Efect of processing method and concentration of mangifera indica leaf

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Abstract: Mangifera indica leaves play a major role in combating antibiotic resistance. They are processed using various methods to yield powder for phytochemical extraction. Present study was conducted to determine effect of processing method and concentration of leaf extract on their antibacterial activity against S. aureus. Four by four factorial design was employed involving two factors namely processing method (mortar and pestle, blender, laboratory mill and stone) and concentration of extract (6.25, 12.5, 25 and 50 mg/ml). Antibacterial activity was determined by disc method. Both factors influenced antibacterial activity, but there was no interaction effect (p = 0.353) of the factors. Highest inhibition zones were recorded for Laboratory mill (14.5mm) and 50 mg/ml concentration (16.7mm). Inhibition zones for other methods were similar (p = 0.624). MIC was 6.25mg/ml while MBC was 12.5mg/ml. The study has provided evidence on effectiveness of laboratory mill compared with other processing methods in Malawi.

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116 International Journal of Herbal Medicine 2018 66: 116-119 E-ISSN: 2321-2187 P-ISSN: 2394-0514 IJHM 2018 66: 116-119 Received: 16-09-2018 Accepted: 20-10-2018 Martin Kalumbi Biomedical Science Department Malawi Adventist University Malamulo College of Health Sciences Campus Thyolo Malawi Correspondence Martin Kalumbi Biomedical Science Department Malawi Adventist University Malamulo College of Health Sciences Campus Thyolo Malawi Effect of processing method and concentration of Mangifera indica leaf extract on their antibacterial activity against Staphylococcus aureus Martin Kalumbi Abstract Mangifera indica leaves play a major role in combating antibiotic resistance. They are processed using various methods to yield powder for phytochemical extraction. Present study was conducted to determine effect of processing method and concentration of leaf extract on their antibacterial activity against S. aureus. Four by four factorial design was employed involving two factors namely processing method mortar and pestle blender laboratory mill and stone and concentration of extract 6.25 12.5 25 and 50 mg/ml. Antibacterial activity was determined by disc method. Both factors influenced antibacterial activity but there was no interaction effect p 0.353 of the factors. Highest inhibition zones were recorded for Laboratory mill 14.5mm and 50 mg/ml concentration 16.7mm. Inhibition zones for other methods were similar p 0.624. MIC was 6.25mg/ml while MBC was 12.5mg/ml. The study has provided evidence on effectiveness of laboratory mill compared with other processing methods in Malawi. Keywords: Mangifera indica processing method concentration antibacterial Staphylococcus aureus 1. Introduction Staphylococcus aureus infection and other bacterial infections have been of great concern over the decades. The organisms have emerged resistance to a great deal of conventional medicine leading to increased cases of infectious diseases prolonged stay in hospitals and death contributing to escalating healthcare costs 12 .Several attempts and efforts have been made to contain antibacterial resistance including use of natural products such as Mangifera indica Mango leaves 3 . In Malawi M. indica continues to play a major role in primary health care and it has antibacterial activity against S. aureus 4 5 6 . M. indica leaves are processed using different methods to yield powder from which bioactive compounds with antibiotic activity such as alkaloids flavonoids tannins steroids gallotannin and mangiferin are extracted 7 8 . However there is no evidence on the effect of different processing methods on antibacterial activity of the leaf extract against disease causing clinical isolates. In Sub-Saharan Africa M. indica leaves are often processed using less costly manual methods to yield powder due to poverty and unreliable electricity. People rely on two simple traditional methods namely grinding by a stone and wooden mortar and pestle methods. In the earlier method leaves are placed in a plastic bag and are ground manually by hammering using a metal hammer or stone. Mortar and pestle involves placing the leaves in a wooden mortar and are ground by hitting using a wooden pestle. These methods are tedious and slow as compared to modern methods. Particle size temperature and pressure are not uniform and cannot be regulated. They also expose the powder to contamination due to broken wooden particles and plastic paper chemicals in a stone method. Laboratory mill and home blender are modern methods designed to achieve rapid grinding rate. Proponents of these modern methods claim that they do not degrade phytochemicals as compared to traditional methods where pressure during grinding is variable. While the opponents claim that temperature in traditional methods is lower as compared to modern methods since temperature influences antibacterial activity 9 10 thereby preserving most of the phytochemicals. It is also believed that extraction rate is higher in modern methods although we were unable to find evidence to support this claim. To address this gap in knowledge the current study was conducted to systematically compare performance of the four techniques with respect to M. indica leaves and S. aureus organism. The primary objective was to establish whether there is a difference in antibacterial activity of M. indica leaf extract against S. aureus processed using different techniques. Another aim was to find out the effect of the concentration of M. indica leaf extract against growth of S. aureus.

