Original article

Screening culinary herbs for antioxidant and a-glucosidaseinhibitory activities

Kai Tze Kee, Marilyn Koh, Li Xuan Oong & Ken Ng*

Department of Agriculture & Food Systems, Melbourne School of Land & Environment, The University of Melbourne, Parkville, Vic., 3010,

Australia

(Received 3 February 2013; Accepted in revised form 24 March 2013)

Summary Twelve commonly consumed culinary herbs were assessed for their potential effect in mitigating oxidative

stress and postprandial hyperglycaemia: River Mint, Vietnamese Mint, Fish Mint, Spearmint, Sweet Basil,

Thai Basil, Coriander, Lemon Verbena, Vietnamese Perilla, Rice Patty, Sawtooth and Rosemary. The

radical scavenging and reducing antioxidant activity of the herbs were quite variable ranging from

31–652 mg Trolox Equivalent to 35–512 mg Ferrous Equivalent per gram dried leaves, respectively. The

herbs were largely inactive against pancreatic a-amylase, but showed strong inhibitions against yeast

a-glucosidase at 100 lg Gallic Acid Equivalent (GAE) per millilitre. Vietnamese Mint is the most potent

herbs with the concentration required for 50% inhibition of activity of 6.9 lg Dried Leaves per millilitre.

In addition, Vietnamese Mint was the only herb that produced significant inhibition of rat intestinal

a-glucosidases, reducing activity to 29.6% at 100 lg GAE mL�1 compared with control.

Keywords Antioxidant, culinary herbs, diabetes, flavonoid contents, ferric reducing antioxidant power, phenolic contents, Trolox equiva-

lent antioxidant capacity, a-glucosidase.

Introduction

Diabetes is a prevalent human health condition world-wide and constitutes a costly health burden to govern-ments, as it reduces the quality and productivity of lifeof affected individuals and contributes to high morbid-ity and mortality (WHO, 2011). There are several typesof diabetes and among these, Type 2 diabetes mellitus(DM) is the most common, accounting for more than80% of all diabetes cases (WHO, 2011). The onset ofDM (prediabetes) occurs when the pancreatic tissues areless responsive to insulin due impaired glucose tolerance(IGT) as a result of glucotoxicity (Tab�ak et al., 2012).Under such conditions, the pancreatic b-cells arestressed to secrete more insulin to maintain blood glu-cose homoeostasis but eventually become impairedresulting in reduce insulin production. Additionally,insulin resistance induces excessive lipolysis by adiposecells resulting in an increase in plasma fatty acids thatcauses decreased insulin response in the skeletal musclesand contributes to weakening of b-cell functions. Thus,full-blown DM is developed.

Both prediabetes and DM are characterised byhyperglycaemia, which leads to a range of adverse

processes in the body such as the glycosylation of extra-cellular proteins, increase in body free radicals load andthe generation of advanced glycation end products as aresult of the cold Maillard Reactions (Gerich, 2003).Researchers have shown that glucose toxicity arisingfrom hyperglycaemia eventually damages the body,resulting in various micro- and macrovascular complica-tions such as retinopathy, neuropathy, nephropathy andsignificantly increases cardiovascular diseases risk(Tab�ak et al., 2012). Thus, the control and managementof hyperglycaemia and postprandial hyperglycaemia(PPHG) are a crucial element in the treatment of diabe-tes (Nyenwe et al., 2011).One of the most effective ways of controlling PPHG

is to suppress starch digestion as it is the main contribu-tor of glucose in the human body from diet. Starchdigestion occurs mainly in the gastrointestinal tract, andit involves the combine action of pancreatic a-amylaseand intestinal a-glucosidases to release absorbable glu-cose monosaccharides (Jones et al., 2011). a-Amylasehydrolyses starch to form linear and branched glucoseoligomers as its major product. As glucose oligomerscannot be absorbed into the bloodstream, absorbablefree glucose monosaccharides are released from theseoligomers by the action of intestinal a-glucosidases. Theinhibition of a-amylase and/or a-glucosidases would*Correspondent: E-mail: ngkf@unimelb.edu.au

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doi:10.1111/ijfs.12166

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retard starch digestion from our diet and reduce post-prandial blood glucose levels (Gerich, 2003). As such, a-glucosidase inhibitor drugs such as acarbose, miglitoland voglibose are often prescribed in combination withother antidiabetic drugs for maximum diabetes treat-ment efficacy (Mizuno et al., 2008).

However, the inhibitory activity of these a-glucosi-dase inhibitors against intestinal a-glucosidases is rela-tively weak compared with their inhibition ofpancreatic a-amylase, resulting in the passage of theundigested starch polymers into the colon where fer-mentation by colonic bacteria occurs (Dehghan-Koo-shkghazi & Mathers, 2004). Starch fermentationcauses gastrointestinal discomfort like flatulence anddiarrhoea in patients, causing many to prematurelyterminate their treatments (Derosa & Maffioli, 2012).A better clinical outcome could be achieved from morespecific a-glucosidase inhibitors with strong inhibitoryactivity against the a-glucosidases but weak or noinhibitory activity against a-amylase, so that the unde-sirable side effects of the starch polymer fermentationare minimised, while still achieving the desired resultof delaying PPHG. In addition, the currently clinicallyprescribed a-glucosidase inhibitors are relatively expen-sive, and the cost is exacerbated by the long-termtreatment regime required for diabetes (Mizuno et al.,2008). Thus, there is a strong interest in herbs andplants as alternative sources of more tolerable andcheaper a-glucosidase inhibitors. Furthermore, the useof cheap, safe and commonly consumed herbs orplants for controlling PPHG especially for those ‘atrisk’ individuals in the early phase of diabetes withIGT is an attractive health management strategy.

