Application of chaste tree twig compound ethylparabe

Abstract

The invention discloses an application of a chaste tree twig compound ethylparaben. The invention further discloses the application of the chaste tree twig compound ethylparaben in preparation of antifungal or antibacterial drugs. The chaste tree twig compound ethylparaben obtained through separation and purification has a remarkable inhibition effect on escherichia coli, staphylococcus aureus and candida albicans, is a potential antifungal drug and is also a potential antibacterial drug, and a scientific basis is provided for further development and utilization of chaste tree twig resources.

Description

[0001] This application is a divisional application of patent application No. CN202310607680.2.

[0002] Original application date: May 26, 2023

[0003] Original application number: 202310607680.2

[0004] Original invention title: A method for extracting and separating compounds from Vitex negundo Linn var. heterophylla (Franch.) Rehd. and its applications Technical Field

[0005] The present invention relates to the technical field of the uses of traditional Chinese medicine chemical components, and specifically relates to the application of a compound ethylparabe from Vitex negundo Linn var. heterophylla (Franch.) Rehd. Background Art

[0006] Vitex negundo Linn var. heterophylla (Franch.) Rehd. is a plant of the genus Vitex in the Verbenaceae family [1] , and its name in Miao medicine is Dulai Gun. Miao doctors believe 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 a 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

[0007] The purpose of the present invention is to provide the application of a compound ethylparabe from Vitex negundo Linn var. heterophylla (Franch.) Rehd. in antifungal and antibacterial drugs.

[0008] The present invention is achieved through the following technical solutions:

[0009] The method for extracting and separating the compound of Vitex negundo Linn var. heterophylla (Franch.) Rehd. described in the present invention includes the following steps:

[0010] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of the whole dry Vitex negundo var. heterophylla plant, crush it, and perform cold extraction with 2 - 4 times the amount of 90 - 95% ethanol for 2 - 4 times, each time for 5 - 9 days. 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;

[0011] (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. Use petroleum ether - ethyl acetate as the eluent and perform gradient elution according to the 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;

[0012] (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. 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;

[0013] (4) Fine separation and purification of chemical components:

[0014] ① Separation and purification of the Fr.C part in the dichloromethane fraction: The Fr.C part is 12 - 14 g of dark green powder. After separation by silica gel column chromatography, use petroleum ether - ethyl acetate as the eluent and perform gradient elution according to the volume ratios of 10:1, 7:1, 5:1. Analyze and combine the components by spotting TLC thin layer plates to obtain 3 components, namely Fr.C 1 、Fr.C 2 、Fr.C 3 , among which Fr.C 2 After separation by silica gel column chromatography and isocratic elution with petroleum ether - ethyl acetate at 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 Then recrystallize with dichloromethane to obtain 18.5 - 19.2 mg of the compound ethylparaben;

[0015] ② Isolation and purification of fraction Fr.I in the ethyl acetate fraction: Fr.I is an orange-yellow powder, 0.25 - 0.32 g. It is separated by polyamide column chromatography and eluted isocratically with ethanol-water with a volume ratio of 1:4. By analyzing and combining components through spotting polyamide thin-layer plates, 2 components are obtained, namely Fr.I 1 、Fr.I 2 ,Fr.I 2 Then it is further separated by gel column chromatography and eluted isocratically with dichloromethane-methanol with a volume ratio of 1:1 to obtain 5.6 - 6.1 mg of compound vitexdoin A.

[0016] The method for extracting and separating the compounds from Vitex negundo var. heterophylla described in the present invention specifically includes the following steps:

[0017] (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 3 times the amount of 95% ethanol by cold soaking 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;

[0018] (2) Isolation 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 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;

[0019] (3) Isolation 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. Perform gradient elution with 10% - 70% ethanol-water. After the absolute ethanol column flushing is completed, use polyamide thin-layer plates 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;

[0020] (4) Fine isolation and purification of chemical components:

[0021] 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 analyzing the combined components by spotting TLC thin layer plates, 3 components were obtained, namely Fr.C 1 , Fr.C 2 , Fr.C 3 , among which Fr.C 2 was separated by silica gel column chromatography, and isocratic elution was carried out with petroleum ether - ethyl acetate at a volume ratio of 7:1. By analyzing the combined components by spotting TLC thin layer plates, 2 components were obtained, namely Fr.C 2-1 , Fr.C 2-2 . Fr.C 2-1 was further recrystallized with dichloromethane to obtain 19 mg of the compound ethylparaben;

