.alpha.-glycosyl hesperidin, and its preparation and uses

.alpha.-Glycosyl hesperidin, a novel hesperidin derivative wherein equimolar or more D-glucose residues are bound to hesperidin via the .alpha.-bond, is formed by a saccharide-transferring enzyme in a liquid containing hesperidin and .alpha.-glucosyl saccharide. The .alpha.-glycosyl hesperidin is easily recovered from the reaction mixture with a synthetic macroporous resin. .alpha.-Glycosyl hesperidin is superior in water-solubility, substantially tasteless and odorless, free of toxicity, and readily hydrolyzable in vivo into hesperidin and D-glucose to exhibit the physiological activity inherent to hesperidin. Thus, .alpha.-glycosyl hesperidin is favorably usable in vitamin P-enriching agents, foods, beverages, tobaccos, foods, pet foods, pharmaceuticals for susceptive diseases, cosmetics and plastics.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a novel .alpha.-glycosyl hesperidin 
sometimes hereinafter referred to as .alpha.-glucosyl hesperidin, and its 
preparation and uses. 
More particularly, the present invention relates to .alpha.-glycosyl 
hesperidin wherein equimolar or more D-glucose residues are bound to 
hesperidin via the .alpha.-bond. 
The present invention also relates to a process to prepare .alpha.-glucosyl 
hesperidin, which comprises allowing a saccharide-transferring enzyme to 
act on a liquid containing hesperidin together with an .alpha.-glucosyl 
saccharide to form .alpha.-glycosyl hesperidin, and recovering the 
.alpha.-glucosyl hesperidin. 
The present invention further relates to foodstuffs including beverages and 
processed foods, pharmaceuticals for susceptive diseases including 
preventive and remedy therefor, and cosmetics including skin-refining 
agent and skin-whitening agent, characterized in that they all contain the 
.alpha.-glycosyl hesperidin obtainable by the process. 
2. Description of the Prior Art 
Hesperidin, whose chemical structure is given below, has been known as a 
yellow pigment and vitamin P with physiological activities such as 
stabilization of blood vessel, prevention of hemorrhage and regulation of 
blood pressure, and used from ancient times in foodstuffs, pharmaceuticals 
and cosmetics. 
##STR1## 
It is known that vitamin P takes part in some physiological activities of 
vitamin C in vivo; for example, in the hydroxylation of proline and lysine 
which are necessary to synthesize collagen as the main element of living 
connective tissues; the oxidation-reduction reaction of cytochrome C 
wherein Fe.sup.+++ is reduced into Fe.sup.++ ; and in the 
immunopotentiation via the increase of leukocyte. These are because 
vitamin P plays a significant role in the maintenance and promotion of 
health in living bodies. 
Nowadays the use of hesperidin is not limited to agents which enrich 
vitamin P as a nutritive element, but is extending in various 
applications. More particularly, because of the chemical structure and 
physiological activities, hesperidin is useful as a yellow coloring agent 
and antioxidant alone or in combination with one or more vitamins, for 
example, in foods, beverages and pharmaceuticals for susceptive diseases 
such as preventive and remedy for circulatory diseases, as well as a 
yellow coloring agent and uv-absorbent in cosmetics such as skin-refining, 
melanin-formation inhibitory agent and skin-whitening agents. 
Hesperidin is, however, hardly soluble in water (only about 1 g in 50 
liters of water or about 0.002 w/v % at ambient temperature). This renders 
its practical use very difficult. 
To improve this low water-solubility, a method wherein dimethyl sulfuric 
acid is allowed to act on hesperidin to convert it into its methyl 
derivative having an increased water-solubility has been proposed. 
The method is, however, unsatisfactory in view of toxicity, safeness and 
economical efficiency because it is carried out by an organic chemical 
procedure using a harmful dimethyl sulfuric acid which renders the 
purification of the resultant derivative very difficult. Another drawback 
is that the methyl derivative has a bitter taste. 
SUMMARY OF THE INVENTION 
Accordingly, the realization of a hesperidin derivative which is free from 
the drawbacks of conventional hesperidin and its derivatives, in 
particular, superior in water-solubility, substantially tasteless and 
odorless, free from toxicity, and exhibits a desired physiological 
activity in vivo has been in strong demand. 
