Background

Atherosclerosis is a chronic inflammatory condition resulting in cardiovascular disease with life-threatening implications. Atherosclerosis is a complex and multifaceted condition marked by the accumulation of plaque within arteries. This can lead to reduced blood flow, contributing to a range of cardiovascular diseases (CVDs). CVDs are one of the leading causes of death worldwide, and their prevalence is rapidly increasing, owing in large part to changes in lifestyle and an increase in the elderly population [1]. Since atherosclerosis is a complex condition affected by numerous risk factors, there are various well-established and innovative strategies to diagnose and manage this disease. During the early phases of atherosclerosis, the primary objective of treatment is on managing significant risk factors such as elevated cholesterol levels, hypertension, and elevated blood sugar. Making alterations to one's diet, engaging in regular physical activity, and quitting smoking are all assumed to play pivotal roles in both the prevention and management of atherosclerosis. The current treatment for management of atherosclerosis includes drugs that have antihypercholesterolemic, antihypertensive, hypoglycemic, and antiplatelet activity. Anti-inflammatory drugs like canakinumab, methotrexate, and colchicine have passed clinical trials but need to be further assessed for their therapeutic efficacy. The typical medications utilized to address hypercholesterolemia are statins solely or in combination with drugs like ezetimibe. These drugs function by inhibiting the hepatic enzyme HMG CoA reductase, a crucial enzyme in the synthesis of cholesterol. The primary advantage of statins lies in their ability to lower low-density lipoproteins (LDL). However, they suffer from adverse effects mainly statin-associated muscle symptoms, myalgias, and glucose intolerance. Additionally, the utilization of statins has been associated with the development of type 2 diabetes mellitus [2,3,4].

Considering the negative effects of synthetic medications, emphasis should be given to embrace natural drugs, which have negligible adverse reactions. Natural origin drugs still continue to be the focus of pharmaceutical research because substitute drug discovery methods have failed to provide lead compounds for treatment of various diseases like metabolic diseases. Terminalia arjuna, also known as Arjuna in Hindi, belongs to the family Combretaceae and is found in India, Mauritius, and Sri Lanka. Arjuna has been documented in numerous ancient Indian medical texts, including Charaka Samhita, Astang Hridayam, and Sushruta Samhita, dating back to the Vedic period. Notably, it was Vagabhatta who first recommended the utilization of stem bark powder for heart-related conditions. Ayurvedic practitioners have employed this plant in the management of cardiovascular ailments [5]. The stem bark, fruits, and even leaves of Arjuna are commonly used in different traditional systems across India for various medicinal properties such as astringent, demulcent, expectorant, styptic, antidysenteric, cardiotonic, and urinary astringent. It is also used in the treatment of ulcers, earaches, and even bark ashes are used for snakebites and scorpion stings [6]. Numerous experimental and clinical studies have effectively showcased Arjuna’s potential as an anti-ischemic agent, antiatherogenic agent, and a strong antioxidant. As per data shared for Scientific Harvesting—Dos and Don’ts by NTFP Centre of Excellence (NCE) established by Government of Tripura, India; the bark of Arjuna tree should not be harvested before 15 years of growth to get quality product with a minimum gap of 2 years for successive harvesting [6,7,8,9,10,11].

The present study includes the callus culture to reduce the harvesting time period. Nanoparticle delivery systems offer the enhanced stability and solubility of encapsulated drug molecules, facilitating their movement across membranes, and extending circulation time to enhance both safety and effectiveness of the medicament [12, 13]. In this study, a multidisciplinary approach was employed to create a nanosuspension from Arjuna callus.

Methods

Preparation of callus

The branch of Arjuna was collected from Dr. Babasaheb Ambedkar Central Nursery Kagal, India, in the month of June, and plant was authenticated form Botanical Survey of India, Pune. The leaves were surface sterilized. Murashige and Skoog agar media with different concentrations of 2, 4,-dichlorophenoxy acetic and kinetin were used for callus induction. The explants were incubated at 25 ± 2 °C under 16-h photo-period of 2000 lx with white light. The relative humidity was maintained at 60–70% during incubation at Seem Biotech Pvt Ltd, Warananagar, India. Sub-culturing was carried out every six weeks. After 6 months, the callus cultures were weighed and callus index was calculated.

