Extraction method and application of green prickleyash extract

Abstract

The invention relates to the technical field of green prickleyash extracts, and particularly discloses a green prickleyash extract extraction method, which comprises: S1, crushing green prickleyash, adding ethanol, carrying out ultrasonic extraction, filtering, and evaporating the obtained filtrate to remove ethanol and water so as to obtain an ethanol extract; s2, dissolving the ethanol extract in acid, filtering, and carrying out first extraction by adopting a fat-soluble extraction agent to obtain first extraction liquid and a first water phase; s3, performing second extraction on the water phase by adopting an alkaloid extraction agent to obtain second extraction liquid and a second water phase; s4, combining the first extraction liquid and the second extraction liquid, and then drying until the mass is constant, so as to obtain the green prickleyash extract. The green prickleyash extract prepared by the method can effectively inhibit the attack of colitis.

Description

Technical Field

[0001] The invention relates to the technical field of green prickly ash extracts, and in particular to an extraction method and application of the green prickly ash extracts. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease. The etiology is still unclear and may be related to multiple factors such as genetics, immunity, environment, and infection. It is more common in people with a family history of the disease, long-term smokers, and those who eat a high-fat diet, and has a low mortality rate. The treatment of this disease is mainly based on drug therapy. Surgery should be considered when drug therapy is ineffective or when serious complications or canceration occur. The disease is characterized by repeated attacks and remissions, which seriously affects the patient's quality of life. However, when the disease is severe or complications occur, it may be life-threatening.

[0003] Green prickly ash (Zanthoxylum schinifolium Siebold & Zucc.) is a shrub of the genus Zanthoxylum of the Rutaceae family. It has a pungent taste and is slightly warm in nature. It has the effects of warming the middle and relieving pain, killing insects and relieving itching, strengthening the stomach, dispelling wind and cold, removing dampness and stopping diarrhea, and promoting blood circulation and menstruation. According to the "Chinese Medicine Dictionary", green prickly ash has the effects of aromatic stomachic, warming the middle and relieving cold, removing dampness and relieving pain, killing insects and detoxifying, and relieving itching and removing fishy smell. It can remove the fishy smell of various meats; promote saliva secretion and increase appetite; dilate blood vessels, thereby playing a role in lowering blood pressure. Taking prickly ash water can remove parasites. It can be seen that traditional Chinese medicine points out that green prickly ash has the effects of strengthening the stomach and relieving pain.

[0004] "Zhang Zecai. Study on the protective effect and mechanism of different extracts of Zanthoxylum bungeanum on ulcerative colitis model mice [D]. Jilin University, 2017." The DSS-mediated UC mouse model was used to explore the preventive and therapeutic effects of Zanthoxylum bungeanum extract on ulcerative colitis, verify the anti-UC effects of Zanthoxylum bungeanum extract, Zanthoxylum bungeanum essential oil, 4-terpineol and other extracts, and speculate their anti-UC mechanisms. Specifically, Zanthoxylum bungeanum extract exerts anti-UC effects by inhibiting TLR4 and its related downstream signaling pathways; Zanthoxylum bungeanum essential oil exerts anti-UC effects by regulating the expression of NF-κB, PPARγ and NLRP3 inflammasome and related proteins, regulating the expression of intestinal tight junction protein ZO-1, regulating the number of Escherichia coli, Lactobacillus and Bifidobacterium; 4-terpineol exerts anti-UC effects by inhibiting the expression of NLRP3 inflammasome and related proteins, regulating the expression of intestinal tight junction protein ZO-1 and occludin, regulating the number of Escherichia coli and Lactobacillus, and inhibiting the increase of serum LPS content.

[0005] However, the anti-UC effects of the three Zanthoxylum bungeanum extracts are different, which may be caused by the different extraction methods of the extracts. Since the effects of the three extracts are not outstanding, the present invention provides a new extraction method to obtain a Zanthoxylum bungeanum extract with better anti-UC effect. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for extracting a green pepper extract, so as to at least achieve an excellent effect of inhibiting the onset of colitis.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for extracting a green prickly ash extract comprises the following steps:

[0009] S1: crushing the green pepper, then adding ethanol for ultrasonic extraction, filtering, and evaporating the obtained filtrate to remove ethanol to obtain an ethanol extract;

[0010] S2: dissolving the ethanol extract in acid, filtering, and then performing a first extraction with a fat-soluble extractant to obtain a first extract and a first aqueous phase;

[0011] S3: performing a second extraction on the aqueous phase using an alkaloid extractant to obtain a second extract and a second aqueous phase;

[0012] S4: combining the first extract and the second extract, and then drying until the mass is constant to obtain the green pepper extract.

[0013] Preferably, the green pepper is crushed to 60 mesh.

[0014] Furthermore, in step S1, the concentration of ethanol is 60-80wt%.

