Corresponding author: Yancho Zarev (
Establishment of
Plant biotechnology approaches could be a successful tool for increasing the yield of pharmaceutically important metabolites such as various flavonoids.
Thus, the object of this study was to establish
For establishment of shoots, seedlings were incubated under sterile conditions, in flasks with solid MS culture medium
Callus cultures were obtained when shoot explants were cultivated on G48 medium (
Actively growing calli were transferred to G48 liquid medium (G48 medium without agar-agar) under light regimen of cultivation and transferred to fresh medium every two weeks. Thus, suspension cultures of
Due to the low quantity of flavonoids in callus and suspension cultures, experiments were made in order to increase the production of flavonoids. For that reason callus cultures were cultivated on modified G48 medium in light regimen of cultivation supplemented with various concentrations of Ca2+ (G48 + ½Ca2+ 220 mg/L and G48 + 2Ca2+ 880 mg/L) and Mg2+ (G48 + ½Mg2+ 185 mg/L and G48 + 2Mg2+ 740 mg/L). For increasing the production of flavonoids within suspension cultures, at the first day of cultivation sterile quercetin solution was applied in three concentrations (10, 20 and 30 mg/mL) under aseptic conditions. Each concentration level consists of three randomized samples and the results are the average of three replicates. In addition, three controls were cultivated without addition of substrate.
The air-dried powdered plant material (0.20 g) was exhaustively extracted with 80% methanol under reflux (3 × 50 mL), the extracts were filtered and concentrated under reduced pressure. The residue was dissolved in 50 mL water and extracted with ethylacetate (3 × 50 mL), and then the ethylacetate fractions were dried and concentrated under reduced pressure. For the purpose of the analysis, those samples were diluted in 80% methanol to reach concentration of 500 µg/mL. The extraction of the plant material for individual quantitation of rutin and camelliaside A was performed as reported before (
For the individual analysis of rutin and camelliaside a method, previously described (
UPLC separations for determination of total flavonoid content were performed on a Hypersil Gold C18 column (1.9
HRESIMS spectra were taken with a LC-MS system consisting of a Q Exactive Plus Orbitrap mass spectrometer with a HRESI ion source (Thermo Fisher Scientific, Bremen, Germany) used in ultra-high resolution mode (70 000, at
Rutin dihydrate CRS (Sigma-Aldrich, Germany) and camelliaside A (isolated previously, 99.8% purity,
Determination of total flavonoids was based on calibration curve of rutin: four different concentrations ranging from 0.12
The total flavonoid amount was quantified using the formula:
Total flavonoids = (Afl * Cst) / (Ast * Ws),
Afl – a peak area of deprotonated molecular ions corresponding to flavonoid derivatives based on their MS spectra, Table
Deprotonated molecular ions observed in LC/MS spectra of callus cultures of
Peak | Formula | tR | MW | |
---|---|---|---|---|
1 | C15H18O8 | 2.16 | 326.1001 | 325.0932 |
2 | C21H28O13 | 2.54 | 488.1529 | 487.1464 |
3 | C16H20O9 | 2.72, 3.04* | 356.1107 | 355.1040 |
4 | C16H28O10 | 3.52, 3.84* | 380.1682 | 379.1615 |
5 | C21H22O11 | 3.14 | 450.1162 | 449.1094 |
6 | C22H24O12 | 3.47 | 480.1267 | 479.1202 |
7 | C16H16O7 | 4.28 | 320.0896 | 319.0827 |
8 | C17H30O10 | 5.06, 5.33* | 394.1839 | 393.1773 |
9 | C22H28O9 | 3.86 | 436.1733 | 435.1665 |
10 | C24H26O13 | 4.21, 4.65*, 7.62*, 10.37* | 522.1373 | 521.1308 |
11 | C19H26O10 | 4.37 | 414.1526 | 413.1460 |
12 | C22H24O11 | 4.55, 4.78*, 6.14*, 6.40* | 464.1318 | 463.1254 |
