Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 56-61

INDO GLOBAL JOURNAL OF

PHARMACEUTICAL SCIENCES
ISSN 2249- 1023

A Comparative Study between Aqueous and Ethanolic Extracts of

Allium Odorum Linn with Reference to its Antioxidant and Alpha-
Amylase Inhibition Activities
Nayan Talukdar 1*, Rujita Devi 1, Indrani Barman 2
1 Programme of Biotechnology, Faculty of Science, Assam down town University, Guwahati, Assam-781026, India
2 Programme of Biochemistry, Faculty of Science, Assam down town University, Guwahati, Assam-781026, India

Address for Correspondence: Nayan Talukdar, Nayan.new16@gmail.com

Received:
13.04.2020 ABSTRACT: Allium odorum Linn belongs to the family Liliaceae which is established as a potent
Accepted: medicinal plant in folkware medicinal system. The plant is native to China as well as Japan and also cultivated
26.11.2020
Published: in North-east part of India as an essential part of culinary. In this study, leaves of this plant were collected
30.03.2021 from Imphal East district of Manipur and extracted in water and ethanol. The extracts were screened for the
Keywords presence of various bioactive compounds and its inhibition activity against α-amylase. Both aqueous and
Allium odorum, ethanol extracts showed presence of bioactive compounds like sterol, tannin, saponin, di-terpines and sugars
phytochemical whereas bioactive compounds like flavonoid, glycoside, and amino acids were present only in ethanol extracts.
screening, α-
Major bioactive compound like alkaloid test showed negative result during the study. The sample was also
amylase
inhibition found to exhibit a maximum of 89.27% and 69.79% of α-amylase inhibition activity in ethanol and water
activity, extract respectively. When sample was subjected to its antioxidant activity it exhibited 84.85% and 65.88% by
antioxidant DPPH method and a maximum of 67.73% and 57.97% by H2O2 method in ethanol and water extract
activity. respectively. Hence, from this study the sample can be used as a potential source for anti-oxidant and anti-
diabetic agent which will help us to substitute some commercially available synthetic drugs near future. ©
2020 iGlobal Research and Publishing Foundation. All rights reserved.

Cite this article as: Talukdar, N.; Devi, R.; Barman, I. A Comparative Study between Aqueous and Ethanolic
Extracts of Allium Odorum Linn with Reference to its Antioxidant and Alpha-Amylase Inhibition Activities.
Indo Global J. Pharm. Sci., 2021; 11(1): 56-61. DOI: http://doi.org/10.35652/IGJPS.2021.111008 .
the plant raw material [3-5]. Plant mainly uses alpha amylase
INTRODUCTION inhibition property to protect themselves from insect. They
Medicinal plants play an important role in human health. follow the mechanism of changing digestive property of alpha
Almost 80% of the world population fully depends on amylases and proteinases in the gut of insects resulting in
medicinal plants for meeting their health care needs [1]. abnormal feeding behaviour [3,6]. Diabetes is a clinical
Bioactive compounds extracted from medicinal plants contain condition recognised by high level of glucose accumulated in
many organic compounds which provide definite the blood as cell does not respond to the insulin that is
physiological action in our body. For an example, phenols and secreted by pancreas. Compounds like metformin, gliclazides,
flavonoids isolated from medicinal plants are reported to have glitazones/vitagliptin/saxagliptin etc are predominantly
positive impact on health and cancer prevention supplied to patient to control the blood glucose level. Hence,
[2].Antioxidants are the chemicals that can prevent or slower plants with alpha amylase inhibition property are always very
cell damage. They have important preventive roles in crucial as there are always needs for new source of
changing the flavour and nutritional quality of food. They also antidiabetic molecule [7-8]. Allium species belongs to the
prevent illness such as different types of cancers, family Liliaceae. It is the plant which is the native of China
cardiovascular and neurological diseases etc. Polyphenols are and Japan. Many natural products have been produced from
the most significant compounds for antioxidant properties of this genus as they are used to cure tumor, cardiovascular

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 Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 56-61
disease, thrombosis, cholesterolmia and hyperglycemia [9-12]. b. Sulphuric acid test- A fraction of extract was treated with
Allium odorum is a small annual plant with leaves that are concentrated sulphuric acid. Appearance of orange colour
small, green, grass-like, narrowly linear, and flattish. Seeds are indicates the presence of flavonoid.
black, depressed, globose or uniform. Fruits are capsule that
open longitudinally along the capsule wall between the c. Lead acetate test- A small amount of extract was treated
partitions of the locule [11,13]. with lead acetate.

