Molecular Analysis of Biofield Treated Sponge and Bitter Gourd
Bitter Gourd and Antidiabeticsksiconnect.icrisat.org/wp-content/uploads/2014/05/Ray...Bitter Gourd...
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Bitter Gourd and Antidiabetics: Phytochemical diversity and variation among germplasm, commercial lines and as affected by planting season, ripening stage and storage length R.-Y. Yang 1 , Y.-L. Tien 1 , P. Hanson 1 , N. Dhillon 1 and D. Ledesma 1 , C.-Y. Hung 2 , Y.-M. Chung 2 and M.-L. Cheng 2 , M.-S. Shiao 2 , M. Pathak 3 , W. Easdown 4 , and T. Omvir Singh 4 1 AVRDC – The World Vegetable Center, Shanhua, Tainan, Taiwan 2 Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan 3 Department of Vegetable Crops, Punjab Agricultural University, India 4 AVRDC – The World Vegetable Center, Hyderabad, India Bitter Gourd Conference Hyderabad, India 20-21 March 2014
Transcript of Bitter Gourd and Antidiabeticsksiconnect.icrisat.org/wp-content/uploads/2014/05/Ray...Bitter Gourd...
Bitter Gourd and Antidiabetics:
Phytochemical diversity and variation among germplasm, commercial lines and as affected by
planting season, ripening stage and storage length
R.-Y. Yang1, Y.-L. Tien1, P. Hanson1, N. Dhillon1 and D. Ledesma1, C.-Y. Hung2, Y.-M. Chung2 and M.-L. Cheng2, M.-S. Shiao2,
M. Pathak3, W. Easdown4, and T. Omvir Singh4
1AVRDC – The World Vegetable Center, Shanhua, Tainan, Taiwan
2Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan 3Department of Vegetable Crops, Punjab Agricultural University, India
4AVRDC – The World Vegetable Center, Hyderabad, India
Bitter Gourd Conference Hyderabad, India
20-21 March 2014
Outline
• Bitter gourd- a vegetable and medicinal plant
• Primary and secondary antidiabetic effects
• Antidiabetic compounds
• Phytochemical diversity and variation: – In selected germplasm accessions
– In selected commercial varieties
– As affected by ripening stage
– As affected by storage length
– As affected by planting season
Bitter gourd • A vegetable
– Popular in India, China, the Philippines, Taiwan, and Japan
– Consumed worldwide, particularly in Chinese and India communities
• A medicinal plant
– Anti-hyperglycemia
– Anti-hyperlipidemia
– Anti-oxidation
– Anti-inflammation
– Anti-microbial pathogens
Source of photos: AVRDC
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Primary effect of anti-diabetics
• Plasma glucose homeostasis
– Decrease carbohydrate digestion & absorption
– Increase insulin secretion
– Increase hepatic glucose utilization
– Increase insulin sensitivity in liver & muscle cells
5
Secondary effects of anti-diabetics
• Reduce inflammation
• Anti-oxidation and reduce/delay oxidative stress
• Increase fatty acid utilization
• Lower blood lipids
• Reduce pre-fat cell proliferation
• Broad-spectrum anti-microbial activity
2014/3/21 6
Anti-hyperglycemic substances in bitter gourd
Saponin fraction
Lipid fraction
Water fraction
Steroid and triterpenoid saponins
•Momordicine I, II, III •Momordicosides Q, R, S, T •Momordicosides A, D, E, F2, I, K, L •Kuguacin A,…, S •Charantin •Cucurbitan B, K… •……
Alkaloids
•Vicine •…
Lipids
•c9, t11 conjugated linoleic acid •c9, t11, t13 conjugated linolenic acid . a- Eleostearic acid -……
Proteins
•p-Insulin-like •v-Insulin-like •Lectin •Mormodin I, II…. •……
Source: literature review, AT, AVRDC
2014/3/21 7
Cucurbitane-type triterpenoids Momordicosides
Q: b-D-glucopyranosyl , H
R: b-D-allopyranosyl, b-D-glucopyranosyl
S: b-D-glucopyranosyl , b-D-glucopyranosyl
T: b-D-xylopyranosyl, b-D-glucopyranosyl
Kuguacin
I : R1,R2: H
