Introduction

Nanobiotechnology is an important and emerging technical tool for the development of an eco-friendly and reliable methodology for the synthesis of nanoscale materials using biological sources [1]. Recently, bionanotechnology is a rapidly advancing area of scientific and technological opportunity that applies the tools and process of nano/microfabrication to build devices for studying biosystems. It concerned with molecular scale properties such as size, shape, and surface morphology and applications of nanostructures and its interface between the chemical, biological, physical, optical, and electronic properties of nano materials [2]. Owing to the medical and pharmaceutical applications, nanoparticles (NPs) of noble metals, such as gold, silver, platinum, and zinc oxide are widely practiced [3]. Nanoparticles produced by plants are more stable and more varied in shape and size in comparison with those produced by other organisms [4]. Among metal oxide nanoparticles, zinc oxide (ZnO) has received much attention in the recent past. ZnO nanostructures are the forefront of research due to their unique properties and wide applications [5].

In particular, utilization of medicinal plant extracts as potential reducing and stabilization agents to synthesis ZnO nanostructures attainment many advantages over conventional physical and highly toxic chemical methods [6]. The advantages of nanostructured ZnO particles over other metal nanoparticles are due to their lower cost, UV blocking properties, high catalytic activity, large surface area, white appearance and their remarkable applications in the field of medicine, environmental remediation, anti-microbial activity and agriculture [7,8,9]. Several recent plants have been successfully employed for efficient and rapid extracellular biosynthesis of ZnO NPs. For examples, Pongamia pinnata [10], Vitex trifolia [11], Ficus benghalensis [12], Punica granatum [13], Trifolium pratense [14], Hibiscus subdariffa [15] and Couroupita guianensis Aubl. [16].

Andrographis paniculata (Burm.f.) Wall. ex Nees (A. paniculata) is an important medicinal plant which belongs to the family Acanthaceae and used as a traditional herbal medicine in Bangladesh, China, Hong Kong, India, Pakistan, Philippines, Malaysia, Indonesia, and Thailand [17, 18]. A. paniculata has been reported to have a broad range of pharmacological effects including anti-cancer [19, 20], anti-diarrheal [21], anti-hepatitis [22], anti-HIV [23], anti-hyperglycemic [24], anti-inflammatory [25], anti-microbial, anti-malarial [26], anti-oxidant [27], cardiovascular [28], cytotoxic [23], hepatoprotective [29] immunostimulatory [30], and sexual dysfunctions [31]. Aqueous extract of A. paniculata was more potent than ethanolic extract in anti-oxidant activities; compared with andrographolide, the aqueous extract also possessed potent anti-oedema and analgesic activities [32]. Thus, in the present study, ZnO NPs were aimed to prepare through green synthesis using the leaf of A. paniculata, which include low cost, nontoxicity, and the ability to prepare compounds with varying morphologies having different prosperities by utilizing reducing and capping potential.

Materials and methods

Preparation of A. paniculata leaf extract

Fresh leaves of A. paniculata were collected from Vellore, Vellore district, Tamil Nadu, India, washed thoroughly with double distilled water and incised into small pieces. From this 10 g were weighed and transferred into a 250-mL beaker containing 100 mL of double distilled water, mixed well and boiled for 2 min. The extract obtained was filtered through Whatman no. 1 filter paper, and the filtrate was collected in 250-mL Erlenmeyer flask and stored at 4 °C for further use.

Synthesis of ZnO NPs

Biosynthesis of ZnO NPs was carried out according to the method of Murali et al. [33] with modifications. About, 10 g of leaf material was extracted with 100 mL of sterile distilled water and filtered through Whatman no. 1 filter paper. The plant extract (about 20 mL) was boiled (60–80 °C) on a magnetic stirrer. When the temperature of the extract reached 60 °C, 2 g of zinc nitrate hexahydrate [Zn(NO3)2·6H2O] was added and allowed to boil until the extract became a paste. The collected plant extract was then heated in a furnace at 400 °C for 2 h. The resultant dried material was powdered and stored in an airtight container for further analysis.

