Hypoglycemic and hypolipidemic composition derived from pandan leaves as well as preparation method and application of hypoglycemic and hypolipidemic composition

Abstract

The invention discloses a composition for reducing blood sugar and blood fat from pandan leaves as well as a preparation method and application of the composition. The pharmaceutical composition is prepared from p-coumaric acid, rutin, kaempferol-3-O-rutinoside, the pandamariactonine-A, the pandamariactone-1 and the pandamariactone-2, and the pharmaceutical composition is prepared from the following raw materials: the p-coumaric acid and the rutin. The composition with a preferable mass ratio has a certain synergistic effect, and on the premise of consistent total dosage of administration, the effects of improving insulin resistance and reducing fat deposition of the composition are superior to those of monomeric compounds such as p-coumaric acid, rutin and kaempferol-3-O-rutinoside, and are also superior to those of a pandan alkaloid composition consisting of pandan actonine-A, pandan actone-1 and pandan actone-2, so that the composition has a good application prospect. It is shown that all the components of the composition have a synergistic effect.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a pandan leaf-derived blood sugar and blood lipid lowering composition, a preparation method and uses thereof. Background Art

[0002] In recent years, diabetes and dyslipidemia have become serious metabolic diseases worldwide. Diabetes is a chronic metabolic disease caused by insufficient or dysfunctional insulin secretion. Dyslipidemia is a significant risk factor for cardiovascular disease and is closely associated with atherosclerosis, coronary heart disease, and stroke. Diabetes and dyslipidemia often coexist and interact, exacerbating insulin resistance and increasing the risk of cardiovascular disease. With changing lifestyles and an aging population, the incidence of diabetes and hyperlipidemia continues to rise, with a trend toward younger individuals.

[0003] Pandanus amaryllifolius Roxb., also known as fragrant pandan, is the only aromatic plant in the Pandanaceae family. Its unique "zongzi-scented" flavor is widely used in food processing. Pandan was introduced to China in the 1950s and has been successfully cultivated in Hainan, Taiwan, Yunnan, and Guangdong. As a typical tropical cash crop, pandan leaves not only have high economic value but also show promising development prospects. Traditional medicine believes that they can be used to treat hyperglycemia, hypertension, gout, and rheumatism.

[0004] Current research on the hypoglycemic and hypolipidemic effects of pandan leaves is limited to preliminary efficacy verification of crude extracts, lacking systematic research on the screening of active sites. The present invention, based on the screening of active sites in pandan leaves, provides an effective hypoglycemic and hypolipidemic composition. Summary of the Invention

[0005] The present invention aims to provide a pandan leaf-derived hypoglycemic and hypolipidemic composition, a preparation method thereof and uses thereof.

[0006] The hypoglycemic and hypolipidemic composition of the present invention contains p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1 and pandamarilactone-2.

[0007] Preferably, the composition contains, by mass, 15-35% p-coumaric acid, 5-20% rutin, 5-20% kaempferol-3-O-rutinoside, 15-20% pandamarilactonine-A, 15-20% pandamarilactone-1, and 15-20% pandamarilactone-2.

[0008] Preferably, the mass ratio of p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 is 33:5:8:19:16:19.

[0009] Preferably, the medicine or health product prepared from the above-mentioned hypoglycemic and hypolipidemic composition contains a pharmaceutically or food-acceptable carrier, solvent, diluent, excipient or other medium, and can be prepared into corresponding powders, granules, capsules, injections, oral solutions or tablets according to different needs.

[0010] The present invention also provides a method for preparing the above-mentioned hypoglycemic and hypolipidemic composition, which comprises uniformly mixing the components.

[0011] The present invention also provides the use of the above-mentioned hypoglycemic and hypolipidemic composition in the preparation of medicines and / or health products with hypoglycemic and hypolipidemic effects.

[0012] The above composition has the effect of alleviating insulin resistance and fat deposition, and thus has application value in lowering blood sugar and blood lipids. Its application includes but is not limited to preparing the composition into a medicine and / or health product with the effect of lowering blood sugar and blood lipids.

[0013] The present invention has the following advantages:

[0014] (1) The present invention systematically screened the active parts of Pandan leaves based on the insulin resistance and fat deposition cell models commonly used in the art, and for the first time identified its active parts for lowering blood sugar and blood lipids.

[0015] (2) By analyzing the components of the active part of pandan leaves that lowers blood sugar and blood lipids, a composite material was constructed using the main compounds in the active part of pandan leaves. The resulting composite material has better activity than the active part of pandan leaves.

