In Vitro Antioxidant and Antiproliferative E cacy of Abrus precatorius Seed Extracts On Hep2c Cells and Its Inhibitory E cacy With Receptors Of Cervical Carcinoma


 Abrus precatorius is a widely distributed tropical medicinal plant with several therapeutic properties, however its seed extract has not been studied against cervical cancer (CaCx) till date. Herein, we have assessed the antioxidant and antiproliferative properties of A. precatorius seed extracts (ethyl acetate and 70% ethanol) prepared from different extraction methods (Soxhlet and maceration) against human cervix carcinoma cells (Hep2C). We observed a significantly higher total flavonoid content of APE (sox) i.e.,112.7±0.127 mg Quercetin Equivalent/g of extract than others; total phenolic content of APA (mac) seed extract was higher i.e.,108.53±0.089 mg Quercetin Equivalent/g of extract and total tannin content of APA (sox) was higher i.e., 98.98± 0.011 mg tannic acid equivalents/g of extract. In addition, tannic acid, rutin and piperine were identified in extracts by HPLC. Furthermore, APA (sox) exhibited the highest radical scavenging activity with an IC50 value of 14.49±0.93µg/ml. APE (mac) showed the most significant antiproliferative activity with IC50 value of 85.90±0.93µg/mL against Hep2c cells. SOD and GST activity was observed as highest in the APA(Mac) extract whereas the catalase activity and GSH content was maximum in the APE(Mac) extract. Further, the MDA content was observed to be the least in APE(Mac) extracts. Docking results suggested maximum binding energy between tannic acid and Her2 receptor compared to doxorubicin (standard). This study provides evidence that A. precatorius seed extracts possess promising bioactive compounds with probable anticancer and antioxidant properties against CaCx which might be utilized as a possible herbal remedy aimed at restricting tumor growth.


Introduction
Cancer is one of the major causes of mortality in both men and women worldwide and its research has garnered attention from the scienti c fraternity all across the globe. Considering the global burden of gynecological cancers, cervical cancer (CaCx) among them ranks as the fourth most common cancer with nearly 5,70,000 new reported cases and 3,11,000 deaths [1]. Despite effective screening measures and treatment modalities, CaCx continues to hold the banner of the leading cause of cancer related mortality among women [2] . Therefore, the development of novel therapeutic drugs with increased e ciency is warranted. Receptors like Glucocorticoids (GCR), Human epidermal growth factor receptor 2 (HER2) and Beta-hexosaminidase subunit beta (HEXB) are reported to be associated with progression of CaCx and have been established as promising therapeutic candidates as corroborated by in-silico analysis [3][4][5] . Out of the 1881 new chemical entities, that had been approved as drugs between 1981 and 2019, the purely synthetic compounds amount for only 24.6%, while a majority of the new drugs were derived from medicinal plants [6]. Herbal plants have made important contributions to the development of anticancer drugs. Numerous natural substances are recognized to be anti-oxidants, cancer preventive agents or even antitumor agents such as paclitaxel [7].
Abrus precatorius L. (Fabaceae) is a plant that spans tropical and subtropical parts of the world. Lowly elevated, dry regions are conducive for its growth. Known for its medicinal value, its leaves, roots and seeds are often exploited for anti-helminthic, anti-diarrhoeal, neuroprotective, anti-dipression, anti-fertility, anti-cataract, anti-arthritic, anti-allergic and anti-emetic purposes. Abrus derived lectins have been widely used in treating various cancers [8]. Evidence advocates that the seeds of this medicinal herb are effective in treating diabetes and chronic nephritis. Moreover, A. precatorius leaves possess a sweetness quotient equivalent to sucrose and are therefore used to sweeten foods in West tropical Africa [9].
The main aim of the study was to prepare A. precatorius seed extracts using soxhlet and maceration methods to identify various phytochemical compounds and evaluate their abilities to function as antioxidant and invitro assessment as anti-proliferative agent in human cervix carcinoma (Hep2c) cells.

