Dillenia indica L.

Sanjeet Kumar
Dillenia indica L.
Common name: Awoo
Botanical name: Dillenia indica L.
Family: Dilleniaceae
Distribution
It is native to Southeastern Asia, Deccan peninsula, Sub-Himalayan tract, West Bengal,
Assam, Meghalaya, Sri
Lanka,Myanmar, China,
Sumatra, Java, Bangladesh, Vietnam, Thailand, Malaysia, Indonesia.

Botany of Dillenia indica

Moderate sized evergreen tree with a dense crown. Trunk rather crooked and irregular. Leaves deep
green, oblong to lanceolate, pubescent beneath. Flowers white, solitary. Sepals, elliptic, thick. Carpels
yellowish green with linear-lanceolate. Fruit indehiscent, yellowish-green. Seeds 5 or more in each
carpel in colourless glutinous pulp, reniform, black.
Medicinal properties
Fruit juice is mixed with sugar is used as cooling beverage against fever. Fresh fruit juice is used as a
cardio tonic. Leaves and bark are used as laxative. Different leaf extracts have anti-inflammatory
activity.
Chemical Compound(s)
It has Lupeol group of triperpene, sucha as Betulinic acid, Flavonol such as myricetin. In stem bark
there are many chemical compounds such as betulin, betulinaldehyde, betulic acid, flavonoids,
dillentin, dihydroisorhamnetin, lupeol, glucosides, B-sitosterol; Wood: betulinic acid, lupeol, sitosterol; Leaf: cycloartenone, flavonoids, n-hentriacontanol, Bsitosterol; Fruit: an arabinogalactan,
betulinic acid, -sitosterol.

Common Use(s)
Fruits are edible in Odisha and seed pulp is used to make jam and jellies.
It is planted as ornamental plants due to beautiful flowers.
Fruit decoction is used to cure dandruff in Manipur.
Leaf paste is used to cure dysentery by Santhal in Odisha.
Posted by Aaditya Sanjeet Kumar at 09:58
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Brazilian Journal of Pharmaceutical Sciences

Print version ISSN 1984-8250

Braz.J.Pharm.Sci.vol.47no.2SoPauloApr./June2011
http://dx.doi.org/10.1590/S1984-82502011000200018

ARTICLE

Antidiabetic and antihyperlipidemic effects ofDillenia

indica (L.) leaves extract

Sunil Kumar; Vipin Kumar*; Om Prakash

Institute of Pharmaceutical Sciences, Kurukshetra University, Haryana, India

ABSTRACT
The present study was carried out to evaluate antidiabetic and antihyperlipidemic effects
of Dillenia indica methanolic leaves extracts in streptozotocin induced diabetic Wistar rats
by administering graded oral doses (250 and 500 mg/kg body weight) for 21 days. The
extract showed significant antidiabetic activity (p<0.001). Furthermore, the decreased
body weight of rats was significantly improved after extract treatments. Daily oral
treatment with the extract for 21 days to diabetic rats, also resulted in significant
reduction in serum cholesterol, triglycerides and serum transaminase levels but HDLcholesterol level was found to be improved (p<0.001) as compared to the diabetic
control group. The extract treatment also showed to enhance serum insulin level in
diabetic rats as compared to the diabetic control group. In conclusion, D. indica leaf
extract might be useful for diabetes mellitus management and other abnormalities
associated with this metabolic disorder.
Uniterms: Dillenia indica/antidiabetic effects/experimental study. Dillenia
indica/antihyperlipidemic effects/experimental study. Antidiabetics/experimental study.
Serum transaminase. Serum cholesterol. HDL-cholesterol level.

RESUMO

Realizou-se o presente estudo para avaliar os efeitos antidiabtico e anti-hiperlipidmico

de extratos metanlicos de folhas de Dillenia indica em ratos wistar com diabetes
induzido por estreptozotocina por meio da administrao de doses orais (250 e 500
mg/kg de peso corporal) por 21 dias. O extrato mostrou atividade antidiabtica
significativa (p<0,001). Alm disso, a diminuio do peso corporal dos ratos foi
significativamente melhorada aps o tratamento com os extratos. O tratamento com
doses orais do extrato por 21 dias aos ratos diabticos tambm resultou em reduo
significativa do colesterol, triglicerdios e nveis de transaminase sricos, mas o nvel de
HDL-colesterol foi melhorado (p<0,001), quando comparado ao grupo controle diabtico.
O tratamento com extrato tambm mostrou aumento do nvel srico de insulina em ratos
diabticos comparativamente ao grupo controle diabtico. Em concluso, o extrato de
folha de D. indica poderia ser til para o controle do diabetes mellitus e de outras
anormalidades associadas a essa disfuno metablica.
Unitermos: Dillenia indica/efeitos antidiabticos/estudo experimental. Dillenia
indica/efeito anti-hiperlipidmico/estudo experimental. Antidiabticos/estudo
experimental. Transaminase srica. Colesterol srico. Nvel de HDL-colesterol.

INTRODUCTION
Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat and protein
metabolism that affects nearly 25% of the population afflicting 150 million people, a
figure set to rise to 300 million by 2025 (Vats et al., 2000; Vetrichelvan et al., 2002).
The disease causes numerous complications such as retinopathy, neuropathy, and
peripheral vascular insufficiencies (Chehade, Mooradian, 2000). Hyperglycemia can be
handled initially with oral synthetic agents and insulin therapy. However, these synthetic
agents produce some serious side effects and are relatively expensive for developing
countries. Therefore, searching for effective, low cost hypoglycemic agents with fewer
side effects is important (Rubin et al., 1994). Since ancient times, diabetes has been
treated orally with several medicinal plants or their extracts based on folklore medicine.
The World Health Organization has also recommended the evaluation of traditional plant
treatments for diabetes (Day, 1998).
Dillenia indica Linn. (Family: Dilleniaceae) is an evergreen tree, 30-80 ft. in height, which
bears large and hard fruit 3-5 in. in diameter and grows in moist and evergreen forests
of India. The ripe fruits are widely used in the flavoring of curries and preparation of jam
and jelly. The acidic juice is sweetened with sugar and used as a cooling drink (The
Wealth of India, 1992). The fruit is said to possess tonic laxative properties and is used
for relieving abdominal pain. The bark and leaves are astringent (Kirtikar, Basu, 2003).
The mixed juices of leaves, bark and fruits are given orally for the treatment of cancer
and diarrhea (Sharma et al., 2001). Traditionally, the plant is also used for treatment of
diabetes (Sood et al., 2005). The literature survey revealed that there is no experimental
evidence of the plant's antidiabetic effect. Therefore, the present work was undertaken

to explore the antidiabetic and antihyperlipidemic potentials of the leaves extract of the
plant.

