Preparation method of ethanol extract of polypodium vulgare and application thereof

Abstract

The application discloses a preparation method of ethanol extract of Drynaria Rhizome and application thereof. The method comprises the following steps: crushing and sieving Drynaria Rhizome, adding ethanol, reflux extraction, combining filtrates, vacuum concentration and drying, and thus the ethanol extract of Drynaria Rhizome is obtained. The in-vitro alpha-glucosidase enzyme inhibition experiment shows that the ethanol extract of Drynaria Rhizome has a significant inhibitory effect on alpha-glucosidase enzyme, and the IC 50 is about 142.68 μ g / mL. Therefore, the ethanol extract of Drynaria Rhizome can be used for preparing a hypoglycemic drug, health care product or functional food, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a type of dragon bone (Polygonum hydropiper). Polypodium vulgare Preparation method and application of L. ethanol extract. Background Technology

[0002] Water keel ( Polypodium vulgare L., also known as Eurasian water dragon, is a perennial fern belonging to the genus L. in the family Polypodiaceae. Its dried rhizome has a long history of use in traditional medicine. The rhizome is rich in various active ingredients such as flavonoids, polyphenols, and triterpenoids, possessing multiple potential pharmacological activities including anti-inflammatory, antioxidant, neuroprotective, and skin-protective effects.

[0003] Alpha-glucosidase is a key carbohydrate metabolism enzyme located at the brush border of the small intestine, hydrolyzing oligosaccharides into glucose. Inhibiting its activity can effectively delay carbohydrate absorption and lower postprandial blood glucose, making it an important strategy for treating type 2 diabetes. While existing hypoglycemic drugs such as acarbose are effective, they have side effects such as bloating and diarrhea. Therefore, developing highly effective and low-toxicity alpha-glucosidase inhibitors from natural products has significant clinical importance and application value.

[0004] Currently, ultrasonic extraction is the most common method for extracting the effective components of Polygonum multiflorum. However, ultrasonic extraction equipment is expensive and has limitations in large-scale industrial production. Reflux extraction, as a traditional thermal extraction method, has advantages such as simple equipment, convenient operation, and ease of large-scale production. However, systematic optimization research on the reflux extraction process of Polygonum multiflorum as a single herb is still lacking, especially reports on the comprehensive investigation of key parameters such as particle size, material-to-liquid ratio, ethanol concentration, extraction time, and extraction times are scarce.

[0005] Currently, regarding Eurasian water keel ( Polypodium vulgare The inhibitory activity of L. ethanol extract against α-glucosidase has not been systematically reported.

[0006] In summary, developing a simple, low-cost method for preparing ethanol extract of *Polygonum hydropiper* suitable for industrial production, and systematically studying its α-glucosidase inhibitory activity and hypoglycemic application, has significant practical value and broad market prospects. Summary of the Invention

[0007] The present invention aims to provide a method for preparing an ethanol extract of *Polyporus umbellatus* and its application. The method involves pulverizing and sieving *Polyporus umbellatus*, adding ethanol, refluxing and extracting, combining the filtrates, concentrating under reduced pressure, and drying to obtain the ethanol extract. In vitro α-glucosidase inhibition experiments show that the ethanol extract of *Polyporus umbellatus* has a significant inhibitory effect on α-glucosidase, with an IC50 value of [missing information]. 50 Approximately 142.68μ g / mL. Therefore, the ethanol extract of *Polygonum hydropiper* of the present invention can be used to prepare hypoglycemic drugs, health products, or functional foods, and has broad application prospects.

[0008] The preparation method of the ethanol extract of *Polygonum hydropiper* according to the present invention is carried out according to the following steps: a. Crush the dried rhizomes of the water dragon bone and pass them through a 60-120 mesh sieve to obtain water dragon bone powder; b. Add an ethanol aqueous solution with a volume concentration of 50%-95% to the water caltrop powder obtained in step a at a mass ratio of 1:10-1:40, and perform reflux extraction for 0.5-3 hours. Filter, and reflux extract the filter residue 1-3 times. Combine the filtrates, concentrate by rotary evaporation under reduced pressure, and freeze-dry, spray-dry or vacuum-dry to obtain the water caltrop ethanol extract.

[0009] The sieve mesh size mentioned in step a is 80-100 mesh.

[0010] The mass ratio mentioned in step b is 1:10-1:20; the volume concentration of the ethanol aqueous solution is 70%-80%; the reflux extraction time is 1-2 hours, and the extraction is repeated 2-3 times.

