Antidiabetic and Antihyperalgesic Effects of a Decoction and Compounds from Acourtia thurberi

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Chemicals and reagents
• General procedures
• Plant material, extraction, and isolation
• Quantification of the main components from the decoction of Acourtia thurberi
• Pharmacological assays
• Statistical analysis
• References

Abstract

The purpose of this research was to examine the preclinical efficacy of a decoction from the roots of Acourtia thurberi as a hypoglycemic, antihyperglycemic, and antihyperalgesic agent using well-known experimental models in mice. Acute oral administration of A. thurberi decoction did not produce toxic effects in mice, according to the Lorke procedure. A. thurberi decoction (31.6–316.2 mg/kg, p. o.) decreased blood glucose levels during acute hypoglycemic and the oral glucose tolerance and oral sucrose tolerance tests, both in normoglycemic and hyperglycemic animals. Phytochemical analysis of A. thurberi roots led to the isolation of perezone (1), a mixture of α-pipitzol (2) and β-pipitzol (3), and 8-β-D-glucopyranosyloxy-4-methoxy-5-methyl-coumarin (4). A pharmacological evaluation of compounds 1–4 (3.2–31.6 mg/kg) using the same assays revealed their hypoglycemic and antihyperglycemic actions. Finally, local administration of A. thurberi decoction (31.6–316.2 µg/paw) and compounds 1–4 (3.2–31.6 µg/paw) produced significant inhibition on the licking time during the formalin test in healthy and hyperglycemic mice, demonstrating their antinociceptive and antihyperalgesic potential, respectively. Altogether, these results could be related to the use of A. thurberi for treating diabetes and painful complaints in contemporary Mexican folk medicine. A suitable UPLC-ESI/MS method was developed and successfully applied to quantify simultaneously compounds 1 and 4 in A. thurberi decoction.

Introduction

According to the International Diabetes Federation, around 415 million people were affected worldwide with T2DM in 2015 and the prevalence is expected to rise beyond 642 million by 2040 [1]. The best treatment for T2DM involves hyperglycemic control using appropriate therapies and a healthy lifestyle. Although metformin remains the most important oral agent for the initial management of T2DM, the current allopathic therapies include also sulfonylureas, α-glucosidase inhibitors, thiazolidinediones, and dipeptidyl peptidase-4 inhibitors. Frequently, these drugs are combined to make the treatment more efficient [2].

A large segment of the world population relies on herbal medicine or a combination of allopathic and herbal products for treating diabetes [3]. Most of these patients consider herbal preparations less toxic, more efficacious, and less expensive than allopathic medications [4]. According to recent reviews, in Mexico there are at least 383 plant species employed for the treatment of T2DM [4], [5]. One of these species is Acourtia thurberi (A. Gray) Reveal & R. M. King (Asteraceae). The decoction prepared from the roots of this plant is highly valued for treating diabetes, gastrointestinal ailments, infections, and pain [4], [6], [7], [8]. A previous phytochemical study on A. thurberi led to the isolation of perezone (1) and a mixture of α-pipitzol (2) and β-pipitzol (3). The presence of alkaloids and sugars was also suggested on the basis of preliminary qualitative tests [9]. From the pharmacological point of view, Alarcón-Aguilar et al. [9] demonstrated that the decoction of the roots modestly reduced glucose levels in normal rabbits and mice.

Thus, as part of our systematic investigation of Mexican medicinal flora as a source of well-accepted alternative treatments for T2DM, the main goal of this work was to demonstrate the alleged antidiabetic properties of A. thurberi using well-known animal models.

Results and Discussion

Acute toxicity studies in animals are essential for any product intended for human use. In addition, the information obtained from these studies is useful for choosing appropriated doses for pharmacological assays and identifying toxicity in target organs. In this work, acute toxicity was studied in mice following the Lorke method [10], which gives reproducible results using a minimum number of experimental animals. Acute administration of AtD did not provoke behavior alterations, macroscopic tissue injury, or weight loss during the 14-day observation period. The estimated LD₅₀ was higher than 5000 mg/kg. Therefore, according to the Lorke criteria, A. thurberi is devoid of acute toxic effects for mice (Table S1, Supporting Information).

