CN115501286A - Camellia seed shell extract and preparation method and application thereof - Google Patents
Camellia seed shell extract and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000000605 extraction Methods 0.000 title abstract description 6
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- 240000001548 Camellia japonica Species 0.000 claims abstract description 55
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000002386 leaching Methods 0.000 claims abstract description 39
- 239000010903 husk Substances 0.000 claims abstract description 34
- 238000001035 drying Methods 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 21
- 238000000502 dialysis Methods 0.000 claims abstract description 15
- 241000252230 Ctenopharyngodon idella Species 0.000 claims abstract description 12
- 241001122767 Theaceae Species 0.000 claims abstract description 8
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- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 7
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- 238000000034 method Methods 0.000 claims description 6
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
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- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/82—Theaceae (Tea family), e.g. camellia
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention provides an oil tea seed husk extract and a preparation method and application thereof, belonging to the technical field of oil tea seed husk extraction. The preparation method of the camellia seed husk extract provided by the invention comprises the following steps: (1) Crushing and drying the camellia seed hulls to obtain camellia seed hull powder; (2) Leaching the camellia seed shell powder by using an ethanol solution to obtain a leaching solution; (3) Removing ethanol from the leaching solution through reduced pressure concentration, and sequentially performing ultrasonic treatment and dialysis treatment to obtain dialysate; (4) And concentrating and drying the dialysate to obtain the camellia seed husk extract. The camellia seed hull extract provided by the invention is safe to CIK cells, can obviously inhibit GCRVJX01 virus, and can be used for treating grass carp hemorrhagic disease.
Description
Technical Field
The invention relates to the technical field of camellia seed husk extraction, in particular to a camellia seed husk extract and a preparation method and application thereof.
Background
Grass carp (Ctenophaggodon idella) is a fish of the genus Ctenopharyngodon of the family Cyprinaceae. The grass carp is fresh and tender in meat quality, is excellent cultured fish, is one of the four traditional freshwater cultured fishes in China, and has higher economic value. However, during the cultivation process, some diseases often occur in the grass carps. The risk of hemorrhagic disease is regarded as serious. The hemorrhagic disease is a viral disease caused by reovirus, generally prevails in 4-10 months per year, and the main harm object is adult fish of 1-2 years old.
The Camellia seed husk extract is obtained by extracting Camellia seed husk. Research shows that when the feed is added with the sasanquasaponin to feed the penaeus vannamei, the activity of serum lysozyme is improved by 52.11 percent, the activity of liver and pancreas superoxide dismutase is improved by 105.52 percent, and the survival time of the penaeus vannamei infected with white spot syndrome virus can be prolonged. The feed is added with sasanquasaponin to feed tilapia, and the lysozyme activity in serum is improved by 4.88 percent compared with that of flavomycin. The activity of the serum lysozyme is an identification reflecting the nonspecific immune function of the fish, and the result shows that the camellia seed husk extract can influence the nonspecific immune function of the fish body, so that the disease resistance is enhanced. However, few studies on the inhibition of aquatic animal viruses and the prevention and treatment of viral diseases by using the camellia oleifera seed husk extract are available at present.
Disclosure of Invention
The invention aims to provide an oil-tea camellia seed shell extract, a preparation method and application thereof, which improve the functional activity of the oil-tea camellia seed extract and prevent and treat grass carp hemorrhagic disease by using the extract.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of an oil tea seed shell extract, which comprises the following steps:
(1) Crushing and drying the camellia seed hulls to obtain camellia seed hull powder;
(2) Leaching the camellia seed shell powder by using an ethanol solution to obtain a leaching solution;
(3) Removing ethanol from the leaching solution through reduced pressure concentration, and sequentially performing ultrasonic treatment and dialysis treatment to obtain dialysate;
(4) And concentrating and drying the dialyzate to obtain the camellia seed husk extract.
Preferably, the drying temperature is 60-80 ℃ and the drying time is 1-2 h.
Preferably, the volume concentration of the ethanol solution is 50-70%, and the volume mass ratio of the ethanol solution to the camellia seed hull powder is 10-20ml.
Preferably, the leaching temperature is 80-90 ℃, and the leaching time is 6-8 h.
Preferably, the ultrasonic power is 300-500W, and the time is 10-20 min.
Preferably, the dialysis bag for dialysis has a molecular weight cut-off of 2 to 20kDa.
Preferably, the temperature of the concentration and drying is 70-80 ℃, and the time is 1-2 h.
The invention also provides the camellia seed husk extract obtained by the preparation method.
The invention also provides application of the camellia seed husk extract in preparing a medicine for treating grass carp hemorrhagic disease.
Preferably, the camellia seed husk extract is used for treating grass carp hemorrhage by inhibiting grass carp reovirus.
The invention adopts the technologies of ethanol extraction, ultrasonic extraction and dialysis treatment to obtain the camellia seed husk extract from camellia seed husks. The camellia seed husk extract provided by the invention is safe to CIK cells, can obviously inhibit GCRV JX01 virus, and can be used for treating grass carp hemorrhage.
