WO1995023604A1

WO1995023604A1 - Cell growth stimulating compositions containing aloesin

Info

Publication number: WO1995023604A1
Application number: PCT/KR1995/000016
Authority: WO
Inventors: Kyeong Ho Kim; Jeong Hill Park; Chung Ki Sung; Jin Woong Kim; Jae Sue Choi; Seung Ki Lee; Yeong Shik Kim; Byeong Hwa Son; Myung Hee Chung; Young In Park; Kyu Won Kim; Chong Kil Lee
Original assignee: Namyang Aloe Co., Ltd.
Priority date: 1994-03-03
Filing date: 1995-03-02
Publication date: 1995-09-08
Also published as: AU1961395A

Abstract

The present invention relates to a cell growth stimulating composition containing aloesin as an active ingredient. More specifically, the present invention relates to a composition for stimulating cell growth, especially for stimulating epidermal cells and hepatic cells, which contains aloesin as an active ingredient. Aloesin is a component of Aloe vera, for which an effect for blocking ultraviolet ray from skin and an activity as mushroom tyrosinase inhibitor have been disclosed in the prior art. However, accordint to the present invention, it has now been identified that aloesin has the novel pharmacological use as a cell growth stimulating agent which has never been disclosed in the prior art.

Description

CELL GROWTH STIMULATING COMPOSITIONS CONTAINING ALOESIN

TECHNICAL FIELD

The present invention relates to a cell growth stimu¬ lating composition containing aloesin as an active ingre¬ dient. More specifically, the present invention relates to a composition for stimulating cell growth, especially for stimulating epidermal cells and hepatic cells, which contains aloesin as an active ingredient.

BACKGROUND ART

Aloe is a medicinal herb belonging to Liliaceae which has been widely and popularly used from ancient times. The record of the first use of aloe as a medicine can be found from a mural painting in Egyptian temple in which Aloe vera is described as the plant of immortality. The specific record of the use of aloe can be found in Sumeri- an clay tablets around 1750 B.C. In addition, Egyptian Book of Remedies around 1550 B.C. describes the use of aloe as an agent for treatment of infections and for skin massage and also the method for preparing aloe as a medi¬ cine. Meanwhile, in A.D. 74 Dr. Disscordes, who is a Greek doctor, discloses in his book, De Materis Media, that Aloe vera is a very effective laxative and has the effect of treating wound, furuncle, chilblained skin, etc., and also alleviating genital ulcers and tonsillitis. In practice, various aloe plants, for example, Aloe perryi, Aloe forex, Aloe barbadenis, etc., have been popularly used for a long time in the form of suntan ointment, suntan powder, cosmetic cream, lotion, shampoo, and the like. Although aloe has been used from long ago, the scien¬ tific study of aloe regarding that its pharmacological activities are derived from what components and through what acting mechanism has not been conducted until compar- atively lately. In practice, as a result of the test for determining. the wound healing effect of aloe on burn patients in the convalescent stage it has been reported that the complications after wound healing, including erythema, pruritus and hypertropic scarring are found only in below 10% of the aloe treatment group whereas in 25% of the control group. In addition, it has been also report¬ ed that when burned albino rabbits are treated by applying the aloe cream to wound portion, the wound is cured with¬ out any scar after 2 weeks, while in the animals treated only with vaseline and gauze the treatment is completed with remaining a big scar after 3 to 4 weeks [see Davis, R.H. , Leitner, M.G., Russo, J.M. and Byrne, M.E., J. of American Pediatric Medical Association, 79: 559-562, 1989] . Further, it has been disclosed that when a par- tially purified aloe extract is intravenously injected into inflammation-induced rat, it shows an effect of reducing pain and inhibiting abdominal vascular permeation [see Beppy, H. , Fujita, K. , Shinzato, M. and Murakami, A., 12th Annual Meeting of Fujita Medical Society Abstract, 4: 51-52, 1980], It has also been reported that in SD male rats in which edema is induced by injection of mustard into their hind legs the injection of Aloe vera extract can inhibit edema by 44% and that in the test for deter¬ mining PMN (polymorph nuclear leukocyte) infiltration in the portion of inflammation induced by subcutaneous injec¬ tion of gelatin into mouse the administration of Aloe vera extract can inhibit PMN infiltration by about 58% [see Davids, R.H. , Kabbai, J.M. and Maro, N.P., Proceedings of the Pennsylvania of Science, 60: 67-70, 1986]. From these results, it can be identified that aloe has various pharmacological activities including wound healing effect, anti-inflammatory activity, anti-ulcer activity, anti- cancer activity ( cell differentiation activity) , anti¬ microbial activity, immunomodulatory activity, cell growth stimulating activity, skin penetrating activity and the like . However , no systematic and definite result of study regarding that each pharmacological activity of aloe plants is derived from what kind of the components has been reported yet.