[0016] (3) The composition with the preferred mass ratio has a certain synergistic effect. Under the premise of the same total dosage, its effect on improving insulin resistance and reducing fat deposition is better than that of the monomer compounds of p-coumaric acid, rutin and kaempferol-3-O-rutinoside, and is also better than the pandan alkaloid composition composed of pandamarilactonine-A, pandamarilactone-1 and pandamarilactone-2, indicating that there is a synergistic effect among the components of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the result graph of the effects of various extracts of Pandan leaves on triglyceride deposition in HePG2 cells;

[0018] Figure 2 This is the result graph of the effects of various extracts from pandan leaves on glucose consumption in HePG2 cells;

[0019] Figure 3 This is the liquid chromatography of the active fraction of Pandan leaves extracted with 95% ethanol and then with ethyl acetate in Comparative Example 1. In the figure, labels 1: pandamarilactonine-A, 2: pandamarilactone-1, 3: pandamarilactone-2, 4: p-coumaric acid, 5: rutin, 6: kaempferol-3-O-rutinoside;

[0020] Figure 4 This is the liquid phase spectrum of the pandan leaf alkaloid composition in Comparative Example 2, marked in the figure as 1: pandamarilactonine-A, 2: pandamarilactone-1, and 3: pandamarilactone-2;

[0021] Figure 5 This is a liquid phase spectrum of the composition constructed in Example 1, where 1: pandamarilactonine-A, 2: pandamarilactone-1, 3: pandamarilactone-2, 4: p-coumaric acid, 5: rutin, 6: kaempferol-3-O-rutinoside;

[0022] Figure 6 This is a liquid phase spectrum of the composition constructed in Example 2, where 1: pandamarilactonine-A, 2: pandamarilactone-1, 3: pandamarilactone-2, 4: p-coumaric acid, 5: rutin, 6: kaempferol-3-O-rutinoside are marked;

[0023] Figure 7 Graph showing the effects of comparative examples, examples, and coumaric acid, rutin, and kaempferol-3-O-rutinoside monomers on triglyceride deposition in HePG2 cells;

[0024] Figure 8 The figures show the effects of the comparative examples, examples, and coumaric acid, rutin, and kaempferol-3-O-rutinoside monomers on the glucose consumption of HePG2 cells; DETAILED DESCRIPTION

[0025] Unless otherwise specified, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.

[0026] 4-Coumaric acid (4-Hydroxycinnamic acid), molecular formula is C9H8O3, CAS number is: 501-98-4, structural formula is:

[0027]

[0028] Rutin, molecular formula is C 27 H 30 O 16 , CAS number: 153-18-4, structural formula:

[0029]

[0030] Kaempferol 3-rutinoside, molecular formula C 27 H 30 O 15 , CAS number: 17650-84-9, structural formula:

[0031]

[0032] Pandamarilactonine-A, molecular formula: C 18 H 23 NO4, CAS number: 303008–80-2, structural formula:

[0033]

[0034] Pandamarilactone-1, molecular formula: C 18 H 23 NO4, CAS number: 152606–63-8, structural formula:

[0035]

[0036] Pandamarilactone-2, molecular formula: C 18 H 23 NO4, CAS number: 1814904–51-2, structural formula:

[0037]

[0038] The essential contents of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0039] Comparative Example 1

[0040] In order to clarify the active parts and components of pandan leaves for lowering blood sugar and blood lipids, a cell experiment system was used to evaluate the effects of different extracted parts of pandan leaves on indicators such as glucose uptake, lipid accumulation, and insulin sensitivity. The active parts with clear effects were screened out, and the components of the active parts were studied.

[0041] 1. Separation of different extraction parts of pandan leaves

[0042] Weigh about 50 g of dried pandan leaf sample (cut into small segments) and place it in a round-bottom flask. Add 1000 mL and 800 mL of water respectively and heat to a slight boiling point for extraction twice, each extraction for 1 hour. Filter and combine the two extracts. After evaporation to dryness, the pandan leaf water extract (Fr1,50 g→9.69 g) was obtained.

[0043] Approximately 250g of dried pandan leaves (cut into small pieces) were weighed and extracted twice with 5L and 4L of water, respectively, heated to a slight boil. Each extraction lasted 1 hour. The extracts were filtered, combined, concentrated, and separated using a macroporous resin. Elution was performed sequentially with water, 20% ethanol, 40% ethanol, and 60% ethanol, with six column volumes of each solvent used. The eluents were evaporated to dryness to yield Fr2-Fr5 (250g → 40.68g, 3.94g, 2.92g, 0.60g, respectively).

