1. INTRODUCTION:

The field of nano is one of emerging technologies in the recent time, pledges quantum leaps not only in materials developed and circuit engineering parts although provides a lot of operations in diagnosis, drugs, energy, biotechnology and safety. The special properties produced by nanoparticles are due to the large in surface to volume ratio which influences the mechanical, thermal and catalytic nature of nanomaterials. The use of plant extracts, microorganisms are harmless, unswerving, inexpensive and environmental friendly approach to synthesize nanoparticles.

¹The role of pharmacological properties of nanoparticles has become one of the significant basic studies in biomedical sciences in recent years ². Distinguish methods are used in the synthesis of Nps including physical, chemical methods have been developed, but biosynthesis has been represented as environmentally friendly synthesis of Nps, whereas other methods have number of challenges. Most of the plants have inherent capacity of reducing the metallic solution into metal Nps, though some plants do have higher capacity than others, due to presence of more amount of unique phytochemicals, which acts an excellent role in the bioreduction and consequently formation of Nps ^3-6. Recently, ZnO Nps have gained significant interest in the cancer treatment ⁷, antibacterial, antifungal ⁸, and antioxidant activities due to their potential pharmacological properties ⁹. Zinc oxide nano-particles have diverse physical and chemical properties compared to the bulk material ¹⁰. The induced catalytic performance of nano-sized ZnO with reference to the commercial analog in catalysis could be the presence of more surface area and causes changes in surface properties. ZnO NPs are used in the making PV cells, photo catalyst, and photoluminescence sensors ¹¹. They also have power over bacterial activity against broad spectrum microbes ¹² and also have capability to absorb UV radiations, microwaves, infrared and radiofrequencies which enables them to be used in ointments and cosmetic creams. Synthesis of ZnO/Ag bimetallic NPs have been performed using chemical and physical routes i.e. reduction by hydrazine hydrate¹³method ; solvothermal method ¹⁴: ultrasonic irradiation ¹⁵. However the synthesis of bimetallic NPs through biogenic approach is lacking. So synthesis of ZnO NPs via green synthesis i.e. utilization of Sphagneticola trilobata root extract as the reducing agent for bioreduction of Zinc acetate into ZnO nanoparticles was designed in the present study. Furthermore, synthesized NPs were characterized and tested for a series of pharmacological assays i.e. anti-oxidant, anti-inflammatory, anti-diabetic assays, and further study focused on chemical remediation of heavy metals and ZnO NPs as plant growth stimulants. The study explores new insight into applications effect of zinc and in nanoparticle configuration.

2. MATERIAL AND METHODS:

2.1 Collection of Plant Material:

Fresh plant parts of Sphagneticola trilobata was collected in agriculture fields, Guntur, Andhra Pradesh, India. Collected plant parts were cleaned with water absorbent paper (wet filter paper). Then it was cut into small pieces and was shade dried. The dried plant material was powdered and the plant powder was preserved.

2.2 Preparation of aqueous leaf, stem and root extracts Sphagneticola trilobata

Ten grams of leaves, stem and root powder was mixed with 100 ml of Milli- Q water and kept in water bath at 60°C for 20 mints. Then the extracts were cooled to room temperature and filtered using Whatmann No. 1 filter paper. The extract was stored in a refrigerator for further studies.

2.3 Green Synthesis of Zinc Oxide nano particles:

The plant parts i.e leaves, stem and root were studied separately for the synthesis of Zinc nano particles. The Zinc Oxide nanoparticles were prepared by using a 0.1M Zinc acetate dehydrates solution slowly mixed with 25 ml of the leaf, stem and root extracts in a magnetic stirrer at 60 °C for 2 h. Then, the yellow colour appeared and allowed to settle for 8 hrs. The particles were separated by centrifugation at 6000 rpm for 20 min and washed with distilled water followed by methanol to remove the unwanted impurities. Finally, powder of Zinc Oxide nanoparticles were acquired after overnight drying the purified precipitant at 80 °C in a hot air oven used for characterization and biological activities.

2.4 Characterization of nanoparticles

The crystalline structure of nanoparticles was characterized through X-Ray diffraction patterns by X-Ray Diffract meter at a scanning rate of 2 u angles/ min at 40 kV and 30 mA current. The scanning range was kept between 10⁰and 80⁰using Nickel monochromatic Cu Kα radiation (u = 1.5406 Å), NaI detector, variable slits and 0.02 scan step size. The crystallite size (D) was calculated using the Debye–Scherrer equation. Scanning electron microscopy (SEM) was performed for morphological characterization and energy dispersive spectroscopy (EDS) for analysis of elemental composition of metal nanoparticles. Fourier transform infrared spectroscopy (FTIR) for charactering the surface chemistry and organic functional groups Bruker (Germany) with model ALPHA.

