Phytochemistry, Bioactivities, Pharmacokinetics and Toxicity Prediction of Selaginella repanda with Its Anticancer Potential against Human Lung, Breast and Colorectal Carcinoma Cell Lines

In this study, we investigated the bioactive potential (antibacterial and antioxidant), anticancer activity and detailed phytochemical analysis of Selaginella repanda (S. repanda) ethanolic crude extract for the very first time using different in vitro approaches. Furthermore, computer-aided prediction of pharmacokinetic properties and safety profile of the identified phytoconstituents were also employed in order to provide some useful insights for drug discovery. S. repanda, which is a rich source of potent natural bioactive compounds, showed promising antibacterial activity against the tested pathogenic bacteria (S. aureus, P. aeruginosa, E. coli and S. flexneri). The crude extract displayed favorable antioxidant activity against both 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50 = 231.6 μg/mL) and H2O2 (IC50 = 288.3 μg/mL) molecules. S. repanda also showed favorable and effective anticancer activity against all three malignant cancer cells in a dose/time dependent manner. Higher activity was found against lung (A549) (IC50 = 341.1 μg/mL), followed by colon (HCT-116) (IC50 = 378.8 μg/mL) and breast (MCF-7) (IC50 = 428.3 μg/mL) cancer cells. High resolution-liquid chromatography–mass spectrometry (HR-LC–MS) data of S. repanda crude extract revealed the presence of diverse bioactive/chemical components, including fatty acids, alcohol, sugar, flavonoids, alkaloids, terpenoids, coumarins and phenolics, which can be the basis and major cause for its bioactive potential. Therefore, achieved results from this study confirmed the efficacy of S. repanda and a prospective source of naturally active biomolecules with antibacterial, antioxidant and anticancer potential. These phytocompounds alone with their favorable pharmacokinetics profile suggests good lead and efficiency of S. repanda with no toxicity risks. Finally, further in vivo experimental investigations can be promoted as probable candidates for various therapeutic functions, drug discovery and development.


Results and Discussion
Medicinal plants have been utilized chiefly as ethnic remedies against various diseases since ancient time. They are extensively contemplated as a source of novel phytochemicals with bioactive potential. In order to treat a variety of diseases, these phytomedicines can also lead in drug discoveries or itself become a potential drug. Hence, it is very essential to determine and estimate the bioactive potential of ethnomedicinal plants by particularizing the phytochemistry and developing them as a potent source for therapeutic agents [19][20][21]. Pteridophytes ethnomedicinal nature makes them as a traditional medicine to cure several complicated health conditions [22][23][24]. Therefore, in the presented work, we evaluated the antibacterial, antioxidant and anticancer properties of S. repanda.

Antibacterial Potential of S. repanda
In recent times, almost all developing and developed countries are going through with a global problem of antibiotic resistance, which is continuously challenging the global healthcare sector. Multidrug-resistant pathogens are emerging and spreading in such a way that it continues to diminish the current antimicrobial therapies. This demands a search for novel antimicrobial substances, preferably from natural sources. Plants are one such example consisting of a variety of bioactive compounds with known therapeutic properties [3]. The microdilution method was used to study the antagonistic potential of S. repanda crude extract against pathogenic bacterial strains S. flexneri, S. aureus, E. coli and P. aeruginosa. Results revealed that S. repanda showed significant antagonistic activity against all the four tested bacterial strains, and are presented in Table 1. The antimicrobial activity of different species of Selaginella was evaluated against various human pathogenic bacteria. The crude extract of S. bryopteris was reported for its antibacterial activity against different Gram-negative and Gram-positive bacteria such as E. coli, E. faecalis, C. tropicalis, S. aureus, C. albicans, C. krusei and K. pneumonia [25]. S. equalifolia and S. involvens has been reported to show antimicrobial activity against different poultry pathogens namely, Klebsiella, Salmonella, Staphylococcus, Proteus and Bacillus [26]. S. inaequalifolia showed good antagonistic activity against S. aureus, E. coli and C. albicans [27]. S. convoluta showed significant antibacterial potential against B. cereus, E. coli, S. enterica, S. marcescens, K. pneumoniae, S. flexneri, E. faecalis and S. aureus [28]. S. tamariscina is known to possess potent activity against oral bacterial pathogens such as P. gingivalis, P. intermedia, S. mutans, S. sobrinus, S. gordonii, F. nucleatum, S. sanguinis, S. anginosus, S. ratti, A. actinomycetemcomitans, S. parasanguinis, S. criceti and S. downei [29]. Our study is also in line with previous antibacterial studies and showed that S. repanda possess antibacterial potential against various human pathogenic bacteria.

