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Journal of Ethnopharmacology 115 (2008) 173183

Review

Animal models to test drugs with potential antidiabetic activity


T.S. Frode a, , Y.S. Medeiros b
a

Department of Clinical Analysis, Center of Health Sciences, Federal University of Santa Catarina,
Campus Universitario, Trindade, 88040-970, Florianopolis, SC, Brazil
b Department of Pharmacology, Center of Biological Sciences, Federal University of Santa Catarina,
Campus Universitario, Trindade, 88049-900, Florianopolis, SC, Brazil
Received 1 March 2007; received in revised form 26 October 2007; accepted 26 October 2007
Available online 4 November 2007

Abstract
Although medicinal plants have been historically used for diabetes treatment throughout the world, few of them have been validated by scientific
criteria. Recently, a large diversity of animal models has been developed to better understand the pathogenesis of diabetes mellitus and new drugs
have been introduced in the market to treat this disease. The aim of this work was to review the available animal models of diabetes and some in
vitro models which have been used as tools to investigate the mechanism of action of drugs with potential antidiabetic properties. In addition, a
MEDLINE/PUBMED search for articles on natural products, pancreatectomy and diabetes mellitus treatment published between 1996 and 2006
was done. In the majority of the studies, natural products mainly derived from plants have been tested in diabetes models induced by chemical
agents. This review contributes to the researcher in the ethnopharmacology field to designs new strategies for the development of novel drugs to
treat this serious condition that constitutes a global public health.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Diabetes mellitus; Animal models; Genetic studies; Natural products

Contents
1.
2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vivo animal models of diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Pharmacological induction of diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Surgical models of diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Genetic models of diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1. Animal strains that spontaneously develop diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Genetically engineered diabetic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Other models of type 2 diabetes to evaluate the reduction of pancreatic -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vitro studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. In vitro studies on insulin secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. Studies using isolated pancreatic islet cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Studies using insulin-secreting cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. In vitro studies on glucose uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174
174
174
178
178
178
178
179
179
179
179
179
179

Abbreviations: ADP, adenosine diphosphate; AE, aqueous extract; AT, acute treatment; ATP, adenosine triphosphate; BB rat, Bio Breeding Laboratories rat;
BuOH, n-butanol fraction; Ca2+ , calcium; CH2 Cl2 , methylene chloride extract; CH3 Cl, chloroform extract; db/db mouse, monogenic model of obesity (leptin
resistant); DNA, deoxyribonucleic acid; DPP-4, dipeptidyl peptidase-4; EtOAc, ethyl acetate fraction; EtOH, ethanolic extract; GK rat, Goto-Kazikasi rat; GLUT,
glucose transporter; Hex, hexane fraction; i.p., intraperitoneal route; i.v., intravenous route; IGFs, insulin-like growth factors; MeOH, methanolic extract; NOD
mouse, non-obese diabetic mouse; Ob/Ob mouse, monogenic model of obesity mouse (leptin deficient); OLETL rat, Otsuka Long-Evans Tokushima Fatty rat; p.o.,
oral route; STZ, streptozotocin; ZK rat, Zucker rat.
Corresponding author. Tel.: +55 48 99614846; fax: +55 48 32440936.
E-mail addresses: saleh@ccs.ufsc.br, taniafrode@zipmail.com.br (T.S. Frode).
0378-8741/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2007.10.038

174

4.
5.
6.

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

Models of diabetes accelerated atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Potential effects of novel antidiabetic drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
The increasing worldwide incidence of diabetes mellitus in
adults constitutes a global public health burden. It is predicted
that by 2030, India, China and the United States will have the
largest number of people with diabetes (Wild et al., 2004). By
definition, diabetes mellitus is categorized as a metabolic disease
characterized by hyperglycemia resulting from defects in insulin
secretion, insulin action, or both. The vast majority of cases of
diabetes fall into two broad etiopathogenetic categories. In one
category, type 1 diabetes, the cause is an absolute deficiency
of insulin secretion. In the other, much more prevalent category, type 2 diabetes, the cause is a combination of resistance to
insulin action and an inadequate compensatory insulin-secretory
response (American Diabetes Association, 2005).
Despite the great interest in the development of new drugs to
prevent the burden of complications associated with this disease
and the raised interest in the scientific community to evaluate
either raw or isolated natural products in experimental studies
(Fig. 1), few of them were tested in humans (Liu et al., 2004;
Vuksan and Sievenpiper, 2005; Johnson et al., 2006; Jung et al.,
2006; Matsui et al., 2006). Nevertheless, natural supplements
are widely used around the world to treat diabetes, but medical
research does not support their effectiveness. In these studies the
low methodological quality, small sample size of tested patients,
and limited number of trials deserve caution in the interpretation of the positive data and require further examination in
high-quality trials (Liu et al., 2004). Nowadays, clinical treatment of diabetes targets both insulin deficiency and resistance
and more recently the prevention of pancreatic -cell function
decline (Hansotia and Drucker, 2005) (Table 1).
The aim of this work was to review the available animal models of diabetes in order to provide adequate tools to investigate
the mechanism of action of drugs with potential antidiabetic
properties. Considering that there is a marked overlap between
type 1 and 2 diabetes mellitus either in human beings or in ani-

