Preparation method of portulaca oleracea alcohol extract and application thereof in treatment of liver fibrosis

Abstract

The present application relates to the field of extraction method of traditional Chinese medicine Portulaca oleracea L and the influence of its extract on mouse liver fibrosis, especially to the research on alkaloids extracted from Portulaca oleracea L and its mechanism of action, belonging to the field of traditional Chinese medicine extraction method and application. The present application provides a separation method of alkaloids derived from Portulaca oleracea L, which adopts SP825L macroporous resin to elute n-butanol layer, and successfully extracts Portulaca oleracea L alkaloids POL-1. The method has only four steps, and the operation method is simple and fast, and the extraction process mainly adopts the principle of similar solubility. In addition, the research shows that the compound POL-1 has the effect of treating liver fibrosis, so the compound POL-1 of the present application can be used as a raw material for new drug development and pharmacological activity research, and also can be used for preparing candidate drugs for treating liver fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction methods and applications, relates to the field of extraction methods of traditional Chinese medicine Portulaca oleracea and the influence of the extract on mouse liver fibrosis, and particularly relates to the research on alkaloids extracted from Portulaca oleracea and the mechanism of action. BACKGROUND

[0002] At present, domestic and foreign experts have conducted in-depth research on the treatment of liver fibrosis from the aspects of chemical drugs, cytokines, genes and the like, hoping to find a suitable treatment method. However, these treatment plans cannot be widely applied in clinical practice due to their shortcomings. Traditional Chinese medicine treatment of chronic diseases has gradually gained international recognition, and relevant research shows that the most commonly used traditional Chinese medicine in clinical practice mainly focuses on promoting blood circulation, anti-inflammation, protecting cell membranes and promoting stem cell regeneration. Research has found that natural extracts extracted from herbal medicines are helpful for anti-fibrosis and become drugs, which is related to their good anti-inflammatory effect. Portulaca oleracea L. is a plant of Portulacaceae, and has the effects of detoxification, anti-inflammation, diuresis and analgesia. Portulaca oleracea contains rich natural therapeutic ingredients, including flavonoids, saponins and alkaloids. It has many pharmacological effects, such as liver protection, antioxidant, anti-inflammatory, antibacterial and the like. The activity research of Portulaca oleracea is in antioxidant, anti-inflammatory and liver protection and the like, and at present, the research on the effect of Portulaca oleracea extract on the treatment of liver fibrosis and the mechanism thereof is not clear, and therefore further exploration is needed. SUMMARY

[0003] In view of the problems existing in the prior art, the purpose of the present application is to provide a method which is simple, fast and environmentally friendly and has high extraction rate, and to explain the protection mode of Portulaca oleracea extract on liver fibrosis. The present application extracts alkaloid components (POL-1) from Portulaca oleracea. It is verified through experiments that the POL-1 treatment of liver fibrosis is achieved by improving the oxidative stress state, reducing inflammation, adjusting the TLR4 / NF-κB, Bcl-2 / Bax and TGF-β1 / Smad2 pathways, which can provide a scientific basis for developing POL-1 as a candidate drug for treating liver fibrosis.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0005] The present application provides an application of alkaloid POL-1 in Portulaca oleracea in the preparation of a medicine for treating liver fibrosis.

[0006] Further, the extraction method of the alkaloid POL-1 in Portulaca oleracea specifically comprises the following steps:

[0007] Step 1: 20.0 kg of dried Portulaca oleracea medicinal materials is crushed with a pulverizer to obtain Portulaca oleracea powder;

[0008] Step 2: The powder obtained in step 1 is soaked in methanol for 2 hours, and then extracted with methanol at a solid-liquid ratio of 1:8 by reflux for 3 times, 3 hours for the first time, 3 hours for the second time, and 3 hours for the third time. After the three times of extraction, the filtrate is obtained by filtering through 8 layers of gauze.

[0009] Step 3: The filtrate obtained in step 2 is concentrated by a rotary evaporator, and the concentrated extract 5 kg is obtained. 3 kg of the extract is dissolved in 2 L of distilled water.

