Multidrug-resistant bacterial infections pose a significant problem for both hospitals and clinics. According to a recent report published by the U.N. agency (WHO), carbapenem-resistant A. baumannii is considered to be the world’s most dangerous superbugs, and the bacteria is listed as the priority-1 (critical) pathogens (Scientific American, 2017). High doses of antibiotics are frequently used to treat resistant microorganisms, although this causes significant side effects. The existence of different secondary metabolites determines the efficacy of therapeutic plants in preventing microbial development. Nano-size silver materials represent a novel antimicrobial approach. the plant metabolites such as polyphenols, terpenoids, alkaloids, etc mediate the bioreduction of silver ions to nanoconjugates.
For the first time, this study is aimed at evaluating the methanolic extract of the root of V. wallichii as a reducing agent for synthesizing Nanoconjugates. This extract could function as a capping and stabilizing agent because it contains significant secondary metabolites and biological constituents including proteins and ethylene groups. Their various activities were investigated using qualitative and quantitative methods including HPLC. Further, nanoconjugates were characterized using DLS, AFM, and SEM–EDX. Its antimicrobial activity was studied.
Phytochemical Screening
The methanolic root extract of V. wallichii has shown the presence of saponins, tannins, flavonoids, alkaloids, phenols, terpenoids, inulin, glycosides, carbohydrates, and reducing sugars as mentioned in Table 1.
As per another study done by Babu et al., (39), Valeriana Jatamansi (V. jatamansi) showed the presence of alkaloids and reducing sugars in the hydro-alcoholic extract of the plant while steroids and saponin were found in the hexane extract of the plant.
Table 1
Phytochemical screening of methanolic V. wallichii root extract
Extract Name
Test Name
|
Methanolic Valeriana wallichii extract
|
Saponins
|
++
|
Tannins
|
+
|
Steroids
|
-
|
Terpenoids
|
+
|
Flavonoids
|
++
|
Napthoquinone
|
-
|
Inulin
|
+
|
Carbohydrates
|
+
|
Alkaloids
|
+
|
Phenol
|
+
|
Starch
|
+
|
Amino acid
|
-
|
Glycosides
|
+
|
Reducing Sugar
|
++
|
Phlobtannins
|
+
|
*Note: “++”: present in higher amount; “+”: present; “-“: absent |
Quantification Of Phytochemical Constituents
None of the scientific papers has reported the presence of antioxidant and polyphenolic content in the methanolic root extract of V. wallichii though some researchers have studied some of these activities for V. Jatamansi which is a completely different species to be considered.
So, for the first time, Total Phenolic, Flavonoid, Terpenoid & Tannin Contents, FRAP, Reducing Power Assay & DPPH antioxidant activity is being reported for this extract.
Total Phenolic Content (Tpc)
TPC in the extract was measured using the Folin-Ciocalteu (F-C) technique with gallic acid used as the standard. TPC was estimated for the extract and represented as mg of gallic acid equivalents (GAE) per g of sample in dry weight (mg/g) using the regression equation of the calibration curve (Y = 0.0087x; R2 = 0.9917) (Graph 1, Supplementary). The mean TPC value observed in the extract was 28.536 ± 0.253 mg GAE/g (Table 2). The result was found to be significant at p < .05 i.e., p = 0.01.
As per the study done by Rawat et al., (40), the TPC of V. jatamansi was maximum in methanolic extract (14.37 mg GAE/g dw). Also, According to Thusoo et al., (41), the highest amount of phenolic compound was observed in the methanolic extract of V. jatamansi root (187.13 ± 6.8 mg GAEs/g) followed by aqueous extract (77.66 ± 2.1 mg GAE/g).
Numerous studies have shown that the presence of phenolic compounds in plants is related to their antioxidant activities. This association is most likely caused by the plants' redox characteristics, which enable them to function as reducing agents, hydrogen donors, and singlet oxygen quenchers.
Table 2
Total phenolic content (TPC) in the methanolic root extract of V. wallichii
S. No
|
Samples
|
100 (µg/ml)
|
200(µg/ml)
|
300 (µg/ml)
|
400 (µg/ml)
|
500 (µg/mL)
|
Mean TPC value (GAE/g)
|
1.
|
Root
|
16.582 ± 0.236
|
28.153 ± 0.236
|
37.846 ± 0.329
|
27.233 ± 0.236
|
32.856 ± 0.236
|
28.536 ± 0.253
|
Total Flavonoid Content (Tfc)
The TFC of the extract was estimated using the regression equation of the calibration curve (Y = 0.0047x; R2 = 0.9955) (Graph 2, supplemental) and represented as mg quercetin equivalents (QE) per g of sample in dry weight (mg/g). The mean TFC value obtained for the extract was 21.568 ± 0.4199 mg QE/g (Table 3). The result was found to be significant at p < 0.0001.
