CN111450100A - Camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus as well as preparation method and application of camellia nitidissima caffeine and chlorogenic acid composition - Google Patents
Camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus as well as preparation method and application of camellia nitidissima caffeine and chlorogenic acid composition Download PDFInfo
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Abstract
The invention provides a camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus, which comprises the following raw materials in parts by weight: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry. The invention also provides a camellia chrysantha caffeine and chlorogenic acid composition for resisting the novel coronavirus, and a preparation method and application thereof. Compared with the prior art, the invention discovers that the caffeine and the chlorogenic acid have good inhibition effect on the novel coronavirus, and the virus inhibition rate is obviously improved under the synergistic effect of the caffeine and the chlorogenic acid, so that the invention initiatively provides a composition which is prepared by compounding the caffeine and the chlorogenic acid as main antiviral active ingredients and compounding auxiliary materials such as mogroside, soluble starch, water-soluble dextrin, citric acid, 10% starch slurry and the like, the composition can be orally taken, and a new formula and a new thought are provided for preventing the novel coronavirus.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus, and a preparation method and application thereof.
Background
Camellia nitidissima C.W.Chi belongs to Theaceae and Camellia, and according to the inspection of Chinese disease prevention and control center nutrition and food safety, the Camellia nitidissima is nontoxic, contains various nutritional and medicinal active ingredients such as tea polyphenols, flavonoids, amino acids, caffeine, chlorogenic acid and the like, and has very high medicinal value. Particularly, the golden camellia contains abundant Chlorogenic acid (Chlorogenic acid) and Caffeine (Caffeine), and the Chlorogenic acid is an important bioactive substance and has the effects of resisting bacteria and viruses, increasing white blood cells, reducing blood pressure and blood fat, scavenging free radicals and the like; caffeine is an alkaloid with high content in tea, has the effects of removing fatigue and exciting nerves when being used properly, and is commonly used for treating neurasthenia and coma resuscitation clinically.
The study on anti-virus effect of Lilising, etc. (Lilising, Wang Yan, etc.. Senecio cannabifolius extract and its effective component J. Chinese medicine basic medicine journal, 2004, 011(8):585-587) shows that both chlorogenic acid and caffeine have strong inhibitory effect on influenza virus (RMI type) and on Newcastle disease virus (NDV type).
However, at present, there is no report that camellia chrysantha chlorogenic acid and caffeine are applied to prevention and treatment of novel coronavirus.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus, and a preparation method and application thereof.
The invention provides a camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus, which comprises the following raw materials in parts by weight: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
Compared with the prior art, the invention discovers that the caffeine and the chlorogenic acid have good inhibition effect on the novel coronavirus, and the virus inhibition rate is obviously improved under the synergistic effect of the caffeine and the chlorogenic acid, so that the invention creatively provides a composition which is formed by compounding the caffeine and the chlorogenic acid as main antiviral active ingredients and compounding auxiliary materials such as mogroside, soluble starch, water-soluble dextrin, citric acid, 10% starch slurry and the like, the composition can be taken orally, and a new formula and a new thought are provided for preventing the novel coronavirus.
Further, 25 parts of camellia nitidissima caffeine, 20 parts of camellia nitidissima chlorogenic acid, 3 parts of mogroside, 20 parts of soluble starch, 6 parts of water-soluble dextrin, 1 part of citric acid and 3 parts of 10% starch slurry.
The invention also provides a preparation method of the camellia nitidissima caffeine and chlorogenic acid composition for resisting the novel coronavirus, which comprises the following steps:
(1) preprocessing golden camellia: selecting old camellia chrysantha leaves or leftovers, cleaning, drying, crushing and sieving to obtain camellia chrysantha tea powder;
(2) respectively extracting Camellia nitidissima caffeine and Camellia nitidissima chlorogenic acid, wherein,
the method for extracting the camellia nitidissima caffeine comprises the following steps: weighing the camellia chrysantha tea powder, adding deionized water, leaching for 2-5 times under the conditions of ultrasound and water bath, filtering, adding equal volume of absolute ethyl alcohol into filtrate, performing first ultrasonic treatment, standing, filtering, and centrifuging to obtain supernatant a; adjusting the pH value of the supernatant a to 7.0-8.0, adding metal salt for secondary ultrasonic treatment, filtering and centrifuging to obtain a supernatant b; adding NaCl into the supernatant b, performing ultrasonic treatment, adding ethyl acetate, performing ultrasonic treatment at the water bath temperature of 35-80 ℃, standing, separating out an ethyl acetate phase, and performing vacuum drying on a product to obtain crude camellia nitidissima caffeine; purifying the crude camellia chrysantha caffeine to obtain camellia chrysantha caffeine;
the method for extracting the chlorogenic acid of the camellia nitidissima comprises the following steps: weighing the camellia chrysantha tea powder, adding 80% ethanol at a material ratio of 1:10-30, and reacting at 25-80 ℃ for 1-4h to obtain crude camellia chrysantha chlorogenic acid; carrying out primary purification treatment on the crude camellia chrysantha chlorogenic acid by adopting macroporous resin; performing secondary purification treatment by adopting semi-preparative liquid chromatography; and finally, carrying out three times of purification treatment: dissolving in water, precipitating, filtering, concentrating the filtrate, and vacuum drying to obtain chlorogenic acid of Camellia Chysantha;
(3) preparing materials: and (3) weighing the camellia chrysantha caffeine and the camellia chrysantha chlorogenic acid prepared in the step (2) according to a certain proportion, and mixing mogroside, soluble starch, water-soluble dextrin, citric acid and 10% starch slurry to obtain the camellia chrysantha caffeine and chlorogenic acid composition.
