# Green Synthesis of Silver Nanoparticles UsingCatharanthus roseus Flower Extracts and the Determination of Their Antioxidant, Antimicrobial, and Photocatalytic Activity

Authors: Mathivathani Kandiah; Kavishadhi N. Chandrasekaran
Journal: Journal of Nanotechnology
 (2021)

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## Abstract

The present study describes the antioxidant, antimicrobial, and photocatalytic activity of silver nanoparticles (AGNPs) synthesized using six varieties ofCatharanthus roseus flower extracts for the first time. Initially, the synthesized AgNPs were visually confirmed by color change. Further, the formation, size, and shape of the synthesized AgNPs were characterized by UV-Vis spectroscopy and scanning electron microscopy (SEM). The SEM image of purple flower AgNPs and the calculated bandgap energies of the synthesized AgNPs showed that the synthesized AgNPs were in the range of 0–30 nm. Qualitative phytochemical analysis revealed the presence of the phytocompounds that were responsible for the capping, formation, bioreduction, and stabilization of AgNPs. The antioxidant ability of the AgNPs and their respective flower extracts were analyzed using TFC, TPC, TAC, DPPH, FRAP, and IC50 assays. The results of the antioxidant assays indicated that the AgNPs showed higher antioxidant activity compared to their respective flower extracts. The synthesized AgNPs showed significant antimicrobial activity against Gram-negative Escherichia coli compared to Gram-positive Staphylococcus aureus assayed using the agar well diffusion method. Furthermore, the photocatalytic activity of the synthesized purple flower AgNPs at two different concentrations 5000 ppm and 333 ppm was analyzed by the removal of methyl orange dye from an aqueous solution under sunlight irradiation in the presence of NaBH4 catalyst. Results indicated that 333 ppm purple flower AgNPs exhibited an efficient photocatalytic activity in the degradation of methyl orange compared to 5000 ppm purple flower AgNPs in 20 minutes. Thus, the results obtained indicated that Catharanthus roseus is an ecofriendly source for the green synthesis of AgNPs which can be used as a novel antioxidant, antimicrobial, and photocatalytic agent; thereby, it can be used in a variety of applications to improve the quality of human life.


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## Body

## 1. Introduction

Nanotechnology is a developing field of research involving the design, fabrication, characterization, and application of particles, materials, or systems in the nanoscale due to the unique size-dependent physical and chemical properties [1]. One of the key areas most attention is drawn to is the synthesizing of different metal nanoparticles [2]. Nanoparticles are microscopic particles with at least one dimension in the nanoscale which is 1–100 nm [3].In this study AgNPs are synthesized as silver unlike other metals such as copper, gold, and nickel that have an increasing interest due to properties such as localized surface plasma resonance, chemical stability, catalytic activity, high conductivity, and cost-effectiveness compared to synthesizing nanoparticles from metals such as gold. Besides AgNPs have proven to be less toxic to mammalian cells than other nanoparticles [4].Due to their surface plasmon resonance properties, AgNPs are used in sensors like fluorescence sensors, colorimetric sensors, and surface-enhanced Raman spectrometry sensors to detect pollutants in the environment. Similarly, due to their antimicrobial and properties, AgNPs can easily penetrate cells through the cell membrane and serve as an antimicrobial agent; therefore, it is used in the treatment of microbes and is also used in coating surgical devices [5]. Furthermore due to the smaller size of AgNPs which contain high number of surface active sites and faster spatial charge, increase in the photocatalytic activity against pollutants such as azo dyes is proved. [6].Although many nanoparticles are synthesized over the past few years using conventional physical and chemical methods, only a few of them are currently used as these methods use harmful chemicals and produce byproducts that are toxic to the environment. Furthermore, these nanoparticles synthesized from these methods cannot be used especially in clinical fields due to the lack of knowledge about health-related issues and toxicity [7]. Therefore, a bottom-up approach “green synthesis” method has been developed as a substitute to defeat the limitations and challenges of the conventional methods (Figure 1).Figure 1
The two approaches and methods of nanoparticle synthesis [8].Green synthesis is the utilization of biological extracts such as plants or microorganisms to synthesize nanoparticles (Figure2). Plant-mediated green synthesis approaches are more favorable and are mainly focused on the current research and fabrication advances since using microorganisms are biohazardous, require special isolation techniques, and are more prone to contaminations [9].Figure 2
Basic steps of the green synthesis method.In the green synthesis of metal nanoparticles, the plant extract is mixed with a metal salt solution. Using phytochemicals present in the plant extracts in different combinations and concentrations including other factors such as pH of the reaction mixture, temperature, concentration, pressure, and reaction time, the electrochemical potential of a metal ion a biochemical reduction takes place.During this reaction, the metal ions are converted from their mono/divalent state to a zero-valent state followed by nucleation and growth to form metal nanoparticles (Figure3). Recent studies reported that various plants contain an extensive range of phytochemicals such as phenols and enzymes that contain important functional groups including hydroxyl and carboxyl which potentially can act as reducing, capping, and stabling agents [10].Figure 3
Mechanism of AgNPs green synthesis.In this study, the flower extracts ofCatharanthus roseus were used to synthesize nanoparticles as previous studies have shown Catharanthus roseus extracts possess a good antioxidant property due to the presence of various polyphenols identified [11] (Figure 4).Figure 4
Antioxidants produced byCatharanthus roseus [12].Catharanthus roseus of the family Apocynaceae is commonly known as periwinkle which is grown as an ornamental flowering plant [12]. Flowers are pentamerous, actinomorphic with variations of colors (white, purple, peach, pink, scarlet, and red) [13]. Different parts of the plant are found to be used worldwide for different purposes. In Sri Lanka, it is widely used in Ayurvedic medicine to treat a variety of diseases. Researchers investigated for the medical properties have discovered the presence of two phytochemicals, Vinblastine and vincristine, which can be used as chemotherapeutics drugs [14].Free radicles are molecules with an unpaired electron in the outer shell that are highly unstable, so they tend to react with organic substrates such as DNA and lipids in the body [15]. In humans, free radicles in excess, generate oxidative stress damaging cell structures which are a risk factor in many diseases such as cancer and neurodegenerative diseases [16]. There are several mechanisms in the body to counter react oxidative stress using antioxidants which are naturally occurring in the body or taken via diet. Studies show that nanoparticles synthesized from various natural sources rich in antioxidants can act as free radical scavengers to replace harmful synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [17].Furthermore, the disposal of organic dyes such as methyl orange is a crucial problem in textile and paper industries since they are nondegradable under normal conditions due to the presence of strong azo bonds (Figure5) [18]. Studies report that the removal of organic dyes using AgNPs is a better alternative option than the common dye removal techniques like redox treatments since it does not produce any harmful byproducts [19].Figure 5
Structure of methyl orange. Methyl orange is an orange organic dye that contains a strong azo bond that is not degradable under normal conditions commonly used in titrations as a pH indicator and textile industries for dyeing and printing.The aim of this research is to synthesize ecofriendly AgNPs using six varieties ofCatharanthus roseus flower extracts and to access their antioxidant, antimicrobial, and photocatalytic activity. The antioxidant activity will be determined using TFC, TPC, TAC, DPPH, FRAP, and IC50 assays. The antimicrobial activity will be determined against a Gram-negative and Gram-positive bacterium, Escherichia coli and staphylococcus aureus, respectively, and the photocatalytic activity will be analyzed by the removal of methyl orange dye. By achieving all the objectives, the AgNPs can be a useful way for recycling flower wastes, treating free radical-mediated diseases, and making a better environment free from hazardous azo dyes.


## 2. Materials and Methods

### 2.1. Materials

#### 2.1.1. Reagents

Acetic acid (CH3COOH) (CAS-7758-99-8), aluminium chloride (AlCl3) (CAS-7446-70-0), ammonium hydroxide (NH4OH), ammonium molybdate ([NH4]6Mo7O24.4H2O), chloroform (CH3Cl) (CAS-67-66-3), copper sulphate (CuSO4) (CAS-7758-98-7), 2, 2-diphenyl-1- picrylhydrazyl (DPPH) (CAS-1898-66-4), ethanol (C2H5OH) (CAS- 64-17-5), ferric chloride (FeCl3) (CAS-7705-08-0) , Folin–Ciocalteu phenol reagent, methanol (CH3OH) (CAS-67-56-1), hydrochloric acid (HCl) (CAS-7647-01-01-0), McFarland solution, , methylene orange (C14H14N3NaO3S) (547-58-0), Molisch’s reagent, Mueller-Hinton agar powder, nutrient agar powder, saline, silver nitrate (AgNO3) (CAS-7761-88-8), sodium acetate (CH3OONa) (CAS 127-09-3), sodium borohydride (NaBH4), sodium carbonate (Na2CO3) (CAS-497-19-8), sodium hydroxide (NaOH) (CAS 1310-73-2), sodium nitrate (NaNO3) (CAS 7631-99-4), sodium sulphate (Na2SO4) (CAS- 7757-82-6), sulphuric acid (H2SO4) (CAS-7664-93-9), and 2, 4, 6-Tris(2-pyridyl)-s-triazine (TPTZ) (CAS-7757-82-6).


