Green synthesis, characterization and bioactivity evaluation of Caesium carbonate nanoparticle using Coleus amboinicus

doi:10.21203/rs.3.rs-1433474/v1

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Green synthesis, characterization and bioactivity evaluation of Caesium carbonate nanoparticle using Coleus amboinicus

https://doi.org/10.21203/rs.3.rs-1433474/v1

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posted 14 Mar, 2022

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The Caesium carbonate nanoparticle was effectively synthesized via green method using Coleus amboinicus leaf extract. The phytochemical screening for the plant extract confirmed the presence of flavonoids, saponins, tannins and phenols. The phytocompounds were responsible for capping and helped in the stabilization of synthesis of Caesium carbonate nanoparticles. The biosynthesised Caesium carbonate nanoparticle was characterized using UV-Spectrophotometer,FTIR (Fourier Transform Infrared Spectroscopy),Scanning Electron Microscopy (SEM) and XRD (X-ray Diffraction).The bio-synthesized nanoparticles exhibited remarkable inhibition against selected gram negative and gram-positive bacteria.

Nanoparticle synthesis

Coleus amboinicus Leaf extracts

FTIR

Caesium Carbonate

Nanoparticles have a wide range of uses in today's world, including medicinal, optical, and electronic disciplines, and so have a burgeoning research field (Bamrungsap et al., 2012). The development of simple, faster, economically and environmentally acceptable nanoparticle synthesis is more highly significant in the field of nanotechnology. Additionally, the production of nanoparticles of various size and shapes has a crucial implication by improvising the experimental techniques (Chen et al., 2003). The properties of many conventional materials change when formed from nanoparticles. Organic compounds including stabilisers, capping, surface ligands, or passivating agents are used to coat inorganic nanoparticles throughout the biosynthesis process (Heuer-Jungemann et al., 2019). In the present study, Coleus amboinicus leaf extract was employed to make Caesium carbonate (Cs₂CO3) nanoparticles, which works as a stabilising or capping agent. This approach is termed as Green synthesis method (Ismail et al., 2016). Green synthesis cuts down on the number of chemicals use(Yadi et al., 2018). In comparison to chemical or micro-mediated synthesis, it is simple, efficient, and environmentally benign, with a short processing time (Sivakumar, 2021). As a result, green synthesis is the ideal option for making nanoparticles. Coleus amboinicus is an excellent stabilising and capping agent (Narayanan & Sakthivel, 2010).

Coleus amboinicus is a semi-succulent perennial plant native to Southern and Eastern Africa belongs to the Lamiaceae family (Dathar, 2019). Its smell and flavours are most similar to oregano. It's widely grown and neutralised across the tropics, where it's used as a traditional medicine. They offer a wide range of therapeutic qualities. Glucosides of luteolin and apigenin are being derived from the leaves. They also contain antibacterial, antifungal and antimicrobial properties (Sathyan et al., 2018; Winter et al., 2020).

Caesium carbonate is an alkali metal with an alternate high surface area in the form of nanoscale elemental powder and suspensions of caesium carbonate. In the process of organic synthesis, they serve as a starting point. It is a crystalline compound with a greater solubility in polar solvents including water, alcohol and DMF and even more increased solubility in organic solvents(Elayaraja & Karunakaran, 2012; Yadi et al., 2018). A caesium source that is insoluble in water can be quickly transformed to other caesium compounds. When dilute acids are used to treat carbonate compounds, CO₂ is released. In most cases, Cs₂Co₃ is instantly available in most amounts(Lei et al., 2019). Hence, the current study was attempted to synthesis and analyse caesium carbonate nanoparticles utilising Coleus amboinicus.

2.1 Chemicals

Calcium carbonate and Caesium chloride were purchased from Sigma-Aldrich, Chennai, India.

2.2 Soxhlet extraction

Coleus amboinicus leaves were collected from neighbouring area. The fresh Coleus amboinicus leaves were dried in shade for a week. The dried leaf sample was then finely powdered using mortar and pestle.5 g of the finely powder of Coleus amboinicus was subjected to Soxhlet extraction using distilled water. After the completion of 32 cycles the extract was concentrated using rotatory evaporator and the percentage yield was calculated (Malathi et al., 2011).

