Application of folium sennae extract in preparation of medicine for preventing and treating helicobacter pylori infection

Abstract

The invention discloses application of a folium sennae extract in preparation of a medicine for preventing and treating helicobacter pylori infection. The folium sennae alcohol extract disclosed by the invention shows an obvious bactericidal effect on multi-drug-resistant helicobacter pylori; when being combined with amoxicillin, the compound has a synergistic antibacterial effect on general helicobacter pylori and helicobacter pylori with drug resistance to amoxicillin. According to the invention, a new drug choice is provided for treatment of patients suffering from mild symptoms of H.pylori infection and drug resistance, and unique advantages are played in graded diagnosis and treatment and drug-resistant H.pylori treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of senna leaf extract in the preparation of drugs for the prevention and treatment of Helicobacter pylori infection. Background Technology

[0002] Helicobacter pylori (H. pylori) is a Gram-negative, spiral-shaped, multi-flagellated microaerophilic bacterium. It is highly adapted to the acidic environment of the stomach and colonizes the human gastric mucosa through mechanisms such as urease-catalyzed urea hydrolysis and single-terminal multi-flagellated motility. After infection with H. pylori, the host's gastric microenvironment and immunity are affected, and the diversity and structure of the gastrointestinal microbiota are altered. This can induce various serious complications such as gastric bleeding, gastric ulcers, and gastric perforation. Some patients may develop peptic ulcers or even gastric cancer. Therefore, H. pylori infection is one of the important public health problems facing the world today. Currently, the primary clinical treatment for Helicobacter pylori eradication is antibiotic therapy. Amoxicillin, a representative β-lactam antibiotic, exerts its antibacterial effect by inhibiting bacterial cell wall synthesis and has long been a core antibacterial component of standard triple or quadruple therapy for H. pylori eradication. In recent years, with continuous optimization of dosing regimens and the rise of combination therapy strategies, dual therapy based on this drug has gradually become one of the important trends in the treatment of H. pylori infection in the digestive field. However, with the increase in antibiotic resistance, amoxicillin resistance has gradually become an issue, affecting the success rate of H. pylori eradication. In addition, adverse reactions such as vomiting and diarrhea caused by antibiotic therapy lead to decreased patient compliance, making the search for complementary and alternative therapies for H. pylori infection increasingly inevitable. Summary of the Invention

[0003] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of senna leaf extract in the preparation of drugs for the prevention and treatment of Helicobacter pylori infection.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides the use of senna leaf alcohol extract in the preparation of drugs for preventing and treating multidrug-resistant Helicobacter pylori infection.

[0006] In some embodiments of the present invention, the multidrug-resistant Helicobacter pylori includes Helicobacter pylori resistant to at least two of clindamycin, metronidazole, levofloxacin, and amoxicillin, such as resistant to amoxicillin and at least one selected from clindamycin, metronidazole, and levofloxacin.

[0007] In some embodiments of the present invention, the multidrug-resistant Helicobacter pylori includes Helicobacter pylori resistant to metronidazole and levofloxacin.

[0008] In some embodiments of the present invention, the multidrug-resistant Helicobacter pylori includes Helicobacter pylori resistant to clindamycin, metronidazole and levofloxacin.

[0009] In some embodiments of the present invention, the multidrug-resistant Helicobacter pylori includes Helicobacter pylori resistant to clindamycin, metronidazole, levofloxacin and amoxicillin.

[0010] In some embodiments of the present invention, the multidrug-resistant Helicobacter pylori includes at least one strain of QYZ003, QYZ004, and ICDC111001.

[0011] In some embodiments of the present invention, the working concentration of the senna leaf alcohol extract is 150–600 μg / mL, such as 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 250 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 350 μg / mL, 380 μg / mL, 400 μg / mL, 420 μg / mL, 450 μg / mL, 480 μg / mL, 500 μg / mL, 520 μg / mL, 550 μg / mL, 580 μg / mL, etc.

[0012] In some embodiments of the present invention, the working concentration is the working concentration in the in vitro culture system.

[0013] In some embodiments of the present invention, the senna leaf alcohol extract contains at least one of flavonoid glycosides, flavonoid aglycones, anthraquinone glycosides, free anthraquinones, and phenolic glycosides.

[0014] In some embodiments of the present invention, the flavonoid glycosides include at least one of verbascoside-2, vitexin, vitexin-2, rutin, kaempferol 3-O-sophoroside, kaempferol-3-O-rutinoside, quercetin 3-gentiobiose, isorhamnetin-3-O-gentiobiose, and rhamnetin 3-neohesperidin.

