CN106420844B - One kind having the active fresh Cordyceps extract of melanoma - Google Patents
One kind having the active fresh Cordyceps extract of melanoma Download PDFInfo
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- CN106420844B CN106420844B CN201610985426.6A CN201610985426A CN106420844B CN 106420844 B CN106420844 B CN 106420844B CN 201610985426 A CN201610985426 A CN 201610985426A CN 106420844 B CN106420844 B CN 106420844B
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Classifications
-
- 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
- A61K2236/30—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, decoction
Abstract
The invention discloses a kind of applications with active fresh Cordyceps extract of melanoma and preparation method thereof and in antitumor field.Fresh Cordyceps extract is prepared by the following method: after fresh cordyceps sinensis is shredded plus water homogenate is centrifuged cordyceps sinensis fresh after homogenate progress low-temperature ultrasonic assisted extraction, takes supernatant, freeze-drying obtains fresh Cordyceps extract.The fresh Cordyceps extract that the present invention obtains has good inhibitory effect to melanoma cells, while highly-safe, has good prospect in medicine.Extract preparation process of the present invention is simple, is easy to be mass produced, use is safe.
Description
Technical field
The present invention relates to Chinese medicines and natural medicine field, and in particular to a kind of preparation method of fresh Cordyceps extract and
Its application in anti-tumor aspect.
Background technique
Cordyceps sinensis is that section ergot fungus cordyceps sinensis bacterium Cordycepssinensis (Berk.) Sacc. colonizes in bat
The complex of stroma and larva corpse on hepialidae insect larvae.Contain a variety of active ingredients in cordyceps sinensis, including nucleosides,
Polysaccharide, sterol, fatty acid, protein, polypeptide and volatile component etc. have antitumor, adjusting immune system, promotion body thin
Cellular damage reparation, free radical resisting, anti-oxidant, anti-aging, protection cardiac muscle and vascular cell improve cardiovascular function, protection liver
Dirty, protection kidney adjusts respiratory system, adjusts hematological system and adjusts the functions such as blood lipid.The high temperature that many consumers use
The edible way for stewing dry cordyceps sinensis, will cause the destruction of heat-sensitive materials, and fresh cordyceps sinensis can retain to greatest extent wherein
Active constituent.
Melanoma is malignant tumour most aggressive in cutaneum carcinoma, and nearly 20 years, worldwide frequency steeply rose,
Increased every year with 3.1% speed.Melanoma is insensitive to chemicotherapy, poor prognosis, is one of the tumour for being most difficult to effectively treat.
Existing research shows that cordyceps sinensis has significant inhibiting effect to kinds of tumor cells, wherein also including melanoma tumors.So
And existing literature report is all to dry in the shade, dry or dry cordyceps sinensis or with cordyceps sinensis artificial fermentation's mycelium for research
Material, and all extracted using higher temperature water or extracted using organic solvent.Such as document " Inhibitory effects of
ethylacetate extract of Cordyceps sinensis mycelium on various cancer cells
In culture and B16melanoma in C57BL-6mice " report the mycelial acetic acid of cordyceps sinensis artificial fermentation
Ethyl ester extract has a good inhibiting effect to MCF-7 breast cancer cell and B16 melanoma cells, and its hot water extract
Then unrestraint acts on.Document " Mycelium cultivation, chemical composition and antitumour
activity of a Tolypocladium sp.fungus isolated from wild Cordyceps sinensis》
Also it is poor to B16 melanoma cell growth inhibiting effect in vitro to report Base in Mycelia of Cordyceps extract.
Fresh cordyceps sinensis can be good at keeping its various active constituent, especially volatilize without the process of drying process
The Large molecule active substance of property ingredient and heat sensitivity.The present invention selects fresh cordyceps sinensis as raw material, controls temperature 15
In~25 DEG C of water carry out ultrasound, freeze thawing, again ultrasound extracting method, can be good at retaining active constituent therein.To mention
The research of active constituent in cordyceps sinensis is taken to provide a kind of new method and thinking.
Summary of the invention
The present invention provides a kind of fresh cordyceps sinensis extraction with anti-tumor activity by furtheing investigate to fresh cordyceps sinensis
Object and its application.
