Evaluating the Efficacy of Benincasa Hispida Seed Extract in Inhibiting the Proliferation of a Human Triple Negative Breast Cancer Cell Line

Abstract

Objective:

This study aimed to investigate the possible therapeutic benefits of Benincasa hispida extract as an adjunctive treatment for breast cancer. While previous studies have highlighted the anticancer effects of various components of Benincasa hispida, including its fruit and leaves, there remains a paucity of research concerning the application of Benincasa hispida seeds in the context of cancer treatment, especially breast cancer.

Methods:

The cytotoxic impact of Benincasa hispida on breast cancer cell lines was assessed via in-vitro analysis employing the MTT assay. The apoptosis levels were assessed by applying the Annexin V assay. Furthermore, an analysis of the cell cycle and quantification of intracellular reactive oxygen species (ROS) were conducted.

Results:

The findings of this study demonstrated noteworthy anti-proliferative properties of the Benincasa hispida extract, highlighting its ability to induce apoptotic cell death and to cause cell cycle arrest at the S-phase. Furthermore, the extract exhibited antioxidant characteristics, influencing reactive oxygen species. The aforementioned attributes present significant advantages in the restoration of disrupted physiological mechanisms within breast cancer cells, thereby suggesting potential therapeutic benefits for the treatment of breast cancer.

Conclusion:

In summary, the findings indicate that Benincasa hispida extract presents a noteworthy potential as a supplementary strategy in the treatment of breast cancer. A more thorough examination of the active components and the intricate mechanisms of action is necessary. Such efforts may ultimately promote the advancement of secure and effective therapeutic strategies for the management of breast cancer.

Introduction

Cancer, characterized by abnormal cell growth, poses a significant global health challenge with an increasing mortality rate. Breast carcinoma is distinguished as the most common malignancy affecting women globally [1,2], and it holds the position of the second leading cause of cancer-related deaths among females [3]. In India, it represents one-fifth of all female cancers and accounts for seven percent of global breast cancer occurrences. As of 2020, it had overtaken lung cancer to become the most commonly diagnosed cancer, with around 2.3 million new cases documented, accounting for 11.7% of all cancer cases [4]. Forecasts in the field of epidemiology indicate that by the year 2030, the number of global cases of breast carcinoma is projected to surpass two million [5]. The conventional approaches to managing breast carcinoma include radiation, chemotherapy, surgical intervention, and hormone therapy. Nevertheless, the effectiveness of chemotherapy agents is often undermined by obstacles including multi-drug resistance, recurrence of the disease, and significant side effects [3,6-8].

The selection of the most suitable treatment strategy is contingent upon various factors, such as the tumor type, disease stage, and the clinical status of the patient [9]. While chemotherapy continues to play a crucial role in the management of cancer, the considerable side effects and the development of resistance, particularly in hormone therapy, pose significant challenges [9,10]. Consequently, there is a growing fascination with alternative anticancer therapies that utilize naturally occurring substances and extracts characterized by their minimal toxicity [10,11]. Current pharmaceutical research focuses on the development of novel and effective anticancer therapies, particularly those derived from natural sources like plants and certain microbial species. These natural origins provide a diverse array of compositions, ecological traits, phytochemical profiles, and ethnopharmacological properties, making them essential in the formulation of anticancer medications.

Belonging to the Cucurbitaceae family, Benincasa hispida is known by a multitude of names, including winter melon, ash gourd, winter gourd, white pumpkin, wax gourd, white gourd, animal oil gourd, gourd melon, and Chinese watermelon. This vegetable crop is cultivated extensively. It holds considerable esteem in Asian countries, valued for its applications in both culinary and medicinal contexts.

[12,13]. Historically, the applications of Benincasa hispida have encompassed a diverse array of medicinal functions, including its roles as a laxative, diuretic, tonic, aphrodisiac, and cardiotonic agent. They were additionally utilized in the management of urinary stones, hematological conditions, psychological disorders including insanity, epilepsy, and schizophrenia, along with jaundice, dyspepsia, fever, and menstrual irregularities [14,15]. The plant is rich in phytochemicals such as alkaloids, flavonoids, tannins, glycosides, phenolic compounds, amino acids, steroids, triterpenoids, and saponins [16], showcasing a diverse pharmacological profile. The polyphenols present in Benincasa hispida exhibit significant antioxidant properties, providing a safeguard against a range of ailments including neurodegenerative disorders, cardiovascular diseases, cancer, liver diseases, and infectious diseases [17]. The tannins found in the plant demonstrate notable inhibitory effects on the activity of pancreatic lipase and the absorption of fats from the intestine [18]. Benincasa hispida is widely utilized in culinary applications, serving both as a vegetable and as an ingredient in confectionery production. Since antiquity, the fruit has been esteemed for its therapeutic properties, attributed to its diverse array of chemical constituents. The composition of these fruits is predominantly characterized by a variety of bioactive compounds, including triterpenoids, flavonoids, glycosides, saccharides, carotenes, vitamins, ß-sitosterol, uronic acid, n-triacontanol, tannins, and an array of amino acids [19-21].

