INTRODUCTION:

On the planet, numerous medicinal plant species are accessible for the wide range of remedial purposes. Some have their immediate impact against various pathogen in an environment. Approximately 1500 plant species utilized as a part of indigenous arrangement of pharmaceutical like Ayurveda, Unani and Siddha¹. In the preliminary antibacterial screening against several pathogenic microorganism, an aggregate of 82 Indian therapeutic plants are customarily utilized².

To treat the irresistible maladies, numerous more medications are utilized however tranquilize wellbeing is a basic worldwide issue.

But, there is a lower adverse impact to plant preparation in contrast with pharmaceuticals which had decreased cost and exceptionally powerful for both the devouring open and national social insurance establishments to take plant medications³. Catharanthus roseus has rich measure of alkaloids which had hypotensive, narcotic and have tranquilizing and hostile to destructive properties. It is additionally been utilized as a part of therapeutic reason like for diminishing muscle torment, dejection of the focal sensory system and wasps stings, instances of nose drain, draining gums, mouth ulcers, sore throats, inside for the treatment of the loss of memory, hypertension, cystitis, gastritis, enteritis, the runs, raised glucose levels⁴, in aversion of tumor⁵.

The therapeutic properties of Neem leaves, fruits, and bark has been utilized as a customary treatment in Ayurveda in India since from medieval period. Neem has pharmacological like hostile to viral, against contagious, against bacterial, against malarial, hostile to ripeness, against pyretic, calming and pain relieving, against ulcerogenic, against hypertensive and against hyperglycemic, neuropharmacological, hostile to dermatophytic, orodental insurance, hepatoprotective impact, immunostimulant, cell reinforcement and against genotoxic, hostile to growth, dangerous, pesticidal exercises. There are numerous more employments of Neem items like toiletry and pharmaceutical utilize, agronomic utilize, used as encourage for poultry, cattle additionally⁶.

A standout amongst the most encouraging and novel helpful specialists are the nanoparticles⁷. In the pathogenic microscopic organisms and parasites, the expanded rate of irresistible infections and the improvement of medication protection at high rate involves genuine concern. The microbial diseases which are related with the grimness and mortality is still stays high⁸. In this way, there is an interest for generation of new normal and inorganic antimicrobial particles of medications to diminish the proportion of microbial diseases. The inorganic antimicrobials, for example, silver and copper were utilized since long circumstances to treat microbial disease in current social insurance framework. As of late, the propelled uses of Nano sciences and nanotechnology are in mechanical solution and therapeutics engineered materials and nourishment bundling items⁹. Measurements of nanoparticles typically running from 1-100 nanometers (nm). They have exceptional properties from their mass proportionate. The properties are changes with diminish in the measurements of the materials to the nuclear level. The one of a kind properties, for example, physical-concoction, optical and organic reasonable for suitable applications. Nanoparticles additionally have the restorative application due to their nanoscale and to their natural functionalization¹⁰. In both in vivo and in vitro biomedical research, vital applications in focused medication conveyance, imaging, detecting and fake inserts¹¹.

These days, green blend technique is utilized for the adjustment of nanoparticles utilizing plant. By and large; by utilizing polymer scattering technique, numerous quantities of nanoparticles are readied. For the arrangement of copper nanoparticles, number of procedures can be utilized which are warm lessening¹³, a topping specialist strategy¹⁴, sono compound decrease¹⁵, metal vapor combination¹⁶, smaller scale emulsion strategies¹⁷, laser light¹⁸, and initiated radiation¹⁹.

In the amalgamation of copper nanoparticles, plant concentrates may act both as diminishing specialists and settling operators²⁰. As indicated by World Health Organization (WHO), 80% of the world's occupants rely upon conventional solution for their health insurance and it is included the utilization of plant extracts and their dynamic compounds²¹. Our body deliver more reactive oxygen species (ROS-e.g., superoxide anion radicals, radicals and hydrogen peroxide) under stress condition than enzymatic antioxidants (e.g., superoxide dismutase-SOD, glutathione peroxidase-GPx, and catalase) and non-enzymatic antioxidants (e.g., ascorbic corrosive vitamin C, α-tocopherol-vitamin E, glutathione, carotenoids and flavonoids), which causes cell harm^22-24 and medical issues^25,26.

