Pandan leaf extract, preparation method and application thereof

Abstract

The present invention relates to a pandan leaf extract and a preparation method and application thereof. The preparation method comprises the following steps: (1) extracting pandan leaves, adding 15 w / w% to 60 w / w% ethanol solution for extraction to obtain an extract; (2) removing ethanol from the extract, and adding an organic solvent for extraction, wherein the organic solvent is ethyl acetate or dichloromethane; and (3) removing the organic phase obtained by extraction and removing the organic solvent to obtain the pandan leaf extract. The pandan leaf extract prepared by the preparation method of the present invention has high α-glucosidase inhibitory activity and can be used as a highly effective inhibitor of α-glucosidase to better prevent and treat related diseases such as diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extracts, and in particular relates to a pandan leaf extract, a preparation method thereof and an application thereof. Background Art

[0002] Pandan leaf, scientifically known as Pandanus amaryllifolius Roxb., also known as colorful leaf and pandan leaf, is a perennial evergreen herb in the Pandanaceae family and genus Pandanus. It is the only plant in the Pandanaceae family with fragrant leaves, naturally exuding a "zongzi" aroma. Its main aroma component is 2-acetyl-1-pyrroline (2AP), the same component of Thai fragrant rice. It plays a key role in Southeast Asian cuisine and is one of the most commonly used natural spices, known as the "herb of the East." Pandan leaf primarily grows in the tropical environments of South and Southeast Asia. Currently, it is primarily cultivated in Hainan Province in my country, with significant plantings also occurring in Yunnan, Guangdong, and Fujian. With the increasing sophistication of pandan leaf cultivation technology and the expanding market for pandan leaf, the pandan leaf industry holds a promising future.

[0003] Pandan leaves are rich in squalene, unsaturated fatty acids, and vitamins, which not only provide them with a unique aroma and flavor but also impart various pharmacological properties. Modern pharmacological research has shown that pandan leaves possess antioxidant, anti-inflammatory, anticancer, antibacterial, hepatoprotective, anti-gout, and neuroprotective properties, possessing significant health and economic value and promising development prospects.

[0004] Diabetes is a common chronic metabolic disease primarily caused by genetic and environmental factors. It is categorized into type 1 and type 2 diabetes. Type 1 diabetes is primarily due to insufficient insulin secretion, requiring daily insulin injections to maintain life. Type 2 diabetes is the most common type of diabetes and is a chronic disease caused by insufficient or ineffective insulin use, accounting for over 90% of all diabetes cases. The typical symptoms of diabetes are known as "three mores and one less": increased drinking, increased eating, increased urination, and weight loss. Diabetes is often accompanied by multiple complications, and if left uncontrolled and treated, it can lead to damage to other organs. The International Diabetes Federation (IDF) reports that currently 463 million people suffer from diabetes, a number projected to increase to 578 million by 2030 and 700 million by 2045. There are many medications available for the treatment of type 2 diabetes. Among them, α-glucosidase inhibitors, with their mechanism of action independent of insulin, offer a relatively good safety profile, pose no risk of hypoglycemia, and do not increase weight. They are particularly suitable for Eastern diabetics whose diets tend to be high in carbohydrates, and are currently a widely used oral hypoglycemic agent.

[0005] α-glucosidase inhibitors competitively inhibit the activity of α-glucosidase in the brush border of the small intestine, slowing the breakdown of carbohydrates into glucose and thus reducing postprandial blood sugar levels. This inhibitory effect reduces intestinal absorption of glucose, helping to improve blood sugar control, while having little effect on fasting blood sugar and blood lipid levels. The main α-glucosidase inhibitors currently on the market include acarbose, miglitol, and voglibose, but these drugs also have side effects such as bloating, abdominal distension, diarrhea, and gastrointestinal spasmodic pain.

[0006] Screening new low-toxic, safe and efficient α-glucosidase inhibitors from natural products has received increasing attention. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide a pandan leaf extract and its use as an α-glucosidase inhibitor, especially in a drug for preventing and treating diabetes, wherein the pandan leaf extract has good α-glucosidase inhibitory activity.

