Anti-Inflammatory Potential of Hexane Extract of Mud Lobster (Thalassina anomala) in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Mud lobsters are crustaceans from the genus Thalassina which are lesser known and seldom seen but are nevertheless an important organism to the mangrove ecosystem. In Malaysia and Thailand, mud lobsters are eaten by locals as treatment for asthma. It is traditionally believed that they are effective in reducing the number of asthma attacks and severity of asthma symptoms. However, the therapeutic potential of mud lobster extract remains unclear and has not been fully elucidated or reported in any scientific study. The objectives of this study are to investigate the anti-inflammatory potential of mud lobster, Thalassina anomala extracts (hexane, chloroform and methanol) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and to identify the potential bioactive compounds involved. An MTT assay was performed to determine the cytotoxicity of the T. anomala extracts on RAW 264.7 macrophages. Nitrite quantification assay and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the ability of the T. anomala extracts to suppress the secretion and expression of nitric oxide (NO), Prostaglandin E2 (PGE2) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in LPS-stimulated macrophages. GC-MS analysis was done to identify putative metabolites. The hexane extract of T. anomala showed anti-inflammatory activity by significantly inhibiting the LPS-induced production of NO, PGE2, interleukin- (IL-) 6, IL-1β and tumour necrosis factor-alpha (TNF-α) in a concentration-dependent manner. Hexane extract treatment with 100 μg/mL has decreased the NO secretion into 37 μM. Meanwhile, hexane extract at concentration of 100 μg/mL able to significantly suppressed PGE2,TNF-α, IL-6 and IL-1β production into 2015 pg/mL, 2406 pg/mL, 460 pg/mL and 9.6 pg/mL, respectively. GC-MS analysis of the hexane extract revealed the presence of 19 putative compounds. The identified compounds were reported to have anti-inflammatory, antioxidant and antibacterial activities. These results suggest that the hexane extract of T. anomala potentially has anti-inflammatory properties and concentration dependently suppressed NO, PGE2 and proinflammatory cytokines’ production in LPS-stimulated macrophages. The findings provide a rational basis of the traditional use of mud lobster for inflammation-associated ailments.


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and IL-1β production into 2015 pg/mL, 2406 pg/mL, 460 pg/mL and 9.6 pg/mL, respectively. GC-MS analysis of the hexane extract revealed the presence of 19 putative compounds. The identified compounds were reported to have anti-inflammatory, antioxidant and antibacterial activities. These results suggest that the hexane extract of T. anomala potentially has anti-inflammatory properties and concentration dependently suppressed NO, PGE 2 and proinflammatory cytokines' production in LPS-stimulated macrophages. The findings provide a rational basis of the traditional use of mud lobster for inflammationassociated ailments.

INTRODUCTION
Mud lobsters (Thalassina spp.) are nocturnal organisms that belong to the Order Decapoda and are rarely seen as their habitat is far underneath the ground. Their existence is however acknowledged by the presence of their mounds. In Malaysia, the Thalassina species have been found in remote localities such as the T. anomala in Penang and Sarawak (De Man 1928), Selangor (Sasekumar 1974) and Terengganu (Hassan et al. 2015) , the T. kelanang in Carey Island and Kuala Langat, Selangor (Moh & Chong 2009;Sasekumar 1974) and the T. gracilis in Kuala Langat, Selangor (Moh et al. 2013).
Mud lobsters are important organisms in the mangrove ecosystem due to its burrowing activities which help bring and recycle nutrients from underground to the upper levels of sediment (Kartika & Patria 2012). The digging activity by this species helps loosen the mud and allow air and oxygenated water to penetrate into the ground. Their nests provide habitat for many mangrove organisms such as the mud shrimp, mud crab, spiders, snakes, snails and flatworms (Macintosh et al. 2002).
In Malaysia and Thailand, mud lobsters are eaten by the local people as a traditional remedy for asthma. Over the years, it is traditionally believed that mud lobsters are able to improve asthma condition (Holthuis 1991;Hassan et al. 2015). According to Holthuis (1991), people in Thailand use the mud lobster as a remedy for asthma, although it is not eaten directly. Instead, mud lobsters are dried, grinded into powder form, and drunk with water or dissolved in alcoholic liquor for a couple of days before being drunk. However, the therapeutic potential of this crustacean has yet to be scientifically documented.
Asthma is a treatable but incurable chronic inflammatory disease characterised by a reversible airway obstruction (Murdoch & Lloyd 2010). Inflammatory mediators are released in allergic asthma and accompanied by inflammation of the airways with increased numbers of inflammatory cells accumulating in the alveolar submucosa (Vane & Botting 1987). Since mud lobsters have been used as a traditional medicine against asthma, present study was conducted to investigate the anti-inflammatory potential of three mud lobster extracts (hexane, chloroform and methanol) towards the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Sample Collection, Species Identification and Sample Extraction
Mud lobsters were purchased from local people in Kuala Tatau, Bintulu (3°05'42.8"N, 112°51'56.0"E), Kuala Balingian, Mukah (3°00'36.9"N, 112°35'26.0"E) and Sarikei (2°07'55.5"N, 111°30'59.8"E), Sarawak. The identification of species was carried out based on the morphological characteristics described in Moh and Chong (2009) and Moh et al. (2013). There are a few morphological features that can be examined to differentiate the species such as carapace, rostrum and abdominal somite (Fig. 1). Samples were transported to the laboratory and oven-dried at 50°C for 24 h until a constant weight was attained. The dried samples were then grinded into powder using an electric grinder. The mud lobster powder was macerated in hexane (MH), chloroform (MC) and methanol (MM) by using 2:5 ratio and vortexed for 30 s before placed in a 50°C water bath for 10 min. Next, the supernatants were centrifuged at 3000 rpm for 5 min and evaporated using a rotary evaporator at 40°C. The concentrated extracts of MH, MC and MM were freeze-dried and stored at 4°C until further investigation.

