JPS63207380A - Production of cultured plant cell containing antioxidative substance - Google Patents

Abstract

PURPOSE:To produce a cell containing antioxidative substance useful as a food and pharmaceutical, by inducing a reproductive cell from a grown sesame plant and culturing the cell in a medium. CONSTITUTION:Slices of sprout, stalk, leaf and/or root of aseptically prepared seedling of sesame are cultured in a solid and/or liquid medium containing carbon source, nitrogen source, vitamins, minerals, etc., and preferably added with phytohormones such as auxin, cytokinin, etc., in dark place at 28-30 deg.C and the induced reproductive callus obtained by this process is subcultured to form a large callus. A stably reproducible cell is grown from the callus and obtained cell is proliferated in a medium containing naphthaleneacetic acid, benzyladenine, gelan gum, etc., and added with natural organic nutrients in light place at 20-37 deg.C, preferably 25-35 deg.C and pH5.6-6.0. The objective antioxidative substance is separated from the proliferated cell.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides sesame seeds (Sesame+num 1ndicu+u
The present invention relates to a method for producing plant cells containing antioxidant substances using cultured cells of L.
It is applied in the pharmaceutical industry.

(Conventional technology and problems) Foods are manufactured from agricultural products, marine products, livestock products, etc. However, during storage and preservation processing of food raw materials and products, food raw materials and products deteriorate due to contamination and putrefaction by microorganisms, chemical and physical effects, etc., reducing their commercial value. For this reason, various food additives have been developed, and methods such as temperature treatment, oxygen removal treatment, vacuum packaging, low temperature storage, and radiation treatment have been developed and 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 maintaining the life of living organisms 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, reducing the value of the product. Not only do
It is known to result in the formation of harmful substances in food.

For example, nutritionally essential unsaturated fatty acids such as linoleic acid and lylunic 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" Vol. 17, p. 106 (1986)).

In order to prevent such peroxidation of lipids, packaging techniques such as removing oxygen from packaging using an oxygen scavenger, vacuum packaging, and nitrogen gas displacement packaging are used. On the other hand, with the development of the chemical industry, synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (Bl-IT) 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

On the other hand, as mentioned above, it is thought that peroxides and carcinogenic substances produced in animal bodies due to the toxic effects of oxygen have a negative effect on animal cells. , cellular aging and, in turn,
It is thought to be related to lifespan (free radical aging theory). Therefore, highly safe, naturally derived antioxidants are used as substances that support the antioxidant defense mechanism in the body, in the technical field of foods, especially health foods and nutritional foods, as well as pharmaceuticals and cosmetics. There are high expectations.

However, natural antioxidants, which are expected to be used in place of synthetic antioxidants that have problems with food pollution, are stable because they originate from plants and animals that are influenced by natural conditions such as weather. Currently, only a small amount of natural vitamin E, vitamin C, etc. has just been put into practical use.

The present invention does not extract from plants in this way, but
Culture containing antioxidant substances uses cultured cells, and this is the first time that we have succeeded in industrially mass producing natural antioxidant substances by mass culturing cultured cells. This is completely unknown and new.

(Problem to be Solved by the Invention) Cultivating and producing plants by agricultural means and extracting useful components from them may be carried out in a non-industrial manner on a laboratory scale. The supply of useful substances derived from plants relies on agricultural methods such as cultivation, which has a limited production efficiency and requires large areas of farmland for mass production.
Cultivation is also easily affected by weather, so there are many difficulties in stably supplying it as an industrial raw material, and there are problems such as the difficulty of producing a certain amount of specific useful ingredients in a planned manner. Therefore, the development of new technology was desired.

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. However, it has not been possible to industrially extract and produce large amounts of antioxidant substances from adult plants.

(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, focusing on proliferative cell masses (callus) artificially induced from plants, and researching technology to produce useful substances by culturing this. Ta.

During this process, we investigated the products contained in callus cells derived from many types of plants, and found that sesame (Sesame)
Not only did they succeed for the first time in cultivating proliferative cells from the plant body of Samum indicum L., but they also discovered for the first time that the proliferated cells contained strong antioxidant substances and that they could be advantageously extracted. As a result of further research based on this new knowledge, the present invention was completed.

