1. Introduction

Nanotechnology is a versatile branch of development and research which is growing at a tremendous rate from the past two decades.Nanoparticles are known for their numerous physical, biological,and pharmaceutical applications[1]. Silver nanoparticles (AgNPs), in particular, are being used as antimicrobial agents[2-5] as they exhibit interesting antibacterial[6-8] and antiplasmodial[9,10] activities.Several chemical methods for synthesis of nanoparticles have been reported[11-14]. However, the green synthesis methods are considered to be more versatile[15,16].

对于农户而言，家庭是其组织生产的基本单位。如果在生产决策角度，将农户的非农就业与农地经营看做是农户成员选择个人从事何种工作的专业化水平问题，那么，在家庭收益或者说效用最大化的理性假设下，这种成员决策在农户家庭层面所最终形成的专业化水平的基础便是尽可能地利用家庭内部成员的分工比较优势。由此，可以将农户的非农就业决策函数表达为Y=F(X1，X2，…，Xn)，其中X1反映的是农户家庭内部的分工比较优势。无疑，值得肯定的是，已有研究根据家庭联合决策的理论启示，将家庭内部分工主要是男女之间的性别分工，成功融入非农就业与农地流转的考察之中。②

The use of green techniques for synthesis of nanoparticles is a rapid, low cost and eco-friendly process. The advantage of using plant extracts for synthesis of nanoparticles is that each plant extract, by virtue of its unique metabolites such as polyphenols,terpenoids and thiols, gives rise to a wide diversity of unique microenvironments. This influences the physico-chemical and biological properties of the AgNPs which are hence formed. Several plants of medicinal importance are commonly prevalent and have been used over centuries[17,18]. Azadirachta indica (neem) and Ocimum sanctum (tulsi) plants are commonly available and each part of these plants has been used as a household remedy against various human diseases from historic times[19]. These plants have also been found to show antimalarial and antiplasmodial[20-22] properties.AgNPs prepared from neem and tulsi leaf extracts individually show good antibacterial and antimicrobial properties. However, the combinatorial properties of the above two extracts in unison are still an unexplored niche.

In the present work, we have studied the antiplasmodial activity of the AgNPs synthesized from the combination of neem and tulsi plant leaf extracts when taken in different proportions. The AgNPs synthesized by novel combination of neem and tulsi plant leaf extracts have proved to be potential antiplasmodial agents.The knowledge of antiplasmodial activities of AgNPs based on the calculated half maximal effective concentration (EC50) values would be helpful to understand their antimalarial properties.

配套MAN B&W ME系列电喷柴油机的主机监控系统见图1，其中：气缸控制单元实施对燃油喷射阀、排气阀、启动控制阀的控制及对曲轴转速和转角的测量；辅助控制单元实施对辅助鼓风机、机带共轨伺服油泵和电动共轨伺服油泵等辅助设备的控制；柴油机控制单元实施对气缸滑油电磁阀的控制，为气缸提供润滑。柴油机数据交换接口单元A与柴油机数据交换接口单元B互相冗余，是ME电喷柴油机电气控制系统对外进行数据交换的接口控制模块。

2. Materials and methods

2.1. Materials

Fresh leaves of neem and tulsi were taken from the botanical garden of Sri Venkateswara College Campus. Silver nitrate was purchased from Merck India Ltd and 1 mM solution of the same was prepared.Other chemicals and solvents used were of analytical grade. All the glassware were washed with distilled water and dried in oven.Antiplasmodial activities were tested at National Institute of Malaria and Research, New Delhi.

2.2. Preparation of leaf extract

Collected leaves were first washed with distilled water and then air dried for 2 h. A total of 20 g of finely cut neem leaves were boiled in 150 mL distilled water for 30 min at 90 ℃ on an electric water bath and filtered through Whatman filter paper to obtain aqueous neem leaf extract. Also, 20 g of finely cut tulsi leaves were processed through the same procedure in order to obtain aqueous tulsi leaf extract. The tulsi extract obtained was centrifuged for 15 min at 5070 g. The prepared extracts were stored at 4 ℃.