MATERIAL AND METHODS

Plant material
D. indica leaves were collected from the campus of Kurukshetra University, Kurukshetra,
India during the month of October, 2009 and were identified by Dr. H.B. Singh, scientist
F& Head, Raw Material Herbarium & Museum, NISCAIR, and New Delhi, India. A voucher
specimen of the plant is preserved in the herbarium (NISCAIR/RHMD/Consult/-200910/1381/182/1).
Extract preparation
The leaves were dried under shade and powdered to coarse particles. The powdered
plant material was defatted with petroleum ether (60-80C) in a Soxhlet extraction
apparatus at 60C and further the same amount of plant material extracted with
methanol. The extract was dried at 45C in a rotary evaporator to produce a semisolid
mass and stored in airtight containers in a refrigerator below 10 C.
Chemicals
Streptozotocin was purchased from Sigma-Aldrich, India. Total cholesterol, High density
lipoprotein (HDL)-cholesterol and triglyceride (TC), serum transaminase were assayed by
an autoanalyser (Erba Chem 7, Mannheim, Germany) using standard kits from Erba
diagnostics Mannheim Gambh, Germany, and Blood glucose level was measured using an
Elegance glucose meter (CT-X10) by Convergent Technologies, Germany. All reagents
used in the study were analytical grade.
Animals
Wistar rats of both sexes (15 male and 15 female rats), weighing about 150-250 g were
used in the study. Animals were maintained under standard environmental conditions i.e.
ambient temperature of 22 2 C and at 45-55% relative humidity for 12 h, each of
dark and light cycle, and fed with a standard pellet rat diet obtained from Ashirwad
Industries, Chandigarh, India and water was supplied ad libitum. All the studies were
conducted in accordance with the Animal Ethics Committee of the University.
Antidiabetic studies
Induction of diabetes
After one week of acclimatization, the rats were subjected to a 12-h fast. Diabetes was
induced with a single injection of streptozotocin (STZ) 60 mg/kg body weight i.p. The
STZ was freshly dissolved in citrate buffer (0.01 M, pH 4.5). The blood glucose level was
checked before and 72 h after streptozotocin injection to confirm the development of

diabetes. The diabetic animals were stabilized for five days and the experiment was
started on the next day (day 0). Only those animals which showed hyperglycemia (blood
glucose levels >250 mg/dL) were used in the experiment.
Experimental design
Overnight fasted rats were divided into five groups and six animals were taken for each
group. The extract was dissolved in Tween 80, 1% v/v in saline and given orally. Group I
(Normal healthy control) received only vehicle (Tween 80, 1% v/v in saline). Group II
served as the diabetic control, Group III and IV received D. indicamethanolic leaves
extract (DIME) 250 and 500 mg/kg.b.w respectively once a day for 21 days and Group V
received (Glibenclamide 10 mg/kg.b.w.) once a day for 21 days and served as the
standard.
Biochemical parameters
Blood glucose was measured with an Elegance glucometer (Frankenberg, Germany) at
weekly intervals i.e. 0, 7, 14 and 21 days after daily administration of extract orally. On
the 21st day all the animals were sacrificed and evaluated for the biochemical status of
serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline
phosphatase (ALP) by autoanalyser using Erba diagnostic kits (Brandely, Maynard, 1972;
Wilkinson et al., 1969). Serum insulin levels were determined using insulin ELISA kits
(Kratzsch et al., 1990).
Antihyperlipidemic effect
Serum total cholesterol and triglycerides were determined by the method of Rifai et al.,
1999. HDL-cholesterol was also evaluated in normal and streptozotocin-induced diabetic
rats by autoanalyser using Erba diagnostic kits (Burstein et al., 1970).
Statistical analysis
Results are presented as mean standard error of mean (S.E.M.) The statistical analysis
involving two groups was evaluated by means of Student's t-test whereas One way
analysis of variance (ANOVA) followed by Dunnet's multiple comparison post-test was
used for statistical comparison between control and the various treated groups.
Statistical significance was accepted at the p <0.05 values.

RESULTS
Effect on blood glucose level and serum insulin
Table I shows the results of blood glucose values in STZ-induced diabetic rats, after the
daily treatment with the methanolic extract of D. indica (DIME 250 and 500 mg/kg,
p.o.), for 21 days. A significant decrease (p<0.001) in blood glucose levels were
observed in the groups treated with both doses of DIME of 48.64 and 55.86%,
respectively, as compared to the same group before treatment. At the end of the

experiment (21st day) blood glucose level was 139.43 2.28 and 124.32 1.75 mg/dL
of the groups treated with the doses of DIME 250 and 500 mg/kg, respectively. Serum
insulin level was also significantly improved by treatment with DIME in the diabetic rats.
Effect on body weight
Body weight was slightly increased in the normal control rats (group I) compared to
initial body weight whereas in the diabetic control rats (group II) there was a significant
decrease in body weight. Glibenclamide (10 mg/kg) as well as the extracts (DIME 250
and 500 mg/kg) treatment significantly (p < 0.05) prevented this reduction in body
weight. Thus, based on weekly observation of the extract-treated diabetic rats, there
were significant weight gains on day 21 compared to day 0 as shown in Table II.
Effect on lipid profile and serum transaminase
In diabetic rats, there was a significant increase in total cholesterol and triglycerides, as
well as a significant decrease in HDL cholesterol in serum compared to that of normal
controls. The standard drugs as well as DIME (250 and 500 mg/kg) plant extracts used
in the experimental study significantly decreased (p < 0.05) the levels of cholesterol and
triglycerides whereas HDL cholesterol level was improved (Table III) after 21 days of
treatment. Also, at the end of study, the serum transaminase, such as AST, ALT and ALP
activity, was significantly elevated in the diabetic control groups. After supplementation
with DIME (250 and 500 mg/kg) and glibenclamide (10 mg/kg), the serum transaminase
level was restored to normal levels (Table III).

