Medical uses of Patchouli leaf extract

Abstract

The present invention belongs to the field of medicine and relates to the medical use of Patchouli leaf extract. Specifically, the present invention relates to the use of Patchouli leaf extract in the preparation of anti-influenza drugs. Patchouli leaf extract has a significant anti-influenza virus effect, low cytotoxicity, and good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the medical application of an extract of Patchouli vulgaris leaves. Background Art

[0002] Influenza virus, also known as flu virus, is an RNA virus that causes influenza in humans and animals and belongs to the family Orthomyxoviridae. Influenza virus causes acute upper respiratory tract infection and spreads rapidly through the air. According to the objects infected by influenza virus, the virus can be divided into human influenza virus, swine influenza virus, equine influenza virus and avian influenza virus. Influenza virus can be divided into four categories according to the antigenicity of its nucleoprotein: influenza A virus, influenza B virus, influenza C virus and influenza D virus.

[0003] Among the three influenza viruses that infect humans, influenza A virus has extremely strong variability and has caused global pandemics many times, followed by influenza B virus, while influenza C virus has very stable antigenicity. The high variability of influenza A virus increases the difficulty of people dealing with influenza. People cannot accurately predict the subtype of the virus that will be prevalent, and cannot carry out preventive vaccination in a targeted manner; in addition, mutations in the viral genome have produced a high level of resistance to anti-influenza virus drugs.

[0004] The immunogenicity of influenza vaccines is generally lower in high-risk groups such as the elderly and immunocompromised subjects. Therefore, anti-influenza drugs are an important option for treating seasonal influenza infection cases. There are currently three main types of anti-influenza virus drugs, namely neuraminidase inhibitors (oseltamivir, laninamivir octanoate, zanamivir, peramivir), M2 protein inhibitors (amantadine, rimantadine) and cap-dependent endonuclease inhibitors (baloxavir). Oseltamivir can inhibit neuraminidase activity and prevent mature viruses from leaving host cells. Amantadine and rimantadine can inhibit influenza virus M2 ion channel inhibitors to block viral replication. Baloxavir can inhibit transcription, making it impossible for viral mRNA to be synthesized, thereby preventing viral replication. In addition to treatment for influenza viruses, more treatments are aimed at the symptoms caused by influenza viruses, including non-steroidal anti-inflammatory drugs, which can relieve influenza symptoms but cannot shorten the course of the disease.

[0005] It is worth noting that the incidence of drug resistance of antiviral drugs currently used in clinical practice is increasing, and their side effects are becoming increasingly severe. During the 2005 influenza outbreak, the US Centers for Disease Control and Prevention recommended that M2 inhibitors should not be used as much as possible due to high drug resistance. In Japan, oseltamivir is prohibited for use in children and adolescents aged 10-19 years due to concerns about neurological side effects.

[0006] Patchouli is the above-ground part of the herbaceous plant Pogostemincablin (Blanco) Benth. or Patchouli Agastacherugosus (Fisch. et Mey) O.Ktze. of the Lamiaceae family. It is a commonly used aromatic dampness-removing Chinese medicine in clinical practice. It is pungent in taste and slightly warm in nature. It has the effects of aromatic dampness-removing, harmonizing the stomach and stopping vomiting, and relieving heatstroke. It is used to treat dampness blocking the middle jiao, abdominal distension and vomiting, abdominal pain and diarrhea, summer dampness and external symptoms, the initial onset of damp-heat, fever and fatigue. It has anti-inflammatory, antibacterial, and gastrointestinal function regulation pharmacological effects. Patchouli is often used as medicine in its dried above-ground part. In addition to being a prescription drug for traditional Chinese medicine, it is also the main component of many Chinese patent medicines, such as Huoxiang Zhengqi Water (pills / capsules), Xiangsha Yangwei Pills, Huodan Pills, and antiviral oral liquid.

[0007] There is still a need to develop new anti-influenza virus drugs. Summary of the invention

[0008] According to the pathogenic characteristics of influenza virus and in combination with the characteristics of traditional Chinese medicine, the inventors designed a corresponding in vitro antiviral pharmacodynamic test based on an in vitro cell virus infection model. Four doses of 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL were designed for the water extract of Patchouli leaves and the alcohol extract of Patchouli leaves, respectively, to observe their effects on influenza A virus H1N1 cytopathic effects, cell survival rate, and inhibition of viral replication, and to evaluate their antiviral pharmacodynamic effects. The inventors were surprised to find that the water extract of Patchouli leaves and the alcohol extract of Patchouli leaves have significant anti-influenza virus effects, low cytotoxicity, and good application prospects. The following invention is thus provided:

[0009] One aspect of the present invention relates to use of Patchouli leaf or Patchouli leaf extract in preparing anti-influenza medicaments.

[0010] In some embodiments of the present invention, in the use, the Patchouli leaf extract is a Patchouli leaf water extract or a Patchouli leaf ethanol extract.

[0011] In some embodiments of the present invention, in the use, the Patchouli leaf water extract is an extract obtained by heating and refluxing Patchouli leaf and water.

[0012] In some embodiments of the present invention, the use, wherein the Agastache rugosa leaf water extract is prepared by a method comprising the following steps:

[0013] (1) mixing Patchouli leaves with water in a ratio of 1 g: 5-30 mL, and heating and refluxing for extraction once or multiple times (e.g., 2 times, 3 times, 4 times, or 5 times);

[0014] (2) filtering, and combining the filtrates if multiple heating and reflux extractions are performed;

[0015] (3) concentrating the filtrate to a viscous state to obtain a viscous product;

[0016] Preferably, the method further comprises the following steps:

[0017] (4) Drying the viscous product to obtain a dry product.

