Application of hippophae rhamnoides leaf extract in preparation of anti-helicobacter pylori product

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly discloses application of a hippophae rhamnoides leaf extract in preparation of an anti-helicobacter pylori product. The sea-buckthorn leaf water extract and the ethanol extract are separated from the dried leaves of sea-buckthorn, the sea-buckthorn leaf water extract and the ethanol extract have strong inhibiting and killing effects on helicobacter pylori antibiotic sensitive strains and drug-resistant strains, and the inhibiting and killing effects of the ethanol extract are better. As a new resource food, the hippophae rhamnoides leaves are safe and reliable in application and have the action characteristics of multiple components, multiple ways and multiple targets, and in addition, the traditional Chinese medicine therapy is low in drug resistance incidence rate, few in adverse reaction, good in palatability and good in patient medication compliance. In-vitro experiments prove that the preparation method of the extract is stable and reliable, the extract has a very strong eliminating effect on helicobacter pylori in-vitro infection, the extract is used as food, the phenomenon that the stomach is injured by bitter cold of traditional Chinese medicines is avoided, the use of antibiotics can be reduced, the drug resistance rate of helicobacter pylori is reduced, and the extract is suitable for supplementary treatment of helicobacter pylori infected people.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of seabuckthorn leaf extract in preparing anti-Helicobacter pylori products. Background Art

[0002] Helicobacter pylori (H.pylori) is a Gram-negative, flagellated, spiral-shaped microaerophilic bacterium that grows in the digestive tract and can adapt to the acidic conditions in the human gastric mucosa. H.pylori is the main cause of chronic gastritis, peptic ulcers, and gastric cancer. Eradicating Helicobacter pylori infection can effectively prevent the occurrence and development of related diseases inside and outside the gastrointestinal tract and reduce the risk of tumors such as gastric cancer.

[0003] At present, the eradication treatment of Helicobacter pylori infection still mainly relies on standard triple therapy (two antibiotics + proton pump inhibitors) and bismuth quadruple therapy (triple therapy + bismuth) based on antibiotics such as clarithromycin, amoxicillin, metronidazole, and levofloxacin. However, with the widespread use of antibiotics worldwide and the long-term high-dose use of triple and quadruple therapy, the resistance rate of Helicobacter pylori to commonly used antibiotics has continued to rise, resulting in a year-on-year decline in the eradication rate of antibiotic therapy. However, due to factors such as antibiotic resistance, the drug combination and drug dose in the Helicobacter pylori eradication treatment program have continued to increase, the course of treatment has been extended, the overall eradication rate is still on a downward trend, and there is a certain recurrence rate, and there are certain side effects during the treatment process. Therefore, finding alternative or supplementary drugs other than antibiotics to treat Helicobacter pylori infection is an important measure to deal with the increasing resistance rate of Helicobacter pylori worldwide. The Chinese medicine diet therapy "Qianjin·Food Therapy Recipe" points out that due to the strong pharmacology of drugs, it is recommended that doctors can first use diet therapy when treating. Therefore, it is more valuable to find natural Chinese herbal products with good anti-Helicobacter pylori activity from food or medicine and food.

[0004] Seabuckthorn (Hipopphae rhamnoides L.) is a hard shrub belonging to the genus Seabuckthorn of the Elaeagnaceae family. It is also known as Elaeagnus angustifolia and sour willow. As a natural Tibetan medicine with a long history, seabuckthorn is widely used in traditional medicine to treat gastrointestinal diseases such as gastritis and gastric ulcers. Among them, seabuckthorn leaves, as an important medicinal and edible part of seabuckthorn, show many advantages compared to seabuckthorn fruits: they are easy to pick and preserve, have extremely rich reserves, and have more significant flavonoid content. These characteristics have made seabuckthorn leaves widely used in Tibet, Qinghai, Inner Mongolia and other regions, especially playing an important role in folk treatment of gastrointestinal diseases. In addition, modern pharmacological studies have confirmed that seabuckthorn leaves have biological activities such as enhancing immunity, anti-oxidation, anti-inflammatory, antiviral and antibacterial effects, but there is no report that they have the effect of resisting drug-resistant Helicobacter pylori. Summary of the invention

[0005] In order to overcome the increase in antibiotic-resistant Hp strains and the lack of effective drugs to inhibit Hp in the above-mentioned prior art, the present invention provides the use of seabuckthorn leaf extract in the preparation of anti-Helicobacter pylori products. The seabuckthorn leaf extract has a strong inhibitory effect on both antibiotic-sensitive and resistant strains of Helicobacter pylori, and can exert an inhibitory or killing effect by destroying the bacterial structure of Helicobacter pylori.