DISCUSSION
Streptozotocin possess diabetogenic properties mediated by pancreatic beta cell
destruction, the cells that normally regulate blood glucose levels by producing the
hormone insulin, and this compound has been widely used to induce diabetes in
experimental animals (Junod, 1969). A 21-day treatment by DIME and glibenclamide in
the STZ-induced diabetic rats significantly reduced the elevated blood sugar levels. This
shows that the extract may not be able to produce the effect by one dose but by
continuous treatment it acts effectively. It is well established that glibenclamide (a long
lasting sulfonylurea) acts mainly by stimulating insulin secretion. The DIME treatment
also increased the insulin level. Thus, one possible antidiabetic mechanism of D.
indica extract may be stimulation of insulin secretion.
STZ also induces oxidative stress or relative overload of oxidants i.e. reactive oxygen
species (Wright, 1999).D. indica leaves are rich in polyphenols and have an in
vitro antioxidant effect (Arbianti, 2007). Various studies have shown that diabetes is
associated with increased formation of free radicals and a decrease in antioxidant
potential.
STZ-induced diabetes is characterized by severe loss in body weight and this reduction is
due to loss or degeneration of structural proteins, as the structural proteins are a known
major contributor to body weight (Chen, Ianuzzo, 1982). A significant weight loss was

observed in the diabetic group which was improved significantly by the DIME in treated
groups.
The most commonly observed lipid abnormalities in diabetes are hypertriglyceridemia
and hypercholesterolemia (Shepherd, 2005; Shirwaikar, 2006) and contribute to
coronary artery disease (Arvind et al., 2005). In fact, lipid abnormalities accompanying
with atherosclerosis is the major cause of cardiovascular disease in diabetes. Therefore,
the ideal treatment of diabetes, in addition to glycemic control, should have a favorable
effect on lipid profiles. Marked increases in total cholesterol, triglyceride levels and
decrease in HDL cholesterol have been observed in diabetic control rats. In the present
study, the total cholesterol and triglycerides increased in the diabetic control group and
were reduced after the 21 days of treatment with DIME whereas the HDL cholesterol
level was significantly increased. This suggests that the extract may inhibit the pathway
of cholesterol synthesis (Rang, Dale, 1999).
Elevated transaminase such as AST, ALT and ALP was observed in diabetic rats and
indicates hepatic damage. The elevated transaminase activities were significantly
reduced by the DIME treatment. Diabetic complications such as increased
gluconeogenesis and ketogenesis may be due to elevated transaminase activity (Ghosh,
Suryawansi, 2001). These results suggest that DIME may also act as a hepatoprotective
agent.
The present study demonstrated that DIME could be useful in the management of
diabetes associated with abnormalities in lipid profiles and transaminase. The study
needs to be validated in human volunteers prior to proposed usage of DIME in other
human volunteers.

CONCLUSION
Based on these results, it may be concluded that the antidiabetic activity of DIME may
sensitize the insulin receptor or stimulate the secretion of insulin from beta cells of Islets
of Langerhans in pancreas of STZ-induced diabetic rats, where this was supported by
improved in vitro antioxidant status. The extract also improved other biochemical
parameters associated with diabetes. Therefore, the present study clearly reveals the
importance of leaves of D. indica as an economical antidiabetic agent. The plant bears
potential for further research to isolate the antidiabetic principle.

REFERENCES
ARBIANTI, R.; UTAMI, T.S.; KURMANA, A.; SINAGA, A. Comparison of antioxidant
activity and total phenolic content of Dillenia indica leaves extracts obtained using
various techniques. Proceedings In: REGIONAL SYMPOSIUM ON CHEMICAL
ENGINEERING, 14., Yogyakarta-Indonesia, 2007. Available at:
<http://staff.ui.ac.id/internal/132206932/publikasi/COMPARISONOFANTIOXIDANTACTIVI
TY_rita_tania.pdf>. Accessed on: 25 mar. 2010.
[ Links ]

ARVIND, K.; PRADEEP, R.; DEEPA, R.; MOHAN, V. Diabetes and coronary artery
diseases. Indian J. Med. Res.,v.116, p.163-176, 2002.
[ Links ]
BRANDELY, D.W.; MAYNARD, J.E.; EMERY, G.; WEBSTER, H. Transaminase activities in
serum of long-term hemodialysis patients. Clin. Chem., v.18, p.1442, 1972.
[ Links ]
BURSTEIN, M.; SCHOLNICK, H. R.; MORFIN, R. Rapid method for the isolation of
lipoproteins from human serum by precipitation with polyanions. J. Lipid Res., v.11,
p.583-595, 1970.
[ Links ]
CHEHADE, J.M.; MOORADIAN, A.D.A Rational approach to drug therapy of type 2
Diabetes Mellitus, disease management. Drugs, v.60, p.95-113, 2000.
[ Links ]
CHEN, V.; IANUZZO, C.D. Doasge effect of streptozotocin on rat tissue enzyme activities
and glycogen concentration. J. Physiol. Pharmacol., v.60, p.1251-1256, 1982.
[ Links ]
DAY, C. Traditional plant treatments for diabetes mellitus: pharmaceutical foods. Br. J.
Nutr., v.80, p.5-6, 1998.
[ Links ]
GHOSH, S.; SURYAWANSI, S.A. Effect of Vinca rosea extracts in treatment of alloxan
diabetes in male albino rats. Indian J. Exp. Biol., v.39, p.748-759, 2001.
[ Links ]
JUNOD, A.; LAMBERT, A.E.; STAUFFACHER, W.; RENOLD, A.E. Diabetogenic action of
streptozotocin: Relationship of dose to metabolic response. J. Clin. Invest., v.48, p.21292139, 1969.
[ Links ]
KRATZSCH, J.; ACKERMANN, W.; LELIACKER, H.; BERCH, W.; KELLER, E. A sensitive
sandwich enzyme immunoassay for measurement of insulin on microtitre plates. Exp.
Clin. Endocrinol., v.95, p.229-236, 1990.
[ Links ]
KRITIKAR, K.R.; BASU, B.D. Indian medicinal plants. Dehradun: Oriental Enterprizes,
2003. v.1, p.75-77.
[ Links ]
RAND, H.P.; DALE, M.M.; RITTER, J.M. Text book of pharmacology. 4.ed. Edinburgh,
London: Churchill Livingstone, 1999. p.301-305.
[ Links ]
RIFAI, N.; BACHORIK, P.S.; ALBERS, J.J. Lipids, lipoproteins and apolipoproteins. In:
BURTIS, C.A.; ASHWOOD, E.R. (Eds.) Textbook of clinical chemistry. Philadelphia: W.B.
Saunders Company,1999. p.809-861.
[ Links ]
RUBIN, R.J.; ALTMAN, W.M.; MENDELSON, D.N. Health care expenditures for people with
diabetes mellitus, 1992. J. Clin. Endocrinol. Metab., v.78, p.809A-809F, 1994.
[ Links ]
SHARMA, H.K.; CHHANGTE, L.; DOLUI, A.K. Traditional medicinal plants in Mizoram,
India. Fitoterapia, v.72, p.146-161, 2001.
[ Links ]