II,III :H, b-D-glucopyranosyl
Momordicine
2014/3/21 8
Cucurbitane-type steroid saponins
b-D-GLC
Sitosterol glucosides Stigmastadienol glucosides
b-D-GLC
4 4
2014/3/21 9
Fatty acids
15,16-dihydroxy-a-eleostearic acid
c9, t11 conjugated linoleic acid (C18:2)
c9, t11, t13 conjugated linolenic acid (C18:3) (a-eleostearic acid)
2014/3/21 10
Proteins
P-Insulin-like V-Insulin-like Lectin Momordin
11 kDa 11 kDa 120 kDa 27.41 kDa
17 amino acids
17 amino acids
547 amino acids
250 amino acids
2014/3/21 11
Bitter Gourd Project
• Goal
– Improved income and quality of life of diabetics in developing countries
• Objectives
– Optimize production of anti-diabetic compounds in bitter gourd through varietal selection, postharvest practices, and preparation methods
– Develop evidence-based dietary strategies using bitter gourd to reduce hyperglycemia (high blood sugar) in type 2 diabetic populations in Asia and Africa
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Commercial variety Field and location Harvest/ maturity Postharvest Food preparation Animal Human
Co
nte
nt, stab
ility and
effect o
f p
hyto
nu
trien
ts in b
itter go
urd
Germplasm
Evidence-based agricultural and dietary strategies for the production and use of bitter gourd for anti-hyperglycemic control
Project approach
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Project partners and study sites
AVRDC-HQ
AVRDC-ESEA
AVRDC-RCSA
KCMC AVRDC-RCA
PAU
NTU
JLU
AUW
Project planning workshop, 3-6 May 2011, AVRDC
PAU: Punjab Agricultural Univ. AUW: Avinashilingam Univ. for Women KCMC: Kilimanjaro Christian Medical Center JLU: Justus-Liebig Giessen University NTU: National Taiwan University
Germplasm evaluation • Location
– AVRDC-HQ, Taiwan
• Plant materials
– AVRDC GRSU
• Exp design:
– RCBD, 3 rep, 8 plants/rep
– 2 years
• Evaluation:
– Horticultural traits
– Antidiabetic compounds
– LC/MS profile fingerprints
Source of photos: N Dhillon, AVRDC-ESEA
LCMS profile of bitter gourd
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NS1020
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00
%
0
100
20130827_NS01 1: TOF MS ES+ BPI
9.65e3
0.59305.109
15.07778.540
9.48496.344
8.00518.327
7.72518.336
5.33476.244
1.63188.072
0.68120.078
2.19529.207
3.451046.803
3.38;455.346 4.43739.4273.82
814.500
7.19523.341
6.27523.341
5.65419.328
6.54437.338
8.84520.343
8.57520.345
8.92523.342
15.05954.608
9.50496.339
11.13524.371
9.88522.355
14.64149.021
11.22353.264
12.02279.230 14.11
568.427
13.78611.467
12.89593.278
15.10778.544
16.43684.54115.65
792.565
16.88782.566
18.45663.45516.91
782.567
16.94782.568
18.43663.463
18.48663.456
15
16
2
1
12
10, 14 11
9
5
6
4
3 13 8
7
16 compounds identified - 7 phosphatidylcholines, 2 fatty acid derivatives (PKG1) - 5 triterpenoids (PKG2) -1 alkaloid glycoside (PKG3) - 1 amino acid
16 16
2014/3/21 17
Anti-hyperglycemic substances in bitter gourd
Saponin fraction
Lipid fraction
Water fraction
Steroid and triterpenoid saponins
•Momordicine I, II, III •Momordicosides Q, R, S, T •Momordicosides A, D, E, F2, I, K, L •Kuguacin A,…, S •Charantin •Cucurbitan B, K… •……
Alkaloids
•Vicine •…
Lipids
•c9, t11 conjugated linoleic acid •c9, t11, t13 conjugated linolenic acid . a- Eleostearic acid -……
Proteins
•p-Insulin-like •v-Insulin-like •Lectin •Mormodin I, II…. •……
Source: literature review, AT, AVRDC
PKG1
PKG2 PKG3
Germplasm trial at AVRDC, Taiwan - Group means, content ranges and the top/bottom 3
accessions
PKG1 PKG2 PKG3 2011 2012 2011 2012 2011 2012
Range 30-60 30-68 0.2 - 9.4 0.2-13.9 29-95 25-103 Top 3 TOT4009 TOT4009 TOT5869 TOT4009 THMC155 THMC155
