Three vegetal extracts were obtained for each plant: in water, 50% water-alcohol and in 96% ethanol. The extacts were designated as AH (for Aesculus hippocastanum), CU (for Curcuma longa) and VV (for Vitis vinifera), and indexed with w (for water), 50 (for 50% ethanol) or 96 (for 96% ethanol). Furthermore, the extracts were analyzed for their composition by HPTLC (main components) and their total phenolic content.

The extracts were further subjected to acid and alkaline hydrolysis, under mild (0.08 M HCl and 0.02 M NaOH) as well as severe conditions (1 M HCl and 2 M NaOH under reflux) in order to examine whether the vegetal products generate hydrolytic products and alter their behavior at the gastric or at intestinal level. The hydrolysates were designated with the letters A (for acid) and B (for alkaline) hydrolysis. The in vitro antioxidant activity of the extracts, as well as that of their hydrolysates was evaluated by DPPH assay.

Jurkat lymphocytes were cultivated under normal and stress conditions (hyperglycemia mimicking diabetes mellitus) for 24 h, and exposed to the extracts for 1 h. They were then washed, and tested for their antioxidant activity. Cell membrane susceptibility to lipid peroxidation induced by cumene hydroperoxide (CuOOH; Sigma-Aldrich, St. Louis, MO, USA) was also examined following treatment with the extracts. The ICAM-1 expression level in the cell culture medium was measured using an ELISA kit (IBL International GmbH, Hamburg, Germany).

Vitis vinifera leaves were harvested from Bicaz (Neamt County, Romania) in July 2014 and Aesculus hippocastanum seeds from Bucharest in September 2014. The plant material was dried in the dark in our laboratory (25°C, 40% relative humidity). Voucher specimens of the two species are available in the herbarium collection at the Department of Pharmaceutical Botany and Cell Biology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania. Curcuma longa rhizome powder was purchased from a local herb store.

A total of 15 g of each plant material was grounded and extracted with 3×150 ml solvent (96-ethanol 96%; 50-ethanol-water 1:1, v:v: w-water) under reflux. The extractive solutions combined were concentrated using a rotary evaporator (RVO 004; Ingos, Prague, Czech Republic), and further dried by lyophilization at −55°C (CoolSafe ScanVac 55 freeze dryer; LaboGene, Lynge, Denmark).

In order to identify the main flavonoid aglycones, we performed acid and alkaline hydrolysis. Acid hydrolysis was performed on 100 mg of each dry extract with 1 M HCl under reflux. Similarly, alkaline hydrolysis was performed on the same quantity from each dry extract with 2 M NaOH under reflux. Following hydrolysis, the flavonoid aglycones were extracted with diethyl ether. The ether was then removed using a rotary evaporator (RVO 004; Ingos), and the residue was dissolved in methanol.

The qualitative analysis of the extracts was performed by HPTLC, using HPTLC silica gel plates [Nano silica gel on TLC plates from Sigma-Aldrich Chemie GmbH (Buchs SG, Switzerland) and 60 F254 HPTLC plates from Merck Millipore (Merck KGaA, Darmstadt, Germany)] and a mobile phase consisting of toluene:ethyl acetate:formic acid 5:3:1 (v:v:v), as previously described (27). The identification of the components was performed using standard substances from Sigma-Aldrich, in two ways: by the similarity of the peak spectrum at the Rf corresponding to the standard (obtained using the facilities of TLC3 Scanner and winCATS software from Camag, Müttenz, Switzerland), and by visual observation of the peaks in UV (366 and 254 nm) following derivatization with 2-aminoethyl diphenyl borate (Roth, Karlsruhe, Germany).

The amount of total phenolics in the plant extracts was determined with Folin-Ciocalteau reagent (obtained from Scharlau Co., Barcelona, Spain) using the method described in the study by González et al (28), as modified by Olaru et al (29). Briefly, to appropriate volumes of dilutions from each sample, 0.6 ml of 1/10 dilution of Folin-Ciocalteau reagent and 2 ml of 15% Na₂CO₃ in water were added and incubated at 50±1°C for 15 min on a water bath (Memmert GmbH & Co. KG, Schwabach, Germany). The absorbance of all samples was then measured at 765 nm using a Halo DB-20–220 UV/visible spectrophotometer (Dynamica, Salzburg-Mayrwies, Germany). A calibration curve using gallic acid was plotted under the same conditions. The results are expressed as micrograms of gallic acid equivalents per milligram of dry weight (µg GAE/mg) of extract. All experiments were performed in triplicate and the means ± standard deviation (SD) and confidence interval (95% CI) were calculated for each sample.