Antioxidant Potential of S. repanda
Free radicals are naturally formed in our body and perform many cellular processes [30]. At a higher concentration, free radicals can be dangerous and can damage the important constituents of the cell, which may include cell membrane, proteins, DNA, etc. Damage to DNA, in particular, may lead to cancer or other severe health conditions. However, in order to prevent this type of cellular damage, antioxidants play an essential role. They are generally described by "mopping up" free radicals, which means they are able to neutralize the electrical charge and can prevent the free radical from receiving electrons from other molecules [30]. It has been reported that plant extract or naturally extracted compounds having antioxidant activity, exhibits anticancer activity in a defined mechanism.
S. repanda antioxidant potential was analyzed against DPPH and H 2 O 2 molecules in comparison to ascorbic acid. The results of antioxidant activity displayed that S. repanda crude extract exhibited remarkable free radical scavenging activity, both against DPPH and H 2 O 2 molecules in a dose dependent manner. The scavenging capacity against DPPH free radicals was higher than H 2 O 2 molecules. The IC 50 values of crude extract of S. repanda for DPPH and H 2 O 2 were 231.6 µg/mL and 288.3 µg/mL, respectively ( Figure 1A,B). The antioxidant potential of different species of Selaginella have been evaluated by different methods; from which, most are based on the determination of free radical scavenging activity. Commonly used methods are, ABTS, DPPH, superoxide anion radical scavenging assays and total phenolic content. Total phenolic content of S. repanda dried powder was found to be 6.51 ± 0.71 mg gallic acid equivalents/g dry matter. Indian Sanjeevani (S. bryopteris) is well known for its protective effect against various stress-induced conditions [31]. The crude extract of S. tamariscina have strong antioxidant property, as its extract can reduce blood sugar levels and also able to act as a lipid peroxide and increases insulin serum [32]. In one study, the aqueous extract of S. involvens, S. delicatula and S. wightii also displayed in vitro lipid peroxidation and varying levels of hydroxyl radical scavenging activity. The 50% inhibition (EC 50 ) for in vitro lipid peroxidation of S. wightii, S. delicatula and S. involvens was 76.6 ± 4, 38.2 ± 1.2 and 2.1 ± 0.1, respectively. Moreover, flavonoids obtained from the S. doederleinii also possess very strong free radical scavenging activity [33]. Similar results were found in our study. S. repanda displayed potent antioxidant activity, indicating its protective role against free radicals. found to be 6.51 ± 0.71 mg gallic acid equivalents/g dry matter. Indian Sanjeevani (S. bryopteris) is well known for its protective effect against various stress-induced conditions [31]. The crude extract of S. tamariscina have strong antioxidant property, as its extract can reduce blood sugar levels and also able to act as a lipid peroxide and increases insulin serum [32]. In one study, the aqueous extract of S. involvens, S. delicatula and S. wightii also displayed in vitro lipid peroxidation and varying levels of hydroxyl radical scavenging activity. The 50% inhibition (EC50) for in vitro lipid peroxidation of S. wightii, S. delicatula and S. involvens was 76.6 ± 4, 38.2 ± 1.2 and 2.1 ± 0.1, respectively. Moreover, flavonoids obtained from the S. doederleinii also possess very strong free radical scavenging activity [33]. Similar results were found in our study. S. repanda displayed potent antioxidant activity, indicating its protective role against free radicals.

