Glycerylphytate compounds with tunable ion affinity and osteogenic properties

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.


Results and Discussion
Synthesis and characterization of GPhy derivatives. The main goal of the present work was to obtain phytate derivatives with improved cytocompatibility without sacrificing the interesting biological properties of PA, and at the same time, to enlarge their applications in the biomedical and pharmaceutical fields. Thus, two phytate derivatives have been prepared by introducing glyceryl moieties in the chemical structure of PA, obtaining two compounds that were named as G 1 Phy and G 3 Phy according to their G content, as it will be explained below. This type of strategy has been previously reported for different polyol-phosphate derivatives synthetized as prodrugs with enhancing bioavailability 26 . Figure 1 displays the synthetic procedure applied in the preparation of GPhy. Based on the work of He et al. 27 who proposed a 5-axial/1-equatorial structure of PA at alkaline solutions, we assumed a similar structure for solid PA. As illustrated in the synthetic scheme, there are different possibilities of reaction between the reactants due to the six phosphate groups in the PA structure and the two hydroxylic groups in the G molecule. In addition, other factors such as the PA:G ratio, the conformation of the cyclohexane ring and steric factors will have influence in the final reaction products 28 . In order to have an insight of the stoichiometry of the two condensates, structural characterization was performed. Table 1 shows the results obtained from Elemental analysis (EA) and ICP. The formulas were deduced taking also into consideration the results of loss of water and percentage of residue at 800 °C obtained from (Thermogravimetric Analysis) TGA of PA and both glycerylphytates. Results indicated an average number of 1 glyceryl residue for the compound obtained with a molar ratio PA:G of 5:1 and an average of 3 glyceryl groups for that obtained with a molar ratio PA:G of 1:7 (G 1 Phy and G 3 Phy compounds, respectively). This showed an agreement between the amount of G in the feed and the conjugation of glycerol moieties with phosphate groups. Additionally, the results suggested that the conjugation reaction was also determined by steric hindrance since the number of glyceryl conjugated moieties increased from 1 to 3 when the PA:G molar ratio varied from 5:1 to 1:7. EDX analysis confirmed the presence of expected elements and the absence of impurities (spectra not shown). The quantification of the elements content is shown in Fig. S1 and results mainly reflected an increase of carbon content in the G 3 Phy sample respect to those of PA and G 1 Phy as it was expected due to the higher G conjugation in the former derivative. These results correlated well with those obtained by EA. Figure 2 shows the infrared spectra of PA and GPhy derivatives. PA spectrum showed a band at 3284 cm −1 due to the stretching vibrations of O-H groups, which suffered a broadening in the spectra of GPhy derivatives with the glyceryl content showing the contribution of the additional OH groups. The same observation was found in bands at 2974 and 2892 cm −1 , corresponding to asymmetric and symmetric stretching vibrations of C-H bonds of inositol rings and glyceryl groups. The intensities of these bands increased in both GPhy compounds, but the rise was considerably remarkable in G 3 Phy due to the higher G conjugation. This feature was confirmed by the band at 1460 cm −1 attributed to δ CH 2 groups in glyceryl groups as we can observe in the region of interest (1500-400 cm −1 ) in Fig. 2 29 . PA spectrum also showed a band at 1192 cm −1 corresponding to stretching vibration of P=O groups, and two bands at 1056 cm −1 and 913 cm −1 , which were attributed to P-O and C-O-P stretching vibrations in COPO 3 groups. Similar assignment for other phytates was given by Ishiguro et al. 30 and Guan et al. 31 , who also assigned the two absorption bands at 1192 and 1056 cm −1 to asymmetric and symmetric stretching vibrations corresponding to P-O in HPO 3 − groups in alkaline conditions. In the synthetized glycerylphytates these absorption bands shifted to lower wave numbers respect to PA. In particular, the band due to υ P=O groups shifted from 1192 cm −1 in the PA spectrum to 1185 cm −1 in the GPhy spectra, and the intense peak at 1056 cm −1 attributed to υ C-O alcohols, υ P-O and P-O-C, and υ C-O glycosidic, moved to 1035 cm −1 in the GPhy spectra. Finally, the absorption band at 970 cm −1 assigned to -PO 3 groups, and peaks at 915 and 851 cm −1 assigned to υ P-O and P-O-C bonds shifted respect to PA. All these findings observed in the GPhy spectra indicated changes in the chemical environment of phosphate groups because of the condensation reaction 27,30,32 .
