Food Chemistry 126 (2011) 172–176

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Food Chemistry

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Quantification of antidiabetic extracts and compounds in bitter gourd varieties

Sandra D. Habicht a,⇑, Veronika Kind a, Silvia Rudloff a, Christian Borsch a, Andreas S. Mueller b, Josef Pallauf c,Ray-yu Yang d, Michael B. Krawinkel a

a Institute of Nutritional Sciences, Justus Liebig University, Giessen, Wilhelmstrasse 20, 35392 Giessen, Germanyb Institute of Agricultural and Nutritional Sciences, Preventive Nutrition Group, Martin Luther University, Halle, Germanyc Institute of Animal Nutrition and Nutritional Physiology, Justus Liebig University, Giessen, Germanyd Nutrition Unit, AVRDC – The World Vegetable Center, Taiwan

a r t i c l e i n f o

Article history:Received 28 June 2010Received in revised form 17 September2010Accepted 22 October 2010

Keywords:Bitter gourdSaponinsConjugated linolenic acidBioactive compounds

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.094

Abbreviations: CLA, conjugated linoleic acid; CLN,⇑ Corresponding author. Tel.: +49 641 99 39 035; f

E-mail addresses: Sandra.D.Habicht@ernaehrung.uMichael.Krawinkel@ernaehrung.uni-giessen.de (M.B.

a b s t r a c t

Several studies have shown blood glucose lowering effects of bitter gourd compounds. Previous experi-ments with db/db mice revealed that the lipid and the saponin extracts are more effective in loweringglycated haemoglobin levels and excessive body weight gain than the hydrophilic extract or the wholefruit.

Therefore, saponin, lipid, and hydrophilic extracts were quantified in bitter gourd varieties using amodified extraction method. Fatty acids were quantified via gas chromatography mass spectrometry.Saponins were quantified photometrically using their haemolytic properties.

White bitter gourd varieties were found to contain significantly lower saponin concentrations (0.25%)compared to green varieties (0.67%). The lipid extract contained high amounts of conjugated linoleic andlinolenic acids (up to 65.89%). Quantification of the most effective antidiabetic compounds in bitter gourdvarieties may help to identify the most effective varieties and to establish the bitter gourd or bitter gourdextracts as effective blood glucose lowering dietary components or supplements.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Momordica charantia (Cucurbitaceae) is a tropical plant withgreen leaves and yellow flowers. It is grown for its edible fruit,the bitter gourd, in African, Asian, and South American countriesas well as in the Caribbean (Basch, Gabardi, & Ulbricht, 2003).Depending on the variety, the immature fruit is white or greenwith a different size or shape. The immature fruit is a cheap vege-table available the whole year at local markets in southern andeastern Asia and tropical Africa (Sekar, Sivagnanam, & Subramani-an, 2005; Sridhar, Vinayagamoorthi, Arul Suyambunathan, Bobby,& Selvaraj, 2008). The bitter gourd is reported to be effective inthe treatment of various diseases in traditional medicine (Baschet al., 2003). It is rich in nutrients such as essential amino acids,vitamin A, carotenoids, folic acid, and vitamin C and the wholeplant contains many bioactive compounds. Momordin I is reportedas having tumour protective effects; momordicines I and II as hav-ing antimicrobial, acylglucosylsterols antimutagenic, and chitinasebacteriostatic effects (Nerurkar, Lee, Motosue, Adeli, & Nerurkar,

ll rights reserved.

conjugated linolenic acid.ax: +49 641 99 39 039.ni-giessen.de (S.D. Habicht),

Krawinkel).

2008; Njoroge & van Luijk, 2004; Yuwai, Rao, Kaluwin, Jones, & Riv-ett, 1991). Saponins, momordicosides K and L as well as momord-icines I and II cause the bitter taste (Harinantenaina et al., 2006;Yasuda, Iwamoto, Okabe, & Yamauchi, 1984).

