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Antioxidant, Antimicrobial and Cytotoxicity Activities of Propolis from Beladin, Sarawak Stingless Bees Trigona itama Extract
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ScienceDirect Materials Today: Proceedings 19 (2019) 1752–1760
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ICCSE 2018
Antioxidant, Antimicrobial and Cytotoxicity Activities of Propolis from Beladin, Sarawak Stingless Bees Trigona itama Extract Syed Ahmad Tarmizi Wan Yusop, Ahmad Hafizi Sukairi, Wan Mazliena Aliana Wan Sabri, Mohd Razip Asaruddin* Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Abstract The natural product produced by stingless bees such as honey, beehive, pollen and propolis are well known for their benefits and has been used as traditional medicine by the Asian community. Trigona itama known as “lebah kelulut” by Malaysia community are widely distributed in Sarawak and T. itama are the main local species used by our local industries involved in commercialization of stingless bee products. Limited research and data on T. itama propolis, which resulted in less demand in industrial sectors than honeybee propolis. Biological activity and chemical constituent of T. itama propolis are influenced on the vegetation in T. itama habitat. Propolis is a by-product of T. itama bees that are produced by a combination of beeswax and resins. This study aims to identify the antioxidant, antimicrobial and cytotoxicity activities of Sarawak T. itama propolis in hexane, ethyl acetate and methanol extracts. Antioxidant activities of T. itama propolis was identified using 2,2-diphenyl-1-picrylhydrazyl (DPPH) solvent and measured using UV spectrophotometer. The antibacterial activity of T.itama propolis was evaluated by using Kirby-Bauer disk diffusion method. The sample of concentration 500, 750 and 1000 ug/mL were treated on the blank disc to determine the antimicrobial activity of propolis. The result from this study shows that all T. itama propolis extract possesses antioxidant activity and methanol extract (EC50 17.18) show the highest antioxidant activity compared to ascorbic acid (EC50 21.05). T. itama propolis extract has anti-bacterial activity against Staphylococcus aureus and Escherichia coli. All T. itama propolis crude extract show a low level of toxicity in Brine shrimp lethality test. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018. Keywords: Stingless bees, Trigona itama, Propolis, Antimicrobial, Antioxidant, Malaysia Stingless bees
* Corresponding author. Tel.: +6082-583136 E-mail address: [email protected]; [email protected]
2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018.
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1. Introduction Trigona itama is the most dominant stingless bee species found in Malaysia. The natural product produced by stingless bees such as honey, beehive, pollen and propolis are well known for their benefits and has been used as traditional medicine by Asian community [1]. Propolis is a by-product of T. itama bees that are produced by a combination of beeswax and resins collected from a variety of plant parts by stingless bees [2]. Propolis can be observed sticky at above room temperature and lower room temperature it becomes brittle and hard [3]. Stingless bees used propolis as sealing agent and defences mechanism to prevent invader entering hive [4]. Studies show that oxidative stress leads to several pathologies condition like cancer, cardiovascular, neurodegenerative and increase the aging process. Oxidative stress is a condition where unbalance free radical (reactive oxygen and nitrogen species production) and antioxidant in the human body [5]. Free radical production exceeds the antioxidant rate of the organism and leads to a condition where free radical species attack DNA, protein and lipid, thus damaging the cell structure and genetic material [6]. Antioxidant in a natural product such as consumable fruit and vegetable categorized as chemopreventive agents where antioxidant provide protection against oxidative stress by scavenging the free radical, inhibiting lipid peroxidation and preventing disease progression [7]. Diet rich in antioxidant from the natural product will bring benefit to human health. The previous study shows that reactive oxygen species (ROS) such as superoxidenanion and hydroxyl radical are scavenged by antioxidant present in propolis [8]. Antibiotic is used to treat infectious disease caused by microorganism infection and the rate of antimicrobial resistance has increased during the past few decades due to the rapid usage of main antibiotic and lack of alternative medicine. Antibiotic is used to treat infectious disease caused by microorganism infection and the rate of antimicrobial resistance has increased during the past few decades due to the rapid usage of main antibiotic and lack of alternative medicine. A microorganism that develops antimicrobial resistance (AMR) is known as a superbug. Superbug can become a global concern because antimicrobial resistance can cause prolonged illness, ineffective treatment and increase risk in a medical procedure. Studies show that antibiotic resistance has increased during the past decade and give adverse side effect to the hospital patient, pharmaceutical industry and community. Presences of active compounds such as flavonoids, phenolic acids, and their esters that lead to biological activities of propolis [9]. Previous research revealed that propolis consists of biological activity that will bring many therapeutic benefits to humans, such as antimicrobial, antitumor, and antioxidant [10]. Lack of information regarding the antioxidant and antimicrobial activity of stingless bees propolis lead to less demand for propolis in industrial industries compared to honeybee propolis. The search for new antioxidant and antimicrobial origin from a natural product with less side effect is the major challenge in the pharmaceutical industry. Therefore, this study was conducted to investigate the antioxidant, antimicrobial and cytotoxicity activity in stingless bees propolis extract. 2.0 Methodology 2.1 General Methods Biological assay (DPPH Radical Scavenging Assay, disk diffusion method and Brine shrimp lethality) was performed to identify the potential activity of antioxidant and antimicrobial of T. itama propolis. T. itama propolis was soaked with hexane, ethyl acetate and methanol solvent were used during the extraction process. The sample was partition into hexane, ethyl acetate and methanol crude extract. Data was calculated and analyzed using GraphPad Prism 8.0. 2.2 Instrument Ultraviolet-Visible (UV) Spectrophotometer at wavelength 517 nm and 625nm (Jasco V-630) was used to determine the absorbance value of the sample for DPPH radical scavenging activity and to the standardized density of bacteria to 0.5 McFarland. Rotary evaporator. 2.3 Materials Hexane, Ethyl Acetate, Methanol (HmBG), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich), ascorbic acid (Sigma Aldrich), Muller Hinton agar, Muller Hinton Broth, dimethyl sulfoxide (DMSO), Benzylpenicillin and imipenem.
