RU2757463C1 - Method for increasing efficiency of cultivating callus culture of chinese magnolia vine (scisandra chinensis (turcz.) baill.), in vitro conditions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology and can be used in the food and pharmaceutical industry for the cultivation of a callus culture of Chinese magnolia vine (Schisandra chinensis (Turcz.) Baill.). The method for cultivating the callus culture of Chinese magnolia vine (Schisandra chinensis (Turcz.) Baill.) includes sterilization of planting material, preparation of a nutrient medium on the mineral basis of Murashige-Skoog (MS) or Gamborg (B-5) with or without the addition of hormones: 6-benzylaminopurine (6-BAP) and naphthylacetic acid (NAA), cultivation under sterile conditions at a photoperiod (light/dark) of 16/8 hours, a temperature of 25±2°С and humidity of 60-70%.

EFFECT: production of biomass of the callus culture of Chinese magnolia vine with a high yield and a high content of biologically active substances of a phenolic nature.

4 cl, 3 tbl

Description

The invention relates to the field of biotechnology and can be used in the food and pharmaceutical industries for the cultivation of the callus culture of Schisandra chinensis (Schisandra chinensis (Turcz.) Baill.).

Recently, medicinal plant materials have received increased scientific interest as an alternative to synthetic pharmaceuticals. Biologically active substances (BAS) of plants play an important role in maintaining and strengthening human health, in the treatment of diseases of cardiovascular, oncological, diabetes mellitus, obesity, etc. [1,2].

Diseases of the cardiovascular system (myocardial infarction, arterial hypertension, stroke, etc.) are the leading cause of death [3-5]. In 2018, they accounted for 46.8% of all deaths in Russia. In 2017, almost 541 thousand people with cancer were identified in Russia. From malignant neoplasms died 290.7 thousand people, which is 15.9% in the overall structure of mortality [6]. Within 1 year, 22.5% of patients die, since about 40% of newly diagnosed malignant neoplasms have stage III-IV of the disease.

Thus, the general picture of the health status of the population reflects the urgent need for the development of domestic science at a more modern level, including the use of all possible reserves in the development of natural components from medicinal plant species to obtain biologically active substances and targeted use in the treatment of serious diseases.

A promising raw material for the pharmaceutical industry is such a medicinal plant as Schisandra chinensis (Turcz.) Baill.).

S. chinensis is a valuable medicinal plant used in traditional and modern Chinese medicine. Included in the International Pharmacopoeia edited by WHO in the list of important medicinal plants [7]. In the Russian Pharmacopoeia, the plant belongs to the classical adaptogens along with the spiny eleutherococcus (Eleutherococcus senticosus (Rupr.) Maxim.), Common ginseng (Panax ginseng CA Mey.), Safflower leuzea (Rhaponticum carthamoides (Willd.) Dittrich) and ...

Biologically active substances of Schisandra chinensis, acting as adaptogens, have a normalizing effect regardless of the nature of pathological changes and nonspecifically increase the body's resistance to stressors of various nature.

For medicinal purposes, seeds and whole fruits are used, in the folk - all the organs of the plant (including the stems and the root system). The following products are produced on the basis of S. chinensis raw materials: oil and extract (liquid) from seeds, extract (dry) from plant fruits, etc. These products are used in the production of food products enriched with biologically active additives [9].

The composition of Schisandra chinensis is represented by sugars, organic acids (citric, malic, fumaric, tartaric), vitamins (C, E, group B, etc.), phenolic acids, lignans, tannins, phytosterols, essential oil [10-12]. The essential oil of S. chinensis fruit is rich in sesquiterpene hydrocarbons. I. V. Krotova and Efremov A.A. in their work, they consider the constituent components of the essential oil of schisandra chinensis. More than 60% of the essential oil is represented by sesquiterpenes; aliphatic, monocyclic, bicyclic terpenes have also been found [13].

