Controlling postprandial hyperglycaemiaat the early pre-diabetes stage is an important preventative strategy against developing full blown diabetes mellitus and in glycemic control for diabetics (DM)^[1]. The WHO advises a controlled and balanced diet with low calorie intake and also active lifestyle with certain degree of exercise to lower the risk of developingDM^[2]. For long term treatment and management of DM, medical approaches usually involve drugs that can be used together with lifestyle interventions for glycemic control and delay the onset of diabetic complications^[3]. Several pharmaceutical drugs are currentlyavailable to treat DMand are clinically prescribed in monotherapy or in combination therapy that usually included α-glucosidase inhibitors (AGIs),to increase efficacy in glycemic control and modulation of postprandial hyperglycaemia^[4-6].

  Synthetic AGIs such as acarbose, voglibose, and miglitol have been available as oral anti-diabetic drugs since early 1990s, and acarbose has become the most commonly prescribed AGI^[7]. Unlike the other oral anti-diabetic drugs, AGIs act locally in the gastrointestinal tract by inhibiting intestinal starch digestion^[8]. However, treatment with AGIs is costly which is made worst by the long-term requirement for the drugs in diabetes^[6]. Furthermore, the very strong inhibitory action of acarbose against pancreatic α-amylase limits starch digestion that promotesundigested starch fermentation in the colon,which resulted inunpleasant gastrointestinal side effects such as increases production of gases, flatulence, abdominal pain, and diarrhoea. These side effects are responsible for significant discontinuation of AGIs therapy by patients^[4,7].

  Flavonoids are a class of plant secondary metabolites with a basic structure of a 15-carbon skeleton consisting two benzene rings (ring A and ring B) and a heterocyclic ring (ring C). Over 9000 flavonoid species have been identified to date and the number is still growing^[8]. Flavonoids are specially enriched in foods like parsley, blueberries, citrus, cocoa, peanut, black and green tea, and are also found in medicinal herbs^[9-11]. Several plant productshave been shown to have potential for use inDM treatment. For instance, grape skin extract has been shown to inhibits mammalian intestinal α-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice^[12]. Green tea extract has been shown tostrongly inhibit rat intestinal α-glucosidase^[9], and the in vitro effects of the combined treatments of green tea flavonoid extracts and acarbose have been shown to produce synergistic effect in inhibiting α-amylase and α-glucosidase at low enzyme concentrations^[13]. All these evidence indicate that flavonoids have great potential in DM prevention and management by controlling postprandial hyperglycaemia.

  In this paper we compare the inhibition of mammalian α-amylase and α-glucosidase by different classes of flavonoids so as to get a better understanding of their contribution to inhibitory activities in plant foods or herbs. We also raised the issue of using starch/3,5-dinitrosalicylic acid reagent in accessing the inhibition of α-amylase by flavonoids.

Experimental Section
Chemicals
   Acarbose (A8980), 3,5-dinitrosalicylic acid (DNS; 128848), ρ-nitrophenyl-α-D-glucopyranoside (ρNPG; N7014), p-nitrophenyl-α-D-maltopentaoside (pNPG5; N1519), porcine pancreatic α-amylase (A4262), rat intestinal acetone powder (I1630) were from Sigma-Aldrich (Castle Hills, NSW).

  The following phenolic acids and flavonoids were also from Sigma-Aldrich (Castle Hills, NSW). Phenolic acids: p-Coumaric acid (C9008), 3,4-dihydroxybenzoic acid (protocatechuic acid; 37580), ferulic acid (128708), 4-hydroxybenzoic acid (240141), sinapic acid (D7927), and 3,4,5-trihydroxybenzoic acid (gallic acid; 398225). Flavonoids:Apigenin (A3145), baicalein (465119), baicalin (572667), chrysin (C80105), daidzein (D7802), (±) -catechin (C1788), (-)-epigallocatechin gallate (E4143), (-)-epigallocatechin (E3768), flavone (F20003), galangin (282200), genistein (G6649), 5-hydroxyflavone (H4405), 6-hydroxyflavone (411035), 7-hydroxyflavone (H4530), isoquercitrin (quercetin 3-O-β-D-glucoside; 17793), kaempferol (K0133), oroxylin A (O0641), myricetin (70050), naringenin (N5893), pinocembrin (P5239), quercetin (Q0125), rutin (quercetin 3-O-β-D-rutinoside; R5143).

  All other chemicals and organic solvents used were of analytical-grade or better. Deionised water (18 mΩ) used was produced using a Synergy UV Millipore System (Millipore).

