Preparation method of a cannabis extract composition for pain relief, stress-related disease prevention, or stress related disease treatment

Abstract

The present invention relates to a method of preparation of a cannabis extract composition comprising a cannabis extract as an active ingredient for relieving pain and preventing or alleviating a stress-involved disease by suppressing a cyclooxygenase (COX)-2 enzyme and preventing dopamine and serotonin from being suppressed.

Claims

1. A method of making a cannabis extract consisting essentially of: a) grinding cannabis to form ground cannabis; b) extracting the ground cannabis by using a first organic solvent to form a first extracted cannabis extract; c) drying the first extracted cannabis extract to form a first dried cannabis product; d) extracting the first dried cannabis product by using a second organic solvent to form a second extracted cannabis product; e) drying the second extracted cannabis product to form a second dried cannabis product; and f) extracting the second dried cannabis with water to form a third cannabis extract, wherein the amounts of the first organic solvent and the second organic solvent are 2 times to 50 times greater than the amount of the ground cannabis product and the first dried cannabis product; in each extracting step, each of the ground cannabis product and the first dried cannabis product are left to stand in the first organic solvent and the second organic solvent for 1 hour to 72 hours and wherein the third extract is further consisting essentially of 20 to 40 parts by weight of an Erigeron annuus (L.) Pers extract, 20 to 40 parts by weight of a Ceratonia siliqua (L.) Taub. extract, and 20 to 40 parts by weight of a Stellaria media extract based on 100 parts by weight of the third cannabis extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates experimental results for the pain relief of a composition according to an exemplary embodiment of the present invention.

(2) FIG. 2 illustrates the results of an experiment for measuring dopamine of a composition according to an exemplary embodiment of the present invention.

(3) FIG. 3 illustrates the results of an experiment for measuring serotonin of a composition according to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION

(4) Hereinafter, the Examples of the present invention will be described in detail such that a person skilled in the art to which the present invention pertains can easily carry out the present invention. However, the present invention can be implemented in various different forms, and is not limited to the Examples described herein.

Preparation Example: Preparation of Extract

(5) Preparation of Cannabis Extract

(6) Cannabis including leaves and flowers was washed thoroughly with running water and then air-dried completely. The dried cannabis was pulverized with a mixer and then prepared as a powder. After 50% ethanol as an extraction solvent was added to the powder sample at a ratio of 1:10 (w:v) and then the powder sample was completely immersed, extraction was performed repeatedly three times at 80° C. under reflux for each 3 hours. The liquid extract was filtered with a Whatman No. 2 filter paper. A cannabis extract (CE) was prepared by concentrating the filtrate at 60° C. under reduced pressure.

(7) 2. Preparation of Other Natural Extracts

(8) An Erigeron annuus (L.) Pers extract (EE), a Ceratonia siliqua (L.) Taub. extract (IE), and a Stellaria media extract (SE) were prepared using a method which is the same as the preparation method of the cannabis extract (CE).

(9) 3. Preparation of Complex Extract

(10) A complex extract was formed by mixing the cannabis extract (CE), the Erigeron annuus (L.) Pers extract (EE), the Ceratonia siliqua (L.) Taub. extract (IE), and the Stellaria media extract (SE) as shown in the following Table 1.

(11) TABLE-US-00001 TABLE 1 MX1 MX2 MX3 MX4 MX5 MX6 CE 100 100 100 100 100 100 EE —  10  20  30  40  50 IE —  10  20  30  40  50 SE —  10  50  30  40  50 (Unit: parts by weight)

Experimental Example 1: Cytotoxicity Experiments

(12) In order to test the toxicity of cannabis extract (CE) and complex extracts (MX2 to MX6), differences in toxicity and side effects during the administration of the complex extracts were confirmed in a rat repetitive dose toxicity experiment.

(13) Six-week-old SD male and female rats were divided into 10 per group (5 males and 5 females), and the complex extracts MX1 to MX6 were administered to the groups, and after each drug was dissolved in a 0.5% MC solution, oral administration once daily at the same morning time was repeated for 13 weeks.

(14) As a single dose, the solution was administered in an amount of 3.75 mg/kg to 5 mg/kg. Thereafter, the mortality rate, general symptoms, weight change, and food and water intake were observed.

