Anti-Inflammatory Activity of Ethanol Extract of Marine Sponge Petrosia Sp. by Supression the Level of Tumor Necrosis Factor-alpha

Adryan Fristiohady¹*, Muhammad Hajrul Malaka¹, Andi Rizqa Wahyuni Safitri¹, Dewo Diha¹, Saripuddin¹,

La Ode Muhammad Julian Purnama¹, Sudarman Rahman¹, Agung Wibawa Mahatva Yodha¹, Dian Munasari¹,

Baru Sadarun², Idin Sahidin¹

¹Faculty of Pharmacy, Halu Oleo University, Kendari, Southeast Sulawesi, Indonesia.

²Faculty of Fisheries and Marine Science, Halu Oleo University, Kendari, Southeast Sulawesi, Indonesia.

*Corresponding Author E-mail: adryanfristiohady@uho.ac.id

ABSTRACT:

Inflammation is the host's protective response to any stimulus that harms the body. Excessive inflammatory process causes tissue damage. Therefore, an anti-inflammatory agent is needed. The use of natural ingredients, especially sea sponges, is an option to reduce the side effects of anti-inflammatory agents. This utilization is related to the discovery of new agents. So, we tested the effect of the ethanol extract of Petrosia sp. as an anti-inflammatory agent. Animal induced with 1% carrageenan and left for 1 hour. After that the animals were divided into 6 groups (n = 4) and given oral treatment, namely: Group I (normal group); Group II (negative group); Group III (ethanol extract of Petrosia sp. Concentration of 0.05mg/ml); Group IV (ethanol extract of Petrosia sp. Concentration 0.1mg/ml); Group V (ethanol extract of Petrosia sp. Concentration 0.2mg/ml); and Group VI (positive group, Diclofenac Sodium). After 1 hour, the animals were measured for edema volume and plasma TNF-α levels. Based on the research conducted, the ethanol extract of Petrosia sp. decreased edema volume and plasma TNF-α levels in inflammatory mice. The concentration of 0.2mg/mL had a significant effect on the negative control used (p <0.05). On the other hand, Petrosia sp. indicates the presence of alkaloids, flavonoids, and steroids. They may play an important role in the anti-inflammatory process. Thus, it can be concluded that the ethanol extract of Petrosia sp. has anti-inflammatory activity.

KEYWORDS: Anti-Inflammatory, Petrosia sp., plethysmometer, TNF-α

INTRODUCTION:

Indonesia is a country with a reasonably high incidence rate for diseases involving inflammatory process, including around 30.3% National prevalence for non-communicable diseases (Asthma, Cancer/Tumors, Stroke, Chronic Kidney Disease, Joint Disease, and Diabetes Mellitus), and 15.4% of them were infectious diseases (ARI, Pneumonia, TB, Diarrhea, Hepatitis, Malaria and Filariasis). Besides, 9.2% of injuries that interfere with daily activities. These injuries are included in diseases with inflammatory reactions¹.

Inflammation is a protective response of the host against physical stimuli, damaging chemicals, or microbiological agents. This stimulation causes the release of inflammatory mediators such as histamine, serotonin, bradykinin, and prostaglandins². It involves a complex process involving the body cells’ work, blood vessels, proteins, and released inflammatory mediators. These mediators are referred to as pro-inflammatory cytokines, including IL-1, IL-6, IL-12, IL-18, TNF-α, and TNF-β. These pro-inflammatory cytokines increase and accumulate, resulting in the cardinal sign of inflammation such as swelling (tumor), redness (rubor), pain (dolor), impaired function (functiolesia)^3,4,5,6.

Although the inflammatory process is a normal process, it can lead to tissue damage if the inflammatory process is uncontrolled. Thus, the anti-inflammatory agent is required. However, long term anti-inflammatory consumption can lead to significant side-effects, such as a steroidal anti-inflammatory or non-steroidal anti-inflammatory. Therefore, the utilization of anti-inflammatory agents sourced from terrestrial and marine natural products is massively studied for discovering novel anti-inflammatory agents with great potency and fewer side effects^5,6,7.

Marine sponges are terrestrial natural products that exhibit many pharmacological activities; one of them is Petrosia sp. It has many pharmacological activities, including cytotoxic agents, anti-malaria, anti-HIV, antivirus, and antibacteria⁸. Petrosia sp. is one of the marine sponges that belong to Demospongiae Class, which dominates among the current Porifera⁹. Several studies of the Demospongiae exhibited anti-inflammatory activity such as Aaptos sp., Callyspongia sp., Melophlus sarasinorum, Xestospongia sp., and many more^10-13. Petrosia sp. might have anti-inflammatory activity. Thus, this study was conducted to evaluate its anti-inflammatory activity by measuring edema volume of rats’ paw with plethysmometer and plasma TNF-α levels witn ELISA-kit TNF-α.

