Siansmalysia
Siansmalysia
Siansmalysia
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B.S. Ashok Kumar*, Saleemulla Khan, Gopi Setty Saran, R. Nandeesh & N.K. Manjunath
ABSTRACT
Nisamalaki Churna is an Ayurveda formulation used for diabetes, which consists of amalaki and haridra. In vitro
antidiabetic screening of Nisamalaki Churna by α-amylase inhibition was carried out by starch iodine method,
dinitrosalicylic acid method (DNSA) and effect on normal rats blood sugar level was studied. Nisamalaki Churna shows
potent α-amylase inhibition IC50 89.44 µg/mL by starch iodine method and IC50100.0 µg/mL by DNS method. Nisamalaki
Churna showed potent decrease in blood glucose level in normal rats.
Abstrak
Nisamalaki Churna adalah formula Ayurveda untuk diabetis yang mengandungi amalaki dan haridra. Penyaringan
antidiabetis in vitro Nisamalaki Churna telah dijalankan. Kesan Nisamalaki Churna terhadap perencatan α-amilase
telah dikaji menggunakan kaedah iodin kanji. Dengan menggunakan kaedah asid dinitrosalisilik (DNSA) kesan terhadap
aras gula dalam darah tikus biasa telah dijalankan. Nisamalaki Churna menunjukkan kesan perencatan α-amilase
yang kuat, IC50 89.44 µg/mL melalui kaedah iodin kanji dan IC50100.0 µg/mL DNS. Nisamalaki Churna menunjukkan
penurunan aras glukos yang kuat dalam tikus biasa.
In Vitro Alpha Amylase Inhibition Assay in polypropylene cage and were fed with standard food
3, 5-Dinitrosalicylic acid method (DNSA) The inhibition pellets (Kamadenu Enterprises, Bangalore) and water ad
assay was performed according to Miller (1959) using libitum. All the studies conducted were approved by the
DNS method. Aqueous extract of Nisamalki churna of institutional animal ethical committee of Sri K.V. College
varied concentrations in 500 μL were added to 500 μL of Pharmacy, Chickballapur, Karnataka, according to
of 0.02 M sodium phosphate buffer (pH6.9 containing 6 prescribed guidelines of CPCSEA, Government of India
mM sodium chloride) containing 0.04 units of α-amylase (Reg. No. 117/2000/CPCSEA).
solution and were incubated at 37°C for 10 min, followed
by addition of 500 μL of a 1% starch solution in 0.02 M ANTIDIABETIC SCREENING IN NON-FASTED RATS
sodium phosphate buffer (pH6.9) all the test tubes. The
Prior to the study, blood samples were collected from all
reaction was stopped with 1.0 mL of 3, 5 DNSA reagent. The
the animals to check random glucose levels. The animals
test tubes were then incubated in a boiling bath water for 5
which had a glucose level >130 gm/dL were excluded from
min and cooled to room temperature. The reaction mixture
the study. Five groups of non-fasted rats, six in each were
was then diluted after adding 10 mL distilled water and
grouped as follows:
absorbance was measured at 540 nm. The control samples
Group I – Control (0.5% w/v CMC) administered;
were also prepared accordingly without any plant extracts
Group II – Glucose control ;
and were compared with the test samples containing
Group III – Standard drug treatment (metformin-
various concentrations of the plant extracts prepared
250 mg/kg, p.o.);
with different solvents. The results were expressed as %
Group IV – Nisamalaki Churna (90 mg/kgp.o.)
inhibition calculated using the formula:
Group V – Nisamalaki Churna (180 mg/kgp.o.)
Abs (Control)-Abs (Extract) The test and standard substances were suspended in
% Inhibition activity = ______________________ × 100.
0.5% w/v carboxy methyl cellulose (CMC). Group II to
Abs (Control)
group V animals were given 5 g/kg glucose additionally
after 30 min of standard drug/ test substance administration.
Starch-iodine Colour Assay Screening of Nisamalaki Blood samples were collected from retrooribatal puncture
Churna for α-amylase inhibitors was carried out according for glucose estimation at various time points like pre dose,
to Xiao et al. (2006) with slight modification based on the 1.0, 2.0, 3.0 and 4.0 h after test substance/ standard drug
starch-iodine test. Aqueous extract of Nisamalki churna treatment. Glucose was estimated by using Lab-India auto-
of varied concentrations in 500 μL were added to 500 μL analyzer with the help of Lab kit enzymatic kit (Nyunai
of 0.02 M sodium phosphate buffer (pH6.9 containing 6 2006; Vogel 2002).
