CN113317328B - Application of campsis grandiflora extract in inhibiting growth of gram-positive bacteria - Google Patents
Application of campsis grandiflora extract in inhibiting growth of gram-positive bacteria Download PDFInfo
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Abstract
The invention discloses an antibacterial component in a campsis grandiflora extract and application of the antibacterial component in inhibiting the growth of gram-positive bacteria. The main antibacterial components in the campsis grandiflora extract comprise acteoside, apigenin-7-O-diglucuronide and pinolenic acid. The research on the in-vitro antibacterial activity of the campsis grandiflora extract shows that the campsis grandiflora extract has excellent antibacterial capability on two representative gram-positive bacteria (listeria monocytogenes and staphylococcus aureus), the campsis grandiflora extract can obviously reduce the listeria monocytogenes and staphylococcus aureus growing on the surface of celery leaves, and the campsis grandiflora extract is proved to be used for fresh-keeping storage of leafy vegetables, reduce the bacterial load in the leafy vegetables and prevent food poisoning or food-borne diseases caused by the bacteria.
Description
Technical Field
The invention belongs to the technical field of extraction and separation of traditional Chinese medicinal materials, and relates to an antibacterial component in a Chinese trumpetcreeper extract and application thereof in inhibiting the growth of gram-positive bacteria, in particular to preparation of the Chinese trumpetcreeper extract and identification of the antibacterial component of the Chinese trumpetcreeper extract, and also relates to application of the Chinese trumpetcreeper extract in inhibiting the growth of gram-positive bacteria.
Background
The food safety belongs to the problem of livelihood, and is a basic guarantee for the harmonious and stable development of the society. Pathogenic microorganisms (especially pathogenic bacteria) are one of the most important factors causing food-borne diseases. Over 200 diseases can be transmitted by food, most of which are caused by eating food or water contaminated with pathogenic bacteria. In any link of the whole process from farmland to dining table such as planting, harvesting, storing, circulating and preprocessing of vegetables and fruits, soil pathogenic bacteria (such as Listeria monocytogenes and Clostridium botulinum) and fecal pathogenic bacteria (such as Escherichia coli, Salmonella and Shigella) or animal pathogenic bacteria (such as Staphylococcus aureus and Campylobacter jejuni) can pollute the vegetables and fruits. In recent years, more and more food-borne disease epidemic situations are related to pollution of fresh fruits and vegetables, so that pathogenic bacteria pollution of the fresh fruits and vegetables is part of routine monitoring in food poisoning traceability investigation. Although chemical antistaling agents are commonly used in fruit and vegetable storage, although the chemical antistaling agents can kill pathogenic bacteria and achieve the purpose of antisepsis and freshness preservation, most chemical synthetic substances have certain influence on human health, and even have the conditions of carcinogenesis, teratogenesis, mutagenesis and the like. In addition, the long-term use of chemical fungicides in certain areas leads to the production of drug-resistant strains, thereby reducing the control effect. The result is: the dosage and the chemical residual quantity of the chemical bactericide are greatly increased, and the threat to the health of human is also increased. With the development of science and technology, people have recognized that chemical preservative preservatives have the defects of harming health, polluting environment and the like, and the research of pollution-free high-efficiency preservative products and technologies is urgently needed to replace the use of chemical bactericides in large quantity.
