Biological properties and Acute Toxicity Study of Copper oxide nanoparticles prepared by aqueous leaves extract of Portulaca oleracea (L)

Atoussi Ouidad¹, Chetehouna Sara¹, Derouiche Samir^1,2

¹Department of Cellular and Molecular Biology, Faculty of Natural Science Sand Life,

University of El Oued, El-Oued 39000, Algeria

²Laboratory of Biodiversity and Application of Biotechnology in the Agricultural Field,

University of El Oued, El-Oued 39000, Algeria

*Corresponding Author E-mail:

ABSTRACT:

The aim of this study is to use the purslan (Portulacae oleracea L) leaves aqueous extract in order to prepare copper nanoparticles and to explore antioxidant and anti-inflammatory activity of aqueous extract of P.oleracea L and CuNPs. The nanoparticles were characterized from structural and morphological point of view by using analytical techniques such as: UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). Antioxidant activity of purslane and CuNPs was studied done FRAP assay also the anti-inflammatory and hemolysis essay were studies. The acute toxicity test was applied in Wistar albino rats. Obtain results show that the SEM analysis revealed that the particle size was found to be ranging under 46 nm. On the other hand, the copper nanoparticles and purslan possessed the reducing capacity when IC50 value was 68,316 µg/ml and 79,675 µg/ml respectively, the anti-inflammatory ability while IC50 value was 77,503 µg/ml and 60,727 µg/ml respectively. In this study, the toxicity test showed no mortality or behavioral change up to 20 mg / kg of albino Wistar rats CuNPs. We concluded that Portulaca oleracea L has potential properties as biocatalyst or natural stabilizers for CuNPs synthesis. Each CuNPs as P.oleracea L leaves extract were have a reducing and anti-inflammatory capacity which can be protect the cells against the degenerative effects of Reactive Oxygen Species (ROS).

KEYWORDS: Copper nanoparticles, Portulaca oleracea L, Spectroscopy, Antioxidant ability.

1. INTRODUCTION:

In recent years, the increasing commercial demand for nanoparticles due of their wide applicability in various areas such as electronics, catalysis, chemistry, energy, medicine [1], correspond the development of efﬁcient green chemistry methods for synthesis of metal nanoparticles which has become a major focus of researchers [2]. It is of interest to biologists, chemists and materials scientists alike, especially in light of efforts to find greener methods of inorganic material synthesis [3] due to the growing need to develop environmentally benign technologies in material synthesis [4]. The biosynthesis for obtaining nanoparticles using naturally occurring reagents such as vitamins, sugars, plant extracts, biodegradable polymers, and microorganisms as reductants and capping agents could be considered attractive for nanotechnology [5]. Copper nanoparticles, from their unique physical and chemical properties and the low cost of preparation, have been of great interest recently [6], Copper nanoparticles have potential applications in diverse fields including biomedicine, electronics, and optics [7]. Known as “vegetable for long life” in Chinese folklore [8]. Portulaca oleracea has been qualified as unique food of future, it is remarkable that no sign of notable toxicity of Purslane has been reported yet[9]. Portulacae oleracea L is a reach source of α-linolenic acid, omega-3 fatty acids, ascorbic acid, β-carotene, α-tocopherols, phenolic alkaloids, glutathione and many other components. Furthermore, experiments have shown anti-inflammatory, anti-bacterial and oxidative stress protective effect [10]. Previous studies indicate the synthesis, characterization, and biological activities of copper nanoparticles and copper nanocrystals in different ways, however, the green synthesis of copper nanoparticles has been little attention in studies [11]. So, the aim of this study was to use Portulacae oleracea L leaves aqueous extract in order to prepare CuNPs by an eco-friendly method and to evaluate antioxidant and anti-inflammatory activity of aqueous extract of purslan and CuNPs.

2. MATERIALS AND METHODS:

2.1. Chemicals:

analytical-grade copper sulfate, sodium hydroxide and other reagents used were obtained from Sigma Aldrich

2.2. Plant materials:

The plant of Portulaca oleracea were collected in August from a Touggourt village in Ouargla state, Algeria. The leaves were washed with distilled water, then dried at room temperature, then grind to powder and stored at room temperature until use.

