SUPPLEMENTARY MATERIAL
Solvent based extraction optimization for efficient ultrasonication assisted ginsenoside recovery
from Panax quinquefolius and P.sikkimensis cell suspension lines.
Tanya Biswas1, P V Ajayakumar2, Ajay Kumar Mathur1 and Archana Mathur1*
1
Division of Plant Biotechnology, 2Analytical Chemistry Division
CSIR-Central Institute of Medicinal & Aromatic Plants; Council of Scientific & Industrial Research; PO
CIMAP; Lucknow-226015 (India)
*Author for correspondence:
Dr Archana Mathur
Plant Biotechnology Division
Central Institute of Medicinal & Aromatic Plants
Council of Scientific & Industrial Research
PO CIMAP, Lucknow-226015 (India)
Tel++ 91-522-2359623, 2357134
Fax++91-522-2342666
Email: archnacimap@gmail.com, a.mathur @cimap.res.in
ABSTRACT
The present study aims at developing an extraction protocol for efficient ginsenoside recovery from cell
suspensions of Panax quinquefolius and P. sikkimensis. Methanol (100%,70% ,30%), Water (40°C , 90°
C), water saturated butanol and butanol saturated water were compared for their ultrasonication assisted
ginsenoside retrieval efficacy. HPLC and HP-TLC analysis revealed 100% Methanol as the best solvent
for maximum retrieval of Rb (diol) and Rg (triol) ginsenosides (P.quinquefolius: Rb: 0.189, Rg: 3.163
mg/g DW; P.sikkimensis: Rb: 0.245, Rg: 4.073 mg/g DW), followed by water (90°C). Methanolic
solutions, especially 70%, proved to be significant retrievers of Rg1 (1.812 and 1.327 mg/g DW in
P.quinquefolius and P.sikkimensis), with poor Re recovery (0.328 and 0.342 mg/g DW). Water saturated
butanol also led to significant ginsenoside extraction (72.4 % of content extracted by Methanol),
selectively in P.quinquefolius, with a less than 50% of total content extracted by methanol, in P.
sikkimensis.
Key words: Panax quinquefolius, Panax sikkimensis, ginsenosides, ultrasonication-assisted extraction,
HPLC, HPTLC
Experimental
Chemicals and standards used
All solvents and reagents used in the experiments were of analytical grade purchased from Merck
Chemicals. The ginsenoside standards Rb1, Rb2, Rc, Rd, Rf, Re, Rg1, Rg2 and Vanillin were obtained
from Sigma Chemicals (St Louis, USA). Aluminium backed Silica gel 60 F254 HP-TLC sheets (Merck)
were used for HP-TLC quantification. The plates were scanned on a TLC scanner (TLC scanner 3,
Camag, Switzerland) for densitometric evaluation of the spots obtained on the chromatogram. All
chromatograms were documented as photographic evidence using UVP Multi-Doc It Imaging System,
USA.
Plant material used
Callus induced from P. quinquefolius (American ginseng, Bonera, J &K, India) and P. sikkimensis
(Lachung, Sikkim, India) root explants are being maintained in the host laboratory and regularly subcultured within a six week cycle, since the past 15 years under optimized media and illumination
conditions (Mathur et al., 2003) . The two cell lines on which this work has been carried out are:
1) A high ginsenoside yielding, fast growing cell line of P. quinquefolius (US PATENT 6326202
B1)
2) An anthocyanin containing cell line of P. sikkimensis (US PATENT 6368860)
Respective suspensions were developed on MS media (Murashige and Skoog, 1962) supplemented with
3.0% sucrose, 0.01% myoinositol, 0.33μM thiamine hydrochloride, 2.5μM pyridoxine hydrochloride,
4.0μM nicotinic acid, 5.4 μM NAA (naphthalene acetic acid) , and 1.2μM Kinetin (Biswas et al., 2014).
The pH of the medium was adjusted to 5.8±0.03 before autoclaving at 1.04 kg/cm2 pressure (121°C) for
15–20 min. The cultures were incubated on a rotatory shaker (100rpm) under diffused light (15μE
m−2s−1) provided by specially designed LED light racks , 16:8-h light/dark photoperiod, 25±3°C, and
60–70% relative humidity.
Sample preparation
Known amount of cells (10 g FW in the present study) were used for qualitative and quantitative
analysis of ginsenosides following a modified procedure of Furuya et al. (1983). Cells were lyophilized
till constant weight in a lyophilizer (Labconco, Free zone 2.5, USA) to determine their dry weights and
were utilized for extraction using the following extractants:
1) Methanol 100% (100% MeOH), 70% aqueous solution (70% MeOH), 30% aqueous solutions
(30% MeOH)
2) Water at 40°C(NW), 90°C (HW), and butanol saturated water(BSW)
3) Water saturated butanol (WSB)
The dried cells were soaked in fixed volume of the above mentioned extractants (20 mL) in
polypropylene centrifuge tubes and subjected to Ultrasonication (120 W US power; tank size:
250x150x150mm), for 30 min in an Ultrasonic Cleaning Bath (Rivotek, India) at 40°C. Following
centrifugation (6000rpm, 5 min), the supernatant was decanted and the procedure was repeated four
times, including an overnight soak, in the respective extractant. The solvent extracts were then processed
downstream as elucidated in Figure S1.
HP-TLC Densitometric/ HPLC quantification
High performance thin layer chromatography (HP-TLC) was carried out as reported previously by the
host laboratory (Biswas et al, 2014). Known amounts of sample (12µL) and authentic (2 µL; 1mg/ml)
were spotted at equal distances from each other on HP-TLC plates. The mobile phase employed for
optimum separation of the mixture comprised of a chloroform: methanol: water::13:7:2 composition
(lower phase). The plates were run in a twin trough (Camag,Switzerland) pre-conditioned in the mobile
phase (1 hour). After separation, the plates were air-dried, visualized after spraying with VanillinSulphuric acid-Ethanol solution (Vanillim-5g ; Ethanol-475mL; conc. H2SO4- 25 mL) and heating the
plates at 110°C on a Plate heater (Plate heater III Camag, Switzerland). For quantitative and qualitative
analysis, the plates were scanned with a TLC Scanner at 575 nm, where clear separation of 6 (Rb1, Rd,
Re, Rf, Rg1, Rg2) ginsenoside standards were achieved with distinct Rf and peak heights. For further
resolution and identification of Rb2 and Rc (not resolved using HP-TLC), HPLC-UV analysis was
performed using Waters symmetry C18, 4.16x 150 mm (3.5 micron) column, 600 E Waters pump and a
2996 photodiode detector. A 717 autosampler injection system was used. The flow conditions were
identical to what was reported previously by the host laboratory (Biswas et al, 2014). The peaks were
identified by matching retention times and corresponding UV spectra. Where required, peak identity was
further confirmed by spiking the sample with the respective authentic. All samples were analyzed at 203
nm using Empower 2 software.
Statistical analysis and content measurement
The ginsenoside content is reported in mg/g DW of plant material, as mean of three independent extracts
of a particular sample. The data was further subjected to ANOVA using CRD (completely randomized
design; Singh and Chaudhary, 1979)
Fig S1. Schematics of the extraction procedure employed for sample preparation. (US refers to
Ultrasonication)
References
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