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Chapter 3
Screening of leaves of Moringa oleifera
germplasm for folic acid, iron and
carotenoids
Screening of phytoconstituents in M. oleifera cultivars
35
Abstract
Experiments were conducted to screen the major phytoconstituents in eight
commercially grown Indian cultivars of Moringa oleifera, supported by assessment of
genetic diversity in studied cultivars. In this regard, six major carotenoids i.e., trans-
luteoxanthin, 13-cis-lutein, trans-lutein, trans-zeaxanthin, 15-cis-β-carotene and
trans-β-carotene, were purified using open column chromatography (OCC) and TLC.
Trans-lutein was found as the major carotenoid in leaves, flowers and fruits and
accounted for 53.6, 32.5 and 52.0% of the total carotenoids, respectively. Among the
eight cultivars screened, the cultivar Bhagya had the maximum amount of trans-
zeaxanthin, trans-β-carotene and total carotenoids. PKM-1 genotype registered
maximum amount of total iron (4.08 mg/100g FW) and ascorbic acid (224.2 mg/100g
FW), whereas, α-tocopherol were recorded maximum in CO-1 cultivar. Maximum
variation (9.1 to 20.9 mg/100g FW) is also recorded for α-tocopherol content among
the studied cultivars.
Among the eight cultivars screened, the maximum total folate content of 167.4
µg/100g FW was recorded in Bhagya cultivar. Folate was purified by immunoaffinity
chromatography using folate binding protein and quantified by RP-HPLC from fresh
leaves of folate rich cultivar. The content of 5-formyl-5,6,7,8-tetrahydrofolic acid
(73.1 µg/100g FW) and 5,6,7,8-tetrahydrofolic acid (32.6 µg/100g FW) were found as
the most dominant forms of folate in fresh leaves of Bhagya cultivar.
In leaves, α-linolenic acid (C18:3, cis-9,12,15) was found in highest quantity
(49-59%) followed by palmitic acid (C16:0) (16-18%), and linoleic acid (18:2, cis-
9,12) (6-13%). Total content of saturated fatty acid (SFA) and unsaturated fatty acid
(UFA) showed a ratio of 0.33 (cv. DHANRAJ) to 0.39 (cv. PKM-2) in leaves, 0.53 in
Screening of phytoconstituents in M. oleifera cultivars
36
flowers and 0.56 in fruits. Similarly, PUFA and MUFA were found in ratios of 5.68
(cv. DHANRAJ) to 9.71 (cv. CO-1) in leaves, 1.11 in flowers and 2.79 in fruits. The
total lipid content was recorded in the range of 1.92% (flowers) to 4.82% (leaves, cv.
CO-1).
Earlier studies on screening of iron, α-tocopherol, ascorbic acid, carotenoids,
folate, fatty acids and other phytoconstituents content in Moringa oleifera is restricted
to unknown cultivars or genotypes. This is the first report of detailed profile of
phytoconstituents in commercially grown cultivars of M. oleifera.
Three DNA marker techniques, i.e., random amplified polymorphic DNA
(RAPD), inter simple sequence repeat (ISSR) and cytochrome P450 gene based
markers were used for the detection of genetic variability in eight Indian cultivars of
M. oleifera, collected from various states of India. Based on the three types of marker
data, the eight cultivars of M. oleifera were grouped into four sub-clusters in a
dendrogram, but without any distinct geographical pattern. This suggests the spread of
planting material and high rates of gene flow through cross pollination.
Screening of phytoconstituents in M. oleifera cultivars
37
3.1. Introduction
In India and other third world countries, a large proportion of the population suffers
from malnutrition, due to lack of appropriate sources of nutrients in the food
consumed. Moringa oleifera L. is a tropical tree and recent investigations have
established it as a unique food plant with medicinal properties and with high levels of
minerals, vitamins and protein. Fresh leaves of M. oleifera have been established as
rich source of carotenoids (Bhaskarachary et al. 1995), tocopherols (Ching and
Mohamed 2001), phenolics and glucosinolates (Bennett et al. 2003) ascorbic acid
(Bineesh et al. 2005), folate (Devi et al. 2008), iron (Anjorin et al. 2010) and fatty
acids (Amaglo et al. 2010a), but no efforts have been made to evaluate these
phytoconstituents in Indian cultivars. Similarly, information of these
phytoconstituents in fruits and flowers of this tree is not available. Knowledge on
nutrient composition in different edible parts and cultivars will be useful to nutritional
experts for selection of nutrient-rich plants for food fortification and proper diet
recommendation. Variability in content of nutrients can be obtained by genetic and
environmental factors (Strålsjö et al. 2003; Kuti and Konuru 2005), so the variability
due to genetic factors can be supported by assessment of genetic diversity (Muluvi et
al. 1999). Therefore, the objective of this study was to quantify the carotenoids, folate,
iron, ascorbic acid, tocopherol and fatty acids in in different cultivars of M. oleifera,
followed by assessment of genetic diversity in studied cultivars.
3.2. Materials and methods
3.2.1. Plant material
Seeds of different Moringa oleifera cultivars, i.e., PKM-1, PKM-2, GKVK-1 and
Dhanraj were collected from University of Agriculture Sciences, Bangalore
Screening of phytoconstituents in M. oleifera cultivars
38
(Karnataka, India). Seeds of cultivars CO-1 and Bhagya (KDM-1) were collected
from Tamil Nadu Agriculture University, Coimbatore (Tamil Nadu, India) and
University of Horticultural Sciences, Bagalkot (Karnataka, India), respectively. Seeds
of Amar-32 cultivar were purchased from Amar seeds Pvt. Ltd., Pune (Maharashtra,
India). The PAVM-1 cultivar was collected from a progressive farmer in the state of
Tamil Nadu, India. The seeds were sown in the field; the soil type was red loam with
fine-silty characteristics. At two months interval after germination, 10 g N-P-K
(15:15:15) were applied to each plant. The fresh leaves from top third petiole, freshly
opened flowers with anthers and tender fruits with pulp and immature seeds were
collected from two year old M. oleifera plants and used in this study.
3.2.1. Estimation of carotenoids
3.2.1.1. Purification of standards used for LC-MS quantification
All organic solvents used for extraction and purification of carotenoids were of
HPLC grade (Rankem, Delhi, India). Standard carotenoids used in LC-MS were
purified from leaves of M. oleifera according to method of Kimura and Rodriguez-
Amaya (2002) with some modifications, as shown in Figure 3.1. Mass spectra of
different purified fractions were recorded using a Waters Alliance 2695 HPLC
(Waters Corporation, Manchester, UK) equipped with an auto-sampler and coupled
with a Waters 2696 photodiode array detector and a Q-TOF Ultima™ mass
spectrometer, utilising the atmospheric pressure chemical ionization (APCI-MS)
interface. For chromatographic separation, a YMC C30 Carotenoid column (250 x 4.6
mm, 5 mm, YMC, Wilmington, NC, USA) was used (Darnoko et al. 2000). The
mobile phase was 81:15:4 methanol: methyl tertiary butyl ether (MTBE): H2O
(solvent A) and 91:9 MTBE: methanol (solvent B). The gradient elution was 0% B to
Screening of phytoconstituents in M. oleifera cultivars
39
66% B for 30 min followed by 0 % B for the next 5 min at a flow rate of 1 ml/min.
PDA spectra was recorded at 200-660 nm. Mass spectra of column eluates were
recorded in APCI +ve mode with Corona (µA): 6.6, cone: 100 V, source temp: 120
°C, APCI probe temp: 500 °C, cone gas flow: 50 L/h, desolvation gas flow: 5000 L/h.
Absorbance of purified carotenoid fractions was recorded in acetone using an
UV- spectrophotometer (Shimadzu Japan, Model UV-18000). Spectral fine structure
is given as %III/II (Britton 1995). The concentrations of individual carotenoids were
determined spectrophotometrically according to Rodriguez-Amaya (2001) and
Kimura and Rodriguez-Amaya (2002). Purity of the purified fractions was determined
by LC-MS (i.e., a chromatogram showing a single peak). The percentage of purity
was calculated as the percentage of the carotenoid peak area relative to total area.
Identification of individual carotenoids was done according to Kimura and Rodriguez-
Amaya (2002) by use of retention time, visible absorption spectrum, spectral fine
structure and mass spectrum obtained in our study with comparison to literature data
(Kamffer et al. 2010). These purified and quantified standard compounds were used
for the quantification of carotenoids in leaves, flowers and fruits of M. oleifera.
Screening of phytoconstituents in M. oleifera cultivars
40
Figure 3.1. Flow chart showing the steps used in purification of carotenoids
standards by OCC and TLC from leaves of M. oleifera.
3.2.2.2. Quantification of carotenoids from leaves, flowers and fruits
The fresh leaves from top third petiole, freshly opened flowers with anthers
and tender fruits with pulp and immature seeds (38.0 cm length and 0.6 cm diameter)
from PKM-1 cultivar were used in carotenoids extraction and quantification. Five
grams of each fresh leaves, flowers and fruits was homogenized in chilled acetone and
the extraction was repeated until the samples became colourless (total volume 50-100
Carotenoid extraction in cold acetone from 50g fresh leaves
Partitioned to the petroleum ether containing 10% (v/v) diethyl ether and washed with water to remove
the traces of acetone
Saponified overnight with 10% KOH in methanol (w/v), washed with water to remove the alkali, and
dried in vacuum rotavapour (T≤ 35°C)
Dried extract was applied to Natural alumina (grade III) packed glass Column (10 cm height x 2.5 cm
i.d.)
Separation of polar and non polar carotenoids by 10 % diethyl ether in petroleum ether (v/v) and 30 %
acetone in petroleum ether (v/v), respectively
Polar compounds
Thin layer chromatography on silica gel with 30 %
acetone in petroleum ether as mobile phase
Rf-0.36
Thin layer
chromatography on
MgO + Kiselguhr (1:1)
with 50 % acetone in
petroleum ether as
mobile phase
Rf-0.54
Thin layer
chromatography on
MgO + Kieelguhr (1:1)
with 50 % acetone in
petroleum ether as
mobile phase
Obtained
Luteoxanthin
isomers (Rf-0.35
and 0.82)
Obtained Trans
lutein (Rf-0.42)
and Trans
zeaxanthin
Obtained 13-Cis-lutein, 15-
Cis-β-carotene and Trans β-
carotene (≥85% pure) from
column eluted fractions
Confirmation of purity by RP-HPLC and Confirmation of carotenoids by Mass spectrometry and
absorbance spectrum
Screening of phytoconstituents in M. oleifera cultivars
41
ml). The extracts were then filtered through a 0.45 µm membrane (Nupore,
Ghaziabad, India). Carotenoids were transferred to petroleum ether (50-100 ml)
containing 10% diethyl ether (v/v) by partitioning and traces of acetone were removed
by repeated water wash (Kimura and Rodriguez-Amaya 2002). The extract was dried
in a vacuum rotary evaporator (Buchi Laboratory Equipment, Switzerland) at 35°C
temperature to complete dryness and subsequently redissolved in 10 ml acetone. A
volume of 20 µl extract was injected into the LC-APCI-MS system without
saponification. Chromatographic conditions were same as used in purification and
LC-APCI-MS analysis.
