Otimização de parâmetros para obtenção de culturas de calos, suspensão e raiz de Ulmária (<i>Filipendula ulmaria</i>) para isolar o maior número de substâncias biologicamente ativas com propriedades geroprotetoras

Dyshlyuk, S.; Vesnina, A. D.; Dmitrieva, A. I.; Kozlova, O. V.; Prosekov, A. Y.

S. Dyshlyuk

Kemerovo State University, Kemerovo, Russia

http://orcid.org/0000-0002-7333-8411

A. D. Vesnina

Kemerovo State University, Kemerovo, Russia

http://orcid.org/0000-0002-4552-7418

A. I. Dmitrieva

Kemerovo State University, Kemerovo, Russia

http://orcid.org/0000-0002-8764-4049

O. V. Kozlova

Kemerovo State University, Kemerovo, Russia

http://orcid.org/0000-0002-2960-0216

A. Y. Prosekov

Kemerovo State University, Kemerovo, Russia

http://orcid.org/0000-0002-5630-3196

*e-mail: irinakrish2000@gmail.com

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Figure 1
The algorithm of the study in this paper.

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Figure 2
Agrobacterium strains used to transform the leaves of seed seedlings into roots: a) Strain No. 1-Agrobacterium rhizogenes 15834 Swiss (Ufa, Russia); b) Strain No. 2-Agrobacterium rhizogenes 15834 Swiss (Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Russia); c) Strain No. 3-Agrobacterium rhizogenes 8196 (Czech Buduyevitsy, Czech Republic).

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Figure 3
Germination process of sterile seeds of meadowsweet (Filipendula ulmaria).

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Figure 4
Callus cell cultures of meadowsweet (Filipendula ulmaria) grown on nutrient medium No. 2.

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Figure 5
Suspension cultures of meadowsweet (Filipendula ulmaria) cells grown on nutrient medium No. 2.

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Figure 6
In vitro root cultures of meadowsweet (Filipendula ulmaria), cultivation duration 5 weeks.

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Figure 7
Results of the study of changes in the growth index of root culture in vitro of meadowsweet (Filipendula ulmaria).

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Figure 8
Composition of BAS in the biomass of callus, suspension and root crops of meadowsweet (Filipendula ulmaria).

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Figure 9
Densitogram of the extract of callus cultures of meadowsweet (Filipendula ulmaria).

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Figure 10
HPLC chromatogram of the extract of callus cultures of meadowsweet (Filipendula ulmaria): 1 – gallic acid, 2 – 4-hydroxybenzoic acid, 3 – 2-methoxyphenol, 4 – pyrocatechin, 5 – 4-venyl-2-methoxyphenol, 6 – benzaldehyde, 7 – spireoside, 8 – avicularin, 9 – avicularin, 10 – m-hydroxybenzoic acid.

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Figure 11
UV spectrum of avicularin.

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Figure 12
Chromatogram of a sample of the extract of callus cultures of meadowsweet (Filipendula ulmaria) under the conditions of amperometric detection with a glass-carbon electrode voltage of 0.85 V.

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Table 1
Composition of the nutrient medium for obtaining sterile seedlings from the seeds of meadowsweet.

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Table 2
Composition of nutrient media that differ in the composition of growth regulators used in the experiment on the selection of optimal parameters for growing callus culture of meadowsweet.

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Table 3
Composition of the nutrient medium for the cultivation of suspension crops of meadowsweet.

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Table 4
Parameters of extraction of biomass of cell cultures of meadowsweet (Filipendula ulmaria).

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Table 5
Results of the study of the growth index of the obtained callus crops grown on various nutrient media.

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Table 6
Results of determining the viability of primary suspension cell cultures, cell units.

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Table 7
Results of the study of the growth characteristics of suspension cultures of meadowsweet cultivated on various nutrient media.

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Table 8
Number of explants formed, transformed with the help of various agrobacteria.

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Table 9
Content of components in the extract of callus cultures of meadowsweet (Filipendula ulmaria) according to HPLC data with matrix.

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Table 10
Content of individual components in samples of alcohol extracts from the biomass of callus cultures of Filipendula ulmaria according to amperometric detection data.

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Table 11
Results of the study of the antioxidant activity of alcoholic extracts of the callus culture of meadowsweet (Filipendula ulmaria).

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Table 12
Antimicrobial properties of extracts from the biomass of the callus culture of meadowsweet (Filipendula ulmaria).

