ORI GINAL RESEARCH

a-Glucosidase inhibitory activity and in vitroantioxidant activities of alcohol-water extract (AWE) ofIchnocarpus frutescens leaves

Chidambaram Kumarappan ÆSubhash Chandra Mandal

Received: 2 November 2007 / Accepted: 12 November 2007 / Published online: 16 December 2007

� Birkhauser Boston 2007

Abstract In this study alcohol–water extract (AWE) of Ichnocarpus frutescenswas studied for its a–glucosidase inhibitory activity and antioxidant properties.

HAE exhibited the rat intestinal a-glucosidase, sucrase, isomaltase, and maltase

activities. Sucrose was administered orally with or without extract to rats at a dose

of 1000 mg/kg. The postprandial elevation in the blood glucose level after the

administration of sucrose with the extract was significantly suppressed when

compared with the control. The antioxidant activity, and 1,1-diphenyl-2-pic-

rylhydrazyl (DPPH) radical, superoxide anion radical scavenging , and hydrogen

peroxide scavenging activities were evaluated to determine the total antioxidant

capacity of the alcohol–water extract. HAE exhibited strong activity in phenyl-

hydrazine–induced hemolysis. The total amount of polyphenol compounds in HAE

was determined as pyrocatechol equivalents per gram of alcohol–water extract.

Keywords a-glucosidase � Ichnocarpus frutescens � Free-radical scavenging �Alcohol-water extract (AWE) � Hemolysis � Antioxidant

Introduction

Type 2 diabetes is an increasingly common disorder, with approximately 150–

300 million people suffering from this debilitating disease worldwide (Zimmet

C. Kumarappan (&) � S. C. Mandal

Division of Pharmacognosy, Pharmacognosy and Phytotherapy Research Laboratory, Faculty of

Engineering and Technology, Jadvapur University, Kolkata 700 032, India

e-mail: ctkumarrx@gmail.com

S. C. Mandal

Department of Pharmaceutical Technology, Faculty of Engineering and Technology, Jadvapur

University, Kolkata 700 032, India

Med Chem Res (2008) 17:219–233

DOI 10.1007/s00044-007-9056-1

MEDICINALCHEMISTRYRESEARCH

et al., 2001). Persistent hyperglycemia, the common characteristic of diabetes,

can lead to various complications, including diabetic nephropathy, retinopathy,

and neuropathy (Wong and Aiello, 2000; Gross et al., 2005; Tesfaye et al.,

2005). There are many articles related to antidiabetic compounds from plants

(Matsui et al., 2006; Yamahara et al., 1981). However, in clinical practice

normalizing blood glucose level is s formidable challenge. Even more difficult is

the control of postprandial hyperglycemia (Mooradian and Thurman, 1999). The

pharmacological agents with greatest effect on postprandial hyperglycemia

include insulin, lispro, amylin analogues, and a-glucosidase (acarbose and

voglibose) inhibitors (Goda et al., 1981). It has been well acknowledged that

plant-derived extracts and phytochemicals are potential alternatives to synthetic

inhibitors against a-glucosidase.

Oxygen and reactive oxygen species (ROS) are among the major sources of

primary catalysts that initiate oxidation in vivo and in vitro. An increasing body of

evidence suggests that free-radical formation and oxidative stress are involved in the

pathogenesis of diabetes and the development of diabetic complications (Baynes

and Thorpe, 1999). The generation of reactive oxygen species (ROS) is increased in

diabetes due to prolonged exposure to hyperglycemia. Insufficient antioxidant

defense mechanisms have been reported in diabetes (Maridonneau et al., 1983).

Studies have shown that several parameters of red blood cell function and integrity

are negatively affected by increased oxidative stress (Rohn et al., 1998). RBCs from

diabetic subjects were more susceptible to oxidative hemolysis and lipid peroxi-

dation than those from normal subjects. However, antioxidant supplements or foods

containing antioxidants may be used to help the human body reduce oxidative

damage and decreases the occurrence of complications in diabetic animals (Bursell

et al., 1999).

