Composition of arctium lappa and lithospermum erythrorhizon, method for preparing same, and use thereof

Abstract

Provided is a composition of Arctium lappa and Lithospermum erythrorhizon and a method for preparing same. The composition comprises an A. lappa extract and an L. erythrorhizon extract, wherein the A. lappa extract accounts for 90%-10% of the composition, in percentage by weight; the L. erythrorhizon extract accounts for 10%-90% of the composition, in percentage by weight. The composition can be used for preparing a pharmaceutical composition for preventing or treating peptic ulcer or for resisting Helicobacter pylori.

Description

Composition of arctium lappa and larkspur, its preparation method and use TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a composition of arctium lappa and larkspur, its preparation method and use of the composition in the prevention or treatment of peptic ulcer and / or anti-helicobacter pylori. BACKGROUND

[0002] Arctium lappa L., also known as fruit, is a biennial herbaceous plant of the family Asteraceae. The plant is 1-2 m high. The stem is erect with purple color, and the upper part is branched. The basal leaves are clustered, large, with long petioles, and the head-shaped inflorescences are numerous with pale red flowers. The arctium lappa root is spindle-shaped, fleshy and straight, with a light brown to brownish color on the outside and a yellowish white color on the inside, with many obvious longitudinal grooves. It tastes slightly bitter and is sticky. Arctium lappa is distributed in Europe, North America and Asia. Later, Japan cultivated many varieties of arctium lappa and regarded it as a high-grade vegetable with excellent nutritional and health values. Arctium lappa fruit is a traditional Chinese medicine with the functions of dispelling wind-heat, promoting rash, benefiting the throat, and detoxifying and reducing swelling. The arctium lappa root is considered a blood purifying agent that can remove toxins from the blood. It is recorded in "Biographies of Famous Doctors" that long-term consumption can make the body light and resist aging. It is also used as a kidney-tonifying and yang-strengthening product in folk medicine. In Europe, the British Pharmacopoeia also records the use of arctium lappa. The root can purify blood, prevent colds and flu, and eliminate toxins that cause skin disorders, treat rheumatism, cystitis, and kidney stones. Some literature indicates that arctium lappa has anti-inflammatory, anticancer, antidiabetic, antimicrobial and viral, antioxidant, liver protection, anti-aging and anti-fatigue activities. The diversity of arctium lappa's effects is attributed to many active ingredients in arctium lappa, including polyphenols (chlorogenic acid, caffeic acid, etc.), flavonoids and glycosides (quercetin, isoflavones, rutin, etc.), lignans (arctiin and aglycone), and polysaccharides (inulin). The nutritional value of arctium lappa root: per 100 grams of fresh vegetable contains about 87 grams of water, 4.1-4.7 grams of protein, 3.0-3.5 grams of carbohydrates, 0.1 grams of fat, and 1.3-1.5 grams of fiber. The carotene content is as high as 390 milligrams, which is 280 times higher than that of carrots. Vitamin C is 1.9 milligrams. Among the mineral elements, calcium is 240 milligrams, phosphorus is 106 milligrams, and iron is 7.6 milligrams, and it also contains other nutrients. In 2002, arctium lappa root was included in the list of health foods in Taiwan Province, China; in the 2018 edition of the Chinese Health Commission's list of raw materials for food and medicine, arctium lappa root is both a common food and a traditional Chinese medicine that can be used for health food.