3.2.3. Estimation of folates
3.2.3.1. Trienzyme extraction of total folate
Total folate was extracted (under subdued light) according to the method of Aiso and
Tamura (1998) with some modification from fresh leaves of different cultivars of M.
oleifera. One gram of fresh leaves was homogenized with 10 ml extraction buffer
containing 0.1mM sodium phosplate buffer (pH 6.1), 1% ascorbic acid (w/v) and
0.1% 2-merceptoethanol (v/v) and proceed to digestion procedure. Similarly a blank
was prepared without adding the sample. Loosely capped 50 ml digestion tubes
containing sample and blank were placed in boiling water bath for 5 min and then
allowed to cool to room temperature (water or ice bath). After cooling, 0.5 ml
charcoal treated rat serum (De Brouwer et al. 2010) and 300 units of α-amylase from
Aspergillus oryzae were added in all sample and blank tubes. Tubes were gently swirl,
tightened caps, and incubated at 37ºC for 4 hours. After 4 hours of incubation, tubes
were kept in boiling water bath for 5 min to deactivate the enzymes and cool to room
temperature (water or ice bath). After cooling, 35 units of Protease from Streptomyces
Screening of phytoconstituents in M. oleifera cultivars
42
griseus (Type XIV) were added, gently swirls, tightened the caps, and incubated
overnight at 37 ºC in water bath. The reason for this order and the time for addition of
enzymes to samples are to allow conjugase and α-amylase to act on respective
substrates before possible enzyme deactivation by protease which has very broad,
non-specific protease activity. To deactivate enzymes after incubation period, 2 ml
extraction buffer was added and heated for 5 min in boiling water bath and cooled to
room temperature. After cooling, centrifuged at 10,000g for 5 min, collected the
supernatant, and mixed 2 ml supernatant in 18 ml water containing 2% extraction
buffer, this sample was used for microbiological assay. For folate purification,
undiluted sample was used. All the samples were stored in -80ºC in amber color tubes
to avoid the light mediated degradation of folate. Total folate was quantified by
microbiological assay, on the same day of extraction.
3.2.3.2. Preparation of glycerol cryoprotected L. casei
To minimize the analysis time and error between the analysis, cryoprotected
Lactobacillus casei (ATCC 7469), were prepared according to Grossowicz et al.
(1981), Pandrangi and LaBorde (2004) and Ortiz-Escobar et al. (2010). Briefly, 20 µl
of L. casei culture from lactobacillus broth was transferred to 10 ml assay medium
(folic acid casei medium) containing 250 mg/L ascorbic acid and 30 ng/L folic acid,
and incubated at 140 rpm and 37°C. After obtaining the constant optical density (OD;
14 to 20 hours after inoculation), the whole culture was inoculated to 20 ml assay
medium containing 250 mg/L ascorbic acid, with no added folic acid, and incubated at
140 rpm and 37°C. After obtaining the constant O.D. (14 to 20 hours after
inoculation), culture was centrifuged and pellets were dissolved in assay medium to
Screening of phytoconstituents in M. oleifera cultivars
43
obtain the OD (550 nm) value 0.2, diluted to equal volume of sterile glycerol and
water (80:20, v/v), aliquoted to 2.0 ml sterile cryo-vials and stored at -80°C.
3.2.3.3. Microbiological assay of total folate
Folic acid is assayed according to the method of AOAC (1990) Four sample extract
levels (0.5, 1.0, 2.0, 4.0 and 5.0 ml) were assayed in triplicate. Volume in each tube is
adjusted to 5 ml with deionized water. Five ml of single-strength assay medium is
added to each tube. Prepared assay tubes, standard curve tubes, blank and enzyme
blank tubes were autoclaved at 121°C for 5 min. For inolculation 1.0 ml glycerol
cryoprotected L. casei culture was diluted with 9.0 ml assay medium to obtain 10
times dilution, each assay tube was inoculated with 50 µl of prepared inoculum. Assay
tubes were incubated at 37ºC for 20 to 24 h, and growth response was measured at
550 nm.
3.2.3.4. Folate purification and HPLC analysis
Folate was purified by immunoaffinity chromatography using folate binding protein
and quantified by RP-HPLC from fresh leaves of folate rich cultivar (Bhagya),
according to method of Konings (1999) with some modifications. Outline of column
preparation with affigel matrix and folate binding protein, and purification procedure
is given in Figure 3.2. Binding capacity of column was analyzed by loading the excess
amount of 5-CH3-THF. The HPLC analysis of purified folate derivatives was
performed using a Shimadzu chromatograph (LC 20-AD HPLC), equipped with dual
pump, florescent detector (RF-20A) , an YMC-Pack ODS-AQ column (250 x 4.6 mm
ID x 5 µ) and YMC ODS-AQ guard column (10 x 5mm ID x 5µ). The separation and
elution accomplished by employing a binary gradient mode using solvent A (0.1%
Trifluoroacetic acid in water; v/v) and solvent B (Acetonitrile) with injection volume
Screening of phytoconstituents in M. oleifera cultivars
44
of 20 µl of the sample and at a flow rate of 0.8 ml/min for 25 min. The solvent system
was run as follows (% solvent A/solvent B): 0 min (10/90), 20 min (50/50), and 25
min (10/90). For the detection and quantification of folates derivatives, florescent
detector was set in dual wavelength mode; 5,6,7,8-tetrahydrofolic acid, 5-Methyl-
5,6,7,8-tetrahydrofolic acid and 5-Formyl-5,6,7,8-tetrahydrofolic acid were detected
at (Ex/Em) 290/360nm. Similarly, 10-Formylfolic acid was detected (Ex/Em) at
360/460nm (Shohag et al. 2011). Folate standards were diluted (Patring et al. 2005)
to obtain a concentration of 1 µg/ml. The actual concentrations of folate standards
solution were checked spectrometrically using the molar extinction coefficients
(Baggott and Johanning 1999; Jastrebova et al. 2003). Quantification of folate
derivatives were based on external calibration curve with a linear range of 5-100
ng/ml for H4-folate and 5-CH3-H4-folate, and 100-1000 ng/ml for 5-HCO-H4-folate
and 10-HCO-folic acid.
Screening of phytoconstituents in M. oleifera cultivars
45
Figure 3.2. Outline of column preparation and folate purification procedure
Screening of phytoconstituents in M. oleifera cultivars
46
3.2.4. Estimation of iron, tocopherol and ascorbic acid
3.2.4.1. Iron
An amount of 10 g of the fresh leaf sample was dried in hot air oven at l05°C for 3
hours. The dried sample was charred until it ceased to smoke, and then ashed in a
muffle furnace at 550°C until a whitish or greyish ash was obtained (3-5 hrs). The ash
was treated with 5 ml concentrated hydrochloric acid, filled the solution through ash
less filter paper (Whatman Ltd, London) and make up the volume (50ml) with 1 N
HNO3.
Total iron content in M. oleifera leaves was estimated by direct aspiration into
the AA-6710F Atomic Absorption Flame Emission Spectrophotometer (Shimadzu)
equipped with ASC-6000 auto sampler. Wavelength was set to 248.3 nm for solutions
with iron concentrations ranging from 0.5 ppm to 2 ppm. Fe+3 solution for atomic
absorption spectrophotometry was used as standard. A calibration curve with at least
4 concentrations of iron within the analytical range was prepared. Concentrations of
iron in test solutions were calculated from the standard curve prepared. For each ash
solution, at least three readings were obtained and the average calculated.
3.2.4.2. Tocopherol and ascorbic acid
Tocopherols were extracted in similar way of carotenoids; one gram of fresh leaves
were homogenized in chilled acetone and the extraction was repeated until the
samples became colorless (total volume 15 ml). The extracts were then centrifuged at
8000g and filtered through a 0.45 µ membrane (Nupore, India). A volume of 20 µL
extract was injected into the HPLC system without saponification. The content of
total carotenoids and α-tocopherol is expressed as mg/100g dry weight.
Ascorbic acid was extracted (under subdued light) according to the method of
Screening of phytoconstituents in M. oleifera cultivars
47
Vanderslice et al. (1990) with some modification, from fresh leaves of Moringa
oleifera. One gram of fresh leaves were homogenized with 2 ml methanol and 10 ml
of cold extraction solution, containing 3% meta phosphoric acid (MPA, w/v), 0.05%
EDTA (w/v) and 0.8% glacial acetic acid (v/v). The slurry was centrifuged for 15 min
at 5000g in a cooling centrifuge (2–4°C), and the supernatant was collected. Samples
in duplicates were filtered through 0.22 µ membranes into amber HPLC vials. The
samples (20 µl) were then directly injected into the HPLC system.
The HPLC system consisted of a Shimadzu chromatograph (LC 20-AD
HPLC), equipped with dual pump and UV detector (SPD 20A). For ascorbic acid
analysis, Cromatopack-C18 column (150 x 4.6 mm I.D, 5 µ particle size) was used.
The mobile phase was 50 mM K2HPO4 (solvent A) and methanol (solvent B). The
gradient elution was 1% B to for 5 minutes, linear gradient of 1% to 30% B for 15
minutes, followed by 30% B in the next 10 minutes at a flow rate of 1 ml/min.
Ascorbic acid was monitored at 254 nm.
For α-tocopherol analysis, an YMC C30 Carotenoid column (250 x 4.6 mm, 5
mm, YMC, Wilmington, NC) was used. The mobile phase used for this column was
81:15:4 methanol: methyl tertiary butyl ether (MTBE): H2O (solvent A) and 91:9
MTBE: methanol (solvent B). The gradient elution was 100% A to 50% A in 45 min
followed by 100% A in the next 10 min at a flow rate of 1 ml/min. α-tocopherol was
monitored at 295 nm.
3.2.5. Estimation of fatty acid methyl esters (FAMEs)
3.2.5.1. Fatty acid extraction and FAME preparation
Total lipids were extracted by the method of Bligh and Dyer (1959) with
minor modifications. Briefly, 0.5 g of dehydrated leaf, flower or fruit sample was
Screening of phytoconstituents in M. oleifera cultivars
48
homogenized with the help of acidified sand in 20 ml chloroform and 10 ml methanol.
After homogenization, 20 ml of 0.85% NaCl (w/v) was added and filtered through
filter paper. Filtrate was allowed to separate into two layers and lower (chloroform)
phase was collected into pre weighted tube, dried in vacuum rotary rotavapour and
total lipid content is then determined gravimetrically. Fatty acid methyl esters
(FAMEs) were prepared by conventional anhydrous methanolic HCl method. Briefly,
4 ml of anhydrous methanolic HCl (5%, v/v) was added into lipid sample in a
graduated glass tube, fitted with refluxing tubes, and refluxed for 3 hours at 60°C in
water bath. After cooling, FAMEs were washed sequentially with 5% NaCl followed
by 2% NaHCO3 and recovered in 20 ml hexane. Hexane was dried up to 1 ml in
vacuum rotary rotavapour, transferred to 2 ml glass GC tubes, dried under nitrogen
gas, and stored at -20 °C in presence of anhydrous sodium sulphate.