Compared with the prior art, the method has the advantages that the camellia nitidissima caffeine and the camellia nitidissima chlorogenic acid are purified from old camellia nitidissima tea leaves and the waste, the utilization rate of camellia nitidissima waste is improved, and the additional value of camellia nitidissima is improved; meanwhile, the invention adopts purification treatment to obtain the refined camellia nitidissima caffeine, the purity of the camellia nitidissima caffeine is as high as 99.5%, and three times of purification treatment are adopted to purify the camellia nitidissima chlorogenic acid, the purity of the camellia nitidissima chlorogenic acid is as high as 99.5%, so that the quality of the purified camellia nitidissima caffeine and the quality of the camellia nitidissima chlorogenic acid meet the requirements of national food or medical specifications.
Further, after the step (3), a step (4) of granulating is also included: after the step (3), a step (4) of granulating is also included: and (4) preparing the camellia chrysantha caffeine and chlorogenic acid composition in the step (3) into camellia chrysantha caffeine and chlorogenic acid granules through a traditional Chinese medicine granulator, wherein the granularity of the granules is 10-50 meshes.
Further, the method also comprises a step (5) of sterilizing after the step (4): the step (4) is followed by a step (5) of sterilization: the caffeine and chlorogenic acid granules of Camellia Chysantha can be used137The radiation of Cs-gamma ray is carried out for 3-6h, and then the radiation of ultraviolet ray is carried out for 16 h. The invention carries out two times of sterilization treatment on the camellia chrysantha caffeine and chlorogenic acid granules, prevents the camellia chrysantha caffeine and chlorogenic acid granules from going bad, and ensures that the quality of the product meets the national food or medicine product specification.
Further, in the step of extracting the camellia nitidissima caffeine in the step (2), the metal salt is any one of calcium chloride, magnesium chloride, zinc chloride and zinc nitrate. Because the golden camellia is rich in tea polyphenol, the invention adopts the coordination of the metal salt and the tea polyphenol to generate a precipitate, thereby removing the tea polyphenol in the golden camellia and further achieving the purpose of purification.
Further, in the step of extracting the caffeine from the camellia chrysantha in the step (2), the purification treatment step is as follows: putting the rough camellia chrysantha caffeine into a container, reversely buckling a surface dish above the container, heating the container under the stirring condition, controlling the temperature at 115-125 ℃, enabling the rough camellia chrysantha caffeine to be sublimated, crystallized and attached to the inner surface of the surface dish, and repeating the steps to obtain the camellia chrysantha caffeine. The invention adopts a sublimation crystallization mode to obtain the high-purity camellia nitidissima caffeine.
Further, in the step (2) of extracting the chlorogenic acid of the golden camellia, the conditions of primary purification treatment are that macroporous resin is adopted for separation and purification, the sample loading concentration is 0.15-0.5mg/ml, the pH is 2.0-4.0, and the mass ratio of the sample injection liquid volume to the macroporous resin is 10-30 ml-L-1The flow rate is 1-5ml/min, and the eluent is 30% ethanol; the conditions of the third purification treatment are as follows: dissolving twice purified Camellia Chysantha chlorogenic acid 1 times in 2, 3, 4, and 5 times of water respectively, precipitating, filtering, concentrating the filtrate, and vacuum drying to obtain Camellia Chysantha chlorogenic acid.
Further, in the step (3), the mass parts of the raw materials are as follows: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
Further, the camellia chrysantha caffeine and chlorogenic acid composition is used for killing novel coronavirus.
