Composition of arctium lappa and arnebia, the preparation method and the use thereof

Abstract

This invention provides a composition of burdock and gromwell and a method for preparing the same, wherein the burdock extract accounts for 80%-20% of the composition by weight; the gromwell extract accounts for 20%-80% of the composition by weight; and the composition can be used to prepare a pharmaceutical composition for the prevention or treatment of peptic ulcers or for the treatment of Helicobacter pylori.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to a composition of burdock and lithospermum and its preparation method, as well as the use of the composition in the prevention or treatment of peptic ulcers and / or Helicobacter pylori. [Previous Technology]

[0002] Burdock (Arctium lappa L.), also known as "Great Burdock Seed" or "Evil Fruit," is a biennial herbaceous plant belonging to the genus Arctium in the family Asteraceae. The plant grows to a height of 1-2 meters. The stem is erect, purplish, and highly branched in the upper part. Basal leaves are clustered, large, and long-stalked. Numerous capitula bear pale red flowers and seeds. The burdock root is spindle-shaped, fleshy, and straight, with a light brown to brownish-red outer surface and numerous distinct longitudinal grooves; the interior is yellowish-white. It has a slightly bitter and sticky taste. Burdock is distributed throughout Europe, North America, and Asia. Later, Japan cultivated various varieties, considering burdock a high-grade vegetable with excellent nutritional and health benefits. Burdock seeds are a traditional Chinese medicine, possessing the effects of dispersing wind-heat, promoting rash eruption and soothing the throat, and detoxifying and reducing swelling. Burdock root is traditionally considered a blood purifier in Chinese medicine, capable of removing toxins from the blood. The *Mingyi Bielu* (Records of Famous Physicians) states that "long-term use can lighten the body and prolong life," and it is also used in folk medicine as a kidney tonic and aphrodisiac. In Europe, the *British Pharmacopoeia* also records the medicinal use of burdock. The root can purify the blood, prevent colds and flu, remove toxins causing skin disorders, and treat rheumatism, cystitis, and kidney stones. Some literature indicates that burdock possesses various pharmacological activities, including anti-inflammatory, anti-cancer, anti-diabetic, antimicrobial and antiviral, antioxidant, liver-protective, anti-aging, and anti-fatigue properties. The diverse effects of burdock are attributed to its many active ingredients, including polyphenols (chlorogenic acid, caffeic acid, etc.), flavonoids and their glycosides (quercetin, isoflavones, rutin, etc.), lignans (arbusculin and its aglycones), and polysaccharides (inulin). Nutritional value of burdock root: Per 100 grams of fresh burdock root, it contains approximately 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. It is rich in carotene (up to 390 mg), 280 times higher than carrots. It also contains 1.9 mg of vitamin C. Among minerals, it contains 240 mg of calcium, 106 mg of phosphorus, and 7.6 mg of iron, as well as many other nutrients. In 2002, Taiwan included burdock root in its list of medicinal and edible raw materials; in the 2018 edition of the National Health Commission of China's catalogue of medicinal and edible ingredients, burdock root is both a common food and a traditional Chinese medicine that can be used in health foods.

