阅读说明：本技术 一种抗香蕉枯萎病小分子化合物的制备方法及其应用 (Preparation method and application of banana vascular wilt resistant small molecular compound ) 是由 王尉 周登博 陈宇丰 谢江辉 张璐 李凯 赵炎坤 井涛 起登凤 于 2021-10-15 设计创作，主要内容包括：本发明的第四个方面是提供一种土曲霉酮的制备方法,从萨姆松链霉菌XJC-2-6发酵液中分离得到。本发明首次采用放线菌发酵分离得到土曲霉酮,进一步研究发现,该化合物能有效拮抗香蕉枯萎病菌4号生理小种,破坏菌丝结构,抑制菌丝生长,EC-(50)、EC-(75)和EC-(95)分别为25.76μg/mL、86.30μg/mL和491.37μg/mL,能够显著降低香蕉枯萎病菌4号生理小种菌体中可溶性总糖含量、可溶性总蛋白含量、脂肪含量、线粒体呼吸链复合体酶I～IV活性等。本发明为制备土曲霉酮提供的新的思路,为枯萎病等植物病害的防治拓展新领域,具有广阔的发展空间和良好的开发应用前景。(The fourth aspect of the invention provides a preparation method of the oxytetracycline, which is separated from the fermentation liquor of streptomyces samsunus XJC-2-6. According to the invention, the tomarone is obtained by fermenting and separating actinomycetes for the first time, and further research shows that the compound can effectively antagonize No. 4 physiological race of banana fusarium oxysporum, destroy hypha structure, inhibit hypha growth and realize EC 50 、EC 75 And EC 95 25.76. mu.g/mL, 86.30. mu.g/mL and 491.37. mu.g/mL, respectively,can obviously reduce the soluble total sugar content, the soluble total protein content, the fat content, the activity of mitochondrial respiratory chain complex enzymes I to IV and the like in the No. 4 physiological race thalli of the banana wilt bacteria. The invention provides a new idea for preparing the oxytetracycline, develops a new field for preventing and treating plant diseases such as blight and the like, and has wide development space and good development and application prospects.)

1. Application of Streptomyces samsunensis XJC-2-6 in preparing oxytetracycline is provided.

2. Application of Streptomyces samsunensis XJC-2-6 fermentation broth in preparation of oxytetracycline is provided.

3. Application of Streptomyces samsunensis XJC-2-6 fermentation broth ethyl acetate extract in preparation of oxytetracycline is provided.

4. The use according to claim 3, wherein the ethyl acetate extract of the Streptomyces samsunus XJC-2-6 fermentation broth is obtained by adding ethyl acetate into the supernatant obtained by ethanol extraction and filtration of the Streptomyces samsunus XJC-2-6 fermentation broth, and extracting and concentrating the ethyl acetate phase.

5. A preparation method of oxytetracycline is characterized in that the oxytetracycline is separated from Streptomyces samadensis XJC-2-6 fermentation liquor.

6. The method of claim 5, comprising the steps of:

(1) inoculating streptomyces samsonii XJC-2-6 into a fermentation culture solution for fermentation culture to obtain a fermentation solution;

(2) adding appropriate amount of ethanol into the fermentation broth for extraction, filtering, collecting supernatant, adding appropriate amount of ethyl acetate for extraction, collecting ethyl acetate phase, and concentrating to obtain ethyl acetate extract;

(3) separating the ethyl acetate extract with silica gel column chromatography, and purifying with CH₂Cl₂Gradient elution with MeOH/MeOH system (v/v: 100% CH)₂Cl₂100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, 100% MeOH), performing antibacterial property test on each obtained component, and performing TLC-direct bioautograph antibacterial activity detection by using Foc TR4 as a target pathogen to obtain a main active component fr.a;

(4) subjecting active component Fr.A to Sephadex LH-20 gel column chromatography, and purifying with CH₂Cl₂Eluting with eluent/MeOH (v/v:2:1), testing antibacterial property of each obtained component, and detecting antibacterial activity by TLC-direct bio-automation to obtain higher active component Fr.A-4;

(5) repeatedly separating and purifying the active component Fr.A-4 by RP-HPLC to obtain the oxytetracycline ketone.

7. The method according to claim 6, wherein in the step (1), the fermentation medium is M6 liquid medium, the inoculation amount is 5%, and the fermentation medium is cultured at 28 ℃ for 8d under 180r/min shaking.

8. Application of the oxytetracycline in antagonizing No. 4 physiological race of banana vascular wilt pathogens.

9. Application of the oxytetracycline ketone in preparation of medicines for preventing and treating diseases caused by No. 4 physiological race of banana vascular wilt.

10. The application of the tulathromone in reducing the soluble total sugar content, the soluble total protein content, the fat content and the activity of mitochondrial respiratory chain complex enzymes I to IV in the physiological race thallus of No. 4 banana wilt pathogen is disclosed.

Technical Field

The invention relates to a preparation method and application of a micromolecular compound, in particular to a preparation method and application of a small molecule compound for resisting banana vascular wilt

Background

Bananas (Musa spp.) belonging to the genus Musa of the family Musaceae (Musaceae) are one of the most valuable primary agricultural products in the world, are the highest-yielding fruits in the world (total yield of 1.48 million tons in 135 countries in 2016), provide staple food for about 4 million people worldwide, and are positioned by the world food and agriculture organization as the fourth largest food crop next to rice, wheat, and corn (duskenoli, 2017; Li et al, 2018). Bananas are also the eighth food crop in the world, and have a very important position in the world due to their rapid growth, abundant nutritional ingredients and high economic value (Dong et al, 2015). However, while the worldwide banana industry is rapidly developing, the occurrence and spread of banana wilt disease also pose the most serious threats and challenges. Banana wilt, also known as banana panama disease and yellow leaf disease, is a devastating soil-borne disease caused by Fusarium oxysporum cubense (Fusarium oxysporum f.sp.cubense, Foc) which destroys banana vascular bundles and causes plant death, is one of the most serious fungal diseases in the world, and is also a main limiting factor for banana production (Zhang et al, 2014).

Banana wilt first appeared in australia and was found and reported in panama in 1896 (Ploetz et al, 2015). In 1935-1939, it was spread in south America in a large area and spread all over the world rapidly. Currently, the disease is prevalent in tropical and subtropical banana growing areas such as latin america, africa, asia, etc., and seriously affects the development of banana industry around the world (Hwang et al, 2004).

