Application of scutellaria baicalensis stem and leaf extract

Abstract

The invention belongs to the field of medicines, and particularly discloses an application of a scutellaria baicalensis stem and leaf extract, and the scutellaria baicalensis stem and leaf extract has a good effect on treating anemia induced by ulcerative colitis and improving erythropoietin.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and specifically discloses the application of an extract from the stems and leaves of Scutellaria baicalensis. Background Technology

[0002] Scutellaria baicalensis is a perennial herb belonging to the genus Scutellaria in the family Lamiaceae. Its root is used medicinally and is a commonly used traditional Chinese medicine. Scutellaria baicalensis is cold in nature and bitter in taste, entering 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, feverish thirst, cough due to lung heat, red and swollen eyes, enteritis, dysentery, and other diseases.

[0003] Scutellaria baicalensis root mainly contains flavonoid derivatives, which act as carriers for its anti-inflammatory and antiviral effects. The flavonoids mainly include baicalin, baicalein, wogonin, wogonin glycoside, and baicalein succinate. Non-flavonoid substances include scutellarin A, benzoic acid, and sitosterol.

[0004] Traditionally, the root of Scutellaria baicalensis is used in traditional Chinese medicine, with an annual production of approximately 20,000 tons in recent years. However, about 40,000 tons of the stems and leaves are discarded each year, resulting in a serious waste of medicinal resources and placing enormous pressure on the ecological environment. The stems and leaves of Scutellaria baicalensis are the above-ground parts used medicinally, and their chemical composition differs significantly from that of the root. The main component is baicalin, and data shows that the stems and leaves also contain two flavonoids, safflowerin and isocaryonin, as well as trace amounts of baicalin, tannins, and resins.

[0005] Currently, there are no traditional Chinese medicine treatments for anemia using Scutellaria baicalensis stem and leaf extracts. Therefore, it is of research significance to develop a drug containing Scutellaria baicalensis stem and leaf extracts that can provide long-term treatment, stabilize the condition, and is inexpensive. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of Scutellaria baicalensis stem and leaf extract, which addresses the shortcomings of the prior art.

[0007] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0008] This invention discloses the application of Scutellaria baicalensis stem and leaf extract in the preparation of drugs for treating anemia.

[0009] The Scutellaria baicalensis stem and leaf extract of the present invention was prepared by the following method:

[0010] Step S1: The stems and leaves of Scutellaria baicalensis are extracted with ethanol solution to obtain an ethanol extract;

[0011] Step S2: The ethanol extract was subjected to column chromatography with a non-polar resin. After adsorption, the sample was eluted with water and then with ethanol solution. The ethanol eluent was collected and concentrated.

[0012] In the preparation of the above-mentioned Scutellaria baicalensis stem and leaf extract, the parameters of each step have the following characteristics:

[0013] In step S1, the volume fraction of the ethanol solution is 45%-95%, preferably 50%-80%, more preferably 60%-70%, and in some embodiments it is 50%, 60%, or 70%.

[0014] In step S1, the extraction is thermal extraction, and in some embodiments it is reflux extraction.

[0015] In step S1, the extraction is performed 1-5 times, 2-4 times in some embodiments, and 3 times in some embodiments.

[0016] In step S1, the volume-to-mass ratio of the ethanol solution to the Scutellaria baicalensis stems and leaves is 8-40 mL:1 g, in some embodiments it is 10-30 mL:1 g, in some embodiments it is 12-20 mL:1 g; or by weight it is 8-40:1, in some embodiments it is 6-32:1, in some embodiments it is 7-20:1, in some embodiments it is 12-20:1 or 10-16:1.

[0017] In step S1, the extracted liquid is filtered, the filtrate is concentrated, and the concentrated liquid is filtered to obtain the ethanol extract; in some embodiments, the filtrate is concentrated under reduced pressure until there is no alcohol odor; in some embodiments, the concentrated liquid is filtered by centrifugation.

[0018] In step S2, the non-polar resin is either 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.

[0019] In step S2, the ethanol extract is loaded with ethanol or an aqueous solution; in some embodiments, the loading rate is 0.24-1.8 BV / h, in some embodiments it is 0.32-1.2 BV / h, in some embodiments it is 0.4-0.6 BV / h, and in some embodiments it is 0.4 BV / h, 0.5 BV / h, or 0.6 BV / h.

[0020] In step S2, the adsorption time is 1.2-9 hours, 1.6-6 hours in some embodiments, 2-3 hours in some embodiments, and 2 hours, 2.5 hours, or 3 hours in some embodiments.

[0021] In step S2, the adsorption is, in some embodiments, static adsorption.

[0022] In step S2, the volume of water elution is 2-9 BV, in some embodiments it is 3-7 BV, in some embodiments it is 4-6 BV, and in some embodiments it is 4 BV, 5 BV, or 6 BV.

[0023] In step S2, the water elution rate is 0.5-6 BV / h, in some embodiments it is 0.75-4.5 BV / h, in some embodiments it is 1-3 BV / h, and in some embodiments it is 1 BV / h, 2.5 BV / h, or 3 BV / h.

