doi : 10.5958/0975-6892.2018.00040.

0
Medicinal Plants
Vol. 10 (3), September 2018, 256-259
IndianJournals.com
A product of Diva Enterprises Pvt. Ltd.

Research Article

Antioxidant and inhibition of α-glucosidase enzyme activity of extract and

their fractions of Ashitaba (Angelica keiskei)

Haryoto1*, Y. Fitriana2, D.A.R.L. Anggraini1 and K.H. Yen2

1
Faculty of Pharmacy, Universitas Muhammadiyah Surakarta, Jl. Ahmad YaniTromol Pos 1, Pabelan Surakarta 57102,
Central Java, Indonesia
2
Faculty of Health Sciences, Universitas Muhammadiyah Mataram, Nusa Tenggara Barat, Indonesia
3
Faculty of Applied Sciences, Universiti Teknologi MARA, 94300, Kota Samarahan, Sarawak, Malaysia

Received: July 19, 2018; Accepted: August 07, 2018

ABSTRACT

Diabetes Mellitus (DM) is a metabolic disorder characterized by hyperglycemic condition. Previous study was suggested that
Ashitaba (Angelica keiskei) extract had the antidiabetic effect on diabetic rats. The active compound was identified as 4 –
hydroxyderricin. This research aimed to determine the antioxidant activity and inhibition potency on α-glucosidase enzymes
of ethanol extract of ashoitaba and their fractions. Ashitaba (A. keiskei) herbs were collected from the local farmers and
extracted by maceration. Fractination was performed by vaccum liquid chromatography using various solvents, while its
mobile phase was hexane-chloroform (6:4; 5:5), hexane-ethyl acetate (9.5:0.5; 9:1; 8:2), and ethanol. The result showed that
the extract, non polar, semi polar and polar fractions had an inhibition activity on α-glucosidase enzyme with IC50 values of
28.63, 18.45, 42.75 and 64.74 µg/mL, respectively. Based on the kinetics study, the activity of the samples showed a mixed
type of inhibition. The antioxidant activity of extract was equal with vitamin E in which IC50 values in sequence were 19.38
± 0.99 and 19.54 ± 0.46 µg/mL. Meanwhile, IC50 values of non polar, semi polar and polar fractions were 256.01 ± 3.79, 41.93
± 0.43, and 116.7 ± 3.45 µg/mL, respectively. The semipolar fraction of Ashitaba was found with the strongest antioxidant
activity compared to other fractions.

Keywords: Ashitaba extract, fractions, DPPH, α-glucosidase

INTRODUCTION active compound belonging to the flavonoid groups. There

Angelica keiskei (Ashitaba) is an annual herb, which origin
has been reported from Japan. It belongs to the Umbelliferae
family (Monma et al., 1990). In Japan, Ashitaba has been
known as vegetable and used as a medicinal plant with the
dark green celery-like leaves. It has been cultivated in several
areas, one of which is in East Lombok precisely in Sembalun
Village, Sumbawa sub-district, Nusa Tenggara Barat,
Indonesia (Sembiring and Manoi, 2011).
Baba et al. (2009) reported that Ashitaba contained the
chalcone concentration of 0.32%. Chalcone is known as
are 20 types of chalconoids that have been identified.
Xanthoangelol (XAG) and 4-hydroxyderricin (4HD) were the
most active types of chalconoids (Akihisa et al., 2011).
Previous researches revealed that Ashitaba has an
antibacterial activity (Inamori et al., 1991), antihypertensives
(Ogawa et al., 2005), antidiabetic (Enoki et al., 2007),
anticarcinogenics (Nishimura et al., 2007), antitumor-agent
(Akihisa et al., 2011) and antidepressants (Kim et al., 2013).
Enoki et al. (2007) showed that 4–hydroxyderricin had
antidiabetic activity on diabetic rats because of lowering
blood glucose levels and inhibition α-glucosidase enzymes.