[0015] Furthermore, in step S1, the solid-liquid ratio of the green pepper and the ethanol is 1:20-30, g / mL.

[0016] Furthermore, in step S1, the ultrasonic extraction time is 30-50 min;

[0017] And / or, the extraction temperature of the ultrasonic extraction is 50-65°C.

[0018] Furthermore, in step S2, the acid is an acidic solution, which can convert the alkaloids in the ethanol extract into alkaloid salts and dissolve them in water.

[0019] It is worth noting that the main purpose of dissolving the ethanol extract in the acid is to separate and purify the alkaloids and remove impurities in the solution, especially those impurities that are insoluble in water.

[0020] Furthermore, in step S2, the acid is preferably hydrochloric acid, and the concentration of the hydrochloric acid is 1-3 wt %.

[0021] Furthermore, in step S2, the plastomeric ratio of the purified extract to the hydrochloric acid is 1:1, g / mL.

[0022] Furthermore, in step S2, the fat-soluble extractant includes ethyl acetate or petroleum ether.

[0023] Preferably, the fat-soluble extractant is ethyl acetate.

[0024] Preferably, the amount of the fat-soluble extractant used is three times the volume of the acid.

[0025] Furthermore, in step S3, before performing the second extraction, the pH of the first aqueous phase needs to be adjusted to 9-10.

[0026] It is worth noting that the purpose of adjusting the pH is to adjust the pH of the aqueous phase extracted by the fat-soluble extractant to alkaline, so as to facilitate the subsequent second extraction.

[0027] Further, in step S3, the alkaloid extractant includes chloroform or dichloromethane;

[0028] Preferably, the alkaloid extractant is chloroform.

[0029] Preferably, the amount of the alkaloid extractant is three times the volume of the acid.

[0030] Furthermore, in step S4, the drying temperature is 50°C.

[0031] Another object of the present invention is to provide a green pepper extract obtained by the above extraction method.

[0032] Another object of the present invention is to provide a use of the green pepper extract in inhibiting the onset of colitis.

[0033] The beneficial effects of the present invention are:

[0034] 1. The extraction method of the present invention has simple process and low cost, and the green pepper extract obtained by extraction has a significant inhibitory effect on UC.

[0035] 2. The green pepper extract obtained by the method of the present invention has a significant inhibitory effect on Bacteroides (Bacteroides); Clostridiales (Clostridium difficile); Dubosiella newyorkensis and other bacteria closely related to the onset of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a bar graph of IL-1β concentration data obtained by microplate reader detection in Example 2;

[0037] Figure 2 The TNF-α concentration data bar graph obtained by the microplate reader in Example 2;

[0038] Figure 3 This is a bar graph of IL-6 concentration data obtained by microplate reader detection in Example 2;

[0039] Figure 4 This is the gray value detection image obtained using ImageJ software in Example 3. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0041] Example 1

[0042] A green pepper extract is prepared, which is specifically put as follows:

[0043] 50 g of green pepper powder (early-maturing nine-leaf green pepper from Weirui Planting Professional Cooperative in Wangcang County, sieved through a 60-mesh sieve) was accurately weighed, and 70% ethanol was added at a solid-liquid ratio of 1:25. Ultrasonic extraction was performed under the conditions of ultrasonic time of 30 min and extraction temperature of 60°C, and filtration was performed. The above ultrasonic extraction steps were repeated 3 times, and the filtrate was combined and rotary distilled to obtain an ethanol extract.

[0044] The ethanol extract was dissolved in a 2% hydrochloric acid aqueous solution at a plastosol ratio of 1:1, the insoluble matter was filtered out, the fat-soluble part was extracted with ethyl acetate (3 times the volume of hydrochloric acid), the pH value of the aqueous phase was adjusted to 9-10 with ammonia water, and then extracted with chloroform (3 times the volume of hydrochloric acid) for 3 times, and the extracts were combined and dried in a constant temperature drying oven at 50°C to constant mass to obtain the green pepper extract.

[0045] Example 2

[0046] Animal experiments were used to verify the anti-UC effect of green pepper extract. The specific methods are as follows:

[0047] C56BL7 mice were taken for 7-8 weeks. The experiment was started after one week of adaptive feeding. The mice were kept in an environment with a room temperature of 23±2℃, a humidity of 55±5%, and a 12h day / night cycle. They were given sterile standard feed and purified water to allow free diet. After 7 days of adaptive feeding, they were randomly divided into 5 groups (n=10): control group (C), model group (DSS), low-dose intervention group (DSS+L), medium-dose intervention group (DSS+M) and high-dose intervention group (DSS+H). During the experiment, except for group C, which was given distilled water, the other experimental groups were given 3% (w / v) DSS drinking water on days 8-14, and allowed to drink freely for 6 consecutive days. Fresh DSS drinking water was replaced at the same time every day to construct an acute UC mouse model. All mice were killed on the 7th day to obtain colon and feces. From the 7th day to the 13th day, the L group (40 mg / kg), the M group (80 mg / kg) and the H group (160 mg / kg) were gavaged with alkaloid solution every day, and the administration volume was 0.1 mL / 10 g body weight. The mice in the C group and the DSS group were gavaged with an equal volume of CMC-Na every day, and each intervention group was gavaged uniformly at 9:00 am every day, once a day, for 6 consecutive days.