13 | C22H22O12 | 4.85, 6.58*, 6.77*, 11.88* | 478.1111 | 477.1046 |
14 | C23H24O13 | 5.27, 8.63* | 508.1217 | 507.1150 |
15 | C28H36O13 | 5.7 | 580.2155 | 579.2088 |
16 | C22H22O11 | 8.59 | 462.1162 | 461.1097 |
17 | C29H38O15 | 6.02 | 626.2210 | 625.2148 |
18 | C21H20O10 | 3.25, 5.52*, 6.44* | 432.1056 | 431.0989 |
19 | C30H38O14 | 6.53 | 622.2261 | 621.2199 |
20 | C25H34O14 | 6.63 | 558.1948 | 557.1885 |
21 | C16H12O6 | 6.96 | 300.0633 | 299.0564 |
22 | C23H24O11 | 7.15 | 476.1318 | 475.1251 |
23 | C23H38O14 | 7.28, 7.46*, 7.74*, 8.07* | 538.2261 | 537.2197 |
24 | C26H34O11 | 4.21, 4.66* | 522.2101 | 521.2035 |
25 | C24H24O13 | 7.85 | 520.1216 | 519.1153 |
26 | C24H24O12 | 9.70, 9.97* | 504.1267 | 503.1204 |
27 | C23H24O12 | 10.14 | 492.1267 | 491.1201 |
28 | C23H22O13 | 10.87 | 506.1060 | 505.0996 |
29 | C29H36O14 | 11.31 | 608.2105 | 607.2044 |
* Isomers
Each experiment was done in triplicate. Results were expressed as mean ± SD. MedCalc 12.3 (MedCalc Software 2012) was used. The one-way analysis of variance was performed to define the statistical significance of the amount found. Probability values of
The highest amount of biomass accumulation (GI = 1.17 ± 0.04) of the plant cells of
Growth index of in vitro cultures of
Rutin was observed in the negative ion mode as a deprotonated ion [M-H]- with
A visual evaluation of the linear regression line plots showed that the method was linear for both standards. The determination coefficient for rutin was
Rutin (8.72 ± 0.09 ng/mg DW) and camelliaside A content (74.65 ± 0.09 ng/mg DW) were proved in the highest amount in shoot cultures on MS medium. Camelliaside A was observed in the lowest amount (2.19 ± 0.09 ng/mg DW) in suspension cultures grown on G48 medium. Rutin was not detected in calli grown on G56 medium in the dark (Fig.
Rutin and Camelliaside A content in
The quantity of rutin reached 0.39 ± 0.04 ng/mg DW in suspension cultures, while callus, cultivated on G48 medium achieved 0.95 ± 0.05 ng/mg DW under light regimen of cultivation and 0.50 ± 0.06 ng/mg DW when cultivated in the dark. Callus cultivated on G56 medium in light achieved 0.73 ± 0.05 ng/mg DW rutin content.
The quantity of camelliaside A reached 7.30 ± 0.11 ng/mg DW in calli, cultivated on G56 medium in the light and 2.88 ± 0.08 ng/mg DW when cultivated on G56 in dark. Callus cultures cultivated on G48 medium achieved 6.93 ± 0.12 ng/mg DW camelliaside A content under light and 7.60 ± 0.10 ng/mg DW when cultivated in dark.
Determination of growth index and total flavonoids content in callus cultures, cultivated on modified MS media supplemented with half and double the amount of Mg2+ and Ca2+
The highest amount of biomass (GI = 0.82 ± 0.03) was observed on callus cultures of
Rutin was observed in the positive ion mode as protonated ion [M-H]- with
Growth index of callus cultures of
LC-MS chromatogram of flavonoids from callus cultures of
The highest concentration of flavonoids content (2.37 ± 1.08 mg/g DW) was observed on callus cultures cultivated on G48 + 2Ca2+ medium, while the lowest concentration (0.98 ± 0.85 mg/g DW) was detected on calli established on G48 + ½Mg2+ medium (Fig.
Total flavonoid contents (mg/g DW) of callus cultures of
The addition of quercetin to the G48 medium of suspension cultures of the plant increased the quantity of total flavonoids (Fig.
Total flavonoid contents (mg/mg DW) in suspension cultures of
In addition, biotransformation of quercetin to its monoglycosylated derivative isoquercitrin was observed (
The medicinal uses of
This study was financially supported by the Council of Medicinal Science at Medical University of Sofia, contract № D-80/2019.