CN111012817A - Preparation method and application of dogwood seed extract - Google Patents
Preparation method and application of dogwood seed extract Download PDFInfo
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- CN111012817A CN111012817A CN201911235807.2A CN201911235807A CN111012817A CN 111012817 A CN111012817 A CN 111012817A CN 201911235807 A CN201911235807 A CN 201911235807A CN 111012817 A CN111012817 A CN 111012817A
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- A61K2236/10—Preparation or pretreatment of starting material
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses a preparation method of a dogwood seed extract, which comprises the following steps: a: pulverizing dry Corni fructus seed into superfine Corni fructus seed powder; b: adding absolute ethyl alcohol into the cornus officinalis seed ultrafine powder, and soaking for 24 hours at the temperature of 50 ℃; c: and (3) placing the soak solution in a rotary evaporator, distilling under reduced pressure, and drying to obtain a crude extract of the dogwood seeds. The dogwood seed crude extract has good bacteriostatic effect on escherichia coli and salmonella, and has no obvious cytotoxicity on rabbit red blood cells. The preparation method of the dogwood seed extract provided by the invention only needs to extract with ethanol in one step, has short extraction time and high extraction efficiency, obtains more dogwood seeds with more effective components, and is less influenced by experimental conditions.
Description
Technical Field
The invention relates to the technical field of plant extraction, and particularly relates to a preparation method and application of a dogwood seed extract.
Background
The dogwood, also called as the medicinal jujube or hollyhock jujube, has the efficacies of enhancing the immunity of the organism, resisting cancer, promoting urination, reducing blood pressure, resisting oxidation and the like. The research finds that the dogwood pulp contains the effective components of dogwood polysaccharide, organic acid, tannin and the like, wherein the dogwood polysaccharide especially has the effects of regulating immunity, resisting aging and the like. Dogwood pulp is also widely used in the food industry. Dogwood seeds are often discarded as a by-product of processing.
Research shows that the extract of dogwood seeds has the effects of relaxing arteries and reducing blood pressure, for example, Chinese patent publication No. 102688275A discloses a dogwood kernel extract and application thereof in preparing a blood pressure reducing composition. Therefore, personnel operating petroleum ether must be specially trained, the operating environment is closed, and the overall ventilation is realized. These strict operating conditions do not make use of the widespread application of this separation technique; the Chinese invention patent (publication No. 103110685A) discloses a dogwood kernel water extract and application of the water extract in preparing a chest-dilating aortic ring composition and a blood pressure reducing composition.
Disclosure of Invention
The invention develops a high-efficiency separation, extraction and purification method of dogwood seeds aiming at recycling waste generated in the existing development and utilization process of dogwood, and the obtained product has good antibacterial activity. The invention widens the utilization range of the dogwoods and prolongs the industrial chain.
A preparation method of a dogwood seed extract comprises the following steps:
a: pulverizing dry Corni fructus seed into superfine Corni fructus seed powder;
b: adding absolute ethyl alcohol into the cornus officinalis seed ultrafine powder, and soaking for 24 hours at the temperature of 50 ℃;
c: and c, placing the soak solution obtained in the step b in a rotary evaporator, distilling under reduced pressure at 50 ℃, and carrying out vacuum freeze drying at low temperature to obtain a dogwood seed crude extract.
Further, the cornus officinalis seed ultrafine powder is 80 meshes.
Further, the feed-liquid ratio of the cornus officinalis seed ultrafine powder to the absolute ethyl alcohol is 1: 20.
Further, the condition of the reduced pressure distillation was 0.09 kPa.
The extracted dogwood seed crude extract has good antibacterial effect on escherichia coli and salmonella, and has no obvious cytotoxicity on rabbit red blood cells. Therefore, the extracted dogwood seed crude extract can be applied to the preparation of the anti-gram-negative bacteria medicament.
The invention has the beneficial effects that:
(1) the method firstly crushes the dogwood seeds, and can effectively shorten the extraction time of absolute ethyl alcohol after crushing;
(2) the invention effectively ensures that the structure of the active substances of the dogwood seeds is not damaged by one-step ethanol extraction of the dogwood seeds, so that a more efficient crude extract of the dogwood seeds can be obtained;
(3) the dogwood seeds are wastes generated in dogwood processing and utilization, and the method is based on industrial wastes, so that the utilization value of the dogwood is further improved, and the industrial chain is prolonged.
(4) The extract obtained by taking waste dogwood seeds as the raw material is basically consistent with the extract obtained by taking dogwood pulp as the raw material which is commercialized at present.
Drawings
FIG. 1 shows the hemolysis rate of erythrocytes of crude extract of Corni fructus seed;
FIG. 2 shows the morphological changes of E.coli before and after the crude extract of Corni fructus seed is treated; wherein A and B represent normal Escherichia coli and Salmonella, respectively, and a and B represent Escherichia coli and Salmonella acted on Corni fructus seed extract, respectively.
