In-vitro antioxidant and anti-proliferative activity of aerial parts of Senecio Laetus Edgew on breast cancer (MCF-7) and colon carcinoma (HCT116) cell lines

Abstract

The present study aimed to perform phytochemical investigation of various fractions of the aerial parts of Senecio laetus Edgew for its bio-constitution and to pharmacologically evaluate these fractions for antioxidant and antiproliferative activities against breast cancer (MCF-7) and colon carcinoma (HCT-116) cell lines. Phytochemical screening and characterization of compounds was carried out as per standard procedures. Extracts were subjected to GC-MS analysis for identification and characterization of constituents. The antioxidant activity was carried out using DPPH, ferric ion reducing power assay, and nitric oxide radical inhibition assays. In vitro antiproliferative activity of the extracts was carried out using MCF-7 (breast cancer) and HCT-116 (colon carcinoma) cancer cell lines by MTT assay, colony formation assay, and wound healing assay. Phytochemical screening showed the presence of alkaloids, flavonoids, carbohydrates, glycosides (cardiac and anthraquinone), tannins, triterpenoids, phenolic compounds, and phytosterols. GC-MS analysis showed the presence of 54 compounds. Ethyl acetate and methanolic extract exhibited the maximum amount of phenolic content compared to dichloromethane and hexane fractions. Antioxidant capacities were shown highest in ethyl acetate and methanolic fractions. The antiproliferative activity was found to be concentration dependent, with dichloromethane fraction more effective than ethyl acetate and hexane fractions against both the MCF-7 and HCT-116 cancer cell lines. The results indicate that S. laetus Edgew has a promising antioxidant and antiproliferative potential as shown by its activity against MCF-7 (breast carcinoma) and HCT-116 (colon carcinoma) cancer cell lines.

Introduction

Cancer has been the major problem in both developed as well as under developing countries & remains as one of the leading causes of death around the globe with lung cancer (LC) being currently the most prevalent malignant tumor with quickest rising incidence and morbidity of all cancers [1, 2]. Colorectal cancer (CRC) is also one of the major causes of cancer-related mortality in the populations of developed countries [3]. The incidence and fatality rate of this type of cancer is rising in developing nations as well, even though the disease is primarily associated with high-income countries [4]. Worldwide breast cancer is the most prevalent malignant tumor, responsible for 30% of all new cases of female malignancies. One in six cancer deaths and one in four cancer cases in females is due to breast cancer [5]. Breast cancer ranks fifth in terms of mortality and is the most diagnosed cancer today thereby surpassing lung cancer [6]. Present treatment options available for breast and colon cancer include surgical treatment, hormone, radiation, and chemotherapy. These treatments have shown improvement in patients, but one of the major drawbacks is their toxicity to the normal cells, which is due to their inability to differentiate between normal and cancerous cells [7]. Plants have a long history of use in the treatment of cancer [8, 9]. The interest in nature as a source of potential chemotherapeutic agents continues [10]. Evidence from World Health Organization (WHO), states that about 65% of the population across the globe prefer to use traditional and herbal medicines to treat diseases. The use of complementary alternative medicines (CAM) has dramatically increased in India along with USA, in the last two decades [11]. One of the key contributors to the development of cancer is oxidative stress. The reason for this is that the reactive oxygen species (ROS) produced by oxidative stress have the ability to damage proteins, lipids, and DNA, which can lead to mutations and other cellular alterations that encourage the growth of cancer. A promising approach to mitigate the occurrence of cancer involves the use of drugs derived from natural sources, particularly those rich in flavonoids and phenolic compounds [12]. These bioactive compounds exhibit potent antioxidant properties, enabling them to neutralize ROS and reduce oxidative stress. By minimizing oxidative stress, flavonoids and phenolic compounds can protect DNA from damage and prevent harmful cellular changes. This protective mechanism not only reduces the likelihood of mutations but also helps maintain cellular integrity, thereby potentially lowering the risk of cancer development. Consequently, natural compounds with antioxidant activity represent a valuable avenue for cancer prevention and therapeutic strategies [13]. Moreover, approximately 60% of antiproliferative agents are derived from medicinal plants and other natural resources, the quest to find newer agents having better efficacy with minimal toxic effects has been inculcating in researchers the desire to investigate the vast pool of plant-based drugs, as there is still a substantial number of plants having antiproliferative potential yet to be fully investigated.

