Preparation of Novel Nanoformulation to Enhance Efficacy in the Treatment of Cardiovascular Disease

Despite many efforts over the last few decades, cardiac-based drug delivery systems are experiencing major problems, such as the effective delivery of the precise amount of a drug. In the current study, an effort has been made to prepare a nano-herbformulation (NHF) to overcome the major problem of conventional intervention. Copper oxide-based NHF was prepared using plant extract of Alternanthera sessilis and characterized using physicochemical techniques such as Transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Dynamic light scattering (DLS), UV-Vis spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). TEM analysis revealed that spherical NHF obtained of size 20–50 nm. In addition, XRD and FTIR confirmed the presence of phytochemicals with biological properties over the surface of copper oxide-based NHF. It was demonstrated that dose-dependent antiapoptotic activity was shown against DOX-induced cardiomyocytes, where ROS levels were significantly reduced to 0.29% from 37.99%. The results of the flow cytometry analysis using PI and Annexin staining further confirmed the antiapoptotic activity of NHF against DOX-induced cardiomyocytes by ROS scavenging. Thus, NHF might be used for cardiovascular disease treatment.


Introduction
Nano-biotechnology combines nanotechnology with biotechnology to improve products and create new technologies. We manufactured several nanoparticle materials, including pigments, cosmetics, and biomedical devices. Nanomaterials such as copper oxide nanoparticles have made possible advances in pharmaceuticals, food packaging, and catalyst manufacturing [1,2]. There are several methods available for synthesizing nanoparticles [3]. To prevent agglomeration, we use toxic chemicals as reducing or stabilizing agents. CuO nanoparticles synthesized by chemical routes are also toxic to acute and chronic aquatic organisms [4]. Nano herb formulation contains a variety of active ingredients. Invade the targets of cells involved in developing various diseases [5]. The reason is that many of these nano-herbal compounds combine metal with many other bioactive compounds. The presence analysis of numerous chemical components to detect these presents a challenge for the chemist and pharmacist. These are due to the presence of the metal compound and the variety of herbal compounds. The present works were approached based on the Siddha principle.

Collection and Identification of Plants
Our nano herbomineral formulation is synthesized using floral species such as Alternanthera sessilis, Azadirachta indica, Sphaeranthus indicus, Nelumbo nucifera, and Michelia champaca. Fresh leaves of Sphaeranthus indicus, Alternanthera sessilis, and Azadirachta indica, free from diseases, were collected from the Vellore institute of technology. Nelumbo nucifera and Michelia champaca were collected from Arulmigu Arunachaleswarar Temple, Thiruvannamalai, Tamilnadu, India.

Optimization and Formulation of Nano Herbomineral in Modified Siddha Approach
The CuONP required for our herboformulation was biosynthesized by a green method in Alternanthera sessilis and optimized using response surface methodology (RSM) [27]. RSM is an optimization approach using statistical analysis to optimize the parameters and determine the optimal conditions for obtaining nano-sized particles. Testing the existing drugs available in the market, followed by understanding the prime relationship based on polynomial mathematics and representing the results at optimum levels, are the main goals of this optimization method, as shown in Figure 1. treatment with nanoparticles being directly incorporated into the genome to either treat/suppress/delete the disease-causing gene and thus cure the ailment with a high success rate commonly known as "target drug delivery" system [23], biosensors [24], cosmetic industry [25], and therapeutics [26].

Collection and Identification of Plants
Our nano herbomineral formulation is synthesized using floral species such as Alternanthera sessilis, Azadirachta indica, Sphaeranthus indicus, Nelumbo nucifera, and Michelia champaca. Fresh leaves of Sphaeranthus indicus, Alternanthera sessilis, and Azadirachta indica, free from diseases, were collected from the Vellore institute of technology. Nelumbo nucifera and Michelia champaca were collected from Arulmigu Arunachaleswarar Temple, Thiruvannamalai, Tamilnadu, India.

