DOI: 10.14457/tu.the.2023.1021
title: Comparison of cytotoxic activity against gastrointestinal cancer cell lines and anti-inflammatory activity of Basella alba L. and Basella rubra L. extracts

description: Basella alba L. and B. rubra L. are medicinal plants in Thailand, that have been traditionally used as food and traditional medicine for treatments of constipation, and peptic ulcer. There was no report on the cytotoxicity on gastrointestinal cancer of these plant extracts. The aim of this study was to investigate and compare cytotoxic activity against gastrointestinal cancer cell lines and anti-inflammatory activity of B. alba and B. rubra extracts and also to determine total phenolic, flavonoid, saponin, carotenoid, and mucilage contents of B. alba and B. rubra.B. alba and B. rubra were extracted by mucilage extraction, decoction, maceration with 95% ethanol, and in vitro digestion. All extracts were examined for cytotoxic activity against gastrointestinal cancer cell lines namely oral epidermoid carcinoma (KB), gastric carcinoma (KATO III), colorectal adenocarcinoma (SW480 and LS 174T), hepatocellular carcinoma (Hep G2), cholangiocarcinoma (KKU-M156) cell lines and two normal cell lines, including HaCat (immortal human epidermal keratinocytes) and MRC-5 (human fetal lung fibroblast cells) by SRB assay. The mucilage, aqueous and digestion extracts had no cytotoxic activity (IC50&gt;100 µg/mL). The extracts of B. alba showed higher cytotoxic activity against cancer cell lines in gastrointestinal than B. rubra. The ethanolic extract of fresh B. alba (FAE) exhibited the highest activity against all cancer cell lines, showed significant differences compared to the other active extracts (p-value &lt; 0.05) and showed moderate cytotoxic activity against KB, KATO III, SW480 and KKU-M156 cell lines with IC50 = 39.31, 33.82, 31.54, 33.79 µg/mL, respectively, as established by NCI, while LS 174T, and Hep G2 cell lines with IC50 = 63.06 and 59.63 µg/mL. Compared to other extracts depending on the cell line groups, KB cells showed the IC50 value of the FAE was not significantly different (p-value &lt; 0.05) from that of the ethanolic extract of dried B. alba (DAE), while KATO III, SW480, LS 174T, and KKU-M156 cells also showed no significant difference (p-value &lt; 0.05) from the DAE and the ethanolic extract of dried B. rubra (DRE). The FAE also showed the highest selectivity index (SI) between HaCaT cell lines against cancer cell lines including KB, KATO III, SW480, LS 174T, KKU-M156, and Hep G2 with SI = 2.39, 2.78, 2.98, 1.49, 2.79 and 1.58, respectively. Similarly, the result of MRC-5 cell lines against all cancer cell lines with SI = 1.80, 2.09, 2.24, 1.12, 2.09, and 1.18. This study demonstrated cytotoxic activity against KB, KATO III, SW480, LS 174T, and KKU-M156 cell lines of Basella species extracts for the first time.The extracts were tested for anti-inflammatory activity, B. alba extracts show higher inhibitory activity against LPS induced NO production in RAW 264.7 cell lines than B. rubra, and the fresh plant had stronger activity than dry plants. The mucilage and aqueous extracts show higher inhibitory activity than the ethanolic extract, while the digestion extracts had no activity (IC50 &gt;100 µg/mL). The aqueous extract of fresh B. alba (FAW) showed the highest NO production inhibitory activity with an IC50 = 4.27 µg/mL, which was not significantly different (p-value &lt; 0.05) from the mucilage extract of dried B. alba (DAM), the mucilage extract of dried B. rubra (DRM), the mucilage extract of fresh B. rubra (FRM), containing IC5 = 4.84, 4.89, and 5.42 µg/mL, respectively followed by the ethanolic extract of fresh marc B. rubra (FREm), the ethanolic extract of dried B. alba (DAE), and the aqueous extract of fresh B. rubra (FRW), with IC50 15.86, 24.98, and 29.63 µg/mL, respectively and Prednisolone which is a positive control had an IC50 = 0.092 µg/mL.The extracts were separated into fractions according to their solubility, i.e., the ethanolic extract of fresh B. alba (FAE) and the ethanolic extract of fresh B. rubra (FRE) because of their good