Enhanced Anti-Bacterial Activity Of Biogenic Silver Nanoparticles Synthesized From Terminalia mantaly Extracts

Figures & data

Table 1 List Of Bacterial Strains Used For Anti-Bacterial Activity

Bacterial Strains	Acronym	Reference No.	Supplier
Streptococcus pneumoniae	S. pneumoniae	ATCC 49619	ATCC
Klebsiella pneumoniae	K. pneumoniae	ATCC 13883	ATCC
Haemophilus influenzae	H. influenzae	ATCC 49247	ATCC
Shigella flexneri	S. flexneri	NR-518	BEI resources
Salmonella enterica	S. enterica^a	NR-13555	BEI resources
Salmonella enterica	S. enterica^b	NR-4294	BEI resources
Salmonella enterica enterica	S. enterica enterica	NR-4311	BEI resources
Staphylococcus aureus	S. aureus	NR-45003	BEI resources

Notes: S. enterica^a (Salmonella enterica subsp. enterica A36 (Serovar Typhimurium) vs S. enterica^b (Salmonella enterica subsp. enterica 2004 Pennsylvania Tomato Outbreak, Serovar Anatum, Isolate 4).

Abbreviations: ATCC, American Type Culture Collection; BEI resources, biodefense and emerging infections research resources repository.

Figure 1 Schematic representation of the AgNP synthesis from TM plant extracts.

Notes: Aqueous and methanolic TM extracts were used to reduce AgNO₃ into AgNPs, color change indicates NP formation.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; TML, TM leaf extract; TMSB, TM stem bark extract; TMR, TM root extract; TML-AgNPs, AgNPs from TM leaf extract; TMSB-AgNPs, AgNPs from TM stem bark extract; TMR-AgNPs, AgNPs from TM root extract; _a, aqueous; _m, methanolic.

Table 2 SPR And DLS Analysis Of TM-AgNPs Synthesized At 25°C And 70°C

TM-AgNPs	NP Parameters
	25°C					70°C
	[Extract] (mg/mL)	λmax (nm)	PD (nm)	Pdi	ZP (mV)	[Extract] (mg/mL)		λmax (nm)	PD (nm)	Pdi	ZP (mV)
aTML-AgNPs	0.78	438	11	0.50	−24	1.56		460	18	0.40	−26
mTML-AgNPs	1.56	474	44	0.60	−22	1.56		458	22	0.80	−29
aTMSB-AgNPs	0.78	464	28	0.22	−34	1.56		460	56	0.46	−37
mTMSB-AgNPs	1.56	480	60	0.62	−12	1.56		458	58	0.53	−12
aTMR-AgNPs	3.12	472	39	0.40	−23	3.125		478	30	0.88	−24
mTMR-AgNPs	0.78	450	83	0.48	−22	0.78		438	67	0.51	−28

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; _a, aqueous extracts; _m, methanolic extracts; TML-AgNPs, AgNPs from TM leaf extracts; TMSB-AgNPs, AgNPs from of TM stem bark extracts; TMR-AgNPs, AgNPs from TM root extracts;SPR, surface plasmon resonance; UV, ultraviolet; PD, particle diameter; ZP, Zeta potential; Pdi, Polydispersity index; DLS, dynamic light scattering.

Figure 2 UV–vis spectral profiles of TM-AgNPs synthesised at 25°C and 70°C.

Notes: AgNPs were synthesized by reducing AgNO₃ with aqueous (A) and methanolic (B) TM extracts. The optical properties of the NPs were measured by UV-vis spectophotometry.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; TML-AgNPs, AgNPs from TM leaf extract; TMSB-AgNPs, AgNPs from TM stem bark extract; TMR-AgNPs, AgNPs from TM root extract.

Table 3 Comparison FTIR Spectra Of TM Extracts And Their Respective AgNPs

aTML				aTMSB
Peak Position In Extracts (cm^−1)	Peak Position In AgNPs At 25 °C (cm^−1)	Shift In Position (cm^−1)	Type Of Chemical Bond	Peak Position In Extracts (cm^−1)		Peak Position In AgNPs at 70°C (cm^−1)	Shift In Position (cm^−1)	Type Of Chemical Bond
1338.32	1384.39	+46.07	C–H methyl rock alkanes	1048.80		----------	----------	C-O Stretch
1640.29	1638.56	−1.73	–C=C–stretch alkenes	1131.16		----------	----------
1876.99	1884.90	+7.91	C=O Anhydrides	1384.03		1384.96	+0.93	C-H methyl rock alkanes
2946.61	2830.64	−115.97	H–C=O:C–H stretch aldehydes	1639.21		1638.68	−0.53	–C=C– stretch alkenes
3530.47	3417.07	−112.48	OH Alcohol, phenols	2016.03		2034.58	+18.55	–C≡C– stretch alkynes
3545.10	3417.99	−127.11		2930.41		2789.28	−141.13	H–C=O:C–H stretch aldehydes
3647.29	3712.96	+65.67		3530.72		3530.35	+0.37	OH Alcohol, phenols
				3544.94		3544.99	+0.05
				3717.06		3731.61	+14.55

Note: Shift values were calculated by subtracting the peak position in TM-AgNPs from the peak position in TM-extracts.

