Journal of Drug Delivery and Therapeutics

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Open Access Full Text Article   Research Article

Impact of ZnO nanoparticles on growth and biochemical characteristics of Macrotyloma uniflorum (LAM.) Verdcourt

Balasundari Ponpandi ¹, Aarthi Jeganathan ¹, Sradha Sajeev ¹, Anju Rani George ¹, Kavimani Thangasamy ¹, Madhu Priya Govindhan Anbazhagan ¹ and Geetha Natesan ²*

¹ Research Scholar, Department of Botany, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India

² Professor, Department of Botany, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India

 

 

1. INTRODUCTION

Nanotechnology is an up-and-coming field of science, whose potential is seen in almost every aspect of life. The field of nanotechnology uses various engineered nanoparticles (NPs) and includes various fields of science under one umbrella, including biology, physics, chemistry, and engineering. The areas starting from drug release to wastewater cleanup, soil remediation, and controlled fertilizers come under the purview of nanotechnology. Nanotechnology deals with the synthesis of nanoparticles with controlled size, shape and delivery of materials at the nanometer scale length¹. Nanoparticles have received much consideration because of their novel properties, which differ from those of bulk materials ^2,3.   

Chemical fertilizers increase human and animal food toxicity and environmental pollution and also, they harmfully affect chemical and physical properties of soil, ground water, fauna and ecosystem ⁴. Thus, it is important to use an alternative soil fertility improvement technique to get high plant growth and yield and at the same time make certain ecological sustainability. The use of a variety of NPs is increasing day by day in the field of agriculture, where NPs have found used as fertilizers and stress reducing agents ⁵. During the past decades, various engineered NPs are included in the different fields of agriculture in the form of nano fertilizer, nano pesticides and nano devices in order to increase the effectiveness and sustainability of agricultural practices ^6,7.

Zinc (Zn) is an indispensable micronutrient for both plants and animals owing to its involvement in several enzyme activities and metabolic functions. Zinc deficiency in plants leads to poor yield levels, while, in humans, it contributes to malnutrition and several ailments ^8,9. Also, it poses serious threats to soil and water ecosystem, if it is present more than permissible limit. The permissible quantity of Zn in the soil is 70 to 400 mg/kg ¹⁰. Zn is absorbed in the form of divalent cations and it is important for various vital physiological processes of plants such as synthesis of protein, maintenance of membrane integrity and production of energy ¹¹, biosynthesis of different plant growth hormones ¹², formation of chlorophyll pigment, regulation of starch biosynthesis and root development and activation of enzymes like dehydrogenases, phosphohydrolases, peptidases and proteases ¹³. 

Nanoparticles based nanofertilizers have exhibited significant effects on various aspects of agriculture including seed germination, plant growth, development, enhanced yield ¹⁴, nutritional enrichment ¹⁵, disease ¹⁶ and plant stress management ¹⁷. Nanofertilzers are found effective to crops by releasing the nutrients on-demand with targeted and controlled release ^{18, 19}.   Nanofertilizers are prepared from organic and inorganic nanomaterials by physical or chemical methods using various inorganic metal oxides such as AgO (silver oxide), MgO (magnesium oxide), ZnO, (zinc oxide) and TiO₂ (titanium oxide) and organic nanomaterials like lipids, polymers and carbon nanotubes. In addition to this, biodegradable, biocompatible and eco-friendly, natural biopolymer chitosan is used to synthesize polymeric NPs based fertilizer ²⁰.

Zinc oxide nanoparticles (ZnO NPs) are extensively synthesized and used nanomaterials worldwide with a diameter of 1–100 nm. Due to their small size, ZnO NPs can be absorbed by the stomata and cuticle of root hair cells and inflowing into various plant tissues and promoting plant growth and yield ²¹. Compared to traditional ZnO fertilizers, ZnO NPs have characteristics such as small size, large specific surface area, easy dissolution and diffusion and allowing plants to quickly absorb the Zn released by ZnO NPs. For instance, the application of ZnO NPs has been found to appreciably enhance growth and yield of various plant species ^22,23,24.

Conventional nanoparticle synthesis methods frequently involve utilizing harmful chemicals and high-energy processes, increasing environmental concerns and producing poisonous by-products. Green nanotechnology is emerging an eco-friendly alternative by using microorganisms, plants and agricultural waste as sources for nanoparticle synthesis ^25,26. ZnO NPs are used in different scientific and industrial applications because of their wide band gap and large excitation binding energy ²⁷. Among the physical and chemical methods of synthesis of ZnO NPs, green synthesis using biological materials appear to be highly flexible due to their simplicity, biocompatible and eco-friendly approaches ²⁸. For the green synthesis of nanoparticles, plants are found to be the most suitable organisms. In addition, plants consist of various primary metabolites (protein, chlorophyll, carbohydrates etc.) and secondary metabolites (phenols, flavonoids, tannins, terpenoids, alkaloids etc.). Different functional groups present in these metabolites can react with inorganic metal ions and convert them into metal nanoparticles with nano ranged size ²⁹. Phenolic compounds present in the green tea leaves (Camellia sinensis (L.) Kuntze) show higher antioxidant property and they are found to be very good reducers of metal ions ³⁰. Plants mediated ZnO NPs synthesis has been reported in various plant species. For example, Calatrophis giganta ³¹, Acalypha indica ³², Parthenium hysterophorus ³³, Hibiscus rosasinensis ³⁴, Azardirachta indica ³⁵, Punica granatum ³⁶, Murraya koengii ³⁷, Atalantia monophylla ³⁸ and Piper betel ³⁹. 

Horse gram (Macrotyloma uniflorum (Lam.) Verdcourt (Syn., Dolichos uniflorus Lam., Dolichos biflorus auct. Non L.) ⁴⁰ belonging to Fabaceae, is protein-rich and found acquiescent for cultivation in dry conditions and marginal soil fertility ⁴¹.   In Tamil it is known as kollu. In ayurvedic medicine, whole seeds of horse gram is consumed for the treatment of renal stones, piles, oedema, etc. ⁴². The seeds have been reported to be a important source of iron, molybdenum ⁴³ and protein ⁴⁴. Various studies have reported their anti-hypercholesterolemic ⁴⁵, anti-microbial ^{46, 47}, anti-helmintic ^{48, 49}, anti-diabetic ⁵⁰, anti-inflammatory ⁵¹, anti-oxidant ^{52, 53} and anti-urolithiatic ^{54, 55, 56} properties. Based on literature, it is understood that so far, no studies have been addressed to study the effects of green synthesized ZnO NPs on various growth parameters of M. uniflorum. Hence, the study was conducted to determine the germination capacity, some morphological and biochemical attributes of M. uniflorum after treatment with various concentrations of phytosynthesized ZnO NPs.

