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Antidiabetic activity of clerodendrum philippinum schauer leaves in

streptozotocin induced diabetic rats

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 Innovare International Journal of Pharmacy and Pharmaceutical Sciences
Academic Sciences
ISSN- 0975-1491 Vol 7, Issue 9, 2015

Original Article
ANTIDIABETIC ACTIVITY OF CLERODENDRUM PHILIPPINUM SCHAUER LEAVES IN
STREPTOZOTOCIN INDUCED DIABETIC RATS

MIHIR K. KAR1*, TRUPTI R. SWAIN2, SAGAR K. MISHRA1

1University Department of Pharmaceutical Sciences (UDPS), Utkal University, Vani Vihar, Bhubaneswar 751004, 2Department of
Pharmacology, S. C. B. Medical College, Cuttack 753007, Odisha, India
Email: [email protected]
Received: 26 Apr 2015 Revised and Accepted: 25 Jul 2015
ABSTRACT
Objective: The present study has been undertaken to evaluate the antidiabetic activity of Clerodendrum philippinum Schauer leaves.
Methods: The fresh leaves were collected from Kuruan village of Jajpur district in the state of Odisha, India and extracted successively with n-
hexane, methanol and water. The effect of extracts at the dose level of 400 mg/kg body weight was studied in normal, glucose loaded and
streptozotocin-induced diabetic rats.
Results: The test extracts showed significant reduction of blood glucose level in normal, glucose loaded and streptozotocin-induced diabetic rats.
Methanol extract demonstrated maximum blood glucose lowering potential as compared to other extracts.
Conclusion: The leaf of Clerodendrum philippinum Schauer is endowed with blood sugar lowering potential in both normal and diabetic rats.
Keywords: Clerodendrum philippinum Schauer, Antidiabetic activity, Streptozotocin.

INTRODUCTION MATERIALS AND METHODS

Clerodendrum philippinum Schauer (Synonym: Clerodendrum Chemicals
fragrans Willd.) belongs to family Verbenaceae, is a semi woody
shrub distributed in southern Asia. It is commonly known as Chinese Streptozotocin (STZ) and glibenclamide were procured from SIGMA-
glory tree, Scent malli and Brajamalli in the state of Odisha, India. It Aldrich, Mumbai. All other chemicals and reagents used were of
grows wild, spreads vegetative and is also grown as ornamental [1, analytical grade.
2]. Various species of Clerodendrum are used as folk and traditional Plant material and extraction
medicines in various parts of the world like India, China, Korea,
Japan, Thailand, Africa etc. and are reported to be used for the The plants were collected from Kuruan village of Jajpur district in
remedial purpose in inflammatory disorders, diabetes, cancers, the state of Odisha, India. The taxonomic identity of the plant was
malaria, fever, etc [3]. confirmed by Dr. K. B. Satapathy, P. G. Department of Botany, Utkal
University and the voucher specimen (SVN-534) was deposited at
Leaves of C. Philistinism has been used as traditional medicine the departmental herbarium. The collected fresh leaves were
for treatment of colic pain and exhibited anti-fungal activity [4]. washed, shade dried, powdered and extracted successively with n-
The dried root is used as an anti-inflammatory [5] and for hexane and methanol by using soxhlet apparatus. Then the marc was
myalgia, tinea and rheumatoid arthritis [6]. The seed is used in extracted with distilled water by the method of continuous hot
constipation [7]. The water and ethanol extracts of leaf show extraction at 60 °C for 6 h [19]. Finally, the extracts were
anti-fungal [8] and ethanol extract possesses antibacterial concentrated by evaporating the solvent using rotary evaporator.
activity [1]. The ethanol extracts of flower exhibits anti-anxiety
The yield of n-hexane, methanol and aqueous extracts were found to
and CNS depressant properties [9]. The leaf juice is used
be 3.81%, 11.75% and 11.82% w/w respectively.
externally for scabies, cuts and burns [10]. The leaf juice mixed
with the equal amount of ‘tulsi’ (Ocimum sanctum) juice is used Qualitative phytochemical screening
to reduce sugar content in blood by tribal and rural people in the
state of Odisha, India [2]. The presence of phyto constituents in the extracts was determined
by standard & prescribed chemical procedure [20-23].
The major chemical constituents reported from Clerodendrum
philippinum are phenolics, flavonoids, terpenoids, steroids, etc. Animals
Flavones such as Cirsimaritin and Sorbifolin were isolated from the
Healthy adult Wistar albino rats of either sex (150-200 g body
leaf and stem [11]. Flavone, 5-7-8-Trihydroxy-4-Methoxy [12] and
weight) procured from the animal house of School of Pharmaceutical
Kaempferol [13] were isolated from the dried leaf. Phenolic
Sciences (SPS), S‘O’A University, Bhubaneswar were used for the
compounds, Acteoside, Leucosceptoside A, Isoacteoside, Methyl and
study, and the experimental protocol was approved by the
Ethyl esters of Caffeic acid, Jinoside, etc. were reported from the
Institutional Animal Ethics Committee vide proposal no. 23/11,
whole plant [13].
dated 24/01/2012 of SPS, S‘O’A University, Bhubaneswar bearing
Toubi et al. [14] isolated Bascoside, Derhamnosyl, Verbascoside, Iso- Registration No. 1171/c/08/CPCSEA.
Verbascoside, and Calceolarioside A from the leaves and also
Acute oral toxicity study
reported the presence of O-Iridoids and O-flavonoids. Triterpenes
(α-Amyrin and Clerodolone) and N-Triacontane were isolated from Healthy adult female Wistar albino rats starved overnight were
different parts of C. philippinum [15]. Steroids (Clerosterol, divided into eight groups, each consisting of four rats and were
Daucosterol, β-Sitosterol, Poriferasterol, Stigmasterol, etc.) were orally fed with the test extracts in increasing dose levels of 500,
also reported from leaves of C. philippinum [15-18]. The present 1000, 2000 and 4000 mg/kg body weight. The acute toxicity study
investigation deals with the evaluation of antidiabetic activity of was carried out according to OECD guidelines. The rats were
various extracts of C. philippinum. observed continuously for 2 h under the following profiles [24].
 Kar et al.
Int J Pharm Pharm Sci, Vol 7, Issue 9, 386-389