[0015] In some embodiments of the present invention, the flavonoid aglycone compound includes at least one of quercetin, kaempferol, isorhamnetin, luteolin, and 5-O-methylquercetin.

[0016] In some embodiments of the present invention, the anthraquinone glycosides include at least one of sennoside A1, sennoside B, sennoside C, sennoside D, sennoside glucoside B, rhein-8-glucoside, and rhein-8-O-β-D-glucopyranoside.

[0017] In some embodiments of the present invention, the free anthraquinone compound includes at least one of rhein, rhein, and phytoestrogen.

[0018] In some embodiments of the present invention, the phenolic glycosides include cassia benzophenone A-2-glucoside and / or cassia benzophenone B-2-glucoside.

[0019] In some embodiments of the present invention, the senna leaf alcohol extract includes the following compounds: kaempferol-3-O-sophoroside, vitexin, quercetin-3-gentiobiose, sennoside B, and sennoside A.

[0020] In some embodiments of the present invention, the senna leaf alcohol extract comprises at least one of the following compounds:

[0021] Cassia phenylephrine A-2-glucoside, Cassia phenylephrine B-2-glucoside, Verbascoside-2, Kaempferol, Kaempferol 3-O-Sophoroside, Kaempferol-3-O-Rutinoside, Vitexin, Vitexin-2, Sennoside A1, Sennoside B, Sennoside C, Sennoside D, Sennoside glucoside B, Quercetin, Quercetin 3-Gentianobiose, 5-O-Methylquercetin, Rutin, Isorhamnetin-3-O-Gentianobiose, Emodin-8-glucoside, Rhein-8-O-β-D-glucopyranoside, Rhamnetin 3-Neohesperidin, Isorhamnetin, Emodin, Luteolin, Emodin, Rhein.

[0022] In some embodiments of the present invention, the senna leaf alcohol extract is a senna leaf ethanol extract; the concentration of the ethanol is 50-75%.

[0023] In some embodiments of the present invention, the preparation method of the senna leaf alcohol extract includes extracting the senna leaves by soaking them in 50-75% ethanol; the number of extractions is 1-5 times, such as 2-4 times; the mass ratio of ethanol to senna leaves is (8-16):1, such as (10-15):1.

[0024] In some embodiments of the present invention, the preparation method of the senna leaf alcohol extract includes grinding senna leaves and soaking them in 50-75% ethanol, heating and refluxing to obtain a filtrate; adding 50-75% ethanol to the filter residue for extraction, combining the filtrates, drying to obtain an extract, and then freeze-drying to obtain a freeze-dried powder.

[0025] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the term "pharmaceuticalally acceptable excipient" includes pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active ingredient. The use of pharmaceutically acceptable excipients in the preparation of pharmaceutical formulations is well known to those skilled in the art. The pharmaceutical formulations of the present invention contain the aforementioned senna leaf alcohol extract as the active ingredient, and combine the senna leaf alcohol extract with pharmaceutically acceptable excipients (such as carriers, excipients, diluents, etc., well known to those skilled in the art) to formulate various formulations, preferably solid and liquid formulations. The formulations of the present invention can be in unit dose form, such as tablets, pills, capsules (including sustained-release or delayed-release forms), powders, suspensions, granules, tinctures, syrups, emulsions, suspensions, injections, and various sustained-release formulations, thus suitable for various forms of administration, such as oral, parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal, or transdermal administration. The carriers, excipients, and diluents are pharmaceutically acceptable and compatible with the active ingredient.

[0027] A second aspect of the present invention provides the use of a composition of senna leaf extract and amoxicillin in the preparation of a drug for Helicobacter pylori infection.

[0028] In this invention, the combined use of senna leaf extract and amoxicillin can significantly reduce the effective antibacterial concentration of both, demonstrating a synergistic effect.

[0029] In some embodiments of the present invention, the mass ratio of senna leaf extract to amoxicillin in the composition is (70-150):1, such as (80-130):1, (90-110):1, 100:1, etc.

[0030] In some embodiments of the present invention, the working concentration of senna leaf extract in the composition is 50-600 μg / mL, such as 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 250 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 350 μg / mL, 380 μg / mL, 400 μg / mL, 420 μg / mL, 450 μg / mL, 480 μg / mL, 500 μg / mL, 520 μg / mL, 550 μg / mL, 580 μg / mL, etc.