2.3. Synthesis of silver nanoparticles

In-vitro parasite cultivation and drug sensitivity assay were performed as follows. The parasite strain (3D7) was collected from NIMR parasite bank, and revived in vitro using Trager and Jensen method in RPMI 1640 medium for 7-8 cycles. The culture of Plasmodium falciparum 3D7 parasites were maintained in human erythrocytes (AB+) at 2% hematocrit in RPMI medium 1640 supplemented with 50 mg/L gentamicin, 5% sodium bicarbonate 10% human serum. Parasites were synchronized with 5% D-sorbitol and maintained in ring stage. ELISA plates were used in triplicates with 20 μM of each sample of AgNPs dissolved in water serially diluted to seven fold concentrations. A total of 180 μL/well of parasites with 0.5% synchronized ring stage parasitemia and 2%fresh RBC hematocrit was added. Further plates were incubated for 72 h at 37 ℃ in CO2 incubator (Flow Laboratories).

2.4. Characterization of the synthesized AgNPs

Revision 25 January 2018

2.2.3 供试品溶液的制备 取和血胶囊内容物各3.0 g，精密称定，置具塞锥形瓶中，加甲醇50 mL，密塞，称定重量，超声处理（功率250 W，频率40 kHz）1 h，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液，即得。

2.5. Antiplasmodial activity

For synthesis of AgNPs, 3 mL aqueous extract of neem leaves was mixed with 27 mL of 1 mM silver nitrate and a vivid color change was observed indicating the formation of AgNPs. Similarly, other samples were prepared by mixing plant extracts of neem and tulsi in different compositions as 100% tulsi (0% neem), 100% neem(0% tulsi), 80% neem (20% tulsi), 60% neem (40% tulsi), 40%neem (60% tulsi) and 20% neem (80% tulsi) namely A, B, C, D, E,F respectively. Keeping the ratio of extract(s) to 1 mM silver nitrate equal to 1:9, the effects of various physico-chemical parameters were examined by varying the reactant concentration, pH and reaction time. Reduction of ionic silver to AgNPs was monitored after diluting a small amount of the sample 20 times. Absorption spectra were recorded with UV/VIS/NIR spectrometer (Systronics-P C Based Double Beam Spectrophotometer - 2202). The excitation source was a 450 Watt CW xenon lamp. Completion of the reaction indicated by a change in color from colorless solution to a brownyellow solution was observed, confirming the formation of AgNPs after 15 min in the presence of NaOH. Effect of pH was studied by varying the pH of all the samples by adding 2 drops of 1 M NaOH and adjusting its pH to 8. The pH of tulsi leaf extract was 7.0 and that of neem leaf extract was 6.5.

ELISA plates were initially pre-coated with 100 μL of 1.0 μL/mL primary IgM antibody (MPFM-55A, Immunology Consultants Laboratories, Inc, Newberg, OR, USA) and incubated at 4 ℃overnight. The plates were further dried and blocked with 200 μL of blocking solution (2% bovine serum albumin in PBS) followed by three times washing with the PBS-Tween 20 (0.05%). The plates were stored at -20 ℃ till the experiment was conducted.

Sample for TEM was prepared by drop coating purified AgNPs on carbon-carbon coated 300 mesh copper TEM grids at AIIMS, New Delhi. TEM measurements were performed on Techai G2 20 S-Twin with accelerating voltage at 200 kV.

The UV-Vis spectrum of silver nanoparticles (Figure 1) of samples A-F was recorded as a function of concentration in the range of 350-550 nm. λmax(nm) range of the synthesized nanoparticles was found to be between 407-427 nm.

第一阶段，人与兽鸟合体形象。这在中国的神话中有许多记载，比如《列子·黄帝》云：“庖牺氏、女娲氏、神农氏、夏后氏蛇首人身，牛首虎鼻。”第二阶段，人兽分离，人是人，兽是兽。这也有两种情况：一种是人兽虽分却相关、相连，另一种人兽彻底分离，不相关不相连。良渚的“神人兽面纹”显然属于第二阶段中的第一种情况。这幅图案中，人的形象是独立的，完整的，兽与鸟并不构成人体中的一部分，只是兽首为人手所执，鸟背为人所骑，因此而显示人与兽鸟相关、相连。