Cell Culture
The murine macrophages cell line, RAW 264.7 was purchased from the American Type Culture Collection (ATCC, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% streptomycin/penicillin at 37°C in a humidified incubator with 5% CO 2 . The cells were sub-cultured in 70%-90% confluent condition to ensure healthy propagation of the cell line.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Colorimetric Assay
RAW 264.7 macrophages were cultured in a 96-well plate (100 µL/well) at a density of 1 × 10 5 cells/well and incubated for 24 h. The macrophages were treated with MH, MC and MM at various gradient concentrations of 15.625, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL, followed by additional incubation for 24 h. Then, 20 µL of MTT solution (5 mg/mL) in phosphate-buffered solution (PBS) was added to each well. The macrophages were further incubated for 3 h. Next, the medium was removed and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the purple formazan crystals. The absorbance was measured using ELx800 Absorbance Microplate Reader (BioTek Instruments Inc., VT, USA) at a wavelength of 570 nm.

Nitrite Quantification Assay
The effect of the mud lobster extract on the production of nitric oxide (NO) was determined using the level of nitrite accumulated in the culture medium. RAW 264.7 macrophages were cultured in 6-well plates at a density of 1 × 10 6 cells/well with 2 mL of cell culture media. The macrophages were incubated for 24 h. The old medium was removed and replaced with fresh medium to maintain the cells. Next, the RAW 264.7 macrophages were treated with different concentrations of MH, MC, MM and a positive control, dexamethasone (0.5 µg/mL). This was followed with the induction of LPS (1 µg/mL) for all samples except in the control. The samples were further incubated for another 24 h. After incubation, 100 µL of collected cell culture medium was mixed with 100 µL of Griess reagent (0.1% NED, 1% sulphanilamide and 2.5% phosphoric acid) and incubated in dark condition at room temperature for 10 min. The absorbance was measured using ELx800 Absorbance Microplate Reader at a wavelength of 540 nm.

Enzyme-linked Immunosorbent Assay (ELISA)
The RAW 264.7 macrophages were cultured in 6-well plates at a density of 1 × 10 6 cells/well for 24 h. The macrophages were then treated with MH and dexamethasone (0.5 µg/mL), followed with the addition of LPS (1 µg/mL) to all macrophages except the control for another 24 h to induce inflammation. The concentration of PGE 2 and cytokine mediators such as TNF-α, IL-6 and IL1β were assayed in the cultured media of macrophages using mouse ELISA kits (R&D Systems Inc., MN, USA) according to the manufacturer's instruction.

GC-MS Analysis
GC-MS analysis of MH was performed using GC-MS (Shimadzu QP2010 Ultra system, Kyoto, Japan) equipped with a capillary column Rxi-5ms (30 m length × 0.25 mm ID × 0.25 µm film thickness). The programmed temperatures were as follows: injector temperature was 250°C, ion-source temperature was 200°C, oven temperature was initially 50°C which was then increased at 3°C/min to 300°C and held for 10 min. Helium was used as the carrier gas at a pressure of 37.1 kPa. The mass spectra of separated components were identified based on data from WILEY and National Institute of Standards and Technology (NIST) libraries.