Deficiency of antioxidants extinguish the reactive free radicals, facilitates the development of degenerative diseases and inflammatory diseases. This problem solved by addition of the diet with antioxidant compounds that are control in natural plant sources²⁷. These natural antioxidants are served as a type of preventive medicine.

In the present study, the two medicinal plants C. roseus and A. indica were experimented to check the antibacterial and antioxidant activities against various pathogenic microorganism through copper nanoparticles which was synthesized by using ethanolic leaves extracts of these two plants.

MATERIALS AND METHODS:

Plant collection:

Fresh leaves of C. roseus and A. indica are collected from the Botanical garden of M B Patel science college, Anand (22.5528° N, 72.9625° E) which are authenticated by Dr. M. A. Patel, Senior Scientist (Agriculture University, Anand). Leaves are cleaned under tap water and then with distilled water and then air dried to it. Now, dried plant material was weighed about 25gm of leaves using electronic weighing balance (Mettler Toledo).

Crude Extract preparation²⁸:

25gm of leaves samples were suspended in screw cap vials with 75ml solvent (ethanol) with a ratio of 1:3 w/v.The suspensions were put in a microwave oven for 30 seconds. The suspensions were not allowed to boil; therefore, after 30 seconds they were cooled to room temperature for a few minutes. The above step was repeated up to seven times in order to obtain the maximum yield of the extracted compounds. The extract should be prepared fresh.

Synthesis of Copper Nanoparticles²⁹:

For the synthesis of copper nanoparticles and for the reduction of copper ions, 5ml of plant leaves extract was mixed with 5ml freshly prepared 0.001M aqueous solution of CuSO₄. It was then kept for incubation for 1h. The change in color was noted. Thus color change indicates reduction and reduced copper nanoparticles were obtained.

Characterization of copper nanoparticles:

pH Analysis²⁹:

The pH was determined by digital pH meter. The pH of the reduced solution with nanoparticles synthesized was noted.

UV-Visible spectra analysis²⁹:

The reduction of pure copper to nanoparticle was monitored by measuring the UV-Vis spectrum, the most confirmatory tool for the detection of Surface Plasmon Resonance property (SPR) of CuNPs, by diluting a small aliquot of the sample in distilled water. UV-Vis spectra analysis was done by using UV-Vis spectrophotometer, Systronics at the range of 350-600nm.

Fourier Transform Infra-Red Spectroscopy (FTIR)³⁰:

FTIR measurements were carried out using FTIR spectroscopy (Perkin-Elmer FTIR apparatus), the range from 4000cm^-1 to 400cm^-1. After complete reduction of CuSO₄ ions by C .roseus and A. indica leaf extracts, 1-2μl drop of this nanoparticle mixture was used for FTIR analysis in transmittance mode.

Cultures used for Anti-microbial testing:

The microorganisms used were as follows: The antibacterial activity has been checked against bacteria like, Escherichia coli, Proteus vulgaris and Staphylococcus aureus.

Antibacterial assay³¹:

Antibacterial assay is performed to check the antibacterial activity of the plant extract and copper nanoparticles against the gram positive and gram negative organisms i.e., E. coli, P. vulgaris, S. aureus. The cultures were maintained on the nutrient agar slant. The nutrient agar is poured into the petri-plates and allowed to solidify. Thereafter it was kept in the incubator. Next day, the bacterial culture was spread on N-agar plates and properly labelled with their respective microorganisms and incubated in inverted position at 37°C for 24hrs for the growth of bacteria. Furthermore, 4 wells in each plate were made with sterile cup borer. The different concentrations of sample were poured in each well and incubated further to check the antibacterial activity against the microorganisms. Next day, observe and measure the clear zone of inhibition was observed and measured.

