Lv et al

Tropical Journal of Pharmaceutical Research July 2018; 17 (7): 1367-1372

ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.

Available online at http://www.tjpr.org

http://dx.doi.org/10.4314/tjpr.v17i7.20
Original Research Article

Effect of hydroalcohol extract of lemon (Citrus limon) peel

on a rat model of type 2 diabetes
Juan Lv1, Lanxiu Cao2*, Min Li12, Rui Zhang3, Fu Bai1, Pengfei Wei4
1
Department of Traditional Chinese Medicine, Department of Prescription, Basic Medical College, Shanxi University of
3
Traditional Chinese Medicine, Xianyang, 712046, Department of Diabetes, Second Affiliated Hospital of Shanxi University of
4
Traditional Chinese Medicine, Department of Radiotherapy, First Affiliated Hospital of Shanxi University of Traditional Chinese
Medicine, Xianyang, 712000, China

*For correspondence: Email: [email protected]

Sent for review: 8 March 2018 Revised accepted: 25 June 2018

Abstract
Purpose: To determine the effect of the hydroalcohol extract of lemon peel (LP) on a rat model of type
2 diabetes (T2D).
Method: The rat model of T2D by injection of streptozotocin was established. The effects of a
hydroalcoholic extract of LP was characterised on a rat model of type 2 diabetes based on body weight,
food intake, fasting blood glucose (FBG), glucose tolerance test, and insulin tolerance test. Antioxidant
activity and oxidative stress were analysed by superoxide dismutase (SOD) and malonaldehyde (MDA)
assays.
Results: In acute toxicity studies, administration of LP extract at 2000 mg/kg orally did not cause any
symptoms of poisoning or death after 14 days. The body weight of rats increased after treatment with
LP extracts. Food intake in diabetic rats decreased with LP extract treatment. Continual treatment with
LP extracts for 35 days significantly reduced blood glucose levels in diabetic rats. Glucose tolerance
improved, and insulin resistance was reduced after treatment with LP extracts. SOD and MDA data
indicate that treatment with LP extract alleviated the oxidative stress in diabetic rats as well as
enhanced the antioxidant activity of liver in a dosage-dependent manner.
Conclusion: LP extract decreased food intake and FBG, but increased body weight in rats. The effect
of LP on T2D is likely related to improved antioxidant activity and reduced oxidative stress. Thus, LP
extract has potentials for the treatment of T2D.

Keywords: Lemon peel, Type 2 diabetes, Antioxidant activity, Glucose tolerance, Insulin tolerance

This is an Open Access article that uses a funding model which does not charge readers or their institutions
for access and distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative
(http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited.

Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus,
International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African
Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals
(DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts

INTRODUCTION patients worldwide [1]. Relative to other

diseases, T2D has an extremely high occurrence
Type 2 diabetes (T2D) is a chronic disorder of of complications and may even contribute to
the metabolism of carbohydrate and lipid, which Alzheimer’s disease and depression [2,3]. Insulin
has long been diagnosed by hyperglycaemia with resistance and increased oxidative stress are
a high fasting blood glucose (FBG) level. generally accepted as underlying the main
Patients with T2D represent 90% of all diabetes pathogenesis of T2D and its complications [4,5].

-----------------------------------------------------------------------------------------------------------------------------------------------------
© 2018 The authors. This work is licensed under the Creative Commons Trop J Pharm4.0
Attribution Res, July 2018; License
International 17(7): 1367
 Lv et al