2.3 Phytochemical screening of the extract

Phytochemical screening was performed to identify the presence of pytochemical compounds such as alkaloids, flavonoids, terpenoids, steroids, phenols, glycosides, saponins and tannins in the Coleus amboinicus leaf extract. These test were performed by following the methods in previous studies. (Igbinosa et al., 2009; Varsha et al., 2013).

2.4 Synthesis of caesium carbonate:

Calcium carbonate of 0.1M and 0.1M caesium chloride solutions were prepared and combined in a proportion of 1:1. A magnetic stirrer was used to stir 25ml of Coleus amboinicus plant extract in a beaker (Ahmad et al., 2003; Paul et al., 2015). The prepared solution mixture is added drop wise to the plant extract, stirring constantly until the desired colour change is achieved. The beaker is then withdrawn from the magnetic stirrer, covered with aluminium foil, and incubated for roughly 24 hours at room temperature of about 27^o C (Mittal et al., 2014). The precipitate that accumulated in the beaker is separated and dried in a hot air oven. Further characterisation was done on the vacuum-dried powder (Shankar et al., 2003).

2.4 Characterization of synthesized nanoparticles:

2.4.1 UV-VIS

UV-Vis spectroscopy (UV-Vis) is a low-cost and simple characterisation approach that is frequently used to characterise nanoscale materials. A Systronic UV-Visible Spectrophotometer 117 with a resolution of 1nm between 200 and 800nm and a scanning speed of 2 nm/sec was used to perform UV-Visible spectroscopy analysis (Becheri et al., 2008; Haiss et al., 2007).

2.4.2 FTIR

FTIR is employed as an analytical method based upon the theory of Fourier transformation. It is important and suitable method for the evaluation of the functional group existing in the compound. The sample was analyzed by an IR light beam through the samples. The FTIR spectrum of the monomer and caesium carbonate nanoparticles was measured mostly on Nicolet 6700 FT-IR model to assure the formation from an ether linkage in between molecules. For FTIR spectroscopic studies, a Jasco Fourier Transform Infrared Spectrometer was employed. Infrared spectra created by the absorption of electromagnetic energy in the frequency range 400 to 4000 cm-1 are Fourier transformed (Bhuyan et al., 2015; Kulkarni & Muddapur, 2014).

2.4.3 SEM ANALYSIS

Scanning electron microscopy is most used method for high-resolution imaging of surfaces. The SEM used electrons for imaging the nanoparticles. The sample was prepared by drop casting 5 µL of the sample on a carbon coated silver grids and air drying. SEM were carried out with Field Emission Scanning Electron microscopy (FESEM) with EDS Carl Zeiss, Sigma. On the carbon coated copper grid, the samples were prepared by dropping a very small amount of the sample on the grid, and to remove excess solution blotting paper was used and then the film on the SEM grid was dried and subjected to analysis(Devi et al., 2012; Srivastava et al., 2013).

2.4.4 XRD

X-ray diffraction (XRD) is a widely used techniques for the characterization of NPs. Typically, XRD analysis reveals the crystalline structure, the nature of the phase, lattice parameters and crystalline grain size. The latter parameter is determined by the broadening of the highest intense peak of an XRD measurement for a specific sample using Scherrer's equation. XRD techniques have the benefit of being routinely used for powdered materials that have been dried from their colloidal solutions, resulting in statistically representative and volume-averaged readings (Mehta et al., 2017).

2.5 Bioactivity of biosynthesized caesium carbonate nanoparticles

2.5.1. Antioxidant Assay

The antioxidant activity of the synthesized caesium carbonate nanoparticles from Coleus amboinicus leaves was determined by DPPH Radical Scavenging Assay and Total Anti-oxidant Assay(Koleva et al., 2002).