Anticancer Potential of S. repanda
Cancer is the main health burden in developing and developed countries, as it is the second leading cause of deaths globally. Different types of treatments are available for the therapy, depending on the stage of cancer and type. Radiotherapy and surgery are efficacious in the treatment of cancer at the earlier stage, while chemotherapy was used when

Anticancer Potential of S. repanda
Cancer is the main health burden in developing and developed countries, as it is the second leading cause of deaths globally. Different types of treatments are available for the therapy, depending on the stage of cancer and type. Radiotherapy and surgery are efficacious in the treatment of cancer at the earlier stage, while chemotherapy was used when the tumor reaches the stage of metastasis. However, the high cost of treatments is associated with the side effects, which accounted for the use of herbal products for the treatment of different types of cancers. Over the past decade, natural products perceived high attention because of their capability as novel therapeutic and preventive agents. About 60% of all orthodox anticancer drugs are directly or indirectly derived from plants [34], which imply that medicinal plants have the ability in leading to the discovery of novel drugs.
Selaginella species have different groups of chemical compounds, which are well known to have a wide-range of biological actions and are a potential source for finding the novel anticancer drugs. As many of the phytochemical compounds obtained from few of these species possess strong cytotoxic activity against various cancer cell lines. S. repanda crude extract was evaluated for its anticancer potential by MTT assay against three different human cancer cell lines, i.e., breast (MCF-7), colon (HCT-116) and lung (A549). Our results revealed that, proliferation of all the three cell lines were inhibited by S. repanda in a dose dependent manner, compared to fluorouracil, which was used as the positive control. The crude extract of S. repanda showed the highest growth inhibitory activity against A549 cells with IC 50 value of 341.1 µg/mL, followed by HCT-116 cells (378.8 µg/mL) and MCF-7 cells (428.3 µg/mL) (Figure 2A). Cytotoxicity test of S. repanda crude extract against noncancerous human normal colon cells (CRL-1831) was also performed and results revealed no cytotoxicity to normal cells ( Figure 2B). the tumor reaches the stage of metastasis. However, the high cost of treatments is associated with the side effects, which accounted for the use of herbal products for the treatment of different types of cancers. Over the past decade, natural products perceived high attention because of their capability as novel therapeutic and preventive agents. About 60% of all orthodox anticancer drugs are directly or indirectly derived from plants [34], which imply that medicinal plants have the ability in leading to the discovery of novel drugs.
Selaginella species have different groups of chemical compounds, which are well known to have a wide-range of biological actions and are a potential source for finding the novel anticancer drugs. As many of the phytochemical compounds obtained from few of these species possess strong cytotoxic activity against various cancer cell lines. S. repanda crude extract was evaluated for its anticancer potential by MTT assay against three different human cancer cell lines, i.e., breast (MCF-7), colon (HCT-116) and lung (A549). Our results revealed that, proliferation of all the three cell lines were inhibited by S. repanda in a dose dependent