388 Yanti. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 388-392. e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage: www.ijbpr.com IJBPR IN VITRO ANTIACNE ACTIVITY OF MARINE SPONGE ACANTHELLA CAVERNOSA EXTRACTS Yanti1*, Cindy1, Vicky Vendy1, Jae-Kwan Hwang2 1 Faculty of Biotechnology, Atma Jaya Catholic University, Jl Jenderal Sudirman 51, Jakarta 12930, Indonesia. 2 Department of Biotechnology, Yonsei University, Yonsei-ro 50, Seodaemun-gu, Seoul 120-749, Korea. ABSTRACT Propionibacterium acnes is recognized as one of common skin microflora that triggers the inflammation in acne. Here, we investigated the potentials of marine sponge Acanthella cavernosa extracts for management of acne. Methanolic, ethanolic, and hexane extracts of A. cavernosa were tested for antimicrobial and antibiofilm activities against P. acnes, as well as antioxidant activity. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were conducted for antibacterial assay. A. cavernosa was also tested for its ability to prevent P. acnes biofilm formation by performing crystal violet assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used for testing antioxidant activity. Ethanolic A. cavernosa extract exerted MIC and MBC values of 125 and 250 μg/ml against P. acnes. At 250 μg/ml, ethanolic A. cavernosa extract was found to inhibit the formation of P. acnes biofilm up to 45%, respectively. All A. cavernosa extracts up to 250 μg/ml only showed slight antioxidant activity (~30%). Among all extracts, ethanolic extract of A. cavernosa demonstrated the optimal antibacterial and antibiofilm potentials against P. acnes in vitro. These results suggest that marine sponge A. cavernosa may be naturally applied for cosmeceuticals targeting acne prevention. Key Words: Acanthella cavernosa, Propionibacterium acnes, Antibacterial activity, Antibiofilm activity, Antioxidant activity. INTRODUCTION Acne vulgaris is a skin disease that very common among teenager. Face, back, and trunk area are the main areas that contain the largest oil glands. Excessive oil on skin triggers acne formation. Microorganisms including Propionibacterium acnes also implicate in acne progression. P. acnes is a Gram-positive, rod shape, nonsporulating, slow growing, and fastidious anaerobic bacterium that belongs to Actinomycetales ordo, and Propionibacteriaceae family. P. acnes is one of the normal skin microflora that mostly resides in the pilosebaceous follicle of the skin (Nakatsuji et al., 2009). Various treatments have been implemented to combat acne vulgaris. Antibiotics have been used for acne therapy for Corresponding Author Yanti Email: yanti@atmajaya.ac.id years. Unfortunately, side effects of antibiotics therapy have been observed and become concerned (Chomnawang et al., 2005). Discovery of natural products as a new leads for antiacne drugs has been a focus in pharmaceutical perspective. Indonesia as a marine country has a diverse marine ecosystem which is inhabited by various organisms, such as fish, crustaceans, algae, corals, marine mammals, molluscs, and sponges. Sponges are member of the kingdom Animalia and phylum Porifera. Sponges are simple multicellular invertebrates that live on solid substrates, including sand and corals. Sponge characteristics can be determined from the large number of pores on its surface that allow water circulated. (Hutomo and Moosa, 2005) reported that there are 830 species of Demospongiae class of marine sponges in Indonesia. For years, marine sponges have been proven as new producers of secondary metabolites with various human therapeutic 389 Yanti. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 388-392. values (Sipkema et al., 2004). Some bioactive compounds from marine sponges have potentials against diseases, such as malaria and inflammation. Although the molecular mechanism of most compounds is still unclear, there is an increased interest in research to find applicable natural compounds from marine sponge (Sipkema et al., 2004). Marine sponge Acanthella cavernosa belongs to ordo Halicondrida, Dictyonellidae family, and Demospongiae class. It is a brightly colored orange tropical marine sponge that commonly can be found in Indonesia and Australian ocean. Genus Acanthella is known as a rich source of new diterpene compounds. For example, kalihinol-A isolated from Acanthella sp. is one of diterpene compound that reported has antimalarial effect (Sipkema et al., 2004; Xu et al., 2012). This study was aimed to screen for potential antibacterial, antibiofilm, and antioxidant activities of marine sponge A. cavernosa extracts for acne