Suaeda vermiculata Aqueous-Ethanolic Extract-Based Mitigation of CCl4-Induced Hepatotoxicity in Rats, and HepG-2 and HepG-2/ADR Cell-Lines-Based Cytotoxicity Evaluations

Abstract

Suaeda vermiculata, an edible halophytic plant, used by desert nomads to treat jaundice, was investigated for its hepatoprotective bioactivity and safety profile on its mother liquor aqueous-ethanolic extract. Upon LC-MS (Liquid Chromatography-Mass Spectrometry) analysis, the presence of several constituents including three major flavonoids, namely quercetin, quercetin-3-O-rutinoside, and kaempferol-O-(acetyl)-hexoside-pentoside were confirmed. The aqueous-ethanolic extract, rich in antioxidants, quenched the DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals, and also showed noticeable levels of radical scavenging capacity in ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) assay. For the hepatoprotective activity confirmation, the male rat groups were fed daily, for 7 days (n = 8/group, p.o.), either carboxyl methylcellulose (CMC) 0.5%, silymarin 200 mg/kg, the aqueous-ethanolic extract of the plant Suaeda vermiculata (100, 250, and 500 mg/kg extract), or quercetin (100 mg/kg) alone, and on day 7 of the administrations, all the animal groups, excluding a naïve (250 mg/kg aqueous-ethanolic extract-fed), and an intact animal group were induced hepatotoxicity by intraperitoneally administering carbon tetrachloride (CCl₄). All the animals were sacrificed after 24 h, and aspartate transaminase and alanine transaminase serum levels were observed, which were noted to be significantly decreased for the aqueous-ethanolic extract, silymarin, and quercetin-fed groups in comparison to the CMC-fed group (p < 0.0001). No noticeable adverse effects were observed on the liver, kidney, or heart's functions of the naïve (250 mg/kg) group. The aqueous-ethanolic extract was found to be safe in the acute toxicity (5 g/kg) test and showed hepatoprotection and safety at higher doses. Further upon, the cytotoxicity testings in HepG-2 and HepG-2/ADR (Adriamycin resistant) cell-lines were also investigated, and the IC₅₀ values were recorded at 56.19±2.55 µg/mL, and 78.40±0.32 µg/mL (p < 0.001, Relative Resistance RR 1.39), respectively, while the doxorubicin (Adriamycin) IC₅₀ values were found to be 1.3±0.064, and 4.77±1.05 µg/mL (p < 0.001, RR 3.67), respectively. The HepG-2/ADR cell-lines when tested in a combination of the aqueous-ethanolic extract with doxorubicin, a significant reversal in the doxorubicin's IC₅₀ value by 2.77 folds (p < 0.001, CI = 0.56) was noted as compared to the cytotoxicity test where the extract was absent. The mode of action for the reversal was determined to be synergistic in nature indicating the role of the aqueous-ethanolic extract.

Keywords: HepG-2; HepG-2/ADR; LC-MS; Suaeda vermiculata; antioxidant; aqueous-ethanolic extract; cytotoxicity; halophyte; hepatoprotective; liver disorders; liver toxicity; mass spectrometry.

Figure 1

Radical scavenging activity of Suaeda vermiculata extracts by (A) 1,1-diphenyl-2-picrylhydrazyl (DPPH), and (B) 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) methods. Values are the mean of three replicates ± SEM. p < 0.0001 using two-way ANOVA, and p < 0.0001 compared to quercetin for all groups at all the measured concentrations for both methods according to multiple comparisons for Tukey’s method.

Figure 2

The hepatoprotective effects of Suaeda vermiculata aq.-ethanolic extract on CCl₄ induced liver toxicity. Percentage protection of CCl₄ induced elevation of AST and ALT enzymes. The protection percentage formula assumes the enzyme level of the CCl₄ administered negative group (carboxyl methylcellulose) at 0% protection and that of intact group (no exposure to CCl₄) at 100% protection as such they are excluded from the graphical representation. In addition, the aq.-ethanolic extract 250 mg/kg CCl₄- the group which did not receive CCl₄- is also excluded from the graphical representation. **** Data differed significantly at p < 0.0001 when compared with the negative control group. Values are expressed as mean ± SEM, n = eight rats per group, CCl₄-: Carbon tetrachloride was not administered. CCl₄+: Carbon tetrachloride-induced liver toxicity.

Figure 3

The dose-response curves of Suaeda vermiculata aq.-ethanolic extract: (A) aq.-ethanolic extract, and (B) doxorubicin (DOX) in wild-type HepG-2, and resistant HepG-2/ADR cell-lines.

Figure 4

(A) The dose-response curves and isobologram analysis of the combination of DOX with 20 µg/mL of Suaeda vermiculata extract in resistant HepG-2/ADR. (B) The isobologram on the right side of the figure showed the synergistic interaction between DOX and the extract. IC₅₀, DOX, and IC₅₀ extract correspond to the IC₅₀ for DOX and extract alone. CI < 1 indicates synergism, CI = 1 indicates additive, and CI > 1 indicates antagonism.

Figure 5

Prospective roles of antioxidants in hepatoprotection.

Figure 6

Sketch representing the role of the plant extract in liver protection.