A mixed extract of neem seeds and neem bark, and preparation method and application thereof

Abstract

The invention discloses a mixed extract of neem fruit and neem bark, and a preparation method and application thereof, belonging to the technical field of extracts. The preparation method comprises the following steps: S01, pretreatment: crushing and sieving neem fruit and neem bark with a mass ratio of 1: (2-4.5), and mixing to obtain a composite powder; S02, extraction: adding a non-polar solvent to the composite powder, soaking, extracting, and filtering; after the filter residue evaporates the solvent, adding a polar solvent to the filter residue, extracting, filtering, and concentrating to obtain a crude extract; S03, purification: purifying the crude extract with a macroporous adsorption resin, concentrating, and drying. The raw materials for preparing the plant-derived antibacterial agent of the invention are derived from neem fruit and neem bark, which cooperate with each other and have a synergistic effect, and show good antibacterial properties against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella typhimurium, Klebsiella pneumoniae and other bacteria.

Description

Technical Field

[0001] The invention relates to a mixed extract of chinaberry seeds and chinaberry bark, a preparation method and application thereof, and in particular to component extraction, component analysis, antibacterial and antitumor activity research of the mixed extract of chinaberry seeds and chinaberry bark, belonging to the technical field of extracts. Background Art

[0002] Since ancient times, plants have been a natural treasure trove of antimicrobial active substances. They have been widely used in food preservation and pharmaceutical excipients, solving many problems in human production and life and promoting the development and utilization of plant resources. Melia toosendanSieb.et Zucc. or Melia azedarach L. family of the Meliaceae family. Melia is widely planted in Sichuan, Hunan, Anhui, Hebei and other provinces in China. Melia is a commonly used Chinese medicinal material. Its bark is used as medicine. It tastes bitter, is cold in nature, and is toxic. It belongs to the liver, spleen, and stomach meridians. It is collected in the herbal medicines of all dynasties and the Chinese Pharmacopoeia. It has the effects of killing insects and curing allergies. At present, the compounds isolated from Melia bark mainly include triterpenes, volatile oils, sterols, and organic acids. Melia bark has the effects of anthelmintic, antiviral, antibacterial, and antitumor. Because of its toxicity, it is often used as a raw material for agricultural pesticides and is a commonly used Chinese medicinal material.

[0003] Melia azedarach bark extract interferes with the metabolic function of roundworms, causing an imbalance in their energy supply and demand. The worms spasm due to contractile fatigue and are then expelled from the body by the host. TSN contained in Melia azedarach bark can act on multiple targets, causing ulceration of the midgut tissue of the worms, interruption of central nervous conduction, and disorders of the worms' digestion and absorption functions, affecting normal physiological and biochemical activities and causing worm lesions. Toosendanin contained in Melia azedarach bark has good anti-tumor activity. TSN can inhibit the growth of MKN-45 and BGC-823 cells. qRT-PCR detection shows that 80nmol·L -1 TSN can inhibit the proliferation of gastric cancer cells by downregulating the expression of MYC gene in cells, increasing the number of CTPS cells, and inhibiting the proliferation of gastric cancer cells. Melia azedarach bark extract has the same antibacterial and bactericidal effects on Escherichia coli and Staphylococcus aureus.

[0004] With the increasing demand for neem bark, the planting area of ​​neem has also increased year by year, and its seeds have caused large-scale waste; therefore, in order to maximize the development and utilization of neem resources, this study conducted relevant research on the mixed extract of neem bark and neem fruit, optimized its extraction method, and analyzed the antibacterial and antitumor activities, providing a scientific basis for the future development of neem-related products. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for preparing a mixed extract of neem seeds and neem bark, which adopts a variety of impurity removal methods, uses non-polar and then polar extraction, and uses macroporous adsorption resin for re-separation and enrichment, and finally separates chemical components such as triterpene glycosides and flavonoid glycosides, which are antibacterial active substances.

[0006] Meanwhile, the invention provides a mixed extract of neem seeds and neem bark.