(I) Behavioral profile: Alertness, restlessness, irritability, and Effect of extracts on glucose loaded hyperglycemic rats
fearfulness.
The rats were ingested with glucose (2 g/kg) in distilled water, 30
(II) Neurological profile: Spontaneous activities, reactivity, touch min following the administration of the test substances by gastric
response, pain response and gait. intubation. The treatments were made similarly as above and the
BGL was measured at 1, 2 and 4 h following the administration of
(III) Autonomic profile: Defecation and urination. test substances.
After a period of 24 h, 72 h and 14 days, the rats were observed for Effect of extracts on STZ-induced diabetic rats
any lethality or death.
The effect of extracts on BGL was studied in STZ-induced diabetic
Induction of diabetes rats on a similar basis, and BGL was estimated at 0, 1, 2, 4, 8 and 10 h
Experimental diabetes was induced by single intra-peritoneal following the treatment.
injection of 55 mg/kg of Streptozotocin (STZ), freshly dissolved in Statistical analysis
cold citrate buffer, pH 4.5. After 5 days of STZ injection, rats with
fasting blood glucose above 250 mg/dl were considered as diabetic The results are expressed as mean±SEM the statistical analysis is
and included for the study [25]. carried out using one-way ANOVA followed by Dunett’s t-test.
Statistical P<0.05 is considered as significant.
The blood glucose level (BGL) was estimated using gluco-monitor
(Contour TS, Bayer HealthCare Limited) by puncturing the tail vein. RESULTS
Effect of extracts on normoglycemic rats Preliminary phytochemical screening
The effect of extracts on BGL was studied in normal rats [26], were The data represented in table 1 depicted the preliminary
divided into five groups of six rats each and fasted for 12 h with free phytochemical investigation reports of the various extracts of C.
access of water, and the treatments were made orally as: Group I: philippinum indicates that the n-hexane extract was found to contain
solvent control (Tween 40+distilled water); Group II: Glibenclamide alkaloids, steroids, triterpenoids; whereas methanol extract shown
(10 mg/kg); Group III: n-hexane extract (400 mg/kg); Group IV: the presence of alkaloids, flavonoids, glycosides, phenolic
methanol extract (400 mg/kg); Group V: aqueous extract (400 compounds, saponins, steroids, triterpenoids; and the aqueous
mg/kg). The BGL was measured at 0, 1, 2, 4, 8 and 10 h following the extract showed the presence of flavonoids, glycosides, phenolic
treatment. compounds, saponins.

