JPS61173774A - Method for cultivating biological tissue containing crude drug component - Google Patents

Abstract

PURPOSE:To feed stably a biological tissue and cell containing a crude drug component, by washing a tissue fragment or cell, etc. to be cultivated with a germ-free isotonic solution containing a nutriment and a hormonal agent, transferring the tissue fragment, etc. to a culture medium and multiplying aseptically the tissue fragment, etc. CONSTITUTION:A cell or tissue of a living body containing a crude drug component such as growing point, terminal bud, bone marrow, cartilage or lymphocyte is isolated, washed with a germ-free isotonic solution containing a nutriment and hormonal agent, transferred to a solid culture medium or liquid culture medium such as agar culture medium, and aseptically multiplied. The cell or tissue can be cultivated with a high growth rate of the cultivated tissue in a large amount in a short time and the pretreatment process can be simplified.

Description

[Detailed Description of the Invention] Example 1 Tissue culture method using the whole plant and the apical bud of a plant containing medicinal components in the stem and leaf parts (Application examples: Assemblage japonica, Heutynia japonica, Hikiokoshi.

Epimedium, celandine, japonica, henruda, porphyry, periwinkle, wasabi, serakok, gennoshoko, saxifrage, periwinkle,
Chillingweed, Swallowtail, Ichiyakusou, Yotsuba Bulbul, Mizorikirishi, Shirone, and Fujibakama.

The apical part of the stems and leaves containing the herbal medicine ingredients shown above is aseptically peeled off under a dissecting microscope and the growing point cells are taken out. The extracted meristem cells were quickly washed with a sterile culture solution with the following composition.
Cultivate under constant temperature conditions in a solid medium containing culture medium.

Typical culture solution composition CG/L) (G/
L) A) Potassium chloride (KCL) 0
．． 08a) Magnesium sulfate (MGSO4) 0
．． 36) Sodium sulfate (NA2SO4) 0
．． 20 steps) Ferric sulfate 0.
025o) monobasic sodium phosphate 0.
0165 force) Lucium nitrate 0
．． 20g) Manganese sulfate 0.
0045ri) Zinc sulfate 0
．． 0015ke) Boric acid
o, ooisco) Potassium iodide
0.00075) Glycine
0.003) Vitamin B1, B6. Nicotinic acid 0.005s) Sucrose
20,000 ce) Indole acetic acid
By aseptically washing the 0.05 growth point tissue cells with a culture solution containing nutrients and hormones, the culture solution surrounds the tissue piece, replenishing the necessary moisture and nutrients, and The action of hormones in the culture medium stimulates the tissues and cells to be cultured, further promoting their growth.

2) Implantation of tissue pieces and culture The pretreated tissue pieces are implanted into an agar medium in a small Erlenmeyer flask in a sterile box. Add 10 grams of agar to the culture solution IL, correct the pH with O and IN NaOH, and after dispensing, pressurize and sterilize.

When implanting tissue pieces, sterilize the flask by holding the mouth of the flask over a flame, and always keep the mouth facing to the side to prevent any bacteria that may fall from above from entering. After implantation, the mouth of the flask is sterilized with a flame and then capped to prevent bacteria from entering from outside.

The container in which the tissue piece was implanted was removed from the sterile box and heated for 20-30 minutes.
It is placed in a constant temperature room adjusted to 25 degrees Celsius, preferably about 25 degrees Celsius.

Lighting is used to differentiate and grow stems and leaves. If you use lighting that emits light with wavelengths that are efficiently absorbed by plants, photosynthesis will be active, and growth will be faster.
Growth rates are similar whether the lighting is continuous or alternating between day and night. In addition,
After 2 to 3 days in a constant temperature room, if the tissue piece was not sufficiently sterilized at the time of transplantation or if it is contaminated with bacteria, bacteria colonies will form, so immediately remove the container in which these bacteria have formed.

3) Formation, division, and division of callus Ten or more days after the start of culture in the incubator, the implanted tissue pieces begin to undergo cell division and the color of the surface of each tissue piece changes. After about 4 weeks, the growing point will grow larger, and as the days pass, the callus will continue to proliferate.However, if left as is, the callus will change color and become hard due to lack of nutrients in the culture medium. The growth stops. Before this happens, divide the callus and transfer it to a fresh agar medium for further vegetative growth. First, divide the 4-5 week old growing point tissue vertically, and this time divide the divided sections into smaller sections than before. Transfer to agar medium in a flask with a slightly larger internal volume. A medium with almost the same composition as the previous one is used, but the hormone agent in this medium is changed from indole acetic acid to kinetin. Alternatively, kinetin may be added while still containing a small amount of indole acetic acid. Kinetin promotes the differentiation of stems and leaves in the cultured tissue. As the number of days passes, stems and leaves grow into the flask, eventually filling the flask. In this way, by dividing and transplanting the plants several times before the flask is full of stems and leaves, it is possible to propagate countless numbers of the same plant from one growing point. Furthermore, when using flasks with large internal volumes.

