INTRODUCTION:

Nanotechnologies emerge from physical, chemical and biological and engineering sciences where, novel techniques are being developed to probe and manipulate single atoms and molecules. In nanotechnology, a nanoparticle (10^-9 m) is defined as a small object that behaves as a whole unit in terms of its transport and properties.¹ Nanoparticles are clusters of atoms and their size range from 1–100 nm².

They can be synthesized using various elemental materials, namely gold, silver, zinc, platinum etc.³ They are the clusters of atoms and their size range from 1–100 nm.³ Zinc oxide nanoparticles have been used in various cutting edge applications like electronics, communication, sensor, cosmetics, environmental protection, biology and medicinal industry.⁴

In literature survey investigate the production of zinc oxide nanostructure such as laser ablation, hydrothermal methods, electro- chemical depositions, sol- gel method, chemical vapor deposition, thermal decomposition and combustion method. Recently, ZnO nanoparticles were prepared by ultrasound, microwave-assisted combustion method, two-step mechanochemical-thermal synthesis, co-precipitation and electrophoretic deposition.^5,6

ZnO nanoparticles have received considerable attention due to their antimicrobial, UV blocking, high catalytic and photochemical activities. Synthesis of ZnO NPs via green method have several merits such as, simple, inexpensive, good stability of nanoparticles, less time consumption, non-toxic byproducts and large-scale synthesis. ZnO NP have their own importance due to their vast area of application in gas sensor, chemical sensor, bio-sensor, cosmetics, storage, optical and electrical devices, window materials for displays, solar cells, and drug-delivery.^7,8

Plant Profile

Family: Leguminoseae

Subfamily: Mimoseae

Binomial name: Albizzia lebbeck

Common name: Shirish

Plant of part used: Stem Bark

Pharmacological activity:

Anti- oxidant, anti- diabetic activity, anti- bacterial activity, anti- inflammatory, anti- asthmatic, anti- fertility activity.^9,10,11

Fig. 1: Albizzia lebbeck

MATERIAL AND METHODS:

Collection and authentication of the plant material:

The plant material was collected from local areas of Karad and was further identified and authenticated by the Department of Botany, Science College, Karad.

Preparation of plant extracts:

The bark of Albizzia lebbeck were cleaned, dried in a hot air oven (50°C), powdered, passed through 60 mesh sieve (BS) and stored in an airtight container at 4°C till further use. Aqueous extracts were prepared by extracting the powders of bark of Albizzia lebbeck with hot water (70°C) in a mechanical shaker (24h), filtered and freeze dried.

Green synthesis of Zinc oxide nanoparticle using Albizzia lebbeck extract:

The aqueous bark extract of Albizzia lebbeck and Zinc acetate was used as precursor for the synthesis of zinc oxide nanoparticles. 20 mg of bark extract was added to 20 ml of distilled water heated at 50⁰C for 10min and 91mM of zinc acetate solution was added drop wise to it with stirring. Reaction temperature was allowed to rise gradually to 60⁰C, 80⁰C and 90⁰C. The reaction mixture became yellowish and cream colored precipitate of zinc hydroxide was formed. After completion of reaction, the solution was allowed to settle for overnight and supernatant liquid was discarded. The cream colored precipitate formed was washed thoroughly with double distilled water and then centrifuged at 16000rpm for 10min. The obtained precipitate was dried in hot air oven at 80⁰C. The above resulting dried precursor was crushed into powder and stored in an airtight container for further analysis.

Characterization:

Optical properties of synthesized ZnO nanoparticles done by UV- visible spectroscopy (Jasco 4600, Japan) with the wavelength range of 200- 800 nm confirm the formation of nanoparticle.FTIR (Jasco 4100, Japan) carried out with the spectral range of 4000- 400 cm^-1.

