CN111603518B

CN111603518B - Application of black tiger extract and macleaya cordata extract in preparation of whitening or blood sugar reducing medicines

Info

Publication number: CN111603518B
Application number: CN202010597409.1A
Authority: CN
Inventors: 阳祁斌; 张胜; 陈伟波; 姚烨林
Original assignee: Central South University of Forestry and Technology
Current assignee: Guangdong Chengbei Biotechnology Co.,Ltd.
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2021-12-31
Anticipated expiration: 2040-06-28
Also published as: CN111603518A; CN113559034B; CN113559034A

Abstract

The invention belongs to the field of biological medicines, finds that the black tiger extract has the function of inhibiting alpha-glucosidase activity and tyrosinase activity, and the macleaya cordata extract has the function of inhibiting the alpha-glucosidase activity, and particularly provides application of the two substances in preparing medicines for whitening and reducing blood sugar.

Description

Application of black tiger extract and macleaya cordata extract in preparation of whitening or blood sugar reducing medicines

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a black tiger extract and a macleaya cordata extract in preparation of medicines for whitening or reducing blood sugar.

Background

Diabetes Mellitus (DM) is a disease caused by the failure of insulin to meet the normal demand of the human body or the resistance of the body to the action of insulin, and belongs to a common endocrine disease. Diabetes is a metabolic disease with multiple causes and is characterized by chronic hyperglycemia, sugar, fat, protein metabolic disorder and the like. The main characteristic is elevated blood sugar. Diabetics can reach as high as 4.63 billion worldwide, with type 2 diabetes accounting for about 90%, and only 1.16 billion in China accounting for 11.6% of the total population. There are many therapeutic mechanisms for diabetes, among which delaying the absorption of glucose in the intestinal tract is one of the main therapeutic mechanisms, and alpha-glucosidase inhibitors such as Acarbose (Acarbose) and Voglibose (Voglibose) are mainly used. Alpha-glucosidase can react with 4-p-nitrotoluene-alpha-glucopyranoside (PNPG) to generate nitrobenzene, the nitrobenzene shows yellow color under alkaline condition, the maximum absorption is about 405nm, and the OD value of the final reactant is in positive correlation with the activity of the alpha-glucosidase in a certain concentration range. The alpha-glucosidase can decompose the sugar with low polymerization degree into glucose, and the blood sugar rise of the diabetic can be slowed down by inhibiting the activity of the alpha-glucosidase, so that the blood sugar is stable, and the purpose of reducing the blood sugar is achieved.

Tyrosine is a functional catalytic substrate of tyrosinase monophenolase, and is a main raw material for finally forming brown melanin and eumelanin. Tyrosinase is closely related to the production of melanin. The most important class of enzymes in the overall process of melanin formation is tyrosinase. The amount of melanin formation is determined by the activity of tyrosinase. Tyrosine is a functional catalytic substrate of tyrosinase and is a main raw material for finally forming brown melanin and melanoidin. The product obtained after the reaction of tyrosinase and tyrosine has maximum absorption at 475m, and the absorbance of the product at 475m can be measured by ultraviolet instrument detection. In the current cosmetic market, most whitening products take tyrosinase activity inhibitors as main ingredients, and with the development of research, new compounds are continuously discovered at a higher speed every year.

The natural medicine has the characteristic of high safety, so that the search of the components for inhibiting the activities of alpha-glucosidase and tyrosinase from the natural medicine is a research hotspot in the field of related medical chemistry. So far, few reports on natural alpha-glucosidase activity inhibitors and tyrosinase activity inhibitors exist, and various traditional Chinese medicinal materials with the functions of reducing blood sugar and whitening skin are in urgent need of development and research.

Disclosure of Invention

Aiming at the defects of the prior art, the further research finds that the black tiger extract has the function of inhibiting the activity of alpha-glucosidase and tyrosinase, and the macleaya cordata extract has the function of inhibiting the activity of alpha-glucosidase, so that a new basis is provided for the development of hypoglycemic drugs and health care products or whitening skin care products.

One of the technical schemes of the invention is as follows:

the invention provides an application of a black tiger extract in preparing an alpha-glucosidase inhibitor.