[0031] In some embodiments of the present invention, the working concentration of amoxicillin in the composition is 0.02–3.0 μg / mL, such as 0.025 μg / mL, 0.03 μg / mL, 0.05 μg / mL, 0.08 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.2 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.8 μg / mL, 2.0 μg / mL, 2.2 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3.0 μg / mL, etc.

[0032] In some embodiments of the present invention, the working concentration is the working concentration in the in vitro culture system.

[0033] In some embodiments of the present invention, the Helicobacter pylori includes susceptible Helicobacter pylori and / or drug-resistant Helicobacter pylori.

[0034] In some embodiments of the present invention, the drug-resistant Helicobacter pylori includes Helicobacter pylori resistant to at least one of clindamycin, metronidazole, levofloxacin, and amoxicillin, such as Helicobacter pylori resistant to amoxicillin. In the present invention, the combined use of senna leaf extract and amoxicillin has a strong bactericidal effect against Helicobacter pylori resistant to amoxicillin.

[0035] In some embodiments of the present invention, the susceptible Helicobacter pylori includes at least one strain of ATCC 700392 and SS1.

[0036] In some embodiments of the present invention, the drug-resistant Helicobacter pylori includes at least one strain of QYZ003, QYZ004, and ICDC111001.

[0037] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0038] In some embodiments of the present invention, the term "pharmaceuticalally acceptable excipient" includes pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active ingredient. The use of pharmaceutically acceptable excipients in the preparation of pharmaceutical formulations is well known to those skilled in the art. The pharmaceutical formulations of the present invention comprise the composition of the senna leaf extract and amoxicillin as the active ingredient, and are formulated into various formulations, preferably solid and liquid formulations, by combining the composition of the senna leaf extract and amoxicillin with pharmaceutically acceptable excipients (such as carriers, excipients, diluents, etc., well known to those skilled in the art). The formulations of the present invention can be in unit dose form, such as tablets, pills, capsules (including sustained-release and delayed-release forms), powders, suspensions, granules, tinctures, syrups, emulsions, suspensions, injections, and various sustained-release formulations, thus suitable for various forms of administration, such as oral, parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal, or transdermal administration. The carrier, excipients, and diluents are pharmaceutically acceptable and compatible with the active ingredient.

[0039] The beneficial effects of this invention are:

[0040] The senna leaf alcohol extract in this invention exhibits significant bactericidal activity against multidrug-resistant Helicobacter pylori; when used in combination with amoxicillin, it shows synergistic antibacterial effects against both common Helicobacter pylori and amoxicillin-resistant Helicobacter pylori. This invention provides a new drug option for the treatment of mild and drug-resistant H. pylori infections, demonstrating unique advantages in tiered diagnosis and treatment and the treatment of drug-resistant H. pylori. Attached Figure Description

[0041] Figure 1 The total ion chromatogram of the senna leaf alcohol extract test sample in Example 1 of the present invention is shown.

[0042] Figure 2 This is the time-bactericidal curve of the combined use of senna leaves and amoxicillin in Example 3 of the present invention. Detailed Implementation

[0043] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0044] The clinical strain of Helicobacter pylori (ICDC111001) was purchased from the Chinese Center for Disease Control and Prevention.

[0045] The Helicobacter pylori standard strain ATCC 700392 was donated by Professor Bi Hongkai of Nanjing Medical University;

[0046] Both QYZ-003 and QYZ-004 were donated by Qingyuan Municipal Hospital of Traditional Chinese Medicine.

[0047] Example 1

[0048] This embodiment studies the antibacterial effect of senna leaf alcohol extract on multidrug-resistant Helicobacter pylori. The specific process is as follows:

[0049] Preparation of senna leaf ethanol extract: Senna leaves were pulverized into coarse powder and passed through a 20-mesh sieve (particle size ≤ 0.85 mm). 8 g of powder was weighed into a 250 mL round-bottom flask, and 10–15 times the amount of 60% ethanol was added for soaking. The mixture was refluxed in a hot water bath for 1 hour, filtered, and the residue was extracted a second time with 10–15 times the amount of 60% ethanol. The extraction was repeated three times in total. The filtrates from the three extractions were combined and concentrated by rotary evaporation at 75 °C. The resulting extract was freeze-dried to obtain a lyophilized powder of the herbal water extract, which was then weighed. The powder was packed into EP tubes and stored in a refrigerator for later use.

