JP4741899B2 - Method for producing blood sugar level increase inhibitor and blood sugar level increase inhibitor - Google Patents

Description

  The present invention is a technique relating to a novel substance obtained from the fruit of tochi after punching, and is particularly effective when applied to the suppression of an increase in blood glucose level by mixing with food. In addition, the present invention has newly found that substances obtained from tochi fruits after punching treatment that have not been thought to have an anti-glycemic activity so far have an anti-glycemic activity and are effective. It is intended for application.

  Traditionally, seeds of Tochinoki (Aesculus turbinata), that is, Tochino fruit (hereinafter sometimes referred to as Tochinomi), were once considered an emergency food during famines, and nowadays Tochigi in various parts of Japan. It is used as a raw material for foods such as Tochino dango.

  Tochinomi contains a lot of saponin and has a strong bitter taste. Therefore, unless a punch for removing saponins is used, tochinomi cannot be used as food. It has been found that the main component of this saponin is a mixture of triterpenoid glycosides called escin. Traditionally, various methods are used in various areas of Japan. As a typical method, a step of exposing the water flea to water, ripening and immersing in wood ash is taken.

  On the other hand, this escin is also contained in the seeds of Aesculus hippocastanum L. Various pharmacological activities such as suppression of blood glucose level increase, alcohol absorption suppression, anti-inflammation, and anti-swelling have been reported. This extract is actually used in medicines and cosmetics for the purpose of treating inflammation and swelling.

  Non-Patent Document 1 describes the use of tochinomi as a food ingredient and the punching in use that are generally performed conventionally. It is described in Non-Patent Documents 2 and 3 that the bitter component of Tochinomi is escin. Furthermore, the pharmacological action and the like of escin are described in Non-Patent Documents 2 to 8.

Non-Patent Document 9 describes the characteristics and the like of the mass of Western Tochi. In addition, Non-Patent Document 10 shows a description of the fruits of Chinese Tochi. Non-Patent Document 11 has a description of “the chemical structure of the saponin component and the inhibitory effect on the increase in blood glucose level of the punched-out torch tree seed”, which has been previously filed.
Toshio Matsuyama, “Acupuncture technique and distribution of wild nuts, especially Tochinomi and acorns”, National Museum of Ethnology Research Report, 2, p 498-528 (1977) Yoshikawa, M., Murakami, T., Matsuda, H., Yamahara, J., Murakami, N., and Kitagawa, I., "Bioactive saponins and glycosides. III. Horse chestnut. (1): The structures, inhibitory effects on ethanol absorption, and hypoglycemic activity of Escins Ia, IIa, Ib, IIb and IIIa from the seeds of Aesculus hippocastanum L. '', Chem. Pharm. Bull., 44, 1454-1464 (1996) Yoshikawa, M., Harada, E., Murakami, T., Matsuda, H., Wariishi, N., Yamahara, J., Murakami, N. and Kitagawa, I., `` Escins Ia, IIa, Ib, IIb and IIIa, bioactive triterpene oligoglycosides, from the seeds of Aesculus hippocastanum L .: Their inhibitory effects on ethanol absorption, and hypoglycemic activity on glucose tolerance test. '', Chem. Pharm. Bull., 42, 1357-1359 (1994) Yuji Yamahara, Hisashi Matsuda, Toshiyuki Murakami, Hiromi Shimada, Masayuki Yoshikawa, “Inhibition of Glucose Levels and Active Ingredients of Medicinal Plants—Structure and Activity of Triterpene Glycosides”, Journal of Japanese-Wan Pharmaceutical Sciences, 13, p295-299 ( 1996) Masayuki Yoshikawa, “Diabetics preventive ingredients of medicinal plants, from the viewpoint of medical foods,” Chemistry and Biology, 40, p172-178 (2002) Guillaume, M. and Padioleau, F., `` Veintomic effect, vascular protection, anitiinflammatory and free radical scavenging properties of horse chestnut extract. '', Arzneimittelforschung, 44, 25-35 (1994) Sirori, CR, `` Aescin: Phamacology, phamacokinetics and therapeutic profile. '', Phamacological Research, 44, 183-193 (2001) Yoshikawa Masayuki, Murakami Toshiyuki, Otsuki Keiko, Yamahara Shinji, Matsuda Hisashi, “Bioactive Saponins and Glycosides. XIII. Western Tochinoki Seed Saponins (3): Escin Ia, IIa, Using High Performance Liquid Chromatography Quantitative analysis of Ib and IIb ", Pharmaceutical Journal, 119, p81-87 (1999) Facino, RM, Carini, M., Moneti, G., Arlandini, E., Pietta, P. and Mauri, P., `` Mass spectrometric characterization of horse chestnut saponins (Escin). '', Org. Mass Spectrometry, 26, 989-990 (1991) Zang, Z., Koike, K., Jia, Z., Nikaido, T., Guo, D. and Zheng, J., `` New saponins from the seeds of Aesculus chinensis '', Chem. Pharm. Bull., 47, 1515-1520 (1999) Hideto Kimura, Ai Watanabe, Mitsuo Chisaka, Tatsuyuki Yamamoto, Ikuo Kimura, Takuya Katsubebe, Kazunari Yokota, "Chemical structure of saponin component of perforated seeds and inhibitory action to suppress increase in blood glucose level", 51, 672-679 (2004).

  The present inventor has been studying the health effects of tochi fruits that have been used since ancient times in various parts of Japan, while being involved in the technological development of food production such as confectionery. It has been found that various pharmacological effects of tochi have been reported, as described in the above-mentioned various documents.

  However, the inventor has noticed a certain fact. In other words, as described above, reports on the pharmacological effects of tochi fruits described in various documents have been researched only on substances obtained from raw tochi fruits. I noticed that it was not a pharmacological action.

  As described above, when using the tochino fruit as a food, it is bitter as it is, so it has to be punched out once. It is considered that a certain amount of saponin is removed by such a punching process. Therefore, the present inventor considered that it is necessary to examine whether the tomato seeds after the punching process from which the saponin has been removed have a pharmacological action as described in the above-mentioned various documents. There is no report on the content and components of saponin present in this torch after punching. From the viewpoint of medical foods, I thought it necessary to study the saponin components of tochinomis actually used as food ingredients and their biological activities.

