The assessment of antidiabetic properties of novel synthetic curcumin analogues: α-amylase and α-glucosidase as the target enzymes

Abstract

Diabetes mellitus is a metabolic disorder characterized by high blood glucose levels and instability in carbohydrate metabolism. For treating diabetes, one important therapeutic approach is reducing the postprandial hyperglycemia which can be managed by delaying the absorption of glucose through inhibition of the carbohydrate-hydrolyzing enzymes, α-amylase (α-Amy) and α-glucosidase (α-Gls) in the digestive tract. In this work, a new class of curcumin derivatives incorporating pyrano[2,3-d]pyrimidine heterocycles was synthesized using a multicomponent reaction between curcumin, aldehydes, and barbituric acid. Using UV-Vis spectroscopic method, the synthetic compounds were assessed for their inhibitory properties against α-Amy and α-Gls enzymes. Also, the antioxidant potential of these compounds was measured spectroscopically and compared with Trolox which is known as a gold standard to measure antioxidant capacity. The results of present study suggest that the curcumin derivatives were able to efficiently inhibit both yeast and mammalian α-Gls. In comparison with the antidiabetic medicine acarbose, the synthetic curcumin derivatives were also capable to inhibit more effectively the yeast α-Gls. The partial inhibitory effects of these compounds against pancreatic α-Amy were also important in the terms of avoiding development of the possible gastrointestinal side effects. Moreover, some of the curcumin derivatives indicated stronger antioxidant activity than Trolox. Overall, these synthetic curcumin analogues might be considered as novel molecular templates for development of efficient antidiabetic compounds with promising inhibitory activities against α-Amy and α-Gls enzymes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40200-020-00685-z.

Introduction

Diabetes mellitus (DM) is currently a serious medical concern. This growing endocrine disturbance is connected with increased incidence and fatality, in addition to high cost of the health care. In 2000, about 171 million cases of DM have been reported, and it was expected that this number would become double (around 366 million) by 2030 [1]. It was demonstrated that DM and its associated problems are linked with the enhancement of the oxidative stress, leading to disproportion between the generations of reactive oxygen species (ROS). The activity of the antioxidant defense system is also affected by DM [2]. Furthermore, the etiology of DM and its complications is controlled by ROS [3]. More importantly, DM causes important defects in insulin production or action, characterized by the hyperglycemia and alteration in the metabolism of carbohydrate, lipid and proteins [4]. As a well-known defect occurring in diabetes, postprandial hyperglycemia may result to a variety of secondary complications such as high venture for the cardiovascular illnesses [5], cataracts, atherosclerosis, retinopathy, nephropathy, neuropathy, and impaired wound healing [6].

Ingesting carbohydrate-rich diets could trigger the high blood glucose level because of the quick absorption of carbohydrates in small intestine. This phenomenon is assisted by the sugar hydrolysing enzymes such as α-glucosidase (α-Gls) which exist in the epithelial mucosa of the small intestine. Cleavage of the glycosidic bonds in the complex carbohydrates is the main task of these enzymes to release the absorbable monosaccharides [7]. In this regard, one of the important therapeutic approaches to treat diabetes is the control of postprandial hyperglycemia through inhibition of α-Gls. In fact, delays in the overall time of carbohydrate digestion in digestive tract are the aim of this treatment approach. On the other hand, sluggishness carbohydrate digestion can decrease the rate of glucose absorption to control the insulin levels and consequently postprandial blood glucose [8, 9].