DPPH is a stable, nitrogen-centered free radical (violet/purple color as a methanol solution). The color of DPPH gradually changes to a pale yellow (reduced form) when DPPH is exposed to free radical scavengers. The decrease in absorbance at 517 nm induced by antioxidants determines the reduction capability of the DPPH radicals. The advantage of this method, which makes it highly appropriate for screening the antioxidant activity of plant extracts, is the fact that it allows the testing of both lipophilic and hydrophilic compounds (30–32) and it is not restricted by the nature of the antioxidants.

As the positive controls of the experiment, ascorbic acid, quercetin and curcumin standard solutions were used. For each of these known antioxidants, the reduction of DPPH was evaluated for different concentrations of the standard: 5 µM-1 mM for quercetin and curcumin, and 5–250µM for ascorbic acid.

For all the prepared extracts, as well as for the products resulting from their hydrolysis, either under acidic (1 M HCl) or alkaline (1 M NaOH) conditions, the DPPH scavenging effect was assessed. For this purpose, we evaluated the decrease in the optical density of the DPPH solution (Sigma-Aldrich) mixed with each sample, for 30 min, at 517 nm. The results were compared with the discoloration curve of DPPH alone, as well as with the positive controls represented by ascorbic acid, curcumin and quercetin, at different levels of exposure. The antioxidant effect was expressed as the scavenged DPPH (%) after 30 min, when exposed to the extracts compared to the values obtained from the spontaneous discoloration of DPPH, according to the recommendations of Molyneux (32), using the following equation: %DPPH inhibition=A0-AsA0×100

where A₀ is the absorbance of DPPH without inhibitor and A_s is the absorbance of the sample (DPPH plus inhibitor).

Experiments were performed using Jurkat cells, from the European Collection of Cell Cultures, at the 3–4 th passage. The Jurkat lymphocytes were grown for 24 h in RPMI-1640 (11.11 mM glucose; Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum at 37°C in 5% CO₂ atmosphere, in 24-well plates (2×10⁵ cells/well). For diabetic conditions, the cells were plated for 24 h in glucose-rich medium (35 mM high glucose, equivalent to hyperglycemia associated with oxidative stress) (33,34).

The prepared cells were exposed for 1 h to the extracts, dissolved in phosphate buffer (saline) at the concentration of 0,1 mg/ml. Following exposure, the cells were washed 3 times with phosphate-buffered saline (PBS; Sigma-Aldrich), pH 7, re-suspended in RPMI at 2×10⁵ cells/ml and used for the assay of lipid peroxidation. Untreated cells served as negative controls and cells treated with quercetin, curcumin and ascorbic acid served as the positive controls. The viability of the cells was examined using trypan blue exlucion assay, but no significant changes were observed (data not shown).

DPPP is a fluorescent probe that has weak fuorescence characteristics in its basic form; however, its oxide, which can be specifically generated in reaction with hydroperoxides, has a strong fluorescence (34,35). Experimental lipid peroxidation of the cell membranes was induced treatment with 10 µM CuOOH, generating an effective production of reactive oxygen species in the cell membrane, which was further quantified with DPPP (36).

DPPP from ThermoFisher Scientific/Invitrogen/Molecular Probes™ (Eugene, Oregon, USA) was dissolved in dimethyl sulfoxide at a concentration of 5 mM. Following the addition of DPPP at a final concentration of 5 µM, the cell suspension was incubated at room temperature for 20 min in the dark. The DPPP-labeled cells were treated with 10 µM CuOOH to induce DPPP oxide formation. The process was assayed by performing fluorescence emission spectrum analysis between 360 and 410 nm, excitation at 351 nm, every min, for 5 min; the fluorescence intensity maximum was measured at 380 nm, as previously described (37) and peroxidation was computed as percentage of the fluorescent signal increase following 5 min of exposure to CuOOH.