[0003] Xinjiang Amebia Root, the root of a perennial herb of the Gentianaceae family, is distributed in the southern and northern slopes of the Tianshan Mountains in Xinjiang, western Tibet, Nepal, Pakistan, Iran, Afghanistan, Kazakhstan, Kyrgyzstan, and other Central Asian regions of the former Soviet Union. It is the original plant of the current edition of the Gentiana root, which is listed in the Chinese Pharmacopoeia, the Chinese Taiwan Provincial Pharmacopoeia, and the Hong Kong Chinese Herbal Medicine Standard. Gentiana root is a commonly used traditional Chinese medicine for detoxification, heat-clearing, and blood-cooling. It was first recorded in the Shennong Bencao Jing as a medicinal material of medium quality. The Chinese Pharmacopoeia records that Gentiana root has the effects of clearing heat and blood, promoting blood circulation and detoxification, and penetrating rash and eliminating spots; it is used for blood heat and toxin, purple and black spots, measles, ulcers, eczema, and scalds. Gentiana root can be used externally and also for internal use as a decoction. Modern pharmacological studies have shown that Gentiana root has antibacterial (Staphylococcus aureus, Staphylococcus albus, Pseudomonas aeruginosa, Escherichia coli, and Shigella dysenteriae) effects, and its naphthoquinone compounds have a wide and good antibacterial effect, mainly inhibiting the growth and reproduction of gram-negative bacteria, gram-positive bacteria, and fungi; anti-tumor, naphthoquinone compounds have an inhibitory effect on various tumors, such as gastric cancer, spontaneous breast tumors, and colon cancer; anti-inflammatory, acetyl gentisic acid has a significant anti-inflammatory effect; antioxidant, its antioxidant effect is equivalent to 35.5% of vitamin C; antiviral; anti-fertility; and liver protection. The main chemical components of Xinjiang Gentiana root include naphthoquinone compounds (gentisic acid and acylarin), monoterpenes, benzoquinones, and phenols; phenolic acids and their salts; aliphatic and ester compounds; and alkaloid compounds (pyrrolizidine). Among them, naphthoquinone compounds are the main active substances of Gentiana root, and pyrrolizidine alkaloids are toxic components.

[0004] Peptic ulcer is a mucosal erosion of the esophagus, stomach, duodenum and other organs caused by gastric juice, which is a deep tissue damage of the digestive tract wall. It is usually a little deeper than erosion (or called skin break). Ulcer occurs in the stomach called gastric ulcer, and occurs in the duodenum called duodenal ulcer. Usually, peptic ulcer refers to gastric or duodenal ulcer, and only a few ulcers occur in the esophagus. According to epidemiological statistics, the population infected with Helicobacter pylori in the healthy population of Taiwan Province of China is more than 10 million people; these infected people are also a high-risk group for gastric adenocarcinoma. Disease surveys show that the Helicobacter pylori infection rate of duodenal ulcer patients is as high as more than 90%, the infection rate of gastric ulcer patients is as high as 70-90%, the infection rate of gastritis patients is also as high as 70-90%, and the infection rate of gastric cancer patients is as high as 50-60%. If a specific antibiotic is used for sterilization and eradication therapy, the recurrence rate of peptic ulcer can be reduced from 75% to 5%. The medical community has confirmed that Helicobacter pylori can cause chronic gastritis, of which 15-20% will develop into gastric ulcer and duodenal ulcer; 1-2% will develop into gastric cancer; and 0.1% will develop into gastric lymphoma. Since Helicobacter pylori plays an important role in the cause of peptic ulcer, if the peptic ulcer cannot be cured or relieved after long-term treatment, the possibility of Helicobacter pylori infection should be considered, and the necessary antibiotic treatment should be used. SUMMARY

[0005] In order to solve the problem of effectively preventing or treating peptic ulcer and / or anti-Helicobacter pylori, the present application provides a composition of Arctium lappa and Lithospermum, including Arctium lappa extract and Lithospermum extract; the Arctium lappa extract accounts for 80%-20% of the weight percentage of the composition; and the Lithospermum extract accounts for 20%-80% of the weight percentage of the composition.

[0006] The composition has better antibacterial effect on Helicobacter pylori than the Arctium lappa extract or the Lithospermum extract alone, and the composition has better repair effect on chemical or physical damage of rat gastric mucosa cell (RGM-1) than the Arctium lappa extract or the Lithospermum extract alone.

[0007] The present application provides a method for preparing a composition of Arctium lappa and Lithospermum erythrorhizon, comprising: grinding Arctium lappa into powder, and soaking and stirring with alcohol to form a first solution, and standing overnight; grinding Lithospermum erythrorhizon into powder, and soaking and stirring with alcohol to form a second solution, and standing overnight; extracting and filtering the first solution and the second solution twice respectively, collecting and combining the filtrates twice respectively to obtain Arctium lappa filtrate and Lithospermum erythrorhizon filtrate; taking a small amount of the Arctium lappa filtrate and the Lithospermum erythrorhizon filtrate to dry, respectively calculating the solid content of the Arctium lappa filtrate and the Lithospermum erythrorhizon filtrate, and according to the solid content of the two filtrates and the weight percentage of the required extract of Arctium lappa and Lithospermum erythrorhizon, taking an appropriate volume of the first solution and the second solution to combine to obtain a combined solution, and drying the combined solution after concentration under reduced pressure to obtain the composition of Arctium lappa and Lithospermum erythrorhizon.

