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ISSN NO: 2231-6876

INDO AMERICAN
JOURNAL OF
PHARMACEUTICAL
RESEARCH

PHYTOCHEMICAL SCREENING, GC-MS ANALYSIS AND ENZYME INHIBITORY


ACTIVITY OF PASSIFLORA FOETIDA L.
Joseph Asir Paulraj, Hemmalakshmi Subharamanian, Priyanga Suriyamoorthy and Devaki
Kanakasabapathi *
Department of Biochemistry, Karpagam University, Coimbatore-641 021, India.
ARTICLE INFO
Article history
Received 16/07/2014
Available online
30/08/2014

Keywords
Passiflora Foetida L.,
Phytochemical, GCMS Analysis, Alpha
Amylase And Alpha
Glucosidase
Inhibitory Activity.

ABSTRACT
Within the medicine system medicinal plants and their derivatives play a cruciall role
to conserve our health. The present study was aimed to identify the potential bioactive
compounds from parts of Passiflora foetida L. through GC-MS analysis. The plant was also
analysed for -amylase and -glucosidase inhibitory activity. The results of phytochemical
screening revealed the presence of
alkaloids, flavanoids, tannins, phenols, steroids,
cardioglycosides, saponins and terpenoids werepresent in almost all the parts of P. foetida
L. In the GC-MS analysis, 27 bioactive compounds were identified in the seed ethanolic
extract of Passiflora foetida L. Maximum -amylase inhibitory activity was recorded in 100
g/ml of aqueous and ethanolic extract of root with an inhibition of 80.3% and 83.3%
respectively. In -glucosidase inhibitory activity, the aqueous extract of seed of 72.3%) P.
foetida showed a maximum inhibition of 72.3% followed by ethanolic extract of root with an
inhibition of 65.7%.This study provide an evidence that the bioactive comp[ounds present in
P. foetida exert -amylase and -glucosidase inhibitory activity and also authenticate its use
in the management of diabetes.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Joseph Asir Paulraj et al. Phytochemical Screening, Gc-Ms Analysis and Enzyme Inhibitory
Activity of Passiflora Foetida L. Indo American Journal of Pharm Research.2014:4(08).

352

Corresponding author
Dr.K.Devaki
Assistant Professor in Biochemistry
Karpagam University
Coimbatore
Tamil Nadu, India
dr.devaki.bc@gmail.com
091-0422-6453777, 6471113-5
91-0422-2980022

Vol 4, Issue 08, 2014.

Joseph Asir Paulraj et. al.