[0006] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0007] Application of seabuckthorn leaf extract in preparing anti-Helicobacter pylori products, wherein the seabuckthorn leaf extract is a water extract or an alcohol extract.

[0008] The preparation method of the seabuckthorn leaf water extract comprises the following steps:

[0009] S1. Crush the dried leaves of seabuckthorn into powder, add 8 to 12 times the amount of water to the seabuckthorn leaf powder, and soak for 30 to 60 minutes;

[0010] S2, placing the water extract containing seabuckthorn leaves in a water bath at 80-100° C. for 0.5-3 h under reflux extraction, and collecting the filtrate;

[0011] S3. The filtrate is concentrated by rotary evaporation to an extract, and then dried to a solid powder to obtain the seabuckthorn leaf water extract.

[0012] The preparation method of the seabuckthorn leaf alcohol extract comprises the following steps:

[0013] S1. Crush the dried leaves of seabuckthorn into powder, add 20 to 40 times the amount of 75% ethanol to the seabuckthorn leaf powder, and soak for 30 to 60 minutes;

[0014] S2, placing the 75% ethanol extract containing seabuckthorn leaves at 55°C for 1-2 hours by ultrasonic extraction, and collecting the filtrate;

[0015] S3. The filtrate is concentrated by rotary evaporation to an extract, and then dried to a solid powder to obtain the seabuckthorn leaf alcohol extract.

[0016] Preferably, the dried leaves of seabuckthorn are made by drying leaves of a deciduous shrub of the genus Hippophae rhamnoides of the family Elaeagnaceae.

[0017] Preferably, the seabuckthorn leaves are crushed into powder, passed through a 30-50 mesh sieve and then soaked.

[0018] Preferably, the extraction with the infusion containing seabuckthorn leaves is repeated 2-5 times, and the filtrates are combined.

[0019] Preferably, the anti-Helicobacter pylori product includes a medicine, a preventive health product or a functional food.

[0020] Preferably, the dosage forms of the anti-Helicobacter pylori product include powder, granules, injection, capsule, tablet, oral solution.

[0021] The beneficial effects of the present invention are:

[0022] The present invention extracts seabuckthorn leaf water extract and alcohol extract from the dried leaves of seabuckthorn, and through the stability study of the extraction process, it is found that the extraction process is stable and reliable. Through the quantitative analysis of the main components and the antibacterial experiment, it is found that the main component content of the seabuckthorn leaf extract is stable, and the standard strains and clinical strains of Helicobacter pylori (including antibiotic sensitive strains and resistant strains) are all strongly inhibited. In addition, the seabuckthorn leaf water extract and alcohol extract can both have a damaging effect on the surface structure of Helicobacter pylori, and can be used in the preparation of anti-Helicobacter pylori products. Seabuckthorn, as a natural Tibetan medicine with a long history, is widely used in traditional medicine to treat gastrointestinal diseases such as gastritis and gastric ulcers. Among them, seabuckthorn leaves, as an important medicinal edible part of seabuckthorn, show many advantages compared with seabuckthorn fruits: it is easy to pick and preserve, the reserves are extremely rich, and the content of flavonoid compounds is more significant, and it has significant anti-regulation of lipid metabolism, anti-inflammatory, antiviral, antibacterial and antioxidant functions, and is safe to eat and mild in effect. Compared with the characteristics of easy drug resistance of antibiotics, seabuckthorn leaves have the advantages of multi-components, multi-target effects, not easy to produce drug resistance, little impact on intestinal flora, mild adverse reactions, safe use, etc. It is suitable for supplementary or alternative treatment of Helicobacter pylori infected patients, especially for patients allergic to antibiotics and infected elderly and children. The present invention broadens the application field of food seabuckthorn leaves, provides a new direction for the treatment of Helicobacter pylori, and can effectively avoid the influence of antibiotic resistance on Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0024] Figure 1 This is the HPLC chromatogram of aqueous and ethanol extracts of seabuckthorn leaves.