In a finding by Thusoo et al., (41), the Aqueous extract of V. jatamansi had a relatively high amount of flavonoids (452.30 ± 12.4 mg QE/g) followed by methanolic extract (257.69 ± 9.8 mg QEs/g). In another study maximum flavonoid content was found in the Acetone extract of V. jatamansi (6.24 ± 0.01) followed by ethanol (6.15 ± 0.01) and methanol (5.58 ± 0.02) (40).
Table 3
Total flavonoid contents (TFC) in the methanolic root extract of V. wallichii.
S. No
|
Samples
|
500 (µg/ml)
|
600 (µg/ml)
|
700 (µg/ml)
|
800 (µg/ml)
|
900 (µg/ml)
|
1000 (µg/ml)
|
Mean TFC value (QE/g)
|
1.
|
Root
|
18.063 ± 0.347
|
19.411 ± 0.437
|
20.546 ± 0.437
|
22.815 ± 0.437
|
23.666 ± 0.437
|
25.015 ± 0.437
|
21.586 ± 0.4199
|
Determination Of Total Terpenoid Content (Ttc)
The total terpenoid content of the extract was estimated using a standard calibration curve (y = 0.0038x, r2 = 0.9913) (Graph 3, supplementary) of Linalool and expressed in mg linalool equivalents/g of extract. The mean TTC value (LE/g) was found to be 26.315 ± 0.324 (LE/g) as presented in Table 4. The result was found to be significant at p < 0.0001. It is being reported for the first time in this extract.
Table 4
Determination of total terpenoid content in the methanolic root extract of V. wallichii.
S. No
|
Samples
|
0.5 (mg/ml)
|
1 (mg/ml)
|
2 (mg/ml)
|
3 (mg/ml)
|
4 (mg/ml)
|
Mean TTC value (LE/g)
|
1.
|
Root
|
11.070 ± 1.749
|
17.736 ± 3.727
|
26.684 ± 2.263
|
37.912 ± 1.102
|
38.175 ± 0.754
|
26.315 ± 0.324
|
Estimation Of Total Tannin Content (Ttc)
The total tannin content of the extract was estimated using a standard calibration curve (y = 0.0218x, r2 = 0.992) (Graph 4, supplementary) of gallic acid and expressed in mg gallic acid equivalents/g of extract. The results show that the extract had a mean TTC of 4.690 ± 0.032 mg gallic acid equivalents/g (GAE/g) of extract as depicted in Table 5. The result was found to be significant at p < 0.0001. We have evaluated this activity for the first time in our extract.
Table 5
Total Tannin Content in the methanolic root extract of V. wallichii.
S. No
|
Samples
|
100 (µg/ml)
|
200 (µg/ml)
|
300 (µg/ml)
|
400 (µg/ml)
|
500 (µg/ml)
|
Mean Reducing Power value (GAE/g)
|
1.
|
Root
|
1.472 ± 0.044
|
3.091 ± 0.067
|
4.673 ± 0.061
|
6.124 ± 0.033
|
8.091 ± 0.016
|
4.690 ± 0.032
|
Ferric Reducing Antioxidant Power (Frap)
The ability of the extract to convert ferric (III) iron to ferrous (II) iron was used to estimate the antioxidant activity as determined by the FRAP technique. It was seen in the extract using a standard calibration curve (y = 0.0023x, r2 = 0.998) (Graph 5, supplementary) of FeSO4 (100–1000 µg). The results were presented as mg Fe (II) equivalents per g of the extract's dry weight. The mean antioxidant activity observed with this method was 70.343 ± 0.117 mg FeSO4/g. (Table 6). The result was found to be significant at p < 0.0001.
A study reported maximum antioxidant activity by FRAP assay in acetone extract of V. jatamansi (6.70 ± 0.20), followed by Methanol (6.47 ± 0.29) (40).
Table 6
Ferric Reducing Antioxidant Power (FRAP) in the methanolic root extract of V. wallichii.