Ichnocarpus frutescens, indigenous to India, contains a wide range of polyphe-

nolic compounds such as simple phenolic acids and flavonoids, whereas it contains

no alkaloids. A survey of the literature revealed that a number of pentacyclic

triterpenoids, and flavonoids been isolated (Lakshmi et al., 1985; Singh and Singh,

1987) The utilization of decoction of leaves of I. frutescens in the treatment of

jaundice and diabetes is noteworthy and it is also one of the plants species used by

the tribes of the Karnataka and Utter Pradesh states to treat diabetes and jaundice

(Parinitha et al., 2004). Some of the constituents of the plant, such as triterpenoids

and flavonoids, were shown to present antidiabetic, antioxidant, and related

biological activities (Vessal et al., 2003). In spite of this reported use, no systematic

clinical experimental studies have been carried out to assess the therapeutic uses of

this species. We examined the inhibitory effect on postprandial glucose levels and

a-glucosidase inhibition profiles of the alcohol–water extract (AWE) of I. frutescensboth in vivo and in vitro. The total phenolic content of the alcohol–water extract

(AWE) was determined and then the antioxidant properties of this extract containing

different concentrations of phenolic compounds were assessed by five model

systems.

220 Med Chem Res (2008) 17:219–233

Rational and Design

Reagents

Butylated hydroxyl toluene (BHT), naringenin, quercetin, pyrocatechol, sucrose,

maltose, phenyhydrazine hydrochloride (PHZ), 1,1-diphenyl-2-picrylhydrazyl

(DPPH), and linoleic acid were obtained from Sigma Chemical Co (St. Louis,

MO, USA). Folin-Ciocalteu Reagent, a-tocopherol, ascorbic acid, polyoxyethylene

sorbitan monolaurate (Tween-20), reduced glutathione, hydrogen peroxide, trichlo-

roacetic acid, aluminium chloride, ferrous chloride, ammonium thiocynate,

dimethyl sulfoxide (DMSO), 2,4-dinitrophenyl hydrazine, thiobarbituric acid

(TBA), and nitroblue tetrazolium (NBT) were purchased from SISCO Reasearch

Laboratories Pvt. Ltd (Mumbai, India). A glucose estimation kit was purchased

from Span Diagnostic Ltd (Mumbai, India). All other chemicals and solvents used

were of analytical grade.

Phytochemistry

Plant materials

Fresh leaves of Ichnocarpus frutescens were collected from the region of Cauvery

River, Thiruchirappalli, India, in February 2004 and authenticated at the Botanical

Survey of India (BSI), Central National Herbarium (CNH), Howrah, India

(reference number CNH/I-I/87/2005-TECH/1326). An authentic voucher specimen

was deposited in the Herbarium of Division of Pharmacognosy, Department of

Pharmaceutical Technology, Jadavpur University, Kolkata, India.

Preparation of alcohol–water extract (AWE)

The leaves were air dried at room temperature without exposure to sunlight and

coarsely powdered. The dried, powdered leaves (200 g) were macerated with 70%

aqueous/ethanol (500 mL) by stirring at room temperature for 7 days. The extract

was filtered before drying using Whatman filter paper no. 2 and the solvent was

removed under vacuum, concentrated in a rotary evaporator at 35 ± 2�C under

reduced pressure (SUPERFIT, India) and then lyophilized, and the resulting powder

extract (yield 23% w/v) was used in the present study. Alcohol–water extract was

stored at –4�C. Thextract was suspended in 5% Tween 80 solution.

Preliminary phytochemical screening

Preliminary phytochemical screening of alcohol–water extract of leaf was carried

out for the detection of phytoconstituents using standard chemical tests (Harborne,

1998).

Med Chem Res (2008) 17:219–233 221

Determination of total phenolic content

The total concentration of phenolics in the alcohol–water extract was determined

according to the previously described method (Singleton et al., 1999). Briefly, 0.1

mL of extract solution (containing 500 lg of extract) was transferred to a 100 mL

Erlenmeyer flask, and the final volume was adjusted to 46 mL by the addition of

distilled water. Afterwards, 1 mL of Folin–Ciocalteu reagent (FCR) was added to

the mixture and after 3 min 3 mL of Na2CO3 (2%) was added. Subsequently, the

mixture was shaken on a shaker for 2 h at room temperature, and then the

absorbance was measured at 760 nm. Pyrocatechol was used as the reference

standard for the calibration curve. The estimation of phenolics in the fractions was

carried out in triplicate, and the results were averaged. The phenolic compound

content was determined as pyrocatechol equivalents using the following linear

equation based on the calibration curve:

A = 0.0034C - 0.058, R2 [ 0.9996,

where A is the absorbance, and C is the pyrocatechol equivalent (lg).

