Introduction

The genus Cocculus is a genus of the Menispermaceae family comprises about 10 species that are scattered in Australia, North America, and Asia [1]. Cocculus hirsutus (L.) W. Theob. (Fig. 1) is one of the member species in Menispermaceae family known as Jal-jammi. It is a climber recognized to grow in India’s tropic and sub-tropical areas. Logesh [1] reported the list of alkaloids, flavonoids, triterpenes, and volatile constituents from different parts of C. hirsutus. The isoquinoline alkaloids like cohirsinine, cohirsitine, jamtinine, d-trilobine, and dl-coclaurine [2,3,4] were found in an ethanolic extract of the whole plant. Decoction of these leaves is used for treating many disorders like dysentery, psoriasis, and urinary problems. The plant parts such as roots and leaves are used as a diuretic and in the treatment of gout. The aerial components of the plant are employed as a diuretic and purgative, and root extract had anaesthetic and anti-inflammatory properties. Decoction made from the leaves of this plant cures psoriasis. Researchers are looking for new anti-diabetic drugs that are both therapeutically effective and free of the side effects. There are very few reports available on the synthesis and characterization of nanoparticles using leaf extract of C. hirsutus [5,6,7]; hence, the contemporary study emphasizes on the synthesis, characterization, and applications of CuNPs.

Fig. 1
figure 1

Cocculus hirsutus plant (leaves)

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Nanotechnology is mainly concerned with the manipulation of atoms and molecules which could be used in various applications such as biomedical engineering. Diosgenin encapsulated PCL-Pluronic nanoparticles were developed by nanoprecipitation method for improving the inhibition of proliferation of brain cancer cells [8]. A process known as the synthesis of nanoparticles using plant extracts could provide a path for production of commercially interesting nanoparticles. Due to its numerous properties such as ultrafine proportions, appreciable pore size, and large precise surface area, nanomaterials have become one of the most attractive sectors in nanotechnology field over conventional methods [9,10,11]. In recent era, research analysts shown their keen interest towards the synthesis of varied nanoparticles using diverse + non-metals/metals such as gold, titanium, chromium, manganese, iron, palladium, silver, and zinc [12,13,14,15,16,17]. Among them, copper nanoparticles (CuNPs) are one of the most important and extensively used nanoparticles. Aside from its noble properties, copper has a wide variety of potential applications in nanotechnology. Its size, chemical stability, conductivity, and numerous catalytic properties, as well as antibacterial and anti-inflammatory properties, make it stand out [18,19,20].

The silver nanoparticles were formed when the aqueous silver ions were reduced in the leaf extract of C. hirsutus. They exhibited a high degree of transparency and were highly effective against pathogens [5]. Currently, plant extracts and microbes like Catharanthus roseus and Moringa oleifera [21,22,23], bacteria [24], fungi [25], and sea weeds [26] are employed to make metal nanoparticles because of their therapeutic potential [27]. Secondary metabolites are prevalent in florae, among these biological materials, and significant medicinal chemicals are encapsulated with nanoparticles throughout the production method. The generation of nanoparticles from medicinally important plants has been the subject of numerous investigations due to varied applications like acaricidal, pediculicidal, and larvicidal activity using Momordica charantia [28]; antimicrobial activity using Amaranthus caudatus [29]; antioxidant, in vitro and in vivo effects to anti-cancerous activity against breast cancer cell line using Clerodendrum infortunatum, Abutilon indicum, and Clerodendrum inerme [30]; antibacterial, photocatalytic activity using Azaridacta intica [31]; cytotoxicity, antibacterial activity using Tabernaemontana divaricata [32]; dye degradation using Kalopanax septemlobus [33]; antibacterial and catalytic activity using Conyza canadensis [34]; antimicrobial activity using Glycosmis pentaphylla [35]; and antioxidant, anti-diabetic, and anti-inflammatory using Andrographis paniculata [36]. The current research focus on the synthesis, characterization of C. hirsutus leaf extract-derived CuNPs, and evaluation of their in vitro antioxidant, antibacterial, and anti-diabetic properties.