*Corresponding author e-mail: haryoto@ums.ac.id

 Antioxidant and inhibition of α-glucosidase enzyme activity of extract and their fractions of Ashitaba
One the examples of this drug is acarbose which could inhibit
maltase, isomaltase, sucrose and glucoamilase enzymes (Luo
et al., 2012), and later on reduce glucose levels in blood
postprandial (Dipiro et al., 2005). In this study, we attempted
to determine the antioxidant and inhibition of α-glucosidase
enzyme of extract and the ethanol fractions of ashitaba.
MATERIALS AND METHODS
965.83g dried powdered sample of Ashitaba (Angelica
keiskei) was macerated with 95% ethanol. The mixture was
filtered and replaced with fresh solvent. The solvent was
removed from the collected filtrate using a rotary evaporator
to obtain a viscous extract. The maceration process was
repeated 3 times.
Fractionation using Vacuum Liquid Chromatography
(VLC)
The condensed extracts were then qualitatively tested by
eluting on thin layer chromatography (TLC) plates prior to
the fractionation with vacuum liquid chromatography
(VLC). The stationary phase employed was silica gel 60 GF254,
while its mobile phase was hexane-chloroform (6:4; 5:5),
hexane-ethyl acetate (9.5:0.5; 9:1; 8:2), and ethanol.
Fractions were kept separately based on the phase of motion
used. The different fraction was then classified by polar,
proportion and non polar polarity by using TLC plate to be
further thickened by using rotary evaporator and water bath.
Antioxidant Activity
2 mL of sample solution was added by 1.0 mL DPPH solution
0.04 mM and methanol to make up to 5 mL. Solution was
incubated for 30 minutes in the dark at room temperature
(Rezaeizadeh et al., 2011). Vitamin E was used as a positive
control in this study. The absorption was measured at the
maximum wavelength of 517 nm.
Determination of % inhibition and IC50 values
The percent of inhibition of DPPH radical from the sample
solution was calculated by the following equation.
Abs. of negative control – Abs. of sample
% inhibition = x 100%
Abs. of negative control
The percent of the inhibition obtained was then
calculated IC50 values through the linear regression equation
with (x) as the sample concentration and (y) as the percent of
inhibition (Molyneux, 2004). IC 50 values showed the
Medicinal Plants, 10(3) September 2018
257
concentrations of samples that could reduce DPPH radicals
by 50%.
Inhibitory activity of the α-glucosidase enzyme
The inhibition activity of α-glucosidase was determined (Rao
et al., 2009) method with some modifications. Sample
solution was made to obtain a series of concentration 12.5,
25, 50, 75 and 100 µg/mL. One micromililiter of sample
solution was mixed with 49 µL buffer phosphat solution pH
6.9 and 25 µLp-NPG substrate (p-nitrophenyl-α-D-
glucopyranoside) for further incubation for 5 minutes at
37oC. Then the solution was added with phosphate buffer
pH 6.9 as much as 25 µL and enzyme α-glucosidase 25 µL.
The mixed solution was incubated for 15 minutes at 37°C.
The solution was subsequently added with 100 µL Na2CO3
2%. Acarbose was used as a positive control with a
concentration of 50 mg/mL obtained from Glucobay tablets
(acarbose). Kinetic release of the substrate was measured using
spectrophotometry with the wavelength of 405 nm.
Kinetics inhibition of enzyme inhibition by active fraction
The 1 µL sample solution with the highest inhibitory
concentration was fed into the microplate, mixed with 49 µL
phosphate buffer pH 6.9 and 25 µL p-NPG (p-nitrophenyl-α-
D-glucopyranoside) subtrates, incubated for 15 minutes at
37°C. Then a 25 µL α-glucosidase enzyme solution was added
and reincubated for 15 minutes at 37°C. After incubation
was completed, 100 µL of 2% Na2CO3 solution was added.
The solution was read by absorbance by using microplate
reader with the wavelength of 05 nm.
RESULTS AND DISCUSSION
The obtained extraction yield was 29.63% and fractionation
was based on interaction between samples with stationary
and mobile phase on liquid vaccum chromatography. Due
to the result of spray reagent to identification of chemical
composition, the extract contained flavonoids, alkaloid and
terpenoid. This research was comparable with research of
Sembiring et al. (2011).
Antioxidant activity showed that ethanol extract of
ashitaba had the lowest IC50 value compared to others (Table
1). The smaller IC50 values indicated a stronger antioxidant
activity. On the other hand, the value of antioxidant activity
index (AAI) was calculated to determine the categories of an
antioxidant. The weak antioxidant activity was when the
AAI value <0.5; Moderate was at 0.5 <AAI <1.0; Strong was
258 Haryoto et al.

Table 1: Antioxidant activity An enzyme inhibition kinetics test was performed to

