CN111265580A - Composition containing grapefruit seed extract, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and discloses a composition containing pomelo seed extract, a preparation method and use thereof. The preparation method comprises the following steps: 1) screening pomelo seeds, quick freezing and then crushing to a certain particle size , using ethanol as the extractant to extract by stirring to obtain a crude extract, and filtering the crude extract to obtain a filtrate; then by recycling ethanol to obtain an extract; 2) adding glycerol to the extract obtained in step 1), Then add water to dilute to obtain a diluent, and add citric acid and sodium citrate to the diluent to obtain the composition of the mixed solution. The composition of the present invention has significant inactivation effect against common viruses and enveloped viruses, At the same time, it also shows excellent bactericidal effect, is safe, non-toxic, and not easy to remain. It can be used to prepare antibacterial and antiviral drugs, food and cosmetic additives, medical flushing liquid or medical spray, which broadens its application range.

Description

A kind of composition containing grapefruit seed extract, preparation method and use thereof

technical field

The invention belongs to the technical field of medicine, and in particular relates to a composition containing pomelo seed extract, a preparation method and use thereof.

Background technique

Viruses are a type of microorganisms that do not have a cellular structure, but have life characteristics such as inheritance and replication. Like all living things, viruses have the ability to inherit, mutate, and evolve. They are very small in size and extremely simple in structure. Highly parasitic, completely dependent on the host cell's energy and metabolic system to obtain the substances and energy required for life activities, leaving the host cell, it is just a large chemical molecule, which stops its activity and can be made into protein crystals for a non-living body , when encountering host cells, it will display typical life characteristics by adsorbing, entering, replicating, assembling, and releasing progeny viruses, so viruses are a kind of primitive life between living things and non-living things.

Influenza A virus is a common influenza virus. Influenza A virus is most likely to mutate. The subtype of influenza A virus is called "avian influenza". Avian influenza is caused by avian influenza virus. It is an acute infectious disease that can infect humans after the virus gene mutates. The symptoms after infection are mainly high fever, cough, runny nose, myalgia, etc. Most of them are accompanied by severe pneumonia. In severe cases, heart, kidney and other organ failure lead to death, the fatality rate is high. The disease can be transmitted through various ways such as digestive tract, respiratory tract, skin damage and conjunctiva, and the movement of people and vehicles is an important factor in the transmission of this disease. At present, broad-spectrum antiviral western medicines are mostly used in epidemic prevention and treatment of viral diseases. Such medicines are easy to develop drug resistance and mutate the virus. The viral mutation of avian influenza is the best proof. Therefore, this type of medicines use has greater limitations.

Therefore, the development of simple and effective pure natural antibacterial and antiviral extract products has become a necessity for daily antiviral protection, although researchers have developed some pure natural products, which can be effective against most common germs. However, for some viruses (such as influenza virus), which have an envelope-virus envelope wrapped in a protein capsid, the currently widely developed pure natural extract preparations are difficult to inactivate the above-mentioned viruses in a targeted manner.

After searching, related applications have been disclosed for the prior art of pure natural extract products, such as the Chinese patent application number 2018105905215, the application dated November 2, 2018 discloses GSE antibacterial rinse solution and its preparation method , which is composed of the following raw materials and their mass percentages: the content of sodium alginate is 0.1%-4.0%; the content of grapefruit seed extract is 0.01-10%; glycine 1%-3% is added with water for injection or purified water to a sufficient amount. The GSE antibacterial rinse solution in this application is non-toxic, irritating, and non-sensitizing to the human body, has good biodegradability, and has a good effect on killing bacteria, viruses, molds and fungi. The main component of the flushing solution, grapefruit seed extract, uses compound biphenol aminobenzene as the main antibacterial and disinfecting component. Although it can be used as a broad-spectrum flushing solution, it is difficult to exert a targeted effect on the enveloped avian influenza virus. effect. For the research on grapefruit seeds, the Chinese patent application number is 201510875346.0, and the application date is 2016.03.23 discloses a natural broad-spectrum antiseptic composition containing grapefruit seed extract and its application, mainly containing grapefruit seed extract compound, a hydroxamic acid, an aromatic alcohol, and one or more polyols. The antiseptic composition uses natural grapefruit seed extract as the main raw material, is compatible with hydroxamic acid which has a good inhibitory effect on fungi and an aromatic alcohol which has a good inhibitory effect on bacteria, is dissolved in an improved functional solvent, and has a stimulating effect. The characteristics of low resistance and high safety not only provide excellent broad-spectrum antibacterial effect, but also the good antioxidant properties of grapefruit seed extract increase the performance and value of the product. The grapefruit seed extract in this application mainly exerts reducing properties and antioxidant properties, and only exhibits good inhibitory activity against Escherichia coli, Staphylococcus aureus, Salmonella, and fungi. The substances with main antibacterial activity come from Aromatic alcohol, the composition of this application is mainly used for the antiseptic application of cosmetics as a preservative composition, and it is difficult to exert a wider antibacterial and antiviral effect.