Molecular Identi cation
The ampli ed DNA was puri ed and sequenced using rbcLa-forward and rbcLa-reverse primers used for PCR. NCBI BLAST_N program was used to compare the nucleotide sequence available in Gen Bank. Sequence analysis con rmed the sample showed 98.8% identity having 81% query cover with previous reported sequences of A. precatorius (NC_047402.1).

Quanti cation of phytochemicals constituents
Total avonoid content in seed extracts of A. precatorius were obtained from standard calibration curve y= 0.010x, r² = 0.984, where x is the concentration and y are the absorbance of quercetin (μg/ml) expressed in mg quercetin equivalents/ g of Extract as shown in Fig 1. APE (sox) extract demonstrated high avonoid content i.e., 112.7±0.127 mg quercetin equivalents/ g of extract, while APE (mac) extract showed 77.57± 0.002 mg quercetin equivalents/ g of extract. Moreover, APA (mac) extract exhibited 41.85± 0.017 mg quercetin equivalents/ g of extract while APA (sox) extract only showed 4.8±0.014 017 mg quercetin equivalents/ g of extract (Table1) (Graph 1, Supplementary). Furthermore, the phenolic content in seed extracts of A. precatorius as obtained from standard calibration curve y= 0.015x, r² = 0.998, where x is the concentration and y are the absorbance of is the absorbance of quercetin (μg/ml) expressed in mg quercetin equivalents/ g of extract as shown in Fig 1. Phenolic content of APA (mac) and APA (sox) seed extracts were found to be nearer to each other i.e., 108.53±0.089 mg quercetin equivalents/ g of extract and 103.98± 0.036 mg quercetin equivalents/ g of extract, respectively. Whereas, APE (sox) and APE (mac) seed extract showed less amount of QE (quercetin equivalents) at 28.40±0.157mg / g of extract and 14.34± 0.045mg / g of extract, respectively (Table1) (Graph 2, Supplementary). Total tannins content in seed extracts of A. precatorius as obtained from standard calibration curve y = 0.0143x, r = 0.9989, where x is the concentration and y are the absorbance of is the absorbance of quercetin (μg/ml) expressed in mg tannic acid equivalents/ g of extract as shown in Fig 1. The results indicated that APA (sox) seed extract contained 98.98± 0.011 mg tannic acid equivalents / g of extract, while tannic acid equivalents value of APA (mac) seed extract was 89.35±0.090mg / g of extract Whereas, APE (sox) and APE (mac) seed extracts showed less amount of tannic acid equivalents at 26.35± 0.045 mg / g of extract and 17.07± 0.052mg / g of extract, respectively (Graph 3, Supplementary ).
Antioxidant capacity was also quanti ed by FRAP assay. FRAP values were obtained against standard calibration curve of FeSO 4 at concentration between 100-1000 µM i.e., y=0.001x, r 2 =0.919 where x is the concentration and y are the absorbance that indicates the e capacity to reduce ferric (III) iron to ferrous (II) iron. APA (mac) and APA (sox) have exhibited reducing capacity value105060.2±0.086 mM Fe (II)/ g dry weight of seed extract as and 95841±0.126 mM Fe (II)/ g dry weight of seed extract, respectively. APE (sox) and APE (mac) have revealed reducing capacity value 5849.39± 0.014mM Fe (II)/ g dry weight of seed extract and 6216.59± 0.005mM Fe (II)/ g dry weight of seed extract, respectively (Graph 4, Supplementary).