Sample IC50 Average AAI* Category determine the mechanism of inhibition of the semipolar
(ppm) Average fraction, competitive or uncompetitive inhibition. Based on
Vitamin E 19.54±0.46 8.022 Very strong kinetic study, Line weaver-Burk plot (Figure 1) showed that
Extract 19.38±0.99 8.098 Very strong the mechanism of inhibition of semipolar fraction was
Polar fraction 116.7±3.45 1.343 Strong mixture type. Mixed type inhibition criteria were based on
Semipolar fraction 41.93±0.43 3.737 Very strong KAP> K and VAP <V values (Illines, 2008).
Non polar fraction 256.01±3.79 0.611 Moderate CONCLUSION
*AAI: antioxidant activity index.
The semipolar fraction of Ashitaba (Angelica keiskei) has
at 1.0 <AAI <2.0 and very strong was at AAI> 2.0 (Scherer strong antioxidant activity and the inhibition of enzyme α-
and Godoy, 2009). glucosidase with IC50 value were 28.63 and 18.45 µg/mL.
Meanwhile, in-vitro α-glucosidase inhibitory activity test
result showed that samples had good activity, especially ACKNOWLEGDMENT
semipolar fraction. The basic measurement of this method
was α-glucosidase enzyme that would hydrolyze p- We are thankful to the Rector Universitas Muhammadiyah
nitrophenol-α-D-glucopyranoside (p-NPG) as a substrate Surakarta, and Rector UiTM Sarawak for fund support.
producing p-nitrophenol and D-glucose. The reaction
REFERENCES
between the enzyme and the substrate would form a yellow
solution from p-nitrophenol in basic condition. IC50 values Akihisa T, Takashi K, Nagai H, Ishii K, Tabata K and Suzuki T
obtained from the semipolar fraction were 18.45 µg/mL, while (2011). 4-Hydroxyderricin from Angelica keiskei Roots
acarbose as positive control had IC50 value of 61.152 µg/mL. Induces Caspase-dependent Apoptotic Cell Death in HL60
Human Leukemia Cells. Journal of Oleo Science, 60(2): 71–
77.
Table 2: IC50 values of Samples
Baba K, Taniguchi M, Shibano M and Minami H (2009). The
Samples IC50 (µg/mL) Components and Line Breeding of Angelica keiskei Koidzumi.
Extract 28.63 Bunseki Kagaku, 58(12): 125.
Polar Fraction 116.16 Dipiro Joseph T, Robert L Talbert, Gary C Yee, Gary R Matzke,
Barbara G Wells and LMichael Posey (2005).
Semipolar fraction 18.45 Pharmacotheraphya pathophysiologic approach. New York :
Non polar fraction 29.23 McGraw-Hill Companies, Inc, pp. 1333-1352.
Acarbose 61.15 Enoki T, Ohnogi H, Nagamine K, Kudo Y, Sugiyama K, Tanabe M,
Kobayashi E, Sagawa H and Kato I (2007). Antidiabetic
activities of Chalcones isolated from a Japanese Herb, Angelica
keiskei. Journal of Agriculture, Food and Chemistry.
0.012 y = 0,0051x + 0,0017 Series2 Series1 Biotechnology Research Laboratories, Takara Bio Inc.
R² = 0.9972 Illanes A ( 2008). Enzyme biocatalysis: Principles and applications,
0.01 Inhibition
Internasional Diabetes Federation Diabetes atlas. (2006). The
0.008 Global burden. 2 August 2011.
Inamori Y, Baba K, Tsujibo H, Taniguchi M, Nakata K and Kozawa
y = 0,0014x + 0,0009
0.006 M (1991). Antibacterial activity of two chalcones,
Vmax

R² = 0.9178
Non Inhibition xanthoangelol and 4-hydroxyderricin, isolated from the root
0.004 of angelica keiskei koidzumi. Chem Pharm Bull., 39(6): 1604–
1605.
0.002
Kim JH, Son YK, Kim GH and Hwang KH (2013). Xanthoangelol
0 and 4-hydroxyderricin are the major active principles of the
-0.5 0 0.5 1 1.5 2 inhibitory Activities against Monoamine Oxidases on Angelica
-0.002 keiskei K. Biomol Ther., 21(3): 234–240.
Kmax Luo L, Ruihan wang, Xian ojunwong, Zhong Jun Ma and Ning Li
(2012). Compound from Angelica keiske with NQO1
Figure 1: The results of kinetics test of semipolar fraction with induction, DPPH scavenging and α-glukosidase inhibitory
0.5% concentration according to line weaver-Burk Plot activities. Food Chemistry, 131(3): 992-998.

Medicinal Plants, 10(3) September 2018

 Antioxidant and inhibition of α-glucosidase enzyme activity of extract and their fractions of Ashitaba
Monma K, Kasahara T, Iguchi M, Tomomatsu T, Murakami Y and
Urano M (1990). Proximate and mineral composition of
Ashitaba (Angelica keiskei) harvested in the Izu Islands, Ann.
Rep. Tokyo Metr. Res., 41: 158–161.
Nishimura R, Tabata K, Arakawa M, Ito Y, Kimura Y, Akihisa T,
Nagai H, Sakuma A, Kohno H and Suzuki T (2007).
Isobavachalcone, a chalcone constituent of Angelica keiskei,
induces apoptosis in neuroblastoma. Biol. Pharm. Bull, 30(10):
1878–1883.
Ogawa H, Ohno M and Baba K (2005). Hypotensive and Lipid
Regulatory Actions of 4-hydroxyderricin, a chalcone from
Medicinal Plants, 10(3) September 2018
259
Angelica keiskei, in stroke-prone spontaneously hypertensive
rats.Clin Exp Pharmacol Physiol, 32(1-2): 19–23.
Rao RR, AK Tiwari, PP Reddy, KSBabu, AZ Ali, Madhusudana K
and Rao JM (2009). New furanoflavonoids, intestinal á-
glucosidase inhibitory and free radical (DPPH) scavenging
activity from antihyperglycemic root extract of Derris indica
(Lam). Journal of Bioorganic Medical Chemistry, 17(14):
5170–5175.
Sembiring BB and Manoi F (2011). Plant quality identification
Ashitaba (Angelica keiskei Koidzumi). Bul. Littro., 22(2): 177–
185.