[0008] The first aspect of the present invention is to provide a method for preparing a pandan leaf extract, comprising the following steps:

[0009] (1) extracting pandan leaves by adding 15 w / w% to 60 w / w% ethanol solution to obtain an extract;

[0010] (2) taking the extract, removing ethanol, and adding an organic solvent for extraction, wherein the organic solvent is ethyl acetate or dichloromethane;

[0011] (3) taking the organic phase obtained by extraction, removing the organic solvent, and obtaining the pandan leaf extract.

[0012] In some preferred embodiments, 30 w / w% to 60 w / w% ethanol solution is added for extraction in step (1).

[0013] More preferably, in step (1), 45 w / w% to 60 w / w% ethanol solution is added for extraction.

[0014] In some embodiments, the mass ratio of the pandan leaves to the ethanol solution in step (1) is 1:5 to 1:15, preferably 1:10 to 1:15, and more preferably 1:10 to 1:12.

[0015] In some embodiments, the extraction temperature in step (1) is 20°C to 60°C, preferably 20°C to 45°C, more preferably 20°C to 30°C; the extraction time is 20h to 35h, preferably 20h to 30h, more preferably 22h to 26h.

[0016] In some embodiments, the mass ratio of the extract to the organic solvent is 1:1 to 1:3, preferably 1:1 to 1:2, and more preferably 1:1 to 1:1.5.

[0017] In some embodiments, the method for removing the organic solvent in step (3) is reduced pressure concentration; preferably, reduced pressure concentration is carried out at 30°C to 40°C; more preferably, reduced pressure concentration is carried out at 32°C to 37°C.

[0018] The second aspect of the present invention is to provide a pandan leaf extract prepared by the preparation method described above.

[0019] The third aspect of the present invention is to provide the use of the pandan leaf extract as described above in the preparation of α-glucosidase inhibitors.

[0020] In some embodiments, the application includes preparing a drug for preventing and / or treating diabetes; preferably, the diabetes is type II diabetes.

[0021] The fourth aspect of the present invention is to provide a drug for preventing and / or treating diabetes, wherein the main active ingredient of the drug includes the pandan leaf extract as described above.

[0022] The present invention has developed a method for preparing a pandan leaf extract. The method involves extracting the pandan leaf using an ethanol solution of an appropriate concentration (15 to 60 w / w%) to obtain an extract, which is then extracted using a specific organic solvent, ethyl acetate or dichloromethane. The resulting pandan leaf extract exhibits excellent α-glucosidase inhibitory activity. In particular, using a 30 to 60 w / w ethanol solution as the extraction solvent and then extracting with ethyl acetate or dichloromethane significantly enhances the α-glucosidase inhibitory effect of the pandan leaf extract.

[0023] The pandan leaf extract prepared by the preparation method of the present invention can be used as a highly effective inhibitor of α-glucosidase to better prevent and treat diabetes and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Comparison of the α-glucosidase inhibitory effects of Pandan leaf extracts 1, 3, 18, and 20.

[0025] Figure 2Comparison of α-glucosidase inhibition of Pandan leaf extracts 3-5.

[0026] Figure 3 Concentration gradient and IC of α-glucosidase inhibition of pandan leaf extract 1 50 .

[0027] Figure 4 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 2 50 .

[0028] Figure 5 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 3 50 .

[0029] Figure 6 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 4 50 .

[0030] Figure 7 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 6 50 .

[0031] Figure 8 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 7 50 .

[0032] Figure 9 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 8 50 .

[0033] Figure 10 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 9 50 .

[0034] Figure 11 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 10 50 .

[0035] Figure 12 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 11 50 .

[0036] Figure 13 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 12 50 .

[0037] Figure 14 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 13 50 .

[0038] Figure 15 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 14 50 .

[0039] Figure 16 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 15 50 .

[0040] Figure 17 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 16 50 .

[0041] Figure 18 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 17 50 .

[0042] Figure 19 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 18 50 .

[0043] Figure 20 Concentration gradient and IC of α-glucosidase inhibition of pandan leaf extract 19 50 .

[0044] Figure 21 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 20 50 .

[0045] Figure 22 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 21 50 .

[0046] Figure 23 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 22 50 .

