WO2004006946A1

WO2004006946A1 - Composition comprising the crude extract of the fruit of morus alba l having neuro-protective activity

Info

Publication number: WO2004006946A1
Application number: PCT/KR2003/001368
Authority: WO
Inventors: Sun-Yeou Kim; Jong-Hoon Ryu; Ho-Cheol Kim; Ae-Jung Kim; Won-Ju Lee; Hyun-Bok Kim
Original assignee: Kyung Hee University
Priority date: 2002-07-10
Filing date: 2003-07-10
Publication date: 2004-01-22
Also published as: AU2003247175A1; KR20040005434A

Abstract

The present invention relates to the pharmaceutical composition and health care food comprising the extract of mulberry (Morus alba L.) having neuronal cell protecting activity. The mulberry extract in the present invention has the neuroprotecting effect and inhibitory effect of brain/neuronal diseases such as brain stroke, cerebral ischemia and the like. Therefore, it is useful as the therapeutics for the prevention or treatment of brain stroke and brain diseases in human or mammal.

Description

COMPOSITION COMPRISING THE CRUDE EXTRACT OF THE FRUIT OF MORUS ALBA L HAVING NEURO-PROTECTIVE ACTIVITY

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates to a composition comprising crude drug extract having neuron-protective activity for preventing and treating brain diseases such as brain stroke and cerebral ischemia in human or mammal.

Background Art

Brain stroke is consisted of two type, i.e., ischemic stroke occurred from ischemic condition of brain tissue caused by intervention or decrease of blood supply to brain, and hemorrhagic stroke occurred from the bleeding of brain blood vessel where the former occupy about 80% among total patient suffered from brain stroke. It has been reported that the cause of damage of brain neuronal cells are the release of excessive excitational neuronal transmitter, the production of free radical, the inhibition of protein synthesis, abnormal expression of gene and the activation of immune response etc., however, there has been not yet developed therapeutically effective agent to protect the damage of brain neuronal cells. The inhibition of cyclooxygenase-2 (COX-2) results in protecting activity of brain neuronal cell due to the inhibition of glutamate release caused by inhibiting the production of PGE₂. Therefore, since many patients suffered from rheumatic disease and pains already have taken COX-2 inhibitor, much interest has been focused to the result of their clinical investigation about the co-relation between the incidence rate of brain stroke and the population of patient taken the drug, which may be new target for investigating effective agents to prevent or treat brain stroke (Iadecola, C. et al., PNAS., 30, ppl294-1299, 2001).

It is known that mulberry, the fruit of Morus alba L and Morus genus belonged to Moraceae, shows reducing effect of blood glucose, moran A, a glycoprotein isolated from Morus alba L., has reducing activity of blood glucose and nitrogen containing monosaccharide substance isolated from the leaves of Morus alba L. inhibits alpha- glucosidase enzyme (Hikino et al; Planta Med., 51, ppl59-160, 1985: Kim et al.; J

However, there have been no disclosure or suggestion about a use of mulberry for treating or inhibiting neuronal diseases such as brain stroke.

The inventors of the present invention have intensively carried out the scientific investigation concerning pharmacological effects and its mechanism of action of a mulberry, in particular a treating or inhibiting activity of brain stroke of the extract of a mulberry. As a result of the investigation, the inventors have discovered that the extract of mulberry shows novel pharmacological effects, especially, its preventing or treating activity for brain stroke and they have finally completed the present invention.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a use of a mulberry extract in the manufacture of a medicament for the prevention or treatment of brain disease such as brain stroke and cerebral ischemia disease.

And, another object of the present invention is to provide a method of treating or preventing brain disease such as brain stroke and cerebral ischemia in a mammal comprising administrating to said mammal an effective amount of above extract, together with a pharmaceutically acceptable carrier thereof.