T2D is characterised by progressive Experimental animals

degeneration of insulin function, accompanied by
β-cell dysfunction that attempts to compensate Healthy male Sprague-Dawley (SD) rats with
for insulin resistance [6]. T2D is often associated weights ranging from 240 to 280 g were used in
with high levels of free radicals [7]and attenuated this study. All rats were raised in the same
antioxidant function [8]. Interestingly, the oxidant environment with a constant humidity of 50 % at
hydrogen peroxide is known to negatively impact 24 °C in a 12-h light/dark cycle. All animals were
glucose transportation capabilities and to raised and observed in specific pathogen free
stimulate insulin signalling elements [9]. Thus, laboratory animal room. The study was approved
oxidant stress may cause insulin resistance in by Animal Ethic Committee of the Affiliated
Hospital of Shanxi University of Traditional
mammalian muscle via the p38 MAPK pathway.
Chinese Medicine (no. EA_20160083), and the
For hundreds of years, traditional treatments for
experiments with rats were in full compliance
patients with T2D have utilised medicinal plants with the European Communities Council
that have few side effects. In recent years, Directive of 24 November 1986 (86/609/EEC)
medicinal plants have played an important part in [13] and with the Guidelines laid down by the NIH
new drug discovery. The most widely used anti- in the US [14].
T2D drug, metformin, was initially developed
from Galega officinalis, a medicinal plant that has Acute toxicity studies
been used for diabetes treatment for several
hundred years [10]. The extract of Galega Acute toxicity analysis of LP extracts was
officinalis provided a predicted chemical structure performed on SD rats according to Organization
for new anti-diabetes drugs. Therefore, there has for Economic Co-operation and Development
been increasing interest in the research on new (OECD) Guidelines for Testing of Chemicals No.
medicinal plants that have anti-diabetes 423. Before acute toxicity analysis, SD rats were
properties with few or no side effects to discover fasted for 5 h with only water available, and then
chemical structures that account for their divided into four groups with eight animals in
therapeutic effect. each group. The rats were administrated with LP
extract at a dosage of 5, 50, 500, and 2000
Lemon, which is grown worldwide, is used for mg/kg orally. The rats were observed individually
ethnomedicinal applications due to the anti- every 24 h for two weeks. General behaviours,
inflammatory and anti-tumour functions of including writhing, gasping, palpitation,
flavonoids and limonene that are contained in the decreased respiratory rate, and mortality were
observed.
plant [11,12]. Natural antioxidants extracted from
plants are attracting more interest for their in vivo
Establishment of rat model ofT2D and animal
radical scavenging activities. The bioactive
grouping
components that are extracted from lemon peel
(LP) depend on the lemon species and the To establish rat model of T2D, rats were adapted
method of extraction. to the new environment for 7 days and then
supplied with a 10 % fructose solution for 14
In this study, the peel of Citrus limon was used days. The rats were then injected with 30 mg/kg
for extraction by the hydroalcoholic method. The streptozotocin (STZ) in citrate buffer, which
anti-diabetic and antioxidant effects of LP extract causes pancreatic β-cell dysfunction. Rats in
were examined by performing experiments in rat control groups were fed with water followed by
model of T2D. injection with citrate buffer only [15]. FBG level
was measured with a glucose meter 7 days after
EXPERIMENTAL T2D induction. Fats with a FBG greater than 250
mg/dL were considered to be diabetic.
Plant material and extraction
The SD rats were divided into four groups:
Lemon (Citrus limon) was purchased from normal control (NC), diabetic control (DC),
Shanxi University of traditional Chinese diabetic + low dose of LP extract (DLDLPE, 250
Medicine. The LP was cleaned and dried at 50 mg/kg), and diabetic + high dose of LP extract
°C for 48 h. After grinding into powder, the lemon (DHDLPE, 500 mg/kg). LP extracts of different
peel was extracted in 50 % water / 50 % ethanol concentrations were administered orally to rats in
for 48 h using a Soxhlet extractor. Water and the DLDLPE and DHDLPE groups via a force-
feeding needle once a day continuously for 35
ethanol in the resultant liquid was removed by
days after establishing TD2 models. For NC and
lyophilizer. The crude extracts were stored at 4
DC groups, saline was administered instead. For
°C.
Trop J Pharm Res, July 2018; 17(7): 1368
 Lv et al

