KR20240021683A - Antimicrobial composition comprising the extracts of pollen of cannabis sativa l. - Google Patents

Abstract

The present invention relates to an antibacterial composition containing an extract of pollen of Cannabis sativa L. as an active ingredient. More specifically, an antibacterial and antifungal composition containing an ethanol extract of pollen produced from male cannabis as an active ingredient. It relates to a broad-spectrum antibacterial composition with excellent activity. As proven through the examples herein, the male cannabis pollen ( Cannabis sativa L.) extract as an active ingredient of the broad-spectrum antibacterial composition of the present invention is used in antibacterial pharmaceutical compositions, health functional foods, cosmetic compositions, and animals. It has excellent effects that can be used for purposes such as feed composition, food, cosmetics or animal feed additives, various detergents, and various cleaning agents. In addition, the pollen extract of male cannabis of the present invention has excellent thermal stability and does not show loss of activity even under acidic conditions of pH 2 and in plasma, so it can be easily processed into various forms such as liquid, cream, powder, pills, and tablets. It can be very useful in the pharmaceutical industry, food industry, livestock industry, health industry, and cosmetics industry.

Description

ANTIMICROBIAL COMPOSITION COMPRISING THE EXTRACTS OF POLLEN OF CANNABIS SATIVA L.}

The present invention relates to an antibacterial composition containing an extract of pollen of Cannabis sativa L. as an active ingredient. More specifically, an antibacterial and antifungal composition containing an ethanol extract of pollen produced from male cannabis as an active ingredient. It relates to a broad-spectrum antibacterial composition with excellent activity.

Humanity has used various antibacterial agents and preservatives to defend against invasion by pathogenic microorganisms and control food spoilage microorganisms. However, while chemical antibacterial agents and preservatives show excellent antibacterial activity against various bacteria and fungi, long-term use causes many side effects such as redness due to allergies, vascular edema, bone marrow suppression, anemia, gastritis, and degenerative brain disease. do. In fact, the United States has banned the use of antibiotics with severe side effects, such as chloramphenicol. Therefore, various studies are in progress on the development of natural antibacterial agents using natural products that are safe and have long-term use experience.

According to the U.S. Center for Disease Control and Prevention, hospital-acquired bloodstream infections are one of the leading causes of death in the United States. In addition, infection with pathogenic bacteria and fungi through skin and soft tissues is recognized as a common medical disease and is understood to be mainly caused by trauma or surgery. Many pathogenic bacterial and fungal infections can be treated successfully with oral antimicrobial agents and local cleansing, although treatment methods vary depending on the severity of the infection. However, more severe or complex infections commonly occur in patients with underlying risk factors (e.g., vascular blood flow abnormalities, diabetes) and/or infections caused by difficult-to-treat or multi-resistant bacteria and fungi. Treatment with antimicrobial agents and surgical removal of invasive tissue may be necessary. For example, vancomycin, a glycopeptide antibiotic, has been used successfully against severe nosocomial infections caused by multi-drug-resistant Gram-positive bacteria, especially MRSA, coagulase-negative Staphylococcus and Enterococcus. However, it has been reported that the isolation of strains resistant to vancomycin is increasing recently, and it is true that treatment of severe infections caused by some of the pathogens with current antibiotics is very limited. Therefore, the need to develop new, more effective antibiotics against the increasing number of resistant bacteria is increasing.

Hemp ( Cannabis sativa L.) is an annual plant of the hemp family. The stem bark is made into hemp and used for clothing, shoes, and crafts. The stem cob is used as paper and building materials, and the seeds are used for food and medicine. Additionally, hemp seed oil is a very valuable plant resource used as oil and cosmetics. The seed coat, flower buds, and leaves of cannabis contain the hallucinogenic ingredient THC (Tetrahydrocanabinol), which has been misused as a hallucinogenic drug. Hemp's origin is Central Asia, and it is currently cultivated in various regions around the world. In oriental medicine, the root of hemp is called hemp root and is used to relieve stagnant blood, treat dystocia, and make stones come out. The stem bark of hemp is used to treat bruises, difficulty urinating, and intestinal pain, and hemp leaves. (Cannabis) was used as an asthma, analgesic, anesthetic, and diuretic.

