Mariscus alternifolius plant was collected in June 2014 from the Botanical garden of the University of Ibadan and identified by Dr Ayodele of the Botany Department, University of Ibadan.

The following equipment and apparatus were used: heating mantle, Clavenger apparatus, electronic weighing balance (OHAUS), Oven (Carbolite), round bottom flask, syringe, Gas chromatograph/GC-MS Agilent 7890B, CE 2021, 2000 series double beam UV-Vis spectrophotometer.

The Clavenger apparatus and a heating mantle set at a thermo-stated temperature of 100^oC were the major apparatus used for extracting the essential oil in accordance with the European Pharmacopoeia, 1996 in a technique known as the hydro distillation method. Freshly collected plant (874 g) of M. alternifolius was air dried for 5 days. This yielded 367 g which was transferred into a 10 litres distillation flask and distilled water was added until the sample was completely submerged. The flask was then placed on a heating mantle and fitted with a glass Clavenger. At a thermo-stated temperature of 100^oC, hydro-distillation was carried out for a period of 3 hours under close monitoring to ensure continuous flow of cold water through the condenser and to ensure that the distillate (essential oil) was not lost due to its volatile nature. After 3 hours, the essential oil which was made to dissolve in a known quantity of n-Hexane, was collected from the receiving arm of the Clavenger into a vial bottle of known weight and stored at 4^oC [14Onocha, P.A.; Oloyede, G.K.; Bamkole, O.A. Volatile Oil Composition, Cytotoxicity and free radical scavenging of leaves and twigs of the Chenille plant (Acalypha hispida). Int. J. Essent. Oil Ther., 2010, 4, 86-89.].

The free radical scavenging activity of the essential oil of M. alternifolius Vahl, was studied using the DPPH free radical scavenging method [11Saleh, M.A.; Clark, S.; Woodard, B.; Deolu-Sobogun, S.A. Antioxidant and free radical scavenging activities of essential oils. Ethn. Dis., 2010, 20(1)(Suppl. 1), S1-S78, 82.
[PMID: 20521390] , 18Oloyede, G.K.; Onocha, P.A.; Soyinka, J.; Oguntokun, O.; Thonda, E. Phytochemical screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Ann. Biol. Res., 2010, 1(2), 114-120.]. Dilutions (5 mg/ml, 25 mg/ml and 100 mg/ml) of 1.5 ml of the essential oil was mixed with a 0.5 mM methanol DPPH solution (2.0 ml) prepared by dissolving 240 mg of DPPH in 1 L of methanol. This was incubated in the dark for 15 min at room temperature and the absorbance at 517 nm was recorded using CE 2021, 2000 series double beam UV-Vis spectrophotometer. In its radical form, DPPH absorbs, but upon reduction by antioxidant specie, its absorption reduces and is decolorized. A blank experiment was carried out by applying the same procedure but without the test material (DPPH and Methanol) and the absorbance values were recorded. The standard ascorbic acid was also examined for its antioxidant activity using the same method. The free radical scavenging activity of the test oil solutions was then calculated [11Saleh, M.A.; Clark, S.; Woodard, B.; Deolu-Sobogun, S.A. Antioxidant and free radical scavenging activities of essential oils. Ethn. Dis., 2010, 20(1)(Suppl. 1), S1-S78, 82.
[PMID: 20521390] , 18Oloyede, G.K.; Onocha, P.A.; Soyinka, J.; Oguntokun, O.; Thonda, E. Phytochemical screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Ann. Biol. Res., 2010, 1(2), 114-120.].

