Protective Effects of Oleanane and Ursane Type Triterpenoids from Origanum majorana Against the Formation of Advanced Glycation Endproducts

Bioassay guided fractionation of a crude methanol extract of leaves of Origanum majorana led the isolation of six new triterpenes (1-6). The structures of the isolates were established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation and by comparison with those previously reported in the literature as 3β,11α,16β,21β-tetrahydroxyolean-12-en-28 oic acid (1), 2α,3β,21β,22α,29α-pentahydroxy-olean-12-en-28-oic acid (2), 3β,15α,21α-trihydroxyolean-olean-12-en-28-oic acid (3), 2α,29α-diacetoxy,3β,23α-dihydroxy-olean-5,12-dien-28oic acid (4), 3β,15α,23α,19α-tetrahydroxyursan-12-ene-28-oic acid (5) and 2α,3β-diacetoxy-ursan-12,19-diene (6), Six other known triterpenes were isolated including oleanolic acid (7), 28-norlup-20(29)-ene-3β,17 β-diol (8), 3β,6β,7βtrihydroxy-20(29)lupine-ene (9), ursolic acid (10), 3β,15α,dihydroxy-30-norurs-12-ene (11), and lupeol (12). All of the isolates were subjected in vitro bioassays to evaluate their inhibitory activity on the formation of advanced glycation end products (AGEs) including AGEs-BSA, methylglyoxal, Amadorin activity, carboxymethyl-lysine, protein carbonyl content, α-dicarbonyl compounds formation, cellular oxidative stress inhibition and protein structural changes were evaluated by Maldi-TOF-MS. Triterpenes 1-5 displayed inhibitory effects on these specific AGEs, more effectively than the positive control, aminoguanidine. Among these, compounds 2, and 1 exhibited the most potential inhibitory activity against AGEs formation. This activity is attributed in part to carbonyl scavenging capacities. This edible plant may be used for controlling oxidative stress and inhibiting the AGE formation, which are implicated in the pathogenesis of diabetic complications (Image 1).


Introduction
Glycation, a nonenzymatic reaction between reducing sugars and lysine residues is an important source of reactive carbonyl species (RCS) that leads to the formation of protein advanced glycation end products (AGEs) such as Nɛ-carboxymethyllysine (CML), methylglyoxal lysine dimers and glyoxal lysine dimers [1]. These adducts are formed in all stages of the glycation process, by Schiff 's bases in early glycation, or degradation of glucose or Amadori products (fructosamine) in the intermediate stages of glycation. Thus, α-dicarbonyl could can be considered very important to understand how glucose can form AGEs by the Maillard reaction [2]. Methylglyoxal and glyoxal are reactive dicarbonyl species (RCS) common intermediates in protein damage. In the presence of protein dicarbonyl compounds, reducing sugars and transition metal ions, can auto-oxidize to form superoxide radical that can subsequently converted to hydroxyl radical which is highly toxic. Lipoxidation, AGEs, reactive oxygen species (ROS) generation can provoke tissue damage y activate inflammation [3]. The hyperglycaemia en diabetic can increase the production of free radicals and reactive oxygen species [4]. Glycation, is increased in hyperglycemic leading to an acceleration of AGEs [5]. Increased oxidative stress and accumulation of AGEs can induce cellular changes producing diseases, such as diabetic, atherosclerosis, retinopathy, neuropathy and nephropathy [6].
Origanum majorana L., is a perennial herb of the mint family (Lamiaceae or Labiatae). Marjoram is used traditionally, as a folk remedy against indigestion, asthma, rheumatism, and headache. Marjoram is used as a spice and its flavour is highly search to consumers worldwide. The spice is valued not only for its flavour but also for its antimicrobial and antioxidant activities [7][8][9]. Because marjoram has been known to possess medicinal effects, it have been used in pharmaceuticals and the industries of cosmetics [10]. In the present study isolation, structures and anti-AGEs activity of derivatives triterpenes of the oleanane and ursene from the leaves of Origanum majorana are described with some comments on the structural requirements for their activity.

