Phytochemical Test

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Journal of Chemical and Pharmaceutical Research __________________________________________________

ISSN No: 0975-7384 CODEN(USA): JCPRC5

J. Chem. Pharm. Res., 2010, 2(4):266-283

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Phytochemical and Pharmacological Evaluation of Leaves of Spinica Oleraceae Linn

V. Gomathi*, R. Kodai, B. Jayakar, Sasi Bhushanarao. Poola

Vinayaka Missions College of Pharmacy

Vinayaka Mission University, Salem, Tamilnadu ______________________________________________________________________________

ABSTRACT Spinica oleraceae is an indigenous, potent, non-toxic traditional medicinal plant in its alcoholic and ethanolic aqueous leaf extract shows significant anti-hyperglycemic and anti-hyperlipedaemic activity. Diabetes mellitus is the most common metabolic disorder and it ranks highly among top ten disorders causing mortality throughout the world. The present study was designed to study the anti-hypeglycemic and anti-hyperlipedaemic activities of Spinica oleraceae using alloxan induced diabetic rats.Alloxan when administered in rabbits reported a specific necrosis of pancreatic islets since then alloxan diabetes has been commonly utilized as an animal model of insulin dependant diabetes which is used to measure anti-diabetic activity.Hyper lipidemia can be caused by genetic predisposition through secondary causes such as drugs,lifestyle etc.At first test for different chemical compounds in the plant leaves is performed and then pharmacological studies such as oral acute toxicity studies invitro and invivo studies on swin albino and sprauge drawly rats of either sex is performed .The effect of ethanolic and aqueous extract of leaves of spinica oleraceae linn in normal fasted rats is studied and finally found that the leaves exhibit considerable anti-hyperglycemic and anti-hyperlipedaemic property

Key Words: Alloxan, Invitro, Hyperlipidemic, Invivo, Diabetes mellitus. ______________________________________________________________________________

V. Gomathi et al J. Chem. Pharm. Res., 2010, 2(4):266-283 _____________________________________________________________________________

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Aim and Objective: The main aim and objective of the present study was to determine the phytochemical and pharmacological evaluation of leaves of spinica oleraceae linn and to focus on anti -hyperglycemic and anti-hyperlipedaemic property of leaves using alloxan induced diabetic rats.

INTRODUCTION

Diabetes mellitus is a group of diseases characterized by an elevated blood glucose level (hyperglycaemia) resulting from defects in insulin secretion, in insulin action, or both. Diabetes mellitus is not a pathogenic entity but a group of etiologically different metabolic defects. Common symptoms of diabetes are lethargy from marked hyperglycaemia, polyuria, polydipsia, weight loss, blurred vision and susceptibility to certain infections. Severe hyperglycaemia may lead to hyperosmolar syndrome and insulin deficiency to life-threatening ketoacidosis. Chronic hyperglycaemia causes long-term damage, dysfunction and failures of various cells, tissues and organs. The word "diabetes" is derived from a Greek word that means, "to siphon or drain off", the most obvious sign of diabetes being excessive urination. "Mellitus" comes from a Latin word that means "sweet". Hyperlipidemia is defined as an increase in the total cholesterol and low densitylipoproteins in the circulating blood lipids and other abnormalities of lipid metabolism.Studies have shown the elevated cholesterol levels are independent and significant risk factor for CHD (Dr.rose etal),(R.Walker et al.Hyperlipidemia is the major risk factor in the initiation and progress of the artherosclerotic lesions.Evidences from studies both in animals and humans indicatethat progression can be solved if elevated serum concentrations of the artherogenic lipoprotein and triglycerides are reduced,whichin turn prevents coronary heart diseases.The term hyperlipidemia signifies high lipid or fat content in blood.Most people are familiar with having high cholesterol,but hyperlipidemia could also refer to having high amounts of triglycerides,phospholipids,or genetic factors,as in certain familial diseases.It may also be caused by secondary factors like certain dietery influences,especially in acquired hyperlipidemia.