(15) As a result, no dead individuals occurred during the experimental period. In view of the experimental results, it was confirmed that the cannabis extract (CE), the Erigeron annuus (L.) Pers extract (EE), the Ceratonia siliqua (L.) Taub. extract (IE), the Stellaria media extract (SE), and a mixture thereof had no toxicity problems.

Experimental Example 2: Effect of Relieving Pain

(16) Cyclooxygenase (COX)-2 Inhibition Ability

(17) The cannabis extract (CE), which was a sample used for the cyclooxygenase (COX)-2 enzyme inhibition ability measurement experiment, was dissolved in a DMSO solution and used. First, after the COX-2 enzyme, heme, and an inhibitory sample were put into a test tube containing a buffer for a reaction (the control group contained the same amount of DMSO instead of the inhibitory sample), the mixture was allowed to react at 37° C. for 10 minutes. After the reaction was completed for 10 minutes, 10 μL of a substrate (arachidonic acid) was added thereto, and the reaction was further performed for 2 minutes. When the reaction was completed, the reaction was terminated by adding 50 μL of a 1 M HCl solution thereto, and a reduction reaction was caused by adding tartaric acid. After each test tube reaction solution was diluted by 2000 fold and 4000 fold, 50 μL of the reaction solution was each added to a 96-well plate (well plate). After 50 μL of each of a tracer and antiserum was added thereto, the resulting mixture was allowed to react at 25° C. for 18 hours. After the reaction was terminated, the well was emptied and washed 5 times using a washing buffer, and then a coloring reagent was added thereto and the resulting mixture was allowed to react for 1 hour and 30 minutes. When the reaction was completed, the absorbance was measured at 405 nm using an ELISA reader.

(18) As a result, the inhibition ability of the cannabis extract (CE) against COX-2 was the same as in Table 2. When each sample contained the same level (1 mg/mL) of the cannabis extract (CE), the sample exhibited 35.75% COX-2 enzyme inhibitory activity, whereas MX2 to MX6 exhibited an inhibitory activity of 58.57%, 60.38%, 62.47%, 61.97% and 60.27%, respectively. The complex extracts (MX2 to MX6) exhibited inhibitory activity by about 1.7 fold or more compared to MX1 which is a cannabis extract.

(19) Therefore, as a result of inhibiting COX-2 enzyme, which is a synthase of prostaglandin (PG), it was confirmed that the complex extracts (MX2 to MX6) had better inhibitory effects than the cannabis extract at the same concentration.

(20) TABLE-US-00002 TABLE 2 COX-2 inhibition ability MX1 35.75 ± 2.45.sup.1 MX2 58.57 ± 8.08.sup.2 MX3 60.38 ± 5.41.sup.2 MX4 62.47 ± 2.41.sup.2 MX5 61.97 ± 9.06.sup.2 MX6 60.27 ± 4.41.sup.2 (Unit: %)

(21) Further, as a result of confirming the result in the complex extracts MX3 to MX5 showing the highest inhibitory effect, it was confirmed that the cyclooxygenase (COX)-2 inhibitory effect was excellent within the above-described range.

(22) Formalin Pain Model Experiment of Cannabis Extract (CE)

(23) An experiment for the effect of a cannabis extract (CE) on the relief of acute temporomandibular joint pain was performed using a temporomandibular joint (TMJ) pain model (Won, K. A., Kang, Y. M., Lee, M. K., Park, M. K., Ju, J. S., Bae, Y. C., et al. Participation of microglial p38 MAPK in formalin-induced temporomandibular joint nociception in rats. J Orofac Pain 2012; 26(2): 132-141.).

(24) When the specific experimental method is described, experimental animals were acclimatized at 10 or more minutes in an experimental plastic bucket prior to pain response evaluation. 30 μl of 5% formalin was injected using an insulin syringe (0.25×8 mm), and it was observed that consciousness was restored within several seconds after the injection of formalin.