The GPhy compounds were subsequently characterized by NMR spectroscopy. The 13 C NMR spectra of G 1 Phy and G 3 Phy and precursors are shown in Fig. S2a. The spectrum of PA showed four peaks at 74.7, 76.8, 77.5 and 78.9 ppm. The assignment of these peaks was performed according to previous work of Crimella et al. 33 as follows: δ 75.3, C 4 and C 6 ; δ 78, C 2 ; δ 78.5, C 1 and C 3 ; and δ 79.3, C 5 . The 13 C-NMR spectrum of G showed a peak at 63.9 ppm due to CH 2 carbons (C α ) and a peak at 73.5 ppm assigned to the CH carbons (C β ). Spectra of both  derivatives were rather similar to each other and revealed notable changes in their signals respect to precursors, indicating the presence of different C atoms after the condensation reaction. 13 Figure S2b shows the 1 H NMR spectra of both GPhy and precursors. Spectrum of G showed two doublet-of-doublet between 3.53 and 3.66 ppm that were assigned to no chemically equivalent H α protons and a multiplet signal between 3.75 and 3.80 ppm assigned to H β protons 34 . Spectrum of G 3 Phy showed modified signals in the range 3.50-3.82 ppm and two new multiplet signals at lower field; one of them was in the range 3.82-3.98 ppm and it was tentatively attributed to H α and H β protons in the groups CH 2 -O-P and CH-O-P of the glyceryl group. The other multiplet appeared in the range 4.05-4.10 ppm and it was attributed to H β protons of CH-O-P groups in the glyceryl moieties. The spectra of G 1 Phy differed somewhat respect to that of G 3 Phy. The main difference lied in that the new multiplet in the range 3.82-3.98 ppm almost disappeared and the signal in the range 3.98-4.10 ppm attributed to resonance of H β protons in glyceryl CH-O-P groups increased 33,35 . Proton NMR analysis of GPhy derivatives varying in glyceryl content suggested that for low G content in the feed, the reactivity of β hydroxylic groups towards phosphate groups would be favoured whereas an increase in the G content would enhance the reactivity of α hydroxylic.
This hypothesis was further studied through 2D DEPT-HSQC NMR analysis. In this concatenated experiment, the carbon multiplicity information obtained from the DEPT-experiment (CH or CH 2 in our products) was detected in the 2D HSQC spectrum and illustrated in different colours (Fig. 3). In the DEPT spectra of both glycerylphytates, carbons in the range 60-67 ppm appeared in red colour (CH 2 ) and carbons in the range 67-75 ppm appeared in blue colour (CH). Considering this information, in particular, in the 2D DEPT-HSQC spectrum of G 3 Phy the intense signal at 3.8/65.8 ppm was attributed to the glyceryl CH 2 -O groups conjugated with PA through CH 2 -O-P bonds and the signal at 3.8/70.7 ppm of lesser intensity, to conjugated CH-O groups of glyceryl moieties forming CH-O-P bonds. In addition, another two intense signals were present in the 2D spectrum, one of them at 3.5/62.2 ppm that could be due to CH 2 -O groups of reacted G, and the other one at 3.5/72.2 ppm due to reacted CH-O groups in the glyceryl moieties. As far as G 1 Phy is concerned, its 2D DEPT-HSQC spectrum showed that the majority of the signals were due to association of protons with CH carbons (blue colour). Moreover, the 2D plot of G 1 Phy showed less intense signals at 3.8/65.8 and 3.5/62.2 ppm as observed in G 3 Phy and instead, intense signals at 4.1/72.4 and 4.1/78.5 ppm. These findings suggest that conjugation reaction preferably proceeds between the β hydroxylic groups of G when this component is at low concentrations in the feed, whereas for higher G contents, the α hydroxylic groups are more likely to react, confirming the hypothesis previously proposed.
