In-vitro Cytotoxicity and In-silico Insights of the Multi-target Anticancer Candidates from Haplophyllum tuberculatum

This study aimed to investigate the anticancer activity of Haplophyllum tuberculatum (Forsk.) aerial parts ethanol extract and fractions and reveal the potential anticancer targets, binding modes, pharmacokinetics, and toxicity properties of its phytoconstituents. MTT assay was used to investigate the anticancer activity. TargetNet, ChemProt version 2.0, and CLC-Pred web servers were used for virtual screening, and Cresset Flare software was used for molecular docking with the 26 predicted targets. Moreover, pkCSM, swiss ADME, and eMolTox web servers were used to predict pharmacokinetics and safety. Ethanolic extracts of H. tuberculatum on HepG2 and HeLa cell lines showed promising activities with IC50 values 54.12 and 48.1 μg/mL, respectively. Further, ethyl acetate fraction showed the highest cytotoxicity on HepG2 and HeLa cell lines with IC50 values 41.7 and 52.31 μg/mL. Of 70 compounds screened virtually, polygamain, justicidin A, justicidin B, haplotubine, kusunokinin, and flindersine were predicted as safe anticancer drugs candidates. They showed the highest binding scores with targets involved in cell growth, proliferation, survival, migration, tumor suppression, induction of apoptosis, metastasis, and drug resistance. Our findings revealed the potency of H. tuberculatum as a source of anticancer candidates that further studies should support.


INTRODUCTION
Cancer is one of the significant health problems in developed and developing countries. It is the second leading cause of death globally 1 . Unfortunately, despite their structural diversity, most currently approved anticancer drugs have drawbacks, mainly due to their harmful effects extended to normal cells, with further consequences on health 2 . Up to date, secondary metabolites continued to be a potential source of anticancer leads and utilized as a source of many therapeutic and preventive anticancer agents.
Interestingly, more than 60 % of currently used anticancer drugs are derived from natural sources 3 .
Haplophyllum tuberculatum (Forsk.) of the Rutaceae family is used in folk medicine for malaria and parasitic infections 4 . Moreover, H. tuberculatum is used for many digestive, gynecological, respiratory, cardiovascular, and CNS disorders 5, 6 . A literature survey showed that H. tuberculatum contains more than 50 phytoconstituents, including polyphenols, alkaloids, lignans, flavonoids, and essential oils 6-10 . Therefore, it is necessary to conduct research to investigate the biological activity, safety, and determination of the appropriate biological target of the phytochemical constituents of H. tuberculatum.
Although identifying drug targets is an essential step in understanding the mechanism of action, it is a challenging task mainly because of its complexity.
Therefore, there is a need to use advanced technologies such as computer-aided drug design tools, including virtual screening molecular docking, to identify drug targets. 11,12 . With this background information, this study aims to investigate the anticancer effect of H. tuberculatum aerial parts ethanol extract and fractions on HeLa (cervical) and HepG2 (liver) cell lines, as well as to reveal the potential anticancer targets, binding modes, pharmacokinetics, and toxicity properties of reported phytoconstituents using virtual computational methods.

Preparation of ethanol extract and fractions
Dried powdered plant materials (60 g) were soaked in 80% ethanol (Merck, Germany) for three days at 25-30°C and then filtered. Extraction was repeated twice, as described previously. The extract was collected through Optical density (OD) was recorded at 570 nm using a microplate reader ELx800 and cell survival fraction was determined using the equation in Formula 1: In which ODs, ODb, and ODc were the optical density of the sample, blank and negative control, respectively 14 .
Data were subjected to analysis using Microsoft Excel software, and the survival fraction was plotted against the concentration. The concentration required for 50% inhibition of cell viability (IC50) was calculated. The extracts that fail to inhibit 50% of cell viability up to 100 µg/mL were considered inactive.

In silico cytotoxicity and drug-likeness prediction
The cytotoxic effect of about 70 compounds reported H.
tuberculatum phytochemical constituents 6-10 were predicted using Cell Line Cytotoxicity Predictor (CLC-Pred) 15 . The probability of phytochemical constituents to be a drug candidate was predicted using the SwissADME webserver 16 .

Virtual screening and molecular docking
Virtual screening for anticancer targets depended on a protocol described by Al-Nour et al. 12 with a modification.

Pharmacokinetics and toxicity prediction
The pkCSM 22 and SwissADME 16 web servers were used to predict intestinal absorption, the apparent volume of distribution, clearance, and CYP-450 enzyme inhibition.
Furthermore, the pkCSM 22 and eMolTox 20 web servers were used to predict the toxicity of phytochemical constituents on major organs (cardiotoxicity, hepatotoxicity, and renal toxicity).

RESULTS AND DISCUSSION
In vitro cytotoxicity assay  Nevertheless, this further suggests that the bioactive compounds were of low to intermediate polarity.

Virtual screening and molecular docking
Once the biological activity was confirmed, identifying the drug target was a valuable subsequent step that leads to further success in the drug development pipeline 11

Pharmacokinetics and toxicity prediction
Web servers were used to predict the promised anticancer phytochemical constituents of H. tuberculatum compounds' toxicity, and results are summarized in

DATA AVAILABILITY
None.