Balangla  (Litsea  cubeba)  plant  is  used  as traditional  medicine  by Dayak  Kenyah tribe in East Kalimantan. It contains active compounds that are efficient to treat many human diseases and believed to have an antibacterial activity. The purposes of this study were to determine  the  phytochemical  compounds  of  the  balangla  leaves  and  to  investigate  the antibacterial  activity  of  ethanol  extract  of  leaves  L.  cubeba.  Respectively,  various  levels  of ethanol extract of L.  cubeba leaves  viz: 10%,  20%,  30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%  were  used  to  examine  its  antibacterial  activity  against  bacteria  gram  positive (Staphylococcus  aureus)  and  gram  negative  (Escherichia  coli)  by  using  pitting  diffusion method.  The  results  indicated  that  alkaloids,  flavonoids,  phenols  and  steroids  have  been found as phytochemical compounds in the ethanol extracts of L. cubeba leaves. Meanwhile, the antibacterial activities of ethanol extracts of L. cubeba leaves against the test organisms had been determined and significantly inhibited the growth of S. aureus and E. coli, forming a  wide  inhibition  zone  (15.91±0.950  mm)  for  S.  aureus  and  (16.23±0.416  mm)  for  E.  coli. Further,  antibacterial  activity  of  (L.  cubeba)  in-vitro  had  been  justified  on  its  utility  in traditional medicines for the treatment of infections of bacterial origin

Manurung, Hetty

Marina, Elvi

Nugroho, Rudy Agung

English

Diponegoro University Institutional Repository

Proceeding of 5th International Seminar on ISSN: 978-602-71169-7-9 New Paradigm and Innovation on Natural Sciences and Its Application (5th ISNPINSA) Hetty Manurung, et al. P a g e  | 5 This Proceeding © Faculty of Sciences and Mathematics Diponegoro University, 2015 Phytochemical Screening and Antibacterial Activity of Leaves Extract Balangla (Litsea cubeba (Lour) Pers.) from Malinau, East Borneo Hetty Manurung1,a, Rudy Agung Nugroho2,b and Elvi Marina3,c 1,2,3 Department of Biology, Faculty of Mathematics and Natural Sciences Mulawarman University, Samarinda. Indonesia ahetty_manroe@ymail.com, brudyagung.nugroho@fmipa.unmul.ac.id, celvimarina92@gmail.com Abstract. Balangla (Litsea cubeba) plant is used as traditional medicine by Dayak Kenyah tribe in East Kalimantan. It contains active compounds that are efficient to treat many human diseases and believed to have an antibacterial activity. The purposes of this study were to determine the phytochemical compounds of the balangla leaves and to investigate the antibacterial activity of ethanol extract of leaves L. cubeba. Respectively, various levels of ethanol extract of L. cubeba leaves viz: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% were used to examine its antibacterial activity against bacteria gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) by using pitting diffusion method. The results indicated that alkaloids, flavonoids, phenols and steroids have been found as phytochemical compounds in the ethanol extracts of L. cubeba leaves. Meanwhile, the antibacterial activities of ethanol extracts of L. cubeba leaves against the test organisms had been determined and significantly inhibited the growth of S. aureus and E. coli, forming a wide inhibition zone (15.91±0.950 mm) for S. aureus and (16.23±0.416 mm) for E. coli. Further, antibacterial activity of (L. cubeba) in-vitro had been justified on its utility in traditional medicines for the treatment of infections of bacterial origin. Keywords: Balangla (Litsea cubeba), Lauracea, Antibacterial, Phytochemical Screening. Introduction Litsea cubeba (Lours.) Pers., locally known as Balangla belongs to the Lauraceae plant family. L. cubeba is a 3- to 10 m evergreen tree or shrub widely distributed in South-eastern Asia, Southern China, Japan, and Taiwan [1, 2]. In Indonesia. L. cubeba distributed