[0050] A concentration gradient of 2560, 1280, 640, 320, 160, 80, and 40 μg / mL was prepared in 96-well plates using a two-fold dilution method. Simultaneously, antibiotics were diluted to a concentration gradient of 0.032, 0.016, 0.008, 0.004, 0.002, and 0.001 μg / mL. 50 μL of the drug solution was added to each well, with three replicates for each concentration. Negative control and growth control wells were also included. H. pylori in the logarithmic growth phase was scraped using a sterile, moistened cotton swab and suspended in broth (BHI) containing 20% ​​fetal bovine serum (FBS). The turbidity of the bacterial suspension was adjusted to the 1 McFarland standard, then diluted 10-fold, and 50 μL was inoculated into each well of the drug-containing 96-well plate. The well plates were placed in a microaerophilic environment (85% N2, 10% CO2, 5% O2) and incubated at 37°C with shaking at 150 rpm for 72 h. After incubation, bacterial growth was observed visually. If the control wells showed a button-like appearance or were significantly turbid, while the turbidity of the drug-treated wells was significantly reduced, then the drug concentration was considered to inhibit bacterial growth, and its MIC was the lowest drug concentration that inhibited visible growth. Clarithromycin was used as a positive control in each experiment, and the measurements were repeated three times to ensure the reliability of the results.

[0051] The results are shown in Table 1:

[0052] Table 1

[0053]

[0054]

[0055] Note: R indicates resistance; MTZ: metronidazole, CLR: clarithromycin, LEF: levofloxacin, AMO: amoxicillin; resistance breakpoint values ​​were determined according to the European Committee for Testing Antimicrobial Susceptibility 2019 (EUCAST 2019) standards.

[0056] As shown in Table 1, the MIC range of senna leaf alcohol extract against multidrug-resistant Helicobacter pylori is 160–320 μg / mL, and a significant bactericidal effect can be observed when the concentration is 320–640 μg / mL.

[0057] Further analysis of the components of senna leaf alcohol extract was conducted, and the specific process was as follows:

[0058] Chromatographic conditions

[0059] Column: YMC-Triart C18, 4.6*150mm, No.: C-153

[0060] Mobile phases: Mobile phase A: formic acid-water (1:1000); Mobile phase B: methanol; Gradient elution

[0061] Elution procedure (gradient elution):

[0062] Table 2

[0063]

[0064] Column temperature: 35℃

[0065] Injection volume: 5.0 μL

[0066] Scanning wavelength: 220nm (200-400nm for 3D scanning)

[0067] Mass spectrometry conditions

[0068] Scan range (m / z): 100-1500 positive and negative ion scan

[0069] Spray voltage: (+) 3500V (-) 3000V;

[0070] Monitoring mode: Full MS;

[0071] Auxiliary airflow velocity: 10 Arb;

[0072] Sheath flow velocity: 50 Arb;

[0073] Evaporation temperature: 300℃;

[0074] Ion transfer tube temperature: 325℃;

[0075] Solution preparation: Take 49.68 mg of 60% ethanol extract of senna leaves, add 3 mL of ultrapure water to dissolve, mix well, and filter through a 0.22 μm aqueous microporous membrane to obtain a sample solution of 16.56 mg / mL.

[0076] The main chemical components of the 60% ethanol extract of senna leaves, which exhibits strong antibacterial activity, were analyzed and identified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The total ion chromatograms (TIC) of the test solution in positive and negative ion modes are shown below. Figure 1 As shown in Table 3, by analyzing fragment ion information from secondary mass spectrometry and comparing it with literature data, a total of 26 compounds were identified, including 17 flavonoids and 9 anthraquinones.

[0077] Table 3

[0078]

[0079]

[0080]

[0081] Example 2

[0082] This embodiment demonstrates an antibacterial test of the combination of senna leaf alcohol extract and antibiotics. The specific process is as follows:

[0083] The standard strain ATCC 700392 was selected as the experimental subject. A combined antimicrobial susceptibility test was performed using the micro-broth checkerboard dilution method. The bacterial suspension, senna leaf extract solution, and antibiotic solution were mixed at a volume ratio of 2:1:1 and inoculated into 96-well plates, with a final total volume of 150 μL per well. The plates were placed in a tri-gas incubator and cultured under microaerophilic conditions at 37°C and 150 rpm for 72 h with shaking. The lowest drug concentration at which the culture medium in the wells became completely clear upon visual inspection was defined as the minimum combined inhibitory concentration (MIC). The FICI (Fractional Inhibitory Concentration Index) was calculated as follows:

[0084] FICI = (MIC FXY combine / MIC FXY alone )+(MIC FXY antibiotics combine / MIC FXY antibiotics alone )

[0085] When FICI ≤ 0.5, it is a synergistic effect; when 0.5 < FICI ≤ 1, it is an additive effect; when 1 < FICI ≤ 4, it is an indifferent effect; when FICI > 4, it is an antagonistic effect.

