A method for extracting antibacterial monomer compounds from Scutellaria baicalensis leaves

Abstract

The present invention provides a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria baicalensis, relating to the technical field of traditional Chinese medicine. The extraction method of the present invention comprises subjecting the leaves of Scutellaria baicalensis to alcohol extraction, followed by further extraction using petroleum ether, ethyl acetate, and n-butanol in sequence, and then isolating three antibacterial monomeric compounds from the leaves of Scutellaria baicalensis based on antibacterial activity in combination with chromatography, including compound 1, luteolin, and gallic acid. The method of the present invention extracts compound 1 from the leaves of Scutellaria baicalensis for the first time and provides a new extraction method for luteolin and gallic acid, which not only provides a new route for obtaining drug sources, but also provides new technical solution support for the further separation and activity evaluation of monomeric compounds in the leaves of Scutellaria baicalensis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a method for extracting antibacterial monomeric compounds from leaves of Scutellaria baicalensis. Background Art

[0002] The leaves of the genus Acacia are sharp. Turpinia arguta ) is a traditional Chinese herbal medicine in my country and is included in the "Chinese Pharmacopoeia 2020 Edition". The leaves of Rhizoma Cibotii have the effects of clearing away heat and detoxifying, relieving sore throat and swelling, antibacterial and anti-inflammatory, promoting blood circulation and relieving pain, and enhancing immunity. They are mostly used clinically for the treatment of abscesses, fever, ulcers, inflammation, etc. Studies have shown that the extract of Rhizoma Cibotii leaves has a strong inhibitory effect on Staphylococcus aureus (including penicillin-resistant strains), which is equivalent to 1 unit of penicillin per milliliter; in addition, existing studies have found that the ethanol extract of Rhizoma Cibotii leaves has high antibacterial activity against Staphylococcus aureus and Vibrio parahaemolyticus. The ethanol extract of Rhizoma Cibotii is rich in 20 types of secondary metabolites such as phenolic acids and flavonoids. It can be seen that the leaves of Rhizoma Cibotii contain chemical components that can effectively inhibit bacteria, but there are still few reports on the extraction, separation and identification of the antibacterial active monomer compounds in the leaves of Rhizoma Cibotii.

[0003] The traditional method for screening active plant ingredients is to first test the activity of crude plant extracts or parts, select the most active parts, and then use chromatography and mass spectrometry to perform precise component separation and identification. In this method, the activity detection of the components is carried out separately from the separation, analysis, and structural identification of the different components. Currently, the traditional screening method is used to isolate the active ingredients of the leaves of Scutellaria baicalensis. Studies have been conducted on the separation and purification of the n-butanol and ethyl acetate parts of the ethanol extract of the leaves of Scutellaria baicalensis. A total of 27 flavonoids were isolated and identified from the leaves of Scutellaria baicalensis. However, there is still a lack of specific solutions to support the further separation and activity evaluation of the monomeric compounds.

[0004] Therefore, there is an urgent need for a technical solution to accurately separate monomer compounds with antibacterial activity from the leaves of Scutellaria baicalensis, which is of great significance to the research in the field of Scutellaria baicalensis medicine. Summary of the Invention

[0005] The present invention aims to provide a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria serrata. The extraction method provided by the present invention, guided by antibacterial activity, combines chromatography to extract substances with antibacterial activity from the leaves of Scutellaria serrata. This method can achieve the extraction of antibacterial monomeric compounds from the medicinal material of Scutellaria serrata. Modern spectral and spectroscopic techniques are used to structurally identify the isolated antibacterial monomeric compounds, which are identified as Compound 1, luteolin, and gallic acid.

[0006] The present invention provides a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria baicalensis, comprising the following steps:

[0007] S1. Extracting the leaves of Scutellaria baicalensis with ethanol and concentrating the extracted extract to obtain an alcohol extract. Extracting the extracted extract with petroleum ether, ethyl acetate, and n-butanol in sequence, separating and concentrating the extracted extract to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract.

[0008] S2. Dissolve the ethyl acetate extract in methanol and apply it to a silica gel column. Gradient elution is performed using a dichloromethane-methanol system with a volume ratio of 50:1, 30:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1, to obtain eight fractions, which are concentrated into an extract. The second fraction is selected as effective fraction A.

[0009] S3. Dissolve the effective fraction A extract in methanol and apply it to an ODS column. Gradient elution is performed using a methanol-water system with volume fractions of 40%, 60%, 80%, 85%, 90%, 95%, and 100% in sequence to obtain four fractions, which are concentrated into an extract. The third fraction is selected as the effective fraction B. The extract of the effective fraction B is evaporated to dryness under reduced pressure to obtain monomer compound 1.

