systematically study the rational and effective utilization of Luo Han Guo flower resources. few.

Contents of the invention

The technical problem to be solved by the present invention is: how to realize efficient separation of components in Luo Han Guo flower.

In order to solve the above-mentioned technical problems, the present invention proposes a method for isolating active ingredients from Luo Han Guo flowers.

The present invention proposes a method for isolating active ingredients from Luo Han Guo flowers, comprising the following steps:

S1. Extracting the Luo Han Guo flower with water steam to obtain the wet flower and the first extract;

S2. Extracting the wet flower with ethanol as a solvent at 25-65°C to obtain a second extract and a first filter residue, concentrating the second extract and separating solid and liquid to obtain a third extract and a wet residue;

S3. Concentrating the third extract and standing still to obtain crystals and crystallization mother liquor;

S4. Put the crystallization mother liquor on a macroporous adsorption resin chromatography column, and sequentially elute with an acid solution, an alkali solution and an alcohol solution to obtain an acid eluent, an alkali eluent and an alcohol eluent, respectively.

Further, in step S2, the wet flower and ethanol with a volume concentration of 50-95% are used as solvents at a mass ratio of 1:3-1:5, and extracted at 25-65°C for 30-60 minutes to obtain the first Two extracts and the first filter residue.

Further, it also includes extracting the first filter residue obtained in step S2 with water as a solvent, wherein the ratio of the first filter residue to water is 1:1-1:5, and the extraction temperature is 80-100°C , the extraction time is 30-120 min; and then filtered to obtain the fourth extract and the second filter residue.

Further, it also includes concentrating the fourth extract to a solid content of 30-40%, adding protease for enzymatic hydrolysis, followed by inactivation, and then adding alcohol solution to precipitate polysaccharides.

Further, it also includes drying the second filter residue until the water content is 30-35%, and then adding nutrients and yeast to ferment to obtain the feed material of Luo Han Guo flower dregs.

Further, in step S1, using the water vapor extraction specifically includes: extracting the Luo Han Guo flowers for 0.1-3 hours with water vapor at a pressure below 0.5 MPa.

Further, in step S1, it also includes separating the first extract from oil and water to obtain essential oil and water phase solution; the oil-water separation includes: cooling the obtained first extract to 5-40°C, and then using oil-water The separator separates the first extraction solution, and the oil phase passes through a chromatographic column filled with a decolorizing agent, and the weight of the decolorizing agent is 0.5-2% of the oil weight.

Further, in step S1, it also includes concentrating the aqueous phase solution to 2%-20% of the weight of the Luo Han Guo flower raw material to obtain Luo Han Guo flower dew.

Further, in step S4, it also includes adjusting the pH value of the alkaline eluent to acidic or neutral, concentrating and performing crystallization and recrystallization to obtain Mogroside; and/or, also including acid washing The pH is adjusted to alkaline by dehydration, and then the alkaloids are obtained by extraction with an organic solvent.

Further, in step S4, it also includes decolorizing the alcohol eluent with silicate to remove impurities, collecting the effluent, recovering the alcohol to the water phase, and then adjusting the pH value to weakly acidic or neutral, concentrating and drying to obtain three Terpene saponins.

Compared with the prior art, the beneficial effects of the present invention include: firstly, the Luo Han Guo flower is extracted with water steam, which evenly destroys the cell structure of the Luo Han Guo flower, which is beneficial to the subsequent extraction of the flavonoids of the Luo Han Guo flower, and obtains the first flavonoid that can be used to separate essential oil and pure dew. The first extract, and then the wet flowers are reflux extracted with ethanol as a solvent at 25-65°C to obtain the second extract. The temperature is lower, and the active components in the flowers can enter the second extract without destroying the structure. Concentrate the second extract and separate the solid and liquid to obtain the third extract and wet residue, most of the polyphenols enter the wet residue, then concentrate the third extract and let it stand to obtain crystals and crystallization mother liquor, the crystal is kaempferol, continue Put the crystallization mother liquor on the macroporous adsorption resin chromatography column, and successively elute with acid solution, alkali solution and alcohol solution in a specific order to obtain respectively the acid eluate containing alkaloids, the alkali eluate containing kaempferi glycosides and the eluate containing Alcohol eluent of triterpene saponins, the first extract containing essential oil and flower dew, polyphenols, kaempferol, acid eluate containing alkaloids, containing kaempferia are sequentially separated from Luo Han Guo flowers by the method proposed by the present invention The alkali eluent of total glycosides and the alcohol eluent containing triterpenoid saponins realize the efficient and comprehensive extraction of Luo Han Guo flowers, and the extraction rate of kaempferol is as high as 93.95%.

In addition, after other steps of treatment, the extraction rate of polyphenols obtained is as high as 97.75%, the extraction rate of alkaloids is as high as 91.91%, the content of mogroside is as high as 98.65%, the extraction rate of triterpenoid saponins is as high as 95.42%, and the extraction rate of polysaccharides is as high as 94.56%.