Optimization and Formulation of Nano Herbomineral in Modified Siddha Approach
The CuONP required for our herboformulation was biosynthesized by a green method in Alternanthera sessilis and optimized using response surface methodology (RSM) [27]. RSM is an optimization approach using statistical analysis to optimize the parameters and determine the optimal conditions for obtaining nano-sized particles. Testing the existing drugs available in the market, followed by understanding the prime relationship based on polynomial mathematics and representing the results at optimum levels, are the main goals of this optimization method, as shown in Figure 1. Initially, plant extract of Alternanthera sessilis and cupric chloride was used to synthesize CuONP (to eliminate the toxicity), which was later optimized to a size of 10 nm. Our formulation is entirely inspired by the prevailing Siddha medicine formulation in which they take pure copper along with the extract of other plants and synthesize the particles for around 32 days to eliminate its toxicity and achieve efficiency. However, here in our formulation methodology, we tried to reduce the synthesis time by opting for suitable technology and following the same flow for formulation. After the optimization of CuONP in a powdered concentration of 300 mg, it is placed in an oil bath. Parallelly 30 mL of another essential extract (i.e.) Nelumbo nucifera (leaf extract), Spharanthus indicus Initially, plant extract of Alternanthera sessilis and cupric chloride was used to synthesize CuONP (to eliminate the toxicity), which was later optimized to a size of 10 nm. Our formulation is entirely inspired by the prevailing Siddha medicine formulation in which they take pure copper along with the extract of other plants and synthesize the particles for around 32 days to eliminate its toxicity and achieve efficiency. However, here in our formulation methodology, we tried to reduce the synthesis time by opting for suitable technology and following the same flow for formulation. After the optimization of CuONP in a powdered concentration of 300 mg, it is placed in an oil bath. Parallelly 30 mL of another essential extract (i.e.) Nelumbo nucifera (leaf extract), Spharanthus indicus (Leaf extract), Azardica indica (flower extract), and Mangolia champaca (flower extract) were prepared. A time interval of 1 h 1.5 mL of the essential extract was added consecutively to the CuO particles placed in the oil bath, ensuring room temperature and rotation. After adding all the extract, the setup is left to formulate for 24 h. After the incubation of 24 h, the vial contents had filtered using a 0.22 µm cellulosic membrane filter, and obtained NHF was converted to powder form with the help of a hot air oven. Moreover, the entrapment efficiency (EE) of phytochemicals in NHF was calculated based on the following Equation (1) % Entrapment efficiency = Total phytochemical − Free phytochemical Total phytochemical × 100 (1) where UV-Vis measurements at 435 nm were used to assess content Vs (Standard Graph)

Physiochemical and Compound Identification
Gas Chromatography-Mass Spectrometry/Mass Spectrometry was used to analyze NHF samples following liquid-liquid extraction with hexane and ethanol followed by ethanol. The extract was analyzed with UV-Vis DRS, X-Ray diffraction, FTIR, SEM, HRTEM, and DLS to determine its size, shape, and morphology. Elemental analysis of NHF was performed through the EDAX method. A different part of the plant was used to synthesize the nano herboformulation.

