The use of β-GIucan delaying the

biosynthesis of yeast and mold under anaerobic condition

Field of Invention

This invention relates to the use of β-glucan having the immune and hyperhepatia activity of the body. Background and purpose of Invention

The function of β-Glucan, the main component of mushroom polysaccharide is being watched and the research of its structure, mechanism of the action and its use has achieved the great progress, and some of them are widely disseminated in the field of medical treatment for the promotion of health. ( -fj fSi i¾—„ β- 4 4 , 1 i , 2 0 0 9 , 3 - 4 ) β-Glucan is a kind of functional polysaccharide from vegetable derivation and included in barley, black yeast, or mushroom and it is examined to decreasing the cholesterol content in the blood or restraining elevation of blood glucose value, treating of the adipositas, anticancer effectiveness, treating effectiveness of hepatic diseases. As the result, a lot of research data have been published that β-Glucan increases the immune function of human and have the treatment effectiveness to al lergic rhinitis and have anticancer effectiveness. |¾ , β- ? > Jo c tlfit t fi ifa t 4 4 , 1 1 , 2

0 0 9 , 1 4 — 1 5 ) With the exeception of the above documents, materials about the pharmaceutical effect of β-G lucan were mentioned in US Patent no. 5.576.015 and 5.504.079.

Many study have been alreadly made against the method to devide β-Gl ucan owing to its effect and disclosed in many documents (US 005599697A. US 005385832A etc.).

Recently, as the effect that imchi. the national diet in the DPR Korea has is verified in various points of view, the preparing method of Kimchi is variously developed and the filed of its use is more and more expanded.

WO2000003608 A l 20000127 disclosed a pulverized Kimchi seasoning composition and it's preparing method and WO2001084942 disclosed the preparing method of Kimchi with silkworm Dong-Chung-Ha-Cho. This Kimchi is beneficial to the health of the body owing to its containging Cordyceps mil itaris, has a good flavour. WO200 I 087078 A I 2001 1 122 disclosed white Kimchi containg opuntia, which is beneficial to the health of the body owing to its containing opuntia and has a peculiar flavour, thereby becoming a globalized food.

Like this, various methods to peculiarize a flavour of Kimchi and raise its nutritional physiological activity by adding the other materials having pharmacological activity in Kimchi are proposed.

This invention relates to the use of β-Glucan delaying the biosynthesis process of yeast and mold in anaerobic condition and provides the methods to apply β-Glucan to vegetable-fermented foodstuffs such as Kimchi.

I Also, this invention provides the methods to apply β-Gl ucan delaying the biosynthesis process of yeast and mold under anaerobic condition to treatment of the disease such as hepatic diseases and adipositas.

Brief description of the invention

This invention mentions that β-Glucan delays selectively only the growth of yeast and mold in anaerobic condition and increases the biosynthesis of B | and B ₂ of yeast more than 5 times.

This invention discloses the method of using of β-Glucan in the fermented foodstuffs such as imchi and unrefined liquor. When making the vegetable-fermented foodstuffs such as Kimchi, 5g of β-Glucan can be mixed in its seasoning. With this usage, Kimchi was tasty and the period to be not sour was extended than comparison.

This invention describes the method of using of β-Glucan in treating of modern diseases such as adipositas and diabetes and hepatic disease.

The mentioned β-Gl ucan is injected oral ly by 0.5g.

To consider the effectiveness of treating the hepatic diseases, the activation of enzyme ""GPT ^" , "GOT", ""ALP ^" , '"CHE ^" as its experimental test indicator and BiL-exponent as the test relating to metabolism of the bile pigment has been observed. And the test of the periphery blood has indicates the counts of red cell, leukocyte, blood platelet, and for observing the functional and morphological changes of the liver, the dimensions of lobus hepatic sinister, lobus hepatic dexter, and spleen have been measured by the fluorescence detector of liver, and as the test of immune function, the weakening rate of lymph cell, CH ₅₀ . lc has been tested. β-Glucan in indices related to all examination enumerated has the effect of obvious improvement. When the patients of diabetes and adipositas has been treated by administering with 0.25g of β-Glucan each time, one hour before breakfast and dinner twice a day. β-Glucan has the obvious improvement in all index under considering.

β-Glucan in this invention can be used as the medicines, or the functional foods in form of capsule.

Detailed Description of the Invention This invention is concretely described by the following examples.

[Example 1 ] Effect of β-Gl ucan on growth of yeast and mould under the anaerobic conditions.

I . Materials and method

For yeast and mould, the experimental liquid was vaccinated on SAVRO culture medium's surface, and cultured at 30°C for 3~5days to calculate the number of colony.

For the bacterium, sample suspension usually was vaccinated on the argar culture medium ^' s surface and cultured at 35 °C for 20 hours to calculate the number of colony.