2.3. Portulacae oleracea L. leaves extract preparation:

The extraction methods described by Derouiche et al (2019) [12]. The aqueous extract was prepared by adding 50 ml of distilled water to 5 g dry leave powder of Portulaca oleracea. After 24 h of maceration at room temperature, the mixture was filtered by filter paper and then dried in a stove.

2.4. Green synthesis of CuONPs:

In order to prepare CuO NPs, 1 to 4 g of the copper nitrate was added into the defined amount of the prepared walnut leaf extract (10-40 mL) and the reaction solutions were mixed using a heater-stirred, adjusted at 500 rpm and 70°C, for 15 min and finally the samples were put in an electric furnace (FM4P, Fanazma Gostar Co., Alborz, Iran) adjusted at 300 to 500°C for 2 h. The obtained powders, as farmed CuO NPs, were then used for further studies [13].

2.5. Characterization:

The characterization of CuNPs were identified by UV-Vis, FTIR spectroscopy and SEM analysis. UV-Vis spectrum of copper Nano-colloidal solution was recorded in the range of 250-500 nm. SEM analysis were performed in order to observe the morphology and surface topography of CuNPs and to determine the average particle size.

2.6. Anti-inflammatory activity:

The anti-inflammatory activity of the Portulaca oleracea and synthesized NPs as a measure of protein denaturation inhibition was studied through in vitro assay. Bovine serum albumin (BSA) solution (1%) was incubated at room temperature for 30 min with or without different concentrations (10–50 µg ml−1) of sample. The pH of the solution was adjusted to 2 using drop-wise addition of concentration HCl. After incubation, the mixture was heated at 72 °C for 30 min. Finally all tubes were cooled for 10 min and the turbidity was measured at a wavelength of 660 nm. Diclofenac was used as standard. The percentage inhibition [(A0-A1/A0) × 100] was calculated and the results was expired by IC50 [14].

2.7. Hemolysis assay:

The Hemolysis assay was done as described by Vinjamuri et al. 5mL of blood was collected from healthy volunteers in the tubes containing 5.4 mg of EDTA to prevent coagulation and centrifuged at 1000 rpm for 10 min at 40C. Plasma was removed carefully and the white buffy layer was completely removed by aspiration with a pipette with utmost care. The erythrocytes were then washed for additional three times with 1X PBS, pH 7.4 for 5 min. Washed erythrocytes were stored at 4oC and used within 6 h for the hemolysis assay. 50 uL of 10 dilutions (100 uL Erythrocytes suspension: 900 uL 1XPBS) of erythrocytes suspension was mixed with 100 uL of test samples (extract of Portulaca oleracea and synthesized NPs) (20-80µg/mL), 100 uL of 1XPBS was used as negative control and 100 uL of 1% SDS as positive controls. Reaction mixture was incubated at 37⁰C water bath for 60 min. The volume of reaction mixture was made upto 1 mL by adding 850 uL of 1XPB. Finally, it was centrifuged at 300rpm for 3min and the resulting hemoglobin in supernatant was measured at 540 nm by spectrophotometer to determine the concentration of hemoglobin. The percentage hemolysis was calculated as % Hemolysis inhibition= 100-[Sample / Control] x 100 [15].

2.8. Antioxidant activity (Ferric reducing ability “FRAP”):

One milliliter of the extract and CuONPs at different concentrations (from 0.007 to 2.5 mg / ml) is mixed with 2.5 ml of a 0.2 M phosphate buffer solution (pH 6.6) and 2.5 ml of a solution of potassium ferricyanide K3Fe (CN) 6 to 1%. The whole is incubated in a water bath at 50 ° C for 20 min then, 2.5 ml of 10% trichloroacetic acid are added to stop the reaction. The tubes are centrifuged at 3000 rpm for 10 min. An aliquot (2.5 ml) of supernatant is combined with 2.5 ml of distilled water and 0.5 ml of an aqueous solution of FeCl3 (ferric chloride) at 0.1%. The absorbance of the reaction medium is read at 700 nm against a similarly prepared blank, replacing the extract or NPs with distilled water which makes it possible to calibrate the device (UV-VIS spectrophotometer). The positive control is represented by a standard of an antioxidant; ascorbic acid, the absorbance of which was measured under the same conditions as the samples. An increase in absorbance corresponds to an increase in the reducing power of the extracts tested [16]

FRAP Value =100− [(control absorption / Sample absorption) ×100] [17]

2.9. Acute toxicity study:

The test was performed using healthy albino rats of Wistar strain weighing between 154 and 181g. The animals were divided into three groups of two rats each and administered 0, 10 and 20 mg/kg of CuNPs orally. Animals were observed after dosing at least once during the first 30 min, periodically during the first 24 h with special attention given during the first 4 h, and daily thereafter for a total of 14 consecutive days [18].