DISCLOSURE OF INVENTION

Thus , the present inventors have taken more scientific approaches by conducting series of pharmacological tests for the components contained in aloe plants . As a re¬ sult , we have identified that aloesin, one of the effec¬ tive components of aloe plants , has an excellent cell growth stimulating activity , and then completed the present invention.

Accordingly, it is an object of the present invention to provide a use of aloesin as an agent for stimulating cell growth, especially growth of epidermal cells and hepatic cells.

It is an another object of the present invention to provide a composition containing aloesin as an active ingredient which stimulates growth of cells, especially epidermal cells and hepatic cells.

BRIEF DESCRIPTION OF DRAWINGS

For a thorough understanding of the nature and objects of the invention, reference should be had to the following detailed description taken in connection with the acco - panying drawings in which:

Figure 1 is the result of Western Blot Analysis which determines the influence of aloesin on the activity of antibody cdk2 in hepatoma cell line sk-Hep-l according to Example 7-2;

Figure 2 is the result of Western Blot Analysis which determines the influence of aloesin on the activity of antibody cyclin E in hepatoma cell line sk-Hep-l according to Example 7-2;

Figure 3 is the result of Western Blot Analysis which determines the influence of aloesin on the activity of antibody cdc25 in hepatoma cell line sk-Hep-l according to Example 7-2; and

Figure 4 is the result of Immune Complex Kinase Assay which determines the influence of aloesin on the activity of antibody cdk2 in hepatoma cell line sk-Hep-l according to Example 7-3.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, a cell growth stimulating composition containing aloesin, which is one of the effective components of aloe plants, as an active ingredient is provided. The composition of the present invention can effectively stimulate the growth of cells, especially epidermal cells and hepatic cells.

Up to date, aloe has been used generally in the form of a dry extract which is obtained by extracting leaves of various aloe plants belonging to Aloe genus such as Aloe vera with water and then evaporating the resulting aqueous extract to dryness. However, since the dry extract of aloe still contains the other effective components of aloe, in addition to certain components having the desired pharmacological effect, it may occasionally cause the undesirable side effects. Accordingly, in the present invention, by selectively isolating aloesin from aloe plants and then identifying its pharmacological effect the effective component of aloe, i.e. aloesin can be utilized without any side effects other than the desired effect.

Aloesin isolated and utilized in the present invention is a component extacted from leaves of Aloe vera and is represented by the following structure [molecular formula: ^C19^H22°9' ^molecular weight: 394] :

Aloesin [ chemical name = 4H-l-benzopyran-4-one-8-β-D-glu- copyranosyl-7-hydroxy-5-methyl-2- ( 2-oxopropyl) ]

In the prior art, it has been reported that aloesin has an effect for blocking ultraviolet ray from skin [see Strickland, F.M. , Pelley, R.P., Hill, D. and Kripke, M.L., J. Investigative Dermatology, 98: 653, 1992]. In addi¬ tion, it has also been disclosed that aloesin has an activity as mushroom tyrosinase inhibitor [see Akira Yagi, Toshimitsu Kanbara and Naoka Morinobu, Planta Medica, 515- 517, 1987]. However, any pharmacological use of aloesin as a medicinal agent has not been reported yet.

According to the present invention, it has been iden¬ tified that aloesin which is obtained by treating aloe plants according to known method and then isolating and purifying aloesin has a potent effect for stimulating cell growth, especially for stimulating growth of skin epithe- lial cells and hepatic cells.

In the present invention, although the dosage of aloesin which can be administered to the patients suffer¬ ing from skin wound can be varied depending on the size and condition of wound, aloesin can be administered gener¬ ally in an amount of O.lmg to 500mg, preferably in an amount of 0.4mg to 125mg, per a day, per kg of body weight of adult man to obtain the desired effect. In addition, in patients suffering from hepatic diseases such as hepa- toma, hepatitis, etc., a suitable daily dosage of aloesin can be varied depending on the severity of diseases and is generally O.lmg to 500mg, preferably 0.4mg to 125mg, per kg of body weight of adult man to provide the desired effect for stimulating the growth of hepatic cells.

