Quantification of andrographolide:The andrographolide
quantification was done through HPLC.
Fresh 30 days old callus (0.1g) was dissolved in 1ml
80% ethanol and crushed by the use of plastic micro
pestle in centrifuge tube. Other hand 30 days old cell
suspension was centrifuged at 5000rpm for 15
minutes. Supernatant was discarded and cells (0.1g)
were re-dissolved in 1ml 80% ethanol. Cell was
digested by the use of cellulose at the rate of 0.3mg/
g of cell [8]. 20 μl of each of dissolved callus and
cell culture solution injected in the HPLC after
centrifugation at 12000 rpm. The andrographolide
standard purchased from Sigma-Aldrich, USA used
in 0.44, 0.22 and 0.11 mg/ml solvent for standard
curve preparation. The HPLC grade
methanol:acetonitrile:water at the ratio of 65:20:15
used as solvent system for HPLC estimation [9].
HPLC analysis was carried out on Younglin
chromatograph, equipped with a vacuum degasser, a
quaternary pump and a UV/vis detection system,
connected to a reversed-phase column (Diamonsil
C18, particle size 5 μm, metachem (150 mm X 4.6
mm) column. The temperature was fixed at 25°C.
The injection volume was 20 μl and elution was
monitored at 223 nm