MATERIAL AND METHODS:

Chemicals and reagents:

Materials:

The material used in this study were sponges Petrosia sp., distilled water (Onemed^®), aluminum foil, 96% ethanol, filtrated paper (Whatmann^®), 0,5% Na-CMC (Food Grade^®), pellet chow, Aqua Pro Injeksi (Otsuka^®), 1% carrageenan, and ELISA Rat TNF-α (Aviva Systems Biology^®). Besides, rotary vacuum evaporator (Buchi^®), tube (Pyrex^®), Eppendorf tube, analytical balances (Precisa^®), and plethysmometer were used in this study.

Animals:

Animals used were Wistar male rats (Rattus novergicus). They were obtained from the animal farm in Surabaya. All experiments involved in animals were according to the ethical committee of Halu Oleo University (No: 16428/UN29.20/PPM/2019). A total of 24 rats were acclimatized for 7 days under a controlled environment (25±1ºC, Rh 55±5%, and 12:12h light: dark cycle. They were allowed to access food and water ad libitum.

Sponge Collection and Extraction:

Marine sponge Petrosia sp. was obtained from Bintang Samudra Marine Edu-Park, located in Soropia, Konawe Regency, Southeast Sulawesi, and it was determined in LPPM Universitas Halu Oleo (no: 101/UN29/Ld202/2020). The sponge was obtained using a cutter and SCUBA Diving set by diving. The collected sample was then wetly sorted and chopped into small pieces, continued by soaking in cold water to precipitate the salts (1 x 24 hrs). The sample was then continued by maceration with 96% ethanol (3 x 24 hrs). The extract was then collected and filtrated with filtrated paper and followed by concentrating the extract using a rotary vacuum evaporator. The concentrated extract was yielded 11.80g (0.34%).

Chemical Screening:

1. Alkaloid test:

Ethanol extract of Petrosia sp. (4mL) was put in a tube and added chloroform (2mL), 10% ammonia (5mL), and 2M H₂SO₄ (10 drops) to form 2 layers of phase. The upper layer was taken and added with Mayer's reagent. Red deposit indicated the presence of alkaloids¹⁴.

2. Flavonoid test:

Ethanol extract of Petrosia sp. (1mL) was added with Magnesium (Mg²⁺) powder and HCl (10 drops). Discoloration extract into red, yellow, or orange was indicated the presence of flavonoids¹⁴.

3. Steroid Test:

Ethanol extract of Petrosia sp. (1mL) was added with chloroform (2mL) and vortexed. Filtrate formed was added with anhydrous acetate and HCl 2 drop each. Discoloration into blue or green indicated steroids' presence¹⁴.

Anti-inflammatory in vivo by Measuring plasma TNF-α Levels:

Each animal was inducted with 1% carrageenan (100 µL) to obtain the anti-inflammatory model. 1% carrageenan was prepared as suspension (w/v) by mixing 0.25g carrageenan in 25ml API and left for 1 hour. Following that, animals were divided into 6 groups (n=4) and treated 1-hour post-treatment orally. The groups were divided as follows:

Group I: as a normal group, not treated any treatment

Group II: as a negative group, given 0.5% Na CMC

Group III: treated with 0.05mg/mL of ethanol extract of Petrosia sp.

Group IV: treated with 0.1mg/mL of ethanol extract of Petrosia sp.

Group V: treated with 2mg/mL of ethanol extract of Petrosia sp.

Group VI: as positive group (1.3406mg)

The anti-inflammatory activity by measuring edema volume of rats’ paw was conducted by measuring the initial volume (V₀) for normal group, the volume of hind paw rats' 1-hour post-induced with 1% carrageenan (V₁), and 2 hours post-treated with extract (V₂). Inflamed volume is the difference between V₂and V₁and presented as a percentage (Kesuma, 2009).

For anti-inflammatory activity assay by measuring TNF- α levels, 1 hour post-treated with extract, animals were sacrificed to collecting 3mL of blood intracardial and put in EDTA-tube. Collected blood was centrifugated 3000 rpm for 15 minutes. Blood assayed with ELISA kit TNF-α.

Data analysis:

Data was statistical analysis by using SPSS with ANOVA (Analysis of Variance) one-way test. p<0.05 value considered as significant in increased levels TNF-α.

