A biological preparation for preventing and treating Panax notoginseng root rot and its application

Abstract

The present invention discloses a novel biological preparation for preventing and treating Panax notoginseng root rot and its application. The main active ingredient of the preparation is Panax notoginseng total saponins. Ginsenoside Rb1 in the Panax notoginseng total saponins inhibits the growth of the Chryseobacterium MA9 strain, which is responsible for Panax notoginseng root rot. This further prevents and treats Panax notoginseng root rot, improves the survival rate of Panax notoginseng seedlings, increases yield, and significantly improves economic benefits. Furthermore, a new approach to preventing and treating Panax notoginseng root rot is provided.

Description

Technical Field

[0001] The present invention belongs to the field of microbial preparations, and in particular relates to a biological preparation for preventing and treating Panax notoginseng root rot and its application. Background Art

[0002] Panax notoginseng (Burkill) FH Chen, the dried root of a perennial herbaceous plant in the Araliaceae family, is extremely demanding in its growing environment. It prefers warm winters and cool summers, with neither extreme cold nor extreme heat, and a humid environment. This translates to low latitudes and high altitudes, which is why successful cultivation trials elsewhere have been limited. Due to limited planting sites and the growing market demand for Panax notoginseng, commercial cultivation is the only way to meet this demand. To prevent continuous cropping problems, Panax notoginseng seedlings must be transplanted after one year of cultivation. Furthermore, Panax notoginseng has a long cultivation lifespan, ranging from three to seven years. Therefore, soil previously planted with Panax notoginseng requires a rest period of at least ten years before replanting. Otherwise, serious diseases are highly likely to develop, making it a particularly susceptible Traditional Chinese Medicine variety to continuous cropping problems. Root rot is the primary cause of continuous cropping problems in Panax notoginseng, and it is estimated that this can reduce yields by over 70%, with some farms even experiencing a total crop failure, severely impacting the healthy development of the Panax notoginseng industry.

[0003] Currently, the primary control measure for Panax notoginseng root rot is the extensive use of chemical pesticides. This severely contaminates the growing soil and leads to excessive levels of pesticide residues in Panax notoginseng, which is inconsistent with the concept of green development. As the plant ages, the disease is becoming more common, and chemical control alone is no longer sufficient to control the occurrence and progression of Panax notoginseng root rot. To address this issue, green and safe biocontrol bacteria and natural product antagonists are gaining increasing attention.

[0004] Some studies have applied a seed coating made with antagonistic microorganisms to the seed surface, enabling rapid nutrient absorption and preventing soil-borne disease infection during germination and seedling growth. A preliminary study on methods to mitigate continuous cropping problems in Panax notoginseng in modern Chinese medicine research and practice describes combining wood ash with EM microbial fertilizer, which improves both plant nutrition and disease resistance. Isolation of Panax notoginseng endophytes and screening of anti-root rot pathogens reported the inhibitory effects of endophytes on Panax notoginseng root rot. Chaetomium spp., identified from Panax notoginseng endophytes, showed strong inhibitory effects against the root rot pathogen Cylindrospermum destructans in laboratory experiments. Endophytes with antagonistic effects against Fusarium solani were isolated from Sophora japonica: Fusarium sp. TRXY-34-1 and Rhexocercosporidium sp. TRXY-46.

[0005] Many substances in natural products also have a significant inhibitory effect on root rot pathogens. Zhao Yameng et al. found that covering the soil with Artemisia annua promoted the growth of Panax notoginseng plants and achieved a 76.6% control rate for Panax notoginseng root rot. GC-MS analysis revealed 58 components in Artemisia annua, with camphor, camphorol, β-caryophyllene, and gemmaene D as the main components. Experiments with in vivo Panax notoginseng showed that a petroleum ether extract from Artemisia annua had a significant inhibitory effect on Panax notoginseng root rot. Many other volatile compounds also have a significant inhibitory effect on Panax notoginseng pathogens. Volatiles from fennel stems and leaves and rapeseed can effectively inhibit the growth of three Panax notoginseng root rot pathogens (Fusarium solani F-3, Phytophthora caerulea D-1 and Cylindrospermum destructans RS006); galangal and tsaoko essential oils can inhibit the in vitro growth of Panax notoginseng-related pathogenic fungi, which may be achieved through the rich linalool and eucalyptol in the essential oils; cumin essential oil can inhibit the in vitro growth of Panax notoginseng pathogenic fungi. Scanning electron microscopy and projection electron microscopy showed that cuminaldehyde and cumin essential oil increased cell permeability and destroyed the integrity of cell membranes.