In other words, the present invention establishes a completely new method for culturing proliferative cells of sesame plants, thereby providing a method for producing large amounts of natural antioxidant substances. In other words, the present invention aims to develop antioxidants derived from safe natural products that do not cause food pollution, and to develop 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 succeeded in solving all of the existing technical issues all at once.

It is already known that sesame plants or sesame oil contain antioxidants such as vitamin E and lignan compounds (Agricultural and Biological Chemistry).
Biological Chemistry) No. 49
Vol. 301 (1985) 1 Journal of the Japan Food Industry Association, Vol. 32, p. 407 (1985)). However, the cultivation of proliferative cells from sesame plants is completely unknown, and the present invention is considered the first. Furthermore, it was previously completely unknown that proliferative cells from sesame plants contain strong antioxidants, and that they could be extracted industrially and efficiently without any alteration or deterioration. , I don't see that suggestion.

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, sprouts are prepared under sterile conditions, and callus cells are induced by culturing the sprouts, one leaf of the stem, and/or root sections in a solid and/or liquid medium. The proliferative callus obtained is subcultured to grow into a large callus.6 The callus is then cultured stationary and/or with stirring in a solid and/or liquid medium to proliferate callus cells.

Various types of media can be used as the culture medium.

As carbon sources, monosaccharides such as glucose and flag-1-saccharides, disaccharides such as maltose and sucrose, and polysaccharides such as oligosaccharides and starch can also be used. As a nitrogen source, nitrate nitrogen such as ammonium nitrate and potassium nitrate,
In addition to ammonia nitrogen such as ammonium sulfate and ammonium tartrate, casamino acids, amino acids, peptones, corn staple liquor, yeast cells, yeast extract, malt extract lactate, etc. can be used.

In addition, vitamins such as nicotinic acid, nicotinamide, thiamine, folic acid, and biotin; inositol, adenylic acid, guanylic acid, and cytidylic acid.

Nucleic acid-related substances such as thymidylic acid and cyclic AMP; minerals such as iron, manganese, zinc, boron, iodine, 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 1.650II
Ig potassium nitrate 1, 9
00 Calcium chloride 440 Magnesium sulfate 370 Potassium phosphate 170 Boric acid
6.2 Manganese sulfate
22.3 Subsulfuric acid
8゜6 iosopotassium
0.83 Sodium Molybdate
0.25 cobalt chloride
0.025 copper sulfate
0.025 Sodium ethylenediaminetetraacetate 37.3 Ferrous sulfate
27.8 Myo-inositol
100 Glycine Pyridoxine dihydrochloride 0.5 Nicotinic acid 0.5 Thiamine hydrochloride 0.1 M
30g water 1.00
0dpH5,7 It is preferable to add auxin and cytokinin to the basic medium, and as the auxin, indoleacetic acid, indolebutyric acid, naphthaleneacetic acid, 2.4 dichlorophenoxyacetic acid, etc. are used as appropriate. 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, potato extract, cornstarch liquor, yeast extract, etc. It is effective to add natural organic nutrients such as malt extract and malt extract. The culture can be carried out at a temperature of 20 to 37°C, preferably 25 to 35°C. The pH of the culture solution is slightly acidic (pH
5,6-6.0) is advantageous for proliferation.

To extract antioxidants from proliferative cells obtained by culturing, cells can be extracted by biological treatment using cellulase or lysozyme, chemical treatment, mechanical or ultrasonic treatment, or a combination of these. It can be recovered by destroying it and extracting it with methanol, ethanol, acetone, chloroform, other organic solvents, water, etc., or a mixture of these organic solvents and water.

To analyze the activity of antioxidants, a method is used in which the amount of autooxidation by air using linoleic acid as a reaction substrate is measured by rhodan iron property. This method is a conventional method used to examine lipid peroxidation. (Accrural &
Biological chemistry
al and Biological Chemistry
y) Vol. 45, p. 735 (1981)) Next, the method of the present invention will be explained in detail step by step.

1. Preparation of aseptically grown sesame seeds Sesame seeds were first immersed in a 75% ethanol aqueous solution for a few seconds, washed with sterile water, and then soaked in 0.1% benzalkonium chloride (
The seeds are immersed in a commercially available disinfectant solution for 2 to 5 minutes to sterilize microorganisms attached to the seeds. After thoroughly washing the seeds again with sterilized water, 1% hypochlorous acid I-lium solution (0.1%
The sesame seeds are completely sterilized by treatment with a fungicide solution (containing the surfactant Tween 20) for 30 minutes.