3.2.5.2. GC-MS analysis of FAMEs
FAMEs were analyzed by GC-MS (PerkinElmer, Turbomass Gold, Mass
spectrometer) equipped with FID using fused silica Rtx-2330 column (Restek made,
30 m × 0.32 mm ID and 0.20 µm film thickness). Injector port and detector were set
up at 230 and 250 °C, respectively, N2 was used as carrier gas. Initially, column
temperature was maintained at 120 °C, followed by increasing to 220 °C in 20 min,
and hold at 220 °C for 10 min. The FAMEs were identified by comparing their
fragmentation pattern and retention time with authentic standards and also with the
NIST library.
Screening of phytoconstituents in M. oleifera cultivars
49
3.2.6. Assessment of genetic diversity
3.2.6.1. DNA extraction, purification and quantification
All the reagents used for DNA extraction, purification and PCR amplification
were of molecular biology grade and purchased from Sigma-Aldrich (Bangalore,
India). Oligos were synthesizes in BioServe Biotechnologies Pvt. Ltd., Hyderabad
(India).
Genomic DNA was extracted from leaves of the different M. oleifera cultivars
by the CTAB method, according to Murray and Thompson (1980) with some
modifications to eliminate phenolics. From each cultivar, one gram of leaves were
ground in liquid nitrogen to a fine powder in a pre-chilled mortar and transferred into
a 30 ml centrifuge tube containing 10 ml of DNA extraction buffer containing 0.1 M
Tris chloride (pH 8.0), 0.02 M EDTA (pH 8.0), 1.4 M NaCl, 2% CTAB (w/v), 2%
polyvinylpyrrolidone (w/v) and 0.2% β-mercaptoethanol (v/v) and incubated in a
water bath at 65˚C for 60 min. The tubes were cooled at room temperature and an
equal amount of chloroform : isoamyl alcohol (24:1) was added, mixed thoroughly by
gentle inversion and finally centrifuged at 10,000 g for 15 min at 20°C. The upper
aqueous layer was transferred to a fresh sterile centrifuge tube and 0.7 vol chilled
isopropanol was added and incubated overnight at -20°C. The tubes were centrifuged
at 10,000 g for 15 min at 4°C and the pellet was washed with 70% ethanol, air dried
and dissolved in 200 µl TE buffer and stored at 4°C. For DNA purification, 5 µl
RNase (10 mg/ml) was added to total isolated DNA (200 µl) and incubated at 37°C
for 60 min. An equal volume of phenol : chloroform : isoamyl alcohol (25:24:1) was
added and mixed gently. The tubes were centrifuged at 10,000 g for 5 min and the
aqueous layer was transferred to fresh eppendorf tube and 1/10 volume of sodium
Screening of phytoconstituents in M. oleifera cultivars
50
acetate (3 M, pH 5.2) and a double volume of chilled absolute ethanol was added.
After 30 min, the mixture was centrifuged at 10,000 g for 5 min and finally, the pellet
was washed with 70% ethanol, dried and dissolved in 200 µl TE buffer. For
quantification of genomic DNA, the absorbance of the DNA samples was measured at
260 nm in a Nano Drop 1000 (Thermo Scientific). After quantification, the quality of
the purified DNA was analysed in a 0.7% (w/v) agarose gel.
3.2.6.2. RAPD, ISSR and Cytochrome P450 based marker analyses
A total of 25 RAPD primers, 10 ISSR primers and 11 Cytochrome P450 based
primers were used for the polymorphism survey. The RAPD assay was carried out in
0.2 ml PCR vials containing 1 X reaction buffer, 200 µM dNTPs mix, 0.4 µM primer,
50 ng DNA template, 1 U Taq DNA polymerase and sterile distilled water to a final
volume of 25 µl. The content was gently mixed by spinning for few sec. The PCR
amplification was performed with a thermocycler (Eppendorf, Germany). The
standardised amplification was performed at an initial denaturation at 94°C for 4 min,
followed by 40 cycles of denaturation at 94°C for 30 sec; primer annealing based on
Tm for 1 min; primer extension at 72°C for 2 min and final primer extension at 72°C
for 10 min. For ISSR analysis, ISSR primers were used and other conditions was the
same as for RAPD. For Cytochrome P450 based markers, PCR amplification were
performed using 0.4 µM forward primer and 0.4 µM reverse primer in a volume of 25
µl with same PCR conditions as that of RAPD. The annealing temperature of each
primer was calculated online by OligoAnalyzer 3.1 (http://www.idtdna.com)
according to the sequence of the oligos.
PCR amplified products were analysed by agarose gel electrophoresis using
1.5% agarose in 1X TAE buffer. The amplicon sizes were measured with a 100-5000
Screening of phytoconstituents in M. oleifera cultivars
51
bp DNA ladder. Respective gels were stained with 10 ppm ethidium bromide
followed by image capturing using a gel documentation system (Herolab, Germany).
The procedure was repeated twice for each DNA set and reproducible primers were
subjected for diversity analysis.
3.2.6.3. Data analysis
The various sizes of amplified products were scored for presence (1) or absence (0) in
the eight cultivars to generate a binary matrix. Binary matrix data were analysed by
the software NTSYS-pc, version 2.11w, to calculate the Jaccard’s similarity
coefficient (using the SIMQUAL programme) and generate a dendrogram based on
the unweighted pair-group method using the arithmetic average (UPGMA) method
(Rohlf 2001). Genetic similarity between cultivars was calculated according to
Jaccard’s similarity coefficient (Jaccard 1980). The computer programme WINBOOT
was used to determine the robustness of the dendrogram, with 2,000 replications
along with Jaccard’s coefficient (Yap and Nelson 1996).
Per cent polymorphisms were calculated for each primer combination
according to the formula: % Polymorphism=p/(m+p), where ‘p’ is total number of
polymorphic bands and ‘m’ is the total number of monomorphic bands of the primer
combination used. Multiplex ratio (MR) for each marker was calculated by using the
formula: MR=(m+p)/n, where ‘p’ is the total number of polymorphic bands, ‘m’ is the
total number of monomorphic bands and ‘n’ is the total number of primer
combinations used (Powell et al. 1996). Polymorphic information content (PIC) was
calculated using the formula: PIC=1-Ʃpi2, where ‘pi’ is the frequency of the ith allele.
Average heterozygosity (Hav) was obtained by taking the average of PIC values
obtained for all the markers. Marker index (MI) was obtained by multiplying the
Screening of phytoconstituents in M. oleifera cultivars
52
average heterozygosity (Hav) with the multiplex ratio (MR).
3.2.7. Statistical analysis
All the samples for carotenoids, folate, ascorbic acid, tocopherol, iron and
fatty acid methyl esters were extracted and analysed in triplicate. Values from
triplicate determinations of each sample were averaged and represented as means with
standard deviations. Data were analysed statistically by the SPSS 17.0 software by
one-way ANOVA and homogenous subsets were determined to separate the mean
values of the different treatments. Means with statistically significantly difference
(different subsets) was marked with different alphabetical letters.
3.3. Results & Discussion
3.3.1. Carotenoids
3.3.1.1. Purification of standards used for LC-MS quantification
Six carotenoids were purified from leaves of M. oleifera by the combined use
of open column chromatography and preparative thin layer chromatography (Figure
3.3). The identification of carotenoids was done by visible spectra, spectral fine
structure and chromatographic behaviour, and confirmed by mass spectroscopy. Mass
spectra of purified carotenoids are shown in Figure 3.4. Percentage of purity, visible
spectra (λmax), spectral fine structures (%III/II) and APCI +ve mass spectra (m/z) of
purified carotenoids is given in Table 3.1. The purity of the purified carotenoids was
between 89% (trans-β-carotene) and 94% (trans-luteoxanthin) and the concentration
of these carotenoids was corrected accordingly. Cis isomers were co-eluted with
trans-β-carotene in alumina column, resulting in decreasing the purity. Similarly, low
purity of β-carotene was observed during the purification of carotenoids compounds
from lettuce (Kimura and Rodriguez-Amaya 2002). Visible spectra (λmax) and spectral
Screening of phytoconstituents in M. oleifera cultivars
53
fine structures (%III/II) obtained in our study was comparable to earlier reports in
other plants (Britton 1976; Davies, 1976; Kamffer et al. 2010). APCI+-MS
fragmentation ions (m/z, 551) obtained for trans-lutein was due to removal of water
molecule (m/z, 18). A similar APCI+-MS fragmentation behaviour of lutein was
reported by Kamffer et al. (2010). According to the guidelines of Davies (1976), 13-
cis-β carotene and 13-cis-lutein were quantified on the basis of the absorption
coefficient of β-carotene and lutein, respectively, due to same chromophore and
molecular weight. Similarly, for luteoxanthin, the absorption coefficient of 2500
(absorbance at a given wavelength of a 1% solution in 1 cm light-path
spectrophotometer cuvette) was used, due to unavailability of an absorption
coefficient (Davies 1976).
Screening of phytoconstituents in M. oleifera cultivars
54
Figure 3.3. Carotenoids eluted from Natural alumina packed open column (A & B),
Numbers showing the increased polarity. S. No- 1 to 6, β-carotene and their isomers;
7-12, Lutein and their isomers; these carotenoids were further purified by preparative
TLC. C- Showing the separation of column eluted polar fraction of carotenoids on
preparative TLC.
Screening of phytoconstituents in M. oleifera cultivars
55
Figure 3.4. Mass spectrum (APCI+) of different carotenoids purified from leaves of
M. oleifera (cv. PKM-1).
Table 3.1. LC-APCI+-MS data of carotenoids extracted and purified from M. oleifera
(cv. PKM-1).
Peak
No.
Carotenoids tR
(min.)
%
purity
λmax (nm)a %
(III/II)
m/z m/z observed
1 Trans-luteoxanthin 8.92 94 401,423,450 89 600 601 [M+H]+, 583 [M-H20+H]+
2 13-cis-lutein 11.44 92 328, 420, 447, 474 15 568 569 [M+H]+
3 Trans-lutein 13.37 93 424, 447, 475 55 568 551 [M+H]+
4 Trans-zeaxanthin 14.77 90 430, 452, 478 17 568 551 [M-H20+H]+
5 15-cis-β-carotene 20.59 93 340, 420, 447, 472 12 536 537 [M+H]+
6 Trans-β-carotene 26.67 89 429, 451, 478 16 536 537 [M+H]+
arecorded in acetone. The %III/II is the ratio between the height of the longest-
wavelength absorption peak (III) and that of the middle absorption peak (II),
multiplied by 100 (Britton 1995).
Screening of phytoconstituents in M. oleifera cultivars
56
Table 3.2. Carotenoids composition (mg/100g FW) of fresh leaves of eight M.
oleifera cultivars, analysed by LC-APCI-MS
Cultivar/Composition PKM-1 PKM-2 GKVK-1 Amar-32 CO-1 Dhanraj Bhagya PAVM-1
Trans-luteoxanthin 5.20b 4.55d 5.00ab 3.92d 5.68a 4.18d 4.73c 2.58e
13-cis-lutein 2.31d 1.58f 2.49c 1.60f 2.23d 1.94e 7.12a 5.80b
Trans-lutein 36.88b 32.76c 39.66b 29.64c 41.16a 34.85b 31.27c 17.60d
Trans-zeaxanthin 5.46c 2.26h 3.95e 3.38f 4.42d 2.87g 13.54a 5.99b
13-cis-β-carotene 0.69a 0.49d 0.69a 0.40e 0.63bc 0.61c 0.66ab 0.47d
Trans-β-carotene 18.27cd 14.68f 18.36c 15.72e 20.77b 17.19d 23.15a 11.86g
Total carotenoidsa
68.81c 56.32e 70.14bc 54.66e 74.90b 61.63d 80.48a 44.30f
Values are the mean of tree replications. Different letters among (cultivars) columns
indicate statistically significant differences between the means (P < 0.05). a sum of all
the individual carotenoids.