Detailed ways

This specific embodiment proposes a method for isolating active ingredients from Luo Han Guo flowers, comprising the following steps:

S1. Extract the flowers of Luo Han Guo with steam, specifically, extract the flowers of Luo Han Guo with water vapor at a pressure of 0.5 MPa or less for 0.1-3 hours to obtain wet flowers and a first extract; cool the first extract to 5-40°C , and then use an oil-water separator to separate the first extract to obtain the essential oil and an aqueous phase solution, and then use a reverse osmosis membrane or a nanofiltration membrane to concentrate the aqueous phase to 2%-20% of the weight of the Luo Han Guo flower raw material to obtain Luo Han Guo flower dew; Specifically, the first extract is passed through a chromatographic column filled with a decolorizing agent, and the weight of the decolorizing agent is 0.5-2% of the oil weight to obtain essential oil and pure dew; the decolorizing agent is anhydrous sodium sulfate, aluminum oxide, chlorine One or more of sodium chloride, calcium chloride, activated carbon, magnesium oxide, magnesium carbonate, silicon oxide, silica gel, attapulgite clay, attapulgite, kaolin, bentonite, diatomaceous earth and perlite; further, the The preferred mass ratio of the decolorizing agent is anhydrous sodium sulfate, magnesium oxide and activated carbon of 1:1:1;

S2. Using the wet flower and ethanol with a volume concentration of 50-95% as a solvent according to the mass ratio of 1:3-1:5, reflux extraction at 25-65° C. for 30-60 minutes to obtain the second extract and the first filter residue , concentrating the second extract and separating the solid and liquid to obtain the third extract and wet residue, sending the wet residue to vacuum drying to obtain a brown solid containing polyphenols; using water as the solvent for the first filter residue by continuous countercurrent extraction method, wherein the ratio of the first filter residue to water is 1:1 to 1:5, the extraction temperature is 80 to 100°C, and the extraction time is 30 to 120 minutes; filter residue; then concentrate the fourth extract to a solid content of 30-40%, add protease for enzymolysis reaction, then inactivate, then add alcohol solution to precipitate polysaccharide precipitation; then dry the second filter residue to The moisture content is 30-35%, and then add nutrients and yeast to ferment to obtain the feed material of Luo Han Guo flower residue; preferably, keep the moisture content of residue at 20-40%, add nutrients according to the wet weight of flower residue 5-20%, Add yeast at 0.03-0.1% of the wet weight of the slag, and ferment naturally for 4-8 days under airtight conditions. Use the boiler exhaust gas as a heat source to dry the fermented flower slag in vacuum for 6-24 hours, crush it through a 40-mesh sieve with a crusher, and obtain the Luo Han Guo flower Slag feed raw material; the nutrient component is preferably corn starch;

The solid-liquid separation process is that the second extraction liquid is passed through the disc centrifuge, the centrifugal liquid is filtered with a ceramic membrane, the retained liquid of the ceramic membrane is passed through the disc centrifuge again, and the centrifugal liquid is sent to the ceramic membrane for filtration. For the first centrifugal slag of the disc centrifuge, 2-3 times the water is fully stirred and mixed, and then sent to the disc centrifuge again, and the centrifugal liquid is sent to the ceramic membrane for filtration, and the second centrifugal slag, the first centrifugal slag and the second centrifugal slag are collected is wet residue;

S3. Concentrate the third extract and let stand to obtain crystals and crystallization mother liquor; collect the third extract obtained by solid-liquid separation and concentrate to a relative density of 1.10-1.25, cool to 0-10°C, crystallize for 5-24h, and filter Afterwards, the crystals were recrystallized, and then vacuum-dried to obtain kaempferol. Preferably, the kaempferol recrystallization solvent is 20-40% ethanol aqueous solution containing 0.1-0.3% sodium bicarbonate;

S4. Put the crystallization mother liquor on a macroporous adsorption resin chromatography column, and sequentially elute with acid solution, alkali solution and alcohol solution to obtain acid eluent, alkali eluent and alcohol eluent respectively; The removal is elution with an acid with a volume concentration of 0.1-1%, and the acid eluate after the acid elution is collected, and the acid is one or more of hydrochloric acid, sulfuric acid or oxalic acid; the alkali elution is with a volume concentration 0.1-1% alkali elution, the alkali is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate; the alcohol solution elution is 30-65% volume concentration The ethanol solution is eluted; the pH of the acid eluent is adjusted to alkaline, and then the alkaloid is obtained by extraction with an organic solvent immiscible with the acid eluent, and the organic phase is collected, concentrated and dried to obtain the alkaloid of Luo Han Guo flower; Adjust the pH value of the alkali eluent to acidic or neutral, crystallize and recrystallize after concentration, filter and dry the crystals to obtain mogrosin, preferably, the recrystallization solvent of mogrosin is 5-25% ethanol containing 0.02-0.1% sodium bicarbonate aqueous solution, filter the obtained mother liquor and dry to obtain kaempferol glycosides, that is, a class of flavonoid glycosides with kaempferol as aglycone; decolorize the alcohol eluate with silicate to remove impurities, collect the effluent, recover the alcohol to the water phase, and then Adjust the pH value to weakly acidic or neutral, concentrate and dry to obtain triterpene saponins, preferably, the alcohol eluent is decolorized using a magnesium trisilicate chromatography column. In some preferred embodiments, after washing the column with acid solution and alkaline solution, 1-10BV of water will be used to wash the column, and these water washing solutions will be selectively combined into the eluent of acid solution or eluent of alkaline solution or directly discharged .