S. laetus (SL) Edgew locally known as “Bagghu” (Asteraceae) and commonly as “cheerful Senecio” is an endemic medicinal herb of North western Himalaya and is widely distributed in Pakistan, India (Jammu & Kashmir, Himachal Pradesh, Uttar Pradesh, Nepal, Sikkim, Bhutan, Arunachal Pradesh), Burma, S. W. China. The plant is found at an altitude of 2400–4000 m [14]. Traditionally the genus Senecio has many species, which have been used in traditional or folklore medicine as anti-inflammatory, antipyretic, antimicrobial, antibacterial and antitubercular [15]. SL is a widespread medicinal herb from the Himalayas that has long been used as a remedy for mouth swelling and sore throat [14]. SL has shown larvicidal potential against Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi [16].

Because of limited data available regarding the antiproliferative potential of the plant, this study was aimed to evaluate the antioxidant and antiproliferative potential of the plant against breast cancer (MCF-7) and colon carcinoma (HCT116) cancer cell lines.

Materials and methods

Collection and authentication of plant

The plant was collectected from Wanihama area of Kashmir valley at an altitude of 1650 m in July, 2019. The collection was done from off the side of road. The collection place was not a wetland or a protected wildlife sanctuary. Additionally the plant grows wild and in abundane in this region, thus not an endangered species, therefore no permission was required for its collection. The plant material was identified and authenticated in the Centre for Biodiversity and taxonomy (CBT), Department of Botany, University of Kashmir, Srinagar under voucher specimen no. 2304-KASH. A sample specimen of collected material was deposited in herbarium for future reference. The plant grows in abundance in this region the plant grows in abundance in this region.

Preparation of plant extract and other fractions

Aerial parts of S. laetus were air dried, powdered and passed through sieve no. 40 and subjected to extraction with Soxhlet extraction unit using methanol as solvent. Extract was filtered, evaporated to dryness using rotary evaporator at 40 °C and subjected to fractionation using various solvents viz. hexane, dichloromethane, and ethyl acetate.

These solvents were selected for extraction as they represent a gradient from non-polar to moderately polar. Also, polarity gradient allows for sequential extraction, where each solvent selectively extracts compounds based on their polarity. Polar compounds dissolve more readily in ethyl acetate while nonpolar compounds dissolve better in hexane. Fractions obtained were concentrated using rotary evaporator and stored in amber colored glass vials for further studies.

Phytochemical analysis

A stock concentration of 1% (w/v) of each fraction was prepared using methanol as solvent for qualitative assessment of different phytochemical constituents like alkaloids, flavonoids, glycosides, phenols, proteins, amino acids, saponins, sterols, tannins, anthraquinone glycosides and reducing sugar by following the standard procedure [17].

GC-MS analysis

The hexane, dichloromethane, and ethyl acetate fractions of SL Edgew were subjected to GC-MS analysis. A capillary column (30 m 0.25 mm 0.25 m) with a FID detector and an gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HHP-5MS phenylmethylsiloxane 5% was used for the GCMS. The oven’s temperature was first kept at 70 °C for one minute. Next, it was gradually raised to 180 °C at a pace of 6 °C per minute for five minutes, and finally, it was raised to 280 °C at a rate of 5 °C per minute for twenty minutes. Injector and detector temperatures were set at 220 °C and 290 °C, respectively. The carrier gas employed was helium, which flowed at a rate of 1 millilitre per minute [18].

Antioxidant activity

Concentrations of 20, 40, 80, 160, and 320 µg/mL of hexane, dichloromethane, ethyl acetate, and methanol extracts were used to determine in vitro antioxidant activity.

