Screening of NO Inhibitor Release Activity from Soft Coral Extracts Origin Palu Bay, Central Sulawesi, Indonesia

Abstract

Background:

As a marine organism, soft corals can be utilized to be various bioactive substances, especially terpenoids and steroids. The soft corals family which produces bioactive generally come from clavulariidae, alcyoniidae, nephtheidae and xeniidae family.

Objective:

To investigate the bioactivity of Nitric Oxide (NO) inhibitor release from soft coral crude extracts of Sinularia sp. (SCA), Nephthea sp. (SCB), Sarcophyton sp. (SCC), Sarcophyton sp. (SCD), Sinularia sp. (SCE) and Sinularia sp. (SCF).

Materials and Methods:

Soft coral is collected from Palu Bay (Central Sulawesi). NO inhibitory release activity measured according to the Griess reaction. Soft corals sample macerated with 1:2 (w/v). Then, Soft coral extracts with the best NO Inhibitor activity partitioned with Dichloromethane, Ethyl acetate, and n-butanol. The bioactive of all crude extracts were identified by GC-MS to find compounds with anti-inflammatory potential.

Results:

Sarcophyton sp. (SCC) and Sinularia sp. (SCF) are able to inhibit NO concentrations of 0.22 ± 0.04 and 0.20 ± 0.04 μM at 20 mg/mL, respectively. The chemical constituents determined and showed the potential as anti-inflammatory in the crude of Sinularia sp. (SCA) were Octacosane (3.25%). In Nephthea sp., (SCB) were Cyclohexene, 6-ethenyl-6-methyl-1-(1-methylethyl)-3-(1-methylethylidene)-,(S)- (0.55%); Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethylidene)-, (1S-cis)- (0.53%); and 1,7,7-Trimethyl-2-vinylbicyclo[2.2.1]hept-2-ene (4.72%). In Sarcophyton sp, (SCC) were Eicosane (0.12%); Nonacosane (10.7%); 14(β)-Pregnane (0.87%); Octacosane 6.39%); and Tricosane (1.53%). In Sarcophyton sp. (SCD) were 14(β)-Pregnane (2.69%); and Octadecane (27.43%). In crude of Sinularia sp. (SCE) were Oleic Acid (0.63%); 7,10-Hexadecadienoic acid, methyl ester (0.54%); 14(β)-Pregnane (1.07%); 5,8,11,14-Eicosatetraenoic acid, ethyl ester, (all-Z)- (4.60%); Octacosane (7.75%); and 1,2-Benzisothiazole, 3-(hexahydro-1H-azepin-1-yl)-, 1,1-dioxide (1.23%). In the crude of Sinularia sp., (SCF) were Oxirane, decyl- (1.38%); Nonacosane (0.57%); Cyclohexanol, 5-methyl-2-(1-methylethenyl)- (0.61%); 14B-Pregnane (0.76%); and Tetratriacontane (1.02%).

Conclusion:

The extract of Sarcophyton sp. (SCC) and Sinularia sp. (SCF) showed the best NO inhibitory release activity. This study is making soft corals from Central Sulawesi, Indonesia can become a potential organism in the discovery and development of bioactive substances anti-inflammatory.

1. INTRODUCTION

Indonesian marine organisms are so diverse and have potential as bioactive producer. Soft corals are well-known as various bioactive substances source, especially terpenoids and steroids with exceptional functionality [1-6]. Soft corals are included in cnidarian phylum, anthozoa class, octocorallia subclass and 60% of bioactive substances reportedly have the potential to produce drug compounds [7-9].

Soft corals are soft-bodied sessile invertebrates, having no physical defense systems that rely on chemical defense system to survive [2]. The percentage of drug raw materials from soft coral found at 11-17% [10]. Soft corals become a source of biologically active molecules and model compounds used in drug material [11, 12].

The soft corals family which produces bioactive generally come from Clavulariidae, Alcyoniidae, Nephtheidae, Xeniidae family. Family Alcyoniidae most reported as bioactive producer [13]. Soft corals were reported to produce a variety of unique bioactive substances, including sesquiterpenoid, diterpenoid and steroids compounds [14]. The bioactive substances isolated from soft corals had anti-inflammatory activity [15, 16].

