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Original article
2021
:14;
202106
doi:
10.1016/j.arabjc.2021.103180

Photocatalytic degradation of Rhodamine B (RhB) dye in waste water and enzymatic inhibition study using cauliflower shaped ZnO nanoparticles synthesized by a novel One-pot green synthesis method

, , , , ,
a Department of Metallurgical and Materials Engineering, Bartin University, Bartin 74100, Turkey
b Department of Biotechnology, Faculty of Science, Bartin University, Bartin 74100, Turkey
c Rectorate of Bartin University, Bartin, Turkey
d Ankara University, Department of Physics, Ankara, Turkey
e Faculty of Basic Sciences, School of Chemistry, Shoolini University, Bajhol, Himachal Pradesh 173229, India

⁎Corresponding authors. shashanka@bartin.edu.tr (Shashanka Rajendrachari), ptaslimi@bartin.edu.tr (Parham Taslimi) parham_taslimi_un@yahoo.com (Parham Taslimi)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

The ZnO nanoparticles (NPs) are considered to be one of the inexpensive and very important bioactive materials widely used in drug delivery, as a photo-catalyst material, antibacterial, bioimaging, anticancer, anti-inflammation, wound healing, and diabetes treatment. Therefore, we have prepared ZnO NPs using Alchemilla vulgaris (Lady’s mantle) leaves and investigated their anti-enzymatic properties. Phases of the ZnO NPs were determined from X-Ray diffraction (XRD) spectra. The shape and size of the green synthesized NPs were investigated by scanning electron microscopy (SEM) and it confirms the cauliflower shape with an average size of 120 nm. But the beads inside the cauliflower shape are 20 nm in size. The prepared ZnO NPs show the cumulative median particle size of 550 nm. The thermogravimetric (TG) and differential thermal analysis (DTA) were performed at different heating rates to study the percentage of weight loss, and activation energy. The activation energy was calculated to be 4.98 kJ/moles using Kissinger method. We also studied the enzyme inhibition effect of ZnO NPs containing biologically active groups on important metabolic enzymes like butyrylcholinesterase(BChE), acetylcholinesterase (AChE) and α-glycosidase (α-Gly) enzymes. The bandgap of ZnO NPs was also studied by UV–Vis spectroscopy via Tauc's method and it was determined to be 3.27 eV. The photocatalytic property of the ZnO NPs was executed against rhodamine B (RhB) under the illumination of an AM 1.5 solar simulator.

Keywords

ZnO nanoparticles
Green synthesis
Alchemilla vulgaris
Metabolic enzyme inhibition
Photocatalytic
Rhodamine B
1

1 Introduction

The recent past had witnessed numerous foremost innovations in the field of nanotechnology with an acceleration speed (Mirzaeia and Darroudi, 2017). Researchers are developing a variety of nanoparticles with improved properties depending upon their types, methods, morphology, size, crystal structures, etc. (Reddy et al., 2012; Rajendrachari et al., 2013; Shashanka, 2017, 2019). The nanomaterials made up of metals, metal oxides (MO’s), nano-graphenes, carbon nanotubes, and nano-composites are the most important nanomaterials can be prepared from different methods with utmost specificity. Among them, the research on MO nanoparticles are marching with significant speed because of their extremely small size, greater surface area, simple preparation methods, unique properties and applications (Hareesha et al., 2019). Generally, they are prepared from chemical, physical and biological methods. But, we have prepared them from biological methods due to its fast, robust, non-toxic nature, and do not require high-priced equipment and special chemicals unlike physical and chemical methods. The plant mediated green synthesise of MO nanoparticles is one of the popular biological method and advantageous due to the wide availability and biodiversity of flora. All the plants consists of phytochemicals such as phenols, flavonoids, aldehydes, and ketones; they converts metal salts into metal oxide nanoparticles due to their reducing power (Shashanka, 2020).

The present method is a one-pot synthesis which improves the reactivity of the reaction in just one pot or glassware or a reactor. This method also reduced the elongated extraction, separation, purification processes. Therefore, one-pot synthesis is becoming more popular and having plenty of advantages over other methods. Hence we have prepared ZnO NP’s using one-pot synthesis green method.

Boroja et al. (2018) studied the amount of different phytochemicals present in Alchemilla vulgaris and reported the high content of phenolic compounds. This enhances their antioxidant capacity, metal chelation, reducing power, enzyme inhibition properties, etc. Therefore, one can expect that the metal oxide nanoparticles prepared from Alchemilla vulgaris (Lady’s mantle) exhibits excellent enzyme inhibition properties. Hence we studied the enzyme inhibition properties of ZnO NP’s in the present paper.

