African Journal of Biotechnology Vol. 10(67), pp. 15184-15194, 31 October, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Correlation between enzymes inhibitory effects and
antioxidant activities of standardized fractions of
methanolic extract obtained from Ficus deltoidea
Elham Farsi1*, Armaghan Shafaei2, Sook Yee Hor1, Mohamed B. Khadeer Ahamed1, Mun Fei
Yam3, Idress Hamad Attitalla4, Mohd. Z. Asmawi1 and Zhari Ismail2
1Department of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Penang-11800,
2Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau
3Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia.
4Department of Botany, Faculty of Science, Omar Al-Mukhatr University, Box 919, Al-Bayda, Libya.
Accepted 5 September, 2011
Recently, there has been increasing interest in Ficus deltoidea (Moracea) due to its chemical
composition and the potential health benefits. The leaves of the plant have been suggested to have
potential antidiabetic effects. Inhibition of carbohydrate-hydrolysing enzymes, such as ?-glucosidase
and ?-amylase is one of the therapeutic approaches to control postprandial hyperglycemia. In this
study, enzymes inhibitory effect and antioxidant properties of different fractions of methanolic extract
obtained from F. deltoidea leaves was evaluated. Further, the possible relationship between
pharmacological properties and phytochemical content of fractions was investigated. The n-butanol
fraction showed significant ?-glucosidase and ?-amylase inhibitory effects (IC50 values 15.1 and 39.42
µg/ml, respectively) along with the remarkable antioxidant activity when compared to the other
fractions. High performance liquid chromatography (HPLC) chemical profiling of the n-butanol fraction
revealed that the contents of isovitexin (24.63 mg/g) and vitexin (8.3 mg/g) were found to be
significantly higher than the other fractions. These results indicate that F. deltoidea could be the
potential source of promising anti-diabetic drug.
Key words: Ficus deltoidea, enzymes, phytochemical, high performance liquid chromatography (HPLC),
Diabetes mellitus is a complex metabolic disorder that
disturbs the metabolism of carbohydrates, fats and
proteins, which is characterized by the elevated plasma
glucose levels (Eliza et al., 2009). Postprandial
hyperglycemia is the main risk factor for the development
of diabetes mellitus type II (Baron, 1998) and is asso-
ciated with microvascular complications in diabetic
*Corresponding author. E-mail: firstname.lastname@example.org. Fax:
individuals (Irene et al., 2000; Lebovitz, 2001). Therefore,
control of postprandial blood glucose level plays key role
in treatment and decrease progression of diabetes
mellitus (Kim et al., 2000). One of the latest therapeutic
approaches to reduce hyperglycemia with diabetes
mellitus is suppression of carbohydrate absorption after
Alpha-glucosidase is a membrane-bound enzyme
located at the epithelium of small intestine, where it
hydrolyzes the final step in digestion of carbohydrates,
whereas, ?-amylase is a salivary enzyme that catalyses
the breakdown of starch into sugars (Ali et al., 2009).
Hence inhibition of these enzymes can significantly
decrease the postprandial increase of blood glucose and
therefore can be an important strategy in the mana-
gement of various carbohydrates metabolic disorders
including type 2 diabetes mellitus (Floris et al., 2005).
Several ?-glucosidase and ?-amylase inhibitors such as
acarbose, trestatin, amylostatin and valiolamine isolated
from microorganisms (Remi and Jean, 2004) have been
used to control the diabetic condition. However, the use
of these drugs as the inhibitors is associated with side
effects including liver disorder, abdominal distention,
flatulence, meteorism and diarrhea. In such circum-
stance, medicinal plants were suggested as alternative
medicine for prevention and treatment of diabetes
because of their negligible side effects (Cheng and
Ficus deltoidea (Moraceae) is an evergreen shrub that
widely occurs in several Southeast countries commonly
Malaysia and Indonesia (Adam et al., 2009). Traditionally,
it is widely used to treat different diseases including
diabetes mellitus, high cholesterol, high blood pressure,
gout, improve blood circulation, pneumonia, diarrhea and
skin infections (Hakiman and Maziah, 2009). Also, F.