2,2-Diphenyl-1-picrylhydrazyl (DPPH)

Free radical scavenging capacity or electron donating ability of different extracts of SL Edgew. was determined using DPPH [19]. DPPH solution (0.004%) was freshly prepared in 95% ethanol. Different concentrations (20, 40, 80, 160 & 320 µg/mL) of extracts (hexane, dichloromethane, ethyl acetate and methanol) were prepared & 0.1 mL of each were mixed with 1.0 mL of DPPH solution and standard ascorbic acid solution separately. The reaction mixture was incubated for 30 min in dark at room temperature and absorbance was measured at 517 nm.

Ferric ion reducing antioxidant power (FRAP) assay

The reducing power was determined according to the method of [20]. Aliquots (2.5 mL) of different concentrations of extracts viz., hexane, dichloromethane, ethyl acetate and methanol ranging from (100–600 µg/mL) were mixed with 2.5 mL phosphate buffer (0.2 M, pH = 6.6) and 2.5 mL potassium ferricyanide (1% w/v), followed by incubation at 50 °C for 20 min in dark. After incubation, 2.5 mL of trichloroacetic acid (TCA) (10% w/v) was added to terminate the reaction and the mixture was subjected to centrifugation at 3000 rpm for 10 min. The upper layer (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL (0.1% w/v) ferric chloride. The reaction mixture was incubated for 10 min at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution. The assay was run in triplicate. Ascorbic acid as positive control was also tested for the reducing power assay in similar manner.

Nitric oxide radical inhibition assay

The nitric oxide scavenging activity of hexane, dichloromethane, ethyl acetate and methanol extracts was determined according to the standard method [21]. In this assay, the solution of 2 mL sodium nitroprusside (SNP) (10 mM) in 0.5 mL phosphate buffer saline (PBS, pH = 7.4) was mixed with 0.5 ml different concentrations of extracts (100–500 µg/mL), and the mixture was incubated at 37°C for 60 min in light. The half quantity of aliquots was taken and mixed with equal quantity of Griess reagent (0.05% N-(1-naphthyl) ethylenediamine dihydrochloride + 0.5% sulfanilic acid + 2.5% phosphoric acid) and the mixture was incubated at 25⁰C for 30 min in dark. The absorbance of pink colour chromophore, generated during diazotization of nitric ions with sulphanilamide and subsequent coupling with naphthyl ethylene diaminedihydrochloride was measured at 546 nm against a blank sample. All the tests were performed in triplicate. Ascorbic acid was used as the reference compound in same concentration range. The percentage nitrite radical scavenging activity of various extracts were calculated.

Estimation of total phenolic content

The total phenolic content of the extracts was determined by the Folin–Ciocalteu method [22]. 200 µL of extract (1 mg/mL) was made up to 3 mL with distilled water and mixed thoroughly with 0.5 mL of Folin–Ciocalteu reagent for 3 min, followed by the addition of 2 mL 20% (w/v) sodium carbonate. The mixture was allowed to stand for 60 min in the dark and absorbance was measured at 650 nm. The total phenolic content was calculated from the calibration curve and expressed as mg/g Gallic acid equivalent (GAE) of dry matter.

Determination of total flavonoid content

The total flavonoid content of extracts was determined by the aluminum chloride colorimetric method, using quercetin as standard. The assay was conducted using 0.5 mL of each extract stock solution and each dilution of standard quercetin taken separately in test tubes. To each test tube 1.5 mL methanol, 0.1 mL aluminum chloride solution, 0.1 mL potassium acetate solution and 2.8 mL distilled water were added and mixed well. Sample blank for all the dilution of standard quercetin and all the extracts were prepared in similar manner by replacing aluminum chloride solution with distilled water. All the prepared solutions were filtered through Whatman filter paper before measuring their absorbance. Absorbance was taken at 415 nm against the suitable blank method [23]. Total flavonoid content was expressed as mg of quercetin equivalents (Q)/g of extract.

Antiproliferative activity

Culturing of cell lines

Cell lines MCF-7 (breast carcinoma) and HCT-116 (colon carcinoma) were procured from Hybridoma Laboratory, National Institute of Immunology, New Delhi. Cells were maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, 100U/mL penicillin and100 mg/mL streptomycin and incubated in a humidified atmosphere of 50 µg/mL CO₂ at 37 °C. The medium was changed every two days or until the cells became confluent and were then used for the experimentation.