in Sumatera, Java, and Borneo. This plant is one of the common medicinal plants in Borneo. L. cubeba can be used as a flavouring or herbal medicine. As a flavouring, it gives a unique flavour resembling that of a mixture of pepper, ginger, and citrus. It is popular as a flavour enhancer in foods, cosmetics, and cigarettes [3, 4]. Balangla by the people of East Kalimantan especially Dayak Kenyah tribe used as traditional medicine to cure for coughs, colds, migraine headaches, skin diseases, pain reliever, diarrhoea, fever, aromatherapy, and even benefit as seasoning (spices) and believed to have an antibacterial activity. Recent study has been reported that an aqueous EtOH extract of the barks of L. cubeba has yielded five novel isoquinoline alkaloids, namely (+)-N-(methoxylcarbonyl)-N-nordicentrin, (+)-N-(methoxycarbonyl)-N-norpredicentrine, (+)-N-(methoxyl-carbonyl)-N-norbul-bodione, and (+)-N-(methoxycarbonyl)-N-norisocorydione, and (+)-8-methoxyl-isolaurenine N-oxide, and one known compound, (+)-N-(methoxycarbonyl)-N-norglaucine [5]. Another study [6] showed that the essential oil in the leaves of L. cubeba containing sineol, sitronellol, oinen alpha, beta-pinene, and limonene sitronellal. Previous studies revealed that essential oil and bioactive compound were shown potential as an anticancer, antioxidant, and antibacterial. Antibacterial compounds usually found in plant parts such as leaves, twigs, bark, and other parts. Antibacterial is useful to eliminate bacteria potentially harmful bacteria to health and pathogens such as Staphylococcus aureus and Escherichia coli. E. coli generate bladder infections and diarrhoea [7] while S. aureus can cause purulent infection. S. aureus led to various types of diseases such as infections of the polikel hair, sweat glands, ulcers, skin infections, and wound infections [8]. The purposes of this study were to determine the phytochemical compounds that found in the balangla leaves and to investigate the ISSN: 978-602-71169-7-9 Proceeding of 5th International Seminar on  New Paradigm and Innovation on Natural Science  and Its Application (5th ISNPINSA) 6 | P a g e  Hetty Manurung, et al.  This Proceeding © Faculty of Sciences and Mathematics  Diponegoro University, 2015 antibacterial activity of ethanol extract of leaves Litsea cubeba. Material and Methods Sample Collection The leaves of L. cubeba were collected locally from Gunung Seribu (Can batu) Mahak Baru, district Sungai Boh Malinau, East Borneo, in December 2014. The plants collected were identified botanically in Plant Anatomy and Taxonomy Laboratory, Biology Department Mulawarman University and deposited in the herbarium for future reference. Preparation of plant material The fresh leaves were washed with tap water and then thoroughly cleaned with distilled water and shade dried for a week. Then the dried leaves (1500 g) were grinded to a fine powder by using blender. The powder was taken and macerated with 95% EtOH. They were kept at room temperature for 5 days. Thereafter the mixtures were filtered by using Whatmann filter paper no .1. The supernatant were pooled together, concentrated in rotary evaporator at 40°C. The dried extract (57.283 g) was used directly for the determination of a presence of phytochemicals and antibacterial activity. Qualitative Analysis of Phytochemicals Phytochemical examinations were carried out for leaves extract L. cubeba as per the standard methods [9].  Test for Alkaloids Dragendorff’s test: A-2 mL of extract was added with 5 mL of chloroform-ammonia 0.005 M, then homogenized and filtered. The filtrate was added with a few drops of 2M sulfuric acid and shaken to form two layers of acids and bases. The layer acid (found on the top layer), added with a few drops of reagent Dragendorff’s. Formation of deposits of red-brown colour indicated the presence of alkaloids.  