Table 2.

Compilation of C. sinensis extraction method and results from a selection of current and pertinent research.

Extraction	Extraction methods	Bioactive compounds or fractions	In vitro/in vivo evaluations	Observations	References
–Sequential water/ethanol	–Water extraction at 100 °C for 3 h –Absolute ethanol extraction –Combination of aqueous and ethanol extracts	–CSE	– In vivo	–Cerebroprotective effect of CSE on ischemic neuronal damage –CSE reduced lactate dehydrogenase activity; indicator of low oxidative stress –Reduced glutathione levels increased in treated groups relative to ischemic group; indicating heightened antioxidant defense system	Liu et al. (2010)
–Ethanol extraction with partitioning	–Ethanol extraction under reflux for 8 h –Partitioned into n-hexane/n-hexane fraction (CSEHH), an n-hexane/MeOH–H2O fraction (CSEHM), an ethyl acetate fraction (CSEE), and a water-soluble fraction (CSEW)	–Identification of 45 known compounds (i.e. ergosteryl-3-O-β-d-glucopyranoside and perlolyrine) –5 Constituents of Cordysinins A–E	– In vitro	–CSEHH, CSEHM and CSEE fractions displayed significant inhibition of superoxide anion generation as well as inhibiting elastase release	Yang et al. (2011)
–80% Medicinal alcohol (ethanol) –Aqueous extraction –Trypsin extraction	–Soaked in 80% ethanol and vacuum concentrated –Mixed in boiling water for 2 h and vacuum concentrated and heated –Trypsin treated and subsequently inactivated –The three extracts combined, homogenized and sterilized at 60	–CSE	– In vivo (Inbred male BN (Brown-Norway) and Lewis rats)	–CSE attenuates ICAM-1 and TNF-α expression levels in transplanted aortas in addition to reducing serum levels of both ICAM-1 and TNF-α –CSE inhibited the proliferative activity of vascular smooth muscle cells by decreasing PCNA expression, and effectively reduced the development of transplant arteriosclerosis, in addition to conferring protective effects on allograft vasculopathy	Zhang et al. (2011a)
–Water-based ethanol extraction	–Extracted in water via decoction –Filtered and precipitated in 95% ethanol –Dissolved CSE was sterilized and diluted in Dulbecco modified eagle medium (DMEM)	–CSE	–In vitro	–CSE was found to suppress hypoxia-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) –The expression of PCDNA was observed as it is an indicator for cell proliferation; CSE inhibited the expression of PCDNA –CSE inhibits c-jun and c-fos oncogenes –Cell proliferation measured by MTT assay	Gao et al. (2010)
–Methanol extraction –Ethyl acetate extraction	–Dry mycelia were extracted with MeOH three times, combined and concentrated under reduced pressure –Residue redissolved in 1:1 MeOH/H2O and aqueous layer extracted with EtOAc and extracts concentrated under reduced pressure –Extract chromatographed on silica gel-flash column with increasing solvent polarity	–Steroidal glycoside 5a,8a-epi-dioxy-24(R)-methylcholesta-6,22-dien-3b-d-glucopyranoside –5a,6a-Epoxy-24(R)-methylcholesta-7,22-dien-3b-ol –Ergosteryl-3-O-b-d-glucopyranoside –22,23-Dihydroergos-teryl-3-O-b-d-glucopyranoside	– In vitro (erythroleukemia K562, T-lymphoblastic Jurkat, promyeloctic leukemia HL-60, malignant melanoma WM1341, and multiple myeloma RPMI 8226)	–Antitumor activity tested on K562 (erythroleukemia), Jurkat (T-lymphoblastic), HL-60 (promyelocytic leukemia), WM1341 (malignant melanoma) and RPMI 8226 (multiple myeloma) malignant cell lines –The glycosylated form of ergosterol peroxide was found to be a greater inhibitor to the proliferation of K562, Jurkat, WM-1341, HL-60 and RPMI-8226 tumor cell lines by 10–40% than its previously identified aglycone, 5a,8a-epidioxy-24(R)-methylcholesta-6,22-dien-3b-ol	Woo Bok et al. (1999)
–Hot water extraction	–Extraction performed at 120 °C for 20 min –Filtered and freeze-dried	–CSE	– In vitro and in vivo (male and female ICR mice and Sprague–Dawley rats)	–Chronic injections of d-galactose subcutaneously into mice induced changes that resembled accelerated aging –CSE increased learning and memory significantly in a dose-dependent manner –Brain morphology showed that CSE ameliorated the ultrastructure of the hippocampus –Results showed that CSE has a mildly beneficial effect on sexual function –CSE appeared to increase antioxidant enzyme function and slow the overall aging progress	Ji et al. (2009)
