In this study, we evaluated the assessed the hypoglycemiant, toxicogenetic and genotoxic effects of nanoencapsulate extracts of Bauhinia forficata Link. Phytochemical evaluations of extracts were carried out, as well as the evaluation using HPLC-MS and of antioxidant capacity in vitro. DM2 was induced in mice with streptozotocin and extracts were given orally for 28 days. That dried extract from infusion (ESIN) had a higher rate of bioactive compounds compared to the dried extract from decoction (ESDC), and higher antioxidant capacity. Glucose levels decreased from 77.26% to 57.79% and 45.15% after supplementation with ESIN (200 and 600 mg/kg/day) and ESDC (600 mg/kg/day), respectively, when compared to the diabetic group treated with metformin (600 mg/kg/day) (21.53%), with an improvement in the glycemic response e recovery of pancreatic β cells. Thus, our study has shown that these extracts exhibit hypoglycemiant activity, with a beneficial effect superior to metformin, as a result they could be considered as potential therapeutic agents for application in pharmaceutical formulations in the treatment of DM2.

Type 2 diabetes mellitus (T2DM) is a highly prevalent disease that affects more than 500 million people worldwide and is estimated to reach 1 billion people by 2050, with diabetes type 2 being responsible for over 90% of all cases. Despite the current knowledge of its risk factors and many public policies with prevention strategies, its incidence keeps increasing globally, which is why research on DM2 and drug development is encouraged in the search for novel alternatives to improve the quality of life for these patients [1,2].

Hyperglycemia is the main characteristic of DM2, caused by a deficiency in insulin due to a dysfunction in pancreatic β cells and resistance to insulin on tissues, with high morbimortality and rise of long-term complications, including retinopathy, nephropathy, damage to peripheral nerves, and cardiovascular diseases, leading to functional impairment and depression on patients, besides the financial burden on the family due to insulin costs, glycemic monitoring, oral antidiabetic medications and other drugs, constituting a relevant economic impact on countries and health systems [3].

Regarding DM2 pathogenesis, concomitant to hyperglycemia and hyperlipidemia, oxidative stress has played a key role, considering that in DM, the mitochondria are the main generators of reactive oxygen species (ROS) [4,5]. The increase in the production of ROS induces alterations in the expression and activity of antioxidant enzymes, making tissues more susceptible to oxidative stress, which leads to the development of diabetic complications [6]. Thus, antioxidants such as flavonoids, which eliminate free radicals, can help prevent or fight against DM2 and its aggravations, since they act on the metabolism of glucose, decreasing glycemia [7-9].

Bauhinia forficata Link is a medicinal plant, belonging to the family Fabaceae, largely used in forms (infusion and decoction) as a hypoglycemia in popular medicine [10]. Several studies reported the pharmacological effects of its leaves associated with the presence of flavonoids, mainly quercetin, and kaempferol. Antidiabetic effects as well as the prevention of aggravations associated to oxidative stress were attributed to a derivative of kaempferitrin [10-22].

Kaempferitrin also induces cytotoxic, antitumor, antiproliferative, and chemopreventive effects in vitro and in vivo against cells derived from human tumors of cervical carcinomas (HeLa), through apoptosis via the intrinsic caspase-dependent pathway and induces cell cycle arrest in the G1 phase [23]. Cechinel-Zanchett et al [24], showed that pre-treatment with the flavonoid kaempferitrin decreased the cytotoxicity of irinotecan in intestinal epithelial cells (IEC-6) and intestinal damage in mice exposed to irinotecan, due to its antioxidant potential, with consequent chemo preventive effect.

The cytotoxic effects of B. forficata extract were also evaluated in non-cancer cells by in vivo and in vitro systems, Artemia salina and human lymphocytes, respectively, showing no toxicity. However, in a human melanoma cell line (FO-1), B. forficata extracts significantly reduced the growth of these cancer cells, inferring a potential selectivity of this medicinal plant in inhibiting the growth of cancer cells [25].

There are some commercially available drugs for the treatment of DM. However, some of them are not accessible to the population, in addition to demonstrating adverse effects on health such as gastrointestinal disorders, hypoglycemia, weight gain, cardiovascular diseases, and urinary tract infections. Thus, B. forficata Link can be a viable alternative as a coadjutant in the treatment of DM2, with minimal side effects, and relatively low cost compared to synthetic medicines [26,27]. Its use as nanoencapsulated dried extracts to be applied in pharmaceutical forms is very promising when compared to fluids, due to lower storage costs and higher concentration and stability of the bioactive substances [28].

However, most studies with this plant species were conducted using fresh leaves or extracts obtained by organic solvents [29,30]. Thus, the present work used dry nanoencapsulated extracts, obtained from aqueous extracts of the leaves of B. forficata, in which the antioxidant activity, as well as the antidiabetic activity, in mice, in addition to its toxicogenetics through hematological, biochemical, and histopathological parameters and its genotoxic effects in multi-organs for application in novel pharmaceutical formulations, as a coadjutant in the treatment of DM2.

