CN106064993A - The culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers., bioconversion mycelium, the mycelial extract of bioconversion and application thereof - Google Patents
The culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers., bioconversion mycelium, the mycelial extract of bioconversion and application thereof Download PDFInfo
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Abstract
The invention discloses the culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers., bioconversion mycelium, the mycelial extract of bioconversion and application thereof.The culture medium of this Hericium erinaceus (Bull. Ex Fr.) Pers. is addition Scutellaria barbata in basal medium, and including basal medium and Herba Scutellariae Barbatae, the culture medium of every 100 parts includes basal medium 70~50 parts, Herba Scutellariae Barbatae 30~50 parts;Described basal medium includes each component of following weight portion: Testa Tritici 490~350 parts, Semen Maydis powder 140~100 parts, bran coat 63~45 parts, white sugar 7~5 parts, water 400~600 parts.This bioconversion mycelium is Hericium erinaceus (Bull. Ex Fr.) Pers. to be accessed above-mentioned culture medium convert the product cultivating gained through mycelia.The mycelial extract of this bioconversion is by Hericium erinaceus (Bull. Ex Fr.) Pers. filament concentrating under reduced pressure again after water carries, last vacuum lyophilization and prepare.The mycelial extract of this bioconversion has anti-gastrointestinal tract cancer effect, and effect is substantially better than the anti-gastrointestinal tract cancer effect of Hericium erinaceus (Bull. Ex Fr.) Pers. filament or Herba Scutellariae Barbatae, can be used for preparing anti-gastrointestinal tract cancer drug.
Description
Technical field
The present invention relates to biological pharmacy technical field, particularly to the culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers., bioconversion mycelium,
Mycelial extract of bioconversion and application thereof.
Background technology
Hericium erinaceus (Bull. Ex Fr.) Pers. is a kind of hedgehog hydnum Cordycepps mushroom (fungus), is used for treating gastroenteropathy at Chinese medicine and alreadys more than 2000
History, a series of micromolecular compounds such as erinacines, be considered to have neuranagenesis, and blood brain screen can be passed through
Barrier repairs damaged nerve tissue.Hericium erinaceus (Bull. Ex Fr.) Pers. also has antiulcer, antiinflammatory, antimicrobial, immunomodulating, improves liver function, defying age,
Reduce blood glucose and blood fat, improve anti-anoxia ability, increase heart output and improve the effects such as remarks circulation, comprising Hericium erinaceus (Bull. Ex Fr.) Pers.
Medical preparation is widely used in China.
Active substance in Hericium erinaceus (Bull. Ex Fr.) Pers. filament the most important thing is polysaccharide and glycoprotein, grinds Hericium erinaceus polysaccharide both at home and abroad at present
Studying carefully and show, Hericium erinaceus (Bull. Ex Fr.) Pers. Polysaccharides has multiple biological activity and pharmacological action, can strengthen the phagocytic function of macrophage, promotes haemolysis
The formation of element, anti-leukopenia, blood sugar lowering, anticoagulation, antithrombotic, mutation and anti-ageing wait for a long time.Therefore, Hericium erinaceus (Bull. Ex Fr.) Pers. Polysaccharides is standby
Paid close attention to by people, become the area researches such as molecular biology, medicine, Food Science in recent years and the focus of exploitation application." China
Pharmacopeia " describe Hericium erinaceus (Bull. Ex Fr.) Pers. filament (seeing the 1614-1615 page of version in 2015), Hericium erinaceus (Bull. Ex Fr.) Pers. filament of the present invention
It it is exactly the Hericium erinaceus (Bull. Ex Fr.) Pers. filament of this standard record.
Currently, carry out screening and finding active component from higher fungus and culture thereof, be applied to antitumor, disease-resistant
Poison and treatment diabetes etc., not only become the developing direction of state key research new drug, be also world exploitation biology
The focus of medicine.But, at present and have not seen the Chinese medicine culture medium as Hericium erinaceus (Bull. Ex Fr.) Pers. novel medicinal bacterium after bioconversion
Filament is applied to the report of gastrointestinal cancer.
Herba Scutellariae Barbatae (formal name used at school: Scutellaria barbata D.Don), another name: Herba Portulacae Grandiflorae (botany voluminous dictionary), Folium Pini
Paeonia suffruticosa, dragon palpus Paeonia suffruticosa, spun gold Cuculus polioephalus, ocean Herba Portulacae, heronsbill, Herba Portulacae Grandiflorae, be horse Labiatae Scutellaria herbaceos perennial.
Herba Portulacae Grandiflorae all herbal medicine, has heat-clearing and toxic substances removing, blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, the function such as anticancer.Sour in the mouth cold in nature, herb is containing multiple
The compositions such as vitamin, trace element and aminoacid.There are removing pathogenic heat from blood and toxic substance from the body, eliminating stasis to stop pain, detumescence and effect of clearing away heat-damp and promoting diuresis.Herba Scutellariae Barbatae
Containing alkaloid, flavonoid glycoside, steroidal and phenols, tannin etc..Its contained rivularin, is a kind of flavone compound
(2,5-dihydroxy-6,7,8-trimethoxy flavones), additionally, also other two flavone compound Chinese Radix Scutellariae element (5,7-dihydroxies
Base-8 methoxy flavone) and 5-hydroxyl-7,8-dimethoxy flavone.Zoopery confirms, Herba Scutellariae Barbatae is to sarcoma 180, Emhorn abdomen
Water cancer, cerebroma 22 etc. all have certain inhibitory action.