Methylthiazolyl diphenyl-tetrazolium bromide (MTT) assay

MTT assay was used as a standard test for determining the effect of various extracts of SL on cell proliferation. Hexane, dichloromethane and ethyl acetate fractions of SL were dissolved in DMSO (dimethyl sulphoxide), filtered, sterilized and then further diluted to attain required concentrations. Cell suspensions containing 2 × 10⁴ cells per well were seeded into 96 well microtiter plate. After 24 h of cell seeding, HCT116 and MCF-7 cells were treated with hexane, dichloromethane, and ethyl acetate extracts at concentrations of 1.9 µg/mL, 3.9 µg/mL, 7.825 µg/mL, 15.625 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, and 500 µg/mL. Control cells were treated with DMSO alone. Each concentration was tested in triplicate. The cells were incubated at 37 °C in a humidified incubator with 5% CO₂ for 48 h. MTT solution was added to the cells at 0.1 mg/mL concentration followed by incubation for 2 h at 37 °C in dark. The supernatant was removed and an equal volume of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 590 nm with a UV-Spectrophotometer. The effect of the samples on the proliferation inhibition on MCF-7and HCT-116 was expressed as the percentage inhibition [24].

Clonogenic assay

HCT-116 and MCF-7 cells were plated (3 × 10⁵ per well) in a 6-well plate and incubated overnight. Next day cells were treated with different concentrations of dichloromethane extract of SL. Following a 72-hour exposure to several doses of dichloromethane extract of SL, viable cells were counted and plated at a density of 200 cells per well on 6 well plate. After wards, cells were cultured in a humidified 5% CO₂ atmosphere for 14 days at 37 °C. The media was then eliminated and cells were given two washings with PBS. The colonies were fixed for ten minutes using 95% ethanol. 0.1% crystal violet was used to stain cells for ten minutes. After ten minutes, plate was rinsed three times with water and colonies were counted [25].

Wound healing assay

MCF-7 and HCT − 116 cells were seeded in 6 well plate at a concentration of 5.5 × 10⁵ in each well. A monolayer of cells was formed after incubating the cells overnight at 37 °C with 5% CO₂. A sterile 200 µl microtip was used to make a scratch in the center of each well to establish a wound. PBS washing was done to wash off detached cells. Following the removal of detached cells, cells were exposed to different concentrations of dichloromethane extract of SL for 24 h, no treatment was given to control group. The cells were analyzed under inverted microscope and photographs were taken at 0 and 24 h [26].

Statistical analysis

Data were analyzed by Sophisticated piece of software by social scientists (SPSS), version 20. Descriptive statistics in the form of frequency distribution and associated percentage were employed for the presentation of demographic details. Comparison between the hemodynamic values of two drugs was analyzed by Student’s t-test and paired t-test (from baseline to 5 min) within each group with confidence interval of 95%. p-values at < 0.05 were taken as significant.

Results

Phytochemical analysis and preliminary screening

The percentage yield of crude extracts of aerial parts of SL Edgew hexane, dichloromethane, ethyl acetate, and methanol was 7.54%, 11.3%, 10.26% and 24.11% respectively. Preliminary screening of extracts showed presence of alkaloids, flavonoids, carbohydrates, glycosides (cardiac and anthraquinone), tannins, triterpenoids, phenolic compounds & phytosterols (Table 1).

Table 1 Preliminary phytochemical screening of various extracts of aerial parts of SL Edgew

GC-MS analysis

The hexane, dichloromethane and ethyl acetate fractions were analyzed by GC-MS respectively. Tables 2, 3 and 4 and supplementary Figs. 1–3 (Figures S1-S3) show the constituents obtained from hexane, dichloromethane and ethyl acetate fractions of the plant respectively, with retention time and percent composition.