Plant material, extract preparation, chemical products and reagents

The leaves of Bauhinia forficata Link were collected during the year 2018, in Piaui, Brazil (5°03’7.2” S 42°77’26.4” W). A voucher specimen for leaves was deposited at Herbarium Graziela Barroso of the University Federal do Piaui, with registration at SISGEN AE1E536, as suggested by Chan, et al. [31]. Leaves were submitted to drying in an industrial stove at a temperature of 40 ºC, for 52 h, and were then pulverized in a mill. Aqueous extracts were obtained through decoction and infusion, in a proportion of 10% (m/v), according to Menezes, et al. [32]. The drying process of the extracts was carried out using a BUCHI B-290 spray-dryer, under the following conditions: inlet temperature 110 °C, flow rate 5.4 mL/min, 15% injection, pressure 0.9 bar and spray at 70%. The concentration of encapsulating agents was 30% (15% colloidal silicon dioxide and 15% maltodextrin), obtained through a factorial design 22, according to Rolim Neto, et al. [33], added to extractive solutions, consisting of 70% of plant material. After nanoencapsulation, the nanocapsulated extracts were named extract infusion extract (ESIN) and decoction dry extract (ESDC) and were previously characterized in a previous study by De Souza, et al. [34], regarding the physical-chemical, spectroscopic parameters, in addition to the bioaccessibility of its phenolic compounds, through in vitro digestion and its antioxidant activity, to identify differences between these extracts. All the chemical products and reagents required for this study were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA), and HPLC reagents were purchased from Merck KGaA (Darmstadt, Germany).

Phenolic compounds and antioxidant capacity

Total polyphenols were analyzed according to the method proposed by Swain and Hills [35]. Total flavonoids were quantified by the aluminum trichloride (AlCl3) method, according to Zhishen, Mengcheng, and Jianming [36]. Antioxidant capacity was measured through the DPPH· (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay, the 2,2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS^•+) radical cation assay, ferric reducing antioxidant power (FRAP) and by the oxygen radical absorption capacity (ORAC) method. The (DPPH) radical scavenging assay was performed according to Kim, et al. [37]. Antioxidant activity, through the ABTS free radical scavenging, was conducted according to Re, et al. [38]. Ferric reducing antioxidant power (FRAP) was assessed using the method proposed by Arnous, et al. [39]. ORAC assay was conducted according to the described methods of Prior, Hoang, Gu [40] and Dávalos, Gómez-Cordovés, Bartolomé [41].

Analysis by high-performance liquid chromatography coupled to mass spectrometry - HPLC-MSⁿ

Main total-polyphenol and flavonoids were separated, identified, and quantified by high-performance liquid chromatography through a photodiode array detector coupled to electrospray ionization sequential mass spectrometry and ion trap analyzer (HPLC-PDA-ESI-IT-MSⁿ), according to Cuyckens and Claeys [42]. Samples were filtered and 20 μL were injected into the HPLC system, in triplicates. The mobile phase consisted of water, 0.1% formic acid (A), and acetonitrile (B) and ran at a flow rate of 0.5 mL.min^-1. Polyphenols and flavonoids were identified using strategies alongside an analysis of the mass spectra obtained by the HPLC-PDA-ESI-IT-MSⁿ, comparison to a database of mass spectrum like the Massbank® (https://massbank.eu/MassBank/) and literature data [43,44].

Animals

A total of 55 male mice (Mus musculus), Swiss strain, weighing between 20 to 30 grams, were obtained from the Central Bioterio of the Federal University of Piaui. Animals were kept in a controlled environment for temperature (23° ± 1°C), in a 12-hour light/dark cycle with free access to water (ad libitum) and to commercial food for rodents. The experiment protocol was approved by the Ethics Committee on Animal Experimentation of UFPI (CEEA/UFPI N° 008/12) and developed following the guidelines of well-being and biosafety in animal experimentation proposed by the Brazilian Society of Science in Laboratory Animals and the National Council for Control of Animal Experimentation.

Toxicogenetic study

Acute toxicity: Acute oral toxicity studies were conducted in agreement with the Guidelines of the Organization for Economic Cooperation and Development (OECD) [45]. Mice were divided into four groups (n = 5), in which two of them received an aqueous solution containing nanoencapsulated dried extracts of the leaves of B. forficata Link subsp. forficata (ESIN2 and ESDC2); another group received a solution solely with the drying adjuvants (ADJV2); and a negative control group (NA), received only distilled water. The administered dose, via orogastric gavage, was 2000 mg/kg of the body weight.

Animals were monitored for the analysis of signs of toxicity through Hippocratic screening, which provides a general estimative for the toxicity of the substance under a state of conscience and general disposition, activity and motor system coordination, reflexes, and activities controlled by the central and autonomic nervous systems according to Malone and Robichaud [46] and Cunha, et al. [47].