The anti-inflammatory properties often isolated and applied to the treatment of fever, pain and inflammatory conditions [17]. From the literature, it was reported that soft corals produce potentially anti-inflammatory compounds by inhibiting and reducing the expression of iNOS protein [18-25]. iNOS (inducible nitric oxide synthase) is one of the pro-inflammatory proteins, which can affect the development of acute to be chronic inflammatory responses and is closely related to the development of chronic human disease, including Alzheimer's [26], atherosclerosis, arthritis [27], diabetes [28], inflammatory bowel disease [29], and cancer [30].

The production of nitric oxide (NO) by iNOS involves in the pathogenesis of inflammatory response [31]. NO concentration by iNOS expressed in macrophage cells is related to phagocytic activity [32]. The inhibitor of NO release in macrophages is one way to study the potential of anti-inflammatory agents. Increased levels of NO in chronic inflammatory affect various palogical conditions [33]. The activity and expression of the iNOS enzyme are related to the production of NO. Therefore, one way of developing anti-inflamma-tory agents is by controlling the levels of NO in macrophages [34].

The oceans of eastern Indonesia hold great potential in the marine organism biodiversity [35]. The great potential of the marine organism is theoretically also a potential for the discovery of bioactive substances from the marine organism [36]. However, there is not so many exploration of soft coral potential bioactive substances. Only 19 new compounds were isolated from soft coral and ascidians and have the potential pharmacological [37]. Based on the description above, this research examines NO-Inhibitor of six soft corals origin Palu Bay, Central Sulawesi, Eastern Indonesia. NO inhibitory release potency was measured by observing the inhibition of LPS-induced NO release. The aim of this research is to investigate the NO inhibitor release of soft corals crude extract Sinularia sp. (SCA), Nephthea sp. (SCB), Sarcophyton sp. (SCC), Sarcophyton sp. (SCD), Sinularia sp. (SCE) and Sinularia sp. (SCF) from Central Sulawesi, Indonesia, which is one oceans with high biodiversity.

2. MATERIALS AND METHODS

2.1. Chemical and Reagents

Dichloromethane (DCM with p.a grade and purity of purchased from Merck), methanol (MeOH p.a, Merck), Ethyl acetate (EtOAc p.a, Merck), n-butanol (BuOH p.a, Merck), dimethyl sulfoxide p.a, (Merck), Dulbecco’s modified Eagle’s medium (DMEM/F12) powder Gibco Life Technologies, 10% Fetal Bovine Serum (FBS), 1% penisilin-streptomicin, Lipopolysaccharide LPS E. coli O111:B4 (List Biological Laboratory, Inc.), Griess Reagent Kit for Nitrite Determination G-7921 (Thermo Fisher Scientific).

2.2. Animal Materials

Soft coral samples were collected from the coastal of Kabonga Besar Village, Donggala District, Palu Bay, Central Sulawesi, Eastern Indonesia at coordinates 43.31 South Latitude and 119.46 East Longitude in December, 2016. Sampling was done with equipment SCUBA at a depth of 3-5 m. Each soft coral sample was rinsed with seawater and immediately stored in ice. After arriving at the laboratory, samples of soft corals were cut into smaller sizes, put into containers and stored in a freezer immediately. This study was approved by Animal care and used committee with ethical clearance number 680-KEP-UB.

2.3. Extraction

A total of 350 g (wet weight) sample was macerated 1:2 w/v with methanol: dichloromethane (1: 1) for 48 hours [18, 38, 39]. Sample was filtered, evaporated (Rotary Vacuum Evaporator EYELA N-1100), dried, weighed and divided into several parts: SCA (4.08 g), SCB (5.21 g), SCC (2.99 g), SCD (8.59 g), SCE (7.12 g), and SCF (5.58 g).

The maceration was performed three times each of the soft coral samples. Soft coral extracts with the best NO inhibitor activity were partitioned with Dichloromethane, Ethyl acetate, and n-butanol for 24 hours. Then, evaporated and weighed, so obtained SCC DCM (0.93 g), SCC EtOAc (0.08 g), SCC BuOH (0.06 g), SCE DCM (2.32 g), SCE EtOAc (0.27 g), SCE BuOH (0.18 g), SCF DCM (1.64 g), SCF EtOAc (0.14 g), SCF BuOH (0.09 g). Each crude extracts were subjected to preliminary phytochemical screening and assays for NO inhibitory activity.

