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Title:
PHARMACEUTICAL COMPOSITIONS COMPRISING AN EXTRACT OF PELVETIA SILIQUOSA FOR THE PREVENTION OR THE TREATMENT OF LIVER DISEASE, DIABETES AND DIABETIC COMPLICATIONS
Document Type and Number:
WIPO Patent Application WO/2003/092710
Kind Code:
A1
Abstract:
The present invention relates to an extract of $i(Pelvetia siliquosa) and pharmaceutical compositions and health foods comprising the same. Particularly, it relates to an extract of $i(Pelvetia siliquosa) prepared by extracting $i(Pelvetia siliquosa) with one or more solvent selected from a group consisting of water, single or mixed ether, ethanol and chloroform, and pharmaceutical compositions and health foods comprising the same. The extract of $i(Pelvetia siliquosa) shows strong effect on liver protection, down-regulation of the blood glucose level, and prevention of diseases related to diabetes mellitus. Moreover, Pelvetia siliquosa is proved to be very safe because it has been used for a long time as oriental plant medication. So, the extract of $i(Pelvetia siliquosa) of the present invention can be use as pharmaceutical compositions or health foods for the prevention or treatment of liver disease or diseases related to diabetes mellitus.

Inventors:
SHIN KUK HYUN (KR)
SHIN YAUNG WOO (KR)
SHIN KYOO YUL (KR)
Application Number:
PCT/KR2002/000863
Publication Date:
November 13, 2003
Filing Date:
May 09, 2002
Export Citation:
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Assignee:
SHIN KUK HYUN (KR)
SHIN YAUNG WOO (KR)
SHIN KYOO YUL (KR)
International Classes:
A23G4/00; A23G4/06; A23L1/30; A23L13/60; A61K36/03; A61P1/16; A61P3/04; A61P3/10; (IPC1-7): A61K35/78; A23L1/30; A23L1/337; A61K38/55; A61P3/04
Foreign References:
JP2000342224A2000-12-12
Other References:
OHTA T. ET AL.: "Alpha-glucosidase inhibitory activity of a 70% methanol extract from ezoishige (pelvetia babingtonii de toni) and its effect on the elevation of blood glucose level in rats", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 66, no. 7, July 2002 (2002-07-01), pages 1552 - 1554, XP055192543, DOI: doi:10.1271/bbb.66.1552
ROZKIN M.L.A. ET AL.: "The anticoagulant and lipolysis-stimulating activity of polysaccharides from marine brown algae", FAMARKOL. TOKSIKOL., vol. 54, no. 5, September 1991 (1991-09-01) - October 1991 (1991-10-01), pages 40 - 42
SHIRAHAMA K.: "Unsaponifiable matter and phosphatides in marine algae fat", J. OF FACULTY AGR. HOKKAIDO IMP. UNIV., vol. 49, 1942, pages 1 - 93
Attorney, Agent or Firm:
Lee, Won-hee (Sung-ji Heights II 642-16 Yoksam-don, Kangnam-ku 135-080 Seoul, KR)
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Claims:
What is claimed is:
1. An extract of Pelvetia siliquosa having strong effects on liver protection, downregulation of the blood glucose level, and prevention of diseases related to diabetes mellitus prepared by extracting Pelvetia siliquosa with one or more solvent.
2. The extract of Pelvetia siliquosa as set forth in claim 1, wherein the solvent is selected from the group consisting of water, single or mixed ether, ethanol and chloroform.
3. The extract of Pelvetia siliquosa as set forth in claim 2, wherein the solvent is single or mixed ether.
4. The extract of Pelvetia siliquosa as set forth in claim 3, wherein the ether is ethyl ether.
5. A pharmaceutical composition for the prevention or the treatment of liver diseases, containing the extract of Pelvetia siliquosa of claim 1 as an effective ingredient.
6. A pharmaceutical composition for the prevention or the treatment of diseases related to diabetes mellitus, containing the extract of Pelvetia siliquosa of claim 1 as an effective ingredient.
7. The pharmaceutical composition for the treatment or the treatment of diseases related to diabetes as set forth in claim 6, wherein the diseases related to diabetes and diabetic complications.
8. A health food for liver protection and down regulation of the blood glucose, containing the extract of Pelvetia siliquosa of claim 1 as an effective ingredient.
Description:
PHARMACEUTICAL COMPOSITIONS COMPRISING AN EXTRACT OF PELVETIA SILIQUOSA FOR THE PREVENTION OR THE TREATMENT OF LIVER DISEASE, DIABETES AND DIABETIC COMPLICATIONS FIELD OF THE INVENTION The present invention relates to an extract of Pelvetia siliquosa and pharmaceutical compositions and health foods comprising the same. Particularly, it relates to an extract of Pelvetia siliquosa prepared by extracting Pelvetia siliquosa with one or more solvent selected from a group consisting water, single or mixed ether, ethanol and chloroform, and pharmaceutical compositions and health foods comprising the same.

