2.6. Identification of compounds

This experiment used a headspace Gas Chromatography-Mass Spectrometry (GC–MS; Shimadzu Corp., Japan) apparatus to distinguish the chemical compounds in dried and pulverized Antidesma bunius (L.) Spreng fruits. Supelco® 24 gauge 100 m Polydimethylsiloxane (PDMS) Solid Phase Microextraction (SPME) fiber was used in this experiment. Other fiber coating was also used such as 50/30 μm 24-gauge Divinylbenzene/Carboxen/Polydimtheylsiloxane (DVB/CAR/PDMS) SPME Fiber if needed throughout the analysis. SPME fibers were baked prior to use according to manufacturer’s instructions. A 500 mL Pyrex® Erlenmeyer flask coated with aluminum foil and parafilm was used as a headspace chamber for the headspace review. Before use, flasks were washed with technical grade acetone and baked for 1 h at 150 °C. After cooling, each sample was put in separate clean flasks and heated to 30 °C–40 °C for 25 min while the PDMS fiber was exposed. After incubation, SPME fiber was immediately injected into the GC–MS. Blank trials of the GC–MS, SPME fiber, and flasks were also carried out in order to eliminate any potential errors during the study. The Shimadzu GC–MS QP2020 was used in this analysis, with a SH-Rxi-5Sil capillary column (30 m x0.25 mm ID x 0.25 m df). The injector port was manually injected with SPME fiber. A 0.75 mm ID Shimadzu® SPME Inlet Liner and a Restek® Merlin Microseal Septa General Purpose Kit were installed in the injector port. MS Settings were set to SCAN Mode (35 m/z to 500 m/z) and acquisition mode to SCAN Mode (35 m/z to 500 m/z). For library matching, libraries from the NIST 2017 Mass Spectral Library and the Wiley Registry 11th edition were used. The temperature program was set to start at 50 °C for 5 min, then increase by 10 °C every 10 min until it reached 200 °C. Desorption of volatiles was carried out at 250 °C in split less mode with Linde® Helium as the carrier gas at a rate of 1 mL/min (99.995 %). The relative quantity of chemical compounds present in each of the extracts of Bignay fruit was expressed as a percentage based on the peak area generated in the chromatogram. Trials were carried out in threes.

Secondary metabolites were detected directly using an ethanolic extract of Antidesma bunius (L.) Spreng fruits. In three runs from three different collected samples, at least ten secondary metabolites were measured qualitatively [24].

To a total of 0.5 mL of ethanolic fruit extract, 2.5 mL 2% hydrochloric acid (HCl) solution was added followed by 0.25 g powdered NaCl. The mixtures were then stirred and filtered. The residues obtained were washed with 2% HCl solution and 2.5 mL distilled water was added to the residue. To 0.5 mL of mixture, 1 mL of Mayer’s reagent was added. The presence of turbidity indicates the presence of alkaloids.

A total of 0.5 mL of ethanolic fruit extract was boiled with 1 mL of 10 % HCl for 5 min. While still hot, the mixture was filtered and allowed to cool followed by addition of equal amount of chloroform (CCl₄). The CCl₄ layer was aspirated into a clean dry test tube using a pipette. Equal volume of 10 % NH₄Cl solution was added into the CCL₄ containing test tube. The mixture was agitated and allowed to separate. The separated aqueous layer was monitored for any color change; delicate rose pink color indicates the presence of anthraquinones.

To 0.5 mL of ethanolic fruit extract, 2.5 mL of CCl₄ was added. The mixtures were vortexed for 1 min and were filtered using Whatman filter paper #1 (Sigma-Aldrich Chemical Co.). To the filtrate, 1.5 mL 85 % sulfuric acid (H₂SO₄) was added. Formation of a blue color at the interface indicates the presence of carotenoids.

To 0.5 mL of the ethanolic fruit extract, 0.5 mL of 10 % sodium hydroxide (NaOH) was added to the mixture. Formation of yellow color indicates the presence of coumarins.

To 0.5 mL of the ethanolic fruit extract, distilled water was added to a final volume of 2.5 mL and was boiled for 5 min. The sample was filtered, and a few drop of 20 % NaOH solution was added to the filtrate. A change of color yellow to colorless indicates the presence of flavonoids after addition of 10 % HCl.

Extracted fruit sample (1 mL) was diluted in 20 mL distilled water. The mixture was shaken in a graduated cylinder for 15 min. Development of stable foam suggests the presence of saponins.

A total of 0.5 mL ethanolic fruit extract was mixed in a 2.5 mL of CCl₄. Equal volume of concentrated H₂SO₄ was then added carefully through the side of the test tube. The presence of steroids showed when upper layer turned red and the sulfuric acid layer showed yellow with green fluorescence.

A 0.5 mL ethanolic fruit extract is boiled in equal amount of distilled water and was filtered prior to addition of 0.2 mL 0.1 % ferric chloride (FeCl₃). A brownish green or blue-black coloration shows the presence of tannins.

A 0.25 mL CCl₄ was added to 0.5 mL ethanolic fruit extract and gradually; 1 mL of concentrated sulfuric H₂SO₄ was added to form a layer. A reddish brown precipitate coloration at the interface formed indicates the presence of terpenoids.

One mL of 70 % laboratory grade ethanol was added to 0.5 mL ethanolic fruit extract followed by addition of 1 mL potassium hydroxide (KOH) to the mixture. Formation of blue color indicates the presence of Quinone.