Dammarane type triterpenes, preparation method thereof and anti-infection application of dammarane type triterpenes

Abstract

The invention discloses a dammarane type triterpenoid compound or a pharmaceutically acceptable salt thereof. The dammarane type triterpenoid compound has a structural formula as shown in a formula I which is described in the specification. The triterpenoids which are extracted and separated from a gynostemma pentaphyllum medicinal material and have a new chemical structure have an obvious anti-infection effect, the application range of gynostemma pentaphyllum can be expanded by developing the triterpenoids into medicines for treating infection-related diseases and symptoms, and the utilization value of gynostemma pentaphyllum plant resources is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to dammarane-type triterpenes, their preparation methods, and their use in anti-infection. Background Art

[0002] With the extensive application of antibiotics, the variation of bacterial structures and morphologies has led to an increasing number of bacteria developing resistance to antibiotics, and the degree of resistance is intensifying day by day. Currently, methicillin-resistant Staphylococcus aureus (MRSA) infections, hepatitis B virus (HBV), and AIDS are listed as the three major intractable infectious diseases worldwide. Methicillin-resistant Staphylococcus aureus is resistant to a variety of antibiotics, and it has strong pathogenicity and poor treatment effects, being one of the main pathogenic bacteria causing hospital clinical infections. Due to the strong pathogenicity and fast transmission speed of methicillin-resistant Staphylococcus aureus (MRSA), and its frequent emergence of multi-drug resistance, its dissemination in hospitals can lead to outbreaks in the wards. When the human body's resistance is low, it takes advantage of the opportunity to invade, causing suppurative inflammation in human tissues and organs and even leading to critical fatal diseases such as meningitis, endocarditis, and septicemia.

[0003] Clinically, although vancomycin can be used to treat infections caused by methicillin-resistant Staphylococcus aureus, in recent years, after vancomycin has been regarded as a conventional antibiotic in more and more hospitals, resistance has gradually emerged. Currently, to slow down the growth rate of methicillin-resistant Staphylococcus aureus resistance, in addition to restricting the abuse of antibiotics to slow down the growth rate of resistant MRSA, it is necessary to research and develop new drugs for treating diseases caused by methicillin-resistant Staphylococcus aureus as a backup to prevent large outbreaks of methicillin-resistant Staphylococcus aureus diseases. Therefore, it is extremely urgent to screen and develop antibiotics that can effectively resist methicillin-resistant Staphylococcus aureus infections.

[0004] Gynostemma pentaphyllum Makino., also known as Gynostemma pentaphyllum, Gospel Herb, etc., is a plant of the genus Gynostemma in the family Cucurbitaceae. Among them, gypenosides are the main active components in Gynostemma pentaphyllum. Pharmacological experiments and clinical applications have shown that it has effects such as reducing blood lipids, anti-tumor, protecting the liver, and enhancing the body's immune function. It is mainly used to treat the proliferation of cancer cells such as liver cancer, lung cancer, uterine cancer, and skin cancer. In addition, it has effects such as nourishing the body, sedation, hypnosis, anti-tension, promoting appetite, reducing cholesterol, and reducing transaminase in the human body.

[0005] When treating bacterial infectious diseases with Chinese herbal medicines, its greatest advantage is that it is not easy to produce drug resistance, and the toxic and side effects of traditional Chinese medicines are relatively small, with a wide range of antibacterial effects. After being absorbed by the body, it can enhance the body's ability to resist pathogenic microorganisms.

[0006] The inventor of the present invention has discovered through research that a class of triterpenoid compounds with a completely new chemical skeleton structure in Gynostemma pentaphyllum have good anti-infection effects and may have broad prospects in the preparation of drugs for preventing or / and treating diseases and disorders related to infection. The triterpenoid compounds in Gynostemma pentaphyllum itself have a wide range of pharmacological activities, are derived from natural plants, are safe and reliable, and there are currently no reports on the use of the triterpenoid compounds involved in the present invention for treating or / and preventing diseases and disorders related to infection. Summary of the Invention

[0007] The main object of the present invention is to provide a drug for treating diseases and disorders related to infection with definite curative effects, few side effects, and natural safety, so as to solve the problems of common drugs in the prior art being prone to drug resistance, having large toxic and side effects, and having a single bacteriostatic effect.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided a dammarane-type triterpenoid compound or a pharmaceutically acceptable salt thereof, and the dammarane-type triterpenoid compound has a structural formula shown in Formula I:

[0009]

[0010] Furthermore, the dammarane-type triterpenoid compound is obtained by hydrolyzing the ethanol extract of Gynostemma pentaphyllum medicinal materials by an acid hydrolysis method.