2. MATERIALS AND METHODS

2.1 Chemicals

Zinc acetate dihydrate (Zn (CH₃COO)₂. 2H₂O, 99%) was procured from Sigma Aldrich and used for the synthesis of ZnO NPs.   Green tea leaves were purchased from a commercial shop and seeds of horse gram variety Paiyur 2 were purchased from TNAU, Coimbatore and used for the present study. Analytical grade reagents and double distilled water were used for the entire study.

2.2 Preparation of aqueous green leaf extract

The aqueous green tea leaf extract was prepared by boiling 10 g green tea leaves using 100 mL distilled water at 60°C for about 20 minutes, until the colour of the aqueous solution changes from watery to light yellow. Then the extract was cooled at room temperature and filtered using Whatman No.1 filter paper and the filtrate was used for making ZnO NPs.

2.3 Synthesis of ZnO NPs

ZnO NPs were synthesized using green tea leaves by following the method of Shah et al. ⁵⁷ with some minor changes. In brief, 45 mL of 0.1M of zinc acetate solution was mixed with 5 mL of green tea leaf extract to prepare ZnO NPs and the mixture was kept under dark condition at room temperature for overnight. After incubation period, the colour changed solution i.e pale yellow to dark brown was filtered using Whatman No. 1filter paper and the filtrate collected on the filter paper was scraped gently and used for further experiments.

2.4 Characterization of synthesized ZnO NPs     

 The green synthesized ZnO NPs were spectroscopically characterized by UV-Visible spectrophotometer (Shimadzu), Fourier Transform Infrared (Perkin Elmer), Zeta sizer (Malvern analytical, Chennai India) and and Field emission scanning electron microscope (JSM 7610F, JOEL, USA). 

2.5 Treatment of horse gram seeds with ZnO NPs

Overnight soaked seeds of horse gram were surface sterilized with 0.1% sodium hypochlorite solution for 10 min. After sterilization, the seeds were thoroughly rinsed with distilled water to remove excess of chlorine and were placed in petriplate containing absorbent cotton. 10 seeds were sown per petriplate. Different concentrations of green synthesized ZnO NPs i.e 10 – 50 mg/L were taken and dissolved in 100 mL distilled water and then they were added to each petriplate separately. The plates were kept at room temperature along with control (without ZnO NPs) and regularly sprayed with respective nano-formulations for15 days. 

2.6 Percentage of seed germination 

The seed germination percentage was determined based on total number of germinated seeds on fifth day of seed germination (ISTA, 1996). It was calculated by means of the following formula:

Germination percentage (GP %) = (Gf/n) × 100

 Where, Gf is the total number of germinated seeds at the last part of experiment and n is the total number of seed used in the test.

2.7 Measurement of shoot and root lengths

Shoot and root lengths for all the treated plants were measured using scale after 15 days of growth.

2.8 Effects of ZnO NPs on biochemical attributes of horse gram

After 15 days of treatment with ZnO NPs, plants were harvested, then dried and ground with mortar and pestle. The whole plant powder was stored at 4^oC for experimental use.

2.9 Estimation of total chlorophyll, protein, carbohydrate contents

The chlorophyll content in the sample was estimated by Arnon’s method⁵⁸. Lowry’s method⁵⁹ was adopted for the estimation of protein concentration. Total carbohydrate content was estimated by anthrone method ⁶⁰. 

2.10 Quantitative phytochemical analysis

The four important secondary metabolites such as flavonoids, tannins, terpenoids and phenols were quantitatively determined. Total phenolic content of the sample was estimated as described by Singleton and Rossi⁶¹ and modified by Gülçin et al.⁶² Total flavonoid content of the sample was measured following a spectrophotometric method of Dewanto et al.⁶³. The total terpenoid content of the sample was determined based on an assay described by Ghorai et al.⁶⁴ with some modifications. The tannins were determined by Folin-Ciocalteu method.

2.11 Statistical analysis

The mean and standard deviation data obtained from measurement of various treatments in three replicates were statistically analyzed using ANOVA (analysis of variance) and Tukey’s multiple comparison tests to examine the significant difference among various concentrations nanoparticles treatment. P values of <0.05 and lower were considered significant. 

3. RESULTS AND DISCUSSION

3.1 Green synthesis of ZnO NPs 

When green tea leaf boiled extract incubated with 0.1 M Zn acetate, the color changed from pale yellow to light brown initially and after overnight incubation under dark condition, it completely turned into deep brown color (Fig.1). Various phytocompounds present in the green tea leaves act as a reducing as well as stabilizing agents during ZnO NPs synthesis and the color change from pale yellow to dark brown was completed well after overnight incubation. No more color change was observed after this incubation. This indicates that the polyphenol contents of green tea leaves are concerned in the green synthesis and stability of ZnO NPs, particularly a water-soluble phytocompound epigallocatechin gallate acts as both a reducing and stabilizing agent ⁶⁵. Due to the excitation of surface plasmon resonance in ZnO NPs, the color change occurred ⁶⁶.

         

(a)                           (b)

Figure 1: (a) shows ZnO synthesis within few hours, (b) shows ZnO synthesis after overnight

3.2 Characterization of green synthesized ZnO NPs

3.2.1 UV-VIS spectrophotometer analysis

Optical properties of ZnO nanoparticles were characterized using UV–Vis spectrophotometer. The UV–Vis absorption curve of ZnO nanoparticles is shown in fig. 2. Zinc oxide formation was confirmed as the absorption peak (lambda max) was found near 380 nm and similar result was shown by Karimzadeh et al.⁶⁷.