The Sarangadhara Samhita, an esteemed Ayurvedic medical text, discusses the application of Benincasa hispida in the treatment of haemorrhage, especially in cases involving lung ulceration and pulmonary complications. Furthermore, the juice derived from the cortical section of the plant has been utilized in the management of diabetes [22]. This investigation explored the anticancer efficacy of an aqueous seed extract derived from B. hispida, specifically targeting breast cancer through the utilization of the MDA-MB231 cell line.

Materials and Methods

Authentication and standardisation of B. hispida (Aqueous) seed Extracts

The desiccated seed specimen of B. hispida was procured from a certified pharmacy and subsequently validated for its authenticity under the expert supervision of a botanist at the Agharkar Research Institute in Pune, culminating in the issuance of an authentication number [AUTH-23-161]. The aqueous extract was prepared, and the physicochemical analysis was conducted at Sudhatatva Pharmacy, located within Dr. Y Patil Ayurvedic College and Research Centre in Pimpri, Pune-18, in accordance with the guidelines outlined in API Volume 4. The extract underwent a thorough assessment concerning its color, aroma, flavor, and solubility. The analysis included assessments of pH, loss on drying, total ash content, and shelf life.

Extract preparation

In order to generate the aqueous extract through the Soxhlet method (Wation DB 50), 50 grams of Benincasa hispida (BH) seed powder were positioned within the thimble of the extractor. Following this, 250 ml of water was introduced into the round-bottom flask (RBF) and subjected to heating for a duration of approximately 6 to 7 hours. The liquid in its condensed form (water) descends from the condenser into the chamber of the Soxhlet extractor, thereby enabling the extraction of soluble constituents from the pulverized seeds. The extraction process ought to persist until the requisite concentration of extract is achieved, which may require multiple cycles. Meticulously detach the thimble that holds the extracted substance from the apparatus. The liquid obtained was subsequently subjected to filtration through filter paper to remove any solid particulates or contaminants. Introduce the filtered extract into a water bath maintained at 80°C to facilitate the acquisition of a dry extract.

Anti-cancer activity of B. hispida

Cell Culture

The human Triple Negative Breast cancer cell line (MDA-MB231) was obtained from the National Centre for Cell Science (NCCS) located in Pune, India. MDA-MB231 cells were cultured as a monolayer in DMEM (GibcoTM), enriched with 10% fetal bovine serum and 1% antibiotic (GibcoTM). The cell lines were cultivated and sustained at a temperature of 37 °C within a humidified environment enriched with 5% CO₂.

Assessment of Cytotoxicity

MDA-MB231 cells, at a density of 1 × 10⁴ cells per well, were introduced into a 96-well plate and subjected to incubation at 37 °C for a duration of 24 hours. Following the incubation period, different concentrations of the aqueous extract of B. hispida, specifically 20, 40, 80, 160, 320, and 640 µg/mL, were introduced in triplicate and subjected to incubation at 37 °C for a duration of 24 hours. Subsequently, the assessment of cytotoxicity was conducted utilizing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) from Himedia. The media from the 96-well plates were removed, after which 20 µL of MTT dye (prepared at a concentration of 5 mg/mL in phosphate buffer saline (PBS), (GibcoTM)) was added to each well, followed by incubation at 37 °C for 4 hours. To facilitate the dissolution of the formazan crystals, 150 µL of dimethyl sulfoxide (DMSO) was introduced. A microplate reader (MultiSkan Go by Thermo Scientific) facilitated the estimation of reduced MTT by quantifying the optical density (OD) at 570 nm [23].

Apoptosis analysis

The FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen-Molecular Probes®) was employed to perform an apoptosis assay, incorporating the IC₅₀ values of BH (142µg/mL) and DOXO (52 µg/mL) [24].