The purpose of the invention is achieved by the following technical solution:
On the one hand, the present invention provides a kind of with the active fresh Cordyceps extract of melanoma, is with fresh winter worm
Summer grass is raw material, and gained is extracted with water under the conditions of temperature is 15~25 DEG C.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
It is using fresh cordyceps sinensis as raw material, water is then centrifuged for, collects through homogenate, ultrasonic extraction under the conditions of temperature is 15~25 DEG C
Supernatant is simultaneously freeze-dried gained.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
Preparation method includes the following steps:
A, fresh cordyceps sinensis water is homogenized, crushed, controlled at 15~25 DEG C；
B, the fresh cordyceps sinensis after homogenate is subjected to ultrasonic extraction, controlled at 15~25 DEG C, then by the fresh winter worm summer
Careless extracting solution is stood in -20 DEG C of freezings；
C, the fresh cordyceps extracting liquid of freezing is taken out, is homogenized and is uniformly dispersed, again ultrasonic extraction, control temperature control
15~25 DEG C are made as, is then centrifuged for, collects supernatant；
D, supernatant is freeze-dried to obtain fresh Cordyceps extract.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
In the step a of preparation method, water consumption be 8-12 times of volume, Homogenization time be 6~8min, homogenate revolving speed be 14000~
16000rpm。
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
In the step b of preparation method, the ultrasonic extraction time is 20~40min, and cooling time is 40~60min.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
In the step c of preparation method, the ultrasonic extraction time is 20~40min again, and centrifugation time is 10~15min, and centrifugation turns
Speed is 8000~10000rpm.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
Preparation method includes the following steps:
A, fresh cordyceps sinensis is crushed, add 8~12 times amount water, be homogenized 6~8min, homogenate revolving speed be 14000~
Fresh cordyceps sinensis is ground into the suspension of fine particle controlled at 15~25 DEG C by 16000rpm；
B, the fresh cordyceps sinensis after homogenate is subjected to 20~40min of ultrasonic extraction, controlled at 15~25 DEG C, then will
Fresh cordyceps extracting liquid is placed in -20 DEG C of refrigerator freezings and stands 40~60min；
C, the fresh cordyceps extracting liquid of freezing is taken out, is homogenized and is uniformly dispersed, again 20~40min of ultrasonic extraction,
Controlling temperature control is 15~25 DEG C, is centrifuged 10~15min, and revolving speed is 8000~10000rpm, collects supernatant；
D, supernatant is freeze-dried to obtain fresh Cordyceps extract.
In some embodiments, one kind of the present invention has the active fresh Cordyceps extract of melanoma,
Preparation method includes the following steps:
A, fresh cordyceps sinensis is crushed, adds the water of 10 times of amounts, be homogenized 7min, homogenate revolving speed is 15000rpm, controls temperature
It is 20 ± 2 DEG C, fresh cordyceps sinensis is ground into the suspension of fine particle；
B, the fresh cordyceps sinensis after homogenate is subjected to ultrasonic extraction 30min, controlled at 20 ± 2 DEG C, then by the fresh winter
Worm summer grass extracting solution is placed in -20 DEG C of refrigerator freezings and stands 50min；
C, the fresh cordyceps extracting liquid of freezing is taken out, is homogenized and is uniformly dispersed, again ultrasonic extraction 30min, controlled
Temperature is centrifuged 12min, revolving speed 9000rpm at 20 ± 2 DEG C, collects supernatant；
D, supernatant is freeze-dried to obtain fresh Cordyceps extract.
On the other hand, present invention offer is of the present invention, and there is the active fresh Cordyceps extract of melanoma to exist
It is applied in preparation anti-melanin tumor medicine.
In some embodiments, of the present invention that there is the active fresh Cordyceps extract of melanoma to prepare
It is applied in anti-melanin tumor medicine, wherein the anti-melanin tumor medicine is the drug for inhibiting Humanmachine tumour B16 cell.
The positive effect of the present invention:
1. the present invention selects fresh cordyceps sinensis as raw material, temperature is controlled at 15~25 DEG C, is surpassed again using ultrasound, freeze thawing
The extracting method of sound can be good at retaining active constituent therein.
2. extract of the present invention has very strong inhibiting effect, IC to B16 melanoma cells50Only 0.02mg/mL, tool
There is boundless application prospect.