Materials and Methods

Collection of Plant Material

The leaves of C. hirsutus were gathered from the Rampathadu village, Pendlimarri Mandal, Kadapa, Andhra Pradesh, India in January 2021. The plant material (leaves) was verified and authenticated by the Department of Botany at the Yogi Vemana University in Kadapa, India. C. hirsutus leaves were thoroughly washed to eliminate debris on the surface of the leaves, rinsed with distilled water and shade dried for 7–8 days, and pulverized into fine powder via electric grinder and used for further experimentation.

Preparation of Leaf Extract

A 10 g of the shade dried leaves was added to 100 mL double distilled water which was then placed in water bath for 15 min at 70 °C. The solution was then filtered using Whatman no.1 filter paper and cooled down to room temperature. The collected extract was then stored at 4 °C further analysis. The leaf infusion can be utilized as reducing agent for the formation of copper nanoparticles (CuNPs).

Phytochemical Analysis of C. hirsutus Leaf Extract

The primary phytochemicals in the C. hirsutus leaf extract were identified using a standard procedure for qualitative phytochemical analysis [37].

Green Synthesis of Copper Nanoparticles

For 2 min, 200 mL copper acetate was mixed well using magnetic stirrer in an Erlenmeyer flask. Then, 20 mL of C. hirsutus leaf extract was added. The obtained infusion was then stirred continuously for another 24 h at RT. During reaction, the colour changed from brown to greenish brown indicates the synthesis of CH-CuNPs and sodium hydroxide was added for controlling the pH, followed by centrifugation process. Finally, the dark greenish CH-CuNPs were collected after the supernatant was decanted and finally monitored by using UV–visible spectrophotometer.

Characterization of Synthesized CH-CuNPs

A Thermo Scientific Evolution 401 UV–Vis spectrophotometer with a resolution of 1 nm was used for UV–vis analysis range between 200 and 700 nm. The vibrational analysis was studied with PerklinElmer (Spectrum Two model), UK, Vertex 70 model Bruker, Germany, and FTIR spectra in the range of 500–4500 cm−1. The structural evidences (size and shape) and elemental composition of the synthesized CuNPs were monitored by SEM (Model: EVO 18; Carl Zeiss, Germany).

Antioxidant Activity of CH-CuNPs

Determination of Total Antioxidant Activity

A modified form of the Pavithra [38] method was used to evaluate total antioxidant content. The standard reagent solution was combined with C. hirsutus leaf extract and CH-CuNPs concentration (50–250 \(\upmu\)g/ml) (28 mM sodium phosphate, 4 mM ammonium molybdate, and 0.6 M sulphuric acid). Incubation was done in closed tubes for 90 min at 95 °C in a thermal block. The absorbance readings were examined at 695 nm once cooled to room temperature. The antioxidant capacity was calculated as a percentage of total antioxidant activity.

H2O2 Scavenging Assay

C. hirsutus leaf extract and CH-CuNPs’ ability to scavenge H2O2 was monitored using a modified protocol from Pavithra [38]. Phosphate buffer is used to make a hydrogen peroxide solution (40 mM) (1 M pH 7.4). In a 40 mM H2O2 solution, different concentrations of sample were added (50–250 \(\upmu\)g/ml). After 10 min, the OD values were monitored at 230 nm. The standard for this experiment was ascorbic acid. The scavenging activity of free radicals was measured using percent inhibition.

Antibacterial Activity of CH-CuNPs Against Pathogenic Bacteria

The antibacterial activity of CH-CuNPs was scrutinized using the agar well method. Various bacterial strains such as MTCC 6571 Staphylococcus aureus, MTCC 443 Escherichia coli, MTCC 3610 Bacillus subtilis, MTCC 6380 Proteus vulgaris, and MTCC 10,248 Salmonella typhi were selected to evaluate the activity of leaf extracts and CH-CuNPs. Various concentrations (25, 50, 75, and 100 µl) of leaf extracts and CH-CuNPs were used. Plates were allowed to incubate at 37 °C for 24 h and the diameter of antibacterial zone was noted [39].

