Kemerovo, Кемеровская область, Россия
Kemerovo, Кемеровская область, Россия
Kemerovo, Кемеровская область, Россия
Kemerovo, Кемеровская область, Россия
Kemerovo, Кемеровская область, Россия
Introduction. In recent years, scientists have been actively searching for medicinal plants containing biologically active substances with geroprotective properties to treat diseases of old age, in particular cancer, diabetes, cardiovascular diseases, and others. Ginseng (Panax ginseng L.) is a promising source of geroprotective compounds. We aimed to select optimal parameters for extracting organic compounds from ginseng callus, suspension, and root cultures and analyze their qualitative composition. Study objects and methods. We studied ginseng callus, suspension, and root cultures, as well as their extracts. Biologically active substances were extracted with 30 to 70% ethanol. Organic compounds were determined by thin-layer chromatography. The results for each plant were archived and analyzed for the presence of quercetin, mangiferin, luteolin, rutin, quercetin-2-D-glucoside, malvidin, as well as caffeic, cinnamic, ferulic, and sinapinic acids. Results and discussion. We developed a procedure for screening solvents and performed a fractional qualitative analysis of biologically active substances extracted from ginseng. As a result, we established the optimal parameters for extracting biologically active substances from the dried biomass of ginseng cultures. In all cases, temperature and the ratio of solvent to biomass were the same (50°C, 1:5). However, the extraction time and ethanol concentration differed, amounting to 60 min and 50% for callus cultures, 30 min and 60% for suspension cultures, and 60 min and 70% for root cultures. The qualitative analysis of organic compounds showed the presence of rutin (0.25), quercetin (0.75), and mangiferin (0.57), as well as caffeic and sinapinic acids in the extracts. Conclusion. Our set of experiments to isolate biologically active substances from ginseng callus, suspension, and root cultures resulted in selecting the optimal extraction parameters and analyzing the extracts for the presence of organic compounds.
Plant cultures, Panax ginseng, ginseng, plant extracts, geroprotective properties, gerontology
INTRODUCTION
Modern medicine and biology are actively searching
for new drugs with geroprotective effects [1–8]. Highly
useful in this regard are extracts of medicinal plants [8, 9].
A common extraction method involves using a reflux
condenser, a Soxhlet extractor, mechanical stirring,
and ultrasound. Soxhlet extraction takes place at
80–90°C and lasts from 20 to 24 h. Such parameters
make it possible to efficiently extract biologically active
compounds, such as saponins [10–13].
Modern extraction methods include ultra-high
pressure extraction (UHPE), ultra-temperature
extraction (UTE), pressurized liquid extraction (PLE),
microwave-assisted extraction (MAE), pressurized
hot-water extraction (PHWE), and supercritical fluid
extraction (SFE) [9–12, 14–17].
Compared to traditional techniques, modern methods
use smaller amounts of solvents, are easily automated,
and take little time. However, they are hardly more
effective than, for example, Soxhlet extraction or mechanical mixing [13, 18]. Moreover, pressurized hotwater
extraction and supercritical fluid extraction are
technically quite difficult to perform [10, 14, 19, 20].
Among medicinal plants with geroprotective properties
are Schisandra chinensis (L.), Scutellaria
baicalensis (L.), Rhodíola rósea (L.), Ginkgo biloba (L.),
and others [21–24]. The most highly valued geroprotective
medicinal plants include Panax ginseng (L.),
Aralia mandshurica (L.), and Eleutherococcus
senticosus (L.) [25]. Since the 1980s, scientists have
known of their antitumor effects [25–27].
Ginseng (Panax ginseng L.) is a slowly growing
perennial plant that is often used as a functional
component and a phytotherapeutic agent to
prevent and treat various diseases, such as cancer,
allergies, inflammatory diseases, and diabetes
mellitus [26–31].
According to scientific literature, ginseng extract is
used as an adaptogen to increase physical performance,
vitality, immunity, as well as resistance to stress and
aging [26, 32–34]. It also lowers total cholesterol and
low-density lipoproteins, thereby improving a blood
lipid profile [31, 32].
