JPH0421475B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention provides a method for extracting antioxidant substances from cultured cells of sesame (Sesamum indicum L.) using hydroalcohol, and an oil-insoluble substance obtained by the method. It relates to water-insoluble antioxidant substances and is applied to the food and pharmaceutical industries. (Conventional technology and problems) Foods are manufactured from agricultural products, marine products, livestock products, etc. However, during the storage and preservation processing of food ingredients and products, microbial contamination and spoilage occur.
Food ingredients and products deteriorate due to chemical and physical effects, reducing their commercial value. For this reason, various food additives have been developed, as well as methods such as temperature treatment, deoxidation treatment, vacuum packaging, low temperature storage, and radiation treatment, and some of them have been put into practical use. Among these types of deterioration of food materials and products, the most problematic is the oxidation or peroxidation reaction of food components caused by oxygen in the air. Oxygen is important for the life support of living things through respiration, but on the other hand, as it is a highly reactive compound, it reacts with various components in food, oxidizing or peroxidizing them.
It is known that not only does it reduce the value of the product, but it also causes the production of harmful substances in food. For example, nutritionally essential saturated fatty acids such as linoleic acid and linolenic acid contained in foods are easily peroxidized by oxygen in the air, producing peroxidized fatty acids and reactive radicals (free radicals). It has also been reported that carcinogenic substances such as malondialdehyde are produced. (“Mutagen and Toxicity” Vol. 5, p. 243 (1982), “Food Packaging”
Volume 17, p. 106 (1986)). To prevent such lipid peroxidation,
Packaging techniques such as oxygen absorbers to remove oxygen from packaging, vacuum packaging, and nitrogen gas displacement packaging are used. With the development of the chemical industry, synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been commonly used. However, as the use of these synthetic antioxidants increases, food pollution increases, leading to major safety issues, and consumers' negative reaction to synthetic antioxidants becomes stronger, leading to a decline in their usage. The current situation is that As mentioned above, it is thought that peroxides and carcinogens that are generated in animal bodies due to the toxic effects of oxygen have a negative effect on animal cells. is thought to be related to cell aging and, ultimately, to lifespan (free radical aging theory). Therefore, highly safe, naturally derived antioxidants are used in foods, especially health foods and nutritional foods, as well as in the technical fields of pharmaceuticals and cosmetics, as substances that support the body's antioxidant defense mechanism. In,
Highly anticipated. However, natural antioxidants, which are expected to be used in place of synthetic antioxidants that pose food pollution problems, are derived from plants and animals that are influenced by natural conditions such as weather, and therefore cannot be supplied stably. difficult,
In addition, the content is very small, extraction is very difficult, and the ingredients change during extraction, so only a few natural vitamins such as vitamin E and vitamin C have been put into practical use. It's just that. The present invention uses cultured cells containing antioxidant substances instead of extracting them from plants, and the natural antioxidant substances are obtained by culturing cultured cells in large quantities and subjecting them to hydroalcohol extraction. This is the first time that we have succeeded in industrially mass-producing a product, but this is completely unknown and new. (Problem to be solved by the invention) As described above, cultivating and producing plants by agricultural means and extracting useful components from them is difficult if carried out in a non-industrial manner on a laboratory scale. ,
The supply of these plant-derived useful substances is through agricultural means, so there is a limit to production efficiency.