BACKGROUND OF THE INVENTION According to the rapid economic growth, westernization and upgraded standard of living, a remarkable physical development has been achieved to meet the western standard. Meanwhile, disease pattern also follows that of western putting adult diseases first, which is resulted from taking excessive high- calorie food, lack of exercise and stress accompanied by working in this industrial society. Liver diseases,

hypertension, diabetes, obesity and hyperlipemia are the well-known examples of adult diseases.

In case of liver disease, Korea has comparatively higher incidence rate comparing to other countries.

Chronic fatigue caused by stress and most xenobiotics do harm the liver. The liver shows the most active metabolism among human organs. The acute or chronic disorder of the liver caused by fatty food, excessive alcohol intake, virus infection, toxic substances including various drugs, and malnutrition can grow to a fatty liver, hepatitis, jaundice, hepatic cirrhosis, and hepatoma. Especially, excessive fatty food taking and alcohol overdrinking can cause a fatty liver led by the accumulation of lipid in the liver tissues. At this time, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and y-glutamyl transpeptidase (y-GTP) in the blood serum are increased (Med. Interne., 20,69-71, 1982; Acta. Med.

Acad. Sci. Hung., 33,309-319, 1976). Liver diseases are growing without pain or subjective symptoms, and usually found after being seriously developed.

The most widely used treatments for liver disease are dietary treatment and medication, and in most cases both treatments are used together. For the medication for liver diseases, various medicines having different mechanisms are used to suit the cases and types of

liver diseases. For example, stimulators of the liver cell regeneration and auxiliary agents of the liver function such as ursodeoxycholic acid, silymarin (Biotech. Therapeutics, 4,263-270, 1993), biphenyl dimethyl dicarboxylate (DDB; Biochem. Biophy. Res. <BR> <BR> <P>Comm. , 103,1131-1137, 1981), glutathione and glycyrrhizin, anti-viral agent such as acyclovir, immune inhibitors such as corticosteroid, 6- mercaptopurine (6-MP), azathioprine can be used.

However, they are not good enough to get rid of causes of the diseases for the fundamental treatment. The fundamental treatment for the liver diseases is very difficult since liver diseases are developed not by one factor but by the complex action of various causes.

Thus, it is required to use various medicines having different mechanisms together for the satisfactory treatment. In addition, the treatment agents for liver diseases have side effects when being used massively or for a long period.

Therefore, the epoch-making pharmaceutical compositions for the liver protection and for the treatment of liver diseases such as hepatitis caused by virus, fatty liver, chronic hepatitis, etc are not developed yet, even though these diseases become a serious social problems.

In order to examine liver protective effects of a

medicine, the CC14 model (Biochemical Pharmacology, 39, <BR> 12,2027-2034, 1990 Japan J. Pharmacol. , 42,19-26, 1986 J. Biological Chemistry, 236,2, 1961) has been used as a general liver disorder model. CCl4 is converted to a toxic metabolite trichloromethyl free radical (CCl3) by cytochrome P-450 inside the liver and this metabolite is combined strongly with membrane protein thiol group of liver microsome to form a lipid radical which is later converted to peroxy radical when oxygen is added thereto. Peroxy radical stimulates peroxydation of liver membranes, resulting in the liver disorders. In other words, CCI4 suppresses protein synthesis of the liver, increases the levels of GOT and GPT in the blood, and causes small leaf-centered death of the liver cells.