[0006] The inventor participated in the first successful isolation of a Chryseobacterium bacterium MA9 from Panax notoginseng root rot plants and conducted preliminary exploration of its pathogenicity, and published an article titled "Identification of Pathogenic Bacteria of Panax notoginseng Root Rot in Yunnan" in the Journal of Southern Agriculture. Summary of the Invention

[0007] The present invention provides a novel biological preparation based on the pathogenic bacteria Chryseobacterium indologenes that can cause the root rot of Yunnan Panax notoginseng. The biological preparation prevents and treats the root rot of Yunnan Panax notoginseng by inhibiting the pathogenic bacteria Chryseobacterium indologenes that causes the root rot of Yunnan Panax notoginseng.

[0008] To achieve the above purpose, the biological preparation of the present invention inhibits the expression of Chryseobacterium MA9 in Panax notoginseng root rot through its main active substance ginsenoside Rb1, thereby achieving the inhibitory effect on Panax notoginseng root rot.

[0009] The specific technical solutions are as follows:

[0010] A biological preparation for preventing and treating Panax notoginseng root rot, comprising an active ingredient and an agriculturally acceptable carrier; wherein the active ingredient is Panax notoginseng total saponins, and its concentration of 125 μg / mL can significantly inhibit MA9.

[0011] Furthermore, the active ingredients of the biological preparation include one or more of ginsenoside Rb1 and / or ginsenoside CK and / or escinoid XVII.

[0012] Application of biological agents in the prevention and control of Panax notoginseng root rot.

[0013] Furthermore, the concentration of ginsenoside Rb1 in the biological preparation is greater than or equal to 62.5 μg / mL.

[0014] Furthermore, the concentration of ginsenoside CK in the biological preparation is greater than or equal to 31.25 μg / mL.

[0015] Furthermore, the concentration of aescin XVII in the biological preparation is greater than or equal to 250 μg / mL.

[0016] Furthermore, the MA9 is the Chryseobacterium MA9 in Panax notoginseng root rot ( Chryseobacterium sp. MA9) strain, the Chryseobacterium MA9 was named Chryseobacterium indologenes, and the preservation number was CCTCC No: M 2024594.

[0017] The working principle of the present invention is introduced:

[0018] The inhibitory mechanism of ginsenoside Rb1 against MA9 is primarily due to the oxidative stress (OS) generated during the interaction between Rb1 and MA9. During oxidative stress, MA9 produces a large amount of reactive oxygen species (ROS), which directly or indirectly attack cellular nucleic acids, proteins, and cell membranes. H2O2, a key ROS with strong oxidative properties, can be decomposed by CAT to produce water and oxygen. Ginsenoside Rb1 inhibits CAT activity in MA9, inhibiting H2O2 decomposition and leading to an increase in intracellular H2O2. Excessive H2O2 ultimately causes oxidative damage to cell membranes and inhibits bacterial growth. MDA, one of the most important products of membrane lipid peroxidation, causes cross-linking and polymerization of macromolecules such as proteins and nucleic acids and is cytotoxic. Its production can further exacerbate membrane damage, leading to cell fragmentation and the release of intracellular substances. Biofilm bacteria are highly resistant to antibiotics and host immune defenses, enhancing their adaptability and pathogenicity in the environment. Crystal violet staining and SEM results showed that during the early stages of biofilm formation (colonization and aggregation), MA9's inherent film-forming ability was weak, and ginsenoside Rb1 further inhibited MA9 biofilm formation. Damage to the cell membrane altered the surface morphology of the MA9 cell membrane, affecting MA9's adhesion to surfaces and, consequently, biofilm formation.

[0019] These results suggest that ginsenoside Rb1 is responsible for the inhibitory effect of Panax notoginseng saponins on MA9 growth. This inhibitory effect may be exerted by inhibiting cellular CAT activity, leading to the accumulation of reactive oxygen species (ROS), which accelerates cell aging and cell membrane damage. Cell membrane aging and damage not only lead to cell fragmentation and the release of endosomes, but also alter cell morphology and properties, and impair the formation of mature biofilms.

[0020] In summary, ginsenoside Rb1 is responsible for the inhibitory effect of Panax notoginseng saponins on MA9 growth. This inhibitory effect, through the suppression of cellular CAT activity, leads to the accumulation of reactive oxygen species in the body, thus accelerating cell aging and cell membrane damage. Cell membrane aging and damage not only lead to cell fragmentation and the release of endosomes, but also alter cell morphology and properties, and impair the formation of mature biofilms.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Provides a new approach to preventing and treating Panax notoginseng root rot;

[0023] (2) A bacterial biological agent was developed, which achieved the functional efficacy of preventing and treating Panax notoginseng root rot by inhibiting the bacterium Chryseobacterium MA9 in Panax notoginseng root rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the morphological characteristics of the MA9 strain;

