Antidiabetic Activity of Phragmites karka (Retz.) in Alloxan
Induced Diabetic Rats.
Bharath C.L*, Bandenawaz Ramadurg
Dept. of Pharmacology, Gautham
college, Bengluru – 32
ABSTRACT:
Diabetes mellitus is the most common
endocrine disorder that impairs glucose homeostasis resulting in severe
diabetic complications including retinopathy, angiopathy,
nephropathy, and neuropathy causing neurological disorders due to perturbation
in utilization of glucose. In the present study diabetes was induced in albino
rat models with alloxan monohydrate. Phragmites karka (Retz), has been claimed to possess antidiabetic properties in Ayurveda
medicine system. The present study was undertaken to screen the hypoglycemic
activity of methanolic, petroleum ether and aqueous,
extracts Phragmites karka
(200mg/kg.,b.w.). The methnolic
and petroleum ether of Phragmites karka (200mg/kg.,b.w.) showed
significant decrease in blood glucose level when compare to aqueous extract of Phragmites karka
extract (200mg/kg.,b.w.). It also showed the signifent response in blood serum parameters (albumin,
urea, creatnine, and total protein) lipid parameters
such as total cholesterol, LDL, HDL, VLDL and TG when compared with diabetic
control.
KEYWORDS:
Alloxan, Phragmites karka,
Diabetes mellitus, Blood glucose, Lipid profile and Blood serum parameters.
INTRODUCTION:
Diabetes mellitus is a chronic disease that occurs
either when the pancreas does not produce enough insulin or when the body
cannot effectively use the insulin it produces. Insulin is a hormone that
regulates blood sugar. Defective insulin secretion is the major cause for
chronic hyperglycemia resulting in impaired function or serious damage to many
of the body’s systems, like eyes, kidneys, nerves, heart and blood vessels [1,
2]. The common signs and symptoms are excessive thirst and urination, weight
loss or gain, fatigue, and influenza–like symptoms. Early diabetes symptoms can
be very mild and often even unnoticeable. Diabetes mellitus is one of the
common metabolic disorders with micro and macro vascular complications that
results in significant morbidity and mortality. It is considered as one of the
five leading causes of death in the world [3, 4].
Diabetes mellitus is a group of syndromes
characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and
proteins and an increased risk of complications from vascular diseases. Most patients
can be classified clinically as having either Type 1 diabetes mellitus (IDDM).
It is an auto immune type I the main cause of this beta cell loss is a T-cell
mediated autoimmune attack. It is also characterized by loss of the
insulin-producing beta cells of the islets of Langerhans
in the pancreas, leading to a deficiency of insulin [5]. Type 2 diabetes
mellitus (NIDDM) is characterized differently due to insulin resistance or
reduced insulin sensitivity, combined with reduced insulin secretion. Variants
in 11 genes significantly associated with the risk of Type 2 diabetes of these
8 genes are responsible for impaired beta-cell function [6]. One such plant is Phragmites karka which
has been used in ayurveda medicine systeme for treating diabetes.
Phragmites
karka is
a perennial reed with long rhizomes and robust, erect culms to 3 m.
The leaves are 15-30 cm long and nearly 2.5 cm broad; inflorescence is a large
plume-like panicle with capillary branches and small, slender spikelets. It is leafy up the panicle. Phragmites
can be easily distinguished from Arundo and Neyraudia by the silky beards at the bases of the lowest
panicle branches, which are absent in the other two. In New
Guinea the reed occurs from near sea-level to at least 2000 m. It thrives
in a rainfall regime from 200 to 5000 mm in swamps (India). It grows in
standing water and is therefore tolerant of flooding. It usually grows in clay
soils ranging from strongly acid (pH 4.5) to slightly alkaline (pH 7.5)[7].
In Ayurveda system of medicine
this plant is reported to posses anti diabetic, diuretic and anti-emitic activity[8]. The present paper narrates and justify
the traditional use of plant with respect to antidiabetic
activity, blood serum and lipid profile parameters of methanolic,
petroleum ether and aqueous extract of
plant of Phragmites
karka in rats.
