_Applied_  
_sciences_  
Evaluation of Two Extraction Methods for the Analysis of Hydrophilic Low Molecular Weight Compounds from _Ganoderma lucidum_ Spores and Antiproliferative Activity on Human Cell Lines  
**[PERSON] 1,\({}^{\intercal}\)\({}^{\copyright}\)**, **[PERSON] 2,3,\({}^{\intercal}\)\({}^{\copyright}\)**, **[PERSON] 4\({}^{\copyright}\)**, **[PERSON] 1\({}^{\copyright}\)**, **[PERSON] 1, **[PERSON] 3,\({}^{\star}\)\({}^{\copyright}\)**, **[PERSON] 1,\({}^{\star}\)\({}^{\copyright}\)**, **[PERSON] 1,\({}^{\copyright}\)** and **[PERSON] 2,3,5,\({}^{\ddagger}\)**  
1 Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy; [EMAIL_ADDRESS] (M.M.S.); [EMAIL_ADDRESS] (M.M.C.); [EMAIL_ADDRESS] (A.C.); [EMAIL_ADDRESS] (D.N.)  
2 Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, 80125 Naples, Italy; [EMAIL_ADDRESS] (V.D.G.); [EMAIL_ADDRESS] (P.A.N.)  
3 Center for Advanced Biomaterials for Health Care @ CRIB, Istituto Italiano di Tecnologia, 80125 Naples, Italy  
4 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy; [EMAIL_ADDRESS]  
5 Department of Chemical, Materials and Industrial Production Engineering (DICMAPI) University of Naples Federico II, 80125 Naples, Italy  
* Correspondence: [EMAIL_ADDRESS] (R.V.); [EMAIL_ADDRESS] (A.A.); Tel.: +39-081-2539179 (A.A.)  
\({}^{\dagger}\)These authors contributed equally to this work.  
\({}^{\ddagger}\)These authors contributed equally to this work.  
Received: 24 May 2020; Accepted: 9 June 2020; Published: 11 June 2020  
Featured Application: In this study two extraction methods for _Ganoderma lucidum_ spores were evaluated for the potential use of extracted compounds in cancer treatments. Our findings showed that the innovative Rapid Solid Liquid Dynamic Extraction (RSIDE) permits the successful extraction of compounds with antiproliferative activity against human cell lines  
**Abstract:** Background: The genus Ganoderma includes about 80 species of mushrooms. _Ganoderma lucidum_ is the best-known fungal species in mycotherapy and likely has the highest number of studies. Numerous bioactive compounds seem to be responsible for its beneficial effects; in particular, triterpenenes, peptidoglycanans and polysaccharides are the main physiologically active constituents. The aim of the present work is to identify the main bioactive components in aqueous extracts of _G. lucidum_ spores obtained by two different extraction processes. Methods: The spores were at first extracted by a Soxhlet apparatus with \(n\)-hexane and the aqueous solutions were submitted to two different procedures: hot water extraction (HWE) and rapid solid-liquid dynamic extraction (RSLDE) using the Naviglio extractor. The extracts were then dialyzed to separate the compounds with higher molecular weight from polar compounds with lower molecular weight. The aqueous extracts and dialyzed fractions were tested on two human cell lines: human colonic epithelial cells (Caco-2) and human colorectal carcinoma cells (HTC-116). Results: GC-MS and NMR data revealed the presence of a mixture of glucose and mannitol in ratio 3.7:1 in the most active fraction. Conclusions: The outside dialysis phase of RSIDE extract seems to be particularly cytotoxic for HTC-116 and, interestingly, solutions with different concentrations of pure mannitol showed cytotoxic activity against this cell line too, although to a lesser extent.  
**Keywords:** spores; extraction techniques; RSIDE; mannitol; NMR; antiproliferative activity; GC-MS