Application of 5-indanol in amomum tsao-ko in antibacterial aspect

Abstract

The invention discloses an application of 5-indanol in amomum tsao-ko in an antibacterial aspect. The natural component 5-indanol is separated and purified from amomum tsao-ko kernels by adopting an ethyl acetate extraction mode, experiments show that the natural component 5-indanol is good in antibacterial activity and can effectively inhibit growth and proliferation of bacteria, and the minimum inhibitory concentration of the natural component 5-indanol to staphylococcus aureus is 62.5 mu g / mL. Further research finds that commercially available 5-indanol also has antibacterial activity and can effectively inhibit growth and proliferation of bacteria. Therefore, the 5-indanol can be used as a novel natural antibiotic for treating bacterial infection.

Description

Technical Field

[0001] This invention belongs to the field of biology, and specifically relates to the application of 5-indanol from cardamom in antibacterial properties. Background Technology

[0002] With the development of modern medicine, antibiotics are one of the main drugs for treating bacterial infections. They can effectively improve the cure rate of diseases and the survival rate of patients. However, the improper use of antibiotics, especially the abuse of antibiotics, has led to the emergence and spread of bacterial resistance, which in turn leads to patients needing to receive higher doses and broader spectrum of antibiotics, or even facing the dilemma of having no drugs available.

[0003] Amomum tsao-ko is a commonly used traditional Chinese medicine with various effects and functions, such as strengthening the stomach and aiding digestion, dispelling cold and dampness, promoting blood circulation and removing blood stasis, and anti-inflammatory and antibacterial properties. Amomum tsao-ko contains a variety of antibacterial active ingredients, mainly including pinenes and cardamomone. Among them, amomum tsao-ko extract has broad-spectrum antibacterial activity, showing significant antibacterial effects against common pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. Current research on the antibacterial active ingredients of amomum tsao-ko mostly focuses on pinenes, terpenes, and diphenylheptanes, but it itself contains a rich variety of natural active ingredients. Therefore, further research is needed on the natural active ingredients of amomum tsao-ko to discover new natural components with antibacterial activity. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of 5-indanol in the preparation of antibacterial products.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] Application of 5-indanol in the preparation of antibacterial products.

[0007] The bacteria in the antibacterial product are bacteria, including at least one of Staphylococcus aureus, Escherichia coli, and Ralstonia solanacearum; more preferably at least one of Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Ralstonia solanacearum ATCC 11696.

[0008] The products mentioned include medicines, such as medicines for treating bacterial infections.

[0009] The 5-indanol can be isolated from cardamom or obtained through conventional commercial purchases; wherein, the method for isolating 5-indanol from cardamom is as follows:

[0010] (1) Ethyl acetate extraction

[0011] The seed coat and kernel of Amomum villosum were separated. The kernel was then ground into powder, sieved, and then fully soaked and extracted with ethyl acetate. The powder was then filtered and concentrated to obtain ethyl acetate extract of Amomum villosum kernel.

[0012] (2) Separation and purification

[0013] The ethyl acetate extract of Amomum villosum seed obtained in step (1) was stirred evenly with 100-mesh silica gel powder. At the same time, 300-400-mesh silica gel powder was stirred and mixed with n-pentane and wet-loaded onto a column. The column was continuously flushed with n-pentane until the silica gel was compacted. Then, gradient elution was used: n-pentane: ethyl acetate = 100:0, 100:10, 100:20, 100:30, 100:40, 100:60, 100:80, 100:100; ethyl acetate: n-pentane = 100:100, 100:50, 100:0. 500 mL of each gradient was collected as a fraction. All fractions were then spotted by TLC. Fractions with the same characteristics were combined. Finally, the combined fraction and the 5-indanol standard were spotted by TLC to verify the 5-indanol.

[0014] The sieving process described in step (1) is preferably sieved through a 60-mesh sieve.

[0015] The ratio of the powder to ethyl acetate in step (1) is 1g:2-4ml; preferably 1g:2ml.