A. thurberi is widely commercialized in contemporary Mexico for the treatment of diabetes. This fact encouraged us to establish its preclinical efficacy as a hypoglycemic and/or antihyperglycemic agent using well-known animal models. The traditional preparation, namely AtD, was tested in both normal and NA-STZ-treated mice using half-log interval doses (31.6, 100, and 316.2 mg/kg), chosen according to a standard protocol of allometric scaling [11]. The NA-STZ model is widely used for evaluating potential antidiabetic drugs since it portrays a similar biochemical blood profile and pathogenesis to T2DM in humans. In addition, this model has been validated using anti-T2D drugs [12]. Oral administration of AtD to normal and NA-STZ mice significantly decreased the blood glucose level at the dose of 316.2 mg/kg ([Fig. 1]). This effect was less than that produced by GLI, an antidiabetic drug. The antihyperglycemic effect of AtD was initially established throughout an OGTT ([Fig. 1]). All doses (31.6–316.2 mg/kg) induced a significant drop in the postprandial peak after the glucose challenge in normal and hyperglycemic mice. The effect was comparable to that induced by MET. Because of the dose of 31.6 mg/kg produced a pronounced effect, a lower dose of the preparation, 10 mg/kg, was also tested. However, the effect was similar to those produced by the higher doses. This fact suggests that the effect induced by AtD during the OGTT is not dose-dependent. Next, when AtD was tested in hyperglycemic mice, the reduction of the postprandial peak was better than that induced by MET. Finally, during an OSTT ([Fig. 1]), the traditional preparation reduced blood glucose in a comparable fashion to ACAR. The latter result prompted us to evaluate the potential inhibitory effect of AtD on yeast α-glucosidase using a spectrophotometric method [13]. The results revealed that AtD inhibited the activity of the enzyme by 97.5 ± 4.0 % (Fig. S1, Supporting Information) in a concentration-dependent manner (IC₅₀ = 566.7 [502.3–639.4] µg/mL), confirming the in vivo results.

Diabetic peripheral neuropathy is among the most debilitating consequences of chronic diabetes, and ultimately affects 50 % or more of all diabetic people [14]. This complication provokes a few sensory abnormalities, including hyperalgesia [15]. Thus, the next step in the investigation was to find out if AtD was effective in a hyperalgesic condition. After two weeks, mice pretreated with NA-STZ developed a hyperalgesic state. Treatment of these mice with 30 µL of formalin solution (1 %) showed a clear increment in the response to chemical stimuli when compared with the formalin-treated normal mice group. These observations demonstrated that pretreatment with NA-STZ reduces the pain threshold to chemical stimulation with formalin (Fig. S2, Supporting Information). As shown in [Fig. 2], local administration of AtD (31.6, 100, and 316.2 µg/paw) attenuated the licking time in both neurogenic and inflammatory phases in hyperglycemic-hyperalgesic mice. At the medium concentration (100 µg/paw) tested, the licking time was reduced in both phases in comparison with the VEH group. The same trends of effects were observed with the reference drug GBP. For the sake of completeness, the antinociceptive effect of AtD (31.6, 100, and 316.2 µg/paw) in normoglycemic mice was also assessed using the formalin test; the results were compared with diclofenac (31.6 µg/paw). At the doses of 31.6 and 100 µg/paw, AtD displayed an important antinociceptive action in both phases of the test ([Fig. 3]). In the second phase, the effect exerted by the three doses was comparable to diclofenac. Subplantar injection of formalin results in flinching and licking or biting behavior during an early acute phase, which resembles acute pain, followed by a second delayed phase representative for tonic pain [16], [17], [18], [19]. The acute phase is observed during the first 5 min after formalin injection, and results essentially from direct chemical activation of nociceptive afferent fibers, mainly the C fibers, which release substance P and glutamate, or direct activation of TRPA1 and TRPV1 channels expressed on nociceptors (peripheral pain pathways) [19]. The second phase or inflammatory phase involves the release of local inflammatory mediators (bradykinin and prostaglandins) and central sensitization mediated by NMDA and substance P receptors, which lead to the activation of supraspinal pathways similar to those activated in the chronic constriction injury model, a neurophatic pain model [20], [21], [22]. Altogether, these results indicated that AtD has antinociceptive and anti-hyperalgesic effects and acts both centrally and peripherally to produce pain relief in normal and hyperglycemic mice.