EXPERIMENTAL SECTION Materials: Collection and Authentication of the Plant Material The plant Spinacia Oleraceae were purchased from salem market, Tamil Nadu, India. The plant was identified and authenticated by the botanist Mr. A .Balsubramanian. (consulant-central siddha research) Executive Director ABS botanical garden, Salem , Tamil Nadu. Extract preparation The fresh leaves of Spinacia Oleraceae collected and dried under shade, sliced into small pieces and ground into powder with mechanical grinder. The powder was passed through sieve no.30 and stored in a container.


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Solvents For Extraction Petroleum ether (60-80˚c) Alcohol (95% v/v) Distilled water with chloroform Extraction procedure The dried powder of leaves of Spinacia Oleraceae was defatted with petroleum ether in a Soxhlet apparatus by hot percolation. The defatted powder material (marc) thus obtained was further extracted with alcohol and fresh powder was used for aqueous extraction by Cold maceration method. The solvent was removed by distillation under low pressure and evaporation. The resulting semisolid mass was vacuum dried by using rotary flash evaporator. Methods: Tests for Carbohydrates and Glycosides Tests for carbohydrates A small quantity of the extracts was dissolved separately in 4 ml of distilled water and filtered. The filtrate was subjected to Molisch’s test to detect the presence of Carbohydrates. Molisch’s Test Filtrate was treated with 2-3 drops of 1% alcoholic α - napthol solution and 2 ml of Con sulphuric acid was added along the sides of the test tube. Appearance of brown ring at the junction of two liquids shows the presence of carbohydrates. Another portion of the extract was hydrolysed with hydrochloric acid for few hours on a water bath and the hydrolysate was subjected to Legal’s and Borntrager’s test to detect the presence of different glycosides. Borntrager’s Test Hydrolysate was treated with chloroform and then the chloroform layer was separated. To this equal quantity of dilute ammonia solution added. Ammonia layer acquires pink color, showing the presence of glycosides.

Test for Alkaloids: A small portion of the solvent free pet ether, alcohol extracts were stirred separately with few drops of dil hydrochloric acid and filtered. The filtrate was tested with various reagents for the presence of alkaloids. A) Mayer’s reagent - Cream ppt B) Dragendroff’s reagent - Orange brown ppt C) Hager’s reagent - Yellow ppt D) Wagner’s reagent - Reddish brown ppt.


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Test for Phytosterol The extract was refluxed with solution of alcoholic potassium hydroxide till complete saponification has taken place. The mixture was diluted and extracted with ether. The ether layer was evaporated and the residue was tested for the presence of phytosterol.

Libermann Burchard Test The residue was dissolved in few drops of dil. Acetic acid; 3 ml of acetic anhydride was added followed by few drops of Conc Sulphuric acid. Appearance of bluish green color shows the presence of phytosterol.

Tests for fixed oils Spot test Small quantities of various extracts were separately pressed between two filter papers. Appearance of oil stain on the paper indicates the presence of fixed oil. Few drops of 0.5N alcoholic potassium hydroxide were added to a small quantity of various extracts along with a drop of phenolphthalein. The mixture was heated on a water bath for 1-2 hours. Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

Test for gums and mucilages Small quantities of the extracts were added separately to 25 ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties for the presence of gums..

Test for saponins The extract was diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. The formation not less than of 1 cm layer of foam shows the presence of saponins. Test for proteins and free amino acids Small quantities of the extracts were dissolved in few ml of water and treated with following reagents. 1. Million’s reagent - Appearance of red color shows the presence of protein and free amino acid. 2. Ninhydrin reagent - Appearance of purple color shows the presence of proteins and free amino acids. 3. Biuret test - Equal volumes of 5% sodium hydroxide solution &1% copper sulphate solution was added. Appearance of pink or purple color shows the presence of proteins and free amino acids.