Preparation of rat intestinal α-Glucosidase
  Mixture of rat intestinal α-glucosidase was prepared by suspending 1 g of freeze-dried rat intestinal acetone powder with 25 mL of PBS. The mixture was maintained chilled in an ice/water bath and sonicated using a tip sonicator with f = 50 Hz (Unisonics, Sydney, Australia) for 8 min. The sonicated mixture was then centrifuged for 30 minutes at 14,000 rpm using a bench top centrifuged and the supernatant containing the α-glucosidasewas recovered, pooled and kept at -20°C until use. Protein content was determined to be 0.21 mg protein/mL using the Bradford method and bovine serum albumin as the protein standard (Sigma-Aldrich).

Assay of α-glucosidase activity
  Rat intestinal α-glucosidase activity was assayed using pNPG substrate and spectroscopic determination of the liberated p-nitrophenol by absorbance measurement as previously described^[9]. 100 μL of phenolic acid or flavonoid inhibitor (10 mM in DMSO) was mixed with 100 μL of rat intestinal α-glucosidase (0.21 mg protein/mL) and 700 μL of buffer (0.12 M potassium phosphate pH 6.8 + 1 % w:w NaCl) in an Eppendorf tube. The mixture was pre-incubated at 37°C for 5 min after which 100 μL of ρNPG (2.5 mM in water) was added and the reaction mixture further incubated at 37°C for 60 min. Termination of reaction was achieved by chilling the tubes in ice/water bath, rather than using NaOH or Tris alkaline buffer to avoid changes in absorbance of mixture due to instability of flavonoids in alkaline conditions. The absorbance of the liberated ρ-nitrophenol was promptly measured at λ 410 nm using a spectrophotometer (Novaspec II, USA). Reaction tubes without added pNPG substrate and with or without inhibitor were used as blank to correct for α-glucosidase extract colour and inhibitor absorbance. The percentage Inhibition of α-glucosidase activity was calculated by using the formula:

Determination of IC50 for α-glucosidase activity
   The IC50s (concentration of inhibitor required to reduce 50% of enzyme activity) for α-glucosidase activity were determined for strong inhibitory compounds by measuring % inhibition of activity at a range of inhibitor concentrations. Enzyme activity versus inhibitor concentration plot was then produced for each inhibitor and data points were fitted by a non-linear regression method (sigmoidal) to fit the non-linear concentration dependent of enzyme-inhibitor interaction at low and high concentrations^[14]. All measurements were done in triplicate and the mean IC50 ± SD were obtained.

Assay of α-amylase activity
  α-Amylase activity was assayed using the chromogenic substrate p-nitrophenyl-α-D-maltopentaoside (PNPG5) as described by Funke and Melzig^[15] with modifications. The assay was adapted to microplate platform using 96 wells microplate. Porcine pancreas α-amylase was diluted to 50 units/mL in buffer (20 mM sodium phosphate buffer pH 6.8 + 7 mM NaCl + 1 mM CaCl₂). PNPG5 was made up to 25 mM in the same buffer. 50 μL of PNPG5, 10 μL of flavonoids (in DMSO), and 190 μL buffer were added to each well, and 50 μL of enzyme was added to initiate the reaction. Absorbance was measured at λ 405 nm using a Multiscan Spectrum microplate reader (Multiskan GO, Thermo Scientific, Australia) at 4 minute intervals for up to 120 minutes. Absorbance for control enzyme activity without inhibitor and for inhibitor background without added enzyme was also acquired. The percentage inhibition of α-amylase activity was calculated by the ratio of the linear gradients of control and extract incubation.

Statistical analysis
  Percentage Inhibition and IC50 values were expressed as mean ± SD from triplicate determinations. Uncertainty of the mean was reported to 2 significant figures according to the European Analytical Chemist Guidelines^[16]. Statistical comparisons of means were performed using one-way analysis of variance (ANOVA) followed by the Fisher's protected Least Significant Difference Test using SAS software version 9.2. Analyses with p < 0.05 were considered to be statistically different.

Results and Discussion

Inhibition of rat intestinal α-glucosidase by flavonoids
  Figure 1 shows the structures of the flavonoids studied, which are flavones (flavone, 5-hydroxyflavone, 6-hydroxy-flavone, 7-hydroxyflavone, chrysin, baicalein, baicalin, oroxylin A), isoflavones (genistein, daidzein), flavonols (galangan, kaempferol, quercetin, myricetin), flavanones (pinocembrin, naringenin), and flavanols ((±)-catechin, (-)-epigallocatechin, (-)-epigallocatechin gallate). Initial screening of flavonoidinhibitory activity against rat intestinal α-glucosidaseas an indication of itsanti-hyperglycemic potential as inhibitor of starch digestion was carried out at a fixed concentration of 1 mM. This is so that comparisons to be made between and within the five subclasses of flavonoids (Table 1). Some hydroxybenzoic acids (4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid) and hydroxycinnamic acids (p-coumaric acid, ferulic acid, sinapic acid) were also screen for inhibitory activity.