Herbs and plants synthesise a variety of secondarymetabolites that possess a variety of roles such asstructure and defence (Rice-Evans, 2001). Phenoliccompounds are among these secondary metabolitesthat are broadly categorised into phenolic acids suchas hydroxybenzoic and hydroxycinnamic acids and po-lyphenols such as flavonoids. Phenolic acids such ascaffeic, q-coumaric, ferulic, gallic and vanillic acids,and flavonoids such as baicalein and quercetin, forexample, can be found in most herbs and plants, whileothers can be found in selected herbs and plants(Rice-Evans, 2001). These phenolic compounds have anumber of beneficial effects owing to their sensory,nutritional and antioxidant properties. Besides, theirhigh antioxidant capacity is linked to the reduction inoxidative damage in the body when consumed andmight thus help to prevent oxidative stress diseases(Ranilla et al., 2010). Many researchers have beeninterested in the use of flavonoids as antidiabetics due totheir efficacious role in mitigating the hyperglycaemiceffects of diabetes, presumably by delaying PPHG byinhibiting starch digestion (Tadera et al., 2006; Ranillaet al., 2010). Compared to flavonoids, hydroxybenzoic

and hydroxycinnamic acids, which are present in abun-dance in herbs, are less studied as a-glucosidase inhibi-tors but more for their antioxidant and antimicrobialproperties (Tundis et al., 2010).In this study, we report on the screening of com-

monly consumed culinary herbs for antioxidant anda-glucosidase inhibitory activities as a possible sourceof functional foods for use in mitigating PPHG and itsassociated oxidative stress.

Materials and methods

Materials

Saccharomyces cerevisiae a-glucosidase (G0660), ratintestinal acetone powder (I1630), porcine pancreatica-amylase (A4268), 4-nitrophenyl-a-D-glucopyranoside(qNPG; N1377), soluble potato starch (S2004), acarbose,quercetin, gallic acid, Folin–Ciocalteu reagent (2M), 3,5-dinitrosalicylic acid (DNS), Na-K tartrate, sodiumcarbonate anhydrous, aluminium chloride hexahydrate(AlCl3�6H2O), potassium acetate, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid (ABTS), potassiumpersul-fate, 6-hydroxy-2, 5, 7, 8-tetramethylchromane- 2-carboxylicacid (Trolox), sodium acetate anhydrous, 2,4,6-tripyridyl-s-triazine (TPTZ), iron[II] dichloride tetrahydrate (FeCl2�4H2O), iron[II] trichloride hydrate (FeCl3�H2O) and iron[II]sulphate heptahydrate (FeSO4�7H2O) were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA). Lowry’sReagents Protein Assay Kit (500-01112) was from Bio-Rad� (UK). All other chemicals and solvents used wereof analytical grade or better. Deionised water(� 18 MO) was produced using a Synergy UV MilliporeSystem (Millipore, Sydney, Australia) and was usedthroughout.

Sample sources

All herbs were obtained fresh from local markets inSpringvale (Victoria, Australia), and four or five lots ofeach herb were purchased from different outlets andmixed for average sampling. The samples were authenti-cated by reference to published sources (referencescited in Table S1). Lipton� Green Tea (Melbourne,Australia) was purchased from a local supermarket. Thecommon names and scientific names of the herbs andtea are listed in Table S1.

Preparation of water extracts

Fresh herbs were thoroughly rinsed with water and airdried at 37 °C using a commercial forced air dehydra-tor (Sunbeam, Sydney, Australia) for 48 h. The dryleaves (DL) were separated from the dry stems, and1 g of DL from each herb was placed in individualSchott glass bottle and was extracted by adding

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100 mL of boiling water and boiled for 5 min. Thehot extracts were then filtered through a WhatmanNo. 4 filter paper to remove insoluble materials andthe filtrates cooled and stored at 4 °C in tightly sealedglass jars until analysis. The commercially obtainedgreen tea dried leaves were similarly extracted andstored.

Determination phenolic content

Phenolic content was determined by the Folin–Ciocal-teu method as described by Waterhouse (2002) withslight modifications. To 100 lL of extract was added100 lL of Folin–Ciocalteu reagent (2M), 1 mL of waterand 300 lL of 20% sodium carbonate solution (v:v).The solution was mixed and then incubated for 30 minat 40 °C. The absorbance of the reduced phospho-molybdotungstate acid was measured at k 765 nm usinga UV-spectrophotometer (Multiskan Spectrum; ThermoElectron Corporation, UK). Gallic acid was used asstandard, and the phenolic content of extracts wasobtained from an absorbance–gallic acid standard curveand was expressed as gallic acid equivalents (GAE).