35 days, we recorded body weight, FBG, and groups were performed by Student’s t-test. P <
food intake of SD rats before and after drug 0.05 indicated statistical significance.
administration.
RESULTS
Glucose tolerance test (GTT)
Acute toxicity
To analyse glucose tolerance in SD rats, GTT
was performed. After rats were fasted for 12 h, In acute toxicity analysis, after 14 days of orally
blood glucose was measured. The rats were administering LP extract at 2000 mg/kg, any
administered with saline or LP extracts 1 h prior symptoms of poisoning or death were not
to glucose administration. After sampling blood observed, indicating that LP extract was not toxic
from the tail vein, glucose was immediately for rats at 2000 mg/kg. Therefore, oral dosages
intraperitoneally injected into rats at a dose of 1 of 250 and 500 mg/kg were safe and feasible for
g/kg. Blood glucose was measured at 0, 0.5, 1, this study.
1.5, and 2 h after glucose administration.

Insulin tolerance test (ITT)

After the GTT was performed on overnight-fasted

rats daily for 4 days. The rats were administered
saline or LP extracts 1 h before insulin
administration. After sampling blood from the tail
vein, a single dose of insulin solution (0.5 U/kg)
was immediately subcutaneously injected into
each rat. Blood glucose was tested at 0, 0.5, 1,
and 1.5 h after insulin injection.

Histological assessment Figure 1: Effect of LP extracts on glucose tolerance.

 represents NC group; ♢ = DC group (T2D rats
After 24 h fixation, pancreas tissues were treated with vehicle); Δ = DLDLPE group (T2D rats
dehydrated, cleared in xylene, and immersed in treated with a low dose of LP extracts);  represents
paraffin for embedding. The embedded tissue DHDLPE group (T2D rats treated with a high dose of
was sliced by microtome, dehydrated, and de- LP extracts)
waxed. The slices were then stained with
haematoxylin, washed with distilled water, Body weight, FBG, and food intake
decolorized using hydrochloric acid ethanol, and
then stained again with eosin. Slices were then Body weight, FBG, and food intake was
dehydrated, dried, and sealed for observation evaluated for 35 days, before and after
under microscope. administration of LP extract or saline. Body
weight was found to decrease after establishing
Assessment of oxidative stress and the rat model of T2D (Table 1). During saline
antioxidant activity administration, the body weight of diabetic rats
continued to decrease. However, after treatment
Superoxide dismutase (SOD) and with LP extracts, the body weight of rats
malonaldehyde (MDA) extractions were increased in the DLDLPE and DHDLPE groups.
performed by phosphate-buffered saline after the A high dose of LP extract (500 mg/kg) increased
liver tissues was ground by liquid nitrogen body weight more effectively than a low dose of
grinding. SOD activity was analysed by a LP extract (250 mg/kg). Food intake also
commercial kit (Beyotime, China) and expressed increased after establishing a model of T2D in
as units/mg protein. MDA content was analysed rats. After treatment with LP extracts, food intake
using a commercialMDA assay kit (Beyotime, decreased in diabetic rats. However, a high dose
China) and expressed as μmol/mg protein. The of LP extract did not reduce food intake more
concentration of total protein was measured by than a low dose of LP extract. The rats in the DC
Bradford protein kit (Beyotime, China) [16]. group had a significantly higher FBG level (p <
0.01) than the NC group. Continuation of
Statistical analysis treatment with LP extracts for 35 days
significantly reduced blood glucose levels of
The data was analysed using SPSS 21 software diabetic rats.
(IBM, USA). The results are presented as mean
± standard deviation. Comparisons between two

Trop J Pharm Res, July 2018; 17(7): 1369

 Lv et al

Table 1: Effect of LP extract treatment on food intake and body weight before and after drug administration in
STZ-induced diabetic rats. NC, Normal rats treated with vehicle alone; DC, T2D rats treated with vehicle alone;
DLDLPE, T2D rats treated with a low dose of LP extracts (250 mg/kg); DHDLPE, T2D rats treated with a high
dose of LP extracts (500 mg/kg)