The bio-functional nanomaterials like ZnO have caught the attention of biomedical researchers due to their antibacterial, anti-inflammation, anticancer, drug delivery, diabetes treatment, bio-safe, biocompatibility, photo-catalytic, structure-dependent, electrical, thermal transport properties, wound healing, and bio-imaging properties (Jiang et al., 2018). All these properties are majorly depends on the particle size, morphology, methods of preparation, orientation and ratio (Knoll and Keilmann, 1999; Liz-Marzán et al., 1996). Plant mediated synthesis of ZnO NP’s is preferred over other biological methods due to the combined biomedical properties of both plant extract and the nanoparticles. In the present paper biomedical properties of Alchemilla vulgaris (Lady’s mantle) further improves the properties of ZnO NP’s. Literatures on the green synthesis of ZnO NP’s by various plant leaves are available. But, no literature is reported on the preparation of the same using Alchemilla vulgaris (Lady’s mantle) leaves extract.

The Alchemilla vulgaris is grows in the mountain regions of Europe, North America, Asia and belongs to the Rosaceae family (Tobyn et al., 2011). The plant is specially cultivated for its medicinal properties like anticoagulant, anti-bleeding, anti-inflammatory, antimicrobial, antioxidant properties, and for its wonderful healing power (Tobyn et al., 2011). There are 541 Alchemilla species were registered in Europe and the Mediterranean; Turkey registered with 74 varieties of Alchemilla species majorly found at the northern and the eastern part of Anatolia.

The plant mediated synthesis of nanomaterials can significantly bring greater green revolution for nanomaterial industries where environmental contamination is an important concern (Gültekin et al., 2016). Many researchers are trying to improve this method by using different variety of flora and reaction conditions. Table 1 depicts the ZnO NP’s prepared from different plants by various researchers followed by their important findings.

Table 1 The ZnO nanoparticles prepared from different plants by various researchers.
Plant name Precursor Average size and morphology Featured study References
Deverra tortuosa Zn(NO3)2·6H2O 9–31 nm and almost spherical shape Cytotoxic Activity (Selim et al., 2020)
Tecoma castanifolia ZnSO4 70–75 nm and spherical shape Antioxidant, bactericidal and anticancer activities (Sharmila et al., 2019)
Passiflora caerulea Zn(CH3CO2)2·2H2O 70 nm and spherical shape Anti-bacterial activity against urinary tract infection (Santhoshkumar et al., 2017)
Solanum torvum Zn(NO3)2·6H2O 34–40 nm and spherical shape Evaluation of the toxicological profile (Ezealisiji et al., 2019)
Hibiscus subdariffa Zn(CH3CO2)2·2H2O 300–400 nm and Cauliflower shape Anti-bacterial and anti-diabetic activity (Bala et al., 2015)
Tectona Grandis Zn(NO3)2·6H2O 54 nm and partially spherical Anti-bacterial, anti-arthritic, anti-oxidant and
in-vitro cytotoxicity activities		 (Senthilkumar et al., 2017)
Sambucus ebulus Zn(CH3CO2)2·2H2O 25–30 nm and spherical Antibacterial activity and a photocatalytic degradation of methylene blue dye pollutants (Alamdari et al., 2020)
Peganum
Harmala 		Zn(CH3CO2)2·2H2O 40 nm and spherical Antibacterial activity (Mehar et al., 2019)
Crocus sativus Zn(CH3CO2)2·2H2O 50 nm and spherical The antibacterial
effect was investigated against food pathogens		 (Rahaiee et al., 2020)
Parthenium
Hysterophorus 		Zn(NO3)2·6H2O 16–45 nm and quasi-spherical, radial and cylindrical shape Antibacterial studies (Datta et al., 2017)
Pelargonium zonale Zn(NO3)2·6H2O 50–60 nm and spherical Antibacterial studies (Vahidi et al., 2019)
Tilia Tomentosa Zn(NO3)2·6H2O 80 nm and spherical Dye-sensitized solar cell applications (Shashanka et al., 2020)
Veronica multifidi Zn(CH3CO2)2·2H2O 10–100 nm and nearly hexagonal and quasi-spherical Antimicrobial and antibiofilm activities (Dogan and Kocabas, 2020)
Alchemilla vulgaris Zn(NO3)2·6H2O 120 nm and cauliflower shape Thermal, optical and enzymatic studies (Present paper)

The prepared ZnO NPs are mainly used to inhibit the growth of various isoenzymes. One of the important biological effects of quinazolinone derivatives is to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in brain which causes Alzheimer's disease (Biçer et al., 2019). Cholinesterase (ChE) enzymes mainly catalyzes the hydrolysis of the neurotransmitter ACh restoring the cholinergic pathway at the end of the nerve transmission in the centric neural system (Erdemir et al., 2019). The α-Glycosidase hydrolyzes the glycosidic bonds and converts di- or oligosaccharides into monosaccharides to help in the breakdown and absorption of sugars (Gulçin et al., 2018; Huseynova et al., 2019). Additionally, the green synthesized ZnO NP’s are a strong potential candidate for inhibiting enzymes. Therefore, we studied the effect of ZnO NP’s in inhibiting the some metabolic enzymes.