[0025] Figure 2 is the growth curve and AUC of MA9;

[0026] Where AF is the growth curve, af is the corresponding AUC; A is Panax notoginseng saponins, B is ginsenoside Rg1, C is ginsenoside Rd, D is ginsenoside Rb1, E is Panax notoginseng saponin R1, F is ginsenoside Re, * indicates significance, *(p<0.05), **(p<0.01), ***(p<0.001);

[0027] Figure 3 It is the changes of OD260 nm, OD280 nm and total protein content in the supernatant;

[0028] Figure 4 is the biofilm formation amount of strain MA9;

[0029] Figure 5 is a scanning electron micrograph of the MA9 strain;

[0030] Note: A and C are SEM images of the control group, and B and D are SEM images of the group treated with 1 mg / mL Rb1. A and B are 5,000×, and C and D are 20,000×. The culture medium in the control group did not contain ginsenoside Rb1, while the culture medium in the experimental group did contain 1 mg / mL Rb1.

[0031] Figure 6 CAT activity, MDA content and T-AOC in cells

[0032] Note: Figure A shows the CAT activity in MA9 cells after treatment with ginsenoside Rb1.

[0033] Figure B shows the MDA activity in MA9 after treatment with ginsenoside Rb1.

[0034] Figure C shows T-AOC in MA9 after treatment with ginsenoside Rb1;

[0035] Figure 7 TIC chromatograms of nine saponins;

[0036] Figure 8 Total ion profiles of saponins in samples at different time points;

[0037] Figure 9 relative abundance of saponins;

[0038] Figure 10 Transformation pathway of ginsenoside Rb1;

[0039] Figure 11 Effects of transformation products on MA9;

[0040] Note: A: Ginsenoside CK, B: Ginsenoside Rh2, C: Ginsenoside F2, D: Aescin XVII, E: Gynostemmaside LXXV, F: Protopanaxadiol, G: Ginsenoside Rg3. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are merely illustrative and are not intended to limit the scope of the present invention. In addition, in the following invention, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0042] Example 1: Inhibitory effect of ginsenoside Rb1 on Chryseobacterium MA9

[0043] Taking Chryseobacterium chryseum MA9 and Panax notoginseng saponins as research objects, we explored saponins that have significant inhibitory effects on MA9, clarified the antibacterial mechanism of saponins on MA9, explored the de-inhibition effect of different β-glucosidase encoded by MA9 on Rb1, and attempted to reveal the adaptive mechanism of MA9 in the pathogenic process of Panax notoginseng.

[0044] 1. Materials

[0045] The strain used in the experiment was Chryseobacterium MA9 ( Chryseobacterium sp. MA9).

[0046] The culture medium used in the experiment was NA medium, the same as above.

[0047] Reagents: (1) Saponins: Panax notoginseng saponins (batch number: AZ20033002), ginsenoside Rb1 (batch number: AZ21090302), ginsenoside Rg1 (batch number: AZ21101585), Panax notoginseng saponin R1 (batch number: AZ21110206), ginsenoside Re (batch number: AZ21090601), ginsenoside Rd (batch number: AZBG0407), ginsenoside Rg3 (batch number: AF 22040901), ginsenoside F2 (batch number: AF2153006), ginsenoside CK (batch number: AF21061457), ginsenoside Rh2 (batch number: AFBL0804), esculoside XVII (batch number: AF21050204), gypenosyl saponin LXXV (batch number: AFBK2104), and protopanaxadiol (batch number: AF21020303) were purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0048] (2) Kits: Catalase (CAT) activity detection kit (batch number: 2307001), malondialdehyde (MDA) content detection kit (batch number: 20230627), total antioxidant capacity (T-AOC) detection kit (batch number: 2306001), β-glucosidase activity detection kit (batch number: 2305001), BCA protein concentration determination kit (batch number: 2307001JH), purchased from Beijing Solebeau Technology Co., Ltd.

[0049] (3) Real-time fluorescence quantitative PCR: Simply P total RNA extraction kit was purchased from Hangzhou Bioray Company; FastKingRT Kit (with gDNase) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Hieff UNICON ®UniversalBlue qPCR SYBR Green Master Mix was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. Primers used in this chapter were designed using NCBI Primer and synthesized by Shanghai Bioengineering. The primer information for each gene is shown in the table below:

[0050] Table 1 Real-time fluorescence quantitative PCR primers

[0051]

[0052] (4) Others: 1% crystal violet solution (batch number: 2305001) and 2.5% glutaraldehyde solution (batch number: 2306001) were purchased from Beijing Solaibao Technology Co., Ltd. Analytical grade methanol, ethanol, and n-butanol were purchased from Xilong Chemical Co., Ltd., and chromatographic grade methanol and acetonitrile were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. 96-well and 6-well plates were purchased from Corning, and 0.22 μm filter membranes were purchased from Biosharp Biotechnology.