In-Vitro Drug Release Study
The cumulative phytochemical release from NHF formulation was performed. The NHF formulation at the concentration of 100.0 µg /mL in a dialysis bag taken in a beaker having the required pH buffer solution. The dialysis bag containing the NHF formulation was kept inside the buffer solution at 37 • C, and samples of the buffer were collected at regular intervals. The amount of drug release was measured by measuring the absorbance of the collected external buffer at 435 nm. The cumulative phytochemical release from NHF was determined by the following Equation (2) Phytochemical release (%) = Phytochemical Released Total Phytochemical × 100 Furthermore, the kinetics of phytochemical release from NHF have been understood using the drug release dynamics using zero order, first order, and Korsmeyer-Peppas Model.
Zero-order is an independent kinetics model where the drug release rate is independent of its concentration and given by the Equation (3) as follows where Q 0 , is the initial amount of drug, Q t is the cumulative amount of drug released at a time "t", K 0 is the zero-order rate constant, and t is the time. Zero-order release kinetics is given by the graph plotted between the cumulative percentage of drugs released ( Q t Q 0 ) versus time. First order kinetics system describes that the drug release rate depends on its concentration and is given by the Equation (4) as follows where Q 0 the initial amount of the drug is, Q t is the cumulative amount of drug released at a time "t", K 1 is the first order rate constant, and t is the time? The rate constant is given by plotting as log cumulative percentage drug remaining versus time where the slop is equal to K 1 2.303 . The Korsmeyer-Peppas kinetics Model is given by the Equation (5). As follows: where the drug fraction released is denoted as F at a time 't', M t is the amount of drug released at a time 't', M is the total amount of drug, K m is the kinetic constant, and n is the diffusion or release exponent. The kinetic constant 'K m ' and n was given by plotting the graph of log (Mt/M) versus log t. The value of n indicates the type of diffusion to be either fickian diffusion (n = 0.45), fickian diffusion (0.45 < n < 0.89), or case-2 relaxation (n = 0.89).

Cell Culture and Assessment of Cell Viability
In Dulbecco's Modified Eagles Medium, H9C2 myocardial cells were purchased from National Centre for Cell Sciences, Pune, India. Under humidified 5% CO 2 atmosphere at 37 • C, the cells at density 1 × 10 4 counts were grown in 10% FBS, 1% penicillin-streptomycin antibiotic solution. MTT assay was used to assess cell viability. To reduce toxicity, we stimulated H9C2 myocardial cells with doxorubicin. NHF at different concentrations was used to reduce the toxicity of myocardial cells. A 96-well cell culture plate was filled with NHF with doxorubicin (5, 10, 20, 30, 40, and 50 µg/mL) for 30 min. The cells were incubated with various NHF Cells concentrations and then set at 37 • C in the dark for 4 h with MTT. After discarding the supernatant, 100 mL DMSO was added to the plates, dissolving the formazan crystals caused by MTT's interaction with the live cells. Microplate readers were used to measuring the absorbance at 570 nm.

Apoptosis and Cell Death Estimation
Following the manufacturer's instructions, FITC Annexin V/Dead Cell Apoptosis Kit was used to analyze apoptosis. Following trypsinization, cells were harvested, maintained at 1 × 10 6 counts, and suspended in Annexin binding buffer containing FITC Annexin and PI for 20 min in the dark. After that, the cells were suspended in a buffer containing propidium iodide for 5 min. An FC500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA.) was used to acquire data for 10,000 events using three cell suspensions (5, 10, and 20 µg/mL).

Intracellular ROS Generation Estimation Using Flow Cytometry
Monitoring intracellular ROS was accomplished using dichloro-dihydro-fluorescein diacetate fluorescent probes. After 24 h of incubation in 6-well plates maintained at 1 × 10 6 counts, cells were treated with NHF with doxorubicin (5, 10, or 20 µg/mL) and incubated for another 24 h. Incubation with DCFH-DA at 37 • C for about 30 min, followed by rinsing with fresh DMEM, was followed by analysis using a flow cytometer after cells were rinsed three times with fresh DMEM. Results were expressed as fold changes in fluorescence intensity based on a minimum of 10,000 events per sample. A comparison was made between the results and the control value.

Statistical Analysis
The experiments were run in triplicate, and the values were analyzed using the ANOVA statistical method. The results were statistically significant when the p-value was <0.05 and was expressed as the mean ± standard error.