2.4. Phytochemical Screening

All crude extracts were subjected to initial phytochemical screening assay to check the presence of secondary metabolites using the standard conventional protocol described by Harborne [39].

2.5. In Vitro NO Inhibitory Release Activity

In vitro anti-inflammatory assay followed instructions of of B.-W with modification [40-42]. Macrophage cells isolated from mice BALB/c were obtained from the Biomedical Center Laboratory, Faculty of Medicine, Brawijaya University. Isolation of macrophages from mice following the instructions of Zhang et al. with modifications [43].

Mice were cervical-dislocated and then placed in supine position, abdominal skin opened and cleaned the peritoneum sheath with 70% alcohol. Then, injected ±10 mL cold DMEM medium into the peritoneal cavity (wait ± 1 minute while pressed-press slowly). After that, the peritoneal fluid aspirated from the peritoneal cavity by tapping with two fingers of internal organs, fluids aspirated by syringe injection, selected in a non-fat and distant part of the intestine. Aspirates collected in a centrifuge tube and centrifuged (Biosan Centrifuge LMC-3000) at 800-1000 rpm at room temperature, 8-10 minutes. Then, the supernatant was removed and added to 1 ml of medium DMEM complete (containing 10% FBS) in pellet obtained.

The macrophage cells placed in TC plate 96 well (1 × 10⁶ cell/well) were suspended in a DMEM medium containing 10% FBS, at 5% CO₂ incubator, 37ºC for 2 hours. Inflammation in macrophages was induced by incubating them for 24 h in a medium containing LPS (0.01 µg/mL) without the presence of test extracts. The crude extracts SCA-SCF (5, 10, 20 mg/mL) were added to the cells 5 min before LPS challenge, respectively. The culture supernatant was collected to calculate the concentration of NO by Griess reaction and read using a microplate reader (Bio-Rad 550) absorbance of 570 nm.

2.6. Gas Chromatography and Mass Spectrometry (GC/MS)

For a quantitative analysis of bioactive profiles from all crude extracts, Hewlett-Packard (HP) 6890 GC MS was used with Agilent 19091S-433 HP-5MS column having 30 m length and 250 µm id. Helium was used as carrier gas at flow rate of 1 mL/min and oven temperature was set at 325°C. The initial oven temperature was 150°C which was held at 1°C/min. It ran for 10°C/min and was later increased to 240°C hold time for 2 min. The total run time was 22 minutes. The scan range was 50 - 550 amu. Structural assignments were based on analysis of fragmentation pattern of mass spectra and direct comparison of mass spectra with profiles in the National Institute of Standards and Technology (NIST) and Wiley library.

2.7. Statistical Analysis

All experimental measurement data were performed in three replicates and expressed as Mean ± SD (n = 3). The results of research were tested for statistical significance with One-way ANOVA. Differences were considered statistically significant at P <0.05. Statistical analysis was completed using The Microsoft Excel 2013 data processing program.

3. RESULTS

The procedure of identification of soft corals samples follows the instructions of Fabricus & Alderslade (2001). Based on monomorphic colony color, interior and surface sclerites, soft coral samples were identified as Sinularia sp. (SCA), Nephthea sp. (SCB), Sarcophyton sp. (SCC), Sarcophyton sp. (SCD), Sinularia sp. (SCE) and Sinularia sp. (SCF) as presented in Fig. (1).

Fig. (1).

Soft corals from Palu Bay, Central Sulawesi.

The phytochemical analysis of all soft corals crude extracts was presented in Table 1. The crude extracts by using solvents dichloromethane: methanol of soft corals were assayed to detect secondary metabolites. The chemical constituent analysis of all crude extracts indicated the presence of saponins, polyphenols (tannins), steroids, triterpenoids, alkaloids, and flavonoids.

Table 1. Phytochemical analysis of all soft corals crude extracts.