[0008] Preferably, the volume percentage concentration of the alcohol is 90% to 95%.

[0009] The present application provides a composition of Arctium lappa and Lithospermum erythrorhizon for use in the preparation of a pharmaceutical composition for preventing or treating peptic ulcer and / or resisting Helicobacter pylori.

[0010] The present application provides a pharmaceutical composition prepared by taking the composition of Arctium lappa and Lithospermum erythrorhizon as an active ingredient, and adding a pharmaceutically acceptable excipient or carrier or auxiliary ingredient.

[0011] The present application also provides a pharmaceutical composition for use in the preparation of a drug for preventing or treating peptic ulcer and / or resisting Helicobacter pylori.

[0012] The composition of Arctium lappa and Lithospermum erythrorhizon provided by the present application has a specific Ultra Performance Liquid Chromatography (UPLC) fingerprint, and the composition has better antibacterial performance on Helicobacter pylori than the extract of Arctium lappa or Lithospermum erythrorhizon alone, and has better repair effect on RGM-1 cell strain chemical or physical damage than the extract of Arctium lappa or Lithospermum erythrorhizon alone. The composition and the pharmaceutical composition prepared by using the composition can effectively prevent or treat peptic ulcer and / or resist Helicobacter pylori. BRIEF DESCRIPTION OF DRAWINGS

[0013] Fig. 1 is a flow chart of the preparation method of the composition of Arctium lappa and Lithospermum erythrorhizon according to the embodiment of the present application;

[0014] Fig. 2 is a schematic diagram of the effect of the composition of Arctium lappa and Lithospermum erythrorhizon according to the embodiment of the present application on the growth of Helicobacter pylori BCRC 15415 with the assistance of antibiotic Clarithromycin;

[0015] Figure 3 is a diagram showing the effect of the composition of the present application of Arctium lappa and L. glabra assisting antibiotic clarithromycin on the growth of H. pylori BCRC 17132;

[0016] Figure 4 is a diagram showing the procedure of the experiment of the present application of indomethacin-induced chemical damage repair of rat gastric mucosa cell (RGM-1);

[0017] Figure 5 is a diagram showing the result of the experiment of the present application of the composition of Arctium lappa and L. glabra on the repair ability of RGM-1 cell strain chemical damage; abbreviation: ppb: parts per billion;

[0018] Figure 6 is a diagram showing the procedure of the experiment of the present application of RGM-1 cell strain physical damage repair;

[0019] Figure 7 is a diagram showing the result of the experiment of the present application of the composition of Arctium lappa and L. glabra on the repair ability of RGM-1 cell strain physical damage; abbreviation: ppm: parts per million;

[0020] Figure 8 is a diagram showing the ultra performance liquid chromatography (UPLC) fingerprint of the composition of Arctium lappa and L. glabra (2:3) of the present application;

[0021] Figure 9 is a diagram showing the ultra performance liquid chromatography (UPLC) fingerprint of the composition of Arctium lappa and L. glabra (1:4) of the present application. DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further described below in combination with the drawings and examples.

[0023] The present application provides a composition of Arctium lappa and L. glabra, which comprises Arctium lappa extract and L. glabra extract, wherein the weight percentage of Arctium lappa extract in the composition is 80%-20%, and the weight percentage of L. glabra extract in the composition is 20%-80%. The composition has better antibacterial effect on H. pylori than the extract of Arctium lappa or L. glabra alone, and the composition has better repair effect on RGM-1 cell strain chemical or physical damage than the extract of Arctium lappa or L. glabra alone.