Additionally, the green synthesized ZnO NPs are also used as a photocatalyst to purify the wastewater from organic toxic pollutants like rhodamine-B. ZnO NPs, which has a direct wide bandgap and a large exciton binding energy (Özgür et al., 2015). Recently, ZnO nanomaterials used in photo-catalytic applications has emerged significantly in the field of environmental applications due to their impressive photo-catalytic activities (Kakarndee and Nanan 2018; Chankhanittha and Nanan 2018, 2021; Juabrum et al., 2019). Among the various techniques of Advanced Oxidation Processes (AOPs) to treat contaminated water, the heterogeneous photo-catalysis process is a promising, efficient, and environmental-friendly process to eliminate toxic pollutants, especially organic compounds which are degraded to harmless products or transform into less toxic (Ahmed and Haider 2018; Chankhanittha et al., 2021). The photo-catalytic property of ZnO materials are utilized in the purification of wastewater from organic toxic pollutants via the heterogeneous photo-catalytic method (Chankhanittha et al., 2019, 2021; Senasu et al., 2021). Bhuyan et al. reported the photo-catalytic activity of biosynthesis of ZnO from Azadirachta indica against methylene blue (MB) under UV irradiation (Bhuyan et al., 2015) and Fu and Fu (2015) investigated biosynthesis of ZnO using leaf extract of Plectranthus amboinicus against methyl red (MR). In this study, by using Alchemilla Vulgaris leaves extract, we prepared ZnO NPs, which were then employed as a photo-catalyst for determining the degradation activity against rhodamine-B (RhB) organic molecule.

2

2 Experimental

2.1

2.1 Chemicals used

Zn(NO3)2·6H2O, NaOH and rhodamine B were purchased from Sigma-Aldrich. All the solutions and plant extract were prepared using RO water.

2.2

2.2 Plant extract of Alchemilla vulgaris

The leaves of Alchemilla vulgaris were purchased from the city market of Bartin, Turkey. Five grams of powdered Alchemilla vulgaris (Lady’s mantle) leaves were weighed and boiled with 100 mL deionized water for 15 min till we get a dark yellow color. Then the solution was filtered to remove any suspended impurities and the aliquots of the solution can be stored at 5˚C. This solution is called as plant extract and it serve as both reducing as well as capping agent. Using this plant extract we have prepared cauliflower shaped ZnO NP’s. Fig. 1 shows the leaves of Alchemilla vulgaris (Lady’s mantle), extract and colloidal solution of ZnO NP’s.

(a) Leaves of Alchemilla vulgaris (Lady’s mantle), (b) Plant extract and (c) Colloidal solution of ZnO NP’s.
Fig. 1
(a) Leaves of Alchemilla vulgaris (Lady’s mantle), (b) Plant extract and (c) Colloidal solution of ZnO NP’s.

2.3

2.3 Synthesise of cauliflower shaped ZnO NP’s

Stir 2.972 g of Zinc nitrate hexahydrate in 100 mL distilled water for 5 min and later added 5 mL of extract till the solution turns yellow. Later, add 1 M NaOH drop by drop till pale yellow color precipitate appears. Centrifuge the solution for 10 min at 7500 rpm and wash the precipitate using water and centrifuge it at least 5 times. Collect the precipitates of ZnO NP’s and dry them at 70˚C for 24 h followed by calcination at 400˚C for 15 min to vaporize the organic impurities. Allow calcined samples to cool down and hand pulverize them using mortar pestle (If needed). Fig. 2 depicts the experimental representation of ZnO NP’s using Alchemilla vulgaris (Lady’s mantle) leaves extract.

The graphical representation of synthesis of cauliflower shaped ZnO NP’s using Alchemilla vulgaris (Lady’s mantle) leaves extract.
Fig. 2
The graphical representation of synthesis of cauliflower shaped ZnO NP’s using Alchemilla vulgaris (Lady’s mantle) leaves extract.

2.4

2.4 Bioactivity studies

BChI and AChI were used as reaction substrate molecules and DTNB was used to determine anticholinesterase activity by Ellman et al., (1961) method (Küçükoğlu et al., 2019; Kuzu et al., 2019). The inhibition effect of glycosidase of ZnO nanoparticle was studied using Tao et al., (2013) process. The 5 μL of the sample and the phosphate buffer (pH 7.4, 75 μL) was combined. After pre-incubation, 50 μL of p-Nitrophenyl-D glycopyranoside (p-NPG) was applied to the phosphate buffer (5 mM, pH 7.4) and re-incubated at 37 °C. The 3 different concentrations of ZnO NP’s were used to de-terminate the Ki values (Taslimi and Gulçin, 2018; Taslimi et al., 2018).

