CN111533680A - Folium Mori extract and folium Mori multicomponent mixture with blood sugar lowering effect prepared from the same - Google Patents

Abstract

The invention relates to a mulberry leaf extract and a mulberry leaf multi-component mixture with the hypoglycemic effect prepared from the mulberry leaf extract. Particularly provides a mulberry leaf DNJ extract which is prepared by the following method: extracting folium Mori with acidic ethanol solution; the concentration of the ethanol solution is 65-75%, and the volume-to-mass ratio of the ethanol solution to the mulberry leaves is 15-30 mL/g. The extraction method provided by the invention is simple to operate and mild in condition, and under the specific extraction condition of the invention, the yield and purity of DNJ in the obtained mulberry leaf DNJ extract are remarkably improved. The mulberry leaf DNJ extract is further used as a raw material, and is mixed with a mulberry leaf flavone extract and a mulberry leaf polysaccharide extract in a specific ratio to prepare a mulberry leaf multi-component mixture, so that the mulberry leaf multi-component mixture has an excellent blood sugar reducing effect and has a good application prospect in preparation of blood sugar reducing medicines.

Description

A kind of mulberry leaf extract and the prepared mulberry leaf multi-component mixture with hypoglycemic effect compound

technical field

The invention belongs to the field of traditional Chinese medicine extraction, in particular to a mulberry leaf extract and the prepared mulberry leaf multi-component mixture with hypoglycemic effect.

Background technique

Mulberry leaves are the leaves of Morus alba L., which contains essential amino acids, vitamins, minerals and other nutrients and flavonoids, alkaloids, polysaccharides, polyphenols and other biological Active ingredient. These include a polyhydroxyalkaloid, 1-deoxynojirimycin (DNJ), which binds to disaccharides such as maltase, sucrase, and lactase in the small intestine, preventing further breakdown of disaccharides Because the monosaccharide is absorbed by the body, it obviously inhibits the phenomenon of sharp rise in blood sugar after meals, and delays the occurrence and development of diabetes. In addition, according to the document "Multi-component synergistic hypoglycemic effect of mulberry leaves, Wang Deping, Jiang Yan, etc.", the mixture of mulberry leaf polysaccharides, flavonoids, and DNJ can reduce oxidative damage in diabetic mice, repair damaged islet cells, and improve the body's insulin. The resistance of effector cells to achieve a synergistic hypoglycemic effect. Therefore, effectively extracting DNJ from mulberry leaves has very important value for preparing medicines for treating diabetes.

At present, there are few research reports on the extraction process of DNJ. The existing extraction processes mainly include microwave-assisted method, dilute acid extraction method, and ultrasonic-assisted method. The document "Orthogonal optimization of 1-deoxynojirimycin acid-water extraction process from mulberry leaves" discloses a method for extracting DNJ from mulberry leaves with an acidic aqueous solution. The temperature is 55°C, the pH=3 hydrochloric acid solution, the material-liquid ratio is 1:20 (g/mL), the extraction time is 1.5h, the yield of DNJ is 0.062%, and the purity is 19.7%. The document "Optimization of Response Surface Method for Ultrasonic-assisted Extraction of 1-Deoxynojirimycin in Mulberry Leaves" discloses an ultrasonic-assisted extraction method using water as a solvent. The temperature was 75℃, the ultrasonic power was 350W, the liquid-material ratio was 16mL/g, and the maximum extraction amount of DNJ was 1.873mg/g.

The above extraction methods all use water as the extraction solvent, which increases the difficulty of removing the solvent during post-processing, and is not suitable for large-scale production; and the DNJ yield in the mulberry leaf extract obtained by the above method is relatively low, which increases the production cost. Therefore, there is an urgent need for an extraction process that can improve the extraction rate and purity of DNJ in mulberry leaf extracts.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a mulberry leaf DNJ extract and the prepared mulberry leaf multi-component mixture with hypoglycemic effect.

The invention provides a DNJ extract of mulberry leaves, which is prepared by the following method: taking mulberry leaves as raw materials and extracting with an acidic ethanol solution; the concentration of the ethanol solution is 65% to 75%, and the ethanol solution The volume-to-mass ratio of the solution to the mulberry leaves is 15-30 mL/g.

Further, the pH of the acidic ethanol solution is 1 to 3, the concentration of the ethanol solution is 70% to 71%, and the volume-to-mass ratio of the ethanol solution to the mulberry leaves is 20 to 26 mL/g, and the mulberry leaves are processed after being pulverized. 60 mesh mulberry leaf powder;

And/or, during the extraction, the extraction temperature is 45-65°C, preferably 50-61°C.

Further, the extraction method is a leaching method, which includes the following steps: using mulberry leaves as raw materials, extracting with an ethanol solution with a pH of 1-3; the concentration of the ethanol solution is 70%-71%, and the extraction temperature is 60- 61° C., the volume-to-mass ratio of the ethanol solution and the mulberry leaves is 20-21 mL/g, the extraction times are 1-2 times, and the extraction time is 148-150 min each time.