This monitoring took place during the first hour after administration of the substances and periodically, in the first 24 h, with special attention during the first 4 h, the experiment was conducted in three moments: the first day (T0), seventh day (T7), and fourteenth day (T14), also, weight assessment was carried out in these three instances. The parameters assessed were the screening of the general activity of the animal, vocal tremor, irritability, force feedback, tail grip, twisting, body tone, strength in grasping, negative coordination, corneal reflex, tremors, convulsions, hypnosis, ptosis (inclination or fall upper eyelid), urination, defecation, piloerection (involuntary erection or bristling of the hair), breathing and mortality as Brito [48] and Silva, et al. [49]. Animals were placed in metabolic cages to monitor feed intake, water, and excreta elimination for 14 days.

Genotoxic assessment: For genotoxic assessment, during the acute toxicity study, a comet assay was carried out in different tissues and peripheral blood. The alkaline version of the comet assay was performed according to Dos Santos, et al. (2012) and Speit, and Rothfuss [50]. 10 μL aliquots of caudal peripheral blood of male mice were collected on the seventh (T7) and the fourteenth (T14) days of treatment and submitted to the comet assay. On the 14th day of treatment, 10 μL aliquots of cell homogenate of bone marrow, pancreas, liver, and peripheral blood were also collected and submitted to the comet assay.

Cell material was mixed with a thin layer of 0.75% low melting point agarose (90 μL) and smeared on pre-coated slides with 1.5% negative control agarose. The slides were then dipped in lysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, pH 10 with the addition of 1% Triton X-100 and 10% DMSO at the time of use) for up to 72 hours at 4 ºC. After this period, slides were incubated in an alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 minutes; then, they were exposed to an electric current of 300 mA and 25 V (0.90 V/cm) for 15 minutes in an electrophoresis chamber.

Finally, slides were neutralized with Tris buffer (0.4 M and pH 7.5) and stained with silver nitrate solution. Results were expressed as damage index (DI) and frequency of damage (%FD). DI was calculated using the formula: DI = Σ (number of cells in a given damage class X damage class), which ranged from 0 to 400. FD was calculated using the following formula: FD = 100 - number of class 0 cells. Bone marrow was collected from the epiphysis with fetal bovine serum, and centrifuged at 1500 rpm. After collection from the liver and pancreas, the organs were macerated in PBS, centrifuged, and subjected to the aforementioned procedure.

Hematological parameters: After the period of the experiment, animals were anesthetized using sodium thiopental (40 mg.kg^-1, intraperitoneally (i.p.)) and received lidocaine pre-analgesia (10 or mg.kg^-1, i.p.). Blood samples were collected by cardiac puncture and conditioned in microtubes with EDTA anticoagulant (tetra acetic acid) at the concentration of 10 mg/dL to analyze the following hematological parameters: erythrocytes, hematocrit, hemoglobin, total leukocyte count, neutrophils, eosinophils, basophils, monocytes and platelets, using the Advia 120/hematology (Siemens) automated hematology analyzer according to Ferreira, et al. [51].

Data for the toxicogenetic study were analyzed using the statistical software Graphpad prism (version 6.0).

Antidiabetic activity

Induction of diabetes: A total of 35 male mice were submitted to diabetes induction through intraperitoneal injection of a freshly made solution of streptozotocin (STZ, Sigma Chemical Company, Saint Louis, MO, USA) [52], at a single dose at a concentration of 150 mg/kg of the weight, dissolved in 0.01 M citrate buffer (pH 4.5), after a 12-hour fasting, according to Salgueiro, et al. [18]. Healthy mice received the same volume of the vehicle (citrate buffer). Seventy-two hours after induction, the glucose levels in the blood of the mice were measured by collecting a drop of blood from the tip of the tail and submitting it to an On-Call Plus glucometer. Mice with fasting blood glucose levels≥ 240 mg/dL were considered diabetic for this experiment.

Experiment design

After the DM2 model was established, mice were divided randomly into seven groups with five animals each, according to Matteucci and Giampietro [53]. The negative control group (NA) and the diabetic group (DA) were only administered distilled water; another diabetic group (DM) was treated using metformin, at a dose of 600 mg/kg/day, and treatment groups received ESIN and ESDC at doses of 200 mg/kg/day (ESIN2 and ESDC2), and 600 mg/kg/day (ESIN6 and ESDC6).

The doses used in this study were determined from the result of the toxicity tests, and also guided by the doses used in previous studies conducted by Da Cunha, et al. [13], and Pereira, et al. [54]. All samples were administered orally through an orogastric cannula, for 28 days, and animals were weighed weekly. Blood samples from the caudal vein were collected every seven days, after a night fasting of eight hours, to estimate blood sugar levels.

Oral Glucose Tolerance Test (OGTT): During the last week of treatment, mice were submitted to OGTT after a night fast of 12 hours, according to Pan, et al. [55] and Zhu, et al. [9]. We administered an oral solution of glucose (aqueous solution at 40%) at a dose of 2 mg/kg body weight. Then, blood was collected from the caudal vein of the mice at times 0, 30, 60, 90, and 120 minutes after administration of glucose, which was conferred using the glucometer.