Parameter NC DC DLDLPE DHDLPE

Body weight (g) Before 269.0±5.2 220.0±7.7 224.0±6.5 225.0±8.7
After 288.0±4.5 170.0±5.2 250.0±3.8 265.0±5.3
Food intake Before 17.5±2.1 30.5±2.5 29.2±1.2 31.5±2.1
(g/rat/day) After 19.2±1.8 33.4±2.2 22.4±1.7 26.3±2.6
FBG Before 100.2±5.7 283.8±7.5 280.6±15.3 277.7±8.6
(mg/dl) After 109.8±10.5 288.4±12.8 180.6±6.5 155.1±12.2

GTT and ITT

In the GTT, the blood glucose level of rats in the

NC, DLDLPE, and DHDLPE groups reached a
maximum value at 0.5 h and then decreased
over time. However, rats in the DC group
reached a maximum blood glucose value at 1 h.
In the ITT, after insulin administration, FBG
initially decreased and then increased in the
DLDLPE and DHDLPE groups, when compared
to the DC group. After insulin administration for 2
h, the FBG level in the DLDLPE group was close
to that of the DC group. In the NC and DHDLPE
groups, FBG reached a minimum value 0.5 h
after insulin administration. In the DC and
DLDLPE groups, FBG level reached a minimum
value 1 h after insulin administration.

Figure 3: Effect of treatment with LP extracts on

pancreatic islet tissues of rats in T2D model and
control groups. NC, Normal rats treated with vehicle
alone; DC, T2D rats treated with vehicle alone;
DLDLPE, T2D rats treated with a low dose of LP
extracts (250 mg/kg); DHDLPE, T2D rats treated with
a high dose of LP extracts (500 mg/kg)

Antioxidant activity and oxidative stress

Figure 2: Effect of LP extracts on insulin tolerance.  The antioxidant activity and oxidative stress were
= NC group; ♢represents DC group (T2D rats treated determined by measuring MDA level and SOD
with vehicle); Δ represents DLDLPE group (T2D rats activity, respectively. The increase of MDA was
treated with a low dose of LP extracts);  = DHDLPE observed in diabetic rats, indicating that the
group (T2D rats treated with a high dose of LP oxidative stress in rat liver significantly increased
extracts) after T2D model establishment (p < 0.05) (Figure
4A). The oxidative stress, as measured by SOD
Histological features activity, was alleviated in diabetic rats after
treatment with a high dose of LP extract.
Haematoxylin-eosin staining results showed However, low doses of LP extract did not have
serious damage to the intra-pancreatic islets and the same effect on oxidative stress as high
the number and size of islets was decreased in doses. The decreased SOD activity in diabetic
STZ-induced diabetic rats (Figure 3A and Figure rats indicated that antioxidant activity decreased
3B). The vacuolation and invasion of in the liver after T2D model establishment, and
intrapancreatic tissue in the diabetic rats were that treatment with LP extract restored
also observed. After treatment with LP extract, antioxidant activity in a dose-dependent manner
the regeneration of intrapancreatic islet cells and (p < 0.05) (Figure 4B).
enhanced restoration of intrapancreatic tissues
were observed (Figure 3 C and Figure 3 D).