M. alternifolius Vahl has been reported to be used locally as an antimicrobial agent. Agar well diffusion method was used to establish this fact [14Onocha, P.A.; Oloyede, G.K.; Bamkole, O.A. Volatile Oil Composition, Cytotoxicity and free radical scavenging of leaves and twigs of the Chenille plant (Acalypha hispida). Int. J. Essent. Oil Ther., 2010, 4, 86-89., 18Oloyede, G.K.; Onocha, P.A.; Soyinka, J.; Oguntokun, O.; Thonda, E. Phytochemical screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Ann. Biol. Res., 2010, 1(2), 114-120.]. The essential oil (0.50 ml) was dissolved into 5 ml of hexane to give 100 mg/ml. Hexane (2.5 ml) was poured into five other test tubes to get the stock solution which was serially diluted until concentration of 6.25 mg/ml was achieved in the fifth test tube. Two other test tubes representing the sixth and seventh test tubes contained hexane and gentamicin/tioconazole as the negative and positive controls for bacteria/fungi, respectively. An overnight culture (0.2 ml) of test micro-organisms was added to 20 ml of cooled molten agar and maintained for 8 hours on nutrient agar slants at 12°C, sub cultured in nutrient broth and preserved at 4-5°C prior to each antimicrobial assay. Pour plate method for bacteria and surface plate method for fungi were employed in the antimicrobial screening [14Onocha, P.A.; Oloyede, G.K.; Bamkole, O.A. Volatile Oil Composition, Cytotoxicity and free radical scavenging of leaves and twigs of the Chenille plant (Acalypha hispida). Int. J. Essent. Oil Ther., 2010, 4, 86-89., 18Oloyede, G.K.; Onocha, P.A.; Soyinka, J.; Oguntokun, O.; Thonda, E. Phytochemical screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Ann. Biol. Res., 2010, 1(2), 114-120.].

In the Agar diffusion pour plate method for anti-bacterial screening, molten sterile agar prepared according to the manufacturer's standard was poured (pour plate) asceptically into sterile petri dishes in triplicate and allowed to set on the bench for 45 minutes. Inoculation of the test organisms from the broth culture into the agar prepared plate was achieved with the aid of flamed and cooled wire loops. The inoculated plates were allowed to solidify. Thereafter, 9mm wells were asceptically bored into the solid nutrient agar using a sterile cork borer. The test solutions of the oil at various concentrations (6.25 - 100 mg/ml) were introduced into each of the designated wells with the aid of sterile syringes in each plate ensuring that no spillage occurred. The same amount of the solvent (hexane) and standard antibacterial agent (gentamicin) were introduced into the 6th and 7th wells to act as negative and positive controls, respectively. The plates were left at room temperature for two hours, to allow diffusion into the medium, turned upside down and thereafter, incubated at 37°C. After 24 hours, the plates were removed from the incubator and diameter of clear zones of inhibition of test organisms and control were measured in millimeters (mm) using a transparent well calibrated ruler (Table 2).

Table 2
Antimicrobial activity of Essential oil of Mariscus alternifolius*.

In the Agar diffusion surface plate method for antifungal screening, sterile sabourand dextrose agar (SDA) prepared according to the manufacturer's standard was poured asceptically into sterile petri dishes in triplicate and allowed to cool down for 45 minutes. 0.2 ml of the fungal strain from the broth culture was spread on the surface (surface plate or spread drop) of the prepared SDA plate using a sterile spreader and allowed to dry and solidify. Subsequently, the same procedure described for antibacterial assay above was carried out. All the plates for the antifungal assay were incubated at 27°C. After 72 hours, the plated were again removed from the incubator and clear zones of inhibition were measured and recorded using the same method as described in the case of antibacterial assay (Table 2).

Minimum inhibitory concentration was determined for all the tested organisms.

Dilution method [19Vanden-Berghe, D.A.; Vlietinck, A.J. Methods in plant biochemistry. Assay for bioactivity. In: Screening methods for antibacterial and antiviral agents from higher plants; Dey, P.M.; Harbone, J.B.; Hostettman, K., Eds.; Academic press: London, 1991; vol. 6, pp. 47-69.] was used for the determination of the minimum inhibitory concentration (MIC) of the essential oil of M. alternifolius. Tubes of nutrient broth containing various concentrations of volatile fractions mentioned in Table 3 were inoculated with microbial strains from the broth cultures. They were incubated in a shaker (120rpm) at 37°C for 24 hours and 27°C for 72 hours for bacteria and fungi, respectively. Control tubes without tested samples were assayed simultaneously. All samples were tested in triplicate. The MIC is defined as the lowest concentration of the test samples at which the organisms do not demonstrate visible growth.

Table 3
Minimum Inhibitory Concentration (MIC) of Essential oil of Mariscus alternifolius*.