S. No
|
Samples
|
50 (µg/ml)
|
100 (µg/ml)
|
200 (µg/ml)
|
300 (µg/ml)
|
400 (µg/ml)
|
Mean FRAP value (FeSO4/g)
|
1.
|
Root
|
15.989 ± 0.128
|
37.927 ± 0.128
|
68.552 ± 0.128
|
102.072 ± 0.128
|
127.177 ± 0.077
|
70.343 ± 0.117
|
Reducing Power Assay
The Reducing Power Assay of the extract was estimated using a standard calibration curve (y = 0.0145x, r2 = 0.989) (Graph 6, supplementary) of Ascorbic acid and expressed in mg ascorbic acid equivalents/g of extract. The reducing power of the extract has shown the mean reducing power, 32.117 ± 0.141 AAE/g (Table 7). The properties of the reducing power can be an indicator of the antioxidant potential of the compound evaluated. The result was found to be significant at p < 0.0001. This is being estimated by us for the first time in this extract.
Table 7
Reducing Power Assay in the methanolic root extract of V. wallichii.
S. No
|
Samples
|
100 (µg/ml)
|
200 (µg/ml)
|
300 (µg/ml)
|
400 (µg/ml)
|
500 (µg/ml)
|
Mean Reducing Power value (AAE/g)
|
1.
|
Root
|
23.354 ± 0.141
|
30.043 ± 0.141
|
30.273 ± 0.141
|
34.871 ± 0.141
|
42.043 ± 0.141
|
32.117 ± 0.141
|
Dpph Free Radical Scavenging Assay
The antioxidant potential of the extract was analyzed by DPPH free radical scavenging assay. The standard was ascorbic acid and its IC50 value was found as 0.817 𝜇g/ml (Graph 7, supplementary). The IC50 value of the extract evaluated in this study was found to be 32.253 ± 0.319 𝜇g/mL as shown in Table 8. (Graph 8, supplementary) The result was found to be significant at p < 0.0001.
Thusoo et al., (41) reported the methanolic extract of V. jatamansi as the most potent antioxidant (IC50 78 ± 2.9 𝜇g/mL). Rawat S. et al., (2016) revealed almost similar antioxidant capacity in different solvent extracts of V. jatamansi studied by him in the range of 9.82 ± 0.08 to 10.88 ± 0.07 mM AAE/100 g dw.
Table 8
DPPH radical scavenging activity of methanolic root extract of V. wallichii.
Concentration (µg/ml)
|
% Inhibition
|
IC50
|
10
|
17.032 ± 0.395
|
32.253 ± 0.319
|
20
|
34.785 ± 0.308
|
|
30
|
49.096 ± 0.250
|
|
40
|
61.961 ± 0.549
|
|
50
|
71.744 ± 0.636
|
|
High-pressure Liquid Chromatography (Hplc)
Polyphenolic flavonoids like quercetin and rutin and Tannins like tannic acid were determined in the methanolic root extract of V. wallichii root by the HPLC method. The retention time of the standard quercetin, rutin, and tannic acid was found to be 3.147 min, 3.817 min, and 3.093 min respectively as shown in Fig. 2 and the retention time of the extract was also found similar to the standards i.e., 3.015 min, 3.110 min., and 3.085 min respectively as compared in Fig. 2 which indicates the presence of both polyphenolic flavonoids and tannins in the extracts. The identified compounds and their corresponding concentrations are listed in Table 9.
In a previous study, Rawat et al., (40) reported Six phenolic compounds: gallic acid, catechin, hydroxyl benzoic acid, caffeic acid, chlorogenic acid, and p-coumaric through HPLC in V. jatamansi but in our study, we have focused on the concentration of quercitin, rutin, and tannic acid.
Table 9
Concentrations of Polyphenolic flavonoids & Tannins against their respective standards in the methanolic root extract of V. wallichii
Standards
|
Peak ID
|
Ret Time
|
Peak Height
|
Peak Area
|
Concentration
|
Quercetin
|
3.147
|
329519.219
|
3898018
|
100
|
Rutin
|
3.817
|
176880.094
|
6469150
|
100
|
Tannic Acid
|
3.093
|
566871.625
|
3475821
|
100
|
Valeriana wallichii methanolic root extract
|
Peak ID
|
Ret Time
|
Peak Height
|
Peak Area
|
Concentration
|
Quercetin
|
3.015
|
39301.43
|
2091349.375
|
99.1981
|
Rutin
|
3.11
|
15727.441
|
585420.625
|
98.9848
|
Tannic Acid
|
3.085
|
4846.437
|
27028.447
|
100
|
Molecular Docking Studies
One of the current and efficient ways of assessing therapeutic compounds for potential targets of A. baumannii's drug resistance is computer-aided screening. The KEGG database searches indicated a few potential drug targets that could be putative therapeutic targets for the development of antibiotics based on the functional involvement of A. baumannii in the amino acid biosynthesis pathway.