Figure 1: Structures of flavonoids studied

Table 1: Inhibition of α-amylase and α-glucosidase activity

* Inhibitory effect of compounds was measured at 1 mMand percentage inhibition was relative to control reaction without added inhibitor.
** Concentration of compound that produced 50% inhibition of activity derived from non-linear Percentage Inhibition verses Inhibitor Concentration plots.
- Not determined.
^a,b,c Values in the same column followed by different superscript were significantly different (P < 0.05) in ANOVA and LSD test.

  None of the hydroxybenzoic and hydroxycinnamic acids was inhibitory (data not shown). Of the flavone series, baicalein was the most potent inhibitor. Apigenin, chrysin, baicalin and oroxylin A were weak inhibitors, andflavone, 5-hydroxyflavone, 6-hydroxyflavone and 7-hydroxyflanovewere not inhibitory. Of the flavonols series, myricetin was the strongest inhibitor while galangan, kaempferol and quercetin were weak inhibitors. Both isoquercitrin and rutin which have sugar attached to the C-ring C3-OH were not inhibitory. Of the flavanols series, epigallocatechin gallate was the strongest inhibitors while epigallocatechin was weak and catechin not inhibitory. The isoflavones genistein and daidzein, and the flavanones pinocembrin and naringenin were weak inhibitors.

  Clearly, the flavonoid structure andthe position and number of hydroxyl groups are determining factors for α-glucosidase inhibition.The primary importance of the A-ring C5-OH, C6-OH and C7-OH group was cleared as shown by the inhibitory effect of the flavones series, which were in accord with previous reports^[17-20]. Modification of any one of the hydroxyl groups in the A-ring diminished inhibitory activity as seen with oroxylin A (methylated C6-OH) and baicalin (glucuronidated C7-OH). Likewise, reduced number of hydroxyl groups in the A-ring also diminished inhibitory activity as seen with flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and chrysin and apigenin (both lacking C6-OH). The three B-ring OH groups in myricetin mitigated for the lack of A-ring C6-OH in the structure for its observed inhibitory activitythat was comparable to baicalein.

  In order to get a better evaluation of the potency of the inhibitory flavonoids, the IC50, which is the concentration of inhibitor required for 50% reduction in activity, were determined (Table 1). Baicalein was the most potent inhibitor with an IC50 of 0.713 mM, followed closely by myricetin at 0.908 mM and (-)-epigallocatechin gallate at 2.208 mM. The most notable feature of the IC50 results was that none of the inhibitory flavonoids were able to completely inhibit α-glucosidase activityat high concentration (Figure 2). Mammalian α-glucosidasesuch as those from rat intestine contains more than one enzyme activity from maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) complexes^[21,22]. MGAM and SI have been shown to utilise the ρNPG substrate with different Km and the inhibitors were suggested to be selective towards one or more of these enzymes, which would explain why complete inhibition of enzyme activity was not observed at higher flavonoid concentration. Thus, it is not possible to obtain meaningful inhibition constants (K_i) from double reciprocal plots of substrate-inhibitor concentrations using such crude extracts. Nonetheless, comparison of IC50 showed that the inhibitory potency of baicalein was closed to that of acarbose, a pharmaceutically prescribed α-glucosidase inhibitor, which has an IC50 of 0.541 mM measured in the same system.

Figure 2: IC50 of flavonoids against rat intestinal α-glucosidase
Activity data points (absorbance) were plotted as mean ± SD (n = 3).

