ISSN: 0975-766X CODEN: IJPTFI Available Online …...Bharath University, BIHER, Chrompet, Chennai, India. Email id: [email protected] Received on 10-05-2016 Accepted on 09-06-2016

S.Latha* et al. International Journal Of Pharmacy & Technology

IJPT| June-2016 | Vol. 8 | Issue No.2 | 12671-12684 Page 12671

ISSN: 0975-766X CODEN: IJPTFI

Available Online through Research Article

www.ijptonline.com SYNERGETIC HYPOGLYCEMIC AND HYPOLIPIDEMIC EFFECT OF HERBAL

FORMULATION OF FLAX SEED, FENUGREEK AND JAMUN SEEDS IN

STREPTOZOTOCIN- NICOTINAMIDE INDUCED DIABETIC RATS S.Latha*

1, R.Vijayakumar

1, *B.R.Senthil Kumar

2, R.Srikumar

3, G.Bupesh

4

1Department of Physiology, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India.

2Department of Physiology, National siddha institute, tambaram,chennai, India.

3Centre of research, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India.

4Central Research Laboratory & Department of Physiology,Sree Balaji medical college and Hospital,

Bharath University, BIHER, Chrompet, Chennai, India.

Email id: [email protected]

Received on 10-05-2016 Accepted on 09-06-2016

Abstract:

The present study was designed to investigate the synergism in hypoglycemic and hypolipidemic effect of hydro alcoholic

extract of Herbal Formulation of Flax seed, Fenugreek and Jamun seeds (FFJ) and their individual drugs in diabetic

animals. Due to the sedentary life style there is a drastic increase in chronic metabolic diseases such as diabetes

mellitus. Even though many synthetic allopathic drugs are in use, most of the affected individuals search for the effective

Herbal formulations because of their high efficacy, less or no side effects. Anti diabetic activity was evaluated in

Streptozocin-Nicotinamide induced diabetic wister rats. The hydro alcoholic extract of herbal formulation of FFJ (1:1:1)

and their individual drugs are given by oral route at doses of 200mg/kgbw for 30 days. The animal weight and food

consumption were measured for every 15 days. On 31st day blood were collected by retero orbital puncture and Blood

glucose, insulin, blood lipids, Haemoglobin(Hb), Glycosylated Hb(HbA1c), were measured on 31st day. Diabetic rats

treated with Herbal Formulation of FFJ (1:1:1) and their individual drugs shown significant (P<0.05) reduction in the

food consumption, blood glucose, blood lipid except HDL cholesterol, glycosylated Hb level and increased body weight,

serum insulin level and Hb level and when compared to diabetic group. The percentage changes occur in Herbal

Formulation FFJ treated group was greater than their individual drugs treated groups. This study concluded the

formulation of FFJ was found to have greater antidiabetic effect compared to individual drugs of FFJ.

Key words: Diabetic, Streptozotocin, Nicotinamide, Glycosylated Hb



Introduction:

Diabetes mellitus is a common present day problem, increasing at an alarming rate both in developing and developed

countries. It is a chronic metabolic disease characterized by hyperglycemia, hyperlipidemia and negative nitrogen balance

resulting from defects of insulin secretion, insulin action or both.1 The major complications of diabetes mellitus are high

blood glucose levels which lead to serious diseases affecting the heart and blood vessels, eyes, kidneys, and nerves.

People with diabetes are also at increased risk of developing infections. Type II DM which accounts 95% of the diabetic

population.

According to IDF report Some 382 million people worldwide are estimated to have diabetes. It may rise to some 592

million people, by 2035.2 it is also a common cause of chronic morbidity and disability among the working population in

the world. The treatment of DM in clinical practice has been confined to use of oral hypoglycemic agents and insulin, the

former being reported to be endowed with characteristic profiles of serious side effects.3

Plants have been commonly used to treat diabetes since ancient times and have served as a good source of medicine.4

many different plants are used individually or in the formulations for the treatment of diabetes mellitus and for its

complications.