[0022] ②Separation and purification of fraction Fr.I in the ethyl acetate fraction: Fraction Fr.I was 0.3 g of orange - yellow powder. It was separated by polyamide column chromatography, and isocratic elution was carried out with ethanol - water at a volume ratio of 1:4. By analyzing the combined components by spotting polyamide thin films, 2 components were obtained, namely Fr.I 1 , Fr.I 2 . Fr.I 2 was further separated by gel column chromatography, and isocratic elution was carried out with dichloromethane - methanol at a volume ratio of 1:1, and 6 mg of the compound vitexdoin A was obtained.

[0023] The mesh number of the silica gel described in step (2) of the present invention is 100 - 200 mesh.

[0024] The mesh number of the polyamide described in steps (3) and (4) of the present invention is 200 - 300 mesh.

[0025] The mesh number of the silica gel described in step (4) of the present invention is 200 - 300 mesh.

[0026] Use of the vitexdoin A and ethylparaben compounds of the present invention in the preparation of antifungal or antibacterial drugs.

[0027] Use of the obtained vitexdoin A and ethylparaben compounds of the present invention in the preparation of drugs for Candida albicans, Escherichia coli, and Staphylococcus aureus.

[0028] The concentration of the compound vitexdoin A described in the present invention is: MIC for Escherichia coli 90 > 1 mg / mL, MIC 50 > 1 mg / mL; MIC for Staphylococcus aureus90 > 1 mg / mL, MIC 50 > 1 mg / mL; MIC against Candida albicans 90 > 1 mg / mL, MIC 50 is 1 mg / mL;

[0029] The concentration of the compound ethylparaben is: MIC against Escherichia coli 90 > 1 mg / mL, MIC 50 is 0.5 mg / mL; MIC against Staphylococcus aureus 90 > 1 mg / mL, MIC 50 > 1 mg / mL; MIC against Candida albicans 90 > 1 mg / mL, MIC 50 is 0.25 mg / mL.

[0030] The preparation of the present invention is a pharmaceutically acceptable preparation prepared by adding pharmaceutically acceptable excipients, and the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation;

[0031] The solid preparation of the present invention is a granule, a capsule, a tablet, a pill; the liquid preparation is an injection preparation, an oral liquid.

[0032] Advantages of the present invention:

[0033] 1. The extraction and separation method of the present invention is simple and easy to implement, and the monomer compounds vitexdoin A and ethylparaben have significant inhibitory effects on Escherichia coli, Staphylococcus aureus, and Candida albicans; it is a potential antifungal drug and also a potential antibacterial drug, providing a scientific basis for the further development and utilization of Vitex negundo var. heterophylla resources.

[0034] 2. The present invention uses Escherichia coli, Staphylococcus aureus, and Candida albicans as test strains to study the in vitro antibacterial activity of the monomer compounds vitexdoin A and ethylparaben. Results: The two 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; MIC against Staphylococcus aureus 90 > 1 mg / mL, MIC 50 is 1 mg / mL; MIC against Candida albicans 90 > 1 mg / mL, MIC 50 is 1 mg / mL; the MIC of ethylparaben against Escherichia coli 50 is 0.5 mg / mL; MIC against Staphylococcus aureus 50is 1 mg / mL; MIC against Staphylococcus aureus 90 > 1 mg / mL, MIC 50 is 1 mg / mL; MIC against Candida albicans 90 > 1 mg / mL, MIC 50 is 0.25 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Hydrogen spectrum of the compound ethylparaben

[0036] Figure 2 : Carbon spectrum of the compound ethylparaben

[0037] Figure 3 : Hydrogen spectrum of the compound vitexdoin A

[0038] Figure 4 : Carbon spectrum of the compound vitexdoin A DETAILED DESCRIPTION OF THE INVENTION

[0039] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0040] Example 1

[0041] Extraction and separation method of vitex compounds:

[0042] (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 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;

[0043] (2) Separation and purification of the 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;

[0044] (3) Isolation and purification of the ethyl acetate fraction: After dissolving 60 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;

[0045] (4) Fine isolation and purification of chemical components:

[0046] ① Isolation and purification of the Fr.C part in the dichloromethane fraction: The Fr.C part was 13.5 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.C 1 、Fr.C 2 、Fr.C 3 Among them, Fr.C 2 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 was further recrystallized with dichloromethane to obtain 19 mg of the compound ethylparaben;

[0047] ② Isolation and purification of the Fr.I part in the ethyl acetate fraction: The Fr.I part was 0.3 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.I 1 、Fr.I 2 Fr.I 2 was further separated by gel column chromatography and isocratically eluted with dichloromethane - methanol at a volume ratio of 1:1, and 6 mg of the compound vitexdoin A was obtained.

