Purslane isoquinoline alkaloid as well as preparation method and application thereof

Abstract

The invention discloses purslane isoquinoline alkaloid as well as a preparation method and application thereof. The purslane isoquinoline alkaloid is shown as a formula 1 or a formula 2, has GLP-1 receptor agonistic activity and can be used for preparing medicines and health-care foods for preventing and treating diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to isoquinoline alkaloids from purslane, their preparation methods, and applications. Background Technology

[0002] Diabetes mellitus is a systemic chronic metabolic disease determined by genetics. It is caused by a relative or absolute deficiency of insulin in the body, leading to disorders in the metabolism of carbohydrates, fats, and proteins. Its main characteristics are hyperglycemia and glycosuria. "Xiao Ke" is the traditional Chinese medicine term for diabetes. In his book *Gu Jin Lu Yan Fang*, Zhen Liyan of the Tang Dynasty defined Xiao Ke as: "Thirst with excessive drinking, frequent urination, and a taste that is sweet like bran without fat; all these are symptoms of Xiao Ke." This disease has now become a global pandemic, ranking third in mortality after cancer and cardiovascular diseases.

[0003] Purslane is a commonly used traditional Chinese medicine. It has a sour taste and cold properties, and is used to stop diarrhea, clear heat and detoxify, stop bleeding, and cool the blood. It is often used in folk medicine to treat diabetes. Records of purslane treating diabetes can be found in many traditional Chinese medicine classics, such as *Ben Cao Shi Yi*, *Ben Cao Zheng Yi*, and *Kai Bao Ben Cao*. However, current research on purslane's blood sugar-lowering effects mainly focuses on its polysaccharide components. Purslane contains various chemical components, such as alkaloids, flavonoids, organic acids, polysaccharides, terpenes, and coumarins. Among these, alkaloids, as important secondary metabolites in plants, possess various pharmacological activities and have received widespread attention from researchers; however, their blood sugar-lowering activity is rarely reported.

[0004] Exploring the theoretical and practical basis for the prevention and treatment of diabetes using components of purslane has significant socio-economic benefits. This invention demonstrates that two isoquinoline alkaloid components in purslane possess GLP-1 receptor agonist activity, enabling them to prevent and treat diabetes. Summary of the Invention

[0005] The present invention aims to provide a purslane isoquinoline alkaloid and its preparation method, and its application in the preparation of drugs and health foods for the prevention and treatment of diabetes.

[0006] The technical solution of the present invention is as follows:

[0007] A purslane isoquinoline alkaloid, as shown in Formula 1 or Formula 2:

[0008]

[0009] The method for preparing the purslane isoquinoline alkaloids shown in Formula 1 or Formula 2 of this invention is carried out according to the following steps:

[0010] (1) Fresh purslane is washed, dried and then crushed and sieved (20 mesh) to obtain purslane powder; the purslane powder is mixed with ethanol solution and stirred and soaked at room temperature for 1-3 hours, then heated to 70-100℃ and refluxed for 0.5-3 hours, then cooled to 30-50℃, filtered, and the filtrate is concentrated under reduced pressure until there is no alcohol smell to obtain extract;

[0011] The preferred ethanol solution is an aqueous solution with a volume fraction of 75% ethanol;

[0012] The preferred mass-to-volume ratio of purslane powder to ethanol solution is 1:7-9, g / mL;

[0013] (2) Disperse the extract obtained in step (1) with water, adjust the pH to 4-5 (adjust with hydrochloric acid), add chitosan, stir at room temperature for 2-6 hours, centrifuge (5000-10000 rpm, 5-10 min, 20℃), take the supernatant, adjust the pH to 7-9 (adjust with sodium hydroxide), add the extractant, stir and extract at 65-75℃ for 20-90 min, cool and let stand to separate the layers, take the upper extract, concentrate and dry under reduced pressure to obtain crude extract of purslane isoquinoline alkaloids;

[0014] The preferred extract is dispersed in 10 to 30 times its volume of water;

[0015] The preferred amount of chitosan is 1 / 200 to 1 / 300 of the mass of purslane powder in step (1);

[0016] The extractant is ethyl acetate;

[0017] (3) The crude extract of purslane isoquinoline alkaloids obtained in step (2) was subjected to amino silica gel column chromatography to separate the purslane isoquinoline alkaloids shown in Formula 1 and Formula 2.