ISSN NO: 2231-6876

INTRODUCTION
Diabetes mellitus is a chromic metabolic disorder associated with long term damages, dysfunctions, failure of organs
[1]
especially the eyes, kidneys, nerves and cardiovascular system . Medicinal plants have played a significant role in various ancient
traditional systems of medicine. They are rich sources of bioactive compounds and thus serve as an important raw material for drug
[2]
production and have become a target for the search of new drugs . Plants secondary metabolites have been implicated for most of
the
[3 ]
detection,
fire investigation,
environmental
analysis,different
explosives
investigation,
ofcombines
unknown
samples.
GC-MS
can
chromatography
and
mass spectrometry
to identify
substances
withinand
a test
Applications
ofthe
GCMS
drug
plants
therapeutic
activities
.Gas chromatographymass
spectrometry
(GCMS)
isidentification
a sample.
method that
featuresinclude
of gasliquid
also be used to detect substances in luggage by airport security and also to identify the abused drugs in human beings. Even though
GC-MS supports for the identification of phytoconstituents in medicinal plants, there are only very few reports are available regarding
this analysis. At this juncture, analysis of Passiflora foetida phytoconstituents with the help of GC-MS provides the finger print of
[4]
secondary metabolites present in this plant .Pancreatic alpha-amylase (PPA) inhibitors offer an effective strategy to lower the levels
[5]
of post-prandial hyperglycemia via control of starch breakdown . Passiflora is the largest genus in the Passifloraceae family and
comprises nearly 500 species. The mostly available wild species are P. edulis, P. incarnata, P. leschenaultia, P. mollissima and P.
subpelta. Passiflora foetida L. (Stinking passion flower) is South American origin, which has been spread to many tropical areas in
[6]
India. It is found in riverbeds, dry forest floors, covering the top thorny shrubs and also growing near hamlets . Many species of
Passiflora have been used in therapeutic practice, but scanty reports are available on Passiflora foetida, which has been used
[7]
in treatment of some disease like anxiety, insomnia, convulsion, sexual dysfunction, cough and cancer . The present study
was carried out with the aim of finding the best source of phytoconstituents among the various parts of P. foetida, with respective to
various extraction procedures using solvents of increasing order of polarity. Further they were analysed for -amylase and glucosidase inhibitory activity. The ethanolic extract of seed was also subjected to GC-MS analysis with a view to find the
phytoconstituents.
MATERIALS AND METHODS
Collection and identification of plant material:
The leaves, root, fruit peel (both ripened & un ripened) & seed of Passiflora foetida L. were collected from Coimbatore
district, Tamilnadu and authenticated by Dr. M. Palanisamy, Botanical Survey of India, Tamilnadu Agricultural University Campus,
Coimbatore. The Voucher No is BSI/SRC/5/23/2012-13/Tech-108. All the parts of plant were washed well with water. They were air
0
dried at 25 C for 10 days in the absence of sunlight and powdered coarsely using a mixer. They were then weighed and kept in an
airtight container and stored in refrigerator for future use.
Sample extractions:
About 50g of powdered plant materials were extracted sequentially with 250ml of different solvents (Petroleum ether,
chloroform, Ethyl acetate, Ethanol and distilled water) by using a separating funnel with occasional shaking for 24 hours. The extract
was concentrated by Rotary flask evaporator. Each time before extracting with the next solvent the residue was dried thorough ly to
remove the solvent used. After extraction the samples were collected and stored in a vial for further studies.
Qualitative estimation of phytoconstituents
Phytochemical screening was carried out to assess the qualitative chemical composition of crude extracts and to identify the
major natural chemical compounds such as steroids, reducing sugars, alkaloids, phenolic compounds, saponins, tannins, flavono ids,
[8, 9]
amino acids, terpenoids and cardioglycosides using commonly employed precipitation and coloration
. General reactions in these
analyses revealed the presence or absence of these compounds in the crude extract.

Page

In vitro inhibitory assay for the -glucosidase activity


[10]
The -glucosidase inhibitory assay was done by the method of Adisakwattana et al.,
with slight modifications. The
substrate (maltose 37mM or sucrose 37mM) and the test compounds (P. foetida L. or acarbose) was dissolved in a 0.1M phosphate
buffer solution (pH 7.0). A crude enzyme solution 20 l and the test compounds 40 l was pre-incubated simultaneously for 10 min.
o
After the pre-incubation period, the substrate 140 l was added and incubated at 37 C for 30 and 60 min, for maltose and sucrose,
respectively. The assay tubes was immediately immersed in boiling water for 10 min. to stop the reaction. Glucose concentration was
determined by glucose oxidase test. The results were expressed as % inhibition calculated using the formula:

352

Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of Passiflora foetida L.


:
GC-MS analysis of the seed ethanolic extract of P. foetida L. was performed using the equipment Thermo GC-Trace Ultra
Version: 5.0, Thermo MS DSQ II. The equipment has a DB 35 MS Capillary Standard Non-polar column with dimensions of 30 mts
x
0.25 mm ID x0.25m film. The carrier gas used is Helium with flow of 1.0 ml/minute.
The injector was operated at 250C and the oven temperature was programmed as follows; 60C for 15minutes, then
o
gradually increased to 280 C for 3 minutes. The identification of components were based on comparison of their mass spectra
with those of Wiley and NBS libraries as well as comparison of their retention indices.