Japanese scholars is found to have 88 kinds of Chinese medicines to swollen when 800 kinds of Chinese medicines being made antitumor activity screening by experiment in vitro
The suppression ratio of tumor cell proliferation is more than 90%, and wherein Herba Scutellariae Barbatae reaches 100% to JTc-26 oncocyte extracorporeal inhibiting rate, and it aligns
Often the suppression ratio of cell is then only 50%.Find when observing the clinical antineoplastic curative effect of these Chinese medicines, even weary to treatment
The late tumor of art, also has and improves symptom, suppression tumor proliferation and extend the effect of patient vitals.Experiment also demonstrates that, uses methylene blue
Test tube method, BANJILIAN has slight inhibitory action to Acute granulocytic leukemia cell;Use Cellular respiration method, to acute myelogenous
Type leukaemia and suppression ratio are more than 75%.Sculellaria barbata formulation is also equal to animal tumors such as W256.U14.S186.EAC, ESC
There is certain inhibitory action.In addition to antitumaous effect, Herba Scutellariae Barbatae is the most antibacterial, diuresis, cough-relieving, the multiple pharmacological effect such as relieving asthma.
Gastrointestinal cancer such as hepatocarcinoma, gastric cancer, colorectal cancer are one of modal cancers, estimate to account for according to ACS
The 25% of all cancers.Gastric cancer sickness rate in the world is positioned at the 4th in cancer, in the highest state-owned incidence rate and death
Rate (death of 50% occurs in China), the treatment of gastrointestinal cancer includes operation, chemotherapy, radiotherapy and exempts from
Epidemic disease therapy.Chemotherapy is most common method, although cannot thoroughly treat.In the decades in past, sending out of molecular biology
Exhibition makes some progress in the treatment of gastric cancer, but drug resistance and toxicity still limit the progress of Therapeutic Method.
Bioconversion " mycelium " refers to utilize the metabolic function of mycelium (including fungus), makes the biology that Organic substance decomposes
Chemical reaction process.With suitable culture medium as nutrition, produce abundant time by mycelial growth metabolism and vital movement
Raw metabolite.Use for reference Chinese medicine prescription thought, using single medicinal material, there is similar or synergistic Chinese medicine as part cultivation
Base carries out mycelium conversion, the bioactive ingredients that different strains mutation " secondary " metabolism is different, then carries out " post-directed training, double
To converting " bio-conversion process, different biological active matter raw material can be obtained, it is therefore an objective to strengthen effect or reduce medicine poison pair
Effect.
Summary of the invention
It is contemplated that overcome the deficiencies in the prior art, interdisciplinary innovation, multidisciplinary in conjunction with microbiology, Chinese materia medica etc.,
There is provided a kind of new raw material (Herba Scutellariae Barbatae) as the medium component of Hericium erinaceus (Bull. Ex Fr.) Pers., i.e. it is an object of the invention to provide a kind of biological turn
Change mycelium, the mycelial extract of bioconversion and application thereof.The preparation method of the mycelial extract of described bioconversion
Simply, cost is relatively low, and extract is used for preparing anti-gastrointestinal tract cancer drug.
Based on above-mentioned purpose, the culture medium of a kind of Hericium erinaceus (Bull. Ex Fr.) Pers. of the present invention, including basal medium and Herba Scutellariae Barbatae, every 100
Part culture medium include basal medium 70~50 parts, Herba Scutellariae Barbatae 30~50 parts;Described basal medium includes following weight
Each component of part:
Testa Tritici 490~350 parts, Semen Maydis powder 140~100 parts, bran coat 63~45 parts, white sugar 7~5 parts, water 400~600 parts.
In the present invention, it is preferred to, described basal medium includes each component of following weight portion:
420 parts of Testa Tritici, Semen Maydis powder 120 parts, bran coat 54 parts, white sugar 6 parts, 500 parts of water.
In the present invention, it is preferred to, the culture medium of every 100 parts includes basal medium 60 parts, Herba Scutellariae Barbatae 40 parts;Described
Basal medium includes each component of following percetage by weight: Testa Tritici 70%, Semen Maydis powder 20%, bran coat 9%, white sugar 1%.This be
Optimal Hericium erinaceus (Bull. Ex Fr.) Pers. Medium Proportion.
Present invention also offers a kind of bioconversion mycelium, by hedgehog hydnum mushroom inoculation to the most described culture medium,
At 20~27 DEG C, cultivate 30~40 days, take out mycelium, dry, obtain bioconversion mycelium.