The perennial nature and polycyclicity of banana wilt disease complicates and limits long-term control measures (Ploetz, 2015). In recent years, due to the great input of pesticides, economic, environmental and food safety concerns are caused, biological control has attracted wide attention in plant pathological control, biological control technology is one of important means for controlling blight, and the biological control technology has the characteristics of high efficiency, safety and the like, and the acquisition of high-efficiency antagonistic microorganisms is the basis of biological control research (Fu et al, 2017). The use of biological control agents has proven to be an environmentally friendly prevention strategy (Xue et al, 2015, Deltour et al, 2017; Fu et al, 2017). Some studies have shown that biological control agents inhibit the growth of Foc under in vitro conditions and in potting experiments (Thangavelu et al, 2004; Mohammed et al, 2011; Gnasekaran et al, 2015; Ho et al, 2015; Sekhar et al, 2015). Chen et al (2018) reported that banana plants inoculated with Fusarium oxysporum No. 4 race treated with Streptomyces in a potting test did not show symptoms of banana wilt such as leaf blight and discoloration inside the plants. In field trials, application of trichoderma harzianum in soil effectively controlled banana wilt with an effect comparable to carbendazim (Thangavelu et al, 2004). Xue et al (2015) screened a potential biocontrol agent from bacillus that plays an important role in controlling banana wilt. Cao et al (2005) found that Streptomyces bananas endogenetic streptomyces has control effect on fusarium oxysporum and can be developed into biological control agents for controlling banana fusarium wilt. The success of biocontrol depends not only on the method of production, but also on the cost involved and the effective biocontrol agents, and furthermore, these biocontrol agents must be able to be stored as dry preparations for long periods of time (Jackson, 1997).

Disclosure of Invention

The invention aims to overcome the defects in the prior art, takes a banana vascular wilt No. 4 physiological race (Foc TR4) as a target pathogenic bacterium, adopts an optimal proportioning culture medium and fermentation conditions to ferment and extract a strain of marine streptomycete to obtain an active crude extract, and adopts modern chromatographic separation technologies such as positive and negative phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, preparative HPLC and the like to research active secondary metabolites. 1 active small molecular monomer compound resisting banana vascular wilt is obtained through coseparation, and the structure of the compound is identified by adopting modern spectrum technologies such as 1D-NMR, 2D-NMR and HR-MS and combining with data information reported by literatures.

The first aspect of the invention provides the application of streptomyces samsonii XJC-2-6 in preparing oxytetracycline.

The second aspect of the invention provides the application of the fermentation liquor of streptomyces samsunus XJC-2-6 in the preparation of the oxytetracycline.

The third aspect of the invention provides the application of the extract of the streptomyces samsonii XJC-2-6 fermentation liquor ethyl acetate in the preparation of the oxytetracycline.

Wherein the ethyl acetate extract of the streptomyces samsunus XJC-2-6 fermentation liquor is obtained by adding ethyl acetate into the supernatant obtained by adding ethanol into the streptomyces samsunus XJC-2-6 fermentation liquor, extracting and filtering, and extracting and concentrating an ethyl acetate phase.

The amount of ethanol to be added is not particularly limited in the present invention, and may be added by experience by those skilled in the art.

The amount of ethyl acetate to be added is not particularly limited in the present invention, and may be added empirically by those skilled in the art.

The fourth aspect of the invention provides a preparation method of the oxytetracycline, which is separated from the fermentation liquor of streptomyces samsunus XJC-2-6.

Preferably, the preparation method comprises the following steps: (1) inoculating streptomyces samsonii XJC-2-6 into a fermentation culture solution for fermentation culture to obtain a fermentation solution; (2) adding appropriate amount of ethanol into the fermentation broth for extraction, filtering, collecting supernatant, adding appropriate amount of ethyl acetate for extraction, collecting ethyl acetate phase, and concentrating to obtain ethyl acetate extract; (3) separating the ethyl acetate extract with silica gel column chromatography, and purifying with CH₂Cl₂Gradient elution with MeOH/MeOH system (v/v: 100% CH)₂Cl₂100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, 100% MeOH), performing antibacterial property test on each obtained component, and performing TLC-direct bioautograph antibacterial activity detection by using Foc TR4 as a target pathogen to obtain a main active component fr.a; (4) subjecting the active component Fr.A to Sephadex LH-20 gel column chromatography, eluting with CH2Cl2/MeOH (v/v:2:1) eluent, testing antibacterial property of each obtained component, and detecting antibacterial activity by TLC-direct bioautoagraphy to obtain a higher active component Fr.A-4; (5) repeatedly separating and purifying the active component Fr.A-4 by RP-HPLC to obtain the oxytetracycline ketone.

Further preferably, in the step (1), the fermentation culture solution is M6 liquid culture medium, the inoculation amount is 5%, and the shaking culture is carried out at 28 ℃ and 180r/min for 8 d.

The amount of ethanol to be added is not particularly limited in the present invention, and may be added by experience by those skilled in the art.

The amount of ethyl acetate to be added is not particularly limited in the present invention, and may be added empirically by those skilled in the art.

The fifth aspect of the invention provides the application of the oxytetracycline in antagonizing No. 4 physiological race of fusarium oxysporum f.sp.cubense.

The sixth aspect of the invention provides the application of the oxytetracycline ketone in preparing medicines for preventing and treating diseases caused by No. 4 physiological races of banana vascular wilt.

The seventh aspect of the invention provides the application of the oxytetracycline ketone in reducing the soluble total sugar content, the soluble total protein content, the fat content and the activity of mitochondrial respiratory chain complex enzymes I to IV in the No. 4 physiological race thallus of the fusarium oxysporum f.sp.

The invention takes banana vascular wilt disease No. 4 physiological microspecies (Foc TR4) as target pathogenic bacteria, ferments streptomyces sempervirens XJC-2-6, extracts to obtain active crude extract, adopts the modern chromatographic separation technologies such as positive and negative phase silica gel column chromatography, Sephadex LH-20 gel column chromatography and preparative HPLC and the like to research active secondary metabolites, finally separates to obtain 1 banana vascular wilt disease resistant active small molecular monomer compound, adopts the modern wave spectrum technologies such as 1D-NMR, 2D-NMR and HR-MS and the like, combines the data information reported by the literature to identify the structure of the compound, namely the compound is the aspergillone (terrein), which is obtained by adopting actinomycetes fermentation and separation for the first time, the compound can effectively antagonize the banana vascular wilt disease No. 4 physiological microspecies, destroy the hypha structure and inhibit the hypha growth, EC (EC)₅₀、EC₇₅And EC₉₅Respectively 25.76 mug/mL, 86.30 mug/mL and 491.37 mug/mL, and can obviously reduce the soluble total sugar content, the soluble total protein content, the fat content, the activities of mitochondrial respiratory chain complex enzymes I to IV and the like in the No. 4 physiological race thalli of the fusarium oxysporum f.sp. The invention provides a new idea for preparing the oxytetracycline, develops a new field for preventing and treating plant diseases such as blight and the like, and has wide development space and good development and application prospects.