[0010] S4. Dissolve the n-butanol extract in distilled water and apply it to a macroporous adsorption resin column. Elute with 30%, 50%, 70%, and 95% ethanol in sequence to obtain 30%, 50%, 70%, and 95% ethanol elution fractions, which are concentrated into extracts. The 50% ethanol elution fraction is selected as the effective fraction C.

[0011] S5. Dissolve the effective fraction C extract in methanol and apply it to a silica gel column. Gradient elution is performed using a chloroform-methanol system with a volume ratio of 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 2:1, and 1:1, to obtain ten fractions, which are concentrated into an extract. The first and eighth fractions are selected as effective fractions D and E, respectively.

[0012] S6. Dissolve the effective fraction D extract in methanol and apply it to a gel column. Use 60% methanol solvent for isocratic elution to obtain a fraction. Perform liquid phase purification on the separated fraction and evaporate to dryness under reduced pressure to obtain monomer compound 2.

[0013] S7. Dissolve the effective fraction E extract in methanol and apply it to a gel column. Use 80% methanol solvent for isocratic elution to obtain two fractions and concentrate them into an extract. Select the second fraction as the effective fraction F. Perform liquid phase purification on the effective fraction F and evaporate to dryness under reduced pressure to obtain monomer compound 3.

[0014] Optionally, when the ethanol extraction of the Scutellaria baicalensis leaves is performed in S1, the volume fraction of ethanol in the ethanol solution is 65%-75%; and / or, the alcohol extraction and concentration are concentrated using a rotary evaporator; and / or, when the ethanol extraction of the Scutellaria baicalensis leaves is performed, the Scutellaria baicalensis leaves are dried and crushed in advance; and / or, when the ethanol extraction of the Scutellaria baicalensis leaves is performed, the mixing ratio of the Scutellaria baicalensis leaves and ethanol is (4-6) kg: (8-12) L.

[0015] Optionally, when the ethyl acetate extract is dissolved in methanol and loaded onto a silica gel column in S2, the volume ratio of silica gel to sample is 2-3:1, and the sample is a methanol solution of the ethyl acetate extract;

[0016] Optionally, the macroporous adsorption resin column in S4 is one of AB-8 macroporous adsorption resin column, D101 macroporous adsorption resin column, and XAD-16 macroporous adsorption resin column;

[0017] Optionally, the liquid phase conditions for liquid phase purification of the separated fractions in S6 are an acetonitrile-ultrapure water system, a concentration of 15%-30%, a flow rate of 8-12 mL / min, a column temperature of 26°C-32°C, and a detection band of 340 nm-360 nm.

[0018] Optionally, the liquid phase conditions for liquid phase purification of the effective fraction F in S7 are a formic acid water-acetonitrile system, a flow rate of 10-14 mL / min, a column temperature of 25°C-30°C, and a detection band of 260 nm-280 nm; wherein the volume fraction of formic acid water is 1%, and / or the system proportion of formic acid water in the formic acid water-acetonitrile system is 86%-90%.

[0019] Alternatively, in the above extraction method, the structure of the monomer compound 1 is as follows:

[0020] .

[0021] Alternatively, in the above extraction method, the structure of the monomer compound 2 is as follows:

[0022] .

[0023] Alternatively, in the above extraction method, the structure of the monomer compound 3 is as follows:

[0024] . BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the extraction method according to Example 1 of the present invention;

[0026] Figure 2 The monomer compound 1 (Compound 1) extracted from Example 1 of the present invention 1H NMR spectrum;

[0027] Figure 3 The monomer compound 1 (Compound 1) extracted from Example 1 of the present invention 13 C NMR spectrum;

[0028] Figure 4 This is a high-resolution mass spectrum of the monomer compound 1 (Compound 1) extracted in Example 1 of the present invention;

[0029] Figure 5 The monomer compound 2 (luteolin) extracted in Example 1 of the present invention 1 H NMR spectrum;

[0030] Figure 6 The monomer compound 2 (luteolin) extracted in Example 1 of the present invention 13 C NMR spectrum;

[0031] Figure 7 The monomer compound 3 (gallic acid) extracted in Example 1 of the present invention is 1 H NMR spectrum;

[0032] Figure 8 The monomer compound 3 (gallic acid) extracted in Example 1 of the present invention is 13 C NMR spectrum;

[0033] Figure 9 This is a scanning electron microscope (SEM) observation image in the antibacterial activity test of Example 1 of the present invention;

[0034] Figure 10 This is a conductivity diagram in the antibacterial activity test of Example 1 of the present invention;

[0035] Figure 11 This is a diagram of alkaline phosphatase (AKP) activity determination in the antibacterial activity detection in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0037] The present invention provides a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria baicalensis, comprising the following steps:

[0038] S1. Extracting the leaves of Scutellaria baicalensis with ethanol and concentrating the extracted extract to obtain an alcohol extract. Extracting the extracted extract with petroleum ether, ethyl acetate, and n-butanol in sequence, separating and concentrating the extracted extract to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract.

