Anti Hyperglycemic Activity of Cichorium intybus, Gymnema sylvestre, Aegle marmelos in Alloxan Induced Diabetic Rats

 

B Eswarapriya2, B Kameswari1*, P Devi1, M Sharmila banu1, B Madhumitha1 and R Meera1

1K.M.College of Pharmacy, Uthangudi, Madurai -625107.

2Department of Biotechnology, St. Michael College of Engineering, Sivagangai, TamilNadu, India

 

ABSTRACT:

The antidiabetic activity of ethanolic fresh leaf extracts of Cichorium intybus, Gymnema sylvestre, Aegle marmelos in alloxan induced diabetic rats. Alloxan induced diabetic in rats (n=6) were administered ethanolic fresh leaf extracts of  Cichorium intybus (0.25g/Kg), Gymnema  sylvestre (0.25g/Kg) , Aegle marmelos (0.25g/Kg)  and standard drug  Glibenclamide (0.07g/Kg) for 7 days. On eight day of the treatment , blood samples were collected by puncturing the retro-orbital plexus under mild ether anesthesia and kept aside for ½ h for clotting. Blood glucose levels were determined by glucose oxidase method. Serum was separated by centrifuging the samples for 20 mins and stored in the refrigerator. The serum was analyzed for blood glucose level, total protein, cholesterol and triglycerides .These three medicinal plants of alcoholic extracts shown significant (P<0.05) reduction in blood glucose level.  However, Cichorium intybus  leaf extracts was highly effective and results are comparable with that of reference drug, glibenclamide. Diabetic animals treated with these three leaf alcoholic extracts shown significant (P<0.05) effect on serum protein, cholesterol and triglyceride level. In comparison to other extracts Cichorium intybus alcoholic leaf extract fraction was found to be more potent in normalizing the blood lipids and protein level in Alloxan induced diabetic rats. It is concluded that Cichorium intybus,  Gymnema sylvestre  and Aegle marmelos  has significant antidiabetic activity as it lowers the blood glucose levels in diabetic rats and increases the glucose tolerance.

 

KEY WORDS: Cichorium intybus, Gymnema sylvestre  and Aegle marmelos, Antidiabetic  activity, alloxan – induced diabetes in rats, Fresh leaf ethanolic extracts.

 

INTRODUCTION:

Diabetes  Mellitus  is  a  syndrome  resulting  from  a  variable  interaction  of  hereditary   an  environmental  factor.  It  also  a  common  disorder  with  high  generation  of  free radical  injury,  mediated  complication.1  In  modern  medicine  no  satisfactory   effective  therapy  is  still  available  to  cure  Diabetes  Mellitus.  In  a  large  number  of  cases  treatment  with  traditional  medicine  in  the  form  of  plant  extracts  have  been  reported  to  give  remarkably  good  results.  Herbal preparations prove good to maintain blood sugar level.  Though  plant  drug  are  frequently  considered  to  be  less  toxic  and  more  free  from  side  effect  than  synthetic  chemical  compounds2.  Many medicinal  plants  have  been  found  possess  principle  useful  for  treating  diseases3.

 

Cichorium intybus has a long tap root and round stem and dark leaves. It is used for curative properties against kapha, pitta, amenorrhea and general ability4. It as hypoglycemic effect also named as “Kasinikeerai”. Gymnema sylvestre is most potent agents to co-interact diabetes. The leaf is bitter, acrid and it has hypoglycemic effect. The common name is “Sugar destroyer”. It acts indirectly through stimulation and secretion of pancreas5. Aegle marmelos a member of genes Aegle belonging to family Rutaceae.

 

The mechanism of Aegle marmelos extract can be used partially to increase the utilization of glucose by direct stimulation of glucose uptake (or) the mediation to enhance the insulin secretion6. It is used as Antidiabetic agents. From literature survey it was evident that the extracts of plants have been reported for its Antidiabetic activity in streptozotocin total protein, cholesterol and triglycerides in diabetic mice at a dose of 0.45 g/ kg body weight 7. In the present investigation has been made to find out the potent fraction responsible for the said activity at a dose of 250mg/ kg (or) 0.25g/kg body weight.    Thus, the present work describes effect of the these three medicinal plants leaf extracts on the blood glucose, total protein, cholesterol and triglyceride level in alloxan induced diabetic rats.

 

MATERIALS AND METHODS:

Collection of plant material:

The three medicinal plants were collected at Aritapatti village, near Madurai. The crude drug was authenticated from the Botany Department Dr. Stephen, American college, Madurai.