Test for Flavonoids A-2 mL crude extract was added with 5 mL of water, boiled for 5 minutes and filtered. Two mL filtrate was added with 0.05 mg of Mg powder and 1 mL chloride acid, then shaken until homogeneous. A yellow or red colouration indicates the presence of flavonoids.  Test for phenols Lead acetate test: To 2 mL of the extract, few mL of 1 % lead acetate solution was added. The formation of bluish black precipitate indicated the presence of tannins and phenolic compounds. Test for saponins Frothing test: A-2 mL of filtrate was diluted with 5 mL of distilled hot water and the mixture was shaken vigorously and observed for persistent foam which lasted for at least 10 mins which indicated the presence of saponins. Test for Terpenoids Liebermann - Burchard’s test: Extract was treated with a few drops of acetic anhydride, boiled and cooled. Concentrated sulfuric acid was added from the sides of the test tube which showed a brown ring at the junction of two layers, and the formation of deep red colour indicated the presence of terpenoids. Test for Antimicrobial Activity Source of Bacterial Two microbial isolates were chosen for antimicrobial investigation: Gram positive bacteria (S. aureus) and Gram negative bacteria (E. coli). Both microbial were purchased from Laboratory of Microbiology, Faculty of Sciences Mulawarman University, Samarinda, Indonesia. Testing for Antibacterial Activity The test organisms were sub cultured by streaking them on nutrient agar, followed by incubation for 20 hr at 37 °C. These were subcultured prior to each experiment and were used for antibacterial. The pitting-diffusion method of two species of bacteria Staphylococcus aureus and Escherichia coli was performed in in-vitro antibacterial activity. Medium that was used in the antimicrobial activity was MHA (Mueller Hinton Agar), an inoculum containing 107 bacteria/mL was incorporated. After the Proceeding of 5th International Seminar on ISSN: 978-602-71169-7-9 New Paradigm and Innovation on Natural Sciences and Its Application (5th ISNPINSA) Hetty Manurung, et al. P a g e  | 7 This Proceeding © Faculty of Sciences and Mathematics Diponegoro University, 2015 suspension of bacteria seep into the media, then created pit in the media using the sterile iron cork borer (6 mm diameter). Using sterilized dropping pipettes, different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% /well) of leaves extract L. cubeba was carefully added into the wells The plates were then incubated at 37 °C for 24–48 hr. Chloramphenicol was used as positive control for antibacterial activities while DMSO was used as the negative control. The diameter of the inhibition zone was measured for antibacterial activities testing. Experiments were performed in triplicate, and the results were presented as the mean values of the diameters of the inhibitory zones from three runs. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone [10, 11]. The diameters of the inhibitory zones value were used as criteria to judge the antimicrobial activity (strong active: the diameters of the inhibitory zones ≥ 20 mm, active: the diameters of the inhibitory zones 10-20 mm, moderately active: the diameters of the inhibitory zones 5-10 mm, not active: the diameters of the inhibitory zones are invisible or ≤ 5 mm) [12]. Results and Discussion Phytochemical Analysis The results of the phytochemical analysis were carried out in extracts of Litsea cubeba. The experiment showed the presence of secondary metabolites such as alkaloids, flavonoids, phenols and steroids. The results of the phytochemical analysis of L. cubeba are shown in Table 1. The presence of alkaloids in leaves extract is likely to be responsible for antimicrobial activity effects observed. Alkaloid is one of the phytochemical compounds in Litsea cubeba that act as an antibacterial activity [13, 14]. Table 1. Qualitative phytochemical