7 For the coli bacillus, sample was vaccinated on the BG LB (containing lactose) culture medium, and cultured at 35°C for 24 hours to decision existence of production of gas and product was vaccinated on the ENDO culture medium, and cultured for 12 hours to calculate the number of colony.

For the lactic acid bacteria, sample was vaccinated on the lactic acid bacteria's culture medium, and cultured at 35°C for 20 hours to calculate the number of yel low colony.

The crude protein by kjeldahl method, total acidity by titrimetry. and vitamin C & vitamin B l by fluorescent method was measured.

Vitamin B2 by AOAC. β-carotin & nitrous acid/nitrate by spectrophotometry, lead, cadmi um, natrium and kalium by atomic absorption method was measured.

Hexachloran by gas chromatography and coarse fibre & moisture by drying method was measured. Experiment on effect of β-Glucan on the biosynthesis of yeast under the anaerobic culture conditions was made in the different concentrations of sugar.

The culture condition is 25 °C(±2), culture time is 48 hours, nutrient source is sugar and mineral salt, and concentration of polysaccharide is 0.02%, experimental germ name is SACCHA RO YSES CEREVISIAE

2. Result

Table 1 shows results of the experiment on β-Glucan delaying the biosynthesis of yeast and mould under the anaerobic culture conditions.

Effect of β-Gl ucan on the biosynthesis of yeast Table I

It shows that growth of the yeast is delayed by β-Gl ucan delaying the biosynthesis of yeast and mould under the anaerobic culture conditions.

The polysaccharides such as carboxylethylglucan, schizopilan. starch, Cardran, Csantan, alginic acid have no effect on the biosynthesis of yeast, in the concentration of 0.02%.

[Example 2] Effect in case that β-G l ucan delaying the biosynthesis of yeast and mould under the anaerobic conditions was added to the vegetable- fermented foodstuffs. K imchi. 1 . Materia! and method

To make seasoning corresponding to the vegetable of l OOKg as stuffs of Kimchi. the polysaccharide, β-Glucan delaying the biosynthesis of yeast and mould under the anaerobic condition of 5g is added hereto and mixed equally, and then Kimchi was completed with these spices. Wherin, seasoning include pulverized red pepper of 5 Kg, garlic of 300g, stone-leek of l OOg, ginger of 50g, salt of 50g, and salted fish of 500g. The above listed pices were mixed equally and kneaded with warm water and then used as seasoning. The composition of the above mentioned seasoning is not limited to above that and it could be modified according to taste of user.

After completion of Kimchi, the vegetable should be soaked in Kimchi ^' s juice.

The control was made without onlyP-Glucan by the same method.

Kimchi has developed its flavour for 40days with keeping at 5~ I 0°C.

2. Result

Table 2 shows the influence in case that β-Glucan delaying the biosynthesis of yeast and mould under the anaerobic condition is added to the vegetable- fermented foodstuffs, Kimchi.

The effect on the microorganism of the vegetable- fermented foodstuffs, "Kimchi ^" of β-Giucan. Table 2

¾ 1 : β-Glucan 2: the control As seen in the Table 2, in Kimchi which β-Glucan is added in the seasoning, the biosynthesis yeast and mould is selectly delayed. And the biosynthesis of vitamin B l & B2 is accelerated quintuple and over. Also, the flavour of the kimchi is good, and unsoured term is extended, and decomposition is prevented.

[Example 3] Effectiveness when β-Gl ucan under anaerobic condition is administered to hepatitis patients.

I . Material and method

β-Glucan is administered by 0.25g each time, one hour before meal twice a day. In control group, "Ariman ^" ™ polysaccharide by 0.25g each time, one hour before meal twice a day. "Ariman ^"" ™ is a trademark name of lipomanan extracted from Acetobacter SP, gram-negative bacterium.

2. The observing items of treating effect

1 ) Clinical symptoms

Pains in the liver region, indigestion, inertia, and loss of appetite, abdominal disorders, diarrhea, constipation, and hemorrhage were noticed as self conscious symptoms. Liver swel ling, spleen swelling, liver beating pain, sclera icterus, palm-rose hash, and dilation of the abdominal be in were noticed in the diagnostic symptom.

2) Experimental test

® The level of enzyme activation was observed with the leverl of GPT, GOT. ALP, CHE.

© Bil indices were observed in tests of bile pigment metaboism.

© The number of erythrocytes, leucocytes, and blood platelets were observed.

® To observe the function of the liver and its morphological changes, the left and right lobe of the liver, and the size of the spleen were measured with Scintiinat-2.

© Test of the rete of lymphocyte juvenility. CH _5U and 1C were carried out to observe immunity functions.

3) The judging criterion of treating effects

Treating effects were judged by comparing the disappearance of clinical symptoms, the degree of improvement and the results of the tests before and after treatment.

Favorable improvement: three or more self-conscious symptoms have disappeared, objective symptoms show improvement, and results of test are near normal.

No change: results of test show no improvement after the beginning of treatment, though some improvement was observed in the clinical symptoms.

