Antiproliferative assay of suma or Brazilian ginseng (Hebanthe eriantha) methanolic extract on HCT116 and 4T1 cancer cell lines, in vitro toxicity on Artemia salina larvae, and antibacterial activity

Abstract Hebanthe eriantha is a medicinal plant used in folk medicine and a subject of commercial interest. The cytotoxicity effects from H. eriantha root extracts on cancerous and normal cells were assessed by the MTT method, and in vitro toxicity was evaluated on Artemia salina. The inhibition of the proliferation of bacteria and MIC values were examined by the disc diffusion and the broth microdilution method, respectively. Human colon cancer HCT116 and mouse breast tumour model 4T1 cells treated with methanolic extract showed a significant decrease in viability of cells with IC50: 272.6 and 88.5 µg/mL at 72h, respectively. The methanolic extract of H. eriantha showed moderate toxicity against A. salina (LC50: 589.4 µg/mL). In antimicrobial activity, the methanolic extract showed the highest inhibitory function against S. aureus and P. vulgaris (17.5 and 16 mm) with MICs of 500 µg/mL. The results confirmed the potential of plant roots as cytotoxic agents. Graphical Abstract


Introduction
Hebanthe eriantha (Poir.)Pedersen (many synonyms, including Iresine erianthos and Pfaffia paniculata (Mart.)Kuntze) belongs to the Amaranthaceae family, however, it was also known as suma or Brazilian ginseng (Costa et al. 2015).H. eriantha has been subjected to the NAPRALeRT database concerning plants utilised for cancer (Graham et al. 2000).So far, there have been many reports on their biological activities, such as anti-tumoral, anti-inflammatory, and analgesic (Moura et al. 2011).Nortriterpenoid saponins (pfaffosides A, B, C, d, e, and F), pfaffic acid, and phytosterols, including stigmasterol and β-sitosterol, were found abundantly in H. eriantha (Nishimoto et al. 1984;Li et al. 2010).H. eriantha has conventional applications against the low immune system, stress, fatigue, inflammation, and intestinal anti-inflammatory activity (Costa et al. 2018).Besides, the H. eriantha root butanol extract increased the survival of mice suffering from the ascitic type of ehrlich tumour carcinoma (Matsuzaki et al. 2006).According to researchers' study, the roots of H. eriantha have anti-neoplastic activities and cancer chemopreventive in the mice hepatocarcinogenesis models, indicating chemoprotection by reducing cell proliferation and inducing apoptosis (Carneiro et al. 2007;da Silva et al. 2010).The purpose of this paper was to evaluate the cytotoxicity of H. eriantha root on HCT116 human colorectal and 4T1 mouse breast cancer cells compared to normal cells.The potential toxicity of extracts was also examined by Artemia salina, and Gram-positive and Gram-negative bacterial strains.To the best of our knowledge, there is no published study on the anti-cancer properties of H. eriantha crude methanolic extract on HCT116 and 4T1 cancer cells, and only a few studies have evaluated its anti-microbial action.The current research is important considering the wide use of H. eriantha and its importance as a global export product, especially in Asian countries.