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2.4 Source of microorganism Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) was used in this study and was collected from UNIMAS Virology Laboratory. E. coli (ATCC 25922) and S. aureus (ATCC 25923) (ATCC 25922) was preserved and stored in selected agar. 2.5 Propolis preparation T. itama propolis was received from Beladin, Sarawak. Propolis was collected into the sterilized container to prevent from contamination and attack from a predator. T. itama propolis was cut into small pieces using a sterile scissor and weighted. Small fragments of propolis were placed in the sterilized beaker and sealed with aluminum foil and parafilm. T. itama propolis was soaked with 1000ml of hexane and left for 72 hours in an orbital shaker. After 72 hours, the T. itama propolis extracts were filtered using filter paper (0.040-0.063mm) and concentrated using a rotary evaporator at 650C and under reduced pressure (100 psi). After the crude extract was obtained, the remaining propolis was extracted in the same manners using ethyl acetate solvent. Remaining propolis was extracted with methanol solvent. The final weight of the T. itama propolis crude extract was weighed and stored at -200C for further tests. 2.6 Antioxidant assay Antioxidant activity of T. itama propolis was measured according to 2,2-diphenyl-1-picrylhydrazyl DPPH Radical Scavenging Assay based on a method established Maskam, et al., [11], with minor modification. T. itama propolis samples were reacted with DPPH in a methanol solution and ascorbic was used as positive control. DPPH stock solution was prepared by dissolving 20 mg of DPPH in 100 ml of methanol and sample stock solution was prepared by dissolving 1 mg of T. itama propolis sample in 1 ml of methanol. The stock solution was transferred to vial based on the concentration of T. itama propolis extract and DPPH (10, 30, 50, 100, 300, 500 µL) and 1 ml of methanol was added into each vial. The vial contains with mixture was shaken vigorously and incubated at room temperature for 30 minutes. After incubation in dark room. The total volume of sample in the vial is 1 mL of sample, 0.8 mL of methanol and 0.5 mL of DPPH solution. The absorbance was measured at 517 nm using UV spectrophotometer. DPPH activity was expressed as IC50, which indicate the concentration of the sample required to inhibit 50% of DPPH free radical. The ability of T. itama propolis extract to scavenge DPPH free radical was calculated using the equation shown below:
Percentage of DPPH radical scavenging activity (100%)=
Abs. control-Abs. sample ×100 Abs. control
2.7 Antibacterial assay Antimicrobial activity of T. itama propolis on S. aureus ATCC 25923 and E. coli ATCC 25922 was examined using the disk diffusion method based on the Kirby-Bauer method [12]. 10 ug of benzylpenicillin disk and IU of imipenem antibiotic disk was used as a positive control in this antibacterial assay. Extract solvent was used as negative control. S. aureus and E. coli were cultured on blood agar and incubated for 24 hours. A single colony of microorganism was transferred to 10 ml of Mueller Hinton Broth for 24 hours at 370C in the incubator. Dilution of the Mueller Hinton Broth of S. aureus and E. coli was performed to the standardized density of bacteria to 0.5 McFarland standard (1 × 108 CFU/ mL) by adjusting the absorbance value (0.08 to 0.10) under 625nm wavelength and measured using spectrophotometer. The swab was streaked on the entire plate for three times and each time the MHA plate was rotated 900 to ensure consistent distribution of bacterial suspension on the MHA surface. T. itama propolis extract was dissolved in extract solvent based on different concentration, ranging from 500, 750 and 1000 ug/mL. 20 µl sample of concentration 500, 750 and 1000 ug/mL were treated on the blank disc. Impregnated disc with T. itama propolis extract was transferred to MHA plate using sterile forceps. The place was incubated at 370C for 24 hours and zones of inhibition of growth around the disc were recorded as data. 2.8 Brine shrimp lethality Potential toxicity of each T. itama propolis crude extract was determined using brine shrimp lethality method, where 6 graded doses (.10µg/mL, 20µg/mL, 100µg/mL, 200µg/mL, 600µg/mL, 1000µg/mL) were used [13]. Artemia salina shrimp was used in this test and eggs were hatched in a beaker filled with saltwater and constant illumination and oxygen supply for 48 hours. The mature shrimp were then used to identify toxicity of propolis sample. The
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stock solution was prepared by dissolving 6 mg of each T. itama propolis extract in 6 ml of methanol solvent. The stock solution was transferred into each of multi-well based on six graded doses. Six graded concentration of methanol solvent was transferred into each multi-well and was used as positive control. Multi-well was left for 24 under fume food for evaporation process. 50µl dimethyl sulfoxide (DMSO) and 1m of water were transferred into each well. Ten brine shrimp were placed into each vial and incubated for 24 hours. Three replicated was performed in this test to ensure the reliability of toxicity test. After 24 hours of incubation, larva were examined using a magnifying glass and the number of survival and death shrimp was counted as a data. Data were transformed to Probit analysis for the determination of LC50 values of each T. itama propolis extract. LC50 of each T. itama propolis extract was obtained by plotting percentage of the dead shrimp against the logarithm of the sample concentration.
Percentage of mortality (%)=
total brine shrimp death ×100 total brine shrimp
2.9 Data and statistical analyses Concentration curves of antibacterial and antioxidant inhibition of T. itama propolis extract were calculated using GraphPad Prism 6.0 (linear regression). LC50 T. itama crude extract was determined by plotting graph of mortality percentage versus log concentration using GraphPad 8.0 Prism (Sigmodal mode). 3.0 Results and Discussion The result of antioxidant, antimicrobial and toxicity activity of T. itama propolis crude extract were shown in Table 1-5 and Figure 1-3 3.1 Antioxidant activity of T. itama propolis TABLE 1. Shows the percentage of DPPH inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid.
Concentration of sample (mg/mL) 10 30 50 100 300 500
Hexane crude extract 32.56 35.8 65.4 76.6 88.4 90.7
Percentage of inhibition (%) Ethyl Acetate crude Methanol crude extract extract 41.77 46.71 46.71 51.97 67.76 66.11 77.6 88.15 83.22 99.4 92.1 99.34
Ascorbic acid 95.9 96.35 96.81 96.13 95.44 95.22
TABLE 2. Shows the EC50 value of inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid.