Fig. 1. Number of papers that tested natural products in experimental models


of diabetes published in the period of 19962006 (source: PUBMED, English
language, accessed in 06-11-2007).

179
180
180
180

mal models, some classical models for each type of this disease
cannot also be sustained (Chaparro et al., 2006; Donath and
Ehses, 2006). However, it is cited, when appropriated along
the text, which category (type) the described model fits better.
The information in this review was obtained by searching
MEDLINE/PUBMED for literature published in English from
1996 to 2006, using terms natural products, pancreatectomy and
diabetes mellitus treatment. The decision of which model of diabetes to use for any particular protocol is mainly influenced by
local resources. Ideally, preclinical experiments should be initially carried out in vivo, and be complemented, when possible,
with in vitro studies to explore and advance in the mechanism
of action of a natural product.
2. In vivo animal models of diabetes mellitus
Diabetes can be induced by pharmacologic, surgical or
genetic manipulations in several animal species. Most experiments in diabetes are carried out on rodents, although some
studies are still performed in larger animals. The classical model
employed by Banting and Best was pancreatectomy in dogs
(Bliss, 2000). It is also described prone strains to diabetes mellitus that have been employed in several researches (Chen and
Wang, 2005; Rees and Alcolado, 2005; Masiello, 2006). Currently, the murine model is one of the most used due to the
availability of over 200 well-characterized inbred strains and the
ability to delete or over-express specific genes through knockout and transgenic technologies (Rees and Alcolado, 2005;
Masiello, 2006).
2.1. Pharmacological induction of diabetes
The majority of studies published in the field of ethnopharmacology between 1996 and 2006 employed these models.
Streptozotocin (STZ, 69%) and alloxan (31%) are by far the
most frequently used drugs and this model has been useful for the
study of multiple aspects of the disease. Both drugs exert their
diabetogenic action when they are administered parenterally:
intravenously, intraperitoneally or subcutaneously. The dose of
these agents required for inducing diabetes depends on the
animal species, route of administration and nutritional status.
According to the administered dose of these agents, syndromes
similar to either type 1, type 2 diabetes mellitus or glucose intolerance can be induced (Lenzen et al., 1996; Mythili et al., 2004).
Protocols are available any where, being critical the pH and type
of buffer employed as well as the preparation of the solution of
either alloxan or streptozotocin in the day of the experiments
(Yu et al., 2000; Gupta et al., 2005a,b; Lei et al., 2005; Babu et
al., 2006; Miranda et al., 2006).

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

175

Table 1
Drugs available to treat diabetes mellitus
Insulin deficiency
Insulin and its analogs
Sulphonylureas, glinides

