1. INTRODUCTION:

Cancer is a condition marked by the invasion of surrounding internal organs or abnormal cells that can migrate to certain other organs and multiply exceeding their respective standard limit. It is 2^nd leading reason of mortality in the world, with a probable 19.3 million new patients and 9.9 million deaths found in 2020¹ Despite the complexity of pathophysiological events, scientists have discovered several types of therapeutic strategies like chemotherapy, hormonal therapy, radiation therapy, surgical therapy, cryotherapy, and immunotherapy for the treatment of cancer^2,3,4,5. But, all the therapies have some limitations in terms of their efficacy against cancer along with some serious side effects ⁶.

Herbal remedies have active ingredients with sequence homology, metabolic precision, chemical variety, and clinical effects, making them potential and safe for a wide range of ailments. Moreover, herbal medicine contains many therapeutic constituents that act by various pathophysiological pathways involved in the progression of cancer ⁷. These medications have some challenges, including poor dissolution, ingestion, quick metabolic, elimination, and distribution of drugs to other tissues, as well as a significant variation in plasma level, and uncertain bioavailability is the major reason for drug failure ⁸. There are various phytoconstituents-based nanoparticles such as resveratrol, Ferulic acid ⁹, Gambogic acid¹⁰, Genistein¹¹, Honokiol, Naringenin¹², Quercetin¹³, Ursolic acid¹⁴, Eugenol¹⁵, Ellagic acid ¹⁶, Epigallocatechin Gallate (EGCG)¹⁷, Curcumin^18,19 have been shown potential effect against several cancers. Additionally, various oils possess a plethora of pharmacological activities like antibacterial, antiviral, antioxidant, and anticancer have been studied for the management of cancer Laurel essential oil²⁰, jasmine oil²¹, Zingiber ottensii essential oil ²² based nanoformulations offer several benefits in cancer treatment via site-specific and target-oriented drug delivery. Furthermore, there has been a greater emphasis on bioactive for cancer, and now researchers emphasize nanotechnology for cancer using nanoparticles in an advanced stage. In literature, several nanoformulations were found compared to currently used anticancer medications, and have shown to be better soluble, stable, efficacious, and have a better biodistribution, among other things. But, much care needs to be taken to formulate targeted formulations that can help improve treatment outcomes while causing minimal tissue harm²³.

Ferula asafoetida is a monoecious, herbaceous, perennial plant belonging to the Umbelliferae family. Asafoetida originated in eastern Iran to Afghanistan, central Afrom where it is exported worldwide. It is not native to India but has been utilized in Indian medicine and household for ages. Ferula asafoetida is extracted from the rhizome and root of the plant. It has several pharmacological activities, such as antioxidant, antifungal, antiviral antidiabetic, cancer chemopreventive, hypotensive, antispasmodic, and molluscicidal ²⁴. Chitosan is an innocuous and excellent biocompatibility polymer produced from crustacean and insect shells, and its safety has been established in both experimental and mammalian models²⁵. It has biodegradable, recyclable, and harmless qualities, making it a suitable polymeric carrier for nanoparticles. It has been observed that it preferentially penetrates cancerous cell barriers and has anti-cancer properties. Furthermore, the antioxidant ability of chitosan is linked to the scavenging of cancer-causing free radicals, which may encourage the etiological state of cancer ²⁶. Hence, in the present study PEG (polyethylene glycol) coated Ferula asafoetida oil chitosan nanoformulation was fabricated to target cancerous cells.

2. MATERIALS AND METHODS:

2.1 Oil and chemicals:

Ferula asafoetida oil (SNN Natural Products, Pacific Computech Pvt. Ltd. India), Chitosan Sodium tripolyphosphate, and PEG were purchased from Hi-Media Laboratories Pvt. Ltd. India, Tween 80 utilized in this investigation was obtained from Sisco Research Laboratories Pvt. Ltd. in India. Fr2 normal (breast epithelial) cell lines, PC-3 (prostate), A549 (lung), and MIAPACA (pancreas) cell lines were obtained from the National Cancer Institute United States.

