Scutellaria baicalensis stem and leaf extract as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine, and particularly discloses a scutellaria baicalensis stem and leaf extract and a preparation method and application thereof, and the scutellaria baicalensis stem and leaf extract is high in effective component content, simple in preparation method and higher in extract yield. The scutellaria baicalensis stem and leaf extract prepared by the method provided by the invention can safely and effectively treat ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a scutellaria baicalensis stem and leaf extract, and a preparation method and application thereof. Background Art

[0002] Scutellaria baicalensis is a perennial herbaceous plant of the genus Scutellaria in the family Lamiaceae. Its root is used for medicinal purposes and is a commonly used Chinese herbal medicine. Scutellaria baicalensis is cold in nature and bitter in taste. It enters the heart, lung, gallbladder, and large intestine meridians. It has the effects of clearing heat, detoxifying, removing dampness, and stopping bleeding. It is used to treat hepatitis, viral colds, upper respiratory tract infections, fever and thirst, lung heat cough, red and swollen eyes, enteritis, dysentery and other diseases.

[0003] The root of Scutellaria baicalensis mainly contains flavonoid derivatives, which are carriers of anti-inflammatory and antiviral effects. Flavonoids mainly include baicalin, baicalin, wogonin, baicalin and baicalin. Non-flavonoid substances include melaleuca A, benzoic acid and sitosterol.

[0004] The root of Scutellaria baicalensis is traditionally used as medicine. In recent years, the annual output is about 20,000 tons, while about 40,000 tons of Scutellaria baicalensis stems and leaves are discarded every year, which not only causes serious waste of Chinese medicine resources, but also brings great pressure to the ecological environment. The stems and leaves of Scutellaria baicalensis are the above-ground parts of the medicinal Scutellaria baicalensis. The chemical composition is quite different from that of the roots. The main component is baicalin. There is evidence that the stems and leaves also contain two flavonoids, safflower and isocarpentin, as well as a small amount of baicalin, tannins, resins, etc.

[0005] Ulcerative colitis is an inflammatory bowel disease, clinically manifested by intestinal ulcers, which damage the body's barrier, and pathogenic factors invade the body, causing an imbalance of inflammatory factors and immune damage, which accelerates the development of the disease. Severe ulcerative colitis may cause colon cancer. At present, the causes of ulcerative colitis are mostly related to congenital heredity, lifestyle habits and other factors. The affected population is not limited to gender and is mostly distributed in the middle-aged and elderly population.

[0006] The treatment of ulcerative colitis is mainly based on Western medicine, including aminosalicylic acid, glucocorticoids and immunosuppressants. Since ulcerative colitis is a chronic inflammatory intestinal disease, the treatment cycle is long and the disease is prone to recurrence. Long-term treatment with Western medicine faces disadvantages such as adverse reactions and high prices. Therefore, it is of research significance to develop a drug that can provide long-term treatment, stabilize the disease and is inexpensive.

[0007] Literature shows that the extract of Scutellaria baicalensis stems and leaves can treat DSS-induced ulcerative colitis by regulating the body's intestinal peroxidation reaction and inflammatory factor levels, thereby restoring the homeostasis of intestinal flora; it can also inhibit the activity of HCT116 cells through anti-inflammation and cell apoptosis, thereby exerting an anti-colon cancer effect. Summary of the invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a scutellaria baicalensis stem and leaf extract in view of the deficiencies in the prior art.

[0009] The technical problem that the present invention needs to solve is to provide a method for preparing the scutellaria baicalensis stem and leaf extract.

[0010] A further technical problem to be solved by the present invention is to provide an application of the above-mentioned Scutellaria baicalensis stem and leaf extract.

[0011] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0012] In a first aspect, the present invention discloses a scutellaria baicalensis stem and leaf extract, which is prepared by the following method:

[0013] (1) extracting the stems and leaves of Scutellaria baicalensis baicalensis with an ethanol solution to obtain an ethanol extract;

[0014] (2) The ethanol extract is subjected to column chromatography using a non-polar resin. After adsorption, the extract is eluted with water and then with an ethanol solution. The ethanol eluate is collected and concentrated.