In the docking analysis, the binding affinities of the identified compounds (Fig. 3) were studied against the A. baumannii receptors as mentioned in Table 10. The docking analysis revealed that tannic acid depicted the maximum binding affinity in the range of − 11.6 kcal/mol to − 13.3 kcal/mol with all the receptors studied. After Tannic acid, rutin and linalool also have shown comparatively higher affinities with the receptors. Details of the binding energy and the active amino acid residues of all the ligands which are interacting against the crucial receptors are mentioned in Table 10. The docked complexes were further visualized for their molecular interactions using the Discovery Studio 2021 client. 2D and 3D structures of the best-docked molecules against all the receptors are shown in Fig. 3.
Table 10
The binding potential (ΔG (Kcal/mol)) of secondary metabolites present in the methanolic root extract of V. wallichii against 4 receptors retrieved from PDB and 2 modeled receptors of A. baumannii using AutoDock Vina
Receptor
|
PDB ID
|
Ligand
|
Docking Score
(kcal/mol)
|
Amino acid residue
|
Hydrolysing Class C Extended- Spectrum –Lactamase (AmpC):
The major gene code for β-lactamase known as amp C from A. baumannii was a prioritized drug target by database search.
|
4QD4
|
Gallic Acid
|
-5.3
|
Met295, Val294, Glu291, Ser290, Asn289, Asn345, Arg342, Cit402
|
Quercetin
|
-6.1
|
Cit402
|
Ascorbic Acid
|
-4.9
|
Tyr224
|
Linalool
|
-7.9
|
Phe123, Pro124, Pro218, Gln122, Tyr224, Cit402, Ser317
|
Rutin
|
-8.3
|
Ser317, Arg342, Asp125, Pro218, Val127, Tyr224, Gln122
|
Tannic Acid
|
-11.6
|
Glu291, Val294, Ser290, Asn289, Asn345, Arg342, Met295, Cit402
|
NADH-dependent enoyl-ACP
reductase (FabI):
FabI has been reported as an important drug target in both methicillin-resistant Staphylococcus aureus and MDR Mycobacterium tuberculosis.
Enolyl-[acyl-carrerir protein] reductase I (FabI), which is involved in the metabolism of biotin and fatty acids, was identified as a possible therapeutic target using the KEGG pathway.
|
4ZJU
|
Gallic Acid
|
-5.9
|
Lys9, Ser237, Gln141, Cys143, Arg140, Asp88
|
Quercetin
|
-6.0
|
Lys9, Cys236, Arg186, Pro238, Asp88, Arg140
|
Ascorbic Acid
|
-4.5
|
Pro238, Cys143, Arg186
|
Linalool
|
-7.5
|
Asp88, Cys236, Arg186
|
Rutin
|
-7.9
|
Gly142, Gln141, Asp88, Arg186, Pro238, Arg140, Lys9
|
Tannic Acid
|
-13.3
|
Ala192, Tyr149, Ile195, Ser93, Ser21, Ala68, Asp66, Val67, Cys65, Gly95, Ile94, Ile22, Lys166
|
Response regulator BfmR (RstA):
Recent studies have revealed that RstA in A. baumannii has been validated as a potential antimicrobial target.
The transcriptional regulatory protein-RstA, one of the main outer membrane proteins involved in this bacteria's two-component system, was thought to be the therapeutic target.
|
5E3J
|
Gallic Acid
|
-4.5
|
Glu33, Ile10, Glu50
|
Quercetin
|
-5.4
|
Ile10, Lys9, Glu50
|
Ascorbic Acid
|
-4.7
|
Lys107, Leu19, Pro108, Val109, Pro111
|
Linalool
|
-6.3
|
Arg123, Asp53, Leu122, Pro8, Arg119
|
Rutin
|
-7.2
|
Ala10, Leu97, Glu98, Arg74, Arg71, His78, Asp102
|
Tannic Acid
|
-12.3
|
Glu50, Arg45, Arg46, Arg42, Lys9, Ile10, Val36, Gln24
|
UDP-N-acetylglucosamine acetyltransferase (LpxA):
LpxA is conserved in almost all
prokaryotes and a crucial enzyme in the biosynthetic pathway of the lipopolysaccharides and reported as a potential therapeutic drug target for many pathogenic
Gram-negative bacteria.