[0003] Xinjiang Lithospermum (Amebia Root), a perennial herb belonging to the genus Amebia in the Boraginaceae family, is used medicinally for its root. It is distributed on the northern and southern slopes of the Tianshan Mountains in Xinjiang, western Tibet, and in Nepal, Pakistan, Iran, Afghanistan, Kazakhstan, Kyrgyzstan, and other Central Asian regions formerly part of the Soviet Union. It is the original plant of Lithospermum in the current versions of the Chinese Pharmacopoeia, the Taiwan Pharmacopoeia, and the Hong Kong Standard for Chinese Herbal Medicines. Lithospermum is a commonly used traditional Chinese medicine for reducing swelling and detoxifying, clearing heat and cooling the blood. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) as a medium-grade medicinal material. The Chinese Pharmacopoeia records that Lithospermum has the effects of clearing heat and cooling the blood, promoting blood circulation and detoxifying, and promoting rash eruption and eliminating spots; it is used for blood heat and toxicity, purplish-black rashes, incomplete measles eruption, ulcers, eczema, burns, etc. Besides external use, Lithospermum can also be decocted and taken internally. Modern pharmacological studies show that *Lithospermum erythrorhizon* possesses antibacterial properties (against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella spp.), with its shikonin compounds exhibiting broad and effective antibacterial activity, primarily inhibiting the growth and reproduction of Gram-negative bacteria, Gram-positive bacteria, and fungi; antitumor activity, with naphthoquinone components showing inhibitory effects on various tumors, such as gastric cancer, spontaneous breast cancer, and colon cancer; anti-inflammatory activity, with acetylshikonin exhibiting significant anti-inflammatory effects; antioxidant activity, with its antioxidant capacity equivalent to 35.5% of vitamin C; antiviral activity; antifertility; and hepatoprotective activity. The main chemical components of *Lithospermum erythrorhizon* from Xinjiang include: naphthoquinone compounds (shikonin derivatives, arcinyl groups); monoterpenoid benzoquinones and phenols; phenolic acids and their salts; aliphatic and ester compounds; and alkaloid compounds (pyrrolizidine derivatives). Naphthoquinone compounds are the main active substances in Lithospermum erythrorhizon, while pyrrolizidine alkaloids are its toxic components.

[0004] Peptic ulcers are deep-seated lesions in the digestive tract walls, such as those of the esophagus, stomach, and duodenum, caused by erosion from gastric juices. They are usually deeper than erosions (or skin abrasions). Ulcers in the stomach are called gastric ulcers, and those in the duodenum are called duodenal ulcers. Generally, peptic ulcers refer to gastric or duodenal ulcers; only a small number occur in the esophagus. Epidemiological statistics show that over 10 million healthy individuals in Taiwan are infected with Helicobacter pylori; these infected individuals are also at high risk of developing gastric adenocarcinoma. Disease surveys show that the infection rate of Helicobacter pylori in duodenal ulcer patients is over 90%, while the infection rate in gastric ulcer patients is 70-90%, in gastritis patients it is also 70-90%, and in gastric cancer patients it is 50-60%. With eradication therapy using specific antibiotics, the recurrence rate of peptic ulcers 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 ulcers or duodenal ulcers; 1-2% will develop into gastric cancer; and 0.1% will develop into gastric lymphoma. Because Helicobacter pylori plays a very important role in the causes of digestive diseases, if peptic ulcers cannot be cured or relieved after long-term treatment, the possibility of Helicobacter pylori infection should usually be considered, and necessary antibiotic treatment should be used. [Summary of the Invention]

[0005] In order to solve the problem of effectively preventing or treating peptic ulcers and / or fighting Helicobacter pylori, the present invention provides a composition of burdock and lithospermum, comprising a burdock extract and a lithospermum extract; the burdock extract accounts for 80%-20% of the weight percentage of the composition; the lithospermum extract accounts for 20%-80% of the weight percentage of the composition.

[0006] The composition exhibits superior antibacterial activity against Helicobacter pylori compared to extracts of burdock or comfrey alone, and the composition also demonstrates superior repair effects on chemical or physical damage to rat gastric mucosa cell line (RGM-1) compared to extracts of burdock or comfrey alone.

[0007] This invention provides a method for preparing a burdock and gromwell composition, comprising: grinding burdock into powder, soaking and stirring it in alcohol to form a first solution, and letting it stand overnight; grinding gromwell into powder, soaking and stirring it in alcohol to form a second solution, and letting it stand overnight; extracting and filtering the first solution and the second solution twice respectively, collecting and combining the two filtrates to obtain a burdock filtrate and a gromwell filtrate; drying a small amount of the burdock filtrate and the gromwell filtrate, calculating the solid content in the burdock filtrate and the gromwell filtrate respectively, and according to the solid content of the two filtrates and the weight percentage of the required extracts of burdock and gromwell, measuring an appropriate volume of the first solution and the second solution, combining them to obtain a combined solution, and concentrating and drying the combined solution under reduced pressure to obtain the burdock and gromwell composition.

