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Phytochemical analysis and Evaluation of
hepatoprotective effect of Maytenus
royleanus leaves extract against anti-
tuberculosis drug induced liver injury in
mice
Maria Shabbir
1,2†
, Tayyaba Afsar
3*†
, Suhail Razak
3
, Ali Almajwal
3
and Muhammad Rashid Khan
2
Abstract
Background: Myrin®-p Forte is an anti-tuberclosis agent that can cause hepatic injuries in clinical settings. Maytenus
royleanus (Celastraceae) is a medicinal plant, possesses antioxidant and anticancer activities. The hepatoprotective
effect of the methanol extract of Maytenus royleanus leaves (MEM) against Myrin®-p Forte induced hepatotoxicity in
mice was investigated.
Methods: Mice were randomly parted into six groups (n= 6). Fixed-dose combination of Myrin®-p Forte (13.5 mg/
kg Rifampicin, 6.75 mg/kg Isoniazid, 36.0 mg/kg Pyrazinamide and 24.8 mg/kg Ethambutol; RIPE] was administered
for 15 days to induce liver injury. In treatment groups MEM (200 mg/kg and 400 mg/kg doses) and Vitamin B6
(180mg/kg) were administered prior to RIPE. Control group received 2% DMSO. Serum liver function tests, DNA
damage, tissue antioxidant enzymes and histopathological alterations were studied. HPLC analysis was performed
to determine the chemical composition using standard compounds.
(Continued on next page)
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* Correspondence: tayyaba_sona@yahoo.com
†
Maria Shabbir and Tayyaba Afsar contributed equally to this work.
3
Department of Community Health Sciences, College of Applied Medical
Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia
Full list of author information is available at the end of the article
Shabbir et al. Lipids in Health and Disease (2020) 19:46
https://doi.org/10.1186/s12944-020-01231-9
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(Continued from previous page)
Results: The quercitin, gallic acid, luteolin, viteixin, apigenin, kaempherol, hyperoside and myricetin contents of all
samples were determined by reverse-phase HPLC. Quercetin (0.217 mg/g dry weight) and luteolin (0.141 mg/g dry
weight) were the major flavonoids identified in MEM. Myrin®-p Forte markedly (p< 0.05) deteriorated lipid profile
and upregulated the concentration of LDH, AST, ALP, ALT and γ-GT in serum along with DNA fragmentation
(37.13 ± 0.47%) and histopathological injuries in hepatic tissues of mice compared with the control group. Myrin®-p
Forte increased (p< 0.001) lipid peroxidation and H
2
O
2
while decreased (p < 0.001) the activity level of CAT, SOD,
POD, GPx, GST, GSR, γ-GT and GSH. Co-administration of MEM (200 mg/kg; 400 mg/kg) or the vitamin B6 (180 mg/
kg) to Myrin®-p Forte administered mice significantly ameliorated LDL, cholesterol, HDL and triglyceride content.
Furthermore, MEM dose dependently corrected serum liver function tests, decrease % DNA fragmentation (17.82 ±
0.35 and 7.21 ± 0.32 respectively), DNA damage. MEM treated protect RIPE induced oxidative damage by enhancing
antioxidants to oxidants balance. Histological examination comprehends biochemical findings.
Conclusion: The antioxidant effects of MEM exerted the hepatoprotective potential against the Myrin®-p Forte
induced hepatotoxicity in mice.
Keywords: HPLC, Liver function tests, DNA damages, Histopathology, Antioxidant, Lipid peroxidation
Background
Tuberculosis (TB) a curable respiratory ailment insti-
gated by Mycobacterium tuberculosis; mostly affecting
the poor countries of Africa and Southeast Asia. Accord-
ing to World Health Organization (WHO), its preva-
lence recorded was 14 million, while 2.38 million deaths
were estimated. Fixed dose combination of Myrin®-p
Forte [contains Rifampicin (13.5 mg/kg), Isoniazid (6.75
mg/kg), Pyrazinamide (36.0 mg/kg) and Ethambutol
(24.8 mg/kg); RIPE]. RIPE has been recommended by
WHO for the intensive phase (2 months) followed by
continuous treatment of Rifampicin and Isoniazid for 4–
6 months [1]. However, this regimen causes hepatic in-
juries in clinical settings [2]. The clinical symptoms of
anti-TB drug appear in nonspecific elevation of transam-
inases to fulminant of liver failure [3]. Hepatic injuries
have been induced with isoniazid and rifampicin in ex-
perimental animals [4]. It is suggested that isoniazid is
metabolized into monoacetyl hydrazine as well as isoni-
cotinic acid; the latter can be activated through meta-
bolic oxidation of cytochrome P-450 to toxic species
causing hepatic damages. Rifampicin aggravated the hep-
atotoxicity due to its high amidase activity and is in-
volved in release of large concentrations of acetyl-
hydrazine from isoniazid [5]. The reactive metabolites of
an acetyl-hydrazine bind with hepatic proteins causing
injuries [6]. Likewise the pyrazinamide is metabolized by
the hepatic xanthine oxidase as well as microsomal ami-
dase and the intermediaries; pyrazinoic acid and 5-
hydroxy pyrazinoic acid are considered to be involved in
hepatotoxicity [7]. Although the exact mechanism and
contributing factors of hepatotoxicity induced with anti-
tuberculosis drug are not clear; reactive oxygen species
(ROS)-mediated oxidative damage is postulated to be
the main factor of lipid peroxidation and consequently
the hepatic injuries. Alterations in the enzymatic and
non-enzymatic entities of the cellular defence mechan-
ism have been reported with the use of anti-TB drug [8].
Hydrazine declines the level of cellular glutathione
(GSH) and suggested to minimize the oxidative defence
mechanism and consequently cause cellular injuries and
death [9].
The plants provide a natural source to treat various as-
pects of diseases. It has been observed that most of the
plant based drugs impart their therapeutic potential by
exhibiting antioxidant activities. The plant extracts com-
prise a range of compounds including alkaloids, glyco-
sides, flavonoids, fatty acids, saponins, sterols, and
others. Polyphenolic compounds of plants are of remark-
able importance because they confer such hydroxyl
groups that show scavenging potential for free radicals
[10]. On account of potent antioxidant properties; dur-
ing current years many species of plants have been eval-
uated for the management and treatment of various
ailments. For this reason, research work is being con-
ducted to suggest an approach that involves certain
agents tending to alleviate the anti-TB drug induced
hepatotoxicity. Maytenus royleanus belonging to the
family Celastraceae is distributed in the lower Himalayas
surrounding Islamabad and on dry sunny slopes of
Kaghan (KPK) Pakistan. Its bark is used by the local
population in gastrointestinal disorders [11]. In our pre-
vious investigations, we have identified caffeic acid, quer-
cetin 3-rhamnoside, triterpenoids by HPLC, flavonoids
and tannins by quantitative phytochemical analysis,
while LC-MS fingerprinting revealed Anthocyanines,
Phenolics, Chlorophylls, Macro and micro constituents
in MEM [10,12]. In vitro antioxidant and anti-lipid per-
oxidation activities of M. royleanus leaves have been re-
corded in our previous studies [13]. Furthermore, we
have observed significant decrease in prostate cancer cell
viability and clonogenic survival with the methanol
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 2 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
extract of M. royleanus leaves. These findings are associ-
ated with a significant inhibition in tumor growth and
decline in serum level of prostate-specific antigen (PSA)
in athymic nude mice [12]. Many species of Maytenus
were explored for their antioxidant and hepato-
protective potential in in vivo system. The methanolic
extract from Maytenus robusta leaves showed hepato-
protective effect in mice and HepG2 cells against CCL4
induced toxicity. Extract reduced the hepatic histological
damages and normalize hepatic biomarkerss. The anti-
oxidant effect of M. robusta in liver tissue promoted the
reduction in lipoperoxides levels increased the reduced
glutathione content and increased the activity of super-
oxide dismutase, catalase, and glutathione-S-transferase
Moreover, the extract reduced hepatic inflammation by
diminishing myeloperoxidase activity, TNF and
interleukin-6 levels by its antioxidant effects [14]. Simi-
larly, Maytenus emarginata ethanol extract possess po-
tent hepatoprotective effect. Treatment of rats with the
Maytenus emarginata extract showed marked decrease
in levels of serum ALP, ALT as well as AST with eleva-
tion of serum protein and albumin [15].