[0018] The specific column chromatography operation is as follows: The crude extract of purslane isoquinoline alkaloids is dissolved in eluent and added to a chromatography column packed with amino silica gel at a flow rate of 0.02-0.08 BV / min. The mass ratio of the crude extract of purslane isoquinoline alkaloids to the amino silica gel packing is 1:20-50 (preferably 1:30). Elution separation is performed with eluent, and 3.5-4 BV and 5.5-6 BV of eluent are collected respectively. The solvent is removed by vacuum evaporation to obtain the purslane isoquinoline alkaloids shown in Formula 1 and Formula 2.

[0019] BV represents the bed volume;

[0020] The eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1.

[0021] The purslane isoquinoline alkaloids shown in Formula 1 or Formula 2 of this invention have GLP-1 receptor agonist activity and can be used to prepare drugs and health foods for the prevention and treatment of diabetes.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the fact that the two purslane isoquinoline alkaloids obtained by isolation have GLP-1 receptor agonist activity and can play a role in preventing and treating diabetes. Attached Figure Description

[0023] Figure 1 Effects of compound 2 on insulin secretion in rats at different glucose concentrations;

[0024] Note: ** indicates a comparison between two groups, P<0.01; *** indicates a comparison between two groups, P<0.001. Detailed Implementation

[0025] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] Example 1

[0027] Weigh 1000g of purslane powder (crushed to 20 mesh) into a round-bottom flask, add 8L of 75% ethanol, stir and soak at room temperature (25℃) for 3 hours, then heat under reflux at 100℃ for 3 hours. Cool the reaction solution to 30℃, filter, and collect the filtrate to obtain the purslane ethanol extract. Concentrate under reduced pressure to 200mL using a rotary evaporator (0.09MPa, 60℃). Disperse the concentrate in 2L of water, adjust the pH to 4 with hydrochloric acid, then add 4g of chitosan, stir at room temperature for 3 hours, and then centrifuge at 20℃ at a high speed of 5000 rpm. Remove the precipitate by stirring for 5 minutes to obtain the supernatant; transfer the supernatant to a round-bottom flask, adjust the pH to 7 with sodium hydroxide, add 1.6 L of ethyl acetate to the round-bottom flask, stir and extract the mixture at 70 °C for 60 minutes, then cool to 40 °C, place in a separatory funnel, and let stand for 30 minutes to separate the layers, obtaining the upper extract and the lower raffinate; concentrate and dry the upper extract under reduced pressure on a rotary evaporator (0.09 MPa, 55 °C) to obtain purslane isoquinoline alkaloid extract.

[0028] The isoquinoline alkaloid extract of Portulaca oleracea was dissolved in 50 mL of a petroleum ether:ethyl acetate (8:1) mixture and added to a 500 mL amino silica gel column (4.0 cm × 400 cm) at a flow rate of 0.022 BV / min for adsorption. After loading, the sample was eluted with a petroleum ether:ethyl acetate (8:1) mixture, and 3.5–4 BV and 5.5–6 BV eluates were collected, respectively. The eluates were concentrated under reduced pressure to remove the solvent, yielding Portulaca oleracea isoquinoline alkaloids 1 (150 mg) and 2 (210 mg).