[0011] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned dammarane-type triterpenoid compound, and the method comprises the following steps:

[0012] (1) Weigh an appropriate amount of Gynostemma pentaphyllum medicinal materials, add ethanol for extraction and then concentrate to obtain the ethanol extract of Gynostemma pentaphyllum medicinal materials;

[0013] (2) Add an acid solution with a certain volume ratio to the ethanol extract of Gynostemma pentaphyllum medicinal materials, reflux for a period of time, filter, and collect the precipitate; and

[0014] (3) Subject the precipitate to silica gel column chromatography, elute with a certain mobile phase, and recrystallize the eluate to obtain the dammarane-type triterpenoid compound.

[0015] Furthermore, in step (1), the volume percentage concentration of the ethanol is 40% to 60%, for example, about 50%.

[0016] Furthermore, in step (1), the volume / mass (L / Kg) ratio of the ethanol to the Gynostemma pentaphyllum medicinal materials is 1 to 5, for example, about 2.

[0017] Furthermore, in step (1), the extraction method is reflux extraction, ultrasonic extraction, and / or shaking extraction, for example, reflux extraction.

[0018] Further, in step (1), the extraction time is 0.5 - 3 h, such as about 2 h.

[0019] Further, in step (1), the concentration is carried out under reduced pressure.

[0020] Further, in step (1), the volume / volume ratio of the ethanol extract of Gynostemma pentaphyllum to the ethanol is 0.05 - 0.2, such as about 0.1.

[0021] Further, in step (2), the acid is selected from one or more of hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, acetic acid, propionic acid, malonic acid, and butyric acid, such as hydrochloric acid.

[0022] Further, in step (2), the volume percentage concentration of the acid is 0.5% - 3%, such as about 1%.

[0023] Further, in step (2), the volume / volume ratio of the acid to the ethanol extract of Gynostemma pentaphyllum is 0.1 - 1, such as about 0.2.

[0024] Further, in step (2), the reflux time is 0.5 - 3 h, such as about 1 h.

[0025] Further, in step (3), the column chromatography is carried out using silica gel with a particle size of 100 - 400 mesh.

[0026] Further, in step (3), the elution mobile phase for the column chromatography is a mixed solvent of cyclohexane - ethyl acetate with a volume ratio of (15 - 35):(0.1 - 2).

[0027] Further, in step (3), the elution mobile phase for the column chromatography is a mixed solvent of cyclohexane - ethyl acetate with a volume ratio of about 25:about 1.

[0028] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above dammarane - type triterpenoid compound.

[0029] Further, the pharmaceutical composition comprises one or more drugs and / or extracts for anti - infection.

[0030] Further, the extract is a traditional Chinese medicine extract.

[0031] Further, the drug is selected from one or more of the following: antibiotics, antifungal drugs, antiviral drugs, anti - parasitic drugs, and traditional Chinese patent medicines.

[0032] Further, the antibiotic is selected from one or more of the following: β-lactam antibiotics, quinolone antibiotics, macrolide antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, glycopeptide antibiotics, oxazolidinone antibiotics, sulfonamide antibiotics, lincomycin antibiotics, polymyxin antibiotics, and cyclic lipopeptide antibiotics.

[0033] Further, the β-lactam antibiotics are cephalosporins, penicillins, cephamycins, carbapenems, monocyclic β-lactams, β-lactamase inhibitors, and / or β-lactamase inhibitor combinations.

[0034] Further, the quinolone antibiotics are levofloxacin, ciprofloxacin, and / or moxifloxacin.

[0035] Further, the macrolide antibiotics are azithromycin, clarithromycin, and / or roxithromycin.

[0036] Further, the aminoglycoside antibiotics are gentamicin, streptomycin, amikacin, and / or netilmicin.

[0037] Further, the tetracycline antibiotics are doxycycline and / or minocycline.

[0038] Further, the glycopeptide antibiotics are vancomycin and / or teicoplanin.

[0039] Further, the oxazolidinone antibiotic is linezolid.

[0040] Further, the lincomycin antibiotics are lincomycin and / or clindamycin.

[0041] Further, the polymyxin antibiotics are polymyxin B and / or colistin sulfate.

[0042] Further, the cyclic lipopeptide antibiotic is daptomycin.

[0043] Further, the antifungal drug is fluconazole and / or voriconazole.

[0044] Further, the antiviral drugs are ribavirin and / or ganciclovir.

[0045] Further, the anti-parasitic drugs are praziquantel and / or albendazole.

[0046] Further, the Chinese patent medicine is Shuanghuanglian Oral Liquid and / or Jinyinhua Oral Liquid.

[0047] Further, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0048] Further, the adjuvant is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained release agent, retention aid, lubricant, dispersant, plasticizer, light shielding agent, and antioxidant.