  Through this research, if a substance showing a health effect is found in the fruit of the tomato after being punched, the substance of the tochi that has been used since ancient times can be made into a material such as a health food by mixing the substance with food. It should be able to be used actively. In addition, such tochi fruits have already been used since ancient times as one of the ingredients, and should be able to be taken with peace of mind.

  It is an object of the present invention to find a substance having a health effect derived from tochi seeds after punching.

  Another object of the present invention is to provide a technology for using a substance having a health effect derived from the seeds of tochi after punching.

  The above and other objects and novel features of the present invention will be apparent from the description of this specification and the accompanying drawings.

  Of the inventions disclosed in the present application, the outline of typical ones will be briefly described as follows.

  In view of the above problems, the present inventor has discovered a substance that has not been known so far while searching for a substance that exhibits a pharmacological action and a health effect after the punching process of Tochi. In other words, we identified saponins of punched processed tostenoids, which are food ingredients, and clarified their contents and chemical structure. It was also clarified that the novel saponins contained in the cut-out Tochinomi have an inhibitory effect on the increase in blood glucose level.

  The present invention has been made based on such a novel substance. That is, the present invention comprises a step of punching out tochi fruits and an extraction step of extracting deacetylescin (deacetylescin) from the tomato fruits after punching processing. And a method for producing a blood glucose level elevation inhibitor. The present invention is a method for producing a blood glucose level increase inhibiting substance, characterized by performing deacetylation treatment at the 22nd carbon position of escin.

  The substance for suppressing blood sugar level elevation of the present invention is characterized by having deacetylescin. In this configuration, the deacetyl escin is obtained by subjecting tomato seeds to an alkali treatment. Alternatively, the deacetyl escin can be obtained by alkali treatment of escin. The alkaline treatment of escin is a deacetylation treatment at the 22nd carbon position of escin.

  In addition, the present inventor has newly found out that a substance obtained from tochi seeds after the punching process, which has been thought to have no blood glucose increase inhibitory action, has a blood glucose increase inhibitory action. That is, the present invention is a method for producing a substance for inhibiting an increase in blood glucose level, comprising: a step of punching out the tomato fruit; and an extraction step of extracting desacyl escin from the tomato fruit after the punching process. is there. The present invention is a method for producing a substance for suppressing an increase in blood glucose level, which comprises performing deacylation treatment at the 21st carbon position of escin and deacetylation treatment at the 22nd carbon position.

  The substance for suppressing blood sugar level elevation of the present invention is characterized by having desacyl escin. In such a configuration, the desacyl escin is obtained by an alkali treatment of tochino fruit, and is administered at 200 mg / kg body weight or more.

  The food of the present invention is characterized by containing a blood glucose level elevation inhibiting substance produced by the above-described method for producing a blood sugar level elevation inhibiting substance, or the above-described blood sugar level elevation inhibiting substance. In such a food, the food is a health food having an effect of suppressing an increase in blood glucose level. Alternatively, the food is a confectionery.

  The food additive of the present invention is characterized by containing a blood sugar level increase inhibiting substance produced by the aforementioned method for producing a blood sugar level rise inhibiting substance, or the blood sugar level rise inhibiting substance described above.

  Among the inventions disclosed in the present application, effects obtained by typical ones will be briefly described as follows.

  The blood sugar level elevation-suppressing substance of the present invention is a substance found in the fruits of tochi after punching, and since the fruits of tochi after punching have been used edible since ancient times, It can be used with peace of mind for general foods, health foods, food additives, etc., and can develop functional foods that exhibit the function of suppressing blood sugar level elevation.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that components having the same function are denoted by the same reference symbols throughout the drawings for describing the embodiment, and the repetitive description thereof may be omitted.

(Embodiment 1)
The blood sugar level elevation inhibiting substance according to the present invention is produced by punching out the tomato fruits and then extracting the tochi fruits that have been punched out by hot water extraction or solvent extraction using alcohol such as ethanol. be able to.

  For example, the step of removing the tomato seeds is to dry the raw tomato seeds to a moisture content of 20% or less, and then immerse and peel the dried tomato seeds in water and expose to water or boil. It is sufficient to immerse or boil it in wood ash or an alkaline agent such as potassium carbonate, sodium hydroxide, lime or the like.

  Tochi fruits that can be used in practicing the present invention are the fruits of Aesculus turbinata. Of course, as long as it is a kind of cypress, it can be applied to, for example, western cypress (Aesculus hippocastanum L.) or cypress seeds planted in China.

  The extraction step after the punching process step may be performed by hot water extraction, for example. Such hot water extraction may be performed, for example, by boiling in hot water with water. What is necessary is just to manage hot water temperature in 50 degreeC or more. The reason for setting this temperature range is that if the temperature is lower than 50 ° C., the extraction efficiency is poor and it takes time.

  The extraction step may use an organic solvent such as alcohol. For example, in the case of extraction using alcohol, 0 (not including 0) to 100% or less of alcohol such as methanol, ethanol, butanol, or propanol may be used.

  The substance thus obtained exhibits suppression of an increase in blood glucose level. Such a blood glucose level increase inhibitor is deacetylescin and has a structure in which an acetyl group bonded to the 22nd carbon of escin is deacetylated and a hydroxyl group is bonded. The structure, pharmacology, and health effects of such substances have been described in detail in experiments described below.

  Further, in the above description, the case of producing a blood glucose level increase inhibiting substance using collected tomato fruits has been described. However, the present inventors have found that a blood glucose level increase inhibiting substance is a deacetyl ester having the chemical structure as described above. Since it is a thin substance, it may be produced by alkaline treatment of escin using escin as a starting material.

  For example, by using potassium carbonate, potassium hydroxide, sodium hydroxide or the like as an alkali agent, escin is hydrolyzed under the conditions of pH 8 to 14, and the functional group bonded to carbon at the 22nd position of escin is obtained. Deacetylation may be performed.

  The blood glucose level elevation-suppressing substance thus obtained may be a single substance having a blood glucose level elevation-inhibiting action or a mixture with other substances.

  Such a blood glucose level elevation inhibiting substance can be used by mixing with food. For example, foodstuffs, such as confectionery and a side dish, are mentioned. In particular, among foods, examples of confectionery include confectionery such as rice cake, dumpling, rice cracker, cookies, and goullets. As food, seasonings may be included.

