CN106860484A - A kind of fresh Cordyceps extract of antitumor activity and its preparation method and application - Google Patents
A kind of fresh Cordyceps extract of antitumor activity and its preparation method and application Download PDFInfo
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Classifications
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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
- A61K36/062—Ascomycota
- A61K36/066—Clavicipitaceae
- A61K36/068—Cordyceps
-
- 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
-
- 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
- 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
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The application in field the invention discloses a kind of fresh Cordyceps extract with antitumor activity and preparation method thereof and in terms of anti-breast cancer, melanoma medicine.Fresh Cordyceps extract is prepared from by the following method：Add water homogenate after fresh Cordyceps sinensis is crushed, and carries out low temperature ultrasonic assisted extraction to Cordyceps sinensis afterwards, is centrifuged, and takes supernatant, concentrates, alcohol precipitation, collects precipitation, and freeze-drying obtains Cordyceps extract.The fresh Cordyceps extract that the present invention is obtained has obvious suppression Cells Proliferation of Human Breast Cancer and melanoma transferance, and active ingredient is clearly, can be used for pre- anti-cancer, treating cancer, with good prospect in medicine；Poisonous and hazardous organic solvent is not introduced in preparation process, safety is used；Preparation process is simple, it is easy to mass produce.
Description
Technical field
The present invention relates to Chinese medicine and natural medicine field, and in particular to a kind of fresh Cordyceps extract of antitumor activity
And preparation method thereof and in terms of anti-breast cancer, melanoma diversion medicaments field application.
Background technology
Cordyceps sinensis is colonized in for section ergot fungus cordyceps sinensis bacterium Cordyceps sinensis (Berk.) Sacc.
The complex of stroma and larva corpse on Hepialidae insect larvae, is the rare Chinese medicine of China's special product, with invigorating the lung and the kidney,
The effects such as hemostasis and phlegm.Modern medicine study shows that Cordyceps sinensis has antitumor, regulation immunity, hypotensive, interior point of regulation
Secrete, it is calm, resist myocardial ischemia and adjust the functions such as heart rate.In recent years, substantial amounts of document report Cordyceps sinensis and its activity
Growth and transfer of the composition to kinds of tumor cells such as stomach cancer, lung cancer, thyroid cancers are inhibited, but wherein to mammary gland
Cancer, melanoma research it is relatively fewer.Meanwhile, when Cordyceps extract sample is prepared more than using boiling, high temperature ultrasonic
Extract or high temperature reflux is extracted, these preparation methods can cause the change of the amount of activated structure of matter, cause the medicine of Cordyceps sinensis
Reason activity is affected.
Melanoma (melanoma) is a kind of strong skin neoplasin of invasion, is substantially risen recently as the incidence of disease,
It is increasingly becoming common cancer.Its progress is rapid, easily shifts, clinical prognosis extreme difference, serious harm human health.B16 is black
Melanoma is to find for 1954, and the knurl is the spontaneous melanoma in pure lines mouse C57BL/6 basal part of the ear skins, there is document report B16
The Lung metastases rate of melanoma is more than 90%.Existing document reports that Cordyceps sinensis is little to Nasopharyngeal neoplasms aspect research, only
There is a small amount of correlative study on aweto mycelium, and research is not goed deep into very much, is extracted or had for a long time using hot water more
Machine solvent extraction.Such as Wang Lin exists《Influence of the Cordyceps sinensis to mouse melanin tumor cell Lung metastases》In report Cordyceps sinensis
Erinaceus mycelium powder extracts 3h with 90~95 DEG C of hot water, can reduce lung surface metastatic nodules, and its dose,optimum is 250mg/kg,
But do not mention the rate of transform and the influence to mouse quality of life for suppressing melanoma.And for example document《Inhibitory
effects of ethylacetate extract of Cordyceps sinensis mycelium on various
cancer cells in culture and B16 melanoma in C57BL-6 mice》Cordyceps sinensis is reported manually to send out
The mycelial ethyl acetate extract of ferment has good inhibiting effect to MCF-7 breast cancer cells and B16 MCs,
And its hot water extract then unrestraint effect.
Breast cancer has turned into the most common malignant tumour of world today women.Exist according to doctor Chen Wanqing《Chinese women mammary gland
Cancer morbidity death and survival state》Described in, according to the data information assessment of national tumour Register, Chinese women breast in 2011
Gland cancer number of the infected about 24.9 ten thousand, the incidence of disease 37.86/10 ten thousand, last decade morbidity is in rising trend.Annual dead about 6.0 ten thousand, extremely
Rate of dying 9.21/10 ten thousand, last decade Death Rate of Breast Cancer is in rising trend.With socio-economic development, female life mode occurs
Significantly change, breast cancer Related Risk Factors generally existing, it is contemplated that the incidence of disease of following one period China's breast cancer more long and
The death rate will also persistently rise.The problems such as producing toxic and side effect and drug resistance to patient due to chemical synthetic drug, screens day
Right medicine has turned into the focus of research as the candidate or ancillary drug of oncotherapy.There are some researches show Cordyceps sinensis is to mammary gland
The growth or transfer of cancer cell have certain inhibitory action.But existing document is usually that will dry, dry or Cordyceps sinensis
Cordyceps sinensis raw material is carried out high-temperature water and boils extraction by zymotic fluid as research object, such as《Activation of innate
immunity to reduce lung metastases in breast cancer》Report using the worm summer in extracting in boiling water winter
The water extract obtained after careless erinaceus mycelium powder 1h growing with certain to 4T1 breast cancer cells when concentration is 3.4mg/mL
Inhibitory action.Document《Cordyceps sinensis is to the induction of Apoptosis of Breast Cancer and the regulation and control of related gene expression》Report the worm summer in winter
Careless fermented liquid is have inhibitory action in 1.0~50 μ g/mL concentration ranges to the propagation of MCF-7 cells in concentration, and concentration is presented
And time dependence.Research shows there is ERs (ER) positive (ER in patient with breast cancer+) and feminine gender (ER+) two species
Type, while also there is (HER2) low expression of EGF 2 with expression two types high.Wherein MCF-7 cells are to retain wild
Raw type P53, ER positive breast carcinoma cell strain, and MDA-MB-453 is the negative breast cancer cell of expression saltant type P53, ER
Strain.Numerous studies show there are 30% patient with breast cancer's HER2 Overexpressions, and this kind of patient tumors grade malignancy is high, multiple
There are early, poor prognosis in hair and transfer, exist to some chemotherapeutics and resist.