analysis of leaves extract L. cubeba No Parameters Leaves extract 1 Alkaloid + 2 Flavonoid + 3 Phenol + 4 Saponin - 5 Steroid +  (+indicates presence, - indicates absence) Antimicrobial Activity Antimicrobial activity is used to test whether the leaf extract has capability to control the growth of the bacterial. The zone of inhibition had been obtained for both bacterials Staphylococcus aureus and Escherichia coli. The result obtained, the zone of inhibition was recorded at ten concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100% in Table 2. Table 2. Antimicrobial activity of leaves extract of Litsea cubeba Sample extract Conc. (%) Zone inhibition (mm) Staphylococcus aureus Escherichia coli 10% 9.58±0.36 9.63±0.32 20% 10.71±0.62 10.65±0.75 30% 11.36±0.41 11.07±0.40 40% 12.40±0.40 12.41±0.18 50% 12,71±0.70 13.62±0.11 60% 13.06±0.29 13.16±0.68 70% 13.63±0.27 14.00±0.37 80% 14.03±0.81 14.56±0.66 90% 13.80±0.69 15.08±0.32 100% 15.91±0.95 15.23±0.41 Chloramphenicol (25%) 18.33±0.10 18.63±0.18 In general Table 2 showed that increasing concentrations of leaf extracts enhanced inhibitory zone on both bacteria. The extracts displayed relative antimicrobial activities against bacterial tested with the diameter of inhibition zones ranging between 9.58±0.36 to 15.91±0.95 mm (S. aureus) and 9.63±0.32 to 18.63±0.18 mm (E. coli). The highest antibacterial activity in both bacteria was found at 100 % concentration and the lowest at 10 % concentration of leaves extract. The results obtained was indicated of the presence of broad spectrum antimicrobial compounds or metabolic toxins in L. cubeba leaves extract that could be exploited in treating infections associated with the aforementioned bacterial. Previous studies on antimicrobial activities of medicinal plants indicated that inhibition zones of 10 mm or ISSN: 978-602-71169-7-9 Proceeding of 5th International Seminar on  New Paradigm and Innovation on Natural Science  and Its Application (5th ISNPINSA) 8 | P a g e  Hetty Manurung, et al.  This Proceeding © Faculty of Sciences and Mathematics  Diponegoro University, 2015 greater were taken to represent good activity of such plants [15, 16]. Conclusion Balangla leaf extract can be used as a source of secondary metabolites such as alkaloids, Flavonoids, Phenol, and steroids. The results suggest that the antibacterial activity of leaves extract L. cubeba may contribute to prevent some of the diseases caused by S. aureus and E. coli. References [1] T.T. Liu, T.S. Yang. Antimicrobial impact of the components of essential oil of Litsea cubeba from Taiwan and antimicrobial activity of the oil in food systems. Int. J. Food Microbiol. 2012, 156, 68–75. [2] C.H. Huang, W.J. Huang, S.J. Wang, P.H. Wu, W.B. Litebamine, a phenanthrene alkaloid from the wood of Litsea cubeba, inhibits rat smooth muscle cell adhesion and migration on collagen. Eur. J. Pharmacol. 2008, 596, 25–31. [3] Y. Wang, Z.T. Jiang R. Li. Complexation and molecular microcapsules of Litsea cubeba essential oil with β-cyclodextrin and its derivatives. Eur. Food Res. Technol. 2009, 228, 865–873. [4] M. Luo, L.K. Jiang, G.L. Zou. Acute and genetic toxicity of essential oil extracted from Litsea cubeba (Lour.) Pers. J. Food Prot. 2005, 68, 581–588. [5] W. Zhang, J-F. Hu, Lv. Wen-Wen, Q-C. Zhao, G-B. Shi. Antibacterial, Antifungal and Cytotoxic Isoquinoline Alkaloids from Litsea cubeba. Molecules 2012, 17, 12950-12960. [6] Y.N. Heryati, A.S. Mindawati, Kosasih. Prosfek Pengembangan Lemo (Litsea cubeba L. Persoon) Di Indonesia. Jurnal Tekno Hutan tanaman. Pusat penelitian Hutan Tanaman. 2009, 2(1):9-17. [7] Books, F.G., Butel, J.S., dan Morse, S.A. 2001. Mikrobiologi Kedokteran (Edisi Terjemahan). Selemba Medika. Jakarta. [8] I. Entjang. Mikrobiologi & Parasitologi untuk Akademi Keperawatan dan Sekolah Tenaga Kesehatan yang Sederajat. 2003. PT. Citra Aditya Bakti.  [9] J.B. Harbone. Phytochemical Methods London.Chapman and Hall Ltd, 1973, pp 49-188. [10] E.G. Trease, W.C. Evans. Textbook of Pharmacognosy, 3 rd edn. Bailliere Tindal, London pp.81-90,268-98,(1989). [11] J. Owolabi, E.K.I. Omogbai, O. Obasuyi. Antifungal and antibacterial activities of ethanolic and aqueous extract of Kigelia african (Bignoniaceae) stem bark. African Journal of Biotechnology, 2007, 6(14):882-85. [12] W.W. Davis, T.R. Stout. Disc Plate Methods of Microbiological Antibiotic Assay. Microbiology. 1971, 22(4): 659-665. [13] T. Feng, Y, Xu, X.N. Cay, Z.Z. Du, X.D. Luo. Antimicrobiolly active isoquinoline alkaloids from Litsea cubeba, Planta med. 2009, 75: 76-79. [14] W.A. Volk, M.F. Wheeler. Basic Microbiology. Philadelphia, Lippincott ,1973. [15] C.A. Bukola, A.A. Onilude. Screening of lactid acid bacteria strains isolated from some nigerian fermented foods for EPS production. World Applied Sciences Journal. 2008, 4(5):741-747. [16] N. Ramesh, M.C.Sathyanarayana, H.Lloyd. Abundance of grey junglefowl Gallus sonneratii at Theni Forest Division, Western Ghats, India: implications for monitoring and conservation. 2011. International Journal of Galliformes Conservation 2, 14-21.   

Phytochemical Screening and Antibacterial Activity of Leaves Extract Balangla (Litsea cubeba (Lour) Pers.) from Malinau, East Borneo

Balangla  (Litsea  cubeba)  plant  is  used  as  traditional  medicine  by  Dayak  Kenyah tribe in East Kalimantan. It contains active compounds that are efficient to treat many human diseases and believed to have an antibacterial activity. The purposes of this study were to determine  the  phytochemical  compounds  of  the  balangla  leaves  and  to  investigate  the antibacterial  activity  of  ethanol  extract  of  leaves  L.cubeba.  Respectively,  various  levels  of ethanol  extract  of  L.cubeba  leaves  viz:  10%,  20%,  30%,  40%,  50%,  60%,  70%,  80%,  90%, 100%  were  used  to  examine  its  antibacterial  activity  against  bacteria  gram  positive (Staphylococcus  aureus)  and  gram  negative  (Escherichia  coli)  by  using  pitting  diffusion method.  The  results  indicated  that  alkaloids,  flavonoids,  phenols  and  steroids  have  been found as phytochemical compounds in the ethanol extracts of L. cubeba leaves. Meanwhile, the antibacterial activities of ethanol extracts of L. cubeba leaves against the test organisms had been determined and significantly inhibited the growth of S. aureus and E.coli, forming a wide inhibition zone (15.91±0.950 mm) for S. aureus and (16.23±0.416 mm) for E. coli. Further, antibacterial  activity  of  (L.cubeba)  in-vitro  had  been  justified  on  its  utility  in  traditional medicines for the treatment of infections of bacterial origin

Proceeding of 5th International Seminar on ISSN: 978-602-71169-7-9 New Paradigm and Innovation on Natural Sciences and Its Application (5th ISNPINSA) Hetty Manurung, et. al. P a g e  | 69 This Proceeding © Faculty of Sciences and Mathematics Diponegoro University, 2015 Phytochemical Screening and Antibacterial Activity of Leaves Extract Balangla (Litsea cubeba (Lour) Pers.) from Malinau, East Borneo Hetty Manurung1,a, Rudy Agung Nugroho1,b and Elvi Marina1,c 1Department of Biology, Faculty of Mathemathics and Natural Sciences, Mulawarman University, Samarinda. Indonesia a hetty_manroe@ymail.com, b rudyagung.nugroho@fmipa.unmul.ac.id, celvimarina92@gmail.com Abstract. Balangla (Litsea cubeba) plant is used as traditional medicine by Dayak Kenyah tribe in East Kalimantan. It contains active compounds that are efficient to treat many human diseases and believed to have an antibacterial activity. The purposes of this study were to determine the phytochemical compounds of the balangla leaves and to investigate the antibacterial activity of ethanol extract of leaves L.cubeba. Respectively, various levels of ethanol extract of L.cubeba