–Hot water extraction	–Whole fruiting bodies boiled in distilled water for 30 min (common preparation) –Dried fruiting bodies ground to fie powder and heated in distilled water at 90 °C for 2 h (max. water soluble material) –Repeated for liquid medium-grown fruiting bodies	–Extract 1 (whole rice- and potato-grown hot water extract) –Extract 2 (ground rice- and potato-grown extract) –Extract 3 (powdered liquid medium-grown hot water extract) –Extract 4A (ground rice- and potato-grown ethanol insoluble extract) –Extract 4B (round rice- and potato-grown ethanol soluble extract)	– In vitro (human colonic HT29 cells) and in vivo	–Rice- and potato-grown and liquid-phase-grown C. sinensis possess pro-migratory, invasive and proliferative activity –Ethanol-soluble fraction showed pro-proliferative activity –Ethanol-soluble and insoluble fractions showed pro-migratory activity – In vivo study displayed protective effect of C. sinensis extract on rat gastric damage induced by indomethacin	Marchbank et al. (2011)
–Water extraction	–Defatted with ethanol and residue suspended in water and treated with hot water at 100 °C –Hot water extract concentrated and added 1 vol. of EtOH –Precipitate used as the crude polysaccharide (CS-P) –CS-P redissolved in water and dialysis performed against distilled water to remove low molecular weight constituents –Liquid within dialysis membrane fractionated into soluble and insoluble polysaccharide (CS-Ps and CS-Pp)	–Polysaccharides –CS-Pp is a 1,3-β-d-glucan containing 1,6-branched chains with mean particle diameter of 1.5 μm	– In vitro (mouse splenocyte cell line C3H/HeJ and macrophage cell line RAW264.7)	–CS-Pp induced the most TNF-α production –FTIR and 13C-NMR analysis revealed the immunostimulating polysaccharide of CS-Pp was 1,3-β-d-glucan –GC and GC/MS analysis revealed CS-Pp contained a 1,6-branched chain sugar –Particle size of CS-Pp has a mean diameter of 1.5 μm contributing to its effectiveness when administered orally	Akaki et al. (2009)
–Hot water extraction	–Sequential fractionating with ethylacetate, methanol and hot water; in increasing order of polarity	–CSE	– In vivo (male ICR mice and Sprague–Dawley rats)	–Swimming endurance capacity of mice administered orally with CSE was prolonged from 75 to 90 min indicating a lessening of fatigue symptoms. –In rats fed CSE, stress symptoms were suppressed by observing weight changes of the adrenal gland, spleen, thymus and thyroid	Koh et al., 2003a, Koh et al., 2003b, Koh et al., 2003c
–Hot water extraction	–Natural and cultured C. sinensis extracted using hot water for 2 hr –Vacuum filtered –Rotary evaporated and lyophilized	–CSE	– In vitro (inhibition of linoleic acid peroxidation; scavenging abilities on DPPH, hydroxyl and superoxide anion radicals; the reducing power and the chelating ability on ferrous ions)	–Extracts showed inhibition of linoleic acid peroxidation, scavenging activities on superoxide anion and hydroxyl radicals and DPPH scavenging activities –Moderate reducing power and ferrous ion chelating activity was also observed	Dong and Yao (2008)
–Hot water extraction	– C. sinensis powder heated in 90 °C for 2 h and filtered	–CSE	– In vivo (male Wistar rats)	–CSE markedly decrease urine protein, BUN and SCr levels in Adriamycin damaged kidneys –CSE attenuated the pathological alteration in rat glomerular sclerosis –Immunohistochemical results show decreased expressions of fibronectin (FN), collagen-IV (Col-IV), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1), and increasing the expression of matrix metalloproteinase-2 (MMP-2) in rats treated with CSE	Song et al. (2010)
–Sequential three-solvent extraction (ethyl acetate, methanol, and 50% aqueous methanol)	–Ethyl acetate extraction with filtrate redissolved in acetone –Residue extraction using 100% methanol followed by 50% methanol with filtrate redissolved in respective extraction solvents	–Ethyl acetate extract –Methanol extract –Aqueous methanol extract	– In vitro	–Extracts from the most polar solvent and least polar solvents contained the lowest antioxidant activity –Not much remains post aqueous methanol extraction –Very hydrophilic solvents are less effective at extracting phenolic compounds which represents a direct correlation with antioxidant activity –Based on ORAC values from 55 medicinal herbs, 15.7%, 39.1%, and 45.2% of the total antioxidant activity measured were attributable to the ethyl acetate, methanol, and aqueous methanol extracts, respectively	Wojcikowski, Stevenson, Leach, Wohlmuth, and Gobe, (2007)
–Ethyl acetate extraction	–Crude ethyl acetate extract further fractioned by silica gel chromatography	–Two new epipolythiodioxopiperazines, named gliocladicillins A and B	– In vitro (breast cancer MCF-7, hepatocellular carcinoma HepG2 and cervical cancer HeLa tumor cell lines – In vivo (C57BL/6J mice)	–Inhibited growth of HeLa, HepG2 and MCF-7 tumor cells –Arrested cell cycle at G2/M phase and induced apoptosis – In vivo studies showed inhibitory effect on cell population growth of melanoma B16 cells in immunodeficient mice	Chen et al. (2009)