3. RESULTS AND DISCUSSION:

Our study reported that the addition of P.oleracea L in the synthesis of CuNPs induced to changes the color from bleu to bluish black color mixture indicating the formation of CuO (Fig.1). Due to phytochemicals compounds present in the aqueous extract of P.oleracea L such as alkaloids, carbohydrates, tannins, phenolic compounds, flavonoids and triterpenoid [19] which reduced copper hydroxide Cu(OH)₂to CuO and formed a colloidal solution. The copper hydroxide was result from the reaction between CuSO₄,5H2O and hydroxyl anion OH^-- produced in aqueous solution [20].

Figure 01: Visual observation of P. oleracea L leaves (A), and powder of CuNPs synthesized

3.1. UV–Vis spectroscopy:

UV-vis spectroscopy is an important technique to determine the formation and stability of metal NPs in aqueous solution [21]. Our results (Fig.02) showed a maximum absorbance in UV-vis spectrum of CuNPs synthesized by leaf aqueous extract of P.oleracea L at 300 nm as confirmed by the Surface Plasmon's resonance (SPR), which is deﬁned as collective excitation of negatively charged ions (electrons) in a conduction band around the surface of synthesized copper nanoparticles. Electrons present in the nanoparticles were conﬁned to have an exact vibration mode because of their particular shape and size [22] This result was very close to other study while they used R.tuberosa leaf extract in biosynthesis of CuNPs and the distinct peak at 327nm [23] .

Figure 02: UV-Vis spectra of purslane aqueous extract (A), copper sulfate (B), mixture of P.oleracea L extract and copper sulfate (C) and CuNPs biosynthesized by purslane aqueous extract(D).

3.2. SEM analysis:

The images of CuNPs biosynthesized by P.oleracea L aqueous extract obtained from SEM determined that this nanoparticle owned spherical shape with the size 45.94 nm, this result as reported by Rajeshkumar S and Rinitha G., 2018 [24], when the copper nanoparticles biosynthesized by Persea americana seed extract and the size range under 90 nm.

Figure 03: SEM images of CuNPs biosynthesized by purslane aqueous extract.

3.3. FT‑IR analysis:

The biosynthesis of CuNPs was characterized by FTIR analysis within the scan range of 800–4000 cm−1 in order to investigate the functional groups responsible for the synthesis and stability of nanoparticles [25] The spectral data study shows that of formation of metal and metal oxide nanoparticles in phytochemically assisted synthesis strictly requires clear FTIR information, showing that phytochemicals present in the plant extract actually assisted in such synthesis. The major peak was observed to be 576 cm-1 should be a stretching of Cu-O. The similar results has been reported in literature where CuO NPs was synthesized using different leaves extracts [26, 27].

Figure 04: FTIR spectrum of powder CuNPs obtained from purslane aqueous extract.

3.4. Anti-inflammatory activity:

Extracting plant materials is the first major step towards testing the biological activities of this plant. When a whole extract is used, there is a good chance for synergism between active components that might be lost when each of these components is isolated. Such it was discovered in several medicinal tests, including those for anti-inflammatory activity [28]. The results obtained in Figure05 show that the inhibition concentration 50 levels of diclofenac, followed by aqueous extract and lastly by cooper nanoparticle are respectively increased, the major finding of this study indicated that purslane aqueous extract showed significant anti-inflammatory activity when compared with the diclofenac sodium as the active control [29]. Study recealed that P. oleracea suppresses inflammation at the vascular level [30]. Also the Copper Nanoparticles have a good anti-inflammatory activity as it demonstrated by Angajala G et al., 2014. [31]

Figure 05: IC50 levels of anti-inflammatory activity of purslane aqueous extract, copper nanoparticles and diclofenac.

