WO2010030082A2 - Mastic extract, and method for extracting same - Google Patents

Abstract

The present invention provides an ethanol extract of mastic and a method for extracting same. The extract of the present invention has a lower toxicity than that of well-known mastic, and has excellent cell regeneration effects and anticancer effects. The extract of the present invention is extracted in ethanol, a solvent which is harmless to a human body, and which has high aqueous solubility, thus making it possible to use the extract of the present invention as additives to drugs, foods, cosmetics, and the like.

Description

Mastic Extract and Extraction Method

The present invention relates to a mastic extract, a preparation method thereof and a product containing the same.

Mastic Resin is a resinous sap extract taken from the Pistachia lentiscus tree native to the Greek island of Hiros. It is a mastic oil, dissolved in mastic resin, mastic gum or vegetable oil. Used as an oil, it is a health food that has been used by Greek and Mediterranean people for more than 5000 years to promote health, especially maintaining the digestive system.

Mastic has a strong antimicrobial activity against pililori, which causes gastric and duodenal ulcers in 1998 [Huwez FU, Thirlwell D, Cockayne A, Ala'Aldeen DA. Mastic gum kills Helicobacter pylori. N Engl J Med. 24; 339 (26): 1946, 1998] In Korea, when Mastic Gum was applied to patients with pylori gastritis, it was reported that the density of pylori decreased and the gastritis was improved [Lim, Lim Hwan, Nam Seung-woo, Myeong Na Hye, Kim Jung Taek, Shin Ji Hyun : The Effect of Mastic Gum on Helicobacter pylori Gastritis, Korean Journal of Gastroenterology 41: 277-283, 2003]. Currently, mastic has been widely known to be effective in gastric and duodenal ulcers and gastritis.

It is also known that mastic has an effect of reducing bacterial growth and oral plaque formation in oral saliva [Takahashi K, Fukazawa M, Motohira H, Ochiai K, Nishikawa H, Miyata T: A pilot study on antiplaque effects of mastic chewing gum in the oral cavity. J Periodontol. 74 (4): 501-505, 2003], and therefore, toothpaste forms containing mastic ingredients are also sold in Korea.

Since the mastic is a poorly soluble substance in aqueous solution, it is difficult to be solubilized, so it is used as a food additive, which is collected naturally, and then pulverized and used as a food additive, or used only as an additive such as a flavor or dressing oil by dissolving it in vegetable oil. It became.

Various studies are underway to overcome this problem, but most studies are focused on dissolving the mastic itself in an aqueous solution. For example, Korean Patent Publication No. 2001-0069608 discloses a method of preparing a mastic gum composition having water dispersibility.

However, since mastic contains not only substances useful for the human body but also substances that may be poisonous, the mastic may be fatal to the human body when the mastic is added to foods and medicines by dissolving all the mastic in oils and the like.

Therefore, the present invention is to provide a high-content, low-toxic mastic extract extracted with ethanol, orally administrable solvent. In addition, to provide a method for manufacturing the same, and pharmaceuticals, food, cosmetics and the like containing the extract.

The present invention provides an ethanol extract of Mastic.

The extract of the present invention is a mastic extract from which a large amount of gum is removed.

In the present invention, mastic means mastic gum and mastic resin, and resin sap collected directly from Pistachia lentiscus tree may be used or purchased commercially.

In addition, in the present invention, the term "ethanol" without any other modification means to include both anhydrous ethanol or hydrous ethanol, hydrous ethanol means ethanol including water, and 00% hydrous ethanol is ethanol content of 00% means (v / v). For example, 30% hydrous ethanol, 50% hydrous ethanol, 70% hydrous ethanol, 80% hydrous ethanol, 90% hydrous ethanol.

The mastic extract of the present invention may be extracted from a mixture of mastic and ethanol mixed in various ratios, for example, may be extracted from a mixture of 20 to 95% by weight of ethanol in 5 to 80% by weight of mastic. have. In addition, the mastic extract of the present invention may be extracted from a mixture of mastic and ethanol 1: 1 to 20 (w / v), 1: 1 (10 / w) extracted from a mixture It is preferable.

The mastic extract of the present invention can be extracted through a conventional extraction method using ethanol, hot water extraction method, supercritical extraction method.

