WO1995029928A1

WO1995029928A1 - D-erythroascorbic acid derivatives and process for preparing the same

Info

Publication number: WO1995029928A1
Application number: PCT/KR1995/000043
Authority: WO
Inventors: Sa Ouk Kang; Seung Rock Lee; Woo Jeong Joo
Original assignee: HAH YUNG CHIL
Current assignee: HAH YUNG CHIL
Priority date: 1994-04-29
Filing date: 1995-04-28
Publication date: 1995-11-09
Anticipated expiration: 1996-10-29

Abstract

D-erythroascorbic acid derivatives of structural formula (V) are used as antioxidant having powerful reducing power and additives for medicine, and food, and for treatment for scurvy, diabetes, cancer, etc., wherein R1 represents hydrogen atom or alcoholic group and R2 represents hydrogen atom or methyl group.

Description

D-ERYTHROASCORBIC ACID DERIVATIVES AND PROCESS FOR PREPARING THE SAME

Background of the Invention

Field of the Invention

The present invention relates to novel reductant and antioxidant, and more particularly, to 5-methyl-5-0- (α-D-xylopyranosyl)-D-erythroascorbic acid, 5-0-(α-D- xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-0-(α- D-glucopyranosyl)-D-erythroascorbic acid and 5-0-(α-D- glucopyranosyl)-D-erythroascorbic acid which are oligomeric D-erythroascorbic acid derivatives having high thermal stability and bio-usefulness.

Description of the Related Arts

L-ascorbic acid, typical example of antioxidants, namely, vitamin C is a substance having the function of reduction and antioxidation, which is mainly biosynthesized from reptiles, amphibians, lower birds, vegetables and fruits. Deficiency of this substance causes scurvy and this substance is widely used as a medicine which increases immunological activity, anti- tumour activity and red blood cells activity, and prevents and alleviates symptom of diabetes and colds. The substance has the functions of germination of seeds, growth of plants and growth promotion of roots thereof, tolerance increasing activity against air pollution materials such as ozone, prevention of the falling of fruits and protection of the disease. Deficiency of L- ascorbic acid causes various abnormal symptoms such as growth stagnation and deformity in fishes. For the above reason, L-ascorbic acid is used for various purposes in agriculture and fisheries such as plants cultivation and fishes raising. As component or additive, L-ascorbic acid prevents discoloration of fruits and acidification of foods, and preserves colour and favour of meat, and is also used as reductant in the process for preparing wine. Thus, L-ascorbic acid is used to preserve natural foods so the amount thereof can be a criteria of quality. Optical reaction such as the above substance is widely used in the fields such as polymerization industries, photography and metal industries and it has also been used for various purposes for cosmetics, tobaccos, blood storage, foods preservation and detergents.

However, the above L-ascorbic acid is easily degrades because it is unstable under heat, and neutral or alkaline aqueous solution. The degraded products cause subsidiary effects, and it also has limitations in use of various purposes because of low solubility in lipid. To solve these problems, various derivatives have been produced and their properties have been studied in living bodies. Metal complex compounds, salts, inorganic esters, fatty acid esters and antibiotic derivatives of ascorbic acid have been produced, and D-erythroascorbic acid in living bodies has been discovered in fungi, such as Candida and yeast. However, their properties are similar to those of ascorbic acid. Ascorbic acid and derivatives having monosaccharide derivatives at C-2 or C-3 carbon atom of ascorbic acid were developed. However, these materials lose typical properties of ascorbic acid because their functional diol groups are transformed so that it can not be used as reductant antioxidant.

Summary of the Invention It is an object of the present invention to provide novel materials that can be used for antioxidant having various usage and they have strong reducing power as described above. The materials of the present invention have high stability and high solubility, and they can be easily transferred between cells. Also, they can be used for energy source. Another object of the present invention is to provide D-erythroascorbic acid derivatives, namely, 5-methyl-5-0-(α-D-xylopyranosyl)-D- erythroascorbic acid, 5-0-(α-D-xylopyranosyl)-D- erythroascorbicacid, 5-methy1-5-0-(α-D-glucopyranosyl)- D-erythroascorbic acid and 5-0-(α-D-glucopyranosyl)-D- erythroascorbic acid.