Using α-Gls inhibitors, it is possible to have a therapeutic strategy to reduce the risks of diabetes. Also, other carbohydrate-mediated diseases, such as hyper lipoproteinemia and obesity can be controlled through α-Gls inhibition approach [10]. However, serious gastrointestinal side effects were observed due to non-selective and strong inhibition of pancreatic α-Amy which is one of the major drawbacks of the available α-Gls inhibitor drugs [11]. Consequently, development of strong α-Gls inhibitors with weak to moderate inhibitory action against α-Amy is an important object in this field of research. More importantly, it was illustrated that the compounds bearing more than one type of therapeutic actions (combination therapy) possesses significant role in the precautionary treatment of diabetic impediments [12]. In order to realize combination therapy in practice, one can use medicines with diverse methods of action. It was reported that compounds with both antioxidant and anti-glycation activities have shown remarkable reactivity in diabetes mellitus treatment [13]. Thus, by the use of known medicinal compounds such as curcumin which has previously shown different biological activities to synthesize new derivatives, it is possible to obtain new combinational therapeutic agents. Curcumin is a yellow color pigment in food industry which displays different biological properties such as antioxidant, anticancer, anti-inflammatory, and anti-HIV integrase [14]. This compound was also recommended by Chinese traditional medical prescriptions to be used against the diabetic complications [15]. Accordingly, in the current study, the effects of novel synthetic curcumin analogues on α-Glc/α-Amy inhibition were evaluated. Also, antioxidant properties and mode of inhibitory action of these curcumin analogues against α-Glc were discussed.

Materials and methods

Materials

In this study, the yeast α-Gls (EC.3.2.1.20), p-nitrophenyl α-D-glucopyranoside (pNPG), porcine pancreatic α-Amy (EC 3.2.1.1), 2,2^′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ampicillin were purchased from Sigma-Aldrich Chemical Company. Dimethyl sulfoxide (DMSO) was used as solvent to prepare the stock solutions of curcumin and its synthetic derivatives. For the dilution of the stock solutions, phosphate buffer was used. Chemicals were purchased from Fluka, Merck and Aldrich Chemical Companies and used as received without further purification. For recording ¹H NMR and ¹³C NMR spectra we used a Bruker Avance DRX (250 MHz) and samples were dissolved in pure deuterated CDCl₃ with tetramethylsilane (TMS) as an internal standard. The reactions were monitored by TLC on silica gel PolyGram SILG/UV254 plates.

Methods

General procedure for the synthesis of compounds L₁–L₇

For the synthesis of curcumin-based pyrano[2,3-d] pyrimidines, we followed a procedure according to our previous reported synthetic methodology [16]. Accordingly, into a mechanically stirred solution of curcumin (1.0 mmol), aldehyde (1.0 mmol), and barbituric acid (1.0 mmol) in EtOH (3.0 mL) the magnetic nanoparticles functionalized sulfanilic acid (MNP-SAA) (8 mol%, 0.05 g) were added at 80 °C. The mixture was stirred within a specified period of time of 6-8 h. After completion of the reaction (controlled by TLC), the mixture was cooled to the room temperature. The MNP-SAA catalyst was separated from the reaction mixture using an external magnetic field. Then, some water was added for precipitation of the reaction mixture. If the precipitate was not observed immediately, it was kept in refrigerator overnight. The obtained precipitate was washed with water and dried in an oven. To obtain the pure product, recrystallization was carried out in n-hexane/ethylacetate solvents. The obtained products were characterized using different spectroscopic techniques and the results were compared with our previous reports in order to confirm the successful synthesis of the ligands and also their acceptable purity [16].

Rat intestinal acetone powder preparation

The acetone powder of rat intestine (25 rats with the bodyweights between 200 ± 20 g) was produced using a method reported previously in literature [17, 18]. The rat’s sacrification was accomplished under common anesthesia and their small intestines were cut from a point just under the entrance of the bile duct to around 6 cm on top of the cecum. The rate intestines were separated into the slighter parts, facilitating the washing process [19]. Subsequently, the intestinal segments were cleaned using 30 mL of cold NaCl (0.9%). All processes were carried out at4°C. The intestinal segments were homogenized using a Potter–Elvehjem type homogenizer in 2 vol of 5 mM phosphate buffer while pH was fixed at7.0.Then, a 10volume of the acetone solution (−20 °C) was added to the homogenates after 1 h.Afterward the mixture was shacked for 10 min, allowed to stand and next centrifuged for 30 min (at1500×g). In this stage the precipitates were washed with cold acetone (12 vol) and the mixture centrifuged for 30 min (at 1500×g). Finally, the precipitate was dried overnight (4 °C) and stored at low temperature (−20 °C).

