Whatman grade no 1 filter paper

Ganoderma spp. obtained from a laboratory stock culture collection (isolated from the bark of Eucalyptus lanceolate from the forested area near Delhi, India), were maintained on malt extract agar (MEA), containing (g/L) malt extract, 20.0; KH₂PO₄, 0.5; MgSO₄•7H₂O, 0.5; Ca(NO₃)₂•4H₂O, 0.5; and agar, 20.0 (pH 5.5), at 30°C. The obtained culture was stored at 4°C and subcultured every 2 wk. A 250-mL Erlenmeyer flask containing 50 mL of sterile malt extract broth (MEB) was inoculated with two mycelial discs (8 mm in diameter) from the periphery of 7 d-old fungal cultures grown on MEA. The inoculum was incubated at 30°C for 5 d under static conditions. The obtained mycelial mat was further homogenized and then inoculated (4% v/v) in 2-L Erlenmeyer flasks containing 500 mL MEB and incubated at 30°C for 5 d in a rotary incubator at 200 rpm. The fungal mass, obtained in the form of pellets, was separated from the culture broth through filtration using pre-weighed Whatman grade no. 1 filter paper. The formed pellets were used as inocula for further experiments. After being thoroughly washed with deionized water, dried at 55°C until a constant mass was achieved, and weighed.

Air-dried powdered leaves of O. quadripartita (100 g) were extracted by maceration in 80% methanol for 72 h with occasional shaking. The extract was filtered using a Whatman grade No-1 filter paper (Whatman Ltd, England) and concentrated under reduced pressure a using rotary evaporator to give a yellow brown amorphous substance (24.47 g) [21 (link)].

Dried herbs and plants were purchase from a local market, Gyeongdong market, and double boiled in 70% ethanol to extract their components. The ratio of dried herbs or plants to solvent (w/v), boiling temperature, and the extraction time were different for each sample, as described in Table S1. Each extract was successively filtered using a Whatman grade no. 1 filter paper and a 0.45 µm pore filter. The filtered extracts were lyophilized, and the lyophilized powders were dissolved in water to prepare stock solutions with the same concentration of 1 mg/mL. The final concentration used to treat the sensor cells was 1 μg/mL.

Genistein is generally soluble in polar organic solvents, and subsequently, the concentration of these solvents is also very important in extracting the maximum amount of genistein from the sample. Initial extraction was performed by taking 1 g of sample in 100 mL of solvent at 70°C for 1 h. Extraction was carried out using six different solvent mixtures (1) Methanol : Water (90 : 10, v/v), (2) Methanol : Water : Dimethyl sulphoxide (90 : 5 : 5 v/v), (3) Ethanol : Water (70 : 30, v/v), (4) Ethanol : Water : Dimethyl sulphoxide (70 : 25 : 5, v/v), (5) Acetonitrile : Water (58 : 42, v/v), and (6) Acetonitrile : Water : Dimethyl sulphoxide (58 : 37 : 5, v/v). Other conditions of extraction were kept constant (extracting temperature 70°C and extraction time 1 hour). Each extract solution was centrifuged at 2000 g, for 10 min, filtered through Whatman filter paper grade No. 1, and concentrated to 10 mL at 80°C in hot air oven. These aliquots of extracts were filtered through a 0.45 μm PVDF syringe filter prior to analysis of genistein by UPLC-APCI-TOF-MS. From each parameter study, the highest yielding factor is considered for further parameter optimization studies.

The previous studies on N. leucophylla already revealed that the extracts obtained by Soxhlet extraction method provided better results with respect to extraction yield, phytoconstituents composition and biological potential. So, in the current study Soxhlet extraction was utilized in order to get different extracts from the dried powdered leaves [3 ,24 ,29 ].
Soxhlet extraction method (SEM): The dried powder (80 gm in case of methanol extract, while 85 gm in case of chloroform and hexane extract) of N. leucophylla was extracted using various solvents with a varied polarity index such as hexane (PI = 0), chloroform (PI = 4.1), and methanol (PI = 5.1). Using 300 mL of the solvent, the extraction was performed at solvent boiling point with an extraction time of 36 h in every case. Further, the extracts were filtered (2 times) using Whatman filter paper (Grade no. 1). The extracts were dried using a rotary evaporator at temperature below 45 °C. Lastly, the % yields of extracts obtained were calculated. For further assessment, the extracts were stored at temperature below 4 °C.