M. alternifolius Vahl essential oil contained 10 constituents totalling 71.91% (Table 1). The percentage yield of the essential oil was 0.47%. Tricosane (19.45%) was the most abundant primary active constituent. Other primary active constituents with relatively high percentages include (Z)-14-nonacosene (13.37%) and Octacosyltriflouroacetate (10.91%). Some derivatives of these primary active constituents and a number of the minor constituents of the essential oil of M. alternifolius have been reported to possess antimicrobial, antioxidant and other properties. The most abundant constituent tricosane, is one of the major constituents of the essential oil of Rosa damascena which has been shown to have strong radical scavenging effect [20Yassa, N.; Masoomi, F.; Rohani Rankouhi, S.E.; Hadjiakhoondi, A. Chemical composition and antioxidant activity of the extract and essential oil of Rosa damascena from Iran, Population of Guilan. DARU. J. Pharm. Sci., 2009, 17, 175-180.]. Nonacosane, a saturated derivative of (Z)-14-nonacosene, another major active constituent of the essential oil has been reported to possess antibacterial activity [21kalaisezhiyen, C.P.; Sasikumar, V. GC- MS Evalution of chemical constituents from methanolic leaf extract of Kedrostis foetidissima (Jacq.). Asian J. Pharm. Clin. Res., 2012, 5(4), 77-81.]. Nonacosane is also one of the major constituents of the seed hexane extract of Hypericum scabrum L [22Shafaghat, A. Antioxidant, antimicrobial activities and fatty acid components of flower, leaf, stem and seed of Hypericum scabrum. Nat. Prod. Commun., 2011, 6(11), 1739-1742.
[PMID: 22224301] ]. and essential oil from the leaves of Moringa oleifera [23Marrufo, T.; Nazzaro, F.; Mancini, E.; Fratianni, F.; Coppola, R.; De Martino, L.; Agostinho, A.B.; De Feo, V. Chemical composition and biological activity of the essential oil from leaves of Moringa oleifera Lam. cultivated in Mozambique. Molecules, 2013, 18(9), 10989-11000.
[http://dx.doi.org/10.3390/molecules180910989] [PMID: 24022760] ] which have been shown to exhibit high radical scavenging activity. Octacosyltriflouroacetate, one of the primary active constituents of the essential oil of M. alternifolius is the trifluoroacetate derivative of n-octacosane. N-octacosane has been confirmed to have significant antioxidant activity [24Hassan, R.A.; Tawfeek, W.A.; Habeeb, A.A.; Mohamed, M.S.; Abdelshafeek, K.A. Investigation of some chemical constituents and antioxidant activity of Asparagus sperngeri. Int. J. Pharm. Pharm. Sci., 2014, 6(11), 46-51., 25Ali, A.; Waly, M.; Bhatt, N.; Al-Saady, N.A. Proximate and Phytochemical composition and antioxidant properties of Indigenous Land races of Omanifenu greek seeds. Afr. J. Tradit. Complement. Altern. Medicines, 2015, 12(2), 149-154.
[http://dx.doi.org/10.4314/ajtcam.v12i2.22] ]. Another derivative cyclooctacosane on the other hand, has been reported to exhibit significant antibacterial activity [26Fan, P.; Manoli, D.S.; Ahmed, O.M.; Chen, Y.; Agarwal, N.; Kwong, S.; Cai, A.G.; Neitz, J.; Renslo, A.; Baker, B.S.; Shah, N.M. Genetic and neural mechanisms that inhibit Drosophila from mating with other species. Cell, 2013, 154(1), 89-102.
[http://dx.doi.org/10.1016/j.cell.2013.06.008] [PMID: 23810192] ]. 2-Decanone, one of the constituents of the study plant has been shown to possess antifungal activities [27Arif, T.; Bhosale, J.D.; Kumar, N.; Mandal, T.K.; Bendre, R.S.; Lavekar, G.S.; Dabur, R. Natural productsantifungal agents derived from plants. J. Asian Nat. Prod. Res., 2009, 11(7), 621-638.
[http://dx.doi.org/10.1080/10286020902942350] [PMID: 20183299] ] while hexadecane, a derivative of the constituent 2-methyl-1-hexadecanol has been reported to be responsible for the anti-bacterial and antioxidant properties of the essential oil of Cestrum noctrunum [28Al-Reza, S.M.; Rahman, A.; Kang, S.C. Chemical composition and inhibitory effect of essential oil and organic extracts of Cestrum nocturnum L. on food-borne pathogens. Int. J. Food Sci. Technol., 2009, 44, 1176-1182.
[http://dx.doi.org/10.1111/j.1365-2621.2009.01939.x] ] and Monochaetia kansensis [29Yogeswari, S.; Ramalakshmi, S.; Neelavathy, R.; Muthumary, J. Identification and Comparative Studies of Different Volatile Fractions from Monochaetia kansensis by GCMS. Global J. Pharmacol., 2012, 6(2), 65-71.]. Eicosane, a derivative of the constituent 1-Eicosene is reported to be one of the main constituents in the Aloe vera extract responsible for high antimicrobial activity against clinical pathogens [30Arunkumar, S.; Muthuselvam, M. Analysis. of Phytochemical Constituents and Antimicrobial Activities of Aloe vera L. against Clinical Pathogens. World J. Agric. Sci., 2009, 5(5), 572-576.] while 9-Eicosene has been shown to have potent antimicrobial property [31Hsouna, A.B.; Trigui, M.; Mansour, R.B.; Jarraya, R.M.; Damak, M.; Jaoua, S. Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat. Int. J. Food Microbiol., 2011, 148(1), 66-72.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2011.04.028] [PMID: 21601302] ]. The presence of these constituents in the essential oil justifies the ethno medicinal uses of the plant.