Based on the hepatoprotective potential of related spe-
cies in animal models, and diversified in vitro and
in vivo pharmacological properties of M. royleanus, the
current experiment was designed to evaluate the pro-
tective effects of methanol extract of M. royleanus leaves
against the anti-TB drug induced hepatotoxicity in mice.
The chemical composition was analysed by HPLC using
standard flavonoid compounds. Various biochemical pa-
rameters, lipid profile, DNA ladder assay and histological
investigations were done to apprehend the protective
impact of plant extract against anti-TB drug induced
hepatotoxicity.
Methods
Plant collection
Leaves of M. royleanus were collected in March 2011
from village Lehtrar, Tehsil Kotli Sattian, District Rawal-
pindi, Pakistan. The plant identity was verified by Dr.
Saleem Ahmad (curator at the Herbarium of Pakistan,
Museum of Natural History, Islamabad). Voucher speci-
men (# 032564) of a plant was submitted at the Herbar-
ium of Pakistan, Museum of Natural History, and
Islamabad.
Preparation of plant extract
Leaves of M. royleanus were dried in an aerated but
shaded area. Dried material was ground by an electric
grinder to obtain 60 μm powder. The methanol extract
was obtained by allowing 3 kg of powder to macerate 3
times in 95% methanol (3 × 2000 ml) for 5 consecutive
days. The supernatants were mixed and filtered. The
solvent was evaporated by rotary vacuum evaporator
(Buchi, R114, Switzerland). The residue was taken to
dryness to obtain a viscous mass as the crude methanol
extract (MEM).
Sample preparation
Stock solutions of quercitin, gallic acid, luteolin, viteixin,
apigenin, kaempherol, hyperoside and myricetin were
prepared in methanol at concentration of 1 mg/ml and
then serially diluted with methanol to get 10, 20, 50, 100
and 200 μg/ml for making the standard calibration curve.
MEM stock was prepared as 10 mg/ml in methanol.
High performance liquid chromatography
HPLC was performed with an Agilent liquid chromatog-
raphy system, consisting of UV-VIS Spectra-Focus de-
tector (220 nm) and injector-auto sampler. Sample (10
mg/ml) were filtered through a 0.45 μm PVDF-filter and
injected in to the HPLC column. The injection volume
was 10 μl and the column was 20RBAX ECLIPSE, XDB-
C18, (5 μm; 4.6 × 150 mm, Agilent USA). The method
involved the use of a binary gradient with mobile phases
containing: solvent A (0.05% trifluoroacetic acid) and
solvent B (0.038% trifluoroacetic acid in 83% acetonitrile
(v/v) with the following gradient: 0–5 min, 15% B in A,
5–10 min, 50% B in A, 10–15 min, 70% B in A. The flow
rate was kept constant at 1 ml/min. Identifications were
based on retention times in comparison with authentic
standards. The crude extract was partitioned three times
with 25% hydrochloric acid and methanol. The percolate
was concentrated in a rotary evaporator and dissolved in
HPLC grade methanol. Calibration curves for standard
analytes at 10, 20, 50, 100 and 200 μg/ml concentrations
were found to be linear (Supplementary File 1). Quantifi-
cation of each constituent was completed by means of
integration of peaks using the external standard scheme.
Animals
Adult BALB/c male mice (25–30 g) were obtained from
National Institute of Health (NIH), Islamabad and
housed at the Primate Facility of Quaid-i-Azam Univer-
sity Islamabad at a temperature of 25 ± 3 °C with a 12 h
dark/light cycle in pathogen free environment. They
were allowed to standard laboratory feed and water. The
study was performed with acceptance (#0173) from the
Institutional Animals Ethics Committee, Quaid-i-Azam
University Islamabad.
Assessment of acute toxicity in mice
The acute toxicity assessment was done as per the
guidelines 425 of the Organization for Economic Co-
operation and Development (OECD) for analysis of che-
micals for acute oral toxicity [16]. Mice were separated
into six groups with four mice per group. MEM was ad-
ministered orally as a single dose to mice at different
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.
dose levels of 250, 500, 1000, 1500, 2000 and 4000 mg/
kg body weight. The animals were examined individually
for the signs of toxicity and behaviour for 24 h; at 15, 30,
60 min and 4 h. Animals were observed continuously for
24 h for behavioral, neurological and autonomic profiles
and after a period of 14 days for any lethality or death
[17].
RIPE induced hepatotoxicity in mice
Mice were separated randomly in 6 groups with 6 mice
in each.
Control (Group I) was treated with vehicle (2% DMSO
dissolve in saline, i.p).
Group II received fixed-dose combination of anti-TB
drugs [rifampicin (13.5 mg/kg), isoniazid (6.75 mg/kg),
pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/
kg); RIPE] suspension in sterile saline solution (0.9%)
daily for 15 days.
Animals of Group III (200 mg/kg) and Group IV (400
mg/kg) received intraperitoneal treatment of MEM dis-
solved in 2% DMSO.
Group V received vitamin B
6
at 180 mg/kg dissolved
in 10% DMSO, 45 min prior to RIPE challenge.
Group VI was treated with vitamin B6 (180 mg/kg).
Group VII with MEM (400 mg/kg) alone once daily
for 15 days.
After the last dose of treatment schedule, animals were
fasted for 12 h and euthanized by decapitation. The liver
was taken out immediately, rinsed in cold saline at 4 °C
and blotted dry. One part of a liver was stored at −70 °C
in liquid nitrogen for the determination of biochemical
as well as DNA fragmentation assays. The other portion
was used for histopathological studies. Blood was with-
drawn through cardiac puncture and sera were separated
without additive by centrifugation (640 g) at 4 °C and
stored at −20 °C for biochemical evaluation.
