|Year : 2018 | Volume
| Issue : 1 | Page : 44-48
Lack of antibacterial activity of aqueous and ethanolic leaf extracts of Ziziphus talanai (Blanco) Merr.
Angelico Garcia Reyes1, Adrian Eustrace P Miclat2, Angelo B Bañares3, Renato A Dela Peña4
1 Department of Natural Sciences and Mathematics, Institute of Arts and Sciences, Mabalacat City College, Mabalacat City, 2010 Pampanga; Biology Department, College of Science, De La Salle University, 2401 Taft Ave., Malate, Manila, 1004 Metro Manila, Philippines
2 Department of Natural Sciences and Mathematics, Institute of Arts and Sciences, Mabalacat City College, Mabalacat City, 2010 Pampanga; Department of Education, Division of Angeles City, Jesus St., Pulong Bulo, Angeles City, 2009 Pampanga, Philippines
3 Department of Natural Sciences and Mathematics, Institute of Arts and Sciences, Mabalacat City College, Mabalacat City, 2010 Pampanga, Philippines; Department of Energy Science and Technology, Energy and Environment Fusion Technology Center, Myongji University, Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do, Korea 170-58
4 Chemistry and Life Sciences Department, Manila Tytana Colleges, Pres. Diosdado Macapagal Blvd., Pasay City, 1300 Metro Manila, Philippines; Science Education Department, Br. Andrew Gonzalez College of Education, De La Salle University, 2401 Taft Ave., Malate, Manila, 1004 Metro Manila, Philippines
|Date of Web Publication||21-Aug-2018|
Angelico Garcia Reyes
Biology Department, College of Science, De La Salle University, 2401 Taft Ave., Malate, Manila, 1004 Metro Manila
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The objective of the study is to investigate the phytochemicals and potential antibacterial activity of aqueous and ethanolic leaf extracts of Ziziphus talanai. Materials and Methods: Aqueous and ethanolic crude leaf extracts were subjected to phytochemical screening and assayed for their antimicrobial activities via paper disc diffusion method against Escherichia coli and Staphylococcus aureus. Results: Glycosides, condensed tannins, and saponins were found present in the two extracts; however, leucoanthocyanins were additionally detected in the aqueous extract. Both extracts exhibited no antibacterial activity against E. coli and S. aureus as indicated by the absence of a zone of inhibition. Conclusion: The results of the present study suggest a limited presence of phytochemicals in the crude aqueous and ethanolic leaf extracts of Z. talanai. Water can extract more phytochemicals from Z. talanai leaves than ethanol. However, the results further suggest that the phytochemicals from the leaves extracted using water and ethanol do not have antibacterial activities against E. coli and S. aureus. The negative results of the antimicrobial assay using the crude leaf extracts may not be generalized as lack of bioactivity of the other parts of Z. talanai.
Keywords: Antibacterial activity, aqueous leaf extract, ethanolic leaf extract, negative results, phytochemicals, Ziziphus talanai (Blanco) Merr.
|How to cite this article:|
Reyes AG, P Miclat AE, Bañares AB, Dela Peña RA. Lack of antibacterial activity of aqueous and ethanolic leaf extracts of Ziziphus talanai (Blanco) Merr. J Pharm Negative Results 2018;9:44-8
|How to cite this URL:|
Reyes AG, P Miclat AE, Bañares AB, Dela Peña RA. Lack of antibacterial activity of aqueous and ethanolic leaf extracts of Ziziphus talanai (Blanco) Merr. J Pharm Negative Results [serial online] 2018 [cited 2019 Feb 17];9:44-8. Available from: http://www.pnrjournal.com/text.asp?2018/9/1/44/239512
| Introduction|| |
Ziziphus talanai (Blanco) Merr., an endemic species in the Philippines and locally known as Balakat tree, has been traditionally used by folks as a herbal plant to treat parasitic infections caused by ringworms and mites, as well as diseases caused by bacteria, like urinary tract infections. Pioneering investigations on Balakat tree revealed that the methanolic bark extract was effective against Gram-positive bacteria but inactive against Gram-negative bacteria and certain fungal species. Aside from its promising antimicrobial activities, the crude ethanolic extract of Balakat tree was demonstrated to have hepatoprotective and reproprotective activities against tetracycline-induced hepatic damage and reproductive dysfunction in mice models. It has been shown that crude extracts of species under the genus Ziziphus exhibited positive antimicrobial activities against test microorganisms, clinical isolates, and pathogenic strains. A previous phytochemical screening of the ethanol leaf extract of Balakat tree revealed the presence of alkaloids, glycosides, and triterpenes in minimal quantities, sterols in moderate level, and flavonoids, saponins, and tannins in abundant concentrations.