In order to determine the active principles of AtD, a preliminary UPLC analysis was performed ([Fig. 4]). This study revealed the presence of four major components that were isolated by conventional phytochemical procedures and characterized as perezone (1), an inseparable mixture of α-pipitzol and β-pipitzol (2 and 3), and 8-β-D-glucopyranosyloxy-4-methoxy-5-methyl-coumarin (4) ([Fig. 5]). The structures were elucidated using one- and two-dimensional NMR spectroscopic techniques and by comparison of their spectroscopic and spectrometric data (see Supporting Information) with those previously described in the literature [23], [24], [25]. This is the first report on the presence of compound 4 in this species.

Compounds 1–4 were tested in the same assays as AtD. In general, the isolates did not display an important hypoglycemic action ([Figs. 6]–[8]). The best effect was observed with compound 1, which was active at the higher dose tested (31.6 mg/kg). However, during the OGTT and OSTT, all compounds were active at all doses tested (3.2, 10, and 31.6 mg/kg). Once more, compound 1 exhibited the best activity, which was comparable to the reference drugs MET (200 mg/kg) and ACAR (5 mg/kg), respectively.

The antihyperglycemic effect observed for compounds 1–4 in vivo during the OSTT suggested that they are α-glucosidases inhibitors, thus, they were tested against yeast α-glucosidase. The results revealed that a mixture of 2 and 3 and compound 4 inhibited yeast α-glucosidase in a concentration-dependent manner (Fig. S1, Supporting Information) with IC₅₀ values of 944.9 (705.4–1265.8) µM and 3.98 (2.5–6.4) µM, respectively (ACAR IC₅₀ was 310.0 [172.7–556.5] µM). The results for compound 1 were not reliable due to color interference during the assay. Altogether, these results indicated that compounds 1 and 4 are the most relevant antihyperglycemic compounds of AtD. As with compound 4, other simple coumarins have demonstrated a significant antihyperglycemic effect in STZ-induced diabetic rats [26] and α-glucosidase inhibitory activity [27].

Compounds 1–4 were also tested as antihyperalgesic agents using the same model as for AtD. The results are summarized in [Figs. 9]–[11]. All samples were very active in the inflammatory phase, and the effect was comparable to that of GBP used as a reference drug.

The participation of opioid, benzodiazepine/GABA, and 5-HT_1 A receptors as targets for the antihyperalgesic effect of 1 and the mixture of 2 and 3 ([Table 1]) was assessed. Pretreatment of animals with naloxone, flumazenil, and WAY 100 635 did not reverse the antihyperalgesic effect observed with compound 1 or the mixture of 2 and 3. Therefore, the mechanism underlying the pharmacological action of these compounds remains an open question. Further work is necessary to explore the involvement of other molecular targets in the mode of action of these metabolites.

Recent studies have shown that 1 exhibited moderate cytotoxicity (8.2–32.2 µM) against several tumor cell lines. The effect was partially associated to the production of ROS [28]. These results prompted us to assay the potential acute toxicity effects of 1 using the Lorke protocol. As in the case of AtD, the administration of 1 at doses not higher than 1000 mg/kg did not provoke behavior alterations, macroscopic tissue injury, weight loss, or death during the 14-day observation period; the LD₅₀ calculated was 1264.9 mg/kg (Table S1, Supporting Information). These finding reveals that the risk of toxicity can be higher upon the consumption of the pure compound.