Test for phenolic compounds and tannins Small quantities of the extracts were taken separately in water and test for the presence of phenolic compounds and tannins was carried out with the following reagents.


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1. Dil. Ferric chloride solution (5%) – Violet color. 2. 1% solution of gelatin containing 10% sodium chloride – White ppt. 3. 10% lead acetate solution - White ppt

Test for flavonoids With aqueous sodium hydroxide solution Blue to violet color (anthocyanins) yellow color (flavones), yellow to orange (flavonones). With conc. Sulphuric acid Yellow orange color (anthocyanins) yellow to orange color (flavones) orange to crimson (flavonones).( Mukherjee)

Table no: 4 preliminary phytochemical screening of dried leaves of spinacia oleraceae

S.no

Constituents

Tests

Pet ether

Alcohol

Aqueous

1

2.

3.

4.

5. 6

7.

ALKALOIDS

STEROLS

CARBOHYDRATES

GLYCOSIDES

FIXED OILS AND FATS

PHENOLIC COMPOUND

Mayer’s test Dragondraff’s test

Hager’s test Wagner’s test

Libermann’s Burchard test Salkowski’s

Molisch’s test Fehling’s test Benedict’s test

Borntrager’s test

legal test keller kiallani test

Spot test Saponification test

Ferric chloride

Biuret test Ninhydrin test

- - - - - - + + + + + + - - - - -

- - - - - -

+ + + +

+ +

+

+

+

+ +

- - - - - -

+ + + +

+ +

+

+

+

+ +


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8.

9.

10.

11.

PROTEIN & AMINO ACIDS

TERPENOIDS & SAPONINS

TANNINS

GUMS & MUCILAGE

FLAVONOIDS

Xanthoprotein test Millon’s test

Foam test

Haemolysis test

Gelatin test FeCl3 test

Precipitation to 90% alcohol

Aqueous NaOH

Conc. H2SO4

- - - - - - - + +

+ + - - - + -

+ +

+ + - - - + -

+ +

Where + =present; - =absent Pharmacological studies Procure of Experimental Animals Swiss albino mice (20-25 g) and Sprague drawly rats (150-200 g) of either sex and of approximate same age used in the present studies were procured from listed suppliers of Sri Venkateswara Enterprises, Bangalore, India. The animals were fed with standard pellet diet (Hindustan lever Ltd. Bangalore) and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of 12 hours of darkness and light. The animals were acclimatized to the laboratory conditions for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The experimental protocols were approved by Institutional Animal Ethics Committee (IAEC No.-P.Col./39/2008)after scrutinization. The animals received the drug treatments by oral route as well as intra peritoneal. Oral acute toxocity studies Organization for Economic co-operation and Development (OECD) regulates guideline for oral acute toxicity study. It is a international organization which work with the aim of reducing both the number of animals and the level of pain associated with acute toxicity testing. Following are the main type of guideline followed by OECD.

� Guideline 420, Fixed dose procedure. (5 animals used) � Guideline 423, Acute toxic class. (3 animals used) � Guideline 425, Up and Down method. (1 animal used)

Acute toxic class, guidelines 423 It is a group sequential procedure using 3 animals of one sex per step three pre identified starting doses is possible.Three animals of the opposite sex are then dosed at the final dose level used with the first sex the method was tested in validating studies with animals.


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Procedure Swiss albino mice with weight ranging (20-25 gm female) were taken for the experiment. The animals were made into a group of 3 each, dose of alcohol and aqueous extracts were given according to the body weight (mg/kg), starting dose of 5 mg /kg was given to the first individual animal, no death was occurred, higher doses were given to next group of animals.(OECD Guideline 423)

TABLE NO: 5: Acute toxicity study of aqueous and alcoholic extracts of leaves of

Spinacia oleraceae Linn based on OECD guidelines

S. No Number of animals Dose in mg/kg Report

1 3 5mg/kg No death 2 3 50mg/kg No death

3 3 300mg/kg No death

4 3 2000mg/kg No death From the observation the alcoholic and aqueous extract of leaves of spinacia oleracea were screened for acute toxicity study by OECD guidelines for determining the LD50. The results showed that LD50 was found to be 2000mg/kg. Therefore its ED50 was found to be 200mg/kg.