3. Results

3.1. UV-Vis spectra analysis

For HRPII ELISA assay, 100 μL of hemolysed lysate of 72 h post infection (hpi) of parasite was transferred to pre-dosed primary IgM antibody coated plate and incubated at RT in humid chamber for 1 h. The content was discarded, plate was washed thrice with PBS-Tween 20 (0.05%) and tap dried. Followed by addition of 100 μL of 0.2 μg/mL concentration of IgG secondary antibody(MPFG-55P, Immunology Consultants Laboratories, Inc. Newberg,OR, USA) dissolved in 2% BSA and 1% Tween 20, IgG added plate was incubated for 1 h at RT in humid chamber, washed three times in PBS/Tween and dried. Further 100 μL 3,3’,5,5’-Tetramethylbenzidine chromogen (Ameresco, US) was added and incubated for 10 min at RT in dark. The reaction was stopped by addition of 50 μL of 1M Sulphuric acid followed by absorbance recording of each plate using an ELISA plate reader (Spectrostar Nano, BMG LABTECH, Germany) at 450 nm[23,24]. EC50 value was calculated by nonlinear regression analysis. The software used for the study was based on a polynomial regression model and was freely available from http://malaria.farch.net.

Figure 1. UV-Vis spectra of silver nanoparticles synthesized from aqueous leaf extracts of neem and tulsi in different combinations.

(A: 100% tulsi, 0% neem; B: 100% neem, 0% tulsi; C: 80% neem, 20% tulsi;D: 60% neem, 40% tulsi; E: 40% neem, 60% tulsi; F: 20% neem, 80% tulsi).

Figure 2. TEM images of AgNPs formed from different combinations of neem and tulsi extracts: Sample A (100% tulsi, 0% neem), B (100% neem, 0% tulsi), C (80%neem, 20% tulsi), D (60% neem, 40% tulsi), E (40% neem, 60% tulsi), and F (20% neem, 80% tulsi).

3.2. TEM analysis

（1）初剪。先反复观看视频，把抖动的、镜头之间的连接部分、不需要的视频镜头全部剔除，凡是可以使用的镜头都尽可能保留，并注明从几分几秒到几分几秒对应哪些解说词。然后按照脚本，把视频、语音编辑好。

Table 1 Particles size, λmax(nm), EC50 and R2 values of AgNPs synthesized from samples A-F.

A 100% tulsi + 0%neem 20.00-32.68426.81.6920.9612 B 100% neem +0% tulsi 4.74-13.33411.60.3131.0000 C 80% neem +20% tulsi 16.48-30.33422.80.3131.0000 D 60% neem +40% tulsi 21.94-22.33420.40.7421.0000 E 40% neem +60% tulsi 19.82-22.29407.61.4370.9848 F 20% neem +80% tulsi 9.38-39.32420.80.5130.9876

3.3. Antiplasmodial studies

To determine the efficacy, the nanoparticles of neem and tulsi alone and in combination were incubated with synchronized ring stage Plasmodium falciparum 3D7 parasites. Each nanoparticle was screened in triplicates till 72 h post infection (hpi) using HRPII ELISA assay.The result indicated that 100% and 80% neem contained nanoparticles showed better value of EC50 about 0.3 μM among other combinations while other showed moderate activity. The EC50 values were mentioned in the Table 1 and belonged to 0.313-1.692 range.

4. Discussion

Reduction of silver ions into metallic silver when exposed to the plant extracts depicted a color change. The Surface Plasmon Resonance phenomena is accountable for the dark yellowish - brown color of AgNPs. For characterisation of the nanoparticles, UV-Vis spectroscopy substantiated to be a suitable technique for the analysis of nanoparticles. Reduction of Ag+ ions in the aqueous medium to atomic silver in the presence of plant leaf extracts was correlated with the UV-Vis spectra.

