2.1. Total Antioxidant Power and Phenolics Content
Both aqueous and methanolic extracts from
C. coccineum were evaluated for their content of phenolics and flavonoids, and for their total antioxidant capacity. The selected ET-based methods were the DPPH assay, ABTS-based assay in order to measure the TEAC (Trolox Equivalents Antioxidant Capacity), and the FRAP method. The selected HAT-based method was the ORAC assay based on pyrogallol red. The full results are summarized in
Table 1. Both extracts showed significant antioxidant capacities, with a few differences between them. ORAC-PYR assay gave in both cases the highest antioxidant capacity value, in accordance with the fact that this method is the only HAT-based, being thus able to evaluate overall antioxidant capacity, while FRAP, TEAC, and DPPH only evaluate the presence of reducing compounds (they are in fact electron-transfer based methods). This is in agreement with previously reported studies about the risk that ET-based assays may underestimate total antioxidant capacity [
11]. Rached
et al. (2010) have measured the antioxidant potential of specimens of
C. coccineum grown in Algeria, using two complementary assays, namely inhibition of DPPH radical and β-carotene bleaching [
12]. Other authors have studied the antioxidant activity in specimens of
C. songaricum, an Asian species of the genus
Cynomorium, also widely used in traditional Chinese medicine and known as “suoyang” [
13,
14].
Table 1.
Total antioxidant capacity of aqueous and methanolic extracts from C. coccineum.
Table 1.
Total antioxidant capacity of aqueous and methanolic extracts from C. coccineum.
Assay | Methanol extract | Water extract |
---|
ORAC-PYR (mM TE/g) | 0.91 ± 0.04 | 1.18 ± 0.06 |
DPPH (mM TE/g) | 0.52 ± 0.01 | 0.50 ± 0.2 |
DPPH (IC50 μg/mL) | 54.20 ± 2.1 | 51.6 ± 3.2 |
TEAC (mM TE/g) | 0.89 ± 0.05 | 0.99 ± 0.11 |
TEAC (IC50 mg/mL) | 0.91 ± 0.08 | 0.89 ± 0.04 |
FRAP (mM TE/g) | 0.58 ± 0.02 | 0.50 ± 0.01 |
FRAP (mmol FeII/g) | 1.35 ± 0.04 | 1.10 ± 0.03 |
Total phenolics (mM GAE/g) | 1.02 ± 0.03 | 0.64 ± 0.02 |
Total flavonoids (mM CE/g) | 0.139 ± 0.002 | 0.128 ± 0.003 |
When data reported in these studies are compared to what we found for the Maltese mushroom grown in Sardinia the results are not similar. In fact, the DPPH-based IC
50 values of our extracts are significantly higher, showing a lower antioxidant capacity than Algerian
C. coccineum and
C. songaricum (4.1 μg/mL and 13.5 μg/mL for Algerian
C. coccineum ethanolic and water extracts, and about 35 μg/mL for
C. songaricum) [
12,
13]. On the contrary, the antioxidant capacity measured with FRAP assay appeared to be higher for our specimens (0.051 and 0.167 mmol Fe
II/g for
C. songaricum methanolic and water extracts respectively [
14]). These conflicting data can be explained by the significant chemical and methodological differences between the two assays. Accordingly, for the full evaluation of the antioxidant capacity of any extracts, the use of a variety of assays is usually recommended [
10,
15]. The total content of phenolics was similar when comparing methanolic and aqueous extracts, and lower if compared with the content reported in other
Cynomorium studies [
12,
14]. Among the total phenolics, the content of flavonoids represents a significant part, and this value for both extracts showed no significant differences if compared with the total flavonoids content observed in
C. coccineum specimens picked in Algeria [
12].