2.5

2.5 Photocatalytic activity

Determining the photocatalytic performance of synthesized zinc oxide particles was executed against rhodamine B (RhB) organic dyes at ambient temperature in atmospheric conditions. The typical photo-catalytic testing dye solution used for determining the photocatalytic activity of synthesized particles was the concentration of 10 mg RhB per Litre. The 40 mg of ZnO NPs were dispersed in 80 mL of a prepared aqueous RhB solution. Prior to illumination under a solar simulator, the dispersed solution was magnetically stirred in the dark for 1 h to confirm the adsorption/desorption equilibrium on the surface of the ZnO nanoparticles. Subsequently, the solution was then irradiated by using an AM1.5 filtered light with a 500 W xenon lamp SHBC XSS-5XD solar simulator under continuously magnetic stirring conditions. At a certain irradiation time interval, 3 mL samples were taken from the solution for the absorption spectra of the solution. After removing synthesized ZnO nanoparticles from an aqueous solution by centrifuge, the absorption characteristics of the solutions at a particular time were determined to measure the degradation rate of RhB organic dye by using a UV–Vis-NIR spectrophotometer.

3

3 Results and discussion

3.1

3.1 Phase analysis:

Fig. 3 depicts the XRD pattern of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves. The planes (1 0 0), (0 0 2) and (1 0 1) were present at 31.82°, 34.47°, and 36.28° diffraction peaks respectively as shown in the figure. This confirms the formation of hexagonal ZnO as confirmed by JCPDF Card No.: 01–070-8072. Even after calcination, the XRD peaks are broadened due to their refined size (Shashanka and Chaira, 2015a, 2015b), and high strain (Gupta et al., 2015; Shashanka et al., 2015; Nayak et al., 2016). The average crystallite size of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves was determined by Scherrer’s formula (Shashanka and Chaira, 2014; Shashanka et al., 2015, 2016);

(1)
D = K λ β cos θ
X-ray diffraction (XRD) pattern of ZnO NP’s synthesized from Alchemilla vulgaris (Lady’s mantle) leaves.
Fig. 3
X-ray diffraction (XRD) pattern of ZnO NP’s synthesized from Alchemilla vulgaris (Lady’s mantle) leaves.

The crystallite sizes of ZnO NP’s were calculated for (1 0 0), (0 0 2) and (1 0 1) planes respectively using equation (1) and it were found to be ∼ 20 nm.

3.2

3.2 Microstructure of ZnO NP’s

The SEM microstructure of biosynthesized ZnO NP’s prepared by Alchemilla vulgaris (Lady’s mantle) leaves is shown in Fig. 4 (a). The SEM microstructure confirms the cauliflower shaped ZnO NP’s with average size of 120 nm. But the beads inside the cauliflower shape exhibit the average particle size of 20 nm. They are monodispersed with no agglomeration due to the natural capping agents like phytochemicals present in the ZnO NP’s. Therefore, no external capping agents were added to reduce the agglomeration; this reduced the use of toxic and expensive chemicals. Hence, this method is considered as one of the less expensive and environmentally friendly methods to prepare ZnO NP’s.

(a) SEM image (b) Energy dispersed spectroscopy (EDS) image of cauliflower shaped ZnO NP’s prepared by Alchemilla vulgaris (Lady’s mantle) leaves.
Fig. 4
(a) SEM image (b) Energy dispersed spectroscopy (EDS) image of cauliflower shaped ZnO NP’s prepared by Alchemilla vulgaris (Lady’s mantle) leaves.

The EDS image of ZnO NP’s prepared by Alchemilla vulgaris (Lady’s mantle) leaves is depicted in Fig. 4 (b). The qualitative and quantitative analysis of ZnO NP’s were carried out using EDS. The atomic % of zinc and oxygen was found to be 50% each; which is very well matches with the theoretical values and they are stoichiometric to each other.

3.3

3.3 UV–visible spectroscopy

Fig. 5 shows the UV–visible spectrum of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves. It uses light in both near UV as well as in near-infrared ranges. Generally, the molecules undergo electronic transitions in the visible range and directly affect the perceived color of the chemicals involved (Shashanka and Ceylan, 2020). The green synthesized ZnO NP’s were sonicated for 2 min in deionized water to get homogeneously dispersed ZnO nanoparticle solution. The sonicated ZnO solution depicts a sharp absorption peak at 362 nm as a result of surface plasmon absorption (SPA). The collective oscillation of the free conduction band electrons results in SPA when electromagnetic radiation colloid with them (Shashanka et al., 2019; Shashanka and Swamy, 2020). The result obtained from UV–Visible spectroscopy in this paper is comparable to the published reports of the authors elsewhere (Shashanka and Swamy, 2020; Shashanka et al., 2020).