[0024] Referring to Figure 1, the preparation method of the composition of Arctium lappa and L. glabra provided by the present application comprises the following steps:

[0025] Step S101 : grind burdock into powder, and soak and stir with alcohol to form a first solution, and let it stand overnight;

[0026] Step S102: grind liquorice into powder, and soak and stir with alcohol to form a second solution, and let it stand overnight;

[0027] Step S103: extract and filter the first solution twice, and collect and combine the filtrates; Step S104: extract and filter the second solution twice, and collect and combine the filtrates;

[0028] Step S105: dry a small amount of the filtrate, and separately calculate the solid content in the burdock filtrate and the liquorice filtrate, and according to the solid content in the two filtrates and the weight percentage of the extract required from burdock and liquorice, take an appropriate volume of the first solution and the second solution, and combine them to obtain a combined solution, and after reducing pressure concentration and drying the combined solution, the composition of burdock and liquorice is obtained.

[0029] The following application examples are given as follows:

[0030] Example

[0031] Take 10 g of dried medicinal material of black burdock from Pingdong, Taiwan Province, China, and 15 g of Xinjiang liquorice from Yili, Xinjiang, grind them into coarse powder respectively. Soak them with 20 times and 10 times weight of 95% volume percentage alcohol respectively, stir for 40 minutes, and let them stand overnight to obtain burdock alcohol solution and liquorice alcohol solution; extract the burdock alcohol solution and the liquorice alcohol solution respectively, and add 95% volume percentage alcohol to the residue obtained after extraction, and extract again; collect and combine the filtrates obtained from the two extractions respectively, dry a small amount of the filtrate, calculate the solid content in the filtrate, and according to the solid content and the weight percentage of the extract required from burdock and liquorice, take an appropriate volume of the burdock alcohol solution and the liquorice alcohol solution, and combine them to obtain a combined solution, and after reducing pressure concentration and drying the combined solution, the composition of burdock and liquorice with different combination ratios is obtained for pharmacological test.

[0032] In order to test whether the composition of burdock and liquorice can effectively prevent or treat peptic ulcer and / or anti-Helicobacter pylori, the following tests need to be carried out:

[0033] 1. Anti-Helicobacter pylori test

[0034] 1.1 Inhibition of Helicobacter pylori growth by the composition of burdock and liquorice

[0035] 1.1.1 Method

[0036] 1.1.1.1 Pharmaceutical reagents and bacterial strains

[0037] Claritromycin and Amoxicillin were purchased from Sigma-Aldrich, BBL™ Mueller Hinton Broth, Difco TM Tryptic Soy Broth and Difco™ Tryptic Soy Agar were purchased from Becton Dickinson, Defib. Sheep Blood was purchased from Creative Lifesciences, Iodonitrotetrazolium chloride (INT) was purchased from Sigma-Aldrich, H. pylori 15415 and H. pylori 17132 were purchased from the Bioresource Collection and Research Center (BCRC) of Taiwan Food Industry Development Institute. The combination of Arctium lappa and Arnebia euchroma was prepared into six samples according to different weight percentages: Sample One, 100% AL; Sample Two, 80% AL + 20% AE; Sample Three, 60% AL + 40% AE; Sample Four, 40% AL + 60% AE; Sample Five, 20% AL + 80% AE; Sample Six, 100% AE (AL represents Arctium lappa extract, AE represents Arnebia euchroma extract).

[0038] 1.1.1.2 Culture of H. pylori

[0039] The H. pylori used in this example were BCRC 15415 and BCRC 17132, purchased from the Bioresource Collection and Research Center (BCRC) of Taiwan Food Industry Development Institute. H. pylori is a microaerophilic bacterium, so Tryptic Soy Broth containing 5% Defib. Sheep Blood was used, and the culture was placed in a microaerophilic gas-producing bag in an anaerobic chamber and incubated in an incubator at 37°C. When subculturing, 0.1-0.5 mL of bacterial solution was added to 5 mL of fresh culture medium for incubation. After the experimental strains were subcultured and scaled up, they were inoculated into Tryptic Soy Broth containing 5% Defib. Sheep Blood.

[0040] 1.1.1.3 Test drug configuration

[0041] Samples One to Six were prepared into samples with a concentration of 100 mg / mL by dissolving the test substances in 100% dimethyl sulfoxide (DMSO).

[0042] 1.1.1.4 Minimum inhibitory concentration (MIC) determination

[0043] The sample detection concentration of the experiment was 0.3 mg / mL, 0.1 mg / mL and 0.03 mg / mL, the positive control group clarithromycin (Clarithromycin) and amoxicillin (Amoxicillin) concentration was 2 μg / mL, and 10 concentrations were obtained by 2-fold serial dilution. The prepared sample and antibiotic were added to the 96-well plate.

