TWI353847B - Sulfur-containing proanthocyanidin oligomer compos - Google Patents

Description

1353847 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a sulfur-containing proanthocyanidin oligomer and a composition thereof, and a method for producing the same, and a use thereof, more specifically, in a plant a method for producing a sulfur-containing proanthocyanidin oligomer having a low molecular weight of proanthocyanidins to such an extent that it is easily absorbed by the intestinal tube of the living body; and treating and preventing the sulfur-containing proanthocyanidin oligomer as an active ingredient The production of active oxygen species and the like are health foods and pharmaceutical compositions such as various lifestyle-related diseases and brain diseases. [Prior Art] Hyperlipemia, hypercholesterolemia, hypertension due to changes in dietary life, excessive intake of fat, changes in the environment, increased exposure to ultraviolet light due to destruction of the ozone layer, and increased environmental pollutants. The so-called lifestyle-related diseases such as diabetes and cancer have increased year by year, and patients with brain diseases such as allergies and dementia have also increased year by year. With the formation of an aging society, diseases such as dementia and Alzheimer's syndrome are increasing. The main cause of these diseases is thought to be related to the active oxygen species produced in the body (for example, Bioorgnic & Medicinal Chemistry, 10 (2002), 2495-2509). However, the complete inhibition or control technology for active oxygen species has not been developed, so effective prevention and treatment techniques for lifestyle-related diseases or brain diseases have not yet been fully established. Natural substances which are physiologically active in plants, especially phenolic compounds, have received particular attention in recent years. Phenols are usually found in tea, vegetables, fruits, and herbs. They have been ingested as foods or hobbies for many years. 6 1353847 is a substance that has no side effects and is considered to have therapeutic and preventive effects. Phenolic compounds are secondary metabolites of plants. They are widespread and abundant in the plant kingdom. They are known for their various physiological activities. In ancient times, they were widely used in the fields of pharmacy and phytochemistry. Attention. For example, polyphenols in tea, especially catechin, have antibacterial, antiviral, anti-mutation, anti-oxidation, inhibit blood pressure, lower blood cholesterol, anti-allergic, anti-intestinal microbes and deodorization. A wide range of physiological activities are known. Polyphenols The most widely contained polyphenols in plants are proanthocyanidins. In order for the original pigment to exhibit various physiological activities as described above, the proanthocyanidin compound must be absorbed into the living body via the intestinal tract. However, the molecular weight of proanthocyanidins is still on the scale of thousands or even tens of thousands. Such a substance having a large molecular weight is extremely difficult to be absorbed through the intestinal tract, and is not absorbed and utilized by the living body even if it is ingested. The present inventors have found that a polyphenol extracted by Qiaomai has a patent application (Japanese Unexamined Patent Publication No. Hei-218786), whose active ingredient is a proanthocyanidin polymer. However, its absorption by the body is still difficult to achieve, and it is still waiting for research and development to improve. SUMMARY OF THE INVENTION In the present invention, a proanthocyanidin compound which is difficult to be absorbed into a living body via a gut tube is changed into a form which is easily absorbed, and a plurality of physiologically active effects of the proanthocyanidin compound are sufficiently exerted to enhance the body. Antioxidant activity for the purpose of providing therapeutic and preventive medicines and health foods for use in living habits or intestinal diseases of reactive oxygen species. The present inventors have found that a low molecular weight sulfur-containing proanthocyanidin oligomer obtained by reacting a plant containing proanthocyanidins or a mixture thereof with an SH group-containing compound is easily absorbed by a living body through an intestinal tube, and is orally ingested. The present invention has been completed on the basis of the fact that various physiological activities can be exhibited. That is, the present invention relates to the following sulfur-containing proanthocyanidin oligomers and compositions thereof useful as pharmaceutical and health food compositions, and methods for their manufacture. 1. A reaction product obtained by reacting a plant containing a proanthocyanidin or an extract thereof with an SH group-containing compound, and concentrating and drying the sulfur-containing sulfonium pigment oligomer as a main component. 2. The sulfur-containing proanthocyanidin oligomer composition according to the above 1, wherein the oligomer is a 2 to 5 oligomer of proanthocyanidin. 3. The sulfur-containing proanthocyanidin oligomer composition according to the above 1, wherein the plant containing the proanthocyanidin is at least one selected from the group consisting of fruits and vegetables, teas, herbs, and wood and bark. 4. The sulfur-containing proanthocyanidin oligomer composition according to the above 1, wherein the SH group-containing compound is selected from the group consisting of cysteine, cystine, glutathione, an amino group containing a peptide, and the like. At least one of the salts. 5. The composition according to any one of the above 1 to 4, which is a pharmaceutical composition for the treatment and/or prevention of lifestyle-related diseases. 6. The composition according to any one of the above 4, which is a pharmaceutical composition for preventing aging. 7. The composition according to any one of the above 1 to 4, which is a health food composition for improving and/or preventing lifestyle-related diseases. 8. The composition according to any one of the above 1 to 4, which is a health food composition for aging prevention 1353847" 9-a sulfur-containing proanthocyanidin oligomer which is a plant containing proanthocyanidins The extract containing the sulfur-containing proanthocyanidin oligomer obtained by reacting the extract with the SH group-containing compound is isolated. 10. The sulfur-containing proanthocyanidin oligomer described in the above 9, wherein the oligomer is a 2 to 5 oligomer of proanthocyanidin. 11. A method for preparing a sulfur-containing proanthocyanidin oligomer composition, comprising: reacting a plant containing proanthocyanidins or an extract thereof with an SH-containing compound under acidic conditions, and then concentrating and drying the reaction solution deal with. 12. A method for producing a sulfur-containing proanthocyanidin oligomer, comprising: reacting a plant containing proanthocyanidins or an extract thereof with an SH-containing compound under acidic conditions, and then concentrating and separating the reaction solution . 13. The method according to the above 11 or 12, wherein the proanthocyanidin-containing plant is at least one selected from the group consisting of fruits and vegetables, teas, grass plants, and wood and bark. 14. The method according to the above 11 or 12, wherein the ligand-containing compound is at least one selected from the group consisting of cysteine, cystine, glutathione, a peptide containing a peptide, and a salt thereof. 15. The method of the above 11 or 12 wherein the acidic condition is the use of a mineral acid, an organic acid or both. 16. The method according to the above 15, wherein the acidic condition is at least one selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, ascorbic acid, and malic acid. 9 1353847 17. A proanthocyanidin compound of the following formula (4):

0H

(4) 18—a proanthocyanidin compound represented by the following formula (5)··

19. A proanthocyanidin compound represented by the following formula (6): 10 1353847

OH

20. A proanthocyanidin compound of the following formula (7):

21. A proanthocyanidin compound of the following formula (8):

OH

11 1353847 DETAILED DESCRIPTION OF THE INVENTION The sulfur-containing oligomer of the present invention is usually a 2 to 5 polymer of proanthocyanidin, and has a molecular weight of usually 1500 or less. 0 This oligomer, that is, a sulfur-containing proanthocyanidin oligomer is preferred. It is prepared by reacting a plant containing proanthocyanidins or an extract thereof with an SH-containing compound under acidic conditions. 5N。 The acid is preferably selected from hydrochloric acid, sulfuric acid, nitric acid and other inorganic acids, or at least one of acetic acid, citric acid, ascorbic acid, malic acid, a concentration of 0. 1N~1. 0 or so, preferably 0. 5N. The proanthocyanidin referred to in the present specification refers to a catechin polymer, specifically, a catechin, an epicatechin, an epigallocatechin, and a table. Epigallocatechin gallate, gallo catechin, gallo catechin gallate, or a component of these . Plants containing proanthocyanidins may, for example, persimmons, pears, grapes, strawberries, bananas, avocados, peaches, lotus roots, kiwi, and other fruits, green tea, black tea, oolong tea, and the like, as well as herbs, wood, and pine. A variety of plants such as bark. These plants containing proanthocyanidins or their extracts (including mash) are suitable. The SH-containing compound used in the present invention may, for example, be cysteine, cystine, glutathione, SH-containing peptide acid or the like, and other natural substances such as succulent onions and garlic. Although mercaptans can be used, since the sulfur-containing proanthocyanidin oligomers of the present invention are used as foods and pharmaceutical compositions, SH-based compounds permitted for foods and medical products are preferably used. 12 1353847 The reaction of a plant containing proanthocyanidins or an extract thereof with an SH-containing compound is carried out at a temperature of from room temperature to 8 (TC, preferably 40 to 60 t for several hours or even one week, preferably 24 to 48). The reaction solvent may be one or a mixture of two or more of water 'methanol, ethanol, etc.' However, water or ethanol is preferably used in consideration of the use thereof for foods and pharmaceuticals. After the reaction, the residue is filtered off, and the filtrate is concentrated and purified by a usual method. That is, the purification of the concentrated liquid can be treated by a membrane treatment method (external filtration or reverse soaking, etc.) or by an absorbent. The absorbent may be a styrene-diethylene absorbing agent, a thiol-based absorbing agent, or a hydrophilic vinyl system. Polymer, modified polymeric glucose gel, polypropylene guanamine gel, reverse phase ruthenium gel, ion exchange resin, etc. When using these adsorbents, they will react with SH-containing compounds in the adsorption zone and have low molecular weight. The polyphenol compound (sulfur-containing proanthocyanidin oligomer) is obtained by dissolving from the adsorption zone using aqueous ethanol, ethanol or acetone to obtain various molecular weight components.