In addition, the mastic extract of the present invention may be liquid or lyophilized by a conventional freeze-drying method to be in powder form.

The present invention also provides a medicament containing the mastic extract of the present invention.

Medicine means, for example, conventional pharmaceutical forms such as injections, tablets, ointments, dressings, bandages, gauze, wherein the mastic extract can be used for normal cell regeneration or anticancer drugs.

In this case, the daily dose or daily dose is preferably 0.1mg to 1000mg, and when administered in a liquid state, preferably administered at a concentration of 10mg / ml to 100mg / ml, but may vary according to the severity, age, sex and complications of the patient. .

The present invention also provides a method of treating cancer by administering the mastic extract of the present invention to a mammal. As used herein, the term “administration” means introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, and the route of administration of the pharmaceutical composition of the present invention is administered via any general route as long as it can reach the target tissue. Can be. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but is not limited thereto.

The present invention also provides the use of the mastic extract of the present invention for producing an anticancer agent.

Pharmaceutical products of the present invention may include additives such as pharmaceutically acceptable diluents, binders, disintegrants, lubricants, pH adjusting agents, antioxidants, dissolution aids, etc., to make the medicines, in addition to the active mastic extract of the present invention The ingredient may contain an anticancer agent.

The present invention also provides a food, beverage, shampoo, soap, toothpaste or cosmetic containing the mastic extract of the present invention.

In addition, the present invention comprises the steps of (a) mixing the mastic with anhydrous ethanol or hydrous ethanol, stirring and dipping; And filtering the supernatant of the dissolved mastic ethanol solution, and concentrating the filtered supernatant under reduced pressure to prepare a mastic extract.

In the manufacturing method of the present invention, step (a) may be performed in an extraction apparatus, a hydrothermal extraction apparatus commonly used by those skilled in the art, or may be performed in a supercritical extraction apparatus.

In addition, the manufacturing method of the present invention provides a manufacturing method further comprising the step of preparing a mastic extract in powder form by freezing drying the mastic extract after step (b).

In the preparation method of the present invention, the mixed amount of ethanol, mastic and ethanol of step (a) is applied in the same manner as described in the mastic extract of the present invention.

In the manufacturing step of the present invention, the stirring in step (a) may be performed at various temperatures, but preferably at 20 to 50 ° C, more preferably at 45 ° C.

The present invention also provides a mastic extract prepared by the method of producing a mastic extract of the present invention.

In addition, the present invention provides a pharmaceutical, food, beverage, shampoo, soap, toothpaste or cosmetic containing the mastic extract prepared by the method of producing a mastic extract of the present invention. The medication is the same as described above.

The mastic extract of the present invention is less toxic than mastic itself, has excellent cell regeneration effect and anticancer effect, is extracted with ethanol which is a solvent harmless to the human body and has high water solubility, and can be used in medicine, food, cosmetics and the like.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Preparation of Liquid Mastic Extract Using Anhydrous Ethanol

To 20 g of mastic (Chios gum mastic), added anhydrous ethanol at a ratio of 1:10 (w / v) and stirred at 50 rpm at 45 ° C. for 3 hours, followed by 1 hour Precipitated at room temperature. Supernatant of mastic ethanol solution was used as Whatman No. After filtering with 2 (Advantec TOYO, Japan), the filtered supernatant was concentrated under reduced pressure with a rotary vacuum evaporator (NN SERIES, Eyela Tokyo, Japan) at 45 ° C. to remove Gum, thereby obtaining a highly pure Mastic extract. 18g was obtained.

Example 2 Preparation of Mastic Extract Powder Using 70% Hydrous Ethanol

Add 50% of 70% hydrous ethanol (v / v) at a ratio of 1:10 (w / v) to 50 g of mastic (Chios gum mastic), 3 hours at 45 rpm at 50 rpm. After stirring, the solution was immersed and extracted at 25 to 30 ° C. for 24 hours, and the supernatant of the mastic ethanol solution was washed with Whatman No. After filtering with 2 (Advantec TOYO, Japan), the filtered supernatant was concentrated under reduced pressure with a rotary vacuum evaporator (NN SERIES, Eyela Tokyo, Japan) at 45 ℃. 450 g of distilled water (H ₂ O) was added as much as the amount of ethanol evaporated during concentration. Mastic solution of the obtained colloidal phase was taken only by supernatant and solidified for 24hr at 0∼ -6 ℃, and then lyophilized in a freeze drying apparatus (manufacturer: Ilshin) for 30 minutes under the atmosphere of -50 ℃ ± 5 ℃, and 40 g of mastic extract was obtained as a white powder. Obtained.