Diabetes is an uncontagious chronic disease and is developed when the insulin secretion in P-cells of pancreas is insufficient or insulin does not work well, by which glucose cannot be used as an energy source and remained in the blood in high concentration which will be excreted in urine later.

According to the causes and the treatment methods of symptoms, diabetes is mainly classified into two groups ; one is insulin dependent diabetes mellitus (IDDM) and the other is non-insulin dependent diabetes

mellitus (NIDDM). In Korea, more than 95% of diabetes patients are NIDDM patients. The importance of dealing with diabetes is not in the disease itself but in its complications such as neuropathy, retinopathy, cataract, nephropathy, etc since they affect patients to carry on normal lives and can even make fetal blow.

The general therapeutic medicines for diabetes are oral alleviator of blood glucose and insulin injection. Insulin injection is required for insulin dependent diabetic patients who have no insulin secretion inside body, diabetic patients caused by pregnancy, and non-insulin dependent diabetes patients whose blood glucose is not regulated by oral alleviator of blood glucose. Meanwhile, non-insulin dependent diabetic patients whose blood glucose is not controlled with just a dietetic treatment and an exercise treatment need to take oral alleviator of blood glucose.

Oral alleviator of blood glucose is classified into two groups; sulfonylureas and biguanides.

Sulfonylureas such as glipizide, gliclazide, gliquidone, glibenclamide and chlorpropamide work to stimulate insulin secretion in pancreas, and thus they do not work for insulin-dependent diabetic patients who have no insulin secretion at all. Pregnant women should avoid taking sulfonylureas since they can cause serious side effects such as birth of malformed child,

abortion and stillbirth. Patients with liver and kidney disorders also need careful considerations on taking sulfonylureas since most sulfonylureas are metabolized in the liver and discharged through kidney.

The mechanism of biguanides like metformin has not been clearly clarified yet, but it is believed not to stimulate insulin secretion in the pancreas and to have weaker blood glucose-lowering effect and lower possibility of causing low-blood glucose compared to sulfonylureas. However, biguanides accompany side effects of digestive system such as nausea, vomiting, diarrhea, eruption, etc, especially in early stage of the treatment and even cause fetal side effects by inducing lactic acidosis, resulting in the limited use thereof only for experimental medicine in the united states.

Considering all those side effects of sulfonylureas or biguanides, it is required to develop much safer alleviator of blood glucose having less side effects for the satisfactory treatment of diabetes.

Pelvetia siliquosa (Tseng et Chang), which belongs to Fucaceae family, is a kind of yellow-brown colored seaweeds living widely throughout southern seashore and western seashore in the Korean peninsula by forming colonies.

Pelvetia siliquosa has been used as an edible herb especially traditionally for the ancestor's worship table. Recently, this herb has been proved to have a medicinal effect when being used as a raw material for alginate, a treatment agent for incurable chronic adult diseases like hypertension, etc and as a healthy food. Yet which ingredient of Pelvetia siliquosa has what effect is not reported and particularly the pharmacological effect on the liver disorder and diabetes is not fully studied.

Therefore, we, the present inventors have studied whether the extract of Pelvetia siliquosa prepared by extracting Pelvetia siliquosa with one or more solvent selected from the group consisting of water, single or mixed ether, ethanol and chloroform can protect liver and suppress diabetes and its complications.

Resultingly, the present inventors have accomplished the present invention by confirming that the extract of Pelvetia Siliquosa has a similar liver protective effect as to silymarin, a widely used as clinically liver protective agent, and a suppressing effect against diabetic complications as well as a similar blood glucose-lowering effects to epalrestat and metformin, which are agents clinically used for the

treatment of diabetes and diabetic complications.

SUMMARY OF THE INVENTION The objective of the present invention is to provide the extracts of Pelvetia siliquosa having the hepato-protective effects, blood glucose-lowering in diabetes and suppression of diabetic complications, and a pharmaceutical composition or a healthy food containing thereof as an effective ingredient.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention provides the extracts of Pelvetia Siliquosa having the hepatoprotective effects, blood glucose-lowering in diabetes and diabetic complication suppression.

The present invention also provides a pharmaceutical composition for the prevention or the treatment of liver disease, diabetes and diabetic complications or a healthy food containing the extracts of Pelvetia siliquosa as an effective ingredient.