The present invention provides D-erythroascorbic acid derivatives having the following structural formula (V).

HO OH

wherein , represents hydrogen atom or alcoholic group and R₂ represents hydrogen atom or methyl group.

The present invention provides the structural formula (I) of 5-methyl-5-0-(α-D-xylopyranosyl)-D- erythroascorbic acid, the structural formula (II) of 5- O-(α-D-xylopyranosyl)-D-erythroascorbic acid, the structural formula (III) of 5-methyl-5-0-(α-D- glucopyranosyl)-D-erythroascorbic acid and the structural formula (IV) of 5-0-(α-D-glucopyranosyl)-D- erythroascorbic acid.

HO OH

HO OH

HO OH

HO OH The above compounds of the present invention having structural formula (I) , (II) , (III) or (IV) can be used as antioxidant. Further the present invention provides a methods for purification of the compounds having structural formula (I) , (II) , (III) or (IV) containing the step of extracting the above compounds from mushroom. It is preferable to select the above fungi from the groups consisting of shiitake, enekitake and oyster fungus.

The present invention provides pharmaceutical composition comprising the effective amount of one of the compounds having the above structural formula (I) , (II) , (III) , (IV) and (V) and a carrier.

5-methyl-5-0-(α-D-xylopyranosyl)-D-erythroascorbic acid and 5-0-(α-D-xylopyranosyl)-D-erythroascorbic acid are the compounds having α-D-xylose at 5-0H group of D- erythroascorbic acid. 5-methyl-5-0-(α-D- glucopyranosyl)-D-erythroascorbic acid and 5-0-(α-D- glucopyranosyl)-D-erythroascorbic acid are the compounds having α-D-glucose at 5-OH group D-erythroascorbic acid. A methyl group is attached to C-5 in 5-methyl-5-0-(α-D- glucopyranosyl)-D-erythroascorbic acid and 5-methyl-5-0- (α-D-xylopyranosyl)-D-erythroascorbic acid. These compounds have reducing power due to enediol group in D- erythroascorbic acid residue. α-D-xylose and α-D- glucose attached to 5-0H group maintain the reduced state which may stabilizes D-erythroascorbic acid, and their hydroxide groups increase solubility thereof.

5-methyl-5-0-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5-0-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5- methyl-5-0-(α-D-glucopyranosyl)-D-erythroascorbic acid and 5-0-(α-D-glucopyranosyl)-D-erythroascorbic acidhave excellent cellular transfer ability because these derivatives use sugar transfer system existing in the membrane so that they can be used as energy source.

It is also preferred that these novel antioxidants are used as medicine for disease treatment of scurvy, diabetes and cancer etc., and used as food additive like L-ascorbic acid.

Brief Description of the Drawings

The accompanying drawings which are incorporated in and constitute a part of this specification, illustrate one embodiment of the invention and, together with the description, serve to explain the principles of the invention.

Fig. 1A is HPLC chromatogram of extract from the fruit body of Pleurotus ostreatus;

Fig. IB is HPLC chromatogram of extract supplemented with L-ascorbic acid; and

Fig. 1C is HPLC chromatogram of extract supplemented with D-erythroascorbic acid;

Fig. 2 is HPLC chromatogram of extract after oxidation by ascorbate oxidase;

Fig. 3A is HPLC chromatogram after separation of Sep-pak C₁₈ cartridge eluent containing P6 component by Deltapak C₁₈ column;

Fig. 3B is HPLC chromatogram after separation of Deltapak C₁₈ column eluent containing P6 component by Aminex HPX-87H column;

Fig. 3C is HPLC chromatogram after separation of Aminex HPX-87H column eluent containing P6 component by Deltapak C₁₈ column;

Fig. 3D is HPLC chromatogram after separation of Deltapak C₁₈ column eluent containing P4 component by Aminex HPX-87H column;