Further, the extraction method is an ultrasonic-assisted acidic ethanol extraction method, which includes the following steps: using mulberry leaves as raw materials, extracting with an ethanol solution with a pH of 2-3 under ultrasonic waves with a power of 320-322W; the ethanol solution The concentration is 70%, the extraction temperature is 50°C, the volume-to-mass ratio of the ethanol solution and the mulberry leaves is 20mL/g, the extraction times are 1-2 times, and the extraction time is 30-31min each time.

Further, the extraction method is a microwave-assisted acidic ethanol extraction method, which includes the following steps: using mulberry leaves as raw materials, extracting with an ethanol solution with a pH of 2-3 under a microwave with a power of 300-306W; the ethanol solution The concentration is 70%, the volume-to-mass ratio of the ethanol solution and the mulberry leaves is 25-26 mL/g, the extraction times are 1-2 times, and the extraction time is 70s each time.

Further, the method further includes a purification step, and the purification method is as follows: the product obtained by extraction is purified by a macroporous resin and/or an ion exchange resin; preferably, the macroporous resin is NKA-9 macroporous resin, and the ion The exchange resin is 001×7 cation exchange resin.

Further, when the macroporous resin was purified, the loading conditions were as follows: the loading concentration was 1.5 mg/mL, pH=8, and the loading flow rate was 1.5 BV/h; the elution conditions were as follows: the eluent was 75% ethanol, The eluent flow rate is 2BV/h;

And/or, when purified by ion exchange resin, the sample loading conditions are as follows: the sample loading concentration is 0.8 mg/mL, pH=2, and the sample loading flow rate is 1.5 BV/h; the elution conditions are as follows: the eluent is 1 mol/L Ammonia, the eluent flow rate is 1BV/h.

The present invention also provides a multi-component mixture of mulberry leaves, the multi-component mixture of mulberry leaves is obtained by mixing the above-mentioned DNJ extract of mulberry leaves, flavonoid extracts of mulberry leaves, and polysaccharide extracts of mulberry leaves; preferably, the In the multi-component mixture of mulberry leaves, the weight ratio of DNJ, flavonoids and polysaccharides is 1:6:8.

Further, the mulberry leaf flavonoid extract is prepared by the following method: using mulberry leaf as a raw material and extracting it with an ethanol solution; the concentration of the ethanol solution is 70%, the extraction temperature is 88°C, and the ethanol solution and The volume-to-mass ratio of mulberry leaves was 30mL/g, and the extraction time was 2.5h;

Or, the mulberry leaf flavonoid extract is prepared by the following method: taking mulberry leaf as raw material, extracting with ethanol solution under the ultrasonic wave with a power of 126W, and obtaining; the concentration of the ethanol solution is 51%, and the extraction temperature is At 70°C, the volume-to-mass ratio of ethanol solution and mulberry leaves was 25mL/g, and the extraction time was 36min;

And/or, the mulberry leaf polysaccharide extract is prepared by the following method: taking mulberry leaf as raw material, extracting with water, and obtaining; the extraction temperature is 82 ° C, and the volume-to-mass ratio of water and mulberry leaf is 17 mL/ g, the extraction time was 74 min.

Further, the preparation method of the mulberry leaf flavonoid extract further includes the following purification steps: the product obtained after extraction is purified by AB-8 macroporous resin; preferably, the loading concentration during purification is 3 mg/mL, and the loading flow rate is is 2BV/h; the eluent is 70% ethanol, and the eluent flow rate is 2BV/h;

And/or, the preparation method of the mulberry leaf polysaccharide extract further includes the following purification steps: precipitating the product obtained after extraction with an ethanol solution; 3 times, the precipitation temperature is 2～4℃, and the precipitation time is 12h;

Preferably, the preparation method of the mulberry leaf polysaccharide extract further includes the following purification steps: purifying the above-mentioned system after precipitation with ethanol solution through AB-8 macroporous resin; preferably, the loading concentration during purification is 0.7 mg/ mL, the sample flow rate was 3BV/h, the eluent was distilled water, and the eluent flow rate was 2BV/h.

Unless otherwise specified, the concentration of the mixed solution of the present invention is the volume concentration, for example, 70% ethanol is an ethanol solution with a volume concentration of 70%.

The present invention provides three methods for preparing mulberry leaf DNJ extracts. Experiments show that: (1) when acid ethanol is used for leaching, under the following specific extraction conditions: ethanol concentration of 70-71%, extraction time of 148-150min, The liquid-to-material ratio is 20-21 mL/g, and the extraction temperature is 60-61 °C, and the yield of DNJ in the obtained extract is as high as 2.939 mg/g; (2) When using ultrasonic-assisted acidic ethanol extraction, the following specific extraction Under the conditions: the ultrasonic power is 320-322W, the extraction time is 30-31min, the liquid-to-material ratio is 2mL/g, and the extraction temperature is 50℃, the yield of DNJ in the obtained extract is as high as 3.203mg/g; (3) Using microwave During the extraction with the auxiliary acidic ethanol extraction method, under the following specific extraction conditions: the microwave power is 300-306W, the extraction time is 70s, and the liquid-material ratio is 25-26mL/g, the yield of DNJ in the obtained extract is as high as 3.097mg/g g.

The invention also utilizes NKA-9 macroporous resin and 001×7 cation exchange resin to purify the obtained DNJ extract of mulberry leaves, and under specific purification conditions, the purity of DNJ in the extract is significantly improved.

