RU2394100C2 - Method for obtaining alkaloids - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves growing tissue culture Rauwolfia serpentina Benth (Rauwolfia serpentina) K-27 strain in a nutrient medium of given composition in the presence of melaphen (melamine salt of bis (hydroxymethyl) phosphinic acid) of 10-17 passages at a concentration of melaphen 1·10^-3 g/l. After that received tissue culture Rauwolfia serpentina Benth is transplanted to the nutrient medium of the same composition but containing very small concentrations of melaphen 1·10^-10-1·10^-15 g/l, mainly at 1·10^-13 g/l and is grown in this medium for a passage not more than 50 days.

EFFECT: increase in the accumulation of alkaloids.

3 cl, 5 dwg, 2 tbl

Description

The invention relates to the field of biotechnology, and in particular to a method for the accumulation of pharmacologically valuable alkaloids of aymalin and yohimbine when growing a tissue culture of Rauwolfia serpentina Benth. (Rauwolfia serpentine).

The initial tissue culture of Rauwolfia serpentina Benth. received in 1964 R.G. Butenko (Butenko R.G. Culture of isolated tissues and physiology of plant morphogenesis. M: Nauka, 1964, p. 272). However, it was characterized by low alkaloids. As a result of directed mutagenesis and optimization of the composition of the nutrient medium, strain K-27 was obtained (Catalog. All-Union Collection of Cell Cultures. L .: Nauka, 1991), which exceeded the intact plant by 2 times the accumulation of alkaloids. This strain is considered as the main source of industrial production of indole alkaloids, including aymalin.

The cultivation of strain K-27 is carried out on a special nutrient medium, the composition of which was developed in the laboratory of cell and plant cultures of the Leningrad Chemical-Pharmaceutical Institute (Vollosovich A.G., Nikolaeva L.A., Puchinina T.N., Pozdnyakova N.N. Nutrient medium for the cultivation of tissue culture Rauwolfia serpentina Benth. - producer of alkaloids. A.S. No. 679625, USSR, cl. C12K 9/00, decl. 3.05.77, publ.

Peak of synthesis of alkaloids by strain K-27 tissue culture Rauwolfia serpentina Benth. when reseeding for 38-40 days, it falls on 65 days of growth, in case of earlier or later reseeding, it shifts by 75-85 days (Kaukhova I.E. Theoretical and experimental foundations of developing effective resource-saving technologies of herbal medicines // abstract of dissertation on Doctor of Pharmaceutical Sciences, St. Petersburg, 2007).

It is known that various chemical additives, including phytohormones, were used to increase the yield of biomass and alkaloids. The addition of auxins, gibberellins, and cytokinins to the tissue culture medium does not affect biomass growth, but sharply inhibits the accumulation of alkaloids (Bereznegovskaya L.N. Metabolism of secondary nitrogen compounds in an isolated plant tissue culture. Tomsk. Publishing House Tomsk University, 1978; Butenko RG Culture of isolated tissues and physiology of plant morphogenesis. M: Nauka, 1964, p. 272; Vollosovich AG, Culture of isolated tissues and plant cells. M: Nauka, 1970, p. 234-235; Vollosovich F. Highlights Mod. Biochemie: Proc. 14 th. Int. Congr. Biochem. Prague. 10-15 July 1988 Vol. 2, Utrecht-tokyo 198 9).

At the same time, it was shown (Chechetkin I.R., Neustrueva S.N., Siyanova N.S., Winter V.G. Stimulation of the formation of alkaloids in tissue culture Rauwolfia serpentina Benth. Under the influence of extreme factors. All-Russian meeting. Forest chemistry and organic synthesis Abstracts of reports (Syktyvkar, 1998) that when 113.5 μM (3 · 10 ^-2 g / l) is introduced into the nutrient medium for 1 passage of abscisic acid possessing the properties of a defoliant and growth inhibitor of many cultures (N. Melnikov . Pesticides. M: Chemistry, 1987, p. 144), leads to an increase in the synthesis of aymalin by 80% compared with the control m However, the disadvantage of abscisic acid is its high cost, since it is obtained by the photochemical oxidation of vitamin A (Muromtseva G.S. et al. Plant hormones gibberillins. M .: Nauka, 1973, 270 pp .; Muromtseva G.S. et al. Plant growth regulators M.: Kolos, 1979, 246 p.).