Chemical Constituents	Soft Corals Species						Standard
	Sinularia sp. (SCA)	Nephthea sp. (SCB)	Sarcophyton sp. (SCC)	Sarcophyton sp. (SCD)	Sinularia sp. (SCE)	Sinularia sp. (SCF)
Saponins	+	-	-	+	+	+	Stable foam formed for 15 minutes
Polyphenols (tannins)	+	+	+	+	-	+	Brown precipitate formed
Steroids	-	+	-	-	+	-	Green or blue colour produced (Lieberman-Buchard)
Triterpenoids	+	-	+	+	-	+	Brown or reddish-brown colour produced (Lieberman-Buchard)
Alkaloids	+	+	+	+	+	+	Orange precipitate formed (Dragendorff)
Flavonoids	+	-	-	-	+	-	Orange, pink or red colour produced

+: Present; -: Absent.

Six soft corals crude extract activity had analyzed as an inhibitor of NO release in macrophages. NO inhibitor activity was then evaluated by LPS-induced NO production on peritoneal mice macrophage cells, as presented in Fig. (2).

Fig. (2).

Inhibitory effects of all soft corals crude extracts on LPS-induced NO production in Mice Peritoneal Macrophages cell. Extracts were tested at 5 - 20 mg/ml. Data are presented as the means ± SD of three replications. Sarcophyton sp. (SCC) and Sinularia sp. (SCF) extracts showed the best NO inhibition activity. Based on statistical analysis, the difference of extract concentration affects the NO concentration, not the different types of soft corals.

Fig. (2) illustrates all soft coral crude extracts, showed the ability to inhibit NO production. Sarcophyton sp. (SCC) and Sinularia sp. (SCF) extracts showed the best NO inhibition activity with the concentration of NO, 0.22 ± 0.04 and 0.20 ± 0.04 μM at 20 mg/mL, respectively. On the other hand, NO concentration on negative control was 9.12 ± 0.07 μM. Sinularia sp. (SCA), Nephthea sp. (SCB) and Sarcophyton sp. (SCD) extracts increases NO concentration at 20 mg/mL. The results of statistical analysis obtain that the different types of soft corals did not affect the concentration of NO, while the difference of extract concentration affected. The different types of soft corals and concentration of the extract did not show any interaction on the concentration of NO. The results of analysis of variance presented in Table 2.

Table 2. Analysis of variance two-factor with replication ANOVA.

Source of Variation	SS	df	MS	F	P-value	F crit
Sample	0.221	9	0.025	1.194	0.351	2.393
Columns	0.978	1	0.978	47.476	0.000	4.351
Interaction	0.109	9	0.012	0.585	0.794	2.393
Within	0.412	20	0.021	-	-	-
Total	1.720	39	-	-	-	-

Furthermore, soft coral extract Sarcophyton sp. (SCC), Sinularia sp. (SCE) and Sinularia sp. (SCF) was partitioned by polarity. Then, evaluated by LPS-induced NO production on peritoneal mice macrophage cells, as presented in Table 3.

Table 3. Inhibitory effects of SCC, SCE, and SCF partitioned extracts on LPS-induced NO production in Mice Peritoneal Macrophages cell. Extracts were tested at 5 - 20 mg/ml. Data are presented as the means ± SD.

Fractions	NO Production (µM)
	5 mg/ml	10 mg/ml	20 mg/ml
Control with LPS	163.38 ± 3.79
Control without LPS	6.00 ± 0.92
SCC DCM	4.85 ± 0.17	6.31 ± 0.21	6.08 ± 0.01
SCC EtOAc	6.48 ± 0.25	6.46 ± 0.38	6.62 ± 0.34
SCC BuOH	8.46 ± 0.17	5.15 ± 0.21	4.77 ± 0.21
SCE DCM	6.54 ± 0.32	6.38 ± 0.17	6.77 ± 0.17
SCE EtOAc	6.38 ± 0.32	6.92 ± 0.17	7.08 ± 0.21
SCE BuOH	4.62 ± 0.17	4.54 ± 0.01	6.38 ± 0.17
SCF DCM	6.08 ± 0.01	7.23 ± 0.27	6.54 ± 0.17
SCF EtOAc	5.07 ± 0.50	5.84 ± 0.66	7.97 ± 0.28
SCF BuOH	6.23 ± 0.34	6.62 ± 0.21	5.69 ± 0.27

Table 3 illustrates SCC, SCE, and SCF partitioned extracts showed the ability to inhibit NO production. Sarcophyton sp. (SCC) DCM and Sinularia sp. (SCF) EtOAc extracts showed the best NO inhibition activity with the concentration of NO 4.85 ± 0.17 and 5.07 ± 0.50 μM at 5 mg/mL, respectively.

