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Insulin Plant(Costus pictus) Extract Restores Thyroid Hormone
Levels in Experimental Hypothyroidism
S. Ashwini, Zachariah Bobby, M. G. Sridhar, C. C. Cleetus
Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
ABSTRACT
Background: The aim of the present study was to investigate the preventive
effect of Costus pictus leaf extract in experimental hypothyroidism.
Materials and Methods: Forty male Wistar rats were randomly divided
into four groups with ten rats in each group: Control(C), hypothyroid(H),
control+extract (C+E), and hypothyroid+extract (H+E). Rats in C group
did not receive any intervention throughout the experimental period. The
rats in the C+E and H+E groups received pretreatment with C.pictus leaf
extract for 4weeks. Subsequently, for the next 6weeks, rats in the H
group received 0.05% propylthiouracil in drinking water while C+E group
received C.pictus leaf extract and H+E group received propyl thiouracil and
C.pictus leaf extract. Results: Hypothyroid group rats exhibited dramatic
increase in thyroid-stimulating hormone (TSH) levels with concomitant
depletion in the levels of thyroid hormones. Treatment with the extract
resulted in remarkable improvement in thyroid prole. Extract produced
10.59-fold increase in plasma free T3, 8.65-fold increase in free T4, and
3.59-fold decrease in TSH levels in H+E group in comparison with H group.
Treatment with the extract ameliorated hypercholesterolemia, decreased
levels of plasma C-reactive protein and tumor necrosis factor alpha,
suppressed tissue oxidative stress and prevented hepatic and renal damage
caused due to thyroid hormone depletion in the H+E group. Pentacyclic
triterpenes alpha and beta amyrins were identied and quantied in the
extract. Conclusions: This is the rst study to reveal that C.pictus extract
has therapeutic potential to restore thyroid hormone levels and prevent
the biochemical complications due to thyroid hormone insufciency in the
animal model of experimental hypothyroidism.
Key words: Costus pictus, hypothyroidism, insulin plant, propylthiouracil,
thyroid hormones
SUMMARY
• The preventive effect of Costus pictus leaf extract in experimental hypothy-
roidism was evaluated in the present study.
• Hypothyroidism was induced in the experimental animals by giving 0.05%
propylthiouracil in drinking water.
• Hypothyroid rats exhibited dramatic increase in thyroid-stimulating hormone
(TSH) levels with concomitant depletion in the levels of thyroid hormones.
• Treatment with Costus pictus leaf extract in hypothyroid rats signicantly
improved the thyroid prole. It also ameliorated hypercholesterolemia, de-
creased the levels of plasma inammatory markers, suppressed tissue oxida-
tive stress and prevented hepatic and renal damage caused due to thyroid
hormone depletion.
• The possible active principles alpha and beta amyrins were identied and
quantied in the extract through LC-MS.
Abbreviations Used: APCI: Atmospheric pressure chemical ionization;
AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; C group:
Control group; C+E group: Control+extract group; C. pictus: Costus
pictus; CRP: C-reactive protein; DPPH: 2,2-diphenyl-1-picrylhydrazyl;
FRAP: Ferric reducing antioxidant power; HDL: High-density lipoprotein;
H group: Hypothyroid group; H+E group: Hypothyroid+extract group;
LDL: Low-density lipoprotein; LC-MS: Liquid chromatography–mass
spectrometry; MDA: Malondialdehyde; PTU: 6-Propyl-2-thiouracil;
SRM: Single reaction monitoring; TSH: Thyroid-stimulating hormone;
TPTZ: 2,4,6-tri-(2-pyridyl)-5-triazine; TBA: 2–Thiobarbituric acid;
TG: Triglyceride; TNFα: Tumor necrosis factor
alpha; TAS: Total antioxidant status
Correspondence:
Dr.Zachariah Bobby,
Department of Biochemistry,
Jawaharlal Institute of Postgraduate
Medical Education and Research,
Puducherry-605006, India.
E-mail:zacbobby@yahoo.com
DOI: 10.4103/0974-8490.199766
INTRODUCTION
Hypothyroidism is one of the most common endocrine diseases.
In the general population, the major cause for hypothyroidism is
autoimmune thyroiditis. It is characterized by decreased serum levels
of thyroid hormones (T3 and T4) and elevated thyroid-stimulating
hormone (TSH).[1] e current mode of treatment for hypothyroidism
is levothyroxine replacement therapy. However, there are certain
limitations associated with levothyroxine replacement therapy. Recent
studies have reported that a signicant number of hypothyroid
patients on levothyroxine replacement therapy experience decreased
neurocognitive function and lead poorer quality of life despite being
biochemically euthyroid.[2,3] Clinically, it has been observed that
since levothyroxine replacement therapy requires lifelong treatment,
it associated with poor compliance in some patients.[4] Hence, there
is an urgent need for more eective therapeutic strategies to treat
hypothyroidism.
Recently, there has been renewed interest in the use of medicinal
plants and their bioactive constituents in the treatment of endocrine
diseases.[5] Costus pictus is one such medicinal plant belonging to the
family of Costaceae. It is commonly known as insulin plant. It is grown
in various parts of India.[6,7] e leaf of this plant is being consumed
by diabetic patients to control their blood glucose levels. Leaf extract
Pharmacogn. Res.