The inhibition assay of yeast and rat α-Gls

The inhibition assay of yeast and rat enzymes was carried out based on our earlier reports with some modifications [17, 18]. The assay was done on micro plate reader (BioTekELx 808 instrument, USA) which is equipped with temperature controller system. The activity of both yeast and mammalian α-Glc was measured with pNPG as a substrate reagent. When the pNPG is hydrolyzed by α-Glc, p-nitrophenol was released as a color agent, which can be monitored by its absorption at 405 nm. The inhibitory action of yeast enzyme (0.2 U/mL) was accomplished in phosphate buffer (100 mM; pH 7.0) at 25 °C for 10 min. The yeast enzyme assay was investigated with enhancement of pNPG concentration in the presence of different molar ratio of curcumin-derived inhibitors in the range of 0–100 μM. To determine the activity of rat α-Gls, the intestinal acetone powder was dissolved in ice cold phosphate buffer (100 mM, pH 7.0) and sonicated for 25 s at 4 °C. Next, the suspension was centrifuged (10,500×g) at 4 °C, for 75 min. Then, the obtained supernatant was dialyzed using 10 mM potassium phosphate buffer (pH 7.0) for 24 h. The activity of rat α-Gls (0.15 U/mL) was obtained by increasing concentration of pNPG substrate (0.1–10 mM) at 37 °C. A mixture including rat α-Gls (0.15 U/mL) and different concentrations of each ligand was pre-incubated for 10 min at 37 °C, prior to adding pNPG. Subsequently, the increase of absorbance was pursued for 30 min (405 nm). Lineweaver–Burk analysis was used to obtain the mode of enzyme inhibition.

The enzyme activity at different concentrations of substrate was used to drew the Dixon plot. In this way, the activity of yeast and mouse enzymes was measured once in the absence of the inhibitors and several times in the presence of constant concentrations of each synthetic compound. To reach to Dixon plot, first the absorption against time was drawn and then the velocity was determined in different concentrations of the inhibitors and converted to 1/V. We also used Dixon and Cortes transforms with the aim to obtain the IC₅₀ values and inhibition constants (Ki). The slope of 1/IC₅₀ vs v₀/v (relative velocity) in Cortes plot demonstrates the amount of inhibition constant [20]. In this study, Acarbose (pharmacological inhibitor) was used as a positive control. Also, all tests were repeated at least for three times.

Pancreatic α-Amy assay

We followed a method which has been previously published to assess the inhibition of α-Amy by the synthetic curcumin derivatives [34]. These synthetic compounds (250 mL, 100 μM) were incubated with porcine α-Amy (250 mL, 0.4 mg/mL). The experiment was done in buffer C (20 mMNaPi, pH 6.9 bearing 6 mM sodium chloride) for 10 min at 25 °C. After pre-incubation, the starch solution (250 mL; 0.1%) which has been already prepared in buffer C was added to each tube. We then incubated the reaction mixture at 25 °C for 10 min. Finally, 1 mL of dinitrosalicylic acid (DNS) was used to stop the reaction progress. These tubes were then incubated for 5 min in a boiling water bath. After cooling down to the room temperature and subsequent to dilution by adding 10 mL distilled water, we measured the absorbance of each sample at 540 nm. The obtained absorption values were compared with that of the control buffer. The results were expressed as percentage of α-Amy inhibition, according to the following equation:

$$\%\mathit{\text{Inhibition}}=\left[\frac{A_{\mathit{\text{Control}}}-A_{\mathit{\text{Sample}}}}{A_{\mathit{\text{Control}}}}\right]\times 100$$ 1

