CN110664908A - Camellia nitidissima hypoglycemic active ingredient and extraction method and activity detection method thereof - Google Patents
Camellia nitidissima hypoglycemic active ingredient and extraction method and activity detection method thereof Download PDFInfo
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Abstract
The invention relates to an extraction method and an activity detection method of a camellia nitidissima blood glucose-reducing active component, and belongs to the technical field of biology. The extraction method of the camellia nitidissima hypoglycemic active ingredient provided by the invention comprises the following steps: soaking camellia chrysantha leaf powder in ethanol; soaking camellia chrysantha leaf powder in ethanol, heating, refluxing, extracting, filtering, evaporating to obtain camellia chrysantha leaf extracting solution; extracting the camellia chrysantha extract by ethyl acetate and evaporating to obtain the camellia chrysantha hypoglycemic active ingredient. The extraction method of the camellia nitidissima blood-glucose-reducing active ingredients provided by the invention can effectively improve the extraction rate of the camellia nitidissima blood-glucose-reducing active ingredients, and the camellia nitidissima blood-glucose-reducing active ingredients extracted by the extraction method of the camellia nitidissima blood-glucose-reducing active ingredients are liquid without pungent smell, are easy to store and have high activity.
Description
Technical Field
The invention relates to a camellia nitidissima blood glucose reducing active ingredient, an extraction method and an activity detection method thereof, and belongs to the technical field of biology.
Background
The golden camellia is a unique rare plant in China, has the reputation of 'pandas of plants' and 'queen of tea family', and has records in history of folk use, except for the records of 'compendium of materia medica', 'Difang Zhi' of Guangdong and Guangxi: camellia nitidissima, evergreen shrubs, grow in barren mountains and greens. The leaves and flowers of the golden camellia can be processed to be famous and precious for clearing heat, treating dysentery, removing fat and reducing blood sugar. In folks in two broad areas, people in millennium widely use the golden camellia tea for making tea or cooking soup to clear away heat and toxic materials, reduce blood fat and blood sugar, promote urination, remove dampness and reduce blood pressure, and the golden camellia has profound folk inheritable bottom-story.
The existing extraction method of the chemical components of the golden camellia mainly takes ethanol reflux extraction and ultrasonic extraction as main steps, but the two extraction methods have no unified standard on factors such as reflux time, ethanol-water ratio and the like. Meanwhile, few researches are carried out on the blood sugar reducing active ingredients of the golden camellia, and the activity of the golden camellia is almost verified in an animal experiment mode. In addition, the existing extraction method cannot ensure that the golden camellia has good extraction rate, wastes more materials and solvents, and in addition, the existing golden camellia active ingredients for reducing blood sugar are detected only singly, so that research data of in vitro experiments are lacked.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an extraction method of a camellia nitidissima hypoglycemic active ingredient.
The invention also aims to provide a detection method of the camellia nitidissima hypoglycemic active ingredient.
The invention also aims to provide the camellia nitidissima hypoglycemic active ingredient extracted by the extraction method.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for extracting blood sugar-reducing active ingredients of golden camellia comprises the following steps:
(1) soaking camellia chrysantha leaf powder in ethanol;
(2) heating, refluxing and extracting the ethanol-soaked camellia nitidissima powder in the step (1), and performing suction filtration and evaporation to obtain a camellia nitidissima extract;
(3) and (3) extracting the camellia chrysantha extract in the step (2) by ethyl acetate and evaporating to obtain the camellia chrysantha hypoglycemic active ingredient.
Preferably, the specific operation of step (1) is: pulverizing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 12-48 h by using 50-90% ethanol by volume concentration to obtain mixed liquid.
Preferably, the specific operation of step (2) is: heating and refluxing the mixed solution obtained in the step (1), extracting for 12-24 h, and performing suction filtration to obtain a first extracting solution, wherein solid precipitates are reserved; and then adding 50-90% ethanol into the solid precipitate, continuously heating and refluxing at constant temperature for 4-8 h, performing suction filtration to obtain a second extract, mixing the first extract and the second extract, and evaporating by using a rotary evaporator to obtain the camellia nitidissima extract.
The invention uses the rotary evaporator for evaporation for many times, and aims to remove ethanol.
