CN112225825A - Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof - Google Patents

Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof Download PDF

Info

Publication number
CN112225825A
CN112225825A CN202010955008.9A CN202010955008A CN112225825A CN 112225825 A CN112225825 A CN 112225825A CN 202010955008 A CN202010955008 A CN 202010955008A CN 112225825 A CN112225825 A CN 112225825A
Authority
CN
China
Prior art keywords
phellinus
polysaccharide
solution
phellinus linteus
fine powder
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202010955008.9A
Other languages
Chinese (zh)
Inventor
王建功
张信岳
李钦
王星晨
王建文
方锦棋
谷丽丽
武柠子
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHUN'AN QIANDAO LAKE SANGDU EDIBLE FUNGUS PROFESSIONAL COOPERATIVES
Original Assignee
CHUN'AN QIANDAO LAKE SANGDU EDIBLE FUNGUS PROFESSIONAL COOPERATIVES
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHUN'AN QIANDAO LAKE SANGDU EDIBLE FUNGUS PROFESSIONAL COOPERATIVES filed Critical CHUN'AN QIANDAO LAKE SANGDU EDIBLE FUNGUS PROFESSIONAL COOPERATIVES
Priority to CN202010955008.9A priority Critical patent/CN112225825A/en
Publication of CN112225825A publication Critical patent/CN112225825A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses an effective component of phellinus igniarius for resisting liver cancer cell proliferation and a preparation method thereof, belonging to the technical field of phellinus igniarius polysaccharide preparation, wherein the preparation of phellinus igniarius polysaccharide comprises the following steps: weighing 50g of phellinus linteus fine powder, sieving the phellinus linteus fine powder by a 50-70-mesh sieve, adding the phellinus linteus fine powder into 1500g of water for hot water extraction, wherein the extraction time is 1-3h, the extraction temperature is 75-95 ℃, extracting twice repeatedly according to the steps, and combining the extracting solutions obtained by the two times; performing microwave pretreatment on the phellinus igniarius fine powder for 4-10min, wherein the microwave power is 350w, adding 0.5% of compound protease and 0.5% of cellulase into a phellinus igniarius fine powder sample after the microwave pretreatment, adjusting the pH value to 6, setting the ultrasonic time to be 15-20min, the ultrasonic temperature to be 50-70 ℃ and the ultrasonic power to be 240w, and extracting for 1.5-3 h; performing rotary evaporation on the aqueous extract of Phellinus linteus polysaccharide at 60-70 deg.C, concentrating to 1/10-1/5 of original volume, adding 80% ethanol into Phellinus linteus polysaccharide solution, precipitating at 4 deg.C for 12-18h, and centrifuging at 5000r/min for 30min to obtain Phellinus linteus polysaccharide solution.