UV-Vis Spectra Studies
Nano Herboformulation (NHF) was prepared as per the protocol described above, where the capacity of phytochemical in NHF was investigated and found to be 305.51 µg/mg with a loading efficiency of around 61.58% in Figure 2. UV-Vis spectroscopy is a widely recognized methodology for studying powder-suspended nanoparticles [28]. At room temperature, the UV-Visible absorption spectra of the green synthesized nanoformulations are presented in Figure 3. As a result of surface plasmon resonance (SPR), the nanoformulations show high absorption below 350 nm and a well-defined absorbance peak at around 280 nm. Copper oxide nanoparticles synthesized from various plant precursors also showed a similar result [29]. nanoformulations are presented in Figure 3. As a result of surface plasmon resonance (SPR), the nanoformulations show high absorption below 350 nm and a well-defined absorbance peak at around 280 nm. Copper oxide nanoparticles synthesized from various plant precursors also showed a similar result [29].

XRD and FTIR Analysis of Nano Herboformulation
XRD technique was used to analyze the microcrystalline structure of nano herboformulation. Characteristic XRD peaks of the nanoformulations were observed at 32.7, 40.2, 46.4, and 57.3, correspondings to 110, 200, 202, and 021 planes, respectively, as shown in Figure 4. This observation indicates the formation of monoclinic CuO. The NP system is seen as a typical result. Their sharp peaks confirm CuO NP. Reveals the crystalline nature of the further confirmation (JCPDS card number 89-2529). The XRD crystal structure provides the lattice parameters, nature of the phase, and crystal grain size [30]. Our Sharer equation is used to determine how broadening the most extreme peak of an XRD measurement is for a model. The average size of the nano herboformulation was 7.70 nm.

XRD and FTIR Analysis of Nano Herboformulation
XRD technique was used to analyze the microcrystalline structure of nano herboformulation. Characteristic XRD peaks of the nanoformulations were observed at 32.7, 40.2, 46.4, and 57.3, correspondings to 110, 200, 202, and 021 planes, respectively, as shown in Figure 4. This observation indicates the formation of monoclinic CuO. The NP system is seen as a typical result. Their sharp peaks confirm CuO NP. Reveals the crystalline nature of the further confirmation (JCPDS card number 89-2529). The XRD crystal structure provides the lattice parameters, nature of the phase, and crystal grain size [30]. Our Sharer equation is used to determine how broadening the most extreme peak of an XRD measurement is for a model. The average size of the nano herboformulation was 7.70 nm.  In FTIR analysis, we were able to classify nano-sized substances by the composition of nano-herboformulation. Figure 5 shows FTIR spectra of nano herboformulation. Nano herboformulation FTIR spectra show Cu-O vibrations at frequencies less than 700 cm −1 [31]. Small peaks (592.17 cm −1 ), O-H stretching vibration (3321.42 cm −1 ), frequency-independent (2927.94 cm −1 ), vibration resulting from C=C stretching (1600.92 cm −1 ), O-H deformation vibration (1359.82 cm −1 ), and bands corresponding to the characteristic frequency of inorganic ions are observed in CuO NPs [32]. Alkane, aromatic, ketone compounds, carboxylic acids, and aromatic nitro compounds are attributed to C-H deformation vibration (1317.38 cm −1 ) and CH3 rocking vibration (1031.92 cm −1 ). Deflation of the capping caused a comparatively high peak in CuO NPs due to C-O stretching [33]. In FTIR analysis, we were able to classify nano-sized substances by the composition of nano-herboformulation. Figure 5 shows FTIR spectra of nano herboformulation. Nano herboformulation FTIR spectra show Cu-O vibrations at frequencies less than 700 cm −1 [31].  [32]. Alkane, aromatic, ketone compounds, carboxylic acids, and aromatic nitro compounds are attributed to C-H deformation vibration (1317.38 cm −1 ) and CH 3 rocking vibration (1031.92 cm −1 ). Deflation of the capping caused a comparatively high peak in CuO NPs due to C-O stretching [33].