Escherichia coli, an extensively studied Gram-negative bacterium, has been used as a model organism in industrial microbiology and biological engineering and a key to advancements in biochemistry, genetics, molecular biology, and physiology. E. coli is a common inhabitant of feces and intestines of reptiles and warm-blooded animals including humans. E. coli is a gut commensal of vertebrates and an adaptable pathogen speculated to be the cause of extraintestinal and intra-intestinal infections leading to the death of more than two million people worldwide. It is estimated that 90% of urinary tract infection cases are caused by E. coli. About 40% to 80% of cases of neonatal meningitis have been reported to be due to E. coli. Some pathogenic strains of E. coli have been reported to cause hemorrhagic colitis with severe abdominal pain, and bloody stool, bloody or nonbloody diarrhea, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, intussusception, gross, and anal dilatation. E. coli had already caused outbreaks in certain countries through contamination of various foods such as ground beef, roast beef, precooked beef, water, cold sandwiches, raw milk, raw potatoes, and Turkey roll with a significant number of deaths. The general transmission patterns in these E. coli-caused outbreaks suggest that the infectious dose is low, making E. coli a life-threatening pathogen. Due to the extensive use of antibiotics as treatments for microbial infections in humans and other animals, commensal microbes, especially E. coli, have been shown to play a vital role in the emergence of antibiotic resistance.
Staphylococcus aureus is a Gram-positive commensal bacterium estimated to have colonized around 30% of the human population. Humans are considered the main reservoir of S. aureus, which typically inhabit their mucous membranes and skin. The colonization rate of S. aureus varies from individual to individual, with higher frequency and rates in people who are hospitalized, regularly using needles, and immunocompromised.S. aureus does not cause infections on healthy individuals; however, it may cause a wide array of serious diseases if it invades the bloodstream or penetrates internal tissues.S. aureus is a common causative agent of bacterial infections in humans. It is considered the leading cause of infective endocarditis, bacteremia and osteoarticular, pleuropulmonary, soft and skin tissue, and prosthetic device-related infections. There have been reported clinical cases which found that S. aureus has caused epidural abscess, meningitis, toxic shock syndrome, urinary tract infection, as well as septic thrombophlebitis. Clinical infections caused by S. aureus may conceivably persist as a common and serious threat to human well-being if proper attention is not given and potential health concerns are neglected. In the past 20 years, the epidemiology of S. aureus has experienced shifts because of (a) increasing rates of healthcare-related infections, and (b) community-associated skin and soft-tissue infection epidemic caused by antibiotic-resistant strains with virulence factors.
There is a growing concern with infections caused by E. coli and S. aureus, which inevitably pose health problems to humans. Preliminary phytochemical screening of Z. talanai ethanolic leaf extract revealed the presence of phytochemicals with antimicrobial potentials. Extensive literature search, however, revealed few to no reports on the antimicrobial potential of Z. talanai against E. coli and S. aureus. Hence, to address the growing need for natural products as treatments for infections caused by E. coli and S. aureus, this study was conducted to evaluate the antimicrobial potentials of the crude aqueous and ethanolic leaf extracts of Z. talanai against E. coli and S. aureus to further inform its use as a medicinal plant in the local communities.
| Material and Methods|| |
Collection of leaves
A total of 10 kg of Z. talanai leaves were collected from a piece of land located in Xevera Subdivision, Barangay Tabun, Mabalacat City, Pampanga, Philippines, on May 2017. The leaves were then separated from branches and washed with tap water to get rid of unwanted materials. Z. talanai leaves were air-dried for 1 week at room temperature in a well-ventilated room without any exposure to solar radiation. The air-dried leaves were cut into small pieces and then ground using Wiley mill.
Leaf samples were brought to Jose Vera Santos Memorial Herbarium, Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines, for taxonomic authentication.
Preparation of aqueous leaf extract
A volume of 500 g of ground leaves were soaked in 11 L of distilled water and heated at 50°C for 20 min. The heated mixture was subsequently filtered using a filter cloth. The solid filtrate was discarded while the liquid filtrate was subjected to phytochemical screening and antimicrobial assay.