[0015] In a second aspect, the present invention discloses a method for preparing a scutellaria leaf extract, comprising the following steps:

[0016] (1) extracting the stems and leaves of Scutellaria baicalensis baicalensis with an ethanol solution to obtain an ethanol extract;

[0017] (2) The ethanol extract is subjected to column chromatography with a non-polar resin, adsorbed, eluted with water, and then eluted with an ethanol solution, and the ethanol eluate is collected and concentrated.

[0018] In the first and second aspects above, the parameters of each step have the following characteristics:

[0019] In step (1), the volume fraction of the ethanol solution is 45%-95%, preferably 50%-80%, more preferably 60%-70%, and in some embodiments, 50%, 60%, or 70%.

[0020] In step (1), the extraction is thermal extraction, and in some embodiments, reflux extraction.

[0021] In step (1), the extraction is performed 1-5 times, in some embodiments 2-4 times, and in some embodiments 3 times.

[0022] In step (1), the volume mass ratio of the ethanol solution to the stems and leaves of Scutellaria baicalensis is 8-40 mL: 1 g, in some embodiments, 10-30 mL: 1 g, in some embodiments, 12-20 mL: 1 g; or the weight ratio is 8-40: 1, in some embodiments, 6-32: 1, in some embodiments, 7-20: 1, in some embodiments, 12-20: 1 or 10-16: 1.

[0023] In step (1), the extract obtained by the extraction is filtered, the filtrate is concentrated, and the concentrated solution is filtered to obtain the ethanol extract; in some embodiments, the filtrate is concentrated under reduced pressure to concentrate the filtrate until there is no alcohol taste; in some embodiments, the concentrated solution is filtered by centrifugation.

[0024] In step (2), the non-polar resin is D101 macroporous resin or AB-8 macroporous resin, and in some embodiments, it is D101 macroporous resin; in some embodiments, the adsorption is static adsorption.

[0025] In step (2), the ethanol extract is loaded in the form of ethanol or aqueous solution; in some embodiments, the loading rate is 0.24-1.8BV / h, in some embodiments, 0.32-1.2BV / h, in some embodiments, 0.4-0.6BV / h, in some embodiments, 0.4BV / h, 0.5BV / h, or 0.6BV / h.

[0026] In step (2), the adsorption time is 1.2-9 h, in some embodiments, 1.6-6 h, in some embodiments, 2-3 h, in some embodiments, 2 h, 2.5 h, or 3 h.

[0027] In step (2), the adsorption is static adsorption in some embodiments.

[0028] In step (2), the volume of the water elution is 2-9 BV, in some embodiments, 3-7 BV, in some embodiments, 4-6 BV, in some embodiments, 4 BV, 5 BV, or 6 BV.

[0029] In step (2), the water elution rate is 0.5-6 BV / h, in some embodiments, 0.75-4.5 BV / h, in some embodiments, 1-3 BV / h, in some embodiments, 1 BV / h, 2.5 BV / h, or 3 BV / h.

[0030] In step (2), the volume fraction of the ethanol solution is 45%-95%, in some embodiments, 55%-85%, in some embodiments, 65%-75%, in some embodiments, 65%, 70%, or 75%.

[0031] In step (2), the volume of the ethanol solution elution is 1-6BV, in some embodiments, 1.5-5BV, in some embodiments, 2-4BV, and in some embodiments, 2BV or 4BV.

[0032] In step (2), the elution rate of the ethanol solution is 0.9-7.5 BV / h, in some embodiments, 1.2-5 BV / h, in some embodiments, 1.5-2.5 BV / h, in some embodiments, 1.5 BV / h, 2 BV / h, or 2.5 BV / h.

[0033] In step (2), the concentration is reduced-pressure concentration; in some embodiments, the concentration is followed by further drying to obtain the Scutellaria baicalensis stem and leaf extract.