Extraction of callus

The callus culture obtained was dried at 50 ± 5 °C in hot air oven and was subjected to ethanolic extraction using Soxhlet apparatus and subsequently dried in vacuum dryer.

Preparation of arjuna callus extract nanosuspension (ACEN)

One gram of extract was mixed with 20 ml ethanol. The solution was injected drop wise in 100 ml aqueous solution of polyvinyl pyrrolidone K30 (PVP K30) and was subjected to mechanical stirring for 6 h. A factorial design was employed, taking into account two independent factors: the concentration of PVP K30 and mixing speed, each varying at three different levels.

Evaluation of nanosuspension

Bruker, ALPHA Infrared Spectrophotometer was used for checking the compatibility of callus extract with the PVP K30. The Horiba Zetasizer was used for determination of particle size and zeta potential [14]. Absorption maxima were determined using a Jasco V-750 UV Spectrophotometer from Japan to calculate % drug content, % drug encapsulation, and % drug release. For the % drug release study, a dialysis bag (dimensions: flat width 28.46 mm, inflated diameter 18 mm; provided by HiMedia Laboratories Pvt, Ltd) was utilized. One milliliter of 10 µg/ml of each ACEN batch was placed in a dialysis bag immersed in 300 mL of diffusion media (phosphate buffer saline—PBS, pH 7.4) in USP Type I dissolution test apparatus. Samples were withdrawn at 5-min intervals over 40 min from the receptor compartment and analyzed at 282 nm. The % drug content, % drug encapsulation efficiency, and % drug release studies were conducted in triplicate [15].

Acceleration stability studies for the optimized batch were carried out according to ICH guidelines for 90 days. It involved a modification in the intermediate storage condition, transitioning from 30 °C ± 2 °C and 60% ± 5% relative humidity (RH). The adequate amount of freeze-dried optimized ACEN batch was subjected to acceleration stability study. The particle size, % drug content, and % drug release study were used as parameters to check the stability of ACEN [16].

Antilipase assay

The enzymatic activity of porcine pancreatic lipase (PPL, type II) was assessed using p-nitrophenyl butyrate (p-NPB) as a substrate. PPL stock solution (1 mg/mL) was prepared in 0.1 mM potassium phosphate buffer (pH 6.0) and stored at -20 °C. Different concentrations 20, 40, 60, 80, and 100 μg/mL of extract and standard drug Orlistat were pre-incubated with PPL for 1 h in a 0.1 mM potassium phosphate buffer (pH 7.2), supplemented with 0.1% Tween 80, at 30 °C. After addition of 0.1 μL of p-NPB, all samples were incubated at 30 °C for 5 min. The absorption for all samples was recorded at 405 nm. The analysis was performed three times for each concentration of sample and standard. The inhibitory activity (I) was calculated using the following formula [17]:

$${\text{\% Inhibition}} = \frac{{{\text{Absorbance}}\;{\text{of}}\;{\text{control}} - {\text{Absorbance}}\;{\text{of }}\;{\text{sample}}}}{{{\text{Absorbance }}\;{\text{of}}\;{\text{control}}}} \times 100$$

Antioxidant activity assay

The antioxidant potential of the nanosuspension was assessed through its ability to scavenge free radicals using DPPH (1,1-diphenyl-2, picryl-hydrazyl) radicals. The standard solutions were prepared by mixing different concentrations of 100 μL of aqueous ascorbic acid solution with 100 μL of a 0.1% methanolic DPPH solution. Similarly, test samples were prepared by mixing different concentrations of 100 μL of an aqueous callus extract solution with 100 μL of the 0.1% methanolic DPPH solution. After mixing, all samples were incubated for 30 min in a dark environment at room temperature. The absorbance was recorded three times at 490 nm [18, 19].

The radical scavenging activity was determined using the following formula:

$${\text{\% }}\;{\text{ radical}}\;{\text{scavenging}}\;{\text{activity }} = \frac{{{\text{Absorbance}}\;{\text{of }}\;{\text{Control}} - {\text{Absorbance}}\;{\text{of }}\;{\text{Test}}\;{\text{ sample}}}}{{{\text{Absorbance }}\;{\text{of}}\;{\text{ Control}}}}{*}100$$

Experimental animals

The protocol was approved by the Institutional Animal Ethics Committee of Appasaheb Birnale College of Pharmacy, Sangli (Approval number-IAEC/ABCP/13/2021–22). Healthy young male Wistar rats aged 6–8 weeks weighing approximately 200–250 g were divided into four groups, with six rats in each group. Prior to the initiation of the experimental procedure, one-week acclimatization period maintaining a temperature of 24 ± 3 °C, a humidity level of 50–60% RH, and a 12-h light/dark cycle, was provided for the rats.