[0024] In step S2, the volume fraction of the ethanol solution is 45%-95%, in some embodiments it is 55%-85%, in some embodiments it is 65%-75%, and in some embodiments it is 65%, 70%, or 75%.

[0025] In step S2, the elution volume of the ethanol solution is 1-6 BV, in some embodiments it is 1.5-5 BV, in some embodiments it is 2-4 BV, and in some embodiments it is 2 BV or 4 BV.

[0026] In step S2, the elution rate of the ethanol solution is 0.9-7.5 BV / h, in some embodiments it is 1.2-5 BV / h, in some embodiments it is 1.5-2.5 BV / h, and in some embodiments it is 1.5 BV / h, 2 BV / h, or 2.5 BV / h.

[0027] In step S2, the concentration is performed under reduced pressure; in some embodiments, the concentration is followed by further drying to obtain a Scutellaria baicalensis stem and leaf extract.

[0028] In some embodiments, the Scutellaria baicalensis stem and leaf extract is prepared by the following method, which includes the following steps;

[0029] Take the stems and leaves of Scutellaria baicalensis, add 10-20 times the volume of 50%-80% ethanol solution for reflux extraction, extract three times, one hour each time. Filter the resulting extracts and combine the three extracts. Concentrate under reduced pressure until there is no alcohol odor, centrifuge and filter to obtain the filtrate. Load the filtrate onto D101 macroporous resin at a flow rate of 0.4-0.6 BV / h, allow it to stand for adsorption for 2-4 hours, elute with 3.5-6 BV of pure water at a flow rate of 1-5 BV / h, and elute with 1.2-5 BV of 65%-75% ethanol solution at a flow rate of 1.5-2.5 BV / h. Collect the 65%-75% ethanol eluent, concentrate under reduced pressure and dry to obtain the Scutellaria baicalensis stem and leaf extract.

[0030] In this invention, there are no specific requirements for the centrifugation parameters. For example, centrifugation can be performed at 50-5000 rpm for 1-30 minutes, or at 3500 rpm for 10 minutes.

[0031] Unless otherwise specified, the ethanol solutions described in this invention are all aqueous solutions of ethanol.

[0032] In this invention, the Scutellaria baicalensis stems and leaves are extracted with an ethanol solution. Unless otherwise specified, this means that the Scutellaria baicalensis stems and leaves are extracted with a coarse powder using an ethanol solution. There are no requirements on the mesh size of the coarse powder; it can be pulverized.

[0033] The diameter-to-height ratio of the column chromatography column described in this invention is 1:2-10, 1:4-8 in some embodiments, and 1:6 in some embodiments.

[0034] The total flavonoid content of the Scutellaria baicalensis stem and leaf extract prepared by this invention is more than 50%, and in some cases it is 50-75%, in some cases it is 55-70%, and in some cases it is 60%-70%.

[0035] The Scutellaria baicalensis stem and leaf extract prepared in this invention has a good therapeutic effect on anemia, and can treat anemia induced by ulcerative colitis and increase erythropoietin.

[0036] The present invention also provides a formulation of the Scutellaria baicalensis stem and leaf extract obtained by the preparation method described above, which is a solid dosage form containing pharmaceutically acceptable excipients, such as the filler mannitol.

[0037] The beneficial effects of this invention are:

[0038] Compared with the prior art, the present invention provides a high content of effective components in the Scutellaria baicalensis stem and leaf extract, a simple preparation method, and a higher extract yield; the Scutellaria baicalensis stem and leaf extract obtained by the present invention can safely and effectively treat anemia and increase erythropoietin (EPO). Attached Figure Description

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0040] Figure 1 Effects of Scutellaria baicalensis stem and leaf extracts on colon length in UC mice

[0041] Figure 2 Effects of Scutellaria baicalensis stem and leaf extracts on histopathological changes in colon tissue of UC mice (HE, ×20).

[0042] Figure 3 Effects of Scutellaria baicalensis stem and leaf extracts on the mRNA expression of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in UC mice

[0043] Figure 4Effects of total flavonoids from Scutellaria baicalensis stems and leaves on the expression of ZO-1, AHR, CYP1A1, CYP1B1, and Trpv6 proteins in the colon tissue of UC mice.

[0044] Figure 5 Cell viability diagram of Scutellaria baicalensis stem and leaf extract.

[0045] ## indicates p < 0.05 compared to the control group; ** indicates p < 0.05 compared to the model group; * indicates p < 0.01 compared to the model group. Detailed Implementation

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0047] In the following examples, the Scutellaria baicalensis stems and leaves are dried products, and the centrifugation is performed at 3000-4000 rpm for 6-15 minutes.

[0048] Example 1: Preparation of Scutellaria baicalensis stem and leaf extract

[0049] Step S1: Take 50g of coarse powder of Scutellaria baicalensis stems and leaves, add 800mL of 60% ethanol solution and reflux for extraction. The extraction is performed 3 times, 1 hour each time. The resulting extract is filtered and the liquids from the three extractions are combined. The mixture is concentrated under reduced pressure until there is no alcohol odor, centrifuged and filtered to obtain the filtrate.

