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Anti diabetic activity by the in vitro alpha amylase and alpha-glucosidase

inhibitory activity of Catharanthus roseus

Article · January 2010

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5(4) : 655-659, 2010

ANTI DIABETIC ACTIVITY BY THE IN VITRO ALPHA AMYLASE

AND ALPHA-GLUCOSIDASE INHIBITORY ACTIVITY OF
CATHARANTHUS ROSEUS

V. MALATHI*, S. SATHANA DEVI AND K. REVATHI1

Department of Biochemistry, Ethiraj College for Women, Chennai - 600 008
1
Department of Zoology, Ethiraj College for Women, Chennai - 600 008
E-mail: [email protected]

KEY WORDS ABSTRACT

Catharanthus roseaus Catharanthus roseus is a plant extensively known for its anti-inflammatory potential. In this study the alcoholic
Anti diabetic activity extract of flower and leaf of Catharanthus roseus was tested for its α-amylase and α-glucosidase inhibitory
α - amylase activity to understand its anti-diabetic potential.Varying concentration of the extracts of leaf and flower were
α-glucosidase inhibitory assayed for their inhibitory action [in vitro] on serum amylase, pancreatic amylase, α–amylase from fermented
activity barley and α- glucosidase. Both extracts of the leaf and flower were found to inhibit the enzymes considerably.
Metformin The leaf extract showed maximum inhibitory action with a concentration of 10 mg/mL [IC50]. IC50 for the flower
extract was found to be at a concentration of 12.5mg/mL. Further the anti-oxidant property of the herb was also
Received on : evaluated by its activity to inhibit lipid per oxidation. Catharanthus roseus extract of leaf and flower exhibit their
19.06.2010 anti-diabetic effect by inhibiting the enzymes which has a main role in carbohydrate metabolism like α-amylase
Accepted on : and α-glucosidase.
27.10.2010

*Corresponding
author

INTRODUCTION continues to confront patients and physicians. The graph of

diabetes related mortality is rising and reducing the life
Diabetes mellitus (DM) is the commonest endocrine disorder expectancy to 5 to 15 years.
that affects more than 100 million people worldwide (6% of
Diabetes mellitus in youth is emerging as a serious clinical
the population) and in the next 10 years it may affect about
entity and its incidence has increased over the year. The Indian
five times more people than it does now (WHO/Acadia, 1992;
diabetic population is predicted to rise to > 80.9 million by
ADA, 1997). In India, the prevalence rate of diabetes is
the year 2030 (Bjork et al., 2003).
estimated to be 1-5 %. Complications are the major cause of
morbidity and mortality in DM. The World Health In recent years, much interest has been focused on biologically
Organization (WHO) predicts that the number of cases active compounds occurring in natural resources. Plants have
worldwide for diabetes is now 150 billion, which will double always been an exemplary source of drugs and many of the
currently available drugs have been derived directly or
by the year 2025.
indirectly from them. The ethno botanical information reports
Diabetes mellitus is a chronic, serious metabolic disorder about 800 plants that may possess anti-diabetic potential
caused by inherited or acquired deficiency in production of (Alarcon-Aguilara et al., 1998). Wide arrays of plant derived
insulin by the pancreas or by the ineffectiveness of the insulin active principles representing numerous chemical compounds
produced resulting in impaired function in carbohydrate, lipid have demonstrated activity consistent with their possible use
and protein metabolism (Quong Luo et al., 2004). Among in the treatment of Non-insulin dependent diabetes mellitus
the two principal form of diabetes, insulin dependent (type~1) (NIDDM) (Bailey and Day, 1989). Among these are alkaloids,
and non insulin dependent (type~2) , Non insulin dependent glycosides, galactomannan, polysaccharides, peptidoglycans,
diabetes accounts for 90% of all cases worldwide, due to the hypoglycans, guanidine, steroids, carbohydrates,
body’s inability to respond properly to the action of insulin glycopeptides, terpenoids, amino acids and inorganic ions.
produced by the pancreas (Notkins, 2002). Non insulin Even the discovery of widely used hypoglycemic drug,
dependent diabetes is becoming a pandemic and despite the metformin came from the traditional approach of using Galega
recent surge in new drugs to treat and present the condition, officinalis.Thus, plants are a potential source of anti-diabetic
its prevalence continues to soar. In spite of great strides that drugs but this fact has not gained enough momentum in the
have been made in understanding and in the management of scientific community.
this disease, serious problems like diabetic retinopathy, Although, oral hypoglycemic agents such as insulin are the
diabetic nephropathy and low extremely amputation main stay of treatment of diabetes and are effective in

