isolation and identification of a hermiterpene ...lamidi et al.[11] has reported the isolation of...

International Journal of Pharmaceutical

Biological and Chemical Sciences e-ISSN: 2278-5191

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS)

| APR-JUN 2015 | VOLUME 4 | ISSUE 2 |54-61 www.ijpbcs.net or www.ijpbcs.com

Research Article

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ISOLATION AND IDENTIFICATION OF A HERMITERPENE GLYCOSIDE

FROM THE FRUIT OF SARCOCEPHALUS LATIFOLIUS (SM) BRUCE 1H. B. Yesufu*,

1A.A. Sani,

1G.T. Mohammed,

2F.I. Abdulrahman ,

2I.Z. Khan

1I. Iliya,

2U. Hamidu.

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy University of Maiduguri, Maiduguri, Nigeria.

2 Department of Chemistry, Faculty of Science, University of Maiduguri, Maiduguri, Nigeria.

*Corresponding Author Email: [email protected]

INTRODUCTION

The research into natural products is preceded by

structural elucidation of the isolated compounds. In

most cases of extraction and isolation of natural

products, the end point is the identification of the

compound or the conclusive structure elucidation of

the isolated compound [1,2,3] If the target compound

is known, it is very easy to compare preliminary

spectroscopic data with literature data or to make

direct comparison with the standard sample. However,

if the target compound is unknown and very complex,

a comprehensive systematic approach involving a

variety of physical, chemical and spectroscopic

techniques is required. Information on the chemistry of

genus or the family of plant under investigation could

sometimes provide additional hints regarding the

possible chemical class of the unknown compound

[4].Sarcocephalus latifolius (sm) Bruce belongs to the

Rubiaceae family which is the largest in the

magnoliopsida class encompassing over 450 genera

and 9000 species used for several ethno-medical

practice [5,6]. In the literature, quite a number of

chemical constituents have been reported from

different part of the plant. Abreu et al. [7] reported the

presence of benzoylated sugars via HPLC. This

include the α and β-D forms of glucose, fructose,

xylose, arabinose and D-erythriol perbenzoate. Isah et

al. [8] reported the isolation of betulinic acid a

‘pentacyclic triterpenoid’ from the stem bark. Pedro

and Antonio [9] reported the presence of a new indole

alkaloid 19-0-ethylangustoline from the stem bark of

the plant. Also, Pedro and Antonio [10] reported

another new indole alkaloid, 21-0- ethyl-strictosamide

from the root of S. latifolius. Lamidi et al.[11] has

reported the isolation of quinovic acid and its

glycosides from the root of S. latifolius. Therefore, it

was in furtherance of the search for more active

principles that the objectives of the study were put

together.

ABSTRACT:

Sarcocephalus latifolius belongs to the family of Rubiaceae. The present work revealed a hermiterpene glycoside “2-

methylbut-(1)-yl-6΄6΄-di-O-α-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-D-glucopyranoside” that was isolated and

characterized from the ethyl-acetate portion of the fruit of S. latifolius, which was established on the basis of chemical and

spectral studies. The Plant material is very prominent for its economic and medicinal benefits. Though, fruits are eaten for its

culinary delight, its medicinal properties have been highlighted in several studies. This study was aimed at unveiling the active

principles responsible for the biological activities reported in the literature of the plant material.

KEYWORDS: Sarcocephalus latifolius (Rubiaceae), fruit, Hermiterpene glycoside, Chemical and Spectral studies.

file:///F:\0000.JOURNALS\002.IJPBCS\001.Volume%204-2015\issue%202\000.%20To%20be%20publish\05.Hassan%20Yesufu-4650476743\[email protected]

1H. B. Yesufu* et al; ISOLATION AND IDENTIFICATION OF A HERMITERPENE....

International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | APR-JUN 2015 |VOLUME 4 | ISSUE 2 | 54-61| www.ijpbcs.net

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MATERIALS AND METHODS

Plant Collection and Identification:

The Fruits of Sarcocephalus latifolius (Temperate

strawberry) used in this study were collected in

September, 2013 from Gaya in Hong LGA, Adamawa

State, Nigeria. The plant specimen was identified by a

plant taxonomist of the Department of Biological

Science, while the voucher specimen No. 544a was

prepared and deposited at the Post-Graduate Research

Laboratory, Department of Chemistry, and University

of Maiduguri.

General Experimental Conditions:

The 1H-nuclear magnetic resonance (NMR) spectra

were recorded in CD3OD on Bruker AM-400 and

AMX-500NMR spectrometers with TMS as an

internal standard using UNIX operating systems at

400– 500MHz, respectively. The 13C-NMR spectra

were recorded in CDCl3 and CD3OD at 125MHz on a

Bruker AMX-500 NMR spectrometer. The Fourier

Transform Infrared (FTIR) spectra were recorded on a

FTIR- 8400S spectrophotometer.