 

Figure 2: UV-VIS spectrum of green synthesized ZnO NPs

3.2.2 Fourier Transform Infrared (FTIR) analysis

FTIR analysis of the synthesized ZnO NPs was carried out at room temperature and a frequency range between 600-4000 cm^-1. The FTIR spectrum (Fig.3) shows the composition of the various bioactive molecules of green tea leaf and their distribution on the surface of the ZnO NPs. There is a broad stretch between 1000 - 4000 cm^-1. The broad stretch indicates the presence of various phytochemicals unique to green tea leaf such as alkyl amine, amides, aromatic ketones, phosphine, carboxyl acid, halogen compound and so on which aid in the stabilization of ZnO NPs. The obtained result was in agreement with the results of Lingegowda et al. ⁶⁸, Lazar et al. ⁶⁹, Hemmalakshmi et al. ⁷⁰.

 

 

 

Figure 3: FTIR spectrum of green synthesized ZnO NPs

 

3.2.3 Zeta Potential 

The Zeta potential of the green synthesized zinc oxide nanoparticles was determined in water as dispersant. The zeta potential of green synthesized ZnO NPs was found to be 8.32 mV as shown in Fig. 4. Zeta potential analysis was carried out to find out the surface charge of biosynthesized ZnO NPs ⁷¹. The result of zeta potential indicates the capping phytocompounds present on the biosynthesized ZnO NPs were mostly comprised of negatively charged groups ⁷². The detected negative charge of the ZnO NPs revealed the electrostatic repulsion between the synthesized nanoparticles ⁷³. A large positive or negative value of zeta potential indicates improved physical colloidal stability due to the electrostatic repulsions between the individual particles. On the other hand, particles with a smaller magnitude of zeta potential may lead to aggregation due to the action of Van der Waals forces ⁷⁴. 

 

 

 

Figure 4: Zeta potential of green-synthesised ZnO NPs

 

3.2.4 SEM analysis

The topography of the green synthesized ZnO NPs was analyzed by SEM ⁷⁵. The image has shown individual semispherical as well as aggregated form of ZnO NPs (Fig.5). Similar result was reported by Wang et al. ⁷⁶ during coffee leaf mediated ZnO NPs synthesis.

 

 

 

Figure 5: SEM micrographs of green-synthesised ZnO NPs synthesized

 

 

3.3 Effects of ZnO NPs on germination, morphological and biochemical attributes of M. uniflorum 

3.3.1 Seed germination percentage

Table 1 shows the data regarding seed germination percent of M. uniflorum treated with different concentrations of ZnO NPs on fifth day of observation. ZnO NPs had significant effect on seed germination percentage (Fig.6 A-D). 10 and 20 mg/L ZnO NPs treated seeds showed the highest germination percentage (100 and 95%, respectively) compared to control seeds (61%). The germination percentage was dropped considerably with the increase of ZnO NPs. The lowest germination percentage was observed in 50 mg/L ZnO NPs treated seeds (40%). There are many reports that the application of nanomaterials at lower doses can enhance seed germination and plant growth ^77,78,79,80. The enhanced plant growth and development is due to the interaction between plant cells and nanoparticles. This interaction leads to changes in various metabolic pathways ²². Small pores are produced when seeds are treated with nanoparticles which in turn boost the water absorption capacity and germination ability of the seeds ^81,82.

Table 1: Germination percentage of M. uniflorum treated with different concentrations of ZnO NPs

Different letters within one column indicate significant difference between treatments

(p<0.01) on DMRT analysis. Values consisted of Mean±SD

 

 

 

 

Figure 6: A- day One; B-Fifth day observation of seedlings of horse gram treated with different concentrations of ZnO NPs; C- Tenth day observation of seedlings of horse gram treated with different concentrations of ZnO NPs; D-Fifteenth day observation of seedlings of horse gram treated with different concentrations of ZnO NPs.

 

 

3.3.2 Shoot and root length of ZnO NPs treated plants

Data pertaining to shoot and root lengths horse gram plants treated with different concentrations of ZnO NPs is presented in the Table 2. 

Increased concentrations of ZnO NPs had noteworthy effect on shoot and root lengths of germinated seedlings of horse gram (Fig.7). With the increase of concentration of NPs, both shoot and root lengths were reduced. The lowest shoot and root lengths were observed in seedlings treated with 50mg/L ZnO NPs (2.26±0.25 for shoot; 3.23±0.25 for root). The highest shoot and root lengths were observed in seedlings treated with 10 mg/L (10.03±0.25 for shoot; 8.46±0.15 for root) followed by 20 mg/L ZnO NPs (9.8.13±0.12 for shoot; 7.02±0.10 for root).
 

 

 

Figure 7: Fifteenth-day-old seedlings of horse gram treated with different concentrations of ZnO NPs used for measurement of shoot and root lengths

 

 

Table 2: Shoot and root length of horse gram treated with different concentrations of ZnO NPs

Treatment (mg/L)	Shoot Length (cm)	Root Length (cm)
Control	4.03±0.55d	2.30±0.30d
10	8.46±0.15b	10.03±0.25a
20	10.03±0.25a	10.63±0.15a
30	7.53±0.15c	4.70±0.20b
40	4.3±0.30d	3.66±1.76c
50	2.26±0.25e	3.23±0.25c

Different letters within one column indicate significant difference between treatments

(p<0.01) on DMRT analysis. Values consisted of Mean±SD.

3.4 Effects of ZnO NPs on biochemical attributes of horse gram

3.4.1 Estimation of total chlorophyll content

Table 3 presents the data regarding chlorophyll contents of horse gram treated with different doses of ZnO NPs. Various concentrations of ZnO NPs had considerable effect on chlorophyll a, chlorophyll b and total chlorophyll contents of germinated seedlings. Compared to control, these contents were gradually increased with the increase of ZnO NPs concentrations. The data revealed that maximum total chlorophyll contents were in plants treated with 10 and 20 mg/L ZnO NPs (17.30±0.005 and 15.85±0.004, respectively). The contents were found reduced gradually from the dose of 30 mg/L ZnO NPs. The amount of chlorophyll a and b in control leaf was less when compared to green synthesized ZnO NPs treated plants. Earlier studies stated that ZnO NPs at lower dose significantly augmented chlorophyll content in Triticum aestivum ⁸³, Cenchrus americanus ⁸⁴, Cicer arietinum ⁸², Cyamopsis tetragonoloba ⁸⁵ and Persicaria hydropiper ⁸⁶. Enhanced chlorophyll contents are due to zinc which acts as a structural and catalytic component of proteins and enzymes and as a co-factor for the characteristic development of pigment biosynthesis ⁸⁷.