In a preferable scheme, the black tiger extract is applied to preparing the hypoglycemic medicament.

Preferably, the black cutworm extract is an alcohol extract and/or an aqueous extract of black cutworm, and the solid-to-liquid ratio is 1: 50-200, further preferred solid to liquid ratio is 1: 100.

in a further preferable scheme, the black tiger leaf extract in the black tiger extract has the best effect, and the application of the black tiger leaf extract in preparing the hypoglycemic medicament is preferable.

The second technical scheme of the invention is as follows:

the invention provides an application of black tiger leaf extract in preparation of tyrosinase inhibitors.

In a preferable scheme, the black tiger leaf extract is applied to the preparation of whitening drugs.

Preferably, the black cutworm leaf extract is an alcohol extract and/or an aqueous extract of black cutworm leaves, and the solid-to-liquid ratio is 1: 50-200, further preferred solid to liquid ratio is 1: 100.

the black tiger extract can be a commercially available existing product. In a further preferred scheme, the alcohol extraction method of the black tiger alcohol extract comprises the following steps: crushing the medicinal materials, sieving the medicinal materials by a sieve of 80-100 meshes, drying the medicinal materials at 40-50 ℃ to constant weight, refluxing the medicinal materials for 30-40min by using petroleum ether at 35-45 ℃, removing fat in the raw materials, recovering the petroleum ether to obtain each sample powder, weighing 1.0000g of the sample powder, adding 100mL-200mL of 75-95% ethanol for reflux extraction for 1-2h, taking a proper amount of extracting solution for centrifugation, and taking the supernatant as a sample alcohol extract;

in a further preferred scheme, the water extraction method of the black tiger aqueous extract comprises the following steps: pulverizing the medicinal materials, sieving with a 80-100 mesh sieve, drying at 40-50 deg.C to constant weight, refluxing with petroleum ether at 35-45 deg.C for 30-40min, removing lipid from the raw materials, recovering petroleum ether to obtain various sample powders, accurately weighing 1.0000g of the sample powders, adding 100-200 mL of distilled water, reflux-extracting for 1-2h, centrifuging appropriate amount of extractive solution, and collecting supernatant as sample water extract.

The third technical scheme of the invention is as follows:

the invention provides an application of macleaya cordata extract in preparation of an alpha-glucosidase inhibitor.

In a preferable scheme, the macleaya cordata extract is applied to preparing the hypoglycemic drug.

Preferably, the macleaya cordata extract is a macleaya cordata alcohol extract and/or a macleaya cordata water extract, and the solid-to-liquid ratio is 1: 50-200, further preferred solid to liquid ratio is 1: 100.

the macleaya cordata extract can be a commercially available existing product. In a further preferred scheme, the extraction method of the macleaya cordata alcohol extract comprises the following steps: crushing the medicinal materials, sieving the medicinal materials by a sieve of 80-100 meshes, drying the medicinal materials at 40-50 ℃ to constant weight, refluxing the medicinal materials for 30-40min by using petroleum ether at 35-45 ℃, removing fat in the raw materials, recovering the petroleum ether to obtain each sample powder, weighing 1.0000g of the sample powder, adding 100mL-200mL of 75-95% ethanol for reflux extraction for 1-2h, taking a proper amount of extracting solution for centrifugation, and taking the supernatant as a sample alcohol extract;

in a further preferred scheme, the extraction method of the macleaya cordata aqueous extract comprises the following steps: pulverizing the medicinal materials, sieving with a 80-100 mesh sieve, drying at 40-50 deg.C to constant weight, refluxing with petroleum ether at 35-45 deg.C for 30-40min, removing lipid from the raw materials, recovering petroleum ether to obtain various sample powders, accurately weighing 1.0000g of the sample powders, adding 100-200 mL of distilled water, reflux-extracting for 1-2h, centrifuging appropriate amount of extractive solution, and collecting supernatant as sample water extract.

Further explanation and description:

the tyrosinase and the alpha-glucosidase are prepared by using a PBS solution.