MATERIAL
AND METHODS:
Plant
material:
The leaves of Phragmites
karka (Retz.) were collected from the Paschimavahani, Mysore, Karnataka, identified and
authenticated by Dr. Rajanna Project Co-ordinator, AICRP on Small Millets, ICAR, UAS, GKVK,
Bangalore, Karnataka, India.
Prepration of
extract:
Methanolic
(MEPK), petroleum ether (PEPK) and aqueous extracts (AEEN) of Phragmites karka (Retz.)
were obtained from Green Chem, Anekal road – 107, Bengluru.
The methanolic, petroleum ether,
and aqueous extracts of Phragmites karka (Retz.) was subjected to the following
investigations.
1. Preliminary phytochemical screening of different extracts.
2. Acute oral toxicity studies
to determine the safety and dose.
3. Antidiabetic
activity.
Experimental
animals:
Albino wistar
rats weighing 150-220g were procured from Biogen,
Bangalore. They were maintained in the animal house of Gautham
College of Pharmacy. Animals were maintained under controlled condition of
temperature at 27o ± 2o C and 12-h light-dark cycles. They were housed in
polypropylene cages and had a free access to standard pellets (Amruth) and water ad libitum.
All the studies conducted were approved by the Institutional Animal Ethical
Committee (IAEC) of Gautham College of pharmacy, Bangalore
(REF-IAEC/003/5/2010) according to prescribed guidelines of Committee for the
Purpose of Control and Supervision of Experiments on Animals
(REF-IAEC/02/06/2012-13), Govt. of India.
Chemicals
used:
Alloxan Monohydrate was purchased
from Sigma Swemed Diagnostics Pvt., Ltd., Bangalore.
All other chemicals and reagents used were of analytical grade.
Drugs:
Standard drug:
Glibenclamide,
Test drug: Plant extract of Phragmites karka (Retz). Oral acute toxicity studies
Acute
Toxicity studies[9]:
Animals:
According to the OECD guideline no. 425, the acute oral toxicity
study was performed. Female Albino rats weighing 150-220g were used for the
study. They were nulliparous and non-pregnant. These
were acclimatized to laboratory condition for one week prior to start of
dosing.
Procedure:
Animals were fasted overnight with water ad libitum. Each animal received a single dose of
2000 mg/kg b.w., p.o. After
administration of extract, food was withheld for 3-4 h. If the animal dies,
conduct the main test to determine the LD50. If the animal survives, dose four
additional animals sequentially so that a total of five animals are tested.
However, if three animals die, the limit test is terminated and the main test
is performed. The LD50 is greater than 2000 mg/kg p.o,
if three or more animals survive. If an animal unexpectedly dies late in the
study, and there are other survivors, it is appropriate to stop dosing and
observe all animals to see if other animals will also die during a similar
observation period. Late deaths should be counted the same as other deaths. The
same procedure was repeated with another set of animals to nullify the errors.
All the animals are kept under observation for 14 days to see any late effects
of the extracts. All the three different extracts were studied as above to
determine the safety and dose of the respective extracts.
EXPERIMENTAL
DESIGN [10,11]:
i. Preparation of alloxan
(ALX) solution:
Alloxan monohydrate dissolved on
0.9% sodium chloride solution.
ii. Experimentally Induced
Diabetes Mellitus:
Female Wistar rats weighing
150-220g were used for this study. The animals were overnight fasted for 16h
before the induction of diabetes. Diabetes was induced by a single dose of 120
mg/kg body weight of alloxan by intraperitoneal
route. After a period of 2 days blood glucose levels were checked by snipping
the tail of alloxan treated fasted rats. Rats showing
the blood glucose levels more than 300 mg/dl is taken into the study.
Diabetic rats were divided into five groups (Group II –
VI). Group I served as vehicle control (Non diabetic rats).
Ř Group-I:
Vehicle control (Non diabetic rats/ normal rats).