[0029]

[0030] Compound NMR data

[0031] Compound 1: ESIMS: m / z 230.1, [M+H] + ; 1 H NMR(600MHz,D2O)δ:3.71(2H,t,J=6.2Hz,H-3),2.77(2H,t,J=6.2Hz,H-4),6.83(1H,s,H-5),7.37(1H,s,H-8),7.47(1H,br s,H-3'),6.80(1H,br s,H-4'),7.96(1H,br s,H-5'); 13 C NMR(150MHz,D2O)δ:156.8(C-1),39.9(C-3),24.9(C-4),116.1(C-5),155.1(C-6),144.0(C-7),1 18.3(C-8),135.1(C-9a),114.3(C-10b),143.8(C-2'),124.5(C-3'),114.2(C-4'),150.6(C-5').

[0032] Compound 2: ESIMS: m / z 222.2 [M+H] + ; 1 H NMR(600MHz,D2O)δ:6.65(1H,s,H-8),6.64(1H,s,H-5),4.35(1H,q,6.8Hz,H-1),3.45(1H,m,H-3a),3.25(1H,m,H -3b),2.89(2H,m,H-4),1.69(2H,m,H-1′),1.69(1H,m,H-2′),0.94(3H,d,5.6Hz,H-3′),0.92(3H,d,5.6Hz,H-4′); 13 CNMR(150MHz,D2O)δ:143.9(C-6),143.0(C-7),124.7(C-10b),123.8(C-9a),115.9(C-5),113. 7(C-8),52.9(C-1),39.1(C-3),24.0(C-4),43.0(C-1′),23.9(C-2′),20.6(C-3′),22.4(C-4′).

[0033] Example 2

[0034] Effects of Purslane Isoquinoline Alkaloids on Fasting Blood Glucose in Diabetic Rats

[0035] Twenty-four male STZ-induced diabetic rats were divided into four groups of six each: a normal control group, a model control group, and a group receiving purslane isoquinoline alkaloids (20 mg / kg intraperitoneally). The rats were administered the drug once daily for 23 consecutive days, and the following experiments were conducted.

[0036] Blood glucose levels were measured by tail clipping of rats in each group before administration and at 7 and 14 days after administration. The effects of purslane isoquinoline alkaloids on rat blood glucose are shown in Table 1. During the experiment, fasting blood glucose levels decreased in all groups, while the decrease was not significant in the model control group. Specifically, purslane isoquinoline alkaloid compounds 1 and 2 showed significant blood glucose reduction one week after administration, with a significant difference compared to the model control group (P<0.05). At two weeks after administration, the hypoglycemic effect of purslane isoquinoline alkaloids was very significant (P<0.01). This indicates that purslane isoquinoline alkaloids can significantly control the increase in fasting blood glucose levels in diabetic rats and have a hypoglycemic effect.

[0037] Table 1. Effects of Purslane Isoquinoline Alkaloids on Blood Glucose in Diabetic Rats (mmol / L) )

[0038]

[0039] Note: *，** Compared with the model control group, P<0.05, P<0.01; ▲ ， ▲▲ Compared with the normal control group, P<0.05, P<0.01.

[0040] Example 3

[0041] Effects of Purslane Isoquinoline Alkaloids on Serum Insulin in Diabetic Rats

[0042] The serum insulin levels in diabetic rats are shown in Table 2. The serum insulin levels in diabetic rats were significantly lower than those in normal rats (P<0.01). After treatment with these compounds, the serum insulin levels of compounds 1 and 2 were significantly increased compared to the model control group (P<0.01). This indicates that isoquinoline alkaloids from Portulaca oleracea have the effect of promoting the secretion of insulin by pancreatic β-cells in diabetic rats.

[0043] Table 2. Effects of Purslane Isoquinoline Alkaloids on Serum Insulin in Diabetic Rats

[0044]

[0045] Note: ** Compared with the model control group, P<0.01; ▲▲ Compared with the normal control group, P<0.01.

[0046] Example 4

[0047] Further experiments were conducted to investigate insulin levels in rats after high-sugar, high-fat treatment of compound 2 (C2) and subsequent intervention in insulin secretion from the pancreas. The results showed that compound 2, an isoquinoline alkaloid from purslane, possesses certain GLP-1R agonist activity. Figure 1 ).