The effect of AgNPs of neem and tulsi alone and in combination suggested that 100% and 80% neem exhibited EC50 value of about 0.3 μM. The observed anti-plasmodial activity could be due to presence of steroids, terpenes, coumarins, flavonoids, phenolic acids,lignans, xanthones and anthraquinones[25]. A simple green synthesis of AgNPs prepared from leaf extract showed effective antiplasmodial properties at concentrations 100% neem with 0% tulsi (Sample B)and 80% neem with 20% tulsi (Sample C). Pure neem and tulsi leaf extract have been reported[20,26] to show insignificant antiplasmodial activity with IC50 values ranging from 35-40 μg/mL whereas AgNPs synthesized by our group shows a manifold increase in antiplasmodial activity. This is probably because of the fact that AgNPs can penetrate more against Plasmodium falciparum owing to their small size and spherical shape. Further, a lot of clinical trials[1,27] are being carried out on antimalarial activity of AgNPs and our results would be helpful to understand the mechanism of their action.

In the present study we have found that plant extracts of neem and tulsi, when taken in different proportions in combination,give rise to novel AgNPs that are potential antiplasmodial agents.This knowledge of antiplasmodial activity of AgNPs based on the reported EC50 values would be helpful in understanding their antimalarial properties as well.

Conflict of interest statement

显效:患者的空腹血糖和餐后2h血糖降至正常水平或是下降40%以上,糖化血红蛋白降至正常水平或下降幅度在30%以上,骨密度明显增加。有效:血糖水平下降幅度在20%~40%,糖化血红蛋白下降10%~30%,骨密度上升不明显,不超过0.06g/cm2。无效:不符合上述标准者。

We declare that we do not have any conflict.

Acknowledgements

TEM technique was employed to visualize the size and shape of AgNPs. Table 1 showed the corresponding sample combinations,particles size obtained, λmax(nm), EC50 and R2 values for the synthesized AgNPs. The optimum particle size was found to be ＜ 40 nm. The TEM images of different samples were illustrated in Figure 2, confirming the formation of AgNPs between size range 4.74-39.32 nm. In addition, the TEM images showed that the nanoparticles were circular and spherical in shape (Figure 2).

The authors are thankful to the University of Delhi for their financial assistance through the Innovation project (SVC 311) and Principal, Sri Venkateswara College for her valuable advice. KCP is thankful to Dept. of Science and Technology for extramural grant(SR/SO/BB-0092/2013).

References

[1] Elechiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, et al. Interaction of silver nanoparticles with HIV-1. J Nanobiotechnol 2005; 3: 6.

[2] Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, et al. Corrigendum to“Antimicrobial effects of silver nanoparticles”. Nanomed Nanotechnol Biol Med 2007; 1: 95-101.

[3] Anandalakshmi K, Venugobal J, Ramasamy V. Characterization of silver nanoparticles by green synthesis method using Pedalium murex leaf extract and their antibacterial activity. Appl Nanosci 2016; 6: 399-408.

[4] Das MR, Sarma RK, Borah SCh, Kumari R, Saikia R, Deshmukh AB, et al. The synthesis of citrate-modified silver nanoparticles in an aqueous suspension of graphene oxide nanosheets and their antibacterial activity.Colloids Surf B Biointerfaces 2013; 105: 128-136.

[5] Sondi I, Salopek-Sondi B. Silver nanoparticles as antimicrobial agent:A case study on E. coli as a model for gram-negative bacteria. J Colloid Interface Sci 2004; 275(1): 177-182.

[6] Paosen S, Saising J, Septama AW, Voravuthikunchaia SP. Green synthesis of silver nanoparticles using plants from Myrtaceae family and characterization of their antibacterial activity. Mater Lett 2017; 209: 201-206.

[7] Rajeshkumar S, Bharath LV. Mechanism of plant-mediated synthesis of silver nanoparticles – A review on biomolecules involved, characterisation and antibacterial activity. Chem-Biol Interact 2017; 273: 219-227.

[8] Li WR, Sun TL, Zhou SL, Ma YK, Shi QS, Xie XB, et al. A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains. Int Biodeter Biodegr 2017; 123: 304-310.

[9] Ponarulselvam S, Panneerselvam C, Murugan K, Aarthi N, Kalimuthu K, Thangamani S. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities. Asian Pac J Trop Biomed 2012; 2: 574-580.

[10] Mishra A, Kaushik NK, Sardar M, Sahal D. Evaluation of antiplasmodial activity of green synthesized silver Nanoparticles. Colloids Surf B Biointerfaces 2013; 111: 713-718.

