Effects of polyphenol-rich traditional herbal teas on obesity and oxidative stress in rats fed a high-fat-sugar diet

Abstract

Hibiscus rosa-sinensis and Zingiber officinalis teas are traditionally used for the therapies of various diseases, including obesity. The present research work was planned to appraise the potential of polyphenol-rich extracts of selected herbal plants in obesity and related biochemical parameters of high-fat–sugar diet-induced obese rats. Three herbal teas were prepared from Hibiscus rosa-sinensis flowers and Zingiber officinalis rhizomes and their mixture (3:1, respectively). Total phenolic contents (TPC) of Hibiscus rosa-sinensis and Zingiber officinalis extracts were found to be 5.82 and 1.45 mg/g of dry plant material, measured as GAE, while total flavonoid contents (TFC) were 9.17 and 1.95 mg/g of dry plant material, measured as CE, respectively. Two doses (250 and 500 mg/kg BW) of each tea were administered and body weight, BMI, kidney, liver, and atherogenic indices, TC, TG, HDL, LDL, VLDL, BT, AST, ALT, AP, SC, MDA, SOD, GSH, and TAC of rats groups were measured. Data showed that higher doses of Hibiscus rosa-sinensis significantly reduced the rat's BMI (0.50 g/cm²) in comparison with the high-fat–sugar diet group (0.79 g/cm²). All treatment groups, especially H-500 group, showed a significant decrease in the elevated kidney and liver weights and atherogenic index in comparison with HFSDC groups. Higher doses of Hibiscus rosa-sinensis significantly decreased the levels of AST, ALT, AP, and SC in comparison with the HFSDC group. A significant decrease in the levels of serum TC, TG, LDL, and VLDL was observed in all the treatment groups in comparison with the HFSDC group. Furthermore, all the teas, especially higher doses of Hibiscus rosa-sinensis, prevented the alterations in MDA, SOD, and GSH levels of experimental groups, thus showing the potential against oxidative stress. It can be concluded from these results that Hibiscus rosa-sinensis teas exhibited strong protective effects against obesity and oxidative stress, especially at higher doses.

Full text

698
|
Food Sci Nutr. 2022;10:698–711.wileyonlinelibrary.com/journal/fsn3
Received: 19 July 2021
|
Revised: 14 November 2021
|
Accepted: 21 November 2021
DOI: 10.1002/f sn3 .2695
ORIGINAL RESEARCH
Effects of polyphenol- rich traditional herbal teas on obesity
and oxidative stress in rats fed a high- fat– sugar diet
Neelam Iftikhar1 | Abdullah Ijaz Hussain1,2 | Shahzad Ali Shahid Chatha1 |
Nazia Sultana1 | Hassaan Anwer Rathore3,4
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2022 The Authors. Food Science & Nutrition published by Wiley Periodicals LLC .
1Department of Chemistry, Government
College University Faisalabad, Faisalabad,
Pakistan
2Central Hi- Tech Lab, Government College
University Faisalabad, Faisalabad, Pakistan
3Department of Pharmaceutical Sciences,
College of Pharmacy, QU Health, Qat ar
University, Doha, Qatar
4Biomedical and Pharmaceutical Research
Unit (BPRU), QU Health, Qatar University,
Doha, Qatar
Correspondence
Hassaan Anwer Rathore, Department
of Pharmaceutic al Sciences, College of
Pharmacy, QU Health, Qatar Universit y,
P.O. Box 2713, Doha, Qatar.
Email: hrathore@qu.edu.qa
Abdullah Ijaz Hussain, Central Hi- Tech
Lab, Government College University
Faisalabad, Faisalabad, Pakistan.
Email: abdullahijaz@gcuf.edu.pk
Funding information
Qatar Universit y
Abstract
Hibiscus rosa- sinensis and Zingiber officinalis teas are traditionally used for the thera-
pies of various diseases, including obesity. The present research work was planned to
appraise the potential of polyphenol- rich extracts of selected herbal plants in obesity
and related biochemical parameters of high- fat– sugar diet- induced obese rats. Three
herbal teas were prepared from Hibiscus rosa- sinensis flowers and Zingiber officinalis
rhizomes and their mixture (3:1, respectively). Total phenolic contents (TPC) of Hibiscus
rosa- sinensis and Zingiber officinalis extracts were found to be 5.82 and 1.45 mg/g of
dry plant material, measured as GAE, while total flavonoid contents (TFC) were 9.17
and 1.95 mg/g of dry plant material, measured as CE, respectively. Two doses (250
and 500 mg/kg BW) of each tea were administered and body weight, BMI, kidney,
liver, and atherogenic indices, TC, TG, HDL, LDL, VLDL, BT, AST, ALT, AP, SC, MDA,
SOD, GSH, and TAC of rats groups were measured. Data showed that higher doses
of Hibiscus rosa- sinensis significantly reduced the rat's BMI (0.50 g/cm2) in compari-
son with the high- fat– sugar diet group (0.79 g/cm2). All treatment groups, especially
H- 500 group, showed a significant decrease in the elevated kidney and liver weights
and atherogenic index in comparison with HFSDC groups. Higher doses of Hibiscus
rosa- sinensis significantly decreased the levels of AST, ALT, AP, and SC in comparison
with the HFSDC group. A significant decrease in the levels of serum TC, TG, LDL,
and VLDL was observed in all the treatment groups in comparison with the HFSDC
group. Furthermore, all the teas, especially higher doses of Hibiscus rosa- sinensis, pre-
vented the alterations in MDA, SOD, and GSH levels of experimental groups, thus
showing the potential against oxidative stress. It can be concluded from these results
that Hibiscus rosa- sinensis teas exhibited strong protective effects against obesity and
oxidative stress, especially at higher doses.
KEYWORDS
BMI, GSH, high- fat diet, kidney index, LDL and HDL, liver index, MDA, nutraceutical, phenolic
acids and flavonoids, SOD

|
699
IFTIK HAR eT A l.
1 | BAC KGROU ND
Incessantly increasing rate of unnecessary weight gain and obe-
sity has become a worldwide concern in the past 20 years (NCD-
RisC, 2019). As per the World Health Organization report, about 650
million people in the world are obese and about 1.9 billion adults are
overweight (World Health Organization, 2017). Obesity is consid-
ered the fifth leading risk factor of mortality as the relative death
rate reaches about 2.8 million every year due to the various com-
plications linked with obesity, including oxidative stress, hypercho-
lesterolemia, hypertension, certain cancers, nonalcoholic fatty liver,
and coronary heart diseases (Gomathi et al., 2008; Goyal & Kadnur,
2006; Gutin, 2020; Saravanan et al., 2014).
Obesity results from excessive deposition of fats in adipose tis-
sue, pancreatic islets, muscles, liver, and other metabolism- involved
organs. Moreover, the frequency of obesity depends on various fac-
tors, including change in lifestyle, genetics, employment and social
status, avoidance of physical activities, and increasing consumption
of high- calorie foods (Goyal et al., 2006). Different practices are in
use, including various types of surgeries, strenuous exercises, use
of herbal formulations, and powerful synthetic drugs such as orli-
stat, rimonabant, and sibutramine to control obesity- related issues
(Chien et al., 2016). However, there are certain difficulties in the
implementation of these therapeutic approaches because of the
busy and easy life routines of the individuals. Furthermore, the high
cost of the conventional drugs, their toxicities, and unavailability in
many rural areas may limit their overall benefits (Chien et al., 2016;
Mahmoud & Elnour, 2013). Hence, convenient and effective anti-
obesity approaches are required to address this issue.
Various medicinal plants and herbs have long been recog-
nized as a chief natural product source and are therapeutically
effective against oxidative stress and obesity (Chien et al., 2016;
Gomathi et al., 2008; Ismail, 2014; Sobhy et al., 2017; Priya &
Veeranjaneyulu, 2016; Sultana et al., 2007). Due to fewer side ef-
fects in comparison with synthetic drugs, herbal teas (hot decoc-
tion, infusions, and herbal drinks), including lemon, hibiscus, ginger,
mint, and cardamom teas, are some of the well- known beverages.
These herbal teas are used not only as refreshment drinks but
also as traditional remedies for various ailments, including obesity
(Chien et al., 2016; Lingesh et al., 2019; Paranjpe et al., 1990; Priya &
Veeranjaneyulu, 2016). Phenolic compounds, due to their antioxidant
potential, have shown various biological and pharmacological activ-
ities (Hussain et al., 2013; Shah et al., 2020). Therefore, polyphenol-
rich plant extracts have gained much interest by pharmacologists
due to various potential biological activities (Shah et al., 2017).
In the present research work, two well- known traditional
teas [Hibiscus rosa- sinensis (Gurhal or Hibiscus tea) and Zingiber
officinale (Adrak or Ginger tea)] were selected after a thorough
ethno- pharmacological survey to investigate, compare, and vali-
date their potential against obesity (Afify & Hassan, 2016; El- Rokh
et al., 2010; Gomathi et al., 20 08; Goyal & Kadnur, 2006; Lingesh
et al., 2019; Paranjpe et al., 1990). A herbal decoction of Hibiscus
rosa sinensis flowers has been traditionally used to reduce body
weight (Lingesh et al., 2019) and Shunth, an ayurvedic formulation
of Zingiber officinale, has been used traditionally for dec ades in the
treatment of obesity (Paranjpe et al., 1990). Some data are avail-
able in the literature covering the antiobesity potential of hibis-
cus and ginger teas (Gomathi et al., 2008; Goyal & Kadnur, 2006).
However, to the best of our knowledge, the effects of Hibiscus rosa-
sinensis and Zingiber officinalis teas in combination and individually
at two incremental doses (250 and 500 mg/kg BW) on reduction
in body weight gain, BMI, and related biochemical parameters in
high- fat– sugar diet- induced obese rats are being reported for the
first time along with complete phenolic profile. Therefore, the aim
of the study was to investigate the phenolic profile and in vivo
antiobesity activity of Hibiscus rosa- sinensis and Zingiber officinale
teas individually and in combination (3:1) using a specific high- fat–
sugar diet- induced obesity model in WKY rats. Body weight (BW),
body mass index (BMI), kidney, liver and atherogenic indices (KI,
LI, AI), biochemical (total cholesterol; triglycerides; high- density
lipoprotein; low- density lipoprotein; very low- density lipoprotein,
bilirubin total; alanine aminotransferase; aspartate aminotransfer-
ase; serum creatinine; alkaline phosphatase) and oxidative stress
(malondialdehyde; superoxide dismutase; reduced glutathione;
total antioxidant capacity) parameters, and histopathological
analysis were performed to confirm the effectiveness of selected
herbal teas.
2 | MATERIALS AND METHODS
2.1 | Collection and identification of plant
materials
The petals of Hibiscus rosa- sinensis L. (hibiscus) and rhizomes of
Zingiber officinale Roscoe (ginger) were collected in the early sum-
mer of 2019 from the Herbal Botanical Garden of Government
College University, Faisalabad, Pakistan. All plant materials were
further identified and authenticated by a taxonomist, by compari-
son with authentic vouchers (Hibiscus rosa- sinensis L. No 230- bot- 19
& Zingiber officinale Roscoe No. 240- bot- 19) of botanical herbarium
of the university. Authenticated samples were stored in polythene
bags and transferred to the Natural Products Research Laboratory,
Government College University, Faisalabad, Pakistan.
2.2 | Reference compounds,
reagents, and chemicals
Phenolic and flavonoid standards (caffeic acid, p- coumaric acid , fer u-
lic acid, chlorogenic acid, gallic acid, sinapic acid, p- hydroxy benzoic
acid, vanillic acid, catechin, myricetin, quercetin, kaempferol), orl-
istat, linoleic acid (60%– 74%), Folin– Ciocalteu reagent, and ascorbic
acid were procured from Sigma Chemical Co. All other chemicals
used were of analytical grade and purchased from Merck, unless
stated otherwise.

