Chemical Compositions of Lonchocarpus cyanescens Benth . , ( Fabaceae ) — Case Study of Its Volatile Oils , and Two Triterpenoids

Leaf and stem essential oils of Lonchocarpus cyanescens Benth., (Fabaceae) were obtained by hydro-distillation, using all-glass apparatus adapted to British Pharmacopeia specifications with yields of 0.03% and 0.17% respectively. The oils were investigated by GC and GC-MS analyses. Results show seven compounds were responsible for 90.4% of leaf; eleven compounds responsible for 97.6% of stem volatile oils. Phytol (62.5%) and hexadecanoic acid (12.4%) dominate the leaf oil; octadecenoic (24.1%) and hexadecanoic acids (17.2%) are predominant in the stem oil. Phytochemical screening of stem, leaf, and root methanol extracts of Lonchocarpus cyanescens reveal presence of following seven classes of metabolites: saponin, tannin, steroid, terpenoid, cardiac glycosides, phlobatannins and flavonoids. Partitioning of methanol extract from dried leaf of it with ethylacetate gave fraction, which was chromatographed using gradient elution. TLC with high Rf was used in monitoring elution. Two white crystalline solids eluted at 5% and 10% ethylethanoate in hexane, were re-crystallized and characterized. They were subjected to Infra-Red spectra, mass spectrometry (ESI technique), H and C-NMR analyses, which confirmed they are triterpenoids. Above mentioned metabolites may be responsible for literature acclaimed bio-activities L. cyanescens display [anti-inflammatory, anti-arthritic, anti-microbial effects]. This paper presents details of above results which are new in literature.

The plant is utilized in traditional medicine.In Senegal, the leaves are used as a condiment eaten with couscous.L. cyanescens leaves and roots are applied as a poultice to treat skin diseases and ulcers; in Sierra Leone and Guinea Bissau, leaves and roots have been considered as a possible cure for leprosy; in Ghana, it is believed that the roots are more effective.Leaves are used as laxative.In Benin, leaf sap is drunk against intestinal disorders and dysentery.A decoction of leafy twigs and roots is given to women during or after childbirth and is also taken as an aphrodisiac.In Nigeria, this decoction is used to treat arthritic conditions, venereal diseases and diarrhea.Ground root is applied to yaws and washing with water containing powdered root helps to cure sores [5,  L. cyanescens leaves have indoxyl which yields indigotin contained in the indigo dyestuff.Oleanane derivatives and glycyrrhetinic acid are responsible for the antiinflammatory properties and relief of peptic ulcers observed in L. cyanescens [2,[12][13][14].The triterpenes act against arthritis [2,11,12,15,16].These natural products are also responsible for the taste effects utilized in flavoring [17].
Bioactivity effects of Lonchocarpus cyanescens have been demonstrated in its anti-inflammatory, anti-arthritic and its relief on ulcer.It has some additional pharmacological properties including antiviral, antifungal, anti-protozoal, and antibacterial activities.The anti-inflammatory activity of L. cyanescens has been confirmed using animal tests and is attributed to the presence of oleanane derivatives and glycyrrhetinic acid [pentacyclic triterpenoids of beta-amyrin type, easily obtained from hydrolysis of glycyrrhizic acid].It also has antitussive properties, effective as expectorant, treatment of ulcer, and flavoring for masking bitter taste of drugs like quinine.The acid in ulcer treatment, inhibits the enzymes that metabolize the prostaglandins, PGE 2a to their inactive metabolites.This increases the level of prostaglandins in digestive system.These prostaglandins inhibit gastric secretion but stimulate pancreatic secretion and mucous secretion in the intestines.This may be the reason why glycyrrhetinic acid is said to relieve peptic ulcers [2,12,15,17].A triterpene component of the plant has been found active against arthritis [2,12,13].The plant is an active anti-arthritic remedy utilized with 3 other plants in Igbo tribal ethnomedicine [11].
Adewuyi et al. [18] reported in their studies on molecular speciation of the triacylglycerol, that seeds oils of L. cyanescens and L. sericeus were rich in K, Na and Fe.Their investigation revealed that linolenic and oleic acids are its dominant fatty acids with neutral lipids.GC-MS analyses of the seed oil showed phytol, sterols, beta-tocopherol and hydrocarbons are in the unsaponifiable matters of the oils; while HPLC showed presence of glycolipids [monogalactosyldiacylglycerol, digalactosyldiacylglycerol, digalactosylmonoacylglycerol and monogalactosylmonoacylglycerol].
We have identified more promising families of Nigerian plants from our collations of standardized questionnaires on anti-malaria herbs utilized in South-western Nigeria ethno-medicine comprising of Ogun, Oyo and Lagos states.Results of our recent studies revealed forty plants were utilized as anti-malaria herb [1].We report our studies on volatile and phytochemical metabolites of one of them, a Fabaceae-Lonchocarpus cyanescens leaf, stem and root.