The present invention is to overcome the drawbacks of prior art. We 
investigated novel hesperidin derivatives by utilizing a biochemical 
procedure. 
As the result, we found that a novel .alpha.-glycosyl hesperidin wherein 
equimolar or more D-glucose residues are bound to hesperidin via the 
.alpha.-bond is formed by allowing a saccharide-transferring enzyme to act 
on a liquid containing hesperidin together with an .alpha.-glucosyl 
saccharide, as well as that the .alpha.-glycosyl hesperidin is superior in 
water-solubility, substantially tasteless and odorless, free from 
toxicity, and readily hydrolyzable in vivo to exhibit the physiological 
activity inherent to hesperidin. 
Furthermore, we established its preparation and uses in foodstuffs, 
pharmaceuticals for susceptive diseases, and cosmetics. Thus, we 
accomplished the present invention. 
We also found that the .alpha.-glycosyl hesperidin formed by the 
saccharide-transfer reaction is easily purified by allowing a reaction 
mixture to contact with a synthetic macroporous resin, and utilizing the 
difference in absorbability thereto. 
Thus, we confirmed that the process according to the invention completely 
overcomes the drawback of prior art, and extremely facilitates the 
commercialization of .alpha.-glycosyl hesperidin.

The following experiments will illustrate .alpha.-glycosyl hesperidin 
according to the invention. 
EXPERIMENT 1 
Preparation of .alpha.-Glycosyl Hesperidin 
Experiment 1(1) 
Saccharide-Transfer Reaction 
One part by weight of hesperidin and 6 parts by weight of dextrin (DE 20) 
were added with 5,000 parts by weight of water, and the mixture was 
dissolved by heating, added with 20 units/g dextrin of cyclomaltodextrin 
glucanotransferase from Bacillus stearothermophilus commercialized by 
Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, allowed to 
react for 18 hours while keeping the mixture at pH 6.0 and 70.degree. C., 
and heated to inactivate the enzyme. Thus, an .alpha.-glycosyl 
hesperidin-containing liquid was obtained. 
Experiment 1(2) 
Purification 
A reaction mixture obtained by the method in Experiment 1(1) was filtered, 
and the filtrate was applied to a column of "HP-10", a synthetic 
macroporous resin commercialized by Mitsubishi Chemical Industries Ltd., 
Tokyo, Japan, at a flow rate of SV 2. The column was then washed with 
water, and applied with 50 v/v % aqueous ethanol, after which the eluate 
was concentrated in vacuo to remove the ethanol, and pulverized to obtain 
a pale yellow .alpha.-glycosyl hesperidin specimen [I] in the yield of 
about 130% against the weight of the starting hesperidin, on the dry solid 
basis (d.s.b.). 
Experiment 1(3) 
Hydrolysis by Amylase 
A small portion of an .alpha.-glycosyl hesperidin specimen [I] obtained by 
the method in Experiment 1(2) was dissolved in water to 1 w/v %, and the 
solution was added with 100 units/g specimen of glucoamylase (EC 3.2.1.3) 
commercialized by Seikagaku Kogyo Co., Ltd., Tokyo, Japan, and allowed to 
react for 5 hours while keeping the solution at pH 5.0 and 55.degree. C. 
The reaction mixture was heated to inactivate the remaining enzyme and 
filtered, after which the filtrate was applied to a column of "HP-10", a 
synthetic macroporous resin commercialized by Mitsubishi Chemical 
Industries Ltd., Tokyo, Japan, at a flow rate of SV 2. As the result, the 
column adsorbed the .alpha.-glycosyl hesperidin and remaining hesperidin, 
while such as glucose and salts flew out through the column without 
causing adsorption. The column was then washed by applying thereto water, 
and further applied with an aqueous ethanol having a stepwisely increasing 
concentration to recover an .alpha.-glycosyl hesperidin-rich fraction 
which was then concentrated in vacuo and pulverized to obtain a pale 
yellow .alpha.-glycosyl hesperidin specimen [II] in the yield of about 70% 
against the weight of the starting hesperidin, d.s.b. 