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

[0009] This invention provides the use of a burdock and gromwell composition for preparing a pharmaceutical composition for the prevention or treatment of peptic ulcers and / or for the treatment of Helicobacter pylori.

[0010] The present invention provides a pharmaceutical composition which is prepared by using the burdock and gromwell composition as the active ingredient, plus a pharmaceutically acceptable excipient or carrier or other auxiliary ingredient.

[0011] The present invention also provides the use of a pharmaceutical composition for preparing a medicament for the prevention or treatment of peptic ulcers and / or for the prevention or treatment of Helicobacter pylori.

[0012] The burdock and lithospermum composition provided by this invention has a specific ultra-high performance liquid chromatography (UPLC) fingerprint spectrum. This composition exhibits superior antibacterial performance against Helicobacter pylori compared to extracts of burdock or lithospermum alone, and its repair effect on chemical or physical damage to RGM-1 cell lines is also superior to extracts of burdock or lithospermum alone. The composition provided by this invention and pharmaceutical compositions prepared using this composition can effectively prevent or treat peptic ulcers and / or Helicobacter pylori infection. [Simplified Explanation of the Diagram]

[0013] Figure 1 is a flowchart of the preparation method of the burdock and gromwell composition of the present invention.

[0014] Figure 2 is a schematic diagram showing the effect of the burdock and lithospermum composition of the present invention, which is an adjuvant antibiotic clarithromycin, on the growth of Helicobacter pylori BCRC 15415.

[0015] Figure 3 is a schematic diagram showing the effect of the burdock and gromwell composition of the present invention, which is an adjuvant antibiotic clarithromycin, on the growth of Helicobacter pylori BCRC 17132.

[0016] Figure 4 is a schematic diagram of the process of the chemical damage repair experiment of rat gastric mucosa cell (RGM-1) induced by indomethacin according to the present invention.

[0017] Figure 5 is a schematic diagram of the test results of the ability of the burdock and gromwell composition of the present invention to promote the repair of chemical damage to RGM-1 cell line; abbreviation: ppb: parts per billion.

[0018] Figure 6 is a schematic diagram of the process of the physical damage repair experiment of the RGM-1 cell line of the present invention.

[0019] Figure 7 is a schematic diagram of the test results of the ability of the burdock and gromwell composition of the present invention to promote the repair of physical damage to RGM-1 cell line; abbreviation: ppm: parts per million.

[0020] Figure 8 is the fingerprint spectrum of the burdock and gromwell composition (2:3) of the present invention by ultra-high performance liquid chromatography (UPLC).

[0021] Figure 9 is the fingerprint spectrum of the burdock and gromwell composition (1:4) of the present invention by ultra-high performance liquid chromatography (UPLC).

Implementation Method

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] This embodiment of the invention provides a composition of burdock and lithospermum, comprising burdock extract and lithospermum extract, wherein the burdock extract accounts for 80%-20% of the composition by weight, and the lithospermum extract accounts for 20%-80% of the composition by weight. This combination exhibits superior antibacterial performance against Helicobacter pylori compared to using either burdock or lithospermum extract alone, and the composition also demonstrates superior repair effects on chemical or physical damage to RGM-1 cell lines compared to using either burdock or lithospermum extract alone.

[0024] Referring to Figure 1, the preparation method of the burdock and gromwell composition provided in this embodiment of the invention includes the following steps:

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

[0026] Step S102: Grind the purple gromwell root into powder, soak and stir it 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 two filtrates;

[0028] Step S104: Extract and filter the second solution twice, and collect and combine the two filtrates;

[0029] Step S105: Take a small amount of the filtrate and dry it. Calculate the solid content in the burdock filtrate and the gromwell filtrate respectively. Based on the solid content of the two filtrates and the weight percentage of the required extracts of burdock and gromwell, measure an appropriate volume of the first solution and the second solution and combine them to obtain a combined solution. After the combined solution is concentrated and dried under reduced pressure, the composition of burdock and gromwell is obtained.