Table 2 GC-MS of hexane fraction

Table 3 GC-MS of dichloromethane fraction

Table 4 GC-MS of ethyl acetate fraction

In-vitro antioxidant activity

The reducing power capability of various extracts using ascorbic acid as standard was found to be appreciable and concentration dependent (Fig. 1).

Fig. 1

Reducing power ability of different extracts using DPPH assay. All values are expressed as mean ± SEM of triplicates. Data was analyzed by two-way ANOVA followed by Tukey’s’ comparisons’ test. Comparison is done with ascorbic acid; P value was found to be p < 0.0001

However, ethyl acetate fraction exhibited higher activity than other fractions. The antioxidant potential was also evaluated by FRAP assay and results are shown in Fig. 2.

Fig. 2

Results of FRAP assay. All values are expressed as mean ± SEM of triplicates. Data was analyzed by two-way ANOVA followed by Tukey’s’ comparisons’ test. Comparison is done with ascorbic acid; P value was found to be p < 0.0001

The nitric oxide radical scavenging activity shown by different extracts was also found to be concentration dependent. With increase in concentration the radical scavenging activity increased. Hexane fraction showed less activity than other three extracts which exhibited almost similar results (Fig. 3).

Fig. 3

Results of nitric oxide radical scavenging activity shown by different extracts. All values are expressed as mean ± SEM of triplicates. Data was analyzed by two-way ANOVA followed by Tukey’s’ comparisons’ test. Comparison is done with ascorbic acid; P value was found to be p < 0.0001

Antiproliferative activity of different extracts of aerial parts of SL Edgew

MTT assay results revealed that the hexane, dichloromethane, and ethyl acetate extract of aerial parts of SL Edgew decreased the percent viability of all the cells but to different extent. The antiproliferative activity shown by different extracts was found to be concentration dependent i.e., with increase in concentration antiproliferative action increased (Figs. 4 and 5).

Fig. 4

Antiproliferative activity of aerial parts of SL Edgew against HCT-116 cell lines. All values are expressed as mean ± SEM of triplicates. Data was analysed by two-way ANOVA followed by Tukey’s’ multiple comparisons’ test. P value = p < 0.0001

Fig. 5

Antiproliferative activity of aerial parts of SL Edgew against MCF-7 cell lines. All values are expressed as mean ± SEM of triplicates. Data was analyzed by two-way ANOVA followed by Tukey’s’ multiple comparisons’ test. P value = p < 0.0001

The IC₅₀ values of hexane, dichloromethane, and ethyl acetate against MCF-7 & HCT-116 cancer cells lines is presented in (Figure S4). Dichloromethane fraction showed the maximum activity and was therefore used for further evaluation by other methods.

Dichloromethane fraction showed the maximum activity and was therefore used for further evaluation by other methods.

Effect on colony formation of MCF-7 and HCT-116 cells

The antiproliferative potential of dichloromethane extract of SL on HCT-116 and MCF-7 cells was further investigated by colony formation assay. Cancer cells have an important property of growing in colonies by encountering adjacent cells. The cancer cells die when this connection is lost. DCM extract of SL reduced the number of colonies in a dose dependent manner in both HCT-116 and MCF-7 cancer cells Fig. 6 (a & b) respectively.

Fig. 6

(a) Inhibition of colony formation of HCT-116 cells in presence of 5 µg/mL, 10 µg/mL and 15 µg/mL of dichloromethane extract of SL or in absence of drug (control). (b) Dichloromethane extract of SL inhibit colony formation of MCF-7 cells at given concentrations compared to control group

The reduction in colony formation for HCT-116 was found to be 102, 43, and 29% at 5 µg/mL, 10 µg/mL and 15 µg/mL, respectively when compared to control having 255 colonies as shown in Fig. 7(a) and for MCF-7 the reduction in colony formation was found to be 45, 28 and 21% at 4 µg/ml, 8 µg/mL and 12 µg/mL, respectively as shown in Fig. 7(b).

