# Rosemary Leaf Extract Inhibits Glycation, Breast Cancer Proliferation, and Diabetes Risks
## 3 Results and Discussion
### Effect of Rosemary Extract on AGEs, CML and Glycation in Sugar-Protein Rich Foods  
AGEs are a group of heterogeneous compounds generated through the Maillard reaction [23]. Generally, they can be cataloged into fluorescent cross-linking AGEs and non-fluorescent non-cross-linking AGEs (such as CML) on the basis of the fluorescence properties and cross-linking structures [23]. Covalently cross-linked protein aggregates are reported to be involved in the development of chronic diabetic complications [24]. The intake of AGEs induces intracellular oxidant stress by the interruption of the calorie restriction on oxidant stress and causes diabetes mellitus or other chronic diseases in biological systems [23]. Therefore, the inhibition of AGEs, CML, and protein glycation formation in both the biological and food systems is critical for reducing the risks of age-related diseases. In this study, the inhibitory efficiency of rosemary extract on the formation of AGEs and CML as well as protein glycation were evaluated by a sugar-protein rich food model. As shown in Figure 2a, the levels of fluorescent intensity representing AGEs of control and all treatments decreased in the first 14 day but increased after day 21. It was because during the early storage of high protein food, amino acids and glucose would form glycosylation products, most of which had no fluorescence absorbance [24]. Furthermore, the reduce of some amino acids with fluorescence property by the Maillard reaction caused the decrement of fluorescence intensity [24]. However, after 21 days, the reaction could further react with free amino groups and thiol groups of protein, forming stable AGEs products. Thus, the fluorescence at the late stage dramatically increased [24]. Two water activities of wa 0.75 and wa 0.56 were used in this study to represent the most common values in food. A water activity higher than 0.80 would shift the reaction equilibrium from the glycated proteins end to the reactants end, which limits the reactants molecular mobility. As shown in Figure 2, the same trend of AGEs formation was observed in the IMF under both the wa 0.75 and 0.56 conditions. However, at day 28 and day 45, more AGEs were produced under the wa 0.75 condition. As indicated in Figure 2a,b, the inhibition of rosemary extract against AGEs formation was in a dose-dependent manner. Compared with the control group, RE3 reduced approximately 52.10% and 32.38% of AGEs at day 28 and day 56, respectively, under the wa 0.75 condition (Figure 2a). Similarly, RE3 reduced about 39.50% and 34.94% of AGEs at day 28 and day 56, respectively, under the wa 0.56 condition (Figure 2b). Generally, the Maillard reaction is relatively more active in the the wa range of 0.4-0.8, due to a dual effect of water. Under wa 0.75, the mobility of reactants was high, because the reactants became concentrated and this limited their diffusion under wa 0.56 [25]. Thus, higher level of AGEs was determined under wa 0.75 than wa 0.56. \begin{table}
\begin{tabular}{c c c} \hline
**Peak No.** & **Compounds** & **Concentration (mg/g DW)** \\ \hline
1 & Protocatechuic acid & \(2.16\pm 0.01\) \\
2 & Caffeic acid & \(0.02\pm 0.00\) \\
3 & Ellagic acid & \(2.04\pm 0.01\) \\
4 & Ferulic acid & \(0.01\pm 0.00\) \\
5 & Rosemarinic acid & \(17.28\pm 0.74\) \\
6 & Carnosol & \(4.45\pm 0.03\) \\
7 & Carnosc acid & \(8.31\pm 0.06\) \\ \hline \end{tabular} DW: Dry weight basis.  
\end{table} 
Table 1: Major phenolic compounds in rosemary extract. The CML accumulated with the increasing storage time under both of the conditions (Figure 3a,b). The inhibition of CML and AGEs may be due to the phenolic compounds in the rosemary extract. For example, rosemarinic acid was capable of inhibiting the formation of both total fluorescent AGEs and nonfluorescent carboxymethyllysine (CML) in a glucose-casein glycation model reported by [PERSON] [26]. There was 46.5% to 75.2% of CML reduction of with addition of carnosic acid [22].