Embelia pandshurica leaf extract as well as preparation method and application thereof

Abstract

The invention relates to a pandan leaf extract as well as a preparation method and application thereof, the preparation method comprises the following steps: (1) taking pandan leaves, and adding 15w / w%-60w / w% of an ethanol solution for extraction to obtain an extracting solution; (2) the extracting solution is taken, ethanol is removed, an organic solvent is added for extraction, and the organic solvent is ethyl acetate or dichloromethane; and (3) taking 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 disclosed by the invention has high alpha-glucosidase inhibitory activity, can be used as an efficient inhibitor of alpha-glucosidase, and can be used for better preventing and treating related diseases such as diabetes mellitus and the like.

Description

Technical Field

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

[0002] Pandanus, also known as Pandanus amaryllifolius Roxb., is a perennial evergreen herbaceous plant of the Pandanaceae family and the genus Pandanus. It is also the only plant in the Pandanaceae family with fragrant leaves. The leaves naturally emit the fragrance of "zongzi fragrance". Its main aroma component is 2-acetyl-1-pyrroline (2AP), which is consistent with the main component of Thai fragrant rice. It plays an important role in Southeast Asian cuisine and is one of the most commonly used natural spices. It is known as "Oriental vanilla". Pandanus mainly grows in tropical environments in South Asia and Southeast Asia. At present, my country mainly introduces and cultivates it in Hainan Province. In addition, it is also planted in large quantities in Yunnan, Guangdong, Fujian and other places. As the cultivation technology of pandanus leaves becomes more and more mature, the market of pandanus leaves continues to expand, and the development prospects of the pandanus leaf industry are very broad.

[0003] Pandan leaves are rich in squalene, unsaturated fatty acids, vitamins and other ingredients, which not only provide pandan leaves with a unique aroma and taste, but also give it a variety of pharmacological effects. Modern pharmacological research shows that pandan leaves have biological activities such as antioxidant, anti-inflammatory, anti-cancer, antibacterial, liver protection, anti-gout and neuroprotection, and have important health value, economic value and development prospects.

[0004] Diabetes is a common chronic metabolic disease, the cause of which is mainly due to genetic and environmental factors. It is divided into type I diabetes and type II diabetes. Type I diabetes is mainly due to insufficient insulin secretion, and insulin injections are required daily to maintain life; type II diabetes is the most common type of diabetes, a chronic disease caused by insufficient insulin use or reduced efficiency, accounting for more than 90% of the total number of people with diabetes. The typical symptoms of diabetes are called "three mores and one less", which refer to drinking more, eating more, urinating more and losing weight. Diabetes is often accompanied by a variety of complications. If not controlled and treated in time, symptoms of damage to other organs will appear. The International Diabetes Federation (IDF) report shows that there are currently 463 million people with diabetes, which is expected to increase to 578 million by 2030 and 700 million by 2045. There are many types of drugs for the treatment of type II diabetes. Among them, the mechanism of action of α-glucosidase inhibitors does not rely on insulin, has good safety, no risk of hypoglycemia, and does not increase weight. It is particularly suitable for Oriental diabetic populations whose dietary structure is biased towards carbohydrates. It is a type of oral hypoglycemic drug currently widely used in clinical practice.

[0005] α-glucosidase inhibitors competitively inhibit the activity of α-glucosidase in the brush border of the small intestine, slowing down the rate at which carbohydrates are broken down into glucose, thereby reducing postprandial blood sugar levels. This inhibitory effect reduces the intestinal absorption of glucose, helps improve blood sugar control, and has little effect on fasting blood sugar and blood lipid levels. The main α-glucosidase inhibitors on the market currently 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) taking pandan leaves, adding 15 w / w% to 60 w / w% ethanol solution for extraction 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 in step (1) for extraction.

[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, and more preferably 20°C to 30°C; the extraction time is 20h to 35h, preferably 20h to 30h, and 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 as 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 use 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 obtained a preparation method of a pandan leaf extract through research. The preparation method first uses an ethanol solution of a suitable concentration (15w / w% to 60w / w%) to extract the pandan leaf to obtain an extract, and then uses a specific organic solvent ethyl acetate or dichloromethane for extraction. The obtained pandan leaf extract has good α-glucosidase inhibitory activity. In particular, when a 30w / w% to 60w / w% ethanol solution is used as an extraction solvent and then combined with ethyl acetate or dichloromethane for extraction, the α-glucosidase inhibitory effect of the prepared pandan leaf extract can be greatly improved.