Drawings

FIG. 1 is a flow chart of the separation and purification of chemical components of Streptomyces 2-6 ethyl acetate extract.

FIG. 2 is the structure of Compound A7.

FIG. 3 is a graph showing the inhibitory effect of Compound A7 on mycelial growth of Foc TR 4.

FIG. 4 shows the effect of compound A7 on morphological changes of Foc TR4 hyphae under scanning electron microscopy, with CK on the left and compound A7 on the right.

FIG. 5 shows the effect of Compound A7 on conidia morphosis of Foc TR4 under transmission electron microscopy, with CK on the left and compound A7 on the right.

FIG. 6 shows the effect of compound A7 on the ultrastructural change of cells in Foc TR4 under a transmission electron microscope, wherein A is a control, and B and C are compound A7 treatments.

FIG. 7 is a graph showing the effect of active compound A7 on the intracellular N-acetylglucosamine content of Foc TR 4.

FIG. 8 shows the effect of active compound A7 on total intracellular sugars of banana vascular wilt disease # 4 race.

FIG. 9 shows the effect of active compound A7 on the content of soluble protein in the bacterial cells of the banana vascular wilt disease No. 4 race.

FIG. 10 shows the effect of active compound A7 on the intracellular fat content of banana vascular wilt disease number 4 race.

FIG. 11 shows the effect of active compound A7 on mitochondrial complex enzyme in No. 4 microspecies of banana vascular wilt.

Detailed Description

The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

1 test Material

1.1 test strains

Streptomyces samsunensis XJC-2-6(Streptomyces samsunensis XJC-2-6) (hereinafter referred to as Streptomyces 2-6) is separated and screened from a sample collected from south China sea coral, and is preserved in China center for type culture collection in 2017 at 10 and 23 months with the preservation number of CCTCC NO: m2017620. The morphological characteristics, culture characteristics, physiological and biochemical characteristics, and the identification of the molecular biological characteristics of 16S rRNA are described in patent numbers: 201711435432.5, patent name: the invention relates to a Chinese patent of streptomyces samsunensis and application thereof, which is not repeated herein.

1.2 test pathogens

Banana fusarium oxysporum f.sp.cubense Race 4(ATCC 76255) (Foc TR 4).

1.3 test Medium

The main media used in this study are shown in Table 1.

TABLE 1 fermentation media and formulations

1.4 Primary reagents

The main reagents used in this study are shown in table 2.

TABLE 2 major Biochemical reagents and sources

1.5 Main Instrument

The main instruments used in this study are shown in Table 3.

TABLE 3 Main instruments

2 test methods and results

All experiments were performed in triplicate, three replicates were set up and data results are expressed as mean ± Standard Deviation (SD). Differences between the mean values obtained in each treatment were assessed by analysis of variance (ANOVA; SAS 9.2), with p <0.05 indicating statistically significant differences.

2.1 Streptomyces fermentation and metabolite extraction

Streptomyces 2-6 is inoculated into ISP2 liquid culture medium, and shake culture is carried out for 4d under the conditions of 28 ℃ and 180 r/min. Inoculating fresh bacterial liquid into a 5L triangular flask filled with 1L M6 liquid culture medium according to the inoculation amount of 5%, and performing shaking culture at 28 ℃ for 8d at 180r/min to obtain 120L of fermentation liquid. Mixing with anhydrous ethanol at a ratio of 1:1(v/v), ultrasonic extracting for 1 hr, filtering, collecting supernatant, and concentrating under reduced pressure at 45 deg.C to obtain 10L. Performing ultrasonic extraction with ethyl acetate at a volume of 1:1(v/v) for 5 times (2L/time, 30min), mixing ethyl acetate phases, concentrating under reduced pressure at 45 deg.C, and removing ethyl acetate to obtain 26.33g of ethyl acetate extract. Activity tests prove that the ethyl acetate extract has obvious bacteriostatic activity on Foc TR4 pathogenic bacteria.

2.2 TLC detection

TLC analysis method, sucking sample with 0.3mm capillary pipette, spotting at 1cm distance from the bottom of GF254 silica gel plate, and developing after solvent evaporation. And (3) expanding the sample by adopting an upright ascending expansion method, adding an expanding agent into the chromatographic cylinder in advance, placing the chromatographic cylinder into a thin-layer chromatography plate after 20min, carrying out chromatography, taking out the silica gel chromatography plate when the front edge of the expanding agent is 1.0cm away from the edge of the silica gel plate, marking the position of the front edge of the solvent, observing the result at 254nm of an ultraviolet analyzer and calculating the Rf value.

2.3 TLC-bioautograph bioactivity assay

And (3) taking the physiological race of banana vascular wilt No. 4 (Foc TR4) as a target pathogen, and detecting the bacteriostatic activity of the metabolite by adopting a TLC-bioautography method. Preparing PDA culture medium, inoculating Foc TR4, culturing at 24 deg.C for 7-10 days, adding 10mL sterile water to elute spore, preparing conidium suspension, adding appropriate amount of PDB liquid culture medium, and making into 3.0 × 10⁵spore/mL spore suspension mix. The crude extract was dissolved in methanol to a concentration of 20mg/mL, and samples of 4. mu.L and 8. mu.L were spotted on TLC plates using calibrated capillaries, onto which spore suspensions (3.0X 10) were uniformly sprayed (Foc TR4)⁵spores/mL) three times, placing the TLC plate in a humid box, 12h light at 25 ℃ in an incubator, 12h dark, exchanging for 4d day and night, when a blank area appears on the TLC plate, indicating that the growth of fungi is inhibited, the crude extract contains antifungal components, and recording the diameter of inhibition zone.

2.4 Ethyl acetate extractum silica gel column chromatography

Adding ethyl acetate into methanolDissolving the extract, adding n-hexane with the same volume, extracting for three times, removing small polar components such as oil and fat in the extract, completely drying the residual methanol layer sample, and adding a small amount of CH₂Cl₂Dissolving (DCM) (adding a little MeOH dropwise when not completely dissolving), weighing equivalent 100-mesh 200-mesh silica gel, adding sample liquid dropwise, uniformly stirring, and keeping for later use after the silica gel sample is completely dried. Taking a small amount of sample, detecting by silica gel Thin Layer Chromatography (TLC), and selecting CH₂Cl₂The extract is subjected to normal phase silica gel column chromatography (200-300 meshes) by using a/MeOH system as an eluent. By CH₂Cl₂Gradient elution with MeOH/MeOH system (v/v: 100% CH)₂Cl₂100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, 100% MeOH), fractions were collected, 10mL was collected per tube, and 7 fractions (fr.a to fr.g) were combined by TLC detection. And (3) performing antibacterial activity detection by adopting a TLC-bioautograph method, wherein the target pathogenic bacteria is Foc TR4, and obtaining an active component Fr.A. The active component methanol is dissolved out and purified by silica gel column chromatography repeatedly.