[0053] 2. Experimental Methods

[0054] (1) Culture of strains

[0055] Remove the stored strain from the -80°C freezer, streak a small amount onto NA solid medium, and culture at 28°C for three consecutive passages. Pick a single colony from the NA solid medium and transfer it to NA liquid medium, incubate at 180 rpm and 28°C.

[0056] (2) Effects of Panax notoginseng total saponins and main monomer saponins on the growth curve of MA9

[0057] Preparation of saponin solution: Dissolve notoginseng total saponins, ginsenoside Rb1, ginsenoside Rg1, notoginsenoside R1, ginsenoside Rd and ginsenoside Re in NA liquid culture medium, filter and sterilize with 0.22 μm filter membrane for later use.

[0058] Culture to OD 600 nm The cell suspension was suspended in NA liquid medium containing different saponins, and the concentration of the bacterial suspension was about 10 6 CFU / mL, 180 rpm, 28℃ culture. Record OD every 10 min. 600 nm .

[0059] 3. Experimental Results

[0060] Notoginseng total saponins are the main active substances of Panax notoginseng, mainly composed of notoginseng saponin R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd. Using the growth curve and area under the curve (AUC) as indicators, we first tested the inhibitory effects of notoginseng total saponins and saponin Rb1 on MA9. Figure 2 ).

[0061] from Figure 2 As shown in Figures A and a, total saponins of Panax notoginseng have a significant inhibitory effect on MA9, and the intensity of the inhibitory effect is positively correlated with the saponin concentration. A concentration of 125 μg / mL of total saponins of Panax notoginseng can significantly inhibit the growth of MA9. Ginsenoside Rb1 is one of the most important active ingredients in total saponins of Panax notoginseng. According to pharmacopoeias, ginsenoside Rb1 accounts for over 30% of the total saponins of Panax notoginseng. Our experimental results show that ginsenoside Rb1 also has a significant saponin concentration-dependent inhibitory effect on MA9, with the same inhibitory trend as total saponins, and the lowest inhibitory concentration is 62.5 μg / mL (Figures D and d). This indicates that saponin Rb1 is the primary inhibitory factor for the inhibitory effect of total saponins of Panax notoginseng on MA9 growth.

[0062] In order to further explore the role of other monomeric compounds in inhibiting the growth of MA9, we used the same method to detect common ginsenosides Rg1, R1, Rd and Re. Figure 2 As shown in the results, ginsenoside Rg1 and notoginsenoside R1, while also highly water-soluble, significantly inhibited MA9 growth only at higher concentrations (4000 μg / mL) compared to notoginsenosides and ginsenoside Rb1 (Figures B, b, E, e). Ginsenoside Rd and ginsenoside Re, on the other hand, exhibited no significant inhibitory effect on MA9 within the assay range due to their poor water solubility, resulting in lower solution concentrations (Figures C, c, F, f).

[0063] In summary, the inhibitory effect of Panax notoginseng saponins on Chryseobacterium MA9 mainly comes from ginsenoside Rb1.

[0064] Example 2: MA9 cell membrane integrity assay

[0065] In order to elucidate the underlying mechanism of ginsenoside Rb1's inhibition on MA9 cell growth, crystal violet staining was used to detect the effect of ginsenoside Rb1 on the integrity of MA9 cell membrane.

[0066] The integrity of the cell membrane is an important basis for bacterial growth, metabolism and reproduction. When the integrity of the cell membrane is affected, intracellular substances will leak out of the cell, including large molecules of nucleic acids and proteins. Nucleic acids and proteins are expressed in OD 260 nm and OD280 nm There is strong absorption at OD 260 nm and OD 280 nm The changes in OD can be used to reflect the leakage of nucleic acids and proteins in cells, and thus to evaluate the integrity of cell membranes. 260 nm and OD 280 nm , can further understand the antibacterial mechanism of ginsenoside Rb1 on MA9 (results as shown in Figure 3 shown).

[0067] Figure 3 The leakage of nucleic acids and proteins in the supernatant is shown.

[0068] Compared with the control group, the OD 260 nm and OD 280 nm were significantly increased (see Figure 3 A and Figure 3 B) indicates that under the action of saponin, the integrity of the MA9 cell membrane was destroyed, resulting in a gradual increase in the amount of nucleic acid and protein leaked into the supernatant, which was most significant at 36 h and 48 h.