UV–visible spectrum of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves.
Fig. 5
UV–visible spectrum of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves.

The bandgap energy of the biosynthesized ZnO NP’s were calculated using the following equation,

(2)
E = h × C λ where E = Bandgap energy

h = Planks constant

C = Speed of light

λ = Cut off wavelength

The intrinsic bandgap energy of 3.43 eV was obtained for cauliflower shaped ZnO NP’s. The presently reported bandgap value of ZnO NP’s is high compared to the published articles elsewhere (Angélica et al., 2016; Senthilkumar et al., 2017; Alamdari et al., 2020). This is due to the nanostructure of the biosynthesized ZnO and this resulted in the gradual decrease in the confined (Davis et al., 2019).

3.4

3.4 FTIR analysis

The spectrum depicts a strong broad peak at 3500 cm−1 which attributes the hydroxyl (O-H) group comprised of stretching and bending vibrations (Mishra et al., 2013). The IR peak at 3730 cm−1 attributed by free hydroxyl group as shown in Fig. 6. The presence of stretching vibrations of polyphenols present in the extract substantiates the capping activity of surface of ZnO NP’s. The presence of small sharp peaks between 2000 and 2200 cm−1 confirms the existence of amino acid (N–H) stretching (Venkateasan et al., 2017). The sharp and intense peaks at 1510 and 1400 cm−1 corresponds to the C = C group and are likely aroused due to the aromatic conjugates of the biomolecules present in the plant extract. IR bands at 1200 cm−1 and 865 cm−1 attributed to the C-O stretching vibration and the tetrahedral coordination of Zn respectively (Jayarambabu et al., 2015).

The FTIR spectrum of biosynthesized ZnO NP’s.
Fig. 6
The FTIR spectrum of biosynthesized ZnO NP’s.

3.5

3.5 Investigation of particle size

The cumulative particle size distribution of ZnO NP’s was successfully studied using particle size analyzer. Added the ZnO NP’s to a pure solvent (water) and sonicated the same for 1 min to get the homogeneous dispersion of nanoparticles and later measured the particle size distribution. Fig. 7 shows the particle size distribution curve of biosynthesized ZnO NP’s. The curve is slightly broad and confirming the different sized ZnO particles ranging from 250 nm to 1 μm. The particle size of maximum number of green synthesized ZnO NP’s were around 550 nm as shown in the graph.

The particle size distribution curve of green synthesized cauliflower shaped ZnO NP’s.
Fig. 7
The particle size distribution curve of green synthesized cauliflower shaped ZnO NP’s.

3.6

3.6 Thermal analysis

Thermal properties of biosynthesized ZnO NP’s were investigated by TG and DTA at 6, 8, and 10˚C/min heating rates between 30 and 1000˚C temperature as shown in Fig. 8 (a), 8 (b) and 8 (c) respectively. The prepared nanoparticles are thermally unstable over a temperature of 30–1000˚C and variation in heating rates. But, showed less weight loss after above 434˚C temperature at all the heating rates; in other words, the prepared ZnO NP’s exhibited a great level of stability at higher temperature (after 434˚C) (Arora et al., 2014). At 6˚C/min, each 0.6% weight loss was observed at 111.5 and 252.1˚C respectively. The resultant weight loss is due to evaporation of water, decomposition of organic, and carbonaceous materials (Shashanka et al., 2020). This resulted in a total weight loss of 2.4% during 6˚C/min heating rate. Similarly, during 8˚C/min heating rate 7.1 and 2.5% of weight loss was observed at 338.1 and 707.7˚C respectively and a total of 10.1% weight loss was observed. At 10˚C/min heating rate, a total of 3.5% weight loss was observed in which 1.7% weight loss at 302.4˚C and 0.9% weight loss at 769.5˚C was recorded respectively. These results confirmed that variation in the heating rate affects the amount of weight loss abruptly over a temperature of 30–1000˚C as shown in the figure. It is also confirmed that 6˚C/min heating rate results in the lesser weight loss and therefore, the biosynthesized ZnO NP’s show greater thermal stability at lower heating rates.

The thermogravimetric analysis (TG) and differential thermal analysis (DTA) curve of biosynthesized nanoparticles at (a) 6 (b) 8, and (c) 10˚C/min heating rates over a temperature range of 30–1000˚C respectively.
Fig. 8
The thermogravimetric analysis (TG) and differential thermal analysis (DTA) curve of biosynthesized nanoparticles at (a) 6 (b) 8, and (c) 10˚C/min heating rates over a temperature range of 30–1000˚C respectively.

