Introduction

Nano-biotechnology was an interdisciplinary field that fell between material science and bionanotechnology (Sarmast and Salehi 2016). In comparison to bulk materials, nanomaterials have unique features that lend themselves to use in the fields of material science, physics, chemistry, environmental clean-up, optics, biology, biomedicine, and semiconductors (Hasnidawani et al. 2016; Mirzaei and Darroudi 2017; Darvishi et al. 2019). Due to their enormous surface area, small sizes, exceptional reactivity, and free-hanging connections, nanoparticles (NPs) possess astonishing and alluring features. These particles are the consequence of an exponential increase in global spending on nanotechnology and nanoscience research (Zhao et al. 2020; Shojaei et al. 2021). Metal oxide NPs have been produced utilizing a variety of processes, including thermal decomposition, solution phase, sonochemical, solvothermal, microwave irradiation, ionic liquid assisted, electrochemical, spray pyrolysis, and sol-gel method, according to numerous papers up to this point (Sodipo and Aziz 2016; dos Santos Araújo et al. 2019). But regrettably, the aforementioned physical and chemical techniques have unique drawbacks including being very expensive and unpleasant (Taghizadeh et al. 2021). The search for a practical and environmentally friendly approach to NPs synthesis led to the development of greener methods. Traditional plant extraction techniques, such as capping, natural reducing, and stabilizing agents, have made significant advancements toward a successful green synthesis process (Sathiyavimal et al. 2021; Waris et al. 2021). Attributed to the prevalence of phytochemicals such as phenolic compounds, flavonoids, alkaloids, organic acids, terpenoids, and others, the process that is mediated by plants is quicker and displays excellent biological activity (Mahendiran et al. 2017; Rehana et al. 2017; Król et al. 2019). Particularly, the use of common medicinal plants results in the creation of substitute medications to treat a variety of illnesses such as bacterial infections, inflammations, diabetes, malignancies, and viral infections (Singh et al. 2020a, b).

T. divaricata is a member of the apocynaceous family and is both a medicinal and cosmetic plant that possesses anti-infection, antiparasitic, antibacterial, antifungal, and anti-inflammatory properties in its crude extract T Chandani (in Hindi), Nandibatle (in Kannada), Crepe Jasmine (in English), Kutampale (in Malayalam), Nandivardhanamu (in Telugu), and Nandhiyavattam are some of its other names (Tamil) (Sivaraj et al. 2014). It is widely distributed in Bangladesh, South Asia, and India and has dichotomous branches that contain herbs or young trees. It produces stunning white fragrant flowers that can occasionally be seen throughout the year (KALAIMAGAL C 2019). The leaves are streamlined, substantial, deep green in colour, and rich in phytochemicals such alkaloids, tannins, flavonoids, phytosterols, phenols, terpenes, and carbohydrates, T. divaricata leaf aqueous extract is used to make herbal medicines (Umamaheswari 2015; Raja et al. 2018). It is well known that plants produce flavonoids in response to microbial diseases. Numerous metal oxide nanoparticles such as CuO (Ananda Murthy et al. 2021), ZnO (Muthuvel et al. 2020), TiO2 (Narayanan et al. 2021), NiO (Anand et al. 2020), CdO (Gowri et al. 2018), and Co3O4 (Kainat et al. 2021) were reported in previous literature for their unique properties and applications. There have been a lot of studies on expensive materials like gold and silver NPs (Rafique et al. 2019), but there have not been sufficient papers on copper nanoparticles presently. According to their biocidal and antibacterial ability against several infections that are resistant to commercially available antimicrobial treatments, copper oxide (CuO) nanoparticles have received considerable attention (Li et al. 2021) (Kumar et al. 2015; Sharmila et al. 2016). By customizing the surfaces of antimicrobial compounds with CuO nanomaterials, the nanosystem built with CuO is not only employed for treating infections but also for detecting pathogens (Jadhav et al. 2018). As a result, its features pave the way for the creation of smart drug delivery systems. Additionally, the CuO metal oxides used to make the organic and inorganic nanocompounds have special qualities like excellent thermal and electrical conductivity, resilience at high temperatures, and high mechanical strength. Also, in the production of superconductors, gas sensors, solar cells, lithography, and magnetoresistance materials, CuO NPs are widely used as a potential material (Jayakumarai et al. 2015; Jose Varghese et al. 2020). Utilizing human cervical (HeLa) cancer cells, the MTT test was used to evaluate the in vitro anticancer efficacy. The in vitro anti-inflammatory and anti-diabetic studies were evaluated through BSA denaturation and α-amylase inhibition technique.

