WO2014014271A1

WO2014014271A1 - Tablet comprising melissa officinalis folium extract for preventing or treating angiogenesis or mmp activity-mediated disease

Info

Publication number: WO2014014271A1
Application number: PCT/KR2013/006393
Authority: WO
Inventors: Eun Jin Yoon; Kyeong Soo Kim; Yong Il Kim; Jae Hyun Park; Jong Soo Woo
Original assignee: Hanmi Pharm. Co., Ltd.
Priority date: 2012-07-18
Filing date: 2013-07-17
Publication date: 2014-01-23
Also published as: KR20140011209A; CN104470507A

Abstract

The present invention relates to a tablet for preventing or treating angiogenesis or matrix metalloproteinase (MMP) activity-mediated disease, the tablet comprising Melissa (Melissa officinalis) folium extract having rosmarinic acid, caffeic acid, and rutin as major active ingredients, and a pharmaceutically acceptable disintegrator. The tablet in which the dissolution rates of the rosmarinic acid or the caffeic acid are 80% or more within 30 minutes has an excellent release rate and is stable under accelerated conditions, and thus the tablet may be used effectively for the prevention or the treatment of angiogenesis or MMP activity-mediated disease, in particular, obesity.

Description

DESCRIPTION

TABLET COMPRISING MELISSA OFFICINALIS FOLIUM EXTRACT FOR PREVENTING OR TREATING ANGIOGENESIS OR MMP ACTIVITY-MEDIATED DISEASE

FIELD OF THE INVENTION The present invention relates to a tablet for preventing or treating angiogenesis or matrix metalloproteinase (MMP) activity-mediated disease, the tablet comprising Melissa officinalis folium extract in which rosmarinic acid, caffeic acid, and rutin are major active ingredients, and a pharmaceutically acceptable disintegrator.

BACKGROUND OF THE INVENTION

Melissa {Melissa officinalis), a perennial herb in a Labiatae family, is also called lemon balm.

Melissa folium extract contains flavonoids, terpene acids, volatile oils, glycosides of alcoholic and phenolic compounds, and caffeic acid derivatives. In particular, flavonoids which are contained abundantly in Melissa officinalis folium include cynaroside, cosmosin, rhamnocitrin, and isoquercitrin; and ursolic acid is contained as terpene acid. Among nonvolatile components, rosmarinic acid is contained, and caffeic acid derivatives are contained in an amount of about 4.7%. Among volatile oils, geraniol, neral, citronellal, and eugenol are contained. Above this, coumaric acid, a kind of polyphenol, and cinnamic acid having weak aromaticity, and so on, are contained in Melissa folium extract.

Roles of major pharmacological components in Melissa folium extract are described as follows:

Rosmarinic acid has strong anti-inflammatory and antipyretic effect, and essential oils contained in folium have been used in the treatment of diseases, in particular, depression, neurogenic headache, memory loss, neuralgia, fever, and the like. Above this, rosmarinic acids are also known to have sedative, antibacterial, antiviral, and antihormonal effects. Currently, dried Melissa folium extract which is known to contain a large amount of rosmarinic acid has been used as a blood circulation enhancer component.

Caffeic acid is contained in coffee and plants, such as fruits, vegetables, herbs, and so on. It is known to act as a carcinogenic inhibitor, and as an excellent antioxidant both in vivo and in vitro. Absorption of caffeic acid is limited when its chlorogenic acid form is esterified to quinic acid, and^* oral administration of high doses of caffeic acid in rats has caused stomach papillomas, and caffeic acid was recognized as a carcinogen in those rats. Administration of high doses of caffeic acid combined with antioxidants showed a decrease in growth of colon tumors.

Rutin is a flavonol glycoside substance, and is widely distributed in nature. Its major physiological activity is a capillary contraction effect, and has been used as a hemostatic; however, the action is not so strong, and temporary. Also, it has a capillary-strengthening effect, and can be used for preventing or treating cerebral hemorrhage, radiation sickness, hemorrhagic diseases, etc.