Hereinafter, the present invention is described in detail.

In one aspect, the present invention provides the extracts of Pelvetia Siliquosa prepared by extracting

Pelvetia Siliquosa with solvent, and having a strong liver protective activity.

The extracts of Pelvetia Siliquosa of the present invention was prepared by extracting Pelvetia siliquosa with one or more solvent selected from the group consisting of water, single or mixed ether, ethanol and chloroform and then pressured concentration was performed. At this time, using single or mixed ether is preferred for the solvent, and ethyl ether is more preferred. Extraction can be performed by the methods of cold precipitation, reflux or ultrasonic wave and ultrasonic extraction is preferred.

The present inventors have investigated whether the extracts of Pelvetia siliquosa of the present invention have a liver protective activity. To do this, the present inventors have tested the suppressing effect of the extracts of Pelvetia Siliquosa on liver diseases induced by CCl4 in rats. As a result, the extracts of Pelvetia Siliquosa were confirmed to have a strong liver protective activity, which was almost equipotent to that of silymarin, a well-known liver protective agent used as a positive control.

The present inventors have also investigated whether the extracts of Pelvetia siliquosa cause to

reduce blood glucose level. Particularly, the present inventors measured the blood glucose-lowering effect and diabetic complications suppressing activity of the extracts of Pelvetia siliquosa against diabetes induced by streptozotocin. As a result, the extracts of Pelvetia siliquosa were confirmed to have a strong blood glucose-lowering activity and suppressive effect against sorbitol accumulation in the tissues such as lenses, sciatic nerves and red blood cells. The blood glucose-lowering activity of the extracts in the present invention was similar to that of epalrestat, a well-known treatment agent clinically used for diabetic diseases used as a positive control.

The present invention further provides a pharmaceutical composition containing the extracts of Pelvetia siliquosa as an effective ingredient for the prevention or the treatment of liver diseases, diabetes and diabetic complications. The diabetic complications include degenerative complications induced by diabetes such as diabetic retinitis, diabetic neurosis, renal disease, artherosclerosis, cardiosclerosis, skin trouble, diabetic food symptom and peripheral blood vessel disorder, which are all resulted from the accumulation of solbitol in the crystalline lens, red blood cells or the sciatic nerves. Thus, the

suppression of the accumulation of sorbitol in the tissues due to the inhibition of aldose reductase activity in the polyol pathway can contribute to inhibit the development of diabetic complications.

Recently, studies have been undergoing to control such diabetic complications as cataract, retinitis, renal disease, etc by suppressing aldose reductase, which is related to the first step in the polyol pathway.

The pharmaceutical composition for the prevention or the treatment of liver and diabetic diseases contains the above extracts of Pelvetia stliquosa'as an effective ingredient. And the pharmaceutical composition of the present invention can be administered orally or parenterally. The pharmaceutical composition of the present invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients. The present invention also includes the pharmaceutical formulations in dosage units, which means that the formulations are presented <BR> in the form of individual doses, e. g. , tablets, coated tablets, capsules, pills, suppositories, or ampules.

In general, it has proved advantageous both in human and in veterinary medicine to administer the

active compound or compounds according to the present invention in total amounts of about 500 mg/adult (about 50 kg) to about 1,000 mg/adult (about 50 kg), preferably 250-300 mg/adult, one to six times every 24 hours, if appropriate, in the form of several individual doses, to achieve the desired results. However, it may be necessary to deviate from the dosages mentioned, and in particular to do so as a function of the nature and body weight of the object to be treated, the nature, and severity of the disease, the nature of the formulation and of the administration of the medicament, and the period or interval within which administration takes place. Thus, in some cases, it can suffice to manage with less than the above-mentioned amount of active compound, while in other cases the above mentioned amount of active compound must be exceeded.

The particular optimum dosage and mode of administration required for the active compounds can be determined by any expert in the field.

The extracts of Pelvetia siliquosa, as the raw material, are a natural medicinal stuff and its safety has been confirmed. The present inventors performed acute toxicity test in rats by administrating the extracts of Pelvetia siliquosa orally. As a result, the extracts of Pelvetia siliquosa are evaluated to be safe since they do not cause any toxic symptoms in rats

treated up to the level of 10 g/kg orally and their estimated LD50 values are higher than 10 g/kg in rats.

