AU2020103248A4

AU2020103248A4 - The preparation and application of antifungal extracts from Oxytropis falcate Bunge

Info

Publication number: AU2020103248A4
Application number: AU2020103248A
Authority: AU
Inventors: Erbu Aga; Changliang He; Yunkai Hu; Dandan HUANG; Fanglong LI; Xiaoxia Liang; Lizi Yin; Jinlin Zhang; Li Zhang
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-01-14
Anticipated expiration: 2028-11-05

Abstract

The invention relates to an effective extract from Oxytropis falcate Bunge with antifungal effect and its preparation method. The effective extract is selected from one of petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract, water extract and their combinations obtained by extraction from the alcohol extract of Oxytropis falcate Bunge. Studies have shown that petroleum ether extract, chloroform extract, ethyl acetate extract, n butanol extract and water extract have obvious antifungal ability, so the above effective extracts can be used to prepare antifungal drugs.

Description

The preparation and application of antifungal extracts from Oxytropis falcate

Bunge

TECHNICAL FIELD

[01] The invention discloses a preparation method of antifungal extract form Oxytropis falcate Bunge and its application in antifungal infection, belonging to the field of traditional Chinese medicine.

BACKGROUND

[02] Fungi are widely distributed microorganisms in nature, and are also common bacteria on the surface of human skin and mucosa. Under normal circumstances, they coexist with human beings. In recent years, with the increasing application of broad-spectrum antibacterial drugs, invasive operations, organ transplantation and chemotherapy drugs, the incidence of clinical fungal infection is on the rise. While antifungal drugs have been widely used in clinical practice for a long time, the drug resistance of fungi is becoming more and more common and the degree of drug resistance is getting higher and higher. Drug resistance has become the main reason for the failure of clinical treatment of antifungal drugs. At present, the structural types of antifungal drugs used clinically are limited, mainly polyene and triple-grade drugs. The mechanism of action and target are relatively single. Moreover, these antifungal drugs have various toxic and side effects and increasingly severe drug resistance problems. Therefore, people are still in urgent need of new antifungal drugs.

[03] The incidence rate of fungal skin diseases in pets (dogs, cats) and precious fur animals (foxes, raccoons, minks, etc.) has increased year by year, becoming a difficult problem in the pet market and fur animal special breeding industry. The main pathogenic sources of the disease are Microsporum canis, Microsporum gypsum and Trichophyton whiskers. It is a highly contagious infectious disease with strong infectivity and zoonosis, which directly poses a serious threat to human health and also causes huge economic losses to the aquaculture industry. However, at present, the treatment of animal fungal skin diseases is mainly the external ointment or spray of chemical antifungal drugs. In the treatment process, there are many problems such as strong toxicity, frequent use, short effect, easy drug resistance, etc. A new antifungal animal drug with high efficiency, non-toxicity and long effect is imminent.

[04] Oxytropis falcate Bunge is a medicinal plant of Oxytropis genus of Leguminosae. It is mainly produced in Qinghai, southern Gansu and western Sichuan of China. It grows in floodplain, sandy land, ravine, hillside, shrub forest and meadow with an altitude of 2700-4300m. It is rich in resources. Tibetan medicine is called "E Da Xia" and is one of the "three anti-inflammatory drugs" of Tibetan medicine. It enjoys the reputation of "King of Herbs". The root, rhizome or whole herb of the medicine are used as medicine, and the nature and taste of the medicine are pungent and cold, with small toxicity, and the meridian return is to the lung and spleen meridians. The medicine has the effects of clearing away heat and toxic materials, promoting granulation and healing sore, reducing swelling and pain, astringing pulse, stopping bleeding, relaxing bowels, etc. It can be taken orally to treat influenza, tonsillitis, laryngitis, tracheitis, anthracnose, leprosy, etc., and applied externally to treat sore, furuncle, swelling and pain, trauma, and bone injury pain. The folk are mostly used for detoxification, analgesia, knife wounds and other treatments with good curative effects. However, due to its toxic reaction, livestock have miscarriage, weak fetus, teratogenesis, etc. after eating, which has become one of the grassland toxic weeds. In recent years, Tibetan medicine Oxytropis falcate Bunge has attracted the attention of researchers due to its unique curative effect and unique ecology of plateau plants, and its research and application have increased year by year. A large number of studies have found that it has analgesic, anti-inflammatory, hemostatic, anti-tumor, expectorant and antiasthmatic effects, but there is no report on antifungal.

