A method for extracting and separating vitex negundo compounds and its application

Abstract

The present invention discloses a method for extracting and separating vitex compounds and their applications. The extraction and separation method includes: (1) crude extraction and separation of medicinal materials: refluxing and extracting the crude powder of medicinal materials with ethanol, and extracting with petroleum ether, dichloromethane, ethyl acetate, and n-butanol; (2) separation and purification of the dichloromethane fraction; (3) separation and purification of the ethyl acetate fraction; (4) fine separation and purification of the active fraction: separating and purifying the extract of the active fraction by methods such as column chromatography, semi-preparative liquid phase, and recrystallization to finally obtain the compounds vitexdoin A and ethylparaben. The two compounds have significant inhibitory effects on Escherichia coli, Staphylococcus aureus, and Candida albicans, and are potential antifungal drugs and potential antibacterial drugs, providing a scientific basis for the further development and utilization of vitex resources.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and separation of traditional Chinese medicine chemical components, and particularly relates to a method for extracting, separating and applying vitex negundo linn var.heterophylla (Franch.) Rehd. compounds. Background Art

[0002] Vitex negundo Linn var.heterophylla (Franch.) Rehd. is a plant of the genus Vitex in the Verbenaceae family [1] , and its Miao medicine name is Dulai Gun. Miao medicine believes that it is bitter in taste, cold in nature, enters the heat meridian, and has the effects of clearing heat and relieving the exterior, promoting diuresis and detoxifying. It is mainly used to treat colds, jaundice, rheumatism, traumatic swelling and pain, sores and scabies [2] . The main chemical components of Vitex negundo Linn var.heterophylla (Franch.) Rehd. include volatile oils, terpenes, flavonoids, steroids, lignans and their derivatives, etc., and have effects in aspects such as anti-inflammatory, analgesic, antitussive and antiasthmatic, antioxidant, antibacterial, antitumor, and enhancing immunity [3～4] . "Guizhou Herbal Medicine" records that taking an appropriate amount of leaves, pounding them into fluff, and taking the juice to apply to the affected area can be used to treat tinea pedis, with obvious effects [5] . At present, there is no report on the research of the material basis of the antibacterial activity of Vitex negundo Linn var.heterophylla (Franch.) Rehd. and the optimization of its extraction process. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for extracting and separating vitex negundo linn var.heterophylla (Franch.) Rehd. compounds;

[0004] Another purpose of the present invention is to provide the application of vitex negundo linn var.heterophylla (Franch.) Rehd. compounds in antifungal and antibacterial drugs.

[0005] The present invention is realized through the following technical solutions:

[0006] The method for extracting and separating vitex negundo linn var.heterophylla (Franch.) Rehd. compounds of the present invention includes the following steps:

[0007] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of dry whole plants of Vitex negundo linn var.heterophylla (Franch.) Rehd., crush them, and extract them with 2 - 4 times the amount of 90 - 95% ethanol by cold soaking for 2 - 4 times, 5 - 9 days each time. Combine the filtrates and recover the solvent under reduced pressure to obtain 994 - 1042 g of extract; Disperse the extract in water, and extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Recover the solvent to obtain 156 - 172 g of petroleum ether fraction, 135 - 150 g of dichloromethane fraction, 54 - 62 g of ethyl acetate fraction, and 210 - 223 g of n-butanol fraction;

[0008] (2) Isolation and purification of the dichloromethane fraction: After dissolving 135 - 150 g of the dichloromethane fraction extract, 100 g of silica gel was weighed and used for sample mixing. The separation was carried out by silica gel column chromatography, with petroleum ether - ethyl acetate as the eluent, and gradient elution was performed at volume ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, and 1:2. After the 1:1 elution of the column was completed, TLC was used for tracking, and the same parts were combined to obtain 8 fractions, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H;

[0009] (3) Isolation and purification of the ethyl acetate fraction: After dissolving 54 - 62 g of the ethyl acetate fraction extract, 50 g of polyamide was weighed and used for sample mixing. The separation was carried out by polyamide column chromatography, with a gradient elution of 10% - 70% ethanol - water. After the elution with absolute ethanol of the column was completed, polyamide thin layer plates were used for tracking, and the same parts were combined to obtain 9 fractions, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, and Fr.Q;

[0010] (4) Fine isolation and purification of chemical components:

[0011] ① Isolation and purification of Fr.C in the dichloromethane fraction: Fr.C was a dark green powder of 12 - 14 g. The separation was carried out by silica gel column chromatography, with petroleum ether - ethyl acetate as the eluent, and gradient elution was performed at volume ratios of 10:1, 7:1, and 5:1. By analyzing and combining the components through TLC thin layer plates, 3 fractions were obtained, namely Fr.C1, Fr.C2, and Fr.C3. Among them, Fr.C2 was separated by silica gel column chromatography with an isocratic elution of petroleum ether - ethyl acetate at a volume ratio of 7:1. By analyzing and combining the components through TLC thin layer plates, 2 fractions were obtained, namely Fr.C 2-1 and Fr.C 2-2 , Fr.C 2-1 was recrystallized with dichloromethane to obtain 18.5 - 19.2 mg of the compound ethylparaben;

[0012] ② Isolation and purification of Fr.I in the ethyl acetate fraction: Fr.I was an orange - yellow powder of 0.25 - 0.32 g. The separation was carried out by polyamide column chromatography, with an isocratic elution of ethanol - water at a volume ratio of 1:4. By analyzing and combining the components through polyamide thin layer plates, 2 fractions were obtained, namely Fr.I1 and Fr.I2. Fr.I2 was further separated by gel column chromatography with an isocratic elution of dichloromethane - methanol at a volume ratio of 1:1 to obtain 5.6 - 6.1 mg of the compound vitexdoin A.

