2(1)55-60 CONTACT : FAJRIN HASAN BASRI fajrinhasan97@gmail.com � International Journal of Applied Biology 55 Abstract Utilization of secondary metabolites from Actynomycetes Mangrove forests can be used as supportive therapy for colon cancer patients that have already through apoptotic testing so it can directly treat the target of illness. This study was prepared in the form of Michrosphere Bioadhesive using secondary metabolite Actynomycetes M10a rhizosphere mangrove. The purpose of this study was to obtain the development of Michosphere Bioadhesive preparations from secondary metabolites Actynomycetes M10a rhizosphere mangrove as supportive therapy for colon cancer patients. Method of the research is base on Laboratory experiment which started with Purification of Actynomycetes isolate, Secondary Metabolite Fermentation, Michrosphere Bioadhesive Formulation and evaluation of Michrosphere Bioadhesive preparation. The concentration of secondary metabolite Actynomycetes M10a rhizosphere mangrove that been used for each formula are 400 μl, 500 μl, and 600 μl, where the percentage of submerged in F1, F2, and F3 are 93,94%, 96,96%, and 94, respectively, 12%. Particular Particle Size Distribution is F1 (3.1μm), F2 (4.4 μm), F3 (3.9 μm). The rest angles obtained are F1 (0.002O), F2 (0.002O), F3 (0.0016O), respectively. Applied efficiency was obtained by F1 (214 nm), F2 (210 nm), F3 (211 nm). Dissolution results show the absorbance results at 266 nm wavelength. From the results obtained that meet the specifications seen in Formula 2. ISSN : 2580-2410 eISSN : 2580-2119 Development Michrosphere Bioadhesive Delivery System from Secondary Metabolit Actynomycetes M10a Rhizosphere Mangrove as Supportive Therapy of Colon Cancer Patients Fajrin Hasan Basri, Andi Nur Annisa Abbas, Annisa Fitri, Herlina Rante Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia Introduction Colon cancer is one of the leading causes of death in both men and women. Patients with colon cancer continue to increase as long as the increasing of smoking habits and bad lifestyle (Gracia, 2011). In Indonesia, colon cancer is included in the third most common cancer attack, after lung cancer and breast cancer. Basic Health Research (Riskesdas) in 2013 states that the prevalence of colon cancer in Indonesia in the productive age population or under 40 years is high enough to reach 30%. Meanwhile, according to data from the American Society Cancer, in 2017 the total new cases of colorectal cancer patients reached 135,430 with an estimated death of 50,260 cases. The main therapies of colorectal cancer include surgery, radiotherapy, and chemotherapy that require a long therapy time OPEN ACCESS International Journal of Applied Biology Keyword Colon Cancer Actynomycetes M10a Rhizosphere mangrove Michosphere Bioadhesive. Article History Received 13 June 2018 Accepted 15 July 2018 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, p-ISSN : 2580-2410 e-ISSN : 2580-2119. Journal homepage : http://journal.unhas.ac.id/index.php/ijoab International Journal of Applied Biology, 2(1), 2018 56 and great maintenance costs. Although very effective for treating various types of cancer, surgery and radiation are only localized therapies. Most patients have a disease that has spread during diagnosis so that local therapy often fails in eliminating overall cancer (Dipiro, 2009).Through several studies one source of anticancer compounds derived from Actynomycetes. Actynomycetes is the most antibiotic and anticancer-producing microbial group compared to molds and bacteria especially those from Rhizosphere. Mangrove forests are a highly productive ecosystem, providing important protection for beaches in tidal areas in the tropics and subtropics. The ecosystem of flora and fauna habitats of various marine, freshwater and terrestrial species. Research conducted by (Wang et al., 2011; Xiao et al., 2009) found that the mangrove environment, especially in rhizosphere sediments containing new actinobacteria and streptomyces avicenniae. The bioactive activity of the rhizosphere increases as a result of exudation of compounds produced by plant roots and through the formation of plant hormones (Berg et al,. 