#### 2.1.2. Instrumentation

Hot-air oven (meditry DHA-9053A), incubator, analytical balance, spectrophotometer (JENWAY 6305), roller mixer, micropipettes, refrigerator, fume hood (Biobased FH1000), and autoclave.


#### 2.1.3. Glassware and Miscellaneous

Conical flask, measuring cylinder, filter funnel, spatulas, test tubes, cuvettes, volumetric flask, Petri dishes, beakers, cotton swabs, watch glass, filter paper, falcon tubes, and Whatman filter papers No.1.


### 2.2. Sample Collection

Five varieties ofCatharanthus roseus flowers (Figure 6) were collected from Diyatha Uyana plant nursery, Battaramulla, Sri Lanka.Figure 6
Five varieties ofCatharanthus roseus flowers and their code names. (LPF) Light pink (a), (RF) red (b), (WPF) white pink dot (c), (WYF) white yellow dot (d), and (PF) purple (e).
(a)(b)(c)(d)(e)


### 2.3. Preparation of Flower Extracts Using Distilled Water

The samples were air-dried and grounded into smaller pieces. To 2 g of each grounded sample, 40 mL of distilled water was added. The samples were incubated at 60°C for 30 min and were filtered using Whatman filter paper No.1 into 50 mL falcon tubes. The samples were stored at 4°C until further use.


### 2.4. Green Synthesis of AgNPs

1 mL of each water extract was mixed with 9 mL of AgNO3 solution and was kept at room temperature (RT) for 3 days. The color change of the samples was observed, and the absorbance was measured from 300 to 620 nm using distilled water as a blank.


### 2.5. SEM and Particle Size Analysis

10 mL of PF AgNPs was centrifuged at 5000 rpm for 10 min and was transferred to wash glass. The sample added to the watch glass was dried completely at 180°C. The dried smear was dissolved by adding 200µL of distilled water and transferred into an Eppendorf tube. The sample was wrapped using aluminum foil and was sent for the SEM analysis. The morphology of PF AgNPs was examined using a Scanning Electron Microscope and the average particle size was analyzed by the Malvern Zetasizer Nano ZS particle size analyzer from the Sri Lanka Institute of Nanotechnology (SLINTEC) using the Hitachi SU6600 SEM.


### 2.6. Phytochemical Analysis

The phytochemical analysis was carried out according to the methodologies shown in Table1.Table 1
Methodology of the phytochemical analysis [20].
PhytochemicalMethodologyExpected positive color resultCarbohydrates2 ml of extract was treated with 1 mL of Molisch’s reagent and a few drops of concentrated H2SO4 were addedFormation of purple or reddish color ringTannins1 mL of extract was added to 2 mL of 5% FeCl3Formation of dark blue or greenish-black colorSaponins2 mL of extract and distilled water was added and shaken in a graduated cylinder for 15 minutesFormation of 1 cm layer of foamTerpenoids0.5 mL of the extract was treated with 2 mL of chloroform and concentrated H2SO4Formation of red-brown color at the interfaceAnthraquinonesFew drops of 10% NH4OH were added to 1 mL of extractFormation of a pink color precipitateSteroidsTo 1 mL of extract, an equal volume of chloroform was added and a few drops of concentrated H2SO4Formation of a brown color ringProteinsTo 1 mL of extract, a few drops of CuSO4 and 1 mL of 10% NaOH were addedFormation of purple or mauve color


### 2.7. Determination of Antioxidant Activity

AgNPs and the water extracts were diluted 1 : 15 to analyze the antioxidant activity. Each analysis was carried out in triplicate.


#### 2.7.1. Determination of Total Flavonoid Content (TFC)

TFC was determined using an AlCl3 colorimetric method adopted from Perera and Kandiah [21]. 1.5 mL of the sample was mixed with 1.5 mL of 2 % AlCl3·6H2O solution and was incubated at RT for 10 minutes. The absorbance of the samples was measured at 415 nm using distilled water as a blank and the results were expressed in µg Quercetin equivalents per 100 g (µg QE/100 g).


#### 2.7.2. Determination of Total Phenolic Content (TPC)

0.1 mL of the sample was mixed with 1.4 mL of 7.5 % Na2CO3 and was incubated for 6 minutes. To the same solution, 1.5 mL of Folin–Ciocalteu reagent was added and incubated at RT for 60 minutes in a dark condition. The absorbance was measured at 765 nm using distilled water as a blank and the results were expressed in g gallic acid equivalents per 100 g (g GAE/100 g).


#### 2.7.3. Determination of Total Antioxidant Capacity (TAC)

TAC was evaluated using a phosphomolybdenum assay adapted from Biwas et al. [22] with modifications. The phosphomolybdenum reagent was prepared by mixing equivalent volumes of 28 mM sodium sulphate and 4 mM ammonium molybdate and 0.6 M sulphuric acid. 3 mL of the sample was mixed with 1 mL of the phosphomolybdenum reagent, and the samples were incubated at 90°C for 90 min. The absorbance was measured at 695 nm using distilled water as a blank and the results were expressed as g ascorbic acid equivalents per 100 g (g AAE/100 g).


#### 2.7.4. Determination of Ferric Reducing Antioxidant Power (FRAP)

The FRAP reagent was prepared by mixing 50 mL of 0.3 M pH 3.6 acetate buffer, 5 mL of 10 mM TPTZ solution prepared in 40 mM HCl, and 5 mL of 20 mM FeCl3·6H2O Solution. 100 µL sample was mixed with 2.9 mL of FRAP reagent and the absorbance was measured at 593 nm using distilled water as a blank for every 1 minute.


#### 2.7.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

1 mL of the sample was mixed with 2 mL of 0.004% DPPH and was incubated for 30 minutes at RT. The absorbance was measured at 517 nm using methanol as a blank. The %DPPH scavenging activity was calculated using the following equation:(1)Inhibition%=Acontrol−AsampleAcontrol×100.


#### 2.7.6. Determination of Median Inhibition Concentration (IC50)

To 1 mL of series of five concentrations, 100%, 80%, 60%, 40%, and 20%, which were prepared using the samples and distilled water, 2 mL of 0.004 % DPPH solution was added and was incubated for 30 mins at RT. The absorbance of all the samples was measured at 517 nm using methanol as a blank. The % DPPH scavenging activity was calculated using equation (1).


### 2.8. Determination of the Antimicrobial Activity

The antimicrobial activity was determined using two types of bacterial strainsStaphylococcus aureus and Escherichia coli using the agar well diffusion technique on Mueller-Hinton agar (MHA). The bacterial culture was evenly spread on the MHA plates using a cotton swab. Three wells were created on the MHA for the negative control (−) and the two replicates of the sample (S1 and S2) as shown in Figure 7. 1 mL of saline was added to the negative control (−) and 1 mL of sample was added to S1 and S2. Gentamycin discs were used as the positive control (+). The plates were incubated at 37°C for 24 hours and the diameter of the zone of inhibition was measured using a ruler.Figure 7
Illustration of the labeling of Petri plates.


### 2.9. Statistical and Particle Size Analysis

10 mL of PF AgNPs was centrifuged at 5000 rpm for 10 min and were transferred to wash glass. The sample added to the watch glass was dried completely at 180°C. The dried smear was dissolved by adding 200µL of distilled water and transferred into an Eppendorf tube. The sample was wrapped using aluminum foil and sent for the SEM analysis. The morphology of PF AgNPs was examined using a Scanning Electron Microscope and the average particle size was analyzed by the Malvern Zetasizer Nano ZS particle size analyzer from the Sri Lanka Institute of Nanotechnology (SLINTEC) using the Hitachi SU6600 SEM.


### 2.10. Determination of the Photocatalytic Activity

1 mL of 5000 ppm PF AgNPs and 1 mL of 0.2 M NaBH4 were added to 100 mL of 2 mM MO. The absorbance of the reaction mixture was measured from 300 to 560 nm using distilled water as a blank every 5 minutes for 20 minutes. The procedure was repeated for 333 ppm PF AgNPs.