Preferably, the specific operation of step (3) is: and (3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate for extraction, wherein the upper layer of the solution obtained after extraction is an organic phase, the lower layer of the solution is a water phase, and after extraction is repeated for three times, the upper layer of organic solution is combined and subjected to rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
Preferably, in the step (1), the solid-liquid mass ratio of the camellia chrysantha powder to the ethanol is 1:10-1:20, in the step (2), the mass ratio of the solid precipitate to the ethanol is 1:10-1:20, and the heating reflux temperature is 75-85 ℃; in the step (3), the mass ratio of the camellia chrysantha extract to the ethyl acetate is 1:1-1: 2.
The invention provides a method for extracting blood sugar-reducing active ingredients of golden camellia, which comprises the following steps: the operation steps of the extraction method are carried out under the conditions of normal temperature and normal pressure; in the step (1), the golden camellia leaf powder is placed into a shaking table to shake continuously when being soaked in 50-90% ethanol; in the step (2), the reflux system cannot be completely closed when heating reflux is carried out; in the step (3), multiple rotary evaporation can be carried out before extraction to ensure that the ethanol component is completely removed; when ethyl acetate was used for extraction, the separation of the aqueous and organic phases was carefully observed.
The camellia nitidissima hypoglycemic active ingredient extracted by the extraction method is provided by the invention.
The method for detecting the camellia nitidissima hypoglycemic active ingredients detects the camellia nitidissima hypoglycemic activity by taking nitro-alpha-D glucopyranoside (NPG) as a substrate in a phosphate buffer solution with the pH of 7.0 and the concentration of 0.01M.
As a preferred embodiment of the detection method of the present invention, the detection method specifically comprises the following steps:
(1) test sample group and positive control group preparation: dissolving the camellia nitidissima blood glucose-reducing active ingredients in dimethyl sulfoxide to prepare a test sample solution with the concentration of 10mg/ml, and dissolving acarbose of a positive control group in dimethyl sulfoxide to prepare a positive control solution with the concentration of 10 mg/ml;
(2) substrate group and enzyme solution preparation: dissolving alpha-glucosidase in phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare enzyme solution with concentration of 17.6 mug/ml; dissolving p-nitro-alpha-D glucopyranoside in a phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare a substrate solution with concentration of 1.5 mg/ml;
(3) adding the test sample solution and the positive control solution in the step (1), the enzyme solution in the step (2) and the 0.01M phosphate buffer solution into 96 holes to form a reaction system;
(4) activating the reaction system in the step (3) at 37 ℃ for 5min, adding 10 mul of substrate, reading at 405nm of an enzyme-labeling instrument for the first time, continuing to react at 37 ℃ for 15min, and reading at 405nm of the enzyme-labeling instrument for the second time to obtain the blood-glucose-reducing activity of the golden camellia.
As a preferred embodiment of the detection method of the present invention, the specific operation of step (3) is: 200. mu.l of the 0.01M phosphate buffer was added to rows 2, 3 and 4 of the second column of the 96-well plate, 180. mu.l of the 0.01M phosphate buffer and 10. mu.l of the enzyme solution were added to rows 5, 6 and 7 of the second column of the 96-well plate, 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the test sample solution were added to rows 2, 3 and 4 of the third column of the 96-well plate, and 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the acarbose solution were added to rows 5, 6 and 7 of the third column of the 96-well plate, respectively.
Compared with the prior art, the invention has the beneficial effects that:
(1) the extraction method of the camellia nitidissima blood-glucose-reducing active ingredients provided by the invention can effectively improve the extraction rate of the camellia nitidissima blood-glucose-reducing active ingredients, and can fully utilize raw materials to enable the raw materials to better play a role;
(2) the camellia nitidissima blood-glucose-reducing active ingredient extracted by the extraction method of the camellia nitidissima blood-glucose-reducing active ingredient is liquid without pungent smell, is easy to store and has high activity;
(3) according to the detection method of the camellia nitidissima blood-glucose-reducing active ingredients, the camellia nitidissima blood-glucose-reducing active ingredients are not verified in an animal experiment mode, but the inhibition activity of the camellia nitidissima blood-glucose-reducing active ingredients on alpha-glucosidase is detected in vitro, so that the blank of the in-vitro determination of the blood-glucose-reducing activity of the camellia nitidissima is made up;
(4) according to the extraction method and the detection method of the camellia nitidissima blood sugar-reducing active ingredients, the whole process flow avoids complex procedures and processes, is simple, effective and low in cost, is suitable for actual production, and has important significance in production of camellia nitidissima related medicine products.