Insulin resistance
Metformin
Glitazones
Alpha-glucosidase inhibitors

The cytotoxic action of these diabetogenic agents is mediated by reactive oxygen species, but both drugs differ in their
mechanism of action (Federiuk et al., 2004; Lei et al., 2005).
Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals.
These radicals undergo dismutation to hydrogen peroxide with
a simultaneous massive increase in cytosolic calcium concentration, which causes rapid destruction of pancreatic -cells
(Szudelski, 2001). The range of the diabetogenic dose of alloxan
is quite narrow and even light overdosing may be generally toxic
and may cause the loss of many animals. This loss is likely
to stem from kidney tubular cell necrotic toxicity, in particular when too high doses of alloxan are administered (Lenzen
et al., 1996). The most frequently used intravenous dose of
alloxan in rats is 65 mg/kg, but when it is administered intraperitoneally (i.p.) or subcutaneously its effective dose must be higher
(Federiuk et al., 2004). For instance, an intraperitoneal dose
below 150 mg/kg may be insufficient for inducing diabetes in
this animal species (Katsumata et al., 1992). In mice, doses vary
among 100200 mg/kg by intravenous route (i.v.) (Machado et
al., 2001; Miranda et al., 2006).
Streptozotocin enters the pancreatic -cell via a glucose
transporter-GLUT2 and causes alkylation of deoxyribonucleic
acid (DNA). Furthermore, STZ induces activation of poly adenosine diphosphate ribosylation and nitric oxide release. As a
result of STZ action, pancreatic -cells are destroyed by necrosis
(Mythili et al., 2004). In adult rats, 60 mg/kg is the most common dose of STZ to induce insulin dependent diabetes (Patel et
al., 2006), but higher doses are also used. STZ is also efficacious
after intraperitoneal administration of a similar or higher dose,
but single doses below 40 mg/kg may be ineffective (Katsumata
et al., 1992). In general, rats are considered diabetic if tail
blood glucose concentrations in fed animals are greater than
200300 mg/dl, 2 days after STZ injection. A model of type 2
diabetes can be induced in rats by either i.v. (tail vein) or i.p.
treatment with STZ in the first days of life. At 810 weeks of age
and thereafter, rats neonatally treated with STZ manifest mild
basal hyperglycemia, an impaired response to the glucose tolerance test, and a loss of pancreatic -cell sensitivity to glucose
(Pascoe and Storlien, 1990). It has been observed that STZ at
first abolished the pancreatic -cell response to glucose, but a
temporary return of responsiveness then appears which is followed by its permanent loss (Mythili et al., 2004). In adult mice,
STZ given in multiple low doses (40 mg/kg, i.v. for 5 days) (Rees
and Alcolado, 2005) induces an insulin dependent diabetes that
is quite similar to the autoimmune forms (islet inflammation and
-cell death) of type 1 diabetes. On the other hand, a single dose
between 60 and 100 mg/kg of STZ (Lei et al., 2005; Sharma
et al., 2006), administered systemically can also cause insulin

Prevention of -cell decline


Amylin analogs
Exenatide, DPP-4 inhibitors

dependent diabetes, but it lacks the autoimmune profile (Yu et


al., 2000).
The potential problem with STZ is that its toxic effects are
not restricted to pancreatic -cells since it may cause renal
injury (Valentovic et al., 2006), oxidative stress inflammation
and endothelial dysfunction (Lei et al., 2005).
The destruction of pancreatic -cells by both drugs is associated with a huge release of insulin which makes animals more
susceptible to severe hypoglycemia that may be lethal. Thus,
following treatment with either STZ or alloxan, animals are fed
with glucose solution (5%) for 1224 h. Afterwards, an increase
of glucose levels is observed in comparison to control animals
due to insulin deficiency. It is also reported that fasted animals
are more susceptible to alloxan effects (Katsumata et al., 1992;
Federiuk et al., 2004) and increased blood glucose in fed animals
provides partial protection (Federiuk et al., 2004). In general,
experimental protocols recommend that administration of either
STZ or alloxan must be done in the fasting period (812 h), followed by addition of glucose solution to avoid hypoglycemia.
Besides rats, dogs and mice, other animal species such as rabbits
and monkeys have been employed to induce diabetes by these
protocols, but rabbits and pigs are more resistant to STZ (Rees
and Alcolado, 2005).
In general, by using these models of diabetes induced by
chemical drugs, the majority of published studies report the
amount of reduction of blood glucose that is always evaluated
after a period of fasting following acute or chronic treatment
with a specific natural product. Comparative studies are carried
out with nondiabetic and/or diabetic animal groups treated with
known antidiabetic drugs, but results do not permit to further
explore the mechanism of action of the studied natural product.
Glucose is measured by standard glucose-oxidase or dehydrogenase assays, mainly by means of commercial meters available
everywhere. Insulin determination is available in experimental
animals by different methodologies (radioimmunoassayRIA
or immunometric assays) (Esmaeili and Yazdanparast, 2004). In
chronic experiments, A1c glycated hemoglobin can be also measured (Gupta et al., 2005b; Sathishsekar and Subramanian, 2005;
Sekar et al., 2005; Shirwaikar et al., 2005; Narendhirakannan et
al., 2006; Santhakumari et al., 2006; Tanaka et al., 2006).
Table 2 displays a list of plants and/or their active compounds
tested in diabetic animals (induced by either STZ or alloxan)
published in the past 2 years.
It is necessary to reemphasize that natural products display
several effects besides lowering blood glucose in these experimental models. In view of the lack of parallel studies of their
toxicity, these models of diabetes induced by either alloxan or
STZ are considered a screening step in the search for drugs for
the treatment of diabetes (Kecskemeti et al., 2002).

