Laborota 4011

A powder of cultivated strain TM02 of L. rhinocerus (LR) was obtained from LiGNO Biotech™ Sdn Bhd, Selangor, Malaysia. This powder was extracted into three fractions with ethanol, cold water and hot water using the maceration technique. Briefly, 100 g of LR powder was macerated with 1 L of ethanol and the extract was placed on the shaker at 4 °C for 24 h. After that, it was filtered by using Whatman® No.2 filter paper and ethanol was removed by rotary evaporation (Heidolph, Laborota 4011) to yield the crude ethanol extract (LRE). Cold water extraction: 100 g of LR powder was suspended in sterile water and placed on the shaker at 4 °C for 24 h. For hot water extraction, sclerotial powder was extracted with water at 95–100 °C for 2 h. After that, the mixture was filtered and freeze dried by lyophilizer (ModulyoD freeze dryer, Thermo Fisher Scientific, Waltham, MA, USA) to give a crude cold-water extract (LRC) and a crude hot-water extract (LRH). Yields of LRE, LRC and LRH were 0.73 g, 11.07 g and 10.13 g, respectively. Before starting the experiment, the crude extract of LRE was dissolved in DMSO, while the crude extracts of LRC and LRH were dissolved in sterile water to make the 100 mg/mL stock solution.

A powder of the cultivated strain TM02 of LR or tiger milk mushroom was obtained from LiGNO Biotech^TM Sdn Bhd, Selangor, Malaysia. The three extractions of LR were ethanol extract (LRE), cold-water extract (LRC), and hot water extract (LRH). One hundred grams of LR powder were macerated with 1 L of ethanol and the extract was shaken at 4 °C for 24 h. After incubation, the extract was filtered using Whatman^® No.2 filter paper and ethanol was removed by rotary evaporation (Heidolph Laborota 4011, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The procedure of cold-water extraction is similar to LRE, but sterile water is used instead of ethanol. Although the powder was boiled in hot water at 95–100 °C for 2 h, both LRC and LRH were filtered and freeze dried in a lyophilizer (ModulyoD freeze dryer, Thermo Fisher Scientific, Waltham, MA, USA). Finally, about 0.73 g, 11.07 g, and 10.13 g were obtained from LRE, LRC, and LRH, respectively.