The extract of the invention is in concentration, the tyrosinase solution is 0.2-0.8 mg/mL; the alpha-glucosidase solution is 0.5-5U/mL in terms of activity. In a preferable scheme, the tyrosinase solution is 0.4mg/mL, and the alpha-glucosidase is 1U/mL.

The extract of the invention has a mixing ratio of the alcohol and/or water extract to the tyrosinase and alpha-glucosidase solution of 1: 0.5-5. Preferably, the mixing ratio of the alcohol and/or water extract to the tyrosinase and alpha-glucosidase solution is 1: 1.

compared with the prior art, the invention has the beneficial effects that:

by the experiments of inhibiting the activity of the alpha-glucosidase by using the alcohol extract and the water extract of medicinal materials, the fact that the water extract of the black cutworm leaves, the alcohol extract of the pericarp and the aqueous solution and the alcohol solution of the macleaya cordata have high inhibition rates on the alpha-glucosidase on the premise that the solid-to-liquid ratio is 1:100 is known, wherein the inhibition rate of the water extract of the black cutworm leaves is 75.51%, the inhibition rate of the alcohol extract of the pericarp is 64.90%, the inhibition effect on the activity of the alpha-glucosidase is most obvious, and the alpha-glucosidase is probably good in hypoglycemic activity. The invention provides a direction basis for the development of new hypoglycemic drugs and provides a data basis for the development and research of natural alpha-glucosidase inhibitors.

Through experiments, the method for inhibiting the tyrosinase activity experiment is optimized, and the optimal reaction condition of the tyrosinase activity inhibition experiment is that the temperature is 37 ℃, the reaction time is 1h, the concentration of tyrosinase is 0.04mg/ml, and the concentration of tyrosine is 0.4 mg/ml. The tyrosinase inhibition effect of different natural components is tested under the condition, and the results show that: the alcohol extract of the black cutworm pericarp has higher inhibition rate on tyrosinase, and the significance of further research and development is prompted. The invention provides a direction basis for the development of natural whitening skin care products and provides a data basis for the development and research of natural tyrosinase inhibitors.

Drawings

FIG. 1 is a Lineweave-Burkf double reciprocal plot;

FIG. 2 is a graph of the slope versus the second reciprocal of acarbose concentration;

FIG. 3 is an absorbance curve of tyrosinase for various reaction times;

FIG. 4 is a tyrosinase dynamics linear equation;

FIG. 5 is a graph of liquid chromatography-mass spectrometry analysis of Kadsura coccinea;

FIG. 6 is a structural formula of chemical components of the leaf part of black tiger identified by LC-MS.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not to be construed as limiting the invention in any way.

Unless otherwise specified, the herbs and reagents used in the present invention are all commercially available.

The detailed preparation process of the black tiger alcohol extract comprises the following steps: respectively placing leaves, stems and fruit peels of the kadsura coccinea into a traditional Chinese medicine grinder for grinding, sieving with a 80-mesh sieve, placing into a drying box for drying at 40 ℃ to constant weight, refluxing for 30min at 40 ℃ by using petroleum ether, removing fat in the raw materials, recovering the petroleum ether, placing the medicinal materials in a ventilated place, accurately weighing 1.0000g of sample powder (leaves/stems/fruit peels) after the raw materials are completely dried, adding 100mL of 95% ethanol, slightly boiling to prevent gelatinization, performing reflux extraction for 1h, placing a proper amount of extracting solution into a centrifuge, setting the rotating speed to 6000r/min, centrifuging for 3min, and taking the supernatant as a sample solution; because the ethanol in the alcohol extract can inhibit the enzyme, the alcohol extract is pretreated by the following specific operations: putting 30mL of alcohol extract into a 100mL round-bottom flask, performing rotary evaporation on the flask in a rotary evaporator until no liquid drops in a condenser, taking down the flask, adding a proper amount of water in batches, washing the flask with water, combining the washing solutions, adding distilled water to make up to 30mL, and filtering with a 0.45-micrometer filter membrane.