Biochemical parameter: Biochemical tests were performed with blood samples, following Ribeiro, et al. [56], in which blood was centrifuged at 3000×g for 10 min at 4 ºC, immediately, and serum was obtained to determine levels of blood glucose, total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine. Assays were conducted using an automatic machine Labmax 240 with commercial systems from LABTEST®. After the death of the animal, the heart, lungs, stomach, liver, pancreas, and kidneys were collected, then cleaned with 0.9% NaCl solution and weighed to calculate the relative index of each organ. The last three organs were fixed with 10% formaldehyde for histopathological analysis.

Histopathological analysis: Proceedings followed by Li, et al. [2] [57] and Wang, et al. [58], in which tissue from the liver, kidneys, and pancreas was dehydrated in crescent series of ethanol (70 – 100%), diaphanized in xylol, followed by inclusion in histological paraffin. The inclusion blocks were sectioned in a standard microtome at a thickness of 3 μm and the sections obtained were stained using hematoxylin and eosin; the stained slides were examined under an electron microscope.

Statistical analysis

Results were presented as the mean ± standard deviation or standard error of the mean (S.E.M.). Data were analyzed by the t-student test for phenol content and antioxidant capacity, analysis of variance (ANOVA), Tukey test for the remaining experiments, and Bonferroni test for genotoxic assessment. These data, as well as correlations and graphs, were analyzed utilizing the program GraphPad Prism (version 6.0), with a 95% confidence level (p < 0.05).

Phenolic compounds and antioxidant capacity

Table 1 displays the quantification of polyphenols, flavonoids, and the antioxidant capacity of nanoencapsulation dried extracts ESIN and ESDC, through the DPPH·, ABTS^•+, FRAP, and by the method ORAC. Total polyphenols and flavonoids content showed a significant statistical difference between extracts, making it possible to infer that ESIN (29.98 mg EAG.g^-1 and 9.78 EC. g^-1) contains more bioactive compared to ESDC (21.49 mg EAG.g^-1 and 3.06 EC. g^-1). Results from this study exhibited superior flavonoid values to the ones obtained by Da Cunha, et al. (2010) for dried extracts of B. forficata leaves in an oven and by spray drying.

Concerning the antioxidant activity of these extracts, we also observed statistically significant differences between them in all assays (DPPH, ABTS, FRAP and ORAC), in which ESIN displayed more activity (101.60 μmol Trolox.g^-1, 8.12 μmol Trolox.g^-1, 118.86 μmol Trolox.g^-1, 1465.63 μmol Trolox.mg^-1) when compared to ESDC (64.72 μmol Trolox.g^-1, 7.11 μmol Trolox.g^-1, 57.68 μmol Trolox.g^-1, 1102.03 μmol Trolox.mg^-1), showing that the types of flavonoids extracted during the infusion process had a better response to oxidative stress (Table 1).

In our study, both showed antioxidant capacity in the ORAC assay which was superior to ascorbic acid (1136.0 μmol trolox eq/g) and to other ethanolic extracts obtained from the same plant species (855.8 μmol trolox eq/g) in a study by de Franco, et al. [59], implying that the process of extraction and nanoencapsulation of the studied extracts preserved the content of phenolic compounds and their antioxidant activity. These results are relevant, as this assay provides some benefits when compared to other antioxidant methods, such as the fact that it is conducted in physiological temperature and pH and the use of fluorescence instead of absorbance reduces the interference of colored compounds present in the samples, as reported by Dávalos, Gómez-Cordovés, Bartolomé [41] and Prior, Hoang, Gu [40].

ESIN and ESDC extracts can be a potential source of antioxidant compounds that play a role in detoxing the body, eliminating reactive oxygen or nitrogen species caused by hyperglycemia, a characteristic of DM, suggesting that this antioxidant capacity contributes positively to the prevention and treatment of this pathology, besides acting as signaling molecules in the mitochondrial endoplasmic reticulum and enzymatic pathways of metabolism. Therefore, it is relevant to assess the content of phenolic compounds as well as the antioxidant activity of plant extracts to verify their antidiabetic activity [60,61].

Analysis by HPLC-PDA-ESI-IT-MSⁿ

During the chromatographic analysis, phenolic acids and their glycosylated derivatives (between 8-10 minutes) and C- and O-glycosylated flavonoids within 10-20 minutes of the chromatogram (Figure 1) were mainly identified in the studied extracts. Da Cunha, et al. [13], Da Silva e Filho [62], Filho [63], and Pizzolatti, et al. [16], in their studies with extracts of B. forficata leaves, also found that phenolic compounds, especially flavonoids, are the main constituents.