Characterization of ZnO NPs

The visual color change observed the preliminary detection for ZnO NPs formation. Reduction of Zn2+ in the prepared mixtures was monitored by UV–Vis spectral analysis from 200 to 700 nm using UV–Vis spectrophotometer (Shimadzu, UV 1601). The crystalline structure of the biosynthesized ZnO NPs was investigated by the X-ray diffraction a using X-ray diffractometer (Perkin-Elmer spectrum). XRD spectrum was recorded from 20° to 80° 2θ angles using CuKα radiation operated at 10 kV and 30 mA. FTIR spectra were obtained in the 400–4000−1 spectral range with a Perkin-Elmer 1725X FTIR spectrometer using the KBr pellet technique and detect surface functional groups. For FTIR measurements, a small amount of powdered ZnO NPs was placed on round-shaped zinc selenide plate for FTIR spectral analysis. The suspension of ZnO NPs was dried and subjected to SEM–EDS using a JEOL MODEL JSM 6360 SEM. The shape, size, and microstructures of the products obtained were analyzed by transmission electron microscopy (Model JEM 3100 LV, JOEL, USA).

Anti-oxidant activity assays

1-Diphenyl-1-2-picrylhydrazyl (DPPH) scavenging assay

A. paniculata leaf extract and synthesized ZnO NPs were screened for free radical scavenging activity by DPPH method [34]. The scavenging activity on the DPPH radical was determined by measuring the absorbance at 517 nm using a UV-spectrophotometer. Radical scavenging activity was calculated using the formula (Eq. (1)):

$$\% I = \frac{{A_{\text{control }} - A_{\text{sample}} }}{{A_{\text{control}} }} \times 100,$$
(1)

where %I is the % of radical scavenging activity, A control is the absorbance of the control sample (DPPH solution without test sample), and A sample is the absorbance of the test sample (DPPH solution with test compound). All tests were performed in triplicate, and the results were averaged.

Reducing power assay

The reducing power was determined according to the method of Oyaizu [35]. Various concentrations of leaf extracts of A. paniculata and biosynthesized ZnO NPs were mixed with 2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. After the addition of 2.5 mL of 10% trichloroacetic acid (w/v), the mixture was centrifuged at 3000 rpm for 10 min. The upper layer was mixed with 5 mL of deionized water and 1 mL of 0.1% of ferric chloride, and the absorbance was measured at 700 nm. A higher absorbance indicates higher reducing power. ascorbic acid was used as a standard.

Nitric oxide (NO) free radical scavenging activity

Nitric oxide has been involved in a variety of biological functions, including anti-microbial, vascular homeostasis, neurotransmission, and anti-tumor activities. Nitric oxide, generated from sodium nitroprusside, was measured spectrophotometrically by Griess reaction. About 100, 200, 300, 400, and 500 µL (1:1 w/v) concentrations of ZnO NPs previously dissolved in DMSO, as well as ascorbic acid (standard compound), was taken in separated tubes and each tube 2.0 mL of sodium nitroprusside in phosphate buffer saline was added. The solution was incubated at room temperature for 150 min. After incubation, 5 mL of the Griess reagent was added, in each tube including control. Methanol was used as a blank. The absorbance was measured at 546 nm on UV–visible spectrometer [36].

$$\%\, {\text{Scavenging reduction}} = \frac{{T_{0} - T}}{{T_{0} }}{ \times } 100 ,$$

where T 0 is the absorbance of control and T is the absorbance of the test sample.

Anti-diabetic potential of NPs (α-amylase inhibitory activity)

In brief, 100 μL of different concentrations of the A. paniculata leaf extract, ZnNo3, ZnO NPs were allowed to react with 200 μL of the α-amylase enzyme (Hi-media Rm 638) and 100 μL of 2 mM phosphate buffer (pH 6.9). After 20 min incubation, 100 mL of 1% starch solution was added. The same was performed for the control where 200 μL of the enzyme was replaced by the buffer. After incubation for 5 min, 500 μL of dinitro salicylic acid reagent was added to both control and test, and were boiled for 5 min in a water bath. The absorbance was recorded at 540 nm, and the result was interpreted regarding the IC50 values (effective concentration showing 50% inhibition activity) [37].