HPLC investigation of the fresh Maltese mushroom extracts revealed that the SFE extract had a chromatographic profile similar to that obtained for the methanol-water extraction (not shown). The chromatograms of both extracts showed a peak, identified as gallic acid, and two zones at about 32–33 and 37–39 min retention time of unidentified peaks. The UV-Vis spectra (200–600 nm) of these peaks revealed similarities with procyanidins and gallic acid derivatives, suggesting that they can be procyanidin oligomers. In the methanol-water extract of whole
C. coccineum extract, the anthocyan cyanidin 3-
O-glucoside was also detected. This is in agreement with that reported by Harborne
et al. [
16] who found the presence of cyanidin 3-
O-glucoside in the red-brown inflorescences of
C. coccineum where, like other anthocyans, it plays the role of attracting insect pollinators. Such investigation [
16] reported that this is the only anthocyan of this plant, but in our extracts another five anthocyans were detected and quantified (
Table 2). Interestingly, the external layer of
C. coccineum is the richest in anthocyans, which are responsible for the intense plant color (
Figure 1). The presence of considerable amounts of gallic acid and cyanidin 3-
O-glucoside in the Maltese mushroom, when compared with other sources [
17], is worthy of note. In fact, these are plant polyphenolic compounds naturally present in the human diet. Their use in dietary supplementation is well documented [
18] as it can improve the antioxidative potential and nutritional and functional qualities of several foods [
19], and is known to reduce the risk of disease [
20,
21].
Table 2.
Phenolic composition of C. coccineum extracts (mg/g *).
Table 2.
Phenolic composition of C. coccineum extracts (mg/g *).
Extract | Gallic acid | Cyanidin 3-O-glucoside | Other anthocyans # |
---|
SFE CO2, whole plant | 1.184 ± 0.079 | 0.009 ± 0.002 | nd |
Solvent, whole plant | 4.573 ± 0.226 | 3.134 ± 0.071 | 0.042 ± 0.004 |
Solvent, external layer | 3.413 ± 0.135 | 11.892 ± 0.676 | 0.507 ± 0.049 |
Solvent, peeled plant | 2.894 ± 0.031 | 0.028 ± 0.002 | tr |
Taken together, these data demonstrate that extracts of
C. coccineum show a significant total antioxidant potential, and that flavonoids reasonably give a contribution to the overall antioxidant capacity. This is quite interesting, since flavonoids seem to be involved in the anti-carcinogenic and anti-mutagenic activities of plant extracts [
22].
2.2. Antioxidant Power in Models of Lipid Oxidation
To better assess the antioxidant activity observed with
in vitro chemical assays, the methanolic extract obtained from
C. coccineum was subsequently tested for its protective effect in a biochemical assay of oxidative stress, namely the thermal (140 °C), solvent-free degradation of cholesterol for 1 h or 2 h. The methanolic extract was chosen as the richest in total phenolics (
Table 1), and also because, in general, methanolic extracts are more active when studying lipid models. Our model of lipid oxidation has been widely used to assess the antioxidant properties of extracts and natural compounds [
23,
24]. The consumption of cholesterol and its transformation in its oxidized products (7-keto and 7β-OH) were measured as markers of the oxidative process. At 140 °C, cholesterol was an oil, and more than 80% of the initial compound disappeared within 1 h of heating, with a significant related increase of the oxysterols 7β-OH and 7-keto, as previously observed [
23,
24].
Figure 2 shows the antioxidant activity obtained in the presence of different amounts (1–50 μg) of extract during cholesterol oxidation for 1 h and 2 h. Antioxidant activity is reported as percentage of cholesterol protection (
Figure 2A), calculated considering the percentage of sterol consumption in the presence of the antioxidant with respect to total cholesterol consumption without antioxidant (100% of consumption or 0% of protection). The extract showed a significant inhibitory activity of cholesterol degradation from 2.5 μg at the 1 h time point (40% protection) to 5 μg at the 2 h time point (70% protection, data not shown).
Figure 2B shows the values (μg) of the oxysterols 7-keto and 7β-OH formed during cholesterol oxidation at 1 h in the absence (0) or in the presence of methanolic extract. The extract (5 μg) efficiently prevented the formation of the oxysterols at 1 h.
Figure 2.
Antioxidant activity (% protection) of different amounts (1–50 μg) of methanolic extract of C. coccineum measured during cholesterol degradation at 140 °C for 1 h (A) and values (μg) of the oxysterols 7-keto and 7β-OH formed in the absence (0, oxidized control) and in the presence of extract (B); a = P < 0.001, b = P < 0.01 versus oxidized control (0), a’ = P < 0.001 for both oxysterols versus 0 (n = 6).
Figure 2.