2.5.1.1 Total Antioxidant Assay

The total antioxidant activity was carried out for the biosynthesised caesium carbonate nanoparticles (Cs₂CO₃).About 0.1ml of sample of different concentration was added to a test tube and followed by the addition of 1ml of the reagent blend containing 0.6M sulphuric acid, sodium phosphate of 28mM and 4mM of ammonium molybdate was added and shaken gently. The whole mixture is incubated in water bath at 95°C for 90 minutes. After the incubation period the samples were cooled down to the room temperature and the optical density was measured at 680nm.The result was expressed in ascorbic equivalence(Saeed et al., 2012; Singh et al., 2018).

2.5.1.2 DPPH Radical Scavenging Assay:

The biosynthesized caesium carbonate nanoparticles (Cs₂CO₃) of different concentration of 800 µl was taken and to that 200 µl of 1mM DPPPH solution was added and shaken up effectively, the incubation period for the mixture is for 30 minutes at room temperature. After the incubation duration the absorbance was measured at 517nm to evaluate IC 50 value (Garcia et al., 2012; Mensor et al., 2001). The DPPH scavenging activity percentage was determined by applying the below mentioned formula.

$$Percentage Inhibition= \frac{{A}_{c}- {A}_{1}}{{A}_{c}} \times 100\dots \dots \dots \dots \dots \dots \dots \dots \dots \left(2\right)$$

Where A_c is the absorbance of control and A₁ is the absorbance of test or standard sample.

2.5.2Anti-inflammatory

Anti-inflammatory assay for the biosynthesized nanoparticles was determined by BSA Inhibition Assay and Protein Inhibitory Assay.

2.5.2.1 BSA Inhibition assay

The inhibition assay was performed for the synthesised nanoparticles. To 100 µl of each sample about 500 µl of BSA (Bovine Serum Album) was added and it was incubated for 10 minutes at room temperature and after that heating at 51°C for 20 minutes and cooled to temperature. The absorbance was measured at 660 nm against acetyl salicylic acid as positive control(Harnett et al., 2005). The percentage of inhibition of protein denaturation was calculated using the following equation:

$$Percentage Inhibition= \frac{{A}_{c}- {A}_{1}}{{A}_{c}} \times 100\dots \dots \dots \dots \dots \dots \dots \dots .. \left(3\right)$$

Where A₁ is the absorbance of the sample and A_c is the absorbance of the positive control.

2.5.2.1 Protease Inhibitory Assay

Protease Inhibitory assay was performed where 100 µl of sample was taken to which 100 ml of BSA(Bovine Serum Albumin) was added and incubated for 5 minutes at room temperature. To this trypsin of 250 µl was added and the mixture was centrifuged. The supernatant was collected after centrifugation and the absorbance was measured at 210nm against the acetyl salicylic acid as positive control (SAMAL & KUMAR, 2015). The percentage inhibition was calculated using the below mentioned formula.

$$Percentage Inhibition= \frac{{A}_{c}- {A}_{1}}{{A}_{c}} \times 100\dots \dots \dots \dots \dots \dots \dots \dots \dots . \left(4\right)$$

Where A₁ is the absorbance of the sample and A_c is the absorbance of the positive control.

2.5.3. Antibacterial Assay

Antibacterial Assay was carried out on a sterilized Muller Hinton agar medium. The medium was poured into a sterilized petri plates and left out for solidification. Gram positive Escherichia coli Bacteria and Gram-negative Bacteria Staphylococcus aureus was normalized to 0.5 McFarland to attain 10⁸ cells/ml. The standardized culture was swabbed on the agar plates. On the inoculated agar plates, wells were punched with a sterile well borer. The prepared sample concentration (30, 60, 90 mg/ml) was applied to each well, and the plates were incubated at 37°C for 24 hours. After the incubation period, the inhibition zone was noticed encircling the wells and the diameter was measured in mm. As a positive control amoxicillin was used and DMSO and water as negative control (Khani et al., 2018; Zhao et al., 2011).