[0010] Step 4: The 2 L aqueous solution obtained in step 3 is first extracted with 2 L of petroleum ether for 3 times. After the layers are separated, the lower aqueous solution of the three times of extraction is combined and concentrated by a rotary evaporator at 35 DEG C to obtain extract A. Then, the extract A is dissolved in 1 L of distilled water, extracted with 2 L of ethyl acetate for 3 times, and after the layers are separated, the lower aqueous solution of the three times of extraction is combined and concentrated by a rotary evaporator at 35 DEG C to obtain extract B. Finally, the extract B is dissolved in 1 L of distilled water, extracted with 1 L of saturated n-butanol for 3 times, and after the layers are separated, the upper saturated n-butanol layer of the three times of extraction is combined and concentrated by a rotary evaporator at 35 DEG C to obtain extract C 650 g. 600 g of the extract C is dissolved in 4000 mL of distilled water, and the obtained solution is eluted by SP825L macroporous resin. The elution steps and the method for judging the end of elution are as follows: flow rate: 1.5 mL / min, eluate is collected every 1 L, elution is stopped until the eluate is light yellow, and then the eluate is concentrated by a rotary evaporator. After concentration, the extract appears, and if there is still extract, continue to elute and collect with distilled water. When there is no extract, elute with 10% ethanol. The elution steps and the method for judging the end of elution with 10% ethanol are the same as those for the end of elution with distilled water. After the end, elute with 30% ethanol, and the elution steps and the method for judging the end of elution with 30% ethanol are the same as those for the end of elution with distilled water. After the end, elute with 50% ethanol, and the elution steps and the method for judging the end of elution with 50% ethanol are the same as those for the end of elution with distilled water. After the end, elute with 70% ethanol, and the elution steps and the method for judging the end of elution with 70% ethanol are the same as those for the end of elution with distilled water. Finally, the extract of 70% ethanol is dissolved in distilled water, and POL-1 is obtained by freeze-drying.

[0011] The application also provides an extraction method of the Portulaca oleracea L. alkaloid POL-1, which comprises the following steps:

[0012] Step 1: 20.0 kg of dried Portulaca oleracea L. medicinal material is crushed by a pulverizer to obtain Portulaca oleracea L. powder.

[0013] Step 2: The powder obtained in step 1 was soaked in methanol for 2 hours, and then extracted with methanol for 3 times at a solid-liquid ratio of 1:8, with 3 hours for the first extraction, 3 hours for the second extraction, and 3 hours for the third extraction. After the three extractions were combined, the mixture was filtered with 8 layers of gauze to obtain the filtrate;

[0014] Step 3: The filtrate obtained in step 2 was concentrated by rotary evaporation instrument. After concentration, 5 kg of extract was obtained, and 3 kg of the extract was dissolved in 2 L of distilled water.

[0015] Step 4: The 2 L aqueous solution obtained in step 3 was first extracted with 2 L of petroleum ether for 3 times. After the layers were separated, the lower aqueous solution of the three extractions was combined and concentrated by a rotary evaporator at 35 ℃ to obtain extract A. Then, extract A was dissolved in 1 L of distilled water and extracted with 2 L of ethyl acetate for 3 times. After the layers were separated, the lower aqueous solution of the three extractions was combined and concentrated by a rotary evaporator at 35 ℃ to obtain extract B. Finally, extract B was dissolved in 1 L of distilled water and extracted with 1 L of saturated n-butanol for 3 times. After the layers were separated, the upper saturated n-butanol layer of the three extractions was combined and concentrated by a rotary evaporator at 35 ℃ to obtain 650 g of extract C. 600 g of extract C was dissolved in 4000 mL of distilled water, and the obtained solution was eluted by SP825L macroporous resin. The elution steps and the method for determining the end of elution were as follows: flow rate: 1.5 mL / min, eluate was collected every 1 L, elution was stopped when the eluate was light yellow, and the eluate was concentrated by a rotary evaporator. After concentration, extract appeared, and if there was still extract, the elution was continued with distilled water. When there was no extract, 10% ethanol was used for elution. The elution steps and the method for determining the end of 10% ethanol elution were the same as those for the end of distilled water elution. After completion, 30% ethanol was used for elution, and the elution steps and the method for determining the end of 30% ethanol elution were the same as those for the end of distilled water elution. After completion, 50% ethanol was used for elution, and the elution steps and the method for determining the end of 50% ethanol elution were the same as those for the end of distilled water elution. After completion, 70% ethanol was used for elution, and the elution steps and the method for determining the end of 70% ethanol elution were the same as those for the end of distilled water elution. Finally, the extract of 70% ethanol was dissolved in distilled water, and POL-1 was obtained by freeze-drying.

