Synergistic Effect of Natural Sweetener on Antidiabetic Potential of Madhujeevan
churna
Vijay R Salunkhe and
Satish B Bhise
Govt.
ABSTRACT
Objective: To
study the effect of dried powder of leaves of S rebaudiana
on antidiabetic potential of developed Madhujeevan churna by using alloxan treated Wistar rats.
Materials and
Methods: Madhujeevan churna was formulated and developed using Aegle marmelos, Curcuma longa,
Azadichta indica, Momordica charantia, Gymnema sylvestre, Salacia reticulata, and Emblica officinalis
as active ingredients and Stevia rebaudiana as natural sweetener and nutraceuticals.
Churna
without Stevia (bitter Churna) and Madhujeevan
churna with Stevia (sweet Churna)
were evaluated for its antidiabetic activity in alloxan induced diabetic rats. Bitter and sweet Churnas were tested for their antidiabetic potential by blood sugar level, average animal
weight and applicable biochemical parameters. Glibenclamide
was used as a standard.
Results: Both Churnas showed antidiabetic potential in comparison with standard Glibenclamide. Both Churnas were significantly superior to control in reducing blood sugar
as well as weight of animals. Madhujeevan Churna
containing natural sweetener showed marked reduction in total cholesterol, HDL
cholesterol, tiglyceride, SGOT, SGPT, VLDL, creatinine and uric acid as compare to standard and bitter Madhujeevan churna.
Conclusion: Sweet churna showed significant
synergistic effect in comparison to Churna without Stevia. Aqueous extract of dried leaves of Stevia rebaudiana
acts as a natural antidiabetic agent.
Keywords: Formulation
development, Antidiabetic activity, Madhujeevan churna, Stevia rebaudiana, Glibenclamide.
INTRODUCTION
Diabetes mellitus is one of the common
metabolic disorder characteristic by hyperglycemia due to absolute or relative
deficiency of insulin and results in significant morbidity and mortality. Lack
of insulin affects the metabolism of carbohydrates, protein and fat and causes
a significant disturbance of water and electrolyte homeostasis1.
Diabetes increases production of tissue damaging oxidative stress. Therefore,
in diabetes the oxidative stress is referred as a case of double jeopardy for
any β cells that survive the disease 2. Management of diabetes
with minimal side effects is still a complicated medical challenge and there is
an increasing demand by patients to use the natural products wit antidiabetic activity, because both insulin and oral
hypoglycemic drugs possess undesirable side effects3. Many Ayurvedic
plants have been reported for their antidiabetic
activity. Some of the typical medicinal plant species have been reported as an antidiabetic by preclinical and clinical trials. Therefore
it was our intention to prepare polyherbal
formulation using reported antidiabetic herbs and to
develop the polyherbal product with a more potent
natural sweetener, S. rebaudiana. The present
research work supports the combined effect of Churna
and synergistic or additive impact of natural sweetener while formulated with
herbal product. The following medicinal plant species have been selected to
develop a sweet antidiabetic polyherbal
formulation.
Table 1 Effect of MC (Without Stevia) and MCS
(with Stevia)
on average body weight of animals.
|
Sr. no. |
Treatment |
Dose |
Average body weight (g) ± SEM |
||||
|
0th day |
7th day |
14th day |
20th day |
35th day |
|||
|
I. |
Vehicle control |
0.2 mla |
200.50 ± 2.84 |
201.83 ± 1.03 |
203.0 ± 1.06 |
205.83 ± 1.34 |
204.8 ± 1.09 |
|
II. |
Diabetic control |
0.2 mlb |
205.50± 4.88 |
175.0 ± 8.1 |
160.33 ± 2.5 |
148.83 ± 1.72 |
145.2 ± 1.18 |
|
III. |
Glibenclamide control |
10 |
206.66 ± 2.23 |
198.0 ± 1.5 |
195.16 ± 2.48 |
192.0 ± 3.96 |
188.6 ± 2.1 |
|
IV. |
MC without Stevia |
Feed |
209.66 ± 2.37 |
177. 50 ± 1.89 |
163.50 ± 3.35 |
160.60± 2.04 |
159.7 ± 2.5 |
|
V. |
MC with Stevia |
feed |
207.3 ± 2.33 |
175. 50 ± 1.5 |
161.4 ± 1.75 |
158.5 ± 2.1 |
155.5 ± 1.9 |
a=
Vehicle (0.5% Tween 80 SEM in normal, b= Alloxan single dose 80 mg/ Kg i.
p. in normal saline on day 0., *P < 0.05 as compared to vehicle
continued on corresponding day., MC- Madhujeevan churna, MCS- Madhujeevan churna
containing Stevia.
