## 4 Discussion  
_Ganoderma lucidum_ spores are a rich source of bioactive compounds which are known to help in improving human health [20; 21]. In the last decades, their possible implication for cancer prevention and treatment has stimulated a huge research activity concerning the exploitation of this part of _G. lucidum_ for medical purposes [11; 22; 23; 24]. For this reason, many extraction techniques were employed to obtain the maximum yield of bioactive compounds from _G. lucidum_[25]. In the present study, two strategies were compared to determine the best way to obtain an extract with antiproliferative activity against human cancer cells from _G. lucidum_ spores (Figure 1). Hence, the sporoderm-broken spores of _G. lucidum_ were extracted with a Soxhlet apparatus in _n_-hexane. The lipophilic fraction was analyzed via NMR and GC-MS [16], while the exhausted material was further processed. In particular, it was split into two fractions, one of which was extracted by hot water extraction (HWE) and the other fraction by rapid solid-liquid dynamic extraction (RSLDE) in order to compare a common with an innovative extraction technique. As it is known, this latter method exploits an extraction principle that allows the optimization of diffusion and osmosis phenomena, increasing the yield and reducing the extraction times compared to the classic extraction phenomena [26]. Using this principle, RSLDE allows to extract at room temperature without the diffusion effect due to temperature [27]. Furthermore, in this study RSLDE was optimized to extract the spores of _G. lucidum_ adding Pyrex(r) solid glass beads in order to avoid the excessive sample compaction during the extraction. Cytotoxicity tests on HWE and RSLDE extracts were performed on two human cancer cell lines: Caco-2 cells show poor tumorigenic or non-tumorigenic features with a non-invasive phenotype [28] and HCT-116 cells display a high invasive ability in vitro [29]. The Caco-2 cells viability was not significantly affected by the exposure of the HWE and RSLDE extracts, but a strong dose- and time-dependent reduction in cell viability was observed for HTC-116. These interesting results triggered us to study the composition of the extracts. The extracts were dialyzed to separate compounds with higher molecular weight from hydrophilic compounds with lower molecular weight. The outside aqueous phases (OUT) of dialysis, as well as the internal phases (IN) of the dialysis tube for both HWE and RSLDE extracts were analyzed via NMR and also evaluated for toxicity on Caco-2 and HTC-116. In particular, among the fractions analyzed, a high reduction in cell viability for Caco-2 cells was shown by the exposure of HWE\({}_{\text{OUT}}\), probably due to the presence of some low molecular weight products that were extracted from thermal degradation. On the other hand, the most active fraction on HTC-116 is the outside dialysis phase of RSLDE extract  
Figure 7: Comparison of proliferative activities on Caco-2 (non-proliferative) and HCT-116 (proliferative) cell lines of RSLDE\({}_{\text{OUT}}\) (containing 1.1 mg mL\({}^{-1}\) of mannitol) and the mannitol concentrations at 1 and 1.2 mg mL\({}^{-1}\) after 24 (**A**) and 48 h (**B**) exposure (\({}^{*}p<0.05\), \({}^{**}p<0.01\)). which was found to be a mixture of glucose and mannitol in ratio 3.7:1. Mannitol is responsible of several functions in many organisms, such as protection from osmotic, saline, and oxidative stress [30]. In fact, a few in vivo studies have investigated the effects of mannitol as a free radical scavenger [31]. Considering that glucose is not a cytotoxic compound [19], some solutions of different concentrations of commercially available mannitol were tested on both cell lines. In addition, HTC-116 cells seem to be more sensitive than Caco-2 and, interestingly, the outside dialysis phase of RSLDE extract was more active on cancer cells than pure mannitol at the same concentration.