Expanding Current Knowledge on the Chemical Composition and Antioxidant Activity of the Genus Lactarius

Despite the presence of toxic compounds in inedible mushrooms, the question whether the chemical nutrients and non-nutrients compositions in edible and inedible Lactarius species are similar remains unanswered. To answer this question, Lactarius citriolens Pouzar and Lactarius turpis (Weinm.) Fr., two inedible species, were studied in order to obtain information about their chemical composition and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. citriolens and L. turpis methanolic extracts were tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). The composition of macronutrients varied among the two species, but the profiles were similar between them and among other Lactarius species; L. citriolens gave the highest energy contribution, saturated fatty acids and organic acids, while the L. turpis sample was richer in free sugars, mono- and polyunsaturated fatty acids, tocopherols and phenolic compounds. L. turpis methanolic extract showed the highest antioxidant activity. The absence of hepatoxicity of the methanolic extracts was confirmed in porcine liver primary cells (in vitro conditions). The present study provided new information about wild L. citriolens and L. turpis, comparing their chemical composition and antioxidant properties with other Lactarius species, and expanding the knowledge about this genus.


Chemical Composition of the Fruiting Bodies
The macronutrients composition of L. citriolens and L. turpis fruiting bodies is presented in Table 1. L. turpis showed the highest level of proteins, ash and carbohydrates, however, the energetic contribution of L. citriolens was superior, due to its higher fat content. It was possible to find some reports about other species of the same genus, namely L. deliciosus [15][16][17][18][19][20][21][22][23], L. hatsudake [19], L. piperatus [16], L. quieticolor [24], L. salmonicolor [13,21], L. sanguifluus [23,25], L. semisanguifluus [23] and L. volemus [19]. All of these species also presented carbohydrates and proteins as major macronutrients, and some of them similar energetic contributions (317-389 kcal/100 g dry weight) [13,15]. Table 1. Macronutrients, free sugars, fatty acids and tocopherols of the fruiting bodies expressed in dry weight basis (mean ± SD). Observing the macronutrient profile of the genus it can be concluded that the two studied species have similar characteristics to the edible species. However, since there are reports of the presence of necatorin, a highly mutagenic compound, in L. turpis, [26], and the possibility of poisoning which manifests by stomach and intestinal troubles [27], L. turpis is nowadays considered non-edible [28].

Lactarius citriolens Lactarius turpis t-Student Test p-value
Regarding L. citriolens, although this species is rarely found, it should be consumed with caution given the lack of information [28].
Analyzing the free sugars composition, mannitol and trehalose were detected in both samples (Table 1; Figure 1A). L. turpis and L. citriolens revealed the highest content in mannitol and trehalose, respectively. L. turpis gave the highest content in total free sugars. As far as we know, there are only some Portuguese studies, all of them from our research group, on the free sugars in Lactarius spp., namely in L. bertillonii [29], L. deliciosus [15][16][17][18], L. hepaticus [30], L. piperatus [16], L. quietus [14], L. salmonicolor [13] and L. vellereus [29]. All the mentioned species presented mannitol and trehalose, with the exception of the report of Fernandes et al. [15] who also found fructose, but in this case in lower amounts (0.18 g/100 g dry weight). In fact, mannitol, a sugar alcohol, and trehalose, an oligosaccharide, are the main representatives sugars present in mushrooms [1,16]. Mannitol, has half the calories of sucrose and because of their mannitol contents, mushrooms are useful for diabetic patients [31]. Trehalose is a common sugar component of most immature sporocarps and it may function as a reserve material, which can be metabolised when the sporocarps are maturing [32].
Concerning tocopherols, both samples presented α-, β-and γ-isoforms (Table 1; Figure 1C). The α-and γ-isoforms were the most abundant in L. turpis. On the other hand, β-tocopherol was present in higher amounts in L. citriolens. This was also the only sample presenting δ-tocopherol. It was L. turpis that revealed the highest content of total tocopherols. To our knowledge, there are only Portuguese studies presenting tocopherols content in Lactarius spp. The main isoform was variable, C and the total content reported varied between 15 µg/100 g to 316 µg/100 g [29,38]. Besides preventing lipid peroxidation, vitamin E appears to exert effects on other cardiovascular risk factors including reduction of platelet adhesion and aggregation [39].
Regarding the organic acids profile, it was possible to identify and quantify four different compounds ( Table 2; Figure 2A), namely oxalic, quinic, malic and fumaric acids. The wild mushroom L. turpis was the one that revealed the highest concentration of oxalic acid, while L. citriolens presented the highest malic and fumaric acids content. Quinic acid was only present in L. citriolens and this mushroom also showed the highest content in total organic acids. Table 2. Organic acids and phenolic compounds of the fruiting bodies expressed in dry weight basis (mean ± SD). As far as we know, there are only two reports concerning organic acids in Lactarius spp. namely, L. deliciosus from different locations within Portugal and L. volemus, with malic acid as the main organic acid present [40,41].