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117 International Journal of Herbal Medicine It is expected that the findings of the study will help to reduce antibacterial potency losses of the plant extracts due to poor processing methods. 2. Materials and Methods 2.1 Source of raw plant materials and organisms Local fresh M. indica Maboloma variety leaves were collected from Malamulo College of Health Sciences in Malawi. The plant’s identity was confirmed by a botanist. S. aureus organisms BGT25N-STA were obtained from Malawi Liverpool Welcome Trust. 2.3 Preparation of M. indica leaf extract Initially fresh leaves were sorted and cleaned from any extraneous material and contaminants using distilled water. They were put in plastic zip lock bags and taken to Malamulo College of Health Sciences Microbiology Laboratory for experimentation. The leaves were cut into pieces and were shade dried for 7 days. After drying they were ground into fine powder using different processing methods mortar and pestle stone home blender and laboratory mill. The bioactive compounds were extracted using the methods of Olasehinde et al. 6 with slight modification. 100g of the powder was soaked into 1000 ml of ethanol for 72 hours at 6 o C. The extract was shaken vigorously and filtered using Whatman filter paper number 1 to obtain 100 concentration. The extract was stored in a refrigerator at 4 o C ready for experiment. 2.4 Determination of antibacterial activity of M. indica leaf extract against S. aureus Antibacterial activity of differently processed leaf extracts was evaluated using agar well disc diffusion method. The extract solutions were prepared at different concentrations of 6.25 mg/ml 12.5 mg/ml 25 mg/ml and 50 mg/ml. Then paper discs were impregnated with extracts and placed onto the Mueller Hinton nutrient agar inoculated with S. aureus 0.5 McFarland’s standard using sterile swab stick 11 . They were incubated at 37 o C for 18 hours and 25 g/l of Amoxillin was used as a control. After incubation the zone of inhibition was measured to the nearest millimeter mm to assess antibacterial activity of different concentrations of leaf extracts. 2.5 Determination of minimum inhibitory concentration MIC and minimum bactericidal concentrations MBC Two-fold serial broth macrodilution method was used to estimate MIC of the extracts. Sequential extract concentrations of 6.25 mg/ml 12.5 mg/ml 25 mg/ml and 50 mg/ml were poured into test tubes containing 2 ml nutrient broth. According to NCCLS 12 standardized inocula of 0.1 ml of S. aureus was added to each tube and were incubated at 37°C for 24 hour. MIC was taken as the least concentration of M. indica leaf extract that showed no observable growth no turbidity. Samples from tubes that showed no visible bacterial growth during MIC determination were inoculated into separate nutrient agar plates. The plates were incubated at 37 o C for 24 hours. The least concentration of the extract that showed no colonies on the surface of the medium after incubation period was regarded as MBC 12 . 2.6 Data analysis Data was analyzed using Statistical Package for Social Sciences SPSS version 22 SPSS Inc. Chicago IL USA at 0.05 significance level. All the laboratory analyses were conducted in triplicates and SPSS was used to generate the mean. ANOVA and general linear model were run. 3. Results and discussion 3.1 Antibacterial activity of differently processed M. indica leaves on S. aureus The results of different processing methods on antibacterial activity of ethanol leaf extracts of M. indica on S. aureus are presented in Table 1. The largest zone of inhibition of 14.5 mm was recorded on laboratory mill method which was significantly higher than other processing methods. Mortar and pestle stone and blender methods achieved similar zone of inhibitions. Table 1: Zone of inhibition of the extract against S. aureus for different processing methods Processing method Mean zone of inhibition mm P-value 0.05 Mortar and Pestle 13.4 a 0.77 Grinding by Stone 12.3 a Blender 13.0 a Laboratory Mill 14.5 b The means with similar superscripts are statistically the same. Antibacterial activity of M. indica leaf extract was due to the presence of gallotannin and mangiferin bioactive compounds 7 . The leaves also contain mangiferin Tannin saponin steroid flavonoid and glycoside which are major phytochemicals that contribute to the inhibitions 8 13 6 14 . These findings suggest that laboratory mill processing method could maximize retention of bioactive compounds and preserve degradation of these compounds during processing of plant leaves into powder. It is likely that higher antibacterial activity of the leaves processed by laboratory mill resulted from production of fine powder which retained more bioactive compounds compared to other processing methods 15 Data on effect of temperature on levels of phytochemicals during production of powder is limited. Thus a laboratory mill method can be cost-effective venture especially in developing countries for retaining the bioactive compounds activity of M. indica leaves. In countries where electricity is not reliable or available people can use simple methods such as stones and pestle-mortar because they can yield similar results to home blender. This research work corroborates with similar findings of Islam et al. 17 who reported that ethanol extract of M. indica leaves have antibacterial properties. 