In earlier studies the existence of an insulin-like protein, firstnamed ‘‘p-insulin’’ later ‘‘polypeptide p’’ was reported as beingresponsible for blood glucose lowering effects of the bitter gourdin types I and II diabetic patients (Khanna & Jain, 1981). Besidesthis peptide, other bioactive compounds of M. charantia are sug-gested as being capable of lowering blood glucose levels. Thesesubstances include glycosides, such as the momordicosides S andT, alkaloids, and triterpenoids (Cheng, Huang, Chang, Tsai, & Chou,2008; Harinantenaina et al., 2006; Tan et al., 2008).

Numerous different methods to extract antidiabetic bittergourd compounds or extracts have been described (Cheng et al.,2008; Miura, Itoh, Iwamoto, Kato, & Ishida, 2004; Roffey, Atwal,Johns, & Kubow, 2007). A comparison of the results of these studiesin terms of the quantity of components and the antidiabetic effectsof the extracts is therefore often not possible. However, data fromthe current literature (Chao & Huang, 2003; Oishi et al., 2007) andour experiments with db/db mice revealed that bitter gourd sapo-nins and lipids are even more effective in lowering glycated hae-moglobin levels and excessive body weight gain than thehydrophilic extract or a whole fruit powder (Klomann, Mueller,Pallauf, & Krawinkel, 2010).

S.D. Habicht et al. / Food Chemistry 126 (2011) 172–176 173

Even though it was not as effective as the saponin or the lipidextract, the hydrophilic extract reduced body weight gain in db/db mice, too.

Thus, the aim of the present study was to quantify the saponinextract, the lipid extract, and the hydrophilic extract in bitter gourdvarieties using the extraction method reported earlier (Klomannet al., 2010) that combines the methods described by Oishi et al.(2007) and Chao and Huang (2003). We further quantified haemo-lytic saponins in the saponin extracts as well as 9c-, 11t-, 13t-con-jugated linolenic acid (CLN) and conjugated linoleic acid (CLA) inthe lipid extracts.

2. Materials and methods

2.1. Bitter gourd material

Fruits of six green bitter gourd varieties (samples 1–6) were ob-tained from a market garden near Frankfurt/Main, Germany. Sam-ple 5 was similar to a variety described as pearl bitter gourd by Chaoand Huang (2003). The other five samples were more or less simi-lar to the variety described as green bitter gourd in Chao and Huang(2003) with differences in size, shape and colour. Fresh fruits werecut, freeze-dried (Gamma 1–20, CHRIST, Osterode, Germany) andground.

Powder of the flesh of another five green (samples 7–11) andthree white varieties (samples 12–14) was obtained from theAVRDC (The World Vegetable Center, Taiwan). Samples 7–14 canbe defined as TOT4234 from Bangladesh (7), NS1020 from Namd-hari seed in India (8), TOT2533 from India (9), best 165 F1 fromEast–West seed company in Thailand (10), Karela from a tradi-tional market in Taiwan (11), Yueh-Hua from Know-you seed inTaiwan (12), Showy from Know-you seed in Taiwan (13), andShowy from a traditional market in Taiwan (14). Samples 11 and14 were purchased from a local market in Shanhua (Taiwan), theothers were planted and obtained from AVRDC fileds.

2.2. Preparation of bitter gourd extracts

The saponin, lipid, and hydrophilic extracts were extractedcombining the methods published by Oishi et al. (2007) and Chaoand Huang (2003).

10 g homogeneous powder from each sample were stirred in150 ml ethyl acetate in the dark for 2 h and filtered. Using a rotaryevaporator (Laborota digitally 4002, Heidolph, Schwabach, Ger-many) the ethyl acetate was evaporated at 35 �C to obtain the lipidextract. The non-ethyl acetate soluble filter residue was stirred in150 ml methanol in the dark for 2 h and filtered. The filtrate wasreduced to dryness in the rotary evaporator at 42 �C and the dryresidue was dissolved in 50 ml of distilled water and 50 ml of n-butanol. The water phase and the n-butanol phase were separatedand then evaporated at 42 �C to obtain the hydrophilic and thesaponin extract.