The macroporous adsorption resin is preferably a styrene-divinylbenzene type resin whose skeleton structure contains one or more groups such as amides, sulfoxides, and cyano groups, and a skeleton structure containing one or more groups such as amides, sulfoxides, and cyano groups. Or a variety of polyacrylic acid resin groups.

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, specific implementations of the present invention will be described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific implementations disclosed below.

Example 1

This embodiment proposes a method for isolating active ingredients from Luo Han Guo flowers, comprising the following steps:

S1, get 10kg of dry male flowers of Luo Han Guo, extract the flowers of Luo Han Guo with a water steam generator, the steam pressure is 0.25MPa, the extraction time is 0.5h, the steam is cooled to 5-10°C with condensed water, and the oil-water mixture (i.e. the first step) is separated with an oil-water separator - extract) to separate to obtain yellow essential oil and aqueous phase solution, the oil phase is filled with a plastic small column of 2g decolorant, wherein the decolorant is composed of diatomaceous earth, anhydrous sodium sulfate, magnesium oxide, a mixture of gac, Obtain 236g of light yellow Luo Han Guo flower essential oil, and 53L of aqueous phase solution obtained by separating the oil-water separator is concentrated to 1L with a 500 Dalton nanofiltration membrane to obtain Luo Han Guo flower dew;

S2. Extract the Luo Han Guo flowers obtained after water vapor extraction (ie wet flowers) with 90% ethanol at 30° C. for 1 hour, and extract twice. Filtrate the second extract and the first filter residue, concentrate the second extract under reduced pressure and vacuum to recover ethanol to the water phase, filter with filter paper between plate and frame, and obtain the third extract and wet residue, and send the obtained wet residue to vacuum drying, - 0.08 to -0.09MPa, vacuum drying at 40-50°C for 4 hours to obtain a brown solid, which was pulverized through an 80-mesh sieve to obtain 537g of brown powder. The polyphenol content was 32.40% as detected by UV spectroscopy. The clear liquid obtained by plate and frame filtration (ie the third extract) is concentrated to a relative density of about 1.1, cooled to 3-5°C, and placed for 10 hours, crystals are precipitated, filtered, the crystals are rinsed with a small amount of cold water, and the filtrate is rinsed with cold water The liquefied liquids are combined into a crystallization mother liquor for use, and the wet crystals are heated and dissolved with 0.1% sodium bicarbonate 20% ethanol aqueous solution that is 5 times the weight of the wet crystals, cooled to 3-5°C, left for 4 hours, and golden yellow crystals are precipitated, filtered and dried in vacuo to obtain 153g of yellow powder, detected by HPLC, the content of kaempferol is 98.35%;

S3. Put the crystallization mother liquor of step S2 on a 10L macroporous adsorption resin chromatography column, the resin model is LX-38, and the column loading flow rate is 2BV/h. After the column loading is completed, wash the column with 2BV of water, drain the effluent, and then use 4BV0.2% sulfuric acid Wash the chromatographic column with aqueous solution, collect the acid eluent, then wash 2BV with water, drain the water wash, then wash the chromatographic column with 4BV of 0.1% sodium hydroxide aqueous solution, collect the alkali eluent, then wash 2BV with water, and wash the chromatographic column with water Drain, and finally elute with 2BV40% ethanol aqueous solution. The above elution flow rate is 2BV/h;

S4. The collected pickling solution was adjusted to pH 10 with aqueous sodium hydroxide solution, and the aqueous solution was extracted three times with chloroform, the first time was 40 L, the second time was 20 L, and the third time was 10 L. Combine chloroform, add a small amount of water, concentrate under reduced pressure to about 12 Baumé, and dry to obtain 59 g of yellow powder. The alkaloid content is 82.70% as detected by UV spectrophotometry;

S5. After adjusting the pH value of the collected alkali eluent to 7 with hydrochloric acid aqueous solution, concentrate it under reduced pressure to 15 Baume, cool to 1-5°C, and place it for 20 hours. Crystals are precipitated, filtered, and the wet crystals are 3 times the weight of wet crystals. Heat and dissolve 0.05% sodium bicarbonate in 10% ethanol aqueous solution, cool to 1-5°C, place for 10h, precipitate golden yellow crystals, filter and vacuum dry to obtain 71g golden yellow powder, the content of mogrosin is 98.4% as detected by HPLC. The mother liquor produced by filtering during the crystallization process of mogrosanthin was concentrated under reduced pressure to 12 degrees Baume, and spray-dried to obtain 352 g of yellow kaempferia powder. The content of mogrosides kaempferol was detected by UV spectrophotometry to be 52.72%.