Materials and methods

Materials and chemicals required

The leaves of Tabernaemontana divaricate were collected from the local region of the southern part of Tamil Nadu, India. The chemicals and solvents used in this research work are of analytical grade. Especially, the highly pure copper (II) nitrate hexahydrate (Cu (NO3)2. xH2O) was bought from Sigma-Aldrich and used as purchased without any such purifications. The Millipore water with ultrapure grade has been utilized throughout the experiment.

Green synthesis of copper oxide nanoparticle utilizing Tabernaemontana divaricate (bio-reductant) leaf extract

The leaves of Tabernaemontana divaricate (T. divaricate) were collected from the local region around Tamil Nadu. The freshly plugged leaves were weighed carefully at about 200 mg and milled sufficiently with an aid of a mortar and pestle. The grounded T. divaricate leaves were then suspended inside a round bottom flask containing 500 mL of water, and fitted in a Soxhlet apparatus under constant water flow. The set-up was heated till boiling, maintained for 6 h, and allowed to attain room temperature normally. Finally, the obtained extract was filtered using (Whatman no. 40) filter paper, leaving behind the residue. For later usage, the T. divaricate extract was gathered and kept in a refrigerator that was maintained at 5 °C.

The 50% of T. divaricate leaf extract was made up to a volume of 250 mL using DI water Cu (NO3)2. H2O designated as the originator of Cu NPs was carefully weighed and prepared as copper nitrate solutions utilizing DI water. The prepared copper nitrate solutions were added dropwise to the T. divaricate leaf extract mixture with constant stirring for 5–6 h at 80 °C. The colour of the solution turns brown as an indication expressing the generation of copper hydroxide (Cu (OH)2) NPs. The obtained solid residue was washed with water and ethanol to remove impurities stuck with the NPs and eventually dried overnight at 80 °C. The schematic representation of CuO NPs synthesis with aid of T. divaricate leaf extract was displayed in Fig. 1, to explain the copper oxide formation with higher oxygen vacancies, CuO NPs composition, and structural phase change. The experiment was carried out under the same condition with varying amounts of Cu originator and named as follows, 1g (TDCO1), 3g (TDCO3), and 5g (TDCO5) respectively.

Fig. 1
figure 1

Scheme on the synthesis of CuO NPs using T. divaricate leaf extract

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Physical characterization of synthesized copper oxide nanoparticles

Powder X-ray diffraction (X’pert PRO analytical diffractometer) with Cu Kα radiation (0.1541 nm) was employed to characterize the crystalline structure, size, and phase confirmation of the T. divaricate leaf extract–induced CuO NPs. Fourier transform infrared (FT-IR) spectroscopy (Perkin-Elmer 1725) was used to confirm the chemical bonding and functional groups of the nanoparticles. Scanning electron microscopy and elemental mapping (SEM, EV018 (CARL ZEISS)) were employed to characterize the nanoparticles average size and size distribution.

Antimicrobial activity

The Gram-positive, Bacillus subtilis (B. subtilis), and Gram-negative, Klebsiella pneumonia (K. pneumonia), two bacterial strains were used to test the synthesized CuO NPs antibacterial efficacy via agar well diffusion method (Tanna et al. 2015). On agar plates, 20 mL of bacterial culture broth was poured using sterile L-rods. The spread plates were left undisturbed for 10 min, and the wells (5 mm in dia.) were made using a sterile gel core. Three different concentrations of CuO NPs suspension (40, 50, and 60 μg/ml) and positive control (50 μg/ml) (Amoxicillin) were made and added to the wells on all plates with the aid of a micropipette. All of the plates were held at 7.3 pH and incubated at 37 °C for 24 h. The inhibition zone (IZ) was evaluated followed by incubation. Three replicates of each screening test were used, and the results were tabulated as mean ± SE (standard error).