700
|
IF TIKH AR eT Al .
2.3 | Preparation of herbal teas
The plant materials were cleaned with distilled water and dried in
a hot air oven for 10 days (IM- 30m Irmeco). The dried plant sam-
ples were ground (mesh size 80) using electric grinder (LG BL 999SP,
Germany) and stored at room temperature (25°C). For the prepara-
tion of tea, a 28- g grinded sample was shaken in 280- ml distilled
water (1:10 ratio) separately for each tea and for the preparation of
combination tea, 21- g hibiscus and 7- g ginger were taken in 280 ml
of distilled water. This mixture was selected on the basis of prelimi-
nary evaluations. Samples were kept in a temperature- controlled
shaker (Gallenkamp) for 24 h at 50°C temperature for continuous
agitation at 180 rpm. Solid residues were separated and the extracts
were dried under reduced temperature and pressure using a rotary
evaporator to get dried extracts. The yield of extracts was calculated
using the formula given below. The material was then saved at −4°C
temperature (Hussain et al., 2013).
2.4 | HPLC analysis for phenolic
acids and flavonoids
Stock solutions of all the standards were newly prepared by dis-
solving 1 mg of each reference compound in 1 ml of methanol.
Working standards (0.4– 100 µg/ml in methanol) were prepared
and the calibration curve of each standard was formed. The hy-
drolysis of herbal tea extracts was done as reported previously
(Hussain et al., 2013). The extracts were passed through a non-
pyrogenic filter (0.45 µm) prior to injection. The HPLC analysis
was performed with Flexar Perkin Elmer System (Perkin Elmer)
equipped with gradient model Flexar pumps system, LC- Shelton
CT, 06484 (USA) UV/Visible detector, column oven and degasser
(DG- 20A5) systems. A hypersil GOLD C18 (250 × 4.6 mm × 5 µm)
column (Thermo Fisher Scientific Inc.) and a nonlinear gradient
(acetonitrile:methanol (70:30) and water with 0.5% glacial acetic
acid) were used. Spectra were recorded at 275 nm and analyses
were identified by matching the retention times and spiking the
samples with standards, whereas quantification was based on an
external standard method.
2.5 | Determination of antioxidant potential of
herbal teas
2.5.1 | Determination of total phenolic (TP) and
total flavonoid (TF) contents
Total phenolic contents (TPC) of selected herbal teas were measured
using Folin– Ciocalteu reagent as reported (Hussain et al., 2013).
The standard curve of gallic acid (10– 80 ppm) was prepared
(y = 0.026x + 0.000, R2 = 0.997) and results were calculated and
reported as mg of phenolic content per gram of dry plant weight,
measured as gallic acid equivalent.
The total flavonoid contents of selected herbal teas were de-
termined using the method reported by Hussain et al. (2013).
The standard curve of catechin (10– 160 ppm) was prepared
(y = 0.006x + 0.015, R2 = 0.999) and results were calculated and
reported as mg of catechin per gram of dry plant weight, measured
as catechin equivalent.
2.5.2 | DPPH radical scavenging assay
2,2- Diphenyl- 1- picrylhydrazyl (DPPH) radical scavenging activity
was performed by the method reported by Hussain et al. (2013).
The extract and BHT solutions (10 µg/ml) were mixed with an equal
volume of 90 μmol/L DPPH solution in methanol. The solution was
incubated for half an hour at 30°C, the absorbance was measured at
517 nm, and the percentage scavenging was calculated as follows:
2.6 | In vivo antiobesity activity
All animal- related experiments were performed with prior approval
and were carried out in agreement with the procedures of the
Institutional Review Board for Animal Studies (Study No. 19680/IRB
No 680), Government College University Faisalabad, Pakistan.
2.6.1 | Animals
Weaning, 3- week- old male Wistar Kyoto (WKY) rats (weighing ap-
proximately 130– 160 g) were purchased from animals’ house of the
University of Veterinary and Animal Sciences, Lahore (UVAAS). Rats
were housed in standard polypropylene cages (41 × 34 × 16 cm) at
25 ± 2°C temperature and 65 ± 5% humidity in a 12- h light/dark
cycle with water ad libitum and standard rat chow freely available to
the animals. The animals were distributed randomly and a maximum
of six rats were kept in each cage.
2.6.2 | Acute oral toxicity study
Organization for Economic Co- operation and Development (OECD)
guidelines- 425 were followed for the acute toxicity study of extract
(OECD, 2001). The rats were fasted 12 h prior to the experiments
with free access to water. The hibiscus and ginger extract were ad-
ministered at doses of 50, 30 0, 500, and 2000 mg/kg/p.o., and the
behavioral change was observed up to 24 h. Both the extracts were
found to be nontoxic up to the maximum dose of 2000 mg/kg body
weight. Doses selected for in vivo antioxidant and antiobesity study
were 250 and 500 mg/kg, respectively.
Yield (
g
100g
)
=Weight of dry extract
Weight of dry plant material ×
100
Scavenging
(%)=
Absorbance of DPPH solution −Absorbance of sample solution
Absorbance of DPPH solution
×
100