The content of the invention
The present invention is furtherd investigate by fresh Cordyceps sinensis, there is provided a kind of fresh Cordyceps sinensis with antitumor activity
Extract and preparation method thereof and the application in terms of antitumor activity.Of the present invention
Worm summer grass extract active component is clear and definite, safe and effective, quality controllable, also has good work(to the transfer of melanoma tumors cell
Effect, with good DEVELOPMENT PROSPECT.Moreover, it relates to have the preparation of the fresh Cordyceps extract of antitumor activity
Method, the method mild condition can to greatest extent protect the active component of extract, and simple to operate, and repeatability is strong, prepare
During do not introduce poisonous and harmful organic solvent, for extract Cordyceps sinensis active component research provide a kind of new method with
Thinking.
The purpose of the present invention is achieved through the following technical solutions：
On the one hand, the present invention provides a kind of fresh Cordyceps extract with antitumor activity, it is characterised in that the fresh winter
Polyoses content is 69.4~76.7% in worm summer grass extract, and protein content is 10.2~14.3%.
In certain embodiments, a kind of fresh Cordyceps extract with antitumor activity of the present invention, wherein,
Polysaccharide of the polysaccharide comprising 3 kinds of molecular weight more than 1KDa, weight average molecular weight is respectively 2.7KDa, 29KDa and 1422KDa；Albumen
Range of molecular weight distributions is primarily present at 10~15KDa and 20~37KDa.
On the other hand, the present invention relates to a kind of fresh Cordyceps extract with antitumor activity of the present invention
Preparation method, the preparation method is comprised the following steps：
A, fresh Cordyceps sinensis is crushed, add water homogenate, crushed, control temperature for 15~25 DEG C；
B, by homogenate after fresh Cordyceps sinensis carry out ultrasonic extraction, control temperature for 15~25 DEG C, supernatant is centrifuged to obtain, so
Fresh Cordyceps sinensis residue is stood in -20 DEG C of freezings afterwards；
C, the fresh Cordyceps sinensis residue that will be freezed take out, and add water and are homogenized and are uniformly dispersed, again ultrasonic extraction, control temperature
It is 15~25 DEG C, is then centrifuged for, obtains supernatant, the supernatant of combining step b；
D, toward centrifugation after supernatant in plus absolute ethyl alcohol to concentration be 60~80% after, stood overnight at 4 DEG C；In 4
Refrigerated centrifuge at DEG C, collects precipitation；
E, precipitation is carried out into freeze-drying, obtain fresh Cordyceps extract.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, wherein, in step a, described fresh Cordyceps sinensis and the w/v of water are 1:8~12g/mL, Homogenization time is 4
~8min, homogenate rotating speed is 14000~18000rpm.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, wherein, in stepb, the ultrasonic extraction time is 20~40min, and ultrasonic power is 400~600W, and centrifugation time is 10
~15min, centrifugal rotational speed is 8000~10000rpm, and freezing time of repose is 20~60min.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, wherein, in step c, the w/v of fresh Cordyceps sinensis residue and water is 1:8~12g/mL, again Homogenization time
It is 4~8min, homogenate rotating speed is 14000~18000rpm, the ultrasonic extraction time is 20~40min again, and ultrasonic power is 400
~600W, centrifugation time is 10~15min, and centrifugal rotational speed is 8000~10000rpm.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, wherein, in step d, the time of refrigerated centrifuge is 15~25min, and the rotating speed of centrifugation is 8000~10000rpm.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, described preparation method comprises the steps：
A, fresh Cordyceps sinensis is crushed, 1 is added by the w/v of fresh Cordyceps sinensis and water:The water of 8~12g/mL, it is even
4~8min of slurry, homogenate rotating speed is 14000~18000rpm, is crushed, and controls temperature for 15~25 DEG C, and fresh Cordyceps sinensis is crushed
Into the suspension of fine particle；
B, by homogenate after fresh Cordyceps sinensis carry out 20~40min of ultrasonic extraction, control temperature for 15~25 DEG C, power is
400~600W, be centrifuged 10~15min, rotating speed be 8000~10000rmp, obtain supernatant, then by fresh Cordyceps sinensis residue in-
20 DEG C of freezings stand 20~60min；
C, the fresh Cordyceps sinensis residue that will be freezed take out, and it is 1 to add w/v:The water of 8~10g/mL, homogenate 4~
8min, homogenate rotating speed is 14000~18000rpm, and is uniformly dispersed, again 20~40min of ultrasonic extraction, and it is 15 to control temperature
~25 DEG C, power is 400~600W, is then centrifuged for 10~15min, and the rotating speed of centrifugation is 8000~10000rpm, is merged twice
Supernatant；
D, toward centrifugation after supernatant in plus absolute ethyl alcohol to concentration be 60~80% after, stood overnight at 4 DEG C；In 4
15~25min of refrigerated centrifuge at DEG C, centrifugal rotational speed is 8000~10000rpm, collects precipitation；
E, precipitation is carried out into freeze-drying, obtain fresh Cordyceps extract.