 

 

Table 3: Total chlorophyll contents of horse gram treated with different concentrations of ZnO NPs

S.No	Concentration (mg/mL)	Chlorophyll a (mg/g)	Chlorophyll b (mg/g)	Total chlorophyll (mg/g)
1	Control	3.07±0.002f	1.56±0.003f	4.64±0.005f
2	10	14.01±0.003a	3.29±0.001c	17.30±0.005a
3	20	11.06±0.002b	4.79±0.002a	15.85±0.004b
4	30	8.86±0.003c	3.56±0.003b	12.43±0.006c
5	40	5.90±0.002d	2.60±0.002e	8.50±0.004d
6	50	5.41±0.012e	2.71±0.003d	8.13±0.015e

Different letters within one column indicate significant difference between treatments (p<0.01) on DMRT analysis. Values consisted of Mean±SD.

 

 

3.4.2 Determination of total protein 

The result presented in the fig. 8 shows the total protein content of germinated seedlings of horse gram treated with different concentrations of ZnO NPs. The plants treated with 10  and 20 mg/L ZnO NPs had significantly higher level of protein compared to control. Protein content was found to be reduced gradually when doses increased from 30 mg/L.   These findings were in accordance with Raliya and Tarafdar ⁸⁵, Tarafdar et al. ⁸⁴ and Mukherjee et al. ⁸⁰ who showed ZnO NPs at lower dose significantly enhanced the total protein content in cluster bean, pearl millet and green pea, respectively. Zinc is essential as structural and catalytic constituent of protein and enzymes for normal growth and development of plants ⁸⁸.

 

 

Figure 8: Total protein content of M. uniflorum treated with different concentrations of ZnO NPs 

 

 

3.4.3 Determination of total carbohydrate

Fig. 9 presents the mean data regarding the effect of ZnO NPs on total carbohydrate content of germinated seedlings of M. uniflorum. The results show that ZnO NPs has a significant rising effect on carbohydrate levels in all the germinated seedlings. The data indicated that maximum carbohydrate contents were in plants treated with 10 and 20 mg/L ZnO NPs 360.0±14.93 and 240.3±1.15, respectively). Similar results were shown in ZnO NPs on growth of sesamum indicum L. ⁸⁹. A higher level of total carbohydrate was noted at a lower dose of ZnO nanoparticles treated with Sesamum indicum ⁸⁹ and Cicer arietinum seedlings ⁸².

 

 

Figure 9: Total carbohydrate content of M. uniflorum treated with different concentrations of ZnO NPs.

 

3.4.4 Quantitative phytochemical analysis

Table 4 presents the mean data regarding the effect of ZnO NPs on four secondary metabolites i.e total phenolic, flavonoid, terpenoid and tannin contents of germinated seedlings of M. uniflorum. The data revealed that maximum level these contents were in plants treated with 10 mg/L ZnO NPs. Generally, the application of NPs in lower concentrations has been found to enhance plant growth and secondary metabolite production⁹⁰.

 

 

Table 4: Quantitative analysis of secondary metabolites of M. uniflorum treated with different concentrations of ZnO NPs

Different letters within one column indicate significant difference between treatments (p<0.01) on DMRT analysis. Values consisted of Mean±SD

 

 

 

 

4. CONCLUSION

This study presents the effect of green synthesized ZnO NPs using green leaves at different concentrations on Macrotyloma uniflorum (kollu) seed germination, shoot and root lengths and some primary and secondary metabolites. Maximum levels of germination percentage, shoot and root lengths, primary metabolites such as total chlorophyll, protein, carbohydrates and secondary metabolites such as total phenols, flavonoids, tannins and terpenoids were noted in 10.0 mg/L ZnO NPs treated plants compared to control. With the increase of concentrations ZnO NPs, all the parameters were found to be decreased. These findings indicate that the application of ZnO NPs at a lower concentration may increase germination, morphological and biochemical attributes of M. uniflorum. Further investigations are required to understand the mechanisms at a molecular level for ZnO NPs to be used as nanofertilizers or growth stimulants for this plant. 

Ethics statement: As the seed materials of Macrotyloma uniflorum (Lam.) Verdcourt variety Paiyur 2 were purchased from TNAU, Coimbatore 641 046, Tamil Nadu, India, ethical permission is not required.

Author contributions: Balasundari Ponpandi and Aarthi Jeganathan carried out the experiments. Both Sradha Sajeev and Anju Rani George written and edited the whole manuscript, Kavimani Thangasamy and Madhu Priya Govindhan Anbazhagan assisted in making figures and other related work. Geetha Natesan designed the experiments and supervised.

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

Acknowledgements: The authors duly acknowledge funding agencies i.e DST-FIST and DST-PURSE, India for providing all the instrumentation facilities for carrying out the research work in the Dept. of Botany, Bharathiar University, Coimbatore, Tamil Nadu, India.

Funding: Not applicable

Availability of data and materials: The authors confirm that the data supporting the findings of this study are available within the articles and its supplementary material. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.

REFERENCES

1. Li YF, Liu ZP, Particle size, shape and activity for photocatalysis on titania anatase nanoparticles in aqueous surroundings, Journal of the American Chemical Society, 2011; 133(39):15743-15752. https://doi.org/10.1021/ja206153v PMid:21879719

2. Baron PA, Deye GJ, Chen BT, Schwegler-Berry DE, Shvedova AA, Castranova V, Aerosolization of single-walled carbon nanotubes for an inhalation study, Inhalation Toxicology, 2008; 20(8):751-760. https://doi.org/10.1080/08958370801975303 PMid:18569097

3. Moghaddam AB, Ganjali MR, Dinarvand R, Razavi T, Saboury AA, Moosavi-Movahedi AA, Norouzi P, Direct electrochemistry of cytochrome c on electrodeposited nickel oxide nanoparticles, Journal of Electroanalytical Chemistry, 2008; 614(1-2):83-92. https://doi.org/10.1016/j.jelechem.2007.11.011