LpxA (one of the major proteins involved in the amino acid sugar and nucleotide metabolism) was prioritised as probable drug target.
|
4E6U
|
Gallic Acid
|
-4.8
|
Glu84, Ile85, Ile55, Thr36
|
Quercetin
|
-5.0
|
Ile35, Gln57, Asn87, Gly86, Glu84
|
Ascorbic Acid
|
-4.2
|
Thr36, Arg54
|
Linalool
|
-6.8
|
Glu84, Lys109, Asp130, Asn87, Ser112
|
Rutin
|
-7.8
|
Thr36, Ile55, Gly38, Glu84, Lys109, Asn87, Ser112
|
Tannic Acid
|
-13.3
|
Lys178, Gly259, Arg262, Ilea260, Ser228
|
Aspartokinase:
Lysine, methionine, and threonine are three essential amino acids, and aspartokinase (EC 2.7.2.4), an enzyme, catalyzes the first stage of their production. The absence of this gene in animals makes this enzyme
a key target for antimicrobials
|
Homology Modeling
|
Gallic Acid
|
-5.6
|
Lys7, Val210, His212, Gly209, Gly10, Ser41
|
Quercetin
|
-5.9
|
Ser41, Gly151, Ser153, Asp154
|
Ascorbic Acid
|
-4.5
|
Gly151, Gly152, Ser153
|
Linalool
|
-8.1
|
Arg150, His212, Gly151, Gly209, Thr173, Tyr172, Asp174, Gly10, Lys7, Asp154, Thr47
|
Rutin
|
-8.8
|
Gly10, Ser41, Asp154, Asp174, Lys7, Lys45
|
Tannic Acid
|
-12.2
|
Tyr222, Asp115, Ile116, Lys165, Ala162
|
Shikimate dehydrogenase:
In Pseudomonas aeruginosa, Pseudomonas putida and Helicobacter pylori, shikimate dehydrogenase (EC 1.1.1.25) have been identified as an interesting target for antimicrobial drugs.
|
Homology Modeling
|
Gallic Acid
|
-6.1
|
Ser17, Asp102, Gln243
|
Quercetin
|
-6.4
|
Gln243
|
Ascorbic Acid
|
-4.6
|
Met214, Tyr216, Leu240, Thr62, Val7
|
Linalool
|
-8.1
|
Ser192, Val63, Ala130, Lys66
|
Rutin
|
-8.5
|
Ser15, Tyr216, Ser194, Thr62, Asp102, Met239, Lys66, Met214, Val63, Ala130, Pro11
|
Tannic Acid
|
-12.7
|
Arg155, Gly129, Asn150, Arg151, Ala127, Lev200, Ser196
|
*Note- values with bold & underlined are depicting the highest binding scores of the ligands on the receptors. |
According to the study reported by S. Skariyachan et al., (42) the binding potential of Limonin, Ajmalicine & Strictamin was studied against the same receptors as evaluated by us in the current study as shown in Table 10.
They showed that Limonin (7,16-Dioxo-7,16- dideoxylimondiol), a natural limonoid commonly present in Citrus sps. demonstrated promising interaction towards modeled aspartokinase with a binding energy of − 8.7 kcal/mol. Likewise, it illustrated a significant binding towards the probable targets such as AmpC and FabI, with binding energies of − 8.4 and − 8.1 kcal/mol respectively. Ajmalicine ((19α)-16, 17- didehydro-19-methyloxayohimban-16-carboxylic acid methyl ester) exhibited promising interaction with Fab1 and aspartokinase with a binding energy of -7.5 and − 7.1 kcal/mol respectively. Strictamin (Akuammilan-17-oic acid methyl ester) displayed a good binding with modeled shikimate dehydrogenase and AmpC with binding energies of − 7.8 and − 7.4 kcal/mol respectively.
However, in our study, the binding energy is quite enhanced compared to the previous study reported, which qualifies natural compounds close to these found in our extract by HPLC or quantitative phytoconstituent methods as potent antimicrobial agents.
