ANTIBACTERIAL AND ANTIVIRUS COMPOSITION COMPRISING Extract of CANNABIS SATIVA L.

Abstract

The present disclosure relates to an antibacterial and antiviral composition containing an extract of Cannabis sativa L. as an active ingredient. More specifically, the present disclosure provides an antibacterial and antiviral composition which contains an extract of the natural product Cannabis sativa L., and thus may exhibit antibacterial and antiviral activities, and when taken or administered, has no side effects and does not cause bacterial or viral resistance.

Claims

1. An antibacterial and antiviral composition containing an extract of Cannabis sativa L. as an active ingredient.

2. The antibacterial and antiviral composition of claim 1, wherein the composition exhibits an antibacterial effect by effectively removing bacteria selected from the group consisting of Streptococcus mutans, Staphylococcus aureus, Candida albicans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus.

3. The antibacterial and antiviral composition of claim 1, wherein the composition has an antiviral effect against A/H1N1/WSN influenza virus.

4. The antibacterial and antiviral composition of claim 1, wherein the extract is obtained by extraction with an extraction solvent selected from the group consisting of water, a C.sub.1 to C.sub.6 lower alcohol, and a mixture thereof.

5. A functional food comprising the antibacterial and antiviral composition according to claim 1.

6. A skin external preparation comprising the antibacterial and antiviral composition according to claim 1.

7. A medicament comprising the antibacterial and antiviral composition according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 shows the results of measuring the cell viability of A549 cells treated with a composition according to one embodiment of the present disclosure before infection (pre-treatment), after infection (post-treatment), and before and after infection (pre-post-treatment) with A/H1N1/WSN influenza virus.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[0081] Hereinafter, examples of the present disclosure will be described in detail so that those of ordinary skill in the art can easily carry out the present disclosure. However, the present disclosure may be embodied in a variety of different forms and is not limited to the examples described herein.

Production Example 1: Production of Extracts

[0082] 1. Production of Cannabis sativa L. Extract

[0083] Cannabis sativa L. including leaves and flowers was washed clean with running water, and then completely dried naturally. The dried Cannabis sativa L. was crushed with a mixer and then prepared into powder. The powder sample was added to a 50% ethanol as an extraction solvent at a ratio of 1:10 (w:v), and then completely immersed in the solvent. Next, the powder sample was extracted under reflux at 80° C. three times for 3 hours each. The extract was filtered through Whatman No. 2 filter paper. The filtrate was concentrated under reduced pressure at 60° C., thereby producing a Cannabis sativa L. extract (CE).

[0084] 2. Production of Other Natural Extracts

[0085] A Vicia heptajuga Nakai extract (VE), a Calluna vulgaris extract (RE) and a Nigella sativa extract (NE) were produced according to the same method as the method for producing the Cannabis sativa L. extract (CE).

[0086] 3. Production of Extract mixtures

[0087] The Cannabis sativa L. extract (CE), the Vicia heptajuga Nakai extract (VE), the Calluna vulgaris extract (RE) and the Nigella sativa extract (NE) were mixed together as shown in Table 1 below to obtain extract mixtures.

TABLE-US-00001 TABLE 1 AM1 AM2 AM3 AM4 AM5 AM6 CE 100 100 100 100 100 100 VE — 10 20 30 40 50 RE — 10 20 30 40 50 NE — 10 50 30 40 50

[0088] (unit: parts by weight)

Test Example 1: Cytotoxicity Test

[0089] To test the toxicity of each of the Cannabis sativa L. extract (CE) (AM1) and the extract mixtures (AM2 to AM6), differences in toxicity and side effects caused by administration of the extract mixtures in repeated-dose toxicity tests for rats were examined.

[0090] 6-week-old male and female SD rats were divided into a plurality of groups, each consisting of 10 rats (5 male rats and 5 female rats), and each of the extract mixtures AM1 to AM6 was administered to the rats. Each of the extract mixtures was dissolved in a 0.5% methylene chloride (MC) solution and then administered orally once at the same time in the morning every day. This administration was repeated for 13 weeks. Each of the extract mixtures was administered once a day at a daily dose of 3.75 mg/kg to 5 mg/kg. Thereafter, mortality, general symptoms, weight changes, and feed and water intakes were observed.