Preparation of ethanolic leaf extract
Five kilogram of ground leaves were soaked in 95% ethanol for 72 h at room temperature. After soaking, the mixture was filtered using a Buchner funnel with gentle suction. The plant residues obtained from filtration were discarded. The liquid filtrate was concentrated under vacuo through rotary evaporation at 50°C. The solid concentrated extract was then subjected to phytochemical analysis and antimicrobial assay. Before antimicrobial assay, the ethanolic leaf extract was dissolved in normal saline solution.
Phytochemical screening was conducted following the procedure according to Guevarra. The aqueous and ethanolic crude leaf extracts of Z. talanai were subjected to phytochemical screening for the presence of eight phytochemicals, namely alkaloids by Mayer/Meyer test, anthraquinones by Borntrager test, cardenolides, and bufadienolides by Keller–Kiliani test, flavonoids by Bate-Smith and Metcalf tests, tannins and polyphenolic compounds by ferric chloride test, and saponins by froth test.
The antimicrobial assay was conducted in triplicates using paper disc diffusion method. Aseptic techniques were strictly observed during the antimicrobial assay. The test organisms used in the present study are E. coli(BIOTECH 1634) and S. aureus (BIOTECH 1582). The inoculum size of each test organism was standardized by comparing the turbidity of the culture broth to that of the 0.5 McFarland standard. The standardized inocula were then plated on Mueller-Hinton agar through cotton swabbing. The plates were then incubated for 24 h. Twelve paper discs (6-mm diameter) were punched out from Whatman No. 1 filter paper and subsequently sterilized. Six sterilized paper discs were immersed in the aqueous leaf extract, and the other six were soaked in the ethanolic leaf extracts. The discs were then placed on inoculated Mueller-Hinton agar plates with the test organisms. Incubation was done at 37°C for 24 h. After incubation, the plates were then examined for the presence of zones of inhibition.
| Results|| |
The extraction procedures performed yielded 11 L of aqueous leaf extract and 140.83 g of ethanolic leaf extract.
Phytochemicals detected in the extracts
The phytochemical screening revealed that the aqueous leaf extract contained glycosides, leucoanthocyanins, condensed tannins, and saponins (four out of eight screened phytochemicals) while the ethanolic leaf extract showed lesser phytochemicals, namely glycosides, condensed tannins, and saponins (three out of eight screened phytochemicals). Alkaloids, anthraquinones, anthocyanins, and hydrolyzable tannins were not detected in the aqueous leaf extract (four out of eight screened phytochemicals). No alkaloids, anthraquinones, anthocyanins, leucoanthocyanins, and hydrolyzable tannins (five out of eight screened phytochemicals) were detected in the ethanolic extract [Table 1]. These suggest that the aqueous solvent is more efficient in extracting a relatively higher number of phytochemicals compared to ethanol.
|Table 1: Results of phytochemical screening of Ziziphus talanai crude extracts for eight various phytochemicals|
Click here to view
In this investigation, the two different extracts from Z. talanai leaves were assayed for their antimicrobial activity against test microorganisms. The antimicrobial susceptibility testing revealed that both crude aqueous and ethanolic leaf extracts of Z. talanai were inactive against E. coli and S. aureus as there was no zone of inhibition observed [Table 2].
|Table 2: Results of antimicrobial activity of Ziziphus talanai crude leaf extracts|
Click here to view
| Discussion|| |
The findings of the current study showed that the crude leaf extracts of Z. talanai have limited number of phytochemicals. Furthermore, it was revealed that the extracts were unable to inhibit the growth of both E. coli and S. aureus. The method used in the antimicrobial assay, which is disc diffusion method, offers various limitations in antimicrobial susceptibility testing, making the method inaccurate to a certain extent. The disc diffusion method is currently widely utilized among microbiological laboratories because it is easy to perform; however, according to Clinical and Laboratory Standards Institute, it should only be used for ciprofloxacin and macrolide resistance screening. To overcome the present limitations, other antimicrobial susceptibility methods such as broth dilution, agar dilution, Epsilometer test, and automated antimicrobial susceptibility testing systems may be employed along with the disc diffusion method to decrease the likelihood of technical errors that can possibly affect the outcomes of the study. The use of these alternative antimicrobial susceptibility testing methods may yield more robust and promising results.