Fig. 3E is HPLC chromatogram after separation of Aminex HPX-87H column eluent containing P4 component by Deltapak C₁₈ column;

Fig. 3F is HPLC chromatogram after separation of Deltapak C₁₈ column eluent containing P5 component by Aminex HPX-87H column;

Fig. 3G is HPLC chromatogram after separation of Aminex HPX-87H column eluent containing P5 component by Deltapak C₁₈ column;

Fig. 3H is HPLC chromatogram after separation of Deltapak C₁₈ column eluent containing P3 component by Aminex HPX-87H column; and

Fig. 31 is HPLC chromatogram after separation of Aminex HPX-87H column eluent containing P3 component by Deltapak C₁₈ column;

Fig. 4A is negative ion thermospray LC-MS spectrum of P4 component;

Fig. 4B is negative ion thermospray LC-MS spectrum of P6 component;

Fig. 4C is negative ion thermospray LC-MS spectrum of P5 component; and Fig. 4D is negative ion thermospray LC-MS spectrum of P3 component;

Fig. 5A is H¹-NMR spectrum of P6 component dissolved in D₂0;

Fig. 5B is H¹-NMR spectrum of P4 component dissolved in D₂0;

Fig. 5C is H¹-NMR spectrum of P5 component dissolved in D₂0; and

Fig. 5D is H¹-NMR spectrum of P3 component dissolved in D₂0;

Fig. 6A is C¹³-NMR spectrum of P6 component dissolved in D₂0;

Fig. 6B is C¹³-NMR spectrum of P4 component dissolved in D₂0;

Fig. 6C is C¹³-NMR spectrum of P5 component dissolved in D₂0; and

Fig. 6D is C¹³-NMR spectrum of P3 component dissolved in D₂0;

Fig. 7A is gas chromatography of the standard carbohydrate (a)rhamnose (t_R 3.62), (b)arabinose (t_R 5.19), (c)xylose (t_R 6.54), (d)mannose (t_R 8.21), (e)galactose (t_R 8.64)and (f)glucose (t_R 9.40);

Fig. 7B is gas chromatogram of sugar residue of P6 component;

Fig. 7C is gas chromatogram of sugar residue of P4 component;

Fig. 7D is gas chromatogram of sugar residue of P5 component; and

Fig. 7E is gas chromatogram of sugar residue of P3 component;

Fig. 8A is absorption spectra of P6 component at pH 1.8 (a) and pH 7.5 (b) ;

Fig. 8B is absorption spectra of P4 component at pH 1.8 (a) and pH 7.5 (b) ;

Fig. 8C is absorption spectra of P5 component at pH 1.8 (a) and pH 7.5 (b) ; and

Fig. 8D is absorption spectra of P3 component at pH 1.8 (a) and pH 7.5 (b) ;

Fig. 9A is absorption spectra of P6 component before (a) and after (b) oxidation by ascorbate oxidase from Cucurbita species at 25 °C for 1 hour;

Fig. 9B is absorption spectra of P6 component before (a) and after (b) oxidation by ascorbate oxidase from Cucurbita species at 25 °C for 1 hour;

Fig. 9C is absorption spectra of P6 component before (a) and after (b) oxidation by ascorbate oxidase from Cucurbita species at 25 "C for 1 hour; and

Fig. 9D is absorption spectra of P6 component before (a) and after (b) oxidation by ascorbate oxidase from Cucurbita species at 25 °C for 1 hour; Fig. 10A and Fig. 10B are colour change from blue to colourless by reduction of 2,6-dichlorophenol indophenol by P6 component;

Fig. IOC and Fig. 10D are colour change from blue to colourless by reduction of 2,6-dichlorophenol indophenol by P4 component; and

Fig. 10E and Fig. 10F are colour change from blue to colourless by reduction of 2,6-dichlorophenol indophenol by P5 component.

Fig. 10G and Fig. 10H are colour change from blue to colourless by reduction of 2,6-dichlorophenol indophenol by P3 component;

Detailed Description of the Embodiments

The present invention will now be described more specifically with reference to the preferred embodiments described below only by way of example.