HPLC analysis
Phytochemicals like polyphenolic avonoids (rutin), tannins (tannic acid), and alkaloids (piperine) were determined in the seed extracts of A. precatorius prepared from different extraction method. The results show that the retention times of rutin, tannic acid and piperine were RT3.817, RT3.093 and RT14.847, respectively as shown in Fig 3. Rutin and tannic acid were identi ed in APA (sox), APA (mac), APE (sox), and APE (mac) seed extracts and their retention time were similar to the standards. Piperine was also identi ed but showed very less peaks in APA (mac), APE (sox) and APE (mac). No peak of piperine was identi ed in APA (sox) seed extract (Fig.4&5). This indicates the presence of both the polyphenolic avonoids and tannins in the seed extracts of A precatorius prepared obtained from different extraction methods.
Cytotoxic activity of seed extracts against Hep2 Cell line A. precatorius seed extracts were evaluated for its antiproliferative potential using MTT assay. Doxorubicin (standard) and the different seed extracts showed variable antiproliferative activity. IC50 values were also determined, lower IC50 values indicating higher the antiproliferative activity. As illustrated in Fig.6C, APE (mac) seed extract exhibited highly signi cant (p<0.005) cytotoxicity against Hep2C cells in a dose-dependent manner with concentration (50-200 µg/ml) with an IC50 value 85.90±0.93µg/ml as compared to the APE (sox) seed extract with an IC50 value 142.80±10.80 µg/ml. Moreover, APA (sox) and APA (mac) seed extracts showed less cytotoxicity against Hep2C cells as compared to APE seed extracts with an IC50 values 374.16±20.53µg/ml and 430 µg/ml, (Fig. 6B) respectively. Comparatively, Doxorubicin, an anticancer drug used in this study as a positive control, demonstrated IC50 value 1.84 µg/ml in a dose-dependent manner with concentration (5-15 µg/ml) (Fig. 6A). Further, upon observation under the microscope, the Hep2c cells after exposure with standard (doxorubicin), APE and APA seed extracts displayed reduced size and abnormally shrunken, compared to untreated cells (Fig. 7).

Antioxidant Enzymes Activity Assay on Hep2C cells
The antioxidant activity of each extract was observed in the treated Hep2C cells with IC50 speci c values to determine the intercellular free radical scavenging ability. We observed the highest SOD enzymatic activity in APA (Mac) i.e., 4.6±0.08 U/min/mg of protein at an IC50 of 430.08±20.46µg/ml compared to the control cells i.e., 2.74±0.08 U/min/mg of protein and other extracts. Similarly, the catalase enzyme activity was observed to be the highest in the APE (Mac) extract with 28.37±0.32 µmoles/min/mg of protein at IC50 of 85.90 ±0.93µg/ml compared to the control i.e., 8.49±0.08 µmoles/min/mg of protein and other extracts. The non-enzyme content such as glutathione and lipid peroxidation were also quanti ed in the cells. The GST activity in the Hep2C cells after treating with the APA(Mac) extract was observed to be the highest (42.16±0.55 µmoles/ min/mg of protein, IC50:430.08 ±20.46) compared to the control (28.5±0. 31  showed a marked decrease in the MDA content (0.041±0.0009 µg/mg of protein) upon exposure to APE(Mac) with IC50 of 85.90 ±0.93µg/ml rather than the extracts and control cells i.e., 0.084±0.002 µg/ mg of protein.