3.5. Hemolysis assay:

Hemolytic assay tests were performed to determine the toxicity of the prepared materials against erythrocytic cells [32]. The results (Fig.06) clearly shows that the inhibition percentage value of purslane aqueous extract higher more than it exhibit in copper nanoparticles. P. oleracea extracts inhibited hemolytic damage induced by AAPH which is associated to antioxidant constituents, the ability of P. oleracea to scavenge free radicals are related to reduction the net concentration of AAPH-derived peroxyl radicals generated during the test [33]. Additionally, the cooper nanoparticles exhibited un hemolytic activity in contrary to what it mentioned by Li Qiang Chen et al., 2013 [34].

Figure 06: Effects of purslane aqueous extract and copper nanoparticles on blood hemolysis.

3.6. Antioxidant assay:

Figure 07, the result show clarify that the inhibition concentration level of purslane aqueous extract is higher more than it presented in copper nanoparticle and ascorbic acid levels. To assess the antioxidant activities of the extracts and copper nanoparticles, we used Ferric-reducing antioxidant power assay, which involves the presence of antioxidants in extract to reduce the ferricyanide complex to the ferrous form [35]. From this result, we can say that purslane aqueous extract has anti-oxidant activity as reported by Ahn E Y et al., 2018 [36]. This property of Portulaca oleracea is attributed to its constituents, such as gallotannins, omega-3 fatty acids, ascorbic acid, α-tocopherols [37]. Furthermore there are a significant antioxidant activity showed by copper nanoparticles [38].

3.7. Sub‑acute toxicity study:

The results obtained from this test (Table 01) showed that normal physiological parameters during the experimental period as the eyes, the sleep and the diarrhea of the albino Wister rats were treated by different dose of CuNPs 0, 10 and 20 mg/kg. In addition, there was no abnormal symptom or adverse effects and no mortality case were noted before 14 days.

Figure 07: FRAP scavenging assay of purslane aqueous extract, copper nanoparticles and ascorbic acid.

Table 01: Sub‑acute toxicity test of CuNPs on physiological parameters of Wister albino rats.

Parameters	0 h		3 h		24 h		Day- 7		Day-14
Parameters	Control	Test	Control	Test	Control	Test	Control	Test	Control	Test
Dead rats	0	0	0	0	0	0	0	0	0	0
Eyes	N	N	N	N	N	N	N	N	N	N
Sleep	N	N	N	N	N	N	N	N	N	N
Diarrhea	N	N	N	N	N	N	N	N	N	N

Test, CuNPs (10 and 20 mg/kg b.w rats) administered rats, N, Normal.

4. CONCLUSION:

In conclusion, these results show that P.oleracea L has potential properties as biocatalyst or natural stabilizers for CuNPs synthesis, which characterized by different methods; UV-VIS spectroscopy, FT-IR spectroscopy and SEM analysis. In addition, the antioxidant and anti-inflammatory activity of CuNPs let us to classify this nanoparticle as one of the important biological and medical molecules that can used in various fields.

5. ACKNOWLEDGEMENTS:

We thank the members of Lab of Faculty of Sciences of Nature and Life, University of El Oued, Algeria, for the permission to utilize the institute facilities to make this work.

6. CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest.

7. REFERENCES:

1. Singh A, Dhaliwal H. Green synthesis of silver nanoparticles using phyllanthus niruri leaf extract and evaluation of their antimicrobial activities. European Journal of Environmental Ecology, 2015; 2(1): 9-13.

2. Iravani S. Green synthesis of metal nanoparticles using plants. Green Chemistry. 2011; 13(10) : 2638-2650.

3. D Philip. Honey mediated green synthesis of gold nanoparticles. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2009; 73(4): 650–653.

4. Harekrishna B, Dipak KB, Gobinda PS, Priyanka S, Sankar PD, Ajay M. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2009; 339(1–3): 134-139.

5. Oxana VK, Rasika D, Boris IK, Victor M. Jiménez P. The greener synthesis of nanoparticles. Trends in Biotechnology, 2013; 31(4): 240-248.

6. Honary S, Barabadi H, Gharaeifathabad E, Naghibi E. Green synthesis of copper oxide nanoparticles using penicillium aurantiogriseum, penicillium citrinum and penicillium waksmanii. Digest Journal of Nanomaterials and Biostructures. 2012; 7(3): 999 – 1005.

7. Rubilar O, Rai M, Tortella G, Diez M C, Seabra A B and Duran N. Biogenic nanoparticles: copper, copper oxides, copper sulphides, complex copper nanostructures and their applications. Biotechnol Lett. 2013; 35(9): 1365-75.

