Assessment of Tunisian Trigonella foenum graecum diversity using seed vitamin B6, B1, B9 and C contents

Abstract

Trigonella foenum graecum is used as spice and forage, and it is known for its therapeutic effects in traditional medicine. The seeds arc rich in vitamins A, B and C. This work aims to study the diversity of 38 Tunisian Trigonella foenum graecum cultivars using seed vitamin B6, B1, B9 and C contents. Cultivars were collected from upper semi arid, sub humid and lower arid Tunisian bioclimatic stages. Vitamin contents of the seeds were analysed by HPLC. A variance analysis was assured with SPSS 16.0. Cultivar classification was obtained after the calculation of Mahalanobis distances between the pairs of cultivars by XIStat software. A principal compound analysis was done by StatBox software to verify the dendrogram clustering. Results showed significant variations in vitamins B6 (P<0.01), B9 (P<0.01), B1 (P<0.01) and C (P<0.01) between the studied cultivars. Dendrogram analysis revealed three clusters. The first cluster had quantities of vitamin B6 near to 0.0189 mg/g, B1 near to 0.0001 mg/g, B9 near to 0.002 mg/g and C near to 0.0025 mg/g. The second one recorded quantities (mg/g) of 0.029 for B6, 0.00026 for B9 and 0.0026 for C and no detectable quantities of B1. The last cultivar cluster reached vitamin contents (mg/g) of 0.068 for B6, 0.00019 for B9 and 0.0014 for C and no detectable quantities of B1. Principal component analysis confirmed this clustering and showed the same three clusters that were obtained by the dendrogram. Significant correlation between Mahalanobis distances matrix and geographic distances matrix was revealed by the test of Mantel (r = 0.222; p = 0.1 x 10(-1) < 0.05).