RESULTS AND DISCUSSION:

Chemical screening is a preliminary study conducted to identify the secondary metabolites contained in natural products, qualitatively. It is an initial step to exploring the products' chemical constituents for discovering novel drugs from derived secondary metabolites¹⁵. According to chemical screening conducted, ethanol extract of Petrosia sp. contains alkaloids, flavonoids, and steroids (Table 1).

In assaying the anti-inflammatory activity of ethanol extract of Petrosia sp, we measured the edema volume of rats’ paw and the levels of TNF-α that induced with 1% carrageenan. Carrageenan induces tissue damage, resulting in acute inflammatory response by stimulating the release of thrombin, histamine, and pro-inflammatory cytokines. TNF-α is a pro-inflammatory cytokine that is involved in the acute inflammatory response. The accumulation of concentration TNF-α will cause the cardinal signs of inflammation, including heat (kalor), swelling (tumor), redness (rubor), pain (dolor), and loss of function (functiolesia)^4,16,17. TNF-α active as an angiogenesis factor and forms new blood vessels and is a growth factor for fibroblasts, which results in the formation of connective tissue in the healing process at the site of injury¹⁸.

Shortly after induction, the animals exhibited inflammatory symptoms, which is a tumor or more known as swelling at the induction's site. The accumulation of fluid on rats' paw leads to edema is caused by an inflammatory response that could last for 5-6 hours. The initial step is the release of histamine, cytokines and 5-Hydroxy tryptamine, followed by kinin like substances and prostaglandin like substances. For measuring the edema volume by using plethysmometer^19,20,21. The measurement of edema volume of rats’ paw was presented in Figure 1.

According to Figure 2, ethanol extract of Petrosia sp. concentration of 0.2mg/mL (Group V) decreased edema volume of rats’ paw by 11%, followed by concentration of 0.1mg/mL (group IV) and 0.05mg/mL (group III) by 9% and 4%, respectively. Meanwhile, Group VI, as a positive control was exhibiting decreased edema volume of rats' paw by 27%. In contrast, Group II as negative control did not show any decreased edema volume of rats' paw (0%); this is due to the negative control used was 0.5% Na-CMC that was not possessing anti-inflammatory activity. Group I was not treated with any treatment; it acts as normal control (0%).

Swelling is caused by the induced localize the pro-inflammatory cytokines, one of them is TNF-α. TNF-α exhibited in plasma or 3 hours during acute inflammation, increasing and reaching the optimum levels in the first hour and gradually decreasing to normal levels in the second and third hours. It also credited for pain in inflammation^3,22. The examination of plasma TNF-α levels of animals was presented in Figure 2.

Although Group V (ethanol extract of Petrosia sp. concentration of 0.2 mg/mL) had better decreased TNF-α levels, compared to group III (ethanol extract of Petrosia sp. concentration of 0.05mg/mL) and group IV (ethanol extract of Petrosia sp. concentration of 0.1 mg/mL). Group V exhibited a significant differenced to group II (negative group). Meanwhile, group III and IV were not significantly different from group II. Group I was becoming baseline for the TNF-α levels. This result demonstrated that ethanol extract of Petrosia sp. able to decreased the plasma TNF-α levels of animals implicated in anti-inflammatory activity.

The presence of alkaloids, flavonoids, and steroids in the Petrosia sp. might synergistically provide anti-inflammatory activity^23,24. Alkaloids are involved in immune response due to its ability to stimulate the proliferation of lymphocytes, macrophages, dendritic cells, eosinophils, neutrophils, and mast cells, which work by inhibiting apoptosis of cells²⁵. Flavonoids act as an anti-inflammatory by inhibiting: (1) the activity of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, (2) leukocyte migration and neutrophil degranulation, (3) the release of histamine by mast cells, and stabilize Reactive Oxygen Species (ROS) by reacting with reactive compounds from free radicals thereby they become inactive^26,27. Moreover, lastly, steroids generally act by inhibiting the enzyme phospholipase via the arachidonic acid pathway that playing a vital role in inflammation²⁸.

Petrosia sp. is known to produce complex polycyclic alkaloid derivatives, namely Manzamine A²⁹. Manzamine A is a β-carboline alkaloid that has inhibitory activity against mitogen-activated protein kinase (MAPK or MAP kinase). Manzamine A decreases single cell formation, cancels cell migration, and restores cells' susceptibility to TRAIL-induced apoptosis in AsPC-1 cells³⁰. Several types of anti-inflammatory compounds currently in the clinical trial phase were isolated from the Petrosia contignata³¹.

CONCLUSION:

According to the study conducted, ethanol extract of Petrosia sp. has anti-inflammatory properties by decreasing the edema volume of rats’ subplantar, and it also decreased TNF-α levels of rats. Concentration of 0.2 mg/mL was the effective concentration compared to concentration of 0.05 and 0.1 mg/mL.