Acute toxicity study

An acute toxicity assessment was conducted on rats in accordance with the OECD guideline 423 using the up-and-down procedure. Three nulliparous and non-pregnant female rats aged 8 to 12 weeks were employed for the study. The rats were fasted overnight before dosing, which extended for an additional 3-h post-dosing. Close monitoring of the animals was carried out for the initial 24 h to detect any signs of toxicity and continued for 72 h to identify potential mortalities. The determined LD50 value was 3000 mg/kg of rat body weight. As a result, a dose of 300 mg/kg body weight was selected for ACEN administration [4, 7, 11].

Preparation of high-fat diet (HFD)

The fine powder of the conventional standard animal pellets was combined with 2% cholesterol, 1% cholic acid, 40% sucrose, and 10% coconut oil. The resulting powdered mixture was blended with an appropriate amount of water to form compact feed spheres. These spheres were then stored in a refrigerator.

Animal test groups

The animals were randomly distributed into four distinct groups, each group comprising of six rats. Group I, designated as normal group, was provided with the standard rat animal pellets. Group II, known as the disease control group, was fed a HFD. Throughout the study, both Group I and Group II received normal saline per day orally. Group III, referred to as the Standard group, received HFD and atorvastatin orally at a dosage of 10 mg/kg body weight of rat per day, while Group IV received HFD and ACEN at a dose of 300 mg/kg body weight per day orally. Upon the conclusion of the 30-day study period, the rats were fasted overnight. Subsequently, the rats were anesthetized using diethyl ether, and blood samples were collected from the retro orbital venous plexus. These blood samples were promptly transferred to sterile plain tubes to conduct biochemical tests [20,21,22].

Atherogenic index

The atherogenic index of serum (AIS) is the measure of the extent of atherosclerotic lesions based on serum lipids. The atherogenic index was calculated using following formula [23]:

$${\text{AIS}} = \frac{{{\text{TC}} - {\text{HDL}}}}{{{\text{HDL}}}}$$

where TC = concentration of total cholesterol in mg/ml. HDL = concentration of HDL in mg/ml.

Histology of the aorta

The rats were killed by cervical decapitation. Their aortas were dissected out carefully. The aortas were stored in 10% formaline solution and sent to a local pathological laboratory for hematoxylin and eosin staining.

Statistical Analysis: The outcomes were presented as the mean accompanied by the standard error of the mean (SEM). Statistical analysis involved the utilization of analysis of variance (ANOVA). A significance level of p < 0.05 was employed as the threshold for determining statistical significance.

Results

Evaluation parameters for callus

The callus cultures were developed with 2% 2,4 D concentrations using leaves explant. Callus index is a parameter used to access the growth rate of the tissue. Callus index was calculated using following formula [24]:

$${\text{Callus}}\;{\text{index}} = \frac{n*G}{N}{*}100$$

where n = total number of callused explants. G = Average weight of callus rating on explant. N = Total number of cultured explants.

The callus index was 54.075 indicating good callus culture growth. The % practical yield for callus extract was 7.45% w/w.

Phytochemical analysis

The liquid chromatography–mass spectrometry (LC–MS) data revealed presence of terpenoids like Asiatic acid, 2-Oxo-5,11(13)-eudesmadien12,8-olide, Cucurbitacin B, Cucurbitacin E, (+)-cis-5,6-Dihydro-5-hydroxy4-methoxy-6-(2-phenylethyl)-2H-pyran-2-on, Ganoderic acid C, Asiatic acid, (3beta,19alpha)-3,19,23,24-Tetrahydroxy-12-oleanen-28-oic acid, while flavonoids like Genistin, Allixin, Quercetin, Liquiritic acid on majority. Figures 1 and 2 are the chromatograms of Arjuna callus extract.

