COMPOSITION CONTAINING EXTRACT OF CANNABIS SATIVA FOR PREVENTING OR TREATING METABOLIC SYNDROME-RELATED DISEASE
20220226406 · 2022-07-21
Inventors
Cpc classification
A23L33/105
HUMAN NECESSITIES
International classification
A23L33/105
HUMAN NECESSITIES
Abstract
The present disclosure relate to a composition for preventing and treating metabolic disease containing a Cannabis sativa extract, and may provide a composition for preventing and treating metabolic disease, which contains an extract of the natural product Cannabis sativa, and thus has little or no side effects when taken or administered, and has an excellent effect of preventing or treating metabolic syndrome by reducing body weight, adipose tissue, blood glucose, triglyceride and cholesterol levels through promotion of AMPK activity and inhibition of the activity of the lipogenic transcription factor SREBP-1c.
Claims
1. A composition for preventing and treating metabolic disease containing a Cannabis sativa extract as an active ingredient.
2. The composition of claim 1, wherein the Cannabis sativa extract contains cannabidiol and terpene.
3. The composition of claim 1, wherein the metabolic disease includes a disease selected from the group consisting of obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis and fatty liver.
4. The composition of claim 1, wherein the composition inhibits an activity of a lipogenic transcription factor by promoting AMPK activity.
5. The composition of claim 4, wherein the lipogenic transcription factor is SREBP-1c (sterol regulatory element-binding protein-1c).
6. A food composition for preventing metabolic disease comprising the composition according to claim 1.
7. A pharmaceutical composition for treating metabolic disease comprising the composition according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0089] 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
[0090] 1. Production of Cannabis sativa Extract
[0091] Cannabis sativa including leaves and flowers was washed clean with running water, and then completely dried naturally. The dried Cannabis sativa was crushed with a mixer and then prepared into powder. 100 g of Cannabis sativa powder was immersed in 1,000 g of ethanol and eluted at 40° C. for 48 hours. Thereafter, the solid was removed by centrifugation, and the remaining supernatant was collected and filtered. The filtrate was subjected to a conventional concentration process under reduced pressure to obtain a Cannabis sativa extract (CE) containing cannabidiol and terpene at a concentration of 0.15 mg/ml.
[0092] 2. Production of Other Natural Extracts
[0093] First, Polypogon monspeliensis was washed, dried and then crushed. The crushed Polypogon monspeliensis was added to a 60% ethanol and extracted for 2 hours. The extract was cooled and then filtered through Whatman filter paper. The filtrate was collected, thus producing a Polypogon monspeliensis extract (PE).
[0094] An Artemisia sylvatica Maxim. extract (AE) and an Aster fastigiatus Fisch extract (OE) were produced according to the same method as the method for producing the Polypogon monspeliensis extract (PE).
[0095] 3. Production of Extract Mixtures
[0096] The Cannabis sativa extract (CE), the Polypogon monspeliensis extract (PE), the Artemisia sylvatica Maxim. extract (AE) and the Aster fastigiatus Fisch extract (OE) were mixed together as shown in Table 1 below to obtain extract mixtures.
TABLE-US-00001 TABLE 1 MT1 MT2 MT3 MT4 MT5 MT6 CE 100 100 100 100 100 100 PE — 10 20 30 40 50 AE — 10 20 30 40 50 OE — 10 50 30 40 50 (unit: parts by weight)
Test Example 1: Cytotoxicity Test
[0097] To test the toxicity of each of the Cannabis sativa extract (CE) (MT1) and the extract mixtures (MT2 to MT6) produced in Production Example 1, differences in toxicity and side effects caused by administration of the extract mixtures in repeated-dose toxicity tests for rats were examined.
[0098] 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 Cannabis sativa extract (CE) (MT1) and the extract mixtures (MT2 to MT6) was administered to the rats. Each of the Cannabis sativa extract and 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 Cannabis sativa extract (CE) and 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.
[0099] As a result, no death occurred within the test period. In view of the above test results, it was confirmed that the Cannabis sativa extract (CE) (MT1) and the extract mixtures (MT2 to MT6) had no toxicity problem.
Test Example 2: Effect of Cannabis sativa Extract on AMPK Activity
[0100] In order to examine the effect of each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6), produced in Production Example 1, on the activity of AMPK, the following experiment was performed. AMPK, a serine/threonine kinase, is activated by decreased ATP levels and increased AMP levels due to intracellular energy depletion, and activation of AMPK inhibits the synthesis of intracellular fat and promotes the degradation of intracellular fat in the human body. Accordingly, AMPK is well known as a therapeutic target against metabolic diseases such as obesity, diabetes, fatty liver, and hyperlipidemia.