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Cite this article as: Ashwini S, Bobby Z, Sridhar MG, Cleetus CC. Insulin
Plant (Costus pictus) extract restores thyroid hormone levels in experimental
hypothyroidism. Phcog Res 2017;9:51-9.
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ORIGINAL ARTICLE
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
52 Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017
of C.pictus can stimulate insulin secretion,[8] can regenerate beta cells
of the pancreas,[9] and has potent antidiabetic activity as evident from
experiments carried out in animal models.[9-12]
Although the antidiabetic eect of C.pictus has been well documented,
the eect of C. pictus extract on thyroid function has not been
explored so far. It has been reported that pentacyclic triterpenes such
as betulinic acid ameliorate experimental hypothyroidism.[13] We
hypothesized that C. pictus extract containing pentacyclic triterpenes
could exert benecial eect in alleviating hypothyroidism. To the best
of our knowledge, this is the rst study to investigate the eect of C.
pictus extract on hypothyroidism. In the present study, the ability of
this plant extract to ameliorate hypothyroidism was studied in propyl
thiouracil(PTU)-induced hypothyroid rat model.
MATERIALS AND METHODS
Chemicals
PTU, 2,2-diphenyl-1-picrylhydrazyl(DPPH) and 2,4,6-tri-(2-pyridyl)-
5-triazine, 2–thiobarbituric acid, alpha-amyrin, and beta-amyrin were
of molecular grade purchased from Sigma-Aldrich (USA). All other
chemicals used were of analytical grade obtained from SRL(India).
Plant material
Fresh leaves of C. pictus were obtained from plants cultivated by the
Department of Horticulture, Jawaharlal Institute of Postgraduate
Medical Education and Research(JIPMER), Puducherry. e identity
of the plant was conrmed by the Botanical Survey of India, Coimbatore
(Authentication Certicate No. BSI/SRC/5/23/2011-12/Tech/630 dated
July 25, 2011).
Preparation of Costus pictus extract
Fresh leaves of C. pictus were shade-dried, powdered and extracted
overnight with 80% methanol as solvent in a shaker. e solvent was
evaporated to dryness using rotational vacuum concentrator(Martin
Christ, Germany) and the nal residue was lyophilized using
lyophilizer(Martin Christ, Germany).
Ferric reducing antioxidant power assay
Ferric reducing antioxidant power(FRAP) assay was carried out based on
the method described by Benzie and Strain.[14] e antioxidant capacity of C.
pictus extract was measured based on the ability to reduce Fe(III)-tripyridyl
triazine compound to Fe(II)-tripyridyl triazine compound. Ten microliters
of C.pictus extract at dierent concentrations was added to 300 µl of FRAP
reagent and thoroughly mixed. e reaction mixture was incubated at 37°C
for 4min. e increase in absorbance at 593 nm was measured. Astandard
curve was generated using dierent concentrations of FeSO4 solutions. e
antioxidant capacity of C.pictus extract was expressed as mmol of ferrous
equivalent Fe(II) per gram of the sample.
2,2‑Diphenyl‑1‑picrylhydrazyl scavenging assay
DPPH assay was carried out based on the method described by
Brand-Williams etal.[15] e free radical scavenging activity of C. pictus
extract was determined from the bleaching of purple-colored methanolic
solution DDPH. Hundred microliters of 0.5 mM freshly prepared DPPH
ethanol solution was added to 100 µL of sample solution in 50% ethanol
at dierent concentrations. e mixture was shaken vigorously and
incubated for 30min in the dark at room temperature. e absorbance
of each reaction mixture was measured at 517 nm. Lower absorbance
of the reaction mixture indicates higher free radical scavenging activity.
e concentration of the extract that scavenges 50% of DPPH(IC50) was
calculated.
Liquid chromatography–mass spectrometry
analysis of Costus pictus extract
e experiment was carried out using an Agilent 1290 Innity
ultrahigh-performance liquid chromatography (HPLC). Shim-pack
XR-ODSIII C18 column was used(dimension: 150 mm×2 mm internal
diameter; particle size: 2 µm). e column oven temperature was 40°C.
e mobile Phase A was 10 mM ammonium acetate in water and mobile
Phase B was methanol. e mobile phase was used in gradient mode
as follows: 0–3min: 50% B, 3–5min: 50–100% B, 5–25min: 100% B,
25–25.1min: 100–50% B, 25.1–30min: 50% B. e ow rate was 0.2
ml/min. e run time was 30min. For detection and quantication,
HPLC system was coupled with a ermo Fisher TSQ Vantage triple
quadrupole mass spectrometer, operated with atmospheric pressure
chemical ionization positive. e discharge current was 4 µA. e
vaporizer temperature was 300°C. e sheath gas and auxiliary(Aux)
gas used were nitrogen. Sheath gas ow rate was 25 Arb. Aux gas ow
rate was 15 Arb. e analysis mode was single reaction monitoring
mode.
Animal experiments
e study was conducted in the Department of Biochemistry, JIPMER,
Puducherry, India, aer obtaining approval from the institutional animal
ethics and scientic advisory committees.