In addition, the present invention uses the mulberry leaf DNJ extract obtained by the above method as a raw material, and the mulberry leaf flavonoid extract and the mulberry leaf polysaccharide extract are in a specific ratio (controlling the weight ratio of DNJ: flavonoid: polysaccharide to 1:6:8 ), the multi-component mixture of mulberry leaves obtained by mixing has excellent hypoglycemic effect, and has a good application prospect in the preparation of hypoglycemic drugs.

The extraction method provided by the invention has simple operation and mild conditions, and under the specific extraction conditions of the invention, the yield and purity of DNJ in the obtained DNJ extract of mulberry leaves are significantly improved. The mulberry leaf DNJ extract of the present invention has a good prospect in preparing a hypoglycemic drug containing only mulberry leaf DNJ or a mixture of mulberry leaf polysaccharides, flavonoids and DNJ as active ingredients.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Detailed ways

The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

NKA-9 macroporous resin was purchased from Hebei Cangzhou Baoen Adsorption Material Technology Company, with a specific surface area of 450-520 and an average pore size of 12.5-13.5nm.

White mulberry (Morus alba L.) mulberry leaves were washed and dried in the shade, pulverized with a universal pulverizer, and passed through a 60-mesh sieve to obtain mulberry leaf powder, which was stored in the dark for later use.

Embodiment 1 adopts acid ethanol extraction method to extract mulberry leaf DNJ

Weigh 2 g of mulberry leaf powder and place it in a flat-bottomed flask, add 42 mL of ethanol with pH=2 adjusted with hydrochloric acid to a concentration of 71%, extract at an extraction temperature of 61° C. for 148 min, and extract twice to obtain the mulberry leaf DNJ extract 1 of the present invention. . The DNJ yield was 2.939 (mg/g).

Embodiment 2 adopts ultrasonic-assisted acid ethanol extraction method to extract mulberry leaf DNJ

Weigh 2g of mulberry leaf powder and place it in a flat-bottomed flask, add 40 mL of pH=2 concentration adjusted with hydrochloric acid to be 70% ethanol, extract for 31 min at an extraction temperature of 50° C. and an ultrasonic power of 322 W, and extract twice to obtain the mulberry of the present invention. Leaf DNJ Extract 2. The DNJ yield was 3.203 (mg/g).

Example 3 Extraction of DNJ from mulberry leaves by microwave-assisted acid ethanol extraction

Weigh 2 g of mulberry leaf powder and place it in a flat-bottomed flask, add 52 mL of pH=2 adjusted with hydrochloric acid to 70% ethanol, extract for 70 s under the condition of 306 W microwave power, and extract twice to obtain the mulberry leaf DNJ extract 3 of the present invention. . The DNJ yield was 3.097 (mg/g).

Example 4 Purification of DNJ extract of mulberry leaves

The DNJ extract 1 of mulberry leaves (DNJ purity was 8.2%) prepared in Example 1 was used as the crude product, and was purified by NKA-9 macroporous resin. The sample loading conditions were as follows: the loading concentration was 1.5 mg/mL, pH=8, and the flow rate was 1.5 BV/h; the eluent was 75% ethanol, and the eluent flow rate was 2 BV/h.

After the above purification process, the DNJ purity of the obtained mulberry leaf DNJ extract was increased from 8.2% to 28.4%, and its purity was increased by 3.5 times.

Example 5 Further purification of mulberry leaf DNJ extract

The purified mulberry leaf DNJ extract (DNJ purity was 28.4%) prepared in Example 4 was used as the crude product, and was further purified by 001×7 cation exchange resin. The sample loading conditions were as follows: sample loading pH=2, concentration 0.8 mg/mL, flow rate 1.5 BV/h; eluent was 1 mol/L ammonia water, elution flow rate was 1 BV/h.

After the above-mentioned further purification process, the DNJ purity of the obtained mulberry leaf DNJ extract was increased from 28.4% to 70.4%, and its purity was further increased by 2.5 times.

Embodiment 6 adopts ethanol extraction method to extract mulberry leaf flavonoids

2 g of mulberry leaf powder was weighed and placed in a flat-bottomed flask, 60 mL of 70% ethanol was added, and the extraction temperature was 88° C. for 2.5 hours to obtain the mulberry leaf flavonoid extract 1 of the present invention. The yield of flavonoids was 4.16%.

Flavonoid yield = flavonoid mass in mulberry leaf flavonoid extract/mulberry leaf powder mass.

Embodiment 7 adopts ultrasonic-assisted ethanol extraction to extract mulberry leaf flavonoids

2 g of mulberry leaf powder was weighed and placed in a flat-bottomed flask, 50 mL of 51% ethanol was added, and the ultrasonic temperature was 70° C. and the ultrasonic power was 126 W and extracted for 36 min to obtain the mulberry leaf flavonoid extract 2 of the present invention. The yield of flavonoids was 3.79%.

Example 8 Purification of mulberry leaf flavonoid extract

The mulberry leaf flavonoid extract 1 (the flavonoid purity was 8.49%) prepared in Example 6 was used as the crude product, and AB-8 macroporous resin was used for purification. The sample loading conditions were as follows: the loading concentration was 3 mg/mL, and the flow rate was 2BV/h; the eluent was 70% ethanol, and the eluent flow rate was 2BV/h.