However, this plant is included in the Red Book of
the Russian Federation and the collection of young roots
is prohibited due to its depletion. In addition to low
seed productivity and relatively slow growth, ginseng
population is irreparably damaged by forest fires and
human activities in its endemic areas [35, 36].
Therefore, a justified solution would be to use the
plant’s cell and organ cultures as an alternative source
of renewable medicinal material [30, 32, 35–37]. In our
study, we used ginseng callus, suspension, and root
cultures – obtained in the early stages of research – as a
source of biologically active substances.
We aimed to select optimal extraction parameters
and perform a qualitative analysis of organic compounds
isolated from ginseng callus, suspension, and root
cultures.
STUDY OBJECTS AND METHODS
Our study objects included callus, suspension, and
root cultures of ginseng (Panax ginseng L.) obtained in
vitro, as well as their extracts.
To determine the optimal parameters for extracting
biologically active substances from ginseng by reflux
extraction, we analyzed several extraction systems
for their effectiveness. A water-ethanol mixture was
selected as an extractant due to its safety (GRAS),
economic efficiency, and the ability to extract a wide
range of biologically active substances from plant
materials [38, 39]. We screened the solvents and
performed a qualitative analysis of organic compounds
(Fig. 1). The percentage of ethanol in the solvents is
indicated in mass fractions. The yield of extracts (%) is
expressed in terms of 100 g of dry raw material.
To extract biologically active substances from
ginseng callus cultures, we placed 3 ± 0.001 g of
dry powdered callus culture in a 50 mL plastic tube
and added 40 mL of 30, 40, 50, 60, or 70% solvent
according to the screening scheme (Fig. 1). The tube
was connected to a reflux condenser. After 60 min
of extraction, we separated the dry mass from the
solution by filtration. To remove suspended particles,
we centrifuged the filtrate at 3900 rpm. Ethanol was
evaporated from a 100 mL pre-weighed flask under
reduced pressure. After evaporation, we weighed the
flask and measured the extract yield.
Then, we dissolved the residue in a minimum
amount of the solvent and determined the qualitative
composition of organic compounds in the extract by
thin-layer chromatography.
The chromatograms for each plant were archived and
analyzed for the presence of quercetin (Sigma-Aldrich,
USA, ≥ 95%), mangiferin (Sigma-Aldrich, USA, ≥ 98%),
luteolin (Sigma-Aldrich, USA, ≥ 98%), rutin (Sigma-
Aldrich, USA, ≥ 94%), quercetin-2-D-glucoside (Sigma-
Aldrich, USA, ≥ 95%), caffeic acid (Sigma-Aldrich,
USA, ≥ 98%), cinnamic acid (Acros Organics, Belgium,
Figure 1 Solvents efficiency in extracting biologically active substances from ginseng
Sample,
3 g
30% ethanol extraction
40% ethanol extraction
50% ethanol extraction
Concentration,
yield measurement,
thin-layer
chromatography
Is the yield
high?
Analysis
of efficiency
in reflux
extraction
High-performance
liquid
c
Figure 1 Solvents efficiency in extracting biologically active substances from ginseng
Sample,
3 g
30% ethanol extraction
40% ethanol extraction
50% ethanol extraction
Concentration,
yield measurement,
thin-layer
chromatography
Is the yield
high?
Analysis
of efficiency
in reflux
extraction
High-performance
liquid
chromatography
60% ethanol extraction
70% ethanol extraction
0
1
2
3
4
5
6
30 40 Total extract yield, %
4
6
8
extract yield, %
9
12
15
extract yield, %
NO
YES
371
Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376
≥ 98%), ferulic acid (Sigma-Aldrich, USA, ≥ 99%),
sinapinic acid (Honeywell, USA, ≥ 95%), and malvidin
(Sigma-Aldrich, USA, ≥ 90%).
To prepare ginseng suspension and root cultures for
the experiment, they were pre-dried to constant weight.
Then, 0.5–2.0 g samples of dried cultures were extracted
with solvents for callus cultures.
Thin-layer chromatography was performed as
described in Pharmacopeia Article 1.2.1.2.0003.15. After
evaporation of the solvent from the total extract, we
dissolved the dry residue in 1 ml of a suitable extractant
(methanol, methylene chloride or acetone) and applied
it to the plate with a glass capillary for thin-layer
chromatography.