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FIG. 1 is a flow chart of an extraction process of the camellia nitidissima hypoglycemic active ingredient.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The embodiment of the invention relates to an extraction method of a camellia nitidissima hypoglycemic active ingredient, which comprises the following steps:
(1) pulverizing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 24 hours by using 70% ethanol with the mass 20 times of that of the golden camellia leaf powder to obtain a mixed solution;
(2) heating and refluxing the mixed solution obtained in the step (1) at 82 ℃ for extraction for 24 hours, carrying out suction filtration on the obtained solution to obtain a first extracting solution, and carrying out solid precipitation for later use; adding 70% ethanol with the mass 20 times of that of the camellia chrysantha powder into the solid precipitate, continuously heating and refluxing at 82 ℃ for extraction for 5 hours, carrying out suction filtration on the obtained solution to obtain a second extracting solution, combining the first extracting solution and the second extracting solution, and evaporating by using a rotary evaporator to obtain a camellia chrysantha extracting solution;
(3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate with equal mass, extracting to obtain a solution, layering the solution, repeatedly extracting for three times, combining the organic solution on the upper layer, and performing rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
Example 2
The embodiment of the invention relates to an extraction method of a camellia nitidissima hypoglycemic active ingredient, which comprises the following steps:
(1) crushing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 48 hours by using 90% ethanol with the mass 10 times of that of the golden camellia leaf powder to obtain a mixed solution;
(2) heating and refluxing the mixed solution obtained in the step (1) at 85 ℃ for 12 hours, filtering the obtained solution to obtain a first extracting solution, and precipitating solids for later use; then adding 90% ethanol with the mass 10 times of that of the camellia chrysantha powder into the solid precipitate, continuously heating and refluxing at 85 ℃ for extraction for 4 hours, carrying out suction filtration on the obtained solution to obtain a second extracting solution, combining the first extracting solution and the second extracting solution, and evaporating by using a rotary evaporator to obtain a camellia chrysantha extracting solution;
(3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate with equal mass, extracting to obtain a solution, layering the solution, repeatedly extracting for three times, combining the organic solution on the upper layer, and performing rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
Example 3
The embodiment of the invention relates to an extraction method of a camellia nitidissima hypoglycemic active ingredient, which comprises the following steps:
(1) pulverizing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 12 hours by using 50% ethanol with the mass 20 times of that of the golden camellia leaf powder to obtain a mixed solution;
(2) heating and refluxing the mixed solution obtained in the step (1) at the temperature of 75 ℃ for 24 hours, filtering the obtained solution to obtain a first extracting solution, and precipitating solid for later use; then adding 50% ethanol with the mass 20 times of that of the camellia chrysantha powder into the solid precipitate, continuously heating and refluxing at the temperature of 75 ℃ for 8 hours, carrying out suction filtration on the obtained solution to obtain a second extracting solution, combining the first extracting solution and the second extracting solution, and evaporating by using a rotary evaporator to obtain a camellia chrysantha extracting solution;
(3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate with the mass being 2 times that of the camellia chrysantha extract, extracting to obtain a solution, layering the solution, repeatedly extracting for three times, combining the organic solutions on the upper layers obtained by three times of extraction, and performing rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
Comparative example 1
The comparative example is an extraction method of a camellia nitidissima hypoglycemic active ingredient in the prior art, and the extraction method comprises the following steps:
(1) pulverizing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 24 hours by using 70% ethanol with the mass 20 times of that of the golden camellia leaf powder to obtain a mixed solution;
(2) heating and refluxing the mixed solution obtained in the step (1) at 82 ℃ for extraction for 24 hours, carrying out suction filtration on the obtained solution to obtain a first extracting solution, and carrying out solid precipitation for later use; adding 70% ethanol with the mass 20 times of that of the camellia chrysantha powder into the solid precipitate, continuously heating and refluxing at 82 ℃ for extraction for 5 hours, carrying out suction filtration on the obtained solution to obtain a second extracting solution, combining the first extracting solution and the second extracting solution, and evaporating by using a rotary evaporator to obtain a camellia chrysantha extracting solution;
(3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by petroleum ether with equal mass, extracting to obtain a solution, layering the solution, repeatedly extracting for three times, combining the organic solution on the upper layer, and performing rotary evaporation to obtain the camellia chrysantha hypoglycemic active ingredient.