3.1 Soxhlet Extraction

After the completion of extraction process the sample was concentrated in rotatory evaporator leaving out a slight yield of extracted sample of about 3.2 g (Suryowati et al., 2015). The yield efficiency was calculated using the formula (1) and found out to be 64%.

$$Yield efficiency= \frac{Final weight of dried sample}{Initial weight taken for extraction} \times 100$$………………….. 1

3.1 Phytochemical screening for the leaf extract

The phytochemical screening was carried out for Coleus amboinicus leaf extract. The plant extract was screened for flavonoids, alkaloids, saponins, phenol, terpenoids, Glycosides, Steroids, and Tannins. The positive results were shown for flavonoids, Alkaloids, saponins, phenol and Terpenoids(Hullatti & Bhattacharjee, 2011).

Table 1

Phytochemical screening of *Coleus amboinicus* leaf extract
S.NO	Phytochemical Screening	Coleus amboinicus aqueous leaf extract
1	Flavanoids	++
2	Alkaloids	+
3	Saponins	+
4	Phenol	++
5	Terpenoids	+
6	Glycosides	-
7	Steroids	-
8	Tannins	++
9	Blank	-

+ Slightly present, ++ Moderately present

3.2 Synthesis and Characterization of caesium carbonate nanoparticles

In the present study synthesis of caesium carbonate nanoparticles by using Coleus amboinicus was carried out. While nanoparticle synthesis protocol was completed, the colour shift was detected, followed by turbidity check. The precipitate collected at the bottom of the beaker after 24 hours of incubation, indicating nanoparticle generation, and was examined using XRD, SEM, FTIR, and UV-VIS Spectrophotometer.

3.2.1 Ultra Violet – Visible Spectroscopy Analysis:

UV-Visible spectroscopy is used to determine the amount of UV-absorbing chemicals in a sample .It is used to elucidate the structure of organic compounds, as well as the presence or absence of unsaturation and heteroatoms. The absence or presence of a band at a specific wavelength is used as proof of the absence or existence of a specific group(Sgro et al., 2001). The presence of Caesium carbonate in the obtained sample is confirmed by the UV-VIS spectroscopy shown below. The absorbance peak for the caesium carbonate nanoparticle was between 200 to 800 nm. From the Fig. 2, the peak at 290nm and 490nm confirmed the presence of Caesium carbonate nanoparticles(Biradar et al., 2011).

3.2.2 Fourier Transform Infrared Spectroscopy Analysis:

Fourier Transform Infrared Spectroscopy is an important analytical technique used to identify compounds. This technique identifies the chemical compounds in a wide of capabilities. The chemical linkages and molecular structures of the biomolecule were identified using the FTIR results (Shimpi et al., 2015). As indicated below in Fig. 3, the FTIR spectra were recorded using an FTIR spectrometer. From the Fig. 3, the absorption band positioned at 3400 cm^− 1 attributed to -OH stretching due to the presence of phenol. The intense band at 2100 cm^− 1 corresponds to the stretching of -C-C and -C-O which is due to the presence of carboxylic acid group. And the band at 1600 and 1400 cm^− 1 arise from C = C and C-OH that are derived either from tannins or terpenes(Li & Mann, 2002).

3.2.3 SEM Analysis:

The structure of the caesium carbonate nanoparticle was found out to be irregular hexagonal in shape, as shown in the SEM image below(Fig. 4). It also assists in determining the particle size range, which is approximately 75-110nm. SEM analysis is used to investigate the morphology of caesium carbonate nanoparticles(Budhiraja et al., 2013).

3.2.4 XRD Analysis:

The use of X-Ray Diffraction Analysis to characterise crystalline materials is a strong non-destructive technique [15]. It is an effective method for determining the crystal structure of materials [28]. It is clear that Coleus amboinicus has recorded the presence of metal alkali caesium carbonate nanoparticle is confirmed by the XRD measurements shown below in Fig. 1.From the Fig. 5, nine prominent peaks were observed which corresponds to the following crystal coordinates such as (001), (011), (111), (012), (112), (200), (113), (202) and (220). Hence thus formed caesium carbonate nanoparticle was found out be multi-planar hexagonal crystalline structure(Ramyadevi et al., 2012).

