Veronica indica extract with blood sugar reducing effect as well as preparation method and application of Veronica indica extract

Abstract

The invention discloses a waltheria indica extract with a blood sugar reducing effect as well as a preparation method and application of the waltheria indica extract. The preparation method comprises the following steps: uniformly mixing and stirring waltheria indica powder and an ethanol water solution, and performing ultrasonic-assisted extraction to obtain a waltheria indica extracting solution; performing macroporous resin column chromatography on the waltheria indica extracting solution, collecting eluent, and performing freeze drying to obtain the waltheria indica extract with the blood sugar reducing effect. The preparation method is simple and convenient to operate, excellent in yield, green, environment-friendly and suitable for industrial production and large-scale extraction. The invention discovers that the waltheria indica extract has a blood sugar reducing function for the first time, and is suitable for preparing the blood sugar reducing preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, processing and application, and particularly relates to a snakewort extract with blood sugar lowering effect, and a preparation method and application thereof. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by elevated blood sugar levels, usually due to insufficient insulin secretion or impaired insulin action. Diabetes is an increasingly serious public health problem worldwide, with an increasing incidence and complications that can lead to cardiovascular disease, kidney disease, vision problems, etc. Therefore, the development of effective hypoglycemic drugs is of great significance for improving the quality of life of diabetic patients and preventing complications.

[0003] The blood sugar lowering active ingredients from natural plants have attracted widespread attention. Compared with the side effects of traditional drugs, natural plant active ingredients not only have good blood sugar lowering effects, but also have higher safety. Therefore, the development of natural plant-derived blood sugar lowering products has become one of the effective measures to lower blood sugar.

[0004] Waltheria indica L. is a plant of the genus Waltheria in the family Sterculiaceae. It is also known as Heta grass, carpet, and grass sycamore. It is mainly distributed in Guangdong, Fujian, Hainan, Guangxi and other places in my country. It is a Chinese medicinal material with roots and stems used as medicine. "Chinese Herbs" and "National Compilation of Chinese Herbal Medicines" record that Waltheria indica has the effects of anti-inflammatory, heat-clearing and detoxifying, and removing dampness and expelling wind. At present, the research reports on the activity of Waltheria indica mainly focus on its anti-inflammatory, antibacterial, and whitening effects. There are no research reports on the hypoglycemic effect of Waltheria indica extract. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a snakewort extract having a blood sugar lowering effect.

[0006] Another object of the present invention is to provide a Herba Coleopterae extract having blood sugar lowering efficacy obtained by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above-mentioned Herba Coleopterae extract having the effect of lowering blood sugar.

[0008] The purpose of the present invention is achieved by the following technical solution: A method for preparing a Herba Coleopterae extract having a blood sugar lowering effect, comprising the following steps:

[0009] (1) mixing the snakewort powder and the ethanol aqueous solution and stirring them uniformly, and performing ultrasonic-assisted extraction to obtain the snakewort extract;

[0010] (2) performing macroporous resin column chromatography on the snakewort extract obtained in step (1), collecting the eluate, and freeze-drying the eluate to obtain a snakewort extract having a blood sugar lowering effect.

[0011] The snakewort powder in step (1) is derived from the whole plant or any part of the snakewort plant.

[0012] The concentration of the ethanol aqueous solution in step (1) is preferably 30 to 70% by volume; more preferably 50% by volume.

[0013] The amount of the ethanol aqueous solution in step (1) is preferably calculated based on a solid-liquid ratio of 15 to 35 mL / g; more preferably, based on a solid-liquid ratio of 20 mL / g.

[0014] The mixing time in step (1) is preferably 30 to 60 minutes, more preferably 30 minutes.

[0015] The extraction conditions described in step (1) are preferably: ultrasonic power of 200-400W, ultrasonic temperature of 40-60°C, and ultrasonic time of 1-2h; more preferably, ultrasonic power of 300W, ultrasonic temperature of 50°C, and ultrasonic time of 1h.

[0016] The macroporous resin described in step (2) is at least one of AB-8, NKA-9, D101, DM301, ADS-17, X-5 and S-8; more preferably NKA-9.

[0017] The column chromatography step described in step (2) is preferably as follows:

[0018] 1) Activate macroporous resin;

[0019] 2) loading the activated macroporous resin filler into a chromatography column;

[0020] 3) loading the extract of the snakewort into a chromatography column filled with a macroporous resin filler for adsorption;

[0021] 4) After the adsorption is completed, the macroporous resin is eluted with an eluent and the eluent is collected.