Cell cycle analysis

The MDA-MB231 cells, whether subjected to treatment with BH and DOXO or left untreated, were harvested 24 hours subsequent to being rinsed with 1X PBS (Phosphate Buffered Saline) and subjected to trypsinization. To the cellular samples, 25 μL of RNase A (20 mg/mL from Invitrogen), 2 mM MgCl2 (from Sigma), and 5-10 μL of 100 µg/mL propidium iodide (from Invitrogen) were introduced. The cells were subsequently incubated at ambient temperature for a duration of 10 to 15 minutes prior to analysis utilizing a FACS Caliber (BD Bioscience) [25].

Estimation of generation of intracellular reactive oxygen species

Flow cytometry was employed to examine the generation of reactive oxygen species utilizing Dichloro-dihydro-fluorescein diacetate (DCFH-DA) [26]. In the subsequent experiment, MDA-MB231 cells were cultivated for a duration of 24 hours within 6-well plates. Upon achieving 70-80% confluence, the cells underwent treatment with BH and DOXO at their respective IC₅₀ concentrations of BH (142 µg/mL) and DOXO (52 µg/mL), along with 10 µM DCFH-DA. Subsequently, they were incubated in a 5 mM PBS buffer for a duration of 2 hours. Flow cytometry utilizing the Beckman Coulter Cytomics FC 500 (kex = 495 nm and kem = 520 nm) was employed to quantify the fluorescence produced from the hydrolysis of DCFH-DA to DCFH [25].

Statistical analysis

All experiments were performed in triplicate, and the outcomes are presented as mean ± standard deviation. The analysis of the data was conducted utilizing GraphPad Prism 8, employing a “Two-way ANOVA” in conjunction with a suitable post-hoc test, with statistical significance established at P < 0.05.

Results

Benincasa hispida (Aqueous) seed Extracts Preparation

The total amount of 2.7 grams of BH aqueous extract was produced from 50.02 grams of seeds that were in a sticky or sticky-like state. A total of thirteen hours was necessary for the synthesis of the extract (Figure 1).

Figure 1.

Images (A to F) Shows the Weight of Dry Seeds of BH, its aq. Extraction by Soxhlet Apparatus and Total Extract Obtained.

Physicochemical Examination

The physicochemical analysis revealed that all parameters conformed to the normal range as stipulated by the Ayurvedic Pharmacopeia of India (API) guidelines, demonstrating an absence of adulteration. The extract possesses a white hue, exhibits a distinctive aroma, has a sweet flavor profile, and is soluble in water. The drying loss of 3.48% suggests that the seeds possess an optimal moisture content, facilitating effective extraction and ensuring an extended shelf life. A pH of 6.09 guarantees a state of equilibrium, security, and possible medicinal uses. The total ash content of 1.78% signifies a high level of purity, whereas the acid-insoluble ash at 0.56% corroborates the presence of minimal siliceous impurities. The water-soluble extractive value of 28.13% indicates a substantial presence of bioactive compounds, including phenolics, flavonoids, tannins, and glycosides, thereby endorsing its application in aqueous-based therapies. The extractive value soluble in alcohol, quantified at 23.17%, indicates a notable abundance of bioactive compounds that are soluble in alcohol. The entirety of these findings is catalogued in Table 1.

Table 1.

Physiochemical Analysis Results of Benincasa hispida Seed Aqueous Extract

Sr No	Parameter	Test Observation
1	Description	Colour-white, taste-sweet, Odour- characteristics
2	Solubility	Soluble in water
3	pH	6.09
4	Loss on Drying	3.48%
5	Total ash	1.78%
6	Acid insoluble ash	0.56%
7	Water soluble extractive	28.13%
8	Alcohol soluble extractive	23.17%

Antiproliferative activity of Benincasa hispida on MDA-MB231 cell line

The antiproliferative effects of Benincasa hispida on the MDA-MB231 cell line are of considerable interest. The aqueous extract of BH was administered at concentrations of 20, 40, 80, 160, 320, and 640 µg/mL, alongside DOXO at concentrations of 5, 10, 20, 40, 50, and 60 µg/mL, to the MDA-MB231 cancer cell line. The cytotoxic impact of BH aqueous extract on the MDA-MB231 cell line exhibited a dose-dependent relationship, yielding an IC₅₀ value of 142±7.54 µg/mL, in contrast to DOXO, which presented an IC₅₀ value of 52±3.41 µg/mL (Figure 2).

Figure 2.