[0044] Weigh about 50 g of dried pandan leaf powder and place it in a round-bottom flask. Add 500 mL of 95% ethanol solution each time and heat in a water bath until it is slightly boiling. Extract twice, each time for 1 hour. Filter, combine the two extracts, and evaporate to dryness to obtain the 95% ethanol extract of pandan leaves (Fr6, 50 g→7.92 g).

[0045] Weigh approximately 100g of dried pandan leaf powder and place it in a round-bottom flask. Add 1000mL of 95% ethanol solution, heating in a water bath until slightly boiling. Extract twice, each time for 1 hour. Filter, combine the two extracts, concentrate until alcohol-free, and transfer to a separatory funnel. Extract with appropriate amounts of petroleum ether, ethyl acetate, and n-butanol three times with each solvent. The remaining liquid is the aqueous fraction. Evaporate the extracts from each fraction to dryness to obtain Fr7-Fr10 (100g → 4.35g, 1.84g, 2.05g, 3.67g, respectively).

[0046] Weigh about 50 g of dried pandan leaf powder and place it in a round-bottom flask. Add 500 mL of ethyl acetate each time and heat in a water bath until it is slightly boiling. Extract twice, each time for 1 hour. Filter, combine the two extracts, and evaporate to dryness to obtain the pandan leaf ethyl acetate extract (Fr11, 50 g→1.08 g).

[0047] 2. Effects of extracts from various parts of Pandan leaves on fat deposition in HepG2 cells

[0048] Each extract was tested separately using the free fatty acid-induced HepG2 cell fat deposition model.

[0049] Preparation of free fatty acids: Weigh 1g and 2g of fatty acid-free bovine serum albumin (BSA), add 5ml of deionized water, and dissolve to obtain 20% and 40% BSA solutions, respectively. Weigh 0.06g of sodium hydroxide (NaOH) and add 10ml of deionized water to dissolve to obtain a 0.15mM NaOH solution. Divide the solution into two equal portions, add 63.47μl of oleic acid to one portion, and 0.025624g of palmitic acid to the other portion. Saponify in a 75°C waterbath for 30 minutes to obtain 40mM oleic acid solution and 20mM palmitic acid solution. Immediately mix the 40% and 20% BSA solutions to obtain 20mM oleic acid and 10mM palmitic acid solutions. Adjust the pH to a value similar to that of the culture medium with hydrochloric acid. After filter sterilization, store at 4°C until use. Before use, mix the oleic acid solution and the palmitic acid solution and dilute with culture medium to the desired concentration. Weigh 1.5 g of BSA, add 10 mL of deionized water, dissolve and obtain a 15% BSA solution as a control solvent, filter and sterilize, and store at 4°C for future use.

[0050] Based on the experimental requirements, a free fatty acid concentration (0.6 mM, oleic acid:palmitic acid = 2:1) with minimal effect on cell viability was selected to induce a cellular fat deposition model. HepG2 cells in the logarithmic growth phase were seeded at an appropriate density in a 24-well cell culture plate and incubated overnight at 37°C in a 5% CO2 incubator. After the cells adhered, they were grouped and treated as follows:

[0051] (1) Solvent control group: DMEM complete medium containing 0.6% BSA and 0.1% DMSO was added;

[0052] (2) Model group: complete culture medium (containing 0.6% BSA and 0.1% DMSO) containing 0.4 mM oleic acid and 0.2 mM palmitic acid, respectively;

[0053] (3) Positive drug group: complete culture medium (containing 0.6% BSA and 0.1% DMSO) containing 0.4 mM oleic acid and 0.2 mM palmitic acid, respectively, and 50 μM lovastatin;

[0054] (4) Pandan leaf extract low, medium, and high dose groups: complete culture medium (containing 0.6% BSA and 0.1% DMSO) containing 0.4 mM and 0.2 mM oleic acid and palmitic acid, respectively, and appropriate concentrations of Pandan leaf extract was added;

[0055] Each group treated cells for 24 hours, and the triglyceride content absorbed by HepG2 cells was detected using a triglyceride detection kit and normalized with the total protein content. The results are shown in the attached figure. Figure 1 shown.

[0056] The experimental results showed that the ethyl acetate extraction part of Fr8, i.e., pandan leaves, extracted with 95% ethanol had the most significant effect in reducing fat deposition.

[0057] 3. Effects of extracts from various parts of Pandan leaves on insulin resistance in HepG2 cells

[0058] Each extract was tested using a high-glucose + palmitic acid-induced insulin-resistant HepG2 cell model. Insulin-resistant cells exhibit decreased glucose uptake and utilization, leading to abnormal glucose metabolism. Therefore, this study used HepG2 glucose consumption as a measurement indicator.