A large number of studies have found that grapefruit seeds and their extracts are rich in essential oils, vitamin E, fatty acid flavonoids and polyphenols, and fatty acid flavonoids have excellent sterilization and disinfection effects. It is of great value to the epidemic prevention work of human beings to be widely used in antibacterial and antiviral applications.

SUMMARY OF THE INVENTION

1. The problem to be solved

Aiming at the defects in the prior art that the pure natural antibacterial and antiviral products have weak inhibitory activity on specific viruses and limit their application scope, the present invention extracts an extract whose main component is fatty acid flavonoids from grapefruit seeds, and adds glycerin. After the active ingredients are stabilized, citric acid and sodium citrate are added to obtain a composition, which provides a natural antiviral product that is effective against common viruses and specific viruses with envelopes that are difficult to inactivate, and has excellent properties. It is safe, non-toxic and not easy to remain. It can be used to prepare antibacterial and antiviral drugs, food additives, medical flushing liquid or medical spray, which broadens its application range.

2. Technical solutions

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

The present invention provides a composition containing grapefruit seed extract, the composition contains grapefruit seed extract, citric acid, sodium citrate and glycerin.

As a further improvement of the present invention, the mass of the citric acid accounts for 0.1%-5% of the mass of the composition, and the mass of the sodium citrate accounts for 0.05%-1% of the mass of the composition.

As a further improvement of the present invention, the described preparation method of the composition containing pomelo seed extract, comprises the following steps:

1) screen and wash the grapefruit seeds, dry them, pulverize to a certain particle size, and extract by stirring ethanol as an extractant to obtain a crude extract, and filter the crude extract to obtain a filtrate; and then recover ethanol to obtain extract;

2) Add glycerin to the extract obtained in step 1), and then add water to dilute to obtain a diluent, and add citric acid and sodium citrate to the diluent to obtain a mixed solution.

As a further improvement of the present invention, the seeds need to be rapidly frozen at -25°C to -80°C for more than 5 hours before crushing.

As a further improvement of the present invention, when adding glycerol to the extract in step 2), the mass ratio of the extract to the glycerol is 1:(1.0-2.5).

As a further improvement of the present invention, in the step 1), ethanol with a mass concentration of 70% is used as the extractant, and the material-to-liquid ratio is 1:(10-60).

The extraction conditions of step 1) are as follows: under the condition that the extraction temperature is 25° C., the extraction time is stirred for 120 minutes.

As a further improvement of the present invention, the pomelo seeds in the step 1) contain grapefruit seeds and/or green pomelo seeds, and/or the pulverized particle size in the step 1) is less than 5 mm.

As a further improvement of the present invention, the composition is used to prepare a virus- and bacteria-inactivated product. The product includes any one of antibacterial and antiviral drugs, food and cosmetic additives, medical rinses or medical sprays.

The composition of the present invention uses pure natural ingredients and is very safe. The recommended method of use as a medical spray is: use an ultra-fine spray device (spray particles smaller than 10 microns) to spray dry mist, and this method can make the ratio of spray to air contact. Large surface area, high efficiency and low operating cost; it is not only used for sanitation management of food factories, supermarkets and various vegetable and fruit stores, sterilization and disinfection of various foods, but also for sterilization and disinfection of various human living spaces ( Such as home, office, school, meeting place, plane, train, high-speed rail, car, ship, restaurant, hotel).