Noteworthy are the data provided in the source (Zhegalova I.V., Siyanova N.S., Neustrueva S.N., Winter V.G. Stimulation of the growth and accumulation of indoline alkaloids in the callus culture of Rauwolfia serpentina Benth by ribonuclease Bacillus Intermedius. Plant resources, issue 2, 1995). Stimulation and accumulation of indoline alkaloids (aymalin) in it was achieved by adding 1 · 10 ^-4 g / l of Bacillus Intermedius ribonuclease (binase) to the nutrient medium. The enzyme is not a growth regulator; its biological effect is associated with the associative properties of the enzyme with membrane structures of a nucleotide nature. The effect of stimulating the growth of biomass is 61%, and the accumulation of alkaloids is 82-121%. However, with prolonged passage, the biomass increase fell from the 22nd passage to 16%, and by the 27th passage it was lower than the control, and the effect of stimulating the formation of alkaloids was 103 and 69%, respectively. Moreover, the high cost of isolation and purification of the enzyme increases the cost of the resulting product.

It is also described that to increase the productivity of tissue culture Rauwolfia serpentina Benth. a low-toxic synthetic plant growth regulator Melafen (melamine salt of bis (oxymethyl) phosphinic acid) was used (RU 2158735 C1, 10.11.2000).

It is known that when applied for 1 passage at concentrations of 1 · 10 ^-3 -1 · 10 · ⁶ g / l, melafen has a stimulating effect on the accumulation of aminal alkaloids by tissue culture of Rauwolfia serpentina Benth., Which was maximal at melafen concentrations of 1 · 10 ^{- 3} -1 · 10 ^-4 g / l of culture medium. At a concentration of 10 ^-4 g / l on day 80, the growth of amimalic alkaloids relative to the control was 60%. (RU 2174555 C1, 10/10/2001). The optimal concentration of melafen, stimulating the accumulation of both amimalins and the total amount of alkaloids, is 1 · 10 ^-3 g / l, and the optimal time for removing biomass when using melafen in passage 1 is 60 days of culture growth. At the same time, the increase in the synthesis of aimalin alkaloids at a concentration of melafen relative to the control was 80%, the amount of alkaloids was 85%, which corresponds to the alkaloids content of 1.3 and 3.8% of air-dry biomass, respectively.

The method described in this source is the closest to the claimed solution and is selected as the prototype.

The objective of the invention is a new method for producing alkaloids, expanding the range of known methods for their preparation.

The problem is solved by the claimed method of producing alkaloids, including the cultivation of tissue Rauwolfia serpentina Benth. strain K-27 on a nutrient medium of a given composition in the presence of melafen (melamine salt of bis (hydroxymethyl) phosphinic acid), followed by the accumulation of the target product, with the tissue culture Rauwolfia serpentina Benth. strain K-27 on a nutrient medium in the presence of melafen is cultivated for a long time - 10-17 passages are grown at a concentration of melafen of 1 · 10 ^-3 g / l, then transplanted onto a nutrient medium of the same composition with ultra-low concentrations of melafen 1 · 10 ^-10 -1 · 10 ^-15 g / l, mainly 1 · 10 ^-13 g / l, and grown for 1 passage.

The technical result of the invention is to increase the yield of amimalin and yohimbine and to shorten the growth cycle of the cell tissue culture Rauwolfia serpentina Benth.

The method is as follows.

In the inventive method using strain K-27. To determine the effectiveness of melafen, the yield of aymalin and yohimbine is increased and the cycle of growing a tissue culture of Rauwolfia serpentina Benth. Is reduced. A qualitative and quantitative composition of alkaloids in the grown tissue culture of Rauwolfia serpentina Benth cells is compared. in the presence of melafen in a concentration of 1 · 10 ^-3 g / l (experience) and without it (control).

The composition of the nutrient medium, the conditions for its sterilization, as well as planting and tissue cultivation, were the same both in the control and in the experimental variants, except that in the control variants there was no melafen in the nutrient medium.