A suitable UPLC-ESI/MS method was next developed to quantify simultaneously the two major active components of AtD. The optimal chromatographic separation conditions were achieved with a reversed-phase BEH C-18 column ([Fig. 4]). The peaks corresponding to 1 and 4 possess an area of 27.6 and 24.5 %, respectively, of the total peak area quantified. Next, the UPLC-ESI/MS method was validated according to the International Conference on Harmonization guidelines in terms of linearity, accuracy, precision, repeatability, specificity, and detection and quantitation limits [29]. The system was linear when tested in the concentration range between 10 to 250 µg/mL for 1 and 1.0 to 100 µg/mL for 4 (r ² > = 0.987 and 0.999, respectively). The coefficients of variation (CV) values were 0.46 and 0.67, respectively, at each concentration level analyzed. Limit of detection (LOD) values were 2.5 and 1.0 µg/mL for 1 and 4, respectively, whereas the limit of quantification (LOQ) values were 10 and 2.5 µg/mL for 1 and 4, respectively. The linearity of the method was tested by a recovery assay. The linear regression equations for 1 and 4 were found to be y = 0.997 x + 0.6052 and y = 1.0035 x − 0.0553, respectively. The recovery ranges for the two standards are expressed as the concentration detected as a percentage of the expected concentration and were found to be 98.4 to 102.0 and 98.3 to 102.0, respectively ([Table 2]). The reproducibility and repeatability of the analytical method were determined in terms of the intermediate precision by analyzing three replicates of six samples of stock solution (75 µg/mL for 1 and 100 µg/mL for 4) on two different days. The relative standard deviation (RSD; n = 6) was calculated for each sample evaluated. The results indicated that their chromatographic pattern was similar, showing in each case the presence of the major peaks. The CV values for accuracy were less than 1.7 and 0.8 % for 1 and 4, respectively, and this method had good repeatability. Subsequently, the proposed method was successfully applied to quantitatively analyze 1 and 4 in AtD, which was found in a ratio of 1.2 : 1 (27.0 ± 0.24 and 20.3 ± 0.21 mg/g, dry matter, respectively). Calculation of the doses of 1 and 4 in AtD, at the doses administrated to mice, showed that they are present in a range of 2.1 ± 0.02 to 21.9 ± 0.2 mg/kg for compound 1, and 1.7 ± 0.02 to 16.5 ± 0.17 mg/kg for compound 4. Thus, because of these results, the doses of AtD given to the mice contained the amounts of compounds 1 and 4 needed to achieve the pharmacological activity, and suggested that the major components in the AtD are indeed the active principles.

In summary, this study highlights the potential of A. thurberi for the development of new herbal remedies for treating diabetes and painful complaints, and will contribute to the rational use of this Mexican plant.

Materials and Methods

Chemicals and reagents

Acetonitrile and water (LC-MS grade), formic acid (HPLC grade), and all solvents (analytical grade) used for chromatographic and phytochemical analysis were supplied from J. T. Baker. Compounds 1 and 4 (purity ≥ 97 %), used as reference compounds, were isolated from AtD. ACAR (≥ 95 %), FMZ (≥ 99 %), GBP (≥ 98 %), GLI (≥ 99 %), metformin hydrochloride (MET, 97 %), naloxone hydrochloride dihydrate (NX, ≥ 98 %), NA (≥ 98 %), STZ (≥ 98 %), WAY 100 635 (WAY, ≥ 98 %), D-(+)-glucose (99.5 %), sucrose (ACS reagent), S. cerevisiae α-glucosidase, formaldehyde, and Tween 80 were purchased from Sigma-Aldrich.

All drugs were dissolved in saline solution (NaCl, 0.9 %) except compound 1, a mixture of 2 and 3, 4, GLI, and FLU, which were suspended in saline solution with 0.05 % Tween 80 (vehicle). For evaluation of antidiabetic potential, the treatments were orally administered using a volume of 0.2 mL/10 g of body weight for assessment of antihyperalgesic activity. The mice were injected into the dorsal surface of the right hind paw with 20 µL of treatment. All treatments were used in fresh preparation and administered 30 min before each evaluation. GLI, MET, ACAR, and GBP were used as reference drugs.

General procedures

Melting points were determined using a Fisher-Johns apparatus and are uncorrected. Thin-layer chromatography analyses were performed on silica gel 60 F₂₅₄ plates (Merck), and visualization of the plates was carried out using a solution of ceric sulfate in 10 % H₂SO₄ (w/v). NMR experiments (¹H, ¹³C, HMBC, HSQC, COSY, and NOESY) were recorded in DMSO-d ₆ or CDCl₃ on a Varian Unity Plus 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C. Tetramethylsilane (TMS) was used as an internal standard. Column chromatography (CC) was accomplished using silica gel 60 (Merck, 70–230 mesh).