Meanwhile, pollen is a reproductive organ of vascular plants that produces seeds. It is a structure surrounded by a sturdy shell to protect the male gametophyte. It is mainly transported by wind or insects and reaches the female gametophyte, fertilizing it, and then forming seeds. There are two types of pollen: insect-mediated pollen and wind-borne pollen. Insect pollen is divided into single-plant pollen such as actinidia pollen and acorn pollen and miscellaneous plant pollen, and wind-maed pollen is pine pollen. Examples include pine pollen and hemp pollen.

Existing research on pollen was mainly focused on the nutritional properties of pollen and the development of extraction conditions and pretreatment methods to improve digestion and absorption by decomposing the sturdy outer shell, and research on antibacterial properties of pollen was very limited only to cattail pollen and pine pollen. It was reported as In addition, to date, most research related to hemp has focused on hemp seeds and hemp leaves, and little research has been conducted on hemp pollen.

Accordingly, the present inventors conducted research to derive the functionality of hemp pollen and completed the present invention.

Republic of Korea Patent Registration No. 10-2158674

The present invention was made to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to provide a broad-spectrum antibacterial composition containing an extract of male cannabis pollen ( Cannabis sativa L.) as an active ingredient. This is what I want to do.

In order to solve the above problems, the present invention provides an antibacterial composition containing an extract of pollen of Cannabis sativa L.

The extract is preferably an ethanol extract.

The antibacterial agent is preferably against gram-positive bacteria, gram-negative bacteria, or fungi.

The composition is preferably selected from the group consisting of pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, additives for animal feed, detergents, and cleaning agents.

As proven through the examples herein, the male cannabis pollen ( Cannabis sativa L.) extract as an active ingredient of the broad-spectrum antibacterial composition of the present invention is used in antibacterial pharmaceutical compositions, health functional foods, cosmetic compositions, and animals. It has excellent effects that can be used for purposes such as feed composition, food, cosmetics or animal feed additives, various detergents, and various cleaning agents. In addition, the pollen extract of male cannabis of the present invention has excellent thermal stability and does not show loss of activity even under acidic conditions of pH 2 and in plasma, so it can be easily processed into various forms such as liquid, cream, powder, pills, and tablets. It can be very useful in the pharmaceutical industry, food industry, livestock industry, health industry, and cosmetics industry.

Figure 1 is a diagram showing the steps for recovering hemp ash from male hemp.
Figure 2 is an optical micrograph of recovered hemp pollen.
Figure 3 shows the results of thin layer chromatography analysis of hemp pollen extract. A: Hemp leaf ethanol extract, B: Male hemp flower bud extract, C: THC (tetrahydrocannabinol) standard, D: CBD (Cannabidiol) standard, E: Hemp pollen ethanol extract.

Hereinafter, the present invention will be described in detail.

In order to verify the antibacterial efficacy of the hemp pollen extract, the inventors of the present invention recovered the flower buds of male hemp, mechanically shook them to release the pollen, and then recovered the pollen by passing it through a 15-mesh sieve. An extract was prepared by adding ethanol to hemp pollen without any additional pretreatment such as heating, and as a result of evaluating the antibacterial and antifungal activity of the extract, broad-spectrum antibacterial activity was confirmed, including gram-positive bacteria, gram-negative bacteria, and fungi. By confirming that the extract has excellent thermal and acid stability while showing no hemolytic activity against human red blood cells, it was confirmed that the extract could be developed as a broad-spectrum antibacterial agent against Gram-positive bacteria, Gram-negative bacteria, and fungal infections. After confirmation, the present invention was completed.

Accordingly, the present invention provides an antibacterial composition comprising an extract of pollen of Cannabis sativa L.

It is preferable that the flowerpot has not been heat treated.

The extract is preferably an ethanol extract.

The antibacterial agent is preferably against gram-positive bacteria, gram-negative bacteria, or fungi.

The composition is preferably selected from the group consisting of pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, additives for animal feed, detergents, and cleaning agents.

Below, the manufacturing method and efficacy test of the male cannabis pollen ( Cannabis sativa L.) extract of the present invention will be described in more detail.

The present invention includes the steps of collecting pollen from male cannabis; Preparing pollen extract through solvent extraction; Evaluation of antibacterial and antifungal activity of the extract; and a step of investigating the stability of the pollen extract.

The "hemp pollen extract" included in the composition of the present invention is obtained by extracting pollen recovered from the flower buds of male hemp with an organic solvent, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating it under reduced pressure. It can be obtained by.

