Determination of bioactive constituents of Rauwolfia vomitoria Afzel (Asofeyeje) roots using gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared spectrometry (FT-IR)

Rauwolfia vomitoria Aftzel belong to the family of Apocynacea with a common name serpent wood and Igbo name as akanta. It is used traditionally to treat hypertension and rheumatoid arthritis. The present study deals with GC-MS determination and FT-IR analysis of methanolic root extract of the named plant. Exactly thirty phytochemical constituents have been identified by comparing the chromatogram peek values of unknown compounds with entries in NIST database. The major bioactive compounds were 2Formyl-9-[-d-ribofuranosyl]hypoxanthine (8.63%), 5-Cyclopropylcarbonyloxypenta-decane (5.58%), 1,2,5-Oxadiazol-3-carboxamide-4,4’-azobis-2,2’-dioxide (4.47%), 1-(5-Bicylco[2.2.1]heptyl)ethylamine (3.66%), 1-Adamantanemethylamine,-methyl(2.22%), Diphenylephrine (1.26%), Imidazole,2-amino-5[(2-carboxy)vinyl](0.85%), Spiro[androst-5-ene-17,1’-Cyclobutan]-2’-one,3-hydroxy-,(3,17)(0.82, and Cyclohexan-1,4,55-triol-3-one-1-carboxylic acid (0.65%). The FTIR spectrum confirmed the presence of alkyl halides, alcohols, phenols, secondary alcohols, tertiary alcohols, aromatic ethers, aldehydes, ketones, aliphatic nitro compound, aromatic organophosphorus compounds, aromatic compounds, carboxylic acid derivatives, alkenes, saturated ketones, and alkanes. Hence, this study offers bases of employing R. vomitoria as herbal alternative for the treatment of various diseases.