Detecting streptomycete 2-6 ethyl acetate extract by silica gel Thin Layer Chromatography (TLC) sample application, selecting CH₂Cl₂the/MeOH system elutes as the mobile phase. Subjecting 26.33g of the crude extract to silica gel column chromatography (200-300 mesh), CH₂Cl₂Gradient elution with MeOH/MeOH system (v/v: 100% CH)₂Cl₂100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1, 100% MeOH), collecting 65 tube fractions, combining 7 components Fr.A (2.0782g), Fr.B (3.0783g), Fr.C (5.5961g), Fr.D (0.7065 g), Fr.E (1.3725g), Fr.F (0.9760g), Fr.G (1.5719g) and Foc TR4 as target pathogens, and detecting the antibacterial activity of TLC-direct bioautoagraph to obtain a main active component Fr.A (2.0782 g).

2.5 active ingredient Sephadex LH-20 gel column chromatography

The active components obtained after silica gel column chromatography, TLC detection and bioactivity determination are dissolved by a small amount of methanol, and are subjected to wet-process sample loading and Sephadex LH-20 gel column chromatography separation (3cm multiplied by 150 cm). By CH₂Cl₂the/MeOH (v/v:2:1) mobile phase was eluted, fractions were collected, 8mL of each tube was collected, and fractions were combined by TLC. Using TLC-bioautographThe method is used for detecting the antibacterial activity, and the target pathogenic bacteria is Foc TR4 to obtain an active component Fr.A-4.

Subjecting active component Fr.A to Sephadex LH-20 gel column chromatography, and purifying with CH₂Cl₂Eluting with eluent/MeOH (2:1), collecting 51 tube fractions, detecting by TLC, mixing to obtain 5 components Fr.A-1 (0.9233g), Fr.A-2(0.1685g), Fr.A-3(0.2759g), Fr.A-4(0.3396g), and Fr.A-5(0.1922 g), and detecting by TLC-direct bioautograph to obtain higher active component Fr.A-4(0.3396 g).

2.6 RP-HPLC separation and purification of active ingredient

The semi-preparation conditions of the high performance liquid chromatography are as follows: agilent 1100, UV/RID detector, column temperature 30 ℃; the column was YMC Pack ODS-A (250X 10mm, 5 μm); the mobile phase is MeOH/H2O; the detection wavelengths are 210, 230,254 and 305nm respectively; manually injecting samples, wherein the sample injection amount is 50 mu L; the flow rate was 0.2 mL/min.

Subjecting to Sephadex LH-20 gel column chromatography to obtain active component Fr.A-4, and repeatedly separating and purifying by RP-HPLC to obtain high purity monomer compound A1(5.1mg, MeOH: H)₂O＝60:40,2mL/min,tR ＝59min)、A5(9.1mg,MeOH:H₂O＝60:40,2mL/min,tR＝59min)、A6(3.2mg, MeOH:H₂O＝60:40,2mL/min,tR＝59min)、A7(15.3mg,MeOH:H₂O60: 40, 2mL/min, tR 30.5 min). And (5) determining A7 as a high-activity compound through antibacterial activity detection, and performing structure identification.

Compound A7 is a dark brown solid with molecular formula C₈H₁₀O₃Melting point 133-]⁺,139,121,109,95,79。¹H-NMR(600MHz,CD₃OD)δ_H 4.68(1H,d,J ＝2.5Hz,H-3),4.08(1H,d,J＝2.5Hz,H-2),6.00(1H,s,H-5),6.44(1H,m,J＝16.0 Hz,1.2Hz,H-6),6.83(1H,dq,J＝16.0Hz,1.2Hz,H-7),1.94(3H,dd,J＝6.5Hz,1.2 Hz,CH₃-8)；¹³C-NMR(150MHz,CD₃OD)δ_C 205.60(C-1),82.41(C-2),78.11(C-3), 170.83(C-4),125.92(C-5),126.42(C-6),141.83(C-7),19.49(C-8)。¹The H-NMR spectrum shows 3 double bond hydrogen signals in the low field region: delta_H 6.83(dq,J＝16.0Hz,1.2Hz,1H)，6.44(m,J＝ 16.0Hz,1.2Hz,1H), 6.00(s, 1H); 2 continuous oxygen hydrogen signal: delta_H4.08(d, J ═ 2.5Hz,1H),4.68 (d, J ═ 2.5Hz, 1H); the high field region has 1 methyl hydrogen signal: delta_H 1.94(dd,J＝6.5Hz,1.2Hz,3H)；³C-NMR and DEPT spectra show that the compound contains 8 carbon atoms in total, and the low field region has 1 carbonyl carbon signal: delta_C205.60, 4 double bond carbon signals: delta_C170.83, 141.83, 125.92, 126.42, identifying compounds containing two double bonds; 2 vicinal oxygen carbon signals: delta_C82.41.11, judged from chemical shifts to be typical ring hydroxyl substitution structures, it is concluded that the compound contains two hydroxyl substitutions; the high field region has 1 methyl carbon signal: delta_C19.49. According to the compound¹H-NMR、¹³C-NMR and DEPT spectra, and looking up the literature for comparison, compound A7 spectral data were found to be consistent with those reported in the literature, and compound A7 was determined to be terrein (Raistrick et al, 1935; Kim et al, 2005; Asfour et al, 2019). The planar structure of the compound A7 was resolved by 2D-NMR spectroscopy, and the nuclear magnetic data of the compound are shown in Table 4 as shown in FIG. 2.

TABLE 4 NMR data (J units: Hz) of Compound A7

¹H-NMR represents 600 MHz;¹³C-NMR represents 150 MHz.