The results are expressed as the means ± SD. Statistical analysis was performed using the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

HPTLC analysis performed on the extracts revealed the presence of certain polyphenols and flavones in the extracts, as presented in Table I.

The results obtained from the analysis of the total flavonoids are presented in Table II. All relative standard deviations (RSD) were <5%.

The main flavonoids and phenolic acids identified in the three extracts were as follows: p-coumaric acid, caffeic acid, quercetin, kaempferol, apigenin and esculetin (Aesculus hippocastanum); ferrulic acid and curcumin (Curcuma longa); caffeic acid, gallic acid, quercetin, luteolin and esculetin (Vitis vinifera). Besides, in the case of Curcuma longa and Vitis vinifera a few other tested standard substances presented positive results: caffeic acid, kaempferol and chrysisn, and ferrulic acid, p-coumaric acid and kaemferol, respectively. However, due to the fact that the UV-VIS spectra (data not shown) of the spots supposed to belong to the above-mentioned substances were not similar to the corresponding standards (possibly due to other unidentified components that co-eluted with the searched phenolic compounds) we decided to mark them as ‘possibly present’ (Table I). Other major spots with a positive reaction for phenolic compounds (data not shown) were observed in all the extracts, but they were not identified on the basis of a standard substance.

The highest yield of total flavonoids from horse chestnut seeds and common grape wine leaves was obtained by extraction with 96% ethanol and was approximately 3.5-fold higher than the content obtained by extraction with water. With the extraction of turmeric with 96 and 50% ethanol, we obtained close values with similar upper limits and slightly different lower limits of 95% CI. The use of ethanol and hydro-ethanol for the extraction of turmeric resulted in a total phenolic content yield of approximately 4-fold higher compared to extraction with water (Table II).

The kinetics of DPPH decoloration for various concentrations of positive controls was tested before the main measurements to establish the reagent concentration and the speed of the reaction (data not shown). We finally found that within 30 min, the reaction was completed (DPPH was scavenged), and the results obtained for the positive controls (curcumin, quercetin and ascorbic acid) are presented in Table III. The results ranged from 2% scavenged DPPH (lowest concentration of quercetin and ascorbic acid tested) to 85% scavenged DPPH (highest concentration of quercetin or ascorbic acid tested). It is interesting that ascorbic acid and quercetin are more potent antioxidants compared with curcumin.

Furthermore, the ability of the extracts and that of their hydrolisates to scavenge DPPH was evaluated. Our results revealed that, in the case of the Aesculus hippocastanum extracts, the antioxidant effects were limited. The discoloration kinetics for the sample solutions (data not shown) was close to that of DPPH alone; therefore, the ability of the extracts to reduce DPPH radicals was rather insignificant.

The aqueous and 96% ethanol extracts did not exhibit antioxidant effects; however, such effects were observed for the 50% ethanol extract. As regards the effects exerted by the hydrolytic products of the Aesculus hippocastanum extracts, the results revealed that the acid, as well as the alkaline hydrolysis of the aqueous extracts and the alkaline hydrolysis products of the 96% ethanol extract exhibited antioxidant effects (Table IV).

The antioxidant activity of the Curcuma longa extracts increased with the increase in the ethanol concentration of the extraction solvent. The alkaline hydrolysis resulted in mixtures with a low antioxidant activity (approximately 20%), but the acid hydrolysis generated a high antioxidnat activity from all the extracts (Table IV).

The antioxidant effects induced by the Vitis vinifera extracts increased with the increase in the ethanolic concentration of the extraction solvent. The alkaline hydrolysis generated, from all the extracts, products with the same type of antioxidant activity (approximately 20–30% inhibition of DPPH). The antioxidant activity of the products resulting from acid hydrolysis was supperior (>60%) in the case of all extracts (Table IV).

In order to extrapolate the results into in vivo models, the antioxidant effects of the extracts were assessed in a model of Jurkat cells, cultivated either in RPMI medium with 11.11 mM glucose (normoglycemic) or in RPMI medium supplemented with glucose up to 35 mM (equivalent with hyperglycemia and oxidative stress). A low susceptibility to lipid peroxidation (%) expressed a greater protective effect against hydroperoxide generation.

