Antioxidative glycoside and antioxidative composition containing the same

##STR1## wherein R is ##STR2## The present invention relates to the antioxidative glycoside of structural formula (A) above-shown, obtained from the culture of growing cells derived from the plant body of sesame (Sesamun indicum L.), can provide the glycoside in large quantities by utilizing sesame, and also relates to an antioxidant comprising at least one substance of the glycoside of structural formula (A) above-shown as effective ingredient, the antioxidant being harmless and effective particularly for prevention of oxidation of foods, drugs, cosmetics, etc.

FIELD OF THE INVENTION 
The present invention relates to glycosides extracted from callus 
artificially derived from the plant body of sesame (Sesamum indicum L.) 
which has an anti-oxidative activity, relates to the process for preparing 
the same and also relates to the use thereof. 
BACKGROUND OF THE INVENTION 
In general, foodstuffs are prepared from agricultural products, marine 
products, livestock products, etc. However, during the course of storage 
or preservation processing of raw food materials or products, raw food 
materials or products are deteriorated by contamination and putrefaction 
with microorganisms or by chemical or physical action to reduce their 
commercial value. Therefore, a variety of food additives have been 
developed and at the same time, methods for a temperature treatment, an 
oxygen absorption treatment, vacuum packaging, low temperature storage, a 
radiation treatment, etc. have been developed and practically used. 
The most serious problem in such deterioration of food materials or 
products is oxidation or peroxidation of food ingredients with oxygen in 
the air. Oxygen maintains life of the living thing through respiration. On 
the other hand, it is known that oxygen is a very reactive compound so 
that it reacts with various ingredients in food to oxidize or peroxidize 
the ingredients thereby to not only reduce their commercial value but also 
form injurious materials in food. It is reported that for example, 
nutritionally required unsaturated fatty acids such as linoleic acid, 
linolenic acid, etc., which are contained in foods, are readily 
peroxidized by oxygen in the air to form peroxidized fatty acids or 
reactive radicals (free radicals) and at the same time, form carcinogenic 
substances such as malone dialdehyde, etc. It is also reported that 
peroxidized lipids formed by peroxidation of unsaturated fatty acid 
molecules in lipids modify protein or nucleic acid in vivo to cause the 
living body in its carcinogenic action, etc. ("Mutagen and Toxicity", vol. 
5, page 243 (1982), "Packaging of Food", vol. 17, page 106 (1986)). 
In order to prevent such peroxidation of lipids, techniques for packaging 
such as removal of oxygen in the package with a free oxygen absorber, 
vacuum packaging, nitrogen gas substitution packaging, etc. have been 
used. On the other hand, synthetic antioxidants, for example, 
butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc. have been 
generally used, backed by development of chemical industries. However, as 
use of such synthetic antioxidants increases, food pollution increases and 
a serious problem occurs in view of safety. It is thus the actual 
situation that the consumer's rejection against synthetic antioxidants has 
been increasing and its amount of use decreases. 
On the other hand, it is considered that peroxides or carcinogenes formed 
in animal living body would adversely affect animal cells due to the toxic 
action of oxygen as described above. Such peroxidation of vital components 
with oxygen would be correlated to aging of cells and thus to a life span 
(theory of free radical aging). Therefore, highly safe antioxidants 
derived from the natural world have been greatly expected to be substances 
for supporting the antioxidative protective mechanism in vivo, not only in 
foodstuffs, especially health maintaining foods or nutrient foods, but 
also in the technical fields of medicines and cosmetics. 
However, the only natural antioxidants that have been expected to be 
practically used in place of synthetic antioxidant involving problems in 
food pollution are vitamin C prepared by chemical synthesis and vitamin E 
(tocopherol) extracted and purified from natural products. 
In order to suitably use such antioxidants derived from the natural world 
for use in foods, drugs, cosmetics, etc., it is important to find natural 
antioxidants having different properties and utilize the antioxidants 
under such conditions that their characteristics are exhibited. 
A variety of compounds having an antioxidative activity are contained in 
spices derived from plants and, spices have been added to food as having 
an action of preserving foods (Packaging of Food, vol. 19, No. 1, page 97, 
1987). However, most of spices exhibit a strong flavor or color. In order 
to add these spices to foods, drugs, cosmetics, etc. as antioxidants or 
the like, such properties have automatically limited their use range. 
Vitamin C is insoluble in fat or lipids in vivo since it is a water soluble 
substance. On the other hand, vitamin E is insoluble in an aqueous 
solution such as blood, etc. and accumulated in lipids in vivo, since it 
is fat-soluble. Such properties of extreme water solubility and fat 
solubility are not considered to be necessarily advantageous, in the case 
of foods, drugs, cosmetics, etc. applied to the living body. In order to 
exhibit their antioxidative activity appropriately in any of lipids and 
aqueous solutions in vivo, natural substances having an intermediate 
property between water solubility and fat solubility are advantageous. 
In addition to vitamin C or vitamin E, extensive investigations have been 
made on properties of natural antioxidants derived from spice plants, and 
reports are made thereon. 
However, natural antioxidants other than natural vitamin E, vitamin C, 
etc., that are expected to be used in place of synthetic antioxidants 
involving problems in food pollution have not been practically utilized, 
because their origins are plants or animals affected by natural conditions 
such as weather, etc. and it is thus difficult to stably supply them, 
their contents are extremely a trace amount, extraction may be made only 
with extreme difficulty, and components change during extraction. 
A potent antioxidative activity is noted in sesame oil obtained from seeds 
of Sesamum indicum L. by lignan compounds modified during the course of 
purification of sesame oil. Thus, sesame oil has been given an important 
position as an excellent edible oil which is not deteriorated by oxidation 
("Grand Modern Encyclopedia", Gakushu Kenkyusha (Jan. 1, 1979), pages 
123-124). 
On the other hand, it is already known that not only sesame oil is 
contained in Sesamum indicum L. contains but also antioxidants such as 
vitamin E or lignan compounds, etc. are contained in the plant body of 
sesame (Agricultural and Biological Chemistry, vol. 49, page 301 (1985)), 
Journal of the Japanese Food Industry Association, vol. 32, page 407 
(1985)). However, it is quite unknown or not even suggested that growing 
cells from sesame plants, especially cells grown at a high temperature can 
be industrially cultivated, a glycoside substance having a potent 
antioxidative activity is contained in these growing cells, and the 
substance can be efficiently extracted and produced industrially without 
causing any denaturation or deterioration. 
SUMMARY OF THE INVENTION 
The present invention has been made to overcome all the demerits 
aforementioned. An intensive many-faceted research resulted in the success 
that a novel antioxidative glycoside represented by the following 
structural formula (A) can be produced industrially in large quantities 
and systematically from culture cells prepared by cultivating growing 
cells (callus) using a synthetic medium, which growing cells are derived 
from the plant body of sesame (Sasamum indicum L.). Thus the present 
invention has been completed. 
##STR3## 
wherein R is 
##STR4## 
Namely, the antioxidative glycoside of formula (A) includes three 
substances: Substance 1, Substance 2 and Substance 3.