Mass production requires large areas of farmland, and cultivation is easily affected by the weather, so there are many difficulties in ensuring a stable supply of industrial raw materials. There were problems such as difficulties in producing it, and there was a desire to develop new technology. Among the active ingredients of these plants, antioxidant substances in particular contain only trace amounts, and extraction itself is extremely difficult, and the ingredients often change during extraction. Antioxidant substances are industrially extracted in large quantities from adult plants,
It was impossible to manufacture it. (Means for Solving the Problems) The present invention has been made to solve these drawbacks all at once. In other words, in order to improve the extraction method from natural products, which are made from plants that are directly affected by natural conditions and whose production efficiency is extremely low, we have conducted research and examination from various angles, and as a result, we have developed an artificial culture method. It was concluded that the method is optimal. Therefore, we conducted thorough research on artificial culture, focused on proliferative cell mass (callus) artificially induced from plants, and researched technology to produce useful substances by culturing this. I went. In the process, we investigated the products contained in callus cells derived from many types of plants, and as a result, we not only succeeded in growing proliferative cells from sesame (Sesamum indicum L.) plants for the first time. discovered for the first time that proliferated cells contain a powerful new antioxidant substance and that it could be advantageously extracted using hydroalcohol. Based on this new knowledge, further research led to the completion of the present invention. It was done. In other words, the present invention provides a method for culturing proliferative cells of sesame plants, extracting the resulting cultured cells with hydroalcohol, and thereby producing a large amount of new natural antioxidant substances. In other words, the present invention is directed to the development of a new antioxidant derived from safe natural products that does not cause food pollution, and the development of a method that can be mass-produced in place of the extraction method from adult plants, which can extract only a very small amount. This is truly groundbreaking, as it has succeeded in solving all of the conventional technical problems in one fell swoop. That is, the present invention uses sesame (Sesamum indicum).
A yellow-brown extract with oil-insoluble and water-insoluble antioxidant properties extracted with hydroalcohol from cultured cells of proliferative cells derived from adult L. It is related to. It has long been known that sesame plants and sesame oil contain antioxidant substances such as vitamin E and lignan compounds (Agricultural and Biological Chemistry). (Chemistry)
Vol. 49, p. 301 (1985), Journal of Japan Food Industry Association,
Volume 32, page 407 (1985)). However, it has been discovered that proliferative cells from sesame plants contain strong antioxidant substances, and that they can be efficiently extracted industrially using hydroalcohol without causing any alteration or deterioration. , is completely unknown so far, and there is no suggestion of it. Antioxidant substances derived from sesame, which have been known for a long time, are contained in sesame seeds, and are obtained by squeezing or extracting them during the production of sesame oil. (large) substance. On the other hand, the antioxidant substance according to the present invention is derived from cultured cells rather than adult plants such as sesame seeds, and is extracted with hydroalcohol, so it has an intermediate property between hydrophilicity and lipophilicity. The conventionally known antioxidant substances derived from adult sesame plants have the following properties:
They are completely different not only in their origins but also in their physical properties (for example, polarity), and substances with such antioxidant properties are completely unknown new substances. To carry out the present invention, it is first necessary to create sesame-derived proliferative cells as follows. As sesame, use buds, roots, or seeds. Then, shoots are prepared under sterile conditions, and the shoots, stems, leaves and/or root sections are cultured in a solid and/or liquid medium to induce callus cells. The proliferative callus obtained is grown into a large callus by subculture. This is then cultured stationary and/or with stirring in a solid and/or liquid medium to proliferate callus cells. As the medium, various media can be used, and as the carbon source, in addition to monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as oligosaccharides and starch sugars can also be used. . Nitrogen sources include nitrate nitrogen such as ammonium nitrate and potassium nitrate, ammonia nitrogen such as ammonium sulfate and ammonium tartrate, as well as casamino acids, amino acids, peptone, corn staple liquor, yeast cells, yeast extract, and malt extract. etc. can be used. In addition, vitamins such as nicotinic acid, nicotinamide, thiamine, folic acid, and biotin; nucleic acid-related substances such as inositol, adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, and cyclic AMP; iron, manganese, zinc, boron, and iodine. ,
Minerals such as potassium, cobalt, magnesium, molybdenum, phosphorus, and copper are also used. An example of the basic medium is shown in Table 1 below. Table 1 Ammonium nitrate 1650mg Potassium nitrate 1900 Calcium chloride 440 Magnesium sulfate 370 Potassium phosphate 170 Boric acid 6.2 Manganese sulfate 22.3 Zinc sulfate 8.6 Iodopotassium 0.83 Sodium molybutate 0.25 Cobalt chloride 0.025 Copper sulfate 0.025 Sodium ethylenediaminetetraacetate 37.3 Ferrous sulfate 27.8 Myo-inositol 100 Glycine 2 Pyridoxine hydrochloride 0.5 Nicotinic acid 0.5 Thiamine hydrochloride 0.1 Sucrose 30g Water 1000ml PH 5.7 It is preferable to add auxin and cytokinin to the basic medium.As auxin, indoleacetic acid, indolebutyric acid, naphthaleneacetic acid,
2,4 dichlorophenoxyacetic acid and the like are appropriately used. Further, as the cytokinin, benzyladenine, kinetin, etc. can be used. Although these plant hormones and cytokinins can be used alone, it is effective to use them in combination.
