Balsam pear (Momordica charantia L.), also known as Lianggua in Chinese, belongs to the Momordica genus of Cucurbitaceae family, is a kind of characteristic medicinal vegetables. The tender fruit of Momordica charantia contains rich nutrients, such as vitamin C, amino acid, crude protein, cellulose, soluble sugar, etc., among which the content of Vc ranks first among melon vegetables, and greatly exceeds that of tomato. Momordica charantia fruit also contains saponins (Keller et al., 2011), polysaccharides (Zhang et al., 2010), flavonoids (Lin et al., 2011), phenols (Huang et al., 2014) and other physiological active ingredients. It has physiological functions such as lowering blood sugar, lowering blood pressure, lowering blood lipid, antioxidant, clearing heat and detoxifying, improving immunity, etc., and has high medicinal value (Yuan, 2006; Li, 2007; Luo and Zhang, 2016). In recent years, with the enhancement of people’s health awareness, Momordica charantia has attracted more and more attention and is cultivated all over the country.

The content of nutrients and active substances in plants often varies from variety to variety, and Momordica charantia is no exception. Du et al. (2014, Chinese Agricultural Science Bulletin, 30(1): 226-231) found that there were significant differences in nutrient composition and physiological active substances of 22 varieties (lines) of Momordica charantia.. Cao et al. (2014) found that there were significant differences in the content of polysaccharides and saponins in Momordica charantia among different varieties. Liu et al. (2017) concluded that there were significant differences in saponin content, antioxidant activity and α-glucosidase inhibition rate in the pulp of 13 different varieties of Momordica charantia. Therefore, it is of great significance to select the varieties of bitter melon which are rich in certain nutrients or physiological active substances and have strong antioxidant activity for consumer’ diet orientation and the development of health products with Momordica charantia as raw materials. At present, the existing studies are limited to the comparison of the content of nutrients and functional substances in different Momordica charantia varieties, and the analysis of antioxidant activity of functional substances. There is no study on the comparison of antioxidant activity in pulp of different Momordica charantia varieties, and the correlation analysis between functional substance content and activity is also few. In this study, the differences of nutrient content, functional substance content and total antioxidant activity in pulp of 25 different varieties (lines) of Momordica charantia L. were compared, and the correlation between functional substance content and activity was analyzed, in order to provide theoretical basis for the breeding of high-quality functional Momordica charantia L. varieties and the development of functional food of Momordica charantia L..

1 Results and Analysis

1.1 Comparison of nutritional constituents in different varieties of Momordica charantia

There were some differences in the nutritional components of different Momordica charantia varieties (Table 1; Table 2), the Vc content of different Momordica charantia varieties (lines) ranged from 56.65 to 158.65 µg/g, with an average value of 85.71 µg/g, among which the highest Vc content was No.10, and the lowest Vc content was No.14. There were significant differences in VC content among different varieties (lines), and the coefficient of variation was 28.47%. The soluble protein content of 25 Momordica charantia varieties (lines) ranged from 4.70 mg/g to 13.30 mg/g, with an average value of 7.98 mg/g, with the highest content being No.9 and the lowest content being No.15, and the coefficient of variation among different varieties (lines) was 29.07%. The cellulose content of different Momordica charantia varieties (lines) varied from 262.28 to 399.46 mg/g, with an average value of 319.04 mg/g, with the highest content being No.14 and the lowest content being No.16. The difference among varieties (lines) was relatively small, and the coefficient of variation was 11.05%. The amino acid content of different Momordica charantia varieties (lines) varied from 0.31 to 2.60 mg/g, with an average value of 1.45 mg/g, with the highest value being No.11 and the lowest value being No.23. The coefficient of variation among varieties (lines) was 43.35%, and the difference was significant. The total acid content of different Momordica charantia varieties (lines) ranged from 0.31 to 0.52%, with an average value of 0.41%, with little difference among varieties (lines) and coefficient of variation of 12.20%.

Table 1 Comparison of nutrient contents in pulp of 25 Momordica charantia varieties (lines) Note: The value marked by different letters are significantly different (P<0.05)

Table 2 Variation range and coefficient of variation of nutrient content of 25 Momordica charantia varieties (lines)

1.2 Comparison of functional components in different varieties of Momordica charantia

1.2.1 Comparison of functional component content, variation amplitude and variation coefficient of different varieties of Momordica charantia