Aloesin which is used in the present invention can be obtained by treating aloe plants according to the method known in this technical field, for example, the method disclosed by Yagi, et al. [see Akira Yagi, Kenji Makino and Itsuo Nishioka, Chem. Pharm. Bull. 25(7), 1771-1776, 1977]. For example, aloesin can be obtained by cleanly washing fresh aloe plants, for example, whole leaves of Aloe vera, with water, extracting the washed plant with a lower alcohol such as methanol, ethanol, propanol, buta- nol, etc., a lower ketone such as acetone, methylethyl ketone, etc., or a mixture thereof, suspending the result¬ ing extract in water, extracting again the suspension with a lower ether such as diethyl ether, etc., a lower ester such as ethyl acetate, etc. , a halogenated hydrocarbon such as carbon tetrachloride, dichloromethane, chloroform, etc., or a mixture thereof, extracting again the aqueous layer with a lower alcohol, and then subjecting the alco¬ hol extract to silica gel column chromatography eluting with a mixed solvent consisting of halogenated hydrocarbon and lower alkanol in the mixing ratio of 2:1 to 100:1, preferably 5:1 to 20:1, or a mixed solvent consisting of lower ester, lower alcohol and water in the mixing ratio of 20:1:0 to 6:4:2 to isolate aloesin in a pure state.

When aloesin is clinically used, aloesin can be admin¬ istered to patients suffering from burn, trauma, etc, who require cell growth stimulating effect, or patients who has a damage of tissue cells caused by hepatoma, hepati¬ tis, etc. , in the form of conventional pharmaceutical preparations according to a conventional manner utilized in the field of pharmaceutical preparation. The pre- ferred pharmaceutical preparations which can be suitably used for this purpose include preparations for oral admin¬ istration such as tablets, hard or soft capsules, solu¬ tions, suspensions, etc., injections for parenteral admin¬ istration such as injectable solutions or suspensions, etc., preparations for topical use such as ointments, creams, gels, lotions, etc., and the like. Such pharma¬ ceutical preparations can also be prepared by utilizing pharmaceutically acceptable carriers conventionally used in this technical field, for example, excipients, binders, disintegrants, lubricants, solubilizing agents, suspending agents, preservatives or extending agents for preparations for oral administration, stabilizers, preservatives, solu¬ bilizing aids, buffers, agents for isotonicity, etc., for injections, or aqueous or oily ointment bases, anti- oxidants, preservatives, extending agents, etc., for topical preparations. The preparation for stimulating growth of epidermal cells and hepatic cells which contains aloesin according to the present invention can be prepared so that a unit dosage form can contain the above-mentioned preferred daily dosage of aloesin or its 1/2, 1/3, 1/4, 1/5 or 1/6. This unit dosage form can be administered 1 to 6 times per day.

The effect of aloesin for stimulating cell growth can be observed according to the determination of cell count using a he ocytometer or Coulter counter as described by Doyle, et al. [see Doyle, A., Griffiths, J.B. and Newell, D.G., Cell & Tissue Culture: Laboratory Procedures, 10E, WILEY (1993)], the quantitative method by means of a dye such as Hoechst 38825, acridine orange, diaminobenzoic acid (DABA) , a method for determination of DNA synthetic rate using a radioisotope [³H]-methyl-thymidine, and recently a method for determination of cell growth without using any radioisotope, for example, methods for deteπai- nation of a cell component BCECF (2 ^■ ,7 '-bis-(carboxy- ethyl)-5(or 6)-carboxyfluoresin acetoxymethyl ester) [see Kolber et al., Journal of Im unological Methods 108: 225- 264, 1988], determination of neutral red uptake which reflects lysosome activity [see Borenfreund & Purener, Journal of Tissue Culture Methods 9: 7-9, 1984] or deter¬ mination of 4-methylumbelliferone phosphate decomposition which reflects alkaline phosphatase activity. Among these methods, a method for determination of certain components including BCECF, neutral red, 4-methylumbellif- erone phosphate, etc. , takes advantage of characteristic features of these components that they contain a uniform amount of enzymes or shows an increased activity in pro¬ portion to the increase of cell counts. However, these methods have some disadvantages in that their sensitivity is lower than that of methods for determination of induc¬ tion rate of a radioisotope [³H] -methyl-thymidine. Accordingly, the following examples of the present inven- tion were conducted utilizing a method for determination of induction rate of [ H]-methyl-thymidine and a MTT assay for determination of activity of cellular reductase to determine the effect of aloesin for stimulating cell growth. In addition, Western Blot Analysis and Immune Complex Kinase Assay were conducted to identify that the cell growth stimulating activity of aloesin is derived from the stimulation of DNA synthesis in cells without any cell damage.