Ř Group-II:
Diabetic rats
Ř Group-III:
Diabetic rats received Glibenclamide (5 mg/kg.b.w.,p.o.)
for 21 days and served as standard
Ř Group-IV:
Diabetic rats received MEPK
(200 mg/kg.b.w.,p.o.) for 21 days.
Ř Group-V:
Diabetic rats received PEPK
(200 mg/kg.b.w.,p.o.) for 21 days.
Ř Group-VI:
Diabetic rats received AEPK
(200 mg/kg.b.w.,p.o.) for 21 days.
Fasting blood glucose levels were measured before the
administration of MEPK, PEPK & AEPK.
It was recorded as 0 day. The blood
glucose levels were checked on 0, 7, 14, and 21 day of the treatment period.
Blood was collected from snipping of the rat tail. Blood glucose levels were
measured by using Sugarchek glucometer
manufactured by Wockhardt.
At the end of the experimental period, all the animals
were sacrificed by survical dislocation and different
organs of the body (heart, pancreas, liver, kidney R & L and spleen ) was
weighed and blood was collected with anti-coagulant and the serum was used for
the estimation of various biochemical parameters like albumin, urea, creatinine,
total protein LDL, HDL, VLDL, TG, TC.
The values are expressed as Mean ± SEM. The data was analysed by using one way ANOVA followed by Dunnett’s test using Graph pad prism software (version
6.01). Statistical significance was set at P ≤ 0.05.
RESULTS
AND DISCUSSION:
Preliminary phytochemical
constituents[12]:
Phytochemical analysis
was carried out by using the standard procedures. Alkaloids, carbohydrates, flavonoids, glycosides, phytosterols/terpenes, proteins and saponins
were qualitatively analysed.
Acute oral toxicity studies:
Single dose administration of MEPK, PEPK & AEPK at
2000mg/kg b.w. showed no mortality in any of the
animals. Hence, 1/10th of the dose (200 mg/kg b.w.)
was selected for the present antidiabetic study.
Antidiabetic
activity:
The effect of repeated oral administration of methanolic, petroleum ether and aqueous extract of the plant of Phragmites
karka (MEPK, PEPK and AEPK) on blood glucose
levels and various organ weight in alloxan-diabetic rats is presented in Table- 1 , and the
effect on body weight and different
serum parameters levels is presented in Table- 2. MEPK and PEPK (200mg/kg.,b.w.) in alloxan-treated diabetic rats caused significant dose
related and duration dependent reduction of blood glucose levels when compared
to AEPK(200mg/kg.,b.w.). Maximum reduction was
observed on day 21. Gradual increase in body weight was also observed. MEPK and
PEPK (200mg/kg.,b.w.) exhibited maximum glucose
lowering effect in diabetic rats and also significant changes in the serum
parameters level when compared with the AEPK (200mg/kg.,b.w.).
Glibenclamide exhibited significant reduction in
blood glucose levels at the end of the study when compared to diabetic control.
Table 1: ALX Model Sheet Blood Glucouse
level and Various Organs Weight.