[0039] S2. Dissolve the ethyl acetate extract in methanol and apply it to a silica gel column. Gradient elution is performed using a dichloromethane-methanol system with a volume ratio of 50:1, 30:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1, to obtain eight fractions, which are concentrated into an extract. The second fraction is selected as effective fraction A.

[0040] S3. Dissolve the effective fraction A extract in methanol and apply it to an ODS column. Gradient elution is performed using a methanol-water system with volume fractions of 40%, 60%, 80%, 85%, 90%, 95%, and 100% in sequence to obtain four fractions, which are concentrated into an extract. The third fraction is selected as the effective fraction B. The extract of the effective fraction B is evaporated to dryness under reduced pressure to obtain monomer compound 1.

[0041] S4. Dissolve the n-butanol extract in distilled water and apply it to a macroporous adsorption resin column. Elute with 30%, 50%, 70%, and 95% ethanol in sequence to obtain 30%, 50%, 70%, and 95% ethanol elution fractions, which are concentrated into extracts. The 50% ethanol elution fraction is selected as the effective fraction C.

[0042] S5. Dissolve the effective fraction C extract in methanol and apply it to a silica gel column. Gradient elution is performed using a chloroform-methanol system with a volume ratio of 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 2:1, and 1:1, to obtain ten fractions, which are concentrated into an extract. The first and eighth fractions are selected as effective fractions D and E, respectively.

[0043] S6. Dissolve the effective fraction D extract in methanol and apply it to a gel column. Use 60% methanol solvent for isocratic elution to obtain a fraction. Perform liquid phase purification on the separated fraction and evaporate to dryness under reduced pressure to obtain monomer compound 2.

[0044] S7. Dissolve the effective fraction E extract in methanol and apply it to a gel column. Use 80% methanol solvent for isocratic elution to obtain two fractions and concentrate them into an extract. Select the second fraction as the effective fraction F. Perform liquid phase purification on the effective fraction F and evaporate to dryness under reduced pressure to obtain monomer compound 3.

[0045] In fact, the present invention provides a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria serrata, using antibacterial activity as a guide, and combines chromatographic methods to extract substances with antibacterial activity from the leaves of Scutellaria serrata. Specifically, the isolates are subjected to an antibacterial activity assay, and preferably 1-3 isolates or the top 20% of isolates from the same batch that perform the assay are further isolated.

[0046] In some embodiments, during step S1, the leaves of the safflower are extracted using an ethanol solution having a volume fraction of 65%-75%. In practice, when extracting the leaves of the safflower with ethanol, the leaves are pre-dried and pulverized. The leaves and ethanol solution can be mixed and extracted at a ratio of (4-6) kg: (8-12) L.

[0047] Specifically, when performing the alcohol extraction and concentration in step S1 to obtain the alcohol extract, a rotary evaporator is used for concentration, and the parameters of the rotary evaporator are 45°C and -0.09 MPa. At this time, the obtained alcohol extract is in a solid-liquid mixed state; when performing the separation and concentration after extraction in step S1, further freeze-drying is required to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract.

[0048] In some embodiments, during step S2, when the methanol solution of the ethyl acetate extract is applied to the silica gel column, the volume ratio of silica gel to sample is 2-3:1, wherein the sample is a methanol solution of the ethyl acetate extract. In practice, during step S2 for gradient elution, the eluted fractions are subjected to TLC (thin layer chromatography) analysis, and the fractions are combined based on the number of detected spots and Rf values. During step S3 for gradient elution, the eluted fractions are subjected to HPLC analysis, and the fractions are combined based on the number of detected main peaks and peak elution times.

[0049] Specifically, when executing step S2 for gradient elution, eight merged fractions are obtained and are named fractions a to h, and the second fraction of these eight fractions, i.e., fraction b, is selected as the effective fraction A; when executing step S3 for gradient elution, four merged fractions are obtained and are named b.1 to b.4, and the third fraction, i.e., b.3, is selected as the effective fraction B.

[0050] Specifically, in step S3, the effective fraction B extract was evaporated under reduced pressure to obtain monomer compound 1, and the structure of the monomer compound was identified as compound 1, which is from the literature: Siswina, T., et al. (2023). "AntifungalConstituents of Piper crocatum and Their Activities as ErgosterolBiosynthesis Inhibitors Discovered via In Silico Study Using ADMET and Drug-Likeness Analysis." Molecules 28(23), and the structure is: .

[0051] In some embodiments, during the execution of step S4, the macroporous adsorption resin column selected is one of AB-8 macroporous adsorption resin column, D101 macroporous adsorption resin column, and XAD-16 macroporous adsorption resin column.