Cell culture procedure and cytotoxicity evaluation (MTT assay method)

The National Centre for Cell Sciences (NCCS), located in Pune, India, is where the human cervical cancer cell type HeLa was acquired. The cells were kept in McCoy’s media that was supplemented with FCS, 2mM l-glutamine, and balanced salt solution (BSS), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 10 mM (4-(2-hydroxyethyl)-1piperazineethane, and 1.5 g/L glucose; furthermore, streptomycin and penicillin were made up to 1mL/L. The cells were kept at 37 °C in a 5% CO2 environment. The test samples were applied to the cells in successive concentrations after 24 h. Firstly, they were dissolved in plain dimethyl sulfoxide (DMSO), and the sample solution was diluted with serum-free medium to twice the desired final maximum test concentration. To give a total of five sample concentrations, an additional four serial dilutions were made. These various sample dilutions were added in aliquots of 100 μL to the relevant wells that already had 100 μL of the medium, resulting in the necessary final sample concentrations. The plates were then incubated for an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity after the addition of the sample (Khan et al. 2019). For all concentrations, triplicates were maintained and the medium containing no samples served as the control.

3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) is a yellow-coloured water-soluble substance that was used to measure the amount of % viable cells, while the number of viable cells was directly correlated with the amount of formazan produced. The succinate-dehydrogenase is a mitochondrial enzyme that converts the MTT into purple-coloured formazan by cleaving the tetrazolium ring. After incubating for 48 h, each well was supplemented with 15 μL of MTT (5 mg/mL) in phosphate-buffered saline (PBS) and allowed to incubate for a period of 4 h at 37 °C.

In vitro anti-inflammatory activity

Utilizing the BSA denaturation procedure, the anti-inflammatory properties of the synthesized TDCO3 NPs were evaluated. In order to confirm the scavenging action, the standard reagent and the NPs were examined for albumin denaturation process inhibitions (Velsankar et al. 2020b). The minimal amount of dimethylformamide (DMF) was slowly dissolved in the crude extract and standard diclofenac sodium, which was then diluted and pH was altered to 7.4 using phosphate buffer. BSA solution (1 mM) was well mixed into 4 mL of the test solution that contains varied sample concentrations (50–500 g/mL). The mixture was then incubated for 15 min at 37 °C. The mixture was kept in a water bath at 70 °C for 30 min to induce the denaturation process.

Anti-diabetic activity

The α-amylase inhibition assay was used to assess the anti-diabetic activity of the synthesized TDCO3 NPs. In particular, 0.5 mg/mL of α-amylase, taken as the standard drug, was incubated for 10 min at 25 °C. The pH was adjusted to 6.9 with the addition of sodium phosphate buffer. The synthesized TDCO3 NPs was added to the above-prepared buffer solution at varying concentration (50–500 μg/mL) and left to incubate for 10 min at 25 °C. Afterward, starch (1%) was supplemented, and incubated at 100 °C inside a water bath, followed by the addition of dinitrosalicylic acid to control the enzymatic reaction. The UV-Visible spectrophotometer was used to read the absorbance at 540 nm after the mixture cooled down to normal temperature (Javed et al. 2017).