It can be grown as a large plant inside. Furthermore, even if the plants are cultured, propagated, and grown using the aforementioned large flask and then transferred to normal soil for cultivation, fully satisfactory results can be obtained. In this way, the present invention also includes a cultivation method in which the plants are grown in a flask until they reach a certain size and then transferred to soil. As a result, the growth rate is significantly faster than when grown in the open field, and the same plant can be divided into many parts, so it is possible to produce plants that grow quickly during cultivation and strains that are highly resistant to pathogens. It is also possible to select.

The present invention further includes application to a method called hydroponics or hydroponic cultivation instead of using soil when transplanting a plant that has been divided, multiplied, and grown in a flask. By transferring the plants that have grown and taken root in the flask to natural light conditions, they perform stronger photosynthesis than under artificial light conditions, and the content of herbal medicine components increases.
Less weight loss and improved yield. Furthermore, when the seedlings that have been elongated and grown according to the present invention are cultivated in a greenhouse using the hydroponic cultivation method, the growth of stems and leaves is remarkable, and the yield can be 2 to 3 times that of soil cultivation. Another advantage of hydroponic cultivation using the present invention, which is not uncommon, is that it eliminates the need to remove soil from the roots and stems. Since the roots and stems grow in the culture solution, they can be cleaned with just a small amount of water.

Example-2 Root, rhizome, tuber, tuber, bulb, rhizome, bulb 1
Tissue culture method using corms, storage roots, stem bark, trunk bark, bark, xylem, etc., which contain herbal medicine components, such as the cambium, plate ends, phosphorus pieces, and budding parts of plants (Application examples: peonies, peonies, buds, etc.) Oriental lily, Morel lily, Morel lily.

Valerian, Amadokoro, Naruko lily, Clara, Chigaya, Corydalis trifoliata, Shirtbeard, Sekishi, Tsu, Nakinagusa, Shinshu rhubarb, Calamus, Amana, Cantorum lily, Fujikobu, Bittersweet.

Tendaiuyaku, 7 Odamo, Serakoku, Murasaki, Scutellaria japonica, Japanese ginseng, Panax ginseng, Bellflower, Yellow-bellied lily, Yellowfin tuna, Yellowfin tuna, Japanese chinensis, Japanese trumpet, Japanese trumpet, Japanese red lily, Japanese tit, Madder, Ibukitranoo, Hinata-no-kozuchi, Onitsutsuda tit, Japanese tit, Big tsuzurafuji, Kouhone, Nirakei , Shishiudo, Actinidia, Rose of Sharon, Horse chestnut ginseng, Myamatobera, Trifolium japonica, Shunsai, Ikebu, Juzdama, Hirohasenega, Kagamigusa, Japanese knotweed, Onidokoro, Tiger lily, Japanese lily, Japanese lily, Japanese lily, Japanese ginseng, Jabulan, Shaku, Jurosou, Byanobu,
Yamadori beetle, Natsuzuisei, Yapkoji, and Okera.

Shi, Uga, Japanese cypress, Japanese yam, Japanese yam, Kusagi kasuzuru, kudzu, Ominaeshi, Gentian, Waremokou, Nodake, Amaryllid lily, Red gourd, Sagiomodaka, Tsudokudami, Sarutoriibara, Tororo mallow, Narcissus, Kanaoi, Rakkyo, Tamasa kids rough ji, Coffisar saw power etc.) Use the pretreatment method shown in Example-1. Thoroughly washed roots, rhizomes, tubers, and scales are soaked in 70% ethanol for a few seconds in a sterile box, washed with sterile culture solution, and then cut into rings.
Divide into several pieces to include the cambium portion and use them as tissue pieces for culture. The culture solution used here has a low concentration of plant hormones, contains gibberellin, or does not contain it.

2) Implanting and culturing tissue pieces Prepare, dispense, and sterilize agar culture solution by the method shown in Example-1, 2). The method for implanting tissue pieces is described in Example-1.
In almost the same way, the implant is sterilized using flame sterilization in a sterile box, and once the implantation is complete, it is incubated at a constant temperature to prevent contamination with bacteria. Lighting is hardly required during constant temperature culture.