2.7 bacteriostatic action of active ingredient A7 on Foc TR4 hyphal growth

The inhibitory activity of Streptomyces metabolites on the growth of banana fusarium oxysporum filaments was evaluated by the growth rate method (Sharma et al, 2016; Sharma et al, 2017). PDA plates with different concentrations of active ingredient were prepared by 2-fold serial dilution by adding active ingredient A7 dissolved in sterile water (10.0mg/mL) to PDA medium at 45-50 deg.C, to final concentrations of 100.0, 50.0, 25.0, 12.5, 6.25, and 3.125. mu.g/mL, respectively, with the same amount of sterile water added as control. Fungal cake of Foc TR4 (phi. 5mm) was inoculated into the center of each plate and incubated at 28 + -2 deg.C until the control hyphae reached the edge of the plate. The average of the vertical diameters of the respective colonies was determined. Each treatment was repeated 3 times. The formula for the inhibition of hyphal growth is as follows (nimachond et al, 2015):

in the formula: c is the average diameter of colonies in the control group, and T is the average diameter of colonies in the treatment group.

The effect of active ingredient A7 on the growth of hyphae of the target pathogen Foc TR4 was determined using the growth rate method at concentrations of 100.0, 50.0, 25.0, 12.5, 6.25 and 3.125. mu.g/mL, as shown in FIG. 3. The virulence regression equation was determined by measuring colony diameter and model calculations, and the EC for Compound A7 was calculated as shown in Table 5₅₀、 EC₇₅And EC₉₅25.76. mu.g/mL, 86.30. mu.g/mL and 491.37. mu.g/mL, respectively. The concentration of the active ingredients is positively correlated with the hypha growth inhibition, and the higher the concentration is, the stronger the inhibition effect is.

TABLE 4 bacteriostatic effects of Compound A7 on Foc TR4

2.8 Effect of active ingredient A7 on the morphology and intracellular ultrastructure of Foc TR4 pathogenic bacteria

Respectively adopting a scanning electron microscope and a transmission electron microscope observation method to research the teratogenicity effect of the streptomycete active ingredient A7 on Foc TR4 pathogenic bacteria hypha, pathogenic bacteria conidium and an intracellular ultrastructure of the pathogenic bacteria.

(1) Scanning electron microscope observation of Foc TR4 pathogen mycelium changes

A0.5 cm cake was applied to the center of the plate containing the active ingredient A7 by using a punch at the edge of the colony of the target pathogen Foc TR 4. After culturing for 5 days at 28 ℃, cutting hypha tips at the edges of bacterial colonies by using a blade and cutting off a culture medium as much as possible, fixing the hypha tips overnight at 4 ℃ by using 2.5% (w/v) glutaraldehyde solution, rinsing the hypha tips for three times by using phosphate buffer solution, dehydrating the hypha tips for once by using 30%, 50%, 70% and 90% of ethanol step by step, dehydrating the hypha tips for two times by using 100% of ethanol, dehydrating the hypha tips for 20min each time, eluting the ethanol for two times by using isoamyl acetate, eluting the ethanol for 30min each time, drying the ethanol in vacuum, and spraying gold for observation.

As can be seen from FIG. 4, the hyphae of the control group had compact and smooth surface, uniform and full shape, good integrity, and a large number of microspores were produced around the hyphae. After being treated by the active ingredient A7, the hyphae of the pathogenic bacteria have rough and uneven surfaces, are shrunk and thinned, and have damaged integrity, and have the phenomena of breakage and rupture, so that the generation of conidia is inhibited. The experiment shows that the compound A7 can destroy the hypha structure of pathogenic bacteria, and further inhibit the growth of the pathogenic bacteria and the generation of conidia.

(2) Scanning electron microscope observation of Foc TR4 pathogen conidiophore change

Preparation of Foc TR4 spore suspension (1X 10)⁶CFU/mL), 5. mu.L of spore suspension was placed on a glass slide with 5. mu.L of EC₅₀The active ingredient A7 was treated at a concentration and incubated for 24h with sterile water as a control. Fixing the glass slide with 2.5% glutaraldehyde at 4 deg.C overnight, rinsing with phosphate buffer solution three times, dewatering with 30%, 50%, 70%, 90% ethanol once step by step, dewatering with 100% ethanol twice each for 20min, eluting with isoamyl acetate twice each for 30min, vacuum drying, spraying gold, and observing.

The effect of active ingredient A7 of Streptomyces 2-6 on conidia of the Foc TR4 pathogen was observed by scanning electron microscopy, as shown in FIG. 5. SEM images show active ingredient treated pathogens, spore deformation, shrinkage, collapse, bending and head swelling, with integrity destroyed, and the appearance of fragmentation and rupture. The control group had plump spores, smooth surface and intact spore morphology. The experiment shows that the compound A7 can destroy the conidium structure of pathogenic bacteria, thereby inhibiting the growth of the pathogenic bacteria.

(3) Transmission electron microscope observation for Foc TR4 pathogenic bacteria cell ultrastructure change

A0.5 cm cake was taken at the edge of the target colony by a punch and inoculated into the center of the plate containing the active ingredient A7. After culturing at 28 ℃ for 5 days, cutting hypha tips at the edges of colonies by using a blade, cutting off the culture medium as much as possible, fixing the hypha tips with 2.5% (w/v) glutaraldehyde solution at 4 ℃ overnight, rinsing the hypha tips with phosphate buffer solution for three times, dehydrating the hypha tips with 30%, 50%, 70% and 90% ethanol step by step once, dehydrating the hypha tips with 100% ethanol twice for 20min each time, and immersing the sample in propylene oxide for 2 times for 20min each time. The samples were prepared in a propylene oxide: after the epoxy resin (1:1) solution is soaked for 1h for embedding, the embedded object is cut into 70nm ultrathin sections by a diamond knife. Sections were stained with uranyl acetate and lead citrate for 30min, respectively, and observed with a transmission electron microscope (Phillips et al, 2003).

The effect of active ingredient A7 of Streptomyces 2-6 on the ultrastructure of the pathogenic bacteria Foc TR4 is shown in FIG. 6. The control group of pathogenic bacteria was observed by transmission electron microscope to have full cell shape, complete structure, complete organelles, complete cell wall, uniform cytoplasm, regular mitochondrial shape and uniform body type (fig. 6A). After the treatment of the active compound, the cell wall of Foc TR4 pathogenic bacteria is obviously thinned, organelles are dissolved and disappear, cell tissues are disintegrated, cell vacuoles are formed, and vesicles appear (figure 6B); cytoplasmic electron density increased, mitochondrial number significantly increased, mitochondrial morphology aberrant, showing rough collapse of surface, lack of matrix, clear visualization of inner and outer membranes, ridge swelling and structural disorder (fig. 6C).