deltoidea is used as aphrodisiac; the fruit is chewed to
relieve headache, toothache, and to treat cold (Sulaiman
et al., 2008; Adam et al., 2007). Several studies showed
that the aqueous extract of whole plant possesses anti-
ulcerogenic properties (Adam et al., 2010; Sulaiman et
al., 2008). In addition, wound healing activity of aqueous
extract of leaves of F. deltoidea was also documented
(Mahmood et al., 2010). Its blood glucose lowering and
insulin stimulating effects were reported successively
(Adam et al., 2007; Aminudin et al., 2007). Recently,
toxicological study on F. deltoidea reported that the plant
contains no toxic elements (Armaghan et al., 2011).
Though F. deltoidea has been used widely in Malaysian
folk medicine, its pharmacological properties and active
principles are still not well understood. This investigation
was therefore undertaken to study the active composition
of fractions prepared by bioassay guided method using ?-
glucosidase and ?-amylase inhibitory assays, and also to
find out the relationship between the phytochemical
contents and the pharmacological properties of fraction of
methanolic extract of F. deltoidea leaves.
MATERIALS AND METHODS
Chemicals and reagents
HPLC grade methanol was purchased from Merck, Germany. Folin-
ciocalteau reagent, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical,
catechin, gallic acid, butylated hydroxyl anisole (BHA), butylated
hydroxytolune (BHT), quercetine (QTN), 2-thiobarbituric acid (TBA),
trichloroacetic acid (TCA), xylenol orange, ammonium molybdate,
sodium phosphate, potassium ferricyanide, ferric chloride,
potassium chloride (KCl), iron(III) chloride, manganese(II) chloride,
ammonium iron(II) sulfate, sulfuric acid, hydrochloric acid, hydrogen
peroxide solution, porcine pancreatic ?-amylase, rat-intestinal
acetone powder, p-nitrophenyl-a-D-glucopyranoside, starch and
Farsi et al. 15185
dinitrosalicylic (DNS) acid were purchased from Sigma Aldrich,
USA. All other chemicals used were either HPLC or analytical
Plant material and preparation of extract and fractions
F. deltoidea (FD) leaves were purchased from HERBagus Sdn.
Bhd. Penang, Malaysia. The specimen (voucher number: 11204)
was deposited at the herbarium of School of Biology, Universiti
Sains Malaysia. The extract was prepared by maceration of oven-
dried (at 37° C) leaves powder successively in petroleum ether,
chloroform, methanol and distilled water at 37° C water bath. The
supernatant was filtered and the solvent evaporated under reduced
pressure and then lyophilized. Methanolic extract was partitioned in
distilled water and n-hexane to obtain the n-heaxne fraction (HF),
the aqueous part was further fractioned with dichloromethane (DF)
and n-butanol (BF), to obtain respective fractions and aqueous
High performance liquid chromatography (HPLC) analysis
The HPLC analysis of different fractions with reference to isovitexin
and vitexin were performed by the methodology of Fu et al. (2007)
on HPLC system equipped with quaternary pump, online degasser,
auto sampler, automatic injector, column heater degasser
photodiode array detector (Agilent Tech, Palo Alto, CA) and
chromatographic separation were performed using Eclipse C18
reverse-phase column (250 mm × 4.6 mm) with flow rate of 1
ml/min at 30° C and sample size of 10 µl. The isocratic mobile
phase constituted of methanol/water/formic acid (33:66.37:0.67,
v/v/v). The sample was monitored with UV detection at 330 nm at
30° C. The chromatographic peaks of the analytes were confirmed
by comparing their retention time and UV spectra with
corresponding reference standards. The results were obtained by
comparison of peak areas (at 330 nm) of fractions with those of
Determination of total soluble phenolics content
Following the method of Slinkard and Singleton (1977), total soluble
phenolic component of Ficus deltoidea was determined. 2.5 ml of
0.2 N Folin-Ciocalteu reagent was mixed with 0.5 ml of sample
solution (1 mg/ml) for 8 min and 2 ml of 75 g/L Na2CO3 was added
to the mixture. The absorbance was read at 760 nm against blank
after 2 h using Fisher scientific multiskan microplate reader (Oxford,
MI, USA). Quantitative measurements were performed based on a
standard calibration curve of 3.1 to 100 µg/ml of gallic acid in
methanol. The total phenolic content was expressed as gallic acid
equivalents in mg/g of dry material.
