The phytochemical screening of different plant extracts revealed a varied combination of these phytochemicals (Table 1) in each plant part. The qualitative analysis of phytochemicals in leaves has shown the presence of alkaloids, phenols and tannins in the extracts derived from methanol, ethanol and water. Flavonoids were screened in aqueous extract only. Glycosides and saponins were present in the methanol and aqueous extracts. Similarly, amino acids, flavonoids and quinones were detected in the aqueous extract of leaves only. The aqueous extract of seed was tested positive for all the phytochemicals except quinones. On the other hand, both the methanol and ethanol seed extracts revealed the presence of alkaloids, glycosides, phenols, tannins and terpenoids only; amino acids, flavonoids and saponins were not detected in these extracts. The bark aqueous extract revealed the presence of the seven phytochemicals viz
. phenols, saponins, tannins, terpenoids, glycosides and amino acids as compared to two (alkaloids and glycosides) in ethanol and methanol extracts.
With increasing awareness among the people towards herbal medicines and side effects of allopathic medicines. a number of experimental studies to explore and select the natural sources of compounds have been carried out in past (Palhares et al., 2015).
In the present study, qualitative analysis of the leaf, seed and bark extracts of B. tomentosa
prepared using three different solvents (methanol, ethanol and water) demonstrated the presence of a good number of active constituents in them overall; however, these extracts from different tissues, when compared, revealed remarkable variation in their phytochemical constitution (Table 1). Maximum of the metabolites could be extracted using water from all the plant parts under study followed by methanol and ethanol. Further, bark extracts were seen to constitute least number of phytochemicals using latter two solvents; however, with water it revealed similar composition like leaf and seed extracts. So, it could be stated that the polarity of a solvent as well as the nature of the metabolites present in a tissue determine the number and amount of the phytochemicals extracted from the plant part. Moreover, the maximum number of active constituents extracted using aqueous conditions, in all the plant parts, indicates towards their highly polar nature as per our study. Further, the presence of flavonoids and amino acids only in the water extracts strengthen the above statement. Our results corroborated the finding of Wen et al., (2007)
and Thenmozhi et al., (2012)
who have also shown the highest amount of the phenolic constituents in the high polar solvents. Further, the selection of the better plant parts and optimization of the extraction and purification process could result in the optimum yield of these active constituent. Here, on the basis of our results, we may suggest the use of B. tomentosa
leaves followed by seeds and bark and water as the solvent system to extract maximum number of phytochemicals for medicinal use.
Broth dilution assay and antimicrobial screening
Table 1: Phytochemical screening of different leaf, seed and bark extracts of B. tomentosa.
MIC values calculated using broth dilution assay for all the extracts against the tested bacterial strains could be seen in the Table 3. Whereas the lowest MIC value (17.6 mg/mL) for leaf aqueous extract was observed for Enterobacillus
, the highest for K. pneumoniae
(87.4 mg/mL). The seed aqueous extract of revealed the lowest (18.4 mg/mL) and the highest MIC value (62.5 mg/mL) for Enterobacillus
and K. pneumoniae
respectively. On the other hand, ethanol seed extract was found to exhibit MIC values against H. influenzae
(18.9 mg/mL) and Enterobacillus
(112 mg/mL) only. Methanol seed extract showed the lowest MIC value against S. thermophilus
and the highest against Enterobacillus.
The bark methanol, aqueous and ethanol extracts were observed to be effective at the MIC values in the range of 65.1-170 mg/mL, 19.4-125 mg/mL and 17.6-160 mg/mL respectively for the tested bacteria. Further, a comparative analysis of the antimicrobial potential of the seed, bark and leaf extracts in different solvents could be seen in the Table 2 and Fig 1a-1c.
Table 2: In-vitro anti-bacterial activity of leaf, seed and bark extracts of B. tomentosa.
Table 3: The MIC of B. tomentosa extracts prepared from different plant parts against selected bacteria.
Fig 1a: Antibacterial activity of ethanol extract of A. Bark, B. Leaf and C. Seed of B. tomentosa.
1- Positive control; 2- Negative control; 3- at 50 mg conc.; 4- 75 mg conc. 5- 100 mg conc.
Fig 1b: Antibacterial activity of Methanol extract of A. Bark, B. Leaf and C. Seed of B. tomentosa.
1- Positive control; 2- Negative control; 3- at 50 mg conc.; 4- 75 mg conc.; 5- 100 mg conc.
Antimicrobial potential of leaf extracts
Fig 1c: Antibacterial activity of Aqueous extract of A. Bark, B. Leaf and C. Seed of B. tomentosa.