[0013] The method for extracting and separating the compounds from Vitex negundo var. heterophylla described in the present invention specifically includes the following steps:

[0014] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of the whole dried Vitex negundo var. heterophylla, pulverize it, and extract it with 3 times the amount of 95% ethanol by cold soaking for 3 times, 7 days each time. Combine the filtrates and recover the solvent under reduced pressure to obtain 1026 g of extract. Disperse the extract in water and extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Recover the solvents to obtain 168 g of the petroleum ether fraction, 144 g of the dichloromethane fraction, 60 g of the ethyl acetate fraction, and 219 g of the n-butanol fraction;

[0015] (2) Separation and purification of the dichloromethane fraction: After dissolving 144 g of the dichloromethane fraction extract, weigh 100 g of silica gel for sample mixing, and separate it by silica gel column chromatography. Use petroleum ether - ethyl acetate as the eluent and perform gradient elution according to the volume ratio of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:2. After the 1:1 column flushing is completed, use TLC for tracking, combine the same parts, and obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H;

[0016] (3) Separation and purification of the ethyl acetate fraction: After dissolving 60 g of the ethyl acetate fraction extract, weigh 50 g of polyamide for sample mixing, and separate it by polyamide column chromatography. Use a gradient elution of 10% - 70% ethanol - water. After the absolute ethanol column flushing is completed, use a polyamide thin layer plate for tracking, combine the same parts, and obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, Fr.Q;

[0017] (4) Fine separation and purification of chemical components:

[0018] ① Separation and purification of the Fr.C part in the dichloromethane fraction: The Fr.C part is 13.5 g of dark green powder. Separate it by silica gel column chromatography, use petroleum ether - ethyl acetate as the eluent, and perform gradient elution according to the volume ratio of 10:1, 7:1, 5:1. Analyze and combine the components by spotting TLC thin layer plates to obtain 3 components, namely Fr.C1, Fr.C2, Fr.C3. Among them, Fr.C2 is separated by silica gel column chromatography and isocratically eluted with petroleum ether - ethyl acetate with a volume ratio of 7:1. Analyze and combine the components by spotting TLC thin layer plates to obtain 2 components, namely Fr.C 2-1 、Fr.C 2-2 ,Fr.C 2-1 Recrystallize with dichloromethane to obtain 19 mg of the compound ethylparaben;

[0019] ② Isolation and purification of fraction Fr.I in the ethyl acetate fraction: Fraction Fr.I is 0.3 g of orange-yellow powder. It is separated by polyamide column chromatography and eluted isocratically with ethanol-water with a volume ratio of 1:4. By analyzing the combined components through spotting on a polyamide thin film plate, 2 components are obtained, namely Fr.I1 and Fr.I2. Fr.I2 is further separated by gel column chromatography and eluted isocratically with dichloromethane-methanol with a volume ratio of 1:1, obtaining 6 mg of compound vitexdoin A.

[0020] In step (2) of the present invention, the mesh number of the silica gel described is 100 - 200 meshes.

[0021] In steps (3) and (4) of the present invention, the mesh number of the polyamide described is 200 - 300 meshes.

[0022] In step (4) of the present invention, the mesh number of the silica gel described is 200 - 300 meshes.

[0023] The application of the vitexdoin A and ethylparaben compounds of the present invention in the preparation of antifungal or antibacterial drugs.

[0024] The application of the obtained vitexdoin A and ethylparaben compounds of the present invention in the preparation of drug preparations against Candida albicans, Escherichia coli, and Staphylococcus aureus.

[0025] The concentration of the compound vitexdoin A of the present invention is: the MIC for Escherichia coli 90 > 1 mg / mL, MIC 50 > 1 mg / mL; the MIC for Staphylococcus aureus 90 > 1 mg / mL, MIC 50 > 1 mg / mL; the MIC for Candida albicans 90 > 1 mg / mL, MIC 50 is 1 mg / mL;

[0026] The concentration of the compound ethylparaben is: the MIC for Escherichia coli 90 > 1 mg / mL, MIC 50 is 0.5 mg / mL; the MIC for Staphylococcus aureus 90 > 1 mg / mL, MIC 50 > 1 mg / mL; the MIC for Candida albicans 90 > 1 mg / mL, MIC 50 is 0.25 mg / mL.

[0027] The preparation of the present invention is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable excipients. The pharmaceutically acceptable preparation is a solid preparation or a liquid preparation;

[0028] The solid preparations of the present invention are granules, capsules, tablets, pills; the liquid preparations are injection preparations and oral liquids.

[0029] Advantages of the present invention:

[0030] 1. The extraction and separation method of the present invention is simple and easy to implement. The monomer compounds vitexdoin A and ethylparaben have significant inhibitory effects on Escherichia coli, Staphylococcus aureus, and Candida albicans; they are potential antifungal drugs and also potential antibacterial drugs, providing a scientific basis for the further development and utilization of Vitex negundo var. heterophylla resources.

[0031] 2. Using Escherichia coli, Staphylococcus aureus, and Candida albicans as test strains, the in vitro antibacterial activity of the monomer compounds vitexdoin A and ethylparaben was studied. Results: Both monomer compounds have significant inhibitory effects on Escherichia coli, Staphylococcus aureus, and Candida albicans. Among them, the MIC of vitexdoin A against Escherichia coli 50 is 0.5 mg / mL; the MIC against Staphylococcus aureus 90 > 1 mg / mL, the MIC 50 is 1 mg / mL; the MIC against Candida albicans 90 > 1 mg / mL, the MIC 50 is 1 mg / mL; the MIC of ethylparaben against Escherichia coli 50 is 0.5 mg / mL; the MIC against Staphylococcus aureus 50 is 1 mg / mL; the MIC against Staphylococcus aureus 90 > 1 mg / mL, the MIC 50 is 1 mg / mL; the MIC against Candida albicans 90 > 1 mg / mL, the MIC 50 is 0.25 mg / mL. Description of the Drawings