The major problem with this herbal formulation is lack of scientific and clinical data providing their safety and efficacy.

According to ayurveda, combination of plant drugs are used to get desired action and to eliminate unwanted side effects.5

In traditional systems, diabetes is better managed by the herbal combination instead of single herb. most of the

formulations contain eight or more drugs in combination, it is very difficult to standardize and dispense. Since FDA

recommends the number of incredients to be three or less than three in any combination6, present study used three herbs

namely flax seed, fenugreek and jamun seeds. Flaxseed (Linum Usitatissimmum), popularly known as linseed. It has

been used in various forms such as flour, oil and seed. The studies conducted on the flax seed proved that it reduces

cardiovascular risks 7 like hypertension,and blood cholesterol, triglycerides level

8,colon carcinogenesis and diabetes.

Fenugreek(trigonella foenum greacum) has been used for diabetes, particularly in India.9 it is used in atherosclerosis,

rheumatism, sugar lowering, blood lipids lowering, appetizer and contain antioxidant activity .[10-15]

Syzygium Cuminii or Eugenia jambolana (black plum or jamun) belongs to the family Myrtaceae. Jamun has been

reported to be used in numerous complementary and alternative medicine systems of India. The efficacy of Eugenia



jambolana has also been tested in preclinical and clinical studies for hypolipidemic, anti-inflammatory, neuropsycho

pharmacological, antiulcer, antibacterial, anti-HIV, antidiarrhoeal, and antihypertensive activities. [16-21]

.

On the basis of the above evidence the present study was designed to evaluate the synergetic action in hypoglycemic and

hypolipidemic effect of heberbal formulation of FFJ and their individual drugs in STZ-nicotinamide induced diabetic

rats.

Materials and methods:

Collection and identification of plant:

Seeds belonging to the herbal formulation of FFJ were collected from the local market, puducherry, India and

authenticated by siddha physician and nodal officer, siddha unit, dept of ISM&H, puducherry. The seeds were dried under

shade and powdered before use.

Extraction of the plant material:

1. Extraction of flax seed:

Powdered flax seeds are defatted by petroleum ether(at 60-80°c )in the soxhlet apparatus. the merc was

hydrolyzed with 1m sodium hydroxide for 1 hr at room temperature by constant rotation followed by extraction

with 50% ethanol then acidified with 1m Hcl upto the PH:2-4.dry the filterate at 50°c .22

2. Extraction of fenugreek and jamun seeds:

Dried powdered seeds were extracted with 50%ethanol using soxhlet apparatus for 20-24 hrs. The extract was

concentrated under vacuum.

Experimental Animal

Wister strain albino rats weighing 180-220gm were used for this study. Before the commencement of the experiments

proper IACE permission was obtained (IAEC NO: JKKMMRF/IAEC/2013/014). Rats were housed under standard

laboratory conditions with food and water provided ad libitum .

Induction of diabetes mellitus:

Streptozotocin (STZ) was dissolved in citrate buffer (pH 4.5) and nicotinamide was dissolved in normal saline. Type II

DM (NIDDM) was induced in overnight fasted Wistar strain albino rats by a single intraperitoneal injection of 60 mg/kg

streptozotocin, 15 min after the i.p. administration of 120 mg/kg of nicotinamide.



Hyperglycemia was confirmed by the elevated glucose levels in plasma, determined at 72 hr after administration. Rats

with fasting blood sugar levels around 160 to 300 mg/dl were selected for the study .23

Experimental groups:

Group 1: Control Animals were administered saline (1ml) orally for 30 day

Group 2: diabetic animal

Group 3: diabetic Animals were administered flax seed extract 200mg/kg bw for 30 days

Group 4: diabetic Animals were administered fenugreek extract 200mg/kg bw for 30 days

Group 5: diabetic Animals were administered jamun seed extract 200mg/kg bw for 30 days

Group 6: diabetic animals were administered herbal formulation of FFJ (1:1:1) of 200 mg/kg bw for 30 days

Group 7: diabetic animals were administered metformin (200mg/kg bw) for 30 days

Sample collection

At the 31st day Blood samples were collected by of retero-orbital plexus puncture for the measurement of plasma glucose,

insulin, serum lipids, haemoglobin and glycosylated hb levels.

