International Conference on Mathematics, Science, and Education2016 (ICMSE 2016)

Antimicrobial and Antioxidant Activitiesof Resins and Essential Oil From (Pinus merkusii, Pinus oocarpa, Pinus insularis) and Agathis (Agathis loranthifolia) Mardho Tillah1,a, Irmanida Batubara1,2,b, Rita Kartika Sari3,c 1Department of Chemistry, Faculty of Mathematics and Natural Science, Bogor Agricultural University, Bogor,16680, . 2Tropical Biopharmaca Research Center, Bogor Agricultural University, Jalan Taman Kencana No 3, Bogor,16128, Indonesia. 3Departement of Forest Products, Faculty of , Bogor Agricultural University, Bogor,16680, Indonesia. Correspondent Author a)[email protected], b)[email protected],c)[email protected]

ABSTRACT

This research aimed to explore the antibacterial and antioxidant activities of Pinus merkusii, Pinus oocarpa, Pinus insularis, Agathis loranthifolia resins and essential oil. Resin was separated by distillation process to get essential oil and the residue was extracted using n-hexane, ethyl acetate (EtOAc), and methanol (MeOH). Antioxidant activity was performed by DPPH (1,1diphenyl-2-picryl hydrazyl) radical scavenging method. The antibacterial activity of resins and essential oil of the samples determined using the disc diffusion method against Staphylococcus aureus and Escherichia coli. The results showed that the yield of resin extract was ranging from 8,44 % to 95,56%. All extracts and essential oil could not inhibit E coli growth but inhibit the S. aureus growth. Resinn-hexane extract from Pinus oocarpa was the most potent as antibacterial activity against S. aureus. All of the samples used had less potential antioxidant activity compared to positive control ascorbic acid.. The potential spot as antioxidant was determined by bioautography and the next step need to separate the active component from the active extract to find the active compounds.