176

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

Table 2
List of studied natural products with putative antidiabetic effects tested in either in vivo or in vitro models in the period of 20052006 (source: PUBMED, English
language)
Plant (family)

Material

Treatment

Drug-induced
diabetes

Results

References

Aegle marmelos (Rutaceae)

EtOH/leaves

i.p., 14 d

STZ-rat

Narendhirakannan et al.
(2006)

AE/seeds
AE/fruits

p.o., 14 d
p.o., 30 d

STZ-rat
STZ-rat

Glucose, glycosylated
hemoglobin, C-peptide,
glucose
tolerance,glycogen, insulin
Glucose
Glucose, insulin

EtOH, isolated
compounds/leaves
EtOH/leaves

p.o., 28 d

db/db mice

Tanaka et al. (2006)

p.o., 21 d

STZ-rat

Glycosylated hemoglobin
A1c
Glucose, lipids

Whole plant, oil


fraction
AE, EtOH/leaves

p.o., 21 d

STZ-rat

Gluose; insulin

Kim et al. (2006)

p.o., 1030 d

STZ-rat

Glucose, lipids, lipidic


peroxidation, glucose
tolerance

Gupta et al. (2005a); Kaleem


et al. (2006)

p.o., 10 d
p.o., 15 d

Glycosylated hemoglobin,
glucose tolerance
Glucose, lipids

Gupta et al. (2005b)

Aloe vera (Liliaceae)

Amaranthus esculantus
(Amaranthaceae)
Annona squamosa
(Annonaceae)

Egyptian Morus alba


(Moraceae)
Eugenia jambolana
(Asteraceae)
Garlic (Allium sativum L.)
(Liliaceae)

Hintonia standleyan
(Rubiaceae)
Hypoxis hemerocallidea
(Hypoxidaceae)
Leonotis leonurus
(Lamiaceae)
Lepidium sativum
(Brassicaceae)
Lycium barbarum
(Solanaceae)
Malmea depressa
(Annonaceae)
Mangifera indica
(Anacardiaceae)
Momordica charantia
(Cucurbitaceae)

Rajasekaran et al. (2004)

AE, BuOH, EtOAc,


Hex/leaves
AE, EtOH,
BuOH/leaves
AE/leaves

p.o., 14 d

Alloxan-rabbit
STZ-rat,
alloxan-rabbit
STZ-rat

p.o., 7 d

STZ-mice

Glucose

Oliveira et al. (2005)

p.o./i.p., AT

STZ-rat

Glucose

Ojewole (2005a)

MeOH/CH2 Cl2 /stem


barks
AE/leaves

p.o.,14 d

STZ-rat

Glucose

Kamtchouing et al. (2005)

p.o., 15 d

STZ-rat

Glucose

Eddouks et al. (2005a)

AAE/stem barks

p.o., 12 d

STZ-rat

Shirwaikar et al. (2005)

AAE, isolated
compounds/stem
barks
AE, EtOH, isolated
compounds/fruitpulp/seeds
EtOH/bulbs

p.o., 10 d

STZ-rat

Glucose, glycosylated
hemoglobin, glycogen,
lipids, oxidative stress
Glucose, lipid
peroxidation, insulin

p.o., AT

STZ-rabbit

Glucose, lipid, glucose


tolerance

Sharma et al. (2006); Ravi et


al. (2005)

p.o., 14 d

STZ-rat

Glucose, lipids, insulin

Eidi et al. (2006)

Garlic oil, isolated


compounds

p.o., 21112 d

STZ-rat
alloxan

Glucose tolerance,
glucose, oxidative stress

Liu et al. (2005, 2006);


El-Demerdash et al. (2005)

MeOH, isolated
compounds/stem
barks
AE/fruits

p.o., AT

STZ-rat

Glucose

p.o., AT

STZ-mice, rat

Glucose

Guerrero-Analco et al.
(2005); Navarrete and Mata
(2005)
Ojewole (2006)

AE/leaves

p.o., AT

STZ-mice, rat

Glucose

Ojewole (2005b)

AE/leaves

p.o., AT, p.o.,15 d

STZ-rat

Glucose

Eddouks et al. (2005c)

Isolated
compound/fruits
AE, EtOH,
BuOH/roots
AE/stem barks

p.o., 2126 d

STZ-rat

p.o., AT

STZ-rat

Glucose, oxidative stress,


GLUT4, insulin
Glucose

Zhao et al. (2005b); Wu et al.