Experimental Section
General experimental procedures IR spectra were obtained on a Perkin-Elmer 1720 FTIR. A Bruker DRX-300 NMR spectrometer, operating at 599. 19 MHz for 1H and 150.86 MHz for l3 C, using the UXNMR software package, was used for NMR experiments; chemical shifts are expressed in δ (ppm) using TMS as an internal standard. DEPT l3 C, ID TOCSY, lH-lH DQF-COSY, and HMBC NMR experiments were carried out using the conventional pulse sequences as described in the literature [11]. HREIMS were measured on a JEOL HX 110 mass spectrometer (JEOL, Tokyo, Japan). Precoated TLC silica gel 60 F254 aluminium sheets from Sigma-Aldrich (St. Louis, USA) were used. Column chromatography was carried out on Silica gel 60 (230-400 mesh, Merck Co. New Jersey, USA); solvents used as eluents were from Fermont (California, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, USA).

Plant material
Fresh leaves of Origanum majorana (Lamiaceae) were collected in the county of Amecameca de Juarez (Mexico State). They were identified by the Herbarium of the Metropolitan Autonomous University-Xochimilco. A representative specimen was kept (No. 7419) for further reference.

In vitro glycation of proteins
Determination of glycative product formations by fluorescence spectroscopy: According to the method of Vinson and Howard [12] the reaction mixture, 10 mg/ml of bovine serum albumin (BSA; Sigma, St Louis, MO, USA) in 50 mM phosphate buffer (PH 7.4) with 0.02% sodium azide to prevent bacterial growth, was added to 0.2 M of glucose. The reaction mixture was then mixed with compounds or aminoguanidine (Sigma, St. Louis, MO, USA). After incubating at 37°C for 15 day the fluorescent reaction products were assayed on a spectrofluorometric detector (BIO-TEK, Synergy HT, U.S.A.; Ex: 350 nm, Em: 450 nm).

BSA-methylglyoxal assay
The assay evaluates the middle stage of protein glycation [13]. BSA and methylglyoxal were dissolved in phosphate buffer (100 mM, pH 7.4) to a concentration of 20 mg/ml and 60 mM, respectively. Compounds were dissolved in the same phosphate buffer. 1 ml of the BSA solution was mixed with 1 ml of methylglyoxal solution and 1 ml of the samples. The mixture was incubated at 37ºC. Sodium azide (0.2 g/l) was used as an aseptic agent. Phosphate buffer was used as a blank. Aminoguanidine and phloroglucinol were used as positive controls. After seven days of incubation, fluorescence of the samples was measured using an excitation of 340 nm and an emission of 420 nm, respectively.

Amadorin activity
Amadorin activity was determined using a post-Amadori screening assay [14]. Lysozyme (10 mg/ml) was incubated with 0.5 M ribose in 0.1 M sodium phosphate buffer containing 3 mM sodium azide, pH 7.4 at 37°C for 24 h. Unbound ribose was removed by dialysis against 0.1 M sodium phosphate buffer, pH 7.4 at 4°C for 48 h with 5-6 changes. Following dialysis, the protein concentration was determined using the Bio-Rad standard protein assay kit based on the Bradford dye-binding procedure [15]. Dialysed ribated lysozyme (10 mg/ml) was reincubated with 10 mg/ml of compound or aminoguanidine or pyridoxamine in 0.1 M sodium phosphate buffer containing 3 mM sodium azide, pH 7.4 at 37°C for 15 days.

Determination of Nε-(carboxymethyl) Lysine (CML)
BSA was incubated with glucose, ribose and fructose for 4 weeks. N ε -CML was determined using a ELISA kit according to the manufacturer´s protocol. The absorbance of sample was compared with the CML-BSA standard providen in the assay kit.

Measurement of α-dicarbonyl compounds formation
100 µl of aliquots of glycated material were incubated at room temperature for 1 h with a reaction mixture containing 50 µl of Girard-T stock solution (500 mM) and 850 µl, of sodium formate (500 mM, pH 2.9). Absorbance was measured at 290 nm using a spectrophotometer (UV Mini 1240, Shimadzu, Kyoto, Japan), and glyoxal contents were calculated using a standard curve for glyoxal. A calibration curve was prepared using 40% glyoxal solution in a similar way [16].