[0007] Meanwhile, the invention provides an application of a mixed extract of neem seeds and neem bark.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A method for preparing a mixed extract of neem seeds and neem bark comprises the following steps:

[0010] S01, pretreatment: crushing and sieving neem seeds and neem bark in a mass ratio of 1: (2-4.5), and mixing to obtain a composite powder;

[0011] S02, extraction: adding a non-polar solvent to the compound powder, soaking, extracting, and filtering; after the solvent is evaporated from the filter residue, adding a polar solvent to the filter residue, extracting, filtering, and concentrating to obtain a crude extract;

[0012] S03, purification: purifying the crude extract with a macroporous adsorption resin, concentrating and drying to obtain a mixed extract of Melia azedarach seeds and Melia azedarach bark.

[0013] In S01, neem seeds and neem bark should be cleaned, dried and then crushed; the drying temperature is 45-60°C; the drying time is 18-24h; the mesh size of the sieve is 100-300 mesh.

[0014] In S02, the non-polar solvent includes any one of petroleum ether, chloroform, cyclohexane or n-hexane, or a combination of at least two of them; the mass ratio of the compound powder to the non-polar solvent is 1:(15-30); the polar solvent includes any one of ethanol, methanol, acetone or water, or a combination of at least two of them; the mass ratio of the filter residue to the polar solvent is 1:(15-30); the immersion temperature is 20-40°C; the immersion time is 2-4h; the extraction is first ultrasonic extraction and then microwave heating extraction; the concentration is vacuum concentration, the vacuum concentration pressure is -0.085--0.09MPa, and the vacuum concentration temperature is 40-55°C.

[0015] The temperature of ultrasonic extraction is 25-35°C, the extraction time is 20-30min, and the ultrasonic power is 75-100W; the sample is then placed in a microwave instrument, and the temperature of microwave heating extraction is 50-95°C; the microwave heating extraction time is 10-20min.

[0016] The macroporous adsorption resin is AB-8, D101, CAD-40, DA201 or DM301.

[0017] The crude extract is concentrated to 1 / 10 of the volume of the polar solvent, and then filtered and loaded. The diameter-to-height ratio of the macroporous adsorption resin column is 1:(5-10), and the column volume is (15-80)×r 2 , r is the diameter of the macroporous adsorption resin column, and the amount of crude extract loaded is (10~30)×r 2 After washing with water to remove impurities, elute with 15-68% ethanol aqueous solution for 5-8 column volumes, collect the eluate, and concentrate in vacuo. The pressure of the vacuum concentration is -0.085 to -0.09 MPa, and the temperature of the vacuum concentration is 40 to 55°C; after vacuum concentration, freeze-vacuum drying is performed. The temperature of the freeze-vacuum drying is -60 to -20°C, and the time of the freeze-vacuum drying is 30 to 50 hours.

[0018] The mixed extract of Melia azedarach seeds and Melia azedarach bark obtained by the preparation method of the invention.

[0019] The mixed extract of chinaberry seeds and chinaberry barks of the invention is used in preparing traditional Chinese medicine products, anti-tumor drugs, cosmetics, health products and pesticides.

[0020] The mixed extract of chinaberry seeds and chinaberry bark of the present invention is used as an antibacterial agent, and the added amount of the mixed extract of chinaberry seeds and chinaberry bark in the antibacterial agent is 0.0076-0.50 g / mL.

[0021] The invention performs LC-MS characterization on the mixed extract of chinaberry fruit and chinaberry bark, performs qualitative analysis on the mixed extract of chinaberry fruit and chinaberry bark in combination with retention index, optimizes toosendanin and ferulic acid conditions by thin layer chromatography, and studies the antibacterial and antitumor effects of the mixed extract.

[0022] The present invention provides a plant-derived antibacterial agent, wherein the raw materials for preparing the plant-derived antibacterial agent are neem fruit and neem bark. Neem fruit and neem bark both have different degrees of antibacterial effects. Neem fruit and neem bark cooperate with each other, and have a synergistic effect and a cost-reducing effect. The present invention controls the quality of neem fruit and neem bark within the range of 1: (2-4.5), and has a high-efficiency broad-spectrum antibacterial effect. If it is lower than this range, that is, the specific gravity of neem fruit is larger, the antibacterial effect on Gram-negative bacteria is relatively poor. If it is higher than this range, the antibacterial effect on Klebsiella pneumoniae is relatively poor.