ACKNOWLEDGEMENT:

We would like to thanks the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia for Hibah Penelitian Dasar Scheme 2019 with Contract no: 519a/UN29.20/PPM/2019.

CONFLICT OF INTEREST:

None declared.

REFERENCES:

1. Riset Kesehatan Dasar (Riskesdas). 2018. Badan Penelitian dan Pengembangan Kesehatan Kementerian RI tahun 2018. http://www.depkes.go.id/resources/download/infoterkini/materi_rakorpop_20 18/Hasil%20Riskesdas%202018.pdf –Accesed September 7^th, 2020

2. Raharjo ST, Dwi BP, Eko M. Pengaruh Penambahan Ekstrak Daun Sirsak (Annona muricata L.) Terhadap Profil Farmakokinetika Piroksikam. URECOL, 2018; 420-427

3. Baratawijdaja KN, Iris R. Imunologi Dasar Edisi 12, Jakarta: Badan Penerbit FKUI. 2018

4. Freire MO, Van Dyke TE. Natural resolution of inflammation. Periodontol 2000, 2013; 63(1):149-164. Doi: 10.1111/prd.12034

5. Aravind R., Bindu A.R., Bindu K., Kanthlal S., Anil Kumar B. Anti-Inflammatory Evaluation of the Cinnamomum malabatrum (Burm. F).Blume Leaves using Carrageenan Induced Rat Paw Oedema Method. Research J. Pharm. and Tech 6(7): July 2013; Page 746-748.

6. Kousalya K, Priya T, Venkatalakshmi P. Studies on the Anti-inflammatory potential of selected medicinal plants in vitro. Research J. Pharm. and Tech. 2020; 13(7): 3147-3150. doi: 10.5958/0974-360X.2020.00556.9

7. Das A, Roy A, Rajeshkumar S, Lakshmi T. Anti-inflammatory Activity of Turmeric Oil Mediated Silver Nanoparticles. Research J. Pharm. and Tech. 2019; 12(7):3507-3510. doi: 10.5958/0974-360X.2019.00596.1

8. Apriyandi RA, Hadisaputri YE. Artikel Ulasan: Aktivitas Kandungan Senyawa Dan Karakteristik Spons Laut Genus Petrosia. Farmaka, 2019; 17(2): 285-295

9. Suparno. Kajian Bioaktif Spons Laut (Porifera: Demospongiae) Suatu Peluang Alternatif Pemanfaatan Ekosistem Karang Indonesia dalam Bidang Farmasi, Bogor. 2005; 1-20

10. Fristiohady A, Wahyuni W, Kalimin WOIL, Purnama LOMJ, Saripudin S, Sahidin I. Anti-Inflammatory Activity of Marine Sponge Aaptos sp. to the Plasma Interleukin-1ß Level in Wistar Male Rats. Pharmacology and Clinical Pharmacy Research, 2019; 4(2): 35-39. Doi: 10.15416/pcpr.v4i2.24269

11. Fristiohady A, Wahyuni W, Malik F, Purnama LO MJ, Sadarun B, 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. Doi: http://dx.doi.org/10.22159/ajpcr.2019.v12i12.34737

12. Wahyuni, Yusuf MI, Malik F, Lubis AF, Indalifiany A, Sahidin I. Efek Imunomodulator Ekstrak Etanol Spons Melophlus sarasinorum Terhadap Aktivitas Fagositosis Sel Makrofag Pada Mencit Jantan Balb/C. Galenica Journal of Pharmacy, 2019; 5(2):147-157. Doi: 10.22487/j24428744.2019.v5.i2.13611

13. Zubaydah WOS, Wahyuni W, Sahidin I, Halik TA, Andriani R, Indalifiany A, Fristiohady A. Anti-Inflammatory Activity Of Pharmaceutical Gel Of Ethanolic Extract From Marine Sponge Xestospongia Sp. Borneo Journal of Pharmacy, 2019; 2(1):1-9. Doi: https://doi.org/10.33084/bjop.v2i1.694

14. Rumagit HM, Max RJR, Sri S. Uji Fitokimia dan Uji Aktivitas Antioksidan Dari Ekstrak Etanol Spons Lamellodysidae herbacea. Jurnal Ilmiah Farmasi, 2015; 4(3): 183-191.

15. Varghese K, Chitra S, Sheriff SAA. Phytochemical Screening and In-vitro Studies on Anti-Inflammatory Properties of Sapindus emarginatus. Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1125-1127.