[0044] 3 to 5 single colonies were picked from the cultured medium to Mueller Hinton medium (M-H medium) with a standard turbidity of 0.5 McFarland (about 1 x 10 8 ~ 2 x 10 8 CFU / mL) as the standard, and the bacterial solution was diluted, mixed, and then diluted to 5 x 10 5 CFU / mL of bacterial solution. Then the bacterial solution (5 x 10 4 CFU / 0.1 mL) was added to the 96-well plate containing the test sample and mixed evenly.

[0045] The 96-well plate was incubated for 24 hours under suitable culture conditions for the strain, and then Iodonitrotetrazolium chloride (INT) was added for reaction. The ELISA reader was used to detect the absorbance value at 492 nm. The increase in absorbance value before and after the addition of INT was significantly lower than that of the control group (control group), which was considered to have the ability to inhibit the growth of H. pylori, and the corresponding sample concentration was the minimum inhibitory concentration.

[0046] 1.1.2 Results

[0047] Active results: For H. pylori BCRC 15415 and BCRC 17132, the sample five composition 20% AL + 80% AE had similar or even better antibacterial ability than burdock (100% AL; sample one) and comfrey (100% AE; sample six) groups, especially for BCRC 17132 strain, the sample five composition 20% AL + 80% AE showed the best performance.

[0048] 1.2 Composition of burdock and comfrey assisted antibiotics to inhibit the growth of H. pylori

[0049] 1.2.1 Method

[0050] 1.2.1.1 Pharmaceutical reagents and bacterial strains

[0051] The operation is the same as that described in 1.1.1.1 above, which is not repeated here.

[0052] 1.2.1.2 Culture of H. pylori

[0053] The operation is the same as that in 1.1.1.2 above and will not be repeated here.

[0054] 1.2.1.3 Test drug configuration

[0055] The operation is the same as that in 1.1.1.3 above and will not be repeated here.

[0056] 1.2.1.4 Auxiliary antibiotic efficacy determination

[0057] The antibiotics clarithromycin and amoxicillin were tested at concentrations of 0.0625 μg / mL and 1 μg / mL, respectively. The test concentrations of the following samples for H. pylori BCRC 15415 were as follows: Sample 1, 100% AL; Sample 2, 80% AL + 20% AE; Sample 3, 60% AL + 40% AE; Sample 4, 40% AL + 60% AE, all diluted to 0.15 mg / mL; Sample 5, 20% AL + 80% AE; and Sample 6, 100% AE, all diluted to 0.05 mg / mL. The test sample concentrations for H. pylori BCRC 17132 were as follows: Sample 1, 100% AL prepared at 0.15 mg / mL; Sample 2, 80% AL + 20% AE; Sample 3, 60% AL + 40% AE; Sample 4, 40% AL + 60% AE; and Sample 6, 100% AE prepared at 0.05 mg / mL; and Sample 5, 20% AL + 80% AE prepared at 0.05 mg / mL and 0.015 mg / mL, respectively. After preparation, the samples were added to a 96-well plate along with the antibiotic.

[0058] Pick 3 to 5 single colonies from the culture medium and add them to Mueller Hinton medium (MH medium) and use 0.5McFarland standard turbidity (about 1×10 8 ~2×10 8 CFU / mL) was used as the standard, and the bacterial solution was diluted, mixed, and then diluted to 5×10 5 CFU / mL of bacterial solution. Then the bacterial solution (5×10 4 CFU / 0.1 mL) was added to a 96-well plate containing the test sample and antibiotics and mixed evenly.

[0059] The 96-well plate was incubated for 24 hours under conditions appropriate for the strain being used. After the addition of INT, the absorbance at 492 nm was measured using an ELISA reader. Samples with significantly lower increases in absorbance before and after the addition of INT compared to the control group were considered to have inhibited H. pylori growth and were considered to have adjunctive antibiotic efficacy at the corresponding sample concentration.