3) Formation, division, proliferation, and division of callus Cambial tissues such as rhizomes being cultured in a thermostatic chamber form callus after 3 to 4 weeks. This callus divides, proliferates, and grows larger as days pass. Once the callus has reached a certain size, it is divided into plants using the method shown in Example 1, 3). The aseptically divided callus is transferred to a new agar medium and allowed to grow vegetatively at a constant temperature.

A flask with a slightly larger internal volume is used as the culture container, and the culture solution has almost the same composition as in Example-1, but the concentration of the hormone contained therein is slightly diluted than in Example-1. Use gibberellin or use one that contains few plant hormones. Indole acetic acid has a suppressive effect on roots and stems at high concentrations, so care must be taken as indole acetic acid has a suppressive effect on roots and stems.As the number of days passes, roots and stems grow into the flask.

Some parts turn green, and some have chloroplasts and pigments.
←Nike h! Before the roots and stems spread throughout the flask like this? - By repeating dividing and transplanting three times, it is possible to propagate many identical plants from one cambium. Callus transplantation, stock division #e
After several weeks, the transplanted callus grows thickly, and a rhizome tissue culture product containing the herbal drug components which is the object of the present invention is completed. In this way, by repeating callus transplantation, division, and enlarged growth, a plant tissue containing the desired herbal drug component can be obtained. After the roots and stems have enlarged and grown, buds and roots will grow from the rhizomes by further supplying nutrients, and eventually they will become a normal plant. Chloroplasts will appear on the leaves that have grown due to light irradiation. Begins to carry out photosynthetic function. The plants grown in this manner can also be grown in a greenhouse using hydroponics or hydroponics as described in Example 1-3).

However, in the method of Example 2, only the roots and stems of plants that contain a large amount of herbal medicine components are cultivated in large quantities in a short period of time, so that they are not allowed to elongate and grow to the entire plant body. It is not necessary.

Example-3 A set of relevant parts of a plant containing crude drug ingredients such as pistil, stamen, petal, flower, style, sac cell, cotyledon, bud, bud, plant gall, plant grain, and embryo bud. Method for culturing fibrous cells (Application examples: saffron, Fuji gall, etc.) l) Pistil, stamen, petal, calyx, style, sac cell, cotyledon, young bud, budding, plant gall, plant grain containing pre-treated herbal medicine ingredients , the young panicle, young flower, young pistil, young stamen, young bud, young bud,
The galls and plant grains are aseptically removed under a dissecting microscope, pretreated and washed by the method of Example-1 or Example-2.

After washing, the tissue is divided into four parts if it is large, or into two parts if it is small, and used as tissue pieces for culture.

2) Implantation of tissue pieces and culture The pre-treated tissue pieces for culture are aseptically loaded into an agar medium in a small Erlenmeyer flask.The composition of the culture solution of the agar medium is the typical one of Example-1. A for those with culture solution composition
TP (adenosine triphosphate) 0.001-5.0g
/L, preferably 0.02g/L, add 10g of agar to the culture solution LL, and add O, INHcI or 0.02g/L. Correct the pH with IN NaOH, and sterilize after dispensing. ATP is required for cell division and promotes cell division. When implanting a tissue piece, perform flame sterilization to prevent contamination with bacteria. Place a lid on the mouth of the flask after implantation, and place a 20-4
0'C1 Place in a thermostatic chamber adjusted to preferably around 30''C.During the initial stage of culture, stem and leaf differentiation does not occur, so no lighting is required.Flasks contaminated with bacteria will be detected after 2-3 days. Therefore, remove it.

3) Formation, division, proliferation, and division of callus Ten or more days after the start of culture in a constant temperature chamber, the transplanted plant pieces undergo cell division and proliferation [At first, the surface of each tissue piece changes color and gradually grows larger. and form a callus. Here, in order to further propagate the same plant, the callus is divided aseptically, and the divided callus is transferred to a new agar medium for vegetative propagation.

Transfer to a flask with a slightly larger internal volume. After dispensing the agar culture solution from 2) above into the flask, sterilize it. Divide the grown callus onto this agar and sterilize one part by flame sterilization. port to. The culture solution may be the same as in 2) above, but some cultures may grow faster if the hormone agent is changed from indole acetic acid to kinetin, so in that case, add kinetin to the flask after 0 days have passed. The callus grows and differentiates into pistils, stamens, petals, stigmas, cotyledons, plant galls, etc. Before the callus is all in the flask, split it several times.
Through transplantation, it is possible to grow an infinite number of plant tissues from one tissue, and it becomes possible to utilize the herbal medicine components contained in these tissues in a wide variety of ways. In this way, the ability to proliferate tissues containing necessary herbal drug components as needed makes it possible to supply crude drug components in large quantities and at low cost.

