INTRODUCTION:

Marine natural resources that can be utilized in the pharmaceutical sector other than fish¹ (Resen et al, 2017) include algae^2-4, bacteria^5-8, mollusk⁹, fungi¹⁰ and sponges¹¹. Southeast Sulawesi is an archipelago province in Indonesia. The region has 651 islands (361 named, 290 unnamed) and 74.25% of the whole province area is oceans¹². Sponge is one of the main biodiversity of this area which can be developed as a source of new chemicals and pharmaceuticals. In the past 50 years, the study on chemical and pharmaceutical aspects of sponges produced various advantaged secondary metabolites, for example, saprol C-methylcetal and orhalquinone were produced by South Pacific marine sponge Xestospongia that displayed a significant inhibition of both human and yeast farnesyltransferase enzymes¹³, and also some sterols have been isolated and identified from Mayotte Xestospongia sp.¹⁴. Furthermore, three new phenolic bisabolene sesquiterpenoid dimers, dysodonols A-C were reported from fungus Aspergillus sp. which was isolated from X. testudinaria. Dysodonol A and C were cytotoxic against HepG-2 and Caski human tumour cell lines¹⁵. In addition, isoquinolinequinone alkaloids also reported from Xestospongia sp.¹⁶, and three brominated polyacetylenes have been isolated from Chinese marine sponge X. testudinaria¹⁷. From Indonesian’s marine, xestosaprol D and E have been isolated and identified from Xestospongia sp. in Turtle Bay Sangalaki¹⁸, four antibacterial alkaloids of aaptamine class have been collected off Jakarta¹⁹, and extract of bacteria of X. testudinaria from Sorong (Indonesia) contained alkaloids and terpenoids/steroids which were active towards some bacteria²⁰, and also an antiplasmodial sterol, kaimanol, has been isolated and identified from West Papua’s Xestospongia sp.²¹.

Three sponges were distributed in the sea of Southeast Sulawesi, have been screened biological activities in Faculty of Pharmacy Universitas Halu Oleo that are Callyspongia aerizusa, Clathria basilana and Xestospongia sp. The activities included antibacterial, antifungal, toxicity and antioxidant. The extract of Xestospongia has good potency as antioxidant^22-24. Because of antioxidant has good correlation with anti-inflammatory activity²⁵. Investigating anti-inflammatory properties of sponges is beneficial for discovering novel agents against inflammatory which might reduce side effects from commercial anti-inflammatory drugs like steroids or nonsteroidal anti-inflammatory drugs (NSAIDs), including gastrointestinal disorder, hypertension, and hyperlipidemia²⁶. Qualitative chemical screening of methanol extract of Xestopongia sp. using some detection reagents has shown the presence of alkaloids, terpenoids/steroids and saponins. The LC-MS/MS analysis revealed that the sponge extract contained xestosaprol C-methylacetate, mutafuran H, fucosterol, isofucosterol, saringosterol, xestosterol, pulchrasterol, pyrophaeophorbide A and spinasterone. The extract also has potency as a radical scavenger²⁷.

As part of our continuing study, the present article reported the antioxidant and anti-inflammatory activities of ethanol extract and steroids of Xestospongia sp. from Southeast Sulawesi.

MATERIAL AND METHODS:

Sample:

Sponge Xestospongia sp. was collected from the Marine Education Centre “Bintang Samudra” of Konawe district, Sulawesi Tenggara Province, Indonesia with geographical location of 3^o53’47”S and 122^o36’43”E. The sample identification was done by Dr. Baru Sadarun, from Faculty of Fisheries and Marine Sciences, Universitas Halu Oleo, Indonesia.

Chemical:

The solvents used were methanol, ethanol, ethyl acetate, n-hexane, chloroform, aquades, and acetone. All of them are distilled technical grade solvents except chloroform is analytical grade. Chromatography works that are thin layer chromatography (TLC), vacuum liquid chromatography (VLC), and radial chromatography (RC) were performed using Kieselgel 60 F₂₅₄ 0,25 mm (Merck), silica 60 G (Merck), and silica gel 60 GF₂₅₄ p.a (Merck), respectively.

Instrumentation:

Elucidation structure of isolated compounds used FTIR (Fourier Transform Infra Red) and NMR (Nuclear magnetic Resonance) Spectroscopy. The infrared spectra of compounds were measured using a Varian 1000 FTIR spectrophotometer (Varian Inc., USA). Meanwhile, the NMR spectra were recorded using an ECP 500 MHz spectrometer (JEOL, Japan). Antioxidant potencies of the samples were calculated based on Absorbance. The Absorbance was measured using a spectrophotometer 5010 (RIELE, Germany).

