CN108392500B - Method for preparing ganoderma triterpene - Google Patents

Method for preparing ganoderma triterpene Download PDF

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CN108392500B
CN108392500B CN201810295221.4A CN201810295221A CN108392500B CN 108392500 B CN108392500 B CN 108392500B CN 201810295221 A CN201810295221 A CN 201810295221A CN 108392500 B CN108392500 B CN 108392500B
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ganoderma
extract
ethanol
triterpene
extraction
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CN108392500A (en
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张劲松
贾薇
汪雯翰
徐锦
唐传红
庄海宁
杨焱
刘艳芳
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Shanghai Academy of Agricultural Sciences
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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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Abstract

The invention discloses a method for preparing ganoderma triterpene, which comprises the following steps: adding 50-100% ethanol into Ganoderma fruiting body, extracting at 55-100 deg.C for 1-3 hr at a liquid-material mass ratio of 30-80:1, and concentrating the extractive solution to obtain Ganoderma alcoholic extract; processing Ganoderma encarpium into 10-80 mesh, adding Ganoderma ethanol extract, mixing, placing into supercritical extraction tank, and performing supercritical CO extraction2Extracting; concentrating the extract at below 65 deg.C, and drying to obtain Ganoderma extract. According to the method for preparing the ganoderma triterpene, the ganoderma triterpene extracted by the method has the inhibition rate of 66.76% on human prostate tumor cell LNCaP and the inhibition rate of 62.69% on 22RV1 when the application concentration is 100 ug/mL.