Determination of flavonoids content
Flavonoids content of samples were determined by AlCl3
colorimetric method (Jamshid et al., 2009). 100 µl of each FD
fractions (1 mg/ml) and concentrations (3.1 to 100 µg/ml) of QTN
(standard reference) were mixed separately with 0.1 ml of 10%
(w/v) AlCl3 solution, 0.1 ml of 1 M potassium acetate solution, 1.5
ml of methanol and 2.8 ml of distilled deionized water. The mixture
was incubated for 30 min at room temperature, and then the
absorbance was read at 415 nm. For blank, 0.1ml of 10% (w/v)
AlCl3 solution was replaced with distilled water. Using the standard
curve, the total flavonoids content in samples were calculated as
milligrams of quercetin equivalents.
15186 Afr. J. Biotechnol.
DPPH radicals scavenging activity
Electron scavenging of samples was measured following the
method described by Yang et al. (2006). 200 µl of DPPH solution
(0.004% w/v) was added to100 ml of different concentrations (3.1 to
100 µg/ml) of methanolic solution of fractions. The mixture was
allowed for 30 min in the dark at room temperature and reduction of
DPPH was measured at 517 nm. The percentage of scavenging
activity was evaluated by comparing with the control (100 µl
methanol + 200 µl DPPH). QNT and BHT were used as reference
standards. The radical scavenging activity was calculated using the
Determination of total antioxidant capacity
Phosphomolybdenum method (Saliha et al., 2010) was used to
evaluate the total antioxidant capacity of different concentrations of
fractions (25, 50 and 100 µg/ml). The assay was based on the
reduction of Mo (VI) to Mo (V) and formation of green
phosphate/Mo (V) complex at acidic pH. 300 µl of sample solution
was mixed with 3 ml of reagent containing 0.6 M sulfuric acid, 28
mM sodium phosphate and 4 mM ammonium molybdate. The
mixture was allowed for 90 min incubation at 95° C in boiling water
bath and the absorbance was read at 695 nm after cooling in room
temperature. 300 µl of methanol was used as blank and the
antioxidant capacity was expressed as the number of equivalents of
Reducing power assay (iron reducing activity)
Following the method described by Oyaizu (1986), reducing power
of fractions was determined by mixing 1 ml methanolic solution of
different concentrations of samples (3.1 to 100 µg/ml) with 2.5 ml of
0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium
ferricyanide and the mixture was incubated for 20 min at 50° C.
Subsequently, 2.5 ml of 10% (w/v) trichloroacetic acid was added
and centrifuged at 3000 rmp for 10 min, and 2.5 ml of the
supernatant was allowed to react for 10 min with 2.5 ml distilled
water and 0.5 ml of 0.1% (w/v) ferric chloride. The absorbances of
the solutions were measured at 700 nm; increase in absorbance
indicated the increase in reducing power capacity of samples. QNT
and BHT were used as reference standards.
In vitro lipid per-oxidation
Preparation of tissue homogenate
Brain and liver homogenates were prepared from 3 months old
male Sprague-Dawley rats (200 to 250 g). 40% (w/v) homogenate
for the ferrous ion oxidation with xylenol orange (FOX method) was
prepared in HPLC-grade methanol. For the thiobarbituric acid, non-
enzymatic lipid peroxidation method, 3.3% (w/v) homogenate was
prepared in 50 mM phosphate buffer (pH 7.4). All solutions were
centrifuged at 5000 rpm for 15 min and the supernatants were used
for the experiment.