Weight of the animal and food consumption were measured for every 15 days once. Measurement of blood glucose: By

using GOD-PAP k it – purchased from agappe diagnostic Ltd, kerala 24

Plasma insulin levels were assayed by using rat

Insulin ELISA enzyme immune assay kit by Mercodia,

Sweden Measurement of total cholesterol and HDL cholesterol and serum triglycreides are done by using the kit

purchased from span cogent diagnostic Ltd. 25

LDL cholesterol are measured by using friedewald’s equation (40)26

(LDL-cholesterol=total cholesterol-triglycerides/5- HDL cholesterol)

Serum VLDL cholesterol concentration was calculated according to Nobert formula

VLDL cholesterol= Triglycerides/5)27.

Haemoglobin by the method of Dacie & Lewis (1975)32

,Glycosylated

Haemoglobin(HbA1c) by the method of sudhakar nayak and pattbiraman(1982), 33

.

Statistical analysis:

All the data were analyzed using one way analysis of variance (ANOVA) followed by tukeys multiple comparison.

Results were expressed as mean ±S.D and the values p<0.05 a were considered statistically significant .



Result:

Table 1: Effect of Herbal formulation and their individual drugs on weight and food consumption in STZ-

Nicotinamide induced diabetic rats.

Groups Weight of the animal (gm) Food consumption (gm)

1st day 15

th day 30

th day 1

st day 15

th day 30

th day

Group1

control)

206.16±10.5 212.33±10.5

aNS

221.16±11.3

a*d

NS 21.03±1.2

23.41±1.24

a*

25.9±2.15

a*d

*

Group2

(diabetic)

203±11.5 193.33±9.79

aNS

b*

187.3±9.9

a*d

NS b*

26.73±1.4

33.68±1.94

a* b

*

40.05±2.17

a*d

*b

*

Group3

(Flax seed extract

(200mg/kg bw))

210.16±10.8 212.8±10.5

aNS

bNS

c*

219.83±11.9

aNS

dNS

bNS

c*

25.43±1.2 24.65±1.30

aNS

bNS

c*

25.61±1.27

aNS

dNS

bNS

c*

Group 4

(FenugreekSeed

extract

(200mg/kg bw))

202±10.8 205.5±9.0

aNS

bNS

c*

209.5±12.1

aNS

dNS

b* c*

25.1±1.4 24.15±1.54

aNS

bNS

c*

25.58±1.39

aNS

dNS

bNS

c*

Group5

(Jamun seed Extract

(200mg/kg bw))

206.5±10.8 210±11.11

aNS

bNS

c*

214±10.0

aNS

dNS

bNS

c*

26.7±1.4 25±1.43

aNS

bNS

c*

26.1±1.26

ANS

dNS

bNS

c*

Group6

(Herbalformulation(

FFJ)

(200mg/kg bw))

208.5±10.7 210.33±11.2

aNS

bNS

c*

218±11.7

aNS

dNS

bNS

c*

26±1.3 24.98±1.47

aNS

bNS

c*

26.38±1.37

ANS

dNS

bNS

c*

Group7(Metformin

(200mg/kg bw)

204.6±11.0

207.66±10.6

aNS

bNS

c*

215.5±11.8

aNS

dNS

bNS

c*

25.7±1.3 24.8±1.40

aNS

b* c*

25.4±1.27

ANS

dNS

bNS

c*

Values are expressed mean±SD of six animals

Symbol * represents the statistical significance (p<0.05) NS-no significance

a- compared with 1st day d-compared with 15

th day

b- compared with group 1 c- compared with group 2

Table 1 represented the effect of herbal formulation FFJ and their individual drugs on weight and food consumption in

control, diabetic and treated groups at 1st, 15th, and 30

th day. When compared with 1

st day no significant weight reduction

was observed in all the groups on 15th

day and in the treated groups on 30th

day. No significant weight reduction was

observed in all the groups on the 30th

day when compared with 15th

day.

