CN108619321A - Application of the Camellia nitidissima seed extract in alpha-glucosidase restrainer - Google Patents

Abstract

The invention discloses the application of camellia japonica seeds in alpha-glucosidase inhibitors, in particular health products and medicines for the prevention and treatment of type II diabetes. The present invention adopts in vitro inhibition of α-glucosidase activity model to investigate the inhibitory effect of the ethyl acetate part, n-butanol part and water layer part of Camellia japonica seed ethanol extract and ethanol extract to α-glucosidase, and through Lineweave‑Burk plots were used to determine the type of inhibition. The preparation method of the present invention is simple to the target product, is easy to operate, has the potentiality of industrialized production, and the inhibitory rate to α-glucosidase is better than the clinical drug acarbose, and can be used for the preparation of α-glucosidase inhibitors in natural products Develop applications.

Description

Application of Camellia Camellia Seed Extract in α-Glucosidase Inhibitor

technical field

The invention relates to the field of medicine and health care products, in particular to the application of camellia camellia seed extract in alpha-glucosidase inhibitors.

Background technique

Type 2 diabetes, also known as non-insulin-dependent diabetes, is a common disorder of glucose and fat metabolism. Diabetes is characterized by high concentrations of blood sugar, which may lead to serious complications in the kidneys, eyes and cardiovascular system. It is chronic Metabolic diseases can cause metabolic disorders such as sugar, protein, and fat, and damage the blood circulation system (Morrison, F., Shubina, M., & Turchin, A. Encounter frequency and serum glu-cose level, bloodpressure, and cholesterol level control in patients with diabetes mellitus [J]. Archives of Internal Medicine. 2001, 171, 1542–50.). Therefore, the treatment of diabetes mainly focuses on reducing blood sugar fluctuations and mitigating complications. Drugs that affect the absorption of carbohydrates will slow down the digestion rate of carbohydrates entering the body. When the human body consumes high carbohydrates such as rice and noodles, their decomposition rate slows down, and blood sugar after meals will not be reduced in a short time. Rising rapidly, the main representative drugs are α-glucosidase inhibitors, amylin, and glucokinase (GK) activator drugs (Liu Xuerui. Extraction of okra polysaccharide and research on its hypoglycemic activity[D]. Tianjin University of Science and Technology, 2017 .).

α-glucosidase is the key enzyme for the digestion and absorption of carbohydrates in the small intestine. Therefore, α-glucosidase inhibitors can delay or inhibit the intestinal absorption of glucose, thereby significantly reducing postprandial blood sugar, as well as protecting islet cell function and It is the drug of choice for the treatment of type II diabetes (Ma Yonglei, Zhang Yuqing, Zhou Lixia, et al. Antioxidation and inhibitory effect on α-glucosidase activity of mulberry bark alcohol extract[J]. Industry Science. 2010, 36(01): 143-7.). There are currently marketed drugs such as acarbose, voglibose, and miglitol as clinical medication for diabetic patients. These listed α-glucosidase inhibitors are all produced by microbial fermentation broth. The target product obtained by biological or chemical synthesis after body material has obvious side effects such as flatulence, diarrhea, and abdominal discomfort (Yu Caiyun, Gao Zhaolan, Chen Tianzi, et al. Research progress of α-glucosidase inhibitors in natural products[J] . Food Industry Science and Technology. 2015, 36(22): 394-9.). Therefore, finding new safe and effective α-glucosidase inhibitors from natural products has become a research hotspot in the prevention and treatment of diabetes. Such as: CN105030914A discloses a preparation method of privet privet tea α-glucosidase inhibitor; CN105213493A discloses the application of rhubarb, kiwi root or Tianjihuang extract in α-glucosidase inhibitor, It can be used to treat or prevent diabetes; CN1565467 discloses the application of Cornus officinalis and its extracts as alpha-glucosidase inhibitors.