On the other hand, put sterilized gauze and water into a sterilized wide-mouth container with a lid (commercially available plastic product), and germinate the sesame seeds thereon to prepare sterile sprouts.

2. Induction of proliferative cell mass (callus) derived from sesame In the basic medium with the composition shown in Table 1, auxin, naphthalene acetic acid (10-" to 10-' M), and 2,4 dichlorophenol were added. Cyacetic acid (10-''-10-'M), as cytokinin, benzyladenine cio-''~1
0-'M), or kinetin (10''5-10-
4M) was added in combination, 0.2% gellan gum was added as a solidifying agent, and after sterilization, the mixture was dispensed into a Petri dish and solidified. Cut pieces of sesame sprouts prepared aseptically are transplanted to this, and cultured in the dark in a thermostatic chamber at a temperature of 28 to 30°C. 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 with the same composition, large callus can be grown.

3. Proliferation culture of callus cells Callus cells induced from sesame sprouts were prepared by adding naphthalene acetic acid (1 to 5 x 10-'M) to a medium with the composition shown in Table 1.
, benzyladenine (1-5X10-sM), gellan gum 0.2%, and 8,000-i.s.
The cells are grown by culturing at 25 to 30°C, preferably 28 to 35°C, in the light of , ooo lux.

4. Extraction and Analysis of Antioxidant Substances Proliferative callus cells are finely crushed using a blender or the like, and then ground together with quartz sand. This was extracted with a solvent such as methanol, dehydrated with anhydrous sodium sulfate, and
Evaporate to dryness at 0-35°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 rhodan iron properties, which are often used to measure the degree of oxidation of fats and oils. In other words, divalent iron ions are oxidized to trivalent iron ions by peroxides produced by autooxidation of fats and oils, which react with ammonium thiocyanate to produce red rhodan iron, and its absorbance is measured. This is a method to determine the amount of peroxide.

The outline of the analysis method is as follows.

Linoleic acid 0.13m□Q in a 50tQ Erlenmeyer flask
, 99.5% ethanol 10mQ, 0.2M phosphate buffer 101! Add appropriate amounts of IQ and the sample dissolved in methanol, and add water to bring the total volume to 25+nfl.
Make it. This was sealed with parafilm, left in a thermostat at 40°C, and measured over time. The measurement will start from 0.
Take 2ffiR, 75% ethanol 9.4 companies, 30%
Amnium rhodanate aqueous solution 0.2 mfl, 2XlO'''
Add 0.2 rrrQ of a 3.5% solution of zM ferrous chloride in hydrochloric acid and measure the absorbance at 500 nanometers after exactly 3 minutes. As a control, we prepared a sample containing only linoleic acid, ethanol, phosphate buffer, and water without adding any sample, and conducted comparative measurements. When there are antioxidants in the sample, the increase in absorbance of the SOO nanometer will be lower than the control.

The present invention will be explained below with reference to Examples and Test Examples.
These are examples and do not limit the invention.

Example 1 (1) Sesame (Sesamum indicum L,)
Prepare the seeds.

After dipping the cloth in 75% ethanol solution for several seconds, it was washed twice with sterilized distilled water. This was immersed in a 0.1% pensulfnium chloride solution (manufactured by Seka Kagaku Sangyo Co., Ltd.) for 2 minutes. After washing thoroughly three times with sterilized distilled water,
Sterilized sesame seeds were prepared by immersing the seeds in a disinfectant solution containing % sodium hypochlorite (Wako Pure Chemical Industries, Ltd.) and 0.1% Tween 20 (Wako Pure Chemical Industries, Ltd.) for 30 minutes and washing with sterilized water.

Put sterilized water and sterilized gauze in a plastic container for plant culture (manufactured by Flow Laboratory), and place on top of it.
Pre-sterilized sesame seeds were sown.

When the seedlings were left in a constant temperature room at 30° C. under 20 watts of fluorescent light for two weeks, sesame sprouts with a length of 5 to 7 cm were obtained.