[0034] In some embodiments, the scutellaria baicalensis stem and leaf extract in the first aspect is prepared according to the following method, and the preparation method in the second aspect comprises the following steps:

[0035] (1) taking the stems and leaves of Scutellaria baicalensis, adding 10-20 times the volume fraction of 50%-80% ethanol solution to reflux extraction, the extraction times are 3 times, each time for 1 hour, filtering the obtained extract, and combining the three extracts, concentrating under reduced pressure until there is no alcohol taste, centrifuging and filtering to obtain a filtrate;

[0036] (2) The filtrate is loaded onto D101 macroporous resin at a flow rate of 0.4-0.6 BV / h, and allowed to stand for adsorption for 2-4 hours. The filtrate is then eluted with 3.5-6 BV of pure water at a flow rate of 1-5 BV / h, and then eluted with 1.2-5 BV of 65%-75% ethanol solution at a flow rate of 1.5-2.5 BV / h. The 65%-75% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Scutellaria baicalensis stem and leaf extract.

[0037] In a third aspect, the present invention discloses the use of the Scutellaria baicalensis stem and leaf extract described in the first aspect or the Scutellaria baicalensis stem and leaf extract prepared by the method described in the second aspect in the preparation of a drug for treating ulcerative colitis.

[0038] The present invention has no specific requirements for the centrifugal parameters, such as centrifugation at 50-5000 rpm for 1-30 min, or at 3500 rpm for 10 min.

[0039] The ethanol solutions described in the present invention, unless otherwise specified, are all ethanol aqueous solutions.

[0040] The scutellaria stem and leaves in the present invention are extracted with an ethanol solution. If there is no special description, the scutellaria stem and leaf coarse powder is extracted with an ethanol solution. There is no requirement for the mesh size of the scutellaria stem and leaf coarse powder, which can be crushed.

[0041] The diameter-to-height ratio of the column chromatography described in the present invention is 1:2-10, in some embodiments, 1:4-8, and in some embodiments, 1:6.

[0042] The scutellaria baicalensis stem and leaf extract prepared by the present invention has a total flavonoid content of more than 50%, 50-75% in some cases, 55-70% in some cases, and 60%-70% in some cases.

[0043] Beneficial effects of the present invention:

[0044] Compared with the prior art, the present invention provides a method for preparing a scutellaria stem and leaf extract, which has a high content of active ingredients, a simple preparation method, and a higher extract yield; the scutellaria stem and leaf extract prepared by the method provided by the present invention can safely and effectively treat ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0046] Figure 1 Effect of Scutellaria baicalensis stem and leaf extract on colon length in UC mice

[0047] Figure 2 Effects of Scutellaria baicalensis stem and leaf extract on pathological changes of colon tissue in UC mice (HE, ×20).

[0048] Figure 3 Effects of Scutellaria baicalensis stem and leaf extract on the mRNA expression of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in UC mice

[0049] Figure 4 Effects of total flavonoids from the stems and leaves of Scutellaria baicalensis on the expression of ZO-1, AHR, CYP1A1, CYP1B1, and Trpv6 proteins in colon tissue of UC mice

[0050] Figure 5 This is a graph of cell viability of Scutellaria baicalensis stem and leaf extract.

[0051] ##: Compared with the control group, P<0.05; **: Compared with the model group, the treatment group p<0.05; *: Compared with the model group, the treatment group p<0.01. DETAILED DESCRIPTION

[0052] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0053] The stems and leaves of Scutellaria baicalensis described in the following examples are dried products, and the centrifugation is performed at 3000-4000 rpm for 6-15 min.