Biochemical studies of serum and lipid profile
In the serum the level of AST, ALT, ALP, γ-GT, LDH
(aspartate transaminase, alanine transaminase, alkaline
phosphatase, gamma glutamyltransferase. Lactate de-
hydrogenase), total protein, albumin and total bilirubin
with AMP diagnostic kits according to the experimental
design of the manufacturer. Furthermore, the amount of
total cholesterol (TC), high-density lipoproteins (HDL),
low-density lipoproteins (LDL) and triglycerides (TG)
were approximated by using standard AMP diagnostic
kits (Stattogger Strasse 31b 8045 Graz, Austria).
Hepatic antioxidant studies
Preparation of homogenate and estimation of protein
The liver sections were homogenized in 50 mM phos-
phate buffer (pH 7.8), the protein content of tissue
homogenate was assessed by using bovine serum albu-
min as standard [18]. The reduced glutathione (GSH)
concentration was also estimated in the homogenate.
Later on, centrifugation of the homogenate at 9000 g(15
min and 4 °C) was done and obtained supernatant was
utilized for the estimation of an activity level of various
enzymes.
Peroxidase (POD) determination The activity level of
the POD enzyme assay was performed by following pre-
vious protocol [19]. The variation in absorbance of the
sample was determined at 470 nm after each 20 s. An al-
teration in absorbance of 0.01unit/min was determined
as one-unit POD level of activity.
Catalase (CAT) assay The activity level of CAT was es-
timated by applying the previously established protocol
[20]. Alteration in absorbance of the sample at a wave-
length of 240 nm followed after every 30 s. Catalase ac-
tivity one unit was defined as an alteration in
absorbance (0.01 unit/min).
Superoxide dismutase (SOD) assay Activity level of
SOD in hepatic tissues was determined by well establish
protocol [21]. Superoxidase dismutase activity was esti-
mated by the use of sodium pyruvate phosphate and
phenazine methosulphate. 150 μL of the supernatant was
mixed with 600 μL of 0.052 mM of sodium pyrophos-
phate buffer (pH 7.0) having 50 μL of 186 mM phenazine
methosulphate as substrate. In order to initiate the reac-
tion 100 μL of 780 μM of NADH was added and after 1
min the reaction was pause by the addition of 500 μLof
acetic acid. Change of color was determined at 560 nm
and SOD activity was evaluated as unit/mg protein.
Glutathione-S-transferase (GST) assay The activity
level of GST was determined by the previously reported
protocol [22]. The reaction formulation was prepared by
the addition of solution comprised of 150 μL of the tis-
sue homogenate to 720 μL of 0.1 mM sodium phosphate
buffer in addition to 150 μL of 1 mM GSH and 14.5 μL
of chloro-2,4-dinitrobenzene (CDNB). The optical dens-
ity of the CDNB conjugate formed was determined at
340 nm with a spectrophotometer. The activity level of
GST was estimated by the molar coefficient of 9.6 ×
10
−3
/M/cm in terms of CDNB conjugate produced per
minute per mg protein.
γ-Glutamyltranspeptidase (γ-GT) assay For the deter-
mination of γ-GT activity level of hepatic samples, the
glutamylnitroanilide was used as substrate according to
the previous protocol [23]. For this purpose, 40 μLof
hepatic supernatant was mixed to 200 μL of 5 mM of
glutamylnitroanilide, 200 μL of 20 mM glycine and
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 4 of 15
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200 μL of 12 mM of MgCl
2
mixed in 185 mM of Tris
HCl buffer. The reaction formulation was maintained at
room temperature for 15 min and the reaction was
stopped by the mixing of 200 μL of 30% trichloroacetic
acid. The optical absorbance of the homogenate after
centrifugation at 2500 gfor 15 min was observed at 400
nm with a spectrophotometer. The activity of γ-GT was
evaluated as nM p-nitroaniline produced per minute per
mg protein by using the molar coefficient of 1.74 × 10
−3
/
M/cm.
Glutathione peroxidase (GPx) assay The GPx activity
level of liver sections was estimated by using previously
reported protocol [24]. The reaction formulation was
prepared by the adding 1.59 mL of 0.2 M of potassium
phosphate buffer (pH 7.4), 0.1 mL of 1 mM of sodium
azide, 0.5 mL of glutathione reductase (1 1 U/mL), 0.1
mL of 0.2 mM of NADPH, 0.01 mL of 0.25 mM of H
2
O
2
,
and 0.1 mL of the liver sample supernatant by means of
reagents like NaN
3
(1 mM), glutathione reductase (1 IU/
mL), NADPH (0.2 mM) and hydrogen peroxide (0.25
mM, 0.01 mL) as reaction substrate. Activity level of
GPx was determined as nM NADPH oxidized per mi-
nute per mg protein by applying molar coefficient of
6.22 × 10
−3
/M/cm.
Glutathione reductase (GSR) assay The activity level
of GSR was estimated by previously developed protocol
[25]. The reaction formulation was done by the addition
of 0.2 mL of liver supernatant in a mixture consisted of
1.56 mL of 0.2 M of potassium phosphate buffer (pH
7.6), 0.1 mL of 0.1 mM of EDTA, 0.05 mL of 2 mM of
oxidized glutathione and 0.1 mM of NADPH (0.1 mL).
The absorbance of the reaction mixture was observed by
spectrophotometer at 340 nm at 30 °C. GSR activity was
calculated in terms of nM NADPH oxidized per minute
per mg protein by using the molar coefficient of 6.22 ×
10
−3
/M/cm.
Reduced glutathione (GSH) assay The concentration
of reduced glutathione in liver samples was observed by
following the previously reported protocol [26]. 1.0 mL
of the liver supernatant was precipitated with 1.0 mL of
4% of sulfosalicylic acid and spun at 1200 gfor 20 min
after placing the sample at 4 °C for 1 h. From the hom-
ogenate 0.1 mL was added to a reaction formulation
which consisted of 2.8 mL 0.1 M of potassium phosphate
buffer (pH 7.4) and 2 mL of 100 mM of dithiobis nitro
benzoic acid (DTNB). The absorbance of the yellow
product was recorded at 412 nm. The quantity of GSH
was estimated as μM GSH/g of liver sample.
Oxidative stress markers
Lipid peroxidation (TBARS) assay Determination of
lipid peroxidation in liver samples was done as previous
procedure [27]. The reaction formulation for the estima-
tion of lipid peroxidation was compiled by adding 0.2
mL of supernatant in the reaction having 0.2 mL of 150
mM of ascorbic acid, 0.02 mL of 100 mM of ferric chlor-
ide and 0.58 mL of 0.1 M of potassium phosphate buffer.
Afterward, incubated in shaking water bath at the
temperature of 37 °C for duration of 1 h. Centrifugation
of the reaction mixture was carried out for 15 min and
the absorbance of the supernatant was observed at 535
nm against a reagent blank. The quantity of thiobarbitu-
ric acid reactant substances (TBARS) was estimated as
nM TBARS per min per mg of tissue by applying a
molar coefficient of 1.56 × 10
−5
/M/cm.