 

Preparation of ethanolic fresh leaf extract:

The leaves were dried under shade and crushed into coarse powder. The dried powdered three plant leaves were used for extraction. Extraction is done separately for three plant leaves. 1Kg of dried powdered leaves were first defatted by using organic solvents such as petroleum ether (60-80 analytical grade). Then after defatting the leaves by using cold extraction method, the leaves were air dried. This air dried leaves were extracted by using ethanol (Soxhlet extraction method) for 48 hours. The solution was evaporated and stored in a desiccator.  Then it was dried in vacuum oven and utilized for animal studies. The alcoholic extracts of these three plants were subjected to preliminary qualitative chemical investigations which indicated the presence of phenolic compounds, alkaloids, steroids, flavanoids and saponins.

 

Animals:

In house bred male albino rats between 170-200g were utilized for study. Animals were housed in polypropylene cages maintained under standard conditions and the animal fed with standard pellet diet and water adlibitum. All the protocols were performed in accordance with the Institutional Animal Ethical committee(IAEC) as per the directions of the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals.

 

Acute toxicity study:8-10

Animals were starved over night and divided into 5 groups (n=6) .They were fed orally with the ethanolic fresh leaf extracts of Cichorium intybus , Gymnema sylvestre, Aegle marmelos in increasing does levels of up to 2000mg/Kg body weight. The behavior of the animal was carefully monitored for 1 day.

1.   CNS activity : Tremors, Convulsions

2.   CVS activity: Palpitation, increase in pulse rate.

3.   Respiratory tract activity  :Bronchial constriction,        difficult in breathing

4.   Reflex action: Loss of writhing reflex.

 

The number of deaths, if any was recorded after 24 and 72 h.

 

Induction of alloxan monohydrate diabetes mellitus:

Diabetes was induced in rats by Intra peritoneal injection of alloxan monohydrate dissolved in normal saline solution, 11pH 4.5 at a dose of 0.14g/ kg. The diabetic state was confirmed after 7 days induction of alloxan by weight loss and hypoglycemia12. Rats with blood sugar level 200-250mg/dl were selected for the study.

 

Collection of blood and determination of serum glucose:

Blood samples were collected by puncturing the retro-orbital plexus under mild ether anesthesia and kept aside for ½ h for clotting. Blood glucose levels were determined by glucose oxidase method13. Serum was separated by centrifuging the samples for 20 mins and stored in the refrigerator. The serum was analysed for blood glucose level (GOD-PAP method)14, total protein (Biuret method)15, cholesterol (CHOD-PAP method)16 and triglycerides (enzymatic method)17 .

 

Experimental design for antidiabetic activity:

The rats were divided into six groups each group of six animals. Group I served as diabetic control received 0.2% w/v CMC, orally and alloxan. Group II served as positive control and received standard Glibenclamide (0.07g/Kg). Group III received alcoholic extract of Cichorium intybus (0.25g/Kg).Group IV received alcoholic extract of Gymnema sylvestre (0.25g/Kg).Group V received alcoholic extract of Aegle marmelos (0.25g/Kg). Group VI served as normal untreated group. The treatment was continued for 7d by administering the respective fractions in 0.2%w/v CMC. On eigth day of the treatment, the blood glucose level and body weight were calculated.

 

statistical analysis

All the values of the experimental results were expressed as mean ± SEM and analysed for ANOVA followed by Dunnett’s test.

 

RESULTS:

Effect of ethanolic fresh leaf extracts of Cichorium intybus, Gymnema sylvestre, Aegle marmelos on blood sugar level and biochemical parameters Cichorium intybus  leaf extracts was highly effective and results are comparable with that of reference drug,  glibenclamide  (Table  1). Diabetic animals treated with these three leaf alcoholic extracts shown significant (P<0.05) effect on serum protein, cholesterol and triglyceride level (Table 2).  In comparison to other extracts Cichorium intybus alcoholic leaf extract fraction was found to be more potent in normalizing the blood lipids and protein level.

 

Effect of ethanolic fresh leaf extracts of Cichorium intybus, Gymnema sylvestre, Aegle marmelos on changes in body weight

Diabetic rats showed a decrease in body weight during the experimental period. This was significantly antagonized by the extracts.

 

DISCUSSION:

Alloxan –induced β-cell cytotoxicity is well established animal model for investigating antidiabetic activity18. From the results it is evident that these three medicinal plants of alcoholic extracts shown  significant (P<0.05)  reduction in blood glucose level Alloxan treated  rats shown substantial weight   loss and  also  affect  carbohydrate,  lipid  and protein metabolism. 


Sl. no

Groups

Before commencement of treatment

After end of the treatment

Normal

Alloxan treated

After extract/ drug treatment

1.