[0018] The viscous product in step (3) or the dried product in step (4) is an aqueous extract of Patchouli leaves.

[0019] Preferably, in step (1), the ratio of Patchouli leaf to water is 1 g: 5-20 mL, 1 g: 5-15 mL, or 1 g: 10 mL.

[0020] Preferably, the time of heating reflux extraction in step (1) is 0.1-5 hours, 0.2-4 hours, 0.3-3 hours, 0.2-2 hours, 0.5-1.5 hours, 0.8-1.2 hours or 1 hour.

[0021] Preferably, the Patchouli leaves are dried Patchouli leaves and are crushed and then passed through a 10-mesh sieve.

[0022] In some embodiments of the present invention, the use described, wherein the Patchouli leaf ethanol extract is an extract obtained by heating and refluxing extraction of Patchouli leaves and ethanol or ethanol solution; preferably, the ethanol solution is 20%-80% ethanol solution, 30%-70% ethanol solution, 40%-70% ethanol solution, 50%-70% ethanol solution, 55%-65% ethanol solution or 60% ethanol solution.

[0023] In some embodiments of the present invention, the use, wherein the ethanol extract of Patchouli Leaves is prepared by a method comprising the following steps:

[0024] (1) mixing Patchouli leaves with ethanol or 10%-90% ethanol solution at a ratio of 1 g: 5-30 mL, and heating and refluxing for extraction once or multiple times (e.g., 2 times, 3 times, 4 times, or 5 times);

[0025] (2) filtering, and combining the filtrates if multiple heating and reflux extractions are performed;

[0026] (3) concentrating the filtrate to a viscous state to obtain a viscous product;

[0027] Preferably, the method further comprises the following steps:

[0028] (4) Drying the viscous product to obtain a dry product.

[0029] The viscous product in step (3) or the dry product in step (4) is an ethanol extract of Patchouli leaves.

[0030] Preferably, in step (1), the ratio of Patchouli leaf to ethanol or ethanol solution is 1 g: 5-20 mL, 1 g: 5-15 mL, or 1 g: 10 mL.

[0031] Preferably, the ethanol solution in step (1) is 20%-80% ethanol solution, 30%-70% ethanol solution, 40%-70% ethanol solution, 50%-70% ethanol solution, 55%-65% ethanol solution or 60% ethanol solution.

[0032] Preferably, the time of heating reflux extraction in step (1) is 0.1-5 hours, 0.2-4 hours, 0.3-3 hours, 0.2-2 hours, 0.5-1.5 hours, 0.8-1.2 hours or 1 hour.

[0033] Preferably, the Patchouli leaves are dried Patchouli leaves and are crushed and then passed through a 10-mesh sieve.

[0034] Preferably, the concentration is rotary evaporation concentration.

[0035] Preferably, the drying is vacuum drying.

[0036] In some embodiments of the present invention, the use, wherein the anti-influenza is the treatment or prevention of influenza.

[0037] In some embodiments of the present invention, the use, wherein the anti-influenza is the inhibition of influenza virus; preferably, the influenza virus is influenza A virus; more preferably, it is influenza A virus H1N1 subtype; particularly preferably, it is influenza A virus H1N1 FM1 strain or influenza A virus H1N1 PR8 strain.

[0038] In some embodiments of the present invention, in the use, Patchouli leaf or Patchouli leaf extract (such as Patchouli leaf water extract or Patchouli leaf ethanol extract) is used as the sole active ingredient.

[0039] Another aspect of the present invention relates to an anti-influenza pharmaceutical composition, which comprises an extract of Patchouli Leaves as an active ingredient, and one or more pharmaceutically acceptable excipients, such as carriers or excipients; specifically, it is in the form of an injection, oral solution, capsule, tablet, granule, pill or extract remix.

[0040] Conventional processing methods can be used to prepare dosage forms such as injection, oral solution, capsule, tablet, granule, pill, and extract remix containing the Patchouli leaf extract and excipients.

[0041] The pharmaceutical composition of the present invention generally contains 0.1-90% by weight of the active ingredient. The pharmaceutical composition can be prepared according to methods known in the art. When used for this purpose, if necessary, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare a suitable administration form or dosage form for human use.

[0042] The pharmaceutical composition of the present invention can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum. The dosage form is, for example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make the unit dosage form into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to make the dosing unit into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to make the dosing unit into a capsule, the active ingredient is mixed with the various carriers mentioned above, and the resulting mixture is placed in a hard capsule or soft capsule. The active ingredient can also be made into a microcapsule, suspended in an aqueous medium to form a suspension, or can be loaded into a hard capsule or made into an injection for use. In order to prepare the dosing unit into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.

[0043] In addition, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0044] The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the influenza to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the route of administration and the number of administrations, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms. The dosage level must be selected based on the specific route of administration, the severity of the condition to be treated, and the condition and previous medical history of the patient to be treated. However, the practice in the art is to start the dosage from a level lower than that required to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained.