[0022] The specific steps of activation described in step 1) are preferably: soaking in anhydrous ethanol for 10 to 18 hours, then washing with distilled water until there is no ethanol smell; soaking in a hydrochloric acid solution with a concentration of 3 to 6% v / v for 4 to 10 hours, then washing with distilled water until the pH is neutral; soaking in a sodium hydroxide solution with a concentration of 3 to 6% w / v for 4 to 10 hours, then washing with distilled water until the pH is neutral; more preferably, soaking in anhydrous ethanol for 12 hours, then washing with distilled water until there is no ethanol smell; soaking in a hydrochloric acid solution with a concentration of 5% v / v for 6 hours, then washing with distilled water until the pH is neutral; soaking in a sodium hydroxide solution with a concentration of 5% w / v for 6 hours, then washing with distilled water until the pH is neutral.

[0023] The diameter-to-height ratio of the macroporous resin filler column in step 2) is 1:5 to 1:15, and more preferably 1:7.5.

[0024] The adsorption flow rate of the sample loading in step 3) is 1-3 mL / min; more preferably 1 mL / min.

[0025] The eluent in step 4) is an ethanol solution; preferably, the concentration is 80-100% ethanol solution; more preferably, the concentration is 90% (v / v) ethanol solution.

[0026] The elution flow rate in step 4) is 1-3 mL / min, preferably 1 mL / min.

[0027] The extent of collection in step 4) is to collect until the eluate becomes clear and colorless, then stop collecting.

[0028] The freeze-drying conditions described in step (2) are: pre-freeze the snakewort extract at -80°C for 12 hours, then quickly transfer it to a freeze dryer and freeze-dry it for 2-3 days.

[0029] A Herba Coleopterae extract with blood sugar lowering effect is prepared by the method.

[0030] The application of the Coleus chinensis extract with hypoglycemic effect in the preparation of hypoglycemic preparations.

[0031] The preparation can be used in the field of food or medicine.

[0032] Compared with the prior art, the present invention has the following advantages and effects:

[0033] (1) The present invention first discovered that the extract of Coleus chinensis has the function of lowering blood sugar. The present invention adopts ultrasonic assisted extraction method and column chromatography method to extract the Coleus chinensis extract with the effect of lowering blood sugar. The extraction process is simple to operate, has good yield, is green and environmentally friendly, and is suitable for large-scale extraction in industrial production.

[0034] (2) The Herba Coleopterae extract prepared by the method of the present invention has a good blood sugar lowering effect and can be used as a blood sugar lowering substance in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a flow chart of the preparation of the Herba Codonopsis pilosulae extract. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0037] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention can be obtained from commercial sources.

[0038] Example 1

[0039] A method for preparing a snakewort extract, Figure 1 As shown, the specific steps include:

[0040] (1) The crushed whole plant of Coleus chinensis was mixed with 50% (v / v) ethanol aqueous solution at a liquid-to-solid ratio of 20:1 (mL / g) for 30 minutes, then placed in an ultrasonic cleaner at 50°C and ultrasonically extracted at 300W for 60 minutes, centrifuged, and the supernatant was taken and concentrated by rotary evaporation to remove ethanol to obtain a concentrated solution. The turbidity of the concentrated solution was adjusted to 55 NTU and pH 4 by a scattered light turbidity meter to obtain a crude extract of Coleus chinensis.

[0041] (2) Activation of resin: first soak the resin in anhydrous ethanol for 12 hours, and wash it with distilled water until there is no ethanol smell; then soak the resin in 5% v / v hydrochloric acid solution for 6 hours, and wash it with distilled water until the pH is neutral; then soak it in 5% w / v sodium hydroxide solution for 6 hours, and wash it with distilled water until the pH is neutral; wherein, the method of solid-liquid separation is suction filtration.

[0042] (3) The prepared crude extract of Coleus chinensis was added to the activated NKA-9, AB-8, D101, and DM301 macroporous resin chromatography columns (diameter-to-height ratio of 1:7.5) in the same volume, and adsorbed at a flow rate of 1 mL / min. After the loading was completed, 90% (v / v) ethanol aqueous solution was used for elution at a flow rate of 1 mL / min, and the eluate was collected until it was clear and colorless. The eluate was then pre-frozen at -80°C for 12 hours, and then quickly transferred to a freeze dryer and freeze-dried for 2-3 days to obtain four Coleus chinensis extracts. Corresponding to NKA-9, AB-8, D101, and DM301 macroporous resins, the Coleus chinensis extracts were named Coleus chinensis extract A, Coleus chinensis extract B, Coleus chinensis extract C, and Coleus chinensis extract D.

