On the other hand, the first-line drug to treat diabetes is Metformin, which has been shown to be effective both in monotherapy and in association with other oral drugs or with insulin (Salazar ÁLvarez, 2011). Additionally, it has been observed that patients treated with Metformin have a lower total and cardiovascular mortality than those treated with other oral drugs or insulin (Cases, 2008). The main mechanism of action of Metformin is the reduction of hepatic glucose production by decreasing hepatic gluconeogenesis, and in smaller proportion also increases the uptake of glucose in the muscle cell (Cases, 2008). Despite the abundant reports of Metformin and chaya as alternatives to treat diabetes, there are few studies focused on studying the possible synergism when combining them. Nowadays, many people usually consume chaya in the form of tea as an adjuvant for diabetes control, which is why we have found it important to study the effect that these extracts may have on Metformin. The objective of the present study was to evaluate the hypoglycaemic power of combination of various doses of Metformin and aqueous extracts of chaya in Long Evans rats induced to diabetes by streptozotocin.

To determine the effect of the combination of aqueous extract of chaya with Metformin, Long Evans rats were used, of both sexes, which presented a weight of 189 ± 30 g at the time of the study. The rats were kept in individual cages with access to food and drink and cleaned daily. The ambient temperature was maintained at 25 ± 3°C respecting circadian cycles of 12 h. Throughout the experiment, the ethical guidelines for experimentation in laboratory animals established by NOM-062-ZOO-1999 "Technical specifications for the production, care and use of laboratory animals" were met. The fulfillment of the ethical aspects was certified by MVZ Gerardo del Campo G. (C.P. 975133-R. SAGARPA   10-0006).   The   chaya  leaves  were  identified  in  the herbarium of the Interdisciplinary Center for Regional Integral Research and Development (CIIDIR) by Dr. Arturo Castro Castro (Voucher num 53,591) as C. aconitifolius (Mill.) I.M. Johnst from Euphorbiaceae family. (The name was confirmed in http://www.theplantlist.org/1.1/browse/A/Euphorbiaceae/Cnidoscolus/, July 5^th 2019).

Fresh leaves were collected from a bush grown in a domestic garden in the city of Durango, Dgo. Mexico (25° 11' 00'' N - 104° 34' 00'' W and 1885 m of elevation). The bush has been cultivated directly on land. It is approximately 2 m high and shows abundant ramifications. The leaves were collected during the summer of 2018. They had an intense green color, lobed and 10 to 15 cm long. The leaves were dried in the shade naturally until a weight loss of 80% (500 ± 170 mg per dried leaf).

Chaya extracts and Metformin

Chaya extracts were obtained by boiling 7.5 mg of dry leaf in 1 L of water for 5 min. This procedure is the one that the population commonly uses. The concentration of this extract was taken as 100%. Dilutions of the extracts were made with water and administered ad libitum. Metformin was also used in tablets of 850 mg of PiSA brand, Code 010.000.5165.00 with registration 2992000 SSA, which were pulverized in mortar and adjusted to the required dose according to the weight. The recommended dose in humans (850 mg per day, Cases 2008) was used as the basis for calculation. Metformin was given daily at a single evening dose.

Treatments

The rats were randomly distributed into five groups of six rats each fed a Roden Chow specific diet of Purina® rodents. Group 1 served as a control group and water ad libitum was administered. The remaining groups were streptozotocin-induced diabetic. Streptozotocin (STZ) is an antibiotic that produces pancreatic islet β‐cell destruction; therefore, it is widely used to induce type 1 and 2 diabetes in rats and mice (Furman, 2015). According to the protocol applied by Aragón and Ospina (2009), the rats were subjected to a 12-h fast, and then, intraperitoneally, they were injected with a single dose of 60 mg/kg of streptozotocin dissolved in a 0.1 M citrate buffer - pH 4.5. After checking the hyperglycemia (time zero) they were treated with combinations of Metformin and aqueous extract of chaya in two treatments as described below. Group 1 did not have any special treatment and remained healthy with food and water. Treatment 1 (T1) lasted two months counting from induction to diabetes, during which the doses specified in Table 1 were administered. At the end of this time, the second treatment (T2) was implemented for one more month as also specified in the same table.

