AU2020104046A4 - A method for extracting anti-tumor compounds from Actinidia eriantha Benth. root - Google Patents
A method for extracting anti-tumor compounds from Actinidia eriantha Benth. root Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C62/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C62/30—Unsaturated compounds
- C07C62/32—Unsaturated compounds containing hydroxy or O-metal groups
Abstract
The present invention relates to a method for extracting 2a,3a,24-trihydroxy-12-en-28
oic acid from Actinidia eriantha Benth. root, which includes ethanol reflux extraction,
macroporous adsorption resin, reversed-phase chromatographic purification, and
recrystallization to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid. The antitumor active
component obtained by the method of the present invention has a purity of more than 95%, a
high reaction yield, a simple preparation method, a high efficiency, a relatively low
environmental pollution, a low preparation cost, and is suitable for industrial production.
Description
A method for extracting anti-tumor compounds from Actinidia eriantha Benth.
root
[01] The invention relates to the field of traditional Chinese medicine preparation, in particular to a method for extracting compounds from Actinidia eriantha Benth. roots.
[02] Cancer, as a disease with high fatality rate, seriously endangers human health. It is predicted that by 2020, there will be 15 million new cases of cancer in the world, and 12 million people will die from the disease. In the field of cancer treatment, natural drugs have attracted more and more attention because of their unique action mechanism, multiple action targets, good tolerance and relatively small toxic and side effects. According to statistics, nearly 50% of chemotherapy drugs used in clinic today are natural products or derived from their derivatives.
[03] Actinidiasinensis, commonly known as "kiwifruit", "kiwi berry" and "Rattan pear", which is rich in plant resources all over the world, with 66 species, most of which grow from Malaysia to eastern Siberia, among which 62 species are found in China, mainly distributed in the south of Qinling Mountains and the east of Hengduan Mountains. Actinidia erianthaBenth,is a kind of Actinidia, also known as Baihuashan Maotao, Maodonggua and Baitengli,It is widely distributed in Zhejiang, Fujian, Jiangxi, Hunan, Guangxi and Guangdong provinces. According to records, it has the effects of clearing away heat and promoting diuresis, promoting blood circulation and detumescence, detoxifying and so on. As one of the traditional she medicine used by ethnic minorities in Zhejiang Province, it is widely used in tumor treatment in She nationality.
[04] Actinidia erianthaBenth., as a traditional national medicine with the basis of clinical treatment of cancer, has relatively few reports on its research, development and utilization at home and abroad. Therefore, it is necessary to use new technologies and methods to systematically study the anti-tumor active substances, extraction methods and pharmacodynamic mechanisms of Actinidia eriantha Benth., so as to provide potential drugs for cancer treatment with definite curative effect and little toxic and side effects.
[05] It has been reported in the literature that a pentacyclic triterpenoid saponin monomer compound, 2a,3 a,24- trihydroxy -12- ene -28- ursolic acid, was extracted and isolated from the root of Actinidia erianthaBenth., its molecular structure is as follows:
[06] OH
[07] In vitro experiments show that it can significantly inhibit the proliferation of human hepatoma cell line HepG2, human prostate cancer cell line pc3, human gastric cancer cell line BGC-823 and human colorectal cancer sw620, and is a potential natural anti-tumor drug. However, the large-scale industrial production process of 2a,3a,24 trihydroxy-12-en-28-oic acid, which is simply and efficiently extracted from the root of Rhinopithecus tomentosa, has not been reported in the literature. At present, the prior art only reports related methods for extracting and separating 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia erianthaBenth. root for identification purposes, which have complicated steps and low efficiency, and the purity and yield do not meet the requirements of industrial mass production.
[08] To solve the above technical problems, the present invention provides a method for efficiently extracting antitumor components 2 a,3a,24- trihydroxy -12- ene -28- ursolic acid from Actinidia erianthaBenth. root.
[09] The invention realize that purpose of the invention by the following method:
[010] (6) Taking the root of Actinidia eriantha Benth., which is crushed into 40 100 meshes;
[011] (7) Refluxing and extracting kiwifruit root powder with ethanol;
[012] (8) The filtrate is decompressed to have no alcohol smell, diluted with water and centrifuged;
[013] (9) Passing the centrifuged filtrate through a macroporous adsorption resin column, sequentially eluting with water and ethanol, collecting the ethanol eluted part, concentrating under reduced pressure, and drying to obtain total saponin;
[014] (10) The total saponin were purified by reversed-phase chromatography and recrystallized to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid.
[015] More preferably, the particle size in step (1) of the method of the present invention is 60 mesh.
[016] More preferably, the volume concentration of ethanol in step (2) of the method of the present invention is 70%.