Meanwhile, angiogenesis refers to the process through which new capillaries form from pre-existing vessels. Under normal circumstances, angiogenesis occurs during embryo development, tissue regeneration and wound healing, and development of corpus luteum, the periodic changes in the female reproductive system, and is strictly regulated during these processes.

For adults, vascular endothelial cells grow very slowly, and do not divide very well in a relative sense as compared to other kinds of cells. However, adult angiogenesis is induced as necessary by hormone signal response, release of pro-angiogenic cytokines from inflammatory cells, activation of hydrolytic enzymes which release isolated angiogenic mediators to extracellular matrix, or stimulation of angiogenic factors. The process of angiogenesis is generally composed of degradation of basement membrane on blood vessels induced by proteases, migration of vascular endothelial cells, lumen formation via proliferation and differentiation of vascular endothelial cells to reconstruct blood vessels, and formation of new capillaries. One of the most important events in the process of angiogenesis is an enzymatic breakdown of basement membrane on blood vessels which surrounds vascular endothelial cells. Enzymes playing an important role therein are the enzymes which belong to matrix metalloproteinase (MMP).

It is known that when angiogenesis is not autonomically regulated, or MMP which plays an important role in angiogenesis is over-activated, blood vessels grow abnormally, and thereby causing various diseases, e.g., cancer growth and metastasis; cardiovascular diseases such as angioma, angiofibroma, vascular deformity, atherosclerosis, angiostenosis, edemic sclerosis and restenosis; opthalmological diseases, such as diabetic retinopathy, macular degeneration (including senescence-related macular degeneration such as age- related macular degeneration), pterygium, retinal degeneration, angiogenesis associated with corneal transplantation, angiogenic glaucoma, angiogenic corneal diseases, retrolental fibroplasias, granular conjunctivitis, corneal ulcer, proliferative vitreous body retinopathy, immature retinopathy, eye inflammation, conical cornea, Sjogren's syndrome, myopia, eye tumors, and rejection in corneal transplantation; obesity, and the like (see Polverini PJ, Critical Reviews in Oral Biology, 6(3), 1995, 230-247; Arup Das, et al, Progress in Retinal and Eye Research, 22, 2003, 721-748; Nick Di Girolamo, et al, IOVS, Vol. 42, No.9, August 2001, 1963-1968; Patricia Lee, et al, Survey of ophthalmology, vol 43, No. 3, Nov-Dec 1998, 245-269; D.B. Holland, et al, British Journal of Dermatology, 150, 2004, 72-81; Anthony H Vagnucci Jr, et al, The Lancet, vol 361, Feb. 15, 2003, 605-608; Berislav V. Zlokovic, Trends in Neuroscience, Vol. 28, No.4, April 2005, 202-208; Jaap G. Neels, et al, The FASEB Journal express article 10. 1096/fj.03-l lOlfje. Published online April 14, 2004; D.L. Crandall, et al, Microcirculation, 4, 1997, 211-232; G. Voros, et al, Endocrinology, 146, 2005, 4545-4554; M.A. Rupnick, et al, PNAS, 99, 2002, 10730-10735; E. Brakenhielm, et al, Circ. Res., 94, 2004, 1579-1588; H.R. Lijnen, et al, Arterioscler Thromb Vase Biol, 22, 2002, 374-379; and D. Demeulemeester, et al, Biochem. Biophys. Res. Commun., 329, 2005, 105-110).

Therefore, development of angiogenesis inhibitors or MMP activity inhibitors can lead to treatment of these diseases.

In this regard, Korean Patent No. 10-550298 discloses that Melissa folium extract has an anti-angiogenic effect through an experiment of suppressing formation of tubes such as capillaries using human umbilical vein endothelial cells (HUVEC), and in vivo assay using a mouse Matrigel model and chorioallantoic membrane (CAM) of chick embryo experiment.

Korean Patent No. 10-473688 further discloses that Melissa folium extract has an inhibitory effect on the activity of MMP (matrix metalloproteinase), which plays an important role in degradation of the basement membrane, which surrounds vascular endothelial cells, the important process of angiogenesis.