Description

Method for preparing ganoderma triterpene
Technical Field
The invention relates to the field of deep processing of edible and medicinal fungi, in particular to a method for preparing ganoderma triterpene.
Technical Field
Ganoderma (Ganoderma lucidum) belongs to Monosporophyceae, Polyporaceae, has effects of invigorating spleen, replenishing qi, strengthening body resistance, consolidating constitution, and prolonging life, is a precious Chinese medicinal material, and has reputation of "immortal grass". Research shows that the lucid ganoderma has the effects of resisting aging, regulating immunity, protecting liver, expelling toxin, inhibiting tumor, resisting bacteria, resisting virus and the like. Ganoderma contains various bioactive components including triterpene, polysaccharide, sterol, alkaloid, etc. Ganoderma triterpene and ganoderan are the main active substances in Ganoderma.
The method for extracting the ganoderma triterpene mainly comprises the following steps: organic solvent extraction, ultrasonic extraction and supercritical carbon dioxide extraction. The traditional extraction method mainly uses an organic solvent to extract triterpenes from lucid ganoderma sporocarp, and the method consumes more organic solvent and takes long time; most of the supercritical technologies are widely applied to the extraction of ganoderma lucidum spore oil, and the supercritical extraction of ganoderma lucidum sporocarp generally has the problems of low extraction efficiency, high cost, low activity and the like. Therefore, there is a need to develop a method for extracting ganoderma triterpene with high extraction rate and low cost.
Disclosure of Invention
The invention aims to provide a method for preparing ganoderma triterpene, which comprises the following steps:
adding 50-100% ethanol into ganoderma lucidum fruiting body, extracting at 55-100 ℃ for 1-3 hours at a liquid-material mass ratio of 30-80:1, and concentrating the extract to obtain ganoderma lucidum ethanol extract;
② processing Ganoderma encarpium into 10-80 mesh, adding the extract ①, mixing, and supercritical CO extracting in a supercritical extraction tank2Extracting;
the supercritical extraction process conditions are as follows: the temperature is 40-70 ℃, the pressure is 20-50Mpa, 50-100% (V/V) ethanol is used as entrainer, the dosage is 4-10mL/g, and the time is 1-3 h;
fourthly, the extract is concentrated and dried under the temperature of 65 ℃ to obtain the ganoderma lucidum extract.
Wherein, in the step (i):
the ganoderma alcohol extract obtained by concentration contains 2-20% of ethanol by mass fraction, wherein the content of the ethanol is calculated by the recovery amount of the ethanol when the extracting solution is concentrated;
wherein, the step two is as follows:
the mass ratio of the ganoderma lucidum fruiting body to the ganoderma lucidum alcohol extract is 10:1-1: 10;
said CO2The purity is 99-100%;
the supercritical extraction process conditions are as follows: the temperature is 40-60 ℃, the pressure is 20-35Mpa, 80-100% (V/V) ethanol is used as entrainer, the dosage is 4-8mL/g, and the time is 1-3 h.
The method combines hot reflux extraction and supercritical extraction, greatly reduces extraction solvent consumption, shortens extraction time, improves extraction efficiency, and protects activity of Ganoderma fruiting body extract.
The invention combines two extraction techniques: firstly, a thermal reflux technology for extracting ganoderma triterpene in a large scale is used for primary extraction, and then a green and efficient supercritical extraction technology is adopted for further extraction. The results show that: the two methods are effectively combined, so that the extraction efficiency of the ganoderma triterpene is improved, the time for hot reflux extraction is shortened, the energy consumption is reduced, the loss of thermosensitive components in the extract is reduced, the activity of the ganoderma extract is improved, and the extracted ganoderma triterpene has high biological activity on prostatic hyperplasia.
The invention relates to a method for extracting ganoderma triterpene, and the extracted ganoderma triterpene has good effect on prostate-related diseases. When the action concentration of the ganoderma triterpene sample is 100ug/mL, the inhibition rate of prostate tumor cells LNCaP is 66.76%, the inhibition rate of 22RV1 is 62.69%, and the inhibition effect is close to 400 mu M of positive drug finasteride.
According to the preparation method of the ganoderma triterpene, the traditional ganoderma alcohol extract and the ganoderma fruit body are mixed uniformly, and the green and efficient supercritical technology is adopted for deeply processing and producing the ganoderma active ingredient, so that the extraction efficiency is greatly improved, the extraction cost is greatly reduced, the activity is also well improved, and the technical support can be provided for new products and raw materials in the fields of nutrition, health care and medicine.
Drawings
FIG. 1 comparison of the inhibitory activities of the Ganoderma lucidum extract of example 1 against LNCaP cells and 22RV1 cells
Wherein F in the abscissa is finasteride
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
Pulverizing fruiting body of Hunong Ganoderma (G.lucidum) No. 1 (Shanghai agricultural academy of sciences) produced in Longquan of Zhejiang, sieving with 20 mesh sieve, weighing 1000g, placing into extraction tank, adding 80% ethanol at liquid-material mass ratio of 40:1, extracting at 85 deg.C for 3 hr, filtering the extractive solution, and concentrating to obtain Ganoderma ethanol extract 88.83g, labeled as I, containing 10% ethanol.
Processing Ganoderma fruiting body into 60 mesh, adding the prepared extractum-shaped Ganoderma ethanol extract I and Ganoderma fruiting body at a mass ratio of 1:1, mixing, weighing 40g of the mixture, placing into supercritical extraction tank, and performing supercritical CO extraction2Extracting under the following process conditions: the temperature is 40 ℃, the pressure is 30Mpa, the entrainer is carried by 90 percent ethanol, the dosage of the entrainer is 6mL/g, and the time is 1 h.