SUMMARY

[05] The object of the invention is to disclose an antifungal extract from Oxytropis falcate Bunge, and its preparation method and application in antifungal infection, so as to solve the above problems existing in the background art.

[06] The technical scheme provided by the invention is as follows:

[07] An active extract of Oxytropis falcate Bunge, characterized in that the active extract is selected from one of petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract obtained by extracting from Oxytropis falcate Bunge alcohol extract or their combination; The active extract is prepare by a method comprising that follow steps: (1) the coarse powder of Oxytropis falcate Bunge was mixed with ethanol, extracted under ultrasonic assisted extraction and filtered, repeating the above extraction with filter residue, filtering, and combining extract solutions; (2) the ethanol extract was concentrated under vacuum concentration to obtain a concentrated solution, the latter was freeze-dried and dispersed with distilled water; (3) gradient extraction is carry out with different polar solvents, petroleum ether, chloroform, ethyl acetate and n-butanol, each solvent is repeatedly extracted, the same organic phase and water phase are combined, and vacuum concentration is carried out to obtain petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract.

[08] The preferred technical scheme of the invention is as follows:

[09] (1) the coarse powder of OxytropisfalcateBunge was mixed with 20 times ethanol for 24h, extracted under ultrasonic aid for 1h, filtered; the filtering residue was repeated the extraction process for 2 times, then filtered, and combined all the extracts; the ethanol extract was concentrated under vacuum concentration at 55°C to obtain a concentrated solution, the latter was freeze-dried and dispersed with distilled water;

[010] (2) gradient extraction with different polar solvents such as petroleum ether, chloroform, ethyl acetate and n-butanol with the same volume was conducted and repeated for twice respectively, after combining the same organic phase and aqueous phase, the concentration under vacuum concentration to obtain petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract.

[011] The ethanol used in the process according to the invention is preferably 60 % ethanol.

[012] The invention also provides a pharmaceutical composition comprising an extract prepared by any of the above methods and a pharmaceutically acceptable carrier.

[013] The composition is optionally present in the form of a suitable pharmaceutical formulation selected from the group consisting of the tablets, capsules, oral liquid, mixture, oral agent, granule, granule, pill, powder, paste, Dan, suspension, solution, injection, powder injection, freeze-dried powder injection, suppository, ointment, hard paste, cream, spray, spray film, dripping pills, dripping pills and / or patches; The tablets are preferably sugar-coated tablets, film-coated tablets, enteric coated tablets or sustained-release tablets; The capsules are preferably hard capsules, soft capsules or sustained release capsules.

[014] The invention further provides the application of various extracts in preparing antifungal infection drugs; The fungus is selected from the group consisting of M. gypseum, T. mentagrophytes and/or M. canis. For this purpose, MIC was used as the activity index in antifungal experiments. The experimental results show that the petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract from Oxytropis falcate Bunge have different effects on external fungal infection, and can be used for the preparation of antifungal drugs.

BRIEF DESCRIPTION OF THE FIGURES

[015] Fig. 1: antifungal activity of total ethanol extract.

[016] Fig. 2: MIC with different polarity.

DESCRIPTION OF THE INVENTION

[017] EXAMPLE 1 Ethanol extract from Oxytropis falcate Bunge and its preparation of different polar part

[018] Oxytropis falcata Bunge (Oxytropis falcata Bunge) was pulverized properly, and the volume ratio of Oxytropis falcata Bunge to solvent was 1:20, soaked in 75% ethanol and extracted by ultrasonic aid for lh, and the filtrate was obtained by extraction and filtration. Repeat extraction of residue once, and combine the two filtrates. Then, the filtrate is concentrated under reduced pressure to obtain paste medicinal material extract, freeze-dried, dispersed with distilled water, sequentially extracted with petroleum ether, chloroform, ethyl acetate and n-butanol, combined with extracts of the same solvent, and vacuum concentrated and dried to obtain petroleum ether extract, chloroform extract, ethyl acetate extract, water extract and n-butanol extract respectively.

[019] EXAMPLE 2 determination of antifungal activity in vitro

[020] Mycelial growth rate method and microdilution method (MIC) were used to determine the antibacterial activity of ethanol extract of medicinal materials in vitro, and the antibacterial activity of ethanol extract of medicinal materials was determined qualitatively and quantitatively.

[021] 1. Experimental strains: M. gypseum, T. mentagrophytes and M. canis.

