Introduction

Cancer is a leading cause of death worldwide, second only to cardiovascular disease (Global Burden of Disease Study 2015). Colorectal cancer (CRC) is the third most common cancer worldwide and the incidence is increasing (Anand et al. 2008). Many drugs in common use have been developed from potent herbal phytochemicals, e.g. pomiferin is a prenylated isoflavonoid derived from Maclura pomifera that have been shown to inhibit the growth of colon cancer (Amin et al. 2009). Thus, some medicinal plants can be strong and beneficial pharmacological effects. The Pikut Trichinthalamaka remedy is a Thai traditional medicine that was “rediscovered” in the Thai Pharmacy scripture and consists of rhubarb (Rheum palmatum L.), myrabulan fruit (Terminalia chebula Retz.) and gamboge (Garcinia hanburyi Hook F.). It has been used in Thai herbal remedies as a purgative and to treat lymphoedema related cancer. Interestingly, recent studies have indicated that a crude extract of R. palmatum significantly inhibited human colorectal cancer cells (LS1034) (Ma et al. 2014). T. chebula fruit has been used traditionally as a laxative, an anti-inflammatory, and to treat ascites and clear mucus from the respiratory tract. The active pharmaceutical ingredient (API) of the fruit is chebulic acid which has moderate cytotoxicity activity against the colon cancer HCT-15 and COLO-205 cell lines (Reddy et al. 2009; Saleem et al. 2002). G. hanburyi, a dry latex and folk medicine for constipation, also has cytotoxic properties against cancer HCT-15 cell; its major component is gambogic acid, the putative API, induces apoptosis in HT-29 human colon cancer cell (Huang et al. 2015).

There are no data on the cytotoxic and antioxidant activities of Pikut Trichinthalamaga remedy. Therefore, we investigated these properties and report our results, herein.

Materials and methods

Plant material

The Pikut Trichinthalamaga remedy consists of equal parts of rhubarb root (Rheum palmatum L.), myrabulan fruit (Terminalia chebula Retz.) and gamboge latex (Garcinia handuryi Hook F.). Gamboge and myrabulan fruit were collected from Tak and Surin provinces Thailand, and rhubarb was purchased from China in September 2013. Further botanical identification was conducted at the Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University.

Extraction

Different parts of the plants were dried using a hot air oven at 50 °C.

Decoction - 500 g of Pikut Trichinthalamaga remedy and each plant materials were separately boiled in distilled water (2 l) for 15 min.

Maceration - 95% ethanol (EtOH) extracts were prepared by maceration of the plant material (200 g) with 95% EtOH (500 ml) at room temperature for 3 days (25 °C). The water extracts were lyophilized after filtration and the organic extracts were vacuum concentrated until dry. All the extracts were stored in airtight containers at −20 °C until analysed.

Solvent partitioning

The extracts were subjected to liquid-liquid partitioning by vacuum liquid chromatography (VLC) using hexane (1000 ml), hexane-chloroform (1000 ml), chloroform (1500 ml), chloroform- methanol (1500 ml) and methanol (2000 ml), in sequence. The fractions were concentrated by rotary evaporation.

Cell culture

LS174T, SW 480 and RAW 264.7 cells were obtained from Assoc. Prof. Dr. Arunporn Itharat, Faculty of Medicine, Thammasat University, Thailand. The normal lung fibroblasts cell line MRC-5 was obtained from Asst. Prof. Pintusorn Hansakul, Faculty of Medicine, Thammasat University, Thailand. The LS174T and MRC-5 cell lines were cultured in Dulbecco’s modified Eagle’s (DMEM) culture medium containing 10% heat-inactivated foetal bovine serum, 50 IU/ml penicillin and 50 μg/ml streptomycin. SW 480 and RAW 264.7 cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 10% heat-inactivated foetal bovine serum, 50 IU/ml penicillin and 50 μg/ml streptomycin. These cells were maintained at 37 °C in a 5% CO2 atmosphere with 95% humidity.