|
Alloxane induced
model in rats |
||||||||
|
Physical Parameters |
||||||||
|
Parameters |
Groups |
|||||||
|
Group-I Vehicle
control |
Group-I ALX
(120mg/kg, b.w.) + Saline |
Group-I ALX
(120mg/kg, b.w.) + Glibenclamide(5mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + MEPK
(100mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + PEPK
(100mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + AEPK
(100mg/kg, b.w.) |
|||
|
Body
weight (g) |
Day
0 |
173.0
± 0.50 |
159.3
± 1.08 |
171.30
± 1.05 |
161.0
± 1.61 |
159.3
± 1.38 |
160.0
± 1.06 |
|
|
Day
7 |
176.3
± 1.64 |
152.8
± 1.30 a*** |
173.2
± 0.79 b*** |
165.70
± 1.60 b*** |
162.0
± 0.89 b*** |
162.20
± 0.87 b*** |
||
|
Day
14 |
108.3
±2.04 |
146.7
± 1.47 a*** |
174.7
± 0.88 b*** |
169.5
± 1.72 b*** |
165.0
± 0.44 b*** |
165.2
± 0.87 b*** |
||
|
Day
21 |
189.2
± 2.41 |
140.80
± 1.70a*** |
184.2
± 0.70 b*** |
174.7
± 1.56 b*** |
168.3
±0.71b*** |
167.7
± 0.98b*** |
||
|
Organ
weight (g) |
Pancreas |
0.79
± 0.01 |
0.48
± 0.04 a*** |
0.74
± 0.04 b*** |
0.70
± 0.02 b*** |
0.65
± 0.02 b** |
0.60
± 0.02 b* |
|
|
Liver |
4.22
± 0.44 |
3.27
± 0.17 b*** |
4.11
± 0.18 b*** |
3.95
± 0.33 b*** |
3.80
± 0.11b* |
3.25
± 0.25 b* |
||
|
Heart |
0.66
± 0.44 |
0.36
± 0.02 a*** |
0.64
± 0.04 b*** |
0.58
± 0.01 b*** |
0.54
± 0.03 b** |
0.47
± 0.01b* |
||
|
Kidney |
R |
0.72
± 0.03 |
0.48
± 0.02 a*** |
0.67
± 0.02 b*** |
0.61
± 0.02 b* |
0.58
± 0.01b* |
0.58
± 0.04 bns |
|
|
L |
0.70
± 0.02 |
0.47
± 0.01 a*** |
0.67
± 0.03 a*** |
0.64
± 0.02 b*** |
0.60
± 0.03b** |
0.56
± 0.02 b* |
||
|
Spleen |
0.80
± 0.02 |
0.53
± 0.01a*** |
0.73
± 0.01 b*** |
0.68
± 0.05b** |
0.62
± 0.01bns |
0.56
± 0.03b* |
||
MEPK – Methnolic
extract of phragmites karka;
PEPK – Petroleum ether extract of phragmites
karka; AEPK – Aqueous extract of Phragmites karka; Values
are expressed as mean ± SEM (n=06)
Data
were analyzed by one way ANOVA followed by Dunnett’st
test. *** P<0.001, ** P<0.01 &
*P<0.05.
a compared with Vehicle control, bcompared with the alloxan
treated group
Table 2 : ALX Model sheet of Body Weight and Various Parameters.
|
Alloxane induced
model in rats |
||||||||
|
Biochemical Parameters |
||||||||
|
Parameters |
Groups |
|||||||
|
Group-I Vehicle
control |
Group-I ALX
(120mg/kg, b.w.) + Saline |
Group-I ALX
(120mg/kg, b.w.) + Glibenclamide(5mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + MEPK
(100mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + PEPK
(100mg/kg, b.w.) |
Group-I ALX(120mg/kg,
b.w.) + AEPK
(100mg/kg, b.w.) |
|||
|
Body
weight(g) |
Day
0 |
173.0
± 0.50 |
159.3
± 1.08 |
108.3
±2.04 |
161.0
± 1.61 |
159.3
± 1.38 |