leaves viz: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% were used to examine its antibacterial activity against bacteria gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) by using pitting diffusion method. The results indicated that alkaloids, flavonoids, phenols and steroids have been found as phytochemical compounds in the ethanol extracts of L. cubeba leaves. Meanwhile, the antibacterial activities of ethanol extracts of L. cubeba leaves against the test organisms had been determined and significantly inhibited the growth of S. aureus and E.coli, forming a wide inhibition zone (15.91±0.950 mm) for S. aureus and (16.23±0.416 mm) for E. coli. Further, antibacterial activity of (L.cubeba) in-vitro had been justified on its utility in traditional medicines for the treatment of infections of bacterial origin. Keywords: Balangla (Litsea cubeba), Lauracea, Antibacterial, Phytochemical Screening. Introduction Litsea cubeba (Lours.) Pers., locally known as Balangla belongs to the Lauraceae plant family. L. cubeba is a 3- to 10 m evergreen tree or shrub widely distributed in Southeastern Asia, Southern China, Japan, and Taiwan [1,2]. In Indonesia. L.cubeba distributed in Sumatera, Java, and Borneo. This plant is one of the common medicinal plants in Borneo. L. cubeba can be used as a flavoring or herbal medicine. As a flavoring, it gives a unique flavor resembling that of a mixture of pepper, ginger, and citrus. It is popular as a flavor enhancer in foods, cosmetics, and cigarettes [3,4]. Balangla by the people of East Kalimantan especially Dayak Kenyah tribe used as traditional medicine to cure for coughs, colds, migraine headaches, skin diseases, pain relieve, diarrhea, fever, aromatherapy, and even benefit as seasoning (spices) and believed to have an antibacterial activity.  Recent study has been reported that an aqueous EtOH extract of the barks of L. cubeba has yielded five novel isoquinoline alkaloids, namely (+)-N-(methoxylcarbonyl)-N-nordicentrin, (+)-N-(methoxycarbonyl)-N-norpredicentrine, (+)-N-(methoxyl-carbonyl)-N-norbulbodione, and (+)-N-(methoxycarbonyl)-N-norisocorydione, and (+)-8-methoxyl-isolaurenine N-oxide, and one known compound, (+)-N-(methoxycarbonyl)-N-norglaucine [5]. Another study [6] showed that the essential oil in the leaves of L.cubeba containing sineol, sitronellol, oinen alpha, beta-pinene, and limonene sitronellal. Previous studies revealed that essential oil and bioactive compound were shown potencial as an anticancer, antioxidant, and antibacterial. Antibacterial compounds usually found in plant parts such as leaves, twigs, bark, and other parts. Antibacterial is usefull to eliminate bacteria potentially harmful bacteria to health and pathogens such as Staphylococcus aureus and Escherichia coli. E. coli generate bladder infections and diarrhea [7] while S. aureus can cause purulent infection. S. aureus led to various types of diseases such as infections of ISSN: 978-602-71169-7-9 Proceeding of 5th International Seminar on  New Paradigm and Innovation on Natural Science  and Its Application (5th ISNPINSA) 70 | P a g e  Hetty Manurung, et. al.  This Proceeding © Faculty of Sciences and Mathematics  Diponegoro University, 2015 the polikel hair, sweat glands, ulcers, skin infections, and wound infections [8]. The purposes of this study were to determine the phytochemical compounds that found in the balangla leaves and to investigate the antibacterial activity of ethanol extract of leaves Litsea cubeba. Material and Methods Sample Collection The leaves of L.cubeba were collected locally from Gunung Seribu (Can batu) Mahak Baru, district Sungai Boh Malinau, East Borneo, in December 2014. The plants collected were identified botanically in Plant Anatomy and Taxonomy Laboratory, Biology Departement Mulawarman University and deposited in the herbarium for future reference. Preparation