Samples Hexane crude extract Ethyl Acetate crude extract Methanol crude extract Ascorbic acid
EC50 32.11 21.05 17.18 30.63
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FIGURE 1. Shows the percentage of DPPH inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid. Log concentration vs Percentage of antioxidant inhibition of T. itama propolis crude extract. 3.2 Antimicrobial activity of T. itama propolis TABLE 3. Shows the mean of inhibition zone of T. itama propolis sample against for S. aureus. Inhibition was measured in mm (millimeter) unit.
Samples
Mean zone of Inhibition (MM) Concentration of T. itama propolis extract
Hexane crude extract Ethyl acetate crude extract Methanol crude extract
500µg/mL
750µg/mL
1000µg/mL
-
5 6
8 4 10
TABLE 4. Shows the means of inhibition zone of T. itama propolis sample against E.coli Inhibition was measured in mm (millimeter) unit.
Samples
Mean zone of Inhibition (MM) Concentration of T. itama propolis extract
Hexane crude extract Ethyl acetate crude extract Methanol crude extract
500µg/mL
750µg/mL
1000µg/mL
-
4 6
8 5 10
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FIGURE 2. (A) and (B) shows the mean of inhibition zone of T. itama propolis sample against for E.coli and S. aureus. Inhibition was measured in mm (millimeter) unit. Data were analyzed using GraphPad 8.0 3.3 Toxicity (brine shrimp lethality) activity of T. itama propolis TABLE 5. Shows the average percentage of mortality and LC50 of T. itama propolis sample
Sample T. itama propolis Crude extract Hexane Ethyl acetate Methanol
10 0 0 0
20 0 0 0
Average percentage of mortality (%) Concentration of sample (mg/mL) 100 200 600 0 20.00 53.33 0 0 13.3 16.67 36.67 70.33
LC50 µg/ml 1000 73.3 37.3 93.33
623.7 670.8 501.2
FIGURE 3. Shows mortality percentage produced by T. itama propolis sample in brine Shrimp lethality test. Log concentration of T.itama propolis crude extract vs mortality percentage of brine shrimp. Data were analyzed using GraphPad Prism 8.0.
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4.0 Discussion In this research, T. itama propolis was studied due to T. itama are widely distributed in Sarawak and T. itama are the main local species used by our local industries involved in commercialization of stingless bees products. Based on previous studies, the active compound in propolis was influenced by a plant that was collected by bees [14]. The propolis used in this study was collected from Beladin, Sarawak and the primary source of resin was harvested from Earleaf acacia that known as “pokok akasia” by Malaysia community. In this study, antioxidant activity was observed in hexane, ethyl acetate and methanol propolis crude extract. Based on this research, Table 1 and 2 shows the ability of T. itama propolis extract scavenge di2,2-diphenyl-1picrylhydrazyl (DPPH) with different concentration. DPPH assay will help to identify the percentage of radical was trapped by the potential antioxidant compound in T. itama propolis extract. Figure 1 shows methanol (EC50 17.18) extract shows the highest percentage of antioxidant compared to hexane (EC50 32.11), ethyl acetate (EC50 21.05) and positive control which is ascorbic acid (EC50 30.63). Methanol extract shows a high percentage of EC50 due to methanol has high efficiency to extract a wide range of active compound in the extract samples [15]. The difference result was produced in this antioxidant result is due to the different type of solvent was used during the extraction process. Antioxidant activity was contributed by the synergistic effect of multiple components present in T. itama propolis extract [16]. High radical scavenging activity produced due to presences of flavonoid and phenolic content that contribute to the antioxidant activity of T. itama propolis [17]. Previous study Bakar [18], shows that honey collected from T. itama also exhibit a high level of DPPH (97.30%) but methanol extract propolis from this study shows the highest percentage of antioxidant (99.34%). Propolis was proven a natural powerful antioxidant product and has a high level of antioxidant compared to bee bread and honey [16]. Propolis classified as powerful natural antioxidant due to the ability of polyphenols and flavonoid that work as an antioxidant agent due to the hydrogendonating ability of their hydroxyl groups and ability to donate electrons to arrest the production of free radicals as a result of oxidative stress [19]. Based on Table 3 and 4, all T. itama propolis extract shows possess of antimicrobial activity against E. coli ATCC 25922 and S. aureus ATCC 25923. All T. itama propolis crude extract shows inhibition of S. aureus ATCC 25923 and E. coli ATCC 25922 at a concentration of 750µg and 1000µg. Figure 2(A) and 3(B) shows the inhibition zone of S. aureus ATCC 25923 and E. coli ATCC 25922 can be observed at a concentration of 750µg and 1000µg T. itama propolis extract. Methanol crude extract shows the highest rate of inhibition at 750µg/mL and 1000 µg/mL compared to other types of T. itama propolis crude extract. On the other side, ethyl acetate crude extract produced the lowest rate of inhibition and no inhibition can be observed on 750µg/mL against S. aureus and E. coli. High inhibition rate in methanol crude extract may indicate more active antimicrobial compound soluble in methanol extract method. Gram-positive and gram-negative bacteria were more sensitive at a concentration of 1000 µg/mL. T T. itama propolis can produce antimicrobial effects due to the active compound from propolis able to inhibit bacterial RNA polymerase and degradation of the bacterial cytoplasmic membrane [20]. Based on Table 5 and Figure 3 brine shrimp lethality study, methanol crude (LC50 501.2 µg/mL) extract shows a high level of toxicity compared to hexane (LC50 623.7 µg/mL) and ethyl acetate extract (LC50 670.8µg/mL). On the opposite site, ethyl acetate crude shows the lowest level of toxicity compared to other T. itama propolis extracts. The previous study shows that sample produce LC50 values above than 250 were considered significantly not active for toxicity effect this indicate that all T. itama extract have a low level of toxicity [21]. In this study, we found that all three types of T. itama propolis extract from Beladin Sarawak shows presences of antioxidant activity and antimicrobial activity against gram-positive and gram-negative bacteria. The different result compared to the previous study of propolis from other countries will represent the uniqueness of Beladin, Sarawak propolis. Variation of toxicity result due to the difference amount of phytochemical compound in T. itama propolis. From this study, we can observed that methanol extract produced a high rate of antioxidant and antimicrobial activity that lead to a high level of toxicity compared to the other extract in this study, indicates that methanol has high efficiency to extract a wide range of active compound in T. itama propolis extract. Different or the same biological active compound are present in all 3 types of T. itama propolis extract that lead to a positive result for antimicrobial, antioxidant and cytotoxicity. Study by Viuda-Martos T et al., [22], shows that the biological activities