Camellia chrysantha, whose botanical name is Camellia chrysantha(Hu)Tuyama, is a Camellia family, Camellia, evergreen shrubs to small trees, and is the same as Camellia, Camellia, Camellia oleifera, Camellia Camellia, and Camellia sasana. It is recorded in "Compendium of Materia Medica". Golden camellia tea is slightly bitter, astringent and flat in nature and flavor, and has the effects of clearing heat and detoxifying, diuretic and detumescence, etc. etc. (Department of Health of Guangxi Zhuang Autonomous Region. Standards of Chinese Medicinal Materials in Guangxi (Volume II) [M]. Nanning: Guangxi Science and Technology Press, 1996.). Modern pharmacological studies have found that its active ingredients also have the ability to inhibit tumor cell growth (Li Ming-Hui, Du Hong-Zhi, Kong Gui-Ju, et al.Nuclear Magnetic Resonance-Based Metabolomics Approach to Evaluate the Prevention Effect of Camellia nitidissima Chi on Colitis- Associated Carcinogenesis [J]. Front Pharmacol, 2017, 8: 447.), anti-allergic (Wang Yongqi, Peng Xiao, Tang Qian, et al. Anti-IgE-mediated type Ⅰ hypersensitivity activity screening of Camellia japonica group plants [J]. Zhongnan Pharmacy .2009, (10): 721-4.), Antioxidation (Niu Guangjun, Xing Jianhong, Zhu Si, etc. Determination of active ingredients and antioxidant activity of Camellia japonica [J]. Journal of Fujian Forestry University, 2015, 35(2): 165-8.), regulating blood lipids (Oku Hisae, Ogawa Yuko, Iwaoka Emiko, et al. Preventive effects of the extract of kinka-cha, a folk tea, on a rat model of metabolic syndrome[J].J Nat Med, 2011, 65(3-4):610-6.) and other effects. Natural golden camellia tea has been made into health medicine "Golden camellia teabag and oral liquid". At present, most of the researches on Camellia japonica focus on the leaves, flowers and other parts, while the research on the seeds of Camellia japonica is less, and the effect of inhibiting α-glucosidase on the seeds of Camellia japonica has not been reported yet.

Contents of the invention

The purpose of the present invention is to provide the application of Camellia camellia seed extract in alpha-glucosidase inhibitors.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

Application of camellia camellia seed extract in alpha-glucosidase inhibitors.

The above-mentioned camellia seed extract is the ethanol extract of camellia seeds, the ethyl acetate part of the ethanol extract of camellia seeds, the n-butanol part of the ethanol extract of camellia seeds or/and the water-soluble part of the ethanol extract of camellia seeds.

The above-mentioned golden camellia seeds are the four seasons golden camellia seeds.

The above ethanol extract of Camellia japonica seeds, the ethyl acetate part of the ethanol extract of Camellia japonica seeds, the n-butanol part of the ethanol extract of Camellia japonica seeds, and the water-soluble part of the ethanol extract of Camellia japonica seeds are prepared by the following preparation methods:

(1) Get dried golden camellia seed pulverizer to pulverize;

(2) After degreasing the petroleum ether, use 3 times the amount of 95% ethanol to heat and reflux to extract 3 times, each time for 3 hours, to obtain Camellia japonica extract;

(3) the Camellia japonica extract is suction-filtered with filter paper, the filtrates are combined, concentrated under reduced pressure to form an extract, and the ethanol extract of Camellia japonica seeds is obtained;

(4) Take the ethanol extract of Camellia japonica seeds and add an appropriate amount of water to suspend, then extract with ethyl acetate and n-butanol successively to obtain the ethyl acetate part, n-butanol part and raffinate water layer part, concentrate and vacuum-dry, The ethyl acetate part, the n-butanol part and the water-soluble part of the ethanol extract are obtained.

Each assay method involved in the present invention is specifically as follows:

1) Determination of p-nitrophenol (PNP) content

The present invention uses 4-nitrophenyl-α-D-glucopyranoside (4-ntrophyl-α-D-glucopyranoside, PNPG) as substrate, because α-glucosidase can be combined with substrate, reacts to produce Nitrophenol, so the enzyme activity, reaction rate, etc. can be monitored by detecting the PNP content.

Precisely weigh PNP, and use phosphate buffer solution (PBS PH6.8) to prepare seven solutions with different concentrations of 0, 50, 100, 200, 300, 400, and 500 μmol/l. Take 152 μl of PNP solutions with different concentrations, add 8 μl of DMSO, and add 80 μl of 0.2mol/l Na ₂ CO ₃ solution into a 96-well plate. After mixing, measure the OD value at 405 nm with a microplate reader, repeat 5 groups, and take the average value . With the OD value as the ordinate and the PNP concentration C as the abscissa, draw a standard curve (accompanying drawing 1). The regression equation OD value=0.0066C+0.1016 (R ² =0.9944) was obtained.