Korean Patent No. 10-903030 discloses a method for increasing efficacy of Melissa folium extract, which includes the steps of: (i) Melissa folium extract is extracted with 50 to 100% ethanol or methanol, followed by drying; (ii) the alcohol extract is suspended in water, fractionated with ethyl acetate, followed by drying; and (iii) the ethyl acetate fraction is resuspended in water, followed by drying to obtain the ethyl acetate fraction of Melissa folium. In addition, examples of hard capsules including Melissa folium extract are disclosed therein; however, when Melissa folium extract is prepared in a capsule formulation, release of active ingredients is not easy due to capsule base compounds, and drug release is nonfavorable due to agglomerization with capsule base compounds. When the Melissa folium extract-containing capsule formulation is left under the condition of high temperature and humidity, the release rates of active ingredients become slower, and therefore, it is not suitable for Melissa folium extract-containing capsule to exhibit desired medicinal effects.

However, the above prior art does not consider sufficiently active ingredients which exhibit medicinal effects derived from Melissa folium extract and suitable formulations to administer to the body.

Therefore, the present inventors have studied the effects of drug metabolism depending on the release rates of major active ingredients derived from Melissa folium extract, and observed that efficacies vary depending on dissolution profiles and formulations of pharmaceutical compositions comprising Melissa folium extract, thereby completing the present invention.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a pharmaceutical formulation including Melissa folium extracts, in which rosmarinic acid, caffeic acid, and rutin are major active ingredients and which has angiogenic inhibitory and abdominal obesity- treating effects, and showing excellent drug metaboli sm . To achieve the above object, the present invention provides a tablet for preventing or treating angiogenesis or MMP activity-mediated disease, wherein the tablet comprises Melissa folium extract in which rosmarinic acid, caffeic acid, and rutin are major active ingredients, and a pharmaceutically acceptable disintegrator, and the dissolution rates of rosmarinic acid or caffeic acid are 80% or more within 30 minutes.

Since tablets in accordance with the present invention are stable even under accelerated conditions, have excellent release rates of active ingredients, and exhibit dissolution profiles which can maximize the effectiveness in treating or preventing angiogenesis or MMP activity-mediated disease, the tablets can be used effectively for the prevention or the treatment of the above diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

FIG. 1 and FIG. 2 show the test results of rosmarinic acid and caffeic acid dissolution from formulations in Examples 2 and 3, and Comparative Example 1 , respectively;

FIGs. 3 to 5 show the results of content stability test for rosmarinic acid, caffeic acid, and rutin from formulations in Examples 2 and 3, and Comparative Example 1, respectively, according to storage period under accelerated conditions;

FIG. 6 and FIG. 7 show the results of dissolution stability test for rosmarinic acid and caffeic acid from formulations in Examples 2 and 3, and Comparative Example 1, respectively, according to storage period under accelerated conditions; and

FIG. 8 shows the changes in rat body weight by administration of formulations in Comparative Examples 2 and 3, and Example 4.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a tablet for preventing or treating angiogenesis or MMP activity-mediated disease, wherein the tablet comprises Melissa folium extract having rosmarinic acid, caffeic acid, and rutin as major active ingredients, and a pharmaceutically acceptable disintegrator, and the dissolution rates of rosmarinic acid or caffeic acid are 80% or more within 30 minutes.

In the present invention, "dissolution rate" refers to the value measured as follows: 900 mL of purified water was used as a dissolution test liquid and the dissolution test was performed at 37 + 0.5 °C, 50 rpm of rotation speed in accordance with Paddle method, Method 2 in the general test methods of the Korean Pharmacopoeia. After a specific time, the dissolution liquid was filtered with a 0.45 μιη filter. Using liquid chromatography (HPLC) analysis method in the general test methods of the Korean Pharmacopoeia, mobile phases were operated at a rate of 1 mL per minute according to concentration gradient condition in mobile phase A (5% formic acid aqueous solution) and mobile phase B (methanol) described in the following. 10 of a sample was injected into an analytical column, e.g., Zorbax Eclipse Plus CI 8 (150 x 4.6 mm) column, and effective ingredients were analyzed to measure at 285 nm wavelength. Concentration gradient in the mobile phase>

Time (min) Mobile phase A (%) Mobile phase B

0 100 0

5 100 0

25 0 100

35 0 100

35.1 100 0

45 100 0

In accordance with one aspect of the present invention, the dissolution rate of rosmarinic acid or caffeic acid in the tablet according to the present invention is 80% or more within 15 minutes (see FIGs. 6 and 7).