[0023] In the preparation method of the antibacterial agent of neem seeds and neem bark of the present invention, multiple impurity removal methods are adopted, S02 uses non-polar extraction followed by polar extraction, and S03 uses macroporous adsorption resin for re-separation and enrichment. After two steps, chemical components such as triterpene glycosides and flavonoid glycosides, which are antibacterial active substances, are selectively separated.

[0024] In the extraction process of the mixed extract of Melia azedarach seeds and Melia azedarach bark, a gradual impurity removal technology of first ultrasonic extraction and then microwave heating extraction is adopted to enrich the antibacterial active ingredients in the extract and improve the relative content of the inhibitor effective component.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The raw materials for preparing the plant-derived antibacterial agent of the present invention are derived from neem seeds and neem bark, which cooperate with each other to have a synergistic effect and show good antibacterial performance against various bacteria such as Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella typhimurium, and Klebsiella pneumoniae.

[0027] (2) The preparation method of the antibacterial agent of the composite extract of Melia azedarach and Melia azedarach bark of the present invention is simple to operate and low in cost. The obtained plant-derived inhibitor comes from natural plants, has a high content of effective substances and few impurities. It has a high inhibition rate and good effect when used as an antibacterial agent. It has high practical value and can be used in common antibacterial products such as traditional Chinese medicines, cosmetics, health products, pesticides, etc., and also has potential medical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the total ion current of the mixed extract of Melia azedarach seeds and Melia azedarach bark;

[0029] Figure 2 This is the thin layer chromatogram of Toosendanin;

[0030] Figure 3 This is the thin layer chromatogram of ferulic acid. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0032] Example 1

[0033] A method for preparing a mixed extract of neem seeds and neem bark comprises the following steps:

[0034] S01, pretreatment: crush and sieve the seeds of chinaberry and the bark of chinaberry in a mass ratio of 1:3, and mix them to obtain a composite powder; the seeds of chinaberry and the bark of chinaberry should be washed, dried and then crushed; the drying temperature is 50°C; the drying time is 20h; the mesh number of the sieve is 200 mesh;

[0035] S02, extraction: add non-polar solvent petroleum ether to the powder of the compound, the mass ratio of the powder of the compound to petroleum ether is 1:20, soak at 30℃ for 3h, after soaking, first ultrasonic extraction and then microwave heating extraction, the temperature of ultrasonic extraction is 30℃, the extraction time is 25min, and the ultrasonic power is 80W; after the ultrasonic extraction is completed, put it into a microwave instrument, the temperature of microwave heating extraction is 75℃; the microwave heating extraction time is 15min, and then filter; after the filter residue evaporates the solvent petroleum ether, add polar solvent 80% ethanol to the filter residue, the mass ratio of the filter residue to 80% ethanol is 1:20, first ultrasonic extraction and then microwave heating extraction, the temperature of ultrasonic extraction is 25℃, the extraction time is 20min, and the ultrasonic power is 90W; after the ultrasonic extraction is completed, put it into a microwave instrument, the temperature of microwave heating extraction is 70℃; the microwave heating extraction time is 12min, filter, vacuum concentrate, the vacuum concentration pressure is -0.085MPa, and the vacuum concentration temperature is 50℃, to obtain a crude extract;

[0036] S03, purification: The crude extract was purified by using macroporous adsorption resin AB-8, concentrated and dried to obtain a mixed extract of Melia azedarach and Melia azedarach bark. The crude extract was concentrated to 1 / 10 of the volume of the polar solvent to obtain a crude extract, which was filtered and loaded with the sample. The diameter-to-height ratio of the macroporous adsorption resin column was 1:8, and the column volume was 50×r 2 , r is the diameter of the macroporous adsorption resin column, and the amount of crude extract loaded is 20×r 2 After washing with water to remove impurities, 6 column volumes of elution were performed with 40% ethanol aqueous solution, the eluate was collected and concentrated under vacuum, the pressure of vacuum concentration was -0.09 MPa, and the temperature of vacuum concentration was 55°C; after vacuum concentration, freeze-vacuum drying was performed, the temperature of freeze-vacuum drying was -50°C, and the time of freeze-vacuum drying was 40h to obtain a mixed extract of neem fruit and neem bark.