Fig. 7

(a) Bar graph shows the number of colonies of HCT-116 at indicated concentrations. (b) Bar graph shows the number of colonies of MCF − 7 at indicated concentrations

Inhibition of migration in HCT-116 and MCF-7 cells

The central characteristic feature of cancer metastasis is migration, the inhibitory effect of dichloromethane fraction of SL on migration of HCT-116 and MCF-7 cells was evaluated using wound healing assay. Dichloromethane extract treatment at 4, 8 and 12 µg/mL significantly inhibited migration of MCF-7 cells when compared to control Fig. 8(a). Dichloromethane fraction (4, 8 and 12 µg/mL) showed wound closure of 28%,18% and 14% after 24 h Fig. 8(b).

Fig. 8

(a) Representative images taken at 0 and 24 h for the wound-healing assay of MCF-7 cells. (b) Wound closure (%) in MCF-7 cells upon treatment with different concentrations of dichloromethane extract of SL in wound healing migration assay

In case of HCT-116, dichloromethane fraction at 5, 10 and 15 µg/mL inhibited migration of cells as shown in Fig. 9(a) and % wound closure at 5, 10 and 15 µg/mL was found to be 45%, 28% and 21% as shown in Fig. 9(b).

Fig. 9

(a) Representative images taken at 0 and 24 h for the wound-healing assay of HCT-116 cells. (b) Wound closure (%) in HCT-116 cells upon treatment with different concentrations of DCM extract of SL in wound healing migration assay

Discussion

In recent years, the use of herbal medicines in cancer treatment has received increasing attention due to their varied phyto-metabolic contents with multiple biological activities. Medicinal plants are of great importance to the health of individuals [27, 28]. The wide variety of plant species contributes to the production of numerous secondary metabolites, which play a crucial role in the development of novel medicines. These metabolites are categorized into groups such as aromatic compounds, glycosides, flavonoids, alkaloids, and terpenoids [29]. Many secondary metabolites themselves have shown potential as antiproliferative agents, leading to the development of new drugs with distinct mechanisms of action against cancer. Several of these compounds have already been successful in the pharmaceutical industry. During the 20th century, significant progress was made in the exploration of natural products for oncology, leading to the discovery of substances that are now integral to cancer treatment. Discoveries like the vinblastine and vincristine (secondary metabolites) from Catharanthus roseus (L.) have acted as a catalyst for further research in this field [29, 30].

Phytochemical analysis conducted on SL has revealed the presence of diverse constituents which are known to exhibit medicinal as well as physiological activities [31]. In the study the phytochemical analysis of the herb revealed the presence of carbohydrates, phenols, tannins, flavonoids, saponins, glycosides, terpenoids and alkaloids, with significant phenolic and flavonoid content which might be responsible for its excellent antioxidant activity.

GC-MS analysis revealed the presence of 54 constituents from all the three extracts (hexane, dichloromethane and ethyl acetate) of aerial parts of SL Edgew. The identified constituents possess some important biological potential for future drug development. The compound cinnamic acid (2-propenoic acid, 3-phenyl) has previously been reported for antiproliferative activity [31]. 2-Propenoic acid, 3 phenyl-methyl ester (methyl cinnamate) has vasorelaxant property [32]. Hexadecanoic acid is used as antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant, antiandrogenic, hemolytic, 5-alpha reductase inhibitor, and antipsychotic. It is also used in muscle weakness, tetany, anemia, diarrhea, pulmonary edema, respiratory failure [33]. 9,12.15-octadecatrienoic acid, methyl ester is reported to have antiarthritic, antiproliferative, hepatoprotective, diuretic, antimicrobial, antiasthmathic activity [34]. Nonacosane is used in tetany, anemia, and pulmonary edema. Octadecanoic acid is reported to have antimicrobial activity. The compounds highlighted in Table 3 such as vitamin E [35], stigmasta-5,22-dien-3-ol [36], lup-20(29)-en-3-one [37] and betulinaldehyde [38] have been evaluated for anticancer activity using different experimental models. Thus, either any of these compounds cinnamic acid, 2-propenoic acid, 3-phenyl, betulinaldehyde, hexadecanoic acid, vitamin E, 9,12.15-octadecatrienoic acid, stigmasta-5,22-dien-3-ol, (3á,22e), Lup-20(29)-en-3-one either alone or in conjunction with other compounds may be responsible for the anti-proliferative, anti-cancer and antioxidant activity.