As the cell lines used in the experiments of the following examples, hepatoma cell line sk-Hep-l, skin epithelial cell line HT-1080, kidney epithelial cell line COS-7 and gastric cancer cell line KATO-111 were obtained from Cancer Institute of Seoul National University in Korea, and then incubated with a culture solution, which is prepared by dissolving DMEM (Dulbecco's Modified Ea¬ gle's Medium, made by Gibco) in 1 liter of deionized water, adjusting the resulting solution to pH 7.4, adding 10% calf fetal serum, 1X10^"7 M insulin and 50mg/l of gentamycine thereto, and then sterilizing the resulting mixture with a Millipore filter, in an incubator of 37°C while maintaining 5% carbon dioxide.

The present invention will be more specifically ex¬ plained in the following examples. However, it should be understood that the present invention is not limited to those examples in any manner.

EXAMPLE 1 : Isolation and purification of aloesin

Whole leaves of Aloe vera were cleanly washed with water, divided into a suitable size and then lyophilized. About lOOg of lyophilized aloe leaves was added to 1 liter of methanol and extracted under reflux with heating. The extracted methanol fraction was concentrated under reduced pressure, and then the residue was suspended in a suitable amount of deionized water. The resulting suspension was transferred to a separatory funnel and then extracted 3 times with 200ml of ethyl acetate each time. The residu- al aqueous layer was extracted 3 times with 200ml of butanol each time. The extracted butanol fractions were combined and then subjected to column chromatography on silica gel eluting with a mixed solvent of ethyl acetate/ ethanol/water (10:1:0.5) or a mixed solvent of chloroform/methanol (5:1) to obtain the desired aloesin- containing fraction. The obtained aloesin-containing fraction was subjected again to silica gel column chroma¬ tography eluting with a mixed solvent of ethyl acetate/methanol/water (10:1:0.2) or a mixed solvent of chloroform/methanol (7:1) to obtain more purified aloesin- containing fraction. This aloesin-containing fraction was recrystallized from ethyl acetate or a mixture of ether and methanol to obtain about 70mg of the desired aloesin.

Aloesin obtained above shows NMR and mass spectrum data which are identical to those disclosed in the refer¬ ence [see John M. Conner, Alexander I. Gray, Tom Reynolds and Peter G. Waterman, Phytochemistry , 29, 941-944, 1990]. specifically, when about 3mg of the obtained aloesin is dissolved in 600μl of DMSO-d_g (99.8%) to measure its NMR spectrum, NMR spectrum of aloesin shows characteristic peaks at 2.20, 2.63, 3.75, 6.07, 6.65 pp . In addition, mass spectrum (EI+, 70eV) of aloesin shows peaks of 394 (molecular ion peak) , 245 (basic peak) , 304, 203, 163, etc.

EXAMPLE 2 : Effect of aloesin for stimulating cell growth on human hepatoma cells

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, made by Gibco ) was dissolved in 1 liter of deionized water . After adjusting the resulting solution with sodium carbonate and hydrochloric acid to pH 7 . 4 , 10% calf fetal serum, 1X10^"7M insulin and 50mg/l of gentamycin were added thereto . The mixture was sterilized through a

Millipore filter to obtain the culture solution . Human hepatoma cell line sk-Hep-l which was obtained from Cancer Institute of Seoul National University was inoculated into the culture solution in the ratio of 1X10⁶ cells per 25cm² of the surface area of T flask and then incubated for 43 hours in an incubator of 37°C while maintaining 5% carbon dioxide . The cultures were transferred to a 24 -well plate , subcultured for one day and then treated with 100 to O . OOOlμg/ml of aloesin . For the control group , the cultures were treated with the same amount of PBS (phos¬ phate buffered saline) instead of aloesin . After 3 6 hours from this treatment, the treated sample was treated w ith ³ H- label led thymidine in the concentrati on o f lμCi /ml . After 12 hours , the medium was removed from each well , and the cells were fixed with methanol and then washed with PBS . The cells were washed two times with 10 % trichloroacetate to remove unreacted radioactive thymidine , dissolved in IN sodium hydroxide and then neutralized with IN hydrochloric acid. Then , the radio- activity introduced into DNA was measured with a scintil¬ lation counter (Pharmacia 1024 ) . The measured result is described in the following Table 1.