Procedure:

Extraction from Xestospongia sp.:

The wet powder (230-270 mesh) of Xestospongia sp. sponge (3.6 kg) was macerated by ethanol (EtOH, 4 L) for 3 x 24 h at room temperature. The ethanol extract was concentrated by vacuum rotary evaporator at low pressure, yielding a dark brown gum (82.53 g). The extract obtained was used for isolation, antioxidant and anti-inflammatory assays.

Isolation of compounds from Xestospongia sp.:

A portion of ethanol extract (50 g) was subjected to a silica gel VLC (5 x 10cm, 150 g), eluting with n-hexane/EtOAc (from 8:2 to 0:10) followed by pure MeOH, to afford five main fractions (F1-F5): F1 (1.3 g), F2 (18.0 g), F3 (14.3 g), F4 (4,4 g) and F5 (7,2 g). Based on TLC chromatogram and weight of fraction, F2 and F3 were potential to be purified. Fraction F2 (18.0 g) was fractionated over silica gel VLC (5 x 5cm, 75 g) with n-hexane/EtOAc (from 7:3 to 0:10) as mobile phase and gave five subfractions (F21-F25). Subfraction F23 (1.0 g) was repeatedly purified using silica gel RC, eluting with CHCl₃/MeOH (9.5:0.5), to afford compound 1 (20 mg). Meanwhile, subfraction F24 (2.2 g) was subjected to silica gel RC and separated using CHCl₃/MeOH (9:1) as a mobile phase, to afford compound 2 (80 mg). Furthermore, fraction F3 (14.3 g) was fractionated using a silica gel VLC, eluting with n-hexane/EtOAc (from 7:3 to 0:10) followed by pure MeOH, to yield four subfractions (F31-F34). Subfraction F32 (1.0 g) was exhaustively purified using silica gel RC with n-hexane/EtOAc (8.5:1.5) and afforded compound 3 (10 mg). Meanwhile, repeated purification of subfraction F33 (2.8 g) through silica gel RC with n-hexane/EtOAc (7.5:2.5) as a mobile phase yielded compound 4 (10 mg).

Structures determination of pure compounds:

The structures of pure compounds were identified by using the FTIR and NMR spectral data. All values obtained were also compared to those reported in the literature.

DPPH Assay:

Antioxidant assay of the isolated compounds were measured by DPPH radical according to the previous study [27]. For screening, samples (100µL) prepared in DMSO with a final concentration of 200 µg/mL in the 96- well microplates were reacted with 0.1 mM of DPPH (100µL) and the plates were incubated for 30 min in dark at room temperature. After that, the absorbances were measured at 540nm using a microplate reader (Thermo Multiskan FC). The percentage scavenging activity (%SA) was calculated using the formula: [(AC–AS)/AC] x 100, where AC is the absorbance of control (solvent and DPPH) and AS is the absorbance of the sample after correction with sample blank. The IC₅₀ values of active samples were determined in eight serial dilutions ranging from 200 to 1.56µg/mL. The assay was performed in triplicates.

Anti-inflammatory Activity by Human Red Blood Cell (HRBC) Membrane Stabilization:

Anti-inflammatory activity of ethanol extract of Xestospongia sp. was evaluated on membrane stabilization of human red blood cell²⁸ . Whole blood was obtained voluntary from the Indonesian Red Cross Society (Palang Merah Indonesia, PMI). The use of human blood was in accordance with the ethical approval by Health Research Ethics Comittee of Universitas Halu Oleo with the number 982/UN29.20/PPM/2018. Blood sample (10mL) was centrifuged at 3000 rpm for 10 min. The supernatant layer was separated from the red blood cell by careful pipetting. The red blood cell further washed with isosaline and re-centrifugated. The separated red blood cell (2 mL) was then suspended in isosaline (18mL). The ethanol extract of sponge solutions and diclofenac sodium as positive control were prepared in isosaline with concentrations ranging from 50 to 3200ppm. The red blood cells solution (0.5mL) in phosphate buffer (1 mL, pH 7.4, 0.15 M) was transferred into tube and added with the extract solution (1 mL) and hyposaline (2 mL), following by homogenization. The mixture incubated at 56°C for 30 min and centrifuged at 5000 rpm for 10 min. The supernatant collected and its absorbance measured spectrophotometrically at 546nm. The percentage of stability of HRBC membrane was calculated according to the following equations:

% Hemolysis = (Optical density of sample / optical density of control) x 100

% Stability = 100 – [(Optical density of sample / optical density of control) x 100]

RESULTS:

Physicochemical property and spectroscopic data of the isolated compounds from Xestospongia sp.:

Compound 1, white crystal. FTIR at ν_max(cm^-1) 3307 (OH); 2925, 2852 (Csp³-H_str); 1506, 1455 (C=C); and 1049 (C-O). Signals of ¹H NMR (CDCl₃, 500 MHz) δ_H (ppm) 1.12 (m, H-1a), 1.79 (m, H-1b), 1.00 (m, H-2a), 1.85 (m, H-2b), 4,53 (1H, m, H-3), 2.29 (2H, d J=7.3 Hz, H-4), 5,34 (1H, m, H-6), 1.50 (m, H-7a), 1.20(m, H-7b), 1.43 (m, H-8), 0.94 (m, H-9), 1.46 (2H, m, H-11), 1.15 (m, H-12a), 2.02 (m, H-12b), 0.97 (1H, m, H-14), 1.50 (2H, m, H-15), 1.22 (2H, m, H-16), 1.08 (m, H-17), 0.67 (3H, s, H-18), 1.00 (3H, s, H-19), 1.97 (1H, m, H-20), 0.93 (3H, d, 6,7, H-21), 1.58 (br d, H-22), 1.66 (m, H-23), 1.77 (m, H-25), 1.34 (m, H-26), 1.34 (m, H-27), 4.75 (d, H-28a), 4.69 (d, H-28b), 0.84 (3H, m, H-29), 0.82 (3H, m, H-30). Signals of ¹³C NMR (CDCl₃, 125 MHz) (δ_C (ppm) 37.4 (C-1), 28.4 (C-2), 72.1 (C-3), 42.4 (C-4), 140.9 (C-5), 121.9 (C-6), 31.9 (C-7), 32.1 (C-8), 50.3 (C-9), 36.7 (C-10), 21.3 (C-11), 39.9 (C-12), 42.5 (C-13), 56.9 (C-14), 24.5 (C-15), 28.4 (C-16), 56.2 (C-17), 12.0 (C-18), 19.6 (C-19), 35.9 (C-20), 18.9 (C-21), 34.8 (C-22), 28,5 (C-23), 155.2 (C-24), 50.3 (C-25), 26,0 (C-26), 24.5 (C-27), 107.3 (C-28), 12.2 (C-29), 12.1 (C-30).

Compound 2, white crystal. FTIR ν_max(cm^-1) 3344 (OH); 2925, 2852 (Csp³-H_str); 1506, 1454 (C=C); 1047 (C-O eter). Signals of ¹H NMR (CDCl₃, 500 MHz) δ_H (ppm) 1.12 (m, H-1a); 1.79 (m, H-1b); 4,53 (m, H-2); 1.22 (2H, m, H-3); 2.29 (d, H-4); 0.9 (2H, m, H-6); 1.4 (s, H-7); 0.61 (3H, s, H-18); 0.75 (3H, s, H-19); 1.03 (3H, s, H-21); 5.10 (s, H-27); 1.11 (2H, s, H-28). Spectra of ¹³C NMR (CDCl₃, 125 MHz) (δ_C (ppm) 37.4 (C-1), 72.2 (C-2), 28.5 (C-3), 42.4 (C-4), 36.7 (C-5), 50.3 (C-6), 31.9 (C-7), 121.9 (C-8), 140.9 (C-9), 42.5 (C-10), 21.3 (C-11), 42.5 (C-12), 39.9 (C-13), 56.9 (C-14), 24.5 (C-15), 28.4 (C-16), 56.2 (C-17), 12.1 (C-18), 19.6 (C-19), 35.9 (C-20), 18.9 (C-21), 34.8 (C-22), 28.5 (C-23), 32.9 (C-24), 26.3 (C-25), 155.9 (C-26), 24.5 (C-27), 107.3 (C-28), 12.2 (C-29), 12.1 (C-30).

Compound 3, white crystal. FTIR at ν_max(cm^-1) 3307(O-H); 2924 (Csp3-H); 1554 (C=C); 1371 C-C and 1057 (C-O ether). Signals of ¹H NMR (CDCl₃, 500 MHz) (δ_H (ppm) 5.82 (1H, dd, J=17.4, 11.0, H-28), 5.36 (1H, broad d, J=5.4, H-6), 5.20 (1H, dd, J=17.4, 1.5, H-29a), 5.15 (1H, dd, J=11.0, 1.5, H-29b), 3.52 (1H, m, H-3), 1.02 (3H, s, H-19), 0.94 (3H, d, J=6.4, H-21), 0.90 (3H, d, J=6.9, H-27), 0.88 (3H, d, J=6.9, H-26), 0.69 (3H, s, H-18). Signals of ¹³C NMR (CDCl₃, 125 MHz) δ_C (ppm) 37.4 (C-1), 31.8 (C-2), 71.9 (C-3), 42.4 (C-4), 140.9 (C-5), 121.8 (C-6), 31.8 (C-7), 32.0 (C-8), 50.2 (C-9), 36.6 (C-10), 21.2 (C-11), 39.9 (C-12), 42.5 (C-13), 56.9 (C-14), 24.4 (C-15), 28.4 (C-16), 55.9 (C-17), 12.1 (C-18), 19.5 (C-19), 36.3 (C-20), 18.9 (C-21), 29.3 (C-22), 28.3 (C-23), 77.8 (C-24), 36.3 (C-25), 16.6 (C-26), 17.7 (C-27), 142.6 (C-28), 113 (C-29).