Disclosure of the invention

In accordance with the present invention, the present invention provides a pharmaceutical composition comprising mulberry extract, as an active ingredient in an amount effective to protect neuronal cell and to treat or prevent human or mammal suffering from brain diseases such as brain stroke and cerebral ischemia, together with a pharmaceutically acceptable carrier.

The present invention also provides a pharmaceutical composition comprising mulberry extract as an active ingredient in an amount effective for prevention and treatment of inflammatory diseases, together with a pharmaceutically acceptable carrier.

Above described mulberry extract can be obtained by extracting dried fruit of Morus alba L. and Morus genus belonged to Moraceae, with polar organic solvent such as water, lower alcohol for example, methanol, ethanol etc or the solvent mixture thereof.

The present invention also provides a use of mulberry extract in the preparation of the medicament to prevent or treat brain disease such as brain stroke and cerebral ischemia of mammal or human.

And the present invention also provides a use of mulberry extract in the preparation of the medicament to prevent or treat inflammatory disease of mammal or human.

Additionally, the present invention also provide a method of treating or preventing brain disease such as brain stroke and cerebral ischemia in a mammal comprising administrating to said mammal an effective amount of above described extract, together with a pharmaceutically acceptable carrier thereof.

The present invention also provide a method of treating or preventing inflammatory disease in a mammal comprising administrating to said mammal an effective amount of above described extract, together with a pharmaceutically acceptable carrier thereof.

An inventive extract may be prepared in accordance with the following preferred embodiment.

Dried mulberry is sliced to pieces. The pieces are mixed with 1 to 15-fold, preferably, 2 to 5 -fold volume of water, lower alcohols such as methanol, ethanol and the like, or the mixtures thereof; and is heated at a temperature ranging from 20 to 100°C, preferably 70 to 100°C, by sonication extraction or reflux extraction, for a period ranging from 0.5 to 2 days, preferably 1 to 24 hours, with 1 to 5 times, preferably 2 to 3 times, to obtain an crude extract thereof. The crude extract is centrifuged, filtered and then lyophilized to obtain an extract powder.

Above prepared mulberry extract can be applied to the serial procedure comprising the steps consisting of concentrating under reduced pressure and subjecting column chromatography eluting with distilled water or lower alcohol to obtain pharmacologically active fractions.

The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the present invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection.

Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation

(solution, suspension, emulsion).

The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

The desirable dose of the inventive extract of the present invention varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.01-lOg/kg, preferably, 1 to 5g/kg by weight/day of the inventive extract. The dose may be administered in single or divided into several times per day. In terms of composition, the composition should be present between 0.01 to 80% by weight, preferably 0.5 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebro ventricular injection.

The present inventors demonstrated that present composition comprising above described mulberry extract of the present invention have preventing or treating activity of brain stroke by accomplishing in vivo experiment already well known in the art, e.g., middle cerebral artery occlusion model test which is consisted of following step i.e., nylon filament is inserted into internal carotid artery to occlude middle cerebral artery and 120 minutes after, the filament is removed again to allow the reperfusion of the artery.

And the present inventors showed the present composition comprising above described mulberry extract of the present invention significantly decreased the COX-2 activity.

Accordingly, it is another object of the present invention to provide a health care food comprising above described extract of the present invention prepared by above processes and a sitologically acceptable additive to protect neuronal cell and to prevent or improve brain diseases such as brain stroke and cerebral ischemia.

Above described composition therein can be added to food, additive or beverage for prevention of brain diseases such as brain stroke and cerebral ischemia. For the purpose of preventing brain stroke diseases, wherein, the amount of above described extract of the present invention in food or beverage may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % of total weight of food for the health food composition and 1 to

30 g, preferably 3 to 10 g on the ratio of lOOmi. of the health beverage composition.

Providing that the health beverage composition of present invention contains above described extract of the present invention as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of lOOm-β of present beverage composition.

The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition.

Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawing, in which;

Fig. 1 shows the protective effect of various concentration of a mulberry extract on the injury of brain tissue in MCAo model experiment,

Fig. 2 represents the increasing effect of ATP content in various concentration of mulberry extract on brain tissue section of Sprague-Dawley rat model,

Fig. 3 presents the inhibiting activity of various concentration of mulberry extract on the nitrogen oxide production induced by lipo-polysaccharide in BV-2 cell.

Best Mode for Carrying Out the Invention

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

Example 1. Preparation of water-soluble extract of a mulberry (1)

1.0kg of dried mulberry powder procured from Applied Entomology Division Department of Sericulture & Entomology, NIAST, was sliced into pieces. 2 L of water was poured to the pieces and subjected to reflux extraction at 100 ^°C for 2 hours at two times. The resulting extracts were filtrated with filter paper (Whatman Co., USA), combined, concentrated under reduced pressure with evaporator (Eyela Co. FD-5N model, Japan) and dried with cryophilization to afford 185.5g of dried water-soluble extract of mulberry, which was used as a sample in further experiment.

Example 2. Preparation of methanol-soluble extract of a mulberry (2)

10kg of dried mulberry powder procured from Applied Entomology Division Department of Sericulture & Entomology, NIAST, was sliced into pieces. 2 L of 85% methanol was poured to the pieces and subjected to ultra-sonification extraction at room temperature for 1 hour at two times. The resulting extracts were filtrated with filter paper (Whatman Co., USA), combined, concentrated under reduced pressure with evaporator (Eyela Co. FD-5N model, Japan) and dried with cryophilization to afford 140.8g of dried water-soluble extract of mulberry, which was used as a sample in further experiment.

Example 3. Preparation of pharmacologically active fractions of a mulberry (3)

To obtain pharmacologically active fractions of a mulberry, the water-soluble extract in Example 1 were subjected to open column chromatography, i.e., the mulberry extract was poured into HP-20 column (Dianion Co. Ltd) with a drop wise manner, eluted with 100%) distillated water and then 100% methanol to obtain two kinds of fractions i.e., 100% water elution and 100% methanol elution respectively.

Reference Example 1 : MCAo experimental model The procedure and experimental method used in Experimental Examples was described as follows:

Method 4-weeks old white male Sprague-Dawley mouse weighing about 150g was procured from Biogenomics Co. Ltd. (Seoul), fasted up to the weight of 300kg providing with free access to water and feed and acclimated to experimental environment.

The mouse was orally anesthetized with 5% isoflurane gas (mixed gas with the gas mixture consisting of 70% N₂O and 30% O gas and 5% isoflurane) and maintained with 1% isoflurane gas during experiment.

The skin at the center of mouse neck was excised and right-handed CCA (common carotid artery) and ECA (external carotid artery) were isolated from neighboring tissue and nerves with care. CCA and ECA were ligated with ligature and ICA (Internal Carotid Artery) and pterygopalatine artery were also isolated and ligated in similar manner to above procedure. ICA near by bifurcation was partially excised with scissors and the blood vessel was pored to insert probe. The probe was made by the step consisting that 23 mm of 4-0 nylon thread was coated with silicone component such as Polysiloxan solidified with activator to increase their adhesive force to blood vessel. ICA was ligated at the point that about 10 mm of coated probe was inserted to reach at 20mm or the less from the inserting point and the resistance was felt. The probe was fixed at the same time preventing regurgitation. The skin excision area was sealed again and the mouse was restored from anesthetized status naturally. Since inserted probe obstruct the blood flow of MCA, the regional ischemia was induced and the mouse was re-anesthetized to remove the probe restoring carotid blood flow after 1 hour 30 minutes. 24 hours after reperfusion, the mouse was killed to cervical dislocation to deliver brain and the brain tissue was examined by microscope. The body temperature was monitored and maintained at 37+ 0.5 ^°C . The area of +5.2, +3.2, +1.2, -0.8, -2.8, -4.8, -6.8, -8.8 mm caudal to the Bregma, were excised and terminal tissue was discarded to obtain 7 numbers of brain slices with the thickness of 2mm using by brain matrix. Optimum amount of 2%> triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium-chloride: TTC) solution diluted with saline solution, was poured into 9 well plates. The brain slices were added thereto and stained at 37°C for 30 minutes. After 30 minutes staining, the plates were fixed by 4% paraformaldehyde for about 3 hours. The stained tissues were photographed by digital camera (Olympus Co. c2500L model, USA) and the images were transferred to computer. The volume of cerebral infarction region was calculated by the OPTIMAS program (Media Cybernetics Co., 6.51 version) and analyzed by image analysis system. To determine the exact volume of cerebral infarction region of right hemisphere excluding edema volume (A), the real volume of cerebral infarction (B) was calculated by following Empirical Formula 1 and 2.

