PHARMACEUTICAL COMPOSITION BASED ON TiQ ₂

NANOMATERIALS AND STEVIA REBAUDIANA BERTONI BACKGROUND OF THE INVENTION

1. Technical Field of the invention.

The present invention relates to a process for the preparation of a hypoglycemic pharmaceutical composition based on T1O2 nanomaterials and Stevia Rebaudiana Bertoni (SrB), and to the delivery of SrB/Ti02 nanomatrices on hyperglycemia and hyperlipidemia in alloxan-induced diabetes mellitus (DM) and diabetes mellitus type 1 (DM1 ) models. 2. Particulars of the Invention

Stevia Rebaudiana Bertoni (SrB) is a small bush native to the northern part of Paraguay and of adjacent zones of Brazil. The plant's leaves have been utilized to sweeten food.

Genus Stevia includes more than 200 species and only two of them contain steviol glycosides, SrB being the variety that contains the sweetest compounds. Glycosides perform numerous important roles in live organisms, they are hydrolyzed in the presence of water and an enzyme, thereby generating important sugars in the metabolism of the plant. The four main glycosides present in SrB are dulcoside A (0.3%), rebaudioside C (0.6%), rebaudioside A (3.8%) and stevioside (91 %). Other minor glycosides include rebaudioside B, D E and F, steviolbioside and rubososide. Rebaudioside A is from 250 to 450 times sweeter when compared with sucrose, thus having the highest sweetening power of all glycosides of these leaves. Glycosides of Stevia have a sweetness without calories and only a small quantity is necessary for sweetening. This aspect, in addition to the fact that it does not induce a glycemic response after its consumption, turns it into a good option for diabetics.

Furthermore, this plant has other beneficial properties such as: antihypertensive, digestive, antibacterial, hypoglycemic, anti-inflammatory, anti- carcinogenic, antioxidant and detoxifying effects, and it is abundantly rich in iron, manganese and cobalt.

The interest in the release of drugs through carriers has considerably increased in recent years; if the carrier has the potential to direct the drug to its place of action, an optimum pharmacological effect would be achieved and adverse effects of the drug would be reduced at the same time. It is a well-known fact that, once delivered to the organism, the distribution of the drug can cause secondary effects in other parts thereof. Therefore, an adequate balance between the beneficial effects produced by the medication and the adverse reactions that can be triggered in different organs should be established. The current pharmacological industry directs its studies to the search of a vehicle capable of transporting the drug to its place of action (target tissue), in order to avoid its adverse effects as much as possible. According to this criterion, currently new drug delivery systems have been developed, such as liposomes, nanomatrices and microparticles, among others, being particular colloidal carriers that are used as drugs releasing systems. Nanoencapsulation in biocompatible inorganic materials with human cell activity is an avantgarde technology to control the releasing process of the drug in the target place. Currently, the sol-gel process has reappeared as a promising platform for immobilization, stabilization and encapsulation of biological molecules such as enzymes, antibodies, microorganisms and a great variety of drugs. The matrices obtained by this method are chemically inert, hydrophilic and in a single step of the synthesis the desirable products are obtained, and furthermore possess high mechanical strength, thermal stability in wide temperature ranges and negligibly adsorb organic solvents in comparison with other organic polymers. An additional advantage is that it provides for viability to the encapsulated molecules, as these matrices act as water reservoirs, thereby helping to maintain the biological activity of the enzymes, antibodies, cells and, in the case of drugs, the hydration level required for the molecule.

The present invention addresses the issue of developing a process for the preparation of a hypoglycemic pharmaceutical composition based on T1O2 nanomaterials and Stevia Rebaudiana Bertoni (SrB), and to the delivery of SrB/Ti02 nanomatrices on hyperglycemia and hyperlipidemia in alloxan-induced diabetes mellitus (DM) and diabetes mellitus type 1 (DM1 ) models.