[0030] The following are application examples, as detailed below:

[0031] Example

[0032] Take 10g of dried black burdock from Pingtung, Taiwan and 15g of Lithospermum erythrorhizon from Yili, Xinjiang, and grind them into coarse powder. Soak them in 20 times and 10 times their weight of 95% alcohol by volume, respectively, stir for 40 minutes, and let stand overnight to obtain burdock alcohol solution and Lithospermum erythrorhizon alcohol solution; extract the burdock alcohol solution and Lithospermum erythrorhizon alcohol solution separately, and add 95% alcohol by volume to the filter residue obtained after extraction to extract again; collect and combine the filtrates obtained from the two extractions, take a small amount of the filtrate and dry it, calculate the solid content in the filtrate, and according to the solid content and the weight percentage of burdock and Lithospermum erythrorhizon extracts required, measure an appropriate volume of burdock alcohol solution and Lithospermum erythrorhizon alcohol solution, combine them, concentrate and dry the combined solution under reduced pressure to obtain combinations of burdock and Lithospermum erythrorhizon in different ratios for pharmacological experiments.

[0033] In order to test whether the combination of burdock and comfrey can effectively prevent or treat peptic ulcers and / or Helicobacter pylori, the following tests need to be conducted:

[0034] 1. Anti-Helicobacter pylori test

[0035] 1.1 The combination of burdock and gromwell root inhibits the growth of Helicobacter pylori.

[0036] 1.1.1, Method

[0037] 1.1.1.1, Pharmaceuticals, Reagents and Bacterial Strains

[0038] Clarithromycin and amoxicillin were purchased from Sigma-Aldrich; BBL™ Mueller Hinton Broth, Difco™ 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; Helicobacter pylori 15415 and Helicobacter pylori 17132 were purchased from the Bioresource Conservation and Research Center (BCRC) of the Taiwan Food Industry Development Research Institute. Six samples were prepared from the composition of burdock and gromwell root in different weight percentages: Sample 1, 100% AL; Sample 2, 80% AL + 20% AE; Sample 3, 60% AL + 40% AE; Sample 4, 40% AL + 60% AE; Sample 5, 20% AL + 80% AE; Sample 6, 100% AE (AL represents burdock extract, AE represents gromwell root extract).

[0039] 1.1.1.2 Culture of Helicobacter pylori

[0040] The Helicobacter pylori strains used in this embodiment are BCRC 15415 and BCRC 17132, purchased from the Biological Resource Preservation and Research Center of the Taiwan Food Industry Research Institute. Helicobacter pylori is a microaerophilic bacterium; therefore, tryptic soy culture medium containing 5% defibrillated sheep blood was used, and the culture was placed in an anaerobic chamber containing a microaerobic gas generator at a temperature of 37°C. For subculture, 0.1-0.5 mL of the bacterial culture was added to 5 mL of fresh culture medium. After subculture and amplification, the experimental strains were inoculated onto Tryptic Soy medium containing 5% Defibrillated Sheep Blood.

[0041] 1.1.1.3, Preparation of Test Drugs

[0042] The test substances of samples one to six were prepared into samples with a concentration of 100 mg / mL using 100% dimethyl sulfoxide (DMSO).

[0043] 1.1.1.4 Determination of Minimum Inhibitory Concentration (MIC)

[0044] The sample concentrations tested in this experiment were 0.3 mg / mL, 0.1 mg / mL, and 0.03 mg / mL. The positive control group, containing clarithromycin and amoxicillin, was serially diluted 2-fold from 2 μg / mL to 10 concentrations for the experiment. The prepared samples and antibiotics were added to a 96-well plate.