4. Camargo JA, Alonso Á, Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment, Environment International, 2006; 32(6):831-849. https://doi.org/10.1016/j.envint.2006.05.002 PMid:16781774

5. Nowack B, The behaviour and effects of nanoparticles in the environment, Environmental Pollution, 2009; 157(4):1063-1185. https://doi.org/10.1016/j.envpol.2008.12.019 PMid:19157660

6. Parisi C, Vigani M, Rodríguez-Cerezo E, Agricultural nanotechnologies: what are the current possibilities?, Nano Today, 2015; 10(2):124-127. https://doi.org/10.1016/j.nantod.2014.09.009

7. Liu R, Lal R, Potentials of engineered nanoparticles as fertilizers for increasing agronomic productions, Science of the Total Environment, 2015; 514:131-139. https://doi.org/10.1016/j.scitotenv.2015.01.104 PMid:25659311

8. Stietiya MH, Wang JJ, Zinc and cadmium adsorption to aluminum oxide nanoparticles affected by naturally occurring ligands, Journal of Environmental Quality, 2014; 43(2):498-506. https://doi.org/10.2134/jeq2013.07.0263 PMid:25602651

9. Srivastava PC, Rawat D, Pachauri SP, Shrivastava M, Strategies for enhancing zinc efficiency in crop plants, Nutrient Use Efficiency: From Basics to Advances, 2015; 87-101. https://doi.org/10.1007/978-81-322-2169-2_7

10. Kabata-Pendias A, Pendias H, Trace Elements in Soils and Plants, Boca Raton, FL, USA: CRC Press, 2010. https://doi.org/10.1201/b10158

11. Hänsch R, Mendel RR, Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl), Current Opinion in Plant Biology, 2009; 12(3):259-266. https://doi.org/10.1016/j.pbi.2009.05.006 PMid:19524482

12. Mansour E, Casas AM, Gracia MP, Molina-Cano JL, Moralejo M, Cattivelli L, Igartua E, Quantitative trait loci for agronomic traits in an elite barley population for Mediterranean conditions, Molecular Breeding, 2014; 33:249-265. https://doi.org/10.1007/s11032-013-9946-5

13. Wassel AH, El-Hameed MA, Gobara A, Attia M, Effect of some micronutrients, gibberellic acid and ascorbic acid on growth, yield and quality of white Banaty seedless grapevines, 8th African Crop Science Society Conference, El-Minia, Egypt, 27-31 October 2007; 547-553.

14. Haydar MS, Ghosh S, Mandal P, Application of iron oxide nanoparticles as micronutrient fertilizer in mulberry propagation, Journal of Plant Growth Regulation, 2021; 1-21. https://doi.org/10.1007/s00344-021-10413-3

15. Elemike EE, Uzoh IM, Onwudiwe DC, Babalola OO, The role of nanotechnology in the fortification of plant nutrients and improvement of crop production, Applied Sciences, 2019; 9(3):499. https://doi.org/10.3390/app9030499

16. Worrall EA, Hamid A, Mody KT, Mitter N, Pappu HR, Nanotechnology for plant disease management, Agronomy, 2018; 8(12):285. https://doi.org/10.3390/agronomy8120285

17. Manzoor N, Ali L, Ahmed T, Rizwan M, Ali S, Shahid MS, Wang G, Silicon oxide nanoparticles alleviate chromium toxicity in wheat (Triticum aestivum L.), Environmental Pollution, 2022; 315:120391. https://doi.org/10.1016/j.envpol.2022.120391 PMid:36223852

18. Nair R, Varghese SH, Nair BG, Maekawa T, Yoshida Y, Kumar DS, Nanoparticulate material delivery to plants, Plant Science, 2010; 179(3):154-163. https://doi.org/10.1016/j.plantsci.2010.04.012

19. Prajapati SK, Maurya SD, Das MK, Tilak VK, Verma KK, Dhakar RC, Dendrimers in drug delivery, diagnosis and therapy: basics and potential applications, Journal of Drug Delivery and Therapeutics. 2016;6(1):67-92 https://doi.org/10.22270/jddt.v6i1.1190

20. Ghormade V, Deshpande MV, Paknikar KM, Perspectives for nano-biotechnology enabled protection and nutrition of plants, Biotechnology Advances, 2011; 29(6):792-803. https://doi.org/10.1016/j.biotechadv.2011.06.007 PMid:21729746

21. Su Y, Ashworth V, Kim C, Adeleye AS, Rolshausen P, Roper C, Jassby D, Delivery, uptake, fate, and transport of engineered nanoparticles in plants: a critical review and data analysis, Environmental Science: Nano, 2019; 6(8):2311-2331. https://doi.org/10.1039/C9EN00461K

22. Venkatachalam P, Priyanka N, Manikandan K, Ganeshbabu I, Indiraarulselvi P, Geetha N, Sahi SV, Enhanced plant growth promoting role of phycomolecules coated zinc oxide nanoparticles with P supplementation in cotton (Gossypium hirsutum L.), Plant Physiology and Biochemistry, 2017; 110:118-127. https://doi.org/10.1016/j.plaphy.2016.09.004 PMid:27622847

23. Hussain A, Ali S, Rizwan M, ur Rehman MZ, Javed MR, Imran M, Nazir R, Zinc oxide nanoparticles alter the wheat physiological response and reduce the cadmium uptake by plants, Environmental Pollution, 2018; 242:1518-1526. https://doi.org/10.1016/j.envpol.2018.08.036 PMid:30144725

24. Srivastav A, Ganjewala D, Singhal RK, Rajput VD, Minkina T, Voloshina M, Shrivastava M, Effect of ZnO nanoparticles on growth and biochemical responses of wheat and maize, Plants, 2021; 10(12):2556. https://doi.org/10.3390/plants10122556 PMid:34961025 PMCid:PMC8708393

25. Karim N, Liu S, Rashwan AK, Xie J, Mo J, Osman AI, Chen W, Green synthesis of nanolipo-fibersomes using Nutriose® FB 06 for delphinidin-3-O-sambubioside delivery: Characterization, physicochemical properties, and application, International Journal of Biological Macromolecules, 2023; 247:125839. https://doi.org/10.1016/j.ijbiomac.2023.125839 PMid:37454997