Statistical Analysis
The results from three independent experiments were summarised and all data are expressed as the mean ± standard deviation (SD). The significant differences were examined using IBM SPSS 20.0 software (SPSS Inc., Chicago, USA), one-way analysis of variance (ANOVA) and Tukey's post hoc test for pairwise comparison. p-values of 0.05 or less were considered as statistically significant.

Identification of Species
Based on the morphological characteristics described in Moh and Chong (2009), it was concluded that the samples collected are T. anomala. The features of the rostrum are useful when differentiating the species. The rostrum of all samples collected were triangular with a shallow median sulcus (groove) that does not extend beyond the adrostrals, properties identical to both T. anomala and T. squamifera. Meanwhile, T. kelanang has a waisted rostrum with an acute tip and a deep median sulcus (groove) that reaches behind the adrostrals. Besides, according to Moh and Chong (2009), the tergite of the first abdominal somite of T. anomala has two petaloid depressions in the form of an inverted V; these features existed in all of the samples collected. This differentiates T. kelanang and T. squamifera which have an inverted Y groove at the first abdominal somite located on the dorsal tergite.

Effect of T. anomala Extracts on Cell Viability
The cytotoxicity effects of T. anomala extracts (hexan, MH; chloroform, MC and methanol, MM) on the RAW 264.7 macrophages were examined using MTT assay. As shown in Fig. 2, all extracts decreased the viability of the treated macrophages in a concentration-dependent manner. Concentrations of each extracts that exhibited more than 80% cell viability were chosen for further antiinflammatory analysis. For MH, 25, 50 and 100 µg/mL concentrations were chosen. Meanwhile for MC, 12.5, 25 and 50 µg/mL concentrations were selected, and for MM, 15, 30 and 60 µg/mL concentrations were chosen for subsequent analysis.

Effect of T. anomala Extracts on NO Production
The anti-inflammatory effect of T. anomala extracts (hexane, MH; chloroform, MC and methanol, MM) was evaluated by assessing the inhibition of NO production in LPS-induced RAW 264.7 macrophages by using the Griess reagent. The results from Figs. 3, 4 and 5 show that MH exhibits a good inhibitory effect on NO production compared to MC and MM. MC and MM did not show any inhibitory activity towards NO. Based on these results, MH was chosen for further antiinflammatory analysis. The untreated control group released a low level of NO, while the LPS induction group without treatment elevated NO production (Fig. 3). In addition, dexamethasone as a positive control was able to suppress NO production significantly.

Effect of Hexane Extract of T. anomala on PGE 2 and Proinflammatory Cytokines Production (TNF-α, IL-6 and IL-1β)
Evaluation of anti-inflammatory activity was based on the ability of T. anomala extracts to suppress the production of PGE 2 and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in LPS-induced RAW 264.7 macrophages. For this analysis, the hexane extract, MH was chosen as it was the only extract that exhibited antiinflammatory activity in the NO analysis. Fig. 6A shows that the MH was able to significantly inhibit PGE 2 production. Figs. 6B, 6C and 6D show that TNF-α, IL-6 and IL-1β productions were significantly elevated in response to LPS induction. However, upon treatment with MH, the levels of TNF-α, IL-6 and IL-1β were reduced significantly. Dexamethasone as a positive control was able to inhibit the production of PGE 2 and proinflammatory cytokines as well.

GC-MS Analysis of Active Compounds
The GC-MS analysis of the hexane extract of T. anomala, MH was conducted due to the presence of anti-inflammatory activity. The GC-MS analysis of MH revealed the presence of 19 putative compounds which account for its various biological activities including anti-inflammatory, antioxidant and anti-bacterial. The putative compounds along with their retention times, molecular formula, molecular weight and percentage of peak area are listed in Table 1.

