Chemical Standardization and Anti-Proliferative Activity of Ardisia Elliptica Fruit Against the HCT116 Human Colon Cancer Cell Line

molecules Article Chemical Standardization and Anti-Proliferative Activity of Ardisia elliptica Fruit against the HCT116 Human Colon Cancer Cell Line Suchanuch Ondee 1, Pongtip Sithisarn 2, Supachoke Mangmool 3 and Piyanuch Rojsanga 1,* 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand; [email protected] 2 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand; [email protected] 3 Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +668-9206-2499 Received: 28 December 2019; Accepted: 21 February 2020; Published: 25 February 2020 Abstract: The present study is intended to carry out the chemical standardization and evaluation of the anti-proliferative activity of A. elliptica fruit extract. A. elliptica fruit powder was extracted with ethanol. The obtained extract was assessed for total phenolic content using the Folin–Ciocalteu method. Moreover, a simple, accurate, and precise reversed phase high-performance liquid chromatographic method was developed and validated to determine the embelin content of A. elliptica fruit extract. Then, the extract and embelin were investigated for their anti-proliferative effect against HCT-116 cells. Finally, the mechanisms of inhibition of the extract and embelin on the mRNA expression of pro-apoptotic genes Bad, Bax, and Caspase-8 and anti-apoptotic genes c-IAP1, Mcl-1, and XIAP were determined by real-time qRT-PCR. The phenolic content and embelin content of the extract were 5.20 0.01 g of gallic acid equivalent per 100 g of dried fruit (g% GAE) and 5.57 0.56 mg/g of ± ± extract, respectively. The extract and embelin showed strong anti-proliferative effects on HCT-116 cells with 50% inhibition concentration (IC ) values of 19.16 1.09 µg/mL and 25.93 1.75 µg/mL, 50 ± ± respectively. The A. elliptica extract exhibited a significant increase in the mRNA level of Bad, Bax, and Caspase-8 and a significant decrease in c-IAP1, Mcl-1, and XIAP. Embelin showed a significant decrease in Mcl-1 and XIAP. Keywords: standardization; Ardisia elliptica; anti-proliferative; embelin; colon cancer; HCT-116; XIAP 1. Introduction Colon cancer, also known as bowel cancer and colorectal cancer, is uncontrolled cell growth in the colon or rectum [1]. Colon cancer is the fourth most common cause of death in industrialized countries and the third most common cancer around the world [2]. Even though there are enhanced diagnostic and therapeutic methodologies available, the morbidity and mortality of colon cancer is still substantial [3]. The limits of cancer screening and a poor prognosis in the advanced stages of colon cancer have caused a great deal of interest in exploring antitumor agents. Due to the reduced toxicity and cost effectiveness, herbal medicine has gradually attracted more consideration as an alternative cancer therapy [4]. There are many phytochemicals that have been identified for their modulating effects on these molecular targets including genistin, resveratrol, allicin, lycopene, capsaicin, curcumin, 6-gingerol, ellagic acid, catechins, cruciferous, ursolic acid, and eugenol [5–8]. Ardisia elliptica Thunb, known in Thailand as Ram Yai or Pi-lung-ga-sa, is a Thai medicinal plant that belongs to the Myrsinaceae family. It is a small-branched shrub tree with smooth and leathery Molecules 2020, 25, 1023; doi:10.3390/molecules25051023 www.mdpi.com/journal/molecules Molecules 2020, 25, 1023 2 of 14 texture leaves and pale violet flowers. Fruits are round, berry-like drupes that turn from red and dark purple to black when they are ripe. The fruits are edible and taste slightly astringent. A. elliptica is commonly found in Sri Lanka, China, Taiwan, and Southeast Asian countries especially: Thailand, Vietnam, Malaysia, Indonesia, and the Philippines [9]. A. elliptica is traditionally used for alleviating chest pains, the treatment of fever, diarrhea, liver poisoning, and parturition complications. The leaves and roots of this plant have traditionally been used in Southeast Asian herbal remedies. The decoction of the leaves of A. elliptica is used for treatment of pain in the region of the heart or to alleviate chest pains [10]. A. elliptica has more potency than aspirin in the inhibition of collagen-induced platelet aggregation by β-xamyrin contained in A. elliptica leaves [11]. The ethanolic extract of A. elliptica fruit exhibited anti-proliferative activity on SKBR3 human breast adenocarcinoma cell lines [12] and showed antioxidant and antidiarrheal activities [13]. There is also a report that highlighted the anticancer potential against liver cancer cells of tea extracts from the leaves of six species of Ardisia species of which A. compressa, A. crenata, and A. japonica promoted a high potential among the tested samples, but with an unclear mechanism of action [14]. There is also a study demonstrating the in vitro antibacterial