In the present invention, it is preferred to, described hedgehog hydnum mushroom bacterial strain is hedgehog hydnum Cordycepps fungus Hericium erinaceus (Bull. Ex Fr.) Pers. Hericium
erinaceum(Bull.ex Fr.)Pers。
In the present invention, it is preferred to, described hedgehog hydnum Cordycepps fungus Hericium erinaceus (Bull. Ex Fr.) Pers. Hericium erinaceum (Bull.ex
Fr.) mycelium of Pers. is grown nonparasitically upon another plant with it drying composite of the solid medium containing Herba Scutellariae Barbatae composition.
Further, present invention also offers a kind of mycelial extract of bioconversion, described extract is by following
Step prepares:
(1) described bioconversion mycelium is carried out water to carry, be soaked in water 2-3 time at 50-70 DEG C, soak 2-5 every time
Hour, the weight of water is 6~10 times of biological transformed bacteria filament weight, obtains extracting solution;
(2) by the extracting solution concentrating under reduced pressure in step (1), by the concentrated solution of concentrating under reduced pressure gained under the conditions of 50-70 DEG C
Evacuation is dried, and obtains the mycelial extract of bioconversion.
In the present invention, it is preferred to, described in the temperature that is soaked in water be 60 DEG C;By the concentrated solution of concentrating under reduced pressure gained 60
Under the conditions of DEG C, evacuation is dried.
Further, present invention also offers the mycelial extract of described bioconversion and prepare anti-gastrointestinal tract cancer
Purposes in medicine.
In the present invention, it is preferred to, described gastrointestinal cancer is the esophageal carcinoma, gastric cancer, colon cancer or hepatocarcinoma.
The mycelial extract of bioconversion of the present invention uses the simple technique that processes to prepare, its antitumous effect
Just reach unforeseeable technique effect, be better than all of natural plants through the antitumous effect of " mycelium " of bioconversion
The Chinese crude drug in source, if further isolated and purified active constituent-enriched, its antitumous effect will more preferably can be used for preparing anti-gastrointestinal
Road cancer drug.
Hericium erinaceus (Bull. Ex Fr.) Pers. is a kind of medicinal fungi, can absorb culture medium Middle nutrition well, thoroughly convert culture medium, produce new
Active substance and metabolite, to this end, Scutellaria barbata is joined in the basal medium of Hericium erinaceus (Bull. Ex Fr.) Pers. by the present invention, as Hericium erinaceus (Bull. Ex Fr.) Pers.
One of medium component, and the ratio of the culture medium that Chinese crude drug Herba Scutellariae Barbatae mixes Hericium erinaceus (Bull. Ex Fr.) Pers. is allocated, final selects
The ratio that medical material Herba Scutellariae Barbatae most preferably mixes is 40%.When Herba Scutellariae Barbatae accounts for 40% ratio, mycelial growth situation is preferable, total flavones,
Content Measurement of Scutellarin is higher.
Hericium erinaceus (Bull. Ex Fr.) Pers. carries out Chinese medicine mycelium conversion to Herba Scutellariae Barbatae, obtains new mycelium.The biology of gained is turned by the present invention
Changing mycelium to extract, the mycelial extract of bioconversion obtained has carried out In Vitro Anti gastroenteric tumor cytoactive sieve
Choosing test, the water product that get sample are with IC50 value and Emax as evaluation index, and result of the test shows: the mycelial extract pair of bioconversion
Gastroenteric tumor cell all has good inhibiting effect, and anti-cancer effect in vitro is by weak being by force: bioconversion mycelium > Herba Scutellariae Barbatae
Medical material > Hericium erinaceus (Bull. Ex Fr.) Pers. filament ≈ Hericium erinaceus (Bull. Ex Fr.) Pers. filament+Herba Scutellariae Barbatae, the mycelial extract anti-gastrointestinal tract cancer effect of this bioconversion is obvious
It is better than the anti-gastrointestinal tract cancer effect of Hericium erinaceus (Bull. Ex Fr.) Pers. filament or Herba Scutellariae Barbatae, and bioconversion mycelial IC50 value is more than independent half
The IC50 value sum of lotus medical material and independent Hericium erinaceus (Bull. Ex Fr.) Pers. filament, Emax is more than independent Herba Scutellariae Barbatae medical material and independent Hericium erinaceus (Bull. Ex Fr.) Pers. filament
Emax sum, enzyme system abundant in this explanation Hericium erinaceus (Bull. Ex Fr.) Pers. is by structural modification or Degradation, the active component in Herba Scutellariae Barbatae
Change into the higher composition of activity, or enhance the yield of active component (such as total flavones) in Herba Scutellariae Barbatae, or nonactive one-tenth
Dividing and change into active component, active component is mutually promoted and is reached to strengthen the purpose of anticancer function;Carry out people gastric cancer (MGC80-simultaneously
3) transplanting inhibition test in nude mice, result of the test shows: the mycelial extract of bioconversion is to people's gastric cancer (MGC80-3)
Tumor bearing nude mice has obvious tumor-inhibiting action.Therefore, the mycelial extract of bioconversion can be used for preparing anti-gastrointestinal tract cancer medicine
Thing.
Water at low temperature of the present invention soaks bioconversion mycelium, water soaking at 50-70 DEG C, without high temperature, can dissolve
Effective ingredient but do not destroy effective ingredient;50-70 DEG C of concentrating under reduced pressure, will not destroy effective ingredient, particularly active enzyme thing
Matter;In order to not affect relevant active substance, under the conditions of present invention preferably employs 60 DEG C, evacuation is dried.