Table 2 Effect of Stevia on antidiabetic
potential of MC.
|
Sr. no. |
Group of Animal |
Dose Mg/ Kg |
Blood glucose conc. |
||||
|
0th day |
7th day |
14th day |
28th day |
35th day |
|||
|
1. |
Normal control |
Vehicle |
85 |
84 |
84 |
85 |
84 |
|
2. |
Diabetic control |
Untreated 80 mg/Kg alloxan |
82 |
205.5 |
215.2 |
218.1 |
208.9 |
|
3. |
Standard G |
Treated 10 mg/kg |
84 |
201.2 |
198.5 |
165.8 |
115.6 |
|
4. |
MC without
Stevia |
Oral |
85 |
202.1 |
185.7 |
171.9 |
128.8 |
|
5. |
MC
with Stevia |
Oral |
84 |
205.3 |
175.2 |
138.6 |
112.5 |
MC- Madhujeevan churna, G- Glibenclamide.
Aegle marmelos: The aqueous extracts of the stem and root bark are used4
to treat malaria, fever, jaundice, and skin diseases such as ulcers, urticaria, and eczema. In pharmacological trials, both the
fruit and root showed antiamoebic and hypoglycaemic activities. The plant is rich in alkaloids,
among which aegline, marmesin,
marmin, and marmelosin are
the major ones. Aqueous leaf extract and methanolic
extract of the root bark of A. marmelos showed
preventive effects on myocardial diseases.
The compounds luvangetin and pyranocoumarin,
isolated from the seeds of A. marmelos,
showed significant antiulcer activity. Essential oil isolated from the leaf
has antifungal activity. The aqueous extract of leaf possesses a hypoglycaemic effect.
Curcuma longa: Curcuma longa contains5 an essential oil
(5%), an alkaloid, starch grains, yellow matter curcumins
and other curcuminoids, turmeric oil (5-8%), turmerol, a coporioc acid (0.1%)
as a free acid, and veleric acid (0.1%) as combined
acid. Distillation of oil yields 2% d-sallinene, 1% µ- phellandrene
and 3% cineol from the lower-fraction. The middle fraction yields 30.5% zingiberene and higher fraction shows mixture of sesquiterpene hydrocarbons and sequiterpene
alcohol (50.5%). The oil contains small amount of sequiterpenes,
µ- and b– pinene, camphor, camphene and µ- and b- curcumins.
Gymnema sylvestre:
A water soluble extract of leaves of Gymnema sylvestre therapy appears to enhance endogenous insulin,
possible by degeneration/ revitalization of the residual β cell in IDDM.6
It also contents a new bitter neutral principle; lbuminous
and colouring matters, calcium oxalate, pararabin, glucose, carbohydrates, some tartaric acid and
organic acid said to be glycoside and to possess antisaccharine
properties and called X (C32
H59 O12 ) and gymnemic acid.7
Azadirachta indica:
A recent study showed that Neem leaf extract is
effective hypoglycemic agent and can act only in presence of suitable stimuli
like glucose load. They suggested that Neem leaf
extract basically act through potentiation of insulin
secretion in response to glucose load and thus, is helpfully NIDDM.8
Salacia reticulata: Anthocyanidines,
catechins, phenolic acids,
quinines, fridooleananes, tritepene
quinine- methides and related triterpenoids
(celastroloids), mangiferin,
gutt- percha, and dulcitol, salacinol and kotalanol9.
Syzygium jambolinum10: Delphinidin- 3 gentiobioside, malvidin- 3 laminaribioside, petunidin- 3 gentiobioside, glucose, fructose, gallic
acid, malic acid, Eugenia triterpenoids
A and B, oleanolic acid (fruits), jamboline,
myricyl alcohol, quercetin
1 nd 3- galloyl glucose
(seeds). A seed has been hypoglycemia and allays thirst in diabetes.
Momordica charantia:
In the clinical evaluation, carries out
at Wagharkar Hospital and Reasearch
Institute, it was found that karela in combination
with other herbal hypoglycemic drugs showed marked reduction in blood glucose
level and no side effect were observed.11
Embelica officinalis12: Emblica
officinalis normally known as amla,
has been used extensively in ancient Indian Ayurveda.