[0016] The soaking time in step (1) is 2 to 4 days; preferably 3 days. During the soaking period, the seeds are shaken once every 8 hours to ensure that the ethyl acetate is in full contact with the cardamom seeds.

[0017] The ethyl acetate extraction in step (1) is preferably performed at least twice.

[0018] The concentration described in step (1) is carried out by rotary evaporation.

[0019] The rotary evaporation concentration temperature is 40–45°C; preferably 40°C.

[0020] The mass ratio of the ethyl acetate extract of cardamom seed kernels to 100-mesh silica gel in step (2) is 1:1 to 1.05; preferably 1:1.03.

[0021] The method for isolating 5-indanol from cardamom further includes a step of identification using GC-MS after step (2).

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] 1. This invention uses ethyl acetate, an organic solvent, to extract and purify a natural component, 5-indanol, from the kernel of Amomum villosum. Experiments have shown that this natural component, 5-indanol, has certain antibacterial activity and can significantly inhibit the growth and proliferation of bacteria (such as Staphylococcus aureus, Escherichia coli, and Ralstonia solanacearum).

[0024] 2. The present invention also found through experiments that commercially available 5-indanol also has antibacterial activity and can effectively inhibit bacteria. Therefore, 5-indanol can be used to treat bacterial infections.

[0025] 3. The 5-indanol isolated by this invention has stronger antibacterial activity (the minimum inhibitory concentration of 5-indanol against Staphylococcus aureus is 62.5 μg / mL, while the antibacterial concentration range of conventional cardamom essential oil is 1-3 mg / mL). Attached Figure Description

[0026] Figure 1 This is a schematic diagram of Staphylococcus aureus activation on LB agar plates (front and back).

[0027] Figure 2 This is a schematic diagram of the activation of Escherichia coli on LB plates (both sides).

[0028] Figure 3 This is a schematic diagram of Ralstonia solanacearum LB agar plate (front and back).

[0029] Figure 4 This is a correlation clustering heatmap of the components identified by GC-MS of Amomum villosum.

[0030] Figure 5 This is a comparison of the fingerprint chromatograms obtained by GC-MS determination of 5-indanol and GC-MS determination of crude extract of Amomum villosum.

[0031] Figure 6 This is a comparison of the TLC verification results of 5-indanol isolated from cardamom and the standard.

[0032] Figure 7 This is a NIST 14 identification result of 5-indanol isolated from cardamom.

[0033] Figure 8 This is a diagram showing the antibacterial test results of 5-indanol isolated from cardamom.

[0034] Figure 9 This is a time-dependent bactericidal curve of 5-indanol against Staphylococcus aureus. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Experimental methods in the following embodiments that do not specify specific experimental conditions are generally obtained according to conventional experimental conditions or conventional culture and separation methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent manufacturers.

[0036] The Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Ralstonia solanacearum ATCC 11696 involved in the embodiments of this invention were all purchased from the American Type Culture Collection (ATCC).

[0037] The cardamom involved in this embodiment of the invention is a high-quality cardamom selected from Lushui City, Nujiang Prefecture, Yunnan Province.

[0038] The organic solvents involved in the embodiments of this invention are specifically analytical grade methanol, ethanol, and ethyl acetate.

[0039] The components and formulations of the culture medium involved in the embodiments of the present invention are as follows:

[0040] ① Components and proportions of LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1 g / L glucose, adjust pH to 7.0, autoclave at 121℃ for 20 min.

[0041] ② Components and proportions of CAMHB medium: 3 g / L beef extract powder, 17.5 g / L acid-hydrolyzed casein, 1.5 g / L soluble starch, 0.05 g / L calcium chloride, pH adjusted to 7.0, autoclaved at 121℃ for 20 min.

[0042] ③ Components and proportions of TSA medium: 15 g / L casein trypsin digest, 5 g / mL soybean papain hydrolysate, 5 g / mL sodium chloride, 15 g / mL agar, pH adjusted to 7.3, autoclaved at 121℃ for 20 min.