[0045] However, it should be recognized that the total daily dosage of the pharmaceutical composition of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex and diet; administration time, route of administration and excretion rate; duration of treatment; drugs used in combination or simultaneously; and similar factors known in the medical field. For example, the practice in the art is to start the administration of a dose lower than the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained. In general, the dosage of the pharmaceutical composition of the present invention calculated as the active ingredient for mammals, especially humans, can be between 100-2000 mg / kg body weight / day, such as between 100-1000 mg / kg body weight / day, such as between 200-500 mg / kg body weight / day, or 300 mg / kg body weight / day.

[0046] In some embodiments of the present invention, in the anti-influenza pharmaceutical composition, the Patchouli leaf extract is the Patchouli leaf water extract described in any one of the present invention or the Patchouli leaf ethanol extract described in any one of the present invention.

[0047] In some embodiments of the present invention, the anti-influenza pharmaceutical composition comprises Patchouli leaves or Patchouli leaf extract (eg, Patchouli leaf water extract or Patchouli leaf ethanol extract) as the sole active ingredient.

[0048] In the present invention, the term "inhibiting influenza virus" or "anti-influenza virus" refers to, but is not limited to, one or more of the following: improving influenza virus cytopathic effect, increasing the survival rate of host cells infected by influenza virus, inhibiting influenza virus replication, and the like.

[0049] In the present invention, unless otherwise specified, the concentration of ethanol is expressed as volume percentage (v / v%).

[0050] In the present invention, unless otherwise specified, Patchouli leaf refers to Patchouli leaf medicinal material or dried Patchouli leaf.

[0051] Advantageous Effects of the Invention

[0052] The present invention achieves one or more of the following technical effects (1) to (4):

[0053] (1) Both the water extract and the alcohol extract of Patchouli leaves have significant inhibitory effects on influenza viruses, especially significantly improving the cytopathic effects of influenza A virus H1N1 FM1 strain and PR8 strain, increasing the survival rate of virus-infected host cells, and effectively inhibiting viral replication;

[0054] (2) The three dose groups of 100 μg / mL, 10 μg / mL, and 1 μg / mL of Agastache rugosa leaf water extract and Agastache rugosa leaf alcohol extract showed a good dose-effect relationship;

[0055] (3) The water extract and alcohol extract of Patchouli leaf have lower cytotoxicity;

[0056] (4) The anti-influenza effect of using the extract of Patchouli leaves is significantly better than the anti-influenza effect of using extracts from other parts of Patchouli (such as the aerial parts, stems, roots or the whole plant). BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A to 1G :Effects of Agastache rugosa leaf water extract on MDCK cell pathogenesis induced by influenza A virus H1N1 FM1 (representative images). Figure 1A Normal control; Figure 1B Model control; Figure 1C Agastache rugosa leaf water extract 100 μg / mL; Figure 1D Agastache rugosa leaf water extract 10 μg / mL; Figure 1E Agastache rugosa leaf water extract 1 μg / mL; Figure 1F Agastache rugosa leaf water extract 0.1 μg / mL; Figure 1G Tamiflu (39.06 μg / mL).

[0058] Figure 2 :Effect of Agastache rugosa leaf water extract on the survival rate of MDCK cells induced by influenza A virus H1N1 FM1. Note: drug concentration μg / mL; compared with the model control group, *P<0.05, ** P<0.01.

[0059] Figure 3 :Effect of Agastache rugosa leaf water extract on the viral load of influenza A virus H1N1 FM1. Note: drug concentration μg / mL; n=4; compared with the model control group, ** P<0.01.

[0060] FIG. 4A to FIG. 4G :Effects of Agastache rugosa leaf aqueous extract on influenza A virus H1N1 PR8-induced A549 cell pathological changes (representative images). Figure 4A Normal control; Figure 4B Model control; Figure 4C Agastache rugosa leaf water extract 100 μg / mL; Figure 4D Agastache rugosa leaf water extract 10 μg / mL; Figure 4E Patchouli leaf water extract 1 μg / mL; F Patchouli leaf water extract 0.1 μg / mL; Figure 4G Tamiflu (39.06 μg / mL).

[0061] Figure 5 :Effect of Agastache rugosa leaf water extract on the survival rate of A549 cells infected with influenza A virus H1N1 PR8. Note: Drug concentration μg / mL; compared with the model control group, ** P<0.01.

[0062] Figure 6 :Effect of Agastache rugosa leaf water extract on the viral load of influenza A virus H1N1 PR8. Note: drug concentration μg / mL; n=4; compared with the model control group, ** P<0.01.

[0063] FIG. 7A to FIG. 7G :Effects of ethanol extract of Patchouli Leaves on MDCK cell pathogenesis induced by influenza A virus H1N1 FM1 (representative images). Fig. 7A Normal control; Figure 7B Model control; Figure 7C Patchouli leaf ethanol extract 100 μg / mL; Fig.7D Patchouli leaf ethanol extract 10 μg / mL; Fig. 7E Patchouli leaf ethanol extract 1 μg / mL; Figure 7F Patchouli leaf ethanol extract 0.1 μg / mL; Figure 7G Tamiflu (39.06 μg / mL).

[0064] Figure 8 :Effect of ethanol extract of Patchouli Leaves on the survival rate of MDCK cells induced by influenza A virus H1N1 FM1. Note: Drug concentration μg / mL; compared with the model control group, * P<0.05, **P<0.01.