[0049] Further, the dosage form of the pharmaceutical composition is ointment, powder, tablet, dripping pill, capsule, film, lozenge, granule, injection or oral liquid.

[0050] Further, the ointment is an ointment or a plaster.

[0051] According to another aspect of the present invention, there is provided the use of the above dammarane-type triterpenoids or the above pharmaceutical composition in the preparation of a drug for anti-infection.

[0052] Further, the infection is a bacterial infection.

[0053] Further, the bacterium is methicillin-resistant Staphylococcus aureus.

[0054] Advantages of the present invention:

[0055] Gynostemma pentaphyllum is the dried whole herb of Gynostemma pentaphyllum (Thunb.) Makino of the genus Gynostemma in the family Cucurbitaceae. It is a perennial climbing herb, mainly distributed in China, Japan, Korea and other countries. Modern pharmacological research shows that its main active ingredient is gypenoside (Gyp). We found that a class of triterpenoids with a new chemical structure isolated from Gynostemma pentaphyllum medicinal materials has obvious anti-infection effects. Developing it into a drug for treating diseases and disorders related to infection can expand the application range of Gynostemma pentaphyllum and improve the utilization value of Gynostemma pentaphyllum plant resources. Description of the Drawings

[0056] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0057] Figure 1 It is a schematic diagram of the hydrogen spectrum of the compound of formula I of the present invention.

[0058] Figure 2 It is a schematic diagram of the carbon spectrum of the compound of formula I of the present invention. Detailed Embodiments

[0059] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0060] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the art in the field of the present invention or the field in which the term is applied. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, the preferred methods, conditions, substances or materials are described herein.

[0061] The present invention is expected to cover all alternatives, variations and equivalents, which may be included in the field of the existing invention as defined by the claims. Those skilled in the art will recognize many methods and substances similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is not limited to the description of methods and substances.

[0062] The present invention will apply the following definitions unless otherwise indicated. For the purposes of the present invention, chemical elements are defined according to the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75th Ed., 1994. Additionally, general principles of organic chemistry are found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March’s Advanced Organic Chemistry", Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, and thus all content incorporates the references.

[0063] The singular forms "a", "an" and "the" as used in the specification and the appended claims include plural referents unless the context clearly dictates otherwise.

[0064] In the present invention, the term "comprising" is synonymous with "including". The terms "comprising", "including", "having", "containing" or any other variation thereof used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or apparatus containing the listed elements need not be limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article or apparatus.

[0065] As described in the background art section, common drugs currently used for treating diseases and disorders related to infections are prone to drug resistance, have large toxic and side effects, and have a single bacteriostatic effect. To solve the above problems, the present invention provides a dammarane-type triterpenoid compound or a pharmaceutically acceptable salt thereof, and the dammarane-type triterpenoid compound has a structural formula shown in Formula I:

[0066]

[0067] The term "salt" used in the present invention refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Suitable salts include all known pharmaceutically acceptable salts, including those formed with organic acids and inorganic acids. Thus, suitable salts include those formed by the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, lactic acid, valeric acid, acetic acid, succinic acid, fumaric acid, maleic acid, methanesulfonic acid, and benzenesulfonic acid.

[0068] In a preferred embodiment, the dammarane-type triterpenoid compound is obtained by hydrolyzing an ethanol extract of Gynostemma pentaphyllum herbs by an acid hydrolysis method.

[0069] According to another aspect of the present invention, a method for preparing the dammarane-type triterpenoid compound is provided, and the method includes the following steps:

[0070] (1) Weigh an appropriate amount of Gynostemma pentaphyllum herbs, add ethanol for extraction, and then concentrate to obtain an ethanol extract of Gynostemma pentaphyllum herbs;

[0071] (2) Add an acid solution with a certain volume ratio to the ethanol extract of Gynostemma pentaphyllum herbs, reflux for a period of time, filter, and collect the precipitate; and

[0072] (3) Subject the precipitate to silica gel column chromatography, elute with a certain mobile phase, and recrystallize the eluate to obtain the dammarane-type triterpenoid compound.

[0073] In a preferred embodiment, the volume percentage concentration of the ethanol in step (1) is 40% to 60%, for example, about 50%.

[0074] In the present invention, when a concentration, ratio, time, particle size, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed individually. For example, when the range "40% to 60%" is disclosed, the described range should be interpreted as including the ranges "40% to 60%", "40% to 55%", "40% to 50%", "40% to 45%", "45% to 60%", "45% to 55%", "45% to 50%", "50% to 60%", "50% to 55%", "55% to 60%", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range, and all numerical values within the range can achieve the effects of the present invention.