(2006)
Andrade-Cetto et al. (2005)

i.p., AT

SZT-rat

Glucose

Ojewole (2005c)

MeOH, isolated
compounds/gourd
AE/leaves

p.o., AT

SZT-mice
SZT-rat
STZ-rat,
alloxan-rat

Glucose

Shetty et al. (2005);


Harinantenaina et al. (2006)
Sekar et al. (2005); Reyes et
al. (2006)

Fruit-pulp
Averrhoa bilimbi
(Oxalidaceae)
Baccharis trimera
(Myrtaceae)
Bryophyllum pinnatum
(Crassulaceae)
Canarium schweinfurthi
(Burseraceae)
Chamaemelum nobile
(Asteraceae)
Coscinium fenestratum
(Menispermaceae)

Kesari et al. (2006)


Kamalakkannan and Prince
(2005)

p.o., 2730 d

Glucose, glycosylated
hemoglobin, oxidative
stress, glycogen

Tan et al. (2005)

Singap et al. (2005)

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

177

Table 2 (Continued )
Plant (family)

Piper betle (Piperaceae)

Material

Treatment

Drug-induced
diabetes

Results

References

AE/seeds

p.o., 30 d

STZ-rat

Sathishsekar and
Subramanian (2005)

Fruits

p.o.,21 d

Alloxan-rat

Glucose, glycosylated
hemoglobin, oxidative
stress, lipid peroxidation
Glucose, lipids

AE, EtOH/leaves

p.o., 30 d

STZ-rat

Glucose, glycosylated
hemoglobin

Arambewela et al. (2005);


Santhakumari et al. (2006)

Yadav et al. (2005)

p.o., AT
Psidium guajava Linn.
(Myrtaceae)
Raphanus sativus
(Brassicaceae)
Retama raetam (Fabaceae)
Salvia ofcinalis (Lamiaceae)
Scoparia dulcis
(Scrophulariacae)

AE/leaves

p.o., AT

STZ-rat

Glucose

Ojewole (2005d)

AE/whole plant

p.o., 21 d

STZ-rat

Glucose, lipids, insulin

Taniguchi et al. (2006)

AE/whole plant
AE/leaves
AE/whole plant

i.v., AT
p.o., 14 d
p.o., 2142 d

STZ-rat
STZ-rat, mice
STZ-rat-in
vitro/in vivo

Maghrani et al. (2005)


Lima et al. (2006)
Latha and Pari (2005);
Pari and Latha (2005, 2006)

Strobilanthes crispus
(Acanthaceae)
Syzygium cordatum
(Myrtaceae)
Syzygium cumini (synonym
Tamarindus indica
(Caesalpiniaceae)
Taxus yunnanensis
(Taxaceae)
Terminalia chebula
(Combretaceae)
Terminalia superba
(Combretaceae)
Trema orientalis (Ulmaceae)
Tremella mesenterica
(Combretaceae)
Triticum repens P. Beauv.
(Gramineae)
Viscum album L.
(Loranthaceae)
Zizyphus spina-christi
(Rhamnaceae)

AE/leaves

p.o., 21 d

STZ-rat

Glucose
Glucose gluconeogenesis
Glucose, lipids,
3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase
activity, oxidative stress,
insulin
Glucose

AE/leaves

p.o., 28 d

STZ-rat

Glucose, hepatic glycogen

Musabayane et al. (2005)

AE, EtOH, BuOH


fraction/leaves and
seeds
AE, MeOH, isolated
compounds/woods
CH3 Cl extract/seeds

p.o., 714 d

STZ-mice, rat

Glucose

Maiti et al. (2005); Oliveira et


al. (2005)

i.p., AT

STZ-rat

Glucose

Banskota et al. (2006)

p.o., AT

STZ-rat

Glucose

Rao and Nammi (2006)

MeOH, CH2 CL2 /stem


barks
AE/stem barks
Isolated
compounds/fruits
AE/rhizomes

p.o., 14 d

STZ-rat

Glucose

Kamtchouing et al. (2005)

p.o., AT
p.o., 14 d

STZ-rat
STZ-rat

Glucose
Glucose

Dimo et al. (2006)


Lo et al. (2006)

p.o., AT

STZ-rat

Glucose

Eddouks et al. (2005b)

AE, EtOH/whole
plant
BuOH, isolated
compounds/leaves

p.o., 7 d

STZ-rat

Orhan et al. (2005)

p.o., AT

STZ-rat

Glucose, lipidic
peroxidation
Glucose, insulin

Fadzelly et al. (2006)

Abdel-Zaher et al. (2005)

AAE: Alcoholic extract, AE: aqueous extract, AT: acute treatment, BuOH: n-butanol fraction, CH2 Cl2 : methylene chloride extract, CH3 Cl: chloroform extract,
EtOAc: ethyl acetate fraction, EtOH: ethanolic extract, GLUT-4: glucose transporter, Hex: hexane fraction, i.p.: intraperitoneal route, MeOH: methanolic extract,
p.o.: oral route, and STZ: streptozotocin.