2.3. Fatty acids

For the gas chromatography mass spectroscopy analyses,methyl esters of cis/trans-isomers of conjugated linoleic acid(CLA) from Sigma (Steinheim, Germany) as well as unmethylated9c-, 11t-, 13t-conjugated linolenic acid (CLN) from Biozol (Eching,Germany) were used as standards. Fatty acids of the bitter gourdlipid extracts and the unmethylated CLN standard were methyl-ated according to the method described by Suzuki, Arato, Noguchi,Miyashita, and Tachikawa (2001). 2.0 ll of the samples or the stan-dards were then injected into an Agilent 6890 N GC with a split ra-tio of 10. A DB-FFAP capillary column (Agilent 122-3262) was used

with a temperature ramp of 80 to 245 �C and 1 ml/min helium ascarrying gas. Impact ionisation and detection via a quadrupolemass selective detector (Agilent 5973 N) followed. NIST02 wasused as the standard spectra database in addition to custom com-pound libraries.

2.4. Saponins

Saponins were quantified via a modification based on the meth-od described by Mackie, Singh, and Owen (1977) using the haemo-lytic effect of saponins. The saponin extract was dissolved indistilled water and 100 ll of this solution were incubated with1 ml fresh EDTA-blood at 30 �C for 30 min. After centrifugation(3000 rpm) for 10 min, haemoglobin was quantified in the super-natant photometrically (Cary 50 Bio, Varian, Australia) at 545 nm.We used a number of three analytical replicates per sample. Theratio of the standard deviation to the mean was for every samplesmaller than 0.05. A calibration curve with Saponin depur fromRoth (Karlsruhe, Germany) was used to quantify saponins. As wequantified saponins using the haemolytic effect of saponins wehave defined saponin concentration in the following as concentra-tion of haemolytic saponins.

2.5. Statistical analysis

After ascertainment of normal distribution and homoscedastic-ity, the t-test (two-sided) for independent samples was used to cal-culate significant differences (P 6 0.05) with the SPSS 17.0 programfor windows.

3. Results

We found mean concentrations of 5.05% lipid extract, 3.68%saponin extract, and 19.95% hydrophilic extract in dried wholefruit powders (samples 1–6). Proportions of the extracts were dif-ferent in the dry matter of the flesh (samples 7–14), namely 0.91%lipid extract, 3.97% saponin extract, and 16.86% hydrophilic ex-tract. The mean concentration of the saponin extract was almostthe same in the flesh and in the whole fruit of the bitter gourd vari-eties, whereas concentrations of both the lipid extract and thehydrophilic extract were lower in the flesh. However, this was onlysignificant for the lipid extract (P 6 0.0001) (Table 1).

Mean concentrations of haemolytic saponins were 0.40%(0.27%–0.66%) on a dry matter basis in whole fruits (samples 1–6) and 0.52% (0.22%–0.79%) on a dry matter basis in the flesh (sam-ples 7–14) of the bitter gourd varieties (Table 1). The mean concen-tration of haemolytic saponins in the saponin extract was 11.77%,ranging from 7.09% to 16.98% (data not shown).

Interestingly, we found a significantly (P 6 0.001) lower saponinconcentration in the fleshy part of the white (samples 12–14) bit-ter gourd varieties (0.25% ± 0.04%) compared to the green (samples7–11) varieties (0.67% ± 0.12%) (Fig. 1). The proportion of the sapo-nin extract was also significantly (P 6 0.05) lower in the flesh ofwhite fruits (3.17% ± 0.06%) than in the flesh of the green ones(4.44% ± 0.85%). As all used whole fruit powders were from greenvarieties, we compared saponin concentrations of the whole fruitpowders to that of the green flesh powders. Mean concentrationof haemolytic saponins was 0.67% ± 0.12% significantly (P 6 0.01)higher in the flesh of green bitter gourd varieties (samples 7–11)in comparison with green whole fruit powders (samples 1–6) con-taining only 0.40% ± 0.14% haemolytic saponins on a dry matter ba-sis (Fig. 1).