Detailed Description
The following examples are intended to better illustrate the technical solutions of the present invention, but not to limit the scope of the present invention.
Example 1 preparation of extract of dogwood seeds
A preparation method of a dogwood seed extract comprises the following steps:
a: pulverizing dry Corni fructus seed into 80 mesh ultrafine powder, wherein the water content of the dry Corni fructus seed is less than 13%;
b: adding absolute ethyl alcohol into the cornus officinalis seed ultrafine powder, wherein the material-liquid ratio is 1: 20, and soaking for 24 hours at 50 ℃;
c: and c, putting the soak solution obtained in the step b into a rotary evaporator, distilling under reduced pressure under the condition of 0.09kPa, and carrying out vacuum freeze drying at low temperature to obtain a dogwood seed crude extract.
Example 2 determination of minimum inhibitory concentration of crude extract of Cornus officinalis seeds against Standard Strain
The test strains were grown overnight in TSB liquid medium (purchased from Solarbio, Beijing Soilebao technologies, Ltd.) and at OD600Measuring the OD value of the cultured bacterial liquid, when the OD value of the bacterial liquid reaches 0.6-0.8, centrifuging the cultured bacterial liquid for 10min at 6000r/min, discarding supernatant after centrifugation, collecting thalli sediment, and then re-suspending bacteria by using 0.01MPBS with the same volume; then using MH culture medium to dilute the bacterial liquid to be detected to 2 x 106CFU/ml. Diluting the dogwood seed extract solution with deionized water to the concentration of 10-320 mu g/mL. Adding 50 of the solution into each hole of the cell culture plateMu L of dogwood seed crude extract diluent and 50 mu L of diluted bacterium liquid with different concentrations are subjected to experiment for 3 holes at each concentration, the dogwood seed crude extract is cultured for 16-20 hours at 37 ℃, and the concentration at which bacteria in the holes can not grow completely (the bottom of the holes can not be turbid by naked eyes) is the minimum inhibitory concentration of the dogwood seed crude extract, and the results are detailed in Table 1.
TABLE 1 bacterial growth at different concentrations
Note: "-" indicates no bacterial growth; "+" indicates that there is bacterial growth
As can be seen from table 1: the minimum inhibitory concentrations of the dogwood seed crude extract on staphylococcus aureus, escherichia coli and salmonella are respectively 160 mug/mL, 80 mug/mL and 40 mug/mL.
EXAMPLE 3 detection of hemolytic Activity of crude extract of Cornus officinalis seeds
Taking 50mL of fresh rabbit blood, centrifuging at 3000rpm for 20min, discarding the supernatant, washing erythrocytes with isotonic PBS buffer solution, repeatedly centrifuging for three times until the supernatant is colorless and transparent, and preparing into rabbit erythrocyte suspension; taking 5 centrifuge tubes of 1.5mL, and numbering 1-5; the crude extract of dogwood seeds of the present invention was added in amounts of 2mg, 1mg and 0.5mg in tubes 1 to 3, and 800. mu.L of 0.01M PBS (pH 7.2) buffer was added in each tube. No. 4 tube added with 800 μ L PBS buffer solution as negative control group; tube 5 was filled with 800. mu.L of 1% Triton X-100 deionized water diluent (which completely hemolyzes erythrocytes) to prepare a positive control. 1-5 tubes each were added with 200. mu.L of erythrocyte suspension. Standing for 3h, centrifuging 1-5 tubes at 1500r/min for 10min, and respectively taking 150 μ L of supernatant to a microplate for absorbance determination; wherein, the positive sample is scanned at full wavelength to determine the maximum absorption wavelength of bovine hemoglobin. The absorbance of each sample was measured at the maximum absorption wavelength, and the degree of hemolysis was calculated by the formula HD ═ a-sample-a anion)/(a-positive-a anion) × 100%.
The results show that: hemoglobin showed a distinct absorption peak at a wavelength of 414 nm. The red blood cells used were counted by red blood cell counting method, and the result showed the concentration of red blood cellsDegree of 4.8X 1013As shown in figure 1, the hemolysis rate of the red blood cells of the crude extract of dogwood seeds is equivalent to that of the negative control under the condition that the concentration of the dogwood seed extract is 1mg/mL, which indicates that the osmotic fragility of the crude extract of dogwood seeds on the red blood cells can be ignored.