Determination of flavones and flavonols

Flavones and flavonols in the alcohol–water extract (AWE) were expressed as

quercetine equivalent. Quercetine was used to make the calibration curve (standard

solutions of 6.25, 12.5, 25.0, 50.0, 80.0, and 100.0 mg/mL in 80% ethanol v/v). 0.5

mL of a product (ethanolic solutions of HAE) was mixed with 1.5 mL 95% ethanol

(v/v), 0.1 mL 10 %(w/v) aluminum chloride, 0.1 mL of 1 mol/L potassium acetate,

and 2.8 mL water. A volume of 10% (w/v) aluminum chloride was substituted by

the same volume of distilled water in blank. After incubation at room temperature

for 30 min, the absorbance of the reaction mixture was measured at 415 nm (Chang

et al., 2002).

Determination of flavanones

Flavanones in the alcohol–water extract (AWE) were expressed as naringenin

equivalent. Naringenin was used to make the calibration curve (standard solution of

0.125, 0.25, 0.30, 0.50, 1.00 and 2.00 mg/mL in methanol). One milliliter of a

product (ethanolic solution of HAE) was separately mixed with 2 mL of 1% 2,4-

dinitrophenylhydrazine (w/v) and 2 mL of methanol at 50�C over a water bath for

50 min. After cooling to room temperature, the solution was mixed with 5 mL 1%

potassium hydroxide (w/v) in 70% ethanol (v/v). Then, 1 mL of the mixture was

taken and centrifuged at 1000 g for 10 min and the supernatant was filtered through

Whatman no. 1 filter paper. The filtrate was adjusted to 25 mL. The absorbance of

the filtrate was measured at 495 nm (Chang et al., 2002).

222 Med Chem Res (2008) 17:219–233

Pharmacology

Animals, feeding, housing conditions, and ethical approval

Swiss albino mice (20–25 g body weight) and Wistar albino rats (180–200 g body

weight) were used in the present study. Animals were collected from the breeding

colony and acclimatized to the laboratory conditions for 2 weeks. They were housed

in macrolon cages under standard laboratory conditions (light period 07:00 to 19:00,

21 ± 2�C, and relative humidity 55–70%). The animals were fed with a commercial

diet from Hindustan Lever Ltd. (Bangalore, India) and had free access to water

during the experiments. The experiments complied with the rulings of the

Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA) New Delhi, India (registration no: 0367/01/C/CPCSEA) and the study

was permitted by the institutional ethical committee of Jadavpur University.

Acute toxicity study

Acute oral toxicity study was performed as per OECD 423 guidelines (OECD 1996;

acute toxic class method), albino rats (n = 6) of either sex selected by a random

sampling technique were used for the acute toxicity study (OECD, 2002). The

animals were fasted overnight, providing only water, after which the extracts were

administered orally at a dose of 5 mg/kg body weight by gastric intubation and

observed for 14 days. If mortality was observed in two out of three animals, then the

dose administered was assigned as toxic dose. If mortality was observed in one

animal, then the same dose was repeated to confirm the toxic dose. If mortality was

not observed, the procedure was repeated for further higher doses such as 50, 300,

and 2000 mg/kg body weight.