[0043] Example 2

[0044] This example is an in vitro hypoglycemic activity evaluation experiment of the Herba Codonopsis pilosulae extract obtained in Example 1, and the steps are as follows:

[0045] (1) Effect on α-amylase activity

[0046] 1mL of different concentrations of snakewort sample or acarbose and 1mL of α-amylase (3U / mL, dissolved in pH 6.8, 0.1mol / L phosphate buffer solution) were mixed evenly, incubated at room temperature for 5min, and then 1mL of soluble starch solution (2mg / mL) was added, incubated at 37℃ for 20min, and 5mL of 3,5-dinitrosalicylic acid (DNS) colorimetric agent (0.4mol / L NaOH solution containing 12% potassium sodium tartrate and 1% 3,5-dinitrosalicylic acid) was added to terminate the reaction, and the enzyme was inactivated in a boiling water bath for 5min, and then cooled to room temperature after removal, and the absorbance was measured at 540nm. Distilled water was used instead of α-amylase as the blank control group, distilled water was used instead of the sample as the negative control group, and acarbose was used instead of the sample as the positive control group. The α-amylase inhibition rate was calculated according to the following formula:

[0047] α-Amylase activity inhibition rate (%) = [1-(negative group-sample group) / blank group]×100.

[0048] Experimental results using IC 50 Value representation.

[0049] (2) Effect on α-glucosidase activity

[0050] 1mL of different concentrations of snakewort sample or acarbose and 1mL of α-glucosidase (0.05U / mL, dissolved in pH 6.8, 0.1mol / L phosphate buffer solution) were mixed evenly, incubated at room temperature for 5min, and then 1mL of PNPG (0.5mmol / L, dissolved in pH 6.8, 0.1mol / L phosphate buffer solution) was added, incubated at 37°C for 30min, and the reaction was terminated with 2mL of 0.2mol / L Na2CO3, cooled at room temperature for 5min, and the absorbance was measured at 405nm. Distilled water was used instead of α-glucosidase as the blank control group, distilled water was used instead of the sample as the negative control group, and acarbose was used instead of the sample as the positive control group. The α-glucosidase inhibition rate was calculated according to the following formula:

[0051] α-glucosidase activity inhibition rate (%) = [1-(negative group-sample group) / blank group] × 100.

[0052] Experimental results using IC 50 Value representation.

[0053] The experimental results are shown in Table 1. As shown in Table 1, the half-inhibitory concentration of the positive control acarbose on α-amylase activity is 2.98 mg / mL, while the half-inhibitory concentration of the snakewort extract A on α-amylase activity is 1.24 mg / mL, which is lower than acarbose; the half-inhibitory concentration of acarbose on α-glucosidase activity is 2.43 mg / mL, while the half-inhibitory concentration of the snakewort extract A on α-glucosidase activity is 1.67 mg / mL, which is lower than acarbose, indicating that the snakewort extract has a good hypoglycemic effect.

[0054] Table 1 Effects of different resins on the hypoglycemic activity of Coptis chinensis extract in vitro

[0055]

[0056] Example 3

[0057] Since the in vitro hypoglycemic effect of Coleus chinensis A is the best, this example is an in vivo hypoglycemic activity evaluation experiment of the Coleus chinensis extract obtained in Example 1, and the steps are as follows:

[0058] (1) Effects on blood glucose in diabetic mice

[0059] Forty 4-week-old mice (purchased from Guangdong Medical Experimental Animal Center) were randomly divided into 4 groups, 10 mice in each group, namely blank control group, model group, positive control group, and snakewort extract group. Except for the blank group, the remaining groups were weighed after fasting for 14 hours without water deprivation, and the mice were intraperitoneally injected with a 10 mg / mL streptozotocin (STZ) solution at a dose of 120 mg / kg. After injection, the mice were free to eat and drink for 5 consecutive days to obtain diabetic mice. After 5 days, the model group and the blank control group were gavaged with normal saline (100 mg / kg·BW), the positive control group was gavaged with acarbose (20 mg / kg·BW), and the snakewort extract group was gavaged with snakewort extract (20 mg / kg·BW). The drug was administered for 4 weeks, fasting for 8 hours without water deprivation, weighing, removing the eyeballs to collect blood, and measuring blood sugar levels.

[0060] The experimental results are shown in Table 2. As shown in Table 2, compared with the blank group, the fasting blood glucose values ​​of the model group mice increased significantly after 4 weeks of administration, indicating that the diabetic mice model was successfully established. Compared with the model group, the fasting blood glucose values ​​of the positive control group and the snakewort extract group mice decreased significantly after 4 weeks of administration, indicating that the snakewort extract has a hypoglycemic effect and has a certain therapeutic effect on diabetic mice.

