Preparation method and application of punicalagin extract

Abstract

The invention provides a preparation method and application of punicalagin extract. According to the method, pomegranate rind is taken as a raw material, pretreatment, extraction, purification and concentration are carried out, the purpose of enriching punicalagin mainly containing an active ingredient in the pomegranate rind is achieved, and finally the punicalagin extract with the content of 30%-90% is obtained. The method has the characteristics of convenience in operation, controllable conditions, economy and high efficiency, and is convenient for rapid enrichment of punicalagin serving as an effective main component in pomegranate rind; the problems that in the conventional process of enriching punicalagin serving as a main component of pomegranate rind, the process is complex, operation is tedious, cost is high, potential safety risks are caused by introduction of more other reagents, and application is difficult to amplify are solved to a certain extent. The pharmacodynamic effect of the punicalagin extract in the anti-colitis aspect is verified through oral administration and enema administration modes, and the medicinal value and application potential of the punicalagin extract serving as a raw material medicine for developing various preparations and dosage forms are disclosed.

Description

Technical Field

[0001] The invention relates to a preparation method of a punicalagin extract and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a nonspecific intestinal inflammation caused by excessive inflammatory response, and has been defined by the World Health Organization as a refractory disease. Typical clinical manifestations are bloody diarrhea, abdominal pain, etc. Pathological manifestations are continuous and diffuse inflammation of the colorectal mucosa and submucosa. Lesions are more common in the colorectum, and the course of the disease is long, prone to recurrence, and has a certain risk of cancer. It has a serious impact on the patient's quality of life and causes long-term physical and psychological damage to the patient. However, the exact cause is still unclear and difficult to cure. In recent years, the incidence of UC has increased year by year worldwide, especially in Asia.

[0003] At present, conventional treatment drugs for ulcerative colitis (UC) have certain limitations and side effects, while natural plant extracts are generally more effective and have fewer side effects. Pomegranate peel is the dried peel of the pomegranate (Punica granatum L.), a plant of the Punicaceae family. It is rich in polyphenol compounds and is a traditional Chinese medicine in my country. It has the effects of astringent diarrhea, hemostasis, and anthelmintic. It is commonly used for chronic diarrhea, chronic dysentery, blood in the stool, prolapse of the anus, metrorrhagia, leucorrhea, and abdominal pain caused by worms. Punicalagin is the main active ingredient in pomegranate peel. It belongs to both polyphenol compounds and glycoside compounds. It has multiple pharmacological effects such as antioxidant, anticancer, antibacterial, antiviral, and anti-inflammatory. It is widely used in the fields of food, daily chemicals, and medicine.

[0004] Most of the existing macroporous resin purification processes for pomegranate peel require the addition of other reagents to adjust the pH value and improve the purification efficiency, which will lead to potential safety risks. In addition, the existing extraction and purification processes basically take the polyphenol content as the ultimate goal of extraction and purification, emphasizing the efficacy of polyphenols. Few of them focus on the content of punicalagin and use it as the main indicator in the macroporous resin purification stage. There are also many preparation processes for high-purity punicalagin, but generally they require complicated and cumbersome processes and special or rare equipment, and require the addition of more toxic and harmful reagents, which consumes a lot of energy. In order to obtain high purity, the consideration of economy and environmental protection has to be abandoned. Summary of the invention

[0005] The purpose of the present invention is to obtain an active part rich in punicalagin, and the present invention proposes a preparation method of punicalagin extract and its application. The method uses pomegranate peel as raw material, and after pretreatment, extraction, purification and concentration, the purpose of enriching the pomegranate peel mainly containing the active ingredient punicalagin is achieved. The punicalagin extract with a punicalagin content of 30%-90% is finally obtained. The method is easy to operate, controllable conditions, and economical and efficient. It is convenient for the rapid enrichment of the effective main component punicalagin in the pomegranate peel, and to a certain extent solves the problems of complex process, cumbersome operation, high cost, introduction of more other reagents, potential safety risks, and difficulty in amplification of application in the conventional enrichment process of the main component punicalagin of the pomegranate peel. The present invention verifies the therapeutic effect of punicalagin extract in anti-colitis through two modes of oral administration and enema, revealing its medicinal value and application potential as a raw material for the development of various preparations and dosage forms.