Ferrous xylenol orange (FOX) method
Lipid peroxidation was carried out at 37° C. The reaction mixture
contained 10 µl of sample solution (3.1 to 100 µg/ml), 10 µl of
Fenton’s reagent (5 µl of 50 mM hydrogen peroxide and 5 µl of 5
mM manganese chloride), and 80 µl of each homogenate
separately. 900 µl of FOX reagent (49 mg of ferrous ammonium
sulfate in 50 ml of 250 mM H2SO4, 0.397 g of BHT, and 0.038 g of
xylenol orange in 950 ml of HPLC grade methanol) was added to
each sample, and left to react for 30 min at room temperature. The
absorbance was read at 560 nm against blank. QTN and BHT were
used as standards. The blank solution was prepared in the same
manner with methanol in place of the test sample (Jiang et al.,
1992). The percentage of inhibition was calculated as:
Thiobarbituric acid non- enzymatic lipid peroxidation assay
Thiobarbituric acid reactive
spectrophotometrically at 535 nm using the method of Igene et al.
(1985) with minor modifications. Briefly, 0.5 ml of tissue
homogenate was added to 1 ml of various concentrations of the
samples solution (3.1 to 100 µg/ml). Peroxidation was initiated by
adding 100 µl of 0.2 mM FeCl3 after incubation for 30 min at 37° C.
The reaction was terminated by the addition of 2 ml TBA-TCA-HCl
reagent (15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric
acid and 0.25N hydrochloric acid) and further heating at 90° C for 15
min in a boiling water bath. The absorbance of supernatant was
measured at 535 nm after centrifugation at 3000 rpm for 10 min. 1
ml of HPLC-grade methanol in place of test sample was used as
the blank. QTN and TBA were used as reference standards. The
result was expressed as percentage of inhibition.
Assay for ?-glucosidase inhibitory activity
In vitro ?-glucosidase inhibition assay was performed according to
the method described by Kwon et al. (2006) with slight
modifications. Crude ?-glucosidase solution was prepated using100
mg of rat-intestinal acetone powder. The powder was suspended in
1 ml of 0.9% saline, and the suspension was sonicated for 30 s.
The supernatant containing the crude ?-glucosidase solution was
separated after centrifugation (5000 g, 30 min, 4° C). 100 µl of rat-
intestinal ?-glucosidase solution was pre-incubated with 50 µl of
sample solution (3.1 to 100 µg/ml) at 37° C for 10 min. After pre-
incubation, 50 µl of 5 mM p-nitrophenyl-a-D-glucopyranoside
solution in 0.1 M phosphate buffer (pH 6.9) was added. The
reaction mixture was incubated at 37° C for 15 min. The reaction
was stopped by addition of 100 µl of Tris –HCl buffer (pH 7) and the
absorbance readings were recorded at 405 nm. The ?-glucosidase
inhibitory activity of fractions was expressed as percentage of
Assay for porcine pancreatic ?-amylase inhibitory activity
Porcine pancreatic ?-amylase inhibition assay was conducted as
substances were determined
Farsi et al. 15187
Figure 1. HPLC chromatogram of the four fractions of metanolic extract of F. deltoidea leaves with detector response at
330 nm. A, Standard; B, n-hexane;C, dichloromethane; D, n-butanol; E, aqueous fraction.
part of the protocol described by Kwon et al. (2006). 500 µl of
sample solution (3.1 to 100 µg/ml) and 500 µl of 0.02 M sodium
phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing
?-amylase solution (0.5 mg/ml) were incubated at 37° C for 10 min.
After pre-incubation, 500 µl of 1% starch solution in 0.02 M sodium
phosphate buffer was added. The reaction mixture was then
incubated at 37° C for 15 min and the reaction was stopped with 1.0
ml of DNS acid; color reagent. The reaction mixture was then
incubated in a boiling water bath for 5 min, cooled to room
temperature, then diluted with 10 ml distilled water and absorbance
was measured at 540 nm.
All assays were performed in three independent experiments and
expressed as mean ± standard deviation (SD).
HPLC analysis of fractions of methanolic extract of F.
HPLC was used to identify and quantify isovitexin and
vitexin in different fractions of methanolic extract of F.
deltoidea leaves. Individual constituents were identified
by comparing their peaks, UV spectra and retention
times, with corresponding reference standards (Figure 1).