We studied the activation energy of ZnO NP’s prepared from Alchemilla vulgaris (Lady’s mantle) leaves using DTA as shown in Fig. 8 (a), 8 (b), and 8 (c) respectively. The green synthesized nanoparticles exhibit exothermic peaks at 666.4, 671.4 and 675.6˚C respectively at 6, 8, and 10˚C/min heating rates. The ZnO nanophase is more stable at high temperatures due to the oxidative decomposition at higher temperatures which resulted in exothermic peaks. These decomposition peaks shift towards higher temperatures with an increase in heating rate as shown in the figure.

The activation energy of the biosynthesized ZnO NP’s were calculated using Kissinger method (Kissinger, 1956). For more detail of method and calculation authors strongly recommend to refer their previous publications (Shashanka and Ceylan, 2020; Shashanka et al., 2020). The Kissinger equation is as follows;

(4)
ln α T P 2 = - E c RT P + C o n s t a n t Where

TP = Decomposition peak temperature,

α = Heating rate

R = Gas constant

Fig. 9 shows activation energy of the green synthesized ZnO NP’s calculated from Kissinger equation and it was found to be 12.92 kJ/moles. Table 2 represents the calculation of activation energy of ZnO NP’s.

The Kissinger plot of green synthesized ZnO NP’s used to calculate the activation energy.
Fig. 9
The Kissinger plot of green synthesized ZnO NP’s used to calculate the activation energy.
Table 2 The calculated values of activation energy of ZnO nanoparticles using Kissinger method.
Type of nanomaterial Heating rate
α
(K/min)		 Peak temperature
TP
(K)		 α T P 2 (X 10-4) l n α T P 2 1000 X 1 T P Activation energy
Ea (KJ/mol)
CuO nanoparticles 279 939.4 3.16 −8.059 1.064 12.92
281 944.4 3.15 −8.062 1.059
283 948.6 3.14 −8.065 1.054

3.7

3.7 Inhibition of metabolic enzymes

AChE and BChE inhibition have been reported for the effective management of AD and the drugs act as cholinesterase inhibitors have recorded various benefits (Zengin et al., 2018). ZnO nanoparticle was very active in inhibiting AChE. The Ki value for AChE was determined to be 5.10 ± 0.47 µM (Table 3). The Ki value for Tacrine (TAC) molecule inhibitting AChE enzyme was determined to be 7.98 ± 1.76 µM. ZnO nanoparticle and TAC values IC50 were: ZnO nanoparticle (6.81 µM, r2: 0.9067) < TAC (10.51 µM, r2: 0.9983) for AChE. Similarly, the Ki values for BChE and the inhibiting property of TAC molecule was found to be 0.28 ± 0.03 and 0.80 ± 0.12 µM respectively (Table 3).

Table 3 The enzyme inhibition results of ZnO nanoparticle against butyrylcholinesterase (BChE), achethylcholinesterase (AChE) and α-glycosidase (α-Gly) enzymes.
Compounds IC50 (µM) Ki (µM)
BChE r2 AChE r2 α-Gly r2 BChE AChE α-Gly
Zn nanoparticle 0.34 0.9348 6.81 0.9067 65.88 0.9460 0.28 ± 0.03 5.10 ± 0.47 70.30 ± 7.92
TAC** 0.88 0.9295 10.51 0.9983 – – 0.80 ± 0.12 7.98 ± 1.76 –
ACR*** – – – – 71.11 0.9406 – – 85.88 ± 9.37

**Tacrine (TAC) was used as a control for AChE and BChE enzymes.

***Acarbose (ACR) was used as a control for α-glycosidase enzyme.

On the other hand, ZnO nanoparticle shown as IC50 and Ki values are 65.88 µM, r2:0.9460, and 70.30 ± 7.92 (Table 3). The α-glycosidase assay exhibited that ZnO nanoparticle has shown an effective α-glycosidase inhibition than acarbose (IC50: 71.11 µM, Ki: 85.88 ± 9.37). Consequently this has prompted the diabetes researchers to look for other potential therapies including α-glycosidase inhibitors (AGIs) to treat T1DM. Recently few reports indeed indicated that AGIs can also treat insulin deficiency caused by T1DM (Taslimi et al., 2017a, 2017b; Gülçin et al., 2019).

3.8

3.8 Optical properties and photocatalytic performance

Fig. 10 represents the optical properties and photo-catalytic testing results of as-synthesized ZnO NPs. In the graphs given in Fig. 10 (a) and 10 (b), the optical behavior of synthesized ZnO NPs, which has critical importance in photocatalytic reactions, has been examined. The wavelength versus absorption spectrum curve given in Fig. 10 (a) is obtained from converting the diffused reflectance (given inset of the graph) data of solid ZnO particles via the Kubelka-Munk (K-M) Reflectance Theory (Kubelka, 1931, 1948). The optical band gap value for as-synthesized ZnO NPs was estimated by using the Tauc method, given by the following equation (Tauc et al., 1966; Tauc, 1974):

(5)
α h ν = A ( h ν - E g ) n
(a) UV–Vis-NIR absorption spectrum of the as-synthesized ZnO derived from the diffused reflectance spectrum (given at inner graphic) (b) the determination of optical band gap value for the as-synthesized ZnO by using Tauc approximation. (Eg, defines an optical band gap of ZnO particles).
Fig. 10
(a) UV–Vis-NIR absorption spectrum of the as-synthesized ZnO derived from the diffused reflectance spectrum (given at inner graphic) (b) the determination of optical band gap value for the as-synthesized ZnO by using Tauc approximation. (Eg, defines an optical band gap of ZnO particles).