Example 4 Method for culturing cells or tissues of animal bodies such as sac, foreskin gland, pancreas, perineal vagina, thyroid gland, digestive gland, etc. (Application examples: musk deer, civet cat, toad, mouse, rat, rabbit.

(dogs, cats, birds, etc.) This method attempts to obtain a large amount of cells containing a large amount of Jacora by culturing the cells or tissues of the glands of Jacora. In addition, by culturing the digestive gland cells of toads and other animals, we can detect the steroid hormones contained in these cells.

The target is to obtain it from within the cell.

l) Pretreatment Tissues and cells containing aromas and herbal medicine components are taken out aseptically, immersed in sterile physiological saline while aerating sterile air, and kept at 37C. After thorough washing with this saline, the culture solution of Tsuza. Cultivate with

2) Animal tissue culture solution When culturing animal tissues and cells, use a culture solution with the primary composition 7) Sodium chloride
9.000 i) Magnesium sulfate
0.360) monosodium phosphate
゛ 0.020 engineering) Sodium glutamate o, oi.

e) Methionine 0.
010 power) Alanine
0.010 K) tryptophan
0.005 lysine)
0.005) Vitamin B, B,
Nicotinic acid 0.005) ATP (
Adenosine triphosphate) 0.005su)
Glucose 0,010
C) Chicken egg pressing juice 0,
010su) Plasma and Watori)
The grafts that have undergone the 0.010 pretreatment in a short period of time are aseptically cultured in a culture solution having the above composition with one aeration. The temperature required for culturing is 35-45°C, preferably 3
Constantly bubble sterile air through the culture at 7'C.

In addition to liquid culture in which oxygen is supplied and the culture solution is stirred, in solid culture using agar, agar is added to the culture solution having the above composition at 1 Og/L, and the tissue pieces are transplanted after dispensing and sterilization.

3) Formation, division, proliferation, and division of callus Ten or more days after the start of culture in the incubator, the implanted tissue pieces begin to actively divide, and the color of the surface of the tissue pieces changes. Approximately 4
After a week, the graft becomes a large mass, which continues to proliferate over the next few days.

However, if the culture continues as it is, the callus will change color or become hard due to lack of nutrients in the culture solution, and growth will stop.

Before this happens, split the callus and transfer it to fresh culture medium.

After 4-5 weeks, the callus undergoes further vegetative growth.
Divide into 2-4 pieces, transfer the divided sections to a new culture medium, and culture with aeration. The M section transferred to a new culture medium absorbs nutrients again and proliferates. just.

At this point, calli that are unsuitable for liquid culture are transferred to an agar medium, and calli are transplanted by flame sterilization.

Animal tissue pieces or animal cells that have grown large in a flask.

It becomes aromatic and contains steroid hormones.

The cells and tissues in the flask are crushed and destroyed to separate, extract, and purify the aromatic and herbal ingredients.

After placing the stems and roots of the plant in sterilized warm or boiling water,
Cooling with sterile water is an effective method because it stimulates the growing point cells and cambium, thereby increasing cell division and killing germs attached near the growing point. It is. In addition, by exposing the ffi tissue piece to infrared rays, ultraviolet rays, X-rays, visible light, etc. during the pretreatment process, the energy of the light rays can stimulate cells and promote proliferation. As a pretreatment, the tissue piece is placed in an electric or magnetic field, and the magnetic energy stimulates and promotes cell division.

Furthermore, immersing a tissue piece in an organic acid solution stimulates the cell walls and cell membranes of the cells in the tissue piece, which in turn activates cell division.

In addition, the purpose of using a medium containing metal ions is to replenish raw elements such as Fe and Mg, which are very important for the formation of chlorophyll and the production of hexaglobin. It is.

Nucleic acids such as vitamins and ATP also play an important role in cell division by supplying energy and acting as coenzymes.

Additionally, administration of hormones has the effect of promoting cell division and increasing cell elongation.

There are elements that cells need while preparing to divide, and these necessary elements are contained in the culture medium, which contains inorganic salts, organic substances, vitamins, nucleic acids, etc. , hormones, etc.