Example 3 Preparation of Supercritical Extract

50 g of mastic (Chios gum mastic) is added to a supercritical extraction unit (manufacturer: Jasco, model name: Jasco SFE), and then the pressure is 250 bar, the temperature is 40 degrees Celsius, and carbon dioxide is present. 500 ml of 70% hydrous ethanol was added thereto, and the mixture was extracted for 5 hours at a flow rate of 3 mL / min. Repeat this step three times and extract all the extracts centrifuged at 12000 rpm for 10 minutes, and then the supernatant was washed with Whatman No. After filtration with 2 (Advantec TOYO, Japan), the filtered supernatant was concentrated under reduced pressure with a rotary vacuum evaporator (NN SERIES, Eyela Tokyo, Japan) at 45 ℃. As concentrated, 450 g of H ₂ O was added as much as the amount of ethanol evaporated. Mastic solution of the obtained colloidal phase was taken only by supernatant and solidified for 24hr at 0∼ -6 ℃, and then lyophilized in a freeze drying apparatus (manufacturer: Ilshin) for 30 minutes at -50 ℃ ± 5 ℃ atmosphere to make 30 g of mastic extract as a white powder. Obtained.

-The cell survival rate described in the table of the experimental example was calculated statistically between each group by t-test using the SPSS 12.0 program.

Experimental Example 1 Confirmation of Low Toxicity Effect on HGF-1 (Human gingival fibroblast) Cells

Human gingival fibroblast (HGF-1) cells (purchased from ATCC) were placed in a 96-well plate at 1 × 10 cells per well.⁴ After dispensing cell, 37 ℃ 5% CO₂ The incubator was incubated for 24 hours. 10 g of the liquid mastic extract prepared in Example 1 was taken, dissolved in 90 g of anhydrous ethanol, and prepared at a concentration of 100 mg / ml (CGM-EtOH solution); As a control, 10 g of mastic (trade name: Chios gum mastic (CGM), manufacturer: Greek Bios Mastic Cultivation Association) was dissolved in 90 g of DMSO and 10% of a solution prepared at a concentration of 100 mg / ml (CGM-DMSO solution). Dilutions were made in cell cultures containing fetal bovine serum (DMEM) to concentrations of 0, 10, 50, and 100 μg / ml. After the cells were treated with 100 µl of the diluted solution, the change in viability after 24, 48 and 72 hours was measured. After a certain time in each concentration group, the treated CGM-DMSO solution and CGM-EtOH solution were removed from the well on a 96-well plate, and then MTT assay {3- (4,5-dimethylthiazol-2yl) at 500 μg / ml concentration. -2,5-diphenyl-2H-tetrazolium bromide assay} 100 μl of each reagent to each well and 37 ℃ and 5% CO₂ The reaction was carried out for 4 hours in the incubator. MTT assay reagent was removed and 150 μl of ethanol and DMSO (dimethylsulfoxide) diluted 1: 1 (v / v) were added to each well and allowed to react at room temperature for 15 minutes. The absorbance of each sample was measured at 570 nm using an ELISA reader (manufacturer: Sunrise Remote Control, model name: Tecan), and thus the CGM-DMSO and CGM-EtOH solutions of HGF-1 cells were measured. The cell hypotoxic effect was measured.

The results are shown in Table 1 below.

Table 1

As a result, in the group treated with 100 μg / ml of CGM-DMSO solution in HGF-1 cells, the cell viability decreased significantly below 57.7% from about 48 hours, but 100 μg / ml of CGM-EtOH solution, which is the extract of the present invention, In the group treated with, the cell survival rate was 137.9% even after 72 hours and no cell death was observed.

Therefore, it is confirmed that the mastic extract of the present invention has an excellent cellular low toxicity effect compared to the known mastic gum.