In vitro assay for cytotoxic activity - Sulphorhodamine B (SRB) assay

The SRB assay was performed according to the method of Skehan et al. 1990 to measure the cellular protein content. The effect of the plant extracts on the proliferation of the human cell lines was determined in a 96-well microplate. For the assay, the densities of each cell line LS174T, SW 480 and MRC-5 were determined to be 3 × 103, 1 × 103 and 5 × 103 cell/well. After 24 h, cells added with different concentrations of the extracts and incubated for 72 h. Then they were washed and incubated cell for 72 h in drug-free medium. The experiment were carried out at 37 °C 5% CO2 atmosphere and 95% humidity. At the end of the incubation, 100 μl of 40% (w/v) cold TCA was added to each well. The plates, which were incubated at 4 °C for 1 h, were washed and 50 μl of 0.4% (w/v) SRB solution in 1% acetic acid was added to each well. After staining, the unbound dye was removed by 1% acetic acid and the plates were air dried. SRB was dissolved in 100 μl per well of 10 mM Tris base, and the absorbance was read at 492 nm. The percentage of inhibition = 100% × (Abs control – Abs sample)/ Abs control.

ABTS radical-scavenging activity assay

Total antioxidant capacity of the extracts was assayed according to the method of Re et al. 1999. ABTS•+ solution was produced by reacting 7 mM ABTS stock solution in distilled water with 2.45 mM potassium persulfate. The mixture was allowed to stand in the dark at room temperature for 12–16 h. The reaction was carried out for 6 min and then the absorbance was measured at 734 nm using a microplate reader. The scavenging activity of the extracts against ABTS•+ was expressed as the EC50 (μg/ml).

Cell-based assay to assess the inhibitory effect on nitric oxide production and the viability of LPS-activated RAW 264.7 macrophages

The inhibitory effect on nitric oxide (NO) production by mouse macrophage RAW 264.7 cells was evaluated using a method modified from that of Tewtrakul and Itharat 2007.

Measurement of NO production was measured using Griess reagent. Briefly, 200 μl of 1 × 105 cells/well were seeded in 96-well microplate at 37°C in a humidified atmosphere containing 5% CO2 for 1 h. After that cells were treated with LPS at 5 ng/ml (final concentration) for 48 h. NO production was determined by measuring the accumulation of nitrite (NO2 −) in the culture supernatant using the Griess reagent at 570 nm.

Inhibition (%) was calculated using the following equation and the IC50 value was calculated graphically using GraphPad 4.0 software in triplicate (Prism, USA). The percentage of inhibition = (Abs control – Abs sample)/ Abs control × 100 where Abs control = Abs control with LPS - Abs control non LPS and Abs sample = Abs sample with LPS – Abs non LPS.

The cell viability was also performed using the MTT assay. Briefly, after 48 h incubation with test samples, MTT solution was added to the wells. After 4 h incubation, the medium was removed, and isopropanol containing 0.04 M HCl was added to dissolve the formazan production in the cells. The optical density of the formazan solution was measured with a microplate reader at 570 nm. The test simples were considered to be cytotoxic when the optical density of the sample-treated group was less than 70% of that in the control (vehicle-treated) group. Cell survival was calculated using the following formula: The percentage of cell survival = 100% ´Abs sample/Abs control.

Phytochemical analysis

High-performance liquid chromatography coupled to electrospray ionisation and quadruple time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) was used to identify compounds.

The HPLC-ESI-QTOF MS/MS analysis was performed on a 6540UHD accurate mass QTOF LC/MS mass spectrometer (Agilent Technologies, Singapore) coupled with an Agilent 1260 infinity series HPLC system (Agilent, Waldbronn, Germany). ESI was performed in both negative and positive ion modes. The following conditions were adopted: drying gas (N2) flow rate of 1.0.0 L/min; drying gas temperature 350 °C; nebulizer pressure 30 psig; capillary voltage of 3.5 kV; skimmer 65 V; octapole RFV 750 V. The m/z ranged from 100 to 1000 amu. The auto fragmentation was settled with collision energy at 10, 20 and 40 V. Chromatographic separation was carried out on a Luna C18(2) column (5 μm, 150 mm × 4.6 mm i.d., Phenomenex, USA) at a flow rate of 0.5 ml/min. The column heater was kept at 35 °C. The mobile phase consisted of a combination of A (0.1% formic acid in water, v:v) and B (0.1% formic acid in acetonitrile, v:v); the linear gradient was from 5 to 95% B at 30 min.

Peak identifications

The compounds were identified mainly by their ESI-QTOF/MS spectra and fragmentation patterns which were compared with published data and a library search: Mass Hunter Metlin metabolite PCD/PCDL (Agilent Technologies), Scifinder, Chemspider and Massbank. Molecular formulae were generated by the Agilent MassHunter Qualitative Analysis Software B 06.0 (Waldbronn, Germany).

