chapter 3 - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36432/8...trans-lutein was found as...

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


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.


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


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


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.


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


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


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


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


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.


45

Figure 3.2. Outline of column preparation and folate purification procedure


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


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


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.


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


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


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


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


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).


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.


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).


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.


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


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


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.


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).


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


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


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.


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).


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


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


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.


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


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).


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).


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

%


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


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

%


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

%


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

%


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

%


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

%


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

%


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

%


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


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.


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,


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).


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


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


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


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


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


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


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.


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


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


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


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).

chapter 3 - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/36432/8...trans-lutein was found as...

Documents