CN105832789A - Method for preparing saponin enriched product through high-pressure microwave assisted semi-bionic extraction - Google Patents

Method for preparing saponin enriched product through high-pressure microwave assisted semi-bionic extraction Download PDF

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CN105832789A
CN105832789A CN201610322779.8A CN201610322779A CN105832789A CN 105832789 A CN105832789 A CN 105832789A CN 201610322779 A CN201610322779 A CN 201610322779A CN 105832789 A CN105832789 A CN 105832789A
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filtrate
pressure
value
saponin
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刘扬
陈志永
马祎博
高开华
聂静
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Weihai Songling Nuokejia Chinese Herbal Medicine Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

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Abstract

The invention relates to a method for preparing a saponin enriched product through high-pressure microwave assisted semi-bionic extraction. The method comprises the steps of 1, taking panax ginseng or panax quinquefolius powder as the raw material, adding buffer solution, regulating pH value to be 3.5-5.5, adding extraction complex enzyme, and conducting enzymolysis and filtration to obtain filter residues 1 and filtrate 1; 2, adding buffer solution to the filter residues 1, regulating pH value to be 1.5-3.0, and conducting high-pressure microwave treatment and filtration to obtain filter residues 2 and filtrate 2; 3, adding buffer solution to the filter residues 2, regulating pH value to be 7.5-8.5, and conducting high-pressure microwave treatment and filtration to obtain filtrate 3; 4, combining the filtrate, adding buffer solution, regulating pH value to be neutral, conducting high-pressure microwave treatment, and then conducting resin treatment, elution, concentration and drying to obtain the saponin enriched product. The method simulates the absorption and transportation process of saponin in gastrointestinal tracts after people take saponin orally, leaching of active ingredients can be achieved easily, saponin extraction efficiency can be improved, and the obtained extract is high in rare saponin content and high in antineoplastic activity.

Description

A kind of method utilizing high-pressure microwave auxiliary-semi-biomimetic method extraction to prepare saponin enriched substance
Technical field
The invention belongs to medical and health technology field, be specifically related to a kind of method utilizing high-pressure microwave auxiliary-semi-biomimetic method extraction to prepare saponin enriched substance.
Background technology
Radix Ginseng (Panax ginseng) and Radix Panacis Quinquefolii (Panax quinquefolius) be precious medicinal plants, Araliaceae, have the kidney invigorating, enrich blood, the function such as cardioprotection, regulation blood glucose blood pressure, enhancing immunity.Ginsenoside be in Radix Ginseng and Radix Panacis Quinquefolii rich in a class main active, be divided into protopanoxadiol saponins, Protopanaxatriol's saponins and oleanolic acid saponins, cental system, hormonal system etc. all shown certain regulation effect.The ginsenoside that in Radix Ginseng and Radix Panacis Quinquefolii, content is higher has Rg1、Re、Rb1、Rc、Rb2And RdDeng, the saponin Rh that some content are relatively low1、Rh2、Rh3、Rg3、Rg5、Rg6、Rk1、Rk3、Rh4、F4, c-k etc. be referred to as secondary ginsenoside or rare ginsenoside.Result of study shows, some secondary ginsenosides have stronger pharmacologically active, such as ginsenoside Rh1、Rg3There is anticancer effect, wherein Rg3Infiltration and the transfer etc. with antitumor cell act on.The rare saponin content of Radix Ginseng and Radix Panacis Quinquefolii is extremely low, is difficult to directly be extracted from medical material by traditional extracting method obtain, it is common that elder generation is enriched with the ginsenoside of rich content from Radix Ginseng and Radix Panacis Quinquefolii, then is degraded and obtain rare ginsenoside.