[0065] Fig. 9 :Effect of ethanol extract of Patchouli Leaves on viral load of influenza A virus H1N1 FM1. Note: drug concentration μg / mL; n=4; compared with the model control group, * P<0.05, ** P<0.01.

[0066] FIG. 10A to FIG. 10G :Effects of ethanol extract of Patchouli Leaves on the cytopathic effect of influenza A virus H1N1 PR8 induced in A549 cells (representative images). Fig. 10A Normal control; Fig. 10B Model control; Fig. 10C Patchouli leaf ethanol extract 100 μg / mL; Fig. 10D Patchouli leaf ethanol extract 10 μg / mL; Fig. 10E Patchouli leaf ethanol extract 1 μg / mL; Fig.10F Patchouli leaf ethanol extract 0.1 μg / mL; Figure 10G Tamiflu (39.06 μg / mL).

[0067] Fig.11 :Effect of ethanol extract of Patchouli Leaves on the survival rate of A549 cells infected with influenza A virus H1N1 PR8. Note: Drug concentration μg / mL; compared with the model control group, ** P<0.01.

[0068] Fig.12 :Effect of ethanol extract of Patchouli Leaves on viral load of influenza A virus H1N1 PR8. Note: drug concentration μg / mL; n=4; compared with the model control group, * P<0.05, ** P<0.01.

[0069] FIG. 13A to FIG. 13B :Toxic effect of Agastache rugosa leaf water extract on MDCK cells cultured in vitro (×20). Fig.13A , Agastache rugosa leaf aqueous extract: stock solution (50 mg / mL); Fig. 13B , Agastache rugosa leaf water extract: TC0 (1:128).

[0070] FIG. 14A to FIG. 14B :Toxic effect of Agastache rugosa leaf water extract on A549 cells cultured in vitro (×20). Fig.14A , Agastache rugosa leaf aqueous extract: stock solution (50 mg / mL); Fig. 14B , Agastache rugosa leaf water extract: TC0 (1:256).

[0071] FIG. 15A to FIG. 15B :Toxic effect of Agastache rugosa alcohol extract on MDCK cells cultured in vitro (×20). Fig.15A, Agastache rugosa leaf ethanol extract: stock solution (50 mg / mL); Fig. 15B , ethanol extract of Patchouli leaves: TC0 (1:128).

[0072] FIG. 16A to FIG. 16B :Toxic effect of alcohol extract of Patchouli sinensis leaves on A549 cells cultured in vitro (×20). Fig.16A , Agastache rugosa leaf ethanol extract: stock solution (50 mg / mL); Fig. 16B , ethanol extract of Patchouli leaves: TC0 (1:256). DETAILED DESCRIPTION

[0073] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0074] 1. Test materials

[0075] 1.1 Cells

[0076] As shown in Table A below.

[0077] Table A

[0078]

[0079] 1.2 Virus strains

[0080] See Table B below.

[0081] Table B

[0082]

[0083] 1.3 Reagents

[0084] See Table C below.

[0085] Table C

[0086]

[0087]

[0088] 1.4 Instruments

[0089] See Table D below.

[0090] Table D

[0091]

[0092]

[0093] 1.5 Drugs under investigation

[0094] The Agastache rugosa leaf water extract prepared in Preparation Example 1.

[0095] Preparation Example 2 prepared Patchouli leaf ethanol extract.

[0096] 1.6 Positive control drug:

[0097] Oseltamivir phosphate capsules (Tamiflu), produced by Delpharm Milano Srl, Italy, and packaged by Shanghai Roche Pharmaceuticals Co., Ltd.; production batch number: M1066; packaging batch number: SH0089; specification: 75 mg / capsule, 10 capsules / box; properties: gray and light yellow capsules, the contents are white to yellow-white powder; storage conditions: stored below 25°C; production date: 2019.06.05, valid until: 2024.06.04.

[0098] 2. General Experimental Methods

[0099] 2.1 Dosage design

[0100] Patchouli leaf extract: Patchouli leaf water extract and Patchouli leaf alcohol extract were calculated according to the extract concentrate, and four doses of 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL were set respectively. During the experiment, the extract concentrate powder was diluted to the corresponding concentration with cell maintenance medium (2% fetal bovine serum + DMEM culture medium).

[0101] Tamiflu: The lowest dilution that does not cause obvious pathological changes on the cells involved in this experiment is determined as the maximum non-toxic concentration (TC0), and TC0 and the next 2-3 concentrations are used as the drug concentrations of the positive control wells. In this experiment, the TC0 of Tamiflu on MDCK and A549 cells was 78.125μg / mL.

[0102] 2.2 Statistical analysis

[0103] GraphPad Prism 6.0 software was used for data analysis. The differences among the groups were analyzed using t-test, and P < 0.05 was considered statistically significant.

[0104]

[0105]

[0106] Preparation Example 1: Preparation of Agastache rugosa leaf water extract

[0107] Patchouli leaf medicinal material was purchased from Tianjin Chinese Medicine Pieces Co., Ltd., produced in Guangdong. Patchouli leaf was crushed by a grinder and passed through a 10-mesh sieve with an inner diameter of 2 mm.

[0108] The preparation steps are as follows:

[0109] (1) Weigh 800 g of Patchouli leaves, mix the Patchouli leaves with pure water at a ratio of 1 g of Patchouli leaves to 10 mL of pure water, heat and reflux for extraction for 1 h, cool to room temperature, and filter to obtain a filtrate.