manner, compared to fluorouracil, which was used as the positive control. The crude extract of S. repanda showed the highest growth inhibitory activity against A549 cells with IC50 value of 341.1 μg/mL, followed by HCT-116 cells (378.8 μg/mL) and MCF-7 cells (428.3 μg/mL) (Figure 2A). Cytotoxicity test of S. repanda crude extract against non-cancerous human normal colon cells (CRL-1831) was also performed and results revealed no cytotoxicity to normal cells ( Figure 2B). One study revealed the anticancer effect of ethanolic and aqueous extracts of S. doederleinii using the brine shrimp lethality test against two cancer cell lines MDAMB231 (breast) and HepG2 (liver). As a result, after 24 h of exposure to 50% lethal concentration (LC 50 ) in the brine shrimp lethality test was found to be >1000 µg/mL. MDAMB231 and HepG2 were found to be the most susceptible with the treatments of ethanol (LC 50 = 306 µg/mL) and aqueous (LC 50 = 329 µg/mL) extracts of S. doederleinii, respectively [35]. The crude extract of S. tamariscina also showed potent anticancer activity against different cancer cell lines. It was found to decrease the metastasis, expression of MMP-2 and 9 (matrix metalloproteinase) and urokinase plasminogen activator in A549 cells and Lewis lung carcinoma [36]; inhibits nucleus antigen cell from stomach epithelium [37]; inhibits gastric cancer cells [37] and induce apoptosis via blockade of fatty acid synthesis in breast cancer [38]; inhibits leukemia cancer cells HL-60 and U937 [39];. Apart from this, amentoflavone was extracted from the crude extract of S. tamariscina and its anticancer efficacy was screened against five different cancer cells, including HL-60 (human leukemia cells), HeLa (human cervical carcinoma cells), PANC-1 (human pancreatic cancer cells), MCF-7 (human breast cancer cells) and BEL-7402 (human hepatoma carcinoma cells). The extract was found to be efficient in the inhibition of the proliferation of all cells with a remarkable inhibition of HL-60 [39].
The cytotoxic effect and apoptosis induction potential of hexane, methylene chloride, ethyl acetate and butanol extracts of S. plana was performed against MCF-7 cells. Different crude extracts of S. plana displayed inhibition of MCF-7 cells with an IC 50 value of 30 µg/mL, 19 µg/mL, 24 µg/mL and 2 µg/mL respectively. Butanol crude extract was found as the highest cytotoxic and apoptotic induction against MCF-7 cancer cells [40]. The cytotoxic and apoptosis activity of three different crude extracts (ethyl acetate, ethanol and aqueous) of S. uncinata, S. tamariscina, S. remotifolia, S. delicatula, S. moellendorfii, S. pulvinata and S. labordei were evaluated using BEL-7402, HT-29 and HeLa cells. In results, S. labordei, S. tamariscina and S. uncinata had a higher inhibition of Bel-7402 and HeLa cells, whereas, S. moellendorfii had moderate inhibition, but S. remotifolia and S. pulvinata had almost no inhibitory activities. The major bioactive compounds responsible for the inhibition for cancer cells were bioflavonoids, detected in the ethyl acetate extracts. Moreover, the efficacy of all three extracts of all the plants on cell inhibition and apoptosis were not the same, they were highly efficient on HeLa cells than HT-29 cells [41]. Our results also revealed similar data, S. repanda possessing anticancer potential against three malignant lineages, HCT-116, MCF-7 and A549 cell lines. Though, all the three cell lines have high metastatic potential, S. repanda was found to inhibit the proliferation of all three malignant cells in a time and concentration dependent manner.