[0045] Take 3 to 5 single colonies from the cultured medium and transfer them to Mueller Hinton medium (MH medium). Dilute the bacterial suspension using 0.5 McFarland standard turbidity (approximately 1*10⁸~2*10⁸ CFU / mL) as the standard, mix well, and then further dilute to a bacterial suspension of 5*10⁵ CFU / mL. Next, add the bacterial suspension (5*10⁴ CFU / 0.1 mL) to a 96-well plate containing the test sample and mix thoroughly.

[0046] The 96-well plate was cultured for 24 hours under suitable culture conditions for the strain used. After the addition of iodonitrotetrazolium chloride (INT), the absorbance was detected using an ELISA reader at 492 nm. Samples showing a significantly lower increase in absorbance before and after the addition of INT compared to the control group were considered to have inhibitory effects on Helicobacter pylori growth, and the corresponding sample concentration was the minimum inhibitory concentration (MIC).

[0047] 1.1.2, Results

[0048] Activity results: For Helicobacter pylori BCRC 15415 and BCRC 17132, the sample five composition 20% AL+80% AE had similar or even better antibacterial ability than the burdock (100% AL; sample one) and lithospermum (100% AE; sample six) groups. In particular, the sample five composition 20% AL+80% performed best against BCRC 17132 strain.

[0049] 1.2. The combination of burdock and lithospermum erythrorhizon assists antibiotics in inhibiting the growth of Helicobacter pylori.

[0050] 1.2.1, Method

[0051] 1.2.1.1, Pharmaceuticals, Reagents, and Bacterial Strains

[0052] The operation is the same as in 1.1.1.1 above, and will not be repeated here.

[0053] 1.2.1.2 Culture of Helicobacter pylori

[0054] The operation is the same as in 1.1.1.2 above, and will not be repeated here.

[0055] 1.2.1.3, Preparation of Test Drugs

[0056] The operation is the same as in 1.1.1.3 above, and will not be repeated here.

[0057] 1.2.1.4, Auxiliary antibiotic efficacy determination

[0058] The tested concentrations of antibiotics Clarithromycin and Amoxicillin were 0.0625 μg / mL and 1 μg / mL, respectively. The tested concentrations of the experimental samples treated with Helicobacter 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 prepared at 0.15 mg / mL; Sample 5, 20% AL + 80% AE; and Sample 6, 100% AE, all prepared at 0.05 mg / mL. The concentrations of the samples used to treat Helicobacter pylori BCRC 17132 were as follows: Sample 1, prepared with 100% AL to a concentration of 0.15 mg / mL; Sample 2, prepared with 80% AL + 20% AE; Sample 3, prepared with 60% AL + 40% AE; Sample 4, prepared with 40% AL + 60% AE; and Sample 6, prepared with 100% AE to a concentration of 0.05 mg / mL. Sample 5, prepared with 20% AL + 80% AE, was prepared to concentrations of 0.05 mg / mL and 0.015 mg / mL. After sample preparation, the samples were added to the 96-well plate along with the antibiotics.

[0059] Take 3 to 5 single colonies from the cultured medium and transfer them to Mueller Hinton medium (MH medium). Dilute the bacterial suspension using 0.5 McFarland standard turbidity (approximately 1*10⁸~2*10⁸ CFU / mL) as the standard, mix well, and then further dilute to a bacterial suspension of 5*10⁵ CFU / mL. Next, add the bacterial suspension (5*10⁴ CFU / 0.1 mL) to a 96-well dish containing the test sample and antibiotics and mix thoroughly.

[0060] The 96-well plate was cultured for 24 hours under suitable culture conditions for the strain used. After the INT reaction was added, the absorbance was detected at 492 nm using an enzyme immunoassay analyzer (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 inhibitory effects on Helicobacter pylori growth, and the corresponding sample concentrations were determined to have adjuvant antibiotic efficacy.