[0110] (2) Repeat the previous step (1) once;

[0111] (3) The filtrates from the two extractions were combined, concentrated by rotary evaporation to a viscous state, and then vacuum dried to obtain an aqueous extract of Patchouli leaf.

[0112] Preparation Example 2: Preparation of Patchouli Leaf Alcohol Extract

[0113] Patchouli leaf medicinal material was purchased from Tianjin Chinese Medicine Pieces Co., Ltd., produced in Guangdong. Patchouli leaf was crushed by a grinder and passed through a 10-mesh sieve with an inner diameter of 2 mm.

[0114] The preparation steps are as follows:

[0115] (1) Weigh 800 g of Patchouli leaves, mix the Patchouli leaves with an ethanol solution having a volume percentage concentration of 60% in a ratio of 1 g of Patchouli leaves to 10 mL of ethanol solution, heat and reflux for extraction for 1 h, cool to room temperature, and filter; obtain a filtrate

[0116] (2) Repeat the previous step (1) once;

[0117] (3) The filtrates from the two extractions were combined, concentrated by rotary evaporation to a viscous state, and then vacuum dried to obtain an aqueous extract of Patchouli leaf.

[0118] Example 1: Effect of Agastache rugosa leaf aqueous extract on cytopathic activity of influenza A virus H1N1 FM1 strain

[0119] 1. Experimental Methods

[0120] Take the MDCK cell culture plate that has grown into a monolayer, pour out the culture medium, rinse the cell surface with cell maintenance medium (2% fetal bovine serum + DMEM culture medium) for 3 times, and then inoculate 100 TCID 50 Influenza A virus H1N1 FM1 virus solution, 100 μL / well, placed in a 37℃ 5% CO2 incubator for 2 hours of adsorption, then discarded the virus solution, added 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL of four dilutions of Patchouli leaf water extract, 100 μL / well, each dilution of the solution set 4 replicates, and set up the following control groups:

[0121] Normal cell control group: only add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well); set up 8 replicate wells;

[0122] Model control group: add virus to infect cells, and after 2 hours of virus adsorption, add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well; set 8 replicate wells; and

[0123] Tamiflu control group: Add virus to infect cells, add Tamiflu solution 100 μL / well after virus adsorption for 2 hours, and the drug concentration is 39.06 μg / mL; set up 8 replicate wells.

[0124] Each group of cells was placed in a 37°C 5% CO2 incubator for continued culture. After 72 hours, the cell pathological changes were observed under an inverted microscope, the pathological results were recorded, the cell survival (CCK8) was tested, and the cell supernatant was aspirated to detect the viral load.

[0125] Cytopathic effect (CPE): refers to the phenomenon that a virus proliferates in large quantities in host cells, causing cell pathology or even death. Specifically, when cytolytic viruses are cultured in vitro, they replicate and proliferate in large quantities in susceptible cells, leading to cell death or cell rounding, shedding, aggregation, etc., which is called cytopathic effect.

[0126] The CPE method refers to the classification based on the cytopathic effect, which evaluates the degree to which the drug improves the virus-induced cytopathic effect, and is used as a basis for evaluating whether the drug has an in vitro antiviral effect. The cytopathic effect is graded according to a 6-level standard:

[0127] -: The cells grow normally and no lesions appear;

[0128] ±: cytopathic effect is less than 10% of the entire monolayer;

[0129] +: cytopathic cells account for less than 25% of the entire monolayer cells;

[0130] ++: cytopathic effects account for less than 50% of the entire monolayer of cells;

[0131] +++: cytopathic factors account for less than 75% of the entire monolayer cells;

[0132] ++++: Cytopathic changes account for more than 75% of the entire monolayer of cells.

[0133] 2. Experimental results

[0134] As shown in Table 1, Figures 1A to 1G , Figure 2 and Figure 3 shown.

[0135] Table 1: Effect of Agastache rugosa leaf water extract on MDCK cell pathological changes induced by influenza A virus H1N1 FM1 (CPE method)

[0136]

[0137] Note: " / " indicates: no drug intervention, only cell maintenance solution; "——" indicates: no lesions. The numbers "1, 2, 3" in Table 1 correspond to the cytopathic criteria "+, ++, +++", and "—" corresponds to the criteria "- or ±". The number represents the number of replicates set in this experimental group. The same below.

[0138] Table 1 and Figures 1A to 1G The results showed that after 72 hours of influenza A virus H1N1 FM1 infection of MDCK cells, the cells aggregated into clusters, shrank until they broke, and necrotized and fell off. After intervention with the water extract of Patchouli leaves, the cell lesions caused by the virus were significantly inhibited, with the best efficacy at 100μg / mL and 10μg / mL. The three doses of 100μg / mL, 10μg / mL, and 1μg / mL showed a good dose-effect relationship.

[0139] Figure 2 The results showed that after 72 hours of influenza A virus H1N1 FM1 infection of MDCK cells, the cells showed obvious pathological changes, even necrosis, and the number of viable cells decreased. After intervention with the water extract of Patchouli leaves, the cell survival rate increased, and the three dose groups of 100μg / mL, 10μg / mL, and 1μg / mL were statistically different from the model control group (p<0.01 or p<0.05).