[0032] Figure 1 : 1H NMR spectrum of the compound ethylparaben

[0033] Figure 2 : 13C NMR spectrum of the compound ethylparaben

[0034] Figure 3 : 1H NMR spectrum of the compound vitexdoin A

[0035] Figure 4 : Carbon spectrum of compound vitexdoin A Detailed implementation manners

[0036] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0037] Example 1

[0038] Extraction and separation method of vitex compounds

[0039] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of dry whole vitex plants, crush them, and extract them with 3 times the amount of 95% ethanol by cold soaking for 3 times, 7 days each time. Combine the filtrates and recover the solvent under reduced pressure to obtain 1026 g of extract; Disperse the extract in water and extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Recover the solvents to obtain 168 g of petroleum ether fraction, 144 g of dichloromethane fraction, 60 g of ethyl acetate fraction, and 219 g of n-butanol fraction;

[0040] (2) Separation and purification of dichloromethane fraction: After dissolving 144 g of the dichloromethane fraction extract, weigh 100 g of silica gel (100 - 200 mesh) and mix the sample. Separate it by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, and perform gradient elution according to the volume ratio of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:2. After the 1:1 column flushing is completed, use TLC to track, combine the same parts and obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H;

[0041] (3) Separation and purification of ethyl acetate fraction: After dissolving 60 g of the ethyl acetate fraction extract, weigh 50 g of polyamide (200 - 300 mesh) and mix the sample. Separate it by polyamide column chromatography, and perform gradient elution with 10% - 70% ethanol - water. After the absolute ethanol column flushing is completed, use polyamide thin layer plate to track, combine the same parts and obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, Fr.Q;

[0042] (4) Fine separation and purification of chemical components

[0043] ① Isolation and purification of fraction Fr.C in the dichloromethane fraction: Fraction Fr.C was 13.5 g of dark green powder, separated by silica gel column chromatography (200 - 300 mesh), using petroleum ether - ethyl acetate as the eluent, with gradient elution at volume ratios of 10:1, 7:1, and 5:1. By analyzing the combined components by spotting TLC plates, 3 components were obtained, namely Fr.C1, Fr.C2, and Fr.C3. Among them, Fr.C2 was separated by silica gel column chromatography, with isocratic elution using petroleum ether - ethyl acetate at a volume ratio of 7:1. By analyzing the combined components by spotting TLC plates, 2 components were obtained, namely Fr.C 2-1 and Fr.C 2-2 , Fr.C 2-1 and then recrystallized from dichloromethane to obtain 19 mg of compound ethylparaben;

[0044] ② Isolation and purification of fraction Fr.I in the ethyl acetate fraction: Fraction Fr.I was 0.3 g of orange - yellow powder, separated by polyamide column chromatography (200 - 300 mesh), with isocratic elution using ethanol - water at a volume ratio of 1:4. By analyzing the combined components by spotting polyamide thin - film plates, 2 components were obtained, namely Fr.I1 and Fr.I2. Fr.I2 was further separated by gel column chromatography, with isocratic elution using dichloromethane - methanol at a volume ratio of 1:1, and 6 mg of compound vitexdoin A was obtained.

[0045] Example 2

[0046] Extraction and isolation method of compounds from Vitex negundo var. heterophylla

[0047] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of dry whole plants of Vitex negundo var. heterophylla, crush them, and extract them with 2 - fold volume of 90% ethanol by cold maceration 2 times, each time for 5 days. Combine the filtrates and recover the solvent under reduced pressure to obtain 994 g of extract. Disperse the extract in water and extract it with petroleum ether, dichloromethane, ethyl acetate, and n - butanol respectively. After recovering the solvents, 156 g of petroleum ether fraction, 135 g of dichloromethane fraction, 54 g of ethyl acetate fraction, and 210 g of n - butanol fraction were obtained;

[0048] (2) Isolation and purification of the dichloromethane fraction: After dissolving 135 g of the dichloromethane fraction extract, weigh 100 g of silica gel (100 - 200 mesh) and mix the sample. Separate it by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, with gradient elution at volume ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, and 1:2. After the 1:1 elution of the column is completed, use TLC for tracking, combine the same parts, and obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H;

[0049] (3) Isolation and purification of the ethyl acetate fraction: After dissolving 54 g of the ethyl acetate fraction extract, 50 g of polyamide (200 - 300 mesh) was weighed and used for sample mixing. The separation was carried out by polyamide column chromatography, eluting with a gradient of 10% - 70% ethanol - water. After the column was flushed with absolute ethanol, polyamide thin layer plates were used for tracking. The same parts were combined to obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, Fr.Q;

[0050] (4) Fine isolation and purification of chemical components:

[0051] ① Isolation and purification of the Fr.C part in the dichloromethane fraction: The Fr.C part was 12 g of dark green powder. It was separated by silica gel column chromatography (200 - 300 mesh), using petroleum ether - ethyl acetate as the eluent, and gradient elution was carried out at a volume ratio of 10:1, 7:1, and 5:1. By analyzing and combining the components through TLC thin layer plates, 3 components were obtained, namely Fr.C1, Fr.C2, Fr.C3. Among them, Fr.C2 was separated by silica gel column chromatography and isocratically eluted with petroleum ether - ethyl acetate at a volume ratio of 7:1. By analyzing and combining the components through TLC thin layer plates, 2 components were obtained, namely Fr.C 2-1 、Fr.C 2-2 ,Fr.C 2-1 Then, it was recrystallized with dichloromethane to obtain 18.5 mg of the compound ethylparaben;

[0052] ② Isolation and purification of the Fr.I part in the ethyl acetate fraction: The Fr.I part was 0.25 g of orange - yellow powder. It was separated by polyamide column chromatography (200 - 300 mesh) and isocratically eluted with ethanol - water at a volume ratio of 1:4. By analyzing and combining the components through polyamide thin layer plates, 2 components were obtained, namely Fr.I1, Fr.I2. Fr.I2 was further separated by gel column chromatography and isocratically eluted with dichloromethane - methanol at a volume ratio of 1:1, and 5.6 mg of the compound vitexdoin A was obtained.