The Chinese has rich Chinese herbal medicine resources, and the types of medicinal plants are about 11020. Many of them have been reported to contain various antibacterial components and have strong antibacterial ability against many bacterial microorganisms. According to incomplete statistics, the Chinese herbal medicines (mainly plant medicines) belong to 78 families, about 196 kinds, wherein 131 kinds of medicines with antibacterial effect, 50 kinds of medicines with antifungal effect, 23 kinds of medicines with antiviral effect and both antibacterial effect and antifungal effect are proved by experiments. The active ingredients separated from the antibacterial Chinese herbal medicine have been proved to have 128 kinds of antibacterial effects, 27 kinds of antifungal effects and 12 kinds of antiviral effects, wherein, some of the active ingredients have antibacterial effects, antifungal effects and antiviral effects. The research result shows that the bacteriostatic or antibacterial active ingredients mainly comprise: terpenoids and derivatives thereof; nitrogen-and sulfur-containing compounds such as alkaloids, amides, and proteins; fatty compounds, mainly long-chain carbon alkanes and fatty acids; aromatic compounds include phenols, flavonoids, quinones, and the like. The botanical fungicide contains composite bacteriostatic components, has multiple action mechanisms on pathogenic bacteria, and can inhibit fungi which have large sporulation quantity, short breeding cycle and easy resistance from multiple aspects, thereby avoiding the problem of drug resistance caused by chemical fungicides. Compared with chemical bactericides, the plant source bactericide is safer, has no side effect, and can obtain cheap and rich raw materials by enlarging cultivation, so that the plant source bactericide is a potential development direction for controlling postharvest diseases of fruits and vegetables.
The Campsis grandiflora is dry Campsis grandiflora (Thub.) K.Schum.) of Bignoniaceae), and has effects of clearing heat, cooling blood, removing blood stasis, resolving hard mass, dispelling pathogenic wind and relieving itching. The campsis grandiflora is distributed in the areas from the Yangtze river basin to the North China, so that the campsis grandiflora is usually cultivated in more provinces. The trumpet creeper is listed as a Chinese product from Shen nong Ben Cao Jing, and has main chemical components of flavonoids, triterpenes and stilbene glycosides. Modern researches show that the campsis grandiflora has the effects of improving blood circulation, improving blood flow, inhibiting thrombosis, resisting free radicals and the like. It is used for treating menoxenia, vertebrobasilar artery insufficiency, cerebral hemorrhage, insomnia, acne, etc. Modern pharmacological experiments prove that the campsis grandiflora can inhibit the thrombosis of rats, the water extraction and alcohol precipitation supernatant extract can improve the microcirculation of aged rats, the methanol extract can obviously improve the reduction of the blood flow of sensitized mice, and the campsis grandiflora has the effects of promoting blood circulation and removing blood stasis. In recent years, pentacyclic triterpene component and two new iridoid are respectively separated from campsis grandiflora leaves, and both have platelet aggregation resisting effect. At present, the research is more focused on the research of the anti-coagulation active site in the campsis grandiflora, but the research on the antibacterial component in the campsis grandiflora is not available.
Disclosure of Invention
The invention aims to provide an antibacterial component in a campsis grandiflora extract and application of the antibacterial component in inhibiting the growth of gram-positive bacteria. The trumpetcreeper extract provided by the invention is brown solid powder in appearance, and can be dissolved in solvents such as water, methanol, ethanol and the like.
The purpose of the invention is realized by the following technical scheme:
the invention relates to an application of a campsis grandiflora extract in inhibiting the growth of gram-positive bacteria.
The invention also relates to the application of the campsis grandiflora extract as an antibacterial agent. The campsis grandiflora extract can be used as a new source of an antibacterial agent and is used for preventing food poisoning and food-borne diseases caused by food-borne pathogenic bacteria. Specifically, the compound can be used as an antibacterial agent for leafy vegetables; can be used for fresh-keeping storage of leafy vegetables, reducing bacterial load in leafy vegetables, and preventing food poisoning or food-borne diseases caused by these bacteria.
As an embodiment of the invention, the bacteriostatic agent is for use in inhibiting the growth of gram-positive bacteria.
As an embodiment of the present invention, the gram-positive bacteria include at least one of listeria monocytogenes, staphylococcus aureus.
As one embodiment of the present invention, the antibacterial active substance in the extract of campsis grandiflora includes acteoside, apigenin-7-O-diglucuronide and pinolenic acid.
