Chemical composition, antitumor and antioxidant effects of four lebanese plants extracts on human pulmonary adenocarcinoma

Abstract Lung cancer leading cause of humans’ cancer-related death remains incurable with current drug therapies having many side-effects. Finding new natural cancer remedies would help design better therapies. This study aims to investigate the anticancer and antioxidant effects of the Levant endemic plants Prango sasperula, Stachys ehrenbergii, Hirtellina lobelia, and Salvia multicaulis on human pulmonary adenocarcinoma. GC/MS and NMR analyzed the bioactive compounds of methanolic extracts of these plants. Their antioxidant effect was measured by ABTS assay. The effect of these extracts on the viability of A549 (Human lung adenocarcinoma cell line) and NHBE (Normal Human Bronchial Epithelial Cells) was examined using MTT and Anchorage-Independent Colony Formation assays. Our findings show an interesting, specific cytotoxic effect of these plant extracts on A549 cells. Additionally, S. ehrenbergii and H. lobelii present a specific high alkylation potential. Data reveal an interesting antitumor potential attributed to their antiproliferative and alkylation actions towards human pulmonary adenocarcinoma. Graphical Abstract


Introduction
Lung cancers are classified into two types: small cell lung cancers (SCLC) and non-small cell lung cancers (NSCLC). SCLC includes around 10%À15% of lung cancers. This lung cancer type is the fastest growing and most aggressive of all kinds. SCLCs spread quickly to too many places of the body and are frequently discovered after expanding vastly.
Concerning NSCLC, it is the most common lung cancer. It has three main types assigned by the type of cells found in the tumor. a) Adenocarcinomas, usually associated with smoking like other lung cancers, but also seen in non-smokers, especially women. b) Squamous cell carcinomas, accounting for about 25% of NSCLC cases where squamous cell cancers arise in the bronchi of the central chest area and often stays within the lung spreading into the lymph nodes and growing quite large, forming a cavity. c) Large cell carcinomas, known as undifferentiated carcinomas, which are the least common type of NSCLC, accounting for 10%À15% of all lung cancers having a high risk of spreading to the lymph nodes and distant sites (Ford et al. 2003).
Today, exploring anticancer and antioxidative agents from plants seems to be one of the fastest-growing areas of research (Gautam et al. 2014;Rowshan and Najafian 2020). Studies showed an anticancer activity of plant extracts against human lung adenocarcinoma but without elucidating the chemical composition of these extracts. As such, this study focuses on the use of some Lebanese wild plants extract Prangos asperula, Stachys ehrenbergii, Hirtellina lobelii and Salvia multicaulis in order to evaluate their treatment potential on human lung adenocarcinoma cell line A549 that was kindly offered by the Department of Biochemistry at the American University of Beirut AUB. Voucher specimen was deposited at the Herbarium of USEK and numbered as follow P. asperula (MNII 265a) and H. lobelii (MNIII 145 b) from "Tannourine" region in the North of Lebanon, S. multicaulis (MNIII 475a) from "Mdayrej" (Zahleh region) and S. ehrenbergii (MNIII 235c) from "Jabal El Makmel" (Arez, North of Lebanon).

Chemical composition
The chemical composition of the plants' extracts was analyzed by GC-MS and results are detailed in the Table S1.

Antioxidant activity
Our results show that the antioxidant IC 50 of P. asperula was 27 mg/ml, S. ehrenbergii's 0.052 g/ml, H. lobelii's 5.4 mg/ml and S. multicaulis' 12 mg/ml. H. lobelii has the lowest IC50 value which means that it presents the highest antioxidant activity followed by S. multicaulis, P. asperula and S. ehrenbergii (Table S2).

Cytotoxicity analysis
The absorbance rates (optic density) of the experimental samples were lower than the negative controls (Fig. S1); this indicates a reduction in the rate of cell proliferation or a reduction in overall cell viability and so an increase in cell inhibition.
All the extracts had variable cytotoxicity effects on A549 and NBHE cells. S. ehrenbergii's extract showed the highest cytotoxicity with 11.57% ± 1.09 of survival rate at a concentration of 3000 mg/mL whereas S. multicaulis showed the lowest cytotoxicity with (41.55% ± 3.94) of cell survival at a concentration of 6000 mg/mL. With the extract of H. lobelii's 27.15% ± 5.03 of the cells survived at a concentration of 3000 mg/mL with S. multicaulis extract (41.55% ± 3.94) of cells survived at a concentration of 6000 mg/mL ( Figure S1). Interestingly all the extracts were not cytotoxic to normal cells (IC 50 values >10000 mg/ml), but caused significant cell death in the human adenocarcinoma cell line A549. The IC50 values of targeted cells are represented in Table S3.

Colony formation assay
Untreated A549 and NHBE cells formed colonies in soft agar at a rate of 78%±3.5 and 59%±12, respectively. All the plant extracts severely inhibited the colony formation of the A549 cells (<15%) in soft agar ( Figure S2), indicating a low survival rate of tumorigenic cancer cells with these extracts. No statistical difference (p > 0.05) was observed in the treated and non-treated NHBE cells.

Alkylating effect (NBP assay)
Results of the NBP assay were used to estimate the mechanism of the DNA damage of many anticancer agents in vitro via DNA alkylating activity (Mojtaba et al. 2011).
No statistical difference was observed during the treatment of A549 with P. asperula and S. multicolis. All the concentrations showed low and medium alkylating activity.
Interestingly, S. ehrenbergii and H. lobelia extracts showed a specific anticancer activity via their alkylation properties. Above 1000 mg/mL, both extracts present high alkylation activities compared to lower concentrations and control cells ( Figure S3).

Conclusion
Since many species are active in different experimental models, natural products derived from plants are a rich source of herbal medicines or biologically active compounds. There is a need for further studies on the standardization or chemical characterization of the extracts used, and especially, a study of each component by itself is required for further research.
Our findings show that P. asperula, S. ehrenbergii, S. multicaulis and H. lobelii may be important candidates for the development of natural medicine against human lung adenocarcinoma. However, despite many studies showing the evaluation of possible mechanisms of action of these compounds, the research presented only preliminary screening data and therefore did not describe any mechanism of action.
Also, new research findings have shown extracts as potential phytotherapeutic adjuvants in advanced lung cancer therapy which could lead to greater safety and benefits for people, contributing to a better access to health care and thereby better quality of life of patients with lung cancer.