EA020503B1 - Culture strain of andrachnoid scullcap root (scutellaria andrachnoides vved.) - scut. andr. (hrc) - producer of vogonoside and acteoside - Google Patents

Abstract

The invention relates to plant physiology and genetic engineering and can be used in pharmaceutical industry as phenol scullcap compounds to which refer such substances as flavones (vogonoside) and phenylethanoides (acteoside) have high antioxidant activity stipulating their exclusive pharmacological value being in use in medical practice. The invention is aimed at producing culture strain of andrachnoid skullcap root (Scutellaria andrachnoides) intensively growing in conditions in vitro at nonehormonal nutrient medium, preserving ability to the synthesis of phenol compounds characteristic to roots of a whole plant and having genetic and biochemical stability. The assigned task is solved by producing a novel culture strain of andrachnoid skullcap root Scut. Andr. (HRC) (Scutellaria andrachnoides Vved.) - a producer of flavone vogonoside and phenylethanoide acteoside.

Description

(72) Inventor:

Kuzovkina Inna Nikolaevna, Prokofieva Maria Yuryevna (Design Bureau), Umralina Anara Rustamovna, Chernysheva Tatyana Petrovna (COP) (56) KUZOVKINA I.N. et al. The formation of flavonoids in ρΚί T-DNA-transformed roots of the Scutellaria baicalensis (Zs1e11apa ba1ca1ei518 Ssogsch) and methods for its regulation. Plant physiology. 2001, t. 48, No. 4, p. 523-528

MALIKOV V.M. et al. Phenolic compounds of plants of the genus 8Cl1c11apa b. Distribution, structure and properties. Chemistry of Natural Compounds, 2002, No. 4, p. 299-324

OLEINIKOV D.N. et al. Phenolic compounds of Scutellaria baicalensis (8si1s11apa ba1sa1ei518 deotf). Chemistry of Plant Raw Materials, 2009, No. 4, p. 89-98

COWAS8 ΟΥ. c1 a1. NRS Os1stpppa0op o £ ΡΗνοηοίάδ ίη Naiu-Koo! CuNitek about £ 8si1s11apa ba1sa1ei518 Oeotf. Skgota1odgar1ya 8irr1etep1, 2004, νοί. 60, pp. 881-885

KUZOVKINA I.N. The cultivation of genetically transformed plant roots: the prospects of use in plant physiology. Plant Physiology, 1992, vol. 39, no. 6, p. 1208-1214

020503 B1

020503 Β1 (57) The invention relates to the field of plant physiology, biotechnology and genetic engineering and can be used in the pharmaceutical industry, since the phenolic compounds of Scutellaria, which include substances such as flavones (vogonoside) and phenylethanoids (acteoside), have a high antioxidant activity, which determines their exceptional pharmacological value, which is used in medical practice. The objective of the invention is to obtain a culture strain of the root of the plant of Scutellaria andrachidifolia (8s1e11apa apbtasbshnbek), intensively growing under conditions of ίη νίίτο on hormone-free nutrient media, preserving the ability to synthesize phenolic compounds characteristic of the roots of the whole plant, and possessing genetic and biochemical stability. The problem is solved by the creation of a new strain of culture of the root of Scutellaria andrachis 8si1. aibt. (IRS) (8si1e11apa aibtasbioMek ννεά.) - producer of flavone vogonoside and phenylethanoid acteoside.

Application area

The invention relates to the field of plant physiology, biotechnology and genetic engineering and can be used in the pharmaceutical and food industry, since the phenolic compounds of Scutellaria, which include substances such as flavones (vogonoside) and phenylethanoids (acteoside), have high antioxidant activity, which determines their exceptional pharmacological value, which is used in medical practice.

State of the art

Andrachiformes mongrel (8cShe11apa aiigasLio1Ye8 Uusy.) Is a perennial endemic herbaceous plant belonging to the family of caudaiformes (Larusaceae) and the genus 8cShe11apa (Flora of the USSR, Moscow, Leningrad, 1954, v. 20, p. 51, p. 51.