Experimental Example 2 Confirmation of Low Toxicity Effect on HaCaT (Human Keratinocytes) Cells of Normal Extracts

        HaCaT (Human Keratinocytes) cells (purchased from ATCC) were placed in a 96-well plate with 1 × 10 cells per well.⁴ After dispensing cell, 37 ℃ and 5% CO₂ The incubator was incubated for 24 hours. 10 g of the liquid mastic extract prepared in Example 1 was taken, dissolved in 90 g of anhydrous ethanol, and prepared at a concentration of 100 mg / ml (CGM-EtOH solution); As a control, 10 g of mastic (brand name: Chios gum mastic (CGM), manufacturer: Greek Bios Mastic Cultivation Association) was dissolved in 90 g of DMSO and 10% of a solution prepared at a concentration of 100 mg / ml (CGM-DMSO solution). Diluted in the cell culture (DMEM) containing fetal bovine serum, it was made to a concentration of 0, 10, 50, 100 μg / ml. After 100 μl of these cells were treated, the change in viability after 24, 48 and 72 hours was measured. After a certain time in each concentration group, the treated CGM-DMSO solution and CGM-EtOH solution were removed from the well on a 96-well plate, and then MTT assay {3- (4,5-dimethylthiazol-2yl) at 500 μg / ml concentration. -2,5-diphenyl-2H-tetrazolium bromide assay} 100 μl of each reagent to each well and 37 ℃ and 5% CO₂ The reaction was carried out for 4 hours in the incubator. MTT assay reagent was removed and 150 μl of ethanol and DMSO (dimethylsulfoxide) diluted 1: 1 (v / v) were added to each well and allowed to react at room temperature for 15 minutes. The absorbance of each sample was measured at 570 nm using an ELISA reader (manufacturer: Sunrise Remote Control, model name: Tecan), which resulted in cell low toxicity of CGM-DMSO and CGM-EtOH solutions, respectively, in HaCaT cells. The effect was measured.

The results are shown in Table 2 below.

TABLE 2

As a result, in the group treated with CGM-DMSO solution at the concentration of 100μg / ml in HaCaT cells, the cell viability was remarkably decreased from 24 hours before, but the CGM-EtOH solution as the extract of the present invention was treated at the concentration of 100μg / ml. One group did not affect cell viability until 48 hours and some cells were killed only after 72 hours.

Therefore, it was confirmed that the extract of the present invention is less toxic than dissolving mastic in DMSO in normal cells, HaCaT cells.

Experimental Example 3 Anticancer Effect of Oral Squamous Carcinoma Cells of the Extracts of the Present Invention

        SCC25 cells (human oral squamous carcinoma cells) (purchased from ATCC) were placed on a 96-well plate and the number of cells per well was 1 × 10.⁴ After dispensing cell, 37 ℃ and 5% CO₂ The incubator was incubated for 24 hours. 10 g of the liquid mastic extract prepared in Example 1 was taken, dissolved in 90 g of anhydrous ethanol, and prepared at a concentration of 100 mg / ml (CGM-EtOH solution); As a control, 10% fetal bovine was prepared by dissolving mastic (trade name: Chios gum mastic (CGM), manufacturer: Greek Hisos Mastic Association) in DMSO at a concentration of 100 mg / ml. Diluted in serum-containing cell culture (DMEM) to a concentration of 0, 10, 20, 50, 100 μg / ml. After 100 μl of these cells were treated, the change in viability after 24, 48 and 72 hours was measured. After a limited period of time in each concentration group, the treated CGM-DMSO and CGM-EtOH solutions were removed from the wells in a 96-well plate, followed by 100 μl of 500 μg / ml MTT assay reagent in each well. ℃ and 5% CO₂ The incubator was incubated for 4 hours. MTT assay reagent was removed and 150 μl of ethanol and DMSO (dimethylsulfoxide) diluted 1: 1 (v / v) were added to each well and allowed to react at room temperature for 15 minutes. The absorbance of each sample was measured at 570 nm using an ELISA reader (manufacturer: Sunrise Remote Control, model name: Tecan), and the anticancer effect of SCC25 cells (human oral squamous carcinoma cells) was measured. The results are shown in Table 3.

TABLE 3

As a result, in the case of CGM-EtOH solution in SCC25 cells, cells were rapidly killed at 88%, 13% and 5% from 24 to 72 hours after 20 μg / ml treatment. It was confirmed. However, CGM-DMSO showed that about 50% of cells died from about 48 hours after 50 μg / ml treatment.