## 2.1. Materials

### 2.1.1. Reagents

Acetic acid (CH3COOH) (CAS-7758-99-8), aluminium chloride (AlCl3) (CAS-7446-70-0), ammonium hydroxide (NH4OH), ammonium molybdate ([NH4]6Mo7O24.4H2O), chloroform (CH3Cl) (CAS-67-66-3), copper sulphate (CuSO4) (CAS-7758-98-7), 2, 2-diphenyl-1- picrylhydrazyl (DPPH) (CAS-1898-66-4), ethanol (C2H5OH) (CAS- 64-17-5), ferric chloride (FeCl3) (CAS-7705-08-0) , Folin–Ciocalteu phenol reagent, methanol (CH3OH) (CAS-67-56-1), hydrochloric acid (HCl) (CAS-7647-01-01-0), McFarland solution, , methylene orange (C14H14N3NaO3S) (547-58-0), Molisch’s reagent, Mueller-Hinton agar powder, nutrient agar powder, saline, silver nitrate (AgNO3) (CAS-7761-88-8), sodium acetate (CH3OONa) (CAS 127-09-3), sodium borohydride (NaBH4), sodium carbonate (Na2CO3) (CAS-497-19-8), sodium hydroxide (NaOH) (CAS 1310-73-2), sodium nitrate (NaNO3) (CAS 7631-99-4), sodium sulphate (Na2SO4) (CAS- 7757-82-6), sulphuric acid (H2SO4) (CAS-7664-93-9), and 2, 4, 6-Tris(2-pyridyl)-s-triazine (TPTZ) (CAS-7757-82-6).


### 2.1.2. Instrumentation

Hot-air oven (meditry DHA-9053A), incubator, analytical balance, spectrophotometer (JENWAY 6305), roller mixer, micropipettes, refrigerator, fume hood (Biobased FH1000), and autoclave.


### 2.1.3. Glassware and Miscellaneous

Conical flask, measuring cylinder, filter funnel, spatulas, test tubes, cuvettes, volumetric flask, Petri dishes, beakers, cotton swabs, watch glass, filter paper, falcon tubes, and Whatman filter papers No.1.


## 2.1.1. Reagents

Acetic acid (CH3COOH) (CAS-7758-99-8), aluminium chloride (AlCl3) (CAS-7446-70-0), ammonium hydroxide (NH4OH), ammonium molybdate ([NH4]6Mo7O24.4H2O), chloroform (CH3Cl) (CAS-67-66-3), copper sulphate (CuSO4) (CAS-7758-98-7), 2, 2-diphenyl-1- picrylhydrazyl (DPPH) (CAS-1898-66-4), ethanol (C2H5OH) (CAS- 64-17-5), ferric chloride (FeCl3) (CAS-7705-08-0) , Folin–Ciocalteu phenol reagent, methanol (CH3OH) (CAS-67-56-1), hydrochloric acid (HCl) (CAS-7647-01-01-0), McFarland solution, , methylene orange (C14H14N3NaO3S) (547-58-0), Molisch’s reagent, Mueller-Hinton agar powder, nutrient agar powder, saline, silver nitrate (AgNO3) (CAS-7761-88-8), sodium acetate (CH3OONa) (CAS 127-09-3), sodium borohydride (NaBH4), sodium carbonate (Na2CO3) (CAS-497-19-8), sodium hydroxide (NaOH) (CAS 1310-73-2), sodium nitrate (NaNO3) (CAS 7631-99-4), sodium sulphate (Na2SO4) (CAS- 7757-82-6), sulphuric acid (H2SO4) (CAS-7664-93-9), and 2, 4, 6-Tris(2-pyridyl)-s-triazine (TPTZ) (CAS-7757-82-6).


## 2.1.2. Instrumentation

Hot-air oven (meditry DHA-9053A), incubator, analytical balance, spectrophotometer (JENWAY 6305), roller mixer, micropipettes, refrigerator, fume hood (Biobased FH1000), and autoclave.


## 2.1.3. Glassware and Miscellaneous

Conical flask, measuring cylinder, filter funnel, spatulas, test tubes, cuvettes, volumetric flask, Petri dishes, beakers, cotton swabs, watch glass, filter paper, falcon tubes, and Whatman filter papers No.1.


## 2.2. Sample Collection

Five varieties ofCatharanthus roseus flowers (Figure 6) were collected from Diyatha Uyana plant nursery, Battaramulla, Sri Lanka.Figure 6
Five varieties ofCatharanthus roseus flowers and their code names. (LPF) Light pink (a), (RF) red (b), (WPF) white pink dot (c), (WYF) white yellow dot (d), and (PF) purple (e).
(a)(b)(c)(d)(e)


## 2.3. Preparation of Flower Extracts Using Distilled Water

The samples were air-dried and grounded into smaller pieces. To 2 g of each grounded sample, 40 mL of distilled water was added. The samples were incubated at 60°C for 30 min and were filtered using Whatman filter paper No.1 into 50 mL falcon tubes. The samples were stored at 4°C until further use.


## 2.4. Green Synthesis of AgNPs

1 mL of each water extract was mixed with 9 mL of AgNO3 solution and was kept at room temperature (RT) for 3 days. The color change of the samples was observed, and the absorbance was measured from 300 to 620 nm using distilled water as a blank.


## 2.5. SEM and Particle Size Analysis

10 mL of PF AgNPs was centrifuged at 5000 rpm for 10 min and was transferred to wash glass. The sample added to the watch glass was dried completely at 180°C. The dried smear was dissolved by adding 200µL of distilled water and transferred into an Eppendorf tube. The sample was wrapped using aluminum foil and was sent for the SEM analysis. The morphology of PF AgNPs was examined using a Scanning Electron Microscope and the average particle size was analyzed by the Malvern Zetasizer Nano ZS particle size analyzer from the Sri Lanka Institute of Nanotechnology (SLINTEC) using the Hitachi SU6600 SEM.


## 2.6. Phytochemical Analysis

The phytochemical analysis was carried out according to the methodologies shown in Table1.Table 1
Methodology of the phytochemical analysis [20].
PhytochemicalMethodologyExpected positive color resultCarbohydrates2 ml of extract was treated with 1 mL of Molisch’s reagent and a few drops of concentrated H2SO4 were addedFormation of purple or reddish color ringTannins1 mL of extract was added to 2 mL of 5% FeCl3Formation of dark blue or greenish-black colorSaponins2 mL of extract and distilled water was added and shaken in a graduated cylinder for 15 minutesFormation of 1 cm layer of foamTerpenoids0.5 mL of the extract was treated with 2 mL of chloroform and concentrated H2SO4Formation of red-brown color at the interfaceAnthraquinonesFew drops of 10% NH4OH were added to 1 mL of extractFormation of a pink color precipitateSteroidsTo 1 mL of extract, an equal volume of chloroform was added and a few drops of concentrated H2SO4Formation of a brown color ringProteinsTo 1 mL of extract, a few drops of CuSO4 and 1 mL of 10% NaOH were addedFormation of purple or mauve color


## 2.7. Determination of Antioxidant Activity

AgNPs and the water extracts were diluted 1 : 15 to analyze the antioxidant activity. Each analysis was carried out in triplicate.


### 2.7.1. Determination of Total Flavonoid Content (TFC)

TFC was determined using an AlCl3 colorimetric method adopted from Perera and Kandiah [21]. 1.5 mL of the sample was mixed with 1.5 mL of 2 % AlCl3·6H2O solution and was incubated at RT for 10 minutes. The absorbance of the samples was measured at 415 nm using distilled water as a blank and the results were expressed in µg Quercetin equivalents per 100 g (µg QE/100 g).


### 2.7.2. Determination of Total Phenolic Content (TPC)

0.1 mL of the sample was mixed with 1.4 mL of 7.5 % Na2CO3 and was incubated for 6 minutes. To the same solution, 1.5 mL of Folin–Ciocalteu reagent was added and incubated at RT for 60 minutes in a dark condition. The absorbance was measured at 765 nm using distilled water as a blank and the results were expressed in g gallic acid equivalents per 100 g (g GAE/100 g).


### 2.7.3. Determination of Total Antioxidant Capacity (TAC)

TAC was evaluated using a phosphomolybdenum assay adapted from Biwas et al. [22] with modifications. The phosphomolybdenum reagent was prepared by mixing equivalent volumes of 28 mM sodium sulphate and 4 mM ammonium molybdate and 0.6 M sulphuric acid. 3 mL of the sample was mixed with 1 mL of the phosphomolybdenum reagent, and the samples were incubated at 90°C for 90 min. The absorbance was measured at 695 nm using distilled water as a blank and the results were expressed as g ascorbic acid equivalents per 100 g (g AAE/100 g).


