CN116478826A - Agilawood endophyte and extract thereof, and preparation method and application thereof - Google Patents

Abstract

The invention providesA plant is isolated from Aquilaria genusAquilaria) Endophyte ribes curriculum vitae of plantBotryosphaeria dothideaKET49 and its extract, and its preparation method and application are provided. Belongs to the field of microorganism. The fungus of the invention is Botrytis cinerea @, which is a fungus of the genus BotrytisBotryosphaeriasp.) KET49, and is stored in China general microbiological culture collection center (CGMCC) No.40491, and the storage date is 2023, 2 and 15. The endophyte is separated from the wood blocks containing the grease of the agilawood plants, the endophyte is subjected to solid fermentation culture, and then the extract PXS209 is obtained through extraction, so that the PXS209 provides a new choice for preparing the anti-dermatophyte drugs or skin care products, and can be used for treating skin diseases caused by dermatophytes.

Description

Agilawood endophyte and extract thereof, and preparation method and application thereof

Technical field:

the invention belongs to the field of microorganisms, and particularly relates to endophytes separated from aquilaria sinensis, in particular to a ribus curriculum (Botryosphaeria dothidea) KET49 and an extract PXS209 thereof, and application thereof in preparing medicines or skin care products with antifungal activity.

The background technology is as follows:

dermatophytes often cause infection of skin and its subcutaneous protein tissue, and are a pathogenic bacterium with a high rate of skin infection in humans and animals [ demand c., contet-Audonneau N., kombila M., miegeville M., berthonneau M., de Vroey c., percebois G.Microsporum gypseum complex in man and animals.journal of medical and veterinary mycology,1992,30 (4), 301-308 ]. The human and animal are susceptible to dermatophytes, and the main infection strains are Microsporum gypseum (Microsporum gypseum), microsporum canis (Microsporum canis) and Trichophyton mentagrophytes (Trichophyton mentagrophytes) and the like [ Zhang pre, shallow mycosis 954 strain analysis, journal of practical hospital clinic, 2006,3 (5): 66 ]. Of these, microsporopsis gypsea is widely present worldwide and is a wide-ranging dermatophyte. The direct contact of the bacterial spores with human and animals is the main way of infecting the bacteria, and the skin infection in animals is mainly characterized by alopecia, erythema, scaling and surrounding crusting and onychomycosis, besides, the symptoms caused by infecting the bacteria can appear special skin lesions, some are psoriasis scab, white pseudomembranous, psoriasis-like lesions, and some can appear eczematoid, dry impetigo, concentric ring-like lesions [ Yan. The skin and lung pattern recognition receptor and cytokine change law observation after the microsporobacteria infection of mice, sichuan university of agriculture, 2019]. With the widespread use of antifungal agents, a number of clinically novel pathogenic bacteria and drug-resistant strains have emerged. Drugs used in conventional treatments include amphotericin B, imidazolone-conazole, terbinafine, itraconazole and the like, which have been reported to have high toxicity in recent years and cause liver and kidney damage after long-term use [ Duckweed, weijing, liu Wanyu, huang Jiangeng, S Liu Qin, zhangyu, wu Sanlan. Development of liver injury caused by triazole antifungal drugs. Journal of Chinese Hospital pharmacy, 2021,41 (14): 1475-1480; wang Rui and Liang Beibei adverse reactions to antifungal agents, J.Chen New Endoconcha 2004,13 (2): 175-178; the adverse reaction analysis of 241 antifungal medicines in 1979-2001 of China, china pharmacy, 2002,13 (12): 744-745. Aiming at the problems of high toxicity and drug resistance of the existing antibacterial western medicines, it is urgent to find a novel antifungal medicine with high efficiency, low toxicity and broad spectrum.

The lignum Aquilariae Resinatum is saussurea lappa Aquilaria sinensis (Lour.) Spreng. Core material containing resin (the edition 2020 of the pharmacopoeia of the people's republic of China). Agilawood is one of the most valuable non-wood products, useful as fragrances, perfumes, traditional medicines and other products. The agalloch eaglewood mainly contains sesquiterpene and 2- (2-phenethyl) chromone components, and recent researches show that the agalloch eaglewood has anti-inflammatory, cytotoxic, anti-tumor, antibacterial, acetylcholinesterase inhibition, neuroprotection, blood glucose reduction and antioxidation effects [ Li W, chen H-Q, wang H, mei W-L, dai H-F.Nature products in agarwood and Aquilaria plants: chemistry, biological activities and biosynthesis. Nature Product Reports,2021,38,528-565]. At present, the research on agilawood is relatively wide, and the function research on secondary metabolites of agilawood endophytes is relatively few. Until now, no report on the endophyte Botryopharia (Botryosphaeria) and the extract thereof, and no report on the antifungal activity of the agalloch eaglewood, is seen in the prior art.