160.0
± 1.06 |
|
|
Day
7 |
176.3
± 1.64 |
152.8
± 1.30 a*** |
173.2
± 0.79 b*** |
165.70
± 1.60 b*** |
162.0
± 0.89 b*** |
162.20
± 0.87 b*** |
||
|
Day
14 |
108.3
±2.04 |
146.7
± 1.47 a*** |
174.7
± 0.88 b*** |
169.5
± 1.72 b*** |
165.0
± 0.44 b*** |
165.2
± 0.87 b*** |
||
|
Day
21 |
189.2
± 2.41 |
140.80
± 1.70a*** |
184.2
± 0.70 b*** |
174.7
± 1.56 b*** |
168.3
±0.71b*** |
167.7
± 0.98b*** |
||
|
Serum
Albumin(g/dL) |
5.46
± 0.14 |
2.82
± 0.26 a*** |
4.98
± 0.09 b*** |
3.90
± 0.13 b*** |
3.40
± 0.04 b** |
3.22
± 0.14 bns |
||
|
Serum
Urea(mg/dL) |
51.10
± 1.38 |
107.2
± 2.60 a*** |
60.29
± 2.14 b*** |
65.63
± 0.78 b*** |
68.36
± 0.45 b*** |
70.50
± 0.59 b*** |
||
|
Serum
Total protein(mg/dL) |
10.75
± 0.83 |
5.01
± 0.11 a*** |
9.03
± 0.19 b*** |
7.98
± 0.12 b*** |
7.18
± 0.09 b** |
6.59
± 0.13 b* |
||
|
Serum
Cretanine(mg/dL) |
0.88
± 0.01 |
1.75
± 0.02a*** |
1.12
± 0.03b*** |
1.49
± 0.04b*** |
1.57
± 0.02b** |
1.68
± 0.03ns |
||
|
Hemoglobin(mg/dL) |
12.57
± 0.19 |
7.46
± 0.21a*** |
12.23
± 0.23 b*** |
11.93
± 0.24 b*** |
11.07
± 0.34 b** |
8.61
± 0.30 b* |
||
|
Lipid
profile (mg/dL) |
TG |
66.67
± 4.15 |
111.6
± 2.52 a*** |
76.09
± 3.32 b*** |
85.51
± 2.15 b*** |
88.41
± 3.10 b*** |
90.58
± 3.05 b*** |
|
|
TC |
90.56
± 1.70 |
129.7
± 2.17 a*** |
94.17
± 1.65 b*** |
99.44
± 0.92 b*** |
102.50
± 1.47 b*** |
105.3
± 1.00 b*** |
||
|
HDL-C |
30.38
± 0.21 |
15.40
± 0.30 a*** |
27.65
± 0.33 b*** |
22.13
± 0.36 b*** |
20.15
± 036 b*** |
18.69
± 0.25 b*** |
||
|
LDL-C |
73.49
± 2.04 |
136.6
± 2.04 a*** |
81.73
± 1.83 b*** |
94.42
± 1.05 b*** |
100.01
± 1.86 b*** |
104.7
± 1.29 b*** |
||
|
VLDL-C |
11.32
± 1.9 |
22.32
± 0.50 a*** |
15.22
± 0.66 b*** |
17.10
± 0.43 b*** |
17.68
± 0.62 b*** |
15.68
± 2.49 b*** |
||
|
SOD
U/mg protein |
17.97
± 1.05 |
8.03
± 0.09 a*** |
16.20
± 0.58 b*** |
15.10
± 0.25 b*** |
13.23
± 0.22 b*** |
10.72
± 0.26 b*** |
||
|
TBARS
(nmoles of MDA/ 100 mg of tissue) |
1.33
± 0.05 |
3.83
± 0.07 a*** |
1.65
± 0.03 b*** |
1.97
± 0.03 b*** |
2.32
± 0.01 b*** |
2.59
± 0.03 b*** |
||
|
GSH
(mM/ 100 mg of tissue) |
45.93
± 0.27 |
24.31
± 0.09 a*** |
47.40
± 0.36 b*** |
49.59
± 0.17 b*** |
52.38
± 0.18 b*** |
34.24
± 0.14 b*** |
||
Values are expressed as mean ± SEM (n=06)
Data were analyzed by one way ANOVA followed by Dunnett’st test.
*** P<0.001, ** P<0.01 & *P<0.05.
a compared
with Vehicle control, compared with the alloxan
treated group
CONCLUSION:
The present study indicated that administration of Methanolic, Petroleum ether & Aqueous extracts of Phragmites karka at
a dose of (200mg/kg.,b.w.) posseses
antihyperglycemic activity in ALX diabetic rats.