|
701
IFTIK HAR eT A l.
2.6.3 | Composition of normal and high- fat– sugar
diet formula
The normal diet (standard rat chow) was purchased locally and con-
tained corn starch (50%), casein (26%), sucrose (9%), cellulose (5%),
corn oil (5%), mineral mixture (4%), and vitamin mixture (1%). The high-
fat and high- sugar diet was prepared in a pellet form and contained
beef tallow (40%), casein (26%), corn starch (15%), sucrose (9%), cel-
lulose (5%), mineral mixture (4%), and vitamin mixture (1%). The high-
fat– sugar diet group animals were also administered coke® (soft drink)
(containing total sugar 10.8 g/100 ml, carbohydrates 10.5 g/100 ml,
and sodium 20 g/100 ml) solution (50:50) in water. Thus, the high- fat
diet was a hyper- caloric diet as compared with the normal diet and
contained more lipids with an energy difference of 4.37 kJ/g. Food
was stored in the dark at 24°C to avoid the oxidation of fat.
2.6.4 | Experimental design
The rats were acclimatized in the animal transit room for 1 week and
were then randomly distributed into the following 11 groups (n = 6
each). The control group was provided water ad libitum, while all
other groups were provided soft drink:water (1:1) solution ad libitum
throughout the study period. The orlistat or water extract solution
of selected plants was given to relevant groups through oral gavage.
Normal Control (NC) group [Received normal feed (approx. 20 g/
rat/day)]
High- fat and sugar diet control (HFSDC) group [Received HFD
(approx. 20 g/rat/day)]
Positive control (PC) group [Received HFSD (approx. 20 g/rat/
day) plus orlistat 250 mg/kg body weight/day for 28 days]
H- 250 group [Received HFSD (approx. 20 g/rat/day) supple-
mented with hibiscus extract (250 mg/kg BW/day for 28 days)]
H- 500 group [Received HFSD (approx. 20 g/rat/day) supple-
mented with hibiscus extract (500 mg/kg BW/day for 28 days)]
G- 250 group [Received HFSD (approx. 20 g/rat/day) supple-
mented with ginger extract (250 mg/kg BW/day for 28 days)]
G- 500 group [Received HFSD (approx. 20 g/rat/day) supple-
mented with ginger extract (500 mg/kg BW/day for 28 days)]
HG- 250 group [Received HFSD (approx. 20 g/rat/day) supplemented
with hibiscus:ginger (3:1) extract (250 mg/kg BW/day for 28 days)]
HG- 500 group [Received HFSD (approx. 20 g/rat/day) supplemented
with hibiscus:ginger (3:1) extract (500 mg/kg BW/day for 28 days)]
2.7 | Observations recorded
2.7.1 | Obesity parameters
Body weight gain (%) and body mass index (BMI) were calculated
as indicators of obesity. Individual body weight of each rat was re-
corded at days 0, 7, 14, 21, and 28 and the average weight gain of
each group was calculated as
BMI was calculated at the end of the experiment by dividing the
rat weight in g by the rat length (from nasal to anal region) in cm2
(Chien et al., 2016).
2.7.2 | Collection of blood sample
At the end of each experiment, the animals were fasted for 12 h but
allowed free access to water. Blood samples were taken (4 ml) from
the right carotid artery, under chloroform anesthesia, into a tube and
centrifuged for 15 min at 30 00 rpm. The clear layer of plasma was
transferred into microcentrifuge tubes, labeled, and stored at −70°C
until biochemical investigation (Ismail, 2014). Upon completion of
blood collection, the animals were euthanized by exsanguinations
under chloroform anesthesia.
2.7.3 | Collection of kidneys and liver
After collecting the blood, kidneys and liver were rapidly dissected and
placed in petri dishes containing normal saline. The organs were cleared
from connective tissues and blood clots, weighed, and then stored in 10%
formalin until histological examination was performed (Chien et al., 2016).
The kidney index (KI) was calculated from body and average kid-
neys’ weights using the equation
Similarly, liver index (LI) was calculated from body and liver
weights using the equation
2.8 | Biochemical investigations
2.8.1 | Estimation of cholesterols
Serum was used for the estimation of following biochemical pa-
rameters using a semi auto analyzer, as reported by Goyal et al.
(2006). Total cholesterol (TC) was estimated by the cholesterol es-
terase method, triglyceride (TGL) was estimated by the glycerol-
3- phosphate oxidase method, high- density lipoprotein (HDL)
cholesterol by the phosphotungstate method using respective di-
agnostic kits obtained from Bayer Diagnostics Ltd, Pakistan. Low-
density lipoprotein (LDL) and very low- density lipoprotein (VLDL)
cholesterols were calculated as per Friedewald's formulae (Goyal
et al., 2006), whereas the atherogenic index was calculated by using
the method described by Muruganandan et al. (2005):
Weight increase
(%)=
Weight at day 28 −Weight at day 0 (g)
Weight at day 0 (g)
×
100
KI
(%)=
Average kidney weight (g)
Rat weight (g)
×
100
LI
(%)=
Liver weight (g)
Rat weight (g)
×
100

702
|
IF TIKH AR eT Al .
2.8.2 | Estimation of liver and kidney functions
Serum creatinine and alkaline phosphate were determined as indica-
tor of kidney function, while serum alanine aminotransferase (ALT),
aspartate aminotransferase (AST), and bilirubin total (BT) levels
were measured as indicators for liver function. All these biochemical
assays were performed on an auto analyzer (Opera, Techicon, Bayer,
and USA) (İşeri et al., 2007).
2.8.3 | Estimation of oxidative stress parameters
The oxidative status of the animals was determined by conducting a
series of tests from the serum samples. Oxidative damage of lipids
was assessed by estimating the lipid peroxidation product, malon-
dialdehyde (MDA), as reported by Ohkawa et al. (1979) with minor
changes. Both nonenzymatic and enzymatic defense against oxida-
tive stress was studied by measuring reduced glutathione (GSH) and
superoxide dismutase (SOD) levels using reported protocols with
minor modifications (Kakkar et al., 1984). The sum of endogenous
antioxidant activity was determined by estimating the total antioxi-
dant capacity (TAC) using the method of Miller et al. (1993).
2.9 | Histopathology of liver and kidney tissues
Collected tissues, such as liver and kidney of all the groups, were
placed in formalin solution (10%) for 3– 5 days. Thereafter, tissues
were subjected to overnight washing in running tap water to remove
tissues from fixative. To remove water from tissues, dehydration
process was carried out in serial dilutions of ethyl alcohol followed
by clearing with xylene. The tissues were embedded in paraffin wax
to make blocks in a special plastic. Tissues were cut into thin sec-
tions (5– 15 µm thickness) using a microtome. Mayer's egg albumin
was used for mounting the tissue sections on labeled glass slides.
Finally, the stained slides were observed using a light microscope for
morphological studies.
2.10 | Statistical analysis
All procedures were performed in three replicates and the results
are presented as mean ± standard deviation of three independent
experiments. Statistical analysis was performed by means of the
statistical package, STATISTICA (Stat Sift Inc.). Data from different
tests were analyzed using one- way and two- way analysis of variance
(ANOVA) followed by the Bonferroni/Dunnett (all mean) post hoc
test and the differences between the means were considered statis-
tically significant at p ≤ .05 (Hussain et al., 2013). Linear regression
analysis and analysis of covariance (ANCOVA) were performed by
using SPSS (Version 16).
3 | RESULTS AND DISCUSSION
3.1 | Extract yields and in vitro antioxidant
potentials
Aqueous extract yields (g/100 g) of Hibiscus rosa- sinensis and
Zingiber officinalis on dry plant material basis are given in Table 1.
The maximum yield of extract (18.41 g/100 g) was found from
Hibiscus rosa- sinensis and the minimum from Zingiber officinalis
(10.22 g/100 g). Aqueous extract yield depends on the amount of
extractable polar compounds in plant materials (Sobhy et al., 2017;
Sultana et al., 2007).
Total phenolic (TP) and total flavonoid (TF) contents of both
the herbal extracts are presented in Table 1. The aqueous extract
of Hibiscus rosa- sinensis showed a higher concentration of TPC
LDL cholesterol (mg
dL
)
=Total serum cholesterol −
HDL cholesterol
−Total serum triglycerides
5
VLDL cholesterol (mg
dL )
=
Total serum triglycerides
5
Atherogenic index
=
Total serum cholesterol −HDL cholesterol
HDL cholesterol
Assays
Herbal Teas
BHTHibiscus Ginger
Extract yield (g/100 g) 18. 41 ± 0.55b10.22 ± 0. 51a- - -
TPC (mg/g of dry plant material, measured
as gallic acid equivalent)
5.82 ± 0.33b1.45 ± 0.08a- - -
TFC (mg/g of dry plant material, measured
as catechin equivalent)
9.17 ± 0.90b1.95 ± 0.13a- - -
DPPH radical scavenging activity (%)
exhibited by 10 µg/ml
59. 0 ± 2.0b53.4 ± 1.4a8 9.1 ± 2.2c
Note: Values are mean ± SD of three independent experiments.
Different superscript letters in the same row represent significant (p ≤ .05) difference among
hibiscus, ginger teas, and synthetic antioxidant (BHT).
TABLE 1 Extract yield, total phenolic,
total flavonoid contents, and radical
scavenging capacity of different herbal
teas