[0043] Example 1: Initial antibacterial screening of cardamom

[0044] The initial antibacterial screening method for cardamom includes the following steps:

[0045] 1. Activation and culture of the strain

[0046] Glycerol tubes containing Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and Ralstonia solanacearum (ATCC 11696) were taken from a -80℃ freezer and streaked onto LB agar plates. Single colonies were picked and placed in Erlenmeyer flasks containing LB liquid medium and incubated at 28℃–40℃ (preferably 37℃) with shaking at 180 rpm for 24 h for activation. Results are as follows: Figure 1 , Figure 2 and Figure 3 As shown.

[0047] 2. Preparation of Amomum tsao-ko extract

[0048] (1) Extraction of active ingredients from Amomum tsao-ko: Amomum tsao-ko was pulverized into powder using a pulverizer, and the obtained powder was sieved through a 60-mesh sieve to obtain fine powder. 200g of the fine powder was weighed and soaked in organic solvents (methanol, ethanol, ethyl acetate) at a material-to-liquid ratio of 1g:10-20ml. Under room temperature conditions, ultrasonic extraction (ultrasonic power 240W, ultrasonic frequency 40KHZ) was performed, followed by standing for 1h. A second ultrasonic extraction (ultrasonic power 240W, ultrasonic frequency 40KHZ) was then performed, and finally, the mixture was allowed to stand for 24h to obtain the first extract. Then, the above operation (repeating the steps of adding organic solvent and ultrasonic extraction) was repeated to obtain the second extract, and the two extracts were combined.

[0049] (2) Preparation of crude extract of Amomum villosum: The extract obtained in step (1) was filtered using a Buchner funnel and a circulating water vacuum pump to remove excess residue and obtain a pure extract. The solvent was removed by rotary evaporation (45°C), and the extract was concentrated to obtain the corresponding methanol extract, ethanol extract and ethyl acetate extract.

[0050] 3. Antibacterial activity of crude extract of cardamom

[0051] (1) Count the three types of bacterial cultures prepared above, and adjust the bacterial culture to 1×10⁻⁶. 8 CFU / mL, then serially diluted to 5.5 × 10⁻⁶. 5 CFU / mL. The MIC experiment was performed using the checkerboard method, with dimethyl sulfoxide (DMSO) as a suspending agent. The volume ratio of DMSO to the culture medium was 10 / 1000 to avoid the influence of DMSO on the antibacterial experiment results. The extracts of cardamom in methanol, ethanol, and ethyl acetate were dissolved to prepare solutions with a concentration of 10 mg / mL.

[0052] (2) Take 50 μL of sterile CAMHB medium and place it in each well. Also add 50 μL of the above-prepared solution to the wells in the first column of the 96-well plate. Dilute the solution by serial dilution up to the wells in the 10th column. Use gentamicin sulfate as a positive control in the 11th column and sterile CAMHB medium as a negative control in the 12th column.

[0053] (3) The minimum inhibitory concentration (MIC) was initially determined based on the absence of bacterial plaques larger than 2 mm at the bottom of the 96-well plate and the absence of turbidity in the solution within the wells. Then, 50 μL of the liquid from each well was aspirated and spread onto a TSA plate. The plate with no sterile growth was considered the minimum bactericidal concentration. The experiment was repeated three times. The results are shown in Table 1.

[0054] Table 1. Minimum inhibitory concentrations of Amomum villosum against Staphylococcus aureus, Escherichia coli, and Ralstonia solanacearum.

[0055]

[0056] Example 2: GC-MS analysis and identification of cardamom components

[0057] The analytical method for the components of cardamom includes the following steps:

[0058] 1. Pretreatment of cardamom for GC-MS testing

[0059] Weigh 0.5 g of the crude extract of acetic acid ethyl ester obtained in Example 1 using a 0.1 g balance, add 5 mL of dichloromethane (containing 5 ppm internal standard: ethyl decanoate), and extract by sonication for 30 min. Then let it stand for 1 h, and perform a second sonication for 30 min. Add 5 mg of anhydrous sodium sulfate, and place in a refrigerator at 4 °C overnight for later use.