[0054] Example 1 Preparation of Scutellaria baicalensis stem and leaf extract

[0055] (1) 50 g of crude powder of Scutellaria baicalensis stems and leaves was added to 800 mL of 60% ethanol solution for reflux extraction. The extraction times were 3 times, each time for 1 hour. The obtained extract was filtered, and the liquids filtered from the three extractions were combined, concentrated under reduced pressure until there was no alcohol taste, and centrifuged to obtain a filtrate;

[0056] (2) The filtrate was loaded onto D101 macroporous resin at a flow rate of 0.5 BV / h, and allowed to stand for adsorption for 3 hours. The filtrate was eluted with 4 BV of pure water at a flow rate of 3 BV / h, and the water washing liquid was discarded. The elution was continued with 4 BV of 70% ethanol at a flow rate of 2 BV / h. The 70% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain 5.63 g of Scutellaria baicalensis stem and leaf extract.

[0057] Example 2 Preparation of Scutellaria baicalensis stem and leaf extract

[0058] (1) 50 g of 20-mesh coarse powder of Scutellaria baicalensis stems and leaves was added with 600 mL of 60% ethanol solution for reflux extraction. The extraction times were 3 times, each time for 1 hour. The obtained extract was filtered, and the liquids filtered from the three extractions were combined, concentrated under reduced pressure until there was no alcohol taste, and centrifuged to obtain a filtrate;

[0059] (2) The filtrate was loaded onto D101 macroporous resin at a flow rate of 0.6 BV / h, and allowed to stand for 2 hours of adsorption. The filtrate was eluted with 5 BV of pure water at a flow rate of 2.5 BV / h, the water washing liquid was discarded, and the elution was continued with 4 BV of 65% ethanol at a flow rate of 1.5 BV / h. The 65% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain 6.12 g of Scutellaria baicalensis stem and leaf extract.

[0060] Example 3 Preparation of Scutellaria baicalensis stem and leaf extract

[0061] (1) 50 g of crude powder of Scutellaria baicalensis stems and leaves was added to 1000 mL of 70% ethanol solution for reflux extraction. The extraction times were 3 times, each time for 1 hour. The obtained extract was filtered, and the liquids filtered from the three extractions were combined, concentrated under reduced pressure until there was no alcohol taste, and centrifuged to obtain a filtrate;

[0062] (2) The filtrate was loaded onto D101 macroporous resin at a flow rate of 0.4 BV / h, and allowed to stand for adsorption for 2.5 hours. The filtrate was eluted with 6 BV of pure water at a flow rate of 1 BV / h, the water washing liquid was discarded, and the elution was continued with 2 BV of 75% ethanol at a flow rate of 2.5 BV / h. The 75% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain 5.99 g of Scutellaria baicalensis stem and leaf extract.

[0063] Comparative Example 1 Preparation of Scutellaria baicalensis stem and leaf extract from patent CN100378089C

[0064] 50g of scutellaria baicalensis stem and leaf were crushed into 20 mesh coarse powder, and after enzyme killing treatment, water was added for extraction twice, and 10 times of water of crude drug amount was added each time, and the extraction was carried out at 100℃ for 1 hour. The two extracts were combined, and the extracts were concentrated under reduced pressure at 70±5℃ to a relative density of 1.02-1.10 (measured at 20℃), and centrifuged to obtain a supernatant, and hydrochloric acid was added to acidify to pH 2-3, and centrifuged. The supernatant was adsorbed by AB-8 macroporous adsorption resin, washed with purified water to neutrality, and then desorbed by 70% ethanol, and the analytical solution was collected, ethanol was recovered and concentrated, and dried under reduced pressure at 70±5℃ to dryness to obtain the product.

[0065] Example 4 Detection of Scutellaria baicalensis stem and leaf extract

[0066] The scutellarin content refers to the scutellarin content in the prepared Scutellaria stem and leaf extract;

[0067] The total flavonoids content refers to the total flavonoids content in the prepared Scutellaria baicalensis stem and leaf extract;

[0068] The transfer rate of scutellarin is the content of scutellarin in the prepared scutellaria stem and leaf extract / the content of scutellariarin in the scutellaria stem and leaf crude powder;

[0069] The yield of the extract is the weight of the scutellaria stem and leaf extract / the weight of the scutellaria stem and leaf crude powder.