Diabetic group showed significant (p<0.05) weight reduction than control as well as with other treated groups on 15th

and

30th

day. Flax seed, fenugreek seed, jamun seed extract and herbal formulation FFJ treated diabetic animals showed



significant increase in weight of the animal when compared with diabetic animals on 15th

and 30th

day and the weight of

the treated groups were almost similar to the control and standard drug metformin treated groups.

The food consumption were increased in control as well as diabetic group animals on 15th

and 30th

day when compared

with 1st and 15

th day but the Herbal formulation FFJ and their individual drug treated groups were shown

significant(p<0.05) reduction in food consumption.

The percentage of food consumption was greater in diabetic group on 15th

(26%) and 30th

day (49.83%) than control

group (11.3%, 23.15%). changes in food consumption was observed in flax seed extract, fenugreek seed extract, jamun

seed extract and Herbal Formulation FFJ treated diabetic animals were not significant when related to control animals.

The food consumption of the treated groups was nearly same as the control and the standard drug metformin treated

groups.

Table 2: Effect of Herbal formulation (FFJ) and their individual drugs on blood glucose and insulin level in STZ-

Nicotinamide induced diabetic rats.

Groups Blood Glucose

(mg/dl)

serum insulin

(μU/ml)

Group1 (control) 111.83±6.08 31.62±1.85

Group2 (diabetic) 292.16±14.17 a* 18.41±1.01 a*

Group3 (Flax seed extract

(200mg/kg bw))

165.16±9.0

a* b*

23.10±1.24

A* b*

Grou4 (Fenugreek Seed extract

(200mg/kg bw))

128.83±7.05

a* b*

25.13±1.1

a* b*

Group5 (Jamun seed Extract

(200mg/kg bw))

135±6.57

a* b*

25.18±1.31

a* b*

Group6(Herbal

formulation(FFJ)

(200mg/kg bw))

116.3±5.75

aNS

b*

29.55±1.93

aNS

b*

Group7(Metformin

(200mg/kg bw))

113.5±5.16

aNS

b*

29.67±1.75

aNS

b*

Values are expressed as mean±SD of six animals

Symbol * represents the statistical significance (p<0.05) NS-no significance

a –comparison with group 1 b-comparison with group 2



The table 2 shown that the blood glucose level has significantly increased in diabetic group than control. The treatment

with herbal formulation FFJ and their individual drugs significantly reduced the blood glucose in diabetic induced rats,

but there is significant difference present in the blood glucose level of flax seed, fenugreek seed and jamun seed extract

treated groups when compared with control.

The greater percentage reduction of blood glucose in herbal formulation FFJ treated group than their individual drugs

treated groups. The Flaxseed, Fenugreek, Jamun seed extract treated groups reduced 43.46%,55.9%,53.7% of blood

glucose from the diabetic group but the herbal formulation FFJ reduces to 60.18% and which was similar to control and

standard drug metformin treated groups.

Blood insulin level given in the table 2 showed significant reduction in the serum insulin level in diabetic group when

compared with control. The treatment with herbal formulations FFJ and their individual drugs increases serum insulin

level significantly (p<0.05).

Apart that there is significant difference present in the blood insulin level of flax seed, fenugreek seed and jamun seed

extract treated animals when compared with control.

The percentage increase in insulin level of herbal formulation treated group (60.49%) was higher than the individual

drugs such as flax, jamun seed (25.4%), fenugreek seed (36.4%) and jamun seed (36.7%) extract treated groups.

Table: 3 Effect of Herbal formulation and their individual drugs on blood lipid level in stz-nicotinamide induced

diabetic rats.