[0061] 1.2.2, Results

[0062] Activity Results: Compared with extracts of burdock and lithospermum alone (Sample 1 and Sample 6), the composition showed greater efficacy in adjuvanting the antibiotic Clarithromycin, enhancing the inhibition of Helicobacter pylori BCRC 15415 and BCRC 17132 growth. The adjuvant effect of the composition in Sample 5 (20% AL + 80% AE) was the most significant, as shown in Figures 2 and 3. Figure 2 shows that the detection concentration used for the compositions was 0.15 mg / mL for Sample 1 (100% AL), Sample 2 (80% AL + 20% AE), Sample 3 (60% AL + 40% AE), and Sample 4 (40% AL + 60% AE). The concentration used for Sample 5 (20% AL + 80% AE) and Sample 6 (100% AE) was 0.05 mg / mL. The concentration of the antibiotic Clarithromycin was 0.625 μg / mL. The results showed that, compared with the groups treated with Clarithromycin alone, the experimental groups of Sample 2 (80% AL + 20% AE), Sample 3 (60% AL + 40% AE), Sample 4 (40% AL + 60% AE), and Sample 5 (20% AL + 80% AE) all significantly inhibited the growth of Helicobacter pylori BCRC 15415 (P < 0.01). It is speculated that the above four combinations have the antibacterial effect of the antibiotic Clarithromycin, and their adjuvant effect is better than that of the crude extracts of burdock and purple gromwell (Sample 1, 100% AL and Sample 6, 100% AE). Figure 3 shows the results of the test on the inhibition of Helicobacter pylori BCRC 17132 growth by the combined adjuvant antibiotic Clarithromycin. The test concentrations of each sample and antibiotic were the same as above. The results show that compared with the antibiotic Clarithromycin alone, the combined sample 5 (20% AL + 80% AE) with antibiotic Clarithromycin significantly inhibited the growth of Helicobacter pylori BCRC 17132 (P<0.05).

[0063] 2. Gastric mucosal cell repair activity test of the combination of burdock and lithospermum.

[0064] 2.1.1 Indomethacin-induced chemical damage repair assay in rat gastric mucosa cell line (RGM-1)

[0065] 2.1.1.1 Test Procedure (See Figure 4)

[0066] 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 in 10 cm cell culture dishes and cultured in a cell incubator at 37°C and 5% CO2. When the cells reached 80-90% confluence, they were washed once with PBS and the PBS was removed. 0.25% trypsin (trypsin-EDTA) was added and incubated at 37°C for several minutes. Cells were observed to detach from the flask wall under a microscope. Fresh culture medium was added and mixed thoroughly to terminate the trypsin enzymatic reaction. The cell suspension was collected into centrifuge tubes. After centrifugation, the supernatant was removed, and the cells were resuspended in DMEM medium. Cell concentration was calculated using a hemocytometer. Cells were seeded at a rate of 5*10³ cells / well in 96-well cell culture dishes at a volume of 100 μL / well and cultured overnight in a cell incubator to allow them to adhere to the dish for later use.

[0067] 2.1.1.1.2 Remove the old culture medium and add DMEM / Ham F12 culture medium containing 0.25mM Indomethacin to the attached cells in the 96-well culture dish at a rate of 100μL / well, and incubate in a cell culture incubator for 5 hours.

[0068] 2.1.1.1.3 Remove the DMEM / Ham F12 culture medium containing 0.25mM Indomethacin, add the sample solution to a 96-well plate at a rate of 100μL / well, and culture in 3 wells (3 replicates) for each group, and incubate in a cell culture incubator for 24 hours.

[0069] 2.1.1.1.4 Cell viability test: Remove the cell culture medium containing the test sample, add 10mM MTT reagent at a rate of 100μL / well to the cells, and react in a cell incubator for 4 hours. Remove the 96-well plate, shake it at 75 rpm for 10 minutes using an orbital shaker, remove the top cover, and read the absorbance at a wavelength of 540nm using a spectrophotometer. Calculate the cell proliferation rate relative to the control group, and use GraphPad Prism 9 software to plot a bar chart based on the mean and standard error of the mean (Average ± SD) for each group.