2.2 GC-MS technique :

The oils of Ferula asafoetida were analyzed using a GC-MS-QP2010 Plus computerized system (Shimadzu Corporation, Kyoto, Japan). AOC-20i auto-injector, AOC-20’s headspace sampler, and a mass selective detector made up the equipment. The capillary column (Rtx-5MS) has dimensions of 30 m (length), 0.25 mm (diameter), and 0.25 m (film thickness), with cross bond, 5 percent diphenyl/95 percent dimethylpolysiloxane as the packing material (Restek Corporation, Bellefonte, USA). The temperature of the ion source was set to 230 °C, and the temperature of the interface was set to 260 °C for 2.5 minutes to generate GC-MS spectra. Electronic impact at 70 eV with an m/z range of 40–650 was used to ionize the sample. Helium (> 99.999 percent) was used as the gas in split mode (10:1) at a flow rate of 1.21 ml/min. The injecting temperature was set at 250 °C, and the injection volume was.1.0μl. The oven temperature was kept at 100°C for 3 minutes before being increased to 280°C at a rate of 10°C/min and held for the following 19 minutes²⁷.

2.3 Method for nanoformulation:

The ionotropic gelation method was employed to formulate Ferula asafoetida oil-loaded chitosan nanoparticles with fewer modifications. Acetic acid solution (2%) with a pH of 5.6 was used for the solution of chitosan (0.1,0.2, or 0.3 percent w/v). Finally, using a magnetic stirrer with chitosan, a freshwater solution of sodium tripolyphosphate (TPP) was added drop by drop with continuous stirring for 60 minutes at 1000 rpm. For coating, PEG (polyethylene glycol) (0.1, 1.5, or 2.0%) was added drop-wise, followed by further stirring at 1000 rpm for 30 minutes. The formulation was centrifuged at 15000 rpm for 40 minutes at 0°C, resulting in the isolation of nanocrystals in the form of granules, which would then be cleaned and re-dispersed in 20 ml of DDW and sonicated for 2 minutes using a probe sonicator (Remi Cool Equipment, Mumbai, India). This solution (2 ml) was subsequently diluted up to ten times before even being sonicated for two minutes to determine Particle size. The leftover suspension was homogenized (Alpha 2-4 LD Plus, CHRIST, Germany) with the addition of D-mannitol (5 percent w/v) as a cryoprotectant to minimize particle aggregation (Yadav et al. 2017). Based on earlier research, the chitosan and PEG ranges were determined and subsequently optimized using a 3² factorial design. 3 levels of both parameter X₁ (i.e., % chitosan; w/v) and X₂ (i.e., % PEG; w/v) were chosen according to the design criteria, and factor levels were coded accordingly. Experiments with various levels of the identified factors were conducted (13 runs). For a comparative investigation, blank nanoparticles (positive control) were fabricated following the ideal parameters but without the inclusion of oil²⁸.

2.4 Particle size analysis:

Dynamic light scattering was employed to assess the particle size of the chitosan-based nanoparticles, whereas electrophoretic light scattering were used to determine the zeta potential.

2.5 Drug entrapment efficiency:

The upper layer of Ferula asafoetida oil formulation was separated and filtered using a 0.22m membrane filter after centrifugation and evaluated at 275nm using a UV-Visible spectrophotometer. To quantify DEE, the concentration of drug in the upper layer was measured using a standard curve, and the concentration of drug dispersed in the upper layer was deducted from the total drug used.

2.6 Optimization (3² factorial design):

Polynomial equations were generated using Design-Expert Software, version 11 (Stat-Ease, Inc. Minneapolis, MN, USA), along with extra interaction terms for selected responses with selected factors. To achieve the effective result for the optimized formulation, there was possible space was located as well an exhaustive grid search was done. The program also provided an effective solution through overlay plots, and the optimized formulation was employed in further in-vitro investigations.