The detailed preparation process of the black tiger aqueous extract comprises the following steps: respectively placing leaves, stems and fruit peels of the kadsura coccinea into a traditional Chinese medicine grinder for grinding, sieving with a 80-mesh sieve, placing into a drying box for drying at 40 ℃ to constant weight, refluxing for 30min at 40 ℃ by using petroleum ether, removing fat in the raw materials, recovering the petroleum ether, placing the medicinal materials in a ventilated place, accurately weighing 1.0000g of sample powder (leaves/stems/fruit peels) after the raw materials are completely dried, adding 100mL of distilled water, slightly boiling to prevent gelatinization, performing reflux extraction for 1h, placing a proper amount of extracting solution into a centrifuge, setting the rotating speed to 6000r/min, centrifuging for 3min, and taking the supernatant as a sample solution; the darker water extract will affect absorbance, so the colored water extract is lyophilized at a constant volume, and then distilled water is added at the same volume to eliminate pigment interference in the water extract.

The detailed preparation process of the macleaya cordata alcohol extract comprises the following steps: accurately weighing commercially available Macleaya cordata extract 1.0000g, adding 100mL 95% ethanol for dissolving, placing appropriate amount of dissolving solution in a centrifuge, setting the rotation speed at 5000r/min, centrifuging for 3min, and taking supernatant as sample solution; because the ethanol in the alcohol extract can inhibit the enzyme, the alcohol extract is pretreated by the following specific operations: and (3) completely evaporating ethanol from a certain amount of ethanol extract by using a rotary evaporator, drying in a drying box at 40 ℃, and dissolving by using distilled water with the same volume to obtain an ethanol extract sample solution.

Detailed preparation process of macleaya cordata water extract: 1.0000g of macleaya cordata extract purchased from the market is accurately weighed, 100mL of distilled water is added for dissolving, a proper amount of dissolving solution is taken and put in a centrifuge, the rotating speed is set to 5000r/min, the mixture is centrifuged for 3min, and the supernatant is taken as a sample solution. The darker water extract will affect absorbance, so the colored water extract is lyophilized at a constant volume, and then distilled water is added at the same volume to eliminate pigment interference in the water extract.

Example 1

1. Alpha-glucosidase activity inhibition assay

1.1. Enzyme inhibition kinetics experiment

PBS buffer solution was prepared by adding 1.361g of potassium dihydrogen phosphate (0.1mol/L potassium dihydrogen phosphate) to 100ml of double distilled water. Solution B (0.1mo/L disodium hydrogen phosphate) 1.78g disodium hydrogen phosphate, and double distilled water to 100m 1. Mixing solution A and solution B at a ratio of 1: 1.

Preparing an alpha-glucosidase mother liquor: 1.0mg of alpha-glucosidase powder with the activity unit of 100U/mg is dissolved in 1.0mL of PBS buffer solution to obtain 100U/mL of alpha-glucosidase mother liquor. 0.10mL of the mother solution was dissolved in 10mL of PBS buffer solution to prepare a 2U/mL solution of α -glucosidase.

Preparing a positive control drug acarbose standard solution: dissolving 0.3398g of acarbose in a 10ml volume bottle by PBS to constant volume to prepare a mother solution of 50.0mmol/L, and performing gradient dilution to obtain standard solutions of 5.0mmol/L, 12.5mmol/L, 25mmol/L, 37.5mmol/L and 50 mmol/L.

Preparation of standard solution of substrate 4-p-nitrophenyl-alpha-D-glucopyranoside (PNPG) (M: 301.25): 0.1505g of PNPG was dissolved in 1.654ml of dimethyl sulfoxide (1.6 ml by pipette and 0.054ml by pipette), and 2.646ml of a phosphate buffer (2.6 ml by pipette and 0.046ml by pipette) having a pH of 6.8 was added to prepare a 116mmol/L PNPG stock solution, which was diluted to a concentration of 1mmol/L, 2mmol/L, 3mmol/L, 5mmol/L and 7.5mmol/L by pipette.