[0050] Step S2: Load the filtrate onto D101 macroporous resin at a flow rate of 0.5 BV / h, allow it to stand for 3 hours for adsorption, elute with 4 BV of pure water at a flow rate of 3 BV / h, discard the water wash, and continue eluting with 4 BV of 70% ethanol at a flow rate of 2 BV / h. Collect the 70% ethanol eluent, concentrate under reduced pressure, and dry to obtain 5.63 g of Scutellaria baicalensis stem and leaf extract.

[0051] Example 2: Preparation of Scutellaria baicalensis stem and leaf extract

[0052] Step S1: Take 50g of Scutellaria baicalensis stem and leaf powder (passed through a 20-mesh sieve), add 600mL of 60% ethanol solution and reflux for extraction. Extract three times, one hour each time. Filter the resulting extract and combine the liquids from the three extractions. Concentrate under reduced pressure until there is no alcohol odor, centrifuge and filter to obtain the filtrate.

[0053] Step S2: Load the filtrate onto D101 macroporous resin at a flow rate of 0.6 BV / h, allow it to stand for 2 hours for adsorption, elute with 5 BV pure water at a flow rate of 2.5 BV / h, discard the water wash, and continue eluting with 4 BV 65% ethanol at a flow rate of 1.5 BV / h. Collect the 65% ethanol eluent, concentrate under reduced pressure, and dry to obtain 6.12 g of Scutellaria baicalensis stem and leaf extract.

[0054] Example 3: Preparation of Scutellaria baicalensis stem and leaf extract

[0055] Step S1: Take 50g of coarse powder of Scutellaria baicalensis stems and leaves, add 1000mL of 70% ethanol solution and reflux for extraction. The extraction is performed 3 times, 1 hour each time. The resulting extract is filtered and the liquids from the three extractions are combined. The mixture is concentrated under reduced pressure until there is no alcohol odor, centrifuged and filtered to obtain the filtrate.

[0056] Step S2: Load the filtrate onto D101 macroporous resin at a flow rate of 0.4 BV / h, allow it to stand for 2.5 hours for adsorption, elute with 6 BV of pure water at a flow rate of 1 BV / h, discard the water wash, and continue eluting with 2 BV of 75% ethanol at a flow rate of 2.5 BV / h. Collect the 75% ethanol eluent, concentrate under reduced pressure, and dry to obtain 5.99 g of Scutellaria baicalensis stem and leaf extract.

[0057] Comparative Example 1: Preparation of Scutellaria baicalensis stem and leaf extract (Patent CN100378089C)

[0058] 50g of Scutellaria baicalensis stems and leaves were pulverized into a 20-mesh coarse powder. After enzyme inactivation treatment, the powder was extracted twice with water, each time adding 10 times the amount of water as crude drug. The extraction was carried out at 100℃ for 1 hour. The two extracts were combined and concentrated under reduced pressure at 70±5℃ to a relative density of 1.02-1.10 (measured at 20℃). After centrifugation, the supernatant was obtained and acidified with hydrochloric acid to pH 2-3. After centrifugation, the supernatant was adsorbed onto AB-8 type macroporous adsorption resin, washed with purified water until neutral, and then desorbed with 70% ethanol. The eluent was collected, the ethanol was recovered, and the solution was concentrated. The solution was dried under reduced pressure at 70±5℃ to dryness to obtain the final product.

[0059] Example 4: Detection of Scutellaria baicalensis stem and leaf extracts

[0060] The content of baicalin was determined by measuring the content of baicalin in the prepared Scutellaria baicalensis stem and leaf extract. The detection method followed the content determination method for medicinal materials in the "Jilin Province Standard for Traditional Chinese Medicines". The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the packing material; methanol-0.02% formic acid solution (28:27) as the mobile phase; and a detection wavelength of 335 nm.

[0061] The total flavonoid content refers to the total flavonoid content in the prepared Scutellaria baicalensis stem and leaf extract. The determination method is ultraviolet spectrophotometry, with baicalin as the reference standard.

[0062] The transfer rate of scutellarin is the content of scutellarin in the prepared extract of Scutellaria baicalensis stem and leaf / the content of scutellarin in the crude powder of Scutellaria baicalensis stem and leaf;

[0063] The extraction rate of the extract is the weight of the extract of Scutellaria baicalensis stem and leaf / the weight of the crude powder of Scutellaria baicalensis stem and leaf.