Antioxidant activity measurement of the curcumin-derived compounds

A T90⁺ UV-Vis spectrophotometer instrument (PG Instrument Ltd., UK) was used to measure the antioxidant activity of the curcumin-derived inhibitors. The antioxidant assay was done based on a decolorization system, recording reduction of ABTS radical cation (ABTS^•+) [21]. The final concentration of ABTS was fixed at 7 mM in double distilled water. As a result of reaction between ABTS and potassium persulfate (2.45 mM), ABTS radical cation (ABTS^•+) was produced. During formation of ABTS^•+, the reaction mixture was kept in the dark condition for 12–16 h before use. Then, the solution of ABTS radical was diluted in 5 mM phosphate buffer (pH 7.4) to obtain an absorbance value of 0.70 ± 0.02 at 734 nm. Also, the solution of the synthetic compound was diluted between 0 and 200 μM in the same buffer. We added 10 μL of each curcumin derivative to one milliliter of ABTS radical solution. At last, the absorbance was read at 734 nm in a kinetic fashion for 6 min. The disappearance of ABTS radical in the presence of each inhibitor was estimated as the percentage inhibition of absorbance at 734 nm.

$${\mathit{\text{ABTS}}}^{•+}\mathit{\text{inhibition}}\left(\%\right)=\left[1-\frac{A_{\mathit{\text{Sample}}}}{A_{\mathit{\text{Control}}}}\right]\times 100$$ 2

The Trolox equivalent antioxidant capacity (TEAC) of the synthetic inhibitors (μM) was determined by comparing their percent inhibition with that of the standard curve for Trolox [22].

Assessment of the effect of curcumin based compounds on human lumen microflora

In this experiment, the sterilized Brain–Heart Infusion (BHI) agar medium was poured into sterile Petri dishes and permits it to solidify. The Lactobacillus plantarum (PTCC 1058) and Escherichia coli (PTCC 1553) were provided by the Persian Microbial Type Culture Collection. After solidification, 100 μL of each overnight culture of the bacteria in BHI broth with a density equivalent to 0.5 McFarland scale was swabbed into the individual plates, using sterile cotton swabs. Then, the wells of 6 mm diameter were made on BHI agar plates by a gel puncture and each compound with concentration of 100 μM at the final volume of 20 μL was poured onto each well of the plates. Ampicillin at the same concentration of compounds and 20 μL BHI broth were used as positive and negative controls, respectively. After incubation at 37 °C for 24 h, the diameter of inhibition zone was measured.

Statistical analysis

The data were analyzed by Graphpad Prism 6.07 software. The differences between multiple groups were compared using parametric t-test with significant p-values (<0.05). The data were analyzed using three independent tests and expressed as mean ± SD.