Table 1. Amount of radioactive thymidine introduced into hepatoma cell line sk-Hep-l DNA depending on aloesin concentration

Concentration dpm/well Percentage to (μg/ml) (M ± S.D.) control group

Control group 1638.4 ± 75.0 100.0

100 1835.5 ± 61.8 106.6

10 1766.3 ± 33.4 115.8

^•

1 I 2228.1 ± 21.7 156.1 ;

0. 1 5352.5 ± 135.8 204.8

0. 01 2337.6 ± 264.3 151.1 | i

1

0. 001 2384.0 ± 197.6 122.7 !

0 0001 1954.1 ± 21.9 106.6

Note : M ± S.D. = Mean ± Standard deviation

EXAMPLE 3 : Effect of aloesin for stimulating cell growth on human epithelial cells

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, made by Gibco) was dissolved in 1 liter of deionized water. After adjusting the resulting solution with sodium carbonate and hydrochloric acid to pH 7.4, 10% calf fetal serum, 1X10 M insulin and 50mg/l of genta ycin were added thereto. The mixture was sterilized through a Millipore filter to obtain the culture solution. Human epithelial cell line HT-1080 which was obtained from Cancer Institute of Seoul National University was inoculated into the culture solution in the ratio of 1X10⁶ cells per 25cm² of the surface area of T flask and then incubated for 48 hours in an incubator of 37°C while maintaining 5% carbon dioxide. The cultures were transferred to a 24-well plate, subcultured for one day and then treated with 100 to lμg/ml of aloesin. For the control group, the cul¬ tures were treated with the same amount of PBS (phosphate buffered saline) instead of aloesin. After 36 hours from this treatment, the treated sample was treated with ³H- labelled thymidine in the concentration of lμCi/ml. After 12 hours, the medium was removed from each well, and the cells were fixed with methanol and then washed with PBS. The cells were washed two times with 10% trichloro- acetate to remove unreacted radioactive thymidine, dis¬ solved in IN sodium hydroxide and then neutralized with IN hydrochloric acid. Then, the radioactivity introduced into DNA was measured with a scintillation counter (Phar¬ macia 1024) . The measured result is described in the following Table 2.

Table 2. Amount of radioactive thymidine introduced into cell line HT-1080 DNA depending on aloesin con¬ centration

Concentration dpm/well i Percentage to (μg/ml) (M ± S.D.) control group

Control group 1577.3 ± 161.9 100.0

100 2363.0 ± 321.0 149.9

25 12993.7 ± 749.6 823.8

5 7663.7 ± 1050.5 485.9 ,

1 4007.3 ± 387.7 254.1

Note : M ± S.D. = Mean ± Standard deviation

EXAMPLE 4 Effect of aloesin for stimulating cell growth on monkey's kidney cell line

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, made by Gibco) was dissolved in 1 liter of deionized water. After adjusting the resulting solution with sodium carbonate and hydrochloric acid to pH 7.4, 10% calf fetal serum, 1X10~⁷M insulin and 50mg/l of gentamycin were added thereto. The mixture was sterilized through a Millipore filter to obtain the culture solution. Mon¬ key's kidney cell line C0S7 which was obtained from Cancer Institute of Seoul National University was inoculated into the culture solution in the ratio of 1X10⁶ cells per 25cm² of the surface area of T flask and then incubated for 43 hours in an incubator of 7°C while maintaining 5% carbon dioxide. The cultures were transferred to a 24-well plate, subcultured for one day and then treated with 100 to O.OOOlμg/ml of aloesin. For the control group, the cultures were treated with the same amount of PBS (phos¬ phate buffered saline) instead of aloesin. After 36 hours from this treatment, the treated sample was treated with ³H-labelled thymidine in the concentration of lμCi/ml. After 12 hours, the medium was removed from each well, and the cells were fixed with methanol and then washed with PBS. The cells were washed two times with 10% trichloroacetate to remove unreacted radioactive thymidine, dissolved in IN sodium hydroxide and then neutralized with IN hydrochloric acid. Then, the radio- activity introduced into DNA was measured with a scintil¬ lation counter (Pharmacia 1024) . The measured result is described in the following Table 3.

Table 3. Amount of radioactive thymidine introduced into DNA of cell line COS7 depending on aloesin concentration

Concentration dpm/well Percentage to

(μg/ml) , (M ± S.D.) control group

Control group 1538.7 ± 142.3 100.0

100 1639.9 ± 91.7 106.6

10 1781.6 ± 166.2 115.8

1 2402.1 ± 57.0 156.1

0.1 3150.5 ± 164.6 204.8

0.01 2325.4 ± 204.3 151.1

0.001 1887.9 ± 52.3 122.7

0.0001 1639.9 ± 91.7 106.6

Note : M ± S.D. = Mean ± Standard deviation

EXAMPLE 5 : Effect of aloesin for stimulating cell growth in primary cultures of rat hepatic cells

Male Sprague-Dawley rat weighing 180 to 220g was anesthetized with ether. The abdomen of rat was incised and then large lobes were removed from left and center of liver according to the known method [see Anderson and

Higgins, Biochem. Biophy. Acta. 129, 445, 1931] to reduce the liver's weight by 1/3. Then, the abdomen was sutured again. After operation, rat was received only 5% dex¬ trose solution without any food.