Microwave applications in the extraction of Effective Component of Chinese Medicine, also brings nonthermal effect for experimental system while improving extraction efficiency, has that extraction time is short, extraction efficiency is high and high repeatability and other advantages.Part chemical bond in Ginsenosides glycosidic bond and parent nucleus side chain belongs to relatively weak chemical bond, it is easy to fracture and restructuring occur.Therefore, microwave technology is utilized can to speed up to the common ginsenoside transformation to rare ginsenoside.Hyperpressure extraction is to act on medicinal liquid by the hydrostatic pressure of 10 ~ 1000MPa at normal temperatures, Extraction solvent is made to penetrate into intracellular, a period of time is kept to make effective ingredient reach release rapidly after dissolution equilibrium under a predetermined, owing to intraor extracellular osmotic pressure difference increases suddenly, intracellular effective ingredient is transferred to, in extracellular extracting solution, reach the purpose of effective component extracting.The employing high-pressure microwave extraction method such as Wang Yutang is directly extracted from Radix Ginseng and is obtained rare ginsenoside; the method makes extraction and degraded one step complete, and the extraction for rare saponin enriched substance provides new method [Wang Yutang, SCI; 2007,28 (12)].
Semi-bionic extraction is to imitate oral administration in gastrointestinal transport process, the method using the water extraction of selected pH value.The extraction process of the method meets the feature of traditional Chinese medical science compatibility, clinical application, can be extracted to greatest extent by the effective substance of Chinese medicine or compound recipe.Having been used up high-temperature water decocting method when semi-bionic extraction just proposes, decocting cooking method can improve extraction efficiency, but will also result in destruction and the volatilization of active component, and impurity component is more simultaneously, is unfavorable for refining.CN102512469A discloses a kind of bionical flooding of Radix Ginseng aerial parts half and extracts technology of preparing, and the method advanced technology is simple, but the method is gentleer, improves the limited use of the rare saponin content of Radix Ginseng.
Summary of the invention
The invention provides a kind of method utilizing high-pressure microwave auxiliary-semi-biomimetic method extraction to prepare saponin enriched substance, the method is beneficial to the leaching of active component, improves saponin extraction efficiency, the extract obtained, and rare saponin content is high, anti-tumor activity is strong.
The present invention is achieved by the following technical solutions:
A kind of method utilizing high-pressure microwave auxiliary-semi-biomimetic method extraction to prepare saponin enriched substance, comprises the following steps:
(1) taking Radix Ginseng or American Ginseng powder is raw material, add buffer, regulation pH value is 3.5 ~ 5.5, adds the enzyme eliminating plant cell wall accounting for raw materials quality 0.3% ~ 0.6%, enzymolysis, filter, obtain filtering residue 1 and filtrate 1 after mixing；
(2) taking filtering residue 1, add buffer, regulation pH value is 1.5 ~ 3.0, fully mixes, and high-pressure microwave processes, and filters to obtain filtering residue 2 and filtrate 2；
(3) taking filtering residue 2, add buffer, regulation pH value is 7.5 ~ 8.5, fully mixes, and high-pressure microwave processes, and filters to get filtrate 3；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds buffer, and regulation pH value be neutral, after high-pressure microwave processes, crosses D101 or AB-8 type macroporous adsorbent resin, eluting, and eluent concentrates, dry be saponin enriched substance.The condition of adsorption and desorption by resin is: preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min；It is the most colourless that ultra-pure water is washed till effluent；65% ~ 70% ethanol water eluting, 5 times of column volumes of consumption, elution flow rate is 1 mL/min, collects eluent, concentrating under reduced pressure, vacuum drying, obtains saponin enriched substance.Vacuum drying temperature is 40 DEG C ~ 55 DEG C.
Preferably, the buffer described in step (1), (2), (3) and (4) is disodium hydrogen phosphate-citrate buffer solution.
Preferably, the enzyme clearing up plant cell wall in step (1) is cellulase, xylanase, pectase and the compound enzyme of 1,4 beta-glucanase composition, 1,4 beta-glucanase >=3,000,000 u/g, cellulase >=400,000 u/g in compound enzyme, xylanase >=2,600,000 u/g, pectase >=20,000 u/g.