Results and discussion

The chemistry of the synthetic curcumin-based derivatives

Diversity oriented synthesis (DOS) is one of the most widely used approaches in organic chemistry for preparing structurally different natural-like and biologically active compounds [23]. By applying this approach, it is possible to synthesize a large numbers of compound libraries using simple starting materials which are highly considered in the medicinal chemistry. Multicomponent reactions (MCRs), as a most widely used method of DOS, is very important in organic synthesis due to its high possibility for preparing of complex molecules using simple and available starting materials [24]. MCRs allow us to synthesize plenty of natural-based compounds, highly considered in the design of biologically active and biocompatible products [25]. When natural organic molecules to be used in MCR, it is possible to synthesize complex natural-like products which are very useful in drug design and synthesis. Along this line, we have disclosed some MCRs including small natural organic molecules such as carbohydrates and nucleosides for the synthesis of diverse biologically active compounds [26, 27]. We have also synthesized an interesting class of natural product-like compounds using curcumin as starting material. Curcumin has been indicated to be most active component of turmeric (Curcuma longa) plant [28]. Many published articles on this compound have described several biological properties ranging from antioxidant, anti-inflammatory to inhibition of angiogenesis [29]. Multiple cellular targets within the cells including different transcription factors, kinases, receptors, inflammatory cytokines and growth factors have been suggested for curcumin [30]. This natural product indicates a limited cellular absorption, poor metabolism and rapid elimination which are major reasons for its poor bioavailability [31]. In order to overcome the mentioned is advantages, different strategies including synthesis of new curcumin analogues with improved bioavailability have been proposed [32]. Those from turmeric and synthetic analogues are two major groups of curcumin derivatives [33]. On the other hand, the curcumin analogues were designed by removing the β-ketone moiety to mono-ketone or changing to heteroaromatic ring and putting different substitutions on the benzene ring, coupling as co-polymer and metal complex [34]. Considering the chemical structure of curcumin, this valuable molecule has high potential to be used as starting material in MCRs for the synthesis of diverse complex molecules with important applications in the medicinal chemistry. Moreover, curcumin structure allows its versatile biological activities in both medicinal chemistry and food science [35]. However, new curcumin-based derivatives open up our hand in finding new medicinal applications of this valuable material. We have recently developed a multicomponent reaction which is applicable in the synthesis of a class of curcumin-based pyrano[2,3-d]pyrimidine analogues (Scheme 1).

Scheme 1.

The chemical structure of synthesized curcumin derivatives

Our initial assessment suggested this new class of curcumin derivatives as good antidiabetic compounds through inhibition of α-Amy and α-Gls with high antioxidant activity [15, 36]. These valuable results prompted us to synthesize other derivatives of these compounds in order to deep our insight in the inhibition activity of designed ligands with change of the molecular structures by selecting type of components. In this way seven new derivatives (L₁–L₇) were synthesized for the assessment of their inhibitory actions against two carbohydrate-hydrolyzing enzymes α-Amy and α-Gls (Schme 1). Other biological properties including antioxidant activity of these compounds were also investigated.

The biological activities of synthetic curcumin-based derivatives

The assessment of biological activity of curcumin-based derivatives

Uncontrolled diabetes will finally culminate in development of many associated complications including heart disease, retinopathy, amputation, neuropathy and hypoglycemia. Due to many adverse effects associated with the long-term consumption of synthetic drugs, new trends are the medications provided by the natural based products which have indicated lesser complications [37]. During the previous decades many efforts have been done with the aim to introduce new drugs with pleiotropic and complementary functions which can be used in the treatment of diabetes mellitus. Curcumin possesses potent anti-inflammatory, antitumor, anti-HIV and antimicrobial properties [38].

The wound healing properties, antioxidant activity [39] and its effect in delaying diabetic cataract [40] suggest curcumin as a valuable precursor in the synthesis of novel medicinal compounds with application in the treatment of diabetes and its complications. The ability of curcumin to inhibit the intestinal carbohydrate hydrolyzing enzyme, α-Gls [41] was the reason behind the synthesis of novel derivatives of this compound with potential application to reduce the rise of postprandial blood glucose in the diabetic patients.

Inhibition parameters of curcumin derivatives against rat and yeast α-glucosidases

The poor clinical outcomes of current anti-diabetic medicine and increasing prevalence of type-II diabetes mellitus are two important reasons that led us to investigate on the new therapeutic approaches focusing to control this metabolic disorder in its initial stages [36]. In the small intestine, two carbohydrate-hydrolyzing enzymes α-Amy and α-Gls are responsible to digest the complex carbohydrates into the absorbable monosaccharides [42]. Therefore, among several types of available medicines, inhibitors of α-Gls which prevent the absorption of monosaccharides in the small intestine are important to prevent diabetes and its complications in the initial stages. Moreover, α-Gls inhibitors have already indicated to suppress secretion of insulin after each meal and this event has less impact on decreasing capacity of insulin secretion by pancreatic β-cells [42]. In the current study, the inhibitory activities of novel curcumin derivatives (L₁–L₇) were examined against both yeast and rat α-Gls enzymes.