2) α-glucosidase activity assay

This test is completed on a 96 microwell plate, add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL DMSO, incubate at 37°C for 15min, add 20μl 2.5mmol/l PNPG, incubate for 15min, then add 80μl 0.2mol /lNa ₂ CO ₃ , measured the OD value at 405nm, and substituted it into the standard song to obtain the enzyme activity as 4.73U.

The enzyme activity unit is defined as the amount of enzyme required to hydrolyze PNPG to produce 1 μmol of PNP per minute under the conditions of 37°C and pH 6.8, which is defined as 1 enzyme activity unit (U).

3) Determination of inhibition of α-glucosidase activity

This monitoring is completed on a 96 microwell plate. Add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL sample solution, incubate at 37°C for 15min, add 20μl 2.5mmol/l PNPG, incubate for 15min, then add 80μl 0.2mol/l Na ₂ CO ₃ , measure the OD value at 405nm with a microplate reader.

A total of 4 groups were set up in the experiment, and each group was repeated three times, and the settings were as follows:

a. Negative control group (buffer + enzyme solution + substrate: buffer 112μl + enzyme solution 20μl + DMSO 8μl, after constant temperature at 37°C for 15 minutes + substrate PNPG 20μl, after constant temperature reaction at 37°C for 15 minutes + Na ₂ CO ₃ 80μl),

b. Blank control group (buffer solution: 112μl buffer + 20μl buffer + 8μl DMSO, 15min at 37°C + buffer 20μl, 15min at 37°C + Na ₂ CO ₃ 80μl),

c. Sample determination group (sample+enzyme solution+substrate: buffer 112μl+enzyme solution 20μl+sample 8μl, after constant temperature at 37°C for 15 minutes + substrate PNPG 20μl, after constant temperature reaction at 37°C for 15 minutes+Na ₂ CO ₃ 80μl),

d. Sample control group (sample + buffer: 112 μl buffer + 20 μl buffer + 8 μl sample, 15 minutes at 37°C + substrate PNPG 20 μl, 15 minutes at 37°C + Na ₂ CO ₃ 80 μl).

Compared with the prior art, the present invention has the following advantages and effects:

1. The three polar parts (ethyl acetate part, n-butanol part, water-soluble part) of the Camellia japonica seed ethanol extract and the ethanol extract extracted from the Camellia japonica seeds are used as α-glucosidase inhibitors, which have anti- The development of drugs, health products and food for the prevention and treatment of type Ⅱ diabetes has broad application prospects;

2. Heating and reflux extraction is a classic method for extracting natural products. It is used to extract Camellia japonica seeds with less process steps, simple operation and mild conditions, and is suitable for industrial production.

Description of drawings

The standard curve of Fig. 1 p-nitrophenol;

Fig. 2 is the activity inhibition curve of positive drug acarbose to α-glucosidase;

Fig. 3 is the activity inhibition curve of α-glucosidase of Camellia japonica seed ethanol extract and three different polar parts of ethanol extract;

Fig. 4 is the inhibitory action kinetic curve of positive drug acarbose;

Fig. 5 is the inhibition kinetic curve of Camellia japonica ethanol extract;

Fig. 6 is the inhibitory action kinetic curve of the ethyl acetate part of Camellia japonica seed ethanol extract;

Fig. 7 is the inhibitory action kinetic curve of the n-butanol part of Camellia japonica seed ethanol extract;

Fig. 8 is the inhibitory kinetics curve of the water-soluble fraction of the ethanol extract of Camellia japonica seeds.

Detailed ways

The present invention will be further described below in combination with specific embodiments.

The preparation of the ethyl acetate part, n-butanol part, water-soluble part of embodiment 1 golden camellia seed ethanol extract and ethanol extract

1) Take 800g of dried Camellia japonica seeds and pulverize them with a pulverizer;

2) After degreasing petroleum ether, heat and reflux extraction with 3 times the amount of 95% ethanol for 3 times, each time for 3 hours, to obtain Camellia japonica extract;

3) The Camellia japonica extract was filtered with filter paper, the filtrates were combined, concentrated under reduced pressure to obtain an extract, and 76 g of the ethanol extract of Camellia japonica seeds was obtained; after taking 71 g of the ethanol extract of Camellia japonica seeds and adding an appropriate amount of water to suspend, successively distilled with ethyl acetate , n-butanol extraction, to obtain ethyl acetate part, n-butanol part and raffinate aqueous layer part, after concentrating, vacuum-dry to obtain ethyl acetate part 12g, n-butanol part 45g, water-soluble part 14g of ethanol extract.

