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24 INNOSC Theranostics and Pharmacological Sciences, 2022, Vol. 5, No. 2 Madhavamurthy et al.
A C

B

Figure 1. The map shows the diversity of orchids in the Western Ghats. (A and B) Western Ghats of India
and their states. (C) Diversity of orchids in Karnataka districts.
Note: Green stars – indicate the areas inhabited by orchids.

2.5.2. Disk diffusion method (0.002–5 mg/mL). Subsequently, a 10 μL bacterial
inoculum suspension was added to the mixture
The antibacterial activity of the extract was tested and incubated at 37±2°C for 24 h. Absorbance
using the disk diffusion method [26]. Briefly, 100 was measured without agitation at 620 nm using
μL (1.5 × 10 CFU/mL) of the bacterial test sample an ELISA plate reader (LabTech 4000). The
8
(24-h-old) was spread uniformly on the surface of MIC was confirmed by adding 10 μL of TTC
the Petri plates containing solidified nutrient agar (2, 3, 5-triphenyl tetrazolium chloride) per well
media using a sterile glass spreader. The sterile disks at a concentration of 2 mg/mL and incubating the
(6 mm) were loaded with 50 μL (3.5 mg) and 75 μL mixture for 30 min. The MIC was determined as
(6.5 mg) of each plant extracts, respectively, from the lowest concentration at which no color changes
the stock (50 mg/mL) and placed equidistantly. The were observed. The experiments were performed in
streptomycin (25 μg/mL) and respective solvents triplicates.
(25 μL) were used as positive and negative controls,
respectively, and plates were incubated at 37±2°C 2.6. Antioxidant activity
for 24 h. After incubation, the zone of inhibition The free radical scavenging activity (RSA) of
was measured, and the experiment was repeated in each extract was evaluated using the DPPH assay,
triplicates. following the method described by Wahab et al. [28].
2.5.3. Minimum inhibitory concentration (MIC) Each extract at varying concentrations (20, 40, 60,
80, and 100 μg/mL) was prepared from the stock
The MIC was determined using the micro-broth of 1mg/mL and tested for antioxidant activity. Four
dilution method [27]. The extracts were prepared at milliliters (4 mg 100/mL in methanol) of DPPH
a concentration of 50 mg/mL in respective solvents. solution was added to each test tube containing
In a 96-well plate, each well was dispensed with extract, and methanol only was used as blank. The
100 μL of nutrient broth, followed by the addition reaction mixture was incubated for 20 min at room
of 100 μL stock solutions that were serially diluted temperature in the dark. The absorbance was measured

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