Antioxidant properties especially radical scavenging activities are very important due to the deleterious role of free radicals in biological systems. The DPPH radical has been widely accepted as a tool for estimating free radical scavenging activities of various compounds and plant extracts. The extract showed a profound capacity to scavenge free radicals. The activity was comparable to known standard antioxidant (ascorbic acid) indicating higher content of antioxidants present in the extracts. The anti-oxidant potential was found to be dose dependent with the maximum effect at 320 µg/mL. The anti-oxidant activity can be attributed to redox properties of phenolic or flavonoid compounds present in the extracts which play an important role in absorbing and neutralizing free radicals and hence terminating the free radical chain reaction.

The reducing capacity of the extracts, another significant indicator of antioxidant activity, was also found to be appreciable for all the four extracts. In the reducing power assay, yellow colour of the test solution changes to green depending upon the reducing power of the test solution. Presence of antioxidant in the solution causes the reduction of Fe³⁺ to Fe²⁺ by donating the electron. The amount of Fe²⁺ complex can then be monitored by measuring the formation of pearl’s blue at 700 nm. Increasing absorbance indicates an increase in reductive ability. The results showed that there was an increase in the reducing power of plant extract as the concentration increased.

Nitric oxide scavenging activity is another parameter for the evaluation of antioxidant activity. The compound, SNP is known to decompose in aqueous solution at physiological pH (7.2) producing nitric oxide. Under aerobic concentration, nitric oxide reacts with oxygen to produce nitrate and nitrite. This leads to reduction of nitrite concentration in the assay media. All the fractions showed significant scavenging activity with ethyl acetate fraction showing highest nitric oxide scavenging activity of 73.213% at a concentration of 500 µg/mL using ascorbic acid as standard.

In this study, we first performed MTT assay to find out the most effective fraction. Dichloromethane fraction showed significant activity in inhibiting the growth of both cancer cell lines, MCF-7 (IC₅₀ 5.74 µg/mL) and HCT116 (IC₅₀ 9.2 µg/mL), in a dose dependent manner. Further assays were then carried out on this most active fraction i.e. dichloromethane fraction. Dichloromethane fraction of SL was able to inhibit various markers associated with cancer genesis including migration, proliferation, metastasis and colony formation. During metastasis, tumor cells with epithelial phenotype move away from original tumour and invade the other organs by moving through lymph and blood. Their clonogenic survival determines the invasiveness of the cancer cells to reach different tissues. The results indicate the presence of promising antiproliferative agents in the fraction. In a dose dependent manner, dichloromethane fraction significantly decreased the migration in the wound healing assay and cologenic survival in both MCF-7 and HCT-116 cancer cells. The results obtained suggest that SL not only inhibits viability of cancer cells but also migration capacities.

Till date, the plant has been evaluated only for few activities viz., antimicrobial, antifungal and insecticidal. This is a first attempt to evaluate it for antiproliferative activity. Our results demonstrate that dichloromethane fraction of aerial parts of SL exhibit selective antiproliferative activity against both the cancer cell lines. The potent antioxidant nature of the extract adds to its anticarcinogenic potential suggesting a promising role in development of antiproliferative therapeutics in future. However, these findings warrant extensive studies to evaluate mechanistic action of antiproliferative and antioxidant principles of the plant.

Conclusion

We conclude that SL Edgew contains a wide variety of secondary metabolites (54 compounds) of which cinnamic acid (2-propenoic acid, 3-phenyl) and 9,12.15-octadecatrienoic acid, methyl ester, and betulinaldehyde have been reported to possess antiproliferative and anti-tumor activity. Hexadecanoic acid and vitamin E are used as antioxidant agents. Presence of these important phytoconstituents may in part explain the antioxidant and antiproliferative action of the herb. However, further studies are required to investigate and isolate the lead compounds present in the plant which can be tested in various in vivo models for authentication of their effectiveness by studying their mechanism of action.