In addition, considering the diversity of active principles


found in the crude plant extracts some studies also include further analysis of lipids (cholesterol, and triglycerides) as well as
additional evaluation of the antioxidant properties of the product.
This can be exemplified with studies with the fruit-pulp, seeds
and leaves extracted from Eugenia jambolona, whose antidiabetic properties are largely recognized by folk medicine in
several countries (Grover et al., 2002). Despite of the lack of
dose standardization, plant environment selection and toxicological studies, different studies have confirmed that the ethanolic
extracts of either the fruit-pulp or seeds of this plant present
hypoglycemic, hypolipemic and antioxidant properties (Sharma
et al., 2003; Pepato et al., 2005). These results suggest that
numerous active principles may be present in the raw extract of

the plant and highlight the need to further advance in their characterization. In line with this hypothesis, Tanaka et al. (2006) had
isolated several compounds with antihyperglycemic properties
from Aloe vera leaves (Tanaka et al., 2006). The identification of
these products explains, in part, why these compounds present
antioxidant, antihyperglycemic, antilypemic properties and even
enhance the process of wound healing in diabetic and nondiabetic animals (Okyar et al., 2001; Sharma et al., 2003, 2006;
Pepato et al., 2005; Rajasekaran et al., 2005; Tanaka et al., 2006).
Due to the nonspecific action of compounds isolated from
extracts of natural products, some studies have aggregated additional in vitro protocols to the in vivo studies such as liver
perfusion to evaluate glucose influx inhibition (Chung et al.,
2006), gastrointestinal absorption methodologies and antiox-

178

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

idant enzymatic systems, liver glycogen level, among others


(Babu et al., 2006; Fadzelly et al., 2006; McAnuff-Harding et al.,
2006). These protocols contribute to extend the analysis of the
antidiabetic effects of a certain natural product. In this context,
liver perfusion methodologies with the simultaneous measurement of glucose influx help to elucidate if the natural product
exerts extra-pancreatic effects like metformin and glitazones. On
the other hand, other studies suggest that inhibition of carbohydrate absorption may be linked to the antidiabetic properties
of the natural product. Thus, inclusion of at least two different
routes of treatment, for instance, i.p. and oral route (p.o.) can
help in the analysis of the possible site of action of a studied
natural product.
Vacor,
dithizone(diphenylthiocarbazone),
and
8hydroxyquinolone may also cause experimental diabetes,
but their use in research is restricted due to their level of toxicity
(Clark et al., 1994).
2.2. Surgical models of diabetes
Another technique used to induce diabetes is the complete
removal of the pancreas. Few researchers have employed this
model in the last years to explore effects of natural products
with animal species such as rats, pigs, dogs and primates (Choi
et al., 2004; Liu et al., 2004; Rees and Alcolado, 2005; Masiello,
2006). Limitations to this technique include (1) high level of
technical expertise and adequate surgical room environment,
(2) major surgery and high risk of animal infection, (3) adequate post-operative analgesia and antibiotic administration, (4)
supplementation with pancreatic enzymes to prevent malabsorption and (5) loss of pancreatic counter regulatory response to
hypoglycemia. More recently, partial pancreatectomy has been
employed, but large resection (more than 80% in rats) is required
to obtain mild to moderate hyperglycemia. In this case, small
additional resection can result in significant hypoinsulinemia
(Masiello, 2006).
Choi et al. (2004) investigated the action or relative glucose uptake in various tissues of 90% pancreatectomized rats
by using either hyperglycemic or euglycemic hyperinsulinemic
clamp methodologies. This experimental design permits to evaluate if the compound has some effect upon both resistance to
and secretion of insulin.
2.3. Genetic models of diabetes
2.3.1. Animal strains that spontaneously develop diabetes
These models permit the evaluation of the effect of a natural product in an animal without the interference of side
effects induced by chemical drugs like alloxan and STZ reported
above. Several recent publications summarized the major
advances in this field (for review see Rees and Alcolado, 2005;
Masiello, 2006) and at the website http://www.informatics.jax.
org).
Similar to the human condition, these strains display complex and heterogeneous characteristics. In some of these models,
insulin resistance predominates in association with obesity, dyslipidemia and hypertension, which provides valuable insights to

Fig. 2. Number of papers that tested natural products in prone strains to diabetes
published in the period of 19962006 (source: PUBMED, English language,
accessed in 06-11-2007).