|
703
IFTIK HAR eT A l.
(5.82 mg/g of dry plant material measured as gallic acid equiva-
lent) and TFC (9.17 mg/g of dry plant material, measured as cate-
chin equivalent). Zingiber officinalis extract showed 1.45 mg/g and
1.95 mg/g TPC and TFC, respectively. Free radical scavenging ac-
tivity of various herbal extract solutions (10 µg/ml) was measured
by the DPPH radical scavenging assay and the results are presented
in Table 1. Hibiscus rosa- sinensis tea exhibited more radical scaveng-
ing activity (59.0%) than ginger tea (53.4%). When compared with
the synthetic antioxidant butylhydroxytoluene (BHT) (89.1%), herbal
teas offered lower radical scavenging capacity. The statistical analy-
sis showed that the difference in TRC, TFC, and DPPH radical scav-
enging capacity of herbal teas was significant (p ≤ .05).
Phenolic compounds are present in almost every plant and have
physiological and morphological importance due to their antioxi-
dant potentials (Kanatt et al., 2014). Hence, it is essential to quantify
phenolic contents and to assess its contribution against oxidative
stress (Oh et al., 2013). Afify and Hassan (2016) evaluated TPC and
TFC from aqueous hibiscus flowers extract, which were 2.35 and
0.31 mg/g of flowers, respectively. Tohma et al. (2017) reported
52.8 µg TPC per mg GAE and 3.9 µg TFC per mg quercetin equiv-
alent in water extract of ginger. The variation in the TPC and TFC
compared with the findings of the previous study might have been
due to the differences in agro- climatic, geographical, and seasonal
conditions. DPPH free radical scavenging capacity increases when
the extract concentration increases due to an increase in the con-
centration of phenolic compounds (Sultana et al., 2007).
3.2 | HPLC analysis for phenolic
acids and flavonoids
Phenolic acids and flavonoids were quantified using the RP- HPLC
method. The developed HPLC method could separate 15 phenolic
acids and four flavonoids simultaneously within 25 min at a flow rate
of 0.8 ml/min (Figure 1). The concentration (mg/100 ml of herbal
tea) of 15 phenolic acids, including gallic acid, chlorogenic acid, sali-
cylic acid, caffeic acid, 4- hydroxy benzoic acid, arbutin, p- coumaric
acid, syringic acid, vanillic acid, sinapic acid and ferulic acid, ellagic
acid, cinnamic acid, benzoic acid, tannic acid, and three flavonoids,
including catechin, myricetin, and quercetin in Hibiscus rosa- sinensis
and Zingiber officinalis tea is presented in Table 2. Gallic acid (55.21
and 17.32 mg/100 ml) was the major phenolic acid found in hibis-
cus and ginger teas and catechin (78.25 and 102.8 mg/100 ml) was
the major flavonoid followed by rutin (15.34 and 83.37 mg/100 ml).
Hibiscus rosa- sinensis extract also contained chlorogenic acid
(14.39 mg/100 ml) and caffeic acid (8.43 mg/100 ml), while ginger
tea contained 4- hydroxybenxoic acid (24.18 mg/100 ml), salicylic
acid (8.99 mg/100 ml), and arbutin (7.66 mg/100 ml). The variation
in the contents of phenolic acids and flavonoids was found to be
significant (p ≤ .05) between the two teas.
Flowers, due to accumulation of phenolic compounds, possess
comparatively higher amounts of flavonoid contents than other
plant parts. Purushothaman et al. (2016) reported that the methanol
extract of hibiscus flower contained quercetin (7.6 μg/g), kaempferol
FIGURE 1 Typical HPLC chromatogram
showing the separation of phenolic acids
and flavonoids from hibiscus extracts

704
|
IF TIKH AR eT Al .
(361 .9 μg/g), and myricetin (50.7 μg/g). Tohma et al. (2017) identified
eight different phenolic acids in the water extract of ginger, among
which p- hydroxybenzoic acid (321.6 mg/kg), ferulic acid (88.8 mg/
kg), and p- coumaric acid (291.4 mg/kg) were more abundant in ly-
ophilized aqueous ginger extract. Our results are also in agreement
with findings in the literature and some variation might be due to
variation in extraction techniques, drying parameters, geographical,
and seasonal conditions of the samples.
3.3 | Effect of herbal teas on various parameters of
high- fat– sugar diet- induced obesity model
3.3.1 | Effect on body weight
The initial and final body weight, percent increase in body weight,
and BMI of control and treated rat groups are presented in Table 3.
The high- fat and sugar diet control (HFSDC) group showed a sig-
nificant (p ≤ .05) increase in the rats’ body weight and BMI, thus
indicating the effectiveness of the high- fat– sugar diet- induced obe-
sity model. The HFSDC group presented an increase of 99.30% of
body weight when compared with the normal diet control group
(40.64%). The BMI of the HFSDC group was 0.79 g/cm2, which was
significantly higher (p ≤ .05) than that of the NC group (0.60 g/cm2).
The results revealed that the oral administration of all herbal teas
and orlistat drug significantly (p ≤ .05) decreased the percent in-
crease in body weight and BMI in all the treatment groups. Among
all treatment groups, the higher dose of hibiscus (H- 500) showed a
less increase in body weight (57.14%) as compared with the HFSDC
group and also less BMI (0.50 g/cm2), and results are comparable to
the positive control group, that is, orlistat group (PC), while the lower
dose of ginger (G- 250) showed the least protective effect against
obesity (Table 3).
A higher dose of mixed tea (hibiscus and ginger 3:1) showed a
57.82% increase in body weight and 0.53 g/cm2 BMI, thus show-
ing more protective effect than ginger tea alone. Overall, the higher
doses of extracts showed a better antiobesity potential as compared
with lower doses in terms of BMI and weight gain.
The cause of obesity is manifold, but the most common cause
is still the dietary factor, especially the consumption of high-
fat and sugar diet (Chien et al., 2016). BMI, which is defined as
“weight in kg/square of height in meters (kg/m2),” is an important
parameter to assess the weight gain and obesity (WHO, 2017). A
BMI of 30 kg/m2 or higher is normally considered obese, while
25 kg/m2 or higher BMI is considered overweight (WHO, 2017).
Consumption of HFSD increases the fat mass due to the augmen-
tation of triglyceride storage in adipose tissues after fatty acid
synthesis in the liver (Hernández- Saavedra et al., 2016). Increase
in the body we igh t in hig h- fat and su gar di et rats gr oup of th e pres-
ent study may be due to the consumption of diet rich in energy in
the form of saturated fats and high calorie drinking water which
would have resulted in fat deposition in various body fat pads of
rats. According to Velez- Carrasco et al. (2008), 5%– 10% reduction
in body weight can have a significant effect on the health status.
However, the reduced body weight in orlistat- treated groups is
because of a selective reduction in body fat, leaving lean mass
unchanged (Mahmoud & Elnour, 2013). The results of the pres-
ent study are in line with the findings of Nammi et al. (2009)
who reported that ethanol extract of Z. officinalis (100– 400 mg/
kg) significantly suppresses body weight gain by 17%– 23% after
3– 4 weeks compared with the high- fat diet group. In another
study, Nazish et al. (2016) reported that administration of 20 mg/
kg aqueous extract of Z. officinalis significantly decreases the body
weight of rats fed a high- fat diet. Similarly, the aqueous extract of
ginger (200 mg/kg) produced an antiobesity effect in obese di-
abetic rats (Ismail, 2014). Mahmoud and Elnour (2013) reported
that the administration of 5% ginger powder to rats fed a high- fat
diet significantly decreases body weight of rats. The reduction in
the rat body weight due to herbal extract treatment may be due
to the inhibitory action of ginger on the absorption of dietary fats
(Gerald et al., 2008). No study is available in the literature on the
antiobesity effect of Hibiscus rosa sinensis tea on rat body weight
reduction; however, the antiobesity effect of Hibiscus sabdariffa
aqueous extract was demonstrated by the significant reduction in
weight gain between the treated and nontreated rat groups, which
was dose dependent (Omar et al., 2018).
TABLE 2 Contents of phenolic acids and flavonoids identified
from aqueous herbal extract by Rp- HPLC
Compounds
Concentration (mg /100 ml of tea)
Hibiscus Ginger
Tannic acid ND 0.21 ± 0.01
Arbutin 1.39 ± 0.05a7. 6 6 ± 0.40b
Gallic acid 55.21 ± 2.70d1 7. 32 ± 0.53a
4- Hydroxybenzoic acid 3.60 ± 0 .19b24.18 ± 1.12d
Catechin 78. 25 ± 3.92 a102.8 ± 3.94b
Chlorogenic acid 14.39 ± 0.70 b8.14 ± 0.42a
Caffeic acid 8.43 ± 0.41d0.83 ± 0.05a
Syringic acid 1.83 ± 0.04c1.49 ± 0.06b
Vanillic acid 1.24 ± 0.07b0.95 ± 0.04a
p- Coumaric acid 3.92 ± 0 .17b2.63 ± 0.11a
Salicylic acid 0.39 ± 0.02a8.99 ± 0.34d
Rutin 15.34 ± 1.01a83. 37 ± 3.39c
Sinapic acid 0.23 ± 0.02a0.22 ± 0.01a
Ferulic acid 1.35 ± 0.05c0.17 ± 0.01a
Ellagic acid 0.89 ± 0.06a1.6 3 ± 0.06c
Cinnamic acid 0.19 ± 0.01a0.33 ± 0.02b
Benzoic acid 4.72 ± 0.30cND
Myricetin 0.20 ± 0.01aND
Quercetin 1.74 ± 0.07aND
Note: Values are mean ± SD of three independent experiments.
Different superscript letters in the same row represent significant
(p ≤ .05) difference between hibiscus and ginger teas.