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of propolis due to phytochemical compounds in propolis. Active organic compound that can be found in propolis is flavonoids, terpenes, beta steroid, phenolic, and aromatic aldehydes [23]. Active biological compound inside propolis help in maintaining the low level of microorganism in the hive and protect the bee’s colony [24]. Variation of result due to the different origin of T. itama propolis. The phytochemical compound in propolis will influence the quality, colour and biological activity of propolis. Further research needs to be performed to identify an active phytochemical compound that contributed to antimicrobial and antioxidant effect from Beladin, Sarawak T.itama propolis. 5.0 Conclusion This study showed that all three types of T. itama propolis extract from Beladin Sarawak shows presences of antioxidant activity and antimicrobial activity against gram-positive and gram-negative bacteria. Methanol extract produced a high rate of antioxidant and antimicrobial activity. All T.itama propolis extract showed a low level of toxicity. Further study needed to perfume to identify a specific compound that leads to positive result in a biological assay. Acknowledgements The author would like to express special appreciation to Dr. Mohd Razip Asaruddin, Beladin community and Universiti Malaysia Sarawak staff for the endless support and guidance and patience throughout the research Declaration of interest The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper. References [1] Rasmussen, C. and Cameron, S.A., 2009. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society, 99(1), pp.206-232 [2] Bankova, V., 2009. Chemical diversity of propolis makes it a valuable source of new biologically active compounds. Journal of ApiProduct and ApiMedical Science, 1(2), pp.23-28. [3] Parolia, A., Thomas, M.S., Kundabala, M. and Mohan, M., 2010. Propolis and its potential uses in oral health. International Journal of Medicine and Medical Science, 2(7), pp.210-215. [4] Popova, M.P., Bankova, V.S., Bogdanov, S., Tsvetkova, I., Naydenski, C., Marcazzan, G.L. and Sabatini, A.G., 2007. Chemical characteristics of poplar type propolis of different geographic origin. Apidologie, 38(3), pp.306-311. [5] Halliwell, B. and Gutteridge, J.M., 2015. Free radicals in biology and medicine. Oxford University Press, USA. [6] Byers, T. and Sedjo, R.L., 2015. Body fatness as a cause of cancer: epidemiologic clues to biologic mechanisms. Endocrine-related cancer, 22(3), pp.R125-R134. [7] Braughler, J.M., Duncan, L.A. and Chase, R.L., 1986. The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation. Journal of Biological Chemistry, 261(22), pp.10282-10289. [8] Campos, J.F., dos Santos, U.P., Macorini, L.F.B., de Melo, A.M.M.F., Balestieri, J.B.P., Paredes-Gamero, E.J., Cardoso, C.A.L., de Picoli Souza, K. and dos Santos, E.L., 2014. Antimicrobial, antioxidant and cytotoxic activities of propolis from Melipona orbignyi (Hymenoptera, Apidae). Food and Chemical Toxicology, 65, pp.374-380. [9] Kurek-Górecka, A., Rzepecka-Stojko, A., Górecki, M., Stojko, J., Sosada, M. and Świerczek-Zięba, G., 2013. Structure and antioxidant activity of polyphenols derived from propolis. Molecules, 19(1), pp.78-101. [10] Paviani, L.C., Fiorito, G., Sacoda, P. and Cabral, F.A., 2013, April. Different solvents for extraction of Brazilian green propolis: Composition and extraction yield of phenolic compounds. In III Iberoamerican Conference on Supercritical Fluid (pp. 1-5). [11] Maskam, M. F., Mohamad, J., Abdulla, M. A., Afzan, A. & Wasiman, I. P.L., 2014. Antioxidant Activity of Rhodomyrtus tomentosa (Kemunting) Fruits and Its Effect on Lipid Profile in Induced-cholesterol New Zealand White Rabbits. Sains Malaysiana, 43(11), pp.1673-1684. [12] Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology, 45(4_ts), pp.493-496. [13] Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.J. and McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta medica, 45(05), pp.31-34. [14] Park, Y.K., Alencar, S.M. and Aguiar, C.L., 2002. Botanical origin and chemical composition of Brazilian propolis. Journal of Agricultural and Food Chemistry, 50(9), pp.2502-2506.
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[15] Brand-Williams, W., Cuvelier, M.E. and Berset, C.L.W.T., 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology, 28(1), pp.25-30. [16] Hamburger, M. and Hostettmann, K., 1991. 7. Bioactivity in plants: the link between phytochemistry and medicine. Phytochemistry, 30(12), pp.3864-3874. [17] Massaro, F.C., Brooks, P.R., Wallace, H.M. and Russell, F.D., 2011. Cerumen of Australian stingless bees (Tetragonula carbonaria): gas chromatography-mass spectrometry fingerprints and potential anti-inflammatory properties. Naturwissenschaften, 98(4), pp.329-337 [18] Bakar, M.F.A., Sanusi, S.B., Bakar, F.I.A., Cong, O.J. and Mian, Z., 2017. Physicochemical and Antioxidant Potential of Raw Unprocessed Honey From Malaysian Stingless Bees. Pakistan Journal of Nutrition, 16(11), pp.888-894. [19] Afroz, R., Tanvir, E.M., Paul, S., Bhoumik, N.C., Gan, S.H. and Khalil, M.I., 2016. DNA damage inhibition properties of sundarban honey and its phenolic composition. Journal of Food Biochemistry, 40(4), pp.436-445. [20] Cushnie, T.T. and Lamb, A.J., 2005. Antimicrobial activity of flavonoids. International journal of antimicrobial agents, 26(5), pp.343-356. [21] Rieser, M.J., Gu, Z.M., Fang, X.P., Zeng, L., Wood, K.V. and McLaughlin, J.L., 1996. Five novel mono-tetrahydrofuran ring acetogenins from the seeds of Annona muricata. Journal of Natural Products, 59(2), pp.100-108. [22] Viuda‐Martos, M., Ruiz‐Navajas, Y., Fernández‐López, J. and Pérez‐Álvarez, J.A., 2008. Functional properties of honey, propolis, and royal jelly. Journal of food science, 73(9), pp.R117-R124. [23] Aga, H., Shibuya, T., Sugimoto, T., Kurimoto, M. and Nakajima, S., 1994. Isolation and identification of antimicrobial compounds in Brazilian propolis. Bioscience, Biotechnology, and Biochemistry, 58(5), pp.945-946. [24] Popova, M., Reyes, M., Le Conte, Y. and Bankova, V., 2014. Propolis chemical composition and honeybee resistance against Varroa destructor. Natural product research, 28(11), pp.788-794.