In certain embodiments, the preparation of a kind of fresh Cordyceps extract with antitumor activity of the present invention
Method, described preparation method comprises the steps：
A, fresh Cordyceps sinensis is crushed, 1 is added by the w/v of fresh Cordyceps sinensis and water:The water of 10g/mL, homogenate
6min, homogenate rotating speed is 16000rpm, is crushed, and controls temperature for 20 ± 2 DEG C, and fresh Cordyceps sinensis is ground into the mixed of fine particle
Suspension；
B, by homogenate after fresh Cordyceps sinensis carry out ultrasonic extraction 30min, control temperature for 20 ± 2 DEG C, power is 400~
600W, is centrifuged 12min, and the rotating speed of centrifugation obtains supernatant for 9000rpm, then that fresh Cordyceps sinensis residue is quiet in -20 DEG C of freezings
Put 40min；
C, the fresh Cordyceps sinensis residue that will be freezed take out, and are homogenized 6min, and homogenate rotating speed is 16000rpm, and is uniformly dispersed,
Ultrasonic extraction 30min again, it is 20 ± 2 DEG C to control temperature, and power is 400~600W, is then centrifuged for 12min, the rotating speed of centrifugation
It is 9000rpm, obtains supernatant, merges the supernatant of collection step b；
D, toward centrifugation after supernatant in plus absolute ethyl alcohol to concentration be 60~80% after, stood overnight at 4 DEG C；In 4
Refrigerated centrifuge 20min at DEG C, centrifugal rotational speed is 10000rpm, collects precipitation；
E, precipitation is carried out into freeze-drying, obtain fresh Cordyceps extract.
On the other hand, the fresh Cordyceps extract with antitumor activity a kind of of the present invention that the present invention is provided
Application in anti-breast cancer or melanoma medicine is prepared.
In certain embodiments, the anti-breast cancer medicines are the medicines for suppressing human breast carcinoma MDA-MB-453 cells, are resisted
MC diversion medicaments are the medicines for suppressing anti-B16 MCs transfer.
The positive effect of the present invention：
1. the present invention provide fresh Cordyceps extract in polyoses content be 65~75%, protein content be 10~
15%, i.e. active component are clear and definite, and quality controllable.
2. the mild condition for preparing fresh Cordyceps extract that the present invention is provided, can to greatest extent protect the anti-of extract
Tumor promotion composition, and it is simple to operate, repeatability is strong.
3. the fresh Cordyceps extract that the present invention is provided is with obvious anti-strong pernicious Cells Proliferation of Human Breast Cancer and resists
MC transferance, and do not introduce poisonous and harmful organic solvent in preparation process, safely and effectively, with good
Prospect in medicine.
4. it is 50~150mg/kg present invention finds the transcellular effective taking dose of anti-B16 melanoma tumors,
Not only it is beneficial at utmost play drug effect, moreover it is possible to save drug cost.
5. extract preparation process is simple of the present invention, it is easy to mass produce, use safety.
Brief description of the drawings
Fig. 1 has the polysaccharide molecular weight distribution chromatogram of the fresh Cordyceps extract of anti-breast cancer activity；
Fig. 2 has the protein electrophorese figure of the fresh Cordyceps extract of anti-breast cancer activity.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Fresh Cordyceps sinensis source：The fresh harvesting of Yichang mountain city Shui Dou Cordyceps sinensis Co., Ltd.
Ultrasonic instrument is originated and parameter：KQ-500DB numerical controls supersonic cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd.), work(
Rate：500w；Supersonic frequency：40khz.
Centrifugation apparatus are originated：272R-3 vast capacities refrigerated centrifuge (Hunan Hunan is from instrument plant)
First, embodiment is prepared
Embodiment 1
Fresh Cordyceps sinensis 10g is taken, is shredded, add water 80mL, be homogenized 4min, homogenate rotating speed is 14000rpm, homogenate control temperature
15 ± 2 DEG C of degree, that is, obtain fresh Cordyceps sinensis fine particle suspension.Cordyceps sinensis after the homogenate that will be obtained carries out ultrasonic extraction
20min, controls ultrasonic temperature for 15 ± 2 DEG C, and power is 400W, and 10min is centrifuged, and rotating speed obtains supernatant for 8000rmp, then will
Fresh Cordyceps sinensis residue is placed in be freezed in -20 DEG C and stands 20min.4min is homogenized again, and homogenate rotating speed is 14000rpm, and is disperseed
Fresh Cordyceps sinensis residue after uniform freezing, repeats ultrasonic extraction 20min, and at 15 ± 2 DEG C, power is 400W to temperature control.Will
Repeating the suspension after ultrasound carries out centrifugation 10min, and rotating speed is 8000rpm, merges supernatant twice.It is toward supernatant plus anhydrous
Ethanol is placed under 4 DEG C of environment and stands overnight to final concentration of 60%, the refrigerated centrifuge 15min at 4 DEG C, and rotating speed is 8000rpm,
Supernatant is abandoned, precipitation is collected.Precipitation is finally carried out into freeze-drying and obtains Cordyceps extract 0.948g, extract yield is
9.48%.