Inhibition kinetics analysis: the same substrate concentration was used for the color reaction with different inhibitor concentrations, and three parallel experiments were performed for each inhibitor concentration. Adding 70uL of phosphate buffer solution into a 96-hole enzyme label plate, then adding 10uL of acarbose solution and 10uL of alpha-glucosidase solution, finally adding 10uL of PNPG solution, shaking for 3min in an enzyme label instrument to uniformly mix, reacting for 15min at 37 ℃, and measuring the absorbance of generated p-nitrophenol every 1min (the rate of generating nitrophenol is the same every 15 min). The initial reaction rate is the absorbance of the nitrophenol formed divided by the corresponding time of formation. Judging the inhibition type of acarbose according to a Lineweave-Burkf double reciprocal mapping method. The abscissa is 1/[ S ] and the ordinate is 1/v, where [ S ] is the substrate PNPG concentration, v is the reaction rate and R is the inhibitor concentration.

1.2. Positive group experiment:

accurately weighing 0.1000g of acarbose, fixing the volume of the acarbose in a 100ml volumetric flask by using deionized water to prepare a solution with the concentration of 1000 mu g/ml, diluting the solution by using a pipette in proportion to obtain a required concentration gradient (0 mu g/ml, 20 mu g/ml, 40 mu g/ml, 60 mu g/ml, 80 mu g/ml and 100 mu g/ml), and performing an alpha-glucosidase activity inhibition experiment by using the concentration gradient.

1.3. Results of the experiment

The experimental data of the enzyme kinetics are shown in table 1:

wherein 1/[ S ] is reciprocal of each substrate concentration, R is inhibitor concentration, 1/v is reciprocal of reaction rate, 1 is reciprocal of reaction rate in different combinations

Table 1 Table of response rate countdown for different combinations

From the double reciprocal diagram, the Lineweave-Burkf double reciprocal diagram of acarbose has the characteristics of competitive inhibition and non-competitive double reciprocal diagram, and therefore, the inhibition type of acarbose is inferred to be composed of competitive inhibition and non-competitive inhibition. The reason why acarbose can inhibit the activity of α -glucosidase is that acarbose can be bound to the active site of α -glucosidase.

And then taking each slope in the Lineweave-Burkf reciprocal diagram as an ordinate and taking the acarbose concentration as an abscissa, and carrying out linear fitting, as shown in FIG. 2.

The acarbose inhibition constant obtained from the intersection of the fitted line and the horizontal axis was 44.66.

The data of the inhibition rate of acarbose to alpha-glucosidase at each concentration and the experimental results are shown in table 2:

TABLE 2 acarbose Activity inhibition results

Tab2 Acarbose activity inhibition results

Acarbose is a α -glucosidase inhibitor and is often used as a treatment for diabetes. According to the data, the acarbose has an obvious inhibition effect on the alpha-glucosidase, the inhibition rate of the acarbose is positively correlated with the concentration of the acarbose, and after the acarbose reaches a certain concentration, the inhibition rate is stable and finally keeps unchanged basically along with the increase of the concentration of the acarbose to a certain value, so that the method for screening the hypoglycemic activity of the traditional Chinese medicine is feasible by using the model for inhibiting the alpha-glucosidase, which is established by taking the acarbose as a positive control substance.

2. Screening experiment for black tiger extract and macleaya cordata extract for inhibiting activity of alpha-glucosidase

2.1. Preparing standard concentration experiment reagent

Preparation of 4-p-nitrophenyl-alpha-D-glucopyranoside (PNPG) solution: 0.1505g of PNPG was accurately weighed and dissolved in 1.654mL of dimethyl sulfoxide, and after complete dissolution, 2.646mL of PBS buffer was added to prepare a substrate solution of 0.116 mol/L. Preparing an alpha-glucosidase mother liquor: 1.0mg of alpha-glucosidase powder with the activity unit of 100U/mg is dissolved in 1.0mL of PBS buffer solution to obtain 100U/mL of alpha-glucosidase mother liquor. 0.10mL of the mother solution was dissolved in 5mL of PBS buffer solution to prepare a 2U/mL solution of α -glucosidase.

PNPG color reaction

Respectively adding water, an extracting solution, a PBS solution and an alpha-glucosidase solution into a 96-well plate of an enzyme labeling instrument, incubating for 10min at 37 ℃, adding a substrate PNPG solution, and reacting for 10min at 37 ℃; then 100. mu.L of 1.0mol/L Na was added₂CO₃Terminating the reaction by the solution; after the reaction was terminated, the OD of the product was measured at 405nm, 3 times in parallel, and the average value was taken. According to equation 1:

the alpha-glucosidase inhibition rate was calculated. Wherein:

i is the alpha-glucosidase inhibition rate; a. the₁Is a blank group, A₂For test group, A₃Is a background group. A. the₁-A₃All are measured od values.