Aggravation: the cl inical symptoms and results of test were aggravated.

The tests were carried once every 1 5 days and the effects before and after treatment were judged by t-examination.

4) Remote observation judgment criterion

Remote observation was assessed by comparing clinical symptoms and results of tests during and after treatment in hospital.

Markedly good: clinical symptoms and results of tests remain unchanged for two years or more.

Good: results of tests remain un-changed, some self-conscious symptoms appear, depending on conditions.

Aggravation; pathological symptoms are noticed in clinical symptoms and results of test in two years after the hospital discharge.

5) Diagnostic criterion of chronic hepatitis

We followed the criteria of diagnosis of virus chronic hepatitis prescribed in volume 5, of complete book of prevention of infectious diseases.

GPI>35, GOT>140, TTT>4, ZTT>14, Alb<58%, GGT>25%

6) Diagnostic standard of progressive hepato-cirrhosis

Diagnostic standard of progressive hepato-cirrhosis referred to the information of document.

[ - The clinics of hepatic diseases, 21-119, 1991 , Kim Ha won,

- Encyclopedia of treating the hepatopathy, 25- 1 14, 1985, the Publishing House of Foreign Language,

- The Internal Medicine Book 12, 150-200, 1981 , the Publishing House of Science Encyclopedia, Kye

Sun Bom

3. The result

1) Changes in self-conscious symptoms

Table 1 shows results of comparison of changes in main self-conscious symptoms before and after treatment.

As shown in Table 1 , the rate of self-conscious symptoms in both groups before treatment was not distinct. After the treatment, all the indices of self-conscious symptoms in the experimental group considerably improved compared to those in the control group. The improvement of pains in the liver area, inertia, indigestion, and diarrhea was extraordinarily significant.

The total rate of improvement in the experimental group was 92.0%, which was 69.4% improvement from the control group (p<0.05). Changes in self-conscious symptoms

*: P<0.05 P: comparison with control

2) Changes of objective symptoms

Table 2 shows results of comparison of changes in objective symptoms before and after treatment.

Change of objective symptoms Table 2

*: PO.05 P: comparison with control

As summarized in Table 2, the difference between the experimental and control groups in the rates of diagnostic symptoms was insignificant before the treatment. The rate of improvement of main diagnostic symptoms after treatment in the experimental group was higher than that in the control group in all indices. The total improvement rate in the experimental group was 83.9%, 12.3% higher than that in the control group.

3) Changes in opinions of clinical experiment

Δ Change in the test opinion of protein metabolic function

© Change in serum protein amount

Change in serum albumin before and after the treatment is summarized in Table 3.

. Change in serum albumin amount Table 3

PI : Comparison with pre and post treatment; P2: Comparison with control

As shown in Table 3, the serum albumin amount in the experimental group before the treatment was 6.8±0.2g/dl, while that in the control was 6.7±0.1g/dl. The post-treatment amounts of serum albumin in the experimental and control groups were 6.9±0.3g/dl and 6.9±0.2g/dl, respectively, whereas the pre and post treatment gap between the groups was different. It was likely to increase the amount of serum albumin.

© Change in serum proteinogram

Change in serum albumin proteinogram before and after treatment in summarized in Table 4.

Change in serum albumin distribution Table 4

* P<0.05 P: Comparison with pre and post treatment, P': Comparison with control

As shown in Table 4, in the experimental group distinctly increases the albumin from 43.1±1.2% before treatment to 51.4±1.7% after treatment (p<0.05). The control group showed tendency to increase, but no marked difference. As far as γ-globulin is concerned, the experimental group experiences a spectacular fall from 34.7±1.5% before treatment to 26.6±1.4% after treatment. The control group had not an obvious change. ® Change in serum colloid reaction

Table 5 shows the change in serum colloid reaction before and after treatment. Change in serum colloid reaction Table

* P <0.05 P: Comparison with pre and post treatment, P': Comparison with control

As shown in Table, in the experimental group, TIT distinctly decreases from 7.5±0.8 before treatment to 4.6±0.7 after treatment (PO.05). In the control group had not an obvious change. ZTT in the experimental group decreases from 16.7±0.6% before treatment to 12.5±0.7% after treatment The change in the control group was insignificant.

Δ Change in serum enzyme activity

Φ Change in transaminase activity

Table 6 shows the change in transaminase activity before and after treatment.

Change in the transaminase Table 6

***P<0.01 P: Comparison with pre and post treatment

P': Comparison with control

As seen in Table 6, in the experimental group and control group saw a fresh improvement of GPT and GOT after treatment as against before treatment (p>0.001 each). A comparative look at the change showed that the experimental group's value was much higher than the control group (p<0.05).

Change in alkali phosphatase and choiinesterase activation.

Table 7 shows the changes in alkali phosphatase and choiinesterase activation before and after treatment.