Then, we placed the plate in a chamber and added
a suitable eluent. When we used silica gel without
modification, chromatography was performed in
the CH2Cl2:MeOH system with a 0–10% methanol
gradient, in increments of 1%. For reversed-phase
chromatography, we used the H2O:MeCN eluent system
with a 0–20% acetonitrile gradient, in increments of 2%,
and 0.1% trifluoroacetic acid as a modifier.
We separated the fractions with high-performance
liquid chromatography (HPLC), using a Prominence
LC-20 chromatograph with diode-array detection
(Shimadzu, Japan) and a 250×4.6 mm Kromasil C18
chromatographic column with 5 μm sorbent particles.
A mixture of water with o-phosphoric acid, pH = 4.6 (A)
and acetonitrile (B) were used as a mobile phase.
The gradient elution modes (% B) were 0–20 and
20–60 min with a gradient change of 10–20% and
20–50%, respectively. The eluent flow rate was
1.0 mL/min; the temperature of the column thermostat
was 35°C. In the preparative accumulation mode, the
eluent was used without the acid.
The instrument was calibrated with caffeine (Sigma-
Aldrich, USA, ≥ 90%).
Nuclear magnetic resonance (NMR) spectra were
recorded on a Varian NMR System 400 spectrometer
with a silent Ceccato OFCS 5/8 SD compressor
(Varian, USA), with DMSO-D6 used as a solvent and
tetramethylsipane as the internal standard.
RESULTS AND DISCUSSION
To analyze the efficiency of various extraction
systems, we obtained average yields of solids in total
extracts. Total extract yields depending on the solvent’s
concentration are presented in Fig. 2.
Based on the results, we selected 50% ethanol as a
solvent to extract biologically active substances from
the dried biomass of ginseng callus cultures by reflux
extraction. Further selection parameters are shown in
Tables 1–2.
According to Table 1, the maximum yield of
biologically active substances extracted from dried
ginseng callus cultures (5.88 ± 0.59%) at 45°C was
provided by a 1:5 ratio of solvent to biomass and the
Figure 2 Solvents efficiency in extracting biologically active
substances from ginseng callus cultures
Table 1 Dry extract yield of biologically active substances from dried ginseng callus culture biomass depending
on extraction time (at 45°C)
Solvent:culture Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
1:1 0.50 ± 0.05 0.81 ± 0.08 1.22 ± 0.12 1.29 ± 0.13 1.38 ± 0.14 1.38 ± 0.14
1:2 0.80 ± 0.08 0.94 ± 0.09 1.35 ± 0.14 1.58 ± 0.16 1.67 ± 0.17 1.71 ± 0.17
1:5 1.20 ± 0.12 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.58
1:10 1.40 ± 0.14 1.98 ± 0.20 2.98 ± 0.30 5.94 ± 0.59 5.97 ± 0.60 6.04 ± 0.60
1:20 1.40 ± 0.14 2.01 ± 0.20 3.01 ± 0.30 5.95 ± 0.60 6.01 ± 0.60 6.07 ± 0.61
Table 2 Temperature selection for extracting biologically active substances from ginseng callus cultures
Temperature, °С Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
25 1.20 ± 0.12 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.58
40 1.55 ± 0.16 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.62
50 1.79 ± 0.18 2.35 ± 0.24 6.98 ± 0.70 7.05 ± 0.71 7.01 ± 0.70 7.12 ± 0.71
80 1.62 ± 0.16 2.14 ± 0.21 6.04 ± 0.60 6.12 ± 0.61 6.14 ± 0.61 6.17 ± 0.62
Analysis
efficiency
in reflux
extraction
High-performance
liquid
chromatography
0
1
2
3
4
5
6
30 40 50 60 70
Total extract yield, %
Ethanol concentration, %
50 60 70
Ethanol concentration, %
372
Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376
extraction time of at least 120 minutes. Noteworthily, a
further increase in duration had no effect on the yield of
biologically active substances.
Next, we optimized the temperature and time of
extraction (Table 2).
We found that the duration of 60 min and a
temperature of 50°C produced the optimal yield of
biologically active substances from the dried biomass of
ginseng callus cultures (6.98 ± 0.70%).