The sulfur-containing proanthocyanidin oligomer thus obtained was confirmed to be a 2-mer to a 5-mer of the proanthocyanidins represented by the following formula (9) by the cis phase HPLC and NMR measurements. 13 1353847 R2

Where: n is an integer of 0 or 1 to 3, R1 is hydrogen or is represented by the following formula

OH

A galloyl group of OH, R2 and R3 each independently represent a hydrogen or a hydroxide group, and R4 is a cysteine, cystine, glutathione, or SH group-containing peptide residue. Since the sulfur-containing proanthocyanidin oligomer is a low molecular substance having a molecular weight of 1,500 or less, it is easily absorbed into the living body through the intestinal tube. It is shown by the following test examples: good DPPH radical elimination, SOD-like activity enhancement, P450 ester peroxidation inhibition, protection against FNT oxidative stress' induced by gelatinized cellulose (amyloid) The oxidized 14 1353847 cells have a neuronal protective effect and a preventive effect on streptozotocin (STS)-induced diabetes, and have higher antioxidant energy than other polyphenol materials. In addition, the monitoring test for the antioxidant index of humans has also been obtained as a data based on the antioxidant effect. Therefore, the composition containing the sulfur-containing proanthocyanidin oligomer of the present invention as an active ingredient not only has an inhibitory effect on the production of lipid peroxidase in the living body but also has an effect on a disease caused by an oxidative barrier of active oxygen, and therefore causes a cause It has a preventive effect on the barriers and aging of various organs produced by lipid peroxide or active oxygen, and has an effect on the treatment and prevention of various diseases caused by it. In addition, inhibition, prevention, and treatment of brain dysfunction such as dysfunction caused by aging of the brain may be effective. At the same time, due to the improvement of brain function, the improvement of learning ability, the reduction of anxiety, and the resolution of insomnia can be expected. Therefore, the composition containing the sulfur-containing proanthocyanidin oligomer of the present invention as an active ingredient can be used as a pharmaceutical composition and a health food composition. The composition of the present invention can be used with ease because it is completely non-toxic. The dosage can be administered by π or non- σ 'oral use. Although the dosage is different depending on age, body weight, symptoms, treatment purpose and administration method, it is usually used once a day for adults. 〇〇mg range. When the composition of the present invention is orally administered, the composition of the present invention is used as a tablet, a capsule, a granule, a powder, and the like, and is prepared as an injection or a coating agent. For the preparation of granules, it is necessary to use appropriate supplements. Additives, such as powders, pastes, flavors, pigments, flavors, etc. [Embodiment] 15 1353847 Hereinafter, the sulfur-containing original flower of the present invention will be specifically described by way of examples and test examples. The pigment oligomer composition, but the invention is not limited by the scope of the examples. Example 1: Production of sulphur-containing proanthocyanidin oligomers from vines (1) Extraction of grape seed phenols 8 kg of dried grape seeds were extracted at room temperature using 80% sterol (30 liters) After the residue was filtered off, the filtrate was concentrated under reduced pressure, and then the concentrate was purified under the following conditions. [Refining conditions] The entire amount of the concentrate is placed in a crucible having a diameter of 200 faces and a height of 30 cm (about 25 liters) filled with DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation) at 1 liter (L). Wash the water. Subsequently, it was eluted with 50 liters of decyl alcohol. The precipitate was concentrated under reduced pressure and lyophilized to obtain 456 g of a powdery composition. (2) Low Molecular Weight Reaction with Cysteine 400 g of polyphenols obtained from the above (1), L-cysteine (manufactured by Wako Pure Chemical Industries, Ltd.) 400 g, ascorbic acid (Pure Chemical Co., Ltd.) 4 g of citric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was mixed with 8 kg of water and 4 liters of water, and reacted at 4 ° C for 48 hours. The reaction solution was introduced into a column filled with a carrier DIAION HHP-20 (manufactured by Mitsubishi Chemical Corporation) with a diameter of 200 ga ga 1 (volume about 25 liters), and the non-adsorbed portion was washed with water of ι liters. 4 〇% ethanol 5 liters of liters was recovered for the dissolved fraction, then concentrated under reduced pressure and lyophilized to obtain 408 g of reddish brown powder which was easily soluble in water, methanol and ethanol. Introducing this powder into a filled carrier 16 1353847

Column of Sephadex LH-20 (manufactured by Parmacia) (5 直径 diameter, 500 高 high, volume 1 liter) in 'distilled with ethanol/water mixture, and according to the method of middle-aged people (in 〇1^ 111, 卩11 has 1*111, publication, 34(2), 633~642 (1986)) The oligomers of the fraction were analyzed and the results were as follows: 19% 21% 11% 16% cysteine binding Epoxy catechin monomer cysteine binding epoxidized catechin dimer cysteine binding epoxidized catechin trimer cysteine binding epoxidized catechin 4~6 polymer ( 3) Purification and separation of cysteine-bound epicatechin monomer by column chromatography using Sephadex LH-20 carrier (colloid) (using water-sterol-acetone as mobile phase) The ketone-acetic acid test solution is sprayed to give a peach-brown color point as a heart essence: the cysteine-binding epicatechin monomer portion is divided into the following compounds (1) to (3).

OH

17 1353847

OH

OH

Determination of the molecular ion peak in the hr-fab-ms (high decomposition high-speed atomic impact mass spectrometry) of the compound of formula (1) (compound 1)