As one embodiment of the present invention, the campsis grandiflora extract is prepared by a method comprising the following steps: weighing and grinding dried campsis grandiflora, extracting with organic solvent combined with ultrasound to obtain an extraction solution, separating and enriching to obtain a campsis grandiflora extract, and drying with a vacuum freeze dryer to obtain campsis grandiflora extract powder.
As an embodiment of the invention, the organic vehicle is 80% (v/v) ethanol; the weight ratio of the trumpetcreeper flower to the organic solvent is 1: 10-1: 20. Preferably, the weight ratio of the campsis grandiflora to the organic solvent is 1: 20.
As an embodiment of the invention, the microwave-assisted extraction method has parameters of 40-60 deg.C and 480W for 1-2 hr. Preferably, the microwave-assisted extraction method has parameters of 40 deg.C and 480W for 1 hr.
As an embodiment of the present invention, the separation method is 900-1000 Xg centrifugation for 10-20 minutes, and the supernatant is collected. Preferably 900Xg for 15 minutes and collecting the supernatant.
As an embodiment of the present invention, the enrichment method is to evaporate and concentrate the supernatant liquid by using a rotary evaporator, and the concentration ratio is 5:1-4: 1. Preferably, the concentration ratio is 4: 1.
As an embodiment of the present invention, the conditions of vacuum freeze-drying are: 4 hours at-50 ℃; 10 hours at-35 ℃; 10 hours at-10 ℃; at 25 ℃ for 24 hours.
By combining all the technical schemes, the invention has the following beneficial effects:
1) according to the invention, when the antibacterial activity of the Chinese herbal medicine extract is researched to obtain an antibacterial agent with better antibacterial capability and antibacterial range, the Chinese herbal medicine campsis grandiflora is found to have obvious antibacterial activity on two representative gram-positive bacteria (staphylococcus aureus and listeria monocytogenes);
2) according to the invention, the extraction efficiency and yield (yield is 19%) of the antibacterial compound in the campsis grandiflora are improved by using an ultrasonic-assisted extraction method;
3) the main antibacterial components of the campsis grandiflora extract, namely acteoside, apigenin-7-O-diglucuronide and pinolenic acid, are identified by HPLC-DAD and UPLC-QTOF-MS, and the composition of the substance for the campsis grandiflora extract to exert the antibacterial activity is determined.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a time-sterilization curve of extracts of Campsis grandiflora at different concentrations against Staphylococcus aureus and Listeria monocytogenes; wherein, a is a time-sterilization curve for staphylococcus aureus, and b is a time-sterilization curve for listeria monocytogenes;
FIG. 2 is a chromatogram of HPLC-DAD detecting the main chemical components in the extract of Campsis grandiflora; wherein A is a chromatogram of an ergot steroid glycoside standard substance, and B is a chromatogram of a campsis grandiflora extract;
FIG. 3 is a UPLC-MS chromatogram of a trumpet creeper extract; wherein A is a chromatogram in a positive ion mode, and B is a chromatogram in a negative ion mode.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that numerous modifications and adaptations can be made by those skilled in the art without departing from the inventive concepts herein. All falling within the scope of the present invention.
Specifically, the campsis grandiflora extract provided by the invention is prepared by the following method:
step one, grinding the dried campsis grandiflora by a grinder, and sieving by a 20-40 mesh sieve to obtain the campsis grandiflora powder.
Step two, adding 80% (v/v) ethanol solution into the trumpet creeper pollen powder obtained in the step one, extracting antibacterial ingredients in the trumpet creeper by combining an ultrasonic-assisted extraction method, centrifuging to obtain supernatant, namely trumpet creeper extract solution, and repeating the operation twice to improve the yield of the trumpet creeper extract; the adding ratio of the campsis extract powder to the ethanol is 1: 10-1: 20; extracting at 480W for 1-2 hr at 40-60 deg.C with ultrasonic extraction parameters; the obtained campsis grandiflora extract solution is centrifuged for 10-15 minutes at the temperature of 900-.