Table 1: Preliminary phytochemical screening of Clerodendrum philippinum Schaur leaf extracts

Extracts Alkaloids Carbohydrates Flavonoids Glycosides Phenolic Proteins Saponins Steroids Triterpenoids
compounds
n- + - - - - + - + +
Hexane
Methanol + + + + + + + + +
Aqueous + + + + + - + - -
‘+’ indicates present, ‘-’indicates absent.

Acute oral toxicity study Effect of extracts on normoglycemic rats

The gross observational results revealed that the extracts of C. The effect of extracts on BGL of normal rats, depicted in table 2,
philippinum leaves did not show any sign of toxicity and mortality up to showed a significant fall when compared with the solvent control
14 d of the study in the dose level of 4000 mg/kg. One-tenth of the group at the end of 10 h. Among them, methanol extract exhibited
observed safety dose was taken for experimental purpose considering highest reduction of BGL with the percentage reduction of 36.28%
the fact it may show the therapeutic effect as well as safe in longer (P<0.001) followed by aqueous extract of 26.62% (P<0.01) and n-
duration of use, as per previously published literature [27, 28]. hexane extract 18.58% (P<0.05).

Table 2: Effect of extracts of C. philippinum leaves on BGL in normal rats

Treatment Blood Glucose Levels (mg/dl) % decrease at 10
0h 1h 2h 4h 8h 10 h
Solvent Control 103.66±5.47 101.5±5.82 102.66±4.21 101.66±4.12 96.16±4.18 96.33 -
(Tween+Water) 4.28
Glibencamide (10 mg/kg) 101.83±5.16 91.5±3.29 77.16±2.38c 68.16±3.02c 58.83±2.78c 53.33±3.77c 47.62
n-Hexane Extract (400 94.16±4.23 101.16±4.69 101. 98.33±4.60 82.66±4.81a 76.66±3.27a 18.58
mg/kg) 83±4.04
Methanol Extract (400 99.66±4.49 98.66±3.01 84.16±3.85b 79.66±3.36b 68.66±3.13c 63.5±4.86c 36.28
mg/kg)
Aqueous Extract (400 102.66±3.92 97.83±4.98 88.83±4.72a 82.33±4.68b 76.5±4.97b 75.33±5.73b 26.62
mg/kg)
F (4, 25) - - 8.04** 11.91** 12.02** 13.01**
Values are expressed in mean±SEM of six rats. One Way ANOVA followed by Dunnet’s t-test. F-value denotes statistical significance at *P<0.05,
**P<0.01 and t-value denotes statistical significance at aP<0.05, bP<0.01 and cP<0.001 respectively, in comparison to the solvent control.

Effect of extracts on glucose loaded hyper glycemic rats of test substances, whereas 52.97% (P<0.001) with the standard.
Methanol extract exhibited maximum reduction of blood glucose and
Methanol and aqueous extracts showed 46.51% and 37.28% better glucose tolerability among all the extracts when compared
(P<0.001) fall of BGL respectively at 4 h following the administration with the solvent control group at the end of 4 h (table 3).

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 Kar et al.
Int J Pharm Pharm Sci, Vol 7, Issue 9, 386-389

Table 3: Effect of extracts of C. philippinum leaves on BGL in glucose loaded rats

Groups and treatments Blood glucose levels (mg/dl) % decrease at 4 h
Pre-treatment Post-treatment
1h 2h 4h
Solvent Control 62.5±2.34 152.83±3.60 138.33±4.34 125.83±4.78 17.66
(Tween+Water)
Glibencamide (10 mg/kg) 72.33±6.91 137.16±3.86a 95.83±4.49c 64.5±3.11c 52.97
n-Hexane Extract (400 mg/kg) 71.83±3.34 151.33±2.83 125.83±4.11 116.16±6.25 23.24
Methanol Extract (400 mg/kg) 62.16±4.19 138.66±3.69a 112.16±4.63b 74.16±3.60c 46.51
Aqueous Extract (400 mg/kg) 74.66±3.33 147.5±3.73 116.16±3.26b 92.5±5.89c 37.28
F (4, 25) - 4.08* 14.24** 28.98**
Values are expressed in mean±SEM of six rats. One Way ANOVA followed by Dunnet’s t-test. F-value denotes statistical significance at *P<0.05,
**P<0.01 and t-value denotes statistical significance at aP<0.05, bP<0.01 and cP<0.001 respectively, in comparison to the solvent control.