For culturing proliferative callus, a medium with the composition shown in Table 1 may be used, but to further improve proliferation, coconut milk, casein hydrolyzate, ginger extract, cornstarch liquor, yeast extract, etc. It is effective to add natural organic nutrients such as malt extract and malt extract. The culture temperature is 20
Cultivation can be carried out at ~37°C, but preferably at 25°C – 35°C.
It is ℃. A slightly acidic pH of the culture solution (PH5.6-6.0) is advantageous for growth. In order to extract antioxidant substances from proliferative cells obtained by culturing, biological treatment using cellulase or lysozyme, chemical treatment, mechanical or ultrasonic treatment, or a combination thereof can be used. The cells can be recovered by destroying the cells and extracting with methanol, ethanol, acetone, chloroform, other organic solvents, water, etc., or a mixture of these organic solvents and water. As the extraction solvent, the latter is particularly advantageous, and it is preferable to use, for example, a hydrous alcohol such as 80% ethanol. The activity of antioxidants is analyzed by measuring the amount of autooxidation by air using linoleic acid as a reaction substrate using the Rodan iron method. This method is a conventional method used to examine lipid peroxidation. (Agricultural and Biological Chemistry)
Chemistry, Vol. 45, p. 735 (1981)). The present invention will now be explained in detail step by step. 1. Preparation of aseptically grown sesame sprouts Sesame seeds were first immersed in a 75% ethanol aqueous solution for a few seconds, then washed with sterilized water, and then 0.1%
Microorganisms attached to the seeds are sterilized by soaking them in benzalkonium chloride (commercially available fungicide) solution for 2 to 5 minutes. After thoroughly washing the seeds with sterilized water, they were treated with a 1% sodium hypochlorite solution (containing 0.1% surfactant Tween 20) for 30 minutes to completely sterilize the sesame seeds. do. Meanwhile, put sterilized gauze and water in a sterilized wide-mouth container with a lid (commercially available plastic product).
Sesame seeds are germinated on this to prepare sterile sprouts. 2. Induction of proliferative callus from sesame seeds Table 1
Naphthaleneacetic acid (10 ^-8 to 10 ^-5 M) or 2,4-dichlorophenoxyacetic acid (10 -8 to 10 ^-5 M) was added as auxin to the basic medium with the composition shown in .
10 ^-5 M), a combination of benzyladenine (10 ^-6 to 10 ^-4 M) or kinetin (10 ^-6 to 10 ^-4 M) as cytokinin, 0.2% dielan gum as a solidifying agent, and sterilization. After that,
It was dispensed into a petri dish and solidified. Cut pieces of sesame sprouts prepared aseptically are transplanted to this, and cultured in a thermostatic chamber at a temperature of 28 to 30°C in the dark. After 2 to 3 weeks of culture, cells proliferate from the cut end of the cut piece of sesame sprouts and form a clump to form a callus. By subculturing this proliferative callus in a medium of the same composition, large callus can be grown. 3 Proliferation and culturing of callus cells Callus cells induced from sesame sprouts were grown in a medium with the composition shown in Table 1 by naphthalene acetic acid (1
~5×10 ^-5 M), benzyladenine (1~5×
10 ^-5 M), transplanted into a medium containing 0.2% dielan gum, and incubated for 25 to 30 h in the light of 8000 to 15000 lux.