The invention comprises the following steps:

the invention aims to provide an endophyte KET49 separated from aquilaria sinensis and an extract PXS209 thereof, a pharmaceutical composition taking the endophyte KET49 as an active ingredient, a preparation method of the pharmaceutical composition and application of the pharmaceutical composition in preparation of anti-dermatophytes drugs or skin care products.

In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

the invention provides an endophyte, which is a Botryosphaeria (Botryosphaeria sp.) KeT49 of a black currant, is separated from an Aquilaria (Aquiaria) plant, is preserved in China general microbiological culture collection center (CGMCC) No.40491, and has a preservation date of 2023, 2 months and 15 days. The endophyte is separated from a wood block containing grease of the trunk of the aquilaria sinensis, and the separation method is as follows: the method comprises the steps of taking fresh aquilaria sinensis trunks containing oil and fat wood blocks, cutting samples into small blocks with the length of 5mm multiplied by 5mm, firstly rinsing with sterile water, then rinsing and sterilizing sequentially with 30-60 s of 75% ethanol, 2-3 min of 3% -5% sodium hypochlorite solution and 30s of 75% ethanol, rinsing with sterile water for three times, wiping with sterile paper, then placing the dried blocks in potato-glucose agar PDA, oxoid, UK culture medium and malt extract agar culture medium MEA, oxoid and UK, slightly pressing, simultaneously taking a small amount of sterile water for the last rinsing in a culture dish to test the surface sterilizing effect, culturing for 2-10 d at the room temperature of 25-28 ℃, after bacterial colonies are formed, carrying out subculture, picking hyphae of single bacterial colonies, transferring to a new culture dish, continuously culturing for 5-7 d at the room temperature, carrying out repeated separation and purification, transferring the obtained bacterial strains to a freeze tube, and preserving for standby.

An endophyte KET49 extract PXS209 prepared by the following method:

(1) Activating strains: inoculating the preserved strain KET49 onto PDA culture medium for activation;

(2) Fermentation culture: inoculating the activated strain in the step (1) into a PDA culture medium, and culturing for 7d at the temperature of 28 ℃ to obtain endophyte KET49 solid fermentation product;

(3) Extracting the endophyte KET49 solid fermentation product with 95% ethanol at 60deg.C under ultrasonic treatment for 3 times, and concentrating to obtain fermented product extract PXS209.

A pharmaceutical composition comprising a therapeutically effective amount of PXS209, an extract of said endophyte key 49, and a pharmaceutically acceptable carrier.

The active ingredients of the skin care product for resisting dermatophytes comprise the endophyte KET49 extract PXS209.

The endophyte KET49 or the extract PXS209 thereof is applied to the preparation of the antifungal drugs.

The endophyte KET49 or the extract PXS209 thereof is applied to the preparation of skin care products against dermatophytes.

The application of the pharmaceutical composition in preparing anti-dermatophyte medicines.

The preparation method of the pharmaceutical composition comprises the following steps:

(1) Activating strains: inoculating the preserved strain KET49 onto PDA culture medium for activation;

(2) Fermentation culture: inoculating the activated strain in the step (1) into a PDA culture medium, and culturing for 7d at the temperature of 28 ℃ to obtain endophyte KET49 solid fermentation product;

(3) Extracting endophyte KET49 solid fermentation product with 95% ethanol at 60deg.C under ultrasonic wave for 3 times, and concentrating to obtain fermented product extract PXS209;

(4) The extract is added into pharmaceutically acceptable carrier.

The pharmaceutical composition provided by the invention comprises the agilawood endophyte KET49 or an extract PXS209 thereof and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semi-solid or liquid diluent, filler and pharmaceutical preparation adjuvant. The pharmaceutically acceptable carrier is not particularly limited, and pharmaceutically acceptable carriers which are well known in the art, nontoxic and inert to human and animals can be selected.

The preparation method of the pharmaceutical composition is not particularly limited, the agilawood endophyte KET49 or the extract PXS209 thereof is directly mixed with a pharmaceutically acceptable carrier, the mixing process is not particularly limited, and the pharmaceutical composition can be obtained by selecting a process well known in the art.

The invention provides the application of the pharmaceutical composition in preparing the antifungal skin medicament, the application method is not particularly limited, and the method well known in the art can be selected.