Drawings
FIG. 1 is a graph showing the inhibitory effect of camellia seed husk extract on GCRV JX01 virus strain at different concentrations in Experimental example 2.
Detailed Description
The invention provides a preparation method of an oil tea seed shell extract, which comprises the following steps:
(1) Crushing and drying the camellia seed hulls to obtain camellia seed hull powder;
(2) Leaching the camellia seed shell powder by using an ethanol solution to obtain a leaching solution;
(3) Removing ethanol from the leaching solution through reduced pressure concentration, and sequentially performing ultrasonic treatment and dialysis treatment to obtain dialysate;
(4) And concentrating and drying the dialysate to obtain the camellia seed husk extract.
The invention crushes and dries the camellia seed shell to obtain the camellia seed shell powder.
In the present invention, the particle size of the pulverized powder is preferably 80 to 120 mesh, and more preferably 100 mesh.
In the invention, the drying temperature is preferably 60-80 ℃, and more preferably 70 ℃; the drying time is preferably 1 to 2 hours, and more preferably 1.5 hours.
After the camellia seed shell powder is prepared, the camellia seed shell powder is leached by an ethanol solution to obtain a leaching solution.
In the present invention, the volume concentration of the ethanol solution is preferably 50 to 70%, and more preferably 60%.
In the invention, the volume mass ratio of the ethanol solution to the camellia seed hull powder is preferably 10-20ml, more preferably 15ml.
In the invention, the leaching temperature is preferably 80-90 ℃, and more preferably 85 ℃; the leaching time is preferably 6 to 8 hours, and more preferably 7 hours.
After preparing the leaching liquor of the ethanol solution, carrying out reduced pressure concentration on the leaching liquor to remove ethanol, and then carrying out ultrasonic treatment and dialysis treatment in sequence to obtain the dialysate.
In the present invention, the leaching solution is preferably obtained by centrifugal separation.
In the present invention, the rotation speed of the centrifugal separation is preferably 800 to 1200rpm, and more preferably 1000rpm; the time for the centrifugal separation is preferably 30 to 50min, and more preferably 40min.
In the present invention, the concentration under reduced pressure is carried out in a rotary evaporator.
In the present invention, the water bath temperature of the rotary evaporator is preferably 50 to 60 ℃, and more preferably 55 ℃. Removing ethanol in the leaching liquor.
In the present invention, the ultrasonic power is preferably 300 to 500W. More preferably 400W; the time of the ultrasonic treatment is preferably 10 to 20min, and more preferably 15min.
In the present invention, the dialysis bag for dialysis preferably has a molecular weight cut-off of 2 to 20kDa, and more preferably 12kDa.
And after preparing the dialyzate, concentrating and drying the dialyzate to obtain the camellia seed husk extract.
In the present invention, the temperature of the concentration and drying is preferably 70 to 80 ℃, and more preferably 75 ℃.
In the present invention, the time for the concentration and drying is preferably 1 to 2 hours, and more preferably 1.5 hours.
The invention also provides the camellia seed husk extract obtained by the preparation method.
The invention also provides application of the camellia seed husk extract in preparing a medicine for treating grass carp hemorrhagic disease.
In the present invention, the camellia seed husk extract is used for treating grass carp hemorrhage by inhibiting grass carp reovirus.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Crushing tea seed shells, sieving with a 100-mesh sieve, taking undersize, and drying at 70 ℃ for 1.5h to obtain the camellia seed shell powder. Mixing camellia seed hull powder with 60% ethanol solution according to the mass volume ratio of 1g:15ml of mixed leaching is carried out, the leaching temperature is controlled to be 85 ℃, the time is 7 hours, and the leaching solution is centrifuged for 40min at 1000rpm to obtain the leaching solution. Placing the leaching solution in a rotary evaporator, and concentrating under reduced pressure at 55 deg.C to remove ethanol in the leaching solution. Ultrasonically treating the ethanol-removed leaching solution with 400W for 15min, dialyzing with 12KDa dialysis bag, and concentrating and drying the filtrate at 75 deg.C for 1.5h to obtain Camellia seed shell extract.
Example 2
Crushing tea seed shells, sieving with a 80-mesh sieve, taking undersize, and drying at 60 ℃ for 2h to obtain the camellia seed shell powder. Mixing the camellia seed shell powder with 50% ethanol solution according to the mass volume ratio of 1g:20ml of mixed leaching is carried out, the leaching temperature is controlled to be 80 ℃, the time is 8 hours, and the leaching solution is centrifuged for 50min at 800rpm to obtain the leaching solution. Placing the leaching solution in a rotary evaporator, and concentrating under reduced pressure at 50 deg.C to remove ethanol in the leaching solution. Treating the ethanol-removed leaching solution with 300W ultrasound for 20min, dialyzing with 20KDa dialysis bag, collecting the permeate, concentrating and drying at 70 deg.C for 1h to obtain Camellia seed shell extract.