Cells are grown by culturing preferably at 28-35°C. 4 Extraction and Analysis of Antioxidant Substances Proliferative callus cells are finely crushed using a blender, etc., and then ground together with quartz sand. This was extracted with a solvent such as aqueous ethanol or methanol, dehydrated with anhydrous sodium sulfate, and then
Evaporate to dryness at ~35 °C. Dissolve in methanol again to obtain a fraction containing antioxidant substances. Next, the method for analyzing antioxidant activity will be described. This method uses linoleic acid as a reaction substrate and utilizes the Rodan iron method, which is often used to measure the degree of oxidation of fats and oils. That is, divalent iron ions are oxidized to trivalent iron ions by peroxides produced by autooxidation of fats and oils, and this reacts with ammonium thiocyanate to produce red rhodan iron, and its absorbance is measured. This is a method to determine the amount of peroxide in fats and oils. The outline of the analysis method is as follows. 0.13ml of linoleic acid in a 50ml Erlenmeyer flask,
99.5% ethanol 10ml, 02M phosphoric acid buffer
Add 10 ml and an appropriate amount of the sample dissolved in methanol, then add water to make the total volume 25 ml. This is sealed with parafilm, left in a constant temperature box at 40°C, and measured over time. To measure, take 0.2 ml from this, add 9.4 ml of 75% ethanol, 30
% ammonium rhodanate aqueous solution 0.2ml, 2x
Add 0.2 ml of a 3.5% hydrochloric acid solution of 10 ^-2 M ferrous chloride,
Measure the absorbance at 500 nanometers after exactly 3 minutes. As a control, we prepared a sample containing only linoleic acid, ethanol, phosphoric acid buffer, and water without adding any sample, and conducted comparative measurements. When there are antioxidants in the sample, the increase in absorbance at 500 nanometers is lower than the control. The present invention will be explained below with reference to Examples and Test Examples, but these are illustrative and do not limit the present invention. Example 1 (1) Sesame (Sesamum indicum L.) seeds were prepared, immersed in a 75% ethanol solution for several seconds, and then washed twice with sterilized distilled water. This was immersed in a 0.1% benzalkonium chloride solution (manufactured by Kanka Kagaku Sangyo Co., Ltd.) for 2 minutes. After washing thoroughly three times with sterilized distilled water, immersed in a disinfectant solution containing 1% sodium hypochlorite (Wako Pure Chemical Industries) and 0.1% Tween 20 (Wako Pure Chemical Industries) for 30 minutes, and then rinsed with sterile water. Sterilized sesame seeds were prepared. Plastic containers for plant culture (flow
Sterilized water and sterilized gauze were placed in a container (manufactured by Laboratories, Inc.), and pre-sterilized sesame seeds were sown on top of it. When I left it for 2 weeks under 20 watts of fluorescent light in a thermostatic room at 30 degrees Celsius, it grew to a length of 5 to 7 cm.
Sesame sprouts were obtained. (2) Prepare medium 2 with the composition shown in Table 1, divide it into equal parts, add 0.2 mL of dielan gum to 100 ml each, and
%, cytokinin and auxin under the experimental conditions shown in Table 2 were added to prepare a proliferative cell mass (callus) induction medium. Do these as usual
Autoclave sterilization was performed at 120°C for 10 minutes. While warm, place 3 of each of these media into 10 cm diameter plastic Petri dishes.
30ml was dispensed onto each sheet and allowed to solidify at room temperature. Sesame seedlings prepared in (1) were then cut into 5-7 mm pieces by aseptic operation, and the stems and leaves were transplanted onto a solid Petri culture medium. The sesame seedlings were sealed with Parfilm to prevent moisture evaporation and left in the dark in a constant temperature room at 28-30°C for 3 weeks to induce callus from the sesame sprout sections, and the results shown in Table 2 were obtained.

[Table] −: No callus induction.
(3) Add 0.2 gellan gum to the basic medium with the composition shown in Table 1.