[0037] The mixed extract of Melia azedarach seeds and Melia azedarach bark obtained by the preparation method of the invention.

[0038] The mixed extract of chinaberry seeds and chinaberry barks of the invention is used in preparing traditional Chinese medicine products, anti-tumor drugs, cosmetics, health products and pesticides.

[0039] The mixed extract of chinaberry seeds and chinaberry bark of the present invention is used as an antibacterial agent, and the added amount of the mixed extract of chinaberry seeds and chinaberry bark in the antibacterial agent is 0.0076-0.50 g / mL.

[0040] A method for analyzing a mixed extract of Melia azedarach seeds and Melia azedarach bark, comprising the following steps:

[0041] 1. LC-MS analysis

[0042] UPLC chromatographic conditions Column: BEH-C 18Chromatographic column (2.1 mm × 50 mm, 1.7 μm); mobile phase: 0.1% formic acid aqueous solution-acetonitrile (60:40); column temperature: 30°C; injector temperature: 15°C; flow rate: 0.3 mL·min -1 ; Injection volume: 1μL.

[0043] Mass spectrometry conditions: electrospray ionization source (ESI); data acquisition in negative ion mode; ion source temperature: 600°C; ion source voltage: -4500V or 5500V, curtain gas 137.89kPa, nebulizer gas and auxiliary gas are both 413.68kPa, multiple reaction monitoring mode (MRM) scanning, scanning range m / z 50~1500. Figure 1 Shown is the total ion chromatogram of a mixed extract of Melia azedarach seeds and Melia azedarach bark.

[0044] The collected raw data were imported into Compound Discoverer 3.1 (CD) software, and the data were filtered by retention time and mass-to-charge ratio. The target ions were then integrated. The molecular formula was then predicted by molecular ion peaks and fragment ions and compared with the mzCloud, mzVault and MassList databases to finally obtain the identification and quantitative results of the data.

[0045] 2. Thin layer identification

[0046] 2.1 Qualitative analysis of toosendanin

[0047] Weigh 3g of mixed extract powder, add 80mL of water, ultrasonically treat for 1.5h, cool, centrifuge, take the supernatant, shake and extract with dichloromethane 3 times, 30mL each time, combine the dichloromethane liquid, evaporate to dryness, add 2mL of methanol to the residue to dissolve it as the test solution. Take the Toosendanin reference substance, add methanol to make a solution containing 1mg per 1mL, as the reference solution. According to the thin layer chromatography test, take 10μL of the above solution and spot it on the same silica gel G 254 On the thin layer plate, chloroform-methanol (16:2) was used as the developing solvent, developed, taken out, dried, and examined at 365nm. Figure 2 As shown, Figure 2 In the figure, the left picture is the developed thin layer plate, and the right picture is the inspection view of the left picture at 365nm. In the left picture, the two points on the left are the toosendanin reference substance, and the two points on the back are the test solution.

[0048] 2.2 Qualitative analysis of ferulic acid

[0049] Weigh 3.02g of mixed extract powder, add 50mL of 1% sodium bicarbonate solution, ultrasonically treat for 10 minutes, centrifuge, take the supernatant and adjust the pH value to 2-3 with dilute hydrochloric acid, shake and extract twice with ether, 20mL each time, combine the ether solution, filter, evaporate, add 1mL of methanol to dissolve the residue, and set aside. Weigh 1.26mg of ferulic acid reference substance, add 1mL of methanol to dissolve, and set aside. According to the thin layer chromatography test, take appropriate amount of ferulic acid reference substance solution and test solution (half capillary tube), respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:1:0.1) as the developing agent, develop 8cm, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference substance.

[0050] The thin layer chromatogram of ferulic acid is as follows Figure 3 As shown in the figure, the two points on the left are ferulic acid reference substances, and the two points on the right are test solution.

[0051] After analysis and detection, the chemical composition of the mixed extract of Melia azedarach and Melia azedarach bark of this embodiment is shown in Table 1. There are many chromatographic peaks in the chromatogram of the mixed extract. The compounds listed in Table 1 are components that have not been reported in Melia azedarach in the literature.