[0047] Figure 24 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 23 50 . DETAILED DESCRIPTION

[0048] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0049] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0051] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0052] Some embodiments of the present invention relate to a method for preparing a pandan leaf extract, comprising the following steps:

[0053] (1) extracting pandan leaves by adding 15 w / w% to 60 w / w% ethanol solution to obtain an extract;

[0054] (2) taking the extract, removing ethanol, and adding an organic solvent for extraction, wherein the organic solvent is ethyl acetate or dichloromethane;

[0055] (3) taking the organic phase obtained by extraction, removing the organic solvent, and obtaining the pandan leaf extract.

[0056] After research, the present invention found that the inhibitory effect of the prepared pandan leaf extract on α-glucosidase can be effectively enhanced by first extracting the pandan leaf with an ethanol solution of an appropriate concentration (15w / w% to 60w / w%) to obtain an extract, and then extracting the extract with a specific organic solvent (ethyl acetate or dichloromethane).

[0057] In the preparation method of the present invention, the concentration of the ethanol solution used as the extraction solvent is very important. After the extract is extracted with 15w / w% to 60w / w% ethanol solution and then extracted with ethyl acetate or dichloromethane, the inhibitory effect of the prepared pandan leaf extract on α-glucosidase can be effectively improved. The IC value of the inhibitory effect of the prepared pandan leaf extract on α-glucosidase is 2. 50The value is lower than 80μg / mL; preferably, 30w / w% to 60w / w% ethanol solution is used for extraction, IC 50 The value is lower than 50 μg / mL; more preferably, 45 w / w% to 60 w / w% ethanol solution is used for extraction, IC 50 If the concentration of the ethanol solution used is not appropriate, even if the extract is further extracted with ethyl acetate or dichloromethane, the inhibitory effect of the prepared pandan leaf extract on α-glucosidase cannot be effectively improved.

[0058] After extracting the extract using an ethanol solution of appropriate concentration, further extraction with a specific organic solvent (ethyl acetate or dichloromethane) can effectively enhance the inhibitory effect of pandan leaf extract on α-glucosidase. Inappropriate extraction solvents (such as petroleum ether) will not only fail to further enhance the inhibitory effect but will instead cause its inhibitory effect to decrease.

[0059] The present invention is further described in detail below with reference to specific embodiments.

[0060] Example 1

[0061] Weigh 40 g of pandan leaf powder, add 30% ethanol (w / w) solution at a solid-liquid ratio of 1:12 (w / w), stir with a glass rod to mix thoroughly, place in a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 1.

[0062] The pandan leaf extract 1 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 1.

[0063] Weigh 100 g of pandan leaf extract 1 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 100 g of pure water and mix well. Then add 100 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layers (organic phases), and obtain the ethyl acetate layer extract. Concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it to remove ethyl acetate to obtain the ethyl acetate layer extract (pandan leaf extract 2).

[0064] Example 2

[0065] Weigh 40 g of pandan leaf powder, add 60% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, place in a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 3.

[0066] The pandan leaf extract 3 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 3.

[0067] Take 100 g of Pandan leaf extract 3 and concentrate it under reduced pressure at 50°C to remove ethanol, add 100 g of pure water and mix well, then add 100 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), obtain an ethyl acetate layer extract and an aqueous layer extract, concentrate the ethyl acetate layer extract under reduced pressure and dry it at 35°C to remove ethyl acetate to obtain an ethyl acetate layer extract (Pandan leaf extract 4); concentrate the aqueous layer extract under reduced pressure at 50°C to remove ethanol, and perform freeze-drying to obtain an aqueous layer extract (Pandan leaf extract 5).

[0068] Example 3

[0069] Weigh 50 g of the pandan leaf extract 3 in Example 2 above and concentrate under reduced pressure at 50° C. to remove ethanol, add 50 g of pure water and mix well, then add 50 g of dichloromethane for extraction, extract three times, use a separatory funnel to separate the liquids, combine the dichloromethane layer (organic phase) to obtain a dichloromethane layer extract and an aqueous layer extract, and concentrate the dichloromethane layer extract under reduced pressure at 35° C. and dry it to remove dichloromethane to obtain a dichloromethane layer extract (pandan leaf extract 6).

[0070] Example 4

[0071] Weigh 40 g of pandan leaf powder, add 45% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, put into a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 7.

[0072] The pandan leaf extract 7 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 7.