Inhibition of porcine pancreatic α-amylase by flavonoids
  Xu, et al. (2012)^[23] raised the issue of reducing activity of some polyphenols with 3,5-dinitrosalicylic acid (DNS) that is commonly employed in the assay of α-amylase activity with starch as substrate. This is because the activity assay depends on the reduction of DNS by liberated glucose in generating the chromogen measured by its absorbance at λ max 540 nm. Furthermore, evidence showed significant higher in vivodigestion rate of high amylose corn starch compared to normal and waxy corn starch in the presence of tea polyphenols, indicating that interactions between polyphenols and amylose which might have enhanced substrate binding to α-amylase^[24]. Another compounding factor of the method unsuitability with polyphenols is the required boiling step in NaOH solution to generate the chromogen, condition in which some polyphenols would be unstable. Flavonoid that contains the catechol or gallate structural element such as flavonols and catechin are known to undergo auto-oxidation and conjugation reactions in alkaline solution^[8]. Indeed, the absorbance of the boiling mixture used in the starch-DNS assay of α-amylase activity in the absence of starch and enzyme is enhanced at varying degree for the different flavonoids (Figure 3), indicating secondary chemical reactions have occurred. The absorbance changes with baicalein, quercetin and catechin were most prominent. Even acarbose produced significant absorbance changes with boiling. The instability of flavonoids in the boiling procedure rendered this method not suitable for measuring α-amylase activity with flavonoids. It raised questions about the validity of reports that used starch-DNS method in evaluating α-amylase inhibitory potential of phenolic acids and flavonoids as documented in some reviews^[5,11,25].

Figure 3: Effect of boiling on the stability of flavonoids in the starch-DNS assay.
Absorbance of mixtures containing buffer (20 mM sodium phosphate buffer pH 6.8 + 7 mM NaCl + 1 mM CaCl₂) and DNS reagent (1% DNS [w:v] + 12% Na-K-tartrate [w:v] in 0.4M in 2:1 ratio [v:v]) before and after boiling (water bath, 20 min). Flavonoid or acarbose was added as DMSO solution to a final concentration of 1mM and 10% DMSO (v:v).

  Hence, an alternative method that employed a chromogenic malto-pentaoside substrate was employed which is similar to the α-glucosidase assay in that liberated p-nitrophenol is measured by absorbance to indicate enzyme activity.

  Figure 4shows the linear enzyme kinetics with time for porcine pancreatic α-amylase in the absence or presence of baicalein, baicalin or acarbosethat can be obtained by this method. The inhibition can be accurately determined by the ratio of the slope gradients of inhibitor over that of control (without inhibitor). The results (Table 1) showed that inhibition of α-amylase by flavonoids mirrored the inhibition of α-glucosidase in that both baicalein and myricetin inhibited the enzyme strongly, but differs in that spectrum of flavonoid inhibition for α-amylase only restricted to these two flavonoids.

Figure 4: Inhibition of porcine pancreatic α-amylase. Enzyme activity was measured in the absence (control) or present of inhibitor (at 1 mM reaction concentration). Activity data points were plotted as mean ± SD.

  None of the hydroxybenzoic and hydroxycinnamic acids inhibited α-amylase activity measured at 1 mM (data not shown). Baicalein was the most potent inhibitor with 41.1% inhibition and that loss of or modification of the A-ring OH groups resulted in diminished or loss of inhibitory activity, as can be seen with the inhibition effect in the flavones series. The primary importance of the A-ring C6-O was highlighted by the fact that apigenin, chrysin and oroxylin A has little or no inhibitory activity while baicalin retained some. Myricetin is the only compound outside of the flavones series that exhibited strong inhibitory activity with 35.9% inhibition, and again this can be attributed to its B-ring hydroxylation feature but its inhibition was weaker than baicalein.

  The proposed structural requirements of flavonoids for inhibiting α-amylase have been raised by Piparo, et al. (2008)^[26] based on molecular modelling. Similar to acarbose, the inhibitory activity of flavonoids was attributed to two interactions: the hydroxyl groups in the flavonoid molecular structure can form hydrogen bonds with the OH groups in active side chains of functional amino acids of the enzyme, and conjugated π-system is likely to be formed between the AC ring system and the indole Trp59 in the enzyme. Both the interactions can hinder the reaction between α-amylase and starch and thus inhibit the starch digestion.

Conclusion

  The structural requirements of phenolic compounds for the inhibition of mammalian pancreatic α-amylase and intestinal α-glucosidase appear similar. The flavonoid structure and the C5, C6 and C7 OH groups of the A-ring are essential elements for inhibitory activity. Baicalein is the most potent bioactive flavonoid that inhibited both α-amylase and α-glucosidase. Baicalein is not a common plant flavonoid but is a major flavonoid from the root of Scutellaria baicalensis Georgi, which is a medicinal plant that has been used in Traditional Chinese Medicine^[27]. The common food flavonol, myricetin, is also a strong flavonoid inhibitor of α-amylase and α-glucosidase activities. Delivery of these two bioflavonoids in concentrated form, for example as capsules, mighthave the potential to reduce postprandial hyperglycaemia with food intake by inhibiting starch digestion.

Conflict of interest
  Author declare no conflict of interest.

References