A previous report has shown that the ethanolic and methanolic bark extracts of Z. talanai exhibited antimicrobial activity against S. aureus, Bacillus subtilis, Mycobacterium phlei, but inactive against E. coli, Pseudomonas aeruginosa, and Candida albicans; however, further purification of the ethanolic extract resulted to the loss of antimicrobial activity against B. subtilis. The ethanolic extract was found to contain a pure compound identified as ceanothic acid, which may account for the antimicrobial activity of the extract. However, in the present study, the crude ethanolic extracts did not exhibit any antimicrobial activity against E. coli and S. aureus. This result suggests that ceanothic acid was either not extracted or was extracted in very low amount insufficient to exhibit the antimicrobial activity. There are various drawbacks with the use of partially purified or crude plant extracts during evaluation of their pharmacological activities. These problems include, but are not limited to, disparities in solvents for phytochemical extraction, differences in extraction protocols, and discrepancy in standardization protocols.
Both leaf extracts of Z. talanai were detected to contain tannins, which are polyphenolic compounds that exhibit antibacterial activities. Yet, tannins and other polyphenolic compounds, which are usually present at high concentrations in plant extracts, may yield false positive and/or negative results in vitro and in vivo because of their tendency to precipitate other important phytochemicals, which may confer antimicrobial extracts, through multipoint hydrogen bonding. Tannins and polyphenols, which are extracted using polar solvents such as ethanol and water, were demonstrated to inhibit enzymes giving false positive and/or negative findings in biological assays. Furthermore, the presence of residual solvents in the Z. talanai extracts may also contribute to the negative antimicrobial findings. The presence of glycosides, leucoanthocyanins, and saponins suggests possible antimicrobial property of the extracts; however, antimicrobial activity was not exhibited by the crude extracts in the present study. If these phytochemicals are the active principles, that is, if they are biologically active, the concentrations of these bioactive compounds may be insufficient in the crude extracts to exhibit antimicrobial activities. Thus, the use of large doses may possibly confer antimicrobial property to the Z. talanai extracts. Alternatively, the glycosides, leucoanthocyanins, and saponins present in the crude extracts may not be the active principles; hence, even though the extracts are employed in high concentrations in antimicrobial assays, antimicrobial activities may not be observed. The detected phytochemicals, which may be active principles, could also exert antagonistic effects against one another, hence, negating the possible antimicrobial activities of the crude extracts. The present study suggests that Z. talanai crude extracts are inactive against E. coli and S. aureus, yet, the leaf extracts may show antimicrobial actions against other test microorganisms not utilized in this study.,
| Conclusion|| |
The results of the present study suggest a limited presence of phytochemicals in the crude aqueous and ethanolic leaf extracts of Z. talanai. Of the two solvents used, water can extract more phytochemicals from Z. talanai leaves than ethanol. Furthermore, the results suggest that the bioactive compounds from the leaves extracted using water and ethanol do not have antibacterial activities against E. coli and S. aureus. The negative results of the antimicrobial assay using leaf extracts, however, may not be generalized as lack of bioactivity of the other parts of Z. talanai. Additional extraction procedures, other solvents and alternative antimicrobial assays may be conducted to validate the findings of the present study.
Financial support and sponsorship
The study was funded by the local city government of Mabalacat City, Pampanga, Philippines.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Anas AR, Villaseñor IM, Matsuura H, Okino T. Anti-Mycobacterium phlei
activity of the bark of Ziziphus talanai
(Blanco) Merrill. Philipp Agric Sci 2009;92:388-91.
Reyes A, Dela Peña R, Sula LF, Bañares A. Histoprotective potentials of ethanol leaf extract of balakat tree (Ziziphus talanai
(Blanco) Merr.) against tetracycline-induced hepatotoxicity and reprotoxicity in male mice (Mus musculus
L.). Int J Pharmacol Toxicol 2016;4:96-104.
Abalaka ME, Daniyan SY, Mann A. Evaluation of the antimicrobial activities of two Ziziphus
species (Ziziphus mauritiana
L. and Ziziphus spinachristi
L.) on some microbial pathogens. Afr J Pharm Pharmacol 2010;4:135-9.
Bañares AB. Phytochemical screening of ethanol leaf extract of balakat tree (Ziziphus talanai
). Department of Natural Sciences and Mathematics, Mabalacat City College, Pampanga, Philippines; 2016.
Tenaillon O, Skurnik D, Picard B, Denamur E. The population genetics of commensal Escherichia coli
. Nat Rev Microbiol 2010;8:207-17.