[0073] Weigh 100 g of pandan leaf extract 7 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 100 g of pure water and mix well. Then add 100 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), and obtain an ethyl acetate layer extract and an aqueous layer extract. Concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it to remove ethyl acetate to obtain an ethyl acetate layer extract (pandan leaf extract 8).

[0074] Example 5

[0075] Weigh 20 g of pandan leaf powder, add 15% ethanol (w / w) solution at a solid-liquid ratio of 1:12 (w / w), stir with a glass rod to mix thoroughly, put into a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 9.

[0076] The pandan leaf extract 9 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 9.

[0077] Weigh 30 g of pandan leaf extract 9 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 30 g of pure water and mix well. Then add 30 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), and obtain an ethyl acetate layer extract and an aqueous layer extract. Concentrate the ethyl acetate layer extract under reduced pressure and dry it at 35°C to remove ethyl acetate to obtain an ethyl acetate layer extract (pandan leaf extract 10).

[0078] Example 6

[0079] Weigh 20 g of pandan leaf powder, add 60% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, put into a 20°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 11.

[0080] The pandan leaf extract 11 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 11.

[0081] Weigh 30 g of pandan leaf extract 11 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 30 g of pure water and mix well. Then add 30 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), and obtain the ethyl acetate layer extract and the aqueous layer extract. Concentrate the ethyl acetate layer extract under reduced pressure and dry it at 35°C to remove ethyl acetate to obtain the ethyl acetate layer extract (pandan leaf extract 12).

[0082] Example 7

[0083] Weigh 20 g of pandan leaf powder, add 60% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, put into a 60°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 13.

[0084] The pandan leaf extract 13 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 13.

[0085] Weigh 30 g of pandan leaf extract 13 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 30 g of pure water and mix well. Then add 30 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase) to obtain an ethyl acetate layer extract and an aqueous layer extract, concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it to remove ethyl acetate to obtain an ethyl acetate layer extract (pandan leaf extract 14).

[0086] Comparative Example 1

[0087] This comparative example adopts the existing method to prepare the pandan leaf extract, comprising the following steps:

[0088] Weigh 10 g of pandan leaf powder, add 100 mL of anhydrous ethanol at a solid-liquid ratio of 1:10 (w / v), stir evenly with a glass rod, and soak in a 37°C water bath for 48 hours. After the extraction, filter with filter paper to obtain pandan leaf extract 15, which is concentrated and dried under reduced pressure at 50°C to obtain pandan leaf extract 15.

[0089] Comparative Example 2

[0090] This comparative example adopts the existing method to prepare the pandan leaf extract, comprising the following steps:

[0091] Weigh 30 g of pandan leaf powder, add 300 mL of 90°C pure water at a solid-liquid ratio of 1:10 (w / v), stir evenly with a glass rod, soak and extract for 15 minutes, filter with filter paper to obtain pandan leaf extract 16, and freeze-dry to obtain pandan leaf extract 16.

[0092] Comparative Example 3

[0093] Weigh 50 g of the pandan leaf extract 3 in Example 2 and concentrate under reduced pressure at 50°C to remove ethanol. Add 50 g of pure water and mix well. Then add 50 g of petroleum ether for extraction, extract three times, use a separatory funnel to separate the liquids, combine the petroleum ether layers (organic phase), and obtain a petroleum ether layer extract and an aqueous layer extract. The petroleum ether layer extract is concentrated and dried under reduced pressure at 35°C to remove the petroleum ether to obtain a petroleum ether layer extract (pandan leaf extract 17).

[0094] Comparative Example 4

[0095] Weigh 40 g of pandan leaf powder, add 75% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, put into a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 18.

[0096] The pandan leaf extract 18 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 18.

[0097] Weigh 100 g of pandan leaf extract 18 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 100 g of pure water and mix well. Then add 100 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), and obtain an ethyl acetate layer extract and an aqueous layer extract. Concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it to remove ethyl acetate to obtain an ethyl acetate layer extract (pandan leaf extract 19).

[0098] Comparative Example 5

[0099] Weigh 40 g of pandan leaf powder, add 95% ethanol (w / w) solution at a solid-liquid ratio of 1:10 (w / w), stir with a glass rod to mix thoroughly, put into a 30°C water bath, stir magnetically (1200 rpm), soak and extract for 24 hours. After the extraction is completed, filter with filter paper to obtain pandan leaf extract 20.

