Hyssopus harzianum extract as well as preparation method and application thereof

Abstract

The invention provides a hyssopus harzianum extract as well as a preparation method and application thereof, and belongs to the technical field of extract preparation. The method for preparing the hyssop officinalis extract comprises the following steps: mixing a whole hyssop officinalis plant with an ethanol solution with the volume fraction of 30-90%, stirring for 1-6 hours at the temperature of 25-60 DEG C, filtering, collecting filtrate, and removing a solvent to obtain a crude extract; and mixing the crude extract, water and ethyl acetate, stirring and extracting for 1-3 hours at 20-45 DEG C, standing, collecting an organic extraction phase, and removing the solvent to obtain the hyssop officinalis extract. The hyssopus officinalis extract prepared by the preparation method disclosed by the invention has remarkable and broad-spectrum antibacterial and antifungal activity, also has remarkable antibacterial activity on various drug-resistant bacteria and drug-resistant fungi, and is expected to become a new scheme for solving the problem of drug resistance of existing antibiotics when being combined with the antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of extract preparation, and particularly relates to an extract of Hyssopus cuspidatus var. cuspidatus, a preparation method thereof and an application thereof. Background Art

[0002] In recent years, the incidence of drug resistance of pathogenic bacteria to various drugs has been steadily increasing. Many pathogenic bacteria have evolved resistance to mainstream antibiotics, and multidrug-resistant bacteria have caused infections that are more difficult to treat and easily lead to death. The emergence of antibiotic-resistant bacteria has a considerable clinical and economic impact. Various studies in recent years have shown that extracts of mixtures extracted from aromatic plants have broad antibacterial activities against human pathogens, including fungi and bacteria. The antibacterial potential of extracts has long been known and has shown good antibacterial effects. The advantage that extracts do not cause drug resistance in pathogenic bacteria is being gradually concerned, and it is expected to become an effective drug for treating pathogenic bacterial infections after antibiotics.

[0003] Hyssopus cuspidatus var. cuspidatus belongs to the genus Hyssopus of the Lamiaceae family. In traditional Uyghur medicine, the whole herb of Hyssopus cuspidatus is used as medicine. The Chinese Pharmacopoeia records that the functions and indications of Hyssopus are to warm the lungs and relieve asthma, dispel cold and relieve cough, and it is generally used to treat respiratory organ diseases of the wet-cold type and mucous type. The volatile oil of Hyssopus is mainly composed of oxygenated monoterpenes, monoterpene hydrocarbons and oxygenated sesquiterpenes. Modern pharmacological studies have found that Hyssopus has pharmacological effects such as anti-asthma and anti-inflammatory effects. At present, there is no in-depth research report on the extract of Hyssopus cuspidatus var. cuspidatus, and there is no relevant research on antibacterial and antifungal effects using the extract of Hyssopus cuspidatus var. cuspidatus. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of an extract of Hyssopus cuspidatus var. cuspidatus which has antibacterial and antifungal effects and has significant antibacterial activities against drug-resistant bacteria and drug-resistant fungi.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing an extract of Hyssopus cuspidatus var. cuspidatus, comprising the following steps: mixing the whole plant of Hyssopus cuspidatus var. cuspidatus with an ethanol solution having a volume fraction of 30% to 90%, stirring at 25°C to 60°C for 1 h to 6 h, filtering, collecting the filtrate, removing the solvent to obtain a crude extract; mixing the crude extract, water and ethyl acetate, stirring and extracting at 20°C to 45°C for 1 h to 3 h, standing to collect the organic extraction phase, and removing the solvent to obtain the extract of Hyssopus cuspidatus var. cuspidatus.

[0007] Preferably, the weight ratio of the whole plant of Hyssopus cuspidatus var. cuspidatus to the ethanol solution having a volume fraction of 30% to 90% is 1:3 to 10.

[0008] Preferably, the rotation speed of the stirring is 100 - 200 rpm.