Plant material, extraction, and isolation

Roots of A. thurberi were collected in The Sierra Tarahumara, Chihuahua in April 2015 and identified by Dr. Robert Bye. A herbarium specimen (37 299) was deposited at the National Herbarium (MEXU), Instituto de Biología, UNAM.

The aqueous extract was prepared by decoction and heating the dried material (10 g) in boiling water (1 L) for 10 min. After filtration, the water was evaporated to dryness under reduced pressure to yield 3.9 g of aqueous extract.

In order to isolate compounds 1–3 in sufficient amounts for biological testing, an organic extract was prepared by macerating dried and ground roots (1 kg) with hexane for 96 h (3 × 5 L). After filtering, the extract was dried in vacuo to yield a brownish residue (61.5 g). Thirty-one g of the hexane extract was fractionated by silica gel CC (500 g) eluting with hexane, hexane-ethyl acetate (95 : 5 → 0 : 100), and ethyl acetate-methanol (100 : 0 → 30 : 70) to yield 22 primary fractions (F₁-F₂₂). From fraction F₄, eluted with hexane-ethyl acetate (95 : 5), 13.3 g of 1 as orange needles were crystallized. From fraction F₅, eluted with hexane-ethyl acetate (93 : 7), a mixture of 2 and 3 (113.1 mg) crystallized spontaneously.

Compound 4 was isolated from an extract prepared by macerating 200 g of dried roots with methanol 72 h (3 × 600 mL). After filtering, the extract was dried in vacuo to yield a brownish residue (24.3 g). Ten g of the methanol extract were subjected to column chromatography on Sephadex eluting with methanol to yield compounds 4 (104.7 mg) and additional amounts of perezone (1). The mixture of α-pipitzol (2) and β-pipitzol (3) was also obtained.

Quantification of the main components from the decoction of Acourtia thurberi

Preparation of stock solutions: Compounds 1 and 4 were accurately weighed and prepared by adding methanol in a volumetric flask and diluted to the appropriate concentration (1 mg/mL). The stock standards solutions for UPLC-ESI/EM determinations were prepared by stepwise dilution of the stock solution with methanol at five concentration levels within the range of 10 to 250 and 1 to 100 µg/mL for 1 and 4, respectively. All solutions were filtered through a 0.2-µm membrane prior to injection. Under the conditions described, the retention times (R_T ) of 1 and 4 were found to be 3.9 and 9.2 min, respectively.

Sample preparation: Decoction from A. thurberi was prepared at the concentration of 500 µg/mL in methanol, and filtered through a 0.2-µm membrane prior to analysis.

Apparatus and chromatographic conditions: UPLC-ESI/MS analyses were performed on a Waters Acquity UPLC-H Class system (Waters) equipped with a quaternary pump, sample manager, column oven, and photodiode array detector (DAD) interfaced with an SQD2 single-quadrupole mass spectrometer detector with an electrospray ion source. MassLynk software version 4.1 was used to control the UPLC-ESI/MS system and for data acquisition and processing. The analytical method was developed using a BEH C-18 column (2.1 × 100 mm, 1.7 µm) (Waters). The mobile phase consisted of 0.1 % formic acid in water (solvent A) and acetonitrile (solvent B) with a gradient elution as follows: 0 min, 15 % B; 2.4 min, 20 % B; 10 min, 100 % B. The column was equilibrated with 15 % B for 1.8 min before the next injection, and detection was carried out using a DAD detector at 270 nm. The flow rate was set to 0.3 mL/min, the injection volume was 3 µL, and the column temperature was maintained at 40 °C using a column oven. For the identification of compounds, the DAD detector scanned from 200 to 400 nm, while the MS parameters were as follows: the cone and capillary voltages were set at 35.0 V and 4.0 kV, respectively, the source temperature was 350 °C, and desolvation flow was 450 (L/hr) for 1 or cone and capillary voltages were set at 40.0 V and 3.0 kV, respectively. The source temperature and desolvation flow were set at 400 °C and 465 (L/hr), respectively, for 4. ESI/MS spectra were obtained using nitrogen as the collision gas within a mass range of m/z 100–1000 (scan duration of 0.5 s). Each sample was analyzed in both positive (ESI⁺) and negative (ESI⁻) modes to provide abundant information for structural identification.