[0069] The total protein content in the culture medium was monitored to verify the accuracy of the experimental results. Figure 3 As shown in Figure C: NA culture medium itself contains protein. During the culture process of MA9 (12-48 h), due to the proliferation of bacteria and the consumption of nutrients, the total protein content in the culture medium showed a gradual downward trend and eventually maintained at a relatively stable state. When ginsenoside Rb1 was added during the bacterial culture process, although the total protein content in the culture medium from 12 to 36 h also showed a downward trend, the increase in total protein content at 36 h (compared to no Rb1 addition) suggested that this phenomenon may be due to the release of protein to the extracellular space after cell disruption. As the culture time extends to 48 h, we can see Figure 3 In Figure C, we observed a continuous increase in the total protein content in the culture medium, which indicated that the damage of MA9 cells by Rb1 was continuous. 260 nm and OD 280 nm The monitoring results are consistent.

[0070] The above experimental results indicate that cell membrane destruction is one of the mechanisms by which ginsenoside Rb1 inhibits the growth of MA9.

[0071] Example 3: Effects of ginsenoside Rb1 on MA9 biofilm

[0072] To further elucidate the inhibitory effect of ginsenoside Rb1 on the growth of MA9, the OD values ​​of the cells were compared with those of the cells stained with crystal violet. 595 nm The effect of drugs on the amount of bacterial biofilm formation was evaluated by the change of ΔH values.

[0073] The specific experimental operations are as follows:

[0074] 1) Culture the activated MA9 cells to OD 600 nm The concentration was about 0.8, and the cells were collected by centrifugation at 12000 g and 4°C for 10 min.

[0075] 2) Resuspend the bacteria in NA liquid medium and adjust the bacterial solution to OD 600 nm 0.5 is reserved.

[0076] 3) Prepare a 2 mg / mL stock solution of ginsenoside Rb1 in NA liquid medium and sterilize by filtration through a 0.22 μm filter membrane.

[0077] 4) Add 100 μL of bacterial culture and 100 μL of ginsenoside Rb1 solution to each well of a 96-well plate. The final concentration of the saponin is 1 mg / mL. Add 100 μL of blank culture medium to the control group.

[0078] 5) Place the 96-well plate in a 28°C incubator for 3 days.

[0079] 6) Remove the 96-well plate from the incubator, remove the bacterial solution from the wells, and rinse three times with sterile water. Add 100 μL of methanol solution to each well and fix at room temperature for 15 minutes. Then remove the methanol solution and air-dry.

[0080] 7) Next, add 100 μL of 1% crystal violet solution to the wells and stain at room temperature for 10 minutes.

[0081] 8) After staining, remove the crystal violet solution from the wells and rinse off the excess dye with running water.

[0082] 9) Dry the microplate, add 200 μL of 95% ethanol solution to each well to dissolve crystal violet, and read the OD value using a microplate reader. 595 nm The absorbance value at .

[0083] The results are as follows Figure 4 As shown, after staining with 1% crystal violet solution, it can be observed that the addition of ginsenoside Rb1 has the effect of inhibiting the synthesis of biofilm, and leads to a significant decrease in the biological content of MA9 formed after three days of culture (the culture medium of the control group does not contain ginsenoside Rb1, the culture medium of the experimental group contains 1 mg / mL Rb1, and the biofilm formed by MA9 is detected by crystal violet staining.).

[0084] Biofilms provide a shelter for bacterial growth, colonization, differentiation, and infection, and are key to bacterial resistance to adverse external factors. Disruption of the biofilm undoubtedly significantly impacts bacterial growth, which is one of the reasons why the addition of ginsenoside Rb1 significantly inhibits the growth of MA9.

[0085] The effect of Rb1 on the morphology of MA9 bacteria was observed using scanning electron microscope (SEM). Figure 5 (A and C are SEM images of the control group, and B and D are SEM images of the 1 mg / mL Rb1-treated group.) As shown, ① the number of bacteria in the control group was large, and they were in an aggregated state, with a smooth and intact bacterial surface (5A, 5C); ② the addition of Rb1 not only reduced the number of bacteria, but also the observed bacterial individuals were mostly in a dispersed state, with no adhesion or adhesion to each other. High-definition electron microscope images showed that the surface of the bacterial individuals was rough, the morphology was irregular, and a few broken cells could be observed (5B, 5D).

[0086] It can be seen that the inhibitory effect of ginsenoside Rb1 on MA9 growth can lead to damage to individual cells and changes in cell morphology, and reduce the degree of adhesion and adhesion between each other, thereby exacerbating the significant decrease in biofilm content within the group.

[0087] Example 4: Effect of ginsenoside Rb1 on the redox system of MA9

[0088] Place a sterile glass slide in a 6-well plate. Add 2 mL of resuspended bacterial suspension and 2 mL of culture medium containing 2 mg / mL Rb1 to each well. For the control group, add 2 mL of blank culture medium. Place the 6-well plate in a 28°C incubator and incubate for 3 days. Remove the glass slide with bacteria, rinse it three times with sterile water, and place it face up on the bottom of a new well plate. Slowly add 4°C pre-chilled 2.5% glutaraldehyde solution along the wall of the wells. Refrigerate at 4°C overnight.