[0009] Preferably, the weight ratio of the crude extract to water is 1:3 - 10.

[0010] Preferably, the weight ratio of the crude extract to ethyl acetate is 1:3 - 10.

[0011] Preferably, the temperature for solvent removal is 10 - 30 °C.

[0012] The present invention also provides a Hyssopus cuspidatus Boriss. extract prepared by the above method.

[0013] The present invention also provides the application of the above method or the above Hyssopus cuspidatus Boriss. extract in the preparation of antibacterial products.

[0014] Preferably, the antibacterial activity includes antibacterial and / or antifungal activities.

[0015] Preferably, the bacteria include at least one of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa, and Acinetobacter baumannii; the fungi include at least one of Candida albicans, Candida tropicalis, and Candida krusei.

[0016] Advantages of the present invention:

[0017] The Hyssopus cuspidatus Boriss. extract prepared by the preparation method of the present invention has significant and broad-spectrum antibacterial and antifungal activities, and also has significant antibacterial activities against a variety of drug-resistant bacteria and drug-resistant fungi. Combining with antibiotics is expected to become a new solution to the existing antibiotic resistance problem. Description of the drawings

[0018] Figure 1 It is the LC-MS analysis chromatogram of the Hyssopus cuspidatus Boriss. extract obtained in Example 7;

[0019] Figure 2 It is the macroscopic observation result of the antibacterial test of the standard strain of Staphylococcus epidermidis S. epidermidis ATCC 12228;

[0020] Figure 3 It is the OD600 graph of the standard strain of Staphylococcus epidermidis S. epidermidis ATCC 12228. Detailed implementation manners

[0021] The present invention provides a method for preparing Hyssopus cuspidatus Boriss. extract, comprising the following steps: mixing the whole plant of Hyssopus cuspidatus Boriss. with an ethanol solution having a volume fraction of 30% to 90%, stirring at 25°C to 60°C for 1 h to 6 h, filtering, collecting the filtrate, removing the solvent to obtain a crude extract; mixing the crude extract, water and ethyl acetate, stirring and extracting at 20°C to 45°C for 1 h to 3 h, standing and collecting the organic extraction phase, removing the solvent to obtain Hyssopus cuspidatus Boriss. extract.

[0022] The present invention has no special limitation on the specific source of Hyssopus cuspidatus Boriss. In the present invention, the weight ratio of the whole plant of Hyssopus cuspidatus Boriss. to the ethanol solution having a volume fraction of 30% to 90% is preferably 1:3 to 10, more preferably 1:4 to 8. In a specific embodiment of the present invention, the volume fraction of the ethanol solution is preferably 40% to 75%. In the present invention, after mixing the whole plant of Hyssopus cuspidatus Boriss. with the ethanol solution having a volume fraction of 30% to 90%, it preferably further includes a soaking step, and the soaking time is preferably 6 to 48 h, more preferably 10 to 30 h. After the soaking is completed, stirring extraction is carried out, and the stirring temperature is preferably 30°C to 50°C, more preferably 35°C to 45°C; the stirring time is preferably 2 h to 5 h, more preferably 3 h to 4 h; the stirring speed is preferably 100 to 200 rpm, more preferably 120 to 180 rpm. The present invention has no special limitation on the specific filtration method, and the conventional filtration methods in the art can be used. In the present invention, the method for removing the solvent is preferably concentration to remove the solvent, and the temperature for removing the solvent is preferably 10 to 30°C, more preferably 15 to 25°C.

[0023] In the present invention, when mixing the crude extract, water and ethyl acetate, the weight ratio of the crude extract to water is preferably 1:3 to 10, more preferably 1:4 to 8; the weight ratio of the crude extract to ethyl acetate is preferably 1:3 to 10, more preferably 1:4 to 8. After mixing, stirring extraction is carried out, and the stirring extraction temperature is preferably 20°C to 40°C, more preferably 22°C to 30°C; the stirring extraction time is preferably 1.5 h to 2.5 h, more preferably 2 h. The present invention has no special limitation on the standing time, and it can be left standing until a liquid separation phenomenon occurs. In the present invention, collecting the organic extraction phase means collecting the ethyl acetate layer.