Full text

56 Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
www.world-food.net
Journal of Food, Agriculture & Environment Vol.7 (1) : 56-61. 2009
WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net
Assessment of Tunisian Trigonella foenum graecum diversity using seed vitamin B6,
B1, B9 and C contents
Nidhal Marzougui
1*
, Ferdaous Guasmi
1
, Mounira Mkaddem
2
, Anissa Boubaya
1
, Abdessalam Mrabet
1
,
Walid Elfalleh
1
, Ali Ferchichi
1
and Mohamed Beji
3
1
Arid and Oases Cropping Laboratory, Arid Area Institute, Medenine 4119, Tunisia.
2
Department of Biology, Laboratory of Plant
Biotechnology, Institute of Applied Sciences and Technologies, Center Urbain Nord, BP 676 Tunis Cedex 1080, Tunisia.
3
Genetic Resources Laboratory, High Institute of Biotechnology of Monastir, Monastir 5000, Tunisia.
* e-mail: marzouguinidhal@yahoo.fr
Received 19 September 2008, accepted 13 December 2008.
Abstract
Trigonella foenum graecum is used as spice and forage, and it is known for its therapeutic effects in traditional medicine. The seeds are rich in vitamins
A, B and C. This work aims to study the diversity of 38 Tunisian Trigonella foenum graecum cultivars using seed vitamin B6, B1, B9 and C contents.
Cultivars were collected from upper semi arid, sub humid and lower arid Tunisian bioclimatic stages. Vitamin contents of the seeds were analysed by
HPLC. A variance analysis was assured with SPSS 16.0. Cultivar classification was obtained after the calculation of Mahalanobis distances between
the pairs of cultivars by XlStat software. A principal compound analysis was done by StatBox software to verify the dendrogram clustering. Results
showed significant variations in vitamins B6 (P<0.01), B9 (P<0.01), B1 (P<0.01) and C (P<0.01) between the studied cultivars. Dendrogram analysis
revealed three clusters. The first cluster had quantities of vitamin B6 near to 0.0189 mg/g, B1 near to 0.0001 mg/g, B9 near to 0.002 mg/g and C near
to 0.0025 mg/g. The second one recorded quantities (mg/g) of 0.029 for B6, 0.00026 for B9 and 0.0026 for C and no detectable quantities of B1. The
last cultivar cluster reached vitamin contents (mg/g) of 0.068 for B6, 0.00019 for B9 and 0.0014 for C and no detectable quantities of B1. Principal
component analysis confirmed this clustering and showed the same three clusters that were obtained by the dendrogram. Significant correlation
between Mahalanobis distances matrix and geographic distances matrix was revealed by the test of Mantel (r = 0.222; p = 0.1x10
-3
< 0.05).
Key words: Trigonella foenum graecum, fenugreek, diversity, Tunisia, seeds, HPLC, classification, test of Mantel, vitamins B6, B1, B9 and C.
Introduction
Trigonella foenum graecum is an annual plant largely cultivated
in the Mediterranean basin and the Middle East countries. In
addition to its dietetic uses, it is known for its therapeutic effects
in traditional medicine
1
. Fenugreek seeds are used as spice and
contain tannic acid, volatile and fixed oils, diosgenin, alkaloids
and vitamins A, B and C
2-3
; leaves are rich in calcium, iron, -
carotene and vitamins
4
.
The water-soluble vitamins B1, B6 and B9 can be present in
food in the phosphorylated form, free or attached to proteins
5
.
Bioavailability of vitamins B is reduced in vegetable origin
foodstuffs compared with animal origin foodstuffs and
strengthened foodstuffs
6-7
. The variety of vitamins B complicates
their analysis. Thus, a high performance liquid chromatography
(HPLC) method roughly detects in the average 40% higher vitamin
B contents than are detected by microbiological methods
8
.
Vitamin C is regarded as being one of the most suitable dietetic
antioxidant agents since it arrives in great quantities in vegetable
food
9-11
. A richness of literature is available on the benefits for the
health of vitamin C. The bioavailability and pharmacokinetic of
vitamin C are well-known, but the datum on the dietetic exposure,
the consideration of the unstable nature of this vitamin and the
applied various procedures to analyze it are rather dubious
12-13
.
HPLC is regarded as simple and rapid method for vitamin B analysis
in foodstuffs
14
. The use of HPLC coupled with ultraviolet detection
is a sensitive and specific method to analyse contents of these
vitamins in foodstuffs
5-15
.
The aim of this study was to evaluate the genetic diversity of 38
cultivars of T. foenum graecum based on their seeds composition
of vitamins B1, B6, B9 and C.
Materials and Methods
Plant material: Thirty eight fenugreek cultivars were collected
from different Tunisian regions (Table 1). Seeds of each cultivar
were used for vitamin analyses. For each vitamin, analyses were
repeated three times for each cultivar.
Preparation of samples for vitamin B analyses: We developed a
fenugreek extraction protocol of the various categories of vitamins
B
16-18
. Fenugreek seeds (1 g) were cooked during 30 min at 95°C in
25 ml of 0.1 N H
2
SO
4
solution. After cooling, the contents were
adjusted into pH 4.5 with 2.5 M sodium acetate. Then 0.05 g of the
enzyme takadiastase was added
19
. The preparation was kept one
night at 35°C, filtered through a micropore filter (0.45 >m) and
diluted with 50 ml of distilled water. A volume of 20 µl of this
filtrate was injected into the injector
20
. The recovery percentage
of the used extraction method reached 79.45% for B6, 73.41% for
B1 and 83.67% for B9.

Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
57
Preparation of samples for vitamin C analysis: Samples (1 g)
were diluted in 30 ml of 1% orthophosphoric acid. The mixture
was vortexed during 1 min. After centrifugation with 1740 rpm
during 15 min at 20°C, the supernatant was filtered through a
micropore filter (0.45 µm) and diluted with 25 ml of orthophosphoric
acid. Handling was quickly carried out to avoid the degradation
of the vitamin C
21
. The recovery percentage of the used extraction
method was 74.86%.
Chromatographic determination: The Knauer apparatus was
equipped with two pumps, a control system, a reversed phase, a
curl injector of 20 >l and an ultraviolet absorbance detector (254
nm). The used column was Eurospher 100 C-18.5 (250 × 4.6 mm
i.d., 5 >m)
22
. The used mobile phase was a mixture of methanol 9
%, 10 ml of crystalline acetic acid and 600 ml of distilled water.
This solvent was added with sulfonic acid pentane and sulfonic
acid octane; the whole was filtered through a 0.45
µm filter and
deaerated by agitation. The prepared solvent was pumped through
the column with a programmed flow between 0.5 and 2.1
ml/min.
Peak surfaces were calculated using an electronic integrator. The
quantitative values were obtained against vitamin standard
injections containing vitamins (mg/50 ml) as follows: 9.7 B6, 4.0
B1, 0.25 B9 and 1.0 C.
Statistical analyses: Vitamin contents of the 38 cultivars were
subjected to one-way variance analysis using SPSS 16.0 (SPSS
Inc., Chicago, IL, USA). Mahalanobis distances matrix for vitamin
contents was generated using XLSTAT version 2008 (Addinosoft
SARL, Paris, France). Matrix values were entered to MVSP 3.1
(Kovach Computing Services, Wales, UK) to establish the
dendrogram. A second cultivars classification was done with
principal component analysis (PCA) using StatBox version 6.4
(Grimmersoft, Paris, France). Correlations between Mahalanobis
distances and geographic distances were evaluated by the test of
Mantel
23
using XLSTAT version 2008 (Addinosoft SARL, Paris,
France).
Results
Vitamins B6, B1, B9 and C analysis: There was a high variation
in vitamin contents between the different analysed fenugreek
cultivars (Fig. 1). In the upper semi arid stage, Cultivar 16 presented
the highest content of vitamin B6 (0.06 mg/g). Seeds of this cultivar
had respectively vitamins B9 and C contents of 1.9 x10
-4
and 1.4x
10
-3
mg/g. Cultivar 30 was rich in vitamin B6 (0.019 mg/g) and
vitamin C (0.017mg/g). Cultivar 4 of Manzel Tmim was the only
one of our collection which presents a detectable quantity of
vitamin B1 (0.1x10
-3
mg/g), the rest presented no detectable
quantities of vitamin B1. In the sub humid stage, Cultivar 28 of
s
the soft alternative;
m
the moderate alternative.
%LRFOLPDWLFVWDJH

/RFDOLW\

&XOWLYDUFRGH (GDSKLF
VXEVWUDWH
$OWLWXGH
P
/DWLWXGH
1
/RQJLWXGH
(
5DLQIDOO
PP\HDU
0DQ]HO7PLP  $UJLOODFHRXV  u u 
%L]HUWH

   u u 
0DWHU

 
/LPHVWRQH
  u u 
0KDPGLD

  &OD\/LPHVWRQH  u u 
6LGL+DPHG

   u u 
%HQ%HFKLU


&DUERQDWHG
OLPHVWRQH  u u 
8SSHU
VHPLDULG
V





1HI]D  0DUO/LPHVWRQH  u u 
6LGL.KLDU   u u 
1HEHXU

   u u 
(ONHI   u u 
7HO(OJR]ODQ   u u 
8SSHU
VHPLDULG
P

%RUM%HUU]LJ


0DUO&OD\
 u u 
6XEKXPLG
P
%HMD

  6DQG\  u u 
/RZHUDULG
P
 0DQ]HO+DELE

  6DQG\ORDP  u u 
Table 1. Location and main ecological traits for the 38 Tunisian Trigonella foenum graecum cultivars.
B6* B1* B9* C*
0
0.02
0.04
0.06
0.08
B6*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
mg/g
Cultivar
Figure 1. Variation in the contents of vitamin B6 between different Trigonella foenum graecum cultivars; Standard deviation corresponds
to three replicates for each cultivar; * Significant variation (ANOVA).