2.7 Transmission electron microscopy (TEM):

TEM at 200 kV and stained negatively with 2% phosphotungstic acid (pH7.0), was used to evaluate the size and shape of the optimized chitosan nanoparticles (Tecnai G20, FEI Inc., USA). Furthermore, produced nanoparticles were dissolution in HPLC water and sonicated for 4 minutes to segregate them.

2.8 Fourier Transform Infrared (FTIR) Spectroscopy:

The FTIR methodology was employed to find if there was any chemical relationship between the chitosan Ferula asafoetida oil and the optimized formulation, which was assessed using the KBr pellet method.

2.9 In-vitro drug release:

A paddle-type (USP Type II) apparatus was used to measure in-vitro release. The optimized formulations were suspended in 5 mL of phosphate buffer (pH 7.4) and put in a dialysis bag (pore size: 2.4 nm, molecular weight:12000-14000, Himedia, India) that had been dipped into distilled water for 1 hour. In the apparatus, the bag was immersed in 300 ml PBS (37±1°C) at 50 pm. A 5 ml sample was obtained at regular intervals and replaced with fresh dissolving media in an equal volume. Zero-order, first-order, Hixson-Crowell, Higuchi, and Korsmeyer Peppas release kinetic models were used to fit in-vitro drug release data.

2.10 Cytotoxicity and anticancer activity:

PC-3, A549, and MIAPACA were developed in tissue culture containers in a growth medium (RPMI-1640) with 10% fetal bovine serum, 100g/ml streptomycin, and 100 units/ml penicillin. MIAPACA was developed in Dulbecco's necessary medium complemented with 10% FBS, 3-mM sodium pyruvate, and 2-mM L-glutamine as 100-units/mL. Cell viability was measured by the thiazolyl blue tetrazolium bromide and Trypan blue assays. 7*103 cells were seeded and differentiated for 48 hours on a 96-well microtitre plate. After 48 hours of treatment, MTT dye (2.5 mg/mL) was further added and incubated at 37 °C for 4 hours; after incubation, the medium was aspirated, and 150L/well DMSO was added and measured at 570nM on a Mutliskan GO plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The IC₅₀ of medicines that demonstrate % inhibition at 10M was computed using the Prism program.

3. RESULTS:

3.1 GC-MS analysis:

The existence of 20 compounds was detected in the GC-MS analysis of Ferula asafoetida oils, with their % area, empirical formula, molecular mass, and residence time all available in the current version of GC-MS (Table 1).

Table 1: Phytochemical compounds measured by GC-MS analysis of Ferula asafoetida oil

S. no.	Compounds	Molecular weight	Molecular formula	Retention time	% area
1	disulfide, bis(1-methyl propyl)	178	C₈H₁₈S₂	7.574	0.45
2	isopropyl formate	88	C₄H₈O₂	11.646	32.00
3	Galvanic acid	398	C₂₄H₃₀O₅	12.379	17.15
4	Ferulic acid	194	C₁₀H₁₀O₅	12.666	14.31
5	terpinene <gamma->	136	C₁₀H₁₆	12.994	0.33
6	dibutylene glycol	162	C₈H₁₈O₃	13.963	3.98
7	Disulfide prop-(Z)-enyl <sec-butyl->	162	C₇H₁₄S₂	17.178	0.60
8	Isopropyl isobutyl disulfide	164	C₇H₁₆S₂	17.767	0.41
9	Disulfide prop-(Z)-enyl <sec-butyl->	162	C₇ H₁₄S₂	17.953	3.48
10	Guaiol	222	C₁₅H₂₆O	18.201	4.07
11	disulfide, bis(1-methyl propyl)	178	C₈H₁₈S₂	20.017	2.97
12	(Z)-1-(But-2-en-1-yl)-2-(sec-butyl)disulfane	176	C₈H₁₆S₂	21.758	0.29
13	α-Ketoisovaleric acid	188	C₈H₁₆O₃Si	26.782	0.65
14	1-(1-(Methylthio)propyl)-2-propyldisulfane	196	C₇H₁₆S₃	29.191	0.31
15	dimethyl mercaptole	136	C₅H₁₂S₂	29.398	1.36
16	Disulfide, methyl 1-(methylthio)propyl	168	C₅H₁₂S₃	29.501	2.29
17	m-Dioxane	88	C₄H₈O₂	36.316	7.18
18	Palmitic Acid	328	C₁₉H₄₀O₂Si	51.855	2.83
19	Linoelaidic acid, trimethylsilyl ester	352	C₂₁H₄₀O₂Si	56.779	1.56
20	Petroselinic acid	354	C₂₁H₄₂O₂Si	57.013	3.76
					99.98