Thermal properties of GPhy derivatives were analysed by Differential Scanning Calorimetry (DSC) and TGA techniques. DSC thermograms revealed a glass transition temperature of 50 °C for both derivatives (thermograms not shown) indicating the amorphous character of these compounds because of the conjugation of glyceryl moieties in their structure. Table S1 summarizes the thermal degradation results of GPhy compounds and their precursors under inert atmosphere. Weight loss of glycerylphytates underwent in three stages in contrast to PA which showed four degradation steps. First stage was due to loss of water, 2 or 3 molecules for G 3 Phy and G 1 Phy, respectively, as explained above in the molecular formula analysis ( Table 1). The second stage involved carbonization of conjugated glyceryl moieties and the dehydration of phytate structure by the decomposition of hydroxylic groups. Finally, the third and last degradation stage of GPhy (T max around 340 °C) corresponded to further decomposition of the phytate moieties (332 °C) 36 . The residues of both derivatives obtained at 800 °C notably decreased in comparison to PA, indicating the content of glyceryl groups after the condensation reaction. Antioxidant properties. PA exhibits a powerful antioxidant activity due to its ion chelating capacity and hence, it is able to prevent Fenton reaction by inhibiting iron-catalysed hydroxyl radical formation 1,14,16,37 . This property has repercussions on the bone mass loss, as it is known that iron-induced oxidative stress is directly linked to the activation of NF-kβ, a key element in osteoclastogenesis 18,21,38,39 . Then, to investigate the antioxidant properties of GPhy compounds, their chelating activity was examined by the ferrozine/FeCl 2 system and compared with that of PA 15 . As it can be observed in Fig. 4a, PA and GPhy antioxidant properties were concentration dependent. Thus, ferrous chelating activity for PA was 33, 60, 73 and 78% at 10, 50, 100 and 300 µg/mL, respectively. Zajdel et al. 16 reported similar results regarding the powerful inhibitory capacity of PA in iron-catalysed oxidative reactions. The GPhy compounds exhibited a significantly lower antioxidant activity than PA at the lowest tested concentration (10 µg/mL). However, their antioxidant activity increased with concentration in both compounds, achieving values of 60% and 54% for G 1 Phy and G 3 Phy respectively, at 100 μg/mL. In particular, the chelating activity of G 3 Phy at all tested concentrations was significantly (p < 0.01) lower compared to PA. G 1 Phy, on the other hand, exhibited similar behaviour to G 3 Phy in the range of 10-100 µg/mL, while antioxidant activity reached to 84% at 300 µg/mL, being no significantly different (p < 0.5) from PA value. These results indicate that antioxidant properties of GPhy compounds measured by ferrozine/FeCl 2 method are not only concentration dependent as occurs with PA, but also composition dependent, showing antioxidant activity in the range of 50-80% for G 3 Phy and G 1 Phy respectively, at the highest studied concentration. This behaviour can be related to the incorporation of glyceryl moieties to PA structure, which reduces the number of ionizable protons and thus chelating properties.
Calcium binding properties. Calcium deficiency plays a crucial role in bone-loss related diseases such as osteoporosis. Although calcium absorption can be affected with age, some dietary factors, and pharmaceutical compounds, can also act as potent chelating agents of calcium ions making them unavailable 40  www.nature.com/scientificreports www.nature.com/scientificreports/ have been focused on the adverse effects of mineral uptake mediated by PA and its strong chelation capacity, concluding that the bioavaibility of these essential minerals can be compromised 40,[42][43][44] . Particularly, magnesium and calcium deficiency has been linked to osteoporosis.