### 2.7.4. Determination of Ferric Reducing Antioxidant Power (FRAP)

The FRAP reagent was prepared by mixing 50 mL of 0.3 M pH 3.6 acetate buffer, 5 mL of 10 mM TPTZ solution prepared in 40 mM HCl, and 5 mL of 20 mM FeCl3·6H2O Solution. 100 µL sample was mixed with 2.9 mL of FRAP reagent and the absorbance was measured at 593 nm using distilled water as a blank for every 1 minute.


### 2.7.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

1 mL of the sample was mixed with 2 mL of 0.004% DPPH and was incubated for 30 minutes at RT. The absorbance was measured at 517 nm using methanol as a blank. The %DPPH scavenging activity was calculated using the following equation:(1)Inhibition%=Acontrol−AsampleAcontrol×100.


### 2.7.6. Determination of Median Inhibition Concentration (IC50)

To 1 mL of series of five concentrations, 100%, 80%, 60%, 40%, and 20%, which were prepared using the samples and distilled water, 2 mL of 0.004 % DPPH solution was added and was incubated for 30 mins at RT. The absorbance of all the samples was measured at 517 nm using methanol as a blank. The % DPPH scavenging activity was calculated using equation (1).


## 2.7.1. Determination of Total Flavonoid Content (TFC)

TFC was determined using an AlCl3 colorimetric method adopted from Perera and Kandiah [21]. 1.5 mL of the sample was mixed with 1.5 mL of 2 % AlCl3·6H2O solution and was incubated at RT for 10 minutes. The absorbance of the samples was measured at 415 nm using distilled water as a blank and the results were expressed in µg Quercetin equivalents per 100 g (µg QE/100 g).


## 2.7.2. Determination of Total Phenolic Content (TPC)

0.1 mL of the sample was mixed with 1.4 mL of 7.5 % Na2CO3 and was incubated for 6 minutes. To the same solution, 1.5 mL of Folin–Ciocalteu reagent was added and incubated at RT for 60 minutes in a dark condition. The absorbance was measured at 765 nm using distilled water as a blank and the results were expressed in g gallic acid equivalents per 100 g (g GAE/100 g).


## 2.7.3. Determination of Total Antioxidant Capacity (TAC)

TAC was evaluated using a phosphomolybdenum assay adapted from Biwas et al. [22] with modifications. The phosphomolybdenum reagent was prepared by mixing equivalent volumes of 28 mM sodium sulphate and 4 mM ammonium molybdate and 0.6 M sulphuric acid. 3 mL of the sample was mixed with 1 mL of the phosphomolybdenum reagent, and the samples were incubated at 90°C for 90 min. The absorbance was measured at 695 nm using distilled water as a blank and the results were expressed as g ascorbic acid equivalents per 100 g (g AAE/100 g).


## 2.7.4. Determination of Ferric Reducing Antioxidant Power (FRAP)

The FRAP reagent was prepared by mixing 50 mL of 0.3 M pH 3.6 acetate buffer, 5 mL of 10 mM TPTZ solution prepared in 40 mM HCl, and 5 mL of 20 mM FeCl3·6H2O Solution. 100 µL sample was mixed with 2.9 mL of FRAP reagent and the absorbance was measured at 593 nm using distilled water as a blank for every 1 minute.


## 2.7.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

1 mL of the sample was mixed with 2 mL of 0.004% DPPH and was incubated for 30 minutes at RT. The absorbance was measured at 517 nm using methanol as a blank. The %DPPH scavenging activity was calculated using the following equation:(1)Inhibition%=Acontrol−AsampleAcontrol×100.


## 2.7.6. Determination of Median Inhibition Concentration (IC50)

To 1 mL of series of five concentrations, 100%, 80%, 60%, 40%, and 20%, which were prepared using the samples and distilled water, 2 mL of 0.004 % DPPH solution was added and was incubated for 30 mins at RT. The absorbance of all the samples was measured at 517 nm using methanol as a blank. The % DPPH scavenging activity was calculated using equation (1).


## 2.8. Determination of the Antimicrobial Activity

The antimicrobial activity was determined using two types of bacterial strainsStaphylococcus aureus and Escherichia coli using the agar well diffusion technique on Mueller-Hinton agar (MHA). The bacterial culture was evenly spread on the MHA plates using a cotton swab. Three wells were created on the MHA for the negative control (−) and the two replicates of the sample (S1 and S2) as shown in Figure 7. 1 mL of saline was added to the negative control (−) and 1 mL of sample was added to S1 and S2. Gentamycin discs were used as the positive control (+). The plates were incubated at 37°C for 24 hours and the diameter of the zone of inhibition was measured using a ruler.Figure 7
Illustration of the labeling of Petri plates.


## 2.9. Statistical and Particle Size Analysis

10 mL of PF AgNPs was centrifuged at 5000 rpm for 10 min and were transferred to wash glass. The sample added to the watch glass was dried completely at 180°C. The dried smear was dissolved by adding 200µL of distilled water and transferred into an Eppendorf tube. The sample was wrapped using aluminum foil and sent for the SEM analysis. The morphology of PF AgNPs was examined using a Scanning Electron Microscope and the average particle size was analyzed by the Malvern Zetasizer Nano ZS particle size analyzer from the Sri Lanka Institute of Nanotechnology (SLINTEC) using the Hitachi SU6600 SEM.


## 2.10. Determination of the Photocatalytic Activity

1 mL of 5000 ppm PF AgNPs and 1 mL of 0.2 M NaBH4 were added to 100 mL of 2 mM MO. The absorbance of the reaction mixture was measured from 300 to 560 nm using distilled water as a blank every 5 minutes for 20 minutes. The procedure was repeated for 333 ppm PF AgNPs.


## 3. Results and Discussion

### 3.1. Green Synthesis of AgNPs

In the preliminary stage, as the temperature is one of the key influence factors in the nanoparticle synthesis, the AgNPs synthesis was done at different temperatures keeping other parameters constant. The optimum condition for all the varieties was at room temperature for 3 days. This may be due to the slow formation and growth at room temperature which usually takes more hours to complete the bioreduction [23]. However, the synthesis at room temperature is greener than using heat, which is a further advantage. It was also observed that the AgNPs were stable even for more than 6 months stored at 4°C. The formation of the AgNPs was initially observed by a color change to a reddish-brown solution (Figure 8). This is due to a phenomenon called the surface plasmon resonance (SPR) which occurs due to the excitation of the surface plasmons present on the outer surface of the AgNPs that get excited when light/UV is incident [24].Figure 8
Green synthesis of AgNPs. (a) Flower extracts mixed with silver nitrate before 3 days and (b) after 3 days at RT.
(a)(b)


### 3.2. Characterization of AgNPs

#### 3.2.1. UV-Visible Spectrophotometric Analysis

The absorption peaks at 440–500 nm (Figure9) further confirmed the formation of AgNPs as they are known to exhibit a UV–Visible absorption peak in the range of 400–500 nm due to SPR [25].Figure 9
UV-Visible spectra of the synthesized AgNPs.