  When mixing with such food, the content of deacetyl escin may be mixed within the range of 0.1 wt% to 50 wt%. However, when mixed into the seasoning, it may be mixed in the range of 0.1% by weight to 20% by weight in consideration of the fact that it is usually used in various dishes and ingested.

  Furthermore, the use as a food additive mixed with food is also considered. Such a food additive may be appropriately mixed with an extender or the like within a range of 100% by weight or less not including 0.

  In addition, application as a health food provided in the form of a liquid, an extract, a capsule, a granule, a pill, a tablet, a syrup and the like is also conceivable. What is necessary is just to adjust so that content in a deacetyl escin may be in the range of 5 weight%-100 weight% at the time of application as this health food.

  Next, as described above, it is possible to obtain deacetylescin as a blood glucose level increase inhibiting substance from the tomato seeds after the punching process, and the chemical structure of the obtained deacetylescin is known. The fact that the structure is deacetylated at the carbon site at the 22nd position of the chemical structure will be described based on experiments.

(1. Experimental material)
In this experiment, tochinomi collected in northern Hyogo Prefecture were used. As the collected Tochinomi, Aceculus turbinata seeds were used. Wood ash was obtained from Makino Wood Industry (Okayama). Pyridine-d ₅ containing 0.05% TMS was purchased from Wako Pure Chemical Industries (Osaka). β-escin, other specialty reagents, methanol for high performance liquid chromatography (HPLC) analysis, and distilled water were obtained from Nacalai Tesque (Kyoto).

(2. A method for punching out Tochinomi)
The dried tomato fruits were dipped in water for 4 days to peel the skin softly, and then 1 kg of tochi fruits (moisture 52.8%, w / w) was boiled with 3 times the amount of water for 1 hour. . A paste-like wood ash solution was prepared by adding 60 ° C hot water (1.4 L) to 1 kg of wood ash in order to remove the boiled tochi fruits. This was dipped in 1 kg of tochi fruits and left for a further 48 hours. Then, after checking the punched state by taste, the fruits of Tochi were washed with tap water. The judgment of the punched state was sensorially evaluated by a skilled worker based on the taste of the bitterness.

(3. Thin-layer chromatography (TLC) analysis of Tochinomisaponin)
TLC plates using silica gel 60F ₂₅₄ (manufactured by MERCK). The developing solvent used was a lower layer of chloroform / methanol / distilled water (65:35:10, v / v). For color development, a 10% sulfuric acid solution was sprayed and heated at 150 ° C. to confirm spots. As a standard substance for TLC, commercially available β-escin (Rf value 0.24) was obtained by alkaline hydrolysis by Yoshikawa et al. Described in Non-Patent Document 2 (desacylescin: Rf value 0.12).

(4. Extraction and quantification of saponin from Tochinomi)
In order to analyze the composition and content of saponins in tochinomi, extraction and purification of saponins from peeled tomato fruits before drilling was performed according to the method of Yoshikawa et al. That is, dried tomato fruits were immersed in water for 4 days to soften the skin, and then the skin was removed. 4 kg of this tochi fruit (water 52.2%, w / w) was immersed in methanol for 1 week and then filtered. The obtained methanol extract was concentrated to dryness to obtain 154 g of a dried product. This extract was applied to a Diaion HP-20 column (length 500 mm x inner diameter 60 mm, manufactured by Nippon Nensui Co., Ltd., Tokyo), and the saccharide fraction was collected with 3000 ml of distilled water and the fraction containing saponin with 3000 ml of methanol. The lipid fraction was eluted with 3000 ml of ethyl acetate. The methanol elution fraction on this column was concentrated to dryness to obtain 56 g of a crude saponin fraction.

  10 g of this crude saponin was applied to a Chromatorex ODS 1024T column (length 320 mm x inner diameter 32 mm, manufactured by Fuji Silysia Chemical Co., Aichi) and eluted in the order of 40% methanol 1000 ml, 90% methanol 1000 ml, 100% methanol 1000 ml. did. The target saponins were recovered in the 90% methanol elution fraction by TLC analysis using commercially available β-escin and desacyl escin as standard substances. When 90% methanol was dried under reduced pressure, 8.45 g of crude crystals were obtained. This operation was repeated, and 47 g of saponins were obtained from 4 kg (moisture 52.2%, w / w) of tochi fruits before punching. The saponins were extracted and quantified by the same method for the tomato seeds (water content 64.8%, w / w) after punching. 11 g of saponins purified using a Dianion HP-20 column and a Chromatorex ODS 1024T column were obtained.

(5. High-performance HPLC analysis and fractionation of tochinomisaponins)
For the HPLC analysis, LC-2010A system manufactured by Shimadzu Corporation and Chromatopack CR8A were used. YMC-Pack ODS AM (AM312-3) column (length 150 mm x inner diameter 6 mm) manufactured by YMC as an analytical column, and YMC-Pack ODS AM (AM 322 manufactured by YMC) as a preparative column. ) A column (length 150 mm × inner diameter 10 mm) was used. Methanol / 10 mM phosphate buffer (pH 2.7) (62:38, v / v) was used as the mobile phase, and the elution rates were 0.8 ml / min for the analytical column and 3.0 ml / min for the preparative column. Adjusted to min. Detection was performed at a wavelength of 203 nm.

(6. Treatment of Escin Ia, IIa, Ib, IIb with potassium carbonate solution and component analysis)
Each component of escin Ia, IIa, Ib, and IIb as the actual component of Tochi before punching was separated and purified by HPLC. The individual components were then allowed to stand at room temperature for 48 hours in a 5% potassium carbonate solution (pH 11.7) prepared to have almost the same alkalinity as when wood ash was immersed. After neutralization with 0.1 M aqueous hydrochloric acid, it was applied to a C18 maxi clean cartridge column (Oltech, Tokyo), washed with 40% methanol, eluted with 70% methanol, filtered through a 0.45 μm membrane filter, and subjected to HPLC analysis. It was.