[0025] Figure 2 The antibacterial curves of seabuckthorn leaf water extract and ethanol extract against Helicobacter pylori (ATCC 700392 and ATCC 43504) (AE: seabuckthorn leaf water extract; HE: seabuckthorn leaf ethanol extract).

[0026] Figure 3These are scanning electron micrographs of the ATCC 700392 strain 8 hours after treatment with seabuckthorn leaf aqueous and ethanol extracts (a: growth control bacteria; b: bacteria treated with seabuckthorn leaf aqueous extract 640 μg / mL-MIC; c: bacteria treated with seabuckthorn leaf ethanol extract 320 μg / mL-MIC; d: strain treated with clarithromycin positive control). DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0028] Example 1 Preparation of Seabuckthorn Leaf Extract

[0029] 1. Preparation of seabuckthorn leaf aqueous extract

[0030] (1) Crush the dried leaves of seabuckthorn into powder, pass through a 40-mesh sieve, add 10 times the amount of water (solid-liquid ratio mg:mL) to the seabuckthorn leaf powder and soak for 30 minutes;

[0031] (2) The water extract containing seabuckthorn leaves was placed in a water bath under reflux extraction at 90°C for 1 h, and the extraction was repeated three times, and the filtrates were combined;

[0032] (3) The filtrate is concentrated by rotary evaporation to an extract, and then freeze-dried to a solid powder.

[0033] 2. Preparation of seabuckthorn leaf ethanol extract:

[0034] (1) Crush the dried leaves of seabuckthorn into powder, pass through a 40-mesh sieve, add 20 times the amount (solid-liquid ratio mg:mL) of 75% ethanol to the seabuckthorn leaf powder, and soak for 30 minutes;

[0035] (2) ultrasonically extracting the 75% ethanol extract containing seabuckthorn leaves at 55°C for 1 h, repeating the extraction three times, and combining the filtrates;

[0036] (3) The filtrate is concentrated by rotary evaporation to an extract, and then freeze-dried to a solid powder.

[0037] (4) Study on the stability of water and alcohol extraction process of seabuckthorn leaves

[0038] The contents of total polyphenols and total flavonoids in three batches of seabuckthorn leaf water extract and three batches of alcohol extract were determined by Folin phenol method and aluminum nitrate colorimetry, respectively.

[0039] The specific steps for determining the total polyphenol content in the water extract and alcohol extract of seabuckthorn leaves by the Folin phenol method are as follows:

[0040] Accurately weigh an appropriate amount of gallic acid standard and add double distilled water to prepare a drug solution of about 0.4 mg / mL (ultrasonic dissolution), pass through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain a control solution. Accurately weigh the seabuckthorn leaf water extract (batches 1-3) and seabuckthorn leaf alcohol extract (batches 4-6) in Example 1, and prepare a drug solution with a concentration of about 50 μg / mL in the same way to obtain a test solution. In a 96-well plate, a 200 μL system is composed of X μL of control solution or test solution + Y μL of ultrapure water + 40 μL of Folin phenol reagent + 57 μL of 35% sodium carbonate solution. After standing at room temperature in the dark for 2 hours, the absorbance is measured at a wavelength of 760 nm. A standard curve is drawn with absorbance as the ordinate and gallic acid concentration as the abscissa. The absorbance of the test sample is substituted into the standard curve to determine and calculate the gallic acid content contained in the sample.