Anti-diabetic Assay of CH-CuNPs

α-Amylase Inhibition Assay

The anti-diabetic activity was estimated by employing C. hirsutus leaf infusion and CH-CuNPs for inhibiting α-amylase activity, as formerly defined by Balan [40]. Varied concentrations (25–100 μg/ml) of leaf infusions and CH-CuNPs were added individually to sodium phosphate (0.02 M) buffer with sodium chloride (6 mM, pH 6.9) and incubated for 20 min at 37 °C. Later, reaction mixture was then added with 1% (250 μl) starch and incubated for another 15 min. Next, the activity was then halted by adding dinitro acid followed by a water bath at 100 °C for 10 min and cooled down. The optical density was measured at 540 nm. Control was created using reaction solutions with varying doses. Negative and positive controls were created using reaction solutions lacking NPs and metformin at various doses (25–100 \(\upmu\)g/ml). The following formula was used to compute the percentage of α-amylase inhibition:

$$\%\;\mathrm{inhibition}=[\frac{\left(\mathrm{At}-\mathrm{Ap}\right)}{\mathrm{At}}]\times 100$$
$$\mathrm{Here\;At}\hspace{0.17em}=\hspace{0.17em}\mathrm{control\;absorbance}$$
$$\mathrm{Ap}\hspace{0.17em}=\hspace{0.17em}\mathrm{sample\;absorbance}$$

α-Glucosidase Inhibition Assay of CH-CuNPs

To evaluate the inhibitory action of Cocculus leaf extract and CH-CuNPs, a slightly modified method described by Viswanathan et al. [41] was followed. Precisely, different concentrations (25–100 μg/ml) of C. hirsutus leaf extract and CH-CuNPs were added individually. To this add sodium phosphate buffer (0.1 M) with NaCl (6 mM) and 0.1 units of α-glucosidase that had been pre-incubated at 37 °C for 10 min. The reaction solutions were then incubated para-nitrophenyl-α-D-glucopyranoside made with sodium phosphate buffer. The reaction was stopped with 50 μl of sodium carbonate (0.1 M Na2CO3), and the activity of α-glucosidase was measured spectrophotometrically at OD 405 nm. Negative and positive controls were formed by means of reaction mixtures lacking NPs and metformin at various doses (25–100 \(\upmu\)g/ml). Using the below mentioned formula, the inhibition percentage was calculated.

$$\%\;\mathrm{inhibition}=[\frac{\left(\mathrm{At}-\mathrm{Ap}\right)}{\mathrm{At}}]\times 100$$
$$\mathrm{Here\;At}\hspace{0.17em}=\hspace{0.17em}\mathrm{control\;absorbance}$$
$$\mathrm{Ap}\hspace{0.17em}=\hspace{0.17em}\mathrm{sample\;absorbance}$$

Statistical Analysis

Statistical analysis was done using a two-way ANOVA. Single asterisk (*) denotes significant difference of positive control with test samples at P < 0.05, double asterisk (**) at P < 0.01, and triple asterisk (***) at P < 0.001.

Results and Discussion

Phytochemical Analysis of C. hirsutus Leaf Extract

Phytochemical analysis of C. hirsutus leaf extract indicates the presence of phenolics, diterpenes, and flavonoids. But among them, phenolics were present at elevated levels.

illustrated in Table 1.

Table 1 Phytochemical analysis of C. hirsutus leaf extract
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Green Synthesis of CH-CuNPs Using the Leaf Infusion of C. hirsutus

Leaf extract of C. hirsutus was mixed with copper acetate solution in 1:4 ratio (v/v) and stirred successively for 24 h at room temperature. A transformation colour change from deep brown to dark greenish-brown visually indicates the formation of CuNPs, which can be determined by UV–vis spectroscopy. Figure 2 shows absorption peak at 355 nm which is due to the activation of surface plasmon resonance (SPR) phenomena [18, 42]. Secondary metabolites such as tannins, saponins, phenol, and alkaloids found in the leaf may act as capping and stabilizing agents, and might be responsible for the reduction of Cu+ to Cu [18,19,20, 43, 44].