prevention and treatment. MATERIALS AND METHODS Sample collection and extraction Marine sponge A. cavernosa (sample code: BA02) was collected from coastal area in Bali (Indonesia) and identified by Dr. Rory A. Hutagalung (Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia). Crude samples were extracted using 3 solvents, i.e. ethanol (70% w/v), methanol (70% w/v), and hexane (100% w/v). Dried sample was mashed using blender, then weighed for 25 g and macerated with 500 ml of each solvent (ethanol, methanol, and hexane). All bottles were vigorously shaken and incubated overnight at room temperature. Then supernatants was transferred into new tubes and 500 ml of each solvent added to the previous bottle and incubated overnight for the second time. After two batch of extraction, all collected supernatants were concentrated using rotary evaporator and vacuum oven to get the crude extract. For preparation of sample stock, each crude extract was weighed for 0.1 g and dissolved with one ml of dimethylsulfoxide (DMSO). Bacterial preparation P. acnes (ATCC 6919) was a gift from Department of Biotechnology, Yonsei University (Korea). The bacterium was cultured in Brain Heart Infusion (BHI) media supplemented by 2% (v/v) fetal bovine serum (FBS). Meanwhile, BHI supplemented by 2% (v/v) FBS and 3% (w/v) sucrose was used for the growth of P. acnes biofilm. P. acnes was cultured anaerobically using anaerobic gas pack in an anaerobic jar at 37 oC for 48 h. Antibacterial activity Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to determine the antibacterial efficacy of marine sponge A. cavernosa extracts (CLSI 2012). Samples and reference drug (tetracycline) were tested within concentration range of 0.25-500 μg/ml using two-fold dilution on BHI broth. P. acnes was grown in BHI supplemented by FBS and diluted in accordance to McFarland standard to achieve final concentration of 10 6 CFU/ml. Sterile BHI broth was used as negative control, and untreated culture as positive control. Tetracycline was used as the reference drug. For treatment, 20 ml of bacterial culture mixed with 200 ml of sample and reference drugs of each concentration on microplate-96 well. Microplate was then incubated overnight at 37oC anaerobically. The absorbance was checked on 595 nm using microplate reader. The inhibition of bacterial growth was measured by comparing its absorbance with untreated bacterial culture. MIC endpoint was defined as the lowest sample concentration for inhibiting >90% of bacterial growth compared to that of the untreated control. All experiments were performed in triplicate and average values was reported as MICs. For defining the MBC endpoint, a 3 µl of MIC result was then spotted on BHI agar and incubated overnight at 37 oC anaerobically. MBC value was determined as the lowest sample concentration for killing the bacteria completely. Antibiofilm activity The preventive effect of marine sponge A. cavernosa extracts against P. acnes biofilm formation was tested with the modified method of (Yanti et al., 2008; 2009). Samples and reference drug (tetracycline) were diluted to various concentrations (5 – 250 μg/ml). Microplate-96 wells were loaded with 50 μl of each testing concentration for 6 h. Then, the excess solutions were removed and followed by overnight air-drying. The next day, P. acnes culture (108 CFU/ml) was added into the microplate-96 well. Sterile BHIS broth was used as negative control, and untreated culture as positive control. Microplates incubated for 3 days at 37oC anaerobically. After that, media was taken out from the wells. Unbound cells were washed using 200 μl of sterile distilled water and air-dried for 1 h. Biofilm was stained with 110 μl crystal violet and gently shaken for 30 min, then was washed with 200 μl distilled water until negative control was cleared. Absolute ethanol (200 μl) was loaded to each well, and then 100 μl of the solution was transferred to new plates. The absorbance of dissolved crystal violet was measured at 595 nm using microplate reader. The percentage of remaining biofilm calculated using the equation (OD595 of the extract – OD595 of the negative control) / (OD595 of the positive control – OD595 of the negative control) X 100%. All experiments were performed in triplicate with two repeats. Antioxidant activity The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to determine the antioxidant activity of marine sponge A. cavernosa extracts according to the method of Zhang et al. (2007) with a slight modification. Samples 390 Yanti. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 388-392. and reference drug (ascorbic acid) were diluted to various concentrations (5 – 250 μg/ml). A 125 μl of each solution was mixed with 50 μl of 0.003M DPPH solution in the microplate-96 well. Then, a 125 μl DMSO mixed with 50 μl DPPH solution was used as positive control, while a 175 μl of DMSO solution was used as negative control. Microplate was incubated for 30 min in the dark room. Absorbance was measured at 515 nm using microplate reader. All experiments were performed in triplicate. Statistical analysis Data were expressed as mean (n = 3) and standard deviation (SD) by computational analysis from triplicate experiments. Statistical analysis of untreated and marine sponge extract-treated bacterial cells and biofilms was performed by analysis of variance (SPSS 11.0 for Windows). The differences between the individual groups were compared using paired Student’s t-test. The level of significance was taken as P < 0.05. RESULTS Antibacterial activity The inhibition of P. acnes growth by marine sponge A. cavernosa extracts was tested within concentration ranges of 0.25 – 500 µg/ml (Table 1). Among all extracts, ethanolic A. cavernosa extract revealed the highest inhibition against P. acnes with MIC value of 125 µg/ml. Meanwhile, both methanol and hexane extracts had MIC values at 250 μg/ml. Further study on MBC assay was determined to check bactericidal activity of A. cavernosa extracts. At 250 µg/ml, ethanol extract showed that P. acnes growth was totally killed. However, both methanol and hexane extracts of A. cavernosa had MBC values of >500 µg/ml against P. acnes. A standard drug of tetracycline exerted MBC value of 0.5 µg/ml against P. acnes. Antibiofilm activity Dose effects of methanol, ethanol, and hexane extracts of marine sponge A. cavernosa on inhibiting P. acnes biofilm formation were tested at various concentrations (5 – 250 µg/ml). The results of the percentage of remaining biofilm after treatment with samples were shown in Table 2. All extracts at lowest concentration (5 µg/ml) showed antibiofilm activity approximately 20% by preventing P. acnes biofilm formation. Interestingly, ethanol extract of A. cavernosa performed similar antibacterial and antibiofilm activities against P. acnes in vitro. Antioxidant activity The results of antioxidant activity of the methanol, ethanol, and hexane crude extracts of marine sponge A. cavernosa at various concentrations (5-250 μg/ml) was shown in Table 3. Among all solvents, methanol extract had the highest antioxidant activity (~30%). However, all extracts showed a slight antioxidant activity compared to ascorbic acid as the reference agent. Table 1. Antibacterial activity of marine sponge A. cavernosa extracts against P. acnes Sample Solvent extraction Methanol Ethanol Hexane Tetracycline Acanthella cavernosa MIC1 (µg/ml) MBC2 (µg/ml) 250 125 250 0.5 >500 250 >500 0.5 High3 [Sample] Inhibition (µg/ml) (%) 0.25 72 ± 3 Moderate4 [Sample] Inhibition (µg/ml) (%) 64 53 ± 13 125 56 ± 5 - Slight5 [Sample] Inhibition (µg/ml) (%) 64 21 ± 6 8 34 ± 1 8 26 ± 5 - 1 MIC (>90%), 2MBC (100%), 3High inhibition (70-90%), 4Moderate inhibiton (40-70%), 5Slight Inhibiton (20-40%). Tetracycline was used as a standard drug. Table 2. Effect of marine sponge A. cavernosa extracts on inhibiting P. acnes biofilm formation Sample Solvent extraction 5 77 ± 3 73 ± 2 83 ± 2 68 ± 6 Methanol Acanthella Ethanol cavernosa Hexane Tetracycline Percentage of remaining biofilm (% ± SD) Concentration (μg/ml) 10 25 50 80 ± 4 79 ± 2 80 ± 5 74 ± 2 75 ± 3 72 ± 3 79 ± 2 80 ± 3 78 ± 5 62 ± 2 32 ± 6 22 ±4 100 76 ± 5 70 ± 1 78 ± 5 15 ± 1 250 59 ± 7 56 ± 6 65 ± 3 9±1 Tetracycline was used as a standard drug. Table 3. Antioxidant activity of marine sponge A. cavernosa extracts Sample Extraction solvent Methanol Acanthella Ethanol cavernosa Hexane Ascorbic Acid 5 3.6 ± 0.2 0.6 ± 0.1 0±0 30.8 ± 7.5 Ascorbic acid was used as a standard drug. Percentage of antioxidant activity (% ± SD) Concentration (μg/ml) 10 25 50 100 6.8 ± 2.8 9.1 ± 4.5 10.6 ± 4.0 13.5 ± 3.7 1.4 ± 0.7 5.0 ± 2.8 7.3 ± 3.2 12.5 ± 2.2 0±0 0±0 6.2 ± 3.2 16.1 ± 1.5 34.9 ± 6.8 37.5 ± 7.2 40.1 ± 7.9 41.2 ± 7.1 250 30.5 ± 2.1 22.2 ± 0.9 19.8 ± 2.0 49.0 ± 8.1 391 Yanti. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 388-392. DISCUSSION Acne vulgaris is a common skin disease that triggered by the activity of hormone. Moreover, microorganism also plays a role on the development of acne. P. acnes is one of the bacteria that involved on the development of acne. In this study, crude extracts of marine sponge A. cavernosa were examined for their potentials as candidates for management of acne vulgaris via antioxidant, antibacterial, and antibiofilm activities against P. acnes. A. cavernosa was extracted with 3 solvents, i.e. ethanol, methanol, and hexane that yield crude extract with polar and non polar fractions. In order to investigate the antibacterial and bactericidal activity of marine sponge A. cavernosa extracts against P. acnes, MIC and MBC assays were used. Our findings showed that ethanol extract demonstrated the highest antibacterial activity against P. acnes (Table 1). Meanwhile, methanol and hexane extracts at higher concentration (250 μg/ml) also demonstrated dual bacteriostatic and bactericidal activities against P. acnes. Bacteriostatic means the compounds are able to inhibit the growth of microorganism, and bactericidal means the compounds act by killing the microorganisms completely. Our results suggest that marine sponge A. cavernosa could act as a potential bactericidal agent against P. acnes. Other study reported that A. cavernosa also possessed antibacterial activity against marine bacterium Vibrio harveyi, a Gram negative pathogenic bacteria that causes mass mortality in shrimp hatcheries. Mass mortality of shrimp by contamination of V. harveyi caused enormous losses to Indonesia two decades ago (Fakhri et al., 2013). Marine sponge of genus Acanthella is also known to exert antibacterial activity due to its kalihinol compounds. (Bugni et al., 2004) demonstrated the ability of kalihinols from A. cavernosa to inhibit bacterial folate biosynthesis of Bacillus subtilis. For many years, antibiotics have been used for acne treatment, either oral antibiotics or topical antibiotics. Several in vitro researches have tested the susceptibility of P. acnes to various antibiotics. It is noted that P. acnes was extremely vulnerable to antibiotics, such as chloramphenicol, clindamycin, and erythromycin (Iinuma et al., 2011). However, due to the increase of antibiotics resistance and its side effects, nowadays, many studies have been focused on the use of natural products with less side effects and safe usage for alternative acne treatment. In vitro study of natural products from Thailand medicinal plants also demonstrated antibacterial and bactericidal activity against P. acnes. Plant extracts of Eupatorium odoratum and Senna alata had MIC and MBC values on 625 μg/ml and 1250 μg/ml against P. acnes. In addition, plant extract Garcinia mangostana at 39 μg/ml significantly inhibited P. acnes growth (Chomnawang et al., 2005). Regarding antibiofilm system, ethanol extract of marine sponge A. cavernosa had the highest antibiofilm activity through preventing P. acnes biofilm formation in vitro (Table 2). Interestingly, ethanol extract of A. cavernosa showed both antibacterial and antibiofilm effects against P. acnes, indicating that A. cavernosa may inhibit P. acnes growth at planktonic cells and biofilms. P. acnes had been reported to form biofilm in vitro. However, the mechanism of P. acnes biofilm formation has not been revealed. Genome sequence of P. acnes showed that this bacterium also encoded hyaluronate lyase that might be known as extracellular polymeric substance (EPS) and quorum sensing molecule autoinducer-2 (AI-2) (Bruggemann et al., 2004). EPS and AI-2 have important role on P. acnes biofilm formation (Coyene et al., 2007). To minimize the side effect of antibiotic usage, natural products derived from plant extracts have been also evaluated to reduce P. acnes biofilm formation. For example, apple extract (Malus pumila) at 500 μg/ml demonstrated ~50% antibiofilm activity against P. acnes (Coyene et al., 2012). Marine sponges potentially produce bioactive compounds for various applications. Several compounds isolated from marine sponges had been reported for their antibiofilm activity. Ageloxime-D from Agelas sp., manoalide from Luffariella variabilis, and pyrroleimidazole alkaloids (PIA) from Agelasidae and Axinellidae are bioactive compounds that have potent antibiofilm activity. (Hertiani et al., 2010) reported that ageloxime-D was able to inhibit Staphylococcus epidermidis biofilm formation. Other research showed anti-attachment activity of PIA was able to interfere with bacterial attachment of V. harveyi (Kelly et al., 2003). However, the mechanisms of antibiofilm activity of ageloxime-D and PIA have not been clearly described. Manoalide has been reported to inhibit biofilm formation by blocking quorum sensing molecules. Marine extract of L. variabillis was effective quorum sensing inhibitors (QSI) toward both Gram positive and Gram negative bacteria such as Pseudomonas