The study was extended by GC-MS analysis of soft coral crude extracts (SCA-SCF). The mass spectrum of each compound compared with that in the NIST and Wiley library. In the crude extract Sinularia sp. (SCA) there were 68 peaks, but only 52 peaks were detected with qualities above 85% and identified in 25 compounds.

Crude extract Nephthea sp. (SCB) there were 64 peaks, but only 35 peaks with qualities above 85% and identified in 33 compounds.

Crude extract Sarcophyton sp. (SCC) there were 31 peaks, but only 23 peaks with quality min 85% and identified in 18 compounds.

In the crude extract Sarcophyton sp. (SCD) there were 33 peaks, but only 26 peaks were detected with qualities above 85% and identified in 15 compounds.

Crude extract Sinularia sp. (SCE) there were 56 peaks, but only 44 peaks with qualities above 85% and identified in 35 compounds. In the crude extract Sinularia sp. (SCF) there were 29 peaks, but only 25 peaks with quality min 85% and identified in 22 compounds.

Based on GC-MS analysis and literature studies, there were 148 compounds identified in all crude extracts (SCA-SCF), and there were 22 compounds that have the potential to be anti-inflammatory. The chemical constituents identified by GC-MS analysis of all crude extracts that indicated as potentially anti-inflammatory presented in Table 4.

Table 4. The chemical constituents probabilities identified by GC-MS analysis of all crude extracts that were indicated as potentially anti-inflammatory.

Compound	Molecular Formula	RT (min)	Area (%)	Quality (min 85)	Library	References
Sinularia sp. (SCA)
Octacosane	C28H58	11.466	3.25	91	NIST	[44]
Nephthea sp. (SCB)
Cyclohexene, 6-ethenyl-6-methyl-1-(1-methylethyl)-3-(1-methylethylid ene)-, (S)-	C15H24	4.745	0.55	90	NIST	[45]
Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl-7-(1-methylethylidene)-, (1S-cis)-	C15H24	5.957	0.53	93	NIST	[46]
1,7,7-Trimethyl-2-vinylbicyclo[2.2.1]hept-2-ene	C12H18	6.334	4.72	90	NIST	[47]
Sarcophyton sp. (SCC)
Eicosane	C20H42	7.357	0.12	86	NIST	[48]
Nonacosane	C29H60	13.352	6.78	98	NIST	[48]
		21.525	3.92	96	NIST
14(β)-Pregnane	C21H36	14.398	0.87	95	Wiley	[49]
Octacosane	C28H58	17.450	6.39	99	NIST	[44]
Tricosane	C23H48	18.250	0.32	95	NIST	[48]
		19.227	1.21	93	NIST
Sarcophyton sp. (SCD)
14(β)-Pregnane	C21H36	6.997	0.11	99	Wiley	[49]
		15.758	0.77	96	Wiley
		15.953	0.98	96	Wiley
		17.302	0.83	97	Wiley
Octadecane	C18H38	10.798	13.06	95	NIST	[44]
		18.273	9.27	97	NIST
		21.977	5.10	95	NIST
Sinularia sp. (SCE)
Oleic Acid	C18H34O2	7.351	0.63	86	NIST	[50]
7,10-Hexadecadienoic acid, methyl ester	C17H30O2	8.740	0.54	90	NIST	[51]
14(β)-Pregnane	C21H36	10.752	0.36	97	Wiley	[49]
		17.296	0.71	95	Wiley
5,8,11,14-Eicosatetraenoic acid, ethyl ester, (all-Z)-	C22H36O2	12.381	4.60	95	NIST	[51]
Octacosane	C28H58	18.256	7.75	91	NIST	[44]
1,2-Benzisothiazole, 3-(hexahydro-1H-azepin-1-yl)-, 1,1-dioxide	C13H16N2O2S	18.759	1.23	91	NIST	[48]
Sinularia sp. (SCF)
Oxirane, decyl-	C12H24O	10.003	1.38	92	NIST	[52]
Nonacosane	C29H60	10.363	0.13	94	NIST	[48]
		12.346	0.44	90	NIST
Cyclohexanol, 5-methyl-2-(1-methylethenyl)-	C10H18O	10.906	0.61	86	NIST	[53]
14(β)-Pregnane	C21H36	11.060	0.26	90	Wiley	[49]
		11.969	0.50	86	Wiley
Tetratriacontane	C34H70	13.678	1.02	95	NIST	[44]