#### 3.2.2. SEM Analysis

The morphology of the PF AgNPs was observed by carrying out an SEM analysis which is a technique used to analyze the morphology. The results revealed that the PF AgNPs were spherical and around 30 nm in size (Figure10).Figure 10
SEM image of the synthesized PF AgNPs. (a) x40 k/scale - 500 nm; (b) x70 k/scale - 500 nm; and (c) x80 k/scale - 500 nm.
(a)(b)(c)


### 3.3. Optical Properties of the AgNPs

Apart from the morphology, the optical properties of the synthesized AgNPs were analyzed by using UV–Visible absorbance spectra. Measuring the bandgap is an important parameter to characterize the conductivity of the nanoparticles. The bandgap is the energy difference between the top valence band (VB) and bottom of the conduction band (CB) and for an electron to move from the VB to the CB, it requires a specific minimum amount of energy known as the bandgap energy based on the which nanoparticles can be classified as semiconductors (<3 eV) or insulators (>4 eV) [26]. The optical edge of LPF AgNPs was observed at 440 nm and the bandgap energy was calculated as 2.82 eV and was classified as a semiconductor by using the following equation, where h is the plank’s constant = 6.626 × 10 −34 J s, C is the speed of light = 3 × 108 m/s, and λ is the cut-off wavelength of LPF AgNPs = 440 × 10−9:(2)E=h×Cλ.Based on the calculated bandgap energies, all the synthesized AgNPs were classified as semiconductors (Table2). According to Singh, Goyal, and Devlal [27], the bandgap energy increases as the size of semiconductor nanoparticles decreases; therefore, it can be deduced that LPF and RF AgNPs are smaller in size compared to WPF, WYF, and PF AgNPs. According to the SEM analysis, PF AgNPs were around 30 nm (Figure 10). This concludes that LPF and RF AgNPs are less than 30 nm in size.Table 2
Classification of the synthesized AgNPs based on bandgap energy.
AgNPs sampleBandgap energy (eV)ClassificationLight pink (LPF)2.82SemiconductorsRed (RF)2.58White pink dot (WPF)2.48White yellow dot (WYF)2.48Purple (PF)2.48


### 3.4. Phytochemical Analysis

Phytochemical analysis revealed the presence of antioxidants for the bioreduction which resulted in stable AgNPs (Table3).Table 3
Phytochemical analysis test results.
PhytochemicalLight pink (LPF)Red (RF)White pink dot (WPF)White yellow dot (WYF)Purple (PF)Positive result color changeCarbohydrate✓✓✓✓✓Tannins✓✗✗✗✓Saponins✓✓✗✓✓Terpenoids✓✓✓✓✓Anthraquinones✗✗✗✗✗—Steroids✓✓✗✗✓Proteins✓✓✓✓✓


### 3.5. Determination of the Antioxidant Activity

Phenolics compounds are phytochemicals produced by all plant species which primarily act as antioxidants as they are effective as reducing agents, hydrogen atom donors, and singlet oxygen scavengers [28]. Flavonoids are the largest group of polyphenolic compounds. Studies indicated that various glycosylated flavonoids, mainly quercetin, isorhamnetin, and kaempferol derivatives were produced in different parts of the Catharanthus roses plant [29].


#### 3.5.1. Total Flavonoid Content (TFC)

The TFC was determined using the aluminum colorimetric method, where the AlCl3 ions form stable acid complexes with C-4 keto group or wither with C-5 or C-3 hydroxyl groups of flavonols and flavones with maximum absorption at 415 nm. Moreover, it also produces acid-labile complexes with the orthodihydroxyl groups in the A- or B- ring of flavonoids. AgNPs contained a higher TFC compared to the water extracts in the following order: WPF = WYF = PF = LPF > RF (Figure 11).Figure 11
Total flavonoid content of water extracts and AgNPs expressed as quercetin equivalents (QE).


#### 3.5.2. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu reagent method, where the phenolic compounds react with Folin–Ciocalteu reagent to form molybdenum and tungsten oxides in an alkaline solution which is blue [30]. AgNPs contained a higher TPC compared to the water extracts in the following order: LPF > WPF = WYF = PF > RF (Figure 12).Figure 12
Total phenolic content of water extracts and AgNPs expressed as gallic acid equivalents (GAE).


#### 3.5.3. Total Antioxidant Capacity (TAC)

TAC was measured using a phosphomolybdenum assay based on the reduction of Mo (VI) to Mo (V) using the electrons donated by antioxidants in the samples to form green phosphate or Mo (V) compounds with maximum absorption of 695 nm. [31]. AgNPs contained a higher TAC compared to the water extracts in the following order: RF > LPF = WPF = WYF > PF (Figure 13).Figure 13
Total antioxidant capacity of water extracts and AgNPs expressed as ascorbic acid equivalents (AAE).


#### 3.5.4. Ferric Reducing Antioxidant Power (FRAP)

To analyze the reducing power, FRAP assay was carried out based on the reduction of ferric tripyridyl triazine complex (Fe3+-TPTZ) to Fe 2+ -tripyridyl triazine, where a blue color compound is produced due to the electron donation by antioxidants at low pH [32]. All the AgNPs took the least time for the free radical scavenging activity compared to the water extracts (Figure 14). PF AgNPs had the highest free radical scavenging activity at 3 minutes followed by WPF = WYF = RF > LPF (Figure 15).Figure 14
FRAP of the water extracts.Figure 15
FRAP of the AgNPs.


#### 3.5.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

The DPPH radical savaging activity is evaluated based on the electron-donating property of the antioxidants to the nitrogen of the DPPH [33]. The odd electron of the nitrogen results in the color change from a purple color to colorless when it pairs up. AgNPs showed a higher % DPPH scavenging activity compared to the water extracts in the following order: LPF = RF = WPF = PF > WYF (Figure 16). The reducing power and the electron-donating property revealed the presence of higher TFC in WPF and WYF.Figure 16
% DPPH scavenging activity of water extracts and AgNPs.


#### 3.5.6. Determination of the Half-Maximal Inhibitory Concentration (IC50)

The IC50 of the water extracts and the AgNPs were calculated to determine the concentration of the sample required to inhibit 50% of the radical (Figures17 and 18). As shown in Table 4 the IC50 was lower in AgNPs compared to the water extracts. According to Perera and Kandiah [21], the TAC is inversely proportional to the IC50; this was also proved by the results obtained as RF AgNPs have the highest TAC and lowest IC50 (Table 4).Figure 17
IC50 of the water extracts.Figure 18
IC50 of the AgNPs.Table 4
IC50 of water extracts and AgNPs.
SampleIC50 of water extractsIC50 of AgNPsLight pink (LPF)23.373.23Red (RF)15.40.03White pink dot (WPF)27.840.12White yellow dot (WYF)3.152.04Purple (PF)29.048.23


### 3.6. Statistical Analysis

The one-way ANOVA analysis indicated that there is a statistically significant difference between the water extracts and AgNPs TFC, TPC, and TAC as theF value > F crit and P value < 0.05.A weak correlation was indicated between TAC and TFC assays when compared to the correlation between TAC and TPC assays (Figure19). Since different phenolic compounds have different antioxidant activities based on their structure according to Kaur and Mondal [34], TAC could also be due to the presence of different types of various phenolic compounds as well as other phytochemicals such as pigments contributing to its antioxidant activity other than flavonoids. This could be also the reason RF AgNPs have the lowest TFC and TPC but show the highest antioxidant capacity. There is a strong correlation between DPPH and FRAP as both assays evaluate the efficiency of electron transference despite the different methods of elevation.Figure 19
Correlation between antioxidant assays.


#### 3.6.1. Determination of the Antimicrobial Activity

A higher antimicrobial activity againstEscherichia coli was shown by AgNPs than that of water extracts in the following order: RF = WYF > WPF > PF > LPF (Figures 20 and 21). However, water extracts showed higher antimicrobial activity against Staphylococcus aureus than AgNPs (Figures 22 and 23). The higher antimicrobial activity of AgNPs against Escherichia coli compared to Staphylococcus aureus can be explained based on their differences in their cell wall. Escherichia coli possesses a thin peptidoglycan cell wall; therefore, the AgNPs can easily enter the bacteria through the cell wall unlike in Staphylococcus aureus [35]. Moreover, since the Staphylococcus aureus cell wall is negatively charged, it tends to bind with more Ag + lowering the interaction of AgNPs to the plasma membrane and protects it from ROS produced by AgNPs [36]. One-way ANOVA results indicated that there is no statistical significance between the antimicrobial activity of the water extracts and AgNPs against Staphylococcus aureus as the F value > F crit andP value < 0.05 and that there is a statistical significance between the antimicrobial activity of the water extracts and AgNPs against Escherichia coli as the F value > F and P value < 0.05.Figure 20
Antimicrobial activity of the water extracts and AgNPs againstEscherichia coli.Figure 21
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstEscherichia coli. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 22
Antimicrobial activity of the water extracts and AgNPs againstStaphylococcus aureus.Figure 23
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstStaphylococcus aureus. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


#### 3.6.2. Determination of the Photocatalytic Activity

Photocatalytic activity is the ability of AgNPs to catalyze a photoreaction under sunlight. When UV/visible light is incident onto the surface of AgNPs that fulfills the bandgap energy, electrons from the CB get excited to the VB and form electron pairs (Figure24):(3)AgNPs+hv⟶e−+h+.Figure 24
Mechanism of AgNPs photocatalysis [37].The holes (H+) and electrons produced by VB and CB react with H2O and O2, respectively, to form OH •, O2•-, and HO2•. These produced radicles together attack the azo bond present in MO bringing about the degradation of the dye-forming intermediates and finally mineralize the dye to a colorless final product as shown in Figure 25. The role of NaBH4 is to produce BH4−1 which acts as an electron relay to provide AgNPs with more electrons to produce more radicles which is why AgNPs have been reported to degrade organic dyes in a short period [37].Figure 25
Decolorization of methyl orange from 0 to 20 minutes: (a) when 5000 ppm PF AgNPs and (b) 333 ppm PF AgNPs were added.
(a)(b)The methyl orange absorption peak at 460 nm was shown to be decreasing with time Figures26 and 27. When 5000 ppm PF AgNPs were used a blue shift of the methyl orange peak (400 nm) was observed as the degradation proceeded. Nagar and Devar [38] proposed degradation pathway could be due to the intermediates produced during the degradation as a result of successive demethylation where homolytic cleavage of nitrogen-carbon bond leads to the substitution of the methyl group by hydrogen (Figure 28).Figure 26
Absorbance of methyl orange with time when 5000 ppm PF AgNPs were added.Figure 27
Absorbance of methyl orange with time when 333 ppm PF AgNPs were added.Figure 28
Proposed mechanism of methyl orange degradation according to Nagar and Devra, 2019.