[M+H]+(m/z) is 410.0925, which is equivalent to an error of 3.6 ppm (within 10 ppm) corresponding to the δ-time value 410.0910 of the molecular formula C18H2〇〇8NS, so the molecular formula of the compound 1 is estimated to be Cl8Hl908NS, and has a sulfur atom of L-cysteine bound to the chemical structure of catechin or epicatechin. In addition, via the compound! The ih_nmR (hydrogen nuclear magnetic resonance spectrum 'refer to Table 1) was determined to be 5 pl〇t〇n signal on all aromatic rings common to catechins or phenoxysamines. 〇9(br.s), (5 4.〇7(m), and 5.86 (d, J=2Hz) confirm the signal group with an oxygen atom or a sulfur atom at the root of one proton. The former two indicate Compound 1 has a stereo-coordination of epicatechin, while the proton signal of 5 3.86 (d, 18 1353847 J=2 Hz) belongs to the 4α coordination, and the sulfur atom is coordinated to the signal corresponding to the thiolysis product. In addition, it is confirmed that there is ABX at 54.13 (1H, dd, J=9, 4 Hz), <5 2.95 (1H, dd, J=15 '9 Hz) and <5 3.43 (1H, dd, J=15, 4 Hz). A signal group of the type, which indicates a partial structure in which a sulfhydryl group of a cysteine has an amidino group. Based on these data, it is presumed that the chemical structure of the compound 1 is 4yS - (SL cysteine) represented by the formula (1) Amino)-(-)-epicatechin. In the 13C-NMR (carbon-13 nuclear magnetic resonance spectrum of the compound 1, see Table 2) (DEPT method), it was confirmed that the carbon number was included and 18 Carbon signal also supports this chemical structure 1H-NMR (see Table 1) of the compound of the formula (2) (refer to Table 1) was determined at 5 4.86 (1H, d, J = 9, 6 Hz), <5 4.22 (1H, dd, J = 9.6, 4.31) ^) and (5 4.23(1^1,0,1=4,31^) confirm the signal group with an oxygen atom or a sulfur atom at the root of a proton. The first two belong to the catechin, 3 /5 coordination, while d 4·23 (1Η, d, J=4, 3Hz) belongs to the 4 Cl! coordination, the sulfur atom is equivalent to the signal of the thiolysis product. Therefore, compound 1 and compound 2 are only 3 The stereotactic coordination is different, and it is presumed that the compound has the structure of the formula (2), 4/3 - (SL-cysteinyl)-( + )-catechin. The compound of the formula (3) (compound 3 The measurement by HR-FAB-MS shows that the molecular ion peak is at [M+H]+(m/z) at 426.0844, which is equivalent to the calculated value of the molecular formula C18H2G09NS corresponding to one more oxygen atom than Compound 1 · 426.0859 with an error of 3.5 The ppm is consistent. It is also known from the measurement of W-NMR (refer to Table 1) that in addition to the equivalent signal of hydrogen at the 2' and 6' positions in the aromatic ring field, 5 6_57 (2H, s) 1 Correspondence, similar. 19 1353847 Therefore, the 13C-NM shown in Table 2 is combined. The signal of R is assigned, and it is presumed that the compound 3 is /, and has the structure of the formula (3) of the gluten-(S_L_cysteine)-(-)-table gallocatechin. Table 1 · • 1H-NMR signal assignment of compounds 1, 2 and 3 (in ppm) ----- H No. Compound 1 Compound 2 Compound 3 2-H 5. 09 (br. s) 4. 86 ( d, 2Hz) 5.13 (s) 3-H 4. 07 (m) 4. 22 (dd, 8. 3, 2Hz) 4. 07 (s) 4-H 3.86(d, 2Hz) 4. 27 (d, 2Hz) 3. 92(s) 6-H 5. 99 (d, 2Hz) 6. 04 (d, 2Hz) 6. 03 (br. s) 8-H 5. 87 (d, 2Hz) 5. 81( d, 2 Hz) 5. 96 (br. s) 2, -H 6. 99 (d, 2 Hz) 6. 94 (br. s) 6. 57 (s) 5, -H 6. 76 (d, 8. 3 Hz) 6. 86 (br. s) 6~H 6. 8l (dd, 8. 3, 2Hz) 6. 86 (br. s) 6. 57 (s) cys 2-H 4. 13 (dd, 9 , 4Hz) 4. 07 (dd, 9, 4Hz) 4. 10 (m) cys 3-3⁄4 2. 95(dd, 15, 9Hz) 3.02(dd,15,9Hz) 3. 00(dd, 15, 9Hz 3. 43 (dd, 15, 4Hz) 3. 55 (dd, 15,4Hz) 3. 45 (dd, 15,4Hz) The compound 1 in the table is in the 1-dg-DzO, while the compound 2 and 3 is the measured value in CD3OD. 20 1353847 Table 2: 丨3C-NMR signal assignment of compounds 1 and 3 (occupies PPm) C No. Compound 1 Compound 3 02 74.7 75.7 -3 70.6 71.9 -*4 41.7 42.9 - 5 156. 1 157.2 -6 94.5 96.2 -7 167. 1 158.2 -8 96.4 99.0 -9 157.7 159.1 -10 99.0 99.6 -1' 130.8 131.1 _2, 115.8 a) 107.1 -3, 144. 6 b) 146.7 -4f 144.7 b) 133.6 -5' 115.9 a) 146.7 _6' 119. 1 107. 1 Cys-1 172. 1 172.9 -2 53.7 54.7 -3 32.9 34.0 The compound 1 in the table is in acetone-heart-〇2〇, and the compound 3 is measured in CDsOD. Also, the values marked a) and b) in the table are exchangeable. (4) Purification and separation of the cysteine-binding epicatechin dimer by the cysteine-binding epicatechin dimer to distinguish the powder 'in the same manner as described in the above (3), and obtain the formula (4) ) the compound shown (compound 4) 〇21 1353847

OH

The determination of the compound 4 in FAB-MS showed that the molecular ion peak [M+H] + (m/z) was at 698, which was considered to have one more catechin than the compound i.

The construction of the unit. Further, although both of the TLc and HPLC measurements (compounds 1 and 4) showed the same behavior, the H-NMR signal of the compound 4 showed a spectrum of a sharp signal mixed in the wide signal group. The ih nmr signal of this characteristic assumes that the binding position of the condensation is 4->6 or 4->8, and the stronger force produces the turning obstacle (4-> 8). In the direct connection of the 4-8 bond combined with each of the benzoquinones from the signal group " tolerate the deformation of the widening. That is, <5 4· 22, 5 4.54, 5 4.96 (each 1H, br. m, c-3, _4, _2) belong to the upper unit (unit) and sharper account for 3.83, (5 3.90 (each 1H) , s, c_4,, -3,), 5.20 (1H, br, m, c-2') belong to the unit of sulfur atom bonding (lower end). It is also observed by molecular model that the lower end unit is less slewing than the upper end unit. Obstacle. In the 5,92 aromatic ring field, it is confirmed that there are e_6, _8, _6, and the three sub-signals are envelopes, and the two units B ring are confirmed between 66 and 5 7 〇9. The double signal (dl〇ublet 22 1353847 signal) of the proton group's combination constant 8·3Ηζ can be regarded as the $coordinate of the lower unit without the rotation obstacle. The remaining signals 5 2.95, 6 3_44 (each Out, 1>]*.111, .)^-〇-3), <5 4·14(1Η' dd, J=9, 4Hz, Cys-C-2) can be presumed to be derived from semi-valine In the 13C-NMR measurement of the compound 4 (see Table 3), it was confirmed that there is a signal group corresponding to the compound 1, thereby supporting the compound 4 having the structure of the formula (4), that is, 4 no (S_L_ half Cystine)_(_)_ epicatechin _ (40,000 8) - (-) - epicatechin. Table 3: 13C-NMR signal assignment of compound 4 (5 ppm) __Cfc. C No. C-2 76.0 C-2f 74.6 '3 72.0 -3' 69.9 -4 36. 1 ~4f 42.0 -5 156. 6 s&gt • 5' 156. 5 a) —6 95.3 -6' 95.3 -7 156.5 A V 156.6 - 8 95.3 A 8' 99.0 *9 156.5 -9' 156. 6 a) -10 99.0 -10, 99.0 〇ring- i 130.7 B ring-1, 130.7 -2 114. 7 b) -2, 115.7 r3 144. 6 e) -3' 144.6 -4 144. 2 c) 144.2 . -5 115.0 w 115.7 -6 119.2 -6' 119.2 Cys-1 172.2 -2 53.7 32.8 Each sample in the table is determined in acetone-d6-D2〇. The chemical shift values of 'with a), b) and c) are exchangeable. 23 (5) Purification of hemi-deacetylated epoxidized catechin trimer The semi-valine-binding polymer powder was purified in the same manner as in the above (1), and the compound represented by the formula (5) was isolated (compound y =

The measurement of Compound 5 in HR-ESI-MS showed that the molecular ion peak [M-H]+(m/z) was 984.1956, which was consistent with the error of 5.5 ppm calculated from the calculated value 984.2011 corresponding to the formula C48H. Therefore, it is presumed that the compound 5 has a molecular formula of C48H43〇2〇NS ' and has a chemical structure in which L-cysteine is combined with a 3-molecular condensate composed of catechin or epicatechin. Further, the 1H-NMR signal of the compound 5 is a broad-band signal, and all three catechin units are linearly condensed in the 4-8 bond, suggesting that l-cysteine binds to the lower unit and is due to the aromatic ring. The signal is displayed as a single signal at 5 5 98, and therefore belongs to a ring-6-H, -8-H 'A' ring-6-H 'A" ring-6-H, respectively. In the lower magnetic field, it is confirmed that 24 1353847 ^f^#(5 6.04,s; 5 6.91,7.01 · s; 57.ll > s) « * - ^ ratio m 3 ' belongs to B ring, Bi ring, The 2h origin signal on the b" ring, which accounts for 6.91 and 57.〇1 (2: The signal of the υ is supposed to be in the middle of the Β, _2·Η is most affected by the transfer of the obstacle and split appears. According to this information, temporarily The planar structure of the putative compound 5 is represented by the formula (5). Example 2: Production of a sulphur-containing proanthocyanidin oligomer from the vine species Example 1 The same - raw material and conditional extraction grape vines are expected to be realized and implemented Example 1 is the same as the conditional purification. The purified product is reacted with glutathione under the following conditions to carry out a low molecular weight. That is, the obtained grape species is expected to be 1 gram, and gluten free (and Prepared by Pure Pure Medicine, 3.0 g, ascorbic acid (manufactured by Pure Chemical Co., Ltd.), 5 g, 50.0 ml of ιν hydrochloric acid, and reacted under thieves for 48 hours, then used to carry DOAPM ΗΡ-20 (Mitsubishi Chemical), MCIgel CHp 2〇 ( Refined by Seitax LH-20 (manufactured by Pharmacia Co., Ltd.), concentrated under reduced pressure and cooled; Donggan did not obtain reddish brown powder 12 〇 1) Glutathione-binding monomer is prepared by using polystyrene gel, Sephadex LH-20 gel and ODS gelatin as a carrier and a mixture of water and methanol as a mobile phase, and refining by column color separation method. Compound 6 represented by formula (6): 25 1353847

The determination of this compound 6 in HR-ESI-MS (High Decomposition Energy Electrospray Ionization Mass Spectrometer) showed a molecular ion peak [M+H]+(m/z) 596.1550, and the calculated value of the equivalent formula CuHwOuNJ was 596.1551. The error of 0.17ppm is consistent. Therefore, it was judged that the compound 6 has a glutathione-like L-semi-deacetylated chemical structure in which a sulfur atom is bonded to catechin or epicatechin. This compound 6 was confirmed to have 5 protons on the aromatic ring common to catechin or epicatechin in the 1H-NMR measurement.