Hydrogen peroxide (H
2
O
2
) assay The previously re-
ported protocol was followed for determination of
hydrogen peroxide in the liver samples [28]. The cocktail
of the reaction comprised of 0.1 mL of supernatant, 0.5
mL of 0.05 M of phosphate buffer (pH 7), 0.1 mL of 0.28
nM phenol red, 0.25 mL of 5.5 nM of dextrose and 8.5
units of horseradish peroxidase. The reaction formula-
tion was placed at room temperature for 60 min
followed by the addition of 0.1 mL of 10 N of NaOH to
terminate the reaction. The absorbance of the super-
natant was observed at 610 nm after centrifugation of
the reaction formulation at 800 gfor 10 min. Amount of
hydrogen peroxide produced was determined as nM
H
2
O
2
per min per mg tissue by a applying reference
curve of hydrogen peroxide oxidized phenol red.
Hepatic histopathological studies Hepatic sections
were dehydrated in ascending order of ethyl alcohol,
shifted to cedar wood oil, fixed in paraffin, thin sections
of 3–5μm were prepared and hematoxylin-eosin was
used for staining purpose. Histopathological studies were
conducted with a microscope (DIALUX 20 EB) and
photographed were captured at 400 × magnification.
DNA damaging studies Liver tissues preserved in liquid
nitrogen were used for the determination of DNA dam-
ages by applying the previous standard experimental de-
sign [26]. Hepatic tissues (100 mg) were homogenized in
10 volumes of TE (pH 8.0) solution (5 mM Tris-HCl; 20
mM EDTA) and 0.2% of Triton X-100. It was spun at
27000 gfor 20 min to attain the pellet (B) and the super-
natant (T). The quantity of DNA in the pellet and the
supernatant was estimated at 620 nm with spectropho-
tometer by using the diphenyl-amine solution.
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%Fragmented DNA ¼Supernatant
Supernatant þPellet 100
DNA ladder protocol+ For the determination of DNA
damages 1.5% agarose gel comprising of 1.0 μg/mL eth-
idium bromide (EtBr) was used. In each well 5 μg of gen-
omic DNA while 0.5 μg the marker DNA was utilized to
determine the DNA damages. Subsequently, the image
of a gel was observed in the gel doc system [29].
Statistical analysis
Data are presented as Mean ± SEM (n= 6). Statistical dif-
ferences between different treatments were calculated by
one-way analysis of variance (ANOVA) followed by
Tukey’s test on Graph pad prism 5 software. Significance
level was set at p< 0.05.
Results
HPLC analysis of MEM
Figure 1indicated the HPLC chromatogram of MEM.
Based on a comparison of the retention times with those
of the standards, major peaks 1 and 2 were identified as
quercetin and luteolin. The other compounds are de-
tected in minor quantities as indicated by low pea areas,
hence quercetin and luteolin are shown to be major fla-
vonoids identified in MEM. The concentration of
quercetin and luteolin in MEM is estimated to be (0.217
mg/g dry weight) (0.141 mg/g dry weight) respectively.
Effect of methanol extract of M. royleanus leaves on liver
and body weight in mice
Anti-TB drug (RIPE) treated group significantly decrease
the liver weight compared to control group. MEM treat-
ment markedly ameliorated the effect of anti-TB drug
on liver weight. Impact of MEM was comparable to the
effect of vitamin B6 on hepatic weight. Similarly, final
body weight was significantly decreased with treatment
of anti-TB drug. MEM treatment significantly amelio-
rated RIPE induced decline in body weight. On the con-
trary, a significant weight gain was observed with the
vitamin B
6
and MEM co-treated groups (Table 1).
Lipid profile
Liver toxins reacts with polyunsaturated fatty acids to
encourage lipid peroxidation by disturbing the lipid pro-
file [30]. RIPE treatment evidently (P< 0.001) augmented
the quantity of total cholesterol, LDL and triglycerides,
while decreasing (P< 0.001) HDL levels as compared to
control group (Table 2). Co-treatment of MEM with
RIPE, dose dependently improved the abnormal lipid
profile. MEM high dose treated group showed similar
shielding action against RIPE induced lipid profile
changes as shown by Vitamin B6 treated group. Animals
Fig. 1 HPLC chromatogram of MEM
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.
treated with AHE alone at 400 mg/kg.bw dose showed
an insignificant difference in results compared to control
group.
Effect of MEM on liver function tests (LFTs)
RIPE administration increased the concentration of
AST, ALT, ALP, γ-GT, and LDH in serum compared to
the control group. The hepatotoxicity induced with RIPE
was ameliorated by the co-administration of MEM to
RIPE administered mice. The protective effects of MEM
on AST, ALT, ALP, GT and LDH were produced in a
concentration dependent manner. The level of AST,
ALT, ALP, γ-GT, and LDH in the serum of MEM and
vitamin B6 alone administered groups remained un-
affected compared to the control group (Table 3).
Effect of MEM on antioxidant enzymes
Effect of MEM on RIPE induced deterioration of tissue
antioxidant enzymes is reveled in Table 4. The activity
level of phase I antioxidants such as CAT, POD and
SOD is decreased (p< 0.05) as compared to the control
group. The protective potential MEM against the tox-
icity induced with RIPE on the phase I antioxidant en-
zymes was evident by significant (p< 0.05) increase in
CAT, POD and SOD compared to RIPE treated group.
Co-administration of vitamin B6 along with RIPE to
mice ameliorated the toxicity of RIPE and increased the
level of CAT, POD, and SOD in liver samples as com-
pared to the RIPE treated group. The protective effects
of MEM at 400 mg/kg dose were comparable (p> 0.05)
to the vitamin B6 treated group for the CAT and SOD
activity while for POD activity level the vitamin B6 +
RIPE group exhibited more protective potential (p<
0.05) to that of the MEM + RIPE co-treated group. Ad-
ministration of vitamin B6 (180 mg/kg) to mice elevated
(p < 0.05) the activity level of POD and SOD while no ef-
fect (p > 0.05) on the activity level of CAT as compared
to the control group. MEM (400 mg/kg) administration
to mice showed significant (p < 0.05) elevation in the ac-
tivity level of CAT and SOD whereas the activity level of
POD was not influenced (p > 0.05) as compared to the
control group.