[0041] The specific steps for determining the total flavonoids content in the water extract and alcohol extract of seabuckthorn leaves by aluminum nitrate colorimetry are as follows:

[0042] Accurately weigh an appropriate amount of rutin standard and add 50% (volume fraction) ethanol solution to prepare a drug solution of about 0.2 mg / mL (ultrasonic dissolution), pass through a 0.22 μm filter membrane, and take the subsequent filtrate to obtain a control solution. Accurately weigh the seabuckthorn leaf water extract (batches 1-3) and seabuckthorn leaf alcohol extract (batches 4-6) in Example 1, and prepare a drug solution with a concentration of about 0.2 mg / mL in the same way to obtain a test solution. In an Ep tube, X μL of control solution or test solution + Y μL of 50% (volume fraction) ethanol solution + 80 μL of 0.25% nitrite reagent + 80 μL of 0.5% aluminum nitrate solution + 40 μL of 0.05% sodium hydroxide solution to form a 1 mL system. After mixing, pipette 200 μL into each 96-well plate, measure the absorbance at a wavelength of 490 nm, draw a standard curve with absorbance as the ordinate and rutin concentration as the abscissa, and substitute the absorbance of the test sample into the standard curve to determine and calculate the rutin content in the sample.

[0043] The results are shown in Tables 1 and 2. The extraction process of the seabuckthorn leaf water extract and the ethanol extract prepared in Example 1 is stable, the extraction yields of the seabuckthorn leaf water extract and the ethanol extract are between 26.09-28.77%, and the total polyphenol and total flavonoid contents of the extracts between batches are similar, indicating that the preparation process is stable and reliable.

[0044] Table 1 Contents of total polyphenols and total flavonoids in the aqueous extract of seabuckthorn leaves

[0045]

[0046] a. Each gram of seabuckthorn leaf water extract contains X mg of total polyphenols with gallic acid (GAE); b. Each gram of medicinal material contains X mg of total polyphenols with gallic acid (GAE); c. Each gram of seabuckthorn leaf water extract contains X mg of total flavonoids with rutin (RT); d. Each gram of medicinal material contains X mg of total flavonoids with rutin (RT).

[0047] Table 2 Contents of total polyphenols and total flavonoids in ethanol extract of seabuckthorn leaves

[0048]

[0049] a. Each gram of seabuckthorn leaf ethanol extract contains X mg of total polyphenols with gallic acid (GAE); b. Each gram of medicinal material contains X mg of total polyphenols with gallic acid (GAE); c. Each gram of seabuckthorn leaf ethanol extract contains X mg of total flavonoids with rutin (RT); d. Each gram of medicinal material contains X mg of total flavonoids with rutin (RT).

[0050] Example 2

[0051] Quantitative analysis experiment of seabuckthorn leaf water extract (AE) and ethanol extract (HE) by HPLC:

[0052] Accurately weigh gallic acid, protocatechuic acid, catechin, caffeic acid, p-coumarin and ferulic acid standards, and prepare them into mother solutions of about 200 μg / mL with methanol. Take appropriate amounts of the mother solutions of the above 6 components, dilute them in a gradient manner, and obtain 24.9, 26.31, 29.26, 30.01, 19.55, and 21.29 μg / mL mixed reference substance stock solutions. Accurately weigh the seabuckthorn leaf water extract (batches 1-3) and seabuckthorn leaf alcohol extract (batches 4-6) in Example 1, and prepare them into a sample solution of about 2 mg / mL with methanol. After ultrasonic dissolution, pass through a 0.22 μm organic phase microporous filter membrane to obtain the test solution.

[0053] The water extracts (three batches, samples AE1-3) and ethanol extracts (three batches, samples HE1-3) of seabuckthorn leaves prepared in Example 1 and mixed standards (gallic acid, protocatechuic acid, catechins, caffeic acid, p-coumarin and ferulic acid) were subjected to HPLC experiments. The results are as follows: Figure 1As shown, in the seabuckthorn leaf water extract prepared in Example 1, the content of gallic acid is: 13.15±0.28 mg / g; the content of protocatechuic acid is: 0.8±0.19 mg / g; the content of catechin is: 2.26±0.01 mg / g; the content of caffeic acid is: 22.71±1.40 mg / g; the content of p-coumarin is: 0.37±0.01 mg / g; and the content of ferulic acid is: 0.47±0.09 mg / g. In the ethanol extract of seabuckthorn leaves prepared in Example 1, the content of gallic acid is 5.66±0.28 mg / g; the content of protocatechuic acid is 4.14±0.18 mg / g; the content of catechin is 2.56±0.22 mg / g; the content of caffeic acid is 28.46±1.48 mg / g; the content of p-coumarin is 0.1±0.03 mg / g; and the content of ferulic acid is 0.81±0.03 mg / g.

