Introduction

In the past decade, around 3 million people were affected by dengue fever due to Aedes ageypti (A. ageypti) and it is the major vector for causing dengue fever. If this vector borne disease left over untreated, it may lead to death in several cases [1]. To overcome the above mentioned drawbacks most of the researchers proposed a protocol for larvicidal and ovicidal activities by using various techniques for killing the A. ageypti [2, 3]. Several techniques and natural products are readily available to treat the threats caused by vectors which also lead to some side effects. But nowadays nanotechnology is one of the frequently growing fields which have wide area of applications [4]. Nanotechnology has a wide range of process for the synthesis of nanoparticles includes chemical, biological and physical [5,6,7,8,9]. The physical and chemical process consists of various drawbacks like usage of sophisticated and expensive instruments, toxic chemicals, involve high temperature and pressure conditions and moreover are time consuming. Biological synthesis is an easiest and simplest process which makes use of microorganisms or plant extract for nanoparticle synthesis. We focused on the biological synthesis which involves plant extract that can act as a reducing agent utilizing plant several phytochemical as secondary metabolites [10,11,12,13,14,15,16,17,18]. It is considered to be an environmentally friendly method and produces less toxic waste when compared to chemical processes.

Here we choose Aristolochia bracteolata (A. bracteolata) leaf powder aqueous extract (ABLPAE) as a source to synthesis C/CuO nano composites [19]. Aristolochia bracteolata is widely distributed in Asian countries like India and China which are utilized for various medicinal purposes. A. bracteolate [20, 21] is a plant belonging to family Aristolochiaceae which has more than 500 genus with several medicinal applications such as anti-bacterial, wound healing, anti-swelling properties, etc. [22, 23].

Both metal and metal oxide nanoparticles can be synthesized using greener methods. Whereas these metal nanoparticles exhibit some airborne diseases and high in size while compared to metal oxide nanoparticles. Henceforth most of the researchers focused on metal oxide nanoparticles synthesis [24]. Copper (II) oxide (CuO) nanoparticles is a p-type semiconductor oxide has a bandgap of 1.2 eV which can be utilized in several applications viz dye degradation, sensors, electrochemical solar cell, pharmacy, chemical, etc., [25,26,27,28,29,30,31,32,33]. Moreover, CuO is a material of electrode with less cost and toxicity with high usage [19, 34].

This manuscript consists of ecofriendly synthesis of C/CuO nano composites using ABLPAE. Further the synthesized C/CuO nano composites were treated towards for its larvicidal and ovicidal efficacy against dengue causing vector A. ageypti. No reports are there on the synthesis of C/CuO nanocomposites using ABLPAE.

Materials and Methods

Materials Utilized

The A. bracteolata leaf powder was purchased from long bazaar market in Vellore. Copper acetate (≥ 98%) was procured from Sigma Aldrich. Fungal strains were procured from Institute of Microbial technology, Chandigarh and throughout the experimental process we utilized double distilled water without any further purification.

Preparation of Extract

The collected A. bracteolata leaf powder 20 g were immersed in 70 mL of double distilled water and placed over the water bath at 60 °C for 30 min. Further the resultant solutions were separated from powder using filter paper. The ABLPAE filtrate was collected and stored in the refrigerator for further use.

Synthesis of C/CuO Nano Composites

We followed the protocol of our earlier report [35] for the synthesis of C/CuO nano composites with slight modifications. 20 mL of ABLPAE and 80 mL of 1 mM of copper acetate were stirred well and kept it in a water bath@ 60 °C for 3 h and the reaction progress was analyzed through main absorption peak [35]. Once the reaction completed the final solution was centrifuged at 3000 rpm for 30 min and the supernatant solution was discarded. Repeat the process of centrifugation thrice with the help of double distilled water and pellet was collected and placed in furnace @ 350° C.

Experimental Design for the Synthesis of C/CuO Nano Composites

The CCD (Central composite design) optimization study has been carried out on three independent factors like Copper acetate [Cu(OAc)2] concentration X1 (3 levels: 0.5, 1and 1.5 mM), Reaction time X2 (3 levels: 2, 3 and 4 h) and Reaction temperature X3 (3 levels: 50, 60 and 70 ◦C). The synthesis of C/CuO nano composites shows the absorption maximum values which can be consider as the process response. Mainly this study explains the relationship between the experimental Vs theoretical values. The ANOVA (analysis of variance) was performed statistically on the variables by Minitab 14. The results for the C/CuO nanocomposites preparation have been summarized in Table 1.

Table 1 The experimental design according to RSM for optimization of C/CuO nanocomposites
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Characterization Techniques

Further the synthesized C/CuO nano composites were subjected for characterization techniques like UV–visible spectrometry (Schimadzu UV–visible spectrophotometer, model UV-1800), XRD (Advance Powder X-ray diffractometer, Bruker, Germany, model D8), Fourier Transform Infrared Spectroscopy (Alpha T Bruker), TEM and Zeta Potential for confirmation, surface morphology and stability analysis.