Determination of flavonoid content

Flavonoid content was determined with AlCl3 asdescribed by Xu et al. (2010) with slight modifications.To 125 lL of extract was added 25 lL of 1 M potassiumacetate, 25 lL of 10% AlCl3�H2O (v:v), 700 lL of waterand 375 lL of methanol. The solution was mixed andincubated for 40 min at room temperature (RT). Theabsorbance of the Al-flavonoid complexes formed wasmeasured at k 415 nm. Quercetin was used as standard,and the flavonoid content of extracts was obtained froman absorbance–quercetin standard curve and wasexpressed as quercetin-equivalents (QE).

Trolox equivalent antioxidant capacity

The free radicals scavenging antioxidant activity of theextracts was measured as the Trolox equivalent anti-oxidant capacity (TEAC) according to Arts et al.(2004). The ABTS radicals were generated by reacting5 mL of 7 mM ABTS with 88 lL of 140 mM potas-sium persulfate in the dark for 16 h at RT. The greensolution was then diluted with methanol until itsabsorbance was approximately 0.8 at k 734 nm. Forthe assay, 1 mL of the diluted ABTS radical solutionwas mixed with 0–100 lL of extract and left at RTfor 15 min before its absorbance measured at k734 nm. A plot of absorbance verse microgram DLwas then obtained for each herb. This method alloweda range of extract concentrations to be taken intoaccount in measuring the radical scavenging capacity,while the concentration of the ABTS radicals kept

constant. A standard curve was obtained using a rangeof Trolox concentrations (0–200 lL of a 0.5 mM solu-tion). The TEAC value was obtained from the ratio ofthe linear gradient of the extract plot to that of the Trol-ox standard plot and expressed as Trolox Equivalent(TE). The linear correlation of the gradients ranges from0.9860 to 0.9997.

Ferric reducing antioxidant power

The reducing antioxidant activity of the extracts wasmeasured as the Ferric Reducing Antioxidant Power(FRAP) according to Benzie & Strain (1996). Threehundred millimolar sodium acetate solution, 10 mM

TPTZ and 20 mM FeCl3�H2O solution were mixedtogether in a 10:1:1 (v:v:v) ratio to make a FRAPreagent. One millilitre of the FRAP reagent was addedto 0–400 lL of extract, and the solution was incubatedfor 10 min at 37 °C. The absorbance of the reducedproduct Fe[II]-[TPTZ]2 was measured at k 593 nm.A plot of absorbance verse microgram DL was thenobtained for each herb. Similar to the TEAC assay, arange of extract concentrations were taken intoaccount in measuring the reducing power, while theconcentration of the electron acceptor kept constant.A Fe[II]-[TPTZ]2 standard curve was obtained using arange of Fe[II] concentration (as FeSO4�7H2O, 200–1000 mM) in the place of FeCl3 in the FRAP reagentto generate the colour. The FRAP value was obtainedfrom the ratio of the linear gradient of the extract plotto that of the Fe(II) standard plot and expressed as Fe[II]-Equivalent (Fe[II]-E). The linear correlation of thegradients ranges from 0.9911–0.9998.

Porcine pancreatic a-amylase assay

A 100 U mL�1 enzyme stock (containing 140 lg pro-tein mL�1) was prepared by diluting the a-amylasesuspension with 20 mM sodium phosphate buffer (pH6.9) containing 6.7 mM NaCl and stored at 4 °C.A 0.5 U mL�1 enzyme solutions (containing0.70 lg protein mL�1) were freshly prepared daily forenzyme activity assay by diluting from enzyme stockwith the phosphate buffer. One unit of enzyme isdefined as activity in liberating 1 lmol of maltose perminute from starch substrate at 37 °C.A series of trials were conducted to identify the opti-

mum conditions for the a-amylase and other enzymeassays. The trials were performed by varying (individu-ally) the enzyme concentration, substrate concentra-tion and incubation time while maintaining all otherconditions constant. Conditions were chosen wherethe incubation time and enzyme concentration weredirectly proportional to enzyme velocity and that thereaction time was not excessive to significantly depletesubstrate or lead to accumulation of high level of

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hydrolysis products. In this assay, however, the starchsubstrate was added in excess.

The following optimal assay conditions were estab-lished: 50 lL of herb extract was mixed with 400 lLof 20 mM sodium phosphate buffer (pH 6.9) contain-ing 6.7 mM of NaCl, followed by 100 lL of enzyme(0.5 U mL�1) and preincubated for 5 min at RT. Fivehundred microlitre of starch solution (0.5% w/v, in20 mM sodium phosphate buffer (pH 6.9) containing6.7 mM NaCl) was then added to the mix and the reac-tion was allowed to proceed for 20 min at 37 °C in awater bath. After which, the reaction was terminatedby the addition of 500 lL of DNS reagent (1% DNS,12% Na-K tartrate, 0.4 M NaOH) and boiled for15 min. The tubes were then cooled to RT and theabsorbance of the chromogenic product of the DNSand reducing sugar reaction was measured at k540 nm. The% enzyme activity was calculated usingthe following equation:

Enzyme activityð%Þ ¼ Abss �AbssbAbsc �Abscb

� 100%;

where Abss = Sample reaction (+ enzyme + starch +extract), Abssb = Sample + starch background (�enzyme+ starch + extract), Absc = Control reaction (+ enzyme +starch �extract) and Abscb = Control background(�enzyme + starch � extract).