[0064] Table 1 Determination of the content and transfer rate of the samples in the examples

[0065]

[0066] Effect of the extract of Scutellaria baicalensis stem and leaf in Example 5 on the cell viability of normal colon epithelial cells (NCM460)

[0067] Normal colon epithelial cells (NCM460) were cultured in RPMI 1640 medium, and the cell density was adjusted to 10

[0077] ,

[0075] ,

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

[0074] , , , , , ,

[0073] ,

[0072] ,

[0071] cells / mL, inoculated into 96-well plates at 100 μL / well. After the cells adhered, the corresponding concentrations of 45 μg / mL of the extracts of Scutellaria baicalensis stem and leaf prepared in Examples 1-3 and Comparative Example 1 prepared with serum-free medium were given, and cultured at 37 °C and 5% CO2. After 24 h, 10 μL / well of MTT solution was added under light protection. After 3 h, 150 μL / well of DMSO solution was added, and the absorbance OD value was detected at 490 nm by an enzyme-labeled instrument.

[0068] From the results of MTT (Table 2, Figure 5 ), it can be seen that the extracts of Scutellaria baicalensis stem and leaf in Examples 1-3 did not cause significant changes in the cell viability of normal colon epithelial cells, while Comparative Example 1 could significantly inhibit the cell viability of normal colon epithelial cells.

[0069] Table 2

[0070] sample Cell viability (%) Example 1 98 Example 2 97 Example 3 95 Comparative Example 1 80

[0071] Effect of the extract of Scutellaria baicalensis stem and leaf in Example 6 on mice with ulcerative colitis (UC) induced by 3% DSS

[0072] 1. Experimental materials

[0073] 1.1 Animals

[0074] 60 male C57bl / 6 mice, weighing (18-20) g, animal license number SCXK (Beijing) 2019-0010, provided by Spf (Beijing) Biotechnology Co., Ltd., and conventionally raised.

[0075] 1.2 Drugs and reagents

[0076] The extract of Scutellaria baicalensis stem and leaf in Example 1 (SBSL);

[0077] Sodium dextran sulfate (batch number 160110) was purchased from MP Biomedicals, USA.

[0078] Sulfasalazine (National Drug Approval Number H31020840) was purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.

[0079] chloroform (batch number 10006818) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0080] Isopropanol (batch number 80109218) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0081] 75% ethanol (batch number 10009218) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0082] RevertAid First-Strand cDNA Synthesis Kit (lot number #K1622) was purchased from Thermo.

[0083] FastStart Universal SYBR Green Master (Rox) (lot number 04 913 914 001) was purchased from Roche.

[0084] 1.3 Instruments

[0085] KZ-II Homogenizer (Kangtao Technology);

[0086] D3024R Tabletop High-Speed ​​Refrigerated Microcentrifuge (DragonLab);

[0087] SLAN Real-Time PCR Detection System (Shanghai Hongshi Medical Instruments);

[0088] SW-CJ-1FD Clean Bench (Suzhou Jingantai);

[0089] NanoDrop2000 Ultra-Micro Spectrophotometer (Thermo);

[0090] FBZ2001-up-p Standard Reagent Type Pure Water System (Qingdao Fullerm Technology Co., Ltd.);

[0091] Ensight Multifunctional Microplate Reader (Platinum Elmer Instruments Co., Ltd.)

[0092] 2. Experimental Methods

[0093] 2.1 Modeling and Drug Administration

[0094] Sixty healthy C57bl / 6 mice were randomly divided into six groups: a normal control group, a model group, low-, medium-, and high-dose SBSL groups (22, 45, and 90 mg / kg), and a SASP positive control group (450 mg / kg), with ten mice in each group. Mice were acclimatized for one week. During the experiment, except for the normal control group, the other experimental groups were given 3% DSS (w / v) in drinking water for seven consecutive days to induce an acute UC model in mice. Fresh DSS water was provided every morning. Simultaneously with modeling, each treatment group received the corresponding treatment drug orally between 8:00 and 10:00 AM, while the control group received the same amount of 0.5% CMC-Na solution once daily for seven consecutive days. The mice's general condition, including hair, diet, and stool, was observed daily. Body weight was recorded, and fecal occult blood tests were performed. The disease activity index (DAI) was scored using the Cooper method.

[0095] 2.2 Animal sampling and sample pretreatment

[0096] Seven days after drug administration, mice in each group were fasted for 24 hours, and samples were collected on day 8. After enucleation and blood collection, the abdominal cavity of mice euthanized by cervical dislocation was opened. Colonic tissue from the ileocecal junction to the anus was taken and placed on ice, its length measured and recorded. The intestinal contents were then rinsed with physiological saline, and a 1cm segment of colon near the anus was harvested and fixed in 10% formalin for paraffin section preparation. The remaining tissue, a 3-4mm section from the middle, was flash-frozen in liquid nitrogen and stored at -80℃ for subsequent experiments. Blood samples were allowed to stand at room temperature for 2-3 hours, then centrifuged at 3500 rpm for 5 minutes. Serum was collected and rapidly stored at -80℃ for subsequent analysis.

[0097] 2.3 Evaluation of colonic inflammation

[0098] DAI score: According to the standard proposed by Cooper et al., the score was determined by combining the percentage decrease in body weight, stool characteristics and fecal occult blood status of mice after 7 days of drug administration, as shown in Tables 3 and 4.