[0048] The specific experiment was conducted as follows: First, 500 μL of 2.4 mmol / L glucose solution was added to each Eppendorf tube. Five islets (uniform in size and with smooth edges) were picked under a stereomicroscope and placed into the Eppendorf tubes, then incubated for 30 min. The supernatant was aspirated and discarded using a pipette (care should be taken not to remove the islets). Then, 500 μL of 2.4 mmol / L, 5.6 mmol / L, and 12.8 mmol / L glucose solutions were added to each group sequentially, and incubated for 30 min. The supernatant was then aspirated into pre-labeled Eppendorf tubes using the same method, mixed, sealed, and stored at 4°C as a control. Subsequently, 500 μL of 2.4 mmol / L glucose solution + 8 μM C2, 5.6 mmol / L glucose solution + 8 μM C2, and 12.8 mmol / L glucose solution + 8 μM C2 were added sequentially, and incubated for 30 min. Finally, remove the Ep tube, aspirate the supernatant into a numbered Ep tube, and perform insulin (INS) radioimmunoassay.

Claims

1. A purslane isoquinoline alkaloid, as shown in Formula 1 or Formula 2:

2. The method for preparing the purslane isoquinoline alkaloids as described in Formula 1 or Formula 2 as claimed in claim 1, characterized in that, Follow these steps: (1) Fresh purslane is washed, dried, crushed and sieved to obtain purslane powder; the purslane powder is mixed with ethanol solution, stirred and soaked at room temperature for 1-3 hours, then heated to 70-100℃ and refluxed for 0.5-3 hours, then cooled to 30-50℃, filtered, and the filtrate is concentrated under reduced pressure until there is no alcohol smell to obtain extract. (2) Disperse the extract obtained in step (1) with water, adjust the pH to 4-5, add chitosan, stir at room temperature for 2-6 hours, centrifuge, take the supernatant, adjust the pH to 7-9, add the extractant, stir and extract at 65-75℃ for 20-90 minutes, cool and let stand to separate the layers, take the upper extract, concentrate and dry under reduced pressure to obtain crude extract of purslane isoquinoline alkaloids; The extractant is ethyl acetate; (3) The crude extract of purslane isoquinoline alkaloids obtained in step (2) was subjected to amino silica gel column chromatography to separate the purslane isoquinoline alkaloids shown in Formula 1 and Formula 2.

3. The preparation method according to claim 2, characterized in that, In step (1), the ethanol solution is an aqueous solution of 75% ethanol by volume.

4. The preparation method according to claim 2, characterized in that, In step (1), the mass-to-volume ratio of purslane powder to ethanol solution is 1:7-9, g / mL.

5. The preparation method according to claim 2, characterized in that, In step (2), the extract is dispersed in 10 to 30 times its volume of water.

6. The preparation method according to claim 2, characterized in that, In step (2), the amount of chitosan used is 1 / 200 to 1 / 300 of the mass of purslane powder in step (1).

7. The preparation method according to claim 2, characterized in that, The operation of column chromatography in step (3) is as follows: The crude extract of purslane isoquinoline alkaloids was dissolved in eluent and added to a chromatography column packed with amino silica gel at a flow rate of 0.02–0.08 BV / min. The mass ratio of the crude extract of purslane isoquinoline alkaloids to the amino silica gel packing was 1:20–50. The sample was eluted with eluent, and 3.5–4 BV and 5.5–6 BV of eluent were collected respectively. The solvent was removed by vacuum evaporation to obtain the purslane isoquinoline alkaloids shown in Formula 1 and Formula 2. BV represents the column volume; The eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:

1.

8. The use of purslane isoquinoline alkaloids as described in Formula 1 or Formula 2 as claimed in claim 1 in the preparation of drugs and health foods for the prevention and treatment of diabetes.

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