DISCUSSION
In this study, we observed three different extracts of T. anomala in order to investigate the organism's anti-inflammatory potential through its ability to inhibit the production of NO, PGE 2 and proinflammatory cytokines in LPS-stimulated RAW 264.7 macrophages. Among the three solvents, the hexane extract showed the highest inhibitory activity. A previous study done by Sindhu and Sherief (2011) reported anti-inflammatory activity among non-polar solvents extracts as well, such as the hexane:isopropanol fractions of Arabian red shrimp, Aristeus alcocki.
In addition, a study done by Khanafari et al. (2007) showed that non-polar solvent extracts from the green tiger prawn, Penaeus semisulcatus exhibited astaxanthin, an anti-inflammatory compound (Farruggia et al. 2015). Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and acts as a potent initiator of inflammation (Meng & Lowell 1997). LPS activates monocytes and macrophages to produce proinflammatory mediators such as NO, PGE 2 and cytokines (Raetz 1990;Guha & Mackman 2001;Cohen 2002). Hence, in the present study, LPS-stimulated RAW 264.7 macrophages were used as an in-vitro inflammation experimental model. The results showed increased levels of pro-inflammatory mediators observed in the LPS induction group without treatment.
The cytotoxicity effects of the three different extracts of T. anomala were determined using MTT assay. As shown in Fig. 2, the hexane extract showed the highest cell viability in comparison to the methanol and chloroform extracts. Concentrations of the extracts which are nontoxic to macrophages were chosen for further anti-inflammatory analysis. The non-toxic concentrations of the extracts were selected to ensure that the anti-inflammatory activity detected was caused by the genuine inhibitory effect of the compounds presented in the extract, and not due to cell death.
NO is a reactive free radical gas which plays a key role in regulating inflammatory responses. It is released in greater amounts during inflammation reactions (MacMicking et al. 1997). The overproduction of NO can lead to numerous inflammation-associated diseases such as cardiovascular disease, hypotension, vasodilatation, induction of apoptosis, and triggering of the pathogenesis of inflammation involving joints, guts and lungs (Sharma et al. 2007;Heo et al. 2014). Previous studies done by Alving et al. (1993); Kharitonov et al. (1995); Massaro et al. (1996) showed increased concentrations of exhaled NO in asthmatic subjects. Therefore, the level of NO may be a useful marker in monitoring asthma and evaluating the effect of anti-inflammatory treatments in reducing asthmatic symptoms (Kharitonov et al. 1994;Pendharkar & Mehta 2008). In the present study, treatments using hexane extract of T. anomala and a positive control drug, dexamethasone were able to supress the production of NO in LPS-stimulated RAW 264.7 macrophages in a concentration-dependent manner (Fig. 3).
Meanwhile, according to Tilley et al. (2003), PGE 2 triggers the bronchoconstrictor effect via the EP1 and EP3 receptors. Previous studies had reported high levels of PGE 2 in the sputum of patients with eosinophilic bronchitis, an airway inflammation disease that cause chronic cough (Brightling et al. 2000;Birring et al. 2004;Sastre et al. 2008). As shown in Fig. 6a, the PGE 2 production in the LPS-stimulated macrophages was reduced after treatment with hexane extract of T. anomala by concentration dependently.
Several studies have reported that pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β are involved in the pathogenesis of asthma as evidenced by their elevated levels in the sputum and bronchoalveolar lavage fluid of asthmatic patients (Kips et al. 1993;Albuquerque et al. 1998;Chung & Barnes 1999;Kips 2001;Mehta & Mahajan 2006;Barnes 2008). In the present investigation, a concentration-dependent decrease can be observed in the levels of TNF-α, IL-6 and IL-1β in LPS-stimulated RAW 264.7 cells after treatment with hexane extract of T. anomala (Figs. 6b-6d).
Based on the findings, the results suggest that the hexane extract of T. anomala has therapeutic potential as an anti-inflammatory agent to alleviate inflammatory respiratory ailments.
Identification of active compounds via GC-MS analysis revealed the presence of 19 putative compounds in the hexane extract of T. anomala as shown in Table 1. The major groups detected are cholesterol and fatty acids. According to Zagryagskaya et al. (2008), cholesterol exhibits anti-inflammatory activity and decreases the leukotriene synthesis in human polymorphonuclear leukocytes. Leukotriene is a substance released during asthma attacks and triggers bronchoconstriction (Berger 1999). Palmitoleic acid is another compound detected in the hexane extract of T. anomala that possess anti-inflammatory activity (Morse 2015;Astudillo et al. 2017). Studies done by Souza et al. (2017) reported that palmitoleic acid exhibits anti-inflammatory potential through its ability to reduce the expression of TNF-α and IL-6 in LPS-stimulated macrophages.

CONCLUSION
In conclusion, results obtained in this study suggested that the hexane extract of T. anomala exhibits anti-inflammatory potential in LPS-stimulated RAW 264.7 macrophages. The non-polar compounds detected in the hexane extract of T. anomala by GC-MS analysis revealed 19 putative metabolites which may contribute to the anti-inflammatory activity. The findings justify the traditional use of mud lobster as remedy for inflammation respiratory ailments.