Five-month-old male Wistar rats were obtained from the institute
central animal house and maintained in polycarbonate cages
under a 12-h light/12-h dark cycle with the standard laboratory rat
chow(35% carbohydrate, 25% proteins, 7% lipids, and 3% vitamins
and minerals) and water available ad libitum. Totally, forty rats were
randomized into four groups with ten rats in each group. To induce
experimental hypothyroidism, 0.05% propylthiouracil was given
in drinking water for 6weeks, which is a well-accepted method for
induction of hypothyroidism in experimental animals.[16-18] C. pictus
extract was given at a dose of 150 mg/kg body weight/day through
oral gavage.[9] is dose was xed based on our pilot study conducted
with dierent doses and results of previously reported toxicity
studies on this plant extract.[19,20] e total duration of the study was
10weeks–rst 4weeks was considered to be Phase 1 and next 6weeks
was considered to be Phase 2. e disease condition was induced in
Phase 2(Group2 and Group4). In the groups that received C.pictus
extract treatment(Group 3 and Group4), this extract was given in
Phase 1(pretreatment) and Phase 2(co-treatment) as our aim was to
see the preventive eect of this extract.
e four groups are as follows:
• Group1:Control(C)–nointerventioninbothPhases1and2
• Group2:Hypothyroid(H)–nointerventioninPhase1and0.05%
propylthiouracil(PTU) in drinking water in Phase 2
• Group3:Control+extract(C+E)–interventionofC.pictus extract
was given in both Phases 1 and 2
• Group4: Hypothyroid+extract (H+E)–C. pictus extract was
given in Phase 1 and 0.05% propylthiouracil(PTU) in drinking
water+C.pictus extract in Phase 2.
Sample collection
Blood samples were collected at the beginning and end of the study.
Plasma was separated and used for the estimation of thyroid prole,
biochemical parameters, and inammatory markers. Body weight, food
intake, and water intake were measured periodically. At the end of the
experimental period of 10 weeks, the animals were sacriced under
anesthesia; liver and kidney tissues were excised, snap frozen in liquid
nitrogen and stored at−80°C for subsequent analysis.
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017 53
Thyroid prole
Plasma free T3, free T4 and TSH were measured using rat-specic ELISA
kits from CUSABIO, Japan. e intra-assay precision and inter-assay
precision for these kits were<15%(precision details were provided by
the manufacturer in the kit).
Measurement of free T3 and free T4 was based on competitive inhibition
enzyme immunoassay technique. For estimation of FT3, standards and
samples were added to microtiter plates precoated with antibody specic
to FT3. en, biotin-conjugated FT3 was added to the microtiter plates.
FT3 present in the sample or standard competed with biotin-conjugated
FT3 to bind to the antibody present in the microtiter plates. Aer a
washing step, avidin-conjugated horseradish peroxidase (HRP) was
added to the wells. is was followed by addition of substrate. Finally,
the color developed was inversely proportional to the amount of FT3
in the sample/standard. Measurement of FT4 was similar to FT3 except
that microtiter plates were precoated with antibody specic to FT4 and
biotin-conjugated FT4 was added to the microtiter plates.
Measurement of TSH was based on quantitative sandwich enzyme
immunoassay technique. Here, antibody specic to TSH was precoated
onto microtiter plates. en, standards and samples were added to the
wells with HRP-conjugated antibody specic for TSH. Following a wash
step, substrate was added to the wells. e color developed was directly
proportional to the amount of TSH present in the sample/standard.
Lipid prole
e following biochemical parameters were analyzed in fasting plasma
sample using a fully automated clinical chemistry analyzer (AU-400,
OLYMPUS, Essex, UK). Total cholesterol was measured using
cholesterol oxidase–peroxidase method(Genuine Bio-systems, Chennai,
India), triglycerides (TGs) using an enzymatic glycerol phosphate
oxidase–peroxidase method (Agappe Diagnostics, Kerala, India), and
high-density lipoprotein(HDL) cholesterol by the cholesterol oxidase–
peroxidase method(Lab-Care Diagnostics, Mumbai, India). Low-density
lipoprotein (LDL)-cholesterol in plasma was calculated using Friedwald
formula.[21]
Glucose tolerance
Glucose tolerance was assessed by method described by Yuan etal.[22]
Blood samples were collected from the rats aer they were maintained
in a fasting state overnight(15 h). Rats were then injected 2.0 g/kg body
weight of glucose(in 1.5 ml saline) intraperitoneally. Blood samples were
collected 2 h aer glucose injection. Plasma glucose was estimated by
the glucose oxidase–peroxidase method(Reckon Diagnostics, Vadodara,
India) in a fully automated clinical chemistry analyzer (AU-400,
OLYMPUS, Essex, UK).
Liver and kidney function tests
e following parameters were analyzed in plasma sample in a fully
automated clinical chemistry analyzer (AU-400, OLYMPUS, Essex,
UK) using diagnostic kits purchased from Pathozyme, Kolhapur, India.
Total protein was estimated using biuret method, albumin by modied
bromocresol green method, aspartate aminotransferase (AST) and
alanine aminotransferase(ALT) by modied International Federation
of Clinical Chemistry method. Urea was estimated by urease-glutamate
dehydrogenase method and creatinine by modied Jae’s method.