[0016] Further, the method for preparing the spironolactone alkaloid POL-1 for treating liver fibrosis.

[0017] Further, the method for preparing the spironolactone alkaloid POL-1 for treating liver fibrosis.

[0018] The Portulaca oleracea extract (POL-1) in the present application is separated from SP825L macroporous resin, and the main component of the Portulaca oleracea extract is alkaloid. Alkaloid is one of the most important organic compounds in plants, and has many medicinal and pharmaceutical uses. Alkaloid is also a very important chemical substance in Portulaca oleracea. Previous reports show that the alkaloid of Portulaca oleracea has antioxidant effect, anti-inflammatory effect, anti-cholinesterase effect, etc. However, the effect of POL-1 on liver fibrosis and its mechanism are not clear, and therefore further exploration is needed.

[0019] Compared with the prior art, the present application has the following beneficial effects.

[0020] The research on the Portulaca oleracea extract (POL-1) in the present application for treating liver fibrosis has not been reported in the existing periodicals. The present application provides an isolation method of alkaloid (POL-1) derived from Portulaca oleracea, and successfully extracts the alkaloid of Portulaca oleracea. The method has only four steps, and the operation method is simple and fast. The extraction process mainly uses the principle of similar solubility. In addition, the research shows that the compound POL-1 has the effect of treating liver fibrosis, and therefore the compound POL-1 of the present application can be used as a raw material for the development of new drugs and the research of pharmacological activity, and can also be used for preparing candidate drugs for treating liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Chemical component analysis of POL-1 by positive mode UPLC-Q-TOF / MS.

[0022] Figure 2 Cell activity determination experiment. Wherein A is the effect of ET and different concentrations of POL-1 on the activity of LX-2 cells; B is the inhibition of POL-1 on the activation of LX-2 cells induced by TGF-β1.

[0023] Figure 3 HE staining experiment results of liver tissue pathological sections of mice in each group and detection results of serum ALT and AST levels. Wherein A is the HE staining diagram of the normal control group; B is the HE staining diagram of the model group; C is the HE staining diagram of the CT group; D is the HE staining diagram of the POL-1H group; E is the HE staining diagram of the POL-1L group; F is the level diagram of ALT in the serum of mice in each group; G is the level diagram of AST in the serum of mice in each group.

[0024] Figure 4Effects of POL-1 on liver histopathology and mRNA levels of collagen I and α-SMA in liver tissues of mice with liver fibrosis. Among them, A is the MT staining diagram of liver tissue sections of the normal control group; B is the MT staining diagram of liver tissue sections of the model group; C is the MT staining diagram of liver tissue sections of the CT group; D is the MT staining diagram of liver tissue sections of the POL-1H group; E is the MT staining diagram of liver tissue sections of the POL-1L group; F is the mRNA level of collagen I in the liver tissues of mice in each group; G is the mRNA level of α-SMA in the liver tissues of mice in each group.

[0025] Figure 5 POL-1 improves oxidative stress in CCl4-induced liver tissues of mice. Among them, A is the SOD level in the liver tissues of mice in each group; B is the GSH level in the liver tissues of mice in each group; C is the MDA level in the liver tissues of mice in each group.

[0026] Figure 6 POL-1 inhibits serum inflammatory factors in CCl4-induced fibrosis mice. Among them, A is the TNF-α level in the serum of mice in each group; B is the IL-6 level in the serum of mice in each group.

