SUPPLEMENTARY MATERIAL
Chemical Characterization of bioactive compounds in Medicago sativa growing in
the desert of Oman
Muhammad Asif Hanif1,2*, Ahmed Yahya Al-Maskari2, Jamal Nasser Al-Sabahi3, Ibtisam
Al-Hdhrami2, Muhammad Mumtaz Khan2, Ahlam Al-Azkawi4, and Abdullah Ijaz
Hussain5
1
Department of Chemistry, University of Agriculture, Faisalabad, Pakistan
2
Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan
Qaboos University, Muscat, Oman
3
Central Instrumental Laboratory, College of Agricultural and Marine Sciences, Sultan
Qaboos University, Muscat, Oman
4
Central Analytical and Applied Research Facility, College of Science, Sultan Qaboos
University, Muscat, Oman
5
Department of Applied Chemistry, Government College University,
Faisalabad, Pakistan.
Author for correspondence*: muhammadasifhanif@ymail.com.00923338362781
Abstract:
Medicago sativa Linn growing in Omani desert were chemically characterized
using Flame Photometry, Inductively Coupled Plasma (ICP), Gas Chromatography Mass Spectrometry and High Performance Liquid Chromatographic (HPLC) analysis.
High performance liquid chromatography (HPLC) analyses were performed to determine
phenolics and flavonoids present in Medicago sativa. Major compounds detected in M.
sativa leaves were protchaechenic acid (3.22%), hydroxyl benzoic acid (1.05%), βphenyl caffate (0.97 %) and kaempferol (0.89 %). Pterostilbene, a cholesterol lowering
compound was detected in M. sativa.
Keywords: Medicago sativa, HPLC, GC, ICP analysis, medical plant
Experimental
Plant Collection: In January 2012, a representative sample of M. sativa was collected
from Nizwa, Oman for the present study. M. sativa samples were carefully identified by
taxonomist (Prof. Mansoor Hameed) and specimens were stored in specimen voucher
placed at Department of Crop Sciences, Sultan Qaboos University, Muscat, Oman. The
voucher specimen number assigned to M. sativa was I002. The collected plant sample
was oven dried at 70 ˚C for 48 hr to remove all the moisture present. The dried samples
were milled using Panasonic blender-grinder.
Wet Digestion and Metal Analysis: One gram of grinded powder sample was placed in a
250 ml digestion tube and 20 ml of nitric acid (HNO3) was added. The mixture was
placed in digester at 180˚C. After 20 min about five milliliter of hydrogen peroxide was
added and heating was continued for more 30 min. After completion of digestion, the
solution was filtered using Whatman filter paper no. 41 and sample was diluted to 50 ml
in a volumetric flask by adding deionized distilled water (DDW). The digested samples
were analyzed to determine Na, K, Li and Ca concentration using Flame Photometric
Analysis (Corniny, Flame Photometric 410) and other metals using ICP analysis (Table
1).
Nitrogen Analysis: Kjeldahl Method was used to determine amount of Nitrogen in the
samples. The sample (0.5-1.0 g) was placed in a digestion tube with a tablet of Kjeltab
catalyst (K2SO4 + CuSO4)/ (K2SO4 +Se). Ten milliliter of H2SO4 was added to digestion
tube. Then digestion tube was placed into a part of digestion block. The digestion was
carried out in a digester (2020 Digester Foss Tecator), at 370 ˚C for 3h. After digestion,
the samples were analyzed using automatic nitrogen analyzer (2300 Kjeltec Analyzer
Unite Foss Tecator).
Extraction of Samples: One hundred grams of sample was extracted using 500 ml of
methanol and hexane.
Gas Chromatographic-Mass spectrometric analysis: The samples extracted using
hexane was analyzed by Gas Chromatographic-Mass spectrometric analysis (GC-MS)
(Perkin Elmer Gas Chromatograph Model # Clarus 600). The oven temperature with a
hold up time of 2 min was programmed from 60-280˚C at a rate of 3˚C/min. Carrier gas
used was helium at a flow rate of 1ml/min. Mass spectra was recorded from 40-500 m/z.
The MS operating parameters were as follows: ionization voltage, 70ev; scan rate,
500amu/s. Each compound was identified by comparison of its spectral data with
reference spectra in the data base (Wiley AccessPak V7, May 2003 and NIST2005
version 2.1.0).
High Performance Liquid Chromatographic-Mass spectrometric (HPLC-MS) analysis:
For HPLC analysis (Model: Waters 1525, USA) samples were diluted using 50%
methanol in water. The used column was a SYMMETRY C18 3.5 um 2.1×100 mm
Column (Waters, USA) with a mobile phase of methanol:water (50:50) at a flow rate
0.250 mL/min. The detection of compounds was done using tandem quadrupole mass
spectrometry. The mode of ionization was negative electron spray ionization (ES-). The
other MS parameters were set as follows: the capillary was -2.81 KV and cone was -35V,
source temperature was 151 ˚C, the desolvation temperature, 350 ˚C; and desolvation gas
flow, 704L/h. LM1& HM1 resolution: 14.0, ion energy (1:1.0), entrance & exit (50),
collision (3), ion energy (2:3.0), LM2 & HM2 resolution (13.0) and multiplier (V) (662).
A mass spectrum was scanned in continuous range from 50 to 1200 amu for 45 min. The
individual compounds were identified using appropriate standards.
Table S1. Mineral composition (mg/g) of M. sativa
Cr
0.20±0
.05
Co
Cd
<0.005
Ba
Pb
1.35±0.0
3
S
Zn
3.57±1
.00
Al
Cu
1.15±0.
12
P
Mn
3.34±0.
01
Na
Fe
38.15±1
.07
K
Mg
1222.5±
3.54
Li
Ni
0.25±0.
78
Ca
B
5.675±0
.04
N (%)
<0.005
0.97±0
.01
1197.5±
3.89
12.1±0
.07
210.5±0
.07
3.52 ±
0.04
0.73 ±
0.01
1.12 ±
0.01
6.75 ±
0.18
0.53 ±
0.10