Compound 4, white amorf. FTIR at ν_max(cm^-1) 3239 (OH); 2912, 2846 (Csp³-H_str); 1505, 1451 (C=C); and 1111 (C-O). Signals of ¹H NMR (CDCl₃, 500 MHz) (δ_H (ppm) 1.41 (2H, m, H-1), 1.60 (2H, m, H-2), 3.91 (1H, m, H-3), 1.71 (2H, m, H-4), 6.22 (1H, d, J=8,5, H-6), 6.50 (1H, d, J=8,5, H-7), 1.54 (1H, m, H-9), 1.46 (2H, m, H-11), 1.35 (2H, m, H-12a), 1,57 (1H, m, H-14), 1.40 (2H, m, H-15), 1.42 (2H, m, H-16), 0.70 (1H, m, H-17), 0.71 (3H, s, H-18), 0.80 (3H, s, H-19), 1.63 (1H, m, H-20), 0.83 (3H, d, 6,7, H-21), 1.38 (2H, m, H-22), 1.36 (2H, m, H-23), 0.82 (1H, t, J=7,4, H-24), 1.27 (2H, m, H-25), 0.84 (3H, d, J=6,7, H-26), 0.84 (3H, d, J=6,7, H-27), 1.10 (3H, s, H-28a), 0.94 (3H, s, H-29). Spectra of ¹³C NMR (CDCl₃, 125 MHz) δ_C (ppm) 34.9 (C-1), 30.3 (C-2), 66.7 (C-3), 37.1 (C-4), 82.3 (C-5), 135.6 (C-6), 131.0 (C-7), 79.6 (C-8), 51.2 (C-9), 37.1 (C-10), 23.6 (C-11), 39.6 (C-12), 44.9 (C-13), 51.7 (C-14), 20.8 (C-15), 28.4 (C-16), 56.5 (C-17), 12.8 (C-18), 18.3 (C-19), 35.9 (C-20), 18.9 (C-21), 34.8 (C-22), 26.5 (C-23), 26.2 (C-24), 23.1 (C-25), 12.5 (C-26), 29.1 (C-27), 19.1 (C-28), 19.8 (C-29).

Data of DPPH test of extract and compounds from Xestospongia sp.:

The IC₅₀ of ethanol extract, isolated compounds and ascorbic acid displayed in Table 1.

Table 1: Radical scavenger activity of the samples

Sample	IC₅₀ (g/mL)
Xestospongia sp. extract	57.2 ± 0.7
Xestosterol (1)	266.1 ± 3.4
Pulchrasterol (2)	246.7 ± 5.5
Saringosterol (3)	198.4 ± 2.8
5α, 24α-Ethylcholest 8α epidioxy-6-en-3β-ol (4)	552.67 ± 4.6
Ascorbic acid (positive control)	27.4 ± 0.5

Data of Anti-inflammatory activity by HRBC method:

The stability of HRBC post treatment with Xestospongia sp. and diclofenac sodium displayed in Table 2.

Table 2: Stability of HRBC Membranes of samples

Concentration	*Xestospongia* sp. (%)	Diclofenac sodium (%)
50 ppm	56%	59%
100 ppm	67%	67%
200 ppm	69%	75%
400 ppm	76%	81%
800 ppm	83%	85%
1600 ppm	87%	87%
3200 ppm	88%	96.30%

DISCUSSION:

Four known compounds were isolated and identified from the ethanol extract of Xestospongia sp. Their structures are displayed in Fig. 1.

Fig. 1: Structure of compounds from the sponge Xestospongia sp.

Compound 1 was isolated as a white crystal. The FTIR spectra of compound 1 at ν_max3307 cm^-1 showed the presence hydroxyl unit which supported by spectra at ν_max 1049 cm^-1for C-O_eter. The wave number at 2925 and 2852 cm^-1 indicated that compound 1 has Csp³–H unit, and alkene units were represented by spectra at ν_max 1506 and 1455 cm^-1. According to the FTIR data, compound 1 has hydroxyl, Csp³–H and alkene units. The presences of all units were supported by ¹H and ¹³C NMR data.

