WO2003024468A1

WO2003024468A1 - Substances extracted from corn which can inhibit the activities of amylase, pharmaceutical compositions and food additives containing the same extracts for treatment or prevention of obesity and diabetes mellitus, and processes for their preparations

Info

Publication number: WO2003024468A1
Application number: PCT/KR2002/001750
Authority: WO
Inventors: Sun-Kyung Kang; Jae-Ho Kim; Hang-Rae Kim; Han-Oh Park; Young-Bae Bang; Yong-Ju Lee; Jeong-Rai Lee; Chil-Mann Jung
Original assignee: Bioneer Corporation
Priority date: 2001-09-19
Filing date: 2002-09-18
Publication date: 2003-03-27
Also published as: KR20030025200A

Abstract

Corn extracts that can inhibit the amylase activities are provided. The corn extracts according to the present invention can be obtained by an extracting process comprising the steps of extracting corn with an extracting solvent selected from water, organic polar solvents and their mixtures, removing solids from the extract solution obtained from the extracting step, and removing the extracting solvent from the extract solution to obtain the corn extract. The corn extracts according to the present invention can inhibit the activities of amylase, a carbohydrate digestion enzyme. Therefore, they can suppress the intake of lower saccharides into the body to prevent the increase of blood glucose level, decrease excessive intake of nutrition in order to be effective in the treatment and prevention of diabetes mellitues and obesity, and induce the feeling of satiety due to the non-digested carbohydrates to obtain the diet effect. Also they are not harmful to the human body because they are extracted from plants for food.

Description

SUBSTANCES EXTRACTED FROM CORN WHICH CAN INHIBIT THE

ACTIVITIES OF AMYLASE, PHARMACEUTICAL COMPOSITIONS AND

FOOD ADDITIVES CONTAINING THE SAME EXTRACTS FOR

TREATMENT OR PREVENTION OF OBESITY AND DIABETES MELLITUS,

AND PROCESSES FOR THEIR PREPARATIONS

FIELD OF THE INVENTION

The present invention relates to corn extracts which can inhibit the activities of

amylase and processes for their preparations. The present invention also relates to

pharmaceutical compositions containing the corn extracts for treatment or prevention of

obesity and diabetes mellitus and food additives containing the corn extracts for

controlling blood glucose level.

BACKGROUND OF THE INVENTION

According to a report of the American National Institutes of Health (NIH) (The

evidence report: clinical guidelines on the identification, evaluation, and treatment of

being overweight and obesity in adults, 1999, NIH), about 97 million Americans were

obese and the number of patients of type II diabetes mellitus associated with obesity,

reached about 15,700,000 individuals.

The World Health Organization (WHO) also presumes that the number of diabetes patients will reach about 0.3 billion in 2025, and the number of patients of type

II diabetes mellitus tightly associated with obesity and unable to control blood glucose

level by insulin is known to be about 5.9% of the total United States population in the

present time (Current Pharmaceutical Design, 2001, 7:417-450). The rapidly increased

obesity and diabetes, particularly diabetes accompanied with obesity, is one of the

chronic diseases that causes enormous economic and social damage.

The obesity seems to be generated from an imbalance between ingestion and

the consumption of energy, although the accurate mechanism for generation of obesity

is not known up to now. Therefore, the development approaches of treatment agents for

treatment of obesity at the present time is mainly classified into three cases (Nutrition,

2000, 16:953-960). The first case is drug therapy that can reduce an appetite to decrease

the amount of food ingestion, mainly stimulants of sympathetic nerve. These types of

drugs approved by the FDA for the treatment of obesity include phentermine, mazindol,

and sibutramine. The type of drugs not yet approved by FDA includes fenproprex. The

second case is to give an effect on digestion of food and absorption of food in the

intestine. The drug associated with this case includes orlist (Xenical™, Roche

Pharmaceuticals) which can suppress the digestion of fat. Also, there are hormones such

as testosterone which is now used as a treatment agent of obesity even though the FDA

had approved it for the usage other than as a treatment agent of obesity and diabetes

mellitus. The third case is a drug that can increase consumption of energy accumulated in the human body. This drug includes ephedrine and caffeine, and also includes β₃-

adrenoreceptor antagonists which are still under development. Overall, medicinal

therapies by drugs to decrease the blood glucose level, inhibit the absorption of sugar,

strengthen the functioning of insulin, or suppress the appetite have been actively

researched up to now for the treatment or prevention of obesity or diabetes mellitus

accompanied with obesity. Also, the drugs for treatment of type II diabetes mellitus

include sulfonylureas, rosiglitazone, and metformin to increase the functioning of

insulin, a hormone to control the blood glucose level. They also include alpha-

glucosidase inhibitors, amylase inhibitors, and dietary fibers to suppress the absorption

of sugar.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide corn extracts

having an effect to inhibit activities of amylase, a digestive enzyme in the intestine, and

largely decreasing the break-up of carbohydrates to lower saccharides absorbable into

the body.

Another object of the present invention is to provide processes for preparation

of corn extract which can inhibit the activities of amylase.

Another object of the present invention is also to provide pharmaceutical

compositions for treatment or prevention of diabetes mellitus or obesity comprising a pharmaceutically effective amount of corn extracts, which are administered to suppress

the formation of lower saccharides absorbable into the body.

Another object of the present invention is still also to provide food additives

comprising the corn extracts, which can be added to food such as bread, noodles, and

boiled rice to provide an effect for preventing diabetes mellitus or obesity.

The present invention provides corn extracts which can inhibit the activities of

amylase.

The corn extracts according to the present invention can be obtained with the

extracting process comprising the steps of extracting corn with an extracting solvent

selected from water, organic polar solvents and their mixtures, removing solids from the

extract solution obtained from the extracting step, and removing the extracting solvent

from the extract solution to obtain corn extract.

The corn used in the present invention is preferably corn kernel or cornhusk,

and the extracting solvent is preferably a lower alcohol with 1~4 of carbons. The

extracting step may be carried out at a temperature of 0~100°C, and the solids removing

step may be carried out by centrifugal separation or filtration.

The present invention may also purify the corn extracts in order to increase the

ratios of effective ingredients inhibiting the activities of amylase in the extracts.