The results also showed an increase in NO production at the concentration of 20 mg/ml; this indicated that there were cytotoxic compounds. The chemical constituents of the GC-MS analysis in Table 5 showed compounds that indicated as cytotoxic.

Table 5. The chemical constituents Probabilities identified by GC-MS analysis of all crude extracts that were indicated as cytotoxic.

Compound	Molecular Formula	RT (min)	Area (%)	Quality (min 85)	Library	References
Sinularia sp. (SCA)
Heptadecanoic acid, 16-methyl-, methyl ester	C19H38O2	10.969	1.60	93	NIST	[54]
1-Hexacosene	C26H52	11.792	2.52	97	NIST
		13.346	0.75	92	NIST
Tricosane	C23H48	12.089	2.16	90	NIST	[55]
		12.232	3.55	91	NIST
		13.581	1.45	91	NIST
		14.175	0.82	95	NIST
		14.838	9.62	95	NIST
		15.044	0.44	96	NIST
		15.947	0.47	90	NIST
Nephthea sp. (SCB)
Cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)- [1S-(1.al pha.2.beta.,4.beta.)]-	C15H24	3.836	1.30	90	NIST	[56]
(-)-Aristolene	C15H24	4.185	0.68	99	NIST	[57]
Hexadecanoic acid, methyl ester	C17H34O2	9.060	1.22	97	NIST	[58]
.beta.-caryophyllene	C15H24	9.226	0.34	92	Wiley	[2]
Octadecanoic acid, methyl ester	C19H38O2	10.969	0.52	97	NIST	[59]
Octadec-9-enoic acid	C18H34O2	11.106	0.97	99	NIST	[60]
5,8,11,14-Eicosatetraenoic acid, ethyl ester, (all-Z)-	C22H36O2	12.386	3.31	94	NIST	[61]
Sarcophyton sp. (SCD)
9,17-Octadecadienal, (Z)-	C18H32O	8.334	0.14	92	NIST	[62]
Hexadecanoic acid, methyl ester	C17H34O2	9.054	1.06	95	NIST	[58]
1-Hexacosene	C26H52	12.781	0.86	94	NIST	[63]
Tricosane	C23H48	12.935	1.53	96	NIST	[55]
		13.164	1.87	92	NIST
Sinularia sp. (SCE)
Cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)- [1S-(1.al pha.2.beta.,4.beta.)]-	C15H24	3.836	0.71	98	NIST	[56]
		9.654	1.01	89	NIST
7-Pentadecyne	C15H28	9.134	0.94	98	NIST	[64]
Tricosane	C23H48	12.986	2.86	91	NIST	[55]
1-Hexacosene	C26H52	14.061	0.76	94	NIST	[63]
		17.181	1.50	90	NIST
		20.302	0.18	95	NIST
Sinularia sp. (SCF)
Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2-butynyl]-	C13H22N2O2	9.060	0.25	91	Wiley	[65]
n-Eicosane	C20H42	10.980	0.41	93	Wiley	[55]
Tricosane	C23H48	11.415	0.35	91	NIST	[55]
		18.250	0.89	95	NIST
1-Hexacosene	C26H52	11.792	3.44	95	NIST	[63]

4. DISCUSSION

The analysis of the bioactive substances component of all soft coral crude extracts showed the presence of saponins, polyphenols (tannins), steroids, triterpenoids, alkaloids, and flavonoids. Phytochemical analysis of Sinularia sp. and Lobophytum sp. also reported the presence of alkaloids, Flavonoids, tannins, steroids, triterpenoids and saponins [38, 66]. Previous research has reported the terpenoid and steroids derivative bioactive compounds of the soft coral genus Lobophytum, Sarcophyton. Nephthea and Sinularia, showing potential anti-inflammatory biological activity. This research also gives information on anti-inflammatory of the soft coral extract of Nephthea sp., Sarcophyton sp., and Sinularia sp.