2.5 Pharmacological studies

2.5.1 Study of antioxidant capacity

2.5.1.1 ABTS radical activity test

The ABTS method used was based on ¹⁶ with slight modifications equal volumes of stock solutions of ABTS⁺ (7 mM) and potassium persulphate (2.45 mM) were mixed and allowed to react for 12–16 h in the dark at room temperature to generate the free radical. Prior to use, this solution was diluted with 60% ethanol to get an absorbance of 0.700 ± 0.020 at 734 nm. An aliquot (5–50µg/ml) of nano particle sample solution was mixed with 1 ml of the working ABTS+ solution and the absorbance was monitored at 734 nm for 6 min using Double Bean UV Visible Spectrophotometer (TECHCOMP–UV 2301, HITACHI 2.2). The measurement was taken in triplicate. % Inhibition = [(AB − AA)/AB] × 100 (Where AB – absorption of blank sample; AA – absorption of tested sample solution).

2.5.1.2 DPPH Inhibition Activity

To assess the scavenging ability on DPPH, each extract (20-200 µg/ml) in water and ethanol was mixed with 1 ml of methanolic solution containing DPPH radicals (0.2 mM). The mixture was shaken strongly and left to stand for 30 min in the dark before measuring the absorbance at 517 nm against a blank. Then the scavenging ability was calculated using the following equation:

% inhibition = (A_blank – A_sample) / A_blank × 100

Where A_blank is the absorbance of the control reaction (containing all reagents except the test compound) and A_sample is the absorbance of the test compound ¹⁷.

2.5.1.3 Hydroxyl radical scavenging activity

According to the scavenging activity of the extract on hydroxyl radical was measured according to a previously described method. In 1.5 mL of each diluted extract, 60 μL of FeCl₃ (1 mmol/L), 90 μL of 1, 10-phenanthroline (1 mmol/L), 2.4 mL of 0.2 mol/L phosphate buffer, pH 7.8 and 150 μL of H₂O₂ (0.17 mol/L) were added respectively. The mixture was then homogenized and incubated at room temperature for 5 min. The absorbance was read at 560 nm against the blank. The percentage of the hydroxyl radical scavenging activity of each extract was calculated from the equation below:

Percentage of hydroxyl radical scavenging activity=[(OD control-OD sample)/OD control] × 100 Where OD is the optical density.

2.5.2 α-Amylase inhibition Activity:

With reference to ¹⁸ the in vitro α-amylase inhibition activity of root extract was determined based on the spectrophotometric assay using Acarbose as the reference compound. The assays were conducted by mixing 80 μL of fixed concentration of synthesized nanoparticles, 20 μL of α-amylase in 40 mmol/L phosphate buffer and 1 mL of 2-Chloro-4-Nitrophenyl-α-D-Maltotrioside (1.0 mg/mL) and incubated at 37 °C for 5 min. The absorbance was measured at 405 nm spectrophotometrically. Correspondingly, a control reaction was carry out without the plant extract. Percentage inhibition was calculated by the expression;

Percentage inhibition = [(Absorbance _Control-Absorbance _Test)/Absorbance _Control] × 100.

2.5.3 Anti-inflammatory activity

2 mL of 1% Bovine serum albumin (BSA) was mixed with 400μL of synthesized nano particles in different concentrations (25-150μg/mL) and the pH of the reaction mixture was adjusted to 6.8 using 1N HCl ¹⁹. The reaction mixture was incubated at room temperature for 20 min and then heated to 55 °C for 20 min in a water bath. The mixture was cooled to room temperature and the absorbance value was recorded at 660 nm. A BSA mixture with 30% methanol solution was used as a control. Diclofenac sodium in different concentrations was used as a standard. The experiment was performed in triplicate. Percentage of inhibition was calculated using the following formula.