Yeast a-glucosidase assay

For the yeast a-glucosidase assay, the pNPG sub-strate was added at 2 9 Km level at 2 mM. The fol-lowing optimal assay conditions were established:100 lL of variable concentration (as GAE mL�1) ofherb or tea extract was mixed with 700 lL of potas-sium phosphate buffer (0.12 M, pH 6.8) followed by100 lL of enzyme (0.18 U mL�1, in 0.12 M potas-sium phosphate buffer) and preincubated for 5 min atRT. One hundred microlitre of qNPG (10 mM, inwater) was then added to the mix, and the reactionwas allowed to proceed for 20 min at 37 °C in awater bath. After which, the reaction was terminatedby the addition of 350 lL of 2 M Na2CO3, and theabsorbance was measured at k 410 nm. One unit ofenzyme activity is defined as the amount of enzymerequired to liberate 1 lmol of p-nitrophenol frompNPG per minute under the assay conditions. The%enzyme activity was calculated using the followingequation:

Enzyme activityð%Þ ¼ Abss �AbssbAbsc

� 100%;

where Abss = Sample reaction (+ enzyme + qNPG+ extract), Abssb = Sample background (�enzyme +qNPG + extract) and Absc = Control reaction (+enzyme + qNPG � extract).

Rat intestinal a-glucosidases assay

One gram of rat intestinal acetone powder was mixedwith 25 mL of potassium phosphate buffer (10 mM

containing 0.9% NaCl) and sonicated for 5 min at4 °C using a tip sonicator and ice water bath. Themixture was then centrifuged at 10 000 g (Eppendorf,model 5415C) for 30 min. The supernatant was recov-ered and used as the enzyme solution for the followingenzyme assay. The protein content of the enzyme prep-aration was determined to be 11.08 � 0.30 mg mL�1

using the Lowry protein determination method.For rat intestinal a-glucosidases assay, the pNPG

substrate was also added at 2 9 Km level at 2.5 mM.The following optimal assay conditions were estab-lished: 100 lL of variable concentration of herb extract(and/or water) was mixed with 730 lL of potassiumphosphate buffer (0.12 M, pH 6.8) followed by 70 lL ofenzyme and preincubated for 5 min at RT. Hundremicrolitre of qNPG (25 mM, in water) was then addedto the mix and the reaction was allowed to proceed for20 min at 37 °C in a water bath. After which, the reac-tion was terminated by the addition of 350 lL of 2 M

sodium carbonated and the absorbance was measuredat k 410 nm (Multiskan Spectrum). The% enzymeactivity was calculated using the following equation:

Enzyme activityð%Þ ¼ Abss �AbssbAbsc �Abscb

� 100%;

where Abss = Sample reaction (+ enzyme + qNPG+ extract), Abssb = Sample + enzyme background (+enzyme � qNPG + extract), Absc = Control reaction(+ enzyme + qNPG � extract) and Abscb = Controlbackground (+ enzyme � qNPG - extract).

Ic50

Active extracts were assessed for their potency by theirIC50 values. This is defined as the concentration of anextract in terms of DL or GAE required for reducing50% of the enzyme activity obtained from an activityversus concentration plot. The data points were fittedinto a nonlinear sigmoid plot to take into accountnonlinear concentration dependent of enzyme-inhibitorinteraction at low and high concentrations (Burling-ham & Widlanski, 2003).

Statistical analysis

For the phenolic and flavonoid contents and the inhibi-tion percentages, data values were expressed as mean�SDof triplicate determinations. Uncertainty of the meanwas reported to two significant figures according to theEuropean Analytical Chemist guidelines (Ellison &Williams, 2012). Statistical comparisons were performedusing one-way analysis of variance (ANOVA) and

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followed by the Fisher’s protected Least Significant Dif-ference Test using SAS software version 9.2. Analyseswith P-values <0.05 were considered to be statisticallydifferent. Linear correlation between two variables wasby linear regression method reporting the linear correla-tion coefficient (R2).

Results and discussion

Phenolic and flavonoid contents

We determined the phenolic and flavonoid contents ofthe herbs to quantify their potential antioxidant andenzyme inhibitory activities in those terms. The phenoliccontents of the hot aqueous extract of the twelve herbwere quite variable, ranging from 8.06–93.8 mg GAE gDL�1 (Table S2). River Mint (93.8 mg GAE g DL�1)and Rice Patty (40.0 mg GAE g DL�1) have signifi-cantly higher level of phenolic content than all the otherherbs, while Rosemary (8.92 mg GAE g DL�1) andFishmint (8.06 mg GAE g DL�1) have the lowest levels.Vietnamese Perilla (27.1 mg GAE g DL�1), Spearmint(24.1 mg GAE g DL�1), Vietnamese Mint (24.0 mg GAEg DL�1) and Lemon Verbena (23.9 mg GAE g DL�1)have similar levels of phenolic content, and, together withRiver Mint and Rice Patty, have higher level of phenoliccontent than Lipton Green Tea (20.2 mg GAE g DL�1)which were measured for the comparison (Table S2).