To ensure a timely and uninterrupted supply of medicinal raw materials, it is necessary, in parallel with traditional methods of reproduction, to widely introduce modern methods of plant biotechnology. Their use significantly speeds up the process of obtaining environmentally friendly raw materials with a high content of biologically active substances. These methods include the cultivation of plant cells and organs (callus, suspension, root cultures) in vitro. Callus cultures are considered as a dedifferentiated mass of cells obtained from explants inoculated in vitro in a medium consisting of relatively higher concentrations of auxins or a combination of equal concentrations of auxins and cytokinins [14]. In plants where the desired metabolites are present in the leaves, the creation of in vitro cultures from the leaves and their use to extract compounds is an ideal alternative.

From the existing prior art, a method is known for increasing the efficiency of in vitro cultivation of silver birch, schisandra chinensis, rhododendron and lilac {RF patent No. 2619177, publ. 05/12/2017). The essence of the method is that plants grown on nutrient media for multiplication (for Schisandra chinensis on Quorin-Lepuavr medium with the addition of sucrose 30 g / l, inositol 100 mg / l, pyridoxine 0.1 mg / l, thiamine 0.1 mg / l, nicotinic acid 0.5 mg / l, 6-benzylaminopurine 0.5 mg / l), transferred to a fresh nutrient medium after 3 weeks, allowing for the presence of no more than two kidneys on one explant. When plants are transferred to a fresh nutrient medium, preference is given to shoots developed from axillary buds, since they are characterized by a higher degree of juvenility than shoots obtained through apical growth.

A significant disadvantage of the proposed method is the lack of variations in nutrient media for the first stage of cultivation, as a result of which it is difficult to assert sufficient efficiency of the method.

Known nutrient medium for reproduction of Schisandra chinensis in vitro (RF patent No. 2440414, publ. 20.01.2012) of the following composition, mg / l: ammonium nitrate 400.0; potassium nitrate 1800.0; magnesium sulfate 360.0; potassium phosphate 270.0; calcium nitrate 1200.0; iron sulfate 27.8; sodium ethylenediaminetetraacetate 37.3; manganese sulfate 1.0; boric acid 6.2; zinc sulfate 8.6; sodium molybdenum 0.25; copper sulfate 0.025; cobalt chloride 0.025; potassium iodide 0.08; thiamine 0.4; pyridoxine 0.5; nicotinic acid 0.5; glycine 2.0; inositol 100.0; casein hydrolyzate 250.0; glucose 15,000.0; agar 7000.0; 6-benzylaminopurine 1.0; indolylbutyric acid 0.1 and water up to 1 liter.

This nutrient medium is aimed at enhancing shoot formation, as a result of which it is not efficient enough for the cultivation of dedifferentiated Schisandra chinensis cells.

Described is a method for reproduction of Schisandra chinensis (patent CN No. 106171991, publ. 07.12.2016), which provides for the following steps: placing sterilized dormant buds of schisandra chinensis in an induction medium to obtain callus; cutting the callus and then placing it in a culture medium to induce embryogenic callus; transfer of embryogenic callus into a liquid medium to induce somatic embryos to obtain globular embryos; transfer of globular embryos to a semi-solid medium for germination of somatic embryos to obtain rooted seedlings; transplanting rooted seedlings into a matrix medium with a mass ratio of perlite and peat at a mass ratio of 1: 2.8-3.2 and transplanting seedlings after cultivation. The components of the induction medium are as follows: Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid 1.8-3.2 mg / l, thidiazuron 0.15-0.25 mg / l, sucrose 28-32 g / l and agar 6 , 5-7.5 g / l.

The disadvantage of the claimed method should be attributed to the insufficient yield of biologically active substances in the biomass of callus, as well as the use of agar in the amount of 7 g, which does not contribute to the creation of the optimal density of the nutrient medium.

In the course of the patent search, no technical solution was found that could be recognized as the closest analogue.

The technical problem solved by using the developed invention is to develop a new effective method for cultivating Schisandra chinensis (Turcz.) Baill.) In vitro.

The technical result obtained when implementing the claimed method is to obtain a biomass of the callus culture of Schisandra chinensis with a high yield and a high content of biologically active substances of a phenolic nature.