Activity level of phase II antioxidant enzymes such as
GSH, GST, GPx, GSR and γ-GT decreased (p < 0.05)
with the RIPE administration as compared to the control
group (Table 3). Toxico-suppressive effects of MEM
against the hepatotoxicity with RIPE were recorded and
the activity level of GSH GST, GPx, GSR and γ-GT in
hepatic samples of mice increased as compared the RIPE
treated group. Co-treatment of vitamin B6 with anti-
Table 1 Protective effect methanol extract of M. royleanus leaves on liver and body weight of mice
Treatments Liver weight (g) Initial body weight (g) Final body weight (g)
Control 6.22 ± 1.36 25.53 ± 0.58 34.7 ± 0.26
RIPE 4.80 ± 0.74
*
25.33 ± 0.46 27.1 ± 0.30
***
RIPE + MEM (200 mg/kg) 5.71 ± 0.97
+
24.83 ± 0.51 31.0 ± 0.39
***, +++, ##
RIPE + MEM (400 mg/kg) 5.98 ± 0.13
+
25.21 ± 0.39 33.1 ± 0.41
+++
RIPE + Vit B
6
5.51 ± 1.81
+
25.11 ± 0.52 33.4 ± 0.29
+++
Vit B6 (180mg/kg) alone 6.28 ± 1.74
+
25.30 ± 0.47 35.1 ± 0.30
+++
MEM (400 mg/kg) alone 6.06 ± 1.13
+
24.71 ± 0.53 34.8 ± 0.37
+++
Values are represented as mean ± SEM (n= 6). Data analyzed by one-way analysis of variance followed by multipl e comparison test. Asterisks *, **, *** represents
significance from control group at p< 0.05, p< 0.001, and p< 0.0001. +, +++ represents significance from RIPE group at p< 0.05 and p< 0.0001 while ###
represents significance difference of RIPE + MEM (200 mg/kg) vs RIPE + MEM (400mg/kg) group at p< 0.0001
Table 2 Protective effects of MEM on Serum Lipid Profile
Treatments Total Cholesterol (mg/dl) Triglycerides (U/L) HDL
(mg/dl)
LDL (mg/dl)
Control 62.37 ± 2.61 54.43 ± 0.74 40.77 ± 2.27 29.1 ± 3.19
RIPE 98.10 ± 1.25
****
134.09 ± 3.35
****
22.78 ± 2.41
****
59.18 ± 1.79
****
MEM (200 mg/kg) + RIPE 80.77 ± 2.98
****++++
72.91 ± 5.93
****++++
31.80 ± 1.09**
++++##
44.31 ± 1.27
****++++
MEM (400 mg/kg) + RIPE 77.40 ± 3.74
****++++
67.81 ± 1.17
****++++
38.78 ± 2.43
++++
33.37 ± 3.69
++++
Vit B6 + RIPE 70.91 ± 2.69
****++++
63.30 ± 3.07**
++++
39.01 ± 1.39
++++
34.01 ± 2.07
++++
Vit B6 63.98 ± 1.37
++++
55.79 ± 0.73
++++
41.01 ± 1.59
++++
28.02 ± 0.31
++++
MEM (400 mg/kg) 65.80 ± 1.11
++++
59.37 ± 5.14
++++
42.11 ± 0.94
++++
29.11 ± 3.11
++++
Values are Mean ± SD (06 number). RIPE, Rifampicin (13.5 mg/kg) , Isoniazid (6.75 mg/kg), Pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/kg). MEM, M.
royleanus leaves methanol extract. *, **, *** indicate significance from the control group at p < 0.05, p< 0.01 and p < 0.0001 probability level, +, ++, +++ indicate
significance from the RIPE group at p< 0.05, p< 0.01 and p< 0.0001, while ### indicate significance of MEM (400 mg/kg) + RIPE group vs MEM (200 mg/kg) + RIPE
group at p < 0.0001 probability level (One-way ANOVA followed by Tukey’s multiple comparison tests)
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 7 of 15
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tuberculosis drug administered mice elevated the activity
level of antioxidant enzymes in hepatic samples in com-
parison to the RIPE administered mice group. Activity
level of GPx and γ-GT with the treatment of MEM (400
mg/kg) alone was statistically similar (p> 0.05) to the
control group while activity level of GST and GSR de-
creased (p< 0.05) as compared to control group.
Effect of MEM on hepatic protein content and oxidative
stress markers
The protective potential of MEM against the RIPE in-
duce oxidative stress and protein content is presented in
Table 5. RIPE administration decreased the soluble con-
centration of protein in hepatic samples of mice com-
pared to the control animals. The mice co-treated with
vitamin B6 and MEM along with RIPE exhibited the in-
crease in soluble protein concentration in tissues com-
pared to the mice treated with anti-tuberculosis drug
alone. The protective ability of the MEM on soluble pro-
tein concentration was in dose dependent manner. Ad-
ministration of vitamin B6 and MEM alone to mice
non-significantly elevated the level of protein compared
to the control mice Treatment with RIPE significantly
increase hepatic oxidative stress as depicted by increased
concentration of TBARS and H
2
O
2
in hepatic tissues
compared to the control group. MEM treatment in com-
bination with RIPE lessened the toxic effects of anti-
tuberculosis drug and decreased the concentration of
TBARS and H
2
O
2
in hepatic samples in a dose
dependent manner. The ameliorative potential of MEM
is equivalent to standard vitamin b
6
treated group.
Effect of MEM on histopathology of liver
The histological architecture of the control group
showed the normal lobular structure of liver (Fig. 2). In
anti-tuberculosis drug treated mice, the histopathology
of the liver was altered and fatty changes were promin-
ent. The lobular structure was disrupted and there was
congestion of blood vessels, a severe degree of
hemorrhage, necrosis with fatty vacuolations. There
were degenerative changes and the chromatin material
showed clumped morphology. The cell membrane hepa-
tocytes in some of the areas were not distinguished.
Treatment of mice with MEM protected the liver from
the toxicity of anti-tuberculosis drug and most of the
changes induced with an anti-tuberculosis drug were ab-
sent from the histopathology. Low level of toxicity was
apparent in the MEM (200 mg/kg) treatment, while at a
higher dose (400 mg/kg) the histopathological architec-
ture of liver was near to the control mice. In the case of
vitamin B6, the histopathological alterations induced
with the anti-tuberculosis drug were recorded at a lower
level and hepatocytes presented a minor level of toxicity.
Administration of vitamin B6 alone to mice caused a
foamy appearance of hepatocytes while in case of MEM
(400 mg/kg) dose to mice an almost normal architecture
of liver was apparent. Furthermore, MEM alone treat-
ment seems to be safe in all organs of mice. The path-
ologist report has been attached in supplementary file 2.
Effect of MEM on DNA damage
Analysis of DNA damage in liver tissues by percent
DNA fragmentation and ladder assay is presented in
Fig. 3(a, b). RIPE administration induce significantly
high percent DNA fragmentation. Co-administration of
RIPE treated rats with MEM significantly attenuated
DNA fragmentation in a dose dependent fashion. The
effect of MEM high dose is similar to Vitamin B6 treated
group (Fig. 3a). DNA ladder assay revealed that the
DNA remain entangled at the base of the well and
showed a sharp single band without degradation and tail
pattern in liver tissues of the control as well as in the
vitamin B6 and MEM alone treated groups (Fig. 3b).
Treatment of mice with RIPE induced DNA damages in
liver tissues and showed continuous pattern of DNA
fragmentation in ladder assay. DNA isolated from the
liver tissue of mice treated with MEM + anti-tuberculosis
drug showed significant protection from DNA damage
as revealed by sharp DNA band similar to untreated
group.