[0027] Figure 7 Expression of TLR4, MyD88, NF-κBp65, Bax, Bcl-2, TGF-β1 and Smad2 proteins in the liver tissues of mice in each group. Among them, A is the Western blotting analysis electrophoresis diagram of mice in each group; B is the TLR4 protein expression amount column chart of the liver tissues of mice in each group; C is the MyD88 protein expression amount column chart of the liver tissues of mice in each group; D is the NF-κBp65 protein expression amount column chart of the liver tissues of mice in each group; E is the Bax protein expression amount column chart of the liver tissues of mice in each group; F is the Bcl-2 protein expression amount column chart of the liver tissues of mice in each group; G is the TGF-β1 protein expression amount column chart of the liver tissues of mice in each group; H is the Smad2 protein expression amount column chart of the liver tissues of mice in each group. DETAILED DESCRIPTION

[0028] The following examples will facilitate the understanding of the present application, but these examples are only for illustrating the present application, and the present application is not limited to these. The operation methods in the examples are all conventional operation methods in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the present application are commercially available.

[0030] Example 1, preparation method of the purslane extract (POL-1) of the present application.

[0031] The extraction method of the purslane extract (POL-1) of the present application, the specific steps are:

[0032] Step 1: Take 20.0 kg of dried purslane medicinal materials, crush them with a pulverizer to obtain purslane powder;

[0033] Step 2: Soak the powder obtained in step 1 in methanol for 2 hours, then extract it by reflux for 3 times according to the solid-liquid ratio of 1:8, with 3 hours for the first extraction, 3 hours for the second extraction, and 3 hours for the third extraction. Combine the three extraction solutions, filter them with 8 layers of gauze to obtain a filtrate;

[0034] Step 3: Concentrate the filtrate obtained in step 2 with a rotary evaporator. After concentration, obtain 5 kg of extract, take 3 kg of the extract, and dissolve it with distilled water. After dissolution, obtain a 2 L aqueous solution;

[0035] Step 4: The 2L aqueous solution obtained in step 3 was first extracted 3 times with 2L of petroleum ether, after layer separation, the lower layer aqueous solution of the 3 extractions was combined, and the lower layer aqueous solution was concentrated by a 35°C rotary evaporator to obtain extract A; then extract A was dissolved with 1L of distilled water, extracted 3 times with 2L of ethyl acetate, after layer separation, the lower layer aqueous solution of the 3 extractions was combined, and the lower layer aqueous solution was concentrated by a 35°C rotary evaporator to obtain extract B; then extract B was finally dissolved with 1L of distilled water, extracted 3 times with 1L of saturated water n-butanol, after layer separation, the upper layer saturated n-butanol layer of the 3 extractions was combined, and the upper layer n-butanol was concentrated by a 35°C rotary evaporator to obtain extract C 650g; 600g of extract C was dissolved with 4000ml of distilled water, and the obtained solution was eluted by SP825L macroporous resin, first eluted with distilled water, the elution step and the method for judging the end of elution: flow rate: 1.5ml / min, collect eluent every 1L, elute to light yellow, pause collection, concentrate by rotary evaporator, after concentration, extract appears, if there is still extract, continue to elute and collect with distilled water, when there is no extract, elute with 10% ethanol. The elution step and the method for judging the end of 10% ethanol elution are consistent with the steps of the end of distilled water elution. After the end, elute with 30% ethanol, the elution step and the method for judging the end of 30% ethanol elution are consistent with the steps of the end of distilled water elution. After the end, elute with 50% ethanol, the elution step and the method for judging the end of 50% ethanol elution are consistent with the steps of the end of distilled water elution. After the end, elute with 70% ethanol, the elution step and the method for judging the end of 70% ethanol elution are consistent with the steps of the end of distilled water elution. Finally, dissolve the extract of 70% ethanol with distilled water, and freeze-dry to obtain POL-1. Whether there is still extract is determined by a rotary evaporator, after there is no extract, elute with 10% ethanol, whether there is still extract is determined by a rotary evaporator, after there is no extract, elute with 30% ethanol, whether there is still extract is determined by a rotary evaporator, after there is no extract, elute with 50% ethanol, whether there is still extract is determined by a rotary evaporator, after there is no extract, elute with 70% ethanol, the eluent of 70% ethanol is concentrated to obtain extract, finally, dissolve the extract of 70% ethanol with distilled water, and freeze-dry to obtain POL-1.