The plant has a limited, almost point distribution area and is found in Kyrgyzstan in the area of the Atoynok and Ferghana ranges at an altitude of 900-1600 m above sea level.

This is a short perennial plant with a height of 4 to 12 cm, which grows in rocky-rocky places, blooms in May-June, bears fruit in July (Abdullaeva M.N. Genus 8cShe11apa b., - Shlemnik. Key to plants of Central Asia, Tashkent, 1987 , t. 9: 13-37; Umralina A.R., Lazkov G.A. Endemic and rare species of plants of Kyrgyzstan (Atlas), 2008, pp. 96-97, Bishkek).

The plant is included in the Red Book of the Kyrgyz Republic. Information on its cultivation is absent. The seeds of the plant as endemic are stored in the seed bank of the Institute of Biotechnology of the National Academy of Sciences of the Kyrgyz Republic.

Scutellaria andrachidifolia is among the ornamental plants. There is no information on the composition of secondary substances in the root system of the plant even in the last extensive summary of Uzbek phytochemists who published the chemical composition of 57 species of Scutellaria (Malikov V.M., Yuldashev M.P. Phenolic compounds of plants of the genus 8cShe11apa. Distribution, structure and properties. Chemistry natural compounds. 2002, v. 4: 299-324, and v. 5: 385-409).

The rare occurrence of the plant and the lack of data on the composition of natural substances of the plant served as the reason for introducing into the culture ίη νίίτο its roots and undifferentially growing cells in order to study the composition of secondary metabolites in the roots of the plant, as well as attempts to clonal micropropagation.

A known method of cultivating the roots of ίη νίίτο is another type of Scutellaria baicalensis (I.N. Kuzovkina, A.V. Guseva, I.E. Alterman, R.A. Karnachuk. The formation of flavonoids in the transformed roots of 8cShe11apa layauyuk and the ways of their regulation, 2001 Plant Physiology, 48: 523-528).

The method consists in carrying out a similar procedure for the genetic transformation of fragments of sterile seedlings of Scutellaria baicalensis with the help of the soil agrobacterium Adgobacterium tuberosum. but in this case another wild agrobacterium strain, A4, was used. The sequence of work on obtaining a strain of culture of the root of Scutellaria baicalensis completely coincided with the sequence of obtaining a strain of culture of the root of Scutellaria baracida, since both works were carried out by employees of the same laboratory. The difference between the method of obtaining a culture strain of the root of Scutellaria andrachidiforum consists in the use of seedlings of a different type of Scutellaria baicalensis as a source of plant material, as well as in the application of another wild strain of soil agrobacteria during genetic transformation (A4). The authors managed to obtain a culture strain of the root of Scutellaria baicalensis, which contains a complex of phenolic compounds, consisting mainly of flavones with high physiological activity (Κονас8 Su., Ki / octa Ι.Ν., δ / oeke Ε., Κιιπ, ίηδζΐο b. NRNS eYuGipipayop ί PauopoLh ίη и у τ τ τ Ь ί ί ί с с с с 11 11 11 11 11 11 ота ота ота 1 1 ота ота ота ота ота 2004 2004 2004 2004 2004.60: 81-85

The approbation of the little-known type of Scutellaria, the andrachidaceous, as an object for introducing into the culture ίη ν егоτο its roots is of great practical and theoretical interest from the point of view of elucidating the composition and quantitative content of phenolic metabolites in its roots.

Almost all types of Scutellaria are vanishing (Scutellaria baicalensis) or endemic (Scutellaria andrachidiforum), which dictates the need to introduce their roots, in which flavones are synthesized, into the ίη νίίτο culture. That is why we have used the method of introducing into the culture of ίη νίίτο the strain of the root culture of another, endemic type of Scutellaria, the Andrachnid scutellaria, tested on the example of Scutellaria Baikal.