Table 3 Protective effects of MEM on liver marker enzymes in serum of mice
Group AST (U/l) ALT (U/l) ALP (U/l) γ-GT (U/l) LDH (U/l)
Control 94.17 ± 3.41 70.46 ± 2.35 142.87 ± 4.65 1.84 ± 0.43 45.34 ± 1.61
RIPE 204.9 ± 6.11
****
211.2 ± 5.13
****
306.47 ± 6.68
****
4.01 ± 0.75
****
141.8 ± 4.17
****
MEM (200 mg/kg) + RIPE 170.29 ± 6.38
****++++####
169.9 ± 4.28
****++++####
252.14 ± 5.31
****++++####
2.98 ± 0.24
*+
110.5 ± 3.68
****++++####
MEM (400 mg/kg) + RIPE 121.15 ± 4.28
****++++
123.0 ± 2.24
****++++
172.13 ± 3.20
****++++
2.16 ± 0.12
++++
73.26 ± 2.34
****++++
Vit B6 + RIPE 109.62 ± 4.4
****++++
98.13 ± 3.13
****++++
161.15 ± 3.98
****++++
2.34 ± 0.57
++++
72.1 ± 2.75
****++++
Vit B6 alone 98.26 ± 4.89
++++
68.14 ± 2.81
++++
143.66 ± 3.63
++++
1.98 ± 0.46
++++
44.32 ± 2.23
++++
MEM (400 mg/kg) alone 93.73 ± 4.36
++++
69.58 ± 2.63
++++
144.35 ± 5.87
++++
1.93 ± 0.79
++++
47.16 ± 1.49
++++
Values are Mean ± SD (06 number). RIPE, Rifampicin (13.5 mg/kg) , Isoniazid (6.75 mg/kg), Pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/kg). MEM, M.
royleanus leaves methanol extract. Vit B6, Vitamin B6. *, **, *** indicate significance from the control group at p < 0.05, p < 0.01 and p < 0.0001 probability level,+,
++, +++ indicate significance from the RIPE group at p< 0.05, p< 0.01 and p< 0.0001, while ### indicate significance of MEM (400 mg/kg) + RIPE group vs MEM
(200 mg/kg) + RIPE group at p < 0.0001 probability level (One-way ANOVA followed by Tukey’s multiple comparison tests)
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 8 of 15
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Table 4 Protective effects of MEM on liver antioxidant status
Treatments CAT (U/min) POD (U/min) SOD
(U/mg protein)
GSH (nM/g tissue) GST (μM/mg protein) GPx (nM/
min/mg
protein)
GSR (nM
/min/mg
protein)
γ-GT (nM
/min/mg
protein)
Control 1.93 ± 0.16 1.99 ± 0.11 9.23 ± 0.21 40.82 ± 2.07 6.84 ± 0.41 123.20 ± 4.22 211.31 ± 5.22 82.36 ± 3.26
RIPE 0.54 ± 0.11
****
1.11 ± 0.14
****
4.15 ± 0.23
****
32.18 ± 1.88
****
3.58 ± 0.45
****
59.28 ± 2.22
****
153.17 ± 4.13
****
61.42 ± 3.45
****
MEM (200 mg/kg) + RIPE 1.83 ± 0.10
++++
1.52 ± 0.13
****+++
6.54 ± 0.17
****++++####
35.51 ± 2.13
**
5.35 ± 0.47
****++++
89.70 ± 3.15
****++++####
177.12 ± 4.03
****++++####
68.74 ± 3.87
**** + #
MEM (400 mg/kg) + RIPE 1.89 ± 0.12
++++
1.72 ± 0.14
*++++
8.35 ± 0.19
****++++
37.71 ± 2.26
+++
5.56 ± 0.34
***++++
102.07 ± 3.03
****++++
199.29 ± 6.20
**++++
76.38 ± 4.21
++++
Vit B6 + RIPE 2.08 ± 0.12
++++
2.04 ± 0.13
++++
8.12 ± 0.24
****++++
36.51 ± 2.05
*+
6.19 ± 0.67
++++
99.24 ± 3.40
****++++
187.30 ± 5.01
****++++
73.54 ± 3.17
**++++
Vit B6 1.88 ± 0.14
++++
2.18 ± 0.15
++++
11.32 ± 0.18
****++++
41.46 ± 2.20
++++
6.36 ± 0.46
++++
118.50 ± 3.21
++++
209.40 ± 6.05
a++++
84.26 ± 4.12
a++++
MEM (400 mg/kg) 2.42 ± 0.17
**++++
1.83 ± 0.17
++++
10.36 ± 0.23
**++++
42.39 ± 1.67
++++
5.62 ± 0.48
**++++
119.25 ± 3.59
++++
201.33 ± 5.02
*++++
85.65 ± 4.65
a++++
Values are Mean ± SD (06 number). RIPE, Rifampicin (13.5 mg/kg), Isoniazid (6.75 mg/kg), Pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/kg). MEM, M. royleanus leaves methanol extract. Vit B6, Vitamin B6. *, **,
*** indicate significance from the control group at p < 0.05, p < 0.01 and p < 0.0001 probability level, +, ++, +++ indicate significance from the RIPE group at p< 0.05, p< 0.01 and p< 0.0001, while ### indicate
significance of MEM (400 mg/kg) + RIPE group vs MEM (200 mg/kg) + RIPE group at p < 0.0001 probability level (One-way ANOVA followed by Tukey’s multiple comparison tests)
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 9 of 15
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Discussion
Anti-TB drugs are the most common group of drugs
that are known to cause severe hepatotoxicity worldwide
and overall, hepatotoxicity attributed to anti-TB drugs
has been reported in 5–28% of people treated with anti-
TB drugs. Up to 20% of the patients receiving RIPE ei-
ther in single or combination therapy develop transient
asymptomatic elevation in liver enzymes, which settle
with continued use of the drug and its hepatotoxicity
manifestation can vary from asymptomatic elevations in
the liver enzymes to fulminant liver failure. Eras of med-
ical surveillance have recognized a range of drugs as well
as host linked aspects which are related to an enhanced
threat of anti-tuberculous drug-prompted liver injury
[8]. Liver biopsy specimens reveal lobular hepatitis, sub
massive to massive necrosis and hydropic degeneration
of hepatocytes in severe cases [31].