[0048] Example 2

[0049] Extraction and separation method of compounds from Vitex negundo var. heterophylla

[0050] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of the whole dried Vitex negundo var. heterophylla plant, crush it, and extract it twice by cold soaking with 2 times the amount of 90% ethanol for 5 days each time. 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. Recover the solvents to obtain 156 g of the petroleum ether fraction, 135 g of the dichloromethane fraction, 54 g of the ethyl acetate fraction, and 210 g of the n-butanol fraction;

[0051] (2) Separation 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 and perform gradient elution according to the 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 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;

[0052] (3) Separation and purification of the ethyl acetate fraction: After dissolving 54 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 using a gradient elution of 10% - 70% ethanol - water. After the absolute ethanol column flushing is completed, use a 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;

[0053] (4) Fine separation and purification of chemical components:

[0054] ① Separation and purification of the Fr.C part in the dichloromethane fraction: The Fr.C part is 12 g of dark green powder. Separate it by silica gel column chromatography (200 - 300 mesh) using petroleum ether - ethyl acetate as the eluent and perform gradient elution according to the volume ratios of 10:1, 7:1, 5:1. Analyze and combine the components by spotting TLC thin layer plates to obtain 3 components, namely Fr.C 1 、Fr.C 2 、Fr.C 3 , among which Fr.C 2 is separated by silica gel column chromatography and isocratically eluted with petroleum ether - ethyl acetate at 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 is further recrystallized with dichloromethane to obtain 18.5 mg of the compound ethylparaben;

[0055] ②Isolation and purification of fraction Fr.I in the ethyl acetate fraction: Fraction Fr.I was 0.25 g of orange-yellow powder, separated by polyamide column chromatography (200 - 300 mesh), eluted isocratically with ethanol-water at a volume ratio of 1:4, and the components were combined by analyzing the polyamide thin layer plate, obtaining 2 components, namely Fr.I 1 、Fr.I 2 ,Fr.I 2 Then it was further separated by gel column chromatography, eluted isocratically with dichloromethane-methanol at a volume ratio of 1:1, and 5.6 mg of compound vitexdoin A was obtained.

[0056] Example 3

[0057] Extraction and separation method of compounds from Vitex negundo var. heterophylla

[0058] (1) Coarse extraction and separation of medicinal materials: Take 50 kg of dry whole plant of Vitex negundo var. heterophylla, crush it, extract it with 3 times the amount of 95% ethanol by cold maceration 4 times, each time for 9 days, combine the filtrates, and recover the solvent under reduced pressure to obtain 1042 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, 172 g of petroleum ether fraction, 150 g of dichloromethane fraction, 62 g of ethyl acetate fraction, and 223 g of n-butanol fraction are obtained;

[0059] (2) Isolation and purification of the dichloromethane fraction: After dissolving 150 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, use petroleum ether-ethyl acetate as the eluent, and perform gradient elution at a 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;

[0060] (3) Isolation and purification of the ethyl acetate fraction: After dissolving 62 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, elute it with 10% - 70% ethanol-water gradient elution. After the absolute ethanol column flushing is completed, use the 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;

[0061] (4) Fine isolation and purification of chemical components:

[0062] ①Separation and purification of fraction Fr.C in the dichloromethane fraction: Fraction Fr.C was 14 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 thin layer plates, 3 components were obtained, namely Fr.C 1 、Fr.C 2 、Fr.C 3 , among which Fr.C 2 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 thin layer plates, 2 components were obtained, namely Fr.C 2-1 、Fr.C 2-2 . Fr.C 2-1 was further recrystallized with dichloromethane to obtain 19.2 mg of the compound ethylparaben;

[0063] ②Separation and purification of fraction Fr.I in the ethyl acetate fraction: Fraction Fr.I was 0.32 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.I 1 、Fr.I 2 . Fr.I 2 was further separated by gel column chromatography, with isocratic elution using dichloromethane - methanol at a volume ratio of 1:1, to obtain 6.1 mg of the compound vitexdoin A.