Organic solvents used in the present invention include water (cold water, hot water), hexene, methylene chloride, acetone, alcohol, anhydrous or hydrous lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), and the above lower alcohols. A mixed solvent of ethanol and water can be used, and hot water or 95% ethanol extraction is preferred.

As a preferred embodiment, it is preferable that the flowerpot of the present invention is not subjected to separate heat treatment. Here, the extract may be sequentially or separately fractionated with organic solvents of hexene, ethyl acetate, and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

The hemp pollen extract of the present invention can be manufactured into powder through a conventional powdering process such as reduced pressure drying, freeze drying, or spray drying. They are not decomposed by various decomposition enzymes in plasma, and remain active even when heat treated at 100°C and at pH 2 in the human stomach.

The hemp pollen extract of the invention exhibits strong antibacterial activity and is used as a material for pharmaceutical compositions related to the prevention or treatment of infection by pathogenic bacteria, health functional foods, cosmetic compositions, animal feed compositions, foods, cosmetics, additives for animal feed, and various cleaning agents. can be used

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a pharmaceutical composition.

The pharmaceutical composition is formulated into various forms such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be used orally or administered through a variety of routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The effective amount of the ingredient with antibacterial activity in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg every day or every other day. It can be administered or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a health functional food.

The health functional food of the present invention can be used in a variety of ways, such as effective foods and beverages that can reduce the possibility of infection from subjects at high risk of infection with pathogenic microorganisms. Foods containing the active ingredient with excellent antibacterial activity of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and may be taken in the form of powders, granules, tablets, capsules, or beverages. It can be used as The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml. In addition to containing the extract as an essential ingredient in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food supplements, such as natural carbohydrates and various flavoring agents, as additional ingredients. Examples of the natural carbohydrates include common sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The flavoring agent may include thaumatin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The ratio of the flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a cosmetic composition.

The cosmetic composition of the present invention can be manufactured using the ingredients having excellent antibacterial activity of the present invention in the form of, for example, essence, nutritional cream, body lotion, rinse, shampoo, body cleanser, lotion, skin, mask pack, etc. It is not limited to this. Manufacturing in the above form can be easily performed according to various manufacturing processes known to those skilled in the art.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as an animal feed composition.

The above-mentioned animals are a group of organisms corresponding to plants and mainly consume organic matter as nutrients and have differentiated digestive, excretory and respiratory organs. Specifically, they include echinoderms, crustaceans, molluscs, fish, amphibians, reptiles, birds, It may be a mammal, preferably echinoderms such as sea urchins or sea cucumbers, arthropods including crustaceans such as crabs, shrimp, and prawns, molluscs such as cephalopods, gastropods, or bivalves, red sea bream, sea bream, cod, and flounder. , it may be fish such as flounder, birds including poultry such as pheasant or chicken, or mammals such as pigs, cows, and goats.

The composition for animal feed may further include grains, vegetable protein feed, animal protein feed, sugar, or dairy products in an antibacterial composition containing the antibacterial ingredient of the present invention. The grain may specifically be ground or crushed wheat, oats, barley, corn and rice, the vegetable protein feed may specifically be composed of rape, soybean and sunflower as main ingredients, and the animal protein feed may specifically be May be blood meal, meat meal, bone meal, and fish meal, and the sugar or dairy product may specifically be a dry ingredient made of various powdered milk and whey powder.

The composition for animal feed may additionally contain ingredients such as nutritional supplements, digestion and absorption enhancers, vocalization accelerators, or disease prevention agents.

The antibacterial composition included in the composition for animal feed of the present invention may vary depending on the purpose and conditions of use of the feed. For example, 0.1 g to 100 g of the antibacterial composition may be included based on 1 kg of final produced feed.

Additionally, the feed composition can be manufactured into a lightly viscous granular or granular material depending on the degree of grinding of the ingredients, and the composition can be supplied as a mesh or formed into a desired discrete shape for further processing and packaging, and storage. For this purpose, it may be subjected to a pelletizing, expanding or extruding process, and for ease of storage, excess water may preferably be removed by drying.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as an additive for food, cosmetics, or animal feed.

The additive is added to provide preservability to food, cosmetics, or animal feed. Since the antibacterial composition is a natural ingredient and is not harmful to the human body, it can be safely applied to food, cosmetics, or animal feed.