NO is a potential mediator of physiological processes, with functions related to cell signaling and vasodilation, protect the organs from ischemic damage, and also shows antimicrobial and antitumor activity [67]. NO is a mediator synthesized by the enzyme NO-synthase (NOS) [68], which can be divided into two types: constitutive isoform (NOS endothelial and neuronal NOS) and an inducible isoform (iNOS). iNOS is regulated by inflammatory mediators (LPS, cytokines) and increased levels of NO by iNOS was directly involved in the pathogenesis of inflammatory response [31].

NO endothelial-derivate induce vascular relaxation (vasodilation) and platelet aggregation and adhesion inhibitors [69]. Some research results report the importance of anti-inflammatory agents that can control NO, as cardioprotective and hypotensive agents [70, 71]. In the chronic inflammatory stage, iNOS produces NO as an inflammatory mediator, causing vasodilation and edema at the site of inflammation [72]. Thus, by inhibiting NO production directly it also inhibits the expression of the iNOS enzyme and this is one way in the treatment of inflammation.

Chemical constituents of the six crude extracts showed the potential of each extract which could be developed as a potential anti-inflammatory agent. In the extract of Sinularia sp. (SCA) there are 3.25% compounds that have the potential as anti-inflammatory agents; Nephthea sp. (SCB) of 5.80%; Sarcophyton sp. (SCC) of 19.61%; Sarcophyton sp. (SCD) of 30.12%; Sinularia sp. (SCE) of 15.82%; and Sinularia sp. (SCF) of 4.34%.

However, there was increased NO concentrations at 20 mg/ml, indicating the presence of substances as NO activators and cytotoxic effects if at high levels (Wanzola et al., 2010). Table 5 shows the chemical constituents GC-MS analysis indicated cytotoxic. There are 23.38% of SCA extracts which are cytotoxic; SCB has 8.34%; SCD has 5.46%; SCE has 7.96%; while SCF has 5.34%. This data confirms that crude extracts of Sarcophyton sp. (SCC) are best developed as an anti-inflammatory agent because from the compound profile the results of a GC-MS analysis are not obtained by compounds that have cytotoxic properties. Compounds with cytotoxic properties can damage cells and can increase NO concentration as an immune response in its role to kill tumor or cancer cells. In the inflammatory response, by inhibiting NO level, it is associated with inhibition of iNOS expression, in this case it can prevent the acute inflammatory response from becoming chronic.

The soft coral genus Sarcophyton is a rich source cembraneterpen [73-76]. About 100 references have reported the results of research on secondary metabolites with various biological variations of the genus Sarcophyton [77]. Previous research has isolated seven compounds from Sarcophyton crassocaule which indicate efficacy as an anti-inflammatory because it can inhibit the expression of iNOS protein in RAW267.7 macrophage cells were stimulated LPS [19]. Six compounds were isolated from S. ehrenbergii also able to reduce the expression of iNOS protein [78]. Soft corals S. pauciplicatum also reportedly able to reduce the expression of iNOS [79].

In the octocorallia subclass of the genus Nephthea, there are a variety of species, which produce various sesquiterpenes, diterpenes, and steroids compounds [15, 80, 81]. Genus Nephthea is a famous coral reef organisms with a wonderful source of terpenoids with various biological activities and widespread throughout the world, especially in the Indo-Pacific region [61, 82]. Previous research has presented the genus Nephthea proven to produce compounds capable of inhibiting the accumulation of iNOS protein expression, such as N. columnaris produces Columnariols A and B [18]; N. erecta produces erectathiol [25]; and N. chabroli produces 4-methylated steroids, nebrosteroids A-E, G-H [83], nebrosteroids M, 19-oxygenatedsteroids, nebrosteroids I-J, K-L [25].