Number group (5 5.84' 5.96, 6.91 (each 1H, 8-, 6-, 2, -H); 3 6 73-6 78 (2H, m, 5'- '6'-H), in <;5 3.90, 3_91, 5.15 (each 1H, 3-, 4-, 5-H) confirmed that the signal group having an oxygen atom or a sulfur atom at the root was estimated to be the same as the compound 1 in the epoxy catechin The 4-position has a sulfur atom arranged in the structure of 4/S. In addition, it is confirmed that it corresponds to a cysteine moiety (5 3·63 (1Η, br.t, J=9, 4Hz, cys-2-H), 3.72 , 3.80 (each 1H d J=17.6 Hz, cys-3-H2), glutamic acid moiety (accounting for im glu-4-H2), 1.78 (2H, m, glu-3-H2), 3.05 (1H , br.d, J=5.4 Hz glu-2-H)) and the signal group of the glycine moiety (5 3_20 (2H, s, gly_2_H2)). This structure consists of 13C-NMR of compound 6 (refer to Table 4). The 25 carbon signals detected by the measurement were also supported. Therefore, the structure of the compound 6 is presumed to be the semi-deacetylated part of the glutathione molecule. The ruthenium atom is bound to the epicatechin 4 A. 4^-(Glycidyloxythio)-(-)-epicatechin as shown in the formula (6). _ C No. C No. C-2 74.9 glu-1 173.3 -3 70.9 -2 55.0 -4 43.3 -3 27.0 -5 156.5 -4 32.4 -6 96.9 _5 174.3 -7 115.9 cys-1 174.3 - 8 95.8 -2 174.3 '9 157.9 -3 34.0 -10 99.7 gly-1 175. 4 131.5 -2 42.7 115.4 -3' 144. 9 * 145.0* -5 '116.7-6' 119.9 Table 4: 13C-NMR signal assignment of compound 6 (in ppm) Each sample in the table was dissolved in cd3od. The chemical shift value with * is exchangeable. Example 3 Preparation of sulfur-containing proanthocyanidin oligomers (1) Extraction of pine bark polyphenols 400 g of pine bark was extracted at room temperature using 8 〇% sterol h5 liters for 3 days, and the filtrate was filtered off and concentrated under reduced pressure. Then, the concentrate was refined under the following conditions.

[Refining conditions I 27 丄 847 The whole amount of the concentrate was placed in a column filled with DIAION Hp_20 (Mitsubishi Chemical Co., Ltd.) having a diameter of 50 mm and a height of 30 cm (about 600 ml), and washed with 2,500 ml of water. Adsorption part. Subsequently, it was extracted with 2 ml of methanol, and the extract was concentrated under reduced pressure and lyophilized to obtain 18 4 g of a powdery composition. (2) Low-molecularization reaction with cysteine The pine bark polyphenol obtained in the above (1) was used as a phenol, cysteine (manufactured by Wako Pure Chemical Industries, Ltd.), 1.7 g, and ascorbic acid (manufactured by Pure Chemical Co., Ltd.) 25 g, 20.0 ml of hydrochloric acid were mixed and placed under 4 〇»c for 48 hours. The reaction solution was introduced into a column (volume about 100 ml) filled with a carrier DIAI〇NHP-20 (manufactured by Mitsubishi Chemical Corporation), and after washing the non-adsorbed portion with 400 ml of water, the dissolved portion was recovered by using 400 ml of methanol, and then decompressed. Concentrate and cool to dry 'Get reddish brown powder 0.95 g. Proanthocyanidins show strong antioxidant activity in test tubes, but do not necessarily exert sufficient effects in the body when ingested orally. The reason for this is that it is difficult to absorb the proanthocyanidins in the intestinal tract due to high molecular weight. On the other hand, in the present invention, the sulfur-containing proanthocyanidin oligomer which is low in molecular weight of the SH-containing substance exhibits an antioxidant activity superior to that of the proanthocyanidin of the polymer in a test tube. Example 4 Manufacture of sulphur-containing proanthocyanidin oligomers from S. chinensis (1) Extraction of sorbitol from phenol Phenolic bark (Yangmeipi) 400 g for 3 days at room temperature using 80% sterol 1.5 liters, and the residue was filtered off. Thereafter, the filtrate was concentrated under reduced pressure, and then the concentrate was purified under the following conditions. [Refining conditions 1 28 1353847 Place the concentrate in a column filled with DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation) with a diameter of 50 liters and a height of 30 cm (about 600 ml), and wash it with 2500 ml of water. section. Subsequently, it was dissolved in 1200 ml of methanol, and the extract was concentrated under reduced pressure and lyophilized to obtain 18.4 g of a powdery composition. (2) Low Molecular Weight Reaction with Cysteine 1.0 g of the mountain peach polyphenol obtained in the above (1), 1.7 g of L-cysteine (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.25 g of ascorbic acid (manufactured by Pure Chemical Co., Ltd.) After mixing 20.0 ml of IN hydrochloric acid, the mixture was reacted at 40 ° C for 48 hours. The reaction solution was introduced into a column filled with a carrier DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation) having a diameter of 25 mm and a height of 150 mm (volume of about 100 ml), and the non-adsorbed portion was washed with 400 ml of water and recovered by using 400 ml of methanol. The fraction was dissolved, then concentrated under reduced pressure and lyophilized to give 0.95 g of red brown powder. (3) Combination of polyphenol and L-cysteine in bayberry skin 1) L-cysteine-binding epigallocatechin gallate monomer in polystyrene gel, Sephadex LH- 20 gel and ODS 矽 gel as a carrier and any mixture of water-sterol-acetone as the mobile phase, and the red-brown powder of the above (2) was repeatedly purified by column chromatography to separate the table. Subcatechin gallate, epicatechin gallate, (4:8) epigallocatechin gallate and compound 3 (compound of formula (3)) The compounds of the following formulas (7) and (8) (compound 7 and compound 8) are isolated. 29 (7) 1353847

OH

OH

The determination of Compound 7 in ESI-MS showed a molecular ion peak [m_h] + (m/z) 575. In addition, the measurement results of WNMR (refer to Table 5) were similar to those of Compound 1 and Compound 3, but it was observed that 3_々(1h, 5 29, s) had a low magnetic field shift of about 1.3 ppm, and in the field of aromatic rings. The 6 salts and 8 digits on the a ring, and the 6.68 and 6.96 digits respectively confirmed the equivalent single signal with 2 points of proton (1 ' 3 ' 4, 5 and 2 red on the benzene) . These signals suggest that the compound 7 is the epigallocate of the epicatechin^ and the information and the table vr r/f ^ v presume that the compound 7 is a structural group having the following formula (7)) - (-) - table tea The catechin has gallate.