[0052] Table 1 Chemical composition of mixed extracts of Melia azedarach seeds and Melia azedarach bark

[0053]

[0054]

[0055]

[0056]

[0057] Application of a mixed extract antibacterial agent of neem seeds and neem bark in the preparation of traditional Chinese medicine products, cosmetics, health products and pesticides. The antibacterial agent has good antibacterial effects on Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella typhimurium and Klebsiella pneumoniae.

[0058] (1) Preparation of bacterial suspension: The above six test bacteria were inoculated into a culture medium (3 g beef extract, 1 g peptone, 20 g agar, 5 g NaCl, 1000 mL distilled water), cultured at 30°C for 24 h, and a single colony was picked and suspended in PDA medium (Shanghai Yuanye Biotechnology Co., Ltd.) at a concentration of 10 6 ~10 8 CFU / mL bacterial suspension for later use.

[0059] (2) Determination of minimum inhibitory concentration: The minimum inhibitory concentration of the mixed extract of Melia azedarach and Melia azedarach bark against the above six pathogenic bacteria was determined by the microdilution method. First, 1.0 g / mL mother liquor (i.e., the mixed extract of Melia azedarach and Melia azedarach bark obtained by ultrasonic extraction followed by microwave heating extraction) was filtered with a 0.22 μm filter membrane, and diluted in turn with the prepared liquid culture medium to 9 concentrations (0.0038, 0.0076, 0.0152, 0.03, 0.06, 0.12, 0.25, 0.50, 1.0 g / mL), and then added to the micro MIC assay plate, 0.1 mL per well; then, the above six bacterial solutions were added, 0.1 mL per well. A positive control (adding 0.1 mL of bacterial solution and 0.1 mL of liquid culture medium) and a negative control (PBS buffer) for toosendanin were set up. At the same time, the comparison solutions were prepared as above. Comparison solution 1 was a neem fruit extract, comparison solution 2 was a neem bark extract, comparison solution 3 was a mixed extract of neem fruit and neem bark at a mass ratio of 1:1.5, comparison solution 4 was a mixed extract of neem fruit and neem bark at a mass ratio of 1:5, and comparison solution 5 was a mixed extract of neem fruit and neem bark obtained only by microwave heating extraction process. The assay plate was cultured in a 37°C incubator for 24 hours until the lowest concentration (minimum inhibitory concentration MIC) when there was no bacterial growth at all was reached. The results are shown in Table 2 below.

[0060] Table 2 Minimum inhibitory concentration MIC

[0061]

[0062]

[0063] The only difference between comparative solution 1 and embodiment 1 is that the medicinal material selected in the pretreatment step is Melia azedarach.

[0064] The only difference between comparative solution 2 and embodiment 1 is that the medicinal material selected in the pretreatment step is Melia azedarach bark.

[0065] The only difference between comparative solution 3 and embodiment 1 is that the medicinal materials selected in the pretreatment step are Melia azedarach seeds and Melia azedarach bark in a mass ratio of 1:1.5.

[0066] The only difference between comparative solution 4 and embodiment 1 is that the medicinal materials selected in the pretreatment step are Melia azedarach seeds and Melia azedarach bark in a mass ratio of 1:5.

[0067] The only difference between comparative solution 5 and embodiment 1 is that only microwave heating extraction process is adopted, the microwave heating extraction temperature is 75° C., and the microwave heating extraction time is 40 min.

[0068] Preferably, the addition amount of the antibacterial agent of the composite extract of Melia azedarach and Melia azedarach bark is 0.0076-0.50 g / mL.

[0069] Preferably, the dosage forms of Chinese medicine products, medicines, cosmetics, health products, pesticides, etc. include but are not limited to granules, tablets, suspensions, emulsions, etc.

[0070] The composite extract of Melia azedarach and Melia azedarach bark of this embodiment is used as an anti-tumor drug. The anti-tumor drug has good anti-tumor effects on human leukemia cell lines (HL-60), human liver cancer cell lines (SMMC-7721), human breast cancer cell lines (MCF-7), human esophageal cancer cell lines (TE-1), and human esophageal squamous cell carcinoma cell lines (KYSE-150), and has a good prospect for medical development.