Assessment of inammatory markers
Inammatory response was assessed by measuring plasma levels of
high-sensitivity C-reactive protein (CRP) and tumor necrosis factor
alpha(TNFα). CRP was measured using sandwich ELISA(Immunology
Consultants Laboratory, Newberg, OR, USA). e microtiter plates
were precoated with anti-CRP antibodies. e samples and standards
were added to these microtiter plates. CRP present in the sample was
bound to the anti-CRP antibody in the microtiter plates. e unbound
proteins were removed by washing. en, HRP-conjugated anti-CRP
antibodies were added. is was followed by addition of the substrate
3,3’5,5’-tetramethylbenzidine (TMB) to detect the bound HRP
conjugate. Finally, the concentration of CRP was determined from the
standard curve.
TNFα was measured using solid phase sandwich ELISA (Diaclone
SAS, Besançon, France). Samples and standards were added to
antibody-coated microtiter wells. Biotinylated polyclonal antibody
specic for rat TNFα was simultaneously added and incubated. Aer
washing, the enzyme–streptavidin–peroxidase was added and nally
bound enzyme was detected using the chromogenic substrate TMB. e
TNFα in each sample was determined from the standard curve.
Assessment of hepatic and renal oxidative stress
Liver and kidney tissues were homogenized using 0.1 M ice-cold
Tris-HCI buer(pH7.5). e homogenate was centrifuged at 14,000×g
for 15 min at 4°C. e supernatant was used for the estimation of
malondialdehyde (MDA) and total antioxidant status(TAS) levels. MDA
was measured according to the method described by Ohkawa etal.[23]
TAS was estimated through FRAP assay.[14] Protein content in the liver
and kidney homogenate was estimated by the method of Lowry etal.[24]
Statistical analyses
All values were expressed as mean±standard deviation. e dierence
in mean values among the groups was analyzed using one-way analysis
of variance (ANOVA) with Bonferroni post hoc test for anthropometric
parameters, tissue oxidative stress markers and plasma inammatory
markers. Two-way repeated measures ANOVA with Bonferroni post
hoc test was used for the analyses of rest of the parameters. All the
analyses were carried out using PRISM 5 soware(GraphPad Soware,
San Diego, CA, USA, [http://www.graphpad.com]). A P < 0.05 was
considered statistically signicant.
RESULTS
Liquid chromatography–mass spectrometry
analysis of Costus pictus extract
We explored for the presence of pentacyclic triterpenes in C. pictus
extract. It was found that C.pictus extract contains pentacyclic triterpenes,
namely alpha and beta amyrins [Table 1 and Figures 1,2]. It contains
0.45 ng of alpha-amyrin/mg of extract and 1.29 ng of beta-amyrin/mg
of extract.
Table1: Liquid chromatography‑mass spectrometry analysis of triterpenes in Costus pictus extract
Molar mass
(g/mol)
Retension
time(min)
Mode of
ionization
Parent
ion(m/z)
Detection
ion(m/z)
Detection
mode
Amount present
(ng/mg of extract)
Alpha amyrin 426.72 23.5 APCI+ve 409 271.246 SRM 0.45
Beta amyrin 426.72 22.3 APCI+ve 409 271.246 SRM 1.29
Estrone(internal
standard for terpenes)
270.36 7.02 APCI+ve 271.2 159.1 SRM
APCI: Atmospheric pressure chemical ionization; SRM: Single reaction monitoring
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
54 Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017
2,2‑diphenyl‑1‑picrylhydrazyl assay
e IC50 value of C. pictus extract for DPPH assay was found to be
38.82±1.26 µg/ml. e IC50 value of positive control ascorbic acid was
found to be 6.73±0.15 µg/ml.
Ferric reducing antioxidant power assay
Antioxidant capacity of C. pictus extract as assessed by FRAP assay was
found to be 2.98±0.03 mmol Fe2+/g and that of positive control ascorbic
acid was found to be 18.76±0.38 mmol Fe2+/g.
Body weight, food intake and water intake
e results of body weight, food intake and water intake is shown in
[Table 2]. In comparison with control, PTU-induced hypothyroid rats
exhibited a signicant decrease in body weight. In comparison with
hypothyroid group, the decrease in body weight was partially prevented
in hypothyroid+extract group. In control+extract group, the body weight
was similar to control group.
e amount of food and water intake was markedly reduced in the
hypothyroid group in comparison with control. It was signicantly
improved in hypothyroid+extract group. e food intake and water
intake were found to be normal in control+extract group.
Thyroid prole
At the beginning of the study, all the groups showed normal thyroid
profile. At the end of the study in hypothyroid group, plasma free
T3 and free T4 levels were decreased 19.92-fold and 15.43-fold,
respectively, and TSH was increased 15.64-fold in comparison with
control [Table 3]. This indicates that a state of severe hypothyroidism
was successfully induced in the experimental animals. Hypothyroid
rats treated with C. pictus extract (hypothyroid+extract group)
exhibited a remarkable improvement in thyroid profile. In
hypothyroid+extract group, plasma free T3 and free T4 levels
were elevated by 10.59-fold and 8.65-fold, respectively, and further
plasma TSH was decreased by 3.59-fold in comparison with
hypothyroid group. These results clearly demonstrate that C.pictus
Table2: Body weight, food intake and uid intake of the experimental
groups
Groups Final body
weight(g)
Food intake
(g/rat/day)
Fluid intake
(ml/rat/day)
Control 328.8±4.34 16.48±1.19 31.96±1.47
Hypothyroid 270.4±5.08a12.37±1.26a18.6±3.12a
Control + extract 324.9±4.68b16.22±1.17b32.2±1.34b
Hypothyroid + extract 297.7±7.32a,b 14.92±1.15a,b 25.16±1.94a,b
Values are expressed as mean±SD. n=10/group. Dierences between the groups
were analyzed using one-way ANOVA with Bonferroni post hoc test. aP<0.05
in comparison to control group; bP<0.05 in comparison to hypothyroid group.
