WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Ohadoma et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 9, 1995-2001 Research Article ISSN 2278 4357

ISOLATION OF THE ACTIVE CONSTITUENT FROM

ETHYLACETATE FRACTION OF LUPINUS ARBOREUS LEAF

Sylvester C. Ohadoma*1 and Felix N. Osuala2

1
Department of Pharmacology, College of Medicine, Imo State University, Owerri, Nigeria.
2
Department of Pharmacognosy, Faculty of Pharmacy, Madonna University, Elele, Nigeria.

ABSTRACT
Article Received on
21 July 2016, Aim: To investigate the pharmacologically active constituents of the
Revised on 11 August 2016, ethyl acetate fraction of Lupinus arboreus leaf for antimicrobial
Accepted on 01 Sep. 2016
DOI: 10.20959/wjpps20169-7661 activity. Methods: The crude methanol extract (CME) of the dried
leaves obtained by 48 hours cold maceration was partitioned to yield n-
hexane fraction (HEF), ethyl acetate fraction (EAF) and methanol
*Corresponding Author
fraction (MEF) and evaluated using modified agar-well diffusion
Dr. Sylvester C.
Ohadoma method. Following the outcome, ethyl acetate fraction was further
Department of fractionated using silica gel column chromatography and the fractions
Pharmacology, College of based on bioactivity-guide, were eluted with gradient mixtures. The
Medicine, Imo State
structure of the isolated active constituent was elucidated using
University, Owerri,
phytochemical and spectral analyses. Results: Chromatography of the
Nigeria.
ethyl acetate fraction over silica gel led to the isolation of a phenolic
acid, identified as ellagic acid. Conclusion: Bioactivity-guided result indicated ellagic acid in
the antimicrobial effect of Lupinus arboreus.
Structure abstract

Fig. 1: L. arboreus leave Fig. 2: Ellagic acid

KEYWORDS: Lupinus arboreus, Chikadoma, antimicrobial activity, ethyl acetate fraction,

Phenolic acid, ellagic acid.

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Ohadoma et al. World Journal of Pharmacy and Pharmaceutical Sciences

INTRODUCTION
The advent of European Scientific methods have led to information on active constituents and
curative actions of medicinal plants. A lot of the folkloric medicinal plants came under
scrutiny, resulting to extraction as well as characterization of their active components.[1]

People of ancient cultures, without knowledge of their active constituents, used medicinal
plants.[2] Plants are known to be sources of secondary metabolites referred to such as natural
products[3], such as terpenoids, alkaloids, tannins, steroids, glycosides, flavonoids and phenol,
accounting for their various uses by humans. The information derived from characterization
of fractions from medicinal plants may make detailed pharmacological studies possible. Also,
it may enable structure-related activity studies, leading to possible synthesis of a more potent
agents with reduced toxicity.[4] The English name of Lupinus arboreus is yellow bush.[5] In
South-eastern Nigeria it is referred to as Chikadoma[6], named after a lead researcher Dr.
Chika Ohadoma, who pioneered extensive work on the novelty study of this plant.[6,4]
Recognised easily as a bushy shrub up to six feet (1.8 m) tall, L. arboreus is planted widely
as ornamental plant with bright yellow sweet-smelling flowers blended with purple white
colours.[7] L. arboreus occurs as an invasive species in Northern California costal dunes.[5,7]
Lupine was employed medicinally without scientifically proven documentation by the
ancients against scabies, scald heads, ulcers, deformities of the skin and other cutaneous
distempers.[8] The leaf extract and fractions of L. arboreus have been reported to have a
plethora of phytochemicals[9], stigmastene 3,6-dione and stigmast steroids[1], ursolic acid and
terponoids[4]; exerts antinociceptive and anti-inflammatory effects[6], as well as antimicrobial
effect[10] in which ethylacetate fraction showed the highest activity. This present study
isolated the active compound of ethylacetate fraction indicated for the antimicrobial effect of
L. arboreus.

MATERIALS AND METHODS

Plant material
The fresh leaves of L. arboreus (Fig.1) were collected from Owerri, Imo State, Nigeria; and
authenticated at the Department of Pharmacognosy, Madonna University, Elele, Nigeria,
where a voucher specimen number M/PC/199/10 has been deposited in the herbarium. The
leaves were air-dried at room temperature for 28 days.

The leaves ground to fine powder (2 kg) were extracted using absolute methanol (Sigma
Aldrich, Germany) for 48 h. After filteration, the crude methanol extract (CME) was

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Ohadoma et al. World Journal of Pharmacy and Pharmaceutical Sciences

concentrated using a rotary evaporator (RV 05 Basic IB, IKA, Staufen, Germany) and further
oven dried and stored in a refrigerator.

Phytochemical screening
Test for phenolics
The fraction (10 ml) was added to ferric chloride solution (0.1 ml). A violet or purple
coloration that disappears on addition of 5 ml of 2- propanol indicates the presence of
phenols.[11,12]

H-NMR spectral analysis

H-NMR (200 MHz) spectra were measured in solution of deuteriochloroform (CDCl3) for the
fraction at ambient temperature using Mercury-200 BB OAUIFE. Chemical shifts were
recorded in parts per million (ppm) relative to tetramethylsilane (TMS) as internal
standard.[13]

Test microorganisms
Standard micro-organisms used were: Bacillus subtilis (NCTC 8326), Pseudomas aeruginosa
(NCTC 6750), Escherichia coli (NCTC 900), Staphylococcus aureus, (NCTC 376).
Klebsiella pneumoniae, Candida albicans, and Aspergillus niger, were standard cultures
obtained from Medical Laboratory Unit of Madonna University Teaching Hospital, Elele,
Nigeria.