Anti-inflammatory activity of ZnO NPs

The anti-inflammatory activity of the biosynthesized nanoparticles to inhibit protein denaturation was studied through in vitro assay. To 50 μL of different concentrations of the NPs, 5 mL of 0.2% w/v BSA was added and was heated at 72 °C for 5 min and then cooled for 10 min. 5 mL of 0.2% w/v BSA solution with 50 μL water was used as control and Diclofenac (100 μg/mL) in water with 5 mL 0.2% w/v BSA solution was used as a standard. The absorbance was measured at a wavelength of 276 nm [38]. The result was interpreted in terms of the IC50 values (effective concentration showing 50% inhibition activity).

Statistical analysis

All experiments were carried out in triplicate and data were analyzed. For the experiments of anti-microbial activity, arithmetic mean values were considered for data analysis. All the statistical analyses were done by SPSS Statistics 18 Release Version 18.0.0, 2009.

Results and discussion

Zinc oxide nanoparticles were synthesized using fresh leaf extract of A. paniculata, which were collected from healthy plants, contains the stabilizing agents such as flavanones, terpenoids and reducing sugars as main constituents. Visual examination confirmed the formation of zinc oxide nanoparticles. The color of the mixture was changed from green-yellow to pale white during the reaction, indicating the synthesis of ZnO NPs. It is suggested that the electron rich functional groups of the leaf extract are responsible for the conversion of ZnO NPs. Thus, it is concluded that the A. paniculata leaf extract acts as reducing and stabilizing agents for the formation of ZnO NPs.

UV–Vis spectroscopy is the most widely used technique for the structural characterization of nanoparticles. The optical absorption of ZnO NPs dispersed in water was recorded using UV–Vis spectrophotometer (Shimadzu, UV 1601) at room temperature in a quartz cuvette with the path length of 1 cm. The absorbance of the reaction mixture was monitored after 24, 48, 72, 96 and 120 h of reaction. The reduction of zinc nitrate to zinc oxide is monitored by UV–Vis spectrum. All the samples exhibit strong UV absorption spectra with the absorption peak ranging from 358 nm due to its surface plasmon resonance and attain a plateau above 3.3 eV (375 nm) (Fig. 1a). This band is ascribed to excitation of valence electrons of ZnO arranged in the nanoparticles (nanocrystal/nanosphere). The shape of the band was symmetrical, suggesting uniform scattering of spherical shape nanoparticles. According to Gupta et al. [39], the absorption edge systematically shifts to the lower wavelength or higher energy with the decreasing size of the nanoparticle. Thus, there is a strong blue shift in the absorption spectra of the ZnO NPs, indicating that particles must be smaller than the Bohr radius of an exciton which is for ZnO [40]. Similar findings have been documented by Vimala et al. [41] who reported that the absorbance spectra of ZnO NPs were recorded between 330 and 370 nm.

Fig. 1
figure 1

a UV–Vis spectra of biosynthesized ZnO NPs from A. paniculata. b FTIR spectrum of the synthesized ZnO NPs