Silica gel columns (120-230 mesh) was used for

column chromatography (CC), pre-coated silica gel

TLC (GF-254, 20 x 20 cm, 0.25mm thick, Merck)

were used to check the purity of the compound and

were observed under ultraviolet (UV) light (250 and

600 nm), while ferric chloride and Iodine vapour were

used as a spraying reagent.

Preparation of Powder and Extract:

The sample was washed with distilled water to remove

the sand particles, air-dried under shade and

pulverized into fine powder. The powdered sample of

S.latifolius (2 kg) was extracted exhaustively in a

sohxlet extractor using 95% ethanol. Then, it was

concentrated in-vacuo at 40oC using a rotary

evaporator. The extract concentrate was weighed and

defatted with Petroleum ether and a dark brown crude

extract(379.2g) was obtained.

Fractionation:

The crude ethanol extract (200.0g) of S. latifolius was

suspended in distilled water (200ml) and partitioned

with chloroform CHCl3 (200ml), ethyl acetate

(EtOAc) (200ml) and normal butanol (n-buOH)

(200ml) to yield SLCF (32.60g), SLEA (60.03.) and

SLNB (20.60g) fractions.

Isolation:

The ethyl acetate fraction of S. latifolius was subjected

to column chromatography and sequentially sub-

fractionated according to standard method [12] with

solvent system of EtOAc / n-buOH [4/1(i), 3/2(ii),

1/1(iii),

2/3(iv), 1/4] mixture with increasing order of polarity.

The fractions of column were continually monitored

using preparative TLC, isolation was further preceded

and the sub-fraction (ii and iii) was selected by

subjecting it to fresh column chromatography using

Sephadex L-H 20. A yellow amorphous solid

compound was formed by eluting the fraction with the

solvent system 100 % methanol which was monitored

by TLC with the solvent n-hexane: ethyl acetate:

methanol : water (2:2:0.5:1). All reagent used were

analar grade.

RESULTS AND DISCUSSIONS

The isolated portion from ethyl acetate fraction of the

S. latifolius was chromatographed over silica gel

(Preparative TLC) to afford a glycoside, which

consisted of a disaccharide sugar (glycone) having a

five carbon saturated moiety. The identification of the

compound ‘’ was established by (FTIR, 1H NMR,

13CNMR) with comparison with corresponding

reported values in the literature [12]. From the isolated

sample (SL1), a deep yellow amorphous solid with Rf

value (0.44) was obtained and melting point of 142 –

143o uncorrected. [13, 2]. The Infra red spectra of the

pure sample (SL1) showed vibrational frequencies for

different organic molecules; OH-stretching

frequencies at 3376.5cm-1, CH3 and CH2- symmetric

and asymmetric stretching frequencies at 2932cm-1,

Saturated C=O stretching frequencies at 1718cm-1 and

1056cm-1 and a tertiary C-H stretching frequencies at

1367cm-1 as shown in Table 1.



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Table 1. Analysis of IR spectra of Compound SL1

Functional Group Wave Number (cm-1)

OH- sym 3376

CH3& CH2 sym 2932

Saturated C=O 1718

OH- bend. 1442

Substituted CH-str 1367

The 13CNMR carried out using a 125 MHz spectrophotometer showed the presence of anomeric carbon at chemical

shift position of 96.815 ppm and 92.601 ppm as shown in Fig 1. This is indicative of reducing disaccharide sugars.

Also, the presence of peaks appearing as doublets at 74.92 ppm, 73.52 ppm, 72.47 ppm, 71.62 ppm. 70.41 ppm and

61.42 ppm supported the fact that the compound contained a glucose moiety [14]. A chemical shift of an outer

terminal sugar carbon (61.42 ppm) to the inner sugar carbon (70.41 ppm), a shift of 9 ppm indicates a linkage of the

monosaccharide unit at 1-6 position [15].

The 1HNMR Spectra of the Compound SL1 glycoside at 500MHz shown in Fig 2 showed anomeric proton at 5.12

ppm and 4.45 ppm while chemical shift positions for a sugar linkage were at 3-4 ppm in the 1HNMR Spectra. The

anomeric proton of SL1 which appeared as doublets at 5.12 ppm and at 4.45 ppm, confirmed the presence of a

disaccharide moiety. In Fig 3, A series of doublet-like signals centered at 3.527 ppm, 3.279 ppm, 3.123 ppm, 3.013

ppm were strong signals for sugar protons. The large overlap of peaks between 3.584 ppm and 3.863 ppm are

indicative of an aldo-hexose (glucose) moiety owing to the integration value of 13 while the overlap of peaks between

3.283 ppm and 3.362 are indicative of an aldo-pentose (xylose) moiety owing to the integration value of 9 [14]. In Fig

4, the presence of an aglycone was shown in 1HNMR Spectrum with chemical shift positions occurring between 0.9

ppm-1.6 ppm. While in Figure 5, an expanded multiplicity of chemical shift protons are shown which were compared

with literature values as shown in Table 3. The Distortionless enhanced by polarization transfer (DEPT) spectra (Fig 5)

was carried out using 100MHz NMR Spectrophotometer. At a magnitude of 135o, the spectra showed the presence of

two (2) methyl carbons, three (3) methylene carbons and clusters of methine carbons which included the anomeric

carbon. In Table 2&3, the observations from the spectra were compared with published literature data.