[0024] The present invention also provides a Hyssopus cuspidatus Boriss. extract prepared by the above method.

[0025] The present invention also provides the application of the above method or the above Hyssopus cuspidatus Boriss. extract in the preparation of antibacterial products.

[0026] In the application of the present invention, the product preferably includes drugs; in the present invention, the dosage forms of the drugs preferably include capsules, tablets, granules, pills, powders, ointments, mixtures, oral liquids or injections, and the present invention has no special limitation on the pharmaceutical excipients for preparing the dosage forms. In the present invention, the antibacterial preferably includes antibacterial and / or antifungal. In the present invention, the bacterial species in the antibacterial preferably include at least one of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa and Acinetobacter baumannii; the Escherichia coli preferably includes drug-resistant strains of Escherichia coli; the Staphylococcus aureus preferably includes drug-resistant strains of Staphylococcus aureus; the Staphylococcus epidermidis preferably includes drug-resistant strains of Staphylococcus epidermidis. In the present invention, the fungi in the antifungal preferably include at least one of Candida albicans, Candida tropicalis and Candida krusei; the Candida albicans preferably includes drug-resistant strains of Candida albicans.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] In the following embodiments, unless otherwise specified, they are all conventional methods.

[0029] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0030] Example 1

[0031] An Hyssopus cuspidatus Boriss. extract is prepared by the following method:

[0032] (1) Mix 100 kg of the whole plant of Hyssopus cuspidatus Boriss. with 300 kg of an ethanol solution with a volume fraction of 90%, stir at 25°C and 100 rpm for 6 h, filter with a plate filter, collect the filtrate, and concentrate to remove the solvent (the concentration is carried out by using a rotary evaporator for vacuum concentration, the concentration temperature is 40°C, and the vacuum degree is 40 Pa) to obtain a crude extract;

[0033] (2) Mix the crude extract, water and ethyl acetate in a weight ratio of 1:3:3, stir and extract at 20°C for 1 h, let it stand for liquid separation, collect the organic extraction phase, and remove the solvent at 10°C (the way of removing the solvent is to use a rotary evaporator, and the vacuum degree is 40 Pa) to obtain the Hyssopus cuspidatus Boriss. extract.

[0034] Example 2

[0035] An Hyssopus cuspidatus Boriss. extract is prepared by the following method:

[0036] (1) Mix 100 kg of the whole plant of *Hyssopus cuspidatus* Boriss. with 1000 kg of an ethanol solution with a volume fraction of 30%, stir at 60 °C and 200 rpm for 1 h, filter with a plate filter, collect the filtrate, and concentrate to remove the solvent (the concentration is carried out by rotary evaporation under reduced pressure, the concentration temperature is 50 °C, and the vacuum degree is 50 Pa) to obtain a crude extract;

[0037] (2) Mix the crude extract, water and ethyl acetate in an amount ratio of 1:10:10 by weight, stir and extract at 45 °C for 3 h, let it stand for liquid separation, collect the organic extraction phase, and remove the solvent at 30 °C (the solvent removal method is by rotary evaporation, and the vacuum degree is 50 Pa) to obtain the extract of *Hyssopus cuspidatus* Boriss.