ScienceDirect Materials Today: Proceedings 19 (2019) 1752–1760
www.materialstoday.com/proceedings
ICCSE 2018
Antioxidant, Antimicrobial and Cytotoxicity Activities of Propolis from Beladin, Sarawak Stingless Bees Trigona itama Extract Syed Ahmad Tarmizi Wan Yusop, Ahmad Hafizi Sukairi, Wan Mazliena Aliana Wan Sabri, Mohd Razip Asaruddin* Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Abstract The natural product produced by stingless bees such as honey, beehive, pollen and propolis are well known for their benefits and has been used as traditional medicine by the Asian community. Trigona itama known as “lebah kelulut” by Malaysia community are widely distributed in Sarawak and T. itama are the main local species used by our local industries involved in commercialization of stingless bee products. Limited research and data on T. itama propolis, which resulted in less demand in industrial sectors than honeybee propolis. Biological activity and chemical constituent of T. itama propolis are influenced on the vegetation in T. itama habitat. Propolis is a by-product of T. itama bees that are produced by a combination of beeswax and resins. This study aims to identify the antioxidant, antimicrobial and cytotoxicity activities of Sarawak T. itama propolis in hexane, ethyl acetate and methanol extracts. Antioxidant activities of T. itama propolis was identified using 2,2-diphenyl-1-picrylhydrazyl (DPPH) solvent and measured using UV spectrophotometer. The antibacterial activity of T.itama propolis was evaluated by using Kirby-Bauer disk diffusion method. The sample of concentration 500, 750 and 1000 ug/mL were treated on the blank disc to determine the antimicrobial activity of propolis. The result from this study shows that all T. itama propolis extract possesses antioxidant activity and methanol extract (EC50 17.18) show the highest antioxidant activity compared to ascorbic acid (EC50 21.05). T. itama propolis extract has anti-bacterial activity against Staphylococcus aureus and Escherichia coli. All T. itama propolis crude extract show a low level of toxicity in Brine shrimp lethality test. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018. Keywords: Stingless bees, Trigona itama, Propolis, Antimicrobial, Antioxidant, Malaysia Stingless bees
* Corresponding author. Tel.: +6082-583136 E-mail address: [email protected]; [email protected]
2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Chemical Sciences and Engineering: Advance and New Materials, ICCSE 2018.
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1. Introduction Trigona itama is the most dominant stingless bee species found in Malaysia. The natural product produced by stingless bees such as honey, beehive, pollen and propolis are well known for their benefits and has been used as traditional medicine by Asian community [1]. Propolis is a by-product of T. itama bees that are produced by a combination of beeswax and resins collected from a variety of plant parts by stingless bees [2]. Propolis can be observed sticky at above room temperature and lower room temperature it becomes brittle and hard [3]. Stingless bees used propolis as sealing agent and defences mechanism to prevent invader entering hive [4]. Studies show that oxidative stress leads to several pathologies condition like cancer, cardiovascular, neurodegenerative and increase the aging process. Oxidative stress is a condition where unbalance free radical (reactive oxygen and nitrogen species production) and antioxidant in the human body [5]. Free radical production exceeds the antioxidant rate of the organism and leads to a condition where free radical species attack DNA, protein and lipid, thus damaging the cell structure and genetic material [6]. Antioxidant in a natural product such as consumable fruit and vegetable categorized as chemopreventive agents where antioxidant provide protection against oxidative stress by scavenging the free radical, inhibiting lipid peroxidation and preventing disease progression [7]. Diet rich in antioxidant from the natural product will bring benefit to human health. The previous study shows that reactive oxygen species (ROS) such as superoxidenanion and hydroxyl radical are scavenged by antioxidant present in propolis [8]. Antibiotic is used to treat infectious disease caused by microorganism infection and the rate of antimicrobial resistance has increased during the past few decades due to the rapid usage of main antibiotic and lack of alternative medicine. Antibiotic is used to treat infectious disease caused by microorganism infection and the rate of antimicrobial resistance has increased during the past few decades due to the rapid usage of main antibiotic and lack of alternative medicine. A microorganism that develops antimicrobial resistance (AMR) is known as a superbug. Superbug can become a global concern because antimicrobial resistance can cause prolonged illness, ineffective treatment and increase risk in a medical procedure. Studies show that antibiotic resistance has increased during the past decade and give adverse side effect to the hospital patient, pharmaceutical industry and community. Presences of active compounds such as flavonoids, phenolic acids, and their esters that lead to biological activities of propolis [9]. Previous research revealed that propolis consists of biological activity that will bring many therapeutic benefits to humans, such as antimicrobial, antitumor, and antioxidant [10]. Lack of information regarding the antioxidant and antimicrobial activity of stingless bees propolis lead to less demand for propolis in industrial industries compared to honeybee propolis. The search for new antioxidant and antimicrobial origin from a natural product with less side effect is the major challenge in the pharmaceutical industry. Therefore, this study was conducted to investigate the antioxidant, antimicrobial and cytotoxicity activity in stingless bees propolis extract. 2.0 Methodology 2.1 General Methods Biological assay (DPPH Radical Scavenging Assay, disk diffusion method and Brine shrimp lethality) was performed to identify the potential activity of antioxidant and antimicrobial of T. itama propolis. T. itama propolis was soaked with hexane, ethyl acetate and methanol solvent were used during the extraction process. The sample was partition into hexane, ethyl acetate and methanol crude extract. Data was calculated and analyzed using GraphPad Prism 8.0. 2.2 Instrument Ultraviolet-Visible (UV) Spectrophotometer at wavelength 517 nm and 625nm (Jasco V-630) was used to determine the absorbance value of the sample for DPPH radical scavenging activity and to the standardized density of bacteria to 0.5 McFarland. Rotary evaporator. 2.3 Materials Hexane, Ethyl Acetate, Methanol (HmBG), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich), ascorbic acid (Sigma Aldrich), Muller Hinton agar, Muller Hinton Broth, dimethyl sulfoxide (DMSO), Benzylpenicillin and imipenem.