The lipid extract was very rich in 9c-, 11t-, 13t-CLN. This fattyacid was present in all the bitter gourd varieties tested. However,the relative amount of 9c-, 11t-, 13t-CLN in the lipid extract varied

Table 1Concentrations of lipid extract, conjugated linoleic acid (CLA), and 9c-, 11t-, 13t-conjugated linolenic acid (CLN), saponin extract, haemolytic saponins, and hydrophilic extract inwhole fruit powders (samples 1–6) or flesh powders of green (samples 7–11) or white (samples 12–14) bitter gourd varieties.

Bitter gourd(sample number)

Lipid extract(% of dry matter)

CLA(% of lipid extract)

9c-, 11t-, 13t-CLN(% of lipid extract)

Saponin extract(% of dry matter)

Haemolytic saponins(% of dry matter)

Hydrophilic extract(% of dry matter)

Whole fruit1 6.40 0.00 4.75 3.22 0.43 17.632 5.35 0.23 22.94 3.02 0.36 19.513 5.79 0.16 22.00 5.16 0.66 24.244 3.98 0.50 32.58 2.9 0.27 21.645 2.97 0.22 9.29 3.61 0.32 16.986 5.84 0.38 65.89 4.13 0.35 19.72Mean 5.05 0.25 26.24 3.68 0.40 19.95

Flesh7 2.04 1.42 33.18 4.78 0.78 11.898 0.79 1.36 33.13 5.34 0.79 17.559 0.57 0.63 21.21 4.99 0.68 16.3210 0.24 0.42 13.13 3.75 0.54 18.4611 1.64 1.44 34.20 3.34 0.57 14.3912 0.63 1.03 27.47 3.22 0.30 15.4113 0.73 1.74 34.90 3.10 0.22 21.5214 0.65 1.16 19.44 3.20 0.24 19.37Mean 0.91** 1.15* 27.08 3.97 0.52 16.86

* Significantly different from whole fruit (P 6 0.001).** Significantly different from whole fruit (P 6 0.0001).

00,10,20,30,40,50,60,70,80,9

whole fruit green flesh white flesh

Hae

mol

ytic

sap

onin

s

[% o

f dr

y m

atte

r]

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

P ≤ 0.001

P ≤ 0.001

Fig. 1. Concentration of haemolytic saponins [% of dry matter] in bitter gourd wholefruit powders (samples 1–6) in comparison to flesh powders of green (samples 7–11) and white (samples 12–14) bitter gourd varieties. Data expressed as Mean ± SD.

174 S.D. Habicht et al. / Food Chemistry 126 (2011) 172–176

markedly within the bitter gourd whole fruits (samples 1–6)(4.75% to 65.89% of total lipids; mean = 26.24%). The fleshy partof the bitter gourd (samples 7–14) had a lower concentration ofthe lipid extract but the relative amount of 9c-, 11t-, 13t-CLNin the lipid extract (mean = 27.08%) was not different from thatof the whole fruit (samples 1–6). However, variance was lower inthe flesh (samples 7–14) and ranged from 13.13% to 34.90% 9c-,11t-, 13t-CLN in the lipid extract (Table 1).

The mean relative amount of CLA in the lipid extract of the bit-ter gourd varieties was 1.15% significantly higher (P 6 0.001) in theflesh (samples 7–14) compared to 0.25% in the whole fruit (sam-ples 1–6). Compared to 9c-, 11t-, 13t-CLN the concentration ofCLA in the bitter gourd was very low (Table 1).