THMC232 THMC195 THMC193 THMC195 THMC168 THMC168 THMC155 TOT4785 TOT4131 TOT4785 TOT4370 TOT4370
Bottom 3 TOT5869 TOT1398 TOT1568 TOT1398 TOT1568 TOT4785 TOT1398 TOT3991 TOT4009 TOT3991 TOT1567 TOT1568 THMC168 THMC232 TOT3991 THMC232 TOT5869 TOT5869
0
20
40
60
80
100
PKG1 PKG2 PKG3
inte
nsi
ty (
E6)
2011
2012
Root mean squared error
2014/3/21 19
Commercial variety multilocation trials
Genotype-Environment Interactions • Plant materials:
– 20 commercial seed company hybrids
• Design: – RCBD, 3 rep, 8-10 plants/rep, 2 years
• Four locations: – Taiwan: AVRDC-HQ, Tainan
– South India: AVRDC-RCSA, Hyderabad
– North India: Punjab Agricultural University, Ludhiana
– Tanzania: AVRDC-RCA, Arusha
• Evaluation: – Horticultural traits
– Anti-diabetic constituents
– LC/MS fingerprints
AVRDC-HQ AVRDC-RCSA
AVRDC-RCA
PAU
Commercial variety trial at AVRDC, Taiwan – ANOVA of PKG1-3 by year and drying method
0
20
40
60
80
100
PKG1 PKG2 PKG3
2011-FD
2012-FD
2011-OD
2012-OD
PK1 PK2 PK3 2011 2012 2011 2012 2011 2012
Replication <.0001 0.1071 <.0001 ns 0.0049 <.0001 Variety 0.0005 0.0001 <.0001 <.0001 <.0001 <.0001 Drying <.0001 <.0001 <.0001 0.0002 <.0001 <.0001 V x D ns ns ns ns ns ns
PKG1 PKG2 PKG3
2011 2012 2011 2012 2011 2012
Freeze dried
Range 28-61 23-53 0.5-13.6 0.9-14.5 51-102 37-97
Top 3 VNR28 Benteng545 NS1024 NS1024 VNR28 NS1020
Amanshri NewMoon BGCT725 BGCT725 NS1020 VNR28
THMC195 THMC195 PreetiITC Amanshri NewMoon PreetiITC
Bottom 3 Zeena BGCT725 JadeDragon JadeDragon JadeDragon I-Taj4625
HighMoon Jasper Jasper HighMoon BGCT385 JadeDragon
Abhishek HighMoon HighMoon Jasper HighMoon BGCT358
Hot air oven dried
Range 24-39 15-31 0.7-16.0 0.7-13.2 53-107 28-89
Top 3 THMC199 THMC199 NS1024 NS1024 VNR28 VNR28
Benteng545 Zeena BGCT725 PreetiITC NewMoon NS1020
Palee THMC195 ARBHT-1 US33 ARBHT-1 NewMoon
Bottom 3 NS1020 NS1020 JadeDragon JadeDragon BGCT385 BGCT358
HighMoon BGCT385 Jasper Jasper HighMoon HighMoon
Abhishek HighMoon HighMoon HighMoon JadeDragon JadeDragon
Commercial variety trial at AVRDC, Taiwan – Content range and the top /bottom 3 varieties
PKG1 PKG2 PKG3 2011 2012 2011 2012 2011 2012
6.2-14.8 1.7-8.8 0.9-18.7 0.2-5.9 3.7-21.3 1.4-26.8 Top 3 BGCT358 NS1024 VNR28 JDragon ARBHT-1 NewMoon
NS1020 BGCT725 NewMoon Ben545 NS1024 VNR28
Jasper US33 Preeti Jasper US33 NS1020
Bottom 3 US33 BGCT358 JDragon HighMoon Jasper BGCT725
BGCT385 JDragon Jasper US33 JDragon Palee
NS1024 Jasper BGCT358 ARBHT-1 HighMoon JDragon
Commercial variety trial at PAU, north India – Content range and the top /bottom 3 varieties
0
5
10
15
20
25
PKG1 PKG2 PKG3
inte
nsi
ty (
10
E6)
2011
2012
Commercial variety trial at AVRDC, Hyderabad – Content range and the top /bottom 3 varieties
PKG1 PKG2 PKG3
2011 2012 2011 2012 2011 2012
Ranges 16-30 19-37 1.6-17.5 2.4-18.2 5-68 6-80
Top 3 BGCT725 Ben545 Amanshri NS451 VNR28 VNR28
Ben545 JDragon NS1024 BGCT725 NS1020 NS1020
HighMoon HighMoon Zeena NS1024 NS451 NewMoon
Bottom 3 VNR28 ARBHT-1 JDragon JDragon HighMoon BGCT358
Preeti BGCT725 Jasper Jasper Ben545 Ben545
Zeena VNR28 HighMoon HighMoon BGCT358 JDragon
0
10
20
30
40
50
PKG1 PKG2 PKG3
inte
nsi
ty (
E+6
)
2011
2012
Commercial variety trial at AVRDC-Arusha Content range and the top /bottom 3 varieties
PKG1 PKG2 PKG3 Ranges 21-39 4-19 17-56 Top 3 Jasper NS1020 VNR28
Palee NS451 NS1020 BGCT358 Zeena NewMoon
Bottom 3 US33 JDragon Zeena Zeena Jasper Abhishek
BGCT725 HighMoon BGCT385
0
10
20
30
40
PKG1 PKG2 PKG3
inte
nsi
ty (
E+6
)
2011
2012
Content ranges of bitter gourd germplasm and commercial variety trials
Trial location D PKG1 PKG2 PKG3