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Characterization of ZnO NPs

FTIR gives an impression on the vibrational and rotational modes of the proposition of a molecule, and hence a consistent technique for identification and characterization of the substance. Figure 1b shows the FTIR spectra of ZnO NPs synthesized by the biological method. Possible biomolecules which are responsible for the reduction of ZnO and capping agent of reduced ZnO NPs through distinct bond vibrations peaks stated at bounded wavenumbers were identified [42]. The bands at 3441 cm−1 indicate the presence of primary and secondary amines, O–H stretching of alcohols, and intramolecular hydrogen bonding [43]). The bands observed at 3002 and 2850 cm−1 have been assigned to asymmetric and symmetric stretching vibrations of C–H. The bands around 1731 and 1601 cm−1 are due to the amide I and amide II regions that are characteristics of proteins/enzymes [44]. The spectrum clearly shows ZnO absorption band near 440 cm−1 [45, 46]. Also, the intense bands observed at 1404, 1274, 1125, 1066, 954 and 891 1020, 1150 and 1380 cm−1 have been assigned to C–H bending, C–N stretching vibrations of aliphatic and aromatic amines, C–O stretching of polysaccharides, alcohols and phenolic groups, respectively. From the FTIR results, it can be concluded that the existence of proteins and metabolites such as polyphenols, alkaloids carboxylic acid and flavonoids is bound to the surface of ZnO NPs that remained despite repeated washing, and these compounds are responsible for the reduction of zinc ions to ZnO NPs. Furthermore, the stability of the synthesized ZnO NPs is expected to be due to the presence of free amino and carboxylic groups that have interacted with the zinc surface. Moreover, the proteins present in the medium prevent agglomeration and aids in the stabilization by forming a coat, covering the metal nanoparticles [43].

XRD pattern of the prepared ZnO NPs has been shown in Fig. 2. The peak positions with 2θ values are of 31.71°, 34.38°, 36.21°, 45.21°, 47.48°, 56.51°, 62.80°, 67.88° and 69.01° which indexed the planes 100, 002, 110, 102, 110,103, 112 and 201, respectively, reflection lines of hexagonal wurtzite ZnO (JCPDS36-1451) (Fig. 2). All of the diffraction peaks were in adaptation to the hexagonal zinc oxide phase by comparison with the data from cards. The narrow and strong diffraction peaks indicate that the product has a well-crystalline particle structure. The peaks in XRD pattern are derived from their particle size and are by the SEM and TEM observations. No characteristic peaks of any impurities were detected, suggesting that a high quality of ZnO NPs was produced. The crystallite size has been estimated from the XRD pattern using the Scherrer equation (Eq. 1):

$$D = K\lambda /\beta \cos \theta ,$$

where K = 0.9 is the shape factor, λ is the X-ray wavelength of CuKα radiation (1.54 Ǻ), θ is the Bragg diffraction angle, and β is the full width at half maximum (FWHM) of the respective diffraction peak. Based on the Scherrer equation, the crystallite size of the ZnO NPs was estimated to be 13.8 nm. The image clearly shows the presence of secondary material capping which may be assigned to bio-organic compounds present in the leaf extract that confirm by the observing sharp reflection in the XRD spectrum. The XRD pattern of bio-synthesized ZnO NPs from leaf extract of Azadirachta indica stated all the peaks are confirmed ZnO hexagonal phase (wurtzite structure) by comparison with JCPDS card no. 89-7102 [47]

Fig. 2
figure 2

X-ray diffraction spectra of biosynthesized ZnO NPs from A. paniculata

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The morphology of the nanostructures obtained from leaves extracts was studied using scanning electron microscopy (SEM). The SEM images of synthesized ZnO NPs exhibit varieties of typical shapes such as spherical and hexagonal nanoparticles. The synthesized ZnO nanoparticles were agglomerated with a particle size ranging from below 96 to 115 nm (Fig. 3a, b). Also, it shows distinguished spherical and hexagonal morphology and generally in random and not uniform, which is very similar to those described in the previous literature [48].

Fig. 3
figure 3

SEM images of ZnO nanoparticles with various concentrations of A. paniculata extract. a 10 μm and b 5 μm

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Transmission electron microscopy images of ZnO NPs of different shapes are spherical, oval and hexagonal in size range of 57 ± 0.23 nm (Fig. 4a, b). Especially, the hexagonal (wurtzite) shape is in agreement with the obtained XRD pattern. The images show the presence of zinc oxide nanoparticles agglomeration owing to high surface energy that usually occurs when synthesis is carried out in an aqueous medium [49]. Owing to the uniform distribution of oxidized metal anions in the three-dimensional polymeric network structure, the agglomeration could be induced by densification resulting in the narrow space between particles [50, 51]. Also, the SAED analysis (Fig. 4c) was proved to be in nanocrystalline nature with SAED rings to be appearing in perfect alignment with the X-ray studies.