655
V. MALATHI et al.,

controlling hyperglycemia, they have prominent side effects amylase, pancreatic amylase, α-amylase from fermented
and fail to significantly alter the course of diabetic barley] was added to the test tube T. The contents of the test
complications (Rang and Dale, 1991). As the knowledge of tube were mixed well and incubated at 37ºC for 10 min. After
heterogeneity of this disorder increases, there is needed to incubation 0.5mL of 2N NaOH was added to the test tube
look for more efficacious agents with lesser side effects. Though T and C.0.2mL of enzymes was added to the control C.5.7mL
development of modern medicine resulted in the advent of of distilled water alone serves as blank.0.2mL of dinitrosalicylic
modern pharmacotherapeutics including insulin, biguanides, acid was added to all the test tubes.
sulfonylureas and thiazolidinediones, there is still a need to The contents were mixed well and kept in a boiling water bath
look for new drugs as no drug (except strict glycemic control for 15minutes.The intensity of reddish orange colour was read
with insulin) has been shown to modify the course of diabetic at 540nm. The percentage of inhibitory action of serum
complications. amylase was calculated from the following formula.
This study is hence aimed at identifying safe herbal remedies O.D = optical density.
for treating diabetes and also to identify their possible O.D of control-O.D of test
mechanism of anti diabetic control. Percentage inhibition = X 100
O.D of control

MATERIALS AND METHODS Effect of Catharanthus roseus on α -glucosidase

The α – glycosidase inhibitory activity of Catharanthus roseus
Materials was tested by the method of Li et al., 2004. Three Test tubes
The leaf and flower [pink coloured flower species] of the plant were taken and labeled as blank, test (T) and control (C) .200μL
Catharanthus roseus were collected from the coastal region of substrate, starch and 200μL of enzyme [α-glucosidase] to
of Kalpakkam, (located on north of Chennai and south of the test tube T and C were added. This was followed by the
Pondycherry) Tamil Nadu, India. The chemicals used for the addition of 0.5mL of extract to T. The contents were mixed
study were from Sigma Chem. (St. Louis, MO, USA). well and incubated at 37ºC for 30 min. Trichloroacetic acid
was added to all the test tube after incubation. The test tubes
Preparation of extract were then centrifuged for 10 min. The liberated glucose from
The Alcoholic extract of leaves and flowers of Catharanthus the substrate by the action of enzyme was reacted with 1.5mL
roseus was prepared by Soxhlet extraction using 50g of the of anthrone reagent. The contents were boiled for 15 min.
dry powder in 200mL of ethanol. The intensity of color developed was read at 640nm. The
Chemical characterization of the extract percentage of inhibitory action of α-glucosidase was calculated
The extract was tested for the presence of alkaloids, flavanoids, from the following formula.
phenol, tannins, saponins etc. Total alkaloids were determined O.D of control-O.D of test
by Dragendroff’s test. Briefly, 1mL of extract, few drops of Percentage inhibition = X 100
O.D of control
Dragendroff’s reagent was added orange colour were noticed
indicating presence of alkaloids and Mayer’s test was also Effect of Catharanthus roseus extract on lipid per oxidation
done to confirm the presence of alkaloids. To the extract 2mL The anti-oxidant potential of the extract of Catharanthus roseus
of Mayer’s reagent was added. The formation of yellow colour leaves and flowers were tested by the ability to inhibit Lipid
precipitate confirmed the presence of alkaloids. Total flavones peroxidation. The lipids in the cell membranes are highly
and flavanones in the extract were determined by using susceptible to per oxidative damage and are broken down
Shinoda test. To the extract, a few magnesium turnings and 1- into number of small units to form malonialdehyde. In Blood
2 drops of concentrated HCl were added, formation of red cells, lipid per oxidation was induced by the addition of 0.1mL
colour showed the presence of flavones. To the extract, of 25μM ferrous sulphate, 10mL of ascorbic acid, 10 mM
10%NaOH was added, yellow to orange color showed the KH2PO4. Three Test tubes were taken and labeled as blank,
presence of flavanones. (Harborne, 1998; Sadasivam and test (T) and control (C). The (T) tubes were incubated at 37ºC
Manickam, 1992; Ogbonnia et al., 2008; Nooman et al., 2008; for one hr with different concentration of extracts. To the test
Mohd. Nawagish and Ahmad, 2007). tubes 1mL of 15% TCA and 0.5mL of 0.375% TBA [thio
Total phenols were determined by ferric chloride test, to the barbituric acid] were added and test tubes were placed in the
extract, few drops of 10% aqueous ferric chloride was added, boiling water bath for 30min. The tubes were centrifuged and
appearance of blue colour indicated the presence of phenols. the supernatant was taken and read at 532 nm.
Effect of Catharanthus roseus extract on serum amylase, The percentage of LPO was inhibition was calculated from
pancreatic amylase and α-amylase from fermented barley the following formula.
O.D of control-O.D of test
The alcoholic extract of leaves and flowers was tested for its Percentage inhibition = X 100
serum amylase inhibitory activity by the Yukihiko hara method, O.D of control
(1990). Three Test tubes were taken and labeled as blank, test
(T) and control (C). To each test tube 2.5mL of phosphate RESULTS AND DISCUSSION
buffer of pH 6.8 was added. 1mL of starch substrate and
sodium chloride was added to all the three test tubes. The test Qualitative phytochemical analysis
tubes were incubated at 37ºC for 10 min. After incubation, C.roseus was found to contain flavones, sugar, phenol, tannins,
0.5mL of extract was added and 0.2 mL of enzyme [serum glycosides and alkaloids. Saponin, steroids, quinones were