Plant Material
The whole shrub of Lonchocarpus cyanescens were collected from Ago-Iwoye, Ogun-State, Nigeria for extraction of its volatile oils.Plant was authenticated at the Herbarium, Department of Botany, University of Ibadan, Ibadan, where voucher samples were deposited and signed certificate of identification was obtained [Prof Ayodele and Mr Donatus O. Esimekhuai].Dr Soladoye M. O. (Plant taxonomists, OOU, Ago-Iwoye) also confirmed the plant.More plant samples for extractions were obtained from a farmland in the University of Ibadan Campus, Nigeria and identified by Dr. L. A. Adebisi of Department of Forest Resources Management, University of Ibadan.

Extractions and Phytochemical Screening of Classes of Metabolites
For essential oils' extractions, the plant was separated into leaf and stem parts.500 g each of leaf and stem were crushed and hydro-distilled for 2.5 h in an all glass Clevenger-type apparatus designed to British Pharmacopeia specifications, with very small quantity of distilled nhexane (0.5 ml), which was removed afterwards and the oils refrigerated.The essential oils were procured in 0.03% and 0.17% yields respectively.Each of the oils had distinct characteristic pleasant smell.Leaf, stem and root cold methanol extracts of Lonchocarpus cyanescens were fractionated using distilled hexane to get methanol and hexane fractions.The leaf residue and stem interface were also obtained as fractions.Fractions were subjected to phytochemical analysis.Standard methods such as N. Lata and V. Dubey (2010) [19] were used in establishing classes of secondary metabolites present in each fraction.

More Extractions and Isolations
More leaf of L. cyanescens (1.5 kg) was dried and exhaustively extracted using cold methanol.The extract was concentrated to give dark green solid (10.8 g).Methanol extract (10 g) was further partitioned between aqueous methanol and ethylacetate to give the ethylacetate fraction.The ethylacetate fraction (2 g) was chromatographed on silica gel column eluting with increasing gradient of n-hexane: ethyl acetate and ethyl acetate: methanol to give seventy 100 ml fractions.Similar fractions as determined by TLC were pooled together giving 10 combined fractions.Fractions 3 and 4 eluted with 5% and 10% ethyl acetate in hexane were subjected to reversed phase HPLC separation using C-18 column and afforded white crystals purified by recrystallization in hexane to give 1 (12 mg, t R = 4.2 min) and 2 (20 mg, t R = 3.2 min).mode and threshold 3000; scan MS ACQ mode; detector FTD; mass range of m/z 40 -400.
Identification of the essential oil components were based on their retention indices (determined with reference to homologous series of n-alkanes), along with comparison of their mass spectral fragmentation patterns by computer matching against in-built data and commercials such as Joulain and Koenig (1998), Adams (1995), and Massada (1976) Libraries, as well as in-house "Başer Library of Essential Oil Constituents" built up by genuine compounds and components of known oils [20][21][22].