|
705
IFTIK HAR eT A l.
3.3.2 | Effect on organ weights
Liver and kidney weights of the control, HFSDC, and all the treat-
ment groups were recorded and are presented in Table 3. The
HFSDC group showed a significant increase (p ≤ .05) in liver and kid-
ney weights as compared with the NC group. The higher doses of all
herbal extracts (500 mg/kg) significantly reduced the organ weights
and kidney and liver indices, whereas lower (250 mg/kg) doses did
not show significant changes in this aspect. To our knowledge, none
of the previous studies have reported the effects of Hibiscus rosa
sinensis tea/extract on the kidney and liver indices. However, Nazish
et al. (2016) reported a decrease in the weight of liver and kidney in
the Z. officinalis- treated albino rat group as compared with control
and high- fat diet- fed rat groups.
3.3.3 | Effect on serum lipid profile
The lipid profile of NC, HFSDC, and all the treatment groups is
presente d in Figure 2. The HFSDC group had significantly (p ≤ .05)
increased serum levels of TC (106.3 mg/dl), TG (96.0 mg/dl),
LDL (62.8 mg/dl), and VLDL (19.2 mg/dl) and had decreased lev-
els of HDL (24.3 mg/dl), when compared with the NC and treat-
ment groups. All the treatment groups showed the protective
effect as measured by levels of serum TC, TG, LDL, VLDL, and
HDL. Maximum protection effect was shown by hibiscus extract
(500 mg/kg BW) and TC, TG, HDL, LDL, and VLDL levels of H- 500
group were found to be 82.4, 55.4, 30.4, 40.9, and 11.1 mg/dl, re-
spec tive ly, which were compar able to the PC grou p. Lowe r dos e of
gi n ger extrac t show e d th e least prot e c t ive effe ct and TC , TG, HDL,
LDL, and VLDL levels of G- 250 group were found to be 84.4, 89.0,
22.4, 44.2, and 17.8 mg/dl, respectively. The atherogenic index of
HFSDC was significantly (p ≤ .05) increased than that of the NC
group and all the treatment groups significantly (p ≤ .05) reduced
the atherogenic index value of all the groups, which showed the
protective effect of hibiscus, ginger, and mixed teas at both 250
and 500 mg/kg doses (Table 4). Overall, all the higher doses of
extracts showed a better protective effect than the lower doses.
There are some reports available in the literature on the effect
of herbal teas on blood cholesterols levels (El- Rokh et al., 2010;
Gomathi et al., 2008; Saluja et al., 1978). The results of the present
study are similar to the findings of El- Rokh et al. (2010) and Ismail
(2014), who found that the aqueous extracts of ginger lowered
serum total cholesterols, triglycerides, and low- density lipoprotein.
The TG- lowering effect of ginger may be due to its ability to en-
hance lipase activity (El- Rokh et al., 2010). Gomathi et al. (2008)
and Sikarwar and Patil (2011) reported that the flowers of hibiscus
significantly reduced the LDL and VLDL cholesterols and increased
the HDL cholesterol level. Hibiscus rosa- sinensis reduced the LDL
cholesterol of monosodium glutamate- induced obesity group from
46.75 to 22.92 mg/dl (Gomathi et al., 20 08). Investigated teas are
rich in flavonoids and phenolic acids and it has been reported that
the hypolipidemic effect of various herbal extracts may be related
to the ability of various phenolic acids and flavonoids by stimula-
tion of thermogenesis and decreased fat accumulation, which can
be related to their pancreatic lipase inhibitory activity (Hernández-
Saavedra et al., 2016). Moreover, flavonoids also inhibit the absorp-
tion of dietary cholesterol and decrease in serum cholesterol (Saluja
et al., 1978). High levels of LDL and TC increase the risk of coronary
heart diseases, while an increase in HDL cholesterol is supportive in
transporting overloaded cholesterol to the liver for excretion in the
bile (Saravanan et al., 2014). The increase in LDL cholesterol may be
due to the reduced expression or activity of the LDL- receptor sites
in response to a high- fat diet treatment (Nammi et al., 2009). The
TABLE 3 Effect of herbal tea and orlistat on the body, kidney and liver weights, body mass, kidney and liver indices of different groups of
obesity rat model
Groups
Body weight
BMI (g/cm2)
Kidney
weight (g) Kidney index (%)
Liver weight
(g) Liver index (%)Initial (g) Final (g) Increase (%)
NC 155 ± 15 218 ± 12 40.64#0.6 0 ± 0.05#1.56 ± 0.24 0.71 6.92 ± 1.11 3.17#
HFSDC 143 ± 14 285 ± 12 99. 3 0 * 0.79 ± 0.05* 1.74 ± 0.17 0. 61* 10.1 ± 1.00 3.54*
PC 145 ± 11 228 ± 21 5 7. 24*#0.57 ± 0.05#1.57 ± 0. 23 0.68#8.13 ± 1.13 3.45
H - 2 5 0 132 ± 14 226 ± 18 71. 2 1*#0 .51 ± 0.03*#1.57 ± 0.23 0.64 7.76 ± 1.01 3.43
H - 5 0 0 147 ± 13 231 ± 11 57.14 *#0.50 ± 0.04*#1.55 ± 0.25 0.67 6. 24 ± 1.09 3.41#
G - 2 5 0 146 ± 10 264 ± 16 80.82* 0.66 ± 0.05#1.71 ± 0 .18 0.63 8.69 ± 1.04 3.4 6*
G - 5 0 0 149 ± 13 244 ± 10 63.75*#0.61 ± 0.05#1.64 ± 0.21 0.66 7.94 ± 1.11 3.41#
H G - 2 5 0 147 ± 12 259 ± 11 76.19*#0. 59 ± 0.04#1 .59 ± 0 .10 0. 61 8.93 ± 0.88 3.45*
H G - 5 0 0 147 ± 11 232 ± 11 57. 8 2*#0.53 ± 0.01*#1.48 ± 0.20 0.64 7.8 8 ± 1.00 3.40#
Note: Values are mean ± standard deviation of six rats of the same group.
Abbreviations: BMI, body mass index; G- 250 and G- 500, ginger 250 and 500 mg/kg BW; H- 250 and H- 500, hibiscus 250 and 500 mg/kg BW;
HFSDC, high- fat and sugar diet control; HG- 250 and HG- 500, hibiscus:ginger (3:1) 250 and 500 mg/kg BW; NC, normal control.
PC, positive control.
*Significant (p ≤ .05) dif ference compared with NC.
#Significant (p ≤ .05) dif ference compared with HFSDC among all the groups.