Effect of extracts on STZ-induced diabetic rats hexane, methanol and aqueous extract at the end of 10 h of the
study, while the standard drug showed the reduction of 68.22%. The
Methanol and aqueous extracts at the dose level of 400 mg/kg body statistical analysis of variance (one-way ANOVA) of all the
weight reduced the BGL significantly, starting from 1 h (P<0.01) to experimental results from the test groups found significant (P<0.01)
the end of 10 h (P<0.001). The standard drug showed a similar effect differences among and in between the groups. The potency order of
during the experiment, and the percentage reductions of the BGL test extracts towards the blood glucose lowering property is found
were calculated as 38.99%, 66.76% and 57.17% with respect to n- to be methanol>aqueous>n-hexane extract (table 4).

Table 4: Effect of extracts of C. philippinum leaves on BGL in STZ-induced diabetic rats

Treatments Blood Glucose Levels (mg/dl) % decrease at
0h 1h 2h 4h 8h 10 h 10 h
Solvent Control 302.33±7.25 298.5±7.45 291.33±8.13 301.83±10.79 293.33±11.13 299.33±12.78 -
(Tween+Water)
Glibencamide 302.66±6.12 243.33±6.08b 164.83±9.08c 124.83±7.43c 99.83±6.83c 96.16±5.68c 68.22
(10 mg/kg)
n-Hexane Extract 295.33±9.85 287.16±9.26 263.83±10.87 241.83±12.06b 192.16±8.26c 180.16±13.06c 38.99
(400 mg/kg)
Methanol Extract 294.33±9.49 247.16±9.58b 191.33±8.15c 133.33±9.99c 103.33±7.13c 97.83±7.95c 66.76
(400 mg/kg)
Aqueous Extract 279.83±10.66 257.33±10.01b 227.16±11.38c 161.83±8.88c 128.66±11.70c 119.83±13.33c 57.17
(400 mg/kg)
F (5, 30) - 8.24** 28.68** 58.93** 77.76** 60.42**
Values are expressed in mean±SEM of six rats. One Way ANOVA followed by Dunnet’s t-test. F-value denotes statistical significance at *P<0.05,
**P<0.01 and t-value denotes statistical significance at aP<0.05, bP<0.01 and cP<0.001 respectively, in comparison to the solvent control.

DISCUSSION known to have α-amylases and α-glucosidases inhibitory activity [35-

37] and hence α-amylases and/or α-glucosidases inhibitory potential
The effect of extracts on normoglycemic rats showed a gradual fall of is expected with the tested extracts. STZ induces activation of poly
BGL in different tested h by all test extracts, in which methanol and adenosine diphosphate ribosylation and nitric oxide release, which
aqueous extract exhibited better effect. It may be suggested that the destroy pancreatic β-cells by necrosis [38]. The possible mechanism
blood glucose lowering effect of extracts after single dose by which extracts mediate its antidiabetic effect could be by
administration may be due to enhancement of peripheral glucose potentiation of pancreatic secretion of insulin from existing β-cells of
uptake [29]. Presence of alkaloids, flavonoids, glycosides, phenolic islets, as was evident by the significant decrease in BGL in the extracts
compounds, saponins, steroids and triterpenoids in methanol treated rats at the 10 h of study.
extract and flavonoids, glycosides, phenolic compounds and
saponins in aqueous extract may contribute the hypoglycemic CONCLUSION
potential of methanol and aqueous extract [30]. After pretreatment
with test extracts, BGL reaches to maximum level, 30 min after The experimental results of the present investigation conclude that
glucose load and then starts falling at the end to the experiment. the leaf extracts of C. philippinum is endowed with antidiabetic
Methanol and aqueous extract showed less BGL as compare to potential.
solvent control, to the response of glucose load and BGL gradually ACKNOWLEDGEMENT
falls down towards normal. Insulin secretion in response to the oral
ingestion of glucose occurs in three phases known as cephalic, The authors wish to thank the HOD, UDPS, Utkal University and
gastric and intestinal phase. The cephalic and gastric phases of Dean, SPS, S‘O’A University, Bhubaneswr for providing the necessary
insulin secretion are part of the initial response to glucose loading, facilities to carry out the research work.
whereas the intestinal response occurs in later stages [31, 32].
CONFLICT OF INTERESTS
The reduction on BGL by the extracts may be due to increased insulin
secretion and increased peripheral glucose utilization [33]. Digestive The authors declare that there are no conflicts of interest
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