Our results showed that acarbose inhibits the yeast alpha-glucosidase enzyme with the IC50 value of 206.57 mM, while the synthetic ligands have a more significant inhibitory effect on this enzyme. Also, acarbose inhibits mouse glucosidase with IC50 value of 26.54, while L1 and L5 ligands inhibit this enzyme with the IC50 values of 106.80 and 46.04 mM, respectively. Lineweaver–Burk and Cortes plots were used to determine the types of inhibition. Moreover, the inhibition constants were also identified by the aid of Dixon plot. As determined by the plots of Lineweaver–Burk and Cortes [20], L₁ and L₅ indicated a competitive mode of inhibition on mouse α-Gls (Fig. 1). Also, against the yeast enzyme, a similar mode of inhibition was observed for the synthetic ligands L₄ and L₆. Other curcumin derivative indicated a mixed-type of inhibition against the yeast enzyme (Fig. 2 and Table 1).

Fig. 1.

The Lineweaver–Burk (a) and Dixon (b) plots of L₁ inhibitor against mouse α- glucosidase. The activity of mouse enzyme was measured with the substrate concentration ranging between 0.1 and 5 mM,in the presence of different concentrations of each inhibitor. The experiments performed in 10 mM phosphate buffer, pH 7.0 at 37 °C for 30 min. This figure represents the plots obtained for inhibitor L₁. The Lineweaver–Burk and Dixon plots of other synthetic compounds are presented in the supporting information (Fig. S1)

Fig. 2.

The Lineweaver–Burk (a) and Dixon (b) plots of L₅ inhibitor against yeast α-glucosidase. The enzyme activity was measured with the substrate concentration ranging between 0.1 and 4 mM in the presence of different concentrations of each inhibitor. The experiments were donein 100 mM phosphate buffer, pH 7.0at 25 °C for 10 min. This figure represents the plots obtained for inhibitor L₅. The Lineweaver–Burk and Dixon plots of other synthetic compounds are presented in the supporting information (Fig. S2)

Table 1.

The IC₅₀,^a Inhibition constants (K_i) values and inhibition mode of the synthetic curcumin derivatives

Entry	IC50 (μM)		Kicb (μM)		Kiub (μM)		Type of inhibition
	Yeast	Mouse	Yeast	Mouse	Yeast	Mouse	Yeast	Mouse
L1	12.41 ± 1.10	106.80 ± 2.10	10.23 ± 0.80	100.29 ± 2.40	37.62 ± 2.70	–	MCc	Cc
L2	61.11 ± 3.50	–	55.62 ± 3.40	–	92.90 ± 3.60	–	MC	–
L3	36.64 ± 2.70	–	39.19 ± 2.00	–	93.10 ± 3.20	–	MC	–
L4	18.20 ± 1.50	–	16.44 ± 1.20	–	–	–	C	–
L5	27.60 ± 1.30	46.04 ± 2.10	19.14 ± 1.30	41.51 ± 0.90	71.68 ± 3.00	–	MC	C
L6	24.10 ± 0.90	–	21.60 ± 3.00	–	–	–	C	–
L7	63.73 ± 2.10	–	59.63 ± 3.20	–	107.00 ± 4.00	–	MC	–
ACd	206.57 ± 4.30	26.54 ± 2.57	166.70 ± 3.81	9.59 ± 2.80	–	–	C	C

^aThe half maximal inhibitory concentration

^bc and u respectively indicate competitive and un-competitive inhibitions

^CMC and C respectively stand for mixed-competitive and competitive inhibitions

^dAcarbose

The inhibition constants (K_ic, K_iu) are also shown in Table 1. However, the competitive inhibition mode of α-Gls is not very desirable because inhibition of this enzyme in the small intestine demands a higher dose of competitive inhibitor to overcome the competition with a carbohydrate rich diet. The Lineweaver–Burk and Dixon plots of other synthetic compounds are presented as the supporting information in Fig. S1 and Fig. S2.