As seen in Table 7 alkali phosphatase was 4.4±0.4 and 4.3±0.3 for both the experimental and control group.

The level of alkali phosphatase in the experimental group fell to 2.1 ±0.2 after treatment (pO.OOl), but the control group did not see a meaningful gap. As for choiinesterase, in the experimental group discovered an 3 increase from pre-treatment 786.1 ±21.6 to post-treatment 967.3± 17.4. The control showed no significant change.

The change of acitivities of alkaliphosphatase and cholinesterase. Table 7

P: comparison between pre and post treatment

P': comparison with control group

Δ Change in serum icterus index

Change in serum icterus index before and after treatment is illustrated in Table 8.

Change in number of serum icterus Table 8

***P<0.001 P: comparison between pre and post treatment;

P': comparison with control group

As shown in Table 8, the serum icterus of both experimental and control groups were reduced remarkably in the indices after treatment compared to before treatment (pO.001).

The experimental group considerably improved compared to those in the control group (PO.05).

Δ Change in the liver scintigram

Table 9 shows comparisons and observations of the changes in the scintigram of liver before and after treatment.

Change in liver scintigram Table 9

*P<0.05 **P<0.01 P: Comparison with the pre-, and post-treatment

P': Comparison with the control group

As clear shown in Table 9, the size of the experimental group's right portion of the liver and left of the liver, spleen after treatment compared with pretreatment (P<0.05, PO.01). The size of the control group's and left livers and spleen showed the trend toward improvement but the trend was not noticeable. There was nothing noticeable between the two groups

Δ Change in test of peripheral blood

Table 10 shows the comparative observations of changes in the test of peripheral blood before and after treatment.

P: Pretreatment comparison P': Comparison with control

As shown in the Table, the number of white and red corpuscles of the experimental and control groups is from 4 I2.9±14.5 to 497.4± I4.4 respectively after treatment compared with pretreatment and increased from 4.1±0.34 to 6.4±0.3. The number of the experimental groups thrombocytes sharply increased from pretreatment 1 1.4±0.12 ^χ 10 ⁴ /mm to 12.3±0.27x l0 ⁴ /mm (p<0.05). There was a considerable difference between the experimental group and control group in the change of the number of white corpuscles and thrombocytes.

A Change in the test value of HBs Ag

The negative test value of HBs Ag means lose, when the test value goes down to less than 64 times from over 128 times it means lowering. Table 1 1 shows the change, when there is no change in the test value fluctuating above 128 times.

As shown in Table 1 1 , the change rate of the experimental group's HBs Ag test value is 48.2 percent, which is much higher than that in the control group, 25 percent.

Change in the value of HBs Ag Table 1 1

*P<0.05 P: Comparison with the contol group ® Change in the test value of immunity function

Table 12 shows comparative observations of the tests of immunity function before and after treatment.

As shown from Table 12, the rate of juvenility the lymphocyte in the experimental group was remarkably improved after treatment compared with pretreatment but where was no special change in the control group. The experimental group 50 percent of complement value has changed noticeably after treatment compared with pretreatment value was no distinct change in the control group. The change of two groups immune complexes has improved noticeably after treatment compared with pretreatment (p<0.01 and p<0.05 each).

The test value changes of immune function Table 12

*P<0.05 **P<0.01 P: Pretreatment comparison

4) Overall results of treatment

® The treatment results

Table 13 shows the effects of two groups' treatment confirmed by the judgment standard.

Overall treatment results Table 13

As shown in the Table, the effect rate of the experimental group was noticeably higher.

© Average treatment days

Table 14 shows the average treatment days of those patients who left hospital after favorable turn.

As shown in the Table, the average treatment days in the experimental group are 32.9±1.9 days, which shorter than those in the control group, which were 43.5±2.6. (P<0.05) The average number of treatment days Table 14

© Remote observation results

Table 15 shows the results of two remote observations of 70 cases completely cared with β-glucan.

Remote observation results Table 15

As Table 15 shows, four of the experimental groups 70 cases (5.7 %) worsened. Among the four cases, one case became active chronic hepatitis and three cases early fattening the unprogressive hepatocirrhosis. Four of the control group 16 cases (25 %) worsened and three cases became active and one case early fattening the unprogressive hepatocirrhosis.

[Example 4] effect of β-Glucan for diabetics

Δ Materials and method

Φ Materials

The method of orally administering β-Glucan one hour before breakfast/dinner twice a day by 0.5g each time. For the control group, administration of Ariman™ polysaccharides before meal twice a day by 0.25g each time.

Analysis facility and tools

Experiment and result processing was administered by using the internationally certified automatic analyzer (Hitachi 912, Japan), automatic analyzer (Elecsys 2010, Japan), automatic analyzer (Sysmex XE-2100, Japan), body composition analyzer (In Body 3.0), computer (HP, Pentium 4) and electronic scale (CAS, accuracy rate of 50g).