Result and discussion

Synthesis of Tabernaemontana divaricate leaf extract–mediated CuO NPs

The 3,7, 11, and 15-tetramethyl-2-hexadecane-1-ol and n-hexadecanoic acid phytochemicals present in the T. divaricate leaf extract promoted the formation of CuO NPs. Figure 2 illustrates the systematic approach to producing CuO NPs using the bio-reductant found in T. divaricate leaf extract. In brief, phytochemicals like 3, 7, 11, and 15-tetramethyl-2-hexadecane-1-ol are well known for their antibacterial and anti-inflammatory capabilities, while n-hexadecanoic acid. These phytochemicals act as bio-reductants because the hydroxyl (-OH) groups found in 3, 7, 11, and 15-tetramethyl-2-hexadecane-1-ol and n-hexadecanoic acid bind to copper nitrate to form a complex that triggers the nucleation process (Pandiyan et al. 2018). The Cu ions are then subjected to reduction (Cu(OH)2) under steady stirring, heating, and changing into the desired shape. The phytochemicals act as a capping agent at this stage, transforming the metal hydroxide into the desired shape (Pandiyan et al. 2019). Additionally, the sample was heated to 80 °C to evaporate the H2O content, resulting in the formation of CuO NPs. Overall, the interaction of naturally occurring phytochemicals and synthesized CuO NPs could significantly increase the cell mortality rate.

Fig. 2
figure 2

Mechanism for the capping and reducing behaviour of phytochemicals in CuO NPs formation

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XRD analysis

The XRD method was used to determine the crystalline size and shape concerning microstrain for all synthesized T. divaricate leaf extract–mediated CuO NPs recorded in between the range of 20–80°. The XRD pattern in Fig. 3 a–c shows the formation of CuO monoclinic phases that matches with standard diffraction data (JCPDS No. 03-065-2309) (Wang et al. 2016). Major diffraction peaks in the spectra can be seen at 2θ = 32.34°, 35.79°, 39.44°, 61.19°, and 65.19°, respectively, which correspond to the (110), (111), (200), (022), and (113) crystal planes. In particular, other low intense peaks with lattice plane values of (112), (200), (312), and (116) were identified as the reflections of tetragonal-phased Cu4O3 NPs (JCPDS No. 033-0480) (Patzschke et al. 2021). As the weight percentage of copper precursors increases, the interference of tetragonal Cu4O3 seems to become more prominent (Fig. 3b). However, a strong intensity peak revealed the monoclinic CuO NPs highly polycrystalline character, which is shown to be higher in the TDCO3 sample. The Willamson Hall plot (W-H plot) was used to calculate the crystalline size and microstrain for the synthesized samples (Fig. 4), as per Eq. (4). Nevertheless, the W-H approach was chosen for this investigation because it calculates the crystallite size with greater accuracy than the Scherrer method and takes into consideration both the broadening of the XRD peak caused by the lattice strain and the broadening that is dependent on the crystallite size (Vijayaprasath et al. 2016; Kumar et al. 2021; Naik et al. 2021).

$${\beta}_O.\mathit{\cos}\uptheta =\frac{K\lambda}{D}+{\beta}_{\varepsilon }.\textrm{sin}\uptheta$$
(4)
$${\beta}_O={\beta}_D+{\beta}_{\varepsilon }$$
(5)
Fig. 3
figure 3

X-ray diffraction pattern of TDCO1 (a), TDCO3 (b), and TDCO5 (c) NPs

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Fig. 4
figure 4

W-H plot of (a) TDCO1, (b) TDCO3, and (c) TDCO5 NPs

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Here, βcosθ is plotted against 4sinθ for all synthesized TDCO1, TDCO3, and TDCO5 NPs which is displayed in Fig. 4a–c. As the slope (m) and y-intercept (c) values of the straight-line equation, y = mx + c (linear fitted line), give the crystal strain (ε) and crystallite size (D), respectively (Xue et al. 2018; Shelke et al. 2022). The estimated crystallite size was 42.6, 42.1 and 48.9 nm for TDCO1, TDCO3, and TDCO5 NPs. Since the broadening caused by the microstrain is taken into account, the calculated D values will be considerably higher than the Scherrer method, whereas calculated microstrain (ε) values were 2.12, 7.54, and 1.07 × 10−3 for TDCO1, TDCO3, and TDCO5 NPs. The estimated microstrain values for the nanoparticles and their corresponding actual crystallite size are inversely related. The synthesized NPs crystal size is decreased owing to the flavonoids available in the leaf extract which functioned as a natural bio-reductant and capping agent (Samari et al. 2019). Typically, the smaller crystalline size allows for a greater possibility of adhesion to the negatively charged cell wall and can accelerate the cell lysis process.