Antioxidant assay:

DPPH (1, 1-diphenyl-2-picrylhydrazine) assay³²:

The 1, 1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging assay was first described by Blois in 1958 and was later modified slightly by numerous researchers. It is one of the most extensively used antioxidant assays for plant samples. DPPH is a stable free radical that reacts with compounds that can donate a hydrogen atom. This method is based on the scavenging of DPPH through the addition of a radical species or an antioxidant that decolorizes the DPPH solution. The antioxidant activity is then measured by the decrease in absorption at 517nm.In this method, a 0.1mM solution of DPPH in methanol was prepared, and 4ml of this solution were added to 1ml of the sample solution (plant extract) in ethanol at varying concentrations of 200, 400, 600, 800 and 1000 μl. After 30 minutes of incubation, the absorbance was measured at 517nm. A large decrease in the absorbance of the reaction mixture indicates significant free radical scavenging activity of the compound. Gallic acid was used as a standard.

The percentage inhibition of DPPH scavenging activity can be find out by the following formula,

% inhibition of DPPH scavenging activity= [(Control O.D-Sample O.D) /Control O.D] *100

Where,

Control O.D: Absorbance of control DPPH at 517nm

Sample O.D: Absorbance of extract with DPPH

FRAP (Ferric Reducing Antioxidant Property)³³:

The FRAP reagent was made [acetate buffer (pH 3.6): 10Mm TPTZ solution in 40mM HCl: 20mM iron (III) chloride solution] in proportions of 10:1:1 (v/v). The freshly prepared FRAP reagent was warmed to 37°C in a water bath. Different concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 ml) of sample extract were added to 1.5ml of the FRAP reagent. The absorbance of the reaction mixture were then recorded at 593nm after 10min of incubation period. The standard curve was constructed using Ascorbic acid solution as standard.

Reducing power determination³²:

The sample in 1ml of ethanol is mixed with a phosphate buffer (5ml, 0.2M, pH 6.6) and potassium ferricyanide (5ml, 1%), and mixture was incubated at 50°C for 20minutes. Next, 5ml of trichloroacetic acid (10%) was added to the reaction mixture and centrifuged at 3000 rpm for 10minutes. The upper layer of the solution (5ml) was mixed thoroughly with distilled water (5ml) and ferric chloride (1ml, 1%), and later the absorbance was measured at 700nm.

A stronger absorbance indicates increased reducing power.

Hydroxyl radical scavenging activity³⁴:

The scavenging activity for hydroxyl radicals was measured with Fenton reaction. The reaction mixture comprised of 60μl of 1.0mM FeCl₂, 90μl of 1mM 1, 10-phenanthroline, 2.4ml of 0.2M phosphate buffer (pH 7.8), and 150μl of 0.17M H₂O₂, and 0.1ml of extract at various concentration of 200, 400, 600, 800, 1000 μl. The reaction was started by adding H₂O₂. After incubation at room temperature for 5min, the absorbance of the mixture at 560nm was measured with UV visible spectrophotometer.

The percentage inhibition of hydroxyl scavenging activity was calculated using the following formula,

% Inhibition of Hydroxyl scavenging activity=

[1- Absorbance (Test) / Absorbance (Blank)]*100

Where,

Absorbance (Test): Absorbance of the test (with extract)

Absorbance (Blank): Absorbance of the control (without extract)

Ferrous ion chelating activity³⁵:

The ability of extracts to chelate ferrous ions was estimated using this method. Different concentrations of the sample ranging from 200-1000μl were added to a solution of 2mM Ferric chloride (0.05ml). The reaction was initiated by the addition of 5mM ferrozine (0.2ml) and the volume of the mixture was finally adjusted to 4ml with methanol, shaken vigorously and left standing at room temperature for 10minutes. After incubation, the absorbance of the solution was measured spectrophotometrically at 562nm.

Ascorbic acid used as a positive control.