Example 2 Evaluation of inhibitory effect on α-glucosidase activity

The method of combining percent inhibition rate (I%) and half inhibitory concentration (IC ₅₀ value) was used for evaluation. _IC50 was calculated using GraphPad Prism software, and the percentage inhibition rate was calculated as follows:

1. Determination of α-glucosidase inhibitory activity of the ethanol extract of Camellia japonica seeds

The ethanol extract of Camellia japonica seeds was prepared with DMSO into sample solutions with five different concentrations of 0.2, 0.5, 1, 1.5, and 2 mg/ml, corresponding to final concentrations of 10, 25, 50, 75, and 100 μg/ml. Set up four groups, namely negative control group, blank group, sample measurement group, and sample control group; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL DMSO to the negative control group in turn, and incubate at 37°C for 15 minutes in a constant temperature incubator , add 20μl 2.5mmol/l PNPG, incubate at 37℃ for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL DMSO in sequence, and incubate at 37℃ for 15min in a constant temperature incubator , add 20μl PBS, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ ; add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL sample solutions with different concentrations in sequence , incubate at 37°C for 15min in a constant temperature incubator, add 20μl 2.5mmol/l PNPG, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL Sample solutions with different concentrations were incubated in a constant temperature incubator at 37°C for 15 minutes, then 20 μl of 2.5 mmol/l PNPG was added, and after incubated in a constant temperature incubator at 37°C for 15 minutes, 80 μl of 0.2 mol/l Na ₂ CO ₃ was added. Then use a microplate reader to measure the OD value at 405nm, and substitute it into the standard curve to calculate the inhibition percentage, as shown in Table 1, and the inhibition curve for the inhibition rate is shown in Figure 3. Using GraphPad Prism software, the IC ₅₀ value of the ethanol extract of Camellia japonica seeds on α-glucosidase was calculated to be 37.43 μg/ml.

2. Determination of the inhibitory activity of the ethyl acetate fraction of the ethanol extract of Camellia japonica seeds on α-glucosidase

The ethyl acetate part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into sample solutions with five different concentrations of 0.2, 0.5, 1, 1.5, and 2 mg/ml, corresponding to the final concentrations of 10, 25, 50, 75, and 100 μg /ml. Set up four groups, namely negative control group, blank group, sample measurement group, and sample control group; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL DMSO to the negative control group in turn, and incubate at 37°C for 15 minutes in a constant temperature incubator , add 20μl 2.5mmol/l PNPG, incubate at 37℃ for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL DMSO in sequence, and incubate for 15min in a constant temperature incubator at 37℃ , add 20μl PBS, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ ; add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL sample solutions with different concentrations in sequence , incubate in a constant temperature incubator at 37°C for 15 minutes, add 20 μl of 2.5mmol/l PNPG, incubate in a constant temperature incubator at 37°C for 15 minutes, then add 80 μl of 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132 μl of PBS to the sample control group in turn, 8 μL of sample solutions with different concentrations were incubated in a constant temperature incubator at 37°C for 15 minutes, 20 μl of 2.5mmol/l PNPG was added, and after 15 minutes of incubation at a constant temperature incubator at 37°C, 80 μl of 0.2mol/l Na ₂ CO ₃ was added. Then use a microplate reader to measure the OD value at 405nm, and substitute it into the standard curve to calculate the inhibition percentage, as shown in Table 1, and the inhibition curve for the inhibition rate is shown in Figure 3. Using GraphPad Prism software, the IC ₅₀ value of the ethyl acetate fraction of the ethanol extract of Camellia japonica seeds on α-glucosidase was calculated to be 335.6 μg/ml.