58 Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
Beja had the highest content of vitamins B6 (0.029 mg/g) and B9
(2.6 x10
-3
mg/g), but no detectable content in vitamin B1 and a
content of 0.8x 10
-3
mg/g of vitamin C. Cultivar 24 (Manzel Habib)
of the lower arid stage contained 8.14x10
-3
mg/g of vitamin B6 and
5.8x10
-4
mg/g of vitamin C. Vitamins B1 and B9 were not detected.
Variance analysis showed significant variations in vitamins B6
(P<0.01), B9 (P<0.01), B1 (P<0.01) and C (P<0.01) contents between
all cultivars (Table 2).
Dendrogram analysis: The dendrogram showed 3 groups of T.
foenum graecum cultivars. The first group was heterogeneous
while it grouped together cultivars 1-9, 11, 12, 14, 15, 17, 18-22, 25-
27, 30-33, 35, 36 and 37 from the upper semi arid stage, cultivars 10,
13, 34 and 38 from the sub humid and cultivars 23 and 24 from the
lower arid stage. These cultivars were characterized by quantities
of vitamin B6 from 0.002 to 0.0189 mg/g, vitamin B1 from 0 to
0.1.10
-3
mg/g, vitamin B9 from 0 to 0.002 mg/g and vitamin C from
8.9x10
-5
to 0.0025 mg/g. The second group reassembled cultivars
28 (sub humid) and 29 (upper semi arid) with quantities of vitamin
B6 from 0.025 to 0.029 mg/g, vitamin C from 8.3x10
-4
to 0.0015 mg/
g, vitamin B9 from 0 to 0.00026 mg/g and no detectable quantities
of B1. The third group included Cultivar 16 from Mater (upper
semi arid), characterized by the highest quantity of vitamin B6
(0.068 mg/g) (Fig. 2).
PCA analysis: The structuring of the 38 T. foenum graecum
cultivars by PCA analysis according to vitamin contents showed
that the first 3 axes absorb 82.03% of the total variance. The test
of Pearson did not reveal a correlation between the analyzed
vitamin contents of the cultivars (Table 3). The plot obtained
according to axes 1-2 (57% of total inertia) confirmed the
dendrogram clustering and showed 3 groups (Fig. 3A). The first
group reassembled Cultivars 1-11, 12, 14, 15, 17-22, 25-27, 30-33,
35, 36 and 37 from the upper semi arid stage, Cultivars 10, 13, 34
and 38 from the sub humid and Cultivars 23 and 24 from the lower
arid stage. The second group reassembled Cultivars 28 (sub humid)
and 29 (upper semi arid), and the third group was formed by
Cultivar 16 from the upper semi arid. The plot obtained according
to axes 1-3 (55% of total inertia) showed the same 3 groups that
were obtained by the dendrogram and the plot obtained according
to axes 1-2 (Fig. 3B).
Correlation between vitamin contents and geographical
distances: The test of Mantel revealed significant correlation
between matrixes of vitamin contents and geographical distances
(r = 0.22 and p = 0.1x10
-3
< 0.05 after 10,000 permutations).
Discussion
In this study, vitamin extraction was carried out by mixed way
using at the same time 0.1 N H
2
SO
4
and the takadiastase. Hot acid
hydrolysis was initially carried out. The enzyme was added after
cooling and the pH was adjusted to 4.5. Incubation at 35°C during
12 h makes it possible to extract several vitamins in the same
sample. The literature confirmed that only one digestion using an
acid and then the enzyme takadiastase is sufficient for the detection
of vitamins B
24
. Our method of analysis was applied for HPLC
vitamins proportioning using fluorometric detectors
25
. The latter
- contrary to the ultraviolet detectors - are expensive, sophisticated
and fragile
26
. The use of HPLC coupled with ultraviolet detection
for the research of the water-soluble vitamins in food was
approved in the literature as being a fast, simple and reliable
method
27-29
.
The results showed significant variations in the contents of
vitamins B6, B1, B9 and C between the different analysed cultivars
(Table 2). Such variations can be justified by the influence of
geographical, edaphic or genetic factors
26
. Indeed, we detected
significant correlations between the vitamin contents and the
geographical distances. The influence of cultivar on quality
parameters and chemical composition of plant fruits was approved
in literature
30
.
The average content of vitamins of the different cultivars were
12.7x10
-3
mg/g for vitamin B6, 0.03x10
-4
mg/g for B1, 0.77x10
-4
mg/g
for B9 and 1.152x10
-3
mg/g for vitamin C. These values are definitely
lower than the daily requirements of vitamins for an adult (1.5-2.0
mg/dayfor vitamin B1, 2-3 mg/day for B6, 15 mg/day for B9 and
DF: Degree of freedom, SC: Sum of squares, MC: Mean square, F: F of Snedecor; P: Probability, **: significant at p < 0.01
Table 2. One-way variance analysis (cultivar effect) of seed contents of vitamins B6, B1, B9 and
C for the selected fenugreek cultivars.
9DULDEOH 6RXUFHRIYDULDWLRQ ') 6& 0& ) 3 6LJQLILFDQFH
,QWHUJURXSV   [

 
,QWUDJURXSV  [

 [

  
%


7RWDO    
[




,QWHUJURXSV  [

 [


,QWUDJURXSV  [

 [

 
%

 7RWDO  [

 
[




,QWHUJURXSV  [

 [

 
,QWUDJURXSV  [

 [

 
%

 7RWDO  [

 
[




,QWHUJURXSV  [

 [

 
,QWUDJURXSV  [

[

 [


&


7RWDO  [

 
[




*Significant value at alpha = 0.05
9LWDPLQ % % % &
%    
%    
%    
&    
Table 3. Correlation matrix of vitamin contents.

Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
59
0 0.0006 0.0012 0.0018 0.0024 0.003 0.0036
3
22
23
2
31
6
27
4
37
13
32
7
26
10
15
38
8
25
34
33
35
5
19
36
12
24
20
18
11
1
17
9
14
30
21
28
29
16
G3
G2
G1
Figure 2. Dendrogram based on Mahalanobis distances for the seeds vitamin contents between the different Trigonella foenum
graecum cultivars.
60 mg/day for vitamin C)
31
. Thus seeds of T. foenum graecum do
not constitute a good source of vitamins B1, B6, B9 and C like all
food of vegetable origin; it is not the case for food of animal
origin
32-33
. Other authors used the leaves of this plant to evaluate
vitamin contents; their results always indicate low quantities of
vitamins
34
.
Diversity studies based on plant chemical and biochemical
composition were approved by some authors working on
Euphorbia esula
35
, Thymus pulegioides
36
and various species
of Origanum
37
. Based on variation in vitamin B1, B6, B9 and C
contents, T. foenum graecum cultivars were separated into 3
groups. The first group included Cultivars 1-15, 17-27, 30-38. The
second group reassembled cultivars 28 and 29, and the third group
(B)
-2
-- axis F1 (30 Z) -->
-1
0
1
2
3
4
5
6
-4 -2 0 2 4
6
G1
G2
G3
-- axis F3 (25%) -->
Observations (axes F1 and F3: 55%)
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-4 -2 0 2 4
G1
G2
G3
-- axis F1 (30 Z) -->
-- axis F2 (27%) -->
(A)
Observations (axes F1 and F2: 57%)
6
Figure 3. Dispersion of Trigonella fooenum graecum cultivars of the principal component analysis based on seed
vitamin contents. A: Cultivars dispersion according to the plan 1-2; B: Cultivars dispersion according to the plan
1-3; G: group.
contained cultivar 16. This structure was different from that
obtained by the morphological and chemical studies which
succeeded also to grouping the studied cultivars into 3 other
groups
38
. Diversity studies of our T. foenum graecum cultivars,
based on morphological descriptors, leaf contents of sodium,
phosphorus, potassium, iron, calcium and magnesium and seed
contents of vitamins B1, B6, B9 and C, led to the same grouping
for 81.57% of the studied cultivars. Thus, the three systems bring
comparable estimates from the genetic relations between the
studied cultivars. Diversity analysis of our cultivars, based on
ISSR markers, succeeded to distinguish 5 different groups (data
not shown) and gave more precise assessment of T. foenum
graecum genetic diversity. The use of molecular markers was
efficient to study the genetic diversity
39-40
.