And step three, evaporating and concentrating the campsis grandiflora extract solution obtained in the step two to a proper volume (the evaporation and concentration ratio is 1/5-1/4 of the original volume) by using a rotary evaporator, and then placing the solution in a refrigerator at the temperature of-80 ℃ for freezing overnight.
Step four, drying the condensed extract of the Chinese trumpetcreeper after being frozen in the step three by a vacuum freeze dryer to prepare the powder of the Chinese trumpetcreeper extract; the conditions of the campsis grandiflora extract concentrated solution vacuum freeze drying are as follows: 4 hours at-50 ℃; 10 hours at-35 ℃; 10 hours at-10 ℃; at 25 ℃ for 24 hours.
And step five, dissolving the campsis grandiflora extract powder obtained in the step four in methanol (the concentration of the methanol solution of the campsis grandiflora extract is 20mg/mL), and detecting main antibacterial components of the campsis grandiflora extract by high performance liquid chromatography-diode array detection (HPLC-DAD) and ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS).
And step six, dissolving the trumpetcreeper extract powder obtained in the step four in DMSO to prepare trumpetcreeper extract solutions with different concentrations (25mg/mL, 50mg/mL, 100mg/mL and 200mg/mL respectively) for detecting the antibacterial activity of the trumpetcreeper extract on Listeria monocytogenes and staphylococcus aureus. The method for evaluating the antibacterial activity of the campsis grandiflora extract comprises the measurement of the diameter DIZ of a bacteriostatic circle, the minimum bacteriostatic concentration MIC and the minimum bactericidal concentration MBC detection and a time sterilization experiment.
Specific examples are as follows:
example 1 preparation of extract of Campsis grandiflora
Grinding dried campsis grandiflora into powder, and sieving with 20-40 mesh sieve. Adding 2 liters of 80% ethanol into 100 grams of trumpet creeper pollen, and performing ultrasonic extraction for 1 hour at 40 ℃ and 480W by an auxiliary ultrasonic extraction method. After centrifugation at 900 deg.C 900X g for 15 minutes, the supernatant was removed and the residue was again extracted with 2 liters of 80% ethanol for 1 hour. Evaporating the supernatant in a rotary evaporator at 40 deg.C to 1/5-1/4, freezing the concentrated extract at-80 deg.C overnight, and drying in a vacuum freeze-drying machine to obtain flos Campsis extract powder. The yield of the campsis grandiflora extract powder is 19%.
Example 2 antibacterial Activity of extracts of Campsis grandiflora
The extract powder of trumpetcreeper was dissolved in dimethyl sulfoxide (DMSO) at various concentrations (25mg/mL, 50mg/mL, 100mg/mL and 200mg/mL) for antimicrobial activity studies. Two representative gram-positive bacteria are selected: staphylococcus aureus (s. aureus ATCC 25923) and listeria monocytogenes (l. monocytogenes ATCC 7644). Individual colonies grown on Tryptone Soy Broth (TSB) agar plates were first inoculated into TSB medium and cultured overnight at 37 ℃ and 250rpm with shaking. Adjusting the bacterial suspension to 1X106The concentration of CFU/mL was used in the following antibacterial experiments.
1. Determination of diameter DIZ of inhibition zone
In order to test the bacteriostatic action of the campsis grandiflora extract on gram-positive bacteria, the invention arranges four campsis grandiflora extracts with different concentrationsThis was filtered through a 0.22 μm filter (25mg/mL, 50mg/mL, 100mg/mL and 200mg/mL) and stored at 4 ℃ until use. All bacteria were diluted to approximately 1X10 with sterile LB medium6CFU/mL, then 100. mu.L of each bacterial suspension was uniformly dispersed on the surface of LB agar plate by sterile glass beads (diameter 6 mm). Placing an Oxford cup (the inner diameter is 6mm, the outer diameter is 7.8mm, and the height is 10mm) on the surface of the agar, then dropwise adding 60 microliters of Chinese trumpetcreeper extracts (25mg/mL, 50mg/mL, 100mg/mL and 200mg/mL) with different concentrations into the Oxford cup, culturing at 37 ℃ for 16-18 hours, and measuring the diameter of the inhibition zone. The experiment was repeated twice in triplicate with three replicates each time using ampicillin (10. mu.L/ml) as a positive control and DMSO (60. mu.L/cup) as a negative control.