[0101] Substrate proteins known to be phosphorylated by AMPK include AMPK, acetyl-CoA carboxylase (ACC), and SREBP-1c (sterol regulatory element-binding protein-1c).
[0102] First, 3T3-L1 adipocytes were cultured, and each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) of the present disclosure was dissolved in DMSO (dimethyl sulfoxide) to a final concentration of 0.2%. The 3T3-L1 adipocytes were treated with each of the solutions. It was confirmed that 0.2% DMSO was not toxic to the cells. A control group was treated with 0.2% DMSO.
[0103] Thereafter, the cells cultured according to the experimental method were collected, lysed, and then placed in a 95-well plate, and the activity of AMPK in the cells was quantified using an AMPK assay kit (CycLex Co. Japan).
[0104] As a result, as shown in Table 2 below, it was confirmed that the AMPK activity in the cells treated with the Cannabis sativa extract (CE) (MT1) increased compared to that in the control group.
TABLE-US-00002 TABLE 2 Control (adipocyte) MT1 MT2 MT3 MT4 MT5 MT6 AMPK activity 1.000 ± 1.344 ± 1.473 ± 1.509 ± 1.772 ± 1.867 ± 1.589 ± (mean ± 0.236 0.003 0.042 0.861 0.132 0.208 0.048 standard deviation)
[0105] Particularly, when comparing between the groups treated with the extract mixtures (MT2 to MT6), respectively, treatment with each of MT3 to MT5 showed the highest rate of AMPK activity promotion, and treatment with each of MT2 and MT6 showed the lowest rate of AMPK activity promotion. Thereby, it could be confirmed that the extract mixtures MT3 to MT5 of the present disclosure had the best effect on the promotion of AMPK activity.
Test Example 3: Effect of Cannabis sativa Extract on SREBP-1c Activity
[0106] After confirming the AMPK activity promotion effect in Test Example 2, the following experiment was performed in order to examine the effect of each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) on the activity of SREBP-1c.
[0107] SREBP (sterol regulatory element-binding protein) is an important transcriptional activator that induces the synthesis of cholesterol and fatty acids in the liver and adipocytes by expressing enzymes related to the biosynthetic pathway of fatty acids and cholesterol, and is classified into three isoforms: SREBP-1a, SREBP-1c, and SREBP-2. Among them, SREBP-1c is most often expressed in tissues such as fat, liver, and muscle, and it is known that ACC1 (acetyl-CoA carboxylase 1), FAS (fatty acid synthatase), SCD1 (stearoyl-CoA desaturase 1) and SREBP-1c, which are major enzymes involved in the synthesis of fat, function as transcription factors that are expressed by themselves. In addition, it is known that phosphorylation of SREBP-1c by AMPK reduces the activity of SREBP-1c.
[0108] According to the above-described experimental method, 3T3-L1 adipocytes were treated with 1,000 μg/ml of each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) for 7 days. For comparison, 3T3-L1 adipocytes were treated with 1,000 μg/ml of metformin (MET), a representative AMPK promoter among commercially available pharmaceuticals. Thereafter, the target DNA (5′-TCACCTGA-3′)-binding activity of SREBP-1c in the cells was measured using a SREBP-1 transcription factor ELISA (Cayman Chemical Co. Ann Arbor, Mich., USA).
[0109] As a result, as shown in
[0110] Thereby, it could be confirmed that the extract mixtures (MT2 to MT6) of the present disclosure inhibited the target DNA-binding activity of the lipogenic transcription factor SREBP-1c and showed stronger inhibitory activity than metformin.
Test Example 4: Effect of Reducing Body Weight Gain Caused by High-Fat Diet Therapy
[0111] High-Fat-Diet Therapy Test Groups
[0112] Each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) of the present disclosure and phloretin was dissolved in 0.5% DMSO, and then was administered orally to each mouse of each test group at a dose of 20 mg/kg every other day for 14 weeks. Another mouse group was administered DMSO in the same manner.
[0113] The body weight of each mouse was measured in units of 0.01 g at the same time every day, and the food intake was measured once a week (every 7 days).
[0114] After 12 weeks of administration of the high-fat diet, adipose tissue was isolated from each mouse and weighed.
[0115] Standard Diet Therapy Test Group
[0116] The remaining one mouse group was administered DMSO in the same manner using standard diet therapy at the same temperature under the same environmental conditions as the above-described high-fat diet therapy, and then the body weight of each mouse was measured. After 12 weeks of administration of the diet, adipose tissue of each mouse was isolated, and the size and weight thereof were measured and used as a negative control.