Embodiment 2
Fresh Cordyceps sinensis 10g is taken, is shredded, add water 100mL, be homogenized 6min, homogenate rotating speed is 16000rpm, homogenate control temperature
20 ± 2 DEG C of degree, that is, obtain fresh Cordyceps sinensis and be ground into fine particle suspension.Fresh Cordyceps sinensis after the homogenate that will be obtained is carried out
Ultrasonic extraction 30min, controls ultrasonic temperature for 20 ± 2 DEG C, and power is 500W, and 12min is centrifuged, and rotating speed obtains supernatant for 9000rpm
, then be placed in fresh Cordyceps sinensis suspension in -20 DEG C of freezings and stand 40min by liquid.6min is homogenized again, and homogenate rotating speed is
Fresh Cordyceps sinensis suspension after 16000rpm, and the freezing that is uniformly dispersed, repeats ultrasonic extraction 30min, controls the ultrasonic temperature to be
20 ± 2 DEG C, power is 500W.The suspension repeated after ultrasound is carried out into centrifugation 12min, rotating speed is 9000rpm, merging is gone up twice
Clear liquid.Toward supernatant plus absolute ethyl alcohol is to final concentration of 70%, it is placed under 4 DEG C of environment and stands overnight, the refrigerated centrifuge at 4 DEG C
20min, rotating speed is 9000rpm, abandons supernatant, collects precipitation.Precipitation is finally carried out into freeze-drying and obtains Cordyceps extract
1.28g, extract yield is 12.8%.
Embodiment 3
Fresh Cordyceps sinensis 10g is taken, is shredded, add water 120mL, be homogenized 8min, rotating speed is 18000rpm, homogenate control temperature 25
± 2 DEG C, that is, obtain fresh Cordyceps sinensis and be ground into fine particle suspension.The fresh Cordyceps sinensis fine particle suspension that will be obtained enters
Row ultrasonic extraction 40min, controls ultrasonic temperature for 25 ± 2 DEG C, and power is 600W, and 15min is centrifuged, and rotating speed must be gone up for 10000rpm
Clear liquid, is then placed in Cordyceps sinensis suspension -20 DEG C of freezings and stands 60min.The winter for being homogenized again and being uniformly dispersed after freezing
Worm summer grass suspension, repeats ultrasonic extraction 40min, and at 25 ± 2 DEG C, power is 600W to temperature control.It is mixed after by repetition ultrasound
Suspension carries out centrifugation 15min, and rotating speed is 10000rpm, merges supernatant twice.Add absolute ethyl alcohol to final concentration toward supernatant
It is 80%, is placed under 4 DEG C of environment and stands overnight, the refrigerated centrifuge 25min at 4 DEG C, rotating speed is 10000rpm, abandons supernatant, is collected
Precipitation.Precipitation is finally carried out into freeze-drying and obtains Cordyceps extract 1.02g, extract yield is 10.2%.
2nd, testing example
The fresh Cordyceps extract anti-breast cancer activity research of test example 1.
(1) cell culture
Human breast carcinoma MDA-MB-453 (Her2+, ER-, PR-) and MCF-7 (HER2-, ER+, PR+/-) cell is placed in
In DMEM culture mediums, cultivated in 37 DEG C, 5% CO2gas incubator.Every daily inverted microscope observation of cell 1 time, every 2 days
Change nutrient solution once.Passage is carried out when the cell growth in blake bottle is to 85~95%.Old nutrient solution is discarded, PBS is used
Rinse 2 times repeatedly, 0.25% pancreatin digestive juice 2mL is added afterwards, terminate disappearing after 4mL nutrient solutions after cell Bian Yuan ﹑ floatings, are added
Change, transfer in 15mL sterile centrifugation tubes, with rotating speed 1000rpm, 4min is centrifuged, supernatant is abandoned, with 1:4 ratios suspend and precipitate
And it is transferred to culture in new sterile culture flask.
(2) medicine ordinance
The accurate fresh Cordyceps extract sample for weighing embodiment 1-3, dissolves and configures one with above-mentioned DMEM culture mediums
Determine the extract solution of concentration.With 3mg/mL as initial concentration, a series of gradient concentrations are diluted to, 4 concentration points are set altogether, respectively
For 3,1.5,0.75,0.25mg/mL, it is thin to MDA-MB-453 and MCF-7 for detecting with 0.22 μm of sterile filters filtration sterilization
The inhibitory action of intracellular growth.
(3) Activity determination
Take the logarithm respectively MDA-MB-453 the and MCF-7 cells in growth period, count, and it is thin to be suspended again with complete medium
Born of the same parents, to suitable concn, 100 μ L/ holes are inoculated into 96 orifice plates to adjustment cell concentration.At 37 DEG C, it is incubated under 5% carbon dioxide conditions
After 24h, the fresh Cordyceps extract of various concentrations is added by 100 μ L/ holes, each concentration sets 3 parallel holes, per the μ of hole 100
L.Blank control group adds culture medium, and 100 μ L set 6 parallel holes per hole；Negative control group addition cell, the every holes of 100 μ L,
6 parallel holes are set.Cell is continued respectively be placed in 37 DEG C, in 5% CO2gas incubator, be incubated 72h.3 groups add
CCK-8, per the μ L of hole 10, is placed in 37 DEG C, is incubated 1~2h in 5% CO2gas incubator.ELIASA determines each hole at 450nm
Absorbance (A) value, calculates the inhibiting rate of drug on tumor cell growth.