S6, the alcohol desorption liquid of collection, passes the chromatographic column decolorization that 100g magnesium trisilicate is housed, collects effluent, concentrates under reduced pressure to 15 degrees Baume, sprays and dries to obtain 517g light yellow powder, detects three through UV spectrophotometry Terpene saponin content is 73.90%;

S7. After flavonoids are extracted, Luo Han Guo flower (i.e. the first filter residue) is extracted with water at a material-to-liquid ratio of 1:4 and an extraction temperature of 100°C for 1 hour, and the fourth extract and the second filter residue are obtained by filtration, and the fourth extract is extracted Concentrate to 5 degrees Baume, add bromelain, adjust pH to 4 with citric acid, enzymolyze at 45°C for 5 hours, heat to boiling, keep for 0.5 hours to inactivate bromelain, concentrate the inactivated solution under reduced pressure to 18 waves Mido, add 3 times the amount of 95% ethanol and stir, filter the polysaccharide after precipitation, rinse with an appropriate amount of 95% ethanol, vacuum dry, grind through an 80-mesh sieve to obtain 211g off-white polysaccharide powder, and detect the polysaccharide content by UV spectrophotometry 81.73%;

S8. Squeeze part of the water from the Luo Han Guo flower dregs (i.e. the second filter residue), keep the moisture in the dregs at about 30-35%, add corn flour with a wet weight of 5% of the dregs, add yeast with a wet weight of 0.03% of the dregs, and seal Under the condition of natural fermentation for 7 days, the fermented flower dregs were dried under vacuum for 12 hours using boiler exhaust gas as a heat source, and crushed by a crusher through a 40-mesh sieve to obtain 6.1KG of Luo Han Guo flower dregs feed raw material.

Example 2

S1. Take 50kg of fresh male flowers of Luo Han Guo, extract the flowers of Luo Han Guo with steam generated by a steam generator, the steam pressure is 0.1 MPa, and the extraction time is 1 h. The steam is cooled to 10-15°C with condensed water, and the oil-water mixture is separated with an oil-water separator to obtain yellow essential oil. The oil phase is passed through a plastic column filled with 10g of decolorizing agent, and the composition of the decolorizing agent is anhydrous sodium sulfate: oxidation Magnesium: Activated carbon = 1:1:1 mixture to obtain 294g of light yellow Luo Han Guo flower essential oil. The 170L water phase obtained by separation by the oil-water separator was concentrated to 1L with a 500 Dalton nanofiltration membrane to obtain Luo Han Guo flower dew.

S2. Extract the Luo Han Guo flowers obtained after steam extraction with countercurrent extraction, the extraction solvent is 80% ethanol, the extraction temperature is 25°C, the ratio of solid to liquid is 1:2, and the extraction time is 0.5h, and the second extraction solution and the first extraction solution are filtered. filter residue. The obtained second extract is concentrated under reduced pressure and vacuum to recover ethanol to the water phase, centrifuged at 4500r/min in a butterfly centrifuge, and the centrifuged liquid passes through a ceramic membrane with a pore size of 50nm. Pass through the butterfly centrifuge again, and then pass through the ceramic membrane, collect the secondary butterfly centrifuge slag and combine with the secondary ceramic membrane retentate, then send it to vacuum drying, -0.08 to -0.09MPa, vacuum drying at 45-55°C for 10h, A brown solid was obtained, which was pulverized and passed through an 80-mesh sieve to obtain 521 g of brown powder, which was detected by UV spectrometry as a polyphenol content of 43.50%. Concentrate the ceramic membrane filter solution to a relative density of about 1.15, cool it to 0-4°C, and place it for 5 hours. Crystals are precipitated, filtered, and the crystals are rinsed with a small amount of cold water. The crystals were heated and dissolved with 0.15% sodium bicarbonate in 40% ethanol aqueous solution 10 times the weight of wet crystals, cooled to 3-5°C, placed for 8 hours, yellow crystals were precipitated, filtered and vacuum-dried to obtain 135g yellow powder, detected by HPLC, kaempferol The content is 98.19%.

S3. Put the crystallization mother liquor in step S2 on a 15L macroporous adsorption resin chromatography column, the resin model is LX-91, and the flow rate on the column is 2BV/h. After the column is completed, wash the column with 2BV of water, drain the effluent, and then use 2BV0.5% Wash the chromatography column with hydrochloric acid aqueous solution, collect the hydrochloric acid eluate, then wash 2BV with water, drain the water wash, then wash the chromatography column with 2BV0.15% potassium hydroxide aqueous solution, collect the potassium hydroxide eluate, and then wash with 2BV water , the washing solution was drained, and finally desorbed with 2BV50% ethanol aqueous solution. The above elution flow rates are all 2BV/h.