2.9 Effect of active ingredient A7 on the physiological metabolism of Foc TR4 pathogenic bacteria

(1) Effect of active ingredient A7 on the cell wall chitinase of pathogenic bacterium Foc TR4

Standard curve of N-acetylglucosamine: a100. mu.g/mL N-acetylglucosamine standard solution was prepared, and the gradient standard solution was diluted to 20, 40, 60, 80, 100. mu.g/mL. Adding 1mL of gradient standard solution into a test tube, adding 0.5mL of potassium borate (0.8mol/L of potassium borate solution is added in 0.8mol/L), boiling in a water bath for 3min, cooling, adding 3mL of DMAB (p-dimethylaminobenzaldehyde) with the mass fraction of 1%, keeping the temperature at 36 ℃ for 20min, cooling, and measuring the absorbance at the wavelength of 544nm by using an ultraviolet spectrophotometer.

100mL of PDB culture medium is added into a 250mL triangular flask, Foc TR4 pathogenic bacteria is inoculated, active ingredients A7 (the concentration is 3.125 mu g/mL, 6.25 mu g/mL, 12.5 mu g/mL, 25.0 mu g/mL and 50.0 mu g/mL) with different concentration gradients are added at the same time, the mixture is subjected to shaking culture at 28 ℃ for 5 days at 180r/min, the mixture is centrifuged at 5000r/min for 15min to collect hyphae, the hyphae is washed with sterile water for 3 times, and then the test is carried out. Weighing 1.0g of hypha, adding 5mL of Tris-HCl, grinding in ice bath, centrifuging at 10000r/min at 4 ℃, and taking supernatant to store at-20 ℃ for later use. Adding 1.0mL of thallus supernatant subjected to different treatments into a clean test tube, adding 0.5mL of potassium borate solution (0.8mol/L), carrying out boiling water bath for 3min, cooling, adding 3mL of DMAB (p-dimethylaminobenzaldehyde) with the mass fraction of 1%, carrying out heat preservation at 36 ℃ for 20min, cooling, and measuring the absorbance at the wavelength of 544nm by using an ultraviolet spectrophotometer. The N-acetylglucosamine content of the chitin hydrolysate was calculated by a standard curve.

The effect of the active ingredient on the change in the intracellular N-acetylglucosamine content of the pathogenic bacterium Foc TR4 is shown in FIG. 7. As the concentration of active compound A7 increased, the N-acetylglucosamine content showed a gradual increase in the concentration. In the compound A7 treatment group, the content of N-acetylglucosamine is increased sharply and the increase slope is larger in the concentration range of 0-25 mug/mL. In the concentration range of 25-50 mug/mL, the rising trend of the content of the N-acetylglucosamine becomes slow, and the increasing slope becomes small. Therefore, 25. mu.g/mL is the concentration node of Compound A7 treatment, and this concentration has an N-acetylglucosamine content of 56.48. mu.g/g. Chitin is the main component of the cell wall of pathogenic bacteria, N-acetylglucosamine is the final product of chitin hydrolysis, and the change of N-acetylglucosamine can reflect the change of the cell wall of pathogenic bacteria. The increase in N-acetylglucosamine content with increasing treatment concentration indicates that the concentration of active ingredient A7 increases, resulting in increased hydrolysis of chitin, cell wall destruction, and increased destruction. The active compound of the streptomycete 2-6 reduces the viability of pathogenic bacteria by decomposing the cell walls of the pathogenic bacteria, thereby achieving the purpose of inhibiting the growth of the pathogenic bacteria.

(2) Effect of active Compound A7 on the Total sugar, protein and fat content of the Foc TR4 pathogen

Determination of total sugar (DNS method): 2mL of a glucose solution with a final concentration of 0, 40, 80, 120, 160, 200. mu.g/mL was prepared, and 1.5mL of 3,5-Dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) reagent was added. Mixing, placing in 100 deg.C constant temperature water bath, keeping the temperature for 5min, cooling to room temperature, adding distilled water to dilute to 20mL, mixing well, and measuring absorbance at 540nm wavelength. The glucose content is plotted on the abscissa and the absorbance is plotted on the ordinate to form a standard curve. 100mL of PDB medium was added to a 250mL Erlenmeyer flask, the Foc TR4 pathogen was inoculated, simultaneously, active ingredient A7 (concentrations of 3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25.0. mu.g/mL, and 50.0. mu.g/mL) was added at different concentration gradients, cultured at 180r/min with shaking at 28 ℃ for 5d, centrifuged at 5000r/min for 15min to collect the mycelia, and the mycelia were rinsed with sterile water and dried. Weighing 1.0g of prepared mycelium, placing in a precooled mortar, adding 6ml of Tris-HCl leaching liquor, grinding in ice bath until homogenate is obtained, and centrifuging at 4 ℃ at 10000r/min for 10 min. Adding 2mL of HCl (6mol/L) into 1mL of supernatant, boiling in a water bath for 30min, cooling by flowing, neutralizing with NaOH (6mol/L) solution to neutrality, adding 1.5mL of DNS into the solution, boiling in a water bath for 5min, adding 1.5mL of DNS into 2mL of supernatant, and boiling in a water bath for 5 min. Taking out, cooling to room temperature, adding distilled water to dilute to 20mL, and mixing uniformly. Absorbance was measured at a wavelength of 540nm and total sugar content was calculated by a standard curve (river et al, 1984).

As can be seen from fig. 8, the soluble total sugar content in the bacterial cells of the pathogenic bacterium Foc TR4 gradually decreased with increasing concentration of active ingredient a7 upon treatment with active compound a7, and there was no significant difference between the low concentration treatment and the high concentration treatment compared to the control. After the treatment of the compound A7 with the concentrations of 3.125 mu g/mL, 6.25 mu g/mL, 12.5 mu g/mL, 25.0 mu g/mL and 50.0 mu g/mL, the total sugar contents of the pathogenic bacteria of Foc TR4 are respectively 1.59 +/-0.04 mg/g, 1.53 +/-0.0 mg/g, 1.47 +/-0.07 mg/g, 1.28 +/-0.02 mg/g and 1.12 +/-0.04 mg/g, while the control is 1.62 +/-0.04 mg/g, and the treatment of the concentration of 3.125 mu g/mL has no significant difference from the control; compared with the control, the soluble total sugar content of the treatment groups is respectively reduced by 2.06%, 5.56%, 9.47%, 20.78% and 30.66%. The saccharides are main carbon sources and energy reserve substances for microbial metabolism, when the concentration of the active ingredient A7 is increased, the growth and metabolism rate of the banana vascular wilt germs is slowed down, the rate of synthesizing energy substances is also slowed down, the energy consumption rate is accelerated,so that the total sugar content is reduced proportionally. The results show that, after treatment with active compound A7, there is a sharp decrease in the total sugar content of the pathogenic bacterium Foc TR4 at 25. mu.g/mL, the concentration being the EC of this compound A7₅₀Regional range of values.