In the present invention, when the pharmaceutical composition is used for preparing an antifungal agent for skin, the content of the composition in the agent is preferably 0.1 to 99%; in the pharmaceutical composition, the content of at least one of the agilawood endophyte KET49 or the extract PXS209 thereof in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a unit weight dose. In the present invention, the prepared medicine may be preferably administered in both injection (intravenous injection, intramuscular injection) and oral administration. Can also be prepared into any medicinal preparation by adopting a conventional method of traditional Chinese medicine preparation.

According to the invention, the solid fermentation product of the agilawood endophyte KET49 is extracted and optimized by using a solvent, and an antifungal experiment of the prepared PXS209 shows that the PXS209 has a certain activity of resisting gypsum microspores. PXS209 provides a new raw material for preparing antifungal cosmetics.

The agilawood endophyte and the extract thereof are used as raw materials to prepare the skin fungus resistant cosmetics, and can be prepared into any preparation of cosmetics. The method of application is not particularly limited, and methods well known in the art may be used. The extract can be made into a lotion, a toner, an emulsion, a cream, a cleansing cream, but this does not limit the scope of the present invention.

Compared with the prior art, the invention has the following advantages:

1. the fungus, ribus curriculum (Botryosphaeria dothidea) KET49, is first isolated from the aquilaria sinensis oil-containing blocks of wood.

2. The antifungal activity evaluation is carried out on the agalloch eaglewood endophyte (Botryosphaeria dothidea) KET49 extract for the first time, and the activity evaluation result shows that the agalloch eaglewood endophyte extract PXS209 has the activity of resisting microspores of gypsum samples.

Preservation description:

classification naming: raspberry grape vine cavity fungus

Latin name: botryosphaeria dothidea

Biological material of the reference: KET49

Preservation mechanism: china general microbiological culture Collection center (China general microbiological culture collection center)

The preservation organization is abbreviated as: CGMCC preservation address: beijing city, the region of Chaoyang, north Chen Xili, no. 1,3, china academy of sciences microbiological institute

Preservation date: 2023, 2 and 15 days

Accession numbers of the preservation center: CGMCC No.40491

Description of the drawings:

FIG. 1 is a schematic diagram of the endophyte (the left panel shows the front surface of endophyte, and the right panel shows the back surface of endophyte).

FIG. 2 shows a phylogenetic tree set up by KET49.

The specific embodiment is as follows:

the following describes the embodiments of the present invention with reference to the drawings, but the present invention is not limited thereto, and only the embodiments are described.

Example 1:

1. separating and purifying the Sclerotinia ribes Botryosphaeria dothidea KET.

Fresh aquilaria sinensis trunks containing oil and fat blocks (healthy blocks are distinguished by burned smell), samples are cut into small blocks (5 mm multiplied by 5 mm), firstly rinsed with sterile water, then rinsed and sterilized by 75% ethanol (30-60 s), 3% -5% sodium hypochlorite solution (2-3 min) and 75% ethanol (30 s) in sequence, rinsed three times with sterile water, dried by sterile paper, placed in potato-glucose agar (PDA, oxoid, UK) culture medium and malt extract agar culture medium (MEA, oxoid, UK) and lightly pressed. And taking a little sterile water for the final rinsing in the culture dish to test the surface disinfection effect. Culturing at room temperature (25-28 ℃) for 2-10 d. After the bacterial colony is formed, subculturing according to the characteristic of the bacterial colony, picking hypha of a single bacterial colony, transferring to a new culture dish, and continuously culturing for 5-7 d at room temperature. The obtained strain is transferred to a freezing tube for storage for later use through multiple times of separation and purification.

The endophyte is obtained through separation, and the morphology of the endophyte is shown in figure 1.

2. Identification of Puccinia striolata Botryosphaeria dothidea KET.

The DNA extraction of aquilaria sinensis endophytic fungi is carried out by adopting a Biospin fungi genome DNA extraction kit-BSC 14S 1. Three pairs of primers ITS-4and ITS-5, LROR and LR5 and Bt2α and Bt2β were selected as sequencing primers, the primer sequences of which are shown in Table 1 below.

Purification and sequencing of DNA was carried out by Kunming Optimago Biotechnology. The sequence of the endophyte base obtained by sequencing is shown as sequence 1-3 in a sequence table. And uploading the obtained sequence, submitting the sequence to a NCBI nucleic acid database for BLAST comparison, and selecting a nucleic acid sequence with high similarity with the sequence for data analysis. The comparison shows that the strain was identified as Botryosphaeria (Botrytis genus), botryosphaeria dothidea (Puccinia rubra), and the established phylogenetic tree is shown in FIG. 2.