In this work, the Ca 2+ binding capacity of GPhy derivatives was studied and compared with that of PA. After dropping a concentrated solution of the tested compound into a 4 mM solution of CaCl 2 , the precipitation of a solid, white or brown for PA and GPhy compounds respectively, was observed. The pellets were isolated and analysed by ICP spectroscopy. In Fig. 4b, the stoichiometry of the Ca-phytate complexes at pH 7.5 is expressed in moles Ca /moles Phy where Phy can be PA, G 1 Phy or G 3 Phy. The stoichiometry of Ca-G 1 Phy complexes was quite similar to that of Ca-PA complexes. Interestingly, a notable decrease in the chelating capacity of G 3 Phy was obtained. This can be explained by the higher content of glyceryl moieties in this derivative in comparison to G 1 Phy. These results confirm that due to the introduction of glyceryl groups, G 3 Phy derivatives show lower chelating activity than PA and they show how this modification can tune their binding ability.
In vitro biological effect of GPhy derivatives. Lipid peroxidation assay on RAW264.7 cells. Lipid peroxidation inhibition was measured in vitro by MDA-TBA adduct formation after the induction of Fenton reaction with the Fe 2+ /H 2 O 2 system on RAW264.7 cells. Figure 5 shows the results of the inhibitory effect on lipid peroxidation for PA and its derivatives at 100 μg/mL concentration. A higher amount of MDA-TBA adduct is indicative of a higher oxidative stress, therefore a 100% MDA-TBA was the maximum lipid peroxidation induced www.nature.com/scientificreports www.nature.com/scientificreports/ in the cells (positive control). When cells were incubated with PA or its derivatives, iron-loaded related stress was reduced due to their strong ability to chelate iron species. A higher MDA concentration was observed for G 3 Phy as compared to PA, which indicated a decrease in the ability to chelate iron ions as consequence of the higher content of glyceryl groups in its structure. Nevertheless, this compound was still able to prevent lipid peroxidation, as previously described for hydrolysis products of PA which maintain three or more phosphate groups 45,46 . Accordingly, G 3 Phy exhibited reasonable antioxidant iron stress properties in RAW264.7 cells in comparison to positive control. On the other side, G 1 Phy showed lipid peroxidation inhibition results comparable to PA, which correlated well with its antioxidant data obtained in previous analysis (Fig. 5). These results demonstrate that G 1 Phy and G 3 Phy maintain and modulate both iron (II) chelation and antioxidant properties of PA in cell cultures using the RAW264.7 cell line.   49 reported that PA extracted from rice bran was not toxic up to 6 mM for 72 h in untransformed cultures of 3T3 cells, yielding < 10% of dead cells 49 , Similar results were reported by de Lima et al. 50 who did not observe PA cytotoxicity at 4.0 mM for 48 h in lymphocytes isolated from healthy human donors. In this work, cytotoxicity (IC 50 ) of PA and derivatives was tested in MSCs cultures using a standardized Alamar Blue assay. Viability of cells treated with G 1 Phy or G 3 Phy increased respect to PA. IC 50 value of PA was 12.53 ± 2.21 mg/mL (14.5 ± 2.55 mM), while IC 50 values of G 1 Phy and G 3 Phy were 22.81 ± 2.64 (23.41 ± 2.7 mM) and 29.81 ± 2.43 mg/mL (27.45 ± 2.23 mM), respectively. These results suggest that larger amounts of glycerylphytate derivatives than of PA could be safely used in different specific biomedical applications.
Osteogenic properties and biomineralization on MSCs. The osteogenic activity of glycerylphytates and PA was investigated in MSCs cultures using Alamar Blue assay. To that end, firstly MSCs cultures were treated with the tested compound at different concentratios for 1, 7 and 14 d. Figure 6 shows that the metabolic activity of treated cells was affected by each tested compound in a different trend depending on dose and time. Cellular viability progressively decreased with concentration and time at 7 and 14 d and the lowest values were obtained at 150-300 µg/mL. G 3 Phy did not affect cell viability and even enhanced it at 300 µg/mL after long-term incubation time (14 d). The explanation of this enhancement is not clear yet but could be related with the less cytotoxicity of G 3 Phy which has an IC 50 value nearly double than that of PA. The behaviour of G 1 Phy approached that of G 3 Phy for a concentration range between 10 and 150 µg/mL and only a deleterious effect was observed when cells were treated for 14 d with the highest tested dose. These findings indicate that composition of the developed glycerylphytates plays a dominant role in their in vitro cytocompatibility and support previous results on their less cytotoxicity respect to PA.