Determination of protein carbonyl content
Protein carbonyl content, a common marker for protein oxidative damage, was measured according to a previous method with minor modifications [17]. Glycated BSA was incubated with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2.5 M HCl at room temperature for 1 h. Afterwards, it was precipitated by 20% (w/v) trichloroacetic acid (TCA), left on ice for 5 min, and centrifuged at 10,000 g at 4°C for 10 min. The pellet was washed three times using 1: 1 (v/v) ethanol-ethyl acetate mixture. The final pellet was dissolved in 6 M guanidine hydrochloride. The absorbance was recorded at 370 nm. The level of protein carbonyl content was calculated by using an absorption coefficient of 22,000 M -1 cm -1 . The results were expressed as nmol carbonyls/mg protein.

Cellular oxidative stress inhibition
Cell-culture and treatment: C2C12 cell purchased from ATCC (Manassas, VA, USA ) was cultured in Dulbecco's modified eagle's medium (DMEM) medium supplemented with 10% Fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cultures were maintained at 37°C in 5% CO 2 incubator. When the cells were about to cover 80% of the flask area, they were disrupted and seeded on 24 well plates. After attaining ~70-80% confluency, the cells were rinsed twice with phosphate buffer saline (PBS) and changed with  Cytoprotective effect against the oxidative stress induced by H 2 O 2 was measured by determining intracellular content of ROS. Intracellular ROS levels were measured employing 2′,7′-deoxyribosedichlorofluorescin-diacetate (DCFH-DA). DCFH-DA is cleaved intracellularly by non-specific esterase and turn to high fluorescent 2,7-dichlorofluorescein (DCF) upon oxidation by ROS, which were analyzed with FACS Aria II Flow Cytometer (BD Bioscience, San Jose, USA). C2C12 cells pretreated with triterpenes were incubated with DCFH-DA at 37°C for 1 h and then read in FACS Aria II.