The present invention also provides a health food containing the extract of Pelvetia siliquosa to protect liver, to lower blood glucose level in the diabetes and to suppress diabetic complications.

For the use thereof as a food, the extract of Pelvetia siliquosa can simply be added as it is or can be used with other foods together following the general food producing process. The mixing rate or amounts depend on the purpose of its use (prevention, health or therapeutic treatment). Generally, when the extract of Pelvetia siliguosa is used for producing foods or drinks, it is recommended to add the extract with the concentration of 40-50 weight% for total raw material and the concentration of 50-60 weight% is more preferred. The effective amount of the extract of Pelvetia siliquosa follows the effective amount of the above pharmaceutical compounds, but it could be lower than the standard dosage when long-term administration is required for controlling health. Over dosage, though, also can be used since the extract of Pelvetia siliquosa has been proved not to have any side effects.

Any kinds of foods containing the extract of Pelvetia siliquosa can be made without limitation. For example, meat, sausage, bread, chocolate, candies,

snacks, cookies, pizza, ramyon and other noodles, gums, dairy products including ice creams, soups, beverages, teas, drinks, alcoholic beverages, vitamin complex, etc are the foods to be made as a health food containing the extract of Pelvetia siliquosa.

EXAMPLES Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on the consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of the extract of Pelvetia siliquosa To remove salt, 2,050 g of Pelvetia siliquosa was put in 20 L of distilled water for 24 hours, and then dried. The dried Pelvetia siliquosa was extracted with ether (10 L) 5 times at room temperature and concentrated in vacuo, from which the extract of Pelvetia siliquosa (28.3 g) was prepared.

Experimental Example 1: The liver protective activity test for liver disease induced by CCl4

Male 11-week old Sprague-Dawley rats were divided into 4 groups (10 rats/group) ; the normal group, the control group, the extract of Pelvetia siliquosa treated group and silymarin treated group. In order to induce acute liver toxicity, rats were injected with the mixture of CC14 and olive oil (mixed by 3: 2 V/V) with 0.6 ml/kg each into peritoneal cavity according to <BR> <BR> Gordis'method (E. Gordis, J. Clin, Inver. , 48,203, 1969). Eighteen hours before, 30 minutes before and 6 hours after CCl4 injection, oral administration was performed to every group with 0. 5% carboxyl methyl cellulose, 300 mg/kg of the extract of Pelvetia siliquosa and 200 mg/kg of silymarin suspended in 0. 5% carboxyl methyl cellulose. Twenty four hours after CC14 injection, the rats were anesthetized with ether and blood sample was obtained by cardiac puncture.

After elimination of blood clots, the blood sample was centrifuged at 3,000 rpm for 10 minutes to obtain the serum. The activity of GOT and GPT, as indices of the liver toxicity, was measured using GOT/GPT kit according to Reitman-Framkel's method (S. Reitman, S.

Frankel, Am. Clin. Pathol., 28,56, 1957).

As a result, the activity of GOT and GPT in the serum remarkably increased in the control group in which only CC14 was injected, but significantly decreased in the extract of Pelvetia siliquosa treated

group compared to the control group. Actually, the extract of Pelvetia siliquosa has been demonstrated to reduce the activity of GOT and GTP in the blood of the rats induced the liver toxicity by treatment with CCI4 and showed liver protective effect as well which is similar to that of silymarin having been used as a liver protective agent for a long time. The test results are shown in Table 1 and 2.

<Table 1> Sample GOT Inhibition rate (Karmen unit) Normal group 32. 9 4. 8 Control group 192.2 + 13. 5 (CC14 treated) Silymarin-treated 118. 8 + 5. 8** 38.2 group The Extract of 135.2 5. 7* 29.7 Pelvetia siliquosa-treated group <Table 2> Sample GPT (Karmen unit) Decreasing rate ( Normal group 15.3 2. 0 Control group 101.7 _ 9. 8 Silymarin-treated 58. 5 6.0** 42.5 group The extract of 62. 7 5. 7** 38.3 Pelvetia siliquosa-treated group

As shown in Table 1 and 2, the extract of Pelvetia siliquosa of the present invention showed an effective liver protective activity in animals having liver diseases induced by CCl4. Therefore, the extract of Pelvetia siliquosa can be effectively used for the prevention or the treatment of the liver disease.