3. Determination of the inhibitory activity of the n-butanol fraction of the ethanol extract of Camellia japonica seeds on α-glucosidase

The n-butanol part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into sample solutions with five different concentrations of 0.2, 0.5, 1, 1.5, and 2 mg/ml, corresponding to the final concentrations of 10, 25, 50, 75, and 100 μg /ml. Set up four groups, namely negative control group, blank group, sample measurement group, and sample control group; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL DMSO to the negative control group in turn, and incubate at 37°C for 15 minutes in a constant temperature incubator , add 20μl 2.5mmol/l PNPG, incubate at 37℃ for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL DMSO in sequence, and incubate for 15min in a constant temperature incubator at 37℃ , add 20μl PBS, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ ; add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL sample solutions with different concentrations in sequence , incubate in a constant temperature incubator at 37°C for 15 minutes, add 20 μl of 2.5mmol/l PNPG, incubate in a constant temperature incubator at 37°C for 15 minutes, then add 80 μl of 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132 μl of PBS to the sample control group in turn, 8 μL of sample solutions with different concentrations were incubated in a constant temperature incubator at 37°C for 15 minutes, 20 μl of 2.5mmol/l PNPG was added, and after 15 minutes of incubation at a constant temperature incubator at 37°C, 80 μl of 0.2mol/l Na ₂ CO ₃ was added. Then use a microplate reader to measure the OD value at 405nm, and substitute it into the standard curve to calculate the inhibition percentage, as shown in Table 1, and the inhibition curve for the inhibition rate is shown in Figure 3. Using GraphPad Prism software, the IC ₅₀ value of the n-butanol fraction of the ethanol extract of Camellia japonica seeds to α-glucosidase was calculated to be 20.11 μg/ml.

4. Determination of the α-glucosidase inhibitory activity of the water-soluble fraction of the ethanol extract of Camellia japonica seeds

The water-soluble part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into sample solutions with five different concentrations of 0.2, 0.5, 1, 1.5, and 2 mg/ml, corresponding to the final concentrations of 10, 25, 50, 75, and 100 μg/ml . Set up four groups, namely negative control group, blank group, sample measurement group, and sample control group; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL DMSO to the negative control group in turn, and incubate at 37°C for 15 minutes in a constant temperature incubator , add 20μl 2.5mmol/l PNPG, incubate at 37℃ for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL DMSO in sequence, and incubate for 15min in a constant temperature incubator at 37℃ , add 20μl PBS, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ ; add 112μl PBS, 20μl 0.2U/ml α-glucosidase, 8μL sample solutions with different concentrations in sequence , incubate in a constant temperature incubator at 37°C for 15 minutes, add 20 μl of 2.5mmol/l PNPG, incubate in a constant temperature incubator at 37°C for 15 minutes, then add 80 μl of 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132 μl of PBS to the sample control group in turn, 8 μL of sample solutions with different concentrations were incubated in a constant temperature incubator at 37°C for 15 minutes, 20 μl of 2.5mmol/l PNPG was added, and after 15 minutes of incubation at a constant temperature incubator at 37°C, 80 μl of 0.2mol/l Na ₂ CO ₃ was added. Then use a microplate reader to measure the OD value at 405nm, and substitute it into the standard curve to calculate the inhibition percentage, as shown in Table 1, and the inhibition curve for the inhibition rate is shown in Figure 3. The results are concentration-dependent, and the dose-effect relationship is very significant. Using GraphPad Prism software, the IC ₅₀ value of the water-soluble fraction of the ethanol extract of Camellia japonica seeds on α-glucosidase was 17.8 μg/ml.

5. Determination of the inhibitory activity of acarbose on α-glucosidase

Acarbose was formulated with DMSO (dimethyl sulfoxide) into sample solutions with five different concentrations of 0.5, 1, 2, 3, and 4 mg/ml, respectively corresponding to the final concentrations of 250, 500, 1000, 1500, and 2000 μg /ml. Set up four groups, namely negative control group, blank group, sample measurement group, and sample control group; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL DMSO to the negative control group in turn, and incubate at 37°C for 15 minutes in a constant temperature incubator , add 20μl 2.5mmol/l PNPG, incubate at 37℃ in a constant temperature incubator for 15min, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL DMSO in sequence, and incubate in a constant temperature incubator at 37℃ After 15 minutes, add 20 μl PBS, incubate at 37°C in a constant temperature incubator for 15 minutes, then add 80 μl 0.2mol/l Na ₂ CO ₃ ; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL samples of different concentrations in sequence Solution, incubate at 37°C for 15min in a constant temperature incubator, add 20μl 2.5mmol/l PNPG, incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to stop the reaction; add 132μl PBS to the sample control group in turn, 8 μL of sample solutions with different concentrations were incubated in a constant temperature incubator at 37°C for 15 minutes, 20 μl of 2.5mmol/l PNPG was added, and after 15 minutes of incubation at a constant temperature incubator at 37°C, 80 μl of 0.2mol/l Na ₂ CO ₃ was added. Then use a microplate reader to measure the OD value at 405nm, and substitute it into the standard music to calculate the inhibition percentage, see Table 1, and the inhibition curve for the inhibition rate is shown in Figure 2. The IC ₅₀ value of acarbose on α-glucosidase calculated by GraphPad Prism software was 484.9 μg/ml.