The extraction of hepatic cells was conducted accord¬ ing to the method of Dickens and Peterson [see Dickens and Peterson, Biochem. Pharmacol. 29, 1231, 1980]. After 24 hours from operation, rat was anesthetized with intraperi- toneal injection of urethane in an amount of lg/kg. The abdomen was shaved to remove hairs and then the shaved portion was disinfected with iodine tincture. The subse- guent experimental procedures were carried out under sterilized condition. The abdomen was incised along the center line and then both ends were cut down to expose the hepatic portal to which a 18 gauge catheter was connected. Inferior vena cava was cut down, and then Hank's balance salt solution was infused in the flow rate of 15 to 20ml per minute through the catheter to remove the blood in liver. After removing blood, the inferior vena cava was ligated, the chest was opened to expose the superior vena cava to which a 16 gauge catheter was connected. The fluid flowing out through the superior vena cava was recycled. This procedure should be practiced while maintaining the temperature of 37°C and under continuously injection of a gas consisting of 95% oxygen/5% carbon dioxide at the rate sufficient to saturate the perfusion. Collagense (Sigma, C1030) was added to the perfusion in the concentration of 0.05% to 0.06% and this mixture was perfused for 15 minutes during which the swelling of liver _Can be visually observed.

The liver was completely extracted, transferred into 60ml of the perfusion and then cut down with a sterilized scissor. The separated cells were filtered with a 250μm nylon membrane. The filtrate was centrifuged at 50Xg for 40 minutes and washed three times with the perfusion. The final precipitate was dispersed in the cell culture solution. The survival rate, of the prepared liver cell dispersion was determined by adding 900μl of the staining solution consisting of 0.4% tryphan blue in physiological saline to lOOμl of the dispersion, standing this mixture for 5 minutes and then counting the stained died cells and the unstained living cells under a microscope and calcu¬ lating their ratio. Then, only the liver cell dispersion having a survival rate of 90% or more was selected, to which the experiment was continuously practiced.

13.8g of Waymouth's culture solution (Waymouth's MB 752/1 Medium, made by Gibco) in a powder state was dis¬ solved in 1 liter of deionized water. The resulting solution was adjusted to pH 7.4 with sodium carbonate, and 1X10~⁷M insulin and 50mg/l of gentamycin were added there¬ to. This mixture was filtered through a Millipore filter to obatin the culture solution. The liver cells prepared above were transferred with this culture solution to a 24- well plate and then incubated for one day in the incubator while maintaining carbon dioxide at the concentration of 5%. After 24 hours, the cultures were treated with 100 to lμg/ml of aloesin. After 36 hours from the sample treatment, the sample was treated with ³H-labelled thymi¬ dine in the concentration of lμCi/ml, and then, after 12 hours, the culture medium was removed from each well. The cells were fixed with methaol and washed with PBS and then washed two times with 10% trichloroacetate to remove unreacted radioactive thymidine. The cells were dis¬ solved in IN sodium hydroxide and neutralized with IN hydrochloric acid, and then the radioactivity introduced into the cells was measured by means of a scintillation counter (Pharmacia 1024) . The result thereof is de¬ scribed in the following Table 4. Table 4. Amount of radioactive thymidine introduced into DNA of primary cultured hepatic cells depending on aloesin concentration

Concentration dpm/well ^! Percentage to (μg/ml) (M ± S . D . ) control group

Control group 2817.3 ± 334.3 100.0

100 13542.3 ± 1163.8 480.3

10 5154.3 ± 32.1 182.8

1 3100.7 ± 131.1 110.0

Note : M ± S . D . = Mean ± Standard deviation

EXAMPLE 6 Effect of aloesin for stimulating cell growth on human hepatoma cells (MTT test.