Peaks number 11, 20, and 23 to 28 showed UV spectra with absorption bands between 254 and 365 nm, characteristic of flavonoids. Peak number 11 presented m/z 447 as its non-protonated ion, and its second-order fragmentation (MS²[447]) gave rise to ion m/z 315 as a product, due to the loss of an O-glycosylated pentose ([M ̶ 132 ̶ H]-), where m/z 315 refers to the aglycone of flavonol isorhamnetin via the break on the Y0 bind, a compound also identified by Farag, et al. [42] in their study with eight Bauhinia species.

Peaks 23 and 24 may be two isomers of kaempferol-O-rhamnosyl-3-O-rutinoside due to sequential fragments similar to peak 20 ((MS²[739]: m/z 593 and (MS3[739-593]: m/z 285), which are shown to be isomeric molecules with each other, having kaempferol such as aglycone and sequential losses of rhamnose and a rutinose. Farias e Mendez [64], Ferreres, et al. [43], Santos, Fortunato, and Spotorno [65], have also identified flavonoid derivatives in extracts of this same plant species, including kaempferol glycosides, whose ions have been identified as flavonoids-O-glycosides.

Toxicogenetic

Acute toxicity: For acute toxicity, a recommended maximum dose of 2000 mg/kg of the body weight was, then mice were observed regarding general behavioral changes, toxicity symptoms, and mortality after treatment during the first 4 hours, which are the most critical; then, during 24 hours and, posteriorly, for 14 days. At the end of the experiment, we did not register any deaths or observable evidence of behavioral toxicity.

Pereira, et al. [53] also conducted an acute toxicity study with a hydroalcoholic extract of B. forficata Link, at this same dose and did not record any deaths, indicating that this extract does not show relative toxicity at the test concentration. Düsman, et al. [66], verified that this plant holds a good potential to be used as a safe medicament, since they showed that its aqueous extract did not exhibit cytotoxic activity on bone marrow cells of Wistar rats, besides contributing to reducing chromosomic damage induced by chemotherapy due to its antimutagenic activity on pre-treatment (71%), simultaneous treatment (91%) and post-treatment (95%) with cyclophosphamide. Pepato, et al. [67], in their study with diabetic and regular rats that received aqueous decoction of B. forficata leaves at the concentration of 150 g/L, orally, for 33 days, also did not observe any measurable toxic effects with the enzymatic markers used.

Systemic toxicity, besides the Hippocratic screening, can be verified through the decrease in consumption of water and food, body weight, and relative weight of organs, according to Teo, et al. [68]. Body weight is one of the most commonly used parameters for toxicological assessments to indicate the often early appearance of toxic effects of a given substance in animal organisms. Lu, et al. [69] considered the gain or loss of body mass of toxicological importance if this alteration is of at least 10%. Acute administration of ESDC2 and ESIN2 did not reduce any of the assessed parameters in comparison with negative control (NA) (Table 2), indicating no significant toxicity of these extracts.

Genotoxic: Results from the genotoxic monitoring conducted during acute toxicity can be seen in Tables 3 and 4.

Index and frequency of DNA damage in the peripheral blood of animals, after seven days of extract administration, demonstrated significant differences concerning negative control (NA); however, we observed that genotoxic DNA damages were repaired after 14 days of administration when compared with the positive control (CPA).

In the bone marrow of mice treated with the extracts, genotoxic effects were observed, at the dose of 2000 mg/kg, in groups ADJV2, ESIN2, and ESDC2, with damage indexes of 161 ± 7.43, 150.60 ± 2.88, and 166 ± 16.10, respectively. We also observed damage in the liver, yet they were inferior to those caused by cyclophosphamide (CPA) (264 ± 44.61) when compared to negative control (NA) (54.80 ± 5.26). However, these effects were not observed in the pancreas of animals of groups treated with extracts ESIN2 (54.20 ± 9.52) and ESDC (61.20 ± 12.07), with no statistical difference concerning negative control (NA) (54.40 ± 5.03).

De Sousa, et al. [70] and Varela‐Barca, Agnez‐Lima, and De Medeiros [71], in their studies with Bauhinia species, have shown divergent results about the mutagenic potential of its extracts, which can be explained by the different chemical compounds that might be present, depending on the type of extract. Hodek, Trefil, Stiborová [72] and Skibola and Smith [73] found that flavonoids may exhibit antioxidant or mutagenic activity depending on the number and the position of the hydroxyl groups on rings A and B, as well as the types of solvents used for the extraction of these compounds.

Effects of ESDC and ESIN on the relative weight of organs: Relative weight of the main organs of mice, treated with a single dose of ESIN and ESDC, did not show significant alterations when compared to negative control (NA). During the 28 days of treatment, we observed a reduction of the pancreas in the following group’s DM, ESIN2, and ESDC6 when compared to NA, due to diabetes. There was no alteration in the liver amongst treated animals, with similar sizes to NA, except for ESIN6 which had an increase, which was also observed for the lungs and the stomach (Table 5).