[0099] Table 3 DAI Scoring Criteria

[0100]

[0101] Table 4 Criteria for Judging Occult Blood Detection

[0102]

[0103] 2.4 Histopathological examination of colon tissue

[0104] Mouse colons were fixed in 10% formalin, dehydrated, embedded in paraffin, and sectioned. After hematoxylin-eosin (HE) and peroxidase-Schiff (PAS) staining, the pathological changes of mouse colon tissue were observed under a microscope.

[0105] 2.5 Effects of Scutellaria baicalensis stem and leaf extracts on the activities of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in UC mice

[0106] Twenty-four hours after the last administration, the mice were weighed, and blood was collected by enucleation. The blood samples were left 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 kit instructions.

[0107] 2.7 The expression levels of ZO-1, Claudin-2, AHR, CYP1A1, CYP1B1, and Trpv6 proteins were determined by qPCR.

[0108] Total RNA was extracted from the colon according to the RNA kit instructions, and the relative expression levels of the target genes were analyzed. Primers for each signal mRNA molecule are shown in Table 5.

[0109] Table 5 Primer sequences

[0110]

[0111] 3. Experimental Results

[0112] 3.1 SBSL alleviates symptoms of DSS-induced ulcerative colitis in mice

[0113] In a mouse model of DSS-induced ulcerative colitis treated with Scutellaria baicalensis stem and leaf extract, mice in the model group gradually developed clinical symptoms such as depression, bradykinesia, bloody stools, diarrhea, and weight loss (Table 6) after continuous ingestion of 3.5% DSS in drinking water. Their disease activity index (DAI) was significantly higher than that of the control group (Table 7). Furthermore, the colon length in the model group was significantly shortened. Figure 1 These results indicate that the mouse acute UC model was successfully established.

[0114] Compared with the model group, the mouse model of ulcerative colitis showed improved coat color, slowed movement, bloody stools, weight loss, and shortened colon length after intervention with Scutellaria baicalensis stem and leaf extract. Compared with the model group, different doses of SBSL significantly increased body weight, and the 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 significantly reduced DAI scores, and the DAI scores were even lower than those of the positive control group (Table 7). Compared with the model group, medium / high doses of SBSL significantly increased colon length, and the colon length of the medium, low, and high dose groups was longer than that of the positive control group. Figure 1 These results indicate that the extract of Scutellaria baicalensis stems and leaves has therapeutic effects on ulcerative colitis.

[0115] Table 6. Effects of each group on the change in body weight of UC mice (ratio of mouse body weight on the current day to body weight on day 1) / %

[0116]

[0117] Table 7 DAI scores for each group

[0118]

[0119] 3.2 Histopathological findings of colon tissue in each group of mice

[0120] Histopathological examination of the colon tissue revealed severe damage to the colonic mucosa in the model group, with extensive loss of crypts, and hemorrhage, edema, and extensive inflammatory cell infiltration in the mucosa and submucosa. However, the colonic glands and other structures were more clearly defined in mice treated with different doses of SBSL. Figure 2 ).

[0121] 3.3 mRNA levels of TNF-α, IL-1β, IL-10, IL-17 and IL-22 in each group of mice

[0122] The results showed that, compared with the control group, the model group had significantly increased levels of TNF-α, IL-1β, and IL-17 (p<0.05), while the levels of IL-10 and IL-22 were significantly decreased (p<0.05), indicating abnormal immune function and significant inflammatory response in the model group mice. Compared with the model group, different doses of SBSL significantly reduced the levels of TNF-α, IL-1β, and IL-17 in mouse plasma (p<0.05), while high / medium doses of SBSL significantly increased the levels of IL-10 and IL-22 in plasma (p<0.05). Figure 3Specifically, when the concentration of SBSL was one-twentieth (22 mg / kg) and one-fifth (90 mg / kg) of the positive control drug, the increase in IL-1β was comparable to that of the positive control drug; when the concentration of SBSL was one-tenth (45 mg / kg) of the positive control drug, the increase in IL-1β was superior to that of the positive control drug. At SBSL concentrations of 45 mg / kg and 90 mg / kg, the increases in IL-10 and IL-22 were superior to those of the positive control drug. At SBSL concentrations of 22 mg / kg and 90 mg / kg, the decreases in IL-17 and TNF-α were comparable to those of the positive control drug; at SBSL concentration of 45 mg / kg, the decreases in IL-17 and TNF-α were superior to those of the positive control drug. These results indicate that SBSL has anti-inflammatory effects and maintains the stability of intestinal immunity.