Antioxidant activity (% protection) of different amounts (1–50 μg) of methanolic extract of C. coccineum measured during cholesterol degradation at 140 °C for 1 h (A) and values (μg) of the oxysterols 7-keto and 7β-OH formed in the absence (0, oxidized control) and in the presence of extract (B); a = P < 0.001, b = P < 0.01 versus oxidized control (0), a’ = P < 0.001 for both oxysterols versus 0 (n = 6).
The scavenging ability of the methanolic extract observed in simple
in vitro chemical assays was thus confirmed in this model of lipid oxidation.
C. coccineum extract protected sterol against free radical attack and inhibited oxysterol formation, showing scavenging ability against peroxyl radicals ROO• [
21,
22]. Cholesterol, present in free and in esterified forms in biological membranes, lipoproteins, and food of animal origin, may undergo oxidation when exposed to oxidative stress, and cholesterol oxidation products, oxysterols, exhibit a host of biological activities of relevance for biomedical research (cytotoxicity, angiotoxicity, and mutagenicity) [
23,
25].
The protective effect of the methanolic extract against the liposome oxidative injury was also evaluated. As an index of the Cu
2+-induced lipid peroxidation, the variation of the levels of unsaturated fatty acids was analyzed, together with the rise in the level of the oxidation product MDA.
Figure 3 shows the values (expressed as μg/mg liposomes) of the main unsaturated fatty acids (18:1, 20:4
n-6, 22:4
n-6, 22:5
n-6, and 22:6
n-3) measured in the control (Ctrl) and following liposome oxidation at 37 °C for 24 h with 5 μM CuSO
4 in the absence (0) and in the presence of different amounts (10, 25, 50 μg/mL) of the extract.
Figure 3.
Values of the main unsaturated fatty acids (18:1, 20:4 n-6, 22:4 n-6, 22:5 n-6, and 22:6 n-3) expressed as μg/mg liposomes (A) and malondialdehyde (MDA) expressed as % of formation (B) measured in the control (Ctrl) and during liposome oxidation at 37 °C for 24 h with 5 μM CuSO4 in the absence (oxidized control, 0) and in the presence of different amounts (10, 25, 50 μg/mL) of methanolic extract of C. coccineum. a = P < 0.001, b = P < 0.01, c = P < 0.05, versus Ctrl; d = P < 0.001, e = P < 0.01, f = P < 0.05 versus oxidized control; (n = 6).
Figure 3.
Values of the main unsaturated fatty acids (18:1, 20:4 n-6, 22:4 n-6, 22:5 n-6, and 22:6 n-3) expressed as μg/mg liposomes (A) and malondialdehyde (MDA) expressed as % of formation (B) measured in the control (Ctrl) and during liposome oxidation at 37 °C for 24 h with 5 μM CuSO4 in the absence (oxidized control, 0) and in the presence of different amounts (10, 25, 50 μg/mL) of methanolic extract of C. coccineum. a = P < 0.001, b = P < 0.01, c = P < 0.05, versus Ctrl; d = P < 0.001, e = P < 0.01, f = P < 0.05 versus oxidized control; (n = 6).
The Cu
2+-mediated degradation of liposomes resulted in a significant decrease of the unsaturated fatty acids 22:6
n-3, 20:4
n-6, and 22:5
n-6 in the oxidized control (0) (
Figure 3A). The consumption of polyunsaturated fatty acids was accompanied by an increase of MDA level in the system (MDA values: 0.46 ± 0.17 and 3.15 ± 0.60 μM in the Ctrl and oxidized control samples, respectively). The addition of the methanolic extract to the system exerted a significant protection, with respect to the oxidized control, against the oxidation of polyunsaturated fatty acids at all the tested concentrations (
Figure 3A), and also significantly reduced the formation of the oxidative product MDA (
Figure 3B).
From our observations, the methanolic extract significantly preserved liposome fatty acids from oxidative damage, maybe protecting the lipid particles by scavenging oxygen radicals and chelating coppers ions present at the aqueous phase [
24]. Liposomes are considered an important membrane model for the study of lipid peroxidation. Much research has been carried out to understand in depth the effects of lipid oxidation in phospholipid membranes to get tools to prevent, modulate and treat the oxidative damage [
26]. The oxidative degradation of unsaturated fatty acids, essential components of biological membranes and LDL, is well known to play a role in the development of tissue damage and in a wide range of pathological events [
23,
25,
26].