Page 31 - ITPS-5-2

25 INNOSC Theranostics and Pharmacological Sciences, 2022, Vol. 5, No. 2
at 517 nm after incubation using a spectrophotometer.
The experiment was performed in triplicates. The
percentage of RSA and the inhibitory concentration
(IC ) were calculated using the following formula:
50
% Scavenging activity =
)
Absorbance (control -Absorbance (sample) × 100
Absorbance (control)
(I)
2.7. Total phenolic content
The presence of phenolic substances was measured
using the FC reagent method, referring to gallic
acid standard [29]. Each plant extract was tested
at a concentration of 100 μg/mL. A combination of
0.5 mL of FC reagent and 1.0 mL of Na CO (20%)
2
3
was added to each test tube and incubated in the
dark for 42 min. The absorbance was measured
at 765 nm using a spectrophotometer (Thermo
Fischer). The experiment was performed in
triplicates. The absorbance of samples was plotted
against the concentration and expressed in terms of
gallic acid equivalence (μg GAE/g of extract).
2.8. Statistical analysis
Data from three replicates were averaged and
analyzed for each experiment. Analysis of variance
was performed using SPSS Inc. 16.0. To determine
the significant effects of treatments, Tukey’s HSD
test was conducted to compare, and separate
treatment means based on F-values (p ≤ 0.05).
3. Results and discussion
3.1. Preliminary phytochemical screening
Plants with a greater number of phytoconstituents
generally exhibit higher medicinal properties. The
phytochemical analysis of tested plant extracts
revealed the presence of various classes of
secondary metabolites in chloroform, methanol,
and aqueous extracts, and the results of the
biochemical test showed the presence of various
classes of phytoconstituents such as alkaloids,
steroids, triterpenes, saponins, tannins, flavonoids,
carbohydrates, resins, proteins, glycosides, and
phenolics in the plant. The results are presented in
Table 1.
©2022 AccScience Publishing
Madhavamurthy et al.
Among the tested plants, D. barbatulum
showed no alkaloids, while B. neilgherrense and
D. Jerdonianum were positive for the majority
of the test in all extracts. These results are
consistent with findings reported by Nagananda
et al. [30], which found alkaloids, flavonoids,
phenols, phytosterols, and other compounds in
the methanol extracts of P. pallida. The majority
of the phytochemical tests yielded the highest
number of positive results in methanol extracts,
followed by aqueous and chloroform extracts in
all the plants. The findings align with previous
reports by Kumari et al. [31] on B. neilgherrensis
and Maridass et al. on D. barbatulum, where the
majority of the phytochemicals were present in
methanolic and aqueous extracts [32]. The study
results indicate that methanol is the most effective
solvent for extracting secondary metabolites
from orchid plants. Methanol has been found
to dissolve the highest number of secondary
metabolites. These findings are consistent with the
previous research conducted by Esmaeili et al.,
who reported that methanol extracted the highest
phytochemical content in in vitro-grown Trifolium
pratense L. [33].
3.2. Antibacterial activity
The antibacterial activity of the crude extracts from
selected orchids was evaluated at 50 mg/mL stock
solution, and the results are presented in Table 2.
The MIC values for each crude extract are shown
in Table S1. Among the tested plant extracts, the
methanolic extract of P. pallida effectively inhibited
E.coli (18.64 mm), P. aeruginosa (15.36 mm), S.
typhi (14.32 mm), S. flexinari(13.24 mm), and B.
subtilis (13.34 mm) (Figure 2). The MIC values
ranged 0.312–2.5 mg/mL. The methanol extract
of D. Jerdonianum showed moderate antibacterial
activity against all the tested bacteria, with inhibition
zones of 9.54, 9.64, 10.24, 11.64, and 8.58 mm,
respectively. The MIC values for D. Jerdonianum
ranged from 0.625–2.5 mg/mL. No antibacterial
activity was observed in the methanolic extracts
of D. barbatulum and Erythrina mysorensis.
The methanol extracts of B. neilgherrense only
showed inhibition against E.coli (8.54 mm) and S.
flexinari (8.12 mm), while chloroform and aqueous
extracts of the other plants showed negative results.
The results align with the previous reports by