The additive may further include other additives appropriately selected depending on the purpose and purpose of use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The additives can be manufactured by powdering, granulating, tableting or liquefying depending on the purpose and purpose of use, and conventional methods can be used for packaging for commercialization.

In addition, the amount of the antibacterial composition among the additives may be appropriately used depending on the purpose of use, application type, application area, application target, etc., and may include, for example, 0.01 parts by weight to 50 parts by weight based on the total composition, but the antibacterial composition The content of the composition is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the purpose of application due to the nature of the product.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a detergent.

The detergent may preferably be a clothing detergent, dishwashing detergent, food detergent, or home appliance detergent. Since the antibacterial composition is a natural ingredient and is not harmful to the human body, it can be more preferably used for cleaning various household products, including kitchen utensils or food cleaners, or for imparting antibacterial properties.

The detergent may further contain selected additives depending on its purpose and use, and may be mixed with other active ingredients such as commonly used detergents or additives such as pigments, surfactants, and preservatives. The detergent can be manufactured by powdering, granulating, tableting or liquefying depending on its purpose and purpose, and conventional methods can be used for packaging for commercialization.

In addition, the amount of the antibacterial composition used among the detergents may be appropriately used depending on the purpose of use, application type, application area, application target, etc., for example, may include 0.01 parts by weight to 50 parts by weight based on the total composition, but the antibacterial effect The content of the composition is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the application purpose due to the nature of the product.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a cleaning agent.

The cleaning agent can be used as a hand, mouth, or feminine cleanser in a simple way to prevent infection of the body by pathogenic microorganisms that are commonly susceptible to infection in daily life. The antibacterial composition is a natural ingredient and is not harmful to the human body, so it can be safely applied even when used as a cleanser for hands, mouth, and women.

The cleaning agent may further contain other additives appropriately selected depending on its purpose and use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The cleaning agent may be manufactured by powdering, granulating, tableting, or liquidizing depending on its purpose and use, but convenient use will be possible in the liquid form.

In addition, the amount of the antibacterial composition used in the cleaning agent may be appropriately used depending on the purpose of use, application type, application area, etc., for example, it may include 0.01 parts by weight to 50 parts by weight based on the total composition, but the content of the antibacterial composition is It is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the purpose of application due to the nature of the product.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

Example 1: Collection of hemp pollen from male hemp flower buds

The flower buds of male hemp grown for about 2 months after sowing were collected in the In-Door hemp cultivation system of Hemp Gro Farm Co., Ltd. in Andong, Gyeongsangbuk-do, and the pollen was mechanically shaken without any separate pretreatment to drain the pollen. The spilled pollen was passed through a 15 mesh sieve. passed through to remove foreign substances. The overall pollen collection process is shown in Figure 1, and the pollen recovery rate compared to flower buds was about 15%.

Meanwhile, the structure of the recovered pollen was confirmed using an optical microscope (Olympus CH-1, Tokyo, Japan) (Figure 2), and there was a groove in the middle of the oval shape (~25μm), which is commonly found in windflowers such as cattail pollen. The wing portion was not confirmed. Therefore, in the case of hemp pollen, which is a wind-borne pollen, it is believed that it scatters and fertilizes due to its small size.

Example 2: Preparation of hemp pollen extract and analysis of its useful components

An ethanol extract was prepared from the hemp pollen collected in Example 1. Specifically, 20 times the amount of ethanol (Deoksan, 95%, Korea) was added to the hemp pollen, extraction was repeated twice at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to prepare an ethanol extract. The extraction yield of hemp pollen and its useful content analysis results are shown in Table 1.

extract Extraction Efficiency
(%) Content (mg/g) Total Polyphenol Total Flavonoids Total Sugar Reducing Sugar hemp flower pot 8.3 41.4±0.1 34.5±0.7 405.3±3.9 6.9±0.3

The extraction efficiency of hemp pollen was about 8.3%, and the total polyphenol content was high at 41.4 mg/g. This is compared to previously reported actinidia pollen (21.5 mg/g), acorn pollen (12.7 mg/g), pine pollen (22.2 mg/g), cattail pollen (16.7 mg/g), and miscellaneous pollen (25.4 mg/g). The content is 16 to 33 times higher. Due to this high polyphenol content, hemp pollen extract showed strong antioxidant activity. Meanwhile, the total sugar content of the hemp pollen extract was very high at 405 mg/g, but the reducing sugar content was relatively very low at 6.9 mg/g, so it is understood that hemp pollen contains a large amount of polysaccharide components that are not easily decomposed. .