[0070] 2.1.1.2, Results

[0071] The burdock and lithospermum composition of the present invention has the ability to promote the repair of chemical damage to RGM-1 cells, and the composition is superior to the crude extracts of burdock or lithospermum used alone. After RGM-1 cell lines are treated with indomethacin, the addition of test samples indicates that the higher the survival rate of RGM-1 cells, the stronger the repair ability of the test samples to chemical damage. As shown in Figure 5, all test samples can significantly improve the survival rate of RGM-1 cell lines, especially the compositions 3:2 (sample 3), 2:3 (sample 4), and 1:4 (sample 5) (P<0.01).

[0072] 2.1.2, Physical damage repair experiment of RGM-1 cell line

[0073] 2.1.2.1 Test Procedure (See Figure 6)

[0074] 2.1.1.2.1 Cell Culture: RGM-1 cells were cultured in DMEM / Ham F12 containing 20% ​​FBS and 1% p / s. Cells were seeded in 10 cm cell culture dishes and cultured in a cell incubator at 37°C and 5% CO2. When the cells reached 80-90% confluence, they were washed once with PBS and the PBS was removed. 0.25% trypsin (trypsin-EDTA) was added and incubated at 37°C for several minutes. Cells were observed to detach from the flask wall under a microscope. Fresh culture medium was added and mixed thoroughly to terminate the trypsin enzymatic reaction. The cell suspension was collected into a centrifuge tube, and the supernatant was removed after centrifugation. The cells were resuspended in DMEM culture medium, and the cell concentration was calculated using a hemocytometer. Cells were seeded at a rate of 2*10⁴ / well into 48-well cell culture dishes, with a volume of 1 mL / well. The cells were cultured in a cell incubator for 48 hours to allow them to adhere to the dish for later use.

[0075] 2.1.1.1.2. Use a 200μL micropipette to make a wound, remove the old culture medium, wash three times with phosphate buffered saline (PBS) to remove the scratched cells, add serum-free DMEM / Ham F12 containing different concentrations of samples at a rate of 1mL / well to a 48-well culture dish for culture, and perform 3 wells (3 replicates) for each group. Incubate in a cell culture incubator for 24 hours, and take pictures under a 4x objective lens at 0 and 24 hours respectively, placing the scratch in the center of the picture.

[0076] 2.1.1.1.3 Result Analysis: Use ImageJ to draw a straight line at the wound boundary and calculate the wound area between cells.

[0077] 2.1.1.1.4. Calculate the wound healing rate. Wound healing rate = (initial scratch area - scratch area at time t) / 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 chart based on the mean and standard error of the mean (Average ± SD) of each group.

[0078] 2.1.2.2 Results

[0079] The burdock and lithospermum composition of the present invention has the ability to promote the repair of physical damage to RGM-1. The composition is superior to or approximately superior to the extracts of burdock or lithospermum used alone, especially the 4:1 (sample 2) and 3:2 (sample 3) compositions, which are superior to the extracts of burdock or lithospermum used alone, as shown in Figure 7. With the initial wound area as 100%, the percentage of the remaining wound area after 24 hours of healing was calculated to determine the composition's ability to repair physical damage to RGM-1. The smaller the percentage of the remaining wound area to the initial area, the higher the wound healing rate, indicating a stronger ability of the composition to promote the repair of physical damage to RGM-1. All compositions demonstrated the ability to promote the repair of physical damage caused by RGM-1, especially compositions 4:1 (Sample 2) and 3:2 (Sample 3), which were superior to crude extracts of burdock or gromwell alone (**p<0.01 indicates a significant difference compared to the untreated group; ##p<0.01 indicates a significant difference compared to the 100% AL group (Sample 1); ++p<0.01 indicates a significant difference compared to the 100% AE group (Sample 6)).