Identification of Phytochemical Compounds of S. repanda Using HR-LC-MS
Different classes of phytochemicals are omnipresent in plants and have been linked with diverse biological activities. This study revealed the presence of diverse groups of chemical compounds, which are known to possess a broad range of bioactivities. After the evaluation of antibacterial, antioxidant and anticancer activities, a comprehensive phytochemical analysis from crude extract of S. repanda was carried out via UHPLC-PDA-ESI-MS/MS. Chromatogram was obtained with both positive and negative run and different types of phytochemicals were identified ( Figure 3). Different classes of metabolites such as sugars, amino acids, vitamins, alkaloids, flavonoids, terpenoids, phenols, etc., were detected (Figures 4-6).
Flavonoids and natural phenolic acids are one of the most prevalent and pharmacologically active groups of plant secondary metabolites. In this study, S. repanda crude extract revealed the presence of phenols like coumarin, chlorogenic acid, 7-hydroxycoumarine, caffeic acid, 4-methoxycinnamic acid, isoferulic acid, formononetin, 2-amino-1,3,4-octadecanetriol and 4-coumaric acid. The other main flavonoids identified in the present study were rutin, vitexin, quercetin, quercetin-3β-D-glucoside, kaempferol, apigenin, luteolin, rhamnetin, glycitein, diosmetin and genistein. They are well known for their potential therapeutic ap-plications including antioxidant, anti-inflammatory, anticancer, cardioprotective effects, etc. These compounds play a crucial role in prevention of cancer through various mechanisms at the molecular level. This may include impeding the signaling pathways, migration, differentiation and proliferation inhibition, gene regulation, carcinogen metabolism, induction of apoptosis via arresting cell cycle, etc. [42].          Alkaloids are nitrogenous compounds, which are widely distributed from prokaryotes to eukaryotes and are well-known for their different biological activities like anti-microbial, anti-HIV, anticancer and antiparasitic [43]. In the present study, 8-hydroxyquinoline, norharman, hordenine and guvacoline were identified. Two terpenoids, citral and pulegone were also identified from the crude extract of S. repanda. Terpenoids possess diverse biological activities including antimalarial, antioxidant, antibacterial, antiviral, anticancer and anti-inflammatory. They are also used for the treatment of various diseases [20]. Apart from this, coumarins, different amino acids, fatty acids, sugars and sugar alcohol were also detected. Detailed list of identified compounds with their mass (m/z), retention time and bioactivities are presented in Table 2.  Therefore, our data suggested that the presence of these diverse biomolecules in S. repanda crude extract is the major reason for its antibacterial, antioxidant and anticancer activities. Our results provide an opportunity to explore further this medicinally treasured pteridophyte. Moreover, for the first time we studied that the S. repanda crude extract had a strong dose dependent cytotoxicity against three malignant cancer cell lines (HCT-116, MCF-7 and A549). Further studies are needed in order to demonstrate the efficacy and potency of specific compound in the crude extract, which might be responsible for the particular biological activity.