Aqueous extract of Phragmites karka (200mg/kg.,b.w.) shows
less effect then the Methanolic and Petroleum ether
extracts (200mg/kg.,b.w.) in reducing the blood
glucose levels. The acute toxicity study indicated that the extracts are devoid
of major toxic effects. Besides this, the drug administered to treat ALX
induced diabetic rats showed a significant reduces in blood glucose levels and
the other serum biomarker levels and also increases the haemoglobin
levels. The extracts also exhibited antioxidant activity in
Methanolic,
Petroleum ether and Aqueous treated diabetic rats.
The reports of histopathology study concluded that the plant
extract treated animal shown significant increased mass of β-cells in the
pancreatic islets. The results showed in Methanolic
extract having some similar to glibenclamide treated
group which was used as reference standard. These observations concluded that
the extracts of the plant Phragmites karka posses antidiabetic and
antioxidant effects. Over all absorved significant
activity may be due to presents of active constituents present in Methnolic, Petroleum ether and Aqueous extract of Phragmites karka.
Further, the work could be extended to evaluate the
effectiveness of the exact active compounds for the treatment of diabetes at
its cellular level to elucidate its exact mechanism for the traditional claim.
ACKNOWLEDGMENT:
The author sincerely thanks to guide Bandenawaz
Ramadurg for rendering his suggestion and helping
them in each and every step of completing this research paper successfully.
REFERENCE:
1. World Health Organization; Fact sheet No
312, November 2009. http://www.who.int/mediacentre/factsheets/fs312/en/index.html
(Cited on 12/6/2013).
2. Susheela T, Padma Balaravi, Jane Theophilus, Narender reddy and Reddy PUM. Evaluation of hypoglycemic and antidiabetic effect of Melia
Dubia fruit in mice. Cur Sci
2008; 94 (9):1191-95.
3. Diabetes statistics: India is the
diabetic capital of the World. Available from:
http://health.savvy-cafe.com/diabetes-statistics-india-is-the-diabetic-capital-of-the-world.
(Cited on 12/6/2013).
4. Bhupesh CM, Kamal S, Nagendra SC Rohit B Kalyani D. anti-diabetic
activity of stem bark of Berberis aristata D.C. in alloxan
induced diabetic rats. Int J Pharmacol
2008;6.p. 1-6.
5. Goodman & Gilman’s. The
pharmacological basis of therapeutics. 10 ed. McGraw Hill: p.1686-1687.
6. Valeriya L, Anna J, Peter A, Nicolo
P, Tiinamaija T. Clinical risk factors, DNA
variants, and the development of type 2 diabetes. N Engl
J Med 2008;359 (21):2220–32.
7. www.fao.org/ag/AGP/AGPC/doc/Gbase/DATA/PF000309.
HTM (Cited on 12/6/2013).
8. http://www.flowersofindia.net/catalog/slides/Tall%20
Reed.html\ (Cited on 12/6/2013).
9. OECD
Guidelines for the Testing of Chemical. Acute Oral Toxicity – Up and Down
Procedure (UDP). ) (Cited on 25/3/2012). 2008 Available from:
http://iccvam.niehs.nih.gov/ SuppDocs/FedDocs/OECD/OEC Dtg425.pdf.
10. Nandhakumar Jothivel, Sethumathi Padhapalyam Pannasamya, Malini Appachi , et al. Anti-diabetic Activity of methanol leaf
Extract of Costus Pictus
D.D ON in Alloxan-induced Diabetic Rats. J Health
Sci. 2007;53 (6)–655-63.
11. Md Shalam, MS Harish, SA Farhana. Prevention of dexamethasone
and fructose induced insulin resistance in rats by SH-01D, a herbal
preparation. Indian J Pharmacol. 2008;38 (6):419-22
12. Kokate CK, Khandelwal KR, Pawar AP, Gohale SB. Practical
Pharmacognosy. 3rd ed. Nirali Prakashan
(Pune); 1995.p. 137-39.
Received on 26.10.2013
Modified on 10.11.2013
Accepted on 14.11.2013
© A&V Publication all right
reserved
Research J. Pharmacology and
Pharmacodynamics. 5(6): November –December 2013, 371-375