Table 10- The binding potential (ΔG (Kcal/mol)) of secondary metabolites present in the methanolic root extract of V. wallichii against 4 receptors retrieved from PDB and 2 modeled receptors of A. baumannii using AutoDock Vina
Cytotoxicity and anti-cancerous activity of methanolic root extract of V. wallichii
The extract was evaluated for its toxicity and anticancerous property using an MTT assay. The extract has shown anticancerous efficacy against Cervical cancer cells (Hep 2C) and has not shown any toxicity on Human Fibroblast cells (L929). In Hep2C cells, the IC50 value was found to be 298.70 µg/ml, however, in L929 cells, the IC50 value was found to be 368.53 µg/ml, as shown in Fig. 4 The extract has shown a selectivity Index of 1.234, which can be effectively used for antiproliferative activity against tumor cells.
None of the researchers have reported the activity of V. wallichii methanolic root extract on the cancerous cell line, however many studies have shown the anticancerous activity of the pure compound isolated from V. wallichii and V. Jatamansi. So, this is being reported for the first time in our study as per our knowledge.
Previous studies have reported that valepotriates isolated from V. wallichii D. C. and V. jatamansi exhibited potent cytotoxic and antitumor activities (43, 44). According To Lin et al (2014),(45) 4 valepotriates isolated from V. jatamansi showed strong activity against metastatic prostate cancer (PC-3M) cells with IC50 values of 1.66, 6.82, 5.75, and 6.22 µM. Wang et al. in 2017(44) evaluated the cytotoxicity of iridoids of V. jatamansi against two human cancer cell lines- lung adenocarcinoma (A 549) and gastric carcinoma cells (SGC 7901) and reported no cytotoxicity effect or very weak cytotoxicity with only two of the compounds. Zhu et al (2019)(46) showed that F3 (a novel fraction isolated from V. jatamansi Jones) exhibited significant growth inhibitory activities on human breast cancer cell lines, MDA-MB-231 cells (IC50 2.91 µg/ml) and MCF-7 cells (IC50 4.23 µg/ml). Han et al.,(47) demonstrated for the first time that valeric acid suppresses liver cancer development. Three liver cancer cell lines with the lowest IC50 were reported (HepG2: 0.948 mM; Hep3B: 1.439 mM: and SNU-449: 1.612 mM). However, IC50 values for liver cancer cell lines SNU-449, Hep3B, and HepG2 were 1.92, 2.15, and 3.27 times higher, respectively, than values for the liver normal cell line THLE-3 (3.097).
The cells were evaluated under an Olympus inverted microscope using a 10X objective lens (magnification 100X). Scale bar: 100 µm.
Characterization of Green Synthesis of Silver Nanoconjugates of V. wallichii methanolic root extract (Vw-AgNCs)
Uv-visible Spectrophotometry
Using a UV-Vis spectrophotometer, the reduction of silver ions into nanoconjugate by V. wallichii methanolic root extract was detected by recording the absorption spectra between the wavelengths of 200–800 nm. AgNC development in the solution was verified by UV-Vis spectral analysis, which revealed that the highest absorption was at 430 nm. (Fig. 5).
Foujdar et al.,(23) showed in the spectral analysis that Punica granatum Peel’s PolyphenolsBased Silver nanoconjugates have maximum absorbance near about 420 nm. Another study also reported similar results that the UV-Vis absorption spectrum of five aqueous leaf extract-mediated silver nano-conjugates has shown a single peak at a wavelength ranging between 402.0 nm to 429.0 nm (48).
Dynamic Light Scattering (Dls)
The DLS technique was used to determine the particle size of the synthesized nanoconjugates. According to the DLS graph of Vw-AgNCs (Fig. 6), the particle size range was between 100 to 120 nm.
The size of nanoparticles obtained in this study is supported by the results of Ghosh et al., (48), who indicated the size of five leaves of aqueous extract-mediated silver nano-conjugates varies from 18.17 nm to 105.7 nm.
Atomic Force Microscopy (AFM) Pattern
AFM was used to determine the parameters of the surface morphology of nanoconjugates. The well-dispersed, nearly spherical shape of the nanoconjugates was confirmed by AFM scans. Figures 7A and 7B show the 2D and 3D pictures of synthesised Vw-AgNCs. The size obtained from Vw-AgNCs was around 12nm (Fig. 7C). The maximum profile of peak height was 69.6573 nm and the average surface roughness was 2.0033 nm. The observed difference in AFM and DLS may be due to the clustering of nanoconjugates in solution form.
A study done by Mishra et al., (49) reported the maximum profile of peak height at 73.43 nm and the average surface roughness at 55.10 nm in green synthesized silver nanoparticles using ethanol leaf extract of Nyctanthes arbor-tristis.