Spectra of ¹H NMR showed 18 signals for 50 protons with chemical shifts less than δ_H 6 ppm. It indicated that compound 1 does not have aromatic ring. The chemical shift at δ_H(ppm) 0.67 (3H, s, H-18) and 1.00 (3H, s, H-19) are characters of steroids which were supported by δ_H (ppm) 4.53 (1H, m, H-3), 5.34 (1H, m, H-6), 4.75 (1H, d, H-28a), and 4.69 (1H, d, H-28b) from a proton at oxygenated carbon, and three olefinic protons consist of a methine and a methylene, respectively. In other word, compound 1 is predicted a steroid which has one hydroxyl group and two double bonds, of which one double bond is located at the end of the carbon chain.

Spectrum of ¹³C NMR shows 30 signals which indicate 30 carbon atoms. Based on DEPT instrument, 30 carbon atoms consist of 5 carbon methyls (CH₃), 13 carbon methylenes (CH₂), 8 carbon methines (CH) and 4 carbon quarteners. According to ¹H and ¹³C NMR data, hydrocarbon formula of compound 1 is C₃₀H₄₉. In accordance with FTIR spectrum at ν_max3307 cm^-1, ¹H NMR at δ_H 4.53 (1H, m, H-3) and ¹³C NMR at δ_C72.1 ppm, compound 1 binds a hydroxyl unit, so that structure formula compound 1 to be C₃₀H₅₀O. The literature review showed that the compound 1 was found earlier on Xestospongia testudinaria [29], which has a very high similarity parameters with ¹H and ¹³C NMR data of xestosterol (Table 3). It can be concluded that compound 1 is xestosterol.

Table 2: Comparison of ¹H and ¹³C NMR data of compound 1^awith reference of xestosterol^b

No. C	δ_Cppm (a)	Type	δ_H(∑H, mult., J in Hz) (a)	δ_Cppm (b)	Type	δ_H(∑H, mult., J in Hz) (b)
1	37.4	CH₂	1.12 (m)	37.0	CH₂	1.13 (m)
1	37.4	CH₂	1.79 (m)	37.0	CH₂	1.84 (m)
2	28.4	CH₂	1.00 (m)	27.8	CH₂	1.02 (m)
2	28.4	CH₂	1.85 (m)	27.8	CH₂	1.85 (m)
3	72.1	CH	4,53 (1H, m)	73.8	CH	4.61 (m)
4	42.4	CH₂	2.29 (2H, 7.3, d)	38.2	CH₂	2.31 (2H, 7.8, d)
5	140.9	C	-	140.0	C	-
6	121.9	CH	5,34 (1H, m)	123.0	CH	5,37 (1H, m)
7	31.9	CH₂	1.50 (m)	31.8	CH₂	1.56 (m)
7	31.9	CH₂	1.20 (m)	31.8	CH₂	1.27 (m)
8	32.1	CH	1.43 (m)	31.9	CH	1.47 (m)
9	50.3	CH	0.94 (m)	50	CH	0.96 (m)
10	36.7	C	-	36.6	C	-
11	21.3	CH₂	1.46 (m)	21	CH₂	1.45 (m)
12	39.9	CH₂	1.15 (m)	39.7	CH₂	1.18 (m)
12	39.9	CH₂	2.02 (m)	39.7	CH₂	2.02 (m)
13	42.5	C	-	42.3	C
14	56.9	CH	0.97 (1H, m)	56.7	CH	1.01 (m)
15	24.5	CH₂	1.51 (2H, m)	24.3	CH₂	1.10 (2H, m)
16	28.4	CH₂	1.22 (2H, m)	28.2	CH₂	1.28 (2H, m)
17	56.2	CH	1.08 (1H, m)	56	CH	1.14 (1H, m)
18	12.0	CH₃	0,67 (3, s)	11.8	CH₃	0,67 (s)
19	19.6	CH₃	1,01 (3, s)	19.4	CH₃	1,02 (s)
20	35.9	CH	1.97 (1H, m)	35.8	CH	1.42 (1H, m)
21	18.9	CH₃	0,93 (3H, d, 6,7)	18.8	CH₃	0,94 (3H, d, 6,6)
22	34.8	CH₂	1.58 (1H, br d)	34.4	CH	1.56 (m)
23	28.5	CH₂	1.66 (1H, m)	29.3	CH	1.76 (m)
24	155.2	C	-	153	C	-
25	50.3	CH	1.77 (m)	50.2	CH	1.79 (m)
26	26,0	CH₂	1.34 (m)	26.3	CH₂	1.36 (2H, m)
27	24.5	CH₂	1.34 (m)	26.4	CH₂	1.36 (2H, m)
28	107.3	CH₂	4.75 (1Ha, d)	109	CH₂	4.76 (d, 1.8)
28	107.3	CH₂	4.69 (1Hb, d)	109	CH₂	4.68 (d, 1.8)
29	12.2	CH₃	0.84 (3H, m)	11.8	CH₃	0, 81 ( t, 7,3)
30	12.1	CH₃	0,82 (3H, m)	12	CH₃	0, 81 ( t, 7,3)

Note : a) Compound 1 b) ²⁹

The structure determination of compounds 2, 3 and 4 have the same way as the structure determination of compound 1 which led to identification of compound 2 as pulchrasterol ³⁰, compound 3 as saringosterol ³¹, and compound 4 as 5α, 24α-ethylcholest 8α epidioxy-6-en-3β-ol ³². These compounds belong to the sterol group. Compounds 1-3 have been identified in the sponge in our previous study by using an LC-MS/MS analysis ²⁷.