The patent application US 2014248382 A1 (LEE ET AL) published on September 4 ^th , 2014, describes: a complex comprising a compound of formula (I), or a vegetable extract comprising the compound or a fraction thereof; and stevioside, or a vegetable extract comprising stevioside or a fraction thereof, and refers to a pharmaceutical composition to prevent or treat a flu virus infection comprising the complex as an active ingredient. Besides, the present invention refers to a food composition to prevent or improve an infection by influenza virus, a quasi-drug virucidal composition, a virucidal food additive and a food comprising the complex as an active ingredient. According to the present invention, the complex comprising a compound of formula (I), or a vegetable extract comprising the compound or a fraction thereof and stevioside, or a vegetable extract comprising stevioside or a fraction thereof, thereby producing a virucidal effect and an effect of inhibiting cell degradation against an influenza virus, as well as the antiviral efficiency in a specific-pathogen-free (SPF) chicken, and therefore they can be employed in the prevention and processing of an infection by the influenza virus. The Mexican patent application No. MX 2014004817 A (MARKOSYAN) published on November 25 ^th , 2014, which corresponds to the document WO2013058870 A1 with international publication date of April 25 ^th , 2013, discloses: a process to produce a highly purified glycosyl stevia composition, characterized because it comprises the following stages: adding starch in water for form a starch suspension, adding a mixture of a-amylase and CGTase in the starch suspension and incubating from approximately 0.5-2 hours at approximately 75°C - 80° C, thereby resulting in a liquid starch suspension, inactivating α-amylase with a low pH heat, cooling the liquid starch suspension and adjusting pH to approximately 5.5-7.0, adding a freely soluble form of steviol glycosides in the liquid starch suspension, thereby resulting in a reaction mixture, adding a second batch CGTase in the reaction mixture and incubating for approximately 12-48 hours at approximately 55°C - 75° C, inactivating CGTase by low heat treatment, discoloring the reaction mixture, cooling the reaction mixture and adjusting the pH approximately from 2.0 to 4,5, precipitating unreacted steviol glycosides, separating precipitated unreacted steviol glycoside crystals from the filtrate, and so on.

Chinese patent application No. CN102512473 (SHI ET AL) published on June 27 ^th , 2012, discloses: a phenol extract of Stevia rebaudiana Bertoni (SRB), which reduces the activity of sugar in blood thereof and application of the phenol extract of Stevia rebaudiana Bertoni for the preparation of products for the reduction of sugar in blood. The phenol extract of Stevia rebaudiana Bertoni is heated and is subjected to reflux for being extracted with a solvent, and the phenol extract of Stevia rebaudiana Bertoni is obtained by chromatography separation and purification. The phenol extract of Stevia rebaudiana Bertoni has a clear effect in reducing sugar in the blood, and can be prepared in any common product. Developments do exist, however. No process exist in the prior art for the preparation of a hypoglycemic pharmaceutical composition based on T1O2 nanomaterials and Stevia Rebaudiana Bertoni (SrB), and for the delivery of SrB/Ti02 nanomatrices on hyperglycemia and hyperlipidemia in alloxan-induced diabetes mellitus (DM) and diabetes mellitus type 1 (DM1 ) models as the one claimed herein.

SUMMARY OF THE INVENTION

An example of an objective of the present invention is to obtain the synthesis separately from T1O2 nanomatrices and T1O2 to 30% volume concentration from the Stevia rebaudiana Bertoni (SRB) extract by means of a reflux system with constant agitation.

Another example of an objective of the present invention is to obtain T1O2 nanomatrices tagged as T1O2-70 through the preparation of a homogeneous solution, which contains butyl alcohol and deionized water. This solution is added to a three-necked glass reactor (flask), which is placed on a grid with integrated heating blanket, with constant stirring and reflux at 70 ^S C. Subsequently, the solution is added with titanium n-butoxide (97%). Ultimately, the solution is submerged in an iced container and the solvent is removed with a rotary evaporator.

Another example of an objective of the present invention is to obtain SRB/T1O2 nanomatrices with 30% by volume, labeled as SrB/TiO ₂ -30-70 and proceeding in a similar manner for obtaining a synthesis of TiO2-70 nanomatrices. A variant is that the homogeneous solution is added with a 30% volume of SRB extract, and performing the same procedure mentioned above.

Yet another example of an objective of the present invention is to characterize the nanomatrices with the following techniques: IR spectroscopy with Fourier Transforms, UV-VIS spectroscopy, x-ray diffraction (XRD) and SEM. Furthermore, another example of an objective of the present invention is to develop a hypoglycemic pharmaceutical composition based on T1O2 nanomaterials and Stevia rebaudiana Bertoni (SRB) to evaluate the hypoglycemic effect of SRB/TiO2-30-70 nanomatrices with alloxan.