[0140] Figure 3 The results showed that after MDCK cells were infected with influenza A virus H1N1 FM1, the viral load in the supernatant increased significantly, and no viral replication was detected in the normal control group (p<0.01). After 72 hours of intervention with the water extract of Patchouli leaves, the viral load in the cell supernatant of the 100μg / mL, 10μg / mL, and 1μg / mL dose groups was significantly reduced, and there were statistical differences compared with the model control group (p<0.01). The virus inhibition rates were 62.25%, 18.37%, and 21.81%, respectively, and the virus inhibition rate of the Tamiflu group was 90.17%.

[0141] Example 2: Effect of Agastache rugosa leaf aqueous extract on cytopathic activity of influenza A virus H1N1 PR8 strain

[0142] 1. Experimental Methods

[0143] Take the A549 cell culture plate that has grown into a monolayer, pour out the culture medium, rinse the cell surface with cell maintenance medium (2% fetal bovine serum + DMEM culture medium) for 3 times, and then inoculate 100 TCID 50Influenza A virus H1N1 PR8 virus solution, 100 μL / well, placed in a 37℃ 5% CO2 incubator for adsorption for 1 hour, discarded the virus solution, added 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL of four dilutions of Patchouli leaf water extract, 100 μL / well, each dilution of the solution set 4 replicates, and set up the following control groups:

[0144] Normal cell control group: only add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well); set up 8 replicate wells;

[0145] Model control group: add virus to infect cells, and after 2 hours of virus adsorption, add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well; set 8 replicate wells; and

[0146] Tamiflu control group: Add virus to infect cells, add Tamiflu solution 100 μL / well after virus adsorption for 2 hours, and the drug concentration is 39.06 μg / mL; set up 8 replicate wells.

[0147] Each group of cells was placed in a 37°C 5% CO2 incubator for continued culture. After 72 hours, the cell pathological changes were observed under an inverted microscope, the pathological results were recorded, the cell survival (CCK8) was tested, and the cell supernatant was aspirated to detect the viral load.

[0148] The cytopathicity judgment standard is as in Example 1.

[0149] 2. Experimental results

[0150] As shown in Table 2, FIG. 4A to FIG. 4G , Figure 5 and Figure 6 shown.

[0151] Table 2: Effect of Agastache rugosa leaf water extract on influenza A virus H1N1 PR8-induced A549 cell pathological changes (CPE method)

[0152]

[0153] Note: " / " indicates: no drug intervention, only cell maintenance solution; "——" indicates: no lesions. The numbers "1, 2, 3" in Table 1 correspond to the cytopathic criteria "+, ++, +++", and "—" corresponds to the criteria "- or ±". The numbers represent the number of replicate wells set in this experimental group.

[0154] Table 2 FIG. 4A to FIG. 4GThe results showed that after 72 hours of influenza A virus H1N1 PR8 infection of A549 cells, the entire cell shrank, became rounded and even broke, and the cells showed large-scale necrosis and shedding, and the cell density decreased. After intervention with the water extract of Patchouli leaves, both the 100μg / mL and 10μg / mL dose groups were able to significantly inhibit the virus-induced cell lesions and showed a good dose-effect relationship.

[0155] Figure 5 The results showed that after 72 hours of influenza A virus H1N1 PR8 infection of A549 cells, cells underwent necrosis and the number of viable cells decreased. After intervention with the water extract of Patchouli leaves, the cell survival rate increased, and the four dose groups of 100μg / mL, 10μg / mL, 1μg / mL, and 0.1μg / mL were statistically different from the model control group (p<0.01).

[0156] Figure 6 The results showed that after A549 cells were infected with influenza A virus H1N1 PR8, the viral load in the supernatant increased significantly, and no viral replication was detected in the normal control group (p<0.01). After 72 hours of intervention with the water extract of Patchouli leaves, the viral load in the cell supernatant of the 100μg / mL and 10μg / mL dose groups was significantly reduced, and there were significant differences compared with the model control group (p<0.01 or p<0.05). The virus inhibition rates were 29.37% and 8.91%, respectively, and the virus inhibition rate of the Tamiflu group was 35.64%.

[0157] Example 3: Effect of the alcohol extract of Patchouli Leaves on the cytopathic effect of influenza A virus H1N1 FM1 strain

[0158] 1. Experimental Methods

[0159] Take the MDCK cell culture plate that has grown into a monolayer, pour out the culture medium, rinse the cell surface with cell maintenance medium (2% fetal bovine serum + DMEM culture medium) for 3 times, and then inoculate 100 TCID 50 Influenza A virus H1N1 FM1 virus solution, 100 μL / well, placed in a 37℃ 5% CO2 incubator for 2 hours of adsorption, then discarded the virus solution, added 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL of four dilutions of Patchouli leaf alcohol extract, 100 μL / well, each dilution of the solution set 4 replicates, and set up the following control groups:

[0160] Normal cell control group: only add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well); set up 8 replicate wells;

[0161] Model control group: add virus to infect cells, and after 2 hours of virus adsorption, add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well; set 8 replicate wells; and

[0162] Tamiflu control group: Add virus to infect cells, add Tamiflu solution 100 μL / well after virus adsorption for 2 hours, and the drug concentration is 39.06 μg / mL; set up 8 replicate wells.

[0163] Each group of cells was placed in a 37°C 5% CO2 incubator for continued culture. After 72 hours, the cell pathological changes were observed under an inverted microscope, the pathological results were recorded, the cell survival (CCK8) was tested, and the cell supernatant was aspirated to detect the viral load.

