The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is the trigger of non-communicable environmental disease (Zeliger, 2015). The ROS and RNS damage biological molecules, in vivo via oxidative stress. Elevated levels of ROS, acting via molecular level toxic effects, are now thought to be responsible for a wide spectrum of diseases, including lipid peroxidation of cellular membranes which causes decrease in membrane  fluidity,  DNA  attack,  adduction, enzyme inhibition, oxidative attack on the central nervous system and cell signaling; all of which have been linked to non-communicable diseases (NCDs) including neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), cancer, cardiovascular disease, diabetes, and others (Basu et al., 1999; Ames et al., 1993; Kovacic and Somanathan, 2012).

Reactive   oxygen   species   (ROS)   are   continuously generated inside the human body while the generated ROS are detoxified by antioxidants present in cells. However, over production of ROS or inadequate antioxidant defences can lead to oxidative damage of various biomolecules including proteins, lipids, lipoproteins and DNA. Free radicals are the major cause of chronic and degenerative diseases such as coronary heart diseases, inflammatory stroke, diabetes and cancer (Scalbert et al., 2005).

An antioxidant is generally defined as any substance that effectively prevents or delays the adverse effects caused by free radicals and the amount of the antioxidant is less than that of the substance to be oxidized (Halliwell and Gutteridge, 1999). An antioxidant significantly delays or prevents oxidation of cell components (susceptible to oxidation) such as proteins, lipids, carbohydrates and deoxyribonucleic acid (DNA). Due to their redox properties, antioxidants act as reducing agents, hydrogen donors, singlet oxygen quenchers and chelating metal (Tung et al., 2009; Lauro and Francis, 2000). Although several synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are commercially available, their toxicity has always been a concern and strong restrictions have been placed on their application in pharmaceuticals. There is therefore a need for more effective, less toxic and cost effective antioxidants. Recently, there has been an upsurge of interest in the therapeutic potential of plant-derived antioxidants in reducing free radical-induced tissue injury and the current trend is to substitute synthetic with naturally occurring antioxidants (Barlow, 1990). Several biologically active compounds of plant origin (phytochemicals) have been found to possess antioxidant, free radical scavenging activity and many are being applied therapeutically for free radical associated disorders (Lee et al., 2000).

The concoctions used in traditional medicine contain many secondary metabolites (SMs) of plant origin such as polyphenolic compounds and polysaccharides which interact synergistically (Eid et al., 2013, 2012; Mulyaningsih et al., 2010; Wink, 2015). The therapeutic benefit of plant-based traditional medicine is often attributed to the antioxidant properties and potential of the constituent phytochemicals (Hertog et al., 1993; Zhang et al., 2001).

In Lesotho, as in many other countries in the world, a system of traditional medicine based on the use of plants, birds, animals, their products and their combinations to treat a broad spectrum of communicable and non-communicable diseases is still being practiced (Padmanabhan and Sujana, 2008).

One of such products used in Lesotho from animals is the fossilized excreta of Procavia capensis (rock hyrax) called hyraceum or “moroto oa pela” in Sesotho. The Basotho use hyraceum to treat respiratory infections, urinary tract or bladder infections, measles and non-communicable diseases such as diabetes mellitus.

The hyraceum is also used in combination with other medicinal plant species to enhance its efficacy (Seleteng-Kose et al., 2015). The strongly aromatic hyraceum is also a well-known Khoikhoi medicine, often used as a post-natal medicine for mothers and babies and a remedy for hysteria and epilepsy (van Wyk, 2008). The fossil is formed from the faeces and urine of P. capensis as the major components which accrete to form dark brown, resin-like masses to which plant material, pollen grains and other digestive remains are trapped. Fossilization occurs with time in arid regions. P. capensis inhabits shelters in rocky outcrops in a variety of biomes and feeds on a variety of grasses, shrubs, tree leaves, fruits and berries including the bark of the tree (Olsen et al., 2008).

The only literature found on the biological activity of hyracei was that on its effect on the GABA-benziodiazepin receptor, which indicated that since the hyracei exhibited a high affinity for GABA-benziodiazepin they could be used to treat epilepsy, a non-communicable disease (Olsen et al., 2008). Other than that, no records were found on the scientific validation of traditional medicinal uses of hyraceum (van Wyk, 2008).

In view of complex nature of hyraceum, composed of animal metabolic waste and plant materials and its use to treat both communicable and non-communicable diseases, the present study was performed and involved the analyses of the crude methanolic (95%) extract of hyraceum for the content of phytochemical and antioxidant activity and the relationship between the total phenolic content and antioxidant activity.