The 1V1XV paw is now heard and belongs to 4 call-(SL-halamine 30 1353847 2) L-cysteine-binding epicatechin gallate dimer compound 8 is shown in HR-ESI-MS The molecular ion peak [MJ]+(m/z) 1032.1517 is consistent with the error of the equivalent formula of the formula C25H3〇012N3Si, which is calculated to be only 0.4ppm, and thus is estimated to have one more epicatechin gallate than the compound 7. The structure of the unit. Further, 1H-NMR of the compound 8 showed that a sharp signal spectrum was mixed in the broad signal group as in the case of the compound 4, and thus it was presumed that it was a 4->condensed type and a signal group of 1Η and 13C-NMR. Compound 4 is the same (see Tables 5 and 6), and is presumed to be 4yS (SL-cysteinyl)-(-)-epetocate gallate having the structure shown in Formula (8). (4/3-8)-(-)- Epicatechin gallate. 31 1353847 Table 5: 1H-NMR signal assignment of compounds 7 and 8 (<5 ppm) H No. Compound 7 Compound 8 C-2-H 5. 37(s) 5. 50 (s) 3-H 5 29 (s) 4. 75(s) 4-H 4. 16 (s) 4. 15 (br. s) A-6-H 6. 08 (s) 5. 87 (br. s) 8-H 6. 01 (d, 2. 2Hz) 5. 90 (br. s) B-2'-H 6. 45(s) 6. 45 (br. s) 6'-H 6. 45 (s) 6. 45 (br. s) gal-2-H 6. 96(s) 6. 96 (br. s) gal_6_H 6. 96(s) 7. 00 (br. s) C-2-H 5. 39 (br s) 3-H 5. 12(br) 4-H 4. 15 (br. s) A~6-H 6. 09 (br· s) 8-H — 6. 57 (br. s) 6' -H 6. 57 (br. s) gal-2-H 7. 00 (br. s) gal-6-H 7. 00 (br. s) cys 2-H 4. 19 (br. m) 4. 07 (dd, 9, 4Hz) cys 3~H2 3. 17 (dd, 15,8Hz) 3. 02 (dd, 15,9Hz) 3. 71 (br. d, 15Hz) 3. 55 (dd, 15, 4 Hz) Compounds 7 and 8 in the table were determined in C--d6D20 at 25 ° C and 40 ° C, respectively. 32 1353847 Table 6: 13C-NMR signal assignment of compound 7 and compound 8 (δ is ppm) C No. Compound 7 Compound 8 C-2 74.0 CC-2 75.0 C,-c-2 73. 5 -3 74.0 -3 75.0 -3 73.5 -4 39. 3 -4 32.4 -4 38.9 -5 156. 2 AC-5 154.8 A' -05 154.8 _6 95. 4 _6 94.7 -6 94. 7 -7 157. 3 -7 156. la ) -7 155. 9 a) -8 97. 0 -8 95.3 -8 95.3 -9 158.9 -9 156.5 -9 156.5 -10 97.9 -10 96.6 -10 96.9 -lr 129.3 B~C~1 131· 9 B' -C -1 131.9 -2' 106.4 -2 106.0 -2 106.0 -3' 145.6 -3 131.9 -3 131.9 -4' 132.9 A 4 119.6 -4 119.6 -5' 145.6 - 5 144.8 _5 144.8 -6, 106.4 A 6 106.0 -6 106.0 gal-C-1 120. 2 gal-C-1 119.6 gal-C-1 119· 6 -2 109. 8 -2 109. 4 b> -2 109.4 -3 146.0 -3 145.0 -3 145.3 -4 139. 2 -4 138.8 -4 138.8 -5 146.0 -5 145.0 -5 145.3 - 6 109. 8 -*6 109. 5 b) - 6 109. 4 -c(=o)o- 167. 0 166. 7 -c(=o)o- 166.7 Cys-1 171.8 〇(=〇)〇- Cys-1 171.0 -2 53. 9 -2 53· 3 -3 33.3 -3 32.6 Compound 7 in the table - Acetone - 'Compound 8 was determined in CDaOD in d6D20. Further, the chemical shift values marked with a) and b) are exchangeable. Test Example 1: DPPH-based elimination The powder before the separation of Example 1 (Substance 1). The monomer portion (substance 2) separated from the substance 1 33 1353847, the dimer and trimer portion (substance 3) separated from the substance 1, and the tetra-hexameric separation obtained from the substance 1 Part (Material 4) 'The raw material of Example 1 (grape seed polyphenol extract) (Comparative Substance 1), and the commercially available catechin (made by Wako Pure Chemical Industries Co., Ltd.) (Comparative Substance 2) The method described was used to determine the elimination of 1,1-diphenyl-2-benzyl acid sulfhydryl (DPPH). Test Method A 100 microliter (//L) DPPH solution (60# L methanol solution) was placed on a 96-well microplate, followed by a sample ethanol solution 100//L and a control solution of ethanol 10 〇eL, from the mixture at room temperature for 30 minutes, and then measure the absorbance at 520 nm, calculate the DPPH-based elimination energy of the sample according to the following formula, and calculate the 50% effective energy and concentration of the DPPH-based elimination energy from the diluted sample of the stage. Concentration (EC50). DPPH group elimination energy (%) = [(1 - absorbance of sample) / absorbance of control liquid] X 100 The 50% effective concentration (EC5G) was calculated from the DPPH group elimination of the concentration of each sample. The results are shown in Table 7. Table 7_ DPPH group elimination of EC5〇 ( /zg/mL) Substance 1 1.62 Substance 2 2.78 Substance 3 1.45 Substance 4 0.77 Comparative substance 1 1.55 Comparative substance 皙 2 1.71 34 1353847 From the above table, DPPH based elimination energy, at low concentration When the substance 4 shows higher. The other samples showed the same activity, and each sample showed high activity at a high concentration. Test Example 2: Active Oxygen-Removing Enzyme (SOD)-like Activity The same substance 1 to 4 as the above Test Example 1 and the comparative substance 1 to 2 were measured using a blood SOD activity measuring device (trade name: SOD Testwako, manufactured by Wako Pure Chemical Industries, Ltd.). SOD-like activity. The SOD-like activity was calculated according to the following formula, and 50% effective concentration (EC50) was calculated from the concentration relationship between the sample and the sample. SOD-like activity (%) = [(1 - absorbance of sample) / controlled absorbance] X 100 EC5 算出 was calculated from the SOD-like activity of the concentration at the stage of each sample, and the results are shown in Table 8. Table 8_ SOD-like activity EC5Q (# g/mL) Substance 1 69.7 Substance 2 84.8 Substance 3 74.6 Substance 4 75.5 Comparative substance 1 70.9 Comparative substance 2 62.0 As shown in the above table, substance 2~4 is higher than comparative substance 2 , SOD-like activity. Test Example 3: Effect on sub-acute FeNTA-induced multi-organ dysfunction modality 35 1353847 The use of a well-known oxidative modality in the body that emits a large amount of reactive oxygen species and induces multiple organ dysfunction Test of FeNTA (iron nitrate, Fe(N〇3)3) with sodium nitrotriacetate, (NTA3Na)). That is, '240 mg of iron citrate non-hydrate (manufactured by Kanto Chemical Co., Ltd.) dissolved in 400 mL of cold water and 390 mg of sodium sulphate monohydrate dissolved in 40 mL of cold water were mixed under ice cooling, and pH was adjusted to 7.5 with IN hydrochloric acid. The whole was prepared to be 100 mL as a FeNTA solution. The FeNTA solution was intraperitoneally administered to a 7-week-old male ddy mouse (average body weight 3 〇g) in a ratio of 1 kg per day to 1 kg in a dose of 22 mg to 5 mg. Substance 1 and comparative substances [usually administered by a ratio of 25 ng (low dose) or 50 mg (high dose) to each body weight of 1 kg per day. Further, the control group was ingested with tap water and divided into a negative control group not administered with FeNTA and a positive control group administered with FeNTA. The beverage 7 was allowed to be freely ingested during the test. The concentration of lipid peroxide (Lp〇) in the serum was determined by blood sampling on the 7th, 21st and 28th days from the start of the administration. On the 28th day after the start of the administration, the rats were dissected and the liver, kidney and brain were taken out to measure the homogenized LPO concentration in the organs. Test results (1) Serum Lp concentration is shown in Figures i(a) and (8). In the low dose, 1 1 > 3 1 on the 21st day, the concentration of LP〇 in the A clear showed an intentional (statistically) decrease in the comparative control group (see Figure 1(A). , the substance f丨 also decreased from the start of the day of the start of the day (as shown in Figure 1 (B)). The comparative substance 显示 also showed a low value on the 21st day of the 36th 353847, but the substance of the present invention showed higher (2) The concentration of L〇 in each organ The concentration of LPO in the liver, kidney, and brain is shown in Fig. 2(A) to (C). It is not confirmed in the liver that there is a concentration of Lp〇 due to administration of FeNTA. Intentional rise, no change in LPO concentration was confirmed in each of the cast groups. There was an increase in the concentration of LP〇 in the renal pelvis and brain due to the administration of FeNTA. However, administration of substance 1 and specific substance 1, LP〇 The concentration is reduced. Both the kidney and the brain are in the dose of 1咼, and the group shows the lowest Lp〇 concentration value, especially when the brain is administered with the comparative substance 1 to show a low value, but the substance i high dose administration group is more interesting than other groups. The low value. Conclusion The results of the above test show that the administration of FeNTA can increase the concentration of LPO in serum. At the same time, substance i can be used lower than comparison j. In the short period of time, it is expected that the substance 1 is easily absorbed by the intestinal tract due to the low molecular weight, and therefore it can be used in a short period of time at a low dosage. Substance i showed strong antioxidant activity in the kidney and brain. Test Example 4: Detection test for humans 34 healthy men and women (average age 41.2 years, 21 males, 13 females) were divided into two groups, as shown in Table 9. As shown above, 500 mg of each of substance 1 and comparative substance 1 was administered for 28 consecutive days, respectively, and blood was collected before and after the start of administration, and the concentration of LP〇 and s〇D in the serum were measured. The results are shown in Table 9. 1353847 Table 9 Average age of males and females LPO initial 値·high SOD initial 値.Low substance 1 41.9 10 : 7 7 people 7 people comparative substance 1 40.5 11 : 6 11 people 8 people results before and after LPO concentration and SOD The activity is shown in Fig. 3 and Fig. 4. Fig. 3(A) shows the LPO concentration of those with normal initial values, Fig. 3(B) shows the LPO value of those with abnormal initial values, and Fig. 4(A) shows the SOD of those with normal initial values. Figure 4 (B) shows the SOD-like activity of those with abnormal initial values. 28 days of continuous administration When comparing substance 1 and substance 1, there is a tendency that the concentration of lp〇 decreases and the activity of SOD increases, and the initial value of the LPO concentration of the initial abnormality is higher (the lp〇 concentration is 8.0 or more) and the s〇D shape. In the case of the lower activity (SOD-like activity 12'0 or less), the concentration of the substance 1 was not only statistically deliberately reduced (see Fig. 3(B)). Similarly, the SOD-like activity was also statistically intentionally increased. (Refer to Fig. 4(B)) ^ It was confirmed that the substance 1 has an improved effect of the biooxidation state than the same amount of the comparative substance 丨. Test Example 5: Neuronal cell protective effect against cell death induced by amyloid-like amyloid (amyi〇id) Alzheimer's disease due to abnormal neurogenetic regeneration has an accumulation of starchy type The senile plaque is characterized by a powdery system of the L dynasty that produces active species such that the nerves are fine (10) due to cytotoxicity and accelerate the Alzheimer's disease. This test is to investigate the cytoprotective effect of the low molecular weight (4) (hereinafter referred to as GSM) nerve 38 1353847 cytotoxicity of the experiments widely used for neurocytotoxicity to confirm the cell death of PC-12 cells by low molecular weight polyphenols ( Apoptosis induced cell injury activity. Test Method PC-12 cell line was cultured in a 10% carbon dioxide atmosphere using DMEM containing 10% heat-inactivated horse serum and 5% half-fetal serum, and the cell density was adjusted to 4×104 cells/300//L. Prepare for hourly incubation, then place in serum-free N-2 medium, add GSM or grape seed polyphenol (hereinafter referred to as GSP) at a concentration of 1, 1.5, 5, 10/zg/mL, in 48-well Cultivate in the pan. (1) Cell viability test After the treatment, it was treated with an MTT solution having a concentration of 1 mg/mL, and the dark blue color of the formazan product was dissolved in a buffer, and the absorbance at 540 to 595 nm was measured. The results are expressed as the ratio (%) of the absorbance to the untreated control cells. Results It was confirmed by MTT assay that GSM has a concentration-dependent inhibition of cell death caused by 3-amyloid. The effect is particularly high in the concentration range of 1 and 1.5 Vg/mL compared to GSP (see Fig. 5). (2) Mitochondria membrane potential measurement test The measurement of the mitochondrial membrane potential uses a fat-soluble cation probe TMRE. Treatment of PC-12 cells (lx 104 cells/mL) with 25#M cold-like amyloid in the presence or absence of GSM or GSP, washed with phosphate buffered saline and at 37 °C It was treated with TMRE (150 nM) for 30 minutes. Then, TMRE which accumulates in the mitochondria of the membrane potential of the dependent mitochondria is detected by fluorescence of an excitation wavelength of 488 nm and an emission wavelength of 590 nm 39 1353847. Results The integrity of the membrane occurred before the mitochondria had signs of cell death. This change occurs in both the inner and outer membranes of the mitochondria, and eventually leads to the release of the membrane breakthrough potential and the membrane permeability of the soluble intermembrane protein such as cytochrome C. When PC-12 cells are exposed to the smectic starch, the mitochondrial membrane penetration potential is rapidly lowered, and this can be visualized by the red fluorescence of the TMRE of the potential-dependent pigment (see Fig. 6(A)). The decrease in the breakthrough potential of the membrane-like starch can be suppressed by the pretreatment of GSM (all), and the effect is higher than that of GSP (see Figs. 6(A) and (B)). (3) Determination of the accumulation of intracellular reactive oxygen species To observe the cumulative amount of reactive oxygen species in the cells, a fluorescent detection element DCF-DA (2',7·-di-hydrogen fluorescein-diacetate vinegar was used. ). Treatment of PC-12 cells (ix 106 cells/3 mL) with amyloid in the presence or absence of gsm or GSP, washed and added with Krebs_ Ringer's solution (a physiological saline solution) DCF-DA. After incubation for 15 minutes at 3 7 C, a confocal laser microscopy using an argon laser was observed at an excitation wavelength of 488 nm and an emission wavelength of 53 Onm. Results In order to understand the oxidative stress at the time of cell death of cells derived from amyloid-like PC-12 cells, the accumulation of reactive oxygen species in