The flavonoid contents of the herbs’ hot aqueousextracts were also quite variable, ranging from 0.40 to8.28 mg Quercetin Equivalent (QE) g DL�1, Table S2).Herbs with high level of phenolic contents such as RiverMint (8.28 mg QE g DL�1) and Rice Patty (5.28 mgQE g DL�1) also have high level of flavonoid contents.The percentages of flavonoid to total phenol rangedfrom a high of 21.2% for Coriander to a low of 4.5%for Rosemary. This indicated that flavonoid compoundscontributed to a significant proportion of the herbs’phenolic contents. Significantly, a number of the herbs(River Mint, Rice Patty, Vietnamese Perilla, Spearmint,Vietnamese Mint, Lemon Verbena and Coriander) havehigher flavonoid content than Lipton Green Tea(2.55 mg QE g DL�1). As the flavonoid determinationmeasures a compound’s capacity to chelate Al3+ ions,only flavonoids containing suitable chelating structuralfeatures (C-ring C4 = O and C-ring C3-OH or A-ringC5-OH pair; B-ring C4′-OH and C3′-OH or C5′-OHpair) such as flavonols and flavanonols are detected(Rice-Evans, 2001). Tea flavonoids, however, are mainlycatechin which does not chelate Al3+ unless it containsB-ring dihydroxy groups (Higdon & Frei, 2003).

TEAC and FRAP

The herbs were evaluated for their antioxidant activityusing two chemical reactions. The radical scavenging

capacities of the herbs’ extracts were determined as theTEAC using APTS radical as the organic radical andwas expressed as TE (Arts et al., 2004). The TEACof the herbs were quite variable ranging from 31 to652 mg TE g DL�1 (Table S2). Notably, River Mint(652 mg TE g DL�1), Spearmint (256 mg TE g DL�1)and Rice Patty (247 mg TE g DL�1) has higher TEACthan Green Tea (223 mg TE g DL�1), the later wellknown for its high antioxidant activity (Higdon & Frei,2003). Coriander (31 mg TE g DL�1), Sawtooth (51 mgTE g DL�1), Rosemary (40 mg TE g DL�1) and Fish-mint (32 mg TE g DL�1), however, have muchlower TEAC than the other herbs and the Green Teacontrol.The reducing power of the extracts were determined

as the FRAP using Fe[III]-[TPTZ]2 complex as theelectron acceptor and expressed as ferrous equivalent(Fe[II]-E) (Benzie & Strain, 1996). River Mint showedthe highest FRAP at 512 mg Fe[II]-E g DL�1, whileCoriander (60 mg Fe[II]-E g DL�1), Sawtooth (63 mgFe[II]-E g DL�1), Rosemary (59 mg Fe[II]-E g DL�1)and Fishmint (33 mg Fe[II]-E g DL�1) were amongthe lowest of the herbs (Table S2). Green Tea(375 mg Fe[II]-E g DL�1) has high FRAP as was pre-viously reported (Higdon & Frei, 2003), but RiverMint has higher FRAP than then Green Tea while allthe other herbs including Spear Mint (119 mg Fe[II]-E g DL�1) and Rice Patty (135 mg Fe[II]-E g DL�1)has lower FRAP. Thus, while the antioxidant activityof the herbs as measured by the TEAC and FRAPassay was similar in that strong herb has high TEACand FRAP value and vice versa for weak herb, theirrelative potency to Green Tea differs. This reflectsthe higher electron donating capacity of the teacatechin relative to their APTS radical scavengingcapacity.The correlations between TEAC values and the phe-

nolic and flavonoid contents of the herbs is shown inFigure S1a and b. Overall, both phenolic and flavo-noid contents were well correlated to radical scaveng-ing capacity, but phenolic content (R2 = 0.9279) has ahigher degree of correlation than flavonoid content(R2 = 0.7283). This points to the fact that althoughflavonoids are a major source of scavenging activity inthe herbs, there are other nonflavonoid phenolic com-pounds present that are also contributing significantlyto radical scavenging capacity.The correlations between FRAP values and the phe-

nolic and flavonoid content is shown in Figure S1cand d. Again, although both phenolic and flavonoidcontents were highly correlated with reducing capacity,phenolic content (R2 = 0.9623) showed a higher corre-lation than flavonoid content (R2 = 0.7028). Thisfurther implies that nonflavonoid phenolic compoundsin the sample extracts have a role in reducing capacityas well as in overall antioxidant activity.

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Inhibition of a-amylase

The herbs were evaluated for their inhibitory activityagainst porcine pancreatic a-amylase as an indicationof their ability to inhibit starch digestion. The inhibi-tory activity was measured at a fixed concentration of100 lg GAE mL�1 to detect inhibitory activity thatmight present in the herbs. Only Vietnamese Mint(17.7%) and Perilla (17.8%) showed inhibition ofactivity compared with control without added extractand that their inhibitions were low at the rather highconcentration of the herbs tested. All the other herbsextracts were inactive. This is not surprising becausethe substrate of a-amylase is starch, and there is arequirement of the enzyme for multiple sugar unitssubstrate for strong substrate binding (Tundis et al.,2010), and leaf materials would lack sugar-basedinhibitors of the enzyme. We also determined theinhibitory activity of acarbose, a pseudotetrasaccharideand a prescribed a-glucosidase inhibitor class antidia-betic drug (Mizuno et al., 2008), as a control assess-ment of our assay which showed strong inhibition ofthe pancreatic a-amylase with an IC50 value of3.67 � 0.21 lM. This value is similar to some previ-ously reported values: 2.71 lM (Kim et al., 2004) and15.5 lM (Kim et al., 2002).