Comparative example 2
The comparative example is an extraction method of a camellia nitidissima hypoglycemic active ingredient in the prior art, and the extraction method comprises the following steps:
(1) crushing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 24 hours by using 40% ethanol with the mass 5 times of that of the golden camellia leaf powder to obtain a mixed solution;
(2) heating and refluxing the mixed solution obtained in the step (1) at 82 ℃ for extraction for 24 hours, carrying out suction filtration on the obtained solution to obtain a first extracting solution, and carrying out solid precipitation for later use; then adding 40% ethanol with the mass 5 times of that of the camellia chrysantha powder into the solid precipitate, continuously heating and refluxing for extraction for 5 hours at the temperature of 82 ℃, carrying out suction filtration on the obtained solution to obtain a second extracting solution, combining the first extracting solution and the second extracting solution, and evaporating by using a rotary evaporator to obtain a camellia chrysantha extracting solution;
(3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate with equal mass, extracting to obtain a solution, layering the solution, repeatedly extracting for three times, combining the organic solution on the upper layer, and performing rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
Test example 1
The test example is an alpha-glucosidase inhibition activity in vitro test of the extracted camellia nitidissima blood glucose reducing active ingredients in examples 1-3 and comparative examples 1-2 of the invention.
The test scheme is carried out according to the detection method of the camellia nitidissima hypoglycemic active ingredient.
The test steps are as follows: (1) test sample group and positive control group preparation: dissolving the camellia chrysantha extracts in the examples 1, 2 and 3 and the comparative examples 1 and 2 in dimethyl sulfoxide respectively to prepare test sample solution groups with the concentration of 10mg/ml, and dissolving the acarbose in the positive control group in the dimethyl sulfoxide to prepare solution with the concentration of 10 mg/ml;
(2) substrate group and enzyme solution preparation: dissolving alpha-glucosidase in phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare 17.6 mu g/ml, wherein the group is enzyme solution, dissolving para-nitro-alpha-D glucopyranoside in phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare 1.5mg/ml solution, and the group is substrate group;
(3) adding the test sample solution group and the positive control group in the step (1), the enzyme solution in the step (2) and 0.01M phosphate buffer solution into 96 holes to form a reaction system; specifically, 200. mu.l of the 0.01M phosphate buffer was added to rows 2, 3 and 4 of the second column of the 96-well plate, 180. mu.l of the 0.01M phosphate buffer and 10. mu.l of the enzyme solution were added to rows 5, 6 and 7 of the second column of the 96-well plate, 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the test sample solution of example 1 were added to rows 2, 3 and 4 of the third column of the 96-well plate, and 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the acarbose solution were added to rows 5, 6 and 7 of the third column of the 96-well plate, respectively; 176. mu.l of the 0.01M phosphate buffer solution, 10. mu.l of the enzyme solution, and 4. mu.l of the test sample solution of example 2 were added to rows 2, 3, and 4 of the fourth column of the 96-well plate, respectively, and 176. mu.l of the 0.01M phosphate buffer solution, 10. mu.l of the enzyme solution, and 4. mu.l of the test sample solution of example 2 were added to rows 5, 6, and 7 of the fourth column of the 96-well plate, respectively; 176. mu.l of the 0.01M phosphate buffer solution, 10. mu.l of the enzyme solution, and 4. mu.l of the test sample solution of example 3 were added to rows 2, 3, and 4 of the fifth column of the 96-well plate, 176. mu.l of the 0.01M phosphate buffer solution, 10. mu.l of the enzyme solution, and 4. mu.l of the test sample solution of comparative example 1 were added to rows 5, 6, and 7 of the fifth column of the 96-well plate, and 176. mu.l of the 0.01M phosphate buffer solution, 10. mu.l of the enzyme solution, and 4. mu.l of the test sample solution of comparative example 2 were added to rows 2, 3, and 4 of the sixth column of the 96-well plate, respectively;
(4) and (3) activating the reaction system in the step (3) for 5min at 37 ℃, adding 10 mul of substrate, recording the first reading at 405nm of an enzyme-linked immunosorbent assay as A, continuing to react for 15min at 37 ℃, and recording the second reading at 405nm of the enzyme-linked immunosorbent assay as B, thus obtaining the blood-glucose-reducing activity of the golden camellia.