2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays
mu.L of MH medium was added to a 96-well plate, and then 100mg/mL of the trumpet creeper extract was twice serially diluted to a final concentration ranging from 0.39mg/mL to 50 mg/mL. Sequentially adding 20 μ L of flos Campsis extract into each well. Then 100. mu.L of a standard bacterial suspension (1X 10)6CFU/mL) was added to each test well to give a final volume of 220 μ L in each well. The well plates were incubated at 37 ℃ for 24 hours. After incubation, 30 μ L of freshly prepared resazurin (0.015%) was added to all test wells and incubated for a further 2 hours so that surviving microorganisms metabolize the blue resazurin dye to pink resazurin. Positive control (bacterial solution + MH broth) and negative control (MH broth only) were also included. MIC was defined as the concentration of extract of campsis grandiflora corresponding to test wells showing no color change. Solutions in wells at concentrations equal to or higher than the MIC value were incubated directly on MH plates, and the lowest concentration at which no colonies grew was defined as MBC. Ampicillin and oxacillin were used as positive controls and the experiment was repeated three times in triplicate.
TABLE 1 bacteriostatic activity of extract of campsis grandiflora on two gram-positive bacteria
Table 1 the results show that: the campsis grandiflora extract shows obvious antibacterial activity to gram-positive bacteria (Listeria monocytogenes and staphylococcus aureus), and MIC values of the campsis grandiflora extract to two gram-positive bacteria are both 5 mg/mL.
Example 3 time-Sterilization test
The two gram-positive bacteria were treated with different concentrations of the extract of campsis grandiflora (1/2 × MIC, MIC × 2 × MIC and 4 × MIC). A time-kill curve was constructed by determining the relationship between lg CFU/mL and time. Fresh celery leaves (10g) were washed three times with distilled water and then dried in a biosafety cabinet for 2 hours to remove water. After drying, celery leaves were exposed to uv light for 30 minutes to eliminate the primary microflora. By containing 107After soaking the celery liquid in the CFU/mL bacterial suspension for 2 minutes, drying the celery liquid for 30 minutes to allow the bacteria to attach. The initial load of bacteria was recorded after drying. The celery leaves attached with the bacteria are respectively immersed in 200mL of sterile distilled water and the campsis grandiflora extract solution with different concentrations for 15 minutes. After the end, the leaf of Brassica oleracea was homogenized in sterile water, and 100. mu.L of the homogenized solution was cultured in TSA medium for 24 hours. The number of bacteria was measured for 3, 6, 9, 12 and 24 hours and the experiment was repeated three times, two in parallel each time.
Table 220 mg/mL campsis extract on celery leaves for 24 hours, the surface bacterial load changes
The results of fig. 1 and table 2 show that: the campsis grandiflora extract treated celery leaves showed a significant reduction in the number of live bacteria attached within 24 hours compared to the control. After the celery leaves are treated by the 2% campsis extract for 24 hours, the number of staphylococcus aureus on the surfaces of the celery leaves is reduced to 4.74 +/-0.40 lg CFU/mL, and the number of listeria monocytogenes is reduced to 4.29 +/-0.10 lg CFU/mL. The campsis grandiflora extract is proved to be capable of reducing bacterial load on celery leaves and reducing the existence of food-borne pathogenic bacteria in fresh leafy vegetables.