[0060] 2. GC-MS testing of cardamom components

[0061] After the above pretreatment, carefully aspirate the supernatant with a syringe, filter the supernatant through a 0.22μm hydrophobic filter membrane, and place the filtered sample in a brown chromatographic bottle containing 2ml of solvent, ensuring that the volume of the sample to be tested in each chromatographic bottle is more than 1ml.

[0062] GC-MS: SHIMADZU GCMS-TQ8040, instrument specifications: triple quadrupole, column: Agilent DB-5MS gas chromatographic column (30m×0.25mm×0.25μm), injection port temperature 250℃, detector temperature 260℃, initial temperature 60℃ held for 2 min, then increased to 260℃ / min at 6℃ / min and held for 15 min. Injection volume: 1μL, split ratio 3:1; MS conditions: EI ion source, ionization energy 70ev, scan range m / z 50~600amu, ion source temperature 200℃, GC-MS interface temperature 250℃;

[0063] After the measurements were completed, a test file of the components of the crude extract of Amomum tsao-ko was obtained. The test file was then calibrated, purified, and discarded using MSDIAL software before identification and analysis. The obtained test file of the crude extract of Amomum tsao-ko was imported into the NIST 14 database. By comparing linear retention indices, components with a retention match rate of over 80% and a retention index difference within ±10 were identified. Cluster heatmap analysis was performed on the identified components to screen out the components with the strongest antibacterial activity found in the ethyl acetate extract of Amomum tsao-ko, mainly 5-indanol, such as... Figure 4 As shown.

[0064] 3,5-Indanol Test

[0065] The same GC-MS method described above was used for determination. The obtained 5-indanol test files and the GC-MS test files of the crude extract of *Amomum villosum* were calibrated, purified, and removed using MSDIAL software before identification and analysis. The samples were imported into the NIST 14 database. By comparing linear retention indices, components with a retention match rate of over 80% and a retention index difference within ±10 were identified. Results are as follows... Figure 5 As shown.

[0066] Example 3: Isolation of 5-indanol from Amomum villosum

[0067] 1. Ethyl acetate extraction

[0068] The seed coat and kernel of Amomum tsao-ko were separated, yielding a total of 2400g of kernels. The kernels were ground into powder and sieved through a 60-mesh sieve to obtain a fine powder. Then, 4800mL of ethyl acetate was added at a ratio of 1g:2ml. The mixture was soaked for 3 days, shaking every 8 hours during the soaking period to ensure that the ethyl acetate was fully in contact with the Amomum tsao-ko kernels.

[0069] The extract was filtered through a Buchner funnel to obtain a pure extract. The extract was then concentrated by rotary evaporation at 40°C. The recovered ethyl acetate was poured back into the container containing the cardamom kernels for a second extraction. Finally, 113.94 g of ethyl acetate extract of cardamom kernels was obtained.

[0070] 2. Silica gel column separation

[0071] The obtained 113.94g of ethyl acetate extract of Amomum villosum seed was added to 110g of 100-mesh silica gel powder at a ratio of approximately 1:1 (mass ratio) and stirred until homogeneous. 900g of 300-400-mesh silica gel powder was weighed and mixed with n-pentane. The mixture was then loaded onto a column using a wet method, continuously flushing the column with n-pentane until the silica gel was compacted. Elution gradients were set as follows: n-pentane:ethyl acetate = 100:0, 100:10, 100:20, 100:30, 100:40, 100:60, 100:80, 100:100; ethyl acetate:n-pentane = 100:100, 100:50, 100:0 (all volume ratios). 500mL of each gradient was collected as a fraction. All fractions were spotted onto a TLC plate, and fractions with similar characteristics were combined. Finally, the combined fraction and 5-indanol standard (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) were subjected to TLC plate testing for verification. The results are as follows: Figure 6 As shown (n-pentane: ethyl acetate = 100: 40).