Carbohydrate tolerance test

For the oral carbohydrate tolerance test, the animals were deprived of food

overnight and administered sucrose orally 2000 mg/kg, with or without HAE at

1000 mg/kg, dissolved in 1 mL of distilled water. Blood was sampled from the tail

vein at 0, 30, 60, and 120 min after carbohydrate administration to measure blood

glucose levels, determined by using glucose estimation strips supplied by

Accucheck, Lifespan, Johnson and Johnson, Germany.

a-Glucosidase inhibitory activity

In order to investigate the inhibitory of HAE , an in vitro a-glucosidase inhibition

test was performed. a-Glucosidase from yeast is used extensively as a screening

material for a-glucosidase inhibitors, but the results do not always agree with those

obtained in mammals. Therefore, we used the mouse small-intestine homogenate as

Med Chem Res (2008) 17:219–233 223

an a-glucosidase solution because we speculated that it would better reflect the in

vivo state. The inhibitory effect was measured using the method slightly modified

from Dahlqvist (1964). After fasting for 20 h, the small intestine between the part

immediately below duodenum and the part immediately above the cecum was cut,

rinsed with ice-cold saline, and homogenized with 12 mL of maleate buffer (100

mM, pH 6.0). The homogenate was used as the a-glucosidase solution. The assay

mixture consisted of 100 mM maleate buffer (pH 6.0), 2% (w/v) each sugar

substrate solution (100 ll), and the sample extract (1–250 lg/mL). It was

preincubated for 5 min at 37�C, and the reaction was initiated by adding the crude a-

glucosidase solution (50 ll) to it, followed by incubation for 10 min at 37�C. The

glucose released in the reaction mixture was determined with the kit described

above. The rate of carbohydrate decomposition was calculated as the percentage

ratio to the amount of glucose obtained when the carbohydrate was completely

digested. The rate of prevention was calculated by the following formula:

Inhibition rat (%) = [{(amount of glucose produced by the positive control) –

(amount of glucose produced by the addition of HAE) – (glucose production value

in blank)/(amount of glucose produced by the positive control)}] 9 100.

In vitro antioxidant activity

Scavenging activity against DPPH radical

Scavenging activity on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals of HAE was

measured according to the method reported by Blois et al. (1958). Each sample

stock solution was diluted to final concentrations of 250, 200, 150, 100, and 50 lg/

mL, and 0.2 mL of methanol and 0.3 mL of various concentrations of the samples in

methanol were mixed in a 10 mL test tube. To this was added 2.5 mL of 75 lM

DPPH (1,1-diphenyl-2-picryl-hydrazyl) in methanol to achieve a final volume of 3

mL. The solution was kept at room temperature for 90 min, and the absorbance at

517 nm was measured. a-Tocopherol was used as a reference compound. The DPPH

(1, 1-diphenyl-2-picryl-hydrazyl) scavenging effect and IC50 values were calculated

using linear regression method.

Superoxide anion scavenging activity

Superoxides were generated (Hyland and Auclair, 1981) by adding 0.25 mL of 5

mM NaOH in 24.75 mL DMSO (1 mL, 1% water, 5 mM NaOH). The reduction of

NBT by superoxide was determined in the presence and absence of HAE at various

concentrations (50, 100, 150, 200, and 250 lg/mL in 0.2 M phosphate buffer pH

7.4). 1.1 mL of HAE solution, 0.1 mL of NBT (0.1 mg/0.1 mL), and 3 mL of

alkaline DMSO were added and mixed in a test tube and absorbance at 560 nm was

immediately noted for 5 min at intervals of 30 s. A graph was plotted between the

scavenging rate and the concentration of HAE to calculate the IC50 value.

224 Med Chem Res (2008) 17:219–233

Scavenging of hydrogen peroxide

The ability of the HAE extracts to scavenge hydrogen peroxide was determined

according to the method of Ruch et al. (1989). A solution of hydrogen peroxide (40

mM) was prepared in phosphate buffer (pH 7.4). The concentration of hydrogen

peroxide was determined by absorption at 230 nm using a spectrophotometer (Jasco

V-530, Japan Servo Co. Ltd., Japan). Extracts (50–250 lg/mL) in distilled water

were added to a hydrogen peroxide solution (0.6 mL, 40 mM). The absorbance of

hydrogen peroxide at 230 nm was determined after 10 min against a blank solution

containing phosphate buffer without hydrogen peroxide. The percentage of

hydrogen peroxide scavenging by the HAE extracts and a standard compound

was calculated as follows:

% Scavenged (H2O2) = [(A0 - A1)/A0] � 100

Where A0 is the absorbance of the control and A1 the absorbance in the presence

of the sample of BL extract and standard.