[0080] 3. Ultra-high performance liquid chromatography (UPLC) fingerprint of the burdock and gromwell root combination

[0081] The preferred burdock and lithospermum composition in the examples was characterized by chemical composition determination using UPLC. The chromatograms show chlorogenic acid, a characteristic chemical component of burdock, and shikonin, a characteristic chemical component of lithospermum.

[0082] UPLC analysis conditions: UPLC was performed using a Shimadzu M30A UHPLC system; column (Waters HSS C18, 1.8μm, 2.1 x 100mm); column temperature was 30℃; flow rate was 0.4mL / min; detection wavelength was UV 320nm.

[0083] Mobile phase system conditions: Mobile phase A is secondary water (0.4% phosphoric acid); Mobile phase B is acetonitrile, with linear gradients of 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), and 24~25 min (23%A~0%A).

[0084] As shown in Figures 8 and 9, the burdock and lithospermum composition simultaneously possesses the characteristic chemical components of both burdock and lithospermum. These include chlorogenic acid from burdock and naphthoquinone compounds from lithospermum. The composition is characterized by: 1) naphthoquinone compounds from lithospermum, three of which have been identified as acetylshikonin, β-acetoxyisovalerylshikonin, and β,β-dimethylaceyshikonin; 2) the absolute configuration of shikonin also includes its mirror isomer, alkannin; 3) chlorogenic acid possesses various biological activities, including antibacterial activity, and therefore, in addition to being a characteristic chemical component of burdock, it can also serve as an indicator component of burdock activity.

[0085] The burdock and lithospermum composition provided in this embodiment of the invention has a specific UPLC fingerprint spectrum. This composition exhibits superior antibacterial performance against Helicobacter pylori compared to extracts of burdock or lithospermum alone, and its repair effect on chemical or physical damage to RGM-1 cell lines is also superior to extracts of burdock or lithospermum alone. The composition provided in this embodiment of the invention can effectively prevent or treat peptic ulcers and / or Helicobacter pylori infection.

[0086] In addition, embodiments of the present invention also provide a pharmaceutical composition, which is prepared by using the above-mentioned burdock and lithospermum composition as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients. This pharmaceutical composition can effectively prevent or treat peptic ulcers and / or Helicobacter pylori infection.

[0087] The specific embodiments described above have further illustrated the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition, comprising: Take a burdock powder, soak and stir it with alcohol to form a first solution, and let it stand overnight; Take a powder of Lithospermum erythrorhizon, soak it in alcohol and stir to form a second solution, and let it stand overnight; extract and filter the first solution and the second solution twice respectively, and collect and combine the two filtrates separately; Calculate the solid content in each of the filtrates; calculate the volumes of the first and second solutions based on the solid content and the weight percentage of the required extracts of burdock and gromwell, and combine them to obtain a combined solution; The composition is obtained by depressurizing, concentrating, and drying the combined liquid, wherein the composition consists of a burdock extract and a gromwell extract, and the burdock extract accounts for 80%-20% of the weight of the composition, and the gromwell extract accounts for 20%-80% of the weight of the composition.

2. A composition prepared by the method described in claim 1.

3. A pharmaceutical composition comprising the composition as described in claim 2 and its pharmaceutically acceptable excipients, carriers or other auxiliary components.

4. Use of the composition as described in claim 2 for the preparation of a pharmaceutical composition for the prevention or treatment of peptic ulcers.

5. Use of the composition as described in claim 2 for the preparation of a pharmaceutical composition for use as an adjunct antibiotic against Helicobacter pylori.

6. The use as described in claim 4 or 5, wherein, The effective dose of this composition is 0.05 mg / mL to 0.15 mg / mL.

7. Use of the composition as described in claim 2 for the preparation of a pharmaceutical composition for treating Helicobacter pylori.

8. The use as described in claim 7, wherein, The effective dose of this composition is 0.015 mg / mL to 0.05 mg / mL.

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