Keywords :Resin; essential oil; antibacterial activity; antioxidant

INTRODUCTION

Pine resin (or pitch) is obtained from the bark of addition, the resin can produce resins (copal) as a floor coniferous . It is mainly produced from Pinus coating varnish and oil. Kopal is a commodity in the tabulaeformi, Pinus massoniana and other species form of a resin produced by tapping a resin depending on the indigenous species in every country. (Agathis). Kopal obtained in this way is referred to as The crude resin is collected by tapping pine trees. copal tapping.4 Kopal is a chemical compound with a produce resin as their main defense mechanism complex composition, insoluble in water, soluble in against pathogens. Resin is released upon tissue injury or organic solvents, crumbly, melt when heated and produced locally at the point of infestation in order to flammable fumes.5 encase the infest-insect and also destroy its symbiotic 1 The aim of this research was to investigate the fungal pathogen. Thick secretions mainly consist of a mixture of terpenoids. Pine resin can be distilled into antimicrobial and antioxidant properties of some resin volatile oleoresin, called oil of turpentine, and the solid and essential oils against Staphylococcus aureus and residue, called resin. Turpentine is mainly composed of Escherichia coli. Antibacterial activity determined using monoterpene (C10) olefin along with several the disc diffusion method and to a test of antioxidant sesquiterpene (C15). On the other hand, resin mainly 2 activity using the free radical scavenging activity of 1,1- contains diterpene (C20) resin acid. Traditionally pine resin has been used to treat diphenyl-2-picrylhydrazyl (DPPH) using both inflammation, as antitussive and analgesic. In Chinese spectrophotometric assay and thin layer chromatography folk medicine and more, pine resin has been used as an (TLC) methods. antiseptic, for the treatment of wounds, burns, and skin diseases as well as maintenance antiulcer.3 METHODS Agathis Salisb loranthifolia naturally spread in Papua Materials New Guinea, New Britain, Indonesia (Maluku, Sulawesi, Resins of pine and Agathis was collected from Kalimantan, Sumatra, Papua), the , and Sukabumi, West Java, Indonesia. The sample was Malaysia. The wood has a high enough quality for veneer distilled to separate the volatile oil and resin. About 200 plywood, pulp, lighters, and household furnishings. In C-4 International Conference on Mathematics, Science, and Education2016 (ICMSE 2016) g resin was extracted by increasing the polarity of (2 g in ethanol) was applied to a TLC Silica gel 60 F254 . solvents (400 mL). First, n-hexane was used as solvent, Chromatography was conducted using n-hexane : ethyl then extraction was continued to the residue using ethyl acetate (73 : 27) as eluent. After elution process the plate acetate (EtOAc), and finally using methanol (MeOH). was sprayed using a DPPH solution (87 mg in ethanol), The yield of all extract and volatile oils are determined 30 min later, the yellow spots from reduced DPPH were after the extract was dried. clearly observed against a purple background. Assays Method .Antimicrobial activities of the resins and essential RESULTS AND DISCUSSION Extraction of pine and agathis resin has been done oils from pine and agathis against Staphylococcus aureus by increasing polarity solvent. The extraction method and Escherichia coli were determined using the agar-disc used is maceration. The extraction method used is easy diffusion method. The bacteria were first incubated at and done at room temperature, so it did not destroy the 37° C for 24 h in nutrient broth. The agar medium was chemical compounds in the sample. Extraction of pine spread with the inoculum. Discs of sterile Whatman and agathis resin yielded shown in Table 1. For pine all paper of 6 mm in diameter are deposited on the plates. samples have been extracted in the n-hexane, there is no Samples in various concentrations was injected in to disc residues found and the extraction process not continue to of sterile Whatman paper. After incubation at 37° C for more polar solvent. For agathis 8,437% can extracted in 24 h, the diameters of inhibition zones were measured in n-hexane, and the residues extracted by EtOAc and mm for the test organisms. Tetracycline was used as a continue with MeOH. The result show that the major positive control and the negative control was dimethyl compound in pine is a non polar compound which can be sulfoxide (DMSO). extracted in n-hexane while for agathis is semi-polar Assay of DPPH scavenging activity by extract which can be extracted in EtOAc. spectrophotometry was conducted according to Batubara Some bacteria are pathogenic, plays an active role et al.6 First, the extracts were dissolved in ethanol, and as a cause of disease. Based on the type of pathogenic different concentrations of each extract were used. In a bacteria can be divided into Gram-positive and Gram- total volume of 200 µL, the assay mixture contained 100 negative bacteria. Staphylococcus aureus are Gram- µl of the extract and 100 μl of DPPH solution (4 mg positive bacteria that cause skin diseases such as boils, DPPH in 100 ml ethanol) were added to each well of a burns and infections; whereas Escherichia coli causes 96-well plate. After 30 min, the absorbance of the acute diarrheal disease in Gram-negative bacteria. The mixture was measured at 514 nm. The positive control antimicrobial activity of resin extract from n-hexane P. was (+)ascorbic acid while ethanol was used as the merkusii, n-hexane P. insularis, n-hexane P. oocarpa, n- blank. The inhibition of DPPH radical was calculated as hexane A. loranthifolia, EtOAc A. loranthifolia, MeOH follows: A. loranthifolia,and essential oils from turpentine P. merkusii, turpentine P. insularis turpentine P. oocarpa 1 − (Asample − Acontrol) against Staphylococcus aureus Gram-positive bacteria Inhibitionwhere,(% A) sample= is the absorbance of the× 100 sample,% (Ablank − Acontrol) and Escherichia coli Gram-negative bacteria as zone Acontrol is the absorbance of (+)ascorbic acid as control inhibition is shown in Table 2. In this research, the and Ablank is the absorbance of ethanol as the blank. Each sample concentration of the samples and positive control antimicrobial activities of the resin extract and essential were tested in triplicate. oil having four different concentrations of 125 to 1000 For antioxidant bioautography was conducted µg/mL, were compared with those of tetracycline used as according to Aranada et al.7 Briefly, 10 µl of each extract positive controls.

Table 1. Yield ectract of pine and agathis Yield (%) Samples n-hexane EtOAc MeOH Pinus merkusii 93,581 - - Pinus oocarpa 76,434 - - Pinus insularis 95,557 - - Agathis loranthifolia 8,437 11,482 10,533

Table 2. Antimicrobial activity of extracts against bacterial strains tested using on agar-disc diffusion method. C-5 International Conference on Mathematics, Science, and Education2016 (ICMSE 2016)

Staphylococcus aureus Escherichia coli Resin / essential oils 1000 500 250 125 1000 500 250 125 n- hexane P. merkusii 7,71 ------n- hexane P. oocarpa 11,20 8,20 ------n- hexane P. insularis 8,20 ------n- hexane A. loranthifolia ------EtOAc A. loranthifolia ------MeOH A. loranthifolia ------Turpentine P. merkusii ------Turpentine P. oocarpa ------Turpentine P. insularis ------Tetracycline nt nt 23,2 nt nt nt 27,1 nt nt : not tested Table 3. Antioxidant activities of resins extract and essential oils Samples IC50 (mg/mL) n- hexane P. oocarpa 154,500 n- hexane P. insularis 99,328 n- hexane P. merkusii 60,203 n- hexane A. loranthifolia 438,551 MeOH A. loranthifolia 313,510 EtOAc A. loranthifolia 245,990 Turpentine P. insularis 359,687 Turpentine P. oocarpa 1194,250 Turpentine P. merkusii 1119,960 Ascorbic acid 0,0052