In the equation above, α, h, ν, A and Eg are extinction coefficient, which is proportional to F(R), Planck constant (J.s), light frequency (s−1), proportionality constant and optical band gap of the material (eV), respectively. The value of the exponent n defines the nature of the electronic transition between the valence band and conduction band for semiconductor. It is well known in the literature that the band type of ZnO is an allowed direct transition (Özgür et al., 2015). Therefore, the estimated optical band gap value for as-synthesized ZnO nanoparticles was found to be 3.27 eV, as represented in Fig. 10 (b), by extrapolating the straight portion to the energy axis at α = 0.

The photocatalytic activities of ZnO NPs were investigated by monitoring the degradation rate of RhB in the presence of ZnO Photo-catalysts under an AM1.5 filtered light solar simulator. The absorption changes of organic dye molecules in an aqueous solution throughout the photo-catalytic test have been demonstrated in Fig. 11 (a) at indicated interval times. As per Beer-Lambert Law, the maximum absorbance value of obtained peaks should gradually decrease depending on the remaining organic molecule concentration in the solution with taking place the photodegradation reactions in the presence of Photo-catalyst under solar irradiation. Therefore, determining the degradation rate of degraded RhB in the photo-catalytic process was calculated using Beer-Lambert equation (Swinehart, 1962):

(6)
A = ε l c where A is absorbance, ε is absorption coefficient, l is the length of the beam in the medium and c is the concentration of the absorbing species in the medium.
(a) UV–vis absorption spectra of photodegraded RhB solutions during photo-catalytic experiment at given time intervals (b) Photodegradation ratio vs irradiation times for as-synthesized ZnO along with the photolysis of RhB, which is calculated by using Beer-Lambert Law at indicated time intervals (30 min.) (C0 indicates the initial concentration of RhB and Ct indicates concentration of RhB at indicated irradiation time intervals).
Fig. 11
(a) UV–vis absorption spectra of photodegraded RhB solutions during photo-catalytic experiment at given time intervals (b) Photodegradation ratio vs irradiation times for as-synthesized ZnO along with the photolysis of RhB, which is calculated by using Beer-Lambert Law at indicated time intervals (30 min.) (C0 indicates the initial concentration of RhB and Ct indicates concentration of RhB at indicated irradiation time intervals).

By utilizing Beer-Lambert Law, the real-time degradation curves of RhB as a function of irradiation time have been given in Fig. 11 (b) via converting absorption values at the indicated time to concentration. For good surveillance of the performance of as-synthesized ZnO Photo-catalysts, photolysis of RhB under solar illumination (black curve), photolysis of RhB in the presence of H2O2 without any catalysts (blue curve), and photodegradation of RhB with synthesized ZnO Photo-catalysts are presented in Fig. 11 (b). Zero minutes in Fig. 11 indicate the starting point of solar irradiation time after establishing the adsorption/desorption equilibrium on the surface of the ZnO nanoparticles in the dark.

Since organic compounds can be decomposed by ultraviolet light, a reference test, shown as a black curve in Fig. 11 (b), was conducted in order to determine the decomposition of RhB for 2 h under solar irradiation and as a result, no degradation could be observed. The powerful oxidizing agent, such as hydroxyl radicals (•OH), are generated to treat contaminated waters in the method of an advanced oxidation processes (AOPs) (Glaze et al., 1987). In process of the photolysis of H2O2 (H2O2/UV), hydrogen peroxide can be photolyzed to lead producing a highly reactive and nonselective oxidizing agent, such as hydroxyl radicals, by radiation at wavelengths of about 250 nm (Guittonneau et al., 1990). The possible photocatalytic mechanism of ZnO powders against RhB dye is shown in Fig. 12. A decolorization of an organic compound by photolysis of H2O2, adding 0.5 mL H2O2 into 80 mL solution in this experiment, is demonstrated as a blue curve in Fig. 11 (b) under the illumination of an AM1.5 solar simulator and the degradation rate of RhB is observed to be about 34% for illumination of 2 h. In the heterogeneous photocatalytic process, upon illumination, electrons (e-) in conduction (CB) and holes (h+) in valence bands (VB) are generated on the solid surfaces of semiconductor Photo-catalysts. The called Reactive Oxygen Species (ROS), such as singlet oxygen (1O2), superoxide anion radical (•O2–), hydrogen peroxide (H2O2) and hydroxyl radical (•OH), are generated through the reactions with the photoinduced holes and electrons in the aquatic media naturally involving oxygen (Nosaka and Nosaka, 2017). The formation of ROS, resulting in taking place these reactions in aquatic media, plays a primary role in the organic degradation process in the heterogenous photocatalysis (Oturan and Aaron, 2014). Adsorption characteristic of as-synthesized ZnO nanoparticles, as seen in absorption vs wavelength graphic in Fig. 11 (a), is as low as about 3% of all RhB in aqueous solution. The degradation rate of RhB in presence of the as-synthesized ZnO Photo-catalysts particles is shown in a red curve [Fig. 11 (b)]. As demonstrated in figure, the degradation rate is seen to be about 75% of the RhB after illumination of 2 h, which is 2.2x more efficient than in the case of degradation by photolysis of H2O2. Table 4 depicts the photodegradation of various dyes using ZnO nanoparticles reported by various researchers in comparison with the present result.