Size and Shape Determination of Nano Herboformulation
The standard physicochemical characterization used for integrated nano-herb formation. As shown in Figure 6A of TEM and Figure 6B of SEM, spherical particles of size ranging between 5-10 nm were identified. The amount of nano-herb formulation differs depending on the solvent medium as plant juices are attached to them. Thus, the cytotoxicity effect and the extent of its medium [34]. SEM showed that the nano herboformulation's nano shape is approximately 300 nm Figure 5B. The shape of nanoparticles in the nano herboformulation plays a significant role in influencing the distinctive process of nanoparticle interaction with living patterns [35]. In in vivo tests, nanoparticles less than 10-20 nm are rapidly distributed over neuronal administration between all organs and tissues, while larger nanoparticles ranging from 50-250 nm are

Size and Shape Determination of Nano Herboformulation
The standard physicochemical characterization used for integrated nano-herb formation. As shown in Figure 6A of TEM and Figure 6B of SEM, spherical particles of size ranging between 5-10 nm were identified. The amount of nano-herb formulation differs depending on the solvent medium as plant juices are attached to them. Thus, the cytotoxicity effect and the extent of its medium [34]. SEM showed that the nano herboformulation's nano shape is approximately 300 nm Figure 5B. The shape of nanoparticles in the nano herboformulation plays a significant role in influencing the distinctive process of nanoparticle interaction with living patterns [35]. In in vivo tests, nanoparticles less than 10-20 nm are rapidly distributed over neuronal administration between all organs and tissues, while larger nanoparticles ranging from 50-250 nm are found in the blood, liver, spleen, and heart [36]. EDX microanalysis is used to create maps of various chemical components. This map is generated using software that evaluates the required element concentration at each imaging stage (X-ray spectra) in scanning mode. EDX analysis confirmed copper oxide nanoparticles and trivial quantities of Cl and K Figure 6 C. The occurrence of copper and EDX microanalysis is used to create maps of various chemical components. This map is generated using software that evaluates the required element concentration at each imaging stage (X-ray spectra) in scanning mode. EDX analysis confirmed copper oxide nanoparticles and trivial quantities of Cl and K Figure 6C. The occurrence of copper and oxygen confirmed the formation of copper oxide nanoparticles in the reaction system. However, minor amounts of other reserves were found, which may be the molecule used in the analysis.

Zeta Potential and Particle Size of Nano Herboformulation
The size and stability of nano herboformulation are essential for medicinal application. To assess the stability of nanofluids, Zeta observes the electrophoretic behavior of the potential analysis fluid [37]. As shown in Figure 7B, a high Zeta potential value represents the stability of nano herboformulations in an aqueous medium. A zeta potential value of −20.58 mV shows colloidal stability. The nanoparticle size and colloidal stability were determined using DLS [38]. The hydrodynamic diameter of the nano herboformulation was determined to be 12.9 nm Figure 7A. oxygen confirmed the formation of copper oxide nanoparticles in the reaction system. However, minor amounts of other reserves were found, which may be the molecule used in the analysis.

Zeta Potential and Particle Size of Nano Herboformulation
The size and stability of nano herboformulation are essential for medicinal application. To assess the stability of nanofluids, Zeta observes the electrophoretic behavior of the potential analysis fluid [37]. As shown in Figure 7B, a high Zeta potential value represents the stability of nano herboformulations in an aqueous medium. A zeta potential value of −20.58 mV shows colloidal stability. The nanoparticle size and colloidal stability were determined using DLS [38]. The hydrodynamic diameter of the nano herboformulation was determined to be 12.9 nm Figure 7A.

