10 grams of the dried plant material was
extracted with 100 ml of methanol and was kept on a magnetic stirrer (Remi) for
24 hours. Thereafter, it was filtered and centrifuged at 5000 g for
15 minutes. The supernatant was collected and the solvent was evaporated to
make the final volume one-fifth of the original volume. It was stored at 4ºC in
airtight bottles for further studies. The same procedure was repeated for
obtaining ethyl acetate, and chloroform extracts.
Isolation and culture of lymphocytes:
Isolation and culture of lymphocytes was
performed by a modification of the method described by Kulkarni et al. (1999). 5 ml of blood was collected from a HIV
positive individual (confirmed by sandwich ELISA using HIV-Check (Xcyton,
India), and also from an uninfected individual based on the procedure described
by Hahn et al. (1984) in EDTA
vacutainers after obtaining informed consent. It was double diluted with RPMI
tissue culture media, layered on 3ml Ficoll-hypaque (Sigma, USA), and
centrifuged at 1500rpm for 15 minutes. The lymphocyte ring obtained was
collected in a separate NUNC sterile tube containing 13 ml RPMI. The contents
of the tube were mixed well and centrifuged at 1000 rpm for 5min. The
supernatant was discarded and the pellet was dissolved in 1ml RPMIC (RPMI
containing 20% FCS). The cell viability was checked using Trypan blue method.
Phytohaemagglutinin (PHA) was added at a concentration of 1µl per 1million
cells. 500 µl of the culture was added to each well of a 24 well NUNC plate.
The culture was incubated at 37 0 C in a 5% CO2 incubator for 24 hrs.
Addition of plant extract:
After 24hrs 1µl of IL-2 (1:100 in RPMIC) was added to each well along with 1µl
of different dilutions (2,3, 4 & 5 mg/ml) of the aqueous extract of C.collinus. One of the wells in which the
standard drug AZT was added was used as the positive control. The plates were
incubated at 37 0 C in a 5% CO2 incubator for 72 hrs. Different dilutions of
the extracts were also added to wells with healthy lymphocytes and incubated at
37 0 C in a 5% CO2 incubator for 72 hrs. Cytotoxicity assay was carried out
using Trypan blue dye , Mishell and Shiigi (1980).