(2) Prepare medium 2Q with the composition shown in Table 1, divide it into equal parts, and add 0.2% gellan gum to each 100 mU.
, and cytokinin and auxin under the experimental conditions shown in Table 2 were added to prepare a proliferative cell mass (callus) induction medium. These were sterilized in an autoclave at 120° C. for 10 minutes in the usual manner.

While still warm, 30 mQ of each of these media were dispensed into three plastic Petri dishes with a diameter of 101, and allowed to solidify at room temperature.

Sesame sprouts prepared in (1) were then cut into 5-7 mm pieces by aseptic operation, and the stems and leaves were transplanted onto a solid medium in a Vetri dish. The pieces were sealed with parafilm to prevent water evaporation and left in a constant temperature room at 28-30°C in the dark for 3 weeks to induce callus from the sesame sprout sections, and the results shown in Table 2 were obtained.

1 +++ 0゜■ + 1 + 0.1+ 1 +++ 0.1+ 1 + 0.1 ++ +: Indicates the amount of callus induction. The more 10 marks, the better.

- Ichiriki J-Less Hakado Kashi 8 (3) Add 0.2% duran gum to the basic medium with the composition shown in Table 1.
, naphthaleneacetic acid 5X10-'M, benzyladenine l
A 600-ml medium Q supplemented with Xl0-'M was sterilized and prepared in the same manner as in (1). This was dispensed into 20 plastic Petri dishes, each with a volume of 30 mΩ, and solidified. The proliferative calli obtained by the induced culture in (1) were transplanted into four cells per Petri dish. 28-30℃, 12
The cells were cultured for 3 weeks in a plant cell culture device with ,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 is N6
B5S-2 (sesame cultured cells subcultured with naphthaleneacetic acid 5X10-'M and benzyladenine 1X10-'M)
It was named.

(4) 0.2% gellan gum in the basic medium with the composition shown in Table 1;
naphthaleneacetic acid 5X10-'M, benzyladenine 1X
A medium IQ to which 5M of 10''' had been added was sterilized and prepared in the same manner as in (1). This was dispensed into 40mQ glass containers for plant cell culture with a diameter of 4cm and a depth of 13cm and solidified. 28 by transplanting 5 to 7II11 of the sesame proliferative cells N5B and S-2 cells grown by subculturing in 3).
Culture was carried out for 3 weeks in a plant cell culture apparatus at ~30°C and 12,000 lux of light. As a result, the average fresh weight of cells grown in the culture container was 4.9 g.

Take 30g of these cells into a mortar, add 3g of quartz sand, grind for 5 minutes, and then add 3g of quartz sand and grind for 5 minutes.
0mM was added and stirred well to extract the antioxidant substance. This was centrifuged (2,500 rpm, 10 minutes) to collect the supernatant, and the cell debris was extracted again by adding 100 mM 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.

Dissolve 100 mg of the extract obtained in the example in 80% ethanol aqueous solution of 100+aQ 1g/1fl
Concentration) This was used as an analysis sample for antioxidant substances. 1m of this
Using M as a sample, the degree of inhibition of acid autooxidation was measured by rhodan iron property. As a result, as is clear from Figure 1, the fraction (11 g) extracted from sesame proliferating cells contained α-tocopherol (0.2 mg) or butylated hydroxyanisole (BHA, 0.2 mg). It was confirmed that it contains highly active antioxidant substances.

(Effects of the Invention) The present invention provides a completely new method of preparing a large amount of proliferative sesame cells in a culture vessel, thereby industrially producing antioxidant substances contained in sesame cells. This also made it possible to mass produce it. Therefore, the method of the present invention has the remarkable effect of being able to systematically supply antioxidant substances that can be used as safe food additives by industrial means without relying on cultivation.

[Brief explanation of the drawing]

FIG. 1 is an oxidation curve of linoleic acid obtained by analyzing the progress of autooxidation using linoleic acid as a reaction substrate using rhodan iron properties. −・−: control group, −0−: α-tocopherol group, −
Δ-nibutylhydroxyanisole group, -・-: Sesame cultured cell group. Agent Patent Attorney Door 1) Parent Male No. IC1l1 Anti-A-Number of days

Claims (1)

[Claims] 1. A method for producing antioxidant-containing cells, which comprises culturing proliferative cells induced from adult sesame (Sesamum indicum L.) plants in a medium.