(4) testing result
Inhibitory action such as table 1 institute of the fresh Cordyceps extract of various concentrations to breast cancer MDA-MB-453 cell growths
Show.
Inhibitory action of the fresh Cordyceps extract of the various concentrations of table 1 to breast cancer MDA-MB-453 cell growths
From the data in table 1, it can be seen that fresh Cordyceps extract resulting in embodiment 1~3 is to breast cancer MDA-MB-453
Cell propagation is respectively provided with good inhibitory action, and metering dependence is presented, when fresh Cordyceps sinensis concentration is in embodiment 2
The effect of preferably suppression breast cancer cell growth is shown during 0.75mg/mL, inhibiting rate is up to 57.4%, with concentration
Raise, inhibitory action is significantly increased.
The fresh Cordyceps extract of various concentrations is as shown in table 2 to the inhibitory action that MCF-7 Breast Cancer Cell grows.
The inhibitory action that the fresh Cordyceps extract of the various concentrations of table 2 grows to MCF-7 Breast Cancer Cell
From the data in table 2, it can be seen that fresh Cordyceps extract resulting in embodiment is to breast cancer MDA-MA-453 cells
With certain inhibitory action, and metering dependence is presented, with the rising of concentration, inhibitory action is significantly increased.
The anti-transcellular research of melanoma tumors in Mice Body of the fresh Cordyceps extract of test example 2.
1. experiment material
1. experimental animal and cell：SPF grades of BALB/C mice (male, 18~20g, 60), this company of Changzhou Cavan carries
For quality certification number：201609149；
B16 melanoma cell strains.
2. Experimental agents：Fresh Cordyceps extract, is prepared by the method for embodiment 2.
3. positive control agent：Cis-platinum.
2. experimental technique
Take BALB/C mice and be placed in mouse injection fixator, tail vein inoculation B16 melanoma cell strains, inoculum density is
1×106/ ml, is inoculated with 100uL.The mouse of inoculated tumour cell is randomly divided into 6 groups, every group 10：Model control group (distillation
Water), positive drug cis-platinum control group (2mg/kg), fresh Cordyceps extract low dose group (50mg/kg), fresh Cordyceps sinensis extract
Thing middle dose group (100mg/kg), fresh Cordyceps extract high dose group (200mg/kg).On the inoculation same day, model control group is given
Give distilled water, gavage gives various dose fresh Cordyceps extract respectively for 4 fresh Cordyceps extract dosage groups, positive drug
It is administered within second day after group inoculation, every 2 days once abdominal cavity injection cis-platinums.Dissect animal within 21st day, calculate the score of lungs tubercle number, solution
Liver, lung are taken, calculating organ coefficient of weighing.
3. experimental result
Influence of the fresh Cordyceps extract of various dose to tumor nodule number is as shown in table 3：
Influence of the fresh Cordyceps extract of the various dose of table 3 to tumor nodule number
| Group | Lung tumors tubercle number | Metastasis suppressor rate |
| Model group | 20.8±3.6 | —— |
| Positive drug group | 5.1±1.8 | 78.6% |
| Fresh Cordyceps extract 50mg/kg | 4.9±1.0 | 79.4% |
| Fresh Cordyceps extract 100mg/kg | 5.9±1.4 | 75.4% |
| Fresh Cordyceps extract 150mg/kg | 8.1±3.3 | 65.8% |
From the data in table 3, it can be seen that the fresh Cordyceps extract of various dose has one to B16 MC Lung metastases
Determine inhibitory action, wherein, when dosage is 50~150mg/kg, suppress the effect highly significant of MC transfer, and turn
Shifting inhibiting rate is suitable with positive drug group, and maximal percentage inhibition is up to 79.4%.
Influence of the fresh Cordyceps extract of various dose to organ coefficient is as shown in table 4：
Influence of the fresh Cordyceps extract of the various dose of table 4 to organ coefficient
| Group | Liver coefficient (mg/kg) | Significant difference |
| Model control group | 52.53±3.49 | —— |
| Positive drug group | 45.08±3.42 | p<0.01 |
| Fresh Cordyceps extract 50mg/kg | 44.76±1.55 | p<0.01 |
| Fresh Cordyceps extract 100mg/kg | 46.11±4.74 | p<0.01 |
| Fresh Cordyceps extract 150mg/kg | 48.84±4.37 | p<0.01 |
From the data in table 4, it can be seen that compared with model control group, the liver coefficient of each fresh Cordyceps extract administration group shows
Work property is reduced, and is illustrated the fresh Cordyceps extract of various dose and is had well to the hepatic metastasis of B16 MCs
Inhibitory action.
Influence of the fresh Cordyceps extract of various dose to mouse death rate is as shown in table 5：
Influence of the fresh Cordyceps extract of the various dose of table 5 to mouse death rate
From the data in table 5, it can be seen that compared with model control group, the mouse death rate of each fresh Cordyceps extract administration group is equal
Significantly reduce, and the mouse coat color of each fresh Cordyceps extract administration group is smooth compared with cis-platinum group shiny black, activity is good,
Illustrate that fresh Cordyceps sinensis can significantly reduce the death rate of mouse, improve the mouse state of mind, improve mouse quality of life.