After the above purification process, the flavonoid purity of the mulberry leaf flavonoid extract was increased from 8.49% to 52.34%, and the purity was increased by 6.16 times.

HPLC method was used to analyze the components of the purified extract of the above AB-8 macroporous resin, and it was found that it mainly contained 5 flavonoids, and the content of each flavonoid was as follows: chlorogenic acid 86.50 mg/g, isoquercitrin 60.94mg/g, astragaloside 35.12mg/g, rutin 23.75mg/g, quercetin 14.87mg/g.

Embodiment 9 adopts water extraction ethanol precipitation method to extract mulberry leaf polysaccharide

(1) Preparation of mulberry leaf polysaccharide extract

The mulberry leaf powder was passed through a 40-mesh sieve, and petroleum ether was degreased. Weigh 2 g of mulberry leaf powder and place it in a round-bottomed flask, add distilled water, and extract under the conditions that the liquid-to-material ratio is 17:1 mL/g, the extraction temperature is 82 °C, the extraction time is 74 min, and the extraction is performed twice. The obtained extract was centrifuged, the supernatant was collected, and concentrated in vacuo for 3 times to obtain a mulberry leaf polysaccharide concentrate.

(2) Ethanol precipitation

Take the mulberry leaf polysaccharide concentrate obtained in step (1), add 3 times the volume of 95% ethanol solution, stand at low temperature (2～4°C) for precipitation for 12h, and separate by high-speed centrifugation (3000r/min, 15min) to obtain mulberry leaf polysaccharide Extract, polysaccharide yield was 14.24%.

Polysaccharide yield = polysaccharide mass in mulberry leaf polysaccharide extract/mulberry leaf powder mass.

Example 10 Purification of mulberry leaf polysaccharide

The ethanol-precipitated mulberry leaf polysaccharide extract prepared in Example 9 was used as the crude product, and AB-8 macroporous resin was used for purification. The sample loading conditions were as follows: the loading concentration was 0.7 mg/mL, and the flow rate was 3BV/h; the eluent was distilled water, and the eluent flow rate was 2BV/h.

After the above purification process, the polysaccharide purity of the obtained mulberry leaf polysaccharide extract was 32.6%, the polysaccharide yield was 19.3%, the polysaccharide retention rate was 56.5%, the deproteinization rate was 68.4%, and the decolorization rate was 50.8%.

Example 11 Preparation of multi-component mixture of mulberry leaves

The mulberry leaf DNJ extract prepared in any one of Examples 1 to 5, the mulberry leaf flavonoid extract prepared in any one of Examples 6 to 8, and the mulberry leaf prepared in any one of Examples 9 to 10 were used. The leaf polysaccharide extracts were mixed, and the weight ratio of DNJ: flavonoids: polysaccharide was controlled to be 1:6:8 to obtain a multi-component mixture of mulberry leaves.

The beneficial effects of the present invention are demonstrated below through experimental examples.

Experimental Example 1 Test of DNJ content and purity in DNJ extract of mulberry leaves

1.1 Preparation of reagents

(1) Preparation of DNJ standard solution: Accurately weigh 5mg of DNJ standard product in a beaker, add a small amount of 0.05mol/L hydrochloric acid to dissolve, add a certain amount of distilled water, transfer it into a 50mL volumetric flask, rinse the beaker three times and move it into the volumetric flask together with the glass rod. Volume to 50mL to obtain 0.1mg/mL DNJ standard solution.

(2) Preparation of wagner reagent: Weigh 1 g I ₂ and 10 g KI, add a small amount of distilled water to dissolve in a beaker, add a small amount of acetic acid to heat, dilute to 100 mL with distilled water, and store in a brown wide-mouth bottle for later use.

1.2 Determine the specific absorption wavelength

DNJ is a piperidine alkaloid, which forms a complex with Wagner's reagent under acidic conditions and can be detected under UV conditions. Take 5 mL of 0.1 mg/mL DNJ standard solution in a 25 mL volumetric flask, add a small amount of wagner reagent, and scan in the wavelength range of 240nm-420nm with an ultraviolet spectrophotometer to determine the specific absorption wavelength of DNJ.

1.3 Draw a standard curve

Pipette 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL of DNJ standard sample solution, respectively, into 5 test tubes with stoppers of different numbers, add an equal amount of iodine-potassium iodide reagent to each stoppered test tube as a chromogenic reagent, and add distilled water to make the sample solution The total volume is 5mL, another test tube with a stopper is added to the same amount of iodine-potassium iodide reagent, and distilled water is gradually added to adjust the solution volume to 5mL, as a blank control. The absorption value of each test tube solution was measured at the maximum absorption wavelength, and the standard curve was drawn with the DNJ concentration as the abscissa and the absorbance as the ordinate.