Figure 3.5. HPLC chromatograms of carotenoids (450 nm) in leaves of M. oleifera
(cv. PKM-1). Peak identification: (1) trans-luteoxanthin, (2) 13-cis-lutein, (3) trans-
lutein, (Chl) Chlorophyll (not quantified), (4) trans-zeaxanthin, (5) 15-cis-β-carotene
and (6) trans-β-carotene.
Screening of phytoconstituents in M. oleifera cultivars
57
Table 3.3. Carotenoid composition (µg/g FW) of flowers and fruits of M. oleifera (cv.
PKM-1), analysed by LC-APCI-MS
Fruits Flowers
Trans-luteoxanthin 1.962 ± 0.08 0.308 ± 0.02
13-cis-lutein 0.835 ± 0.04 0.513 ± 0.03
Trans-lutein 15.425 ± 0.61 1.055 ± 0.05
Trans-zeaxanthin 2.109 ± 0.09 0.791 ± 0.04
13-cis-β-carotene 0.150 ± 0.01 1.340 ±0.06
Trans-β-carotene 9.186 ± 0.35 1.438 ± 0.07
Total carotenoids 29.668 5.445
Values are the mean ± S.D. of tree replications
3.3.1.2. Quantification of carotenoids from leaves, flowers and fruits
The gradient elution system applied in this study for the quantification of
carotenoids provided good resolution, precision and repeatability (Figure 3.5). Fresh
leaves of different cultivars, flowers and fruits of M. oleifera were analysed by LC-
MS for luteoxanthin (RT-9.8min), 13-cis-lutein (RT-14.4min), trans-lutein (RT-
16.9min), trans-zeaxanthin (RT-19.2min), 13-cis-β-carotene (RT-30.0min) and trans-
β-carotene (RT-42.5 min). Two peaks were marked as chlorophyll, since these were
disappeared after saponification. The study revealed significant differences (P<0.05)
among the leaves of cultivars for all the carotenoids studied (Table 3.2). Among the
eight cultivars screened, Bhagya (KDM-1) was found to contain the maximum
amount (mg/100g FW) of trans-zeaxanthin (13.54) and total carotenoids (80.48),
followed by CO-1, which had the maximum amount of trans-luteoxanthin (5.68) and
trans-lutein (41.16). The content of trans-zeaxanthin showed the largest variation of
the carotenoids (2.26-13.54 mg/100 g fresh weight). Similarly, 13-cis-β-carotene was
Screening of phytoconstituents in M. oleifera cultivars
58
recorded maximum (0.69 mg/100g FW) in cv. PKM-1. Leaves contained 23.2 and
126.5 times higher amounts of total carotenoids than fruits and flowers, respectively
(Table 3.3). Trans-lutein was found to be the major carotenoid in leaves, flowers and
fruits, accounting for 53.6, 32.5 and 52.0% of the total carotenoids, respectively.
Up to 90% of the carotenoids in the diet and human body are represented by α-
carotene, β-carotene, lycopene, lutein and cryptoxanthin (Gerster 1997). Carotenoids
play an important role in human diet due to its potent antioxidants activity and
preventive role against number of diseases (Nakazawa et al. 2009; Kuhnen et al.
2009). Lutein and β-carotene contents in leaves of M. oleifera found in this study is in
agreement with such studies carried out earlier (Nambiar and Seshadri 2001; Liu et al.
2007b). However, in the present study a detailed analysis of different types of
carotenoids were investigated and such information is very vital in food fortification
development program. Carotenoids were quantified without saponification, as
saponification is unnecessary for leafy vegetables which are low-lipid materials and
essentially free of carotenol esters. Saponification is also a source of artefact
formation and degradation of carotenoids (Rodriguez-Amaya 2001). The gradient
elution programme used in the present study allowed the separation of all carotenoids
within 35 minutes, compared to earlier reports as described by Kimura and
Rodriguez-Amaya (1999), Darnoko et al. (2000) and Lakshminarayana et al. (2005)
evaluated the neoxanthin, violaxanthin, lutein, zeaxanthin and β-carotene content in
Indian leafy vegetables, including M. oleifera. However in the present investigation,
distribution pattern of six major carotenoids were described in fruits, flowers and
among the leaves of eight cultivars of M. oleifera. Lakshminarayana et al. (2005)
opined that dominance of lutein and absence of α-carotene in green leafy vegetables
Screening of phytoconstituents in M. oleifera cultivars
59
may be due to complete conversion of α-carotene to lutein. A similar phenomenon
might have been taken place in the leaves of M. oleifera also. There appear to be no
previous reports on the presence of luteoxanthin in M. oleifera. However, the presence
of luteoxanthin is reported in variety of plants including lichens (Czeczuga and
Kantvilas 1990), pumpkin (Matus et al. 1993) and chrysanthemum (Kishimoto et al.
2004). Presence of small amounts of luteoxanthin in a sample may also be due to
epoxide formation from violaxanthin at low pH during extraction (Kamffer et al.
2010). According to the guidelines of Britton and Khachik (2009), Moringa
carotenoids can be classified as very high, as it contain ≥2 mg/100 g (Fresh weight) of
lutein, beta-carotene and zeaxanthin.
Nutrient composition of fruits and vegetables are influenced by several factors,
including genetic makeup of the plant. Significant differences for carotenoid content
is well studied among cultivars in many fruits and flowers including pumkin
(Murkovic et al. 2002), Acerola (Malpighia punicifolia) (De Rosso and Mercadante
2005), tomato (Kuti and Konuru 2005), and Capsicum annuum (Topuz and Ozdemir
2007). Similarly, significant variation was observed for all the carotenoids studied in
the present investigation in M. oleifera which is not recorded earlier. Among the eight
cultivars of M. oleifera, leaves of Bhagya and CO-1 cultivar were found to be superior
with respect to content of individual and total carotenoids. The significant differences
could indicate that these cultivars could be exploited for their nutritive value.
Screening of phytoconstituents in M. oleifera cultivars
60
In summary, six carotenoids were purified from M. oleifera by use of open
column chromatography and thin layer chromatography. Among leaves of eight
cultivars screened, highest content of carotenoids were recorded in Bhagya (KDM-1)
and CO-1 cultivars. Trans-lutein was found to be the major carotenoid in leaves,
flowers and fruits, followed by trans-β-carotene. The methodology used in the present
investigation for extraction, purification and quantification of carotenoids is
simplified, which can be used in screening of carotenoids in other plants. Results also
explore the M. oleifera leaves as a rich source of carotenoids, which is significant for
its implications in malnutrition program to alleviate the vitamin A deficiency. This is
the first report of detailed carotenoid composition of fruits and flowers of Moringa
oleifera, and quantitative variation in carotenoid content among the commercially
cultivated Indian cultivars.
3.3.2. Folate
The study revealed significant differences (P<0.05) among the cultivars for total
folate content (Table 3.4). Among eight cultivars screened, Bhagya found most
promising to contain maximum amount of total folate (167.4 µg/100 g FW). 5-HCO-
H4 (73.1 µg/100g FW) and H4 folate (32.6 µg/100g FW) was found to be the most
dominant form of folate in fresh leaves of BHAGYA cultivar (Table 3.5, Figure 3.6).
Significant differences were recorded for total folate content in leaves of 30 days old
seedling (113.64 µg/100g FW; cv. Bhagya), and leaves of two year old M. oleifera
plants (167.4 µg/100g FW; cv. Bhagya).
Screening of phytoconstituents in M. oleifera cultivars
61
Table 3.4. Total folate content of fresh leaves of eight Moringa oleifera cultivars,
analyzed by microbiological assay
Cultivar name Total folate (µg/100g FW)
PKM-1 127.3±5.5d
PKM-2 134.4±6.1d
GKVK-1 137.7±6.4d
Dhanraj 142.7±11.2c
CO-1 142.8±4.3c
Amar 32 154.4±8.9b
Bhagya 167.4±11.3a
PAVM-1 130.2±10.2d
Values are mean ± S.D. of three replicates. Different letters indicate statistically
significant differences between the means (P < 0.05).
Table 3.5. Folate content of fresh leaves of Moringa oleifera (c.v. Bhagya), purified
by immunoaffinity chromatography and analyzed by HPLC
S/No Folate form Folate (µg/100g FW)
1 5,6,7,8-Tetrahydrofolic acid (H4 folate) 32.63±2.3
2 5-Methyl-5,6,7,8-tetrahydrofolic acid (5-CH3-H4 folate) 21.12±2.4
3 10-Formylfolic acid (10-HCO-folic acid) 4.23±1.2
4 5-Formyl-5,6,7,8-tetrahydrofolic acid (5-HCO-H4
folate) 73.16±6.3
5 Total folate 131.14
Values are mean ± S.D. of three replicates
Screening of phytoconstituents in M. oleifera cultivars
62
Figure 3.6. HPLC chromatograms of standards and purified folates form M. oleifera
leaves. (1) 5,6,7,8-Tetrahydrofolic acid (H4 folate); (2) 5-Methyl-5,6,7,8-
tetrahydrofolic acid (5-CH3-H4 folate); (3) 10-Formylfolic acid (10-HCO folic acid)
and (4) 5-Formyl-5,6,7,8-tetrahydrofolic acid (5-HCO-H4 folate).
In the present investigation significant variation in total folate content among
various cultivars were recorded and also different forms of folates in leaves of M.
oleifera were purified and quantified. Total folate content in fresh leaves of unknown
cultivar of M. oleifera from India and Fijian have been reported to vary from 83 to
101 µg/100 g fresh weight (Devi et al. 2008; Vishnumohan et al. 2009), however in
our study total folate content was recorded between 127.3 (cv. PKM-1) to 167.4 (cv.
Bhagya) µg/100 g fresh weight, which shows the significant variation (p˂0.05). H4-
folate, 5-CH3-H4-folate, 5-CHO-H4-folate and 10-HCO-folic acid are major forms of
folate present in cereal grains (Pfeiffer et al. 1997), fruits, yeast (Patring et al. 2005),
vegetables (Hefni et al. 2010) and legumes (Yarbaeva et al. 2011). Different forms
(derivatives) of folates in Moringa oleifera leaves have not been studied earlier. In the
present study, we have recorded the 5-CHO- H4-folate as the major form of folate
Screening of phytoconstituents in M. oleifera cultivars
63
present in Moringa leaves (c.v. Bhagya), which accounted for 55.8% of total folate,
followed by H4-folate (24.9%), 5-CH3-H4-folate (16.1%) and 10-HCO-folic acid.
Total folate quantified by HPLC (131.14 µg/100g FW) was shown 27.6% lower than
microbiological assay, which can be explained by lack of folate standards for all
folate forms and loss of folate during purification. These results are in agreement with
previous studies, who reported folate contents determined by HPLC to be 20–52%
lower when determined by HPLC (Ruggeri et al. 1999; Konings 1999).