As a further improvement of the present invention, the virus includes one or more of avian influenza virus, Newcastle disease virus and infectious bursal disease virus.

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) The composition containing the pomelo seed extract of the present invention is to obtain an extract whose main component is fatty acid flavonoids by extracting the pomelo seeds, and glycerol is added to the extract to facilitate the main component of fatty acid flavonoids. The stability in the extract can effectively protect the main component. Finally, it is obtained by adding citric acid and sodium citrate. All components in the composition are safe and non-toxic, and have a broad-spectrum anti-virus effect. Common viruses can be rapidly inactivated, and at the same time, it can also achieve rapid inactivation of viruses with envelopes that are difficult to inactivate. In addition, it also has excellent bactericidal effect on bacteria, and can be used to prepare various antibacterial and antiviral Medicines, food and cosmetic additives, medical rinses or medical sprays, the excellent sterilization and disinfection effect of the composition of the present invention greatly broadens its application range.

(2) the composition containing grapefruit class seed extract of the present invention, its mechanism of action of virus inactivation is different from vaccines and antibiotic drugs used in the prior art, specifically as follows: the structure of the virus has a DNA or RNA encapsulation The capsule and the spikes protruding from the capsule. Vaccines and antibiotics used in the prior art belong to targeted drugs that are aggressive to spikes, and once the characteristics of spikes change, vaccines and antibiotics will lose their effect. The composition of the present invention is obtained by combining the extract of grapefruit seeds, glycerin, citric acid and sodium citrate. membrane moisture, thereby destroying the protein membrane. The same principle destroys not only viral envelopes, but also bacterial and mold cell membranes. Therefore, it is possible to utilize the rapid lysis of the cell membrane of pathogenic bacteria and the outermost envelope of the enveloped virus, which can exert an effective effect on a variety of viruses without being affected by the characteristics of the spike. Especially for viruses such as avian influenza virus, Newcastle disease virus, infectious bursal disease virus that are difficult to inactivate, it shows excellent inactivation effect. The inactivation of pathogens provides a suitable pH environment, which further strengthens the inhibitory effect on viruses and pathogenic bacteria.

(3) The preparation method of the composition containing the citron seed extract of the present invention, adding glycerin to the extract to ensure that the main component fatty acid flavonoids obtained by extraction can stably exist in the extract system, and make the extract It is not easy to volatilize, so as to obtain more effective components in the final extract, and last for a longer time, which is more conducive to the exertion of sterilization and antivirus effect. In addition, the method of the present invention needs to perform freezing treatment at -25°C to -80°C before the seeds are pulverized, so that a large amount of fatty acid flavonoids can be aggregated in the core part of the seeds, and the seeds can be processed in a frozen state. Crushed, easy to extract more fatty acid flavonoids in ethanol way, and retain more active ingredients.

Detailed ways

The present invention will be further described below with reference to specific embodiments.

Example 1

The composition containing grapefruit seed extract of the present invention, the preparation method comprises the following steps:

1) Screen the grapefruit seeds, wash them, freeze the seeds at -25℃～-80℃ for 5 hours after drying, and then pulverize them. , add 70% ethanol as an extractant, extract under stirring conditions at 25°C, keep the ratio of material to liquid at 1: (10-60), obtain a crude extract after 120 minutes of extraction, and filter the crude extract to obtain a filtrate ; Then, by recycling ethanol, an extract is obtained;

2) adding glycerol to the extract, the glycerol is added according to the ratio of the extract to the glycerol at a ratio of 1: (1.0-2.5), and then adding water for dilution to obtain a dilution, in the dilution Citric acid and sodium citrate are added to obtain a mixed solution, wherein the mass of the citric acid accounts for 0.1%-5% of the mass of the mixed solution, and the mass of the sodium citrate accounts for 0.05%-1% of the mass of the mixed solution.

The above mixed solution is subjected to high temperature sterilization to prepare an antiviral product. The above mixed solution can be used after being directly diluted with water, or it can be prepared into a dry product by drying, and it can be diluted during use.