the cells was measured using DCF-DA permeable to the cell membrane. Within the cell, DCF-DA is hydrolyzed to DCF by the ester 1 activity of the cell and reacts with the peroxide to form a fluorescent substance. The PC-12 cells treated with the amylin-like treatment were visualized by staining with DCF pigment (see Fig. 7). The cumulative amount of intracellular reactive oxygen species resulting from the cytoplasmic amyloid is reduced by GSM, and its effect is higher than that of GSP (see Figs. 7(A) and (B)). (4) Intracellular glutathion concentration The glutathione concentration in the cells was measured using a commercially available device (trade name: BIOXYTECH GSH-400; manufactured by OXIS Research, Inc.), in the presence of GSM or GSP or PC-12 cells were cultured in the absence of cultured and treated with cold-amyloid. After being neutralized in a solution of metaphosphoric acid, a solution of the pigmented hydrochloric acid was added to the solution obtained by centrifugation, and 30 was added after stirring. The sodium hydroxide solution was incubated at 25 ° C for 10 minutes, and then centrifuged to obtain a transparent solution, and the absorbance at 400 nm was measured. In addition, the protein content was determined using a BCA protein analyzer, and the glutathione concentration per unit weight of the measured protein was compared with the untreated control. The result is that the cold-amyloid cells die with oxidative damage. The intracellular glutathione concentration of the amyloid-treated cells is lowered. GSP-treated cells, although their intracellular glutathione concentrations returned to normal levels, but GSM-treated patients showed a level of intracellular glutathione that exceeded normal levels, demonstrating its high antioxidant activity in cells. (Refer to Figure 8). (5) Lipid peroxide concentration 41 1353847 The lipid peroxide concentration was measured using a commercially available apparatus (BIOXYTECH LPO-586, manufactured by OXIS Research, Inc., USA). The PC-12 cells cultured in the presence or absence of GSM or GSP and treated with amyloid were recovered, neutralized in 20 mM trihydrochloric acid buffer containing 0.5 mM butylated hydroxytoluene, and then centrifuged. A solution of 10.3 mM of N-mercapto-2-phenylindole in acetonitrile was added to the obtained supernatant, followed by incubation with 45% hydrochloric acid at 45 ° C for 60 minutes. After cooling, the mixture was centrifuged and the absorbance at 590 nm of the clear liquid was measured. The protein content was also determined using a BCA protein assayer, and the peroxidic lipid concentration per unit weight of the measured protein was compared to the untreated control. Results The malondialdehyde (MDA) produced by the peroxidized peroxidized lipid of the cell membrane produced by the /5-amyloid treatment was expressed. By inhibiting the peroxidation of lipids produced by stone-like starch by GSM or GSP, the effect is higher in GSM and the lipid peroxide is suppressed below the untreated control level (see Fig. 9). Summary GSM can reduce the accumulation of reactive oxygen species in nerve cells in PC-12 cells, which are representative cells of nerve cells, thus inhibiting the death of nerve cells caused by stone-like amyloid toxicity, which is more effective than GSP. The cytotoxicity of the cytotoxicity of the cells is related to the oxidative damage of the cells. GSM can inhibit oxidative damage to cells, and its effect is higher than that of GSP. GSM relies on its anti-oxidation to inhibit the onset and progression of Alzheimer's disease, which is closely related to the death of neuronal cells derived from /3 -starch. Therefore, 42 1^^3847 can be used to suppress arz by GSM. The onset and progression of Haimer's disease. Test Example 6: Prophylactic effect against streptozotocin (STZ)-induced diabetes To investigate the preventive effect of substance 1 on STZ-induced diabetic mice, the use of multiple low dose (MLD) equivalent to STZ was used. The initial morbid modality of induced diabetes. Methods The body weight, blood glucose level and blood LPO value of Jla:ddy mice (male, 7 weeks old) were determined, and the groups were equally divided as follows (where n is the number of mice): (1) Negative control group: STZ (-) (η =: 5, 1 cage) (2) Positive control group: STZ(+) (η=5, 3 cages) (3) Substance 1 Group: STZ ( + ) + substance 1 ( η = ι〇, 2 cages) (4) Comparative substance 1 group: STZ (+) + comparative substance i (η = 10, 2 cages) STZ was administered intraperitoneally twice at a dose of 20 mg/kg for 4 days, and then administered at a dose of 40 mg/kg for 4 consecutive days. In the abdominal cavity. In the meantime, the test rats were allowed to freely ingest 6% of the powdered baits mixed with the substance 1 or the comparative substance 1 (calculated from the intake of the food for one day, the intake of the substance 1 or the comparative substance 1 of one day was about 100 mg/kg) . The period of investment is from 5 days before the start date of STZ to the 65th day. Blood and urine were collected during administration. Glucose, urine sugar, urine egg, blood pulse (BUN) and blood LPO/TEAC (Trolox equivalent antioxidant capacity) were measured. The results are shown in Figures i〇~14. Results 43 1353847 U) When the blood glucose level was fasting, in diabetes, as shown in Fig. ίο, the blood glucose level showed a high value on fasting, but the blood glucose values of the positive control group compared with the negative control group on the 35th, 49th and 66th days after STZ administration. High, multiple doses of STZ were confirmed by low doses to form a mild diabetic modality. The blood glucose level of substance 1 group and comparative substance group 1 showed a deliberately lower value than the positive control group, while the blood glucose level decreased effect, substance 1 was higher than the comparative substance 1 (2) urine sugar value, diabetes, urine sugar excretion The amount increased, and the amount of sugar excretion in the urine was significantly increased by STZ treatment. 'The mouse has induced diabetes, as shown in FIG. In the substance 1 group and the comparative substance group i, an effect of improving the urine sugar value increased by STZ was observed, and on the 49th day, there was a statistically significant difference between the positive control group and the positive control group, and the substance i showed a comparison with the comparative substance i. A slightly lower value. (3) Urine protein value In diabetes, protein excretion in the urine increases. As shown in Fig. 12, substance 1 and comparative substance 1 were able to inhibit leakage of protein in urine due to STZ, and both substances were intentionally reduced by the amount of protein in the positive control group on the 49th day after S'Z administration. 1 is more predictive than the comparative substance 1 in the early stage of the morbid state. (4) Antioxidant index (4-1) Blood LPO (peroxidized lipid) value As shown in Fig. 13, the substance 1 group showed a lower tendency than the positive control group and the comparative substance i group. Comparative substance 1 44 1353847 (4-2) TEAC value in blood TEAC value has been widely used as an antioxidant index. The TEAC method is a method for relatively estimating the antioxidant strength by converting the antioxidant activity of a compound into the antioxidant energy of Trolox of a tocopherol derivative. As shown in Fig. 14, 'the antioxidant activity in blood on day 49 and day 66, the comparative substance group 1 and the positive control group showed approximately the same antioxidant energy, but the substance 1 administration group showed a high TEAC value indicating resistance. The oxidation energy is the strongest. Conclusion STZ's low-dose multiple-dose mode can induce mild diabetes in mice. Compared with high-dose administration mode, the difference between individuals with blood glucose values is small. Therefore, it is considered to be extremely suitable for reviewing the preventive effect of diabetes and inhibiting diabetes. The blood sugar level and the urine sugar value which are increased with the progress of the disease state have the effect of preventing the disease of the sugar spread. For STZ-induced diabetes, it will produce a unique radical in the sputum cells, and the cells will be killed by the injury of the cells. This is caused by the substance i and the comparative substance 5 days before the STZ administration. It can improve the antioxidant activity of the body in advance, so it can alleviate the damage of STZ to the cells. From the results of LPO and TEAC in blood, substance 1 can intentionally increase the antioxidant energy in the blood. The basis of nitric oxide and reactive oxygen species is related to the fact that the pancreatic islet-dependent islet of the insulin-dependent diabetes mellitus (islet) is a well-known fact. The inhibition of cell damage by these free radicals can improve the diabetes section has been temporarily clarified by human trials. Therefore, the substance can be used as a healthy person to prevent diabetes and the initial progression of diabetes mellitus. 45 Formulations using β test Example 7. / Ozone-induced potassium acute kidney dysfunction comparison test 1 for substance 1 and other antioxidant aggregation The antioxidant properties of phenolic materials were compared with the following potassium bromate-induced acute kidney impairment in rats. Methods Wistar rats (male, 12 weeks old) were grouped as follows: Negative control group: potassium bromate (-) (n=5) Positive control group: potassium bromate (+) (n=5) Substance 1 group (Vegetable seed origin) ): Potassium bromate ( + ) + substance l (n = 5) Substance 5 groups (from pine bark): potassium bromate + substance 5 (n = 5) Comparative substance 2 group (catechin): potassium bromate ( + ) + comparative substance 2 (n=5) Comparative substance 3 groups (Ginkgo biloba extract): desert acid oblique (+) + comparative substance 3 (n=5) Comparative substance 4 group (pine bark extract): potassium bromate (+) + comparative substance 4 (n=5) Comparative substance 5 group (cocoa extract): Potassium bromate (+) + comparative substance 5 (n=5) 〇Bromoacid was administered to the abdominal cavity once at a dose of 6 〇mg/kg (body weight). Substance 1, substance 5 and each comparative substance are! The 〇 mg/kg dose is administered once a week before the start of cesium bromide (Day 0) (one day 7) to 翌曰 every day. The bromine acid was cast in the same time, and it was administered 30 minutes after the indifference. Blood was collected from the jugular vein on Day 7, Day 3 and Day 2. The concentration of polyphenols in the blood, lipid peroxide concentration, TEAC, urea nitrogen and 46 1353847 creatinine concentrations were determined. RESULTS: The substance 7 and the comparative substance of potassium bromate induced acute kidney disorder. The fruit was based on the antioxidant effect. For this purpose, the blood concentration of 1 degree of agriculture and blood; = emulsification energy (TEAC) and blood lipid peroxide concentration were used as Antioxidant index, and check the concentration of urea nitrogen in blood and the concentration of creatinine in blood as pathological indicators of renal impairment. The results are shown in Figure 15~19 ^ (ΌAntioxidant index (1-1) concentration of polyphenol in blood The values are shown in the figure. The initial concentration of the concentration of potassium in the pre-existing blood is in the white group, but the substance 1 and the comparative substances are administered to the inferior G after 7 days. On the day of the day (the start of the day), the substance group showed the highest value, followed by the comparison substance group 2. It was also confirmed that the substance 1 can cause the blood: polyphenol concentration to rise 'the rate of increase is higher than the comparison substance. (1-2) Antioxidant energy (TEAC) in the enterprise As shown in Figure 16, one of the substances 1 and each of the comparative substances was injected before the week, so that the antioxidant energy in the blood showed a rising tendency, which was caused by the substance ι. The anti-oxidation increase is intentional for each comparative substance administration group. (1-3) Peroxide lipid concentration in blood\ Each of the comparative substances was administered to inhibit the release of bromic acid before the week, and the concentration of lipid peroxide in the blood was increased. As shown in Fig. 17, the effect was highest in the substance group 1 and the intention was shown for each comparative substance f. Low value. (2) Illness index (2-1) Urea nitrogen concentration in blood 47 U53847 As shown in Figure 18, 'the concentration of urea nitrogen in the blood is intentionally increased from no treatment by potassium bromate. Substance 1 and The comparison of the administration of the compound and the inhibition of the increase in the concentration of urea nitrogen in the blood caused by the administration of potassium bromate, and the increase in the concentration of urea nitrogen in the blood caused by the administration of the substance 1 are intentional for the administration of the comparative substances. 1) The concentration of creatinine in the blood is shown in Fig. 19, and the concentration of creatinine in the blood is intentionally increased compared with no treatment by potassium bromate. The administration inhibition of substance 1 and each comparative substance is administered by potassium t-acid. The increase in the concentration of creatinine in the blood caused by the increase in the concentration of creatinine in the blood caused by the administration of substance 1 is intentional for the administration of each comparative substance. Summary 匕Comparative substances 3, 4, 5 The material of the polymer of the original flower pigment is rich in anti-oxidation in the test officer The activity is low, but the substance which is low in molecular weight when ingested in the body shows high antioxidant activity. The comparative substance 2 is a monomer (catechin), but the activity of the substance i is high, and the amount of the substance is observed. The substance is low in molecular weight, so it is easily absorbed into the two S organisms to show high antioxidant activity. Before the substance f1 is administered