(7. Instrument analysis)
JEOL JNM-A400 FT-NMR (400 Mz) manufactured by JEOL Ltd. was used for ¹ H-NMR and ¹³ C-NMR analysis. The sample was dissolved in pyridine-d ₅ containing 0.05% TMS and subjected to measurement. For mass analysis, an ion trap mass spectrometer model LCQ Deca XP manufactured by ThermoQuest was used, and analysis was performed by an electrospray ionization (ESI) method in a positive ion mode. The water content was analyzed using an infrared moisture meter EB-340MOC manufactured by Shimadzu Corporation.

(8. Glucose tolerance test with mice)
Male ICR mice were purchased from Nippon SLC (Shizuoka). Six-week-old male ICR mice were used for the glucose glucose tolerance test after 2 weeks pre-feeding using solid feed MF for experimental animals from Oriental Yeast Co., Ltd. (Tokyo) as feed. Blood was collected from the tail vein of mice fasted for 16 hours and blood glucose level was measured. Then, the saponin fraction which refine | purified the extracted methanol extract of a tomato seed with the Dianion HP-20 column and the Chromatorex ODS 1024T column was used as a test substance. The sample was suspended in 0.3 ml of physiological saline and orally administered using a stomach tube so that the mouse body weight would be either 0 or 300 mg / kg body weight.

  The test mice were 5 in the 0 mg sample group per 1 kg body weight and 6 in the 300 mg sample group. 30 minutes after administration of the sample, 0.5 kg of glucose dissolved in 0.1 ml of physiological saline per 1 kg of mouse body weight was orally administered using a stomach tube. After this glucose load, 0.5, 1, and 2 hours later, blood was collected from the tail vein of the mouse and the blood glucose level was measured. For the measurement of blood glucose level, Glutest Ace R (Sanwa Chemical Laboratory, Aichi) was used.

(9. Statistical processing)
The blood glucose level was expressed as mean ± standard deviation. Comparison of the blood glucose level of each saponin administration group with respect to the test substance 0 mg / kg body weight group was tested by Dunnett's method, and P <0.05 was considered significant.

(10. Experimental results and discussion)
a) About the saponin content in the tochi fruit before and after the punching process For the purpose of grasping the residual amount of saponin in the tochi fruit after punching, in the above manner, the tochi fruit before the punching process and the punching process. The saponin was extracted from the latter by the same operation, and the weights of the saponin fractions purified by the Dianion HP-20 column and the Chromatorex ODS 1024T column were compared.

  When these saponin fractions were analyzed by TLC, one major spot was confirmed at the same position as commercially available β-escin in the saponin fractions prior to punching. Further, in the saponin fraction after punching, a spot corresponding to desacyl escin, a spot corresponding to escin, and a main spot between escin and desacyl escin were confirmed. As a result, it was confirmed that the composition change of saponins occurred before and after punching.

  Comparing the weight of these saponin fractions, it was found that 47 g from 4 kg of tomato seeds (moisture 52.2%, w / w) before punching, and 64.8%, w / w) 11 g of saponins were obtained from 4 kg. Therefore, when the amount of saponin per dry solid weight of tochi seeds is calculated from the moisture content, 1.0% (w / w) per ton solid weight before punching treatment and 0.3% (w / w) was found to be contained, and it was confirmed that about 30% of the saponins remained even after the punching treatment.

b) About chemical change of saponin component in tochi seeds by punching process In order to investigate the chemical change of saponins in tochi seeds by punching process, saponin fraction extracted from tochi seeds before and after punching process is used. It was subjected to HPLC analysis. As shown in FIG. 1, as a result, the elution position of the tochino saponin before the punching process coincided with the retention times of the four components contained in the commercially available β-escin.

These elution patterns were consistent with the report of Yoshikawa et al. Furthermore, each peak of i, ii, iii, and iv in FIG. 1 (A) was fractionated and subjected to mass analysis in positive ion mode by ¹ H-NMR, ¹³ C-NMR, and ESI method. The results of the analysis agreed with the literature values described in Non-Patent Documents 2 and 9. From this, it was confirmed that the four peaks i, ii, iii, and iv in FIG. 1B correspond to escin Ia, IIa, Ib, and IIb, respectively.

  On the other hand, as shown in FIG. 1C, the tochino saponin component after the punching treatment showed new peaks of i ′, ii ′, iii ′ and iv ′. The desacyl escin hydrolyzed by the acyl group at the C21 position and the acetyl group at the C22 position of escin by the method of Yoshikawa et al. Described in Non-Patent Document 2 elutes around 6 minutes under the HPLC conditions of FIG. It was done. The peaks observed this time are considered to be that the acyl group at the C21 position or the acetyl group at the C22 position was eliminated from escin Ia, IIa, Ib, and IIb, respectively, by the alkali component of wood ash.

  Assuming that the acyl group at the C21 position of escin is cleaved, escin Ia and Ib, and IIa and IIb should have the same structure, and the peak should be two. Such a result was not obtained. Therefore, it was estimated that i ', ii', iii ', and iv' were those in which the acetyl group at the C22 position of escin Ia, IIa, Ib, and IIb was eliminated.

  In order to confirm this, the individual components of escin Ia, IIa, Ib, IIb derived from tochi fruits before punching were separated by HPLC and allowed to stand in 5% potassium carbonate solution (pH 11.7). . This reaction product was neutralized, subjected to pretreatment with a C18 maxiclean cartridge column and analyzed by HPLC. As shown in FIGS. 2 (A) to (D), escin Ia, IIa, Ib and IIb were respectively used. It was confirmed that i ′, ii ′, iii ′ and iv ′ were generated. From the above, it was considered that the peaks of i ', ii', iii ', and iv' were obtained by deacetylating each of escin Ia, IIa, Ib, and IIb. Therefore, it was confirmed that the acetyl group at the C22 position was selectively desorbed as shown in FIG.

  This is because the acetyl group at the C22 position of escin is sterically susceptible to hydroxide nucleophilic attack compared to the acyl group at the C21 position, and the acyl group at the C21 position has an α, β-conjugated carbonyl structure. This is probably because the nucleophilic attack of the hydroxide ion on the carbonyl carbon of the acyl group was prevented.