I. Detection of D-erythroascorbic acid derivatives

1 g of Pleurotus ostreatus was immersed in 1 ml of 95 % methanol containing 0.1 % dithiothreitol, stored for 1 hour at 20 ^βC, pulverized by glass bead of diameter of 0.5 mm in bead beater for 10 seconds three times, respectively, and centrifuged at 10,000 X g for 30 minutes.

0.5 ml of the supernatant was concentrated to 0.1 ml by speed vacuum concentrator and diluted by adding

0.4 ml of doubly deionized water. 0.9 ml of diluted extract was dissolved in 0.01 ml of doubly deionized water and analyzed by High Performance Liquid Chromatography (HPLC) . The result was shown in Fig. 1A. 0.09 ml of above diluted extract was dissolved in 0.01 ml of doubly deionized water, added to 4 μg/ml of L- ascorbic acid aqueous solution and 4 μg/ml of D- erythroascorbic acid, respectively, and analyzed by HPLC. The results were shown in Fig. IB and Fig. 1C. 0.01 ml (2 units) of ascorbate oxidase aqueous solution from Cucurbita was added to 0.09 ml of the above diluted extract, reacted at 25 °C for 40 minutes and analyzed by HPLC. The result was shown in Fig. 2.

For HPLC analysis was used a HPLC system equipped with two ODS hypersil columns (Hewlett Packard, 100 X 4.6 mm) , where the mobile phase was 0.1 % trifluoroacetic acid at the flow rate of 0.8 ml/min. The detector was Waters 460 electrochemical detector and applied voltage was 800 mV. Peaks of from PI to P6 components were exhibited in Fig. 1A and the peaks of PI and P2 components indicate L-ascorbic acid and D- erythroascorbic acid, respectively. And properties of PI to P6 components were similar to those of L-ascorbic acid.

II. Isolation of D-erythroascorbic acid derivatives

1. Preparation of extract

0.5 ml of 95 % aqueous methanol solution containing 0.1 % dithiothreitol was added to 1 g of Pleurotus ostreatus, proportionally, extracted for 1 hour at - 40 °C, pulverized by mixer three times for 30 minutes, respectively, and extracted with cotton cloth followed by centrifugation at 10,000 X g at 4 °C for 15 minutes.

The above centrifugated supernatant was passed through a column containing Dowex 50-X8 resin to remove metal ions. Metal ions and methanol were removed by evaporation (rotary velocity: 120 rpm, pressure; -76 cmHg, temperature: 38 ^βC) , and then freezed by ultra-low temperature freezer (Revco Co.) and dried by dry up freezer-dryer (Dura Co.)

2. Isolation by HPLC equipped Deltapak C₁₈ column

The dried samples were dissolved in 0.1 % trifluroacetic acid aqueous solution and centrifugated at 10,000 X g at 4 °C for 30 minutes. The supernatant were equilibrated with triply deionized water and pigments were removed by passing Sep-pak C₁₈ cartridge. The fractions containing reductone of the samples passed through Sep-pak C₁₈ cartridge were combined and injected on Deltapak C₁₈ HPLC column (Water associate Co. 15 μm, 30 mm X 30 cm) equilibrated with 0.1 % trifluoroacetic acid aqueous solution, and eluted with 0.1 % trifluoroacetic acid aqueous solution at the flow rate of 20 ml/min by chromatography system (Waters Delta Prep 4000) equipped with detector (Waters 484 model) , and assayed by measuring absorbance at 245 nm. The result was shown in Fig. 3A. Various peaks were shown in Fig. 3A and four peaks containing reductone were P3, P4, P5, and P6 components.

3. Isolation by HPLC equipped with Aminex HPX-87H column

The P6, P4, P5, P3 components of components separated by Deltapak C₁₈ were injected on HPX-87H HPLC column (Bio-rad Co. 300 X 7.8 mm) with injector (Waters U6K) and eluted with 0.005 M H₂S0₄ of the flow rate of 0.7 ml/min by Waters Delta Prep 4000 chromatography system. Absorbance at 245 nm was measured by detector (Waters 440 model) . The results were shown in Fig. 3B, Fig. 3D, Fig. 3F, and Fig. 3H.