Validation procedure for chromatographic method: The method was validated for the linearity, LOD, LOQ, accuracy, precision, and repeatability.

Calibration curves, limit of detection- and limit of quantitation: Standard calibration curves for quantifying 1 and 4 were obtained by plotting concentration (µg/mL) against response. Methanol solutions of each standard at five different concentrations were prepared in triplicate (1: 10 to 250 µg/mL; 4: 1.0 to 100 µg/mL). Peak area (y) and concentration (x) for each compound were subjected to regression analysis to calculate the calibration equations and correlation coefficient (r) using the software Origin version 8.0. The lowest concentration of the working solutions was diluted with methanol to a series of appropriate concentrations for the determination of LOD and LOQ for each compound [29].

Accuracy: The linearity of the method was tested by recovery, assaying independently three amounts of 1 and 4. At each level, compounds 1 and 4 were added simultaneously to the AtD. Each sample was injected twice and analyzed according to the method previously described. The method accuracy was established by analyzing different concentrations of the two samples (50, 100, and 200 µg of 1) or (50, 100, and 150 µg of 4) in triplicate. All compounds were added to the decoction (500 µg/mL), concurrently, and analyzed according to the developed method. The mean percentage recoveries for 1 and 4 were found to be between 98 and 102 % by means of Fisherʼs F-test [29].

Precision and repeatability: The repeatability and the inter-day intermediate precision of six identical samples were analyzed according to the above-described method on two different days and by two different analysts in triplicate. The RSD and CV were calculated for each day.

Pharmacological assays

Animals: ICR male mice, weighing between 20 and 25 g, were purchased from Centro UNAM-Envigo (Envigo RMS) and kept in an environmentally controlled room maintained at 22 ± 1 °C with an alternating 12-h light/dark natural cycle and free access to a standard rodent pellet diet (Teklad 2018S, Envigo) and water ad libitum. All studies were conducted according to the principles and guidelines of the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999) with the approval of the Institutional Ethical Committee for the Use of Animals in Pharmacological and Toxicological Testing, Facultad de Química, UNAM (FQ/CICUAL/132/16 and FQ/CICUAL/136/16; both protocols were approved on March 8, 2016).

Acute oral toxicity study: The acute oral toxicity of AtD and 1 was conducted according the Lorke procedure in mice [10]. Male mice were divided into seven groups of three animals. The control group received vehicle (NaCl 0.9 %; 0.2 mL/10 g) and the other six groups received the treatments in two phases; in the first one, mice were treated with doses of 10, 100, and 1000 mg/kg, while in the second phase, the animals received doses of 1600, 2900, and 5000 mg/kg. In both stages, the animals were kept under observation and the body weight was recorded for 14 consecutive days. After this period of time, the toxic effects or macroscopic injuries were observed in the lungs, heart, stomach, and intestines, which removed under dissection.

Nicotinamide-streptozotocin induced experimental type 2 diabetes in mice: The animal model for the T2DM study was based on a single intraperitoneal injection of 50 mg/kg of NA followed by 130 mg/kg of STZ in 100 mM citrate buffer (pH 4.5) 15 min later. Afterwards, food and water were supplied ad libitum [30]. After 7 days, the blood glucose level was determined using a glucometer (One Touch Ultra 2, Johnson & Johnson) and mice having a blood glucose ≥ 200 mg/dL were considered hyperglycemic and selected for the study.

Acute hypoglycemic test: AHT was carried out to assess the reduction of basal blood glucose levels induced by the decoction and compounds isolated from A. thurberi under controlled fasting conditions. Animals were not fasted until the beginning and during the experiment (9 h). The test was carried out in normoglycemic and hyperglycemic mice. In both cases, the animals were randomly divided into five groups containing six mice each. Control groups (I and II) were orally administered with vehicle and GLI (15 mg/kg), respectively. Groups III–V were treated either with AtD or compounds (1–4) at the logarithmic doses of 31.6, 100, and 316 mg/kg or 3.2, 10, and 31.6 mg/kg, respectively. Blood samples were taken from the tail vein at 0, 1.5, 3, 5, 7, and 9 h after to the administration by a small incision at the end of the tail.