At this time, the total polyphenol content was measured by adding 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution to 400 μl of the extraction sample solution, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. To determine the total flavonoid content, each sample was extracted by stirring with methanol for 18 hours, 4ml of 90% diethylene glycol was added to 400μl of the filtered extraction sample solution, 40μl of 1N NaOH was added again, and the absorbance was measured at 420nm after reacting for 1 hour at 37°C. Rutin was used as a standard reagent. Reducing sugars were quantified using the DNS method, and total sugars were quantified using the phenol-sulfuric acid method.

Example 3: Evaluation of antibacterial and antifungal activity of hemp pollen extract

The antibacterial activity of the hemp pollen extract prepared in Example 2 was evaluated, and the results are shown in Table 2. The strains used to evaluate antibacterial activity were Escherichia coli KCTC 1682, Proteus vulgaris KCTC 2433, Pseudomonas aeruginosa KACC 10186, and Salmonella typhimurium KCTC 1926 as Gram-negative bacteria, and Staphylococcus aureus KCTC 1916 and Staphylococcus epidermidis ATCC 12228 as Gram-positive bacteria. , Listeria monocytogenes KACC 10550 and Bacillus subtilis KCTC 1924 were used. Meanwhile, to evaluate antifungal activity, Saccharomyces cerevisiae IF0 0233 and Candida albicans KCTC 1940, the causative agent of candidiasis fungal infection, were used.

First, in the case of antibacterial activity evaluation, each bacteria was inoculated into Nutrient broth (Difco Co., USA) and cultured at 37°C for 24 hours, then each strain was adjusted to OD ₆₀₀ 0.1 and incubated on Nutrient agar (Difco Co.). , USA), 100 μl of each sample was spread on sterilized petridish (90 mm, Green Cross, Korea) containing medium, and 5 μl of each sample was added to sterile disc-paper (diameter 6.5 mm, Whatsman No. 2) and incubated at 37°C for 24 hours. In the case of fungi, the antibacterial activity was evaluated by measuring the size of growth-inhibiting rings after culturing at 30°C for 24 hours in the same manner using Sabouraud dextrose (Difco Co., USA). As a control, the antibacterial agent ampicillin and the antifungal agent miconazole (Sigma Co., USA) were used at a concentration of 1μg/disc each, and the size of the growth-inhibiting ring was calculated by measuring the diameter of the area where growth was not visible to the naked eye. It was measured in mm and presented as a representative result after evaluation three or more times.

Sample/chemicals
(mg/disc) Clear zone(mm) Gram positive Gram negative Yeast LM S.E. SA B.S. EC PA PV S.T. CA S.C. Hemp pollen extract (0.5) 10 9 7 11 7 7 8 7 8 7 Ampicillin 29 28 10 21 12 10 30 16 - - Miconazole (0.01) - - - - - - - - - - LM: Listeria monocytogenes, BS: Bacillus subtilis, SE: Staphylococcus epidermidis, SA: Staphylococcus aureus, EC: Escherichia coli, PA: Pseudomonas aeruginosa, PV: Proteus vulgaris, ST: Salmonella typhimurium, SC: Saccharomyces cerevisiae, CA: Candida albicans, -: No growth inhibition.

As a result, as shown in Table 2, ampicillin, which is used as an antibacterial agent in clinical practice, showed a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria even at 0.01 mg/disc, and against Staphylococcus aureus , Pseudomonas aeruginosa , and Escherichia coli. It showed a relatively weak growth inhibitory ring (12 mm) and no antifungal activity. On the other hand, miconazole, which is used clinically as an antifungal agent, strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans even at 0.01 mg/disc. Meanwhile, hemp pollen extract showed excellent antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi at a concentration of 0.5 mg/disc. Antibacterial activity has been reported in ethanol extracts from existing pine pollen and cattail pollen (Pyo Soo-jin, 2020. Journal of the Korean Society of Beekeeping 35: 65-73), but this was found only for Gram-positive bacteria and is different from the ethanol extract of hemp pollen. .