S4. The collected hydrochloric acid washing solution was adjusted to pH 8 with aqueous sodium hydroxide solution, and the aqueous solution was extracted with dichloromethane 3 times, 40 L for the first time, 20 L for the second time, and 10 L for the third time. Combine dichloromethane, add a small amount of water, concentrate under reduced pressure to about 12 Baumé, and dry to obtain 72 g of yellow powder. The alkaloid content is 80.42% as detected by UV spectrophotometry.

S5. After adjusting the pH value of the collected potassium hydroxide eluent to 6 with aqueous hydrochloric acid solution, concentrate under reduced pressure to 14 Baume, cool to 1-5°C, and place it for 15 hours. Crystals are precipitated, filtered, and wet crystals with 2 times wet Heat and dissolve 0.02% sodium bicarbonate in 15% ethanol aqueous solution of crystal weight, cool to 1-5°C, place for 10h, precipitate golden yellow crystals, filter and vacuum dry to obtain 83g golden yellow powder, and the content of mogrosin is 98.65% through HPLC %. The mother liquor produced during the crystallization process of Mogrosvenin was filtered, concentrated under reduced pressure to 13 degrees Baume, and spray-dried to obtain 425g of golden yellow kaempferia powder. The content of mogrosides kaempferol was detected by UV spectrophotometry to be 50.40%.

S6, the collected 50% ethanol desorption solution is decolorized through a chromatographic column equipped with 100g attapulgite clay, the effluent is collected, the effluent is collected, concentrated under reduced pressure to 15 degrees Baume, and spray-dried to obtain 579g light yellow powder, which is subjected to UV The content of triterpene saponins detected by spectrophotometry was 71.83%.

S7. The Luo Han Guo flower after flavonoid extraction (i.e. the first filter residue) is extracted at a material-to-liquid ratio of 1:5 and an extraction temperature of 90° C. for 1 hour. The filtered extract is concentrated to 5 degrees Baume, and bromelain and Aspergillus niger are added. Protease mixture, citric acid to adjust the pH to 4, enzymolysis at 45°C for 2 hours, heat to boiling, keep for 0.5 hours to inactivate the enzyme, concentrate the inactivation solution under reduced pressure to 20 Baume degrees, add 4 times the amount of 95 Stir with % ethanol, filter the polysaccharide after precipitation, rinse with an appropriate amount of 95% ethanol, vacuum dry, and pulverize through an 80-mesh sieve to obtain 204g of white polysaccharide powder. The polysaccharide content is 90.68% as detected by UV spectrophotometry.

S8. Squeeze part of the water from the Luo Han Guo flower dregs, keep the water in the dregs at about 30-35%, add sugarcane honey with a wet weight of 5% of the dregs, add yeast with a wet weight of 0.1% of the dregs, and ferment naturally for 5 days under airtight conditions , using boiler tail gas as heat source, vacuum-dried the fermented flower dregs for 24 hours, crushed them through a 40-mesh sieve with a crusher, and obtained 5.4KG of Luo Han Guo flower dregs feed material.

Example 3

S1. Take 50kg of fresh male flowers of Luo Han Guo, extract the flowers of Luo Han Guo with steam generated by a water steam generator, the steam pressure is 0.2 MPa, and the extraction time is 1.5 h. The steam is cooled to 20-25°C with condensed water, and the oil-water mixture is separated with an oil-water separator to obtain yellow essential oil. The oil phase is passed through a plastic column filled with 15g of decolorizing agent, wherein the decolorizing agent is composed of anhydrous sodium sulfate and active The mixture of white clay, obtains 280g light yellow Luo Han Guo flower essential oil. The 260L water phase separated by the oil-water separator was concentrated to 1L with a 500 Dalton nanofiltration membrane to obtain Luo Han Guo flower dew.

S2. The Luo Han Guo flowers obtained after steam extraction were extracted by countercurrent extraction, the extraction solvent was 90% ethanol, the extraction temperature was 40°C, the ratio of solid to liquid was 1:3, and the extraction time was 40 minutes. Concentrate the extract under reduced pressure and vacuum to recover ethanol to the water phase, filter with plate and frame filter paper, and send the filter residue to vacuum drying at -0.08 to -0.09MPa, 55-60°C for 8 hours to obtain a brown solid, which is crushed through a 80-mesh sieve 506 g of brown powder was obtained, and the content of polyphenols detected by UV spectrometry was 45.40%. The plate and frame filtrate is concentrated to a relative density of about 1.2, cooled to 5-10°C, and placed for 15 hours, crystals are precipitated, filtered, and the crystals are rinsed with a small amount of cold water. 10 times the weight of wet crystals in 20% ethanol was heated and dissolved, cooled to 3-5°C, and left for 2 hours to precipitate golden yellow crystals, which were filtered and vacuum-dried to obtain 149 g of yellow powder, which was detected by HPLC with a kaempferol content of 95.21%.