See Table 1 for the ethanol extracts of Camellia japonica seeds and the effects of different polar parts on the inhibition of α-glucosidase, and see Table 2 for the inhibitory activities of positive drugs and different samples on α-glucosidase.

Table 1

Compared with the control group, ** is P<0.001, *** is P<0.0001, **** is P<0.00001, n=3

Table 2

As can be seen from Table 1 and Table 2, the inhibitory activity of the ethanol extract of Camellia japonica seeds and the ethyl acetate part, n-butanol part and water layer part of the ethanol extract to α-glucosidase is stronger than that of the positive control drug A Carbose, wherein the inhibitory effect on α-glucosidase water layer > n-butanol > Camellia japonica seed ethanol extract > ethyl acetate, and the IC ₅₀ value of the water layer is extremely small, the effect on α-glucose The inhibitory effect on sidase activity was the strongest, and the inhibition rate reached 79.23% at 50 μg/ml.

Example 3 Inhibition Types of α-Glucosidase Inhibition

According to the Lineweave-Burk mapping method, with 1/[S] as the abscissa and 1/V as the ordinate, draw the inhibition kinetic curves of the samples respectively, determine the inhibition type of the sample according to the position of the intersection point of the straight lines, and determine the inhibition type of the sample according to the position of the straight line intersection, according to the straight line when no sample is added Obtain the K _m value of α-glucosidase with the intersecting point=-1/K _m of x axis, then according to non-competitive inhibition kinetic equation: 1/V ' _max =1/V _max (1+[I]/K _i ) and the competitive inhibition kinetic equation: 1/K _m '=1/{K _m (1+[I]/K _i )}, respectively calculate the K _i value of the sample.

1. Types of inhibition of α-glucosidase inhibitory effect of the ethanol extract of Camellia japonica seeds

The ethanol extract of camellia japonica seeds was prepared with DMSO into two sample solutions with different concentrations of 0.2 and 0.5 mg/ml, corresponding to the final concentrations of 10 and 25 μg/ml, respectively. Set up two groups, namely the sample measurement group and the sample control group; add 112μl PBS, 20μl 0.2U/ml α-glucosidase, and 8μL sample solutions of different concentrations to the sample measurement group, incubate at 37°C for 15min in a constant temperature incubator, and add 20μl Different concentrations of PNPG (0.625, 1.25, 2.5, 5, 10mmol/l) were incubated in a constant temperature incubator at 37°C for 15 minutes, and then 80 μl of 0.2mol/l Na ₂ CO ₃ was added to terminate the reaction; the sample control group was sequentially added with 132 μl of PBS, 8 μL of sample solutions with different concentrations were incubated at 37°C for 15 minutes in a constant temperature incubator, and 20 μl of PNPG (0.625, 1.25, 2.5, 5, 10 mmol/l) were added respectively. mol/l Na ₂ CO ₃ . Then use a microplate reader to measure the OD value at 405nm, draw the inhibition kinetics curve (accompanying drawing 5), determine that the inhibition type of the Camellia japonica seed alcohol extract is non-competitive inhibition, and then calculate according to the non-competitive inhibition kinetic equation The K _i value was obtained to be 13.94 μg/ml.