Larvicidal and Ovicidal Activity of C/CuO Nano Composites

Dengue larva`s and eggs were collected by Zoologist Dr. K. Elumalai, from the Cooum river, Saidapet, Chennai. The collected larva`s were utilized for larvicidal activity and eggs were utilized for ovicidal activity with the help of green synthesized C/CuO nano composites at various concentrations like 15, 30, 60 and 120 ppm. The experiment was carried out using recyclable paper cups with five replicates of different concentrations of C/CuO against 20 larvae. Further the mortality rate was calculated for both larvicidal and ovicidal with the help of MANOVA and LSD-DMRT test. LC50 and LC90 values are also calculated and it represents the value of mean ± S.D after five replications. Distilled water with larvae in the absence of nanocomposite was taken as the control for the experiment. In ovicidal activity a commercially available insecticide Neem Azal was utilized as control [35]. The eggs were placed in petri dishes with different concentrations of C/CuO nano composites. The petri dishes were kept under laboratory conditions and the ovicidal rate was recorded at different intervals of time with five replicates.

Results and Discussion

Optimization of Synthesis of C/CuO Nano Composites with RSM Methodology

The CCD experimental design utilized to optimize the absorption maximum values of the C/CuO nano composites. Here experimental models of the CCD fitted with a second order polynomial equation (a). The regression equation (a) as follow as,

$$\begin{aligned} \text{Y} & = \, 0.407874 \, + \, 0.0482756 \, \left( {\text{X}_{1} } \right) \, + \, 0.0475831 \, \left( {\text{X}_{2} } \right) \, + \, 0.0251373 \, \left( {\text{X}_{3} } \right) \, - \, 0.0487517 \, \left( {\text{X}_{1*} \text{X}_{1} } \right) \\ & \quad - 0.0252895 \, \left( {\text{X}_{2*} \text{X}_{2} } \right) \, - 0.0216674 \, \left( {\text{X}_{3*} \text{X}_{3} } \right) - 0.00885464 \, \left( {\text{X}_{1*} \text{X}_{2} } \right) \, \\ & \quad + \, 0.00955087 \, \left( {\text{X}_{1*} \text{X}_{3} } \right) - 0.0209725 \, \left( {\text{X}_{2*} \text{X}_{3} } \right) \\ \end{aligned}$$

The ANOVA results were summarized in Table S1. From that ANOVA table, the model should highly significant due to the low p value (< 0.001). The F value or Fisher variance ratio was high in 10.94 compared to the theoretical F-value. The greater the F value, denotes that the present model shows high significance, and data values were fit with experimental values (Fig. S1). The quadratic model was showed that the regression coefficient (R2) values, in this study the R2 values were 83.2% indicates that model high significant. In final identification from the ANOVA table was the lack of fit test. From that table the one variables shown the p value of 0.443 implied that the lack of fit was not significant relative to the pure error.

The 2D contour plots and 3D surface plots represented the interactive relationship between the independent variables shown in Fig. S2 & S3. The whole relationships between factors and response can be better understood by examining the 2D contour plots and 3D surfaces plots. The absorption maximum values of C/CuO nanocomposites rises as the initial copper acetate concentration increases from 0.5 to 1.5. Generally, time and temperature also affects the adsorption maximum values. The effects of the copper acetate concentration, temperature and reaction time on the response are shown in Fig. S3. According to Fig. S3, it is clear indicates that the absorption values rise as the reaction temperature increases from 2 to 3 h. The results emphasize that, it was possible to obtain maximum absorption value by determining the optimal reaction conditions.

Characterization of C/CuO Nano Composites

The reaction mixture of ABLPAE filtrate and copper acetate, which turned from blue to black color, was analyzed using electronic absorption spectroscopy at the wavelength between 200 and 400 nm. The results indicated the peak absorbance around at 274 nm, with an optimum time of which results in conversion of metal salts into metal oxide nanoparticles with the help of secondary metabolites present in the ABLPAE were demonstrated in Fig. S4.

Further the synthesized C/CuO nano composites were studied to find out its average crystalline size with the help of X-ray diffractometer studies. All diffraction peaks are in good agreement with monoclinic CuO (JCPDS No. 05-0661) and carbon peak was also present (Fig. 1) [32]. Further, by using FWHM values with d = 2.2836 and 2θ value of 35.24 was substituted in Debay Scherrer`s formulae (DSF) to identify the crystalline size of eco-friendly synthesized C/CuO nano composites36. DSF stated to be (D = kλ/β cosθ), whereas crystalline size was mentioned as D, (0.94) is Scherrer Constant, which is denoted by k, λ value is obtained from Bragg’s Eq. (2dsinθ = nλ) λ is the wave length, β is FWHM. θ indicates the angle diffraction. The results indicated the ecofriendly synthesized C/CuO nano composites with the average crystalline size of 43.24 nm.