[Empirical Formula 1] The volume of infracted hemisphere tissue induced cerebral ischemia (mm²) =

(The volume of left hemisphere to which ischemia was not induced)-(The volume of right hemisphere which ischemia was induced but has no tissue infarction) [Empirical Formula 2] The volume percentage of total infracted hemisphere tissue induced cerebral ischemia (%) = (The volume of infracted hemisphere to which ischemia was induced)-(The volume of left hemisphere to which ischemia was not induced) x 100

In order to confirm the preventing or treating activity of the extract and fractions of the present invention for brain stroke, the MCAo (Middle Cerebral Artery occlusion) experiment model was performed by following procedure disclosed in the literature (Nagasawa H. and Kogure K.; Stroke, 20(8), pρl037-1043, 1989), which is well known to appropriate experimental model in the development of brain stroke treating agent since the action mechanism of this model is most similar to that of human.

Experimental Example 1 :

The inhibition effect of mulberry water extract on ischemic stroke (1) The inhibition activity of mulberry water extract on ischemic stroke, was confirmed by MCAo model disclosed in Reference Example 1 in dose-dependent manner.

The mulberry water extract was administrated before about 5 minutes of cerebral reperfusion in two administration ways. At about 2 hours, the water extract was administrated again in lOOmg/kg dose and another is administrated once in 200mg/kg dose.

In accordance with the procedure disclosed in Test Example 1, the extent of cerebral infarction (%) was determined by above empirical formula 2 and the result was shown in Table 1. Fig. 1 represents the comparison between different treatments for the volume of cerebral infarction (mm²).

Experimental Example 2:

The inhibition effect of mulberry water extract on ischemic stroke (2) Excepting that 150mg/kg of mulberry methanol extract was administrated before about 5 minutes of cerebral reperfusion in Test Example 1, all the procedure was prosecutes in similar to Experimental Example 1 and the result was shown in Table 1.

Ebselen (E3520, Sigma Aldrich Co.) and Baicalein (46511-9, Aldrich Co.) known to be effective in cerebral stroke were used as positive control. As shown in Table 1, it is confirmed that mulberry water and methanol extract have inhibition effect on cerebral infarction similar to those of positive controls. [Table 1]

Experimental Example 3:

The inhibition effect of mulberry extract on ischemic stroke (3) The brain slices were prepared from Sprague-Dawley rat weighing between 180 and

200g (Jungang Lab Animals Inc., Seoul) by the method disclosed in the literature (Miyakawa et al.; Yamazata Med., 3, ppl3-26, 1985). The hippocampus tissue slices was prepared from the brain of rat killed by cervical dislocation in medium of ACSF (Artificial Cerebral Spinal Fluid; 124 mM NaCl, 5mM KC1, 1.25mM NaH₂PO₄, 2mM MgSO₄, 2mM CaCl , 23mM NaHCO₃, lOmM glucose) equilibrated with 95% oxygen gas in 15 minutes and cut into 400 μm section with microtome (Macllwain). The sections were transferred to ACSF equilibrated with 95% O₂ and 5% CO gas and left alone at room temperature for 1 hour in order that the section recovers functionally.