Radical Scavenging Activity:

All samples were weakly active compared to ascorbic acid as the positive control (Table 1). Interestingly, the ethanol extract of Xestospongia sp. was more active than the isolated compounds, indicating other potent antioxidant compounds from this extract ²⁷. Thus, further investigation on these antioxidant compounds from Xestospongia sp. would be a great research opportunity.

Structurally, the antioxidant potency of the isolated compounds is thought to be caused by the presence of double bonds in their structures. Saringosterol showed the lowest IC₅₀ value (198.4 ± 2.8 mg/mL) which might be due to the presence of two hydroxyl units in its structure. Meanwhile, the presence of a peroxide bridge in the structure of 5α, 24α-ethylcholest 8α epidioxy-6-en-3β-ol might reduce the scavenging activity of the compound.

The presence of antioxidant potency of compounds that have been successfully isolated can support the antioxidant activity of Xestospongia crude extracts. However, in this case, the extract is more active than isolated compounds as an antioxidant. This is because there are still many minor compounds that have not been isolated such as xestosaprol C methylacetate, mutafuran H, fucosterol, isofucosterol, pyrophaeophorbide A and spinasterone ²⁷. These compounds are thought to have higher antioxidant activity especially xestosaprol C methylacetate and pyrophaeophorbide A. The two compounds have conjugated double bonds and hydroxyl units and also pyrophaeophorbide A is an alkaloid. Because of Xestospongia extract is more active than the isolated compound and the number (weight) of the isolated compound is very small, the in-vivo anti-inflammatory test is only carried out on Xestospongia extract.

Anti-inflammatory Activity:

The anti-inflammatory activity of the ethanol extract of Xestospongia sp. in stabilizing the human red blood cells (HRBC) membrane was in dose-dependent manner with percentage stability of 56 to 88% at concentration ranging from 50 to 3200 ppm. The activity was comparable (p>0.05) to diclofenac sodium with percentage stability of 59 to 96% at same respective concentrations and was significantly difference (p<0.05) when compared with the negative control. The results showed that the ethanol extract of Xestospongia sp. stabilized the membrane of HRBC, suggesting its anti-inflammatory activity. The effect of the ethanol extract and diclofenac sodium on membrane stability of human red blood cells is displayed in Table 2 and Fig. 2.

The previous study showed that methanolic extract of Xestospongia testudinaria has anti-inflammatory activity by its protective effect against stress oxidative and pro-inflammatory cytokines ³³. Xestosterol, pulchrasterol, sarangosterol and 5α, 8α epidioxy 24α-ethylcholest-6-en-3β-ol suspected provide anti-inflammatory activity ³⁴. They are belonging to the sterol group, which benefits as anti-inflammatory agent. Sterols act as anti-inflammatory by inhibits phospholipase A2, an enzyme who plays a role in the inflammation process via arachidonic acid pathway^28,33.

Fig. 2.: Percentage of stablity human red blood cell (HRBC) membranes (data presented in mean (n=3))

CONCLUSION:

Four steroids were succesfully isolated from the ethanol extract of Xestospongia sp. that are (1) xestosterol, (2) pulchrasterol, (3) saringosterol, and (4) 5α, 8α epidioxy 24α-ethylcholest-6-en-3β-ol. The extracts and compounds showed antioxidant activity against DPPH radicals, which the extract was more active than the isolated compounds. The ethanol extract of Xestospongia sp. also exhibited anti-inflammatory activity by stabilizing red blood cell membranes. To support the extract activities and to find antioxidant and anti-inflammatory compounds, it is recommended that minor compounds in Xestospongia sp which are active as antioxidants and anti-inflammatory can be isolated and determined their structures by increasing the number of samples.

ACKNOWLEDGEMENT:

We would like to thank to Ministry of Research, Science, Technology and Higher Education of Republic of Indonesia for Research grant with scheme Hibah Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) in 2018, Project No: 536/UN29.20/PPM/2018.

CONFLICT OF INTEREST:

None declared.

REFERENCES:

1. Resen AK, Alfekaiki DF, Othman NR. The Chemical Composition of Some Marine Fishes from the Iraqi Marine Waters. Research J. Science and Tech. 2017; 9(2), 231-233.