Wherein, the reading measured by the microplate reader in the experimental step (4) is specifically marked by that the average of the first readings of the 5 th, 6 th and 7 th rows of the second column of the 96-well plate is marked as AAir conditionerThe average of the second reading is marked BAir conditionerThe average number of first readings with acarbose solution added is marked ATo pairThe average of the second readings is BTo pairThe average number of first readings of the solution added to the test sample set is marked ATest forThe average of the second reading is marked BTest for(ii) a The test results are shown in table 1.
The calculation method comprises the following steps: inhibition rate of test sample group 1- [ (B)Test for-ATest for)/(BAir conditioner-AAir conditioner)]*100％
Inhibition rate of positive control group 1- [ (B)To pair-ATo pair)/(BAir conditioner-AAir conditioner)]*100％
TABLE 1 in vitro test of alpha-glucosidase inhibitory activity of Camellia nitidissima hypoglycemic active ingredient
| Test sample | Inhibition rate at 10mg/mL | Extraction rate |
| Example 1 | 94.9％ | 9.85％ |
| Example 2 | 84.4％ | 7.99％ |
| Example 3 | 88.1％ | 7.63％ |
| Comparative example 1 | 0 | 0.79％ |
| Comparative example 2 | 69.8％ | 4.13％ |
| Positive control group | 42.1％ | / |
From the experimental results in table 1, it can be seen that in the methods for extracting camellia nitidissima blood-glucose-reducing active ingredients described in example 1, comparative example 1 and comparative example 2, the extraction rates of the camellia nitidissima blood-glucose-reducing active ingredients are respectively 9.85%, 0.79% and 4.13%, and it can be seen that the extraction rates of the camellia nitidissima blood-glucose-reducing active ingredients described in example 1 are higher than those of comparative example 1 and comparative example 2. When the concentrations of the camellia nitidissima blood-glucose-reducing active ingredients in the example 1, the comparative example 1 and the comparative example 2 are all 10mg/mL, and the inhibition rates of the camellia nitidissima blood-glucose-reducing active ingredients on the alpha-glycosidase activity are 94.9%, 0 and 69.8%, respectively, it can be seen that the inhibition rates of the camellia nitidissima blood-glucose-reducing active ingredients in the example 1 on the alpha-glycosidase activity are higher than those in the comparative example 1 and the comparative example 2. Therefore, the extraction method of the camellia nitidissima blood-glucose-reducing active ingredients provided by the invention can effectively improve the extraction rate of the camellia nitidissima blood-glucose-reducing active ingredients, and the camellia nitidissima blood-glucose-reducing active ingredients extracted by the extraction method of the camellia nitidissima blood-glucose-reducing active ingredients provided by the invention have high inhibition rate on alpha-glycosidase activity, namely the camellia nitidissima blood-glucose-reducing active ingredients have high activity.
In conclusion, the extraction method of the camellia nitidissima blood-glucose-reducing active ingredients provided by the invention can effectively improve the extraction rate of the camellia nitidissima blood-glucose-reducing active ingredients, and can utilize raw materials to enable the raw materials to better play a role; the camellia nitidissima blood-glucose-reducing active ingredient extracted by the extraction method of the camellia nitidissima blood-glucose-reducing active ingredient is liquid without pungent smell, is easy to store and has high activity; according to the method for detecting the camellia nitidissima blood-glucose-reducing active ingredients, the camellia nitidissima blood-glucose-reducing active ingredients are not verified in an animal experiment mode, the inhibition activity of the camellia nitidissima blood-glucose-reducing active ingredients on alpha-glucosidase is detected in vitro, and the blank of in-vitro measurement of the blood-glucose-reducing activity of the camellia nitidissima is made up.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. The extraction method of the camellia nitidissima hypoglycemic active ingredient is characterized by comprising the following steps:
(1) soaking camellia chrysantha leaf powder in ethanol;
(2) heating, refluxing and extracting the ethanol-soaked camellia nitidissima powder in the step (1), and performing suction filtration and evaporation to obtain a camellia nitidissima extract;
(3) and (3) extracting the camellia chrysantha extract in the step (2) by ethyl acetate and evaporating to obtain the camellia chrysantha hypoglycemic active ingredient.