4. Discussion

In herbal medicine various plants and their extracts are used forthe treatment of diabetes mellitus. Only a few of these plants, forexample the bitter gourd, have proven antidiabetic effects in cellculture, rodents, and humans (Hui, Tang, & Go, 2009; Krawinkel& Keding, 2006). Amongst other substances saponins and lipidsare considered as exerting the antidiabetic effects of the fruit (Chao

& Huang, 2003; Oishi et al., 2007). In our animal trial with db/dbmice we could show that the lipid and the saponin extracts aremore effective in lowering glycated haemoglobin levels or bodyweight gain than the hydrophilic extract or the whole fruit powderof the bitter gourd (Klomann et al., 2010). Extracts were preparedcombining the methods reported by Chao and Huang (2003) andOishi et al. (2007). Here we used this extraction method to quantifythese extracts in different bitter gourd varieties. An extractionmethod like this offers the opportunity to extract different frac-tions from one sample and makes future studies analysing bioac-tive effects of the bitter gourd or other medicinal plants morecomparable. In addition we used the saponin extract to measureconcentrations of haemolytic saponins and the lipid extract tomeasure 9c-, 11t-, 13t-CLN and CLA.

Quantification of saponins, conjugated fatty acids as well assaponin and lipid extracts in bitter gourd varieties may help to findvarieties most effective in the prevention and treatment of diabe-tes mellitus that should be preferred for cultivation and use.

Oishi et al. (2007) found a concentration of 1.3% saponin extract(on a dry matter basis) in M. charantia powder after extraction withn-butanol and 15.4% saponins in the saponin extract. Our resultssuggest a higher concentration of the saponin extract (mean3.68%) in bitter gourd whole fruits (samples 1–6) with 11.77% hae-molytic saponins in the saponin extract. Differences regardingthese findings may result from differences regarding the extractionmethods and the different varieties investigated as Oishi et al.(2007) do not provide any information about variety or plant partused. Nevertheless, the results are of the same magnitude indicat-ing that bitter gourd is a vegetable rich in saponins. For examplecharantin is one important steroidal saponin in the bitter gourdwith beta-sitosterol and stigmasterol as aglycones (Dans et al.,2007; Harinantenaina et al., 2006).

We found a mean concentration of 0.40% haemolytic saponinsin bitter gourd fruits (samples 1–6) and 0.52% haemolytic saponinsin bitter gourd flesh (samples 7–14). Green bitter gourd varietiesare with 0.40% ± 0.14% haemolytic saponins (on dry matter basis)in the whole fruit (samples 1–6) and with 0.67% ± 0.12% haemo-lytic saponins (on dry matter basis) in the flesh (samples 7–11)comparable to other saponin rich food plants such as chickpea(0.56% saponins on dry matter basis), soybean (0.43% saponinson dry matter basis), lentils (0.37%–0.46% saponins on dry matterbasis), and other legume plants (Fenwick & Oakenfull, 1983).

S.D. Habicht et al. / Food Chemistry 126 (2011) 172–176 175

However, data are not directly comparable because of the differentmethods used to quantify saponins.

Our results indicate that the flesh of white bitter gourd varieties(samples 12–14) has a lower amount of saponins than the flesh ofgreen ones (samples 7–11), namely 0.22%–0.30% haemolytic sapo-nins on a dry matter basis compared to 0.54%–0.79%. The lowersaponin concentration of white bitter gourd varieties may resultin a lower effectiveness of these varieties in preventing and treat-ing diabetes mellitus compared to green varieties. This assumptionneeds further in vitro and in vivo studies comparing the blood glu-cose lowering effect of white and green fruit powders.

We also found that the mean concentration of haemolytic sap-onins was with 0.67% significantly (P 6 0.01) higher in the flesh ofgreen varieties (samples 7–11) compared to the saponin concen-tration of the green fruits (samples 1–6) with only 0.40%. This leadsto the conclusion that the saponin concentration seems to be high-er in the fleshy part of the fruit than in the seeds. However, furthermeasurement of saponin concentrations in bitter gourd seeds andflesh is needed to confirm this.

In addition to its high concentration of saponins, the bitter gourdis one of a few edible fruits that contain conjugated fatty acids(Suzuki et al., 2001). Identifying bitter gourd varieties rich in oiland 9c-, 11t-, 13t-CLN will help to focus on varieties most effectivein preventing and treating diabetes and diabetic complications.