1.4 Determination of DNJ content

(1) Take a certain amount of sample solution in a 25mL volumetric flask, add wagner reagent, constant volume, shake, measure its absorbance at the maximum absorption wavelength, calculate the corresponding sample concentration from the standard curve, and calculate DNJ according to the following formula: Rate (i.e. DNJ content):

C: standard curve DNJ concentration (μg/mL)

n: dilution factor

V: sample volume (mL)

W: mulberry leaf mass (g)

1.5 Determination of DNJ purity

Take the mulberry leaf DNJ extract to be tested and calculate according to the following formula:

C: standard curve DNJ concentration (mg/mL)

n: dilution factor

V: sample volume (mL)

M: weight of the sample to be tested after drying (g)

1.6 Experimental results

Using the above method, the standard curve of DNJ was obtained: y=0.1641x-0.0013, R ² =0.9991. The DNJ yields in each example were calculated respectively, as shown in Table 1. The invention adopts the ultrasonic-assisted acidic ethanol method to extract DNJ, and the yield is 3.203 mg/g, which is 9.0% higher than that of the acidic ethanol method; The method yield increased by 5.4%.

As shown in Table 2, the DNJ purity of the DNJ extract of mulberry leaves purified by NKA-9 macroporous resin in Example 4 was increased from 8.2% to 28.4%, and the purity was increased by 3.5 times. Example 5 The DNJ purity of the DNJ extract of mulberry leaves after further purification by 001×7 cation exchange resin was increased from 28.4% to 70.4%, and the purity was further increased by 2.5 times.

Calculation results of DNJ yield of each embodiment of table 1

sample Extraction Method DNJ yield (mg/g) Example 1 Acidic ethanol extraction 2.939 Example 2 Ultrasonic-assisted acid ethanol extraction 3.203 Example 3 Microwave-assisted acidic ethanol extraction 3.097

Calculation result of DNJ purity of each embodiment of table 2

sample Extraction Method DNJ purity Example 1 Acidic ethanol extraction 8.2% Example 4 NKA-9 macroporous resin purification 28.4% Example 5 001×7 cation exchange resin purification 70.4%

Experimental example 2 Test of flavonoid content and purity in mulberry leaf flavonoid extract

2.1 Determination principle of aluminum nitrate-sodium nitrite method

After the flavonoids react with aluminum nitrate, the flavonoid-aluminum salt complex ions formed are yellow, and the color depth is proportional to the flavonoid content, which can be quantitatively determined.

2.2 Draw the standard curve

Weigh 10.00 mg of rutin standard, dissolve it in 65% ethanol solution, dilute to 50 mL, and prepare a 0.2 mg/mL rutin standard solution. Pipette 0, 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, and 4.0 mL of rutin standard solution into a 10 mL volumetric flask, add 0.5 mL of NaNO ₂ (1:20) solution, shake well for 6 min, add 0.5 mL of Al ( NO ₃ ) ₃ (1:10) solution, shake well for 6 minutes, add 4 mL of 1 mol/L NaOH solution, 65% ethanol solution to volume, shake well and place for 10-20 minutes, take the first tube of solution as blank control, at 510 nm Absorbance was measured at the wavelength. Draw the standard curve with the concentration of rutin standard solution as the abscissa and the absorbance as the ordinate.

2.3 Determination of flavonoids in mulberry leaves

Take 0.5mL of the sample solution to be tested in a volumetric flask, operate according to the method of drawing a standard curve, and calculate the flavonoid content (ie the flavonoid yield) according to the formula. The formula is as follows:

In the formula: C—standard curve flavonoid concentration (mg/mL)

n—dilution factor

V—sample volume (mL)

W—mulberry leaf mass (g)

2.4 Determination of the purity of mulberry leaf flavonoids

Take the mulberry leaf flavonoid extract to be tested, and calculate the purity of flavonoids according to the following formula.

In the formula: C—standard curve flavonoid concentration (mg/mL)

n—dilution factor

V—sample volume (mL)

M—weight of the extract after freeze-drying (g)

2.5 Experimental results

Using the above method, the standard curve of rutin was obtained: A(y)=10.949(x)+0.0138, R ² =0.9991. The flavonoid yield of the extract obtained in Example 6 was calculated to be 4.16%, and the flavonoid yield of the extract obtained in Example 7 was 3.79%.

In the flavonoid extract purified by AB-8 macroporous resin in Example 8, the purity of flavonoids increased from 8.49% to 52.34%, and the purity was increased by 6.16 times.

Experimental Example 3 Test of polysaccharide content and purity in mulberry leaf polysaccharide extract

3.1 Determination of polysaccharide retention rate

The polysaccharide content was determined by the phenol-sulfuric acid method, and the polysaccharide retention rate was calculated as follows.

3.2 Determination of deproteinization rate

The protein content was determined by the Coomassie brilliant blue method. Preparation of Coomassie Brilliant Blue G-250: Weigh 100 mg of Coomassie Brilliant Blue G-250, dissolve in 50 mL of 90% ethanol, add 100 mL of 85% (W/V) phosphoric acid, and finally dilute to 1000 mL with distilled water. To prepare 100μg/mL bovine serum albumin, take 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, and 0.6mL in test tubes, add 0.15M NaCl solution to 1mL, and add 5mL of pre-prepared test tubes. Mass brilliant blue solution, after standing for 10min, measure the absorbance at 595nm, and draw a standard curve. Calculate the deproteinization rate according to the following formula.