Determination of total antioxidant activity

The total antioxidant activity of HAE was determined using the thiocynate method

(Jayaprakasha et al., 2001). Briefly, for stock solution, 10 mg HAE was dissolved in

10 mL ethanol. 100 and 200 lg/mL concentrations of HAE in 2.5 mL of potassium

phoshate buffer (0.04 M, pH 7.0) were added to 2.5 mL linoleic acid emulsion in

potassium phosphate buffer; the 5.0 mL control consisting of 2.5 mL linoleic acid

emulsion and 2.5 mL potassium phosphate buffer. The mixed solution was

incubated in a glass flask and in the dark at 37�C. After incubation, the mixture was

stirred for 3 min and the peroxide value was determined by reading the absorbance

at 500 nm in a spectrophotometer after reaction with FeCl2 and thiocynate at

intervals during incubation. The percentage inhibition of lipid peroxidation was

calculated by the following equation:

% Inhibition = 100 - (absorbance of sample)/(absorbance of control) � 100

Effect on phenyl hydrazine (PHZ)-induced hemolysis

Isolation of erythrocytes All experiments were performed with human blood.

Healthy human blood was collected in acid-citrate dextrose solution. The packed

erythrocytes were isolated by centrifugation at 3000 g for 10 min at 4�C. The

plasma and buffer coat were removed by aspiration and cells thus obtained were

washed thrice with phosphate buffer saline, pH 7.4 and a suspension of packed cell

were prepared in the same buffer.

Incubation of erythrocyte with oxidants/antioxidants A suitable amount of

erythrocyte cell suspension was incubated with or without 1mM PHZ only, at

Med Chem Res (2008) 17:219–233 225

37�C, in a shaker water bath. To study the effect of antioxidants, HAE of various

doses (6.25–100 lg/mL) was tested. The ascorbic acid concentration in the 1 mL

incubation mixture was followed (4.4–70.4 lg/mL). At the end of the incubation the

cells were collected by centrifugation and lysed with 5 mM sodium phosphate

buffer, pH 8.0 (1:10) and suspensions were centrifuged at 10,000 g for 1 h. The

resulting supernatant or hemolysate was taken and the percentage inhibition of lipid

peroxidation calculated as (Buege and Aust, 1978).

% Inhibition = 100 - (absorbance of sample)/(absorbance of control) � 100

Results

Preliminary phytochemical screening of the alcohol–water extract of leaf was

carried out for the detection of phytoconstituents using standard chemical tests.

Triterpenoids, flavonoids, simple phenolic acids, steroids, and tannins were detected

in the alcohol–water extracts. Chromatography on silica gel 60 with chloroform,

methanol as mobile phase, in a saturated chamber, allows baseline separation of the

target compounds. The polyphenolic profile can be visualized with Fast Blue Salt B

reagent.

The alcohol–water extract of Ichnocarpus frutescens did not cause any mortality

up to 2000 mg/kg and were considered as safe (OECD, 2002). Total phenolic

contents of alcohol–water extract were expressed as mg of pyrocatechol equivalent

per gram of dry weight of alcohol–water extract. 1000 lg of alcohol–water extract

were used to determine the amount of total polyphenolic contents. The level of total

polyphenolic compounds was 100.51 mg of pyrocatechol equivalent per gram of

alcohol–water extract. The present study showed the flavonoid content determined

by two independent colorimetric methods, one for the determination of flavones and

flavonols and other for determination of flavanones, as reported by earlier. The

contents of total flavonoids in the alcohol–water extract of I. frutescens were

expressed as the sum of two complementary methods for the determination of

flavones, flavonols and flavonones and the results found to be 17.8 mg of quercetin

and naringenin equivalent per gram of alcohol–water extract. The major types of

phenolic constituents identified in the leaves of I. frutescens were simple phenolic

acids, flavonol, flavones, flavonones, and flavonoid glycosides.

To test our hypothesis that I. frutescens lowers postprandial blood glucose, the

glycemic response after single oral sucrose ingestion was examined in Wistar albino

rats. At 30 and 60 min after sucrose administration, the rise of blood glucose was

significantly suppressed in rats when the HAE was given orally 3 min before

sucrose administration (Fig. 1). It should be noted that most effective inhibition was

achieved at 30 min when the rise of blood glucose was compared to control. The

inhibitory effect was diminished gradually as time went on and reverted to non

significance at 120 min.