[0011] Application of the ethanol extract of *Polygonum hydropiper* obtained by the method in the preparation of hypoglycemic drugs.

[0012] The application of the ethanol extract of *Polygonum hydropiper* in drugs that inhibit α-glucosidase activity in hypoglycemic agents.

[0013] The application of the ethanol extract of *Polygonum hydropiper* obtained by the method in the preparation of hypoglycemic health products or functional foods. Attached Figure Description

[0014] Figure 1 The concentrations used in this invention are 500, 250, 125, 62.5, and 31.25. μ Water caltrop ethanol extract and acarbose (200 g / mL) μ Inhibition rate of α-glucosidase (g / mL); Figure 2 This is the logarithmic concentration-inhibition rate regression curve of the ethanol extract of *Polygonum hydropiper* inhibiting α-glucosidase according to the present invention. Detailed Implementation

[0015] To further understand the present invention, the present invention will be described in detail below with reference to the embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments. Example 1

[0016] a. Obtaining water keel ( Polypodium vulgare50g of dried rhizome (L.) was crushed and passed through an 80-mesh sieve to obtain water dragon bone powder; b. Add 750g of 75% ethanol aqueous solution to the water caltrop powder obtained in step a at a mass ratio of 1:15, reflux for 1.5 hours, filter, and then reflux extract the filter residue twice more. Combine the three filtrates, concentrate them into an extract by rotary evaporation under reduced pressure, and then freeze-dry to obtain about 5.8g of water caltrop ethanol extract. Example 2

[0017] a. Obtaining water keel ( Polypodium vulgare 50g of dried rhizome (L.) was crushed and passed through a 60-mesh sieve to obtain water dragon bone powder; b. Add 2000g of 50% ethanol aqueous solution to the water caltrop powder obtained in step a at a mass ratio of 1:40, reflux for 0.5 hours, filter, concentrate by vacuum rotary evaporation to extract, and then freeze dry to obtain water caltrop ethanol extract. Example 3

[0018] Take water dragon bone ( Polypodium vulgare 50g of dried rhizome (L.) was crushed and passed through a 120-mesh sieve to obtain water dragon bone powder; Add 500g of 95% ethanol aqueous solution to the water caltrop powder obtained in step a at a mass ratio of 1:10, reflux for 3 hours, filter, and then reflux extract the filter residue 3 times. Combine the 4 filtrates, concentrate them into an extract by vacuum rotary evaporation, and then spray dry to obtain the water caltrop ethanol extract. Example 4

[0019] Take water dragon bone ( Polypodium vulgare 50g of dried rhizome (L.) was crushed and passed through a 100-mesh sieve to obtain water dragon bone powder; Add 1000g of 95% ethanol aqueous solution to the water caltrop powder obtained in step a at a mass ratio of 1:20, reflux for 2 hours, filter, reflux the filter residue once more, combine the two filtrates, concentrate to extract by rotary evaporation under reduced pressure, and then dry under vacuum to obtain water caltrop ethanol extract. Example 5

[0020] Determination of the inhibitory activity of the ethanol extract of *Polygonum hydropiper* against α-glucosidase: Experimental materials and reagents: α-Glucosidase (Sigma-Aldrich), p-nitrophenyl-β-D-glucopyranoside (pNPG, Sigma-Aldrich), positive control acarbose (Sigma-Aldrich), phosphate buffer (PBS, 0.1 mol / L, pH 6.8), dimethyl sulfoxide (DMSO), and ethanol extract of *Polygonum hydropiper* (prepared according to Example 1).