[0038] Example 3

[0039] An extract of *Hyssopus cuspidatus* Boriss. is prepared by the following method:

[0040] (1) Mix 100 kg of the whole plant of *Hyssopus cuspidatus* Boriss. with 500 kg of an ethanol solution with a volume fraction of 75%, stir at 45 °C and 150 rpm for 2 h, filter with a plate filter, collect the filtrate, and concentrate to remove the solvent (the concentration is carried out by rotary evaporation under reduced pressure, the concentration temperature is 50 °C, and the vacuum degree is 50 Pa) to obtain a crude extract;

[0041] (2) Take 1 kg of the crude extract and place it in 4 kg of water and stir evenly, then add 4 kg of ethyl acetate, stir and extract at 30 °C for 1 h, then let it stand and separate the liquid, collect the organic extraction phase, and remove the solvent at a temperature of 20 °C (the solvent removal method is by rotary evaporation, and the vacuum degree is 50 Pa) to obtain 200 g of refined product (denoted as the extract of *Hyssopus cuspidatus* Boriss.). After calculation, the refined yield is 20%.

[0042] Example 4

[0043] The difference from Example 3 lies in that step (2) is different, and the rest are the same as in Example 3. Step (2) of this example is as follows: Place 1 kg of the crude extract in 6 kg of water and stir evenly, then add 6 kg of ethyl acetate, stir and extract at 20 °C for 2 h, then let it stand and separate the liquid, collect the organic extraction phase, and remove the solvent at a temperature of 10 °C to obtain 350 g of refined product (denoted as the extract of *Hyssopus cuspidatus* Boriss.). After calculation, the refined yield is 35%.

[0044] Example 5

[0045] The difference from Example 3 lies in step (2), and the rest is the same as in Example 3. Step (2) of this example is as follows: Place 1 kg of the crude extract in 10 kg of water and stir evenly. Then add 10 kg of ethyl acetate and stir and extract for 2 h at 20 °C. Then let it stand and separate the liquid phases. Collect the organic extraction phase and remove the solvent at a temperature of 10 °C to obtain 365 g of the refined product (denoted as the extract of Hyssopus cuspidatus Boriss). After calculation, the refined yield is 36.5%.

[0046] Example 6

[0047] The difference from Example 3 lies in step (2), and the rest is the same as in Example 3. Step (2) of this example is as follows: Place 1 kg of the crude extract in 6 kg of water and stir evenly. Then add 6 kg of ethyl acetate and stir and extract for 2 h at 35 °C. Then let it stand and separate the liquid phases. Collect the organic extraction phase and remove the solvent at a temperature of 10 °C to obtain 320 g of the refined product (denoted as the extract of Hyssopus cuspidatus Boriss). After calculation, the refined yield is 32%.

[0048] Example 7

[0049] An extract of Hyssopus cuspidatus Boriss is prepared by the following method:

[0050] (1) Mix 100 kg of the whole plant of Hyssopus cuspidatus Boriss with 600 kg of an ethanol solution with a volume fraction of 90%, stir at 45 °C and 100 rpm for 3 h, filter with a plate filter, collect the filtrate, and concentrate to remove the solvent (the concentration is carried out by using a rotary evaporator for vacuum concentration, the concentration temperature is 50 °C, and the vacuum degree is 50 Pa) to obtain 40 kg of the crude extract;

[0051] (2) Place 40 kg of the crude extract in 240 kg of water and stir evenly. Then add 240 kg of ethyl acetate and stir and extract for 2 h at 20 °C. Then let it stand and separate the liquid phases. Collect the organic extraction phase and remove the solvent at a temperature of 10 °C (the way of removing the solvent is to use a rotary evaporator, and the vacuum degree is 50 Pa) to obtain 14 kg of the refined extract of Hyssopus cuspidatus Boriss.

[0052] Example 8

[0053] LC-MS analysis of the extract of Hyssopus cuspidatus Boriss obtained in Example 7

[0054] Prepare the extract of Hyssopus cuspidatus Boriss obtained in Example 7 into a clear and transparent solution of 1 mg / ml with a methanol aqueous solution with a volume fraction of 50% as the analysis sample. Use a reversed-phase chromatographic column (2.1×5 mm) as the analytical column, mobile phase A: 0.1% formic acid water, mobile phase B: 0.1% formic acid acetonitrile, and perform gradient elution according to the program shown in Table 1:

[0055] Table 1 Gradient elution program

[0056] Time / min ％A ％B initial 100 0 2 100 0 10 90 10 28 0 100 30 0 100 30.1 100 0

[0057] The mass spectrometry conditions were as follows: ESI source, positive and negative ion modes, full scan, scan time: 2 - 30 min, scan range: 100 - 1500 Da, scan time: 0.5 sec, data format: continuum.