Abbreviations: FTIR, Fourier-transform infrared; AgNPs, silver nanoparticles; TM, Terminalia mantaly; _aTML, aqueous TM leaf extracts; _aTMSB, aqueous TM stem bark.

Figure 3 FTIR spectra of selected TM-AgNPs compared to their respective TM extracts.

Notes: FTIR spectra of (A) _aTML extract, (B) _aTML-AgNPs synthesized at 25°C, (C) _aTMSB extract and (D) _aTMSB-AgNPs synthesized at 70°C.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; _aTML, aqueous TM leaf extract; _aTMSB, aqueous TM stem bark; FTIR, Fourier-transform infrared.

Figure 4 HRTEM micrographs and SAED patterns of selected TM-AgNPs.

Notes: The size, shapeand SAED patterns of _aTML-25°C and _mTML-70°CAgNPs were analyzed by HRTEM. Scale bar from 20 to 50 nm.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; aTML, aqueous TM leaf extract; aTMSB, aqueous TM stem bark; HRTEM, high resolution transmission election microscope; SAED, selected area electron diffraction.

Figure 5 (A,B) EDX spectra of AgNPs synthesized from _aTML and _mTML AgNPs.

Notes: Silver nanocrystallites display an optical absorption band peak at approximately 3 keV (red arrow), which is typical of the absorption of metallic silver nanocrystallites due to their SPR.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; aTML, aqueous TM leaf extract; aTMSB, aqueous TM stem bark; EDX, Energy Dispersive X-ray.

Table 4 Comparison Of The Bacterial Susceptibility To TM Extracts And AgNPs

Treatment	Bacterial Strains
	S. enterica^a	S. pneumoniae	S. aureus	K. pneumoniae	S. enterica enterica	S. enterica^b	S. flexneri	H. influenzae
aTMSB	–	–	–	–	–	–	–	✓
aTMSB-AgNPs-25°C	–	–	–	✓	–	–	✓	✓
aTMSB-AgNPs-70°C	–	✓	✓	✓	✓	✓	✓	-
mTMSB	–	✓	–	–	✓	–	✓	✓
mTMSB-AgNPs-25°C	–	–	–	–	–	–	–	✓
aTML	–	✓	–	–	–	–	–	-
aTML-AgNPs-25°C	✓	✓	✓	✓	✓	✓	✓	✓
aTML-AgNP-70°C	✓	✓	✓	✓	✓	✓	✓	✓
aTMR	–	✓	–	–	–	–	–	✓
mTMR	–	✓	–	–	–	–	–	✓
mTMR-AgNPs-25°C	–	✓	–	–	–	–	–	–

Notes: Eight bacterial strains were treated with TM extracts and AgNPs for 24 hrs. Inhibitory effects of TM samples were visually assessed to identify the strains that were susceptibility to TM extracts and AgNPs. The _aTM-AgNPs were the most active and inhibited growth of all bacterial strains, compared to the crude extracts and other AgNPs. S. enterica^a (Salmonella enterica subsp. enterica A36 (Serovar Typhimurium) vs S. enterica^b (Salmonella enterica subsp. enterica 2004 Pennsylvania Tomato Outbreak, Serovar Anatum, Isolate 4).

Abbreviations: AgNPs, Silver nanoparticles; TM, Terminalia mantaly; _a, represents aqueous extracts; _m, represents methanolic extracts; TML, TM leaf extracts; TMR, TM root extracts; TMSB, TM stem bark extracts; S. pneumoniae, Streptococcus pneumoniae; S. enterica enterica, Salmonella enterica enterica; H. influenzae, Haemphilus influenzae, S. flexineri, Shigella flexineri; K. pneumoniae, Kbesiella pneumoniae; S. aureus, Staphylococcus aureus, S. enterica- Salmonella enterica; -, no bacterial activity; ✓, bacterial activity.

Figure 6 Screening of the antibacterial effects of TM extracts and AgNPs against a panel of bacterial strains.