In conclusion, Bhagya genotypes were found superior with respect to total
folate content. The significant genotypic effects would indicate higher degree of
heritability in these genotypes that could be exploited within breeding programs to
improve the nutritive status of this plant, and also can be used in folate rich food
formulation. This is the first report of composition of folate derivatives in Moringa
oleifera leaves. Result of present investigation will be useful to improve the
nutritional quality of Moringa oleifera plant through ecofriendly approach.
3.3.3. Iron, tocopherol and ascorbic acid
Fresh leaves of six Moringa oleifera cultivars were analyzed for ascorbic acid and α-
tocopherol by HPLC (Figure 3.7), and total iron by atomic absorption spectroscopy.
The study revealed significant differences (P <0.05) among the genotypes for all the
nutrients studied. Among six genotypes screened, PKM-1 genotype registered
maximum amount of total iron (4.08 mg/100g FW) and ascorbic acid (224.2 mg/100g
FW), whereas, α-tocopherol was recorded maximum in CO-1 cultivar (Table 3.6).
Maximum variation (9.1 to 20.9 mg/100g FW) was also recorded for α-tocopherol
content among the studied cultivars.
Screening of phytoconstituents in M. oleifera cultivars
64
Figure 3.7. HPLC chromatograms (UV, 298nm) of tocopherols standards (a), α-
tocopherol in leaves of M. oleifera (b), ascorbic acid standard (UV, 254nm) (c), and
ascorbic acid in Moringa leaves (d).
Screening of phytoconstituents in M. oleifera cultivars
65
Table 3.6. Content of iron, α-tocopherol and ascorbic acid in fresh leaves of M.
oleifera cultivars
Cultivar Iron (Fe) α-tocopherol Ascorbic acid
PKM-1 4.08a 17.1b 224.2a
PKM-2 3.12cd 11.6c 224.1a
GKVK-1 3.47bc 17.4ab 195.8b
Dhanraj 3.42c 10.8c 189.0bc
CO-1 3.35c 20.9a 168.3c
Amar 32 3.88ab 9.1cd 165.4c
Values (mg/100g FW) are mean of three replicates. Different letters indicate
statistically significant differences between the means (P < 0.05).
Vitamin E consists of four tocopherols (α, β, γ and δ-tocopherol) and four
tocotrienols (α, β, γ and δ- tocotrienols), of which, α -tocopherol is the most
biologically active form in humans (Omenn 1996). In the present study, higher levels
of α-tocopherol was recorded in leaves, this may contribute to beneficial aspects of
Moringa leaves, as vitamin E has importance in human health, mainly in the
prevention of coronary heart disease (Ros 2009), breast cancer (Zhang et al. 2009),
and protection against nicotine induced oxidative stress in the brain (Das et al. 2009).
Similarly, higher level of ascorbic acid in leaves of M. oleifera is also very beneficial
for consumers. In our experiment, ascorbic acid level was found between 165.4 to
224.23 mg/100g fresh weight, which is similar to earlier report in fresh leaves of
Moringa oleifera (Marcu 2005). Total iron content in fresh leaves have been reported
from 4 to 40 mg/100 g fresh weight (Nambiar and Seshadri 2001; Marcu 2005; Yang
et al. 2006), however in our study total iron content was recorded between 3.12 to
4.08 mg/100 g fresh weight. α-tocopherol content recorded in our study (9.1 to 20.9
mg/100g FW) was also similar to the findings of Yang et al. (2006) and Ching and
Screening of phytoconstituents in M. oleifera cultivars
66
Mohamed (2001). Earlier studies on screening of iron, α-tocopherol, ascorbic acid and
other phytoconstituents content in Moringa oleifera is restricted to unknown cultivars
or genotypes. This is the first report of detailed profile of phytoconstituents in
commercially grown cultivars of M. oleifera. In conclusion PKM-1 and CO-1 were
found superior with respect to the nutrients studied in present investigation.
3.3.4. Fatty acid methyl esters
In present study, 15 fatty acids were identified by GC-FID/MS in leaves, flowers and
fruits (immature pods) of M. oleifera (Table 3.7 and 3.8). The GC-FID
chromatograms with identified peaks are provided in Figure 3.8. In leaves, α-linolenic
acid (C18:3, cis-9,12,15) was found in highest quantity (49-59%) followed by
palmitic acid (C16:0) (16-18%), and linoleic acid (18:2, cis-9,12) (6-13%).
Palmitoleic (C16:1, cis-9), stearic (C18:0), oleic (C18:1, cis-9) and erucic acid
(C22:1) were accounted 1 to 5% in leaves of different cultivars. Significantly higher
amounts of palmitic and linoleic acid were recorded in fruits (24.33 & 16.91%) and
flowers (24.25 & 15.48%) compared to leaves. In general, leaves accounted far rich
content of polyunsaturated fatty acid (PUFA, 63.22-67.43%) and poor in total
saturated fatty acid (SFA, 24.59-27.95%) compared to fruits and flowers. SFA and
UFA (unsaturated fatty acid) showed a ratio of 0.33 (cv. DHANRAJ) to 0.39 (cv.
PKM-2) in leaves, 0.53 in flowers and 0.56 in fruits. Similarly, PUFA and MUFA
were found in ratio of 5.68 (cv. DHANRAJ) to 9.71 (cv. CO-1) in leaves, 1.11 in
flowers and 2.79 in fruits. The total lipid content was recorded in range of 1.92%
(flowers) to 4.82% (leaves, cv. CO-1). The Mass spectrum and fragmentation pattern
of the identified FAMEs were found concordant with authentic standards and also
with the NIST library (Figure 3.8). Mass spectrum of caprylic acid (C8:0), capric acid
Screening of phytoconstituents in M. oleifera cultivars
67
(C10:0), tridecylic acid (C13:0) and erucic acid (C22:1) were not obtained from
flowers, fruits and leaves of M. oleifera. This may be due to the presence of these
fatty acids at very low concentrations. Quantification of these four fatty acids were
done on the basis of retention time with standards.
Least but significant influence of genotypic factor was recorded for fatty acid
composition in leaves of M. oleifera. Maximum amount of α-linolenic acid (59.52%),
palmitic acid (18.39%), linoleic acid (13.63%), UFA (75.41%) and PUFA (67.43%)
were recorded in the leaves of AMAR-32, PKM-1, PKM-2, DHANRAJ and CO-1
cultivars, respectively. In general, leaves of PKM-1, PKM-2, CO-1 and DHANRAJ
cultivars were found superior over others in relation to fatty acid profile.
Screening of phytoconstituents in M. oleifera cultivars
68
Figure 3.8. GC-FID chromatograms of fatty acid methyl esters in leaves, flowers and
fruits of Moringa oleifera (cv.PKM-1). Peak numbers are correspondent to Table 3.7.
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
0.00
0.25
0.50
0.75
1.00
1.25
1 2 3
5
6
4 7
8
9
10
11
12
13
14
15
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.00.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
1 2 3
5
6
4 7
8
9
10
11
12
13
14
15
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
0.0
2.5
5.0
7.5
1 2 3 5
6
4 7 8 9
10
11
12
13 14 15
Flowers
Fruits
Leaves
Screening of phytoconstituents in M. oleifera cultivars
69
Table 3.7. Composition of fatty acids in leaves, flowers and fruits of M. oleifera (cv.
PKM-1)*
S/No Fatty acid methyl ester Fruits Flowers Leaves
1 Caprylic acid (C8:0) 0.12c 0.21b 0.57a
2 Capric acid (C10:0) 0.02b 0.07a 0.01c
3 Lauric acid (C12:0) 0.17b 0.20a 0.12c
4 Tridecylic acid (C13:0) 0.17b 0.05c 0.33a
5 Myristic acid (C14:0) 1.93a 1.45b 0.93c
6 Palmitic acid (C16:0) 24.33b 24.25b 18.39a
7 Palmitoleic acid (C16:1, cis-9) 1.49b 0.27c 2.52a
8 Stearic acid (C18:0) 6.23a 5.87b 4.32c
9 Oleic acid (C18:1, cis-9) 9.81b 24.09a 3.30c
10 Linoleic acid (C18:2, cis-9,12) 16.91a 15.48b 11.01c
11 Arachidic acid (C20:0) 0.47b 0.43b 1.06a
12 Linolenic acid (C18:3, cis-9,12,15) 30.42b 18.82c 54.27a
13 Behenic acid (C22:0) 0.54c 0.76a 0.65b
14 Erucic acid (C22:1) 5.68b 6.58a 0.95c
15 Lignoceric acid (C24:0) 1.72a 1.47c 1.57b
16 Total saturated fatty acids (SFA) 35.69a 34.76a 27.95b
17 Total unsaturated fatty acids (UFA) 64.31b 65.24b 72.05a
18 Total mono unsaturated fatty acids (MUFA) 16.98b 30.94a 6.77c
19 Total poly unsaturated fatty acids (PUFA) 47.33b 34.29c 65.28a
20 PUFA: SFA 1.33b 0.99c 2.34a
21 PUFA: MUFA 2.79b 1.11c 9.64a
22 SFA: UFA 0.56a 0.53a 0.39b
23 Total lipids 2.83b 1.92c 4.67a
*Values are % of total fatty acids (n=03). Different alphabet letters indicate the
statistical significant difference within the leaves, flowers and fruits between the
columns (p< 0.05).
Screening of phytoconstituents in M. oleifera cultivars
70
Table 3.8. Composition of fatty acids in leaves of different cultivars of M. oleifera*
Fatty acid methyl ester BHAGYA DHANRAJ PAVM-1 AMAR-32 C0-1 GKVK-1 PKM-1 PKM-2
1 Caprylic acid (C8:0) 0.09e 0.05f 0.02g 0.32c 0.40b 0.24d 0.57a 0.33c 2 Capric acid (C10:0) 0.01f 0.04b 0.01g 0.03c 0.04a 0.02d 0.01f 0.02e 3 Lauric acid (C12:0) 0.39a 0.20c 0.07h 0.15d 0.33b 0.14e 0.12f 0.11g 4 Tridecylic acid (C13:0) 0.38b 0.34d 0.36c 0.39b 0.51a 0.39b 0.33d 0.32d 5 Myristic acid (C14:0) 0.92c 1.62a 0.80d 1.34b 0.69e 0.69e 0.93c 0.62f 6 Palmitic acid (C16:0) 17.47bcd 17.27cd 17.22cd 18.19ab 17.99abc 17.99abc 18.39a 16.93d 7 Palmitoleic acid (C16:1, cis-9) 2.70c 2.60cd 3.17a 2.87b 3.13a 2.63cd 2.52d 2.55d 8 Stearic acid (C18:0) 3.73d 3.04e 4.37b 2.96e 3.87cd 3.99c 4.32b 5.24a 9 Oleic acid (C18:1, cis-9) 3.37cd 3.30de 3.74b 2.96f 3.14e 3.52c 3.30de 4.87a 10 Linoleic acid (C18:2, cis-9,12) 10.49c 6.46d 11.37b 6.47d 11.38b 11.34b 11.01b 13.63a 11 Arachidic acid (C20:0) 0.48d 0.35e 0.16g 0.29f 0.17g 0.64c 1.06b 2.11a 12 Linolenic acid (C18:3, cis-9,12,15) 55.22bc 57.67ab 55.63bc 59.52a 56.05bc 55.02c 54.27c 49.58d 13 Behenic acid (C22:0) 0.76a 0.56e 0.67b 0.47f 0.53e 0.64c 0.65bc 0.41g 14 Erucic acid (C22:1) 2.45c 5.39a 1.04f 2.97b 0.67g 1.39e 0.95f 2.11d 15 Lignoceric acid (C24:0) 1.53a 1.12d 1.37b 1.08d 1.10d 1.36b 1.57a 1.18c 16 Total saturated fatty acids (SFA) 25.77cd 24.59d 25.05cd 25.22cd 25.63cd 26.10b 27.95a 27.27a 17 Total unsaturated fatty acids (UFA) 74.23b 75.41a 74.96a 74.78b 74.37b 73.90cd 72.05cd 72.73cd 17 Total mono unsaturated fatty acids (MUFA) 8.52c 11.28a 7.95d 8.80c 6.94f 7.54e 6.77f 9.53b 18 Total poly unsaturated fatty acids (PUFA) 65.72abc 64.13bc 67.01ab 65.98abc 67.43a 66.36abc 65.28abc 63.21c 19 PUFA: SFA 2.55b 2.61ab 2.68a 2.62ab 2.63ab 2.54b 2.34c 2.32c 20 PUFA: MUFA 7.72d 5.68f 8.43c 7.50d 9.71a 8.80b 9.64a 6.63e 22 SFA: UFA 0.35c 0.33cd 0.33cd 0.34cd 0.34cd 0.35c 0.39a 0.37b 23 Total lipids 4.59b 4.71a 4.62a 4.60a 4.82a 4.55b 4.67a 4.60a
* Values are % of total fatty acids (n=03). Different alphabet letters indicate the statistical significant difference between the cultivars (p< 0.05).