Scanning electron microscopy (SEM)
The results of the SEM investigation are shown in Fig. 8 below at magnifications of 30000X and 50000X. The size of the Vw-AgNCs ranged between 70 and 700 nm (Fig. 8). The data obtained demonstrate that the bioactive constituents from the V. wallichii methanolic root extract on the AgNCs, which is indicated by the formation of clusters at several points. The samples used to examine the morphology of Vw-AgNCs may have been agglomerated, therefore showing larger particle sizes than that obtained from DLS & AFM.
Proposed antibacterial mechanism of Vw-AgNCs against A. baumannii
The SEM micrographs indicate deformity in A. baumannii cells treated with Vw-AgNCs resulting in ruptured cells, where in some cases, the outer cell membrane was intact but in others, membrane distortion was observed and the cells have lost their shape and structural integrity. Additionally, interactions with the exposed peptidoglycan caused by these spherical Vw-AgNCs causes the N-acetyl glucosamine (NAG) and N-acetyl muramine (NAM) to lose their integrity (NAG). This insertion and penetration of synthesized Vw-AgNCs lead to oozing out of the cytoplasmic constituents, resulting in cell death and complete killing (Fig. 8). The grey dots in the background display green synthesized AgNPs.
Several studies have reported that most biogenic AgNPs have a spherical geometry, with a size range of 5 to 500 nm with antimicrobial activities reported against them (50, 51).
Energy Dispersive X-ray Spectrum (Edx)
Silver elements were detected in the sample, according to the EDX analysis of the Vw-AgNCs. The binding energies of silver, whose weight and atomic percentage are 7.62 and 1.31%, respectively, are represented by the peaks about 3 keV. The result shows the peaks as depicted in Fig. 9 for the presence of the silver element. The high atomic and weight percentages of organic elements, such as C and O, revealed the possible presence of organic compounds, such as polysaccharides, phenols, and proteins; The occurrence of other peaks (Ca, Si, Mg, Na, Al, Cl, etc) was presumed to relate with the glass underneath, which held the sample and Au was because of Gold Sputtering. Also, different elements detected by EDX analysis, such as Si, Al, and Cl, were reported to act as capping agents of the biogenic AgNPs according to a previous study (52, 53).
Another study also reported a similar result after the synthesis of biogenic AgNPs using Ferula asafoetida that demonstrated the presence of Ag peaks at 3 keV with weight and atomic percentages of 11.6% and 7.7%, respectively. Also, high atomic and weight percentages of organic elements, C, N, and O, were present in their study as well (50). A.K. Mishra et al, (49) reported similar results in the study done by them and revealed the existence of metallic silver in biosynthesized nanoparticles with a sharp peak at 3 keV. They also showed the occurrence of other peaks (Ca, Si, Mg, and Na) as were in our study. Also, He et al, (54) revealed the presence of silver element in synthesized Ag NPs using A. katsumadai seed aqueous extract (AKSE) with a peak at 3 keV.
Antimicrobial Activity
Using the well diffusion technique, the potential antibacterial efficacy of Vw-AgNCs against MDR- A. baumannii was investigated. The findings were compared to those obtained for AgNPs synthesised using the chemical method. The Vw-AgNCs were showing higher zones of inhibition than chemical AgNPs which proved it to exhibit good antibacterial activity against it. The highest zone of inhibition was observed to be 21 mm ± 0.450 at 20µl of Vw-AgNCs. Among standard antibiotics tested, Ampicillin and Vancomycin were found to be resistant and the zone of inhibition of Tetracyclin and Gentamicin was 22mm & 14mm respectively, which was similar to the green synthesized AgNPs. Vw-AgNCs have proved to be the potential drug candidate against the clinical strain of MDR- A. baumannii as they have shown remarkably promising results as compared to standard antibiotics as using Vw-AgNCs may be one way for combating microbial resistance. Unlike standard antimicrobial agents, low doses of silver nanoconjugates can act as more potent dosages in the treatment of diseases as shown in our results too (Table 11, Fig. 10) (55). Plant extract by itself, on the other hand, lacked any antibacterial effect.
However, it is to be assumed that the plant's root extract utilized has antibacterial properties as the capped methanolic root extract of V. wallichii is getting enhanced by silver as shown in the results.