Glucose-free 6 sections were transferred to ACSF equilibrated with 95% O₂ and 5% CO gas and made to oxygen gas fee status in low oxygen chamber (Forma Scientific Co. USA) to be experimental ischemic status for 20 minutes.

180 i of PCN buffer solution (12mM NaCl, 5mM KC1, lOmM NaHCO₃) was added thereto and the tissues were broken up by ultra-membrane sonicator (Brunson Co. USA). And 120 βl of neutralization buffer solution was added thereto and centrifugation was prosecuted at the speed of 12000 rpm for 7 minutes(Beckman Co.). Resulting supernatant was transferred to e-tube and the ATP content of the solution was determined by luminometer (Lumat Lb 9051, Berthold USA).

The result of calculating ATP content in hippocampus tissue treated with 1, 10, 50 and 100 μi/ A of mulberry extract, was shown in Fig. 2. As shown in Fig. 2, it is confirmed that even lower concentration of mulberry extract treatment group such as 1 μ /ml has increasing effect of reduced ATP content induced by ischemia.

Experimental Example 4:

The free radical scavenging effect of mulberry extract In order to confirm the effect of mulberry water extract on the production of free radical, one of the cause of brain neuronal cell injury, the scavenging effect on DPPH activity was determined to confirm anti-oxidative activity of mulberry extract.

1 mg/ml of mulberry extract was diluted with DPPH(l,l-diphenyl-2-picrylhydrazyl, 1.5xl0^"4M) methanol solution and stirred vigorously. And then the solution was left alone for 30 minutes in air and the content of remaining DPPH in the solution was determined at 520nm.

The experimental procedure was used as the method disclosed in the literature (Vivot, E. et al.; J. Ethnopharmacology, 76, pp65-71, 2001).

As the scavenging activity of DPPH increases, so the anti-oxidative activity increases. The DPPH scavenging activity of mulberry water extract was expressed as percentage (%) for that of untreated group and the result was shown in Table 2.

As shown in Table 2, The DPPH scavenging activity of mulberry water extract shows higher activity than that of Ebselen and similar activity to that of Baicalein control group, which confirms that mulberry water extract has potent anti-oxidative activity also.

[Table 2]

Experimental Example 5:

Inhibitory effect of the Mulberry extract on NO production It has been reported that the cerebral ischemia is caused by the inflammation of microglial cells.

To observe the protective effect of the mulberry extract on inflammation when the brain is damaged, BV-2 mouse microglial cell line (Weill Medical College of Cornell University, U.S.A.) was cultured, stimulated by LPS and treated with mulberry extract, followed by determining the amount of NO production to confirm the suppression of inflammation.

BV-2 cells were co-treated with 10 /g to lOOO tg . of lipopolysaccharides (LPS, Difco Co.) and respective lμg/ t, 5μgM and 10βg/τ of mulberry extract for 20 hours. After treatment, culture supernatants were transferred to the new tube. 50μi of supernatant was mixed with 50//I of Griess reagent in 96-well plate and incubated at room temperature for 10 minutes. In order to quantify the amount of nitrite released to the medium (Choi, J. j. and Kim, W. K.; J. Neurosci. Res., 54, pp870-875, 1998), the absorbance of the medium was measured on a microplate reader at 550nm (Molecular device, U.S.A.). NaNO₂ was used as standard to calculate NO ^" concentration. hi the result of the treatment of 1, 5 and lOβg/mi mulberry extract to BV-2 cell, pretreated with lOOng/mi. LPS to lead significant increase of NO, it was confirmed that the NO production was decreased by treatment of inventive mulberry extract.