[0048] On the 13th day, the mice were killed and the colons were collected. After adding PBS, the colons were homogenized and then centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was collected and stored at -80°C. ELISA kits (all purchased from Eli Rytter Biotech, TNF-α product number is E-EL-M3063, IL-6 product number is E-EL-M0044, and Il-1β product number is E-EL-M0037) were used to detect the content of cytokines (TNF-α, IL-1β) in the supernatant. The detection and analysis were performed using an enzyme marker, and the anti-inflammatory effect of TAZS on DSS-treated mice was explored by measuring the concentrations of proinflammatory cytokines (TNF-α, IL-6 and IL-1β). Figure 1-3 As shown in the figure, compared with the Control group, the concentrations of TNF-α, IL-6, and IL-1β in colon tissue of the Model group were significantly increased (P<0.05). TAZS intervention significantly reduced the concentrations of TNF-α, IL-6, and IL-1β in colon tissue (P).

[0049] Example 3

[0050] Take the colon tissue collected after the mice were killed on the 13th day as mentioned above (stored in a -80°C refrigerator), then homogenize and lyse in RIPA lysis buffer. Centrifuge the homogenate at 4°C and 12000rpm for 10 minutes to obtain the supernatant. Use the BCA protein detection kit to determine the protein concentration according to the manufacturer's instructions. After separating the proteins at 75V for 30min by 10% SDSPAGE, modify the parameters to 120V, and then transfer to a 0.45μm PVDF membrane at 200mA. Afterwards, block with 5% skim milk at room temperature for 60 minutes, and incubate with the corresponding primary antibody (ZO-1, Occludin) on a shaker at 4°C overnight. After washing three times with TBS buffer containing 0.1% Tween-20, incubate with the corresponding secondary antibody at room temperature for 1 hour. Finally, after washing three times with TBST, the chemiluminescence method was used for detection on the chemiluminescence imaging system, and the grayscale value of the results was analyzed and calculated using ImageJ software, as shown in the figure. Figure 4 shown.

[0051] It can be seen that DSS treatment led to a significant decrease in ZO-1 antibody and Occludin (both purchased from ABcam). Compared with the model group, the concentrations of occludin and ZO-1 proteins in the colon of rats in the TAZS intervention group were significantly increased (P<0.01). These results indicate that TAZS intervention effectively prevented the reduction of tight junction proteins caused by DSS.

[0052] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for extracting a green pepper extract, characterized in that: The following steps are involved: S1: crushing the green pepper, then adding ethanol for ultrasonic extraction, filtering, and evaporating the obtained filtrate to remove ethanol and water to obtain an ethanol extract; S2: dissolving the ethanol extract in acid, filtering, and then performing a first extraction with a fat-soluble extractant to obtain a first extract and a first aqueous phase; S3: performing a second extraction on the aqueous phase using an alkaloid extractant to obtain a second extract and a second aqueous phase; S4: combining the first extract and the second extract, and then drying until the mass is constant to obtain the green pepper extract.

2. The extraction method according to claim 1, characterized in that: In step S1, the concentration of ethanol is 60-80wt%.

3. The extraction method according to claim 2, characterized in that: In step S1, the solid-liquid ratio of the green pepper and the ethanol is 1:20-30, g / mL.

4. The extraction method according to claim 1, characterized in that: In step S1, the ultrasonic extraction time is 30-50 min; And / or, the extraction temperature of the ultrasonic extraction is 50-65°C.

5. The extraction method according to claim 1, characterized in that: In step S2, the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 1-3wt%.

6. The extraction method according to claim 5, characterized in that: In step S2, the plastomeric ratio of the purified extract to the acid is 1:1, g / mL.

7. The extraction method according to claim 1, characterized in that: In step S3, before performing the second extraction, the pH of the first aqueous phase needs to be adjusted to 9-10.

8. The extraction method according to claim 1, characterized in that: The fat-soluble extractant includes ethyl acetate or petroleum ether; And / or, the alkaloid extractant comprises chloroform or dichloromethane.

9. A green pepper extract, characterized in that: The extract is obtained by extraction using any one of the extraction methods described in claims 1-8.

10. The use of the Zanthoxylum bungeanum extract obtained by the extraction method according to any one of claims 1 to 8 or the Zanthoxylum bungeanum extract according to claim 9, characterized in that: Used to suppress the onset of colitis.

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