Furthermore, another example of an objective of the present invention is to encapsulate the Stevia Rebaudiana Bertoni (SrB) extract in T1O2 nanomaterials for evaluating the hypoglycemic effect of SRB/TiO2-30-70 nanomatrices with alloxan.

The above objects are realized by a process to prepare Stevia Rebaudiana Bertoni (SrB) extract nanocapsules in T1O2 nanomaterials, wherein it comprises the steps of: a) obtaining the separate synthesis of T1O2 and T1O2 nanomatrices to 30% volume concentration of the Stevia Rebaudiana Bertoni (SrB) extract by: b) preparing an homogeneous solution with a reflux system with constant stirring to obtain T1O2 nanomatrices, labeled as TiO2-70; and c) preparing an homogeneous solution with a reflux system with constant stirring by adding 30% by volume of the SrB extract to the homogeneous solution to obtain the SrB/Ti0 ₂ nanomatrices, labeled as SrB/TiO ₂ -30-70.

Other features and advantages will become apparent from the following detailed description, taken together with the attached drawings, which illustrate by way of example the characteristics of various embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be completely understood by the detailed description given below and in the attached drawings, which are given only by way of illustration and example and therefore do not limit the aspects of the present invention. In the drawings, identical reference numbers identify similar elements or actions. The sizes and relative positions of the elements in the drawings are not necessarily drawn to scale. For example, the forms of the various elements and angles are not drawn to scale, and some of these elements are enlarged and located arbitrarily to improve the understanding of the drawing. In addition, the particular forms of the elements as drawn do not intend to convey any information concerning the real shape of the particular elements and only have been selected to facilitate its recognition in the drawings, wherein:

Figure 1 graphically shows IR spectra of the SrB extract and of ^" ΠΟ2-70 and SrB/TiO2-30-70 nanomatrices in accordance with an embodiment of the present invention;

Figure 2 graphically shows IR spectra of the 200 to 800 nm UV.VIS spectra of the SrB extract and of ^" ΠΟ2-70 and SrB/TiO2-30-70 nanomatrices in accordance with an embodiment of the present invention;

Figure 3 graphically shows IR spectra of the 600 to 800 nm UV.VIS spectra of the SrB extract and of ^" ΠΟ2-70 and SrB/TiO2-30-70 nanomatrices in accordance with an embodiment of the present invention; Figure 4 graphically shows the diffractograms of the ^" ΠΟ2-70 and SrB/Ti02-

30-70 nanomatrices in accordance with an embodiment of the present invention;

Figure 5 shows a micrograph of ^" ΠΟ2-70 nanomatrices in accordance with an embodiment of the present invention.

Figure 6 shows a micrograph of the ^" ΠΟ2-30-70 nanomatrix in accordance with an embodiment of the present invention;

Figures 7A and 7B graphically show the effect of the delivery of the SRB/TiO2-10-70 and SrB/TiO2-30-70 nanomatrices on the temporary course of the glucose concentration with alloxan-induced DM1 in accordance with an embodiment of the present invention;

Figure 8 graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on weight reduction in rats with alloxan-induced DM, in accordance with an embodiment of the present invention;

Figure 9A graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on glucose concentration (mg/dL) in plasma delivered with alloxan, in accordance with an embodiment of the present invention;

Figure 9B graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on glycosylated hemoglobin concentration (%) delivered with alloxan, in accordance with an embodiment of the present invention;

Figure 9C graphically shows the effect of the delivery of SRB/T1O2 nanomatrices on the concentration of insulin (μΙΙ/mL) delivered with alloxan;

Figure 10A graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on cholesterol concentration (mg/dL) delivered with alloxan, in accordance with an embodiment of the present invention;

Figure 10B graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on triglyceride concentration (mg/dL) delivered with alloxan, in accordance with an embodiment of the present invention; and

Figure 10C graphically shows the effect of the delivery of the SRB/T1O2 nanomatrices on HDL concentration (mg/dL) delivered with alloxan, in accordance with an embodiment of the present invention;

DETAILED DESCRIPTION OF THE INVENTION Several aspects of the present invention are described in more detail below, with reference to the attached drawings (figures, diagrams and graphs), in which the variations and the aspects of the present invention are shown. Several examples of aspects of the present invention may, however, be realized of many different forms and should not be construed as limitations to the variations in the present invention; on the other hand, the variations are provided so that this description is complete in the illustrative embodiments, and the scope thereof is fully conveyed to those skilled in the art.

