R ESEARCH ARTICLE doi: 10.2306/scienceasia1513-1874.2021.068 Evaluation of anti-inflammatory, cytotoxic, anti-H. pylori, antioxidant activities, and phytochemical compositions of Shirakiopsis indica (Willd.) Esser Khwanchanok Mokmueda, Bhanuz Dechayontb, Pathompong Phuakleeb, Chantubpapa Liplungb, b c a, Harit Muangpoolsawad , Nitra Nuengchamnong , Nuntika Prommee ∗ a Division of Applied Thai Traditional Medicine, Faculty of Public Health, Naresuan University, Phitsanulok 65000 Thailand b Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Pathum Thani 12120 Thailand c Science Laboratory Centre, Faculty of Science, Naresuan University, Phitsanulok 65000 Thailand ∗Corresponding author, e-mail: [email protected] Received 10 Jan 2021 Accepted 4 Jun 2021 ABSTRACT: Shirakiopsis indica (Willd.) Esser (Sa-Mor-Ta-Lay, in Thai), a herbal plant belonging to the family Euphorbiaceae, has traditionally been used to treat gastrointestinal symptoms. The objectives of this study were to investigate the bioactivities (cytotoxic, antioxidant, anti-inflammatory, and anti-Helicobacter pylori) of S. indica extracts that influence the treatment of gastric inflammation. The chemical constituents of the extracts were also analyzed by performing LC-ESI-QTOF-MS/MS; and they are tentatively identified as Sapintoxin A, Sapintoxin B, Sapintoxin C, Saipinin, and a few other phorbol esters. We found that the ethanolic extract of S. indica exhibited the highest cytotoxicity against the Kato III gastric cancer cell line and the highest inhibition of nitric oxide production with the IC50 values of 3.21 0.77 and 12.99 2.42 µg/ml, respectively. The water extract of S. indica exhibited higher anti- · H. pylori activity than± that of the ethanolic± extract, and exhibited strong antioxidant activity on DPPH and ABTS + assay with the IC50 values of 8.169 0.65 and 20.03 1.33 µg/ml, respectively. Our results give new science-based evidence that S. indica fruit extract has± a potential for treatment± of gastritis. Pharmacological activities and toxicity in animal models should be further investigated. KEYWORDS: Shirakiopsis indica, anti-inflammatory, cytotoxic, antioxidant, anti-H. pylori INTRODUCTION classified by International Agency for Research on Cancer (IARC), World Health Organization (WHO). Gastritis is the inflammation of gastric mucosa. In response to the H. pylori infection, the human The pathological base of gastritis is dystrophy, in- body mechanism reacts to the inflammation site flammation, and unregeneration of gastric mucosa and generates reactive oxygen species (ROS) and causing atrophy as the outcome of these symp- reactive nitrogen species (RNS), i.e., nitric oxide toms [1]. The stomach has mechanisms to protect and superoxide [4]. Nowadays, the treatment of against hydrochloric acid and toxic agents. For gastritis varies according to the cause, but the pri- example, gastric mucosa can control the acid and mary problem has not been solved. Various herbs pepsin enzyme production, and the thickness of the have been used in Thai traditional medicine to treat gastric mucous coating can prevent acid toxicity gastritis and peptic ulcers [5, 6]. The most common in the stomach [2]. The inflammation of gastric herb used for treating gastrointestinal symptoms mucosa or gastritis is a disorder of the stomach. is the fruit of Shirakiopsis indica (Sa-Mor-Ta-Lay There are two main causes of gastritis: Helicobacter in Thai). However, bioactivities of this herb have pylori (H. pylori) infection and non-steroidal anti- never been reported. The objectives of this study inflammatory drugs (NSAIDs). H. pylori is a grade I were to investigate the bioactivities of S. indica carcinogenic bacterium that causes gastric cancer in that influence the treatment of gastric inflammation, humans [3], the most common non-erosive gastritis including cytotoxic activity against Kato III gastric www.scienceasia.org 2 ScienceAsia 47 (2021) cancer cell line using the Sulforhodamine B (SRB) 10% heated fetal bovine serum, 50 IU ml of peni- · / assay, antioxidant activity by DPPH and ABTS + cillin, and 50 µg/ml of streptomycin. The cells cul- assay, proinflammatory cytokines, i.e., nitric oxide tured were maintained at 37 °C in 5% CO2 incubator (NO) by Griess assay, tumor necrosis-alpha (TNF- (Shel lab, USA) at 95% humidity. alpha), interleukin-6 (IL-6) using an ELISA kit assay, Bacterial strains and culture conditions and anti-H. pylori using disc diffusion and minimal inhibitory concentration (MIC) assay. In addition, H. pylori (ATCC® 43504, USA) was obtained from the chemical constituents of S. indica fruits were the American Type Culture Collection. It was cul- analyzed by performing LC-ESI-QTOF-MS/MS. tured on Columbia agar supplemented with 5% sheep’s blood (RPD, Thailand) and incubated at MATERIALS AND METHODS 37 °C under microaerobic conditions (N2, 85%; O2, 5%; CO , 10%) using a gas generating kit. The Sample collection 2 bacteria were sub-cultured every three days in an Dried fruits of Shirakiopsis indica (Willd.) Esser anaerobic