[0036] The chemical composition of POL-1 was analyzed using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS; Thermo U3000 mass spectrometer (Agilent 1290 system; Thermo Fisher Scientific, Massachusetts, USA). Conditions: flow rate: 0.7 mL / min, sample size: 10 μL, solvent system: A: 0.1% formic acid + water, B: formic acid + 0.1% acetonitrile.

[0037] Table 1 and Figure 1 The main chemical components in POL-1 were shown, 20 compounds were identified by comparing with related literatures and mass spectrometry database of natural products, and for NO. 1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 17, 19 and 20, they were alkaloids, which showed that the components eluted by SP825L macroporous resin were mainly alkaloids.

[0038] Table 1 UPLC-Q-TOF / MS analysis to determine the main components in POL-1

[0039] .

[0040] Example two, the effect of the Portulaca oleracea extract (POL-1) of the application on treating liver fibrosis.

[0041] I. Experimental method

[0042] (1) 40 male mice were randomly divided into normal control group (NC), model group (CCl4), CT group (positive drug colchicine tablets 0.1 mg / kg), and two dose POL-1 groups (POL-1H, 200 mg / kg; POL-1L, 50 mg / kg), 8 mice in each group. Except for the NC group, the rest of the groups were given 20% CCl4 / olive oil (1:4 dilution) for 6 weeks, 3 times a week for 6 weeks. The NC group was injected intraperitoneally with the same volume of olive oil at the same time. From the 7th week of modeling, the CT group and the high / low dose POL-1 group were given 0.1 mg / kg, 200 mg / kg and 50 mg / kg respectively, daily for 4 weeks. The NC group was given the same amount of normal saline by gavage for 4 weeks.

[0043] (2) Cell activity assay: LX-2 cells were inoculated in 96-well plates, and an appropriate amount of culture medium was added, and cultured to a certain density. They were divided into normal control group (NC), 0.5 mmol / L ethanol (ET) group and experimental group respectively treated with different concentrations (0, 20, 30, 50, 100, 150, 300, 500 and 800 μg / mL) of POL-1. According to the instructions of CCK-8 kit, cell toxicity was determined. The effect of POL-1 on TGF-β1-induced cell proliferation was detected by CCK-8 method. The experiment was divided into 6 groups: normal control group (NC), TGFβ1 induction group and 4 experimental groups (TGFβ1, ET, 30, 50 and 100 μg / mL of POL-1). Then the cell number was adjusted to 5×10 3NC group was replaced with complete medium, and the rest of the groups were replaced with complete medium containing 10 ng / mL TGF-β1. After 24 h, the cells in the experimental groups were cultured in ET and POL-1 medium at the specified concentrations. After 24 h, the medium was discarded, and CCK-8 (10 μL) reagent was added to each well to detect the absorbance at 450 nm.

[0044] (3) The liver tissue was fixed with 10% formalin and paraffin-embedded. The tissue was cut into 5 μm sections. The sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome (MT). The sections were detected by bright-field microscopy.

[0045] (4) Whole blood was stored in 2 mL EP tubes at 4°C for 2 h, and centrifuged at 3000 rpm for 15 min. The supernatant was the serum sample for subsequent serum transaminase determination. The serum ALT and AST levels were determined using a kit according to the manufacturer's instructions.

[0046] (5) Total RNA was extracted from liver tissue using TRIzol reagent (Invitrogen). The kit reverses the RNA into cDNA. RT-PCR detection was performed on the samples using SYBR Green Master Mix. The mRNA transcription expression levels of Collagen I, α-SMA and GAPDH were detected (Table 2).