It is a member of small genus Emblica (family Euphorbiacae) which is commonly found in
Stevia rebaudina: It is rich in 13 terpenes and flavonoids. Stevia contains a
complex mixture of diterpenes, triterpenes,
stigmasterol, tannins and volatile oil. The
constituents responsible for Stevia's sweetness were documented in 1931, when eight novel
plant chemicals called diterpenic glycosides (5-14%)
were discovered and named as Stevioside, Dulcoside and Rebaudioside A, B,
C, D and E. (2, 3, 4 , 5). Stevia has been
used for centuries as a natural sweetener and may be helpful in treating
diabetes.
These crude drugs
have been used to treat diabetes. Most of these ingredients contain flavanoids and phenolic contents.
Flavanoids are also reported to possess antioxidant
potency 14. It was interested to look over either additive or
synergistic effect of all ingredients together.
The present study supports use of Stevia
rebaudiana
as a natural sweetener with nutraceutical. S. rebaudiana contains sterioside
and rubaudioside as diterpene
glycosides15. These are 300 times sweeter than sugar. It also
contains sequiterpene lactones16 that are
responsible for bitter after taste. S. rebaudiana acts as anti-diabetic17,
anti-hypertensive18, antihyperlipidemic19, anti-yeast, antibacterial20,
anticaricinogenic21 and antifungal. It is considered as GRAS by
USFDA22.
Table 3 The effect of MC (Without Stevia), MCS
(with Stevia)
and Glibenclamide on lipid profile of animals as
biochemical parameters.
|
Parameter |
Control Group I |
Diabetic Group II |
Glibenclamide Group
III |
MC Group IV |
MCS Group V |
|
Total cholesterol |
71.7± 2.1 |
66.8± 1.8 |
64.9± 2.7 |
66.27 ±1.5 |
61.2 ±2.2 |
|
HDL cholesterol |
49.4 ±2.4 |
52.7 ± 1.1 |
48.3 ± 3.8 |
55.6 ±2.1 |
54.72 ±2.2 |
|
Triglyceride |
438.6 ±1.3 |
441.8 ±1.5 |
162.9 ±3.3 |
134.3 ±01.8 |
132.8 ± 1.4 |
|
SGOT |
321.8 ± 2.7 |
325.8 ± 2.5 |
218.4 ±1.5 |
251.1 ± 1.7 |
248.2 ± 1.5 |
|
SGPT |
112.8 ± 1.5 |
11.5 ± 1.7 |
105.6 ± 1.4 |
97.5 ± 1.3 |
95.3 ± 1.4 |
|
VLDL |
85.5 ± 0.9 |
88.5 ± 1.1 |
31.8 ± 2.1 |
26.8 ± 1.5 |
25.5 ± 1.7 |
|
Creatinine |
4.3 ± 0.4 |
4.6 ± 0.5 |
4.1 ± 0.2 |
4.8 ± 0.3 |
4.5 ± 0.5 |
|
Uric acid |
3.5 ± 0.3 |
3.8 ± 0.4 |
3.1 ± 0.25 |
3.2 ± 0.2 |
2.9 ± 0.5 |
HDL:
High Density Lipid level, SGOT: Serum Glutamate oxaloacetic transaminase, SGPT:
Serum Glutamate pyruvic transaminase, VLDL:
Very Low Density Lipoprotein
Churnas developed without Stevia
contain some bitter principles which acts as anti-diabetic. Such formulations
are unacceptable and unpalatable to consumers. In this study antioxidant
potential with taste masking of final product was optimized. Therefore, it was
our intention to develop the bitter formulations with sweet Stevia
and to study comparative antidiabetic activity.
MATERIALS AND METHODS:
Materials: Alloxan and Glibenclamide were obtained from Sigma Co. St. Louis, Mo,
USA. The powdered materials of crude drugs were procured from Satara Ayuvedic Arkshala, Satara. These are
authenticated by Professor B. D. Patil. Botany Dept.
S. G. M. College of Science, Karad (Authentication
voucher number- SGM-KARAD(M.S.,
Method: All the crude materials were powdered and passed
through sieve no. 22 to form a moderately coarse with fine powder. The
following formula was developed using suitable quantity of S. rebaudiana as natural sweetener and nutraceutical. Each 100 gm of Madhujeevan
churna contains-
G. sylvestre 15gm
C. longa 15gm
S. zambolanum 15gm
S. chineses 10gm
A. marmelos 10gm
M. charantia 10gm
A. indica 15gm
S. rebaudiana 10.0%
Antidiabetic
activity23,24
Animal were housed under standard laboratories
conditions 12 hrs. dark and light cycle was maintained. Animal had access to
food and water ad libitum. After a washout period of
7 days the animals received Alloxan in dose of 125
mg/kg IP to induce hypoglycemia25, and the drug treatment restarted
on the same dose.