Fig. 3B, Fig. 3D, Fig. 3F, and Fig. 3H show main peaks as P6, P4, P5 and P3. H₂S0₄ was removed by Deltak C₁₈ column again. The results were shown in Fig. 3C, Fig. 3E, Fig 3G, and Fig 31. It was confirmed that each compound separated by HPLC equipped Deltapak C₁₈ column was a pure compound by Fig. 3C, Fig. 3E, Fig. 3G and Fig. 31.

III. Structure analysis of D-erythroascorbic acid derivatives

1. Element analysis of D-erythroascorbic acid derivative

The amounts of carbon, hydrogen, oxygen, nitrogen and sulphur of P4 component sample isolated in the above process II were determined by elemental analyzer (Carlo Erba Co. EA1108) three times at the combustion temperature of 1,000 °C. These results are presented in the following Table 1.

Table 1

Element Found(%) Calculated for C₁₀H₁₄O₉(%)

carbon 41.38 ± 0.15 43.17

hydrogen 5.16 ± 0.10 5.07

oxygen 50.99 ± 0.24 51.76

nitrogen 0 0

sulphur 0 0

sum 97.53 ± 0.49 100.00

2. Mass analysis

Molecular mass was measured by measuring negative ion thermospray LC-MS of these eluted components using mass spectrometer (VG Quattro quadrupole mass spectrometer) with ion source temperature of 250 °C and probe temperature of 200 °C. The mobile phase was 0.1 M ammonium acetate containing 30 % methanol at the flow rate of 0.3 ml/min. The results were shown in Fig. 4A, Fig. 4B, Fig. 4C, and Fig. 4D.

As shown in Fig. 4A of the spectrum of negative ion thermospray LC-MS, the value of m/z indicated (M-H)^" was 277, 149, 145, etc. This established the molecular mass of P4 component was 278. As shown in Fig. 4B of the spectrum of negative ion thermospray LC-MS, the value of m/z indicating (M-H)^" was 141, 149, 291, etc. This established the molecular weight of P6 component was 292.

As shown in Fig. 4C of the spectrum of negative ion thermospray LC-MS of P5 component, the value of m/z indicating (M-H)^" was 141, 179, 321, etc. This indicated that the molecular weight of P5 component was 322. And as shown in Fig. 4D of the spectrum of negative ion thermospray LC-MS, the value of m/z indicating (M-H)^" was 145, 173, 237, , 307, etc. This indicated that the molecular weight of P3 component was 308.

3. Structure analysis by H¹-NMR

Each of P6, P4, P5 and P3 components as dissolved in 0.5 ml of D₂0 and analyzed by H¹-NMR (Bruker Co. AMX- 500 spectrophotometer) in the condition of temperature of 303 K, resonance frequency of 500 MHz, accumulation number of 4, acquisition time of 1.57 seconds, and pulse width of 48.0 seconds. The results were shown in Fig. 5A, Fig. 5B, Fig. 5C, and Fig. 5D.

Fig. 5A suggested that the signals at 4.91 ppm, 4.18 pp , and 1.39 ppm were assigned to be protons of H- 4, H-5, and CH₃-5, which were originated from 5-methyl- D-erythroascorbic acid moiety. The signals at 4.97 ppm, 3.65 ppm, 3.57 ppm, 3.50 ppm, 3.47 ppm, and 3.40 ppm were originated from α-D-xylopyranose residue. And the signal at 4.97 ppm (J, ₂ = 3.58) indicated that the structure of sugar was α form and that glycosidic bond was formed between hydroxyl groups of C-1 of sugar residue and C-5 position of D-erythroascorbic acid. Fig. 5B suggested that the signals at 5.06 ppm, 4.03 ppm, and 3.95 ppm were assigned to be protons of H- 4, H-5e, and H-5a and J_5ef5a = 12_a08 was germinal coupling constant which was originated from 5-methyl-D- erythroascorbic acid moiety. The signals at 4.89 ppm, 3.67 ppm, 3.58 ppm, 3.55 ppm, 3.50 ppm, and 3.45 ppm were originated from α-D-xylopyranose residue. And the signal at 4.89 ppm (J, ₂ = 3.58) indicated that the structure of sugar was α form and that glycosidic bond was formed between hydroxyl groups of C-1 of sugar residue and C-5 position of D-erythroascorbic acid.