study some events that are observed in human type 2 diabetes


mellitus. Conversely, some strains like Ob/Ob mouse may maintain euglycemia due to a robust and persistent compensatory
pancreatic -cell response, matching the insulin resistance with
hyperinsulinemia. On the other hand, the db/db mouse rapidly
develops hyperglycemia since their pancreatic -cells are unable
to maintain the high levels of insulin secretion required throughout life. Thus, food intake is important in determining the
severity of the diabetic phenotype and restriction of energy
intake reduces both the obesity and hyperglycemia seen in this
strain of mice. Another example is the spontaneously diabetic
Goto-Kakizaki rat which is a genetic lean model of type 2 diabetes originating from selective breeding over many generations
of glucose-intolerant nondiabetic Wistar rats (Chen and Wang,
2005). Regarding type 1 diabetes models, the NOD mouse typically presents hyperglycemia between 12 and 30 weeks of age,
whereas in BB rats it occurs around 12 weeks of age. One great
advantage of these models is that they can also be employed as
model of atherosclerosis which represents the long-term complication of diabetes mellitus and tested against several natural
products (Wu and Huan, 2007).
As it is shown in Fig. 2, the NOD mouse followed by the ZK
rat are the most used to test natural products.
Other prone strains to type 1 diabetes mellitus include New
Zealand white rabbit, Kreesbond dog, Chinese hamster and
Celebes black ape. However, they have not been employed in
studies to evaluate natural products to treat diabetes, except
in preclinical trials of exenatide (incretin analog) (Rees and
Alcolado, 2005).
2.3.2. Genetically engineered diabetic mice
In this case, rodents may be produced to over (transgenic)
or under (knockout)-express proteins thought to play a key part
in glucose metabolism. For a sound review on this topic see
Masiello (2006) and Clee and Attie (2006).
Although significant advances in this field have arisen in
recent years, especially with the advent of transgenic mice, there
have been no studies carried out involving natural products and
these models. Certainly, the high costs restrict their study in
sophisticated protocols which explore mechanisms of potential
therapeutic agents that either stimulate pancreatic -cell growth
or inhibit pancreatic -cell death (Meiton, 2006).

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

2.4. Other models of type 2 diabetes to evaluate the


reduction of pancreatic -cell mass
The importance of the progressive loss of pancreatic -cell
reduction in the course of type 2 diabetes has been the focus
of therapeutic targets in the development of novel and potential drugs acting by enhancing pancreatic -cell growth and/or
survival (Hansotia and Drucker, 2005; Masiello, 2006). Experimental reduction of pancreatic -cell mass by either surgical or
chemical means has been reported above, but these models are
limited due to the variety amount of residual pancreatic -cell
content. On the other hand, experimental studies carried out predominantly in rodents have demonstrated that incretins, IGFs,
hepatocyte growth factor, pituitary adenylate cyclase-activating
polypeptide are capable of enhancing pancreatic -cell mass in
the field of experimental diabetes (Stoffers et al., 2003). Hence,
prolonged administration of these agents may expand pancreatic
-cell mass, leading to increased insulin secretion and improved
glycemic control (Hansotia and Drucker, 2005).
A method to induce diabetes in adult rats is to mimic the
unfavorable intrauterine environment, which in humans leads
to low-birth weight and is supposed to confer high risk for
the development of diabetes in adult age (Holemans et al.,
2003). This model known as intrauterine growth retardation by
uteroplacental insufficiency in the rat is based on the premise
that uterine malnutrition may also increase the risk of diabetes
amongst offspring in later life (Simmons et al., 2001). This
has been achieved by several means, including bilateral uterine artery ligation (Simmons et al., 2001; Boloker et al., 2002),
at 19 days of gestation, i.e. 3 days before term. The diabetogenic effects of manipulating the intrauterine environment are
probably mediated by a permanent programming of the developing offspring, e.g. by the mechanism of imprinting. It should
also be pointed out that the increased risk of diabetes continues into subsequent generations, which in turn, suggests that
changes also affect the germ cell line (Reusens and Remacle,
2001).
Finally, animal models with increased pancreatic -cell apoptosis have also been developed, besides those cited in Section
2.3 (Matveyenko and Butler, 2006).
3. In vitro studies
3.1. In vitro studies on insulin secretion
Conventional antidiabetic agents can affect several pathways of glucose metabolism such as insulin secretion, glucose
uptake by target organs as well as nutrient absorption. Recently,
incretins (Hansotia and Drucker, 2005), and transcription factors such as peroxisome proliferator-activated receptorsPPAR
(Rosenson, 2007) are targets of modern therapy. Insulin receptor, glucose transporters, however, has not been yet the focus of
antidiabetic therapy. Although few studies using natural products have been published (Iwashima et al., 2001; Storling et
al., 2005; Zhao et al., 2005a), these methodologies may serve
as complementary tools to explore findings obtained in in vivo
models.