[0047] Table 2 RT-PCR primer sequence list

[0048] .

[0049] (6) Oxidative stress parameter evaluation: Take an appropriate amount of mouse liver tissue, add ice physiological saline to wash off residual blood, and put it into a tissue homogenizer to prepare homogenate. Centrifuge at 3000 r · min-1 to obtain supernatant, and determine the contents of SOD, GSH and MDA.

[0050] (7) ELISA kit was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum.

[0051] (8) The liver tissue was taken, and Western blotting analysis was performed on TLR4, MyD88, NF-κBp65, Bax, Bcl-2, TGF-β1 and Smad2 proteins. All antibodies were diluted at 1:1000 at 4°C overnight, and GAPDH was used as a loading control.

[0052] II. Experimental results.

[0053] Figure 2A showed that CCK8 assays of 0.5 mmol / L ethanol (ET) and POL-1 had no cytotoxic effect on LX-2 cells. We selected POL-1 containing 70% ethanol as the research subject to investigate its effect on LX-2 cells, finding that dried POL-1 may retain ethanol. Assay, POL-1 contains approximately 3% ethanol, with a volumetric molar concentration of approximately 0.43 mmol / L; therefore, we selected 0.5 mmol / L ethanol (ET) as the carrier group. POL-1 treatment at 150 μg / mL showed inhibitory effects on LX-2 cells; therefore, we selected 30 μg / mL, 50 μg / mL, and 100 μg / mL doses of POL-1 to study the effects on LX-2 cell proliferation. Figure 2 The CCK8 assay showed that ET neither inhibited nor promoted the proliferation of LX-2 cells induced by 10 ng / mTGF-β1; while POL-1 inhibited the proliferative capacity of LX-2 cells stimulated by 10 ng / mTGF-β1. These results suggest that POL-1 has a certain therapeutic effect on animal liver fibrosis models.

[0054] Figure 3 AE showed H&E staining results, indicating that CCl4 injection caused pathological changes in the liver, including steatosis, necrosis, and fibrotic septa. Figure 3 B). The CCl4 plus POL-1 group significantly reduced liver damage (B). Figure 3 (D and E). In addition... Figure 3 F and G showed that, compared with the CCl4 group, POL-1 reduced serum ALT and AST levels. These results indicate that POL-1 has a protective effect on the liver.

[0055] Figure 4 AE results showed that CCl4 significantly increased collagen deposition, while POL-1 treatment significantly reduced collagen deposition in the liver. Furthermore, Figure 4 F and G show the PCR results, indicating that POL-1 significantly inhibited the mRNA levels of collagen I and α-SMA. These results suggest that POL-1 alleviates liver damage in mice and further demonstrates its role in inhibiting liver fibrosis.

[0056] Figure 5 AC assay was used to detect the effects of POL-1 on the levels of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in mouse liver. The CCl4 group significantly decreased SOD and GSH levels, while MDA activity was increased compared to the control group, indicating impaired antioxidant system. POL-1 significantly increased SOD and GSH levels and decreased MDA levels, thereby enhancing antioxidant capacity. These results demonstrate that POL-1 has a significant inhibitory effect on CCl4-induced oxidative damage in mouse liver.

[0057] Figure 6 A and B show that the serum TNF-α and IL-6 proinflammatory factor content of the CCl4 group is significantly higher than that of the blank group. At the same time, POL-1 can reduce the serum TNF-α and IL-6 levels. In summary, POL-1 inhibits CCl4-induced inflammation in mouse liver fibrosis.

[0058] In Figure 7 , CCl4 induced the protein expression of TLR4, MyD88, NF-κBp65, Bax, TGF-β1 and Smad2 to be significantly up-regulated, but down-regulated Bcl-2 protein in mouse liver. However, treating CCl4 mice with POL-1 reduced the proteins of TLR4, MyD88, NF-κBp65, Bax, TGF-β1 and Smad2, but up-regulated Bcl-2 protein in mouse liver. Our study shows that POL-1 treatment inhibits active fibrosis in mice by regulating the expression of related proteins on the TLR-4 / NF-κB, Bcl-2 / Bax and TGF-β1 / Smad2 pathways.