3.2 Antibacterial activity of different concentrations of M. indica leaf extracts against S. aureus. Data on antibacterial activity of different concentrations of M. indica leaf extract on S. aureus is provided in Table 2. The mean zone of inhibition ranged from 10 mm to 16.7 mm and increased significantly with increase in extract concentration. However there was no significant difference between concentrations of 6.25 and 12.5 mg/ml. Similarly concentrations of 25 and 50 mg/ml were statistically the same. Table 2: Antibacterial activity of M. indica leaf extract against S. aureus Concentrations w/v Mean zone of inhibition mm P-Value 0.05 6.25 10.0 a 0.000 12.5 12.1 a 25 14.5 b 50 16.7 b

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118 International Journal of Herbal Medicine The means with different superscripts are significantly different at alpha 0.05.Values are mean zones of inhibition of triplicates. These findings show that increasing concentration from 6.25 mg/ml to 12.5 mg/ml did not affect antibacterial activity of the leaf extract formulations but from 12.5 mg/ml to 25 mg/ml since there were more bioactive compounds in higher concentrations against S. aureus. Interestingly activity of 25 mg/ml and 50 mg/ml concentrations were significantly the same. Application of M. indica leaf extract does not require increasing concentration of extract from 25 mg/ml which in any way does not significantly enhance its efficacy in the treatment of patients. This trend of zone of inhibition was not influenced by different processing methods that the leaves were subjected to before extraction of bioactive compounds Table 3. The implication of the findings is that people from poor resource and even industrialized countries can cost effectively use 25 mg/ml concentration to treat S. aureus infection hence the results were similar to 50 mg/ml concentration’s activity. Observed antibacterial activity of 25 mg/ml leaf extract concentration corroborates published report by Shabani and Sayadi 5 . 3.3 Effect of processing method concentration of the extract and their combined effect on antibacterial activity Processing method and concentration of the extract had an effect p 0.000 on antibacterial activity of M. indica leaf extract against S. aureus. However there was no interaction effect p 353 of these two factors on antibacterial activity Table 3.0. Table 3: Effect of processing method concentration and their interaction on antibacterial activity Tests of Between-Subjects Effects Dependent Variable: Zone of inhibition Source Type III Sum of Squares df Mean Square F Sig Partial Eta Squared Corrected Model 342.979 a 15 22.865 8506.687 9.910 100.576 1.280 1.104 20.708 7704.170 8.975 91.088 1.159 .000 .000 .000 .000 .353 .907 .996 .457 .895 .246 Intercept 8506.687 1 Methods 29.729 3 Concentration 301.729 3 Methods Concentration 11.521 9 Error 35.333 32 Total 8885.000 48 Corrected Total 378.312 47 Methods Concentration interaction between processing method and concentration of the extract. R Squared .907 Adjusted R Squared .863. Absence of interaction between processing method and concentration of the extract means that zone of inhibitions for the processing methods were not influenced by the concentration of the leaf extract and vice versa. In other ways these two factors presented their effects independent of each other. As such one would choose any processing method and any concentration in order to maximize the results since there was no significant combined effect of the two factors in influencing antibacterial activity of M. indica leaf extract. 3.4 MIC and MBC of M. indica leaf extract against S. aureus The MIC was observed at 6.25 mg/ml while the MBC was 12.5 mg/ml Figure 4. Table 4: MIC and MBC of the extract against S. aureus Leaf extract Concentration MIC MBC 6.25 N P 12.5 N N 25 N N 50 N N Key: N no observable growth P observable growth. Although MIC was observed at 6.25 mg/ml the actual MIC could be lower than this because this was the lowest concentration of the extract tested against S. aureus. Most importantly MIC and MBC values against S. aureus were low and this means that M. indica leaves have the potential to treat any infection associated with S. aureus effectively. The MIC of M. indica leaf extract observed in this study differs with earlier findings where MIC ranging from 62.5 mg/ml to 75 mg/ml were recorded for S. aureus 6 17 . These differences are due to variation in solvents temperature strains of S. aureus concentration of extract and varieties of M. indica used in the experiments. 