SUPPLEMENTARY MATERIAL
HPLC analysis of harringtonine and homoharringtonine in the needles of Cephalotaxus
griffithii alkaloid fraction and cytotoxic activity on chronic myelogenous leukemia K562
cell.
Dinesh Singh Moirangthem a,b, Jagat Chandra Borah a, Laishram Surbala a, b, Mohan Chandra
Kalita b, Narayan Chandra Talukdar a*
Affiliation
a
Institute of Bioresources and Sustainable Development, Department of Biotechnology,
Government of India, Takyelpat Institutional Area, Imphal - 795001, Manipur, India.
b
Department of Biotechnology, Gauhati University, Gopinath Bordoloi Nagar, Guwahati -
781014, Assam, India.
*Corresponding author
Email: nctalukdar1@gmail.com
Abstract
Harringtonine (HT) and homoharringtonine (HHT) are Cephalotaxus alkaloids having
good antileukemic activity. The aims of this research were to (1) determine the content of HT
and HHT present in Cephalotaxus griffithii needles alkaloid fraction (CGAF) and (2)
compare antiproliferative activity of CGAF, with that of HT and HHT on human chronic
myelogenous leukemia K562 cell. The concentration of HT and HHT was found to be 122.14
± 7.84 and 16.79 ± 1.69 mg per gram of CGAF, respectively. Treatment of K562 cells with
CGAF, HT and HHT decreased the viable cells in a dose and time dependent manner.
Interestingly, the maximum cell death was found in CGAF (P < 0.05), with IC50 value which
was 3 to 4.6 fold lower than those of HT and HHT. However, HT and HHT did not differ
significantly in the IC50 values. Our results indicate that HT content in the needles of C.
griffithii is higher than HHT and alkaloids other than HT and HHT in CGAF is
predominantly responsible for K562 cell death.
Keywords
Cephalotaxus griffithii, harringtonine, homoharringtonine, leukemia, K562.
1. Experimental
1.1. Chemicals
All solvents used were HPLC grade and purchased from Rankem. HT and HHT were
purchased from Chromadex. MTT was purchased from Himedia Laboratories Pvt. Ltd.
Vincristine (purity >98% by HPLC) and all the cell culture chemicals were purchased from
Sigma-Aldrich.
1.2. Plant material
The needles were collected from Kangchup Hills, Manipur, India (N24°52´10ʺ
E093°46´12ʺ) at an elevation of 1534.66 m above sea level in January, 2012. The specimen
was identified by Dr. Biseshwori Thongam, Plant Systematics and Conservation Laboratory,
Medicinal, Aromatic and Horticultural Plant Resources Division, Institute of Bioresources
and Sustainable Development, Manipur, India and by Dr. S.K. Verma, National Bureau of
Plant Genetics Resources, Meghalaya, India. A voucher specimen (IBSD/C/102) has been
deposited to the IBSD herbarium.
1.3. Extraction and isolation of crude alkaloid
Dried pulverised needles (250 g) were extracted with MeOH at room temperature
exhaustively to give the extract (22 g). The extract was treated with 3% tartaric acid (440 ml)
to adjust at pH 2 and partitioned with EtOAc (3 × 220 ml). Then, the aqueous phase was
treated with saturated Na2CO3 solution and extracted with CHCl3 (3 × 220 ml). CHCl3soluble materials were concentrated to obtain CGAF (136 mg) (Morita et al. 2005).
1.4. Quantification of HT and HHT by RP-HPLC
HPLC analysis was performed using a Waters HPLC system equipped with a binary
pump (Waters 1525), a photodiode-array detector (Waters 2996) and sample injector with a
20 µl loop. Evaluation and quantification were made on Empower pro data system.
Separation was performed using an XTerra MS C18 column (5 µm, 4.6 mm × 250 mm,
Waters, Milford, Massachusetts, USA). The mobile phase was acetonitrile–0.1%
trifluoroacetic acid (1:3) in isocratic mode. The flow-rate was 0.4 ml/min and monitored at
254 nm.
1.5. Cell culture
The K562 cell line was procured from the National Centre for Cell Science (India). The
cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (FBS) and 1% antibiotic antimycotic solution (10,000 U/ml penicillin, 10 mg/ml
streptomycin sulphate, and 25 µg/ml amphotericin-B), and maintained at 37 °C in a
humidified atmosphere with 5% CO2/95% air.
1.6. MTT reduction assay
Cytotoxicity analysis was determined using the MTT assay as reported by Mosmann
(1983). K562 cells were plated at a density of 2 × 105 cells/well in 96-well culture plates and
incubated for 24 h. The cells were then exposed to increasing concentrations of the test
samples (20-640 µg/ml), and incubated for 24 and 48 h. After incubation, the contents were
replaced with 100 µl of MTT dissolved in serum-free medium (1.2 mM) after which the
plates were further incubated for 3 h. The contents were then replaced with equal amounts of
DMSO to solubilise the formazan grains formed by viable cells. Finally, the absorbance was
read at 570 nm using a multi-well plate reader (Thermo, Multiskan spectrum). The viability
percentage was calculated by using the formula below:
Viability % = Absorbance of test sample × 100 / Absorbance of solvent control.
1.7. Statistical analysis
The values are presented as mean ± SD. Statistical analysis was performed by one-way
ANOVA supplemented with Tukey’s HSD post hoc test. Values at P < 0.05 were considered
to indicate statistical significance.
References
Morita H, Yoshinaga M, Kobayashi J. 2005. Cephalezomines G, H, J, K, L. and M, new
alkaloids from Cephalotaxus harringtonia var. nana. Tetrahedron. 58: 5489-5495.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods. 65: 55–63.
Figure S1. Typical HPLC chromatogram of the harringtonine, homoharringtonine,
Cephalotaxus griffithii needles alkaloid fraction (CGAF) and spiked CGAF using
acetonitrile–0.1% trifluoroacetic acid (1:3) in isocratic mode on an XTerra MS C18 column
(5 µm, 4.6 mm × 250 mm). Detection was carried out at 254 nm (A) Harringtonine (elution
time -11.7 min) (B) homoharringtonine (elution time-14.8 min) (C) CGAF and CGAF spiked
(1428 µg CGAF + 286 µg HT + 570 µg HHT per ml).