[0060] 1.2.2 Results

[0061] Active results: Compared with the extracts of Arctium lappa and L. glabra alone (sample one and sample six), the composition was more effective in assisting antibiotic Clarithromycin to inhibit the growth of H. pylori BCRC 15415 and BCRC 17132, and the assisting effect of sample five composition 20% AL + 80% AE was the most significant, as shown in Figures 2 and 3. Figure 2 shows that the compositions of sample one 100% AL, sample two 80% AL + 20% AE, sample three 60% AL + 40% AE, and sample four 40% AL + 60% AE were tested at a concentration of 0.15 mg / mL, sample five 20% AL + 80% AE and sample six 100% AE were tested at a concentration of 0.05 mg / mL, and antibiotic Clarithromycin was selected at a concentration of 0.625 μg / mL. The results showed that compared with the group treated with Clarithromycin alone, the experimental groups of sample two 80% AL + 20% AE, sample three 60% AL + 40% AE, sample four 40% AL + 60% AE, and sample five 20% AL + 80% AE significantly inhibited the growth of H. pylori BCRC 15415 (P < 0.01), suggesting that the above four compositions had the effect of assisting antibiotic Clarithromycin in antibacterial action, and the assisting effect was better than that of the crude extracts of Arctium lappa and L. glabra (sample one 100% AL and sample six 100% AE). Figure 3 shows the test results of the composition assisting antibiotic Clarithromycin in inhibiting the growth of H. pylori BCRC 17132, and the test concentrations of each sample and antibiotic were the same as above. The results showed that compared with antibiotic Clarithromycin alone, the composition of sample five 20% AL + 80% AE combined with antibiotic Clarithromycin significantly inhibited the growth of H. pylori BCRC 17132 (P < 0.05).

[0062] 2. Gastric mucosal cell repair activity test of the composition of Arctium lappa and L. glabra

[0063] 2.1.1 Indomethacin-induced chemical damage repair test of rat gastric mucosa cell (RGM-1)

[0064] 2.1.1.1 Test procedure (see Figure 4)

[0065] 2.1.1.1.1 Cell Culture: RGM-1 cells were cultured in DMEM / Ham F12 medium containing 20% ​​FBS and 1% p / s. Cells were seeded into 10 cm cell culture dishes and incubated in a cell culture incubator at 37°C and 5% CO2. After the cells grew to 80% to 90% confluence, they were washed once with PBS, after which the PBS was removed. 0.25% trypsin (trypsin-EDTA) was added and incubated at 37°C for several minutes. Cells were observed under a microscope to detach from the flask wall. Fresh culture medium was added and mixed thoroughly to terminate the trypsin enzyme reaction. The cell suspension was collected into a centrifuge tube, centrifuged, and the supernatant removed. The cells were then dissolved in DMEM culture medium. The cell concentration was calculated using a hemocytometer. The cells were plated at a concentration of 5 × 10 3 The number of cells per well was seeded into a 96-well cell culture plate with a volume of 100 μL / well, and cultured in a cell culture incubator overnight to allow them to adhere to the plate for use.

[0066] 2.1.1.1.2 Remove the old culture medium and add 100 μL / well of DMEM / Ham F12 culture medium containing 0.25 mM Indomethacin to the attached cells in the 96-well culture plate and culture in a cell culture incubator for 5 hours.

[0067] 2.1.1.1.3 Remove the DMEM / Ham F12 culture medium containing 0.25 mM Indomethacin, add the sample solution at 100 μL / well to a 96-well plate for culture, with 3 wells (3 replicates) for each group, and culture in a cell culture incubator for 24 hours.

[0068] 2.1.1.1.4 Cell viability test: Remove the cell culture medium containing the test sample and add 10 mM MTT reagent to the cells at a volume of 100 μL / well. Incubate in a cell culture incubator for 4 hours. Remove the 96-well plate and shake it at 75 rpm on an orbital shaker for 10 minutes. After that, remove the lid and read the absorbance value at a wavelength of 540 nm using a spectrophotometer. Calculate the cell proliferation rate relative to the control group, and plot a bar graph using GraphPad Prism 9 software based on the mean and standard error of the mean (Average ± SD) for each group.

