Bitter Gourd Conference Hyderabad, India 20-21 March 2014 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
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
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 4
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 5
Anti-hyperglycemic substances in bitter gourd Proteins p-insulin-like v-insulin-like Lectin Mormodin I, II. Water fraction Alkaloids Vicine 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 Saponin fraction Lipids c9, t11 conjugated linoleic acid c9, t11, t13 conjugated linolenic acid. a- Eleostearic acid - Lipid fraction 2014/3/21 6 Source: literature review, AT, AVRDC
Cucurbitane-type triterpenoids Kuguacin Momordicine Momordicosides I : R1,R2: H Q: b-d-glucopyranosyl, H R: b-d-allopyranosyl, b-d-glucopyranosyl II,III :H, b-d-glucopyranosyl S: b-d-glucopyranosyl, b-d-glucopyranosyl T: b-d-xylopyranosyl, b-d-glucopyranosyl 2014/3/21 7
Cucurbitane-type steroid saponins Sitosterol glucosides Stigmastadienol glucosides 4 4 b-d-glc b-d-glc 2014/3/21 8
Fatty acids c9, t11 conjugated linoleic acid (C18:2) c9, t11, t13 conjugated linolenic acid (C18:3) (a-eleostearic acid) 15,16-dihydroxya-eleostearic acid 2014/3/21 9
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 10
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 2014/3/21 11
Project approach Content, stability and effect of phytonutrients in bitter gourd Germplasm Commercial variety Field and location Harvest/ maturity Postharvest Food preparation Animal Evidence-based agricultural and dietary strategies for the production and use of bitter gourd for antihyperglycemic control Human 2014/3/21 12
Project partners and study sites JLU PAU AVRDC- RCSA AUW AVRDC- ESEA NTU AVRDC-HQ AVRDC-RCA KCMC PAU: Punjab Agricultural Univ. AUW: Avinashilingam Univ. for Women KCMC: Kilimanjaro Christian Medical Center JLU: Justus-Liebig Giessen University NTU: National Taiwan University Project planning workshop, 3-6 May 2011, AVRDC 13
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 NS1020 20130827_NS01 0.59 100 305.109 15 16 compounds identified - 7 phosphatidylcholines, 2 fatty acid derivatives (PKG1) - 5 triterpenoids (PKG2) -1 alkaloid glycoside (PKG3) - 1 amino acid 6 7 1: TOF MS ES+ BPI 9.65e3 2 8.00 518.327 4 9.48 496.344 9.50 496.339 15.05 954.608 15.07 778.540 15.10 778.544 1 8.84 520.343 15.65 792.565 16.43 684.541 0.68 120.078 16 1.63 188.072 2.19 529.207 13 3.45 1046.803 3.38;455.346 3.82 814.500 4.43 739.427 5.33 476.244 5.65 419.328 6.27 523.341 6.54 437.338 7.72 518.336 11.13 524.371 10, 8.57 7.19 523.341 520.345 14 11 8.92 9 523.342 12 3 9.88 522.355 5 8 11.22 353.264 12.02 14.64 149.021 13.78 279.230 14.11 611.467 568.427 12.89 593.278 16.88 782.566 16.91 782.567 16.94 782.568 18.45 663.455 18.43 663.463 18.48 663.456 0 Time 1.00 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 2014/3/21 15
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Anti-hyperglycemic substances in bitter gourd Proteins p-insulin-like v-insulin-like Lectin Mormodin I, II. Water fraction Alkaloids Vicine PKG3 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 Saponin fraction PKG2 Lipids c9, t11 conjugated linoleic acid c9, t11, t13 conjugated linolenic acid. a- Eleostearic acid - Lipid fraction PKG1 2014/3/21 17 Source: literature review, AT, AVRDC
intensity (E6) 100 80 60 40 20 Germplasm trial at AVRDC, Taiwan - Group means, content ranges and the top/bottom 3 accessions Root mean squared error 2011 2012 0 PKG1 PKG2 PKG3 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
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- RCSA AVRDC-RCA PAU AVRDC-HQ 2014/3/21 19
Commercial variety trial at AVRDC, Taiwan ANOVA of PKG1-3 by year and drying method 100 80 60 40 20 0 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
Commercial variety trial at AVRDC, Taiwan Content range and the top /bottom 3 varieties 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
intensity (10E6) 25 20 15 10 5 Commercial variety trial at PAU, north India Content range and the top /bottom 3 varieties 0 PKG1 PKG2 PKG3 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 2011 2012
intensity (E+6) Commercial variety trial at AVRDC, Hyderabad Content range and the top /bottom 3 varieties 50 40 30 2011 20 2012 10 0 PKG1 PKG2 PKG3 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
intensity (E+6) Commercial variety trial at AVRDC-Arusha Content range and the top /bottom 3 varieties 40 30 20 10 2011 2012 0 PKG1 PKG2 PKG3 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
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
Intensity (E+6) 120 100 80 60 40 20 0 Bioactive compounds of NS1020 as affected by fruit ripening stage and drying method 120 100 80 16 day 60 18 day 40 20 day 20 0 PK1 PK2 PK3 Freeze dry Oven dry PK1 PK2 PK3 PK1 PK2 PK3 PK1 PK2 PK3 16 day 18 day 20 day
Intensity (E+6) Bioactive compounds as affected by storage time length 120 NS1020 100 80 60 40 S-0 day S-1 day S-2 day 20 0 PK1 PK2 PK3 PK1 PK2 PK3 16 Day 18 Day
12 10 8 6 4 2 0 Nutrient contents of NS1020 by fruit ripening stage and drying method Dry matter 1.00 0.80 Freeze dry 0.60 Oven dry 0.40 0.20 0.00 16 Day 18 Day 20 Day 16 Day 18 Day 20 Day 1.20 Carotenoids Freeze dry Oven dry 120 Vitamin C 1200 Antioxidant Activity 100 1000 80 60 40 800 Freeze dry 600 Oven dry 400 Freeze dry Oven dry 20 200 0 16 Day 18 Day 20 Day 0 16 Day 18 Day 20 Day
Nutrient contents of NS1020 as affected by storage length 12 Dry matter 100 Vitamin C 10 80 8 6 4 2 S-0 day S-1 day S-2 day 60 40 20 S-0 day S-1 day S-2 day 0 16 Day 18 Day 0 16 Day 18 Day 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Carotenoids 16 Day 18 Day S-0 day S-1 day S-2 day 1400 1200 1000 800 600 400 200 0 Antioxidant Activity 16 Day 18 Day 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
The PCA score plot of green bitter gourd grown in different seasons Spring Winter Summer 32
% powder 27 Fig. Base peak chromatogram of of M. charantia (NO 76, spring) 100 0 4 5 7 1 9 2 8 3 6 29 The 30 most affected metabolites were 30 identified: 27 -Amino acids -Alkaloids -Fatty acids 16 -Lysophosphatidylcholines -Lysophosphatidylethanols 17 -Triterpenoids 21 1.00 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 19 24 18 25 26 13 23 12 22 28 15 11 14 10 20 Time 33
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