The contents of saponins in different varieties of Momordica charantia were significantly different (Table 3; Table 4), the average content was 5.02 µg /g, and the coefficient of variation was 45.62%. The saponins content of No.24 Momordica charantia was the highest (10.27 µg/g), and that of No.7 Momordica charantia was the lowest (1.97 µg/g), with a difference of more than five times. The content of polysaccharide in fruits of different varieties of Momordica charantia ranged from 23.49 to 60.01 mg/g, with an average content of 38.36 mg/g and a coefficient of variation of 25.47%. The highest content was No.21 and the lowest was No.9. The average flavonoid content in fruits of different varieties of Momordica charantia was 2.64 mg/g and the coefficient of variation was 21.97%. The highest flavonoid content was 4.11 mg/g in No.10 and 1.76 mg/g in No.20. The variation of total phenol content in Momordica charantia fruit ranged from 5.29 to 10.21 mg/g, the average content was 7.68 mg/g, and the coefficient of variation was 18.49%. The highest content was No.14, and the lowest content was No.1.

Table 3 Comparison of saponins, polysaccharides, flavonoids and total phenolic content in different Momordica charantia varieties Note: The value marked by different letters are significantly different (P<0.05)

Table 4 Variation range and coefficient of variation of saponins, polysaccharides, flavonoids and total phenolic content in different varieties of Momordica charantia

1.2.2 Distribution of saponins, polysaccharides, flavonoids and total phenols in different varieties of Momordica charantia

The saponins content in the flesh of 25 Momordica charantia varieties was approximately normal distribution, and the saponins content of 72.0% Momordica charantia varieties ranged from 1.96 to 6.75 µg/g. The distribution of polysaccharide content in flesh was approximately skewed, and the number of varieties in 23.49~45.61 mg/g accounted for 80% of the total number of varieties. The distribution of flavonoids in the flesh of Momordica charantia was also skewed, with about 80% of varieties distributed in 1.76~3.17 mg/g. The total phenol content in the flesh of Momordica charantia was approximately normal distribution, and about 60% varieties were distributed in 6.10~10.0 mg/g (Figure 1).

Figure 1 Variety distribution of saponins, polysaccharides, flavonoids and total phenolic content in Momordica charantia

1.3 Comparison of antioxidant capacity of different varieties (lines) of Momordica charantia

There were significant differences in the total antioxidant capacity of different Momordica charantia varieties (lines) (Table 5; Table 6). The antioxidant capacity of DPPH, ABTS^+· and FRAP in pulp of Momordica charantia were significantly different among different varieties (lines) (P<0.05). The DPPH antioxidant values of the 25 cultivars ranged from 1.25 to 2.59 μmol Trolox/g, with an average value of 1.73 μmol Trolox/g, and the coefficient of variation was 19.65%. Among them, the DPPH antioxidant capacity was the strongest in No.22, and the weakest in No.6. The ABTS^+· antioxidant values of 25 varieties (lines) ranged from 40.36 to 92.89 μmol Trolox/g, the mean value was 67.38 μmol Trolox/g, and the coefficient of variation was 17.25%. The highest ABTS^+· antioxidant capacity was No.14, and the lowest was No.1. The FRAP antioxidant value of 25 varieties (lines) ranged from 5.26 to 21.42 μmol Trolox/g, with an average value of 11.62 and a coefficient of variation of 31.50%. The FRAP antioxidant capacity was highest in No.14 and lowest in No.22.

Table 5 Total antioxidant capacity of different varieties of Momordica charantiac Note: The value marked by different letters are significantly different (P<0.05)

Table 6 Variation range and coefficient of variation of antioxidant capacity in different varieties of Momordica charantia

1.4 Correlation analysis between functional component content and total antioxidant capacity of Momordica charantia

There was no significant positive correlation between saponin content and antioxidant capacity of DPPH in Momordica charantia, but no significant negative correlation between saponin content and antioxidant capacity of ABTS^+· and FRAP. Polysaccharide content was not significantly positively correlated with the antioxidant capacity of DPPH, was not significantly positively correlated with the antioxidant capacity of ABTS^+· and was significantly negatively correlated with the antioxidant capacity of FRAP (r=-0.581, P<0.05). There was no significant negative correlation between flavonoid content and antioxidant capacity of DPPH in Momordica charantia, but no significant positive correlation between flavonoid content and antioxidant capacity of ABTS and FRAP. The total phenol content in the flesh of Momordica charantia was significantly correlated with the three kinds of antioxidant capacity, and was significantly negatively correlated with the antioxidant capacity of DPPH (r=-0.436, P<0.05), and was significantly positively correlated with the antioxidant capacity of ABTS^+· (r=0.795, P<0.01). It was positively correlated with the antioxidant capacity of FRAP (r=0.853, P<0.01) (Table 7).