of plant material The fresh leaves were washed with tap water and then thoroughly cleaned with distilled water and shade dried for a week. Then the dried leaves (1500 g) were grinded to a fine powder by using blender. The powder was taken and macerated with 95% EtOH. They were kept at room temperature for 5 days. Thereafter the mixtures were filtered by using Whatmann filter paper no .1. The supernatant were pooled together, concentrated in rotary evaporator at 40°C. The dried extract (57.283 g) was used directly for the determination of a presence of phytochemicals and antibacterial activity. Qualitative Analysis of Phytochemicals Phytochemical examinations were carried out for leaves extract L. cubeba as per the standard methods [9].  Test for Alkalooids Dragendroff’s test: A-2 mL of extract was added with 5 mL of chloroform-ammonia 0.005 M, then homogenized and filtered. The filtrate was added with a few drops of 2M sulfuric acid and shaken to form two layers of acids and bases. The layer acid (found on the top layer), added with a few drops of reagent Dragendroff’s. Formation of deposits of red-brown color indicated the presence of alkaloids.  Test for Flavonoids A-2 mL crude extract was added with 5 mL of water, boiled for 5 minutes and filtered. Two mL filtrate was added with 0.05 mg of Mg powder and 1 mL chloride acid, then shaken until homogeneous. A yellow or red colouration indicates the presence of flavonoids.  Test for phenols Lead acetate test: To 2 mL of the extract, few mL of 1 % lead acetate solution was added. The formation of bluish black precipitate indicated the presence of tannins and phenolic compounds. Test for saponins Frothing test: A-2 mL of filtrate was diluted with 5 mL of distilled hot water and the mixture was shaken vigorously and observed for persistent foam which lasted for atleast 10 mins which indicated the presence of saponins. Test for Terpenoids Liebermann - Burchard’s test: Extract was treated with a few drops of acetic anhydride, boiled and cooled. Concentrated sulfuric acid was added from the sides of the test tube which showed a brown ring at the junction of two layers, and the formation of deep red color indicated the presence of terpenoids. Test for Antimicrobial Activity Source of Bacterial Two microbial isolates were chosen for antimicrobial investigation: Gram positive bacteria (S. aureus) and Gram negative bacteria (E. coli). Both microbial were purchased from Laboratory of Microbiology, Faculty of Sciences Mulawarman University, Samarinda, Indonesia. Testing for Antibacterial Activity The test organisms were sub cultured by streaking them on nutrient agar, followed by incubation for 20 hr at 37 °C. These were subcultured prior to each experiment and were used for antibacterial. The pitting-diffusion method of two species of bacteria Staphylococcus aureus and Escherichia coli was ferformed in in-vitro antibacterial Proceeding of 5th International Seminar on ISSN: 978-602-71169-7-9 New Paradigm and Innovation on Natural Sciences and Its Application (5th ISNPINSA) Hetty Manurung, et. al. P a g e  | 71 This Proceeding © Faculty of Sciences and Mathematics Diponegoro University, 2015 activity. Medium that was used in the antimicrobial activity was MHA (Mueller Hinton Agar), an inoculum containing 107 bacteria/mL was incorporated. After the suspension of bacteria seep into the media, then created pit in the media using the sterile iron cork borer (6 mm diameter). Using sterilized dropping pipettes, different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% /well) of leaves extract L.cubeba was carefully added into the wells The plates were then incubated at 37 °C for 24–48 hr. Chloramphenicol was used as positive control for antibacterial activities while DMSO was used as the negative control. The diameter of the inhibition zone was measured for antibacterial activities testing. Experiments were performed in triplicate, and the results were presented as the mean values of the diameters of the inhibitory zones from three runs. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone [10,11]. The diameters of the inhibitory zones value were used as criteria to judge the antimicrobial activity (strong active: the diameters of the inhibitory zones ≥ 20 mm, active: the diameters of the inhibitory zones 10-20 mm, moderately active: the diameters of the inhibitory zones 5-10 mm, not active: the diameters of the inhibitory zonesare invisible or ≤ 5 mm) [12]. Results and Discussion Phytochemical Analysis The results of the phytochemical analysis were carried out in extracts of Litsea cubeba. The experiment showed the presence of secondary metabolites such as alkaloids, flavonoids, phenols and steroids. The results of the phytochemical analysis of L.cubeba are shown in Table 1.  Tabel 1. Qualitative phytochemical analysis of leaves extract L.cubeba No Parameters Leaves extract 1 Alkaloid + 2 Flavonoid + 3 Phenol + 4 Saponin - 5 Steroid +  (+indicates presence, - indicates absence) The presence of alkaloids in leaves extract is likely to be responsible for antimicrobial activity effects observed. Alkaloid is one of the phytochemical compounds in Litsea cubeba that act as an antibacterial activity [13,14]. Antimicrobial Activity Antimicrobial activity is used to test whether the leaf extract has capability to control the growth of the bacterial. The zone of inhibition had been obtained for both bacterials Staphylococcus aureus and Escherichia coli. The result obtained, the zone of inhibition was recorded at ten concentrations of 10,20,30,40,50,60,70,80,90,and 100% in Table 2. In general Table 2 showed that increasing concentrations of leaf extracts enhanced inhibitory zone on both bacteria. The extracts displayed relative antimicrobial activities against bacterial tested with the diameter of inhibition zones ranging between 9.58±0.36 to 15.91±0.95 mm (S. aureus) and 9.63±0.32 to 18.63±0.18 mm (E. coli).  Tabel 2. Antimicrobial activity of leaves extract of Litsea cubeba Sample extract Conc.(%) Zone inhibition (mm) Stapylococcus aureus Escherichia coli 10% 9.58±0.36 9.63±0.32 20% 10.71±0.62 10.65±0.75 30% 11.36±0.41 11.07±0.40 40% 12.40±0.40 12.41±0.18 50% 12,71±0.70 13.62±0.11 60% 13.06±0.29 13.16±0.68 70% 13.63±0.27 14.00±0.37 80% 14.03±0.81 14.56±0.66 90% 13.80±0.69 15.08±0.32 100% 15.91±0.95 15.23±0.41 Chloramphenicol (25%) 18.33±0.10 18.63±0.18 The highest antibacterial activity in both bacteria was found at 100 % concentration and the lowest at 10 % concentration of leaves extract. The results obtained was indicated of the presence of broad spectrum antimicrobial compounds or metabolic toxins in L.cubeba leaves extract that could be exploited in treating infections associated with the aforementioned bacterial. Previous ISSN: 978-602-71169-7-9 Proceeding of 5th International Seminar on  New Paradigm and Innovation on Natural Science  and Its Application (5th ISNPINSA) 72 | P a g e  Hetty Manurung, et. al.  This Proceeding © Faculty of Sciences and Mathematics  Diponegoro University, 2015 studies on antimicrobial activities of medicinal plants indicated that inhibition zones of 10 mm or greater were taken to represent good activity of such plants [15,16]. Conclusion Balangla leaf extract can be used as a source of secondary metabolites such as alkaloids, Flavanoids, Phenol, and steroids. The results suggest that the antibacterial activity of leaves extract L. cubeba may contribute to prevent some of the diseases caused by S. aureus and E. coli. References T.T. 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Phytochemical Screening and Antibacterial Activity of Leaves Extract Balangla (Litsea cubeba (Lour) Pers.) from Malinau, East Borneo

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