Use of an elecampane extract and its sesquiterpene lactone compounds in preparing products for preventing and treating pathogens

Abstract

The present invention provides a use of an Inula extract and its sesquiterpene lactone compounds in preparing products for preventing and treating pathogens, belonging to the field of natural drug chemistry. The use of the Inula extract and / or sesquiterpene lactone compounds in preparing products for preventing and treating pathogens; the sesquiterpene lactone compounds are Inula lactone and / or Iso-Inula lactone; the pathogens include: agricultural bacterial pathogens, agricultural fungal pathogens, and human pathogens. The Inula extract and its sesquiterpene lactone compounds involved in the present invention have the advantages of being green and efficient, low toxicity, having a broad antibacterial spectrum, low residue, and not easy to produce resistance; they have application value in preventing and treating common pathogens.

Description

Technical Field

[0001] The invention relates to the field of natural drug chemistry, and in particular to use of an inula root extract and sesquiterpene lactone compounds thereof in preparing products for preventing and treating pathogens. Background Art

[0002] Plant pathogens are extremely harmful to agriculture and can cause serious plant diseases and crop yield reduction. Once crops are infected by these plant pathogens, not only will the natural growth of the plants be seriously affected, but it will also directly affect the appearance, yield and quality of agricultural products. In order to prevent and control these diseases, in addition to using necessary protective measures in agricultural production, the use of agrochemicals to prevent and control their pathogens is also particularly critical.

[0003] At the same time, human pathogens (i.e., pathogenic bacteria) threaten human health and cause inestimable losses to the national economy. What is more worrying is that with the long-term use of antibiotics, the growing antibiotic resistance has become one of the biggest public health threats in the 21st century. In Europe, about 33,000 people die each year from antibiotic-resistant bacterial infections; it is estimated that by 2050, antibiotic resistance will cause nearly 10 million deaths per year worldwide. Therefore, it is urgent to provide antibacterial products that can prevent and treat pathogens.

[0004] Natural products have always been an important source in drug development. In the past two decades, more than one-third of FDA-approved drugs are natural products and their derivatives. Compared with chemically synthesized compounds, natural products have some unique properties, such as higher structural complexity, more novel skeletons, better drug-like properties, etc. Therefore, natural products are a treasure trove that deserves special attention and have great potential for drug development.

[0005] Inula helenium L. is a perennial herbaceous plant of the genus Inula in the Asteraceae family. Its dried root is a traditional Chinese medicine with the effects of strengthening the spleen and stomach, promoting qi and relieving pain. Inula helenium is grown in many regions of my country and has rich medicinal resources. According to studies, Inula helenium contains many types of chemical components, including terpenes, flavonoids, steroids, sugars, etc., among which sesquiterpene lactone compounds are its main active ingredients, such as inula lactone, isoinula lactone, inula ene lactone, etc. Inula helenium has good anti-inflammatory, anti-tumor, antibacterial, analgesic and other pharmacological activities. Therefore, it is of great technical significance to further develop and expand the application field of Inula helenium and study and develop the potential application value of Inula helenium and its active ingredients in the prevention and control of plant pathogens and human pathogens. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a use of an Inula extract and its sesquiterpene lactone compounds in the preparation of products for preventing and treating pathogens; in particular, the use of an Inula extract and / or sesquiterpene lactone compounds in the preparation of products for preventing and treating pathogens.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] Use of an Inula extract and its sesquiterpene lactone compounds in preparing a product for preventing and treating pathogens, and use of the Inula extract and / or sesquiterpene lactone compounds in preparing a product for preventing and treating pathogens;

[0009] The inula extract is a 95% ethanol extract of inula; the extract is prepared by extracting the inula root with an ethanol solution with a volume concentration of 95%.

[0010] The sesquiterpene lactone compound is inulinolactone and / or isoinulinolactone;

[0011] The chemical structural formula of the inulin is:

[0012] ;

[0013] The chemical structural formula of the isoinulin is:

[0014] ;

[0015] The pathogens include: agricultural bacterial pathogens, agricultural fungal pathogens, and human pathogens;

[0016] The agricultural bacterial pathogen is Xanthomonas oryzae, the pathogen of rice bacterial blight.

[0017] The agricultural fungal pathogen is at least one of the following: Sclerotiniasclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus and Aspergillus niger.