[0123] 3.4 Expression of ZO-1, AHR, CYP1A1, CYP1B1, and Trpv6 proteins in colon tissue of mice in each group

[0124] Compared with the normal group, ZO-1 protein expression in the colonic tissue of the model group decreased (P < 0.05); compared with the model group, ZO-1 protein expression increased in the positive control group and the SBSL-treated group, and the increase was greater than that of the positive control drug at SBSL concentrations of 45 mg / kg and 90 mg / kg. This indicates that Scutellaria baicalensis stem and leaf extract can maintain intestinal protein homeostasis. Compared with the normal group, AHR, CYP1A1, and CYP1B1 protein expression decreased in the colonic tissue of the model group (P < 0.05); compared with the model group, AHR, CYP1A1, and CYP1B1 protein expression increased in the SBSL-treated group, indicating that Scutellaria baicalensis stem and leaf extract can activate AhR, thereby activating AhR downstream proteins CYP1A1 and CYP1B1. Meanwhile, compared with the normal group, Trpv6 protein expression increased in the colonic tissue of the model group (P < 0.05); compared with the model group, Trpv6 protein expression decreased in the SBSL-treated group (P < 0.05). Figure 4 The results indicate that the intestinal barrier function of Scutellaria baicalensis stem and leaf extract can improve the elevated clinical indicators of ulcerative colitis. The intestinal barrier is one of the important lines of defense for immune homeostasis in the body and is also a measure of the efficacy of drug treatment for ulcerative colitis. The above results suggest that SBSL can repair the damaged intestinal epithelial barrier, thereby treating ulcerative colitis.

[0125] Example 7: Animal Experiment Study on the Effects of Scutellaria baicalensis Stem and Leaf Extract on Anemia

[0126] 1. Animals

[0127] 140 male C57BL / 6J mice, with a body weight of (20±2) g, were purchased from Spf(Beijing) Biotechnology Co., Ltd. The animals were housed in the SPF-class animal laboratory of Jiangsu Suzho Pharmaceutical Group Co., Ltd. Biological Pharmaceutical Co., Ltd. The room temperature was (20±2) °C, and the relative humidity was 40% - 60%. During the experiment, the management and regulations of the animal laboratory of Jiangsu Suzho Pharmaceutical Group Co., Ltd. were strictly followed. The animal license number was SCXK(Su) 2022 - 0006, and the ethical approval number was 2023111601.

[0128] 2. Establishment of the anemia model

[0129] Referring to and improving the method in the literature [WIRTZ S, POPP V, KINDERMANN M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation [J]. Nature protocols, 2017, 12(7): 1295 - 309.6], a UC mouse model was established using Oxa. The entire modeling process was divided into three stages: pre-sensitization (d1 - 2), the first enema (d8), and the second enema (d12). One day before pre-sensitization, the hair on the back of the mouse's neck at 1.5×1.5 cm was removed. On d1 - 2, 0.15 ml of 3% Oxa solution (a mixed solution of acetone and olive oil with a volume ratio of 4:1) was applied to the shaved area for sensitization. On d7, the mice were fasted but allowed to drink water for 24 h. On d8, the first enema was performed using 1% Oxa solution (50% absolute ethanol). On d12, the second enema was performed. The enema operation was as follows: A 1 ml syringe was connected to a soft catheter with a diameter of 1 mm as the enema device. The end of the hose was slowly inserted into the anus of the mouse about 4 cm, and the syringe was gently pushed to drip 0.1 ml of 1% Oxa solution into the colon. After staying for 1 min, the hose was slowly withdrawn, and the anus was gently pressed with absorbent cotton. The mouse was always kept with its head down to prevent the liquid from flowing out. After 2 min, it was placed in the cage for normal feeding. After 24 h of enema, loose stools and positive fecal occult blood test in the mice indicated the successful establishment of the UC mouse model.

[0130] 4. Grouping and administration

[0131] The 90 successfully modeled mice were randomly divided into a model (Model) group, a vehicle (Vehicle) group, a positive drug (SASP, sulfasalazine enteric-coated tablets, batch number 22220605, specification: 0.25 g / tablet) group (450 mg·kg -1 ), and low, medium, and high-dose groups of total flavonoids from Scutellaria baicalensis stems and leaves (L, M, H-SSTF, Scutellaria baicalensis stem and leaf extract in Example 1) (26, 52, 104 mg·kg -1Six groups (n=15) were established. An additional 15 mice were designated as the normal group, receiving no treatment during the modeling period. Mice were administered the drug via gavage for 7 consecutive days (days 9-15). The normal and model groups were administered an equal volume of physiological saline, while the vehicle group was administered an equal volume of 0.5% CMC-Na solution. During the drug administration period, the disease activity index (DAI) was assessed using the following criteria: ≤1% body weight loss, normal stool, negative fecal occult blood test: 0 points; 1%–5% body weight loss, soft but formed stool, positive fecal occult blood test: 1 point; 5%–10% body weight loss, semi-loose stool, mild bloody stool: 2 points; 10%–15% body weight loss, loose stool, moderate bloody stool: 3 points; ≥15% body weight loss, severe loose stool, severe bloody stool: 4 points.

[0132] 5. ELISA method for detecting the levels of TNF-α, EPO, SI, IL-10, SF, Hb, Hepcidin, and sTfR in mouse serum.