Fig. 2
figure 2

UV–Visible spectrum of CH-CuNPs using leaf extract of C. hirsutus. The inset figure illustrates visually observed colour change from deep brown to dark greenish-brown

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FT-IR Spectral Analysis of CH-CuNPs

The key peaks, wavenumbers, and interpretation of the probable functional groups are displayed in Fig. 3 and Table 2 of the FT-IR spectra of CH-CuNPs. The FT-IR data further indicates that C. hirsutus phytochemicals or functional groups are responsible for the reduction and stabilization of CH-CuNPs. The absorption bands of CH-CuNPs at 853 and 1525 cm−1 correspond to aromatic C–H, C = C functional groups. Similarly, the CH-CuNPs absorption bands at 1011 and 1397 cm−1 due to C–F stretch correspond to alkyl and aryl groups, respectively. Bands of absorption for the CH-CuNPs at 2307 and 2366 cm−1 due to strong C–H stretch correspond to Alkyne. Bands of absorption for the CH-CuNPs at 3801 and and 3671 cm−1 due to O–H stretch correspond to alcohol. The phytochemical analysis of C. hirsutus extract reveals the elevated levels of phenolics. Thus, the bio-reduction of Cu+ to CH-CuNPs could be attributed to the aromatic functional groups of leaf extract [20].

Fig. 3
figure 3

FT-IR spectra of synthesized of CH-CuNPs using the leaf extract of C. hirsutus

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Table 2 FT-IR analysis and probable functional groups of CH-CuNPs
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SEM and EDX Spectral Analysis of CH-CuNPs

The shape and size of the CH-CuNPs were examined by Scanning Electron Microscopy (SEM) at 500 nm magnification shown in Fig. 4A. CH-CuNPs had a sheet-like form and a dimension of 63.46 nm on average. Energy dispersive X-ray analysis (EDX) was used to investigate the elemental proportions of the green synthesized CH-CuNPs. Figure 4B displays the EDX spectra of the Cu metal, which indicate the percent composition and a significant elemental peak at 1 and 8 keV. Biomolecules that were employed to cap the CH-CuNPs also showed up as tiny peaks. Inset Fig. 4B shows the percentages of Cu and other biomolecules.

Fig. 4
figure 4

A SEM image of synthesized CH-CuNPs using the leaf extract of C. hirsutus. B EDX analysis of CH-CuNPs. Inset figure gives information regarding elemental composition of synthesized nanoparticles

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Antioxidant Activity of Synthesized CH-CuNPs

Total Antioxidant Activity

C. hirsutus leaf extract and CH-CuNPs were subjected to determine the total antioxidant activity by phosphomolybdenum method. CH-CuNPs showed the higher % of total antioxidants activity compared to C. hirsutus leaf extract. Increasing concentration of CH-CuNPs increases the % of total antioxidants activity. The % of total antioxidant activity of CH-CuNPs ranges from 61% ± 0.55 to 84% ± 0.79. At 250 µg/ml of concentration, CH-CuNPs shows the highest % of inhibition of about 84% ± 0.79 and shown in Fig. 5A.

Fig. 5
figure 5

Antioxidant activity of C. hirsutus leaf extract and CH-CuNPs. A Hydroxy radical scavenging activity. B Total antioxidant activity. Single asterisk denotes significant difference of positive control (ascorbic acid) with test samples at P < 0.05; double asterisk at P < 0.01; and triple asterisk at P < 0.001

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The basic principle behind this method is the subsequent conversion of Mo(IV) to Mo(V), which shows its maximum absorbance at 695 nm. The more the antioxidant activity, the higher the absorption.

Hydrogen Peroxide Scavenging Assay

C. hirsutus leaf extract and CH-CuNPs were subjected to H2O2 free radical scavenging. CH-CuNPs showed the elevated % of H2O2 free radical scavenging activity compared to C. hirsutus leaf extract. Increasing concentration of CH-CuNPs increases the percentage of free radical scavenging activity. At 250 µg/ml of concentration, CH-CuNPs shows highest % of inhibition of about 71% depicted in Fig. 5B.

Antimicrobial Activity of CH-CuNPs

Antibacterial activity of C. hirsutus leaf extract and CH-CuNPs was determined by well diffusion technique against G+ve positive (B. subtilis and S. aureus) and G−ve negative (E. coli, P. vulgaris, S. typhi) pathogenic bacteria. CH-CuNPs showed elevated zone of inhibition compared to the C. hirsutus leaf extract. Increasing the concentration of CH-CuNPs increases the zone of inhibition. The produced CuNPs presented significant antimicrobial efficacy against bacterial strains. The results of the zone of growth inhibition were ranged between 9.6 ± 0.17 and 27.86 ± 0.52 mm from Fig. 6A–E. The highest inhibition zone (Fig. 6A) was observed in B. subtilis (27.86 ± 0.52 mm) at 100 µg/mL concentration and least was found in S. aureus (9.6 ± 0.17 mm) as depicted in Fig. 6D, respectively.