Notes: Eight bacterial strains were treated with TM extracts and AgNPs for 24 hrs. Inhibitory effects of TM samples are expressed based on the number of strains that showed growth inhibition. ***P < 0.001.

Abbreviations: AgNPs, silver nanoparticles; TM, Terminalia mantaly; _a, represents aqueous extracts; _m, represents methanolic extracts; TML, TM leaf extracts; TMR, TM root extracts; TMSB, TM stem bark extracts.

Table 5 MIC For Crude TM Extracts And Its Derivate AgNPs On Selected Bacterial Strains

Treatment	MIC (µg/mL)
	S. enterica^a	S. pneumoniae	S. aureus	K. pneumoniae	S. enterica enterica	S. enterica^b	S. flexneri	H. influenzae
aTMSB	>500	>500	>500	>500	>500	>500	>500	125
aTMSB-AgNPs-25 °C	>12.50	>12.50	>12.50	6.24	>12.50	6.24	>12.50	3.12
aTMSB-AgNPs-70 °C	>12.50	6.24	12.50	6.24	12.50	12.50	>12.50	>12.50
mTMSB	>500	>500	>500	>500	>500	>500	125	>500
mTMSB-AgNPs-25 °C	>12.50	>12.50	>12.50	>12.50	>12.50	>12.50	>12.50	6.24
aTML	>500	>500	>500	>500	>500	>500	>500	>500
aTML-AgNPs-25 °C	6.24	6.24	6.24	6.24	>12.50	6.24	>12.50	3.12
aTML-AgNP-70 °C	>12.50	6.24	12.50	6.24	>12.50	6.24	>12.50	>12.50
aTMR	>500	>500	>500	>500	>500	>500	>500	500
mTMR	>500	>500	>500	>500	125	>500	>500	>500
mTMR-AgNPs25 °C	>12.50	>12.50	>12.50	>12.50	>12.50	>12.50	>12.50	>12.50
Ampicillin	32	4	8	16	32	16	32	16

Notes: S. enterica^a (Salmonella enterica subsp. enterica A36 (Serovar Typhimurium) vs S. enterica^b (Salmonella enterica subsp. enterica 2004 Pennsylvania Tomato Outbreak, Serovar Anatum, Isolate 4).

Abbreviations: AgNPs, Silver nanoparticles; TM, Terminalia mantaly; _a, represents aqueous extracts; _m, represents methanolic extracts; TML, TM leaf extracts; TMR, TM root extracts; TMSB, TM stem bark extracts; S.pneumoniae, Streptococcus pneumoniae; S. enterica enterica, Salmonella enterica enterica; H.influenzae, Haemphilus influenzae, S. flexineri, Shigella flexineri; K. pneumoniae, Kbesiella pneumoniae; S. aureus, Staphylococcus aureus; S. enterica, Salmonella enterica.

Figure 7 Growth inhibitory activities of _aTML-AgNPs-25°C against selected bacterial strains.

Notes: Growth curves of (A) S. pneumoniae, (B) S. enterica enterica, (C) H. influenza, (D) S. flexneri, (E) K. pneumoniae, and (F) S. enterica^a (Salmonella enterica subsp. enterica A36 Serovar Typhimurium) after 24 hr treatment. **P < 0.01, ***P < 0.001.

Abbreviations: AgNPs, Silver nanoparticles; TM, Terminalia mantaly; _aTML-AgNPs, AgNPs synthesized from aqueous leaf extracts from TM; S. pneumoniae, Streptococcus pneumoniae; S. enterica enterica, Salmonella enterica enterica; H. influenzae, Haemphilus influenzae, S. flexineri, Shigella fLexineri; K. Pneumoniae, Klebsiella pneumoniae; S. aureus, Staphylococcus aureus; S. enterica, Salmonela enterica.

Figure 8 Growth inhibitory activities of _aTMSB-AgNPs-70°C against selected bacterial strains.

Notes: Growth curves of (A) S. pneumoniae, (B) S. enterica enterica, (C) H. influenzae, (D) S. flexneri, (E) K. pneumoniae, and (F) S. aureus after 24 hr treatment. ***P < 0.001.

Abbreviations: AgNPs, Silver nanoparticles; TM, Terminalia mantaly; _aTMSB-AgNPs, AgNPs synthesized from aqueous Stem Bark extracts from TM; S. pneumoniae, Streptococcus pneumoniae; S. enterica enterica, Salmonella enterica enterica; H. influenzae, Haemphilus influenzae, S. flexineri, Shigella flexineri; K. pneumoniae, Klebsiella pneumoniae; S. aureus, Staphylococcus aureus; S. enterica, Salmonella enterica.