In addition, the tablet in the present invention comprises Melissa folium extract in an amount of 10 to 90% by weight, based on the total weight of the tablet. In one embodiment of the present invention, the tablet of the present invention comprises Melissa folium extract in an amount of 50% by weight to 90% by weight, based on the total weight of the tablet.

The tablet containing Melissa folium extract of the present invention can make the release of active ingredients easy, and thus can release 80% or more of active ingredients such as rosmarinic acid or caffeic acid within 30 minutes, and besides, even if it is left under the accelerated conditions of high temperature and humidity, release rates of its ingredients hardly change and are stable, thus it can exhibit excellent medicinal effects. Medicines prepared with natural product extracts are characterized in that active ingredients are admixed therein, and thus, formulations having rapid disintegration ability are favorable so that effective ingredients may exert their medicinal effects simultaneously. Therefore, a pharmaceutical composition comprising Melissa folium extract may be in a form of tablet, similar to the present invention.

In the present invention, Melissa folium extract may be prepared in accordance with the following procedure using conventional extraction methods which use ethanol. First, Melissa folium is reflux-extracted with 10 to 40 times its amount by weight of 50 to 100% ethanol, preferably 70 to 80% ethanol, and more preferably 75% ethanol, and then filtered to complete a first concentration procedure. Subsequently, a procedure for fractionation of the above extract with a mixture of ethyl acetate and water, e.g., ethyl acetate: water = 1 : 1 (v/v), is repeated two times. The resulting ethyl acetate layer from the above procedures is collected, concentrated secondarily, dried and pulverized to prepare Melissa folium extract according to the present invention. The extract prepared above is used as a dried ethyl acetate-extract of Melissa folium in the present invention.

Dried Melissa folium, non-dried Melissa folium, or a mixture thereof may be used. For effective extraction, Melissa folium may be cut into small pieces to use.

According to the present invention, the Melissa folium extract comprises 5% by weight or more of rosmarinic acid, 0.5% by weight or more of caffeic acid, and 1% by weight or more of rutin, based on the total weight of the extract, and may comprise preferably 5 to 10% by weight of rosmarinic acid, 0.5 to 1% by weight of caffeic acid, and 1 to 2% by weight of rutin. In accordance with one aspect of an exemplary embodiment of the present invention, the dried ethyl acetate-extract of Melissa folium according to the present invention comprises about 5.5% by weight of rosmarinic acid, about 0.65% by weight of caffeic acid, and about 1.42% by weight of rutin (see Table 2)·

Also, the Melissa folium extract may comprise rosmarinic acid and caffeic acid at a weight ratio of 1 :0.1 to 1 :0.2, caffeic acid and rutin at a weight ratio of 1 : 1 to 1 :4, more specifically at a weight ratio of 1 :1 to 1 :3, and rosmarinic acid and rutin at a weight ratio of 1 :0.1 to 1 :0.3.

In addition, the tablet according to the present invention comprises a pharmaceutically acceptable disintegrator so as to achieve the aforementioned dissolution rate, i.e., the dissolution rates of 80% or more of rosmarinic acid or caffeic acid within 15 minutes. The disintegrator may be selected from the group consisting of sodium starch glycolate (SSG), crospovidone, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and a mixture thereof, and may be comprised in an amount of 1 to 10% by weight, based on the total weight of the tablet.

The tablet according to the present invention may further comprise a pharmaceutically acceptable additive selected from the group consisting of excipients, lubricants, binders, and a mixture thereof, and may be prepared according to conventional methods, by mixing the dried ethyl acetate-extract of Melissa folium and the pharmaceutically acceptable additive, and then performing direct compression, or tablet pressing in a dry or wet way.