Such a process for purification of the extract may comprise the steps of dissolving or suspending the extract in water, contacting the aqueous solution or

suspension of the extract with an organic non-polar solvent which cannot be mixed with

water, to distribute the ingredients in the aqueous solution or suspension of the extract

which do not have the amylase inhibition activities, removing the solution of organic

non-polar solvent, contacting the aqueous solution or suspension of the extract with an

organic polar solvent which cannot be mixed with water, to distribute the effective

ingredients in the aqueous solution or suspension of the extract which have the amylase

inhibition activities, and recovering the solution of the organic polar solvent and

removing the organic polar solvent from the solution to obtain partially purified corn

extract.

The purification process of the extract may be carried out by chromatography.

Namely, the ingredients not having the amylase inhibition activities can be removed

from the extract by chromatography.

The present invention also provides pharmaceutical compositions for treatment

or prevention of diseases related with the amylase activities comprising the corn

extracts extracted by the above-mentioned processes and pharmaceutically acceptable

carriers. Wherein, the disease related with the amylase activities in particular is obesity

or diabetes mellitus, and the composition according to the present invention is

preferably in the form suitable to medicinal oral administration.

The present invention also provides food additives comprising the corn extract extracted by the above-mentioned processes. The food additives of the present invention

are for controlling the blood glucose level of the food-ingesting person at the time of

food ingestion. Therefore, the food additives of the present invention should be added to

the food containing polysaccharides (disaccharides or more).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail.

amylase, and their preparation processes.

The corn subject to the extraction in the present invention is an annual plant

belonging to Zea mays [order Cyperales, family Zea, genus mays] such as poaceae, corn,

maize, mealie, Indian corn and the like. Therefore, the corn used in the present

invention may be any kind classified into Zea mays. The amylase-activity-inhibitory

corn extract of the present invention is extracted from corn kernels, corn seed, or

cornhusks, but it may also be extracted from corn stalks and corncobs.

The extract of the present invention includes effective ingredients having the

activity to inhibit the activities of amylase, and is extracted from Zea mays. Preferably

water or organic polar solvents or their mixtures may be used as the extracting solvent.

A preferable extracting solvent is water or alcohol, preferably, lower alcohol with 1~4

of carbons. More preferably, methanol or ethanol may be used. Furthermore, other organic polar solvents with appropriate polarity may be used. Such polar solvents may

include diethyl ether, dichloromethane (also referred as methylene chloride), acetone,

ethyl acetate, and acetonitrile. These extracting solvents can be used alone and in a

mixture.

The extraction can be conducted with conventional methods. Conventionally,

the extraction is conducted by placing corn and an extracting solvent in a closed vessel

and stirring them. The extraction may be carried out at a temperature of 0~100°C,

preferably 30~55°C, during a sufficient time period that the extract is sufficiently

extracted, usually for 1 hour ~ 1 month. In this example, as the temperature increases,

the time period of extraction becomes less. However, if the temperature is too high, the

effective ingredients may decompose or change by some chemical reactions. Therefore,

preferably a suitable temperature is maintained. The corn used in the extraction, for

example, the corn kernel or husk can be used wholly, but preferably should be used in

the form of pieces or in a shattered form in order to conduct the extraction effectively.

After the extraction, solids are removed from the extract solution containing the

extracting solvent. The means for removing the solids may include generally known

means, for example, centrifugal separation, filtration, and decantation. The extracting

solvent can be removed from the solids-removed extract solution to obtain the corn

extract of the present invention. In this case, the removal of extracting solvent may be

performed with conventional means, which may include atmospheric distillation, vacuum distillation by vacuum centrifugal evaporator and the like, lyophilization, and

exposure to dried nitrogen or air. At the time of the removal of extracting solvent, heat

may or may not be added according to conditions. However, the addition of excessive

heat is not preferable.

In order to remove the ingredients not having the inhibitory activity against the

activities of amylase from the corn extract obtained by the above-mentioned extraction,

further purification may be carried out.

One of these purification methods is chromatography. Such chromatography

can be carried out by suitably selecting a kind of chromatography, an eluent, and eluting

conditions. For example, ineffective ingredients can be removed from the corn extract

obtained above with silica gel column chromatography (Merck, 9385, 70-230 mesh)

using an appropriate eluent, for example, the mixed solvent of ethylene chloride,

methanol and distilled water. At this time, the eluent may be set up such that its polarity

goes higher gradually with the eluting time. Also, in order to further purify the corn

extract purified partially as above or to obtain single active ingredient, a

chromatography of Sephadex LH20 (Pharmacia, 17-0090-01) with loading, for example,

an active fraction of the corn extract obtained above and using methanol as an eluent

may be carried out, and then a reverse silica gel chromatography with loading an active

fraction and using 30%~10% acetonitrile as an eluent may be carried out. In order to

purify a single active ingredient, recrystallization using a solvent such as the mixture of acetonitrile and methanol, for example, may be carried out to obtain a pure active

compound.

Also, in order to increase safety in the further purification of the corn extract,

no organic solvents harmful to human body are used, but ethanol (spirituous alcohol)

and water can be only used to purify. One of these examples is to use an anion exchange

resin. Some of the materials to inhibit the activities of amylase in the corn extract of the

present invention have one or more carboxyl groups or similar functional groups.

Therefore, an anion exchange resin can be used to purify the materials. In more detail,

the corn extract of the present invention may be adjusted to have a weak alkalinity, for

example pH 8~9. At this time, the same volume of ethanol as that of the extract may be

used to dissolve the com extract. This solution of the corn extract can be passed through

the anion exchange resin equilibrated with aqueous solution having neutral or weak

alkaline hydrogen ion concentration (pH), for example pH 8~9, to combine the active

ingredients having the corresponding functional groups to the anion exchange resin.

Then, the anion exchange resin can be washed with distilled water or tap water to

remove the ingredients not combined with the resin. The active ingredients combined

with the resin can then be eluted with appropriate concentration of acid aqueous

solution or salt aqueous solution. At this time, the pH of the acid aqueous solution is not

limited if it is in the acid range, but it is preferable that the pH should be 4~6 only in

consideration of activity. The concentration of the salt aqueous solution may be in the range of 0.001-0.5 M, although it can vary with the kinds of salts. The further

purification using the anion exchange resin can be generally carried out with column

chromatography, but also carried out with other means.