3. extract of the present invention is active skull cap components, securely and reliably, it is that development efficacy is good and that toxic side effect is small is novel
Anti-tumor drug provides excellent basis.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention 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 source and parameter: KQ-500DB numerical control supersonic cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd.), function
Rate: 500w；Supersonic frequency: 40khz.
Centrifugation apparatus source: 272R-3 vast capacity refrigerated centrifuge (Hunan Hunan is from instrument plant)
Embodiment 1
Fresh cordyceps sinensis 10g is taken, is shredded, the water of 10 times of amounts, high-speed homogenization 7min are added, homogenate revolving speed is 15000rpm, control
20 ± 2 DEG C of temperature processed, fresh cordyceps sinensis is ground into fine particle suspension, ultrasound assisted extraction 30min, controlled at 20
± 2 DEG C, fresh cordyceps extracting liquid is then placed in -20 DEG C of refrigerator freezings and stands 50min taking-up, is homogenized and is uniformly dispersed, then
Secondary ultrasonic extraction 30min, control temperature control are centrifuged 12min, revolving speed 9000rpm at 20 ± 2 DEG C, collect supernatant, freezing
Dry fresh Cordyceps extract.
Embodiment 2
Fresh cordyceps sinensis 10g is taken, is shredded, the water of 8 times of amounts, high-speed homogenization 6min are added, homogenate revolving speed is 14000rpm, control
15 ± 2 DEG C of temperature, fresh cordyceps sinensis is ground into fine particle suspension, ultrasound assisted extraction 20min, controlled at 15 ±
2 DEG C, fresh cordyceps extracting liquid is then placed in -20 DEG C of refrigerator freezings and stands 40min taking-up, is homogenized and is uniformly dispersed, again
Ultrasonic extraction 20min, control temperature control are centrifuged 10min, revolving speed 8000rpm at 15 ± 2 DEG C, collect supernatant, and freezing is dry
It is dry to obtain fresh Cordyceps extract.
Embodiment 3
Fresh cordyceps sinensis 10g is taken, is shredded, the water of 12 times of amounts, high-speed homogenization 8min are added, homogenate revolving speed is 16000rpm, control
25 ± 2 DEG C of temperature processed, fresh cordyceps sinensis is ground into fine particle suspension, ultrasound assisted extraction 40min, controlled at 25
± 2 DEG C, fresh cordyceps extracting liquid is then placed in -20 DEG C of refrigerator freezings and stands 60min taking-up, is homogenized and is uniformly dispersed, then
Secondary ultrasonic extraction 40min, control temperature control is centrifuged 15min, revolving speed 10000rpm at 25 ± 2 DEG C, collects supernatant, cold
Dry fresh Cordyceps extract is lyophilized.
Embodiment 4
Dry cordyceps sinensis 10g is taken, is shredded, the water of 10 times of amounts, high-speed homogenization 7min are added, homogenate revolving speed is 15000rpm,
Dry cordyceps sinensis is ground into fine particle suspension by 20 ± 2 DEG C of temperature of control, and ultrasound assisted extraction 30min controls temperature
It is 20 ± 2 DEG C, dry cordyceps extracting liquid is then placed in -20 DEG C of refrigerator freezings and stands 40min taking-up, is homogenized and disperses
Even, ultrasonic extraction 30min, controls temperature at 20 ± 2 DEG C, is centrifuged 12min, revolving speed 9000rpm again, collects supernatant, cold
Being lyophilized dry must dry Cordyceps extract.
Embodiment 5
Dry cordyceps sinensis 10g is taken, is shredded, the water of 10 times of amounts, high-speed homogenization 7min are added, homogenate revolving speed is 15000rpm,
Dry cordyceps sinensis is ground into fine particle suspension, ultrasound assisted extraction 30min, water-bath control by 60 ± 2 DEG C of temperature of control
Temperature is 60 ± 2 DEG C, and dry cordyceps extracting liquid is then placed in -20 DEG C of refrigerator freezings and stands 40min taking-up, is homogenized and simultaneously divides
It dissipates uniformly, ultrasonic extraction 30min again, temperature controlled water baths are centrifuged 12min at 60 ± 2 DEG C, revolving speed 9000rpm, in collection
Clear liquid is freeze-dried to obtain dry Cordyceps extract.