[0053] Example 3

[0054] Extraction and separation method of compounds from Vitex negundo var. heterophylla

[0055] (1) Coarse extraction and separation of medicinal materials: 50 kg of dry whole plants of Vitex negundo var. heterophylla were taken, crushed, and cold - soaked and extracted 4 times with 3 times the amount of 95% ethanol for 9 days each time. The filtrates were combined and the solvent was recovered under reduced pressure to obtain 1042 g of an extract. The extract was dispersed in water and extracted with petroleum ether, dichloromethane, ethyl acetate, and n - butanol respectively. After recovering the solvents, 172 g of the petroleum ether fraction, 150 g of the dichloromethane fraction, 62 g of the ethyl acetate fraction, and 223 g of the n - butanol fraction were obtained;

[0056] (2) Isolation and purification of the dichloromethane fraction: After dissolving 150 g of the dichloromethane fraction extract, 100 g of silica gel (100 - 200 mesh) was weighed and used for sample mixing. The separation was carried out by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, and gradient elution was performed according to the volume ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:2. After the column was flushed with 1:1 eluent, TLC was used for tracking, and the same parts were combined to obtain 8 fractions, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H;

[0057] (3) Isolation and purification of the ethyl acetate fraction: After dissolving 62 g of the ethyl acetate fraction extract, 50 g of polyamide (200 - 300 mesh) was weighed and used for sample mixing. The separation was carried out by polyamide column chromatography, and gradient elution was performed with 10% - 70% ethanol - water. After the column was flushed with absolute ethanol, polyamide thin layer plates were used for tracking, and the same parts were combined to obtain 9 fractions, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, Fr.Q;

[0058] (4) Fine isolation and purification of chemical components:

[0059] ① Isolation and purification of Fr.C part in the dichloromethane fraction: The Fr.C part was 14 g of dark green powder. The separation was carried out by silica gel column chromatography (200 - 300 mesh), using petroleum ether - ethyl acetate as the eluent, and gradient elution was performed according to the volume ratios of 10:1, 7:1, 5:1. By analyzing and combining the components by spotting TLC thin layer plates, 3 components were obtained, namely Fr.C1, Fr.C2, Fr.C3. Among them, Fr.C2 was separated by silica gel column chromatography and isocratically eluted with petroleum ether - ethyl acetate with a volume ratio of 7:1. By analyzing and combining the components by spotting TLC thin layer plates, 2 components were obtained, namely Fr.C 2-1 and Fr.C 2-2 and Fr.C 2-1 Then, recrystallization with dichloromethane was carried out to obtain 19.2 mg of the compound ethylparaben;

[0060] ② Isolation and purification of Fr.I part in the ethyl acetate fraction: The Fr.I part was 0.32 g of orange - yellow powder. The separation was carried out by polyamide column chromatography (200 - 300 mesh), and isocratically eluted with ethanol - water with a volume ratio of 1:4. By analyzing and combining the components by spotting polyamide thin layer plates, 2 components were obtained, namely Fr.I1, Fr.I2. Fr.I2 was further separated by gel column chromatography and isocratically eluted with dichloromethane - methanol with a volume ratio of 1:1, and 6.1 mg of the compound vitexdoin A was obtained.

[0061] Example 4

[0062] Take any one or two of ethylparaben and vitexdoin A as raw materials, add pharmaceutically acceptable excipient dextrin, granulate to obtain granules.

[0063] Example 5

[0064] Take any one or two of ethylparaben and vitexdoin A as raw materials, add pharmaceutically acceptable excipient dextrin, mix evenly, fill into capsules to obtain capsules.

[0065] Example 6

[0066] Take any one or two of ethylparaben and vitexdoin A as raw materials, add pharmaceutically acceptable excipient dextrin, granulate, and tableting to obtain tablets.

[0067] Example 7

[0068] Take any one or two of ethylparaben and vitexdoin A as raw materials, add pharmaceutically acceptable excipient dextrin, mix evenly, pill, and dry to obtain pills.

[0069] Example 8

[0070] Take any one or two of ethylparaben and vitexdoin A as raw materials, add 10 times the amount of injection water, mix evenly, filter, sterilize to obtain injections.

[0071] In order to further verify the feasibility and effectiveness of the present invention and screen out the best solution, the inventor conducted a series of experiments as follows:

[0072] 1. Instruments and materials

[0073] 1.1 Experimental instruments

[0074] Instrument JOEL 5937MSD type mass spectrometer (Agilent Technologies, USA); 600MHz superconducting nuclear magnetic resonance spectrometer (Bruker, Germany); JEOL-400 type superconducting nuclear magnetic resonance spectrometer (JEOL Ltd., Japan); WFH-308B type three-color ultraviolet analyzer (Shanghai Jingke Industrial Co., Ltd.); Metter-Toledo electronic balance (Metter-Toledo, Switzerland); semi-preparative high performance liquid chromatography (Shimadzu, Japan).

[0075] 1.2 Experimental materials

[0076] The experimental medicinal materials were collected from Tianzhu County, Guizhou Province and identified by Associate Professor Jiang Hong of Guizhou University of Traditional Chinese Medicine as the whole plant of Vitex negundo L. of the genus Vitex in the Verbenaceae family. Thin layer chromatography GF254 Prefabricated plate (Qingdao Specco Separation Material Co., Ltd.), polyamide film (Taizhou Sijia Biochemical Plastic Factory), chromatography silica gel (Qingdao Ocean Chemical Co., Ltd.), Sephadex LH-20 dextran gel (Amersham Biosciences, Sweden), polyamide powder for chromatography (Chengdu Yuannuo Tiancheng Technology Co., Ltd.), MCI resin (Beijing Huideyi), SiliaSphere C 18 Reverse phase packing (Beijing Green Baicao Technology Development Co., Ltd.); other reagents were of analytical grade.