[0086] The results are shown in Table 4 as follows:

[0087] Table 4

[0088]

[0089] Note: MTZ: Metronidazole, CLR: Clarithromycin, LEF: Levofloxacin, AMO: Amoxicillin

[0090] It can be seen that the alcoholic extract of senna leaf shows an indifferent effect with other antibiotics, but shows an additive effect with amoxicillin and a synergistic antibacterial effect on the standard strain of Helicobacter pylori.

[0091] Example 3

[0092] This example studies the bactericidal effect of the alcoholic extract of senna leaf combined with amoxicillin on drug-resistant Helicobacter pylori. The specific process is as follows:

[0093] Select the H. pylori amoxicillin-resistant strains isolated clinically for the experiment. Scrape the H. pylori in the logarithmic growth phase, resuspend it in 20% FBS - BHI, adjust the turbidity to the 1 McFarland standard, and after diluting 10 times, mix the bacterial liquid and the medicinal liquid at a volume ratio of 1:1 and add them into a 6-well plate. The drug concentrations in each well are 160 μg / mL alcoholic extract of senna leaf, 1.6 μg / mL amoxicillin, and 160 μg / mL alcoholic extract of senna leaf + 1.6 μg / mL amoxicillin. Place the well plate in a triple-gas incubator and culture it at 37°C, microaerophilic, and 150 rpm with shaking. Sampling is carried out at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h respectively. Take 50 μL of the culture solution from each well, dilute it 10-fold serially (10 - 10 8 ) and then coat it on a blood agar plate respectively, culture it in an incubator for 4 - 5 d, take it out, photograph it with a colony counter and record the number of single colonies. The number of H. pylori is expressed as Lg(CFU / mL), and a time-killing curve is drawn. The criterion for judging the synergistic effect is that compared with the most effective single-drug group, the decrease in CFU / mL in the combined group is ≥ 2 - log10. The results are as Figure 2 shown.

[0094] For the H. pylori amoxicillin-resistant strains, using amoxicillin at the MIC concentration alone cannot completely kill the bacteria within 72 h, while combining it with the alcoholic extract of senna leaf can achieve complete bactericidal effect within 48 h. It is particularly noteworthy that at 36 h, the number of colonies in the combined group decreased by 2.513 - log10 compared with the group using the alcoholic extract of senna leaf alone, indicating that the two have a synergistic antibacterial effect.

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of senna leaf alcohol extract in the preparation of drugs for the prevention and treatment of multidrug-resistant Helicobacter pylori infection.

2. The application according to claim 1, characterized in that: The multidrug-resistant Helicobacter pylori includes Helicobacter pylori resistant to at least two of clindamycin, metronidazole, levofloxacin, and amoxicillin.

3. The application according to claim 1, characterized in that: The working concentration of the senna leaf alcohol extract is 150–600 μg / mL.

4. The use of a composition of senna leaf alcohol extract and amoxicillin in the preparation of a drug for Helicobacter pylori infection.

5. The application according to claim 4, characterized in that: In the composition, the mass ratio of senna leaf extract to amoxicillin is (70-150):

1.

6. The application according to claim 4, characterized in that: In the composition, the working concentration of senna leaf extract is 50–600 μg / mL; in the composition, the working concentration of amoxicillin is 0.02–3.0 μg / mL.

7. The application according to claim 4, characterized in that: The Helicobacter pylori mentioned includes susceptible Helicobacter pylori and / or drug-resistant Helicobacter pylori.

8. The application according to any one of claims 1 to 7, characterized in that: The senna leaf alcohol extract contains at least one of flavonoid glycosides, flavonoid aglycones, anthraquinone glycosides, free anthraquinones, and phenolic glycosides.

9. The application according to any one of claims 8, characterized in that: The senna leaf alcohol extract is a senna leaf ethanol extract; the concentration of the ethanol is 50-75%.

10. The application according to claim 1 or 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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