[0089] The fixed slides were rinsed three times in 0.1M phosphate buffer (pH 7.0), fixed with 1% osmium sulfate for 1-2 hours, carefully removed, and rinsed three times in 0.1M phosphate buffer. The slides were then dehydrated in varying ethanol concentrations (20, 30, 50, 70, 90, and 100%) and dried in a critical point dryer. Gold powder was then sprayed onto the slides, and bacterial growth was observed using a scanning electron microscope.

[0090] CAT activity, MDA content and T-AOC determination

[0091] NA liquid culture medium containing different concentrations of ginsenoside Rb1 (0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL) was prepared. A control medium without Rb1 was used. An overnight culture of MA9 bacteria was inoculated into the medium at a 1% inoculum and incubated at 180 rpm at 28°C. Five mL of the culture medium was collected at 6 and 24 hours, respectively, and centrifuged at 12,000 g for 10 minutes at 4°C to collect the cells. Intracellular catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) were determined.

[0092] The results are as follows Figure 6 As shown:

[0093] Depend on Figure 6 As shown in Figure 1, ginsenoside Rb1 significantly reduced intracellular CAT activity compared to the control group. At 6 hours of culture, the bacteria were in the early logarithmic growth phase, with rapid metabolic growth, and Rb1 had the greatest inhibitory effect on CAT activity. As the culture time extended to 24 hours, the bacteria entered the stationary phase and their overall metabolic level decreased. While bacterial CAT activity increased, Rb1 still had an inhibitory effect on CAT compared to the control group. The inhibitory effect of Rb1 on CAT was most pronounced at 0.5 mg / mL, while the inhibitory effect was weakest at 2 mg / mL.

[0094] In summary, ginsenoside Rb1 has a sustained inhibitory effect on CAT activity, and its inhibitory effect depends on the metabolic activity of bacteria.

[0095] Depend on Figure 6 As shown in Figure B, compared to the control group, intracellular MDA levels increased significantly at both 6 and 24 hours after ginsenoside Rb1 treatment, indicating that Rb1's effects on aging and damage are continuous. As the degree of damage deepened, MDA levels in the 0.5 mg / mL and 1 mg / mL groups were higher at 24 hours than at 6 hours. In the 2 mg / mL and 5 mg / mL Rb1 groups, MDA levels were similar at 6 and 24 hours.

[0096] In summary, the inhibition of CAT activity leads to the accumulation of reactive oxygen species in the body, accelerating cell aging and cell membrane damage.

[0097] Although MA9 reduces the oxidative damage of Rb1, the antioxidant system in the cell will participate in self-rescue and increase some total antioxidant capacity ( Figure 6 C), which can delay the cell damage process to a certain extent and demonstrate the cell's self-protection mechanism, but it cannot fundamentally reverse the fact that the cell is ultimately damaged.

[0098] In summary, ginsenoside Rb1 dominates the inhibitory effect of total ginsenosides on MA9 growth. This inhibitory effect is exerted by inhibiting the decrease in cellular CAT activity, leading to the accumulation of reactive oxygen species in the body, thereby accelerating cell aging and cell membrane damage. Cell membrane aging and damage not only lead to cell fragmentation and the release of endosomes, but also alter cell morphology and properties, and impair the formation of mature biofilms.

[0099] Example 5: Biotransformation of ginsenoside Rb1 by Chryseobacterium MA9

[0100] 1. Materials and Methods

[0101] Accurately weigh 0.2000 g of ginsenoside Rb1 in a 100 mL volumetric flask, dissolve it in NA liquid medium and dilute to a volume of 2 mg / mL. Activated MA9 was cultured to OD 600 nm The concentration of the bacterial suspension was about 10. 10 CFU / mL. Equal volumes of bacterial suspension and culture medium containing different Rb1 concentrations were mixed to a final Rb1 concentration of 1 mg / mL. The mixture was incubated on a shaker at 180 rpm and 28°C. Samples were collected at 1, 3, 6, 12, 18, 24, 36, and 48 hours. A control group contained Rb1 solution without bacterial suspension.

[0102] At each time point, take 1 mL of bacterial culture and add 1 mL of water-saturated n-butanol solution. Vortex to mix thoroughly, then centrifuge at 12,000 g at 4°C for 10 min to collect the n-butanol phase. Repeat three times. Combine the extracts, evaporate to dryness on a rotary evaporator under reduced pressure, reconstitute with chromatography-grade methanol, filter sterilize with a 0.22 μm filter, and store in a liquid phase bottle at 4°C.