Statistical analysis

All experiments were carried out in triplicate. Statistical analysis was performed using GraphPad 4.03 software (Prism, USA) and presented as means ± SD (standard deviation). The EC50 and IC50values were calculated from the dose-response curve.

Results

Results of extraction

The extract yields obtained from the decoction and maceration methods are shown in Table 1. Pikut Trichinthalamaga remedy extract by decoction method gave a higher yield of the extract (71.93%) compared to the other extracts.

Table 1 The percentage yields and in vitro cytotoxicity activities (IC50 μg/ml ± SD) of the Pikut Trichinthalamaga remedy and its component extracts against colon cancer cell lines using the SRB assay (n = 3)
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Results of In vitro assay for cytotoxic activity

By the SRB assay, the remedy and most of its component extracts displayed cytotoxic activity against SW 480 and LS174T cell lines at markedly variable IC50 values (μg/ml) (Table 1). GBE and PTE showed specific cytotoxic activity against SW 480 cell line with mean IC50 values of 0.51 ± 0.04 and 0.51 ± 0.02 μg/ml, and LS174T cell line with mean IC50 value with 0.39 ± 0.01 and 0.51 ± 0.01 μg/ml, respectively. On the other hand, the RBW was not cytotoxic in the tested concentrations against these cell lines.

Results of ABTS radical-scavenging activity assay

ABTS radical-scavenging activity of Pikut Trichinthalamaga remedy and its component extracts were compared with two standard antioxidants, BHT and Trolox. The highest inhibition for ABTS radical-scavenging activity was exhibited by MBW (similar value as Trolox and BHT) followed by RBE, PTW and RBW.

Results of cell-based assay assessing the inhibitory effect on nitric oxide production and viability of LPS-activated RAW 264.7 macrophages

NO concentration in the culture medium was determined by the Griess reaction assay. The results of inhibitory activity against LPS induced NO production of extracts are shown in Table 2. GBE and PTE showed inhibitory activities of NO production in induced cells with mean IC50 values of 0.50 ± 0.05 and 0.62 ± 0.05 μg/ml, respectively. These results were supported by the cell viability experiment (cell viability >80%). These extracts showed higher activity than the positive control (Indomethacin) (Table 2).

Table 2 Antioxidant activities of the Pikut Trichinthalamaga remedy and its components extracts measured by chemical-based (ABTS assay) and cell-based assays (NO assay) (n = 3)
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Results of In vitro assay for cytotoxic activity from solvent partitioning

The ethanolic extract of Pikut Trichinthalamaga remedy (PTE) were partitioned with hexane, hexane–chloroform, chloroform, chloroform-methanol and methanol and showed potent cytotoxic and antioxidant activities. The chloroform fraction demonstrated the highest activity against LS174T and SW 480 cell lines with mean IC50 values of 0.45 ± 0.04 and 0.51 ± 0.07 μg/ml, respectively. As for the antioxidant activity, the chloroform-methanol fraction exhibited higher free radical scavenging than the other fractions (Table 3).

Table 3 Percentage yield and in vitro cytotoxicity activity of VLC fractions from ethanolic extracts of Pikut Trichinthalamaga remedy against colon cancer cell lines using the SRB assay (n = 3)
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Results of phytochemical analysis

The identification of compounds in the chloroform fraction by HPLC–ESI-Q-TOF-MS operating in both negative and positive modes are given in Table 4. These compounds are summarized along with their retention time, m/z experimental, error (ppm), molecular formulae generated by the software for the detected deprotonated and protonated molecules, classification order in the list of possibilities (sorted with library search), MS/MS fragments and their proposed assignments. The phytochemical screening revealed that the CHCl3 fraction contained emodin, forbesione, gaudichaudionic acid and gaudichaudionol (Table 4).

Table 4 Identification of chloroform fractions from the Pikut Trichinthalamaga remedy by (+/−) ESI Q-TOF-MS/MS
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Discussion

Pikut Trichinthalamaga remedy, rhubarb, myrabulan fruit, gamboge, and fractions of different polarities from active extracts were tested for cytotoxic activity against colorectal cancer cells. To our knowledge, our study is the first report on the cytotoxicity activity of Pikut Trichinthalamaga remedy. Previous work has shown that gambogic acid, the primary active component of gamboge, induces apoptotic cell death of human colorectal cancer cell lines (HCT116, LOVO and SW-116 cells) (Deng et al. 2013) and that myrabulan fruit extract displayed cytotoxic activity against human colorectal cancer cell lines (HCT-15 and COLO-205) with IC50 values of 20.3 ± 0.23 and 18 ± 0.2186 μM, respectively (Reddy et al. 2009).