706
|
IF TIKH AR eT Al .
increase in LDL- cholesterol may be due to the reduced expression
or activity of the LDL- receptor sites in response to treatment groups
(Nammi et al., 2009). Therefore, lowering the LDL- cholesterol level
may be an important factor in lowering the serum total cholesterol
level in rats fed a high- fat diet. The reduction of LDL- cholesterol by
herbal teas treatments could be due to prevention of the suppres-
sive action of high- fat diet on the LDL- receptor site. The change in
lipid profile levels induced by a high- fat diet might be due to the
activation of gastric lipases and enhanced intestinal fat absorption,
while an increase in triglyceride levels was due to the dietary cho-
lesterol that reduced fatty acid oxidation which, in turn, increased
the levels of hepatic triglycerols (Saravanan et al., 2014). The more
the atherogenic index, the higher is the risk of oxidative damage of
kidneys, liver, heart, aorta, and coronaries due to fatty infiltration,
plaque, foam cells, and/or lipids (Mehta et al., 2003).
3.3.4 | Effect on plasma levels of liver and
kidney enzymes
Liver parameters like bilirubin total (BT), alanine aminotransferase
(ALT), aspartate aminotransferase (AST) and kidney parameters like
serum creatinine (SC) and alkaline phosphatase (AP) of all the control
and treatment groups were studied and are presented in Table 4. Rats
fed on HFSD for 4 weeks had significantly increased serum levels
of AST and decreased total bilirubin level, when compared with the
normal control group, thus showing the effectiveness of the model.
A decline in the levels of ALT and AST and an increase in the BT were
recorded in all the treatment groups. The major effect appeared in
the H- 500 group when compared with the PC group. Alkaline phos-
phate (AP) and serum creatinine (SC) significantly (p ≤ .05) increased
in the HFSDC group in comparison with the control group. All the
extracts showed a protective effect and the increased levels of the
AP and SC decreased in all treated groups. The maximum protective
effect was recorded in H- 500 groups which were comparable to the
PC group (Table 4). Ginger extract showed a better effect in the SC
level as compared with others.
In obesity, generally, oxidative stress is increased causing an
increase in the bilirubin consumption leading to reduced serum
bilirubin level (Karadag et al., 2017). Ismail (2014) also reported
that the ginger water extract significantly decreased the elevated
serum levels of liver enzymes (ALT, AST) in obese rats. Sanadheera
et al. (2021) reported decreased ALT level in pregnant diabetic
Wistar rats fed Hibiscus rosa- sinensis aqueous extract at a dose of
100– 400 mg/kg. In another study, Jenko- Pražnikar et al. (2013)
reported that the serum bilirubin levels were negatively associated
with abdominal obesity. Chang et al. (2012) reported that serum bil-
irubin levels were inversely associated with LDL, TC, and TG and
positively associated with HDL.
3.3.5 | Effect on oxidative stress parameters
The effects of hibiscus, ginger, and mixed extracts on the levels
of malondialdehyde (MDA), superoxide dismutase (SOD), reduced
glutathione (GSH), and total antioxidant capacity (TAC) of all the
treatments and control groups were assessed and are presented in
Table 5. The level of MDA in the serum of rats of high- fat and sugar
diet control (HFSDC) group was 6.97 nmol/L, which is significantly
(p ≤ .05) higher than that of the NC group (2.73 nmol/L), thus show-
ing the oxidative stress in tested animals. All the treatment groups,
including the PC group, showed a significant (p ≤ .05) decrease in
the alleviated levels of MDA. Daily administration of hibiscus, gin-
ger, and mixed teas effectively prevented the generation of MDA in
rat groups and the best effect (3.17 nmol/L) was shown in hibiscus:
ginger- 500 group (500 mg/kg BW/d) followed by hibiscus- 500 group
(3.19 nm ol/L) and ginger- 50 0 grou p (3 .29 nmo l/L). The prote ctive ef-
fect is even better than that of the PC group (3.21 nmol/L). The serum
SOD level in the HFSDC group was found to be 120.4 U/ml, which
is significantly (p ≤ .05) lesser than that of the NC group (158.9 U/
ml). Maximum protective effect was observed in the HG- 500 group
followed by H- 500 group and the levels of SOD were found to be
142.2 and 135.2 U/ml, respectively. A significant (p ≤ .05) reduction
in the level of GSH was recorded in the HFSDC group (123.2 mg/L)
as compared with the NC group (160.3 mg/L), which leads to oxi-
dative stress. All the treatment groups showed the protective ef-
fect against oxidative stress, and the level of GSH in H- 500 group
was 167.6 mg/L followed by the HG- 500 group (161.4 mg/L), H- 250
FIGURE 2 Effect of treatment on
the lipid profile of different rat groups.
HDL, high- density lipoprotein; LDL,
low- density lipoprotein; TC, total
cholesterol; TG, triglycerides; VLDL,
very low- density lipoprotein. *Significant
(p ≤ .05) difference compared with NC.
#Significant (p ≤ .05) difference compared
with HFSDC among all the groups
0
20
40
60
80
100
120
140
NC HFSDCPCH-250 H-500G-250 G-500 HG-250 HG-500
Lipid Profile (mg/dl)
Groups
TC(mg/dl) TG(mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl)
*
**
**
*
*
*
*
*
****
**
*
##
#
#
#
##
#
#
#
#
#
#
#
#
#

|
707
IFTIK HAR eT A l.
group (161.1 mg/L), and the G- 500 group (158.9 mg/L). The total an-
tioxidant capacity was significantly (p ≤ .05) de creas ed in the HFSDC
group (1.36 mmol/L) as compared with the NC group (1.93 mmol/L).
The TAC of different treatment groups is mentioned in Table 5. All
the treatment groups, except G- 250, significantly (p ≤ .05) increased
the TAC values. The best protective effect was shown by mixed tea
groups (HG- 500 and HG- 250) and the value of TAC was 1.85 and
1.71 mmol/L, respectively. The H- 500 group also showed an in-
crease in the TAC value (1.83 mmol/L).
Lipid peroxidation is a key biomarker of evaluation of oxidative
stress and the determination of MDA reflects the degree of lipid
peroxidation, and that indirectly reflects cellular damage (Hosen
et al., 2015). The alleviation in the level of MDA in the HFSDC group
may be due to the lipid peroxidation that leads to oxidative stress
and the decrease in the MDA level in the treatment groups may be
due to the antioxidant potential of hibiscus and ginger teas that re-
tard lipid peroxidation. SOD is metalloenzyme that catalyzes the dis-
mutation of superoxide radicals and decreased levels of SOD show
TABLE 4 Effect of herbal tea and orlistat treatment on the biochemical parameters of different groups of obesity rat model
Groups
Liver parameters Kidney parameters
Atherogenic
indexBT (mg/dl) AST (µ/L) ALT) (µ/L) AP (µ/L) S C (mg /dl)
NC 0.43 ± 0.05#63.0 ± 5.0#81. 2 ± 5.3 143 ± 12#0.43 ± 0.06#1.37 ± 0.08#
HFSDC 0.26 ± 0.02* 98.4 ± 4.8* 82 .1 ± 4.0 164 ± 9*0.55 ± 0.04* 3.37 ± 0.1 5*
PC 0.37 ± 0.03#62. 5 ± 5.8#62 .1 ± 3.9*#152 ± 11 0.41 ± 0.06#1.63 ± 0.10*#
H - 2 5 0 0.33 ± 0.03*#79.9 ± 3.2*#74.7 ± 4.6 146 ± 15 0.46 ± 0.03#2.16 ± 0.11*#
H - 5 0 0 0.38 ± 0.03#66.6 ± 2.6#69. 0 ± 3.1*#142 ± 11#0.45 ± 0.04#1.71 ± 0. 11*#
G−250 0.37 ± 0.03#8 7. 3 ± 4.7*#7 9.0 ± 4.4 147 ± 16 0.43 ± 0.03#2 .77 ± 0.18 *#
G - 5 0 0 0.41 ± 0.03#75. 5 ± 3.3*#78.6 ± 2.8 146 ± 15 0.38 ± 0.03#2 .05 ± 0.19*#
H G - 2 5 0 0.35 ± 0.03#82.0 ± 4.1*#7 7.1 ± 4.0 148 ± 11 0.44 ± 0.06#2.44 ± 0.10*#
H G - 5 0 0 0.38 ± 0.04#70.3 ± 6.0#70. 3 ± 2.7*#14 3 ± 10 #0.39 ± 0.03#1.99 ± 0.09*#
Note: Values are mean ± standard deviation of six rats of the same group.
Abbreviations: ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BT, bilirubin total; G- 250 and G- 500,
ginger 250 and 500 mg/kg BW; H- 250 and H- 500, hibiscus 250 and 500 mg/kg BW; HFSDC, high- fat and sugar diet control; HG- 250 and HG- 500,
hibiscus:ginger (3:1) 250 and 500 mg/kg BW; NC, normal control; PC, positive control; SC, serum creatinine.
*Significant (p ≤ .05) dif ference compared with NC.
#Significant (p ≤ .05) dif ference compared with HFSDC among all the groups.
Groups MDA (nmol/L) S OD (U/ml) GSH (mg/L)
TAC
(mmol/L)
NC 2.73 ± 0.20#158.9 ± 8 .1#160 .3 ± 11 .0 #1.93 ± 0.20#
HFSDC 6.97 ± 0 .41* 120.4 ± 9.0* 123.2 ± 10.0* 1.36 ± 0.09*
PC 3.21 ± 0.18*#1 37.1 ± 7. 7 * 148.9 ± 9. 31 #1.71 ± 0.19#
H - 2 5 0 3.34 ± 0.32*#135.0 ± 9.4* 161 .1 ± 10 .8 #1.68 ± 0.09#
H - 5 0 0 3.19 ± 0.19*#13 9. 3 ± 8.3*#16 7. 6 ± 10.0#1.83 ± 0.16 #
G - 2 5 0 3.41 ± 0.24*#1 29.1 ± 9. 5* 146.3 ± 9.01#1.49 ± 0.08*
G−500 3.29 ± 0.38*#135.2 ± 9.0* 158.9 ± 13.7#1.68 ± 0 .18#
H G - 2 5 0 3.32 ± 0. 21*#136.7 ± 9. 6* 145.8 ± 9.13#1.71 ± 0.20#
H G - 5 0 0 3.17 ± 0. 27*#142 .2 ± 8.2#161.4 ± 10 .7#1.87 ± 0.19#
Abbreviations: G- 250 and G- 500, ginger 250 and 500 mg/kg BW; GSH, reduced glutathione; H-
250 and H- 500, hibiscus 250 and 500 mg/kg BW; HFSDC, high- fat and sugar diet control; HG- 250
and HG- 500, hibiscus:ginger (3:1) 250 and 50 0 mg/kg BW; MDA, malondialdehyde; NC, normal
control; PC, positive control; SOD, superoxide dismutase; TAC, total antioxidant capacity.
*Significant (p ≤ .05) dif ference compared with NC.
#Significant (p ≤ .05) dif ference compared with HFSDC among all the groups.
TABLE 5 Effect of herbal tea and
orlistat treatment on the oxidative stress
parameters of different groups of obesity
rat model
FIGURE 3 Histopathological microscopic image showing the morphological changes of (a) liver and (b) kidney of different rat groups.
G- 250 and G- 500, ginger 250 and 500 mg/kg BW; H- 250 and H- 500, hibiscus 250 and 500 mg/kg BW; HFSDC, high- fat diet control; NC,
normal control; PC, positive control