[0071] (1) Cell culture: HL-60, SMMC-7721, MCF-7, TE-1 or KYSE-150 cells were cultured in a medium containing 10% fetal bovine serum, 100 μg / mL penicillin and streptomycin at 37.5°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for subsequent experiments.

[0072] (2) Detection of cell proliferation activity: The MTT method was used for detection. The above cells in the logarithmic growth phase were taken and the cell density was adjusted to 1×10 5 / mL, 100μL per well was inoculated in a 96-well plate, cultured overnight, and after the cells adhered to the wall, 100μL of 25, 50, 100, 200, 300, 400, 500μmol / L of culture medium of compound extracts of Melia azedarach and Melia azedarach bark was added. After 48h of culture, 20μL of 5mg / mL MTT reagent was added to each well and incubated for 2h; the supernatant was discarded, 200μL of dimethyl sulfoxide was added to each well, and the solution was fully dissolved by shaking for 10min. The absorbance OD value was detected at a wavelength of 570nm using an enzyme marker. 25μmol / L of Toosendanin was added to the positive drug group, and a blank group and a control group were set up at the same time. Each group had 6 replicate wells and continued to culture for 24h. The cell survival rate was calculated by the following formula: Cell survival rate (%) = (OD 给药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%. SPSS23.0 software was used to calculate the half inhibitory concentration (IC 50 ). At the same time, the above-mentioned comparative solutions 1 to 5 were subjected to parallel testing, and the results are shown in Table 3 below.

[0073] Table 3 Half Inhibitory Concentration

[0074]

[0075] Example 2

[0076] The difference between this embodiment and embodiment 1 is only that:

[0077] A method for preparing a mixed extract of neem seeds and neem bark comprises the following steps:

[0078] S01, pretreatment: crush and sieve the seeds of chinaberry and the bark of chinaberry in a mass ratio of 1:2, and mix them to obtain a composite powder; the seeds of chinaberry and the bark of chinaberry should be washed, dried and then crushed; the drying temperature is 50°C; the drying time is 20h; the mesh number of the sieve is 200 mesh;

[0079] S02, extraction: add non-polar solvent chloroform to the composite powder, the mass ratio of the composite powder to chloroform is 1:15, soak at 20℃ for 2h, after soaking, first ultrasonic extraction and then microwave heating extraction, the temperature of ultrasonic extraction is 25℃, the extraction time is 20min, and the ultrasonic power is 75W; after ultrasonic extraction, put it into a microwave instrument, the temperature of microwave heating extraction is 50℃; the microwave heating extraction time is 10min, and then filter; after the filter residue evaporates the solvent chloroform, add polar solvent acetone to the filter residue, the mass ratio of the filter residue to acetone is 1:15, first ultrasonic extraction and then microwave heating extraction, the temperature of ultrasonic extraction is 35℃, the extraction time is 30min, and the ultrasonic power is 100W; after ultrasonic extraction, put it into a microwave instrument, the temperature of microwave heating extraction is 95℃; the microwave heating extraction time is 10min, filter, vacuum concentrate, the air pressure of vacuum concentration is -0.09MPa, and the temperature of vacuum concentration is 40℃, to obtain a crude extract;

[0080] S03, purification: The crude extract was purified by using macroporous adsorption resin D101, concentrated and dried to obtain a mixed extract of Melia azedarach and Melia azedarach bark. The crude extract was concentrated to 1 / 10 of the volume of acetone to obtain a crude extract, which was filtered and loaded with the sample. The diameter-to-height ratio of the macroporous adsorption resin column was 1:5, and the column volume was 15×r 2 , r is the diameter of the macroporous adsorption resin column, and the amount of crude extract loaded is 10×r 2 After washing with water to remove impurities, elute with 15% ethanol aqueous solution for 5 column volumes, collect the eluate, and concentrate in vacuo. The pressure of the vacuum concentration is -0.085MPa and the temperature of the vacuum concentration is 40°C. After vacuum concentration, freeze-vacuum drying is performed at a temperature of -60°C and a time of freeze-vacuum drying is performed for 30 hours to obtain a mixed extract of neem fruit and neem bark.