3.5. Anti-Oxidant Assay

Anti-oxidant Assay was carried out for caesium carbonate nanoparticles (Cs₂CO₃) synthesized from Coleus amboinicus leaf extract.

3.5.1. Total Antioxidant Assay

The antioxidant characteristics of caesium carbonate nanoparticles was evaluated by total antioxidant assay. The biosynthesized Cs₂CO₃ nanoparticles showed a valuable antioxidant property. Ascorbic acid was used as standard and graph was plotted. From the standard graph ,the concentration of the unknown sample was 90.86 µg/ml (Fig. 6)(Dudonne et al., 2009).

Table 2

Total antioxidant Assay of Standard (Ascorbic Acid)
S.No	Concentration	Absorbance at 680nm
1	10	0.63
2	50	0.1
3	100	0.114
4	250	0.179
5	500	0.38

3.5.2 .DPPH Radical Scavenging Assay

The substantial antioxidant activity of caesium carbonate nanoparticles (Cs₂CO₃) was determined by DPPH assay having IC 50 value as 84.64 ( Table 4).Ascorbic acid was used as standard having IC 50 value 28.01 (Table 3). The synthesized nanoparticles showed efficient anti-oxidant activity. The in-vitro antioxidant study of the biosynthesized caesium carbonate nanoparticles (Cs₂CO₃) from Coleus amboinicus leaf extract has an effective antioxidant activity relating to DPPH radical scavenging(Jadid et al., 2017).

Table 3

DPPH Assay of Standard ( Ascorbic Acid)
S.No	Concentration	Absorbance at 517nm	Percentage Inhibition(%)	IC 50 value
1	10	0.159	43.27	28.01
2	50	0.402	56.06
3	100	0.389	57.48
4	250	0.299	67.32
5	500	0.121	86.77

Table 4

DPPH Assay of Biosynthesized caesium carbonate nanoparticles
S.No	Concentration(µg/ml)	Absorbance at 517nm	Percentage Inhibition (%)	IC 50 value
1	10	0.657	32.61	84.64
2	50	0.518	46.87
3	100	0.395	59.48
4	250	0.267	72.61
5	500	0.103	89.43

3.6.Anti-inflammatory Assay

Anti-inflammatory assay of biosynthesized caesium carbonate nanoparticles was determined by BSA Inhibitory Assay and Protease Inhibitory assay.

3.6.1. BSA Inhibitory Assay

BSA Inhibitory Assay was used to evaluate e the anti-inflammatory activity of Biosynthesized caesium carbonate nanoparticles. The percentage of inhibition of denaturation of protein was determined using the above-mentioned formula. The anti-inflammatory activity of the caesium carbonate nanoparticles from leaf extract of Coleus amboinicus with reference to percentage inhibition of protein denaturation exhibited that the synthesized nanoparticle was able to inhibit protein denaturation in a concentration reliant aspect (Fig. 7 and Table 5)(Matsuura et al., 2002).

Table 5

BSA Inhibitory Assay of Biosynthesized Cs2CO3 nanoparticles
S.NO	Concentration	OD at	Percentage Inhibition (%)
1	10	0.615	34.29
2	50	0.59	36.96
3	100	0.573	38.78
4	250	0.554	40.81
5	500	0.539	45.61

3.6.2 Protease Inhibitory Assay

Protease Inhibitory assay was used to determine the percentage of inhibition of protein denaturation by the biosynthesised Cs₂CO₃ nanoparticles. The synthesized nanoparticles showed an effective protein denaturation percentage. Based on the varying concentration the percentage of inhibition of protein by the nanoparticles had considerable effect (Rothan et al., 2014).The percentage inhibition of the synthesized nanoparticles was effective at 500 µg/ml ( Table 6 and Fig. 8)

Table 6

Protease Inhibitory Assay of Biosynthesized Cs₂CO₃nanoparticles
S.NO	Concentration	OD	Percentage Inhibition (%)
1	10	0.638	32.62
2	50	0.571	39.70
3	100	0.548	42.13
4	250	0.532	43.82
5	500	0.506	46.56