GC-MS Analysis of Nano Herboformulation
The presence of a compound responsible for a different type of plant reacts with copper oxide. The Gas chromatography-mass spectrometry of nano herboformulation showed 13 peaks that were identified after comparing the mass spectra with Library used NIST Version-2011, indicating the presence of 13 phytocompounds. Figure 8 represents the formulation's possible components (based on the NIST Library). The compounds are listed in Table 1. along with their peak area and RT molecular weight. Represents compound from 1H-Inden-1-one,2,3-dihydro 5,6-dimethody-3-methyl (RT 10.07), performed experiments revealed that 10e has an anti-β amyloid effect can reduce ROS, LDH, and MDA also a positive posing impact on TAC [39]. Similarly, 9-octadecenoic acid resists doxorubicin and cytarabine-induced oxidative stress. Oleic acid (RT), as reported in our present findings (RT 20.46) compound had more favorable effects similar effects on non-HDL-cholesterol concentrations and fasting LDL-cholesterol in comparison to hexadecenoic acid [40].

GC-MS Analysis of Nano Herboformulation
The presence of a compound responsible for a different type of plant reacts with copper oxide. The Gas chromatography-mass spectrometry of nano herboformulation showed 13 peaks that were identified after comparing the mass spectra with Library used NIST Version-2011, indicating the presence of 13 phytocompounds. Figure 8 represents the formulation's possible components (based on the NIST Library). The compounds are listed in Table 1. along with their peak area and RT molecular weight. Represents compound from 1H-Inden-1-one,2,3-dihydro 5,6-dimethody-3-methyl (RT 10.07), performed experiments revealed that 10e has an anti-β amyloid effect can reduce ROS, LDH, and MDA also a positive posing impact on TAC [39]. Similarly, 9-octadecenoic acid resists doxorubicin and cytarabine-induced oxidative stress. Oleic acid (RT), as reported in our present findings (RT 20.46) compound had more favorable effects similar effects on non-HDL-cholesterol concentrations and fasting LDL-cholesterol in comparison to hexadecenoic acid [40].  We have also studied the in-vitro phytochemical release study of NHF formulati nearly 94.2 % of the phytochemical release ( Figure 9A). The phytochemical release kinet data from NHF formulation were also fitted to the different kinetics models (

In-Vitro Phytochemical Release Study of NHF Formulation
We have also studied the in-vitro phytochemical release study of NHF formulation, nearly 94.2 % of the phytochemical release ( Figure 9A). The phytochemical release kinetics data from NHF formulation were also fitted to the different kinetics models (Table 2). It was found best to follow the Korsmeyer-Peppas model, and hence, the release of the drug must be mediated by diffusion mechanism, especially non-fickian diffusion (0.45 < n < 0.89) type of transport mechanism ( Figure 9B-D).

Cell Viability, Oxidative Stress, and Apoptosis in Doxorubicin-Induced h9c2 Cell Li
The study of different cell biology techniques, the analysis of reactive oxygen the analysis of apoptosis, and the identification of morphological changes. Usin cells to induce doxorubicin treatment, we explored the role of nano herbal form in reducing cardiotoxicity. We then examined the protective capacity of the nan formulations in myocardial cells from H9C2.

Dose-Response Curve Using MTT Assay-Cell Viability Assay
H9C2 cardiomyoblast cells viability to various concentrations of doxoru duced nano herboformulation was studied for 24 h. Dose-dependent cytotoxicity served with doxorubicin treatment (Figure 10). DOX-induced cells showed stable icity after NHF treatment and higher mitochondrial oxidative pressure after NH ment. An MTT assay can distinguish membrane lysis from non-membrane lysis b ing MTT reductase activity.
Cell viability was found to be increased in NHF-treated cells in a concentra pendent manner Figure 10. Cells were treated with (1 μM) of doxorubicin to indu toxicity in H9C2 cells and with NHF at different concentrations (5,10,20,30,40 μg/mL) to revert the induced toxicity. As shown in the graph, we found 5, 10 μg/mL concentrations to have high % cell viability; thus, we chose this concentr further in-vitro studies. NHF of higher concentrations (5, 10, 20 μg/mL). Based o studies, doxorubicin and levosimendan increased cell endurance and prevented o