[0006] The preparation method of the punicalagin extract of the present invention is carried out according to the following steps:

[0007] a. Crush or grind the dried pomegranate peel and pass it through a 30-100 mesh sieve to obtain pomegranate peel lumps or powder;

[0008] b. The pomegranate peel block or powder obtained in step a is added with a 30%-90% ethanol solution with a solid-liquid ratio of 1:5-1:30 for extraction at a temperature of 40-90° C. for 0.5-5 h, extracted 1-4 times, filtered, and the extracts are combined;

[0009] c. The extract obtained in step b was concentrated by rotary evaporation at 50°C to an alcohol-free state, and purified water was added to adjust the concentration to 0.05-0.4 g / mL of the crude drug solution as the loading solution for resin purification;

[0010] d. The pretreated macroporous resin is loaded into a glass column as a purification device with a diameter-to-height ratio of 1:4-1:10, 1-8 BV of the loading solution obtained in step c is added, the sample is loaded at a flow rate of 1-4 BV / h, static adsorption is performed for 0.5-5 h, and then 3-10 BV of purified water is used for elution at a flow rate of 1-7 BV / h to wash and remove impurities, wherein the macroporous resin model is Amberlite XAD16N, AB-8, D101, SA-3, HPD100, HPD300, HPD450, HPD600, HPD750 or HPD826;

[0011] e. Wash the product in step d with water to remove impurities, and then elute with 1-5BV of 5-30% ethanol solution at a flow rate of 1-7BV / h for secondary impurity removal, and then elute with 1-5BV of 15-70% ethanol solution at a flow rate of 1-7BV / h, and concentrate the eluate under reduced pressure, and vacuum dry or freeze-dry to obtain the punicalagin extract.

[0012] The punicalagin extract obtained by the method is used in preparing medicine for treating colitis.

[0013] The invention discloses a preparation method of a punicalagin extract and application thereof. The punicalagin extract obtained by the method has a content of punicalagin, a main active ingredient, as high as 30-80%.

[0014] The punicalagin extract obtained by the method of the present invention is prepared into a tablet for oral use, wherein the tablet contains 0.25 g of punicalagin extract per tablet, and the dosage is: twice a day, one tablet per time;

[0015] The punicalagin extract is prepared into capsules for oral use, and the capsule contains 0.16 g of punicalagin extract per capsule. The dosage is: three times a day, one capsule per time.

[0016] The punicalagin extract is prepared into a suppository for rectal administration, the suppository contains 0.12 g of punicalagin extract per piece, and the dosage is: twice a day, one piece per time;

[0017] The punicalagin extract is prepared into a gel for rectal administration, which contains 0.10 g of punicalagin extract per vial. The dosage is: twice a day, one vial each time.

[0018] The punicalagin extract of the present invention or the pharmaceutical preparation prepared by using the punicalagin extract as a raw material can significantly inhibit the development of inflammation, improve the local pharmacological state, and can be used in medicines for diseases related to inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 It is the HPLC chromatogram of punicalagin standard;

[0021] Figure 2 is the HPLC chromatogram of punicalagin extract;

[0022] Figure 3 The bar graph is the adsorption rate and resolution rate of punicalagin in pomegranate peel extract by different types of macroporous resins;

[0023] Figure 4 The following are the pathological sections of the mouse colon tissue under the oral administration model of different doses of punicalagin extract;

[0024] Figure 5 The results of colon pathological scoring of mice in the oral administration model of punicalagin extract at different doses;

[0025] Figure 6 The changes of TNF-α, IL-6 and IL-1β levels in the serum of mice under the oral administration model after the action of different doses of punicalagin extract;

[0026] Figure 7 The following are the pathological sections of the mouse colon tissues under the enema administration model with different doses of punicalagin extracts;

[0027] Figure 8 The results of colon pathological scoring of mice in the enema administration model with different doses of punicalagin extracts;

[0028] Fig. 9 The changes in TNF-α, IL-6 and IL-1β levels in the serum of mice under the enema administration model treated with different doses of punicalagin extract. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments.