If water and ethanol (spirituous alcohol) are only used in the extraction of corn

and the further purification of the present invention, the obtained com extract is quite

safe. Therefore, this method may be particularly suitable when the corn extract of the

present invention is used as a food additive, and it is economical because it does not

generate an increase in cost related to excess process management to guarantee safety.

Another purification method is to use the difference of distribution coefficient

according to the properties of polarity and solubility. The ingredients (effective

ingredients) having inhibition activity against the activities of amylase contained in the

corn extract of the present invention have higher solubility for the polar solvents having

an appropriate polarity between water and non-polar solvents than for water and the

non-polar solvents. Also, the ingredients (ineffective ingredients) not having inhibition

activity against the activities of amylase are contained in the com extract of the present

invention, and some of these ingredients are dissolvable in water or non-polar solvents.

Therefore, based on the characteristic that solubility of chemical material for polar

solvents and non-polar solvents varies with its polarity, the ineffective ingredients

readily dissolvable into the non-polar solvents and water can be removed from the com

extract of the present invention by using a separate apparatus. In order to remove the ineffective ingredients readily dissolvable into the non-

polar solvents, firstly the corn extract obtained by the extraction process is dissolved or

suspended in water. At this time, in order to dissolve the extract in water perfectly, a

large amount of water or heat may be needed. As a result inconvenience during

manipulation occurs. Thus, the next step may proceed on the condition that the extract

is not dissolved in water perfectly. The aqueous solution or suspension of the extract is

then contacted with an organic non-polar solvent which cannot be mixed with water.

This process can be carried out in a separate apparatus. Then, the ineffective ingredients

having non-polar property contained in the aqueous solution or suspension of the extract

are dissolved in the non-polar solvent. Namely, they are distributed more in the non-

polar solvent. After that, the solution of the non-polar solvent is removed, and water can

be removed from the aqueous solution to obtain the partially purified corn extract of the

present invention. Through these procedures, the ineffective ingredients readily

dissolvable into the non-polar solvent can be removed from the corn extract obtained by

the extraction process.

The organic non-polar solvent used in the present invention is preferably an

organic solvent which has the polarity below petroleum ether on the polarity table

generally provided for fractionation and which cannot be mixed with water. The

particularly preferable non-polar solvent is hexane. The polarity order of organic

solvents depicted on the polarity table generally provided for fractionation is schematically as follows:

Cyclohexane < n-pentane < hexane < isooctane < petroleum ether < xylol <

isopropyl ether < toluene < diethyl ether < chloroform < dichloromethane <

tetrahydrofuran < acetone < dioxane < ethyl acetate < n-propanol < n-butanol < ethanol

< methanol < water (The order is described from non-polar solvent to polar solvent.)

See a more detailed polarity table for the polarity of the solvents not described here.

More detailed and systematic polarity of solvents can be obtained from the

solvent polarity parameter, particularly, normalized solvent polarity parameter proposed

by Dimroth and Reichardt (See Christian Reichant, "Solvents and solvent effects in

organic chemistry", 2^nd Ed., 1988 published by VCH Verlagsgesellschaft mbH). The

solvent polarity parameter (E_T) can be obtained from calculating, by means of

mathematic formula (1), transition energy for longest-wavelength solvatochromic

absorption band of pyridinium-N-phenoxide betain dyes of chemical formulas (1) and

(2) below.

[Chemical formula 1]

[Chemical formula 2]

[Mathematic formula 1]

E_jJ(kcal . mo ¹) = h . c . vJV_Λ = 2.859 . 1(T³ . v/cm^≡Z

In the mathematic formula 1, is Plank's constant, c is the velocity of light, ^v

is a wavenumber of photon generating electronic excitation, and N_A is Avogadro's

number.

That E_T value for methanol is 55.4 means that the transition energy needed to

excite 1 mole of the dye dissolved in methanol from electronic ground state to first

excited state is 55.4 kcal. The measurement is conventionally carried out under the

conditions of 25°C and 1 bar.

In addition, the pyridinium-N-phenoxide betain dye of chemical formula (1)

dissolves in water a little, does not dissolve well in polar solvents, and does not dissolve

in non-polar solvents at all. Therefore, in order to solve this solubility problem, the hydrophobic betain dye of chemical formula (2) substituted with five of tert-butyl group

is further used as a secondary reference probe.

It is also convenient to use the normalized solvent polarity parameter (Eτ^N)

normalized with mathematical formula (2) from the solvent polarity parameter (E_T).

This normalization uses water and tetramethylsilane (TMS) as extreme reference

solvents.

[Mathematic formula 2]

_N ^(solvent) -Sτ(TMS) -g_τ(solvent) -30.7 ^τ ~ £ (water) -£_T(TMS) ^"" 32.4

The solvent polarity parameters for several solvents can be obtained from the

above calculation. The solvent polarity parameters for main solvents are shown on

Table 1 below.

Table 1

The non-polar solvent used in the present invention may be a solvent having a

polarity ranging from 0.15 or less, preferably 0.1 or less of solvent polarity parameter

calculated from experimentally measured transition energy for longest-wavelength

solvatochromic absorption band of pyridinium-N-phenoxide betain dyes. Examples of

preferable non-polar solvents may include n-hexane, cyclohexane, n-pentane, n-octane,

petroleum ether, toluene, and the like.

Also, in order to remove the ineffective ingredients readily dissolvable in water,

the aqueous solution or suspension of the corn extract obtained from the extraction process is contacted with an organic polar solvent that cannot be mixed with water.

Then, the ineffective ingredients readily dissolvable in water are more distributed in

water, while the effective ingredients readily dissolvable in the polar solvent are more

distributed into the polar solvent. Therefore, after removal of the aqueous solution, the

polar solvent can be removed from the solution of the polar solvent to obtain the

partially purified corn extract. Through these procedures, the ineffective ingredients

readily dissolvable into water can be removed from the corn extract obtained from the

extract process.