SD: Standard deviation; ANOVA: Analysis of variance
Table3: Eect of Costus pictus extract on thyroid prole in the experimental groups
Parameter Time period Control Hypothyroid Control + extract Hypothyroid + extract
Free T3(pMol/L) End 4.78±0.49 0.25±0.05ax 5.18±0.23a,b,x 2.55±0.21a,b,x
Basal 4.75±0.42 4.77±0.40 4.73±0.47 4.71±0.49
Free T4(pMol/L) End 18.85±2.20 1.29±0.31a,x 21.06±2.34a,b,x 10.53±0.82a,b,x
Basal 18.76±2.27 19.09±2.18 18.88±2.14 18.71±2.09
TSH(µIU/ml) End 1.75±0.27 26.83±1.38a,x 1.69±0.24b7.52±0.53a,b,x
Basal 1.76±0.30 1.72±0.25 1.77±0.26 1.75±0.26
Values are expressed as mean±SD. n=10/group. Dierences between the groups were analyzed using two-way repeated measures ANOVA with Bonferroni post hoc
test. aP<0.05 in comparison to control group of the same period; bP<0.05 in comparison to hypothyroid group of the same period; xP<0.05 in comparison to basal
values of the same group. SD: Standard deviation; ANOVA: Analysis of variance; TSH: yroid stimulating hormone; Free T3: Free triiodothyronine; Free T4: Free
thyroxin
extract has therapeutic potential to restore thyroid hormone levels in
experimental hypothyroidism.
Control rats administered with C.pictus extract(control+extract group)
showed a small increase in plasma free T3 and free T4, and this increase
was statistically signicant in comparison with control. However, no
signicant dierence was seen in plasma TSH levels between control and
control+extract groups.
Glucose tolerance
At the end of the study, there was no signicant dierence in fasting
plasma glucose in hypothyroid group; however, 2 h postglucose load
value was signicantly elevated in this group in comparison with control
group [Table 4]. Treatment with the extract brought back the elevated
2 h postglucose load value to normal levels in hypothyroid+extract
group. Administration of the extract to control rats(control+extract
group) resulted in normal fasting and 2 h postglucose load levels. Unlike
currently used hypoglycemic drugs, administration of C.pictus extract
Figure 1: High‑performance liquid chromatography chromatogram
of (a) estrone (Internal standard for analysis of terpenes) (b) Costus
pictus extract
b
a
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017 55
Table4: Eect of Costus pictus extract on glucose and lipid prole in the experimental groups
Parameter Time period Control Hypothyroid Control + extract Hypothyroid + extract
Fasting glucose(mg/dl) End 76.2±7.93 81.2±15.77 74.3±7.01 78.4±8.88
Basal 73.4±7.88 72.3±7.83 75.6±5.93 74.5±6.13
2 h postglucose load values during IPGTT
(mg/dl)
End 113.8±12.09 148.9±19.68a,x 110.3±8.96b119.5±11.37b
Basal 115.8±13.64 119.8±11.18 116.2±14.61 114.2±11.41
Total cholesterol(mg/dl) End 61.6±5.66 88.2±4.69a,x 63.2±4.96b68.8±5.90a,b
Basal 61.2±5.41 62.1±4.48 61.6±4.06 63.2±6.49
LDL cholesterol(mg/dL) End 17.5±4.44 47.9±5.36a,x 19.2±7.87b26.9±7.78a,b,x
Basal 18.2±6.11 18.7±5.14 18.9±6.10 18.6±4.75
HDL cholesterol(mg/dL) End 27.1±3.84 24.1±4.56 27.5±4.06 26.1±4.63
Basal 26.7±3.53 27.0±4.67 26.2±4.26 28.1±4.65
TG(mg/dl) End 84.8±6.80 79.1±7.06 82.6±6.29 79.7±6.11
Basal 81.5±7.60 82.2±6.56 82.4±7.55 82.3±5.42
Values are expressed as mean±SD. n=10/group. Dierences between the groups were analyzed using two-way repeated measures ANOVA with Bonferroni post
hoc test. aP<0.05 in comparison to control group of the same period; bP<0.05 in comparison to hypothyroid group of the same period; xP<0.05 in comparison to
basal values of the same group. TG: Triglyceride; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; IPGTT: Intraperitoneal glucose tolerance test;
SD: Standard deviation; ANOVA: Analysis of variance
Figure2: Liquid chromatography–mass spectrometry spectrum of alpha‑amyrin and beta‑amyrin
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
56 Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017
to normal control rats did not result in hypoglycemia, but it maintained
the glucose levels.
Lipid prole
Hypothyroid group showed a profound elevation in both total
cholesterol (1.4-fold increase) and LDL-cholesterol(2.8-fold increase) in
comparison with control at the end of the study [Table 4]. In comparison
with hypothyroid group, hypothyroid+extract group exhibited a marked
reduction in total cholesterol and LDL cholesterol by 1.3-fold and
1.9-fold, respectively. However, there was no signicant dierence seen
in plasma TG and HDL cholesterol levels in hypothyroid group at the
end of the study.