Pharmacokinetic and Toxicity (ADMET) Profiles of Identified Phytoconstituents from S. repanda Ethanolic Crude Extract
It is well known that good drug activity may be destroyed due to inadequate AD-MET (absorption, distribution, metabolism, excretion and toxicity) properties. In addition, unwanted pharmacokinetics and toxicity are important reasons for the failure of drug discovery in the clinical phase, which is very costly. In order to decide, the possibility of S. repanda ethanolic crude extract to become or not become a good candidate for suitable drug, ADMET parameters have been assessed using in silico tools (Table 3). Interestingly, all identified phytoconstituents were found to meet the Lipinski's rule of five, and some of them such as hordenine, 4-coumaric acid and Diosmetin follows also Ghose, Veber and Egan filters with most of them displaying a good bioavailability score (55-85%). The solubility as an important feature for the absorption of the molecule and its distribution in the body given by the values of aqueous solubility indicates most of the compounds were highly soluble in water and the others are soluble.
To be a transdermal drug delivery, the skin permeability, which defines the rate of a chemical penetrating across the stratum corneum, will be checked to improve its efficacy. A molecule will perfectly penetrate the skin if log Kp was higher than −2.5 cm/h. All phytocompounds seem to possess moderate to good skin penetrability among them, those of scopoletin, sedanolide, (-)-Caryophyllene oxide and hordenine. The Caco-2 cell line is composed of human epithelial colorectal adenocarcinoma cells. Caco-2 permeability can predict the intake of oral drugs as Caco-2. All compounds displayed moderate to potent (log Papp values >0.90 cm/s) Caco-2 permeability values. Among them, scopoletin, sedanolide, (-)-Caryophyllene oxide and hordenine were predicted to have the strongest Caco-2 permeability. P-gp is a key to estimate active efflux through biological membranes and known as the most important member among ATP-binding cassette transporters or ABC-transporters used to protect the central nervous system (CNS) from xenobiotics. None of the selected phytocompounds were P-gp inhibitor/substrate. Intestinal absorption was also assessed and most of the identified compounds were highly absorbed by the intestine. The skin permeation as given by LogKP was predicted and results demonstrated that identified compounds might be moderately or highly promoted to penetrate through the skin, which confirmed their drug-like properties. Many of them such as Sedanolide, and (-)-Caryophyllene oxide with log BB (logarithm value of brain to plasma concentration ratio) greater than 0.3 were subjected to have more potential to cross brain blood barrier (BBB). Only fewer compounds like arachidonic acid, hexadecanamide and hordenine have the ability to penetrate to CNS. (-)-Caryophyllene oxide and hordenine are amongst the phytoconstituents that will be more distributed with distribution volume logVDss of 0.564 L/kg and 0.887 L/kg in the tissues, respectively.
Human cytochrome P450 (CYP) isoforms involved in drug metabolism in liver were also generated. Amongst them, CYP3A4 is the major and most clinically relevant drugmetabolizing enzyme in the human body. Its inhibition could lead to drug toxicity, drugdrug interactions and other adverse effects. Some of them were non-inhibitors/substrate of any isoenzymes like sedanolide, however as a good result, more than 92% were found to be non-inhibitors of CYP3A4, isoenzyme responsible for the metabolism of about 60% of xenobiotics including drugs, carcinogens, steroids and eicosanoids.
To determine the excretion routes, the total clearance (CLTOT) for both hepatic and renal and renal organic cation transporter 2 (OCT2) substrate as expressed in log ml/min/kg were predicted. Results showed that about 98% of the identified phytocompounds exhibited a positive total clearance values and can be easily excreted. The toxicity profile of all the identified phytoconstituents from S. repanda ethanolic crude extract has been predicted based on AMES toxicity, hepatotoxicity, hERG potassium channel inhibition and skin sensitization parameters. Results outlined that, out of the fifty four identified compounds, only three have a deviated mutagenic and hepatic toxicity potential, which means that about 95% are devoid of any risk of toxicity, 100% have no hERG I inhibition and 75% exerted no skin-sensitive effects.
To achieve more information for better bioavailability and drug-likeness of the identified compounds, we present an example of the top four suitable phytocompounds that can be delivered more as a potent candidate for drug discovery (Figure 7). The results of the bioavailability radar have been depicted by the lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25 and flexibility: no more than 9 rotatable bonds with the colored zone defined the desired physicochemical space for good oral bioavailability indicating that they possess good drug-likeness properties. tified compounds, we present an example of the top four suitable phytocompounds that can be delivered more as a potent candidate for drug discovery (Figure 7). The results of the bioavailability radar have been depicted by the lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å 2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25 and flexibility: no more than 9 rotatable bonds with the colored zone defined the desired physicochemical space for good oral bioavailability indicating that they possess good drug-likeness properties. An example of the BOILED-Egg model (Brain or IntestinaL EstimateD permeation method) (Figure 8) prediction of gastrointestinal (GI) absorption and BBB permeation of the same selected compounds have been exploited. Results shows that the compounds appeared with a red point in the yellow ellipse having high probability of brain penetration and are non-substrate of P-gp (PGP-).
An example of the BOILED-Egg model (Brain or IntestinaL EstimateD permeation method) (Figure 8) prediction of gastrointestinal (GI) absorption and BBB permeation of the same selected compounds have been exploited. Results shows that the compounds appeared with a red point in the yellow ellipse having high probability of brain penetration and are non-substrate of P-gp (PGP-). Figure 8. BOILED-Egg model of the volatile constituents using the Swiss ADME predictor.