Table 7 Correlation between content of functional components and total antioxidant capacity in Momordica charantia fruit Note: *: Indicates significant correlation at 0.05 level; **: Indicates significant correlation at 0.01 level

2 Discussion

As a vegetable with the same origin as medicine and food, Momordica charantia has attracted more and more people's attention and love (Du et al., 2014, Chinese Agricultural Science Bulletin, 30(1): 226-231). A large number of studies have shown that different varieties and different habitats have a great impact on the nutritional quality of Momordica charantia (Xiang et al., 2000; Zhang et al., 2011; Jie et al., 2011). Du et al. (2014, Chinese Agricultural Science Bulletin, 30(1): 226-231) found that there were great differences in the nutritional composition of 22 different varieties (lines) of Momordica charantia. The average Vc content, cellulose content and titratable acid content were 8.34 mg/(100 g·FW), 2.33% and 10.24 mmol/(100 g·FW), respectively. Li et al. (2007) determined that the protein content of fresh Momordica charantia in the three producing areas was similar, and the Vc content was greatly affected by the producing area. In this study, the nutrient content of fresh Momordica charantia pulp of 25 varieties (systems) was determined. The nutrient content of different varieties (systems) was significantly different. The average contents of Vc, soluble protein, cellulose, amino acid and total acid were 85.71 µg/g, 7.98 mg/g, 319.04 mg/g, 1.45 mg/g and 0.41%, respectively. The average content of Vc was similar to that of Du et al. (2014, Chinese Agricultural Science Bulletin, 30(1): 226-231) showed similar results. The variation range of each nutrient component was large, and the coefficient of variation of amino acid content was the largest, which was 43.35%, followed by soluble protein content and Vc content, which were 29.07% and 28.47% respectively, which was consistent with the results of previous studies (Li et al., 2007; Zhang et al., 2011). As a fresh vegetable, Momordica charantia requires high content of Vc, soluble protein and amino acid, while low content of cellulose and total acid. After comprehensive comparison of 25 Momordica charantia varieties (lines), variety 10 Momordica charantia has the highest Vc content, low total acid and cellulose content. As a type of white Momordica charantia, bitter taste is moderate and crisp. Secondly, the varieties (lines) with higher Vc content were No.1, No.6, No.15, No.23 and No.24, which were suitable for cultivation as fresh edible vegetables.

Saponins, polysaccharides, flavonoids and total phenols are the main functional components of momordica balmomordica, which have various physiological activities such as lowering blood sugar, reducing weight, antioxidant, clearing heat and detoxifying. The content of functional components in different varieties of Momordica charantia was also different (Tian et al., 2008; Huang et al., 2008; Zhang et al., 2016; Yang et al., 2009; Jiang et al., 2020). Liu et al. (2017) compared the content of saponins in pulp of 13 different varieties of Momordica charantia, and the range was 0.52~1.20 g/100g DW, with an average value of 0.79 g/100g DW. Cao et al. (2014) determined the polysaccharide and saponin contents of 41 Momordica charantia varieties, and the polysaccharide contents ranged from 5.24 to 92.40 mg/g, and the saponin contents ranged from 5.21 to 52.21 mg/g. Huang et al. (2011) compared the different contents of total phenol and total flavone among 14 Momordica charantia varieties, and the total phenol ranged from 175.27 to 413.79 mg GAE/100 g DW, with an average value of 292.96 mg GAE/100 g DW, and the coefficient of variation was 22.10%. Flavonoid content ranged from 8.97 to 18.22 mg CE/100 g DW, with an average value of 11.73 mg CE/100 g DW, and the coefficient of variation was 22.80%. In this study, the contents of saponins, polysaccharides, flavonoids and total phenols in the flesh of 25 different varieties (lines) of Momordica charantia showed significant differences, and the variation was also large, with an average value of 5.02 µg/g, 38.36 mg/g, 2.64 mg/g and 7.68 mg/g, respectively. The average content of saponins was slightly lower than that of Liu et al. (2017), but higher than that of Cao et al. (2014) and Jiang et al. (2020). The variation of polysaccharide content was less than that determined by Cao et al. (2014), and the mean contents of flavonoids and total phenols were higher than those determined by Huang et al. (2011). Among the four functional components, saponin content had the highest coefficient of variation (45.62%), while total phenol content had a smaller coefficient of variation (18.49%). In conclusion, the content of functional components in the pulp of Momordica charantia was greatly affected by regional, variety, environment and other factors. Among the 25 varieties (lines), the varieties (lines) with the highest contents of saponins, polysaccharides, flavonoids and total phenols were No.24, No.21, No.10 and No.14, respectively, which could be cultivated as special varieties for processing.