Fig. 5C suggested that the signals at 4.76 ppm, 4.07 ppm, and 1.23 ppm were assigned to be protons of H- 4, H-5, and CH₃-5, and were originated from 5-methyl-D- erythroascorbic acid moiety. The signals at 4.85 ppm, 3.67 ppm, 3.61 ppm, 3.37 ppm, 3.36 ppm, 3.34 ppm, and 3.29 ppm were originated from α-D-glucopyranose residue. And the signal at 4.85 ppm (J, ₂ = 3.70) indicated that the structure of sugar was α form and that glycosidic bond was formed between hydroxyl groups of C-1 of sugar residue and C-5 position of D-erythroascorbic acid.

Fig. 5D suggested that the signals at 4.91 ppm, 3.87 ppm, and 3.81 ppm were assigned to be protons of H- 4, H-5e, H-5a, and germinal coupling constant of J_{5e 5a} = 1.86 was originated from D-erythroascorbic acid moiety. The signals at 4.77 ppm, 3.66 ppm, 3.60 ppm, 3.46 ppm, 3.36 ppm, and 3.25 ppm were originated from α- D-glucopyranose residue. And the signal at 4.77 ppm (J₁₂ = 3.70) indicated that the structure of sugar was α form and that glycosidic bond was formed between hydroxyl groups of C-1 of sugar residue and C-5 position of D-erythroascorbic acid.

Consequently, P3 component of the above structure was determined as 5-0-(α-D-glucopyranosyl)-D- erythroascorbic acid.

4. Structure analysis by C¹³-NMR

Each of P6, P4, P5, P3 components was dissolved in 0.5 ml of D₂0 and analyzed by C¹³-NMR (Bruker Co. AMX-500 spectrophotometer) in the condition of temperature of 303 K, resonance frequency of 400 MHz, acquisition time of 1.2452 sec, pulse width of 19.0 μsec, sweep width of 26,300 Hz, and operation magnetic frequency of 126 MHz. The results were shown in Fig. 6A, Fig. 6B, Fig. 6C, and Fig. 6D.

Fig. 6A shows that the No. of carbon atoms is 11 and the signals at 173.790, 117.759, 156.563, 79.245, 70.000 and 15.504 ppm represent the signals from carbons of 5-methyl-D-erythroascorbic acid and the signals at 96.499, 73.337, 71.389, 69.498, and 60.215 ppm represent the signals from carbons of the α-D-xylopyranose residue.

The results establish that P6 component is 5- ethy1-5-0-(α-D-xylopyranosyl)-D-erythroascorbicacidas below.

HO OH

Fig. 6B shows that the No. of carbon atoms is 10 and the signals at 173.582, 118.213, 155.572, 76.279, and ₆5.₃57 ppm represent the signals from carbons of D- erythroascorbic acid residue and the signals at 98.964, 7₁.703, 73.449, 69.707, and 61.765 ppm represent the signals of carbons of α-D-xylopyranose residue. The results established that P4 component is 5-0-(α-D- xylopyranosyl)-D-erythroascorbic acid as below.

HO OH

Fig. 6C shows that the No. of carbon atoms is 12 and the signals at 173.743, 117.711, 156.501, 79.235, 69.478, and 15.316 ppm represent the signals from carbons of 5-methyl-D-erythroascorbic acid and the signals at 96.136, 73.059, 72.299, 71,344, 69.168, and 60.215 ppm represent the signals from carbons of α-D- glucopyranose residue. The results established that P5 component is 5-methyl-5-0-(α-D-glucopyranosyl)-D- erythroascorbic acid as below.