Unless otherwise defined, all the technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which aspects of the present invention belong. The methods, processes, and examples provided in this document are for illustrative purposes only and are not intended to be limiting.

To the extent that the methods and developments are capable of reproducing the magnitudes reported in experiments, they can be still be considered for modeling various natural processes.

The present invention refers to a hypoglycemic pharmaceutical composition based on T1O2 nanomaterials and Stevia Rebaudiana Bertoni (SrB), produced through the synthesis of T1O2 and T1O2 to 30% volume concentration of the SrB extract.

The antihyperglycemic effect of Stevia of the present invention is based on:

1. Effect on glucose absorption: 1 mM of steviol may inhibit glucose absorption by 40%. Steviol causes a decrease in glucose absorption by the intestinal tissue. This takes place by a reduction of the ATP content in intestinal mucosa as a consequence of a decrease of the enzymatic activity linked with phosphorylation and morphological changes of intestinal cells. 2. Effect on glucose synthesis: This effect is mediated by the action of steviol on phosphoenolpiruvate carboxykinase (PEPCK) enzyme, an enzyme that limits the speed of gluconeogenesis upon the liver. Therefore, the stevioside decreases the gluconeogenesis on the liver by suppressing the gene expression of PEPCK, thereby leading to a decrease in the glucose level in plasma. However, this effect does not occur in a normal situation (without the presence of hyperglycemia), as Stevia leaves contain a variety of active compounds and which one shows the effect has not yet been identified. 1. Methodology.

A separate synthesis of the T1O2 and T1O2 nanomatrices to 30% volume concentration of the SrB extract (labeled as SrB/TiO2-30-70) is made, using a reflux system with constant stirring. All nanomatrices are synthesized under the same conditions separately.

2. Encapsulation.

In the synthesis process (separately) the SrB extract in T1O2 nanomatrices was encapsulated, which help to obtain quicker therapeutic effects with a single delivery, for evaluating the hypoglycemic effect of SrB/TiO2-30-70 nanomatrices.

3. T1O2 nanomatrices. A homogeneous solution is prepared containing butyl alcohol and deionized water. This solution is added to a three-necked glass reactor (flask), which is placed on a grid with integrated heating blanket, with constant stirring and reflux at 70 ^S C. Subsequently, the solution is added with titanium n-butoxide (97%). Afterwards, the solution is submerged in an iced container and the solvent is removed with a rotary evaporator. Therefore, the synthesis of T1O2 nanomatrices, labeled as T1O2-70, are obtained.

4. SrB-Ti02 nanomatrices.

A similar process is followed for obtaining the synthesis of TiO2-70 nanomatrices. The variant is that the homogeneous solution is added 30 vol % of the SrB extract, and the same procedure is performed as above, to obtain the nanoreservoirs of SRB/TiOi0 ₂ with 30% by volume, labeled as SrB/TiO ₂ -30-70.

The nanomatrices are characterized by the following techniques: IR with Fourier transforms, UV-VIS, DRX and SEM.

5. Example.

Assessment of biological activity of Stevia Rebaudiana Bertoni in alloxan- induced diabetic rats.

16 adult male rats of the strain Long Evans from "Claude Bernard" stock of the Autonomous University of Puebia were used, weighing approximately 120g to 180g, from which four experimental groups of n = 4 (c form, A, B and C) were formed.