Accompanying drawing explanation
Fig. 1 is anti-gastric cancer MGC80-3 transplanted tumor curative effect and animal toxicity figure in HB-4 body;
Fig. 2 is that HB-4 affects figure to people's gastric cancer transplanting tumor bearing nude mice tumor body tumor suppression;Wherein, note: D after administration10;A. model pair
According to group;B.HB-4 low dose group;Dosage group in C.HB-4;D.HB-4 high dose group;E. fluorouracil group;F. fluorouracil+HB-
4 groups;G HB-4 (2000mg/kg) group;
Fig. 3 is that people's gastric cancer is transplanted tumor bearing nude mice tumor body internal anatomy by HB-4;
Fig. 4 be in HB-4 body anti-gastric cancer MGC80-3 transplanted tumor weight loss rate affect result figure.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with specific embodiment, to this
Bright further description.
Embodiment 1
A kind of mycelial extract of bioconversion, its preparation method comprises the steps:
(1) agricultural byproducts Testa Tritici 490g, Semen Maydis powder 140g, bran coat 63g, white sugar 7g, and Chinese crude drug Herba Scutellariae Barbatae coarse powder are taken
(Herba Scutellariae Barbatae) 300g, mixing, add 500g water and mix thoroughly, obtain the culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers..Bottle again, sterilizing, access Hericium erinaceus (Bull. Ex Fr.) Pers.,
Convert under the conditions of 27 DEG C and cultivate 40 days, take out mycelium, dry, obtain bioconversion mycelium;
(2) bioconversion mycelium is taken, 60 DEG C of water soakings 2 times, to soak 2 hours every time, the weight of water be biology transformed bacteria
6 times of filament weight, obtain extracting solution;
(3) the extracting solution concentrating under reduced pressure obtained after extracting, takes out the concentrated solution of concentrating under reduced pressure gained under the conditions of 60 DEG C
Vacuum drying, obtains extract.
Embodiment 2
A kind of mycelial extract of bioconversion, its preparation method comprises the steps:
(1) agricultural byproducts Testa Tritici 420g, Semen Maydis powder 120g, bran coat 54g, white sugar 6g, and Chinese crude drug Herba Scutellariae Barbatae coarse powder are taken
(Herba Scutellariae Barbatae) 400g, mixing, add 500g water and mix thoroughly, obtain the culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers.;Bottle again, sterilizing, access Hericium erinaceus (Bull. Ex Fr.) Pers.,
Convert under the conditions of 20 DEG C and cultivate 40 days, take out mycelium, dry, obtain bioconversion mycelium;
(2) bioconversion mycelium is taken, 60 DEG C of water soakings 3 times, to soak 3 hours every time, the weight of water be biology transformed bacteria
8 times of filament weight, obtain extracting solution;
(3) the extracting solution concentrating under reduced pressure obtained after extracting, takes out the concentrated solution of concentrating under reduced pressure gained under the conditions of 60 DEG C
Vacuum drying, obtains extract.
Embodiment 3
A kind of mycelial extract of bioconversion, its preparation method comprises the steps:
(1) agricultural byproducts Testa Tritici 350g, Semen Maydis powder 100g, bran coat 45g, white sugar 5g, and Chinese crude drug Herba Scutellariae Barbatae coarse powder are taken
(Herba Scutellariae Barbatae) 500g, mixing, add 500g water and mix thoroughly, obtain the culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers.;Bottle again, sterilizing, access Hericium erinaceus (Bull. Ex Fr.) Pers.,
Convert under the conditions of 25 DEG C and cultivate 30 days, take out mycelium, dry, obtain bioconversion mycelium;
(2) bioconversion mycelium is taken, 60 DEG C of water soakings 3 times, to soak 4 hours every time, the weight of water be biology transformed bacteria
10 times of filament weight, obtain extracting solution;
(3) the extracting solution concentrating under reduced pressure obtained after extracting, takes out the concentrated solution of concentrating under reduced pressure gained under the conditions of 60 DEG C
Vacuum drying, obtains extract.