[0072] 3. GC-MS identification

[0073] Fractions with TLC spotting results identical to those of the standard were identified by GC-MS, and the name and structure of the compound were determined according to the Nist database. The specific method is as follows:

[0074] GC-MS: SHIMADZU GCMS-TQ8040, instrument specifications: triple quadrupole, column: Agilent DB-5MS gas chromatographic column (30m × 0.25mm × 0.25μm), injection port temperature 250℃, detector temperature 260℃, initial temperature 60℃ held for 2 min, then increased to 260℃ / min at 6℃ / min and held for 15 min. Injection volume: 1μL, split ratio 3:1; MS conditions: EI ion source, ionization energy 70ev, scan range m / z 50~600amu, ion source temperature 200℃, GC-MS interface temperature 250℃.

[0075] The obtained 100:40 gradient fraction test files were calibrated, purified, and removed using MSDIAL software before identification and analysis. The samples were then imported into the NIST 14 database. By comparing linear retention indices, components with a retention match rate of over 80% and retention indices within ±10 were identified. Figure 7 The fraction was identified as 5-indanol (Formula I).

[0076]

[0077] 4. Verification of antibacterial effect

[0078] Staphylococcus aureus (ATCC 6538) was streaked onto LB agar plates after being removed from a -80℃ freezer. Single colonies were picked and placed in Erlenmeyer flasks containing CAMHB liquid medium and incubated at 28℃–40℃ with shaking at 180 rpm for 24 h to activate the colonies. The cultured bacterial suspensions were then counted, and the culture volume was adjusted to 1×10⁻⁶. 8 CFU / mL, then serially diluted to 5.5 × 10⁻⁶. 5 CFU / mL. Spread the diluted bacterial suspension evenly onto a TSA plate using a cotton swab. Place drug sensitivity test discs soaked in different concentrations of 5-indanol solution onto the plate, incubate at 37°C for 24 hours, and observe the size of the inhibition zones.

[0079] The results showed that the inhibition zone size of 5-indanol was 27.2 mm at a concentration of 125 μg / mL; the inhibition zone size was 25.3 mm at a concentration of 62.5 μg / mL, while no inhibition zone appeared at concentrations below 62.5 μg / mL. This indicates that the minimum inhibitory concentration of 5-indanol is 62.5 μg / mL. Figure 8 ).

[0080] Example 4: Antibacterial activity of 5-indanol

[0081] The method for evaluating the antibacterial activity of 5-indanol includes the following steps:

[0082] 1. Activation and culture of bacterial strains

[0083] Take the glycerol tubes of Staphylococcus aureus (ATCC 6538) from the -80℃ freezer and streak them onto LB agar plates. Pick a single colony and place it in an Erlenmeyer flask containing LB liquid medium. Incubate at 28℃~40℃ with shaking at 180 rpm for 24 h to activate the colony.

[0084] 2. Adjust the concentration of the bacterial solution to be tested.

[0085] Count the cultured bacteria separately and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL, then serially diluted to 5.5 × 10⁻⁶. 5 CFU / mL, for later use.

[0086] Preliminary screening of the antibacterial activity of 3,5-indanol

[0087] (1) Weigh 2 mg of 5-indanol (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), add 1 ml of dimethyl sulfoxide (DMSO) to dissolve it, and prepare a solution with a concentration of 2 mg / mL for later use. Prepare CAMHB liquid medium and TSA solid medium for later use.

[0088] (2) Take 50 μL of sterile CAMHB liquid culture medium and place it in each well. Also add 50 μL of the prepared solution to the wells in the first column of the 96-well plate. Dilute the solution by two-fold serial dilution until the wells in the 10th column. Use gentamicin sulfate as a positive control in the 11th column and sterile CAMHB liquid culture medium as a negative control in the 12th column.