Effects of ESDC and ESIN on the hematological parameters: Hematological parameters were analyzed, as they are important criteria to assess the extension of homeostasis and alterations resulting from pathological and toxicity processes, as reported by Okonkwo, et al. [74]. We observed a decrease in neutrophils and an increase in lymphocytes of all groups treated with the maximum dose of 2000 mg/kg/weight when compared to NA. This is justified by the fact that bioactive compounds, at high doses, go from antioxidants to pro-oxidants, which can cause inflammation and induce apoptosis, although this latter effect can be beneficial when applied in the treatment of neoplasms, according to Cerqueira, De Medeiros and Augusto [75].

During the 28-day treatment, leucocytes increased in groups DM, ESIN6, and ESDC2, an alteration that can be attributed to diabetes, which is a chronic inflammatory disease, as reported by Asmat, Abad, and Ismail [6]. Lymphocytes and neutrophils also showed alterations on DN, showing that this group, treated with metformin (600 mg/kg/weight), was the one that displayed more alterations (Table 6).

Effects of ESDC and ESIN on weight, blood glucose levels, and oral glucose tolerance: Figure 2 shows the effect of extracts ESIN and ESDC on body weight, which did not differ statistically between treated groups concerning the negative control group (NA), both on day 1 and day 28. A study conducted by Martínezmartínez, et al. [76] with diabetic mice showed that these animals also did not exhibit a significant weight gain during treatment with decoction from B. forficata leaves.

Fasting blood glucose levels of all experimental groups are shown in Figure 3. Blood glucose levels in the blood of diabetic groups showed a significant elevation in comparison to normal mice (NA) (p < 0.05), indicating the successful establishment of the DM2 model. After 4 weeks of supplementation with extracts ESIN and ESDC, blood glucose levels in groups ESIN2 (200 mg/kg/day), ESIN6 (600 mg/kg/day), ESDC6 (600 mg/kg/day) reduced significantly by 77.26%, 57.79% and 45.15%, respectively, when compared to the diabetic group treated with metformin (600 mg/kg/day), which only reduced its glycemia by 21.53% at the end of 28 days and did not demonstrate any statistical difference concerning group DA.

Amongst the groups treated with extracts, only group DSDC2 (200 mg/kg/day) did not obtain a reduction in blood sugar levels, being similar to the group with no treatment (DA). These results show the efficacy of nanoencapsulated extracts of B. forficata leaves in reducing blood sugar levels, in diabetic mice, at the two tested doses of the tested extracts ESIN and ESDC, except for the dose of 200 mg/kg/day of the latter, with both extracts showing blood glucose levels similar to those of non-diabetic group (NA) (p > 0.05), confirming that they are efficient in the treatment of DM2.

These results are relevant, as these extracts can be used by diabetic patients associated with a synthetic drug or to the use of insulin, to improve their efficacy and lead to treatment of DM2 using lower doses of synthetic drugs and hormones, as a result reducing side-effects, such as those caused by metformin for example (nausea, diarrhea, flatulence, inappetence and stomach pain), according to Cobble e Peters [77] and De Souza, et al. [27]. Additionally, extracts of some Bauhinia species can even improve gastrointestinal symptoms, mainly diarrhea, as reported in the study by Ahmed, et al. [78].

The beneficial effects of ESIN and ESDC extracts in the treatment of DM2 may be related to the various flavonoid glycosides, quercetin and kaempferol found in B. forficata leaves, as they act at multiple sites of carbohydrate metabolism in glucose regulatory pathways, improving insulin sensitivity, due to phosphorylation of the insulin receptor substrate (IRS), increasing insulin release, glucose uptake and glycogen biosynthesis, in addition to increasing adiponectin secretion, as already stated by Cazarolli, et al. [79], Cazarolli, et al. [80], Ren, et al. [81], Santos, Fortunato, Spotorno [65] and Sharma, Balomajumder, Roy [82]. As quercetin was not identified in the chromatographic analysis in our study, we can infer that the reduction in glycemia was due to kaempferol.

The hypoglycemic effect of these nanoencapsulated extracts can also be explained by the antioxidant action of the phenolic compounds released from the nanocapsules that act by removing or inactivating the peroxyl radicals (ROO•), formed during the initiation or propagation of the oxidation reaction, through the donation of atoms of hydrogen to these molecules, interrupting the chain reaction, inhibiting oxidative stress and its consequences [34].

The flavonoids present in these nanoencapsulation extracts (ESIN and ESDC), also modulate the gene expression of cytokines and enzymes such as α-amylase, α-glucosidase, lipase [58], in addition, to activate PPAR-γ, which promotes adipogenesis, as it induces the synthesis and storage of fatty acids and also inhibits the expression of inflammatory genes since iNOS suppresses the transcription factors AP-1 and NF-kB, modulates the MAPK activity and influences glucose uptake [83-86].

Alkhalidy, et al. [11] observed that a single daily dose of kaempferol, orally, can significantly improve hyperglycemia and increase glucose tolerance in insulin-deficient mice, which is associated with the increase in elimination and uptake of glucose, particularly oxidation in the muscular tissue and suppression of hepatic glucose production.