Cell Viability, Oxidative Stress, and Apoptosis in Doxorubicin-Induced h9c2 Cell Line
The study of different cell biology techniques, the analysis of reactive oxygen species, the analysis of apoptosis, and the identification of morphological changes. Using H9C2 cells to induce doxorubicin treatment, we explored the role of nano herbal formulations in reducing cardiotoxicity. We then examined the protective capacity of the nano herboformulations in myocardial cells from H9C2. 3.7.1. Dose-Response Curve Using MTT Assay-Cell Viability Assay H9C2 cardiomyoblast cells viability to various concentrations of doxorubicin-induced nano herboformulation was studied for 24 h. Dose-dependent cytotoxicity was observed with doxorubicin treatment (Figure 10). DOX-induced cells showed stable cytotoxicity after NHF treatment and higher mitochondrial oxidative pressure after NHF treatment. An MTT assay can distinguish membrane lysis from non-membrane lysis by changing MTT reductase activity. fundamentally distinct nanoparticles impose unique biological and toxic effects [42]. Cells treated with NHF for 24 h showed no significant toxic effects. Co-treatment using a similar cocktail formula did not show a significant improvement in cell viability, which is also noticeable compared to free dox (p > 0.05) [43]. The experiments were carried out in triplicates, and the calculated values are mean ± SD. On the contrary, NHF at (5, 10, 20 μg/mL) dosage increased the viability of H9C2 cells to near normal.

NHF in Oxidative Stress Induced by DOX-Treatment in H9c2 Cells
Purine nucleotide acts as an intermediate to DOX metabolize quinone by flavoproteins. Doxorubicin damaged approximately 37.99% of the cells. After DOX treatment of NHF cell damage, DOX-induced H9C2 cells showed a low level of (0.83%, 0.56%, and 0.29%) (Figure 11 C-E) compared to control cells. Figure 11 A-B shows how NHF affects Dox-induced ROS production within cells. Here we have treated doxorubicin-induced toxicity with NHF at 5, 10, and 20 μg/mL Concentrations. Cell viability was found to be increased in NHF-treated cells in a concentrationdependent manner Figure 10. Cells were treated with (1 µM) of doxorubicin to induce cytotoxicity in H9C2 cells and with NHF at different concentrations (5,10,20,30,40, and 50 µg/mL) to revert the induced toxicity. As shown in the graph, we found 5, 10, and 20 µg/mL concentrations to have high % cell viability; thus, we chose this concentration for further in-vitro studies. NHF of higher concentrations (5, 10, 20 µg/mL). Based on earlier studies, doxorubicin and levosimendan increased cell endurance and prevented oxidative stress-induced cell death in rodent hepatocytes [41]. Evaluation of the cytotoxic effects due to various nanoparticles for cells H9C2, A549, HEK293, and MCF-7 shows that fundamentally distinct nanoparticles impose unique biological and toxic effects [42]. Cells treated with NHF for 24 h showed no significant toxic effects. Co-treatment using a similar cocktail formula did not show a significant improvement in cell viability, which is also noticeable compared to free dox (p > 0.05) [43]. The experiments were carried out in triplicates, and the calculated values are mean ± SD. On the contrary, NHF at (5, 10, 20 µg/mL) dosage increased the viability of H9C2 cells to near normal.