The organic polar solvents used in the present invention are preferably organic

solvents which have the polarity between diethyl ether and butanol on the polarity table

generally provided for fractionation and which cannot be mixed with water, or their

mixtures. The particularly preferable polar solvents are ethyl acetate, butanol, and their

mixture. Also, the polar solvent used in the present invention may be a solvent having a

polarity ranging from 0.1-0.9, preferably 0.2-0.8 of solvent polarity parameter

solvatochromic absorption band of pyridinium-N-phenoxide betain dyes. The examples

of preferable polar solvents may include dichloromethane (methylene chloride; MC),

chloroform, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether,

tetrahydrofuran, ethyl acetate, ethyl acetoacetate, ethyl benzoate, and the like.

The removal of the ineffective ingredients readily dissolvable in the organic non-polar solvent and the removal of the ineffective ingredients readily dissolvable in

water as described above are preferably carried out in order. If needed, one of them may

be carried out. Also, in order to remove the ineffective ingredients readily dissolvable in

water, the corn extract may be dissolved in the polar solvent and then contacted with

water.

The com extract or partially purified corn extract obtained by the method as

described above can inhibit the activities of amylase in the intestine to suppress the

absorption of lower saccharides and to prevent an increase of the blood glucose level,

and induce the feeling of satiety due to the indigested carbohydrate. Also it is not

harmful to human body or animal because it is extracted from the edible plant.

Accordingly, the corn extract of the present invention can be used for treatment

or prevention of diseases related with amylase activities. The present invention provides

a pharmaceutical composition for treatment or prevention of diseases related with the

amylase activities comprising the corn extract or the partially purified corn extract

obtained by the method as described above and a pharmaceutically acceptable carrier.

The disease related with the amylase activities is obesity or diabetes mellitus,

particularly diabetes mellitus accompanied with obesity or hyperglycemia.

The pharmaceutical composition of the present invention comprises the corn

extract as an effective ingredient inhibiting the activities of amylase. Such corn extract

is conventionally formulated in combination with a carrier. The pharmaceutical composition of the present invention may be used in the form of solid formulation such

as particle and tablet, and in the form of liquid formulation.

In the preparation of the formulation of the pharmaceutical composition

containing the corn extract of the present invention, the corn extract may be mixed with

any carrier, excipient, and diluent. The diluted composition is preferably inserted into a

capsule, sachet or other type carrier. These formulations may include, for example,

tablet, pill, granule, powder, sachet, alexil preparation, suspension, emulsion, solution,

syrup, aerosol, soft or hard gelatin capsule, and sterilized powder. Particularly, the

formulation suitable for oral administration is preferable.

The examples of suitable carrier, excipient, and diluent used in the

pharmaceutical composition of the present invention may include aluminum salts,

phenoxyethylethanol, water, physiological saline solution, lactose, dextrose, sorbitol,

mannitol, calcium silicate, cellulose, methyl cellulose, amorphous cellulose,

polyvinylpyrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium

stearate, and mineral oil.

The formulation of the pharmaceutical composition of the present invention

may further include filler, anticoagulant, lubricant, wetting agent, perfume, emulsifier,

antiseptic and the like.

The effective amount referred to in this specification means a minimal amount

to reduce the amount of lower saccharides absorbed from the intestine into the body. A dose of the extract of the present invention administered for subject can be controlled in

consideration of the administration route and the administered individuals.

The pharmaceutical composition of the present invention may be administered

to an individual at least once a day. A unit dose means a unit amount physically

separated for suitable unit administration to the administered human being and other

mammals. Each of the unit doses contains a pharmaceutically acceptable carrier and the

extract of the present invention exhibiting the treatment effect.

Regarding an adult, it is preferable that the unit dose for oral administration

should contain O.lg or more of the com extract of the present invention. The

pharmaceutically effective dose of the com extract of the present invention is 0.1 to lOg,

preferably, 0.5 to 5g per one time in oral administration. However, the dose may vary

depending on the weight of the patient, the degree of seriousness of obesity and diabetes

mellitus, the used extract, and the supplementary effective ingredients included.

. Everyday total doses can be divided into several sub-doses, and if necessary,

they can be administered in sequence. Therefore, the range of doses should not limit the

scope of the present invention in any ways.

When the corn extract of the present invention is eaten with food containing

polysaccharides over disaccharides, particularly carbohydrates, it can also suppress

digestion of carbohydrates and thus reduce the amount of lower saccharides absorbed

into the body to suppress the increase of blood glucose level. Therefore, the corn extract of the present invention can be used as a food additive for controlling the blood glucose

level of a food ingesting person at the time of food ingestion. Namely, the present

invention provides a food additive for controlling the blood glucose level of the food-

ingesting person at the time of food ingesting comprising the com extract or the

partially purified corn extract.

The food additive of the present invention can be added into the food

containing polysaccharides over disaccharides, particularly carbohydrates such as

breads, cookies, cool drinks, chewing gums, toothpastes, dental cleaners, butters,

noodles, and rice. The food additive of the present invention does not need to be in the

form of a food additive, but it may be in the form of being separately ingested at the

time of food ingestion or before or after food ingestion. Namely, if a food ingesting

person can add the food additive of the present invention into food at the time of food

ingestion and eat it, or if he can eat it separately at the time of food ingestion or before

or after food ingestion thereby suppressing the digestion and • absorption of food,

particularly carbohydrates in the intestine although it is not in the form of a food

additive, this situation is in the scope of the present invention. Thus, the food additive of

the present invention may include the formulation to exhibit the substantially same

effect as the form to be added into food.

Furthermore, the composition comprising the com extract or the partially

purified corn extract of the present invention doesn't mean only an approved preparation as a medicine, but it may be understood to mean a concept including

conventionally functional food or health-indicated food.

The following examples are provided for better illustration of the present

invention, but nothing therein should be taken as a limitation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the change of blood glucose level measured after

starch and the corn extract are administered into Spraue-Dawley rat.

FIG. 2 is a graph showing the change of blood glucose level measured after

starch and the corn extract are administered into ICR mouse.