656
 ANTI DIABETIC ACTIVITY OF CATHARANTHUS ROSEUS

found to be absent (Table 1). The investigation of the Table 1: Qualitative phytochemical analysis of Catharanthus roseus
Phytochemical constituent show that ethanol extract of S. N. Phyto-chemicals tested Inference
Catharanthus roseus contains Flavonoids. Flavones and iso- 1 Flavones P
flavonoid, glycosides form the ingredient of many Ayurvedic 2 Steroids A
formulation used for the treatment of diabetes and also it 3 Anthraquinones A
stimulates insulin secretion by pancreatic islet cells. It also 4 Phenol P
controls blood glucose and modulates the metabolism of 5 Tannins P
glucose and blood lipid and decrease outputs of lipid 6 Saponins A
7 Glycosides P
peroxidation and scavenge the free radicals in non-insulin
8 Reducing sugar P
dependent diabetic rats.The flavonoids also act as anti- 9 Alkaloids P
inflammatory agents. The Phytochemical analysis of 10 Quinones A
Catharanthus roseus shows a positive result for tannins. The P=Present ; A= Absent
Tannoids inhibit aldose reductase in-vitro and prevent
hyperglycemia induced lens opacification on organ culture. metabolism and improves antioxidant status in streptozotcin
It also inhibit sorbitol formation in the lens and might counter nicotinamide induced Diabetic rats. Catharanthus roseus stem
the polyol pathway induced oxidative stress. Thus tannoids extract in graded doses caused a significant increase in
are effective in delaying development of diabetic cataract in enzymatic antioxidants such as catalase, superoxide
rats (Suryanarayana, 2007). dismutase, glutathione synthetase peroxidase and enzymatic
antioxidants such as ascorbic acid, Ceruloplasmin and
The high Phenolic content of the extract support the anti-
tocopherol. (Punitha et al., 2005).
amylase activity. The phenolic substances have ability to
interact with and / or inhibit proteins / enzymes (Rohan et al., The α-amylase inhibitory activity of Catharanthus roseus
2002). In-vitro, α-amylase inhibitory activity of Catharanthus roseus
The Phytochemical analysis of Cathranthus roseus shows was tested. α−amylases from three different sources was used
the positive result for alkaloids. The Alkaloids present in this namely pancreatic α-amylase, serum α-amylase and α-amylase
extract was found to lower the blood sugar level and act on from fermented barley (supposed to be rich source of α-
hemostatics. It also lowers the number of white cells in blood. amylase).
So, it is also used as an anti-cancer drug. They work by Concentrations of the range 10mg, 12.5mg, 16 and 80 mg/mL
preventing mitosis in metaphase, these alkaloids binds to of the ethanol extract of leaf and flower was tested on the
tubulin, thus preventing the cell from making the spindles it amylases from the sources mentioned above.
needs to divide. This is different from the action of taxol which
interferes with cell division by keeping the spindles from being The leaf extract showed a significant pancreatic amylase
broken down. inhibitory activity of 63% at a concentration of 80mg/mL. For
a similar concentration the flower extract showed 15%
Analysis of the Phytochemical constituents support the
inhibition compared with the commercial drug (Glycomet -
strong anti-diabetic activity of the herb Cathranthus roseus.
Metformin tablet - α-amylase inhibitor) which showed 85%
Antioxidant property of Cathranthus roseus inhibition. IC50 of the flower extract was found to be 16mg/mL
The alcoholic extract of leaf and flower of Catharanthus roseus (Fig. 2).