Description

Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof
Technical Field
The invention relates to the technical field of preparation of phellinus igniarius polysaccharides, and in particular relates to an effective component of phellinus igniarius for resisting liver cancer cell proliferation and a preparation method thereof.
Background
Phellinus linteus is a general name for a class of medicinal fungi, and is mainly parasitic on trunks of mulberry trees, poplar trees, Syringa amurensis, pine trees, white birch and the like. Phellinus igniarius has very wide pharmacological action as an important medicinal bacterium, and is mainly shown in the following aspects: anti-mutation effect; enhancing the immunity of the organism; anti-tumor effect; anti-fibrosis and lipid peroxidation. China is a large export country of wild medicinal fungi, but the research on phellinus igniarius is still in the initial stage at present, and the technology is not mature. As the limited phellinus igniarius resources in China are excessively collected, the wild phellinus igniarius resources are exhausted, so that the phellinus igniarius resources are difficult to become stable industrial product sources, and the market requirements can not be met. Therefore, the production of phellinus linteus fruiting bodies using artificial cultivation techniques instead of wild phellinus linteus is the best way to overcome the limitation of biomass using wild resources. Phellinus linteus fruiting body can not be directly eaten generally, or if the Phellinus linteus fruiting body is directly eaten, the health care and treatment effects are not easy to achieve, and the efficacy can be improved only by concentrating or purifying the effective components.
As an important bioactive substance, the fungus polysaccharide has done much work on the aspects of preparation, structure, synthesis, pharmacology, clinical science and the like of the fungus polysaccharide at home and abroad, and the biological activity of the fungus polysaccharide in various aspects has been developed and applied, and various fungus preparations such as ganoderma lucidum polysaccharide, polyporus polysaccharide, lentinan, pachyman and the like have obtained good clinical effects.
Disclosure of Invention
The invention aims to provide an effective component of phellinus igniarius for resisting liver cancer cell proliferation and a preparation method thereof, the preparation efficiency of phellinus igniarius polysaccharide is high, and the sugar yield of phellinus igniarius polysaccharide is increased.
In order to solve the technical problems, the technical scheme of the invention is as follows:
an effective component of phellinus igniarius for resisting the proliferation of liver cancer cells, wherein the effective component is phellinus igniarius polysaccharide.
The preparation method of the phellinus igniarius polysaccharide comprises the following steps:
weighing 50g of phellinus linteus fine powder, sieving the phellinus linteus fine powder by a 50-70-mesh sieve, adding the phellinus linteus fine powder into 1500g of water for hot water extraction, wherein the extraction time is 1-3h, the extraction temperature is 75-95 ℃, extracting twice repeatedly according to the steps, and combining the extracting solutions obtained by the two times;
performing microwave pretreatment on the phellinus igniarius fine powder for 4-10min, wherein the microwave power is 350w, adding 0.5% of compound protease and 0.5% of cellulase into a phellinus igniarius fine powder sample after the microwave pretreatment, adjusting the pH value to 6, setting the ultrasonic time to be 15-20min, the ultrasonic temperature to be 50-70 ℃ and the ultrasonic power to be 240w, and extracting for 1.5-3 h;
performing rotary evaporation on the aqueous extract of Phellinus linteus polysaccharide at 60-70 deg.C, concentrating to 1/10-1/5 of original volume, adding 80% ethanol into Phellinus linteus polysaccharide solution, precipitating at 4 deg.C for 12-18h, and centrifuging at 5000r/min for 30min to obtain Phellinus linteus polysaccharide solution;
mixing n-butanol and chloroform to prepare a sevag solution, mixing the phellinus igniarius polysaccharide solution and the sevag solution in a separating funnel, violently shaking for 30min, standing for 3h, layering, removing supernatant, and repeating the steps until no white foam exists at the junction of the extracting solution and the sevag solution;
adding activated carbon with a volume 2-5% of that of the polysaccharide solution into the phellinus igniarius polysaccharide solution, decoloring at 40 ℃ for 1h, and repeating the operation twice;
dissolving Phellinus linteus polysaccharide with deionized water to make Phellinus linteus polysaccharide solution concentration be 10mg/mL, slowly injecting along chromatography column tube wall, and eluting with deionized water.
Preferably, the flow rate of the deionized water for elution is 0.2 mL/min.
Preferably, the sevag solution is prepared from n-butanol and chloroform in a volume ratio of 1: 4, mixing and preparing.
Preferably, the volume ratio of the phellinus igniarius polysaccharide solution to the sevag solution is 3: 1 were mixed in a separatory funnel.
By adopting the technical scheme, the sugar yield of the phellinus igniarius polysaccharide can be effectively improved due to the adoption of microwave pretreatment, and the polysaccharide is extracted by adopting the complex enzyme, so that the complex enzyme dissolves out a large amount of polysaccharide by dissolving cell walls, the reaction condition is mild, and the extraction time is short.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
The preparation method of the phellinus igniarius polysaccharide comprises the following steps:
weighing 50g of phellinus linteus fine powder, sieving the phellinus linteus fine powder by a 50-mesh sieve, adding the phellinus linteus fine powder into 1500g of water for hot water extraction, wherein the extraction time is 1h, the extraction temperature is 75 ℃, extracting twice repeatedly according to the steps, and combining the extracting solutions obtained twice;
performing microwave pretreatment on the phellinus igniarius fine powder for 4min, wherein the microwave power is 350w, adding 0.5% of compound protease and 0.5% of cellulase into a phellinus igniarius fine powder sample after the microwave pretreatment, adjusting the pH value to 6, setting the ultrasonic time to be 15min, the ultrasonic temperature to be 50 ℃ and the ultrasonic power to be 240w, and extracting for 1.5-3 hours;
performing rotary evaporation on the aqueous extract of Phellinus linteus polysaccharide at 60 deg.C, concentrating to 1/10 of original volume, adding 80% ethanol into Phellinus linteus polysaccharide solution, precipitating at 4 deg.C for 12h, and centrifuging at 5000r/min for 30min to obtain Phellinus linteus polysaccharide solution;