These four groups are induced diabetes mellitus type 1 (DM1 ) with alloxan, kept in separate boxes with water and food and with periods of light/darkness 12/12 hours. The weight of the experimental subjects are is recorded at the beginning of the study. Rats are weighted on days 5, 10, 15 and 30 in order to watch the development of the DM1 clinical picture. During the course of evolution of DM1 and when rats display glycaemia of between 160-200 mg/dL, the behavior of the subjects is observed and delivered the 0.1 M citrate buffer pH = 4.5 alone and with SRB/TiO2-30-70 nanomatrices. The control group (c) is delivered 1 g/kg weight of 0.1 M citrate buffer pH =

4.5. Group A is delivered 45 mg/kg weight of alloxan in 0.1 M citrate buffer pH = 4.5. Group B is delivered a dose of 45 mg/kg by weight of alloxan in citrate buffer pH 4.5 and then a dose of 1 g/kg by weight intraperitoneal^ (i.p.) of SRB/T1O2-3O- 70 nanomatrices in accordance with Gregersen 2004. Group C is delivered a dose of 45 mg/kg by weight of alloxan in citrate buffer pH 4.5 and then a dose of 1 g/kg by weight i.p. of SRB/TiO2-30-70 nanomatrices.

Determination of glucose Blood glucose determination is performed using a Roche digital glucometer, based on the glucose oxidase reaction, said reaction occurring between blood glucose and glucose oxidase. Measurements of blood glucose concentrations for rodents are made in the short term (0, 4, 8 and 24 hours) and long term (5, 10, 15 and 30 days), after i.p. delivery of nanomatrices 1 g/kg by weight, when they are fasted for 6 hours.

Laboratory determinations

Once the long term study is run, on day 30th, two hours after the final determination of glucose, cardiac puncture is performed to each of the subjects, placing each sample in yellow tubes (thereby preventing blood clotting) for further processing and obtaining the following tests: measurement of insulin, glucose, glycated hemoglobin, triglycerides, total cholesterol, HDL. Statistical analysis With the data obtained from short and long term glucose measurements, a glucose kinetic graph is made in function of time (days) using the statistical treatment of an analysis of variance (one-way ANOVA Bonferroni post hoc); this statistical tool is likewise applied to the weight control data of the subjects and for other determinations (insulin, glycated hemoglobin, triglycerides, total cholesterol, HDL and weight) to better observe the behavior of the subjects. The differences with ^* p< 0.5, ^** p<0.01 and ^*** p<0.001 (Control vs. alloxan); ^# p< 0.5, ^## p<0.01 and ^### p< 0.001 (alloxan vs. SrB/TiO ₂ -30-70) are considered significant. RESULTS

IR spectroscopy

Figure 1 graphically shows FTIR spectra of T1O2 and SrB/TiO2-30 nanocatalysts. The IR spectrum of the T1O2 nanoreservoir that displays an absorption band at 3339.1 cm ¹ , thereby corresponding to the enlargement vibration mode of an VO-H type, which identifies hydroxyl groups (OH ), water(H- OH), of the solvent (butanol, R-OH) and of the hydroxylated matrix (Ti-OH). These functional groups are present in the titanium dioxide pores formed during the first gelation stage. The asymmetrical enlargement vibration modes VC-H are located at 2939.2 cm ¹ , which correspond to chemical species C-H of methyl (Ti- O-CH3) and ethoxy groups (≡Ti-OCH2CH3) which did not react during the T1O2 synthesis. At 1635.9 cm ¹ , the flexural vibration modes (VOH), of hydroxyl groups of water present on the surface of the T1O2 nanomaterial are presented. It is mainly associated with the humidity of the nanomaterial, of the solvent and with the deformation 6HOH of coordinated water. The flexural vibration modes of OH groups from ethoxide in the form of unreacted chemical species≡Ti-OEt are located at 1448 cm ^"1 . In this interval (from -1400 cm ^"1 to -1300 cm ^"1 ) of electromagnetic radiation the symmetrical flexural vibrations vcoo, asymmetrical deformation VC-H and scissors-like deformation 6CH3 are found. At 1290.7 cm ¹ , the oxidation bending type vibration modes vc=o of the material are located, and they are generally linked with impurities present during the material condensation process and with the ethoxy groups (Ti-OEt), which are unreacted still and with the oxidation of some methoxy groups. Wave numbers from 1 1 18.7 cm ¹ to 1006.5 cm ^"1 absorption correspond to the flexural vibration modes of C-C symmetrical groups (vc-c), CH2 (VCH2) and C-0 (vc-o), which correspond to methoxy-bridging species, as well as to solvents, products and byproducts of the synthesis reaction of the material. Around the nearby infrared region, the flexural vibration modes ντί-o from metal-oxygen interactions at 745.3 cm ¹ , 640.2 cm ¹ and 498.8 cm ^"1 are located.