Detailed Description
The invention provides a camellia nitidissima caffeine and chlorogenic acid composition (hereinafter referred to as camellia nitidissima caffeine and chlorogenic acid composition) for resisting novel coronavirus, which comprises the following raw materials in parts by weight: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
Based on the formula, the invention also provides a preparation method of the camellia nitidissima caffeine and chlorogenic acid composition for resisting the novel coronavirus, which comprises the following steps:
(1) preprocessing golden camellia: selecting old tea leaves or leftovers of the golden camellia, cleaning, drying, crushing, and sieving with a 20-40 mesh sieve to obtain the golden camellia tea powder.
(2) Respectively extracting the camellia chrysantha caffeine and the camellia chrysantha chlorogenic acid.
The method comprises the steps of weighing 2-15g of camellia chrysantha tea powder, adding 30-300m L deionized water, extracting for 2-5 times under the conditions of ultrasound and water bath, filtering, adding equal volume of absolute ethyl alcohol into filtrate, carrying out primary ultrasonic treatment under the condition of 300-500w ultrasound for 5-20min, standing for 30-120min, filtering, centrifuging to obtain supernatant a, adjusting the pH value of the supernatant a to be 7.0-8.0, adding metal salt (any one of calcium chloride, magnesium chloride, zinc chloride and zinc nitrate) for secondary ultrasonic treatment to obtain tea polyphenol precipitate, filtering, centrifuging to obtain supernatant b, adding NaCl solution accounting for 0.1-0.4% of the total weight of the solution into the supernatant b, carrying out ultrasound for 2-10min under the condition of 300-500w, adding 50-550m L ethyl acetate, carrying out ultrasound treatment for 15-60min under the temperature of 35-80 ℃ of the water bath, separating out 30-90min, separating out separation on an ethyl acetate phase, covering a product on the surface of the crude camellia chrysantha coffee product under the temperature of 30-60 ℃ and drying the surface of the crude camellia chrysantha coffee crystal, carrying out sublimation treatment under the temperature of the crude camellia chrysantha coffee alkali, carrying out a vacuum condition of 2-6h, and carrying out purification step of a suspension of a camellia chrysantha suspension, and a suspension of a camellia chrysantha suspension under the camellia chrysantha suspension of a camellia chrysantha suspension under the.
The method for extracting the chlorogenic acid of the golden camellia comprises the steps of weighing 4-6g of golden camellia tea powder, adding 80% ethanol at a material ratio of 1:10-30, reacting for 1-4h at 25-80 ℃ to obtain crude golden camellia chlorogenic acid, performing primary purification treatment, namely separating and purifying by adopting macroporous resin, wherein the sample loading concentration is 0.15-0.5mg/ml, the pH value is 2.0-4.0, and the mass ratio of the sample injection liquid volume to the macroporous resin is 10-30 ml-L-1The flow rate is 1-5ml/min, and the eluent is 30% ethanol; and (3) secondary purification treatment: performing secondary purification on the primarily purified camellia chrysantha chlorogenic acid by adopting semi-preparative liquid chromatography, and performing vacuum drying to obtain secondarily purified camellia chrysantha chlorogenic acid; and (3) three times of purification treatment: by secondary purificationThe volume of the camellia chrysantha chlorogenic acid is 1 time, 2 times, 3 times, 4 times and 5 times of water are respectively added for dissolving, and the camellia chrysantha chlorogenic acid is obtained by precipitation, filtration, filtrate concentration and vacuum drying, and the purity is more than 99.5%.
(3) Preparing materials: the camellia chrysantha caffeine and chlorogenic acid composition is obtained by weighing camellia chrysantha caffeine and camellia chrysantha chlorogenic acid according to a certain proportion, and mixing mogroside, soluble starch, water-soluble dextrin, citric acid and 10% starch slurry. Wherein the weight parts of the raw materials are as follows: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
(4) And (3) granulating: and (4) preparing the mixed powder of the camellia chrysantha caffeine and chlorogenic acid composition in the step (4) into camellia chrysantha caffeine and chlorogenic acid granules through a traditional Chinese medicine granulator, wherein the granularity of the granules is 10-50 meshes. Wherein the aperture of the punching plate is 1.50mm, the frequency of a roller motor is 40-50HZ, the frequency of a feeding motor is 30-50HZ, the pressure of an oil cylinder is 16-24bar, and the granulating mode is dry granulating. The camellia nitidissima caffeine and chlorogenic acid composition can be prepared into oral liquid, buccal tablets and other forms of oral medicines.