Fig. 1
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The chromatogram of callus extract for positively charged compounds

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Fig. 2
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Chromatogram of callus extract for negatively charged compounds

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Evaluation of ACEN

As per Figs. 3, 4, and 5, the Fourier transform infrared (FTIR) graphs confirmed the compatibility of Arjuna callus extract with polymer PVP K30.

Fig. 3
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FTIR of callus extract

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Fig. 4
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FTIR of physical mixture of callus extract and PVP K30

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Fig. 5
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FTIR of nanosuspension of callus extract

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The size of the nanoparticles ranged from 304.4 to 544.1 nm, with a polydispersity index between 0.37 and 0.42. Additionally, the zeta potential ranged from -25.6 to -45.6, indicating that the nanosuspension is stable [25].

The % drug release study revealed that the N4 batch exhibited the lowest release, with 91.58 ± 0.06% over 35 min. On the other hand, the N9 batch demonstrated the highest % drug release with 99.40 ± 0.008% over 35 min among all batches. The % drug release data were fitted to standard release equations—zero order, first order, Higuchi model, Hixson, and Kor's peppas. The linear correlation coefficient R square values for all batches are summarized in Table 5. All batches followed first order of kinetics.

Table 7 confirms the optimization of Arjuna callus extract nanosuspension batch.

Stability studies were done for optimized batch N9 as per ICH guideline. Acceleration stability study intermediate storage condition was changed from 30 ± 2 °C and 60 ± 5% RH, and results are shown in Table 8 and Fig. 15. There was slight increase in particle size from 340 to 347.9 nm on day 90. The % drug content and % drug release were almost unchanged.

Antilipase assay

The IC50 for standard drug Orlistat was 53.58, while for ACEN was 78.69. The highest % inhibition of pancreatic lipase enzyme for Orlistat was 83.63 at 100 µg/ml, while for ACEN was 78.69 at 100 µg/ml with p value < 0.0001.

Antioxidant activity

The IC50 value for the standard drug sample, ascorbic acid, was 137.57, while for ACEN, it was 42.49. Remarkably, ACEN exhibited a percentage inhibition surpassing that of ascorbic acid, and this trend was observed to intensify with higher concentrations. The p-value of 0.0009 represents the statistical significance of these results [26].

Animal study

According to Fig. 18, the body weight of rats increased in all groups throughout the experiment, except for the normal group. The control group exhibited the most significant increase in body weight on the 15th and 30th days when compared to the normal group. The body weights of the standard and ACEN-treated groups were lower than the control group, but were similar to the standard group.

Figure 19 shows the lipid profile of all groups. The control group exhibited an increase in total cholesterol (TC) levels compared to the normal group. However, there was a slight decrease in TC levels in both the ACEN-treated and standard groups when compared to the control group. The control group showed a decrease in TG levels, while the ACEN-treated and standard groups maintained stable levels, similar to the normal group. The control group had lower levels of HDL, while the ACEN-treated group had slightly higher levels. Low-density lipoprotein (LDL) level was increased in the control group but was maintained in ACEN-treated group as that of normal. Very low-density lipoprotein (VLDL) levels were elevated in the control group but remained relatively stable in the ACEN-treated and standard groups, approaching levels similar to the normal group. The data underwent ANOVA, and it confirmed its statistical significance with a p-value of less than 0.0001.

Atherogenic index (AIS)

The AIS for the ACEN group was less than the control group and almost similar to that of standard group. In a study involving rats that fed with HFD diet for 30 days, the AIS increased from 0.373278 in the normal group to 1.48455 in the control group (p < 0.0001). However, in the ACEN group, the AIS was 1.09205, which was like the standard group, with a value of 1.0563.

Histology of the aorta

In the control group, there was a noticeable narrowing of the aortic lumen due to lipid deposition. Conversely, in rats treated with ACEN, the aortic lumen appeared almost identical to that of the normal group. Additionally, the standard group's rats exhibited more significant atherosclerosis-related narrowing of the aortic lumen compared to the ACEN-treated group (Figs. 21, 22, 23, 24).

Discussion

Phytochemical analysis

The callus cultures are known to exhibit the phytoconstituents that are not present in mother plants naturally [27]. The LC–MS data showed the presence of terpenoids like (3beta, 19alpha)-3,19,23,24-tetrahydroxy-12-oleanen-28-oic acid, 3-[4-Hydroxy-3-(3-methyl-2-butenyl)phenyl]-2-propenal which are absent in Arjun tree naturally [8] (Table 1).