[0077] 2. Extraction and separation

[0078] 2.1 Extraction and crude separation of medicinal materials

[0079] 50 kg of dried whole plant of Vitex negundo was crushed and extracted with 3 times the amount of 95% ethanol for 3 times, each time for 7 days, and the filtrates were combined and the solvent was recovered under reduced pressure to obtain 1026 g of extract. The extract was dispersed in water and extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively, and the solvent was recovered to obtain 168 g of petroleum ether, 144 g of dichloromethane, 60 g of ethyl acetate and 219 g of n-butanol.

[0080] After dissolving the extract (144 g) of the dichloromethane part, weigh 100 g of silica gel (100-200 mesh) and mix with the sample, separate it by silica gel column chromatography, and elute it with petroleum ether-ethyl acetate (20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:2) gradient. After the 1:1 column flushing is completed, use TLC tracking, combine the same parts and obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H.

[0081] After dissolving the ethyl acetate extract (60g), weigh 50g of polyamide (200-300 mesh) and mix with the sample, separate it by polyamide column chromatography, and elute with 10%-70% ethanol-water gradient. After the column is flushed with anhydrous ethanol, use a polyamide film plate for tracking, combine the same parts and obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, and Fr.Q.

[0082] 2.2 Fine separation and purification of chemical components

[0083] 2.2.1 Separation and purification of dichloromethane fraction

[0084] Isolation and purification of Fr.C fraction

[0085] Part Fr.C is a dark green powder (13.5 g). It was separated by silica gel column chromatography (200 - 300 mesh), eluted with a gradient of petroleum ether - ethyl acetate (10:1, 7:1, 5:1). By analyzing the TLC plates and combining the components, 3 components were obtained, namely Fr.C1, Fr.C2, and Fr.C3. Among them, Fr.C2 was further separated by silica gel column chromatography (200 - 300 mesh), eluted isocratically with petroleum ether - ethyl acetate (7:1). By analyzing the TLC plates and combining the components, 2 components were obtained, namely Fr.C 2-1 、Fr.C 2-2 ,Fr.C 2-1 Compound 18 (19 mg) was obtained by recrystallization with dichloromethane.

[0086] 2.2.2 Separation and purification of the ethyl acetate fraction

[0087] Separation and purification of Part Fr.I

[0088] Part Fr.I is an orange - yellow powder (0.3 g). It was separated by polyamide column chromatography (200 - 300 mesh), eluted isocratically with ethanol - water (1:4). By analyzing the polyamide thin - film plates and combining the components, 2 components were obtained, namely Fr.I1 and Fr.I2. Fr.I2 was further separated by gel column chromatography, eluted isocratically with dichloromethane - methanol (1:1), and compound 29 (6 mg) was obtained.

[0089] 2.2.3 Carbon - 13 nuclear magnetic resonance

[0090] Compound 18: White square crystals, soluble in organic solvents such as chloroform. The molecular formula is: C9H 10 O3. In 1H NMR (400 MHz, CDCl3), δ: 7.93 (1H, d, J = 8.7 Hz, H - 3,5), 6.85 (2H, d, J = 8.5 Hz, H - 2,6) are the signals of the hydrogen protons on the benzene ring AABB, δ: 4.33 (2H, q, J = 7.1 Hz, H - 8) is the signal of the hydrogen proton on the carbon adjacent to the ester group, 1.36 (3H, t, J = 7.1 Hz, H - 9) is the signal of the hydrogen proton on the methyl group; in 13 13C NMR (100 MHz, CDCl3), δ: 167.1 (C - 7) is the carbon signal of the ester group, δ: 160.4 (C - 4), 132.1 (C - 2,6), 122.8 (C - 1), 115.4 (C - 3,5) are the carbon signals on the benzene ring, δ: 61.1 (C - 8) is the carbon signal on the carbon adjacent to the ester group, 14.5 (C - 9) is the carbon signal on the methyl group. The above data are basically consistent with the literature [6] reports. Therefore, this compound was determined to be ethylparaben. The compound's 13The signals of the carbon-13 nuclear magnetic resonance spectrum (13C NMR) are shown in Table 1. See Figure 1 - Figure 2 .

[0091]

[0092] Compound 18 ethylparaben

[0093] Table 1 of Compound 18 13 13C NMR (100 MHz, CDCl3) data

[0094]

[0095]