[0164] The cytopathicity judgment standard is as in Example 1.

[0165] 2. Experimental results

[0166] As shown in Table 3, FIG. 7A to FIG. 7G , Figure 8 and Fig. 9 shown.

[0167] Table 3: Effect of ethanol extract of Patchouli leaves on MDCK cell pathological changes caused by influenza A virus H1N1 FM1 (CPE method)

[0168]

[0169] Note: " / " indicates: no drug intervention, only cell maintenance solution (2% fetal bovine serum + DMEM medium) is added; "——" indicates: no lesions. The numbers "1, 2, 3" in Table 1 correspond to the cytopathic judgment criteria "+, ++, +++", and "—" corresponds to the judgment criteria "- or ±". The number represents the number of replicate wells set in this experimental group.

[0170] Table 3 FIG. 7A to FIG. 7G The results showed that after 72 hours of influenza A virus H1N1 FM1 infection of MDCK cells, the cells aggregated into clusters, shrank until they broke, and necrotized and fell off. After intervention with the alcohol extract of Patchouli leaves, the cell lesions caused by the virus were significantly inhibited, and the drug efficacy was best at 100, 10, and 1 μg / mL. The three doses of 100 μg / mL, 10 μg / mL, and 1 μg / mL showed a good dose-effect relationship.

[0171] Figure 8 The results showed that after 72 hours of infection of MDCK cells with influenza A virus H1N1 FM1, the cells showed obvious pathological changes, even necrosis, and the number of viable cells decreased. After intervention with the alcohol extract of Patchouli leaves, the cell survival rate increased, and there were statistically significant differences between the 100μg / mL and 10μg / mL dose groups and the model control group (p<0.01 or p<0.05).

[0172] Fig. 9The results showed that after MDCK cells were infected with influenza A virus H1N1 FM1, the viral load in the supernatant increased significantly, and no viral replication was detected in the normal control group (p<0.01). After 72 hours of intervention with the alcohol extract of Patchouli leaves, the viral load in the cell supernatant of the 100μg / mL, 10μg / mL, and 1μg / mL dose groups was significantly reduced, and there were statistical differences compared with the model control group (p<0.01 or p<0.05). The virus inhibition rates were 57.34%, 8.48%, and 27.56%, respectively, and the virus inhibition rate of the Tamiflu group was 90.17%.

[0173] Example 4: Effect of the alcohol extract of Patchouli Leaves on the cytopathic effect of influenza A virus H1N1 PR8 strain

[0174] 1. Experimental Methods

[0175] Take the A549 cell culture plate that has grown into a monolayer, pour out the culture medium, rinse the cell surface with cell maintenance medium (2% fetal bovine serum + DMEM culture medium) for 3 times, and then inoculate 100 TCID 50 Influenza A virus H1N1 PR8 virus solution, 100 μL / well, placed in a 37℃ 5% CO2 incubator for adsorption for 1 hour, discarded the virus solution, added 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL of four dilutions of Patchouli leaf alcohol extract, 100 μL / well, each dilution solution set 4 replicates, and set up the following control groups:

[0176] Normal cell control group: only add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well); set up 8 replicate wells;

[0177] Model control group: add virus to infect cells, and after 2 hours of virus adsorption, add the same volume of cell maintenance solution as that of the drug-added group, 100 μL / well; set 8 replicate wells; and

[0178] Tamiflu control group: Add virus to infect cells, add Tamiflu solution 100 μL / well after virus adsorption for 2 hours, and the drug concentration is 39.06 μg / mL; set up 8 replicate wells.

[0179] Each group of cells was placed in a 37°C 5% CO2 incubator for continued culture. After 72 hours, the cell pathological changes were observed under an inverted microscope, the pathological results were recorded, the cell survival (CCK8) was tested, and the cell supernatant was aspirated to detect the viral load.

[0180] The cytopathicity judgment standard is as in Example 1.

[0181] 2. Experimental results

[0182] As shown in Table 4, FIG. 10A to FIG. 10G , Fig.11 and Fig.12 shown.

[0183] Table 4: Effect of ethanol extract of Patchouli Leaves on the cytopathic effect of influenza A virus H1N1 PR8 on A549 cells (CPE method)

[0184]

[0185] Note: " / " indicates: no drug intervention, only cell maintenance solution (2% fetal bovine serum + DMEM medium) is added; "——" indicates: no lesions. The numbers "1, 2, 3" in Table 1 correspond to the cytopathic judgment criteria "+, ++, +++", and "—" corresponds to the judgment criteria "- or ±". The number represents the number of replicate wells set in this experimental group.

[0186] Table 4 FIG. 10A to FIG. 10G The results showed that after 72 hours of influenza A virus H1N1 PR8 infection of A549 cells, the entire cell shrank, became rounded and even broke, and the cells showed large-scale necrosis and shedding, and the cell density decreased. After intervention with the alcohol extract of Patchouli leaves, both the 100μg / mL and 10μg / mL dose groups could significantly inhibit the virus-induced cell lesions, and showed a good dose-effect relationship.

[0187] Fig.11 The results showed that after influenza A virus H1N1 PR8 infected A549 cells for 72 hours, a large number of cells underwent necrosis and the number of viable cells decreased. After intervention with the alcohol extract of Patchouli leaves, the cell survival rate increased, and the three dose groups of 100, 10, and 1 μg / mL were statistically different from the model control group (p<0.01).