mixing n-butanol and chloroform to prepare a sevag solution, mixing the phellinus igniarius polysaccharide solution and the sevag solution in a separating funnel, violently shaking for 30min, standing for 3h, layering, removing supernatant, and repeating the steps until no white foam exists at the junction of the extracting solution and the sevag solution;
adding the phellinus igniarius polysaccharide solution into activated carbon with the volume being 2 of that of the polysaccharide solution, decoloring for 1h at the temperature of 40 ℃, and repeating the operation twice;
dissolving Phellinus linteus polysaccharide with deionized water to make Phellinus linteus polysaccharide solution concentration be 10mg/mL, slowly injecting along chromatography column tube wall, and eluting with deionized water;
the flow rate of the deionized water for elution is 0.2 mL/min;
the sevag solution is prepared from n-butanol and chloroform in a volume ratio of 1: 4, mixing and preparing;
the volume ratio of the phellinus igniarius polysaccharide solution to the sevag solution is 3: 1 were mixed in a separatory funnel.
Example 2
The preparation method of the phellinus igniarius polysaccharide comprises the following steps:
weighing 50g of phellinus linteus fine powder, sieving with a 70-mesh sieve, adding into 1500g of water for hot water extraction, wherein the extraction time is 3 hours, the extraction temperature is 95 ℃, extracting twice repeatedly according to the steps, and combining the extracting solutions obtained by the two times;
performing microwave pretreatment on the phellinus igniarius fine powder for 10min, wherein the microwave power is 350w, adding 0.5% of compound protease and 0.5% of cellulase into a phellinus igniarius fine powder sample after the microwave pretreatment, adjusting the pH value to 6, setting the ultrasonic time to be 20min, the ultrasonic temperature to be 70 ℃ and the ultrasonic power to be 240w, and extracting for 3 hours;
performing rotary evaporation on the aqueous extract of Phellinus linteus polysaccharide at 70 deg.C, concentrating to 1/5 of original volume, adding 80% ethanol into Phellinus linteus polysaccharide solution, precipitating at 4 deg.C for 18h, and centrifuging at 5000r/min for 30min to obtain Phellinus linteus polysaccharide solution;
mixing n-butanol and chloroform to prepare a sevag solution, mixing the phellinus igniarius polysaccharide solution and the sevag solution in a separating funnel, violently shaking for 30min, standing for 3h, layering, removing supernatant, and repeating the steps until no white foam exists at the junction of the extracting solution and the sevag solution;
adding activated carbon with a volume of 5% of that of the polysaccharide solution into the phellinus igniarius polysaccharide solution, decoloring for 1h at the temperature of 40 ℃, and repeating the operation twice;
dissolving Phellinus linteus polysaccharide with deionized water to make Phellinus linteus polysaccharide solution concentration be 10mg/mL, slowly injecting along chromatography column tube wall, and eluting with deionized water;
the flow rate of the deionized water for elution is 0.2 mL/min;
the sevag solution is prepared from n-butanol and chloroform in a volume ratio of 1: 4, mixing and preparing;
the volume ratio of the phellinus igniarius polysaccharide solution to the sevag solution is 3: 1 were mixed in a separatory funnel.
Human hepatoma cells HepG2 were cultured in DMEM medium containing 10% FBS by volume at 37 ℃ with 5% CO2Subculturing under the condition, when the cell fusion degree is about 80%, performing cell subculture, taking cells in logarithmic growth phase during experiment, detecting the survival rate of the cells by adopting MTT method, and adjusting the cell to be 5 × 104Inoculating 100 μ L/mL of the extract into 96-well plate, setting blank group, normal control group, Phellinus linteus fine powder polysaccharide solution with different concentrations, and Phellinus linteus mycelium polysaccharide solution with different concentrations, adding 100 μ L of the above solutions into each well, and treating with 5% CO at 37 deg.C2Culturing in incubator for 24h and 48h, respectively, adding 5g/L MTT20 μ L into each well, culturing for 4h, removing supernatant, adding 150 μ L DMSO into each well, placing in shaking table at room temperature for 10min, measuring OD value at 490nm of enzyme labeling instrument, and calculating cell survival rate and half Inhibition Concentration (IC)50) Survival rate (%) ═ (OD)Sample (I)-ODBlank space)/(ODIs normal-ODBlank space) 100, observing cell forms by an inverted microscope, treating cells in the same way, observing by the inverted microscope after the Phellinus linteus fine powder polysaccharide solution and the Phellinus linteus mycelium solution with different concentrations act for 24 hours and 48 hours, and taking pictures to record cell forms of each group, wherein the times of the objective lens are 20 times;
the results are shown in table 1, which shows that the survival rates of the phellinus igniarius fine powder polysaccharide solution and the phellinus igniarius mycelium polysaccharide solution for 24 hours and 48 hours are high for human liver cancer cells, the MTT result shows that the cell survival rate is reduced along with the increase of the concentration of the phellinus igniarius fine powder polysaccharide solution and the phellinus igniarius mycelium polysaccharide solution and the prolonging of the action time, the cell number is reduced, the adherence is not tight, the cell falls off partially, the volume is reduced, the morphological damage degree is gradually deepened, the concentration of the phellinus igniarius fine powder polysaccharide solution and the phellinus igniarius mycelium polysaccharide solution reaches 6.4mg/mL when the action time is 24 hours, the cell proliferation inhibiting effect (P is less than 0.05) is obvious for human liver cancer HepG2 cells, and the cell proliferation inhibiting effect (P is less than 0.05) is obvious for human liver cancer HepG2 cells when.
The fine powder acts on IC of human liver cancer HepG2 cells for 24h and 48h507.896 and 5.580mg/mL respectively, 3.978-15.67 mg/mL and 3.775-8.249 mg/mL respectively at 95% confidence interval, and IC of Phellinus linteus mycelium polysaccharide solution for 24h and 48h508.413 and 6.051mg/mL respectively, and the 95% confidence intervals are 4.511-15.690 mg/mL and 3.924-9.332 mg/mL respectively.
TABLE 1
Figure BDA0002678302630000041
Note: p <0.05, P <0.01 to placebo (0.0mg/mL)
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (5)