Inhibition of yeast a-glucosidase

Yeast a-glucosidase has been used in vast number ofstudies to screen for a-glucosidase inhibitors in foods(Xu, 2010). Similar to porcine pancreatic a-amylase, ascreening at a fixed concentration of 100 lg GAE mL�1

was initially conducted to access relative potency, and awide range of potency was observed (Fig. S2). Amongthe culinary herbs analysed, Vietnamese Mint and RiverMint produced complete inhibition at this concentra-tion. Inhibition by the other herbs ranged widely, from9.6 to 94.7%. Green Tea was also very effective in inhib-iting yeast a-glucosidase as was previously reported byothers (Toshima et al., 2010), with complete inhibitionat the 100 lg GAE mL�1 concentration tested. Theresults reflect on the different composition or proportionof phenolic compounds present in the different herbsand tea, because there is a structure–activity relation-ship with phenolic compounds in the inhibition of yeasta-glucosidase (Xu, 2010).

To quantify the inhibition potency, the concentrationof the herb required for achieving 50% inhibition of themaximum enzyme activity (IC50) of the more potentsamples were determined and expressed as weight ofdried leaves (lg DL mL�1) and GAE (lg GAE mL�1)(Table S3). The results showed significantly different val-ues between the herbs. Vietnamese Mint was the mostpotent of all the herbs, with an IC50 of 6.9 lg DL mL�1

or 0.165 lg GAE mL�1 and was of similar potency to

Green Tea (IC50 of 5.2 lg DL mL�1 or0.104 lg GAE mL�1). River Mint (29.5 lg DL mL�1

or 2.7 lg GAE mL�1) and Spearmint (50 lg DL mL�1

or 1.19 lg GAE mL�1) were also strong inhibitors ofyeast a-glucosidase. These three herbs comparedfavourably with some reported foods with strong inhibi-tion against yeast a-glucosidase, such as Norton grapeskins (10.5 lg dried materials (DM) mL�1; Zhanget al., 2011), Dinkum raspberries (16.8 lg DM mL�1;Zhang et al., 2010) and edible brown algae(22 lg DM mL�1; Iwai, 2008).It was clear that measuring inhibition at fixed con-

centration provides rather limited information as canbe seen with Spearmint and Rosemary, with gave IC50

values of 1.19 lg and 22.6 lg GAE mL�1 but pro-duced only 94.7 and 83.2% inhibition at 100 lg GAEmL�1, respectively. It indicated that both the Spear-mint and Rosemary extracts could not achieved com-plete inhibition of the enzyme activity even atconcentration in excess of their IC50 values.

Inhibition of rat intestinal a-glucosidases

The a-glucosidase activities in human starch digestionare contributed by two small intestinal mucosalbrush-border enzyme-complexes: maltase–glucoamy-lase (EC 3.2.1.20 and 3.2.1.3) and sucrase–isomaltase(EC 3.2.148 and 3.2.10) (Jones et al., 2011). Themajority of the maltase activity is contributed by thesucrase–isomaltase complex due to its higher relativeabundance, but short, linear oligomers such as malto-triose and maltotetrose are preferentially hydrolysedby the maltase–glucoamylase complex. The rat intesti-nal a-glucosidases, unlike yeast a-glucosidase, aremammalian enzymes similar to human and are thusmore relevant for use in identifying a-glucosidaseinhibitors from foods that might be effective in inhibit-ing starch digestions in the gastrointestinal tract (Simet al., 2010). Furthermore, using a common pNPGsubstrate, all the a-glucosidases activities are assayedeven though it does not provide information on theirrelative inhibitions.Among the herbs, only Vietnamese Mint showed a

reasonable level of inhibition of the rat intestinala-glucosidases, with 29.6% inhibition at 100 lg GAEmL�1 (Fig. S3). Due to the weak inhibition of the herbs,IC50 can only be determined accurately for VietnameseMint which gave a value of 7.75 mg DL mL�1 or186 lg GAE mL�1 (Table S3). Thus, Vietnamese Mint isweaker than Norton grape skin (IC50 ~400 lg DM mL�1

measured with pNPG; Zhang et al., 2011), guava leaves(IC50 970 lg DM mL�1 and 1.28 mg DM mL�1

measured with maltose and sucrose, respectively; Mai &Chuyen, 2007) and Finger Millet seed coat (IC50

16.9 lg GAE mL�1 measured with pNPG; Shobanaet al., 2009) in inhibiting rat intestinal a-glucosidases.