SHEPHERD, J. Does statin monotherapy address the multiple lipid abnormalities in type-2
diabetes? Atheroscler Suppl., v.6, p.15-19, 2005.
[ Links ]
SHIRWAIKAR, A.; RAJENDRAN, K.; BARIK, R. Effect of aqueous bark extract of Garuga
pinnata Roxb. Instreptozotocin-nicotinamide induced type II diabetes mellitus. J.
Ethnopharmacol., v.107, p.285-290, 2006.
[ Links ]
SHIRWAIKAR, A.; RAJENDRAN, K.; PUNITHAI, S.R. Antidiabetic activity of alcoholic stem
extract of Coscinium fenestratum in streptozotocin nicotinamide induced type-2diabetic
rats. J. Ethnopharmacol., v.97, p.369-364, 2005.
[ Links ]
SOOD, S.K.; BHARDWAJ, R.; LAKHANPAL, T.N. Ethnic Indian plants in cure of
diabetes. Jodhpur: Scientific Publishers (India), 2005. p.59.
[ Links ]
THE WEALTH OF INDIA RAW MATERIAL. New Delhi: Council of Scientific & Industrial
Research, 2003. v.3, p.64-65.
[ Links ]
VATS, R.K.; KUMAR, V.; KOTHARI, A.; MITAL, A.; RAMACHANDRAN, U. Emerging targets
for diabetes. Curr. Sci.,v.88, p.241-247, 2000.
[ Links ]
VETRICHELVAN, T.; JAGADEESAN, M.; UMA DEVI, B.A. Antidiabetic activity of alcohol
extract of Celosia argenteaLinn. seeds in rats. Biol. Pharm. Bull., v.25, p.526-528, 2002.
[ Links ]
WILKINSON, J.H.; BOUTWELL, J.H.; WINSTEN, S. Evaluation of a new system for the
kinetic measurement of serum alkaline phosphatase. Clin. Chem., v.15, p.487-495,
1969.
[ Links ]
WRIGHT, J.R.; ABRAHAM, C.; DICKSON, B.C.; YANG, H.; MORRISON, C.M.
Streptozotocin dose response curve in tilapia, a glucose-responsive teleost fish. Gen.
Comp. Endocrinol., v.114, p.431-440, 1999.
[ Links ]

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1984-82502011000200018

Effect of Glycolic Extract of Dillenia indica L.

Combined With Microcurrent Stimulation on
Experimental Lesions in Wistar Rats

Wednesday, 05/11/11 | 10627 reads

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Author(s):
Ketylin F. Migliato, BSPS, PhD; Mateus A. Chiosini, BSPS; Fernanda A. Mendona, BSPS, PhD;
Marcelo A. Esquisatto, PhD; Hrida R. Salgado, BSPS, PhD; Glucia M.T. Santos, PhD
Issue:
Volume 23 - Issue 5 - May 2011

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Abstract: This study evaluated the wound healing activity of a glycolic extract of Dillenia
indica (GED) prepared from the mature fruits of the plant applied alone or in combination with
microcurrent stimulation to skin wounds surgically induced on the back of Wistar rats. Methods.
The animals were randomly divided into six groups: (A) negative control group; (B) group
receiving microcurrent application (MC; [10 mA/2 mins]); (C) group treated with GED; (D) group
treated with an emulsion containing GED; (E) group treated with GED and MC, and (F) group
treated with the emulsion containing GED and MC. Tissue samples were obtained 2, 6, and 10
days after injury and underwent structural and morphometric analysis. Results. There were
observed differences in wound healing among the various treatments when compared to the
control group. The combination of microcurrent plus extract or microcurrent plus emulsion
containing GED was advantageous in all of the studied parameters (P < 0.05) when compared to
the other groups with positive effects seen regarding newly formed tissue, number of fibroblasts,
and number of newly formed blood vessels. The morphometric data confirmed the structural
findings. Conclusion. Microcurrent application alone or combined with GED exerted significant
effects on wound healing in this experimental model. This was probably due to the efficacy of
microcurrent application since the extract alone did not significantly accelerate the healing
process.D indica fruit extract most likely participates in the wound healing process as a result of
its anti-inflammatory properties.
Dillenia indica L. (family Dilleniaceae) originated in tropical Asia and has acclimated in Brazil for
more than a century. The plant has a thick trunk and a fissured bark surface. The branches have
leaves that are concentrated on the terminal region. The flowers are large and white in color and
bloom between December and April. The fruits that appear between April and August are large,
fleshy, and greenish-yellow in color and contain small, flattened seeds surrounded by a
gelatinous substance.14 D indica is used as an antipyretic and cardiotonic drug and for the
treatment of rheumatoid arthritis and anti-inflammatory processes.58 Munit et al9 identified the
presence of triterpenes and flavonoids in a phytochemical study of the crude extract of D
indica leaves. Abdille et al2 observed that the extract of D indica fruit contains significant amounts
of phenolic compounds with expressive antioxidant activity. Flavonoids, tannins, and other