General Experimental Procedure
Melting points were measured on a Gallenkamp melting point apparatus.Infra-Red spectra were measured in KBr on a Nicolet Avalar 320 FT-IR spectrometer.Reversed phase HPLC separation was carried out using C-18 column on an Agilent model 1100, 25 × 250 mm, 5 mm particle size with a flow rate of 4.0 ml·min −1 and monitored at 210 -290 nm.Mass spectrometry was obtained by ESI technique and NMR spectra ( 1 H, 13 C) were determined on a Bruker WP 200SY spectrometer (500 MHz for 1 H and 125 MHz for 13 C) in dimethylsulphoxide and deuteromethanol.Thin layer chromatographic separations were carried out on Merck precoated silica gel sheets (60 F 254 ) 0.25 mm thickness and spots were viewed by spraying with conc.H 2 SO 4 /Vanillin mixture with heating.Glass column of varying sizes (depending on weight of extract) and silica gel (70 -230 mesh) were used for open column chromatography.

Gas Chromatography-Mass Spectrometric [GC-MS] Investigations of the Leaf and Stem Essential Oils
Volatile oils from leaf (L) and stem bark (B) were obtained by hydro-distillation, using an all-glass apparatus adapted to British Pharmacopeia specifications and gave good yields of 0.03% and 0.17% respectively.The oils were analyzed using GC-MS (see Figures 1 and 2 for chromatograms).Seven prominent compounds in leaf make up 90.4% of it, while eleven most abundant compounds in stem are responsible for 97.6% of the oil.Phytol (62.5%) and hexadecanoic acid (12.4%) dominate the leaf oil (Table 1); while octadecenoic (24.1%) and hexadecanoic acids are predominant in the stem oil (Table 2).Alcohols (64.1%) dominate the leaf oil, while acids (49.6%) are predominant in stem.The two oils contain hydrocarbons [L 6.2%; B 10.0%], acids [L 12.4%; B Leaf and stem essential oils were subjected to GC analyses on GC-2010[AOC-20i] gas chromatograph.Column oven temperature was 60˚C, injection temperature of 250˚C, split injection mode, at 100.2 kPa; column flow of 1.61 ml/min; 1.0 split ratio; oven temperature programming was 60˚C (for 5 min), and at the rate of 5˚/min till 140˚C, 15˚/min till 280˚C.
The GC-MS analyses were performed on GC-MS QP 2010 Plus.Ion source and interface temperature 250˚C; solvent cut time 2.5 min; with relative detector gain      running of the GC and GC-MS, which we paid for; 3) Mac-Akhatur Foundation for Dr I. A. Oladosu.
We acknowledge the assistance of 1) DOM students [Osikoya OF; Nwabueze CC & Quadri HB] in collections, isolations and general laboratory work on plant samples; 2) Dr Kunle of NIPRD, Abuja for supervising1, R = H 2, R = CH 3 Chemical Compositions of Lonchocarpus cyanescens Benth., (Fabaceae)-Case Study of Its Volatile Oils,

Table 2 ] . Table 1 . Chemical composition of the essential oil of Lonchocarpus cyanescens leaf. Peak No a MS [Base peak + most abundant peaks] b Identified compound c %TIC d Retention time [min] e Calculated RI f
a According to the rention time from GC; b [m/e] values of base peak 1st stated, and other most prominent ions; c see identification of components; d Total ion concentration in %; e Retention time in minutes; f Retention Index determined with reference to homologous series of n-alkanes; g unidentified component.

Table 2 . Chemical composition of the essential oil of Lonchocarpus cyanescens stem. Peak No a MS [Base peak + most abundant peaks] b Identified compound c %TIC d Retention time [min] e Calculated RI f
Total ion oncentration in %; e Retention time in minutes; f Retention Index determined with reference to homologous series of n-alkanes; g unidentified component.c a According to the rention time from GC; b [m/e] values of base peak 1st stated, and other most prominent ions; c see identification of components; d

Table 4
). Phytochemical analyses of the leaf ethylacetate extract yielded two triterpenoids [1 and 2] (see Table5).We suggest these reported metabolites are responsible for the acclaimed bio-activities L. cyanescens is known to have [anti-inflammatory, anti-arthritic, anti-microbial, anti-tussive and anti-ulcer effects].This is reported for the first time.Work is ongoing on other aspects of L. cyanescens.massspectrometry(ESItechnique), 1 H-NMR and 13 C-NMR analyses, which showed they are triterpenoids (Table5).Results of 1 H-NMR and13C-NMR indicate the compounds 1 and 2 have the following structures