of different concentrations such as 10mg, 12.5mg, 25mg and The extract of leaf and flower showed significant serum amylase
50 mg/mL were tested for their ability to inhibit Lipid inhibitory activity when compared to pancreatic amylase. The
peroxidation. The leaf extract was found to show 26% leaf extract showed a significant serum amylase inhibitory
inhibition at a concentration range of 50 mg/mL. The flower activity of 80% at a concentration of 10mg/mL. While the
extract was found to show a significant inhibition of 47% at a flower extract showed a 50% inhibitory activity at a
concentration of 50 mg/mL. IC50 of the flower extract was concentration of 12.5mg/mL and 16mg/mL (Fig.3).The
found to be 50 mg/mL. percentage inhibition showed by the commercial drug for
From the results, it is clear that the ethanolic extract of leaves similar concentration were 90%, 80% and 60% respectively.
and flowers of Catharanthus roseus possesses significant The α-amylase from fermented barley seeds were also tested
antioxidant properties. The flower extract was found to possess with varying concentration of the leaf and flower extract of
much more significant antioxidant properties than the leaf Catharanthus roseus. The leaf extract showed an inhibitory
extract. activity of 61% at a concentration of 80mg/mL. While for a
Antioxidant property of Catharanthus roseus assessed by similar concentration, the flower extract showed 66%
the ability to inhibit lipid per oxidation inhibition (Fig. 4).
From the results, it is evident that the ethanolic extract of leaves
The antioxidant property of the extract could be explained by
and flowers had potent á -amylase inhibitory activity
its rich content of Flavonoids (Table 1), which has the ability comparable to the commercial drug Metformin.
to decrease the output of lipid peroxidation and scavenge α-amylase begins the process of starch digestion. It takes starch
free radicals. chains and break them into small pieces with two or three
Earlier studies were also found to confirm this property of glucose units. Some phenolic compounds in sweet potato,
Cathranthus roseus(Fig.1).Alcoholic stem extract of strawberry, Raspbery, Olive oil, pears, coca and Lentils are
Catharanthus roseus was found to regulates carbohydrate reported to be effective human α-amylase inhibitors. (Matsui

657
V. MALATHI et al.,

50 70
%inhibition of leaf %inhibition of Flower %inhibition of leaf
45 %inhibition of flower
60
40

35 50

%inhibition
30 40
%inhibition

25
30
20

15 20

10 10

5
0
10 12.5 80
0
Concentration (mg/mL)
10 12.5 25 50
Concentration (mg/mL) Figure 4: α-Amylase inhibitory activity (Fermented Barley)
Figure 1: Antioxidant property of Catharanthus roseus assessed by
the ability to inhibit lipid per oxidation %inhibitio n 0f leaf %inhibitio n 0f flo wer %inhibitio n 0f metfo rmin

%inhibitio n o f leaf %inhibitio n o f flo wer %inhibitio n o f metfo rmin

100

90
90
80
80 %inhibition
70
70
60
60
50

50 40
%inhibition

40 30

20
30
10
20
0
10 10 12.5 16 25
Concentration (mg/mL)
0
10 12.5 16 80 Figure 5: α-Glucosidase inhibitory activity
Concentration (mg/mL)
reduced. The α-amylase inhibitory action of Catharanthus
Figure 2: α-Amylase inhibitory activity (pancreas)
roseus might play a role in diabetic treatment.
%inhibition of leaf %inhibition of flower %inhibition of metformin α-glucosidase inhibitory activity
100 The in-vitro α-glucosidase inhibitory activity of ethanolic
90 extract of leaf and flower of Catharanthus roseus was tested.
80 Varying concentration of the extract 10, 12.5, 16 and 25mg/
70 mL were taken and assayed for the α-glucosidase inhibitory
%inhibition