Antioxidant activity is an important physiological activity of Momordica charantia, which is closely related to functional components of Momordica charantia (Huang et al., 2011; Yuan et al., 2006; Cao et al., 2014; Liu et al., 2017). The antioxidant capacity of different varieties of Momordica charantia was different. Liu et al. (2017) found that the total antioxidant capacity index of total saponins of 13 different varieties of Momordica charantia ranged from 2 747.46 to 15 584.07 μmol Trolox/g, with an average of 8 879.48 μmol Trolox/g, and the coefficient of variation was 34.91%. The total saponins were not the main contributors to antioxidant activity of Momordica charantia. Huang et al. (2011) evaluated the antioxidant capacity of Momordica charantia samples by using FRAP method, DPPH and IC50 value of ABTS^+· scavenging capacity. The ranges were 272.16~713.32 mg TE/100g DW, 11.43~34.14 mg GAE/100 g DW, and 21.57~119.71 mg GAE/100 g DW, respectively, which fully reflected the antioxidant effects of the samples. Total phenol content was positively correlated with FRAP value and negatively correlated with IC50 value of ABTS^+· scavenging ability and DPPH, respectively. In this study, DPPH method, ABTS^+· method and FRAP method were used to study the antioxidant capacity of 25 varieties (lines). The antioxidant values ranged from 1.25 to 2.59 μmol Trolox/g, 40.36 to 92.89 μmol Trolox/g, 5.25 to 21.42 μmol Trolox/g, and the coefficients of variation were 19.65%, 17.25% and 31.50%, respectively. The antioxidant capacity of pulp was much lower than that of total saponins and phenols of extracts (Huang et al., 2011; Liu et al., 2017), which is also the main reason for developing industrial extraction methods of functional components of Momordica charantia and processing them into various health products. In this study, the contents of total saponins and flavonoids were not significantly correlated with the antioxidant capacity of Momordica charantia pulp, while polysaccharide content was negatively correlated with the antioxidant capacity of FRAP (r=0.581, P<0.05), and total phenol content was significantly correlated with the three antioxidant capacities. It was significantly negatively correlated with the antioxidant capacity of DPPH (r=-0.436, P<0.05), and significantly positively correlated with the antioxidant capacity of ABTS and FRAP (r=0.795, P<0.01; r=0.853, P<0.01), which was consistent with the findings of Huang et al. (2011), Du et al. (2009), and Kriengsak et al. (2006). The results showed that the total phenol content in the pulp of Momordica charantia was closely related to the antioxidant capacity of Momordica charantia, indicating that the total phenol was one of the important contributions to the antioxidant capacity of Momordica charantia.

There were significant genotypic differences in the contents of nutrients, functional components and antioxidant values in the pulp of 25 Momordica charantia varieties (lines), and the total phenolic content in the pulp was the main contribution of antioxidant activity of Momordica charantia. In the breeding and cultivation of new varieties of Momordica charantia, fresh food and medicinal processing varieties should be distinguished, and corresponding varieties should be selected according to different uses, which is conducive to giving full play to the advantages of varieties and commercial value.

3 Materials and Methods

3.1 Test materials

The 25 bitter melon materials used in this experiment were provided by the research group of Horticulture Institute of Shanghai Academy of Agricultural Sciences engaged in the breeding of bitter melon varieties (Table 8), among which ‘Qingqiu No.1’ was the self-bred variety, ‘Taiwanqingpi’, ‘Abao No.3’, ‘Fujian’, ‘Yuezhen’, ‘Pingguo’, ‘Baiyu’ and ‘Jufengcuilv’ were purchased hybrids, and the remaining 17 were breeding hybrids.

Table 8 Basic fruit characters of 25 Momordica charantia varieties (lines)

3.2 Test methods

The experiment was conducted at Zhuanghang Comprehensive Test Station of Shanghai Academy of Agricultural Sciences. On February 25, 2020, plump seeds of each Momordica charantia variety were selected and soaked for 15 h, then seeds were artificially consumed, and then placed in an incubator at 30 ℃ to promote germination. When the buds were 0.5 cm long, they were sown in 50 hole trays, and the seedbed temperature was set at 30 ℃. On March 17, the seedlings with 2 leaves and 1 heart were planted in the greenhouse with a plant spacing of 45 cm and a row spacing of 1 m. The climbing net was used to build an arch frame. Field management of all varieties is basically unified. The middle and upper female flowers were selected for pollination in the morning on the day of flowering. After pollination, the pollination date was marked with a label. 18 d after pollination, perfectly good commercial Momordica charantia fruits free of pests and diseases were selected and brought back to the laboratory, the flesh was cut, removed and seeded, and the middle flesh was selected, part of the fruit was dried in a 65 ℃ oven for use, and the other part was stored in a -40 ℃ refrigerator for use. Fruits from 6 plants of each variety were mixed and sampled.