Sit: Antioxidant energy exerts antioxidant activity. As a result, it is possible to suppress an increase in the concentration of lipid peroxide in the blood of the cause of administration, and to suppress renal dysfunction = qi and sarcosine hepatic leakage into the blood. The effect is better than that of each of the Ξ f , , , , , , , , 四 四 四 四 四 四 四 本 本 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 The safety of the substance is based on a single resistance and toxicity test to assess safety. For doses of 2.5, 5.0, 7.5, and 10.0 per kilogram (kg), 8, 7, 3, and 18 ddy mice (9 weeks old, male) were orally administered. After the administration, the LC50 was calculated by observing the change of action and the death. Results The 50% lethal concentration of substance 1 (1^50) was 5. (^/1^(body weight) (95% reliable limit 3.5~6.4 g/kg), no abnormal action after administration and the end of the experiment. The abnormality of the anatomical results. Therefore, it was confirmed that the substance 1 was used as a highly safe substance in the food system. In the aforementioned test example 4 (test test for humans), the opinions of the subjects were collected by questionnaire, and the persons concerned with the sleep were listed in Table 10 shows the five-stage evaluation method for fatigue, head sobriety and stomach comfort. The results are shown in Table 11. Table 10 Group age and gender use opinion substance 1 49 Male sleeping, wake up with improved substance 1 43 Male sleep The quality is improved (sleep well) Substance 1 57 Men get up in the morning to loosen the substance 1 27 Women get up in the morning and have a sense of fatigue 1 34 Men can't get up in the morning, still want to sleep, compare substance 1 43 Men can't sleep well, get up Difficulty 49 1353847 Table 11 Questionnaire scores according to the five paragraphs Assessment of the degree of fatigue of the fatigue of the stomach Comparison of the substance 1 2.8 (3.2) 3.7 (3.3) 3.9 (3.5) Substance 1 3.2 (2.9) 3.7 (3.3) 3.8 ( 3.5) The larger the value, the better the situation, in parentheses before the vote Numerical value 50 1353847 [Simplified description of the drawings] Fig. 1 is a composition of a sulfur-containing proanthocyanidin oligomer according to Example 1 of the present invention (a powder before separation; a substance D and a raw material thereof (grape seed phenol extract; Comparison of the blood of the mouse of the f1) ί 巾 L LPO concentration line chart; Figure 1 (A) shows the low dose group, Figure 1 (B) shows the results of the high dose group. Figure 2 is the contribution The sulfur-containing proanthocyanidin oligomer composition (substance 1) of the present invention is a line of the LPO concentration in the liver (A), the kidney (B) and the brain (C) of the mouse of the comparative substance Fig. 3 is a graph showing the composition of the first embodiment of the present invention (substance 1) and the raw material of the first embodiment (a graph of the concentration of Lp〇 in the serum of a human of a comparative substance), wherein Fig. 3(A) shows the initial concentration of LPO. Fig. 3(b) shows the result of an abnormal initial value of the LPO concentration. Fig. 4 shows the composition of the composition (substance 1) of the first embodiment of the present invention and the raw material (comparative substance 1) of the first embodiment. A graph of the concentration of S〇D in serum; wherein Figure 4(A) shows that the initial value of SOD concentration is normal, and Figure 4(B) shows the knot of abnormal initial value of SOD concentration. Fig. 5 is a graph showing the inhibitory effect of the substance 1 of the present invention and the comparative substance 1 on PC-12 cell death caused by point-like amyloid. Fig. 6 (A) and (B) respectively show the substance 1 of the present invention. Photographs and graphs showing the inhibitory effect of the substance i on the decrease of the mitochondrial membrane potential caused by the /3 -star type of starch "Figure 7 (A) and (B) show the pair of the substance 1 and the comparative substance 1 of the present invention, respectively. Photographs and graphs of the effect of 51 1353847 on the accumulation of reactive oxygen species in PC_12 cells caused by the call-like amyloid. Fig. 8 is a graph showing the effect of the intracellular antioxidant activity of the substance 丨 and the comparative substance 本 of the present invention. Figure 9 shows an embodiment of the invention! The substance 丨 and the comparative substance 图表 are caused by the inhibition effect of the cell membrane peroxidation of the stone-like starch. Figure ίο shows the substance i and comparative substance of the present invention! A graph showing the effect of STZ-induced diabetic rats on fasting blood glucose levels. Fig. 11 is a graph showing the effect of the substance i of the present invention and the comparative substance i on the reduction of the urine sugar value of STZ-induced diabetic rats. Fig. 12 is a graph showing the effect of the substance 丨 and the comparative substance 本 of the present invention on the reduction of the urine protein value of STZ-induced diabetic rats. Fig. 13 is a graph showing the effect of the substance 丨 and the comparative substance 本 of the present invention on the decrease in the LPO value in the blood of the stz-induced diabetic mouse. Fig. 14 is a graph showing the effect of the substance 1 of the present invention and the comparative substance 1 on the increase in the TEAC value (antioxidant energy) in the blood of a diabetic rat. Figure 15 shows the substance of the present invention! A graph showing the effect of increasing the concentration of polyphenols in the blood of rats with acute renal impairment induced by bromate. Fig. 16 is a view showing the acute renal impediment induced by the substance i of the present invention. A graph of the rising effect of antioxidant energy (TEAC) in blood. JintuΠ indicates that the substance 1 of the present invention is used to induce acute kidney impediment in rats. A graph showing the effect of increasing the lipid peroxide concentration in the blood. Fig. 18 shows the acute kidney impediment induced by the substance 1 of the present invention. A graph showing the effect of increasing the concentration of urea nitrogen in the blood. Figure 19 shows the inhibitory effect of the substance i of the present invention on the increase in the concentration of bismuth citrate in the blood of the dysentery. [Main component symbol description] 53

