Callitris columellaris, an evergreen tropical plant commonly called white cypress-pine, belongs to the family Cupressaceae. It is a distinct aromatic plant with several medicinal applications to cure many ailments [1]. The primary essential oil from this plant has been shown to have considerable antioxidant and antiinflammatory properties [2]. Much interest has recently been focused on the development of drugs from natural origins and screening of plants has led to the discovery of novel therapeutics [3].

Hydrodistillation which is one of the major and common methods of extracting natural and neat essential oils of plant materials involves some physicochemical processes hydrodiffusion and hydrolysis. The main shortcoming of hydrodistillation is that complete extraction is not possible; during distillation, part of the essential oil becomes dissolved in condensate or distillation water, which unless recovered, is lost as this water is discarded. Some phytocompounds are usually returned or recycled and retained in the flask as retentate and not collected along with the primary essential oil. The high-boiling and somewhat water-soluble oil and non-volatile constituents cannot be completely collected. Oxygenated components such as phenols have a tendency to dissolve in the warm water, so their complete removal by distillation is not possible. Important phytocompounds are more soluble in hydrosol or retentate. For these reasons, attempts were made to recover the dissolved essential oil from distillation water employing different techniques [4,5]. Solvent extraction was found to be highly effective, providing an essential oil with a composition enriched in terpenoids. Solvent extraction is able to produce essential oils whose fragrance would otherwise be destroyed or altered during hydrodistillation. Solvent extraction is also used on delicate plants to produce higher amounts of essential oils. Solvent extraction is also useful method for capturing the total spectrum of essential oil constituent in plant.

To the best of our knowledge, no literature on the recovered secondary metabolites of post-hydrodistilled C. columellaris leaf and their free radical scavenging potentials have been reported so far. The present research was therefore undertaken for the first time.

Plant materials and extraction of phytochemicals

The leaves of the C. columellaris were collected from their natural habitats in northern part of Nigeria. The sample was identified at the Forest Research Institute of Nigeria (FRIN), Ibadan.

The retentate of the hydrodistilled sample was collected, moisture content reduced and re-extracted with methanol to obtain secondary essential oil extract. The extract was concentrated and stored at low temperature in vial [6].

GC and GC-MS analysis

GC and GC-MS analysis of the secondary essential oil was carried out with capillary column (30 m × 0.25 mm, 0.25 μm film thickness) interfaced with mass detector operating in the EI+ mode (Ionization energy: 70 eV) and ultra-high purity helium (flow rate: 1 ml/min) was used as carrier gas. Temperature program were used as follows: Initial column oven temperature of 60°C (hold: 2 min) programmed at a rate of 3°C/min to a final temperature of 220°C (hold: 5 min). Diluted samples 0.1 μl was injected in the splitless mode. The mass spectra were recorded over 40-500 amu that revealed the total ion current (TIC) chromatograms. Temperature program was used as the same as described above for GC analysis. The temperatures of the injector, transfer line and ion source were maintained at 210°C, 210°C and 200°C, respectively. The identification of the compounds was based on a comparison of their retention indexes determined relative to the retention time of aliphatic hydrocarbons and of the mass spectra with those of authentic compounds by means of NIST library mass spectral database and literature [7].

In vitro DPPH free radical scavenging assay

The antioxidant activity of the leaf secondary essential oil extract of C. columellaris was determined using the stable radical DPPH assay. Briefly, 1.0 ml of the leaf C. columellaris retentate essential oil extract (10, 100 and 1000 μgml^-1) in methanol was added to 1.0 ml of a 0.004% w/v methanol solution of DPPH. The mixture was shaken vigorously and the absorbance was monitored at 517 nm after 30 minutes of incubation, when the reaction reached a steady state. Ascorbic acid was used as reference compound. The inhibition percentage of radical scavenging activity was calculated by using following formula [8].

I% = [(A_blank - A_sample)/A_blank] × 100

Where A_blank is the absorbance value of the control reaction (containing all reagents except the test compound) and A_sample is the absorbance values of the test compounds.

Chemical composition of the recovered secondary essential oil

The secondary oil extract obtained from the post-hydrodistilled leaf retentate sample of C. columellaris was about 1.0% and analysed by GC and GC-MS. Altogether, 77 constituents representing 99.30% of the total oil compositions were identified (Table 1). The study revealed m-cymene (8.40%), γ-4-dimethylbenzenebutanal (7.30%), 4β-17-(acetyloxy)-kauran-18-al (7.0%), cis-8-isopropylbicyclo [4.3.0] non-3-ene (4.30%),7- methoxymethyl-2,7- dimethylcyclohepta -1,3,5-triene (3.40%) and DL-E-nuciferol (3.40%) as the most abundant compounds. We also identified some new phytocompounds which were not reported in the previous study on the primary essential oil of this plant. Notable qualitative and quantitative differences in the chemical composition of the secondary essential oil observed compared to the report of the primary essential oil from the same plant sample where 4-terpineol (11.0%), α-curcumene (8.0%), (+)-Epibicyclosesquiphellandrene (4.0%), β-cubebene (4.0%), γ-cadinene (4.0%), R-(+)-cuparene (4.0%) and 1S-α-pinene (3.0%) are main components [2]. These differences between the primary and secondary extract occurred due to relatively higher solubility of some compounds in warm water during hydrodistillation and difference in method of extraction among other factors.

Table 1: Composition of the Recovered Secondary Essential Oil of C. columellaris.

Antioxidant: Free radical scavenging properties

The leaf retentate secondary essential oil extract of C. columellaris was subjected to screening for its possible antioxidant properties using DPPH assay. DPPH is a stable free radical which can readily experience reduction in the presence of an antioxidant. In this study, the ability of samples to scavenge DPPH radical was determined on the bases of their concentrations providing 50% inhibition (IC₅₀). DPPH involves single electron transfer (SET) and hydrogen atom transfer reactions (HAT) [9].

The IC₅₀ values of the extract and reference compound (ascorbic acid) were given in Table 2. The extract gave excellent radical scavenging activity in comparison to the ascorbic acid as standard drug. The oil extract was able to inhibit the formation of DPPH radicals in a concentration dependent manner. The percentage inhibitions of the secondary metabolites at various concentrations (10, 100 and 1000 μgml^-1) were 23, 45 and 68 % respectively; with the IC₅₀ values of 70 μgml^-1 in comparison to ascorbic acid which gave 58, 85 and 96 as the percentage inhibitions with IC₅₀ value of 10 μgml^-1.

Table 2: Percentage Inhibitions and IC₅₀ of the DPPH Free Radical Scavenging of the Recovered Leaf Secondary Oil Extract of C. columellaris.

Plants are potential sources of natural antioxidants. They produce various antioxidative compounds to counteract reactive oxygen species [10]. Antioxidants are also used as ingredients in dietary supplements in other to maintaining good health and preventing diseases such as cancer and coronary heart disease. Moreover, antioxidants have many industrial uses, such as preservatives in food and preventing the degradation of rubber and gasoline [11].

In this study, a method has been described for recovering the dissolved secondary metabolites from post-hydrodistillation retentate sample and the extract exhibited the presence of high amount of useful phytocompounds, which could be considered as the principal substances behind the antioxidant potentials of this plant.

Therefore, on the basis of present study, it can be said that the retentates of hydrodistillation should not be discarded as usually done. It could be re-extracted to regain the phytocompounds left behind to minimize the loss of valuable components which are pharmacologically useful.