Embodiment 6
Fresh Cordyceps extract anti tumor activity in vitro research
(1) cell culture
Several tumour cells frozen and the human normal fibroblast (HFL-1 cell) as control are added after recovering
Corresponding culture medium is placed in 37 DEG C, cultivates in 5% carbon dioxide incubator.When cell in culture bottle grows into 85%~95%
Carry out cell passage.Old culture solution is discarded, PBS repeated flushing 2 times, 0.25% pancreatin digestive juice 2mL is added, cell Bian Yuan ﹑ is thin
After intercellular spaces become larger, 4mL culture solution is added and terminates digestion, 1000r/min is centrifuged 4min, supernatant is abandoned, with the transfer of 1:4 ratio
It is cultivated into new sterile culture flask.
(2) medicine ordinance
It accurately weighs sample, culture medium configuration extraction object solution is added, be diluted to the initial concentration of 12mg/mL, 0.22 μm
Sterile filters filtration sterilization, then a series of gradient concentrations (totally 10 concentration) i.e. 6.00mg/mL, 3.00mg/ is diluted to by 2 times
mL、1.50mg/mL、0.75mg/mL、0.33mg/mL、0.16mg/mL、0.08mg/mL、0.04mg/mL、0.02mg/mL、
0.01mg/mL。
(3) Activity determination
Each subject cell of logarithmic growth phase, with 3~4 × 104The density of a/mL is inoculated in 96 orifice plates, every hole 100
μL.At 37 DEG C, 5%CO2Insulating box in be incubated for for 24 hours after, abandon supernatant, each embodiment gained cordyceps sinensis be added by 100 holes μ L/
Extract is incubated for 72h, and every hole is added 10 μ L of CCK-8 reagent, is placed in 37 DEG C of incubators and is incubated for 1-2h, examines at microplate reader 450nm
Absorbance (A) value for surveying each hole, calculates fresh cordyceps sinensis to the IC of each tumour cell50Value.
(4) testing result
Influence of the Cordyceps extract to each tumor cell proliferation is as shown in table 1:
Influence of 1 Cordyceps extract of table to each tumor cell proliferation
From the data in table 1, it can be seen that the fresh Cordyceps extract of embodiment 1 is to melanoma, lymph cancer, lung cancer, liver cancer and cream
Adenocarcinoma cell plays the role of its proliferation of certain inhibition, and inhibitory effect is better than dry Cordyceps extract；Fresh cordyceps sinensis
Extract is most strong to the inhibiting effect of Melanoma B16 cell, IC50Only 0.020mg/mL.
The influence that different temperatures Cordyceps extract is proliferated Melanoma B16 cell is as shown in table 2:
The influence that 2 different temperatures extract of table is proliferated Melanoma B16 cell
| Different temperatures Cordyceps extract | IC50(mg/mL) |
| 1 extract of embodiment | 0.020 |
| 4 extract of embodiment | 0.036 |
| 5 extract of embodiment | 0.333 |
From the data in table 2, it can be seen that 1,4,5 Cordyceps extract of embodiment has centainly Melanoma B16 cell proliferation
Inhibiting effect, wherein embodiment 1,4 extract inhibitory effects are obviously good compared with 5 extract of embodiment.Show that different temperatures is extracted
The influence that object is proliferated Melanoma B16 cell is different, and extract anti-melanin tumor activity of the temperature control at 20 ± 2 DEG C is bright
The aobvious extract controlled higher than temperature at 60 ± 2 DEG C, it is seen that it is intracorporal anti-that cordyceps sinensis can be more effectively retained under cryogenic conditions
Tumor promotion ingredient.
Claims (4)
1. one kind has the active fresh Cordyceps extract of melanoma, which is characterized in that preparation method includes following
Step:
A, fresh cordyceps sinensis being crushed, adds the water of 8~12 times of amounts, be homogenized 6~8min, homogenate revolving speed is 14000~16000rpm,
Controlled at 15~25 DEG C, fresh cordyceps sinensis is ground into the suspension of fine particle；
B, the fresh cordyceps sinensis after homogenate is subjected to 20~40min of ultrasonic extraction, controlled at 15~25 DEG C, then by the fresh winter
Worm summer grass extracting solution is placed in -20 DEG C of refrigerator freezings and stands 40~60min；
C, the fresh cordyceps extracting liquid of freezing is taken out, is homogenized and is uniformly dispersed, again 20~40min of ultrasonic extraction, controlled
Temperature control is 15~25 DEG C, is centrifuged 10~15min, and revolving speed is 8000~10000rpm, collects supernatant；
D, supernatant is freeze-dried to obtain fresh Cordyceps extract.