Concentrations in the samples were estimated based on
15188 Afr. J. Biotechnol.
Table 1. Phytochemical contents of fractions obtained from F. deltoidea leaves extract.
Total phenolics (mg/g)
211.05 ± 0.004
273.82 ± 0.002
416.155 ± 0.008
72.36 ± 0.002
aHF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF, aqueous fractions. The results are expressed as
mean ± S.D.
Table 2. IC50 values (?g/ml) of fractions of methanolic extract of F. deltoidea leaves obtained from different antioxidant assays.
Test samplea HF DF
DPPH 51.07 35.43
Reducing power 116.37 71.24
Total antioxidant capacity 152.5 120.2
FOX method Brain 73.99 53.58
Brain 87.01 51.03
?- glucosidase inhibitors 42.61 37.12
?- amylase inhibitors 94.50 68.9
aHF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction and AF, aqueous fraction; QTN, Quercetine; BHT, butylated
hydroxytoluene; bND, not determined.
the calibration curves of isovitexin and vitexin in the
range of 5 to 200 µg/ml, and the quantitative percentage
of dry weight of bioactive markers (isovitexin and vitexin)
was calculated from the following formulas: Y=23.90X –
65.442 (R2 =0.9992, n=6), Y=28.305X-28.245 (R2 =9982,
n=6), respectively, where Y is the peak area of the
analyte and X was the concentration of the analyte
(µg/ml) (Table 1).
According to HPLC chromatogram, it could be noticed
that the n-butanol fractions contained highest amount of
isovitexin and vitexin than compared to the other
fractions. The chromatographic profile indicated that BF
contained other chemical constituents beside isovitexin
The results of the phenolics and flavonoids content of the
fractions of methanol extract from F. deltoidea leaves are
shown in Table 1. The results reveal that the phenolics
content of the n-butanol (416.15 mg/g) was statistically
higher than that of the other fractions. Also, the
flavonoids content of the fractions depicted in Table 1
indicate that n-butanol fraction (1.6 ± 0.03 mg/g) had
Total flavonoids (mg/g)
0.44 ± 0.02
1.29 ± 0.05
1.6 ± 0.03
0.16 ± 0.01
3.81 ± 0.05
2.46 ± 0.03
24.63 ± 0.04
1.91 ± 0.06
4.05 ± 0.08
3.33 ± 0.04
8.3 ± 0.03
4.27 ± 0.04
Liver 88.45 56.38
Liver 105.3 81.5
higher flavonoid content than other tested samples. The
order of total flavonoids and phenolics content in the
samples was given as; n-butanol> dichloromethane> n-
hexane > aqueous fractions.
Antioxidant activities fractions of metanolic extract of
F. deltoidea leaves
The antioxidant efficacy of fractions of metanolic extract
of F. deltoidea leaves were evaluated by in vitro lipid
peroxidation, DPPH radical scavenging, reducing power
and total antioxidant capacity assays. The degree of lipid
peroxidation in tissue homogenates was assessed by two
different methods. In Ferrous xylenol orange (FOX)
method, Fenton’s reagent (H2O2
peroxidation inducer, and the resulting ferrous xylenol
orange, under acidic conditions that reveals the presence
of lipid hydroperoxide was measured spectrophoto-
metrically at 560 nm. In the other method; lipid
peroxidation was determined by measuring the level of
TBARS using the TBA-TCA-HCl reagent at 532 nm. QTN
was found to be more effective than the fractions in
curbing the generation of lipid peroxides in both method,
whereas, the n-butanol fraction of F. deltoidea exhibited
+ Mn) was used as
Farsi et al. 15189
Figure 2. DPPH radical scavenging activity of fractions of metanolic extract of F. deltoidea leaves. Values are
expressed as percentage of inhibition per µg/ml of test sample. HF, n-Hexane fraction; DF, dichloromethane fraction;
BF, n-butanol fraction; AF, aqueous fractions; QTN, Quercetine; BHT, butylated hydroxytolune.
significant anti-lipidperoxidation activity in a dose
dependent manner (Table 2, Figures 5 and 6).