The excipient may be selected from the group consisting of microcrystalline cellulose (MCC), light anhydrous silicic acid, mannitol, lactose, calcium dihydrogen phosphate, starch, low-substituted hydroxypropyl cellulose, and a mixture thereof, and may be comprised in an amount of 7 to 87% by weight, based on the total weight of the tablet.

The lubricant may be selected from the group consisting of stearic acid, metal salts of stearic acid such as magnesium stearate and calcium stearate, talc, sucrose fatty acid esters, hydrogenated vegetable oil, high melting point waxes, glyceryl fatty acid esters, glycerol dibehenate, and a mixture thereof, and may be comprised in an amount of 0.2 to 5% by weight, based on the total weight of the tablet.

The binder may be selected from the group consisting of hydroxypropyl cellulose (HPC-L), hydroxypropyl methylcellulose, polyvinylpyrrolidone, copovidone, and a mixture thereof, and may be comprised in an amount of 1 to 10% by weight, based on the total weight of the tablet. The tablet according to the present invention can be used for the prevention or the treatment of angiogenesis-related diseases or MMP-mediated diseases, e.g., a disease selected from the group consisting of cancer growth and metastasis; cardiovascular diseases, such as angioma, angiofibroma, vascular deformity, atherosclerosis, angiostenosis, edemic sclerosis and restenosis; opthalmological diseases such as diabetic retinopathy, macular degeneration (including senescence-related macular degeneration such as age- related macular degeneration), pterygium, retinal degeneration, angiogenesis associated with corneal transplantation, angiogenic glaucoma, angiogenic corneal diseases, retrolental fibroplasias, granular conjunctivitis, corneal ulcer, proliferative vitreous body retinopathy, immature retinopathy, eye inflammation, conical cornea, Sjogren's syndrome, myopia, eye tumors, and rejection in corneal transplantation; and obesity, and preferably obesity.

Therefore, the present invention provides a method for preventing or treating angiogenesis or matrix metalloproteinase (MMP) activity-mediated disease in a subject, comprising administering the aforementioned tablet to the subject in need thereof, e.g., a mammal, including human.

EXAMPLES

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner. Example 1: Analyses of Components and Ratio in Dried Ethyl

Acetate-Extract of Melissa Folium

To extract effective ingredients from raw Melissa folium, extracts were prepared as follows and used for component analysis.

Specifically, 200 kg of dried leaves of Melissa officinalis was reflux- extracted with 75% ethanol amounting to 20 times the weight thereof for 4 hours, and filtered through a cartridge filter, and concentrated firstly under reduced pressure. The thus obtained concentrate (amount: 50 kg) was fractionated with a mixture of ethyl acetate and water (l : l(v/v)) (amount: 50 kg), which was repeated two times. Subsequently, the ethyl acetate layer was collected, concentrated secondarily under reduced pressure, dried and pulverized to obtain an ethyl acetate extract of Melissa folium (amount: 8.2 kg). The above procedures were performed in triplicate to determine reproducibility for the extract, and the components comprised in the Melissa folium extract and the ratio of components were analyzed as follows.

The Melissa folium extract was analyzed by a liquid chromatography (HPLC). 10 ΐ. of the ethyl acetate extract of Melissa folium prepared above was injected into a Zorbax Eclipse Plus CI 8 (150 4.6 mm) column, and then, mobile phase A (5% formic acid aqueous solution) and mobile phase B (methanol) were eluted according to the ratio described in the following Table 1 at a flow rate of 1 mL/min, and the eluted effective ingredients were analyzed at 285 nm.

[Table 1]

As a result of analyzing effective ingredients in the Melissa folium extract, the Melissa folium extract contained on average about 5.5% by weight of rosmarinic acid, about 0.65% by weight of caffeic acid, and about 1.42% by weight of rutin, as shown in the following Table 2. Based on the analysis results, it is confirmed that rosmarinic acid and caffeic acid may be present at a weight ratio of 1 :0.1 to 1 :0.2, caffeic acid and rutin may be present at a weight ratio of 1 :1 to 1 :3, and rosmarinic acid and rutin may be present at a weight ratio of 1 :0.1 to 1 :0.3 in the Melissa folium extract.