S3. Put the crystallization mother liquor in step S2 on a 10L macroporous adsorption resin chromatography column, the resin model is D101C, and the column flow rate is 2BV/h. After the column is completed, then wash the chromatography column with 3BV1% oxalic acid aqueous solution, and collect the oxalic acid eluent. Then wash the chromatographic column with 3BV1% potassium carbonate aqueous solution, collect the potassium carbonate eluate, and finally desorb with 1.5BV65% ethanol aqueous solution. The above elution flow rates are all 2BV/h.

S4. The collected oxalic acid eluate was adjusted to pH 9 with aqueous calcium hydroxide solution, and the aqueous solution was extracted with ethyl acetate for 3 times, 40 L for the first time, 20 L for the second time, and 10 L for the third time. Combine the ethyl acetate, add a small amount of water, concentrate under reduced pressure to about 12 Baume, and dry to obtain 62 g of yellow powder. The alkaloid content is 72.92% as detected by UV spectrophotometry.

S5. After adjusting the pH value of the collected potassium carbonate eluent to 5 with hydrochloric acid aqueous solution, concentrate under reduced pressure to 12 Baumé, cool to 1-5°C, and stand for 18 hours. Crystals precipitate, filter, and use 3 times wet crystals for wet crystals. Heat and dissolve 0.08% sodium bicarbonate in 5% ethanol aqueous solution by weight, cool to 1-5°C, place for 10h, precipitate golden yellow crystals, filter and vacuum dry to obtain 65g golden yellow powder, and the content of Mogroside is 98.22% as detected by HPLC . The mother liquor produced during the crystallization process of mogroside flavins was filtered, concentrated under reduced pressure to 15 degrees Baume, and spray-dried to obtain 417g of golden yellow kaempferia glucosides powder. The content of mogrosides kaempferia glycosides was detected by UV spectrophotometry to be 49.72%.

S6, the collected 65% ethanol desorption solution is decolorized through a chromatographic column equipped with 100g attapulgite clay, the effluent is collected, the effluent is collected, concentrated under reduced pressure to 18 degrees Baume, and spray-dried to obtain 672g light yellow powder, which is subjected to UV The content of triterpene saponins detected by spectrophotometry was 63.90%.

S7. The Luo Han Guo flower after extracting flavonoids is extracted twice at a material-to-liquid ratio of 1:5, at an extraction temperature of 85°C, for 2 hours, and the extract is passed through a ceramic membrane with a pore size of 100nm, and the membrane is passed through an ultrafiltration of 100,000 Daltons Membrane, the filter solution was concentrated under reduced pressure to 18 Baume, added 6 times the amount of 90% ethanol and stirred, the polysaccharide was precipitated, filtered, rinsed with an appropriate amount of 90% ethanol, vacuum-dried, and crushed through an 80-mesh sieve to obtain 104g of white polysaccharide powder , the content of polysaccharide detected by UV spectrophotometry is 91.75%.

S8. Squeeze part of the water from the Luo Han Guo flower dregs, keep the water in the dregs at about 35-40%, add starch with a wet weight of 20% of the dregs, and yeast with a wet weight of 0.08% of the dregs, and ferment naturally for 8 days under airtight conditions. The fermented flower dregs were dried in vacuum for 24 hours using boiler exhaust gas as a heat source, and crushed by a crusher through a 40-mesh sieve to obtain 7.1KG of Luo Han Guo flower dregs feed material.

Comparative example 1

T1. Take 10 kg of dried Luo Han Guo flowers and extract them twice with 40 L of 95% ethanol at 40° C. for 1 hour. Filtrate the extract, concentrate under reduced pressure and vacuum to recover ethanol to the water phase, filter with plate and frame with filter paper, and send the obtained wet residue to vacuum drying, -0.08 to -0.09MPa, vacuum drying at 40-50°C for 10h, to obtain a brown sticky paste Solid, cannot be dried into powder. The clear liquid obtained by plate and frame filtration is concentrated to a relative density of about 1.1, cooled to 3-5°C, and placed for 10 hours, crystals are precipitated, filtered, and the crystals are rinsed with a small amount of cold water, and the filtrate and the cold water rinse are combined to form a crystal mother liquor for use , the wet crystals were heated and dissolved with 20% ethanol aqueous solution that was 15 times the weight of the wet crystals, cooled to 3-5°C, placed for 4 hours, yellow crystals were precipitated, filtered and vacuum-dried to obtain 148g yellow powder, and the content of kaempferol was 90.41% as detected by HPLC .

T2. Put the crystallization mother liquor in step 1 on a 15L macroporous adsorption resin chromatography column, the resin model is LX-91, and the flow rate of the column is 2BV/h. After the column is completed, wash the column with 2BV of water, drain the effluent, and then oxidize with 0.15% hydrogen at 2BV Wash the chromatographic column with potassium aqueous solution, collect the eluent of potassium hydroxide, wash with 2BV of water, drain the washing liquid, then wash the chromatographic column with 2BV of 0.5% hydrochloric acid aqueous solution, collect the eluent of hydrochloric acid, wash 2BV with water, wash with water The liquid was drained, and finally desorbed with 2BV50% ethanol aqueous solution. The above elution flow rates are all 2BV/h.