[0059] It can be seen that the purslane extract (POL-1) of the present application has a certain therapeutic effect on CCl4-induced mouse liver fibrosis; and can effectively reduce the oxidative stress level and inflammatory factor release in CCl4-induced mice, and is concentration-dependent. Our study also shows that POL-1 treatment inhibits active fibrosis in mice by regulating the expression of related proteins on the TLR-4 / NF-κB, Bcl-2 / Bax and TGF-β1 / Smad2 pathways.

[0060] In summary, the present application provides an extraction method of purslane (POL-1), which sequentially uses methanol extraction, petroleum ether, ethyl acetate and n-butanol solvent extraction. The n-butanol layer is eluted with SP825L macroporous resin to obtain a 70% ethanol component, and POL-1 is obtained. The method is simple, fast, and the yield of the compound obtained by the method is high. It is extracted from the roots of the traditional Chinese medicine purslane, and has the effect of treating liver fibrosis, so the POL-1 of the present application can be developed as a new drug of traditional Chinese medicine as a natural product, and has a broad prospect.

[0061] The above only describes the preferred embodiments of the present application, and is not used to limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting alkaloid components from purslane, specifically comprising the following steps: Step 1: Take 20.0 kg of dried purslane medicinal material, pulverize it with a pulverizer to obtain purslane powder; Step 2: Soak the powder obtained in Step 1 in methanol for 2 hours, and then reflux extract it 3 times at a solid-liquid ratio of 1:

8. The first extraction lasts for 3 hours, the second extraction lasts for 3 hours, and the third extraction lasts for 3 hours. Combine the three extracts and filter them through 8 layers of gauze to obtain the filtrate. Step 3: Concentrate the filtrate obtained in Step 2 using a rotary evaporator. After concentration, 5 kg of extract is obtained. Take 3 kg of the extract and dissolve it in distilled water to obtain 2 L of aqueous solution. Step 4: Extract the 2L aqueous solution obtained in Step 3 three times with 2L of petroleum ether. After separation, combine the lower aqueous layers from the three extractions and concentrate them using a rotary evaporator to obtain extract A. Then, dissolve extract A in 1L of distilled water and extract it three times with 2L of ethyl acetate. After separation, combine the lower aqueous layers from the three extractions and concentrate them using a rotary evaporator to obtain extract B. Finally, dissolve extract B in 1L of distilled water and extract it three times with 1L of saturated water and n-butanol. After separation, combine the upper saturated n-butanol layers from the three extractions and concentrate them using a rotary evaporator to obtain extract C. 650g and 600g of extract C were dissolved in 4000mL of distilled water. The resulting solution was eluted with SP825L macroporous resin. First, distilled water was used for elution until the solution turned pale yellow, at which point collection was stopped. The solution was then concentrated using a rotary evaporator. After concentration, extract was obtained. If extract was still present, it was eluted and collected with distilled water. When no extract remained, it was eluted with 10%, 30%, 50%, and 70% ethanol, respectively. Each time, elution was stopped until the solution turned pale yellow, and collection was stopped before proceeding to the next concentration of ethanol. After elution with 70% ethanol, the 70% ethanol extract was dissolved in distilled water and then freeze-dried to obtain the purslane alkaloid fraction.

2. The extraction method as described in claim 1, characterized in that, In step 4, the elution conditions for SP825L macroporous resin were: flow rate 1.5 mL / min, and the eluent was collected every 1 L.

3. The extraction method as described in claim 1, characterized in that, The therapeutic effect of the alkaloid components in purslane on liver fibrosis is achieved by improving oxidative stress, reducing inflammation, and modulating the TLR4 / NF-κB, Bcl-2 / Bax, and TGF-β1 / Smad2 pathways.

4. The use of the purslane alkaloid component prepared by the method of claim 1 in the preparation of a drug for treating liver fibrosis.

Images