Oncocalyxone A Functions As an Anti-Glycation Agent In Vitro

Advanced glycation endproducts (AGE) are the result of post-translational changes to proteins, which ultimately compromise their structure and/or function. The identification of methods to prevent the formation of these compounds holds great promise in the development of alternative therapies for diseases such as diabetes. Plants used in traditional medicine are often rich sources of anti-glycation agents. Therefore, in this study, we investigated the anti-glycation activity of one such compound, Oncocalyxone A (Onco A). Using spectrofluorimetric techniques, we determined that Onco A inhibits AGE formation in a concentration-dependent manner. Its IC50 value (87.88 ± 3.08 μM) was almost two times lower than the standard anti-glycation compound aminoguanidine (184.68 ± 4.85 μM). The excellent anti-glycation activity of Onco A makes it an exciting candidate for the treatment of diseases associated with excessive accumulation of AGE. However, additional studies are necessary to identify its mechanism of action, as well as the in vivo response in suitable model organisms.


Introduction
Glycation is a non-enzymatic protein modification that occurs when proteins react with sugar molecules and/or metabolized intermediates, such as glyoxal or methylgloxal. These reactions occur both in vitro and in vivo. The first step of the process, the Maillard reaction, leads to the irreversible formation of advanced glycation end products (AGE), which ultimately interact with proteins resulting in damage and degradation. The accumulation of AGE plays an important role in age-related disorders and heart failure due to diabetic complications [1].
AGE arise as the result of post-translational protein modifications [2], and these changes may promote protein-protein interactions through the formation of intra-and intermolecular bonds, ultimately resulting in dysfunctional protein structure and/or function. In the blood, significant accumulation of AGE alters protein dynamics, and this has been attributed to premature aging and the increasing incidence of chronic diseases [3]. Indeed, there is a large body of work indicating that nonenzymatic glycation is involved in other disorders as well, such as arthritis, atherosclerosis, chronic renal failure, Alzheimer's disease, nephropathy, neuropathy, and cataracts [4]. Therefore, controlling the formation of these harmful intermediaries via anti-glycation agents has far-reaching implications in the treatment of various diseases.
There have been several proposed mechanisms of action of these anti-glycation agents, including: (1) the elimination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mainly hydroxyl radicals and superoxide radical anions, to decrease oxidative stress and reduce the generation of carbonyl or dicarbonyl groups; (2) the elimination of Amadori products and Schiff bases by preventing the formation of carbonyl and dicarbonyl compounds; (3) the chelation of free metals as the formation of AGE are related to the presence of transition metal ions; (4) the inhibition of the formation of an Amadori product in the final stage of glycation; (5) by breaking protein crosslinks promoted by AGE; (6) the occlusion of cellular AGE receptors (RAGE), which can reduce oxidative stress and inflammation in biological systems, and ultimately, prevent the late stages of glycation. Among the mechanisms described above, the first four are documented for naturally occurring anti-glycation products, while the final two are more commonly attributed to therapeutic agents such as aspirin, aminoguanidine, and diethylenetriaminepentaacetic acid. Antioxidant compounds are of particular interest as candidates as anti-glycation agents, given that they inhibit the formation of naturally occurring AGE, providing a strong therapeutic prospect toward delaying and preventing diabetic complications. Still, the mechanisms that account for the inhibitory pathways described above are poorly understood [5].
ROS and RNS play important roles in the non-enzymatic glycation of proteins [6]. In recent years, several natural products have been characterized as having anti-glycation activity, and often this correlates with antioxidant properties [7]. There are reports that many food products containing antioxidant compounds are able to eliminate ROS and RNS generated during glycation, as well as prevent the auto-oxidation of reducing sugars and Amadori products, and both are favorable ways for the inhibition of AGE [8].
The characterization of biological compounds identified in plant extracts is still in its infancy, due in large part to the significant biodiversity that exists, especially among Brazil's fauna. The isolation and possible development of an anti-glycation biocompound from these plants would be a highly relevant biotechnological discovery with significant importance for individuals worldwide [9].
The Auxemma oncocalyx Taub (Boraginaceae) is a plant native to Brazil's Northeast region, and it is quite common in the state of Ceará. Popularly known as "pau branco," it is used locally as an astringent and for the treatment of wounds [10]. Chemical studies of A. oncocalyx Taub resulted in the isolation and characterization of various quinones. The first compound isolated from the ethanolic extract was oncocalyxone A [rel-8α-hydroxy-5-hydroxymethyl-2-methoxy-8α, β-methyl-7, 8, 8a, 9-tetrahydro-1 4-anthracenedione] (Onco A ; Fig 1), which is characterized as a red solid, and most abundant quinone in this fraction [11,12].
Onco A exhibited various activities in vivo, including cytotoxic, analgesic and antioxidant functions, and inhibitory effects on inflammation, edema, platelet aggregation and tumorigenicity [10,13,14,15]. Additionally Onco A is known to inhibit cell growth by damaging DNA [15,16]. Strikingly, [17] reported that Onco A isolated from leaves of Prenanthes sarmentosus exerted anti-diabetic effects in rats with alloxan-induced diabetes. The animals had significantly lower blood glucose levels in the treated rats than the controls and an overall increase in body weight.
With the above in mind, the objective of this study was to characterize the inhibition of AGE formation and potential anti-glycation activity of Onco A in vitro.