Inhibition of α-Amy by curcumin-based derivatives

The large insoluble starch break into the smaller oligosaccharide molecules is made by the aid of α-Amy which prominently found in the pancreatic juice and saliva [43]. The inhibition of this enzyme also results in the reduction of glucose release but its complete inhibition can initiate undesirable effects such as intestinal disorder because of the gas production by gut microflora [44]. Because of this unwanted effect, the partial inhibition of α-Amy is desirable for modulating the rate of monosaccharide release from the complex polysaccharide such as starch [45]. In the current study the synthetic curcumin derivatives were also used to study their inhibition against porcine pancreatic α-Amy.

The curcumin derivatives indicated a lower inhibition ability against alpha-amylase enzyme when compared to acarbose, which reduces activity of this enzyme to 42% of its original activity.

The activity of α-Amy was measured according to the procedure which indicated in Scheme 2.

Scheme 2.

The enzymatic process catalyzed by α-amylase enzyme. This scheme indicates the details of α-amylase activity measurement

The results have shown thatα-Amy activity was reduced to the maximum level of 15% in the presence of L₄ and other curcumin derivatives demonstrated lower inhibitory action. The weak inhibitory action of the synthetic ligands compared to Acarbose (42% inhibition) against α-Amy is highly desirable in terms of their reduced susceptibility for possible development of the intestinal disorders (Fig. 3).

Fig. 3.

The inhibitory action of curcumin-based derivatives against porcine pancreatic α-amylase. α-amylase (0.5 mg/ml) was pre-incubated with each synthetic inhibitor for10 min and then with starch solution for 30 min, at 25 °C. After that, the reaction mixture was incubated in a boiling water bath for 5 min in the presence of dinitrosalicylic acid. Subsequent to cooling down to room temperature, the mixture was diluted by adding distilled water and then its absorbance was measured at 540 nm. The measured absorbance was compared with that of the control experiment and presented as percentage of α-amylase inhibition. This experiment was done in 20 mM sodium phosphate buffer, pH 6.9, containing 6 mM sodium chloride

The assessment of antioxidant activity of synthetic curcumin derivatives

Chronic hyperglycemia activates glucose metabolism and associates with significant production of reactive oxygen species (ROS) and increasing oxidative stress in body. Under this situation the poor ability of the antioxidant defense system to neutralize the mass production of oxidative agents may result in appearance of diabetes complications [46]. Oxidative stress associated with hyperglycemia is believed to have an important role in the etiology of diabetes and its complications [47]. Moreover, oxidative stress is highly capable to trigger inflammatory cascade mediated many chronic disorders such as cancer, asthma, arthritis, and other diseases [48]. Therefore, those synthetic inhibitors displaying good antioxidant activity and improved anti-inflammatory properties are highly acceptable as antidiabetic medicines to be used by diabetic patients [49]. Curcumin-based heterocycle compounds contain electron-donating groups, therefore indicating antioxidant properties [50]. In the current study, the antioxidant activity of curcumin-based heterocycle compounds was determined using decolorization of the ABTS⁺ and the obtained results were shown in Figs. 4 and 5. The antioxidant capacity of the synthetic compounds was also calculated as TEAC which indicated in Table 2.

Fig. 4.

The effects of time on suppression of the absorbance of 3-ethylbenzothiazoline-6-sulfonic acid (ABTS^•+). The experiments were performed in different concentrations of each ligand (1.25–10 μM). The curves represent the effects of the specific antioxidant on the suppression of the absorbance of the ABTS•⁺ at 734 nm. The symbols used are as the following: Control ABTS^•+ radical cation (●), L₁(■), L₂(▲), L₃(▼), L₄(♦), L₅(○), L₆(□), L₇(∆)

Fig. 5.