phenolic substances are constituents of plants with antioxidant activity, mainly by acting as
radical scavengers of oxygen. The presence of flavonoids in phytotherapeutic agents has been
shown to favor the wound healing process in experimental models.10,11 Wound healing is a
complex biological process that occurs in response to tissue damage due to trauma or surgical
procedures. The wound healing process can be divided into three phases: inflammatory,
proliferative, and remodeling. Technological advances facilitated the emergence of a wide variety
of wound healing treatments. The application of low amperage electrical stimuli has been shown
to modify the healing process in living organisms, especially factors that delay or impair this
process.1216 Several investigators have studied the effects of electrical stimulation using different
amplitudes and frequencies and observed modifications in the cellular and tissue responses in
experimentally induced wounds.1719 Stimulation of live cells with low-intensity electrical currents
directly affects the membrane potential and is associated with changes in ion gradients across
the cell membrane causing an increase in the synthesis of ATP followed by increased protein
synthesis.12,20,21 The objective of this study was to investigate the effects of a glycolic extract
of Dillenia indica L. fruits and an emulsion containing this extract, either with
or without microcurrent stimulation, on the healing of surgically-induced
wounds in Wistar rats.

Methods
Mature fruits of D indica L. were collected in February 2009 on the Pinhal Farm in Limeira (So
Paulo, Brazil) in the morning. Material was collected from the branches of the same tree for
deposition of a voucher specimen, which was deposited by Vincius Castro Souza, Curator of the
Herbarium of the Department of Biological Sciences, ESALQ-USP, Piracicaba Campus (So
Paulo) under the number ESA 55549. Preparation of plant extracts. Mature fruits were
collected and dried in an oven under circulating air at 45C for weight stabilization. The fruits
were then ground in a knife mill. The extract was obtained by turbo extraction of 50 g of the
sample in 500 mL 70% (w/w) alcohol for 15 minutes followed by filtration in a rotary evaporator
under reduced pressure at a maximum temperature of 40C until complete elimination of the
organic solvent. The turbo extraction technique does not allow the temperature to exceed 40C.
This procedure was performed because it was considered relatively harmless regarding the
extraction of different components. The sample was then lyophilized until all water was removed.
For the physicochemical quality control tests, 10 g of mature fruit was dried at room temperature
in the dark. Later, 1.0 g this material was subjected to infrared heat (110C) for 1 hour and was
weighed afterward. This procedure was performed every hour until the weight did not vary more
than 0.25%. Values are expressed as percentage (w/w). The average of determinations was
three.22,23 For microbiological analysis of the lyophilized D indica extract, total microorganism
count, and counts of the pathogens Salmonella sp, Escherichia coli, Staphylococcus aureus,
and Pseudomonas aeruginosa were determined according to the methods of Migliato et al 24 and
Pinto et al.25 Phytochemical screening. Preliminary phytochemical analysis of the D
indica glycolic extract was performed in triplicate according to Simes et al 26 and Harbone.27 A
portion of 100 mg of the extract was used for each reaction. The presence of flavonoids was
determined using 1% aluminum chloride solution in methanol, concentrated hydrochloric acid,
magnesium turnings, and potassium hydroxide solution. Anthraquinones were analyzed by the
Borntrager reaction and saponins by the observation of persistent and abundant foam

production. To analyze tannins, the lyophilized extract was dissolved in water and tannins were
identified by reaction with 2.5% gelatin, 1% iron salts, and 10% lead acetate. Total alkaloids were
assayed using the reagents of Dragendorff, Bouchardat, Mayer, and Bertrand. Preparation of
an emulsion containing the glycolic extract of Dillenia indica. The lyophilized D
indica extract was solubilized in propyleneglycol:water (1:1) and incorporated into an emulsion
containing the following: Butylated hydroxytoluene (BHT [0.05%]), Ethylenediamine tetraacetic
acid (EDTA [0.1%]), Lanaxan (2%), Polawax (14%), Phenonip (0.5%), propyleneglycol
(3%), D indica glycolic extract (5%), and distilled water qsp (30%). Animals. Male Wistar rats
(Rattus norvegicus) weighing 250 g350 g were housed individually in cages at a constant
temperature (23C 2C). The rats had free access to food and water and were subjected to a
12-hour light/dark cycle. The average weight and behavior during this experiment did not differ
significantly at the end of the study. Linear incision wound model. A trichotomy was
performed on the back of the animal 48 hours before surgical intervention. After local asepsis
with 0.4% chlorhexidine digluconate, the animals were anesthetized by intraperitoneal injection of
xylazine hydrochloride (20 mg/kg body weight) and ketamine hydrochloride (50 mg/kg). After the
position was marked with a dermographic pen and pachymeter, a 2-cm long and 0.2-cm deep
surgical incision was made in the craniocaudal direction. The incision was not sutured. In view of
the similar genetic background of the animals28,29 and according to the Ethics Committee of
Uniararas (protocol number 809/2006), nine animals were used per group: (A) negative control
group receiving sterile saline; (B) group receiving microcurrent application (MC; [10 mA/2 min]);
(C) group treated with the glycolic extract of D indica (GED); (D) group treated with the emulsion
containing GED; (E) group treated with the GED and MC (10 mA/2 min), and (F) group treated
with the emulsion containing GED and MC (10 mA/2 min), according to the protocol of Mendona
et al.16 A transcutaneous electrical stimulator (Physiotonus Microcurrent, Bioset, Rio Claro, So
Paulo, Brazil) was used for electrical stimulation. The device was set to microgalvanic-continuous
mode with the intensity at 10 mA/2, frequency 0.3 Hz, and was used for 2 minutes. The
applications were performed using two metal electrodes with a spherical tip (10 mm) positioned
on the wound. The treatments were started 24 hours after surgical intervention and were
continued daily for 10 days. Collection and preparation of wound samples for structural
analysis. At days 2, 6, and 10 after the injury, three animals in each group were killed under
anesthesia. The total area (approximately 120 mm2160 mm2) of the wound was removed and
submitted for structural and morphometric analysis. Each sample was removed and fixed in 10%
formalin in Millonig buffer (pH 7.4) for 24 hours at room temperature. Next, the specimens were
washed in buffer and processed for embedding in Paraplast . Longitudinal sections (7 m) were
stained with hematoxylin & eosin for routine histology and with picrosirius-hematoxylin in order to
view collagen fibers. The specimens were examined and documented using a Leica DM 2000
photomicroscope at the Laboratory of Micromorphology, Hermnio Ometto University Center,
Uniararas. Morphometric analysis. Cross-sections of the mid-region of the experimental
wound were used for the determination of the following morphometric parameters: tissue repair
area (x103 m2), total number of cells (fibroblastic and inflammatory cells [n/103 m 2]), number of
newly formed blood vessels (n/103 m2), and thickness of the regenerating epithelium (m). For
this purpose, three samples were randomly selected among the sections obtained. All images
were captured and digitalized using a Leica DM 2000 photomicroscope. The measurements were
made on the digitalized images using the Leica Image Measure and Sigma Scan Pro 6.0
programs. The results were compared by ANOVA and Tukeys post-hoc test with the level of
significance set at 5%.30 The results were entered into spreadsheets (Biostat for Windows).