In vitro antidiabetic activity of the prepared nanoparticle¹⁴:

Inhibition of α-amylase enzyme activity:

Alpha-amylase inhibitory activity was carried out using maltose as the reference compound. The A. lebbeck bark extract and their synthesized ZnO NP at a various concentration of (1-9µg/ml) was prepared in 1.0ml of 2M phosphate buffer (pH 6.9) containing enzyme buffer of 0.5 ml added into the NPs suspension and the reaction mixtures were then incubated at 37°C for 2hr. After incubation, 2ml of 3,5- dinitrosalicylic acid reagent was transferred into all the mixture dispersions and heated for 5min, thereafter the mixture was cooled down and the solution was made up to 25.0ml with distilled water and filtered and the absorbance was measured at 550nm. A sample blank and 4.0ml of Acarbose standard were run simultaneously with the samples. The values were expressed as mg of maltose released/g sample. %Inhibition was calculated according to the formula,

Abs of control ₅₅₀ – Abs. of sample₅₅₀

% Inhibition =^{----------------------------------------------------------------------------------------------------------------------------------------------------------------------}X 100

Abs of control₅₅₀

Glucose uptake by yeast cells:

10% (V/V) baker’s yeast suspension was prepared in distilled water and then centrifuged at 21000 rpm for 5 mins until the supernatant was clear. Various concentrations of A. lebbeck bark extract, synthesized ZnO NPs (1µg/ml, 3µg/ml, 5µg/ml, 7µg/ml and 9µg/ml) were prepared. 1ml of glucose solution was added and incubated for 10 minutes at 37°C. The solution was again incubated at 37ºC for 60min, then 100 µl yeast suspension was added. After 60min incubation, the solution were centrifuged at 3800rpm for 5min. The glucose content in the supernatant was determined at 540nm. Metformin was used as standard to compare the activity. And % inhibition of glucose uptake by yeast cell calculated using the formula,

Abs of Control₅₄₀ – Abs of Sample₅₄₀

% Increase in = ^{----------------------------------------------------------------------}X100

Glucose uptake Abs of Control₅₄₀

In-vitro antioxidant activity of the prepared nanoparticles^12,13:

DPPH free radical scavenging activity:

Free radical scavenging activity of the prepared nanoparticles was evaluated by using its ability to trap the 2,2- diphenyl- 1- picrylhydrazyl (DPPH) free radicals. The working solution of prepared NPs sample and 0.002% of DPPH was prepared in methanol separately and then 1ml of this solution was mixed with 1ml of NP sample solution and standard solution separately. Methanolic solution of DPPH and various concentrations of the test substances were kept in dark for 30min. Optical density was measured at 517nm using Shimadzu Spectrophotometer. Methanol (1ml) with DPPH solution (0.002%, 1ml) was used as blank. Ascorbic acid was used as standard in 1-100 μg/ml solution. The optical density was recorded and % inhibition was calculated using the formula given below:

Abs of Control₅₁₇ – Abs of Sample₅₁₇

% Inhibition of = ^{---------------------------------------------------------------------------------------------------------------------------------------------------------------------}^-- X 100

DPPH activity Abs of Control₅₁₇

Reducing power ability:

Different concentrations of nanoparticle samples were mixed with 2.5ml of phosphate buffer (0.2M, pH 6.6) and 2.5 ml of (1%) potassium ferricyanide. After addition the mixture was incubated at 50^oC for 20min. After incubation, reaction mixture was cooled and 2.5ml of (10%) Trichloroacetic acid was added to the mixture. It was then centrifuged at 3000 rpm for 10 min. 2.5 ml upper layer of the supernatant was mixed with 2.5ml of distilled water and 0.5ml of freshly prepared (0.1% FeCl₃) ferric chloride. The final solution was allowed to stand for 10min. Finally, the absorbance of the reaction mixture was read spectrophotometrically at 700nm against blank and ascorbic acid was used as reference standard.

Fig. 2: Schematic representation of Synthesized ZnO NPs their Characterization and in vitro antioxidant, antidiabetic activity

Fig. 2 shows schematic representation of synthesize ZnO nanoparticles using Albizzia lebbeck aqueous bark extract and their characterization, in vitro antioxidant and in vitro antidiabetic activity which was successfully carried out.