[017] More preferably, in step (2) of the method of the present invention, after soaking in 70% ethanol for 0.5 hours, the extraction is carried out twice, with 10 times of 70% ethanol for 1 hour at the first time and 8 times of 70% ethanol for 0.5 hours at the second time.
[018] More preferably, the filtrate in step (3) of the method of the present invention is concentrated under reduced pressure until it has no alcohol smell, and diluted with water to be equivalent to 0.25g crude drug /ml.
[019] More preferably, the macroporous adsorption resin in step (4) of the method of the present invention is an HPD450 macroporous adsorption resin column.
[020] More preferably, in step (4) of the method of the present invention, water, % ethanol by volume and 70% ethanol by volume are sequentially used for elution, and 70% ethanol eluate is collected.
[021] More preferably, in step (4) of the method of the present invention, 5 column volumes of water, 3 column volumes of 40% ethanol and 3 column volumes of % ethanol are sequentially used for elution at an elution flow rate of 1.5-2 column volumes h- 1, 70% ethanol eluate is collected, concentrated under reduced pressure until the density is 1.05-1.10 at 65 °C, and dried under reduced pressure below 60 °C.
[022] More preferably, the elution flow rate in step (4) of the method of the present invention is 2 column volume h- 1 .
[023] More preferably, in the step (4) of the method of the present invention, the density is 1.05-1.10 at 65 °C, and the density is reduced and dried below 60 °C..
[024] More preferably, the reverse phase chromatography purification conditions in step (5) of the method of the present invention are as follows: the loading amount is 1g intermediate /40mL column volume, the volume concentration of ethanol is 5% %, and elution is 1-5bv/h.
[025] More preferably, the reverse phase chromatography purification conditions in step (5) of the method of the present invention are as follows: the loading amount is Ig intermediate /40mL column volume, the ethanol volume concentration is 15%, and the elution is 3bv/h.
[026] More preferably, in step (5) of the method of the present invention, an ethanol-water system is used for recrystallization.
[027] More preferably, the recrystallization conditions in step (5) of the method of the present invention are as follows: ethanol dissolves the crude chemical compound prepared by reversed-phase chromatography until it is just saturated, then water accounting for 1/3 of the volume of ethanol is added, and the mixture is left at room temperature for 24h, and filtered to obtain crystals of 2a,3a,24-trihydroxy-12-en-28-oic acid.
[028] The invention has positive and beneficial effects:
[029] According to the invention, the anti-tumor active monomer component 2a,3a,24-trihydroxy-12-en-28-oic acid of Actinidia erianthaBenth. root is obtained by reflux extraction, macroporous adsorption resin separation, reversed-phase chromatography purification and recrystallization in sequence, and the purity can reach over 95%, the yield is high, the method is simple and convenient, the efficiency is high, the environmental pollution is small, the preparation cost is low, and the method is suitable for industrial production.
[030] The present invention will be further explained with examples below, but the embodiments of the present invention are not limited to this. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the ethanol concentrations are all volume concentrations.
[031] Example 1 Preparation of total saponin Al from Actinidia erianthaBenth. root
[032] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia eriantha Benth. roots comprises the following specific steps:
[033] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[034] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[035] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[036] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, eluting with 5 column volumes of water, 3 column volumes of 40% ethanol and 3 column volumes of 70% ethanol in turn at an elution flow rate of 2 column volumes h- 1, collecting 70% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65°C, and drying under reduced pressure below °C to obtain the total filtrate
[037] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin Al was determined by HPLC, and the calculated yield was 94.7%.
[038] Example 2 Preparation of total saponin A2 from Kiwifruit Root
[039] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia eriantha Benth. roots comprises the following specific steps:
[040] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[041] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[042] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[043] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, sequentially eluting with 5 column volumes of water, 3 column volumes of 20% ethanol and 3 column volumes of 50% ethanol at an elution flow rate of 2 column volumes h- 1, collecting 50% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65 °C, and drying under reduced pressure below 60 °C to obtain total saponin
[044] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin A2 was determined by HPLC, and the calculated yield was 85.7%.
[045] Example 3 Preparation of total saponin A3 from Actinidia erianthaBenth. root
[046] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia eriantha Benth. roots comprises the following specific steps:
[047] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[048] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[049] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[050] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, eluting with 5 column volumes of water, 3 column volumes of 20% ethanol, and 3 column volumes of 90% ethanol in turn at an elution flow rate of 2 column volumes h-1 , collecting 90% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65°C, and drying under reduced pressure below °C to obtain the final product
[051] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin A3 was determined by HPLC, and the calculated yield was 82.9%.