The composition of the nutrient medium:

A. Macrosoli, g / l

Potassium nitrate 0.4 Ammonium nitrate 2.5 Magnesium sulfate 1.3 Ammonium phosphate monosubstitution. 0.3 Ammonium sulfate 0.2 Ammonium phosphate disubstituted. 0.2 Calcium nitrate 0.9

B. Microsalt, mg / l

Boric acid 12.0 Manganese Sulfate 22.0 Zinc sulphate 8.6 Sodium Molybdate 0.25 Copper sulphate 0,025 Cobalt chloride 0.1 Trilon B 38,0 Potassium iodine 0.83

B. Other components, g / l

Melafen 1 · 10 ^-3 Sucrose one hundred Thiamine bromide 5 Agar agar 5

Melafen is added to the nutrient medium at a concentration of 1 · 10 ^-3 g / l immediately before autoclaving. Sterilization of objects is carried out at an overpressure of 0.7 atm for 40 minutes.

Pieces of a 40-day callus of tissue culture Rauwolfia serpentina Benth are used as seed. weighing about 2 g. Callus cultivation is carried out in the dark at a temperature of 27 ° C in 250 ml jars. To obtain statistically reliable results, each experiment is passaged in jars in an amount of 10 pieces. Culture analysis is carried out on the 30th, 50th, 70th and 90th days of callus growth.

The content of aminalin alkaloids is determined according to the method proposed by Shah and modified by Vollosovich (Vollosovich A.G. Method for the quantitative determination of alkaloids of the indoline group in tissue culture Rauwolfia serpentina Benth. / A.G. Vollosovich, L.A. N. Nikolayeva, N.N. Pozdnyakov and other // Plant resources. - 1977. - T.13. - No. 1. - S.127-132). To determine the total content of alkaloids, the technique developed by Neustrueva et al. (Neustrueva S.N. Influence of binase on the dynamics of the main metabolic processes of tissue culture Rauwolfia Serpentina Bants. / S.N. Neustrueva, A.V. Kunin, N.S. Siyanova, I.V. Zhiganova, V.G. Winter, G.N. Mardanova // Scientific notes of Kazan State University, natural sciences. - 2005. - T. 147. - book 2. - S.149-160). Qualitative and semi-quantitative analysis of alkaloids (aminalin and yohimbine) is carried out by high performance liquid chromatography (HPLC).

It was found that long-term cultivation of tissue culture Rauwolfia serpentina Benth. on a nutrient medium with melafen leads to the fact that after 10 passage the culture responds with an increased accumulation of alkaloids in comparison with experiments on a single application of melafen (RU 2174555 C1, 10.10.2001).

The figure 1 presents the results of the accumulation of alkaloids during the cultivation of tissue culture for 17 passages (more than 1.5 years) on a nutrient medium containing melafen in a concentration of 1 · 10 ^-3 g / l. These results show that in the control culture, the peak of accumulation of alkaloids occurs on the 90th day of culture growth, and under the influence of melafen it shifts by 50 days. As can be seen from figure 1, under the influence of melafen, the level of accumulation of the total amount of alkaloids on day 50 is approximately the same as in the 90-day control, and amalina alkaloids on day 50 under the action of melafen are higher than in the 90-day control culture. Moreover, the level of accumulation of amimalic alkaloids in a culture long-passaged on melafen is significantly higher by 50 days than with a single application of melafen to the medium of the corresponding concentration established earlier (RU 2174555 C1, 10.10.2001). So, already on the 50th day, the culture of the 17th passage is characterized by the content of amimalin alkaloids of 2.2%, and the total amount of alkaloids is 3.4% of the air-dry mass, which corresponds to a 200% increase in accumulation of amimalin alkaloids and 100% of the total alkaloids more than in the control culture.

The figure 2 presents the HPLC profile of 50-day old samples:

(a) control; b) 17 passage on a medium with melafen in a concentration of 1 · 10 ^-3 g / l.

HPLC showed that passaging the culture on a medium with melafen 1 · 10 ^-3 g / l over a long time clearly led to a significant increase in the accumulation of amimalin, which is well illustrated by the data on the quantitative analysis of aminal alkaloids of the test sample, shown in table 1.