Nanomaterials with SrB to 30% volume of the extract have the same enlargement vibration modes: (VO-H) and (VC-H) and flexural mode: (VO-H) , (6HOH) , (vcoo ), (VC-H) , (6CH3), (VC-C) , (VC-O) and (vn-o) as those observed in the T1O2 nanomatrix. More pronounced and wide vibration peaks are observed, these tend to move slightly to farther regions of IR.

UV-VIS spectroscopy

Figure 2 graphically shows UV-VIS spectra of TiO ₂ -70 and SrB/TiO ₂ -30-70 nanoreservoirs and of the SrB extract. UV-VIS spectrum of T1O2 presents a forbidden band energy of 376.5 nm. As the concentration (by volume) of the SrB extract in the T1O2 nanoreservoir increases, the curve of the SrB/Ti02 nanomatrix gradually moves towards lower energy regions of the electromagnetic spectrum; from a wavelength of 376.5 nm (for TiO ₂ -70) up to 406.2 nm (for SrB/TiO ₂ -30-70), as shown in table 1 . Table 1 shows the optical and electronic properties from UV- VIS spectra of (TiO2-70 and SrB/TiO2-30-70) nanomatrices obtained from fine chemical synthesis, from where:

Nanomatrices λ (nm) Eg (eV) v (Hz) x 10 ¹⁴ Spectral zone

TiO ₂ -70 376.5 3.29 7.97 UV-VIS SrB/TiO ₂ -30- 406.2 3.0 7.4 UV-VIS (violet)

70

Table 1 Data of optical and electronic properties of TiO2-70 and SrB/TiO2-30-70 nanomatrices.

In the UV-VIS spectra of SrB/TiO2-30-70 nanomatrices an absorption curve is observed at 670 nm, in the visible region. This coincides with the maximum absorption curve of the Stevia Rebaudiana Bertoni extract, having a wavelength of 671 .3 nm, this absorption band being associated with electronic transitions.

Figure 3 graphically shows the nanoreservoirs with a concentration greater than 1 μΙ_ of stevioside (SrB/TiO ₂ -3-70, SrB/TiO ₂ -10-70, SrB/TiO ₂ -20-70 and SrB/TiO2-30-70), and an absorption curve at 670 nm, in the visible region, can be observed. This absorption may correspond to η→π ^* transitions of α, β insaturated ketones and to the presence of cyclic oxypolyene groups of steviol. The greatest intensity band located in the UV-VIS spectrum of the Stevia extract corresponds to the electronic transitions of η→ττ ^* from the stevioside.

X-rav diffraction

In figure 4, x-ray diffractograms from TiO2-70 and SrB/TiO2-30-70 nanomatrices are graphically shown. Atomic dispersion factors from International Tables for X-Ray Crystallography were used for the analysis. According to the diffractograms presented in figure 4, all the nanomatrices obtained (TiO2-70 y SrB/TiO ₂ -30-70) are amorphous. Figure 5 shows the micrograph of TiO2-70 nanomatrices, with a traced area of 25 000X to 100 000X. TiO2-70 presents a form of agglomerate particles with an average particle diameter of 100nm. Figure 6 shows the micrograph of the SrB-TiO2-30-70 nanomatrix. The T1O2 nanomatrix with 30 μΙ_ SrB (SrB/TiO2-30) presents a sweep area of 1000X to 5000X, the average particle area being 50 nm. Evaluation of the biological activity of SrB/TiO2-30-70 nanomatrices in rats with DM1.

The effect of SrB/Ti0 ₂ - 10-70 and SrB/TiO ₂ -30-70 nanomatrices on the glucose levels in rats treated with alloxan during 30 days was assessed by quantifying glucose in a short term (0, 4, 8 and 24 hours) and in a long term (5, 10, 15 and 30 days) with the dry chemistry method.

The results indicate that the group treated with alloxan short term significantly increased glucose levels at 4 hours with 76%, at 8 hours with 76% and at 24 hours with 93% in the control group. Long-term, the results show an increase of 81 % (fifth day), 88% (tenth day), 93% (fifteenth day) and 60% (thirty- day).