(5) And (3) sterilization: the caffeine and chlorogenic acid granules of Camellia Chysantha can be used137The radiation of Cs-gamma ray is carried out for 3-6h, and then the radiation of ultraviolet ray is carried out for 16 h.
The present invention is described in detail below by way of examples 1 and 2.
Example 1
(1) Preprocessing golden camellia: selecting old tea leaves or leftovers of the golden camellia, cleaning, drying, crushing, and sieving by a 20-mesh sieve to obtain the golden camellia tea powder.
(2) Respectively extracting camellia nitidissima caffeine and camellia nitidissima chlorogenic acid:
the method comprises the steps of weighing 5g of camellia chrysantha tea powder, adding 100m L deionized water, leaching for 3 times under the conditions of ultrasound and water bath, filtering, adding equal volume of absolute ethyl alcohol into filtrate, carrying out first ultrasonic treatment under the condition of 400w ultrasound for 5min, standing for 40min, filtering, centrifuging to obtain supernatant a, adjusting the pH value of the supernatant a to 7.0-8.0, adding 2.5g of calcium chloride, carrying out second ultrasonic treatment to obtain tea polyphenol precipitate, filtering, centrifuging to obtain supernatant b, adding NaCl10.02g into the supernatant b, carrying out ultrasonic treatment under the condition of 400w for 5min, adding 200m L ethyl acetate, carrying out ultrasonic treatment under the condition of 400w water bath temperature of 40 ℃ for 30min, standing for 50min, separating an ethyl acetate phase, carrying out vacuum drying at 40 ℃ for 4h to obtain crude camellia chrysantha caffeine, purifying the crude camellia chrysantha caffeine, carrying out purification treatment to obtain the crude camellia chrysantha caffeine, placing the crude camellia chrysantha caffeine in a coarse caffeine cup, covering, placing the coarse camellia chrysantha coffee alkali beaker, stirring, turning over the coarse caffeine in the coarse caffeine, placing the coarse coffee cup, heating the coarse coffee cup, placing the coarse caffeine, controlling the coarse coffee cup, placing the coarse coffee cup, stirring, placing the coarse caffeine, controlling the coarse coffee cup, placing the coarse coffee cup.
The method for extracting the chlorogenic acid of the golden camellia comprises the steps of weighing 3g of golden camellia tea powder, adding 80% ethanol at a material ratio of 1:20, reacting for 2 hours at 35 ℃ to obtain crude golden camellia chlorogenic acid, and performing primary purification treatment, namely separating and purifying by adopting macroporous resin, wherein the sample loading concentration is 0.2mg/ml, the pH value is 3.0, and the mass ratio of sample injection liquid volume to macroporous resin is 20 ml-L-1The flow rate is 2ml/min, and the eluent is 30% ethanol; and (3) secondary purification treatment: secondly, performing secondary purification on the primarily purified camellia chrysantha chlorogenic acid by adopting semi-preparative liquid chromatography, and performing vacuum drying to obtain secondarily purified camellia chrysantha chlorogenic acid; and (3) three times of purification treatment: dissolving twice purified chlorogenic acid of Camellia Chysantha in 2, 3, 4, and 5 times of water respectively at volume of 1 time, precipitating, filtering, concentrating the filtrate, and vacuum drying at 40 deg.C to obtain chlorogenic acid of Camellia Chysantha with purity of above 99.5%.
(3) Preparing materials: the camellia chrysantha caffeine and chlorogenic acid composition is obtained by weighing camellia chrysantha caffeine and camellia chrysantha chlorogenic acid according to a certain proportion, and mixing mogroside, soluble starch, water-soluble dextrin, citric acid and 10% starch slurry. Wherein the weight parts of the raw materials are as follows: 25 parts of camellia nitidissima caffeine, 20 parts of camellia nitidissima chlorogenic acid, 3 parts of mogroside, 20 parts of soluble starch, 6 parts of water-soluble dextrin, 1 part of citric acid and 3 parts of 10% starch slurry.
(4) And (3) granulating: and (4) preparing the mixed powder of the camellia chrysantha caffeine and chlorogenic acid composition in the step (3) into camellia chrysantha caffeine and chlorogenic acid granules through a traditional Chinese medicine granulator, wherein the granularity of the granules is 20 meshes. Wherein the aperture of the punching plate is 1.50mm, the frequency of a roller motor is 50HZ, the frequency of a feeding motor is 40HZ, the pressure of an oil cylinder is 20bar, and the granulating mode is dry granulating. The camellia nitidissima caffeine and chlorogenic acid composition can be prepared into oral liquid, buccal tablets and other forms of oral medicines.