FIG. 3 is a graph showing the change of blood glucose level measured after

starch and the active compound 3 obtained by further purification of the corn extract are

administered into ICR mouse.

FIG. 4 is a graph showing the change of blood glucose level measured after

cornstarch and the corn extract are administered into Spraue-Dawley rat.

FIG. 5 is a graph showing the change of blood glucose level measured after

wheat flour and the com extract are administered into Spraue-Dawley rat.

FIG. 6 is a graph showing the inhibition effect of the corn extract obtained by

purification of the cmde extract of comhusk using ion-exchange eluting method (eluent:

pH 2-6.5) by anion exchange resin, against porcine pancreatic amylase. FIG. 7 is a graph showing the inhibition effect of the com extract obtained by

0.01-0.5 M of NaCl aqueous solution) by anion exchange resin, against porcine

pancreatic amylase.

EXAMPLES

Example 1

Extraction of amylase inhibition activity material from corn

In order to obtain active extracts having inhibition effect against alpha-amylase,

a carbohydrates digestion enzyme from com, com kernel (hereinafter referred to as "C")

and comhusk or corn bran (hereinafter referred to as "CH") was purchased. The C was

broken up into the form of powder. The CH was used in the next process as is.

5 kg of C powder and 15 kg of CH were respectively placed into 10 L and 30 L

of methanol for one day. In order to do effective extraction, they were placed in hot

baths of 37°C, and stirred with a stirrer. After 24 hours, they were placed without

stirring to allow solids to settle to the bottom. They were filtered with a funnel mounted

with absorbent cotton to remove the sunken and floating solids. Each methanol of the

filtrates was evaporated with a rotary evaporator, and the amounts of extracts obtained

were measured. The extracts were recovered with 500 mL of tertiary distilled water per

100 g of the extract. The extract discovered with water was firstly mixed with the same amount of

hexane (hereinafter referred to as "H", of DAEJUNG Chemicals & Metals Co. Ltd.,

Korea) and placed into a separation funnel. After being stirred, it was placed without

stirring for 24 hours. Then, the upper layer of hexane was separated. The water layer

was further fractionated 2-3 times with hexane. The hexane fractions were then

concentrated using an evaporator and dried in a vacuum oven to remove the solvent

perfectly.

The remaining water layer was mixed a second time with the same amount of

methylene chloride (hereinafter referred to as "MC", of DAEJUNG) and placed into a

separation funnel. After being stirred, it was placed without stirring for 24 hours. Then,

the lower layer of MC was separated. The water layer was further fractionated 2-3

times with MC. The MC fractions were then concentrated using an evaporator and dried

in a vacuum oven to remove the solvent perfectly.

The remaining water layer was mixed a third time with the same amount of

ethyl acetate (hereinafter referred to as "EA", of DAEJUNG) and placed into a

the upper layer of EA was separated. The water layer was further fractionated 2-3 times

with EA. The EA fractions were then concentrated using an evaporator and dried in a

vacuum oven to remove the solvent perfectly.

The remaining water layer was mixed a fourth time with the same amount of butanol (hereinafter referred to as "Bu", of DAEJUNG) and placed into a separation

funnel. After being stirred, it was placed without stirring for 24 hours. Then, the upper

layer of Bu was separated. The water layer was further fractionated 2-3 times with Bu.

The Bu fractions were then concentrated using an evaporator and dried in a vacuum

oven to remove the solvent perfectly.

The final remaining water layer was concentrated using an evaporator and dried

in a vacuum oven to remove the water.

Each of the fractions was dissolved in dimethyl sulfoxide (Sigma, D-5879) with

the concentration of 800 mg/ml and stored at -20°C, in order to be used later in

experiments in vitro and in vivo for inhibition activity against the activities of amylase.

Example 2

Further purification of com extract

The EA fraction obtained in the example 1 was chromatographed using silica

gel (Merck, 9385, 70-230 mesh) in the manner that the mixture ratio of methylene

chloride, methanol and distilled water was started at 50:3:0.2 and the polarity of mixture

eluent was gradually increased.

The EA fraction was divided into 7 sub-fractions according to polarity, and

each of the sub-fractions was tested in vitro and in vivo for the inhibition activity against

the activities of amylase. The results showed that the inhibition activity against the activities of amylase increased at the third and fourth sub-fractions.

The active sub-fractions were respectively re-fractionated by chromatography

of sephadex LH20 (Pharmacia, 17-0090-01) with methanol as an eluent. The re-

fractionated sub-fractions were then tested in vitro and in vivo. Active compounds

having 100-300 of molecular weight were separated from the active sub-fractions

confirmed by the test through a reverse silica gel chromatography with 30%~10% of

acetonitrile (hereinafter referred to as "MeCN) as an eluent gradient and a

recrystallization using 5-20% of MeCN and methanol.

The sub-fractions obtained in the purification procedures were tested in vitro

and in vivo for the inhibition activity against the activities of amylase, and the final

obtained sub-fractions and active compounds were dissolved in dimethyl sulfoxide

(Sigma, D-5879) with the concentration of 100 mg ml and stored at -20°C, in order to

be used later in the tests.

Example 3

Experiment in vitro for amylase activities

The fractions fractionated with several organic solvents having several

polarities from the extracts of com kernel and comhusk were tested for the inhibition

level against the activities of amylase. Porcine pancreatic amylase (Sigma, A-6255,

USA) was used as an amylase, and para-nitrophenyl-A-d-maltoside (hereinafter referred to as "pNPM", of Sigma, N-5885) was used as a substrate. 40 mM (final concentration)

of the substrate of pNPM and the extract were placed in a microtiter plate with amylase

reaction buffer (20 mM phosphate buffer, pH 6.9, 20 mM NaCl, 0.1 mM CaCl₂). After

it was left for 1 hour at 37°C, porcine pancreatic amylase was added to it such that the

final concentration became 56 ug/ml, and reacted for 1 hour at 37°C. After 1 hour of

reaction, the absorbance was measured at 405 nm (reference 650 nm) using

spectrophotometry (ELISA reader; Molecular Device Inc., USA). This experiment was

carried out 3 times.

The inhibition activity is calculated as follows:

Amylase inhibition activity (%) = [(absorbance by the substrate - absorbance

by the substrate and the extract) / (absorbance by the substrate)] X 100.