[0096] Compound 29: Yellow powder. Soluble in organic solvents such as methanol, and its molecular formula is: C 19 H 18 O6. In 1 1H NMR (600 MHz, MeOD), δ: 9.30 (1H, s, H-2α) is the aldehyde hydrogen proton signal, there are 5 hydrogen proton signals in the aromatic region, δ: 7.29 (1H, s, H-1) is the hydrogen proton signal of the unsubstituted double bond, δ: 6.95 (1H, s, H-8), 6.72 (1H, s, H-5) are the para hydrogen proton signals of the unsubstituted benzene ring, δ: 6.61 (1H, d, J = 2.1 Hz, H-2'), 6.58 (1H, d, J = 8.2 Hz, H-5'), 6.29 (1H, dd, J = 8.3, 2.0 Hz, H-6') are the hydrogen proton signals of the ABX system on another benzene ring, δ: 3.49 (1H, dd, J = 13.7, 7.2 Hz, H-3α), 3.22 (1H, dd, J = 10.2, 3.4 Hz, H-3), 3.18 (1H, dd, J = 12.3, 7.5 Hz, H-3β) are the coupling signals of the two hydrogen proton signals on the chiral carbon atom and the hydrogen proton signal on the adjacent carbon; δ: 3.72 (3'-OCH3) is the hydrogen proton signal on the methoxy group In 13In the \(^{13}\)C NMR (150 MHz, MeOD) spectrum, δ: 194.8 (C-2α) is the carbon signal of the aldehyde group. There are 14 carbon signals in the aromatic region. δ: 149.8 (C-1), 135.3 (C-2) are the carbon signals on the double bond. δ: 118.3 (C-5), 150.1 (C-6), 145.5 (C-7), 117. (C-8), 124.8 (C-9), 133.2 (C-10) are the carbon signals on the benzene ring. δ: 137.5 (C-1'), 111.9 (C-2'), 148.4 (C-3'), 145.3 (C-4'), 115.8 (C-5'), 120.7 (C-6') are the carbon signals on another benzene ring. δ: 56.1 (3'-OCH3) is the carbon signal of the methoxy group. The above data is consistent with the literature [7] report. Therefore, this compound was determined to be vitexdoin A. The \(^{13}\)C NMR carbon spectrum signals of this compound and the literature are shown in Table 2. See 13 . Figure 3 - Figure 4 .

[0097]

[0098] Table 2 \(^{13}\)C NMR (150 MHz, MeOD) data of compound 29

[0099]

[0100]

[0101] 3. Study on the antibacterial activity of the chemical constituents of Vitex negundo L.

[0102] 3.1 Instruments and materials

[0103] 3.1.1 Experimental instruments

[0104] Biological clean safety cabinet (Model: BHC-1300IIA / B2, Manufacturer: Suzhou Purification Equipment Co., Ltd.); Shaking incubator (Model: BS-1EA, Manufacturer: Changzhou Huapuda Mathematical Instrument Co., Ltd.); Desktop constant temperature oscillator (Model: LY20-92C, Manufacturer: Shanghai Longyue Instrument and Equipment Co., Ltd.); Vertical pressure steam sterilizer (Model: LDZX-30KBS, Manufacturer: Shanghai Shen'an Medical Instrument Factory), Microplate reader (Model: Multiskan FC, Manufacturer: Thermo Scientific).

[0105] 3.1.2 Experimental reagents

[0106] Nutrient broth medium (Lot No.: 022010, Manufacturer: Guangdong Huankai Microbial Science and Technology Co., Ltd.), nutrient agar medium (Lot No.: 022020, Manufacturer: Guangdong Huankai Microbial Science and Technology Co., Ltd.), Sabouraud agar medium (Lot No.: 510D021, Manufacturer: Solarbio Science & Technology Co., Ltd.), 0.5% glucose broth medium (Lot No.: 901F031, Manufacturer: Solarbio Science & Technology Co., Ltd.), 96-well plates.

[0107] 3.1.3 Experimental materials

[0108] Monomeric compounds from different polar parts of Vitex negundo L.: 18 - ethylparaben, 29 - vitexdoin A.

[0109] 3.1.4 Experimental strains and cells

[0110] Candida albicans (Lot No.: 29343), Escherichia coli (Lot No.: Jm109), Staphylococcus aureus (Lot No.: 2021.2.28).

[0111] 3.2 Experimental procedures

[0112] 3.2.1 Preparation of culture media

[0113] Nutrient broth medium: Weigh 18 g of the powder, add it to 1000 mL of distilled water, heat to boiling until dissolved, dispense, and sterilize at 115 °C under high pressure for 15 min for standby.

[0114] Nutrient agar medium: Weigh 33 g of the powder, add it to 1000 mL of distilled water, heat to boiling until dissolved, dispense, and sterilize at 115 °C under high pressure for 15 min for standby.

[0115] Sabouraud agar medium: Weigh 70 g of the powder, add it to 1000 mL of distilled water, heat to boiling until dissolved, dispense, and sterilize at 115 °C under high pressure for 15 min for standby.

[0116] 0.5% glucose broth medium: Weigh 23 g of the powder, add it to 1000 mL of distilled water, heat to boiling until dissolved, dispense, and sterilize at 115 °C under high pressure for 15 min for standby.

[0117] 3.2.2 Cultivation of bacteria and fungi

[0118] In a sterile operating table, pour the sterilized nutrient broth agar medium into a sterile Petri dish. After solidification, smear the Escherichia coli and Staphylococcus aureus bacterial solutions on the Petri dish respectively and incubate at 37 °C for 24 h. Pour the sterilized Sabouraud agar medium into a sterile Petri dish. After solidification, smear the Candida albicans bacterial solution on the Petri dish and incubate at 37 °C for 24 h.

[0119] Take a sterile centrifuge tube, add 5 mL of nutrient broth medium, pick one Escherichia coli and one Staphylococcus aureus colony into the nutrient medium, and culture them in a shaker at 37 °C for 24 h. Then, aspirate 50 μL of the bacterial solution into 5 mL of nutrient broth medium and shake again in a shaker at 37 °C for 2.5 h. Take a sterile centrifuge tube, add 5 mL of broth medium, pick one Candida albicans colony into the nutrient medium, and culture it in a shaker at 37 °C for 24 h. Then, aspirate 50 μL of the bacterial solution into 5 mL of broth medium and shake again in a shaker at 37 °C for 2.5 h to make it in the logarithmic growth phase.

[0120] 3.2.3 Determination of bacteriostatic rate

[0121] Adopt the nutrient broth dilution method [8] , use an ultraviolet spectrophotometer to measure the absorbance value of the above-mentioned bacterial solution at 600 nm for counting, dilute the bacterial solution to 5×10^5 - 5×10^6 cfu / mL in a sterile operating table for standby. First, aspirate 100 μL of different monomer compounds at 1 mg / mL into a 96-well plate, and then add 100 μL of the bacterial solution. Use an enzyme-labeled instrument to measure its absorbance value (sample OD0) at 600 nm respectively, and then culture it in a constant temperature incubator at 37 °C for another 24 h, and then use an enzyme-labeled instrument to measure its absorbance value (sample OD 24 ). Calculate its inhibition rate.