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Green Tea, which inhibited activity by 52.3% at100 lg GAE mL�1 showed higher inhibition than allthe herbs measured at the same concentration of pheno-lic materials (Fig. S3). It was also about twice as potentas Vietnamese Mint, with an IC50 of 4.64 mg DL mL�1

or 92.8 lg GAE mL�1 (Table S3). This value can becompared with the values of 296 lg GAE mL�1 for agreen tea and 58 lg GAE mL�1 for a black teaobtained by Koh et al. (2010) determined with the sameenzyme and substrate.

Overall, the results showed that the herbs wereeither inactive or had much weaker inhibitory activityagainst rat intestinal a-glucosidases than against yeasta-glucosidase. This can be explained by the fact thatthe mammalian (rat) enzymes belong to Family II ofthe a-glucosidases, while the yeast enzyme belongs toFamily I group which differs in primary structure andinhibitor binding (Chiba, 1997). Furthermore, theenzyme activities of rat intestinal a-glucosidases arecontributed by four enzyme activities (maltase, isomal-tase, sucrase and glucoamylase), while the yeast a-glucosidase expresses only the maltase activity. Thereis a possibility that the Vietnamese Mint and GreenTea specifically inhibited the maltase activity, whichresulted in their strong inhibition of the yeast a-gluco-sidase but weak inhibition of the rat intestinal a-gluco-sidases. As there are other enzyme activities in the rata-glucosidases complex, inhibiting maltase activityalone does not result in the ‘complete’ inhibition ofactivities as the other nonmaltase activities can stillhydrolyse the pNPG substrate in the reaction (Hoganet al., 2010). Furthermore, the possibility exists thatthe yeast and rat enzymes might also show differencesin their maltase inhibitor binding strength.

Conclusions

The vast differences in the inhibitory potencies of theherbs on the yeast and rat intestinal a-glucosidaseshighlighted the importance of enzyme system used toscreen foods, plants and herbs or pure compounds fora-glucosidase inhibitors. While inhibition of the ratenzymes by the active herbs were much weaker thanthe yeast enzyme, it is inhibition of the rat enzymesthat has physiological relevance with regard to theinhibition of starch digestion in humans for thepurpose of mitigating postprandial hyperglycaemia.Among the herbs, only Vietnamese mint showed somepromises as a useful antihyperglycaemic herb becauseit inhibited rat intestinal a-glucosidases but not pan-creatic a-amylase at 100 lg GAE mL�1. This concen-tration of the herb can be easily achieved in thegastrointestinal tract with intake of g quantity of herbsor concentrated extracts during meals.

References

Arts, M.J.T.J., Sebastiaan, D.J., Voss, H.P., Haenen, G.R.M.M. &Bast, A. (2004). A new approach to assess the total antioxidantcapacity using the TEAC assay. Food Chemistry, 88, 567–570.

Benzie, I.F.F. & Strain, J.J. (1996). The ferric reducing ability ofplasma (FRAP) as a measure of “antioxidant power”: the FRAPassay. Analytical Biochemistry, 239, 70–76.

Burlingham, B.T. & Widlanski, T.S. (2003). An intuitive look at therelationship of Ki and IC50: a more general use for the Dixon plot.Journal of Chemical Education, 80, 214–218.

Chiba, S. (1997). Molecular mechanism in a-glucosidase and glu-coamylase. Bioscience, Biotechnology, and Biochemistry, 61, 1233–1239.

Dehghan-Kooshkghazi, M. & Mathers, J.C. (2004). Starch digestion,large-bowel fermentation and intestinal mucosal cell proliferationin rats treated with the [alpha]-glucosidase inhibitor acarbose. TheBritish Journal of Nutrition, 91, 357–365.

Derosa, G. & Maffioli, P. (2012). Efficacy and safety profileevaluation of acarbose alone and in association with other antidia-betic drugs: a systematic review. Clinical Therapeutics, 34,1221–1236.

Ellison, S.L.R. & Williams, A. (2012). Eurachem/CITAC guide:Quantifying Uncertainty in Analytical Measurement, 3rd edn. www.eurachem.org.

Gerich, J.E. (2003). Clinical significance, pathogenesis, and manage-ment of postprandial hyperglycemia. Archives of Internal Medicine,163, 1306–1316.

Higdon, J.V. & Frei, B. (2003). Tea catechins and polyphenols:health effects, metabolism, and antioxidant functions. CriticalReviews in Food Science and Nutrition, 43, 89–143.

Hogan, S., Zhang, L., Li, J., Sun, S., Canning, C. & Zhou, K.(2010). Antioxidant rich grape pomace extract suppresses postpran-dial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase. Nutrition & Metabolism, 7, 71.

Iwai, K. (2008). Antidiabetic and antioxidant effects of polyphenolsin brown alga Ecklonia stolonifera in genetically diabetic KK-Ay

Mice. Plant Foods for Human Nutrition, 63, 163–169.Jones, K., Sim, L., Mohan, S. et al. (2011). Mapping the intestinalalpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase. Bioorganic & MedicinalChemistry, 19, 3929–3934.

Kim, S.H., Kwon, C.S., Lee, J.S., Son, K.H., Lim, J.K. & Kim, J.S.(2002). Inhibition of carbohydrate-digesting enzymes and ameliora-tion of glucose tolerance by Korean medicinal herbs. Journal ofFood Science and Nutrition, 7, 62–66.