Experimental Example 6:

Inhibitory effect of the Mulberry extract on inflammation

To test the anti-inflammatory activity of the mulberry extract, it was determined the effect of inventive extract on cyclooxygenase-2 (COX-2) activity. As the expression of COX-2 is enhanced by various inflammatory molecules, we had intended to assess the specific antagonistic function of inventive extract in COX-2 related inflammation.

COX-2 is known to show selectively increased expression in the cells activated by LPS and cytokines. Therefore, after adding LPS to mouse alveolar macrophage to increase COX-2 expression, thromboxaneB₂ was measured

Sprague-Dawley rats were housed in the environment minimized the exposure of airborne bacteria and bronchoalveolar lavage was prepared from their alveolar macrophage according to the method disclosed in the literature (Chandler, D. B and Fulmer, J. D.; J. of Immunol., 139, pp893-898, 1987). Cell viability was assayed by the trypan blue dye exclusion method. Cells showing more than 90% viability were diluted in RPMI1640 medium (Gibco BRL Co.) and laid on microtiter plate.

Asprin (500 μM, Sigma Aldrich Co.) for selectively inhibiting COX-1, LPS (lOβgf mi, Difco Co.) and FBS (fetal bovine serum) were treated to the above-prepared cells and Nabumethone (Handok Pharmaceuticals Co.) for control group and mulberry extract for experimental group was added thereto at the concentration of lOppm and subsequently incubated for 16 hours.

After removing the medium, the cells were incubated again in the fresh medium containing 30μM arachidonic acid (Sigma Aldrich Co.) for 10 minutes. Whole medium in each well was transferred to the new tube, stored at -20°C and used for radio-immuno assay as a sample to measure the amount of TXB₂. Amount of protein was measured with Bradford reagents using BSA as a standard.

To quantify the amount of TXB₂, antiserum and ³H-TXB₂ were added to the each sample. After binding TXB₂ in the sample or ³H-TXB₂ to antiserum competitively, free TXB₂ was precipitated by adding charcoal dextran and the precipitates were removed.

Scintillation cocktail was added to complex of ³H-TXB₂ and antiserum for radioactivity measurement. According to the standard calibration curve, the amount of TXB₂ in each sample was calculated and again it was converted to the production of TXBS per protein mg for the level of COX-2 activity (Fu, J. Y. et al.; J. Biol. Chem., 265, ppl6737-16740, 1990). Antiphlogistic drug, Nabumethone was used as a control for mulberry extract. COX-2 activity of 10 ppm inventive extract showed about 0.55 compared with that of lOppm Nabumethone regarded as 1, therefore it was confirmed that inventive mulberry extract inhibited the COX-2 activity (Table 3). Above results are presented by comparing mulberry crude extract and single compound Nabumethone.

[Table 3] COX-2 inhibitory effect of mulberry extract

Mulberry extract was verified to be effective to prevent or treat brain stroke, cerebral ischemia and brain neuronal diseases generated by inflammation of neuronal cell.

As described above, it is confirmed that mulberry extract prepared by the present invention shows therapeutic and protective effect for brain stroke and thus, it is useful for anti-brain stroke drug or health care food.

Experimental Example 7. Toxicity test

Methods (T)

The acute toxicity tests on ICR mice (mean body weight 25±5g) and Sprague-

Dawley rats (235+ lOg, Jung-Ang Lab Animal Inc.) were performed using the extract of the Example 1. Four group consisting of 10 mice or rats was administrated orally intraperitoneally with 250mg/kg, 500mg/kg, lOOOmg/kg and 5000mg/kg of test sample or solvents (0.2 mi, i.p.) respectively and observed for 2 weeks.

Methods (2)

The acute toxicity tests on ICR mice and Sprague-Dawley rats were performed using the extract of the Example 1. Four group consisting of 10 mice or rats was administrated intraperitoneally with 25mg/kg, 250mg/kg, 500mg/kg and 725mg/kg of test sample or solvents (0.2 mi, i.p.), respectively and observed for 24 hours. Results

There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender. These results suggested that the extract prepared in the present invention were potent and safe.

Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not hmited to them. The representative preparation examples were described as follows.

Preparation of powder

Dried powder of Example 1 50mg

Lactose lOOmg

Talc lOmg

Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet

Dried powder of Example 1 50mg

Corn Starch lOOmg Lactose lOOmg

Magnesium Stearate 2mg

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule Dried powder of Example 1 50mg

Corn starch lOOmg

Lactose lOOmg

Magnesium Stearate 2mg Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of injection

Dried powder of Example 1 50mg

Distilled water for injection optimum amount PH controller optimum amount

Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2mi ample and sterilizing by conventional injection preparation method.

Preparation of liquid

Dried powder of Example 1 0.1~80g

Sugar 5~10g

Citric acid 0.05-0.3%

Caramel 0.005-0.02%

Vitamin C 0.1-1%

Distilled water 79-94%

CO₂ gas 0.5-0.82%

Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

Preparation of health care food Extract of Example 1 lOOOmg Vitamin mixture optimum amount

Vitamin A acetate 70μg Vitamin E l.Omg Vitamin Bi 0.13mg Vitamin B₂ 0.15mg

Vitamin B6 0.5mg Vitamin B12 0.2μg Vitamin C 10mg Biotin 10μg Amide nicotinic acid 1.7mg

Folic acid 50μg

Calcium pantothenic acid 0.5mg Mineral mixture optimum amount

Ferrous sulfate 1.75mg Zinc oxide 0.82mg

Magnesium carbonate 25.3mg Monopotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate lOOmg Magnesium chloride 24.8mg The above-mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of health beverage Extract of Example 1 lOOOmg

Citric acid lOOOmg

Oligosaccharide lOOg

Apricot concentration 2g

Taurine lg Distilled water 900m-β

Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 ^°C for 1 hour, filtered and then filling all the components in lOOOmβ ample and sterilizing by conventional health beverage preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Industrial Applicability

The composition comprising the mulberry extract according to the present invention shows protecting effect of neuronal cell and preventing or treating effect for of brain stroke and cerebral ischemia. Therefore, it is useful in the prevention or treatment of brain stroke and cerebral ischemia in human or mammal.

Claims

1. A pharmaceutical composition comprising mulberry extract as an active ingredient in an amount effective to protect neuronal cell and to prevent and treat human or mammal suffering from brain diseases together with a pharmaceutically acceptable carrier.

2. A pharmaceutical composition comprising mulberry extract as an active ingredient in an amount effective for prevention and treatment of inflammatory diseases, together with a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 1 or 2 wherein said mulberry extract is extracted with the solvent selected from the group consisting of water, lower alcohol and the mixture thereof.

4. The pharmaceutical composition according to claim 3 wherein said lower alcohol is methanol.

5. The pharmaceutical composition according to claim 1 or 2 wherein said mulberry extract is prepared by the sonication or reflux extraction.

6. The pharmaceutical composition according to claim 5 wherein said mulberry extract is containing the fraction obtained by the serial procedure comprising the steps consisting of concentrating the extract under reduced pressure and subjecting column chromatography and eluting with distilled water or lower alcohol.

7. The pharmaceutical composition according to claim 1 or 2 wherein said brain disease is brain stroke or cerebral ischemia.

8. The pharmaceutical composition according to claim 1 or 2 wherein said composition is containing the extract between 0.5 to 50% by weight based on the total weight of the composition.

9. A use of mulberry extract as set forth in claim 1 in the manufacture of the medicament to prevent or treat brain stroke and cerebral ischemia.

10. A health care food comprising the extract as set forth in claim 1 and a sitologically acceptable additive to protect neuronal cell and to prevent or improve brain disease such as brain stroke and cerebral ischemia.

10. The health care food according to claim 9, wherein said health care food is provided as beverage type.