26. Xu Y, Rashwan AK, Osman AI, Abd El-Monaem EM, Elgarahy AM, Eltaweil AS, Rooney DW, Synthesis and potential applications of cyclodextrin-based metal-organic frameworks: a review, Environmental Chemistry Letters, 2023; 21(1):447-477. https://doi.org/10.1007/s10311-022-01509-7 PMid:36161092 PMCid:PMC9484721

27. Chaudhuri SK, Malodia L, Biosynthesis of zinc oxide nanoparticles using leaf extract of Calotropis gigantea: characterization and its evaluation on tree seedling growth in nursery stage, Applied Nanoscience, 2017; 7(8):501-512. https://doi.org/10.1007/s13204-017-0586-7

28. Kajbafvala A, Ghorbani H, Paravar A, Samberg JP, Kajbafvala E, Sadrnezhaad SK, Effects of morphology on photocatalytic performance of zinc oxide nanostructures synthesized by rapid microwave irradiation methods, Superlattices and Microstructures, 2012; 51(4):512-522. https://doi.org/10.1016/j.spmi.2012.01.015

29. Naseer M, Aslam U, Khalid B, Chen B, Green route to synthesize zinc oxide nanoparticles using leaf extracts of Cassia fistula and Melia azadarach and their antibacterial potential, Scientific Reports, 2020; 10(1):1-10. https://doi.org/10.1038/s41598-020-65949-3 PMid:32493935 PMCid:PMC7270115

30. Dhanemozhi AC, Rajeswari V, Sathyajothi S, Green synthesis of zinc oxide nanoparticle using green tea leaf extract for supercapacitor application, Materials Today: Proceedings, 2017; 4(2):660-667. https://doi.org/10.1016/j.matpr.2017.01.070

31. Vidya C, Hiremath S, Chandraprabha MN, Antonyraj ML, Gopal IV, Jain A, Bansal K, Green synthesis of ZnO nanoparticles by Calotropis gigantea, Int J Curr Eng Technol, 2013; 1(1):118-120.

32. Gnanasangeetha D, Thambavani DS, Biogenic production of zinc oxide nanoparticles using Acalypha indica, Journal of Chemical, Biological and Physical Sciences (JCBPS), 2013; 4(1):238.

33. Sri Sindhura K, Prasad TNVKV, Panner Selvam P, Hussain OM, Synthesis, characterization and evaluation of effect of phytogenic zinc nanoparticles on soil exo-enzymes, Applied Nanoscience, 2014; 4:819-827. https://doi.org/10.1007/s13204-013-0263-4

34. Devi RS, Gayathri R, Green synthesis of zinc oxide nanoparticles by using Hibiscus rosa-sinensis, Int J Curr Eng Technol, 2014; 4(4):2444-2446.

35. Oudhia A, Kulkarni P, Sharma S, Green synthesis of ZnO nanotubes for bioapplications, Int J Adv Engg Res Studies, 2015; IV(II):280-281.

36. Mishra V, Sharma R, Green synthesis of zinc oxide nanoparticles using fresh peels extract of Punica granatum and its antimicrobial activities, International Journal of Pharma Research and Health Sciences, 2015; 3(3):694-699.

37. Divyapriya S, Sowmia C, Sasikala S, Synthesis of zinc oxide nanoparticles and antimicrobial activity of Murraya koeiniggi, 2014.

38. Vijayakumar S, Mahadevan S, Arulmozhi P, Sriram S, Praseetha PK, Green synthesis of zinc oxide nanoparticles using Atalantia monophylla leaf extracts: Characterization and antimicrobial analysis, Materials Science in Semiconductor Processing, 2018; 82:39-45. https://doi.org/10.1016/j.mssp.2018.03.017

39. Harikrishnan A, Ramalingam B, Nadeem A, Ramachandran B, Veena VK, Muthupandian S, Eco-friendly synthesis of zinc oxide nanoparticles (ZnOnps) from Piper betel leaf extract: spectral characterization and its application on plant growth parameters in maize, fenugreek and red gram, Materials Technology, 2024; 39(1):2298547. https://doi.org/10.1080/10667857.2023.2298547

40. Chahota RK, Sharma SK, Sharma TR, Kumar NARESH, Kapoor C, Induction and characterisation of agronomically useful mutants in horsegram (Macrotyloma uniflorum), Indian J Agric Sci, 2013; 83(10):1105-1109.

41. Kiranmai K, Gunupuru LR, Nareshkumar A, Reddy VA, Lokesh U, Pandurangaiah M, Sudhakar C, Expression analysis of WRKY transcription factor genes in response to abiotic stresses in Horsegram (Macrotyloma uniflorum (Lam.) Verdc.), 2016. https://doi.org/10.4236/ajmb.2016.64013

42. Singh M, Upadhyaya HD, Bisht IS, Genetic and genomic resources of grain legume improvement, Newnes, 2013.

43. Kadam SS, Salunkhe DK, Maga JA, Nutritional composition, processing, and utilization of horse gram and moth bean, Critical Reviews in Food Science & Nutrition, 1985; 22(1):1-26. https://doi.org/10.1080/10408398509527407

44. Bravo L, Siddhuraju P, Saura-Calixto F, Composition of underexploited Indian pulses. Comparison with common legumes, Food Chemistry, 1999; 64(2):185-192. https://doi.org/10.1016/S0308-8146(98)00140-X

45. Kumar DS, Prashanthi G, Avasarala H, Banji D, Antihypercholesterolemic effect of Macrotyloma uniflorum (Lam.) Verdc (Fabaceae) extract on high-fat diet-induced hypercholesterolemia in Sprague-Dawley rats, Journal of Dietary Supplements, 2013; 10(2):116-128. https://doi.org/10.3109/19390211.2013.790334 PMid:23725525

46. Ram AJ, Bhakshu LM, Raju RV, In vitro antimicrobial activity of certain medicinal plants from Eastern Ghats, India, used for skin diseases, Journal of Ethnopharmacology, 2004; 90(2-3):353-357. https://doi.org/10.1016/j.jep.2003.10.013 PMid:15013201

47. Kawsar SMA, Serajuddin M, Huq E, Nahar N, Ozeki Y, Biological investigation of Macrotyloma uniflorum Linn. extracts against some pathogens, J Biol Sci, 2008; 8(6):1051-1056. https://doi.org/10.3923/jbs.2008.1051.1056

48. Philip A, Athul PV, Charan A, Afeefa TP, Anthelmintic activity of seeds of Macrotyloma uniflorum, Hygeia, 2009; 1(1):26-27.