[0070] Table 1 Content and transfer rate determination of samples in the embodiment

[0071]

[0072] Example 5 Effect of Scutellaria baicalensis stem and leaf extract on cell viability of normal colon epithelial cells (NCM460)

[0073] Normal colon epithelial cells (NCM460) were cultured in RPMI 1640 medium at a cell density of 10 5 / mL, inoculated in a 96-well plate at 100 μL / well, and after the cells adhered, given a serum-free culture medium with a corresponding concentration of 45 μg / mL of the Scutellaria baicalensis stem and leaf extract prepared in Examples 1-3 and Comparative Example 1, cultured at 37°C, 5% CO2, after 24 hours, added 10 μL / well MTT solution in the dark, and added 150 μL / well DMSO solution after 3 hours, and the absorbance OD value was detected at 490 nm by a microplate reader.

[0074] According to the results of MTT (Table 2, Figure 5 ) It can be seen that the treatment with the Scutellaria baicalensis stem and leaf extracts in Examples 1-3 did not cause significant changes in cell viability, while Comparative Example 1 could significantly inhibit cell viability.

[0075] Table 2

[0076] sample Cell viability (%) Example 1 98 Example 2 97 Example 3 95 Comparative Example 1 80

[0077] Example 6 Effect of Scutellaria baicalensis stem and leaf extract on 3% DSS-induced ulcerative colitis (UC) mice

[0078] 1. Experimental Materials

[0079] 1.1 Animals

[0080] Sixty male C57bl / 6 mice, weighing (18-20) g, were provided by Sibeifu (Beijing) Biotechnology Co., Ltd. under animal license number SCXK (Beijing) 2019-0010 and were raised under conventional conditions.

[0081] 1.2 Drugs and reagents

[0082] Example 1 Scutellaria baicalensis stem and leaf extract (SBSL);

[0083] Dextran sulfate sodium (lot number 160110) was purchased from MP Biomedicals, USA;

[0084] Sulfasalazine (National Medicine Standard No. H31020840) was purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.;

[0085] Chloroform (batch number 10006818) was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0086] Isopropanol (batch number 80109218) was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0087] 75% ethanol (batch number 10009218) was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0088] RevertAid First Strand cDNA Synthesis Kit (Lot #K1622) was purchased from Thermo;

[0089] FastStart Universal SYBR Green Master (Rox) (lot number 04 913 914 001) was purchased from Roche.

[0090] 1.3 Instruments

[0091] KZ-II homogenizer (Kangtao Technology);

[0092] D3024R desktop high-speed refrigerated microcentrifuge (DragonLab);

[0093] SLAN fluorescence quantitative PCR detection system fluorescence quantitative PCR instrument (Shanghai Hongshi Medical Equipment);

[0094] SW-CJ-1FD clean bench (Sujing Antai);

[0095] NanoDrop2000 ultra-micro-volume spectrophotometer (Thermo);

[0096] FBZ2001-up-p standard reagent type pure water meter (Qingdao Fulham Technology Co., Ltd.);

[0097] Ensight multi-function microplate reader (PerkinElmer Instrument Co., Ltd.).

[0098] 2. Experimental Methods

[0099] 2.1 Modeling and drug administration

[0100] Sixty healthy C57bl / 6 mice were randomly divided into 6 groups, namely normal control group, model group, SBSL low, medium and high dose (22, 45, 90 mg / kg) groups, SASP positive control group (450 mg / kg), 10 mice in each group. The mice were adaptively fed for 1 week. During the experiment, except for the normal control group, the other experimental groups were given 3% DSS (w / v) drinking water for 7 consecutive days to induce acute UC model in mice. Fresh DSS water was replaced every morning. At the same time as modeling, each treatment group took the corresponding therapeutic drug orally at 8:00-10:00 in the morning, and the control group was given the same amount of 0.5% CMC-Na solution once a day for 7 consecutive days. The general conditions of mice, hair, diet, stool, etc. were observed daily, the body weight of mice was recorded, and fecal occult blood test was performed, and the disease activity index (DAI) was scored according to the Cooper method.