3.2 Design and development of PEG-coated Ferula asafoetida oil-loaded chitosan nanoparticles:

Design expert for optimization:

In this study, a 3² factorial design was employed to find the optimal amount of chitosan and PEG based on particle size and DEE. Following the selected design, thirteen formulations were created, and the influence of two components, chitosan concentration (A) and PEG concentration (B), on the two response variables, particle size, and DEE, was investigated. The particle size was found to be between 400 and 650nm, while the DEE was found to be between 40.5 and 58.8% for all batches (Table 2).

Response analysis was carried out using the polynomial equations generated, and analysis of variance was carried out with the program Design-Expert® by statistical metrics such as the total of degrees of freedom, mean sum of squares, and F value. The p-value, F value, correlation coefficient (R²), and adjusted correlation coefficient were used to confirm the importance of the created model (adj R²).

The polynomial equations for the answers (particle size and DEE) found using regression analysis are as follows:

Particle size = +530 + 100 × A + 6.67 × B (1)

DEE = +50.37 + 7.10 × A + 0.75 × B (2)

The favorable and unfavorable symbols preceding the coefficients in the above equations denote their additive influence on the responses, respectively. The p-value was employed to determine the relevance of the models. Df=2, F-value=59.87, p-value=0.0001, R²=0.9229, and Adjusted R²= 0.9075 are some of the different particle size values. The importance of the model is demonstrated by the p-value. The model's importance is also shown by its strong R2 score. The impact variance is greater than the error variance, as indicated by the F-value. Df=2, F-value=17.26, p-value =0.0006, R2=0.7754 and Adjusted R2 = 0.7305 are the results for DEE. The p-value indicates that the model is statistically significant. The optimum batch was found by the desirability technique for numerical optimization and preferred responses, such as minimal particle size (400-650nm) and maximum entrapment efficiency (40.5-58.8 %).

Design-Expert software recommended -0.043 (coded value) for chitosan and 0.99 (coded value) for PEG for an optimal formulation, resulting in a formulation with a size of 532.31 nm and a DEE of 50.81 percent. The % mean error among predicted and measured values for particle size and DEE was found to be 3.60 percent for particle size and 2.96 percent for DEE, showing that the Ferula asafoetida oil optimization approach was successful. 3D surface plots were used to assess the impact of preparation on response variables. Both of the indicated parameters were shown to have a significant impact on the response variables. When the chitosan concentration was raised from -1 to 1 (coded value), the particle size rose (Fig. 1). With a drop in PEG concentration, a modest rise in particle size was noted (Fig. 1). Both chitosan and PEG contribute to increased particle size, according to the coefficients in equation 1. Because an increase in chitosan resulted in improved DEE, concentration 1(coded value) had the greatest DEE (Fig. 2). PEG, on the other hand, was enhanced by DEE, although it had a smaller influence on DEE than chitosan (Fig. 2). Equation 2 shows a similar effect of both components, with coefficients contributing to an increase in DEE. The expected parameters for the optimized batch are shown in Figure (particle size 532.462nm and DEE 50.99 percent).