The HAE obtained from I. frutescens leaves was determined for the inhibitory

activity against a-glucosidase isolated from male Swiss albino mice’s small

intestine. HAE exhibited sucrase and maltase inhibitory activity in dose dependent

226 Med Chem Res (2008) 17:219–233

manner as shown Fig. 2. In the experiment in vivo, the HAE 1000 mg/kg

significantly inhibited blood glucose elevation. The IC50 values of HAE were found

to be 335.25 lg/mL, 323.54 lg/mL and 453.54 lg/mL, for the maltase, sucrose, and

isomaltase activity, respectively.

The free-radical scavenging activity of the HAE and a-Tocopherol was observed

in the presence of DPPH radical, superoxide oxide and hydrogen peroxide radicals

(Figs. 3, 4, and 5). Reduction of these two free radicals can be observed by the

decrease in absorbance at 517, 560, and 230 nm, respectively. The DPPH,

superoxide oxide, and hydrogen peroxide radical scavenging capacity of the HAE

0

20

40

60

80

100

120

140

160

0 30 60 120

Time (h)

Control HAE (1000 mg/kg)

Blo

od g

luco

se (

mg/

dl)

Fig. 1 Effect of HAE on blood glucose levels in carbohydrate administration. Ten-week-old male SwissAlbino mice were orally administered sucrose at a dose of 2000 mg/kg alone or with HAE 1000 mg/kg.The mice had been deprived food for 24h before administration. Blood samples were taken at 0, 30, 60,and 120 min after loading. Each point represents the mean ± standard error on the mean (SEM) (n = 6)

0

10

20

30

40

50

60

70

80

90

10 20 40 80 160 320 640

Concentration (µg/ml)

Inhi

bitio

n ra

te (

%)

Maltose 500 Mm

Sucrose 500 Mm

Isomaltose 500 Mm

Fig. 2 Dose response curve for the inhibitory effect of HAE on the activityies of maltase, sucrose, andisomaltase from mouse small intestine

Med Chem Res (2008) 17:219–233 227

were found have IC50 values of 194.06 lg/mL,167.46 lg/mL, and 192.47 lg/mL,

respectively, with respect to the reference compound a-tocopherol (147.91 lg/mL,

152.75 lg/mL, and 133.09 lg/mL), used as a positive control. The superoxide oxide

and hydrogen peroxide free radicals scavenging activity of alcohol–water extract

was shown to be strongly concentration dependent.

Figure 6 shows a decrease in absorbance of control after an initial increase. In

control, the absorbance has increased up to 1.25 at 35 h of incubation. This is due to

the oxidation of linoleic acid, which generates hydroperoxides, which are then

decomposed to many secondary oxidation products. The total antioxidant activity of

HAE increased with increasing concentration. The different concentrations of HAE

(100 and 200 lg/mL) exhibited higher antioxidant activities and the percentage

inhibition of HAE on peroxidation in the linoleic acid emulsion system at 40 h was

87.38 % and 89.11%, whereas the standard antioxidant a-tocopherol exhibited

94.01% inhibition on peroxidation of linoleic acid emulsion.

Treatment of human erythrocytes with 1 mM PHZ as an oxidant causes damage

to erythrocytes, which are assessed by measurement of lipid peroxidation. The in

vitro effect of PHZ is to hemolyze erythrocytes, which was observed in this study;

elevation in the treated samples compared with the control demonstrated that PHZ is

a strong oxidant. The results showed that increasing the dose of HAE (6.25–100 lg/

mL reaction mixture) in the incubation medium checked maximum inhibition

observed with 100 lg/mL of HAE. Like HAE, ascorbic acid is a well-known

antioxidant, which is commonly available in various fruits and vegetables.

Exogenous application of increasing doses of ascorbic acid starting 4.48 lg to

70.4 lg of reaction mixture protects PHZ-treated erythrocytes to some extent.