RESULTS AND DISCUSSION:

UV- visible analysis of synthesized ZnO NPs:

UV- visible analysis of prepared Zinc oxide nanoparticles was carried out with wavelength range of 200- 800nm. For measurement of UV- visible analysis prepared ZnO NPs were dispersed in distilled water. Distilled water was used as a reference standard. Fig. 3 shows the absorption peak at 213nm indicating the formation of nanoparticles.

Fig.3: UV- visible analysis of synthesized ZnO NPs

FTIR of Synthesized ZnO NPs:

The IR Spectra of synthesized Zno nanoparticle (Fig.4), showed prominent peak at 3563.81cm^-1, 3405.67 cm^-1, 3191.61 cm^-1 corresponding to O-H Stretching Hydrogen bonded, N-H Stretching Pri, Sec, Ter amine and C-H Stretching aromatic ring respectively. Sharp peak at 1739.48cm^-1 and 1367.28cm^-1 correspond to C=O Stretching saturated ester and C-H Bending alkane respectively. Weak peak obtained at 1101cm^-1 are due to the presence of C-N Stretching aliphatic. Ignorable peak was obtained at 703.89 cm^-1, it suggested C-Cl from alkyl halide.

Table.1: % inhibition of α- amylase activity

Concentration (µg/ml)	% Inhibition *(A. lebbek)*	% Inhibition (ZnO NPs)	% Inhibition (Standard Acarbose)
1	73.33	54.66	46.66
3	68.00	34.66	41.33
5	60.00	30.66	38.66
7	52.00	26.66	34.66
9	49.33	33.33	38.66

The in vitro α-amylase study proved that the green synthesis of Zinc oxide nanoparticles have significant α-amylase inhibitory activity. Maximal a-amylase inhibition of A.lebbeck and their synthesized ZnO NPs was found to be 73.33% and 54.66% at a concentration of 1µg/ml as compared to the standard Acarbose 46.66%. The resulting % inhibition value plotted against various different concentration and IC₅₀ value was calculated by linear regression analysis. Table 2 shows IC₅₀ value of A. lebbeck bark extract, ZnO NPs and standard Acarbose which was found to be 4.91, 9.61 and 3.91 respectively. Fig.5 shows inhibition of α-amylase against concentration. Fig.6 IC₅₀of α- amylase highlights the inhibition of α- amylase activity.

Table. 2: IC₅₀ of α-amylase activity

Samples	IC₅₀
A. lebbeck	4.91
ZnO NPs	9.61
Standard (Acarbose)	3.91

Fig.5: Percentage inhibition of α- amylase activity

Fig. 6: IC₅₀ of α- amylase activity

7.4.2 Glucose uptake by yeast cell:

Table. 3: % inhibition of Glucose uptake by yeast cell

Concentration (µg/ml)	% inhibition (A. lebbeck)	% inhibition ZnO NPs	% inhibition (Standard Metformin)
1	62.5	94.16	89.16
3	55	81.66	77.5
5	47.5	72.5	74.16
7	35.83	60	64.16
9	29.16	57.5	54.16

The amount of glucose in the medium was estimated and Metformin serves as marker of the glucose uptake by yeast cell. The % inhibition of glucose uptake by yeast cell of A.lebbeck extract, their synthesized ZnO NPs were obtained to be 62.5% and 94.16% respectively at the concentration 1mg/ml while %inhibition of Standard Metformin was obtained to be 89.16% at the same concentration. Glucose uptake by yeast cell activity is in dose dependent manner and is proportionate to the sample concentration. It means the percent increase in the glucose uptake by yeast cell was inversely proportional to the glucose concentration while decreasing with increasing the concentration of glucose. Fig. 7 shows Glucose uptake by yeast cell activity.