[0052] In some embodiments, during the execution of step S6, the liquid phase conditions for liquid phase purification of the separated fraction are an acetonitrile-ultrapure water system, a concentration of 15%-30%, a flow rate of 8-12 mL / min, a column temperature of 26°C-32°C, and a detection band of 340 nm-360 nm.

[0053] In some embodiments, during step S7, the liquid phase conditions for liquid phase purification of the effective fraction F are a formic acid-water-acetonitrile system, a flow rate of 10-14 mL / min, a column temperature of 25° C.-30° C., and a detection wavelength of 260 nm-280 nm.

[0054] Specifically, when the effective fraction F is subjected to liquid phase purification in step S7, the volume fraction of formic acid water is 1%.

[0055] In some embodiments, when the effective fraction F is subjected to liquid phase purification in step S7, the proportion of formic acid water in the formic acid water-acetonitrile system is 86%-90%.

[0056] In fact, when executing step S5 for gradient elution, the eluted and separated fractions are subjected to TLC detection (thin layer chromatography detection), and the fractions are merged based on the number of detected spots and Rf values; when executing step S7 for gradient elution, the eluted and separated fractions are subjected to HPLC detection, and the fractions are merged based on the number of detected main peaks and peak elution time.

[0057] Specifically, when executing step S5 for gradient elution, ten merged fractions are obtained and named fractions 1 to 10, respectively. The first fraction and the eighth fraction of these ten fractions are selected as effective fraction D and effective fraction E, respectively, that is, fraction 1 is effective fraction D, and fraction 8 is effective fraction E; when executing step S7 for gradient elution, two merged fractions are obtained and named a.1 and a.2, respectively, and the second fraction, i.e., a.2, is selected as effective fraction F.

[0058] Specifically, in step S6, the effective fraction D extract was evaporated under reduced pressure to obtain monomer compound 2, and the structure of the monomer compound was identified as luteolin, with the structure:

[0059] .

[0060] Specifically, in step S7, the effective fraction F extract was evaporated to dryness under reduced pressure to obtain monomer compound 3, and the structure of the monomer compound was identified as gallic acid, with the structure:

[0061] . Example 1

[0062] See also Figure 1 This embodiment 1 provides a method for extracting antibacterial monomeric compounds from the leaves of Scutellaria baicalensis, comprising the following steps:

[0063] S1. 5.0 kg of dried Scutellaria serrata leaves were crushed, passed through a 40-mesh sieve, and extracted with 10 L of 70% ethanol solution at room temperature for three times, each soaking for 24 h. The extracts were combined and filtered, and the filtrate was concentrated using a rotary evaporator to obtain a Scutellaria serrata leaf extract without ethanol odor, obtaining 500 g of alcohol-extracted extract. This alcohol-extracted extract was in a solid-liquid mixed state and was also referred to as Scutellaria serrata leaf concentrate. The alcohol-extracted extract was extracted with 1 L of petroleum ether, ethyl acetate, and water-saturated n-butanol solution, followed by separation and concentration to obtain 20.75 g of petroleum ether extract, 35.8 g of ethyl acetate extract, 23.24 g of n-butanol extract, and 25.26 g of the raffinate aqueous phase extract. 0.5 g of each extract was dissolved in pure methanol to prepare 25 mg / mL and 50 mg / mL extract solutions for antibacterial activity assay (results are shown in Table 1.1).

[0064] S2. Dissolve 35.8 g of ethyl acetate extract in pure methanol to obtain 5 mL of methanol solution of ethyl acetate extract of the sample, and adsorb and mix the sample with thin layer chromatography silica gel. The volume ratio of silica gel to sample is 2.5:1. Remove methanol by rotary evaporation. Use wet column packing and dry sample loading. Use dichloromethane-methanol system with volume ratios of 50:1, 30:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1 for gradient elution. Adjust the flow rate to 10 mL / min. Collect fractions every 50 mL. Use dichloromethane-methanol system with a volume ratio of 8:1 as the developing solvent. Perform TLC on the fractions. According to the test results, fractions with similar chromatographic spot number and relative migration value Rf (the ratio of the distance from the sample point to the center of the chromatographic spot to the distance from the sample point to the solvent front) are combined into one fraction to obtain eight fractions a to h, which are concentrated and freeze-dried into extracts. Take 0.1 g of each extract and dissolve it in pure methanol to prepare 5 The antibacterial activity of the fraction solutions with a concentration of mg / mL was determined (results are shown in Table 1.2), and fraction b was selected as the effective fraction;