(25) The position of the joint cavity was inferred from the lower inferior boundary surface of the zygomatic arch and mandibular condyle site using a digital exploration, and the site where the injection needle penetrated the joint capsule and touched the lower jaw was recognized to be in the joint cavity. Through a preliminary experiment, the position of the temporomandibular joint space was confirmed by injecting the same amount of a 1 w % evans blue dye as formalin into a separate animal. After the injection of formalin, rubbing or scratching the TMJ site and the face was considered a pain scale, a cumulative recording was performed consecutively 9 times for 45 minutes at an interval of 5 minutes, and an evaluation was performed by dividing the recorded results into a primary pain behavior response (0 to 10 minutes) and a secondary pain behavior response (11 to 45 minutes).

(26) As a result, in an experiment using an animal (Rat), it could be seen that in a formalin-induced acute pain model administered to the temporomandibular joint, a change in pain behavior response upon administration of a cannabis extract (CE) appeared. For the primary pain behavior response, the cannabis extract-administered pain behavior response did not have a significant difference among a formalin injection group, a control group (veh+formalin injection group), and a cannabis extract (CE) injection group (30 μm, 50 μm, and 100 μm), but was effective for adjusting pain at the elapsed time point of 20 to 35 minutes corresponding to the second pain behavior response. These results are illustrated in FIG. 1, and through the result, it could be seen that the cannabis extract (CE) had a pain relieving effect.

(27) Formalin Pain Model Experiment of Complex Extract

(28) In order to investigate the effect of the complex extracts (MX2 to MX6) on the pain relief, the same experiment as the experiment of the cannabis extract (CE) was performed, and the results thereof are shown in comparison with the relative effect on cannabis extract (CE).

(29) For the pain relief effect compared to the cannabis extract (CE), the CE was set at an index of 5, and the temporomandibular joint pain relief of MX2 to MX6 was evaluated as an index of 1 to 10. It can be said to mean that the higher the index is, the better the pain relief effects are.

(30) TABLE-US-00003 TABLE 3 CE MX2 MX3 MX4 MX5 MX6 Temporomandibular joint 5 6 7 8 7 6 pain model (Unit: Index)

(31) As shown in Table 3 above, it was confirmed that the complex extracts MX2 to MX6 relieved pain equivalent to or greater than the cannabis extract (CE). This can be said to mean that when the natural complex extract is used, the effect in which pain is reduced is excellent due to the complex action of each ingredient, compared to when only the cannabis extract (CE) is used.

Experimental Example 3: Effects of Preventing and Improving Stress

(32) 4-week-old ICR male mice {SAMTAKO Bio Korea, Korea} were subjected to an adaptation time of 1 week, and then bred in a space where lighting is turned on and off repeatedly in a unit of 12 hours, and an indoor temperature of 18 to 23° C. and a humidity of 60% were maintained. As a feed, a solid feed was supplied, and there was no restriction on the feed or grade other than the process of inducing stress.

(33) After the complex extracts (MX1 to MX6) in the Preparation Example were dissolved in distilled water, the resulting solutions were orally administered (40 mg/kg) continuously once a day at 14:00 to 15:00 for one month. The experimental group consisted of a total of three groups, which are a control group to which only stress was applied, a group to which the complex extracts (MX1 to MX6) were administered, and a normal control group to which neither stress nor extract was given. Five experimental animals were used for each experimental group, and stress was induced every day for 4 weeks from day 7 day after administration.

(34) The stress-inducing method is used by modifying the method of Wilner et al. (Reduction of sucrose preference by chronic unpredictable mild stress, 1987), and specifically, stress is induced by creating a variety of unexpected mentally stressful situations such as fasting, dietary restriction after fasting (feeding a small amount of feed), suspension of water supply, provision of empty jugs after suspension of water supply, tilted breeding farm, breeding of a number of experimental animals in a breeding farm, sparkling light, cold room, and continuous light.

(35) After the test animal in which the stress was induced was sacrificed by dislocation of the cervical vertebra, the brain was removed and the hippocampus tissue was put into a cooling tube and rapidly cooled. Each tissue sample was treated with 0.4 M perchloric acid and sonicated, and then centrifuged under a condition of 12,000 rpm (4° C.) using a centrifuge, and used for analysis. The analysis was carried out using a high performance liquid chromatography with electrochemical detection (HPLC-ECD) method with a slight modification of the method such as Qi.