2. Sangeetha1 J, Gayathri1 S, Rajeshkumar S. Antimicrobial assessment of marine brown algae Sargassum whitti against UTI pathogens and its phytochemical analysis, Research J. Pharm. and Tech. 2017;10(6), 1905-1910.

3. Jenifer P, Balakrishnan CP, Chidambaram SP. In-vitro Antioxidant activity of Marine Red Algae Gracilaria foliifera. Asian J. Pharm. Tech. 2017; 7(2), 105-108.

4. Murugesan S, Bhuvaneswari S, Mahadeva Rao USM, Sivamurugan V. Screening of Phytochemicals and Antibacterial Activity of Marine Red Alga Portieria hornemannii (Lyngbye) P. C. Silva, Research Journal of Pharmacology and Pharmacodynamics. 2017; 9(3): 131-136.

5. Thirumurugan D and Vijayakumar R. Exploitation of Antibacterial Compound Producing Marine Actinobacteria against Fish Pathogens Isolated from Less Explored Environments. Research J. Science and Tech. 2013; 5(2), 264-267.

6. Manikandan R and Vijayakumar R. Screening of Antifouling Compound Producing Marine Actinobacteria against Biofouling Bacteria Isolated from Poultries of Namakkal District, South India. Research J. Science and Tech. 2016; 8(2), 83-89.

7. Chelvan Y, Chelvan T, Pushpam AC, Karthik R, Ramalingam K, Vanitha MC. Extraction and Purification of Antimicrobial Compounds from Marine Actinobacteria. Research J. Pharm. and Tech. 2016; 9(4),381-385.

8. Maheswari P, Mahendran S, Sankaralingam S, Sivakumar N. In vitro Antioxidant activity of Exoploysaccharide extracted from Marine Sediment Soil Bacteria. Research J. Pharm. and Tech. 2019; 12(9), 4496-4502.

9. Kanchana S and Arumugam M. Alternative exploration of hyaluronic acid from marine superstore. Asian J. Pharm. Res. 2014; 4 (4), 169-173.

10. Gopi K. and Jayaprakashvel M. Bioactive Potential of Marine Endophytic Fungi associated with Plants in Marine Ecosystem. Research J. Pharm. and Tech. 2017; 10(10), 3635-3636.

11. Sudayasa IP, Fristiohady A,Wahyuni, Sadarun B, Bafadal M, Purnama LOMJ, Rahmatika NS, Kalimin WOIL, Sahidin. Effect of Extract Marine Sponge Xestospongia sp. and Aaptos sp. on the Plasma IL-1β Level in Inflammatory Model Rats: Time Dependent. Research J. Pharm. and Tech. 2020; 13(11), 1-4.

12. Tan KG, Amri M, Ahmad NB and Merdikawati N. 2015 Annual Competitiveness Analysis And Development Strategies For Indonesian Provinces. World Scientific Publishing Co. Ptc. LTD. 2016

13. Longeoan A, Copp BR, Roue M, Dubois J, Valentin A, Petek S, Debitus C and Bourguet-Kondracki M. New bioactive helenaquinone derivatives from South Pacific marine sponges of the genus Xestospongia. Bioorganic & Medicinal Chemistry, 2010; 18(16), 6006-6011

14. Gauvin A, Smadja J, Aknin M, and Gaydou E.-M. Sterols composition and chemotaxonomic consideration in relation to sponges of the genus Xestospongia. Biochemical Systematic and Ecology. 2004; 32(5), 469-476

15. Sun L-L, Shao C-L, Chen J-F, Guo Z-Y, Fu X-M, Chen M, Chen Y-Y, Li R, de Voogd NJ, She Z-G, Lin Y-C, and Wang C-Y. New bisabolane sesquiterpenoids from a marine-derived fungus Aspergillus sp. isolated from the sponge Xestospongia testudinaria. Bioorganic & Medicinal Chemistry Letter. 2012, 22(3): 1326-1329.

16. He W-F, Li Y, Feng M-T, Gavagnin M, Mollo E, Mao S.-C, and Guo Y.-W. New Isoquinolinequinone Alkaloids From the South China Sea Nudibranch Jorunna Funebris and Its Possible Sponge-Prey Xestospongia Sp. Fitoterapia. 2014; 96, 109–114

17. Yang M, Liang LF, Yao LG, Liu HL, and Guo YW. A new brominated polyacetylene from Chinese marine sponge Xestospongia testudinaria. Journal of Asian Natural Products Research. 2019, 21(6):573-578

18. Millan-Aguinaga N, Sorea-Mercado IE, and Williams P. Xestosaprol D and E from the Indonesian marine sponge Xestospongia sp. Tetrahedron Letter, 2010, 51(4): 751-753.