Embodiment 4 Herba Scutellariae Barbatae mixed ratio is tested
Basal medium feeds intake in terms of siccative, and ratio is fixed as: Testa Tritici 70%, Semen Maydis powder 20%, bran coat 9%, white sugar 1%
(not including water).The present invention is using Chinese crude drug Herba Scutellariae Barbatae as one of medium component, and the ratio that Chinese crude drug Herba Scutellariae Barbatae mixes is respectively
Accounting for 30%, 40%, the 50% of Hericium erinaceus (Bull. Ex Fr.) Pers. culture medium total amount, contrived experiment scheme, always feed intake 5000g, presses above-described embodiment respectively
1, the step preparation culture medium described in embodiment 2, embodiment 3, the 500g mixing that adds water, sterilizing, inoculates, cultivates, parallel control, trains
The condition of supporting is completely the same, observes mycelial growth situation, growth cycle, pollution condition, the speed of growth, after digging bottle in incubation
Drying is weighed calculated yield, and cellulase in detection sample, Quantitative Determination of Ergosterol (investigate convert the most thorough), total flavones contain
Amount, Content Measurement of Scutellarin, dried sample is respectively labeled as HB-1, HB-2, HB-3.Result of the test statistics is as shown in table 1 below:
Table 1
Packet numbering | HB-1 | HB-2 | HB-3 |
Material gross weight (g) before cultivating | 5000g | 5000g | 5000g |
Contamination ratio (%) in incubation | 9.5% | 8.0% | 15.2% |
Growth cycle (my god) | 33 days | 34 days | 39 days |
The speed of growth (mm/ days) | 4.85mm/ my god | 4.71mm/ my god | 4.10mm/ my god |
Cellulase content (U/g) | 188U/g | 194U/g | 154U/g |
Quantitative Determination of Ergosterol (mg/g) | 0.80mg/g | 0.85mg/g | 0.68mg/g |
General flavone content | 1.22% | 1.55% | 1.62% |
Content Measurement of Scutellarin | 0.16% | 0.30% | 0.35% |
Result of the test represents: when Herba Scutellariae Barbatae accounts for 50%, pollution rate is high, the speed of growth slow, growth cycle length, cellulase wheat
Angle sterol content is low, and bioconversion is the most thorough;And Herba Scutellariae Barbatae the speed of growth is very fast, growth cycle is short when accounting for 30%, but total flavones,
Content Measurement of Scutellarin is low;When Herba Scutellariae Barbatae accounts for 40% ratio the most suitable, mycelial growth situation is preferable, takes into account each advantage, for
Good ratio.
Embodiment 5 In Vitro Anti gastroenteric tumor cytoactive screening test
1. test material
1.1 cell strain
Human esophagus cancer cell Eca-109, gastric carcinoma cells MGC80-3, human colon cancer cell LoVo are purchased from the Chinese Academy of Sciences
Sea cell bank, high-transfer human liver cancer cell strain MHCC97-H is purchased from Fu Xiang bio tech ltd, Shanghai.
1.2 test sample
Sample number into spectrum: HTJ represents that Hericium erinaceus (Bull. Ex Fr.) Pers. filament, BZL represent Herba Scutellariae Barbatae Chinese crude drug, and HTJ+BZL represents the hedgehog hydnum of 60%
Mycelium+40% Herba Scutellariae Barbatae Chinese crude drug, HB-3, HB-4, HB-5 represent Herba Scutellariae Barbatae mixed ratio in the culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers. respectively
It it is the bioconversion mycelium of 30%, 40%, 50%.The operation of above-mentioned sample parallel is handled as follows: 60 DEG C of water soakings 3 times,
Soaking 4 hours, the weight of water is 10 times of biological transformed bacteria filament weight every time;The extracting solution decompression obtained after extracting is dense
Contracting, is dried the concentrated solution of concentrating under reduced pressure gained evacuation under the conditions of 60 DEG C, obtains five kinds of extracts, respectively numbered HTJ,
BZL, HTJ+BZL, HB-3, HB-4, HB-5.
1.3 positive reference substance
Cisplatin for injection (DDP), is produced by Qilu Pharmaceutical Co., Ltd., lot number: 1100331DB;10mg/ bottle.
1.4 main agents
DMEM in high glucose culture medium, HyClone company, lot number NXJ0709,500ml/ bottle;Hyclone, HyClone company,
Lot number NXC0582,500ml/ bottle;Tetrazolium bromide, sigma company, lot number MKBH7489V, 1g/ bottle;Dimethyl sulfoxide, traditional Chinese medicines grouping of the world economy
Learn reagent company limited, lot number 20120705,500ml/ bottle;Trypsin, Beijing prosperity Bioisystech Co., Ltd of ancient cooking vessel state, batch
Number 04D10151,25g/ bottle.
1.5 key instrument
CJ-1F type Medical purification workbench (center number 058, Suzhou Feng Shi laboratory animal equipment company limited);Desk-top
Low speed centrifuge (center number 101, Kai Da industry development company limited of Hunan Province);DMIL type inverted microscope (center number
027, Leica);3111 types CO2Incubator (center number 147, Thermo);MR-96A microplate reader (center number 007, Shenzhen
Mai Rui);Aseptic syringe filter (millipore).
2. test method
2.1 cells are cultivated and inoculation
Eca-109, MGC80-3, MHCC97-H, LoVo tumor cell line is at 37 DEG C, 5%CO2Saturated humidity environment under,
DMEM in high glucose complete medium containing 10% hyclone is cultivated, passes on.Disappear with 0.25% tryptic digestive juice during inoculation
Change, add cell culture medium and blow and beat uniformly cell suspension, cell is made 5 × 104The cell suspension of/ml, every hole 100 μ l inoculates
In 96 orifice plates.
2.2 test sample preparation and CAs
Weigh a certain amount of test sample, be configured to the maximum concentration working solution of 5000 μ g/ml with DMEM complete medium, use
The aseptic syringe filter filtration sterilization of 0.22 μm, is configured to the working solution of 1500,500,150,50,15 μ g/ml the most successively.