(36) Effect of Preventing Suppression of Dopamine

(37) Dopamine is a precursor of norepinephrine as a neurotransmitter in the central nervous system, and is involved in cognition and attention concentration, reward, regulation of motor function, and the like. Since it is known that dopamine secretion is inhibited in a chronic stress situation, the stress prevention and improvement effects caused by the composition of the present invention were confirmed through a dopamine measurement experiment.

(38) As a result of measuring the dopamine concentration using the high performance liquid chromatography with electrochemical detection (HPLC-ECD) method, the value (% of control) compared with the dopamine concentration of the normal control group was shown as an average and standard error. The administration group of the cannabis extract (CE, MX1), the administration groups of the complex extracts (MX2 to MX6), and the stress control group were compared by verifying the significance with a statistical treatment using a Student's T-test, and p<0.05 was considered to be significant and displayed. The results are illustrated in FIG. 2.

(39) Referring to FIG. 2, it can be seen that the concentration of dopamine measured from the hippocampus of the test animals subjected to stress is remarkably lower than that in the normal control group. However, it can be seen that in the groups to which the cannabis extract (CE) and the complex extracts (MX2 to MX6) were administered, the concentration of dopamine was maintained higher than that in the stress control group.

(40) In particular, it can be said to mean that when the natural complex extracts MX3 to MX5 are used, the effect of suppressing the reduction of dopamine is excellent due to the complex action of each ingredient, compared to when only the cannabis extract (CE, MX1) is used.

(41) Therefore, in the case of the above range, it can be seen that the effect of preventing or alleviating stress is shown by suppressing the reduction of dopamine caused by stress.

(42) Effect of Preventing Suppression of Serotonin

(43) As a result, the value (% of control) compared with the serotonin concentration of the normal control group was expressed as the mean and standard error. The administration group of the cannabis extract (CE, MX1), the administration groups of the complex extracts (MX2 to MX6), and the stress control group were compared by verifying the significance with a statistical treatment using a Student's T-test, and p<0.05 was considered to be significant and displayed. The results are illustrated in FIG. 3.

(44) Referring to FIG. 3, it can be seen that the concentration of serotonin measured from the hippocampus of the test animals subjected to stress is remarkably lower than that in the normal control group. However, it can be seen that in the groups to which the cannabis extract (CE) and the complex extracts (MX2 to MX6) were administered, the concentration of serotonin was maintained higher than that in the stress control group.

(45) In particular, it can be said to mean that when the natural complex extracts MX3 to MX5 are used, the effect of suppressing the reduction of serotonin is excellent due to the complex action of each ingredient, compared to when only the cannabis extract (CE, MX1) is used.

(46) Therefore, in the case of the above range, it can be seen that the effect of preventing or alleviating stress is shown by suppressing the reduction of serotonin caused by stress.

Experimental Example 4: Preference Test

(47) Sensory Evaluation Test

(48) Tea beverages were prepared by diluting MX1 and the complex extracts MX3 to MX6. The tea beverages were sampled by 10 tasters, and the taste and aroma were expressed by an index of 1 to 10, and the average values (applied to a rounding of 0.5) are shown in the following Table 4. The higher the number of the index is, the higher the preference is.

(49) TABLE-US-00004 TABLE 4 MX1 MX2 MX3 MX4 MX5 MX6 Taste 6.0 6.0 6.5 7.0 7.0 6.0 Aroma 6.0 6.5 6.5 7.0 7.5 7.0 Overall preference 6.0 6.0 7.0 7.0 7.0 6.5 (average) (Unit: Index)

(50) Referring to Table 4, it can be seen that in the case of MX1, the unique taste and aroma reduced the preference using the cannabis extract alone, and in the case of using the mixture of MX2 to MX6, the preference is enhanced while the taste and aroma peculiar to the cannabis extract are neutralized by other natural extracts. In particular, in the case of using MX3 to MX5, it was confirmed that the aroma was highly evaluated and the preference was also enhanced significantly.

(51) Therefore, in the case of using the complex extracts MX3 to MX5, it is possible to provide a functional food with higher aroma and taste in terms of preference, which has excellent effects of relieving pain and preventing or alleviating a stress-involved disease.

(52) While preferred embodiments of the present invention have been described in detail hereinabove, it is to be understood that the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using basic concepts of the present invention, which are defined in the following claims also fall within the scope of the present invention.