Table 1 Phytoconstituents of callus and their pharmacological activity
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Evaluation of ACEN

Considering Fig. 3, 4, and 5, the alkyn –OH stretching at 3300, alkyl C–H stretch at 2925 and 2855, C=O stretch at 1746 and 1642, C–O at 1000–1300 were preserved in the mixture IR spectra, indicating the compatibility of the callus extract with PVP K 30 (Table 2).

Table 2 Batches of nanosuspension
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Considering two independent factors concentration of polymer PVPK30 (1,1.5, and 2%) and mixing speed (1000, 1500, and 2000) at three levels each, 9 batches were analyzed using design of experiment software version 6 (Fig. 6). The analysis employed a linear model (Table 3) to examine the correlation between polymer concentration and mixing speed regarding particle size of ACEN. We confirmed the significance of the data input with an F-value of 6.16. In Table 4, we observed a negative coefficient for both polymer concentration and mechanical stirring speed. A positive sign denotes a synergistic effect, while a negative sign indicates an antagonistic effect. The interaction plots (Figs. 7, 8, 9, 10, 11) illustrated that the relative slopes of factors—particle size, polydispersity index, zeta potential, % drug content and % drug concentration—displayed a significant interrelation with both polymer concentration and mechanical stirring speed. The % drug content across all formulations ranged from 96.2 ± 0.38 to 99.75 ± 0.46%. Additionally, the range of % drug entrapment was between 94.67 ± 0.02 and 98.92% ± 0.03. This discrepancy from the % drug content suggests the possibility of drug dissolution in the aqueous phase.

Fig. 6
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Summary of design of experiment software analysis

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Table 3 ANOVA table for particle size of nanosuspension
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Table 4 Effect of polymer concentration and speed of mixing on particle size
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Fig. 7
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2D contour plot for particle size analysis of nanosuspension batches

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Fig. 8
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2D contour plot for polydispersity index analysis of nanosuspension batches

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Fig. 9
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2D contour plot for zeta potential analysis of nanosuspension batches

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Fig. 10
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2D contour plot for % drug content analysis of nanosuspension batches

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Fig. 11
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2D contour plot for % drug entrapment efficiency analysis of nanosuspension batches

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We found that all batches N1 to N9 followed first-order kinetics (Table 5). As per Fig. 12, we found that the smaller the particle size, the better is the drug release, which can result in higher solubility.

Table 5 Summary of R square values for nanosuspension batches
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Fig. 12
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% Drug release study

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The analysis of variance (ANOVA) identified 10 potential solutions based on the criteria outlined in Table 6. The combination of 2% PVP K30 and a mechanical stirring speed of 2000 rpm scored 0.917 desirability. The batch optimization is evident from Table 7. The transmission electron microscopy image displayed the nanoscale dimensions and encapsulation of Arjuna callus extract within a polymer matrix (Figs. 13, 14).

Table 6 Constrains for optimization of nanosuspension
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Table 7 Comparison of theoretical value and practical value of optimized nanosuspension
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Fig. 13
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2D contour plot for desirability for nanosuspension batch optimization

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Fig. 14
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Transmission electron microscope image of ACEN

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The N9 sample did not change physical appearance throughout the acceleration stability study. As per results discussed in Table 8 and Fig. 15, we found that there was slight increase in the size of nanoparticle, but it was within acceptable limit. The slight increase in particle size of ACEN did not affect the % drug content of % drug release.

Table 8 Acceleration stability studies for N9 batch
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Fig. 15
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% Drug release studies of N9 batch for acceleration stability studies

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Antilipase assay

Hyperlipidemia exhibits strong associations with various metabolic conditions, including diabetes, atherosclerosis, and hypertension. Within the duodenum, pancreatic lipase plays a pivotal role in the digestion and absorption of lipids, breaking down triacylglycerols into monoacylglycerols and fatty acids. The pancreatic lipase inhibitors have a potential to induce hypolipidemic effects, offering a promising atherosclerosis management [28, 29]. We found that the % inhibition of lipase enzyme by ACEN was concentration dependent (Fig. 16). The antilipase activity of ACEN might be due to presence of 4-Hydroxy-L-threonine, Genistin, Cucurbitacin B, Ganoderic acid C, Asiatic acid, Ellagic acid, Campesteryl ferulate. This could be one of the antiatherosclerotic mechanisms of ACEN.