179

3.1.1. Studies using isolated pancreatic islet cell lines


Several in vitro assays are available to study different steps
of insulin secretion. It is known that insulin secretion occurs
when pancreatic -cells utilize glucose to generate adenosine triphosphate (ATP) from adenosine diphosphate (ADP)
(Affourtit and Brand, 2006). The resulting increase in cytoplasmic ATP/ADP ratio closes ATP-sensitive potassium channels,
causing depolarization of the plasma membrane, which activates
voltage-dependent Ca2+ channels. This results in elevation of the
intracellular Ca2+ concentration which triggers insulin secretion
(Ashcroft and Rorsman, 2004). In type 2 diabetes, pancreatic
-cells exhibit atypical ion channel activity and an abnormal pattern of insulin secretion (Ashcroft and Rorsman, 2004). These
pathways can be studied with isolated pancreatic -cells from
either control or diabetic rat or mouse that can be obtained by
collagenase digestion technique, followed by adequate separation and transference to appropriated culture medium (Zhao et
al., 2005a; Storling et al., 2005). Afterwards, the experimental
protocol is assayed.
3.1.2. Studies using insulin-secreting cell lines
Bioengineered technologies have provided new opportunities
to improve and establish more appropriate cultured cell lines to
help to facilitate studies of mechanisms of both insulin secretion
and -cell dysfunction, being also the target to the study of natural products. The most widely used insulin-secreting cell lines
are RIN, HIT, beta-TC, MIN6 and INS-1 cells (Poitout et al.,
1996). These cell lines release mainly insulin and small amounts
of glucagon and somatostatin. Although the behaviour of none of
these cell lines perfectly mimics primary -cell physiology, they
are extremely valuable tools for the study of molecular events
underlying -cell function (Poitout et al., 1996).
3.2. In vitro studies on glucose uptake
Adipose tissue is considered a key link between obesity and
type 2 diabetes by promoting the development of lipotoxicity,
i.e. cell damage as a consequence of elevated intracellular lipid
concentrations and insulin resistance (Lelliott and Vidal-Puig,
2004). Insulin resistance either at the adipocyte or skeletal muscle levels contribute to hyperglycemia. However, adipocytes
from different sites of the body may have different biological
or pathological effects.
Pathways related to insulin resistance may be studied in cell
lines of adipocytes such as murine 3T3-L1 cells (Karalee et al.,
2001) and rat L6 muscle engineered to over-express GLUT4
(Maddux et al., 2001) and may be employed as tools to evaluate
the effects of natural products upon glucose uptake.
4. Models of diabetes accelerated atherosclerosis
Accelerated cardiovascular disease is a leading cause of both
morbidity and mortality in diabetic patients (Wu and Huan,
2007). Aggressive therapy of dyslipidemia is necessary, since
the risk of myocardial infarction is the same as in nondiabetic
patients with previous myocardial infarction (Haffner et al.,
1998). Currently, rats and mice are the most widely used mod-

180

T.S. Frode, Y.S. Medeiros / Journal of Ethnopharmacology 115 (2008) 173183

els to study diabetes and atherosclerosis. Albeit, diabetic mice


do not exhibit a high degree of atherosclerosis unless hyperglycemia is associated with severe hyperlipidemia, a fat diet is
also present in these protocols. Models of diabetic nephropathy,
a microvascular complication have also been developed (Breyer
et al., 2005; Wu and Huan, 2007). For a review of these models
and other informations see the website: www.mmpc.org.
5. Potential effects of novel antidiabetic drugs
At present no drug is able to arrest the progressive loss
of pancreatic -cells which occurs in type 2 diabetes mellitus. According to the United Kingdom Prospective Diabetes
StudyUKPDS results, at the time of type 2 diabetes mellitus
diagnoses, 50% of pancreatic -cell function had already been
lost (Kan, 2001). Thus, the efficacy and side effect of marketed
oral antidiabetic drugs still need to be optimized. The recent
introduction in the market of incretin analogs opened a new
field to evaluate drugs with putative properties that may cause
both proliferation and maturation of human pancreatic -cells
(Hansotia and Drucker, 2005).
Currently, a standard model of experimental diabetes to study
effects of drugs, which could help in preventing the progressive
loss of pancreatic islet function remains to be established.
6. Concluding remarks
In spite of the worldwide use of herbs and medicinal plants,
the effective treatment of diabetes with phytochemicals has not
been validated with scientific criteria which may support their
substitution for the current therapy. Although some studies have
been published with raw natural products they have neither shed
light on the mechanisms of action of these products nor have
they shown a potential to be employed as new therapeutic drugs.
This implies that several models are necessary to called for, in
addition to the demonstration that a putative natural product
exerts antihyperglycemic activity. Thus, by focusing on other
targets of pancreatic islet cell dysfunction, new models may
help to elucidate effects of medicinal plants employed in the
treatment of diabetes mellitus.
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