Table 2. Comparison of SL1 (Proton chemical shift values) with Literature Values.

Chemical Shift Multiplicities (ppm) of

Compound SL1

Chemical Shift Multiplicities (ppm) of

disaccharide compound from Literature [9;10;11]

Anomeric Protons

4.45 5.12 d 1H 4.38 H-1΄

4.86 4.86 d 1H 4.33 H-1΄΄

Sugar Protons

4.12 4.11 dd, 1H 4.10 H-6΄b 3.88 3.86 dd, 1H 3.88 H-5b΄΄

3.79 3.75 dd, 1H 3.76 H-6΄a

3.48 3.46 m, 1H 3.44 H-5΄

3.38 3.37 m, 1H 3.37 H-4΄ 3.51 3.50 ddd, 1H 3.50 H-4΄΄

3.34 3.33 m, 1H 3.34 H-3΄΄

3.32 3.33 m, 1H 3.32 H-3΄ 3.30 3.29 m, 1H 3.25 H-2΄΄

3.22, 3.23 m, 1H 3.24 H-5a΄΄



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3.19, 3.17 m, 1H 3.20 H-2΄

AGLYCONE

SL1(Spectra Data) (Literature Data)

0.90 0.90 CH3-R

1.35 1.33 R2CH2

1.56 1.5-1.6 R3CH

Fig 1.

13CNMR Spectra of SL1 glycoside in CD3OD at 125 MHz

Anomeric

Carbon

β

pp

m α

pp

m



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Fig.2. 1HNMR Spectra of SL1glycoside in CD3OD at 500 MHz

Fig 3. 1HNMR Spectra of SL1 glycone in CD3OD at 500 MHz



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Fig 4. 1HNMR Spectra of SL1 glycone in CD3OD at 500 MHz

Fig.5 DEPT Spectra of SL1 in CD3OD at 100MHZ



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CH

CH3

CH3CH2

O

O

O

OH

HO

OH

OH

OH

H

O

OH

OH

HO

OOH

OHOH

HO

CH21'2'

3'4'

5'

6'

1''

2''

3''

4''

5''

1'2'

3'4'

5'6'

1''

2''

3''

4''

5''

H H

1 2

3

4

O

H 5

O

Fig.6 Compound (SL1) (C27H56022), [2-methylbut-(1)-yl- 6΄,6΄-di-O-(α-D-xylopyranosyl)-β-D-

glucopyranosyl-(1→4)-D-glucopyranoside)] The mass spectra study of SL1 was carried out using

Electron Spray Ionization Mass Spectrometer. On

fragmentation, SL1 gave the parent ion peak at 731

gmol-1. In ESIMS, fragmentation pattern characterized

by loss of one or more protons are often observed,

hence the actual molecular weight was calculated as

732 gmol-1 which represent the molecular weight of

the compound SL1 and corresponds to its elemental

composition which is a key information in structural

elucidation. The molecular formula (Fig 6) was

therefore analysed as C27H56O22. Loss of water

molecules, M-2H20 (32) and fragmentation

characteristic of saturated hydrocarbon, C2H5 (29) lead

to the peak at 667 gmol-1. Being the most intense peak,

it is referred to as the base peak in the spectrum.

CONCLUSION

Considering the above data collectively, the spectra

information suggested a glycoside which consist of a

large sugar moiety linked to a simple terpene aglycone

through O- glycosylation arising from a methylene

group at chemical shift 76.6 ppm [15,16]. Comparing

the informations obtained from the spectral data of the

isolated compound (SL1) having molecular weight

C27H56022 with literature data, it was concluded that the

compound (SL1) is a Hemiterpene glycoside [2-

methylbut-(1)-yl-6΄,6΄-di-O-(α-D-xylopyranosyl)-β-D-

glucopyranosyl-(1→4)-D-glucopyranoside]. Though,

this class of compounds (Hemiterpene glycoside) had

been reported in the Rubiaceae (Morinda citrifolia);

this is the first report on the isolation and

characterization of this glycosidic compound from the

specie Sarcocephalus (Rubiaceae).

ACKNOWLEDGMENT

The University of Maiduguri for providing enabling

resources and environment for the research. Prof I.M

Hussaini of the University of Maiduguri for his

excellent contribution to the work. Also, the

University of Maryland, USA and University of

Bradford, UK for helping out with the spectral studies

and also Mr. Fine Akawo for his assistance at the

Chemistry Research Laboratory, University of

Maiduguri.

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[5] Chouldhary, K.D., Chouldary, M.D. and Baruah , M.

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*Corresponding author Email address:

a_ahmed @ut.edu.sa

isolation and identification of a hermiterpene ...lamidi et al.[11] has reported the isolation of...

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