Groups Total

cholesterol

(mg/dl)

Triglycerides

(mg/dl)

HDL

cholesterol

(mg/dl)

LDL-

cholesterol

(mg/dl)

VLDL-

cholesterol

(mg/dl)

Group 1 (control) 106.83±4.53 80.83±4.21 65.33±4.03 25.33±1.36 16.16±0.8

Group 2 (diabetic) 200.16±9.98a*

133.66±7.2 a* 30.33±1.36 a

* 143.1±8.3 a

* 26.73±1.4a

*

Group3(Flaxseed

extract(200mg/kgb)

115±4.6

a* b*

94.66±5.1

a*b*

55.50±3.14

a* b*

40.56±2.05

a*b*

18.93±1.0

a*b*

Group4(Fenugreek

Seed extract

(200mg/kg bw)

114.83±5.3

a* b*

94.16±5.0

a*b*

53.16±3.06

a*b*

42.83±2.25

a*b*

18.83±1.0

a* b*

Group5(Jamunseed

Extract

114.66±5.12

a* b*

98.5±5.08

a* b*

54.66±3.20

a*b*

40.30±2.38

a*b*

19.70±1.0

a* b*



(200mg/kg bw)

Group6(Herbal

formulation(FFJ)

(200mg/kg bw)

107.16±4.95

ans

b*

85.0±5.0

ans

b*

63.83±3.54

ans

b*

26.33±1.23

aNS

b*

17.03±1.0

ans

b*

Group7(Metformin

(200mg/kg bw)

106.33±4.32

ans

b*

84.3±4.8

ans

b*

63.33±3.61

ans

b*

26.13±1.37

aNS

b*

16.86±0.9

ans

b*

Values are expressed as mean±SD of six animals, comparison between groups and the statistical significance are as in

table2

The serum lipid level given in the table 3 shown that the Total cholesterol, Triglycerides, LDL and VLDL levels were

significantly increased and HDL cholesterol was significantly decreased in diabetic group when compared with control.

The treatment with herbal formulation (FFJ) and their individual drugs treatment significantly reduced the Total

cholesterol, Triglycerides, LDL and VLDL cholesterol and significantly increased the HDL cholesterol in diabetic

induced animals. the percentage reduction of the Total cholesterol of the individual drugs such as flax seed, fenugreek,

jamun seed extracts treated groups were 42.5%, 42.63% 42.71%, Triglycerides were 29.1%,29.55%,26.3%, LDL-

Cholesterol were 71%,70.06%,71.83% and VLDL-Cholesterol were 29.17%,29.55%,26.30% and the percentage increase

in HDL-cholesterol would be 82.9%,75.27%,80.21% respectively from the diabetic group animal. But the percentage

reduction of total cholesterol, triglycerides, LDL, VLDL-cholesterol was high in herbal formulation treated animals

(46.46%, 36.40%, 81.59%, 36.90%) and percentage increase of HDL cholesterol is 110.4% from the diabetic group

animal and the changes were similar to the control and standard drug Metformin treated group.

Table 4: Effect of Herbal formulation FFJ and their individual drugs on Haemoglobin and Glycosylated

haemoglobin.

Groups Haemoglobin

(mg/dl)

Glycosylated

haemoglobin(HbA1c) (%)

Group1 (control) 15.41±0.72 4.41±0.25

Group2 (diabetic) 12.70±0.69 a* 9.33±0.30 a*

Group3 (Flax seed extract)

(200mg/kg bw))

14.70±0.69

aNS

b*

5.9±0.31

a*b*



Group 4 (Fenugreek Seed

extract)

(200mg/kg bw))

14.63±0.71

aNS

b*

4.98±0.30

a*b*

Group5 (Jamun seed Extract)

(200mg/kg bw))

14.76±0.68

aNS

b*

4.95±0.32

a*b*

Group6 (Herbal

formulation(FFJ)

(200mg/kg bw))