Example 3
Crushing tea seed shells, sieving with a 120-mesh sieve, taking undersize materials, and drying at 80 ℃ for 1h to obtain the camellia seed shell powder. Mixing the camellia seed shell powder with 70% ethanol solution according to the mass volume ratio of 1g: mixing 10ml for extraction, controlling the extraction temperature at 90 deg.C for 6h, and centrifuging at 1200rpm for 30min to obtain the extract. Placing the leaching solution in a rotary evaporator, and concentrating under reduced pressure at 60 deg.C to remove ethanol in the leaching solution. Subjecting the ethanol-removed leaching solution to ultrasonic treatment with 500W for 10min, dialyzing with 8KDa dialysis bag, collecting the filtrate, concentrating and drying at 80 deg.C for 2 hr to obtain Camellia seed shell extract.
Experimental example 1
Taking out the frozen CIK cells from the liquid nitrogen, carrying out water bath at 37 ℃ until the cells are completely dissolved, centrifuging at 900r/min for 5min, quickly transferring to a sterile super clean bench, discarding the frozen stock solution, adding a fresh sterile M199 culture solution containing 10% fetal calf serum, and culturing in an incubator at 28 ℃ until the cells adhere to the wall. Passage 3 times according to the conventional method. The cell concentration was adjusted to 1X 10 5 Each/ml. The diluted CIK cells were plated in 96-well plates, 0.1ml without wells, and incubated overnight in a 28 ℃ incubator. Adding 0.1ml of the Camellia seed husk extract prepared in example 1 at various concentrations to each wellAnd (3) solution. The configuration method comprises the following steps: the camellia seed husk extract prepared in example 1 was prepared to 1. Mu.g/ml, 3. Mu.g/ml, 7. Mu.g/ml, 12. Mu.g/ml, 20. Mu.g/ml, and 30. Mu.g/ml with sterile M199 culture solution. And a blank control group and a cell normal group are arranged and cultured for 72h at 28 ℃. And determining the survival rate of the CIK cells by adopting an MTT colorimetric method.
Cell survival% = [ (experimental group OD value-blank control group OD value)/(cell normal group OD value-blank control group OD value) ] ×%.
The results are shown in Table 1.
TABLE 1 toxicity test results of Camellia seed husk extract on CIK cells
| Concentration of | 1μg/ml | 3μg/ml | 7μg/ml | 12μg/ml | 20μg/ml | 30μg/ml |
| Cell viability | 99.9％ | 99.6％ | 99.1％ | 98.3％ | 97.5％ | 96.8％ |
As can be seen from the detection results in Table 1, the influence of the camellia seed husk extract of 1-30 mug/ml on the activity of CIK cells is small, so that the camellia seed husk extract provided by the invention is safe for the CIK cells.
Example 2
Transferring CIK cells to a 6-well plate, adding 100 μ l of GCRV JX01 virus when the cells substantially overgrow the whole well plate, incubating at 25 deg.C for 1h, removing supernatant, and adding Camellia seed husk extract (extracted in example 1) M199 culture solution at concentrations of 0, 5, 10, 20, and 30 μ g/ml, respectively. The virus titers at various concentrations were measured 72h after infection, and the results are shown in FIG. 1.
As shown in figure 1, the camellia seed hull extract can effectively antagonize GCRV JX01 virus.
The examples show that the camellia seed hull extract provided by the invention is safe for CIK cells, can obviously inhibit GCRV JX01 virus, and can be used for treating grass carp hemorrhagic disease.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of an oil tea seed husk extract is characterized by comprising the following steps:
(1) Crushing and drying the camellia seed hulls to obtain camellia seed hull powder;
(2) Leaching the camellia seed shell powder by using an ethanol solution to obtain a leaching solution;
(3) The leaching liquor is subjected to reduced pressure concentration to remove ethanol, and ultrasonic treatment and dialysis treatment are sequentially carried out to obtain dialysate;
(4) And concentrating and drying the dialysate to obtain the camellia seed husk extract.
2. The method of claim 1, wherein the drying is carried out at a temperature of 60 to 80 ℃ for 1 to 2 hours.
3. The preparation method according to claim 2, wherein the volume concentration of the ethanol solution is 50 to 70%, and the volume mass ratio of the ethanol solution to the camellia seed hull powder is 10 to 20ml.
4. A process according to claim 3, wherein the leaching is carried out at a temperature of 80 to 90 ℃ for a time of 6 to 8 hours.
5. The preparation method according to claim 4, wherein the ultrasonic power is 300-500W and the time is 10-20 min.
6. The method according to claim 5, wherein the dialysis bag for dialysis has a molecular weight cut-off of 2 to 20kDa.
7. The process according to any one of claims 1 to 6, wherein the concentration and drying are carried out at a temperature of from 70 to 80 ℃ for a period of from 1 to 2 hours.
8. An oil-tea camellia seed husk extract obtained by the production method according to any one of claims 1 to 7.
9. Use of the camellia seed husk extract according to claim 8 in the preparation of a medicament for treating hemorrhagic disease of grass carp.
10. The use as claimed in claim 9, wherein the camellia seed husk extract is used for the treatment of grass carp bleeding disease by inhibiting grass carp reovirus.
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