The sulfuric acid-phynol method of test example 3. determines polyoses content in fresh Cordyceps extract
(1) making of standard curve
0.1mg/mL glucose standards solutions 0,0.4,0.8,1.2,1.6,2.0mL are drawn respectively in the colorimetric cylinder of 25mL
In, 2.0mL is diluted to ultra-pure water, 5% phenol 1mL is separately added into, shake up, the 5mL concentrated sulfuric acids are added, shake up immediately, place
30min.Negate should after the μ L of solution 200 in ELISA Plate, parallel three parts, using ELIASA at 490nm mensuration absorbance value, with
Concentration is abscissa, and light absorption value is ordinate, obtains final product standard curve.
(2) measure of sample
A certain amount of fresh Cordyceps extract is taken, ultrapure water dissolves are added, 25mL is shaken up and be settled to.Precision is drawn
0.2mL sample prepare liquids, add water and complement to 2mL, are measured according to the method for standard curve making process, obtain test sample sample
Solution polyoses content concentration.
(3) measurement result
Determination of polysaccharide result is as shown in table 3 in fresh Cordyceps extract.
The fresh Cordyceps extract determination of polysaccharide result of table 6
| Test sample | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Polyoses content (%) | 74.9 | 76.7 | 69.4 |
As shown in Table 6, fresh Cordyceps extract contains more polysaccharide component, and polyoses content is 69.4~76.7%.
Polysaccharide molecular weight measure of spread in the fresh Cordyceps extract of test example 4
(1) chromatographic condition
Chromatographic column is TSKgel Super Multipore PW-H (150mm × 6.0mm, 8 μm), and mobile phase is 0.1mol/
L ammonium acetates, isocratic elution 20min, flow velocity is 0.4mL/min, and column temperature is 35 DEG C, and sample size is 15 μ L, ELSD detectors, carrier gas
It is nitrogen, nebulizer gas pressure is 3.5bar, and drift tube temperature is 60 DEG C, Gain：6Filter：6.Using Agilent GPC
CIRRUS DAS is analyzed.
(2) foundation of standard molecular weight curve
The glucan reference substance about 0.5mg that molecular weight is respectively 5000,25000,80000,150000,410000 is weighed,
It is dissolved in water, makes concentration be 0.5mg/mL.The glucan reference substance solution difference sample introduction analysis of different molecular weight is taken, it is poly- with each Portugal
The weight average molecular weight logarithm of sugar is ordinate, and retention time carries out linear regression analysis, obtains molecular weight calibration curve for abscissa.
(3) test sample is determined
Weigh fresh Cordyceps sinensis dried frozen aquatic products appropriate, about 2.0mg, plus ultrapure water dissolves, make concentration about 2mg/mL.Sample introduction point
Analysis, the molecular weight distribution of each chromatographic peak of application data analysis software calculates each component analyzed with area normalization method
Ratio in chromatographic peak.
(4) testing result
Above-mentioned glucan control series product solution difference sample introduction is taken, the retention time of eluting peak is recorded, by GPC special-purpose softwares
Standard curve is drawn, the logarithm value with standard molecular weight is carried out as ordinate by abscissa of the retention time of corresponding chromatographic peak
Linear regression, obtains regression equation y=13.85-1.419x, R2=0.9946.According to GPC special-purpose softwares draw standard curve and
The retention time of test sample calculates the weight average molecular weight and its molecular weight distribution of sample each component using GPC special-purpose softwares.The fresh winter
The polysaccharide molecular weight of worm summer grass extract is distributed chromatogram as shown in figure 1, being followed successively by fresh Cordyceps extract (a from the bottom up
It is 1422KDa, b is 29KDa, and c is 2.7KDa), glucan reference substance 5KDa, glucan reference substance 25KDa, glucan reference substance
80KDa, glucan reference substance 150KDa, glucan reference substance 410KDa.
As shown in Figure 1, various polysaccharide molecules are contained in Cordyceps sinensis sample.From measurement result, embodiment 1~3
Be respectively provided with Cordyceps extract 3 kinds of molecular weight more than 1KDa main peak, weight average molecular weight be respectively 1422KDa, 29KDa,
2.7KDa, weight average molecular weight is respectively 1.85,1.40,1.41, and the corresponding color of each component with the ratio PD of number-average molecular weight
Ratio shared by spectral peak area is respectively 7.65%, 19.12%, 47.04%.
Protein content determination in the fresh Cordyceps extract of test example 5
(1) preparation of coomassie brilliant blue staining agent
Weigh Coomassie brilliant G-250 100.0mg and be dissolved in the ethanol of 50mL 95%, add the phosphoric acid of 100mL 85%, spend
Ionized water is diluted to 1000mL, and after fully mixing, filter paper filtration is obtained final product.
(2) preparation of need testing solution
By fresh Cordyceps extract test sample powder it is accurate it is weighed after be transferred in 50mL centrifuge tubes, add deionized water
20.0mL, seals, and is placed on turbula shaker after vortex 30s, is transferred in precooling mortar, then the mortar on ice chest
20min.15min is centrifuged under the conditions of 4 DEG C afterwards, rotating speed is 13500rpm, takes supernatant and obtains final product.