[0059] The preparation method of palmitic acid mother liquor is the same as above.

[0060] HepG2 cells in the logarithmic growth phase were seeded at an appropriate density in a 24-well cell culture plate and incubated overnight at 37°C in a 5% CO2 cell culture incubator. After the cells adhered to the wall, the cells were grouped and treated as follows:

[0061] Solvent control group: DMEM low-glucose basal medium (glucose concentration 5 mM) containing 0.1% BSA and 0.1% DMSO was added;

[0062] Model group: high-glucose basal medium (glucose concentration 25 mM, containing 0.1% BSA and 0.1% DMSO) containing 0.1 mM palmitic acid was added;

[0063] Positive drug group: high-glucose basal medium (glucose concentration 25 mM, containing 0.1% BSA and 0.1% DMSO) containing 0.1 mM palmitic acid and 1 mM metformin was added;

[0064] Low-, medium-, and high-dose groups of pandan leaf extract: high-glucose basal medium (glucose concentration 25 mM, containing 0.1% BSA and 0.1% DMSO) containing 0.1 mM palmitic acid and appropriate concentrations of pandan leaf extract was added;

[0065] After 24 hours of treatment, the cells in each group were replaced with basal medium containing 10 mM glucose and cultured for 12 hours. The glucose consumption was measured and normalized with the cell viability test results. The results are shown in the attached figure. Figure 2 shown.

[0066] Compared with the model group, Fr4 (the fraction extracted from Pandan leaves with water and eluted with macroporous resin and eluted with 40% ethanol), Fr8 (the fraction extracted from Pandan leaves with 95% ethanol and ethyl acetate), Fr9 (the fraction extracted from Pandan leaves with 95% ethanol and n-butanol), and the positive control metformin group increased glucose consumption in HepG2 cells. Fr8 and Fr9 showed comparable effects on increasing glucose consumption. Combined with the results of lipid-lowering experiments, Fr8 (the fraction extracted from Pandan leaves with 95% ethanol and then ethyl acetate) was identified as the active fraction in reducing fat deposition and alleviating insulin resistance.

[0067] 4. Analysis of active components of Pandan leaves

[0068] UFLC-Triple TOF-MS / MS was used to qualitatively analyze the pandan leaves extracted with 95% ethanol and then ethyl acetate, and to quantitatively analyze six major compounds. The liquid chromatography conditions were a C18 column (Accucore C18 150×2.1 mm, 2.5 μm, SN: A266008, ACE, UK), with a column temperature of 40°C, a 0.1% formic acid solution as mobile phase A, and methanol as mobile phase B. The gradient elution rate was: 0 to 30 min, A: 10% to 70%; 30 to 35 min, A: 70% to 100%; 35 to 45 min, A: 100%; and a flow rate of 0.3 mL / min. Mass spectrometry was performed using an electrospray ionization (ESI) source, information-dependent acquisition (IDA), and dynamic background subtraction (DBS). Detection was performed in both positive and negative ion modes. ESI source parameters: GS1 55 psi, GS2 55 psi, CUR 35 psi, TEM 550°C, positive mode ISVF 5500 V, negative mode ISVF-4500 V. Compound-related parameters were DP 80 V, CE 35 eV, CES 15 eV. Both the nebulizer and auxiliary gases were nitrogen. The mass-to-charge (m / z) range was 50–1500 Da. 1.2 Software collects data.

[0069] A total of 27 compounds were detected in the active part of pandan leaves, i.e., the part extracted with 95% ethanol and then with ethyl acetate, mainly alkaloids, flavonoids, and organic acids, as shown in Table 1.

[0070] Table 1

[0071]

[0072]

[0073] The active part of Pandan leaves, which is extracted with 95% ethanol and then with ethyl acetate, contains 6 main compounds with high content. The HPLC chromatogram is shown in the attached Figure 3 Quantitative analysis of these six compounds by liquid chromatography-mass spectrometry revealed the following: p-coumaric acid 7.82%, rutin 1.33%, kaempferol-3-O-rutinoside 1.96%, pandamarilactonine-A 4.50%, pandamarilactone-1 3.86%, and pandamarilactone-2 4.55%. The mass ratio of these compounds was approximately 33:5:8:19:16:19, and this ratio was subsequently used to construct the composite.

[0074] Comparative Example 2

[0075] The pandan alkaloid composition was constructed with a mass ratio of pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 of 19:16:19. The difference between the comparative example and the preferred composition is that it only contains alkaloids. The HPLC chromatogram is shown in the attached Figure 4 .