[0133] Twenty-four hours after the last administration, blood was collected by enucleation. After standing at room temperature for 30 minutes, the blood was centrifuged at low temperature and high speed. The supernatant was collected, and the levels of TNF-α, EPO, SI, IL-10, SF, Hb, Hepcidin, and sTfR were measured strictly according to the kit instructions. Statistical analysis methods: P-values ​​for GO analysis and KEGG enrichment analysis in network pharmacology were performed using the Metascape database. Experimental data were analyzed using GraphPad Prism 9.5.1 software. One-way ANOVA was used for comparisons between groups, and results are expressed as mean ± standard deviation. P < 0.05 indicates that the difference is statistically significant.

[0134] 6. RT-qPCR method for detecting the relative expression levels of EPOR, ERFE, and FPN mRNA in mouse spleen tissue and Hepcidin and FPN mRNA in liver tissue.

[0135] 100 mg of mouse spleen and liver tissue were rapidly and thoroughly homogenized. Total RNA was extracted from colon tissue using the TRIzol method, and cDNA was synthesized by reverse transcription. β-actin was used as an internal reference gene. The relative mRNA levels among the groups were calculated (Tab 1). All primers used in the experiment were synthesized by Jiangsu Enzyme Immunoassay Co., Ltd. The primers for each signal mRNA molecule are as follows.

[0136] Primer sequence

[0137] primer sequence

[0138]

[0139] 6. Effects of SSTF on serum IL-10, TNF-α, EPO and Hb in UC mice

[0140] Inflammatory factors and anemia markers in the serum of mice in each group were detected. Compared with the Normal group, the serum TNF-α level of Model mice was significantly increased (P<0.01), while the levels of IL-10, EPO, and Hb were significantly decreased (P<0.01). Compared with the Model group, the M and H-SSTF groups decreased the serum TNF-α level (P<0.01) and increased the serum EPO, Hb, and IL-10 levels (P<0.05, 0.01, respectively) (Tab 3).

[0141] Table 3 Effects of SSTF on TNF-α, IL-10 and EPO in UC mice

[0142] Tab 3 Effects of SSTF on TNF-а, IL-10 and EPO in UC mice

[0143]

[0144] ## P < 0.01 vs Normal group; # P < 0.05 vs Normal group; ** P < 0.01 vs Modelgroup; * P < 0.05 vs. Model group.

[0145] ## P < 0.01 compared with the normal group; ** P < 0.01 compared to the model group; * P < 0.05 compared to the model group.

[0146] 7. Effects of SSTF on serum SI, SF, sTfR, and Hepcidin in UC mice

[0147] Serum iron metabolism parameters were measured in mice of each group. Compared with the Normal group, the levels of Hepcidin, SF, and sTfR in the serum of mice in the Model group were significantly increased (P<0.01), while the level of SI was significantly decreased (P<0.01). Compared with the Model group, different doses of SSTF increased the level of SI in the serum of mice (P<0.01) and decreased the levels of Hepcidin, SF, and sTfR in the serum (P<0.01) (Tab 4).

[0148] Table 4. Effects of SSTF on Hb, SI, SF, sTfR, and Hepcidin in UC mice.

[0149] Tab 4 Effects ofSSTF on Hb, SI, SF, sTfR and Hepcidin in UC mice

[0150]

[0151] ## P < 0.01 vs Normal group; ** P < 0.01 vs Model group; * P < 0.05 vs Modelgroup.

[0152] ## P < 0.01 compared with the normal group; ** P < 0.01 compared to the model group; * P < 0.05 compared to the model group.

[0153] 8. Effects of SSTF on the expression of EPOR, ERFE, and FPN in spleen tissue and Hepcidin and FPN in liver tissue of UC mice.

[0154] Compared with the Normal group, the Model group mice showed decreased levels of EPOR, ERFE, and FPN in spleen tissue and decreased FPN level in liver tissue (P<0.05), while increased Hepcidin level in liver tissue (P<0.01). Compared with the Model group, the SASP and H-SSTF groups showed increased EPOR, ERFE, and FPN mRNA expression in spleen tissue and increased FPN expression in liver tissue (P<0.05)(P<0.01), while decreased Hepcidin expression in liver tissue (P<0.05) (Tab 5)(Tab 6). This indicates that SSTF can activate the EPO / EPOR signaling pathway, promote ERFE expression, thereby inhibiting Hepcidin expression, ultimately stabilizing FPN expression, maintaining stable iron metabolism, promoting iron mobilization, and improving anemia and iron metabolism indicators.

[0155] Table 5. Expression of EPOR, ERFE, and FPN in spleen tissue of mice in each group as detected by RT-qPCR.

[0156] Tab 5 RT-qPCR detection of EPOR, ERFE, FPN expression in spleen tissue of mice in each group

[0157]

[0158] ## P < 0.01 vs Normal group; ** P < 0.01 vs Model group; * P < 0.05 vs Modelgroup.

[0159] ## P < 0.01 compared with the normal group; ** P < 0.01 compared to the model group; * P < 0.05 compared to the model group.