[0030] Example 1

[0031] a. Grind the dried pomegranate peel and pass it through a 30-100 mesh sieve to obtain pomegranate peel powder;

[0032] b. 20 g of the pomegranate peel powder obtained in step a was added with 400 mL of 50% ethanol solution with a solid-liquid ratio of 1:20 for extraction at 70 ° C. The extraction time was 3 h, and the extraction was repeated twice, filtered, and the extracts were combined;

[0033] c. The extract obtained in step b was concentrated by rotary evaporation at a temperature of 50°C to an alcohol-free state, and purified water was added to adjust the concentration to 0.1 g / mL crude drug solution as the loading solution for resin purification;

[0034] d. Load the pretreated XAD16N macroporous resin into a glass column as a purification device with a diameter-to-height ratio of 1:4-1:10, add 3BV of the loading solution obtained in step c, load at a flow rate of 2BV / h, statically adsorb for 2h, and then elute with 8BV of purified water at a flow rate of 3BV / h to wash and remove impurities;

[0035] e. Wash the product in step d with water to remove impurities, and then elute with 3BV of 10% ethanol solution at a flow rate of 2BV / h for secondary impurity removal. Then elute with 2BV of 30% ethanol solution at a flow rate of 2BV / h, concentrate the eluate under reduced pressure, and freeze-dry to obtain the punicalagin extract.

[0036] The obtained punicalagin extract was determined by high performance liquid chromatography (HPLC) using an external standard method under the following chromatographic conditions, and the punicalagin content was 66%;

[0037] Chromatographic conditions: X-Bridge C18 (5 μm, 4.6×250 mm) column, mobile phase A (methanol) and B (0.1% formic acid solution), gradient elution mode: 0-20 min (95% B→90% B), 20-50 min (90% B→80% B), 50-65 min (80% B→50% B), 65-70 min (50% B), 70-80 min (95% B). The column temperature was set to 30°C, the detection wavelength was 254 nm, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The HPLC spectrum of the punicalagin standard was measured under the chromatographic conditions as shown below. Figure 1 As shown in FIG. 1 , the HLPC fingerprint of the punicalagin extract was as follows: Figure 2 shown.

[0038] Example 2

[0039] a. Crush 20 g of dried pomegranate peel to obtain pomegranate peel lumps;

[0040] b. The pomegranate peel block obtained in step a was added with 300 mL of 30% ethanol solution with a solid-liquid ratio of 1:5 for extraction at a temperature of 40° C. for 0.5 h. The extraction was performed once and filtered to obtain an extract;

[0041] c. The extract obtained in step b was concentrated by rotary evaporation at 50°C to an alcohol-free state, and purified water was added to adjust the concentration to 0.05-0.4 g / mL of the crude drug solution as the loading solution for resin purification;

[0042] d. Load the pretreated XAD16N macroporous resin into a glass column as a purification device with a diameter-to-height ratio of 1:4-1:10, add 1BV of the loading solution obtained in step c, load at a flow rate of 3BV / h, statically adsorb for 0.5h, and then elute with 3BV of purified water at a flow rate of 1BV / h to wash and remove impurities;

[0043] e. Wash the product in step d with water to remove impurities, and then elute with 1BV of 30% ethanol solution at a flow rate of 1BV / h for secondary impurity removal, and then elute with 5BV of 70% ethanol solution at a flow rate of 7BV / h, concentrate the eluate under reduced pressure, and obtain the punicalagin extract after vacuum drying or freeze drying.

[0044] The obtained punicalagin extract was determined by high performance liquid chromatography (HPLC) using an external standard method under the following chromatographic conditions, and the punicalagin content was 55%.

[0045] Example 3

[0046] a. Grind 20 g of dried pomegranate peel and pass it through a 30-100 mesh sieve to obtain pomegranate peel powder;

[0047] b. The pomegranate peel powder obtained in step a was extracted with a 70% ethanol solution at a solid-liquid ratio of 1:15 at a temperature of 60° C. for 2 h, three times of extraction, filtered, and the extracts were combined;

[0048] c. The extract obtained in step b was concentrated by rotary evaporation at 50°C to an alcohol-free state, and purified water was added to adjust the concentration to 0.2 g / mL crude drug solution as the loading solution for resin purification;

[0049] d. Load the pretreated HPD300 macroporous resin into a glass column as a purification device with a diameter-to-height ratio of 1:4-1:10, add 6BV of the loading solution obtained in step c, load at a flow rate of 3BV / h, statically adsorb for 1h, and then elute with 5BV of purified water at a flow rate of 4BV / h to wash and remove impurities;

[0050] e. Wash the product from step d with water to remove impurities, then elute with 3BV of 20% ethanol solution at a flow rate of 5BV / h for secondary impurity removal, then elute with 4BV of 70% ethanol solution at a flow rate of 5BV / h, concentrate the eluate under reduced pressure, and dry in vacuo to obtain the punicalagin extract.