[0069] 2.1.1.2 Results

[0070] The combination of Arctium lappa and Lithospermum erythrorhizon of the present application has the ability to promote the repair of RGM-1 chemical injury, and the combination is superior to the crude extract of Arctium lappa or Lithospermum erythrorhizon alone. After the RGM-1 cell strain is treated with Indomethacin, the test sample is added, and the higher the survival rate of RGM-1, the stronger the repair ability of the test sample to the chemical injury. As shown in FIG. 5, each test sample can significantly improve the survival rate of the RGM-1 cell strain, and the effects of the combination 3:2 (sample three), 2:3 (sample four), and 1:4 (sample five) are better (P<0.01).

[0071] 2.1.2 RGM-1 cell strain physical injury repair test

[0072] 2.1.2.1 Test procedure (see FIG. 6)

[0073] 2.1.2.1.1 Cell culture: RGM-1 cells are cultured in DMEM / Ham F12 containing 20% FBS and 1% p / s, and the cells are inoculated in a 10 cm cell culture dish and cultured in a cell culture incubator at 37°C and 5% CO2. After the cells grow to 8 to 9 full, the cells are washed once with PBS, the PBS is removed, 0.25% trypsin-EDTA is added at 37°C for several minutes, the cells are observed under a microscope to peel off from the bottle wall, fresh culture solution is added and mixed evenly, the trypsin enzyme reaction is terminated, the cell suspension is collected into a centrifuge tube, the supernatant is removed after centrifugation, and the cells are resuspended in DMEM culture solution. The cell concentration is calculated by a hemocytometer, and the cells are inoculated into a 48-well cell culture dish at a concentration of 2x10 4 / well, and the volume is 1 mL / well. The cells are cultured in a cell culture incubator for 48 hours to adhere to the plate for use.

[0074] 2.1.2.1.2 A 200 μL micropipette is used to draw a wound, the old culture solution is removed, and phosphate buffered saline (PBS) is used to wash 3 times to remove the cells drawn. Serum-free DMEM / Ham F12 containing different concentrations of samples is added to the 48-well culture dish at a volume of 1 mL / well for culture, and each group is made in 3 wells (3 repeats). The cells are cultured in a cell culture incubator for 24 hours, and the wound is placed in the center of the photo under a 4x objective lens at 0 and 24 hours, respectively.

[0075] 2.1.2.1.3 Result analysis: a straight line is drawn on the wound boundary using Image J, and the wound area between the cells is calculated.

[0076] 2.1.2.1.4 Calculate the wound healing rate, wound healing rate = (initial scratch area - t time scratch area) / initial scratch area. Calculate the wound healing rate of the cells relative to the control group, and use GraphPad Prism 9 software to draw a bar graph according to the average value and standard deviation (Average ± SD) of each group.

[0077] 2.1.2.2 Results

[0078] The combination of arctium lappa and lithospermum root in the embodiment of the present application has the ability to promote the repair of physical damage to RGM-1. The composition is better than or similar to the extract of arctium lappa or lithospermum root alone, especially the composition 4:1 (sample two) and 3:2 (sample three) is better than the extract of arctium lappa or lithospermum root alone, as shown in Figure 7. Each group takes the initial wound area as 100%, calculates the percentage of the remaining wound area after the wound healing of each treatment group after 24 hours to the initial area, judges the repair ability of the composition to the physical damage of RGM-1, and the smaller the percentage of the remaining wound area to the initial area, the higher the wound healing rate, which represents the stronger repair ability of the composition to the physical damage of RGM-1. Each composition has the ability to promote the repair of physical damage to RGM-1, especially the composition 4:1 (sample two) and 3:2 (sample three) is better than the crude extract of arctium lappa or lithospermum root alone (**p≤0.01 indicates significant difference compared with the untreated group; ##p≤0.01 indicates significant difference compared with the 100% AL group (sample one); ++p≤0.01 indicates significant difference compared with the 100% AE group (sample six)).

[0079] 3. Ultra performance liquid chromatography (UPLC) fingerprint of the combination of arctium lappa and lithospermum root

[0080] The chemical composition characteristics of the better combination of arctium lappa and lithospermum root in the embodiment were determined by UPLC method. In the chromatogram, the characteristic chemical composition of arctium lappa, chlorogenic acid, and the characteristic lithospermum component, shikonin, were shown.

[0081] UPLC analysis conditions: UPLC is Shimadzu M30A UHPLC system; column (Waters HSS C18, 1.8 μm, 2.1 x 100 mm); column temperature is 30℃; flow rate is 0.4 mL / min; detection wavelength is UV 320 nm.