The amylase inhibition activities for the fractions of the extract of com kernel

and comhusk and the separated active compounds are shown on Tables 2 and 3. In

comparison with the fractions of other organic solvents, the fraction of ethyl acetate (C-

EA, CH-EA) had high activity. It respectively had about 50% and 73% of inhibition

activities for corn kernel and comhusk. The yield of ethyl acetate fraction of comhusk

(CH-EA, hereinafter referred to as "com extract") was 0.1% (w/w) on the basis of the

dried weight of the starting material, i.e., comhusk.

The results show that the com extract of the present invention is effective in

inhibiting the activities of amylase, a carbohydrate digestion enzyme. Table 2.

Amylase inhibition activities of organic solvent fractions of the extract from

corn kernel.

C-H: hexane fraction of the extract from corn kernel, C-MC: methylene

chloride fraction of the extract from com kernel, C-EA: ethyl acetate fraction of the

extract from corn kernel, C-Bu: butanol fraction of the extract from corn kernel, C-W:

water fraction of the extract from corn kernel, Amylase: porcine pancreatic amylase.

Table 3.

Amylase inhibition activities of organic solvent fractions of the extract from

cornhusk.

CH-H: hexane fraction of the extract from cornhusk, CH-MC: methylene

chloride fraction of the extract from cornhusk, CH-EA: ethyl acetate fraction of the

extract from cornhusk, CH-Bu: butanol fraction of the extract from cornhusk, CH-W:

water fraction of the extract from cornhusk, Amylase: porcine pancreatic amylase Table 4.

IC₅₀ value for compounds separated from EA fraction of cornhusk.

Extract 1-10: Compounds having 100-300 of molecular weight separated from

CH-EA fraction (hereinafter referred to as "active compounds"), Amylase: porcine

pancreatic amylase.

In the meantime, the fractions of organic solvents were obtained with the same

method as example 1 from 302g of the corn extract obtained from the extraction of

cornhusk by the extraction process. In these results, the hexane fraction was 64.4g, the

methylene chloride (MC) fraction was 12.84g, the ethyl acetate (EA) fraction was 3.94g,

the butanol (Bu) fraction was 114g, and the water (W) fraction was 106.65g. It can be

observed from these results and the results of inhibition activities of Table 3 that the

ineffective ingredients not inhibiting the activities of amylase are considerably

contained in the water and hexane layers, and the effective ingredient inhibiting the

activities of amylase are considerably contained in the organic polar solvents. Therefore,

the above results show that the corn extract of the present invention can be further

purified using the polarities of effective ingredients and ineffective ingredients

contained in the com extract and their differences of solubility for each of solvents. It can also be observed from schematic evaluation that ethyl acetate is good in the

selectivity for effective ingredients, but is not good in solubility; butanol is not as good

in the selectivity for effective ingredients compared with ethyl acetate, but is good in

solubility; and methylene chloride ranks between ethyl acetate and butanol in both

selectivity and solubility. Accordingly, the use of their mixture solvents will increase

the efficiency to remove the ineffective ingredients for amylase activity inhibition from

the corn extract.

Example 4

Effect of corn extract to decrease blood glucose level in Spraue-Dawley rat

after a meal.

The Spraue-Dawley (SD) rats (DAEHAN Biolink Co. Ltd., Korea) used in this

example were males aged six weeks and weighing 150~200g. The breeding conditions

were as though 12 hours periodical illumination was performed (lighting for 09:00 -

21:00, shielding the light for 21:00 - 09:00), and temperature and humidity were

respectively kept in the ranges of 22~24°C and 40-60%.

In order to evaluate the effect of the com extract of the present invention

administered with starch to decrease the blood glucose level after a meal, the extract of

ethyl acetate fraction (CH-EA) with good amylase inhibition activity in vitro test

confirmed by Example 2 was used. Starch was used in such treated form that potato starch (sigma, S-2630) was dissolved in physiological saline solution (0.9% NaCl)

cooked in a double boiler, and then an excipient (2% Tween-80) was added to this. The

corn extract was used with the addition of starch and excipient.

Eighteen hours prior to administering the composition of the present invention

prepared as above, SD rats were starved with only a supply of water. In order to

measure the blood glucose level in fasting condition, the tail-tip of a rat was cut and the

blood was collected using a capillary tube with anticoagulant to obtain plasma. In order

to know the effect of the composition of the present invention to decrease the blood

glucose level after a meal, the rats were divided into a group of 4 individuals to be

administered only starch and a group of 4 individuals to be administered starch and the

corn extract. The starch and the corn extract were orally administered with lg and

100-400 mg per 1kg of SD rat's weight, respectively. Blood collection was performed

at 30 and 60 minutes after administration.

The blood glucose level was measured from absorbance at 490 nm with

spectrophotometry using Trinder kit (Sigma, 315-500) using enzyme colorimetry. The

blood glucose level was calculated on the basis of standard solution. The difference of

blood glucose level between two groups was tested with unpaired Student's t-test.

The changes of the blood glucose level obtained in this example are depicted in

FIG. 1. The decrease of blood glucose level having significance (p<0.001) was shown at

30 minutes and 60 minutes after the corn extract (CH-EA) was administered into SD rats with the amount of 400 mg/kg.

Example 5.

Effect of com extract to decrease blood glucose level in ICR mouse after a meal.

The ICR mice (DAEHAN Biolink Co. Ltd., Korea) used in this example were

males aged 6 weeks and weighing 25~30g. The breeding conditions were same as that

of Example 4.

In order to evaluate the effect of the com extract and the active compound 3 of

the present invention separated by further purification administered with starch to

decrease the blood glucose level after a meal, the compositions for oral administration

were prepared as that of Example 4.