[0051] The content of the punicalagin extract was determined by high performance liquid chromatography (HPLC) using an external standard method under the following chromatographic conditions. The content of punicalagin was 48%.

[0052] Example 4

[0053] a. Crush 60 g of dried pomegranate peel to obtain pomegranate peel lumps;

[0054] b. The pomegranate peel block obtained in step a was extracted with a 90% ethanol solution with a solid-liquid ratio of 1:30 at a temperature of 90° C. for 5 h, and the extraction was repeated 4 times, filtered, and the extracts were combined;

[0055] c. The extract obtained in step b was concentrated by rotary evaporation at a temperature of 50°C to an alcohol-free state, and purified water was added to adjust the concentration of the crude drug solution to 0.4 g / mL as the loading solution for resin purification;

[0056] d. Add 50 mL of the sample solution obtained in step c to the pretreated ten macroporous resins of Amberlite XAD16N, AB-8, D101, SA-3, HPD100, HPD300, HPD450, HPD600, HPD750, and HPD826, respectively, and adsorb overnight at room temperature for 12 h at 100 r / min. Take the solution and determine the adsorption rate of punicalagin by HPLC external standard method;

[0057] The resin after adsorption was repeatedly rinsed with water to remove the water, and 100 mL of 95% ethanol was added. The resin was desorbed overnight at room temperature for 12 h at 100 r / min. The solution was taken and the desorption rate of punicalagin was determined by HPLC external standard method.

[0058] The adsorption and desorption rates of punicalagins in pomegranate peel extract by ten macroporous resins were as follows: Figure 3 As shown, all the resins tested have a certain adsorption and desorption capacity for punicalagin. The adsorption capacity and desorption efficiency of the macroporous adsorption resins HPD600, HPD750 and AB-8 for punicalagin are at a medium level, while the adsorption capacity and desorption efficiency of the four types of resins XAD16N, HPD300, D101 and SA-3 for punicalagin are relatively strong.

[0059] Example 5

[0060] The following experimental examples further illustrate the medicinal effects of the punicalagin extract of the present invention on colitis:

[0061] The efficacy experiment of the punicalagin extract of the present invention on the colitis mouse model after oral administration is as follows:

[0062] 48 male Balb / c mice, weighing 18-22g, were selected and randomly divided into 6 groups according to their body weight, with 8 mice in each group, namely normal control group, model control group, positive control (Changyanning tablets), low-dose group of punicalagin extract (PPP-L, 100g / kg), medium-dose group of punicalagin extract (PPP-M, 200g / kg), and high-dose group of punicalagin extract (PPP-H, 400g / kg). Except for the normal control group, the mice in the other groups freely drank drinking water containing 2.5% dextran sodium sulfate solution (DSS) for modeling for 10 consecutive days. Oral administration began on the day of modeling. The normal control group and the model control group were gavaged with sterile water, and the mice in the other groups were gavaged with the corresponding drugs for 10 consecutive days. After the last administration, the mice were replaced with normal drinking water, fasted but not watered for 16h, and processed and collected the next day;

[0063] Colon tissue was fixed in 10% neutral formalin solution, embedded in paraffin, stained with hematoxylin-eosin (HE), sliced, and histopathologically examined and scored for the degree of lesions. Figure 4 and Figure 5 As shown in the results, compared with the model group, the intestinal mucosal damage in the low-dose and medium-dose groups of punicalagin extract was significantly repaired, the congestion of the submucosal layer was significantly alleviated, the infiltration area of ​​inflammatory cells was significantly reduced, and the pathological histological score was significantly reduced (P < 0.01), which was significantly better than the high-dose group.