TABLE 1 sequencing primers

Primer name	Sequence (5 '- -3')
		ITS-4	TCC TCC GCT TAT TGA TAT GC
ITS-5	GGA AGT AAA AGT CGT AAC AAG G
		LROR	ACC CGC TGA ACT TAA GC
LR5	TCC TGA GGG AAA CTT CG
		Bt2α	GGT AAC CAA ATC GGT GCT GCT TTC
Bt2β	ACC CTC AGT GTA GTG ACC CTT GGC

3. Fermentation of the Sclerotinia ribes Botryosphaeria dothidea KET.

After the strain KET49 isolated above was activated on PDA medium, the same size cake was removed with plastic tube, one cake was placed in each 50mL dish containing PDA solid medium (15 mL), and the uninoculated PDA-containing dish was set as a blank (number: KET 49-1), and after 7d of incubation in a constant temperature incubator (28 ℃) for preparation of the extract.

4. Preparation of PXS209 extracted from Sclerotinia ribis Botryosphaeria dothidea KET 49.rubrum.

43.1g of endophyte, namely, the Sclerotinia ribis praecox Botryosphaeria dothidea KET solid fermentation product (containing a culture medium), is extracted by ultrasonic for 3 times with 95% ethanol at 60 ℃, and 67.6mg of a fermentation extract (number: PXS 209) is obtained by concentration.

Example 2

Antifungal experiments with PXS209.

1. Experimental strains

Microsporopsis gypseum (Microsporum gypseum) CBS118893 was purchased from the national academy of medical science and fungus deposit.

2. Experimental reagent

Ceftazidime is purchased from Shanghai leaf biotechnology Co., ltd, penicillin G sodium is purchased from Biosharp; terbinafine hydrochloride, DMSO, purchased from Sigma; MH broth, LB broth, available from guangdong cycikai microbiology limited; agar powder and chloramphenicol were purchased from Scientific Research Special.

3. Experimental method

Diluting the sample to be tested by taking 96-well culture plate, adding fungus liquid into each well, and final concentration is 5×10 ⁵ CFU/mL, incubated at 25℃for 5 days, and absorbance at 625nm was measured by an ELISA. The experiments were also run with medium blank, fungal control and terbinafine hydrochloride control.

4. Experimental results

TABLE 2 PXS209 inhibition of microsporum gypseum

Experimental results show that the agilawood endophyte extract PXS209 has the activity of resisting microspores of gypsum samples.

Pharmaceutical formulation examples 1-7:

in the following preparation examples, conventional reagents are selected and preparation is performed according to the conventional method, and the application example only embodies that the agilawood endophyte KET49 or the extract PXS209 thereof can be prepared into different preparations, and specific reagents and operations are not particularly limited:

1. dissolving lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 in DMSO, adding water for injection according to conventional method, fine filtering, packaging, and sterilizing to obtain injection with concentration of 0.5-5mg/mL.

2. Dissolving lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 in DMSO, dissolving in sterile injectable water, stirring to dissolve, filtering with sterile suction filter funnel, sterile fine filtering, packaging in ampoule, lyophilizing at low temperature, and sealing under sterile condition to obtain powder for injection.

3. Adding excipient into lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 at a ratio of 9:1, and making into powder.

4. Adding excipient into lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 at a mass ratio of 5:1, granulating, and tabletting.

5. The agilawood endophyte KET49 or the extract PXS209 thereof is prepared into oral liquid according to a conventional oral liquid preparation method.

6. Adding excipient into lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 at a ratio of 5:1, and making into capsule.

7. Adding excipient into lignum Aquilariae Resinatum endophyte KET49 or its extract PXS209 at a mass ratio of 5:1, and making into granule.

Cosmetic preparation examples 1 to 4

1. Whitening cream formula (W%) containing agilawood endophyte KET49 or extract PXS209 (hereinafter collectively referred to as active ingredient):

the cosmetic with the formula is prepared according to a conventional method for preparing cosmetics.

2. Emulsion formulation (W%) containing agalloch endophyte KET49 or its extract PXS209 (hereinafter collectively: active ingredient):

the cosmetic with the formula is prepared according to a conventional method for preparing cosmetics.

3. Cosmetic water:

aqueous phase: sodium polyacrylate 0.35 g, glycerin 4 g, 1, 3-butanediol 2.5 g, vitamin B5.5 g, arbutin 0.5 g, EDTA-Na ₂ 50mg; proper amount of deionized water was added to 100ml. The preparation method comprises the following steps: 0.3 g of sodium polyacrylate is dissolved in 70ml of water and stirred to swell thoroughly. Slowly adding the rest ingredients in the prescription, and stirring continuously; then 0.5 g of agilawood endophyte KET49 or extract PXS209 of the invention is added and stirred to be uniform. Deionized water, adding to 100ml, and packaging.