0

20

40

60

80

100

50 100 150 200 250

Concentration (µg/ml)

Perc

enta

ge r

adic

al s

cave

ngin

g ac

tivity

HAE Alpha tocopherol

Fig. 3 DPPH (1,1,diphenyl-2-picryl-hydrazyl) radical scavenging activity of hydroalcoholic extract of I.frutescens. HAE: hydroalcoholic extract. Each value represents the mean ± SEM of triplicateexperiments

228 Med Chem Res (2008) 17:219–233

HAE was added to the reaction mixture in amounts of 50 lg/mL to 250 lg/mL. A

control was also run under the same conditions without HAE. A dose-dependent

increase in screening of superoxide radicals present was observed. The IC50 value

was found to be 167.46 lg/mL and 152.95 lg/mL for HAE and ascorbic acid,

respectively.

0

20

40

60

80

100

0 50 100 150 200 250

Concentration (µg/ml)

Perc

enta

ge s

cave

ning

HAE Alpha tocopherol

Fig. 4 Superoxide radical scavenging activity of hydroalcoholic extract of I. frutescens by alkalineDMSO method. HAE: hydroalcoholic extract, DMSO: dimethyl sulfoxide. Each value represents themean ± SEM of triplicate experiments

0

20

40

60

80

100

0 50 100 150 200 250

Concentration (µg/ml)

Perc

enta

ge s

cave

ngin

g

HAE Alpha tocopherol

Fig. 5 Hydrogen peroxide scavenging activity of hydroalcoholic extract of I. frutescens (HAE) and a-tocopherol. Each value represents the mean ± SEM of triplicate experiments

Med Chem Res (2008) 17:219–233 229

Discussion

Agents with a-glucosidase inhibitory activity have been useful as oral

hypoglycemic agents for the control of hyperglycemia in patients with diabetes.

These drugs inhibit the digestion of disaccharides, and thus absorption of

glucose, eliciting attenuated postprandial blood glucose levels. There are many

natural sources with a-glucosidase inhibitory activity. These studies suggest that

preventing an excessive postprandial rise of blood glucose level by a-glucosidase

inhibition from natural resources is effective in real life as well. HAE effectively

inhibit sucrase activity and rise of blood glucose level in rats after sucrose

administration. It has recently been reported that polyphenols inhibited glucose

transporter of small-intestinal epithelial cells (Hashimoto et al., 1994). In

addition Thomson et al. (1984) have indicated the possibility that polyphenols

control the rise in blood glucose level when humans were fed with a fixed

amount of carbohydrate with foods, because a negative correlation was indicated

between the polyphenol content and glycemic index. Additionally some

flavonoids and polyphenols as well as sugar derivatives were found to be

effective on the inhibitory activities of a-glucosidase (Haraguchi et al., 1996;

Yoshikawa et al., 1998). It appears that this effect is associated with polyphenols

in HAE. Perhaps these results indicated that suppression of the postprandial

glucose level by HAE was mainly due to disaccharidase inhibition.

The DPPH radical scavenging method is a standard procedure applied to the

evaluation of antiradical activity. DPPH, a stable free radical with a characteristic

absorption at 517 nm, was used to study the radical scavenging effects of extracts.

As antioxidants donate protons to these radicals, the absorption decreases. The

decrease in absorption is taken as a measure of the extent of radical scavenging.

HAE showed dose-dependent DPPH radical scavenging activity. The effect of the

free-radical scavenging activity of HAE on DPPH radicals is thought to be due to th

hydrogen-donation ability of polyphenols from I. frutescens.

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10 15 20 25 30 35 40

Time (h)

Abs

orba

nce

(500

nm

)

HAE 100µg/ml

HAE 200µg/ml

Control

alpha tocopherol

Fig. 6 Total antioxidant activity of the hydroalcoholic extract of I. frutescens and a-tocopherol in thelinoleic acid emulsion determined by the thiocyanate method. HAE: hydroalcoholic extract. Control:linoleic acid emulsion without sample