Since PA has been suggested to be involved in osteogenesis 3,5 , ALP activity, an early marker of osteoblast differentiation, was determined in MSCs treated with the compounds. Figure 7 depicts ALP activity of MSCs treated with 10 μg/mL PA or glycerylphytate compounds for 7 and 14 d in complete or differentiation LG-DMEM media. At the two incubation periods, ALP activity was higher in MSCs cultured in differentiation LG-DMEM than in complete LG-DMEM, either they were untreated or treated with compounds. Interestingly, a different response to the compounds was observed depending on the type of medium. In complete LG-DMEM, ALP activity of cells treated with PA or its derivatives slightly increased respect to control, except for the case of G 1 Phy, in which ALP remained unaffected after 7 d. However, a different behaviour was observed in MSCs cultured in differentiating medium. ALP activity of cells treated with PA or G 1 Phy for 7 d was similar to control, while treatment for 14 d resulted in lower ALP activity. Cells incubated with G 3 Phy showed an interesting behaviour as ALP activity was slightly lower at 7 d respect to control and PA, but at 14 d it reached higher values than those found for PA and G 1 Phy.
The mechanism by which PA participates in osteogenic processes is not well understood, but we can elucidate from these results that bioavailability of phosphate residues may be quite relevant. Furthermore, Addison et al. 3 proposed that the high negative charge of PA molecule also has an impact on cellular interaction and diffusion across the membrane. For this reason, a possible explanation can lie in the enhanced cell-material interactions for G 3 Phy compound due to its major organic content. This hypothesis can be supported by the fact that this kind of modifications has been traditionally used for the protection of active phosphate groups, as those from PA 26 . An improved cytocompatibility would also enhance the metabolic effects that PA can trigger, as it has been demonstrated in this work. Thus, we can conclude that composition of GPhy compounds can tune the differentiation process of MSCs in comparison to PA itself. www.nature.com/scientificreports www.nature.com/scientificreports/ Gene expression of osteogenic markers by RT-PCR. As previously mentioned, treatment of MSCs with PA or GPhy for 14 in complete LG-DMEM d enhanced ALP activity (Fig. 7b). Thus, further studies investigated whether these compounds regulate the expression, at the mRNA levels, of osteogenic differentiation markers in MSCs. Results showed that incubation of MSCs with PA for 14 d in complete LG-DMEM increased COL1A expression (Fig. 8a). Interestingly, COL1A mRNA levels in MSCs treated with G 1 Phy or G 3 Phy were higher respect to PA suggesting that GPhy compounds could potentiate the deposition of a collagen matrix, which nucleates mineral deposition during osteogenesis. On the other side, a previous study reported that treatment of hUC-MSCs with 4 µM PA for 14 d under osteogenic conditions increased ALPL expression 24 . Data obtained in the present work indicated that incubation of MSCs with higher doses (10 μm) of PA in complete LG-DMEM induced an increase in ALPL mRNA levels, suggesting that PA could promote the acquisition of osteoblastic features in MSCs populations even in the absence of osteogenic inductors. Notably, G 1 Phy and G 3 Phy also increased ALPL expression in MSCs and ALPL transcript levels in MSCs treated with G 1 Phy were higher than those obtained in G 3 Phy or PA treated cells (Fig. 8b). It was not surprising that higher ALPL mRNA levels in G 1 Phy treated MSCs were not associated with increased ALP activity levels (Fig. 7b), as gene expression at the transcriptional levels does not necessarily correlate with protein activity, which is regulated by multiple post-transcriptional and post-translational mechanisms. Taken together, our results showed that PA and its derivatives might enhance MSC differentiation toward the osteoblastic lineage by regulating the expression of genes involved in matrix formation and maturation.

conclusions
In this paper, we report two new glycerylphytate derivatives (G 1 Phy, and G 3 Phy) which have different content of glyceryl groups. The reaction conditions allow to modulate chelation properties of the compound through the amount of glyceryl moieties incorporated. The new derivatives have improved cytocompatibility and osteogenic properties in comparison to PA in MSCs. Besides, both of them possess antioxidant and in vitro lipid peroxidation inhibition properties, which are essential features in tissue remodelling processes.