Fig. 6D shows that the No. of carbon atoms of P3 component is 11 and the signals indicated that P3 component is 5-0-(α-D-glucopyranosyl)-D-erythroascorbic acid. The results establish that P3 component is 5-0- (α-D-glucopyranosyl)-D-erythroascorbic acid as below.

HO OH

5. Gas chromatography

The isolated P6, P4, P5, P3 components were dissolved in each 2 N trifluoroacetic acid solution sealed with nitrogen, respectively, and incubated at 121 "C for 3 hours to hydrolyze. The hydrolyzed components were concentrated under reduced pressure, and 10 mg/ml of NaBH₄ was added, respectively and reduced for 12 hours at 40 "C. To the reduced components were added three times volume of methanol, concentrated under the reduced pressure at 40 °C, and 0.5 ml of acetic anhydride was added, respectively and acetylated for 3 hours at 121 "C.

Gas chromatography (Hewlett Packard Co. , HP 5890) was performed to analyze samples. Each of 2 μl of the samples was injected in SP - 2380 column (Supeco Co. , 30 m, 0.2 μ ) by injector at 210 °C, where helium was used as a carrier gas at a flow rate of 9 - 9.2 ml/min to separate sugars, and gas chromatography was performed on a HP 5890 gas chromatography (Hewlett Packard) equipped with a FID (Flame Ionization Detector) . Glucose, galactose, mannose, rhamnose, xylose and arabinose were used as standards (Fig.7A).

Chromatogram of P6 component was drawn in Fig. 7B. As shown in Fig.7A and Fig.7B, the retention time of sugar moiety in P6 component is equal to that of xylose. Therefore it was confirmed that P6 component had xylose. Fig.7C exhibited chromatograms of the mixtures of xylose and the samples, which reaffirmed that P6 component had xylose.

Chromatogram of P4 component was drawn in Fig. 7D. As shown in Fig.7A and Fig.7D, the retention time of sugar moiety in P4 component is equal to that of xylose. Therefore it was confirmed that P6 component has xylose. Fig.7E exhibited chromatograms of the mixtures of xylose and the samples, which reaffirmed that P4 component had xylose.

Chromatogram of P5 component was drawn in Fig. 7F. As shown in Fig.7A and Fig.7F, the retention time of sugar moiety in P5 component is equal to that of glucose. Therefore it was confirmed that P5 component had glucose. Fig.7G exhibited chromatograms of the mixtures of glucose and the samples, which reaffirmed that P5 component had glucose.

Chromatogram of P3 component was drawn in Fig. 7H.

As shown in Fig.7A and Fig.7H, the retention time of sugar moiety in P3 component is equal to that of glucose. Therefore it was confirmed that P3 component had glucose.

IV. Properties of D-erythroascorbic acid derivatives

1. Absorption spectrum Each of the isolated P6, P4, P5, and P3 components was dissolved in 0.1 % trifluoroacetic acid of pH 1.8 and 0.1 M phosphate buffer solution of pH 7.5 respectively, and the absorbances from 400 nm to 200 nm with absorption spectrophotometer (Shimadzu Co. Model 265) were obtained and the results were shown in Fig. 8A, Fig. 8B, Fig. 8C, and Fig. 8D.

As shown in Fig. 8A, P6 component showed the maximum absorbance at 243.2 nm and 265.8 nm in the solutions of pH 1.8 and pH 7.5, respectively and isosbestic point was at 249.0 nm.

As shown in Fig. 8B, P4 component showed the maximum absorbance at 242.0 nm and 264.2 nm in the solutions of pH 1.8 and pH 7.5, respectively and isosbestic point was at 248.0 nm.

As shown in Fig. 8C, P5 component showed the maximum absorbance at 243.2 nm and 265.8 nm in the solutions of pH 1.8 and pH 7.5 respectively and isosbestic point was at 249.0 nm.