NHF in Oxidative Stress Induced by DOX-Treatment in H9c2 Cells
Purine nucleotide acts as an intermediate to DOX metabolize quinone by flavoproteins. Doxorubicin damaged approximately 37.99% of the cells. After DOX treatment of NHF cell damage, DOX-induced H9C2 cells showed a low level of (0.83%, 0.56%, and 0.29%) (Figure 11 C-E) compared to control cells. Figure 11 A-B shows how NHF affects Doxinduced ROS production within cells. Here we have treated doxorubicin-induced toxicity with NHF at 5, 10, and 20 µg/mL Concentrations.
The formation of the DOX-iron complex stimulates ROS generation. The results in hydrogen peroxide (H 2 O 2 ) and superoxide ions [44]. Moreover, H 2 O 2 is degraded by low molecular weight clay irons into common reaction hydroxyl radical [45]. Hyper generation of ROS leads to antioxidant pressure, which is activated by DOX and stimulates the apoptotic signaling layer in cardiomyocytes [46]. Figure 11 F complementary graph of ROS. As shown in the image, all treated groups showed reduced ROS levels compared to control cells. The ROS levels were drastically reduced in NHF treatment.

NHF in Apoptosis Induced by DOX-Treatment in H9c2 Cells
Here we have seen apoptosis assay for NHF. As shown in Figure 12A, the Control and doxorubicin-treated cell are the same as before. Figure 12C-E showed cells treated with 5, 10, and 20 µg/mL Figure 12C concentration showed late apoptosis cells. Figure 12D showed fewer late apoptotic cells treated with 10 µg/mL. Figure 12E shows 20 µg/mL concentration when we found reduced apoptosis cells compared to all other treatments, and Figure 12F complementary graph of apoptosis. The formation of the DOX-iron complex stimulates ROS generation. The results in hydrogen peroxide (H2O2) and superoxide ions [44]. Moreover, H2O2 is degraded by low molecular weight clay irons into common reaction hydroxyl radical [45]. Hyper generation of ROS leads to antioxidant pressure, which is activated by DOX and stimulates the apoptotic signaling layer in cardiomyocytes [46]. Figure 11 F complementary graph of ROS. As shown in the image, all treated groups showed reduced ROS levels compared to control cells. The ROS levels were drastically reduced in NHF treatment.

NHF in Apoptosis Induced by DOX-Treatment in H9c2 Cells
Here we have seen apoptosis assay for NHF. As shown in Figure 12 A, the Control and doxorubicin-treated cell are the same as before. Figure 12 C-E showed cells treated with 5, 10, and 20 μg/mL Figure. 12 C concentration showed late apoptosis cells. Figure. 12 D showed fewer late apoptotic cells treated with 10 μg/mL. Figure 12 E shows 20 μg/mL concentration when we found reduced apoptosis cells compared to all other treatments, and Figure 12   The results of NHF with doxorubicin-induced antioxidant pressure are a significant factor in toxicity. The potential role of oxidative stress in mediation with apoptosis induction and DNA damage has been documented [47]. At the end of these studies, our results demonstrate that NHF oxidative stress, DNA damage, and apoptosis triggers are lower The results of NHF with doxorubicin-induced antioxidant pressure are a significant factor in toxicity. The potential role of oxidative stress in mediation with apoptosis induction and DNA damage has been documented [47]. At the end of these studies, our results demonstrate that NHF oxidative stress, DNA damage, and apoptosis triggers are lower than in previous reports [48].

Conclusions
Cardiovascular disease poses a significant threat to society as it holds the highest mortality rate. Here, we have encountered the formation of nano herbal has shown good progress in cardiac function. From this study, it seems possible to shunt CVD harmlessly and healthily using Nano herbo-formulation with reduced toxicity and increased therapeutic support. The overall analysis of the relationship between nano herboformulation revealed by the transformation of value in H9C2 and zebrafish is given with doxorubicin, confirming the reduced toxicity while using nano herboformulation. It provides the exact measures and the role of ROS and the apoptotic analysis in H9C2. Overall, this gives a clear output for further expanding the nano herboformulation in the treatment of CVD. In future studies, the most common preclinical model, the nano herboformulations showing the highest potential in this in vitro model, can be further assessed in mice. It is possible to reduce research time and costs associated with high numbers of mice using this approach. The addition of this animal model would strengthen and improve the approval process for nano herboformulation-based cardiac and cancer treatments.