Oral glucose and sucrose tolerance tests: Normoglycemic or hyperglycemic mice were also divided into five groups (I–V) of six mice each. After 4 h fasting (morning fast), the animals of groups I and II (vehicle and reference drug, respectively) as well as those of groups III–V were administered orally with the treatments (AtD or compounds 1–4) at the doses of 31.6, 100, and 316 mg/kg or 3.2, 10, and 31.6 mg/kg, respectively. Thirty min after administering the treatments (compounds or controls), an oral glucose (2 g/kg) or sucrose (3 g/kg) load was given to each animal. Blood glucose levels were determined at 30, 60, 90, and 120 min post-administration of the carbohydrate load (postprandial). The references drugs employed for the OGTT and OSTT were MET (200 mg/kg) and ACAR (5 mg/kg), respectively. The percentage of glycemic variation (%) was determined as previously described [30].

Formalin induced-hyperalgesia in nicotinamide-streptozotocin mice: The formalin assay was assessed according to Hunskaar and Hole [31]. After 2 weeks of induction with NA-STZ, the hyperglycemic animals were divided into five groups (I–V) containing six animals each. Groups I and II (vehicle and reference drug, respectively) as well as groups III–V were administered subcutaneously on the dorsal surface of the right hind paw with 20 µL of the treatments (AtD or compounds 1–4) at doses of 31.6, 100, and 316 µg/paw or 3.2, 10, and 31.6 µg/paw, respectively. Thirty min after treatment administration, the mice received an injection of 40 µL of formalin (1 %) by using a microsyringe with a 30-gauge needle. All animals were put into a chamber and observed. Only licking or biting of the injected paw was defined as a hyperalgesic response. On the basis of the response pattern, two distinct periods of intensive licking activity were identified and scored separately. The first period (early phase) was recorded 0–5 min after the injection of formalin and the second period (late phase) was recorded 10–30 min after administration.

Assay for α-glucosidase inhibition: The decoction (AtD), compounds 2–4, and ACAR (reference drug) were dissolved in methanol or phosphate buffer solution (PBS; 100 mM, pH 7). Aliquots of 0–10 µL of testing samples were incubated for 10 min with 20 µL of enzyme stock solution [1 U/mL in PBS of yeast α-glucosidase (αGHY), Sigma-Aldrich]. After incubation, 10 µL of substrate [4-nitrophenyl-β-D-glucopyranoside (pNPG), 10 mM] were added and incubated for 20 min at 37 °C. Then, absorbance was measured in a BIO-RAD microplate reader model 680 at 405 nm [13]. Three replicates were made for each assay. The inhibitory activity was determined as a percentage in comparison to the negative control. The concentration required to inhibit activity of the enzyme by 50 % (IC₅₀) and the 95 % confidence interval were calculated by regression analysis using Origin 8.0 software.

Statistical analysis

Data are expressed as the mean ± standard error mean. Statistical significance differences were ascertained by means of two-way ANOVA followed by Bonferroniʼs test for temporal courses to analyze the antihyperglycemic effect, while one-way ANOVA followed by Dunnettʼs test was used for concentration-response curves to analyze the antihyperalgesic effect. GraphPad Prism software (version 5.0) was used for statistical analysis. A value of p ≤ 0.05 was considered statistically significant.

The authors declare that there are no conflicts of interest

Acknowledgments

This research was supported by grants from Dirección General de Asuntos de Personal Académico-UNAM (DGAPA, IN−217 516) and Consejo Nacional de Ciencia y Tecnología (CONACyT grants 205 195 and 219 765). The authors wish to thank Araceli Pérez-Vásquez, Georgina Duarte, Marisela Gutiérrez, Rosa Isela del Villar, Minerva Monroy, and Ramiro del Carmen for their technical assistance. A. L. Martínez acknowledges a fellowship from Programa de Becas Posdoctorales from DGAPA-UNAM.