[0018] The human pathogenic bacteria is at least one of the following: Staphylococcus aureus Newman and Candida albicans.

[0019] Furthermore, the product includes: a pharmaceutical composition and an antibacterial agent.

[0020] A pharmaceutical composition for preventing and / or treating related diseases caused by the aforementioned pathogenic bacteria is prepared by using at least one of the extract of Inula japonica and its sesquiterpene lactone compounds as an effective ingredient or main effective ingredient.

[0021] An antibacterial agent for the aforementioned pathogenic bacteria is prepared by using at least one of the extract of Inula japonica and its sesquiterpene lactone compounds as an effective component or a main effective component.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses the extract of Inula officinalis and its sesquiterpenoid lactone compounds to treat agricultural bacterial diseases caused by Xanthomonas oryzae ACCC11602, the pathogen of rice bacterial leaf blight, Pectobacterium atroseptica ACCC19901, the pathogen of potato black shank disease; agricultural fungal diseases caused by Sclerotinias clerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus ACCC32601, Aspergillus niger ACCC30005; and Escherichia coli Escherichiacoli ATCC25922, Staphylococcus aureus Newman , and Candida albicans, and both showed a certain inhibitory effect. It can be effectively used in the preparation of products for the prevention and treatment of pathogens, further expanding the application field of Inula officinalis, and has great application value and application prospects in the prevention and treatment of common pathogens.

[0024] (2) The present invention uses the extract of Inula officinalis and its sesquiterpene lactone compounds as natural antibacterial agents, which are easy to prepare and have the advantages of being green, highly effective, low toxicity, having a broad antibacterial spectrum, low residue and not prone to resistance. In addition, Inula officinalis is widely distributed in my country and has abundant resources. It is green, highly effective and not prone to drug resistance. It has application value in preventing and treating common pathogens and is worthy of further research and development.

[0025] (3) The extract of Inula officinalis and its sesquiterpene lactone compounds of the present invention have good antibacterial activity against agricultural bacterial pathogens (Xanthomonas oryzae, the pathogen of rice bacterial leaf blight), human pathogens (Staphylococcus aureus Newman, Candida albicans), and agricultural fungal pathogens (Sclerotinia sclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus, and Aspergillus niger), and can be used in the preparation of products for preventing and controlling pathogens; or as an active ingredient or main active ingredient, in the preparation of products (such as pharmaceutical compositions, antibacterial agents, etc.) for preventing and / or treating related diseases caused by the aforementioned pathogens. DETAILED DESCRIPTION

[0026] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0027] This embodiment provides a use of an Inula extract and its sesquiterpene lactone compounds in preparing a product for preventing and treating pathogens, particularly the use of the Inula extract and / or sesquiterpene lactone compounds in preparing a product for preventing and treating pathogens.

[0028] The Inula extract is a 95% ethanol extract of Inula;

[0029] The sesquiterpene lactone compound is inulinolactone and / or isoinulinolactone.

[0030] The chemical structural formula of the inulin is:

[0031] ;

[0032] The chemical structural formula of the isoinulin is:

[0033] ;

[0034] Furthermore, the pathogens include: agricultural bacterial pathogens, agricultural fungal pathogens, and human pathogens;

[0035] Furthermore, the agricultural bacterial pathogen is: Xanthomonas oryzae, the pathogen of rice bacterial blight;

[0036] Further, the agricultural fungal pathogen is at least one of the following: Sclerotiniasclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus, Aspergillus niger;

[0037] Furthermore, the human pathogen is at least one of the following: Staphylococcus aureus Newman, Candida albicans;

[0038] Furthermore, the product includes: a pharmaceutical composition and an antibacterial agent.

[0039] A pharmaceutical composition for preventing and / or treating related diseases caused by the aforementioned pathogenic bacteria is prepared by using at least one of the extract of Inula japonica and its sesquiterpene lactone compounds as an effective ingredient or main effective ingredient.

[0040] An antibacterial agent for the aforementioned pathogenic bacteria is prepared by using at least one of the extract of Inula japonica and its sesquiterpene lactone compounds as an effective component or a main effective component.

[0041] Example 1

[0042] Determination of the antibacterial activity of Inula officinalis extract and its sesquiterpenoid lactone compounds against agricultural bacterial pathogens:

[0043] 1) Test agents: Inula extract, Inula lactone, and Isoinula lactone.