230 Med Chem Res (2008) 17:219–233

In the present study peroxidative change along with other parameters in oxidative

stress-induced erythrocyte in vitro has been reported. Exogenous application of

increasing doses of HAE starting from 6.25 lg to 100 lg/mL of reaction mixture

protects MDA formation in PHZ-treated erythrocytes to some extent. Phenyl

hydrazine (PHZ), an oxidative toxic agent belonging to hydrazine family, causes

intoxication and leads to severe hemolytic anemia and generates reactive oxygen

species (Misra and Fridovich, 1976). PHZ in the presence of hemoglobin

autooxidizes to form hydrogen peroxide, ultimately producing hydroxyl radicals

that initiate the peroxidation of unsaturated fatty acids in endogenous phospholipids

(Jain and Hochstein, 1979). Herein we observed increased lipid peroxidation, and

turbidity by in vitro treatment of erythrocyte with PHZ. All these observations

indicate involvement of free-radical species by PHZ in vitro. Our data on HAE

experiments show that increasing concentrations of a HAE containing polyphenols

gradually attenuates the level of MDA induced by PHZ in erythrocytes.

Polyphenolics from some fruits and vegetables have also been shown to enhance

red blood cell resistance to oxidative stress in vivo and in vitro (Youdim et al.,2000).

Superoxide radicals were generated according to the alkaline DMSO method

described by Hyland et al. (1981). It has been suggested that the role played by

superoxide is more important for inflammation (Petrone et al., 1980). Table 1 shows

that HAE is a potent scavenger of superoxide radicals. Scavenging is dose-

dependent. We have also studied HAE against oxidative agents and hydrogen

peroxide and suggested that HAE can be an important antioxidant component in

modulating oxidative stress. In conclusion, the major finding of the present study is

that HAE possesses direct antioxidant activity against various free radicals. If we

extrapolate these in vitro results to the in vivo situation, we can assume that the

alcohol–water extract of I. frutescens can interfere at distinct levels in the radical

Table 1 Antioxidant activity and a-glucosidase inhibitory effect of hydroalcoholic extract (HAE) of

I.frutescens on in vivo and in vitro models, as expressed (lg/mL) by inhibitory concentration (IC50). Each

value represents the mean ± SEM of three replicates

Method Inhibitors (lg/mL) Inhibitory concentration ( IC50) (lg/mL)

DPPH radical HAE 194.06

a-tocopherol 147.91

Superoxide radical HAE (50–250) 167.46

a-tocopherol (50–250) 152.95

Hydrogen peroxide radical HAE (50–250) 192.17

a-tocopherol (50–250) 133.89

PHZ-induced hemolysis HAE (62.5–100) 179.84

Ascorbic acid (4.4–70.4) 205.50

a-Glucosidase inhibition

Maltase (2% w/v) 335.25

Sucrase (2% w/v) HAE (10–640) 323.54

Isomaltase (2% w/v) 453.44

Med Chem Res (2008) 17:219–233 231

chain reaction, thus exerting a synergistic effect in mitigating the tissue damage that

occurs during inflammatory diseases.

Based on the FTC method, we found that the amount of peroxide in the initial

stage of lipid peroxidation is greater than the amount of peroxide in the secondary

stage. Furthermore, secondary products such as melondialdehyde are not stable for a

long period of time, and will be converted into alcohol and acids that cannot be

detected spectrophotometrically (Ottolenghi, 1979).

Polyphenolic flavonoids are possible candidates that might explain the antiox-

idant activity of this extract. Leaves are reported to contain high levels of flavonoids

(17.8 mg/g of dry alcohol–water extract). Phenolic constituents are very important

in plants because of their scavenging ability due to their hydroxyl groups. In

addition, it has been reported that phenolic compounds are associated with

antioxidant activity and play an important role in stabilizing lipid peroxidation. The

results of this study show that the alcohol–water extract has significant a-

glucosidase inhibitory activity and antioxidant activity in well-characterized

standard methods in vitro. The various antioxidant mechanism of HAE may be

attributed to its strong abilities as a scavenger of DPPH, superoxide, and hydrogen

peroxide free radicals. However, the components responsible for these activities of

HAE are currently unclear. Therefore, it is suggested that further work be performed

on the isolation and identification of active constituents of HAE. Also in vivo

studies are warranted to investigate I. frutescens as an antioxidant in various

oxidative complications.

Acknowledgements The authors would like to thank All India Council of Technical Education

(AICTE), New Delhi, India for providing financial support to carry out this work.

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