Example 4: identification of antibacterial components in campsis grandiflora extract
HPLC-DAD (high Performance liquid chromatography-diode array detection)
The extract of Campsis grandiflora was dissolved in methanol (20mg/mL) before HPLC analysis. The analysis was performed using a Shimadzu LC-20AR HPLC system equipped with a diode array detector. The separation was carried out at 35 ℃ using a Shim-Pack GIS-C18 reverse phase column (4.6X 250 mm, 5 μm). The mobile phase was 0.1% (v/v) formic acid (A) and 100% acetoformic acid (B). Gradient condition is 0-70 minutes, 10-80% B; 80-100% B for 70-71 minutes; 71-74 minutes, 100% B; 74-76 minutes, 100-20% of B, 76-86 minutes and 20% of B. The flow rate was 0.6mL/min and the amount of sample was 20. mu.L. The peak of the HPLC was identified by comparing the retention time with the uv spectrum.
UPLC-QTOF-MS (ultra high Performance liquid chromatography-quadrupole mass Spectrometry in flight)
The samples were separated on a BEH C18 column (2.1 mm. times.100 mm, 1.7 μm) set as follows: column temperature: 45 ℃; flow rate: 0.4 mL/min. The mobile phase consisted of 0.1% (v/v) formic acid (A) and an acetoacetate (B) (containing 0.1% (v/v) formic acid). Gradient elution was performed as follows: 0 min, 5% B; 3 minutes, 20% B; 10 min, 100% B. The loading was 1. mu.L. Mass analysis was performed using a Vion IMS QTOF mass spectrometer equipped with an electrospray ion source (ESI). The parameters of the mass spectrometry were as follows: capillary voltage, 2kV (positive ion mode), 2kV (negative ion mode); cone voltage, 40V; the flow rate of dissolved gas is 900L/h; the flow rate of the conical gas is 50L/h; the dissolved gas temperature, 450 ℃; source temperature, 115 ℃; the collision energy is 6 eV/20-45 eV; the collection range is 50-1000 m/z.
Table 3 UPLC-QToF-MS identification of major phytochemicals in Campsis extract
TABLE 4 bacteriostatic activity of 3 main components of the extract of campsis grandiflora
The results in table 3 and fig. 2 and 3 show that: according to HPLC-DAD and UPLC-QTOF-MS result analysis, the main compounds in the campsis grandiflora extract are ergosterin, apigenin-7-O-diglucuronide and pinolenic acid. Table 4 shows. Acteoside is a main antibacterial substance in the campsis grandiflora extract, and plays a role in inhibiting the growth of staphylococcus aureus and listeria monocytogenes.
Comparative example 1
The campsis grandiflora extract is prepared by the extraction method in CN201010576491 in the present comparative example, namely, 1 liter of ethanol with the weight concentration of 70% is added into 100 g of campsis grandiflora pollen, the campsis grandiflora pollen powder is soaked for 30 minutes, extracted for 1 hour, and filtered to obtain supernatant; adding 1 liter of ethanol with the weight concentration of 70 percent into the precipitate, extracting for 1 hour, and filtering to obtain supernatant; mixing the two supernatants, placing in an evaporation dish, concentrating in a water bath, and vacuum drying to obtain flos Campsis extract powder with yield of 10%. The antibacterial activity of the campsis extract (A extract) obtained by the method is compared with that of the campsis extract (B extract) in the invention, and the result is shown in Table 5, the antibacterial activity of the campsis extract in the invention on staphylococcus aureus is obviously superior to that of the campsis extract obtained by the method in the comparative example, and the MICs of the two extracts on staphylococcus aureus are respectively 5mg/mL and 10 mg/mL; MICs for Listeria monocytogenes were all 5 mg/mL. The campsis grandiflora extract obtained by the invention can effectively inhibit the growth of staphylococcus aureus and listeria monocytogenes. Therefore, the method obviously improves the yield of the campsis grandiflora extract, and the campsis grandiflora extract obtained by the method has stronger antibacterial activity.