Another portion of the .alpha.-glycosyl hesperidin specimen [I] was 
hydrolyzed similarly as above except that the gluco-amylase was replaced 
with .beta.-amylase (EC 3.2.1.2) commercialized by Seikagaku Kogyo Co., 
Ltd., Tokyo, Japan, and the resultant hydrolysate was purified, 
concentrated and pulverized similarly as above to obtain a pale yellow 
.alpha.-glycosyl hesperidin specimen [III] in the yield of about 70% 
against the weight of the starting hesperidin, d.s.b. 
EXPERIMENT 2 
Characterization of .alpha.-Glycosyl Hesperidin 
Experiment 2(1) 
Improvement of Water-Solubility 
An .alpha.-glycosyl hesperidin-containing solution prepared by the method 
in Experiment 1(1) using the saccharide-transfer reaction, and a control 
solution which had been prepared similarly except that the enzyme was 
inactivated by heating prior to its use were allowed to stand at 4.degree. 
C. for 2 days. As the result, in the control the sedimentation of 
hesperidin led to a white turbidity, while the solution containing 
.alpha.-glycosyl hesperidin left transparent. 
Accordingly, the .alpha.-glycosyl hesperidin formed by the 
saccharide-transfer reaction has an extremely improved water-solubility. 
Experiment 2(2) 
Solubility in Solvents 
.alpha.-Glycosyl hesperidin specimens were readily soluble in water, 0.1N 
sodium hydroxide and 0.1N hydrochloric acid; soluble in methanol and 
ethanol; and hardly soluble in ether benzene and chloroform. 
Experiment 2(3) 
Uv-Absorption Spectrum 
A small portion of an .alpha.-glycosyl hesperidin specimen was dissolved in 
0.1N sodium hydroxide solution for the determination of its uv-absorption 
spectrum. Either of the specimens [I], [II] and [III] exhibited an 
absorption peak at about 286 nm as intact hesperidin. 
Experiment 2(4) 
Infrared Absorption Spectrum 
The infrared absorption spectra of .alpha.-glycosyl hesperidin specimens 
were determined by the KBr tablet method. FIGS.1 and 2 show the infrared 
absorption spectra of the specimens [I] and [II] respectively. 
Experiment 2(5) 
Stability Against Hydrolysis 
(a) .alpha.-Glycosyl hesperidin specimens are hydrolyzable by 
.alpha.-glucosidase (EC 3.2.1.20) derived from pig liver into hesperidin 
and D-glucose. 
(b) Not hydrolyzable by .beta.-glucosidase. 
Experiment 2(6) 
Thin-Layer Chromatography 
(a) Analytic procedure 
Thin-layer plate: "Kieselgel 60 F254" commercialized by Merck & Co., Inc., 
Rahway, N.J., USA 
Developing solvent: n-butanol:acetic acid:water=4:2:1 
Color-developing agent: 1 w/w % ceric sulfate in 10 w/w % aqueous sulfuric 
acid solution 
(b) Results 
Analysis of the .alpha.-glycosyl hesperidin specimens revealed that the 
specimen [I] exhibited spots at Rf 0.48, 0.34, 0.22, 0.16, 0.10, 0.04 and 
starting point in addition to a spot at Rf 0.69; the specimen [II], a spot 
at Rf 0.48; and the specimen [III], spots at Rf 0.48 and 0.34. 
The above described physicochemical properties suggest that the substance 
exhibiting a spot at Rf 0.48 in the specimens [I], [II] and [III] is 
.alpha.-glucosyl hesperidin wherein 1 mole of D-glucose residue is bound 
to 1 mole hesperidin via the .alpha.-bond; the substance exhibiting a spot 
at Rf 0.34 in the specimens [I] and [III], .alpha.-diglucosyl hesperidin 
wherein 2 moles of D-glucose is bound to 1 mole of hesperidin via the 
.alpha.-bond; and the substance exhibiting a plurality of spots at not 
higher than Rf 0.22 in the specimen [I], .alpha.-oligoglucosyl hesperidin 
wherein 3 moles or more D-glucose residues are bound to hesperidin via the 
.alpha.-bond. 
As described above, the .alpha.-glycosyl hesperidin according to the 
invention wherein equimolar or more D-glucose residues are bound to 
hesperidin via the .alpha.-bond is a novel, satisfactorily-high 
water-soluble hesperidin derivative which is hydrolyzable by 
.alpha.-glucosidase to exhibit the physiological activity inherent to 
hesperidin when ingested. 