In current investigation the commonly used anti-
tuberculosis (antiTB) drugs; pyrazinamide (PZA), isonia-
zid (INH), ethambutol (ETB) and rifampicin (RMP) were
administered to mice in order to investigate toxic conse-
quences of combination therapies on liver. The outcome
of the present study represents the induction of severe
hepatocellular injuries as evidenced by disturbed liver
profile and upregulation of AST, γ-GT, ALP, ALT, and
LDH in the serum of mice. Hepatotoxicity induced with
the anti-TB drug was ameliorated by co-administration
of MEM and the level of LDL, cholesterol, triglycerides,
AST, ALT, ALP, γ-GT, and LDH in the serum of mice
decreased in dose-dependent manner suggesting the
protective ability of MEM. The protective effect of MEM
might be due to the presence of bioactive metabolites
(Table 6). As earlier reports signify that quercetin and
luteolin improve lipid profile and inhibit inflammatory
cytokines [32,33]. Administration of MEM (400 mg/kg)
alone to mice did not induce an alteration in the level of
hepatic biomarkers enzymes, signifying the safe effect of
MEM. The co-administration of vitamin B6 to the anti-
TB drug administered mice decreased the level of AST,
ALT, ALP, γ-GT, and LDH in the serum of mice sug-
gesting the protective effects on liver function. The ad-
ministration of Vitamin B
6
alone to mice did not induce
a noteworthy alteration in the level of ALT, ALP, γ-GT
and LDH in serum compared to control animals. How-
ever, administration of vitamin B6 causes a non-
significant escalation in the level of AST in the serum of
mice compared to the control group. The increase in
AST might be attributed to the deficiency of vitamin B6
in mice [34]. Induction of hepatic injuries with the anti-
TB drug is multifaceted, but the major mechanism
seems to be oxidative stress induced generation of free
radicles. In this investigation, the anti-TB drug to mice
shifts the dynamic equilibrium of metabolism towards
the oxidative stress as observed by an increase of hepatic
TBARS and H
2
O
2
while a decrease in hepatic GSH and
the antioxidant enzymes. Insufficient hepatic level of
CAT, SOD, and GPx was unable to scavenge the exces-
sive generation of lipid hydroperoxides. These reactive
intermediate metabolites play a crucial role in develop-
ing oxidative stress and consequently cause hepatic dam-
ages [35]. Amongst the cell based antioxidants GST,
CAT, POD, GR, SOD, and GPx are extensively explored
for their significant job in defense mechanism. Superoxi-
dase is an exceptionally active antioxidant enzyme that
catalyzes the dismutation reaction of superoxides to
H
2
O
2
and O
2
whereas CAT is a ubiquitous enzyme but
mainly rich in the liver and is engaged in a breakdown
of H
2
O
2
to water. In the GSH reaction system, GSH is
oxidized to GSSG by the help of GPx which transformed
back to GSH by the reducing power of GSR. GSH also
works as a cofactor for GST that is present equally in
the cytosol and endoplasmic reticulum, essentially en-
gage in catalyzing the production of GSH electrophile
conjugate therefore, detoxifying xenobiotics to generate
irreversible compounds. It is detected that lipids
peroxidation can induce a genetic increase of fibrogenic
cytokines by commencing the generation of collagen
and stimulating liver stellate cells [36]. Enhanced
Table 5 Protective effects of MEM on liver protein and oxidative stress markers
Treatments Protein (μg/mg tissue) TBARS (nM of MDA/mg protein) H
2
O
2
(nM/min
/mg tissue)
Control 1.32 ± 0.16 6.09 ± 1.30 5.38 ± 0.42
RIPE 0.73 ± 0.12
****
18.65 ± 3.72
****
8.88 ± 1.04
****
MEM (200 mg/kg) + RIPE 1.10 ± 0.15
++
13.65 ± 1.82
****++#
6.98 ± 0.80
**+++
MEM (400 mg/kg) + RIPE 1.21 ± 0.13
++++
9.78 ± 1.31
*++++
5.78 ± 0.58
++++
Vit B6+ RIPE 1.16 ± 0.18
+++
9.43 ± 1.55
c++++
6.54 ± 0.66
++++
Vit B6 1.41 ± 0.18
++++
5.80 ± 0.80
e++++
4.95 ± 0.60
++++
MEM (400 mg/kg) alone 1.38 ± 0.15
++++
7.07 ± 1.79
d++++
5.44 ± 0.52
++++
Values are Mean ± SD (06 number). RIPE, Rifampicin (13.5 mg/kg) , Isoniazid (6.75 mg/kg), Pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/kg). MEM, M.
royleanus leaves methanol extract. Vit B6, Vitamin B6. *, **, *** indicate significance from the control group at p < 0.05, p < 0.01 and p < 0.0001 probability level,+,
++, +++ indicate significance from the RIPE group at p < 0.05, p < 0.01 and p < 0.0001, while ### indicate significance of MEM (400 mg/kg) + RIPE group vs MEM
(200 mg/kg) + RIPE group at p < 0.0001 probability level (One-way ANOVA followed by Tukey’s multiple comparison tests)
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 10 of 15
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Complement system activation is known for tissue injur-
ies and free radicals activates complement system. More
complement activation causes more C3b generation and
that can lead to tissue injuries [37] . In the current re-
search, the level of TBARS and H
2
O
2
content moved in
the direction of control after treatment with MEM. This
refurbishment may be accompanied with improvement
of the antioxidant enzymes. The decrease of TBARS and
increase of GSH in hepatic samples have been deter-
mined with the co-administration of Bombax ceiba
extract to anti-TB drug administered rats [38]. Our find-
ings are relevant to other observations about hepatic tis-
sue [39]. Methanol extract of M. emarginata has
antioxidant potential evaluated by SOD, DPPH, ABTS,
iron chelating and free radical NO quenching assays
[40]. Another study revealed that M. krukovii hydro-
alcoholic extract of bark possessed an inhibitory poten-
tial against the mutation causing activity (promutagens)
of 2-aminoanthracenein in both T98 and T100 strains
on the other hand showed poor activity towards
Fig. 2 H & E stain; × 400. (I): Representative section of liver from the control group showing the normal architecture, (II): RIPE treated group (III)
MEM (200 mg/kg) + RIPE treated group, (IV): MEM (400 mg/kg) + RIPE treated group, (V): Vitamin B6 (180 mg/kg) + RIPE treated group, (VI): Vitamin
B6 (180 mg/kg) treated group and (VII): MEM (400 mg/kg) treated group
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 11 of 15
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Fig. 3 Lane from left to right; L, 100 bp ladder (low molecular weight DNA marker) (I) Control group (II) RIPE treated group (III) MEM (200 mg/
kg) + RIPE treated group (IV) MEM (400 mg/kg) + RIPE treated group, (V) Vitamin B6 + RIPE treated group, (VI) Vitamin B6 alone treated group (VII)
MEM (400 mg/kg) alone treated group. MEM: Maytenus royleanus methanol extract. RIPE: 13.5 mg/kg Rifampicin, 6.75 mg/kg Isoniazid, 36.0 mg/kg
Pyrazinamide and 24.8 mg/kg Ethambutol
Table 6 Extraction yield, TPC, TFC, and chemical constituents in M.roelyanus leaves extract (MEM)
Analysis (MEM extract) Observations (References)
Extraction yield (%) 12.2% [11]
TPC (mg gallic acid equivalent/g dry sample) 76 ± 2.7 [11]
TFC (mg rutin equivalent/g dry sample) 63.5 ± 1.84 [11]
LC-MS Compound Fingerprinting Glucosinolates, Anthocyanines, Phenolics, Chlorophylls, Macro and micro
constituents
[11]
HPLC-DAD (Identification of compounds with reference to
standards)
Caffeic acid
Quercetin 3 rhamnoside
[12]
TPC Total Phenolic content, TFC Total flavonoid content. Information derived from our previous lab investigations
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 12 of 15
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mutagens such as sodium azide and 2-nitrofluorene. M.
krukovii demonstrated scavenging ability depending on
the concentration of dose. A potential antioxidant activ-
ity against the monocation 2, 2′-azinobis (3-ethylbenzo-
thiazoline-6-sulfonic acid) and HOCl- was shown by the
ethanol extract of M. ilicifolia root [41].