DDP DMEM complete medium is configured to the working solution that concentration is 50,15,5,1.5,0.75,0.15 μ g/ml.Treat cell attachment
After use the working solution containing variable concentrations test sample and DDP instead, each concentration sets 3 multiple holes.After dosing after 48 hours, often
Hole adds MTT solution (culture medium is made into 5mg/ml, keeps in Dark Place after filtration) 20 μ l, and by supernatant sucking-off after 4 hours, every hole adds
MTT lysate DMSO 150 μ l, shakes and treats that Formazan is completely dissolved, and microplate reader 492nm measures OD value.
3. result of the test
Seeing as in table 2 below, each sample all has good inhibiting effect to 4 kinds of tumor cells, with HB-4 best results.
The different HTJ culture In Vitro Anti gastroenteric tumor cytoactive the selection result of table 2
From Table 2, it can be seen that Anticancer Activity in vitro is by weak being by force: bioconversion mycelium > Herba Scutellariae Barbatae medical material > hedgehog hydnum
Mycelium ≈ Hericium erinaceus (Bull. Ex Fr.) Pers. filament+Herba Scutellariae Barbatae, and bioconversion mycelial IC50 value is more than independent Herba Scutellariae Barbatae medical material with independent
The IC50 value sum of Hericium erinaceus (Bull. Ex Fr.) Pers. filament, Emax is more than independent Herba Scutellariae Barbatae medical material and the Emax sum of independent Hericium erinaceus (Bull. Ex Fr.) Pers. filament, in conjunction with
In the result explanation Hericium erinaceus (Bull. Ex Fr.) Pers. of embodiment 4, abundant enzyme system may pass through structural modification or Degradation, the work in Herba Scutellariae Barbatae
The property Viability higher composition of conversion, or enhance the yield of active component (such as total flavones) in Herba Scutellariae Barbatae, or non-
Active component changes into active component, and active component is mutually promoted and reached to strengthen the purpose of anticancer function.
People's gastric cancer (MGC80-3) is transplanted inhibition test in nude mice by embodiment 6HB-4 sample extraction thing
1. experiment material
1.1 medicine
1.1.1 test sample
HB-4 sample extraction thing, specification: 126.1g/ bag;Lot number: 20150723, limited by Hunan new banknotes-system medicine share
Company provides.
1.1.2 positive reference substance
Fluorouracil Injection (lot number: FA150603, Shanghai Xudong Hipu Medicine Co., Ltd's product), faces the used time and uses
0.9% sodium chloride preparation.
1.2 laboratory animal
Male BALB/c-nu nude mouse, 90, SPF level, body weight 12~15g, Hunan Si Laike scape reaching laboratory animal has
Limit company provides, laboratory animal production licence number: SCXK (Hunan) 2014-0003;In Hunan Province's drug safety evaluation is studied
Heart barrier environment B district laboratory rearing, the Quality of Experimental Animals quality certification number: NO43004700004931, laboratory animal uses to be permitted
Can the number of card: SYXK (Hunan) 2014-0008.Ambient temperature 20 DEG C~26 DEG C during experiment, humidity 40%~70%.
1.3 cell strain
Gastric carcinoma cells (MGC80-3) is purchased from Chinese Academy of Sciences's cell bank, at Drug Safety Evaluation Center of Hunan Province's cell
Indoor Secondary Culture;DMEM high glucose medium (lot number: 041012-UY, Corning Products);Hyclone (lot number:
NYL1009, HyClone Products).
1.4 key instrument
AUY-220 analytical balance (Japan's Shimadzu);Electronic digital indicator (the positive Feng Wujin in Yongkang).
2. research contents
2.1 pre-stage test results
HB-4 extract single oral gavage gives ICR mice Cmax 0.15g/ml, maximum volume 40ml/kg (quite
In 6g/kg), the same day 1 time, have no obvious acute toxic reaction.
2.2.HTJ-4 the impact on people's transplanted gastric cancer model of nude mice bearing tumor
2.2.1 the cultivation of gastric carcinoma cells (MGC80-3)
Add 10% new-born calf serum by DMEM culture medium, and add each 50U/ml of mycillin, at 5%CO2Cell training
Support in case and cultivate.Passing in the ratio of 1:4, when passing on, cell enters exponential phase, at the bottom of covering with bottle 70~80%.
2.2.2 modeling packet
Take the logarithm people's gastric cancer (MGC80-3) cell of trophophase, wash 2 times with PBS, then with serum-free DMEM resuspended after count
Number, adjusting cell concentration is 1 × 107The Cell sap of individual/ml is inoculated on the right side of 90 nude mouses under axillary fossa, every nude inoculation
0.2ml (i.e. 2 × 106Individual cell/only), prepare lotus tumor nude mouse.Observe tumor bulk-growth situation, with vernier caliper measurement tumor body
Long (a) and wide (b), by tumor cubature formula V=1/2 × a × b2Calculate tumor volume, select tumor tumor volume to reach 200mm3
Above and without the lotus tumor nude mouse of diabrosis, taking into account body weight by tumor volume and carry out random packet, be divided into 7 groups, packet administration is shown in Table
3.Model control group gavage gives distilled water, and giving volume is 0.2ml/10g, fluorouracil treated animal intraperitoneal injection, note
Beam amasss 0.1ml/10g, and remaining is respectively organized gavage and gives respective volume medicinal liquid.