Our results show a total lipid content of the whole fruit (sam-ples 1–6) varying from 2.97% to 6.40% of dry matter in different bit-ter gourd varieties. This is similar to results reported by Chuanget al. (2006). They found a total lipid content of 3.7%.

We found a significantly higher concentration of the lipid ex-tract in the whole fruits (samples 1–6) in comparison to the bittergourd flesh (varieties 7–14), indicating that in M. charantia the oilis mainly located in the seeds, whereas the fruit-flesh has a low to-tal lipid content. This assumption is also confirmed by data re-ported by Suzuki et al. (2001). They found up to 41% total lipidsin the wet weight of bitter gourd seeds, depending on maturation,and only up to 0.2% total lipids in the flesh of the fruits.

The mean concentration of CLA was with 0.25% of total lipidsvery low in the whole fruit powders of bitter gourd varieties (sam-ples 1–6), but with 1.15% of total lipids significantly higher in theflesh (samples 7–14) (Table 1). This leads to the conclusion thatCLA seems to be located in the flesh and/or the skin – and not inthe seeds of the bitter gourd and confirms findings reported by Su-zuki, Abe, and Miyashita (2004). They could not even detect CLA inthe seeds. However, the seeds are very rich in CLN like 9c-, 11t-,13t-CLN (Dhar et al., 2007; Suzuki et al., 2004). Our results demon-strate that this fatty acid represents up to 65.89% of total lipids inbitter gourd whole fruits. However, the amount of 9c-, 11t-, 13t-CLN varied within the fruits examined. The mean concentrationwas 26.24% of total lipids, which is comparable to the results ofChuang et al. (2006) who analysed 17.2%–27.6% in the oil of differ-ent fruits. But, in contrast to our results they reported only 7.6% of9c-, 11t-, 13t-CLN in the oil of the bitter gourd flesh, whereas wefound 13.13%–34.90%.

However, the seeds are the main source of 9c-, 11t-, 13t-CLNbecause of the higher concentration of total lipids in the seeds. Re-ported data on 9c-, 11t-, 13t-CLN concentration in the seedoil ofthe bitter gourd range from 28.7% (Chuang et al., 2006) to 57.7%(Dhar et al., 2007) and 61.6% (Suzuki et al., 2004). Thus, the amountof 9c-, 11t-, 13t-CLN definitely varies between different fruits ofthe bitter gourd, persumably depending on the stage of matura-tion. In our study, varieties 1, 2, and 6 had a very similar phenotype(not shown), but exhibited large differences in the amount of 9c-,11t-, 13t-CLN (Table 1). Furthermore, it is well known that totallipids and the amount of CLN increase during maturation of thefruit (Suzuki et al., 2001). Therefore, we conclude that for the con-centration of 9c-, 11t-, 13t-CLN and the fatty acid profile of the bit-

ter gourd maturity is more important than the variety. Because ofthe proven biological effect of the bitter gourd oil and its high con-centration in the seeds of the bitter gourd, it can be concluded thatthe seeds should not be removed during food preparation.

5. Conclusions

It must be assumed that different bioactive compounds are in-volved in the hypoglycaemic effect of the bitter gourd. However,saponins and lipids seem to be of relevance for the antidiabetic ef-fects. Therefore, bitter gourd varieties with high amounts of thesesubstances might be most effective in the prevention and treat-ment of diabetes mellitus and thus should be preferred for cultiva-tion and use. The white bitter gourd varieties have a low saponinconcentration and may be less effective. Total lipids and fatty acidcomposition seem to depend more on the maturity of the fruitsthan on differences between varieties. Hence, green bitter gourdvarieties that are not too immature and gently processed can beconsidered for the prevention and treatment of diabetes mellitus.

Acknowledgements

This work was funded by the Federal Ministry for EconomicCooperation and Development (Germany) and the Dannon Insti-tute Nutrition for Health (Germany).

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