[0101] 2.2 Animal sampling and sample pretreatment

[0102] After 7 days of drug administration, mice in each group were fasted for 24 hours and samples were collected on the 8th day. After removing the eyeballs to collect blood, the abdominal cavity of the mice killed by cervical dislocation was cut open, and the colon tissue from the ileocecal junction to the anus of the mice was taken and placed on ice, and its length was measured and recorded. Then, after flushing the intestinal contents with normal saline, 1 cm of the colon near the anus was cut and fixed in 10% formalin for paraffin section preparation. The remaining tissue was taken from the middle section of 3-4 mm and quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The blood sample was allowed to stand at room temperature for 2-3 hours, then centrifuged at 3500 rpm for 5 minutes, and the serum was taken and quickly stored at -80°C for subsequent testing.

[0103] 2.3 Evaluation of colonic inflammation

[0104] DAI score: According to the standard proposed by Cooper et al., the score was calculated by combining the percentage of body weight loss, stool characteristics and fecal occult blood of mice 7 days after administration, as shown in Tables 3 and 4;

[0105] Table 3 DAI scoring criteria

[0106]

[0107] Table 4 Criteria for occult blood detection

[0108]

[0109] 2.4 Colonic histopathological examination

[0110] The mouse colon was fixed in 10% formalin, dehydrated, embedded in paraffin, and sliced. After hematoxylin-eosin (HE) and peroxy acid Schiff (PAS) staining, the pathological changes of the mouse colon tissue were observed under a microscope.

[0111] 2.5 Effects of Scutellaria baicalensis stem and leaf extract on the activities of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in UC mice

[0112] 24 hours after the last administration, the mice were weighed, their eyeballs were removed and blood was collected. The blood samples were allowed to stand at room temperature for 2-3 hours and then centrifuged at 4000 rpm for 5 minutes. The serum was collected and the activities of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in the serum were detected according to the instructions of the kit.

[0113] 2.7 qPCR determination of ZO-1, Claudin-2, AHR, CYP1A1, CYP1B1, and Trpv6 protein expression levels

[0114] Total colon RNA was extracted according to the instructions of the RNA kit, and the relative expression level of the target gene was analyzed. The primers for each signal mRNA molecule are shown in Table 5.

[0115] Table 5 Primer sequences

[0116]

[0117]

[0118] 3. Experimental results

[0119] SBSL alleviates symptoms of DSS-induced ulcerative colitis in mice

[0120] In the DSS-induced ulcerative colitis mouse model treated with Scutellaria baicalensis stem and leaf extract, after continuous intake of 3.5% DSS drinking water, the mice in the model group gradually developed clinical symptoms such as depression, slow movement, bloody stools, diarrhea, and weight loss (Table 6), and their disease activity index (DAI) was significantly higher than that of the control group (Table 7). In addition, the colon length of the model group was significantly shortened ( Figure 1 ). These results indicated that the mouse acute UC model was successfully established.

[0121] Compared with the model group, the ulcerative colitis mouse model was restored after the intervention of the Scutellaria stem and leaf extract. Compared with the model group, different doses of SBSL can significantly increase body weight. Compared with the positive control group, the body weight change was comparable to that of the positive control group at a dose of 90 mg / kg (Table 6). Compared with the model group, different doses of SBSL can significantly reduce the DAI score. Compared with the positive control group, the DAI score is lower (Table 7). Compared with the model group, medium / high doses of SBSL can significantly increase colon length. Compared with the positive control group, the colon length of the medium, low and high dose groups is longer than that of the positive control group (Table 7). Figure 1 ); These results suggest that the extract of Scutellaria baicalensis stems and leaves has therapeutic efficacy in treating ulcerative colitis.

[0122] Table 6 Effects of each group on the weight change of UC mice (ratio of mouse weight on the day / weight on the first day) / %

[0123]

[0124] Table 7 DAI scores of each group

[0125]

[0126] 3.2 Colonic tissue pathology of mice in each group

[0127] The results of colon histopathological examination showed that the colon mucosal structure of the model group was severely damaged, with extensive loss of crypts, hemorrhage, edema and a large number of inflammatory cell infiltration in the mucosa and submucosa. However, the colon glands and other structures of mice treated with different doses of SBSL were clearer ( Figure 2 ).