Molecular Docking Analysis
Molecules characterized from HPLC were identi ed and docked to cervical cancer speci c targets such as Human Epidermal Growth Factor Receptor 2 (HER2), Hexosaminidase Subunit Beta (HEXB) and Glucocorticoid receptor (GCR). Molecular docking was performed followed by MM-GBSA re nement using Doxorubicin as a control molecule.
The results are given in Table 3. It was found from the study that tannic acid has scored the highest docking score of -112.53 Kcal/mol with HER2 ( Fig.10). Piperine revealed a similar docking score of -42.11Kcal/mol and -42.15 Kcal/mol for Glucocorticoid receptor and HER2 respectively (Fig. 11). However, piperine resulted in a lower dock score of -29.74 Kcal/mol for HEXB. In the binding pocket of HER2, piperine forms one conventional and three aromatic hydrogen bonds. The Hydrogen bond formation occurs between the HN of Leu436 Tyr409 and Leu 436 (Fig. 9).
The ADME and Druglikeliness properties The in-silico ADME and druglikeliness prediction revealed that all the compounds have potential of being hERG blocker, while piperine can be hepatotoxic and may potentially have harmful reproductive effect. Also, it was observed among all the compounds tannic acid has the lowest drug score of 0.17 which is considered as very less for considering a compound as a lead molecule in clinical trials. Rutin and Tannic acid may also cause drug induced liver damage. Since results are based on a prediction model future in vivo studies are needed for validation (Table 4).    [1]. Currently, the recommended therapeutic regimens include chemotherapy, radiation therapy, and surgery [10]. However, they present several limitations including side effects or ineffectiveness. Therefore, it is important to search for new novel therapeutic agents or drug candidates that are naturally synthesized which will speci cally act on the cancer cells without affecting the normal cells. Plant extracts and their bioactive compounds play a signi cant role in prevention of cancer and many more diseases. Phytochemicals have proved to be an excellent reservoir of polyphenols, tannins, avonoids, alkaloids, terpenes, etc. [11]. Recently, more attention has been placed on tannins with the utilization of some herbs such as Phyllanthus emblica, Sanguisorba o cinalis, as well as red wine with considerable tannins [12]. Plant-derived chemotherapeutic agents such as cisplatin, carboplatin, paclitaxel, ifosfamide, curcumin, camptothecin, taxol, and combretastatin have been used widely against cervical carcinoma [13]. Keeping all the above points in mind present study was designed to evaluate ethyl acetate and 70% ethanol seed extracts of A. precatorius obtained by different extraction methods as a potential therapy against cervical carcinoma by evaluating its antioxidant capacity and in-vitro anti-proliferative activity as well as binding a nity of its polyphenolic avonoids (rutin) and tannins (tannic acid) against receptors mediating signaling pathways of cervical carcinoma. Here, we reported that the content of major avonoids of A. precatorius was signi cantly higher in APE (sox) seed extract (112.7±0.127 mg Quercetin Equivalent/g of extract) as compared to the other extracts. Similarly, the total phenolic content in A. precatorius was higher in APA (mac) seed extract (108.53±0.089 mg Quercetin Equivalent/g of extract) in comparison to the other extracts. Moreover, the total tannin content of APA (sox) amounting to 98.98± 0.011 mg tannic acid equivalents/g of extract was higher than that of other extracts. As corroborated previously, less content of avonoids, phenolics and tannins of A. precatorius have been reported. Another study by Jing et. al., reported that polarity of the solvents used for extraction plays an important role in the concentration of phenols and avonoids [14]. HPLC can be used to identify, separate and quantify, phytochemicals [15].The most important compounds identi ed in our study are polyphenolic avonoids (rutin), tannins (tannic acid) and alkaloids (piperine). All these compounds contained in the seed extracts of A. precatorius may account for its antioxidant capacity. To evaluate antioxidant capacity, DPPH free radical scavenging was examined with 70% ethanolic and ethyl acetate seed extracts where APA (sox) seed extract exhibited excellent radical scavenging activity with an IC50 value of 14.49±0.93µg/ml as compare to other extracts. (Figure 2).
Previously, Gul et al., observed antioxidant potential of leaf extracts of A. precatorius, with an IC50 value of 60.67 ± 1.03 µg/ml [16]. The radical scavenging activity can be explained by different composition of each extract as there are compounds (polyphenolic avonoids and phenolics) that react quickly with DPPH to get reduced due to the formation of nonradical [17]. Therefore, as corroborated by previous studies, polyphenols and tannins could be bene cial for human health, owing to their antioxidant property [18,19]. We have reported for the rst time about the potential anticancer activity of A. precatorius seed extracts on Hep2c cells and its inhibitory e cacy with receptors of Human cervical carcinoma. In order to evaluate A. precatorius as a potential therapy for cervical cancer, all seed extracts prepared by using soxhlet & maceration method extracts were assayed against Hep2c Cells. All seed extracts exhibited anti-proliferative activity in a dose-dependent manner. However, our results revealed that the most promising extract was A. precatorius ethyl acetate seed extract (APE (mac)) which had an IC50 value 85.90±0.93µg/mL in the Hep2c cell line. Previous studies on vulpinic acid reported an IC50 value 34.4µM against cervical carcinoma cell line [20] which may be attributed to the type of solvents and methods of extractions used. The enzymatic activity and non-enzyme content were determined in Hep2C cells. Our current study revealed that SOD and GST activity was observed as highest in the APA(Mac) extract whereas the catalase activity and GSH content was maximum in the APE(Mac) extract. Further, the MDA content was observed to be the least in APE(Mac) extracts. Polyphenolic avonoids, tannins and alkaloids possesses potent anticancer activities with multiple mechanisms, such as apoptosis, cell cycle arrest, and inhibition of invasion and metastases [12]. However, to understand the exact mechanism of speci city against cervical cancer cells, further in-depth and extensive investigations are required. The antiproliferative effect of A. precatorius seed extracts may be attributed to the suppression of multiple oncogenic signaling pathways and tumor-promoting factors. In this analysis, we studied the following receptors like Glucocorticoids, Her2 and HEXB known to be associated with proliferation of cervical cancer [3][4][5]. Piperine, rutin and tannic acid exhibits a myriad of medicinal bene ts such as antioxidant, anti-in ammatory, anticancer and neuroprotective effects [21][22][23]. In this study, tannic acid (TA) has shown maximum binding energy against Her2 receptor rather than the standard drug doxorubicin. As far as the previous studies were concerned compounds like Tangeretin, wogonin, quercetin, and other avonoids have shown less binding energy to the GC and Her2 receptor with respect to present studies [24,25].