49. Sree VK, Soundarya M, Ravikumar M, Reddy TR, Devi NKD, In vitro screening of Macrotyloma uniflorum extracts for antioxidant and anthelmintic activities, Journal of Pharmacognosy and Phytochemistry, 2014; 3(4).

50. Gupta LH, Badole SL, Bodhankar SL, Sabharwal SG, Antidiabetic potential of α-amylase inhibitor from the seeds of Macrotyloma uniflorum in streptozotocin-nicotinamide-induced diabetic mice, Pharmaceutical Biology, 2011; 49(2):182-189. https://doi.org/10.3109/13880209.2010.507633 PMid:21043992

51. Giresha AS, Narayanappa M, Joshi V, Vishwanath BS, Dharmappa KK, Human secretory phospholipase A2 (sPLA2) inhibition by aqueous extract of Macrotyloma uniflorum (seed) as anti-inflammatory activity, IJPPS, 2015; 7:217-222.

52. Singh R, Singh MK, Chandra LR, Bhat D, Arora MS, Nailwal T, Pande V, In vitro antioxidant and free radical scavenging activity of Macrotyloma uniflorum (Gahat dal) from Kumauni region, International Journal of Fundamental and Applied Science, 2012; 1(1):7-10. https://doi.org/10.59415/ijfas.v1i1.19

53. Panda V, Suresh S, Gastro-protective effects of the phenolic acids of Macrotyloma uniflorum (horse gram) on experimental gastric ulcer models in rats, Food Bioscience, 2015; 12:34-46. https://doi.org/10.1016/j.fbio.2015.07.004

54. Das I, Gupta SK, Ansari SA, Pandey VN, Rastogi RP, In vitro inhibition and dissolution of calcium oxalate by edible plant Trianthema monogyna and pulse Macrotyloma uniflorum extracts, Journal of Crystal Growth, 2005; 273(3-4):546-554. https://doi.org/10.1016/j.jcrysgro.2004.09.038

55. Chaitanya DAK, Kumar MS, Reddy AM, Mukherjee AM, Sumanth MH, Ramesh A, Anti urolithiatic activity of Macrotyloma uniflorum seed extract on ethylene glycol induced urolithiasis in albino rats, J Innov Trends Pharm Sci, 2010; 1:216-226.

56. Atodariya U, Barad R, Upadhyay S, Upadhyay U, Anti-urolithiatic activity of Dolichos biflorus seeds, Journal of Pharmacognosy and Phytochemistry, 2013; 2(2):209-213.

57. Shah M, Fawcett D, Sharma S, Tripathy SK, Poinern GEJ, Green synthesis of metallic nanoparticles via biological entities, Materials, 2015; 8(11):7278-7308. https://doi.org/10.3390/ma8115377 PMid:28793638 PMCid:PMC5458933

58. Arnon DI, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris, Plant Physiology, 1949; 24(1):1. https://doi.org/10.1104/pp.24.1.1 PMid:16654194 PMCid:PMC437905

59. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, Total protein estimation by Lowry's method, J Biol Chem, 1951; 193:265. https://doi.org/10.1016/S0021-9258(19)52451-6 PMid:14907713

60. Hedge JE, Hofreiter BT, Whistler RL, Carbohydrate chemistry, Academic Press, New York, 1962; 17:371-380.

61. Singleton VL, Rossi JA, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, American Journal of Enology and Viticulture, 1965; 16(3):144-158. https://doi.org/10.5344/ajev.1965.16.3.144

62. Gülçin I, Bursal E, Şehitoğlu MH, Bilsel M, Gören AC, Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey, Food and Chemical Toxicology, 2010; 48(8-9):2227-2238. https://doi.org/10.1016/j.fct.2010.05.053 PMid:20685228

63. Dewanto V, Wu X, Adom KK, Liu RH, Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity, Journal of Agricultural and Food Chemistry, 2002; 50(10):3010-3014. https://doi.org/10.1021/jf0115589 PMid:11982434

64. Ghorai N, Chakraborty S, Gucchait S, Saha SK, Biswas S, Estimation of total terpenoids concentration in plant tissues using a monoterpene, Linalool as standard reagent, 2012. https://doi.org/10.1038/protex.2012.055

65. Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M, Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract, Biotechnology Progress, 2006; 22(2):577-583. https://doi.org/10.1021/bp0501423 PMid:16599579

66. Sutradhar P, Saha M, Synthesis of zinc oxide nanoparticles using tea leaf extract and its application for solar cell, Bulletin of Materials Science, 2015; 38(3):653-657. https://doi.org/10.1007/s12034-015-0895-y

67. Karimzadeh MR, Soltanian S, Sheikhbahaei M, Mohamadi N, Characterization and biological activities of synthesized zinc oxide nanoparticles using the extract of Acantholimon serotinum, Green Processing and Synthesis, 2020; 9(1):722-733. https://doi.org/10.1515/gps-2020-0058

68. Lingegowda DC, Kumar JK, Prasad AD, Zarei M, Gopal S, FTIR spectroscopic studies on Cleome gynandra-comparative analysis of functional group before and after extraction, Romanian Journal of Biophysics, 2012; 22(3-4):137-143.

69. Lazar G, Ureche D, Ifrim IL, Stamate M, Ureche C, Nedeff V, Lazar IM, Effects of the environmental stress on two fish populations revealed by statistical and spectral analysis, Environmental Engineering & Management Journal (EEMJ), 2012; 11(1). https://doi.org/10.30638/eemj.2012.016

70. Hemmalakshmi S, Priyanga S, Devaki K, Fourier Transform Infra-Red Spectroscopy Analysis of Erythrina variegata L, Journal of Pharmaceutical Sciences and Research, 2017; 9(11):2062-2067.