Preferably, in step (1), the temperature of enzymolysis is 50 ~ 60 DEG C, and the time is 1 ~ 2 h；
The pressure of step (2) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min；
The pressure of step (3) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min；
The pressure of step (4) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min.
The useful achievement of the present invention is:
1. the invention provides a kind of method utilizing high-pressure microwave auxiliary-semi-biomimetic method extraction to prepare saponin enriched substance, high-pressure microwave auxiliary and the use in conjunction of semi-bionic extraction, it is possible to the synchronization realizing the rare saponin extraction of Radix Ginseng and conversion is carried out.The bionical acid of the many employings of prior art half carries out enzyme digestion reaction after extracting, the present invention carried out enzyme digestion reaction before semi-biomimetic method extracts, advantageously in the extraction of saponin component, extracting solution in enzymolysis step is put in high-pressure microwave aid system simultaneously, promote ginsenoside's conversion to rare ginsenoside, improve rare saponin conversion ratio.
2. application semi-bionic extraction, meets the feature that traditional Chinese medical science compatibility, clinical application and oral administration are transported in human gastrointestinal tract and absorbed, can strengthen gained rare saponin extract absorbing in human body.The H of human gastric juice is simulated when pH value is the condition of 1.5 ~ 3.0+Concentration, under high-pressure microwave assists, ginsenoside hydrolyzes, and cyclization generates panoxadiol and the derivant of panaxatriol, obtains rare saponin；When pH value is the condition of 7.5 ~ 8.5, simulate ginsenoside's transhipment absorption process in human body intestinal canal, strengthen oral drugs absorbing in intestinal, improve drug bioavailability；Microwave pressure is set to 400 ~ 600 KPa, beneficially ginsenoside's degraded, convert to rare ginsenoside.
3. during extracting, add the enzyme that can clear up plant cell wall, carry out decomposing destruction to the effective ingredient of Chinese herbal medicine cell wall, be conducive to the leaching of active component, and macromolecular substances can be made to change into small-molecule substance, the more conducively extraction of ginsenoside, improves extraction efficiency.The buffer solution added, nontoxic, stable chemical nature.
4. gained Radix Ginseng or Radix Panacis Quinquefolii saponin extract, rich in rare ginsenoside, its anti-tumor activity is significantly higher than commercially available common Radix Ginseng or Radix Panacis Quinquefolii saponin.
Detailed description of the invention
Embodiment 1
(1) Radix Ginseng powder is taken, adding disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 4.0, adds cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.3% of Radix Ginseng powder quality, water enzyme digestion at 60 DEG C, water bath time is 1 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 3.0, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 400 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 2 and filtering residue 2 after extraction；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 7.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 400 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 3 after extraction；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds disodium hydrogen phosphate-citrate buffer solution, and regulation pH value is neutral, and high-pressure microwave processes, and pressure is 400 KPa, and the time is 10 Min, filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 65% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, baking temperature is 40 DEG C, obtains the Radix Ginseng extract rich in rare saponin.
Gained is 75.3% rich in content of ginsenoside in the Radix Ginseng extract of rare saponin.
Embodiment 2
(1) Radix Ginseng powder is taken, adding disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 5.0, adds cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.6% of Radix Ginseng powder quality, water enzyme digestion at 50 DEG C, water bath time is 2 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 1.5, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 600 KPa, the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, obtain filtrate 2 and filtering residue 2；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 8.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 600 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 3 after extraction；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds disodium hydrogen phosphate-citrate buffer solution, and regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 Min, filtrate crosses D101 type macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, baking temperature is 55 DEG C, obtains the Radix Ginseng extract rich in rare saponin.
Gained is 79.2% rich in content of ginsenoside in the Radix Ginseng extract of rare saponin.