The activity of fractions to reduce DPPH radical to
diphenylpicylhydrazine was detemined by the ability of
the fractions in quenching the DPPH free radical which
inturn declines the absorbance at 517 nm. The results
given in Table 2 illustrate that n-butanol fraction exhibited
a significant dose-dependent DPPH scavenging activity,
as the free radical was drastically quenched by the
fraction with IC50 9.74 µg/ml. Dichlromethyl fraction also
showed significant activity with IC50 35.43 µg/ml when
compared to the control. On the other hand, the n-hexane
and aqueous fractions demonstrated moderate DPPH
quenching activity with IC50 51.07 µg/ml and 62.3 µg/ml,
respectively (Figure 2). The results of reducing power
and total anti-oxidant capacity assays are depicted in
Figures 3 and 4, respectively. Consistently, n-butanol
fraction of F. deltoidea exhibited significant activity in both
assays, whereas other fractions showed less activity
(Table 2). Moreover, the n-butanol fraction demonstrated
the activity on a par with the standard reference BHT in
almost all assays.
The inhibitory activity of F. deltoidea fractions on ?-
Table 2 shows the inhibitory effects of F. deltoidea
fractions on ? -amylase activity. As shown in Figure 7, the
highest percentage of inhibition was observed in n-
butanol fraction with IC50 39.42 µg/ml. Likewise, the ?-
amylase inhibitory effect of acarbose (positive control)
and the inhibitory effect of F. deltoidea fraction on ? -
amylase increased with increasing concentrations. On
the basis of increasing order of IC50, the sequence of
enzyme inhibitory activity were found to be: n-butanol>
dichloromethane >n-hexane >aqueous fractions.
The inhibitory activity of F. deltoidea fractions on ?-
Spectrophotometrically standard procedure was used to
assess the inhibitory potency of four different fractions of
F. deltoidea leaves against ?-glucosidase by measuring
hydrolysis of p-nitrophenyl glycoside. (Figure 8) As
15190 Afr. J. Biotechnol.
Figure 3. Reducing power of fractions of metanolic extract of F. deltoidea leaves as compared to QTN. Values were the
average of triplicate experiments. HF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF,
aqueous fractions; QTN, quercetine; BHT, butylated hydroxytoluene.
Figure 4. Total antioxidant capacity assessed by measuring formation green phosphate/Mo (V) complex
of acidic pH at 695 nm in the presence of fractions of metanolic extract of F. deltoidea leaves. Values
are as expressed as µg/ml of quercetin equivalents.
Farsi et al. 15191
Figure 5. Effect of concentrations (3.1 to 100 µg/ml) of fractions of metanolic extract of F. deltoidea leaves in inhibition of lipid peroxidation
generated by Fentons Reagent in brain (A) and liver (B) homogenate (40% w/v) in the Fox method during 60 min at 37°C. The values are
expressed as percentage of inhibition per µg/ml of fraction. HF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF,
aqueous fractions; QTN, quercetine; BHT, butylated hydroxytoluene.
Figure 6. Effect of concentrations (3.1 to 100 µg/ml) of fractions of metanolic extract of F. deltoidea leaves on TBARS content generated by
FeCl3 in brain (A) and liver (B) homogenate (3.3% w/v) in the TBA method. The values are expressed as percentage of inhibition per µg/ml
of fraction. HF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF, aqueous fractions; QTN, Quercetine; BHT,
shown in Table 2, all fractions possessed concentration-
dependent inhibitory activity on mailman ?-glucosidase,
while the most potent tested sample was found to be n-
butanol fraction with IC50 15.1 µg/ml. Based on the
increasing order of IC50 values on the enzyme inhibitory
activity, the order of the fractions is given as: n-butanol>
dichloromethane >n-hexane >aqueous fractions.
Inhibition of ?-glucosidase and ?-amylase is considered
to be efficient strategy in the treatment of carbohydrate
metabolic disorders including diabetes mellitus type II
(Floris et al., 2005), cancer (Olden et al., 1991) and
HIV(Saul et al., 1983). The various beneficial effects of
these inhibitors are due to crucial role of enzymes in
metabolism of carbohydrate (Remi and Jean, 2004).