It is considered that when the weight ratio of rosmarinic acid, caffeic acid, and rutin is appropriately balanced, and is maintained to be constant, the ethyl acetate extract of Melissa folium can exhibit desirable medicinal effects.

It is also considered to be preferable for the reproducibility of medicinal effects that 5.0% by weight or more of rosmarinic acid, 0.5% by weight or more of caffeic acid, and 1.0% by weight or more of rutin are contained, based on the total weight of the Melissa folium extract, and this suggests that it is important to manage the contents continually and reproducibly to maintain the reproducibility of medicinal effects.

[Table 2]

Example 2: Preparation of Tablets Comprising Melissa Folium Extract - (1)

According to the amounts (unit: mg) described in the following Table 3, tablets were prepared by admixing microcrystalline cellulose (MCC, Avicel PH101 , FMC bio polymer) and light anhydrous silicic acid (Aerosil 200, Degussa) as excipients, sodium starch glycolate (SSG, YUNG ZIP Chem) as a disintegrator, and magnesium stearate (S-Mg, NOF Corporation) as a lubricant to the dried ethyl acetate-extract of Melissa folium prepared in Example 1 , and then performing direct compression.

Example 3: Preparation of Tablets Comprising Melissa Folium Extract - (2)

According to the amounts (unit: mg) described in the following Table 3, granules were prepared by mixing the dried ethyl acetate-extract of Melissa folium prepared in Example 1 with MCC and SSG, mixing the mixture homogeneously with a binder liquid, hydroxypropyl cellulose (HPC-L, Shin- Etsu) dissolved in water, and then granulating the resulting mixture. The prepared granules were dried at 50 °C for 2 hours, passed through a 20-mesh sieve, mixed with light anhydrous silicic acid as an excipient and S-Mg as a lubricant, and then compressed to prepare a tablet. Comparative Example 1: Preparation of Capsules Comprising Melissa Folium Extract According to the amounts (unit: mg) described in the following Table 3, capsules were prepared by mixing the dried ethyl acetate-extract of Melissa folium prepared in Example 1 with MCC and light anhydrous silicic acid as excipients and S-Mg as a lubricant, and then, filling the mixture into Size No. 0 capsules.

[Table 3]

Comparative Example 2: Preparation of Tablets for Rat Administration Not Including Melissa Folium Extract

Tablets prepared in Examples 2 and 3 have doses and sizes which are applicable to real humans (about 60 kg). For a body weight measurement experiment through rat administration, tablets for rat administration not including Melissa folium extract were prepared in a ratio which is nearly proportional to the ratio of a rat body weight (about 250 g) to a human body weight.

According to the composition described in the following Table 4, tablets for rat administration were prepared by the same methods as in Example 3, except that the dried ethyl acetate-extract of Melissa folium is not added.

[Table 4]

Comparative Example 3: Preparation of Capsules for Rat Administration Including Melissa Folium Extract

According to the amounts (unit: mg) described in the following Table 5, capsules for rat administration were prepared by mixing the dried ethyl acetate- extract of Melissa folium prepared in Example 1 with MCC and light anhydrous silicic acid as excipients and S-Mg as a lubricant, and then, filling the mixture into Size No. 5 capsules.

Example 4: Preparation of Tablets for Rat Administration Including Melissa Folium Extract

According to the amounts (unit: mg) described in the following Table 5, tablets for rat administration were prepared by the same method as in Example 3.

[Table 5]

Comparative

Components Example 4

Example 3

Test Example 1: Rosmarinic Acid and Caffeic Acid Dissolution Test

Dissolution tests for rosmarinic acid and caffeic acid, major active ingredients of Melissa folium extract, were performed on capsules of Comparative Example 1 and tablets of Examples 2 and 3.