3.6.3 Antibacterial Assay

Well diffusion method was used to study the antibacterial activity of Cs₂CO₃ nanoparticles. The antibacterial property of Cs₂CO₃ nanoparticle was investigated by growing Gram positive Escherichia coli Bacteria and Gram-negative Bacteria Staphylococcus aureus with different concentrations of caesium carbonate nanoparticles [46]. Results thus acquired were tabulated below in Table 7 and Fig. 9.The zone of inhibition indicates utmost antibacterial activity of the test sample prepared. The inhibition zone of bacteria by Cs₂CO₃ nanoparticle from Coleus amboinicus leaf extract shows maximum inhibition for Staphylococcus aureus at 60 mg/ml, with 20 mm and minimum with 30 mg/ml(Mart\’\inez-Castañon et al., 2008).

Table 7

Anti-bacterial Assay of biosynthesized Cs₂CO₃ nanoparticles.
S.No	Cs₂CO₃ (mg/ml)	Zone of Inhibition Escherichia coli	Zone of Inhibition Staphylococcus aureus
1	30	2mm	5mm
2	60	20mm	22mm
3	90	6mm	8mm
4	Std 30	17mm	16mm

The nanoparticles are synthesized rapidly in physical and chemical methods. Nowadays, using biological approaches to synthesise nanoparticles is an essential part of biotechnology(Beyene et al., 2017). The current research is based on the biological production of Caesium carbonate nanoparticles, which reduces the need of chemicals and is thus environmentally beneficial. Caesium carbonate has recently been used in a range of medical applications, allowing for more efficient and effective Caesium carbonate nanoparticle manufacturing. The biosynthesized nanoparticles from Coleus amboinicus leaf extract exhibited distinct antioxidant activity. In Total Antioxidant Activity the concentration of the unknown sample was determined as 90.86 µg/ml and in DPPH radical scavenging assay the biosynthesized nanoparticles exhibited IC 50 value as of 84.64 and in anti-inflammatory activity the percentage of inhibition was about 40% for 500 µg/ml. The biosynthesized caesium nanoparticles have exhibited a significant anti-bacterial activity where the zone of inhibition was 17mm for staphylococcus aureus and 15mm for Escherichia coli. Hence Green synthesised Caesium chloride nanoparticle with high bioactivity can be taken into consideration for developing targeted and personalised bio therapeutics.

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No competing interests reported.

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Green synthesis, characterization and bioactivity evaluation of Caesium carbonate nanoparticle using Coleus amboinicus

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Chemicals

2.2 Soxhlet extraction

2.3 Phytochemical screening of the extract

2.4 Synthesis of caesium carbonate:

2.4 Characterization of synthesized nanoparticles:

2.4.1 UV-VIS

2.4.2 FTIR

2.4.3 SEM ANALYSIS

2.4.4 XRD

2.5 Bioactivity of biosynthesized caesium carbonate nanoparticles

2.5.1. Antioxidant Assay

2.5.1.1 Total Antioxidant Assay

2.5.1.2 DPPH Radical Scavenging Assay:

2.5.2.1 BSA Inhibition assay

2.5.2.1 Protease Inhibitory Assay

2.5.3. Antibacterial Assay

3. Result And Discussion

3.1 Soxhlet Extraction

3.1 Phytochemical screening for the leaf extract

3.2 Synthesis and Characterization of caesium carbonate nanoparticles

3.2.1 Ultra Violet – Visible Spectroscopy Analysis:

3.2.2 Fourier Transform Infrared Spectroscopy Analysis:

3.2.3 SEM Analysis:

3.2.4 XRD Analysis:

3.5. Anti-Oxidant Assay

3.5.1. Total Antioxidant Assay

3.5.2 .DPPH Radical Scavenging Assay

3.6.1. BSA Inhibitory Assay

3.6.2 Protease Inhibitory Assay

3.6.3 Antibacterial Assay

Conclusion

References

Competing Interests

Status:

Version 1