%, naphthaleneacetic acid 5 x 10 ^-5 M, and benzyladenine 1 x 10 ^-5 M. 600 ml of the medium was sterilized and prepared in the same manner as in (1). This was dispensed into 20 plastic Petri dishes, each containing 30 ml, and solidified. The proliferative calli obtained by the induced culture in (1) were transplanted into four cells per Petri dish. Culture was carried out for 3 weeks in a plant cell culture device at 28-30°C and 12,000 lux of light. Cell aggregates with good proliferative properties were selected, and subculture was repeated four times using these as seed cells. In this way, stably proliferating cultured cells were grown. this cell
It was named N ₅ B ₅ S-2 (sesame cultured cells subcultured with naphthaleneacetic acid 5×10 ⁻⁵ M and benzyladenine 1×10 ⁻⁵ M). (4) Add 0.2% dielan gum to the basic medium with the composition shown in Table 1.
Medium 1 containing 5×10 ⁻⁵ M of naphthalene acetic acid and 1×10 ⁻⁵ M of benzyladenine was sterilized and prepared in the same manner as in (1). This was dispensed into 40ml glass containers for plant cell culture with a diameter of 4cm and a depth of 13cm, and solidified. Transplant 5-7 mm squares of the sesame proliferative N ₅ B ₅ S-2 cells grown by subculturing in step (3) into a plant cell culture device at 28-30°C and 12,000 lux light. Culture was performed for 3 weeks. As a result, the average fresh weight of the cells grown in the culture vessel is
It was 4.9g. 30 g of these cells were taken in a milk drum, 3 g of quartz sand was added thereto, and the mixture was ground for 5 minutes. After that, 100 ml of an 80% ethanol aqueous solution was added and stirred thoroughly to extract antioxidant substances. This is centrifuged (2500 rpm/
The supernatant was collected, and the cell residue was extracted again by adding 100 ml of 80% ethanol aqueous solution. The extracts from three aqueous ethanol solutions were collected and evaporated to dryness in a rotary evaporator at 40°C to obtain 1.9 g of a yellowish brown extract. Test Example 1 The excellent antioxidant effect of the antioxidant substance prepared according to the present invention was confirmed as follows. 80% of 100ml of 100mg of extract obtained in Example
It was dissolved in an ethanol aqueous solution (1 mg/ml concentration) and used as an analysis sample of an antioxidant substance. Using 1 ml of this sample as a sample, the degree of inhibition of acid autooxidation was measured by the Rodan iron method. As a result, as is clear from Figure 1, the fraction (1 mg) extracted from sesame proliferating cells contained α-tocopherol (0.2 mg) or butylhydroxyanisole (BHA, 0.2 mg).
It was confirmed that it contained highly active antioxidant substances equivalent to 1.0 mg). (Effects of the Invention) The present invention prepares a large amount of proliferative sesame cells in a culture container, and extracts the obtained cultured cells with hydroalcohol. Moreover, it has made it possible to industrially mass-produce a new type of water-insoluble antioxidant substance. Therefore, according to the present invention, a novel antioxidant substance that can be used as a safe food additive can be systematically supplied by industrial means without relying on cultivation. It is.

[Brief explanation of drawings]

FIG. 1 is an oxidation curve of linoleic acid obtained by analyzing the progress of autooxidation using linoleic acid as a reaction substrate using the Rodan iron method. −●−: Control group, −○−: α-tropherol group, −−△−−: Butylhydroxyanisole group, −○†檗Г 祒淒殕楮挧Χ茵

Claims (1)

[Scope of Claims] 1. Oil-insoluble, water-insoluble antioxidant extracted with hydroalcohol from cultured cells of proliferative cells derived from adult sesame (Sesamum indicum L.) plants. Yellow-brown extract. 2. Oil-insoluble, water-insoluble antioxidant characterized by culturing proliferative cells derived from adult sesame (Sesamum indicum L.) plants in a medium and extracting the cultured cells with hydroalcohol. A method for producing a yellow-brown extract with sexual properties.