Table 1. Quantitative indicators of the accumulation of alkaloids in samples of a 50-day tissue culture Test samples Amalina alkaloids in premium raw materials,% The amount of alkaloids in premium raw materials,% The control 0.48 1.25 Melafen 1 · 10 ^-3 g / l 2,34 3.42

Significant accumulation of yohimbine was also revealed in comparison with the control culture. As can be seen from figure 2, the accumulation level of both aimalin and yohimbine under the action of melafen increases compared with the control culture - for aminalin about 3 times, for yohimbine - about 2 times.

Thus, with long-term cultivation of the culture of Rauwolfia serpentina Benth. on a nutrient medium with melafen, a shortening of the biotechnological cycle of obtaining the alkaloids we need (aymalin and yohimbine) and the accumulating effect of stimulating the synthesis of these alkaloids are observed.

After prolonged cultivation of a tissue culture, Rauwolfia serpentina Benth. on a culture medium with melafen at a concentration of 1 · 10 ^-3 g / l, the tissue culture is transplanted and grown for 1 passage on a culture medium containing ultra-low concentrations of melafen 1 · 10 ^-10 -1 · 10 ^-15 g / l.

Pieces of a 40-day callus of tissue culture Rauwolfia serpentina Benth are used as seed. weighing about 2 g. Callus cultivation is carried out in the dark at a temperature of 27 ° C in 250 ml jars. To obtain statistically reliable results, each experiment is passaged in jars in an amount of 10 pieces. Culture analysis is carried out on 30, 50, 70 and 90 days of callus growth.

To identify the effects of melafen when changing the concentration from 1 · 10 ^-3 g / l to ultralow, a tissue culture after prolonged passage on nutrient medium with melafen in a concentration of 1 · 10 ^-3 g / l (passage 17) is transferred to an environment with ultralow concentrations (1 · 10 ^-10 -1 · 10 ^-15 g / l, which in terms of molar concentration is 3.9 · 10 ^-13 -3.9 · 10 ^-18 M) and on a pure nutrient medium (control).

The composition of the nutrient medium indicated earlier, the conditions for its sterilization, as well as planting and tissue cultivation both in the control and in the experimental versions were the same, except that there was no melafen in the control medium.

In this experiment, 2 types of control are used - a tissue culture of Rauwolfia serpentina Benth. Grown on a medium without melafen (indicated as “K” in figure 3) and a tissue culture of Rauwolfia serpentina Benth. Transplanted after growing for 17 passages on a medium with melafen at a concentration of 10 ^-3 g / l on a medium without melafen (in figure 3 it is indicated as "K with chalk 1 · 10 ^-3 g / l").

The figure 3 presents the results of the accumulation of alkaloids in a tissue culture of cells of Rauwolfia serpentina Benth., Transplanted after cultivation for 17 passages on a medium with melafen in a concentration of 1 · 10 ^-3 g / l on a medium without melafen. These results indicate that the culture, seeded from a medium with melafen at a concentration of 1 · 10 ^-3 g / l on a pure nutrient medium, continues to synthesize an increased amount of alkaloids, which may be due to the residual effect of melafen remaining after re-cultivation in a piece of uterine culture, or with the mutagenic effect of melafen, accumulating during its long-term use.

Next, the results of experiments conducted on a culture transplanted onto a nutrient medium with melafen in ultra-low concentrations are compared with a control grown for a long time on a medium with melafen at a concentration of 1 · 10 ^-3 g / l and transplanted onto a medium without melafen.

The figure 4 presents the accumulation of alkaloids on days 50, 70 and 90 in a tissue culture of Rauwolfia serpentina Benth., Transplanted after cultivation for 17 passages on a medium with melafen in a concentration of 1 · 10 ^-3 g / l on a medium with melafen in ultra-low concentrations - 1 · 10 ^-10 -1 · 10 ^-15 g / l.

The results show that in many experimental variants there is an increase in the synthesis of alkaloids throughout the growth of the culture, especially by 50 days at a concentration of melafen of 1 · 10 ^-13 g / l The 70-day culture was characterized by a decrease in the accumulation of alkaloids, and on day 90, the accumulation of alkaloids in the culture under the influence of melafen again greatly exceeded the control, but was not much more than on the 50th day of tissue culture growth.