Animals treated with SrB/TiO ₂ -10-70 and SrB/TiO ₂ -30-70 nanomatrices, the animals displayed a decrease in glucose levels. Results indicate that after 4 hours a reduction in glucose concentration of 4% and 30% was observed, at 8 hours glucose levels decreased by 19% and 25% and finally at 24 hours the glucose concentration was lower by 32% and 41 % compared with the hyperglycemic group delivered alloxan (see Figures 7A and 7B).

Figures 7A y 7B show the micrograph of the effect of delivery of SrB/Ti02- 10-70 and SrB/TiO2-30-70 nanomatrices on the temporary course (in hours and days) of glucose concentration in rats with alloxan-induced DM1 . Rats were delivered alloxan (150 mg/kg) and later the SrB/TiO ₂ -10-70 and SrB/TiO ₂ -30-70 nanomatrices. The plotted data represent mean ± SEM (standard error of the mean). (One/way variance analysis (ANOVA), post test Bonferroni), ^** p< 0.01 ; ^*** p< 0.001 (control vs. alloxan) ^# p< 0.05; ^### p < 0.001 (alloxan vs. SrB (10 and 30 μΙ_).

To quantify the average weight during the days when the assay is developed, the results indicate that the group treated with alloxan reports a decreased weight when compared with the groups treated with alloxan + SrB / TiO ₂ -10-70 of 14% (fifth day), 46% (tenth day), 94% (fifteenth day) and 187% (thirtieth day). The group treated with alloxan + SrB/TiO ₂ -30-70 was 22% (fifth day), 50% (tenth day), 88% (fifteenth day) and 144% (thirtieth day). The comparative analysis of the results revealed a significant difference (see figure 8).

Figure 8 graphically shows the effect of the delivery of SRB/T1O2 nanomatrices on the weight reduction in rats with alloxan-induced diabetes mellitus (DM). Rats were delivered alloxan (150 mg/kg) and later the SrB/Ti02- 10-70 and SrB/TiO2-30-70 nanomatrices. The plotted data represent mean ± SEM (one-way ANOVA, post test Bonferroni,) ^# p< 0.05; ^### p< 0.001 (alloxan vs. SrB/Ti0 ₂ - 10-70 and SrB/TiO ₂ -30-70). The concentration of glucose and glycosylated hemoglobin of alloxan treated groups + SrB/TiO2-10-70 in respect of the group with alloxan + T1O2 have a lower concentration of glucose of 28% and 40% glycosylated hemoglobin; whereas the group treated with alloxan + SrB/TiO2-30-70 has 28% glucose and 40% glycated hemoglobin 30 days after starting the treatment (See Figures 9A and 9B). Likewise, the concentration of insulin in the group treated with alloxan + SrB/Ti0 ₂ - 10-70 and alloxan + SrB/TiO ₂ -30-70 were increased between 137% and 645% respectively, when compared to the alloxan+Ti02 group. (See Figure 9C).

Figures 9A, 9B and 9C show the effect of the delivery of SRB/T1O2 nanomatrices on the concentration of glucose, glycosylated hemoglobin and insulin in rats delivered alloxan. Quantification was performed with plasma 30 days after the delivery of SRB/TiO ₂ -10-70 and SrB/TiO ₂ -30-70 nanomatrices (1 g/kg) in rats treated with alloxan (150 mg/kg). In the graph contained in Figure 9A glucose concentration (mg/dl) in plasma is shown and in the graph of Figure 9B the percentage of glycated hemoglobin is shown, whereas the graph of Figure 9C shows the concentration of insulin (μΙΙ/mL). The plotted data represent the mean ± SEM. (One-way ANOVA, post test Bonferroni), ^** p< 0.01 ; ^*** p< 0.001 (control vs. alloxan) ^## p< 0.01 ; ^### p < 0.001 (alloxan vs. SrB/TiO ₂ -10-70 and SrB/TiO ₂ - 30-70). The results show a decrease in the group treated with alloxan + SrB/TiO ₂ -

10-70 of 46% cholesterol and 84% triglycerides and for the group treated with alloxan + SrB/TiO ₂ -30-70 a 53% reduction of cholesterol and 80% of triglycerides with respect to the animals treated with alloxan + TiO ₂ . The statistical analysis indicated a significant difference (see Figures 10A and 10B). Likewise, the concentration of HDL in the group treated with alloxan + SrB/TiO ₂ -10-70 and alloxan + SrB/TiO ₂ -30-70 were increased between 1 19% and 120% respectively, a difference that turned out significant when compared to the alloxan+TiO ₂ group. (See Figure 10C). Figures 10A, 10B and 10C graphically show the effect of the delivery of