14.98±0.73

aNS

b*

4.6±0.22

aNS

b*

Group7(Metformin

(200mg/kg bw))

13.65±0.67

aNS

b*

4.9±0.26

a*b*

Values are expressed as mean±SD of six animals, comparison between groups and the statistical significance are as in

table 2

Table 4 shown that the decresed Haemoglobin level and increased glycosylated Haemoglobin level in diabetic group than

control and drug treated groups. Though the glycosylated haemoglobin level in flax seed, fenugreek seed and jamun seed

extract treated groups and in the standard drug metformin treated group were decreased compared to Diabetic group,

these groups also showed significant changes when compared with control . The changes in Hb, HbA1c level in the herbal

formulation treated group was similar to control. the percentage increase of Hb(18.75%), and decrease of HbA1c(50.7%)

in herbal formulation FFJ treated group was higher than the percentage increase of Hb (16.51%,15.98%,17.04%) and

decrease of HbA1c (36.7%,46.6%,46.9%) in flaxseed extract, fenugreek extract and jamun seed extract treated groups.

Discussion:

The result of the present study shown flax seed, fenugreek, jamun seed extracts and herbal formulation FFJ reduces blood

glucose, blood lipids, HbA1c, and increased plasma insulin and HDL cholesterol level. This indicating that the bio active

compounds present in these drugs might be responsible for the changes occur in glucose and lipid levels. The increase in

HbA1c level is one of the common indicator of hyperglycemia.34, 35

treatment with herbal formulation and their individual

drugs significantly reduces HbA1c which might be due to improved glycemic control by the treatment. the presence of

bioactive constituents of flaxseeds have the potential of reducing the various diseases such as cardiovascular ailments,

strokes, diabetes, cancer etc.36,37,38

evidences shows that SDG in the flax seed has the ability to prevent or delay the the

development of type I and II diabetes in diabetes-prone rats, probably by decreasing lipid level and oxidative stress .39,40

It was observed that 4-hydroxyisoleucine in the fenugreek seeds has insulin tropic activity which exert hypoglycemic



effects by stimulating glucose-dependent insulin secretion from pancreatic beta cells, 41,42

as well as by inhibiting the

activities of alpha-amylase intestinal enzyme involved in carbohydrate metabolism. Studies also reported that an impaired

secretion and sensitivity of insulin may be responsible for high triglycerides level as the insulin stimulated the synthesis

of adipose tissue by lipoprotein lipase.43

The sapogenins in the fenugreek decrease the triglycerides and total cholesterol

level 44

by increasing biliary cholesterol excretion.45,46

Jamun Seeds contain glycosides, an alkaloid- jambosine, gallic

acid, ellagic acid, corilagin and related tannins. 47

The β-sitoterol and jambulin in syzygium cumini prevent the drastic

conversion of starch into sugar.48

The reduction of body weight in diabetic rats observed in this study may be due to

muscular atrophy and loss of tissue proteins49,50

and it is reversed by treatment with herbal formulations and their

individual drugs this could be due to increased sensitivity of the cell to insulin that prevent protein catabolism. This study

also shows hypolipidemic and hypoglycemic activity of the herbal formulation FFJ was more pronounced than the

individual drugs this proved the synergetic activity exist between the active phytochemicals present in the individual

drugs.

Conclusion: The Hydroalcoholic extract of the Herbal Formulation FFJ exert synergism in glucose and lipid lowering

activity than the individual extracts in diabetic induced animals. It will be a effective alternative for treating diabetes

mellitus than the individual drugs when they are used alone. Even though the synergetic activity exists between the

individual drug extract when given combinely the exact mechanism of action is not known. So further study is

recommented to elicit the exact mechanism.

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Corresponding Author:

S.Latha*,

Email: [email protected]

ISSN: 0975-766X CODEN: IJPTFI Available Online …...Bharath University, BIHER, Chrompet, Chennai, India. Email id: [email protected] Received on 10-05-2016 Accepted on 09-06-2016

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