Plant hormones and ATP are added to speed up cell division and encourage the growth of neighboring cells.

The purpose of the present invention is to culture cells and tissues that are large, fast, and contain large amounts of herbal drug components.

The method is the present invention. Therefore, the ability to quickly culture cells and tissues containing herbal drug components in large quantities using the method of the present invention will greatly benefit humanity.

Patent Applicant: Nippon Securities Co., Ltd. Mikio Suda Procedures Amendment Book 1986
June 12, 1985 Patent Application No. 1327
No. 1 2. Title of the invention Method for cultivating biological tissue containing herbal medicine components 3. Person making the amendment Relationship to the case Patent applicant 5. Subject of the amendment At the beginning of the specification 3. Describe the detailed description of the invention to do.

6. Contents of the amendment As shown in the attached sheet■ 5.1ゝ＼i＼も・（Dark--- 3.Detailed description of the invention The present invention relates to a method for culturing biological tissue for the purpose of stably culturing organs, tissues, and cells in large quantities.

Traditionally, plants containing herbal medicinal ingredients were collected from the mountains and fields as medicinal herbs.

The L part was created through cultivation. In addition, animal organs containing herbal medicine ingredients were collected from wild animals. This method of collection that relies on wild animals and plants means that the quantities are unstable, small, and irregular, making it difficult to obtain what you need when you need it.

The present invention provides a stable supply of biological tissues and cells containing herbal medicine components without relying on such unstable supply,
This is a method for culturing biological tissue that was developed to be inexpensive and quickly available.

A feature of the present invention is that tissue pieces or cells to be cultured can be cultured.

After washing with a sterile solution containing nutrients and hormones, they are transferred to a solid medium such as an agar medium and grown aseptically. Some tissue pieces or cells of biological species grow and proliferate better in a liquid medium than in a solid medium such as an agar medium, so in that case, a liquid medium is used.

Claims (1)

[Scope of Claims] 1. Growing points, sprouts, budding,
Yobud, cell, stamen, pistil, style, petal,
Calyx, cambium, stem, root tip, liver, kidney, bone marrow, fibroblast, tumor, cancer, cartilage, heart muscle, smooth muscle, striated muscle, neuroblast, lymphocyte, sperm cell, egg cell, protein cell Cultivation of biological tissues characterized by isolating cells and tissues such as hizou cells, applying pretreatment, and then aseptically dividing, proliferating, and growing them in a liquid or solid medium containing the following nutrients. Law. 2. The method for culturing biological tissue according to claim 1, characterized in that the tissue piece is immersed in sterilized warm or boiling water as a pretreatment, and then cooled with sterilized water. 3. The method for culturing biological tissue according to claim 1, wherein the tissue piece is irradiated with ultraviolet rays, infrared rays, X-rays, visible light, etc. as a pretreatment. 4. The method for cultivating biological tissue according to claim 1, which comprises placing the tissue piece in an electric field as a pretreatment. 5. The method for culturing biological tissue according to claim 1, characterized in that the tissue piece is placed in a magnetic field as a pretreatment. 6. The method for culturing biological tissue according to claim 1, wherein the tissue piece is immersed in an organic acid solution as a pretreatment. 7. The method for culturing biological tissue according to claim 1, characterized in that an inorganic salt medium containing metal ions (hereinafter referred to as inorganic salt medium) is used as the medium. 8. It is characterized in that it is used as a medium by adding organic substances (saccharides as a carbon source, amino acids, proteins, yeast exudate, cell exudate, natural product extracts, peptone, etc.) as a carbon source to a medium containing inorganic salts. A method for culturing biological tissue according to claim 1. 9. The method for culturing biological tissue according to claim 1, which comprises adding organic substances and vitamins to a medium containing inorganic salts. 10 Add organic substances, vitamins, hormones, and nucleic acids (AMP, ADP, ATP, NAD) to a medium containing inorganic salts, etc.
, DPN, TPN, FAD, CoA, nucleotide, nucleic acid derivative). 11. The method for culturing biological tissue according to claim 1, which comprises adding organic substances, vitamins, and hormones to the medium containing inorganic salts. 12. The method for culturing biological tissues according to claim 1, which comprises adding organic substances and hormones to a medium containing inorganic salts. 13. The method for culturing biological tissue according to claim 1, which comprises adding organic substances, nucleic acids, and hormones to a medium containing inorganic salts. 14. The method for culturing biological tissue according to claim 1, wherein the tissue piece is immersed in the culture solution of 7-13 above as a pretreatment.