ed to the medium and shake-cultured at 35.degree. C. under a fluorescent 
light under the shake number of 60 rpm. The culture cells corresponding to 
5 flasks cultured for 7 days were recovered by aseptic operation and 
inoculated in the plant cell culture tank. Culture conditions of the plant 
cell culture tank were: stirring number of 30 rpm, pH of 5.7.+-.0.1, 
aerial amount of 1.5 liter/min, light exposure of 8,000 lux, and 
temperature of 35.degree. C. for 10 days. After completion of the culture, 
the culture broth was centrifuged to recover the cells. On a dry weight 
basis, 43 g of the cells were obtained. 
Using 500 g of the sesame cells obtained by culture, the antioxidant was 
extracted and purified. That is, 1.9 liter of ethanol was added to the 
sesame cells. While stirring with a homogenizer, extraction was performed 
for 20 minutes. Then, the cells and the extract were separated from each 
other through a filter. After 2 liters of 80% ethanol water was added to 
the cell debris, extraction was performed for 20 minutes in a similar 
manner. The cell debris was then isolated through a filter and further 
extracted with 2 liters of 80% ethanol. 
The extracts were combined and concentrated at 40.degree. C. under reduced 
pressure to give 11.4 g of brown crude extract. 
Amberlite XAD-II used for adsorption chromatography was packed in a 
glass-made column having a diameter of 5 cm and a length of 30 cm. Water 
was passed through the column to equilibrate. To the column was overlaid 5 
g of the extract in such a state that the resin was adsorbed on the upper 
part of the column. By stepwise elution with water and with methanol while 
sequentially increasing its concentration, the desired substance was 
eluted. The fraction eluted with 60% methanol was collected and 
concentrated at 40.degree. C. under reduced pressure to give 210 mg of the 
yellowish brown intermediary product. High performance liquid 
chromatography of this product was repeated until single peak was 
obtained. As the result, there were obtained, as the purified substances, 
6 mg of Substance 1, 13 mg of Substance 2 and 18 mg of Substance 3. 
The antioxidative activity of the three Substances was determined by the 
rabbit erythrocyte membrane ghost method. The respective antioxidative 
activities were as potent as butylhydroxyanisole (BHA). 
TEST EXAMPLE 
Excellent antioxidative activity of the antioxidant prepared by the present 
invention was confirmed as follows. 
The extract, 100 mg, obtained in EXAMPLE 1 was dissolved in 100 ml of 80% 
ethanol aqueous solution (in a concentration of 1 mg/ml) to make an 
analytical sample of the antioxidative activity. Using 1 ml of the above 
solution as a sample, the degree of prevention of linoleic acid from 
autoxidation was determined by the rhodan iron method. As the result, it 
was confirmed that the antioxidant having a high activity equivalent to 
that of .alpha.-tocopherol (0.2 mg) or butylhydoxyanisole (BHA, 0.2 mg) 
was contained in the sample (1 mg). extracted from the sesame culture 
cells, as is also clear from FIG. 19. 
INDUSTRIAL APPLICABILITY 
The glycoside of the present invention having the antioxidative activity 
exhibits an excellent antioxidative property and is extracted from the 
medium obtained by culturing sesame cells grown at a high temperature. 
Therefore, according to the present invention, antioxidative glycosides 
derived from natural matters can be supplied systematically in large 
quantities. Thus, the present invention may be utilized as foods, drugs 
and cosmetics.