After extraction, the extract was collected from the discharge port of the separation vessel, concentrated and dried at 65 ℃ to obtain 4.09g of ganoderma lucidum extract with an extraction rate of 10.2%. According to 2015 pharmacopoeia, oleanolic acid is used as a reference, and the content of ganoderma triterpene is measured to be 35.91%.
A method for calculating the content of ganoderma triterpene (a method for measuring the content of ganoderma triterpene in 2015 pharmacopoeia):
1. preparation of control solution A proper amount of oleanolic acid control was precisely weighed and added with methanol to prepare a solution containing 0.2mg per 1 mL.
2. Preparation of a standard curve reference substance solutions of 0.1, 0.2, 0.3, 0.4 and 0.5mL are precisely measured, the reference substance solutions are respectively placed in 15mL test tubes with plugs, volatilized to be dry and cooled, a newly prepared vanillin glacial acetic acid solution (0.5 g is precisely measured and glacial acetic acid is added to dissolve the vanillin glacial acetic acid solution into 10 mL) of 0.2mL and perchloric acid of 0.8mL are precisely added, the mixture is uniformly shaken, the mixture is heated in a 70 ℃ water bath for 15 minutes and is immediately placed in the ice water bath to be cooled for 5 minutes, the mixture is taken out, ethyl acetate of 4mL is precisely added, the mixture is uniformly shaken, corresponding reagents are used as blanks, an ultraviolet-visible spectrophotometry method (general rule 0401) is adopted, the absorbance is measured at the 546nm wavelength, and the absorbance is used as a vertical coordinate and the concentration is used as a horizontal.
3. Preparing a test solution, precisely weighing 2g of prepared ganoderma lucidum extract powder, placing the powder into a conical flask with a plug, adding 50mL of ethanol, carrying out ultrasonic treatment (power 140W and frequency 42kHz) for 45 minutes, filtering, placing the filtrate into a 100mL measuring flask, washing the filter and filter residue by using a proper amount of ethanol in turn, adding ethanol into the same measuring flask together with the washing solution to scale, and shaking up to obtain the ganoderma lucidum extract.
4. The determination method comprises precisely measuring 0.2mL of test solution, placing in 15mL test tube with plug, measuring absorbance by the same method from "volatilizing", reading out oleanolic acid content of the test solution from the standard curve, and calculating.
The product contains triterpene and sterol, and oleanolic acid (C) calculated on dry product30H48O3) Not less than 0.50% in terms of weight.
Example 2
Pulverizing fruiting body of Hunong Ganoderma (G.lucidum) No. 1 (Shanghai agricultural academy of sciences) produced in Longquan of Zhejiang, sieving with 10 mesh sieve, weighing 1000g, placing into extraction tank, adding 90% ethanol at liquid-material mass ratio of 40:1, extracting at 90 deg.C for 2h, filtering, and concentrating to obtain 81.49g of Ganoderma ethanol extract labeled as II containing 5% ethanol.
Processing Ganoderma fruiting body into 80 mesh, adding the prepared extractum-shaped Ganoderma ethanol extract II and Ganoderma fruiting body at a mass ratio of 1:1, mixing, weighing 40g of the mixture, placing into supercritical extraction tank, and performing supercritical CO extraction2Extracting under the following process conditions: the temperature is 50 ℃, the pressure is 35Mpa, the entrainer is carried by 90 percent ethanol, the dosage of the entrainer is 6mL/g, and the time is 1.5 h.
After extraction, the extract was collected from the discharge port of the separation vessel, concentrated and dried at 65 ℃ to obtain 5.37g of ganoderma lucidum extract with an extraction rate of 13.4%. According to 2015 pharmacopoeia, oleanolic acid is used as a reference, and the content of ganoderma triterpene is 38.43 percent.
Example 3
Inhibition assay for LNCaP cells
The cell concentration was 54one/mL of LNCaP (purchased from cell resource center of Shanghai Life sciences, China academy of sciences) suspension of tumor cells 180. mu.L was added to a 96-well plateAfter 48h, 20 μ L of the sample to be tested (Ganoderma extract obtained by four methods of hot reflux extraction, Soxhlet extraction, ultrasonic extraction and cold soaking extraction in example 1) was added, DMSO was used as negative control, finasteride was used as positive control, each sample was repeated 3 times, and CO concentration of 5% at 37 deg.C2After 48h of incubation under the conditions, the old medium was aspirated off and 180. mu.L of colorless 1640 medium (Thermo Fisher Scientific, USA) and 20. mu.L of 0.075mg/mL Alamar Blue reagent (Thermo Fisher Scientific, USA) was added, after changing color, absorbance was measured at 570 and 600nm using an enzyme linked immunosorbent assay, and the effect of the sample on cell proliferation was calculated using the formula.
Figure BDA0001618565530000041
When the action concentration of the ganoderma lucidum extract sample in the example 1 is 100ug/mL, the inhibition rate of LNCaP cells is 66.76%.
Example 4
22RV1 cell inhibition rate test
The cell concentration was 5 × 104one/mL of 22RV1 tumor cells (purchased from cell resource center of Shanghai Life sciences of Chinese academy of sciences) suspension 180. mu.L was added to a 96-well plate, and after 24 hours, 20. mu.L of the extract sample of example 1 was added, with DMSO as a negative control and finasteride as a positive control, in 3 replicates per sample, at 37 ℃ and 5% CO concentration2After 72h incubation under these conditions, the old medium was aspirated off and 180. mu.L of colorless 1640 medium (Thermo Fsher Scientific, USA) and 20. mu.L of 0.075mg/mL Alamar Blue reagent (Thermo Fisher Scientific, USA) was added, and after color change absorbance was measured at 570 and 600nm using an enzyme linked immunosorbent assay and the effect of the sample on cell proliferation was calculated using the formula.
When the action concentration of the ganoderma lucidum extract sample in example 1 is 100 mug/mL, the inhibition rate of the prostate tumor cells 22RV1 is 62.69%.