[022] 2. Experimental method

[023] 1) Configuration of test subjects: The five extracts prepared in Example 1 (i.e. Petroleum ether extract, chloroform extract, ethyl acetate extract, water extract and n-butanol extract) were dissolved with 500ulDMSO, diluted to 10mg/ml with distilled water, and added to the melted PAD medium (40-50. Degree. C.) at a ratio of 1: 9 to make the final concentration 1mg/ml.

[024] 2) preparation of culture medium:

[025] Preparation of potato agar medium (PDA): Wash fresh potatoes, peel them, cut them into small pieces, weigh 200g, boil them in 1 L distilled water for 15-30 min, filter them with three layers of gauze, and supplement the distilled water reduced by evaporation to 1 L. Add 20 g of glucose and 20 g of agar to the filtrate, heat, after the agar is completely melted, pack it in a 250 mL conical flask while hot, plug it with cotton plugs, wrap it in newspaper, and sterilize it in a high-pressure steam sterilization pot at 121 °C for 30 min. After sterilization is completed, transfer to aseptic console, and wait for the culture medium temperature to drop to about 50 °C. Pour the sterile plate, shake it evenly, lay it flat, make PDA plate, and store it at 4 °C after solidification for later use.

[026] Preparation of potato liquid culture medium (PDB): Wash fresh potatoes, peel them, cut them into small pieces, weigh 200g, put them into 1 L distilled water, boil them for 15-30min, filter them with three layers of gauze, supplement the distilled water reduced by evaporation to 1 L, and add 20g glucose to the filtrate. Sterilize and store at 4 °C for later use.

[027] Preparation of Sasha's glucose agar medium (SDA): Weigh 10 g peptone, g glucose and 20 g agar in a large beaker, fix the volume of distilled water to 1 L, heat, after the agar melts, split it into 250 mL conical flask while hot, plug it with cotton stopper, wrap it in newspaper, and sterilize it in a high-pressure steam sterilization pot at 121 °C for 30 min. Transfer to aseptic console, pour the plate when the temperature of the culture medium drops to 50 °C, shake it evenly, lay it flat, cool it and store it at 4 °C for later use.

[028] A medicate plate is prepared by pour 1 mL of that medicinal material solution obtained in 1) and about 9mL of the culture medium cooled to about 50 DEG C into a sterilized petri dish to prepare a medicated plate with a concentration of 5 mg/mL. When the culture medium is cooled to 50 DEG C, 1 mL of sterile volume water and about 9 mL of culture medium are added to prepare a control plate of the medicated plate, and the control plate of the medicated plate is stored at 4 DEG C after coagulation for later use.

[029] Preparation of fungus plate: Add 2 mL of fungus suspension to 100 mL of culture medium that has been cooled to about 50 °C, mix well quickly, then pour aseptic plate, stand horizontally for solidification, and store at 4 °C for later use.

[030] 3) Preparation of bacteria suspension: The microsporum gypsum, trichophyton and microsporum canis needed for the test were taken out from the refrigerator, and the three fungi were inoculated on SDA plate with aseptic inoculation ring respectively, and cultured at 27 °C for 2 weeks to activate the fungi so that the colonies were evenly covered on the culture medium. 2 mL of sterilized normal saline was sucked to rinse the hyphae and spores on the surface of the colony, and the rinsing solution containing hyphae and spores was counted with a blood cell counting plate, and the concentration was modulated to 105 ~ 106 CFU/mL to obtain the bacterial suspension for later use.

[031] 4) Determination of mycelium growth rate

[032] Microsporum gypsum, Trichophyton mentagrophytes and Microsporum canis were inoculated on PDA plate at 27 °C and cultured for 5 days. Using a sterilized perforator, a 0.4 cm diameter bacterial cake was cut from the edge of the PDA plate inoculated with the tested bacteria, and the bacterial cake was connected to the drug containing culture medium obtained under 2), with the side with hyphae facing down, and 3 bacterial cakes were inoculated into each culture dish. At the same time, the control plate under 2) is subjected to control treatment. Repeat for 3 times and culture in a constant temperature incubator at 27 °C. After 2-6 days of culture, the colony diameter of the tested fungi on different concentrations of drug-containing medium was measured by cross method every day, and the inhibition rate of each drug solution treatment on mycelium linear growth was calculated compared with the control. According to the following formula, the bacteriostasis rate is obtained: colony growth diameter (cm)= average measured diameter-0.4 bacteriostasis rate (%)= (blank control colony diameter-treated colony diameter)+ blank control colony diameter x 100