2. The extraction method according to claim 1, wherein the specific operation of the step (1) is: pulverizing golden camellia leaves to obtain golden camellia leaf powder, and soaking the golden camellia leaf powder for 12-48 h by using 50-90% ethanol by volume concentration to obtain a mixed solution;
the specific operation of the step (2) is as follows: heating and refluxing the mixed solution obtained in the step (1), extracting for 12-24 h, and performing suction filtration to obtain a first extracting solution, wherein solid precipitates are reserved; adding 50-90% ethanol into the solid precipitate, continuously heating and refluxing at constant temperature for 4-8 h, vacuum filtering to obtain second extractive solution, mixing the first and second extractive solutions, and evaporating with rotary evaporator to obtain Camellia Chysantha extractive solution;
the specific operation of the step (3) is as follows: and (3) fully oscillating and shaking the camellia chrysantha extract obtained in the step (2) by ethyl acetate for extraction, wherein the upper layer of the solution obtained after extraction is an organic phase, the lower layer of the solution is a water phase, and after extraction is repeated for three times, the upper layer of organic solution is combined and subjected to rotary evaporation to obtain the camellia chrysantha blood glucose reducing active ingredient.
3. The extraction method according to claim 2, wherein in the step (1), the solid-liquid mass ratio of the camellia chrysantha leaf powder to the ethanol is 1:10-1:20, in the step (2), the mass ratio of the solid precipitate to the ethanol is 1:10-1:20, and the temperature of heating reflux is 75-85 ℃; in the step (3), the mass ratio of the camellia chrysantha extract to the ethyl acetate is 1:1-1: 2.
4. The camellia nitidissima hypoglycemic active ingredient extracted by the extraction method according to any one of claims 1 to 3.
5. The method for detecting the camellia nitidissima hypoglycemic active ingredient according to claim 4, wherein the camellia nitidissima hypoglycemic activity is detected in a phosphate buffer solution with pH of 7.0 and concentration of 0.01M and with p-nitro-alpha-D glucopyranoside as a substrate.
6. The detection method according to claim 5, wherein the specific operation steps of the detection method are as follows:
(1) test sample group and positive control group preparation: dissolving the camellia nitidissima blood glucose-reducing active ingredients in dimethyl sulfoxide to prepare a test sample solution with the concentration of 10mg/ml, and dissolving acarbose of a positive control group in dimethyl sulfoxide to prepare a positive control solution with the concentration of 10 mg/ml;
(2) substrate group and enzyme solution preparation: dissolving alpha-glucosidase in phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare enzyme solution with concentration of 17.6 mug/ml; dissolving p-nitro-alpha-D glucopyranoside in a phosphate buffer solution with pH of 7.0 and concentration of 0.01M to prepare a substrate solution with concentration of 1.5 mg/ml;
(3) adding the test sample solution and the positive control solution in the step (1), the enzyme solution in the step (2) and the 0.01M phosphate buffer solution into 96 holes to form a reaction system;
(4) activating the reaction system in the step (3) at 37 ℃ for 5min, adding 10 mul of substrate, reading at 405nm of an enzyme-labeling instrument for the first time, continuing to react at 37 ℃ for 15min, and reading at 405nm of the enzyme-labeling instrument for the second time to obtain the blood-glucose-reducing activity of the golden camellia.
7. The detection method according to claim 6, wherein the specific operation of step (3) is: 200. mu.l of the 0.01M phosphate buffer was added to rows 2, 3 and 4 of the second column of the 96-well plate, 180. mu.l of the 0.01M phosphate buffer and 10. mu.l of the enzyme solution were added to rows 5, 6 and 7 of the second column of the 96-well plate, 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the test sample solution were added to rows 2, 3 and 4 of the third column of the 96-well plate, and 176. mu.l of the 0.01M phosphate buffer, 10. mu.l of the enzyme solution and 4. mu.l of the acarbose solution were added to rows 5, 6 and 7 of the third column of the 96-well plate, respectively.
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