708
|
IF TIKH AR eT Al .
HFSDCPC
PCHFSDC
H-250
(a)
(b)

|
709
IFTIK HAR eT A l.
the sign of oxidative stress (Wang et al., 2016). GSH is an important
antioxidant present in the body and plays a role in tissue injury and
damage due to free radicals and peroxides (Hosen et al., 2015). SOD
and GSH levels were decreased significantly in the HFSDC group
as compared with the normal control group. This drop in SOD and
GSH levels indicates oxidative stress in the rats due to destruction
of H2O2 clearance and validates the notion of hydroxyl radical (•OH)
formation (Hosen et al., 2015; Wang et al., 2016). All the treatment
groups including the herbal extract gro ups restored the reduced lev-
els of SOD and GSH, thus showing the potential against oxidative
stress and refurbishment of lipid peroxidation. Previous reports also
presented that oxidative stress distorted the activity of the endog-
enous enzymes which play a major role in scavenging toxic free rad-
icals (Bakır et al., 2016; Hosen et al., 2015). TAC is the total effect
of various antioxidant compounds and their systemic interactions.
Clinically, TAC has been widely used to assess oxidative stress and
serum antioxidant depletion (Rice- Evans & Miller, 1997). Decreased
TAC in the HFSDC group rats may be because of either increased
oxidative stress or decreased availability of antioxidants. This imbal-
ance was restored with the administration of hibiscus, ginger, and
mixed teas, through decreasing free radical generation and increas-
ing antioxidant levels.
3.4 | Histopathological evidence
An acute study showed that all the animals were healthy, agile, with
no redness of the eyes, no vocalization, no signs of loss of hair, no
moribund and hunchbacked signs after oral administration of ex-
tracts. Microscopic examination of the liver and kidney tissues of
rats of all groups showed regular architectures with unnoticeable
differences in the histological and cellular structures of all the or-
gans except rats of the HFSDC group (Figure 3). Livers of all the
treatment groups showed no ballooning, nuclei were of normal
shape, and no inflammatory cells were present. Similarly, kidneys of
all the groups showed normal glomerulus, tubules, and parenchyma.
Mild ballooning and fat droplets were observed in ballooned hepato-
cytes in the HFSDC group. Histopathological evaluation of biopsy
specimens remains the authentic and reproducible diagnostics tool
for the fatty liver or other organs (Takahashi & Fukusato, 2014).
Hepatocellular ballooning is a sign of hepatocellular injury and is il-
lustrated as swollen hepatocytes with rarefied cytoplasm (Takahashi
& Fukusato, 2014).
4 | CONCLUSION
In conclusion, the use of hibiscus and ginger teas individually and in
mixture form at doses of 250 and 500 mg/kg body weight showed
a marked reduction in weight gain and oxidative stress in rats fed
a high- fat and high- sugar diet. The 500 mg/kg dose showed more
pronounced effects, which were comparable to the antiobesity drug
(orlistat). The investigated teas contained a rich source of phenolic
acids and flavonoids, especially gallic acid and catechin. Based on
these results, it is concluded that selected herbal infusions may
have a therapeutic potential and can be used as antiobesity agents.
Further studies should investigate the effects of individual flavo-
noids and phenolic acids on the biochemical parameters in obesity
and elucidate the mechanism of action at the molecular level.
ACKNOWLEDGEMENTS
The authors acknowledge the services provided by the Central Hi-
Tech Lab, Government College University Faisalabad, Pakistan for the
characterization of compounds and Department of Pharmaceutical
Sciences for assistance in in vivo analysis. In addition, the authors ac-
knowledge the startup research grant provided by Qatar University
to the main corresponding author, Hassaaan A. Rathore. The publi-
cation of this article was funded by the Qatar National Library.
CONFLICT OF INTEREST
The authors declare that they do not have any conflict of interest.
ETHICAL APPROVAL
All animal- related experiments were performed with the prior ap-
proval of the Institutional Review Board for Animal Studies (Study
No. 19680/IRB No 680), Government College University Faisalabad,
Pakistan.
DATA AVAILAB ILITY STATE MEN T
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Neelam Iftikhar https://orcid.org/0000-0002-9861-3184
Shahzad Ali Shahid Chatha https://orcid.
org/0000-0002-5913-2281
Hassaan Anwer Rathore https://orcid.org/0000-0002-1154-9395
REFERENCES
Afify, A. E. M. R ., & Hassan, H. M. M. (2016). Free radical scavenging
activity of three different flowers: Hibiscus rosa- sinensis, Quisqualis
indica and Senna surattensis. Asian Pacif ic Journal of Tropical
Biomedicines, 6, 771– 777.
Bakır, M., Geyikoglu, F., Colak, S., Turkez, H., & Bakır, T. O. (2016).
Hosseinigouzdagani, M. The carvacrol ameliorates acute
pancreatitis- induced liver injury via antioxidant response.
Cytotechnology, 68, 1131– 1146.
Chang, Y., Ryu, S., Zhang, Y., Son, H. J., Kim, J. Y., Cho, J., & Guallar, E.
(2012). A cohort study of serum bilirubin levels and incident non-
alcoholic fatty liver disease in middle aged Korean workers. PLoS
One, 7, e37241.
Chien, M. Y., Ku, Y. H., Chang, J. M., Yang, C. M., & Chen, C. H. (2016).
Effects of herbal mixture extracts on obesity in rats fed a high- fat
diet. Journal of Food and Drug Analysis, 24, 594– 601.
ElRokh, E.- S., Yassin, N. A. Z., El- Shenawy, S. M. A., & Ibrahim, B. M.
M. (2010). Antihypercholesterolaemic effect of ginger rhizome
(Zingiber officinale) in rats. Inflammopharmacology, 18, 309– 315.
Gerald, B., Badrelidin, H., Musbah, O., & Abderrahim, N. (2008). Some
phytochemical, pharmacological and toxicological properties of