Fig. 6
figure 6

Antibacterial activity of C. hirsutus leaf extracts and their CuNPs along with positive control (Ampicillin). A Bacillus subtilis; B Escherichia coli; C Proteus vulgaris; D Staphylococcus aureus; E Salmonella typhi. Double asterisk denotes significant difference of positive control with test samples at P < 0.01 and triple asterisk indicates P < 0.001

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The Cu+ ions in the CuNPs can inhibit bacterial growth by penetrating the bacterial cell wall. The presence of Cu+ ions in the CuNPs can stimulate the growth of bacteria by penetrating their cell walls. These ions can also prevent the bacterial cells from producing cytoplasmic fluid. The zone of inhibition was slightly higher in G+ve bacteria, compared to G−ve bacteria. Premanathan [45] and Vijayakumar [46] backed up our findings, claiming that the difference in inhibition of growth between Gram-positive and Gram-negative bacteria was attributable to differences in cell wall composition. Our findings are consistent with Gupta [47] and Devi et al.’s [48] publications.

Anti-diabetic Activity of CH-CuNPs

The breakdown of the α-amylase and the gastrointestinal enzyme α-glucosidase are resulted products in the production of disaccharides and oligosaccharides [40]. Type 2 diabetes is a clinical disorder characterized by hyperglycaemia and frequent urination. It is usually triggered by the lack of insulin. It is a chronic illness characterized by high levels of glucose and insulin secretion. It is caused by the impaired insulin action and the excessive production of hepatic glucose [23, 49]. As a result of the ineffectiveness of insulin, blood glucose levels in diabetic individuals remain elevated. As a result, blocking α-amylase and α-glucosidase enzymes is important to manage blood glucose levels.

Currently, numerous medicines are offered to inhibit the α-amylase and α-glucosidase enzymes like acarbose, miglitol, and voglibose with some extent of negative side effects. The current study focuses on the production of CH-CuNPs as an alternative. The percentage inhibition of the enzymes α\(-\)amylase (Fig. 7A) and α\(-\)glucosidase (Fig. 7B) by C. hirsutus leaf extract and CH-CuNPs. Unlike the pattern found with C. hirsutus leaf extract, the percentage inhibition by CH-CuNPs was more with increasing NPs content. For α-amylase, the percentage of inhibition ranged from 25.60 percent 0.17 (25 \(\upmu\)g/ml) to 64.5 percent 0.11 (100 \(\upmu\)g/ml), and for α-glucosidase, the percentage of inhibition ranged from 20.6 percent 0.17 (20 \(\upmu\)g/ml) to 68.5 percent 0.11 (100 \(\upmu\)g/ml). The α-glucosidase inhibition rate was higher than the α-amylase inhibition rate. The above results are consistent with Badole [50], Sangameswaran [51], Sarkar [52], and Vinotha [53].

Fig. 7
figure 7

The effect of aqueous leaf extract of C. hirsutus and CH-CuNPs on α-amylase and α-glucosidase inhibition. A α-Amylase inhibition percentage, B α-glucosidase inhibition percentages, positive control (Metformin). Triple asterisk indicates a significant increase in relation with the untreated control (enzyme) at P < 0.001

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Conclusion

Only a few studies focused on the green synthesis and evaluation of pharmacological activities of C. hirsutus. Therefore, the current study aimed to synthesized CuNPs by leaf infusion of C. hirsutus in a simple, non-toxic, and ecogenic process. The resultant biogenic CuNPs were characterized and sheet-like structure morphology was observed from SEM images. The synthesized plant based nanoparticles have the highest scavenging activities in both PMA and H2O2 assays and presented significant antimicrobial efficacy against bacterial strains and anti-diabetic efficacy in both the \(\alpha\)-amylase and \(\alpha\) -glucosidase inhibition assay.