[033] 5) Determination of Minimum Inhibitory Concentration (MIC)

[034] The minimum inhibitory concentration of Oxytropis falcate Bunge ethanol extract was determined. Take aseptic 96-well plate, first add 1OOL PDB medium to each well, then add 1OOL of medicinal material solution under item 2) to each well in column 1 and column 2 respectively, and mix evenly; 2-11 hole are diluted by double ratio: 100 L liquid is taken from that second row to the third row, and mixed evenly; Take 100 L of liquid from Column 3 to Column 4, and so on to Column 11. After 11 rows are evenly mixed, 100 L of liquid is taken out and discarded. Finally, 100 L of bacterial suspension was added to each well. At this time, the concentrations of 2 to 11 columns of samples are 2500, 1250, 625,312.5, 156.25, 78.13, 39.06, 19.53, 9.77 and 4.88 g/mL. The first column is blank control and the 12th column is growth control. The 96-well plate was cultured in a constant temperature incubator at 27 °C for 48 h, and MIC was determined by the lowest concentration of fungus-free growth observed by naked eyes. Repeat the experiment 3 times for each sample.

[035] 3. Experimental result

[036] 3.1 The antifungal activity results of the total ethanol extract are shown in Figure 1: The total ethanol extract shows broad-spectrum antifungal activity at the test concentration.

[037] 3.2 Determination of Minimum Inhibitory Concentration (MIC mg/ml): The MIC determination results of antifungal active polar parts are shown in Fig. 2. The results show that the minimum inhibitory concentration of chloroform parts to Microsporum canis is 2.5 mg/mL, that to Microsporum gypsum is 2.5 g/mL, and that to Trichophyton mentagrophytes is 5g/mL.

[038] The experimental results of antifungal activity showed that different solvent extracts of Oxytropis falcate Bunge have different degrees of inhibitory effects on Microsporum gypsum, Trichophyton mentagrophytes and Microsporum canis, and can be used as antifungal drugs.

[039] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[040] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An active extract from OxytropisfalcateBunge, characterized in that the active extract is selected from one or a combination of petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract, water extract obtained by extraction from the alcohol extract Oxytropisfalcate Bunge; The active extract is prepared by a method following the steps: (1) the coarse powder of Oxytropis falcate Bunge was mixed with ethanol, extracted under ultrasonic assisted extraction and filtered, repeating the above extraction with filter residue, filtering, and combining extract solutions; (2) the ethanol extract was concentrated under vacuum concentrationto obtain a concentrated solution, the latter was freeze-dried and dispersed with distilled water; (3) gradient extraction is carry out with different polar solvents, petroleum ether, chloroform, ethyl acetate and n-butanol, each solvent is repeatedly extracted, the same organic phase and water phase are combined, and vacuum concentration is carried out to obtain petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract.

2. The extract according to claim 1, wherein the method preferably comprises the following steps: (1) the coarse powder of Oxytropisfalcate Bunge was mixed with 20 times ethanol for 24h, extracted under ultrasonic aid for 1h, filtered; the filtering residue was repeated the extraction process for 2 times; then filtered, and combined all the extracts; 2) the ethanol extract was concentrated under vacuum concentration at 55°C to obtain a concentrated solution, the latter was freeze-dried and dispersed with distilled water; (3) gradient extraction with different polar solvents such as petroleum ether, chloroform, ethyl acetate and n-butanol with the same volume was conducted and repeated for twice respectively, after combining the same organic phase and aqueous phase, the concentration under vacuum concentration to obtain petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract and water extract.

3. An extract according to claim 2, wherein the ethanol is preferred 60-90% ethanol.

4. A pharmaceutical composition was comprised by the extract of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, characterized in that the composition is present in the form of a suitable pharmaceutical preparation, the preparation form is selected from the tablets, capsules, oral liquid, mixture, oral agent, granule, granule, pill, powder, paste, Dan, suspension, solution, injection, powder injection, freeze-dried powder injection, suppository, ointment, hard paste, cream, spray, spray film, dripping pills, dripping pills and / or patches.

6. The pharmaceutical composition of claim 5, wherein the tablet is selected from the group consisting of sugar-coated tablets, film-coated tablets, enteric-coated tablets, or sustained-release tablets; The capsules are selected from the group consisting of hard capsules, soft capsules or sustained release capsules.

7. Use of the extract according to any one of claims 1 to 3 or the composition according to any one of claims 4 to 6 in the preparation of an antifungal drug.