[0103] Chromatographic conditions: ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm); column temperature 40°C; injection volume 10 µL; flow rate 0.30 mL / min; binary gradient elution with mobile phase A: aqueous solution and mobile phase B: acetonitrile. The gradient elution program is shown in the table below.

[0104] Table 2 Ultra-high performance liquid chromatography gradient elution conditions

[0105]

[0106] Using the gradient elution conditions listed in Table 2, all substances showed good response and separation. Using negative ESI scanning mode, primary and secondary mass spectrometry scans were performed on each of the nine reference substances, acquiring specific MRM data. The parameters were adjusted appropriately to maximize the signal, ultimately determining the optimal mass spectrometry parameters (Table 3).

[0107] Table 3 Mass spectrometry analysis parameters of nine saponin components

[0108]

[0109] The above optimized chromatographic and mass spectrometric conditions were used to measure the standard solution and extract the total ion chromatogram (TIC) (see Figure 7 As can be seen from the figure, the peaks of ginsenoside Rg3 and esculoside LXXV (G-LXXV), ginsenoside CK and protopanaxadiol (PPD) overlap, and the peaks of other saponins are well separated from each other. However, by combining the parent ion and product ion in the mass spectrometry analysis parameters, ginsenoside Rg3 and esculoside LXXV (G-LXXV), as well as ginsenoside CK and protopanaxadiol (PPD) can be separated.

[0110] Note: The concentrations of ginsenoside Rb1 were 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL, respectively. The control group did not contain ginsenoside Rb1. The treatment times were 6 h and 24 h, respectively.

[0111] 2. Experimental Sample Analysis

[0112] The analytical method established in this experiment was used to detect and analyze the nine saponins in the samples collected above, and the content of each saponin in the samples was calculated. The typical total ion chromatogram of saponins in each sample is shown in Figure 8 . Since the CK group did not contain bacterial solution, ginsenoside Rb1 could not be converted, and only one peak of ginsenoside Rb1 was detected. As time went on, new peaks appeared at 2.83 min and 3.44 min at 1 h. The peak at 2.83 min corresponded to ginsenoside Rd, and the peak at 3.44 min corresponded to ginsenoside Rg3 and gypenoside LXXV. At 18 h, a small peak was seen at 3.00 min, which was aescin XVII. At 36 h, a new small peak was found at 3.26 min (ginsenoside F2) and 4.10 min (ginsenoside CK and protopanaxadiol).

[0113] Figure 9The bar chart of the percentage of each saponin content more fully demonstrates the changes in each component. As can be seen from Figure A, the content of ginsenoside Rb1, ginsenoside Rg3, ginsenoside Rd, and gypenoside LXXV gradually decreased, the content of esculoside XVII first decreased and then increased, and the content of the other four saponins gradually increased.

[0114] From T1 to T12, there are a total of five types of saponins in the sample. In addition to the reaction substrate ginsenoside Rb1, the other four are ginsenoside Rg3, ginsenoside Rd, esculoside XVII, and gypenoside LXXV. Saponins ginsenoside F2 and CK were already generated at 18 hours, but the content was extremely low, so the corresponding peaks were not seen in the total ion graph T18 above. They were not seen until T36. Ginsenoside Rh2 and protopanaxadiol PPD were already generated at T36, and the content was also extremely low ( Figure 9 A).

[0115] From T1 to T12, ginsenoside Rb1 was the most abundant of the five saponins, followed by ginsenoside Rd, ginsenoside Rg3, gypenoside LXXV, and esculoside XVII. This suggests that during the early stages of transformation, the β-glucosidase in MA9 preferentially removes the glucose at C20 of Rb1. At T18, the newly formed ginsenosides CK and F2 had the lowest contents, while the order of the other saponins remained unchanged. However, at T24, the order of contents shifted to ginsenoside Rb1 > ginsenoside Rd > esculoside XVII > ginsenoside Rg3 > ginsenoside F2 > gypenoside LXXV > ginsenoside CK, at which point the contents of esculoside XVII and ginsenoside F2 began to rise.

[0116] At T36, the order of content was ginsenoside Rb1>ginsenoside Rd>escin XVII>ginsenoside F2>protopanaxadiol PPD>ginsenoside Rg3>ginsenoside CK>gypenoside LXXV>ginsenoside Rh2, and the contents of ginsenoside F2, ginsenoside CK and escin XVII increased further. At the end of the reaction, at T48, the highest content was escin XVII, followed by escin XVII>ginsenoside F2>ginsenoside Rb1>ginsenoside Rd>ginsenoside CK>protopanaxadiol PPD>ginsenoside Rg3>ginsenoside Rh2>gypenoside LXXV ( Figure 9 B).