[0065] S3. Dissolve 4 g of fraction b extract in pure methanol to obtain 5 mL of methanol solution of fraction b extract, apply it to ODS column, use wet loading method, use methanol-water system with volume fractions of 40%, 60%, 80%, 85%, 90%, 95%, and 100% for gradient elution, adjust the flow rate to 5 mL / min, collect fractions every 20 mL, and detect the fractions by HPLC. The chromatographic conditions are: Wsters C18 column, column temperature 30℃, mobile phase of 80% methanol-water mixed solvent, flow rate 0.8 mL / min, sample volume 20 μL, detection band set to 190 nm~800 nm, merge fractions with the same number of main peaks and similar elution time to obtain four fractions b.1 to b.4, and concentrate them into extracts, take 0.1 g of each extract and dissolve it in pure methanol to prepare 2.5 The antibacterial activity of the fraction solution with a concentration of mg / mL was determined (results are shown in Table 1.2). Fraction b.3 was selected as the effective fraction. The extract of fraction b.3 was evaporated to dryness under reduced pressure to obtain 3 mg of monomer compound 1, which was then structurally determined to be compound 1.

[0066] S4. Dissolve 23.24 g of n-butanol extract in distilled water to obtain 5 mL of n-butanol extract aqueous solution. Apply the solution to an AB-8 macroporous adsorption resin column and elute with 30%, 50%, 70%, and 95% ethanol, sequentially. 30%, 50%, 70%, and 95% ethanol elution fractions were obtained and concentrated and freeze-dried to obtain extracts. Dissolve 0.1 g of each extract in pure methanol to prepare 5 mg / mL fraction solutions for antibacterial activity determination (results are shown in Table 1.3). The 50% ethanol elution fraction was selected as the effective fraction.

[0067] S5. Dissolve 6 g of the 50% ethanol fraction extract in methanol to obtain 5 mL of the 50% ethanol fraction extract in methanol. Apply the solution to a silica gel column and perform gradient elution using a chloroform-methanol system with a volume ratio of 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 2:1, and 1:1, in sequence. Adjust the flow rate to 15 mL / min, collect fractions every 50 mL, and perform TLC analysis on the fractions using a chloroform-methanol system with a volume ratio of 4:1. Based on the analysis results, fractions with similar numbers of chromatographic spots and Rf values ​​are combined to obtain ten fractions 1 to 10, which are concentrated and freeze-dried to form extracts. Dissolve 0.1 g of the extract in pure methanol to prepare solutions of each fraction at a concentration of 1.25 mg / mL for antibacterial activity determination (results are shown in Table 1.3). Select fractions 1 and 8 as the effective fractions.

[0068] S6. Dissolve 100 mg of the effective fraction 1 extract in pure methanol to obtain 5 mL of the methanol solution of the fraction 1 extract. Apply the solution to Sephadex LH-20 gel column chromatography and use 60% methanol solvent for isocratic elution to obtain one fraction. The separated fraction is subjected to liquid phase purification under the following liquid phase conditions: gradient elution with acetonitrile-ultrapure water system, increasing the concentration from 15% to 30% over a period of 0-20 min, flow rate of 10 mL / min, detection wavelength of 350 nm, column temperature of 30°C, combine the eluates from 12-15 min, evaporate to dryness under reduced pressure to obtain 5 mg of monomer compound 2, which is confirmed to be luteolin by structural analysis.

[0069] S7. Dissolve 300 mg of effective fraction 8 extract in pure methanol to obtain 5 mL of methanol solution of fraction 8 extract, apply it to a gel column, use 80% methanol solvent for isocratic elution, obtain two fractions a.1 and a.2 and concentrate them into extracts, take 0.1 g of each extract and dissolve it in pure methanol to prepare a fraction solution with a concentration of 1 mg / mL for antibacterial activity determination (results are shown in Table 1.3), select fraction a.2 as the effective fraction, and perform liquid phase purification on fraction a.2. Liquid phase conditions: 0.1% formic acid water-acetonitrile (88:12), 30 min, flow rate 12 mL / min, detection band 270 nm, column temperature 25℃-30℃, combine the eluents of 7-10 min, evaporate to dryness under reduced pressure to obtain 8 mg of monomer compound 3, perform structural analysis, and determine it to be gallic acid.

[0070] The antibacterial activity was determined using the inhibition zone method in the above steps, and the test strain was Staphylococcus aureus ( S. aureus ) and Vibrio parahaemolyticus ( V.para ), specifically, Staphylococcus aureus was cultured in NA medium, and the test bacteria concentration was 10 5CFU / mL, culture temperature 37 ℃, antibacterial culture time 16-17 h; Vibrio parahaemolyticus selected NA medium with 3% NaCl, test bacteria concentration was 10 5 CFU / mL, culture temperature 37°C, antibacterial culture time 16-17 h. The antibacterial activity determination performed in Example 1 is shown in the table:

[0071] Table 1.1 Antibacterial activity of different extracts from the leaves of Scutellaria baicalensis

[0072] ,

[0073] Table 1.2 Antibacterial activity of different fractions separated by ethyl acetate column chromatography

[0074] ,

[0075] Table 1.3 Antibacterial activity of different fractions separated by n-butanol column chromatography

[0076]

[0077] Structural inspection

[0078] Using modern spectroscopy techniques such as 1 H NMR spectroscopy, 13 C NMR nuclear magnetic spectrum and high-resolution mass spectrometry were used to identify the structure of the separated monomer compounds and obtain relevant spectra. The spectra were interpreted according to the instrument's built-in professional software and relevant spectrum interpretation knowledge, and the molecular formula and structural formula of the compounds were analyzed in combination with relevant literature.