[0091] As a result, no death occurred within the test period. In view of the above test results, it was confirmed that the extract mixtures AM1 to AM6 had no toxicity problem.

Test Example 2: Antibacterial Effect

[0092] In order to measure the antibacterial activities of the Cannabis sativa L. extract (CE) and extract mixtures AM1 to AM6 produced in Production Example 1, a modification of the agar diffusion method (Piddock, L. J. V, Techniques used for the determination of antimicrobial resistance and sensitivity in bacteria, J. Appl. Bacteriol, 68, p. 307, 1990) using a paper disk was performed as follows.

[0093] First, for antibacterial activity analysis, Escherichia coli (KCTC7039), Bacillus subtilis (KCTC1021), Candida albicans (KCTC7270), Streptococcus mutans (KCTC3065) and Staphylococcus aureus (KCTC1621) were obtained from the Korea Collection for Type Cultures (KCTC), the Korea Research Institute of Bioscience and Biotechnology, the Korea Advanced Institute of Science and Technology, and used in the following experiment. The bacterial strains were used after subculture (Candida albicans: 24° C., Bacillus subtilis: 30° C., Streptococcus mutans: 37° C., Staphylococcus aureus: 37° C., and Escherichia coli: 37° C.).

[0094] One loop of each of the prepared strains was taken and inoculated in 5 ml of saline, and 1 ml of each of the active solutions was inoculated into a 4 to 5-mm-thick medium (Candida albicans—YM Agar (271210. BD), Bacillus subtilis—Nutrient Agar (213000 BD), Streptococcus mutans—Brain Heart (241830 BD), Staphylococcus aureus—Tryptic Soy Agar (236950 BD), Escherichia coli—Nutrient Agar (213000 BD); incubator: EYELA Multi-Thermo incubator MTI-202), and then spread evenly with a bent glass rod. Then, a 6.0-mm sterile filter paper disc (Whatman AA Disc) was placed on each of the media, and a sample solution consisting of a 1:5:4 (v/v/v) mixture of each of the extract mixtures AM1 to AM6, 95% ethanol and distilled water was injected at concentrations of 500 μg/ml, 1000 μg/ml, 1500 μg/ml, 2000 μg/ml and 2500 μg/ml.

[0095] The solvent of each injected extract mixture was volatilized, and incubation was performed at 37° C. for 48 hours. Then, the diameter of the inhibition zone formed around each paper disc was measured. In addition, as a control, butylated hydroxyanisole (BHA) (SIGMA), which is currently widely used as a synthetic antioxidant, was used, and measurement for the control was performed in the same manner as described above

[0096] The results of the measurement are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Inhibition zone (mm) Concentration (mg/ml) AM1, CH AM2 AM3 AM4 AM5 AM6 BHA Bacillus subtilis 2.5 7.6 8.2 10.4 20.6 24.8 19.3 16.6 Escherichia coli 2.5 7.0 10.0 17.6 36.0 38.6 23.4 18.4 Candida albicans 2.5 3.0 3.0 5.0 7.6 8.2 7.0 16.0 Staphylococcus aureus 2.5 3.0 4.0 4.0 6.8 6.2 5.0 16.0 Streptococcus mutans 2.5 2.0 3.0 5.0 8.4 15.4 6.8 3.5

[0097] (unit: mm)

[0098] As shown in Table 2 above, BNA used as the control exhibited some degree of antibacterial effects, but exhibited little antibacterial effect against S. mutans.

[0099] It was confirmed that the extract mixtures AM1 to AM6 of the present disclosure exhibited overall antibacterial effects, and had little antibacterial effect against C. albicans and S. aureus, but AM4 and AM5 exhibited excellent antibacterial effects against B. subtillis and E. coli.

Test Example 3: Antiviral Effect

[0100] 1. Cell Culture

[0101] MDCK Cell Line

[0102] MDCK cells (Madin-Darby canine kidney cell line, No: 10034), which are epithelial-like cells established from the kidneys of Cocker Spaniel species, were cultured in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin in a cell culture chamber at 37° C. under 5% CO.sub.2.