2005). (Rante et al., 2016) has isolated actynomycetes M10a from Rhizosphere Mangrove which has activity against colorectal cancer cell WiDr through apoptotic mechanism. Therefore, innovation development of bioadhesive michrosphere formulation of secondary metabolites is expected to increase colorectal anticancer activity without giving side effects that can damage normal cells and the tendency of drug resistance to patients. Microsphere is a drug delivery system that has advantages among others because of its size is very small (smaller than the size of blood cells) so that it can be given directly orally or through blood tissue directly to the center of the pain. However, this microsphere system is limited because of the short contact time with the absorption area. It is therefore necessary for a system to be in contact for longer with absorption membrane. This development is done through a Microsphere bioadhesive delivery system (Parmar, 2010). Bioadhesive is a drug delivery system that extends the density of live dosage at the application site or extends the absorption time and facilitates the tight contact between the preparation and the absorption surface so as to improve the performance of drug therapy. This causes a longer stay time of drug molecules to tend to high local concentrations. This strategy can be applied to drug delivery systems targeting the colon. Various polymers used for bioadhesive systems are polycarbophils, polyurethanes, polyethylene oxide and polypropylene oxide (Parmar, 2010).This study aims to develop a bioengineous michrosphere formula from secondary metabolite actynomycetes M10a Rhizosphere Mangrove anticancer as supportive for colon cancer patients. Materials and Methods Equipment and Materials The tools used are horn spoons, stirring rods, basins, porcelain cups, parchment paper, scissors, watch glasses, erlenmeyer flasks, measuring cups, pumpkin shapes, beaker, pH meters, rotary evaporators, ovens, analytical scales, microsphere size analyzer, UV- Visible spectrophotometer, glass preparation, dissolution tool, spoit, and viscometer. The ingredients used are Actynomycetes Isolate, aquadest, Acetic Acid, 96% ethanol, polycarbophyls, paraffin liquid, span 80, petroleum ether, cyanoacrylic glue, rat, tissue, gloves, Spectrophotometric Uv-Vis, aluminum foil, Whatmann no. 1 and No. 44, silica gel, plastic wrap, and mask. International Journal of Applied Biology, 2(1), 2018 57 Research Prosedures 1. The Process of Rejuvenating Actynomycetes Bacteria Actynomycetes isolates were grown into sna medium (starch nitrate agar). As much as 1 ose isolate actynomycetes from new medium sna kemedium until obtained pure isolate, and then incubated for 24 hours. 2. Fermentation (production of metabolite compounds) The isolate obtained was transferred to the starter kemedium then transferred to a 500 ml erlenmeyer containing 100 ml of the same medium. Fermentation was carried out at 28oc for 11 days under the conditions of the teragity at a rate of 150 rpm. 3. Formulation and Evaluation of Biooadhesive Microsphere No. Equipment Formula F1 F2 F3 1. Metabolite Sekunder Actynomycetes 400 µl 500 µl 600 µl 2. Ethyl Cellulose 2 g 2 g 2 g 3. Aseton 20 ml 20 ml 20 ml 4. Span 80 1,5 g 1,5 g 1,5 g 5. Paraffin liquid (ad) 100 ml 100 ml 100 ml Used Actynomycetes bacteria, then mixed with ethyl cellulose that has been dissolved with acetone to form a mixture A. Separately mixed liquid paraffin with span 80 then homogenized for 10 minutes and formed a mixture of B. After that, mixed A to a mixture of B while being sensed at 2500 rpm for 30 minutes. The final mixture is filtered using a filter paper, and the formed microsphere is washed with n-hexane until it is non- greasy. 4. Evaluation of Microsphere Bioadhesive (Pendyala, yamini. 