[0064] Blood was collected from the eyeballs and centrifuged at 3500 r / min for 15 min to separate serum. The levels of inflammatory factors TNF-α, IL-6, and IL-1β in the serum were detected. Figure 6 As shown in the figure; compared with the model control group, the IL-1β levels in the serum of each mouse in the drug-treated group were significantly reduced (P<0.01), among which the IL-1β levels in the low- and medium-dose groups of punicalagin extract were more significantly reduced;

[0065] The results of histopathological examination and serum inflammatory factor determination together indicate that, under the oral administration mode, the punicalagin extract prepared by the method of the present invention has a good therapeutic effect on DSS-induced colitis model mice.

[0066] Example 6

[0067] The efficacy experiment of the punicalagin extract of the present invention on the colitis mouse model by enema administration is as follows:

[0068] 40 male Balb / c mice, weighing 18-22g, were selected and randomly divided into 5 groups according to their body weight, with 8 mice in each group, namely normal control group, model control group, positive control (mesalazine suppository), low-dose group of punicalagin extract (PPP-L, 50g / kg), and high-dose group of punicalagin extract (PPP-H, 100g / kg). Except for the normal control group, the mice in the other groups freely drank drinking water containing 2.5% DSS for modeling for 10 consecutive days. Enema administration began on the day of modeling. Sterile water was administered by enema to the normal control group and the model control group, and the mice in the other groups were administered with corresponding drugs by enema for 10 consecutive days. After the last administration, the mice were replaced with normal drinking water, fasted but not watered for 16h, and processed and collected the next day;

[0069] Colon tissue was fixed in 10% neutral formalin solution, embedded in paraffin, stained with HE, sliced, and histopathologically examined and scored for the degree of lesions. Figure 7 and Figure 8 As shown in the figure, compared with the model group, the intestinal mucosal damage in the low-dose group of punicalagin extract was significantly repaired, the congestion of the submucosal layer was significantly relieved, the infiltration area of ​​inflammatory cells was significantly reduced, and the pathological histological score was significantly reduced (P < 0.01), and the therapeutic effect on colitis was more obvious;

[0070] Blood was collected from the eyeballs and centrifuged at 3500r / min for 15min to separate serum. The changes in serum inflammatory factors TNF-α, IL-6, and IL-1β levels were detected. The results are as follows Fig. 9 As shown in the figure; compared with the model control group, the IL-1β levels in the serum of mice in the positive control group and the low-dose punicalagin extract group were significantly reduced (P<0.01), and the IL-1β level in the positive control group decreased more significantly;

[0071] The results of histopathological examination and serum inflammatory factor determination together indicate that, under the enema administration mode, the punicalagin extract prepared by the extraction and purification method of the present invention has a good medicinal effect on DSS-induced colitis model mice.

Claims

1. A method for preparing a punicalagin extract, characterized in that: Follow these steps: a. Crush or grind the dried pomegranate peel and pass it through a 30-100 mesh sieve to obtain pomegranate peel lumps or powder; b. The pomegranate peel block or powder obtained in step a is added with an ethanol solution of 30%-90% with a solid-liquid ratio of 1:5-1:30 for extraction at a temperature of 40-90° C. for 0.5-5 h, extracted 1-4 times, filtered, and the extracts are combined; c. The extract obtained in step b was concentrated by rotary evaporation at a temperature of 50°C to an alcohol-free state, and purified water was added to adjust the concentration to 0.05-0.4 g / mL crude drug solution as the loading solution for resin purification; d. The pretreated macroporous resin is loaded into a glass column as a purification device with a diameter-to-height ratio of 1:4-1:10, 1-8 BV of the loading solution obtained in step c is added, the sample is loaded at a flow rate of 1-4 BV / h, static adsorption is performed for 0.5-5h, and then 3-10 BV of purified water is used for elution at a flow rate of 1-7 BV / h to wash and remove impurities, wherein the macroporous resin model is Amberlite XAD16N, AB-8, D101, SA-3, HPD100, HPD300, HPD450, HPD600, HPD750 or HPD826; e. Wash the product in step d with water to remove impurities, and then elute with 1-5 BV of 5-30% ethanol solution at a flow rate of 1-7 BV / h for secondary impurity removal, and then elute with 1-5 BV of 15-70% ethanol solution at a flow rate of 1-7 BV / h, concentrate the eluate under reduced pressure, and vacuum dry or freeze-dry to obtain the punicalagin extract.

2. Use of the punicalagin extract obtained according to the method of claim 1 in the preparation of a drug for treating colitis.

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