Comparing the data presented in figures 1 and 4, it can be seen that the accumulation of alkaloids under the influence of melafen in ultra-low concentrations significantly exceeds that in the culture, which for 17 passages was grown on a medium with melaphen 1 · 10 ^-3 g / l. So, the total accumulation of alkaloids on the 50th day of culture growth was 4.8% of air-dried biomass versus 3.5% in callus grown on melafen 1 · 10 ^-3 g / l (37% increase), and aymalin alkaloids - 2 , 8% against, respectively, 2.2% (27% increase). Moreover, relative to the control culture, the increase in accumulation of the total amount of alkaloids on the 50th day of growth of the culture was 460%, and amalina alkaloids 300%.

The figure 5 presents the HPLC profile of 50-day old samples:

(a) a control with melafen 1 · 10 ^-3 g / l;

(b) on medium with melafen in a concentration of 1 · 10 ^-13 g / l from passage 17 on medium with melafen in 10 ^-3 g / l.

HPLC data (Figure 5) suggests that under the influence of ultra-low concentrations of melafen in a tissue culture, seeded after prolonged growth on a medium with melaphen 1 · 10 ^-3 g / l on a medium with melafen 1 · 10 ^-13 g / l, occurs significant accumulation of aymaline - the peak of aimalin is 2 times higher and significantly wider than in the control, which indicates the predominance of aimalin in the total amount of aimalin alkaloids presented in table 2.

Table 2 presents quantitative indicators of the accumulation of alkaloids in samples of a 50-day-old tissue culture.

table 2 Quantitative indicators of accumulation of alkaloids in samples of a 50-day tissue culture of the river snake Test samples Amalina alkaloids in premium raw materials,% The amount of alkaloids in premium raw materials,% Control with chalk 1 · 10 ^-3 g / l 0.76 2.02 Melafen 1 · 10 ^-13 g / l 2.86 4.75

Also on the chromatogram (figure 5), a not very high but broadened peak of yohimbine is observed, which indicates its significant amount. Thus, a significant decrease in the used concentrations of melafen (to ultralow) in the long-term cultivated on melafen culture, an intense surge in the synthesis of alkaloids, mainly aymalin, appears.

Thus, the claimed method of increasing the yield of amimalin and yohimbine and shortening the growth cycle of a rauwolfia snake tissue culture culture, comprising using melafen as a constant component of a nutrient medium at a concentration of 1 · 10 ^-3 g / l, followed by 10-17 passages by growing callus on a medium with melaphene concentration of 1 × 10 ^-10 -1 × 10 ^-15 g / l, causes a sharp rise in the level of alkaloid accumulation (total amount to 460% of alkaloids and alkaloid aymalinovyh - up to 300%) and narrowing the spectrum of the synthesized alkaloids towards Aime lin and yohimbine, and also to reduce the cycle biotechnological culture growing to reach its maximum accumulation of alkaloids to 50 days, which eventually leads to a cheaper product obtained.

Similar effects of melafen, manifested during its prolonged use, as well as when its concentration is reduced to very small, can occur when alkaloids are obtained using other plant tissue cultures.

Claims (3)

1. The method of producing alkaloids, involving the cultivation of tissue culture Rauwolfia serpentina Benth. strain K-27 on a nutrient medium of a given composition in the presence of melafen (melamine salt of bis (hydroxymethyl) phosphinic acid) followed by the accumulation of the target product, characterized in that the tissue culture is Rauwolfia serpentina Benth. strain K-27 is grown for 10-17 passages at a concentration of melafen of 1 · 10 ^-3 g / l, followed by transfer to a nutrient medium of the same composition with ultra-low concentrations of melafen 1 · 10 ^-10 -1 · 10 ^-15 g / l, and growing on it for 1 passage. 2. The method according to claim 1, characterized in that the use of melafen in an optimal ultra-low dose equal to 1 · 10 ^-13 g / L. 3. The method according to claim 1, characterized in that 1 passage is mainly grown on a nutrient medium at ultra-low concentrations of melafen for not more than 50 days.

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