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Vol 4, Issue 08, 2014.

Joseph Asir Paulraj et. al.

ISSN NO: 2231-6876

Abs (control)-Abs (extract)


Inhibition activity (%) = --------------------------------------------*100
Abs (control)
The IC50 values (inhibitor concentration at which 50% inhibition of the enzyme activity occurs) of the P. foetida L. extracts were
determined from plots of percent inhibition vs inhibitor concentration.
In vitro inhibitory assay for the -amylase activity:
[11]
The -amylase inhibitory activity was determined according to standard procedure
. Briefly, the total assay mixture
containing 200 l of 0.02M sodium phosphate buffer, 20 l of enzyme, and the plant extrac ts in the concentration range 10100g/ml were incubated for 10 min at room temperature followed by addition of 200 l of 1% starch in all the test tubes. The
reaction was terminated with addition of 400 l of 3, 5 dintrosalycylic acid (DNSA) color reagent, then placed in boiling water bath
for 5 minutes, cooled at room temperature and diluted with 15 ml of distilled water and the absorbance measured at 540nm. The
control sample s
were also prepared accordingly without any plant extracts and were compared with the test samples containing various concentrations
of the plant extracts prepared with DMSO. The results were expressed as % inhibition calculated using the formula:
Abs (control)-Abs (extract)
Inhibition activity (%) = --------------------------------------------- x 100
Abs (control)
The IC50 values (inhibitor concentration at which 50% inhibition of the enzyme activity occurs) of the P. foetida L. extracts
were determined from plots of percent inhibition vs inhibitor concentration.
Statistical analysis:
Results are expressed as the Mean SD of three individual experiments.
RESULT AND DISCUSSION
Phytochemical screening of Passiflora foetida L.
The Phytochemical screening of the plant extract of fruit peel (both ripen and un-ripen), leaf, seed and root were carried and
the results are given in the table 1- a, b, c, d & e.
Phytochemicals play an important role in plant defence against prey, microorganism, stress as well as interspecies
protections and these plant components have been used as drugs for millennia. Hence, phytochemical screening serves as the initial
[12]
step in predicting the types of potential active compounds from plants . Preliminary phytochemical screening of the present study
revealed the presence of alkaloids, flavanoids, tannins, phenols, steroids, cardioglycosides, carbohydrate, oils and fats, saponins and
terpenoids which were mostly present in all the plant parts of various extract of P. foetida L. Based on the preliminary
phytochemical analysis ethanolic and aqueous extract of P. foetida L. parts were showed the presence of five important bioactive
compounds, namely phenol, flavanoid, steroids, glycosides and saponins. These compounds were reported to have
hypocholesterolemic and antidiabetic prop erties [13]
.
Table 1: Phytochemical screening of various parts of Passiflora foetida L. with different solvents
Table 1(a): Un ripen peel.
Extracts
Petroleum ether
Chloroform
Ethyl acetate
Ethanol
Aqueous

AL
+
+
+
+

SA
+
+
+
++
++

TP
+
+
+
++
++

FL
+
+
+
++
++

ST
+
+
+
++
++

CG
+
+
+
++
++

OF
+
+
+
+
+

TN
+
+
-

AP
+
+
+
+
++

CHO
+
+
+
++
++

OF
+
+
+
+
+

TN
+
+
-

AP
+
+
+
+
+

CHO
+
+
+
+
++

AL
+
+
+
+

SA
+
+
+
+
++

TP
+
+
+
+
++

FL
+
+
+
++
++

ST
+
+
+
++
++

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CG
+
+
+
++
++

Page

Extracts
Petroleum ether
Chloroform
Ethyl acetate
Ethanol
Aqueous

352

Table 1(b): Fully ripen peel.

Vol 4, Issue 08, 2014.