#### 3.6.3. Methyl Orange Peak

This shift was also observed using 333 ppm PF AgNPs and the degradation was completed in 20 minutes as the absorption of methyl orange was almost zero (Figure28). The absence of the SPR peak of PF AgNPs (500 nm) can be due to the overlapping of the methyl orange peak or due to lack of homogeneity when the absorbance was measured as AgNPs tend sediment at the bottom of the beaker.The kinetics of the methyl orange photocatalytic degradation was studied by plotting ln (Ct/Co) against reaction time (Figures29 and 30). According to the rate constant calculated from the gradients 5000 ppm PF AgNPs and 333 ppm PF AgNPs were found to be 0.082 and 0.1136, respectively. Therefore, 333 ppm PF AgNPs were proposed to have an efficient photocatalytic degradation ability compared to that of 5000 ppm PF AgNPs.Figure 29
Rate constant graph of 5000 ppm PF AgNPs.Figure 30
Rate constant graph of 333 ppm PF AgNPs.The efficiency of photocatalytic degradation decreased when a higher concentration of PF AgNPs (5000 ppm) was used. According to Bansal and Sud, this might be due to the formation of intermediate moieties that can interfere with the degradation process [39].


## 3.1. Green Synthesis of AgNPs

In the preliminary stage, as the temperature is one of the key influence factors in the nanoparticle synthesis, the AgNPs synthesis was done at different temperatures keeping other parameters constant. The optimum condition for all the varieties was at room temperature for 3 days. This may be due to the slow formation and growth at room temperature which usually takes more hours to complete the bioreduction [23]. However, the synthesis at room temperature is greener than using heat, which is a further advantage. It was also observed that the AgNPs were stable even for more than 6 months stored at 4°C. The formation of the AgNPs was initially observed by a color change to a reddish-brown solution (Figure 8). This is due to a phenomenon called the surface plasmon resonance (SPR) which occurs due to the excitation of the surface plasmons present on the outer surface of the AgNPs that get excited when light/UV is incident [24].Figure 8
Green synthesis of AgNPs. (a) Flower extracts mixed with silver nitrate before 3 days and (b) after 3 days at RT.
(a)(b)


## 3.2. Characterization of AgNPs

### 3.2.1. UV-Visible Spectrophotometric Analysis

The absorption peaks at 440–500 nm (Figure9) further confirmed the formation of AgNPs as they are known to exhibit a UV–Visible absorption peak in the range of 400–500 nm due to SPR [25].Figure 9
UV-Visible spectra of the synthesized AgNPs.


### 3.2.2. SEM Analysis

The morphology of the PF AgNPs was observed by carrying out an SEM analysis which is a technique used to analyze the morphology. The results revealed that the PF AgNPs were spherical and around 30 nm in size (Figure10).Figure 10
SEM image of the synthesized PF AgNPs. (a) x40 k/scale - 500 nm; (b) x70 k/scale - 500 nm; and (c) x80 k/scale - 500 nm.
(a)(b)(c)


## 3.2.1. UV-Visible Spectrophotometric Analysis

The absorption peaks at 440–500 nm (Figure9) further confirmed the formation of AgNPs as they are known to exhibit a UV–Visible absorption peak in the range of 400–500 nm due to SPR [25].Figure 9
UV-Visible spectra of the synthesized AgNPs.


## 3.2.2. SEM Analysis

The morphology of the PF AgNPs was observed by carrying out an SEM analysis which is a technique used to analyze the morphology. The results revealed that the PF AgNPs were spherical and around 30 nm in size (Figure10).Figure 10
SEM image of the synthesized PF AgNPs. (a) x40 k/scale - 500 nm; (b) x70 k/scale - 500 nm; and (c) x80 k/scale - 500 nm.
(a)(b)(c)


## 3.3. Optical Properties of the AgNPs

Apart from the morphology, the optical properties of the synthesized AgNPs were analyzed by using UV–Visible absorbance spectra. Measuring the bandgap is an important parameter to characterize the conductivity of the nanoparticles. The bandgap is the energy difference between the top valence band (VB) and bottom of the conduction band (CB) and for an electron to move from the VB to the CB, it requires a specific minimum amount of energy known as the bandgap energy based on the which nanoparticles can be classified as semiconductors (<3 eV) or insulators (>4 eV) [26]. The optical edge of LPF AgNPs was observed at 440 nm and the bandgap energy was calculated as 2.82 eV and was classified as a semiconductor by using the following equation, where h is the plank’s constant = 6.626 × 10 −34 J s, C is the speed of light = 3 × 108 m/s, and λ is the cut-off wavelength of LPF AgNPs = 440 × 10−9:(2)E=h×Cλ.Based on the calculated bandgap energies, all the synthesized AgNPs were classified as semiconductors (Table2). According to Singh, Goyal, and Devlal [27], the bandgap energy increases as the size of semiconductor nanoparticles decreases; therefore, it can be deduced that LPF and RF AgNPs are smaller in size compared to WPF, WYF, and PF AgNPs. According to the SEM analysis, PF AgNPs were around 30 nm (Figure 10). This concludes that LPF and RF AgNPs are less than 30 nm in size.Table 2
Classification of the synthesized AgNPs based on bandgap energy.
AgNPs sampleBandgap energy (eV)ClassificationLight pink (LPF)2.82SemiconductorsRed (RF)2.58White pink dot (WPF)2.48White yellow dot (WYF)2.48Purple (PF)2.48


## 3.4. Phytochemical Analysis

Phytochemical analysis revealed the presence of antioxidants for the bioreduction which resulted in stable AgNPs (Table3).Table 3
Phytochemical analysis test results.
PhytochemicalLight pink (LPF)Red (RF)White pink dot (WPF)White yellow dot (WYF)Purple (PF)Positive result color changeCarbohydrate✓✓✓✓✓Tannins✓✗✗✗✓Saponins✓✓✗✓✓Terpenoids✓✓✓✓✓Anthraquinones✗✗✗✗✗—Steroids✓✓✗✗✓Proteins✓✓✓✓✓


## 3.5. Determination of the Antioxidant Activity

Phenolics compounds are phytochemicals produced by all plant species which primarily act as antioxidants as they are effective as reducing agents, hydrogen atom donors, and singlet oxygen scavengers [28]. Flavonoids are the largest group of polyphenolic compounds. Studies indicated that various glycosylated flavonoids, mainly quercetin, isorhamnetin, and kaempferol derivatives were produced in different parts of the Catharanthus roses plant [29].


### 3.5.1. Total Flavonoid Content (TFC)

The TFC was determined using the aluminum colorimetric method, where the AlCl3 ions form stable acid complexes with C-4 keto group or wither with C-5 or C-3 hydroxyl groups of flavonols and flavones with maximum absorption at 415 nm. Moreover, it also produces acid-labile complexes with the orthodihydroxyl groups in the A- or B- ring of flavonoids. AgNPs contained a higher TFC compared to the water extracts in the following order: WPF = WYF = PF = LPF > RF (Figure 11).Figure 11
Total flavonoid content of water extracts and AgNPs expressed as quercetin equivalents (QE).


### 3.5.2. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu reagent method, where the phenolic compounds react with Folin–Ciocalteu reagent to form molybdenum and tungsten oxides in an alkaline solution which is blue [30]. AgNPs contained a higher TPC compared to the water extracts in the following order: LPF > WPF = WYF = PF > RF (Figure 12).Figure 12
Total phenolic content of water extracts and AgNPs expressed as gallic acid equivalents (GAE).