Statistical analysis
Data are presented as means ± S.E.M. Statistical comparisons between groups were performed using the Student's t-test. The data were analyzed using the SPSS package (Version 10.0, SPSS, SPSS Inc., Chicago, IL, USA). The values at a p<0.05 and b p<0.01 were considered to be statistically significant difference.
Due to the lack of UV chromophore group in the structure of pentacyclic triterpenes, this study was not conducted.
Compound 1 showed strong absorption bands at 3491, 1710 and 1645 cm -1 in the IR spectrum indicative of the presence of hydroxyl, carbonyl and double bond groups respectively. The mass spectrum of 1 exhibited a weak molecular ion at m/z 504, which was in agreement with the formula C 30 H 48 O 6 and seven degrees of unsaturation. This was also supported with analysis of 1 H-and 13 C-NMR and the HMQC spectrum, data which were assigned to 1 as shown in Table 1. This compound displayed in DEPT experiments signals for thirty carbons which were distinguished as seven methyls, seven methylenes, eight methines (four oxygenated, three aliphatic, and one vinyl) and eight quaternary carbons (one oxygenated, six aliphatic, and one vinyl). The 13 C NMR spectrum showed the presence of a trisubstituted double bond at δ C 123.56 and δ C 141.56 (Table 1) together with the signals of 1 H NMR spectrum for seven singlet methyl groups (δ H 0.84, 0.82, 0.91, 1.07, 0.98, 1.19, 1.03) consistent with an 12-oleanene-28 oic acid carbon skeleton [25]. The presence of four oxygen bearing carbon atom in the molecule were indicated for signals at δ H 79.10, 76.59, 75.52 and 72.67 in the 13 C NMR.
A cross peaks of two methyl protons, H3-23 (δ H 0.84) and H3-24 (δ H 0.82), correlated with C-3 (δ C 79.10) carbon showed a hydroxyl group located at C-3 (δ C 79.10). In the 1H NMR spectrum of 1 the doublet of doublet at δH 3.54 and doublet at δH 4.40 were indicative of the deshielded protons attached to the two of the oxygen bearing methine carbons C-3 (δ C 79.10) and C-11 (δ C 72.67), respectively. The oxygenated doublet of doublets at δH 3.54, which correlated to the methine carbon at δ C 72.67 in the HETCOR spectrum, was assigned to H-11. The trans diaxial coupling constants J = 10.7 and 9.0 Hz for a double doublet (δ H 3.54) and doublet (δ H 4.40) respectively, suggested that both the 3-OH and 11-OH groups should be equatorial. This was also supported on the observed COSY coupling with the olefinic proton doublet resonating at δ H 5.32 (H-12) and the proton doublet absorbing at δ H 1.71 (H-9).
Compound 4 was obtained as amorphous powder. The molecular formula was C 33 H 48 O 8 on the basis of HRESI-MS. The IR spectrum showed the presence of hydroxyl (3461 cm -1 ), carbonyl (1735 cm -1 ), and double bond (1643 cm -1 ) groups absorption bands. 13 C NMR and DEPT spectra revealed thirty-three signals assigned to seven methyl groups, nine methylenes, six methynes and eleven quaternary carbon, with a degree of unsaturation of ten. The IR spectrum contained typical aborptions of hydroxyl (3405 cm -1 ) and carbonyl (1732 cm -1 ) groups, and a double bond (1668 cm -1 ) function. The 13 C-and DEPT spectral data indicated the presence of thirty carbons assigned to six methyl groups, eight methylenes including one hydroxy methyl, nine methynes including one olefinic and three hydroxyl groups, seven quaternary carbon including one carbonyl. These data are in agreement with the molecular formula indicated that 5 was ascribed to be derivate of ursane triterpenoids. The location of the C-3 hydroxy group, C-23 hydroxy group, C-28 acid group and C-12 double bond function were determined via an HMBC and NOESY experiments indicated the similarity with those of 4 (Table  1) showed a common 3β,23α-diol-urs-12-ene-28 oic acid nucleus for the triterpene. The HMBC correlations between H-15 (δ H 4.19) with C-8 (δ C 41.26), C-17 (δ C 42.70) and C-27 (δ C 13.14); H 2 -16 (δ H 1.57/2.0) with H 3 -27 (δ H 1.07) and C-15 (δ C 68.41) indicated that the second hydroxy group was located at C-15. The configurations of the hydroxy groups at C-3 and C-15 were assigned as β and α-orientations respectively, due to the trans-diaxial coupling constants of H-3 (J =11.  Table 2). Therefore compound 5 was concluded to be 3β,15α,23α,19α-tetrahydroxyursan-12-ene-28-oic acid (Figure 1). Triterpene 6 was isolated as a colorless solid. The molecular formula C 34 H 52 O 4 was established by its 13 C NMR and MS data, contained double bond, and acetoxyl groups attributable to the IR absorption bands at 1606, and 1732 cm -1 respectively. The 13 H NMR spectrum confirmed that is a triterpene skeleton with an ursolic acid type with a double bond at C-12-C-13, and two carboxyl group at δ C 172.31, and δ C 174. 19. The large coupling constant (J 2.3 ) of 10.3 Hz is typical to an antiperiplanar (axial-axial) relationship between H-2 and H-3 (Table 2)  In this study triterpenes 1-6 were evaluated for their ability to retard glycation reaction between glucose and albumin (Table 1). Among them, triterpenes 2 and 1 exhibited the most potential inhibitory activity against AGEs formation, with IC 50 values of 39 µM, 26 µM and 82 µM respectively. Compounds 3, 4 and 5 also showed stronger inhibitory activities (IC 50 values ranging from 128 to 200 µM) than a well known positive control, aminoguanidine (IC 50 value of 959 µM). However 6 showed very little activity (IC 50 value of 1301) than that produced by the AG.