Conclusion
The present study concludes that both the extraction methods (maceration & Soxhlet) were effective to obtain a maximum amount of biologically active phytochemicals. This information is supported by the results of HPLC of phytochemicals of the extracts and can be further use for the isolation of bioactive compounds from seeds of A. precatorius which might be utilized as a possible herbal remedy against restricting growth of cervical carcinoma in near future. This plant seed has promising compounds to be tested as potential anticancer and antioxidant drugs for treatment of cervical cancer. However, further investigations need to be done either to isolate the antioxidant compounds or to determine the in vivo biological activity of these extracts, cervical cancer models are needed for preclinical trials. Genomic DNA isolation and molecular identi cation Genomic DNA was isolated from seeds of A. precatorius using Cetyltrimethylammonium bromide (CTAB) method [26]. The genomic DNA was ampli ed using rbcLa-forward primer (ATGTCACCACAAACAGAGACTAAAGC) and rbcLareverse primer (GTAAAATCAAGTCCACCRCG). PCR ampli cation was carried out in Veriti model of Applied Biosystem Thermo cycler [27].

Cell culture
Human cervix carcinoma Hep2C cell line was obtained from National Centre for Cell Sciences (NCCS), Pune, India.

Preparation of Extracts
Seeds were washed with distilled water to remove dirt and soil particles, followed by drying and grinding to form powder and used throughout the study.

Soxhlet Extraction Method
Alcoholic extracts were prepared by soxhlet method using ethyl acetate and 70% ethanol as solvent. 10g of seed powder was placed inside a thimble made from thick lter paper and loaded into the soxhlet extractor. The Soxhlet extractor was placed onto the ask containing the solvent (500 mL) equipped with a condenser. The extractor was then allowed to heat to re ux for 16 h at 70 °C. Extracts were ltered twice through a Whatman No.1 paper lter and concentrated to the dry mass with the aid of rotary evaporator [28].

Maceration Extraction Method:
10g of seed powder was dissolved in 100ml of solvent (Ethyl acetate and 70% Ethanol) and then kept in the dark for 7 days at room temperature. The conical asks of the extract were covered with cotton plugs to avoid the evaporation. After 7 days of incubation, they were ltered with muslin cloth followed by Whatman No.1 lter paper and concentrated to the dry mass with the aid of rotary evaporator [29].
The dried extracts were dissolved in absolute dimethyl sulfoxide (DMSO) as 50 mg/mL and diluted with phosphatebuffered saline (PBS, pH 7.4) to give nal concentrations.