FT-IR spectrum analysis

FT-IR spectroscopy was conducted to identify the bio-reductant that was specifically attached to the CuO NPs surface. The Fourier transform infrared spectra of green synthesized TDCO1, TDCO3, and TDCO5 NPs in the 4000–400 cm−1 region are shown in Fig. 5a–c using the KBr pellet method. The FT-IR spectrum of TDCO1 NPs shows the characteristic peak at 3388, 2842, 2343, 1630, 1381, 1119, 883, and 489 cm−1 (Fig. 5a). The broad intense peak around 3388 and 1630 cm−1 is consistent with the -OH stretching and bending vibrations of absorbed water molecules by the synthesized NPs (Daphedar and Taranath 2018). The peak at 2924 cmz−1 indicates the asymmetric -CH stretching vibration and that of corresponding -CH symmetric stretching was found at 2842 cm−1 (Ghasemi et al. 2020). The peak 2343 cm−1 was assigned to the CO2 absorption (Singh et al. 2019), similarly observed in the FT-IR spectrum of TDCO3 and TDCO5 samples. The peak appeared at 1119 cm−1 attributed to the stretching frequency of C-O. The 883 cm−1 absorption peak corresponds to the C=C bending vibration of alkene, confirming 3, 7, 11, and 15-tetramethyl-2-hexadecane-1-ol presence.

Fig. 5
figure 5

FT-IR spectrum of TDCO1 (a), TDCO3 (b), and TDCO5 (c) NPs

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These phytochemical-contained functions serve as effective reducing, capping, and precipitating agents for the synthesis of NPs. The metal oxides are responsible for the absorption peak within the 600 and 300 cm−1 range (Jagathesan and Rajiv 2018). The absorption band observable at 489 cm−1 was assigned to the Cu-O bond confirming the formation of CuO NPs. All the characteristic peaks observed above for TDCO1 NPs are similar to the FT-IR spectrum of TDCO3 and TDCO5 NPs (Fig. 5b and c). TDCO3 NPs Cu-O stretching band shown in Fig. 5b, however, exhibits a minor shift towards a lower wavenumber at 478 cm−1 resembling a change in NPs size which is consistent with the demonstrated XRD findings.

Surface morphology elucidation by SEM analysis

The scanning electron microscopy (SEM) analysis at two distinct magnifications, 1 μm and 200 nm, for the synthesized TDCO1, TDCO3, and TDCO5 NPs revealed the surface morphology, particle size, and distribution (Fig. 6). The size of the grains in the synthesized NPs decreased as a consequence of the bio-reductants phytol and n-hexadecanoic acid. It is interesting to observe that the T. divaricate leaf extract not only functioned as a capping agent but also as a reducing agent by providing -OH and -CO groups, which reduced particle size (Shayegan Mehr et al. 2018; Vasantharaj et al. 2019). It is noted that TDCO1 and TDCO3 NPs displayed in Fig. 6a–d has oval- and round-shaped grains with smooth surface. However, once the concentration of copper (Cu-5g) increases, the grain also starts to expand unevenly and the surface morphology appears to be more agglomerated (Fig. 6e and f), which was clearly seen for TDCO5 NPs.

Fig. 6
figure 6

SEM images at two different magnifications 1 μm and 200 nm of TDCO1 (a and b), TDCO3 (c and d), and TDCO5 (e and f) NPs

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The chemical composition of the prepared TDCO3 NPs was analyzed through EDAX analysis; the consequences of which are shown in Fig. 7. Figure 7a clearly revealed the presence of only Cu and O elements without any impurities in the TDCO3 NPs. To further verify the presence of these two elements, we conducted EDAX elemental mapping analysis, which is shown in Fig. 7b. This analysis further confirmed the presence of copper and oxygen elements in the TDCO3 NPs.

