Cell suspension

Cell suspension culture is a type of culture in which single cells or small aggregates

of cells multiply while suspended in agitated liquid medium. In callus callus culture method,
the callus proliferates as an unorganised mass of cells. So it is very difficult to follow many
cellular events during its growth and developmental phases. To overcome such limitations of
callus culture, the cultivation of free cells as well as small cell aggregates in a chemically de-
fined liquid medium as a suspension is made. This technique also implied on Andrographis
Paniculata to extract andrographolide.
Initiation of cell suspension of Andrographis Paniculata
A friable callus obtained from vegetative phase leaf bits of Andrographis is used to initiate
the suspension culture for secondary metabolite production. Those vegetative phase leaf bits
inoculated in a solid MS medium(4) fortified with 3% sucrose and plant growth hormones
combinationof 2,4-Dichlorophenoxy acetic acid and Napthalene acid. This composition of
medium is the best for cell suspension for maintenance of callus culture. Then the callus cells
of 0.5/1.00 g fresh weight have to be carefully transferred to 100ml Erlenmeyer
flask,containing liquid callus induction medium. This transfer have to be done aseptically in
laminar flood hood. The cells then have to cultured on a gyratory platform shaker with an
orbital motion stroke of 2-4cm in continuous light of less than 2000 lux for 15 days. Then a
known volume of uniformly dispersed suspension culture have to transfer plastic centrifuge
tube and centrifuged at 2000rpm. The andrographolide is estimated by using an HPLC. The
cell is harvested by centrifuging at 5000rpm and extract is filtered through filter paper (grade
80 g/m2) then the culture was homogenized directly with 1ml 80% ethanol. The mixture was
grinded with the micro pestle and shacked well and digested with cellulase. Figure 4.1 shows
the techniques in diagram.

Quantification of Biomass Content

Growth of the suspension culture was quantified on the basis of initial weight and final
weight. The specific growth rate was calculated from (X – Xo) versus the time (t) plot, where
Xo is the initial biomass at the time of inoculation, X is the final biomass after t time. The
extract production is well based and lies on the growth hormones in the medium. The
biomass content of Andrographis is variate with the different concentration growth hormones
that we added in the culture medium. Table 1 shows the andrographolide variation results
with the growth hormone concentrations based on an experiment made by researcher Sharma.

Table 1

As per the results, the more the concentration of growth hormones in culture medium, the
more the biomass content and andrographolide extraction we get. The latest researches show
that the andrographolide content from the extraction is high when the suspension is treated
with salicylic acid. There are formation of some new peaks obtained from the HPLC, when
the salicylic acid treated suspension culture is used. This shows that the salicylic acid is a
factor that having an implication on Andrographis paniculata cell suspension to extract
andrographolide.