A study conducted by Da Cunha, et al. [13] with extracts of the leaves of B. forficata Link, dried by spray drying and oven drying and obtained using extraction solvents ethanol and water (1:2) as solvents, observed similar results to our present study, when finding a reduction in fasting glucose levels, after 7 days of treatment, at the dose of 200 mg/kg/body weight in STZ-induced diabetic mice.

A study on the antidiabetic activity in eight Bauhinia species was conducted by Farag, et al. [42] using the alpha-glucosidase inhibition assay, the results showed that the distinct species studied hold a potential antidiabetic effect, as they slow down the digestion of carbohydrates, resulting in lower post-prandial glucose levels and thus, being able to help in avoiding high peaks of glucose levels on DM2 patients. Ferreres, et al. [43] also confirmed the use of Bauhinia species as antidiabetic agents.

On the other hand, Volpato, et al. [87] orally administered, in pregnant and non-pregnant mice, an aqueous extract of B. forficata leaves, at doses of 500 mg/kg, 600 mg/kg and 1000 mg/kg, for 21 days, and found that this extract was not toxic at the tested doses but did not modify maternal hyperglycemia. Salgueiro, et al. [18], in their research with diabetic mice, treated diabetic mice daily for 21 days with the infusion from leaves of B. forficata subs. pruirosa, at the concentration of 313 mg/kg/weight and also did not observe a reduction in the blood glucose level in these animals, suggesting that kaempferitrin, the main substance responsible for hypoglycemic action, may have been lost during the preparation process of the infusion or is absent in this Bauhinia subspecies. These controversial results can also be justified by the differences in the experimental model used, types and methods of extract and/or fraction preparation, doses, and administration time, besides the genetic constitution of the species, environmental factors, and seasonal variations [76].

Effects of ESIN and ESDC on glucose tolerance in STZ-induced diabetic mice were shown in Figures 4 and 5. Oral glucose supplementation resulted in a gradual increase in blood glucose level within 30 min for all groups, remaining at an elevated level for the next 90 min, with animals from DSIN2 (168 mg/dL) already showing a decrease in blood glucose levels which is statistically different from the diabetic group without treatment (DA) (550 mg/dL) and with blood glucose levels values similar to group NA (149.33 mg/dL), that was permanent until the end of the experiment (T120), while glucose levels were slightly reduced for both (ESIN2 = 157.0 mg/dL and NA = 136.5 mg/dL), suggesting that glucose was being metabolized by insulin, reducing blood glucose levels in these groups.

Concerning animal groups treated with ESDC, ESDC6 displayed a better glycemic response with blood glucose levels of 287.3 mg/dL, when compared to DSDC2 (399.5 mg/dL). It was confirmed in T28, with fasting blood glucose levels, that there was a reduction in blood glucose levels of animals treated with ESIN and ESDC, at the doses of 200 and 600 mg/kg/day except ESDC2.

Effects of ESDC and ESIN on lipid parameters, hepatic enzymes, and kidney function

Total cholesterol (TC) levels for all groups were statistically similar concerning NA (117 mg/dL ± 5.95). Triglyceride levels (TG) of groups DA (72.40 mg/dL ± 6.33), DSIN6 (97.67 mg/dL ± 6.48), DSDC2 (88.40 mg/dL ± 6.75), and DSDC6 (83.40 mg/dL ± 6.62) showed levels similar to those of NA, with no observed significant statistical differences between the treated groups, only DM and DSIN2 displayed elevated levels compared to NA (Figure 6).

Corroborating this study, Damasceno, et al. [88], Pepato, et al. [89], and Pepato, et al. [90], when it comes to hypocholesterolemic and antilipidemic, activities results have not been significant. However, De Sousa Lino, et al. [70] verified a reduction of lipid parameters in rats treated with ethanolic, aqueous, and hexane extract of the leaves of B. forficata Link at doses of 200 and 400 mg/kg/day for 7 days.

Figure 7 shows the effects of ESIN and ESDC on the hepatic enzymes of diabetic mice. Activities of the liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) in the groups treated with the extracts did not demonstrate statistically significant changes when compared to non-diabetic mice (NA), displaying an absence of signs of hepatic toxicity. In contrast, Salgueiro, et al. [18], in a study with diabetic rats, found a significant increase in AST when compared to the negative control group, but ALT levels were not altered with B. forficata infusion treatment.

Urea and creatinine levels did not show a significant statistical difference between the treated groups after 28 days of administration of extracts ESIN and ESDC when compared to the negative control group (NA), which shows that the renal function of these animals had no alterations throughout the treatment (Figure 8). However, Mollica, et al. [91], in their study with an infusion from the leaves of plant species Juglans regia L., observed an increase in levels of urea and creatinine in rats treated at a dose of 25 mg/kg, and a decrease at the dose of 50 mg/kg, when compared to the negative control group.

Effects of ESDC and ESIN on the morphology of the liver, kidneys, and pancreas

Effects of ESDE and ESIN on the morphology of the liver, kidneys, and pancreas of diabetic mice were evaluated through histopathological analysis and are shown in Figures 9-11.