Liver and kidney function tests
e results of liver and kidney function tests is shown in [Table 5]. At the
beginning of the study, all the experimental groups exhibited normal liver
and kidney functions. At the end of the study, plasma AST levels were
found to be signicantly increased in hypothyroid rats in comparison
with control and treatment with extract prevented the increase in AST
levels. ere was no signicant dierence seen in plasma ALT, total
protein, and albumin levels between the experimental groups.
Plasma urea and creatinine levels were elevated in the hypothyroid group
in comparison with control. In hypothyroid+extract group, plasma urea
and creatinine levels were signicantly decreased in comparison to
hypothyroid group.
ese results demonstrate that hypothyroid rats exhibited impairment
in hepatic and renal function; treatment with C.pictus extract partially
prevented hepatic and renal damage as indicated by AST, urea, and
creatinine levels.
Inammatory markers
In comparison with control, hypothyroid group showed a drastic increase
in plasma TNFα and CRP levels by 4.21-fold and 1.46-fold, respectively.
Treatment with the extract signicantly attenuated the increase in TNFα
and CRP in hypothyroid+extract group in comparison with hypothyroid
group [Figures 3 and 4].
Hepatic and renal oxidative stress markers
In both liver and kidney tissues, hypothyroid rats displayed signicant
elevation in MDA levels and signicant reduction in TAS levels in
comparison with control [Figures 5 and 6]. In hypothyroid+extract
group, the lipid peroxidation was found to be suppressed as indicated
by decrease in MDA levels; in addition, TAS levels were enhanced in
comparison with hypothyroid group. MDA and TAS levels were found
Table5: Eect of Costus pictus extract on parameters of liver and kidney function in the experimental groups
Parameter Time period Control Hypothyroid Control + extract Hypothyroid + extract
AST(U/L) End 85.4±2.76 102.8±4.66a,x 87.1±2.81b89.4±3.75a,b,x
Basal 86.7±3.06 83.2±3.49 85.2±3.58 84.8±2.90
ALT(U/L) End 50.5±3.31 54.2±4.49 52.2±3.77 52.1±3.48
Basal 49.7±4.27 51.8±4.49 51.5±4.14 50.1±4.75
Total protein(g/dl) End 7.3±0.32 7.21±0.28 7.42±0.35 7.33±0.36
Basal 7.32±0.25 7.26±0.30 7.34±0.33 7.29±0.24
Albumin(g/dl) End 3.26±0.27 3.23±0.20 3.36±0.32 3.3±0.30
Basal 3.23±0.22 3.24±0.24 3.32±0.23 3.41±0.33
Urea(mg/dL) End 24.2±3.82 43.5±7a,x 25.3±4.14b31.5±3.72a,b,x
Basal 24.6±3.34 25.1±3.21 26±4.16 24.6±3.86
Creatinine(mg/dL) End 0.52±0.10 1.03±0.25a,x 0.50±0.11b0.68±0.12a,b,x
Basal 0.52±0.09 0.51±0.12 0.49±0.10 0.51±0.10
Values are expressed as mean±SD. n=10/group. Dierences between the groups were analyzed using two-way repeated measures ANOVA with Bonferroni post hoc
test. aP<0.05 in comparison to control group of the same period; bP<0.05 in comparison to hypothyroid group of the same period; xP<0.05 in comparison to basal
values of the same group. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; SD: Standard deviation; ANOVA: Analysis of variance
to be similar between control group and control+extract group. To
summarize the results, treatment with extract ameliorated hepatic and
renal oxidative stress seen in hypothyroid rats.
DISCUSSION
e present study was conducted to evaluate the eect of C.pictus extract
in experimental hypothyroidism. Severe hypothyroidism was induced
in rats by administration of 0.05% PTU in drinking water for 6weeks.
Plasma levels of free T3 and free T4 were signicantly decreased and TSH
levels were dramatically increased in hypothyroid group. is conrmed
the successful induction of hypothyroidism in the experimental groups
under study. PTU is a reversible goitrogen. It induces hypothyroidism
by inhibiting crucial enzymes required for thyroid hormone synthesis,
namely thyroperoxidase and peripheral deiodinase.[25] Inhibition of
these enzymes impairs the iodination of tyrosyl residues and coupling
of iodotyrosyl residues to form iodothyronine.[26,27] Treatment with the
extract in hypothyroid+extract group produced 10.59-fold increase in
plasma free T3, 8.65-fold increase in free T4, and 3.59-fold decrease in
TSH in comparison with hypothyroid group. Since PTU is a reversible
goitrogen, withdrawal of PTU can restore the levels of thyroid hormones.