Our study was in accordance with the findings of Abootalebi et al., (50) which revealed that the antimicrobial potential of biogenic NPs (Fer@AgNPs) was significantly higher than chemical AgNPs. Peiris et al., (56) reported green synthesized silver nanomaterials exhibited antibacterial efficiency & an inhibition zone diameter of 12 mm against A. baumannii. The diameter of the zone of inhibition of biosynthesized nanoparticles using ethanol leaf extract of Nyctanthes arbor-tristis against A. baumannii was 13-15mm, which increased with the increase in the concentration of nanoparticles as per the study done by Mishra et al. (49).
The conjugation of NPs with the antibacterial components of the extract may be the cause of the improved antimicrobial impact of the green synthesized AgNCs compared to chemical AgNP. The antimicrobial mechanism of green synthesized AgNCs is still unknown but several studies propose that the interaction of silver nanoconjugates with microorganisms gives rise to the release of silver ions which are potential destroyers of microbial cells due to their ability to deactivate enzymes in microbial cells and membrane permeability disruption leading to lysis and apoptosis (57). Furthermore, Sulfur and phosphorus, two essential elements of DNA, interact with silver ions in ways that affect DNA replication, cell growth, and even microbial cell death. They can prevent protein production by denaturing ribosomes in the cytoplasm (58). Moreover, it has the potential to harm human cells and has significant side effects. As a result, in the current investigation, we additionally assessed the extract's impact on a normal human cell line as well as a malignant human cell line as a measure of its cytotoxic effect and potential as an anticancer drug. Several studies have proven that phytoconstituents like polyphenolic compounds are safe for healthy cells while showing cytotoxicity against cancerous cells.
Table 11
Zone of inhibition (mm) as measured by the Agar Well-Diffusion method
S.No.
|
Components
|
Zone of inhibition (mm)
|
MDR- A. baumannii
|
1
|
TGMC-50 (100 µl)
|
-
|
2
|
AgNO3-5mM (100 µl)
|
-
|
3
|
D.W. (100 µl)
|
-
|
4
|
Blank-media (100 µl)
|
-
|
5
|
Chem AgNP (50 µl)
|
-
|
6
|
S5 (100 µl)
|
-
|
7
|
5A TG SNP- 100 µl
|
17.4 mm ± 0.600
|
8
|
5A TG SNP- 75 µl
|
17.5mm ± 0.200
|
9
|
5A TG SNP- 50 µl
|
16.5mm ± 0.300
|
10
|
5A TG SNP- 25 µl
|
17mm ± 0.400
|
11
|
5A TG SNP- 20 µl
|
21 mm ± 0.450
|
12
|
5A TG SNP- 15 µl
|
20 mm ± 0.450
|
13
|
5A TG SNP- 10 µl
|
19.5 mm ± 0.100
|
14
|
5A TG SNP- 5 µl
|
18 mm ± 0.500
|
15
|
5A TG SNP- 4 µl
|
14mm ± 0.350
|
16
|
5A TG SNP- 3 µl
|
16mm ± 0.250
|
17
|
5A TG SNP- 2 µl
|
15.5mm ± 0.150
|
18
|
5A TG SNP- 1 µl
|
11.5mm ± 0.200
|
19
|
Ampicillin
|
-
|
20
|
Vancomycin
|
9mm ± 0.30
|
21
|
Gentamycin
|
14mm ± 0.25
|
22
|
Tetracycline
|
22mm ± 0.15
|
Note: TGMC-50: V. wallichii methanolic root extract 50mg/ml; 5A TG SNP: 5mM synthesized silver nanoconjugate of V. wallichii methanolic root extract; D.W.: Distilled water; S5: Chem AgNP 5mM.
Figure 10a) Negative Control- Mac Conkey Control- Mac Conkey b) Bacterial Control- MDR- A. baumannii. c) Positive Control: Plate having wells with I) TGMC-50: 50 mg/ml V. wallichii methanolic root extract.; II) AgNo3-5: 5mM AgNo3.; III) D.W.: Distilled Water.; IV) Blank: Empty well, showing no Zone of Inhibition. d) Positive Control: Plate having wells with I) Chem AgNPs: Chemically synthesized AgNPs.; II) S5- 100% Methanol; e) Vw-AgNCs: Volume- 25, 50, 75 & 100 µl; f) Vw-AgNCs: Volume- 5, 10, 15 & 20 µl; g) Vw-AgNCs: Volume- 1, 2, 3 & 4 µl; h) Standard Antibiotics: Ampicillin, Vancomycin, Tetracycline, and Gentamicin.