[0075] In a preferred embodiment, in step (1), the ratio of the volume / mass (L / Kg) of the ethanol to the Gynostemma pentaphyllum medicinal material is 1 to 5, for example, about 2.

[0076] In a preferred embodiment, in step (1), the extraction method is reflux extraction, ultrasonic extraction, and / or shaking extraction, for example, reflux extraction.

[0077] In a preferred embodiment, in step (1), the extraction time is 0.5 to 3 h, for example, about 2 h.

[0078] In a preferred embodiment, in step (1), the concentration is carried out under reduced pressure.

[0079] In a preferred embodiment, in step (1), the ratio of the volume / volume of the Gynostemma pentaphyllum ethanol extract to the ethanol is 0.05 to 0.2, for example, about 0.1.

[0080] In a preferred embodiment, in step (2), the acid is selected from one or more of hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, acetic acid, propionic acid, malonic acid, and butyric acid, for example, hydrochloric acid.

[0081] In a preferred embodiment, in step (2), the volume percentage concentration of the acid is 0.5% to 3%, for example, about 1%.

[0082] In a preferred embodiment, in step (2), the ratio of the volume / volume 1 of the acid to the Gynostemma pentaphyllum ethanol extract is 0.1 to 1, for example, about 0.2.

[0083] In a preferred embodiment, in step (2), the reflux time is 0.5 to 3 h, for example, about 1 h.

[0084] In a preferred embodiment, in step (3), the column chromatography uses silica gel with a particle size of 100 to 400 mesh.

[0085] In a preferred embodiment, in step (3), the elution mobile phase of the column chromatography is a mixed solvent of cyclohexane - ethyl acetate with a volume ratio of (15 - 35):(0.1 - 2).

[0086] In a preferred embodiment, in step (3), the elution mobile phase of the column chromatography is a mixed solvent of cyclohexane - ethyl acetate with a volume ratio of approximately 25: approximately 1.

[0087] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21; "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0088] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned dammarane - type triterpenoids.

[0089] In a preferred embodiment, the pharmaceutical composition comprises one or more drugs and / or extracts for anti - infection.

[0090] In a preferred embodiment, the extract is a traditional Chinese medicine extract.

[0091] In a preferred embodiment, the drug is selected from one or more of the following: antibiotics, antifungal drugs, antiviral drugs, anti - parasitic drugs, and traditional Chinese patent medicines.

[0092] In a preferred embodiment, the antibiotic is selected from one or more of the following: β - lactam antibiotics, quinolone antibiotics, macrolide antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, glycopeptide antibiotics, oxazolidinone antibiotics, sulfonamide antibiotics, lincomycin antibiotics, polymyxin antibiotics, and cyclic lipopeptide antibiotics.

[0093] In a preferred embodiment, the β - lactam antibiotics are cephalosporins, penicillins, cephamycins, carbapenems, monocyclic β - lactams, β - lactamase inhibitors, and / or β - lactamase inhibitor combinations.

[0094] In a preferred embodiment, the quinolone antibiotic is levofloxacin, ciprofloxacin, and / or moxifloxacin.

[0095] In a preferred embodiment, the macrolide antibiotic is azithromycin, clarithromycin, and / or roxithromycin.

[0096] In a preferred embodiment, the aminoglycoside antibiotic is gentamicin, streptomycin, amikacin, and / or netilmicin.

[0097] In a preferred embodiment, the tetracycline antibiotic is doxycycline and / or minocycline.

[0098] In a preferred embodiment, the glycopeptide antibiotic is vancomycin and / or teicoplanin.

[0099] In a preferred embodiment, the oxazolidinone antibiotic is linezolid.

[0100] In a preferred embodiment, the lincomycin antibiotic is lincomycin and / or clindamycin.

[0101] In a preferred embodiment, the polymyxin antibiotic is polymyxin B and / or colistin sulfate.

[0102] In a preferred embodiment, the cyclic lipopeptide antibiotic is daptomycin.

[0103] In a preferred embodiment, the antifungal drug is fluconazole and / or voriconazole.

[0104] In a preferred embodiment, the antiviral drug is ribavirin and / or ganciclovir.

[0105] In a preferred embodiment, the anti-parasitic drug is praziquantel and / or albendazole.

[0106] In a preferred embodiment, the Chinese patent medicine is Shuanghuanglian oral liquid and / or Jinyinhua oral liquid.

[0107] In a preferred embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0108] The above dammarane-type triterpenoid compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient are used in an effective dose for producing a pharmaceutical composition. The dose of the active compound may vary depending on the route of administration, the age and weight of the patient, the nature and severity of the disease to be treated, and similar factors.