Fig. 4
figure 4

a, b TEM image of ZnO NPs synthesized using A. paniculata leaf extract. c Selected electron diffraction patterns of ZnO NPs

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The average number of zinc atoms per nanoparticle may be calculated from high-resolution TEM analysis (Liu et al. [59]). The HRTEM images of the synthesized silver nanoparticles show the average particle size of about 57 ± 0.23 nm (D, nm). Assuming a spherical, oval and hexagonal shape, the average number of zinc atoms (N) for each type of nanosphere was calculated by the following equation, where ρ is the density for hcp zinc (7.13 g/cm3 = 7.13 × 10−21 g/nm3), and M stands for atomic weight of zinc (63.3800 g/mol):

$$N = \frac{\pi }{6}\frac{\rho D}{M}^{3 } N_{\text{A}} = 35.4592D^{3} .$$

On applying this above equation, the average number of silver atoms per synthesized nanoparticles was found to be in the range from 6,487,617.529 to 6,646,603.832 atoms.

The free radical scavenging potential of using the DPPH assay is explored for the anti-oxidant activity of the only plant extract and biologically synthesized ZnO NPs. Green synthesized ZnO NPs have almost similar anti-oxidant activity with only plant extract (Fig. 5a). The maximum inhibition (61.32, 57.71%) for biologically synthesized ZnO NPS and plant extract was observed with very low concentration, i.e., 500 μg/mL. Discoloration of violet DPPH to yellow clearly demonstrated the effect of NPs as an antioxidant. Ascorbic acid is used as a standard. During the synthesis of the ZnO NPs, these metabolites are pooled into the NPs. They adsorbed onto the surface of the ZnO NPs. Considering the high surface area to volume ratio, it appears that these ZnO NPs show a high tendency to interact with and reduce DPPH. Therefore, it is possible to propose on Eq. (2):

$${\text{H}}{-}O{-}\left( {{\text{of}}\,{\text{A}}} \right)\, + \,{\text{DPPH}}\, \to \,{\text{O=CH}}{-}\left( {{\text{of}}\,{\text{AA}}} \right)\, + \,1\text{-}1\;{\text{diphenyl}} - 2{\text{-picrylhydrazine}} .$$
(2)
Fig. 5
figure 5

Anti-oxidant activity of green-synthesized ZnO NPs by a DPPH assay, b reducing power assay, c nitric oxide free radical scavenging activity

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Here hydroxyl of AA quenched the activity of DPPH by donating its electron.

In the functioning of all biosystems, the antioxidants play an important role due to the interaction of biomolecules with molecular oxygen; the free radicals are generated in biological systems [52], which results in the degradation of biomolecules. A crucial role is played by the antioxidants in scavenging these toxic free radicals, thereby terminating the human body’s oxidative damage. This study clearly indicates that ZnO NPs synthesized by Ocimum tenuiflorum can act as potential antioxidants. Anti-oxidant activity of ZnO NPs ascribed due to smaller particle grain size and other reason may be a phenomenon of transfer of electron density from oxygen atom to odd electron located at nitrogen atom in DPPH which results in decreasing n → π* transition intensity at 517 nm [53].

Reducing power assay indicates that they donate an electron and can reduce the oxidized intermediates of lipid peroxidation process; they act as primary and secondary antioxidants. In the present study, various concentrations of crude extract, biologically synthesized ZnO NPs were taken from 50 to 300 μg and the yellow color of the test solution changes to various shades of green and blue color due to the presence of reducers causing the conversion of Fe3+ complex to ferrous form by measuring 700 nm (Fig. 5b). The reducing power was found to be increased with the increase in the concentration of the ZnO NPs, indicating the presence of some compounds in the ZnO NPs acts as both electron donors and could react with free radicals and to terminate free radical chain reactions [54].