(D Measurement on human body and observation method

Δ Measurement indices

Weight was measured once a week at noon by wearing a layer of undergarment (weight scale of l OOg sensitivity level passed through national authorization was used). Waist and hip measurements were taken with measuring tape around navel and the most prominent part around the hips while the subject person removed clothing, comfortably relaxed both arms and exhaled. AObservation method

- For subjective symptoms, the volume and frequency of urination, feeling of heaviness and breathlessness were the main symptoms to observe. In addition, target patients were observed 2~3 times a week for the conditions, such as feeling of unpleasantness in the heart, headache, heaviness in the head, feeling of soreness in the limps and thirst, etc. The results were checked as either (+) or (-) in the result sheet. New symptoms to occur in the course of treatment were recorded in the record chart. For treatment, supervised administration of drug was administered 1~2 times. With focus placed on onsite treatment, it was ensured to produce no changes in the level of diet and exercise in the entire course of treatment.

- Experimental test

o Test for post-prandial blood glucose level

After administering drug to type-2 diabetics, the target patients were fed 1 OOg cooked rice. Blood glucose level was measured by obtaining blood sample from the tip of finger when the subject person was in empty stomach in the morning as well as 1 hour and 2 hours after feeding cooked rice.

o Test for glucose tolerance

Test for glucose tolerance was administered both before and after administering 0.2g of drug per day for 3 months, 6 months and 1 year. Blood glucose level was measured by obtaining blood sample from the tip of finger when the subject person was in empty stomach in the morning as well as 1 hour and 2 hours after feeding cooked rice (the same amount as above).

o Blood analysis

Blood biochemical indices, such as serum, cholesterol and triglyceride level, etc. were measured by using the automatic analyzer of Hitachi 912. After using serum and insulin automatic analyzer of Elecsys 2010, the HOMA-R (homeostasis model of assessment insulin resistance) was calculated with the following equation.

HOMA-R=FIxFG/22.5; FI: Fasting serum insulin (μυ/ml)

FG: Fasting glucose level (mg/dl)

Hematological test was administered with automatic analyzer Sysmex XE-2100.

- Processing of results

o According to the value of blood glucose obtained after loading of glucose, the area below the curve of blood glucose and its increase were calculated with the following equation.

Area below the curve of blood glucose: S(area of blood glucose) = 1/4 (a+ab+c);(mg/dl h)

Increase in the area below the curve of blood glucose:

o 2 ^s (area of blood glucose) =t [1/a (a+ab+c)-a] ;( mg/dl-h) t: Time of the last blood sample collection (h)

a: Blood glucose level at the beginning of experiment (in empty stomach) (mg/dl)

b: Blood glucose level at t/2 (mg/dl)

c: Blood glucose level at t (mg/dl)

@ Results of treatment and bases of observation

Bases of evaluation per weight were categorized as the following in accordance with the degree of weight reduction and improvement of the condition.

Significant improvement: Reduction of weight by 2.0kg or more and simultaneous reduction of waist measurement was achieved in the course of treatment. Significant improvement was achieved in the subjective conditions.

Improvement: Reduction of weight by 1.0kg ~1.9kg and simultaneous reduction of waist measurement were achieved in the course of treatment. Relative improvement was achieved in the subjective conditions.

No change: There has been no change experienced in the weight and waist measurement during the course of treatment (± 1.0kg or less)

Results of experiment were calculated in computer (HP, Pentium 4) with ±SD. Also, significance level was measured with t-test method.

2. Results

1) Effect in obesity treatment

Changes in weight and blood serum composition in the obese.

After 7-day stabilization period, cross-test was administered by using β-Glucan and Ariman™ polysaccharides for the period of 6 months each. The result indicated changes in weight as shown in Table 16.

Changes in weight of the obese Table 16

Compared to the control group *P<0.05 n=59

As shown in the Table, average increased by 7.16kg among the obese in the 6-month control period (administration of Ariman™ polysaccharides). However, during the 6-month experimental period (administration of β-Glucan), the average weight increased by 0.44kg. In other words, β-Glucan decreased the average weight of the obese by 6.71 kg during the period of 6 months. Blood biochemical indices and blood pressure during the period of experiment were as shown in Table 17.

Blood biochemical indices of the obese administered with compound pharmaceuticals Table 17(n=59)

With the exception of cholesterol level increased during stabilization period, there was no difference found between control and experimental groups in terms of triglyceride, HDL-cholesterol level, blood glucose level and GOT/GPT level.

i

J There was no difference, either, in systolic pressure and diastolic pressure.

Changes in weight and serum composition of the obese

j Δ Effect exerted on post-prandial blood glucose level

j Postprandial blood glucose level and increase in blood glucose level were as shown in Table 18.

I Post-prandial blood glucose level and increase in blood glucose level Table 18

* p<0.05 Significant difference by comparison, n=23

Conditions of experiment: After 2 days of stabilization and fasting of 12 hours, target people were fed bread made with flour (lOOg flour). After administering the pharmaceutical for 6 months, blood sample was obtained from the tip of finger before feeding and both 1 and 2 hours after feeding to analyze blood glucose level.