Fig. 7
figure 7

(a) EDAX spectra and (b) elemental mapping analysis of TDCO3 NPs

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Antimicrobial activity

The synthesized TDCO1, TDCO3, and TDCO5 effective bacterial inhibition properties were tested against two basic pathogens, namely, K. pneumoniae as a gram-negative (G−) and B. subtilis as a gram-positive (G+) strain. Interestingly, K. pneumoniae (G-) is a rod-like-shaped thick cell walled (thickness = 160 nm) bacillus that belongs to the family Enterobacteriaceae and the genus Klebsiella. K. pneumoniae is an enteric bacterium found in 5% of healthy human digestive tracts and may also live in the mouth and skin (Doorduijn et al. 2016). Although there are few cases of infections occurring naturally, K. pneumoniae can cause pneumonia in guinea pigs and other living organisms (Amako et al. 1988). The photographic images of K. pneumoniae cultured on Petri dishes which was treated with varying concentrations of NPs (40, 50, and 60 μg/mL) are displayed in Fig. 8a–c.

Fig. 8
figure 8

Antibacterial activity for TDCO1, TDCO3, and TDCO5 NPs with amoxycillin against (G+) B. subtilis (a–c) and (G−) K. pneumoniae (d–f) bacterial strains

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B. subtilis (G+) is a rod-shaped bacterium having a single lipid bilayer membrane that serves as a permeability barrier with a thick cell wall (thickness = 33 nm). It is found commonly in soil and is regarded as a non-pathogenic bacterium (Dadd and Paulton 1968). The B. subtilis bacteria inoculated with varying concentrations of synthesized NPs (40, 50, and 60 μg/mL) are shown in Fig. 8d–f. Table 1 shows that the inhibited zone (IZ) results encounter an elevated value whenever the test material concentration increases which reveals information about the synthesized CuO NPs dose-dependent activity. The bar diagram representation (Fig. 9a and b) justifies that TDCO3 NPs delivered better-inhibited zone values than the other two NPs; here, well-known antibiotic amoxicillin was utilized as a positive control for both bacterial strains. Based on previous studies, we suggested a hypothesis for the enhanced antimicrobial activity of synthesized NPs (Mendes et al. 2022). G− bacteria contain a thin layer between two membranes of peptidoglycan which is responsible for its good antibacterial resistance. Additionally, the membranes dissociated carboxyl groups causing the cell surface to charge negatively. Contrarily, CuO NPs have a positive charge because of the positive charges of the Cu2+ ions and the electrostatic gradient variations across the negative membrane that could severely damage the cell membrane. Subsequently, negative charges on the cell surface are due to the lipoteichoic acid in the membrane and the teichoic acid in the peptidoglycan layer (Ahmed et al. 2021). Teichoic and lipoteichoic acids chelate Cu2+ ions which are then transported across membrane proteins by passive diffusion.

Table 1 Zone of inhibition (ZI) value for TDCO1, TDCO3, and TDCO5 NPs against (G+) B. subtilis (a) and (G−) K. pneumoniae (b) pathogens
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Fig. 9
figure 9

Bar diagrammatic representation of zone of inhibition for TDCO1, TDCO3, and TDCO5 NPs against (G+) B. subtilis (a) and (G−) K. pneumoniae (b) bacterial strains

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As already known, CuO is a transition metal oxide and semiconductor with a broadband gap (3.25 eV) and electron-hole pairs are created when the radiation energy exceeds the CuO band gap. The produced electrons are promoted to the conduction band (CB) and significantly an oxidizing character is acquired by the hole that is produced in the valence band (VB) which oxidizes water molecules or hydroxide anions and produces powerful oxidizing species (Kumar et al. 2019; Abdo et al. 2021). Furthermore, CuO in its Cu2+ form severely damages the cytoplasmic membrane, due to its particular affinity for sulfur, and the Cu2+ ion inhibits the glycolytic enzyme by oxidizing thiol groups (Fig. 10). Importantly, bacterial cell division is a complicated and dynamic process that begins with the polymerization of the FtsZ protein in order to build the divisome which will control all the processes involved in cell division as well as the production and remodelling of the cell wall (Gnanasekaran et al. 2021). CuO NPs work by stopping FtsZ-GTPase activity which in turn induces cell filamentation stops cell division and promotes cell lysis. According to previously published papers, the antibacterial activity of the as-prepared TDCO3 nanoparticles was compared and is shown in Table 2.