Joseph Asir Paulraj et. al.

ISSN NO: 2231-6876

Table 1(c): Leaf.


Extracts
Petroleum ether
Chloroform
Ethyl acetate
Ethanol
Aqueous

AL
+
+
+
++
+

Seed.CG
SA Table
TP 1FL(d):ST
+
+
+
+
+
1+ (e): +Root.+
+ Table
+
+
+
+
+
+
+
+
++ ++ ++
++ ++ ++ ++ ++

OF
+
+
+
+
+

TN
+
+
-

AP
+
+
+
+
+

CHO
+
++
+
+
++

Extracts
Petroleum ether
Chloroform
Ethyl acetate
Ethanol
Aqueous

AL
+
+
+
+

SA
+
+
+
++
+

TP
+
+
+
+
++

FL
+
+
+
++
++

ST
+
++
+
++
+

CG
+
++
+
++
+

OF
+
+
+
+
+

TN
+
+
-

AP
+
+
+
+
+

CHO
+
+
+
++
+

Extracts
Petroleum ether
Chloroform
Ethyl acetate
Ethanol
Aqueous

AL
+
+
+
++
+

SA
++
+
+
++
+

TP
+
+
+
+
++

FL
+
+
+
++
++

ST
+
+
+
++
++

CG
+
+
+
+
++

OF
+
+
+
+
+

TN
+
+
-

AP
+
+
+
+
+

CHO
+
+
+
++
++

AL Alkaloids
CH Carbohydrates
ST Steroids
CG Cardioglycosides
FL Flavanoids
SA Saponin
TP Tannin & Phenolic compounds
OF Oils & Fats
AP Amino acids & Proteins
TN Terpenoids
+ Present
- Absent
Percentage of yield:
The percentage yields of P. foetida L. for various solvents are given in the table 2. Among the extracts, aqueous and
ethanolic extract of P. foetida L. fruit peel (both ripen & un ripen), leaf, seed and root has the highest yield whereas, the ethyl acetate
has the lowest yield when compared to other solvents like petroleum ether and chloroform.

% of yield in various solvents


Petroleum ether Choloroform

Ethyl acetate

Ethanol

Aqueous

UR peel
FR peel
Leaf
Seed
Root

0.78
1.38
2.62
1.40
0.88

0.52
0.74
1.24
0.94
0.58

6.96
5.08
3.18
5.70
3.40

9.72
9.74
5.08
9.30
2.66

0.70
1.02
1.82
2.00
0.86

Gas chromatography-mass spectroscopy (GC-MS) analysis of Passiflora foetida


L.:
In the GC-MS analysis, 27 bioactive compounds were identified in the ethanolic extract of Passiflora foetida L. seed (Table 3
& Fig 1). The identification of compounds is based on the peak area, molecular weight and molecular formula. The GC -MS spectrum
shows the presence of more long chain hydrocarbons. When the number of carbon atoms increases in the molecule, hydrophilicity is

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P. foetida L. parts

352

Table 2: Percentage yield of P. foetida L. in various extracts.

Vol 4, Issue 08, 2014.


Joseph Asir Paulraj et. al.
ISSN NO: 2231-6876
reduced and the lipophilicity is increased. Increased lipophilicity of a drug decreases its transport across intestinal epithelial cells
[14]
.In

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the present study, the GC-MS analysis of ethanolic extract of Passiflora foetida L. seed revealed the presence of Dodecanoic acid,
Tetradecanoic acid, n- Hexadecanoic acid, 9,12, Octadecanoic acid (Z, Z) , Oleic acid, Stearic acid, palmitic acid and Linolenic acid.
Among the identified compounds, Dodecanoic acid, Tetradecanoic acid and n- Hexadecanoic acid has the property of antioxidant and
antimicrobial activities. n- Hexadecanoic acid has the property of larvicidal effect and 9,12, Octadecadienoic acid (Z, Z) has the
[15]
property of anti- inflammatory and antiarthritic activity
. P. foetida L. seed showed 61.1 to 74.8% of total linolenic acid and
[16]
linoleic acids which resembles safflower seed . Mono saturated fatty acids like oleic acid & stearic acid has the ability to lower the
[17]
blood glucose level when its consumed as food
. So the analysis indicates that seed of P. foetida L. contains fatty acids which
have beneficial functions.
Table 3: Phytocompounds identified in the ethanolic extract of Passiflora foetida L. seed by GC-analysis.
Name of the compound