[0100] The pandan leaf extract 20 was concentrated under reduced pressure at 50° C. to remove ethanol, and then freeze-dried to obtain the pandan leaf extract 20.

[0101] Weigh 100 g of pandan leaf extract 20 and concentrate it under reduced pressure at 50°C to remove ethanol. Add 100 g of pure water and mix well. Then add 100 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layer (organic phase), and obtain the ethyl acetate layer extract and the aqueous layer extract. Concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it to remove ethyl acetate to obtain the ethyl acetate layer extract (pandan leaf extract 21).

[0102] Comparative Example 6

[0103] Weigh 20g of pandan leaf powder, add pure water at a solid-liquid ratio of 1:15 (w / w), stir with a glass rod to mix thoroughly, place in a 30°C water bath, stir magnetically (1200rpm), soak and extract for 24h. After the extraction, filter with filter paper to obtain pandan leaf extract 22. Take part of the extract for freeze-drying to obtain pandan leaf extract 22.

[0104] Weigh 30 g of pandan leaf extract 22, add 30 g of ethyl acetate for extraction, extract three times, use a separatory funnel to separate the liquids, combine the ethyl acetate layers (organic phase), obtain ethyl acetate layer extract and aqueous layer extract, concentrate the ethyl acetate layer extract under reduced pressure at 35°C and dry it, remove ethyl acetate, and obtain ethyl acetate layer extract (pandan leaf extract 23).

[0105] α-Glucosidase inhibition activity test

[0106] The pNPG method was used to detect the inhibitory effect of the pandan leaf extract prepared in the above examples and comparative examples on α-glucosidase. α-glucosidase can hydrolyze 4-nitrophenyl-α-D-pyranoglucoside (pNPG) to p-nitrophenol (pNP). pNP has strong ultraviolet absorption under alkaline conditions. The pNP concentration in the reaction solution was detected using a microplate reader to reflect the strength of the extract in inhibiting the activity of α-glucosidase.

[0107] Prepare 0.1 mol / L potassium phosphate buffer and adjust the pH to 6.8. Dilute α-glucosidase to 0.73 U / mL using potassium phosphate buffer. Prepare pNPG into 5 mmol / L solution using potassium phosphate buffer. Weigh pandan leaf extracts 1, 3, 18 and 20, respectively, dissolve them in DMSO to prepare 200 mg / mL stock solutions, and dilute them with pure water to a final concentration of 1 mg / mL sample, which contains 5% DMSO (the final concentration of the sample in the reaction system is 0.5 mg / mL). 1 μmol / L acarbose is used as a positive control.

[0108] 50 μL of each pandan leaf extract sample was added to a 1.5 mL EP tube, with two replicate wells set up for each sample. In addition, a sample control group, a blank reaction group, and a blank control group were set up. The composition of each experimental group was as follows:

[0109] Sample reaction group: 50 μL pandan leaf extract + 50 μL α-glucosidase;

[0110] Sample control group: 50 μL pandan leaf extract + 50 μL potassium phosphate buffer;

[0111] Blank reaction group: 50 μL pure water (containing 5% DMSO) + 50 μL α-glucosidase;

[0112] Blank control group: 50 μL pure water (containing 5% DMSO) + 50 μL potassium phosphate buffer.

[0113] The above reaction system was mixed and incubated in a 37°C water bath for 20 minutes. Then, 50 μL of 5 mmol / L pNPG was added to each tube, mixed and incubated in a 37°C water bath for 10 minutes. After incubation, 1 mL of 0.2 mol / L sodium carbonate solution was added to each tube to terminate the reaction. 200 μL of the reaction system was pipetted into a 96-well plate and the UV absorbance (OD value) at a wavelength of 450 nm was measured on a microplate reader. The enzyme activity inhibition rate was calculated as follows:

[0114]

[0115] The experimental results were compared using GraphPad Prism 8.0.

[0116] The results are as follows Figure 1 As shown, from Figure 1 The results show that at a concentration of 0.5 mg / mL, the α-glucosidase inhibitory activity of extracts obtained using different ethanol concentrations varied significantly, indicating that the concentration of the ethanol solution used as the extraction solvent significantly affects the α-glucosidase inhibitory activity of the pandan leaf extract. Furthermore, the pandan leaf extract obtained by direct vacuum concentration and freeze-drying of the ethanol extract exhibited lower α-glucosidase inhibitory activity, significantly lower than that of acarbose.