Trop J Pharm Res, July 2018; 17(7): 1370

 Lv et al

persistent hyperglycaemia destroys antioxidant

balance by reducing antioxidant levels and
producing reactive oxygen species [21,22], which
is consistent with the result of this study (Figure
4). The results suggest that treatment with LP
extract may alleviate T2D symptoms by reducing
oxidative stress and restoring antioxidant activity.
Figure 4: Effect of LP extracts on MDA (A) and SOD
(B)of liver tissues; #p < 0.05 compared with NC group; CONCLUSION
*p < 0.05 compared with DC group. NC, Normal rats
treated with vehicle alone; DC, T2D rats treated with The findings of this study show that treatment of
vehicle alone; DLDLPE, T2D rats treated with a low type 2 diabetic rats with LP extract ameliorates
dose of LP extracts (250 mg/kg); DHDLPE, T2D rats T2D by reducing food intake and FBG, and
treated with a high dose of LP extracts (500 mg/kg)
increasing body weight. Glucose tolerance and
insulin tolerance are also improved after LP
DISCUSSION administration. The effect of treatment with LP
extract is related to increased antioxidant activity
Lemon has been used as a traditional medicine, and reduced oxidative stress.
likely due to the anti-inflammatory and anti-
tumour functions of flavonoids and limonene that DECLARATIONS
are contained in the plant [11,12]. In this study,
the anti-diabetic and antioxidant activities of Conflict of Interest
extracts of lemon peel was evaluated. Type 2
diabetes is a chronic disorder of energy
No conflict of interest associated with this work.
metabolism, which is diagnosed by
hyperglycaemia with a consistently high FBG
Contribution of Authors
level. In this study, STZ-induced T2D rat model
was used to assess the effect of treatment with
LP extract on T2D.The acute toxicity of LP We declare that this work was done by the
extract and the effect of LP extract on FBG, body authors named in this article and all liabilities
weight, food intake, GTT, ITT, histological pertaining to claims relating to the content of this
changes, and oxidative stress were tested. article will be borne by the authors. Lanxiu Cao
designed all the experiments and revised the
Reduced body weight is a representative paper. Juan Lv, Min Li, Rui Zhang and Fu Bai
symptom for T2D in rats [17]. The establishment performed the experiments, and Pengfei Wei and
of a T2D rat model caused reduced body weight Juan Lv wrote the paper.
in this study. Reduction of body weight was
rescued in DLDLPE and DHDLPE groups, REFERENCES
indicating that treatment with LP extracts
increased the body weight of diabetic rats. 1. Mohammed A, Md S. Butanol fraction of Khaya
Treatment with LP extracts also reduced food senegalensis root modulates β-cell function and
intake and FBG in diabetic rats. These results ameliorates diabetes-related biochemical parameters in
suggest that LP extracts may alleviate typical a type 2 diabetes rat model. J Ethno pharmacol
T2D symptoms. In previous studies, the anti- 2014;154(3): 832-838.
diabetes mechanisms of natural plant medicines
2. Pouwer F, Beekman AT, Nijpels G, Dekker J, Snoek FJ,
were found to be involved in the maintenance of
Kostense P, Heine R, Deeg D. Rates and risks for co-
glucose homeostasis, gastrointestinal glucose
morbid depression in patients with Type 2 diabetes
absorption, insulinotropic actions, and promoting
mellitus: results from a community-based study.
pancreatic β-cell regeneration [18-20].
Diabetologia 2003; 46(7): 892-898.

Furthermore, the results of GTT and ITT showed 3. Ahtiluoto S, Polvikoski T, Peltonen M, Solomon A,
that treatment with LP extracts improved glucose Tuomilehto J, Winblad B, Sulkava R, Kivipelto M.
tolerance and reduced insulin resistance. Diabetes, Alzheimer disease, and vascular dementia A
Histological analysis showed that LP extracts population-based neuropathologicstudy. Neurology
restored the structure of and helped regenerate 2010; 75(13): 1195-1202.
intra-pancreatic islets. SOD and MDA analyses 4. Pistrosch F, Passauer J, Fischer S, Fuecker K, Hanefeld
indicated that treatment with LP extract alleviated M, Gross P. In type 2 diabetes, rosiglitazone therapy for
oxidative stress in diabetic rats’ and restored insulin resistance ameliorates endothelial dysfunction
antioxidant activities in the liver in a dose- independent of glucose control. Diabetes Care 2004;
dependent manner. It was reported that 27(2): 484-490.