(5) And (3) sterilization: the caffeine and chlorogenic acid granules of Camellia Chysantha can be used137The radiation quantity of the Cs-gamma rays is 6kGY after 4 hours of radiation, and the radiation sterilization quality is ensured by selecting the Bacillus pumilus with strong radiation resistance as an indicator for ionizing radiation sterilization; further irradiating with ultraviolet rays for 16 h.
Example 2
(1) Preprocessing golden camellia: selecting old tea leaves or leftovers of the golden camellia, cleaning, drying, crushing, and sieving by a 30-mesh sieve to obtain the golden camellia tea powder.
(2) Respectively extracting camellia nitidissima caffeine and camellia nitidissima chlorogenic acid:
the method for extracting the caffeine of the golden camellia comprises the steps of weighing 10g of golden camellia tea powder, adding 200m L deionized water, extracting for 4 times under the conditions of ultrasound and water bath, filtering, adding equal volume of absolute ethyl alcohol into filtrate, carrying out first ultrasonic treatment under the condition of 500w ultrasound for 10min, standing for 45min, filtering, centrifuging to obtain supernatant a, adjusting the pH value of the supernatant a to 7.0-8.0, adding 6.15g of zinc chloride, carrying out second ultrasonic treatment to obtain tea polyphenol precipitate, filtering, centrifuging to obtain supernatant b, adding NaCl0.04g into the supernatant b, carrying out ultrasound for 5min under the condition of 400w, adding 400m L ethyl acetate, carrying out ultrasound for 20min under the condition of 500w water bath temperature of 45 ℃, standing for 60min, separating an ethyl acetate phase, carrying out vacuum drying for 4.5h at the temperature of 35 ℃ to obtain crude caffeine, purifying the caffeine, carrying out purification treatment to obtain caffeine, placing the crude caffeine in a golden camellia crystal pot, turning over the crude caffeine over, placing the golden camellia crystal in the golden camellia crystal pot, carrying out edge turning over on the crude caffeine, carrying out edge turning over, carrying out heating, carrying out edge turning over on the above steps, and carrying out the steps to obtain the above crude caffeine crystal, wherein the temperature is controlled by metal is controlled by the temperature of the step.
The method for extracting the chlorogenic acid of the golden camellia comprises the steps of weighing 5g of golden camellia tea powder, adding 80% ethanol according to a material ratio of 1:25, reacting for 3.5 hours at 40 ℃ to obtain crude chlorogenic acid of the golden camellia, and performing primary purification treatment, namely separating and purifying by adopting macroporous resin, wherein the sample loading concentration is 0.3mg/ml, the pH value is 2.5, and the mass ratio of sample injection liquid volume to the macroporous resin is 18 ml-L-1The flow rate is 2.5ml/min, and the eluent is 30% ethanol; and (3) secondary purification treatment: secondly, performing secondary purification on the primarily purified camellia chrysantha chlorogenic acid by adopting semi-preparative liquid chromatography, and performing vacuum drying to obtain secondarily purified camellia chrysantha chlorogenic acid; and (3) three times of purification treatment: dissolving twice purified Camellia Chysantha chlorogenic acid 1 times in 2, 3, 4, and 5 times of water respectively, precipitating, filtering, concentrating the filtrate, and vacuum drying to obtain Camellia Chysantha chlorogenic acid with purity of above 99.5%.
(3) Preparing materials: the camellia chrysantha caffeine and chlorogenic acid composition is obtained by weighing camellia chrysantha caffeine and camellia chrysantha chlorogenic acid according to a certain proportion, and mixing mogroside, soluble starch, water-soluble dextrin, citric acid and 10% starch slurry. Wherein the weight parts of the raw materials are as follows: 30 parts of camellia nitidissima caffeine, 20 parts of camellia nitidissima chlorogenic acid, 3.5 parts of mogroside, 25 parts of soluble starch, 6 parts of water-soluble dextrin, 1.5 parts of citric acid and 3 parts of 10% starch slurry.
(4) And (3) granulating: and (4) preparing the mixed powder in the step (3) into camellia chrysantha caffeine and chlorogenic acid granules through a traditional Chinese medicine granulator, wherein the granularity of the granules is 30 meshes. Wherein the aperture of the punching plate is 1.50mm, the frequency of a roller motor is 45HZ, the frequency of a feeding motor is 40HZ, the pressure of an oil cylinder is 19bar, and the granulating mode is dry granulating. The camellia nitidissima caffeine and chlorogenic acid composition can be prepared into oral liquid, buccal tablets and other forms of oral medicines.