Screening of phytoconstituents in M. oleifera cultivars
71
Figure 3.9. GC-FID mass spectrum of major fatty acid methyl esters identified in
leaves, flowers and fruits of Moringa oleifera (cv.PKM-1), and comparison with
standard fatty acids.
39 49 59 69 79 89 99 109 119 129 139 149 159 169 179 189 199 209 219 229 239 249 259m/z0
100
%
ramesh4 1665 (11.402) Cm (1657:1678) Scan EI+ 5.86e774
4341 55
5444
6957
596760
71
87
75
83
8179
1711439788 129101
115123 138
157144166
183172
214186
194 211199 236 255
239
C12:0
39 49 59 69 79 89 99 109 119 129 139 149 159 169 179 189 199 209 219 229 239 249 259m/z0
100
%
5.52e774
43
41
55
44 54
6957
5967
61
71
87
75
83
8179
143
9788 101129111 115
135
199
185157144171168
194
242211
200236
214257
C14:0
43 63 83 103 123 143 163 183 203 223 243 263 283m/z0
100
%
1.20e755
41
43
54
4453
51
69
67
57
59
60
74
71
83
81
79
9784
87
95
93
98
110
109
101
236123
112194152
138129 151 192153
165 183195
207 218 225
237
268238257250 284
C16:1, Cis-9
37 47 57 67 77 87 97 107 117 127 137 147 157 167 177 187 197 207 217 227 237 247 257 267 277m/z0
100
%
7.33e774
43
4155
44 54
6957
59
6760
87
75
83
81
1439788 129
101111
115
135
270227
185171157144 167
199194 213
239229
255271
C16:0
48 68 88 108 128 148 168 188 208 228 248 268 288m/z0
100
%
1.64e767
55
41
43 54
5344
51
57
59
65
81
6879
77
8295
83
93
96
109
97
108
105
123111
115150135125
139294
164153
263178220
183236
295
C18:2, cis-9,12
38 48 58 68 78 88 98 108 118 128 138 148 158 168 178 188 198 208 218m/z0
100
%
2.26e674
43
41
55
44
5445
57
69
6759
65
87
8375
8179
191
979588
143129115111
101 125135
171
157
183214192
206
32 42 52 62 72 82 92 102 112 122 132 142 152 162 172 182 192 202 212 222 232 242 252m/z0
100
%
1.80e774
43
41
55
44 54
57 69
59
6760
71
87
75
83
8179
143
1299788 101
115111
125 135
199
157144 185171168
242211200 243
35 45 55 65 75 85 95 105 115 125 135 145 155 165 175 185 195 205 215 225 235 245 255 265 275m/z0
100
%
1.97e655
43
41
4454
53
45
69
5767
59
65
74
83
81
75
9784
87
95
9391
98
111
109
101
123
112236152124
137129 141
194
192165
153183
179 195213 268
37 47 57 67 77 87 97 107 117 127 137 147 157 167 177 187 197 207 217 227 237 247 257 267 277m/z0
100
%
2.15e874
43
41 55
5444
695759
6760
87
75
83
81
270143
1299788
93
101115
125 139
227185171
157144167
199194 213
239229 269
271
49 69 89 109 129 149 169 189 209 229 249 269 289m/z0
100
%
8.72e78167
55
41
54
43
53
45
59
66
60
79
68
69
77
82
95
83
93
87
96
109
10897
105
123
111294135
125150
139164
152263178
220234
265 292295
48 68 88 108 128 148 168 188 208 228 248 268 288m/z0
100
%
ramesh4 4035 (23.355) Cm (4032:4039) Scan EI+ 1.44e779
67
55
41
43
54
53
45
57
59
65
69
77
71
81
95
93
82
87
108
10796
105
101
109
121
110
115
135123
129
149137 236163151 173 178
191 292263257284
C18:3, cis-9,12,15
48 68 88 108 128 148 168 188 208 228 248 268 288m/z0
100
%
2.23e879
67
5541
43
53
51
59 66
60
77
74
81
93
91
82
95108
107
105
101
121109
119135
122
131149
137 292173163 236191178223
217203
261249
C12:0
C14:0
C16:1, Cis-9
C16:0
C18:2, cis-9,12
C18:3, cis-9,12,15
MS of fatty acid standards MS of fatty acid from M. oleifera leaves
Screening of phytoconstituents in M. oleifera cultivars
72
Figure 3.9. (Continued) GC-FID mass spectrum of major fatty acid methyl esters
identified in leaves, flowers and fruits of Moringa oleifera (cv.PKM-1), and
comparison with standard fatty acids.
29 49 69 89 109 129 149 169 189 209 229 249 269 289 309m/z0
100
%
ramesh4 4053 (23.446) Cm (4048:4060) Scan EI+ 2.04e755
41
43
54
5344
51
69
67
57
59
66
74
83
81
79
9784
8795
93
98
111
108
264123
112 222180137
129152 166
179220183
194 207 236223
246 257
265
296266 292
C18:1, cis-9
31 51 71 91 111 131 151 171 191 211 231 251 271 291 311m/z0
100
%
ramesh4 4162 (23.996) Cm (4156:4167) Scan EI+ 9.97e774
43
4155
5444
6957
59
65
87
75
83 143
9788 101129111115 135
298199
185157144171167 181
255
213200 241227267257
299
C18:0
45 65 85 105 125 145 165 185 205 225 245 265 285 305 325m/z0
100
%
8.78e774
43
41
55
4454
69
59
61
87
75
83
81
326143
9788 129
101 111125 135
283199185171157
144195
227213 241 255 269 295
327
C20:0
39 59 79 99 119 139 159 179 199 219 239 259 279 299 319 339 359m/z0
100
%
ramesh4 5487 (30.678) Cm (5481:5494) Scan EI+ 5.79e774
43
41
55
44
69
59
87
75
83
81
354
14397
88 129101 111116
135
199185157144
171311255
213 241227 269 297283 323
355
C22:0
33 53 73 93 113 133 153 173 193 213 233 253 273 293 313 333 353 373 393m/z0
100
%
ramesh4 6100 (33.770) Cm (6094:6108) Scan EI+ 2.58e774
43
41
57
44
45
69
67
60
87
75
83
81
382
14397
95 11198129
135
199185171157144
339283241
207227 255 269 297 325311 351
383
C24:0
29 49 69 89 109 129 149 169 189 209 229 249 269 289 309m/z0
100
%
6.53e755
41
43
54
53
44
69
67
57
59
66
74
83
81
79
84
8797
95
93
98
110
109
108
264
123
112222180
137220
181194
223235 246
265
296266292
278
31 51 71 91 111 131 151 171 191 211 231 251 271 291 311m/z0
100
%
1.06e874
43
41
55
5444
5769
59
65
87
75
8381
298143
9788 129101
111115 135
199
185157144171 181
255
213200 241227267
299
38 58 78 98 118 138 158 178 198 218 238 258 278 298 318 338 358m/z0
100
%
ramesh5 4861 (27.512) Cm (4856:4867) Scan EI+ 6.57e674
43
41
55
44
54
69
67
60
87
75
83326
14397
88 111101 129
135
283199185171157144 227
213 241 255 269 295327
355
38 58 78 98 118 138 158 178 198 218 238 258 278 298 318 338 358m/z0
100
%
ramesh5 5516 (30.816) Cm (5509:5523) Scan EI+ 8.14e674
43
41
57
44
54
69
67
60
87
75
83
81
354
14397
95 111101 129
135
149
199167
157185
311255207 213
241227 269 279 297 323
355
33 53 73 93 113 133 153 173 193 213 233 253 273 293 313 333 353 373 393m/z0
100
%
ramesh5 6161 (34.068) Cm (6149:6173) Scan EI+ 1.32e774
43
41
57
44
54
69
59
87
75
83
81
382
14397
88 129111
135
199185
157144
171339207
283241227 255 269 297 325311 351
383
C18:1, cis-9
C18:0
C20:0
C22:0
C24:0
MS of fatty acid standards MS of fatty acid from M. oleifera leaves
Screening of phytoconstituents in M. oleifera cultivars
73
α-linolenic acid (ALA) plays important role in human diet, since it’s the precursor for
long chain n-3 fatty acids including docosahexaenoic acid (DHA) (Barceló-Coblijn
and Murphy 2009). Recommended dietary intakes of α-Linolenic acid for adult males
and females are 1.6 and 1.1µg/day, respectively (Otten et al. 2006). Flaxseed,
Walnuts and Canola oil are considered as rich and natural source of ALA, which
contain 28.8, 9.1 and 9.1% ALA, respectively (Barceló-Coblijn and Murphy 2009).
Compared to this, in present investigation, total lipids extracted from M. oleifera (cv.
PKM-1) leaves, flowers and fruits are found to contain 54.27, 18.82, and 30.42% of
ALA. These results are in agreement with the study of (Amaglo et al. 2010), wherein
similar content of ALA in leaves, flowers and fruits (56.4, 23.0 and 26.2%,
respectively) of M. oleifera was recorded. Moringa seed oil has been extensively
characterized for the highest presence of oleic acid (65-75%), palmitic acid (5-10%)
and stearic acid (5-10%) (Lalas and Tsaknis 2002; Abdulkarim et al. 2005; Amaglo et
al. 2010). The seeds of M. oleifera are rich in oleic acid and poor in linoleic and
linolenic acid content compared to flowers and fruits. This shows that, leaves are the
superior source of ALA & PUFA compared to seeds, flowers and fruits (Tsaknis et al.