Presence of quercetin and luteolin might be respon-
sible or the protected afforded by MEM. Previous
studies indicated that quercetin is responsible for sub-
stantial defense in contradiction of INH and RFP-
induced toxicity in rats liver demonstrated a decrease
in AST and ALT potentials, an upsurge in total anti-
oxidant activity, and normal histopathological picture
of the rats liver [42]. The vitamin B6 was used as
standard antioxidant compound in our study and the
effect of MEM was shown to be equivalent to vitamin
B6. The protectective effect of Vitamin B6 has been
already validated in previous researches. Single dose
of vitamin B6 directed right after surgery helped in
improved oxidative parameters and inflammatory
markers in liver. It results in significant reduction of
oxidative stress in liver of mice. The immediate ad-
ministration of vitamin B6 in mice contributed to-
wards neutrophil reduction in liver and helped in
diminishing the oxidative damage to protein and
lipids in liver [43].VitaminB6alsomodulatesthe
kynurenine pathway, sphingosine-1-phosphate, and
nuclear factor-kappa B thus lowering inflammation
[44]. Hence, the mechanism of vitamin B6 is via regu-
lating the oxidative stress in peripheral organs i.e.
liver and lungs etc. Hence we speculated that simi-
larly to Vitamin B6, MEM might have rescued liver
injury through modulating oxidative stress in mice
liver. Quercetin and luteolin being one of the major
phytochemical constituents of M. royleanus might be
responsible for the hepato-preventive effects.
The histopathology is unswerving technique for evalu-
ating the ability of test samples at tissue level, addition-
ally its provides correlation between the functions of
serum biomarkers, tissue enzyme levels and morpho-
logical alterations [30]. Noteworthy alterations in liver
function tests (LFTs) predominantly epitomizes the fi-
brosis in liver. Fibrosis not only intrudes the normal
morphology but also interferes the flow of blood to pre-
clude the transport of nutrients to liver tissues. Liver
histology of anti-TB administered group showed marked
histopathological alterations in liver structure, dissol-
ution of hepatic cords, which give the impression of
empty vacuoles aligned by strands of necrotic hepato-
cytes, nuclear disintegration, vacuolar degeneration,
apoptotic cell death, fibrosis and collagen deposition in
some parts. Our finding is in parallel with previous re-
ports on the toxic effect of anti-TB drug induced liver
toxicity. They observed extreme hepatocyte hypertrophy
characterized by a notable increase in cell size accom-
panied by binucleate hepatocytes with enlarged hepato-
cyte nuclei [45,46]. The liver tissue samples of MEM
treated groups exhibited diminished necrosis, slight in-
flammatory cells without damage to cell membrane sig-
nifying its protective potential. The observed
hepatoprotective effect might be correlated to the pres-
ence of bioactive compounds in MEM most specifically;
quercetin and luteolin. Previous studies also indicated
the hepatoprotective action of quercetin and luteolin
against Thioacetamide induced biochemical and histo-
logical changes in rat liver. The mechanism of protection
is through modulating oxidative stress and augmentation
of antioxidant enzymes [47,48]. A recent study indicated
that luteolin protect liver injury by inhibition of inflam-
matory biomarkers [49].
Binding of free radicals can contribute towards the
macromolecular injuries like DNA to induce muta-
tion, lipids to induce membrane damages and pro-
teins to change their function. In the current research
the hepatic DNA assaulted by anti-TB drug generated
free radicals evidenced by the higher level of hepatic
damages and in DNA ladder assay. Alternatively, co-
treatment of MEM substantially decreased the %
DNA fragmentation that was demonstrated by DNA
ladder assay banding pattern. Analogous findings were
presented in a study of [50] while investigating the
scavenging potential of Sonchus arvensis in response
to carbon tetrachloride intoxicated rat liver. The ob-
served hepatoprotective effect might attribute to the
occurrence of phytochemicals as well as various bio-
logically active secondary metabolites i.e. quercetin
and luteolin that are the major compounds identified
by HPLC [10,12].
Conclusion
The outcomes of the current study demonstrated that
anti-TB drug induced a harmful effect on the liver by af-
fecting DNA, lipids, and protein, together with generat-
ing oxidative stress. MEM demonstrated hepato-
protective ability by preventing oxidative DNA damage,
by enhancing the potentials of antioxidant enzymatic as
well as non-enzymatic levels besides regulating the
histological alterations. Serological studies for liver func-
tion tests also proved the protective consequence of
MEM against anti-TB drug caused deteriorations. These
protective effects might be credited by the occurrence of
various antioxidant phytochemicals. Future investiga-
tions exploring the mechanisms underlying the patho-
genesis of anti-TB drug should be performed using
human tissue and samples whenever possible, so that
the novel findings can be translated readily into clinical
applications.
Shabbir et al. Lipids in Health and Disease (2020) 19:46 Page 13 of 15
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Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12944-020-01231-9.
Additional file 1: Pathology report.
Additional file 2: Figure S1. Standard calibration curve of standard
compounds.
Abbreviations
MEM: Maytenus royleanus; RIPE: Rifampicin (13.5 mg/kg), Isoniazid (6.75 mg/
kg), Pyrazinamide (36.0 mg/kg) and Ethambutol (24.8 mg/kg) ;
MDA: Malonyldialdehyde; CAT: Catalase; TBARS: Thiobarbituric acid reactive
substances; SOD: Superoxide dismutase; GPx: Glutathione peroxidase;
H
2
O
2
: Hydrogen peroxide; NO: Nitric oxide; CYP2E1: Cytochrome P450 2E1
Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship
of Scientific Research at King Saud University, KSA for its funding the
research group (RGP- 193). We acknowledge Higher Education Commission
(HEC) of Pakistan for awarding indigenous scholarship for PhD research to
the first author.
Consent to publication
Not applicable.
Authors’contributions
MS and TA made equal contribution in writing the manuscript and analyzing
the data. SR, MRK and AA made substantial contribution in editing and
revising the manuscript for intellectual content. All authors read and
approved the final manuscript.
Funding
We are grateful to the Deanship of Scientific Research at King Saud
University for its funding of this research through Research Group Project
number 193. Funding body has role in design and interpretation of data.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article. The raw used and/or analyzed during the current study
can be available from the corresponding author on reasonable request.
Ethics approval and consent to participate
This investigation involved testing on mice, and the experimental protocol
for the testing on animal was approved (Bch#0173) by the ethical board of
Quaid-i-Azam University, Islamabad, Pakistan.
Competing interests
The authors declare that they have no competing interest.
Author details
1
Atta-ur-Rahman School of Applied Biosciences, NUST, Islamabad, Pakistan.
2
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam
University, Islamabad 45320, Pakistan.
3
Department of Community Health
Sciences, College of Applied Medical Sciences, King Saud University, Riyadh,
Kingdom of Saudi Arabia.
Received: 29 December 2019 Accepted: 9 March 2020
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