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2.4 Source of microorganism Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) was used in this study and was collected from UNIMAS Virology Laboratory. E. coli (ATCC 25922) and S. aureus (ATCC 25923) (ATCC 25922) was preserved and stored in selected agar. 2.5 Propolis preparation T. itama propolis was received from Beladin, Sarawak. Propolis was collected into the sterilized container to prevent from contamination and attack from a predator. T. itama propolis was cut into small pieces using a sterile scissor and weighted. Small fragments of propolis were placed in the sterilized beaker and sealed with aluminum foil and parafilm. T. itama propolis was soaked with 1000ml of hexane and left for 72 hours in an orbital shaker. After 72 hours, the T. itama propolis extracts were filtered using filter paper (0.040-0.063mm) and concentrated using a rotary evaporator at 650C and under reduced pressure (100 psi). After the crude extract was obtained, the remaining propolis was extracted in the same manners using ethyl acetate solvent. Remaining propolis was extracted with methanol solvent. The final weight of the T. itama propolis crude extract was weighed and stored at -200C for further tests. 2.6 Antioxidant assay Antioxidant activity of T. itama propolis was measured according to 2,2-diphenyl-1-picrylhydrazyl DPPH Radical Scavenging Assay based on a method established Maskam, et al., [11], with minor modification. T. itama propolis samples were reacted with DPPH in a methanol solution and ascorbic was used as positive control. DPPH stock solution was prepared by dissolving 20 mg of DPPH in 100 ml of methanol and sample stock solution was prepared by dissolving 1 mg of T. itama propolis sample in 1 ml of methanol. The stock solution was transferred to vial based on the concentration of T. itama propolis extract and DPPH (10, 30, 50, 100, 300, 500 µL) and 1 ml of methanol was added into each vial. The vial contains with mixture was shaken vigorously and incubated at room temperature for 30 minutes. After incubation in dark room. The total volume of sample in the vial is 1 mL of sample, 0.8 mL of methanol and 0.5 mL of DPPH solution. The absorbance was measured at 517 nm using UV spectrophotometer. DPPH activity was expressed as IC50, which indicate the concentration of the sample required to inhibit 50% of DPPH free radical. The ability of T. itama propolis extract to scavenge DPPH free radical was calculated using the equation shown below:
Percentage of DPPH radical scavenging activity (100%)=
Abs. control-Abs. sample ×100 Abs. control
2.7 Antibacterial assay Antimicrobial activity of T. itama propolis on S. aureus ATCC 25923 and E. coli ATCC 25922 was examined using the disk diffusion method based on the Kirby-Bauer method [12]. 10 ug of benzylpenicillin disk and IU of imipenem antibiotic disk was used as a positive control in this antibacterial assay. Extract solvent was used as negative control. S. aureus and E. coli were cultured on blood agar and incubated for 24 hours. A single colony of microorganism was transferred to 10 ml of Mueller Hinton Broth for 24 hours at 370C in the incubator. Dilution of the Mueller Hinton Broth of S. aureus and E. coli was performed to the standardized density of bacteria to 0.5 McFarland standard (1 × 108 CFU/ mL) by adjusting the absorbance value (0.08 to 0.10) under 625nm wavelength and measured using spectrophotometer. The swab was streaked on the entire plate for three times and each time the MHA plate was rotated 900 to ensure consistent distribution of bacterial suspension on the MHA surface. T. itama propolis extract was dissolved in extract solvent based on different concentration, ranging from 500, 750 and 1000 ug/mL. 20 µl sample of concentration 500, 750 and 1000 ug/mL were treated on the blank disc. Impregnated disc with T. itama propolis extract was transferred to MHA plate using sterile forceps. The place was incubated at 370C for 24 hours and zones of inhibition of growth around the disc were recorded as data. 2.8 Brine shrimp lethality Potential toxicity of each T. itama propolis crude extract was determined using brine shrimp lethality method, where 6 graded doses (.10µg/mL, 20µg/mL, 100µg/mL, 200µg/mL, 600µg/mL, 1000µg/mL) were used [13]. Artemia salina shrimp was used in this test and eggs were hatched in a beaker filled with saltwater and constant illumination and oxygen supply for 48 hours. The mature shrimp were then used to identify toxicity of propolis sample. The
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stock solution was prepared by dissolving 6 mg of each T. itama propolis extract in 6 ml of methanol solvent. The stock solution was transferred into each of multi-well based on six graded doses. Six graded concentration of methanol solvent was transferred into each multi-well and was used as positive control. Multi-well was left for 24 under fume food for evaporation process. 50µl dimethyl sulfoxide (DMSO) and 1m of water were transferred into each well. Ten brine shrimp were placed into each vial and incubated for 24 hours. Three replicated was performed in this test to ensure the reliability of toxicity test. After 24 hours of incubation, larva were examined using a magnifying glass and the number of survival and death shrimp was counted as a data. Data were transformed to Probit analysis for the determination of LC50 values of each T. itama propolis extract. LC50 of each T. itama propolis extract was obtained by plotting percentage of the dead shrimp against the logarithm of the sample concentration.