The antimicrobial showed that resin extract from antioxidant compared with control positive (ascorbic P. oocarpa has the diameter inhibition from acid). Previous study found that the essential oil from concentration of 500 µg/mL, while the P. insularis and P. fresh fruits of P. roxburghii showed only negligible merkusii has diameter inhibition from 1000 µg/mL. This radical scavenging activity.9 Salem10 reported that total phenomenom shows the resin extract of P. oocarpa is the antioxidant activities (TAA%) of the essential oils of P. most active as antibacterial against Staphylococcus roxburghii Sarg. from wood (82 ± 2.12%) and bark (85 ± aureus than all samples. Meanwhile the Escherichia coli 1.24%) were higher than that of tannic acid and essential bacteria samples showed no activity. This goes along oils of needles (50±2.24%) was lower tnan that of tannic with research conducted by Savluchinske-Feio et al8 who acid. reported that resin of Pinus ponterosa effective against Qualitative test of the extract of pine resin, agathis Gram-positive bacteria Bacillus subtilis (ATCC 9372) and essential oil conducted to determine content of the and Brevibacterium ammoniagenes (ATCC 6872) with a extract compounds that have antioxidant activity. paper disc method. Instead, the resin of Pinus nigra Qualitative test is done by thin layer chromatography and ineffective in testing fungi and Gram-negative bacteria. DPPH reagent to detect compounds that have the ability Antioxidant activities of all the extracts were to reduce DPPH by sample and gives a yellow color on analyzed by a DPPH free radical assay using the spot are active as an antioxidant. From the spectrophotometric. IC50 value indicated the bioautography assay as shown in Figure 1 resin extract n- concentration of sample that can inhibit 50 percent hexane from P. oocarpa, P. insularis and P. merkusii reduction of DPPH radical (Table 3) the lowest IC50 displayed a strong antioxidant activity compared with value means the most active sample as antioxidant. resin extract n-hexane A. loranthifolia, MeOH A. Extract also analyzed by a DPPH free radical assay using loranthifolia, EtOAc A. loranthifolia and all essential oil. TLC. Rf value of resin extract from n-hexane P. The result showed that resin extract from n- merkusii, n-hexane P. insularis, n-hexane P. oocarpa, n- hexane P. merkusii has the lowest IC50 value, its mean hexane A. loranthifolia, EtOAc A. loranthifolia, MeOH that resin extract from n- hexane P. merkusii are the A. loranthifolia,and essential oils from turpentine P. most potent as antioxidant compare with the other merkusii, turpentine P. insularis turpentine P. oocarpa sample. All the samples showed the less potential of ranged between 0,15-0,93 (Table 4) before sprayed by

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Figure 1. Chromatogram bioautografi antioxidants; using a detection reagent DPPH (a) visible light (b) 254 nm (c) 366 nm before sprayed by DPPH; (d) visible light after sprayed by DPPH; PM: n- hexane P. merkusii; PI: n- hexaneP. insularis; POn- hexane P. oocarpa; AH: n- hexaneA. loranthifolia; AE: EtOAc A. loranthifolia; AM: MeOH A. loranthifolia; TM: Terpentin P. merkusii; TI: Terpentin P. insularis; TO: Terpentin P. oocarpa Table 4. Rf value of resins extract and essential oils in 254nm, 366nm and sprayed by DPPH Samples Rf Visible 254 nm 366 nm DPPH n-hexane P. merkusii 0,15 - √ - - 0,21 - √ - - 0,33 - √ - - 0,40 - √ - - 0,65 - √ - √ 0,70 - √ - - 0,74 - √ √ (red) √ 0,93 - √ - √ n-hexane P. Insularis 0,07 - √ - - 0,18 - √ - √ 0,20 - √ - √ 0,33 - √ - - 0,40 - √ - - 0,67 - √ √ (red) √ 0,76 - √ - √ 0,92 - √ - √ n-hexane P. oocarpa 0,08 - √ - - 0,18 - √ - √ 0,21 - √ - √ 0,34 - √ - - 0,40 - √ - - 0,65 - √ √ (red) - 0,71 - √ - √