The loading order and volume are shown in table 3:

TABLE 3 sample adding table for experimental groups

Tab3 Sample addition table of experimental group

2.3. Results and analysis of the experiments

2.3.1. The results of the measurement of the α -glucosidase inhibitory activity of macleaya cordata extracts are shown in table 4:

TABLE 4 inhibition of the Activity of Macleaya cordata extracts

Tab4 The results of activity inhibition of Macleaya cordata(willd.)R.Br

From the data in table 4, it is clear that the aqueous solution and the alcoholic solution of macleaya cordata have α -glucosidase inhibition rates exceeding 20%, have high inhibitory activity, and have potential value for the development of hypoglycemic agents.

2.3.2. The results of the measurement of the α -glucosidase inhibitory activity of the black tiger extract are shown in table 5:

TABLE 5 Activity inhibition results of Kadsura coccinea extract

Tab5 The results of activity inhibition of Kadsura coccinea(Lem.)A.C.Smith

As can be seen from the data in Table 5, the water extract and the alcohol extract of Kadsura coccinea have high inhibitory effect on alpha-glucosidase, and the relationship between the extracts of different parts of Kadsura coccinea and the inhibitory rate of alpha-glucosidase is as follows: leaf water extraction, peel alcohol extraction, leaf alcohol extraction, stem water extraction, stem alcohol extraction and peel water extraction. The inhibition rate of the black cutworm leaf water extract on enzyme is up to 75.51%, and the inhibition rate of the alcohol extract of the pericarp on alpha-glucosidase is up to 64.90%, so that the black cutworm leaf water extract has good hypoglycemic activity and has extremely high value in the aspect of developing new hypoglycemic drugs.

Example 2

1. Tyrosinase activity inhibition assay

1.1. Establishment of tyrosinase inhibition experiment method

1.1.1. Preparation of experimental reagent

The L-tyrosine solution is prepared by dissolving 0.04g tyrosine in 100ml PBS buffer solution.

The preparation of tyrosinase solution comprises dissolving 0.004g of tyrosinase in 100ml of PBS buffer.

The vitamin C solution is prepared by dissolving 0.08g vitamin C in 100ml deionized water.

Preparing vitamin C with different concentration gradients, namely adding deionized water into the prepared 0.8mg/ml vitamin C solution, and diluting the solution in a gradient manner to obtain the required concentration (0.2mg/ml, 0.4mg/ml and 0.6 mg/ml).

1.1.2. Ultraviolet spectrophotometric detection method

Taking 2.0ml of PBS2, 0.5ml of L-tyrosine solution and 2.0ml of tyrosinase solution, shaking up in a test tube, placing in a constant-temperature water bath, keeping at 37 ℃, heating in water bath for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h and 3.0h respectively, detecting the reaction degree of tyrosinase under the condition of 475nm, keeping the gradient condition of each concentration unchanged, repeating the experiment, and determining the optimal reaction condition of the enzyme according to the change of the enzyme reaction. By equation 2:

I₁＝[1-(A₃-A₄)/(A₁-A₂)]×100％ (2)

and calculating the inhibition rate. Wherein A1(2.0ml PBS +0.5ml L-tyrosinase solution +2.0ml tyrosinase solution) control and A2(2.5ml PBS +2.0ml tyrosinase solution) were blanks, and A3 and A4 were absorbance of samples with different concentration gradients and vitamin C.