Fig. 10
figure 10

Graphical representation of antibacterial and anticancer activity of CuO NPs

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Table 2 Comparison of antimicrobial activity test results for various nanoparticles
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In vitro anticancer analysis (HeLa cell)

The synthesized TDCO1, TDCO3, and TDCO5 NPs anticancer activity was assessed against the HeLa cell line which is displayed in Fig. 11. This study was assessed with varying concentration (0, 5, 10, 25, 50, and 100 μg/mL−1) of the test material. Hereby, the anticancer results presented in the form of bar diagram suggested that TDCO1 (Fig. 12a) shows 99.4, 81.2, 62.5, 47.1, 33.2, and 24.3 %, TDCO3 exhibits 99.5, 71.4, 56.8, 41.2, 35.6, and 27.2%, and TDCO5 delivered 98.2, 81.2, 62.5, 47.1, 33.2, and 24.3% of cell lysis corresponding to 0, 5, 10, 25, 50, and 100 μg/mL−1 concentration. The percentage cell viability was evaluated to be minimal at 100 μg/mL−1 dose concentration for all the synthesized CuO NPs. Moreover, the IC50 values were found by taking linear regression which suggested that TDCO3 demonstrated a lower IC50 value of about 18.2 μg/mL−1 than TDCO1 (IC50 = 21.5 μg/mL−1) and TDCO5 (IC50 = 19.6 μg/mL−1) NPs (Fig. 12b). The key mechanisms underpinning TDCO3 NPs improved cytotoxicity and increased activity were ROS production to be the production of ROS, oxidative stress, and the activation of apoptosis, all of which play a crucial role in the death of HeLa cancer cells (Arumugam et al. 2021).

Fig. 11
figure 11

Optical microscope images of Hela cells inoculated with different concentrations (0, 5, 10, 25, 50, and 100 μg/mL−1) of TDCO1 (i), TDCO3 (ii), and TDCO5 (iii) NPs

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Fig. 12
figure 12

Bar diagram representation of anticancer activity (a), IC50 value of TDCO1, TDCO3, and TDCO5 NPs, anti-inflammatory activity (c), and anti-diabetic activity of TDCO3 NPs

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The crystalline size, surface shape, surface charge distribution, test material dose, and dissolution kinetics of CuO NPs are some examples of physical characteristics that play a crucial role in the cytotoxic effect. The cytotoxic effects of TDCO3 NPs are more prominent due to their small size (12.1 nm), and they can easily enter HeLa cells to create the ROS that causes cell death. Figure 10 depicts the process through which CuO NPs cause cytotoxicity due to the evenly distributed CuO NPs that enter into the cells easily and kill the HeLa cancer cells. Another significant factor in the lethality of HeLa cancer cells is the surface charge. Owing to the Vander walls interaction between negatively charged cell membranes, positive charged Cu+, and Cu2+ ions could cause greater harm than negative charges (Pandiyan et al. 2019; Ansari and Asiri 2021). The attraction of the positively charged CuO NPs to the negatively charged cell membrane is what causes this charging effect. The CuO NPs interact with the negatively charged surface of cancer cells, causing cell membrane leakage and making cancer cells more hazardous (Mousavi-Kouhi et al. 2021). Additionally, the dissolving Cu+ and Cu2+ ions will cause effective cytotoxicity, which will cause the enzyme system to be disrupted. As a result, CuO NPs synthesized by T. divaricate leaf extract had excellent anticancer activity against HeLa cells. Utilizing plant extracts also has the advantages of being secure and environmentally beneficial. Finally, CuO NPs have the potential to function as a more potent and effective anticancer therapeutic agent.