The exposure of the cells to the extracts did not lead to a reduction in cell viability (data not shown) in both models. The results also revealed that the Aesculus hippocastanum extract did not prove effective in reducing the generation of membrane lipoperoxides; these results confirmed the effects obtained with the DPPH experiments. The antioxidant effects of the Vitis vinifera extracts were higher compared to those exerted by the Curcuma longa extracts, but only for the extracts prepared in alcoholic medium. It should be noted that the reduction in hydroperoxide generation was greater in the extracts prepared with an increased concentration of ethanol, for the normoglycemia cell model.

For cells exposed to hyperglycemia and oxidative stress, the susceptibility to lipid peroxidation in the control sample was greater compared to the one obtained with 11.11 mM glucose medium, as prevoiusly shown (34). The efficacy of the Vitis vinifera and Curcuma longa extracts was in the same range as that observed for the positive controls (quercetin, curcumin and ascorbic acid). The antioxidant efficacy of the Curcuma long extrats was significantly greater compared to that of all the other extracts in the hyperglycemic medium (Table V).

The results demonstrated that, under normal glucose conditions, there were no significant changes in the level of ICAM-1 in the medium (Fig. 1A). Under hyperglycemic conditions, the level of ICAM-1 in the untreated cells was higher compared to the cells in normal glucose medium (Fig. 1B). All the tested Curcuma longa extracts induced a significant decrease in ICAM-1 expression in the cell medium, similar to that induced by the standard curcumin (10 µM) solution. The Aesculus hippocastanum and Vitis vinifera extracts had limited anti-inflammatory effects.

In conclusion, in the present study, we prepared three types of extracts (in water, 50% ethanol and 96% ethanol) from three different vegetal products: Aesculus hippocastanum (semen), Vitis vinifera L. folium (both indigenous plants in Romania) and Curcuma longa L. rhizoma, available on the Romanian market.

The obtained extracts had different phenolic contents, depending on the plant and type of extract, ranging from 0.4 to 39 µg phenolics/mg total extract. The greatest amount of phenolics was found in the Cucurma longa extracts, while the lowest, in the Aesculus hippocastanum extacts. HPTLC analysis revealed the following components in the extracts: quercetin, kaempferol, coumaric acid, caffeic acid, apigenin and esculetin in the Aesculus hippocastanum extracts; ferrulic acid and curcumin in the Cucuma longa extracts; and quercetin, ferrulic acid and gallic acid, kaempferol, luteolin, and esculetin in the Vitis vinifera extacts.

The evaluation of hydrolisis resistance proved that, for all the extracts, no significant chemical changes were obtained under mild hydrolisis conditions (artificial gastric juice and artificial intestinal fluid).

The antioxidant effects of the extracts and their hydrolitic products were assessed by DPPH assay. The results revealed that the Aesculus hippocastanum extracts had a low antioxidant efficacy. Both the Curcuma longa and Vitis vinifera extracts had a high antioxidant activity; the products resulting from alkaline hydrolisis were significantly more efficient in scavenging DPPH radicals compared to their acid hydrolitic products.

The antioxidant effects induced by the extracts on the cell membrane were evaluated as the susceptibility to lipid peroxidation in two different models: Jurkat cells grown in normal glucose (11.11 mM) and high glucose (35 mM) medium. The activity of the Curcuma long extracts was significantly greater under both experimental conditions. The Vitis vinifera extracts also proved to be effective in normoglycemic medium, but not under hyperglycemic conditions.

Exposure to hyperglycemic conditions induced an increase in the ICAM-1 level compared to normal glucose conditions. All the tested Curcuma longa extracts induced a significant decrease in ICAM-1 expression in the cell medium, similar to that induced by the standard curcumin (10 µM) solution. The Aesculus hippocastanum and Vitis vinifera extracts had limited anti-inflammatory effects.

These results could be translated into clinical practice and demonsrated that Curcuma longa extracts may be effective both in the prevention of diabetes mellitus and in attenuating the associated complications, particularly cardiovascular complications.