[0021] Experimental methods: Using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, the inhibitory activity of the ethanol extract of *Polygonum hydropiper* against α-glucosidase was evaluated by measuring the absorbance change of the product p-nitrophenol at 405 nm. The total volume of the reaction system was 100 mL. μ L, the specific operation is as follows: Take 2 μ L was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 500, 250, 125, 62.5, and 31.25%, respectively. μ The sample of *Polygonum hydropiper* ethanol extract was tested at a concentration of g / mL, and 71.5 g / mL was added. μ L of phosphate buffer solution at pH 6.8 and 1.5 μ L-α-glucosidase was added to bring the final enzyme concentration in the reaction system to 0.03 U / mL. After incubation at room temperature for 10 min, 25 μL of the enzyme was added. μ 10 mmol / L of pNPG substrate was added, shaken to mix, and reacted at 37℃ for 30 min. The absorbance of each well was then measured at 405 nm. Simultaneously, an enzyme activity group was set up with dimethyl sulfoxide (DMSO) instead of the sample, and the enzyme activity control group was set up with dimethyl sulfoxide (DMSO) instead of the sample and no enzyme added. The drug control group was set up without enzyme, and the rest was the same as the drug control group, with acarbose as a positive control. Each group had 3 replicates. The inhibition rate (1%) was calculated using the following formula: I% = [1 - (absorbance of the drug-treated group - absorbance of the drug-treated control group) / (absorbance of the enzyme-activated group - absorbance of the enzyme-activated control group)] × 100%; A linear regression was performed with the logarithm of the sample concentration on the x-axis and the inhibition rate on the y-axis to calculate the half-maximal inhibitory concentration (IC50). 50 )value; Experimental results: The inhibitory activities of the ethanol extract of *Polygonum hydropiper* and the positive control acarbose on α-glucosidase were determined, and the results are shown in Table 1. Figure 1 , Table 1: Inhibition rate of α-glucosidase by different concentrations of ethanol extract of Polygonum multiflorum and acarbose

[0022] The ethanol extract of *Polygonum hydropiper* was measured at 500, 250, 125, 62.5, and 31.25 mg / L. μ The inhibition rates at concentrations of g / mL were 92.63%±6.46%, 63.13%±3.56%, 47.16%±1.35%, 21.89%±5.28%, and 4.40%±5.07%, respectively, showing a clear concentration dependence; the positive control acarbose showed inhibition rates at 200 g / mL.μ The inhibition rate at a concentration of g / mL was 45.81% ± 0.40%; The results showed that the ethanol extract of *Polygonum hydropiper* obtained by the method described in this invention had significant α-glucosidase inhibitory activity, and its inhibitory effect was superior to that of acarbose at the same concentration at 125 °C. μ At a concentration of 10 g / mL, the inhibition rate of ethanol extract of *Polygonum aviculare* was higher than that of acarbose at 200 g / mL. μ The inhibition rate at a concentration of g / mL can be used to prepare hypoglycemic drugs or functional foods. A linear regression was performed with the logarithm of the sample concentration on the x-axis and the inhibition rate on the y-axis, yielding the regression equation y = 0.7229x - 1.0574 (R²). 2 =0.9913), the half-maximal inhibitory concentration (IC50) of the ethanol extract of *Polygonum hydropiper* against α-glucosidase was calculated. 50 The value is 142.68. μ g / mL Figure 2 ); The results showed that the ethanol extract of *Polygonum hydropiper* prepared in this invention possessed good in vitro hypoglycemic activity, which was superior to that of the positive control acarbose. Therefore, the ethanol extract of *Polygonum hydropiper* can be used in the preparation of drugs, health products, or functional foods for the prevention or treatment of diabetes and for lowering postprandial blood glucose.

Claims

1. A method for preparing an ethanol extract of *Polygonum hydropiper*, characterized in that, Follow these steps: a. Crush the dried rhizomes of the water dragon bone and pass them through a 60-120 mesh sieve to obtain water dragon bone powder; b. Add an ethanol aqueous solution with a volume concentration of 50%-95% to the water caltrop powder obtained in step a at a mass ratio of 1:10-1:40, and perform reflux extraction for 0.5-3 hours. Filter, and then reflux extract the filter residue 2-4 times. Combine the filtrates, concentrate by rotary evaporation under reduced pressure, and freeze-dry, spray-dry or vacuum-dry to obtain the water caltrop ethanol extract.

2. The method for preparing a water caltrop ethanol extract as described in claim 1, characterized in that, The sieve mesh size mentioned in step a is 80-100 mesh.

3. The method for preparing a water caltrop ethanol extract as described in claim 1, characterized in that, The mass ratio mentioned in step b is 1:10-1:20; the volume concentration of the ethanol aqueous solution is 70%-80%; the reflux extraction time is 1-2 hours, and the extraction is repeated 2-3 times.

4. The use of the ethanol extract of *Polygonum hydropiper* obtained by the method described in claim 1 in the preparation of hypoglycemic drugs.

5. The application as described in claim 4, characterized in that, The application of the ethanol extract of *Polygonum hydropiper* in drugs that inhibit α-glucosidase activity in hypoglycemic agents.

6. The application as described in claim 4, characterized in that, The application of the ethanol extract of *Polygonum hydropiper* obtained by the method in the preparation of hypoglycemic health products or functional foods.

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