Percent Inhibition of MDA-MB231 by BH Aqueous Extract at Concentrations of 20-640 µg/mL and DOXO at Concentrations of 5 -60 µg/mL

Assessment of the influence of BH on the apoptotic process in MDA-MB231 cells

An analysis of apoptosis was conducted on both control and treated MDA-MB231 cells over a 24-hour period at the IC₅₀ concentration of BH, which is 142µg/ml. Upon exposure to the IC₅₀ of BH for a duration of 24 hours, 06.51±6.96 % of cells exhibited characteristics of early apoptosis, whereas 60.17±0.04 % of cells demonstrated features indicative of late apoptosis (Figure 3a). DOXO prompted early apoptosis in 21.2 ± 1.61% of cells at the IC₅₀ value, while late apoptosis was observed in 37.56 ± 1.37% of cells after a duration of 24 hours (Figure 3b). Upon comparing the effects of BH and DOXO, it was revealed that BH exhibited a more pronounced late apoptosis effect than DOXO, while DOXO demonstrated a similar impact in both early and late apoptosis stages (Figure 3c).

Figure 3.

BH-Induced Apoptosis Using Flow Cytometry (A) It depicts cells treated with BH at IC₅₀ (IC₅₀ (142µg/ml) after 24 hours, and (B) depicts cells treated with DOXO at IC₅₀ (52 µg/L) and after 24 hours. (C) Data represented as Mean+SD and, evaluating the apoptotic phases for the respective concentrations of BH and DOXO.

Evaluation of the influence of BH on the regulation of the cell cycle in MDA-MB231 cells

The findings indicated that 58.7±6.59% of cells subjected to the BH IC₅₀ concentration (142µg/mL) for a duration of 24 hours were halted in the S phase, whereas 9.67±3.74% were allocated to the G1 phase, and 22.0±8.7% were observed in the G2/M phase (Figure 4). DOXO resulted in the accumulation of 23.80±2.81% of cells in the G1 phase, 58.6±1.22% of cells in the S phase, and 17.0±2.35% in the G2/M phase after a 24-hour exposure at the IC₅₀ concentration of 52 µg/mL. Ultimately, BH demonstrated cell cycle arrest during the S-phase at the IC₅₀ concentration, with DOXO exhibiting analogous results to those of BH. The data presented above illustrates that BH has the capacity to function as an inducer of apoptosis, in addition to serving as a cell growth inhibitor or S-phase blocker in breast cancer.

Figure 4.

The Regulatory Effect of BH & DOXO on Cell Cycle Distribution in MDA-MB231. The cells were treated with specific concentration of BH & DOXO for 24 h. (A) Cell treated with BH at IC50 (142µg/ml) after 24 h, (B) represents the cells treated with DOXO at IC₅₀ (52 µg/L) after 24 h. (C) Data presented as Mean+SD and analyzed, determining the different phases of cell cycle for the respective concentrations of BH and DOXO.

Measurement of intracellular reactive oxygen species:

Initially, we assessed the distribution of fluorescence intensity both in the presence and absence of BH and DOXO. The administration of BH IC₅₀ to MDA-MB231 cells led to a notable reduction in intracellular ROS generation, while the application of DOXO IC₅₀ values produced a modest decrease in intracellular ROS generation, suggesting that BH exhibits commendable antioxidant properties (Figure 5).

Figure 5.

Effect of BH on Intracellular ROS Production, Determining Production of Intracellular ROS for the Respective Concentrations of BH and DOXO. (A) Cell treated with BH at IC₅₀ (142µg/ml) after 24 h, (B) represents the cells treated with DOXO at IC₅₀ (52 µg/L) after 24 h. (C) Data presented as Mean+SD and analyzed, determining the Intracellular ROS production for the respective concentrations of BH and DOXO.

Discussion

Breast carcinoma is distinguished as the most common cancer affecting women worldwide [1,2], occupying the position of the second leading cause of cancer-related mortality among females. Nonetheless, the effectiveness of chemotherapy agents is often limited by obstacles such as multi-drug resistance, the recurrence of disease, and significant adverse effects [6,7]. As a result, a multitude of scholarly investigations are focusing on both conventional and alternative methodologies. Benincasa hispida is widely utilized in culinary applications, serving both as a vegetable and as an ingredient in confectionery manufacturing. The fruit boasts a lengthy history of medicinal use, owing to its diverse array of chemical components.

