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Nephroprotective Potentials of Ethanol Leaf Extract of Balakat Tree (Ziziphus talanai (Blanco) Merr.) Against Gentamicin-Induced Nephrotoxicity in Male ICR Mice



This study evaluated the anecdotal claims of Balakat tree as a nephroprotective agent against nephrotoxicity using gentamicin-induced male ICR mice.
Ziziphus talanai
)(Blanco) Merr.)
Mus muculus
Oliver Y. Galang1
, Angelico G. Reyes1 and Lourdes Fatima S. David1
1 Department of Natural Science and Mathematics, Institute of Arts and Sciences, Mabalacat City College, Mabalacat City, Pampanga,
2010 Philippines
Nephrotoxicity is one of the major causes of renal dysfunction when body exposed to a drug or toxin (Porter and Bennett, 1981). It is con-
sidered the ninth most leading cause of death worldwide (WHO, 2016). In the Philippines, it has been reported as the seventh leading
cause of death due to a lethargic lifestyle which triggers diabetes and hypertension (DOH, 2014). Until now there are no applicable semi-
synthetic drugs created to treat kidney disease; the only efficient treatments for kidney diseases are dialysis and renal replacement.
Renal dysfunction develops due to increased production of reactive oxygen species on the kidneys induced lipid peroxidation which leads to
depletion of endogenous antioxidants (Kim et al., 2006)
Gentamicin is an aminoglycoside antibiotic produced by Micronospora purpurea used to treat nosocomial infections specifically gram
negative bacteria including pseudomonas, proteus and serratia (Silan et al., 2007). However, it can escalate the blood serum creatinine and
blood urea nitrogen which are the reliable base parameters for renal function (Adelman et al., 1981).Furthermore, there are studies that
show that exogenous antioxidants are needed to protect kidneys against various drugs that have an indication of nephrotoxicity.
Balakat tree (Ziziphus talanai)(Blanco)Merill.) is an endemic plant in the Philippines used as a traditional medicine at San Remigio, Antique,
Philippines against kidney problems including UTI (Anas et al., 2009). Recent studies have shown that Ziziphus talanai possessed hepato-
protective and reproprotective potentials (Reyes et al., 2016).
In addition, preliminary phytochemical screening of ethanol leaf extract of Ziziphus talanai exhibited lower concentrations of triterpenes, al-
kaloids and glycosides; moderate concentration of sterols; and higher concentrations of flavonoids, tannins, and saponins (Bañares, 2016).
There is a paucity of data concerning the property and mechanism of gentamicin on kidneys and as well as nephroprotective potentials of Z.
talanai. The present study evaluated the anecdotal claims and nephroprotective potentials of Z. talanai ethanol leaf extract against gen-
tamicin-induced nephrotoxicity using male ICR mice models.
Collection of leaves
Balakat Extract
Garlic Soft Gel
Administration of Treatments
T0 Treated with 1ml /20g bw distilled
T- Treated with 0.1ml /20g bw genta-
micin alone.
T+ Treated with 0.2ml/20g bw garlic
Soft Gel Supplement.
T1 Treated with 0.3ml/20g bw Bala-
kat extract after 1 hr 0.1ml gen-
tamicin was administered.
Extraction of kidneys and Blood Serum Histological observations
Data Analysis
Figure 1. Statistical Variances of
Creatinine Level
Figure 2. Means of Blood Urea Nitro-
gen Level.
Figure 3. Histological Observations of
Normal Group (RT) normal Renal tu-
bular, (G) intact Glomerulus.
Figure 4. Histological Observations of
intoxicated group treated with gen-
tamicin alone.
Figure 5. Histological Observations of
garlic soft gel supplement exhibited
(G) intact glomerulus, and (RT) normal
Renal tubular.
Figure 6. Histological viewing treated
with Balakat extract and gentamicin .
(G) intact glomerulus, (RT) normal re-
nal Tubular, moderate (IH) intertubular
This research study was conducted to assess the morphological changes, body weight, kidney weight, blood serum creatinine and
BUN level, and histological observations in terms of glomerulus, renal tubular desquamation, mononuclear cell permeation, inter-
tubular hemorrhage and hyaline cast formation among male ICR mice treated with gentamicin, garlic soft gel and Ziziphus talanai
ethanol extract.