Embodiment 3
(1) Radix Panacis Quinquefolii sample is taken, pulverized 40 mesh sieves, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 3.5, the extraction compound enzyme clearing up plant cell wall of American Ginseng powder quality 0.3% is added after mixing, enzyme is cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, extract 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, water enzyme digestion at 60 DEG C, water bath time is 1 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 3.0, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 400 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 2 and filtering residue 2 after extraction；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 7.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 400 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 3 after extraction；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds disodium hydrogen phosphate-citrate buffer solution, and regulation pH value is neutral, and high-pressure microwave processes, and pressure is 400 KPa, and the time is 10 Min, filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 65% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 73.1% rich in content of ginsenoside in the Radix Panacis Quinquefolii extract of rare saponin.
Embodiment 4
(1) Radix Panacis Quinquefolii sample is taken, pulverized 60 mesh sieves, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 5.5, add after mixing and extract compound enzyme, enzyme is cellulase, xylanase, pectase and the compound enzyme of 1,4 beta-glucanase composition, 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.5% of American Ginseng powder quality, water enzyme digestion at 50 DEG C, water bath time is 2 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 1.5, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 600 KPa, the time is set as 10 min, takes out extraction pot, extracting solution in filtering tank, obtain filtrate 2 and filtering residue 2 after extraction；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution, regulation pH value is 8.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank, and microwave pressure is set as 600 KPa, the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, obtain filtrate 3；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds disodium hydrogen phosphate-citrate buffer solution, and regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 Min, filtrate crosses D101 type macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 78.6% rich in content of ginsenoside in the Radix Panacis Quinquefolii extract of rare saponin.
Comparative example 1
(1) Radix Ginseng powder is taken, disodium hydrogen phosphate-citrate buffer solution is added by the solid-liquid ratio of 1:10, regulation pH value is 5.0, add after mixing and extract compound enzyme, enzyme is cellulase, xylanase, pectase and the compound enzyme of 1,4 beta-glucanase composition, extract 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.5% of Radix Ginseng powder quality, water enzyme digestion at 50 DEG C, water bath time is 2 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, regulation pH is neutral, adds water by the solid-liquid ratio of 1:10, fully mixes, be placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, extract continuously twice, merging filtrate, filter to get filtrate 2；
(3) merging filtrate 1 and filtrate 2, regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 Min, filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Ginseng extract rich in rare saponin.
Gained is 68.4% rich in content of ginsenoside in the Radix Ginseng extract of rare saponin.
Comparative example 2
(1) Radix Ginseng powder is taken, disodium hydrogen phosphate-citrate buffer solution is added by the solid-liquid ratio of 1:10, regulation pH value is 5.0, add after mixing and extract compound enzyme, enzyme is cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, extract 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, water enzyme digestion at 50 DEG C, water bath time is 2 h, the consumption of enzyme is the 0.5% of Radix Ginseng powder quality, filters to obtain filtering residue 1 and filtrate 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 1.5, fully mixes, supersound extraction 1 h, filters to obtain filtering residue 2 and filtrate 2；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 8.5, fully mixes, supersound extraction 1 h, obtains filtrate 3.
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds disodium hydrogen phosphate-citrate buffer solution, and regulation pH value is neutral, and filtrate crosses D101 type macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 73.2% rich in content of ginsenoside in the Radix Ginseng extract of rare saponin.
Comparative example 3
(1) taking Radix Ginseng powder, add water by the solid-liquid ratio of 1:10, after mixing, at 50 DEG C, water-bath is extracted, and water bath time is 2 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 1.5, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, obtain filtrate 2 and filtering residue 2；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 8.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Taking out extraction pot after extraction, in filtering tank, extracting solution obtains filtrate 3；
(4) merging filtrate 1, filtrate 2 and filtrate 3, regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 min, and filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Ginseng extract rich in rare saponin.
Gained is 65.3% rich in content of ginsenoside in the Radix Ginseng extract of rare saponin.