1. An effective component of phellinus igniarius for resisting the proliferation of liver cancer cells, which is characterized in that: the effective component is phellinus igniarius polysaccharide.
2. The method for preparing Phellinus linteus polysaccharide effective component for resisting hepatocarcinoma cell proliferation according to claim 1, wherein the preparation of Phellinus linteus polysaccharide comprises the following steps:
weighing 50g of phellinus linteus fine powder, sieving the phellinus linteus fine powder by a 50-70-mesh sieve, adding the phellinus linteus fine powder into 1500g of water for hot water extraction, wherein the extraction time is 1-3h, the extraction temperature is 75-95 ℃, extracting twice repeatedly according to the steps, and combining the extracting solutions obtained by the two times;
performing microwave pretreatment on the phellinus igniarius fine powder for 4-10min, wherein the microwave power is 350w, adding 0.5% of compound protease and 0.5% of cellulase into a phellinus igniarius fine powder sample after the microwave pretreatment, adjusting the pH value to 6, setting the ultrasonic time to be 15-20min, the ultrasonic temperature to be 50-70 ℃ and the ultrasonic power to be 240w, and extracting for 1.5-3 h;
performing rotary evaporation on the aqueous extract of Phellinus linteus polysaccharide at 60-70 deg.C, concentrating to 1/10-1/5 of original volume, adding 80% ethanol into Phellinus linteus polysaccharide solution, precipitating at 4 deg.C for 12-18h, and centrifuging at 5000r/min for 30min to obtain Phellinus linteus polysaccharide solution;
mixing n-butanol and chloroform to prepare a sevag solution, mixing the phellinus igniarius polysaccharide solution and the sevag solution in a separating funnel, violently shaking for 30min, standing for 3h, layering, removing supernatant, and repeating the steps until no white foam exists at the junction of the extracting solution and the sevag solution;
adding activated carbon with a volume 2-5% of that of the polysaccharide solution into the phellinus igniarius polysaccharide solution, decoloring at 40 ℃ for 1h, and repeating the operation twice;
dissolving Phellinus linteus polysaccharide with deionized water to make Phellinus linteus polysaccharide solution concentration be 10mg/mL, slowly injecting along chromatography column tube wall, and eluting with deionized water.
3. The method for preparing the active ingredient of phellinus linteus for resisting the proliferation of hepatoma cells according to claim 2, wherein the preparation method comprises the following steps: the flow rate of the deionized water for elution is 0.2 mL/min.
4. The method for preparing the active ingredient of phellinus linteus for resisting the proliferation of hepatoma cells according to claim 2, wherein the preparation method comprises the following steps: the sevag solution is prepared from n-butanol and chloroform in a volume ratio of 1: 4, mixing and preparing.
5. The method for preparing the active ingredient of phellinus linteus for resisting the proliferation of hepatoma cells according to claim 2, wherein the preparation method comprises the following steps: the phellinus igniarius polysaccharide solution and the sevag solution are mixed according to the volume ratio of 3: 1 were mixed in a separatory funnel.
CN202010955008.9A 2020-09-11 2020-09-11 Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof Withdrawn CN112225825A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010955008.9A CN112225825A (en) 2020-09-11 2020-09-11 Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010955008.9A CN112225825A (en) 2020-09-11 2020-09-11 Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof

Publications (1)

Publication Number Publication Date
CN112225825A true CN112225825A (en) 2021-01-15

Family

ID=74116754

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010955008.9A Withdrawn CN112225825A (en) 2020-09-11 2020-09-11 Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof

Country Status (1)

Country Link
CN (1) CN112225825A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114306398A (en) * 2022-02-08 2022-04-12 中华全国供销合作总社济南果品研究院 Application of phellinus igniarius fermented extract
CN114591448A (en) * 2022-04-01 2022-06-07 浙江省林业科学研究院 Phellinus igniarius sporophore mannogalactan and preparation and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114306398A (en) * 2022-02-08 2022-04-12 中华全国供销合作总社济南果品研究院 Application of phellinus igniarius fermented extract
CN114591448A (en) * 2022-04-01 2022-06-07 浙江省林业科学研究院 Phellinus igniarius sporophore mannogalactan and preparation and application thereof

Similar Documents

Publication Publication Date Title
CN101702984B (en) Phellinus igniarius mycelium liquid fermentation method by utilizing fungal elicitor for improving yield of flavone of phellinus igniarius
CN105820956B (en) One plant of Antrodia camphorata bacterial strain and Antrodia camphorata liquid state fermentation method
CN112225825A (en) Phellinus igniarius anti-hepatoma cell proliferation effective component and preparation method thereof
CN106967775B (en) Method for preparing diosgenin through biocatalysis and microbial inoculum used by same
CN1687429A (en) Chinese traditional medicine containing glycosides transformed by ferment of microbe, and method for producing relevant aglycone preparation
CN105559068B (en) It is a kind of to utilize composition for eating the acquisition of medicine fungi fermentation radix tetrastigme and preparation method thereof
CN112608949B (en) Preparation method and application of pseudo-ginseng flower extract
CN105580638A (en) Method for promoting antrodia camphorata liquid state fermentation growth and triterpene synthesis
CN102363796A (en) Method for producing glycyrrhetinic acid through microbial fermentation transformation
CN102703558B (en) Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini
CN100567318C (en) Nucleoside active matter in the artificial culture Cordyceps militaris (L.) Link. and its production and use
CN109468359B (en) Ginsenoside Rk6Preparation method of (1)
CN111485012A (en) Method for preparing glycyrrhetinic acid monoglucuronide by liquorice fermentation
CN113383840B (en) Selenium-rich vine tea
CN109673393A (en) A kind of production method of peacilomyce hepiahi bacterium filament
KR20120085956A (en) Method for manufacturing of fermented ginseng or fermented red ginseng
CN111763705B (en) Preparation method and application of ginsenoside composition
CN105535035A (en) Inonotus obliquus fermentation culture composition and preparation method thereof
CN105483029B (en) It is a kind of to synthesize 11 α, the method for 15 α-diOH- canrenones using gibberella CA3-1
CN105483028B (en) It is a kind of to synthesize 11 α, the method for 15 α-diOH- 16ALPHA,17ALPHA-epoxyprogesterones using gibberella
KR20200095208A (en) Co-culture method of mushroom mycelium
CN116391568B (en) Phellinus linteus strain culture medium and preparation method thereof
JP3302853B2 (en) Culture method of medicinal carrot callus
CN115057949B (en) Active polysaccharide compound extracted from dragon fruit stems and preparation method and application thereof
CN109456383B (en) 20(R) -ginsenoside Rh1Preparation method of (1)

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication
WW01 Invention patent application withdrawn after publication

Application publication date: 20210115