% Inhibition =

Control O.D = Optical density of control

Sample O.D = Optical density of test sample

3. RESULTS AND DISCUSSIONS:

3.1 Zno synthesis

ZnO nanoparticles were synthesized by one step approach at room temperature using aqueous leaf stem and root extracts of Sphagneticola trilobata. The reaction mixture of root extract for ZnO nanoparticles exhibited dark yellow color while for leaf and stem showed comparatively less color than root. The current primary phytochemicals investigation of aqueous extract from Sphagneticola trilobata revealed that the root extract have more potential phytochemicals which are involved in formation of ZnO nano paricles from zinc acetate, Table 1and Figure.1. The color changed by metallic nanoparticles is due to the coherent excitation of all “free” electrons which are released by the phenolic compounds present in the root extracts. The reducing capacity depends on the amount of hydrolysable tannins, polyphenols and flavonoids present in the plant extracts. A lot of literature has been reported to till date on biological synthesis of nano particles using microorganisms including bacteria, fungi and plants; because of their antioxidant or reducing properties usually responsible for the reduction of metal compounds in their respective nanoparticles. The use of plant extracts for this purpose is potentially advantageous over microorganisms due to the ease of improvement, the less biohazard and elaborate process of maintaining cell cultures ²⁰.

Table 1: Zno synthesis from the extracts of Sphagneticola trilobata from zinc acetate

S. No	Plant part	Amount synthesized
1	Leaves	21.3 mg
2	Stem	15.8 mg
3	Root	24.7 mg

Figure 1: Zno synthesis from the extracts of Sphagneticola trilobata from zinc acetate

3.2 Characterization of nanoparticles

The characterization of nanoparticles is usually done based on their shape, size, surface area and dispersion. Plant mediated synthesis of nanoparticles has now a days become popular because these biosynthesized nanoparticles, which are uniform and stable in nature. Thus, it could be used for its ample applications in various fields ^21-22. The FT IR spectra of synthesized zinc nano particles confirm the functional groups involved in the bio synthesis of Zinc nano particles. The IR frequencies observed at a wave number of 3144 cm^-1, 1665 cm^-1 and 1640 cm^-1 represents the C=O stretching, N-H stretch and N-H bending vibrations in the amide and amine groups. This confirms that the nitro compounds in the root extract of Sphagneticola trilobata are actively participated in the formation nano particles. The FT IR spectra of the synthesized nano particles were given in Figure 2. SEM images confirmed that zinc nanoparticles are in nano range where the size of nanoparticle was found in the range of 65-80 nm. The shape of the nanoparticle was found ir regular and complex. SEM images are shown in Figure 3. EDS results in Figure 4 clearly indicate the presence of zinc approximately 41.4% in the synthesized nanomaterial. Thus proves the formation of zinc and phytoconstituents from the plant root extracts are attached together and formed into complex mixture with irregular zinc nanopartilces. The crystalline nature of the synthesized nano particles was confirmed by XRD analysis in Figure 5. The sharp diffraction peak emerges at 2θ angles of green synthesized NP, corresponds to 111, 200, 221 and 311 crystal planes was observed and the nano particles are in face centered cubic structure and the 40 % of zinc of metal content was observed in the nano particles.

Figure 2: FT IR spectrum of Zinc Nano particles

Figure 3: SEM analysis results

Figure 4: EDS analysis results

Figure 5: XRD analysis results

3.3 Pharmacological studies

3.3.1 Study of antioxidant capacity

Antioxidant agents including enzymatic and non-enzymatic substances regulate the free radical formation. Free radicals are causing cellular damage including brain damage, atherosclerosis and cancer. The free radicals are generated by reactiveoxygen species (ROS) such as superoxide dismutase, hydrogen peroxides and hydrogen radicals. Biomolecules such as proteins, glycoprotein, lipids, fatty acids, phenolics, flavonoids and sugars strongly controlled the free radical formation. The scavenging power of enzymatic and non-enzymatic antioxidants is useful for the management of various chronic diseases such as diabetes, cancer, AIDS, nephritis, metabolic disorders and neuro degenerative. Biosynthesized ZnO Nps was investigated for their antioxidant capacity through ABTS radical activity test, DPPH Inhibition Activity test and Hydroxyl radical scavenging activity followed by anti-inflammatory and anti-diabetic activities.