1999; Mohammed et al. 2003), and can be used as a significant source of ALA. In
view of health benefits, M. oleifera leaves contain low amount of saturated fatty acid
and high mono and poly unsaturated fatty acid content, which can enhance the health
benefits of Moringa based products. Green leafy vegetables have been established as
potential source of ALA, Vidrih et al. (2009) recorded ≥50% of ALA in fatty acid
extracted from tarragon (Artemisia dracunculus), green lettuce, zucchini (Cucurbita
pepo), and broccoli.
Screening of phytoconstituents in M. oleifera cultivars
74
Although, the fresh leaves of M. oleifera have been established as rich source
of polyunsaturated fatty acids, specially, α-linolenic acid (ALA; C18:3, All cis-
9,12,15) which accounted >50% of total lipids in the leaves (Sena et al. 1998;
Freiberger et al. 1998; Amaglo et al. 2010b; Moyo et al. 2011), this is the first report
of fatty acid profile in fruits, flowers and leaves of commercially grown cultivars of
M. oleifera. Similarly the fatty acid profile was verified by Mass Spectrum analysis,
which has not been reported earlier in Moringa. Sánchez-Machado et al. (2010)
characterized the fatty acid profile of leaves, flowers, and immature pods of unknown
genotype/cultivar of M. oleifera grown in Northwest Mexico, and recorded the similar
content of palmitic acid in flowers (23.43%) and leaves (23.28%), but in our study,
significantly lower amount of palmitic acid (16.93-18.39%) was recorded in leaves of
all the cultivars compared to flowers (24.25%). This shows the significant influence
of geographical locations on fatty acid content. Especially, leaves of PKM-1, PKM-2,
CO-1 and DHANRAJ cultivars were found distinguished in fatty acid profile
compared to other. Though, γ-linolenic acid (0.20%) and other fatty acid in leaves of
South African ecotype were recorded (Moyo et al. 2011), however in Indian cultivars
γ-linolenic acid was not present. Lalas and Tsaknis (2002) also observed significant
differences in Iodine value, free fatty acid content and saponification value among
seed oil extracted from Indian (PKM-1) and Mbololo cultivar from Kenya. Apart from
genotypes, year, growing region and climatic conditions also influence the fatty acid
content in the plants (Mohammed et al. 2003; Geleta et al. 2011: Yada et al. 2013).
Werteker et al. (2010) recorded the significant negative correlations between
temperature and the content of linolenic, linoleic acid on the whole quantity of fatty
acid in oil seed crops, including rapeseed, soybeans and sunflowers. Similarly,
Screening of phytoconstituents in M. oleifera cultivars
75
drought is one of the main factors that increase the C22:0 and C18:1 content and oil
yield etc., as is evident in M. oleifera seed (Anwar et al. 2006). The results showed
that genotypic factors significantly influence the fatty acid composition in leaves of
Moringa oleifera, and it’s also found to vary among the different edible parts of the
plant.
In summary, 15 fatty acids were identified by GC-FID/MS in leaves, flowers
and fruits of Indian cultivars of M. oleifera. Leaves contain low amount of saturated
fatty acid (24-27%) and high mono and poly unsaturated fatty acid (73-76%)
compared to fruits and flowers. Especially, leaves of PKM-1, PKM-2, CO-1 and
DHANRAJ cultivars are rich in unsaturated fatty acids, and can enhance the health
benefits through Moringa based functional food products.
3.3.5. Genetic diversity
3.3.5.1 RAPD analysis
Randomly amplified polymorphic DNA assays were performed with 25 random
primers. Of these, 17 produced polymorphic and reproducible bands and were
selected for further screening. In an assay of the 17 RAPD primers in eight cultivars,
76 bands were scored in the size range of 0.3-3.0 kb, with an average of 4.47 bands
per primer. Of the 76 bands, 37 were polymorphic (Table 3.9), with 48.68%
polymorphisms. RAPD-15 was found most efficient with 87.50 % polymorphism out
of 8 bands. A representative RAPD profile obtained by primer RAPD-10 and 11 in
the 8 cultivars of M. oleifera is shown in Figure 1a and 1b. The polymorphic
information content (PIC) values, a measure of informativeness of a marker, were
found in the ranged from 0.49 (RAPD-17) to 0.817 (RAPD-15) with a mean of 0.726
(average heterozygosity).
Screening of phytoconstituents in M. oleifera cultivars
76
Figure 3.10. Profile of eight M. oleifera cultivars, (1) PKM-1, (2) PKM-2, (3)
GKVK-1, (4) Dhanraj, (5) Amar32, (6) CO-1, (7) Bhagya and (8) PAVM-1 by Cyt
P450, ISSR and RAPD markers. (a) RAPD-10, (b) RAPD-11, (c) ISSR-7, (d) ISSR-12,
(e) Cyt P450 (Cyt 2F & Cyt 2R) and (f) Cyt P450 (Cyt 3F & Cyt 3R).
L 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 L
(e) (f)
500 Bp 1000 Bp
3000 Bp
L 1 2 3 4 5 6 7 8
(d) (c)
500 Bp
1000 Bp
3000 Bp
L 1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 L L 1 2 3 4 5 6 7 8
(a) (b)
3000 Bp
1000 Bp
500 Bp
Screening of phytoconstituents in M. oleifera cultivars
77
Table 3.9. Summary of genetic diversity obtained by RAPD primers
S/no Primer
name
Primer sequence Tm value
of primer
(ºC)
Range of
marker
(kb)
Polymorphic
bands/total no.
of bands
Per cent
polymorphism
PIC
1 RAPD-1 CCACACTACC 37 1.2-0.4 1/3 33.33 0.661
2 RAPD-2 CGGCCACTGT 37 1.1-0.3 1/3 33.33 0.553
3 RAPD-3 CGGCCCCGGC 37 1.5-0.3 1/5 20.00 0.775
4 RAPD-4 CGGAGAGCGA 37 1.1-0.3 3/5 60.00 0.772
5 RAPD-5 GACGGAGCAG 37 1.2-0.3 5/6 83.33 0.801
6 RAPD-6 GAAGAACCGC 37 0.8-0.3 2/5 40.00 0.757
7 RAPD-7 GACGGATCAG 37 1.0-0.5 1/3 33.33 0.653
8 RAPD-8 CGGAGAGCCC 37 2.0-0.8 1/4 25.00 0.746
9 RAPD-9 GGGTAACGCC 37 1.5-0.3 4/5 80.00 0.799
10 RAPD-10 AATCGGGCTG 37 1.5-0.4 1/5 20.00 0.764
11 RAPD-11 CAATCGCCGT 37 1.0-0.4 3/5 60.00 0.763
12 RAPD-12 GAACGGACTC 37 1.5-0.5 1/4 25.00 0.741
13 RAPD-13 GTGTGCCCCA 37 1.1-0.5 1/4 25.00 0.749
14 RAPD-14 TGTCTGGGTG 37 1.25-0.3 2/5 40.00 0.794
15 RAPD-15 AGGGCGTAAG 37 1.3-0.3 7/8 87.50 0.817
16 RAPD-16 CCCGGCATAA 37 1.2-0.9 2/4 50.00 0.71
17 RAPD-17 TCTCCGCTTG 37 3.0-1.0 1/2 50.00 0.49
Average 37/76 48.68 0.726
Table 3.10. Summary of genetic diversity obtained by ISSR primers
Sl
no
Primer
name
Primer
sequence
(5' to 3')
Tm value
of primer
(°C)
Range of
marker (kb)
Polymorphic
bands/total no
of bands
%
polymorphism
PIC
1 ISSR4 (GC)6A 56.0 0.9-0.4 2/4 50.00 0.744
2 ISSR6 (CT)6A 40.0 0.6-0.2 4/6 66.66 0.818
3 ISSR7 (TGT)3GC 42.8 1.2-0.3 1/6 16.66 0.822
4 ISSR8 (TGT)3 GA 40.0 1.3-0.3 2/5 40.00 0.799
5 ISSR11 (TCT)4G 45.5 2.5-0.25 3/6 50.00 0.807
6 ISSR12 (TCT)4C 47.0 1.5-0.5 5/8 62.50 0.871
Average - - - 17/35 48.57 0.810
Screening of phytoconstituents in M. oleifera cultivars
78
Table 3.11. Summary of genetic diversity analysis obtained by Cytochrome P450
based primers
Sl no
Primer name
Primer sequence (5' to 3') Tm value of primer (°C)
Range of marker (kb)
Polymorphic bands/total no of bands
% polymorphism
PIC
1 CYP1A1
(F)GCCAAGCTTTCTAACAATGC
(R)AAGGACATGCTCTGACCATT
52 0.5-0.2 2/2 100.0 0.444
2 CYP2C19 (F)TCCTTGTGCTCTGTCTCTCA
(R)CCATCGATTCTTGGTGTTCT
52 1.0-0.4 1/2 50.00 0.473
3 Cyt02 (F)CGGCTTGCCTCATGGA
(R)GAGAAATAGGTGCGTGGA
52 1.0-0.1 2/7 28.57 0.838
4 Cyt03 (F)GACCCAAGCAACGTCA
(R)GACCCAAGCCAACGTCA
52 1.2-0.4 3/5 60.00 0.739
5 Cyt06 (F)ACGTGCCACTCTGCAA
(R)ACCCTAGGCTAAGGTGGA
52 1.0-0.2 2/5 40.00 0.790
6 Cyt07 (F)GGGCCATAACCCACGA
(R)ATTGGAGCGCCGGTGA
52 1.1-0.2 1/4 25.00 0.742
7 Cyt08 (F)CCTGTACGACCCAAGCA
(R)TGGCCCACATATTCACCA
52 0.9-0.2 1/5 20.00 0.799
Average - - - 12/30 40.00 0.689
Screening of phytoconstituents in M. oleifera cultivars
79
Table 3.12. Jaccard similarity coefficients among eight cultivars of M. oleifera
generated from binary matrix of RAPD, ISSR and Cytochrome P450 based marker
Rows/Cols PKM-1 PKM-2 GKVK-1 DHANRAJ AMAR-32 CO-1 BHAGYA PAVM-1
PKM-1 1.000
PKM-2 0.852 1.000
GKVK-1 0.867 0.857 1.000
DHANRAJ 0.837 0.847 0.856 1.000
AMAR-32 0.798 0.846 0.831 0.875 1.000
CO-1 0.746 0.748 0.775 0.760 0.776 1.000
BHAGYA 0.746 0.776 0.776 0.746 0.748 0.824 1.000
PAVM-1 0.689 0.742 0.702 0.768 0.770 0.769 0.803 1.000
Table 3.13. Comparison of RAPD, ISSR and Cyt P450 based molecular markers in
evaluating genetic diversity of M. oleifera
Molecular markers RAPD ISSR Cyt P450 based
No of genotypes 8 8 8
Total no. of bands 76 35 30
Polymorphic bands 37 17 12
Total no. of assays 17 6 7
Percentage polymorphism 48.68 48.57 40.00
Multiplex ratio (MR) 4.47 5.83 4.29
Average heterozygosity (Hav) 0.726 0.810 0.689
Marker index 3.25 4.73 2.95
Screening of phytoconstituents in M. oleifera cultivars
80
PKM-1
PKM-2
GKVK-1
DHANRAJ
AMAR-32
CO-1 BHAGYA
PAVM-1
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F3
(3
.46
%)
F2 (5.71 %)
Variables (axes F2 and F3: 9.17 %)
Figure 3.11. UPGMA cluster analysis showing the relationship and diversity among
eight cultivars of M. oleifera, produced by RAPD, ISSR and Cyt P450 based markers.