Percentage of mortality (%)=
total brine shrimp death ×100 total brine shrimp
2.9 Data and statistical analyses Concentration curves of antibacterial and antioxidant inhibition of T. itama propolis extract were calculated using GraphPad Prism 6.0 (linear regression). LC50 T. itama crude extract was determined by plotting graph of mortality percentage versus log concentration using GraphPad 8.0 Prism (Sigmodal mode). 3.0 Results and Discussion The result of antioxidant, antimicrobial and toxicity activity of T. itama propolis crude extract were shown in Table 1-5 and Figure 1-3 3.1 Antioxidant activity of T. itama propolis TABLE 1. Shows the percentage of DPPH inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid.
Concentration of sample (mg/mL) 10 30 50 100 300 500
Hexane crude extract 32.56 35.8 65.4 76.6 88.4 90.7
Percentage of inhibition (%) Ethyl Acetate crude Methanol crude extract extract 41.77 46.71 46.71 51.97 67.76 66.11 77.6 88.15 83.22 99.4 92.1 99.34
Ascorbic acid 95.9 96.35 96.81 96.13 95.44 95.22
TABLE 2. Shows the EC50 value of inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid.
Samples Hexane crude extract Ethyl Acetate crude extract Methanol crude extract Ascorbic acid
EC50 32.11 21.05 17.18 30.63
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FIGURE 1. Shows the percentage of DPPH inhibition activity of hexane, ethyl acetate and methanol T. itama propolis crude extract against ascorbic acid. Log concentration vs Percentage of antioxidant inhibition of T. itama propolis crude extract. 3.2 Antimicrobial activity of T. itama propolis TABLE 3. Shows the mean of inhibition zone of T. itama propolis sample against for S. aureus. Inhibition was measured in mm (millimeter) unit.
Samples
Mean zone of Inhibition (MM) Concentration of T. itama propolis extract
Hexane crude extract Ethyl acetate crude extract Methanol crude extract
500µg/mL
750µg/mL
1000µg/mL
-
5 6
8 4 10
TABLE 4. Shows the means of inhibition zone of T. itama propolis sample against E.coli Inhibition was measured in mm (millimeter) unit.
Samples
Mean zone of Inhibition (MM) Concentration of T. itama propolis extract
Hexane crude extract Ethyl acetate crude extract Methanol crude extract
500µg/mL
750µg/mL
1000µg/mL
-
4 6
8 5 10
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FIGURE 2. (A) and (B) shows the mean of inhibition zone of T. itama propolis sample against for E.coli and S. aureus. Inhibition was measured in mm (millimeter) unit. Data were analyzed using GraphPad 8.0 3.3 Toxicity (brine shrimp lethality) activity of T. itama propolis TABLE 5. Shows the average percentage of mortality and LC50 of T. itama propolis sample
Sample T. itama propolis Crude extract Hexane Ethyl acetate Methanol
10 0 0 0
20 0 0 0
Average percentage of mortality (%) Concentration of sample (mg/mL) 100 200 600 0 20.00 53.33 0 0 13.3 16.67 36.67 70.33
LC50 µg/ml 1000 73.3 37.3 93.33
623.7 670.8 501.2
FIGURE 3. Shows mortality percentage produced by T. itama propolis sample in brine Shrimp lethality test. Log concentration of T.itama propolis crude extract vs mortality percentage of brine shrimp. Data were analyzed using GraphPad Prism 8.0.
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4.0 Discussion In this research, T. itama propolis was studied due to T. itama are widely distributed in Sarawak and T. itama are the main local species used by our local industries involved in commercialization of stingless bees products. Based on previous studies, the active compound in propolis was influenced by a plant that was collected by bees [14]. The propolis used in this study was collected from Beladin, Sarawak and the primary source of resin was harvested from Earleaf acacia that known as “pokok akasia” by Malaysia community. In this study, antioxidant activity was observed in hexane, ethyl acetate and methanol propolis crude extract. Based on this research, Table 1 and 2 shows the ability of T. itama propolis extract scavenge di2,2-diphenyl-1picrylhydrazyl (DPPH) with different concentration. DPPH assay will help to identify the percentage of radical was trapped by the potential antioxidant compound in T. itama propolis extract. Figure 1 shows methanol (EC50 17.18) extract shows the highest percentage of antioxidant compared to hexane (EC50 32.11), ethyl acetate (EC50 21.05) and positive control which is ascorbic acid (EC50 30.63). Methanol extract shows a high percentage of EC50 due to methanol has high efficiency to extract a wide range of active compound in the extract samples [15]. The difference result was produced in this antioxidant result is due to the different type of solvent was used during the extraction process. Antioxidant activity was contributed by the synergistic effect of multiple components present in T. itama propolis extract [16]. High radical scavenging activity produced due to presences of flavonoid and phenolic content that contribute to the antioxidant activity of T. itama propolis [17]. Previous study Bakar [18], shows that honey collected from T. itama also exhibit a high level of DPPH (97.30%) but methanol extract propolis from this study shows the highest percentage of antioxidant (99.34%). Propolis was proven a natural powerful antioxidant product and has a high level of antioxidant compared to bee bread and honey [16]. Propolis classified as powerful natural antioxidant due to the ability of polyphenols and flavonoid that work as an antioxidant agent due to the hydrogendonating ability of their hydroxyl groups and ability to donate electrons to arrest the production of free radicals as a result of oxidative stress [19]. Based on Table 3 and 4, all T. itama propolis extract shows possess of antimicrobial activity against E. coli ATCC 25922 and S. aureus ATCC 25923. All T. itama propolis crude extract shows inhibition of S. aureus ATCC 25923 and E. coli ATCC 25922 at a concentration of 750µg and 1000µg. Figure 2(A) and 3(B) shows the inhibition zone of S. aureus ATCC 25923 and E. coli ATCC 25922 can be observed at a concentration of 750µg and 1000µg T. itama propolis extract. Methanol crude extract shows the highest rate of inhibition at 750µg/mL and 1000 µg/mL compared to other types of T. itama propolis crude extract. On the other side, ethyl acetate crude extract produced the lowest rate of inhibition and no inhibition