2011 2012 2011 2012 2011 2012 Germplasm
(25) s Taiwan FD 30-60 30-68 0.2-9 0.2-14 29-95 25-103
Commercial (23)
s Taiwan FD 28-61 23-53 1-14 1-15 51-102 37-97
Commercial (23)
s Taiwan OD 24-39 15-31 1-16 1-13 53-107 28-89
Commercial (20)
n India OD 6-15 2-9 1-19 0.2-6 4-21 1-27
Commercial (20)
s India OD 16-30 19-37 2-18 2-18 5-68 6-80
Commercial (20)
n Tanzania OD 21-39 4-19 17-56
D: drying method; FD: freeze drying; OD: hot air oven drying
Postharvest study
• Three varieties: NS1020, US22, Palee
• Planting year: 2013
• Treatments:
– 3 harvest dates (16-, 18- 20- day after flowering)
– 2 drying methods (freeze dried, hot air oven dried)
– 3 storage length (0, 1, 2, days)
• Analysis: LCMS, selected nutrients
Bioactive compounds of NS1020 as affected by fruit ripening stage and drying method
0
20
40
60
80
100
120
PK1 PK2 PK3 PK1 PK2 PK3 PK1 PK2 PK3
16 day 18 day 20 day
Inte
nsi
ty (
E+6
)
Freeze dry Oven dry
0
20
40
60
80
100
120
PK1 PK2 PK3
16 day
18 day
20 day
Bioactive compounds as affected by storage time length
0
20
40
60
80
100
120
PK1 PK2 PK3 PK1 PK2 PK3
16 Day 18 Day
Inte
nsi
ty (
E+6
)
NS1020
S-0 day
S-1 day
S-2 day
Nutrient contents of NS1020 by fruit ripening stage and drying method
0
2
4
6
8
10
12
16 Day 18 Day 20 Day
Dry matter
Freeze dry
Oven dry
0.00
0.20
0.40
0.60
0.80
1.00
1.20
16 Day 18 Day 20 Day
Carotenoids
Freeze dry
Oven dry
0
20
40
60
80
100
120
16 Day 18 Day 20 Day
Vitamin C
Freeze dry
Oven dry
0
200
400
600
800
1000
1200
16 Day 18 Day 20 Day
Antioxidant Activity
Freeze dry
Oven dry
Nutrient contents of NS1020 as affected by storage length
0
2
4
6
8
10
12
16 Day 18 Day
Dry matter
S-0 day
S-1 day
S-2 day
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
16 Day 18 Day
Carotenoids
S-0 day
S-1 day
S-2 day
0
20
40
60
80
100
16 Day 18 Day
Vitamin C
S-0 day
S-1 day
S-2 day
0
200
400
600
800
1000
1200
1400
16 Day 18 Day
Antioxidant Activity
S-0 day
S-1 day
S-2 day
Compounds affected by planting seasons
• Two varieties: green, white
• Design: RCBD, 3 rep, 2 years, 3 seasons
• Location: AVRDC-Taiwan
• LCMS profiling: UPLC-TOF-MS
• Analysis: Metabolomics - pattern recognition
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Spring Winter
Summer
The PCA score plot of green bitter gourd grown in different seasons
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powder 27
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
%
0
100
6
4 5 7
3
2 8
12 22
11 14
10
18 25
17
16
13
15
29
26 23
21
20
19 24
30
28
27
1 9
Fig. Base peak chromatogram of of M. charantia (NO 76, spring)
The 30 most affected metabolites were identified: -Amino acids -Alkaloids -Fatty acids -Lysophosphatidylcholines -Lysophosphatidylethanols -Triterpenoids
Conclusions
• Varietal differences were found for all three compound groups (fatty acids, triterpenoids, alkaloids).
• Among germplasm, variation within fatty acids, triterpenoids, and alkaloid were 4-fold, 10-70-fold, 3-fold, respectively.
• Magnitudes of variation within fatty acids, triterpenoids, and alkaloid were similar for commercial varieties.
• Opportunities exist to improve all three compound groups, especially triterpenoids.
Conclusions
• Longer storage led to decreased fatty acids and vitamin C contents, and increased carotenoids and antioxidant activities, but not triterpenoids and alkaloids
• Metabolite types and amounts in bitter gourd fruits were affected by seasons. The 30 most affected metabolites were identified and belonged to the groups of amino acids, alkaloids, fatty acids, lysophosphatidylcholines, lysophosphatidylethanols, and triterpenoids