One, adopt the composition of the present invention to carry out the experiment of virus inactivation as follows:

The viruses tested in this example are avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV).

In order to evaluate the synergistic virus inactivation effect produced by the addition of citric acid and sodium citrate in combination with the grapefruit seed extract, a comparative experiment was set up. In the comparative experimental group, citric acid and sodium citrate were not added in step 2). , all other conditions being the same.

Among them, for the dilution operation in the above step 2), the experimental group set up experiments with two concentrations respectively: 100-fold and 500-fold dilution groups were respectively set to test the activity on the selected virus.

The two dilution concentrations of the composition solution obtained in step 2) and the two dilution concentrations of the control group solution (without adding citric acid and sodium citrate) were mixed with the test virus AIV, NDV and IBDV virus for a period of time, and then titrated. For the remaining viruses, the specific test experiment includes the following steps:

a) Mix the composition solution (400 μL) and 100 μL virus suspension in a microtube, incubate the tube at room temperature (25°C ± 2°C) for 0, 5, 10, 30s, wherein the incubation time of IBDV virus is up to 30 minutes.

b) Add 500 μL of stop solution to stop the reaction.

As a positive control, 100 μL of the above virus was inoculated into 400 μL of PBS, and after incubation for 30 seconds (the incubation time of IBDV virus was up to 30 minutes), the reaction was terminated with 500 μL of stop solution.

The various remaining viruses were titrated as follows: MDCK (Madin Darby Canine Kidney) cells were prepared in 96-well cell culture plates for the titration of avian influenza virus AIV. At 5 days post inoculation (dpi), cytopathic effects (CPE) were observed and supernatants were examined for hemagglutinin (HA) activity using 0.5% chicken erythrocytes.

For titration of NDV, chicken embryonic fibroblasts were prepared in 96-well cell culture plates. At 5dpi, chicken embryo fibroblasts were observed by the same method to detect hemagglutinin activity.

For IBDV titration, chick embryonic fibroblasts were grown in growth medium on 60 mm cell culture dishes. At 5dpi, chicken embryo fibroblasts were observed by the same method to detect hemagglutinin activity.

Virus titers at 50% tissue culture infectious dose (TCID ₅₀ )/ml were calculated according to the experimental results by the Behrens-kaerbel method. The reduction factor (RF) is calculated, and the calculation of RF adopts the following formula: RF=tpc-ta. "tpc" is the virus titer/amount from the untreated sample, recorded as log ₁₀ TCID ₅₀ /ml, or log ₁₀ PFU/ml, or log ₁₀ CFU/ml; and "ta" is the recovered virus titer/amount , recorded as log ₁₀ TCID ₅₀ /ml, or log ₁₀ PFU/ml, or log ₁₀ CFU/ml. If rf>3, it can be considered that the inactivation of the virus is effective.

The experiment was done in triplicate and the mean was calculated. Table 1 shows the comparison results between the composition of the present invention and the control group at a 100-fold dilution concentration.

Table 1 The comparison results of the composition of the present invention and the comparative group at a 100-fold dilution concentration

Table 2 Comparison results of the composition comparison group of the present invention at a 500-fold dilution concentration

The results show that the composition of the present invention can inactivate AIV, NDV and IBDV viruses in a shorter sensitization time than the control group without adding citric acid and sodium citrate, and has a better inactivation effect. live effect.

Example 2

The composition of the present invention was prepared according to the preparation method of Example 1, except that the dilution factor in step 2) was 300 times, and the restaurant kitchen (about 50 square meters in area) was sterilized by using a common sprayer: the number of sprays 30ml per square meter, the total dosage is 1500ml.

The detection parts are: food storage room, the surrounding area of chopping board, dish making table, etc.; measuring instrument: Lumitester PD30; the detection value at the beginning of June 6, 2019 was 12578RLU, which dropped to 3110RLU after 5 minutes of spraying, and 891RLU after 10 minutes of stopping. (Below 1000RLU conforms to the general miscellaneous bacteria standard in the hygiene standard of Japanese food processing and catering industry). After a week of continuous observation, it can be kept below 1000RLU. After September 3rd, spray 15ml per square meter before closing every day, and continue to monitor for three months. Generally, the value of miscellaneous bacteria has been kept below 300RLU.