Normal control (non- diabetic)

181.1±1.24

180.0±1.01

84.5±3.5

_

87.5±1.5

2.

Diabetic control (untreated)

175.5±0.82*

171.0±1.92*

88.33±0.87*

206.0±1.15*

_

3.

Glibenclamide (0.07 g/Kg)

185.6±1.67*

183.2±1.05*

86.33±0.88*

205.0±1.14*

92±1.15*

4.

Alcoholic extract of Cichorium intybus (0.25g/Kg)

187.2±1.87*

184.3±1.45*

87±1.15*

203.66±0.66*

99.33±1.33*

5.

Alcoholic extract of  Gymnema sylvestre (0.25g/Kg)

190.5±1.58*

184.6±4.9*

84.66±0.88*

203.0±1.15*

97.66±1.20*

6.

Alcoholic extract of Aegle Marmelos (0.25g/Kg)

196.4±1.41*

190.2±1.39*

86.7±0.88*

204.0±1.15*

106.66±1.27*

TABLE I: EFFECT OF LEAF EXTRACTS OF CICHORIUM INTYBUS, GYMNEMA SYLVESTRE AND AEGLE MARMELOS ON BODY WEIGHT AND BLOOD SUGAR LEVEL

The observations are given as mean ±SEM ,n=6, *p< 0.05 compared with diabetic control

 

 

TABLE 2: EFFECT  OF  LEAF  EXTRACTS  OF  CICHORIUM   INTYBUS,  GYMNEMA  SYLVESTRE  AND AEGLE MARMELOS ON BIOCHEMICAL PARAMETERS

Groups

Biochemical parameters

Cholesterol (mg/dl)

Triglyceride (mg/dl)

Totalprotein (g/dl)

Normal control (non- diabetic)

62.5±6.2

115.1±1.5

8.1±0.2

Diabetic control (untreated)

174.3±1.3

205.2±2.3

5.3±2.2

Glibenclamide (0.07g/Kg)

69.1±1.4

132.3±1.0

8.9±0.1

Alcoholic extract of Cichorium intybus (0.25g/Kg)

51.87±1.8

105.0±5.1

6.7±0.2

Alcoholic extract of Gymnema sylvestre (0.25g/Kg)

56.3 ±2.6

115.1±2.5

7.2±0.1

Alcoholic extract of Aegle marmelos (0.25g/Kg)

60.4±1.2

108.2±3.4

7.3±0.2

The observations are given as mean ±SEM , n=6, p< 0.05 compared with diabetic control group.

 

 

All three extracts were found to be effective in restoring the body weight of animals to the normal value. In comparison to other extracts Cichorium intybus alcoholic  leaf  extract fraction was found to be more potent in normalizing the blood lipids  and protein level.  Proximate chemical analysis reveals the presence of phenolic compounds, alkaloids,  steroids,  flavanoids  and  saponins  in   Gymnema sylvestre  and  Aegle marmelos  where as   Cichorium intybus  alcoholic  leaf  extract fraction  was found to be very rich in flavonoid content.  Thus,  flavanoids  from  Cichorium intybus  may be responsible for its antidiabetic potency19.  This study ,  therefore,  indicates  Cichorium intybus  alcoholic  leaf  extract  as a potent  fraction highly responsible for antihyperglycemic effect  in alloxan induced diabetes.

 

CONCLUSION:

The ethanolic fresh leaf extracts of Cichorium intybus , Gymnema sylvestre ,Aegle marmelos has anti diabetic activity as it lowers serum glucose levels in diabetic rats and significantly increases glucose tolerance . It also increases the body weight of  diabetic rats.Hence long term studies of ethanolic fresh leaf extracts of Cichorium intybus , Gymnema sylvestre ,Aegle marmelos and its isolated compounds are necessary to elucidate the exact mechanism of action so as to develop it as a potent anti diabetic drug.

 

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12.     R .Rom an Romos  ,  A .Lara Lemus,  F .Alarc,  Floras Saeny. Hypoglycemic activity of some anti-diabetic plants.  Arch med res .,1992;23:105.

13.     Varley Harold. Practical clinical biochemistry. 4th  ed. Delhi, India; publishers and distributors;.1988,p.84 .

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15.     R.T .Burkhardt  ,  J.G. Batsakis  .An interlaboratory comparison of serum total protein  analyses.  Am  J Clin  Pathol .,1978;70:508-10.

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Received on 11.09.2009

Accepted on 30.10.2009     

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Research J. Pharmacology and Pharmacodynamics 2(1): Jan. –Feb. 2010: 75-77