Fig.no. 6 OECD Guideline


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Hypoglycemic study in normal fasted rats Methodology To investigate the hypoglycemic effect of the alcohol and aqueous extract, the fasted rats were divided into 7 groups of 6 each.

Group 1 - Normal untreated rats Group 2 - Diabetic control Group 3 - Diabetic rats treated with 0.5 mg/kg of Glibenclamide Group 4 - Diabetic rats treated with 200mg/kg of Alcohol extract Group 5 - Diabetic rats treated with 400mg/kg of Alcohol extract

Group 6 - Diabetic rats treated with 200 mg/kg of aqueous extract Group 7 - Diabetic rats treated with 400 mg/kg of aqueous All animals were drug administered with the help of stomach tube. Blood samples were collected for the measurement of blood glucose level from the tail vein at 0,1,2 and 3 hrs.The blood glucose level was determined using an electronic glucometer The results were recorded and represented in Table no: 5 In vivo Anti-diabetic Study Duration of experiment: 15days(3days for diabetic induction +12days for treatment with drug.) Induction of diabetes Animals were allowed to fast 24 hrs prior to injection with freshly prepared aqueous solution of alloxan monohydrate (150mg/kg, i.p.). After 72 Hrs, rats with marked hyperglycemia (fasting blood glucose >300mg/ dl) were selected to determine the efficacy of alcohol extract of the plant.

Determination of the anti-hyperglycemic activity The alloxan induced diabetic rats were divided into 7groups of 6 each. Group 1 - Normal untreated rats Group 2 - Diabetic control Group 3 - Diabetic rats treated with 0.5 mg/kg of Glibenclamide Group 4 - Diabetic rats treated with 200mg/kg of Alcohol extract Group 5 - Diabetic rats treated with 400mg/kg of Alcohol extract

Group 6 - Diabetic rats treated with 200 mg/kg of aqueous extract Group 7 - Diabetic rats treated with 400 mg/kg of aqueous extract

Blood samples were collected for the measurement of blood glucose level from the tail vein at 0, 1, 2 and 3 hrs The blood glucose level was monitored by using electronic glucometer (one touch Horizon) The results were recorded and represented in Table no: 6(Rajesh singh et al 2007) The blood samples were collected in morning 1 hr after drug administration on day 1, 3,6,12. The results were recorded and represented in Table no:7


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Body Weight Test The Body weight of experimental animal was measured during the period of study of antihyperglycemic activity. In diabetes mellitus the body weight loss was common symptoms. Diabetic control group animals loss their body weight continuously but the treated group after some time they start to regain their body weight. The effect of the plant drug on body weight also performed for initial day and final day. The results were recorded and represented in Table no:8 Statistical Treatment Statistical evaluation was done using one-way analysis of Variance (ANOVA) followed by newmanns keul comparison test. P values <0.05 were Considered signific Table no 6: Effect ethanolic and Aqueous extracts of leaves of Spinacia oleracea linn after 3

hours treatment by using normal fasted rats

Group Treatment

BLOOD GLUCOSE LEVEL AT DIFFERENT HOURS IN mg/dl

Initial 1hours 2 hours 3 hours

I Normal control

5%cmc solution 10 ml/kg

82.6±5.56 81.83±2.87 83.5±3.38 82.5±2.83

II Standard treated glibenclamide

(0.5mg/kg) 84.4±3.6 83.5±3.65* 82.6±1.16*** 84.1±2.26***

III Alcoholic Ext ( 200 mg/kg )