As shown in the Table, β-Glucan produced continued effect to lower blood glucose level from 6 months after administration. However, there was no difference observed in the case of administering Ariman™ polysaccharides.

Δ Effect on weight

Weight reduction was found as shown in Table 19.

.Change in weight per month in the obese (kg) Table 19

***p<0.001, **p<0.01 significant difference from the control group, n=38

The degree of weight reduction can be divided into the categories shown in Table 20.

Degree of weight reduction in the obese Table 20

n=38

As shown in the Table, average weight reduction occurred from the 4* month after administration of β-Glucan. The weight reduced by 6.1 kg after 8 months of administration.

Changes in weight, BM1, waist measurement and hip measurement are as shown in Table 22.

Changes in measurements of the obese Table 22

**p<0.001, ***p<0.001 , n=38

After administration of β-Glucan, distinctive reduction in BMI and weight was observed.

Δ Effect on clinical symptoms

Subjective symptoms of the target people changed after administration of β-Glucan as shown in Table 23.

As shown in the Table, the major symptoms of breathlessness and feeling of heaviness experienced by target patients significantly decreased. In other symptoms, improvement of 60% or more was achieved in the conditions. Changes in subjective symptoms (n=38) Table 23

Δ Effect on blood biochemical indices

Blood biochemical indices of the target people administered with β-Glucan and Ariman polysaccharides for 6 months are as shown in Table 24.

As shown in the Table, β-Glucan significantly lowered serum fat level, blood glucose level, serum cholesterol level and uric acid content in the obese compared to before administration. It also significantly improved liver function indices, such as GGT, GOT and GPT. However, there were no marked changes observed in blood biochemical indices in the control group with administration of Ariman™ polysaccharides.

Blood biochemical indices of the obese (n=38) Table 24

Compared to before experiment *p<0.05, **p<0.01 , cip<0.1

Δ Effect of the pharmaceutical on changing of physical composition

Results of physical composition analysis measured on the target people before and after administration of pharma-ceuticals for 6 months are as shown in Table 25.

As shown in the Table, the experimental group with β-Glucan administration displayed significant reduction in body and increase in muscle mass and protein content after experiment. However, there was no marked difference found in the control group.

Change in physical composition of the targets of clinical experiment after administration Table 25

Compared to before experiment

(D Overall result of treatment

In summary, the obesity prevention effects of the pharmaceuticals are as shown in Table 27.

Overall treatment result (rate of efficiency) Table 27

As shown in the Table, the rate of efficiency displayed by the experimental group with administration of β-Glucan was of 90% or higher.

2) Effect on diabetes treatment

© Effect on glucose metabolism indices of type-2 diabetics according to the period of administration Δ Change in fasting glucose level and protein glycation index

Fasting blood glucose level and protein glycation values measured on type-2 diabetics administered with each pharmaceutical for 6 months are as shown in Table 28.

As shown in the Table, β-Glucan significantly lowered the fasting blood glucose level and protein glycation indices in type-2 diabetics in the 4 ^th and 6 ^th month of the treatment Change in glucose metabolism indices of type-2 diabetics Table 28

Significant difference compared to before treatment *p<0.05, **p<0.01

Δ Effect on serum insulin and insulin resistance

Fasting serum insulin values measured on patients as of the above are shown in Table 29.

Changes in serum insulin value and ΗΟΜΑ-γ index in type-2 diabetics Table

Significant difference compared to before treatment *p<0.05

As shown in the Table, β-Glucan significantly lowered fasting serum insulin value and ΗΟΜΑ-γ index in type-2 diabetics after 6 months of treatment.

(2) Effect on clinical symptoms of type-2 diabetics

The results of observing changes in subjective symptoms experienced by some patients after intake of β-Glucan for 6 months are shown in Table 30.

Changes in subjective symptoms after intake of β-Glucan Table 30

As shown in the Table, the typical subjective symptoms of the diabetics, such as lethargy, thirst and large volume of urine, were significantly reduced after treatment.

© Effect on serum lipid of type-2 diabetics Changes in the level in serum lipid of type-2 diabetics after 6-month administration of β-Glucan were observed. As shown in Table 3 1 , triglyceride and cholesterol content in serum significantly decreased.

Effect on serum l i id of obese-t e diabetics Table 3 1

( ) rate of decrease compared to before treatment, significant difference compared to before treatment, **P<0.0 l . ** *P<0.00 l

@ Evaluation of treatment efficiency

Effect of β-Glucan on treatment of diabetes is evaluated as shown in Table 32.

As shown in the Table, β-Glucan produced proximately 80% of treatment effect on diabetes.