In vitro anti-inflammatory analysis

The synthesized TDCO3 NPs anti-inflammatory activity was assessed through the BSA denaturation technique, and the bar diagram representing percentage inhibition at different NPs dosages is displayed in Fig. 12c. Moreover, the TDCO3 NPs were supplemented at five different concentrations 10, 50, 100, 250, and 500 μg/mL; similarly, Diclofenac sodium was also performed. The values obtained for TDCO3 NPs were 20.11, 40.27, 52.44, 65.83, and 85.66% for 10, 50, 100, 250, and 500 μg/mL concentrations. The percentage inhibition acquired for Diclofenac sodium (standard) was at 22.33, 45.67, 70.08, 58.67, and 95.55% for 10, 50, 100, 250, and 500 μg/mL, respectively. Besides, the anti-inflammatory activity was observed maximum of 85.66% for the highest concentration of 500 μg/mL. The obtained inhibition percentage was close to Diclofenac sodium which showed 95.55% at 500 μg/mL concentration. Protein denaturation is a general process that results in the loss of biological activity by degrading secondary and tertiary protein structures (Muthulakshmi and Sundrarajan 2020). A variety of factors, including salts or alcohol, might change the solubility of albumin. In contrast to infectious activity like antibacterial activity, it is attractive to assess the defensive action like an anti-inflammatory potential (Veerasingam et al. 2020; Velsankar et al. 2020a). This analysis demonstrated high inhibition towards BSA denaturation which in turn expressed that TDCO3 NPs functioned as an effective anti-inflammatory drug.

In vitro anti-diabetic drug

The most prevalent condition affecting persons between the ages of 40 and 60 is diabetes mellitus, which is brought on by insufficient insulin production and rising blood glucose levels. Anti-diabetic medications are taken to treat diabetes and manage insulin shortage (Nasab et al. 2020; Sundrarajan and Muthulakshmi 2021). Numerous studies have been done to identify viable anti-diabetic medications (Kazmi et al. 2021). In this current research, we have aimed to figure out whether TDCO3 NPs could function as an effective anti-diabetic drug via inhibiting α-amylase with a standard drug as “acarbose.” The bar diagrammatic representation of percentage inhibition versus TDCO3 and acarbose concentration (10–500 μg/ml) is shown in Fig. 12d. Notably, at a higher concentration of 500 μg/ml TDCO3 NPs and acarbose exhibited maximum scavenging activity as 81.18 and 87.71% as inhibition percentage, respectively. Moreover, the obtained anti-diabetic results suggested that TDCO3 NPs delivered scavenging activity close to the standard acarbose and could be utilized as a potential anti-diabetic drug.

Conclusion

The T. divaricate leaf extract was successfully used to biosynthesize CuO NPs using inexpensive, environmentally responsible, and less dangerous chemical techniques. The development of monoclinic and tetragonal phases of CuO NPs with a crystallite size of 12.1 nm was suggested by the powder XRD pattern. The bands of functional groups involved in the Phyto-mediated green synthesis of CuO NPs, including Cu and O bonding, were visible in the FT-IR spectrum. The morphology of the CuO NPs seemed to be round and oval in the scanning electron micrographs. TDCO3 NPs exhibit excellent antibacterial and anticancer action compared to TDCO1 and TDCO5 NPs. The TDCO3 NPs have therefore functioned as a more potent drug against the K. pneumoniae (G−) and B. subtilis (G+) strains, and they ensured the HeLa cells were most significantly cytotoxic. The anti-inflammatory analysis revealed possible effective activity on egg albumin inhibition by TDCO3 NPs. Synthesized TDCO3 NPs anti-diabetic properties showed that they would work well as a medication to treat diabetes. We conclude that CuO NPs are a promising material for use in biomedical applications.