MATERIALS AND METHODS Test for saponin

Sample collection and sample preparation A few mg of the test residue was diluted with distilled water in
Leaves of A. odorum were collected from Imphal East District a test tube and shaken vigorously and heated in a water bath
of Manipur in the month of March 2017 and was identified for 5 min. Development of frothing indicates the presence of
based on its vernacular name. The leaves were rinsed with tap saponin.
water followed by distilled water and dried in the shade. The
dried samples were grinded into the fine powder with the help
Test for glycoside
of a grinder and kept in an air tight container for further use.
Anthraquinone glycoside (Borntrager`s test )- The extract
solution is boiled with dilute sulphuric acid, filtered and to the
Sample preparation
filtrate chloroform was added and shaken well. The organic
An amount of 200 grams of dried powder was placed in
layer is separated to which ammonia is added.
Soxhlet apparatus for extraction using ethanol as solvent. The
solvent was then evaporated and crude extract were collected
Development of red colour of the ammonical layer indicates
for further use [3]. In another setup, an amount of 200 grams
the presence of glycoside.
of fine powder f the sample were mixed in cold water an kept
in deep freezer for 72hr and centrifuge at 3000rpm. Solvent
Cardiac glycoside (Keller-Killiani test)- Extract (0.5g) was
free crude were collected for further investigation [14].
shaken with distilled water (5mL). To this glacial acetic acid
(2mL) containing a few drops of ferric chloride was added
Screening for Phytochemicals [15-20]
followed by sulphuric acid (1mL) along the side of the test
The crude extract was screened for the presence of different
tube. Formation of ring at the interface gives positive result.
bioactive compounds. 20mg/mL solvent concentration of the
crude extract was prepared and further used for the tests
Test for diterpenes
Extract was dissolved in water and treated with 3-4 drops of
Test for sterol
copper acetate solution.
Salkowski test- The crude extract (100mg) was shaken with
chloroform (2mL) followed by addition of concentrated
Appearance of ceramal green colour indicates the presence of
sulphuric acid (2mL) along the side of the test tube. Reddish
diterpenes.
brown colour development indicates the presence of sterol.
Test for protein
Test for alkaloid
a. Biuret test- A few mg of the residue was taken in water and
Few mg about (15mg) of extract was stirred with1% HCl
1mL of 4% copper sulphate was added to it. Violet ring
(6mL) on water bath for 5min and filtered.
development indicates the presence of protein.
a. Mayer`s test- To a portion of filtrate, Mayer`s reagent
b. Xanthoproteic test- A little residue was taken with 2mL of
(potassium mercuric iodide solution) ( 1mL ) was added.
water and 0.5mL of concentrated nitric acid was added to it.
Cream colour precipitation indicates the presence of alkaloid.
Yellow colour development indicates the presence of protein.
b. Wagner`s test- Potassium iodide ( 2g ) and iodine ( 1.27g )
were dissolved in distilled water ( 5mL ) and the solution was Test for amino acid
diluted to 100 mL distilled water. Few drops of this solution Ninhydrin test- The ninhydrin reagent is 0.1% w/v solution
were added to the filtrate. Brown colour precipitation indicates of ninhydrin in n-butanol. A little of this reagent was added to
the presence of alkaloid. the test extract. Development of violet colour indicates the
presence of amino acid.
Test for tannins
The extract was stirred with distilled water (10mL) and filter. Test for sugar
A few drops of 5% ferric chloride were then added. Green a. Molisch test- A few mg of the test extract was placed in the
colour development indicates the presence of tannins. test tube containing .5mL of water and it was mixed with 2mL
of concentrated sulphuric acid added from the side of the
Test for flavonoid
inclined test tube so that the acid formed a layer beneath the
a. Alkaline reagent test- The crude extract was treated with
aqueous solution without mixing. Development of violet ring
few drops of sodium hydroxide solution. Formation of intense
indicates the presence of sugar.
yellow, which become colourless on addition dilute acid
indicates the presence of flavonoids.
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 Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 56-61
b. Barfoed test- The reagent was prepared by dissolving Absorbance was then measured at 765nm. Results were
13.3g of crystalline neutral copper acetate in 200mL of 1% expressed as mg gallic acid equivalents (GAE)/g sample.