Cytotoxic effect of H. eriantha root methanolic extracts
The incubation of HCT116 and 4T1 cancer cells was performed considering various concentrations of the crude methanolic extract to study the growth-inhibitory activity of H. eriantha roots.The methanolic extract significantly decreased the viability in HCT116 and 4T1 cancer cells treated with the extracts compared to human embryonic kidney cells (HeK293) (Figure S1A-C).However, the decrease in the viability of cells was time and dose-dependent.The methanolic extract exhibited marked inhibition of proliferation against 4T1 cells with IC 50 : 88.5 µg/mL at 72h (Table S1).Moreover, compared with cancerous cells, methanolic extract displayed lesser cytotoxicity in HeK293 non-cancerous cells with IC 50 value of 649.8 µg/mL, and this is a good option for using the plant root extract.5-fluorouracil as a standard drug demonstrated the lowest IC 50 compared to extracts (Table S1).The study by da Silva et al. (2005), showed the anticarcinogenic activities of H. eriantha root on preneoplastic hepatic lesions.Other authors reported the cytotoxic activity of butanol extract of H. eriantha root against human mammary adenocarcinoma cells (MCF7) (Nagamine et al. 2009).The data of the current study revealed a more cytotoxic effect for H. eriantha, as Nagamine's study presented that the difference in the inhibitory effect of butanol extract against MCF7 started from 400 mg/mL, while in the present study, the difference between the viability of cancerous and non-cancerous cells began from 62.5 µg/mL and 250 µg/ mL in 4T1 and HCT116, respectively.In Costa et al. (2015) study, H. eriantha crude extract was not able to inhibit trinitrobenzenesulfonic acid-induced intestinal inflammation, although, it reduced colonic inflammation in a mouse model, suggesting its therapeutic potential.The selectivity index exhibits the selectivity of tested extract between cancer and normal cells.The higher the magnitude of the selectivity index, the greater is its selectivity.In general, H. eriantha methanolic extract showed good selectivity against 4T1 and HCT116 at 72h (>2) (Singh et al. 2019).Generally, researchers use Hebanthe species as an adjunct therapy for some types of cancer now, although information on the nature of its crude extracts is still insufficient.In this regard, the results of the present study are noticeable.According to the uSA National Cancer Institute, the IC 50 should be <30 μg/mL, which is a crude extract to be used as a suitable substance for further purification (Rajkumar et al. 2009).Although the IC 50 s of the methanolic extracts against 4T1 were higher than the suitable dose, they were still significant.In general, the findings persuade us to continue assessing the biological activities, especially the cytotoxic effect of the H. eriantha extracts.

Toxicity assay of H. eriantha extracts on A. salina
This study evaluated the general toxicity of H. eriantha root methanolic extract against A. salina, taking the percentage of lethality as a bioassay indicator for methanolic extract toxicity.The nauplii lethality (%) at concentrations of 31.25 to 1000 μg/mL of methanolic extracts was 26.7% to 60%, indicating the toxicity of methanolic extract (Figure S2).Therefore, if LC 50 detected for extracts is more than 1000 µg/mL, it will be non-toxic (Ruebhart et al. 2009).The methanolic extract of H. eriantha root showed moderate toxicity against brine shrimp larvae (LC 50: 589.4 µg/mL).

Antibacterial assay
The present study revealed the antibacterial activity of the methanolic extract of H. eriantha root against Gram-positive and Gram-negative bacterial strains (Table S2).As shown by the extract's antimicrobial function, the methanolic extract had the highest inhibitory activity against Staphylococcus aureus (17.5 mm) and Proteus vulgaris (16 mm).The methanolic extract (100 mg/mL) showed the same inhibitory effect compared to gentamicin (10 μg/mL) against S. aureus.However, the methanolic extract showed the lowest inhibitory effects on Escherichia coli (9.5 mm).The lowest MICs of H. eriantha root methanolic extracts were determined at 500 µg/mL concentration against S. aureus and P. vulgaris.Generally, these results indicated an acceptable antibacterial activity of methanolic extracts on the bacterial growth process after 24 h (Table S3).In this regard, the hydroalcoholic extract of H. eriantha showed antimicrobial activity against S. aureus with an inhibition zone of 11 and 21 mm, at concentrations of 250 and 500 mg/mL, respectively (Fontanive et al. 2010).Whilst, the inhibition zones of 17.5 mm were shown by the methanol extract of H. eriantha against S. aureus at a concentration of 100 mg/mL.Therefore, we can introduce H. eriantha root methanolic extract as an option for in vitro treatment of Gram-positive and Gram-negative bacteria due to its valuable MICs.

Conclusion
The present study was the first in vitro cytotoxic investigation focussing on H. eriantha root methanolic extract.The findings of two various types of cancer cells (HCT116 and 4T1) provided robust evidence on the cytotoxicity of the plant, indicating the inhibitory effects of H. eriantha root methanolic extract on pathogenic microorganisms and confirming its contribution as a cytotoxic agent.The results also highlighted that it is important to evaluate the therapeutic properties of H. eriantha root for human health protection.