Male ICR mice treated with ethanol leaf extract of Ziziphus talanai mitigated nephrotoxicity caused by the gentamicin on the kid-
neys. ICR male mice treated with Balakat extract were not irritated, showing red-brown kidney color. Based on the statistical analy-
sis of results, the kidney weight increased significantly, body weight escalated significantly and blood serum analysis such as
creatinine and Blood urea nitrogen were decreased significantly.
Furthermore, the effects of Ziziphus talanai ethanol leaf extract based on the longitudinal histopathology of the kidneys exhibited
intact glomerulus, normal renal tubular and moderate intertubular hemorrhage.
In this study, the observations concerning the effects of gentamicin and Ziziphus talanai ethanol leaf extract were supported by the
related studies. Gentamicin can cause nephrotoxicity due to increased production of free radicals which leads to the depletions of
endogenous antioxidants (Apple et al., 1978). The ethanol leaf extract of Ziziphus talanai has been shown to have histoprotective
potentials against tetracycline-induced hepatoxicity and reprotoxicity (Reyes et al., 2016). The present study provides additional
findings on the nephroprotective potentials of the ethanol leaf extract of Ziziphus talanai against drug-induced nephrotoxicity.
These pharmaceutical potentials are attributable to the bioactive compounds contained in Ziziphus species (Abalaka et al., 2011).
Based on the results, Ziziphus talanai ethanol leaf extract has a potential as nephroprotective agent. Thus, more clinical experi-
ments are recommended that may be undertaken by pharmaceutical companies to create semi-synthetic drugs to cure chronic
kidney disease in the near future.
Based upon the statistical results and histopathological observations, it can be concluded that Ziziphus talanai (Blanco) (Merril.) ethanol leaf ex-
tract exhibited nephroprotective potentials against drug-induced nephrotoxicity due to the bioactive compounds possessed by the Balakat tree
(Ziziphus talanai). Ziziphus related species contain flavonoids and polyphenols which have the capability to scavenge reactive oxygen species
produced by the various drugs including gentamicin which induced nephrotoxicity. Further studies are recommended to elucidate the mechanism
of Z. talanai’s nephroprotective activity.
I would like to express my deepest gratitude to my Alma matter (Mabalacat City College) for funding this activity and to the faculty members spe-
cially; To Mam fhat, Mam Due and Mr, Angelico for the unending support, guidance, motivations and encouragement.
To my Family and to all MCC BSBiology Majors thank you all.
1. Abalaka, M. E, Mann, A and Adeyemo S. O (2011). Studies on in-vitro antioxi-
dant and free radicalscavenging potential and phytochemical screening of
leaves of Ziziphus mauritiana L. and Ziziphus spinachristi L. compared
with Ascorbic acid. Journal of Medical Genetics and Genomics Vol. 3(2),
pp. 28 34.
2. Anas, Andrea Roxanne J. et al. (2009).Anti-Mycobacterium phlei activity of
the bark of Ziziphus talanai (Blanco) Merrill. Philipp Agric Scientist, 92(4):
3. Appel, G.B. and Neu , H.C.: Gentamicin in 1978. Ann. Intern. Med. 89:528-
538, 1978.
4. Porter GA and Bennett WM. Nephrotoxic acute renal failure due to common
drugs. American journal of Physiology, 1981; 241(7): F1-F8.