(5) And (3) sterilization: the caffeine and chlorogenic acid granules of Camellia Chysantha can be used137The radiation of Cs-gamma rays is carried out for 5 hours, the radiation dose is 2-35kGY, and the radiation sterilization quality is ensured by selecting the Bacillus pumilus with strong radiation resistance as an indicator for ionizing radiation sterilization; further irradiating with ultraviolet rays for 16 h.
The following verification experiments prove the efficacy of the camellia nitidissima caffeine and chlorogenic acid composition of examples 1 and 2 in resisting novel coronavirus.
(1) Designing primers to obtain a first generation recombinant virus: the RNA of the novel coronavirus COVID-19 is taken as a template, cDNA is synthesized by reverse transcriptase, and the COVID-19S, M, E gene is obtained by PCR technology by taking the cDNA as the template. And constructing pMD-18T recombinant plasmids of the three genes, and identifying. And further constructing pFastBac of the three genesTMThe Dual recombinant plasmid is identified by PCR, double digestion and sequencing. Will pFastBacTMAnd respectively transforming the Dual recombinant plasmids into DH10Bac competent cells, carrying out shake culture for 12-16 h, and carrying out PCR detection. Selecting positive bacterial colonies, amplifying bacteria, extracting Bacmid DNA, obtaining three recombinant rBacmid DNAs, respectively transfecting Sf9 cells with the three recombinant rBacmid DNAs, and harvesting supernate, namely the first-generation recombinant virus.
(2) Obtaining second and third generation recombinant viruses: sf9 cells were infected with 100. mu.l of the first generation recombinant virus, and left at 28 ℃ for 2h, during which the flask was gently shaken to allow the virus to uniformly adsorb the cells. The supernatant was removed, 2ml of complete medium was added, and after culturing at 28 ℃ for 72 hours, the supernatant was collected to obtain a second generation recombinant virus. And (4) continuously passaging the harvested viruses according to the method to obtain third-generation recombinant viruses. Three recombinant baculoviruses were designated rB-S, rB-M, rB-E, respectively, and plaque purification and plaque counting were performed.
(3) Infecting Sf9 cells with purified virus, culturing, collecting supernatant and cells respectively, detecting the expression of recombinant protein by Western-blotting, hemagglutination test and indirect immunofluorescence test, infecting Sf9 cells with three purified recombinant baculovirus rB-S, rB-M or rB-S, rB-M, rB-E together, culturing for about 5 days in a 28 ℃ incubator, collecting infected cells, blowing off adherent cells with PBS, washing for three times, crushing by ultrasonic waves, centrifuging for 10min at 4 ℃, 12,000g, taking supernatant, ultracentrifuging by sucrose density gradient, purifying COVID-19V L P, identifying constituent proteins of V L Ps by Western-blotting, and observing the form of V L Ps by a transmission electron microscope.
(4) Adding Camellia Chysantha caffeine and chlorogenic acid composition, dissolving purified COVID-19 virus-like particles (V L Ps) in PBS solution as virus stock solution, and diluting virus solution (10) with gradient COVID-19 respectively-1、10-2、10-3.....10-9) Preparation, followed by viral inoculation (37 ℃, 5% CO)2Incubator), discard growth medium, wash with PBS solution, add 10PFU/ml virus solution 100 u l (including l% bovine serum albumin PBS solution) placed at 4 degrees C adsorption for 1 h. Washing the adsorbed cells with PBS solution, adding 0.5-3ml of PBS dispersion of the composition of caffeine and chlorogenic acid of Camellia Chysantha prepared in the invention of example 1 and example 2 (taking the parameters of example 1 as example), wherein the concentration of the composition of caffeine and chlorogenic acid is 0.005-0.30mg/ml, and washing the cells with PBS solution at 37 deg.C and 5% CO2Culturing in an incubator for 8-72h, collecting culture solution of different periods, washing dead cells with PBS, measuring live cells by MTT (thiazole blue) colorimetry, measuring virus propagation amount by Plaque Forming Unit (PFU), and observing cytopathic effect (cytopathic effect) under a microscope at different periods (8h, 10h, 18h,24h,36h,48h and 72h) until the produced CPE is not developed any more under the continuous action of the virus of