[0044] Among them, the Inula extract is a 95% ethanol extract of Inula, which is obtained by extracting the Inula root with a volume concentration of 95% ethanol solution as the extraction solvent through a conventional plant component extraction method (such as alcohol extraction); in this embodiment, the Inula extract is extracted by the following method: the Inula root is crushed by a grinder, passed through a 40-mesh sieve, and the Inula powder is obtained, which is weighed; a volume concentration of 95% ethanol solution is added at a ratio of 10:1 (extraction solvent / Inula powder, v / w), heated to reflux, and refluxed for three times, each extraction time is 2 hours, and the filtrate is filtered under reduced pressure, and the filtrate is combined; the filtrate is concentrated under reduced pressure at 60°C to an extract without alcohol odor to obtain an Inula extract, which is weighed and stored in a cool place at room temperature for later use.

[0045] 2) Test bacteria: Xanthomonas oryzae ACCC11602, the pathogen of rice bacterial blight, and Pectobacterium atroseptica ACCC19901, the pathogen of potato black shank.

[0046] Among them, Xanthomonas oryzae ACCC11602, the pathogen of rice bacterial blight, and Pectobacterium atroseptica ACCC19901, the pathogen of potato black shank, were purchased from China Agricultural Culture Collection Center (ACCC).

[0047] 3) Test method:

[0048] The strain used in the test experiment of this embodiment is a strain frozen in 30% glycerol at a temperature of -80°C in the laboratory.

[0049] Take out the frozen strains, streak them on the NB solid medium of plant bacteria (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L; pH 7.0; sterilized at 121℃ for 20min), and culture them at 28℃ until single colonies grow; pick the single colonies on the solid medium and culture them on the NB solid medium of plant bacteria (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L; pH 7.0; sterilized at 121℃ for 20min), shake and culture them at 28℃, 180rpm constant temperature shaker until the logarithmic growth phase. Dilute each strain in the logarithmic growth phase with the corresponding liquid culture medium to 10 6 CFU / mL, obtain bacterial culture and set aside.

[0050] Dissolve the aforementioned safflower extract in DMSO, then add it to the liquid culture medium, mix well, and prepare a drug-containing liquid culture medium with a concentration of 800 μg / mL; dissolve safflower lactone and isosafflower lactone in DMSO, then add them to the liquid culture medium, mix well, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of 10 6 CFU / mL of bacterial culture was added to the wells of a 96-well plate, and the final concentration of the control extract of 400μg / mL, and the final concentration of 100μg / mL of 100μg / mL of 100μL bacterial solution containing an equal amount of DMSO was used as the control group. The 96-well plate was cultured in a constant temperature incubator at 28℃ (37℃) for 24-48h until the bacterial solution of the control group grew out, and the OD value (OD 600 ). In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows:

[0051] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0052] Inhibition rate = [(corrected OD value of bacterial solution in control culture medium - corrected OD value of drug-containing culture medium) / corrected OD value of bacterial solution in control culture medium] × 100%.

[0053] All the above experiments were repeated three times. The inhibition rates of the extracts of Inula japonica against the tested bacteria are shown in the following table:

[0054]

[0055] The inhibition rates of scutellaria lactone and iso-scutellaria lactone on each tested bacterial species are shown in the following table:

[0056]

[0057] It can be seen from the contents of the above table that the Inula extract, Inula lactone and Isoinula lactone used in this embodiment show excellent antibacterial activity against Xanthoceras oryzae; when the Inula extract is administered at a concentration of 400 μg / mL, the inhibition rate of Xanthoceras oryzae exceeds 96.8%; when the Inula lactone is administered at a concentration of 100 μg / mL, the inhibition rate of Xanthoceras oryzae exceeds 98.5%.

[0058] Example 2

[0059] Determination of the antibacterial activity of sesquiterpenoid lactone compounds in Inula officinalis against human pathogens:

[0060] 1) Test agents: Inula lactone and Isoinula lactone.

[0061] 2) Test bacteria: Escherichia coli ATCC25922, Staphylococcus aureus Newman, Candida albicans ATCC24433.