Hence, the experiment was designed such that in H+E group aer Phase
a
b
ab
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
CHC+
EH
+E
Plasma TNF alpha levels (pg/ml)
Figure3: Eect of Costus pictus extract on plasma tumor necrosis factor
alpha levels measured at the end of the study. Dierences between the
groups were analyzed using one‑way analysis of variance with Bonferroni
post hoc test. a= P < 0.05 in comparison to control group, b=P < 0.05
in comparison to hypothyroid group; C: Control group; H: Hypothyroid
group; C+E: Control+extract group; H+E: Hypothyroid+extract group
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017 57
1 (period of pretreatment with C. pictus extract), in Phase 2 along
with the C. pictus extract PTU administration was continued until
the end. Animals in hypothyroid+extract group showed a signicant
improvement in thyroid prole despite continued PTU administration
in Phase 2. ese results prove that C. pictus extract has therapeutic
potential in improving the thyroid hormone levels in experimental
hypothyroidism.
is is the rst study to reveal the eect of C.pictus extract in ameliorating
hypothyroidism. It is possible that C. pictus extract upregulates the
expression of key enzymes involved in thyroid hormone synthesis,
namely thyroperoxidase and 5’deiodinase, or increases the activity
of these enzymes, thereby stimulating the thyroid gland to secrete
thyroid hormones. However, the possibility that the reason for the
improvement in thyroid prole in C.pictus extract treated hypothyroid
rats due to antagonizing eect of C. pictus extract on PTU cannot be
excluded. To conclusively prove the aforementioned mechanism,
further investigations on expression and activity of thyroperoxidase and
5’deiodinase in the thyroid gland and metabolism of PTU on treatment
with C.pictus extract needs to be carried out.
Previous studies have shown that C.pictus extract has good antioxidant
property.[28] Our ndings are in agreement with these studies as indicated
by the results of DDPH assay, FRAP assay, tissue MDA and TAS.
C.pictus extract also showed good anti-inammatory eect. We found
that administration of C. pictus extract signicantly reduced plasma
TNF-α and CRP levels in hypothyroid rats. C. pictus extract, being a
good antioxidant and anti-inammatory agent, could help to repair the
damage caused by PTU in the thyroid gland.
It has been reported in previous studies that pentacyclic triterpenes
such as betulinic acid alleviates experimental hypothyroidism. It
reduces TSH levels and improves T3 and T4 levels in PTU-induced
hypothyroid rats.[13] Hence, we explored for the presence of similar
pentacyclic triterpenes in C. pictus extract. We found that C. pictus
extract contains alpha and beta amyrins both of which belong to the
family of pentacyclic triterpenes. Alpha and beta amyrins possess potent
anti-inammatory, antioxidant, hepatoprotective, and anti-nociceptive
eects.[29-32] Further, it has been reported that alpha and beta amyrins
inhibit nuclear factor-kappa B (NF-kβ) activation.[33] Betulinic acid
alleviates experimental hypothyroidism by preventing the activation of
NF-kβ. Activation of NF-kβ interferes with T3-dependent induction of
5’-Deiodinase gene expression, leading to impairment in the production
of thyroid hormones.[34,35] Since alpha and beta amyrins in C. pictus
extract are also pentacyclic triterpenes, structurally similar to betulinic
acid and inhibit NF-kβ activation, it is possible that amyrins ameliorate
experimental hypothyroidism through similar mechanism.
Plethora of human and animal studies have proved that hypothyroidism
is associated with elevated plasma total cholesterol levels.[36-39] It
has been reported that PTU-induced hypothyroid rats exhibited
hypercholesterolemia with no signicant increase in plasma TG
levels.[40,41] Our results are in agreement with these studies. In the
present study, treatment with C.pictus extract resulted in remarkable
reduction in total cholesterol as well as LDL-cholesterol levels in
hypothyroid rats. Hypocholesterolemic eect of C.pictus extract could
be attributed to the presence of pentacyclic triterpenes such as alpha
and beta amyrins in the extract. Santos etal. have shown that alpha and
beta amyrins exert potential antihyperglycemic and antihyperlipidemic
eects.[42] It has been reported that pentacyclic triterpenes possess
hypolipidemic eect by downregulation of lipogenic genes such as acety
l-CoA carboxylase, stearoyl-CoA desaturase 2, glycerol-3-phosphate
acyltransferase, and acyl-CoA cholesterol acyltransferase.[43,44]
C.pictus extract could alleviate hypercholesterolemia in PTU-induced
hypothyroid rats through similar mechanism. We have reported in our
earlier studies with C.pictus extract that it contains signicant amount
of phenolic compounds and avonoids.[45] Apart from amyrins,
phenolic compounds and avonoids could also be responsible for the
hypolipidemic activity exhibited by C. pictus extract in hypothyroid
rats.
Liver and kidney are important target organs of thyroid hormones.