Therefore, it was confirmed that the extract of the present invention has a higher anticancer activity than the known mastic DMSO solution.

Experimental Example 4 Confirmation of Anticancer Effect of Melanoma Cells

G361 cells (human melanoma cells) (purchased from ATCC) were placed in a 96-well plate at 1 × 10 cells per well.⁴ After dispensing cell, 37 ℃ and 5% CO₂ The incubator was incubated for 24 hours. 10 g of the liquid mastic extract prepared in Example 1 was taken, dissolved in 90 g of anhydrous ethanol, and prepared at a concentration of 100 mg / ml (CGM-EtOH solution); Mastic as a control (brand name: Chios gum mastic (CGM), manufacturer: Greek Bios Mastic Grower Association) 10 g dissolved in 90 g DMSO (100 mg / ml) solution (CGM-DMSO solution) was diluted in cell culture medium (DMEM) containing 10% fetal bovine serum, and 0, 10, 50, 100 μg / ml Made to concentration. After 100 μl of these cells were treated, the change in viability after 24, 48 and 72 hours was measured. After a limited period of time in each concentration group, the treated CGM-DMSO and CGM-EtOH solutions were removed from the wells on a 96-well plate. ℃ CO₂ The incubator was incubated for 4 hours. MTT assay reagent was removed and 150 μl of ethanol and DMSO (dimethylsulfoxide) diluted 1: 1 (v / v) were added to each well and allowed to react at room temperature for 15 minutes. The absorbance of each sample was measured at 570 nm using an ELISA reader (manufacturer: Sunrise Remote Control, model name: Tecan), and the anticancer effects of G361 cells (human melanoma cells) were measured. The results are shown in Table 4.

Table 4

As a result, in the G361 cells, even when a very small amount of 10 μg / ml of CGM-EtOH solution was treated for 48 hours, the cancer cell survival rate was 39.5%, but many cancer cells were killed. Under the conditions, the cancer cell survival rate was 188.3% and it was confirmed that cancer cell death was only a little.

Therefore, the extract according to the present invention in cancer cells than the known mastic gum It was confirmed that it has excellent anticancer activity at low concentration.

Claims (21)

Methanol extract. The extract of claim 1, wherein a large amount of gum has been removed. The extract of claim 1, wherein the ethanol is 70% hydrous ethanol. The extract of claim 1, wherein the ratio of mastic and ethanol is extracted from 1: 1 to 20 (w / v). The extract of claim 4, wherein the ratio of mastic and ethanol is extracted at 1:10 (w / v). The extract of claim 1, wherein the ethanol extraction is supercritical extraction with ethanol. The extract of claim 1, wherein the extract is lyophilized to form a powder. A pharmaceutical product containing the extract of any one of claims 1 to 6. The pharmaceutical product of claim 8, wherein the medicine is an injection, tablet, ointment, dressing, bandage, gauze. Food, beverages, shampoos, soaps, toothpastes or cosmetics containing the extract of any one of claims 1 to 6. A method of treating cancer by administering an extract of any one of claims 1 to 6 to a mammal. Use of an extract of any one of claims 1 to 6 for the manufacture of an anticancer agent. (a) mixing the mastic with ethanol, stirring and dipping; And (b) filtering the supernatant of the mastic ethanol solution, and concentrating the filtered supernatant under reduced pressure to produce a mastic extract. The method of claim 13, wherein step (a) is performed in a supercritical extraction device. The method of claim 14, further comprising, after step (b), freezing-drying the mastic extract to prepare a mastic extract in powder form. The method according to any one of claims 13 to 15, wherein the ethanol of step (a) is 70% functional ethanol. The method according to any one of claims 13 to 15, wherein the ratio of the mastic and ethanol of step (a) is extracted at 1: 1 to 20 (w / v). 18. The method of claim 17, wherein the ratio of mastic and ethanol in step (a) is extracted at 1:10 (w / v). A pharmaceutical article containing a mastic extract prepared by the method for producing a mastic extract according to any one of claims 13 to 15. 20. A medicament according to claim 19, wherein the medicament is an injection, tablet, ointment, dressing, bandage, gauze. A food, beverage, shampoo, soap, toothpaste or cosmetic containing a mastic extract prepared by the method for producing a mastic extract according to any one of claims 13 to 15.