Improvement effect on diabetes by β-Glucan Table 32

[example 5] Toxicity experiment of β-G l ucan delaying the biosynthesis of yeast and mold under anaerobic condition

I . Materials and method

1 ) Materials

β-Glucan was used as test material. Also, 72 ICR/JCL adult white rats and 130 mice were bred in the animal cage in Pyongyang Medical University were randomly used as experimental subjects with no specification in sex.

2) Method

© Creating a chronic liver poisoning model

Chronic liver poisoning was incurred by subcutaneously injecting 10% of carbon tetrachloride and O. I 2ml/ 10g of corn oil to the rats and 50% of carbon tetrachloride and 0.25ml/ 100g of corn oil to the mice, both through the dorsal area, once in three days and 10 injections per administration.

Measurement and Method

- The effect on the activity of serum transaminase For the purpose of testing the effect of the experimental drug to the liver function in chronic hepatitis intoxication, at the same time of initiation of toxication. city water may be administered at the amount of I .Om/l Og in control(normal control, toxication control)group and the polysaccharide which delays the biosynthesis of the yeast and mold under the anaerobic condition in experimental group may be administrated at the different dosage once daily for 30 days in test group. Experimental animals were beheaded on the 3 1 st day from the toxication, gathered the blood and measured the GPT and GOT level by Lightman-Prangkel method.

- The effect on fat content in the l iver tissue

The toxic dose of carbon tetrachloride was injected into the white rates. At the same time, the control group (normal, poisoned) was given with tap water at a dose of 0. I ml/ I OOg. Various doses of drug were given to experimental group once a day for 30 days. The animals were instantly sacrificed through cervical dissection on the 3 1 ^s ' day of the experiment. Final ly, the liver was removed and completely desiccated in a hot-air drier for 24 hours.

The desiccated liver was grinded into fine pieces with a mortar and I g of the liver powder was placed into a soxhiet device to extract fat from the water. After fat was completely extracted by using ether solution, and the powder was dried and then weighted.

Fat content in l iver tissue was calculated by deducting the remnant weight after extracting fat from the total liver weight.

- The effect on pigment degradation function of l iver

The toxic dose of carbon tetrachloride was injected into the white rats. At the same time, the control group (normal, poisoned) was given with tap water at a dose of 0. I ml/ I OOg. Various doses of drug were given to experimental group once a day for 30 days. 0. I ml/ I OOg of 2% bromphenol (BPB) was injected through tail vein. After 20 minutes blood form each animal was collected through cervical dissection. The concentration of BPB in the blood serum was measured by colorimetric titration.

- The effect on the detoxification of white rats ^" l iver

In the group of white mice, 0. 1 ml/ 1 Og of tap water was given to the control group and various doses of drug were given to the experimental group for 10 days. The animals, then, were treated with LD50 ( 160mg/kg) of ammonium chloride through the thoracic subcutaneous injection.

After 72 hours later, the number of the survived animals and died animals was obtained and compared between the compared and experimental groups.

2. Results

(T) The effect on the activity of serum Iransaminase

56 white mice were divided into 7 groups of 8 mice. The result of the experiment was evaluated by comparing the indexes of GPT and GOT, measured through Light-Prankel method, in the blood serum.

The results were as follows.

As shown in the above Table, the activity of serum GOT in the poisoned control group (87.6±4.3) was significantly higher than that in the normal control group (26.9±2.1 ).

The serum GPT activity in the experimental group to which the dosage of 20mg/kg was given, was significantly lower than those in the poisoned control group (p<0.01 ).

The effect of drug on the serum transaminase activity Table 33

* p<0.01

Regarding the GOT activity, the poisoned control group (90.4±6.7) was distinctly higher than the normal control group (43.6±5.8), and experimental group to which the dosage of 20mg/kg was given was lower than the poisoned control group (pO.01).

The effect on galactose metabolic function

32 white mice were divided into 4 groups of 8 mice.

The toxic dose of carbon tetrachloride was injected into the white mice. At the same time, the control group (normal, poisoned) was given with tap water. Various doses of the drug (0.1 ml/1 Og, 40mg/kg, and 60mg/kg) were given to the experimental group for 30 days. Then, the galactose metabolic treatment function was evaluated on the 31 ^st day of the experiment.

The level of blood sugar was obtained 30 minutes before and 30 minutes after giving galactose and the difference was compared between the control group and experimental group.

The res ults of experiment were as Table 34.

As seen in the above Table 34, the level of blood sugar in the poisoned control group (82.7±5.1mg%) distinctly increased compared to that in the normal control group (59.2±2.91). The effect of drug on the galactose metabolic function of white mice Table 34

* pO.01

The level of blood sugar in the experimental group, to which 40mg/kg and 60mg/kg of drugs were given, was 6 l .6±4mg/dl and 63.1±3.7mg/dl, respectively. In addition, the level in the experimental group decreased significantly compared to that in the poisoned control group (p<0.01). The blood sugar level in the experiment group decreased close to that in the normal control group.