Experimental Example 2: Anti-diabetic and anti-diabetic complication activity test <2-1> Measurement of blood plasma glucose Male 11-week old Sprague-Dawley rats were divided into 4 groups (10 rats/group); the normal group, the control group, the extract of Pelvetia siliquosa treated group and epalrestat treated group. In order to induce diabetes, the rats were administered streptozotocin (66 mg/kg) dissolved in 0.05 M citrate into peritoneal cavity. Two weeks after the induction of diabetes, 200 mg/kg of saline, the extract of Pelvetia siliquosa, and epalrestat were administered orally to each group, respectively. Two weeks later, the rats were anesthetized with ether, and then blood, crystalline lens and sciatic nerves were removed.

Divided the blood samples to measure blood glucose level and sorbitol content in red blood cells. Blood samples were centrifuged to obtain serum, and then blood glucose level was measured with glucose-oxidase

kit.

As a result, the group treated with streptozotocin only showed the increase in the blood glucose levelcompared to those of the normal group, but the extract of Pelvetia siliquosa treated group caused remarkable decrease in the blood glucose level compared to those of the control group. Particularly, the extract of Pelvetia siliquosa showed blood glucose lowering effect, which is similar to that of epalrestat, a clinical agent for the treatment of the diabetic diseases, which is classified as an aldose reductase inhibitor. The results were shown in Table 3.

<Table 3> Group Blood glucose Decreasing rate (g/L) (%) Normal group 1.2 0. 08 Control group 5. 7 0. 33 (streptozotocin treated) Epalrestat 3. 9 0. 24** 31.6 treated group Extract of 4. 0 + 0. 30* 29.6 Pelvetia siliquosa treated group <2-2> Glucose tolerance test Male 11-week old rats were divided into the control group, the extract of Pelvetia siliquosa

treated group and metformin treated group (10 rats/group). Before the glucose tolerance test, the rats were fasted for 16 hours. Then, saline, 300 mg/kg of Pelvetia siliquosa extract and 250 mg/kg of metformin were orally administered to each rat. 30 minutes later, 3 g/kg of glucose was orally administered to every group. Blood samples were obtained from orbital veniplex and blood glucose was measured 0,60, 120,180 minutes after glucose load.

As a result, two hours after glucose administration, it was observed that the blood glucose levels were remarkably decreased in the extract of Pelvetia siliquosa treated group compared to the control group, which means the extract of Pelvetia siliquosa has glucose tolerance effect. Such blood glucose-lowering effect was similar to that of metformin, a hypoglycemic agent belonging to biguanides.

The results were shown in Table 4.

<Table 4> Group 0 hour 1 hour 2 hour 3 hour Control group 55.5 1.9 160.3 + 189. 7 + 132. 1 4.8 4.0 3.4 Metformin 54. 5 2.2 140. 1 + 122. 8 139. 6 treated group 7. 0* 1. 4** 4.5 Extract of 47. 6 + 3. 8 191. 5 139. 3 + 124. 3 Pelvetia 12.9 8.8** 4.4 siliquosa treated group

<2-3> Aldose reductase inhibitory effect The present inventors have prepared zymogen according to Hayman's method with slight modification to measure the aldose reductase inhibitory effect.

Particularly, the crystalline lenses of rats were removed and homogenized after adding phosphate buffer according to the wet weight of the lenses. After centrifugation thereof at 4 C, obtained the supernatant and saturated thereof with ammonium sulfate until its concentration reached 40%. Added ammonium sulfate to the obtained supernatant by centrifugation again to 70%, and then stirred for about 1 hour. Obtained the precipitates by centrifugation and suspended thereof in the minimum amount of buffer solution followed by dialysis for one day, from which the zymogen was prepared.

The zymogen was reacted with DL-glyceraldehyde as a substrate, and the decreasing rate of NADPH observance degree at 340 nm was measured. At this time, tetramethylene glutarate (TMG) was used as a standard aldose reductase inhibitor.

As a result, it was confirmed that the extract of Pelvetia siliquosa exhibited an inhibitory effect on rat lens aldose reductase in a dose-dependent manner.