The packet of table 3 animal and dose design
3. evaluation of result
3.1 animal survival quality and time-to-live are evaluated
During test, every day observes the diet of animal, drinking-water, behavioral activity and the situation of tumor body, each treated animal of itemized record
Body weight change and death condition.
3.2 tumor volumes, the mensuration of body weight
In process of the test, from the beginning of packet, being administered, weekly with vernier caliper measurement 2 times and record tumor length (a), wide
(b), body weight;Calculate the difference of tumor growth curve between gross tumor volume more each group, analysis body weight change trend.
Gross tumor volume: V=1/2 × length (a) × wide (b) is calculated according to below equation2
3.3 carry out therapeutic evaluation according to gross tumor volume
According to below equation calculating relative tumour volume (RTV) and Relative tumor rate of increase T/C%:
RTV=Vt/V0
Vt: every day measures gross tumor volume, V0: initial tumor volume (before administration)
RTV meansigma methods × 100% of the RTV meansigma methods/matched group of T/C%=administration group
Relative tumor suppression ratio=100%-Relative tumor the rate of increase (T/C%)
If Relative tumor suppression ratio < 60%, for invalid;If Relative tumor suppression ratio >=60%, through statistical procedures
P < 0.05 is effective.
3.4 carry out therapeutic evaluation according to tumor weight
After off-test, put to death animal at the end of experiment, peel off tumor body, weigh and take pictures.According to tumor re-computation suppression ratio.
Inhibition rate of tumor growth=(model control group average tumor weight-administration group average tumor weight)/model control group average tumor weight ×
100%, < 40% is invalid to inhibition rate of tumor growth;Inhibition rate of tumor growth>=40%, and<0.05 for having through statistical procedures P
Effect.
3.5 statistical method
SPSS16.0 software is used to carry out statistical analysis.Measurement data all uses mean ± standard deviationRepresent, system
Meter data uses SPSS17.0 software to carry out statistical disposition.Compare between many groups variance neat time one factor analysis of variance, two-by-two
Relatively, t inspection is used.P 0.05 represents statistically significant, and P 0.01 represents that the difference checked is very significant.
4. result
The activity of 4.1HB-4 extract anti-lotus MGC80-3 Transplanted Gastric Carcinoma Mus
The continuous gavage of nude mice 19 days, fluorouracil associating HB-4 group has 1 animal death, the outward appearance body of remaining nude mice occur
Levy, the general status of behavioral activity and animal shows no obvious abnormalities, and has no the most abnormal internal organs through gross anatomy perusal,
As shown in Figure 1.
The impact on people's human gastric cancer in nude mice tumor volume of the 4.2HB-4 extract
As shown in table 4 and Fig. 1, HB-4 extract low dosage, fluorouracil can substantially reduce D after administration4、D10~D19Lotus tumor
Nude mice tumor volume, compares with model control group and has significant difference (P < 0.05 or P < 0.01);HB-4 extract high dose energy
Substantially reduce D after being administered4、D13~D19Tumor bearing nude mice tumor volume (P < 0.05);Fluorouracil associating HB-4 can substantially reduce to
D after medicine10~D16Tumor bearing nude mice tumor volume (P < 0.05);HB-4 extract (2000mg/kg) can substantially reduce D after administration4、
D10、D16Tumor bearing nude mice tumor volume (P < 0.05).
As shown in table 5 and Fig. 2, Fig. 2 for be administered after D10, HB-4 on people's gastric cancer transplant tumor bearing nude mice tumor body affect figure.
The basic, normal, high dosage of HB-4 extract, fluorouracil and fluorouracil associating HB-4 extract, HB-4 extract (2000mg/kg)
During administration, Relative tumor suppression ratio is respectively as follows: D4Relative tumor suppression ratio 45.8%, 27.2%, 62.3%, 62.7%,
43.4%, 58.9%;D7Relative tumor suppression ratio 31.6%, 16.1%, 53.0%, 38.3%, 38.4%, 36.0%;D10Phase
To tumor control rate 46.1%, 31.9%, 50.0%, 58.1%, 44.7%, 50.4%;D13Relative tumor suppression ratio 36.9%,
16.2%, 39.8%, 49.9%, 37.2%, 31.4%;D16Relative tumor suppression ratio 33.8%, 16.0%, 39.6%,
61.2%, 47.4%, 45.1%;D19Relative tumor suppression ratio 40.9%, 36.1%, 41.2%, 44.3%, 45.7%,
36.2%.
The table 5HTJ-4 impact on human gastric cancer in nude mice Relative tumor suppression ratio
The impact on people's human gastric cancer in nude mice tumor weight of the 4.3HB-4 extract
As shown in table 6 and Fig. 3, the basic, normal, high dosage of HB-4 extract, fluorouracil group, fluorouracil associating HB-4 extracts
Thing group, HB-4 extract (2000mg/kg) inhibition rate of tumor growth be respectively 20.6%, 9.7%, 6.4%, 27.0%,
20.7%, 1.3%.