  In addition, when comparing FIG. 1 (B) and FIG. 1 (C), the reason why the abundance ratio of escins differs before and after the punching process is that the tigloyl group at the C21 position of i ′ and ii ′ is , Iii 'and iv' had less steric hindrance effect than the angeloyl group, and it was assumed that i 'and ii' were hydrolyzed and converted to desacyl escin over iii 'and iv'.

c) Chemical structure of escin-derived saponin produced by punching process In order to confirm the chemical structure of i 'to iv' in Figure 2 generated by punching process, each was isolated by HPLC, mass spectrometry, ¹ H Structural analysis was performed by -NMR and ¹³ C-NMR. As a result of mass spectrometry, as shown in Table 1, a molecular ion peak corresponding to the elimination of the acetyl group at the C22 position of escins was obtained. ¹ H-NMR analysis confirmed the presence of the tigloyl group at the C21 position of escin Ia and IIa and the angeloyl group of Ib and IIb as shown in Table 2.

Furthermore, as a result of the ¹³ C-NMR analysis shown in Table 3, a signal corresponding to a tigloyl group at the C21 position or an angeloyl group was detected as compared with the data for escins before punching. On the other hand, ¹ H-NMR and ¹³ C-NMR signals indicating the acetyl group at the C22 position of escins after punching were not observed. From the above results, i 'and ii' in HPLC are iii ', and iv' is a hydrolyzed acetyl group at the C22 position of escin Ia, IIa, Ib, and IIb as shown in FIG. It was confirmed that. As a result, the chief saponin of Tochino fruit that was punched and edible was identified as a compound in which the acetyl group at the C22 position was hydrolyzed by escins by the alkali component of wood ash.

  That is, i 'is 21-O-tigloylprotoaescigenin 3-O- [β-D-glucopyranosyl- (1-2)] [β-D-glucopyranosyl- (1-4)]-β-D-glucuronopyranosyl acid, ii' 21-O-tigloylprotoaescigenin 3-O- [β-D-xylopyranosyl- (1-2)] [β-D-glucopyranosyl- (1-4)]-β-D-glucuronopyranosyl acid, iii 'is 21-O- angeloylprotoaescigenin 3-O- [β-D-glucopyranosyl- (1-2)] [β-D-glucopyranosyl- (1-4)]-β-D-glucuronopyranosyl acid, and iv 'is 21-O-angeloylprotoaescigenin 3- It was determined as O- [β-D-xylopyranosyl- (1-2)] [β-D-glucopyranosyl- (1-4)]-β-D-glucuronopyranosyl acid.

  Of these four saponins, i 'and iii' have the same chemical structure as Aesculioside A and Aesculinoside B isolated from Chinese Aceculus chinenis, as described in Non-Patent Document 10. Chinese Tochinomi is said to be a raw material for herbal medicine called “Sha Luo Zi”, which is used for the purpose of healthy stomach and analgesia. However, as far as we know, there are no reports on saponins having the chemical structures ii 'and iv'.

d) About the inhibitory effect on the blood glucose level of the untreated Totonomi saponin The saponins obtained from the untreated Tochino fruit were subjected to a glucose tolerance test using male ICR mice to suppress the increase in the blood glucose level. Examined. As a result, as shown in FIG. 4, the 300 mg / kg administration group of tochinomisaponins that had been subjected to punching treatment was 0 mg / kg at both the glucose load 0.5 hour (P <0.01) and 1 hour (P <0.05). The value was significantly lower than that of the administration group.

  In the figure, ◯ indicates control (n = 5), and ● indicates 300 mg / kg body weight (n = 6) derived from tochinomis after punching treatment.

  In previous studies, as described in detail in Non-Patent Documents 2 to 5, the inhibitory action of escin Ia, IIa, Ib, IIb contained in Western seedlings (Aesculus hippocastanum L.) was suppressed. Has been. According to this, the acyl group at the C21 position and the acetyl group at the C22 position are essential structures for exhibiting the activity. However, in this report, only escins having both an acyl group at the C21 position and an acetyl group at the C22 position, and desacyl escin I and II from which both the acyl group and the acetyl group have been removed by hydrolysis, are investigated. .

  However, at this time, the action of the deacetylated product at the C22 position derived from the punched tomato seeds found by the experiment of the present inventor has not been studied at all. As a result of analysis by HPLC, a peak corresponding to desacyl escin was confirmed in the sample used for the test this time.

  However, Non-Patent Documents 2 and 5 report that desacyl escin I and II show no inhibitory effect on the increase in blood glucose level even when administered at 100 mg / kg body weight in a test using rats. Therefore, it was determined that desacylescin in the sample had no effect on the suppression of the increase in blood glucose level.

  Further, in the analysis by the above HPLC, trace peaks corresponding to escin Ib and IIb were also observed. Of the saponins in the sample, escin Ib was calculated to be 1.24% and IIb to be 0.87%. In this study, when the untreated Tochino saponins were administered to mice, a significant blood glucose level inhibitory effect was confirmed at 300 mg / kg, of which 6.3 mg was a mixture of escin Ib and IIb. It corresponds to.

  Therefore, for the purpose of investigating the involvement of the mixture of escin Ib and IIb in the inhibitory effect on the increase in blood glucose level, 6.3 mg / kg of this mixture was administered per kg of mouse body weight, and the inhibitory effect on the increase in blood glucose level was examined. As a result, the effects of escin Ib and IIb were not significantly different from those in the 0 mg / kg administration group.

  Based on the above results, the active body exhibiting the inhibitory effect on the increase in blood glucose level this time is saponins having a structure in which the acetyl group at the C22 position is eliminated, i ′ and ii ′ are saponins including iii ′ and iv ′. It can be said. Yoshikawa et al., As described in Non-Patent Document 5, with regard to the mechanism of action of the inhibitory activity of triterpene glycosides such as escin and gymnemic acid on the blood sugar level increase inhibitory action, the slow movement of sugar from the stomach to the jejunum and the inhibition of glucose absorption in the small intestine It has been reported that it is also involved in the enhancement of small intestinal motility. Therefore, it is surmised that the deacetylated escins i 'and ii' function in the same mechanism of action for iii 'and iv', but further research is necessary for details. In the future, it is expected that further new actions will be found by examining various biological activities.