and antioxidant effects of methanol extracts from the leaves and the fruits of A. elliptica. This report also suggested the presence of phenolics and flavonoids in these extracts of A. elliptica [15]. The phytochemical compounds contained in leaves of A. elliptica are bauerenol, α-amyrin, β-amyrin, and bergenin [16,17]. Syringic acid, isorhamnetin, β-amyrin, quercetin, and anthocyanin have been isolated from the fruit [18]. A. elliptica fruits contain a quinone derivative, embelin, as a major constituent. Myricetin, quercetin, norbergenin, kaempferol, quercetin 3-0-β-d-glucopyranoside, and gallic acid were also reported [19]. The safety of the extract of A. elliptica fruit was evaluated in an animal model. The oral administration of ethanolic extract of A. elliptica fruits at the dose of 5 g/kg promoted no acute toxicity in mice, while the subchronic toxicity study in Wistar rats, at doses of 20–2000 mg/kg/day, also did not promote any toxicity [20]. Even though A. elliptica fruit has many potential activities such as antimicrobial, antioxidant, and anti-proliferative activities, the effects of phytochemicals contained in A. elliptica fruit extract against colon cancer cells have not been fully defined. The present study aimed to determine the total phenolic and embeline contents as chemical parameters for the standardization of A. elliptica fruit extract, and to investigate the effects of the extract as well as embelin on the inhibition of cell proliferation in HCT-116 cells. 2. Results 2.1. Determination of Total Phenolic Contents in A. elliptica Fruit Extracts by Folin-Ciocalteu Method The total phenolic content was calculated from a standard calibration curve of standard gallic acid and expressed as miligram of gallic acid equivalent per 1 g of dried extract (mg GAE/g). The total phenolic contents of the A. elliptica fruit were 52.0 0.1 mg GAE/g dried extract. ± 2.2. Phytochemical Analysis of A. elliptica Fruit Extract by Thin Layer Chromagography (TLC) A. elliptica fruit extract was phytochemically analyzed using two different solvent systems. The extract exhibited thin layer chromagography (TLC) fingerprints with the presence of chromatographic bands that corresponded to some phenolics and flavonoids, as shown in Figure1. There were chromatographic bands that corresponded to gallic acid and embelin at retardation factor (Rf) values of 0.46 and 0.62 in solvent system 1 and 0.26 and 0.46 in solvent system 2, respectively. Moreover, there were chromatographic bands at Rf values of 0.68 and 0.52 in solvent system 1 and 2, respectively that appeared as yellow fluorescence bands after spraying with natural product/polyethylene glycol (NP/PEG) spray reagent and detected under UV 366 nm and chromatographic bands at Rf values of 0.63 and 0.42 in solvent system 1 and 2, respectively that appeared as orange fluorescence bands after spraying with NP/PEG spray reagent and detected under UV 366 nm. The presences of these chromatographic bands suggested the presences of flavonoids. Molecules 2020, 25, 1023 3 of 14 underMolecules UV2020 366, 25, 1023nm. The presences of these chromatographic bands suggested the presences3 of of 14 flavonoids. FigureFigure 1 1.. ThinThin layer layer chromagography chromagography (TLC) (TLC) chromatogram chromatogram of Ardisia elliptica fruit extract; 1 = gallicgallic acid,acid, 2 2 == embelin,embelin, 3 3= =ArdisiaArdisia elliptica elliptica fruit fruit extract, extract, adsorbent: adsorbent: silica silica gel gelGF254. GF254. Solvent Solvent system: system: 1 = acetate:glacial1 = acetate:glacial acetic acetic acid:formic acid:formic acid:hexane acid:hexane (15:2:2:10, (15:2:2:10, v/v/vv/v/v),/ v2/ v=), ethyl 2 = ethyl acetate:toluene:formic acetate:toluene:formic acid (9:10:2,acid (9:10:2, v/v/v).v /detection:v/v). detection: A = UV A =254UV nm, 254 B nm,= UV B 366= UV nm, 366 and nm, C and= NP/PEG C = NP under/PEG underUV 366 UV nm. 366 Band nm. identificationBand identification system system 1: gallic 1: acid gallic (Rf acid = 0.46), (Rf = embelin0.46), embelin (Rf = 0.62). (Rf =Band0.62). identification Band identification system 2: system gallic acid2: gallic (Rf = acid 0.26), (Rf embelin= 0.26), (Rf embelin = 0.46). (Rf = 0.46). 2.3. Phytochemical Analysis of A. elliptica Fruit Extract by Liquid Chromatography-Mass Spectrometry 2.3.(LC-MS) Phytochemical Analysis of A. elliptica Fruit Extract by Liquid Chromatography-Mass Spectrometry (LC- MS) The peaks of gallic acid, quercetin, kaempferol, and embelin in A. elliptica fruit extract were identifiedThe peaks by LC-MS of gallic technique. acid, quercetin, It was found kaempferol, that the LC-MS and embelin peak at the in retentionA. elliptica time fruit of 0.77extract min were with identifiedmolecular by mass LC-MS (negative technique. mode) It of was 168.95 foundm/z andthatfragment the LC-MS ion peak of 124.95 at the and retention 79.1 m/z timecorresponded of 0.77 min to withgallic molecular acid.

Load more