acetic acid solution The test residue was dissolved in water
and heated with a little of the reagent. Green colour Statistical analysis
development indicates the presence of sugar. Bioactive activity namely antioxidant and α-amylase
inhibition activity for both the extracts were expressed as
Test for phenol mean ± standard deviation (SD) of three replicates with the
Extract was treated with 3-4 drops of 10% ferric chloride help of software.
solution. Formation of bluish black colour indicates the
presence of phenol.
RESULT AND DISCUSSION
Alpha-amylase Inhibition Activity [21,22] Phytochemical Screening
A quantity of 0.1g of starch was dissolved in 100mL of acetate The plant medicinal value relies in the active phytochemicals
buffer (16mM) to prepare 0.1% starch solution. Similarly, that generates certain physiological impact on humans. A
27.5 mg of alpha amylase was dissolved in 100 mL of distilled study on Allium sativum showed the rich presence of
water to prepare the enzyme solution. Sodium potassium alkaloids, reducing sugar, flavonoids, glycosides, cardiac
tartarate solution and 3, 5 dinitro salicylic acid solution at 96 glycosides, tannin and phenolic compounds, saponins, amino
mM were mixed to prepare the colorimetric reagent. Different acid & triterpenoids in aqueous and methanolic extract of
concentration (200, 400, 600, 800, 1000µg/mL) of extract garlic leaves [27]. In the present study, the phytochemical
were placed in different test tubes. To each test tube starch screening of crude extract of Allium odorum also revealed the
(1mL), amylase (1mL) was added and incubated for 15mins. presence of various secondary metabolites. Water extracts
After incubation DNS (1mL) was added and heated at 100°C showed the presence of sterol, tannin, saponin, diterpenes,
and observed the colour change. The absorbance was taken at sugars, flavonoids, whereas ethanol extract showed positive
540nm against a blank. The percentage of α-amylase results for sterol, tannin, saponin, glycoside, diterpenes, amino
inhibition was calculated using acids, sugars and phenol. Alkaloid was absent in both ethanol
and water extract (Table 1). Therefore, the presence or
absence of such compounds depend largely on the extent of
accumulation, the amount of plant material used and the
Antioxidant Assay (23, 24, 25) analytical method employed
DPPH Method TABLE 1: Result of Phytochemical screening for both the
The antioxidant activity of ethanol and water extracts of the extracts.
plant extract were assayed according to the standard method SL. PHYTOCHEMICALS RESULTS
with slight modification. DPPH (2,2-diphenyl picryl hydrazyl) NO. ETHANOL WATER
is composed of stable free radical molecules and is purple in 1 Sterol +ve +ve
colour. The antioxidant molecules (present in the test sample)
2 Alkaloid -ve -ve
react with DPPH and after the incubation time, the purple
colour gets converted to yellow colour. Stock of the plant 3 Tannin +ve +ve
sample was made by dissolving 1mg of sample in 1 mL of 4 Flavonoid +ve +ve
distilled water. The absorbance was taken at 540nm against 5 Saponin +ve +ve
suitable blank using Ascorbic acid as a standard. 6 Glycoside +ve -ve
H2O2 Method 7 Diterpenes +ve +ve
A concentration of 40mM hydrogen peroxide was prepared in 8 Amino acid +ve -ve
phosphate buffer of pH 7.4. Different concentration of 9 Sugar +ve +ve
samples and distilled water were added to H202 (0.6 mL, 10 Phenol +ve - ve
40mM). The absorbance was taken at 230nm against a blank
+=present; -ve=absent
using Ascorbic acid as a standard. In both cases percentage of
scavenging activity was calculated using the following
equation: In vitro alpha amylase inhibition activity
Although there are reports of alpha amylase inhibitory activity
for six Allium species [28], very few reports are available for
A.odorum in Manipur. In our present study, both the extracts
Determination of Total Phenolic Content (25, 26) showed an increase in dose dependent concentration with
Phenolic content is determined by Folin Ciocalteu method. An inhibition activity of 89.27% at 1000 μg/mL and 69.79% at
aliquot of 0.5mL of extract (1mg/mL) was mixed with 2.5mL 1000 μg/mL for ethanol and water extracts respectively, which
Fc reagent (previously diluted with distilled water 1:10) and is quite strong as compared to the results of [28]. A positive
2mL (75%) of Na2CO3. The tubes were vortex for 15 sec and control was maintained and percentage of inhibition for both
allowed to stand for 30min at 40°C for colour development. the extracts were tabulated (Table 2). The IC50 values for