TABLE 5 antibacterial Activity of the extracts of Campsis grandiflora obtained by the two methods
In conclusion, the campsis grandiflora extract has an obvious inhibition effect on gram-positive bacteria through an in vitro bacteriostasis test, and the main antibacterial component in the campsis grandiflora extract is identified by combining HPLC-DAD and UPLC-QTOF-MS, so that a foundation is laid for determining the antibacterial mechanism of the campsis grandiflora extract. The campsis grandiflora is a new source for developing the antibacterial agent, can be used for preventing food poisoning and food-borne diseases caused by food-borne pathogenic bacteria, and has great popularization value.
The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (6)
1. Use of a flos Campsis extract in preparing medicine for inhibiting the growth of gram-positive bacteria; the gram-positive bacteria comprise at least one of listeria monocytogenes and staphylococcus aureus; the antibacterial active substance in the campsis grandiflora extract comprises acteoside, apigenin-7-O-diglucuronide and pinolenic acid;
the campsis grandiflora extract is prepared by a method comprising the following steps: weighing and grinding dried campsis grandiflora, extracting with organic solvent and microwave to obtain extractive solution, separating and enriching to obtain campsis grandiflora extract, and drying with vacuum freeze dryer to obtain campsis grandiflora extract powder; the organic solvent is ethanol with the volume concentration of 80%.
2. The use as claimed in claim 1, wherein the weight ratio of the trumpetcreeper flower to the organic solvent is 1: 10-1:20.
3. Use according to claim 1, wherein the microwave-assisted extraction method has parameters of 40-60 ℃ and 480W for 1-2 hours.
4. The use as claimed in claim 1, wherein the separation process is 900-1000-gCentrifuging for 10-20 min, and collecting supernatant.
5. The use according to claim 1, wherein the enrichment method is to evaporate and concentrate the supernatant using a rotary evaporator, the concentration ratio being 5:1-4: 1.
6. Use according to claim 1, characterized in that the conditions of vacuum freeze-drying are: 4 hours at-50 ℃; 10 hours at-35 ℃; 10 hours at-10 ℃; at 25 ℃ for 24 hours.
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| CA2451144A1 (en) * | 2001-06-21 | 2003-01-03 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing a plant extract containing plant powder |
| MX369176B (en) * | 2012-06-04 | 2019-10-23 | Univ Autonoma Del Estado De Hidalgo Star | Phytochemical compositions that can be used as disinfectants and preservatives for food. |
| CN104922169A (en) * | 2014-03-17 | 2015-09-23 | 孟玉蓉 | Preparation method of flos campsis extract |
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| CN106614798A (en) * | 2016-12-22 | 2017-05-10 | 重庆医学检验试剂研究所 | Composition, application in microorganism removal thereof and disinfecting fluid |
| CN107213074A (en) * | 2017-07-18 | 2017-09-29 | 佛山实瑞先导材料研究院（普通合伙） | A kind of antiinflammatory and its application |
| CN107788024A (en) * | 2017-10-31 | 2018-03-13 | 沿河安发刺梨生态开发有限公司 | One kind preventing and treating Rosa roxburghii pest and disease damage medicament and preparation method thereof |
| CN107880084B (en) * | 2017-11-02 | 2021-07-09 | 中国人民解放军陆军第七十一集团军医院 | Method for extracting high-purity verbascoside from campsis plant flowers |
| US20210260145A1 (en) * | 2018-07-18 | 2021-08-26 | Jeonnam Bioindustry Foundation | Pharmaceutical composition for alleviating eye fatigue, containing, as active ingredients, luteolin-7-o-diglucuronide and apigenin-7-o-diglucuronide isolated from perilla frutescens (l.) britton var. acuta (thunb.) kudo leaf extract |
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