Example 4: THC detection in hemp pollen extract

Thin Layer Chromatography (TLC) was performed on the cannabis pollen extract obtained in Example 2 to evaluate whether it contained THC, a hallucinogenic addictive substance. At this time, Kieselgel 60F254 (Merck CO., Germany) was used as the support thin film, and hexane:acetone (75:25 v/v) was used as the developing solvent. After development, the solvent was removed by heating and a 0.5% Fast blue B salt solution was applied to detect cannabinoid substances. At this time, 2 μg/spot each of THC (C in Figure 3) and CBD (D in Figure 3) were used as standard products. The detection samples used were male hemp leaf ethanol extract, male hemp flower bud extract, and hemp pollen extract, and each was dripped at 500 μg/spot.

As a result, as shown in Figure 3, THC and CBD were not detected in the hemp pollen extract (Figure 3E). However, various cannabinoid substances, including significant amounts of THC and CBD, were detected in hemp leaf extract and male hemp flower bud extract (Figure 3, A and B). In the case of the hallucinogenic substances THC (tetrahydrocannabinol) and CBD (cannabidiol), which are the main components of cannabis, strong antibacterial activity has been reported (Napiroon et al., 2021. PeeJ 9: e11446), but the above results do not support the excellent properties of cannabis pollen extract. It is suggested that the antibacterial activity is independent of THC and CBD.

Therefore, it was confirmed that the hemp pollen extract showed strong antibacterial activity without containing THC, a toxic hallucinogenic substance, and thus had high practical usability.

Example 5: Human Red Blood Cell Hemolytic Activity of Hemp Pollen Extract

To evaluate the acute toxicity potential of cannabis pollen extract, human red blood cell hemolytic activity was evaluated. At this time, hemolytic activity was evaluated according to an existing report (Son Ho-yong, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292). Simply, 100 μl of human red blood cells washed three times with PBS were placed in a 96-well microplate. 100 μl of sample solutions of various concentrations were added and reacted at 37°C for 30 minutes. Afterwards, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. It was measured in . DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

(%)Hemolysis = [(Abs. S - Abs. C) / (Abs. T - Abs. C)] × 100

Abs. S: Absorbance of sample addition port,

Abs. C: Absorbance of DMSO added group,

Abs. T: Absorbance of Triton X-100 addition.

Sample/Control Concentration (mg/ml) Human red blood cell hemolytic activity (%) Distilled water - 0.2±0.3 Solvent (DMSO) - 0.5±1.2 Triton X-100 1.0 100.0±0.1 Empothera Mystery
(Amphotericin B) 0.1 99.6±1.0 0.05 99.6±1.2 0.025 98.3±0.8 0.0125 96.9±0.0 0.0063 89.1±1.5 0.0032 61.0±0.1 0 0.0±0.1 Hemp Pollen Extract 1.0 0.8±0.2 0.5 0.5±0.1

As shown in Table 3, DMSO and water used as controls had no hemolytic activity, and triton In addition, in the case of amphotericin B, which is used as an anticancer and antifungal agent, it was confirmed that more than 50% of red blood cells were hemolyzed at a concentration of 0.0063g/ml.

Meanwhile, the cannabis pollen extract did not cause red blood cell hemolysis at all up to a concentration of 1 mg/ml, confirming that it had no acute toxicity or red blood cell hemolytic activity. These results suggest that the hemp pollen extract of the present invention can safely replace antithrombotic agents that have side effects such as aspirin in the future without separate hemolytic activity.

Example 6: Evaluation of plasma, acid and heat stability of hemp pollen extract

The ethanol extract of cannabis pollen obtained in Example 2 was confirmed for its antithrombotic activity, plasma stability, heat stability, and acid stability. The extract showed no decrease in antithrombotic activity even when heat-treated at 100°C for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, and treated in plasma for 1 hour. Therefore, it was confirmed that the ethanol extract of hemp pollen contains anti-thrombotic active substances with acid resistance and heat resistance, confirming that it has high practical usability.

So far, the present invention has been examined focusing on its embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims, not the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (4)

An antibacterial composition comprising an extract of pollen of Cannabis sativa L.
The antibacterial composition according to claim 1, wherein the extract is an ethanol extract.
The antibacterial composition according to claim 1, wherein the antibacterial agent is against gram-positive bacteria, gram-negative bacteria, or fungi.
The method according to any one of claims 1 to 3, wherein the composition is selected from the group consisting of pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, additives for animal feed, detergents, and cleaning agents. An antibacterial composition that is intended to be.