[0079] The monomer compound (Compound 1) prepared in Example 1 was observed to be yellow crystals, with a quasi-molecular ion peak (mass-to-nuclear ratio) of m / z 319 [M+H]+, a molecular weight of 356.54, and a chemical formula of C 21 H 43 ON, the structural formula is as follows:

[0080] .

[0081] The monomer compound (Compound 1) prepared in Example 1 was characterized by H NMR spectroscopy (400 MHz, Chloroform-d). Figure 2 As shown, the NMR carbon spectroscopy (101 MHz, CDCl3) was performed as shown in Figure 3 As shown, the molecular formula of compound 1 was characterized using a high-resolution mass spectrometer (Agilent, USA, model 1290II-6470). Figure 4 The specific data are as follows:

[0082] 1H NMR (400 MHz, Chloroform-d) δ 5.41 (t, J = 7.0 Hz, 1H, H-2), 4.15 (d, J = 7.1 Hz, 2H, H-1), 1.99 (t, J = 8.3 Hz, 2H, H-4), 1.67 (s, 3H, H-18),1.65 – 1.53 (m, 3H, H-5, 15, 19), 1.34 – 1.19 (m, 18H, H-6~14), 0.88 – 0.80(m, 12H, H-16, 17, 20, 21);

[0083] 13 C NMR (101 MHz, CDCl3) δ 140.49 (C-3), 123.21 (C-2), 59.58 (C-1), 40.02 (C-4), 39.52 (C-14), 37.58 (C-6), 37.51 (C-8), 37.44 (C-11), 36.81 (C-9), 32.94 (C-5), 32.08 (C-19), 29.51 (C-10), 28.13 (C-15), 25.28 (C-7), 24.95(C-13), 24.62 (C-12), 22.87 (C-17), 22.78 (C-16), 19.90 (C-21), 19.87 (C-20),16.33 (C-18).

[0084] The monomer compound luteolin prepared in Example 1 was observed to be a pale yellow amorphous powder with a quasi-molecular ion peak (mass-to-nuclear ratio) of m / z 287 [M+H]+, a molecular weight of 448.38, and a chemical formula of C 21 H 20 O 11 , the structural formula is as follows:

[0085] .

[0086] The monomer compound luteolin prepared in Example 1 was characterized by nuclear magnetic hydrogen spectrum (600 MHz, DMSO-d6). Figure 5 As shown, the NMR carbon spectroscopy (151 MHz, DMSO-d6) was performed as shown. Figure 6 The specific data are as follows:

[0087] 1H NMR (600 MHz, DMSO-d6) δ12.98(1H,s,5-OH),10.82(1H,s,7-OH),9.91(1H,s,4'-OH),9.50(1H,s,3'-OH),7.44(1H,d,J=2.3 Hz,8.3 Hz,H-6'),7.40(1H,d,J=2.3 Hz,H-2'),6.90(1H,d,J=8.3 Hz,H-5'),6.68(1H,d,J=2.2 Hz,H-8),6.45(1H,s,H-3),6.20(1H,d,J=2.2 Hz,H-6);

[0088] 13 C NMR(151 MHz,DMSO-d6)δ164.59(C-2),104.16(C-3),182.13(C-4),161.93(C-5),99.29(C-6),164.35(C-7),94.31(C-8),157.75 (C-9),103.33(C-10),121.96(C-1'),113.82(C-2'),146.2(C-3'),150.16(C-4'),116.48(C-5'),119.46(C-6'),70.87( C-2"), 77.57(C-3"), 67.41(C-4"), 76.4(C-5"), 51.67(C-6").

[0089] The monomer compound gallic acid prepared in Example 1 was observed to be a white powder. Its quasi-molecular ion peak (mass-to-nuclear ratio) was m / z 161 [M+Na]+, its molecular weight was 170.12, its chemical formula was C7H6O5, and its structural formula was as follows:

[0090] .

[0091] The monomer compound gallic acid prepared in Example 1 was characterized by nuclear magnetic resonance spectroscopy (600 MHz, CD3OD). Figure 7 As shown, the nuclear magnetic carbon spectrum (150 MHz, CD3OD) was performed as shown in Figure 8 The specific data are as follows:

[0092] 1 H NMR(600MHz, CD3OD)δ:6.98(2H,s,H-2);

[0093] 13C NMR (150 MHz, CD3OD) δ: 170.26 (COOH), 120.89 (C-1), 109.94 (C-2,6), 144.38 (C-3,5), 137.94 (c-4).