Results
Quality assurance is an important factor to be considered in the production
of medications, cosmetics, and phytotherapeutic agents from the planning to
the eventual release of the product to the consumer. Therefore,
physicochemical (Table 1) and microbiological quality control tests of the D indica glycolic extract
were performed. Analysis of total microorganism count showed no growth of viable fungal or
bacterial colonies. None of the four pathogens investigated (Salmonella sp, E coli, S aureus,
and P aeruginosa) was detected in the lyophilized extract. Preliminary phytochemical analysis of
the glycolic extract of D indica fruits showed the presence of flavonoids, saponins,
anthraquinones, and tannins; alkaloids were not detected. Structural and morphometric
analysis of wound repair. Tissue repair was studied in the different groups by comparing
inflammatory (leukocytosis, hemorrhage, and exudate) and proliferative processes (fibroblastic
hyperplasia, epithelization, and angiogenesis), and tissue reorganization. Temporal differences in
tissue repair were observed among the different treatments. In the control group (group A), the
proliferative phase was observed on day 6 after injury and tissue reorganization was detected on
day 10. In the group receiving MC (group B), the size of the tissue repair area and the total
number of cells were higher than in the control group on day 6 of treatment. On day 10, the
wound area was completely re-epithelialized with the observation of dermis filled with fibrous
tissue, reorganized collagen fibers, and compacted fibril elements (Figures 1, 2). The findings
obtained for the group treated with GED (group C) were similar to those observed for the control
group in terms of the repair of epidermis and dermis for samples collected on days 6 and 10. A
significant increase in the tissue repair area and the total number of cells was observed on days
6 and 10 after experimental injury in the group simultaneously receiving MC and GED (group E)
when compared to the other groups. In this group, the dermal wound area was filled with fibrous
tissue on day 10 and the collagen fibers were reorganized and compacted, findings indicating
tissue repair similar to that observed in animals receiving only MC. No significant difference in the
repair of the lining epithelium was observed during application of different treatments (Figures 1,
2). In the group treated with the emulsion containing GED (group D), the tissue repair area and
total number of cells on days 6 and 10 after injury were similar to those of the control group. On
day 10, the epidermis was completely repaired (Figures 3, 4). A significant increase in tissue
repair area and the total number of cells was observed on days 6 and 10 in the group
simultaneously receiving MC and the emulsion containing GEC (group F) when compared to
group D. However, the collagen fibers of the dermis were poorly compacted. A significantly
larger number of newly formed vessels per tissue area (103 m2) were observed in groups B, E,

and F (Figure 5).

Epithelial thickness in
the tissue repair area
compared to control

Discussion

did not differ among groups when

(Figure 6).

Phytochemical analysis of the glycolic extract of D indica fruits identified the presence of
flavonoids, among other components. These compounds and their derivatives are known to
increase vascularization and to delay the process of cell necrosis due to their anti-inflammatory,
antifungal, and antioxidant properties.31,32 Studies have shown that most of the antioxidant activity
of plant extracts is the result of compounds, such as flavonoids, tannins, isoflavones, flavones,
anthocyanins, catechin, and other phenolic compounds.33,34 Flavonoids are especially important in
the tissue repair process because of their astringent and antimicrobiological properties. 6,35 The
same properties can be attributed to tannins, which were identified in the present study in the
glycolic extract of D indica . According to Jorge Neto et al36 and Bedi and Shenefelt,37 tannins
precipitate proteins in damaged tissues forming a protective lining that favors repair and reduces
wound permeability and exudation. Suntar et al38 reported that an aqueous extract of Colutea
cilicica (Boiss. & Bal. fruit) was effective during the inflammatory and proliferative phases of the
wound healing process in rats. The authors identified flavonoids in the C cilicica extract and
suggested that these substances participate in the healing process together with other
phytochemical components of the plant. In the present study, although flavonoids were identified
in the D indica extract, no significant effects on the acceleration of tissue repair were observed
when the extract or the emulsion containing the extract was applied alone to skin wounds
induced on the back of rats. Although the efficacy of flavonoids in the acceleration of the tissue
repair process has been reported by different investigators, 31,32 the present results indicate that the
presence of these compounds in the D indica extract did not have this effect on the healing
process. Analysis of the glycolic extract of D indica fruit did not reveal the presence of alkaloids.
Similarly, Shome et al6 characterizing the phytochemical composition of hexane, chloroform,
ethanol, and aqueous extracts of D indica fruits, demonstrated the presence of triterpenes and
the absence of alkaloids in all extracts. Yeshwante et al39 observed significant anti-inflammatory
activity of a methanol extract of D indica leaves on edema induced in rats and mice. The authors
suggested that this process is the result of the inhibition of prostaglandin biosynthesis, confirming
the use of D indica in folk medicine. In the present study, application of the D indica extract or of
the emulsion containing the extract to skin wounds surgically induced in rats did not significantly
accelerate the tissue repair process. Positive effects were only observed when microcurrent
stimulation was applied simultaneously. These findings suggest that the extract of D indica fruit
possesses the same properties as the leaf extract studied by Yeshwante et al, 39 which showed
efficacy in treating the effects of inflammatory processes, but did not accelerate wound healing.
Microcurrent application was found to be effective in terms of the parameters analyzed, with
positive effects on the area of newly formed tissue, number of fibroblasts and number of newly
formed blood vessels, but not on epithelial thickness, when compared to the control group and to
the groups receiving only the D indica extract and the emulsion containing the extract. These
findings agree with those reported in different studies demonstrating that low-intensity electrical
currents stimulate wound healing.1719 Becker17 reported that electrical currents are present in all
biological systems and may promote repair and growth after injury. According to this author, a
specific injury stimulus induces another repair stimulus. The author also demonstrated that the
membrane electrical potential is altered in injured tissues. The injury signal gradually decreases
in parallel to the repair process and ceases when the latter is complete. The voltage peaks
immediately after injury and gradually decreases as the wound heals, a fact leading to the
concept that current flows may be defective in chronic wounds and that the application of
electrical currents to wounds may stimulate healing.15,40 Biedeback41 proposed that
transmembrane currents open voltage-controlled calcium channels in fibroblasts, inducing ATP