Using the 300-fold dilution of the control group without adding citric acid and sodium citrate under the same conditions, the effect can only reach about 50%-60% of the composition of the present invention.

Example 3

The present embodiment is an embodiment of using the product of the present invention to carry out household indoor disinfection:

The composition of the present invention is prepared according to the preparation method of Example 1, the difference is: the dilution factor in step 2) is 300 times, and it is prepared as a common spray, which is used for household indoor disinfection: use a common sprayer to sterilize the household indoor (with an area of approx. 120 square meters) for sterilization: the amount of spray is 10 ml per square meter, and the total amount is 1200 ml. The detection parts are: kitchen, bathroom, living room, bedroom, storage room, etc.; measuring instrument: Lumitester PD30; the highest detection value was 5167RLU at the beginning of November 12, 2019, and it dropped to 982RLU after 5 minutes of spraying, and dropped after 10 minutes. To 532RLU (below 1000RLU meets the hygiene standards of Japanese food processing and catering industry). After continuous observation for a week, it can be kept below 600RLU. After December 3rd, spray 10ml per square meter every 3 days and continue monitoring for one month. Generally, the value of miscellaneous bacteria has been kept below 200RLU. Using the 500-fold dilution of the control group without adding citric acid and sodium citrate under the same conditions, the effect can only reach about 50%.

Claims (10)

1. A composition containing grapefruit seed extract is characterized in that: the composition comprises seed extract of Citrus Grandis, citric acid, sodium citrate and glycerol.

2. The composition comprising a grapefruit seed extract as claimed in claim 1, characterized in that: the citric acid accounts for 0.1-5% of the composition by mass, and the sodium citrate accounts for 0.05-1% of the composition by mass.

3. A method for preparing a composition containing a grapefruit seed extract as claimed in claim 1 or 2, characterized in that: the method comprises the following steps:

1) screening grapefruit seeds, crushing to a certain particle size, extracting by using ethanol as an extracting agent in a stirring manner to obtain a crude extract, and filtering the crude extract to obtain a filtrate; then recovering ethanol to obtain an extracting solution;

2) adding glycerol into the extracting solution obtained in the step 1), adding water for dilution to obtain a diluted solution, and adding citric acid and sodium citrate into the diluted solution to obtain a mixed solution.

4. A method of preparing a composition comprising grapefruit seed extract as claimed in claim 3, characterized in that: the seeds need to be subjected to rapid freezing treatment for more than 5 hours at-25 ℃ to-80 ℃ before being crushed.

5. The method for preparing a composition containing grapefruit seed extract as claimed in claim 4, characterized in that: when the glycerol is added into the extracting solution in the step 2), mixing the extracting solution and the glycerol according to the mass ratio of 1: (1.0-2.5) in proportion.

6. A method for preparing a composition containing grapefruit seed extract as claimed in claim 3 or 4, characterized in that: when ethanol is used as an extracting agent in the step 1), the material-liquid ratio is kept to be 1: (10-60).

7. The method for preparing a composition containing grapefruit seed extract as claimed in claim 6, characterized in that: the grapefruit seeds in the step 1) contain grapefruit seeds and/or green grapefruit seeds, and/or the crushing particle size in the step 1) is less than 5 mm.

8. Use of a composition comprising a grapefruit seed extract as claimed in claim 1 or 2, characterized in that: the composition is used for preparing products for inactivating viruses and bacteria.

9. The use of the composition comprising grapefruit seed extract as claimed in claim 8 in the preparation of antibacterial and antiviral products, characterized in that: the product comprises any one of antibacterial and antiviral drugs, food additives, cosmetic additives, medical washing liquid or medical spray.

10. The use of the composition comprising grapefruit seed extract as claimed in claim 8 in the preparation of antibacterial and antiviral products, characterized in that: the virus comprises any one or more of avian influenza virus, newcastle disease virus and infectious bursal disease virus.