Numbers at the node indicate bootstrap values.
Figure 2.12. Representation of principal component analysis (PCA) for the eight
cultivars of M. oleifera, produced by RAPD, ISSR and Cyt P450 based primers.
Jaccard Similarity Coefficient
0.75 0.78 0.81 0.84 0.88
PKM-1
GKVK-1
PKM-2
DHANRAJ
AMAR-32
CO-1
BHAGYA
PAVM-1
Cluster 1
Cluster 2
Sub-cluster 1A
Sub-cluster 1B
Sub-cluster 2A
Sub-cluster 2B
94.4
82.3
64.2
37.5
45.5
61.8
Screening of phytoconstituents in M. oleifera cultivars
81
3.3.5.2. ISSR analysis
Inter-Simple Sequence Repeat (ISSR) assays were performed with 10 ISSR primers.
Of these, 6 gave polymorphic and reproducible bands and were selected for further
screening. In an assay of the 6 ISSR primers on the eight cultivars, 35 bands were
scored in the size range of 0.2-2.5 kb, with an average of 5.83 bands per primer. Of
the 35 bands, 17 were polymorphic (Table 3.10), with 48.57% polymorphism. ISSR-6
was found most efficient with 5 polymorphic bands out of 8 bands (62.50 %). A
representative ISSR profile obtained by primer ISSR-3 and 6 in the eight cultivars of
M. oleifera is shown in Figure 1c and 1d. The PIC values were found in the ranged
from 0.744 (ISSR-1) to 0.871 (ISSR-6) with a mean of 0.810.
3.3.5.3. Cytochrome P450 based markers analysis
Cytochrome P450 based marker assays were performed with 11 set of Cyt P450 based
(forward and reverse) primers. Of these, 7 produced polymorphic and reproducible
bands and were selected for further screening. Tests of 7 sets of Cyt P450 based
primers on the eight cultivars gave 30 bands in the size range of 0.1-1.2 kb, with an
average of 4.28 bands per primer. Of the 30 bands, 12 were polymorphic (Table 3.11),
with 40.00% polymorphisms. Cyt 03 F with Cyt 03 R was found to be most efficient
with 3 polymorphic bands out of 5 bands (60.00 %). A representative profile obtained
by primer Cyt 02 F with Cyt 02 R and Cyt 03 F with Cyt 03 R in the eight cultivars of
M. oleifera is shown in Figure 1e and 1f. The PIC values were recorded in the ranged
from 0.444 (CYP1A1F with CYP1A1R) to 0.838 (Cyt 02 F with Cyt 02 R) with a
mean of 0.689.
3.3.5.4. Statistical analysis of binary data
Jaccard’s similarity coefficient, which reflects the genetic similarity among the
Screening of phytoconstituents in M. oleifera cultivars
82
cultivars, was calculated using binary data based on the RAPD, ISSR and cytochrome
P450 based markers and is shown in Table 3.12. The Jaccard’s similarity coefficient
ranged from 0.689 (between PKM-1 & PAVM-1) to 0.875 (between Dhanraj and
Amar-32). The dendrogram, constructed according to the UPGMA method and based
on the RAPD, ISSR and Cyt P450 based markers data, grouped the eight cultivars into
two separate clusters (Figure 3.11). The principal component analysis of RAPD, ISSR
and Cyt P450 based markers revealed that the genotypes belonging to a particular sub-
cluster were grouped together in the PCA plots (Figure 3.12). The coefficient of
correlation (Pearson’s correlation coefficients) was determined by a mantel test using
matrices generated by these markers. The correlation coefficient between the ISSR
and Cyt P450 based markers, between the RAPD and Cyt P450 based markers and
between the ISSR and RAPD markers was 0.115, 0.097 and 0.0013, respectively. The
correlation coefficient between Cyt P450 based markers and the combined RAPD,
ISSR and Cyt P450 based markers, between the ISSR and the combined RAPD and
ISSR and between the Cyt P450 based marker, the RAPD markers and the combined
RAPD, ISSR and Cyt P450 based markers was 0.557, 0.237 and 0.580, respectively.
The three molecular markers, ISSR, RAPD and Cyt P450, were compared on the basis
of percentage polymorphism, multiplex ratio (MR), average heterozygosity (Hav) and
marker index (Table 3.13). In relation to per cent polymorphisms, ISSR markers
scored higher (48.57%) than RAPD (48.68%) and cytochrome P450 based markers
(40%). The Marker index (MI), which is a measure to evaluate the overall usefulness
of a marker system considering all parameters, was highest for ISSR (4.726),
compared to RAPD (3.246) and Cyt P450 based markers (2.954), making the former a
highly efficient marker for the genetic diversity studies in M. oleifera.
Screening of phytoconstituents in M. oleifera cultivars
83
The eight cultivars of M. oleifera were grouped into two major clusters using a
Jaccard similarity coefficient of 0.75. Cluster 1 comprises 5 cultivars; PKM-1, PKM-
2, GKVK-1, Dhanraj and Amar-32. Cluster 2 contains 3 cultivars; CO-1, Bhagya and
PAVM-1. Bootstrap analysis from the binary data was used to determine the
robustness of the dendrogram and confidence values as percentages at each node.
Very high bootstrap values (94.4 and 82.3) were obtained from the major nodes
(Figure 3.11), indicating the robustness of the markers for assessing and correlating
these groups. .
Information on genetic variability of wild and cultivated plants is important
for identification, conservation and cultivar development (Demir et al. 2010) and
DNA markers are the most appropriate to study the genetic diversity among a set of
genotypes or cultivars. In the present study, the combination of non-functional
(RAPD and ISSR) and functional (Cyt P450 based) marker systems were used to
provide wider genome coverage and, therefore, will be a better indicator of the genetic
relationships among the eight cultivars.
Assessment of genetic diversity in M. oleifera accessions has been reported
using Amplified fragment length polymorphism (AFLP) (Muluvi et al. 1999) and
RAPD markers (Muluvi et al. 1999; Muluvi et al. 2004; Mgendi et al. 2010). Muluvi
et al. (1999) analysed the genetic diversity by AFLP among 140 accessions of M.
oleifera, collected from south India, Southern Malawi and Kenya and found the
highest levels of genetic diversity within the Indian genotypes. They used only one
cultivar (PKM-1) from India whereas the other genotypes were from natural
populations. In our study, we used eight commercially used Indian cultivars, among
these 40.00% (by ISSR markers) to 48.68% (by RAPD markers) polymorphisms were
Screening of phytoconstituents in M. oleifera cultivars
84
recorded. This is the first report on genetic diversity among commercially grown
Indian cultivars. Similarly, Mgendi et al. (2010) investigated the genetic diversity by
RAPD markers between and within cultivated and non-cultivated provenances of M.
oleifera from coastal regions of Tanzania and found that wild provenances were more
diverse than cultivated. In cultivated plants, genetic changes are slowed down because
of reduced competition and natural selection, due to optimum environmental
conditions (Manoko et al. 2008). However, there is lack of information for Indian
cultivated genotypes since India is the centre of origin for M. oleifera. Similarly, there
is a need to evaluate the efficiency of other molecular markers for proper assessment
of genetic diversity in M. oleifera, particularly markers based on Cyt P450 genes to be
exploited for assessing the intra-species diversity.
In the present study, we used RAPD, ISSR and Cyt P450 based markers to
analyse the genetic diversity between eight Indian cultivars M. oleifera. The three
molecular markers used detected an average of 46.80% polymorphisms (genetic
diversity) among the cultivars. RAPD and ISSR revealed an average of 48.68 and
48.57% polymorphism across all cultivars. The Jaccard’s similarity coefficient was
calculated based on RAPD, ISSR and cytochrome P450 markers and it was found in
the ranged from 0.689 to 0.875. Similar values of this coefficient were obtained
between cultivars of close relatives of M. oleifera, i.e., papaya and Phaseolus vulgaris
(Stiles et al. 1993; Maciel et al. 2003).
In the cluster analysis, constructed according to the UPGMA method and
based on the three sets of marker data, the cultivars were not clustered according to
their geographical origin. This may be due to spread of planting materials and high
rates of gene flow through cross-pollination since this plant is highly cross-pollinated
Screening of phytoconstituents in M. oleifera cultivars
85
and different cultivars were collected from different states of India, i.e., Karnataka,
Tamil Nadu, Maharashtra and Kerala. These states are located in close proximity of
each other. Several studies have reported similar observations in other plants e.g.,
Andrographis paniculata and Olea europaea (Padmesh et al. 1999; Virginie et al.
2002), whereas some other studies reported the grouping of genotypes in dendrogram
on the basis of their geographical collection centers (Das et al. 2007; Panwar et al.
2010). The cluster (dendrogram) was bootstrapped by the WINBOOT program (Yap
and Nelson 1996) with 2000 replications as suggested by Hedges (1992) and the
values were obtained in terms of percentages were high (94.4 and 82.3) at major
nodes, suggesting the robustness of the dendrogram.
The comparative analysis of RAPD, ISSR and Cyt P450 based marker systems
revealed ISSR to be the best marker as it generated highest percentage of
polymorphisms, marker index (MI), average heterogeneity (Hav) and multiplex ratio
(MR). In the present study, Cytochrome P450 based markers were not found very
efficient, due to low value of polymorphic information content (PIC) compared to
RAPD and ISSR markers, but these new markers have been found very efficient in
evaluation of diverse species such as rice and various endemic wild plants (Tanaka et
al. 2001; Yamanaka et al. 2003). Similarity Pearson’s correlation coefficients was
found very low (R2= 0.115, between ISSR and Cyt P450 based markers), showing a
very poor fit of similarity matrix between two markers. This indicates that the two
sets of markers target different regions of the genome. Similar results of low levels of
correlation between RAPD and ISSR markers were observed by Lalhruaitluanga and
Prasad (2009) in Melocanna baccifera. However, Panwar et al. (2010) observed the
high value of coefficient of correlation between RAPD and Cyt P450 gene based
Screening of phytoconstituents in M. oleifera cultivars
86
markers.The dendrogram and PCA plots generated from the binary data matrices of
the three marker systems were found highly concordant to each other. In dendrogram,
all the eight cultivars were grouped in four major sub-clusters, similar pattern of
grouping was recorded in PCA plot. The similar type of agreements between
dendrogram and PCA plot were reported by Panwar et al. (2010).
The high genetic diversity (variability) found in the present study is directly
linked with higher levels of biodiversity, which is useful for food security,
productivity, and ecological sustainability. Significant variability can be utilised in
breeding programmes to produce high yielding nutritionally superior cultivars with
better adaptations to different climatic conditions. In India, major breeding
programmes are on-going for the development of fast growing, disease resistant and
high pod yielding cultivars of M. oleifera. Thus, cv. Bhagya (KDM-1) has recently
been developed by University of Horticultural Sciences, Bagalkot (Karnataka) and it
is gaining popularity among farmers, due to its high yield of pods. Similarly, in future,
attention is required to develop cultivars for high foliage yield, since Moringa leaves
are a rich and affordable source of major nutritional and nutraceutical
phytoconstituents (Oduro et al. 2008).
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