T3. After adjusting the pH value of the collected potassium hydroxide eluent to 6 with aqueous hydrochloric acid solution, it was concentrated under reduced pressure to 15 Baume, cooled to 1-5°C, and left for 20 hours. No crystals were precipitated, and spray-dried to obtain 624g of golden yellow kaempferia. Glycoside powder, the total glycoside content of Mogros grosvenori kaempferi is 26.72% as detected by UV spectrophotometry.

T4. The collected hydrochloric acid washing solution was adjusted to pH 8 with aqueous sodium hydroxide solution, and the aqueous solution was extracted 3 times with dichloromethane, 40 L for the first time, 20 L for the second time, and 10 L for the third time. Combine dichloromethane, add a small amount of water, concentrate under reduced pressure to an extract, and dry to obtain 18 g of yellow powder. The alkaloid content is 89.64% as detected by UV spectrophotometry.

T5, the collected 50% ethanol desorption solution is decolorized through a chromatographic column equipped with 100g attapulgite clay, the effluent is collected, concentrated under reduced pressure to 15 degrees Baume, and spray-dried to obtain 452g light yellow powder, which is detected by UV spectrophotometry The content of triterpene saponins is 77.63%.

Comparative example 2

T1. Take 50kg of fresh male flowers of Luo Han Guo, extract them twice with 90% ethanol at 80°C for 3 hours, and the ratio of solid to liquid is 1:5. Filtrate the extract, concentrate under reduced pressure and vacuum to recover ethanol to the water phase, filter with plate and frame with filter paper, and send the obtained wet residue to vacuum drying, -0.08 to -0.09MPa, vacuum drying at 40-50°C for 10h, to obtain a brown sticky paste Solid, unable to be dried into powder, the polyphenol content detected by UV spectrometry is 32.4%. The clear liquid obtained by plate and frame filtration is concentrated to a relative density of about 1.1, cooled to 3-5°C, and placed for 10 hours, crystals are precipitated, filtered, and the crystals are rinsed with a small amount of cold water, and the filtrate and the cold water rinse are combined to form a crystal mother liquor for use , the wet crystals were heated and dissolved with 20% ethanol aqueous solution that was 15 times the weight of the wet crystals, cooled to 3-5°C, placed for 4 hours, yellow crystals were precipitated, filtered and vacuum-dried to obtain 154g yellow powder, and the content of kaempferol was 86.15% as detected by HPLC .

T2. Put the crystallization mother liquor in step 1 on a 15L macroporous adsorption resin chromatography column, the resin model is LX-91, and the flow rate of the column is 2BV/h. After the column is completed, wash the column with 2BV of water, drain the effluent, and then desorb with 2BV50% ethanol aqueous solution , then wash the chromatography column with 2BV0.5% hydrochloric acid aqueous solution, collect the hydrochloric acid eluate, then wash 2BV with water, drain the water washing liquid, and finally wash the chromatography column with 2BV0.15% potassium hydroxide aqueous solution, collect potassium hydroxide to elute solution, and then washed with 2BV water, and the washing solution was drained. The above elution flow rates are all 2BV/h.

T3, the collected 50% ethanol desorption solution is decolorized through a chromatographic column equipped with 100g attapulgite clay, the effluent is collected, concentrated under reduced pressure to 15 degrees Baume, and spray-dried to obtain 1054g yellow powder, which is tested by UV spectrophotometry for three The terpene saponin content is 35.54%.

T4. The collected hydrochloric acid washing solution was adjusted to pH 8 with aqueous sodium hydroxide solution, and the aqueous solution was extracted 3 times with dichloromethane, 40 L for the first time, 20 L for the second time, and 10 L for the third time. Combine dichloromethane, add a small amount of water, concentrate under reduced pressure to the extract, the weight of the extract is too small to be ignored.

T5. After the collected potassium hydroxide eluate was adjusted to a pH value of 6 with aqueous hydrochloric acid, it was concentrated under reduced pressure into an extract. The weight of the extract was too small to be ignored.

The content and extraction rate of each extract of table 1 embodiment 1-3 and comparative example 1-2

The dried flower raw material that embodiment 1 and comparative example 1 are used, all the other used fresh flower raw materials.

Comparative examples 1 and 2 did not carry out the water vapor extraction of the S1 step in the embodiment, and the acid solution washing in the S3 step in the embodiment, the alkali solution washing, and the sequence of the alcohol solution washing were adjusted, and neither S5 nor Step S6.

It can be seen from Table 1 that because the comparative example did not carry out the steam treatment of step S1, the polyphenols of Luo Han Guo flowers could not be dried into powder, and it was difficult to form products. product. The S5 step of the embodiment was not carried out, so two products of mogroside and kaempferenin were lost.