Fig. 16
figure 16

Antilipase assay for ACEN

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Antioxidant activity

Antioxidants play a pivotal role in the treatment of atherosclerosis through a range of mechanisms. These mechanisms encompass the inhibition of LDL oxidation, the reduction production of reactive oxygen species, the suppression of cytokine secretion, the prevention of atherosclerotic plaque formation, prevention of platelet aggregation, the hindrance of mononuclear cell infiltration, the enhancement of endothelial function and vasodilation, the promotion of nitric oxide (NO) bioavailability, the modulation of adhesion molecule expression (e.g., VCAM-1 and ICAM-1) on endothelial cells, and the mitigation of foam cell formation. The effectiveness of these diverse antioxidant actions remains an open question, but it appears that a multi-antioxidant approach may offer a more effective strategy for antioxidant therapy. We found the ACEN shows better antioxidant activity that standard drug ascorbic acid (Fig. 17). The presence of various compounds, including Allixin, Genistin, Ellagic acid, Quercetin, Paeonilactone C, (5x,6x)-5,6-Epoxyergosta-7,22-dien-3-ol, Campesteryl ferulate, and others, is likely responsible for the observed antioxidant activity of ACEN [19, 30].

Fig. 17
figure 17

Antioxidant activity assay of ACEN

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Animal study

Figure 18 summarizes the body weight of animals during the study (Fig. 18). We discovered that ACEN was more effective than the standard drug atorvastatin at decreasing TC, TG, HDL, LDL, and VLDL (Fig. 19). Ellagic acid plays a role in lipid metabolism by promoting cholesterol efflux and reducing LDL uptake. The terpenoids, such as Ellagic acid and Asiatic acid, are known for their potential in managing hyperlipidemia. The potential cause of ACEN's lipid-lowering effect could be attributed to the presence of these terpenoids. [31,32,33,34,35,36,37].

Fig. 18
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Summary of body weight of animal groups at 0, 15, and 30 days

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Fig. 19
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Summary lipid profile of animal groups

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Atherogenic index of serum

The AIS is a measure of the potential risk of developing atherosclerosis. When the AIS is higher, there is a greater potential for atherosclerosis and an increased likelihood of cardiovascular disease. It is important to note that the AIS is influenced by the levels of HDL. When HDL levels are higher, the atherogenic index is lower, reducing the risk of atherosclerosis [23]. From Fig. 20, we found that the AIS of ACEN (1.05 ± 0.035) was slightly lesser than the AIS of standard drug atorvastatin (1.06 ± 0.053) emplacing the antiatherosclerotic potential of ACEN (Figs. 21, 22, 23, 24).

Fig. 20
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Summary atherogenic index of serum

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Fig. 21
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Rat aorta in control group

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Fig. 22
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Rat aorta in normal group

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Fig. 23
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Rat aorta in ACEN group

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Fig. 24
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Rat aorta in standard group

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Histology of the aorta

The histopathological results confirmed the antiatherosclerotic potential of ACEN. The presence of phytoconstituents in ACEN like Ellagic acid, Quercetin, Calophyllum B, (25R)-5beta-spirostan-3beta ol Liquiritic acid known for their lipid-lowering, antioxidant, and anti-inflammatory properties, may be responsible for its antiatherosclerotic effect. The histopathological analysis demonstrated that nanotherapeutics exhibited superior efficacy when compared to the standard drug Atorvastatin, highlighting the enhanced effectiveness of the callus extract attributed to its nanoformulation. Altogether, the comprehensive study has affirmed ACEN's potential in atherosclerosis treatment.

Conclusion

A thorough analysis of phytochemical composition of the callus extract predominantly revealed the presence of flavonoids and terpenoids, endowed with antioxidant, hypolipidemic, and anti-inflammatory activity. The callus culture extract was then transformed into a nanosuspension to achieve better efficacy of the medicament. The optimized nanosuspension formulation was subjected to comprehensive in vitro and in vivo studies, aimed to confirm its antiatherosclerotic potential. The study findings suggest that ACEN has the potential to be a natural-origin drug for the treatment of atherosclerosis.