Quanti cation of Phytochemical Constituents
Total avonoid content was measured by Aluminium Chloride Spectrophotometric method. Absorbance was measured against the prepared blank at 510 nm and results were represented as quercetin equivalents (mg QE)/g of extract. Similarly, total phenolic content and total tannins content was quanti ed by Folin-Ciocalteau method. Absorbance of mixture was measured at 725 nm. Final results were represented as quercetin equivalents (mg QE)/g of extract and tannic Acid equivalents (mg QE)/g of extract, respectively [28, 30,31]. All the concentrations were calculated using a standard calibration plot.

Antioxidant Assay
Antioxidant potential in the seed extracts was determined by electron transfer assay i.e. (2,2-Diphenyl-1picrylhydrazyl) Radical scavenging assay (DPPH) and Ferric reducing antioxidant power (FRAP) assay. DPPH free radical scavenging assay was performed to measure the hydrogen donating or radical scavenging ability. Brie y, 0.04mM DPPH radical solution was prepared in methanol and then 900 μL of this solution was mixed with 100 μL of extract solution containing different concentrations of seed extracts. The absorbance was measured at 517 nm after 30 min of incubation. Methanol (95%), DPPH solution and were used as blank, and control respectively. Quercetin was used as the standard.50% inhibitory concentrations (IC50 values) of the extracts were calculated from graph as concentration versus percentage inhibition. Radical scavenging activity was expressed as percentage of inhibition. Measurements were taken in triplicate. The IC50 of the extract and standards were determined graphically [32].

High Pressure Liquid Chromatography
The presence of phenolic compounds and alkaloids in the prepared extracts was screened against standards rutin, tannic acid and piperine by HPLC. The analysis was performed using a C-18 reversed phase column (Phenomenex, Gemini 5 μ, 250 mm length × 4.6 mm internal diameter). The mobile phase consisted of methanol: 0.1% orthophosphoric acid (77:23) for quercetin, methanol: water (50:50) for tannic acid and 1% acetic Acid: acetonitrile (52:48) for piperine were chosen for the separation at a constant ow rate of 1 mL/min. The column temperature was set to 38°C and the injection volume was 20 μL. The wavelengths were set to 370 nm for the detection of rutin, 280 nm for tannic acid, and 343nm for piperine. Retention time was observed by using HPLC [28,35].
The percent inhibition was calculated by using the following formula: The molecular docking was performed on cervical cancer targets using molecules found in HPLC. The X-ray crystallographic structures of targets were obtained from Protein Data Bank (https://www.rcsb.org/search) using PDB ID:3LMY (HEXB), 5MY6(4LSJ), and HER2 (5MY6). Molecular docking was performed using Schrödinger Maestro Suite 2020-3 (Schrödinger, LLC, New-York, NY) [38, 39]. All the structures were optimized prior to docking, any structural inconsistencies were recti ed using Schrodinger's in-built protein preparation wizard module. HEX-B and GCR both have co-crystallized ligands which were chosen to act as the center of the receptor grid. In case of HER2 active site prediction was made using SiteMap module, active sites with a score <0.9 were rejected. The internal grid size x*y*z was xed 15*15*15 Å. Further, MM-GBSA re nements were carried out on the docked poses of each isoform with a exible residue distance of 5.0 Å.

ADMET Analysis and Drug Score
The computational ADME/TOX analysis for identi ed compounds for 23 molecular descriptors were computed using ADMETLAB (http://admet.scbdd.com/) [40]. Additional Descriptors like mutagenic and drug score were computed using Osiris Property Explorer [41].

Statistical analysis
The statistical data were represented as mean ± standard deviation. Statistical analysis was performed using oneway analysis of variance (ANOVA). A probability value of ≤ 0.05 was considered as statistically signi cant. All analyses were performed using IBM-SPSS statistical software (version 13.0).     Molecular docking of tannic acid and its interaction with amino acids of HER2 receptor of cervical carcinoma.