In summary, the chelation activity and biological properties of glycerylphytate compounds can be tuned by the organic content. This approach can offer a library of compounds with different composition to be used as an alternative to PA in biomedical and pharmaceutical applications related with stress oxidative phenomenon and low bone mass density.

Methods
Synthesis of GPhy derivatives. PA sodium salt hydrate (PA) and glycerol (G) were purchased from Sigma-Aldrich and used without further purification. The synthesis of GPhy compounds was carried out at 120 °C in bulk by reacting PA with the corresponding G volume for 12 h. The reaction products were dissolved in water, precipitated twice in 2-propanol, dried under reduced pressure to remove 2-propanol trace and lyophilized. Two ATR-FTIR spectra were obtained on a Perkin-Elmer (Spectrum One) spectrometer equipped with an ATR accessory for all samples. EA was performed with an elemental LECO model CHNS-932 microanalyzer. EDX spectrometry analysis was performed with a Bruker XFlash model with detector 5030. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were carried out in a 4300 DV Perkin-Elmer plasma emission spectrometer using a Gemcone (Perkin-Elmer) nebulizer. DSC experiments were carried out on a DSC model 8500 (PerkinElmer). Three heating-cooling cycles were analysed between 25 °C and 180 °C with a scanning rate of 10 °C/min under nitrogen at 20 mL/min flow rate. From the thermograms, the glass transition temperature (T g ) was determined as the midpoint of the transition. TGA was performed in a thermogravimetric analyzer TGA Q500 (TA instruments) apparatus, under dynamic nitrogen at a heating rate of 10 °C/min in a range of 40-800 °C.
Formation of ions chelate complexes. The iron chelate complexes formation was studied by the method of Dinis et al. 15 PA and GPhy compounds at various concentrations (10-300 µg/ml) in dH 2 O (0.4 mL) were added to a solution of 2 mM FeCl 2 (0.05 mL, Sigma Aldrich). The mixture was shaken vigorously and left at room temperature for 15 min under darkness conditions. Then, 0.2 mL of 5 mM ferrozine (Sigma Aldrich) solution was added, and the total volume was adjusted to 4 mL with ethanol. Absorbance of the solution was measured spectrophotometrically at 562 nm with a NanoDrop ™ spectrophotometer. 10 mM EDTA solution and dH 2 O were used as positive and negative controls, respectively. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± standard deviations (SD). Mean + SD values are relative to untreated MSCs, which were given an arbitrary value of 1. Analysis of variance (ANOVA) of the results for tested samples was performed with respect to untreated cells at significance levels of *p < 0.05, and with respect to PA treated cells at significance levels of # p < 0.05.
The binding capacity of PA and GPhy derivatives to calcium ions was examined with an adapted method from Saw et al. 51 . Particularly, aliquots of 50 µl at a concentration of 396 mg/mL of each compound were added to a 20 mL of a 4 mM solution of CaCl 2 in intervals of 2 min until achieving a final concentration of 5.27 mg/mL of each compound in the solution. The pH was controlled by preparing all the solutions in Tris-HCl 0.1 M buffer pH 7.5. The pellet was isolated by centrifuging at 4 °C and 8000 rpm (Eppendorf centrifuge 5810 R model) for 10 min, and washed with dH 2 O. The final pellet was digested at 65 °C with 65% v/v HNO 3 . Finally, the phosphorus and calcium content of these pellets in 5% v/v HNO 3 solution were measured by ICP. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD. cell culture. Human mesenchymal stem cells from bone marrow (MSCs, Innoprot) were grown and maintained in Mesenchymal Stem Cell Medium Kit (Innoprot) at 37 °C in a humidified atmosphere of 5% CO 2 . Cell culture media was refreshed every 48 h. For subsequent experiments, MSCs were cultured in Low Glucose Dulbecco's Modified Eagle Medium (LG-DMEM), supplemented with 20% fetal bovine serum (FBS), 200 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin (complete LG-DMEM). Differentiation medium consisted of complete LG-DMEM supplemented with dexamethasone (100 nM), ascorbic acid (50 μg/mL), and β-glycerolphosphate (10 mM). All the experiments were performed using MSCs at passages 4 to 8.