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(1)

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(2)

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(3)

Components of the nutrient medium	Quantity per 1 liter of distilled water
Macrosols 20x MS, ml	50.00
Microsols 20x SH, ml	1.00
Fe-EDTA, ml	5.00
Sucrose, ml	30.00
Nicotinic acid, mg	5.00
Pyridoxine, mg	0.50
Thiamine, mg	5.00
Inositol, mg	100.00
2,4-D, mg	1.00
Kinetin, mg	0.10
Agar, g	20.00

Components of the nutrient medium	Value per 1 liter of distilled water
Components of the nutrient medium	Nutrient medium No. 1	Nutrient medium No. 2	Nutrient medium No. 3
Macro salts MS 20x, ml	50.00	50.00	50.00
Micro salts SH 20x, ml	1.00	1.00	1.00
Fe-EDTA, ml	5.00	5.00	5.00
Thiamine, mg	5.00	5.00	5.00
Pyridoxine, mg	0.50	0.50	0.50
Nicotinic acid, mg	5.00	5.00	5.00
Sucrose, g	30.00	30.00	30.00
Inositol, mg	100.00	100.00	100.00
2,4-D, mg	–	1.00	2.00
Kinetin, mg	–	0.10	0.20
Agar, g	20.00	20.00	20.00

Components of the nutrient medium	Value per 1 liter of distilled water
Components of the nutrient medium	Nutrient medium No. 1	Nutrient medium No. 2	Nutrient medium No. 3
^**on (Murashige and Skoog, 1962).Macro salts MS 20x, ml	50.00	50.00	50.00
^****on (Schenk and Hildebrandt, 1972).Micro salts SH 20x, ml	1.00	1.00	1.00
Fe-EDTA, ml	5.00	5.00	5.00
Thiamine, mg	5.00	5.00	5.00
Pyridoxine, mg	0.50	0.50	0.50
Nicotinic acid, mg	5.00	5.00	5.00
Sucrose, g	30.00	30.00	30.00
Inositol, mg	100.00	100.00	100.00
2,4-D, mg	–	1.00	2.00
Kinetin, mg	–	0.10	0.20

No.	Callus cultures of meadowsweet (Filipendula ulmaria)
No.	Temperature, °C	Volume fraction of ethanol, %	Extraction time, h
1	30	70	2
2	30	70	4
3	30	30	6
4	30	70	6
5	50	70	2
6	50	30	6
7	50	70	6
8	70	70	6
9	35	70	5

Medium	Duration of the subcultivation cycle, day	Dry biomass growth index
No. 1	28–32	8.5–10.3
No. 2	28–32	9.7–13.6
No. 3	28–32	9.5–12.9

Name of the medicinal plant	Nutrient medium No. 1	Nutrient medium No. 2	Nutrient medium No. 3
Meadowsweet vyazolistnaya (Filipendula ulmaria)	23.600±0.001	46.700±0.020	9.800±0.001

Nutrient medium	Х_max, g/l	v, %	µ _dw, day^-1	I _dw
No. 1	7.52–11.96	72–80	0.10–0.13	7.63 – 8.93
No. 2	7.65–12.32	72–85	0.11–0.15	7.80–9.13
No. 3	7.2–11.2	70–79	0.11–0.12	7.35–8.36

Strain	Total number of explants used in experiments, pcs	Number of explants with roots, pcs	% explants
No. 1	36	1	2.778
No. 2	36	3	8.333
No. 3	36	1	2.778

Peak no.	Holding time, min	Component Name	Quantitative content, mg / ml
1	5.25	gallic acid	6.70±0.21
2	6.50	4-hydroxybenzoic acid	11.87±0.76
3	6.90	2-methoxyphenol	4.52±0.50
4	7.41	pyrocatechin	5.33±0.50
5	8.11	4-venyl-2-methoxyphenol	4.51±0.50
6	9.62	benzaldehyde	2.17±0.20
7	10.07	spireoside	15.39±0.72
8	12.30	avicularin	21.33±0.86
9	12.59	hyperoside	9.80±0.50
10	14.67	salicylic acid	3.47±0.14

Holding time, min	Component Name	Quantitative content, mg / ml
5.41	gallic acid	6.50±0.36
6.68	4-hydroxybenzoic acid	11.91±0.76
7.15	2-methoxyphenol	4.54±0.40
7.73	pyrocatechin	5.31±0.71
8.33	4-venyl-2-methoxyphenol	4.52±0.50
9.25	benzaldehyde	2.21±0.05
10.26	spireoside	15.41±0.86
12.51	avicularin	21.3±0.72
15.47	salicylic acid	9.85±0.50
19.64	quercetin	3.52±0.14

Extract no.	Antioxidant activity, mg / g
1	0.3490±0.0022
2	0.3920±0.0022
3	0.3730±0.0025
4	0.3380±0.0027
5	0.3910±0.0025
6	0.3780±0.0023
7	0.3520±0.0031
8	0.3790±0.0027
9	0.3700±0.0017