Kim, Y.M., Wang, M.H. & Rhee, H.I. (2004). A novel a-glucosidaseinhibitor from pine bark. Carbohydrate Research, 339, 715–717.

Koh, L.W., Wong, L.L., Loo, Y.Y., Kasapis, S. & Huang, D.(2010). Evaluation of different teas against starch digestibility bymammalian glycosidases. Journal of Agricultural and Food Chemis-try, 58, 148–154.

Mai, T.T. & Chuyen, N.V. (2007). Anti-hyperglycemic activity of anaqueous extract from flower buds of Cleistocalyx operculatus(Roxb.) Merr and Perry. Bioscience, Biotechnology, and Biochemis-try, 71, 69–76.

Mizuno, C.S., Chittiboyina, A.G., Kurtz, T.W., Pershadsingh, H.A.& Avery, M.A. (2008). Type 2 diabetes and oral antihyperglycemicdrugs. Current Medicinal Chemistry, 15, 61–74.

Nyenwe, E.A., Jerkins, T.W., Umpierrez, G.E. & Kitabchi, A.E.(2011). Management of type 2 diabetes: evolving strategies for thetreatment of patients with type 2 diabetes. Metabolism Clinical andExperimental, 60, 1–23.

Ranilla, L.G., Kwon, Y., Apostolidis, E. & Shetty, K. (2010). Phe-nolic compounds, antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia and

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Antidiabetic herbs K. T. Kee et al.1890

hypertension of commonly used medicinal plants, herbs and spicesin Latin America. Bioresource Technology, 101, 4676–4689.

Rice-Evans, C. (2001). Flavonoid antioxidants. Current MedicinalChemistry, 8, 797–807.

Shobana, S., Sreerama, Y.N. & Malleshi, N.G. (2009). Compositionand enzyme inhibitory properties of finger millet (Eleusine coracanaL.) seed coat phenolics: mode of inhibition of a-glucosidase andpancreatic amylase. Food Chemistry, 115, 1268–1273.

Sim, L., Willemsma, C., Mohan, S., Naim, H.Y., Pinto, B.M. &Rose, D.R. (2010). Structural basis for substrate selectivity inhuman maltase-glucoamylase and sucrase-isomaltase n-terminaldomains. The Journal of Biological Chemistry, 285, 17763–17770.

Tab�ak, A.G., Herder, C., Rathmann, W., Brunner, E.J. & Kivim€aki,M. (2012). ‘Prediabetes: a high-risk state for diabetes development.Lancet, 379, 2279–2290.

Tadera, K., Minami, Y., Takamatsu, K. & Matsuoka, T. (2006).Inhibition of a-glucosidase and a-amylase by flavonoids. Journal ofNutritional Science and Vitaminology, 52, 149–153.

Toshima, A., Matsui, T., Noguchi, M. et al. (2010). Identification ofa-glucosidase inhibitors from a new fermented tea obtained by tea-rolling processing of loquat (Eriobotrya japonica) and green tealeaves. Journal of the Science of Food and Agriculture, 90, 1545–1550.

Tundis, R., Loizzo, M.R. & Menichini, F. (2010). Natural productsas a-amylase and a-glucosidase inhibitors and their hypoglycaemicpotential in the treatment of diabetes: an update. Mini-Reviews inMedicinal Chemistry, 10, 315–331.

Waterhouse, A.L. (2002). Determination of total phenolics. CurrentProtocols in Food Analytical Chemistry, I1.1.1–I1.1.8.

World Health Organization(2011). Diabetes. Available at: http://www.who.int/mediacentre/factsheets/fs312/en/index.html. (Accessed19 April 2012).

Xu, H. (2010). Inhibition kinetics of flavonoids on yeast a-glucosi-dase merged with docking simulations. Protein & Peptide Letters,17, 1270–1279.

Xu, M.L., Wang, L., Hu, J.H., Lee, S.K. & Wang, M.H. (2010).Antioxidant activities and related polyphenolic constituents of themethanol extract fractions from Broussonetia papyrifera stem barkand wood. Food Science and Biotechnology, 19, 677–682.

Zhang, L., Li, J., Hogan, S., Chung, H., Welbaum, G.E. & Zhou,K. (2010). Inhibitory effect of raspberries on starch digestiveenzyme and their antioxidant properties and phenolic composition.Food Chemistry, 119, 592–599.

Zhang, L., Hogan, S., Li, J. et al. (2011). Grape skin extract inhibitsmammalian intestinal a-glucosidase activity and suppresses post-prandial glycemic response in streptozocin-treated mice. FoodChemistry, 126, 466–471.

Supporting Information

Additional Supporting Information may be found inthe online version of this article:Figure S1. Linear correlation coefficient between

TEAC/FRAP and phenolic/flavonoid content.Figure S2. Inhibition of yeast alpha-glucosidase

activity.Figure S3. Inhibition of rat intestinal alpha-glucosi-

dase activity.Table S1. Common names and scientific names used

in the study.Table S2. Phenolic content, flavonoid content,

TEAC and FRAP.Table S3. IC50 of water extracts against yeast and

rat intestinal alpha-glucosidases.

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Antidiabetic herbs K. T. Kee et al. 1891