SRB/TiO ₂ nanomatrices on the concentration of cholesterol, triglycerides and HDL in rats delivered alloxan. Quantification was performed with plasma 30 days after the delivery of SRB/TiO ₂ (1 g/kg) in rats treated with alloxan (150 mg/kg). In the graph contained in Figure 10A, cholesterol concentration (mg/dl) is shown, and in the graph of Figure 10B the concentration of triglycerides of (mg/dL) and the graph of Figure 10C shows the HDL concentration (mg/dL). The plotted data represents the mean ± SEM (one-way ANOVA and Bonferroni post-test, ^** p< 0.01 ; ^*** p <0.001 (control vs. alloxan). ^### p< 0.001 (alloxan vs. SrB/TiO ₂ -10-70 and SrB/TiO ₂ -30-70).

Conclusion. The effect of the delivery of SRB/T1O2 nanomatrices on hyperglycemia and hyperlipidemia in an experimental alloxan-induced diabetes model was addressed. A toxic agent that reproduces the biochemical changes present in DM1 ; which has been used to assess the effectiveness of new drugs with hypoglycemic properties. The assessment of glycaemia and lipidemia allows to determine the degree of metabolic damage that exists in the experimental animals. The results show that the treatment with SrB/TiO2-10-70 and SrB/TiO2- 30-70 in a single delivery led to a hypoglycaemic and antihyperlipidemic activity in alloxan-induced diabetic rats, in respect of the group delivered alloxan + T1O2- V, obtaining better results in the group treated with SrB/TiO2-30-70. On the other hand, the group delivered the vehicle + TiO2-V showed glucose and lipid concentrations within the values physiologically deemed as normal in experimental animals. The results provide the first evidence of the metabolic recovery induced by an acute delivery of T1O2 nanomatrices with SrB versus the pancreatic toxicity caused by alloxan. They also provide a significant improvement in the levels of glucose, glycosylated hemoglobin and insulin in respect of the long-term hyperglycemic group. The results indicate that the mechanism of action of SRB is similar to sulfonylureas, due to the fact that blocks K ⁺ in β-pancreatic cells, depolarizes the membrane and promotes the opening of voltage-dependent Ca ²⁺ channels. Wherein the increase in intracellular Ca ²⁺ is crucial to induce insulin secretion from β-pancreatic cells and consequently causes the decrease of glycemia until biologically normal values are reached. The treatment given to the diabetic rats with T1O2 nanomatrices with SrB

(single dose) constantly stimulates the secretion of insulin in the remnant β- pancreatic cells. In consequence, the resulting insulin from the treatment with SrB promoted the decrease in the concentration of glucose and glycosylated hemoglobin in plasma of diabetic animals. Lipids play an important toxic role in DM1 , usually the lipid concentration increases in this pathology. The graphs reveal that the delivery of T1O2 nanomatrices with SrB in diabetic rats normalized the lipid profile, which shows the hypolipidemic effect of SRB; however, the molecular mechanism by which the effect occurs is unknown. Other studies indicate that SrB also exerts antioxidant and anti-apoptotic effects. The use of a controlled-release system of SRB is effective in restoring the metabolism of carbohydrates and lipids in a DM1 model.

The description of the invention is given by way of example only and is not intended in limiting in any way the scope of the invention. The previous specification is provided to allow any skilled artisan realize the various achievements described herein. Various modifications to these embodiments will be readily apparent to the skilled artisans, and the generic principles defined herein can be applied to other embodiments. Therefore, it is not intended that the claims are limited to the embodiments shown here, but they must be granted a full scope consistent with the wording of the claims, where reference to an element in particular is not intended to mean "one and only one", unless specifically stated, but rather "one or more". All the structural and functional equivalents of the elements of the various embodiments described throughout this specification, which are known or that will be subsequently known by artisans in the art are expressly incorporated herein by reference, and are intended to be covered by the claims. Therefore, it must be understood that numerous and varied modifications can be made without departing from the spirit of the present invention.