[0109] The pharmaceutical composition of the compound of the present invention can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac and intracranial injection or infusion, or administration by means of an implantable reservoir. Among them, oral, topical, intraperitoneal or intravenous administration methods are preferred.

[0110] In a preferred embodiment, the pharmaceutical composition of the present invention contains at least one pharmaceutically acceptable excipient in an amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.% based on the total weight of the pharmaceutical composition.

[0111] In the present invention, the term "pharmaceutically acceptable" herein refers to a substance, such as a carrier or diluent, that does not abolish the biological activity or properties of the compound and is relatively non-toxic, e.g., administering a substance to an individual does not cause unwanted biological effects or interact in a harmful manner with any of its contained components.

[0112] In the present invention, the term "pharmaceutically acceptable excipient" refers to carriers and / or excipients that are compatible (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects) with the subject and the active ingredient pharmacologically and / or physiologically, and which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0113] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, opacifying agents and antioxidants.

[0114] Those skilled in the art will know how to select specific chemical substances within the scope of the above excipients. For example, the diluent can be selected from one or more of the following: powdered sugar, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate, and calcium carbonate. The wetting agent can be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polysorbate such as polysorbate 80, cetyl alcohol, glycerol fatty acid ester (such as triacetin, glycerol monostearate, and the like), polyoxyethylene fatty acid ester, polyethylene glycol fatty acid ester, sodium lauryl sulfate, sorbitan fatty acid ester, sucrose fatty acid ester, polyoxyethylene ether, benzalkonium chloride, polyoxyethylene castor oil, and sodium dioctyl sulfosuccinate. The binder can be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol, and methyl cellulose. The disintegrant can be selected from one or more of the following: carboxymethyl cellulose, calcium carboxymethyl cellulose salt, and sodium carboxymethyl cellulose. The flavoring agent can be selected from one or more of the following: sorbitol, glucose, mannose, sucrose, and lactose. The dispersant can be selected from one or more of the following: sodium croscarmellose, sodium starch glycolate, and pregelatinized corn starch. The plasticizer can be dibutyl sebacate and / or various citric acid esters. The sustained-release agent can be selected from one or more of the following: sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, hydroxyethyl cellulose, gum arabic, gelatin, and shellac. The antioxidant can be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite, and sodium thiosulfate. The lubricant can be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid, and colloidal silica.

[0115] These excipients are preferably pharmaceutically inert, or may have a synergistic or enhancing effect to enhance the therapeutic activity of the pharmaceutical composition, and the above excipients are only illustrative. The excipients actually used in the present invention are not limited to the above excipients and can be adjusted according to actual circumstances, and all can achieve the effects of the present invention.

[0116] In a preferred embodiment, the dosage form of the pharmaceutical composition is an ointment, powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0117] In a preferred embodiment, the ointment is an ointment or a plaster.

[0118] The above drugs, excipients, and dosage forms of the present invention are only illustrative. In actual use, they are not limited to those listed above. As long as they can be used in combination with the pharmaceutical composition of the present invention, they are within the protection scope of the present invention.

[0119] According to another aspect of the present invention, there is provided the use of the dammarane-type triterpenoid compound or the pharmaceutical composition in the preparation of a medicament for anti-infection.

[0120] According to another aspect of the present invention, there is provided the use of the dammarane-type triterpenoid compound or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating infection.

[0121] In a preferred embodiment, the infection is a bacterial infection.

[0122] In a preferred embodiment, the bacterium is methicillin-resistant Staphylococcus aureus.

[0123] In the present invention, the term "treatment" also includes "prevention" unless there is a specific indication to the contrary. The terms "treatment(al)" and "treatment(ly)" should be understood accordingly.

[0124] In the present invention, the term "treatment" includes alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; improving or preventing potential metabolic syndromes; inhibiting the occurrence of a disease or symptoms, such as controlling the development of a disease or condition; alleviating a disease or symptoms; reducing a disease or symptoms; relieving the complications caused by a disease or symptoms, or preventing or treating the signs caused by a disease or symptoms. As used herein, a pharmaceutical composition or a pharmaceutical preparation, after administration, can improve a disease, symptom or condition, especially the severity thereof, delay the onset, slow down the progression of the disease, or reduce the duration of the disease. Whether administered regularly or temporarily, continuously or intermittently, it can be attributed to or related to the administration.

[0125] The present invention also provides the above composition or the above pharmaceutical preparation for preventing and / or treating infection in a subject, such as a bacterial infection.

[0126] The present invention also provides a method for preventing and / or treating infection in a subject, such as a bacterial infection, comprising administering to the subject an effective amount of the above composition or the above pharmaceutical preparation.

[0127] In the present invention, the term "subject" is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses or cows, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects representing a bacterial infection model. Preferably, the subject is a human.