Measurement of protein content: 100mL of PDB medium was added to a 250mL Erlenmeyer flask, the Foc TR4 pathogen was inoculated, simultaneously, active ingredient A7 (concentrations of 3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25.0. mu.g/mL, and 50.0. mu.g/mL) was added at different concentration gradients, cultured at 180r/min with shaking at 28 ℃ for 5d, centrifuged at 5000r/min for 15min to collect the mycelia, and the mycelia were rinsed with sterile water and dried. Weighing 1.0g of prepared mycelium, placing into a precooled ground body, adding 6ml of Tris-HCl leaching liquor, grinding in ice bath until homogenate is obtained, and centrifuging at 4 ℃ at 10000r/min for 10 min. 0.1mL of the supernatant was added with 0.9mL of distilled water, and then 5mL of Coomassie Brilliant blue G-250 solution was added, mixed well, left to stand for 5min, and the absorbance was measured at 595nm, and the protein content was calculated by referring to a standard curve (Bradford, 1976).

As is clear from FIG. 9, the soluble protein content in the bacterial cells of the pathogenic bacterium Foc TR4 gradually decreased with increasing concentration of active ingredient A7, all after treatment with active compound A7. After the compound A7 with the concentrations of 3.125 mu g/mL, 6.25 mu g/mL, 12.5 mu g/mL, 25.0 mu g/mL and 50.0 mu g/mL is treated, the content of the soluble protein of the pathogenic bacteria of Foc TR4 is respectively 2.70 +/-0.06 mg/g, 2.37 +/-0.11 mg/g, 2.20 +/-0.12 mg/g, 2.06 +/-0.12 mg/g and 1.74 +/-0.06 mg/g, while the content of the soluble protein of the pathogenic bacteria of the control group is 2.81 +/-0.10 mg/g, and all the concentration treatments are obviously different from the control group; the soluble total protein content of the treated group was reduced by 3.78%, 15.54%, 21.86%, 26.67%, 37.91%, respectively, compared to the control.

Measuring the fat content: determination of fat content methods of the literature (old, 2002) were performed: 100mL of PDB medium was added to a 250mL triangular flask, the Foc TR4 pathogen was inoculated, simultaneously, active ingredients A7 (concentrations of 3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25.0. mu.g/mL and 50.0. mu.g/mL) were added at different concentration gradients, cultured at 180r/min with shaking at 28 ℃ for 5 days, centrifuged at 5000r/min for 15min to collect the mycelia, and the mycelia were rinsed with sterile water. Accurately weighing 0.5g of mycelium in a filter paper cylinder, sealing two ends of the paper cylinder, and placing in a Soxhlet fat extractor. Connecting a constant-weight liposuction bottle, and adding anhydrous ether (with a boiling range of 30-60 ℃) from the upper end of a condenser. Heating and extracting for 12-16 h on a water bath at 50-60 ℃. After extraction, residual ether is steamed in a water bath, and then dried in an oven at 100-105 ℃ for 8h to constant weight. The calculation formula is as follows:

the results of the effect of active compound A7 on the fat content in the bacterial cells of Foc TR4 pathogen are shown in FIG. 10. After treatment with compound A7 at concentrations of 3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25.0. mu.g/mL and 50.0. mu.g/mL, the fat contents of the pathogenic bacteria Foc TR4 were 5.37%, 5.24%, 5.14%, 4.79% and 3.25%, respectively, compared to 5.43% in the control group, and the fat contents were not significantly different from the control group under low concentration (3.125. mu.g/mL and 6.25. mu.g/mL) treatment with compound A7; and the fat content in pathogenic bacteria is obviously reduced by 11.72 percent and 40.09 percent respectively when the treatment is carried out at higher concentrations of 25 mu g/mL and 50 mu g/mL. Thus, a concentration of 25 μ g/mL of active compound a7 can have a significant effect on target pathogen biosynthesis.

(3) Effect of active ingredients on mitochondrial respiratory chain (ETC) Complex enzyme I-IV Activity

Extraction and pretreatment of mitochondria

And (4) centrifuging to collect hyphae treated by active ingredients with different concentrations. Hyphal mitochondria were extracted by sucrose differential centrifugation (Mizutani et al, 1995), 5g of the mycelia were washed three times with a pre-cooled HEPES-Trisbuffer (20mM, pH 7.2), then the mycelia were resuspended in 3 volumes of pre-cooled mitochondrial extraction buffer (250mM sucrose, 10mM KCl, 5mM EDTA, 20mM HEPES-Tris, pH 7.2, 1.5mg/mL BSA), placed in 50mL centrifuge tubes, and disrupted by vortexing with glass beads (Fishbein et al, 1993). Homogenizing at 4 deg.C, centrifuging at 4000r/min for 15min, collecting supernatant, and discarding precipitate. Taking the supernatant, continuing to centrifuge at 12000r/min, centrifuging at 4 ℃, discarding the supernatant, washing the precipitate with a BSA-free mitochondrial extraction buffer, and centrifuging at 10000r/min at 4 ℃ for 20 min. The precipitate was a crude mitochondrial particle extract (Barrientos, 2002).

Resuspend mitochondrial particles in 400. mu.L of pre-cooled mitochondrial preservation buffer (2mM HEPES, 0.1mM EGTA, 250mM sucrose, pH 7.4) leaving 60-70. mu.L and complex enzyme III activity assay. After 10. mu.L of the mitochondrial solution was taken and protein concentration was measured by the Bradford method (Bradford M, 1976) using BSA as a standard protein, the protein concentration was adjusted to 1mg/mL with a mitochondrial preservation buffer and placed on ice for later use, or was stored by quick freezing with liquid nitrogen at-20 ℃ after being dispensed. Before measuring the enzyme activity, the mitochondrial solution was repeatedly frozen and thawed 3 times at liquid nitrogen/room temperature.

Centrifuging the residual 330-340 mu L of solution at 4 ℃ at 10000r/min for 20min, resuspending the precipitate in 1mL of hypotonic buffer solution, centrifuging the precipitate at 4 ℃ at 10000r/min for 20min, and then resuspending the precipitate in 300 mu L of hypotonic buffer solution. Used for complex enzyme I, complex enzyme II and complex enzyme IV. Taking 10 mu L of mitochondrial solution, using BSA as standard protein, measuring the protein concentration by a Bradford method, adjusting the protein concentration to 1mg/mL by using hypotonic buffer solution, placing on ice for later use, or quickly freezing by liquid nitrogen after subpackaging, and storing at-20 ℃. Before the mitochondrial complex enzyme detection, the solution is repeatedly frozen and thawed for 3 times at the liquid nitrogen/room temperature and then is subjected to subsequent detection. The complex enzyme I-IV is measured at 37 ℃, the total volume of the reaction system is 1mL, and the addition amount of mitochondrial protein is 20-40 mu g. The method for detecting the activity of mitochondrial complex enzymes I to IV is carried out according to a known literature method (Spinazzi, et al, 2012).