[0128] 3.3 mRNA levels of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in each group of mice

[0129] The results showed that compared with the control group, the levels of TNF-α, IL-1β, and IL-17 in the model group were significantly increased (p<0.05), while the levels of IL-10 and IL-22 were significantly decreased (p<0.05), indicating that the immune function of the mice in the model group was abnormal and there was a significant inflammatory response. Compared with the model group, different doses of SBSL could significantly reduce the levels of TNF-α, IL-1β, and IL-17 in mouse plasma (p<0.05), and high / medium doses of SBSL could significantly increase the levels of IL-10 and IL-22 in plasma (p<0.05) ( Figure 3 ). Among them, when SBSL was one twentieth (22 mg / kg) and one fifth (90 mg / kg) of the concentration of the positive control drug, the level of IL-1β increase was equivalent to that of the positive drug, and when SBSL was one tenth (45 mg / kg) of the concentration of the positive control drug, the level of IL-1β increase was better than that of the positive drug; when the concentration of SBSL was 45 mg / kg and 90 mg / kg, the levels of IL-10 and IL-22 increase were better than those of the positive control drug; when the concentration of SBSL was 22 mg / kg and 90 mg / kg, the levels of IL-17 and TNF-α decrease were equivalent to those of the positive drug, and when the concentration of SBSL was 45 mg / kg, the levels of IL-17 and TNF-α decrease were better than those of the positive control drug; these results show that SBSL has anti-inflammatory effects and maintains the stability of intestinal immunity.

[0130] 3.4 Expression of ZO-1, AHR, CYP1A1, CYP1B1, and Trpv6 proteins in colon tissues of mice in each group

[0131] Compared with the normal group, the expression of ZO-1 protein in the colon tissue of the model group decreased (P < 0.05); compared with the model group, the expression of ZO-1 protein in the positive drug administration group and the SBSL drug administration group increased, and the increased level was better than that of the positive control drug when the SBSL concentration was 45 mg / kg and 90 mg / kg. This shows that the stem and leaf extract of Scutellaria baicalensis can maintain intestinal protein homeostasis. Compared with the normal group, the expression of AHR, CYP1A1, and CYP1B1 proteins in the colon tissue of the model group decreased (P < 0.05); compared with the model group, the expression of AHR, CYP1A1, and CYP1B1 proteins in the SBSL drug administration group increased, indicating that the stem and leaf extract of Scutellaria baicalensis can activate AhR, thereby activating the downstream proteins of AhR CYP1A1 and CYP1B1. At the same time, compared with the normal group, the expression of Trpv 6 protein in the colon tissue of the model group increased (P < 0.05); compared with the model group, the expression of Trpv6 protein in the SBSL drug administration group decreased ( Figure 4). This shows that the intestinal barrier of Scutellaria baicalensis stem and leaf extract can improve the rise of clinical indicators of ulcerative colitis. The intestinal barrier is one of the important lines of defense for the body's immune stability and is also a measure of the efficacy of drugs in the treatment of ulcerative colitis. The above results show that SBSL can repair the damaged intestinal epithelial barrier and thus treat ulcerative colitis.

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A scutellaria baicalensis stem and leaf extract, characterized in that Prepared by the following method: (1) extracting the stems and leaves of Scutellaria baicalensis baicalensis with an ethanol solution to obtain an ethanol extract; (2) The ethanol extract is subjected to column chromatography using a non-polar resin. After adsorption, the extract is eluted with water and then with an ethanol solution. The ethanol eluate is collected and concentrated.