The dose–response curve obtained by analysis a range of concentration of curcumin-derived compounds at the selected time points (1, 4 and 6 min). These plots represent the percentage inhibition of the absorbance of ABTS^•+ solution as a function of concentration of the curcumin-derived compounds

Table 2.

Comparison between antioxidant activity as trolox equivalent antioxidant capacity (TEAC) (μM) at specific time points

Ligand	TEAC±SD
L1	1.82 ± 0.07
L2	0.73 ± 0.02
L3	1.39 ± 0.09
L4	1.20 ± 0.10
L5	2.00 ± 0.09
L6	0.82 ± 0.06
L7	1.17 ± 0.10

With the exception of L₂ and L₆, other curcumin derivatives indicated either comparable or higher antioxidant activity than the standard compound. The highest antioxidant activity was also observed for L₁ and L₅ which can be explained with the feature of electron-donating substitutions in their chemical structures.

The effect of curcumin based compounds effect on human lumen microflora

The gut bacteria play important functions to regulate the development of intestine epithelium, maintaining mucosal homeostasis and repair, improving absorption of nutrients from food, and contribute to the innate immune system [51]. Today, over use of several antibiotics and other medicines cause important adverse effects on the intestinal normal flora resulting important disturbance and complication of Gut. Therefore, the aim of this part was to evaluate the effect of these compounds on major bacteria existing in gut. To evaluate effect of these compounds, the inhibition zone in well diffusion assay was performed against two important types of gut bacteria. The concentration of these curcumin derivatives and reference compound (ampicillin) was fixed at 100 μM (Fig. 6). As shown in Fig. 6, the inhibition zone was not evident for curcumin derivatives against L. plantarum.

Fig. 6.

The effect of curcumin derivatives was assessed against Lactobacillus plantarumand Escherichia coli using well diffusion assay. The concentration of each compound was 100 μM. The upper panel belongs to Lactobacillus plantarum while the lower panel stands Forescherichia coli. Ampicillin (100 μM) and Brain–Heart Infusion (BHI) broth were also used as positive and negative controls, respectively

In the case of Escherichia coli, only L₁ indicated an effect with the inhibition zone of 11 ± 0.1 mm, comparable with that of ampicillin (100 μM) as 15 ± 0.3 against Lactobacillus plantarum and 12 ± 0.5 against Escherichia coli. The little difference can be attributed to the cell wall composition of these bacterial target cells. Overall, our assessment confirmed that the curcumin derivatives except L₁ have no significant effect on the microflora existing in the intestinallumen. Therefore, the oral administration of these synthetic compounds for treatment of diabetes may have little ecological disturbance in the normal intestinal microflora.

Conclusion

In brief, with the future rise in diabetic population worldwide, the search for active compounds with α-Gls inhibitory activity from medicinal plants has become a very meaningful task. In the current study, we have synthesized and assessed the novel curcumin-based heterocycle compounds for their inhibitory action against α-Gls and α-Amy. In fact, this study was done with the aim to search for the novel compounds with possible application in controlling the postprandial hyperglycemia in diabetic patients. Among seven curcumin derivatives which have been examined in the current study, only compounds L₁ and L₅with the reasonable α-Gls inhibitory activity, poor α-Amy inhibition and important antioxidant properties were introduced as potentially important anti-diabetic agents. Therefore, these compounds can be potentially used for the more convenient management of postprandial hyperglycemia in diabetic patients. Moreover, these compounds can be evaluated for potential pharmacological activity against other metabolic diseases.

Author contributions

RY, FP and AK conceived and designed research. FP and MN synthesized and characterized synthetic compounds. FH and ZT conducted biological experiments. FP and RY analyzed data and wrote the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by NIMAD (grant number 964854).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.