Some of the constituents have also been shown to possess other medicinal properties beyond the generic anti-microbial and free radical scavenging activities. Tricosane is known to provoke behavioral response in several insect predators and parasitoids while octacosane and (Z)-14-nonacosene (a component of the sex pheromones) play an important role in the control of insects [32Uebel, E.C.; Schwarz, M.; Sonnet, P.E.; Miller, R.W.; Menzer, R.e. Evaluation of the mating Stimulant Pheromones of Fannia canicularis, F. pusio and F. femoralis as attractants. Fla. Entomol., 1978, 61(3), 139-143.
[http://dx.doi.org/10.2307/3494228] ]. Nonacosane has been reported to possess nematicidal activity [21kalaisezhiyen, C.P.; Sasikumar, V. GC- MS Evalution of chemical constituents from methanolic leaf extract of Kedrostis foetidissima (Jacq.). Asian J. Pharm. Clin. Res., 2012, 5(4), 77-81.], antihypertensive, vasodilatory, angiotensin AT2 receptor antagonist and saluretic activities [33Mallikadevi, T.; Paulsamy, S.; Karthika, K.; Jamuna, S. In vitro and in vivo Anti-inflammatory activity of whole plant methanolic extract of Mukia maderaspatana (L.) M.Roem. (cucurbitaceae). Int. J. Pharm. Pharm. Sci., 2012, 4(5), 435-439.]. Hentriacontane, a constituent of M. alternifolius has also been shown to possess anti-cancer, anti-inflammatory [34Kim, S.J.; Chung, W.S.; Kim, S.S.; Ko, S.G.; Um, J.Y. Antiinflammatory effect of Oldenlandia diffusa and its constituent, hentriacontane, through suppression of caspase-1 activation in mouse peritoneal macrophages. Phytother. Res., 2011, 25(10), 1537-1546.
[http://dx.doi.org/10.1002/ptr.3443] [PMID: 21394806] ], anti-tuberculosis [35Liu, Y.W. Antituberculous constituents of Gentiana rhodantha. Zhong Yao Tong Bao, 1985, 10(12), 33-34.
[PMID: 2940022] ] and weak cytotoxic activities [36Quintanilla-Licea, R.; Morado-Castillo, R.; Gomez-Flores, R.; Laatsch, H.; Verde-Star, M.J.; Hernández-Martínez, H.; Tamez-Guerra, P.; Tamez-Guerra, R.; Rodríguez-Padilla, C. Bioassay-guided isolation and identification of cytotoxic compounds from Gymnosperma glutinosum leaves. Molecules, 2012, 17(9), 11229-11241.
[http://dx.doi.org/10.3390/molecules170911229] [PMID: 22996346] ].

M. alternifolius Vahl displayed a higher free radical scavenging activity at 5 mg/ml than ascorbic acid when absorbance measurement was done at 517 nm. Fig. (1) shows the % inhibition of the sample and standard as calculated from the absorbance results obtained [18Oloyede, G.K.; Onocha, P.A.; Soyinka, J.; Oguntokun, O.; Thonda, E. Phytochemical screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Ann. Biol. Res., 2010, 1(2), 114-120.]. At 5 mg/ml, 97.95% inhibition was observed for the plant essential oil. This was slightly higher than that of ascorbic acid (97.88%). However, at higher concentrations of 25 mg/ml and 100 mg/ml tested, the plant oil exhibited % inhibition of 97.85 and 97.92 respectively. These slightly lower values contradict the usual trend of high % inhibition with high concentration. Percentage inhibition of Ascorbic acid on the other hand, increased with increasing concentration. Nevertheless, the fact that the plant oil exhibited a higher % inhibition than the standard ascorbic acid used at a low concentration (5 mg/ml) is a clear indication of its high free radical scavenging activity.