The photocatalytic mechanism of ZnO powders against RhB dye.
Fig. 12
The photocatalytic mechanism of ZnO powders against RhB dye.
Table 4 Photodegradation of various dyes using ZnO reported by various researchers.
Samples Morphology Energy band gap (eV) Material used to photodegradation Photodegradation efficiency (%) References
PVA capped ZnO Spherical 3.57 RR141 azo dye 42 (Kakarndee and Nanan 2018)
ZnO nanoparticles Hexagonal 3.20 RR141 azo dye
 95 (Chankhanittha and Nanan 2018)
SDS-capped ZnO
 Hexagonal 3.20 RR141 azo dye
 90 (Juabrum et al., 2019)
ZnO/Bi2MoO6 Flower 3.25 Rhodamine B (RhB)
 92 (Chankhanittha and Nanan 2021)
ZnO/CdS nanocomposite Spherical 3.24/2.41 RR141 azo dye
 80 (Senasu et al., 2021)
ZnO/CdS nanocomposite Spherical 3.24/2.41 ofloxacin (OFL) antibiotic, 73 (Senasu et al., 2021)
ZnO nanocatalyst Mixed shape Malachite green 96 (Maureen et al., 2019)
ZnO nanoparticles Spherical 3.30 Methylene Blue and Rhodamine
B		 (Blažeka et al., 2020)
ZnO nanoparticles Cauliflower 3.27 Rhodamine B (RhB)
 75 (Present paper)

4

4 Conclusion

Successfully synthesized cauliflower-shaped ZnO NPs by a simple, robust, and environmentally friendly one-pot biological method using Alchemilla vulgaris (Lady’s mantle) leaves. The biosynthesized ZnO NPs exhibited broad XRD peaks even after calcination and this confirms its nano-crystallinity with an average size of 20 nm. SEM analysis confirmed the cauliflower shape of prepared ZnO NPs with an average size of 120 nm and each beads inside the cauliflower shape exhibit the average particle size of 20 nm. We have not used any capping or reducing agents externally. The IR band at 865 cm−1 attributed the tetrahedral coordination of Zn. The presence of stretching vibrations of polyphenols present in the extract substantiates the capping activity on the surface of ZnO NPs. The high percentage of prepared ZnO NPs exhibits a cumulative particle size of 550 nm. The TG analysis confirmed the abrupt variation in the total weight loss of 2.4%, 10.1% and 3.5% was observed at 6, 8, and 10˚C/min heating rates respectively and this confirms that, the biosynthesized ZnO NPs shows greater thermal stability at lower heating rates. The green synthesized ZnO NPs show exothermic peaks at 666.4, 671.4 and 675.6˚C respectively at 6, 8, and 10˚C/min heating rates. The decomposition peak of ZnO NPs shifts towards higher temperatures with an increase in heating rate where ZnO nanophase becomes more stable. The activation energy of the cauliflower-shaped ZnO NPs was calculated by Kissinger method and it was found to 12.92 kJ/moles. The biosynthesized ZnO NPs showed excellent inhibition effects on AChE, BChE, and α-glycosidase enzymes activities even at low micromolar concentrations. We believe that these results may be useful in the synthesis of new metabolic enzymes inhibitors and in the development of drugs for the treatment of some diseases. The bandgap value was calculated by two different methods and the values were found to be slightly different from each other, as determined 3.43 and 3.27 eV, respectively.. The cauliflower-shaped ZnO photocatalysts synthesized by Alchemilla Vulgaris leaves extract exhibited good photocatalytic activity in the degradation of organic molecules (RhB) in aqueous media under AM1.5 solar illumination.

Acknowledgment

The authors gratefully acknowledge Bartin University Scientific Research Projects Unit, Turkey for providing financial support to conduct the research (Project number: 2019-FEN-A-006).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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