3.3.1.1 ABTS radical activity test

In the present investigation, the commonly accepted assay ABTS was used for the evaluation of antioxidant activity of plant root extracts the results of these analyses are given in Table 2. The relative antioxidant ability to scavenge the radical ABTS+ has been studied for ascorbic acid, root based Zn Nps and aqueous root extracts and the antioxidant power was measured by studying decolorization. The ABTS values for root based Nps ranged from. 4.79 to 86.88 %, the results are comparable to the ascorbic acid and for aqueous root extracts and IC50 values were calculated. From the results it is to be noted that Root based ZnNPs have IC50 value of 27.60 µg/ml which is nearer to the value of Ascorbic acid i.e 25.48 and the IC50 value of Aqueous Root extract is 41.76. The antioxidant effect of Zno nanoparticles was stronger than other synthetic commercial standard used. The ABTS radical cation decolorization assay can measure the relative antioxidant ability to scavenge the radical ABTS as compared with BHT, and is an excellent tool for determining the antioxidant capacity of hydrogen-donating antioxidants. The blue and green ABTS radical cation be generated prior to adding up antioxidant containing samples prevents interference, which stable absorbance was achieved, by adding the ethanolic extract of Sphagneticola trilobata and the scavenging ability measured in terms of discolorization ²³

Table 2: ABTS radical activity test of Root extract

S No	Concentration in µM	Ascorbic acid		Root based ZnNPs		Aqueous Root extract
S No	Concentration in µM	Absorbance	% ABTS Activity	Absorbance	% ABTS Activity	Absorbance	% ABTS Activity
1	5	0.658	7.19	0.675	4.79	0.695	1.97
2	10	0.593	16.36	0.602	15.09	0.651	8.18
3	15	0.501	29.33	0.534	24.68	0.603	14.95
4	20	0.422	40.48	0.458	35.40	0.548	22.71
5	25	0.319	55.01	0.369	47.95	0.499	29.62
6	30	0.265	62.62	0.298	57.97	0.439	38.08
7	40	0.132	81.38	0.157	77.85	0.372	47.53
8	50	0.068	90.41	0.093	86.88	0.294	58.53

3.3.1.2 DPPH Inhibition Activity

An examination of Table 3 reveals that the total antioxidant activity measured by DPPH method, ranged from 3.63 to 86.64 µg/ml. which is comparable to standard ascorbic acid. The results from the antioxidant assay showed that root extract of plant can scavenge the radical to a certain extent. The free radical scavenging activity of ethanolic extract of Sphagneticola trilobata and also that of ascorbic acid was evaluated through its ability to quench the synthetic DPPH radical. There are many methods for evaluating the antioxidant activity of both natural and artificial compounds. The DPPH analyze constitutes a fast and low cost method that has regularly been used for evaluation of the antioxidative potential of various natural products. ²⁴Therefore, in the present study, Sphagneticola trilobata was screened for its possible antioxidant and radical scavenging activity by DPPH. The radical scavenging reaction of ascorbic acid with DPPH was essentially instantaneous; the reaction of DPPH with Sphagneticola trilobata was also fast but slower compared to that with ascorbic acid. It is usually noticeable as discoloration of ethanolic extract of plant samples from purple to yellow; hence, DPPH is widely used to evaluate the free radical scavenging capacity of antioxidants ²

Table 3: DPPH Activity test of Root extract

S No	Concentration in µg/ml	Ascorbic acid	% DPPH inhibition activity of Nano particle synthesized
1	5	5.84	3.63
2	10	15.95	20.75
3	15	38.91	31.52
4	20	60.05	47.99
5	25	85.99	68.22
6	30	97.02	86.64

3.3.1.3 Hydroxyl radical scavenging activity

The potential of an ethanolic root extract of Sphagneticola trilobata to inhibit hydroxyl-radical-mediated deoxyribose damage was assessed at a concentration of 5 µM to 100 µM. The sample exhibited minimum activity of 3.02 % at 5 µM and maximum activity of 93.15 at 100 µM, showing that the hydroxyl radical scavenging activity occurred in a dose-dependent manner Table 4. The results indicate the scavenging potential against hydroxyl radicals. Superoxides are produced from molecular oxygen by oxidative enzymes as well as via nonenzymatic reactions such as auto-oxidation by catecholamines.²⁸ Superoxide anions play an important role in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radical and singlet oxygen, which induce oxidative damage in lipids, protein and DNA ^29-30 The superoxide scavenging activity of Sphagneticola trilobata was investigated, because the extract has the potential to scavenge superoxide anions.