710
|
IF TIKH AR eT Al .
ginger (Zingiber officinalis): A review of recent research. Food and
Chemical Toxicology, 46, 409– 420.
Gomathi, N., Malarvili, T., Mahesh, R., & Begum, V. H. (20 08). Lipids low-
ering effect of Hibiscus rosa- sinensis flower petals on monosodium
glutamate (MSG) induced obese rats. Pharmacology, 1, 400– 409.
Goyal, R. K., & Kadnur, S. V. (2006). Beneficial effect s of Zingiber offici-
nale on goldthioglucose induced obesity. Fitotera pi, 77(3), 160– 163.
Gu tin , I. (2 020 ). Educ ation al diffe ren ces in mor talit y assoc iate d wit h cen-
tral obesity: Decomposing the contribution of risk and prevalence.
Social Science Research, 90, 102445.
Hernández- Saavedra, D., Pérez- Ramírez, I. F., Ramos- Gómez, M., Mendoza-
Díaz, S., Loarca- Piña, G., & Reynoso- Camacho, R. (2016). Phytochemical
characterization and effect of Calendula officinalis, Hypericum perfora-
tum, and Salvia officinalis infusions on obesity- associated cardiovascu-
lar risk. Medicinal Chemistry Research, 25, 163– 172.
Hosen, M. B., Islam, M. R., Begum, F., Kabir, Y., & Howlader, M. Z. (2015).
Oxidative stress induced sperm DNA damage, a possible reason
for male infertility. Iranian Journal of Reproductive Medicines, 13,
525– 532 .
Hussain, A. I., Rathore, H. A., Sattar, M. Z. A., Chatha, S. A. S., Ahmad, F.
U. D., Ahmad, A., & Johns, E. J. (2013). Phenolic profile and antiox-
idant activity of various extracts from Citrullus colocynthis (L.) from
the Pakistani flora. Industrial Crops and Products, 45, 416– 422.
İşeri, S., Ercan, F., Gedik, N., Yüksel, M., & Alican, I. (2007). Simvastatin
attenuates cisplatin- induced kidney and liver damage in rats.
Toxicology, 230, 256– 264.
Ismail, N. S. (2014). Protective effects of aqueous extracts of cinnamon
and ginger herbs against obesity and diabetes in obese diabetic rat.
World Journal of Dair y and Food Sciences, 9, 145– 153.
Jenko- Pražnikar, Z., Petelin, A., Jurdana, M., & Žiberna, L. (2013). Serum bili-
rubin levels are lower in overweight asymptomatic middle- aged adults:
An early indicator of metabolic syndrome? Metabolism, 62, 976– 985.
Kakkar, P., Das, B., & Viswanathan, P. N. (1984). A moditometric assay of
superoxide dismutase. Indian Journal of Biochemistry and Biophysics,
21, 131– 132.
Kanatt, S. R., Chawla, S. P., & Sharma, A. (2014). Antioxidant and radio-
protective activities of lemon grass and star anise extracts. Food
Bioscience, 6, 24– 30.
Karadag, F., Sengul, C. B., Enli, Y., Karakulah, K., Alacam, H., Kaptanoglu,
B., Kalkanci, O., & Herken, H. (2017). Relationship between
serum bilirubin levels and metabolic syndrome in patients with
schizophrenia spectrum disorders. Clinic Psychopharmacology and
Neuroscience, 15, 153.
Khadiga G. Abd Elaleem, E. A . S., & Abd Elaleem, H. G. (2017). Potential
Antibacterial Activity of Hibiscus rosa- sinensis Linn fl ow ers ex tra cts .
International Journal of Current Microbiology and Applied Sciences, 6,
1066– 1072.
Lingesh, A., Paul, D., Naidu, V., & Satheeshkumar, N. (2019). AMPK acti-
vating and anti adipogenic potential of Hibiscus rosa- sinensis flower
in 3T3- L1 cells. Journal of Ethnopharmacology, 233, 123– 130.
Mahmoud, R. H., & Elnour, W. A . (2013). Comparative evaluation of the
efficacy of ginger and orlistat on obesity management, pancreatic
lipase and liver peroxisomal catalase enzyme in male albino rats.
European Review for Medical and Pharmacology Sciences, 17, 75– 83.
Mehta, L. K., Balaraman, R., Amin, A. H., Bafna, P. A., & Gulati, O. D. (2003).
Effect of fruits of Moringa oleifera on the lipidprofile of normal and
hypercholesterolaemic rabbits. Journal of Ethnopharmacology, 86,
191– 195.
Miller, N. J., Rice- Evans, C., Davies, M. J., Gopinathan, V., & Milner, A.
(1993). A novel method for measuring antioxidant capacity and its
application to monitoring the antioxidant status in premature neo-
nates. Clinical Science, 84, 407– 412.
Muruganandan, S., Srinivasan, K., Gupta, S., Gupta, P. K., & Lal, J. (2005).
Effect of mangiferin on hyperglycemia and atherogenicit y in strep-
tozotocin diabetic rats. Journal of Ethnopharmacology, 97, 497– 501.
Nammi, S., Sreemantula, S., & Roufogalis, B. D. (2009). Protective effects
of ethanolic extract of Zingiber officinale rhizome on the develop-
ment of metabolic syndrome in high- fat diet- fed rats. Basic and
Clinical Pharmacology and Toxicology, 104, 366– 373.
Nazish, I., Ansari, S. H., Arora, P., & Ahmad, A. (2016). Antiobesity activit y
of Zingiber officinalis. Pharmacognosy Journal, 8, 440– 446.
NCD Risk Factor Collaboration (NCD- RisC) (2019). Rising rural body-
mass index is the main driver of the global obesity epidemic in
adults. Nature, 569, 260– 264.
OECD test guidelines 425 (2001). Guidelines for testing of chemicals.
Guidelines, acute oral toxicit y- up- and- down procedure.
Oh, J., Jo, H., Cho, A. R ., Kim, S. J., & Han, J. (2013). Antioxidant and
antimicrobial activities of various leafy herbal teas. Food Control,
31, 403– 409.
Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in an-
imal tissues by thiobarbituric acid reaction. Analytical Biochemistry,
95, 351– 358.
Omar, M. H., Shamsahal, N., Muhammad, H., Ahmad, W. A. N. W.,
& Wasim, M. I. (2018). Anti- obesity haematological effects of
Malaysia Hibiscus sabdariffa L. aqueous extract on obese Sprague
Dawley rats. Functional Foods in Health and Disease, 8, 340– 352.
Paranjpe, P., Patki, P., & Patwardhan, B. (1990). Ayurvedic treatment of
obesity: A randomized double- blind, placebo- controlled clinical
trial. Journal of Ethnopharmacology, 29, 1– 11.
Priya, J., & Veeranjaneyulu, C. (2016). Comparison of herbal teas for
obesity. International Journal of Research in Pharmacology and
Pharmacotherapeutics, 5, 82– 93.
Purushothaman, A., Meenatchi, P., S, S., Sundaram, R., & Saravanan, N.
(2016). Quantification of total phenolic content, HPLC analysis of
flavonoids and assessment of antioxidant and anti- haemolytic activi-
ties of Hibiscus rosa- sinensis L. flowers in vitro. International Journal of
Pharma Research and Health Sciences, 4, 1342– 1350.
Rice- Evans, C., & Miller, N. (1997). Measurement of the antioxidant sta-
tus of dietary constituents, low density lipoproteins, and plasma.
Prostaglandins, Leukotrienes & Essential Fatty Acids, 57, 499– 505.
Saluja, M. P., Kapil, R. S., & Popli, S. P. (1978). Studies in medicinal plants:
Part VI. Chemical constituents of Moringa oleifera Lam. (hyb rid vari-
ety) and isolation of 4- hydroxymellein. Indian Journal of Chemistry-
Section B (IJC- B), 16B, 104 4– 1045.
Sanadheera, S., Subasinghe, D., Solangaarachci, M. N., Suraweera, M.,
Suraweera, N. Y., & Tharangika, N. (2021). Hibiscus rosa- sinensis
L. (red Hibiscus) tea, can it be used as a home- remedy to control
diabetes and hypercholesterolemia? Biology, Medicines & Natural
Product Chemistry, 101, 59– 65.
Saravanan, G., Ponmurugan, P., Deepa, M. A., & Senthilkumar, B. (2014).
Anti- obesity action of gingerol: Effect on lipid profile, insulin,
leptin, amylase and lipase in male obese rats induced by a high- fat
diet. Journal of the Science of Food and Agriculture, 94, 2972– 2977.
Shah, I., Shah, M. A., Nawaz, M. A., Pervez, S., Noreen, N., Cruz, C. V.,
Khan, F., Blundell, R., Briffa, J., Azzopardi, J., & Niaz, K. (2020).
Analysis of other phenolics (capsaicin, gingerol and alkylresorc-
inols). In S. M Nabavi, M. Saeedi, S.F. Nabavi & A . Sanches Silva
(Eds.), Recent advances in natural products analysis (pp. 255– 271).
Amsterdam: Elsevier Inc.
Shah, M. A., Jakkawanpital, C., Sermwittayawong, D., &
Panichayupakaranant, P. (2017). Rhinacanthins- rich extract en-
hanced glucose uptake and inhibits adipogenesis in 3T3- L1 adipo-
cytes and L6 myotubes. Pharmacognosy Magazines, 13, 817– 821.
Sikar war, M. S., & Patil, M. B. (2011). Antihyperlipidemic ef fect of ethan-
olic extract of Hibiscus rosa- sinensis flowers in hyperlipidemic rats.
RGUHS Journal of Pharmaceutical Sciences, 1, 117– 120.
Sultana, B., Anwar, F., & Przybylski, R. (2007). Antioxidant activity
of phenolic components present in barks of Azadirachat indica,
Terminalia arjuna, Acacia nilotica, and Eugenia jambolana Lam. trees.
Food Chemistry, 104, 1106– 1114.

|
711
IFTIK HAR eT A l.
Takahashi, Y., & Fukusato, T. (2014). Histopathology of nonalcoholic
fatty liver disease/nonalcoholic steatohepatitis. World Journal of
Gastroenterology, 20, 15539– 15548.
Tohma, H., Gülçin, İ., Bursal, E., Gören, A. C., Alwasel, S. H., & Köksal,
E. (2017). Antioxidant activity and phenolic compounds of ginger
(ZingiberofficinaleRosc.) determined by HPLC- MS/MS. Journal of
Food Measurement and Characterization, 11, 556– 566.
Velez- Carrasco, W., Merkel, M., Twiss, C. O., & Smith, J. D. (2008). Dietary
methionine effects on plasma homocysteine and HDL metabolism
in mice. The Journal of Nutrition and Biochemistry, 19, 362– 370.
Wang, C., Chan, J. S. Y., Ren, L., & Yan, J. H. (2016). Obesity reduces
cognitive and motor functions across the lifespan. Neural Plasticity,
2016, 1– 13. https://doi.org/10.1155/2016/2473081
World Health Organization (WHO) (2017). World Health Organization
Factsheets, Obesity and overweight. 2017. https://www.who.int/
n e w s - r o o m / f a c t - s h e e t s / d e t a i l / o b e s i t y - a n d - o v e r w e i g h t
How to cite this article: Iftikhar, N., Hussain, A. I.,
Chatha, S. A. S., Sultana, N., & Rathore, H. A. (2022). Effects of
polyphenol- rich traditional herbal teas on obesity and
oxidative stress in rats fed a high- fat– sugar diet. Food Science
& Nutrition, 10, 698– 711. https://doi.org/10.1002/fsn3.2695