Fig. 1
figure 1

XRD spectrum of C/CuO nano composites

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The Fourier transform infrared (FTIR) spectra of the C/CuO nanocomposites, CuO NPs and the plant extract samples were recorded to provide direct evidence for interactions between CuO and Carbon atoms. Spectrum in Fig. 2, the frequency of the Cu–O stretching vibration experienced a peak shift from 686 to 645 cm−1, with intensity increasing again that indicated the existence of CuO, and the broad band at 3454 cm−1 in C/CuO composites clearly demonstrated the existence of C. The peak observed at 3236 cm−1 is indexed to the stretching and bending modes of hydrogen-bonded hydroxyl groups of plant extract. The strong peak at 1728 cm−1 shows the presence of C = O bond. The sharp band at 1041 cm−1 and 1031 corresponds to C–O–C of carbon material. The peak at 678 and 686 cm−1 can be assigned to Cu–O vibration, which confirms the existence of CuO in the final material.

Fig. 2
figure 2

FTIR of C/CuO nano composites

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Morphological studies were done using Transmission Electron Microscope (TEM), which results in the formation of spherical agglomerated particles which is given in (Fig. 3a–c). From the, Selected Area Electron Diffraction (SAED) pattern value clearly evident that the formation of crystalline patterns of hkl values (002), (111), (200), (202), (211), (220), (113) and (222) which were calculated by unitary method (Fig. 3d).With the help of Fig. 3b and image J software the particle size histogram was analyzed and the obtained results inferred that the synthesized particle was said to be in the average size of 43 ± 2 nm (Fig. S5). Eco-friendly synthesized C/CuO nano composites were subjected for EDAX spectroscopy to identify the elements present in the material (Fig. S6). The percentage of Copper (Cu) is more 58.7%, Oxygen (O) is the second highest quantity 27.8% and carbon (C) is found in good amount (13.5). The crystalline size and particle sizes were compared with commercially synthesized CuO nanoparticles to ensure the advantage of the greener method. In general, Table S2 showed that almost all the CuO nanoparticles from chemical methods exhibited higher crystalline size as well as particle size [36,37,38,39,40,41]. Hence, it was clearly evident that the present greener protocols were easily produced the particles with high crystalline nature and lower particle size.

Fig. 3
figure 3

a–c TEM images of C/CuO nano composites, d SAED pattern of C/CuO nano composites

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ABLPAE extract mediated synthesis of C/CuO nano composites were subject to test its consistency with the help of Horiba nanoparticle analyzer which results in high stability of zeta potential value to be 65.5 mV were clearly stated in Fig. S7.

Larvicidal Activity of Synthesized C/CuO Nano Composites

The collected fourth instar larvae of A. ageypti were utilized for larvicidal study against various concentrations of C/CuO nano composites like 15, 30, 60, 120 ppm. Larvicidal activity completely depends on the dosage of the agent which is clearly evident that the increment of dosage concentration influenced the mortality rate also in the increasing order. The mortality % of various concentrations was clearly listed in Table 2. Further we calculated the LC50 and LC90 with UCL, LCL values by using LSD calculations and χ2 values also stated to be significant at P < 0.05.

Table 2 Larvicidal activity of synthesized C/CuO nano composites
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Comparison of Larvicidal Activity

The larvicidal activity of the green synthesized C/CuO nano composites against A. ageypti was compared with other nanoparticles in the literatures [42]. The larvicidal efficacy of Ag nanoparticles was only performed against A. ageypti in the literatures [42]. Moreover, the mortality percentage was significantly higher compared to that of other nanoparticles in the literature.

Ovicidal Activity of C/CuO Nano Composites

Further the synthesized C/CuO nano composites were subjected to act as an ovicidal agent against eggs of A. ageypti. Here we utilized a commercial available insecticide Neem Azal as a standard. The synthesized C/CuO nano composites show higher the mortality % of 98.4 ± 2.9 in a higher concentration of 120 ppm related to the standard. The ovicidal activity also results in dose dependent reactions which were clearly illustrated in Table 3.

Table 3 Ovicidal activity of C/CuO nano composites
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Conclusion

Aqueous extract of A. bracteata was utilized for the synthesis of C/CuO nano composites and optimization was achieved by the RSM method. The spherical shaped nanocomposites with an average particle size of 43 ± 2 nm were subjected for biological studies to check their larvicidal and ovicidal activity. The results revealed a dose dependent reaction that is, higher the concentration, higher the mortality rate with χ2 values also found to be significant at P < 0.05. Overall this manuscript concluded and proves that the C/CuO nano composites prepared using A. bracteata extract can be utilized as a larvicidal and ovicidal agent.