(3) foundation of standard curve
A. standard curve making：Take 7 teat glasses, sequentially add 0,0.01,0.02,0.04,0.06,0.08,
The BSA standard liquids (2.0mg/mL) of 0.10mL, 0.10mL is supplemented to deionized water.Add 5.0mL Coomassie brilliant blues dye
Toner (is mixed) immediately after often adding a pipe.Each concentration be arranged in parallel 3 groups.After 10min is heated in 25 DEG C of water-baths, let cool to
Room temperature.Draw during 200 μ L solution add ELISA Plate hole, with 0mL BSA standard liquids as blank, in being surveyed under 595nm wavelength
Determine absorbance.With BSA concentration as abscissa, light absorption value is ordinate, and standard curve is obtained.
B. test sample sample protein matter content is determined：The sample solution 0.10mL of above-mentioned steps (2) configuration is drawn in glass
In test tube, 5.0mL coomassie brilliant blue staining agent is added, be well mixed.Every part of test sample sample be arranged in parallel 3 groups.Then 25
After heating 10min in DEG C water-bath, let cool to room temperature.Draw during 200 μ L solution add ELISA Plate hole, in being determined under 595nm wavelength
Absorbance, calculates protein content in sample.
(4) testing result
Fresh Cordyceps extract protein content determination result is as shown in table 4.
The Cordyceps extract protein content determination result of table 7
| Test sample | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Protein content (%) | 12.1 | 10.2 | 14.3 |
As shown in Table 4, fresh Cordyceps extract protein content is 10.2~14.3%.
Protein molecular weight measure of spread in the fresh Cordyceps extract of test example 6
(1) preparation of test sample protein sample
By fresh Cordyceps extract powder it is accurate it is weighed after be transferred in 50mL centrifuge tubes, add deionized water
20.0mL, seals, and is transferred in precooling mortar after being placed on turbula shaker the 30s that is often vortexed, the mortar on ice chest
30min.15min is centrifuged at 4 DEG C afterwards, rotating speed is 13500rpm, and supernatant is protein solution.Take supernatant samples solution
200 μ L to 2mL centrifuge tubes, 2 × Loading of addition Buffer [0.25M Tris-HCl pH 6.8,10% (m/v) SDS,
30% (v/v) glycerine, 0.03% (v/v) bromophenol blue] the μ L of solution 200, it is well mixed, the μ L of 0.1M DTT solution 8 mixings are added,
In 80 DEG C of heating water bath 10min, centrifugation (12000rpm, 5min) is taken out, supernatant is loading sample.
(2) SDS- polyacrylamide gel electrophoresises (SDS-PAGE) detection molecular weight of albumen
A. glue：After assembling glass mold, according to the form below first prepares separation gel, separation gel filled above after filling one layer go from
Sub- water with completely cut off air and eliminate bubble.Concentration glue is filled after glue polymerization to be separated, loading comb, glue polymerization to be concentrated are plugged after filling well
Afterwards can loading (wherein 10%AP and TEMED are added before needing encapsulating).
The separation gel of table 8 and concentration glue allocation list
| Separation gel (12%, 20mL) | Concentration glue (5%, 5mL) | |
| Distilled water | 6.6 | 2.77 |
| 30% acrylamide | 8.0 | 0.83 |
| Tris-HCl (1.5M, pH=8.80) | 5.0 | - |
| Tris-HCl (0.5M, pH=6.80) | - | 1.26 |
| 10%SDS | 0.2 | 0.05 |
| 10%AP | 0.2 | 0.05 |
| TEMED | 0.008 | 0.005 |
B. loading：The μ L of sample solution 20 that aspiration step (1) is handled well, are added in loading hole, while loading hole aside
2 μ L molecular weight of albumen Marker of middle injection.
C. electrophoresis：5 × SDS- electrophoretic buffers are poured into electrophoresis tank, and [0.125M Tris-HCl pH 6.8,1.25M are sweet
Propylhomoserin, 0.5% (w/v) SDS)], to connect with the mains, setting voltage is 80V, treats that bromophenol blue is migrated to separation gel in concentration glue interface
When voltage is adjusted to 140V, when bromophenol blue only has 1cm or so from bottom, close power supply, terminate electrophoresis；
D. dye：By gel carefully from glass plate in taking-up placement dyeing disk, after gently washing three times with deionized water,
Add Coomassie brilliant blue dye liquor, to shaking table on (rotating speed 100rpm) stained over night；
E. develop the color：Coomassie brilliant blue dye liquor is outwelled, with the net gel surface dyeing liquor of pure water rinsing, 3 × 10min.Ensure
The basic washes clean of gel surface dyeing liquor.
F. take pictures：Gel after colour developing is placed in gel imaging instrument and takes pictures and carry out data processing.
(3) experimental result
Cordyceps extract protein electrophorese figure is as shown in Figure 2.
As shown in Figure 2, molecular weight of albumen distribution is primarily present in 10~15KDa and 20 in fresh Cordyceps extract
At~37KDa.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.All publications or patent document cited in the present invention all will be used as this hairs
Bright bibliography.
Claims (10)
1. a kind of fresh Cordyceps extract with antitumor activity, it is characterised in that polysaccharide in fresh Cordyceps extract
Content is 69.4~76.7%, and protein content is 10.2~14.3%.
2. the fresh Cordyceps extract with antitumor activity according to claim 1, it is characterised in that the polysaccharide
Polysaccharide comprising 3 kinds of molecular weight more than 1KDa, weight average molecular weight is respectively 1422KDa, 29KDa and 2.7KDa；The albumen
Range of molecular weight distributions is primarily present at 10~15KDa and 20~37KDa.