  From the results of the above-mentioned experiment in the present invention, 1) the punched tochi fruits used as foods contained 0.3% saponins per dry solid weight, and 2) the punched tochi fruits The main saponins were four types of components with the structure in which the acetyl group at the C22 position of escins was eliminated. 3) Using the saponin fraction separated from the cut-out tochi fruits, When a glucose tolerance test using mice was performed, the 300 mg / kg addition group significantly inhibited the increase in blood glucose level compared to the 0 mg / kg control group at 0.5 hours and 1 hour after glucose load. It has been clearly confirmed that the present inventors have found for the first time that the edible tomato seeds that have been extracted with wood ash liquid contain a new functional food factor produced during food processing.

(Embodiment 2)
In the present embodiment, unlike the above-described embodiment, the blood glucose level inhibitory action of each saponin isolate obtained after the punching treatment was examined.

  In the above-described embodiment, it has been shown that the chemical conversion product of escin is produced by punching out the water flea. That is, they identified deacetylescin Ia, IIa, Ib, and IIb in which the acetyl group at the C-22 position of each oleanane skeleton of escin Ia, IIa, Ib, and IIb was hydrolyzed. In addition, it was clarified that the saponin fraction containing these deacetyl escins suppresses the increase in blood glucose level. These deacetyl escins were contained in a considerable amount in the tochino actually used for food.

  To date, the food functionality of the individual components contained in these deacetylescin fractions has not been studied so far. In the present embodiment, a glucose tolerance test was conducted in mice using isolates of deacetylescin Ia, IIa, Ib, IIb and related substances, and their structure-activity relationship was examined. At the same time, the possibility of an inhibitory effect on digestive enzymes using polysaccharides or disaccharides as a substrate on the inhibitory action on the increase in blood glucose level exhibited by Tochinomi-derived saponins was also examined.

(1. Experimental material)
We used toss collected in the northern part of Hyogo Prefecture. Β-escin as a standard product was purchased from Wako Pure Chemical (Osaka). Other reagents were obtained from Nacalai Tesque (Kyoto) or Wako Pure Chemical (Osaka).

(2. Extraction, separation, and purification of saponin from Tochinomi)
Extraction, separation and purification of saponins were performed from the peeled toskin before punching according to the method of Yoshikawa et al. That is, the dried torch was immersed in water for 4 days to soften the skin, and then the skin was removed. 4 kg of this Tochinomi was soaked in methanol for another week and then filtered. The methanol extract of the obtained filtrate was concentrated to dryness to obtain 154 g of a dried product.

  This extract was applied to a Dianion HP-20 column (length 500 mm x inner diameter 60 mm, manufactured by Nippon Nensui, Tokyo), and the saccharide fraction was collected with 3000 ml of distilled water, and the fraction containing saponin with 3000 ml of methanol. The lipid fraction was eluted with 3000 ml of ethyl acetate. The methanol elution fraction in this column was concentrated and dried to obtain 56 g of a crude saponin fraction.

  10 g of this crude saponin was applied to a Chromatorex ODS 1024T column (length 320 mm x inner diameter 32 mm, manufactured by Fuji Silysia Chemical, Aichi) and eluted in the order of 40% methanol 1000 ml, 90% methanol 1000 ml, 100% methanol 1000 ml. . The 90% methanol elution fraction was dried under reduced pressure to obtain 8.45 g of crude crystals. This operation was repeated 6 times, and 47 g of saponins were obtained from 4 kg of the tocinomi before punching. 11 g of saponins were obtained in the same manner with respect to 4 kg of tochinomis after punching.

  In order to purify the individual components from these saponin fractions, they were subjected to reversed-phase high performance liquid chromatography (HPLC). From 3 g of the saponin fraction before extraction, escin Ia 255 mg, IIa 126 mg, Ib 169 mg and IIb 149 mg were isolated. Moreover, 58 mg of deacetylescin Ia, 34 mg of IIa, 102 mg of Ib, and 80 mg of IIb were obtained from 3 g of the saponin fraction after the punching treatment.

  For the HPLC analysis, LC-2010A system manufactured by Shimadzu Corporation and Chromatopack CR3A were used. This analyzer was equipped with a YMC-Pack ODS AM (AM 322) column (length 150 mm x inner diameter 10 mm) manufactured by YMC, and methanol / 10 mM phosphate buffer (pH 2.7) (62:38, v / v) was used as the mobile phase, and elution was performed at an elution rate of 3 ml / min, and detection was performed at a wavelength of 203 nm.

  The chemical structure of the isolate was confirmed by mass spectrometry using 1H-NMR, 13C-NMR, and positive ion mode electrospray ionization (ESI).

(3. Thin-layer chromatography (TLC) analysis of Tochinomisaponin)
For separation of saponins, TLC plates of silica gel 60F ₂₅₄ (Merck, Darmstadt, Germany) were used, and the lower layer of the chloroform / methanol / water (65:35:10, v / v) mixture was used as the developing solvent. It was. For detection of the compound on the TLC plate, color was developed by spraying a 10% sulfuric acid solution and heating at 150 ° C. As a TLC standard substance, commercially available β-escin (Rf value 0.24) or desacyl escin (Rf value 0.12) obtained by alkaline hydrolysis of β-escin by the method of Yoshikawa et al. Was used.

(4. Preparation of desacyl escin (I + II) used for glucose tolerance test)
The reagent β-escin was subjected to the reverse-phase HPLC described above, and the eluted escin Ia, IIa, Ib, IIb was recovered, and desacyl escin (I + II) was obtained by alkaline hydrolysis by the method of Yoshikawa et al. .

(5. Glucose tolerance test with mice)
Male ICR mice purchased from Japan SLC (Shizuoka) were used as experimental animals. A 6-week-old mouse was used as a feed, fed with a solid feed MF for experimental animals from Oriental Yeast Co., Ltd. (Tokyo), and pre-bred for 1 week before being used for a glucose sugar tolerance test. After collecting blood from the tail vein of a mouse fasted for 16 hours and measuring the blood glucose level, as a test substance, the saponin fraction derived from tochinomi per mouse weight 1 kg before or after the punching process, Alternatively, the isolated component was administered.

  That is, the saponin fraction before or after the punching treatment was 200 mg, and the desacyl escin (I + II) was 100 mg or 200 mg. Further, escin Ia, IIa, Ib, IIb and deacetyl escin Ia, IIa, Ib, IIb, which were isolated purified preparations, were prepared to be 100 mg each.