[0054] Example 3

[0055] 1. Antibacterial test of water and ethanol extracts of seabuckthorn and seabuckthorn leaves

[0056] According to the seabuckthorn leaf extraction method in Example 1, seabuckthorn water-ethanol extract was prepared in the same manner, and the MIC of seabuckthorn and seabuckthorn leaf water-ethanol extract against the standard strain of Helicobacter pylori ATCC 700392 was detected by microbroth dilution method (MIC refers to the lowest concentration of a drug that can inhibit the growth of microorganisms). The specific experimental method is as follows:

[0057] Using clarithromycin as a positive control, multiple drug concentration gradients (2560μg / mL, 1280μg / mL, 640μg / mL, 320μg / mL, 160μg / mL) were prepared in broth culture medium containing 20% ​​FBS, 50μL of drug-containing or drug-free broth culture medium was aspirated into a 96-well plate, and 50μL of diluted bacterial solution (final turbidity was approximately 1×106CFU / mL) was added, and the plate was placed in a three-gas incubator and cultured at 37°C and 150rpm for 72h. The drug concentration when the bacterial turbidity was significantly reduced was the MIC, and the measurement was repeated 3 times.

[0058] From the results in Table 3, it can be seen that the MIC of seabuckthorn water and ethanol extracts are both greater than 2560 μg / mL, exceeding the upper limit of the drug concentration. Although there are literature reports that seabuckthorn fruit has a certain anti-Hp activity, the anti-Hp activity is poor. In comparison, the anti-Hp MIC of seabuckthorn leaf extract is much lower than that of seabuckthorn fruit, and has better antibacterial activity.

[0059] Table 3 Minimum inhibitory concentration (MIC) of seabuckthorn and seabuckthorn leaf aqueous and ethanol extracts against standard strain ATCC 700392

[0060]

[0061] 2. Antibacterial test of water extract and ethanol extract of seabuckthorn leaves from different origins

[0062] According to the microbroth dilution method of Example 3, the MIC of the water extract and ethanol extract of seabuckthorn leaves extracted from different origins were determined. From the results in Table 4, it can be seen that the MIC of the water extract and ethanol extract of seabuckthorn leaves from different origins in Xinjiang and Inner Mongolia against the standard strain of Helicobacter pylori ATCC 700392 were 640 μg / mL and 320 μg / mL, indicating that seabuckthorn leaves from different origins have good inhibitory effects on Helicobacter pylori, and the origin of seabuckthorn leaves has little effect on the anti-Helicobacter pylori activity of seabuckthorn leaves.

[0063] Table 4 Minimum inhibitory concentration (MIC) of seabuckthorn leaves from different origins against standard strain ATCC 700392

[0064]

[0065] 3. Antibacterial test of seabuckthorn leaf aqueous extract (AE) and ethanol extract (HE)

[0066] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the aqueous and alcohol extracts of seabuckthorn leaves prepared in Example 1 against standard strains of Helicobacter pylori (ATCC 43503, ATCC 700392, SS1) and clinical strains (ICDC11101, CS01, QYZ-001, QYZ-003, QYZ-004) were detected by microbroth dilution method. Note: (MIC refers to the minimum concentration at which a drug can inhibit the growth of microorganisms, and MBC refers to the minimum concentration at which a drug can kill microorganisms). The specific experimental method is as follows:

[0067] Using clarithromycin as a positive control, prepare multiple drug concentration gradients (80μg / mL, 160μg / mL, 320μg / mL, 320μg / mL, 640μg / mL) in broth medium containing 20% ​​FBS, pipette 50μL of drug-containing or drug-free broth medium into a 96-well plate, add 50μL of diluted bacterial solution (final turbidity is about 1×106CFU / mL), place in a three-gas incubator, shake and culture at 37°C, 150rpm for 72h, the drug concentration when the bacterial turbidity is significantly reduced is the MIC, repeat the measurement 3 times. Take out the 96-well plate that has been cultured for 72h, pipette 100μL of the liquid in the MIC, 2MIC, 4MIC, 8MIC drug concentration wells and evenly spread it on a fresh blood plate, take out and observe the results after 72h of culture, so that the initial inoculation concentration of the bacterial solution is reduced (10 3 CFU / mL) is the drug concentration at which MBC is measured. The test results are shown in Table 3.