Male Wistar albino rats (150-
200gm) were randomly divided into five groups with six animals in each group.
Group I (Normal control): Carboxyl methyl cellulose 1%
w/v (0.5 ml/100g of body weight) was administered orally.
Group II (Diabetic Control): Alloxan
(125 mg/kg of body wt.) injected intraperitonially
and kept without any treatment to study the diabetic nature of rat.
Group III (standard): Alloxan
(125 mg/kg of body wt.) injected intraperitonially
and Glibenclamide (10 mg/kg of body wt.) administered
orally.
Group IV Madhujeevan
churna (MC): Alloxan
(125 mg/kg of body wt.) injected intraperitonially
and feeding of (MC) by oral route.
Group V Madhujeevan
churna containing Stevia
(MCS): Alloxan (125 mg/kg of body wt.) injected intraperitonially and (MCS) by oral route.
The control group was injected 0.85% saline intraperitonially. Diabetes was induced by intraperitonial injection of alloxan
at a dose of 125 mg/kg body weight. The Alloxan solution
was freshly prepared, kept on ice and injected immediately. Non fasting blood
samples were collected via tail vein and used for measuring blood glucose level
by the glucose oxidase-peroxidase (GOD/POD) method.
Only these rats, which showed blood glucose levels above 250 ng %, were considered diabetic and selected for study. The
urine glucose was monitored using gluco test strips.
Mode for feeding:
The rats were administered the materials twice a day
for a period of 4 weeks. The powders were orally fed at a dosage of 20 mg/kg
body weight and Glibenclamide at a dosage of 0.1%
mg/kg body weight. The rats were weighted everyday throughout the study. Fasting blood was collected from the tail
vein once a week and glucose estimated. In the case of Glibenclamide
and that extract which shows the best hypoglycemic activity, blood was
collected from the animals by retro-orbital bleeding, at the end of the 4th
week, sera separated and used for the determination of bio-chemical parameters,
cholesterol, HDL, VLDL, triglycerides,
SGOT, SGPT, creatinine and uric acid.
\Group I comprising
of six animals was served as control. The control group was injected 0.85%
saline intraperitonealy. Group II was treated with alloxan at a dose of 125 mg/ kg body weight. Group III was
treated with Alloxan. No fasting blood samples were
collected for measurement of blood glucose level and from second day Glibenclamide at dose of 0. Mg/ Kg body weight was given.
Group IV was treated with alloxan and from second day
Madhujeevan churna
(without Stevia) was fed. Rats were
administered with this Churna twice a day
group V was treated with alloxan and Madhujeevan churna (with
Stevia). The same mode of feeding was
maintained up to 35 days.
RESULTS:
Madhujeevan churna (without Stevia)
showed reduced blood glucose level by 55% at the end of first weak which
because 51% at end of 4th weak. Madhujeevan
churna containing Stevia
showed increased antihypotensive activity. Blood
sugar level was reduced by 65% at the end of first week which became 62% at the
end of 4th week. The antidiabetic
potential of both Churnas was
comparable and significant to that of standard Glibendamide.
The superior effects were observed in case of group V in comparison with group
III. Effect seems to reach maximum after 15 days of treatment and remains
constant in third week. The decreased blood sugar level by test samples and
that of standard was significant as shown in table 2.
Vehicle control animals were found to be stable in
their body weight. But diabetic rats showed significant reduction in body
weight in 21 days. Alloxan caused body weight
reduction, which is reversed by Madhujeevan
churna after 7 days of treatment. The oral
feeding of Madhujeevan churna
for 4 weeks resulted in an increase in body weight which was 6 and 23% in
diabetic and control animals, respectively. The Madhujeevan
Churna with Stevia
also led to a gain in body weight being 4 % in diabetics and 25% in controls.
And increase in body weight of 4% in diabetics and 21% in controls was seen
upon administration of the Madhujeevan churna with Stevia. In
case of Glibenclamide, there was no net gain in the
weight of diabetic animals, but the control shows a 24% increase. The decreased
level of average weight of animals is shown in table 1. Thus the diabetic
animals feed the extract showed less weight gain than the corresponding
controls. This observation and the decrease in body weight observed in
uncontrolled diabetes might be the result of protein wasting due to
unavailability of carbohydrates for utilization as an energy source.