[0160] Table 6. Expression of FPN and Hepcidin in liver tissue of mice in each group detected by RT-qPCR

[0161] Tab 6 RT-qPCR detection of FPN, Hepcidin expression in liver tissue of mice in each group

[0162]

[0163] ## P < 0.01 vs Normal group; ** P < 0.01 vs Modelgroup; * P < 0.05 vs. Model group.

[0164] ## P < 0.01 compared with the normal group; ** P < 0.01 compared to the model group; * P < 0.05 compared to the model group.

[0165] The above results show that the extract of the present invention can increase EPO levels and has a good effect on the treatment of anemia.

[0166] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Use of a Scutellaria baicalensis stem-leaf extract in the preparation of a medicament for treating anemia, wherein the Scutellaria baicalensis stem-leaf extract is prepared by the following method: Step S1: extracting Scutellaria baicalensis stems and leaves with an ethanol solution to obtain an ethanol extract; Step S2: subjecting the ethanol extract to column chromatography with a non-polar resin, adsorbing and then eluting with water and an ethanol solution, collecting the ethanol eluate, and concentrating. In step S1, the volume fraction of the ethanol solution is 45-95%, preferably 50-80%, and further preferably 60-70%; preferably, the extraction is hot extraction, preferably reflux extraction; preferably, the extraction is performed 1-5 times, preferably 2-4 times, and further preferably 3 times. In step S1, the volume-to-mass ratio of the ethanol solution to the Scutellaria baicalensis stems and leaves is 8-40 mL: 1 g, preferably 10-30 mL: 1 g, and further preferably 12-20 mL: 1 g; or the weight ratio is 8-40: 1, preferably 6-32: 1, preferably 7-20: 1, and further preferably 12-20: 1 or 10-16:

1.

2. Use according to claim 1, characterized in that, In step S1, the extract obtained by extraction is filtered, the filtrate is concentrated, and the concentrate is filtered to obtain the ethanol extract; preferably, the filtrate is concentrated under reduced pressure until the filtrate has no alcohol smell; preferably, the concentrate is filtered by centrifugation.

3. Use according to claim 1, characterized in that, In step S2, the non-polar resin is D101 macroporous resin or AB-8 macroporous resin, and preferably D101 macroporous resin; preferably, the adsorption is static adsorption.

4. Use according to claim 1, characterized in that, In step S2, the ethanol extract is loaded with an ethanol or water solution; preferably, the loading rate is 0.24-1.8 BV / h, preferably 0.32-1.2 BV / h, and further preferably 0.4-0.6 BV / h; preferably, the adsorption time is 1.2-9 h, preferably 1.6-6 h, and further preferably 2-3 h.

5. The use according to claim 1, characterized in that, In step S2, the water elution volume is 2-9 BV, preferably 3-7 BV, and further preferably 4-6 BV; preferably, the water elution rate is 0.5-6 BV / h, preferably 0.75-4.5 BV / h, and further preferably 1-3 BV / h.

6. Use according to claim 1, characterized in that, In step S2, the volume fraction of the ethanol solution is 45-95%, preferably 55-85%, and further preferably 65-75%; preferably, the ethanol solution elution volume is 1-6 BV, preferably 1.5-5 BV, and further preferably 2-4 BV; preferably, the ethanol solution elution rate is 0.9-7.5 BV / h, preferably 1.2-5 BV / h, and further preferably 1.5-2.5 BV / h; preferably, the concentration is under reduced pressure; and preferably, further drying is performed after concentration.

7. The use according to claim 1, characterized in that, The method is as follows: ​ 8. The use according to claim 1, characterized in that, ​ 9. Use according to claim 1, characterized in that, ​ The Scutellaria baicalensis Georgi stems and leaves are added with 10-20 times of 50%-80% ethanol solution to reflux extraction, the extraction times is 3 times, 1 hour each time, the obtained extract is filtered, and the three times of extract are combined, concentrated under reduced pressure to no alcohol taste, centrifugal filtration to obtain filtrate; the filtrate is added to D101 macroporous resin, the loading speed is 0.4-0.6 BV / h, the adsorption is static for 2-4 hours, eluted with 3.5-6 BV of pure water, the elution speed is 1-5 BV / h, eluted with 1.2-5 BV of 65%-75% ethanol solution, the elution speed is 1.5-2.5 BV / h, the 65%-75% ethanol eluate is collected, concentrated under reduced pressure and dried to obtain Scutellaria baicalensis Georgi stems and leaves extract.

10. The use as claimed in claim 1, characterized in that The anemia is ulcerative colitis-induced anemia; further, the drug can promote erythropoietin. 11.A solid preparation of Scutellaria baicalensis Georgi stems and leaves extract, which is prepared by the following method: Step S1: Scutellaria baicalensis Georgi stems and leaves are extracted with ethanol solution to obtain ethanol extract; Step S2: the ethanol extract is subjected to column chromatography with non-polar resin, after loading and adsorption, eluted with water and ethanol solution, the ethanol eluate is collected and concentrated.

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