[0064] Example 4

[0065] Take any one compound or both of ethylparaben and vitexdoin A as raw materials, add the pharmaceutically acceptable excipient dextrin, granulate to obtain granules.

[0066] Example 5

[0067] Take any one compound or both of ethylparaben and vitexdoin A as raw materials, add the pharmaceutically acceptable excipient dextrin, mix well, and fill into capsules to obtain capsules.

[0068] Example 6

[0069] Take any one compound or both of ethylparaben and vitexdoin A as raw materials, add the pharmaceutically acceptable excipient dextrin, granulate, and press into tablets to obtain tablets.

[0070] Example 7

[0071] Take any one or two of ethylparaben and vitexdoin A as raw materials, add pharmaceutically acceptable excipient dextrin, mix well, make pills, and dry to obtain pills.

[0072] Example 8

[0073] Take any one or two of ethylparaben and vitexdoin A as raw materials, add 10 times the amount of injection water, mix well, filter, sterilize to obtain an injection.

[0074] To further verify the feasibility and effectiveness of the present invention and screen out the best solution, the inventors conducted a series of experiments as follows:

[0075] 1. Instruments and materials

[0076] 1.1 Experimental instruments

[0077] JOEL 5937MSD type mass spectrometer (Agilent Technologies, USA); 600 MHz 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).

[0078] 1.2 Experimental materials

[0079] 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 GF 254 Precast plate (Qingdao Puke Separation Materials Co., Ltd.), polyamide film (Taizhou Sijia Biochemical Plastics Factory), chromatographic silica gel (Qingdao Ocean Chemical Co., Ltd.), SephadexLH-20 dextran gel (Amersham Biosciences, Sweden), chromatographic polyamide powder (Chengdu Yuannuo Tiancheng Technology Co., Ltd.), MCI resin (Beijing Huideyi), SiliaSphere C 18 Reverse phase packing material (Beijing Greengrass Science and Technology Development Co., Ltd.); the rest of the reagents are all of analytical grade.

[0080] 2. Extraction and separation

[0081] 2.1 Medicinal material extraction and crude separation

[0082] 50 kg of dry whole Vitex negundo L. was crushed and extracted with 3 times the amount of 95% ethanol by cold maceration for 3 times, 7 days each time. 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. After recovering the solvents, 168 g of petroleum ether fraction, 144 g of dichloromethane fraction, 60 g of ethyl acetate fraction, and 219 g of n-butanol fraction were obtained.

[0083] After dissolving the dichloromethane fraction extract (144 g), 100 g of silica gel (100 - 200 mesh) was weighed and mixed with the sample, and then separated by silica gel column chromatography. Gradient elution was carried out with petroleum ether - ethyl acetate (20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 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 components, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H.

[0084] After dissolving the ethyl acetate fraction extract (60 g), 50 g of polyamide (200 - 300 mesh) was weighed and mixed with the sample, and then separated by polyamide column chromatography. Gradient elution was carried out with 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 components, namely Fr.I, Fr.J, Fr.K, Fr.L, Fr.M, Fr.N, Fr.O, Fr.P, and Fr.Q.

[0085] 2.2 Fine separation and purification of chemical components

[0086] 2.2.1 Separation and purification of dichloromethane fraction

[0087] Separation and purification of Fr.C part

[0088] The Fr.C part was a dark green powder (13.5 g), which was separated by silica gel column chromatography (200 - 300 mesh) and eluted with petroleum ether - ethyl acetate (10:1, 7:1, 5:1) gradient. By analyzing and combining the components by spotting TLC thin layer plates, 3 components were obtained, namely Fr.C 1 , Fr.C 2 , Fr.C 3 , among which Fr.C 2 was separated by silica gel column chromatography (200 - 300 mesh) and eluted isocratically with petroleum ether - ethyl acetate (7:1). By analyzing and combining the components by spotting TLC thin layer plates, 2 components were obtained, namely Fr.C 2-1 , Fr.C 2-2 , Fr.C 2-1 was recrystallized with dichloromethane to obtain compound 18 (19 mg).