Murine RAW 264.7 macrophage cell line (Innoprot) were grown and maintained in DMEM supplemented with 10% FBS, 200 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin, at 37 °C in a humidified atmosphere of 5% CO 2 . Cell culture media was refreshed every 48 h and these cells were used at passages 5 to 6 for all the experiments.
Lipid peroxidation assay. Lipid peroxidation inhibition was tested in RAW264.7 cells, as macrophages are the major sources of oxidative stress and are used to evaluate the antioxidant function of dietary natural compounds 52 . 2 × 10 6 RAW264.7 cells were seeded in 6-well plates and incubated for 24 h with the corresponding solution of PA, G 1 Phy or G 3 Phy at final concentration of 100 μg/mL. For positive (lipid peroxidation induction but without any studied agent) and negative (no lipid peroxidation induction) controls, the same volume of dH 2 O than for the studied samples was added. Lipid peroxidation was induced by incubating the samples with 50 μM Fe 2 SO 4 ·7H 2 O and 200 μM H 2 O 2 solution at 37 °C for 4 h in order to trigger the Fenton reaction 53 . Lipid peroxidation was evaluated using the Lipid Peroxidation Assay Kit (abcam), following the manufacturer instructions. The percentage (%) of lipid peroxidation inhibition was calculated respect to the positive control, considering it as the 100% of MDA producer. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD.
Cytotoxicity. Cytotoxicity of PA and GPhy compounds was evaluated in MSCs cultures using the Alamar Blue assay. A stock solution of the corresponding tested compound in DMEM lacking FBS and containing 2% w/v D-sorbitol was prepared and successive dilutions were tested (5, 15, 20, 30, 50 mg/mL). 9000 of MSCs were seeded in 96-well plates and incubated for 24 h. Then, the medium was replaced and the cells were treated with the tested compounds for 24 h. A 10% v/v Alamar Blue (Invitrogen) solution was prepared in DMEM without phenol red and the plates were incubated at 37 °C for 3 h. Medium was collected and, after laser excitation at 590 nm, emitted fluorescence at 530 nm was quantified using a Biotek Synergy HT plate reader (Biotek Synergy HT spectrophotometer). cell viability. Alamar Blue assay was also used to analyse cell viability after incubation of MSCs with the tested compounds for 3, 7 and 14 d of. For this experiment, 104 of MSCs were seeded in 48-well plates and cultured in Mesenchymal Stem Cell Medium Kit for 24 h. Then, the medium was replaced, and the cells were treated with the tested solutions at various concentrations in complete LG-DMEM (10, 50,100, 150, and 300 µg/mL), and media containing the corresponding compounds were refreshed every 2 d. Medium was collected and, after laser excitation at 590 nm, emitted fluorescence at 530 nm was quantified using a Biotek Synergy HT plate reader (Biotek Synergy HT spectrophotometer). The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD. ALP activity quantification. ALP activity was evaluated after 3, 7 and 14 d of incubation of MSCs with the tested compounds. Total DNA amount was measured using the PicoGreen dSDNA quantitation kit (Molecular Probes). Determination of the ALP/DNA ratio is indicative of the amount of ALP activity per cell. For this experiment, 10 4 of MSCs were seeded in 48-well plates and cultured in Mesenchymal Stem Cell Medium Kit for 24 h. Then, the medium was replaced, and the cells were treated with PA and its derivatives solutions at 10 µg/mL. The media containing the corresponding compounds were refreshed every 2 d. Negative and positive controls correspond to cells cultured in complete or differentiation LG-DMEM media, respectively. The experiments were conducted in triplicate for each sample and the data obtained were expressed as mean values ± SD.
Analysis of differential gene expression by reverse transcription (RT) and real-time quantitative pcR (qpcR). For this experiment, 10 6 of MSCs were seeded in 6-well plates and cultured in complete