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, made by Gibco) was dissolved in 1 liter of deionized water. After adjusting the resulting solution with sodium carbonate and hydrochloric acid to pH 7.4, 10% calf fetal serum, 1X10^"7M insulin and 50mg/l of gentamycin were added thereto. The mixture was sterilized through a Millipore filter to obtain the culture solution. Human hepatoma cell line sk-Hep-l which was obtained from Cancer

Institute of Seoul National University was inoculated into the culture solution in the ratio of 1X10⁶ cells per 25cm² of the surface area of T flask and then incubated for 48 hours in an incubator of 37°C while maintaining 5% carbon dioxide . The cultures were transferred to a 96-well plate at the ratio of 10^J cells per well , subcultured for one day and then treated with 100 to 1.5μg/ml of aloesin. For the control group, the cultures were treated with the same amount of PBS (phosphate buffered saline) instead of aloesin. After 48 hours from this treatment, 50μl of 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (5mg/ ml) was added to the treated sample and the mixture was reacted for 4 hours at 37°C to produce the insoluble formazan precipitate which was then centri- fuged to remove the supernatant. The produced formazan precipitate was dissolved with addition of lOOμl of di- methylsulfoxide (DMSO) . The absorbance of the solution at 570nm as an index of the amount of produced formazan was measured by means of an automatic plate reader. The measured result is described in the following Table 5.

Table 5. MTT analysis in human hepatoma cell line sk-Hep-l depending on aloesin concentration

Concentration Absorbance (O.D.) Percentage to (μg/ml) (M ± S.D.) control group

Control group 0.477 ± 0.034 100.0

1.5 0.417 ± 0.029 87.2

3 0.429 ± 0.023 89.3

6 1.329 ± 0.060 278.6

12 2.101 ± 0.054 440.5

25 1.849 ± 0.192 387.6

100 1.388 ± 0.085 291.1 Note : M ± S.D. = Mean ± Standard deviation

EXAMPLE 7 : Cell growth stimulating activity of aloesin

The following experiment was conducted to identify that the cell growth stimulating activity of aloesin is derived from the stimulation of DNA synthesis in cells without any cell damage.

1. Preparation of the extract of cell protein

Hepatoma cell line sk-Hep-l which was obtained from Cancer Institute of Seoul Ntional University was spread over a 10mm culture dish in the concentration of 8-10xl0⁵ cells per dish and then incubated in DMEM (Dulbecco's Modified Eagle's Medium, made by Gibco) for 24 hours. Cells were treated with 4mM hydroxyurea for 14 hours to synchronize the cells. Then, the medium in the culture dish was replaced with aloesin-DMEM medium and the cells were incubated again for 24 hours. After removing the medium, the cells were washed two times with cold PBS, harvested using lOOμl, per dish, of RIFA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15% NaCl, ImM phenylmethyl-sulfonylfluoride, 0.2mM EDTA, 50mM NaF, 0.2mM Na₃V0₄, 30mM β-glycerophosphate, 100 kallikrein inactivat¬ ing units [KIU] aprotinin) , and then centrifuged with 12000rpm to separate the supernatant. The separated supernatant was used as the cell lysate which is the test protein in the following Western Blot Analysis and Immune

Complex Kinase Assay.

2. Western Blot Analysis

20μg of the cell lysate obtained in the above 1 was mixed with 5μl of SDS sample buffer (200mM tris, pH 6.8, 400mM DTT, 8% SDS, 0.4% Bromophenol blue, 20% Glyceroi) , denatured at 100°C for 5 minutes, and then subjected to electrophoresis with 12% SDS-polyacrylamide mini gel. The protein was transferred to PVDF membrane using a transfer apparatus. The protein-blotted PVDF membrane was completely dried at room temperature, incubated in 5% nonfat dry milk solution for one hour and then washed three times with TBST solution (50mM Tris, pH 7.4, 150mM NaCl, 0.05% Tween 20) for 5 minutes each time, and primary antibodies cdk2 , cyclin E and cdc25 which were diluted with TBST solution at the ratio of 1:2000, 1:5000 and 1:5000, respectively, were added thereto. Then, the PVDF membrane was incubated for one hour, washed three times with TBST solution for 5 minutes each time, and then incubated with peroxidase-linked secondary antibody (anti- mouse Ig) solution diluted with TBST solution at the ratio of 1:15000 for one hour. The membrane was washed again four times with TBST solution for 5 minutes each time, and then reacted with a mixed solution of the same amounts of reagent 1 and reagent 2 of ECL (Enhance Chemilumines- cence) immunoblotting kit (made by Amersham) . Then, the membrane was wrapped in a plastic wrap and subjected to the measurement of che iluminescence using AGFA X-ray film. The results are shown in Figures 1 to 3.