Lactarius citriolens Lactarius turpis t-Student test p-value
Concerning phenolic acids, the studied samples revealed the presence of gallic and p-hydroxybenzoic acids, and also the related compound cinnamic acid (Table 2; Figure 2B). Gallic acid was only found in L. turpis. However, it was L. citriolens that presented the highest amounts of p-hydroxybenzoic acid and of the related compound cinnamic acid. Analyzing these results we can conclude that the wild L. turpis sample was richer in phenolic acids compared to L. citriolens. It is difficult to establish a profile for the genus as for what concerns phenolic compounds. In fact, the phenolic compounds recorded in different species are not the same and the main phenolic compounds also vary among different species. For example, p-hydroxybenzoic acid was the main phenolic acid in L. deliciosus [42], L. salmonicolor [43] and L. volemus [14]. However, ο-coumaric [23], homogentisic [44] and tannic [45] acids were also reported as the main phenolic compounds in L. deliciosus and L. volemus samples. For L. volemus, protocatechuic acid was also reported as the main phenolic acid [46]. The same phenolic acid was in major abundance in L. vellereus [29]; catechin was also found in L. deterrimus [47] and L. vellereus [48]; and ο-coumaric was the major phenolic acid in L. semisanguifluus [23]. p-OH-Phenylacetic acid was the main phenolic acid in L. sanguifluus [23], however Puttaraju et al. [45] reported tannic acid as the principal compound for the same species. Regarding the related compound cinnamic acid, it was found in L. aurantiacus [43], L. bertillonii [29] and L. quietus [14]. In fact, Lactarius spp. is a genus rich in phenolic compounds, which have been reported to display different health benefits.

Antioxidant Activity of the Methanolic Extracts and Confirmation of Non-Toxicity
Analyzing the results of the antioxidant potential (Table 3), L. turpis methanolic extract revealed the highest reducing power evaluated through the Folin-Ciocalteu and ferricyanide/Prussian blue assays. The same sample also revealed the highest radical scavenging activity, since it presented the lowest EC50 value for DPPH assay. L. turpis also showed the highest lipid peroxidation inhibition in the β-carotene/linoleate and TBARS assays.  The highest total antioxidant activity in the genus, measured through Folin-Ciocalteu assay, was reported by Orhan and Üstün [49] in a L. deliciosus sample (51.27 mg GAE/g). L. deliciosus (EC50 = 500 µg/mL; [49]) and L. bertillonii (EC50 = 1.63 mg/mL; [29]) gave the highest reducing power. Regarding radical scavenging activity, the highest capacity was described by Unekwu et al. [50] for a L. deliciosus sample (EC50 = 300 µg/mL). Finally, regarding the lipid peroxidation inhibition, the highest antioxidant activity was obtained in L. deliciosus (IC50 = 148.0 µg/mL) measured by β-carotene/linoleate assay [34] and L. bertillonii (EC50 = 1.21 mg/mL) through TBARS assay [29]. Both of our samples present lower EC50 values in the TBARS assay being L. turpis the species with the highest antioxidant potential of the genus measured by this biochemical assay. Antioxidants can help the endogenous defense system, playing an important role as potential protective agents, reducing oxidative damage by free radicals so, preventing events related to aging and diseases, such as atherosclerosis, diabetes, cancer and cirrhosis [4].
As the methanolic extracts displayed antioxidant activity, it was important to evaluate their cytotoxicity against liver cells, which are considered the best in vitro model for studies of human cytotoxicity. Despite of the reported presence of toxic compounds in L. turpis fruiting bodies, the prepared extracts did not reveal any toxicity in PLP2 liver primary culture; the positive control ellipticine gave a GI50 (sample concentration that inhibited 50% of the net cell growth) = 2.06 ± 0.03 µg/mL. This could indicate that the toxic compounds were not present in these extracts. Nevertheless, it should be highlighted that an in vitro assay was performed, and that under in vivo conditions the microbiota that inhabit our intestines can readily convert molecules, some of which becoming toxic. The samples were analysed for moisture, proteins, fat, carbohydrates and ash using the standard procedures [51]. The crude protein content (N × 4.38) of the samples was estimated by the macro-Kjeldahl method; the crude fat was determined by extracting a known weight of powdered sample with petroleum ether, using a Soxhlet apparatus; the ash content was determined by incineration at 600 ± 15 °C. Total carbohydrates were calculated by difference. Energy was calculated according to the following equation: Energy (kcal) = 4 × (g protein + g carbohydrate) + 9 × (g fat).