As shown in table 7 and Fig. 4, the basic, normal, high dosage of HB-4 extract, fluorouracil group, fluorouracil associating HB-4 extracts
The maximum tumour transplatation rate of thing group, HB-4 extract (2000mg/kg) is 46.1%, 36.1%, 62.3%, 32.7%, 47.4%,
58.9%.Model control group, the basic, normal, high dosage of HB-4 extract, fluorouracil group, fluorouracil associating HB-4 extract group,
HB-4 extract (2000mg/kg) weight limit rate of descent is respectively 3.4%, 3.4%, 5.0%, 3.8%, 8.9%, 5.8%,
3.7%.
The table 6HTJ-4 impact on human gastric cancer in nude mice tumor weight
Note: compare with model control group,▲P < 0.05,▲▲P<0.01。
Anti-gastric cancer MGC80-3 transplanted tumor curative effect and animal toxicity in table 7HTJ-4 body
Result above shows, in HB-4 i.e. culture medium, Herba Scutellariae Barbatae mixed ratio is that the bioconversion mycelium of 40% is to people's stomach
Cancer (MGC80-3) tumor bearing nude mice has obvious tumor-inhibiting action.HB-4 extract uses the simple technique that processes to prepare, its
Antitumous effect just reaches unforeseeable technique effect, is better than all of through the antitumous effect of " mycelium " of bioconversion
The Chinese crude drug of natural botanical source, if further isolated and purified active constituent-enriched, its antitumous effect will more preferably can be used for
Prepare anti-gastrointestinal tract cancer drug.
Those of ordinary skill in the field are it is understood that the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples;Under the thinking of the present invention, above example
Or can also be combined between the technical characteristic in different embodiments, and there is the different aspect of the present invention as above
Other change of many, in order to simple and clear they do not provide in details.Therefore, all within the spirit and principles in the present invention,
Any omission of being made, amendment, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. the culture medium of a Hericium erinaceus (Bull. Ex Fr.) Pers., it is characterised in that include basal medium and Herba Scutellariae Barbatae, in the culture medium of every 100 parts
Including basal medium 70~50 parts, Herba Scutellariae Barbatae 30~50 parts;Described basal medium includes each component of following weight portion:
Testa Tritici 490~350 parts, Semen Maydis powder 140~100 parts, bran coat 63~45 parts, white sugar 7~5 parts, water 400~600 parts.
The culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers. the most according to claim 1, it is characterised in that described basal medium includes following weight
Each component of part:
420 parts of Testa Tritici, Semen Maydis powder 120 parts, bran coat 54 parts, white sugar 6 parts, 500 parts of water.
The culture medium of Hericium erinaceus (Bull. Ex Fr.) Pers. the most according to claim 1, it is characterised in that the culture medium of every 100 parts includes basis
Culture medium 60 parts, Herba Scutellariae Barbatae 40 parts;Described basal medium includes each component of following percetage by weight: Testa Tritici 70%, Semen Maydis
Powder 20%, bran coat 9%, white sugar 1%.
4. a bioconversion mycelium, it is characterised in that by hedgehog hydnum mushroom inoculation to such as any one of claim 1-3 institute
In the culture medium stated, at 20~27 DEG C, cultivate 30~40 days, take out mycelium, dry, obtain bioconversion mycelium.
Bioconversion mycelium the most according to claim 4, it is characterised in that described hedgehog hydnum mushroom bacterial strain is hedgehog hydnum Cordycepps
Fungus Hericium erinaceus (Bull. Ex Fr.) Pers. Hericium erinaceum (Bull.ex Fr.) Pers.
Bioconversion mycelium the most according to claim 5, it is characterised in that described hedgehog hydnum Cordycepps fungus Hericium erinaceus (Bull. Ex Fr.) Pers.
The mycelium of Hericium erinaceum (Bull.ex Fr.) Pers. is grown nonparasitically upon another plant with it solid culture containing Herba Scutellariae Barbatae composition
The drying composite of base.
7. the mycelial extract of bioconversion, it is characterised in that described extract is prepared by following steps:
(1) the bioconversion mycelium as described in any one of claim 4-6 being carried out water to carry, be soaked in water at 50-70 DEG C 2-3
Secondary, to soak 2-5 hour every time, the weight of water is 6~10 times of biological transformed bacteria filament weight, obtains extracting solution;
(2) by the extracting solution concentrating under reduced pressure in step (1), the concentrated solution of concentrating under reduced pressure gained is taken out very under the conditions of 50-70 DEG C
Empty dry, obtain the mycelial extract of bioconversion.
The mycelial extract of bioconversion the most according to claim 7, it is characterised in that described in the temperature that is soaked in water
It it is 60 DEG C;The concentrated solution of concentrating under reduced pressure gained evacuation under the conditions of 60 DEG C is dried.
9. the purposes in preparing anti-gastrointestinal tract cancer drug of the mycelial extract of bioconversion described in claim 7 or 8.
Purposes the most according to claim 9, it is characterised in that described gastrointestinal cancer be the esophageal carcinoma, gastric cancer, colon cancer or
Hepatocarcinoma.
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