Establishment Of Cell Cultures For Narciclasine And Tazettine Production And Pharmacological Evaluation Of Hymenocallis Littoralis (Jacq.) Salisb
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Date
2015-06
Authors
Noormi, Rosli
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Abstract
A study was carried out to determine narciclasine and tazettine distribution in
vivo intact plants from juvenile and flowering stages; and in vitro cultures namely callus,
cell suspension, root and treated root cultures of Hymenocallis littoralis (Jacq.) Salisb
plant. Phytohormone 2,4-D at 2.0 mg/L was the best auxin for the callus induction and
growth proliferation in all different types of explants. Half continuous light with full
strength of MS medium, pH 5.5 and sucrose at 3 % (w/v) was the best condition for
callus growth and proliferation. The initiation of Hymenocallis littoralis cell suspension
cultures in bulb explants was carried out by using 2.0 mg/L 2,4-D. Cell suspension
cultures at four weeks cultivation period with half continuous light, full strength of MS
medium, pH 5.5, sucrose at 3 % (w/v) and 2.0 mg/L 2,4-D produced the highest amount
of fresh weight biomass. Roots could be induced from in vitro root culture using MS
medium supplemented with 1.4 mg/mL IAA. Highest biomass production for root
proliferation was obtained at fourth week under half continuous light with full strength of
MS medium, 4% (w/v) sucrose and pH 6. The histology of control and treated root
demonstrated the present of clear vascular bundle system in treated root which indicated
it can be easily multiply within short period of time. Twelve (12) samples crude extracts
of different explants from juvenile and flowering stages of Hymenocallis littoralis (Jacq.)
Salisb wild plants were used to screen the antimicrobial and antioxidant activities. In
antimicrobial assay, flowering stage explants showed pronounced inhibition of microbe’s
growth of the cultures compared to the juvenile stage with different range on inhibition
zone. Roots from the flowering stage produced highest activity against Bacillus subtilis
(zone of inhibition was 29.0 + 0.04 mm). Best MIC was obtained at 6.25 mg/mL from
the juvenile roots and flowering old leaves against Micrococcus spp.; flowering old
leaves and stems against Escherichia coli hospital strain; and juvenile old leaves,
flowering old leaves and stems against Candida albicans. For time kill study results,
growth profile curve shows the fungicidal activity exhibited by H. littoralis flowering old
leaf extract against C. albicans. H. littoralis flowering old leaf extract inhibited both
growth of C. albicans and E. coli at different incubation durations with extract was more
fungicidal against C. albicans compared to bactericidal against E. coli. SEM analysis for
H. littoralis treated leaf extract against C. albicans and E. coli after 16 h of treatment
resulted distorted cells with the presence of invaginations and convolutions incidence.
Higher degree of collapsed cells was obtained at 20h. The antioxidant activities presented
in all 12 samples of Hymenocallis littoralis juvenile and flowering stages based on the
results obtained from DPPH, ABTS and FRAP methods. Young leaf at juvenile stage
produced highest antioxidant activities (DPPH (44.2%), ABTS (99.64%) and FRAP (630
mgFeSO4/g DW extract)) and the total phenolic content (1119 mg Garllic acid/g DW
extract). Results for analgesic activity based on hot plate and tail flick assays
demonstrated significant analgesic properties of Hymenocallis littoralis (Jacq.) Salisb
plant’s extracts. Mice treated with 400 mg/kg flowering root extracts produced 65.37 %
of analgesic activity and was the highest analgesic activity within in all the tested
samples. For toxicity studies, the results obtained showed that toxicity of the plant
extracts increases accordingly with the extract concentrations. Chloroform methanolic
juvenile root extract [bioactivity by LC50 :-1.118 (6 h), -3.583 (12 h), -4.245 (18 h), 0 (24
h)] exhibited the highest toxicity among all extracts. Results on wound healing activity
indicated that juvenile bulb extract [56.40 + 2.640 d (1 μg/mL), 150.21 + 2.44c,d (10
μg/mL)] was the most effective for the wound healing activity based on scratch assay
with Hs-27 cell lines within 12 hours compared to control treatment, negative group and
other types of explant’s extracts. Qualitative analysis using TLC revealed that
narciclasine and tazettine production was highest using in vivo root of Hymenocallis
littoralis (Jacq.) Salisb plant from juvenile and flowering stages. Callus culture produced
the highest narciclasine and tazettine under in vitro condition. The results of HPLC
analysis based on chloroform methanolic extracts confirmed that flowering stage
produced the highest concentration of narciclasine (74.07+ 0.017 mg/g DW tissue).
Meanwhile, callus cultures produced highest narciclasine (11.87 + 0.021 mg/g DW
tissue) under in vitro condition. The highest concentration of tazettine was detected in
flowering root (7.264 + 0.003 mg/g DW tissue). Similarly, callus culture contained
highest tazettine (5.456 + 0.003 mg/g DW tissue) under in vitro condition. As a
comparison, the content of narciclasine was significant tested in all samples higher at ten
times compare to the content of tazettine. The production of narciclasine and tazettine in
vitro will be enhanced with large scale through bioreactor technology system.
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Keywords
Narciclasine , Pharmacological Evaluation