Twenty hours prior to administering the composition of the present invention

prepared as above, ICR mice were starved with only a supply of water. In order to

measure the blood glucose level in fasting condition, the blood was collected at the

retro-orbital venousplexus using a capillary tube with anticoagulant. In order to know

the effect of the composition of the present invention to decrease the blood glucose

level after a meal, the mice were divided into a group of 5 individuals to be

administered only starch, a group of 5 individuals to be administered starch and the corn

extract, and a group of 5 individuals to be administered the active compound 3 and

starch. The starch and the corn extract were orally administered with lg and 100-400 mg per 1kg of ICR mouse's weight, respectively. Blood collection was performed as in

Example 4 at 30, 60 and 120 minutes after administration. The blood glucose level was

measured using enzyme colorimetry as in Example 4, and the results were analyzed as

in Example 4

The changes of blood glucose level obtained in this example are depicted in

Figs. 2 and 3. The decreases of blood glucose level having significance (p<0.001) were

shown at 30 minutes after the com extract (CH-EA) and the active compound 3 were

administered into ICR mice with the amount of 100-400 mg/kg.

Example 6

Effect of corn extract to decrease blood glucose level in use of several grains

The SD mice and breeding conditions used in this example were same as those

of Example 4. In order to evaluate the effect of the corn extract of the present invention

administered with grains to decrease the blood glucose level after a meal, the

compositions for oral administration were prepared as those of Example 4. Cornstarch

and wheat flour normally sold as food were used as grains.

In order to know the effect of the composition of the present invention to

decrease the blood glucose level after a meal, the mice were divided into groups of 5

individuals to be administered only grains, and groups of 5 individuals to be

administered grains and the corn extract. The grains and the corn extract were orally administered with 0.5g and 400 mg per 1kg of SD rat's weight, respectively. Blood

collection was performed as above at 30, 60 and 120 minutes after administration. The

blood glucose level was measured using enzyme colorimetry as in Example 4, and the

results were analyzed as in Example 4.

The changes of blood glucose level obtained in this example are depicted in

Figs. 4 and 5. The decreases of blood glucose level having significance (p<0.001,

p<0.01) were shown after the com extracts were administered into SD rat with the

amount of 400 mg/kg.

Example 7

Further purification using anion exchange resin (Elution with acid aqueous

solution)

In order to obtain the active extracts having abilities to inhibit the activities of

amylase, 10 kg of cornhusk, was extracted with the mixed solvent of water and 95%

ethanol (spirituous alcohol) at 40°C for 12 hours. The inhibition effects of the comhusk

crude extracts against porcine pancreatic amylase treated with water and 95% ethanol

mixed in a various ratio are shown on Table 5. The obtained com extract solution was

filtered with Whatman No. 1 filter paper, and water and ethanol were evaporated with a

rotary evaporator from it to obtain the corn cmde extract. The same volume of first

distilled water or tap water was added to this corn crude extract, and this extract solution was stirred at a temperature of 40~80°C for 1 hour and filtered with Whatman

No. 1 filter paper. The filtrate was again concentrated to obtain the concentrated extract,

to which the same volume of spirituous alcohol (95% ethanol) was added. After the

extract solution was stirred for 1 hour, it was filtered with Whatman No. 1 filter paper.

The hydrogen ion concentration (pH) of filtrate was adjusted to pH 7-8 with IN NaOH

aqueous solution. The pH-adjusted extract solution was contacted with anion exchange

resin (Diaion SA 10 AP resin) equilibrated with pH 7-8 of aqueous solution in a reactor.

In order to remove the materials not combined to the resin, the resin was washed with

first distilled water or tap water, and then the washing solution was removed from the

reactor. After that, aqueous solution adjusted to pH 2-6.5 with diluted hydrochloric acid

(0.5N HC1) was added into the reactor of anion exchange resin. The solution was stirred

for 2 hours, placed without stirring for 1 hour, and then recovered. The solution was

continuously neutralized with diluted sodium hydroxide (IN NaOH), and then filtered

with Whatman No. 1 filter paper. The filtrate was concentrated to obtain the cornhusk

extract. At this time, the yield of cornhusk extract was 1% when using pH 2 of aqueous

solution. The inhibition levels of the obtained extracts against porcine pancreatic

amylase are shown in FIG. 6.

Table 5.

Inhibition effect of cornhusk cmde extract against porcine pancreatic amylase treated with water and spirituous alcohol mixed in a various ratio.

In the above table, the ethanol is spirituous alcohol (95% ethanol).

Example 8

Further purification using anion exchange resin (Elution with salt aqueous solution)

ethanol (spirituous alcohol) at 40°C for 12 hours. The inhibition effects of the cornhusk

cmde extracts against porcine pancreatic amylase treated with water and 95% ethanol

mixed in a various ratio are shown on Table 5. The obtained corn extract solution was

rotary evaporator from it to obtain the com crade extract. The same volume of the first

distilled water or tap water was added to this corn crade extract, and this extract

solution was stirred at a temperature of 40~80°C for 1 hour and filtered with Whatman

to which same volume of spirituous alcohol (95% ethanol) was added. After the extract solution was stirred for 1 hour, it was filtered with Whatman No. 1 filter paper. The

hydrogen ion concentration (pH) of filtrate was adjusted to pH 7-8 with IN NaOH

reactor. After that, 0.01-0.5 M of NaCl aqueous solution was added into the reactor of

anion exchange resin. The solution was stirred for 2 hours, placed without stirring for 1

hour, and then recovered. Continuously, the solution was filtered with Whatman No. 1

filter paper. The filtrate was concentrated to obtain the cornhusk extract. At this time,

the yield of cornhusk extract was 1% when using 0.5 M of NaCl aqueous solution. The

inhibition levels of the obtained extracts against porcine pancreatic amylase are shown

in FIG. 7.

INDUSTRIAL APPLICABILITY

The com extracts according to the present invention can inhibit the activities of

amylase, a carbohydrate digestion enzyme. Therefore, they can suppress the intake of

lower saccharides into the body to prevent the increase of blood glucose level, decrease

an excessive intake of nutrition to be effective in the treatment and prevention of

diabetes mellitus and obesity, and induce the feeling of satiety due to the non-digested carbohydrates to obtain the diet effect. Also they are not harmful to human body

because they are extracted from plants for food.

Therefore, the corn extract of the present invention can be used in

pharmaceutical usage for the treatment and prevention of obesity or diabetes mellitus,

and also usage as a food additive which can inhibit the digestion of carbohydrates to

control the blood glucose level of the ingesting person and to induce the feeling of

satiety.