1.1.3. Enzyme-linked immunosorbent assay method

In order to control the reaction time and other variables and reduce the experimental error, the following experiment adopts a microplate reader detection method:

a1(80uL PBS +50uL L-tyrosine solution +80uL tyrosinase solution) and A2(130uL PBS +80uL tyrosinase solution) were used as blank controls. A3(60uL PBS +50uL L-tyrosine solution +80uL tyrosinase solution +20uL vitamin C) and A4(110uL PBS +80uL tyrosinase solution +20uL vitamin C) are different concentration gradients (0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml) of vitamin C absorbance, each set having three wells. Setting the incubation condition of the microplate reader to be 475m, setting the vibration mode to be single, setting the temperature to be 37 ℃, setting the reaction time to be 1h, then incubating, scanning and detecting under the wavelength of 475m, recording data, and calculating the inhibition rate by using a formula 1.

1.2. Results and analysis of the experiments

1.2.1. Absorbance and reaction time length data results

The experimentally determined absorbance data of tyrosinase for different reaction durations are shown in table 6:

TABLE 6 absorbance of tyrosinase for different reaction times

Tab6 Absorbance of tyrosinase at different reaction times

The standard curve of fig. 3 was plotted against the experimental data of table 6.

As can be seen from Table 6 and FIG. 3, the reaction rate becomes significantly slower after 1h, during which the reactants are fully reacted, so that the optimum reaction time for the experiment was found to be 1 h.

1.2.2. Effect of different concentrations of vitamin C on tyrosinase Activity

The data of the inhibition rates of vitamin C to tyrosinase at different concentrations measured in the experiment are shown in Table 7:

TABLE 7 inhibitory Effect of vitamin C on tyrosinase Activity at various concentrations

Tab6 Effects of different concentrations of vitamin C on inhibition of tyrosinase activity

The enzyme kinetic linearity diagram is shown in FIG. 4. As shown in fig. 4, the greater the concentration of vitamin C, the greater the inhibition of tyrosinase activity. The slope of the regression line is about 97, the y axis intersects 5.56, and the linear equation is that y is 5.66+97 x. Therefore, tyrosinase activity is positively correlated with inhibitor concentration.

2. Screening experiment for tyrosinase activity inhibition by black tiger extract and macleaya cordata extract

2.1. Preparing standard concentration experiment reagent

Preparing a vitamin C solution: 0.08g of vitamin C is dissolved in 100ml of deionized water.

2.2. Drip plate setting

TABLE 8 ratio of experimental reagents

Tab8 Reagent ratio

The ratio of the reagents required by the experimental group is shown in table 8, the standard reagent is dripped into the porous plate by the pipette according to the ratio, and three experimental items and three comparison items are set for each group in order to reduce experimental errors. And setting the incubation condition of the microplate reader as a single vibration mode, setting the temperature to be 37 ℃, setting the reaction time to be 1h, then incubating, scanning and detecting under the condition that the wavelength is 475m, and recording data. Absorbance of different extract samples and vitamin C (0.8mg/ml), each group was set with three wells, and the average was taken;

2.3. reaction and detection conditions

Setting incubation conditions of the microplate reader to be 475m, setting a vibration mode to be single time, setting the temperature to be 37 ℃, setting the reaction time to be 1h, then incubating, scanning and detecting under the wavelength of 475m, and recording data;

2.4. calculation of tyrosinase inhibition

Data are recorded and processed, and the inhibition ratio is calculated by formula 2.

2.5. Results and analysis of the experiments

2.5.1. The inhibitory effect of black tiger extract and macleaya cordata extract on tyrosinase activity is shown in table 9:

TABLE 9 tyrosinase inhibitory Activity assay results

Tab9 Determination results of tyrosinase inhibitory activity

2.5.2. Summary of the Experimental results

From the data, it can be seen that: the aqueous extract and the alcohol extract of the black tiger leaves, stems and fruit peels have the inhibition effect on the tyrosinase activity, and the relationship between the extracting solution of each part of the black tiger and the tyrosinase inhibition rate is as follows: leaf water extraction, leaf alcohol extraction, peel water extraction, stem alcohol extraction and peel alcohol extraction. The inhibition rate of the water extract of each part on tyrosinase is higher than that of the corresponding alcohol extract, and the water extraction mode is more suitable for the purpose. Particularly, the inhibition rate of the aqueous extract of the black cutworm leaves on tyrosinase is up to 77.35%, the inhibition activity is obvious, and the black cutworm leaf extract has high potential in the development of whitening products.