2012) a. Testing the particle size distribution Observed microsphere under an optical microscope then calculated the particle size of the microsphere. b. Determination of Break Angle The rest angle is determined by inserting the michrosphere sample into a funnel that has been covered in the bottom, then flattening the surface of the sample. Place the semilog paper just below the funnel. Then the funnel cap is opened, recorded and calculated the break angle of each formula by the following formula c. Determination of Percent Rendamen The microsphere obtained from each formula is weighed, then calculated percent of rendamen by considering the weight of the active ingredient and the polymer by using the following formula: % Rendament = (practice weight) / (theory weight) x 100% d. Applied Efficiency A sample of 50 mg was dissolved with artificial intestinal fluid with a pH ± 8 then filtered and measured its wavelength according to the standard. e. In-vitro Wash-off Test Testing In-vitro wash off Test is done by using organs of animal intestine try the mice (Mus musculus) which I then used the intestinal organs to see the bioadhesive michrosphere can be attached to the intestinal organs. International Journal of Applied Biology, 2(1), 2018 58 f. In-vitro Dissolution Test Dissolution test using basket method at 37 ± 0.5oC with rotation speed 100 rpm using PH 8 Posfat buffer as dissolution medium. Samples of the microsphere 100 mg were used in this test. Samples samples are periodically taken over a period of time and analyzed by spectrophotometric method. Results and discussion After extracting the Actynomicetes isolate, a microsphere bioadhesive formulation was obtained by percent rendamen for each of the formulas, 93.94% (F1), 96.96% (F2), and 94.12% (F3).From the measurement of the particle size distribution, it was found that there was no significant difference of the three tested formulas. The mean particle diameter of F1 (3.1μm), F2 (4.4 μm), and F3 (3.9 μm). These results indicate that the concentration of secondary metabolites Actynomicetes does not affect the resulting formula in which of the three formulas and all form in microspheres. The particle size distribution graph of F2 can be seen in figure 1. Figure 1: Particle size distribution Based on the graph, the higher cumulative percentage is 34% in 3 µm particle size. That means most of particle size that found in F2 has 3 µm. The smallest particle shown the good form of microsphere that have been prepared. On the measurement of the velocity of flow and the rest angle, the flow rate is 0.105 g / s (F1), 0.064 g / s (F2), 0.095 g / s (F3) and the fixed angle is 0.002o (F1), 0.002o (F2) , and 0,0016o (F3). A silent angle below 25° represents a good flow rate so it is predicted to produce a good release rate of the drug when formulated. Maximum wavelength determination results show that the highest uptake of the formula with in vitro gastric medium without enzim is at 266 nm wavelength. Of the three formulas showed insignificants rate of dissolution. Further measurements were made on each of the formulas which obtained the highest absorbance of F1 (214 nm), F2 (210 nm), F3 (211 nm). In the 2 3 4 5 6 13.7 34 29.7 18.3 4.3 1 2 3 4 5 Total of particle Particle Size (µm) The Particle Size Dstribution of F2 Particle size that shown in F2 Cumulative percentage of particle size International Journal of Applied Biology, 2(1), 2018 59 formula 2 the highest absorbance is at 210 nm wavelength indicates the same wavelength as the references so that shown 100% absorption efficiency is found in formula 2. The spectrophotometric graph is shown in Figure 2. Figure 2. Applied Efficiency of F2 Bioadhesive capability is also tested in microsphere bioadhesive formula. In vitro wash test was performed by orally administered in mice (Mus musculus). Method that used is to indicate the microshphere attached to the intestine so it was predicted that the bioadhesive properties were obtained in all three formulas. Conclusions The results obtained by using secondary metabolite Actynomycetes M10a are 400 μl, 500 μl, and 600 μl, respectively, where the percentage of rendamen in F1, F2, and F3 are 93,94%, 96,96%, and 94, respectively, 12%. Particular Particle Size Distribution is F1 (3.1μm), F2 (4.4 μm), F3 (3.9 μm). The rest angles obtained are F1 (0.002O), F2 (0.002O), F3 (0.0016O), respectively. Applied efficiency was obtained by F1 (214 nm), F2 (210 nm), F3 (211 nm). The dissolution results showed absorbance results at 266 nm wavelengths. While for invitro wash off test result using Mucus organs organ (Mus musculus) it was found that the Microsphere bioadhesive preparation could be attached to the intestinal organ of mouse. Based on the evalution results, F2 is the good formula of Microspher bioadhesiveActynomycetes M10a Rhizosphere mangrove. 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