16. Aldi Y, Frisky N, Dian H. Aktivitas Beberapa Subfraksi Herba Meniran (Phyllantus niruri Linn.) terhadap Aktivitas dan Kapasitas Fagositosis Makrofag. Scientia, 2015; 5(2): 92-96.

17. Jennifer F, Thomas AK, Fernandes R, Subrhamanyam EVS. Anti-inflammatory activity of Ethanolic Extract of the Roots of the Plant Caesalpinia sappan Linn. Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 99-101.

18. Suryanis I, Eva D, Yusrawati, Eryati D. Perbedaan Rerata Kadar Resistin dan TNF-α antara Wanita Hamil yang Obesitas dengan yang Memiliki Berat Badan Normal pada Preeklamasi Berat Awitan Lambat. Jurnal Kesehatan Andalas, 2018; 7(1): 112-117

19. Rikomah SE, Deah M. Uji Efektivitas Antiinflamasi Ekstrak Kulit Buah Pisang Kepok (Musa acuminate x balbisiana’saga’) pada Mencit Putih Jantan (Mus musculus). Jurnal Ilmiah Farmacy, 2019; 6 (1): 183-190.

20. Jijith US, Jayakumari S. An Apparatus for the determination of rat paw Edema during In vivo Evaluation of Anti-inflammatory agents. Research J. Pharm. and Tech 2020; 13(5): 2373-2375. doi: 10.5958/0974-360X.2020.00426.6

21. Das RJ, Duragkar NJ, Umekar MJ, Katolkar P. Evaluation of Anti-inflammatory Activity of Different Extracts of Alpinia galanga. Research J. Pharm. and Tech 2017; 10(10):3301-3304. doi: 10.5958/0974-360X.2017.00585.6

22. Deepa B, Acharya S, Holla R. Evaluation of antiarthritic activity of Coriander seed essential oil in Wistar albino rats. Research J. Pharm. and Tech 2020; 13(2): 761-766. doi: 10.5958/0974-360X.2020.00144.4

23. Kurnia D, Prima HR. Aktivitas Farmakologi Dan Perkembangan Produk Dari Lidah Buaya (Aloe vera L.). Jurnal Pharmascience, 2019; 06(01): 38-49.

24. Shastry VM, Patil VR, Chaudhari RY. Evaluation of Acute and Chronic Anti-Inflammatory Activity of Whole Plant of Calotropis gigantea in Various Experimental Animal Models. Research J. Pharm. and Tech. 4(5): May 2011; 815-818.

25. Smoak KA, Cidlowski JA. Corticosteroid in Uveitis Management: Mechanism of Action, Clinical Application and Side Effects. Journal of Indonesian Medical Assocciation, 2008

26. Hidayati NS, Shanti L, Ahmad DS. Kandungan Kimia Dan Uji Antiinflamasi Ekstrak Etanol Lantana camara L. pada Mencit Putih (Rattus novergicus L.) Jantan. Bioteknologi, 2008; 5(1):10-17.

27. Mohd. Tauqeer A, Itankar PR. Evaluation of Anti-inflammatory Activity of Flemingia strobilifera linn. Fabaceae. Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; 865-867.

28. Zaini M, Agung B, Khoerul A. Uji Efek Antiinflamasi Ekstrak Etanol Herba Lampasau (Diplazium esculentum Swartz) terhadap Mencit Jantan yang Diinduksi Karagenin-λ. Jurnal Pharmascience, 2016; 3(2):119-130.

29. El Sayed KL, Kelly M, Kara UK, Ang KH, Katsuyama I, Dunbar DC, Khan AA, Hamann MT. New Manzamine Alkaloids with Potent Activity against Infectious Disease. J Am Chem Soc, 2001; 123(9):1804-1808

30. Mioso R, Marante FJT, Bezerra RDS, Borges FVP, Santos BVDO, Laguna IHBD. Cytotoxic Compunds Derived from Marine Sponges. Molecules, 2017; 22(2):208.

31. Rasyid A. Biota Laut Sebagai Sumber Obat-Obatan. Oseana, 2008; 33(1):11-18

Received on 10.09.2020 Modified on 07.10.2020

Research J. Pharm. and Tech. 2021; 14(8):4435-4439.

DOI: 10.52711/0974-360X.2021.00770

Chemical Screening	Reagent	Results	Conclusion
Alkaloids	Mayer	Formed the white deposits (Mofitarini et al., 2019)	Positive
Flavonoids	Mg²⁺+HCl	Discoloration into yellow/ orange (Wahyuni et al., 2019)	Positive
Steroids	Chloroform + Anhydrous acetate + H₂SO₄	Discoloration into a reddish brick (Nugrahani et al., 2016)	Positive