[0076] Example 1

[0077] The composition was constructed by mixing p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 in a mass ratio of 33:5:8:19:16:19. The HPLC chromatogram is attached. Figure 5 .

[0078] Example 2

[0079] The composition was constructed by mixing p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 in a mass ratio of 1:1:1:1:1:1. The HPLC chromatogram is attached. Figure 6 .

[0080] The free fatty acid-induced HePG2 cell fat deposition model and the high sugar + palmitic acid-induced HePG2 cell insulin resistance model were used to evaluate the activities of the pandan leaf 95% ethanol extraction followed by ethyl acetate extraction of the Fr8 fraction in Comparative Example 1, Comparative Example 2, Example 1, Example 2, as well as p-coumaric acid, rutin, and kaempferol-3-O-rutinoside in reducing fat deposition and alleviating insulin resistance. The results of triglyceride detection in cells are shown in the attached figure. Figure 7 , the cell glucose consumption capacity is shown in the attached Figure 8The experimental results showed that the active fraction of Pandan leaves in Comparative Example 1, Comparative Example 2, Example 1, Example 2, as well as p-coumaric acid, rutin, and kaempferol-3-O-rutinoside can effectively reduce fat deposition in HePG2 cells and alleviate insulin resistance.

[0081] The inhibition rate of fat deposition in HePG2 cells and the promotion rate of glucose consumption in HePG2 cells when the administration concentration of each composition or monomer was 100 μg / ml are shown in Table 2.

[0082] Table 2

[0083] Serial number Test samples Fat deposition inhibition rate Glucose consumption promotion rate 1 Active fraction of pandan leaves in comparative example 1 13.73±2.41％ 13.06±2.77％ 2 Comparative Example 2 19.75±3.70％ 24.60±3.93％ 3 Example 1 38.52±4.93％ 55.41±5.75％ 4 Example 2 30.86±3.11％ 45.63±6.96％ 5 p-Coumaric acid 25.12±3.97％ 27.29±4.33％ 6 Rutin 31.18±3.53％ 51.29±5.84％ 7 Kaempferol-3-O-rutinoside 27.69±5.49％ 39.39±5.06％

[0084] From the above results, it can be seen that the present invention is based on the free fatty acid-induced HePG2 cell fat deposition model and the high sugar + palmitic acid-induced HePG2 cell insulin resistance model commonly used in the art, and screens out the blood sugar and lipid-lowering active part of pandan leaves. The composition constructed by the main components of the active part, coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 can effectively reduce fat deposition and improve insulin resistance. The fat deposition inhibition rate and glucose consumption promotion rate of the composition with the preferred mass ratio (33:5:8:19:16:19) are significantly better than those of the pandan leaf active part, the pandan leaf alkaloid composition, and the monomer compounds of p-coumaric acid, rutin, and kaempferol-3-O-rutinoside, indicating that there is a synergistic effect between the components of the composition.

[0085] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A blood sugar and blood lipid lowering composition, characterized in that: Contains p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1 and pandamarilactone-2.

2. The hypoglycemic and hypolipidemic composition according to claim 1, characterized in that By mass fraction, it contains p-coumaric acid 15-35%, rutin 5-20%, kaempferol-3-O-rutinoside 5-20%, pandamarilactonine-A 15-20%, pandamarilactone-1 15-20% and pandamarilactone-2 15-20%.

3. The hypoglycemic and hypolipidemic composition according to claim 1, characterized in that The mass ratio of p-coumaric acid, rutin, kaempferol-3-O-rutinoside, pandamarilactonine-A, pandamarilactone-1, and pandamarilactone-2 is 33:5:8:19:16:

19.

4. The hypoglycemic and hypolipidemic composition according to claim 1, characterized in that The medicine or health care product prepared from the hypoglycemic and hypolipidemic composition comprises a pharmaceutically or food-acceptable carrier, solvent, diluent, excipient or other medium.

5. The hypoglycemic and hypolipidemic composition according to claim 1, characterized in that The medicine or health care product prepared from the hypoglycemic and hypolipidemic composition can be prepared into corresponding powders, granules, capsules, injections, oral solutions or tablets according to different needs.

6. A method for preparing the hypoglycemic and hypolipidemic composition according to claim 1, 2, 3, 4 or 5, characterized in that: Mix all ingredients evenly.

7. Use of the hypoglycemic and hypolipidemic composition according to claim 1, 2, 3, 4 or 5 in the preparation of medicines and / or health products having hypoglycemic and hypolipidemic effects.

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