To achieve this technical result, it is proposed to use a method of cultivating a callus culture of Schisandra chinensis (Schisandra chinensis (Turcz.) Baill.), Including sterilization of planting material, preparation of a nutrient medium on a mineral basis Murashige-Skoog (MS) or Gamborg (B-5) with the addition of or without hormones - 6-benzylaminopurine (6-BAP) and naphthylacetic acid (NAA), cultivation under sterile conditions with a photoperiod (light / dark) of 16/8 hours, a temperature of 25 ± 2 ° C and a humidity of 60-70%.

The invention is illustrated by the following examples (table 1):

Example 1. At the first stage, the planting material is sterilized. For this, the leaf plates of Schisandra chinensis are washed with detergent and placed in a 0.1% solution of mercuric chloride (HgCl ₂ ) for 1 min for sterilization. After sterilization, the material is washed three times for 20 minutes in distilled sterile water. The leaf blades are cut with a scalpel into 5 × 5 mm segments and used as explants.

At the second stage, an agar nutrient medium is prepared. For this, all components, with the exception of vitamins and hormones, are diluted in 1.0 l of distilled water, brought to a boil, and autoclaved at 121 ° C for 20 minutes. After the medium has cooled down under sterile conditions, vitamins and hormones are added through a membrane filter. The prepared nutrient medium is poured into Petri dishes (diameter 60 mm). To cultivate the callus biomass of Schisandra chinensis (Schisandra chinensis (Turcz.) Baill.), A hormone-free nutrient medium of the following composition (per 1 liter of bidistilled water) is used: mineral base according to B-5, sucrose 30.0 g / l, casein hydrolyzate 500.0 mg / l, inositol 100.0 mg / l, thiamine 10.0 mg / l, pyridoxine 1.0 mg / l, nicotinic acid 1.0 mg / l, bacteriological agar 20.0 g / l.

At the third stage, sterile leaf explants are placed on a frozen agar nutrient medium to initiate callus development. The cultivation is carried out in sterile conditions of a climatic chamber with a photoperiod (light / dark) of 16/8 hours, a temperature of 25 ± 2 ° C and a humidity of 60-70% of the air. When reseeding, callus is divided into 3-7 parts, depending on the growth.

Example 2. Similar to example 1, but 3.0 mg of 6-BAP and 1.5 mg of NAA are additionally introduced into the nutrient medium.

Example 3. Similar to example 1, but in the composition of the nutrient medium as a source of mineral components, a base according to B-5 is used.

Example 4. Similar to example 3, but 3.0 mg of 6-BAP and 1.5 mg of NAA are added to the nutrient medium.

The results of the analysis of the growth index of the callus biomass of Schisandra chinensis (Turcz.) Baill.) Are presented in Table 2.

It is shown that the maximum biomass yield is observed when using a nutrient medium containing a mineral base according to Murashige-Skoog. The average growth index on the medium with a mineral base MS was 21.075, which is 1.26 times higher than the indicator of the medium with mineral salts according to Gamborg. At the same time, hormones have a significant effect on the growth of the culture, in particular the combined use of 6-BAP and NAA (in this case, the growth index was 23.11).

The results of studying the content of biologically active substances of phenolic nature in the callus culture of Schisandra chinensis (table 3) are directly proportional to the results of studying the growth index of callus cultures. The maximum accumulation of biologically active substances is observed when using the nutrient medium according to example 4. Compared with the content of biologically active substances in the starting material (leaf plates of schisandra chinensis), the callus culture according to example 4 accumulates biologically active substances 1.34 times more.

Thus, the claimed method provides a high biomass accumulation and a high content of biologically active substances of phenolic nature in the callus culture of Schisandra chinensis (Turcz.) Baill.).

Bibliography

1. Cicero, A.F.G. Food and plant bioactives for reducing cardiometabolic disease: How does the evidence stack up? / A.F.G. Cicero, A. Colletti // Trends in food science & technology. - 2017. - Vol. 69, P. B. - P. 192-202.

2. Delivery of bioactives in food for optimal efficacy: What inspirations and insights can be gained from pharmaceutics? / E. Nowak, Y.D. Livney, Z. Niu, H. Singh // Trends in food science & technology. - 2019. - Vol.91. - P. 557-573.