[0058] The results were as Figure 1 shown. The identified compounds are shown in Table 2.

[0059] Table 2 Identification results of active substances in the extract of Hyssopus cuspidatus obtained in Example 7

[0060]

[0061] Example 9

[0062] Determination of the minimum inhibitory concentration (MIC) of the extract of Hyssopus cuspidatus obtained in Example 7:

[0063] It was carried out with reference to the drug susceptibility standard experimental protocol of the Clinical and Laboratory Standards Institute (CLSI) in 2023.

[0064] (1) When the extract of Hyssopus cuspidatus obtained in Example 7 was used alone, the effects on the minimum inhibitory concentrations of the standard strain of Escherichia coli E. coli ATCC 25922, the drug-resistant strain of Escherichia coli E. coli ATCC 35218, the standard strain of Staphylococcus aureus S. aureus ATCC 25923, the drug-resistant strain of Staphylococcus aureus S. aureus ATCC 33591, the standard strain of Staphylococcus epidermidis S. epidermidis ATCC 12228, the drug-resistant strain of Staphylococcus epidermidis S. epidermidis ATCC 51625, Streptococcus pyogenes S. pyogenes BNCC 185918, Enterococcus faecalis E. faecalis ATCC 29212, Pseudomonas aeruginosa P. aeruginosa ATCC 27853, and Acinetobacter baumannii A. baumannii ATCC 19606 were detected. The specific method was as follows:

[0065] Pretreatment of the test extract: The extract of Hyssopus cuspidatus obtained in Example 7 was filtered and sterilized through a 0.22 μM filter membrane, diluted to 64 mg / mL with sterile water containing 2% Tween - 80, and ultrasonically treated in an ice - water bath at 30 Hz for 10 min for standby.

[0066] Preparation of the test bacterial suspension: Take a single colony and dilute it with sterilized 0.45% normal saline to a bacterial suspension of 0.5 MacFarland units, that is, the bacterial concentration is about 1×10 8 CFU / mL. Dilute the above bacterial suspension 1000 times with tryptic soy broth (TSB) to obtain the test bacterial suspension, with a concentration of about 1×10 6 CFU / mL.

[0067] Design of the antibacterial experiment for bacteria and determination methods of MIC and OD600: The MIC was determined by the microbroth dilution method (96-well plate method). Place 200 μL of the test Hyssopus cuspidatus Boiss. extract in the culture wells and serially dilute it (the final concentrations of the Hyssopus cuspidatus Boiss. extract are 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031 mg / mL) so that each well contains 100 μL of the Hyssopus cuspidatus Boiss. extract. Add 100 μL of the test bacterial suspension and inoculate it into a 96-well sterile culture plate. TSB is used as the negative control. Set three replicates for each group and incubate it in an incubator at 37°C for 12 h. Read the positive and negative control wells. The positive wells are clear and the negative control wells are turbid. The lowest concentration at which the medium is clear and no bacterial growth is observed with the naked eye is determined as the minimum inhibitory concentration of the drug. The results are shown in Table 3, and the visual observation results of the standard strain of Staphylococcus epidermidis S. epidermidis ATCC 12228 are as Figure 2 shown.

[0068] Meanwhile, the absorbance of the obtained 96-well plate was detected. The absorbance at 600 nm was selected for detection and plotted using GraphPad Prism 10. The OD600 results of the standard strain of Staphylococcus epidermidis S. epidermidis ATCC 12228 are shown in Figure 3 .