Example 3

In order to determine the chemical components with the blood sugar reducing and whitening functional activities in the black tiger extract, further analysis and identification are carried out.

1. Analysis of chemical composition of black tiger leaf

According to the fingerprint spectrum of each part of the black tiger, the detected peak of the black tiger leaves under the liquid phase condition is the most, and the chemical components are the most complicated, so the leaf alcohol extract is used for sample injection LC-MS (liquid chromatography-mass spectrometer) analysis.

1.1. Experimental methods

Analyzing the chemical components of the black tiger leaves by using a liquid chromatography-mass spectrometer. And (3) passing the alcohol extract of the black cutworm leaves through a 0.22 mu m microporous filter membrane, and performing LC-MS analysis on a sample.

Chromatographic conditions are as follows: gradient eluting with Eclipse Plus C18 chromatographic column (250mm × 4.6mm, 5 μm) with acetonitrile (A) -0.1% formic acid (B) as mobile phase for 0-5 min, 12% A; 5-10 min, 12% A → 20% A; 20 percent in 10 to 20minA; 20-25 min, 20% A → 30% A; 25-30 min, 30% A; 30-35 min, 30% A → 80% A; 35-60 min, 80% A → 90% A, sample injection amount of 10uL, flow rate of 0.8 mL/min^-1. Mass spectrum conditions: the HESI ion source has a sheath gas rate of 40mL/min, an auxiliary gas rate of 10mL/min, a spray voltage of 4.0kV positive ions and 3.2kV negative ions, a capillary temperature of 300 ℃, an S-lens of 50 percent, a scanning mode of Fullms/dd-ms2 top5 and a scanning range of 100-1000 m/z.

1.2. Composition of results

The liquid chromatography-mass spectrometry analysis chart of the black tiger leaf alcohol extract is shown in fig. 5, and 8 components are identified in total by relative molecular mass, namely rutin, luteolin 4' -O glucoside, 3,5, 7-trihydroxy-3 ',4',5' -trimethoxy flavone, uridine 5' -triphosphate, BQ-123, (-) -11-9-carboxyl-delta-9-tetrahydrocannabinol, 3-hydroxyl-6, 2',3' -trimethoxy flavone and (3-methylbutane-2-oxyl) -hexadecyloxy biphenyl silane. See table 10. Wherein the content of rutin and 3-hydroxy-6, 2',3' -trimethoxy flavone is the highest. The structural formula of the 8 components is shown in figure 6.

TABLE 10 Mass Spectrometry of chemical Components contained in leaves

Tab10 Mass Spectrometric Analysis of Chemical Components in leaf

While the present application has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes can be made without departing from the true spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, and method to the objective, spirit and scope of the present application. All such modifications are intended to be included within the scope of the claims of this application.

Claims

1. The black tiger extract is an alcohol extract and/or an aqueous extract of black tiger, and the solid-to-liquid ratio is 1: 50-200, the alcohol extraction method comprises the following steps: pulverizing the medicinal materials, sieving with a 80-100 mesh sieve, drying at 40-50 deg.C to constant weight, refluxing with petroleum ether at 35-45 deg.C for 30-40min, removing lipid from the raw materials, recovering petroleum ether, placing the medicinal materials in a ventilated place, weighing 1.0000g of sample powder after the raw materials are completely dried, adding 100-200 mL of 75-95% ethanol, reflux-extracting for 1-2h, centrifuging an appropriate amount of the extract, and collecting the supernatant which is the extract of Kadsura coccinea (L.) Gaertn;

the extraction method of the water extraction comprises the following steps: pulverizing the medicinal materials, sieving with a 80-100 mesh sieve, drying at 40-50 deg.C to constant weight, refluxing with petroleum ether at 35-45 deg.C for 30-40min, removing fat from the raw materials, recovering petroleum ether, placing the medicinal materials in a ventilated place, accurately weighing 1.0000g of sample powder after the raw materials are completely dried, adding 100-200 mL of distilled water, reflux-extracting for 1-2h, centrifuging appropriate amount of extractive solution, and collecting the supernatant as aqueous extract of Kadsura coccinea;

the medicinal material is leaf, stem or pericarp of black tiger.