INTRODUCTION
Developing countries most especially the rural areas are constantly being inundated by infectious diseases and attempts to manage these ailments with conventional drugs have posed mankind with a lot of health consequences.Researches over the years have shown that over dependence on drugs as sole remedies for treating diseases have led to malfunction of important organs of the body such as the liver, the kidney, eyes, *Corresponding author.E-mail: okerekecstan@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License lungs and the brain.Sequel to this, attentions are now being shifted to the use of medicinal plants in the treatment of infectious diseases.The economic realities of the developing countries, where most people hardly can afford good meal, housing, clothing and the expensive pharmaceutical products for health-care, has forced many to resort to alternative complementary medicine.Again, the development of drug-resistant strains of microorganisms and autoimmune problems make it imperative for a continued search for new drugs from natural products (Vaitheeswarn and Edward, 2014).Cowan (1999) reported that plants still represent large untapped sources of structurally novel compounds that might serve as lead for the development of novel drugs.Traditional systems of medicines are prepared from a single plant or combinations of number of plants.The efficacy depends on the use of proper plant part and its biological potency which in turn depends upon the presence of required quantity and nature of secondary metabolite in a raw drug (Savithramma et al., 2010;Vinoth et al., 2011).Herbal medicines are safer than synthetic medicines because the phytochemicals in the plant extract target the biochemical pathway.These bioactive compounds that contribute to the pharmacological efficacy of medicinal plants have recently been identified with relatively expensive and often laborious techniques such as gas chromatography (GC) combined with specific detections schemes (Eisenhauer et al., 2009).The specific detection schemes could be mass spectrometry (MS) or Fourier transform infrared spectrometry (FTIR).Gas chromatography combined with mass spectroscopy (GC-MS) can identify pure compounds present at less than 1 ng biological specimen and quantification purpose (Liebler et al., 1996).The unknown organic compounds in a complex mixture can be determined by interpretation and also by matching the spectra with reference spectra (Hites, 1997).FT-IR spectroscopy has demonstrated to be a reliable and sensitive method for finding out the functional groups present in plant samples using IR region in the range of 400 to 4000 cm -1 . For most common plant compounds, the spectrum of an unknown compound can be identified by comparison to a library of known compounds (Griffiths and Haseth, 1986).
Nigeria is rich in diverse plant resources, therefore screening for active compounds residents in plants with effective protection and treatment of different diseases and infections should be pursued.Rauwolfia vomitoria belongs to the family Apocynacea and its common names include serpent wood, poison's devil pepper and swizzler stick.The plant is a shrub or tree which grows over 10 m tall of the forest and common in secondary growth throughout most of African regions.Sometimes due to the flower and the sweet scented fruits and it is sometime planted as an ornamental shrub.In Nigerian local languages, R. vomitoria is called asofeyeje (Yoruba), akanta (Igbo), wada (Hausa), mmoneba (Efik) and utoenyin (Ibibio) (Mecha et al., 1980;Ekutudo, 2003;Ehiagbonare, 2004).The plant is of different species: the Indian species (Rauvolfia serpentina) and the African species (R. vomitoria) (Kutalek and Prinz, 2007).The roots, root bark, leaves and stem-bark are employed in herbal medicine for the treatment of several ailments.Kutalek and Prinz (2007) reported that the plant is used in ethnomedicine against snake bites, fever and nervous disorders.They further reported that a watery solution of the bark of R. vomitoria can be used against such parasites as lice and scabies.R. vomitoria is employed in the treatment of rheumatoid arthritis (Fapojuwomi and Asinwa, 2013).Sharma (2004) added that the root of R. vomitoria is good for the treatment of snake bites, insect stings, nervous disorders, mania, epilepsy, intractable skin disorders such as psoriasis, excessive sweating, itching, hypertension, sedative, uterine contraction in child birth and gynecological ointment for the treatment of menopausal disorders.Prajapati et al. (2007) observed that R. vomitoria is a sedative, hypnotic, good for reducing blood pressure, good for treating insanity, anthelmintic, an antidote to snake venom, anti-anxiety agent and stimulant to central nervous system.A decoction of the root of the plant could be given during labour pains to increase uterine contraction.Odugbemi (2008) also reported that R. vomitoria (Asofeyeje) is good for the treatment of hypertension, insomnia, nervous disorder, jaundice, fever, diarrhea, dysentery, scabies, mental disorders, anthelmintics and malaria.
Reserpine is an alkaloid derived from R. vomitoria and is the major reason for the medicinal use of the plant in India.Reserpine is derived from the root bark of the plant and was isolated in 1952 as purified alkaloid for the treatment of hypertension.Reserpine irreversibly binds to the storage vesicles of neurotransmitters, particularly norepinephrine, serotonin and dopamine.Eventually, catecholamine depletion occurs because of the body's inability to store these neurotransmitters.Therefore, the study employed gas chromatography combined with mass spectrophotometry and Fourier transform infrared (GC-MS/FT-IR) technique to determine the phytochemicals present and the functional groups they possess, respectively.

Collection and identification of plant
R. vomitoria roots were collected from a bush near Abia State University.The collected specimen was taken to the Forest Herbarium, Ibadan (FHI), Nigeria for proper identification.The specimen already was existing in the FHI.It has the serial No. 72.

Preparation of extract for GC-MS
The roots of R. vomitoria were washed to remove sand and humus, and then allowed to dry under shade.The roots were first chopped into small particles and ground to powder using a grinder.Five Okereke et al.   grams of the plant powder was transferred to a round bottom flask and treated with methanol in a soxhlet apparatus.The temperature was maintained between 45 and 47°C and lasted for 24 h.The extract was collected and evaporated using vacuum distillation unit.
The methanolic extract so obtained was then subjected to GC-MS.

FT-IR spectroscopic analysis
The FT-IR spectra of the samples were determined using a Fourier transform infrared spectrometer (Thermo Scientific Nicolet iS5) iDI Transmission Accessory.Approximately, 2 mg of the solid sample of R. vomitoria was ground with 200 mg of dried potassium bromide (KBr) to form a homogenous powder, which was compressed into a thin pellet by using 15Tons hydraulic press.KBr pellet holder was then recorded for FT-IR measurement in the wave number range from 4000 to 400 cm -1 using 16 scan.