[0128] An "effective amount" of the pharmaceutical composition or preparation used in the present invention can achieve the desired therapeutic and / or prophylactic effect. The amount effective for this use will depend, for example, on the pharmaceutical composition, the mode of administration, the stage and severity of the disease being treated, the individual body weight and overall health status, and the judgment of the prescribing physician. The dose can be administered once a week, or once every two days or daily, or even several times a day. The dose unit can be administered over a short period (e.g., several weeks to several months) or a longer period (several months to several years).

[0129] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or the conditions recommended by the manufacturer.

[0130] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0131] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All the features disclosed in this patent specification can be used in combination with any composition form. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0132] Examples

[0133] Preparation Examples

[0134] 25 Kg of Gynostemma pentaphyllum medicinal materials were reflux-extracted with 50 L of 50% ethanol for 2 hours, concentrated under reduced pressure to 5 L, added with 1 L of 1% hydrochloric acid aqueous solution, refluxed for 1 hour, filtered, the precipitate was collected, subjected to silica gel column chromatography, eluted with cyclohexane-ethyl acetate (25:1), and recrystallized to obtain 1.5 g of Gypensapogenin V.

[0135] The 1H NMR spectrum of Gypensapogenin V (as Figure 1 shown) and the 13C NMR spectrum (as Figure 2 shown) data:

[0136]

[0137] 11H NMR (400 MHz, CDCl3) δH: 5.91 (t, J = 4.2 Hz, 21-H, 1H), 4.41 (d, J = 4.7 Hz, 1-H, 1H), 3.86 (m, 3-H, 1H), 3.14 (td, J = 11.1, 6.2 Hz, 14-H, 1H), 1.28 (s, 3H), 1.27 (s, 3H), 1.16 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H).

[0138] 13 13C NMR (100 MHz, CDCl3) δC: 201.05 (C-21), 150.01 (C-24), 132.82 (C-10), 130.04 (C-5), 106.90 (C-23), 83.97 (C-3), 74.35 (C-25), 73.87 (C-1), 48.36 (C-14), 47.14 (C-17), 44.99 (C-13), 39.17 (C-4), 38.319 (C-8), 37.53 (C-9), 32.62 (C-19), 32.09 (C-20), 32.00 (C-15), 31.19 (C-26), 27.12 (C-28), 26.57 (C-16), 26.46 (C-7), 25.97 (C-12), 25.30 (C-11), 24.01 (C-2), 22.53 (C-18), 20.28 (C-6), 19.01 (C-22), 15.09 (C-29), 12.64 (C-30).

[0139] Pharmacological activity evaluation

[0140] Determination of in vivo antibacterial activity of Gypensapogenin V

[0141] 1 Experimental purpose

[0142] To study the in vivo antibacterial activity of Gypensapogenin V by using the mouse infection protection experiment.

[0143] 2 Experimental materials

[0144] 2.1 Experimental drugs

[0145] 2.1.1 Positive control drug

[0146] Shuanghuanglian Oral Liquid, Henan Tailong Pharmaceutical Co., Ltd., batch number 150528052.

[0147] 2.1.2 Test drug

[0148] Gypensapogenin V was prepared according to the above-mentioned examples.

[0149] 2.2 Experimental animals

[0150] 2.2.1 Source, species, strain, certificate: ICR mice, SPF grade, purchased from the Experimental Animal Center of Zunyi Medical University, animal production license number: SCXK(Qian)2019-0001.

[0151] 2.2.2 Body weight: 18 - 22 g.

[0152] 2.2.3 Gender: Half male and half female.

[0153] 2.2.4 Quantity: 60.

[0154] 2.2.5 Feeding: The animals were raised at 25 ± 3 °C, relative humidity 60 - 75%, with a 12-hour day-night light-dark cycle, free access to food and water, and used for experiments after one week of adaptive feeding.

[0155] 2.3 Experimental materials

[0156] 2.3.1 Culture medium

[0157] MH broth medium, MH agar medium, Beijing Sanyao Science and Technology Development Company, prepared according to the formula and autoclaved.

[0158] 2.3.2 Test strains

[0159] According to the results of in vitro antibacterial experiments, 1 strain of methicillin-resistant Staphylococcus aureus (MRSA), numbered MRSA1, was selected, provided by the Microbiology Laboratory of Jiangsu Provincial People's Hospital after clinical isolation and species identification, and stored in this laboratory at -80 °C.

[0160] 3 Experimental methods

[0161] 3.1 Animal grouping

[0162] The 60 mice were randomly divided into 6 groups, with 10 mice in each group.

[0163] ① Blank control group: Intragastrically administered with the solvent 0.5% CMC-Na solution.