For the dissolution test, 900 mL of water was used as a dissolution test medium and the dissolution test was performed at 37 + 0.5 °C, 50 rpm of rotation speed in accordance with Paddle method, Method 2 in the general test methods of the Korean Pharmacopoeia. After 5, 10, 15, 30, 45, 60, 90, and 120 minutes from the start of the dissolution test, the dissolution medium were taken and filtered through a 0.45 μηι filter, and subsequently, the dissolution rates of rosmarinic acid and caffeic acid were measured by the same method as HPLC analysis method described in Example 1. The results are shown in FIGs. l and 2.

As shown in Figs. 1 and 2, the release rate and the final dissolution rate of each active ingredient were higher in tablets prepared in Examples 2 and 3 as compared to capsules prepared in Comparative Example 1. In addition, it was found that both tablets prepared by a direct dry-tableting process in Example 2 and tablets prepared by a wet granulation process in Example 3 released 80% or more of rosmarinic acid and caffeic acid within 30 minutes.

Test Example 2: Test for Amounts of Effective Components under Accelerated Conditions Capsules prepared in Comparative Example 1, and tablets prepared in Examples 2 and 3 were packed in HDPE bottles, and kept in the accelerated conditions (40 °C and 75% relative humidity) for 6 months, and then, the amounts of major active ingredients of the Melissa folium extract, i.e., rosmarinic acid, caffeic acid and rutin, were determined to confirm stability of formulations. After 1, 3 and 6 months of storage under the accelerated conditions, the amounts of rosmarinic acid, caffeic acid, and rutin were measured, and the results are shown in FIGs. 3 to 5.

Consequently, there was no significant difference in stability between capsule formulation and tablet formulations prepared by the direct dry- tabletting and the wet granulation, and the amount of each component did not change significantly. In particular, the tablet according to the present invention met the standard of 5.0% by weight or more of rosmarinic acid, 0.5% by weight or more of caffeic acid, and 1.0% by weight or more of rutin, and accordingly, it was confirmed that they are stable under the accelerated storage condition as well.

Test Example 3: Determination of Changes in Dissolution under Accelerated Conditions

Capsules prepared in Comparative Example 1, and tablets prepared in Examples 2 and 3 were packed in HDPE bottles, and kept in the accelerated conditions (40 °C and 75% relative humidity) for 6 months, and then, the dissolution rates of rosmarinic acid and caffeic acid were measured by the same way as in Test Example 1. After 15, 30, and 60 minutes from the start of the dissolution test, medium samples were taken to evaluate the dissolution rates, which were then compared with the initial value to determine the degree of change, and the results are shown in FIGs. 6 and 7.

As shown in FIGs. 6 and 7, capsules prepared in Comparative Example

1 showed a slow initial release rate, and when they were kept under the accelerated conditions, the release rate slowed down consistently over time. It is considered that the release rate of the Melissa folium extract was affected by capsules. Since there is a possibility that the medicinal effects could be changed as dissolution slows down, a capsule is considered to be a pharmaceutically undesirable formulation. All tablets of the present invention showed no significant changes in release rate and were stable.

Test Example 4: Administration to Animals and Measurement of Body Weight Change

To examine in vivo efficacy of the Melissa folium extract on high-fat diet-induced obesity, the following experiment was carried out. After a one- week acclimation period, 7-week-old rats were divided into groups of 3 animals per group. The rats fed with 45 kcal% high- fat feed for 8 weeks, while being administered with the tablets of Comparative Examples 2 and 3, and Example 4 one time per day (a daily dose of the Melissa folium extract: 10 mg) using zoned. Then, body weights of the rats were measured, and the results are shown in FIG. 8. Comparison and analysis of the mean body weight of individuals showed that each result was significant in every case (p<0.05).

As shown in FIG. 8, body weight of the group administered with the tablet not including the Melissa folium extract prepared in Comparative Example 2 increased rapidly over time. Meanwhile, the group administered with the capsule comprising the Melissa folium extract prepared in Comparative Example 3 showed the relatively high amount of body weight gain as compared to the group administered with the tablet prepared in Example 4. This result is considered to be interrelated with the dissolution test results in Experimental Example 1, and suggests that the release rates of rosmarinic acid and caffeic acid, which are effective components of the Melissa folium extract, play an important role in exhibiting the medicinal effects of the Melissa folium extract. In other words, if the Melissa folium extract is prepared in a capsule formulation as prepared in Comparative Example 3, and pharmaceutically active ingredients are not released well, the amounts of the components absorbed into a body would be small, and thus the medicinal effects would not be exhibited.