resynthesis, activation of protein kinase mechanisms to synthesize new cellular protein, and DNA
replication necessary for mitotic cell division. Electrical microcurrent has been used in the
treatment of chronic wounds.4244 Lee et al45 using a 100 nA current 3 A in the treatment of
chronic wounds and ulcers associated with chronic diseases and found that the application of
such currents supposedly provides electrons to tissues and saturated free radicals, facilitating
tissue repair. Mendona et al16 suggested that microcurrent application to tissue injuries might be
used as a coadjuvant to accelerate the healing process. Variations in cell metabolism, as well as
fibroblast proliferation, neovascularization, and collagen deposition in the wound area have been
observed after microcurrent application.16,46 The combined application of a microcurrent and the D
indica extract or emulsion containing the extract was advantageous in terms of all parameters
studied when compared to the control group and to either treatment alone. The simultaneous
application of physical and phytotherapeutic agents to wounds has been shown to be effective in
both skin repair and reduction of the inflammatory process. The use of electrical current on
transdermal facilitating the transfer of various substances is known in the literature as
iontophoresisa noninvasive technique that ensures the penetration levels of higher
concentrations of therapeutic substances when compared to passive diffusion. In iontophoresis,
the current is widely used since it generates a unidirectional flow of electrons and constant during
the application, triggering the desired therapeutic effects. 4749 Maia-Filho et al50 investigated the
effects of simultaneous application of ultrasound and Aloe vera gel on an experimental model of
induction of tendinitis in rats and demonstrated that this type of treatment is effective in terms of
both skin repair and reduction of the inflammatory process. Also Soares 51 demonstrated that the
combination of antioxidant agents and photodynamnic or low-amperage electrical therapy
accelerates wound healing in experimental wounds in rats. In addition, the simultaneous
application of Aloe vera and microcurrent was effective in the treatment of open wounds
potentiating wound healing in Wistar rats.16 Similar effects were observed in the present study.
The combination of the D indica glycolic extract or emulsion containing the extract and
microcurrent stimulation exerted significant effects on the repair area, total number of cells, and
total number of newly formed vessels in the wound area. However, these alterations were not
significant when the extract was applied alone. This fact suggests that the positive effects on the
acceleration of tissue repair were due to the action of microcurrent stimulation, which has been
shown to be effective in accelerating wound healing. 15,16 However, the D
indica fruit extract probably participates in the wound healing process as a
result of its anti-inflammatory properties.

Conclusion
The present results demonstrated that the D indica glycolic extract or the emulsion containing
the extract was not effective in accelerating the tissue repair process in skin wounds surgically
induced in Wistar rats. However, microcurrent application alone or combined with glycolic extract
exerted significant effects on wound healing in the experimental model. This finding was probably
due to the efficacy of microcurrent stimulation since this treatment alone accelerated the healing
process.