[0122] Select the above-mentioned monomer compounds with certain bacteriostatic activity, use the double serial dilution method to dilute the extracts of different polar parts to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0313 mg / mL respectively. Add 100 μL of the bacterial solution and solutions of different polar parts with different concentration gradients into a 96-well plate respectively. Use an enzyme-labeled instrument to measure its absorbance value (sample OD0) at 600 nm respectively, and then culture it in a constant temperature incubator at 37 °C for another 24 h, and then use an enzyme-labeled instrument to measure its absorbance value (sample OD 24 ). Calculate the bacteriostatic rate using the formula. Use berberine and chlorogenic acid as positive controls, and 1% DMSO aqueous solution as a negative control.

[0123] Formula: Bacteriostatic rate = [1 - (sample OD 24 - sample OD0) / (blank OD 24 - blank OD0)] * 100%

[0124] 3.3 Experimental results and discussion

[0125] The bacteriostatic rates of different monomer compounds (1 mg / mL) against the tested strains are shown in Table 3. The MIC 50 , MIC 90The statistical results are shown in Tables 4 and 5. The antibacterial rates of monomeric compounds with certain antibacterial activities against the tested strains are shown in Tables 6 and 7.

[0126] From the following results, the strains in the negative group could grow normally, indicating that the 1% DMSO aqueous solution had no inhibitory effect on the growth of bacteria and fungi. The positive group had a certain inhibitory effect on the growth of bacteria and fungi, indicating the feasibility of this experiment.

[0127] The results showed that the 1% DMSO aqueous solution had little effect on the growth of the strains. The MIC of vitexdoin A against Escherichia coli 90 > 1 mg / mL, MIC 50 > 1 mg / mL had certain antibacterial activity against Escherichia coli; the MIC of ethylparaben against Escherichia coli 90 > 1 mg / mL, MIC 50 was 0.5 mg / mL and had certain antibacterial activity against Escherichia coli; the MIC of ethylparaben against Staphylococcus aureus 90 > 1 mg / mL, MIC 50 was 1 mg / mL and had certain antibacterial activity against Staphylococcus aureus; the MIC of vitexdoin A against Candida albicans 90 > 1 mg / mL, MIC 50 was 1 mg / mL and had certain antibacterial activity against Candida albicans. The MIC of ethylparaben against Candida albicans 90 > 1 mg / mL, MIC 50 was 0.25 mg / mL and had strong antibacterial activity against Candida albicans.

[0128] Table 3 Antibacterial rates of different monomeric compounds (1 mg / mL) from Vitex negundo L. var. heterophylla (Franch.) Rehd. against the tested strains

[0129] Table 4 MIC 90 、MIC 50 values of different monomeric compounds against Escherichia coli and Staphylococcus aureus strains

[0130]

[0131] Table 5 MIC 90 、MIC 50 values of different monomeric compounds against Candida albicans strains

[0132]

[0133] Table 6 Antibacterial rates of different monomeric compounds against Escherichia coli and Staphylococcus aureus

[0134]

[0135]

[0136] Table 7 Bacteriostatic Rates of Different Monomeric Compounds against Candida albicans

[0137]

[0138]

[0139] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

[0140] References

[0141] [1] Editorial Committee of Flora Reipublicae Popularis Sinicae, Chinese Academy of Sciences. Flora Reipublicae Popularis Sinicae, Vol. 65, Part 1 [M]. Beijing: Science Press, 1982.03: 141.

[0142] [2] Qiu Dewen, Du Jiang. Chinese Herbal Medicine, Miao Medicine Volume [M]. Guiyang: Guizhou Science and Technology Press, 2005: 482 - 483.

[0143] [3] Ding Guoyu, Hu Ping. Research Progress on Chemical Constituents and Biological Activities of Vitex negundo L. var. heterophylla (Franch.) Rehd. [J]. Journal of Shenyang Medical College, 2020, 22(2): 162 - 164, 173.

[0144] [4] Yu Lili. Study on Chemical Constituents of Xanthoceras sorbifolium Bunge Seed Meal and Vitex negundo L. var. heterophylla (Franch.) Rehd. [D]. Liaoning: Shenyang Pharmaceutical University, 2012.

[0145] [5] Guizhou Institute of Traditional Chinese Medicine. Guizhou Herbal Medicine, Volume 2 [M]. Guiyang: Guizhou People's Publishing House, 1970: 824 - 825.

[0146] [6] Li Huaqiang. Study on Chemical Constituents and Pharmacognostic Identification of Fructus Viticis Negundo [D]. Shanghai: Shanghai Normal University, 2015.

[0147] [7] Zhang Qingjian, Ni Gang, Yu Dequan. Study on Chemical Constituents of Vitex canescens Kurz [J]. China Journal of Chinese Materia Medica, 2009, 34(10): 1305.

[0148] [8]Department of Health Legislation and Supervision, Ministry of Health. Disinfection Technical Specifications [S]. Beijing: Ministry of Health of the People's Republic of China, 2002, 96-97.