Therefore, the most appropriate formulation for the Melissa folium extract was found to be the tablet formulation, from which rosmarinic acid and caffeic acid, the major ingredients of the Melissa folium extract, are released 80% or more within 30 minutes, thereby rapidly exhibiting the medicinal effects, and which show little changes in dissolution during storage.

Claims

What is claimed is:

1. A tablet for preventing or treating angiogenesis or matrix metalloproteinase (MMP) activity-mediated disease, wherein the tablet comprises

Melissa (Melissa officinalis) folium extract having rosmarinic acid, caffeic acid, and rutin as major active ingredients, and a pharmaceutically acceptable disintegrator, and the dissolution rates of rosmarinic acid or caffeic acid thereof are 80% or more within 30 minutes.

2. The tablet of claim 1, wherein the dissolution rate of the rosmarinic acid or the caffeic acid is 80% or more within 15 minutes.

3. The tablet of claim 1, wherein the Melissa folium extract is prepared by reflux-extracting Melissa folium with 50 to 100% ethanol, followed by filtering and concentrating; collecting an ethyl acetate layer produced by repeating a procedure of fractionation of the obtained concentrate with ethyl acetate and water two times; and subsequently, concentrating, drying, and pulverizing the ethyl acetate layer.

4. The tablet of claim 1 , wherein the Melissa folium extract comprises 5 to 10% by weight of the rosmarinic acid, 0.5 to 1% by weight of the caffeic acid, and 1 to 2% by weight of the rutin, based on a total weight of the extract.

5. The tablet of claim 1, wherein the Melissa folium extract comprises the rosmarinic acid and the caffeic acid at a weight ratio of 1 :0.1 to 1 :0.2.

6. The tablet of claim 1 , wherein the Melissa folium extract comprises the caffeic acid and the rutin at a weight ratio of 1 : 1 to 1 :4.

7. The tablet of claim 1, wherein the Melissa folium extract comprises the rosmarinic acid and the rutin at a weight ratio of 1 :0.1 to 1 :0.3.

8. The tablet of claim 1, wherein the pharmaceutically acceptable disintegrator is selected from the group consisting of sodium starch glycolate, crospovidone, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and a mixture thereof.

9. The tablet of claim 1, wherein the pharmaceutically acceptable disintegrator is comprised in an amount of 1 to 10% by weight, based on a total weight of the tablet.

10. The tablet of claim 1, which further comprises a pharmaceutically acceptable additive selected from the group consisting of excipients, lubricants, binders, and a mixture thereof.

11. The tablet of claim 10, which comprises 10 to 90% by weight of the Melissa folium extract, 1 to 10% by weight of the disintegrator, 7 to 87% by weight of the excipient, 0.2 to 5% by weight of the lubricant, and 1 to 10% by weight of the binder, based on a total weight of the tablet.

12. The tablet of claim 1, wherein the angiogenesis or MMP activity- mediated disease is a disease selected from the group consisting of cancer growth and metastasis; angioma, angiofibroma, vascular deformity, atherosclerosis, angiostenosis, edemic sclerosis and restenosis; diabetic retinopathy, macular degeneration, pterygium, retinal degeneration, angiogenesis associated with corneal transplantation, angiogenic glaucoma, angiogenic corneal diseases, retrolental fibroplasias, granular conjunctivitis, corneal ulcer, proliferative vitreous body retinopathy, immature retinopathy, eye inflammation, conical cornea, Sjogren's syndrome, myopia, eye tumors, and rejection in corneal transplantation; and obesity.

13. The tablet of claim 12, wherein the angiogenesis or MMP activity- mediated disease is obesity.

14. A method for preventing or treating angiogenesis or matrix metalloproteinase (MMP) activity-mediated disease in a subject, comprising administering the tablet of claim 1 to a subject in need thereof.