Table 4: Hydroxyl radical scavenging activity

S No	Concentration in µM	Ascorbic acid		Root based ZnNPs		Aqueous Root extract
S No	Concentration in µM	Absorbance	% Activity	Absorbance	% Activity	Absorbance	% Activity
1	5	0.825	4.18	0.835	3.02	0.844	1.97
2	10	0.781	9.29	0.794	7.78	0.794	7.78
3	15	0.713	17.19	0.729	15.33	0.747	13.24
4	20	0.658	23.58	0.685	20.44	0.706	18.00
5	25	0.593	31.13	0.604	29.85	0.656	23.81
6	30	0.512	40.53	0.549	36.24	0.613	28.80
7	35	0.427	50.41	0.482	44.02	0.574	33.33
8	40	0.346	59.81	0.365	57.61	0.511	40.65
9	50	0.241	72.01	0.271	68.52	0.439	49.01
10	75	0.116	86.53	0.168	80.49	0.384	55.40
11	100	0.028	96.75	0.059	93.15	0.306	64.46

3.3.2 α-Amylase inhibition Activity

Root based Zno Nps (at a concentrations 200 μg/mL) showed 88.2416% inhibitory effects on the α-amylase activity with an IC₅₀ value 102.67μg/mL Table 5. Many herbal extracts have been reported to have antidiabetic activities and are used in Ayurveda for the treatment of diabetes. It is mainly a-amylase inhibitory components are present in ethanolic extract of S. amaranthoides ²⁰. Likewise, studied that the nanoparticles are potent therapeuticagent to control diabetes with few side effects. The clinical studies in mice successfully control the sugar level of 140 mg/dL silver nanoparticles treated group.

Table 5: α-Amylase inhibition Activity

S No	Concentration in µg/ml	α-amylase inhibition activity
S No	Concentration in µg/ml	Acarbose	Root based ZnNPs	Aqueous Root extract
1	10	5.50162	3.55987	0.863
2	20	12.4056	10.2481	3.77562
3	40	21.7907	18.2309	11.8662
4	60	33.8727	27.8317	16.2891
5	80	41.4239	37.0011	25.027
6	100	56.7422	50.7012	33.8727
7	150	85.0054	73.4628	41.4239
8	200	94.0669	88.2416	56.2028

3.3.3 Anti-inflammatory activity

Present study of Table 6 indicated that the extract represented dose-dependent inhibition of BSA denaturation. The IC₅₀ value was determined to be 121.58μg/mL respectively. The IC₅₀ value obtained for the standard drug was 111.98μg/mL. Protein denaturation is a well-implicated cause leading to the inflammatory response of cells ³¹ BSA was used as a reagent for the assay. 60% of total protein content in animal serum is constituted from BSA alone, and is commonly used in cell culture, particularly when protein supplementation is necessary and the other components of serum are unwanted. BSA undergoes denaturation when exposed to heat, and expresses antigens associated with Type III hypersensitive reaction, which are related to diseases such as glomerulonephritis, rheumatoidarthritis, systemic lupus erythematosus, and serum sickness ³². Thus, inhibition of BSA denaturation assay was used to evaluate the anti-inflammatory potential of the methanolic crude extract of Kalanchoe pinnata.

Table 6: Albumin Denaturation Inhibition Assay

S No	Concentration in µg/ml	% Albumin Denaturation Inhibition
S No	Concentration in µg/ml	Diclofenac sodium	Root based ZnNPs	Aqueous Root extract
1	25	10.539523	5.6461731	0.7528231
2	50	13.801757	12.923463	5.6461731
3	75	31.994981	28.732748	14.55458
4	100	47.427854	41.40527	27.728984
5	125	61.355082	56.587202	37.139272
6	150	72.898369	68.506901	46.800502
7	200	84.441656	78.920954	56.21079

4. CONCLUSION:

A simple one-pot green synthesis of stable Zinc nanoparticles using Sphagneticola trilobata Lin. root extract at room temperature was reported in this study. Synthesis was found to be efficient in terms of reaction time as well as stability of the synthesized nanoparticles which exclude external stabilizers/reducing agents. It proves to be an eco-friendly, rapid green approach for the synthesis providing a cost effective and an efficient way for the synthesis of zinc nanoparticles. Therefore, this reaction pathway satisfies all the conditions of a 100% green chemical process. The synthesized zinc nanoparticles showed efficient anti-oxidant (DPPH, ABTSand hydroxyl radical) activity, anti-diabetic and anti-inflammatory activities.

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Received on 20.11.2019 Modified on 06.01.2020

Research J. Pharm. and Tech. 2020; 13(12):5972-5978.

DOI: 10.5958/0974-360X.2020.01042.2