an antiviral product. Setting a blank control group (the camellia nitidissima caffeine and chlorogenic acid composition are replaced by PBS liquid with the same volume) under the same condition, and recording detailed anti-COVID-19 virus detailed data of different camellia nitidissima caffeine and chlorogenic acid compositions. According to the method described in this patent, the COVID-19 virus was incubated at 37 ℃ and the virus PFU peaked after 10 hours. Research data show that the oral preparation has no obvious effect of inhibiting COVID-19 virus within the concentration range of 0.005-0.01mg/ml by taking 0.5ml of the composition of the caffeine and the chlorogenic acid of the golden camellia as an example. The composition of caffeine and chlorogenic acid in Camellia Chysantha has significant inhibitory effect on COVID-19 virus within the concentration range of 0.01-0.30mg/mlThe control was effective and had the best effect against COVID-19 virus at a concentration of 0.16mg/ml the data showed that the blank control had a COVID-19 virus titer of 16.71 × 104PFU/ml, and COVID-19 virus titer observations of 14.77 × 10 when the concentrations of the Camellia Chysantha caffeine and chlorogenic acid composition are 0.015mg/ml, 0.04mg/ml, 0.10mg/ml, 0.16mg/ml, 0.23mg/ml and 0.30mg/ml, respectively4PFU/ml、10.18×104PFU/ml、2.31×104PFU/ml、 0.92×104PFU/ml, 0.97 × 104PFU/ml and 1.01 × 104PFU/ml. The effect of all experimental groups on the COVID-19 virus is obviously better than that of a blank control group, wherein under the condition of the optimal concentration of 0.16mg/ml, the PFU value of the COVID-19 virus of the experimental group added with the camellia chrysantha caffeine and chlorogenic acid composition is only about 5.5 percent of that of the blank control group, and the activity of the COVID-19 virus is greatly reduced. The experimental results also fully show that the camellia nitidissima caffeine and chlorogenic acid composition prepared in the embodiment 1 and the embodiment 2 can effectively resist novel coronavirus.
Compared with the prior art, the invention discovers that the caffeine and the chlorogenic acid have good inhibition effect on the novel coronavirus, and the virus inhibition rate is obviously improved under the synergistic effect of the caffeine and the chlorogenic acid, so that the invention creatively provides a composition which is formed by compounding the caffeine and the chlorogenic acid as main antiviral active ingredients and compounding auxiliary materials such as mogroside, soluble starch, water-soluble dextrin, citric acid, 10% starch slurry and the like, the composition can be taken orally, and a new formula and a new thought are provided for preventing the novel coronavirus. According to the method, the camellia nitidissima caffeine and the camellia nitidissima chlorogenic acid are purified from old camellia nitidissima tea leaves and the waste, so that the utilization rate of camellia nitidissima waste is improved, and the additional value of camellia nitidissima is improved; meanwhile, the invention adopts purification treatment to obtain the refined camellia nitidissima caffeine, the purity of the camellia nitidissima caffeine is as high as 99.5%, and three times of purification treatment are adopted to purify the camellia nitidissima chlorogenic acid, the purity of the camellia nitidissima chlorogenic acid is as high as 99.5%, so that the quality of the purified camellia nitidissima caffeine and the quality of the camellia nitidissima chlorogenic acid meet the requirements of national food or medical specifications.
The present invention is not limited to the above-described embodiments, and various modifications and variations of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.
Claims (10)
1. A camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus is characterized in that: the composite material comprises the following raw materials in parts by weight: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
2. The Camellia nitidissima caffeine chlorogenic acid composition against novel coronaviruses as claimed in claim 1, which is characterized in that: 25 parts of camellia nitidissima caffeine, 20 parts of camellia nitidissima chlorogenic acid, 3 parts of mogroside, 20 parts of soluble starch, 6 parts of water-soluble dextrin, 1 part of citric acid and 3 parts of 10% starch slurry.