From the result shown in Figures l to 3 , it can be seen that the protein synthesis increases according as the amount of aloesin used increases, and therefore, the activities of cdk2, cyclin E and cdc25 increase depending on the concentration of aloesin. Thus, it can be appar- ently identified that the cell growth stimulating activity of aloesin is derived from its effect of increasing DNA protein synthesis without any cell damage.

3. Immune Complex Kinase Assay

lOOμg of the cell lysate isolated from aloesin-treated hepatoma cell sk-Hep-l obtained in the above 1 was pre- purified with BSA precoated-Sepharose, and the supernatant was separated and then incubated with cdk2 antibody (5μl) . Immuno-precipitate was obtained by adding the protein A bead and washed three times with RIFA buffer and then three times with two-fold HI kinase assay buffer (lOOmM Tris,pH 7.6, 20mM MgCl₂, 2mM DTT, lμg/ml antipain, leupep- tin, pepstatin) . Then, HI kinase assay of the immuno- precipitate was conducted by reacting the immuno- precipitate with buffer solution (50μM ATP, lOμCi [r- ³²P]ATP) containing 0.6mg/ml of histone HI and, after 12% SDS-PAGE, measuring the phosphorylation of histone HI by means of autoradiography. The result is shown in Figure 4.

From the result shown in Figure 4, it can be seen that the activity of cdk2 which stimulates the DNA synthesis increases depending on the concentration of aloesin.

From the above experimental results, it can be identi¬ fied that aloesin increases the activities of cdk2, cyclin E and cdc25 to stimulate the synthesis of DNA, and ulti¬ mately exhibits an excellent cell growth stimulating activity.

EXAMPLE 8 : Composition Examples

Composition 1 (Soft capsule)

The soft capsules containing 5mg of aloesin per cap¬ sule were prepared using the following components accord¬ ing to the method for capsule preparation described in Korea Pharmacopeia. Component Content (mg/cap.)

Aloesin 5

Gelatin 140

Glycerin 60 Paraoxymethylbenzoate o.3

Paraoxypropylbenzoate o.3

Soybean oil 444.4

Total 650 mg

Composition 2 (Tablet)

The tablet preparations containing 5mg of aloesin per tablet were prepared using the following components ac¬ cording to the method for tablet preparation described in Korea Pharmacopeia.

Component Content (mg/tab.)

Aloesin 5

Lactose 250 Magnesium stearate 5

Calcium carboxymethylcellulose 25

Light anhydrous silicic acid 115

Total 400 mg

Composition 3 (Injection)

The injections containing 2mg of aloesin per ampoule were prepared using the following components according to the method for injection preparation described in Korea Pharmacopeia. Component Content (mg/amp . )

Aloesin 2

Sodium citrate 500

Water for injection added to the total amount of 5ml

Composition 4 (Ointment)

The ointment preparations containing 5mg of aloesin per lg were prepared using the following components ac¬ cording to the method for ointment preparation described in Korea Pharmacopeia.

Component Content (mg/g)

Aloesin 5

Light liquid paraffin 150 Stearyl alcohol 80

Cetostearyl alcohol 13

Propylene glycol 70

Sorbitan monostearate 30

Polyoxyethylene sorbitan monostearate 40 Butylated hydroxytoluene 0.4

Paraoxymethylbenzoate 0.9

Paraoxybutylbenzoate 0.9

Purified water q.s,

Total 1 g

Claims

WHAT IS CLAIMED IS :

1. A cell growth stimulating composition containing aloesin as an active ingredient.

2. The cell growth stimulating composition of claim 1, further comprising one or more pharmaceutically ac¬ ceptable carrier.

3. The cell growth stimulating composition of claim 1, wherein the composition is formulated in the form of pharmaceutical preparations.

4. The cell growth stimulating composition of claim 3, wherein the pharmaceutical preparation is a unit dosage form formulated so as to be administered 0.1 to 500mg of aloesin, per day, per kg of body weight of adult man.

5. The cell growth stimulating composition of claim 4, wherein the pharmaceutical preparation is a unit dosage form formulated so as to be administered 0.4 to 125mg of aloesin, per day, per kg of body weight of adult man.

6. The cell growth stimulating composition of claim 3 , wherein the pharmaceutical preparation is in the form of tablet, hard or soft capsule, solution, suspension, injectable solution or suspension, ointment, cream, gel or lotion.

7. The cell growth stimulating composition of claim 1, wherein the composition is an agent for treatment of skin wound.

8. The cell growth stimulating composition of claim l, wherein the composition is an agent for treatment of hepatic diseases .