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0,80 - √ - √ 0,92 - √ - √ n-hexane A. Loranthifolia 0,24 - √ - - 0,32 - √ - - 0,46 - √ - √ 0,68 - √ √ (red) √ 0,78 - √ - √ EtOAc A. Loranthifolia 0,22 - √ - √ 0,42 - √ - - 0,64 - √ √ (red) √ MeOHA. Loranthifolia 0,23 - √ - - 0,31 - √ - - 0,44 - √ - - 0,65 - √ √ (red) √ TurpentineP. merkusii 0,48 - √ - - 0,68 - √ - - 0,79 - √ - √ TurpentineP. insularis 0,69 - √ - - 0,82 - √ - √ TurpentineP. oocarpa 0,68 - √ - - 0,82 - √ - √ - no spot found √ spot found

DPPH in 254nm. In chromatogram that seen under UV oocarpa were investigated. The results indicated that light at 366 nm (Figure 1.c) resin extract from P. resin extract from n-hexane P. oocarpa is the most merkusii (0,74), n-hexane P. insularis (Rf 0,67), n- potential as antibacterial compared with other sample. hexane P. oocarpa (Rf 0,65), n-hexane A. loranthifolia For antioxidant extract n-hexane P. merkusii has the

(Rf 0,64), EtOAc A. loranthifolia (Rf 0,64), MeOH A. lowest IC50 value compared with other extract. Red spots loranthifolia (Rf 0,65) showed a red spot. It is suspected on the chromatogram seen under UV light at 366nm that resins extract contain terpenoids compound. suspected is a terpenoids group. After sprayed by DPPH (Figure 1.d) there are 3 spots (Rf 0,65; 0,74 and 0,93) for n- hexane P. merkusii, BIBLIOGRAPHY 5 spots (Rf 0,18; 0,20; 0,67; 0,76 and 0,92) for n- 1. Termentzi A, Fokialakis N, Skaltsounis AL. Natural hexaneP. insularis and 5 spots (Rf 0,18; 0,21; 0,71; 0,80 resins and bioactive natural products there of as and 0,92) for n- hexane P. oocarpa active as an potential anitimicrobial agents. Current antioxidant. It is characterized by the appearance of Pharmaceutical Design. 17. 1267-1290 (2011). yellow color on the chromatogram bioautografi. While 2. Norin T. Some aspects of the chemistry of the order . Phytochemistry. 11.1231-1242 (1972). on n- hexaneA. loranthifolia, EtOAc A. loranthifolia, 3. Feliciano AS, Gordaliza M, Salinero MA, Corral MeOH A. loranthifolia yellow spots are disguised, JMM. Abietane acids: sources, biological activities, indicating samples inactive as an antioxidant. For and therapeutic uses. Planta Med. 59.485-490. essential oils turpentineP. merkusii, turpentineP. (1993). insularis, turpentineP. oocarpa with Rf value 0,79; 0,82 4. Santoso G. Pengembangan Metode Penyadapan and 0,82 respectively, there is a faint yellow spots that Kopal Melalui Penerapan Teknik Sayatan [disertasi]. Bogor: Program Pascasarjana, Institut signaled it’s less antioxidant activity. Pertanian Bogor. (2006) CONCLUSION 5. Ando Y, Wiyono B. 1988. Sifat-sifat kopal manila In this study, the antimicrobial and antioxidant dari Pekalongan Timur dan Banyumas Barat. Jurnal activities of resin extract essential oil from from n- Penelitian Hasil Hutan 5(6): 353-356. (1988). hexane P. merkusii, n-hexane P. insularis, n-hexane P. 6. Batubara I, Mitsunaga T, Ohashi H, Screening Antiacne Potency of Indonesian Medicinal : oocarpa, n-hexane A. loranthifolia, EtOAc A. Antibacterial, loranthifolia, MeOH A. loranthifolia,turpentine P. Lipase Inhibition, and Antioxidant Activities. J merkusii, turpentine P. insularis and turpentine P. Wood Sci.55.230–235.(2009)

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7. Aranada RZ, Lopez LAP, Arroyo JL, Garza BAA, of essential oil of Pinus roxburghii from Kashmir, Torres NW. Antimicrobial and Antioxidant india. International Journal of Pharmacological Activities of Plants from Northeast of Mexico. Research. 4. 61-64 (2014). Evidence-Based Complementary and Alternative Salem MZM, Ali HM, Basalah MO. Essential Oils Medicine. 1-6.(2011). from Wood, Bark, and Needles of Pinus roxburghii 8. Soderberg TA, Holm S, Gref Rm Hallmans G. Sarg from Alexandria, Egypt: Antibacterial and Antibacterial effects of zinc oxide, , and resin Antioxidant Activities. BioResources. 9. 7454- acids with special reference to their interactions. 7466(2014). Scand J Plast Reconstr Hand Surg. 25.19-24 (1991) 9. Qadir M, Shah WA, Banday JA. GC-MS analysis, Antibacterial, Antioxidant and Anticancer activity

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