Table 2: Factors and responses of various batches of Ferula asafoetida oil chitosan nanoparticles according to experimental design

Std	Run	Factor 1 A: Chitosan (mg)	Factor 2 B: PEG (%)	Response 1 Particle size (nm)	Response 2 DEE (%)
11	1	0	0	550	50.2
5	2	0	0	545	54.8
4	3	-1	0	450	40.5
6	4	1	0	600	57.9
9	5	1	1	625	54.6
3	6	1	-1	650	58.8
13	7	0	0	525	52.4
1	8	-1	-1	400	42.7
7	9	-1	1	425	45.5
12	10	0	0	500	53.5
2	11	0	-1	520	45.3
10	12	0	0	540	47.4
8	13	0	1	560	51.2
Coded level −1 0 +1 X1=A: Chitosan (%) 0.1 0.2 0.3 X2=B: PEG (%) 1.0 1.5 2.0

Fig. 1: 3D response surface curve showing the effect of chitosan and PEG on the particle size of Ferula asafoetida oil

Fig. 2: 3D response surface curve showing the effect of chitosan and PEG on drug entrapment efficiency of Ferula asafoetida oil

Fig. 3: Overlay plot resenting the predicted parameters for FOCNo

3.3 Transmission electron microscopy (TEM):

The spherical form and size of the particles were revealed by TEM analysis of FOCNo, which was smaller than that recorded by the zeta sizer (Fig. 4), which determined the diameter of the particles.

Fig. 4: TEM of FOCNo

3.4 Fourier Transform Infrared (FTIR) Spectroscopy:

The alkane C-H stretching is illustrated at 2931 cm-1. At 902 cm-1, out-of-plane bending owing to =C-H may be seen. At 661 cm-1, the presence of four or more -CH2 indicates a long open chain in Ferula asafoetida oil fatty acids. Near 3412 cm-1, a large peak was found, indicating the existence of a hydrogen-bonded –OH group. Aromatic C=C is seen at 1457 cm-1, which corresponds to the methylene group's CH2 bending. The alkene C=C peak was found at 1651 cm-1. The existence of C-O stretch in alcohols, esters, and carboxylic acids (1296 cm-1, 1090 cm-1, 1009 cm-1) can be seen in the peaks between 1000 and 1300 cm-1. C-O-H bending seems like a wide and weak peak at 1296 cm-1, which coincides with the peak. At 1376 cm-1, the methyl group (CH3) exhibits its typical bending absorption. C-O stretching at 1090 cm-1 shows the existence of a hydroxyl group in the form of a saturated secondary alcohol. Due to O-H stretching vibrations, the FTIR spectra of chitosan reveal a prominent wide band at 3423 cm-1. At 2927 cm-1, C-H stretching vibrations were found, while N-H stretching vibrations blend with O-H stretching vibrations in the 3600-3150 cm-1 range. At 1637 cm-1, amine exhibits N-H bending (scissoring), while C-N stretching was seen between 1025 and 1200 cm-1. At 1030 cm-1, the C-O-C peak in the glucopyranose ring was found, while the particular bands of the (14) glycosidic bridge showed at 1155 cm-1 (Fig. 5). Because their distinctive bands are found in the FTIR spectra, chemical interaction between the drug and the polymer during the formulation development.