3. the preparation method of the fresh Cordyceps extract with antitumor activity described in a kind of any one of claim 1~2,
The preparation method is comprised the following steps：
A, fresh Cordyceps sinensis is crushed, add water homogenate, crushed, control temperature for 15~25 DEG C；
B, by homogenate after fresh Cordyceps sinensis carry out ultrasonic extraction, control temperature for 15~25 DEG C, supernatant is centrifuged to obtain, then will
Fresh Cordyceps sinensis residue stands in -20 DEG C of freezings；
C, the fresh Cordyceps sinensis residue that will be freezed take out, and add water and are homogenized and are uniformly dispersed, again ultrasonic extraction, and it is 15 to control temperature
~25 DEG C, it is then centrifuged for, obtains supernatant, the supernatant of combining step b；
D, toward centrifugation after supernatant in plus absolute ethyl alcohol to concentration be 60~80% after, stood overnight at 4 DEG C；In at 4 DEG C
Refrigerated centrifuge, collects precipitation；
E, precipitation is carried out into freeze-drying, obtain fresh Cordyceps extract.
4. the preparation method of the fresh Cordyceps extract with antitumor activity according to claim 3, wherein, in step
In rapid a, described fresh Cordyceps sinensis and the w/v of water are 1:8~12g/mL, Homogenization time is 4~8min, and homogenate turns
Speed is 14000~18000rpm.
5. the preparation method of the fresh Cordyceps extract with antitumor activity according to claim 3, wherein, in step
In rapid b, the ultrasonic extraction time is 20~40min, and ultrasonic power is 400~600W, and centrifugation time is 10~15min, and centrifugation turns
Speed is 8000~10000rpm, and freezing time of repose is 20~60min.
6. the preparation method of the fresh Cordyceps extract with antitumor activity according to claim 3, wherein, in step
In rapid c, described fresh Cordyceps sinensis residue and the w/v of water are 1:8~12g/mL, again Homogenization time be 4~
8min, homogenate rotating speed be 14000~18000rpm, again the ultrasonic extraction time be 20~40min, ultrasonic power be 400~
600W, centrifugation time is 10~15min, and centrifugal rotational speed is 8000~10000rpm.
7. the preparation method of the fresh Cordyceps extract with antitumor activity according to claim 3, wherein, in step
In rapid d, the time of refrigerated centrifuge is 15~25min, and the rotating speed of centrifugation is 8000~10000rpm.
8. the preparation method of the fresh Cordyceps extract with antitumor activity according to claim 3, the preparation
Method is comprised the following steps：
A, fresh Cordyceps sinensis is crushed, 1 is added by the w/v of fresh Cordyceps sinensis and water:The water of 10g/mL, is homogenized 6min,
Homogenate rotating speed is 16000rpm, is crushed, and controls temperature for 20 ± 2 DEG C, and fresh Cordyceps sinensis is ground into the suspension of fine particle；
B, by homogenate after fresh Cordyceps sinensis carry out ultrasonic extraction 30min, control temperature for 20 ± 2 DEG C, power is 400~
600W, is centrifuged 12min, and rotating speed is 9000rmp, obtains supernatant, and then fresh Cordyceps sinensis residue is stood in -20 DEG C of freezings
40min；
C, the fresh Cordyceps sinensis residue that will be freezed take out, and it is 1 to add w/v:The water of 10g/mL, is homogenized 6min, and homogenate turns
Speed is 16000rpm, and is uniformly dispersed, again ultrasonic extraction 30min, and it is 20 ± 2 DEG C to control temperature, and power is 400~600W,
12min is then centrifuged for, the rotating speed of centrifugation is 9000rpm, obtains supernatant, the supernatant of combining step b；
D, toward centrifugation after supernatant in plus absolute ethyl alcohol to concentration be 60~80% after, stood overnight at 4 DEG C；In at 4 DEG C
Refrigerated centrifuge 20min, centrifugal rotational speed is 10000rpm, collects precipitation；
E, precipitation is carried out into freeze-drying, obtain fresh Cordyceps extract.
9. the fresh Cordyceps extract with antitumor activity described in a kind of any one of claim 1~8 is anti-in preparation
Application in breast cancer or melanoma cell diversion medicaments.
10. application according to claim 9, wherein, the anti-breast cancer medicines are to suppress human breast carcinoma MDA-MB-453
The medicine of cell, melanoma cell diversion medicaments are the medicines for suppressing anti-B16 MCs transfer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109717348A (en) * | 2019-03-08 | 2019-05-07 | 中国科学院西北高原生物研究所 | A method of the fresh-keeping cordyceps sinensis of controlled atmospheric packing containing argon gas |
| WO2023236700A1 (en) * | 2022-06-07 | 2023-12-14 | 香港理工大学 | Use of functionalized nano-selenium hydrosol in anti-tumor aspect |
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| P. H. LEUNG ET AL: "Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 宋林霞: "冬虫夏草抗肿瘤作用的研究进展及展望 ", 《安徽农业科学》 * |
| 王爱丽: "北冬虫夏草浸提液抗肿瘤细胞活性研究 ", 《生物化工》 * |
| 赵丰丽: "预处理对水提取醇沉法提取虫草多糖效果的影响研究", 《食品工业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109717348A (en) * | 2019-03-08 | 2019-05-07 | 中国科学院西北高原生物研究所 | A method of the fresh-keeping cordyceps sinensis of controlled atmospheric packing containing argon gas |
| WO2023236700A1 (en) * | 2022-06-07 | 2023-12-14 | 香港理工大学 | Use of functionalized nano-selenium hydrosol in anti-tumor aspect |
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