EXPERIMENT 3 
Acute Toxicity 
An .alpha.-glycosyl hesperidin specimen [I], prepared by the method in 
Experiment 1(2), was orally administered to 7 week-old dd mice for acute 
toxicity test. As the result, no mouse died when administered with up to 5 
g of the specimen, and higher dose was difficult. 
These confirmed that the specimen was extremely low in toxicity. An 
.alpha.-glycosyl hesperidin specimen [II], prepared by the method in 
Experiment 1(3), was tested similarly as above to obtain the same result, 
confirming that the toxicity of this specimen was extremely low. 
The following Examples A and Examples B will illustrate the preparation and 
uses of .alpha.-glycosyl hesperidin according to the invention. 
EXAMPLE A-1 
.alpha.-Glycosyl Hesperidin 
One part by weight of hesperidin was dissolved in 4 parts by weight of 1N 
sodium hydroxide, and the solution was neutralized by the addition of 
0.01N hydrochloric acid solution, added with 4 parts by weight of dextrin 
(DE 10), promptly added with 20 units/g dextrin of cyclomaltodextrin 
glucano-transferase derived from Bacillus stearothermophilus 
commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, 
Japan, and allowed to react for 24 hours under stirring conditions while 
keeping the solution at pH 6.0 and 75.degree. C. Thin-layer 
chromatographic analysis of the reaction mixture revealed that about 70% 
of the hesperidin was converted into .alpha.-glycosyl hesperidins such as 
.alpha.-glucosyl hesperidin, .alpha.-diglucosyl hesperidin, 
.alpha.-triglucosyl hesperidin, .alpha.-tetra-glucosyl hesperidin and 
.alpha.-pentaglucosyl hesperidin. Thereafter, the reaction mixture was 
heated to inactivate the remaining enzyme and filtered, after which the 
filtrate was deionized and purified with ion exchanges (H- and OH-forms), 
and concentrated in usual manner to obtain a syrupy .alpha.-glycosyl 
hesperidin product additionally containing .alpha.-glucosyl saccharides in 
the yield of about 90% against the weight of the starting materials, 
d.s.b. 
The product is favorably usable as a highly-safe, natural yellow coloring 
agent, antioxidant, stabilizer, quality-improving agent, preventive, 
remedy and uv-absorbent in foods, beverages, tobaccos, feeds, pet foods, 
pharmaceuticals for susceptive diseases, cosmetics and plastics, as well 
as in vitamin P-enriching agents. 
EXAMPLE A-2 
.alpha.-Glucosyl Hesperidin 
One part by weight of a syrupy .alpha.-glycosyl hesperidin product 
additionally containing .alpha.-glucosyl saccharides, prepared in 
accordance with the method in Example A-1 with a slight modification, was 
dissolved in 4 parts by weight of water, and the solution was added with 
100 units/g syrup solid of glucoamylase (EC 3.2.1.3) commercialized by 
Seikagaku Kogyo, Co., Ltd., Tokyo, Japan, and allowed to react at 
50.degree. C. for 5 hours. Thin-layer chromatographic analysis of the 
reaction mixture revealed that the .alpha.-glycosyl hesperidin was 
converted into .alpha.-glucosyl hesperidin. 
Thereafter, the reaction mixture was heated to inactivate the remaining 
enzyme and filtered, after which the filtrate was applied to a column of 
"HP-10", a macroporous synthetic resin commercialized by Mitsubishi 
Chemical Industries Ltd., Tokyo, Japan, at a flow rate of SV 2. As the 
result, the column adsorbed the .alpha.-glucosyl hesperidin and remaining 
hesperidin both present in the reaction mixture, while glucose and salts 
flew out through the column without causing adsorption. The column was 
then washed by applying thereto water, and further applied with an aqueous 
ethanol having a stepwisely increasing concentration to recover an 
.alpha.-glucosyl hesperidin-rich fraction which was then concentrated in 
vacuo and pulverized to obtain a powdery .alpha.-glucosyl hesperidin in 
the yield of about 60% against the weight of the starting hesperidin, 
d.s.b. 
Acid hydrolysis of the .alpha.-glucosyl hesperidin led to the formation of 
1 mole of rhamnose and 2 moles of D-glucose per 1 mole hesperidin, while 
an .alpha.-glucosidase, obtained by the extraction from pig liver and 
partial purification, hydrolyzed .alpha.-glucosyl hesperidin into 
hesperidin and D-glucose. 