[0062] Among them, Escherichia coli ATCC25922, Staphylococcus aureus Newman, and Candida albicans ATCC24433 were all purchased from Beina Biotechnology (Beijing Beina Chuanglian Biotechnology Research Institute).

[0063] 3) Test method:

[0064] Escherichia coli and Staphylococcus aureus were cultured in hydrolyzed casein medium (MH medium). The specific preparation method of hydrolyzed casein medium is as follows: weigh 38g of MH agar powder and add it to 1L of distilled water, heat and stir until completely dissolved, and then sterilize it by high-pressure steam at 121°C for 30min; after sterilization, cool it to room temperature and dispense it into sterilized culture dishes, and finally store it in a 4°C refrigerator for later use.

[0065] The method for determining the antibacterial activity against Escherichia coli and Staphylococcus aureus is to dissolve scutellaria lactone and iso-scutellaria lactone in DMSO, then add them to the liquid culture medium, mix them evenly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of 10 6 CFU / mL of bacterial culture was added to the wells of a 96-well plate, and the final concentration of the control extract of 400μg / mL, and the final concentration of 100μg / mL of 100μg / mL of 100μL bacterial solution containing an equal amount of DMSO was used as the control group. The 96-well plate was cultured in a constant temperature incubator at 28℃ (37℃) for 24-48h until the bacterial solution of the control group grew out, and the OD value (OD 600 ). In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows:

[0066] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0067] Inhibition rate = [(corrected OD value of bacterial solution in control culture medium - corrected OD value of drug-containing culture medium) / corrected OD value of bacterial solution in control culture medium] × 100%.

[0068] Candida albicans was cultured in yeast peptone glucose medium (YEPD medium). The specific preparation method of yeast peptone glucose medium is as follows: weigh 10g of yeast extract, 20g of peptone, 20g of glucose, and 15g of agar, add them to 1L of distilled water, heat and stir until completely dissolved, and then sterilize by high-pressure steam at 121°C for 30min; after sterilization, cool it to room temperature and dispense it into sterilized culture dishes, and finally store it in a 4°C refrigerator for later use.

[0069] The mycelial growth rate method was used to determine the antibacterial activity of Candida albicans. Specifically, each test agent was added to the sterilized culture medium cooled to 50°C according to a certain concentration gradient, mixed evenly, and diluted into a series of concentrations to prepare a drug-containing culture medium. One bacterial plate (diameter d=5mm) was inoculated in each culture dish, and 3 replicates were set for each concentration. The colony diameter (mm) of each concentration treatment was determined by the cross method when the control colony grew more than 2 / 3 of the culture dish at 25°C, and the inhibitory rate of the agent on the mycelial growth was calculated. The statistical analysis of the difference was performed using the statistical software SAS8.1 (Statistical Analysis System 8.1). The calculation of the inhibition rate was carried out according to the following formula, and the experimental results were averaged and the standard deviation (SD) was calculated. All inhibition rates were expressed in the form of the average value of the inhibition rate ± SD, and the numbers were retained to two decimal places.

[0070] Inhibition rate = [(blank control mycelium growth diameter - mycelium growth diameter) / (blank control mycelium growth diameter - bacterial cake diameter)] × 100%;

[0071] All the above experiments were repeated three times. The inhibition rates of scutellaria lactone and iso-scutellaria lactone on each tested bacterial species are shown in the following table:

[0072]

[0073] It can be seen from the above table that the inulin lactone used in this example exhibits antibacterial activity against human pathogens; in particular, it exhibits excellent antibacterial activity against Staphylococcus aureus, with an inhibition rate of more than 90.5%.

[0074] Example 3

[0075] On the basis of Example 1 and Example 2, the drug-containing liquid culture medium of Inula extract and Inula lactone was diluted in a 96-well plate by a two-fold dilution method to obtain 50 μL of drug-containing culture medium of a series of concentrations, and then the inhibition rate corresponding to the series of concentrations was determined according to the same test method in Example 1 and Example 2. The lowest concentration with an inhibition rate greater than 90% was defined as MIC, and the MIC values ​​of Inula extract and Inula lactone against Xanthomonas oryzaeACCC11602, the pathogen of rice bacterial blight, and Staphylococcus aureus Newman were determined. The specific determination results are shown in the following table:

[0076]

[0077] It can be seen that the inula extract and inula lactone of the present invention show antibacterial activity against Xanthomonas oryzae ACCC11602, the pathogen of rice bacterial blight, and Staphylococcus aureus Newman; among them, inula lactone has the best antibacterial activity against the pathogen of rice bacterial blight, with an MIC value of 100 μg / mL, showing significant antibacterial activity.