3.5 In-vitro drug release:

More than 82% of the drug was released from FAOCN during 32 hours, indicating a sustained release pattern from the formulation. About 18.72% of the drug was released in the first two hours, indicating drug release from the onset of dissolution. The correlation coefficients (R²) for all the kinetics models were 0.931, 0.530, 0.997, 0.661, and 0.599, respectively, when data were fitted into release kinetic equations (mathematical models). The Higuchi model, which indicates the release of the drug via a matrix system that is a diffusion-controlled mechanism dependent on Fick's law, was demonstrated to be the preeminent model for the formulation with an R2 value of 0.997. As a result, it was discovered that Ferula asafoetida released from the optimized formulation followed the diffusion-controlled release pattern. As a consequence, the optimized formulation's diffusion-controlled release of Ferula asafoetida was discovered. Preliminary drug release could be due to faster drug molecule migration from the FOCN_O surface, and subsequent sluggish drug release could be because of chitosan solvation in the dissolution fluid and diffusion of the entrapped drug by the swollen polymer matrix along with erosion at the matrix system's boundary layer.

3.6 Cytotoxicity and anticancer activity:

The MTT assay was used to measure the test chemical's cytotoxicity. It's being used to assess cell viability by using mitochondrial dehydrogenases. (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), a water-soluble tetrazolium salt produces a yellowish color. The conversion of dissolved MTT into insoluble purple formazan by breaking the tetrazolium ring by mitochondrial dehydrogenase enzymes demonstrates cell viability. Using a GO plate reader, this solution is further solubilized into DMSO. The FDA defines IC50 as the concentration of a medication required for 50% inhibition in in-vitro research. Furthermore, IC50 is a pharmacological concentration at which 50% of the cell population dies. For initial screening, a cut off of 50% cell growth inhibition was used as a cut-off for chemical toxicity against cell lines. In the present, the cytotoxic test of Ferula asafoetida oil was studied by MTT assay using human normal breast epithelial cell line (Fr2), human adenocarcinoma prostate cell line (PC3), adenocarcinoma human alveolar epithelial cell line of lung cancer (A549), and cell line of pancreatic cancer (MIAPACA) (Table 3).

Table 3: Cytotoxicity study of Ferula asafoetida oil and their optimized nanoformulations using MTT assay on Fr2, PC3, A549 and MIAPACA

Test compound	Breast epithelial (Fr2)	Prostate cancer (PC3)	Lung cancer (A549)	Liver cancer (MIAPACA)
	IC50 µg/ml	IC50 µg/ml	IC50 µg/ml	IC50 µg/ml
Ferula asafoetida oil (20 mg)	28.40	12.07	9.72	9.76
FOC (10mg)	47.40	16.63	13.78	18.13

4. DISCUSSION:

GC-MS analysis is an effective technique to identify the total phytoconstituents in oils. In the present study, the GC-MS study of Ferula asafoetida oil indicated the presence of 20 compounds, some of which have been pharmacologically described for their medicinal benefits in literature. PEG (polyethylene glycol) coated Ferula asafoetida oil encapsulated chitosan nanoparticles were formulated for the treatment of anti-cancer in the present study. The difference between the observed value and the predicted value for particle size and entrapment efficiency of optimized Ferula asafoetida oil chitosan nanoparticles (FOCNo) was determined to be 3.60 % and 2.96 %, respectively. FTIR spectroscopy of FOCNo demonstrated the encapsulation of Ferula asafoetida oil in the chitosan and the absence of any chemical interaction. Spherical shape nanoparticulate of Ferula asafoetida oils was confirmed by TEM analysis. The Higuchi model with R² = 0.997 was considered an appropriate model for the release of nanoparticles of Ferula asafoetida oil from the chitosan capsules by following diffusion-controlled release. Cytotoxicity and anti-cancer activity of Ferula asafoetida oil and FOCNo was evaluated using MTT assay on human cell lines like breast epithelial cell line (Fr2), human adenocarcinoma prostate cell line (PC3), adenocarcinoma human alveolar epithelial cell line of lung cancer (A549), and cell line of pancreatic cancer (MIAPACA). Ferula asafoetida oil and FOCNo were studied on normal breast epithelial cell lines (Fr2) to determine the safe concentration for further preclinical studies. It has been found that the IC50 value of Ferula asafoetida oil and FOCNo 28.4 µg/ml and 47.4µg/ml respectively shows that oil formulation is safer as compared to pure oil. The IC50 value of Ferula asafoetida oil at the concentration of 20mg was 12.07µg/ml, 9.72 µg/ml, and 9.76µg/ml against PC3, A549, and MIAPACA, respectively. The IC50 value of FAOCN at the concentration of 10mg was 16.63µg/ml, 13.78µg/ml, and 18.13µg/ml against PC3, A549, and MIAPACA, respectively. These results showed excellent cytotoxic and anticancer activity in Ferula asafoetida oil-loaded nanoformulation at the concentration of 10mg as compared to pure oils at a concentration of 20mg, which indicates the better penetration of oil into cancerous cells due to nanonization.