Plant Collection, Storage and Sequence Deposition
S. repanda whole plant was collected from the wild regions of Gujarat state, India during the period of July-August 2020. The plant was identified by the molecular sequencing method, plus by its taxonomic characters ( Figure 9). The voucher specimen (BVBRC035) was deposited at Bapalal Vaidya Botanical Garden, Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India. rbcL gene nucleotide sequence (accession number MT795925) was deposited to NCBI. The whole plant was dried in an oven, followed by grinding into a fine powder and was then stored airtight containers. The percentage yield of the plant extract was calculated according to the following formula: Percentage yield of the extract = Weight of extract (g) Weight of plant material (g) × 100 (1) Figure 8. BOILED-Egg model of the volatile constituents using the Swiss ADME predictor.

Plant Collection, Storage and Sequence Deposition
S. repanda whole plant was collected from the wild regions of Gujarat state, India during the period of July-August 2020. The plant was identified by the molecular sequencing method, plus by its taxonomic characters ( Figure 9). The voucher specimen (BVBRC035) was deposited at bapalal vaidya botanical garden, department of biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India. rbcL gene nucleotide sequence (accession number MT795925) was deposited to NCBI. The whole plant was dried in an oven, followed by grinding into a fine powder and was then stored airtight containers. The percentage yield of the plant extract was calculated according to the following formula: Percentage yield of the extract = Weight of extract (g) Weight of plant material (g) × 100 (1)

S. repanda Crude Extraction
Ethanol (85%) was used for soaking of 20 g S. repanda powder for 24 h at 37 • C with vigorous shaking. Whatman no. 1 filter paper was used for filtering the ethanol phase mixture and then concentrated using a rotary evaporator to get the dried residue. Stock solution of crude ethanolic extract was then further used for performing various biological activities such as antibacterial, antioxidant and anticancer.

S. repanda Crude Extraction
Ethanol (85%) was used for soaking of 20 g S. repanda powder for 24 h at 37 °C vigorous shaking. Whatman no. 1 filter paper was used for filtering the ethanol p mixture and then concentrated using a rotary evaporator to get the dried residue. S solution of crude ethanolic extract was then further used for performing various biolo activities such as antibacterial, antioxidant and anticancer.

Microdilution Method
Antibacterial activity of S. repanda crude extract was performed in 96-well microtiter plates against pathogenic bacteria, as described previously [83]. The inoculums were prepared from a 6 h Muller-Hinton broth (MHB) culture, and suspensions were adjusted to 0.5 McFarland turbidity standards (10 8 CFU/mL). S. repanda crude extract was diluted to twofold ranging from 1000 to 0.48 µg/mL (80 µL as final volume) with a DMSO concentration >1%. Afterward, 20 µL of bacterial suspensions and 100 µL of MHB were loaded onto microtiter plates. Plates were then incubated at 37 • C for 24 h. At the end of the incubation period, microtiter plates were read using a spectrophotometer at 620 nm. Chloramphenicol, a standard antibiotic, was used as a positive control. MHB + DMSO was used as a vehicle control, and MHB alone was used as a sterility control. MIC was recorded as the plant extract with the lowest concentration and has shown absolute inhibition of observable growth [84].
3.4. Antioxidant Assays 3.4.1. Determination of Free Radical Scavenging Effects of Antioxidants Using the DPPH Method S. repanda crude extract antioxidant activity was measured against DPPH free radicals in relation to radical scavenging capability [85]. Crude extract of varied concentrations (100-500 µg/mL) were mixed in the tubes containing 2 mL of DPPH solution (6 × 10 −5 M) in dimethyl sulfoxide (DMSO), followed by dark incubation for 1 h. After incubation, a decrease in absorbance was measured at 517 nm. As a standard, ascorbic acid was used; as a blank, DMSO was used; and as a control, DPPH solution without crude extract was used. Calculation of the percentage of scavenging of DPPH free radicals was then estimated as follows: DPPH scavenging activity (%) = (A 0 − A 1 )/A 0 × 100 (2) whereas, A 0 = absorbance of the control (3a)  [19]. One milliliter of crude extract (100-500 µg/mL) was mixed with 1 mL of 2 mM H 2 O 2 solution, prepared in 0.1 M phosphate buffer (pH 7.4). The tubes were then incubated for 10 min at room temperature, and absorbance was measured at 230 nm. Absorbance was determined against a blank solution (phosphate buffer without H 2 O 2 ). Standard ascorbic acid was used as positive control. Calculation of the percentage of scavenging of H 2 O 2 was then estimated as follows: whereas, A 0 = absorbance of the control (5a) A 1 = absorbance of the extract/standard (5b)