[0094] Antibacterial activity detection

[0095] The present invention uses the inhibition zone method to determine the antibacterial activity of the three isolated monomer compounds, and studies the effects of the monomer compounds on the cell morphology and cell membrane permeability of Staphylococcus aureus and Vibrio parahaemolyticus through scanning electron microscopy, conductivity measurement, enzyme activity measurement and other methods.

[0096] 1. Inhibition zone method

[0097] The antibacterial activities of the three monomeric compounds extracted in Example 1 as determined by the inhibition zone method are summarized in Table 1.4.

[0098] Table 1.4 Antibacterial activity of three monomeric compounds extracted from the leaves of Scutellaria baicalensis

[0099]

[0100] 2. Scanning electron microscopy (SEM) observation

[0101] Staphylococcus aureus and Vibrio parahaemolyticus were cultured to mid-logarithmic phase (10 7 CFU / mL) were incubated with 0 mg / mL and 0.5 mg / mL luteolin at 37°C for 0, 4, 8, and 12 hours, respectively. Bacterial pellets were centrifuged at 8000 g for 5 minutes to obtain bacterial pellets, which were then washed twice with sterile PBS. The bacterial pellets were resuspended in sterile water containing 2.5% glutaraldehyde and incubated overnight at 4°C to fix the cells. After another centrifugation at 8000 g for 5 minutes, the cells were dehydrated with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) at 15-minute intervals. After freeze-drying for 13 hours, the cells were covered with cathodic spray coating, and cell morphology was observed using high-resolution field-emission scanning electron microscopy.

[0102] The results are as follows Figure 9As shown in the figure, 0 hA, 4 hA, 8 hA, and 12 hA represent the results of treating Staphylococcus aureus with 0.5 mg / mL luteolin for 0 h, 4 h, 8 h, and 12 h, respectively. 0 hB, 4 hB, 8 hB, and 12 hB represent the results of treating Vibrio parahaemolyticus with 0.5 mg / mL luteolin for 0 h, 4 h, 8 h, and 12 h, respectively. As can be seen from the figure, untreated S. aureus (0 hA) exhibits a regular spherical shape with a smooth surface and clear boundaries. However, compound-treated S. aureus exhibits convexities and concavities on its surface, with some cells rupturing, contents leaking out, and blurred cell boundaries. Normal V. parahaemolyticus exhibits a regular arc shape (0 hB), while compound-treated cells exhibit deformed and concave shapes, completely disrupting bacterial morphology, with numerous cells clumping and adhering together. This demonstrates that the compound disrupts the cellular morphology of the pathogenic bacteria. This morphological abnormality is primarily due to membrane disruption and content leakage.

[0103] 3. Conductivity measurement

[0104] Staphylococcus aureus was cultured to the logarithmic phase (10 7 CFU / mL), centrifuged at 4000 g for 8 min, and then the bacterial pellet was collected and resuspended in sterile PBS. The experimental group (MIC (minimum inhibitory concentration)) was treated with 0.5 mg / mL luteolin, while the control group (no luteolin) was used. The cells were incubated at 37°C for 0, 4, 8, and 12 hours, followed by centrifugation at 6000 g for 5 min. The supernatant was collected and the conductivity was measured using a conductivity meter.

[0105] The results are as follows Figure 10 As shown in the figure, with the increase of treatment time, the conductivity of the untreated control group changed slightly, while the conductivity difference of Staphylococcus aureus in the MIC group increased, indicating that after the test bacteria were treated with the compound, the permeability of the cell membrane was changed, and the Na in the cells was conducted. + , K + Leakage, conductivity difference increases.

[0106] 4. Determination of Alkaline Phosphatase (AKP) Activity

[0107] Staphylococcus aureus was cultured in LB liquid medium until the logarithmic phase, and the bacterial culture was diluted to 10 7 CFU / mL, the culture medium was aspirated and added to a sterile centrifuge tube and centrifuged at 8000 g for 5 min to collect the bacterial precipitate, which was washed twice with sterile PBS and resuspended with 0.5 mg / mL of luteolin. This group was used as the experimental group MIC group, and the group without monomer compound was used as the control group. The cells were incubated at 37°C for 24 h, and the supernatant was centrifuged every 2 h to detect the alkaline phosphatase content using a kit.