[0068] As shown in Table 5, the seabuckthorn leaf extract has a certain inhibitory or killing effect on the standard strains and clinical strains of Helicobacter pylori. The MIC of the seabuckthorn leaf water extract to each strain is between 160 and 640 μg / mL, and the MBC is 2 to 8 MIC, showing a bactericidal effect. The MIC of the seabuckthorn leaf alcohol extract to each strain is between 80 and 320 μg / mL, and the MBC is 2 to 8 MIC, showing a bactericidal effect.

[0069] Table 5 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of seabuckthorn leaf water extract and ethanol extract against various Helicobacter pylori strains in vitro

[0070]

[0071] Note: R stands for resistant, S stands for sensitive; MTZ: metronidazole, CLR: clarithromycin, LEF: levofloxacin, AMO: amoxicillin; the resistance breakpoint values ​​were determined according to the European Committee on Antimicrobial Susceptibility Testing 2019 (EUCAST 2019) standards.

[0072] 4. Antibacterial curves of seabuckthorn leaf water extract and ethanol extract against Helicobacter pylori

[0073] The time-inhibition curves of seabuckthorn leaf water extract and ethanol extract were drawn by measuring the bacterial solution OD600 nm (AE: seabuckthorn leaf water extract; HE: seabuckthorn leaf ethanol extract). The specific test method is as follows:

[0074] (1) The aqueous extract and ethanol extract of seabuckthorn leaves were prepared by the two-fold dilution method with multiple concentration gradients, such as MIC, 1 / 2MIC, and 1 / 4MIC. The heart extract without drug was used as the growth control group.

[0075] (2) Taking ATCC 43504 and ATCC 700392 as representatives, take out the strains from the incubator, scrape the bacterial moss with a moistened sterile cotton swab and put it into brain heart infusion solution, and adjust the turbidity of the bacterial suspension to 1 McF with brain heart infusion solution containing 20% ​​FBS.

[0076] (3) According to the volume ratio of bacterial suspension to drug solution / brain heart extract without drug of 1:1, 2 mL was taken from each well and inoculated into a 12-well plate, which was placed in a three-gas incubator and cultured with shaking at 150 rpm in a microaerobic environment at 37 °C. At 0, 12, 24, 36, 48, 60, and 72 h, 100 μL of culture solution was taken out of the 96-well plate to measure the OD600 nm. The larger the absorbance value, the more bacteria there are and the worse the antibacterial effect of the drug.

[0077] The results are as follows Figure 2 As shown. Figure 2It can be seen that 48-72 hours is the rapid growth period of Helicobacter pylori, and the antibacterial effect of seabuckthorn leaf water extract and ethanol extract is most significant at 72 hours compared with the growth control group. Seabuckthorn leaf water extract and ethanol extract can inhibit the growth of Helicobacter pylori in the range of 1 / 4-1MIC, and the antibacterial effect is dose-dependent.

[0078] Example 4

[0079] Observation of the effects of aqueous and ethanol extracts of seabuckthorn leaves on the morphology of ATCC 700392 strain using scanning electron microscopy

[0080] Take the strain ATCC 700392 with 1McF turbidity and culture it to the logarithmic growth phase. Take 1mL of bacterial liquid and add it to the medium containing or not containing drugs (drug concentration: sea buckthorn leaf water extract AE 640μg / mL-MIC, sea buckthorn leaf ethanol extract HE 320μg / mL-MIC, clarithromycin 0.008μg / ml-MIC), incubate for 24 hours, collect the bacteria by centrifugation, wash twice with PBS, add 2.5% glutaraldehyde electron microscope fixative, fix overnight at 4℃, then dehydrate with 30%, 50%, 70%, 80%, and 95% series of ethanol, replace by tert-butanol immersion, dry, mount, spray with gold, and use for scanning electron microscopy observation. Observe the smoothness and damage of the bacterial surface to determine the effect of sea buckthorn leaf water extract and ethanol extract on bacterial morphology. The results are as follows Figure 3 shown.