60 activity.
50 The leaf extract showed a significant α-glucosidase inhibitory
40 activity of 53% at a concentration of 12.5mg/mL. For a similar
30 concentration, the flower extract also showed significant
20
inhibitory activity of 59% in comparison with the commercial
10
drug metformin which showed about 73% inhibition for similar
0
concentration (Fig. 5).
10 12.5 16
Concentration (mg/mL) From the result, it is evident that ethanolic extract of leaf and
Figure 3: α-Amylase inhibitory activity (Serum) flower of Catharanthus roseus has significant á-glucosidase
inhibitory activity comparable to the commercial drug
et al., 2001). Flavonoids and anthocyacin are also reported to Metformin.
have inhibitory activity against α-amylase (Matsui et al., 2001). α-glucosidase is one of the numbers of glucosidases located
Metformin is one such commercial drug which exerts glycemic in the brush border surface membrane of intestinal cells and
control by its α-amylase inhibitory activity. Thus metformin is a key enzyme of carbohydrate metabolism (Caspary, 1978).
improves glucose tolerance in patients with type 2 diabetes. α-glucosidase inhibitory activity block the actions of á-
The most common side effect of Metformin are upper glucosidase enzyme in the small intestine which is the rate
respiratory tract infection, diarrhea, edema and headache. By limiting step in the conversion of Oligosaccharide and
the α-amylase inhibitory activity the rate of digestion of disaccharide to monosaccharide, necessary for gastro
carbohydrate and the consequent absorption of glucose in intestinal absorption.

658
 ANTI DIABETIC ACTIVITY OF CATHARANTHUS ROSEUS

From the results, it can be concluded that ethanolic extract of inhibits post prandial hyper glycemic in obese zucker rats. Life Scis.
leaf and flower of Canthranthus roseus can be excellent choice 75: 1735-1746.
of drug with α-glucosidase inhibitory activity and can thus Matsui, T., Ueda, T., Oki, T., Sugita, K., Terahara, N. and Matsumoto,
reduce the rate of digestion and absorption of carbohydrates. K., 2001. Glucosidase inhibitory action of natural acylated
anthocyanins.1. survey of natural of pigments with potent inhibitory
In summary, this work shows that the alcoholic extract of leaf activity. J. Agri. Foof Chem. 49: 1948-1951.
and flower of Catharanthus roseus has adequate antidiabetic
Mohd. Nawagish, Ansar, S. H. and Ahmad, S.2007. Preliminary
potential by its ability to inhibit α-amylase and α-glucosidase.
Pharmacognostical Standardisation of Lawsonia inermis Linn. Seeds.
When compared to the flower extract the leaf extract was found Research J. Botany. 2(3): 161-164.
to significantly inhibit α-amylases. While, the α-glucosidase
Notkins, A. L. 2002. Immunolgiz and genetic factors in typical
inhibitory activity of the leaf and flower extract was almost the Diabetes. J. Biochem. 227(46): 43545-43548.
same. Our result provide a more detailed view of the anti
Nooman, A., Khalaf, Ashok, K., Shakya, Atif Al-Othman., Zaha El-
diabetic properties of canthranthus roseus and it is first of its
Agbar. and Husni Farah. 2008. Anttioxidant activity of some common
kind to reveal the α-amylase and α-glucosidase inhibitory plants. Turk. J. Biol. 32: 51-55.
activity. The significant α-amylase and α-glucosidase inhibitory
Ogbonnia, S., Adekunle, A. A., Bosa, M. K. and Envuru, V. N. 2008.
activity of the leaf and flower extract of Catharanthus roseus Evaluation of acute and subacute toxicity of Alstonia congensis Engler
together with its potent antioxidant potential can make it the (Apocynaceae) bark and Xylopia aethiopica (Dunal)A. Rich
future safe drug of choice in diabetes treatment. Future studies (Annonaceae) fruits mixtures used in the treatment of diabetes. African
will address the molecular mechanisms by which the plant J. Biotechnology. 7(6): 701-705.
and its active compounds regulate glucose homeostasis. Punitha, I. S. R., Rajendaran, K., Shrwaikar, A. and Shrwaikar, A.2005.
Alocholic stem extracts of V.roseus regulates Carbohydrate
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