[0078] Example 4

[0079] Determination of the antibacterial activity of Inula officinalis extract and its sesquiterpenoid lactone compounds against agricultural fungal pathogens:

[0080] 1) Test agents: Inula extract, Inula lactone, and Isoinula lactone.

[0081] Wherein, the Inula extract is a 95% ethanol extract of Inula, and the preparation method is the same as that in Example 1.

[0082] 2) Tested fungi: Sclerotinia sclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus ACCC32601, Aspergillus niger ACCC30005.

[0083] Among them, Sclerotinia sclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus ACCC32601, and Aspergillus niger ACCC30005 were all purchased from China Agricultural Culture Collection Center (ACCC).

[0084] 3) Test method:

[0085] The mycelial growth rate method was used to determine the antibacterial activity of the above-mentioned test strains. Specifically, each test agent was added to the sterilized culture medium cooled to 50°C according to a certain concentration gradient, mixed evenly, and diluted into a series of concentrations to prepare a drug-containing culture medium. One bacterial plate (diameter d=5mm) was inoculated in each culture dish, and 3 replicates were set for each concentration. When the control colony grew more than 2 / 3 of the culture dish at 25°C, the colony diameter (mm) of each concentration treatment was determined by the cross method, and the inhibitory rate of the agent on the mycelial growth was calculated. The statistical analysis of differences was performed using the statistical software SAS8.1 (Statistical Analysis System 8.1). The calculation of the inhibition rate was carried out according to the following formula, and the experimental results were averaged and the standard deviation (SD) was calculated. All inhibition rates were expressed in the form of the average value of the inhibition rate ± SD, and the numbers were retained to two decimal places.

[0086] Inhibition rate = [(blank control mycelium growth diameter - mycelium growth diameter) / (blank control mycelium growth diameter - bacterial cake diameter)] × 100%;

[0087] All the above experiments were repeated three times. The inhibition rates of the extract of Inula japonica against the tested bacteria at a concentration of 500 μg / mL are shown in the following table:

[0088]

[0089] The inhibition rates of scutellaria lactone and iso-scutellaria lactone against various tested bacteria except Aspergillus niger and Aspergillus flavus at a dosage concentration of 100 μg / mL are shown in the following table:

[0090]

[0091] It can be seen from the above table that the inulin lactone used in this example exhibits excellent antibacterial activity against agricultural fungal pathogens.

[0092] Furthermore, the half effective concentration (EC 50 ), the specific results are shown in the following table:

[0093]

[0094] It can be seen that the extract of Inula officinalis and its sesquiterpene lactone compounds of the present invention exhibit excellent antibacterial activity against the aforementioned agricultural fungal pathogens; among them, Inula officinalis lactone has the best antibacterial activity, EC 50 The values ​​were between 6.48 and 84.62 μg / mL, showing significant antibacterial activity.

[0095] In summary, the Inula extract and its sesquiterpene lactone compounds of the present invention have good antibacterial activity against agricultural bacterial pathogens (Xanthomonas oryzae, the pathogen of rice bacterial leaf blight), human pathogens (Staphylococcus aureus Newman, Candida albicans), and agricultural fungal pathogens (Sclerotinia sclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae, Phytophthora Capsici, Aspergillus flavus, Aspergillus niger), and can be used in the preparation of products for preventing and controlling pathogens; or as an effective ingredient or main effective ingredient, to prepare products (such as pharmaceutical compositions, antibacterial agents, etc.) for preventing and / or treating related diseases caused by the aforementioned pathogens.

[0096] Unless otherwise specified, all percentages used in the present invention are by mass.

[0097] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of sesquiterpene lactone compounds in Inula japonica in preparing products for preventing and treating pathogens, characterized in that: The sesquiterpene lactone compound is inulin lactone; The pathogen is selected from: Xanthomonas oryzae, the pathogen of rice bacterial blight, and Sclerotinia sclerotiorum, the pathogen of rapeseed; The product is a pharmaceutical composition.

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