4. Repairing cream:

prescription: 6 g of glycerin, 1.5 g of carbomer, 1.5 g of triethanolamine, 6 g of propylene glycol and 0.2 g of ethyl nilate, and the total weight of the agilawood endophyte KET49 or extract PXS 209.5 g of the agilawood endophyte KET49 is 100 g. The preparation method comprises the following steps: adding carbomer into deionized water, stirring, standing overnight, swelling, adding glycerol, and regulating pH with triethylamine to increase gel matrix viscosity. Mixing tanshinone extract, propylene glycol and deionized water. Adding carbomer gel, adding 0.5% essential oil and ethyl Nib, mixing, stirring, adding distilled water, and grinding.

The sequence table of the invention:

<110> Beijing, department of eaglewood science and technology Co., ltd

<120> an agilawood endophyte, and extract, preparation method and application thereof

<210>1

<211>579

<212>DNA

<213> artificial sequence

<400>1

<110> Beijing, department of eaglewood science and technology Co., ltd

<120> an agilawood endophyte, and extract, preparation method and application thereof

<210>2

<211>939

<212>DNA

<213> artificial sequence

<400>2

<110> Beijing, department of eaglewood science and technology Co., ltd

<120> an agilawood endophyte, and extract, preparation method and application thereof

<210>3

<211>469

<212>DNA

<213> artificial sequence

<400>3

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. An endophyte, characterized in that the endophyte is a ribus curriculum Botrytis championii Botryosphaeria dothideaKET49 stored in China general microbiological culture Collection center with accession numberThe preservation date is 2023, 2 and 15 days.

2. The endophyte of claim 1, wherein the endophyte is separated from a fat-containing wood block of the trunk of aquilaria sinensis, and the separation method comprises the following steps: the method comprises the steps of taking fresh aquilaria sinensis trunks containing oil and fat wood blocks, cutting the samples into small blocks of 5mm, 5mm and 5mm, rinsing with sterile water, sequentially rinsing with 30-60 s% of 75% ethanol, 2-3 min of 3-5% sodium hypochlorite solution and 30 s% of 75% ethanol for sterilization, rinsing with sterile water for three times, wiping with sterile paper, placing the dried blocks in potato-glucose agar PDA, oxoid, UK culture medium and malt extract agar culture medium MEA, oxoid and UK, slightly pressing, taking a small amount of sterile water for the last rinsing in a culture dish at the same time, culturing at the temperature of 25-28 ℃ for 2-10 d, subculturing after bacterial colonies are formed, taking single bacterial colonies, transferring the single bacterial colonies to a new culture dish, continuously culturing at the temperature for 5-7 d, performing multiple separation and purification, and transferring the obtained bacterial strains to a storage tube for standby.

3. The endophyte KET49 extract PXS209 is characterized by being prepared by the following method:

(1) Activating strains: inoculating the strain KET49 in the preserved state according to claim 1 onto PDA culture medium for activation;

(2) Fermentation culture: inoculating the activated strain in the step (1) into a PDA culture medium, and culturing at 28 ℃ to 7d to obtain endophyte KET49 solid fermentation product;

(3) Extracting the endophyte KET49 solid fermentation product with 95% ethanol at 60deg.C under ultrasonic treatment for 3 times, and concentrating to obtain fermented product extract PXS209.

4. A pharmaceutical composition comprising a therapeutically effective amount of the endophyte KET49 extract PXS209 of claim 3, and a pharmaceutically acceptable carrier.

5. An antifungal skin care product comprising the endophyte KET49 extract PXS209 of claim 3 as an active ingredient.

6. Use of endophyte KET49 of claim 1 or extract PXS209 of claim 3 for the preparation of an antifungal agent against skin.

7. Use of endophyte KET49 of claim 1 or extract PXS209 of claim 3 for the preparation of an anti-dermatophyte skin care product.

8. Use of the pharmaceutical composition of claim 4 for the preparation of an antifungal agent against skin.

9. A process for the preparation of a pharmaceutical composition according to claim 4, characterized in that it comprises the following steps:

(1) Activating strains: inoculating the strain KET49 of claim 1 in a preservation state onto PDA culture medium for activation;

(2) Fermentation culture: inoculating the activated strain in the step (1) into a PDA culture medium, and culturing at 28 ℃ to 7d to obtain endophyte KET49 solid fermentation product;

(3) Extracting endophyte KET49 solid fermentation product with 95% ethanol at 60deg.C under ultrasonic wave for 3 times, and concentrating to obtain fermented product extract PXS209;

(4) The extract PXS209 is added to a pharmaceutically acceptable carrier.