[0061] Table 2 Effect of snakewort extract on blood glucose levels in diabetic mice

[0062]

[0063] (2) Effect on glucose tolerance in diabetic mice

[0064] After 4 weeks of administration, blood glucose levels were measured after fasting for 6 hours (i.e., 0 hours). Then, the snakewort extract group and the acarbose group were given the corresponding drugs snakewort extract and acarbose, respectively, and the control group and the model group were given the same volume of normal saline. After 15 minutes, each group of mice was orally given 3g / kg of starch solution, and then blood was collected from the tail tip at 1 and 2 hours to measure blood glucose levels. The area under the blood glucose curve (AUC) was calculated as follows:

[0065] A=0.5×(X+2Y+Z)

[0066] In the formula: A is the area under the blood glucose curve, mmol / L; X is the blood glucose value at 0h, mmol / L; Y is the blood glucose value at 1h, mmol / L; Z is the blood glucose value at 2h, mmol / L.

[0067] The experimental results are shown in Table 3. As shown in the table, compared with the model group, the blood glucose values ​​and AUC of the mice in the snakewort extract group and the positive control group were significantly reduced 1h and 2h after starch gavage, and there was no significant difference in AUC in the snakewort extract group compared with the positive control group. It can be seen that the snakewort extract can reduce the glucose tolerance value of diabetic mice and improve insulin resistance.

[0068] Table 3 Effect of snakewort extract on glucose tolerance in diabetic mice

[0069]

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a snakewort extract having a hypoglycemic effect, characterized in that The steps include: (1) mixing the snakewort powder and the ethanol aqueous solution and stirring them uniformly, and performing ultrasonic-assisted extraction to obtain the snakewort extract; (2) performing macroporous resin column chromatography on the snakewort extract obtained in step (1), collecting the eluate, and freeze-drying the eluate to obtain a snakewort extract having a blood sugar lowering effect.

2. The preparation method according to claim 1, characterized in that: The concentration of the ethanol aqueous solution in step (1) is 30 to 70% by volume; The amount of the ethanol aqueous solution described in step (1) is calculated based on a solid-liquid ratio of 15 to 35 mL / g.

3. The preparation method according to claim 1, characterized in that: The mixing time in step (1) is 30 to 60 minutes; The extraction conditions described in step (1) are: ultrasonic power of 200-400W, ultrasonic temperature of 40-60°C, and ultrasonic time of 1-2h.

4. The preparation method according to claim 1, characterized in that: The macroporous resin in step (2) is at least one of AB-8, NKA-9, D101, DM301, ADS-17, X-5 and S-8; The steps of column chromatography described in step (2) are as follows: 1) Activate macroporous resin; 2) loading the activated macroporous resin filler into a chromatography column; 3) loading the extract of the snakewort into a chromatography column filled with a macroporous resin filler for adsorption; 4) After the adsorption is completed, the macroporous resin is eluted with an eluent and the eluent is collected.

5. The preparation method according to claim 4, characterized in that: The specific steps of activation described in step 1) are: soaking in anhydrous ethanol for 10 to 18 hours and then washing with distilled water until there is no ethanol smell; soaking in a hydrochloric acid solution with a concentration of 3 to 6% v / v for 4 to 10 hours and then washing with distilled water until the pH is neutral; soaking in a sodium hydroxide solution with a concentration of 3 to 6% w / v for 4 to 10 hours and then washing with distilled water until the pH is neutral.

6. The preparation method according to claim 4, characterized in that: The diameter-to-height ratio of the macroporous resin filler column in step 2) is 1:5 to 1:15; The adsorption flow rate of the sample loading in step 3) is 1 to 3 mL / min; The eluent described in step 4) is an ethanol solution; The elution flow rate in step 4) is 1-3 mL / min; The extent of collection in step 4) is to collect until the eluate becomes clear and colorless, then stop collecting.

7. The preparation method according to claim 6, characterized in that: The ethanol solution is an ethanol solution with a concentration of 80-100%.

8. The preparation method according to claim 1, characterized in that: The freeze-drying conditions described in step (2) are: pre-freeze the snakewort extract at -80°C for 12 hours, then quickly transfer it to a freeze dryer and freeze-dry it for 2-3 days.

9. A Herba Coleopterae extract having blood sugar lowering effect, characterized in that: It is prepared by the method according to any one of claims 1 to 8.

10. Use of the Herba Coleopterae Extract with hypoglycemic effect according to claim 9 in the preparation of hypoglycemic preparations.

Images