*2.2. Quantification of Phenolic Content and Antioxidant Activities of B. glabra Nodal Segments and In Vitro-Induced Calli*

#### 2.2.1. Planting Materials and Preparation of Extract

The nodal segments of conventionally propagated plants of *B. glabra* were collected from the Faculty of Agriculture, Universiti Putra Malaysia. In addition, the treatment of 7.5 μM 2,4-D + 1.5 μM BAP under dark and light incubation conditions which exhibited the highest biomass accumulation in vitro were obtained after 16 weeks in the Tissue Culture Laboratory, Faculty of Agriculture, Universiti Putra Malaysia were selected as a suitable treatment and quantified for phenolic content and antioxidant activities. The collected samples were oven-dried at a temperature of 55 ◦C for 48 h or until the weight remained constant. The dried nodes and in vitro-induced calluses were used to determine phenolic content and antioxidant properties. Furthermore, the extraction was conducted following the method employed by Hakiman and Maziah [33] with minor modifications. Dried nodes and in vitro-induced callus samples of *B. glabra* were ground using a commercial blender (Brand: Panasonic). An amount of 1 g dry weight of each sample was weighed and placed in a 150 mL conical flask. Each solvent, such as distilled water, ethanol, acetone, and hexane, received a total volume of 50 mL, and the flasks were covered with aluminum foil. The samples were placed in conical flasks on an orbital shaker at room temperature for 1 h in the dark. The samples were filtered with Whatman No. 1 filter paper and the extract was used for further analysis.

#### 2.2.2. Total Phenolic Acids Content

Total phenolic acid content was evaluated following a method proposed by Singleton and Rossi [34]. To begin, test tubes were filled with 0.5 mL of extracts and 4.5 mL of distilled water. Then, 0.5 mL of Folin–Ciocalteu phenol reagent was added and thoroughly mixed using a vortex machine. After 5 min, 5 mL sodium carbonate (7%) was added. By adding 2 mL of distilled water, the final volume was adjusted to 12.5 mL. At room temperature, the reaction mixtures were incubated for 90 min. At 750 nm, the absorbance was measured. By establishing a standard curve of absorbance against various amounts of gallic acid, the total phenolic acids content was measured. The total phenolic acids content of the extract was measured in milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW).

#### 2.2.3. Total Flavonoids Content

Total flavonoids content was generated using the method established by Marinova et al. [35]. First, 0.5 mL of extracts was added to 2 mL of distilled water in test tubes. After that, 150 μL of 5% sodium nitrite was added to the mixture, which was then incubated for 5 min after 150 μL of 10% aluminum chloride was added. After that, 1 mL of 1 M sodium hydroxide and 1.2 mL distilled water were added at the sixth minute. A spectrophotometer was used to measure absorbance at 510 nm after the mixture was properly mixed. To assess the level of the total flavonoid content of the extract, a standard curve of absorbance against various concentrations of rutin was established. The extract's total flavonoids concentration was calculated as mg rutin equivalents per gram dry weight of the sample (mg RE/g DW).

## 2.2.4. DPPH Free Radical Scavenging Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was determined to quantify the ability of the DPPH molecule to neutralize free radicals by donating a hydrogen atom. Thus, the color shift from purple to violet is visible. DPPH free radical scavenging assay was performed according to the procedure explained by Wong et al. [36]. The DPPH was first generated in methanol at a concentration of 0.1 mM, and the initial absorbance of methanolic DPPH was measured with a spectrophotometer at 515 nm. The extracts were then combined in 1.5 mL of 0.1 mM methanolic DPPH solution. After shaking the mixture and incubating it at room temperature for 30 min, the absorbance was measured at 515 nm. The control was Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), and the DPPH value was measured in mg Trolox equivalent per gram dry weight of the sample (mg TE/g DW)