Molecular formula

Molecular
Weight (amu)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

2.743
3.838
4.536
6.411
8.246
10.163
12.409
17.033
17.822
18.482
20.904
21.041
22.572
23.012
26.442
27.366
27.722
27.988
28.207
28.738
30.129

C15H12N4O6
299.091
C6H5CH=CH2
104.063
Si(OC2H5)4
208.113
CH3(CH2)9CH=CH2 168.188
C18H36
252.282
C16H32
224.25
C16H30O
238.23
C14H24O3
240.173
C16H32O2
256.24
C18H36O2
284.272
C19H34O2
294.256
C19H36O2
296.272
C20H38O2
310.287
C20H40O2
312.303
C18H32O2
280.24
C12H24O
184.183
C24H40Osi
372.285
C17H36
240.282
C19H38O4
330.277
C14H26O
210.198
C13H16N2O5
280.106

0.075
0.315
0.083
0.083
0.153
0.11
0.137
0.514
0.917
0.312
0.138
0.101
0.119
0.174
0.183
0.091
0.128
0.128
0.192
0.156
0.21

22
23
24
25
26
27

30.459
30.99
31.905
33.743
34.95
35.096

Benzhydrazide, N2-(2-methoxy-5-nitrobenzylideno)Styrene
Tetraethyl orthosilicate
1-Dodecene
9-Octadecene, (E)7-Hexadecene, (Z)cis-11-Hexadecenal
Oxacyclotetradecane-2,11-dione, 13-methyln-Hexadecanoic acid
Palmitic acid
Linolelaidic acid,
11-Octadecenoic acid, methyl ester, (Z)Ethyl Oleate
Ethyl Stearate
9, 12- Linoleic acid
methyl nonyl acetaldehyde
Pregna-3,5-dien-20.alpha.-ol, O-trimethylsilyl
Heptadecane
Palmitic acid -monoglyceride
9,12-Tetradecadien-1-ol, (Z,E)4-(3,4,5,6-Tetrahydroxy-2-oxo-hexylamino)benzonitrile
9,17-Octadecadienal, (Z)15-Hydroxypentadecanoic acid
E-11-Hexadecenoic acid, ethyl ester
(R)-(-)-14-Methyl-8-hexadecyn-1-ol
gamma.-Tocopherol (Vit E)
Acetic
acid,
1-methyl-3-(2,2,6-trimethylbicyclo[4.1.0]hept-1-yl)-propenyl ester

C18H32O
C15H30O3
C18H34O2
C17H32O
C28H48O2
C16H26O2

0.201
0.137
0.137
0.192
0.128
0.174

264.245
258.219
282.256
252.245
416.365
250.193

Peak Width
50% (min)

353

RT
(min)

Page

No

Figure 1: GC-MS Chromatogram of ethanolic extract of Passiflora foetida. L seed

Values are expressed as MeanSD of three individual experiments

Figure 2(a) -glucosidase inhibitory activity of ethanolic extract of Passiflora foetida L

Page

353

-glucosidase inhibitory activity of ethanolic & aqueous extract of Passiflora foetida