Comparative example 4
(1) Radix Panacis Quinquefolii sample is taken, pulverize and sieve, disodium hydrogen phosphate-citrate buffer solution is added by the solid-liquid ratio of 1:10, regulation pH value is 5.0, add after mixing and extract compound enzyme, enzyme is cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, extract 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.5% of American Ginseng powder quality, water enzyme digestion at 50 DEG C, water bath time is 2 h, filters to get filtrate 1 and filtering residue 1；
(2) taking filtering residue 1, regulation pH is neutral, adds water by the solid-liquid ratio of 1:10, fully mixes, be placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, extract continuously twice, merging filtrate, filter to get filtrate 2；
(3) merging filtrate 1 and filtrate 2, regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 Min, filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 67.3% rich in content of ginsenoside in the Radix Panacis Quinquefolii extract of rare saponin.
Comparative example 5
(1) Radix Panacis Quinquefolii sample is taken, pulverize and sieve, disodium hydrogen phosphate-citrate buffer solution is added by the solid-liquid ratio of 1:10, regulation pH value is 5.0, add after mixing and extract compound enzyme, enzyme is cellulase, xylanase, pectase and the extraction compound enzyme of 1,4 beta-glucanase composition, extract 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g, the consumption of enzyme is the 0.5% of American Ginseng powder quality, water enzyme digestion at 50 DEG C, water bath time is 2 h, filters to obtain filtering residue 1 and filtrate 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 1.5, fully mixes, supersound extraction 1 h, filters to obtain filtering residue 2 and filtrate 2；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 8.5, fully mixes, supersound extraction 1 h, filters to get filtrate 3.
(4) merging filtrate 1, filtrate 2 and filtrate 3, regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 min, and filtrate crosses D101 type macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 70.5% rich in content of ginsenoside in the Radix Panacis Quinquefolii extract of rare saponin.
Comparative example 6
(1) taking Radix Panacis Quinquefolii sample, pulverize and sieve, add water by the solid-liquid ratio of 1:10, after mixing, at 50 DEG C, water-bath is extracted, and water bath time is 2 h, filters to obtain filtering residue 1 and filtrate 1；
(2) taking filtering residue 1, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 1.5, fully mixes, is placed in extraction pot, is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, obtain filtrate 2 and filtering residue 2；
(3) taking filtering residue 2, add disodium hydrogen phosphate-citrate buffer solution by the solid-liquid ratio of 1:10, regulation pH value is 8.5, fully mixes, and is positioned in microwave sample processing system after being sealed by tank.Microwave pressure is set as 600 KPa, and the time is set as 10 min.Take out extraction pot, extracting solution in filtering tank after extraction, obtain filtrate 3；
(4) merging filtrate 1, filtrate 2 and filtrate 3, regulation pH value is neutral, and high-pressure microwave processes, and pressure is 600 KPa, and the time is 10 min, and filtrate crosses D101 macroporous adsorbent resin, preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min.It is the most colourless that ultra-pure water is washed till effluent, 70% ethanol water eluting, collects eluent, and 5 times of column volumes of consumption, elution flow rate is 1 mL/min.Eluent concentrating under reduced pressure, vacuum drying, obtain the Radix Panacis Quinquefolii extract rich in rare saponin.
Gained is 67.1% rich in content of ginsenoside in the Radix Panacis Quinquefolii extract of rare saponin.
The assay of Radix Ginseng total saponins:
With ginsenoside Re as reference substance (offer of Shanghai Qi Ming biomedical company), after vanillic acid-glacial acetic acid colour developing, be at 555 nm, to carry out absorbance measurement with spectrophotography at wavelength, with absorbance (Y), ginsenoside's mass (X) carried out linear regression, regression equation be Y= 4.9821X+0.0003(R=0.9996).According to saponin content in regression equation calculation extract.
Cell toxicant is tested:
Experimental tumor cell line: K-562, the former leukaemia of the chronic marrow of people；Hela, human cervical carcinoma cell.Example 2, embodiment 4, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5 and the sample of comparative example 6 preparation, Radix Panacis Quinquefolii saponin and ginsenoside are purchased from Jilin ginseng industry company limited.