The percentage of inhibition was calculated using the following formula:

% Inhibition= [1-(As/Ac)]*100

Where,

Ac= the absorbance of the control containing ferric chloride and ferrozine only

As= the absorbance in the presence of extract

RESULTS AND DISCUSSION:

Characterization of Copper nanoparticles:

pH Analysis:

The pH of the reduced solution with copper nanoparticles using plant extract of C. roseus and A. indica were 4.10 and 3.58 respectively. These present obtained pH of copper nanoparticles from both plant extracts were higher than the reported pH of previous study of copper nanoparticles synthesized from ginger extract²⁹. The capping has found to be done between CuSO₄ and these leaf extracts.

UV-Visible spectra analysis:

Formation of copper nanoparticles in plant extract was confirmed by using UV-Visible spectral analysis. Results showed that the reduction of copper ions and formation of copper nanoparticles was completed after 1hr incubation at room temperature. The formation of light green color from dark green indicated the reduction of copper ions in both plant extracts (Figure-1 and 2). Absorption spectra of copper nanoparticles formed in the solution showed absorbance peak at around 400nm for the both plant extract (Figure-3 and 4). But in the previous study, absorption maxima of copper nanoparticles is at 570nm²⁹ which indicated as the copper nanoparticles were formed but in slightly lower amount.

Figure 1- Color changes after 1hr Incubation from addition of 0.1M Copper sulphate solution in C.roseus leaves extract

Figure 2Color changes after 1hr Incubation from addition of 0.1M Copper sulphate solution in A. indica leaves extract

Fourier Transform Infra-Red Spectroscopy (FTIR):

FTIR has become apparent as a powerful tool for understanding the involvement of functional and biological groups present in the C. roseus and A.indica leaf extract responsible for the reduction of copper ions and also the capping reagent responsible for the stability of the bio reduced copper nanoparticles³⁰. FTIR spectra of C. roseus and A. indica leaves extract derived nanoparticles shown in fig. 5 and fig. 6 respectively.

Figure 3 The absorption maxima of copper nanoparticles synthesized from C.roseus at 400nm

Figure 4 The absorption maxima of copper nanoparticles synthesized from A.indica at 400nm

The FTIR spectra for extract of Catharanthus roseus leaves produced vibration bands in between range of 3000-3500 cm^-1at 3329cm^-1which may be due to overlapping of O-H and amine N-H stretching bands is near by the range 3358 cm^-1 as supported in previous study³⁶.The peak at 2981.15 cm^-1 indicates aliphatic C-H stretching which is mostly at 2878 cm^-1.The peak at 1639.08 cm^-1 indicated the N-H banding and C-H banding which was nearly at 1658 cm^-1. The peak at 1044.13 cm^-1 indicated the C-O stretching and 1085.43 cm^-1 symbolized N-H bending which confirms capping of copper nanoparticles surface by the N-H group of polymer.

Table 1 Zone of inhibition of antibacterial activity of ethanolic extract of Catharanthus roseus

Bacterial strains

Escherichia coli

Staphylococcus aureus

Proteus vulgaris

Concentration

50µl

100µl

150µl

200µl

50µl

100µl

150µl

200µl

50µl

100µl

150µl

200µl

Ethanol

1.2cm

1.4cm

1.1cm

1.2cm

3.0cm

1.6cm

1.5cm

1.3cm

1.2cm

CuSO₄

2.1cm

1.3cm

1.4cm

2.4cm

1.9cm

1.7cm

2.7cm

1.2cm

2.8cm

2.0cm

2.2cm

CuNP

2.8cm

1.5cm

1.2cm

1.3cm

2.7cm

1.6cm

1.2cm

2.0cm

1.7cm

2.4cm

2.0cm

2.1cm

Extract

1.2cm

1.7cm

2.2cm

1.6cm

1.8cm

Table 2 Zone of inhibition of antibacterial activity of ethanolic extract of Azadirachta indica

