Bitter Gourd Conference Hyderabad, India March AVRDC The World Vegetable Center, Shanhua, Tainan, Taiwan 2

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1 Bitter Gourd Conference Hyderabad, India 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

2 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,

3 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

4 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

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 5

6 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

7 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

8 Cucurbitane-type steroid saponins Sitosterol glucosides Stigmastadienol glucosides 4 4 b-d-glc b-d-glc 2014/3/21 8

9 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.

10 Proteins P-Insulin-like V-Insulin-like Lectin Momordin 11 kda 11 kda 120 kda kda 17 amino acids 17 amino acids 547 amino acids 250 amino acids 2014/3/21 10

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 2014/3/21 11

Food preparation Animal Evidence-based agricultural and dietary strategies for

12 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.

13 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

14 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

15 % LCMS profile of bitter gourd NS _NS 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 9.65e ; , Time /3/21 15

16 16

17 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

18 intensity (E6) Germplasm trial at AVRDC, Taiwan - Group means, content ranges and the top/bottom 3 accessions Root mean squared error PKG1 PKG2 PKG3 PKG1 PKG2 PKG Range 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

8-10 plants/rep, 2 years Four locations: Taiwan: AVRDC-HQ, Tainan South

Ludhiana Tanzania: AVRDC-RCA, Arusha Evaluation: Horticultural traits

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- RCSA AVRDC-RCA PAU AVRDC-HQ 2014/3/21 19

20 Commercial variety trial at AVRDC, Taiwan ANOVA of PKG1-3 by year and drying method PKG1 PKG2 PKG FD 2012-FD 2011-OD 2012-OD PK1 PK2 PK Replication < <.0001 ns <.0001 Variety <.0001 <.0001 <.0001 <.0001 Drying <.0001 <.0001 < <.0001 <.0001 V x D ns ns ns ns ns ns

21 Commercial variety trial at AVRDC, Taiwan Content range and the top /bottom 3 varieties PKG1 PKG2 PKG Freeze dried Range 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 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

22 intensity (10E6) Commercial variety trial at PAU, north India Content range and the top /bottom 3 varieties 0 PKG1 PKG2 PKG3 PKG1 PKG2 PKG 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

23 intensity (E+6) Commercial variety trial at AVRDC, Hyderabad Content range and the top /bottom 3 varieties PKG1 PKG2 PKG3 PKG1 PKG2 PKG Ranges 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

24 intensity (E+6) Commercial variety trial at AVRDC-Arusha Content range and the top /bottom 3 varieties PKG1 PKG2 PKG3 PKG1 PKG2 PKG3 Ranges Top 3 Jasper NS1020 VNR28 Palee NS451 NS1020 BGCT358 Zeena NewMoon Bottom 3 US33 JDragon Zeena Zeena Jasper Abhishek BGCT725 HighMoon BGCT385

25 Content ranges of bitter gourd germplasm and commercial variety trials Trial location D PKG1 PKG2 PKG Germplasm (25) s Taiwan FD Commercial (23) s Taiwan FD Commercial (23) s Taiwan OD Commercial (20) n India OD Commercial (20) s India OD Commercial (20) n Tanzania OD D: drying method; FD: freeze drying; OD: hot air oven drying

26 Postharvest study Three varieties: NS1020, US22, Palee Planting year: 2013 Treatments: 3 harvest dates (16-, day after flowering) 2 drying methods (freeze dried, hot air oven dried) 3 storage length (0, 1, 2, days) Analysis: LCMS, selected nutrients

27 Intensity (E+6) Bioactive compounds of NS1020 as affected by fruit ripening stage and drying method day day 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

28 Intensity (E+6) Bioactive compounds as affected by storage time length 120 NS S-0 day S-1 day S-2 day 20 0 PK1 PK2 PK3 PK1 PK2 PK3 16 Day 18 Day

29 Nutrient contents of NS1020 by fruit ripening stage and drying method Dry matter Freeze dry 0.60 Oven dry Day 18 Day 20 Day 16 Day 18 Day 20 Day 1.20 Carotenoids Freeze dry Oven dry 120 Vitamin C 1200 Antioxidant Activity Freeze dry 600 Oven dry 400 Freeze dry Oven dry Day 18 Day 20 Day 0 16 Day 18 Day 20 Day

30 Nutrient contents of NS1020 as affected by storage length 12 Dry matter 100 Vitamin C S-0 day S-1 day S-2 day S-0 day S-1 day S-2 day 0 16 Day 18 Day 0 16 Day 18 Day Carotenoids 16 Day 18 Day S-0 day S-1 day S-2 day Antioxidant Activity 16 Day 18 Day S-0 day S-1 day S-2 day

31 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

32 The PCA score plot of green bitter gourd grown in different seasons Spring Winter Summer 32

33 % powder 27 Fig. Base peak chromatogram of of M. charantia (NO 76, spring) The 30 most affected metabolites were 30 identified: 27 -Amino acids -Alkaloids -Fatty acids 16 -Lysophosphatidylcholines -Lysophosphatidylethanols 17 -Triterpenoids Time 33

34 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, 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.

35 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

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