Claims (3)

1. A method for preparing ganoderma triterpene is characterized by comprising the following steps:
adding 50-100% ethanol into ganoderma lucidum fruiting body, extracting at 55-100 ℃ for 1-3 hours at a liquid-material mass ratio of 30-80:1, and concentrating the extract to obtain ganoderma lucidum ethanol extract;
② processing Ganoderma encarpium into 10-80 mesh, adding the extract ①, mixing, and supercritical CO extraction2Extracting; the mass ratio of the ganoderma lucidum fruiting body to the ganoderma lucidum alcohol extract is 1: 1;
the supercritical extraction process conditions are as follows: the temperature is 40-70 ℃, the pressure is 20-50Mpa, 50-100% (V/V) ethanol is used as entrainer, the dosage is 4-10mL/g, and the time is 1-3 h;
fourthly, the extract is concentrated and dried under the temperature of 65 ℃ to obtain the ganoderma lucidum extract.
2. The method for preparing ganoderma triterpene according to claim 1, wherein in the step (i):
the ganoderma alcohol extract obtained by concentration contains 2-20% of ethanol by mass fraction.
3. The method for preparing ganoderma triterpene according to claim 1, wherein the step (II) comprises:
the mass ratio of the ganoderma lucidum fruiting body to the ganoderma lucidum alcohol extract is 1: 1;
said CO2The purity is 99-100%;
the supercritical extraction process conditions are as follows: the temperature is 40-60 ℃, the pressure is 20-35Mpa, 80-100% (V/V) ethanol is used as entrainer, the dosage is 4-8mL/g, and the time is 1-3 h.
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CN109394799B (en) * 2018-12-28 2021-05-28 南京中科药业有限公司 Natural pharmaceutical composition containing ganoderma lucidum extract and preparation method and application thereof
TWI703121B (en) * 2019-03-15 2020-09-01 喬璞科技有限公司 Method of purifying triterpenoid-based compound
CN111346118B (en) * 2020-03-31 2022-07-05 福建仙芝楼生物科技有限公司 Method for subcritical water extraction and separation of ganoderma triterpene extract
CN113150867B (en) * 2020-12-01 2023-05-12 中科健康产业集团股份有限公司 Preparation method of ganoderma lucidum extract oil rich in ganoderma lucidum triterpenes
CN115919908A (en) * 2022-12-20 2023-04-07 中科健康产业集团江苏药业有限公司 Ganoderma spore oil-containing composition for improving tumor inhibition rate and preparation method thereof

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