[0108] There is alkaline phosphatase between the bacterial cell wall and cell membrane, which cannot leak out of the bacteria. Therefore, its activity cannot be measured under normal conditions. The concentration of alkaline phosphatase in the bacterial suspension can reflect the integrity of the cell wall. Figure 11 As shown in the data, there was no significant change in the alkaline phosphatase activity of Staphylococcus aureus in the control group, while the alkaline phosphatase activity in the experimental bacterial suspension of the MIC group increased. With the increase of treatment time, the alkaline phosphatase content in the bacterial suspension increased significantly. The alkaline phosphatase activity in the bacterial suspension was the highest at 8-10 h, reaching 8-10 (King's units / 100 mL), indicating that the degree of bacterial cell wall rupture was the greatest at this time, which may be due to normal bacterial lysis and death.

[0109] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. A method for extracting antibacterial monomeric compounds from the leaves of Scutellaria baicalensis, characterized in that: The following steps are involved: S1. Extracting the leaves of Scutellaria baicalensis with ethanol and concentrating the extracted extract to obtain an alcohol extract. Extracting the extracted extract with petroleum ether, ethyl acetate, and n-butanol in sequence, separating and concentrating the extracted extract to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract. S2. Dissolve the ethyl acetate extract in methanol and apply it to a silica gel column. Gradient elution is performed using a dichloromethane-methanol system with a volume ratio of 50:1, 30:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1, to obtain eight fractions, which are concentrated into an extract. The second fraction is selected as effective fraction A. S3. Dissolve the effective fraction A extract in methanol and apply it to an ODS column. Gradient elution is performed using a methanol-water system with volume fractions of 40%, 60%, 80%, 85%, 90%, 95%, and 100% in sequence to obtain four fractions, which are concentrated into an extract. The third fraction is selected as the effective fraction B. The extract of the effective fraction B is evaporated to dryness under reduced pressure to obtain monomer compound 1. S4. Dissolve the n-butanol extract in distilled water and apply it to a macroporous adsorption resin column. Elute with 30%, 50%, 70%, and 95% ethanol in sequence to obtain 30%, 50%, 70%, and 95% ethanol elution fractions, which are concentrated into extracts. The 50% ethanol elution fraction is selected as the effective fraction C. S5. Dissolve the effective fraction C extract in methanol and apply it to a silica gel column. Gradient elution is performed using a chloroform-methanol system with a volume ratio of 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 2:1, and 1:1, to obtain ten fractions, which are concentrated into an extract. The first and eighth fractions are selected as effective fractions D and E, respectively. S6. Dissolve the effective fraction D extract in methanol and apply it to a gel column. Use 60% methanol solvent for isocratic elution to obtain a fraction. Perform liquid phase purification on the separated fraction and evaporate to dryness under reduced pressure to obtain monomer compound 2. S7, dissolving the effective fraction E extract in methanol and applying it to a gel column, isocratically eluting with 80% methanol solvent to obtain two fractions, which were concentrated into an extract. The second fraction was selected as the effective fraction F, and the effective fraction F was subjected to liquid phase purification and evaporated to dryness under reduced pressure to obtain monomer compound 3; Wherein, the structure of the monomer compound 1 is as follows: ； The structure of the monomeric compound 2 is as follows: ； The structure of the monomeric compound 3 is as follows: 。 2. The extraction method according to claim 1, wherein When the ethanol extraction of the leaves of Scutellaria baicalensis as described in S1 is performed, the volume fraction of ethanol in the ethanol solution is 65%-75%; and / or, the alcohol extraction and concentration are concentrated using a rotary evaporator; and / or, when the ethanol extraction of the leaves of Scutellaria baicalensis is performed, the leaves of Scutellaria baicalensis are dried and crushed in advance; and / or, when the ethanol extraction of the leaves of Scutellaria baicalensis is performed, the mixing ratio of the leaves of Scutellaria baicalensis and ethanol is (4-6) kg: (8-12) L.

3. The extraction method according to claim 1, wherein When the ethyl acetate extract is dissolved in methanol and loaded onto a silica gel column in S2, the volume ratio of silica gel to sample is 2-3:1, and the sample is a methanol solution of the ethyl acetate extract.

4. The extraction method according to claim 1, wherein The macroporous adsorption resin column in S4 is one of AB-8 macroporous adsorption resin column, D101 macroporous adsorption resin column and XAD-16 macroporous adsorption resin column.

5. The extraction method according to claim 1, wherein The liquid phase conditions for liquid phase purification of the separated fractions in S6 were an acetonitrile-ultrapure water system with a concentration of 15%-30%, a flow rate of 8-12 mL / min, a column temperature of 26°C-32°C, and a detection band of 340 nm-360 nm.

6. The extraction method according to claim 1, characterized in that The liquid phase conditions for liquid-phase purification of the effective fraction F in S7 are a formic acid water-acetonitrile system, a flow rate of 10-14 mL / min, a column temperature of 25°C-30°C, and a detection band of 260 nm-280 nm; wherein the volume fraction of the formic acid water is 1%, and / or the system proportion of formic acid water in the formic acid water-acetonitrile system is 86%-90%.

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