[0117] Next, extracts 3-5 of Pandan leaf were dissolved in DMSO to prepare a 200 mg / mL stock solution, which was then diluted with purified water to a final concentration of 1 mg / mL. The solution contained 5% DMSO (for a final sample concentration of 0.5 mg / mL in the reaction system) and 1 μmol / L acarbose as a positive control. The α-glucosidase inhibition activity was tested and compared graphically as described above.

[0118] The results are as follows Figure 2 As shown, from Figure 2 The results show that at a concentration of 0.5 mg / mL, pandan leaf extract 4 exhibited the strongest α-glucosidase inhibitory activity, almost on par with acarbose and significantly higher than pandan leaf extract 3. Pandan leaf extract 5 exhibited the weakest α-glucosidase inhibitory activity, suggesting that ethanol extraction followed by ethyl acetate extraction significantly enhances the α-glucosidase inhibitory effect of pandan leaf extracts.

[0119] Furthermore, the 200 mg / mL stock solution (DMSO solution) of Pandan leaf extracts 1-4 and 6-23 was diluted with pure water to a concentration gradient sample with a final concentration of 0.001 mg / mL-10 mg / mL (containing 5% DMSO). The α-glucosidase inhibitory activity was tested and compared according to the above-mentioned α-glucosidase inhibitory activity test, and the IC 50 value.

[0120] The results are as follows Figures 3 to 24 As shown in Table 1:

[0121] Table 1

[0122]

[0123]

[0124]

[0125] From Examples 1 to 7, it can be seen that the pandan leaf extract prepared by the preparation method of the present invention has a good inhibitory effect on α-glucosidase, IC 50 The value was lower than 80 μg / mL, especially when the extraction was performed after using 45 w / w% ethanol solution (Example 4) and 60 w / w% ethanol solution (Example 2), IC 50 The values ​​were as low as 37.03μg / mL and 27.03μg / mL, respectively.

[0126] The preparation of existing pandan leaf extract usually uses anhydrous ethanol or water as the extraction solvent for extraction (Comparative Examples 1-2). The obtained pandan leaf extract has a poor inhibitory effect on α-glucosidase. 50 The values ​​were 2.32 mg / mL and 1.02 mg / mL, respectively. The preparation method of the present invention first optimizes the extracting solution as the extraction solvent, and uses 15w / w% to 60w / w% ethanol solution for extraction. The inhibitory effect of the pandan leaf extract obtained by directly concentrating and drying the extracting solution on α-glucosidase is enhanced. For example, the IC50 values ​​of the pandan leaf extract obtained by directly concentrating and drying the extracting solution using 15%w / w%, 30w / w%, 45w / w% and 60w / w% ethanol solution are 673.7μg / mL (pandan leaf extract 9), 848.9μg / mL (pandan leaf extract 1), 536.2μg / mL (pandan leaf extract 7) and 444.2μg / mL (pandan leaf extract 3), respectively. Furthermore, the inhibition effect of α-glucosidase of the prepared pandan leaf extract can be greatly improved by combining the extraction with a specific organic solvent (ethyl acetate and dichloromethane) on the basis of selecting a suitable extraction solvent for extraction; the IC values ​​of the pandan leaf extract obtained by extracting the extract obtained by ethyl acetate after the extract obtained by 15% w / w%, 30w / w%, 45w / w% and 60w / w% ethanol solution are as follows: 50 The values ​​were 78.19 μg / mL (Pandan leaf extract 10), 49.03 μg / mL (Pandan leaf extract 2), 37.03 μg / mL (Pandan leaf extract 8) and 27.03 μg / mL (Pandan leaf extract 4), respectively. 50The value was significantly reduced, and the inhibitory effect of α-glucosidase was greatly improved.