[0089] 2.2.2 Isolation and purification of the ethyl acetate fraction

[0090] Isolation and purification of fraction Fr.I

[0091] Fraction Fr.I was 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 components through spotting on polyamide thin-layer plates and combining the fractions, 2 components were obtained, namely Fr.I 1 、Fr.I 2 ,Fr.I 2 Then it was further separated by gel column chromatography, eluted isocratically with dichloromethane-methanol (1:1), and compound 29 (6 mg) was obtained.

[0092] 2.2.3 Carbon-13 nuclear magnetic resonance

[0093] Compound 18: White square crystals, soluble in organic solvents such as chloroform. The molecular formula was: C 9 H 10 O 3 . In 1H NMR (400 MHz, CDCl 3 ), δ: 7.93 (1H, d, J = 8.7 Hz, H-3,5), 6.85 (2H, d, J = 8.5 Hz, H-2,6) were the signals of the hydrogen protons on the benzene ring AABB, δ: 4.33 (2H, q, J = 7.1 Hz, H-8) was the signal of the hydrogen proton on the carbon adjacent to the ester group, 1.36 (3H, t, J = 7.1 Hz, H-9) was the signal of the hydrogen proton on the methyl group; in 13 13C NMR (100 MHz, CDCl 3 ), δ: 167.1 (C-7) was the carbon signal on the ester group, δ: 160.4 (C-4), 132.1 (C-2,6), 122.8 (C-1), 115.4 (C-3,5) were the carbon signals on the benzene ring, δ: 61.1 (C-8) was the carbon signal on the carbon adjacent to the ester group, 14.5 (C-9) was the carbon signal on the methyl group. The above data were basically consistent with the literature [6] reports, so this compound was determined to be ethylparaben. The 13C NMR signals of this compound and the literature are shown in Table 1. See 13 . Figures 1 - 2 .

[0094]

[0095]

[0096] Table 1 13C NMR (100 MHz, CDCl 13 ) data of compound 18 3 )

[0097]

[0098] Compound 29: Yellow powder. Soluble in organic solvents such as methanol, and its molecular formula is: C 19 H 18 O 6 . In 1 H NMR (600 MHz, MeOD), δ: 9.30 (1H, s, H-2α) is the proton signal of the aldehyde group hydrogen, there are 5 proton signals in the aromatic region, δ: 7.29 (1H, s, H-1) is the proton signal of the unsubstituted hydrogen on the double bond, δ: 6.95 (1H, s, H-8), 6.72 (1H, s, H-5) are the proton signals of the unsubstituted para-hydrogens on the 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 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 proton signals on the chiral carbon atom and the proton signal on the adjacent carbon; δ: 3.72 (3'-OCH 3 ) is the proton signal of the methoxy group. In 13 C NMR (150 MHz, MeOD) spectrum, δ: 194.8 (C-2α) is the aldehyde group carbon signal, 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'-OCH 3 ) is the carbon signal of the methoxy group. The above data are consistent with the literature [7] report, so this compound is determined to be vitexdoin A. The 13 C NMR nuclear magnetic resonance carbon spectrum signals of this compound and the literature are shown in Table 2. See Figures 3 - 4 .

[0099]

[0100] 13C NMR (150 MHz, MeOD) data of Compound 29 in Table 2

[0101]

[0102] 3. Study on the Antibacterial Activity of Chemical Constituents from Vitex negundo var. heterophylla

[0103] 3.1 Instruments and Materials

[0104] 3.1.1 Experimental Instruments

[0105] Biological clean safety cabinet (Model: BHC-1300IIA / B2, Manufacturer: Suzhou Purification Equipment Co., Ltd.); Oscillating incubator (Model: BS-1EA, Manufacturer: Changzhou Huapuda Mathematical Instrument Co., Ltd.); Tabletop constant temperature oscillator (Model: LY20-92C, Manufacturer: Shanghai Longyue Instrument 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).

[0106] 3.1.2 Experimental Reagents

[0107] Nutrient broth medium (No.: 022010, Manufacturer: Guangdong Huankai Microbial Science and Technology Co., Ltd.), Nutrient agar medium (No.: 022020, Manufacturer: Guangdong Huankai Microbial Science and Technology Co., Ltd.), Sabouraud agar medium (No.: 510D021, Manufacturer: Solarbio Science & Technology Co., Ltd.), 0.5% Glucose broth medium (No.: 901F031, Manufacturer: Solarbio Science & Technology Co., Ltd.), 96-well plates.