79.6±4.31 78.83±4.8* 78.1±3.2** 78.8±1.33**

IV AlchoholicExt ( 400 mg/kg )

80.1±6.21 81.6±3.9* 82.1±2.6*** 80.7±2.1***

V Aqueous Ext. ( 200 mg/kg )

83.6±2.7 82.3±2.93* 84.8±1.88*** 82.7±2.1**

VI

Aqueous Ext. (400mg/kg)

79.7±1.4 80.3±3.48* 79.6±2.61*** 83.6±1.07***

Values are represented as mean ± SD (n=6) Newmann’s keul comparision test(p< 0.01) is used p < 0.05 and ** p < 0.01 and *** p < 0.001vs control


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Table No: 7: Anti Hyperglycemic Activity Effect ethanolic and Aqueous extracts of leaves of Spinacia oleracea linn on blood glucose level on alloxan induced rats after 3 hours treatment

Group Treatment

BLOOD GLUCOSE LEVEL AT DIFFERENT HOURS AFTER TREATM ENT mg/dl

Initial 1hours 2 hours 3 hours

I Normal control 5%cmc solution 10 ml/kg

81.33±32.81 83.66±13.5 82.66±3.38 87.5±13.12

II Diabetic control.

( 10 ml/kg ) 332.0±31.8 333.33±12.92 z 330.33±6.78z 331.0±12.16 z

III Standard treated

glibenclamide (0.5mg/kg) 299.66±6.00 259.83±9.65*

160.5±2.78***

120.33±4.26***

IV Alloxan+Alcoholic Ext

( 200 mg/kg ) 313.66±4.31 280.66±6.51* 169.16±6.72** 142.3±6.81**

V Alloxan+AlchoholicExt

( 400 mg/kg ) 321.66±3.76 263.83±3.6* 165.33±5.64*** 128.16±6.84***

VI Alloxan+Aqueous Ext.

( 200 mg/kg ) 293.33±11.1 270.66±9.83* 185.8±2.8*** 137.16±4.91**

VII Alloxan+Aqueous

Aqueous Ext. (400mg/kg)

304.66±6.41 265.66±5.88* 170.26±6.52*** 123.16±10.07***

Values are represented as mean ± SD (n=6) Newmann’s keul comparision test(p< 0.01) is used p < 0.05 and ** p < 0.01 and *** p < 0.001vs diabetic control

TABLE NO: 8: Effect of aqueous and alcoholic extracts of leaves of Spinacia oleraceae Linn, on blood glucose level on alloxan induced rats after 12 days of treatment in mg/ dl

S. No Treatment Blood glucose level in mg/dl

Initial 3st day 6th day 9th day 12th day

1 Normal control

5% cmc solution 10 ml/kg81.05±31.833 83.8±11.32 76.63±15.28 79.83±21.39 85.6±15.79

2 Diabetic control

l 50mg/kg 314.5±23.1 322.16±10.9z 324.33±19.35z 341.66±15.38z 339.16±19.51z

3 Glibenclmide (0.5mg/kg)

310.50±6.01 271.88±3.32* 235.0±9.02** 171.7. ±3.88*** 130.0±13.39***

4 Alloxan+Alcoholic Ext.

( 200 mg/kg ) 308.66±4.26 281.58±6.82* 243.0.±40.93* 215.0±11.68 155.56±7.09***

5 Alloxan+Alcoholic Ext

( 400 mg/kg ) 310.166±3.86 274.83±6.17** 241.44±12.50* 205.0±5.87*** 144.33±13.87***

6 Alloxan+Aqueous Ext.

( 200 mg/kg ) 312.66±11.13 277.56±10.53** 256.16±51.74* 210±12.62*** 157.16±10.63*

7 Alloxan+Aqueous Ext.

( 400 mg/kg ) 301.0±10.36 272.66±5.88** 246.16±9.52* 88.830±9.77***z 143.75±10.48***