[0081] The mixed extract of Melia azedarach seeds and Melia azedarach bark obtained by the preparation method of the invention.

[0082] The mixed extract of chinaberry seeds and chinaberry barks of the invention is used in preparing traditional Chinese medicine products, anti-tumor drugs, cosmetics, health products and pesticides.

[0083] The mixed extract of chinaberry seeds and chinaberry bark of the present invention is used as an antibacterial agent, and the added amount of the mixed extract of chinaberry seeds and chinaberry bark in the antibacterial agent is 0.0076-0.50 g / mL.

[0084] Example 3

[0085] The difference between this embodiment and embodiment 1 is only that:

[0086] A method for preparing a mixed extract of neem seeds and neem bark comprises the following steps:

[0087] S01, pretreatment: crush and sieve the seeds of chinaberry and the bark of chinaberry in a mass ratio of 1:4.5, and mix them to obtain a composite powder; the seeds of chinaberry and the bark of chinaberry should be washed, dried and then crushed; the drying temperature is 50°C; the drying time is 20h; the mesh number of the sieve is 200 mesh;

[0088] S02, extraction: add a mixed solution of cyclohexane and n-hexane in a volume ratio of 1:1 to the compound powder, the mass ratio of the compound powder to the mixed solution of cyclohexane and n-hexane is 1:30, soak at 40℃ for 4h, after soaking, first perform ultrasonic extraction and then microwave heating extraction, the temperature of ultrasonic extraction is 35℃, the extraction time is 30min, and the ultrasonic power is 100W; after the ultrasonic extraction is completed, put it into the microwave instrument, the temperature of microwave heating extraction is 95℃; the microwave heating extraction time is 20min, and then filter; evaporate the residue After the solvent cyclohexane and n-hexane are mixed, polar solvent methanol is added to the filter residue, the mass ratio of the filter residue to methanol is 1:30, ultrasonic extraction is first performed and then microwave heating extraction is performed, the ultrasonic extraction temperature is 30°C, the extraction time is 25min, and the ultrasonic power is 75W; after the ultrasonic extraction is completed, it is placed in a microwave instrument, and the microwave heating extraction temperature is 95°C; the microwave heating extraction time is 20min, filtered, vacuum concentrated, the vacuum concentration pressure is -0.085MPa, and the vacuum concentration temperature is 55°C to obtain a crude extract;

[0089] S03, purification: the crude extract was purified by using macroporous adsorption resin DM301, concentrated and dried to obtain a mixed extract of Melia azedarach and Melia azedarach bark. Specifically, the crude extract was concentrated to 1 / 10 of the volume of the polar solvent methanol to obtain the crude extract, which was filtered and loaded with the sample. The diameter-to-height ratio of the macroporous adsorption resin column was 1:10, and the column volume was 80×r 2 , r is the diameter of the macroporous adsorption resin column, and the amount of crude extract loaded is 30×r 2After washing with water to remove impurities, 8 column volumes of elution were performed with 68% ethanol aqueous solution, the eluate was collected, and vacuum concentrated. The pressure of vacuum concentration was -0.09 MPa and the temperature of vacuum concentration was 40°C. After vacuum concentration, freeze-vacuum drying was performed. The temperature of freeze-vacuum drying was -20°C and the time of freeze-vacuum drying was 50 hours to obtain a mixed extract of neem fruit and neem bark.

[0090] The mixed extract of Melia azedarach seeds and Melia azedarach bark obtained by the preparation method of the invention.

[0091] The mixed extract of chinaberry seeds and chinaberry barks of the invention is used in preparing traditional Chinese medicine products, anti-tumor drugs, cosmetics, health products and pesticides.

[0092] The mixed extract of chinaberry seeds and chinaberry bark of the present invention is used as an antibacterial agent, and the added amount of the mixed extract of chinaberry seeds and chinaberry bark in the antibacterial agent is 0.0076-0.50 g / mL.

[0093] Example 4

[0094] The only difference between this embodiment and embodiment 1 is that the macroporous adsorption resin is CAD-40.