The product is favorably usable as a yellow coloring agent, antioxidant, 
stabilizer, quality-improving agent, preventive, remedy and uv-absorbent 
in foods, beverages, tobaccos, pharmaceuticals for susceptive diseases, 
and cosmetics, as well as in an agent directed to enrich a 
highly-purified, readily water-soluble vitamin P. 
EXAMPLE A-3 
.alpha.-Glycosyl Hesperidin 
One part by weight of hesperidin was dissolved in 500 parts by weight of 
water at pH 9.5 by heating, and the solution was mixed with another 
solution which had been separately prepared by dissolving 10 parts by 
weight of dextrin (DE 8) in 10 parts by weight of water by heating, after 
which the resultant mixture was added with 30 units/g dextrin of 
cyclomalto-dextrin glucanotransferase, and allowed to react for 40 hours 
under stirring conditions while keeping the mixture at pH 8.2 and 
65.degree. C. 
Thin-layer chromatographic analysis of the reaction mixture revealed that 
about 80% of the hesperidin was converted into .alpha.-glycosyl 
hesperidin. 
Thereafter, the reaction mixture was heated to inactivate the remaining 
enzyme and filtered, after which the filtrate was applied to a column of 
"XAD-7", a synthetic macroporous resin commercialized by Rohm and Haas 
Co., Philadelphia, USA, at a flow rate of SV 1.5. 
As the result, the column adsorbed the .alpha.-glycosyl hesperidin and 
remaining hesperidin both present in the reaction mixture, while dextrin, 
oligosaccharides and salts flew out through the column without causing 
adsorption. 
The column was then washed by applying thereto water, and further applied 
with 50 v/v % aqueous methanol to elute the .alpha.-glycosyl hesperidin 
and hesperidin which were then concentrated and pulverized to obtain a 
powdery .alpha.-glycosyl hesperidin in the yield of about 120% against the 
weight of the starting hesperidin, d.s.b. 
The product is favorably usable as a highly-safe, natural yellow coloring 
agent, antioxidant, stabilizer, quality-improving agent, preventive, 
remedy and uv-absorbent in foods, beverages, tobaccos, feeds, pet foods, 
pharmaceutical for susceptive diseases, cosmetics and plastics, as well as 
in the use directed to enrich a readily water-soluble vitamin P. 
EXAMPLE A-4 
.alpha.-Glycosyl Hesperidin 
Example A-4 (1) 
Preparation of .alpha.-Glucosidase 
Mucor javanicus IFO 4570 was inoculated and cultivated at 30.degree. C. for 
44 hours under aeration-agitation conditions in 500 parts by weight of a 
liquid culture medium which contained water together with 4.0 w/v % 
maltose, 0.1 w/v % potassium phosphate monobasic, 0.1 w/v % ammonium 
nitrate, 0.05 w/v % magnesium sulfate, 0.05 w/v % potassium chloride, 0.2 
w/v % polypeptone and 1 w/v % calcium carbonate which had been sterilized 
by heating and sterilely added to the water immediately before the 
innoculation. After completion of the cultivation, the mycelia was 
collected from the culture, added with 500 parts by weight of 4M urea in 
0.5M acetate buffer (pH 5.3) per 48 parts by weight of the wet mycelia, 
allowed to stand at 30.degree. C. for 40 hours and centrifuged. The 
supernatant was dialyzed against flowing water overnight, added with 
ammonium sulfate to 0.9 saturation, and allowed to stand at 4.degree. C. 
overnight, after which the resultant sediment was collected, suspended in 
50 parts by weight of 0.01M acetate buffer (pH 5.3) and centrifuged. The 
supernatant was recovered and used is an .alpha.-glucosidase specimen. 
Example A-4(2) 
Preparation of .alpha.-Glycosyl Hesperidin 
Five parts by weight of hesperidin was dissolved in 100 parts by weight of 
0.5N sodium hydroxide solution by heating, and the resultant solution was 
adjusted to pH 9.5 by the addition of 0.01N hydrochloric acid solution, 
added with 20 parts by weight of dextrin (DE 30), promptly added with 10 
parts by weight of an .alpha.-glucosidase prepared by the method in 
Example A-4(1), and allowed to react for 40 hours under stirring 
conditions while keeping the solution at pH 8.5 and 55.degree. C. 