8. Xueqin X, Lishuang Y and Guonan Ch. Determination of flavonoids in Portulaca oleracea L. by capillary electrophoresis with electrochemical detection. Journal of Pharmaceutical and Biomedical Analysis, 2006; 41: 493–499.

9. Ahangarpour A, Lamoochi z , Moghaddam H and Mansouri S. Effects of Portulaca oleracea ethanolic extract on reproductive system of aging female mice. International Journal of Reproductive Biomedicine. 2016; 14(3): 205–212.

10. Noorbakhshnia M, Karimi-Zandi L. Portulaca oleracea L. prevents lipopolysaccharide-induced passive avoidance learning and memory and TNF-α impairments in hippocampus of rat. Physiology & Behavior, 2016; doi: 10.1016/j.physbeh.2016.11.027

11. Mahmoud VH, Khaksarian M, Ebrahimi K, Shiravand S, Jahanbakhsh S, Niazi M and Nadri S. Antinociceptive effects of green synthesized copper nanoparticles alone or in combination with morphine. Journal Pre-proof, 2020; doi.org/10.1016/j.amsu.2019.12.006

12. Derouiche S, Azzi M, Hamida A. Phytochemical Analysis and Antioxidant Property of Rhizome extracts aqueous of Phragmites australis in Alloxan Diabetic Rats. Asian Journal of Pharmacy and Technology. 2019, 9(4): 249-252.

13. Marjan A, Navideh A. Green synthesis of copper oxide nanoparticles using Juglans regia leaf extract and assessment of their physico-chemical and biological propertie. Green Process Synth. 2019; 8: 557–567.

14. Krishnan V, Loganathan C, Rama B, Thayumanavan P. Comparison of biological activities of selenium and silver nanoparticles attached with bioactive phytoconstituents: green synthesized using Spermacoce hispida extract. Adv. Nat. Sci: Nanosci. Nanotechnol. 2018; 9: 015005.

15. Vinjamuri S, Syeda A, Sowmya S, Jaya SV. Evaluation of Hemolytic Activity, Atpase Inhibitory Activity andAntitumor Activity of TLC Extract of Lemon Grass (CymbpogonCitratus). international Journal of Pharmacognosy and Phytochemical Research. 2015; 7(4);785-788.

16. Oyaizu M. Studies on products of browning reaction-Antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition. 1986; 44, 307–315.

17. Yazdani E, Talebi M and Zarshenas M M. Evaluation of possible antioxidant activities of barberrysolid formulation, a selected formulation from Traditional Persian Medicine (TPM) via various procedures. Biointerface Research in Applied Chemistry. 2019; 9(6): 4517-4521.

18. Derouiche S, Azzi M, Hamida A. Effect of extracts aqueous of phragmites australis on carbohydrate metabolism, some enzyme activities and pancreatic islet tissue in alloxan induced diabetic rats. Int J Pharm Pharm Sci. 2017; 9(6): 54-58.

19. Derouiche S, Abbas K, Djermoune M. Polysaccharides and ascorbic acid content and the effect of aqueous extract of Portulaca oleracea in highfat diet-induced obesity, dyslipidemia and liver damage in albino wistar rats. Algerian journal of arid environment. 2017; 7(2): 16-26.

20. Vidovix TB, Quesada HB, Diogo Januário EF, Bergamasco R, Salcedo Vieira AM, Green synthesis of copper oxide nanoparticles using Punica granatum leaf extract applied to the removal of methylene blue. Materials Letters. 2019; 257: 126685.

21. Nasrollahzadeh M, Sajadi SM, Rostami-Vartooni A, Bagherzadeh M, Safari R. Immobilization of copper nanoparticles on perlite: Green synthesis, haracterization and catalytic activity on aqueous reduction of 4-nitrophenol. Journal of Molecular Catalysis A: Chemica., 2015; 400: 22-30.

22. Sharma P, Pant S, Dave V, Tak K, Sadhu V, Reddy KR. Green synthesis and characterization of copper nanoparticles by Tinospora cardifolia to produce nature-friendly copper nano-coated fabric and their antimicrobial evaluation. Journal of Microbiological Methods. 2019; 160: 107–116.