[0069] Table 3 MIC values of the Hyssopus cuspidatus Boiss. extract against bacteria

[0070]

[0071]

[0072] (2) To detect the effect of the Hyssopus cuspidatus Boiss. extract obtained in Test Example 7 on the minimum inhibitory concentration of the standard strain of Candida albicans C. albicans ATCC 90028, the drug-resistant strain of Candida albicans C. albicans ATCC 64550, Candida tropicalis C. tropicalis ATCC 750, and Candida krusei C. krusei ATCC 6258 when used alone, the specific method is as follows:

[0073] Pretreatment of the test extract: The same as the extract pretreatment in (1).

[0074] Preparation of the test bacterial suspension: Take a single colony and dilute it with sterilized 0.45% normal saline to a bacterial suspension of 0.5 McFarland units, that is, the bacterial concentration is about 1×10 8 CFU / mL. Dilute the above bacterial solution 1000 times with Sabouraud dextrose broth (SDB) to obtain the test bacterial suspension with a concentration of about 1×10 6 CFU / mL.

[0075] Design of the antifungal experiment and determination of MIC: The MIC was determined by the microbroth dilution method (96-well plate method). Add 200 μL of the test Hyssopus cuspidatus Boriss. extract to the culture wells and perform serial two-fold dilutions (the final concentrations of the Hyssopus cuspidatus Boriss. extract are 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031 mg / mL) so that each well contains 100 μL of the Hyssopus cuspidatus Boriss. extract. Then add 100 μL of the test bacterial suspension and inoculate it into a 96-well sterile culture plate. SDB is used as the negative control. Each group is set with three replicates and incubated in an incubator at 37°C for 12 h. Read the positive and negative control wells. The positive wells are clear and the negative control wells are turbid. The lowest concentration at which the medium is clear and no fungal growth is observed with the naked eye is determined as the minimum inhibitory concentration of the drug. The results are shown in Table 4.

[0076] Table 4 MIC values of Hyssopus cuspidatus Boriss. extract against fungi

[0077]

[0078] From the above results, it can be seen that the Hyssopus cuspidatus Boriss. extract prepared by the preparation method of the present invention has both antibacterial and antifungal effects, and also has high antibacterial effects against bacterial drug-resistant strains and fungal drug-resistant strains, showing broad-spectrum antibacterial properties.

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing hard-pointed Hyssopus officinalis extract, characterized in that: The method comprises the following steps: mixing the whole plant of Hyssopus sibiricus with an ethanol solution with a volume fraction of 30% to 90%, stirring at 25°C to 60°C for 1h to 6h, filtering, collecting the filtrate, removing the solvent, and obtaining a crude extract; mixing the crude extract, water and ethyl acetate, stirring and extracting at 20°C to 45°C for 1h to 3h, standing and collecting the organic extract phase, removing the solvent, and obtaining a Hyssopus sibiricus extract.

2. The method according to claim 1, characterized in that The weight ratio of the whole plant of hard-tipped Hyssopus officinalis to the ethanol solution with a volume fraction of 30% to 90% is 1:3 to 10.

3. The method according to claim 1, characterized in that The stirring speed is 100-200 rpm.

4. The method according to claim 1, characterized in that: The weight ratio of the crude extract to water is 1:3-10.

5. The method according to claim 1, characterized in that The weight ratio of the crude extract to ethyl acetate is 1:3-10.

6. The method according to claim 1, characterized in that The temperature for removing the solvent is 10-30°C.

7. A hard-pointed Hyssop extract, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. Use of the method according to any one of claims 1 to 6 or the Hyssopus officinalis extract according to claim 7 in the preparation of antibacterial products.

9. The use according to claim 8, characterized in that: The antimicrobial includes antibacterial and / or antifungal.

10. The use according to claim 9, characterized in that: The bacteria include at least one of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enterococcus faecalis, Pseudomonas aeruginosa and Acinetobacter baumannii; the fungi include at least one of Candida albicans, Candida tropicalis and Candida krusei.

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