In the present study, the Madhujeevan
churna with Stevia
lowered the elevated SGOT levels in diabetic by 21% with no effect in control
while Glibenclamide lowered the elevated SGOT level
by 46% also with no effects in controls. SGPT levels are lowered by 14% in
diabetics and 22% control by Madhujeevan churna with Stevia and
12% in diabetes by Glibenclamide with no effects with
control. Total cholesterol along with HDL level remained unchanged in diabetic
compared to the other groups. The triglyceride level observed to be elevated in
untreated diabetes but reduced by both Madhujeevan
churnas (69% in diabetes and 32% in controls) as
well as Glibenclamide (65% in diabetes and 32% in
controls) as well as Glibenclamide (65% in diabetes
and 32% in controls) as well as Glibenclamide (65% in diabetes and 18% in controls) showing
their beneficial effects. The elevated VLDL levels in the untreated diabetes
were also reduced by extract and Glibenclamide. The
values of lipid profiles of animals during study are shown in table 3.
DISCUSSION:
The results suggest the beneficial effects of Madhujeevan churna with
Stevia in improving the imbalance in
lipoprotein metabolism are also comparable to those Glibenclamide.
Control animals fed with Madhujeevan churna, with Stevia
and Glibenclamide do not excrete any glucose in urine
excluding any harmful effect. But none of these Churnas
were able to inhibit or reverse to any significant content the excretion of
glucose in the urine of the diabetics. Considering the above results obtained Madhujeevan churna with
Stevia was chosen for a further detailed study
of biochemical parameters. Glibenclamide, a synthetic
drug was used as a reference for comparison.
Abnormalities in lipoprotein are very common in both
NIDDM and IDDM. Although lipoprotein alteration appears to the intrinsic part
of these disorders, such alterations are also induced by diabetes associated
complications such as obesity and renal disease. The kidneys maintain optimum
chemical composition of body fluids by acidification of urine and removal of
metabolite waste such as urea, uric acid, creatinine
and ions. During renal disease, the concentration of these metabolites
increases in blood. In this study, the levels of uric acid and creatinine did not appear to increase any of the groups.
This indicates the absence of any significant kidney damage. Thus both Madhujeevan churna with
Stevia and Glibenclamide
reduced the increase level of SGOT and SGPT observed in untreated diabetes in
the present study. This might suggest the protective action of the extract and Glibenclamide in reversing any organ damage due to
induction of experimental diabetes that is manifested by elevation in the level
of SGOT an SGPT.
CONCLUSION:
Every ingredient of developed Madhujeevan
churna without Stevia
(MC) has its own antidiabetic
potential. The antidiabetic activity may be due to flavanoids, phenolic moieties or
other antioxidant principles. Combined effect of each and every crude drug of
bitter Churna has a significant effect on
blood glucose level and other biochemical parameters. Decreased level of blood
sugar, reduced amount of HDL, Triglyceride, VLDL, SGOT and SGPT indicates that
the bitter MC possesses improved antidiabetic
potential in comparison to a standard Glibenclamide.
Unpleasant and bitter taste of MC was demasked
by 10% Stevia powder which was most palatable
and acceptable for animals. Madhujeevan churna containing Stevia
(MCS) have a better impact on blood sugar level and other
biochemical contents. There was a valuable percentage reduction in blood sugar
level of animals those are treated with MCS. Lipid profile of blood
containing SGOT and SGPT contributes the most probable mechanism of Stevia formulated with MC. An
effective reduced level of elevated HDL, triglyceride and VLDL of MCS is
greater than MC. Thus the synergistic effect of active principles of Stevia on antidiabetic potential of bitter Madhujeevan
churna highlights the importance of natural
sweetener and nutraceuticals in polyherbal
antidiabetic formulation.
It concluded that both Madhujeevan
churna have good antidiabetic
activity. But, Madhujeevan churna containing Stevia
have better antidiabetic potential then Churna without Stevia.
ACKNOWLWDGEMENT:
We are
thankful to Dr. S. B. Bhise, Principal Govt. College
of Pharmacy, Karad for provision of their research
laboratory facilities and Manager, Satara Ayurvedic Arkshala, Satara for providing
crude materials. We thank to laboratory in- charge, Krishna Institute of
Medical Sciences, Karad, Dist. Satara
for providing animal.
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Received on
17.09.2009
Accepted on
20.10.2009
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Journal of Pharmacognosy and
Phytochemistry. 1(3): Nov. – Dec. 2009, 204-208