Bacterial strains

Escherichia coli

Staphylococcus aureus

Proteus vulgaris

Concentration

50µl

100µl

150µl

200µl

50µl

100µl

150µl

200µl

50µl

100µl

150µl

200µl

Ethanol

1.4cm

1.9cm

1.2cm

1.4cm

1.2cm

2.5cm

1.3cm

1.2cm

1.3cm

1.2cm

CuSO₄

1.8cm

2.7cm

1.8cm

1.3cm

1.8cm

2.8cm

3.0cm

2.0cm

1.8cm

2.0cm

CuNP

2.2cm

3.1cm

1.7cm

2.2cm

2.0cm

1.4cm

3.0cm

2.4cm

2.5cm

2.2cm

Extract

2.0cm

2.3cm

1.8cm

2.3cm

1.7cm

1.8cm

1.5cm

1.7cm

1.6cm

Figure 7Antibacterial activities of copper nanoparticles synthesized by using C. roseus leaf extract; A. E. coli (50µl), A1. E. coli (100µl), A2. E. coli (150µl), A3. E. coli (200µl); B. S. aureus (50µl), B1. S. aureus (100µl), B2. S. aureus (150µl), B3. S. aureus (200µl); C. P. vulgaris (50µl), C1. P. vulgaris (100µl), C2. P. vulgaris (150µl), C3. P. vulgaris (200µl)

Antioxidant assay:

DPPH activity:

The DPPH radical scavenging activity of the ethanolic leaf extract was found to be 32.5% and 78.9% sample of C. roseus and A. indica leaf extract respectively. In both the plant extracts, 1 ml of extract was effective DPPH activity (Table 3). The free radicals gets deactivate and forms nonreactive species, once receiving the hydrogen ion and this is done by the DPPH radical. The antioxidant compounds reduce the purple colored DPPH radical to yellow. The absorbance decreased at 517nm which makes the confirmation of the reduction capability of DPPH radicals induced by the antioxidant⁴³(Wang et al. 2008). Due to the medicinal properties alongside exclusive secondary metabolites of these plant extracts makes it more radical scavenging active.

Table 3 DPPH activity of C. roseus and A. indica extracts

Concentration of extract	Absorbance at 517nm
	Gallic acid	*C. roseus* extract	*A. indica* extract
0.2	0.040	3.312	4.321
0.4	0.042	3.76	3.872
0.6	0.6	4.5	3.381
0.8	0.8	4.12	4.04
1.0	1.0	4.47	4.08

FRAP (Ferric Reducing Antioxidant Property):

The FRAP is versatile and can be readily applied to alcohol extracts of plants. The reduction of ferric (III) iron to ferrous (II) iron is the trade mark of this assay and the highest antioxidant activity was found in the concentration of 0.6 ml and 0.2 ml extracts of C.roseus and A.indica respectively (Table 4). Therefore, the ability to reduce ferric (III) iron to ferrous (II) iron was highest in these concentrations ofC.roseus and A.indica respectively. However, the other concentrations did also effectively show the antioxidant activities in both the plant extracts. This suggests that the active principles does have the potential to reduce the effect of oxidative stress faced by the plant. The FRAP antioxidant capacity was also highest in C. roseus as depicted by Moon et al. (2018)⁴⁴.

Table 4 FRAP activity of C. roseus and A. indica extracts

Concentration of extract	Absorbance at 593nm
	Gallic acid	*C. roseus* extract	*A. indica* extract
0.2	2,883	4.217	4.324
0.4	3.55	3.91	3.906
0.6	3.118	4.6	3.927
0.8	3.35	4.295	4.084
1.0	3.211	4.441	4.081

Reducing power determination:

Reducing power of C .roseus and A. indica leaf extracts increased with increase in concentration. The ethanolic extract showed more effective reductivity. The reduction in Fe (III) is considered as the ultimate indicator of electron donating activity which involves in the mechanism of antioxidant action of phenolic content⁴⁵ and is associated with the presence of reductones which exhibits its antioxidant action by breaking the radical chain by donating hydrogen atom⁴⁶s. Here, the ethanolic extracts showed highest activity in a dose dependent manner which is due to the presence of these reductones (Table 5).