Claims (1)

1353847 , Patent No. 93,712,234, the application for the amendment of the patent application, the revised page without the scribe line, the three-part tenth, the scope of the patent application: 1. A composition containing a sulfur-containing proanthocyanidin oligomer as a main component, A reaction of a proanthocyanidin plant or an extract thereof with at least one SH group-containing compound selected from the group consisting of cysteine, cystine, glutathione, SH group containing a peptide and salts thereof Liquid, concentrated And the 2~5 oligomer of the sulfur-containing proanthocyanidin obtained by drying is characterized as a main component. 2. The composition according to claim 1, wherein the plant containing the proanthocyanidin is at least one selected from the group consisting of fruits and vegetables, teas, herbs, woods and bark. 3. A health food composition comprising the composition of any one of claims 2 and 2. 4. A sulfur-containing proanthocyanidin oligomer, which comprises: a plant containing proanthocyanidins or an extract thereof and a peptide selected from the group consisting of cysteine, cystine, glutathione, and SH group And at least one of the SH groups of the salts thereof contains a sulfur-containing proanthin 2-5 oligomer obtained by separating the reaction solution obtained by the reaction of the compound. 5. A method for producing a sulfur-containing proanthocyanidin oligomer, comprising: a plant containing a proanthocyanidin or an extract thereof under acidic conditions and a patent application No. 93 1353847 No. 93022234 The instruction sheet is modified in triplicate from at least one SH group containing cysteine, cystine, glutathione, SH group containing a peptide and salts thereof, at room temperature to 80 ° C. The reaction is carried out for several hours or even one week, and then the reaction solution is concentrated and dried. 6. A method for producing a sulfur-containing proanthocyanidin oligomer, comprising: causing a plant containing proanthocyanidins or an extract thereof under acidic conditions and selected from the group consisting of cysteine, cysteic acid, glutathione, The SH group contains a peptide and a salt of at least one of the SH groups containing the compound at room temperature to 8 Torr. The reaction is carried out for several hours or even one week at a temperature of 〇, and then the reaction solution is concentrated and separated. 7. The method of claim 5, wherein the plant containing proanthocyanidins is at least one selected from the group consisting of fruits and vegetables, teas, grass plants, and wood and bark. 8. The method of claim 5, wherein the acidity condition is a mineral acid, an organic acid or both. 9. The process of claim 8, wherein the acidic condition is at least one selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, ascorbic acid, and malic acid. 10·—The proanthocyanidin compound represented by the following formula (4): 55 1353847 Patent Application No. 93022234 Supplementary, revised, unlined instruction sheet, revised in triplicate OH 11. A proanthocyanidin compound represented by the following formula (5): 0H (5) 12. A proanthocyanidin compound represented by the following formula (6): 56 1353847 Patent Application No. 93222234 Supplementary, amended, unlined instruction sheet correction sheet in triplicate 0H 13. - Proanthocyanidin compounds of the following formula (8): 0H 57