In their research, Abdullah et al. illustrated that the seed extract of Benincasa hispida exhibits inhibitory properties in scavenging activity, reduction of metallic elements, and beta-carotene bleaching assays. Furthermore, it was observed that the seed extract demonstrated the highest total phenolic content when compared to the skin and pulp extracts of Benincasa hispida [27]. This indicates that Benincasa hispida fruits may represent a valuable source of natural antioxidant compounds, potentially serving as an alternative to synthetic antioxidants [28].

Some studies indicate that the liquid and methanolic extracts of Benincasa hispida exhibit significant activity in a dose-dependent manner when compared to ascorbic acid. The pronounced eliminative efficacy of B. hispida liquid extract was observed at 87% concentration (100 μg/mL), while the alcoholic extract demonstrated a slightly higher efficacy at 88% concentration (100 μg/mL) [29,30]. The current investigation demonstrated anti-proliferative activity at a concentration of 142µg/mL. BH demonstrated anti-proliferative activity primarily through mechanisms associated with late apoptosis. BH predominantly exhibited cell cycle arrest at the S-phase.

Gill et al. investigated the capabilities of the methanolic extract of Benincasa hispida seeds (MEBH) in neutralizing free radicals, as well as its anti-inflammatory and analgesic effects. The evaluation of the free radical scavenging capacity of MEBH was conducted through the DPPH (1,1-diphenyl-2-picryl-hydrazyl) and hydrogen peroxide (H2O2) methodologies, demonstrating notable activity in a dose-dependent fashion when juxtaposed with ascorbic acid. MEBH demonstrated its peak radical scavenging activity of 79.8% at a concentration of 300 μg/mL, alongside an H2O2 scavenging effect of 63.7% at a concentration of 200 μg/mL. The evidence indicates that MEBH may function as a natural antioxidant in the management of inflammation and pain [31]. The findings presented here substantiate previous research, illustrating the antioxidant properties of BH through its capacity to diminish Reactive Oxygen Species (ROS).

Nadira Binte Samad and colleagues conducted an investigation into the effects of water extracts obtained from the dried seeds of Benincasa hispida on antioxidant activity, examining its relationship with the total phenolic and flavonoid contents in vitro. The researchers determined that the dried seeds of Benincasa hispida may serve as promising natural antioxidants within the food industry [32]. The results of these findings are consistent with those observed in our own study.

Hamouda et al. undertook a study aimed at evaluating the toxicity of biosynthesized AgNPs on the human cervical neoplastic cell line (HeLa) as well as on traditional human primary osteoblasts cell line. The characterization of the AgNPs was conducted through the application of UV–Vis spectrometry, dynamic light scattering (DLS), FTIR, and various microscopy techniques. Their findings underscored the therapeutic potential of AgNPs synthesized from B. hispida, attributed to their minimized side effects, indicating a range of possible therapeutic applications [33].

At concentrations between 10 and 1000μg/mL, proteins derived from the fruit, seed, and root demonstrated a cytotoxic effect on Artemia salina that was contingent upon the concentration levels. The median lethal concentration (LC50) values for the extracts of fruit, seed, and root were determined to be 44, 41, and 50 μg/mL, respectively [34]. Furthermore, this study demonstrated that the root proteins inhibited the proliferation of HeLa and K-562 cells by 28.50% and 36.60%, respectively. A further investigation revealed that the methanolic extract derived from the entire plant (5-50μg/mL) displayed cytotoxic effects on A. salina, yielding an LC50 value of 45.187μg/mL [35]. Furthermore, the aqueous seed extract (20-800μg/mL) exhibited no cytotoxic effects on HUVECs and normal fibroblast (NIH/3T3) cells. In a distinct study, the aqueous extract (1–20μg/mL) demonstrated a reduction in the activation of cell adhesion molecules by impeding monocyte adhesion, lowering Reactive Oxygen Species (ROS) levels, and inhibiting the nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB) in high glucose (25mM) induced HUVECs cells [36].

In summary, this research indicates that Benincasa hispida seeds could represent a valuable adjunctive therapeutic avenue for breast cancer treatment. Additional investigation is necessary to explore the underlying mechanisms of action, possible adverse effects, and ideal dosage protocols of Benincasa hispida seed extract in relation to breast cancer therapy. This investigation may ultimately enhance the array of alternatives accessible to healthcare professionals and individuals facing this illness.

Author Contribution Statement

Monali Kanase: Methodology, Data curation, Software, Formal analysis, Vaibhav Ladke: Conceptualization, Formal analysis, Investigation, Validation, Writing original draft. Nilima Dharkar: Methodology, Data curation, Formal analysis, Writing - review & editing. All authors approved the submitted manuscript.