[0095] Example 5

[0096] The only difference between this embodiment and embodiment 1 is that the macroporous adsorption resin is DA201.

[0097] It should be understood that in order to streamline the present disclosure and aid in understanding one or more of the various inventive aspects, in the above description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together into a single embodiment, or description thereof. However, this disclosed method should not be interpreted as reflecting the intention that the claimed invention requires more features than those expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in less than all of the features of the previously disclosed embodiments. Therefore, the claims that follow the detailed description are hereby expressly incorporated into the detailed description, with each claim itself serving as a separate embodiment of the present invention.

[0098] Although the present invention has been described according to a limited number of embodiments, it will be apparent to those skilled in the art, with the benefit of the above description, that other embodiments may be envisioned within the scope of the invention thus described. In addition, it should be noted that the language used in this specification is selected primarily for readability and teaching purposes, rather than for explaining or defining the subject matter of the present invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the present invention is illustrative, not restrictive, with respect to the scope of the present invention, which is defined by the appended claims.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a mixed extract of Melia azedarach seeds and Melia azedarach bark, characterized in that: The following steps are involved: S01, pretreatment: crushing and sieving neem seeds and neem bark in a mass ratio of 1: (2-4.5), and mixing to obtain a composite powder; S02, extraction: adding a non-polar solvent to the compound powder, soaking, extracting, and filtering; after the solvent is evaporated from the filter residue, adding a polar solvent to the filter residue, extracting, filtering, and concentrating to obtain a crude extract; S03, purification: purifying the crude extract with a macroporous adsorption resin, concentrating and drying to obtain a mixed extract of Melia azedarach seeds and Melia azedarach bark; In S01, neem seeds and neem bark should be cleaned, dried and then crushed; the drying temperature is 45-60℃; the drying time is 18-24h; the sieve mesh is 100-300 mesh; In S02, the non-polar solvent includes any one of petroleum ether, chloroform, cyclohexane or n-hexane or a combination of at least two; the mass ratio of the compound powder to the non-polar solvent is 1: (15-30); the polar solvent includes any one of ethanol, methanol, acetone or water or a combination of at least two; the mass ratio of the filter residue to the polar solvent is 1: (15-30); the soaking temperature is 20-40°C; the soaking time is 2-4h; the extraction is first ultrasonic extraction and then microwave heating extraction; the concentration is vacuum concentration, the vacuum concentration pressure is -0.085 to -0.09MPa, and the vacuum concentration temperature is 40-55°C; The temperature of ultrasonic extraction is 25~35℃, the extraction time is 20~30min, and the ultrasonic power is 75~100W; the sample is then placed in a microwave instrument, and the temperature of microwave heating extraction is 50~95℃; the microwave heating extraction time is 10~20min; The macroporous adsorption resin is AB-8, D101, CAD-40, DA201 or DM301; The crude extract is concentrated to 1 / 10 of the volume of the polar solvent, and then filtered and loaded. The diameter-to-height ratio of the macroporous adsorption resin column is 1: (5-10), and the column volume is (15-80) × r 2 , r is the diameter of the macroporous adsorption resin column, and the amount of crude extract loaded is (10~30)×r 2 After washing with water to remove impurities, elute with 15~68% ethanol aqueous solution for 5~8 column volumes, collect the eluate, and concentrate under vacuum. The pressure of vacuum concentration is -0.085~-0.09MPa and the temperature of vacuum concentration is 40~55℃; after vacuum concentration, freeze-vacuum dry at a temperature of -60~-20℃ and a time of freeze-vacuum drying for 30~50h.

2. A mixed extract of Melia azedarach seeds and Melia azedarach bark obtained according to the preparation method of claim 1.

3. Use of the mixed extract of Melia azedarach seeds and Melia azedarach bark according to claim 2 in the preparation of antibacterial Chinese medicinal products, antitumor drugs, cosmetics and pesticides.

4. The use of the mixed extract of Melia azedarach and Melia azedarach bark in the preparation of an antibacterial agent according to claim 2, characterized in that: The addition amount of the mixed extract of Melia azedarach seeds and Melia azedarach bark in the antibacterial agent is 0.0076~0.50g / mL.

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