Claims

1. A method for extracting and separating vitex negundo compounds, characterized in that, It includes the following steps: (1) Crude extraction and separation of medicinal materials: Take 50 kg of the whole dried Vitex negundo var. heterophylla plant, crush it, and extract it with 2 - 4 times the amount of 90 - 95% ethanol by cold maceration for 2 - 4 times, 5 - 9 days each time. Combine the filtrates and recover the solvent under reduced pressure to obtain 994 - 1042 g of extract. Disperse the extract in water and extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Recover the solvents to obtain 156 - 172 g of the petroleum ether fraction, 135 - 150 g of the dichloromethane fraction, 54 - 62 g of the ethyl acetate fraction, and 210 - 223 g of the n-butanol fraction; (2) Separation and purification of the dichloromethane fraction: After dissolving 135 - 150 g of the dichloromethane fraction extract, weigh 100 g of silica gel for sample mixing, and separate it by silica gel column chromatography using petroleum ether - ethyl acetate as the eluent, with gradient elution at volume ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:

2. After the 1:1 column flushing is completed, use TLC for tracking, combine the same parts, and obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H; (3) Separation and purification of the ethyl acetate fraction: After dissolving 54 - 62 g of the ethyl acetate fraction extract, weigh 50 g of polyamide for sample mixing, and separate it by polyamide column chromatography with gradient elution of 10% - 70% ethanol - water. After the absolute ethanol column flushing is completed, use a polyamide thin layer plate for tracking, combine the same parts, and obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, Fr.Q; (4) Fine separation and purification of chemical components: ①Separation and purification of fraction Fr.C in the dichloromethane fraction: Fraction Fr.C is 12 - 14 g of dark green powder, separated by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, with gradient elution at volume ratios of 10:1, 7:1, and 5:

1. By analyzing the combined components through TLC thin layer plates, 3 components are obtained, namely Fr.C1, Fr.C2, and Fr.C3. Among them, fraction Fr.C2 is separated by silica gel column chromatography, with isocratic elution using petroleum ether - ethyl acetate at a volume ratio of 7:

1. By analyzing the combined components through TLC thin layer plates, 2 components are obtained, namely Fr.C 2-1 and Fr.C 2-2 , Fr.C 2-1 and then recrystallized with dichloromethane to obtain 18.5 - 19.2 mg of the compound ethylparaben; ② Separation and purification of the Fr.I part in the ethyl acetate fraction: Fr.I is 0.25 - 0.32 g of orange-yellow powder. It is separated by polyamide column chromatography and isocratically eluted with ethanol - water at a volume ratio of 1:

4. Analyze and combine the components by spotting on a polyamide thin layer plate to obtain 2 components, namely Fr.I1 and Fr.I2. Fr.I2 is further separated by gel column chromatography and isocratically eluted with dichloromethane - methanol at a volume ratio of 1:1 to obtain 5.6 - 6.1 mg of compound vitexdoinA.

2. The method for extracting and separating the vitex negundo l. compound according to claim 1, characterized in that, Specifically, it includes the following steps: (1) Crude extraction and separation of medicinal materials: Take 50 kg of the whole dried Vitex negundo var. heterophylla plant, crush it, and extract it with 3 times the amount of 95% ethanol by cold maceration for 3 times, 7 days each time. Combine the filtrates and recover the solvent under reduced pressure to obtain 1026 g of extract. Disperse the extract in water and extract it with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Recover the solvents to obtain 168 g of the petroleum ether fraction, 144 g of the dichloromethane fraction, 60 g of the ethyl acetate fraction, and 219 g of the n-butanol fraction; (2) Isolation and purification of the dichloromethane fraction: After dissolving 144 g of the dichloromethane fraction extract, 100 g of silica gel was weighed and used for sample mixing. The separation was carried out by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, and gradient elution was carried out at volume ratios of 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, and 1:

2. After the column was flushed with 1:1 eluent, TLC was used for tracking, and the same parts were combined to obtain 8 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H; (3) Isolation and purification of the ethyl acetate fraction: After dissolving 60 g of the ethyl acetate fraction extract, 50 g of polyamide was weighed and used for sample mixing. The separation was carried out by polyamide column chromatography, with gradient elution using 10% - 70% ethanol - water. After the column was flushed with absolute ethanol, polyamide thin layer plates were used for tracking, and the same parts were combined to obtain 9 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, and Fr.Q; (4) Fine isolation and purification of chemical components: ①Separation and purification of fraction Fr.C in the dichloromethane fraction: Fraction Fr.C was 13.5 g of dark green powder. It was separated by silica gel column chromatography, using petroleum ether - ethyl acetate as the eluent, and gradient elution was carried out at volume ratios of 10:1, 7:1, and 5:

1. By spotting TLC plates to analyze and combine the components, 3 components were obtained, namely Fr.C1, Fr.C2, and Fr.C3. Among them, fraction Fr.C2 was separated by silica gel column chromatography, with isocratic elution using petroleum ether - ethyl acetate at a volume ratio of 7:

1. By spotting TLC plates to analyze and combine the components, 2 components were obtained, namely Fr.C 2-1 and Fr.C 2-2 , Fr.C 2-1 was further recrystallized from dichloromethane to obtain 19 mg of the compound ethylparaben; ② Isolation and purification of Fr.I in the ethyl acetate fraction: Fr.I was 0.3 g of orange - yellow powder. It was separated by polyamide column chromatography, with isocratic elution using ethanol - water at a volume ratio of 1:

4. By analyzing and combining the components through spotting polyamide thin layer plates, 2 components were obtained, namely Fr.I1 and Fr.I2. Fr.I2 was further separated by gel column chromatography, with isocratic elution using dichloromethane - methanol at a volume ratio of 1:1, and 6 mg of compound vitexdoinA was obtained.

3. The method for extracting and separating the vitex negundo compound according to any one of claims 1 or 2, characterized in that, The mesh number of the silica gel described in step (2) is 100 - 200 mesh.

4. The method for extracting and separating the vitex negundo compound according to any one of claims 1 or 2, characterized in that, The mesh number of the polyamide described in steps (3) and (4) is 200 - 300 mesh.

5. The method for extracting and separating the vitex negundo compound according to any one of claims 1 or 2, characterized in that, The mesh number of the silica gel described in step (4) is 200 - 300 mesh.

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