3.3 Test items and methods

3.3.1 Determination of nutrient content

The nutrient content was determined by the kit of Suzhou Comin Biotechnology Co., LTD.: Vitamin C reacted with solid blue salt B in acetic acid solution to form yellow oxalhydrazid-2-hydroxybutyllactone derivatives, and the absorbance was determined at the maximum absorption wavelength of 420 nm. Soluble protein content was determined by Coomassie blue staining G-250 staining. Cellulose is heated and decomposed into β-glucose under acidic conditions. β-glucose can be dehydrated to produce β-furfural compounds under the action of strong acid. β-furfural compounds can be dehydrated and condensed with anthrone to produce furfural derivatives. The content of free amino acid was determined by the colorimetry of ninhydrin solution. Titrable acid content was determined by acid-base neutralization titration.

3.3.2 Determination of functional component content

The contents of saponins, polysaccharides, flavonoids and total phenols were determined by dried Momordica charantia and fruit jerky samples. The kits were purchased from Suzhou Comin Biotechnology Co., LTD. The saponins were extracted by ultrasonic wave, and the total saponins were determined by vanillin - perchloric acid chromogenic system. Polysaccharide was extracted by water purification and precipitation method, and the total polysaccharide content was determined by phenol-sulfuric acid method. Flavonoid content was determined by AL(NO₃)₃-NaNO₂ complex method. In alkaline nitrite solution, flavonoid and aluminum ions formed a red complex with characteristic absorption peak at 510 nm. Flavonoid content of sample can be calculated by measuring the absorbance value of sample extract at 510 nm. Phenols are reduced by tungstate molybdate under alkaline conditions to produce blue compounds, which have characteristic absorption peaks at 760 nm. The total phenolic content of the sample can be obtained by measuring the absorption value at 760 nm.

3.3.3 Determination of total antioxidant capacity

The total antioxidant capacity of Momordica charantia pulp was determined by the kit of Suzhou Comin Biotechnology Co., LTD. Determination of the antioxidant capacity of FRAP (Ferric reducing/antioxidant power). The ability of the antioxidant to reduce Fe³⁺ -tripyridine triazine ((Fe³⁺-TPTZ)) to produce blue Fe²⁺-TPTZ in an acidic environment reflects the total antioxidant capacity. The sample solution was thoroughly mixed with the working solution for 20 min, and the absorption value of 593 nm was determined. The total antioxidant capacity of the sample was expressed by the amount of antioxidant Trolox obtained from the standard curve. ABTS^+· (2,2 '-diazo-di-3-ethylbenzothiazolin-6-sulfonic acid, 2,2' -azinobis-3-ethylbenzothiazoline 6-sulphonic acid diammonium salt) determination of antioxidant capacity. When ABTS^+· solution is added into the sample solution, the antioxidant components in the sample solution can react with ABTS^+· and make the reaction system fade. Changes in absorbance were measured at 734 nm, and the antioxidant capacity of the antioxidants was quantified using Trolox as a control system. Determination of antioxidant capacity of DPPH (1, 1-diphenyl-2-Picrylhydrazine radical,1,1-diphenyl-2-picrylhydrazy). DPPH is a stable free radical, soluble in polar solvents such as methanol and ethanol, with maximum absorption at 515 nm. A decolorization reaction occurs when antioxidants are added to DPPH solution, so changes in absorbance can be used to quantify the antioxidant capacity of the antioxidants using Trolox as a control system. All three antioxidant capacities were measured in μmol Trolox/g fresh weight.

3.4 Data processing

EXEL and SPSS19.0 were used for data processing and analysis. The contents of saponins, polysaccharides, flavonoids and total phenols in Momordica charantia were distributed according to (Maximum - Minimum)/N. The histogram was made, and the percentage of the number of varieties in the total sample was calculated. Bivariate correlation analysis was performed between functional components and antioxidant capacity of Momordica charantia, and differences among varieties were compared by Tukey method.

Authors’ contributions

ZHM and DXT participated in the experimental design; ZHM conducted data analysis and paper writing; JHJ, YJZ, HLZ and CJW participated in the experimental design and result analysis. DXT made the final revision of the paper. All authors read and approved the final manuscript.

Acknowledgments

This research was jointly supported by Shanghai Science and Technology Agricultural Development Project (HNKCZ[2020] No.1-4) and Excellent Team Building Project of Shanghai Academy of Agricultural Sciences (NKC 2017[B-06]).

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