The results indicate the drug acted positively on the sugar metabolic function of the liver of rats.

The effect on fat content in liver tissue

32 mice were divided into 4 groups of 8 rats.

The toxic dose of carbon tetrachloride was injected into the white rates. At the same time, the control group (normal, poisoned) was given with tap water at a dose of l .Oml/lOOg. Various doses of drugs were given to experimental group once a day for 30 days. Then, the effect on the fat content of the liver was evaluated on the 31 ⁵¹ day of the experiment.

lg of liver tissue was used to test the effect on the fat content of the liver.

The results of test were as follows.

The effect of drug on the fat content in liver tissue of rats Table 35

*p<0.0

As shown in Table 35, the index of fat content in the liver tissue (lg) was 39.7± 1.8mg/g in the poisoned control group, which increased distinctly compared to that in the normal control group.

In the experimental group, the fat contents in the liver tissue were 26.2±1.8mg/g and 25.2±1.8mg/g, which decreased distinctly compared to that in the poisoned control group.

The result indicates the drug acted positively on the fat metabolic function of the liver of rats. (4) Effect on pigment degradation function of liver

40 rats were divided into 5 groups of 8 rats.

The toxic dose of carbon tetrachloride was injected into the white rates. At the same time, the control group (normal, poisoned) was given with tap water at a dose of l .Oml/lOOg. 40mg kg, 60mg/kg and 80mg/kg of the drug were given to the experimental group once a day for 30 days.

Then, the effect on the pigment degradation function of the liver was observed on the 31 ^a day of the experiment.

The result was evaluated by comparing the BPB concentration in a blood serum 20 minutes after BPB injection between the control and experimental groups.

The results of test were as follows.

As shown in Table 5, the index of the BPB concentration in a blood serum of the poisoned control group was 3.54±0.25mg/%, which increased distinctly compared to that in the normal control group.

In the experimental group, the BPB concentrations of the blood serum were 3.14±0.26mg/%, 1.63±0.15mg/% and 1.53±0.01 mg/% depending on the dosage given, which are significantly lower than that in the poisoned control group (3.54±0.25mg /%). (pO.01)

The effect on the function of secreting liver pigment Table 36

*p<0.01

The result indicates the drug enhances the pigment degradation function of the rats' liver.

(D Effect on the detoxification of liver

30 white mice were divided into 3 groups of 10 mice.

The result was evaluated by obtaining the number of the survived animals and died animals and compared them between the control and experimental groups in 72 hours after the injection of ammonium chloride.

As seen in Table 37, in the experimental group treated with 60mg/kg and 80mg/kg of the drug, the dead animals were 4 (40%) and 3 (30%) respectively, which were significantly lower than that of the control group [6(60%)]. The result indicates the drug enhances the detoxification function of the liver of rats. The effect on the detoxification of white rats' liver Table 37

(D Acute toxicity experiment

12 white mice, 18~22g in weight, were divided into 4 groups of 3 mice. Each mouse was given with the drug at various dosages ( l OOmg/lOg, 500mg/10g, lOOOmg/lOg and 2000mg/10g). After 48 hours, no subject was found dead.

In addition, no significant change in hair and behavioural movement of the white mice was identifiable after treated with the drug.

[Example 6] Clinical safety of β-Glucan

The quantity 5 times larger than that of the general administration was given to 24 healthy individuals for 15 days. Subjective symptoms and results of observation are as shown in Table 38.

As shown in the Table, there was no change in healthy people's subjective symptoms even after large quantity of drug administration. Although some experienced increase in the volume of urine, this conditions disappeared within 2~3 days.

In some cases, there were positive results of achieving improvement in terms of watery feces and constipation.

Changes in subjective symptoms after administration of acute drug (M±SD) Table 38

Also, as the result of observing hematological test results both before and after drug administration, there was no significant change identified in all of the indices (Table 39) Changes in blood indices after administration of acute drug Table 39

As shown in Table, there were no changes observed between before and after drug administration in test indices of hematosis, such as the percentages of red blood corpuscles and leucocytes.

Changes in blood biochemical indices after administration of acute drug Table40

As shown in Table 40, there were no changes observed in blood biochemical indices after administration of acute drug, such as in indices for liver function (GOT, GPT, BILT, GGT, LDH), indices for kidney function (CREA, UREA) and indices for protein metabolism (ALB, TP, CHE). Prior to entering the clinical phase, to evaluate the safety of β-Glucan delaying the biosynthesis of yeast and mold under anaerobic condition(standard 0- 1433-2001 ) the quantity 5 times larger than that of the general administration was given to 24 healthy individuals for 15 days.

As shown in the Table 38. there were no changes in biochemical indices and subjective symptoms.

After administration, there would be found the trend of improvement in property of stool. These result shows that this formulation gives affirmative effect to the function of digestive system.

Also, since indices such as indices of liver function and renal function were within normal range, toxicity of the formulation was not regarded.