2. Types of inhibition of α-glucosidase inhibition by the ethyl acetate fraction of the ethanol extract of Camellia japonica seeds

The ethyl acetate part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into two sample solutions with different concentrations of 0.2 and 0.5 mg/ml, corresponding to the final concentrations of 10 and 25 μg/ml, respectively. Set up two groups, add 112 μl PBS, 20 μl 0.2 U/ml α-glucosidase, 8 μL sample solutions with different concentrations in sequence, incubate at 37°C for 15 minutes in a constant temperature incubator, add 20 μl different concentrations of PNPG (0.625, 1.25, 2.5, 5. 10mmol/l), incubate at 37°C in a constant temperature incubator for 15min, then add 80µl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132µl of PBS, 8µL of different concentrations of sample solutions to the sample control group, in a constant temperature incubator at 37°C After incubating for 15 minutes, 20 μl of different concentrations of PNPG (0.625, 1.25, 2.5, 5, 10 mmol/l) were added, and after incubation for 15 minutes at 37° C. in a constant temperature incubator, 80 μl of 0.2 mol/l Na ₂ CO ₃ was added. Use microplate reader to measure OD value under 405nm subsequently, draw inhibition kinetics curve (accompanying drawing 6), determine that the inhibition type of the ethyl acetate part of golden camellia seed ethanol extract is non-competitive inhibition, then according to non-competitive inhibition The K _i value can be calculated from the kinetic equation to be 131.02μg/ml.

3. Types of inhibition of α-glucosidase inhibition by the n-butanol fraction of the ethanol extract of Camellia japonica seeds

The n-butanol part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into sample solutions with two different concentrations of 0.2 and 0.5 mg/ml, corresponding to the final concentrations of 10 and 25 μg/ml, respectively. Set up two groups, add 112 μl of PBS, 20 μl of 0.2 U/ml α-glucosidase, 8 μL of different concentrations of sample solutions to the sample measurement group, incubate at 37°C for 15 minutes in a constant temperature incubator, and add 20 μl of different concentrations of PNPG (0.625, 1.25, 2.5 , 5, 10mmol/l), incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL of different concentrations of sample solutions to the sample control group, and place in a constant temperature incubator for 37 After incubating at ℃ for 15 min, 20 μl of PNPG of different concentrations (0.625, 1.25, 2.5, 5, 10 mmol/l) were added, and after incubating at 37 °C for 15 min in a constant temperature incubator, 80 μl of 0.2 mol/l Na ₂ CO ₃ was added. Use microplate reader to measure OD value under 405nm subsequently, draw inhibition kinetics curve (accompanying drawing 7), determine that the inhibition type of the n-butanol part of camellia japonica seed alcohol extract is non-competitive inhibition, then according to non-competitive inhibition The K _i value can be calculated as 9.13μg/ml according to the kinetic equation.

4. Inhibition types of the water-soluble fraction of the ethanol extract of Camellia japonica seeds on the inhibitory effect of α-glucosidase

The water-soluble part of the ethanol extract of Camellia japonica seeds was prepared with DMSO into two sample solutions with different concentrations of 0.2 and 0.5 mg/ml, corresponding to the final concentrations of 10 and 25 μg/ml, respectively. Set up two groups, add 112 μl of PBS, 20 μl of 0.2 U/ml α-glucosidase, 8 μL of different concentrations of sample solutions to the sample measurement group, incubate at 37°C for 15 minutes in a constant temperature incubator, and add 20 μl of different concentrations of PNPG (0.625, 1.25, 2.5 , 5, 10mmol/l), incubate at 37°C for 15min in a constant temperature incubator, then add 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; add 132μl PBS, 8μL of different concentrations of sample solutions to the sample control group, and place in a constant temperature incubator for 37 After incubating at ℃ for 15 min, 20 μl of PNPG of different concentrations (0.625, 1.25, 2.5, 5, 10 mmol/l) were added, and after incubating at 37 °C for 15 min in a constant temperature incubator, 80 μl of 0.2 mol/l Na ₂ CO ₃ was added. Use microplate reader to measure OD value under 405nm subsequently, draw inhibition kinetics curve (accompanying drawing 8), determine that the inhibition type of the water-soluble part of Camellia japonica seed alcohol extract is non-competitive inhibition, then according to non-competitive inhibition kinetics Equation can be calculated its K _i value is 6.58μg/ml.