As for antioxidant activity, the ABTS assay is an electron transfer (ET) − based assay and radicals can react with both hydrophilic and lipophilic antioxidants. Myrabulan fruit extracted by absolute alcohol had antioxidant of with antioxidant EC50 values of 1.4 ± 0.0173 and 1.7 ± 0.023 μM by DPPH and ABTS assays, respectively in earlier work (Reddy and Reddanna 2009). In spite of offering advantages of simplicity and cost efficiency, DPPH and ABTS chemical assays do not reflect the complexity of in vivo models (Honzel et al. 2008) whereas cell-based assays do. They incorporate cellular functions such as antioxidant uptake, cellular distribution, and metabolism. Thus, NO is employed in cell-based system. NO is a free radical synthesized from L-arginine and oxygen by several isoforms of nitric oxide synthetase (iNOS) (Alderton et al. 2001). iNOS is regulated by pro-inflammatory mediators and the excessive production of NO by iNOS has been implicated in the pathogenesis of the inflammatory response (Yang et al. 2010). Gamboge has a strong inhibitory activity on NO production and anti-inflammatory properties. When was prepared by percolation with ethyl acetate, gamboge inhibited the acute phase of inflammation in ethyl phenylpropiolate-induced ear edema and carrageenin-induced hind paw edema in rats (Panthong et al. 2007).

The phytochemical screening revealed that the CHCl3 fraction contained emodin, forbesione, gaudichaudionic acid and gaudichaudionol. Emodin, the main bioactive component of rhubarb (R. palmatum), has cytotoxic activity in several types of cancer cell lines (Hsu et al. 2011), including the human tongue squamous cancer cell line (SCC-4) (Lin et al. 2010; Lin et al. 2009), murine leukemia cell line (WEHI-3) (Chang et al. 2011), hepatocellular carcinoma cell line (Mahlavu, PLC/PRF/5 and HepG2) (Jing et al. 2002; Wang et al. 2012), and colorectal cancer cell (SW 480, SW620 and HT22) (Ahn et al. 2016; Pooja and Karunagaran 2014). Moreover, previous data indicated that emodin (50 μM) significantly down regulated Wnt signalling in human colorectal cancer cells. Wnt signalling is involved in the regulation and differentiation of colorectal cancer cells (Way et al. 2014). Emodin inhibits the transcriptional activity and expression of β-catenin, TCF/LEF and p300 complex. The suppression of MMP-2, MMP-9, snail, vimentin, and vascular endothelial growth factor in cancers is thought to be associated with the high metastatic potential of melanoma cells. Emodin also significantly down regulates the NF-κB pathway via its reduced DNA binding activity in colorectal cancer cells (Suboj et al. 2012; Pooja and Karunagaran 2014).

Reactive oxygen species (ROS) are also important for apoptosis. In a previous study, emodin increased ROS production but did not show cytotoxic or genotoxic effects, confirming its pro-oxidant properties which has been found to increase the sensitivity of tumour cells to anticancer drugs (Yang et al. 2004). However, the action of ROS is short because they are rapidly removed by enzymatic and non-enzymatic antioxidants. Current study demonstrated that emodin significantly increased radical-scavenging enzymes such as GSH (Cui et al. 2014). So, emodin may play a role in maintaining the pro-oxidant–antioxidant balance.

Forbesione, an extract of G. hanburyi, has cytotoxicity against cholangiocarcinoma cell lines and induces apoptosis by down-regulating the Bcl-2 protein but also regulates the expression of Bax and AIF proteins, leading to the activation of caspase-9 and caspase-3 and DNA fragmentation (Hahnvajanawong et al. 2014).

Conclusions

This is the first report of the cytotoxic and antioxidant activities of Pikut Trichinthalamaga remedy extracts. The ethanolic extract of Pikut Trichinthalamaga remedy (PTE) displayed a high cytotoxicity against SW 480 and LS-174 T cell lines. Furthermore, PTE also inhibited both chemical and cellular based antioxidant activities. These encouraging results support further work on the potential anticancer properties of Pikut Trichinthalamaga remedy as an anticancer drug. Such work should include determining their underlying molecular mechanisms of action, preclinical pharmacology and toxicokinetics to ascertain its safety before pharmaceutical development.