The inhibitory effects of triterpenes on methylglyoxal-mediated protein glycation were evaluated. Compounds 2, 1, and 3 exhibited significant inhibition, and their IC 50 values were 201, 188, and 224 µM respectively, compared to AG (IC 50 , 335 µM). compounds 4 and 5 showed strong inhibition with IC 50 values of 300 and 242 µM respectively (Table 3). Methylglyoxal is a dicarbonyl intermediates as mediators of advanced glycation endproduct formation and are known to react with arginine, cysteine and lysine residues in proteins to form glycosylamine protein crosslinks [32,33].
A G. K. peptide containing a lysine residue was incubated with D-ribose for 24 h. This model system was used to evaluate the inhibitory effects of triterpenes on protein cross-linking. As shown in Table 3, compound 2 exhibited substantial anti-cross-linking activities. At a concentration of 10 mM, the inhibitory effect of compound 2 and pyridoxamine was 78% and 64%, respectively (Table 3).
For a period of 24 h exposure of lysozyme to ribose produced glycated protein rich in Amadori but not advanced glycation adducts [32]. Lysozyme is widely used for investigation of glycation-induced crosslinking. Triterpenes inhibit cross-linked advanced glycation endproducts and also have Amadorin activity to concentration of 50 µg/ml (Table 3). Pyridoxamine, used as positive control may inhibit at multiple stages of advanced glycation endproduct formation. This contrasts with other inhibitors, such as aminoguanidine which has no Amadorin activity [34].
These results indicated that for each stage of protein glycation compound 2 had the most potent inhibitory effect of all the compounds isolated from O. majorana. These observations suggested that the compounds can potentially inhibit the glycoxidative modification of proteins.
N ε -CML, is a glyco-oxidation product which is not fluorescent and not reactive, is produced from the oxidative degradation of Amadori products [32]. CML is an indicator of the advanced stages of the Maillard reaction. Table 4 shows the effect of 1-6 on N ε -CML level in glycated BSA after 4 weeks of incubation.  . Comparing with the percent reduction in PCO of 1-5 were more effective than AG at the same concentration.
The ability of triterpenes 1-5 to inhibit α-dicarbonyl compounds formation is showed in Table 5. The effect of 1-5 on glyoxal content showed all isolated have less inhibitory effect than that produced by the quercetin used as positive standard. Among the isolated compounds 2 had stronger inhibition activity.
The ability of the triterpenes 1-5 to decrease the oxidative stress in cells was evaluated by intracellular oxidative stress induced by H 2 O 2 in C2C12 cells. The oxidative stress reduction is showed in Table 5. The capacity of triterpenes (50 μg/ml) to reduce the oxidative stress was compared with that of ascorbic acid (25 μg/ml). The triterpenes 1-5 were less potent than ascorbic acid.
Glycation of triterpenes was monitored by MALDI-MS through the increase in the molecular weight of protein as a result of glucose adducts formation with the honnone. Figure 2 shows the typical mass spectra of positive (a) and mass spectra of BSA. In Figure 3, we can observe a typical spectrum of glycated protein where glycation of the protein was performed by incubation with 220 mM glucose for 30 d at 37°C in the absence of reducing conditions and glycated protein with 1 (a). The major peak at m/z 67925.163 corresponds to native BSA whereas the second peak at m/z 3999.656 corresponds to a diglycated form of BSA. A third minor peak at m/z 5397.765 is also observable and should correspond to a monoglycated form. Figure 3b, shows a different mass profile, especially in terms of relative abundance of each specie. There are two additional peaks in the spectrum besides the peak corresponding to native BSA. The major peak, at m/z of 5397.765, is the protonated Data are mean ± standard deviation (n = 3) a p<0.05 when compared to BSA ; b p<0.05 when compared to BSA + Glu, BSA + Fruc and BSA + Rib. Table 4: Effect of triterpenes 1-6 on non-fluorescence N ε -CML level and protein carbonyl content. Values represents mean ± SD (n = 3).   In all screening compound 2 displayed the most potent activity whereas compounds 1, 3, 5 and 4 gave significantly activity, instead compound 6 has much weaker activity considered inactive. This results suggested that the loss of the hydroxyl groups caused a significant loss of activity, revealing also the importance of the carboxyl group since no activity was observed in compound 6 which lacks this group. In general, the most active derivatives were oleanane followed by urseno. Interestingly, acetyl groups greatly diminished activity. Structural variations involving the hydroxyl groups substituted rings resulted in considerable improvement in inhibited AGEs.
In conclusion, according to the data obtained in this study Origanum majorana leaves extract contain various triterpenoids which showed excellent effect anti-glycation and they are effective inhibiting cellular oxidations. In particular, 2α,3β,21β,22α,29α-pentahydroxyolean-12-en-28-oic acid (2) displaying potent activity when compared to aminoguanidine a known glycation agent.