The results were shown in Table 5.

<Table 5> Concentration Decreasing 50% inhibition Group (U g/ml) rate (%) concentration (ii g/ml) TMG 1. 86 87. 4 0. 19 42. 4 0. 67 0. 02 22. 4 Extract of 100 95. 9 Pelvetia 10 49. 4 23.7 siliquosa 5 31.4

<2-4> Measurement of solbitol content in the tissues In order to measure the sorbitol content in red blood cells, the blood samples obtained in <2-1> were pretreated as follows. 5 ml of blood was centrifuged to obtain precipitates. The precipitates were washed three times with cold saline. Took 1 ml therefrom and added 3 ml of 6% perchloric acid. Centrifuged thereof again to precipitate proteins. After neutralization of the supernatant by adding 1 ml of 2.5 M potassium carbonate, centrifuged to obtain protein-free filtrate and measured sorbitol content therefrom according to the Clements method. Particularly, mixed 0.5 ml of supernatant with 1 ml of reaction mixture (glycine buffer pH 9.4, 0.2 mM nicotineamide adenosine dinucleotide, 0.64 U sorbitol dihydrogenase) and allowed to react for 25 minutes. After the reaction

was completed, the sorbitol content in red blood cells was measured by flulorophotometer. The isolated crystalline lens and sciatic nerve were freeze-dried, and then homogenized with 1. 5 ml of distilled water.

Centrifuged thereof and then treated the obtained supernatant by the same method mentioned above to measure sorbitol content in crystalline lens and sciatic nerves.

As a result, sorbitol content in red blood cells was markedly increased in the group treated with streptozotocin only (the control group), but remarkably decreased in the extract of Pelvetia siliquosa treated group, comparing to the normal group. Particularly, the extract of Pelvetia siliquosa showed a significant sorbitol reducing effect similar to epalrestat, clinical agent for the treatment of the diseases related to diabetes mellitus. The results were shown in Table 6.

<Table 6> Group Solbitol in RBC Decreasing (nmol/mg dry wt. ) rate (%) Normal group 63.2 + 3. 9 _ Control group 403.6 61. 3- Epalrestat 108.7 + 11.6** 75.0 treated group

Extract of 145.3 12.8** 66.6 Pelvetia siliquosa treated group Sorbitol content in crystalline lens was also markedly increased in the group treated with streptozotocin only (the control group), but remarkably decreased in the extract of Pelvetia siliquosa treated group, compared to the normal group. Particularly, the extract of Pelvetia siliquosa showed a significant sorbitol reducing effect in crystalline lens similar to epalrestat, clinical drug for the treatment of the disease related to diabetes mellitus. The results were shown in Table 7.

<Table 7> Group Solbitol in Decreasing rate crystalline lens (nmol/mg dry wt.) Normal group 0.05 0. 02 Control group 0. 80 0. 04 Epalrestat 0.38 0. 05* 53.5 treated group Extract of 0.38 0.10* 52.5 Pelvetia siliquosa treated group Sorbitol contents in the sciatic nerves were also markedly increased in the group treated with streptozotocin only (the control group), but remarkably

decreased in the extract of Pelvetia siliquosa treated group, compared to the normal group. Particularly, the extract of Pelvetia siliquosa showed a significant sorbitol reducing effect in the sciatic nerves similar to epalrestat, a clinical agent for the treatment diabetic diseases. The results were shown in Table 8.

<Table 8> Group Sorbitol in Decreasing rate sciatic nerve (nmol/mg dry wt.) Normal group 2. 0 0. 3 Control group 10.4 1. 4 Epalrestat 4.3 0. 5** 58. 2 treated group Extract of 7. 2 0. 5* 31. 0 Pelvetia siliquosa treated group Considering all the results shown in Table 3-8, the extract of Pelvetia siliquosa of the present invention can be said to be effectively used for the prevention or the treatment of diabetic diseases including diabetic complications since it was confirmed that the extract effectively reduced blood glucose of diabetic animal models induced by streptozotocin (IDDM), and showed glucose tolerance effect (NIDDM) and the inhibitory effect of aldose reductase and the accumulation of the sorbitol contents in the tissues,

which are crucial factors causing diabetic complications.