[0117] 3. Transformation Pathway Analysis

[0118] During the early stages of transformation (T0 to T12), the bacteria rapidly remove one or two glucose residues from the C3 and C20 groups of the ginsenoside Rb1 molecule to facilitate growth. This occurs primarily at C20, where the glucose is removed to form ginsenosides Rd and Rg3, with less removal of the C3 glucose. At C3, the bacteria preferentially hydrolyze the inner glycosidic bond, removing two glucose residues to form gypenosides LXXV, while producing less abundant esculoside XVII.

[0119] Ginsenosides F2 and CK begin to appear during the mid-transformation phase (T18 and T24). F2 originates from Rd and G-XVII, which further remove a glucose molecule at C3 and C20, respectively; CK originates from F2 and G-LXXV, which further remove a glucose molecule at C3 and C20, respectively. Rh2, another downstream product of F2, is not formed at this stage, indicating that MA9 preferentially hydrolyzes the glucose at C3 of F2 to form CK.

[0120] In the late stages of transformation (T36 and T48), ginsenoside Rh2 and protopanaxadiol began to be detected, indicating that most of the glucose molecules had been hydrolyzed and utilized in the early stages, and only the last glucose molecule could be removed at this time.

[0121] Combining the entire transformation process, we can speculate the hydrolysis process of ginsenoside Rb1 by Chryseobacterium MA9: In the early stage of the reaction, MA9 preferentially hydrolyzes a large amount of glucose on the outside of C20 to form Rd. On C3, MA9 will directly hydrolyze a small amount of the inner glycosidic bond, remove two molecules of glucose, and form G-LXXV. As time goes on, MA9 begins to hydrolyze the outer glucose on C3 of Rb1 to form G-XVII. G-XVII and Rd are both precursors of F2, and the increase in their content leads to the subsequent increase in the content of F2. MA9 preferentially hydrolyzes the glucose on C3 of F2 to form CK. As the glucose is used by bacteria, Rb1 can eventually be degraded into protopanaxadiol ( Figure 10 ).

[0122] Example 6: Effect of transformation products on MA9

[0123] According to the above transformation pathway analysis, there are 8 transformation products of ginsenoside Rb1 (ginsenoside Rd, ginsenoside CK, ginsenoside Rh2, ginsenoside F2, esculoside XVII, gypenoside LXXV, protopanaxadiol, ginsenoside Rg3). Among them, ginsenoside Rd is not only one of the main components of Panax notoginseng total saponins, but also one of the transformation products of ginsenoside Rb1. The antibacterial effect of Rd has been studied before, and the remaining 7 transformation products are studied. The AUC value calculated according to the growth curve is plotted on the vertical axis, and the results are shown in the figure. Figure 11As can be seen from the figure, ginsenoside CK can inhibit MA9 at 31.25 μg / mL ( Figure 11 A) Aescin XVII has an inhibitory effect on MA9 only at 250 μg / mL ( Figure 11 D), other saponins had no significant inhibitory effect on the growth of MA9. Figure 8 and Figure 9 It can be seen that ginsenoside CK is only formed in the late stage of the transformation process (36 h and 48 h), and its proportion is very small. Although esculoside XVII has a high proportion at 48 h, its inhibitory effect requires a higher concentration. Therefore, their inhibitory effects on MA9 during the transformation process are limited.

[0124] It should be understood that the above-described specific embodiments of the present invention are merely illustrative of the invention or explanation of the principles of the present invention, and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents of such scope and metes and bounds.

Claims

1. A biological agent for preventing and treating Panax notoginseng root rot, characterized in that: The biological preparation comprises an active ingredient and an agriculturally acceptable carrier; the active ingredient is a total saponin of Panax notoginseng that can inhibit MA9, and the concentration of ginsenoside Rb1 in the biological preparation is greater than or equal to 62.5 μg / mL; the MA9 is a pathogenic bacterium Chryseobacterium MA9 ( Chryseobacterium sp. MA9) strain, the Chryseobacterium MA9 was named Chryseobacterium indologenes, and the preservation number was CCTCC No: M 2024594.

2. A biological preparation for preventing and treating Panax notoginseng root rot according to claim 1, characterized in that: The Panax notoginseng total saponin components capable of inhibiting MA9 also include one or more of ginsenoside CK and aescin XVII.

3. Use of the biological preparation according to claim 2 in preventing and treating Panax notoginseng root rot.

4. The use according to claim 3, characterized in that The concentration of ginsenoside CK in the biological preparation is greater than or equal to 31.25 μg / mL.

5. The use according to claim 3, characterized in that The concentration of aescin XVII in the biological preparation is greater than or equal to 250 μg / mL.

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