1- Positive control; 2- Negative control; 3- at 50 mg conc.; 4-75 mg conc.; 5- 100 mg conc.
Overall, all the leaf extracts showed significant zone of inhibition against all the tested bacterial strains at 75 and 100 mg concentrations except K. pneumoniae
which was inhibited only by methanol and ethanol extracts at the same concentrations; however, at 50 mg concentration, only the Enterobacillus
were found to be inhibited by all the leaf extracts. Among the leaf extracts, water extracts presented the maximum zone of inhibition against Enterobacillus
(8 mm) and H. influenzae
(7.6 mm) with MIC values of 17.6 mg/mL and 21.5 mg/mL respectively at 100 mg concentration. The minimum was recorded in Micrococcus
(2 mm; MIC value = 29.3 mg/mL) and H. influenzae
(2.4 mm; MIC value = 125 mg /mL) with methanol leaf extract.
Anti-microbial potential of seed extracts
In case of seed, water and methanol extracts revealed their potential against Enterobacillus
, K. pneumoniae
, S. thermophilus
and H. influenzae
at 75 and 100 mg concentrations. However, the ethanol extract of seed worked against only Enterobacillus
at 100 mg concentration and against H. influenzae
at both 75 and 100 mg concentrations.
At 100 mg concentration, the maximum zone of inhibition (10 mm) was seen for Enterobacillus
using aqueous extract (MIC of 18.4 mg/mL) and minimum zone of inhibition (3.07 mm) for Enterobacillus
with ethanol extract (MIC of 112 mg/mL).
Anti-microbial potential of bark extracts
Extracts prepared in water showed the highest inhibition zone as compared to ethanol and methanol extracts for S
and K. pneumoniae
at 75 mg concentration and for Enterobacillus
at 100 mg concentration. Aqueous and ethanol extracts at 75 and 100 mg concentration revealed the highest zone of inhibition as compared to methanol extracts for Micrococcus
. For H. influenza,
water extract showed the maximum zone of inhibition (7.6 mm with MIC = 20.2 mg/mL) and the minimum inhibition zone was seen for K. pneumoniae
(MIC value of 102 mg/mL) in water extract at 100 mg concentration.
Being the reservoir of pharmacologically important secondary metabolites, the plants have been exploited for the discovery of such natural compounds having potential in the health care industry for novel drug development properties (Djahida and Houcine, 2021)
. In the present study, all the extracts from different plant parts viz.
leaves, bark and seed revealed anti-bacterial potential against one or other kind of bacteria at different concentrations; however, comparatively more at higher concentration (100 mg). It marked towards the presence of these secondary metabolites in all the plant parts in variable amount. Different action mechanisms viz.
cell lysis, interaction with genetic material and resulting in ineffective transcription and coagulation of cell contents after penetrating the cell wall have been suggested for their antimicrobial activities (da Silva et al., 2016; Hayek et al., 2013).
On comparison, overall effect of the aqueous extracts from all the plant parts was seen to be more pronounced on the three tested bacterial strains viz
(Gram +ve), Micrococcus
(Gram +ve) and H. influenzae
(Gram -ve) followed by methanol and ethanol extracts. In contrast, the two other strains K. pneumonia
(Gram -ve) and S. thermophilus
(Gram +ve) were inhibited more by the methanol extracts followed by aqueous and ethanol extracts. A number of other studies on the effects of different plant extracts for their antimicrobial potential demonstrated the both contradictory as well as similar results (Mishra et al., 2013; El-Moula et al., 2019; Chalghoumi et al., 2020).
These differences in the antibacterial activities of the plant parts can be due to differences in the phytochemical composition of the part used and the solvents employed to extract these. The ability of showing very well antibacterial activities by crude aqueous leaf extract at different concentrations correspond to its good MIC values which made it more potent for bacterial inhibition in our results. However, in a report by da Silva et al., (2016),
a weak positive relation was shown between MIC value and the bactericidal action of the plant extract.
Further, our study also revealed the Gram +ve bacteria to be more susceptible than Gram-ve at lower doses of the plant extracts (50 mg and 75 mg). But at higher doses, both strains were affected equally. Other studies have reported the former to be more susceptible than latter at higher concentrations of the extract used (Briers and Lavigne, 2015
; da Silva et al., 2016).
This intrinsic resistance in Gram-ve bacteria towards the lower doses of these plant extracts may be attributed to the fact that the multi-layered cell wall of the Gram-ve prevents the passage of active components inside it; moreover, the overexpression of efflux pumps (EPs) have also been reported to work efficiently in repelling the entry of these active compounds inside the cytoplasm (Venter et al., 2015).