Thin-layer chromatographic analysis of the reaction mixture revealed that 
about 60% of the hesperidin was converted into .alpha.-glycosyl 
hesperidin. 
Thereafter, the reaction mixture was purified, concentrated and pulverized 
similarly as in Example A-3 to obtain a powdery .alpha.-glycosyl 
hesperidin product in the yield of about 110% against the weight of the 
starting hesperidin, d.s.b. 
Similarly as the product in Example A-3, the product is feasible as a 
highly-safe, natural yellow coloring agent, antioxidant, stabilizer, 
quality-improving agent, preventive, remedy and uv-absorbent, as well as 
an agent directed to enrich a readily water-soluble vitamin P. 
EXAMPLE B-1 
Hard Candy 
Fifteen hundred parts by weight of "MABIT.RTM.", a hydrogenated maltose 
syrup commercialized by Hayashibara Shoji, Inc., Okayama, Japan, was 
heated, concentrated to a moisture content below about 2%, and mixed to 
homogeneity with 15 parts by weight of citric acid, 1 part by weight of an 
.alpha.-glycosyl hesperidin powder obtained by the method in Example A-3 
and a small amount of lemon flavor, after which the mixture was molded and 
packaged in usual manner to obtain a hard candy. 
The product is a yellow colored, vitamin P-enriched, low-cariogenic and 
low-caloric lemon candy. 
EXAMPLE B-2 
"Fuki-No-Mizuni (Boiled Bog Rhubarb)" 
Fresh bog rhubargs were pared, cut into short sticks, soaked in a diluted 
saline, and boiled down in a liquid which contained an .alpha.-glycosyl 
hesperidin syrup obtained by the method in Example A-1 and "Aoiro Ichi-go 
(Blue No.1)", a green coloring agent, to obtain a freshly green 
"fuki-no-mizuni)". 
The product pleases the eyes when arranged in Japanese traditional 
cuisines, as well as exhibiting physiological activity as a dietary fiber. 
EXAMPLE B-3 
"Gyuhi (Starch Paste)" 
One part by weight of waxy rice starch was mixed with 1.2 parts by weight 
of water, and the mixture was mixed to homogeneity with 1.5 parts by 
weight of sucrose, 0.7 parts by weight of "SUNMALT.RTM.", a crystalline 
.beta.-maltose commercialized by Hayashibara Co., Ltd., Okayama, Japan, 
0.3 parts by weight of starch syrup and 0.2 parts by weight of an 
.alpha.-glycosyl hesperidin syrup obtained by the method in Example A-1 
while gelatinizing by heating. Thereafter, the resultant was molded and 
packaged in usual manner to obtain "gyuhi". 
The product is a Japanese-style confectionery with excellent flavor and 
biting properties, which looks like "kibi-dango (millet dumpling)". 
EXAMPLE B-4 
Mixed Sweetener 
A mixed sweetener was obtained by mixing 100 parts by weight of honey, 50 
parts by weight of isomerized sugar, 2 parts by weight of "kurozato 
(unrefined sugar)" and 1 part by weight of an .alpha.-glycosyl hesperidin 
powder obtained by the method in Example A-4. 
The product is a vitamin P-enriched sweetener, and suitable for health 
food. 
EXAMPLE B-5 
Cream Filling 
A cream filling was obtained by mixing in usual manner 1,200 parts by 
weight of "FINETOSE.RTM.", a crystalline .alpha.-maltose commercialized by 
Hayashibara Co., Ltd., Okayama, Japan, 1,000 parts by weight of 
shortening, 10 parts by weight of an .alpha.-glycosyl hesperidin powder 
obtained by the method in Example A-3, 1 part by weight of lecithin, 1 
part by weight of lemon oil and 1 part by weight of vanilla oil to 
homogeneity. 
The product is a yellow colored, vitamin P-enriched cream filling which is 
excellent in taste, flavor, melting and biting properties. 