### 3.5.3. Total Antioxidant Capacity (TAC)

TAC was measured using a phosphomolybdenum assay based on the reduction of Mo (VI) to Mo (V) using the electrons donated by antioxidants in the samples to form green phosphate or Mo (V) compounds with maximum absorption of 695 nm. [31]. AgNPs contained a higher TAC compared to the water extracts in the following order: RF > LPF = WPF = WYF > PF (Figure 13).Figure 13
Total antioxidant capacity of water extracts and AgNPs expressed as ascorbic acid equivalents (AAE).


### 3.5.4. Ferric Reducing Antioxidant Power (FRAP)

To analyze the reducing power, FRAP assay was carried out based on the reduction of ferric tripyridyl triazine complex (Fe3+-TPTZ) to Fe 2+ -tripyridyl triazine, where a blue color compound is produced due to the electron donation by antioxidants at low pH [32]. All the AgNPs took the least time for the free radical scavenging activity compared to the water extracts (Figure 14). PF AgNPs had the highest free radical scavenging activity at 3 minutes followed by WPF = WYF = RF > LPF (Figure 15).Figure 14
FRAP of the water extracts.Figure 15
FRAP of the AgNPs.


### 3.5.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

The DPPH radical savaging activity is evaluated based on the electron-donating property of the antioxidants to the nitrogen of the DPPH [33]. The odd electron of the nitrogen results in the color change from a purple color to colorless when it pairs up. AgNPs showed a higher % DPPH scavenging activity compared to the water extracts in the following order: LPF = RF = WPF = PF > WYF (Figure 16). The reducing power and the electron-donating property revealed the presence of higher TFC in WPF and WYF.Figure 16
% DPPH scavenging activity of water extracts and AgNPs.


### 3.5.6. Determination of the Half-Maximal Inhibitory Concentration (IC50)

The IC50 of the water extracts and the AgNPs were calculated to determine the concentration of the sample required to inhibit 50% of the radical (Figures17 and 18). As shown in Table 4 the IC50 was lower in AgNPs compared to the water extracts. According to Perera and Kandiah [21], the TAC is inversely proportional to the IC50; this was also proved by the results obtained as RF AgNPs have the highest TAC and lowest IC50 (Table 4).Figure 17
IC50 of the water extracts.Figure 18
IC50 of the AgNPs.Table 4
IC50 of water extracts and AgNPs.
SampleIC50 of water extractsIC50 of AgNPsLight pink (LPF)23.373.23Red (RF)15.40.03White pink dot (WPF)27.840.12White yellow dot (WYF)3.152.04Purple (PF)29.048.23


## 3.5.1. Total Flavonoid Content (TFC)

The TFC was determined using the aluminum colorimetric method, where the AlCl3 ions form stable acid complexes with C-4 keto group or wither with C-5 or C-3 hydroxyl groups of flavonols and flavones with maximum absorption at 415 nm. Moreover, it also produces acid-labile complexes with the orthodihydroxyl groups in the A- or B- ring of flavonoids. AgNPs contained a higher TFC compared to the water extracts in the following order: WPF = WYF = PF = LPF > RF (Figure 11).Figure 11
Total flavonoid content of water extracts and AgNPs expressed as quercetin equivalents (QE).


## 3.5.2. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu reagent method, where the phenolic compounds react with Folin–Ciocalteu reagent to form molybdenum and tungsten oxides in an alkaline solution which is blue [30]. AgNPs contained a higher TPC compared to the water extracts in the following order: LPF > WPF = WYF = PF > RF (Figure 12).Figure 12
Total phenolic content of water extracts and AgNPs expressed as gallic acid equivalents (GAE).


## 3.5.3. Total Antioxidant Capacity (TAC)

TAC was measured using a phosphomolybdenum assay based on the reduction of Mo (VI) to Mo (V) using the electrons donated by antioxidants in the samples to form green phosphate or Mo (V) compounds with maximum absorption of 695 nm. [31]. AgNPs contained a higher TAC compared to the water extracts in the following order: RF > LPF = WPF = WYF > PF (Figure 13).Figure 13
Total antioxidant capacity of water extracts and AgNPs expressed as ascorbic acid equivalents (AAE).


## 3.5.4. Ferric Reducing Antioxidant Power (FRAP)

To analyze the reducing power, FRAP assay was carried out based on the reduction of ferric tripyridyl triazine complex (Fe3+-TPTZ) to Fe 2+ -tripyridyl triazine, where a blue color compound is produced due to the electron donation by antioxidants at low pH [32]. All the AgNPs took the least time for the free radical scavenging activity compared to the water extracts (Figure 14). PF AgNPs had the highest free radical scavenging activity at 3 minutes followed by WPF = WYF = RF > LPF (Figure 15).Figure 14
FRAP of the water extracts.Figure 15
FRAP of the AgNPs.


## 3.5.5. Determination of 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity

The DPPH radical savaging activity is evaluated based on the electron-donating property of the antioxidants to the nitrogen of the DPPH [33]. The odd electron of the nitrogen results in the color change from a purple color to colorless when it pairs up. AgNPs showed a higher % DPPH scavenging activity compared to the water extracts in the following order: LPF = RF = WPF = PF > WYF (Figure 16). The reducing power and the electron-donating property revealed the presence of higher TFC in WPF and WYF.Figure 16
% DPPH scavenging activity of water extracts and AgNPs.


## 3.5.6. Determination of the Half-Maximal Inhibitory Concentration (IC50)

The IC50 of the water extracts and the AgNPs were calculated to determine the concentration of the sample required to inhibit 50% of the radical (Figures17 and 18). As shown in Table 4 the IC50 was lower in AgNPs compared to the water extracts. According to Perera and Kandiah [21], the TAC is inversely proportional to the IC50; this was also proved by the results obtained as RF AgNPs have the highest TAC and lowest IC50 (Table 4).Figure 17
IC50 of the water extracts.Figure 18
IC50 of the AgNPs.Table 4
IC50 of water extracts and AgNPs.
SampleIC50 of water extractsIC50 of AgNPsLight pink (LPF)23.373.23Red (RF)15.40.03White pink dot (WPF)27.840.12White yellow dot (WYF)3.152.04Purple (PF)29.048.23


## 3.6. Statistical Analysis

The one-way ANOVA analysis indicated that there is a statistically significant difference between the water extracts and AgNPs TFC, TPC, and TAC as theF value > F crit and P value < 0.05.A weak correlation was indicated between TAC and TFC assays when compared to the correlation between TAC and TPC assays (Figure19). Since different phenolic compounds have different antioxidant activities based on their structure according to Kaur and Mondal [34], TAC could also be due to the presence of different types of various phenolic compounds as well as other phytochemicals such as pigments contributing to its antioxidant activity other than flavonoids. This could be also the reason RF AgNPs have the lowest TFC and TPC but show the highest antioxidant capacity. There is a strong correlation between DPPH and FRAP as both assays evaluate the efficiency of electron transference despite the different methods of elevation.Figure 19
Correlation between antioxidant assays.


### 3.6.1. Determination of the Antimicrobial Activity

A higher antimicrobial activity againstEscherichia coli was shown by AgNPs than that of water extracts in the following order: RF = WYF > WPF > PF > LPF (Figures 20 and 21). However, water extracts showed higher antimicrobial activity against Staphylococcus aureus than AgNPs (Figures 22 and 23). The higher antimicrobial activity of AgNPs against Escherichia coli compared to Staphylococcus aureus can be explained based on their differences in their cell wall. Escherichia coli possesses a thin peptidoglycan cell wall; therefore, the AgNPs can easily enter the bacteria through the cell wall unlike in Staphylococcus aureus [35]. Moreover, since the Staphylococcus aureus cell wall is negatively charged, it tends to bind with more Ag + lowering the interaction of AgNPs to the plasma membrane and protects it from ROS produced by AgNPs [36]. One-way ANOVA results indicated that there is no statistical significance between the antimicrobial activity of the water extracts and AgNPs against Staphylococcus aureus as the F value > F crit andP value < 0.05 and that there is a statistical significance between the antimicrobial activity of the water extracts and AgNPs against Escherichia coli as the F value > F and P value < 0.05.Figure 20
Antimicrobial activity of the water extracts and AgNPs againstEscherichia coli.Figure 21
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstEscherichia coli. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 22
Antimicrobial activity of the water extracts and AgNPs againstStaphylococcus aureus.Figure 23
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstStaphylococcus aureus. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.6.2. Determination of the Photocatalytic Activity