3. A preparation method of a camellia nitidissima caffeine and chlorogenic acid composition for resisting novel coronavirus is characterized by comprising the following steps:
(1) preprocessing golden camellia: selecting old camellia chrysantha leaves or leftovers, cleaning, drying, crushing and sieving to obtain camellia chrysantha tea powder;
(2) respectively extracting Camellia nitidissima caffeine and Camellia nitidissima chlorogenic acid, wherein,
the method for extracting the camellia nitidissima caffeine comprises the following steps: weighing the camellia chrysantha tea powder, adding deionized water, leaching for 2-5 times under the conditions of ultrasound and water bath, filtering, adding equal volume of absolute ethyl alcohol into filtrate, performing first ultrasonic treatment, standing, filtering, and centrifuging to obtain supernatant a; adjusting the pH value of the supernatant a to 7.0-8.0, adding metal salt for secondary ultrasonic treatment, filtering and centrifuging to obtain a supernatant b; adding NaCl into the supernatant b, performing ultrasonic treatment, adding ethyl acetate, performing ultrasonic treatment at the water bath temperature of 35-80 ℃, standing, separating out an ethyl acetate phase, and performing vacuum drying on a product to obtain crude camellia nitidissima caffeine; purifying the crude camellia chrysantha caffeine to obtain camellia chrysantha caffeine;
the method for extracting the chlorogenic acid of the camellia nitidissima comprises the following steps: weighing the camellia chrysantha tea powder, adding 80% ethanol at a material ratio of 1:10-30, and reacting at 25-80 ℃ for 1-4h to obtain crude camellia chrysantha chlorogenic acid; carrying out primary purification treatment on the crude camellia chrysantha chlorogenic acid by adopting macroporous resin; performing secondary purification treatment by adopting semi-preparative liquid chromatography; and finally, carrying out three times of purification treatment: dissolving in water, precipitating, filtering, concentrating the filtrate, and vacuum drying to obtain chlorogenic acid of Camellia Chysantha;
(3) preparing materials: and (3) weighing the camellia chrysantha caffeine and the camellia chrysantha chlorogenic acid prepared in the step (2) according to a certain proportion, and mixing mogroside, soluble starch, water-soluble dextrin, citric acid and 10% starch slurry to obtain the camellia chrysantha caffeine and chlorogenic acid composition.
4. The method for preparing Camellia nitidissima caffeine chlorogenic acid composition resisting novel coronavirus according to claim 3, wherein the method comprises the following steps: after the step (3), a step (4) of granulating is also included: and (4) preparing the camellia chrysantha caffeine and chlorogenic acid composition in the step (3) into camellia chrysantha caffeine and chlorogenic acid granules through a traditional Chinese medicine granulator, wherein the granularity of the granules is 10-50 meshes.
5. The method for preparing Camellia nitidissima caffeine chlorogenic acid composition resisting novel coronavirus according to claim 4, wherein the method comprises the following steps: the step (4) is followed by a step (5) of sterilization: the caffeine and chlorogenic acid granules of Camellia Chysantha can be used137The radiation of Cs-gamma ray is carried out for 3-6h, and then the radiation of ultraviolet ray is carried out for 16 h.
6. The method for preparing Camellia nitidissima caffeine chlorogenic acid composition resisting novel coronavirus according to claim 3, wherein the method comprises the following steps: in the step of extracting the camellia nitidissima caffeine in the step (2), the metal salt is any one of calcium chloride, magnesium chloride, zinc chloride and zinc nitrate.
7. The method for preparing Camellia nitidissima caffeine chlorogenic acid composition resisting novel coronavirus according to claim 3, wherein the method comprises the following steps: in the step of extracting the camellia nitidissima caffeine in the step (2), the purification treatment step is as follows: putting the rough camellia chrysantha caffeine into a container, reversely buckling a surface dish above the container, heating the container under the stirring condition, controlling the temperature at 115-125 ℃, enabling the rough camellia chrysantha caffeine to be sublimated, crystallized and attached to the inner surface of the surface dish, and repeating the steps to obtain the camellia chrysantha caffeine.
8. The method for preparing Camellia nitidissima caffeine and chlorogenic acid composition resisting novel coronavirus according to claim 3, wherein in the step of extracting Camellia nitidissima chlorogenic acid in the step (2), the conditions of primary purification treatment are that macroporous resin is adopted for separation and purification, the sample loading concentration is 0.15-0.5mg/ml, the pH is 2.0-4.0, and the mass ratio of the sample injection liquid volume to the macroporous resin is 10-30 ml-L-1The flow rate is 1-5ml/min, and the eluent is 30% ethanol; the conditions of the third purification treatment are as follows: dissolving twice purified Camellia Chysantha chlorogenic acid 1 times in 2, 3, 4, and 5 times of water respectively, precipitating, filtering, concentrating the filtrate, and vacuum drying to obtain Camellia Chysantha chlorogenic acid.
9. The method for preparing Camellia nitidissima caffeine chlorogenic acid composition resisting novel coronavirus according to claim 3, wherein the method comprises the following steps: in the step (3), the mass parts of the raw materials are as follows: 15-40 parts of camellia nitidissima caffeine, 10-30 parts of camellia nitidissima chlorogenic acid, 2-6 parts of mogroside, 15-35 parts of soluble starch, 5-10 parts of water-soluble dextrin, 0.5-3 parts of citric acid and 2-5 parts of 10% starch slurry.
10. Use of the Camellia nitidissima caffeine chlorogenic acid composition against a novel coronavirus according to any one of claims 1-9, wherein: the camellia chrysantha caffeine and chlorogenic acid composition is used for killing novel coronavirus.
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