bioactivity and from the TLC fingerprint test, the ethanolic extract can separate more substances than the water extract. The chloroform mix ethyl acetate fraction FAE [FAE(C:E)] showed the highest activity against all cancer cell lines except the SW480 cell lines, that the chloroform fraction of FRE [FRE(C)] displayed higher activity than FAE(C:E) with IC50 = 5.21 µg/mL. FAE(C:E) fraction showed good cytotoxic activity against KB, KATO III, SW480, LS 174T, KKU-M156, and HepG2 cell lines with IC50 = 5.22, 6.04, 5.36, 5.90, 6.00, and 5.59 µg/mL, respectively, as established by NCI. Compared to other fractions of B. alba and B. rubra extracts depending on the cell line groups, KATO III, SW480, and KKU-M156 cells showed IC50 value of the FAE (C:E) fraction was not significantly different (p-value &lt; 0.05) from that of FRE(C) fraction. Interestingly, the FRE(C) showed a good SI value between HaCaT cell lines against cancer cell lines including KATO III, SW480, LS 174T, and KKU-M156 cells with SI value = 7.81, 9.30, 2.05, and 4.22, respectively. While the result of the MRC-5 cell lines against the cancer cell lines including KATO III, SW480, and KKU-M156 cells with SI value = 3.90, 4.64 and 2.11. If interested in further study, this fraction should be studied because it is less toxic to normal cells. The results of inhibitory activity against LPS induced NO production of the fraction of ethanolic extract of fresh B. alba and B. rubra showed that three fractions showed potent inhibitory activity, namely the FAE(C:E), the hexane fraction of FRE [FRE(H)] and the FRE(C). The FRE(H) fraction exhibited the highest potent inhibitory activity with IC50 value of 16.04 µg/mL, which showed significant differences compare to the other active fraction. (p-value &lt; 0.05) follow by FAE(C:E), and FRE(C), with IC50 value = 43.20 and 51.72 µg/mL. Phytochemical evaluation of B. alba and B. rubra in terms of total phenolic, flavonoid, and saponin contents revealed that the ethanolic extract showed higher contents than the mucilage, aqueous, and digestion extracts. B. alba also showed higher total phenolic, flavonoid, and mucilage contents than B. rubra, while total saponin and carotenoid contents of B. rubra were higher than that of B. alba. The ethanolic extracts of the marc of dried B. alba (DAEm) had the highest phenolic contents of 38.60 in terms of Gallic acid equivalents (GAE)/g of dry extract. The total flavonoid contents were evaluated in terms of Quercetin/g of dry extract and was the highest in the ethanolic extract of dried B. alba (DAE) i.e. 352.58. The ethanolic extract of the marc of fresh B. rubra (FREm) had the highest saponin contents with 187.76 mg DE/g extract. B. rubra had total carotenoid contents higher than B. alba with 3.44 and 2.58 mg BE/g of fresh sample that was evaluated in terms of β-carotenoid equivalents while B. alba had a percentage yield of total mucilage content higher than B. rubra with 2.44 and 1.97%.In conclusion, the extraction of B. rubra by maceration with 95% ethanol and fractionated with chloroform showed the best potential cytotoxic activity against four cancer cells, namely KATO III, SW480, LS 174T, and KKU-M156 cell lines, and it also had anti-inflammatory activity by inhibiting the production of nitric oxide. To study further, this fraction of the extract is suitable for study. For eating according to Thai consumption by boiling or blanching fresh B. alba and B. rubra might help prevent or reduce inflammation in the body. Because the results of the extraction test by boiling with water showed good anti-inflammatory activity. And eating fresh B. alba can also help prevent cancers in the gastrointestinal tract such as oral, stomach, colon, liver and bile duct cancers better than B. rubra.These results provide basic information about the biological activity including cytotoxic activity against gastrointestinal cancer, and the anti-inflammatory activity of Basella species extracts. However, further investigations on chemical markers, in vivo pharmacological activity testing, and toxicity testing are needed for future development as a product.