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• 15 Peltier A, Goutman SA, Callaghan BC. Painful diabetic neuropathy. BMJ 2014; 348: g1799
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• 19 McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A 2007; 104: 13525-13530
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• 20 Vissers KCP, Geenen F, Biermans R, Meert TF. Pharmacological correlation between the formalin test and the neuropathic pain behaviour in different species with chronic constriction injury. Pharmacol Biochem Behav 2006; 84: 479-486
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• 21 Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: characteristic biphasic pain response. Pain 1989; 38: 347-352
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• 22 Sawynok J, Liu XJ. The formalin test: characteristics and usefulness of the model. Rev Analg 2004; 7: 145-163
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• 23 Hernández-Carlos B, Burgueño-Tapia E, Joseph-Nathan P. A new coumarin from Perezia hebeclada . Magn Reson Chem 2003; 41: 962-964
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  CrossrefPubMed
• 25 Zepeda LG, Burgueño-Tapia E, Pérez-Hernández N, Cuevas G, Joseph-Nathan P. NMR-based conformational analysis of perezone and analogues. Magn Reson Chem 2013; 51: 245-250
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• 29 International Conference on Harmonization. Text on validation of analytical procedures. Harmonized tripartite guideline Q2(R1). International Conference on Harmonization, Geneva, 1–13. Available at http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf Accessed April 11, 2016
  PubMed
• 30 Ovalle-Magallanes B, Medina-Campos ON, Pedraza-Chaverri J, Mata R. Hypoglycemic and antihyperglycemic effects of phytopreparations and limonoids from Swietenia humilis . Phytochemistry 2015; 110: 111-119
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• 31 Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987; 30: 103-114
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Correspondence

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• 18 Coderre TJ, Vaccarino AL, Melzack R. Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 1990; 535: 155-158
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  CrossrefPubMedSearch in Google Scholar
• 21 Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: characteristic biphasic pain response. Pain 1989; 38: 347-352
  CrossrefPubMedSearch in Google Scholar
• 22 Sawynok J, Liu XJ. The formalin test: characteristics and usefulness of the model. Rev Analg 2004; 7: 145-163
  PubMedSearch in Google Scholar
• 23 Hernández-Carlos B, Burgueño-Tapia E, Joseph-Nathan P. A new coumarin from Perezia hebeclada . Magn Reson Chem 2003; 41: 962-964
  CrossrefPubMedSearch in Google Scholar
• 24 Pérez-Hernández N, Gordillo-Roman B, Arrieta-Baez D, Cerda-García-Rojas CM, Joseph-Nathan P. Complete ¹H NMR assignment of cedranolides. Magn Reson Chem advance online publication 1 July 2015
  CrossrefPubMed
• 25 Zepeda LG, Burgueño-Tapia E, Pérez-Hernández N, Cuevas G, Joseph-Nathan P. NMR-based conformational analysis of perezone and analogues. Magn Reson Chem 2013; 51: 245-250
  CrossrefPubMedSearch in Google Scholar
• 26 Pari L, Rajarajeswari N. Efficacy of coumarin on hepatic key enzymes of glucose metabolism in chemical induced type 2 diabetic rats. Chem Biol Interact 2009; 181: 292-296
  CrossrefPubMedSearch in Google Scholar
• 27 Zhao DG, Zhou AY, Du Z, Zhang Y, Zhang K, Ma YY. Coumarins with α-glucosidase and α-amylase inhibitory activities from the flower of Edgeworthia gardneri . Fitoterapia 2015; 107: 122-127
  CrossrefPubMedSearch in Google Scholar
• 28 Abreu PA, Wilke DV, Araujo AJ, Marinho-Filho JDB, Ferreira EG, Ribeiro CMR, Pinheiro LS, Amorim KW, Valverde AL, Epifanio RA, Costa-Lotufo LV, Jimenez PC. Perezone, from the gorgonian Pseudopterogorgia rigida, induces oxidative stress in human leukemia cells. Rev Bras Farmacogn 2015; 25: 634-640
  CrossrefPubMedSearch in Google Scholar
• 29 International Conference on Harmonization. Text on validation of analytical procedures. Harmonized tripartite guideline Q2(R1). International Conference on Harmonization, Geneva, 1–13. Available at http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf Accessed April 11, 2016
  PubMed
• 30 Ovalle-Magallanes B, Medina-Campos ON, Pedraza-Chaverri J, Mata R. Hypoglycemic and antihyperglycemic effects of phytopreparations and limonoids from Swietenia humilis . Phytochemistry 2015; 110: 111-119
  CrossrefPubMedSearch in Google Scholar
• 31 Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987; 30: 103-114
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material