Example 3 morphological Observation of crude extract of Cornus officinalis seeds after action on Escherichia coli or Salmonella
Preparing escherichia coli and salmonella suspensions: according to the fact that the minimum inhibitory concentration of the dogwood seed crude extract to escherichia coli and salmonella is 80 mug/mL and 40 mug/mL respectively, a corresponding dogwood seed crude extract solution with the concentration of 80 mug/mL and 40 mug/mL is prepared from the dogwood seed crude extract by adopting PBS (phosphate buffer solution) with pH7.2 and 10mM, namely, a corresponding dogwood seed crude extract solution with the concentration of 0.75mL is prepared from the dogwood seed crude extract by adopting PBS (phosphate buffer solution) with the pH of 7.2 and 10mM aiming at escherichia coli and salmonella thalli; then respectively sucking 2mL of escherichia coli and salmonella which are freshly cultured to logarithmic phase, centrifuging the thalli for 15min under the condition of 6000r/min, resuspending the thalli obtained after centrifugation by using a corresponding equal volume of a dogwood seed crude extract solution of 80 mug/mL and 40 mug/mL, and incubating the suspended thalli for 30min at 37 ℃ to respectively obtain escherichia coli and salmonella suspensions acted by the dogwood seed crude extract;
bacteria (Escherichia coli and salmonella) which grow normally and are not added with the dogwood seed extract are taken as normal control; taking 3mL of bacterial suspension of normally cultured bacteria in logarithmic growth phase, namely escherichia coli, salmonella and dogwood seed crude extract after the action, firstly centrifuging for 5min under the condition of 3000r/min, and removing supernatant; then respectively adopting PBS buffer solutions with pH7.2 and 10mM to wash the thalli for three times, finally respectively adopting 3mL and 5% glutaraldehyde solutions to resuspend, and respectively fixing the thalli obtained after suspension for 24 hours at the temperature of 4 ℃; then centrifuging for 10min under the condition of 3000r/min, collecting thallus precipitates, then performing gradient dehydration (the dehydration time is 20min each time) by adopting ethanol diluted by 30-100% PBS solution, dehydrating twice (the dehydration time is 20min each time) by using absolute ethanol, after the last dehydration is completed, dropwise adding the obtained bacteria liquid on a circular cover glass respectively, then performing vacuum freeze drying, sputtering gold by an ion sputtering instrument after drying, and finally observing the surface structure change of a sample under a scanning electron microscope, wherein the detailed view is shown in figure 2.
As can be seen from fig. 2: compared with the normal escherichia coli morphological structure, the bacteria treated by the dogwood seed crude extract are greatly changed: after the crude extract of the dogwood seeds acts on bacterial cells, the rupture of bacterial cell walls can be caused, the integrity of a bacterial outer membrane structure is further damaged, the loss of contents of bacteria is finally caused, and the bactericidal activity is exerted.
In conclusion, the dogwood seed extract product does not produce hemolytic property, has a wide antibacterial spectrum, and has a high-efficiency antibacterial effect on gram-negative bacteria. Therefore, the dogwood seed crude extract can be preferably applied to the preparation of the medicine for resisting gram-negative bacteria diseases.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. The preparation method of the dogwood seed extract is characterized by comprising the following steps:
a: pulverizing dry Corni fructus seed into superfine Corni fructus seed powder;
b: adding absolute ethyl alcohol into the cornus officinalis seed ultrafine powder, and soaking for 24 hours at the temperature of 50 ℃;
c: and c, placing the soak solution obtained in the step b in a rotary evaporator, distilling under reduced pressure at 50 ℃, and carrying out vacuum freeze drying at low temperature to obtain a dogwood seed crude extract.
2. The method as claimed in claim 1, wherein the ultrafine powder of cornus officinalis seeds is 80 mesh.
3. The method as claimed in claim 1, wherein the ratio of the fine powder of cornus officinalis seeds to the absolute ethyl alcohol is 1: 20.
4. The method of claim 1, wherein the distillation under reduced pressure is carried out under 0.09 kPa.
5. Use of the crude extract of dogwood seeds prepared in claim 1 against gram negative bacteria.
6. Use according to claim 5, wherein the gram-negative bacteria are Escherichia coli and/or Salmonella.
7. The use of the crude extract of dogwood seeds prepared in claim 1 for the preparation of a medicament against gram-negative bacteria.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112608395A (en) * | 2021-01-05 | 2021-04-06 | 海南大学 | Separation and purification method of dogwood seed polysaccharide |
| CN113545427A (en) * | 2021-07-24 | 2021-10-26 | 江苏海洋大学 | Preparation method and application of fish bait for resisting marine pathogenic vibrios |
| CN114557367A (en) * | 2022-02-25 | 2022-05-31 | 南京中医药大学 | Cornus officinalis kernel wood vinegar and application thereof in preparation of antibacterial lotion or disinfectant |
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Cited By (4)
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| CN113545427A (en) * | 2021-07-24 | 2021-10-26 | 江苏海洋大学 | Preparation method and application of fish bait for resisting marine pathogenic vibrios |
| CN114557367A (en) * | 2022-02-25 | 2022-05-31 | 南京中医药大学 | Cornus officinalis kernel wood vinegar and application thereof in preparation of antibacterial lotion or disinfectant |
| CN114557367B (en) * | 2022-02-25 | 2023-09-12 | 南京中医药大学 | Cornus officinalis kernel wood vinegar and application thereof in preparation of antibacterial lotion or disinfectant |
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