3. Boytsov, S. Regional challenges and opportunities in cardiovascular research: The Russian Federation "SWOT" analysis / S. Boytsov, F. Van de Werf // American heart journal. - 2011. - Vol. 161, no. 3. - P. 427-430.

4. Risk of cardiovascular disease morbidity and mortality in frail and pre-frail older adults: Results from a meta-analysis and exploratory meta-regression analysis / N. Veronese, E. Cereba, B. Stubbs, M. Solmi et al. // Aging Research Reviewsio - 2017. - Vol. 35. - P. 63-73.

5. Global burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019 study / G. Roth, G. Mensah, C. Johnson, G. Addolorato et al. // Journal of the American College of Cardiology. - 2020. - Vol. 76, 1.25 .-- P. 2982-3021.

6. Healthcare in Russia. 2019: Stat. Sat. / Rosstat. - M. - 2019 .-- 170 p.

7. Accumulation of dibenzocyclooctadiene lignans in agar cultures and in stationary and agitated liquid cultures of Schisandra chinensis (Turcz.) Baill / A. Szopa, A. Kokotkiewicz et al. // Applied Microbiology and Biotechnology. - 2016. - 100. - P. 3965-3977.

8. Medicinal Plants from the 14th edition of the Russian Pharmacopoeia, recent updates / A.N. Shikov, I.A. Narkevich, E.V. Flisyuk, V.G. Luzhanin, O.N. Pozharitskaya // Journal of Ethnopharmacology. - 2020. - 113685. - 112 p.

9. Pharmacokinetics of Schisandra chinensis lignans / V.М. Kosman, M.V. Karlina, O. N. Pozharitskaya et al. // Reviews on clinical pharmacology and drug therapy. - 2015 .-- T. 13 (4). - S. 3-21.

10. Comparative Studies on Polyphenolic Composition, Antioxidant and Antimicrobial Activities of Schisandra chinensis Leaves and Fruits / A. Mocan, G. Crisan et al. // Molecules. - 2014. - 19. - P. 15162-15179.

11. Pharmacokinetics of Schisandra chinensis lignans / V.М. Kosman, M.V. Karlina, O. N. Pozharitskaya et al. // Reviews on clinical pharmacology and drug therapy. - 2015 .-- T. 13 (4). - S. 3-21.

12. Phytochemical and biotechnological studies on Schisandra chinensis cultivar Sadova No. 1 - a high utility medicinal plant / A. Szopa, M. Klimek-Szczykutowicz et al. // Applied Microbiology and Biotechnology. - 2018. - 102. - P. 5105-5120.

13. Krotova, I.V. Investigation of the chemical composition of the fruits of Schisandra chinensis / I.V. Krotova, A.A. Efremov // Chemistry of vegetable raw materials. - 1999. - No. 4. - S. 131-133.

14. Srivastava, P. Chapter 30 - Herbal medicine and biotechnology for the benefit of human health / P. Srivastava, M. Singh, R. Chaturvedi // Models in Discovery and Translation. - 2020. - P. 613-629.

Claims (4)

1. A method of increasing the efficiency of cultivation of a callus culture of Schisandra chinensis (Schisandra chinensis (Turcz.) Baill.) In vitro, including sterilization of planting material, preparation of a nutrient medium, cultivation under sterile conditions with a photoperiod (light / dark) of 16/8 hours, temperature 25 ± 2 ° C and humidity 60-70%, while the nutrient medium has the following composition: mineral base according to Murashige-Skoog, sucrose 30.0 g / l, casein hydrolyzate 500.0 mg / l, inositol 100.0 mg / l, thiamine 0.1 mg / l, pyridoxine 0.1 mg / l, nicotinic acid 0.5 mg / l, 6-BAP 3.0 mg / l, NAA 1.5 mg / l, bacteriological agar 20, 0 g / l. 2. The method according to claim 1, characterized in that 3.0 mg of 6-BAP and 1.5 mg of NAA are additionally introduced into the nutrient medium. 3. The method according to claim 1, characterized in that the base according to B-5 is used as a source of mineral components in the nutrient medium. 4. The method according to claim 3, characterized in that the composition of the nutrient medium is injected with 3.0 mg of 6-BAP and 1.5 mg of NAA.