Investigation of the profile of phenolic compounds in the leaves and stems of Pandiaka heudelotii using gas chromatography coupled with flame ionization detector

Abstract The profile of phenolic compounds in the leaves and stems of Pandiaka heudelotii was investigated using gas chromatography coupled with flame ionization detector. The leaves and stems had high flavonoids and benzoic acid derivatives content, and moderate levels of lignans and hydroxycinnamates. Twenty‐eight known flavonoids were detected, which consisted mainly of kaempferol (41.93% in leaves and 47.97% in stems), (+)‐catechin (17.12% in leaves and 16.11% in stems), quercetin (13.83% in leaves and 9.39% in stems), luteolin (7.34% in leaves and 7.71% in stems), and artemetin (6.53% in leaves and 4.83% in stems). Of the six known hydroxycinnamates detected, chlorogenic acid (80.79% in leaves and 87.56% in stems) and caffeic acid (18.98% in leaves and 12.30% in stems) were the most abundant, while arctigenin (77.81% in leaves and 83.40% in stems) and retusin (13.82% in leaves and 10.59% in stems) were the most abundant of the nine known lignans detected. Twelve known benzoic acid derivatives were detected, consisting mainly of ellagic acid (65.44% in leaves and 72.89% in stems), p‐hydroxybenzoic acid (25.10% in leaves and 18.95% in stems), and vanillic acid (8.80% in leaves and 7.30% in stems). The rich phytochemical profile of the leaves and stems is an indication of their ability to serve as sources of nutraceuticals.


O R I G I N A L R E S E A R C H
Investigation of the profile of phenolic compounds in the leaves and stems of Pandiaka heudelotii using gas chromatography coupled with flame ionization detector Mercy O. Ifeanacho | Catherine C. Ikewuchi | Jude C. Ikewuchi

| INTRODUCTION
The wide use of phytonutrients reflects a fact that nutrition science has advanced beyond the treatment of deficiency syndromes to the reduction of disease risk. Food is no longer evaluated only in terms of macronutrient and micronutrient levels, but their contents of some biologically active compounds are becoming more important (Zhao, 2007).
In addition to providing macro-and micro-nutrients, vegetables are rich sources of bioactive phytochemicals, and other compounds that support human health and nutrition (Radovich, 2011;Sinha, Hui, Evranuz, Siddiq, & Ahmed, 2011). Pandiaka heudelotii (family: Amaranthaceae) is a wild vegetable commonly consumed in southern Nigeria. It is used for the preparation of soup, and boiled for tea. Despite the use of this plant as both food and medicine, we found no information in the biochemical literature regarding its phytochemical and phenolic compounds composition. Therefore, this study investigated the phenolic compounds composition of the leaves and stems of Pandiaka heudelotii with a view to providing information on their potential as sources of nutraceuticals.
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| Determination of benzoic acid derivatives composition
Benzoic acid derivatives were extracted by the two-stage process described by Andary et al. (2013). The concentrated extract (2.0 ml) was transferred to a 5.0 ml glass vial. It was then saturated with sodium chloride salt before adding 250.0 μl of ethyl acetate to it. The mixture was agitated manually for 10 min at room temperature and later centrifuged for 15 min at 2500 rpm, before removing the organic phase to a 1-ml vial. The extraction was repeated twice and the organic phases were combined. Aliquot of 50.0 μl of N,O-bis (trimethylsilyl) trifluoroacetamide was added, and the mixture was manually agitated for 2 min at room temperature for derivatization. The column type was a capillary HP-1 with the dimension (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detector temperatures were 250°C and 320°C, respectively. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 193.1 kPa and 220.6 kPa. The oven was programmed as follows: initial temperature at 60°C for 5 min, ramping at 15°C/min for 15 min, and attained temperature maintained for 1 min, followed by a second ramping at 10°C/min for 4 min.

| Determination of the flavonoids composition
The flavonoids extraction was carried out according to the method of Millogo-Kone et al. (2009). One gram of the dried ethanol and aqueous extracts were weighed into 100 ml of distilled water in a 250-ml conical flask and boiled for 10 min. To this was added 100 ml of boiling methanol/water (70:30, v:v) mixture. The mixture was allowed to macerate for about 2 hr, and then filtered with Whatman filter paper No.1. The filtrate was concentrated to 5 ml for gas chromatographic analysis. The column was a capillary HP INNOWax (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C.
Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 151.7 kPa and 241.3 kPa. The oven was programmed as follows: initial temperature of 50°C, first ramping at 8°C/min for 20 min, attained temperature maintained for 4 min, followed by a second ramping at 12°C/min for 4 min, and maintaining attained temperature for 4 min.

| Determination of hydroxycinnamates composition
The hydroxycinnamates extract was prepared as described by Ortan, Popescu, Gaita, Dinu-Pîrvu, and Câmpeanu (2009), and subjected to gas chromatographic analysis. The column was HP-5 (30 m × 0.32 mm × 0.25 μm film thickness). The samples were introduced via an all-glass injector working in the split mode, with nitrogen as the carrier gas, at a flow rate of 1 ml/min. The injection and detector temperatures were 260°C and 300°C, respectively. The oven temperature was programmed at the start of the run from 170°C to 250°C at 5°C/min.

| Determination of the lignans composition
The lignan extract was prepared as reported by Chapman, Knoy, Kindscher, Brown, and Niemann (2006), and subjected to gas chromatography. The column was ZP-5 (30 m × 0.32 mm × 0.25 μm film thickness), detected at 300 nm. One microliter of sample was injected. The conditions for the GC were initial oven temperature of 40°C, injector 250°C, transfer line 280°C, a solvent delay of 2 min; and ramped temperature at 10°C/min to a final temperature of 230°C, which was held for 1 min.

| Determination of tannins composition
The tannin extract was prepared as reported by Luthar (1992), and subjected to gas chromatographic analysis. The column was a capillary HP-5 (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250 and 320°C. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier gas. The hydrogen and compressed air pressures were 173.1 kPa and 275.8 kPa.
The oven was programmed to an initial temperature at 120°C, before ramping at 10°C/min for 20 min.
They had moderate total (448.4 mg/kg dw in leaves and 334.3 mg/ kg dw in stems) hydroxycinnamates' contents (Table 3). Six known hydroxycinnamates were detected, including chlorogenic acid (80.87% in leaves and 87.6% in stems), caffeic acid (19.0% in leaves and 12.3% in stems), and chicoric acid (0.1% in leaves and 0.1% in stems). The remaining less than 0.2% consisted of p-coumaric acid, p-coumarin, and scopoletin. Tannic acid was the only compound detected in the tannins fraction.
The total lignans contents of the leaves and stems were 496.6 mg/ kg dw and 914.6 mg/kg dw, respectively (
From the foregoing, it can be seen that the leaves and stems of Pandiaka heudelotii contain a variety of biologically active phytochemicals. The beneficial roles of these bioactive phytochemical constituents can be harnessed in the diet, making them important tools for nutritional therapy. This, therefore, emphasizes the potential of the leaves as a candidate for use as functional food.