3.3 Determination of decolorization rate

The mulberry leaf crude polysaccharide solution with a concentration of 1% was prepared, and the visible-ultraviolet full-wavelength scanning was performed on it. The results showed that the solution had no maximum absorption wavelength. According to the principle of complementary color, the color of the solution is the complementary color that it absorbs light. Since the polysaccharide solution is orange-yellow before and after decolorization and after dilution, the solution mainly absorbs visible light in the blue band. Therefore, the wavelength of 450 nm in the center of this band is selected as the detection wavelength, and the absorbance of the solution is measured. And calculate the decolorization rate according to the following formula.

3.4 Determination of the purity of mulberry leaf polysaccharide after purification

Take the mulberry leaf polysaccharide extract to be tested, and calculate the polysaccharide purity according to the following formula:

In the formula: C—standard curve polysaccharide concentration (mg/mL)

n—dilution factor

V—sample volume (mL)

M—weight of the extract after freeze-drying (g)

3.5 Experimental results

After the AB-8 macroporous resin purification process, the polysaccharide purity of the mulberry leaf polysaccharide extract obtained in Example 10 was 32.6%, the polysaccharide yield was 19.3%, the polysaccharide retention rate was 56.5%, the deproteinization rate was 68.4%, and the decolorization rate was 50.8%. .

Experimental Example 4 Process Screening of Extracting Mulberry Leaf DNJ with Acidic Ethanol

1. Experimental method

With reference to the process of Example 1, the ethanol concentration, extraction time, liquid-to-material ratio, and extraction temperature were changed according to Table 3, and the DNJ extract of mulberry leaves was extracted respectively, and the DNJ yield under each process was calculated according to the method of Experimental Example 1. Each process condition was repeated 5 times, and the average value was taken.

2. Experimental results

The results are shown in Table 3. It can be seen that when using acidic ethanol to extract mulberry leaves, when the ethanol concentration is 70-71%, the extraction time is 148-150 min, the liquid-material ratio is 20-21 mL/g, and the extraction temperature is 60- At 61°C, the yield of DNJ in the obtained extract was the highest.

Table 3 Technological screening experiment results of extracting DNJ from mulberry leaves by acid ethanol method

Experimental Example 5. Screening of the extraction of DNJ from mulberry leaves by ultrasonic-assisted acid ethanol extraction

1. Experimental method

With reference to the process of Example 2, the ultrasonic power, extraction time, liquid-to-material ratio, and extraction temperature were changed according to Table 4, and the mulberry leaf DNJ extract was extracted respectively, and the DNJ yield under each process was calculated according to the method of Experimental Example 1.

2. Experimental results

The results are shown in Table 4. It can be seen that when the mulberry leaves are extracted by ultrasonic-assisted acid ethanol extraction, when the ultrasonic power is 320-322 W, the extraction time is 30-31 min, the liquid-to-material ratio is 20 mL/g, and the extraction temperature is At 50℃, the yield of DNJ in the obtained extract was the highest.

Table 4 The results of the process screening experiment for the extraction of DNJ from mulberry leaves by ultrasonic-assisted acid ethanol extraction

Experimental Example 6 Process Screening of Extraction of Mulberry Leaf DNJ by Microwave-Assisted Acidic Ethanol Extraction

1. Experimental method

With reference to the process of Example 3, the microwave power, extraction time, and liquid-to-material ratio were changed according to Table 5, and the DNJ extract of mulberry leaves was extracted respectively, and the DNJ yield under each process was calculated according to the method of Experimental Example 1.

2. Experimental results

The results are shown in Table 5. It can be seen that when the mulberry leaves are extracted by the microwave-assisted acidic ethanol extraction method, when the microwave power is 300-306 W, the extraction time is 70 s, and the liquid-material ratio is 25-26 mL/g, the obtained extraction Among them, the yield of DNJ was the highest.

Table 5 The results of the process screening experiment of extracting DNJ from mulberry leaves by microwave-assisted acid ethanol extraction

Experimental Example 7 Characterization of hypoglycemic effect of multi-component mixture of mulberry leaves

1. Experimental method

Experimental drug: the multi-component mixture of mulberry leaves prepared in Example 11; the positive drug is metformin.

Establishment of diabetic rat model: healthy male SD rats were taken, fed with high-fat diet for 6 weeks, and then intraperitoneally injected with streptozotocin solution (30 mg/kg·BW) at one time. The 11.1mmol/L index confirmed that the diabetic rat model was successful.

Modeled SD male rats were fed (gavage) with mulberry leaf multi-component mixture (100 mg/kg·d·BW) or positive drug (185 mg/kg·d·BW) for a total of 6 weeks of treatment. Modeled SD male rats fed the same volume of normal saline were used as the model control group, and normal SD rats fed the same volume of normal saline were used as the blank control group.

After 6 weeks of treatment, test the blood sugar level and insulin level of the rat; test the total cholesterol, triglyceride, LDL-C and HDL-C in the serum of the rat; test the antioxidant enzyme in the serum of the rat: superoxide dismutase Activity of SOD, glutathione peroxidase GSH-Px, catalase CAT, lipid peroxidized malondialdehyde MDA level; test adipocytokines in rat serum: free fatty acid, TNF-α, lean The serum levels of urea nitrogen and creatinine were measured, and the levels of urea nitrogen and creatinine in the serum of the rats were measured; the liver tissue, pancreas tissue and kidney tissue sections of the rats were observed.