In the examples, the high-temperature and high-pressure water vapor treatment of the S1 step is carried out, which evenly destroys the cell structure of the Luo Han Guo flower, which is conducive to improving the subsequent extraction speed of the flavonoids of the Luo Han Guo flower, so it takes less time, less solvent consumption, and low extraction temperature. efficient. In comparative example 1 without water vapor treatment, under the same extraction conditions, the extraction rate of the obtained kaempferol and triterpene saponins is about 10-15% less than that of the examples. In comparative example 2, by increasing the extraction temperature and prolonging the extraction time, the extraction rate is increased. The extraction rate of embodiment could be reached only after solvent consumption,

In Comparative Example 1, the steps of washing with acid solution and washing with alkali solution in step S3 in the embodiment were changed to the next position, which caused some alkaloid components to enter the alkali washing solution, although the alkaloid content in the subsequent pickling solution was somewhat different. improved, but the extraction rate decreased from 70-90% to 29%. Due to the high impurities in the alkaline washing solution, the mogroside could not be crystallized, so one product was missing, and the impurity components entered the total glycosides of kaempferia, resulting in The content of total glycosides of kaempferia is low.

Comparative Example 2 puts the alcohol solution washing in the S3 step in the embodiment into the acid solution washing, and the front of the alkali solution washing process, resulting in acid solution washing, and the components of the alkali solution washing are mostly eluted by the alcohol solution, and alkaloids, Both mogroside and kaempferenin have entered into the triterpenoid saponin components, resulting in the loss of 3 products, and the content of triterpenoid saponins has also been reduced to 35%.

It can be seen that without water vapor extraction and/or without elution according to the elution sequence proposed by the present invention, it is not conducive to the comprehensive extraction and utilization of Luo Han Guo flowers.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

Claims

A method for isolating active ingredients from Luo Han Guo flowers, characterized in that it comprises the following steps:

S1. Extracting the Luo Han Guo flower with water steam to obtain the wet flower and the first extract;

S2. Extracting the wet flower with ethanol as a solvent at 25-65°C to obtain a second extract and a first filter residue, concentrating the second extract and separating solid and liquid to obtain a third extract and a wet residue;

S3. Concentrating the third extract and standing still to obtain crystals and crystallization mother liquor;

S4. Put the crystallization mother liquor on a macroporous adsorption resin chromatography column, and sequentially elute with an acid solution, an alkali solution and an alcohol solution to obtain an acid eluent, an alkali eluent and an alcohol eluent, respectively.
The method according to claim 1, characterized in that, in step S2, the wet flower and ethanol with a volume concentration of 50-95% are used as solvents according to the mass ratio of 1:3-1:5, at 25-65 Extract at ℃ for 30-60 minutes to obtain the second extract and the first filter residue.
The method according to claim 1, further comprising extracting the first filter residue obtained in step S2 with water as a solvent, wherein the ratio of the first filter residue to water is 1:1-1 :5, the extraction temperature is 80-100° C., and the extraction time is 30-120 min; then filter to obtain the fourth extract and the second filter residue.
The method according to claim 3, further comprising concentrating the fourth extract to a solid content of 30-40%, adding protease for enzymatic hydrolysis, then inactivating, and then adding alcohol solution to precipitate Polysaccharide precipitation.
The method according to claim 3, further comprising drying the second filter residue until the water content is 30-35%, and then adding nutrients and yeast to ferment to obtain the feed material of Luo Han Guo flower dregs.
The method according to claim 1, characterized in that, in step S1, using the water vapor extraction specifically comprises: extracting the Luo Han Guo flowers for 0.1-3 hours with water vapor at a pressure below 0.5 MPa.
The method according to claim 1, characterized in that, in step S1, further comprising separating the first extraction liquid from oil and water to obtain an essential oil and an aqueous phase solution; said oil-water separation includes: separating the obtained first extraction liquid Cool to 5-40° C., and then use an oil-water separator to separate the first extract, and the oil phase passes through a chromatographic column filled with a decolorizing agent, and the weight of the decolorizing agent is 0.5-2% of the oil weight.
The method according to claim 7, characterized in that, in step S1, further comprising concentrating the aqueous phase solution to 2%-20% of the weight of the Luo Han Guo flower raw material to obtain Luo Han Guo flower dew.
The method according to claim 1, characterized in that, in step S4, further comprising adjusting the pH value of the alkali eluent to acidic or neutral, concentrating and performing crystallization and recrystallization to obtain mogroside; and Or, it also includes adjusting the pH of the acid eluent to alkaline, and then extracting with an organic solvent to obtain the alkaloid.
The method according to claim 1, characterized in that, in step S4, it also includes decolorizing the alcohol eluent with silicate to remove impurities, collecting the effluent, recovering the alcohol to the water phase, and then adjusting the pH value to Weakly acidic or neutral, concentrated and dried to obtain triterpene saponins.