As shown in Fig. 8C, P3 component showed the maximum absorbance at 244.4 nm and 264.6 nm in the solutions of pH 1.8 and pH 7.5 and isosbestic point was at 247.5 nm.

2. Reactivity toward ascorbate oxidase

Each of the isolated P6, P4, P5, and P3 components was dissolved in 0.1 M phosphate buffer solution, respectively and 5 units of ascorbate oxidase from Cucurdita species was added and reacted at 25 °C for 1 hour. Fig. 9A, Fig. 9B, Fig. 9C and Fig. 9D showed the change of absorbance to time in the range of wavelength of from 400 nm to 200 nm. Fig. 9A, Fig. 9B, Fig. 9C and Fig. 9D indicated that P6, P4, P5, and P3 components were oxidized by ascorbate oxidase, so the enediol as a functional group was conserved in the P6, P4, P5, and P3 components like D-erythroascorbic acid.

3. Reduction of 2,6-dichlorophenol indophenol

Each of the isolated P6, P4, P5, and P3 components was dissolved in 0.1 M phosphate buffer solution, respectively and reacted with 2,6-dichlorophenol indophenol (DCPIP) aqueous solution to examine decolorization and the results were shown in Fig. 10A to Fig. 10H. They established that all of P6, P4, P5, and P3 components had the ability to reduce 2,6- dichlorophenol indophenol.

As known in above examples, novel antioxidants, D- erythroascorbic acid derivatives of this invention, namely 5-methyl-5-0- (α-D-xylopyranosyl) -D- erythroascorbic acid, 5-0-(α-D-xylopyranosyl)-D- erythroascorbicacid, 5-methyl-5-0-(α-D-glucopyranosyl)- D-erythroascorbic acid and 5-0-(α-D-glucopyranosyl)-D- erythroascorbic acid have high reducing activity. Therefore they can be used as additive for medicine or food and for treatment of scurvy, diabetes, cancer, etc.

Also, they can be used as substituent or complement to ascorbic acid for the industries based on polymer synthesis and photosynthesis techniques, and manufacturing process for cosmetics, detergents, and foods.

Claims

What is claimed is:

1. A 5-methyl-5-0-(α-D-xylopyranosyl) -D- erythroascorbic acid represented by the following structural formula (I) .

HO OH

2. A use of said compound of claim 1 as antioxidatant or reductant.

3. A process for preparing said compound of claim 1, comprising the step of extracting from fungi.

4. A pharmaceutical composition comprising the effective amount of said compound of claim 1 and a carrier.

5. A 5-0-(α-D-xylopyranosyl)-D-erythroascorbic acid represented by the following structural formula (II) .

HO OH

6. A use of said compound of claim 5 as antioxidatant or reductant.

7. A process for preparing said compound claim 5 comprising the step of extracting from fungi.

8. A pharmaceutical composition comprising the effective amount of said compound of claim 5 and a carrier.

9. A 5-methyl-5-0- (α-D-glucopyranosyl ) -D- erythroascorbic acid represented by the following structural formula (III) .

10. A use of said compound of claim 9 as antioxidatant and reductant.

11. A process for preparing said compound of claim 9 comprising the step of extracting from fungi.

12. A pharmaceutical composition comprising the effective amount of said compound of claim 9 and a carrier.

13. A 5-0-(α-D-glucopyranosyl)-D-erythroascorbic acid represented by the following structural formula (IV).

14. A use of said compound of claim 13 as antioxidatant and reductant.

15. A process for preparing said compound of claim 13 comprising the step of extracting from fungi and.

16. A pharmaceutical comprising the effective amount of composition said compound of claim 13 and a carrier.

17. A D-erythroascorbic acid derivatives represented by the following structural formula (V)

HO OH

wherein R₁ represents hydrogen atom or alcoholic group and R₂ represents hydrogen atom or methyl group.

18. A use of said compound of claim 17 as antioxidatant and reductant.

19. A process for preparing said compound of claim

17 comprising the step of extracting from fungi.

20. A pharmaceutical composition comprising the effective amount of said compound of claim 17 and a carrier.