References

1. Alzugaray D, Alzugaray K. Enciclopdia de Plantas Brasileiras. So Paulo, Brazil: 3rd ed.

1988. 2. Abdille MH, Singh RP, Jayaprakasha GK, Jena BS. Antioxidant activity of the extracts
from Dillenia indica fruits. Food Chemistry. 2005;90:891896. 3. Lorenzi, H, Souza, HM, Torres,
MAV, Bacher, LB. rvores Exticas no Brasil madeireiras, ornamentais e exticas. Nova Odessa,
SP: Instituto Plantarum; 2003. 4. Barroso, GM, Morim, MP, Peixoto, AL, Ichaso, CLF. Frutos e
sementes: morfologia aplicada sistemtica de dicotiledneas. Viosa: UFV. 1999. 5. Shome U,
Khanna KR, Sharma PH. Pharmacognostic studies on Dillenia indica Linn. I. Leaf. Proc Indian
Acad Sci. 1979;88:3548. 6. Shome U, Khanna KR, Sharma, PH. Pharmacognostic studies
on Dillenia indica Linn. II. Fruit and seed. Proc. Indian Acad Sci. 1980;89:91104. 7. Yeshwante,
SB, Juvekar, AR, Nagmoti, DM, et al. Anti-inflammatory activity of methanolic extracts of Dillenia
indica L. leaves. J Young Pharm. 2009;1(1):6366. 8. Zauli-Nascimento RC, Pereira MA,
Cardoso LGV, Silva JMSF, Carvalho JCT, Fiorini JE. Atividade antimicrobiana e determinao da
concentrao inibitria e concentrao bactericida mnima de Dillenia indica L. (Flor de abril).
In: XX Reunio Anual da FeSBe - Federao de Sociedades de Biologia Experimental. guas
de Lindia-SP; 2005. 9. Munit A, Tareq SM, Apu AS, Basak D, Islam MS. Isolation and
identification of compounds from the le Dillenia indica Linn. Bangladesh Pharma J. 2010;13(1):1
7. 10. Quettier-Deleu C, Gressier B, Vasseur J, et al. Phenolic compounds and antioxidant
activities of buckwheat (Fagopyrum esculentum oench) hulls and flour. J Ethnopharmacol.
2000;72:3542. 11. Peruchi, CMS, Acevedo, RA, Franco, SL. Efecto del propoleos en la
cicatrizacin de lesions subcuaneas inducidas en el dorso del ratones, estudo histolgico.
Revista de La /facultad de Odontologia (Santiago, Chile). 2001;19:2336. 12. Cheng N, Van Hof
H, Bocks E. The efect of electric currents on ATP generation, protein synthesis, and membrane
transport in rat skin. Clin Orthop Relat Res. 1982;17:26472. 13. Cheng K, Goldman RJ. Electric
fields and proliferation in a dermal wound model: cell cycle kinetics. Bioelectromagnetics.
1998;19(2):6874. 14. Houghton PE, Kincaid CB, Lovell M, Campbell KE, Keast DH, Woodbury
MG, Harris KA. Efect of electrical stimulation on chronic leg ulcer size and appearance. Phys
Ther. 2003;83(1):1728. 15. Kloth LC. Electrical stimulation for wound healing: a review of
evidence from in vitro studies, animal experiment, and clinical trials. Int J Low Extrem Wounds.
2005;4(1):2344. 16. Mendona FAS, Passarini Jr JR, Esquisatto MAM, Mendona JS, Franchini
CC, Santos GMT. 2009. Efects of the application of Aloe vera (L.) and microcurrent on the
healing of wounds surgically induced in Wistar rats (Rattus norvegicus). Acta Cir Bras.
2009;24:150155. 17. Becker R, Selden G. The Body Electric. New York, NY; William Morrow
and Co.: 1985. 18. Basset CA. Beneficial efects of electromagnetic-fields. J Cell Biochem.
1993;51:387393. 19. Bayat M, Asgari-Moghadam Z, Maroufi M, Rezaie FS, Rakhshan M.
Experimental wound healing using microamperage electrical stimulation in rabbits. J Rehabil Res
Dev. 2006;43:219228. 20. Santos VNS, Ferreira LM, Horibe EK, Duarte IS. Electric microcurrent
in the restoration of the skin undergone a trichloroacetic acid peeling in rats. Acta Cir Bras.
2004;19:466469. 21. Valle KKR, Reis LL, Bouvent JJ, Shida CS. Efeito da aplicao de
microcorrente eltrica na restaurao de pele de ratos exposta ao de radicais livres.
21 CBEB. 2008;247249. 22. Farmacopia Brasileira. 4th ed. So Paulo, Brazil. Ed Atheneu;
2000. 23. Mello JCP, Petrovick PR. Quality control of Baccharis trimera (Less) DC (Asteraceae)
hydroalcoholic extracts. Acta Farm Bonaerense. 2000;19:211215. 24. Migliato KF, Moreira
RRD, Mello JCP, Sacramento LVS, Corra MA, Salgado HRN. Controle da qualidade do fruto de
Syzygium cumini (L.) Skeels. Braz J Pharmacogn. 2007;17:94101. 25. Pinto TJA, Kaneko TM,
Ohara MT. Controle Biolgico de Qualidade de Produtos Farmacuticos, Correlatos e
Cosmticos. 2nd ed. So Paulo, Brazil: Atheneu; 2003. 26. Simes CMO, Schenkel EP,

Gosmann G, Mello JCP, Mentz la, Petrovick PR. Farmacognosia: da Planta ao Medicamento. 5th
ed. Porto Alegre: Editora da UFRGS; 2003. 27. Harborne JB. Phytochemical Methods: A Guide
to Modern Techniques of Plant Analysis. 2nd ed. London: Chapman and Hill; 1998. 28. National
Institutes of Health. Guide for the Care and Use of Laboratory Animals. NIH Publication.
1996;8223. 29. Gill TJ, Smith GJ, Wissler RW. The rats as an experimental animal. Science.
1989;245(4915):269276. 30. Miller RG. Simultaneous Statistical Inference. 2nd ed. New York,
NY: Springer Verlag; 1981. 31. Santos SC, Mello JCP. In: Simes CMO, Schenkel EP, Gosmann
G, Mello JCP, Mentz LA, Petrovick PR, eds. Farmacognosia: da Planta ao Medicamento. 5th ed.
Porto Alegre: Editora da UFRGS; 2003. 32. Nayak SB, Sandifer S, Maxwell A. Evaluation of the
wound healing activity of ethanolic extract of Morinda citrifolia L. leaf. Avid Based Compl Alter
Med. 2009;6:351356. 33. Chalone MP, Hopi AI, Volutella HJ, et al. Antioxidant activity of plant
extracts containing phenolics compounds. J Agric Food Chem. 1999;47(10):39543962. 34.
Addison L, Goody JS, Ferrier LAM, Stedman JR, Brandi MGL. Proparacaine e caracterizao de
extratos gliclicos enriquecidos em taninos a partir das cascas de Stryphnodendron adstringens
(Mart.) Coville (Barbatimo). Rev Bras Farmacol. 2002;12:734. 35. Pesin I, Koca U, Keles H,
Kupeli Akkol E. Wound healing activity of Rubus sanctus Schreber (Rosaceae): preclinical study
in animal models. Evid Based Complement Altern Med. 2009 Sept 15. [Epub ahead of print]. 36.
Jorge Neto J, Fracasso JF, Neves MCLC, Santos LE, Banuth VL. Tratamento de lcera varicosa
e leses de pele com Calendula officinalis e/ou com Stryphnodendron barbatiman (vellozo)
martius. Ver Cienc Farm. 1996;17:181186. 37. Bedi MK, Shenefelt PD. Herbal therapy in
dermatology. Arch Dermatol. 2002;138:232242. Ketylin F. Migliato, BSPS, PhD is from the
1University of Central Paulista, So Carlos, SP, Brazil and the Graduate Program of
Pharmaceutical Sciences, Paulista State University, Araraquara, SP, Brazil; Mateus A. Chiosini,
BSPS, Fernanda A. Mendona, BSPS, PhD, Marcelo A. Esquisatto, PhD, and Hrida R.
Salgado, BSPS, PhD are from the Graduate Program of Pharmaceutical Sciences, Paulista State
University, Araraquara, SP, Brazil; Glucia M.T. Santos, PhD is from the Graduate Program of
Biomedical Sciences, Herminio Ometto University Center, Araras, SP, Brazil Address
correspondence to: Glucia M.T. Santos, PhD Hermnio Ometto University Center
UNARARAS Graduate Program of Pharmaceutical Sciences Av. Dr. Maximiliano Baruto, 500 Jd.
Universitrio 13607339 Araras, SP, Brazil
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