aeruginosa. QSIs are molecules that able to prevent biofilm formation by jamming intracellular communication (Stowe et al., 2011). For antioxidant system, all extracts of marine sponge A. cavernosa only showed less activity (Table 1). However, we found that sample extracts possesed a dosedependent relationship. The increasing concentration may affect the antioxidant activity of A. cavernosa. The highest antioxidant activity (~30%) was shown by methanol extract (250 μg/ml) contained polar compounds. One example of polar antioxidant is phenol (Sultana et al., 2009). Meanwhile, other study reported that marine sponge extracts of Agelas oroides and Ircinia fasiculata at higher concentration (1000 μg/ml) had 20% antioxidant activity (Orhan et al., 2012). CONCLUSION Selection of solvent extraction may affect the potential antioxidant, antibacterial, and antibiofilm 392 Yanti. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 388-392. potentials from marine sponge A. cavernosa for management of acne. Methanolic A. cavernosa extract showed the highest antioxidant activity. Ethanolic extract demonstrated the highest antibacterial and antibiofilm activities against P. acnes through inhibiting P. acnes growth and preventing P. acnes biofilm formation in vitro. ACKNOWLEDGEMENT This research was financially funded by Joint Research Program between Department of Biotechnology, Yonsei University (Korea) and Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia (2012-2014). REFERENCES Bruggemann H, Henne A, Hoster F, et al. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science. 2004; 305: 671-673. Bugni TS, Singh MP, Chen L, et al. Kalihinols from two Acanthella cavernosa sponges: inhibitors of bacterial folate biosynthesis. Tetrahedron. 2004; 60: 6981-6988. Chomnawang MT, Surassmo S, Nukoolkarn VS, et al. Antimicrobial effects of Thai medicinal plants against acne-inducing bacteria. Journal of Ethnopharmacology. 2005; 101: 330-333. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standards, 9th ed., Clinical Laboratory Standards Institute, Wayne, 2012: M07-a9. Coyene T, Brackman G, Rigole P, et al. Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds. Phytomedicine. 2012; 19: 409-412. Coyene T, Peeters E, Nelis HJ. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Research in Microbiology. 2007; 158: 386-392. Fakhri M, Hariati AM, Prajitno A. In vitro antibacterial activity of sponge Acanthella cavernosa against Vibrio harveyi. Journal of Applied Environmental and Biological Sciences. 2013; 3: 1-5. Hertiani T, Edrada ER, Ortlepp S, et al. From anti-fouling to biofilm inhibition: new cytotoxic secondary metabolites from two Indonesian Agelas sponges. Bioorganic and Medicinal Chemistry. 2010; 18: 1297-1311. Hutomo M, Moosa MK. Indonesian marine and coastal biodiversity: present status. Indian Journal of Marine Sciences. 2005; 34: 88-97. Iinuma K, Noguchi N, Nakaminami H, et al. Susceptibility of Propionibacterium acnes isolated from patients with acne vulgaris to zinc ascorbate and antibiotics. Journal of Clinical, Cosmetic and Investigational Dermatology. 2011; 4: 161-165. Kelly SR, Jensen PR, Henkel TP, et al. Effects of Caribbean sponge extracts on bacterial attachment. Aquatic Microbial Ecology. 2003; 31: 175-182. Nakatsuji T, Kao MC, Fang JY, et al. Antimicrobial property of lauric acid against Propionibacterium acnes: its therapeutic potential for inflammatory acne vulgaris. Journal of Investigative Dermatology. 2009; 129: 2480-2488. Orhan IE, Ozcelik B, Konuklugil B, et al. Bioactivity screening of the selected Turkish marine sponges and three compunds from Agelas oroides. Records of Natural Products. 2012; 6: 356-367. Sipkema D, Franssen MCR, Osinga R, et al. Marine sponges as pharmacy. Marine Biotechnology. 2004; 7: 142-162. Stowe SD, Richard JJ, Tucker AT, et al. Anti-biofilm compounds derived from marine sponges. Marine Drugs. 2011; 9: 20102035. Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 2009; 14: 2167-2180. Xu Y, Lang JH, Jiao WH, et al. Formamido-diterpenes from the South China Sea sponge Acanthella cavernosa. Marine Drugs. 2012; 10: 1445-1458. Yanti, Rukayadi Y, Kim KH, et al. In vitro anti-biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria. Phytotherapy Research. 2008; 22: 308-312. Yanti, Rukayadi Y, Lee KH, et al. Activity of panduratin A isolated from Kaempferia pandurata Roxb. against multi-species oral biofilms in vitro. Journal of Oral Science. 2009; 51: 87-95. Zhang WW, Duan XJ, Huang HL, et al. Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae). Journal of Applied Phycology. 2007; 19: 97-108.