  Each preparation was suspended in 0.3 ml of physiological saline and orally administered using a stomach tube. In each test, 5 mice were used per group. Thirty minutes after administration of the sample, 0.1 ml of physiological saline containing 0.5 g of glucose per 1 kg of body weight of the mouse was orally administered using a stomach tube. At 0.5, 1, and 2 hours after this glucose load, blood was collected from the tail vein of the mouse and the blood glucose level was measured using Glutest Ace R (Sanwa Chemical Laboratory, Aichi).

  The blood glucose increase value is obtained by subtracting the blood glucose level before administration of the saponins from the blood glucose level at the time of collection of the sample after glucose loading. Then, the blood glucose elevation value of each saponin-administered group was compared with the control 0 mg / kg body weight test sample, and a significant difference was tested by Dunnett's method. Data are shown as mean ± standard error. The case of P <0.05 was considered significant.

  In addition, although desacyl escin (I + II) was prepared as mentioned above, the mixing ratio of desacyl escin I and II was 58:42.

(6. Experimental results and discussion)
a) TLC analysis of saponins of Tochinomi Tochinomi was extracted with methanol before or after punching, and the resulting extract was purified with Dianion HP-20 and Chromatorex ODS 1024T column. These saponin fractions were confirmed by TLC on silica gel G60 as shown in FIG.

  As a result, spots corresponding to the standard β-escin were confirmed from the chisel before punching. On the other hand, after the punching process, deacetylescin and desacylescin were recognized as main products. A trace amount of escin, a major component of natural Tochinomi, was detected.

  As shown in FIG. 5, escin and deacetylescin derived from Tochinomi collected by reverse-phase HPLC showed one spot each on the TLC plate for use in measuring the inhibitory activity of carbohydrase.

  In each of the spots in FIG. 5, 1 is a standard β-escin, 2 is a purified desacyl escin, 3 is a saponin fraction before punching, 4 is a saponin fraction after punching, 5 shows escin collected by HPLC, and 6 shows deacetylescin collected by HPLC.

b) Inhibition of blood glucose level increase by saponin component isolated from Tochinomi 30 minutes before the glucose tolerance test, various amounts of saponin component derived from Tochinomi are administered to mice and then glucose is administered. Then, the inhibitory effect on the increase in blood glucose level was examined.

  FIG. 3 shows the rate of increase in blood glucose level of each administration group relative to the group not administered saponin 30 minutes after glucose loading. As shown in FIG. 6 (A), the administration group of the saponin fraction before and after the punching treatment significantly showed an inhibitory effect on the increase in blood glucose level, as shown in FIG. 6 (A). Comparing the two at the dose of 200 mg / kg body weight, the saponin fraction derived from the natural totiss before the punching process had a stronger inhibitory effect than the saponin fraction derived from the edible tossies after the punching process. It was.

  In addition, for desacylescin (I + II), a significant inhibitory effect was not obtained at a dose of 100 mg / kg body weight, consistent with the report of Yoshikawa et al. However, the administration of 200 mg / kg body weight significantly suppressed the increase in blood glucose level, contrary to the initial expectation. This is the result that desacyl escin, which was previously thought to have no inhibitory effect on blood glucose level in the administration system up to 100 mg / kg body weight, actually shows an inhibitory effect on blood glucose level in the administration system of 200 mg / kg body weight or more. Thus, the present invention has been clarified for the first time.

  Next, four types of deacetyl escin derived from edible tochinomis after extraction (Figure 6B) isolated by various chromatography and HPLC, and four types of escin from natural totinomis before extraction (Fig. 6B). The effect of 6C) was examined.

  When deacetylescin Ia, IIa, Ib, IIb and escin Ia, IIa, Ib, IIb isolates were each administered at 100 mg / kg, the rate of increase in blood glucose level 30 minutes after glucose loading was When the saponin non-administration group was 100%, deacetylescin Ia, IIa, Ib, and IIb were 85, 84, 75, and 71%, respectively, whereas escin Ia, IIa, Ib, and IIb were 46, 53, respectively. 71, 46% increase.

  From this, it has been clarified that deacetylescins have an action of significantly suppressing an increase in blood glucose level, although the inhibitory activity is weaker than that of escins. In other studies so far, there has been no report examining an inhibitory effect on an increase in blood glucose level using an isolated component of deacetyl escins derived from an edible tochinomi.

  From the above results, escins> deacetylescins> desacylescins in the order of strong inhibitory action on the increase in blood glucose level of saponins of tochinomi, and the acyl group at position 21 and acetyl group at position 22 of escin It was found to be essential for exerting an inhibitory effect on the increase in the amount. In 1 hour and 2 hours after the glucose load, no significant difference was observed in any of the administration groups compared to the non-administration group.

  Although the escins and deacetylescins derived from this tochinomi exhibited α-amylase inhibitory activity, the inhibitory activity was very weak. The main mechanism of action for suppressing the increase in blood glucose level observed in animal experiments using mice appears to be inhibition of glucose absorption in the small intestine.

  In other words, when a glucose tolerance test was performed in mice using isolates of each saponin component derived from tochinomis before or after punching, the four types of edible tochinomis after punching were found. All of the deacetylescins showed the same activity to suppress the increase in blood glucose level. The activity of suppressing the increase in blood glucose level of escin-related substances tended to increase in the order of escins> deacetyl escins> desacyl escins. From this, it became clear that the acyl group at the 21st position and the acetyl group at the 22nd position of escins are greatly related to the strength of the inhibitory activity on the increase in blood glucose level.

  Desacylescin, which is a substance for suppressing blood sugar level increase in such a configuration, may be a single substance having a suppressive action for increasing blood sugar level or may be a mixture with other substances.

  Such a blood glucose level elevation inhibiting substance can be used by mixing with food. For example, foodstuffs, such as confectionery and a side dish, are mentioned. In particular, among foods, examples of confectionery include confectionery such as rice cake, dumpling, rice cracker, cookies, and goullets. As food, seasonings may be included.

  In the case of mixing with such food, the content of desacyl escin may be mixed within the range of 80 wt% or less not including 0. In addition, when it is mixed into the seasoning, it may be mixed in the range of 80% by weight or less not including 0, considering that it is usually used for various dishes and ingested.