2. Experimental results

After 6 weeks, the blood glucose levels and insulin levels of the rats in the mulberry leaf multi-component mixture group were only 26.06% and 53.30% of those in the model control group (P<0.05), which were comparable to those in the positive drug metformin control group (P>0.05). Good hypoglycemic effect.

After 6 weeks, the total cholesterol, triglyceride, LDL-C and HDL-C in the serum of the rats in the mulberry leaf multi-component mixture group were 79.10%, 49.50%, 74.24% and 1.33 times that of the model control group, respectively (P< 0.05), and there was no significant difference with the blank control group and the positive control group (P>0.05), indicating that the multi-component mixture of mulberry leaves can effectively improve the lipid metabolism disorder in diabetic rats.

After 6 weeks, the antioxidant enzymes in the serum of rats in the mulberry leaf multi-component mixture group: the activities of superoxide dismutase SOD, glutathione peroxidase GSH-Px, and catalase CAT were the same as those in the model group. 1.26 times, 1.34 times, and 1.36 times (P<0.05), and the lipid peroxidation MDA level was 84.06% of the model group (P<0.05), and there was no significant difference with the positive control group (P>0.05). . The multi-component mixture of mulberry leaves improves the oxidative stress state in diabetic rats by increasing the activity of antioxidant enzymes and inhibiting lipid peroxidation in diabetic rats.

After 6 weeks, the levels of adipocytokines: free fatty acids, TNF-α, leptin and inflammatory factor C-reactive protein in the serum of the rats in the mulberry leaf multi-component mixture group decreased to 68.63%, 88.25%, 84.15% of those in the model control group. and 41.03% (P<0.05), the level of adipocytokines adiponectin increased to 1.16 times that of the model group (P<0.05), and there was no significant difference between the two groups (P>0.05). Mulberry leaf extract can effectively down-regulate the abnormally high expression of adipocytokines and C-reactive protein in diabetic rats, improve insulin resistance, and activate the expression of adiponectin to enhance insulin sensitivity.

After 6 weeks, the serum levels of urea nitrogen and creatinine in the mulberry leaf multi-component mixture group were 71.31% and 62.23% of the model group, respectively (P<0.05), and were close to the positive control group, with no statistical difference. (P>0.05).

After 6 weeks, the hepatic fat vacuoles of the rats in the mulberry leaf multi-component mixture group were significantly reduced, the cells were arranged in an orderly manner, the morphology was relatively intact, and the hepatic lobule structure was intact. Islet cells have clear borders and regular arrangement, and the number of islet cells increases. The glomerular volume was slightly increased, the renal tubules did not show hyalinization, the thickening of the glomerular and renal tubular basement membranes was significantly reduced, and the pathological state of the renal tissue improved. It is suggested that mulberry leaf extract can improve liver steatosis caused by high-fat diet combined with intraperitoneal injection of streptozotocin in diabetic rats, repair damaged pancreatic islet cells, and reduce kidney damage caused by persistent hyperglycemia.

Therefore, the multi-component mixture of mulberry leaves prepared by the present invention can effectively reduce the oxidative stress damage of liver, pancreas and other tissues by increasing the activity of antioxidant enzymes in the body of diabetic rats, improving the state of insulin resistance, and enhancing insulin sensitivity. It can improve the blood sugar level of diabetic rats, and improve the lipid metabolism disorder in rats, with excellent hypoglycemic effect.

To sum up, the present invention provides a DNJ extract of mulberry leaves, which is obtained by using mulberry leaves as raw materials and extracted with an acidic ethanol solution; the concentration of the ethanol solution is 65% to 75%, and the volume of the ethanol solution and mulberry leaves is The mass ratio is 15 to 30 mL/g. In this method, the extraction method can be leaching, ultrasonic-assisted acidic ethanol extraction or microwave-assisted acidic ethanol extraction. Under the specific extraction conditions of the present invention, the yield and purity of DNJ in the obtained mulberry leaf DNJ extract are significantly improved. The mulberry leaf DNJ extract of the present invention has a good prospect in preparing a hypoglycemic drug containing only mulberry leaf DNJ or a mixture of mulberry leaf polysaccharides, flavonoids and DNJ as active ingredients.

In particular, the mulberry leaf multi-component mixture provided by the present invention is composed of mulberry leaf DNJ extract, mulberry leaf flavonoid extract and mulberry leaf polysaccharide extract in a ratio of 1:6:8, which can improve antioxidant enzymes in diabetic rats by increasing the It can effectively reduce the oxidative stress damage of the liver, pancreas and other tissues, improve the state of insulin resistance, and enhance insulin sensitivity, thereby effectively reducing the blood sugar level in diabetic rats, and improving the lipid metabolism disorder in rats, with excellent hypoglycemic effect, application bright future.

Claims (10)

1. A mulberry leaf DNJ extract is characterized in that: the preparation method comprises the following steps: extracting folium Mori with acidic ethanol solution; the concentration of the ethanol solution is 65-75%, and the volume-to-mass ratio of the ethanol solution to the mulberry leaves is 15-30 mL/g.

