CN103446152A - Application of Lycojaponicumin C in anti-Helicobacter pylori drug - Google Patents

Abstract

An in-vitro activity experiment shows that Lycojaponicumin C has high anti-Helicobacterpylori (Hp) activity. The Lycojaponicumin C can be applied to treatment of acute gastritis, chronic gastritis, stomach diseases, duodenal ulcer and the like and preparation of drugs for treating the acute gastritis, the chronic gastritis, the stomach diseases, the duodenal ulcer and the like. The application of the Lycojaponicumin C in preparation of an anti-Helicobacterpylori drug is disclosed for the first time. Due to the fact that the skeleton type is a novel skeleton type and the high anti-Helicobacterpylori inhibitory activity of the Lycojaponicumin C is previously unimagined, the possibility that other compounds give any revelation does not exist, and the Lycojaponicumin C has outstanding substantive characteristics and meanwhile has a remarkable improvement in prevention and treatment of Helicobacterpylori infection obviously.

Description

The application of Lycojaponicumin C in Anti-helicobacter pylori drugs

Technical field

The invention belongs to biological pharmacy technical field, relate in particular to the application of Lycojaponicumin C in preparing Anti-helicobacter pylori drugs.

Background technology

Helicobacter pylori (Helicobacter pylori, Hp) is a kind of Gram-negative spiral bacteria.Studies show that, helicobacter pylori is the main pathogenesis of acute and chronic gastritis and Stomach duodenum ulcer, and may be relevant with the morbidity of gastric cancer stomach function regulating mucosa-associated lymphoid tissue (MALT) malignant lymphoma.Recently, World Health Organization (WHO) is classified as I class carcinogen by Hp, and it plays a leading role in the gastric cancer development.The scheme that at present popular treatment Hp infects is to take the triple therapy that proton pump inhibitor (PPI) adds two kinds of antibiotic (clarithromycin, amoxicillin, tetracycline, metronidazole etc. select two kinds) simultaneously.The main factor that affects triple therapy is considered to the drug resistance of Hp to antibacterial; Another serious problems are that proton pump inhibitor can bring out dyspepsia, and a large amount of antibacterial cause the serious destruction of flora in digestive tract.Therefore, find the active kind new medicine thing of efficient, safe anti-Hp and become an important and urgent task.

The compound L ycojaponicumin C the present invention relates to is one and within 2012, delivers (Wang, X.J.et al., 2012.Lycojaponicumins A-C, Three Alkaloids with an Unprecedented Skeleton from Lycopodium japonicum.Organic Letters14 (10), 2614-2617.) New skeleton compound, this compound has brand-new framework types, purposes for the Lycojaponicumin C the present invention relates in preparation treatment Anti-helicobacter pylori drugs belongs to open first, because framework types belongs to brand-new framework types, and it suppresses active unexpectedly strong for helicobacter pylori, there do not is the possibility that is provided any enlightenment by other compounds, possesses outstanding substantive distinguishing features, control for helicobacter pylori infections simultaneously obviously has significant progress.

Summary of the invention

The objective of the invention is to study the application of Lycojaponicumin C in Anti-helicobacter pylori drugs.

Described compound L ycojaponicumin C-structure is as shown in formula I:

The experiment in vitro of Lycojaponicumin C shows, Lycojaponicumin C has very strong anti Helicobacter pylori activity, and paper disk method shows that its antibacterial circle diameter is 29mm (ATCC43504).Show that with agar dilution it can suppress the growth of 5 random clinical strains (Hp001, Hp003, Hp004, Hp018 and Hp036) and 1 reference culture (ATCC43504) fully, minimal inhibitory concentration (MIC) is 1.0 μ g/ml.Make positive control with ampicillin, its Cmin (MIC) that 6 strain test bacterium are suppressed fully is 0.5 μ g/ml.

This result of study shows, the energy force rate ampicillin of the inhibition helicobacter pylori activity of Lycojaponicumin C is strong, explanation for the diseases such as the closely-related acute and chronic gastritis of helicobacter pylori, duodenal ulcer, Lycojaponicumin C is the compound of a great exploitation potential for its.It can be directly used in the treatment of corresponding disease and the preparation of related drugs.

The purposes of the Lycojaponicumin C the present invention relates in preparation treatment Anti-helicobacter pylori drugs belongs to open first, because framework types belongs to brand-new framework types, and it suppresses active unexpectedly strong for helicobacter pylori, there do not is the possibility that is provided any enlightenment by other compounds, possess outstanding substantive distinguishing features, the control for helicobacter pylori infections simultaneously obviously has significant progress.

The specific embodiment

The preparation method of compound L ycojaponicumin C involved in the present invention is referring to document (Wang, X.J.et al., 2012.Lycojaponicumins A-C, Three Alkaloids with an Unprecedented Skeleton from Lycopodium japonicum.Organic Letters14 (10), 2614-2617.).

The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not subject to any restriction of specific embodiment, but be limited by claim.

Embodiment 1: the preparation of compound L ycojaponicumin C tablet involved in the present invention:

Get 20 and digest compound Lycojaponicumin C, add conventional adjuvant 180 grams that prepare tablet, mix, conventional tablet machine is made 1000.

Embodiment 2: the preparation of compound L ycojaponicumin C capsule involved in the present invention:

Get 20 and digest compound Lycojaponicumin C, add the conventional adjuvant for preparing capsule as starch 180 grams, mix, encapsulatedly make 1000.

Further illustrate its pharmaceutically active below by pharmacodynamic experiment.

