Full Text: 1 Introduction Neolamarckia cadamba synonyms Kadam, native to South Asia and South East Asia (Krisnawati et al., 2011).  The Charaka Samhita and Sushruta Samhita narrates the ethnomedicinal uses of N. cadamba bark as an analgesic, for strengthening body, against snake bite, diarrhea, dysentery, leucorrhoea and spermatorrhoea (Sircar, 1992; Slkar et al., 1992). Further, various parts of N. cadamba i.e. leaf, bark, fruit and flower has been traditionally used by many tribes and communities in India against fever, cough, sour throat, infections and inflammation (Pandey & Negi, 2016).  Phytochemical analysis of different parts of N. cadamba detects the presence of chlorogenic acid, β-sitosterol, indole alkaloids and cadambine which shows anti-inflammatory, antimutagenic, antipyretic, antidiabetic, antidiarrheal and antioxidant activities (Gupta et al., 1980; Li & Chang, 2005; Alam et al., 2008;   Kumar et al., 2015a; Verma et al., 2019; Mondal et al., 2020). Methanolic extract of N. cadamba bark contains number of secondary metabolites such as cadambagenic acid, saponin B, Glycosides A & B, Phelasin A & B and 3β-dihydrocadambine (Sahu et al., 1999; Sahu et al., 2000; Chandel et al., 2011). Presence of phenolics compounds, alkaloids and saponins in different extracts of N. cadamba bark attributes to its antimicrobial, antidiabetic, antiepileptic activity (Gurjar et al., 2010; Patel et al., 2011; Nagakannan et al., 2011). Chronic persistence of free radicals (reactive oxygen species; ROS and reactive nitrogen species; RNS) lead to oxidative stress, responsible for disorders related to nervous system, metabolic disorders like diabetes mellitus and several form of cancers (Huy et al., 2008). Traditionally bark of N. cadamba has been used against inflammation, fever, cough and wound. To scientifically validate ethno-medicinal uses of bark of N. cadamba, present study has been designed to determine in vitro antioxidant and anticancer efficacy of hydromethanolic (HM) extract of N. cadamba bark.  2 Materials and Methods 2.1 Used chemicals and cell line used Experimental animals 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, butylated hydroxyl toluene (BHT), RPMI-1640 medium, sulforhodamine (SRB) and TCA were procured from Sigma Aldrich, USA. N1S1 rat hepatoma cancerous cell line was obtained from Tata Memorial centre, Mumbai.  2.2 Plant material Neolamarckia cadmba bark was collected from Mathura and Vrindavan areas of Uttar Pradesh, India. Plant was identified and authenticate by Dr A S Updadhye, Agharkar Research Institute, Pune with voucher deposition No. L-084. Dried bark powder of N. cadamba was used in preparation of hydo-methanolic (HM) extract. 2.3 Extract Preparation Dried powder of N. cadamba bark was subjected to soxhlet extraction. Hydro-methanolic (80:20, v/v) solvent was allowed to boil at 65^° C for 7-8 hrs. Solvent was evaporated at temperature and reduced pressure using rotary evaporator. Lastly dark brown crystals were obtained and used in further studies. 2.4 Preliminary Phytochemical analysis of HM extract of N. cadamba bark Qualitative analysis of hydro-methanolic extract of N. cadamba bark was done as per standard protocol (Debela, 2002). HM extract of N. cadamba bark were subjected to preliminary phytochemical screening to detect the presence of steroids, triterpenoids, glycosides, carbohydrates, alkaloids, flavonoids, tannins, phenolics, proteins, amino acids, gum, mucilages, fats and oils.   2.5 Evaluation of in vitro antioxidant potential of HM extract of N. cadamba bark In vitro antioxidant activity of HM extract of N. cadamba bark was evaluated by 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging assay method (Blois, 1958). Different concentrations (0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml) of HM extract of N. cadamba bark were used for antioxidant activity. From various concentrations, 0.1 ml HM extract were mixed with 3.0 ml of DPPH (0.1 mM). Mixture was incubated at 20^° C for 40 minutes in dark. Optical density (OD) was determined at 517 nm with ethanol and water (1:1) as blank. Readings were taken in triplicate. Determination of percentage inhibition of DPPH was calculated by using formula given by Gulcin et al., (2005). Percent inhibition of DPPH = OD_{control (DPPH)}- OD_{test (HM extract)}/OD_{control (DPPH)} x 100 Standard used were butylated hydroxyl toluene (BHT) and ascorbic acid were used as standards. 