Values are represented as mean ± SD (n=6) ; Newmann’s keul comparision test(p< 0.01) is used *p < 0.05 and ** p < 0.01 and *** p < 0.001vs diabetic control . x,y,z compared vs control


276

Table No: 9: Body Weight Test

Effect of alcoholic and aqueous extracts of dried leaves of Spinacia oleracea linn after 12 days treatment with drug

values are represented as mean ± SD (n=6) ; Newmann’s keul comparision test(p< 0.01) is used *p < 0.05 and ** p < 0.01 and *** p < 0.001vs diabetic control; x,y,z compared vs control

Effect Of Alcoholic And Aqueous Extracts Of Dried Leaves Of Spinacia Oleracea Linn on

blood glucose level of alloxan induced rats After 3 hours Treatment

S. No Treatment Initial body wt.

in gm 3rd day 6th day 9th day 12th day

1 Normal control 200.83±8.16 211.0±4.18 213.56±4.84 214.66±4.08 214.30±3.65

2 Diabetic control 153.5±6.12 151.16±4.08 148.33±8.49z 133.33±2.05 z 120.16±6.05 z

3 Standard treated glibenclamide

(0.5mg/kg) 160.50±5.47 156.83±6.64∗ 158.66±4.08*∗ 159.16±3.76*∗ 161.66±3.76***

4 Alloxan+Alcoholic

ext.200 mg/kg 160.33±8.94 141.83±4.08 45.66±4.08** 149.00±6.08* 150.16±6.05**

5 Alloxan+Alcoholic

ext. 400 mg/kg 151.6±7.52 143.16±10.68 144.80±8.36** 153.1±7.36 158.0±6.12***

6 Alloxan+Aqueous

ext.200 mg/kg 154.66±10.20 150.16±8.66∗ 145.83±9.35*∗ 144.05±9.17∗ 147.83±8.21***

7 Alloxan+Aqueous

ext.400 mg/kg 200.16±8.83 175.16±6.83∗ 170.5±6.83∗* 172.3±8.21∗ 175.16±7.36***


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Fig. 8 Effect Of Alcoholic And Aqueous Extracts Of Dried Leaves Of Spinacia Oleracea Linn on blood glucose level of alloxan induced rats After 12 Days Treatment.

D a t a T a b l e - 4

cont Dia con Al200 Al400 Aq200 Aq400 STD0

1 0 0

2 0 0

3 0 0

4 0 0c o n tD i a c o nA l2 0 0A l4 0 0A q 2 0 0A q 4 0 0S T D

G R O U P S

Blo

od g

luco

se le

vel i

n

mg/

dl

10.5. Antihyperlipidaemic Activity:

Table no. 10 Antihyperlipidaemic effect of aqueous and alcoholic extracts of Spinacia oleraceae Linn on alloxan induced diabetic rats after 12 days of treatment

S. No

Groups

cholestrol

mg/dl

TGL mg/dl

HDL mg/dl

LDL mg/dl

VLDL mg/dl

1

Normal control

124.66±.12.02

132.6±5.9

33.66±2.51

81.5±4.63

24.83±1.034 2

Diabetic control

146.16±3.52z

186.83±6.01z

25.3±3.2z

70±8.58z

40.66±3.56z

3

Standard treated

glibenclamide (0.5mg/kg)

110.16±10.60*

126.83±7.51**

38.6±1.15**

74.83±4.16**

22.83±2.66**

4

Alcoholic ext.200 mg/kg

132±3.53*

140.16±8.21*

32.83±4.8*

73.83±6.11*

27±6.5*

5

Alcoholic ext. 400 mg/kg

124.66±7.78**

132.33±6.504**

30.66±4.58**

71.66±2.51**

24.16±4.87**

6

Aqueous ext.200 mg/kg

147.33±4.28*

154.66±7.55*

31.33±2.5*

76.1±4.04*

26±3.37**

7

Aqueous ext.400 mg/kg

124.16±1.98

140.2±6.00*

30.83±1.14**

74.83±3.61***

24.5±4.86****

Histopathology Of Rats Pancreas SLIDE A Normal control [5% CMC solution,10ml/kg p.o] The islets are normal. The architecture is preserved. The acini are lined by round to oval cells with moderate cytoplasm and small round to oval nuclei. .