Individual Compounds
Free sugars were determined by a high performance liquid chromatograph (HPLC) system coupled to a refraction index (RI) detector as previously described by the authors [13]. Fatty acids were determined after a transesterification procedure as described previously by the authors [13]. The fatty acids profile was analyzed using a gas chromatographer equipped with a flame ionization detector (GC-FID). Tocopherols were determined following a procedure previously described by the authors [13]. Analysis was performed by HPLC (equipment described above), and a fluorescence detector. Organic acids were determined by ultra-fast liquid chromatography (UFLC) coupled with a photodiode array detector (PDA) as previously described by the authors [41]. Phenolic acids determination was performed using the UFLC mentioned above, as previously described by Barros et al. [42].

Extract Preparation
Each lyophilized sample (1 g) was extracted by stirring with methanol (40 mL) for 1 h and subsequently filtered through Whatman No. 4 paper. The residue was then extracted with methanol (20 mL) for 1 h. The combined methanolic extracts were evaporated at 40 °C (Büchi R-210 rotary evaporator, Flawil, Switzerland) to dryness and re-dissolved in: (a) methanol for antioxidant activity assays (20 mg/mL) and (b) distillated water for the toxicity assay in porcine liver primary cells (8 mg/mL).

Antioxidant Activity Assays
The antioxidant activity of the methanolic extracts was evaluated by DPPH radical-scavenging activity, reducing power (Folin-Ciocalteu and ferricyanide/Prussian blue assays), inhibition of β-carotene bleaching in the presence of linoleic acid radicals and inhibition of lipid peroxidation using TBARS in brain homogenates. Trolox was used as positive control [13].

Toxicity for Porcine Liver Cells
The assay was performed with a cell culture prepared from a freshly harvested porcine liver (PLP2), and by applying sulphorhodamine assay. A complete procedure was previously described by the authors [52]. Ellipticine was used as positive control.

Statistical Analysis
Three samples were used for each preparation and all the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The results were analyzed using a t-student test and this treatment was carried out using the SPSS v. 22.0 program.

Conclusions
Overall, L. citriolens revealed the highest energetic contribution, saturated fatty acids content and organic acids concentration, while L. turpis was richer in free sugars, mono-and polyunsaturated fatty acids, tocopherols and phenolic compounds. L. turpis methanolic extract showed the highest antioxidant activity in all the in vitro assays. The studied extracts did not show toxicity in porcine liver primary cells. The edible species of the Lactarius genus are considered healthy foods due to their low content in calories and fat, but richness in proteins and carbohydrates. The macronutrients profiles of L. citriolens and L. turpis are similar to the ones reported for those edible species, despite the presence of some toxic compounds that make them not recommended for consumption. Nevertheless, they contain several interesting molecules with bioactive potential, namely antioxidant activity, which can be isolated to be used in drugs or nutraceuticals. This study provides new data concerning chemical characterization and bioactivity of L. citriolens and L. turpis.