5. Reyes, Angelico G. Renato A. Dela Peña, Jr , Lourdes Fatima I. Sula, An-
gelo B. Bañares (2016 ). Histoprotective potentials of ethanol leaf extract
of balakat tree (Ziziphus talanai(blanco) merr.) against tertracycline- in-
duced hepatoxicity and reprotoxicity in male mice (Mus muculus). Interna-
tonal Journal of Pharmacology and Toxicology .4(2) 96-104
Air-drying of leaves
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Full-text available
Hepatic dysfunction can develop due to ROS and lipid peroxide and may lead to primary liver cancer. Hepatic dysfunction may also develop from tetracycline, an antibiotic with a broad-spectrum of bacteriostatic mechanisms, as it can induce hepatotoxicity and reprotoxicity. In addition, liver tumors secrete hormones that can result to testicular atrophy. Thus, there is a need to address liver and testicular damage. Preliminary phytochemical analysis indicated the presence of bioactive compounds with histoprotective potentials in Balakat tree (Ziziphus talanai), which is used in Antique against urinary tract infection. Twelve male mice were divided into four groups of three mice each. Treatments were administered via intragastric gavage technique for 30 days. T0 is the negative control group treated only with water at a dose level of 0.5 ml/20 g body weight (bw). T+ is the positive control group treated only with tetracycline at a dosage level of 0.5ml/20 g bw. T1 and T2 received 0.3 ml/20 g bw and 0.5 ml/20 g bw Z. talanai ethanol leaf extract, respectively.Histopathological evaluation revealed that T+ mice showed hepatotoxicity based on central vein rupture, fibrosis, vacuolations, cytoplasmic degeneration, lymphocyte infiltration, small amount of glycogen deposits and fibrous septa. Reprotoxicity was also observed as indicated by formation of irregularly shaped tubules, narrowing of tubule diameter, widening of the lumen, germinal epithelium exfoliation, reduction in tubules’ cross-sectional areas, absence of germ cell bundles and blood-testis barrier destruction. T1 and T2 mice showed normal liver and seminiferous tubule architectural design as compared to those of T+ mice. Thus, the Z. talanai leaf extract mitigated the hepatotoxicity and reprotoxicity effects of tetracycline causing an improvement in the hepatological and testicular parameters. The ethanolic leaf extract of Z. talanai (Blanco) Merr. Contains bioactive compounds with histoprotective potentials.
The bark of Ziziphus talanai (Blanco) Merrill, a tree endemic to the Philippines, is traditionally used in Antique to cure kidney problems such as urinary tract infections (UTI), and skin diseases such as scabies and ringworm. Its antimicrobial activity was screened against several microorganisms to have a more comprehensive anti-infective profile of the plant. The MeOH extract of Z. talanai bark exhibited antimicrobial activity against Gram-positive Mycobacterium phlei, S. aureus and B. subtilis at 20,000 μg and 2,000 μg but was inactive against Gram-negative E. coli, P. aeruginosa, C. albicans and S. cerevisiae. It has minimal antifungal activity against T. mentagrophytes. The MeOH extract of Z. talanai is therefore inactive against uncomplicated UTI, which is mostly caused by E. coli. Further bioassay- guided isolation and subsequent spectral analysis yielded ceanothic acid, whose activity is specific to M. phlei at 100 μg. M. phlei causes chronic ambulatory peritoneal dialysis- associated peritonitis.
For a decade gentamicin has been used extensively because of its antimicrobial efficacy and the relatively low prevalence of clinical toxicity. Recently the more frequent appearance of resistant organisms, reports of increased nephrotoxicity and ototoxicity, and the development of newer aminoglycoside antibiotics have raised doubts about the continued use of this agent. This paper reassesses the role of gentamicin. It is clear that an appreciation of the pharmacokinetics and the clinical use of gentamicin as well as an understanding of the patterns of toxicity in animals and humans can lead to more efficient treatment with this antimicrobial agent. Despite ample competition from a number of newer aminoglycoside antibiotics, gentamicin will probably continue to be used widely in the near future.
Antibiotics are the most common drugs implicated in clinical reports of drug-induced nephrotoxicity. The experimental basis for proposed mechanisms of acute renal failure in association with three groups of antibiotics--aminoglycoside, cephalosporin, and amphotericin B--are reviewed in detail. Proposed mechanisms of antibiotic-induced acute renal tubular necrosis involve either altering plasma membrane permeability or interference with cellular energy derived from mitochondria, For either aminoglycoside or cephalosporin antibiotics, cellular accumulation followed by interruption of mitochondrial respiration is the concept that has greatest support, although the possibility of an induced phospholipidosis involving intracellular lysosomes cannot be excluded. Altered renal tubular cell permeability due to the incorporation of amphotericin B into the pore structure of the plasma membrane is consistent with in vivo observation in either clinical or experimental examples of nephrotoxicity with this agent. The metal cis-platinum, used in treatment of neoplastic disease, has a clearly defined incidence of clinical nephrotoxicity with little insight as to cellular mechanisms. A possible mediation involving cis-platinum reducing the protein-bound sulfhydryl group of renal tissue has been proposed. With the ever increasing potency of modern pharmacologic agents come a rising risk of serious toxic side effects.