[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 The α-glucosidase inhibition effect of pandan leaf extracts 1, 3, 18, and 20 was compared.

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

[0026] Figure 3 Concentration gradient and IC for 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] Fig. 9 Concentration gradient and IC for α-glucosidase inhibition of pandan leaf extract 8 50 .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0048] In order to facilitate the 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 disclosure of the present invention more thorough and comprehensive.

[0049] The experimental methods in the following examples without specifying specific conditions are usually 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 in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0051] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is 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. In addition, throughout the 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) taking pandan leaves, adding 15 w / w% to 60 w / w% ethanol solution for extraction 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] The present invention has been found through research 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 a suitable 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. The use of 15w / w% to 60w / w% ethanol solution as an extract after extraction with ethyl acetate or dichloromethane can effectively improve the inhibitory effect of the prepared pandan leaf extract on α-glucosidase. The IC value of the inhibitory effect of the prepared pandan leaf extract on α-glucosidase is 50The value is lower than 80 μg / mL; preferably, 30 w / w% to 60 w / 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 well improved.

[0058] After using an ethanol solution of appropriate concentration to extract the extract, 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 lead to a decrease in its inhibitory effect.

[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 in 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 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) to obtain an 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 an 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 in 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, 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 100g of pandan leaf extract 3 and concentrate it under reduced pressure at 50°C to remove ethanol, add 100g of pure water and mix well, then add 100g of ethyl acetate for extraction, extract 3 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 4); concentrate the aqueous layer extract under reduced pressure at 50°C to remove ethanol, and freeze-dry it to obtain an aqueous layer extract (pandan leaf extract 5).

[0068] Example 3

[0069] Weigh 50 g of the pandan leaf extract 3 in the above-mentioned Example 2, concentrate it 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 3 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, 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 in 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, 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) 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 8).

[0074] Example 5

[0075] Weigh 20 g of pandan leaf powder, add 15% ethanol (w / w) solution in 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 layers (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 10).

[0078] Example 6

[0079] Weigh 20 g of pandan leaf powder, add 60% ethanol (w / w) solution in 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, 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 layers (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 12).

[0082] Example 7

[0083] Weigh 20 g of pandan leaf powder, add 60% ethanol (w / w) solution in 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, 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 layers (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 in a solid-liquid ratio of 1:10 (w / v), stir evenly with a glass rod, and soak in a 37°C water bath for extraction for 48 hours. After the extraction, filter with filter paper to obtain pandan leaf extract 15, concentrate and dry 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 in 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 the above embodiment 2, concentrate it under reduced pressure at 50°C, remove ethanol, add 50 g of pure water and mix well, then add 50 g of petroleum ether for extraction, extract 3 times, use a separatory funnel to separate the liquids, combine the petroleum ether layer (organic phase) to obtain a petroleum ether layer extract and an aqueous layer extract, concentrate the petroleum ether layer extract under reduced pressure at 35°C and dry it, remove the petroleum ether, and 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 in 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, 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) 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 19).

[0098] Comparative Example 5

[0099] Weigh 40g of pandan leaf powder, add 95% ethanol (w / w) solution in 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 (1200rpm), soak and extract for 24h. After the extraction, 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) 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 21).

[0102] Comparative Example 6

[0103] Weigh 20g of pandan leaf powder, add pure water in a solid-liquid ratio of 1:15 (w / w), stir with a glass rod to fully mix, put into 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) to obtain an ethyl acetate layer extract and an aqueous layer extract, and concentrate the ethyl acetate layer extract under reduced pressure at 35°C to dryness, remove ethyl acetate, and obtain an ethyl acetate layer extract (pandan leaf extract 23).