Object of the invention

The objective of the invention is to obtain a culture strain of the root of the plant of Scutellaria andrachidifolia (8cSe11apa πηφ ^ ΐηο ^ δ), intensively growing under the conditions of ίη νίίτο on hormone-free nutrient media, preserving the ability to synthesize phenolic compounds characteristic of the roots of the whole plant, and possessing genetic and biochemical stability.

The solution of the problem

The problem is solved by the creation of a new strain of culture of the root of Scutellaria andrachis 8si1. amg. (NCC) (8si1e11apa aηy ^ acbηο ^ ееδ Uusy.) Is a potential producer of flavone vogonoside and phenylethanoid acteoside.

The strain of culture of the root of Scutellaria andrachidiformes (8si1e11apa aηy ^ acbηο ^ ееδ Uusy.) Was deposited in

- 1 020503

Collections of genetically transformed plant roots at the Federal State Budgetary Institution of Science Institute of Plant Physiology of the Russian Academy of Sciences under the designation §сЕЕйиг. (NKS) - producer of vogonoside and acteoside.

The certificate of deposit is presented in the application materials.

The novelty of the invention - the first strain was obtained §si (. Aiig. (NKS) of the culture of andleriformes root (5> cxc11apa apygasNpoFsz Umey.) - producer of flavone vogonoside and phenylethanoid acteoside.

The essence of the invention is that as a result of the genetic transformation of seedlings of Scutellaria andrachidiforum using a wild strain A4 of soil agrobacterium, a new plant system is obtained - a strain §с. (. Ayp. (NKS)) of root culture, which is capable of stable year-round growth under ίη νίίτο and to the synthesis of a significantly larger amount of vogonoside and acteoside than the content of these substances in the slowly growing and difficultly renewable roots of the whole plant of Scutellaria andrachidae.

The implementation of the invention

Upon receipt of a strain of culture of the root of Scutellaria andrachidus 8sie apyg. (NCC) (5> si1c11apa apygasppo1js Umei.) By ρΚί T-DNA transformation, sterile seedlings of this plant grown from sterile seeds are used with the first two true leaves. Sterilization of seeds defatted with ethyl alcohol is carried out with concentrated sulfuric acid for 3 minutes, after which they are thoroughly washed with sterile distilled water. Sterilized seeds are placed in Petri dishes on Street agar medium and kept for 4-5 days at 26 ° C in the dark (Smirnov A.M. Growth and metabolism of isolated roots in a sterile culture. M .: Nauka, 1970, p. 92). Non-contaminated sprouted seeds are transferred to biological tubes on a nutrient medium of the same composition. Juvenile plants are grown at a temperature of 26 ° C in the light (12-hour illumination with fluorescent lamps). After the appearance of a pair of true leaves, the aerial part of the seedlings is separated, cut into segments, which, after carefully pricking the insulin syringe with a needle, are placed in the liquid nutrient medium of Murashige and Skoog (MS) containing a suspension of a 48-hour soil bacterium. For inoculation of explant seedlings, a wild (unmodified) strain of soil agrobacterium Adgobacillus gp / oddsc (ATCC 15834) is used. After incubation with bacteria, the treated explants are washed with a sterile medium and transferred to a hormone-free MS agar medium, but with the addition of an antibiotic (claforan - 500 mg / ml medium) to eliminate the bacteria. After 3-4 weeks, plagiotropically growing roots appear on the cotyledons and first leaves in the wound areas, which is a morphological sign of a successful genetic transformation (Fig. 1). These transformed sites are separated and transplanted onto a Hamburg agar medium (B5) containing a reduced dose of antibiotic (250 mg / ml medium). After 2-3 passages (the period between passages is 4-6 weeks) and the complete elimination of soil agrobacteria from the culture is achieved, the obtained roots are transferred to Petri dishes with agar medium of the same composition, but without antibiotic, and grown in the dark at 25 ° C. The exclusion of an antibiotic from the culture medium leads to stimulation of root growth (Fig. 2). Long-term cultivation of a strain of root culture on an agarized nutrient medium helps to consolidate the signs of rhizogenesis and the formation of an intensely branching mass of thin roots. Transferring the stably growing root culture obtained after one and a half year cultivation (10 passages) on an agarized medium to a liquid nutrient medium of the same composition (but without agar), that is, under conditions of an immersed rocking culture (90 rpm), contributes to a significant improvement in root growth and the formation of a dense mass of thin interwoven roots of yellowish-beige color (Fig. 3). To maintain continuous growth under ίη νίίτο conditions, the strain of Scutellaria root culture 8ciu apyg obtained in this way. (NKS) needs regular transplants into a fresh liquid culture medium every 8 weeks. The size of the inoculum during transplants is about 1 g of wet weight of roots per 100 ml of culture medium.