Physiologically, testosterone is a male sex hormone that is converted into dihydrotestosterone (active) via an enzyme Type 2 5α-reductase. On binding with dihydrotestosterone (DHT), the androgen receptor gets detached from heat shock protein and displaced to the nucleus, where it binds to cofactor ARA-70 and regulates the expression of the target gene, leading to elevate the level of PSA, cell survival, and proliferation. Moreover, AR (Androgen receptor) has been reported to regulate the mitogenic signaling mechanism like PI3K/AKT/mTOR (phosphatidylinositide 3-kinases/AKT/ mechanistic TOR) and MAPK (mitogen-activated protein kinase) pathway in which activated PI3K stimulates the change of membrane-bound (phosphatidylinositol-(4,5)-bisphosphate) (PIP2) to (phosphatidylinositol-(3,4,5)-triphosphate) PIP3 which further serves as a secondary messenger and facilitate the initiation of PDK1 (PI3K-dependent kinase-1). It leads to the stimulation of AKT and mTOR which is responsible for the activation of the apoptosis process. The signaling of PIP3 is regulated by PTEN (phosphatase tensin homolog) which is having antagonistic approach towards PI3K activity, therefore mutation in PTEN leads to uncontrolled cell proliferation leading to the progression of androgen-independent cancer. In the literature, ferulic acid is reported to suppress cell proliferation by upregulating the expressions of pro-apoptotic genes and by downregulating the expressions of BCL2 and XIAP in addition to cell cycle G0/G1 phase arrest (Eroglu et al. 2015). Pharmacologically, guaiol significantly triggered autophagic cell death by targeting both mTORC1 and mTORC2 signaling pathways (Yang et al. 2018). Interestingly, galbanic acid reduces the growth of cancer cells by decreasing the expression of androgen receptors. Phytochemically, in this study GC-MS analysis of Ferula asafoetida oil also showed the presence of these constituents. Ferula asafoetida oil showed a potential anti-cancer effect so, we can be hypothesized that these anticancer potential phytoconstituents can contribute by following the same mechanism of action due to the presence of all these pharmacological active phytoconstituents (Fig. 6).

Fig. 6: Possible mechanism of action of phytoconstituents present in ferula asafoetida oil

5. CONCLUSION:

The present study demonstrates that PEG-coated chitosan optimized nanoformulation of Ferula asafoetida oil showed potential IC50 value against Fr2, PC3, A549, and MIAPACA cell lines in comparison to Ferula asafoetida oil at its half of the concentration, which shows its effectiveness in the cancerous cell with the significant drug release pattern. Moreover, Ferula asafoetida oil possesses protective phytoconstituents like galvanic acid, ferulic acid, and guaiol identified in GCMS analysis and reported to have therapeutic effective cancerous cells. So, Ferula asafoetida oil encapsulated chitosan nanoparticles hold considerable anticancer potential. The use of FAOCN should develop as one of the potent approaches in the treatment of various cancer and, hence more studies are required to understand the exact molecular mechanism.

6. CONFLICT OF INTEREST:

There is no conflict of interest.

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Received on 17.12.2023 Revised on 04.10.2024

Accepted on 21.03.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):241-249.

DOI: 10.52711/0974-360X.2026.00034

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