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 Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 56-61
ethanol and water extract was found to be 253.63 μg/mL and H2O2 Method
575.10 μg/mL respectively. H2O2 assay of T. zeylanicum extract exhibited a dose-
dependent hydrogen peroxide inhibition where methanolic
Antioxidant activity extract possessed good ability of scavenging with IC₅₀ values
Antioxidant activity by DPPH Method 0.122 mg/ml [30]. But our study with A.odorum showed good
The extracts showed noticeable antioxidant activity through scavenging ability with ethanolic extract. IC50 value for
DPPH methods. Antioxidant value was found to be maximum ethanol and water extract was found to be 73.08 μg/mL and
(84.85%) at 200 μg/mL which is quite effective as against the 115.95 μg/mL respectively. Ascorbic acid was used as a
inhibition of 82.2% for 80% ethanolic garlic extract studied by standard while a separate positive control was also maintained.
[29]. IC50 value for ethanol and water extract was found to be
56.12 μg/mL and 143.86 μg/mL respectively. Ascorbic acid
was used as a standard while a separate positive control was
also maintained.

TABLE 2: α-Amylase inhibition activity for both the extracts.

Ethanol Water
Conc. Of Absorbance Absorbance
Sl. IC50 value
sample IC50 value
No. % %
(Values inhibition (μg/mL) (Values inhibition
(μg/mL) represent represent (μg/mL)
mean±SD, n=3) mean±SD, n=3)
1 200 0.266± 0.57 45.15 0.517±0.56 33.54
2 400 0.201±0.55 58.55 0.473±0.48 39.20
3 600 0.136±0.55 72.16 253.63 0.368±0.50 52.69 575.10
4 800 0.094±0.51 80.61 0.305±0.52 60.79
5 1000 0.052±0.53 89.27 0.235±0.52 69.79

TABLE 3: Antioxidant activity by DPPH method.

Ethanol Water
Conc. Of Absorbance Absorbance
Sl. IC50 value IC50 value
sample % %
No. (Values (Values
(μg/mL) inhibition (μg/mL) inhibition (μg/mL)
represent represent
mean±SD, mean±SD,
n=3) n=3)
1 50 0.211±0.80 49.27 0.324±0.72 24.29
2 100 0.154±0.86 62.98 56.12 0.267±0.76 37.61 143.86
3 150 0.100±0.93 75.96 0.207±0.77 51.63
4 200 0.063±0.87 84.85 0.146±0.84 65.88

TABLE 4: Antioxidant activity by H2O2 Method.

Ethanol Water
Conc. Of Absorbance Absorbance
IC50 value IC50 value
Sl. sample
No. (Values %
(Values % inhibition (μg/mL) (μg/mL)
(μg/mL) represent represent inhibition
mean±SD, n=3) mean±SD,
n=3)
1 50 0.343±0.13 47.55 0.533±0.18 40.11
2 100 0.303±0.18 53.66 0.427±0.23 52.02
3 150 0.254±0.14 61.16 73.08 0.410±0.20 53.93 115.95
4 200 0.211±0.16 67.73 0.375±0.24 57.97

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 Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 56-61
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