4. Conclusion and recommendations The study has demonstrated that laboratory mill method is the most effective method compared to pestle and mortar stone and home blender methods of processing M. indica leaves into powder as a means of retaining more bioactive compounds activity of M. indica leaves against S. aureus in Malawi. But we cannot rule out the use of traditional methods which demonstrated that they are capable of retaining bioactive compounds. Both processing method and concentration of the extract influenced antibacterial activity of M. indica leaves against S. aureus although combined effect was not observed. Additional work is encouraged to determine the levels of bioactive compounds following preparation of extracts using different processing methods. 5. Acknowledgments Thanks to Precious Jali George Selemani Malawi Liverpool Welcome Trust Bvumbwe Research Station Chancellor College Chemistry Laboratory staff and Malamulo Mission Hospital and College Laboratory staff for your immense contribution in the analysis and collection of the samples. 6. References 1. WHO. Worldwide country situation analysis: response to antimicrobial resistance. Geneva Switzerland 2015. 2. Obasohan EE Agbonlahor DE. Obano EE. Water pollution: A review of microbial quality and health concerns of water sediment and fish in the aquatic ecosystem. African Journal of Biotechnology. 2010 94:423-427. 3. Parvez M. Pharmacological Activities of Mango

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119 International Journal of Herbal Medicine Mangifera indica: A Review. Journal of Pharmacognosy and Phytochemistry. 2016 53:01-07. 4. Wachtel-Galor S Benzie IFF. Herbal medicine: An introduction to its history usage regulation current trends and research needs. Edn 2 Boca Raton CRC Press/Taylor Francis 2011. 5. Shabani Z Sayadi A. The Antimicrobial in Vitro Effects of Different Concentrations of Some Plant Extracts Including Tamarisk March Acetone and Mango. Journal of Applied Pharmaceutical Science. 2014 5:75-79. 6. Olasehinde GI Sholotan KJ Openibo JO Taiwo OS Bello OA Ajayi JB et al. Phytochemical and Antimicrobial Properties of Mangifera indica Leaf extracts. Covenant Journal of Physical Life Sciences CJPL. 2018 61:55-63. 7. Engels C Schieber A Ganzle MG. Inhibitory Spectra and Modes of Antimicrobial Action of Gallotannins from Mango Kernels Mangifera indica L.. Applied and Environmental Microbiology. 2011 777:2215-2223. 8. Oti Wilberforce JO Nkechinyere EO. Phytochemical Screening and Antimicrobial Activity of Leaves Extracts of Mangifera indica and Carica papaya. Int. J Curr. Microbiol. App. Sci. 2017 69:3253-3259. 9. Ranjan S Dasgupta N Saha P Rakshit M Ramalingam C. Comparative study of antibacterial activity of garlic and cinnamon at different temperature and its application on preservation of fish. Advances in Applied Science Research. 2012 31:495-501. 10. Ahmed MA Ravi S Ghogare P. Studies on antimicrobial activity of spices and effect of temperature and ph on its antimicrobial properties. IOSR Journal of Pharmacy and Biological Sciences. 2015 101:99-102. 11. Olasehinde GI Okolie ZV Oniha MI Adekeye BT Ajayi AA. In vitro antibacterial and antifungal activities of Chrysophyllum albidum and Diospyrosmonbuttensis leaves. J Pharm. Phytothera. 2016 81:1-7. 12. National Committee for Clinical Laboratory Standards NCCLS. Performance standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement. NCCLS document M100-S9. National Committee for Clinical Laboratory Standards Wayne PA 2008. 13. Nwankwo IU Osaro-Mathew RC. Assessment of the phytochemical components of Mangifera indica leaf and Musa paradisiaca roots extracts and their antibacterial activity against some common pathogenic bacteria. IOSR Journal of Pharmacy and Biological Sciences. 2014 91:08-11. 14. Mohammed AH Na’inna SZ Yusha’u M Salisu B Adamu U Garba SA. Phytochemical Screening and Antibacterial Activity of Mangifera indica Extracts. UMYU Journal of Microbiology Research. 2016 11:23-28. 15. Luca-Gonzalez R Fernandez-Lopez J Perez-Alvarez JA Viuda-Martos M. Effect of partical size on phytochemical composition and antioxidant properties of two persimmon flours from Diospyros kaki Thumb. Vars. Rojo Brillate and Triumph co-products. Journal of Science Food and Agriculture. 2018 982:504-510. 16. Islam MR Mannan1 MA Kabir1 MHB Islam A Oliva KJ. Analgesic anti-inflammatory and antimicrobial effects of ethanol extracts of mango leaves. J Bangladesh Agril. Univ. 2010 82:239-244. 17. Doughari J Manzara S. In vitro antibacterial activity of crude leaf extracts of Mangifera indica Linn. Afr. J Microbiol. Res. 2008 2:67-72.

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