19. Calcul L, Longeon A, Mourabit A, Guyot M, and Bourguet-Kondracki M. Novel alkaloids of aaptamine class from an Indonesian marine sponge of the genus Xestospongia sp. tetrahedron. 2003, 59(34): 6539-6544

20. Cita YP, Suhermanto A, Rajasa OK, and Sudharmono P. Antibacterial activity of marine bacteria isolated from sponge Xestospongia testudinaria from Sorong, Papua. Asia Pacific Journal of Tropical Medicine, 2017; 7(5), 450-454.

21. Murtihapsari, Salam S, Kurnia D, Darwati, Kadarusman, Abdullah FF, Herlina T, Husna MH, Awang K, Shiono Y, Azmi MN, Supratman U. A new antiplasmodial sterol from Indonesian marine sponge, Xestospongia sp., Natural Product Research. 2019; 1-8

22. Fristiohady A, Wahyuni, W, Kalimin WOIL, Purnama LOMJ, Saripuddin S, and Sahidin I. Anti-Inflammatory Activity of Marine Sponge Aaptos sp. to the Plasma Interleukin-1β Level in Wistar Male Rats. Pharmacology and Clinical Pharmacy Research, 2019; 4(2), 35–39

23. Fristiohady A, Wahyuni W, Malik F, Purnama LOMJ, Sadarun B, and Sahidin I. Anti-Inflammatory Activity Of Marine Sponge Callyspongia Sp. And Its Acute Toxicity. Asian Journal of Pharmaceutical and Clinical Research, 2019; 12(12), 97–100

24. Wahyuni W, Fristiohady A, Malaka MH, Malik F, Yusuf MI, Leorita M, Sadarun B, Saleh A, Musnina WOS, Sabandar CW and Sahidin I. Effects of Indonesian marine sponges ethanol extracts on the lipid profile of hyperlipidemic rats. Journal of Applied Pharmaceutical Science, 2019; 9(10), 001–008

25. Diaz P, Jeong SC, Lee S, Khoo C and Koyyalamudi SR. Antioxidant and anti-inflammatory activities of selected medicinal plants and fungi containing phenolic and flavonoid compounds. Chin Med. 2012; 7(26), 1-9

26. Katzung B and Trevor A. Basic & Clinical Pharmacology (13th ed.). Mc. GrawHill. 2014

27. Sahidin I, Sabandar CW, Wahyuni, Hamsidi R, Mardikasari SA, Zubaydah WOS, Sadarun B, Musnina WOS, Darmawan A, and Sundowo A. Investigation of Compounds and Biological Activity of Selected Indonesian Marine Sponges. The Natural Products Journal, 2020; 10(3), 312–321.

28. Chippada SC, Volluri SS, Bammidi SR, and Meena V. In Vitro Anti Inflammatory Activity Of Methanolic Extract Of Centella Asiatica By Hrbc Membrane Stabilisation. Rasayan J Chem, 2011; 4(2), 457–460.

29. Pham N, Butler M, Hooper J, Moni R, and Quinn R. Isolation of Xestosterol Esters of Brominated Acetylenic Fatty Acids From the Marine Sponge Xestospongia Testudinaria. J Nat Prod ., 1999; 62(10), 1439–1442

30. Crist BV, Li X, Bergquist PR, and Djerassi C. Sterols of marine invertebrates. 44. Isolation, structure elucidation, partial synthesis, and determination of absolute configuration of pulchrasterol. The first example of double bioalkylation of the sterol side chain at position 26. J. Org. Chem, 1983; 48(24), 4472–4479

31. Gauvin A, Smadja J, Aknin M, Faure R, and Gaydou E.-M. Isolation of bioactive 5α,8α-epidioxy sterols from the marine sponge Luffariella cf. variabilis. Canadian Journal of Chemistry, 2000; 78(7), 986–992.

32. Zhou X, Lu Y, Lin X, Yang B, Yang X, and Liu Y. Brominated Aliphatic Hydrocarbons and Sterols From the Sponge Xestospongia Testudinaria With Their Bioactivities. Chem Phys Lipids, 2011; 164(7), 703–706.

33. El-Shitany NA, Shaala LA, Abbas AT, Abdel-Dayem UA, Azhar EI, Ali SS, van Soest RWM, and Youssef DTA. Evaluation of the Anti-Inflammatory, Antioxidant and Immunomodulatory Effects of the Organic Extract of the Red Sea Marine Sponge Xestospongia Testudinaria Against Carrageenan Induced Rat Paw Inflammation. PLoS ONE, 2015; 10(9), e0138917

34. Ericson-Neilsen W, and Kaye AD. Steroids: Pharmacology, Complications, and Practice Delivery Issues. Ochsner J, 2014; 14(2), 203–207.

Received on 08.12.2020 Modified on 26.06.2021

Research J. Pharm.and Tech 2022; 15(4):1487-1493.

DOI: 10.52711/0974-360X.2022.00247