[0117] Isolation of Adipose Tissue
[0118] The adipose tissues isolated from the high-fat-diet therapy test groups and the standard diet therapy test group were subjected to histological examination using a hematoxylin and eosin (H&E) staining method.
[0119] Specifically, each adipose tissue was embedded in paraffin, frozen, sectioned to a thickness of 8 μm using a cryocut microtome, and then mounted on a slide glass. Each of the slides having the section mounted thereon was deparaffinized by 5 minutes of immersion in xylene, and hydrated using ethanol at gradually decreasing concentrations (100%-95%-85%-70% for 2 minutes each).
[0120] Thereafter, each slide was washed with water to remove the remaining ethanol, and stained with hematoxylin for 6 minutes. Then, each slide was immersed in and taken out of a mixed solution of 1% hydrochloride-ethanol (HCl-EtOH), and this process was repeated three times so that the hematoxylin was sufficiently absorbed into the tissue. Then, the slide was immersed in and taken out of 0.5% ammonia water, and this process was repeated 10 times, thereby fixing the stain.
[0121] The tissue section stained with hematoxylin was stained again with eosin for 1 minute and dehydrated using ethanol at increasing concentrations (70%-85%-95%-100% for 2 minutes each).
[0122] Effect on Weight Loss
[0123] Each of the dehydrated tissue slides was washed clean by 5 minutes of immersion in xylene, and then completely dried at room temperature. Then the section of the tissue was observed under a microscope, and the body weight was measured.
[0124] As a result, as shown in
[0125] This suggests that administration of each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) of the present disclosure has the effect of suppressing weight gain. It was confirmed that this effect of suppressing weight gain was not an effect attributable to a difference in food intake, from the fact that there was no difference in food intake between the test groups.
Test Example 5: Effects of Cannabis sativa Extract on Blood Glucose, Triglyceride and Cholesterol Levels
[0126] Effect on Blood Glucose and Triglyceride Levels
[0127] In order to examine the effect of each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) of the present disclosure, confirmed in Test Examples 2 to 4, on a living body in a high-fat diet-induced obesity mouse model, the following experiment was performed.
[0128] The collected blood was clotted and then centrifuged at 8000 rpm for 10 minutes, and the serum was collected. The levels of glucose and triglyceride in the serum were measured using a blood biochemical analyzer (Modular analytics, Hitachi, Japan).
[0129] As a result, as shown in
[0130] Effect on Cholesterol Level
[0131] The collected blood was clotted and then centrifuged at 8000 rpm for 10 minutes, and the serum was collected. The levels of LDL-cholesterol and total cholesterol in the serum were measured using a blood biochemical analyzer (Modular analytics, Hitachi, Japan).
[0132] As a result, as shown in
[0133] Thereby, it could be confirmed that each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) had the effect of treating metabolic diseases, such as obesity, diabetes, hypertriglyceridemia and hypercholesterolemia, by reducing body weight, adipose tissue and liver weight and lowering the levels of glucose, triglyceride, LDL cholesterol and total cholesterol in blood.
Test Example 6: Palatability Test
[0134] Tea beverages were prepared by diluting each of the Cannabis sativa extract (CE) (MT1) and extract mixtures (MT2 to MT6) of the present disclosure. 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
[0135] Table 3 below, a higher score indicates higher palatability.
TABLE-US-00003 TABLE 3 MT1 MT2 MT3 MT4 MT5 MT6 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) (unit: score)
[0136] Referring to Table 3 above, it can be seen that, in the case of MT1 composed of the Cannabis sativa extract (CE) alone, the palatability was lowered due to the unique taste and flavor of the Cannabis sativa extract, and in the case of the mixtures MT2 to MT6, the palatability increased while the unique taste and flavor of the Cannabis sativa extract were neutralized by the other extracts.
[0137] In particular, it was confirmed that, in the case of MT3 to MT5, the effect of preventing and treating metabolic disease was excellent, and the palatability greatly increased while the taste and flavor were highly evaluated.
[0138] Therefore, each of the extract mixtures MT3 to MT5 according to the present disclosure may provide a functional food having an excellent effect on the prevention and treatment of metabolic disease while having higher flavor and taste palatability.
[0139] As described above, the present disclosure may provide a composition for preventing and treating metabolic disease, which contains an extract of the natural product Cannabis sativa as an active ingredient, and thus has little or no side effects when taken or administered, and has an excellent effect of preventing or treating metabolic syndrome by reducing body weight, adipose tissue, blood glucose, triglyceride and cholesterol levels through promotion of AMPK activity and inhibition of the activity of the lipogenic transcription factor SREBP-1c.
[0140] 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.