mM sodium chloride) containing 0.04 units of α-amylase
solution and were incubated at 37°C for 10 min. Then 500
STATISTICAL ANALYSIS
μL soluble starch (1%, w/v) was added to each reaction
well and incubated at 37°C for 15 min. 1 M HCl (20 μL) The values were expressed as mean ± SEM (n=5) for each
was added to stop the enzymatic reaction, followed by group. The significant difference between groups was
the addition of 100 μL of iodine reagent (5 mM I2 and 5 determined using one-way ANOVA followed by Dunett’s
mM KI). The colour change was noted and the absorbance test. P value less than 0.05 was considered significant.
was read at 620 nm on a microplate reader. The control
reaction representing 100% enzyme activity did not contain RESULTS AND DISCUSSION
any plant extract. To eliminate the absorbance produced
by plant extract, appropriate extract controls without the
IN VITRO α-AMYLASE INHIBITION ASSAY OF
enzyme were also included. Inhibition of enzyme activity
NISAMALAKI CHURNA
was calculated as:
Drugs that inhibit carbohydrate hydrolyzing enzymes have
Inhibition of enzyme activity (%) = (C-S) / C × 100, been demonstrated to decrease postprandial hyperglycemia
and improve impaired glucose metabolism without
where S is the absorbance of the sample and C is the promoting insulin secretion of NIDDM patients. The results
absorbance of blank (no extract). of in vitro studies showed that Nisamalaki Churna inhibits
α-amylase activity. Natural health products of vegetable
origin were clearly indicated as a promising avenue for
ANIMALS
the prevention of chronic diseases (Punitha & Manoharan
Male Wistar rats weighing 150-250 g were acclimatized to 2006). Figures 1 and 2 show that Nisamalaki Churna
the experimental room at temperature 23 ± 2°C, controlled significant inhibition of α-amylase enzyme. IC50 values
humidity conditions (50-55%) and 12 h light and 12 h dark of Nisamalaki Churna by DNSA and Starch Iodine method
cycle. They were caged with a maximum of two animals were 89.44 and 100.0 μg/mL, respectively.
627
% Inhibition
Concentration (μg/mL)
Figure 1. Effect of Nisamalaki Churna on α-amylase inhibition activity
by Starch Iodine method
% Inhibition
Concentration (μg/mL)
Figure 2. Effect of Nisamalaki Churna on α-amylase
inhibition activity by DNS method
Control
Glucose control
Metformin (250 mg/kg)
Nisamalaki Churna (90 mg/kg)
Nisamalaki Churna (180 mg/kg)
Blood Glucose Level (mg/dL)
Time in hours
Figure 3. Effect of Nisamalaki Churna on blood glucose levels of non fasted rats
628
ANTIDIABETIC SCREENING IN NON-FASTED RATS Ageratum conyzoides in rats. African Journal of Traditional,
The oral hypoglycaemic effect of Nisamalaki Churna was Complementary and Alternative Medicines 3(3): 76-79.
studied in non-fasted Wistar rats. The Nisamalaki Churna Punitha, R. & Manoharan, S. 2006. Antihyperglycemic
and antilipidperoxidative effects of Pongamiapinnata
significantly reduced (p< 0.05 – 0.01) hyperglycemia at the
(Linn.) Pierre flowers in alloxan induced diabetic rats. J.
dose levels of 90 and 180 mg/kg, compared with glucose Ethanopharmacol. 105: 39-46.
treated group (group-2). The glucose control group showed Vogel, G.H. 2002. Drug Discovery and Evaluation
significant (p< 0.01) increase in glucose levels after 30 Pharmacological Assays. 2nd ed. Berlin Heidelberg,
min of glucose administration in all the animals when Germany: Springer-Verlag.
compared with normal group animals. The hypoglycemic Xiao, Z., Storms, R. & Tsang, A. 2006. A quantitative starch-
effect of Nisamalaki Churna comparably equivalent to the iodine method for measuring alpha-amylase and glucoamylase
standard metformin (Figure 3). The results of this study activities. Anal. Biochem. 351(1): 146-148.
indicated that Nisamalaki Churna showed appreciable
antidiabetic activity. This study supports the ayurvedic use
of Nisamalaki Churna. B.S. Ashok Kumar* & N.K. Manjunath
Department of Pharmacognosy
Sri K.V. College of Pharmacy
ACKNOWLEDGEMENT Chickballapur, Karnataka
Authors are thankful to Sri K.V. NaveenKiran, Chairman, India
Sri K.V.College of Pharmacy, Chickballapur, Karnataka,
Saleemulla Khan
India, for providing facilities to carry out this work.
Department of Pharmacognosy
ManipalCollege of Pharmaceutical Sciences
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Miller, G.L. 1959. Use of Dinitrosalicylicacid reagent for *Corresponding author; email: ashok4vani@gmail.com
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