GC-MS analysis
GC-MS analysis on the methanolic extract was carried out using Agilent 7890A-5975C GC-MS system, employing the following conditions.HPS-Column (30×0.25 m), operating in electron impact mode at 70 eV; carrier gas flow (a constant) = 1 ml/min.Injection volume was set at 0.5 L, at split ratio of 10:1.The injection temperature was 250°C and ion source temperature 280°C.Oven temperature for 2 min at the initial 70°C and to 280°C at 15°C/min rate for 5 min.The mass spectra was measured at 70 eV.

Identification of components
Interpretation of mass spectrum obtained from GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 82,000 patterns.The spectrum of the unknown component was compared with the spectra of the known components stored in the NIST library.The name, molecular weight, molecular formula and structure of the components of the test materials were ascertained.

Identification of functional groups
The FTIR spectrum was used to identify the functional groups of the active components present in plant sample based on the peaks values in the region of IR radiation.When the plant extract was passed into FTIR, the functional groups of the components were separated based on its peaks ratio.

RESULTS
The results of GC-MS/FT-IR and biological activities of identified compounds of methanol extract of roots of R. vomitoria are presented in Figures 1 and 2 and Tables 1 to 3. A total of thirty compounds were identified from the methanol root extract of R. vomitoria.The identification of the phytochemical compounds was confirmed based on the peak area, retention time and molecular formula (Figure 1 and Table 1).According to Duke's phytochemical and ethnobotanical databases (1998), the biological activities of some of the identified compounds are presented in Table 2.The results of FT-IR spectroscopic analysis revealed the presence of certain functional groups present in methanol extract of roots of R. vomitoria (Figure 2 and Table 3).The absorption at 3333.26 cm -1 is due to the O-H stretching of strong bond present in the extract.The band at 2897.74 cm -1 is due to C-H 3 , C-H 2 and C-H bend of alkanes; the band at 1731.98 cm -1 showed saturated ketones; the band at 1636.36 cm -1 showed alkene C=C bend; the band at 1593.90 cm -1 showed carboxylic acid derivatives C=O bend; the band at 1504.67 cm -1 showed aromatic C=C bend; the bend at 1458.43 cm -1 showed aromatic organophosphorus compounds P-C bend; the bend at 1422.55 cm -1 showed alcohol and phenol O-H in-plane bend, the band at 1370.30 cm -1 showed aliphatic nitro compound N-O bending; the bend at 1321.59 cm -1 showed A-CH 3 bending, the band at 1236.47 cm -1 showed aromatic ethers C-O bend; the band between 1155.99 and 1105.72 cm -1 showed tertiary and secondary alcohol C-O bends; the band at 1029.02 cm -1 showed fluoroalkanes C-X bend; the band at 897.97 cm -1 showed alkenes =C-H and =CH 2 out of plane bending; the band at 661.80 cm -1 showed alcohol phenol bends and the bands between 595.22 and 558.27 cm -1 showed bromoalkane C-X bend.

DISCUSSION
The techniques of GC-MS/FT-IR have proved effective and more reliable in the determination of chemical compounds present in plants.Earlier reports elucidated the biological activities of Cyclohexan-1,4,5-triol-3-one-1-  (2008) observed that 1-Adamantanemethylamine,-methyl-otherwise known as Rimantadine, inhibits in vitro replication of influenza A virus isolates from each of the three antigenic sub-types (H1N1, H2N2 and H3N3) that have been found in man.From the result of the functional group analysis, it can be inferred that alkyl halides, alcohols, phenols, secondary alcohols, tertiary alcohols, aromatic ethers, aldehydes, ketones, aliphatic nitro compound, aromatic organophosphorus compounds, aromatic compounds, carboxylic acid derivatives, alkenes, saturated ketones and alkanes might be responsible for the various medicinal properties of the root of R. vomitoria.

Conclusion
In the present study, the FTIR and GC-MS spectral

Table 1 .
List of compounds identified at various retention times from methanolic extract of roots of Rauwolfia vomitoria by GC-MS.

Table 2 .
Biological activities of phytochemical compounds identified in methanol extract of roots of Rauwolfia vomitoria.

Table 3 .
FTIR analysis of methanolic extract of roots of Rauwolfia vomitoria.
O-H bending in plane (Alcohols and phenols)