60 Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
Conclusions
Diversity analysis using the contents of seeds in vitamins B6, B1,
B9 and C showed 3 groups of Trigonella foenum graecum cultivars
in Tunisia. Vitamin contents of the seeds presented significant
correlation with the geographical distances. Chemical composition
of leaves essential oils is envisaged as another criterion of
variability between the selected cultivars. When associated with
morphological, chemical, biochemical and genetic results these
data are destined to help for more efficient conservation programs
and industrial uses of Trigonella foenum graecum cultivars.
Acknowledgements
We gratefully acknowledge all personnel and technicians of Arid
and Oases Cropping Laboratory, especially Mr. Belgacem
Lachiheb for expert technical assistance.
References
1
Bin-Hafeez, B., Haque, R., Parvez, S., Pandey, S., Sayeed, I. and
Raisuddin, S. 2003. Immunomodulatory effects of fenugreek (Trigonella
foenum graecum L.) extract in mice. Int. Immunopharmacol. 3:257-
265.
2
Jayaweera, D.M.A. 1981. Medicinal Seedling. III Royal Botanic Garden
leaves, Peradeniyra, Srilanka, 57 p.
3
Petit, P.R., Sauviaire, Y.D., Hillaire-Buys, D.M., Leconte, O.M., Baissac,
Y. and Baissac, G. 1995. Steroid saponins from fenugreek seeds:
Extraction, purification and pharmacological investigation on feeding
behaviour and plasma cholesterol. Steroids 10:674-680.
4
Sharma, R.D., Sarkar, A. and Hazra, D.K. 1996. Hyolipidaemic effect of
fenugreek seeds: A chronic study in non-insulin dependent diabetic
patient. Photother. Res. 10:332-334.
5
Ball, G.F.M. 1998. Bioavailability and analysis of vitamins in foods.
Chapman and Hall, London, New York, 87 p.
6
Kabir, H., Leklem, I. and Miller, L.T. 1983. Comparative vitamin B-6
bioavailability from tuna, whole wheat bread and peanut to ridge in
humans. J. Nut. 113:2412 -2420.
7
Leklem, J.E., Miller, L.T., Perera, A.D. and Pefers, D.E. 1980.
Bioavailability of vitamin B-6 from wheat bread in humans. J. Nut.
110:1819-1828.
8
Schoonhoven, J., Schrijver, J., Berg, H. and Haenen, G.R.M. 1994.
Reliable and sensitive high-performance liquid chromatographic method
with fluorometric detection for the analysis of vitamin B-6 in foods
and feeds. J. Agric. Food Chem. 42:1475-1480.
9
Moyer, R.A., Hummer, K.E., Finn, E.C., Frei, B. and Wrolstad, R.E.
2002. Anthocyanins, phenolics, and antioxidant capacity in diverse
small fruits: Vaccinium, Rubus, and Ribes. J. Agric. Food Chem.
50:519-525.
10
Medina, I., Tombo, I., Satue-Gracia, M., German, J.B. and Frankel,
E.N. 2002. Effects of natural phenolic compounds on the antioxidant
activity of lactoferrin in liposomes and oil-in-water emulsions. J. Agric.
Food Chem. 50:2392-2399.
11
Sellappan, S., Akoh, C.C. and Krewer, G. 2002. Phenolic compounds
and antioxidant capacity of Georgia-grown blueberries and blackberries.
J. Agric. Food Chem. 50:2432-2438.
12
Welch, R.W., Wang, Y., Crossman, J.A., Park, J.B., Kirk, K.L. and
Levine, M. 1995. Accumulation of vitamin C (ascorbate) and its
oxidized metabolite dehydroascorbic acid occurs by separate
mechanisms. J. Biol. Chem. 270:12584-12592.
13
Levine, M., Conry-Cantilena, C., Wang, Y., Welch, R.W., Washko, P.W.,
Dhariwal, K.R., Park, J.B., Lazarev, A., Graumlich, J.F. and King, J.
1996. Vitamin C pharmacokinetics in healthy volunteers: Evidence for
a recommended dietary allowance. J. AOAC Int. 89:172-189.
14
Kall, M.A. 2003. Determination of total vitamin B6 in foods by isocratic
HPLC: A comparison with microbiological analysis. Food Chem.
2:315-327.
15
Ball, G.F.M. 1994. Water soluble vitamin assays in human nutrition.
Chapman and Hall, London, New York, 109 p.
16
Arella, F., Lahely, S., Bourguignon, J.B. and Hasselmann, C. 1996.
Liquid chromatographic determination of vitamins B [1] and B [2] in
foods: A collaborative study. Food Chem. 56:81-86.
17
Blake, C.J. and Konings, E.J.M. 2005. Committee on Food Nutrition:
Fat-soluble vitamins: water-soluble vitamins. J. AOAC Int. 88:325-
330.
18
Karatas, F.A., Cansiz, H.C. and Serbetci, Z. 2007. Determination of
folic acid level in some foodstuffs. Chem. Nat. Compd. 43:310-312.
19
Vinas, P., Lopez Erroz, C., Balsalobre, N. and Hernandez Cordoba, M.
2003. Reversed-phase liquid chromatography on an amide stationary
phase for the determination of the B group vitamins in baby foods. J.
Chromat. A 10:77-84.
20
Toma, R.B. and Tabekhia, M. 1979. High performance liquid
chromatographic analysis of vitamins in rice and rice products. USA J.
Food Sci. 44:263-268.
21
Arella, F., Deborde, J.L., Bourguignon, J.B. and Hasselmann, C. 1997.
High performance liquid chromatographic determination of L-ascorbic
acid and total vitamin C in foodstuffs: Interlaboratory study. Ann.
Falsif. Expert. Chim. Toxicol. 90:217-233.
22
Collmer, K. and Davies, L. 1974. Separation and determination of
vitamins B1, B2, B6 and nicotinamid in commercial vitamins
preparation using high performance cation exchange chromatography.
Chromat. 11:635-644.
23
Mantel, N. 1967. Detection of disease grouping and a generalized
regression approach. Cancer Res. 27:209-220.
24
Vinas, P., Lopez-Erroz, C., Balsalobre, N. and Hernandez-Cordoba,
M. 2003. Reversed-phase liquid chromatography on an amide
stationary phase for the determination of the B group vitamins in
baby foods. J. Chromat. A 1007:77-84.
25
Ndawa, S., Bergaentzléa, M., Aoudé-Wernerb, D. and Hasselmann C.
2000. Extraction procedures for the liquid chromatographic
determination of thiamin, riboflavin and vitamin B6 in foodstuffs.
Food Chem. 71:129-138.
26
Voahanginirina, R. 2001. Les vitamines du groupe B de quelques variétés
de riz malgaches en provenance de Marovoay et de Tulear: Mise au
point de la méthode et détermination quantitative. Food Agri. Res.
Council 22:157-165.
27
Albala Hurtado, S., Veciana Nogues, M.T., Izquierdo Pulido, M. and
Marine Font, A. 2005. Determination of water-soluble vitamins in
infant milk by high-performance liquid chromatography. J. Chromat.
A 78:247-253.
28
Konings, E.J. 2006. Water-soluble vitamins. J. AOAC Int. 89:285-
288.
29
Heudi, O., Kilinc, T. and Fontannaz, P. 2005. Separation of water-
soluble vitamins by reversed-phase high performance liquid
chromatography with ultra-violet detection: Application to
polyvitaminated premixes. J. Chromat. A 1070:49-56.
30
Cordenunsi, B.R., Oliveira do Nascimento, J.R., Genovese, M.I. and
Lajolo, F.M. 2002. Influence of cultivar on quality parameters and
chemical composition of strawberry fruits grown in Brazil. J. Agric.
Food Chem. 50:2581-2586.
31
Subcommittee on the Tenth Edition of the Recommended Dietary
Allowances, Food and Nutrition Board, Commission on Life Sciences,
National Research Council 1989. Recommended dietary allowances.
10
th
edn.
32
Kabir, H., Leklem, I. and Miller, L.T. 1983. Comparative vitamin B-6
bioavailability from tuna, whole wheat bread and peanut to ridge in
humans. J. Nut. 113:2412-2420.
33
Leklem, J.E., Miller, L.T., Perera, A.D. and Pefers, D.E. 1980.
Bioavailability of vitamin B-6 from wheat bread in humans. J. Nut.
110:1819-1828.

Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009
61
34
Yadav, S.K. and Sehgal, S. 1997. Effect of home processing and storage
on ascorbic acid and beta-carotene content of Bathua (Chenopodium
album) and fenugreek (Trigonella foenum graecum) leaves. Plant Food
Hum. Nut. 50:239-247.
35
Torell, J.M. and Evans, J.O. 1998. Chemical characterization of leafy
spurge (Euphorbia esula L.) by Curie-point pyrolysis-gas
chromatography-pattern recognition. J. Anal. Appl. Pyrol. 14:223-
236.
36
Loziene, K., Vaiciuniené, J. and Venskutonis, P.R. 2003. Chemical
composition of the essential oil of different varieties of thymus (Thymus
pulegioides) growing wild in Lithuania. Bioch. Systematic Ecol.
31:249-259.
37
Schulz, H., Ozkan, G., Baranska, M., Kruger, H. and Ozcan, M. 2005.
Characterization of essential oil plants from Turkey by IR and Raman
spectroscopy. Vib. Spectrosc. 39:249-256.
38
Marzougui, N., Ferchichi, A., Guasmi, F. and Beji, M. 2007.
Morphological and chemical diversity among 38 Tunisian cultivars of
Trigonella foenum graecum L. J. Food Agric. Environ. 5(3&4):248-
253.
39
Rakhee, S.D., Meena, D.L., Lal, B.C., Prabhakar, K.R. and Vidya, S.G.
2004. Assessment of genetic diversity in Trigonella foenum graecum
and Trigonella coerulea using ISSR and RAPD markers. BMC Plant
Biol. 4:1471-2229.
40
Zoghlami, N., Chrita, I., Bouamama, B., Gargouri, M., Zemni, H.,
Ghorbel, A. and Mliki, A. 2007. Molecular based assessment of genetic
diversity within barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia.
Sci. Hortic. 113:134-141.