[11] Mousavi-Kamazani M, Salavati-Niasari M, Goudrazi M, Zarghami Z.Hydrothermal synthesis of CdIn2S4 nanostructures using new starting reagent for elevating solar cells efficiency. J Mol Liq 2017; 242: 653-661.

[12] Goudrazi M, Mir N, Mousavi-Kamazani M, Bagheri S, Salavati-Niasari M. Biosynthesis and characterization of silver nanoparticles prepared from two novel natural precursors by facile thermal decomposition methods. Sci Rep 2016; 6: 32539.

[13] Goudrazi M, Salavati-Niasari M. Controllable synthesis of new Tl2S2O3 nanostructures via hydrothermal process; characterization and investigation photocatalytic activity for degradation of some anionic dyes. J Mol Liq 2016; 219: 851-857.

[14] Wang Y, Liu W, Liu W, He P, Fan Z, Wang X, et al. Synthesis of SnAgCu nanoparticles with low melting point by the chemical reduction method.Microelectron Reliab 2017; 78: 17-24.

[15] Mallikarjuna K, Narasimha G, Dillip GR, Praveen B, Shreedhar B,Lakshmi CS, et al. Green synthesis of silver nanoparticles using ocimum leaf extract and their characterisation. Dig J Nanomater Bios 2011; 6:181-186.

[16] Bar H, Bhui DK, Sahoo GP, Sarkar P, De SP, Mishra A. Green synthesis of silver nanoparticles using latex of Jatropha curcas. Colloids Surface A 2009; 339: 134-139.

[17] Zhang XF, Liu ZG, Shen W, Gurunathan S. Silver nanoparticles:Synthesis, characterization, properties, applications, and therapeutic approaches. Int J Mol Sci 2016; 17(9): 1534.

[18] Rafique M, Sadaf I, Ra fique MS, Tahir MB. A review on green synthesis of silver nanoparticles and their applications. Artif Cells Nanomed Biotechnol 2017; 45: 1272-1291.

[19] Verkerk RHJ, Wright DJ. Biological activity of neem seed kernel extract and synthetic azadirachtin against larvae of Plutella xylostella. Pestic Sci 1993; 37: 83-91.

[20] Mishra A, Kaushik NK, Sardar M, Sahal D. Evaluation of antiplasmodial activity of green synthesized silver nanoparticles. Colloids Surf B Biointerfaces 2013; 111: 713-718.

[21] Ponarulselvam S, Panneerselvam C, Murugan K, Aarthi N, Kalimuthu K, Thangamani S. Synthesis of silver nanoparticles using leaves of Catharanthus roseus and their antiplasmodial activities. Asian Pac J Trop Biomed 2012; 2: 574-580.

[22] Kaur R, Kaur H. Plant derived antimalarial agents. J Med Plants Stud 2017; 5: 346-363.

[23] Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C. Histidinerich protein Ⅱ: A novel approach to malaria drug sensitivity testing.Antimicrob Agents Chemother 2002; 46:1658-1664.

[24] Sharma S, Mishra N, Valecha N, Anvikar AR. Comparison of WHO Mark Ⅲ and HRP Ⅱ ELISA for in vitro sensitivity of P. falciparum. J Vector Borne Dis 2016; 53: 341-347.

[25] Tona L, Cimanga RK, Mesia K, Musuamba CT, De Bruyne T, Apers S, et al. In vitro antiplasmodial activity of extracts and fractions from seven Medicinal Plants used in the Democratic Republic of Congo. J Ethnopharmacol 2004; 93(1): 27-32.

[26] Inbaneson SJ, Sundaram R, Suganthi P. In vitro antiplasmodial effect of ethanolic extracts of traditional medicinal plant Ocimum species against Plasmodium falciparum. Asian Pac J Trop Med 2012; 5: 103-106.

[27] Chung I, Park I, Seung-Hyun K, Thiruvengadam M, Rajakumar G. Plantmediated synthesis of silver nanoparticles: Their characteristic properties and therapeutic applications. Nanoscale Res Lett 2016; 11(1): 40.