[0164] ② High-dose group of the test drug Gypensapogenin V: Intragastrically administered with 21.0 mg / kg / d Gypensapogenin V solution.

[0165] ③ Medium-dose group of the test drug Gypensapogenin V: Intragastrically administered with 10.5 mg / kg / d Gypensapogenin V solution.

[0166] ④Low-dose group of the test drug Gypensapogenin V: Gypensapogenin V solution was administered by gavage at a dose of 5.25 mg / kg / d.

[0167] ⑤Positive drug Shuanghuanglian Oral Liquid group: Shuanghuanglian Oral Liquid was administered by gavage.

[0168] ⑥Model control group: 0.5% CMC-Na solution was administered by gavage as the solvent.

[0169] 3.2 Construction of a mouse systemic infection model

[0170] One strain of MRSA (MRSA1) was taken from a -80°C ultra-low temperature freezer and activated on an MH agar slant, then transferred to an MH broth medium and cultured with shaking at 37°C and 150 r / min for 12 h. The bacteria were collected by centrifugation at 4000 r / min for 10 min, appropriately diluted with sterile normal saline, and the OD value was measured and viable bacteria were counted to determine the concentration of the bacterial solution. The bacterial solution was serially diluted 10-fold with 5% gastrin prepared with normal saline into 10 9 ,10 8 ,10 7 ,10 6 CFU / mL bacterial solution, and a MRSA systemic infection model was established by intraperitoneal injection at 0.5 mL / animal. The minimum bacterial solution concentration that caused all mice to die was determined as the minimum lethal dose (100% MLD).

[0171] 3.3 In vivo protection test on MRSA-infected mice

[0172] The dosing amounts of the high, medium, and low-dose groups of Gypensapogenin V were 21.0 mg / kg, 10.5 mg / kg, and 5.25 mg / kg respectively, and the Shuanghuanglian group was 10 ml / kg. Mice in each group were administered by gavage at a volume of 0.1 ml / 10 g once a day for 5 consecutive days. The model control group and the blank control group were administered an equal volume of solvent. At 60 min after dosing on the 4th day, a lethal challenge was performed by intraperitoneal injection of the minimum lethal concentration bacterial solution at 0.5 ml / animal to establish a MRSA systemic infection model. The blank control group was given an equal volume of normal saline. The death situation of mice in each group was observed continuously for 7 d.

[0173] 4 Statistical treatment methods

[0174] The number of surviving mice after 7 d was used as the final statistical result. The significance of the difference in animal mortality rates between the Gypensapogenin V, Shuanghuanglian groups and the model control group was compared using statistical software by the T-test method, and P < 0.05 was considered statistically significant.

[0175] 5 Experimental results

[0176] 5.1 Construction of a mouse model of systemic infection

[0177] A mouse model of systemic MRSA infection was successfully constructed. All the mice in the systemic infection model died within 48 h when the bacterial suspension concentration was 1×10 9 CFU / mL.

[0178] 5.2 In vivo protection test on mice infected with MRSA

[0179] All the mice in the uninfected blank control group survived, and 90% of the mice in the model control group died within 48 h after infection (i.e., on the 5th - 6th day of the experiment), indicating that the model was established. Compared with the model group, the survival rates of the mice in the high, medium, and low dose groups of Gypensapogenin V and the Shuanghuanglian group showed an upward trend within 3 days after infection (P<0.05).

[0180] Table 1 Protective effect of Gypensapogenin V on the death of mice infected with methicillin - resistant Staphylococcus aureus

[0181]

[0182] *Compared with the model control group, *P<0.05.

[0183] 6 Experimental conclusions

[0184] Gypensapogenin V has a certain protective effect on the systemic infection of mice caused by MRSA, and its effect is equivalent to that of Shuanghuanglian.

[0185] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. At the same time, changes or deformations made by those skilled in the art based on the idea of the present invention, within the specific implementation manner and application scope of the present invention, all belong to the protection scope of the present invention. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A dammarane-type triterpenoid compound or a pharmaceutically acceptable salt thereof, characterized in that, The dammarane - type triterpenoid compound has the structural formula shown in Formula I:

2. The dammarane-type triterpenoid compound according to claim 1, wherein The dammarane - type triterpenoid compound is obtained by hydrolyzing the ethanol extract of Gynostemma pentaphyllum herbs through an acid hydrolysis method.

3. A method for preparing the dammarane-type triterpenoid compound according to claim 1 or 2, characterized in that, The method comprises the following steps: (1) Weigh an appropriate amount of Gynostemma pentaphyllum herbs, add ethanol for extraction, and then concentrate to obtain the ethanol extract of Gynostemma pentaphyllum herbs; (2) Add an acid solution with a certain volume ratio to the ethanol extract of Gynostemma pentaphyllum herbs, reflux for a period of time, filter, and collect the precipitate; and (3) Subject the precipitate to silica gel column chromatography, elute with a certain mobile phase, and recrystallize the eluate to obtain the dammarane - type triterpenoid compound.