71. Hamk M, Akçay FA, Avcı A, Green synthesis of zinc oxide nanoparticles using Bacillus subtilis ZBP4 and their antibacterial potential against foodborne pathogens, Preparative Biochemistry & Biotechnology, 2023; 53(3):255-264. https://doi.org/10.1080/10826068.2022.2076243 PMid:35616319

72. Barzinjy AA, Azeez HH, Green synthesis and characterization of zinc oxide nanoparticles using Eucalyptus globulus Labill. leaf extract and zinc nitrate hexahydrate salt, SN Applied Sciences, 2020; 2(5):991. https://doi.org/10.1007/s42452-020-2813-1

73. Al-Kordy HM, Sabry SA, Mabrouk ME, Statistical optimization of experimental parameters for extracellular synthesis of zinc oxide nanoparticles by a novel haloalaliphilic Alkalibacillus sp. W7, Scientific Reports, 2021; 11(1):10924. https://doi.org/10.1038/s41598-021-90408-y PMid:34035407 PMCid:PMC8149680

74. Agarwal H, Nakara A, Menon S, Shanmugam V, Eco-friendly synthesis of zinc oxide nanoparticles using Cinnamomum Tamala leaf extract and its promising effect towards the antibacterial activity, Journal of Drug Delivery Science and Technology, 2019; 53:101212. https://doi.org/10.1016/j.jddst.2019.101212

75. Srujana S, Anjamma M, Alimuddin, Singh B, Dhakar RC, Natarajan S, Hechhu R. A Comprehensive Study on the Synthesis and Characterization of TiO2 Nanoparticles Using Aloe vera Plant Extract and Their Photocatalytic Activity against MB Dye. Adsorption Science & Technology. 2022;2022 https://doi.org/10.1155/2022/7244006

76. Wang Q, Mei S, Manivel P, Ma H, Chen X, Zinc oxide nanoparticles synthesized using coffee leaf extract assisted with ultrasound as nanocarriers for mangiferin, Current Research in Food Science, 2022; 5:868-877. https://doi.org/10.1016/j.crfs.2022.05.002 PMid:35647560 PMCid:PMC9133588

77. Prasad TNVKV, Sudhakar P, Sreenivasulu Y, Latha P, Munaswamy V, Reddy KR, Pradeep T, Effect of nanoscale zinc oxide particles on the germination, growth and yield of peanut, Journal of Plant Nutrition, 2012; 35(6):905-927. https://doi.org/10.1080/01904167.2012.663443

78. Hu C, Liu Y, Li X, Li M, Biochemical responses of duckweed (Spirodela polyrhiza) to zinc oxide nanoparticles, Archives of Environmental Contamination and Toxicology, 2013; 64:643-651. https://doi.org/10.1007/s00244-012-9859-z PMid:23271345

79. Mousavi Kouhi SM, Lahouti M, Ganjeali A, Entezari MH, Comparative effects of ZnO nanoparticles, ZnO bulk particles, and Zn2+ on Brassica napus after long-term exposure: changes in growth, biochemical compounds, antioxidant enzyme activities, and Zn bioaccumulation, Water, Air, & Soil Pollution, 2015; 226:1-11. https://doi.org/10.1007/s11270-015-2628-7

80. Mukherjee A, Sun Y, Morelius E, Tamez C, Bandyopadhyay S, Niu G, Gardea-Torresdey JL, Differential toxicity of bare and hybrid ZnO nanoparticles in green pea (Pisum sativum L.): a life cycle study, Frontiers in Plant Science, 2016; 6:1242. https://doi.org/10.3389/fpls.2015.01242 PMid:26793219 PMCid:PMC4710101

81. Song U, Jun H, Waldman B, Roh J, Kim Y, Yi J, Lee EJ, Functional analyses of nanoparticle toxicity: a comparative study of the effects of TiO2 and Ag on tomatoes (Lycopersicon esculentum), Ecotoxicology and Environmental Safety, 2013; 93:60-67. https://doi.org/10.1016/j.ecoenv.2013.03.033 PMid:23651654

82. Hajra A, Mondal NK, Effects of ZnO and TiO2 nanoparticles on germination, biochemical and morphoanatomical attributes of Cicer arietinum L., Energy, Ecology and Environment, 2017; 2(4):277-288. https://doi.org/10.1007/s40974-017-0059-6

83. Solanki P, Laura JS, Effect of ZnO nanoparticles on seed germination and seedling growth in wheat (Triticum aestivum), Journal of Pharmacognosy and Phytochemistry, 2018; 7(5):2048-2052.

84. Tarafdar JC, Raliya R, Mahawar H, Rathore I, Development of zinc nanofertilizer to enhance crop production in pearl millet (Pennisetum americanum), Agricultural Research, 2014; 3(3):257-262. https://doi.org/10.1007/s40003-014-0113-y

85. Raliya R, Tarafdar JC, ZnO nanoparticle biosynthesis and its effect on phosphorous-mobilizing enzyme secretion and gum contents in Clusterbean (Cyamopsis tetragonoloba L.), Agricultural Research, 2013; 2(1):48-57. https://doi.org/10.1007/s40003-012-0049-z

86. Hussain F, Hadi F, Rongliang Q, Effects of zinc oxide nanoparticles on antioxidants, chlorophyll contents, and proline in Persicaria hydropiper L. and its potential for Pb phytoremediation, Environmental Science and Pollution Research, 2021; 28(26):34697-34713. https://doi.org/10.1007/s11356-021-13132-0 PMid:33655481

87. Balashouri P, Effect of zinc on germination, growth and pigment content and phytomass of Vigna radiata and Sorghum bicolor, J Ecobiol, 1995; 7:109-114.

88. Broadley MR, White PJ, Hammond JP, Zelko I, Lux A, Zinc in plants, New Phytologist, 2007; 173(4):677-702. https://doi.org/10.1111/j.1469-8137.2007.01996.x PMid:17286818

89. Narendhran S, Rajiv P, Sivaraj R, Toxicity of ZnO nanoparticles on germinating Sesamum indicum (Co-1) and their antibacterial activity, Bulletin of Materials Science, 2016; 39(2):415-421. https://doi.org/10.1007/s12034-016-1172-4

90. Karimi N, Behbahani M, Dini G, Razmjou A, Enhancing the secondary metabolite and anticancer activity of Echinacea purpurea callus extracts by treatment with biosynthesized ZnO nanoparticles, Advances in Natural Sciences: Nanoscience and Nanotechnology, 2018; 9(4):045009. https://doi.org/10.1088/2043-6254/aaf1af