Trop J Pharm Res, July 2018; 17(7): 1371

 Lv et al

5. Maritim AC, Sanders RA. Diabetes, oxidative stress, and 15. Wilson RD, Islam MS: Fructose-fed streptozotocin-
antioxidants: a review. J Biochem Mol Toxicol 2003; injected rat. an alternative model for type 2 diabetes.
17(1): 24-38. Pharmacol Rep 2012; 64(1): 129-139.
6. Stumvoll M, Goldstein BJ, Haeften TWV: Pathogenesis of 16. Bradford MM. A rapid and sensitive method for the
type 2 diabetes mellitus. Med Clin N Am 2004; 88(4): quantitation of microgram quantities of protein utilizing
787-835. the principle of protein-dye binding. Anal Biochem1976;
7. Baynes JW. Role of oxidative stress in development of 72(1-2): 248-254.
complications in diabetes. Diabetes 1991; 40(4): 405- 17. Haidari F, Shahi MM, Zarei M, Rafiei H, Omidian K. Effect
412. of green tea extract on body weight, serum glucose and
8. Saxena AK, Srivastava P, Kale RK, Baquer NZ. Impaired lipid profile in streptozotocin-induced diabetic rats. A
antioxidant status in diabetic rat liver. Effect of vanadate. dose response study. Saudi Med J 2012; 33(2): 128-
Biochem Pharmacol 1993; 45(3): 539-542. 133.
9. Archuleta TL, Lemieux AM, Saengsirisuwan V, Teachey 18. Gin H, Rigalleau V. Post-prandial hyperglycemia. post-
MK, Lindborg KA, Kim JS, Henriksen EJ. Oxidant prandial hyperglycemia and diabetes. Diabetes
stress-induced loss of IRS-1 and IRS-2 proteins in rat Metab2000; 26(4): 265-272.
skeletal muscle: role of p38 MAPK. Free Radical Biol 19. Musabayane CT, Bwititi PT, Ojewole JA. Effects of oral
Med 2009; 47(10): 1486-1493. administration of some herbal extracts on food
10. Cragg GM, Newman DJ. Natural products: a continuing consumption and blood glucose levels in normal and
source of novel drug leads. Biochim Biophys Acta 2013; streptozotocin-treated diabetic rats. Methods Find Exp
1830(6): 3670-3695. ClinPharmacol2006; 28(4): 223-228.
11. Jr ME, Kandaswami C, Theoharides TC. The effects of 20. Islam MS, Choi H. Dietary red chilli (Capsicum frutescens
plant flavonoids on mammalian cells: implications for L.) is insulinotropic rather than hypoglycemic in type 2
inflammation, heart disease, and cancer. Pharmacol diabetes model of rats. Diabetes Res ClinPract2008;
Rev 2000; 52(4): 673-751. 22(8): 1025-1029.
12. Huang YS, Suchen H. Polymethoxy flavones are 21. Aragno M, Mastrocola R, Catalano MG, Brignardello E,
responsible for the anti-inflammatory activity of citrus Danni O, Boccuzzi G. Oxidative stress impairs skeletal
fruit peel. Food Chem 2010; 119(3): 868-873. muscle repair in diabetic rats. Diabetes2004; 53(4):
13. European Union Commission Regulation. EC 1082-1088.
86/609/EEC. Off. J. Eur. Union 1986, L358, 1–28. 22. Nishikawa T, Edelstein D, Du XL, Yamagishi S,
14. National Institutes of Health. Guidelines regarding the Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates
care and use of animals for experimental procedures PJ, Hammes HP. Normalizing mitochondrial superoxide
(LIN-DL-11) NIH Publication No.8 5–23, revised 1985 production blocks three pathways of hyperglycaemic
damage. Nature 2000; 404(6779): 787-790.

Trop J Pharm Res, July 2018; 17(7): 1372