The results of the invention

To date, the strain of culture of the root of Scutellaria andrachidae. 8 sie apyg. (NCC) (8si1s11apa apygasipoFez Umei.) Grows stably, forming due to intensive branching a mass of tightly interwoven roots (Fig. 4). To maintain continuous growth of the root culture during the 6-week interval between transplants, a portion of fresh nutrient medium (25-50 ml) is regularly added to the culture flasks, which prolongs the growth activity of isolated growing roots and transfers it to prolonged growing conditions. With a depletion of the nutrient medium, the roots quickly darken, while releasing secondary metabolites formed in them into the nutrient medium, which cause intoxication and death of the culture.

The strain of the root culture of Scutellaria andrachidiformes 8siE apyg. (NCC) possesses all the signs of rM T-DNA of transformed roots, namely, intensive and stable plagiotropic growth on nutrient media of a simple composition that does not contain phytohormones, the ability to synthesize opins, which are a marker of transformation, as well as genetic and biochemical stability. In their anatomy, morphology and biochemistry, such roots are comparable to the roots of juvenile plants, since they do not have secondary growth, which contributes to thickening of the roots.

Chemical analysis of secondary metabolites of the strain of root culture §сi1. upbg (IR) of andrecocephalic scutellaria (8c1e11ag1a aibgaeobioMev Umeb.) Confirms that the strain retains the ability to synthesize secondary metabolites characteristic of the roots of the whole plant. The main secondary metabolites of the roots of the seedlings and the strain of culture of the root of Scutellaria andrachnidum 8ei1. upbg (NKS) are phenolic compounds - flavones of the baiklein and wogonin groups, as well as phenylethanoid acteoside. High performance liquid chromatography (HPLC) profile of a methanol extract of a strain of culture of the root of Scutellaria andrachiformes 8ei1. upbg (NCC) bears great resemblance to the profile of the corresponding extract of the roots of Scutellaria seedlings by the retention time (K,) of the detected substances (Figs. 5 and 6). As a result of the preparative separation of the methanol extract of the strain of root culture §сi1. upbg (NCC) andracocephalic scutellaria ((8c1e11ag1a aibgasbioMev Umeb.) 2 dominant substances were isolated and purified, which on the basis of H and ¹³ C NMR spectrometry identified flavone vogonoside and phenylethanoid acteoside. The structural formulas of the isolated and identified secondary metabolites are presented (Fig. 7 and 8.) The quantitative content of vogonoside and acteoside was determined in the strain of culture of the root of Scutellaria andrachiforidae 8si1. apbg (NKS), which turned out to be comparable with the content of these metabolites in the roots of seedlings and in The established fact testifies to the validity of the inclusion of the strain of culture of the root of Scutellaria andrachidae 8si1. apbg (NKS) to the category of producers of these valuable natural substances of great interest for medical practice.

Claims (1)

CLAIM The culture of the root of the Andrakhnovidnas skullcap root (8sile11ag1a apbtaspoMev Memes.), Deposited in the Collections of Genetically Transformed Plant Roots (CGTPR) at FSBUN IHR RAS under the designation §ci1. apb (NCC) - producing vogonoside and acteoside.