Hypothyroidism leads to perturbed liver and kidney function.[46,47]
In the present study, plasma levels of AST, urea and creatinine were
signicantly elevated in hypothyroid rats. Plasma ALT level showed an
increase, but it was not signicant. e increase in levels of transaminases
in hypothyroidism is due to inadequate levels of thyroid hormones and
decreased hepatic clearance.[47] e raise in plasma urea and creatinine
levels in hypothyroid rats is due to reduced glomerular ltration rate
and decreased tubular secretion of creatinine.[46,48] In the present study,
a
b
b
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
CHC+
EH
+E
Plasma CRP levels (µg/ml)
Figure 4: Eect of Costus pictus extract on plasma C‑reactive protein
levels measured at the end of the study. Dierences between the groups
were analyzed using one‑way analysis of variance with Bonferroni post
hoc test. a =P < 0.05 in comparison to control group, b=P < 0.05 in
comparison to hypothyroid group; C: Control group; H: Hypothyroid
group; C+E: Control+extract group; H+E: Hypothyroid+extract group
a
b
b
u
v
v
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
CHC+
EH
+E
Tissue MDA (µMol/mg of protein)
Liver
Kidney
Figure 5: Eect of Costus pictus extract on tissue malondialdehyde
levels measured at the end of the study. Dierences between the groups
were analyzed using one‑way analysis of variance with Bonferroni post
hoc test. a = P < 0.05 in comparison to control group of liver tissue,
b=P < 0.05 in comparison to hypothyroid group of liver tissue, u=P < 0.05
in comparison to control group of kidney tissue, v=P < 0.05 in comparison
to hypothyroid group of kidney tissue; C: Control group; H: Hypothyroid
group; C+E: Control+extract group; H+E: Hypothyroid+extract group
S. ASHWINI, et al.: Insulin Plant Restores yroid Hormone Levels in Experimental Hypothyroidism
58 Pharmacognosy Research, Volume 9, Issue 1, January‑March, 2017
it was observed that oxidative stress was seen in the liver and kidney
tissues of hypothyroid rats as indicated by increased MDA and decreased
TAS in these rats. e presence of oxidative stress further augments the
hepatic and renal damage caused due to thyroid hormone insuciency.
Treatment with C. pictus extract signicantly decreased the levels of
AST, urea and creatinine in hypothyroid rats compared to untreated
group, further hepatic and renal oxidative stress were also found to be
decreased on treatment with extract. Previous studies have suggested that
exogenous antioxidants can alleviate hepatic and renal damage caused
in experimental hypothyroidism.[49-52] e protective eect of C.pictus
extract against hypothyroidism-induced kidney and liver damage could
be attributed to its antioxidant eect and its ability to restore thyroid
hormone levels.
In the present study, we found that body weight, food intake and water
intake were signicantly reduced in hypothyroid rats. ese results are
in line with earlier studies.[16,17,51] e reduction in water intake is due to
impaired ability to excrete water load in hypothyroidism.[53] e reduction
in food intake could be due to impairment in energy metabolic process
and decrease in basal metabolic rate as reported by previous studies.[54,55]
Treatment with C.pictus extract improved both food and water intake.
e benecial eect exhibited by the extract on the aforementioned
anthropometric parameters is mainly due to improvement in thyroid
prole caused by the extract. Although induction of hypothyroidism by
PTU is a well-accepted method of creating hypothyroidism in experimental
animals, it is still associated with certain limitations. is model fails
to produce increase in body weight as seen in human hypothyroidism.
is has been reported in earlier studies.[16,17,51] We have also observed
the same in our study. Despite this limitation, this model is still used
worldwide till date because PTU-induced hypothyroid rat model mimics
most of the main features of human hypothyroidism. yroidectomized
hypothyroid model was not used because in thyroidectomized rats there
is danger of removal of associated parathyroid glands resulting in tetany.
Although this is the rst study to report the benecial eect of C.pictus on
PTU-induced hypothyroidism, the exact mechanism at molecular level
has not been explored in the same. Further investigations at molecular
level are needed to conclusively prove the exact mechanism by which
C.pictus extract exerts its benecial eect with respect to thyroid prole
in experimental hypothyroidism.
CONCLUSIONS
e present study has revealed that C. pictus extract ameliorates
experimental hypothyroidism. Treatment with C. pictus extract in
PTU-induced hypothyroid rats increased plasma free T3, free T4 levels
and decreased TSH levels. Further, the extract exhibited antioxidant and
anti-inammatory eects, improved plasma lipid prole and partially
prevented hepatic and renal damage in hypothyroid rats. Pentacyclic
triterpenes alpha and beta amyrins were identied and quantied in the
extract. Amyrins could be responsible for the aforementioned benecial
eects exhibited by C. pictus extract in experimental hypothyroidism.
However, the exact molecular mechanism through which C. pictus
extract ameliorates hypothyroidism warrants further investigations.
C.pictus extract has the potential to emerge as a novel therapeutic agent
in the treatment of hypothyroidism.
Acknowledgement
We acknowledge metabolomics facility at Centre for cellular and
molecular platforms (C-CAMP), Bengaluru, India, for the LC-MS
analysis of C.pictus extract. We acknowledge technical services of
Ms.Padma Ramakrishnan, Technology associate, C-CAMP and
Dr.Kannan Rangiah, Technology manager, C-CAMP.
Financial support and sponsorship
We are grateful to Jawaharlal Institute of Postgraduate Medical Education
and Research (JIPMER), Puducherry, India for providing nancial
assistance in the form of intramural research grant.
Conicts of interest
ere are no conicts of interest.
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ABOUT AUTHOR
Dr. Zachariah Bobby is Professor and Head in Department of Biochemistry, JIPMER, Puducherry. He has more than
20years of teaching and research experience. His areas of research interest include molecular basis of metabolic syndrome,
post-menopausal state, chronic renal failure, hypothyroidism and effects of treatment with medicinal plant extracts like
green tea, amla (Indian goose berry), soy isoavones and insulin plant in alleviating the complications of some the above
mentioned pathological conditions in experimental animal models. He has more than 75 publications.
Dr. Zachariah Bobby