[0082] Mobile phase system condition: mobile phase A is water (0.4% phosphoric acid); mobile phase B is acetonitrile, linear gradient is 0-5.5 min (93% A-93% A), 5-10 min (93% A-45% A), 10-20 min (45% A-25% A), 20-24 min (25% A-23% A), 24-25 min (23% A-0% A).

[0083] As shown in FIG. 8 and FIG. 9, the composition of Arctium lappa and Alkanna tinctoria has the characteristic chemical components of both Arctium lappa and Alkanna tinctoria. Chlorogenic acid from Arctium lappa and naphthoquinone compounds from Alkanna tinctoria. It is characterized in that: 1) naphthoquinone compounds from Alkanna tinctoria, three of which are identified as acetylshikonin, β-acetoxyisovalerylshikonin, β, β-dimethylaceyshikonin; 2) the absolute configuration of shikonin also includes its mirror image isomer alkannin; 3) chlorogenic acid has various biological activities such as antibacterial activity, so in addition to being a characteristic chemical component of Arctium lappa, it can also be used as an active indicator component of Arctium lappa.

[0084] The composition of Arctium lappa and Alkanna tinctoria provided by the embodiment of the present application has a specific UPLC fingerprint. The composition has better antibacterial performance on Helicobacter pylori than the extract of Arctium lappa or Alkanna tinctoria alone, and has better repair effect on chemical or physical damage of RGM-1 cell strain than the extract of Arctium lappa or Alkanna tinctoria alone. The composition provided by the embodiment of the present application can effectively prevent or treat peptic ulcer and / or resist Helicobacter pylori.

[0085] In addition, the embodiment of the present application also provides a pharmaceutical composition, which is prepared by taking the above-mentioned composition of Arctium lappa and Alkanna tinctoria as an active ingredient, and adding pharmaceutically acceptable adjuvants or auxiliary ingredients. The pharmaceutical composition can effectively prevent or treat peptic ulcer and / or resist Helicobacter pylori.

[0086] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only for specific embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A composition characterized in that, The composition comprises Arctium lappa extract and Arnebia euchroma extract; the Arctium lappa extract accounts for 90%-10% of the weight percentage of the composition; and the Arnebia euchroma extract accounts for 10%-90% of the weight percentage of the composition.

2. The composition of claim 1, wherein, The Arctium lappa extract accounts for 80%-20% of the weight percentage of the composition; and the Arnebia euchroma extract accounts for 20%-80% of the weight percentage of the composition.

3. A process for the preparation of a composition according to claim 1, characterized in that, The composition comprises: Arctium lappa powder is taken and soaked and stirred with alcohol to form a first solution, which is left overnight; Arnebia euchroma powder is taken and soaked and stirred with alcohol to form a second solution, which is left overnight; The first solution and the second solution are respectively extracted and filtered twice, and the filtrates are respectively collected and combined; The content of solid in each filtrate is calculated; According to the content of solid and the weight percentage of the required Arctium lappa extract and Arnebia euchroma extract, the volume of the first solution and the second solution is calculated, and the solutions are combined to obtain a combined solution; and The combined solution is reduced in pressure, concentrated, dried to obtain the composition; The Arctium lappa extract accounts for 90%-10% of the weight percentage of the composition; and the Arnebia euchroma extract accounts for 10%-90% of the weight percentage of the composition.

4. The method of claim 3, wherein, The Arctium lappa extract accounts for 80%-20% of the weight percentage of the composition; and the Arnebia euchroma extract accounts for 20%-80% of the weight percentage of the composition.

5. A pharmaceutical composition comprising a compound of the formula: ###0002### or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition comprises the composition according to claim 1 or 2 and pharmaceutically acceptable excipients, carriers or auxiliary ingredients thereof.

6. Use of the composition according to claim 1 or 2 for preparing a pharmaceutical composition for preventing or treating peptic ulcer.

7. Use according to claim 6, characterized in that, The effective dose of the composition is 0.05 mg / mL-0.15 mg / mL.

8. Use of the composition according to claim 1 or 2 for preparing a pharmaceutical composition for resisting Helicobacter pylori.

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