Determination of sensitivity test and inhibitory zone diameter (IZD)

The modified agar-well diffusion technique was employed.[12] Each of the test
microorganisms was streaked on the surface of the different sterile sensitivity agar media.
Wells were bored on the agar media using sterile cork borer of 6mm diameter. Exactly 2
drops of the extract prepared as described earlier were accordingly put into the wells and then
allowed to stand for 30 min for proper diffusion. The temperature and time of incubation was
37oC for 24 h.

Column chromatographic separation of CME

Two hundred grams of activated silica gel (70-230 mesh) was packed to two-third the length
of a glass column. One hundred grams of the dry methanol extract was dissolved in methanol:
water mixture (1:2 v/v) and introduced into the column. The column was eluted with 1.5 L
hexane, 1.2 L ethyl acetate, and 1.0 L methanol in succession to yield hexane (HEF), ethyl

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Ohadoma et al. World Journal of Pharmacy and Pharmaceutical Sciences

acetate (EAF) and methanol (MEF) fractions. The fractions HEF, EAF and MEF were
screened for antimicrobial activity.

Isolation and purification of the antimicrobial active constituents

A portion (20 g) of the EAF was separated using silica gel column chromatography. The
column was eluted with gradient mixtures: ethyl acetate; ethyl acetate: methanol; ethyl
acetate: methanol: acetic acid. Aliquot of 20 mL was collected and monitored with
phytochemical reactions, TLC and spectral analysis. Similar fractions were combined to get
fractions F1 F4. The fractions were screened for antimicrobial activity. EAF fraction F1 (1-
64) eluted with ethyl acetate on concentration, yielded the isolation of 25 mg compound. The
compound F1 was purified with PTLC on silica gel plate (0.5 nm) developed with ethyl
acetate as mobile phase. The structure of the isolated compound was elucidated using a
combination of phytochemical analysis and spectral analysis.

RESULTS
The results of the antimicrobial activity of the various solvent fractions of L. arboreus leaves
which showed varying degree of activity; with ethyl acetate fraction exhibiting broad
spectrum antibacterial and antifungal effect which were significantly (p < 0.01) higher than
other fractions. Results of other fractions are not shown here. Chromatography of the ethyl
acetate fractions over silica gel afforded four fractions (F1 F4). The fractions F1 F4
exhibited broad spectrum antimicrobial activity.

However, fractions F1 consistently and significantly (p < 0.01) showed higher antimicrobial
activity compared with the activity exhibited by the other fractions (Table I). The first
fraction (F1) which is of interest in this report, was eluted with gradient mixture of
ethylacetate: methanol: acetic acid (1:0:0) and crystallized as 25 mg solid, 1HNMR (CD3OD,
200 MHz): 7.05 (2H s); D: 1.07, m.p: 42.5oC, b.p: 181oC, soluble in alcohol, water, ether,
chloroform, benzene, glycerol, fixed or volatile oil and alkali. This phenolic acid was
identified as ellagic acid (Fig.2) and agreed with physical data[14]; and did not turn pink or red
under influence of light, suggesting its purity.[15,16]

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Ohadoma et al. World Journal of Pharmacy and Pharmaceutical Sciences

Table 1: Antimicrobial activity [IZD (mm) and IZD2 (mm)2] of column ethyl acetate
fractions of L. arboreus leaves
Column S. B. E. K. P. C. A.
fractions aureus subtilis coli pneumoniae aeruginosa albicans niger
IZD IZD IZD IZD IZD IZD IZD
F1 18** 15** 10** 14** 5 5 4
F2 10 9 8 7 4 2 1
F3 9 7 6 7 3 1 3
F4 9 7 8 5 3 2 2
** P<0.01: Values significantly higher when compared to the other fractions.

DISCUSSION
The active compound of ethyl acetate fraction responsible for the antimicrobial activity was
crystallized as amorphous solid, and tested positive for phenolic acid[11,12]. The results of
other fractions are not emphasized because they have been discussed extensively in previous
work.[10] Structurally, phenolics are classified based on substitution of one or more hydroxyl
groups and in some cases methyl groups as well, in the aromatic ring[17]. NMR spectra (2H s)
supported by signal at 7.05 identified the tetrahydroxy aromatic compound as ellagic acid.
This is in consonance with the report of Kar.[15] Accordingly, bioactivity-guided result
indicated ellagic acid in the antimicrobial effect of this plant. Prominently, it confirmed and
extrapolated as well as offered basis for previous report on the antimicrobial activity of L.
arboreus leaf.[10] This finding was of interest because the broad spectrum antimicrobial
activity justified its use in the management of wound abscesses and other wound infections.
Also, the strong activity of the compound against microorganisms implicated in
gastrointestinal (E. coli), urinary tract (S. aureus) and respiratory (K. pneumoniae) infections
remains notable. Significant activity against C. albicans an organism frequently implicated
in the pathogenesis of oral thrush[18], clearly justify the use of extracts of L. arboreus leaves
in the management of oral thrush. Though ellagic acid is well known for such activity, this is
the first time it is being isolated from this plant.

CONCLUSION
This study has isolated, characterized and identified ellagic acid, - a phenolic acid as an
active compound indicated for antimicrobial effect of L. arboreus.

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