5. Types of inhibition of acarbose on α-glucosidase inhibition

Acarbose was prepared with DMSO into sample solutions with two different concentrations of 5 and 10 mg/ml, respectively corresponding to final concentrations of 200 and 500 μg/ml. Four groups were set up, the negative control group was sequentially added with 112 μl PBS, 20 μl 0.2 U/ml α-glucosidase, 8 μL DMSO, incubated at 37°C for 15 min in a constant temperature incubator, and added 20 μl of PNPG of different concentrations (0.625, 1.25, 2.5, 5, 10mmol/l), incubated at 37°C for 15min in a constant temperature incubator, and then added 80μl 0.2mol/l Na ₂ CO ₃ to terminate the reaction; the blank group was sequentially added 132μl PBS, 8μL DMSO, incubated at 37°C for 15min in a constant temperature incubator, and added 20μl PBS, incubate at 37°C for 15 minutes in a constant temperature incubator, then add 80 μl 0.2mol/l Na ₂ CO ₃ ; add 112 μl PBS, 20 μl 0.2U/ml α-glucosidase, 8 μL sample solutions of different concentrations in a constant temperature incubator Incubate at 37°C for 15min, add 20μl of different concentrations of PNPG (0.625, 1.25, 2.5, 5, 10mmol/l), and incubate at 37°C for 15min in a constant temperature incubator, then add 80μl of 0.2mol/l Na ₂ CO ₃ to stop the reaction ; Add 132 μl of PBS, 8 μL of sample solutions of different concentrations to the sample control group, incubate at 37°C for 15 minutes in a constant temperature incubator, add 20 μl of PNPG (0.625, 1.25, 2.5, 5, 10mmol/l) in a constant temperature incubator at 37°C After incubation for 15 min, add 80 μl of 0.2 mol/l Na ₂ CO ₃ . Then use a microplate reader to measure the OD value at 405nm, draw the inhibition kinetics curve (accompanying drawing 4), determine that the type of acarbose inhibition is competitive inhibition, and then calculate its _K value according to the competitive inhibition kinetic equation It is 1.14mg/ml.

The present invention studies the inhibition types of the positive drug acarbose, the ethanol extract of Camellia japonica seeds and their three different polar parts to inhibit α-glucosidase through enzyme inhibition kinetic experiments, and determines that acarbose is the competitive Sexual inhibition, its K _i value is 1.14mg/ml, the alcohol extract of Camellia japonica seeds and the ethyl acetate part, n-butanol part, and water layer part of the alcohol extract are different from acarbose, which are all non-competitive inhibition. It shows that it changes the structure of the active site by combining with sites other than the active site of α-glucosidase, so that the enzyme activity decreases, and its K _i values are 13.94, 131.02, 9.13, and 6.58 μg/ml, respectively. The invention provides a scientific basis for the research, development and application of camellia camellia seeds as alpha-glucosidase inhibitors.

Claims (4)

1. application of the Camellia nitidissima seed extract in alpha-glucosidase restrainer.

2. application of the Camellia nitidissima seed extract as described in claim 1 in alpha-glucosidase restrainer, which is characterized in that Camellia nitidissima seed extract is Camellia nitidissima seed alcohol extract, the ethyl acetate portion of Camellia nitidissima seed alcohol extract, Camellia nitidissima seed The n-butanol fraction of alcohol extract or/and the water portion of Camellia nitidissima seed alcohol extract.

3. application of the Camellia nitidissima seed as claimed in claim 1 or 2 in alpha-glucosidase restrainer, which is characterized in that institute It is four seasons Camellia nitidissima seed to state Camellia nitidissima seed.

4. application of the Camellia nitidissima seed extract as claimed in claim 2 in alpha-glucosidase restrainer, feature exist In the n-butanol portion of, Camellia nitidissima seed alcohol extract, the ethyl acetate portion of Camellia nitidissima seed alcohol extract, Camellia nitidissima seed alcohol extract Divide, the water portion of Camellia nitidissima seed alcohol extract is made for following preparation method：

(1) dry Camellia nitidissima seed pulverizer is taken to crush；

(2) the 95% ethyl alcohol heating and refluxing extraction 3 times measured with 3 times after petroleum ether degreasing, each 3h obtains golden flower tea extracts；

(3) golden flower tea extracts are filtered with filter paper, merging filtrate, are concentrated under reduced pressure into medicinal extract, are obtained Camellia nitidissima seed alcohol extract；

(4) after taking Camellia nitidissima seed alcohol extract that suitable quantity of water is added to be suspended, ethyl acetate, extracting n-butyl alcohol is successively used, ethyl acetate is obtained Partly, n-butanol fraction and raffinate aqueous fraction, are dried in vacuo after concentration, obtain ethyl acetate portion, the n-butanol portion of alcohol extract Divide, water portion.