The scavenging effect increased with the increasing concentrations of test samples. From the results, the ZnO NPs showed maximum scavenging power of 62% at 500 μL (1:1 w/v) concentrations. Similarly, NO scavenging activity was performed with ZnO NPs and ascorbic acid as standard. The reductive potential of nanoparticle was exhibited on dose dependent manner (Fig. 5c). From the graph, when the concentration increases, the percentage of scavenging is also increasing linearly for tested nanoparticles and ascorbic acid (standard). The results showed the maximum activity of ZnO NPs was 62% at 500 μL (1:1 w/v) concentration. All the graphs were plotted with standard values of ascorbic acid. Antioxidants can also react with NO to form toxic radicals such as hydroxyl radicals by peroxy nitrite [55]. Due to the scavenging power, most of the antioxidants are used in the management of diseases such as neurodegenerative diseases, cancer, AIDS, and so on. Even though the ZnO NPs showed better antioxidant nature, a detailed study on its mechanism is needed for the commercialization of these materials in future.

The synthesized NPs exhibited moderate α-amylase inhibition activity, and the results are presented in terms of the IC50 values (Fig. 6a). The IC50 values of the ZnO NPs (121.42 μg/mL) were lower than that of the A. paniculata leaf extract, ZnNO3, (149.65 and 178.84 μg/mL), respectively, that confirmed that the ZnO NPs displayed better anti-diabetic potential in terms of the α-amylase inhibition activity. Zinc oxide (ZnO) nanoparticles (NPs) have been synthesized using Hibiscus subdariffa leaf extract has indicated that small-sized ZnO NPs, stabilized by plant metabolites had better anti-diabetic effect on streptozotocin (STZ)-induced diabetic mice than that of large-sized ZnO particles [15]. ZnO NPs treatment indicates inhibitory effects on glycogenolysis and gluconeogenesis, mechanisms that are active during the fasted state. Interestingly, zinc is reported to regulate glucagon secretion from pancreatic cells [56].

Fig. 6
figure 6

Bio-potential of zinc oxide nanoparticles a anti-diabetic potential; b anti-inflammatory activity

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Maximum inhibition activity of 66.78 μg/mL IC50 value was observed from the ZnO NPs followed by A. paniculata leaf extract, (75.42 μg/mL IC50 value), and ZnNO3 (91.33 μg/mL IC50 value). Diclofenac, which was taken as the standard reference compound, exhibited 62.55 μg/mL IC50 value (Fig. 6b). Nagajyothi et al. [57] utilized the root extract of Polygala tenuifolia to synthesize the ZnO NPs and their antioxidants, as well as anti-inflammatory activity, were performed with the help of scavenging agent DPPH free radical assay and LPS-stimulated RAW 264.7 murine macrophages, respectively. To determine the biomedical potential of ZnO NPs, Thatoi et al. [58] fabricated the ZnO NPs using the aqueous extracts of two mangrove plants, viz. Heritiera fomes and Sonneratia apetala and revealed that the ZnO NPs have greater potential for anti-inflammation (79%) in comparison with silver nanoparticles (69.1%) and hence have potential to be utilized in the biomedical field.

Conclusion

In the present work, ZnO NPs were biosynthesis through green approach, inexpensive, pollution-free and eco-friendly approach for anti-oxidants, anti-diabetic and anti-inflammatory activity using A. paniculata extract. The synthesis of ZnO NPs was characterized using UV–Vis, FTIR, XRD, SEM, TEM and SAED analysis. The results proved the formation of ZnO NPs with distinguished spherical and hexagonal morphology. The particles obtained have been found to be predominantly spherical and the particle size could be controlled by varying the concentrations of leaf broth solution. The ZnO NPs were formed due to the influence of proteins and metabolites such as polyphenols, alkaloids carboxylic acid and flavonoids, especially due to the presence of free amino and carboxylic groups that have interacted with the zinc surface. Anti-oxidant, anti-diabetic and anti-inflammatory studies results proved that the synthesized ZnO NPs can be used to reduce the sugar level and to inflammations. Biosynthesized ZnO NPs prepared from A. paniculata are expected to have notable applications in pharmaceutical and biomedical fields such as drug delivery and in cosmetic industries.