② influence on the Activity of mitochondrial respiratory chain Complex enzyme I

Complex enzyme I (rotenone-sensitive NADH-decyl ubiquinone oxidoreductase): the measurement was carried out by measuring the decrease in absorbance at 340nm caused by the oxidation of NADH, using decyl ubiquinone as an electron acceptor and NADH as an electron donor. mu.L of purified water was added to a 1mL cuvette, 8. mu.L of the pretreated mitochondrial solution (1mg/mL) was added, and the mixture was incubated at 37 ℃ for 2 min. mu.L of potassium phosphate buffer (0.5M, pH 7.5), 60. mu.L of fatty acid-free BSA (50mg/mL), 30. mu.L of KCN (10mM), and 10. mu.L of ADH (10mM) were added, respectively. In the other cuvette of the parallel test 10. mu.L of rotenone (1mM) was added, the rotenone-insensitive compomer activity was determined and the rotenone-sensitive compomer enzyme I activity was quantified after subtraction. Blending with pure waterThe volume of the reaction mixture was adjusted to 994. mu.L. The cuvette sealed with the Parafilm was tumbled and the reaction mixture was mixed well and monitored at 340nm for a baseline of 2 min. The reaction was initiated by adding 6. mu.L of decyl ubiquinone (10mM), and the decrease in absorbance at 340nm within 2min was measured (NADH molar extinction coefficient. epsilon.: 6.2 mM)^-1·cm^-1). Calculating the formula:

l: optical path length (cm); v ═ sample volume (mL); [ prot ] ═ sample protein concentration (mg/mL)

(iii) Effect on the Activity of mitochondrial respiratory chain Complex enzyme II

Complex enzyme II (decyl ubiquinone succinate DCPIP reductase, succinate-ubiquinone oxidoreductase): DCPIP was used as an electron acceptor and succinic acid as an electron donor, and the decrease in the absorbance at 600nm due to the reduction of DCPIP was measured. mu.L of purified water was taken and added to a 1mL cuvette, 50. mu.L of potassium phosphate buffer (0.5M, pH 7.5), 20. mu.L of fatty acid-free BSA (50mg/mL), 30. mu.L of KCN (10mM), 50. mu.L of succinic acid (400mM), 8. mu.L of mitochondrial protein (1mg/mL) and 145. mu.L of DCPIP (0.15 mg/mL) were added, respectively, and the volume of the reaction mixture was adjusted to 995. mu.L with purified water. The reaction mixture was mixed by inverting the cuvette sealed with a Parafilm and incubating at 37 ℃ for 10min, and finally 2min at 600nm monitoring baseline. The reaction was started by adding 5. mu.L of decyl ubiquinone (10mM), and the decrease in absorbance at 600nm within 3min was measured (DCPIP extinction coefficient. epsilon. times.19.1 mM-1. cm-1). Calculating the formula:

influence on the Activity of mitochondrial respiratory chain Complex enzyme III

Complex enzyme III (panthenol Cyt c reductase, decyl panthenol Cyt c oxidoreductase): the measurement of the increase in the absorbance at 550nm caused by the reduction of oxidized Cyt c was carried out by using oxidized Cyt c as an electron acceptor and decyl panthenol as an electron donor. 730. mu.L of purified water was added to a 1mL cuvette, and 50. mu.L of potassium phosphate buffer (0.5M, pH 7.5), 75. mu.L of oxidized Cyt c (1mM), 50. mu.L of KCN (10mM), 20. mu.L of DTA (5mM, pH 7.5), 10. mu.L of Tween 20 (2.5%) and 8. mu.L of mitochondrial protein (1mg/mL) were added, respectively. In parallel test cuvette, in addition to the above reagents, 10. mu.L of antimycin A (1mg/mL) was added to determine the activity of the complex enzyme III insensitive to antimycin A, and the activity of the complex enzyme III insensitive to antimycin A was quantified after subtraction. The volume of the reaction mixture was adjusted to 990. mu.L with pure water. The cuvette sealed with the Parafilm was tumbled and the reaction mixture was mixed well and monitored at 550nm for a baseline of 2 min. The reaction was initiated by the addition of 10. mu.L of decyl panthenol (10mM), and the increase in absorbance at 550nm within 2min was measured immediately after the reaction mixture was rapidly mixed (reduced Cyt c extinction coefficient. epsilon. times.18.5 mM-1. cm-1). Calculating the formula:

influence of activity of mitochondrial respiratory chain complex enzymes I-IV

Complex enzyme IV (Cyt c oxidase): reduced Cyt c was used as an electron donor, and the decrease in the absorption value at 550nm due to oxidation of reduced Cyt c was measured. 400. mu.L of purified water was added to a 1mL cuvette, and 250. mu.L of potassium phosphate buffer (100mM, pH 7.0) and 50. mu.L of reduced Cyt c (1mM) were added, respectively. The cuvette sealed with the Parafilm was tumbled and the reaction mixture was mixed well and monitored at 550nm for a baseline of 2 min. The volume of the reaction mixture was adjusted to 992. mu.L with pure water. The reaction was initiated by adding 8. mu.L of mitochondrial protein (1mg/mL), and the decrease in absorbance at 550nm within 3min was measured (reduced Cyt c extinction coefficient. epsilon.: 18.5 mM-1. cm-1). Calculating the formula:

to investigate the effect of active compounds on mitochondrial electron transport chain complex enzyme activity, Foc TR4 was treated with compound A7 at concentrations of 3.125, 6.25, 12.5, 25 and 50 μ g/mL, respectively, and the activity of ETC complex enzymes I-IV was tested (FIG. 11). Compared with a control group, the activity of ETC complex enzymes I-IV is increased under low-concentration treatment; however, as the treatment concentration increases, the activity or content thereof gradually decreases without exception. The activity of compomers III-III increases to a maximum and begins to decrease at a concentration of 6.25. mu.g/mL and the activity of compomer IV increases to a maximum and begins to decrease at a concentration of 3.125. mu.g/mL with treatment of Compound A7. Compared with the control, the activity of the complex enzymes I to IV is reduced by 31.10 percent, 47.87 percent, 51.27 percent and 59.34 percent respectively at the concentration of 50 mu g/mL.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.