2.6 In vitro anticancer activity of HM extract of N. cadamba bark Determination of in vitro anticancer activity of HM extract of N. cadamba bark was carried out at Tata Memorial centre, Mumbai using N1S1 rat hepatoma cell line according to standard procedure (Vichai & Kirtikara, 2006). Cell suspension (100 µL) in triplet was inoculated in 96 well microtiter plate. Plate was incubated at 37^° C in CO₂ incubator (5% CO₂ & 100% RH) for 24 hrs. Different doses (10, 20, 40 & 80 µg/ml) of HM extract and Adriamycin (standard) were added into wells of microtiter plate. Plate was again allowed to incubate at 37^° C for 48 hours in CO₂ incubator. Termination of reaction was done by adding 30% chilled TCA. This was followed by staining of cells by adding 0.4% sulforhodamine (SRB) in 1% acetic acid solution. Elution of SRB dye bounded to cell protein was carried out by adding Tris base. Absorbance was measured at 510 nm by ELISA reader. Percent growth = Mean absorbance test/Mean absorbance control × 100 Percent of control cell growth =    Mean absorbance sample
 – mean absorbance day 0  × 100 Mean absorbance neg control
– mean absorbance day 0 Percent growth inhibition = 100 - % of control cell growth Percent cell killed = 100 – mean absorbance sample/mean absorbance day 0 × 100 2.7 Statistical analysis Data analysis was done using one way analysis of variance (ANOVA) using SPSS version 20.0 software and DMRT at p < 0.01 to determine significant differences among treated means. Values are expressed as mean ± SEM. 3 Results and Discussion Percentage yield of N. cadamba HM extract was reported 14 percent. Results of preliminary phytochemical analysis revealed the presence of alkaloids, steroids, glycosides, triterpenoids, carbohydrates, flavonoids, tannins, phenols, fats, oils,  gums, mucilage’s where as proteins and amino acids were absent (Table 1). Presence of these secondary metabolites cause inhibition of microbial growth (Negi, 2012) and does offer various ethnomedicinal uses of N. cadamba bark (Pandey & Negi, 2016). Present study concerns with evaluation of in vitro antioxidant potential of N. cadamba bark extract. DPPH free radical scavenging method was used to determine percentage inhibition of free radicals in presence of 0.025, 0.050, 0.1 and 0.2 mg/ml HM extract of N. cadamba bark. Results of study suggested significant (p < 0.01) antioxidant efficacy of N. cadamba bark extract at different doses, when compared with ascorbic acid and BHT (Table 2). Maximum percentage inhibition was reported at 0.20 mg/ml HM extract. Dose dependent inhibition of free radical scavenging activity was found. Result of current study are in agreement with previous findings, suggesting the presence of phenolics compounds in bark extract might caused scavenging of free radicals (Chandel et al., 2011). Antioxidant compounds counter free radicals mediate oxidative stress in the cell (Blokhina et al., 2003; Mondal et al., 2020). Present study also revealed the presence of phenols during phytochemical screening of N. cadamba bark, which might probably responsible for its significant antioxidant activity. Result of current study can be concluded that HM extract of N. cadamba is good enough to overcome oxidative stress and serves as therapeutic agents for treating radical related pathological damage.  Four concentrations (10, 20, 40 and 80 μg/ml) of HM extract of N. cadamba bark were screened using N1S1 rat hepatoma cell line. Adriamycin was used as standard cytotoxic agent. Different concentrations of HM extract of N. cadamba bark were found to be effective against N1S1 rat hepatoma cell line (Table-3). Dose dependent decrease in percentage cell growth of rat hepatoma was reported in current study. LC50, TGI and GI50 of HM extract was reported 75.92, 46.73 and 17.46 µg/ml respectively (Table 4). Previous studies also suggested cytotoxic activity of N. cadamba against different human cancer cell lines (Singh et al., 2013; Kumar et al., 2015b; Dolai et al., 2016). Current study depicts the presence of phenolic compounds in HM extract of N. cadamba bark, responsible for antiproliferative activity (Fatima et al., 2016). Conclusion Present study concluded significant antioxidant activity of different doses of HM extract of N. cadamba bark. Study also revealed anticancer efficacy of bark extract. There is a need to isolate and molecular characterization of active ingredients present in hydro-methanolic extract of N. cadamba bark. Acknowledgements We are thankful to Director, Institute of Applied Sciences and Humanities, GLA University, Mathura for providing necessary facility. Conflicts of Interest The authors declare that there is no conflict of interest. Author’s contribution Vishal Khandelwal  and Pradeep Kumar Choudhary contributed significantly and equally. ORCID ID Vishal Khandelwal: 0000-0003-4321-8125 ORCID ID Pradeep Kumar Choudhary: 0000-0003-4816-455X Funding None. Data Availability All datasets generated or analyzed during this study are included in the manuscript. Ethics Statement Ethical clearance was obtained with IAEC approval vide GLAIPR/CPCSEA/IAEC/2014/Biotech/02.