Fig. (1) DPPH Scavenging Activity of Mariscus alternifolius Plant (MAP) Oil and Ascorbic acid (ASC).

The essential oil of M. alternifolius Vahl as revealed in this study possesses antioxidant activity. Antioxidants are compounds that can neutralize free radicals by accepting or donating an electron to eliminate the unpaired condition [37Sies, H. Oxidative stress: oxidants and antioxidants. Exp. Physiol., 1997, 82(2), 291-295.
[http://dx.doi.org/10.1113/expphysiol.1997.sp004024] [PMID: 9129943] -39Oloyede, G.K.; Farombi, E.O. Antioxidant properties of Crinum ornatum bulb extracts. World J. Chem., 2010, 5(1), 32-36.]. They have been reported to have curative effect on Parkinson’s disease, cancer, coronary and other heart related conditions. They also find application in the industry as preservatives in food and cosmetics [11Saleh, M.A.; Clark, S.; Woodard, B.; Deolu-Sobogun, S.A. Antioxidant and free radical scavenging activities of essential oils. Ethn. Dis., 2010, 20(1)(Suppl. 1), S1-S78, 82.
[PMID: 20521390] , 12Warnke, P.H.; Becker, S.T.; Podschun, R.; Sivananthan, S.; Springer, I.N.; Russo, P.A.; Wiltfang, J.; Fickenscher, H.; Sherry, E. The battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired infections. J. Craniomaxillofac. Surg., 2009, 37(7), 392-397.
[http://dx.doi.org/10.1016/j.jcms.2009.03.017] [PMID: 19473851] , 40Dabelstein, W.; Reglitzky, A.; Schütze, A.; Reders, K. Automotive Fuels. In: Ullmann's Encyclopedia of Industrial Chemistry; Wiley-VCH: Weinheim, 2007.
[http://dx.doi.org/10.1002/14356007.a16_719.pub2] ].

Zones of inhibition of the essential oil of Mariscus alternifolius measured during the antimicrobial screening are presented in Table 2.

The result of the antimicrobial screening of the essential oil of M. alternifolius revealed its inhibitory effect on the growth of all the bacteria: S. aureus, E. coli, B. substilis, P. aeruginosa, S. typhi, K. pneumoniae and on two of the fungi: C. albicans and A. niger used in this assay. No inhibitory effect was observed for P. notatum and R. stolonifer. The minimum inhibitory concentration (MIC) (Table 3) varied with organism: 25 mg/ml for S. aureus and E. coli, 50 mg/ml for B. substilis, S. typhi and C. albicans and 100 mg/ml for P. aeruginosa, K. pneumoniae and A. niger and are comparable to essential oils of other origins [28Al-Reza, S.M.; Rahman, A.; Kang, S.C. Chemical composition and inhibitory effect of essential oil and organic extracts of Cestrum nocturnum L. on food-borne pathogens. Int. J. Food Sci. Technol., 2009, 44, 1176-1182.
[http://dx.doi.org/10.1111/j.1365-2621.2009.01939.x] , 41Akudor, G.C.; Onyewenjo, S.C.; Anyalewechi, N.A.; Essien, A.D.; Akpan, J.L.; Okoroafor, D.O.; Okere, M.O. In vitro antimicrobial activity of leaf extract of Berlina grandiflora Hutch. and Dalz. Afr. J. Microbiol. Res., 2011, 5(11), 1358-1360.
[http://dx.doi.org/10.5897/AJMR10.692] , 42Boussaada, O.; Ammar, S.; Saidana, D.; Chriaa, J.; Chraif, I.; Daami, M.; Helal, A.N.; Mighri, Z. Chemical composition and antimicrobial activity of volatile components from capitula and aerial parts of Rhaponticum acaule DC growing wild in Tunisia. Microbiol. Res., 2008, 163(1), 87-95.
[http://dx.doi.org/10.1016/j.micres.2007.02.010] [PMID: 17482441] ]. These results indicate that the essential oil of the plant M. alternifolius possesses antibacterial activity.