Next, we determined the optimal parameters to
obtain total extracts from the dried biomass of ginseng
suspension cultures with various solvent concentrations
(Fig. 3).
Based on the results, we selected 60% ethanol as
the most optimal solvent to obtain total extracts of
biologically active substances from the dried biomass of
ginseng suspension cultures using the reflux extraction
method. Further selection parameters are shown in
Tables 3–4.
We found that the maximum yield of biologically
active substances extracted from dried ginseng
suspension cultures (8.78 ± 0.88%) at 45°C was provided
by a 1:5 ratio of solvent to biomass and the extraction
time of at least 120 minutes.
According to the results, the optimal parameters for
extracting biologically active substances from ginseng
with 60% ethanol (extract yield of 8.95 ± 0.90%)
were 50°C, 30 min extraction, and a 1:5 solvent-tobiomass
ratio.
At the next stage, we optimized the parameters
for obtaining total extracts from in vitro ginseng root
cultures. The total extract yield depending on the solvent
is shown in Fig. 4.
According to Fig. 4, 70% ethanol produced the
highest yield of biologically active substances from
the dried biomass of ginseng root cultures by reflux
extraction. Further selection parameters are shown in
Tables 5–6.
According to the results, the duration of 30 to
180 min and the solvent-to-biomass ratio of 1:5 and
1:10 provided the maximum yield of biologically active
substances from the dried biomass of ginseng root
cultures. In particular, the yield of 11.98% was produced
at a ratio of 1:10 at 45°C during 30–60 min.
Table 3 Dry extract yield of biologically active substances from dried ginseng suspension culture biomass depending
on extraction time (at 45°C)
Solvent:culture Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
1:1 2.50 ± 0.25 2.51 ± 0.25 2.62 ± 0.26 2.92 ± 0.29 2.98 ± 0.30 2.83 ± 0.28
1:2 2.80 ± 0.28 2.94 ± 0.29 2.75 ± 0.28 2.85 ± 0.29 2.76 ± 0.28 2.91 ± 0.29
1:5 2.93 ± 0.29 8.78 ± 0.88 8.80 ± 0.88 8.68 ± 0.87 8.95 ± 0.90 8.21 ± 0.82
1:10 5.40 ± 0.54 8.98 ± 0.90 8.98 ± 0.90 8.94 ± 0.89 8.97 ± 0.90 8.84 ± 0.88
1:20 6.34 ± 0.63 8.61 ± 0.86 8.51 ± 0.85 8.95 ± 0.90 8.71 ± 0.87 8.77 ± 0.88
Figure 3 Solvents efficiency in extracting biologically active
substances from ginseng suspension cultures
High-performance
liquid
chromatography
70% ethanol extraction
0
1
2
30 40 50 Total extract Ethanol concentration, 0
2
4
6
8
40 50 60 70
Total extract yield, %
Ethanol concentration, %
0
3
6
9
12
15
30 40 50 60 70
Total extract yield, %
Ethanol concentration, %
Table 4 Temperature selection for extracting biologically active substances from ginseng suspension cultures
Temperature, °С Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
25 2.20 ± 0.22 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.58
40 2.55 ± 0.26 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.62
50 2.79 ± 0.28 8.95 ± 0.90 8.40 ± 0.84 8.75 ± 0.88 8.21 ± 0.82 8.32 ± 0.83
80 3.62 ± 0.36 7.56 ± 0.76 7.34 ± 0.73 7.12 ± 0.71 7.14 ± 0.71 7.17 ± 0.72
Figure 4 Solvents efficiency in extracting biologically active
substances from ginseng root cultures
liquid
chromatography
70% ethanol extraction
0
1
30 40 50 Total Ethanol concentration, 0
2
4
6
8
30 40 50 60 70
Total extract yield, %
Ethanol concentration, %
0
3
6
9
12
15
30 40 50 60 70
Total extract yield, %
Ethanol concentration, %
373
Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376
CONCLUSION
We developed a solvent screening procedure and
performed a qualitative analysis of biologically active
substances extracted from ginseng (Panax ginseng L.).