Gordon DM, Cowling A. The distribution and genetic structure of Escherichia coli
in Australian vertebrates: Host and geographic effects. Microbiology 2003;149:3575-86.
Russo TA, Johnson JR. Medical and economic impact of extraintestinal infections due to Escherichia coli
: Focus on an increasingly important endemic problem. Microbes Infect 2003;5:449-56.
Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln K, et al
. Epidemiology of symptomatic urinary tract infection in childhood. Acta Paediatr Scand Suppl 1974;252:1-20.
Robbins JB, McCracken GH Jr., Gotschlich EC, Orskov F, Orskov I, Hanson LA, et al. Escherichia coli
K1 capsular polysaccharide associated with neonatal meningitis. N Engl J Med 1974;290:1216-20.
Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli
O157:H7, other enterohemorrhagic E. coli
, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98.
Doyle MP, Schoeni JL. Isolation of Escherichia coli
O157:H7 from retail fresh meats and poultry. Appl Environ Microbiol 1987;53:2394-6.
Otto M. Staphylococcus
colonization of the skin and antimicrobial peptides. Expert Rev Dermatol 2010;5:183-95.
Taylor TA, Unakal CG. Staphylococcus aureus
. In: Abai B, Abu-Gosh A, Acharya AB, Acharya U, Adigun R, Aeby TC, et al
., editors. Stat Pearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2017.
Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus
infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015;28:603-61.
Nair N, Biswas R, Götz F, Biswas L. Impact of Staphylococcus aureus
on pathogenesis in polymicrobial infections. Infect Immun 2014;82:2162-9.
Chiller K, Selkin BA, Murakawa GJ. Skin microflora and bacterial infections of the skin. J Investig Dermatol Symp Proc 2001;6:170-4.
Pozzi C, Waters EM, Rudkin JK, Schaeffer CR, Lohan AJ, Tong P, et al.
Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus
device-associated infections. PLoS Pathog 2012;8:e1002626.
David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus
: Epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 2010;23:616-87.
Rasmussen RV, Fowler VG Jr., Skov R, Bruun NE. Future challenges and treatment of Staphylococcus aureus
bacteremia with emphasis on MRSA. Future Microbiol 2011;6:43-56.
Naber CK. Staphylococcus aureus
bacteremia: Epidemiology, pathophysiology, and management strategies. Clin Infect Dis 2009;48 Suppl 4:S231-7.
Plaza M, Amigo-Benavent M, del Castillo M, Ibáñez E, Herrero M. Facts about the formation of new antioxidants in natural samples after subcritical water extraction. Food Res Int 2010;43:2341-8.
Guevara BQ, editor. A Guidebook to Plant Screening: Phytochemical and Biological. Revision ed. Manila: UST Publishing House; 2005.
Abreu OA, Sánchez I, Barreto G, Campal AC. Poor antimicrobial activity on seven Cuban plants. J Pharm Negat Results 2017;8:11-4.
Lehtopolku M, Kotilainen P, Puukka P, Nakari UM, Siitonen A, Eerola E, et al.
Inaccuracy of the disk diffusion method compared with the agar dilution method for susceptibility testing of Campylobacter
spp. J Clin Microbiol 2012;50:52-6.
Bhattacharya S, Ahmed KM, Kundoor V. Do medicinal plants possess significant activities? J Pharm Negat Results 2010;1:27-8.
Mandal SC, Mandal V, Das AK. Essentials of Botanical Extraction: Principles and Applications. USA: Academic Press; 2015.
Taylor JL, Rabe T, McGraw LJ, Jager AK, van Staden J. Towards the scientific validation of traditional medicinal plants. Plant Growth Regul 2001;34:23-37.
Milugo TK, Omosa LK, Ochanda JO, Owuor BO, Wamunyokoli FA, Oyugi JO, et al.
Antagonistic effect of alkaloids and saponins on bioactivity in the quinine tree (Rauvolfia caffra
sond.): Further evidence to support biotechnology in traditional medicinal plants. BMC Complement Altern Med 2013;13:285.
Jain N, Sharma M. Insignificant antidermatophytic activity of Brassica campestris
oil. J Pharm Negat Results 2014;5:22-4.
Patil V, Vadnere GP, Patel N. Absence of antimicrobial activity in alcoholic extract of Santalum album
linn. J Pharm Negat Results 2011;2:107-9.
[Table 1], [Table 2]