Claims

1. A process for preparation of a substance extracted from corn which

can inhibit the activities of amylase, the process comprising the steps of:

extracting corn with an extracting solvent selected from water, organic polar

solvents and their mixtures;

removing solids from the extract solution obtained from the extracting step; and

removing the extracting solvent from the extract solution to obtain corn extract.

2. The process according to claim 1, wherein the com is corn kernel or

corn husk.

3. The process according to claim 1, wherein the organic polar solvents

are lower alcohols with 1-4 of carbons.

4. The process according to claim 1, wherein the extracting step is

carried out at a temperature of 0~100°C.

5. The process according to claim 1, wherein the solids removing step is

to remove the solids by centrifugal separation or filtration and to obtain the supernatant

or the filtrate.

6. The process according to claim 1 further comprising the step of

purifying the extract.

7. The process according to claim 6, wherein the step of purifying the

extract comprises:

dissolving or suspending the extract in water;

contacting the aqueous solution or suspension of the extract with an organic

polar solvent or non-polar solvent which cannot be mixed with water, to distribute the

ingredients of the extract; and

recovering the solution or suspension of the organic polar solvent or the

aqueous solution and removing the organic polar solvent or water from the solution to

obtain partially purified corn extract.

8. The process according to claim 6, wherein the step of purifying the

extract comprises:

dissolving or suspending the extract in water;

contacting the aqueous solution or suspension of the extract with an organic

non-polar solvent which cannot be mixed with water, to distribute the ingredients in the

aqueous solution or suspension of the extract which do not have the amylase inhibition activities;

removing the solution of organic non-polar solvent;

contacting the aqueous solution or suspension of the extract with an organic

polar solvent which cannot be mixed with water, to distribute the effective ingredients

in the aqueous solution or suspension of the extract which have the amylase inhibition

activities; and

recovering the solution of the organic polar solvent and removing the organic

polar solvent from the solution to obtain partially purified corn extract.

9. The process according to claim 7 or 8, wherein the organic polar solvent is

an organic solvent having a polarity ranging from 0.1-0.9 of solvent polarity parameter

solvatochromic absorption band of pyridinium-N-phenoxide betain dyes of chemical

formula (1) and (2), and which cannot be mixed with water.

10. The process according to claim 9, wherein the organic polar solvent is

selected from the group consisting of dichloromethane (MC), chloroform, 1-butanol, 1-

pentanol, 1-hexanol, cyclohexanol, 1-dodecanol, diethyl ether, tetrahydrofuran, ethyl

acetate, ethyl acetoacetate, ethyl benzoate and their mixtures.

11. The process according to claim 7 or 8, wherein the organic non-polar

solvent is an organic solvent having a polarity ranging below 0.15 of solvent polarity

parameter calculated from experimentally measured transition energy for longest

wavelength solvatochromic absorption band of pyridinium-N-phenoxide betain dyes of

chemical formula (1) and (2), and which cannot be mixed with water.

12. The process according to claim 11, wherein the organic non-polar

solvent is selected from the group consisting of n-hexane, cyclohexane, n-pentane, n-

octane, petroleum ether, toluene and their mixtures.

13. The process according to claim 6, wherein the step of purifying the

extract is to remove the ingredients not having the amylase inhibition activities mixed in

the extract by chromatography.

14. The process according to claim 13, wherein an anion exchange resin is

used in the chromatography.

15. The process according to claim 14 comprising the steps of:

contacting the com extract with the anion exchange resin;

washing the anion exchange resin with water to remove the materials not combined to the resin;

eluting active ingredients combined to the anion exchange resin with an eluent;

and

concentrating the eluted active ingredients to obtain partially purified corn

extract.

16. The process according to claim 15, wherein the eluent is an aqueous

acid solution or an aqueous salt solution.

17. A substance extracted from corn which can inhibit activities of

amylase, the substance being prepared by a process comprising the steps of:

extracting corn with an extracting solvent selected from water, organic polar

solvents and their mixtures;

18. The substance according to claim 17, wherein the corn is corn kernel

or com husk.

19. The substance according to claim 17 partially purified by a further purification process comprising the steps of:

dissolving or suspending the extract in water;

contacting the aqueous solution or suspension of the extract with an organic

ingredients of the extract; and

recovering the solution or suspension of the organic polar solvent or the

obtain partially purified com extract.

20. The substance according to claim 17 partially purified by a further

purification process comprising the steps of:

dissolving or suspending the extract in water;

contacting the aqueous solution or suspension of the extract with an organic

aqueous solution or suspension of the extract which do not have the amylase inhibition

activities;

removing the solution of organic non-polar solvent;

contacting the aqueous solution or suspension of the extract with an organic

in the aqueous solution or suspension of the extract which have the amylase inhibition activities; and

recovering the solution of the organic polar solvent and removing the organic

polar solvent from the solution to obtain partially purified com extract.

21. The substance according to claim 17 partially purified by a further

purification process using an anion exchange resin.

22. The substance according to claim 21, wherein the further purification

process comprises the steps of:

contacting the corn extract with the anion exchange resin;

washing the anion exchange resin with water to remove the materials not

combined to the resin;

eluting active ingredients combined to the anion exchange resin with an eluent;

and

concentrating the eluted active ingredients to obtain partially purified corn

extract.

23. The substance according to claim 22, wherein the eluent is an aqueous

acid solution or an aqueous salt solution.

24. A pharmaceutical composition for treatment or prevention of diseases

related with the amylase activities comprising the corn extract according to one of

claims 17-23 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition according to claim 24, wherein the

disease related with the amylase activities is obesity or diabetes mellitus.

26. The pharmaceutical composition according to claim 24, wherein the

composition is in the form suitable to medicinal oral administration.

27. A food additive for controlling blood glucose level of a food-ingesting

person during the time of food ingesting comprising the corn extract according to one of

claims 17-23.

28. The food additive according to claim 27, wherein the food additive is

added to the food containing polysaccharides (disaccharides or more).

29. The food additive according to claim 27, wherein the food additive is

for the suppression or prevention of an increase of body weight during the time of food

ingesting.