[0105] α-Glucosidase Inhibitory Activity Test

[0106] The pNPG method was used to detect the inhibitory effect of the pandan leaf extract prepared in the above embodiments and comparative examples on α-glucosidase. α-glucosidase can hydrolyze 4-nitrophenyl-α-D-pyranoglucoside (pNPG) into p-nitrophenol (pNP). pNP has strong ultraviolet absorption under alkaline conditions. The pNP concentration in the reaction solution was detected using an ELISA instrument, which reflects 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, 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] Take 50 μL of each pandan leaf extract sample and add it to a 1.5 mL EP tube. Set up 2 replicate wells for each sample. In addition, set up a sample control group, a blank reaction group, and a blank control group. The composition of each experimental group is 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 the 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 ultraviolet absorption value (OD value) at a wavelength of 450 nm was detected on an ELISA 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 It can be seen from the results that under the condition of an action concentration of 0.5 mg / mL, the α-glucosidase inhibitory activity of the extracts obtained by extraction with different concentrations of ethanol varies greatly, indicating that the concentration of the ethanol solution as the extraction solvent will significantly affect the α-glucosidase inhibitory activity of the pandan leaf extract obtained by extraction. In addition, the pandan leaf extract obtained by direct vacuum concentration and freeze drying of the ethanol extract has a low inhibitory activity on α-glucosidase, which is significantly lower than acarbose.

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

[0118] The results are as follows Figure 2 As shown, from Figure 2 It can be seen from the results that under the condition of an action concentration of 0.5 mg / mL, the inhibitory effect of pandan leaf extract 4 on α-glucosidase is the strongest, almost equal to acarbose, and significantly higher than pandan leaf extract 3, while the inhibitory effect of pandan leaf extract 5 on α-glucosidase is the weakest. This indicates that extraction with ethyl acetate after ethanol extraction can significantly improve the α-glucosidase inhibitory effect of pandan leaf extract.

[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), and the α-glucosidase inhibition activity experiment was performed and compared, 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] It can be seen from Examples 1 to 7 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 after extraction with 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 existing pandan leaf extract is usually prepared by using anhydrous ethanol or water as an extraction solvent (Comparative Examples 1-2). The obtained pandan leaf extract has a poor inhibitory effect on α-glucosidase. 50 The values ​​are 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, uses 15w / w% to 60w / w% ethanol solution for extraction, and the inhibitory effect of the pandan leaf extract obtained by direct concentration and drying of the extracting solution on α-glucosidase is enhanced. For example, the IC50 values ​​of the pandan leaf extract obtained by direct concentration and drying of 15%w / w%, 30w / w%, 45w / w% and 60w / w% ethanol solution extracts 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 inhibitory 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 value 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 is 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 inhibition 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 well 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 extracts obtained by the three extracts after extraction with ethyl acetate were 0.0447 W / W / W. 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 ethanol solution of appropriate concentration, the choice of extraction solvent has a very important influence on the inhibitory effect of α-glucosidase of the pandan leaf extract obtained. From the comparison between 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 a pandan leaf extract, and its IC value for inhibiting α-glucosidase is 50 The value is 444.2 μg / mL (Panlan leaf extract 3); and the extract is extracted using ethyl acetate (Example 2), dichloromethane (Example 3) and petroleum ether (Comparative Example 3) as extraction solvents to obtain the Panlan leaf extract that inhibits 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 compared with the pandan leaf extract prepared without extraction, the use of ethyl acetate and dichloromethane as solvents for extraction can significantly improve the α-glucosidase inhibition effect of the prepared pandan leaf extract. The corresponding IC 50 The IC value of pandan leaf extract inhibiting α-glucosidase was greatly reduced by using petroleum ether as the extraction solvent. 50 The value not only did not decrease, but 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 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 15w / w% to 60w / w% ethanol solution to extract pandan leaves to obtain an extract, and then further extracts with ethyl acetate or dichloromethane, which 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-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a pandan leaf extract, characterized in that: The following steps are involved: (1) taking pandan leaves, adding 15 w / w% to 60 w / w% ethanol solution for extraction 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, characterized in that In step (1), 30 w / w% to 60 w / w% ethanol solution is added for extraction.

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

4. The preparation method according to claim 1, characterized in that: 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, characterized in that: 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, characterized in that: 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 as claimed in claim 7 in the preparation of α-glucosidase inhibitors.

9. The use according to claim 8, characterized in that The application includes preparing a drug for preventing and / or treating diabetes; preferably, the diabetes is type II diabetes.

10. 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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