Experimental Example 3: Acute toxicity test in rats via oral administration Male 6-week old specific pathogen-free (SPF) SD line rats were used in the tests for acute toxicity.

The extract of Pelvetia siliquosa prepared in the Example 1 was suspended in 0. 5% methylcellulose solution and orally administered once to 5 rats per group at the dosage of 1 g/kg, 5 g/kg and 10 g/kg.

Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood performed, and any abnormal signs in the gastrointestinal organs of the chest and the abdomen checked with eyes in autopsy.

The results showed that the test compounds did not cause any specific clinical symptoms, weight change, or death in rats. No hematological or biochemical changes were observed in the blood, and in autopsy.

Therefore, the extract of Pelvetia siliquosa of the present invention used in this experiment are evaluated to be safe substances since it do not cause any toxic changes in rats up to the level of 10 g/kg and its ED50 values could not be calculated.

Manufacturing Example 1: Preparation of soft capsules Soft capsules were prepared containing 100.0 mg of the extract of Pelvetia siliquosa produced in the above Example 1,175. 0 mg of soybean oil, 45.0 mg of yellow beeswax, 127.5 mg of palm hardened oil, 21.0 mg of soybean phospholipid, 212.0 mg of gelatin, 50.0 mg of glycerin, 76.0 mg of D-sorbitol, 0.54 mg of paraoxymethylbenzoic acid, 0.90 mg of paraoxypropylbenzoic acid, 0.56 mg of methylvanillin and a proper amount of yellow food color 203 per capsule according to the manufacturing process of soft capsule of the Korean Pharmacopoeia.

Manufacturing Example 2: Preparation of tablets 100.0 mg of the extract of Pelvetia siliquosa produced in the above Example 1,90. 0 mg of cornstarch, 175 mg of lactose, 15.0 mg of L-hydroxypropylcellulose, 5.0 mg of polyvinylpyrolidone and a proper amount of ethanol were mixed homogeneously and granulized by wet- granulizing method. After adding 1.8 mg of magnesium stearin thereof, tablets were prepared containing 400 mg of the mixture per tablet.

Manufacturing Example 3: Preparation of capsules 100.0 mg of the extract of Pelvetia siliquosa produced in the above Example 1,83. 2 mg of cornstarch,

175.0 mg of lactose and 1.8 mg of magnesium stearin were mixed homogeneously. And then, capsules were prepared containing 360 mg of the mixture per capsule.

Manufacturing Example 4: Preparation of foods and drinks The present inventors have prepared foods and beverage containing the extract of Pelvetia siliquosa as an effective ingredient as follows.

<4-1> Preparation of chewing gum Chewing gum was prepared to have the composition as follows; the extract of Pelvetia siliquosa 0. 24- 0. 64%, gum base 20%, sugar 76. 36-76. 76%, fruit flavor 1%, water 2%.

<4-2> Preparation of beverage Beverage was prepared to have the composition as follows; the extract of Pelvetia siliquosa 0. 48-1.28 mg, honey 522 mg, thioctic acid amide 5 mg, nicotinic acid amide 10 mg, sodium riboflavin hydrochloric acid 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, ortho acid 50 mg, water 200 ml.

<4-3> Preparation of sausage Sausage was prepared to have the composition as

follows; the extract of Pelvetia siliquosa 0.24-0. 64%, pork 63. 6%, chicken meat 27. 5%, starch 3. 5%, soybean protein 1. 7%, salt 1. 62%, glucose 0. 5%, other additives (glycerine) 0.94-1. 34%.

INDUSTRIAL APPLICABILITY As shown above, the extract of Pelvetia siliquosa has a strong liver protective activity, blood glucose lowering activity and diabetic complications suppressing activity. The extract of Pelvetia siliquosa can be effectively used for the prevention or the treatment of liver diseases and diseases related to diabetes mellitus since the Pelvetia siliquosa, a raw material of the extract, is a naturally occurring seaweed which has a good absorption efficiency but exhibit no harmful effects at all when being used as a pharmaceutical composition for the prevention or the treatment of liver diseases and diseases related to diabetes mellitus or a health food.

Those skilled in the art will appreciate that the concepts and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention.

Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.