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SLIDE B Diabetic control:- (alloxan monohydrate 150 mg/kg, i.p) The islets showed different morphology i.e. circular shape of the islet is disrupted, limiting membranes between the islet and the surrounding acinar tissue is dissolved. SLIDE C Standard ( Glibenclamide 0.5 mg/kg) The islets show depletion of cells. There is a mild infinite of lymphocytes at some foci. The acini are lined by round to oval cells with moderate cytoplasm and small round to oval nuclei. SLIDE D Alcoholic extract 200 mg/kg:- The architecture is partially affected. The islets are normal. The acinar normal. There is a mild and diffuse in filtrate of lymphocytes within the stroma. SLIDE E Alcoholic extract 400 mg/kg:- The architecture is normal. The islets show depletion of the acinar cells. The acinar cells show moderate cytoplasm and round to oval nuclei. There is no evidence of inflammation. SLIDE F Aqueous extract 200 mg/kg:- The islets show depletion of cells. The architecture is preserved. The acini are lined by round to oval cells with moderate cytoplasm and small round to oval nuclei. SLIDE G Aqueous extract 400 mg/kg The islets are normal. The architecture is preserved. The acini are lined by round to oval cells with moderate cytoplasm and small round to oval nuclei.

A. Normal Control B. Diabetic Control

B. Standard D. Alcoholic Ext 200mg/kg


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E. Alcoholic Ext 400mg/kg F. Aqueous Ext 200mg/kg

G. Aqueous Ext 400mg/kg

Histopathology of Rats Pancrease Histopathology Of Rats Kidney SLIDE A1 Normal control:- [5% CMC solution,10ml/kg p.o] The glomeruli appear normal. The tubles are normal and lined by a single layer of cuboidal cells. SLIDE B1 Diabetic control:- [5% CMC solution ,10 ml/kg p.o] The glomeruli shows hypercellularity.The tubules shows cloudy swelling of the lining cuboidal cells. Stroma shows small focal hemorrhages and a diffuse infiltrate lymphocytes and plasma cells. SLIDE C1 Standard treated:- [Glibenclamide 0.5 mg/kg ] The glomeruli shows mild hypercellularity.The tubules shows foamy vacuolation of the tubular cells. The stroma is normal. SLIDE D1 Alcoholic ext 200 mg/kg:- The glomeruli shows mesangial hypercellularity.The tubules shows cloudy swelling.The stroma shows a diffuse infiltrate lymphocytes and plasma cells. SLIDE E1 Alcoholic ext 400 mg/kg:-


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The glomeruli shows mesangial hypercellularity.The tubules are normal.The stroma shows a diffuse infiltrate lymphocytes and plasma cells SLIDE F1 Aqueous ext 200 mg/kg:- The glomeruli shows mesangial hypercellularity and focal glomerulosclerosis. The tubules are normal. The stroma is normal. SLIDE G1 Aqueous ext 400 mg/kg:- The glomeruli appear normal. The tubles are normal and lined by a single layer of cuboidal cells.The stroma appears normal. A1) Normal Control B1) Diabetic Control

C1) Standard D1) Alcoholic Ext 200mg/kg

E1) Alcoholic Ext 400mg/kg F1) Aqueous Ext 200mg/kg

Histopathology of Rats Kidney


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RESULTS AND DISCUSSION Extraction of plant material: The dried leaves of Spinacia Oleracea Linn was extracted with petroleum ether and alcohol by soxhlot apparatus and water by cold maceration. The percentage yield of plant extracts as follows

Petroleum ether _____________0.85 % w/v Alcohol_____________________7.69 % w/v Water ______________________ 8 % w/v

The percentage yield of aqueous extract of leaves of SPINACIA OLERACEA was found to be greater than petroleum ether and alcohol extracts.