[0127] In the preparation method of the present invention, the selection of the extraction solvent is very important. It is preferred to use 15w / w% to 60w / w% ethanol solution for extraction and then use ethyl acetate or dichloromethane for extraction. If the concentration of the ethanol solution used is not appropriate, even if the extract is further extracted with ethyl acetate or dichloromethane, the inhibitory effect of the prepared pandan leaf extract on α-glucosidase cannot be effectively improved. For example, in Comparative Examples 4 and 5, 75% w / w% and 90w / w% ethanol solutions were used for extraction, respectively, and in Comparative Example 6, pure water was used for extraction. The IC values ​​of the pandan leaf extract obtained after ethyl acetate extraction of the three extracts were 0.01%. 50 The values ​​were 119.9 μg / mL (Pandan leaf extract 19), 299.5 μg / mL (Pandan leaf extract 21) and 98.65 μg / mL (Pandan leaf extract 23), which were significantly higher than the Pandan leaf extract prepared by the present invention.

[0128] After extraction with an appropriate concentration of ethanol solution, the choice of extraction solvent has a significant impact on the α-glucosidase inhibitory effect of the pandan leaf extract obtained. From the comparison of Examples 2 to 3 and Comparative Example 3, it can be seen that after extraction with a 60w / w% ethanol solution, the extract is directly concentrated under reduced pressure to obtain the pandan leaf extract, which has an IC of α-glucosidase inhibition. 50 The value was 444.2 μg / mL (Pandan leaf extract 3); and the extract was extracted using ethyl acetate (Example 2), dichloromethane (Example 3) and petroleum ether (Comparative Example 3) as the extraction solvent to obtain the Pandan leaf extract that inhibited α-glucosidase IC 50 The values ​​were 27.03 μg / mL (Pandan leaf extract 4), 33.79 μg / mL (Pandan leaf extract 6), and 479.6 μg / mL (Pandan leaf extract 17), respectively. It can be seen that the use of ethyl acetate and dichloromethane as solvents for extraction can significantly improve the α-glucosidase inhibitory effect of the prepared Pandan leaf extracts compared with those prepared without extraction. The corresponding IC 50 The IC of the pandan leaf extract obtained by using petroleum ether as the extraction solvent to inhibit α-glucosidase was greatly reduced. 50 The value not only did not decrease, but actually increased. The results show that an inappropriate extraction solvent not only fails to further improve the α-glucosidase inhibitory effect of the ethanol solution extract, but instead leads to a decrease in its inhibitory effect. The present invention preferably uses ethyl acetate and dichloromethane to extract the 15w / w% to 60w / w% ethanol solution extract, especially ethyl acetate, which can greatly improve the α-glucosidase inhibitory effect of the prepared pandan leaf extract.

[0129] In summary, the preparation method of the present invention first uses an ethanol solution with a concentration of 15w / w% to 60w / w% to extract the pandan leaves to obtain an extract, and then further extracts with ethyl acetate or dichloromethane. This can greatly enhance the α-glucosidase inhibitory effect of the prepared pandan leaf extract, and can more effectively prevent and treat related diseases including diabetes.

[0130] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a pandan leaf extract, characterized in that: The following steps are involved: (1) extracting pandan leaves by adding 15 w / w% to 60 w / w% ethanol solution to obtain an extract; (2) taking the extract, removing ethanol, and adding an organic solvent for extraction, wherein the organic solvent is ethyl acetate or dichloromethane; (3) taking the organic phase obtained by extraction, removing the organic solvent, and obtaining the pandan leaf extract.

2. The preparation method according to claim 1, wherein In step (1), 30 w / w% to 60 w / w% ethanol solution is added for extraction.

3. The preparation method according to claim 2, wherein In step (1), 45 w / w% to 60 w / w% ethanol solution is added for extraction.

4. The preparation method according to claim 1, wherein The mass ratio of the pandan leaves to the ethanol solution in step (1) is 1:5 to 1:

15.

5. The preparation method according to claim 1, wherein The extraction temperature in step (1) is 20° C. to 60° C., and the extraction time is 20 h to 35 h.

6. The preparation method according to claim 1, wherein The mass ratio of the extract to the organic solvent is 1:1 to 1:

3.

7. The pandan leaf extract obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the pandan leaf extract according to claim 7 in the preparation of α-glucosidase inhibitors.

9. The use according to claim 8, characterized in that The application includes preparing medicines for preventing and / or treating diabetes.

10. The use according to claim 9, characterized in that The diabetes is type 2 diabetes.

11. A drug for preventing and / or treating diabetes, characterized in that: The main active ingredient of the medicine includes the pandan leaf extract as claimed in claim 7.

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