4. The method according to claim 3, wherein In step (1), the volume percentage concentration of the ethanol is 40% - 60%, for example, about 50%; Preferably, in step (1), the volume / mass (L / Kg) ratio of the ethanol to the Gynostemma pentaphyllum herbs is 1 - 5, for example, about 2; More preferably, in step (1), the extraction method is reflux extraction, ultrasonic extraction, and / or shaking extraction, for example, reflux extraction; Also preferably, in step (1), the extraction time is 0.5 - 3 h, for example, about 2 h; Especially preferably, in step (1), the concentration is vacuum concentration; Specifically preferably, in step (1), the volume / volume ratio of the Gynostemma pentaphyllum ethanol extract to the ethanol is 0.05 - 0.2, for example, about 0.

1.

5. The method according to claim 3, wherein In step (2), the acid is selected from one or more of hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, acetic acid, propionic acid, malonic acid, and butyric acid, for example, hydrochloric acid; Preferably, in step (2), the volume percentage concentration of the acid is 0.5% - 3%, for example, about 1%; More preferably, in step (2), the volume / volume ratio of the acid to the Gynostemma pentaphyllum ethanol extract is 0.1 - 1, for example, about 0.2; Also preferably, in step (2), the reflux time is 0.5 - 3 h, for example, about 1 h.

6. The method according to claim 3, characterized in that, In step (3), the column chromatography uses silica gel with a particle size of 100 - 400 mesh; Preferably, in step (3), the elution mobile phase for the column chromatography is a mixed solvent with a volume ratio of cyclohexane - ethyl acetate of (15 - 35):(0.1 - 2); More preferably, in step (3), the elution mobile phase for the column chromatography is a mixed solvent with a volume ratio of cyclohexane - ethyl acetate of about 25: about 1.

7. A pharmaceutical composition comprising the dammarane - type triterpenoid compound according to claim 1 or 2; Preferably, the pharmaceutical composition comprises one or more drugs and / or extracts for anti - infection; More preferably, the extract is a traditional Chinese medicine extract.

8. The pharmaceutical composition according to claim 7, wherein The drug is selected from one or more of the following: antibiotics, antifungal drugs, antiviral drugs, anti - parasitic drugs, and traditional Chinese patent medicines; Preferably, the antibiotics are selected from one or more of the following: β - lactam antibiotics, quinolone antibiotics, macrolide antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, glycopeptide antibiotics, oxazolidinone antibiotics, sulfonamide antibiotics, lincomycin antibiotics, polymyxin antibiotics, and cyclic lipopeptide antibiotics; More preferably, the β-lactam antibiotics are cephalosporins, penicillins, cephamycins, carbapenems, monocyclic β-lactams, β-lactamase inhibitors and / or β-lactamase inhibitor combinations; Still more preferably, the quinolone antibiotics are levofloxacin, ciprofloxacin and / or moxifloxacin; Still more preferably, the macrolide antibiotics are azithromycin, clarithromycin and / or roxithromycin; Still more preferably, the aminoglycoside antibiotics are gentamicin, streptomycin, amikacin and / or netilmicin; Still more preferably, the tetracycline antibiotics are doxycycline and / or minocycline; Still more preferably, the glycopeptide antibiotics are vancomycin and / or teicoplanin; Still more preferably, the oxazolidinone antibiotic is linezolid; Still more preferably, the lincomycin antibiotics are lincomycin and / or clindamycin; Still more preferably, the polymyxin antibiotics are polymyxin B and / or colistin sulfate; Still more preferably, the cyclic lipopeptide antibiotic is daptomycin; Even more preferably, the antifungal drugs are fluconazole and / or voriconazole; Even more preferably, the antiviral drugs are ribavirin and / or ganciclovir; Even more preferably, the anti-parasitic drugs are praziquantel and / or albendazole; Even more preferably, the Chinese patent medicines are Shuanghuanglian oral liquid and / or Jinyinhua oral liquid.

9. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, light-blocking agents and antioxidants; More preferably, the dosage form of the pharmaceutical composition is an ointment, powder, tablet, dripping pill, capsule, film, lozenge, granule, injection or oral liquid; Still more preferably, the ointment is an ointment or a plaster.

10. Use of the dammarane-type triterpenoid compound according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of a medicament for anti-infection; Preferably, the infection is a bacterial infection; More preferably, the bacterium is methicillin-resistant Staphylococcus aureus.

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