Data analysis

Data was expressed as means ± standard deviations of three replicate determinations using Microsoft excel 2013. Differences between controls and treatment groups were determined using Student’s t-test. P-values of less than 0.05 were considered statistically significant using the IBMSPSS statistics, version 20 software. Regression equations and graphs were used for the

Qualitative phytochemical profile of the hyraceum extract

The yield of the hyraceum crude methanol extract from the whole mass of hyraceum was 6.17%. In Table 1 is presented the qualitative biochemical profile of hyraceum extract obtained from the various tests. Colour intensities of the hyraceum solutions (ranging from absence of antioxidant (-) to high presence (+++) for the most intense colour reaction, gave a semi-quantitative indication for the presence of different classes and relative the amounts of phytochemicals in the crude extract (Lu et al., 2014; Chukwudi and Yusha’u, 2016). Most of the classes of compounds identified in the hyraceum were phenolic in nature (flavonoids, alkaloids, tannins, terpenoids, saponins, phenols, polyphenols, anthocyanins, quinones, phlobatannins, anthraquinones and coumarins) and amino acids. No sterols, cardiac glycosides, reducing sugars, fatty acids and proteins were detected in the hyraceum extract. The highest colour intensities (+++) indicating a high presence were exhibited  by  terpenoids, saponins, polyphenols, quinones, phlobatannins and coumarins followed by anthocyanins (++). The lowest colour intensities, indicating a low presence (+), were given by flavonoids, alkaloids, tannins, phenols, anthraquinones, and amino acids.

Quantitative determination of total phenolics content (TPP)

In Figure 1 is presented the gallic acid calibration curve for determination of total phenolics of the hyraceum extract. The regression equation obtained was y = 0.053x - 0.033, R²=0.971; where y was the mean absorbance of the sample at 760 nm and x, the concentration established from the gallic acid calibration curve. The total phenolic content of the hyraceum extract was found to be 37.339 mg GAE/g dry weight (37.339 mg GAE/g DW). 

DPPH free radical scavenging assay

The results of the DPPH assay are presented in Table 2. A dose-dependent decrease in absorbance was observed which also reflected a dose-dependent increase in the percentage scavenging of the DPPH free radical for both the hyraceum and L-ascorbic acid. The highest concentration (3000 µg/ml) of the hyraceum crude extract and the reference compound L-ascorbic acid gave the lowest absorbance values at 517 nm  while the lowest concentration (0 µg/ml), being the negative control, gave the highest absorbance as shown.

The qualitative phytochemical screening of HM revealed the presence of different classes of phytochemicals in the crude methanolic (95%) extract of the hyraceum. There was a high presence (+++) of terpenoids, saponins, polyphenols, quinones, phlobatannins and coumarins (Table 1) in the solution of the hyraceum based on the intensity  of  the  colour  in  the  colorimetric  tests,  which indicated that these phytochemicals were most abundant. Other phytochemicals that were detected at smaller quantities were anthocyanins with a moderate presence (++), while flavonoids, alkaloids, tannins, simple phenols, anthraquinones and amino acids exhibited a low presence (+). Sterols, cardiac glycosides and cardenolides, reducing sugars, proteins and fatty acids were not detected and hence assumed to be absent or below detection limit of the methods used. With the exception of terpenoids, all the  phytochemicals  detected in the hyraceum extract were phenolic in nature even though they vary in structure. Terpenoids are highly conjugated molecules also with antioxidant properties just like phenolic compounds. According to Bang et al. (2015) phytochemicals    such    as     carotenoids,     terpenoids, ascorbates, reducing carbohydrates and tocopherols contribute to the antioxidant activity of natural products. The antioxidant activity of the crude methanolic (95%) extract of HM could therefore be attributed to the antioxidants detected in the hyraceum solution (Table 1). Phenolic compounds have antioxidant activity because they can donate hydrogen atoms to neutralize free radicals and the phenolic compounds form will be stabilized by resonance. Tannins act as antioxidants because of their ability to stabilize lipids and inhibition of lipoxygenase. Hydrolysable tannins have anti-ischemic activity. Alkaloids, particularly indole, are efficient at inhibiting the chain reaction of free radicals. All types of phenolic compounds are reported to exhibit antioxidant activity but to different degrees (Ahmad et al., 2014) and flavonoids are reported to exhibit antitumour, anti-inflammatory. The antioxidant activity of phenolic compounds is mainly due to their redox properties which make them act as reducing agents, hydrogen donors, singlet oxygen quenchers and also may have a metallic chelating potential (Rice-Evans et al., 1996). Synergism among the antioxidant compounds in the crude extract makes the antioxidant activity is not only dependent on the concentration but also on the structure and interaction between the antioxidant compounds (Djeridane et al., 2006).