The histopathological profile of the negative control group (NA) displayed a negative control hepatic architecture, with negative control morphology of hepatocytes and hepatic sinusoids organized neatly (Figure 9A). However, group DA exhibited hepatocellular changes, with a decrease between sinusoids (Figure 9B).

In groups DSIN2, DSIN6, and ESIN2, some Kupffer cells were observed around the central vein, the characteristics of which were in agreement with the negative control histology, but there was a mild to moderate loss of liver architecture with some non-radially disposed hepatocytes (Figures 9D, E and F). In the DSDC2, DSDC6, and ESDC2 groups, in addition to what was observed in the other treated groups, fat droplets an increase in vascularization, and the presence of red blood cells in the space of Disse were observed (Figures 9G, H, and I). It is noteworthy that the changes observed in the histopathological analysis did not alter the levels of hepatic enzymes investigated.

Histopathological analysis of the kidneys of diabetic mice showed a renal cortex with renal corpuscles and their glomeruli, as well as proximal and distal tubules, with intact epithelial lining of the glomerular basement membrane and tubular epithelium, besides blood vessels similar to the negative control group (NA), none of the studied groups had alterations in these organs (Figure 10), in consonance with results obtained for urea and creatinine levels, which also did not show changes when compared with the negative control group, confirming that the renal function of these animals was not altered.

The histopathological study of the diabetic mice pancreas revealed that induction of diabetes with STZ caused necrotic alterations and karyolysis, with a decrease of the islets of Langerhans and pancreatic β cells observed in the untreated diabetic (DA) group. The group treated with metformin (DM) had a little more β cells than the DA group but inferior to the NA group. In the DSIN2, DSIN6, and ESIN2 groups, a complete recovery of the pancreatic β cells as well as the pancreatic parenchyma was observed. Groups DSDC2, DSDC6, and ESDC2, have shown to be similar to groups treated using ESIN; however, in group ESDC2, besides an increase in β cells, we also verified a slight decrease in α cells, concerning NA (Figure 11).

Since DM2 is a disorder that arises from the bad functioning of the endocrine pancreas, in which there was a loss or progressive dysfunction of insulin-producing pancreatic β cells, that may be due to a resistance to insulin and glucolipotoxicity, histopathological analyses of this organ are relevant, as already stated by Bakhti, Böttcher, Lickert [92] and Defronzo, et al. [93]. The theory that the main cause of DM2 is related to secretion and action of insulin has been recently confirmed by Chen, et al. [94], although other factors are also relevant, such as the activation of intracellular stress response pathways and inter-organ communication networks mediated by inflammatory molecules (cytokines) and peptide hormones. As there is still no treatment that can stop or revert the progress of this disease, results from this study are promising, since, as observed, the ESIN and ESDC extracts promoted the recovered pancreatic β cells, constituting possible alternatives for the treatment of DM2.

Alves, et al. [95] affirmed that research on natural active substances has grown over the past years, focused on the search for novel drugs with higher therapeutic activity, lower toxicity, better biocompatibility, and greater accessibility to the population who, due to cultural aspects, well accepts phytotherapy. The market of therapeutic products made from bioactive compounds present in medicinal plants presents a good perspective. Thus, studies such as this are relevant for the development of safe natural medicines that are efficient in the treatment of diabetes mellitus.

Chromatographic analysis has shown that the nanoencapsulation technique has preserved polyphenols, flavonoids, and their O-glycosylated derivatives in the dried infusion and decoction extracts of Bauhinia forficata Link, besides a significant antioxidant capacity, are potentially responsible for its beneficial effect in the regeneration of pancreatic β cells, with a decrease in blood glucose levels in diabetic mice superior to that of metformin. During the acute toxicity assays, extracts demonstrated genotoxic effects on bone marrow and on the liver, which were repaired after 14 days of treatment. However, these effects were not observed in the pancreas and peripheral blood. Nanoencapsulated extracts promoted the regeneration of pancreatic β cells, decreasing blood glucose levels in diabetic mice, with a superior beneficial effect to metformin, showing therapeutic potential for application in pharmaceutical formulations with antidiabetic activity.

Credit author statement

Bárbara Verônica Cardoso de Souza: Conceptualization, Writing - Original Draft, Writing - Review and Editing, formal analysis, data curation, responses. Alessandra Braga Ribeiro: Data Curation, methodology. Rita de Cássia Meneses Oliveira: Methodology, validation, resources. Julianne Viana Freire Portela: Research, review. Ana Amélia de Carvalho Melo Cavalcante: Writing - Review, methodology, data curation, formal analysis. Esmeralda Maria Lustosa Barros: Research. Luís Felipe Lima Matos: Research. Tarsia Giabardo Alves: Research. Maria do Carmo de Carvalho e Martins: Conceptualization, methodology, formal analysis. Lívio César Cunha Nunes: Project management – supervision, conceptualization, methodology, review.

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