Table 5 Reducing power activity of C. roseus and A. indica extracts

Concentration of extract	Absorbance at 700 nm
	Ascorbic acid	*C. roseus* extract	*A. indica* extract
0.2	3.764	0.758	0.645
0.4	7.099	1.559	1.688
0.6	8.787	2.14	1.696
0.8	9.226	2.627	2.018
1.0	9.721	5.86	3.414

Hydroxyl Radical Scavenging Activity:

At a concentration of 0.2 ml, the scavenging activity of ethanolic extract in extracts of C. roseus was found to be 94.32% (Table-6). But, above the concentration of 0.2 ml the percentage reduced drastically. This ethanolic extract was found to be more effective in quenching the hydroxyl radicals produced in the reaction mixture. The hydroxyl radical can induce oxidative damage to DNA, lipids and proteins³⁵. The hydroxyl radical scavenging is to scavenge or protect the deoxy ribose from the hydroxyl radical. In this assay, 2-deoxy-2-ribose was oxidized when exposed to hydroxyl radicals generated by Fenton-type reaction³³. Likewise the C. roseus, the extracts of A. indica at a concentration of 0.2 ml, the scavenging activity of ethanolic extract was found to be 89.74% (Table-6). This ethanolic extract was found to be more effective in quenching the hydroxyl radicals produced in the reaction mixture.

Table 6 Hydroxyl radical scavenging activity of C. roseus and A. indica extracts

Concentration of extract	Absorbance at 560 nm
	Ascorbic acid	*C. roseus* extract (% inhibition)	*A. indica* extract (% inhibition)
0.2	3.71	0.214 (94.32)	0.117 (89.74)
0.4	7.07	0.452 (52.65)	0.325 (64.0)
0.6	8.72	0.651 (30.56)	0.411 (20.92)
0.8	9.06	0.921 (29.31)	0.512 (19.72)
1.0	9.1	1.016 (9.35)	0.765 (33.07)

Ferrous Ion Chelating Activity:

The results showed that the ethanolic extract of C. roseus at 0.8ml concentration tends to have an effective capacity for ion binding with an inhibition of 33.8% (Table-7). The ferrous ion chelating activity by C. roseus was estimated using ferrozine. The chelating effects of C. roseus leaf extract on ferrous ions increased with increasing concentrations. But after 0.8ml concentration it started decreasing. In case of the ethanolic extract of A. indica at 0.4ml concentration produced an effective ability for ion binding with an inhibition of 98.1% (Table-7).Unlike that of C. roseus, after the concentration of 0.4 ml of A. indica extract, the values of ferrous ion chelating activity gradually decreased.

Table 7 Ferrous ion chelating activity of C. roseus and A. indica extracts

Concentration of extract	Absorbance at 562 nm
	Ascorbic Acid	*C. roseus* extract (% inhibition)	*A. indica* extract (% inhibition)
0.2	3.52	1.225 (19.6)	1.271 (16.6)
0.4	6.53	1.317 (13.6)	0.029 (98.1)
0.6	7.21	1.115 (26.8)	1.001 (34.3)
0.8	8.71	1.009 (33.8)	1.495 (29)
1.0	9.28	1.120 (26.5)	1.159 (24)

CONCLUSION:

In conclusion, the synthesis of copper nanoparticles from leaves of two medicinal plants i.e. C. roseus and A. indica was demonstrated. The pH, spectrophotometric and FTIR analysis confirmed the formation of copper nanoparticle. Even the lowest concentration of 50 µl concentration of CuNP prepared from the leaves extract of C. roseus could create a 2.8cm and 2.7 cm zone of inhibition against E. coli and S. aureus respectively. However, 100 µl concentration of CuNP required to obtain a zone of inhibition of 2.4 cm against P. vulgaris. But, in case of A. indica, the 100 µl CuNP could able to inhibit the growth of E. coli, S. aureus and P vulgaris and produced 3.1 cm, 3.0 cm and 2.5 cm zone of inhibition respectively. This copper nanoparticle can able to create these zone of inhibition might be due to the active principles found in these two plants. Moreover, the antioxidant activities also justified the above statement. Thus, this study finds an avenue for the use of this copper nanoparticles for the betterment of ecological medicinal purposes.

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Received on 29.04.2018 Modified on 29.05.2018

Asian J. Res. Pharm. Sci. 2018; 8(2):81-90.

DOI: 10.5958/2231-5659.2018.00016.4