Delay in digestion of carbohydrate plays important role in
the control of postprandial hyperglycemia, hyper in-
sulinemea, as well as in decreasing risk of cardiovascular
disease (Maki, 2004; Misra et al., 2010).
Assessment of enzymes inhibitory activity of the four
different fractions of methanolic extract of F. deltoidea
15192 Afr. J. Biotechnol.
Figure 7. Porcine pancreatic ?-amylase inhibitory effect of different concentrations (3.1 to 100 µg/ml) of fractions of
metanolic extract of F. deltoidea leaves measured at 540 nm and expressed as percentage of inhibition by µg/ml of test
sample. HF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF, aqueous fraction.
Figure 8. Mammalian ?- glucosidase inhibitory activity of different concentrations (3.1 to 100 µg/ml) of fractions of
metanolic extract of F. deltoidea. Leaves were quantified by measuringthe released p-nitrophenol at 400 nm and
expressed as percentage of inhibition by µg/ml of test sample. HF, n-Hexane fraction; DF, dichloromethane
fraction; BF, n-butanol fraction; AF, aqueous fractions.
Farsi et al. 15193
Table 3. The R values (correlation coefficients) between antioxidant activities and ?-glucosidase, ?-amylase and lipid peroxidation in
different systems of assessment.
Total antioxidant a
aHF, n-Hexane fraction; DF, dichloromethane fraction; BF, n-butanol fraction; AF, aqueous fraction.
leaves showed that n-butanol fraction exhibited dose
dependent ?-glucosidase and ?-amylase inhibitory
potency which was more pronounced than the other
fractions of the methanolic
assessment of antioxidant capacity of fractions of the
methanolic extract of F. deltoidea leaves proved that
again n-butanol fraction showed higher antioxidant
activity among all fractions, which was consistence with
the result of phytochemical analysis that presented the
higher amount of phenolics and flavonoids content in n-
butanol fraction. HPLC analysis also confirmed the
presence of higher phenolic compounds particularly the
C-glycosylflavones, such as isovitexin and vitexin in n-
butanol fraction of the methanolic extract.
Since the last decade, the antioxidant activity of
phenolic and flavonoid compounds has received much
attention, as it is well documented that antioxidant activity
of these compounds can help in preventing some serious
pathological and chronic conditions (Rakesh et al., 2001).
Furthermore, they are also capable of inhibiting carbohy-
drate digestive enzymes due to their protein binding
ability (Griffiths and Moseley, 1980; Hara and Honda,
1992). The results of antioxidant activies showed that the
fraction rich in phenolics and flavonoids content had
stronger antioxidant activities. Further quantitative
analysis showed there was positive correlation between
total antioxidant activity and phenolic content (R value:
0.993). These results therefore support the role of
phenolic compounds in antioxidant activity, which is in
agreement with other studies (Hiroyuki et al., 2001;
Matsui, 2001). Fraction with higher phenolic and
flavonoid contents, as well as higher total antioxidant
activity, also showed higher enzyme inhibitory activity.
The results of this study are in agreement with other
research that reported high antioxidant activity and ?-
glucosidase inhibitory effect of phenolic rich extract (Esra
et al., 2004). Furthermore, these results reveal a direct
correlation between antioxidant
glucosidase and ?-amylase inhibitory activities (Table 3).
Isovitexin and vitexin from F. deltoidea have been
identified to be responsible for enzymes’ inhibitory effect.
This is the first report of ?-glucosidase and ?-amylase
extract. More also,
inhibitory compounds from F. deltoidea. Isovitexin is C-
glycosyl flavones that are found to have ?-glucosidase
inhibitory, which may reduce postprandial hyperglycemia
and diabetic complication. In addition, this fraction offers
other advantages including antioxidant activity and
reducing lipid peroxidation. The fraction can also reduce
the harmful effects of oxidative stress in diabetics,
therefore aiding in the management of diabetes.
The authors are grateful to the School of pharmaceutical
Sciences, Universiti sians Malaysia for providing financial
and technical supports.
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