L.:
Diabetes mellitus (DM) is a common endocrine system disease that causes metabolic disorders and which leads to multiple
organ damage syndrome. Inhibition of -glucosidase (EC 3.2.1.20) and -amylase (EC 3.2.1.1) enzymes are involved in the digestion
of carbohydrates, can significantly decrease the postprandial increase of blood glucose after a mixed carbohydrate diet and therefore
can be an important strategy in the management of postprandial blood glucose level in type 2 diabetic patients and borderline
[18]
patients . Alpha-glucosidase inhibitors inhibit maltase and sucrase in intestine, consequently delaying the absorption of sugar from
[19]
the gut and hence decrease the postprandial hyperglycemia
. Phenols and flavonoids inhibit amylase, sucrase and also Sodium
Glucose Transporter- 1 (SGLUT- 1) of intestinal brush border cells which reduce the absorption of glucose that ultimately reduce the
[13]
hyperglycemia .
The present study demonstrated that ethanolic & aqueous extract of P. foetida L. have - glucosidase inhibitory activity. The
percent inhibition at 100, 80, 60, 40 and 20 g/ml concentrations of P. foetida L. extract showed a concentration-dependent increase in
percent inhibition (Fig 2a,2b). The highest concentration, 100 g/ml showed a maximum inhibitory activity in ethanolic ext ract of
root (65.7%) and aqueous extract of seed (72.3%). Intestinal -glucosidase is a glucosidase acting as a key enzyme for carbohydrate
digestion, located at the epithelium of the small intestine. - glucosidase has been recognized as a therapeutic target for the
[20]
modulation of postprandial hyperglycemia, which is the earliest metabolic abnormality that occurs in Type 2 diabetes mellitus .

Values are expressed as MeanSD of three individual experiments

Figure 2(b): -glucosidase inhibitory activity of aqueous extract of Passiflora foetida L:

Values are expressed as MeanSD of three individual experiments

Figure 3(a): -amylase inhibitory activity of ethanolic extract of Passiflora foetida L:

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353

Alpha amylase inhibitory activity of ethanolic & aqueous extract of Passiflora foetida L.:
Several -amylase inhibitors including acarbose, voglibose and miglitol are clinically used in the management of diabetes,
but their prices are high and clinical side effects also occur. Natural products are still the most available source of -amylase
inhibitors. Therefore, screening of alpha-amylase inhibitor in medicinal plants has received much attention. These medications are
most useful for people who have just been diagnosed with type 2 diabetes and who have blood glucose levels only slightly above the
[21]
level considered serious for diabetes
.
The in vitro - amylase inhibitory studies demonstrated that ethanolic & aqueous extract of P. foetida L. has - amylase
inhibitory activity. The percentage inhibition of P. foetida L. extract showed a concentration-dependent increse in percentage
inhibition (Fig 3a, 3b). In this study the highest concentration, 100 g/ml of ethanolic extract of root (80.3%) and aqueous extract of
root (83.3%) showed maximum inhibitory activity.

Values are expressed as MeanSD of three individual experiments

Figure 3 (b): -amylase inhibitory activity of aqueous extract of Passiflora foetida L:


CONCLUSION
The aqueous and ethanolic extract of Passiflora foetida L. showed more number of secondary metabolites such as steroids,
flavonoids, tannins and phenol, cardioglycosides, saponins, oils and terpenoids. In the GC-MS analysis, 27 bioactive compounds were
identified in the seed ethanolic extract of Passiflora foetida L. and some of them are reported to decrease the blood glucose level
when consumed. The reduction in post prandial hyperglycemia is evidenced by the in vitro -glucosidase and -amylase inhibitory
studies of P. foetida L and this supports its usage in the management of Type 2 diabetes.
ACKNOWLEDGMENT
We are thankful to Chancellor, Advisor, Vice Chancellor and Registrar of Karpagam University for providing faci lities and
encouragement.

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CONFLICT OF INTEREST DECLARATION


We declare that we have no conflict of interest.

Vol 4, Issue 08, 2014.

Joseph Asir Paulraj et. al.

ISSN NO: 2231-6876

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