The optimal parameters for extracting biologically
active substances (organic solvent, ratio of solvent to
biomass, time, and temperature) were 50% ethanol, 1:5
ratio, 60 min, 50°C for ginseng callus cultures; 60%
ethanol, 1:5 ratio, 30 min, 50°C for suspension cultures;
and 70% ethanol, 1:5 ratio, 60 min, 50°C for root
cultures, respectively.
Table 5 Dry extract yield of biologically active substances from dried ginseng root culture biomass depending
on extraction time (at 45°C)
Solvent:culture Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
1:1 2.35 ± 0.24 2.51 ± 0.25 2.62 ± 0.26 2.92 ± 0.29 2.98 ± 0.30 2.83 ± 0.28
1:2 2.38 ± 0.24 2.94 ± 0.29 2.75 ± 0.28 2.85 ± 0.29 2.76 ± 0.28 2.91 ± 0.29
1:5 2.30 ± 0.23 11.78 ± 1.18 11.80 ± 1.18 11.68 ± 1.17 11.95 ± 1.20 11.21 ± 1.12
1:10 5.34 ± 0.53 11.98 ± 1.200 11.98 ± 1.20 11.94 ± 1.19 11.97 ± 1.20 11.84 ± 1.18
1:20 6.54 ± 0.65 11.61 ± 1.16 11.51 ± 1.15 11.95 ± 1.20 11.71 ± 1.17 11.77 ± 1.18
Table 6 Temperature selection for extracting biologically active substances from ginseng root cultures
Temperature, °С Extract yield depending on duration, %
10 min 30 min 60 min 120 min 180 min 360 min
25 3.20 ± 0.32 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.58
40 8.55 ± 0.86 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.62
50 7.79 ± 0.78 11.95 ± 1.20 12.40 ± 1.24 11.75 ± 1.18 11.21 ± 1.12 11.32 ± 1.13
80 6.62 ± 0.66 11.56 ± 1.16 11.34 ± 1.13 11.12 ± 1.11 11.14 ± 1.11 10.17 ± 1.02
Table 7 Optimal parameters for extracting biologically
active substances from the dried biomass of ginseng callus,
suspension, and root cultures
Type of
ginseng
culture
Organic
solvent
Ratio of
solvent to
biomass
Time,
min
Temperature,
°С
Callus 50% ethanol 1:5 60 50
Suspension 60% ethanol 1:5 30 50
Root 70% ethanol 1:5 60 50
Figure 5 Qualitative analysis of ginseng flavonoids
mAU
Figure 6 NMR spectrum of mangiferin isolated from ginseng
extracts
glucopyranosyl
Based on the results in Table 6, we selected the
following parameters for extracting biologically active
substances from the dried biomass of in vitro ginseng
root cultures: extraction at 50°C during 30 min at a
1:5 ratio of solvent to dried biomass. These parameters
produced a yield of 11.95 ± 1.20%. We recommend to
use 70% ethanol as a solvent.
Thus, we determined the optimal parameters (time,
temperature, organic solvent, ratio of solvent to biomass)
for extracting biologically active substances from
ginseng callus, suspension, and root cultures (Table 7).
The qualitative analysis of standard compounds
and biologically active substances extracted from dried
ginseng callus, suspension, and root cultures showed
the presence of rutin, quercetin, quercetin-glycoside,
mangiferin, luteolin, apigenin, and caffeic acid.
The fractions were separated by preparative
HPLC (Fig. 5).
As a result, we isolated a basic substance with
a retention time of 24 min, which was identified as
mangiferin (Fig. 6).
Thus, rutin (0.25), quercetin (0.75), and mangiferin
(0.57) were major biologically active substances found in
the extracts of in vitro ginseng callus, suspension, and
root cultures. We also identified caffeic and sinapinic
acids in the extracts.
The qualitative analysis of the extracts of ginseng
callus, suspension, and root cultures showed the
presence of rutin (0.25), quercetin (0.75), and mangiferin
(0.57) as predominant components. The extracts also
contained caffeic and sinapinic acids.
Thus, the extracts obtained by water-ethanol
extraction from ginseng callus, suspension, and root
cultures can be used as biologically active ingredients in
the production of functional geroprotective foods.
CONTRIBUTION
The authors were equally involved in writing
the manuscript and are equally responsible for
plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
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