2. The extract as claimed in claim 1, wherein: the pH value of the acidic ethanol solution is 1-3, the concentration of the ethanol solution is 70-71%, the volume-mass ratio of the ethanol solution to the mulberry leaves is 20-26 mL/g, and the mulberry leaves are mulberry leaf powder which is crushed and then sieved by a 60-mesh sieve;

and/or the extraction temperature is 45-65 ℃ during extraction, and preferably 50-61 ℃.

3. Extract according to claim 1 or 2, characterized in that: the extraction method is an extraction method and comprises the following steps: extracting mulberry leaves serving as a raw material by using an ethanol solution with the pH value of 1-3; the concentration of the ethanol solution is 70% -71%, the extraction temperature is 60-61 ℃, the volume-mass ratio of the ethanol solution to the mulberry leaves is 20-21 mL/g, the extraction times are 1-2 times, and the extraction time is 148-150 min each time.

4. Extract according to claim 1 or 2, characterized in that: the extraction method is an ultrasonic-assisted acidic ethanol extraction method, and comprises the following steps: taking mulberry leaves as a raw material, and extracting the mulberry leaves by using an ethanol solution with the pH of 2-3 under the ultrasonic wave with the power of 320-322W; the concentration of the ethanol solution is 70%, the extraction temperature is 50 ℃, the volume-to-mass ratio of the ethanol solution to the mulberry leaves is 20mL/g, the extraction times are 1-2 times, and the extraction time is 30-31 min each time.

5. Extract according to claim 1 or 2, characterized in that: the extraction method is a microwave-assisted acidic ethanol extraction method and comprises the following steps: taking mulberry leaves as a raw material, and extracting the mulberry leaves by using an ethanol solution with the pH of 2-3 under the microwave with the power of 300-306W; the concentration of the ethanol solution is 70%, the volume-mass ratio of the ethanol solution to the mulberry leaves is 25-26 mL/g, the extraction times are 1-2 times, and the extraction time is 70s each time.

6. The extract according to any one of claims 1 to 5, wherein: the method also comprises a purification step, wherein the purification method comprises the following steps: purifying the extracted product by macroporous resin and/or ion exchange resin; preferably, the macroporous resin is NKA-9 macroporous resin, and the ion exchange resin is 001 × 7 cation exchange resin.

7. The extract as claimed in claim 6, wherein: when the macroporous resin is used for purification, the loading conditions are as follows: the sample concentration is 1.5mg/mL, the pH value is 8, and the sample flow rate is 1.5 BV/h; the elution conditions were as follows: the eluent is 75% ethanol, and the flow rate of the eluent is 2 BV/h;

and/or, when the purified product is subjected to ion exchange resin, the loading conditions are as follows: the sample concentration is 0.8mg/mL, the pH value is 2, and the sample flow rate is 1.5 BV/h; the elution conditions were as follows: the eluent is 1mol/L ammonia water, and the flow rate of the eluent is 1 BV/h.

8. A mulberry leaf multi-component mixture is characterized in that: the mulberry leaf multicomponent mixture is obtained by mixing the mulberry leaf DNJ extract as claimed in any one of claims 1 to 7 with a mulberry leaf flavone extract and a mulberry leaf polysaccharide extract; preferably, in the mulberry leaf multi-component mixture, the weight ratio of DNJ, flavone and polysaccharide is 1:6: 8.

9. The multi-component mulberry leaf mixture of claim 8, wherein: the mulberry leaf flavone extract is prepared by the following method: extracting folium Mori with ethanol solution; the concentration of the ethanol solution is 70%, the extraction temperature is 88 ℃, the volume-mass ratio of the ethanol solution to the mulberry leaves is 30mL/g, and the extraction time is 2.5 h;

or, the mulberry leaf flavone extract is prepared by the following method: extracting folium Mori as raw material with ethanol solution under ultrasonic wave of power 126W; the concentration of the ethanol solution is 51%, the extraction temperature is 70 ℃, the volume-to-mass ratio of the ethanol solution to the mulberry leaves is 25mL/g, and the extraction time is 36 min;

and/or the mulberry leaf polysaccharide extract is prepared by the following method: extracting folium Mori with water; the extraction temperature is 82 ℃, the volume-to-mass ratio of water to mulberry leaves is 17mL/g, and the extraction time is 74 min.

10. The multi-component mulberry leaf mixture of claim 9, wherein: the preparation method of the mulberry leaf flavone extract also comprises the following purification steps: purifying the extracted product by AB-8 macroporous resin; preferably, the sample loading concentration during purification is 3mg/mL, and the sample loading flow rate is 2 BV/h; the eluent is 70% ethanol, and the flow rate of the eluent is 2 BV/h;

and/or the preparation method of the mulberry leaf polysaccharide extract further comprises the following purification steps: precipitating the product obtained after extraction with ethanol solution; preferably, the concentration of the ethanol solution is 95%, the volume of the ethanol solution is 3 times of that of the polysaccharide extract, the precipitation temperature is 2-4 ℃, and the precipitation time is 12 hours;

preferably, the preparation method of the mulberry leaf polysaccharide extract further comprises the following purification steps: purifying the system precipitated by the ethanol solution by AB-8 macroporous resin; preferably, the sample concentration during purification is 0.7mg/mL, the sample flow rate is 3BV/h, the eluent is distilled water, and the eluent flow rate is 2 BV/h.