[0089] (3) The minimum inhibitory concentration (MIC) was initially determined based on the absence of bacterial plaques larger than 2 mm at the bottom of the 96-well plate and the absence of turbidity in the solution within the wells. Then, 50 μL of the liquid from each well was aspirated and spread onto a TSA plate. The plate with no sterile growth was considered the minimum bactericidal concentration. The experiment was repeated three times. The results are shown in Table 2.

[0090] Table 2. Minimum inhibitory concentration of 5-indanol against Staphylococcus aureus

[0091] drug Minimum inhibitory concentration (μg / mL) 5-Indanol 62.50

[0092] 4. Determination of the sterilization curve of 5-indanol time

[0093] (1) Prepare CAMHB liquid culture media with concentrations of 0, 0.5MIC, MIC, 2MIC and 4MIC of 5-indanol for later use.

[0094] (2) The cultured bacterial solution (Staphylococcus aureus ATCC 6538) was diluted from 1×10⁻⁶ to 10⁻⁶. 8 CFU / ml, serially diluted to 5.5 × 10⁻⁶ 5 After adding CFU / ml to the drug-containing culture medium, incubate at 28℃~40℃ in Erlenmeyer flasks, with each Erlenmeyer flask containing 30ml of the test liquid.

[0095] (3) Samples were taken at time points of 0, 0.5, 1.5, 3, 4, 8, and 12, and evenly spread onto TSA plates. Colony counts were calculated. A bactericidal curve was plotted with the logarithm of colony-forming units per milliliter as the ordinate and time as the abscissa. Starting at 0 h, when the antibiotic was added to the test bacterial suspension, a decrease in bacterial survival count ≥ 3 log CFU / mL was considered an inhibitory effect of the antibiotic on the test bacteria at that concentration. The experiment was repeated three times. Results are as follows: Figure 9 As shown.

[0096] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

Application of 1,5-indanol in the preparation of antibacterial products.

2. The application according to claim 1, characterized in that: The bacteria mentioned are bacteria.

3. The application according to claim 2, characterized in that: The bacteria mentioned are at least one of Staphylococcus aureus, Escherichia coli, and Ralstonia solanacearum.

4. The application according to claim 1, characterized in that: The 5-indanol was isolated from cardamom, and the specific steps are as follows: (1) Ethyl acetate extraction The seed coat and kernel of Amomum villosum were separated. The kernel was then ground into powder, sieved, and then fully soaked and extracted with ethyl acetate. The powder was then filtered and concentrated to obtain ethyl acetate extract of Amomum villosum kernel. (2) Separation and purification The ethyl acetate extract of cardamom seed obtained in step (1) is stirred evenly with 100-mesh silica gel powder. At the same time, 300-400-mesh silica gel powder is stirred and mixed with n-pentane and wet-loaded onto a column. The column is continuously flushed with n-pentane until the silica gel is compacted. Then, gradient elution is used, with n-pentane:ethyl acetate = 100:0, 100:10, 100:20, 100:30, 100:40, 100:60, 100:80, 100:

100. Ethyl acetate: n-pentane = 100:100, 100:50, 100:0; 500 mL of each gradient was collected as a fraction, and all fractions were then spotted by TLC. Fractions with the same characteristics were combined, and finally, the combined fractions and 5-indanol standards were verified by TLC to obtain 5-indanol.

5. The application according to claim 4, characterized in that: The ratio of the powder to ethyl acetate in step (1) is 1g:2-4ml.

6. The application according to claim 4, characterized in that: The mass ratio of the ethyl acetate extract of cardamom seed kernels to 100-mesh silica gel in step (2) is 1:1 to 1.

05.

7. The application according to claim 4, characterized in that: The soaking time described in step (1) is 2 to 4 days.

8. The application according to claim 4, characterized in that: The concentration described in step (1) is carried out by rotary evaporation; The rotary evaporation concentration temperature is 40–45°C.

9. The application according to claim 4, characterized in that: The sieving mentioned in step (1) refers to sieving through a 60-mesh sieve; The ethyl acetate extraction in step (1) is performed more than twice.

10. The application according to claim 1, characterized in that: The product in question is a medicine.

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