Methods
A. oncocalyx Taub was collected at Pentencoste City in the state of Ceara, which is located in Northeast Brazil. The species was identified by Professor Afranio Gomes Fernandes from the Department of Biology of the Federal University of Ceara, where a voucher was deposited at the Prisco Bezerra Herbarium under the registration number 18459. OncoA was isolated as a deep red powder, mp 207-208°C, from the ethanol extract of the heartwood of A. oncocalyx as previously described [11]. The structure of this secondary metabolite was unambiguously established by spectrometric techniques such as IR, MS and a combination of 1D and 2D NMR methods. Its structural identity and purity (93.26%) were confirmed by thin layer chromatography (comparison with authentic sample), mixed melting point and 1H-and 13C-NMR spectral analysis [18].
Fluorescence readings and controls were normalized to corresponding blanks to exclude baseline fluorescence. The fluorescence (F) corrected for the negative control (F negative control ) and experimental treatments (F experimental corrected ) were used to determine the percentage of inhibition of the formation of AGE using the following formula [9]: Inhibition Percentage ¼ ðF negative control À F experimental corrected = F negative control Þ The IC 50 values, defined as the amount of compound (μM) necessary to reduce 50% of AGE formation as compared to the negative control, were determined using regression analysis. The anti-glycation activity observed in the present study is in agreement with the findings of Khan et al. [19]. While testing derivatives of N-aroyl-isatin, the group reported that of their 15 compounds tested, 6 displayed anti-glycation activity with IC 50 values ranging from 18.01 to 171.3 μM. These values are notably lower than aminoguanidine (268.7 μM). Additionally, Khan et al. [20] analyzed 27 bis-Schiff bases derived from isatins and found three with excellent anti-glycation activity. These compounds had IC 50 values in the range of 243.95-291.14 μM, which exceeded the standard used in their study, rutine (294.46 μM). In fact, these values were over two times higher than those obtained for Onco A, reinforcing again the significance of the anti-glycation activity reported here, Table 1.

Results and Discussion
Additionally, Onco A was found to be twice as effective as ethyl heamatommate, the most active compound (IC 50 = 220.55 μM) isolated from Parmotrema cooperi by Choudhary et al. [21], and three times as effective as the standard compound, rutin (IC 50 = 294.5 μM), used in their study. The authors reported that the presence of carbonyl and hydroxyl groups might be responsible for the anti-glycation activity of this series of compounds. Magadula et al. [22] found that morelloflavona, an isolated biflavonoid from Garcinia volkensis, was a very effective inhibitor of AGE formation with an IC 50 a little lower than that of Onco A (78 μM), according to Table 1.
Muhammad et al. [23] discovered that a derivative of cinnamic acid, the 2,4-dihydroxy-5-methoxy-cinaminic acid, isolated from the plant Viola betonicifolia moderately inhibited the formation of AGE, with IC 50 value of 355 μM. Note this is three times greater than the IC 50 of Onco A (87.88 ± 3.08 μM). Park et al. [24] have reported that three compounds isolated from Lespedeza maximowiczi are actually more active than Onco A, with IC 50 values ranging from 5.4-20.6 μM.  Regarding the molecular mechanism of Onco A, it is possible that its anti-glycation effects are via ROS inhibition, as this compound has been identified in previous antioxidant studies from Ferreira et al. [10]. The authors demonstrated that the quinone fraction of A. oncocalyx, consisting mainly of Onco A, has hepatoprotective effects in the CCl 4 -induced hepatotoxicity rat model, where both free radicals and lipid peroxidation are involved. They clarified that these hepatoprotective effects were mediated by the fraction's antioxidant function, which was verified by surveying the plasma activity of glutamate-pyruvate transaminase (GTP) and glutamate-oxalate-transaminase (GOT). However, this may not be the sole anti-glycation mechanism presented by Onco A.
Importantly, Ferreira et al. [14] identified the anti-platelet aggregation activity of Onco A, which is associated with increased levels of cGMP-independent nitric oxide (NO) and blockade of the glycoprotein GP Ibα. Accumulation of AGE in tissues, and the corresponding dysfunction in platelet aggregation, may promote diabetic complications such as retinopathy, neuropathy, and renal dysfunction [25]. Yamagishi et al. [26] examined the effects of AGE on the production of prostacyclin (PGI2), an antithrombogenic prostanoid, and the inhibitor of endothelial plasminogen activator 1 (PAI-1). They found that increased accumulation of AGE promotes platelet aggregation and PAI-1 expression, while also inhibiting prostacyclin expression, in endothelial cells. This suggests that AGE have the ability to trigger platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis, and ultimately, the development of diabetic vascular complications [26]. It appears likely that the inhibitory effects of Onco A on platelet aggregation are related to its anti-glycation activity, as there is an intimate association with excessive formation of AGE and the stimulation of platelet aggregates.
Thus, it is possible that the three major functions attributed to Onco A, antioxidant, anti-diabetic, anti-platelet aggregation are associated to the anti-glycation properties of the compound.

Conclusion
Onco A demonstrated potent inhibitory effects on glucose/fructose induced protein glycation, and this superior activity suggests it may potentially serve as a viable treatment targeted at preventing diabetic complications, as well as slowing the onset and progression of other AGErelated diseases. However, these findings should be taken with some caution, given the moderate cytotoxicity of the compound.
Still, it seems reasonable to propose that the dramatic effects Onco A has on reactive oxygen and platelet aggregation are associated with the anti-glycation activity demonstrated in this study, although the signaling mechanisms remain poorly understood. For these reasons, future studies in model organisms are both appropriate and necessary to further understanding of the benefits of Onco A.