jar. (1000 g) were obtained from a local herbal medicine shop “Charoensuk Osod” in Nakorn Antioxidant activities Pathom, Thailand. Species identification, Verifica- DPPH radical scavenging assay tion Number 05698, was verified by the Herbarium DPPH was measured by the method of Ya- in the Department of Biology, Faculty of Science, masaki et al 7 with a slight modification. DPPH so- Naresuan University, Thailand, and this study was [ ] lution (Fluka, USA) in absolute ethanol was freshly approved by the Biosafety Committee of University prepared and kept in a dark bottle protected from of Thammasat, Thailand under Biosafety Level 2 light. A 100 µl each of the water and ethanolic (BSL II, Number 011 2019). / extracts and butylated hydroxytoluene (BHT) so- lutions of various concentrations (1, 10, 50, and Sample extraction and preparation 100 µg/ml) were placed in each well of a 96 well Water extraction microplate. After that, a 100 µl of the DPPH so- lution was added to each well, and the plate was Dried S. indica fruits (92.49 g) were boiled in 500 incubated in the dark at room temperature for 30 ml of distilled water for 15 min, and the aqueous min. The radical scavenging activity was expressed extract was filtered through a Whatman filter paper as the number of antioxidants necessary to decrease No. 1. Then, the filtrate was freeze dried using the initial DPPH absorbance by 50% (IC ). The lyophilizer (Lyophilization systems Inc, USA). 50 antioxidant activity was calculated by its ability to inhibit the DPPH radical formation according to Ethanolic extraction Eq. (1): Dried S. indica fruits (208.09 g) were crushed with in a mortar and macerated with 1 l of 95% ethanol (AbsControl AbsSample) % Inhibition 100 (1) for 3 days. The extract was filtered through What- = − AbsControl × man filter paper No. 1 and re-macerated twice. Then, the combined extract was dried using a rotary where AbsControl = absorbance of absolute ethanol; evaporator (Buchi, Switzerland). AbsSample = absorbance of fruit extract. · ABTS + radical scavenging assay Cell culture The herbal extracts of 1, 10, 50, and 100 µg ml · / Human and murine macrophage cell lines concentrations were diluted. Then, ABTS + solu- · The Kato III gastric cancer cell line was obtained tion was prepared by diluting 7 mM ABTS + stock from the Riken Cell Bank (Tsukuba, Japan; cat- solution in distilled water with 2.45 mM potassium alog no. RCB2088). It was cultured in RPMI persulfate and then mixed at room temperature for 1640 medium supplemented with 10% heated fetal 12–16 h. The solution was diluted with distilled wa- bovine serum, 50 IU ml of penicillin, and 50 µg ml ter to obtain an absorbance at 734 nm in the range / / · of streptomycin. For the anti-inflammatory activity, of 0.68–0.72. A 10 µl aliquots of diluted ABTS + the murine macrophage RAW 264.7 cells (ATCC® solution and 90 µl of one of the S. indica extracts TIB-71TM VA, USA) were cultured in DMEM (Dul- or standard Trolox were added into each well of the becco’s Modified Eagle Medium) supplemented with 96 well microplate, and mixed well. Then, the plate www.scienceasia.org ScienceAsia 47 (2021) 3 was incubated for 6 min, and the absorbance (Abs) MTT assay for potential cytotoxicity was measured at 734 nm by a microplate reader 8 . [ ] An MTT assay [10] was used to assess cytotoxicity The antioxidant (radical scavenging) activity was · of the S. indica extracts on RAW 264.7 cell line. calculated as percent inhibition of ABTS + radical 5 The cells (1 10 cells/well) were incubated in 96 formation according to Eq. (2): well microplates× with various extract concentrations (1, 10, 30, 50, and 100 µg/ml) for 48 h. Then, (AbsControl AbsSample) % Inhibition = − 100 (2) the thiazolyl blue tetrazolium bromide (MTT) so- AbsControl × lution (Sigma-Aldrich, USA) was added to these wells and incubated for another 4 h. Next, the where AbsControl = absorbance of absolute ethanol; medium was removed, and isopropanol buffer (RCI AbsSample = absorbance of fruit extract. The radical scavenging activity was expressed as the number Labscan, Thailand) with 0.04 M HCl was added to of antioxidants (µg ml) necessary to decrease the dissolve the formazan produced in the incubated · / initial ABTS + absorbance by 50% (IC ). cells. The absorbance of the formazan solution was 50 measured with a microplate reader (Bio Tek, USA) Anti-inflammatory activity on nitric oxide at 570 nm. A dose-response curve was obtained by production and cytotoxicity plotting the % inhibition values versus the extract Anti-inflamatory activity on nitric oxide concentrations. The extracts were considered non- production toxic when cell survival was more than 70%. Cell survival was followed by determining % toxicity The anti-inflammatory activity of S. indica on nitric according to Eq. (4): oxide production in murine macrophage RAW 264.7 cell line was determined by Griess assay [9–11]. (Abscontrol Absextract) The RAW 264.7 cells were diluted with the medium (%) Toxicity = − 100 (4) Abscontrol × to obtain an optical density of 1 105.