The phytochemicals identified in the hyraceum listed in Table 1 were also identified in the beaver castoreum, another complex product of animal metabolic waste and plant materials which exudes from the castor sacs of the mature North American beaver (Castor canadensis) and the European beaver (Castor fiber) (Müller-Schwarze, 2003).

Pyrolysis gas chromatography mass spectrometry analysis of hyraceum identified nitrogen-containing aromatic compounds, notably benzamide (Carr et al., 2010). The solvent-extractable lipids of hyraceum comprised homologous suites of long-chain n-alkanes (C₂₄–C₃₄) and n-alkanols (C₁₆–C₂₆) characteristic of higher plant leaf waxes, along with an abundance of animal-derived sterols, higher plant sterols and terpenoids as well as benzamide (Carr et al., 2010).  As early as 1879, Green and Parker (1879) analyzed hyraceum and found that about 56% of it was soluble in water, the remaining insoluble material was organic and composed of woody fibre, sand and other inorganic substances as mixtures of various salts such as soda and lime (having the highest proportion among the salts). The organic matter contained traces of urea together with uric, hippuric, and benzoic acids due to the fact that hyraceum is partly derived from urine and faecal matter (Green and Parker, 1879). No study was found on the phytochemical composition of the hyraceum with which to compare the results.

Only the total phenolics content of hyraceum crude extract was determined in this study. The determined value of total phenolics (Figure 1) of the hyraceum extract of 37.339 mg GAE/g dry sample of the hyraceum was much higher than the highest value of 1.699 mg GAE/g observed with cow dung, another complex product of animal metabolic waste and plant materials by Jirankalgikar et al. (2014) most probably due to the fossilized nature of the hyraceum.

The hyraceum extract was tested for antioxidant activity using the DPPH free radical and the hydrogen peroxide (H₂O₂) scavenging assays.

As shown in Table 2, at 3000 µg/ml the highest percentage inhibition of the DPPH free radical and the EC₅₀ for the hyraceum (HM) extract were 55.21±0.42% and 5.983 µg/ml respectively and for L-ascorbic acid (AA) were 97.16±0.16% and 0.4293 µg/ml respectively. The EC₅₀ is the concentration of extract that causes a 50% decrease in the initial concentration of the DPPH free radical (Do et al., 2014).  A lower IC₅₀ represents higher antioxidant activity (Do et al., 2014; Proestos et al., 2013). Jarald et al. (2008) observed an EC₅₀ of 5.10 µg/ml in studies with cow urine while Jirankalgikar et al. (2014) observed an EC₅₀ range of between 12.810 and 41.554 mg/ml with cow dung samples collected at different times of the day in a six day period of study. In the same study by Jirankalgikar et al. (2014), the EC₅₀ of L-ascorbic acid was 20.13 µg/ml. The EC₅₀ of the hyraceum extract of 5.983 µg/ml obtained in the current study is similar to that obtained by Jarald et al. (2008) of 5.10 µg/ml in studies with cow urine but lower than the values of between 12.810 and 41.554 mg/ml obtained in studies with cow dung by Jirankalgikar et al. (2014).

In this study, hydrogen peroxide scavenging activity was estimated by the replacement titration method. As shown in Table 3, at 3000 µg/ml the highest percentage scavenging of H₂O₂ and the EC₅₀ for the hyraceum (HM) extract were 73.61±8.56% and 5.059 µg/ml, respectively and for L-ascorbic acid (AA) were greater than 77.78±4.81% and 1.666 µg/ml, respectively. No literature on hydrogen peroxide scavenging activities by mammalian metabolic waste products was found for comparison with the results of this study. However, in a study by Vinodhini and Lokeswari (2014) the methanolic leaf extract of Toona ciliate scavenged a maximum of 55.69   ± 1.04% of hydrogen peroxide.

The colorimetric tests in the present study detected the major phytochemical components of the hyraceum extract as terpenoids, saponins, polyphenols, quinones, phlobatannins and coumarins and the minor phytochemicals as flavonoids,  alkaloids,  tannins,  simple phenols, anthocyanins, anthraquinones and amino acids. Sterols, cardiac glycosides and cardenolides, reducing sugars, proteins and fatty acids were not detected. The total phenolic content of the hyraceum extract was 37.339 mg GAE/gram dry weight. The hyraceum  extract

was more effective at scavenging H₂O₂ (73.61±8.56%) than the DPPH free radical (55.21±0.42%). Bioactive compounds in HM could potentially be exploited in further studies as potential antioxidants of therapeutic value in preference to synthetic antioxidants as they have negative side-effects in the body.

The authors have not declared any conflict of interests.

This work was supported by a Research grant provided by the Research and conference committee, National University of Lesotho, 2014.