EXAMPLE B-6 
Tablet 
Twenty parts by weight of ascorbic acid was mixed to homogeneity with 13 
parts by weight of crystalline .beta.-maltose, 4 parts by weight of 
cornstarch and 3 parts by weight of an .alpha.-glucosyl hesperidin 
obtained by the method in Example A-2, and the resultant was tabletted 
with a 20 R punch, diameter of 12 mm. 
The product is an easily swallowable vitamin composition containing 
ascorbic acid and .alpha.-glucosyl hesperidin, wherein the ascorbic acid 
is excellently stable. 
EXAMPLE B-7 
Capsule 
Ten parts by weight of calcium acetate monohydrate, 50 parts by weight of 
magnesium L-lactate trihydrate, 57 parts by weight of maltose, 20 parts by 
weight of an .alpha.-glucosyl hesperidin obtained by the method in Example 
A-2, and 12 parts by weight of a .gamma.-cyclodextrin inclusion compound 
containing 20% eicosapentaenoic acid were mixed to homogeneity, and the 
mixture was fed to a granulator, and then encapsulated in gelatine to 
obtain capsules, 150 mg each. 
The product is favorably usable as a high-quality blood cholesterol 
lowering agent, immunopotentiator and skin-refining agent in preventive 
and remedy for susceptive diseases, as well as in foodstuffs directed to 
the maintenance and promotion of health. 
EXAMPLE B-8 
Ointment 
One part by weight of sodium acetate trihydrate, 4 parts by weight of 
DL-calcium lactate and 10 parts by weight of glycerine were mixed to 
homogeneity, and the mixture was added to another mixture of 50 parts by 
weight of vaseline, 10 parts by weight of vegetable wax, 10 parts by 
weight of lanolin, 14.5 parts by weight of sesame oil, 1 part by weight of 
an .alpha.-glycosyl hesperidin obtained by the method in Example A-4 and 
0.5 parts by weight of peppermint oil, and mixed to homogeneity to obtain 
an ointment. 
The product is favorably usable as a high-quality sun-screening, 
skin-refining agent, skin-whitening agent and promoter for healing injury 
and burn. 
EXAMPLE B-9 
Injection 
An .alpha.-glucosyl hesperidin obtained by the method in Example A-2 was 
dissolved in water, and sterilely filtered in usual manner to obtain a 
pyrogen-free solution which was then distributed to 20 ml glass vials to 
give an .alpha.-glucosyl hesperidin content of 50 mg, dried in vacuo and 
sealed to obtain the captioned product. 
The product is intramuscularly and intravenously administrable alone or in 
combination with vitamins and minerals. The product requires no cold 
storage, and exhibits an excellently high solubility in saline when in 
use. 
EXAMPLE B-10 
Injection 
Six parts by weight of sodium chloride, 0.3 parts by weight of potassium 
chloride, 0.2 parts by weight of calcium chloride, 3.1 parts by weight of 
sodium lactate, 45 parts by weight of maltose and 2 parts of an 
.alpha.-glucosyl hesperidin obtained by the method in Example A-2 were 
dissolved in 1,000 parts by weight of water, and sterilely filtered in 
usual manner, after which 250 ml aliquots of the pyrogen-free solution 
were distributed to sterilized plastic vessels to obtain the captioned 
product. 
The product is capable of supplementing, in addition to vitamin P, calorie 
and minerals, therefore is favorably usable in the restoration of health 
during and before suffering from diseases. 
EXAMPLE B-11 
Intubation Nutrient 
Twenty four gram aliquots of a compound consisting of 20 parts by weight of 
crystalline .alpha.-maltose, 1.1 parts by weight of glycine, 0.18 parts by 
weight of sodium glutamate, 1.2 parts by weight of sodium chloride, 1 part 
by weight of citric acid, 0.4 parts by weight of calcium lactate, 0.1 part 
by weight of magnesium carbonate, 0.1 part by weight of an 
.alpha.-glycosyl hesperidin obtained by the method in Example A-3, 0.01 
part by weight of thyamine and 0.01 part by weight of riboflavin were 
packed in laminated aluminum bags, and heat-sealed to obtain the captioned 
product. 
In use, one bag of the product is dissolved in about 300-500 ml of water, 
and the solution is favorably usable as an intubation nutrient directed to 
oral and parenteral administration to the nasal cavity, stomach and 
intestine. 