Experimental technique: extracorporeal anti-tumor cell experiment adds Radix Ginseng extract sample by various dose.
Experimental result:
Experimental result shows, application this method embodiment 2 and embodiment 4 gained are rich in the Radix Ginseng of rare saponin and Radix Panacis Quinquefolii extract, and content of ginsenoside is higher than being used alone high-pressure microwave assisted extraction method and semi-bionic extraction；Gained is significantly higher than high-pressure microwave assisted extraction method rich in the Radix Ginseng of rare saponin and K-562 and the Hela cell strain inhibitory activity of Radix Panacis Quinquefolii extract and semi-bionic extraction be used alone the Radix Ginseng prepared and Radix Panacis Quinquefolii saponin enriched substance (P< 0.05).
Embodiment is only the present invention preferably detailed description of the invention; but protection scope of the present invention is not limited thereto; any those familiar with the art is in the technical scope of present disclosure; according to technical scheme and inventive concept equivalent or change in addition thereof, all should contain within protection scope of the present invention.

Claims (6)

1. one kind utilizes the method that saponin enriched substance is prepared in high-pressure microwave auxiliary-semi-biomimetic method extraction, it is characterised in that comprise the following steps:
(1) taking Radix Ginseng or American Ginseng powder is raw material, add buffer, regulation pH value is 3.5 ~ 5.5, adds the enzyme clearing up plant cell wall accounting for raw materials quality 0.3% ~ 0.6%, enzymolysis, filter, obtain filtering residue 1 and filtrate 1 after mixing；
(2) taking filtering residue 1, add buffer, regulation pH value is 1.5 ~ 3.0, fully mixes, and high-pressure microwave processes, and filters to obtain filtering residue 2 and filtrate 2；
(3) taking filtering residue 2, add buffer, regulation pH value is 7.5 ~ 8.5, fully mixes, and high-pressure microwave processes, and filters to get filtrate 3；
(4) merging filtrate 1, filtrate 2 and filtrate 3, adds buffer, and regulation pH value be neutral, after high-pressure microwave processes, crosses D101 or AB-8 type macroporous adsorbent resin, eluting, and eluent concentrates, dry be saponin enriched substance.
Method the most according to claim 1, it is characterised in that the buffer described in step (1), (2), (3) and (4) is disodium hydrogen phosphate-citrate buffer solution.
Method the most according to claim 1, it is characterized in that, the enzyme clearing up plant cell wall in step (1) is cellulase, xylanase, pectase and the compound enzyme of 1,4 beta-glucanase composition, 1,4 beta-glucanase >=3,000,000 u/g in compound enzyme, cellulase >=400,000 u/g, xylanase >=2,600,000 u/g, pectase >=20,000 u/g.
Method the most according to claim 1, it is characterised in that
In step (1), the temperature of enzymolysis is 50 ~ 60 DEG C, and the time is 1 ~ 2 h；
The pressure of step (2) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min；
The pressure of step (3) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min；
The pressure of step (4) mesohigh microwave treatment is 400 ~ 600 KPa, the time is 10 min.
Method the most according to claim 1, it is characterised in that in step (4), the condition of adsorption and desorption by resin is: preadsorption 1 hour, crosses post effluent and heavily adsorbs 1 time, adsorption flow rate 2.0 mL/min；It is the most colourless that ultra-pure water is washed till effluent；65% ~ 70% ethanol water eluting, 5 times of column volumes of consumption, elution flow rate is 1 mL/min, collects eluent, concentrating under reduced pressure, vacuum drying, obtains saponin enriched substance.
Method the most according to claim 1, the vacuum drying temperature described in step (4) is 40 DEG C ~ 55 DEG C.
CN201610322779.8A 2016-05-16 2016-05-16 Method for preparing saponin enriched product through high-pressure microwave assisted semi-bionic extraction Pending CN105832789A (en)

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Application publication date: 20160810