Total Phenolic Content of S. repanda
The concentration of total phenolics in the extract was analyzed by the Folin-Ciocalteu colorimetric assay [86]. Firstly, crude extract (0.2 mL), deionized water (0.8 mL) and Folin-Ciocalteu reagent (0.1 mL) was incubated at room temperature for 3 min and then 0.3 mL of Na 2 CO 3 (20% w/v) was added and the mixture was incubated at room temperature for 2 h. Absorbance of the mixture was read at 765 nm. A standard curve of gallic acid from 0 to 100 mg/L was prepared. Total phenolic content was expressed in mg gallic acid equivalents/g dry matter.

Cytotoxicity and Anticancer Assay (MTT Assay)
Cytotoxicity of S. repanda crude extract was performed against human normal colon cells (CRL-1831) and anticancer potential was performed against human lung (A549), breast (MCF-7) and colon (HCT-116) cancer cell lines. Cells were obtained from the NCCS, India. They were propagated in a humidified (5% CO 2 ) atmosphere at 37 • C, and were maintained in 25 cm 2 flask containing DMEM supplemented with 10% FBS. They were grown up to 80% confluence and were seeded in 96-well plates at a density of more than 1 × 10 5 cells per well with incubation conditions mentioned above. Cells were then stained with 0.4% Trypan Blue and viable cell numbers were calculated using a hemocytometer. However, assay was performed as triplicate wells for each concentration. Different concentration of S. repanda crude extract (100-500 µg/mL) was then used to treat the cells for 24 h. Followed by washing with PBS solution and subjected with 100 µL of MTT solution (3-(4,5dimethylthiazolyl-2)-2,5 diphenyltetrazoliumbromide) (5 mg/mL), followed by incubation for 4 h. Finally, the medium was removed and 100 µL of DMSO was added to solubilize the formazan crystals. ELISA reader was then used to determine the amount of formazan crystal by measuring the absorbance at 570 nm. Fluorouracil (5FU) was used as a positive control. All assays were done in triplicate and 50% cytotoxic concentration (IC 50 ) was calculated [5].

HR-LC-MS Analysis
Phytochemistry of S. repanda crude extract was analyzed using UHPLC-PDA-Detector Mass Spectrophotometer (HR-L=CMS 1290 Infinity UHPLC System), Agilent Technologies ® , Santa Clara, CA, USA. The liquid chromatographic system consisted of the HiP sampler, binary gradient solvent pump, column compartment and quadrupole time of flight mass spectrometer (MS Q-TOF) with the dual Agilent Jet Stream Electrospray (AJS ES) ion source. Of the sample 10 µL was injected into the system, followed by separation in the SB-C18 column (2.1 mm × 50 mm, 1.8 µm particle size). Solvent A (1% formic acid in deionized water) and solvent B (acetonitrile) were used as solvents. Flow rate of 0.350 mL/min was used, while, MS detection was performed in MS Q-TOF. Compounds were identified via their mass spectra and their unique mass fragmentation patterns. Compound Discoverer 2.1, ChemSpider and PubChem were used as the main tools for the identification of the phytochemical constituents [5].

Statistical Analysis
Results are expressed as mean ± SD of the number of experiments performed. A Student's t-test for paired or unpaired values was performed and a p-value of <0.05 was considered statistically significant. Statistical analysis was conducted with software GraphPad Prism Version 7.03.

Conclusions
From this current work, it was concluded that S. repanda possessed strong antibacterial activity against different pathogenic bacterial strains of human interest, promised a source of antioxidant compounds and offered a worthy understanding about its anticancer potential. Moreover, S. repanda can also be used as a possible ingredient, nutraceutical or functional food, which requires further exploration, in vivo pharmacological and toxicological studies, to prove the unexplored beneficial aspects of S. repanda significant medicinal properties. In silico results indicated a good pharmacokinetic and safety profile of the phytochemical constituents, which make S. repanda ethanolic crude extract a potential drug candidate for the treatment of many diseases.