EXAMPLE B-12 
Bath Liquid 
A bath liquid was obtained by mixing 21 parts of DL-sodium lactate, 8 parts 
by weight of sodium pyruvate, 5 parts by weight of an .alpha.-glycosyl 
hesperidin obtained by the method in Example A-1 and 40 parts by weight of 
ethanol with 26 parts by eight of refined water and appropriate amounts of 
coloring agent and flavoring agent. 
The product is suitable for skin-refining agent and skin-whitening agent, 
which is diluted by 100-10,000-folds in bath water when in use. In this 
case, bath water is replaceable with cleansing liquid, astringent and 
moisture liquid. 
EXAMPLE B-13 
Milky Lotion 
One half part by weight of polyoxyethylene behenyl ether, 1 part by weight 
of polyoxyethylene sorbitol tetra-oleate, 1 part by weight of oil-soluble 
glyceryl monostearate, 0.5 parts by weight of pyruvic acid, 0.5 parts by 
weight of behenyl alcohol, 1 part by weight of avocado oil, 1 part by 
weight of an .alpha.-glycosyl hesperidin obtained by the method in Example 
A-3 and appropriate amounts of vitamin E and antiseptic were dissolved by 
heating in usual manner, and the solution was added with 1 part by weight 
of L-sodium lactate, 5 parts by weight of 1,3-butylene glycol, 0.1 part by 
weight of carboxy-vinyl polymer and 85.3 parts by weight of refined water, 
emulsified with a homogenizer, added with an appropriate amount of 
flavoring agent, and mixed by stirring to obtained the captioned product. 
The product is favorably usable as a high-quality sun-screening, 
skin-refining agent and skin-whitening agent. 
EXAMPLE B-14 
Cosmetic Cream 
Two parts by weight of polyoxyethylene glycol mono-stearate, 5 parts by 
weight of self-emulsifying glycerine monostearate, 2 parts by weight of an 
.alpha.-glucosyl hesperidin powder obtained by the method in Example A-2, 
1 part by weight of liquid paraffin, 10 parts by weight of glyceryl 
trioctanate and an appropriate amount of antiseptic were dissolved in 
usual manner by heating, and the mixture was added with 2 parts by weight 
of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 66 parts by 
weight of refined water, emulsified with a homogenizer, added with an 
appropriate amount of flavoring agent, and mixed by stirring to obtained 
the captioned product. 
The product is favorably usable as a high-quality sunscreen cream, 
skin-refining agent and skin-whitening agent. 
As described above, the .alpha.-glycosyl hesperidin of the invention, 
wherein equimolar or more D-glucose residues are bound to hesperidin via 
the .alpha.-bond, is superior in water-solubility, substantially tasteless 
and odorless, free of toxicity, and readily hydrolyzable in vivo into 
hesperidin and D-glucose to exhibits the physiological activity inherent 
to hesperidin. 
The .alpha.-glycosyl hesperidin is economically superior and easily 
commercializable because it is easily produceable by a biochemical 
procedure wherein a saccharide-transferring enzyme is allowed to act on a 
liquid containing hesperidin together with an .alpha.-glucosyl saccharide. 
Furthermore, we found that hesperidin can be allowed to react at an 
increased initial concentration and this facilitates the formation of 
.alpha.-glycosyl hesperidin at a high concentration. Also was found that 
in the purification of a reaction mixture, the .alpha.-glycosyl hesperidin 
can be recovered by allowing the reaction mixture to contact with a 
synthetic macroporous resin. These render the large-scale production of 
.alpha.-glycosyl hesperidin very easy. 
Since the .alpha.-glycosyl hesperidin thus obtained is characterized in 
that it exhibits a satisfactorily-high water-solubility, light-resistance, 
stability and physiological activity, it is favorably usable as a yellow 
coloring agent, antioxidant, stabilizer, preventive, remedy, uv-absorbent 
and deterioration-preventing agent in foods, beverages, tobaccos, feeds, 
pet foods, pharmaceuticals for susceptive diseases, cosmetics including 
skin-refining agent and skin-whitening agent, and plastics, as well as in 
a highly-safe, natural vitamin P-enriching agent. 
Accordingly, the present invention is extremely significant in food, 
beverage, cosmetic, pharmaceutical and plastic industries in view of the 
establishment of industrial-scale production and practical uses for 
.alpha.-glycosyl hesperidin.