[0108] 3.1.3 Experimental Materials

[0109] Monomeric compounds from different polar parts of Vitex negundo var. heterophylla: 18 ethylparaben, 29 vitexdoin A.

[0110] 3.1.4 Experimental Strains and Cells

[0111] Candida albicans (No.: 29343), Escherichia coli (No.: Jm109), Staphylococcus aureus (No.: 2021.2.28).

[0112] 3.2 Experimental Procedures

[0113] 3.2.1 Preparation of Media

[0114] Nutrient broth medium: Weigh 18 g of the powder, add it to 1000 mL of distilled water, heat to boiling and dissolve, dispense, sterilize at 115 °C under high pressure for 15 min, and set aside.

[0115] Nutrient agar medium: Weigh 33 g of the powder, add it to 1000 mL of distilled water, heat and boil until dissolved, dispense, sterilize at 115 °C under high pressure for 15 min, and set aside.

[0116] Sabouraud agar medium: Weigh 70 g of the powder, add it to 1000 mL of distilled water, heat and boil until dissolved, dispense, sterilize at 115 °C under high pressure for 15 min, and set aside.

[0117] 0.5% glucose broth medium: Weigh 23 g of the powder, add it to 1000 mL of distilled water, heat and boil until dissolved, dispense, sterilize at 115 °C under high pressure for 15 min, and set aside.

[0118] 3.2.2 Cultivation of bacteria and fungi

[0119] In a sterile operating table, pour the sterilized nutrient broth agar medium into a sterile Petri dish. After solidification, smear 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 Candida albicans bacterial solution on the Petri dish and incubate at 37 °C for 24 h.

[0120] Take a sterile centrifuge tube, add 5 mL of nutrient broth medium, pick one colony of Escherichia coli and Staphylococcus aureus into the nutrient medium, and culture at 37 °C in a shaker for 24 h. Then aspirate 50 μL of the bacterial solution into 5 mL of nutrient broth medium and shake again at 37 °C in a shaker for 2.5 h. Take a sterile centrifuge tube, add 5 mL of broth medium, pick one colony of Candida albicans into the nutrient medium, and culture at 37 °C in a shaker for 24 h. Then aspirate 50 μL of the bacterial solution into 5 mL of broth medium and shake again at 37 °C in a shaker for 2.5 h to make it in the logarithmic growth phase.

[0121] 3.2.3 Determination of bacteriostatic rate

[0122] 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, and set aside. First, aspirate 100 μL of 1 mg / mL different monomer compounds into a 96-well plate, and then add 100 μL of the bacterial solution. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure its absorbance value (sample OD 0 ), and then culture it in a 37 °C constant temperature incubator for another 24 h, and use an ELISA reader to measure its absorbance value (sample OD 24 ). Calculate its inhibition rate.

[0123] Select the above monomeric compounds with certain antibacterial activities. Using the double serial dilution method, 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, and 0.0313 mg / mL respectively. Add 100 μL of the bacterial solution and solutions of different polar parts with different concentration gradients into 96-well plates respectively. Use a microplate reader to measure the absorbance values (sample OD 0 ) at 600 nm. Then, culture them in a constant temperature incubator at 37 °C for another 24 h, and use a microplate reader to measure the absorbance values (sample OD 24 ). Calculate the antibacterial rate using the formula. Use berberine and chlorogenic acid as positive controls, and 1% DMSO aqueous solution as a negative control.

[0124] Formula: Antibacterial rate = [1 - (sample OD 24 - sample OD 0 ) / (blank OD 24 - blank OD 0 )] * 100%

[0125] 3.3 Experimental Results and Discussions

[0126] The antibacterial rates of different monomeric compounds (1 mg / mL) against the test strains are shown in Table 3. The MIC 50 , MIC 90 statistical results are shown in Tables 4 and 5. The antibacterial rates of monomeric compounds with certain antibacterial activities against the test strains are shown in Tables 6 and 7.