[052] Example 4 Preparation of total saponin A4 from Actinidia eriantha Benth.root
[053] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid fromActinidia eriantha Benth.roots comprises the following specific steps:
[054] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[055] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[056] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[057] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, eluting with 5 column volumes of water, 3 column volumes of 20% ethanol, and 3 column volumes of 70% ethanol in turn at an elution flow rate of 2 column volumes h-1 , collecting 70% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65°C, and drying under reduced pressure below °C to obtain the total filtrate
[058] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin A4 was determined by HPLC, and the calculated yield was 81.3%.
[059] Example 5 Preparation of total saponin A5 from Actinidia erianthaBenth. root
[060] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia eriantha Benth. roots comprises the following specific steps:
[061] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[062] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[063] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[064] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, eluting with 5 column volumes of water, 3 column volumes of 40% ethanol and 3 column volumes of 90% ethanol in turn at an elution flow rate of 2 column volumes h-1 , collecting 90% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65°C, and drying under reduced pressure below °C to obtain the total filtrate
[065] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin A5 was determined by HPLC, and the calculated yield was 80.9%.
[066] Example 6 Preparation of total saponin A6 from Actinidia erianthaBenth. root
[067] A method for extracting total saponin containing 2a,3a,24-trihydroxy-12 en-28-oic acid from Actinidia eriantha Benth.roots comprises the following specific steps:
[068] (1) Taking the root of Actinidia erianthaBenth., which is crushed into 60 meshes;
[069] (2) Soaking Actinidia erianthaBenth. root powder in 70% ethanol for 0.5 hours, and extracting twice, wherein the first time is extracted with 10 times of 70% ethanol for 1 hour, and the second time is extracted with 8 times of 70% ethanol for 0.5 hours;
[070] (3) The filtrate is decompressed to have no alcohol smell, diluted to 0.25g crude drug /mL by adding water, and centrifuged;
[071] (4) Passing the centrifuged filtrate through HPD450 macroporous adsorption resin column, eluting with 5 column volumes of water, 3 column volumes of 40% ethanol, and 3 column volumes of 50% ethanol in turn at an elution flow rate of 2 column volumes h-1 , collecting the 50% ethanol eluate, concentrating under reduced pressure until the density is 1.05-1.10 at 65°C, and drying under reduced pressure below 60°C to obtain the total filtrate
[072] The 2a,3a,24-trihydroxy-12-en-28-oic acid in total saponin A6 was determined by HPLC, and the calculated yield was 79.8%.
[073] By comparing the above examples 1-6, it can be found that when the filtrate is eluted with macroporous adsorption resin column, it is eluted with 3 columns of 40% ethanol and 3 columns of 70% ethanol in turn, and the content of 2a,3a,24-trihydroxy 12-en-28-oic acid in total saponin Al is the highest.
[074] Example 7 purification method P1 of total saponin Al from Actinidia erianthaBenth. root
[075] Total saponin Al from Actinidia erianthaBenth. root prepared in Example 1 were purified by reversed-phase chromatography, and the chromatographic conditions were as follows: the loading amount was Ig intermediate /40mL column volume, the ethanol concentration was 15%, and the elution was 3bv/h. Ethanol dissolves the crude product of 2a,3a,24-trihydroxy-12-en-28-oic acid prepared by reversed-phase chromatography until it is just saturated, then water accounting for 1/3 of the volume of ethanol is added, and the solution is left at room temperature for 24h, and then filtered to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid crystals. The yield of this step is 87.4% and the purity of the compound is 96.8%.
[076] Example 8 purification method P2 of total saponin Al from Actinidia erianthaBenth. root
[077] Total saponin Al from Actinidia erianthaBenth. root prepared in Example 1 were purified by reversed-phase chromatography, and the chromatographic conditions were as follows: the loading amount was Ig intermediate /40mL column volume, the ethanol concentration was 30%, and the elution was 3bv/h. Ethanol dissolves the crude product of 2a,3a,24-trihydroxy-12-en-28-oic acid prepared by reversed-phase chromatography until it is just saturated, then water accounting for 1/3 of the volume of ethanol is added, and the solution is left at room temperature for 24h, and then filtered to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid crystals. The yield of this step is 71.9% and the purity of the compound is 95.6%.
[078] Example 9 purification method P3 of total saponin Al from Actinidia erianthaBenth. root
[079] Total saponin Al from Actinidia erianthaBenth. root prepared in Example 1 were purified by reversed-phase chromatography, and the chromatographic conditions were as follows: the loading amount was ig intermediate /40mL column volume, the ethanol concentration was 10%, and the elution was 3bv/h. Ethanol dissolves the crude product of 2a,3a,24-trihydroxy-12-en-28-oic acid prepared by reversed-phase chromatography until it is just saturated, then water accounting for 1/3 of the volume of ethanol is added, and the solution is left at room temperature for 24h, and then filtered to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid crystals. The yield of this step is 68.1% and the purity of the compound is 95.0%.