Sinularia genus has proven to be a rich source of bioactive steroids [1, 4]; diterpenoids [84-86], sesquiterpenoids [87-89] and cembranoids [90, 91] with various biological activities [92]. The many previous studies have reported that compounds isolated from soft coral genus Sinularia have been shown to inhibit or reduce the pro-inflammatory protein expression of iNOS and NO production in macrophages, among others Cembrane-based diterpenoids compound were isolated from S. triangular [84]; Secosterol isolated from S. granosa [23]; Sinularioside from Sinularia sp. origin of Bunaken Marine Park North Sulawesi [93]; Crassarosterosides A and C were isolated from S. crassa [94]; Eight diterpenoid compounds from S. flexibilis [95]; Crassarines F and H were isolated from S. crassa which significantly inhibited iNOS protein [96]; 11-dehydrosinulariolide, sinulariolide and 11-epi-sinulariolide acetate from S. discrepans [97]; Thioflexibilolide A was isolated from S. flexibilis [98]; Gyrosanolides A-C and gyrosanin A were isolated from S. gyrosa [16, 99]. Flexibilisolide A and flexilarin from S. granosa and S. querciformis [100]; (+)-11,12-epoxysarcophytol A potentially anti-inflammatory of S. gibberosa [101]. Grandilobatins D was isolated from S. grandilobata [102].

Chemical constituents from the results of GC-MS analysis showed variations in chemical compounds with bioactivity potential from soft coral extracts. These compounds are thought to interact with each other to provide biological effects. Bioactive substances from natural products can work in synergy between compounds with one another [103]. Natural products can work through multi-compound and multi-target synergistic modes [104].

The chemical compounds identified with the GC-MS analysis in soft coral extracts also show potential as antioxidants. Sarcophyton sp. (SCC) there are 13.39%, i.e. 1-Hexadecanethiol [105]; 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexame-thyl-, (all-E)- [106]; 2-Dodecen-1-yl(-) succinic anhydride [107]; and Octacosane [44]. Sinularia sp. (SCF) has 19.81%, i.e. Cyclohexanol, 5-methyl-2-(1-methylethenyl)- [53]; Nonadecane [108]; Bacchotricuneatin c [59]; Tetrapentacontane, 1,54-dibromo-; Hexatriacontane [109]; and Longifolenaldehyde [110]. These compounds can become a natural anti-inflammatory-mechanism of action of bioactive antioxidants, namely by providing electrons, so that free radical molecules are unstable. Bioactive antioxidants can prevent oxidative stress through the scavenging of free radicals. Inflammation can be inhibited by preventing stress oxidative in cells [111]. The author also determined the antioxidant potential (Scavenging DPPH Method) of the six soft coral extracts in other research. The results showed that all six extracts could scavenge DPPH radicals. This research has presented at the International Conference on Fisheries and Marine, Airlangga University October 6^th, 2018 (The manuscript is also temporary the process of publishing proceedings).

The NO is a signaling molecule that has an important role in the pathogenesis of inflammation. Excessive NO production by iNOS was detected in several inflammatory diseases [112]. NO is considered a pro-inflammatory mediator that induces inflammation because of excessive production under abnormal conditions [113]. Therefore, one pathway discovery of an anti-inflammatory agent by observing inhibition of NO levels produced by iNOS pro-inflammatory proteins [114-116].

CONCLUSION

Based on the assay results, extract of Sarcophyton sp. (SCC) and Sinularia sp. (SCF) showed the best NO inhibitory release activity. Purification and characterization of compounds from both can then be done so that the compounds that act as anti-inflammatory can be known. This study is exclusively making soft corals from Central Sulawesi, Indonesia can become a potential organism in the discovery and development of bioactive substances anti-inflammatory.

LIST OF ABBREVIATIONS

iNOS

Inducible Nitric Oxide Synthase

NO

Nitric Oxide

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All the reported experimental procedures on animals were approved by the Animal Care and Used Committee with ethical clearance number 680-KEP-UB, Indonesia.

HUMAN AND ANIMAL RIGHTS

No humans were used in the study. All the reported experiments on animals were in accordance with the Committee for the update of the Guide for the Care and Use of Laboratory Animals.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data supporting the findings of the article is available in the [Universitas Brawijaya, Doctoral Dissertation: Wendy Alexander Tanod, supervised by Yenny Risjani] at [http://ub.ac.id], reference number [In press, 2019].

FUNDING

Thanks to the Ministry of Research, Technology and Higher Education who have provided scholarship and research dissertation doctoral grants in 2018 (No. 1170/K9/KT.03/2018).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.