[0188] Fig.12 The results showed that after A549 cells were infected with influenza A virus H1N1 PR8, the viral load in the supernatant increased significantly, and no viral replication was detected in the normal control group (p<0.01). After 72 hours of intervention with the alcohol extract of Patchouli leaves, the viral load in the cell supernatant of the 100μg / mL and 1μg / mL dose groups was significantly reduced, and there was a significant difference compared with the model control group (p<0.01 or p<0.05). The viral load in the cell supernatant of the 10μg / mL dose group showed a downward trend, and there was no statistical difference compared with the model control group (p>0.05). The virus inhibition rates of the two dose groups of Patchouli leaves alcohol extract 100 and 1μg / mL were 16.37% and 9.64%, respectively, and the virus inhibition rate of the Tamiflu group was 35.64%.

[0189] The experimental results of Examples 1 to 4 above show that the Agastache rugosa leaf water extract and the Agastache rugosa leaf alcohol extract have significant inhibitory effects on influenza virus, can significantly improve the cytopathic effects of influenza A virus H1N1 FM1 strain and PR8 strain, increase the survival rate of virus-infected host cells, effectively inhibit virus replication, and the three dosage groups of 100 μg / mL, 10 μg / mL, and 1 μg / mL show a good dose-effect relationship. It can be seen that the Agastache rugosa leaf water extract and the Agastache rugosa leaf alcohol extract have significant antiviral effects on influenza virus, the effectiveness of the water extract and the alcohol extract are not much different, both have particularly significant pharmacodynamic effects, and have good application prospects.

[0190] Example 5: Cytotoxicity test

[0191] 1. Experimental Methods

[0192] The tested drugs (the original concentration of Agastache rugosa leaf water extract and Agastache rugosa leaf alcohol extract were both 50 mg / mL) were diluted with culture medium in multiple ratios of (1:2) to (1:2048), and then added to the MDCK and A549 cell culture plates (96 wells) that had grown into a monolayer, 100 μL / well, and 4 replicate wells were made for each dilution. At the same time, a normal cell control group (no virus and no drug, 4 wells) was set up. The culture plates were placed in a 37°C, 5% CO2 incubator for culture, and the cell pathological changes were observed under an inverted microscope every day. The lowest dilution multiple at which the cells did not show obvious pathological changes was determined to be the maximum non-toxic concentration (TC0), and the 50% cytotoxic concentration (TC) was calculated according to the Reed-Muench method. 50 ).

[0193] 2. Experimental results

[0194] like FIG. 13A to FIG. 16B shown.

[0195] The results showed that after 72 hours of exposure to Agastache rugosa leaves water extract and Agastache rugosa leaves alcohol extract, the TC0 on MDCK cells was 390.63 μg / mL, and the TC 50 The TC0 of A549 cells was 195.31 μg / mL, and the TC 50 The results showed that the water extract and alcohol extract of Patchouli leaves showed low cytotoxicity to MDCK (NBL-2) and A549 cells.

[0196] The water extract and alcohol extract of Patchouli leaf showed low cytotoxicity on MDCK and A549 cells, with TC0 ranging from 195.31 to 390.63 μg / mL and TC 50 It is between 281.17 and 561.01 μg / mL. It has been reported that the TC0 of Tamiflu on A549 and MDCK cells is 78.125 μg / mL, and the TC 50The values ​​of both were 140.92 μg / mL. It can be seen that the Patchouli leaf water extract and Patchouli leaf alcohol extract of the present invention have lower cytotoxicity.

[0197] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of Patchouli leaf extract as the sole active ingredient in the preparation of an anti-influenza drug, wherein: The Patchouli leaf extract is a Patchouli leaf water extract or a Patchouli leaf ethanol extract; in, The Patchouli leaf water extract is an extract obtained by extracting Patchouli leaf and water through heating and refluxing, and is prepared by a method comprising the following steps: (1) Mix Patchouli leaves with water in a ratio of 1 g: 5-30 mL, and heat and reflux for extraction once or multiple times; (2) Filtration. If multiple heating and reflux extractions are performed, the filtrates are combined; (3) concentrating the filtrate to a viscous state to obtain a viscous product; and (4) drying the viscous product to obtain a dry product; The Patchouli leaf ethanol extract is an extract obtained by heating and refluxing Patchouli leaf with ethanol or an ethanol solution, and is prepared by a method comprising the following steps: ① Mix Patchouli leaves with ethanol or 10%-90% ethanol solution in a ratio of 1 g: 5-30 mL, and heat under reflux for extraction once or multiple times; ② Filter. If multiple heating and reflux extractions are performed, combine the filtrates; ③ Concentrating the filtrate to a viscous state to obtain a viscous product; and ④ Drying the viscous product to obtain a dry product; The anti-influenza agent is to inhibit influenza virus, and the influenza virus is influenza A virus.

2. The use according to claim 1, wherein The influenza virus is influenza A virus H1N1 subtype.

3. The use according to claim 1, wherein The influenza virus is influenza A virus H1N1 FM1 strain or influenza A virus H1N1 PR8 strain.

4. An anti-influenza A virus pharmaceutical composition comprising an extract of Patchouli Leaves as the sole active ingredient and one or more pharmaceutically acceptable excipients, wherein: The Patchouli leaf extract is the Patchouli leaf water extract or the Patchouli leaf ethanol extract as described in claim 1.

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