The pharmacological evaluation of Lycojaponicumin C

1) strains tested

Helicobacter pylori (Helicobacter pylori, Hp) reference culture ATCC43504 is purchased from U.S.'s strain and preserves center (American Type Culture Collection, ATCC).15 strain Hp clinical strains are picked up from Jiangsu Prov. People's Hospital Gastroenterology dept., clinical laboratory of Jiangsu TCM Hospital and Nanjing Children's Hospital Dndoscope Laboratory and are accepted gastroscopic patient; In continuous gastroscopy to the patient of peptic ulcer, duodenal bulbar inflammation or gastritis verrucosa, first through the RUT experiment, be defined as the Hp positive, get again antral gastric mucosa 1-2 piece, after chopping, be inoculated in containing 8% horse serum, trimethoprim (trimethropin, TMP) in the Columbia selectivity agar culture medium of 1.25g/L, polymyxin (polymyxin) B2500U/L, vancomycin (vancomycin) 10mg/L, in 37 ℃ of (5%O under micro-oxygen environment ₂, 10%CO ₂and 85%N ₂) cultivate 72 hours.Collect antibacterial, through the smear Gram’s staining, after oxidase, catalase and urease are accredited as the positive, the pure culture of going down to posterity, obtained strains is as experimental strain.

2) strain culturing

We adopt micro-aerobic bag (purchased from Shanghai Medical Univ) to carry out the strain culturing of HP, and it can produce the needed micro-aerobic environment of Hp by chemical reaction.

3) biological activity determination

Adopt paper disk method (Microbiological paper method) to measure the inhibitory action of compound to helicobacter pylori, measure the minimum inhibitory concentration (minimal inhibitory concentration, MIC) of test sample with agar dilution.

I. paper disk method experiment

(A) prepare culture medium by the Columbia culture medium for preparing after high pressure steam sterilization, be cooled to 50-60 ℃, add 8% horse serum or Sheep Blood, mix in the culture dish that is poured into sterilizing, every ware 7-10ml, culture medium thickness is 1.5mm (sterile working).

(B) switching experimental bacteria (be coated with bacterium) with microscale sampler get diluted 10 ⁸cFU/ml (1OD ₆₆₀=10 ⁸cFU/ml) the bacteria suspension 0.1ml of Hp spreads upon suitable culture dish surface equably.Be inverted in 37 ℃ of drying bakers and take out after 15min, purpose makes the agar surface drying, standby.

(C) pasting the sample scraps of paper gets 6 μ l testing samples (mass concentration 2mg/ml) with microscale sampler and injects on the round filter paper of sterilizing.With the aseptic nipper tweezer containing the scraps of paper of sample and the blank scraps of paper of contrast, by sterile working respectively the scraps of paper be close to containing the bacterio-agar surface, paste at a certain distance a piece of paper sheet.Every kind of bacterium is cooked 3 wares, and acquired results is asked its meansigma methods.

(D) cultivate each plate is placed in to micro-aerobic bag, sealing, open gas generator, then be placed in 37 ℃ of incubators and cultivate 72h.

(E) after surveying antibacterial circle diameter taking-up flat board, measure respectively each scraps of paper size of antibacterial circle diameter on every side.With reference to the result of matched group, can draw the result of testing sample sensitive experiment.Triplicate.

II. agar dilution is measured MIC

(A) at first the preparation of medicine flat board becomes dimethyl sulfoxide for compound (DMSO) solution preparation of test the mother solution of 0.5mg/ml, then, with the sterilized water dilution, finally is made into 10.0,8.0,6.0,4.5,4.0,3.5,3.0,2.5,2.0,1.5,1.0,0.5, with the concentration series of 0.25 μ g/ml, the concentration of DMSO in medium is less than 1%.The test compounds solution that 1ml is prepared separately adds 1ml in the 9ml Colombia culture medium of 50 ℃ and is incubated in the horse serum of 50 ℃ and fully mixes with being incubated, and casts in culture dish cooling.

(B) switching experimental bacteria (be coated with bacterium) with microscale sampler draw diluted 1 * 10 ⁸the bacteria suspension 0.1ml of CFU/ml Hp spreads upon the culture dish surface equably, is inverted in 37 ℃ of drying bakers and takes out after 15min, and purpose makes the agar surface drying, standby.

(C) determine that MIC (contains culture dish to be measured: 85%N at micro-aerobic bag ₂, 10%CO ₂and 5%O ₂) in, be incubated 37 ℃ and cultivate 72 hours, observe the Hp growing state, with blank group contrast, take and there is no the sample of bacteria growing least concentration fully as minimum inhibitory concentration (minimal inhibitory concentration, MIC) value.Positive control is ampicillin (Ampicillin).

3, the pharmacological results of Lycojaponicumin C

Experiment in vitro shows, Lycojaponicumin C has very strong anti Helicobacter pylori activity, and paper disk method shows that its antibacterial circle diameter is 29mm (ATCC43504).Show that with agar dilution it can suppress the growth of 5 random clinical strains (Hp001, Hp003, Hp004, Hp018 and Hp036) and 1 reference culture (ATCC43504) fully, minimal inhibitory concentration (MIC) is 1.0 μ g/ml.Make positive control with ampicillin, its Cmin (MIC) that 6 strain test bacterium are suppressed fully is 0.5 μ g/ml.

Conclusion: the ability that Lycojaponicumin C suppresses the helicobacter pylori activity only than a little less than ampicillin some, explanation for the diseases such as the closely-related acute and chronic gastritis of helicobacter pylori, duodenal ulcer, Lycojaponicumin C is the compound of a great exploitation potential for its.It can be directly used in the treatment of corresponding disease and the preparation of related drugs.

Claims (1)

1.Lycojaponicumin the application of C in Anti-helicobacter pylori drugs, described compound L ycojaponicumin C-structure as formula Ishown in:

formula I.