Throughout the essay, no deaths were recorded in any of the groups. Figure 1 shows the results obtained whereas time zero (To) indicates the start of the  experiment  once hyperglycemia was verified by induction of diabetes. Treatment T1 covers days 0-60 whereas Treatment 2 covers day 60-90. Group 1 (healthy control) presented, throughout the experiment, an average glycemia of92.3 ± 10.1 mg/dl. However, the rest of the groups always maintained a hyperglycemia. In spite of this, it is noteworthy that Groups 3 and 5 showed a tendency to decrease glycaemia during Treatment 1. The ANOVA and Tukey tests used indicated that there was no significant difference between Group 3 and the control group (P>a), so it could be inferred that the 75% combination of chaya + 7.5 mg/kg of Metformin seems to have a therapeutic effect, but a synergistic effect cannot be inferred.

On the other hand, the rest of the combinations seem to have no beneficial effect. During T2 treatment, in which Metformin and extracts were individually and separately administered, glycemia decreased by almost half with respect to the initial value at time zero. However, the applied Student's T test did not show differences between the T1 and T2 treatments for any of the groups. The water consumption in the control group did not present significant differences between the T1 and T2 treatments (21 ± 8 and 24 ± 6 ml/day respectively). However, in the rest of the groups, water consumption was significantly decreased during T2 (77 ± 5 and 55 ± 10 ml/day respectively).

Thirty-two compounds present in the extracts were identified. Table 2 shows these compounds. The ionic abundance of each is presented in percentage in relation to the most abundant registered ion, Kaempferol-3-O-rutinoside (reference ion).

Figure 2 shows two of the chromatograms representative of HPLC-MS/MS analyzes. Panel A shows Kaempferol-3-O-rutinoside (Molecular weight 594.52), which was taken as a reference ion because it has the highest ionic abundance. Panel B shows the mass of arachidonic acid and its formula.

On the other hand, Caballero et al. (2017) has explained that oxidative stress and glycosylation of mitochondrial proteins involve the transcriptional factor NF-kB, NADPH-oxidase and the pro-apoptotic gene BAX. He explained that the NADPH generated from the metabolism of glucose plays an important role in oxidative stress through the reduction of hydrogen peroxide whose enzymatic mechanisms are associated with NF-kB, and whose expression increases in hyperglycemia. Metformin blocks these mechanisms by decreasing the expression of NF-kB and blocking the kinases involved in the activation of gluconeogenesis in the liver (Millán, 2003; Rena et al., 2017). It was observed that some of the phenols present in the extract like naringenin interfere in this action of Metformin in a similar way to that described by Yoshida et al. (2017) behaving  as  partial  agonists  in these sites, as it was noticed in this study.

Until now, the described mechanisms of action of Metformin include the biochemical part, the action in the liver cells and at the intestinal level, but many of them remain unknown (Rodulfo et al., 2017). Although a deep search was done, many reports on the mechanisms of action between Metformin and phenolic compounds in the TLR receptors or in the activation of the nuclear factor KB and its role as enzymatic inhibitor were not found, so it is necessary to go deeper into this area. Thus, although many natural sources such as chaya have a proven hypoglycaemic power, we recommend caution in their use when combined with Metformin because Chaya could inhibit the therapeutic effect of Metformin.

The results obtained suggest a possible antagonistic effect when combining aqueous extracts of Chaya (commonly used in Mexican populations) with Metformin (a medicine widely used in the treatment of diabetes), so it is recommended to extend the study and alert the physician so that this is taken into consideration. Future researches are recommended in the future about the pharmacodynamics and interaction at the molecular level of the combinations as well as verify the behavior of other biomarkers.

The authors declare that there is no conflict of interest