23. Vasantharaj S, SathiyavimaL S, Saravanan M, Senthilkumar P, Gnanasekaran K, Shanmugavel M, Manikandan E, Pugazhendhi A. Synthesis of ecofriendly copper oxide nanoparticles for fabrication over textile fabrics: Characterization of antibacterial activity and dye degradation potential. Journal of Photochemistry & Photobiology, B: Biology. 2019; 191: 143–149.

24. Rajeshkumar S, Rinitha G. Nanostructural characterization of antimicrobial and antioxidant copper nanoparticles synthesized using novel Persea americana seeds. Open Nano, 2018; 3: 18-27.

25. Fritea L, Laslo V, Cavalu S, Costea T and Simona Ioana Vicas S I. Green biosynthesis of selenium nanoparticles using parsley (petroselinum crispum) leaves extract. Studia Universitatis Vasile Goldis Arad, Seria Stiintele Vietii. 2017; 27(3): 203-208.

26. Hassanien R, Dalal Z. Mostafa H, Al-Hakkani F. Biosynthesis of copper nanoparticles using aqueous Tilia extract: antimicrobial and anticancer activities. Heliyon, 2018; 4(12): e01077.

27. Habib U, Cecilia DW, Maizatul SS. Green synthesis of copper nanoparticle using ionic liquid-based extraction from Polygonum minus and their applications, Environmental Technology, 2019; 28, 3705-3712.

28. Azab A, Nassar A, Azab AN. Anti-Inflammatory Activity of Natural Products. Molecules, 2016; 21(1321):19.

29. Mubashir HM, Bahar A, Mir SH, Zargar BA, Tabasum N. Portulaca oleracea L. A Review. Journal of Pharmacy Research. 2011; 4(9): 3044-3048.

30. Derouiche S, Degachi O, Gharbi K. Phytochemistry analysis and modulatory activity of Portulacae oleracea and Aquilaria malaccensis extracts against High-fructose and high-fat diet induced immune cells alteration and heart lipid peroxidation in Rats. International Research Journal of Biological Sciences. 2019; 8(4): 6-11.

31. Angajala G, Pavan P and Subashin R. One-step biofabrication of copper nanoparticles from Aegle marmelos correa aqueous leaf extract and evaluation of its anti-inflammatory and mosquito larvicidal efficacy. The Royal Society of Chemistry Advances, 2014; 4: 51459–51470.

32. Ashfaq M, Verma N and Khan S. Copper/zinc bimetal nanoparticles-dispersed carbon nanofibers: A novel potential antibiotic material. Materials Science and Engineering C 59, 2015; 9 (2016) 938–947.

33. Karimi G, Aghasizadeh M, Razavi M, Taghiabadi E. Protective effects of aqueous and ethanolic extracts of Nigella sativa L. and Portulaca oleracea L. on free radical induced hemolysis of RBCs. DARU Journal of Pharmaceutical Sciences, 2011; 19(4): 295-300.

34. Chen LQ, Kang B, Ling J. Cytotoxicity of cuprous oxide nanoparticles to fish blood cells: hemolysis and internalization. J Nanopart Res. 2013; 15(1507): 9.

35. Lim YY, Quah EPL. Antioxidant properties of different cultivars of Portulaca oleracea. Food Chemistry. 2007; 103(3): 734–740.

36. Ahn EY, Lee YJ, Park J, Chun P, Park Y. Antioxidant Potential of Artemisia capillaris, Portulaca oleracea, and Prunella vulgaris Extracts for Biofabrication of Gold Nanoparticles and Cytotoxicity Assessment. Nanoscale Research Letters. 2018; 13(1): 1-14.

37. Zhou YX, Xin HL, Rahman K, Wang SJ, Peng C, Zhang H. Portulaca oleracea L.: A Review of Phytochemistry and Pharmacological Effects. BioMed Research International, 2015: 1-11.

38. Ljaz F, Shahid S, Ahmad Khan S, Waqar A, Zaman S. Green synthesis of copper oxide nanoparticles using Abutilon indicum leaf extract: Antimicrobial, antioxidant and photocatalytic dye degradation activities. Tropical Journal of Pharmaceutical Research. 2017; 16 (4): 743-753.

Received on 01.02.2020 Modified on 07.03.2020

Asian J. Pharm. Res. 2020; 10(2):89-94.

DOI: 10.5958/2231-5691.2020.00017.9