Typical strains	C^* * C-control-disks with a feeding medium of MPA	Ampicillin	Sample no.
	C^* * C-control-disks with a feeding medium of MPA	Ampicillin	1	2	3	4	5	6	7	8	9
	Growth inhibition zones, mm
Escherichia coli	0	24.5±	19.9±0.5	21.4±0.6	20.2±0.1	19.8±	21.2±	19.9±	19.8±	20.3±	20.1±
ATCC 25922	0	0.5	19.9±0.5	21.4±0.6	20.2±0.1	0.8	0.9	0.7	0.5	0.6	0.5
Candida albicans	0	18.3±	14.1±0.1	18.7±0.2	10.4±	10.1±	16.6±	12.1±	12.4±	13.4±	11.3±
Y-3108	0	0.2	14.1±0.1	18.7±0.2	0.2	0.1	0.4	0.1	0.1	0.7	0.2
Bacillus cereus B-6644	0	18.6±	18.6±0.6	18.8±0.5	18.0±	18.5±	19.0±	18.7±	18.5±	18.9±	18.7±
Bacillus cereus B-6644	0	0.4	18.6±0.6	18.8±0.5	0.7	0.0	0.0	0.8	0.8	0.8	0.7
Pseudomonas aeruginosa B6643	0	17.2±	18.9±0.7	19.0±0.4	18.9±	18.8±	19.3±	19.0±	18.9±	19.4±	19.1±
Pseudomonas aeruginosa B6643	0	0.2	18.9±0.7	19.0±0.4	0.5	0.7	0.5	0.7	0.7	0.8	0.7
Enterococcus faecium 17OM39	0	23.9±	19.1±0.2	19.8±0.6	19.4±0.5	19.0±0.7	19.7±0.8	19.2±0.5	19.1±0.7	19.6±0.6	19.4±0.5
Enterococcus faecium 17OM39	0	0.1	19.1±0.2	19.8±0.6	19.4±0.5	19.0±0.7	19.7±0.8	19.2±0.5	19.1±0.7	19.6±0.6	19.4±0.5
Klebsiella pneumoniae CECT 143	0	19.5±	19.2±0.5	19.8±0.8	19.5±0.7	19.1±0.5	19.9±0.1	19.2±0.3	19.1±0.5	19.8±0.5	19.4±0.7
Klebsiella pneumoniae CECT 143	0	0.4	19.2±0.5	19.8±0.8	19.5±0.7	19.1±0.5	19.9±0.1	19.2±0.3	19.1±0.5	19.8±0.5	19.4±0.7
Helicobacter pylori J99	0	18.2±	19.3±0.6	19.6±0.4	19.5±0.8	19.0±0.5	19.9±0.5	19.5±0.7	19.2±0.7	19.7±0.1	19.5±0.5
Helicobacter pylori J99	0	0.2	19.3±0.6	19.6±0.4	19.5±0.8	19.0±0.5	19.9±0.5	19.5±0.7	19.2±0.7	19.7±0.1	19.5±0.5
Streptococcus viridans ATCC 11563	0	24.6±	18.7±0.9	18.9±0.7	18.8±0.5	18.6±0.8	19.1±0.6	18.8±0.9	18.6±0.8	19.0±0.5	18.8±0.8
Streptococcus viridans ATCC 11563	0	0.6	18.7±0.9	18.9±0.7	18.8±0.5	18.6±0.8	19.1±0.6	18.8±0.9	18.6±0.8	19.0±0.5	18.8±0.8
Streptococcus bovis D ATCC 49147	0	24.2±	18.5±0.9	19.8±0.5	18.9±0.7	18.4±0.9	19.1±0.8	18.6±0.8	18.5±0.5	19.0±0.5	18.9±0.9
Streptococcus bovis D ATCC 49147	0	0.2	18.5±0.9	19.8±0.5	18.9±0.7	18.4±0.9	19.1±0.8	18.6±0.8	18.5±0.5	19.0±0.5	18.9±0.9
Porphyromonas gingivalis ATCC 33277	0	16.7±	18.6±0.8	18.9±0.7	18.8±0.5	18.4±0.2	19.3±0.8	18.7±0.5	18.6±0.8	19.1±0.6	18.8±0.5
Porphyromonas gingivalis ATCC 33277	0	0.5	18.6±0.8	18.9±0.7	18.8±0.5	18.4±0.2	19.3±0.8	18.7±0.5	18.6±0.8	19.1±0.6	18.8±0.5
Acinetobacter baumannii ATCC 19606	0	16.3±	18.4±0.9	18.6±0.8	18.5±0.7	18.3±0.5	18.9±0.1	18.2±0.5	18.3±0.4	18.7±0.6	18.5±0.4
Acinetobacter baumannii ATCC 19606	0	0.5	18.4±0.9	18.6±0.8	18.5±0.7	18.3±0.5	18.9±0.1	18.2±0.5	18.3±0.4	18.7±0.6	18.5±0.4
Streptococcus intermedius	0	24.3±	18.3±0.4	18.8±0.9	18.5±0.5	48.2±0.2	18.8±0.2	18.5±0.8	18.2±0.7	18.8±0.8	18.6±0.5
TYG 1620	0	0.2	18.3±0.4	18.8±0.9	18.5±0.5	48.2±0.2	18.8±0.2	18.5±0.8	18.2±0.7	18.8±0.8	18.6±0.5

[1] *e-mail: irinakrish2000@gmail.com

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Otimização de parâmetros para obtenção de culturas de calos, suspensão e raiz de Ulmária (Filipendula ulmaria) para isolar o maior número de substâncias biologicamente ativas com propriedades geroprotetoras

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