  Furthermore, the use as a food additive mixed with food is also considered. Such a food additive may be appropriately mixed with an extender or the like within a range of 100% by weight or less not including 0.

  In addition, application as a health food provided in the form of a liquid, an extract, a capsule, a granule, a pill, a tablet, a syrup and the like is also conceivable. What is necessary is just to adjust so that content of a desacyl escin may become the range of 100 weight% or less which does not contain 0 in the case of application as this health food.

(Embodiment 3)
In the present embodiment, the bitterness of the Tochinomi saponin fraction before and after the punching process was compared based on a sensory test.

  The tochinomi saponin (B) purified by the Dianion HP-20 column and the Chromatorex ODS 1024T column before or after the punching process was dissolved in distilled water and adjusted to a concentration of 1.2 mg / ml.

  A solution (1.2 mg / ml) having the same concentration was prepared for the saponin fraction before and after the punching treatment, and a sensory test was performed. As a result, all subjects showed that the saponin fraction before the punching treatment had a considerably stronger bitter taste than the saponin fraction after the punching treatment.

  The concentration at which the subjects were able to discriminate bitterness was examined, and it was found that all subjects could be discriminated up to a 1000-fold dilution of the 1.2 mg / ml saponin solution before the punching process, that is, a concentration of 1.2 mg / ml. Therefore, for a solution having the same bitterness as that of the 1.2 mg / ml saponin solution before the punching treatment, the 1.2 mg / ml saponin solution after the punching treatment was sequentially diluted, that is, 1.2, They were selected according to taste from 2.4, 12, and 120 mg / ml solutions.

  As a result, 1 out of 8 people who had the same bitterness in the 1.2 mg / ml saponin solution before punching treatment and the saponin solution between 2.4 and 12 mg / ml after punching treatment 3 out of 8 people were equivalent to the later 12 mg / ml saponin solution, and 4 out of 8 people evaluated the bitterness of the saponin solution between 12 and 120 g / ml after punching treatment. It was.

  As a result of such sensory test, the saponin fraction after the punching treatment contains deacetyl escin and desacyl escin as main components, so that the acyl group at the C-21 position of escin and the C-22 position It was revealed that when the organic acid ester side chain such as an acetyl group is hydrolyzed by alkali, the bitterness is reduced to about 1/10 or more.

  The saponin derived from edible tonomi after the punching process has a weaker inhibitory effect on the increase in blood glucose level than that before the punching process, but the bitterness is considerably reduced. It can be said that there is a big merit as.

  Compared with the saponin fraction derived from the natural tochinomi before the punching process, the saponin fraction derived from the edible tochinomi after the punching process clearly showed a reduction in bitterness at the same concentration.

  Saponins hydrolyzed by escin organic acid ester contained in edible tochinomi have greatly reduced bitterness compared to escin, and also have an inhibitory effect on the increase in blood glucose level. More preferable as a material.

  As mentioned above, the invention made by the present inventor has been specifically described based on the embodiment. However, the present invention is not limited to the embodiment, and various modifications can be made without departing from the scope of the invention. Needless to say.

  INDUSTRIAL APPLICABILITY The present invention can be used in the fields of health foods, functional foods, and the like that show an increase in blood sugar level.

(A)-(C) are charts showing the analysis results of reversed-phase HPLC, (A) is about commercially available β-escin, (B) is about saponins derived from tochi fruits before punching treatment, (C) It is an analysis result about the saponin derived from the Tochi fruit after the punching process. It is the chart which shows the analytical result of reverse phase HPLC of the alkaline reaction substance of escin Ia, IIa, Ib and IIb, (A) upper part is escin Ia, (B) upper part is escin IIa, (C) upper part is escin Ib. The upper row of (D) relates to escin IIb, and the lower row of each relates to after the reaction after alkali treatment with 5% potassium carbonate. It is explanatory drawing which shows the chemical structure of the saponin derived from the Tochino fruit before and after the punching process. It is explanatory drawing which shows the blood glucose level raise effect in the mouse | mouth of the saponin derived from the Tochi fruit after a punching process. It is explanatory drawing which shows the result of the TLC analysis of the saponin fraction derived from the Tochinomi before and after the punching process. (A) is an explanatory diagram showing an inhibitory effect on blood glucose level such as desacyl escin, (B) is an explanatory diagram showing an inhibitory effect on blood glucose level of deacetylescin, and (C) is an explanatory diagram showing an inhibitory effect on blood glucose level of escin.

Claims (10)

A step of punching out the fruits of Tochi,
And a step of extracting deacetylescin from the tomato seeds after the punching process. A method for producing a blood sugar level increase inhibitor comprising deacetylescin as an active ingredient . A step of punching out the fruits of Tochi,
And a step of extracting desacyl escin from the tomato seeds after punching, and a method for producing a blood glucose level increase inhibitor comprising desacyl escin as an active ingredient . A method for producing a blood glucose level increase inhibitor comprising deacetylescin as an active ingredient, wherein the deacetylation treatment is performed at the 22nd carbon position of escin . Desacyl escin and deacetyl escin as active ingredients , characterized by performing deacylation treatment at the 21st carbon position of escin and deacetylation treatment at the 22nd carbon position Manufacturing method. Blood glucose increase inhibitor containing deacetyl S. Singh as an active ingredient. The blood sugar level increase inhibitor according to claim 5,
The deacetyl S. Singh, inhibiting an increase in blood glucose level agent characterized by being obtained by alkaline treatment the fruit of chestnut. The blood sugar level increase inhibitor according to claim 5,
The deacetyl S. Singh, inhibiting an increase in blood glucose level agent characterized by being obtained by alkaline treatment escin. The blood sugar level increase inhibitor according to claim 7,
The alkali treatment, blood sugar increase inhibitor, which is a deacetylation process of the 22-position carbon position of escin. Blood glucose increase inhibitor comprising as an active ingredient Desashiruesushin. The blood sugar level increase inhibitor according to claim 9,
The Desashiruesushin is obtained by alkaline treatment of solid chestnut, increase in blood glucose level inhibitor which comprises administering an above 200 mg / kg body weight.

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