[080] Example 10 purification method P4 of total saponin Al from Actinidia erianthaBenth. root
[081] Total saponin Al from Actinidia erianthaBenth. root prepared in Example 1 were purified by reversed-phase chromatography, and the chromatographic conditions were as follows: the loading amount was lg intermediate /40mL column volume, the concentration of ethanol was 15%, and elution was lbv/h. Ethanol dissolves the crude product of 2a,3a,24-trihydroxy-12-en-28-oic acid prepared by reversed-phase chromatography until it is just saturated, then water accounting for 1/3 of the volume of ethanol is added, and the solution is left at room temperature for 24h, and then filtered to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid crystals. The yield of this step is 70.8% and the purity of the compound is 95.3%.
[082] Example 11 purification method P5 of total saponin Al from Actinidia erianthaBenth.root
[083] Total saponin Al from Actinidia erianthaBenth. root prepared in example 1 were purified by reversed-phase chromatography under the following chromatographic conditions: ig intermediate /40mL column volume, 15% ethanol volume concentration, 5BV/h elution. Ethanol dissolves the crude product of 2a,3a,24- trihydroxy-12-en-28-oic acid prepared by reversed-phase chromatography until it isjust saturated, then water accounting for 1/3 of the volume of ethanol is added, and the solution is left at room temperature for 24h, and then filtered to obtain 2a,3a,24 trihydroxy-12-en-28-oic acid crystals. The yield of this step is 75.3% and the purity of the compound is 94.8%.
[084] By the comparison of Examples 7-11, it can be found that when the ethanol concentration is 15% and the flow rate is 3BV/h, the yield of 2a,3a,24-trihydroxy-12 en-28-oic acid crystals obtained after purification of total saponin is the highest.
[085] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.
[086] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable
Claims (10)
1. The method for extracting antitumor component 2a,3a,24-trihydroxy-12-en-28
oic acid from Actinidia erianthaBenth. root, which is characterized by comprising the
following steps:
(1) Taking the root of Actinidia eriantha Benth., which is crushed to 40-100
meshes;
(2) Refluxing and extracting kiwifruit root powder with ethanol;
(3) The filtrate is decompressed to have no alcohol smell, diluted with water and
centrifuged;
(4) Passing the centrifuged filtrate through a macroporous adsorption resin
column, sequentially eluting with water and ethanol, collecting the ethanol eluted part,
which is concentrated under reduced pressure, and drying to obtain total saponin;
(5) The total saponin are purified by reversed-phase chromatography and
recrystallized to obtain 2a,3a,24-trihydroxy-12-en-28-oic acid.
2. The method according to claim 1, wherein the volume concentration of ethanol
in step (2) is 70%.
3. The method according to claim 2, which is characterized in that, in step (2), after
soaking in 70% ethanol for 0.5 hours, extraction is carried out twice, with 10 times of
% ethanol for 1hour at the first time and 8 times of 70% ethanol for 0.5 hours at the
second time.
4. The method according to claim 1, which is characterized in that in step (3), the
filtrate is concentrated under reduced pressure until it has no alcohol smell, and diluted
by adding water until it is equivalent to 0.25g crude drug /ml.
5. The method according to claim 1, wherein the macroporous adsorption resin in
step (4) is an HPD450 macroporous adsorption resin column.
6. The method according to claim 5, characterized in that in step (4), water, 40%
ethanol by volume and 70% ethanol by volume are sequentially used for elution, and
% ethanol eluate is collected.
7. The method according to claim 6, characterized in that, in step (4), 5 column
volumes of water, 3 column volumes of 40% ethanol and 3 column volumes of 70%
ethanol are sequentially used for elution at an elution flow rate of 1.5-2 column volumes
h- 1, and 70% ethanol eluate is collected and concentrated to a density of 1.05-1.10 at
600 C.
8. The method according to claim 1, characterized in that the reverse phase
chromatography purification conditions in step (5) are as follows: the loading amount
is Ig of intermediate /40mL column volume, the ethanol volume concentration is 5%
%, and the elution is 1-5bv/h..
9. The method according to claim 8, characterized in that the reverse phase
chromatography purification conditions in step (5) are as follows: the loading amount
is Ig intermediate /40mL column volume, the ethanol volume concentration is 15%,
and the elution is 3bv/h..
10. The method according to claim 1, wherein in step (5), an ethanol-water system
is used for recrystallization.
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