2. having the active fresh Cordyceps extract of melanoma as described in claim 1, which is characterized in that it is prepared
Method includes the following steps:
A, fresh cordyceps sinensis is crushed, adds the water of 10 times of amounts, be homogenized 7min, homogenate revolving speed is 15000rpm, controlled at 20
± 2 DEG C, fresh cordyceps sinensis is ground into the suspension of fine particle；
B, the fresh cordyceps sinensis after homogenate is subjected to ultrasonic extraction 30min, controlled at 20 ± 2 DEG C, then by the fresh winter worm summer
Careless extracting solution is placed in -20 DEG C of refrigerator freezings and stands 50min；
C, the fresh cordyceps extracting liquid of freezing is taken out, is homogenized and is uniformly dispersed, again ultrasonic extraction 30min, control temperature
At 20 ± 2 DEG C, it is centrifuged 12min, revolving speed 9000rpm, collects supernatant；
D, supernatant is freeze-dried to obtain fresh Cordyceps extract.
3. having the active fresh Cordyceps extract of melanoma anti-in preparation as described in claim 1-2 any one
It is applied in melanoma drug.
4. as claimed in claim 3 there is the active fresh Cordyceps extract of melanoma to prepare melanoma medicine
Application in object, which is characterized in that the anti-melanin tumor medicine is the drug for inhibiting Humanmachine tumour B16 cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN1822847A (en) * | 2003-07-14 | 2006-08-23 | 小林制药株式会社 | Plant worms mycelium extracat fraction and composition for oral intake |
| CN105380963A (en) * | 2015-12-09 | 2016-03-09 | 郑州莉迪亚医药科技有限公司 | Cordyceps sinensis active soft capsule and production technology thereof |
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| CN1822847A (en) * | 2003-07-14 | 2006-08-23 | 小林制药株式会社 | Plant worms mycelium extracat fraction and composition for oral intake |
| CN105380963A (en) * | 2015-12-09 | 2016-03-09 | 郑州莉迪亚医药科技有限公司 | Cordyceps sinensis active soft capsule and production technology thereof |
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| Title |
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| Myceliumcultivation,chemicalcompositionandantitumouractivityofaTolypocladiumsp.fungusisolatedfromwildCordycepssinensis;P.H.Leung等;《JournalofAppliedMicrobiology》;20060831;第101卷(第2期);第275页摘要，第277页左栏第1-2段 * |
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Effective date of registration: 20200526 Address after: Room 302, building 32, No. 368, Zhenan Middle Road, Chang'an Town, Dongguan City, Guangdong Province Co-patentee after: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Patentee after: Dongguan dongyangguang Cordyceps research and Development Co.,Ltd. Address before: 523808 Guangdong city of Dongguan province Hubei Songshan Industrial Park Industrial Road No. 1 Co-patentee before: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Patentee before: SUNSHINE LAKE PHARMA Co.,Ltd. |
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Denomination of invention: Fresh Cordyceps sinensis extract with anti melanoma activity Effective date of registration: 20210923 Granted publication date: 20191112 Pledgee: Shenzhen branch of China Import and Export Bank Pledgor: Dongguan dongyangguang Cordyceps research and Development Co.,Ltd.|YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: Y2021980009771 |
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Date of cancellation: 20220926 Granted publication date: 20191112 Pledgee: Shenzhen branch of China Import and Export Bank Pledgor: Dongguan dongyangguang Cordyceps research and Development Co.,Ltd.|YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: Y2021980009771 |
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Denomination of invention: A fresh Cordyceps sinensis extract with anti melanoma activity Effective date of registration: 20220929 Granted publication date: 20191112 Pledgee: Shenzhen branch of China Import and Export Bank Pledgor: Dongguan dongyangguang Cordyceps research and Development Co.,Ltd.|YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: Y2022980017022 |
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