Photocatalytic activity is the ability of AgNPs to catalyze a photoreaction under sunlight. When UV/visible light is incident onto the surface of AgNPs that fulfills the bandgap energy, electrons from the CB get excited to the VB and form electron pairs (Figure24):(3)AgNPs+hv⟶e−+h+.Figure 24
Mechanism of AgNPs photocatalysis [37].The holes (H+) and electrons produced by VB and CB react with H2O and O2, respectively, to form OH •, O2•-, and HO2•. These produced radicles together attack the azo bond present in MO bringing about the degradation of the dye-forming intermediates and finally mineralize the dye to a colorless final product as shown in Figure 25. The role of NaBH4 is to produce BH4−1 which acts as an electron relay to provide AgNPs with more electrons to produce more radicles which is why AgNPs have been reported to degrade organic dyes in a short period [37].Figure 25
Decolorization of methyl orange from 0 to 20 minutes: (a) when 5000 ppm PF AgNPs and (b) 333 ppm PF AgNPs were added.
(a)(b)The methyl orange absorption peak at 460 nm was shown to be decreasing with time Figures26 and 27. When 5000 ppm PF AgNPs were used a blue shift of the methyl orange peak (400 nm) was observed as the degradation proceeded. Nagar and Devar [38] proposed degradation pathway could be due to the intermediates produced during the degradation as a result of successive demethylation where homolytic cleavage of nitrogen-carbon bond leads to the substitution of the methyl group by hydrogen (Figure 28).Figure 26
Absorbance of methyl orange with time when 5000 ppm PF AgNPs were added.Figure 27
Absorbance of methyl orange with time when 333 ppm PF AgNPs were added.Figure 28
Proposed mechanism of methyl orange degradation according to Nagar and Devra, 2019.


### 3.6.3. Methyl Orange Peak

This shift was also observed using 333 ppm PF AgNPs and the degradation was completed in 20 minutes as the absorption of methyl orange was almost zero (Figure28). The absence of the SPR peak of PF AgNPs (500 nm) can be due to the overlapping of the methyl orange peak or due to lack of homogeneity when the absorbance was measured as AgNPs tend sediment at the bottom of the beaker.The kinetics of the methyl orange photocatalytic degradation was studied by plotting ln (Ct/Co) against reaction time (Figures29 and 30). According to the rate constant calculated from the gradients 5000 ppm PF AgNPs and 333 ppm PF AgNPs were found to be 0.082 and 0.1136, respectively. Therefore, 333 ppm PF AgNPs were proposed to have an efficient photocatalytic degradation ability compared to that of 5000 ppm PF AgNPs.Figure 29
Rate constant graph of 5000 ppm PF AgNPs.Figure 30
Rate constant graph of 333 ppm PF AgNPs.The efficiency of photocatalytic degradation decreased when a higher concentration of PF AgNPs (5000 ppm) was used. According to Bansal and Sud, this might be due to the formation of intermediate moieties that can interfere with the degradation process [39].


## 3.6.1. Determination of the Antimicrobial Activity

A higher antimicrobial activity againstEscherichia coli was shown by AgNPs than that of water extracts in the following order: RF = WYF > WPF > PF > LPF (Figures 20 and 21). However, water extracts showed higher antimicrobial activity against Staphylococcus aureus than AgNPs (Figures 22 and 23). The higher antimicrobial activity of AgNPs against Escherichia coli compared to Staphylococcus aureus can be explained based on their differences in their cell wall. Escherichia coli possesses a thin peptidoglycan cell wall; therefore, the AgNPs can easily enter the bacteria through the cell wall unlike in Staphylococcus aureus [35]. Moreover, since the Staphylococcus aureus cell wall is negatively charged, it tends to bind with more Ag + lowering the interaction of AgNPs to the plasma membrane and protects it from ROS produced by AgNPs [36]. One-way ANOVA results indicated that there is no statistical significance between the antimicrobial activity of the water extracts and AgNPs against Staphylococcus aureus as the F value > F crit andP value < 0.05 and that there is a statistical significance between the antimicrobial activity of the water extracts and AgNPs against Escherichia coli as the F value > F and P value < 0.05.Figure 20
Antimicrobial activity of the water extracts and AgNPs againstEscherichia coli.Figure 21
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstEscherichia coli. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Figure 22
Antimicrobial activity of the water extracts and AgNPs againstStaphylococcus aureus.Figure 23
Antimicrobial activity of synthesized AgNPs and their respective flower extracts againstStaphylococcus aureus. (a) Light pink flower extract. (b) Light pink AgNPs. (c) White yellow flower extract. (d) White yellow AgNPs. (e) White pink flower extract. (f) White pink AgNPs. (g) Purple flower extract. (h) Purple AgNPs. (i) Red flower extract. (j) Red AgNPs.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.6.2. Determination of the Photocatalytic Activity

Photocatalytic activity is the ability of AgNPs to catalyze a photoreaction under sunlight. When UV/visible light is incident onto the surface of AgNPs that fulfills the bandgap energy, electrons from the CB get excited to the VB and form electron pairs (Figure24):(3)AgNPs+hv⟶e−+h+.Figure 24
Mechanism of AgNPs photocatalysis [37].The holes (H+) and electrons produced by VB and CB react with H2O and O2, respectively, to form OH •, O2•-, and HO2•. These produced radicles together attack the azo bond present in MO bringing about the degradation of the dye-forming intermediates and finally mineralize the dye to a colorless final product as shown in Figure 25. The role of NaBH4 is to produce BH4−1 which acts as an electron relay to provide AgNPs with more electrons to produce more radicles which is why AgNPs have been reported to degrade organic dyes in a short period [37].Figure 25
Decolorization of methyl orange from 0 to 20 minutes: (a) when 5000 ppm PF AgNPs and (b) 333 ppm PF AgNPs were added.
(a)(b)The methyl orange absorption peak at 460 nm was shown to be decreasing with time Figures26 and 27. When 5000 ppm PF AgNPs were used a blue shift of the methyl orange peak (400 nm) was observed as the degradation proceeded. Nagar and Devar [38] proposed degradation pathway could be due to the intermediates produced during the degradation as a result of successive demethylation where homolytic cleavage of nitrogen-carbon bond leads to the substitution of the methyl group by hydrogen (Figure 28).Figure 26
Absorbance of methyl orange with time when 5000 ppm PF AgNPs were added.Figure 27
Absorbance of methyl orange with time when 333 ppm PF AgNPs were added.Figure 28
Proposed mechanism of methyl orange degradation according to Nagar and Devra, 2019.


## 3.6.3. Methyl Orange Peak

This shift was also observed using 333 ppm PF AgNPs and the degradation was completed in 20 minutes as the absorption of methyl orange was almost zero (Figure28). The absence of the SPR peak of PF AgNPs (500 nm) can be due to the overlapping of the methyl orange peak or due to lack of homogeneity when the absorbance was measured as AgNPs tend sediment at the bottom of the beaker.The kinetics of the methyl orange photocatalytic degradation was studied by plotting ln (Ct/Co) against reaction time (Figures29 and 30). According to the rate constant calculated from the gradients 5000 ppm PF AgNPs and 333 ppm PF AgNPs were found to be 0.082 and 0.1136, respectively. Therefore, 333 ppm PF AgNPs were proposed to have an efficient photocatalytic degradation ability compared to that of 5000 ppm PF AgNPs.Figure 29
Rate constant graph of 5000 ppm PF AgNPs.Figure 30
Rate constant graph of 333 ppm PF AgNPs.The efficiency of photocatalytic degradation decreased when a higher concentration of PF AgNPs (5000 ppm) was used. According to Bansal and Sud, this might be due to the formation of intermediate moieties that can interfere with the degradation process [39].


## 4. Conclusion

In conclusion, the green synthesis of AgNPs from all six varieties ofCatharanthus roseus was at RT for 3 days. SEM of the PF AgNPs and bandgap energies calculated revealed that all the synthesized AgNPs were found to be in the range of 0–30 nm. The antioxidant assay results indicated that overall AgNPs show a higher antioxidant activity compared to their respective water extracts. Moreover, significant antimicrobial activity was shown by AgNPs against Escherichia coli compared to Staphylococcus aureus which concluded that the AgNPs were more effective against Gram-negative bacteria. The photocatalytic activity of 333 ppm PF AgNPs was found to be efficient for the photodegradation of methyl orange in 20 minutes compared to 5000 ppm PF AgNPs. Since the synthesized AgNPs are ecofriendly and biocompatible, these AgNPs have the potential to offer their application in biomedical and related fields to improve the quality of life.



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*Source: 5512786-2021-05-29.xml*