Preliminary phytochemical studies: The various extracts of dried leaves were subjected for phytochemical screening which shows the presence of different compounds in plant extracts. Petroleum ether extract- glycosides, carbohydrates, fixed oil & fats, Alcoholic extract – Alkaloids, glycosides, flavonoids, carbohydrates, saponins, tannins, phenolic compounds, fixed oil & fats, phytosterols. Aqueous extract- Alkaloids, glycosides, flavonoids, carbohydrates, saponins, tannins, phenolic compounds.

Acute oral toxicity study: The lethal dose (LD 50) of the alcoholic and aqueous extracts of dried of leaves of SPINACIA OLERACEA . were determine by OECD guideline (423 guideline)

1. The LD50 of alcoholic and aqueous extracts were found to be 2000mg/kg kg and therefore its ED50 is 200mg/kg.

Hypoglycemic activity: In present study, the hypoglycemic activity of plant extracts of leaves of SPINACIA OLERACEA were studied for the decrease in blood glucose level (BGL) in normal fasted rats which was compared with the control animal group and standard treated group. The alcoholic and aqueous extracts treated groups showed then insignificant reduction in blood glucose levels

Antihyperglycemic Activity: The alcoholic and aqueous extracts of leaves of SPINACIA OLERACEA LINN showed the reduction in Blood Glucose Level in alloxan treated rats. As in the alloxan treated group the BGL was high due to cytotoxic effect of alloxan on the β-cell of islet of langarhans. The alcoholic and aqueous extracts decrease the BGL level during the study period. The BGL level of control group was compared with treated group.


282

Body weight test: The Body weight of experimental rats were measured during the period of study of antihyperglycemic activity. In diabetes mellitus the body weight loss was common symptoms. Diabetic control group animals loss their body weight contineouslly but the treated group after some time they start to regain their body weight.

The both alcoholic and aqueous extracts shows the significant regain of body weight during study period.

Evaluation Of Anti Hyperlipidimic Activity As in the alloxan treated group the HDL was high and LDL was high due to cytotoxic effect of alloxan.The both alcoholic and aqueous extracts shows the significant Anti Hyperlipidimic Activity Evaluation of antioxidant activity Alcoholic extract of leaves of SPINACIA OLERACEA shows the significant antioxidant activity.

SUMMARY AND CONCLUSION

The plant Spinacia oleraceae belonging to the family Chenopodiaceae was studied for their phytochemical and pharmacological evaluation in order to get a new antidiabetic drug from natural source.

2. The extractive value of aqueous and alcoholic extracts of leaves of Spinacia oleraceae Linn was found to be 8% w/v and 7.69% w/v respectively.

3. Phytochemical screening of the aqueous and alcoholic extracts of leaves of Spinacia oleraceae showed the presence of flavanoids, carbohydrates, glycosides, saponins, tannins, phenolic compounds, protein and amino acids.

4. The LD50 value of the aqueous and alcoholic extracts of leaves of Spinacia oleraceae was found to be 2000mg/kg and therefore its ED50 is 200mg/kg.

5. The aqueous and alcoholic extracts of leaves of Spinacia oleraceae exhibited significant antihyperglycemic activity.

6. The aqueous and alcoholic extracts of leaves of Spinacia oleraceae exhibited significant antihyperlipidemic activity.

7. The aqueous and alcoholic extracts of leaves of Spinacia oleraceae exhibited significant regain of body weight during study period, when compared to standard drug.

8. The alcoholic extract of leaves of Spinacia oleraceae exhibited significant antioxidant activity.

Hence this work gives some scientific proof for medicinal value of the selected plant.

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