can be observed on 750µg/mL against S. aureus and E. coli. High inhibition rate in methanol crude extract may indicate more active antimicrobial compound soluble in methanol extract method. Gram-positive and gram-negative bacteria were more sensitive at a concentration of 1000 µg/mL. T T. itama propolis can produce antimicrobial effects due to the active compound from propolis able to inhibit bacterial RNA polymerase and degradation of the bacterial cytoplasmic membrane [20]. Based on Table 5 and Figure 3 brine shrimp lethality study, methanol crude (LC50 501.2 µg/mL) extract shows a high level of toxicity compared to hexane (LC50 623.7 µg/mL) and ethyl acetate extract (LC50 670.8µg/mL). On the opposite site, ethyl acetate crude shows the lowest level of toxicity compared to other T. itama propolis extracts. The previous study shows that sample produce LC50 values above than 250 were considered significantly not active for toxicity effect this indicate that all T. itama extract have a low level of toxicity [21]. In this study, we found that all three types of T. itama propolis extract from Beladin Sarawak shows presences of antioxidant activity and antimicrobial activity against gram-positive and gram-negative bacteria. The different result compared to the previous study of propolis from other countries will represent the uniqueness of Beladin, Sarawak propolis. Variation of toxicity result due to the difference amount of phytochemical compound in T. itama propolis. From this study, we can observed that methanol extract produced a high rate of antioxidant and antimicrobial activity that lead to a high level of toxicity compared to the other extract in this study, indicates that methanol has high efficiency to extract a wide range of active compound in T. itama propolis extract. Different or the same biological active compound are present in all 3 types of T. itama propolis extract that lead to a positive result for antimicrobial, antioxidant and cytotoxicity. Study by Viuda-Martos T et al., [22], shows that the biological activities
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of propolis due to phytochemical compounds in propolis. Active organic compound that can be found in propolis is flavonoids, terpenes, beta steroid, phenolic, and aromatic aldehydes [23]. Active biological compound inside propolis help in maintaining the low level of microorganism in the hive and protect the bee’s colony [24]. Variation of result due to the different origin of T. itama propolis. The phytochemical compound in propolis will influence the quality, colour and biological activity of propolis. Further research needs to be performed to identify an active phytochemical compound that contributed to antimicrobial and antioxidant effect from Beladin, Sarawak T.itama propolis. 5.0 Conclusion This study showed that all three types of T. itama propolis extract from Beladin Sarawak shows presences of antioxidant activity and antimicrobial activity against gram-positive and gram-negative bacteria. Methanol extract produced a high rate of antioxidant and antimicrobial activity. All T.itama propolis extract showed a low level of toxicity. Further study needed to perfume to identify a specific compound that leads to positive result in a biological assay. Acknowledgements The author would like to express special appreciation to Dr. Mohd Razip Asaruddin, Beladin community and Universiti Malaysia Sarawak staff for the endless support and guidance and patience throughout the research Declaration of interest The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper. References [1] Rasmussen, C. and Cameron, S.A., 2009. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society, 99(1), pp.206-232 [2] Bankova, V., 2009. Chemical diversity of propolis makes it a valuable source of new biologically active compounds. Journal of ApiProduct and ApiMedical Science, 1(2), pp.23-28. [3] Parolia, A., Thomas, M.S., Kundabala, M. and Mohan, M., 2010. Propolis and its potential uses in oral health. International Journal of Medicine and Medical Science, 2(7), pp.210-215. [4] Popova, M.P., Bankova, V.S., Bogdanov, S., Tsvetkova, I., Naydenski, C., Marcazzan, G.L. and Sabatini, A.G., 2007. Chemical characteristics of poplar type propolis of different geographic origin. Apidologie, 38(3), pp.306-311. [5] Halliwell, B. and Gutteridge, J.M., 2015. Free radicals in biology and medicine. Oxford University Press, USA. [6] Byers, T. and Sedjo, R.L., 2015. Body fatness as a cause of cancer: epidemiologic clues to biologic mechanisms. Endocrine-related cancer, 22(3), pp.R125-R134. [7] Braughler, J.M., Duncan, L.A. and Chase, R.L., 1986. The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation. Journal of Biological Chemistry, 261(22), pp.10282-10289. [8] Campos, J.F., dos Santos, U.P., Macorini, L.F.B., de Melo, A.M.M.F., Balestieri, J.B.P., Paredes-Gamero, E.J., Cardoso, C.A.L., de Picoli Souza, K. and dos Santos, E.L., 2014. Antimicrobial, antioxidant and cytotoxic activities of propolis from Melipona orbignyi (Hymenoptera, Apidae). Food and Chemical Toxicology, 65, pp.374-380. [9] Kurek-Górecka, A., Rzepecka-Stojko, A., Górecki, M., Stojko, J., Sosada, M. and Świerczek-Zięba, G., 2013. Structure and antioxidant activity of polyphenols derived from propolis. Molecules, 19(1), pp.78-101. [10] Paviani, L.C., Fiorito, G., Sacoda, P. and Cabral, F.A., 2013, April. Different solvents for extraction of Brazilian green propolis: Composition and extraction yield of phenolic compounds. In III Iberoamerican Conference on Supercritical Fluid (pp. 1-5). [11] Maskam, M. F., Mohamad, J., Abdulla, M. A., Afzan, A. & Wasiman, I. P.L., 2014. Antioxidant Activity of Rhodomyrtus tomentosa (Kemunting) Fruits and Its Effect on Lipid Profile in Induced-cholesterol New Zealand White Rabbits. Sains Malaysiana, 43(11), pp.1673-1684. [12] Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology, 45(4_ts), pp.493-496. [13] Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.J. and McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta medica, 45(05), pp.31-34. [14] Park, Y.K., Alencar, S.M. and Aguiar, C.L., 2002. Botanical origin and chemical composition of Brazilian propolis. Journal of Agricultural and Food Chemistry, 50(9), pp.2502-2506.
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