Fig. 7: Percentage inhibition of glucose uptake by yeast cell

In vitro antioxidant activity of synthesized ZnO NP:

DPPH radical scavenging assay:

Table. 4: % inhibition of DPPH scavenging assay

Concentration (µg/ml)	% Inhibition
Concentration (µg/ml)	*(A.* *lebbeck*)	(ZnO NPs)	(Standard Ascorbic)
1	89.16	66.66	90.42
3	81.66	47.61	80.95
5	75	4.76	66.66
7	68.33	23.80	57.14
9	62.5	14.28	66.66

The hydrogen donating ability of A. lebbeck, ZnO NPs, and Ascorbic acid was estimated in the presence of DPPH stable radical. Absorption peak was measured at 517 nm was used to studied radical scavenging activity. The resulting maximum %inhibition was found that 89.16%, 66.66% and 90.42% for A. lebbeck, ZnO NPs and Standard Ascorbic acid respectively. Table.5 shows IC₅₀ value of A. lebbeck, Optimized ZnO NPs, it was found to be 12.60, 7.038 which is comparable to the IC₅₀ value of ascorbic acid i.e. 11.28. The lowest IC₅₀ value of optimized ZnO NPs indicated, Zinc oxide nanoparticles have higher DPPH free scavenging activity as compared to A. lebbeck bark extract and Standard Ascorbic acid. Fig.8 shows DPPH radical scavenging activity at 517nm.

Table. 5: IC₅₀ of DPPH scavenging assay

Samples	IC₅₀
A.lebbeck	12.60
ZnO NPs	7.038
Standard (Ascorbic acid)	11.28

Fig. 8: Percentage inhibition of DPPH scavenging activity

Fig. 9: IC₅₀ of DPPH scavenging activity

Reducing power ability:

Table.6: Absorbance of reducing power ability assay

Concentration (µg/ml)	Absorbance
Concentration (µg/ml)	*(A.* *lebbeck)*	(ZnO NPs)	(Standard Ascorbic)
1	0.025±0.003	0.022±0.003	0.053±0.002
3	0.034±0.002	0.03±0.03	0.058±0.001
5	0.04±0.02	0.038±0.004	0.035±0.002
7	0.049±0.002	0.044±0.002	0.07±0.01
9	0.047±0.002	0.05±0.020	0.075±0.002

Reducing power ability assay measures the electron capacity of an antioxidant. In this assay reduction of ferric ion (Fe³⁺) to ferrous ion (Fe²⁺) and the amount of (Fe²⁺) is measured by intensity which absorbed at 700nm. Resulting absorption value of A. lebbeck and ZnO NPs was compared to the Standard Ascorbic acid. Maximum absorption value of A. lebbeck, ZnO NPs was found to be 0.049± 0.002 and 0.044± 0.002 at 7µg/ml while maximum absorption value of 0.075± 0.002 was obtained at conc. 9µg/ml for Ascorbic acid. Fig.10. shows Total reducing power ability at 700nm.

Fig. 10: Reducing power ability of synthesized ZnO nanoparticle

CONCLUSION:

The present investigation conclusively reports an eco-friendly approach for synthesis of zinc oxide nanoparticles. From this study, it was evident that the extract of A. lebbeck can be used to synthesize nanoparticles using green chemistry methods for various applications. The results of antioxidant and antidiabetic activity concluded that the ZnO nanoparticles synthesized using extract of A. lebbeck exhibited higher activity due to the presence of various phytoconstituents. The obtained results are significant, but detailed mechanistic investigations are needed to establish better models for application in antidiabetic treatment.

AKNOWLEDGEMENT:

The authors are thankful to Dr. C. S. Magdum; Pricipal and Dr. S. K. Mohite; Vice Principal for providing research facilities necessary for the research work.

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Received on 18.04.2019 Accepted on 30.04.2019

Asian J. Pharm. Tech. 2019; 9(2):93-98.

DOI: 10.5958/2231-5713.2019.00016.3