2. A method for preparing a scutellaria baicalensis stem and leaf extract, characterized in that: Prepared by the following method: (1) extracting the stems and leaves of Scutellaria baicalensis baicalensis with an ethanol solution to obtain an ethanol extract; (2) The ethanol extract is subjected to column chromatography with a non-polar resin, adsorbed, eluted with water, and then eluted with an ethanol solution, and the ethanol eluate is collected and concentrated.

3. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (1), the volume fraction of the ethanol solution is 45%-95%, preferably 50%-80%, and more preferably 60%-70%; preferably, the extraction is hot extraction, preferably reflux extraction; preferably, the number of extractions is 1-5 times, preferably 2-4 times, and more preferably 3 times.

4. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (1), the volume mass ratio of the ethanol solution to the stems and leaves of Scutellaria baicalensis is 8-40mL:1g, preferably 10-30mL:1g, and more preferably 12-20mL:1g; or the weight ratio is 8-40:1, preferably 6-32:1, preferably 7-20:1, and more preferably 12-20:1 or 10-16:

1.

5. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (1), the extract obtained by the extraction is filtered, the filtrate is concentrated, and the concentrated solution is filtered to obtain the ethanol extract; preferably, the filtrate is concentrated under reduced pressure to concentrate the filtrate until there is no alcohol taste; preferably, the concentrated solution is filtered by centrifugation.

6. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (2), the non-polar resin is D101 macroporous resin or AB-8 macroporous resin, preferably D101 macroporous resin; preferably, the adsorption is static adsorption.

7. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (2), the ethanol extract is loaded in the form of ethanol or aqueous solution; preferably, the loading rate is 0.24-1.8 BV / h, preferably 0.32-1.2 BV / h, and more preferably 0.4-0.6 BV / h; preferably, the adsorption time is 1.2-9 h, preferably 1.6-6 h, and more preferably 2-3 h.

8. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (2), the volume of water elution is 2-9BV, preferably 3-7BV, and more preferably 4-6BV; Preferably, the water elution rate is 0.5-6 BV / h, preferably 0.75-4.5 BV / h, and more preferably 1-3 BV / h.

9. The Scutellaria baicalensis stem and leaf extract according to claim 1 or the preparation method according to claim 2, characterized in that: In step (2), the volume fraction of the ethanol solution is 45%-95%, preferably 55%-85%, and more preferably 65%-75%; preferably, the volume of the ethanol solution elution is 1-6BV, preferably 1.5-5BV, and more preferably 2-4BV; preferably, the elution rate of the ethanol solution is 0.9-7.5BV / h, preferably 1.2-5BV / h, and more preferably 1.5-2.5BV / h; preferably, the concentration is reduced pressure concentration; preferably, further drying is performed after concentration.

10. The Scutellaria baicalensis stem and leaf extract or the preparation method according to any one of claims 1 to 9, characterized in that: The method is: (1) taking the stems and leaves of Scutellaria baicalensis, adding 10-20 times the volume fraction of 50%-80% ethanol solution to reflux extraction, the extraction times are 3 times, each time for 1 hour, filtering the obtained extract, and combining the three extracts, concentrating under reduced pressure until there is no alcohol taste, centrifuging and filtering to obtain a filtrate; (2) The filtrate is loaded onto D101 macroporous resin at a flow rate of 0.4-0.6 BV / h, and allowed to stand for adsorption for 2-4 hours. The filtrate is then eluted with 3.5-6 BV of pure water at a flow rate of 1-5 BV / h, and then eluted with 1.2-5 BV of 65%-75% ethanol solution at a flow rate of 1.5-2.5 BV / h. The 65%-75% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Scutellaria baicalensis stem and leaf extract.

11. The Scutellaria baicalensis stem and leaf extract or the preparation method according to any one of claims 1 to 10, characterized in that: The total flavonoid content in the scutellaria baicalensis stem and leaf extract is more than 50%, further 55%-75%, preferably 60%-70%.

12. Use of the scutellaria baicalensis stem and leaf extract according to any one of claims 1 to 11 or the scutellaria baicalensis stem and leaf extract prepared by the method according to any one of claims 1 to 11 in the preparation of a drug for treating ulcerative colitis.

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