Ruellia tuberosa L. (herb) commonly called ‘cracker plant’, often grown for ornamental purposes is an Acanthaceae: The 3rd largest tropical family of dicotyledonous plants with about 2500  species,  most  of which have medicinal values (Burkill, 1985). It has tuberous roots with leaf, stem, fruit and flower parts, is native to America, but widely spread in dry and hilly regions. R. tuberosa is traditionally used as diuretic,  antipyretic, analgesic, anti-hypertensive, anthelmentic, abortifacient, emetic, in curing bladder disease, kidney disorder, bronchitis, gonorrhoea and syphilis (De Fillpps et al., 2004). There are reports that it possess anti-oxidant, anti-microbial, anti-cancer, anti-nociceptive, anti-inflammatory and gastro-protective activities (De Fillpps et al., 2004; Chothani et al., 2010; Agnihotri et al., 2012; Andhiwal et al., 1985; Alam et al., 2009). The plant has emetic properties and serves as substitute for ipecacuanha plants, in the treatment of bladder stones. Its leaf decoction is used in treatment of bronchitis, and its tuberous roots are ingredient in health tonic (De Fillpps et al., 2004; Agnihotri et al., 2012; Samy et al., 2015). It displayed ulcer protective activity in male Wistar rats (Sri Kumar and Pardhasaradhi, 2013). Leaf extracts of R. tuberosa L. were reported to control lipid peroxide level and help strengthen antioxidant potential in diabetic rats (Manikandan et al., 2010). R. tuberosa root extracts displayed anti-oxidant activity, which were comparable with standards (Chothani and Mishra, 2012).

Preliminary phytochemical screening of ethyl acetate extract of R. tuberosa reveals presence of saponin, tannins, and flavonoids (SriKumar and Pardhasaradhi, 2013). Five bioactive flavonoids cirsimaritin, cirsiliol 4′-glucoside, cirsimarin, sorbifolin, and pedalitin, with three other metabolites betulin, vanillic acid, and indole-3-carboxaldehyde were isolated and reported from ethylacetate extracts of Ruellia tuberosa. First two flavonoids showed cytotoxicity against KB cell line with the IC₅₀ values of 30.05 and 17.91 µg/mL, and the third flavonoid was cytotoxic against HepG2 cell line with an IC₅₀ value of 38.83 µg/mL (Lin et al., 2006). Apigeninglucuronides have also been isolated from Ruellia (Subramanian and Nair, 1972, 1974). Subramanian and Nair reported that R. tuberosa leaves have only traces of apigenin and luteolin, but its flowers contain malvidin-3,5-diglucoside in appreciable amount (Subramanian and Nair, 1974).

Also the flower buds have maximum proportion of flavonoids yielding 3% of apigenin-7-O-glucuronide. Other flavones identified were apigenin-7-O-glucoside, apigenin-7-O-rutinoside and luteolin-7-O-glucoside (Subramanian and Nair, 1974). Aerial parts of R. tuberosa yielded 21-methyldammar-22-en-3β, 18, 27-triol atriterpenoid (Singh et al., 2002). Identified and reported essential oil components are sources of important data and information on the plant and its family classifications (Baser and Buchbauer, 2010). Twenty-five compounds were reported from GC-MS analysis of ethanol extract from tuber of R. tuberosa (%): Lupeol (68.14), stigmasterol (8.89), α-sitosterol (3.99), sucrose (2.24), 3α-bromo-cholest-5-ene     (2.24),    2-methyl-octadecane (2.10), 2-methyl-nonadecane (1.93), 2-methyl-eicosane (1.79), hexacosane (1.43) and heptacosane (1.29) as its prominent compounds (Rajendra et al., 2014).

Previously we examined and reported volatile chemical compositions on three Acanthaceae: Brillantaisia patula (leaf and stem), Hypoestes phyllostachya (leaf and stem), Asystasia gangetica (aerial, seed and root) (Moronkola et al., 2009a, 2009b, 2011). This study examines the composition of volatile content of leaf, stem, root, fruit, and flower of R. tuberose L. also an Acanthaceae, from Nigeria, which have not been earlier reported in literature.

Plant material

Fresh samples of R. tuberosa were collected from Ibadan, Oyo State, Nigeria, on 15^thOctober 2014. The plant was authenticated in the herbarium, Department of Botany, University of Ibadan, Ibadan, where some voucher samples have been deposited, with voucher number UIH - 22426. Collection of the sample was done during the day time. The plant was separated into leaf, stem, root, fruit, and flower parts.

Extraction of the essential oils

Each separated parts (leaf, stem, root, fruit, and flower) of R. tuberosa was crushed and hydro-distilled for 3 to 3½ h in an all glass Clevenger-type apparatus designed to British Pharmacopeia specifications and the oils refrigerated until analyses. Essential oils from the pulverized air-dried plant materials were procured in 0.09 to 0.36% yields (Table 1). Each of the oils had distinct characteristic pleasant smell. 

Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses

Composition of the essential oils was determined by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890N gas chromatography hyphenated with an Agilent system mass detector Triple Quad 7000A in EI mode at 70 eV (m/z range 40 - 600 amu) with an ion source temperature of 250°C and an Agilent ChemStation data system. GC column was equipped with an HP-5MS column (30 m × 250 µm × 0.25 µm) a split-split less injector heated at 200°C and a flame ionization detector (FID) at 230°C. Oven temperature was programmed as follows: Initial temperature 40°C for 5 min, increased 5°C/min to 180°C for 6 min and then 10°C/min to 280°C for 12 min. Helium was the carrier gas at flow rate of 1 mL/min. Injection volume was 2.0 µL (split ratio 1:20).

The components were identified by comparison of their mass spectra with NIST 1998 library data of the GC-MS system as well as by comparison of their retention indices (RI) with the relevant literature data (Adams, 2007). The relative amount of each individual component of the essential oil was expressed as the percentage of the peak area relative to the total peak area. RI value of each component was determined relative to the retention times of a homologous n-alkane series with linear interpolation on the HP-5MS column.

Results of the yields (0.09 to 0.36%) of extracted essential oils from five parts of R. tuberosa L. (leaf, stem, root, fruit, and flower), with their physical examinations are presented in Table 1. GC and GC-MS analysis of the oils afforded the identification of 24 compounds in leaf, 15 in stem, 42 in root, 60 in fruit and 6 in flower. Results of the 109 identified compounds are presented in Table 2. Table 3 has results of the comparison of the eleven classes of compounds found in the five R. tuberosa essential oils studied.

Volatile oils from five parts of R. tuberosa L. (leaf, stem, root, fruit, and flower) were obtained by hydro-distillation. Oils were procured in 0.09 to 0.36% yields, each having characteristic distinctive notes: Leaf oil (0.31% yield) had a pleasant herbal to leafy note; stem oil (0.36%) possessed slightly choking but pleasant smell; root oil (0.09%) had woody odour; fruit (0.14 %) with fruity note, while the flower oil (0.25%) had a floral note (Table 1).

Compounds identified in each are listed in Table 2. A total of 109 volatile compounds had been identified in the five essential oils from the Nigerian R. tuberosa. Our results revealed leaf oil contain 24 compounds, which make-up 86.95 % of it; stem oil has 15 compounds (93.96 % of it); root oil with 42 compounds being 91.49% of it; fruit oil contain 60 compounds (89.68% of it); flower oil has 6 compounds responsible for 95.06%. Dominant compounds (%) in each essential oil are: Leaf (E-phytol 21.06, tributylacetyl citrate 19.44, heptacosane 7.55); stem (m-xylene 33.83, heptacosane 16.57, p-xylene 9.67); root (heptane 22.25, heptacosane 12.89, borneol 12.48); fruit (hexacosane 15.43, sextone 13.12, heneicosane 11.14) and flower (tributylacetyl citrate 67.78, 2-methyl-2-pentanol 10.15, 1-methyl-1-cyclopentanol 6.90).

The oils are generally good sources of sextone, β-linalool and alcohols, with monoterpenoids more abundant than sesquiterpenoids. Important classes of compounds in Nigerian R. tuberosa volatiles are monoterpenes, monoterpenoids, sesquiterpenes, sesquiterpenoids, hydrocarbons, aromatics, esters, alcohols, sulphur compounds, ketones and aldehydes. Percentage of each of this is shown in Table 3, for the five R. tuberosa essential oils.

Each of the identified 109 compounds plays important role in the vast ethno-medicinal uses and biological activities demonstrated by R. tuberosa (De Fillpps et al., 2004; Chothani et al., 2010; Agnihotri et al., 2012; Andhiwal et al., 1985; Alam et al., 2009, Samy et al., 2015). High content of linalool in R. tuberose oils present anticonvulsant and hypnotic activities. This was reported for Ocimum basilicum essential oils (Ismail,2006). Limonene, which is in appreciable amount inR. tuberosa root and fruit oils, and citral compound (in leaf oil) have sedative and stimulant effects, they may be responsible for R. tuberosa application as antinociceptive and anticonvulsant. These activities have been observed and reported in the essential oils of Lippia alba and Satureja hortensis (Vale et al., 2002; Viana et al., 2000; Hajhashemi et al., 2002). β-caryophyllene and other sesquiterpenes are abundant in R. tuberose leaf, root and fruit essential oils, known to be responsible for high anticancer properties, hence responsible for anti-cancer effects of R. tuberose (Sylvestre et al., 2005, 2006). Limonene (in root and fruit oils), with perillyl compounds and other monoterpenoids in R. tuberose essential oils are agent with special benefit as anti-tumor (Jahangir and Sultana, 2007; Low-Baselli et al., 2000). 

Our study on the five volatile oils of R. tuberosa L. from Nigeria resulted in the identification of a total of 109 compounds. Monoterpenoids are more abundant than sesquiterpenoids, with the oils rich in esters, alcohols, aromatics and carbonyls. The oils are good sources of sextone, β-linalool and alcohols. We have presented the volatile constituents in R. tuberosa (leaf, stem, root, fruit, and  flower)  which  have  not  been  earlier   reported   in literature

The authors have not declared any conflict of interests.

The authors acknowledge the use of J-laboratory facilities, University of Ibadan, Nigeria in essential oils’ isolations, and TWAS-UNESCO Associateship Scheme (3240278157) awarded to Dr. Aboaba that afforded the GC-MS analyses in H. E. J Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Pakistan.