[0103] A549 Cell Line

[0104] A549 cells (human lung adenocarcinoma epithelial cell line, ATCC, No. CCL-185), which are epithelial cells established from human lung cancer cells, were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) containing 10% FBS, 1% penicillin/streptomycin and 1% non-essential amino acids in a cell culture chamber at 37° C. under 5% CO.sub.2.

[0105] 2. Culture of Influenza Virus

[0106] Construction of Reporter Influenza Virus

[0107] A plasmid DNA set required for construction of influenza virus (A/H1N1/WSN) was obtained from UCLA (USA) and transfected into 293T cells and MDCK cells together with a reporter plasmid expressing green fluorescent protein (GFP), thereby constructing reporter influenza virus expressing green fluorescence. MDCK cells were infected with the constructed influenza virus, and the viral infectivity of the cells was determined based on the expression level of GFP.

[0108] Multiplication Culture of Influenza Virus in Fertilized Eggs

[0109] Specific pathogen-free (SPF) fertilized eggs were grown at 37° C. for 10 days, and then a dilution of the above-constructed A/H1N1/WSN influenza virus was injected into the allantoic cavity, and the virus was multiplied at 37° C. for 2 days, followed by collection of the allantoic fluid (containing the virus).

[0110] 3. Cell Viability Test

[0111] Since infection with influenza virus shows cytotoxicity, treatment with an antiviral agent may exhibit the effect of reducing cytotoxicity. Thus, before infection (pre-treatment), after infection (post-treatment) and before and after infection (pre-post-treatment) (MOI=0.5) with A/H1N1/WSN influenza virus, A549 cells were treated with each of AM1 to AM6, and after 3 days, the viability of the cells was measured by MTT assay.

[0112] As a result, as shown in FIG. 1, it was confirmed that the cytotoxicity of the cells treated with each of AM1 to AM6 according to the present disclosure decreased compared to that of the cells to which the virus was administered alone.

Test Example 4: Palatability Test

[0113] 1. Sensory Evaluation Test

[0114] Tea beverages were prepared by diluting each of the Cannabis sativa L. extract (CE) and the extract mixtures AM2 to AM6. Each of the tea beverages were tasted by 10 panelists, and the taste and flavor thereof were scored on a 10-point scale (1 to 10). The average values of the scores (any fraction of 0.5 or more is rounded up to the next higher whole number) are shown in Table 3 below. In the scores in Table 3 below, a higher score indicates higher palatability.

TABLE-US-00003 TABLE 3 AM1 AM2 AM3 AM4 AM5 AM6 Taste 6.0 6.0 6.5 7.0 7.5 6.0 Flavor 6.0 6.5 6.5 7.0 7.5 7.0 Overall palatability 6.0 6.0 7.0 7.0 7.5 6.5 (average)

[0115] (unit: score)

[0116] Referring to Table 3 above, it can be seen that, in the case of AM1 composed of the Cannabis sativa L. extract (CE) alone, the palatability was lowered due to the unique taste and flavor of the Cannabis sativa L. extract, and in the case of the mixtures AM2 to AM6, the palatability increased while the unique taste and flavor of the Cannabis sativa L. extract were neutralized by the other extracts.

[0117] In particular, it was confirmed that, in the case of AM3 to AM5, the palatability greatly increased while the flavor was highly evaluated.

[0118] Therefore, each of the extract mixtures AM3 to AM5 according to the present disclosure may provide a functional food having an excellent antibacterial and antiviral effect while having higher flavor and taste palatability.

[0119] As described above, the present disclosure may provide an antibacterial and antiviral composition which contains an extract of Cannabis sativa L. as an active ingredient, and thus exhibits antibacterial and antiviral activities, and when taken or administered, has no side effects and does not cause bacterial or viral resistance.

[0120] Although the preferred embodiments of the present disclosure have been described in detail above, the scope of the present disclosure is not limited thereto, and various modified and improved forms made by those skilled in the art on the basis of the basic concept of the present disclosure defined in the appended claims also fall within the scope of the present disclosure.