[0127] From the following results, the strains in the negative group can all grow normally, indicating that the 1% DMSO aqueous solution has no inhibitory effect on the growth of bacteria and fungi. The positive group has a certain inhibitory effect on the growth of bacteria and fungi, indicating the feasibility of this experiment.

[0128] The results show that the 1% DMSO aqueous solution has almost no effect on the growth of the strains. The MIC 90 of vitexdoin A against Escherichia coli > 1 mg / mL, MIC 50 > 1 mg / mL, showing certain activity against Escherichia coli; the MIC 90 of ethylparaben against Escherichia coli > 1 mg / mL, MIC 50 is 0.5 mg / mL, showing certain activity against Escherichia coli; the MIC 90 of ethylparaben against Staphylococcus aureus > 1 mg / mL, MIC 50 is 1 mg / mL, showing certain activity against Staphylococcus aureus; the MIC 90> 1 mg / mL, MIC 50 is 1 mg / mL and has a certain activity against Candida albicans. The MIC of ethylparaben against Candida albicans 90 > 1 mg / mL, MIC 50 is 0.25 mg / mL and has a strong activity against Candida albicans.

[0129] Table 3 Antibacterial rates of different monomeric compounds of Vitex negundo (1 mg / mL) against test strains ( n = 6)

[0130]

[0131] Table 4 MIC of different monomeric compounds against Escherichia coli and Staphylococcus aureus strains 90 , MIC 50 values

[0132]

[0133]

[0134] Table 5 MIC of different monomeric compounds against Candida albicans strains 90 , MIC 50 values

[0135]

[0136] Table 6 Antibacterial rates of different monomeric compounds against Escherichia coli and Staphylococcus aureus ( n = 9)

[0137]

[0138]

[0139] Table 7 Antibacterial rates of different monomeric compounds against Candida albicans ( n = 9)

[0140]

[0141] Although the present invention has been described in detail in the foregoing 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 required by the present invention.

[0142] References

[0143] [1]Editorial Committee of Flora Reipublicae Popularis Sinicae, Chinese Academy of Sciences. Flora Reipublicae Popularis Sinicae, Vol. 65, Fasc. 1 [M]. Beijing: Science Press, 1982.03: 141.

[0144] [2]Qiu Dewen, Du Jiang. Chinese Materia Medica, Miao Medicine Volume [M]. Guiyang: Guizhou Science and Technology Press, 2005: 482 - 483.

[0145] [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.

[0146] [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.

[0147] [5]Guizhou Institute of Traditional Chinese Medicine. Guizhou Herbal Medicine, Volume 2 [M]. Guiyang: Guizhou People's Publishing House, 1970: 824 - 825.

[0148] [6]Li Huaqiang. Study on Chemical Constituents and Pharmacognostic Identification of Fructus Viticis Negundo [D]. Shanghai: Shanghai Normal University, 2015.

[0149] [7]Zhang Qingjian, Ni Gang, Yu Dequan. Study on Chemical Constituents of Vitex negundo L. var. incana (Thunb.) Hand.-Mazz. [J]. China Journal of Chinese Materia Medica, 2009, 34(10): 1305.

[0150] [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. Application of the vitex negundo compound ethylparaben, characterized in that, the application of the compound ethylparaben in the preparation of antifungal or antibacterial drugs.

2. The application of the vitex negundo compound ethylparaben according to claim 1, characterized in that, the application of the compound ethylparaben in the preparation of drugs against candida albicans, drugs against escherichia coli, and drug preparations against staphylococcus aureus.

3. The application of the vitex negundo compound ethylparaben according to claim 2, characterized in that, The concentration of the compound ethylparaben is: the MIC against Escherichia coli 90 > 1 mg / mL, the MIC 50 is 0.5 mg / mL; the MIC against Staphylococcus aureus 90 > 1 mg / mL, the MIC 50 > 1 mg / mL; the MIC against Candida albicans 90 > 1 mg / mL, the MIC 50 is 0.25 mg / mL.

4. The application of the vitex negundo compound ethylparaben according to any one of claims 1 or 2, characterized in that, the preparation is a pharmaceutically acceptable preparation prepared by adding pharmaceutically acceptable excipients, and the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation.

5. The application of the vitex negundo compound ethylparaben according to claim 4, characterized in that, the solid preparation is a granule, a capsule, a tablet, or a pill; the liquid preparation is an injection preparation or an oral liquid.

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