[0081] Depend on Figure 3 It can be seen that the overall shape of the bacteria in the control group is spiral, curved or rod-shaped, the bacterial surface is smooth and the surface membrane structure is not damaged. The overall morphology of the strains in the treatment group is similar to that in the control group, but the surface morphology of the strains in the treatment group has changed significantly, such as the shrinkage of the bacteria and the damage of the bacterial surface membrane structure, indicating that the MIC seabuckthorn leaf water extract and ethanol extract can inhibit or kill Helicobacter pylori by destroying the bacterial morphology.

[0082] In summary, the present invention separates and obtains seabuckthorn leaf water extract and ethanol extract from the dry leaves of seabuckthorn, a plant of the genus Hippophae rhamnoides of the family Elaeagnaceae. The preparation process of the present invention is stable and has high yield. It has been found that seabuckthorn leaf water extract and ethanol extract have strong inhibitory and killing effects on antibiotic-sensitive strains and resistant strains of Helicobacter pylori, and the inhibitory and killing effect of ethanol extract is better. Further studies have found that seabuckthorn leaf water extract and ethanol extract have damaging effects on the surface structure of Helicobacter pylori. Seabuckthorn leaf is a new resource food, safe to eat, and mild in effect. Compared with the characteristics of easy resistance to antibiotics, seabuckthorn leaf has the advantages of multi-component, multi-target effect, not easy to produce resistance, little effect on intestinal flora, mild adverse reactions, and safe use. It is suitable for supplementary or alternative treatment of Helicobacter pylori infected persons, especially for patients allergic to antibiotics and infected persons of the elderly and children.

[0083] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Use of seabuckthorn leaf extract in the preparation of anti-Helicobacter pylori products, wherein the seabuckthorn leaf extract is a water extract or an alcohol extract; The preparation method of the seabuckthorn leaf water extract comprises the following steps: S1. Crush the dried leaves of seabuckthorn into powder, add 8 to 12 times the amount of water to the seabuckthorn leaf powder, and soak for 30 to 60 minutes; S2, placing the water extract containing seabuckthorn leaves in a water bath at 80-100° C. for 0.5-3 h under reflux extraction, and collecting the filtrate; S3, concentrating the filtrate by rotary evaporation to an extract, and then drying to a solid powder to obtain a seabuckthorn leaf water extract; The preparation method of the seabuckthorn leaf alcohol extract comprises the following steps: S1. Crush the dried leaves of seabuckthorn into powder, add 20 to 40 times the amount of 75% ethanol to the seabuckthorn leaf powder, and soak for 30 to 60 minutes; S2, placing the 75% ethanol extract containing seabuckthorn leaves at 55°C for 1-2 hours by ultrasonic extraction, and collecting the filtrate; S3. The filtrate is concentrated by rotary evaporation to an extract, and then dried to a solid powder to obtain the seabuckthorn leaf alcohol extract.

2. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of an anti-Helicobacter pylori product, characterized in that: The dried leaves of seabuckthorn are prepared by drying the leaves of a deciduous shrub of the genus Hippophae rhamnoides of the family Elaeagnaceae.

3. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of an anti-Helicobacter pylori product, characterized in that: The seabuckthorn leaves are crushed into powder, passed through a 30-50 mesh sieve and then soaked.

4. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of an anti-Helicobacter pylori product, characterized in that: The extraction of the infusion containing seabuckthorn leaves is repeated 2-5 times, and the filtrates are combined.

5. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of an anti-Helicobacter pylori product, characterized in that: The anti-Helicobacter pylori products include medicines, preventive health products or functional foods.

6. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of an anti-Helicobacter pylori product, characterized in that: The dosage forms of the anti-Helicobacter pylori product include powder, granules, injection, capsule, tablet and oral solution.

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