plants

Article
Evaluation of Growth, Yield, and Biochemical Attributes of
Bitter Gourd (Momordica charantia L.) Cultivars under Karaj
Conditions in Iran
Akram Valyaie 1 , Majid Azizi 2, * , Abdolkarim Kashi 3 , Ramaraj Sathasivam 4 , Sang Un Park 4,5, * ,
Akifumi Sugiyama 6 , Takashi Motobayashi 7 and Yoshiharu Fujii 7

1 Department of Horticulture, Faculty of Agriculture and Natural Resources, Karaj Branch,

Islamic Azad University, Karaj 3149968111, Iran; akram_vlyy@yahoo.com
2 Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad,
Mashhad 9177948978, Iran
3 Department of Horticulture, Faculty of Agriculture and Natural Resources Campus, University of Tehran,
Karaj 7787131587, Iran; Kashi@ut.ac.ir
4 Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu,
Daejeon 34134, Korea; ramarajbiotech@gmail.com
5 Department of Smart Agriculture Systems, Chungnam National University, 99 Daehak-ro, Yuseong-gu,
Daejeon 34134, Korea
6 Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Kyoto 611-0011, Japan;
akifumi_sugiyama@rish.kyoto-u.ac.jp
7 Department of International Environmental and Agricultural Science, Tokyo University of Agriculture and
Technology, Tokyo 183-8509, Japan; takarice@c.tuat.ac.jp (T.M.); yfujii@cc.tuat.ac.jp (Y.F.)



* Correspondence: azizi@um.ac.ir (M.A.); supark@cnu.ac.kr (S.U.P.); Tel.: +98-513-880-5753 (M.A.);


+82-42-821-5730 (S.U.P.)
Citation: Valyaie, A.; Azizi, M.;
Kashi, A.; Sathasivam, R.; Park, S.U.; Abstract: Vegetative and reproductive characteristics, fruit yield, and biochemical compounds of
Sugiyama, A.; Motobayashi, T.; Fujii,
six bitter melon cultivars (Iranshahr, Mestisa, No. 486, Local Japanese, Isfahan, and Ilocano) were
Y. Evaluation of Growth, Yield, and
evaluated under Karaj conditions in Iran. The phytochemical properties of the cultivars were
Biochemical Attributes of Bitter
evaluated using both shade-dried and freeze-dried samples at three fruit developmental stages
Gourd (Momordica charantia L.)
(unripe, semi-ripe, and ripe). There were significant differences in the vegetative and reproductive
Cultivars under Karaj Conditions in
Iran. Plants 2021, 10, 1370. https://
characteristics among cultivars, where cv. No. 486 was superior to most vegetative attributes. The
doi.org/10.3390/plants10071370 fruit yield of cultivars varied from 2.98–5.22 kg/plant. The number of days to male and female
flower appearance ranged from 19.00–25.33 and from 25–33 days, respectively. The leaf charantin
Academic Editor: Mukhtar Ahmed content was in the range of 4.83–11.08 µg/g. Fruit charantin content varied with developmental
stage, drying method, and cultivar. The highest charantin content (13.84 ± 3.55 µg/g) was observed
Received: 25 May 2021 at the semi-ripe fruit stage, and it was much higher in the freeze-dried samples than the shade-dried
Accepted: 30 June 2021 samples. Cultivar No. 486 had the highest (15.43 ± 2.4 µg/g) charantin content, whereas the lowest
Published: 5 July 2021 charantin content (8.51 ± 1.15 µg/g) was recorded in cultivar cv. Local Japanese. The highest total
phenol content (25.17 ± 2.27 mg GAE/g) was recorded in freeze-dried samples of ripe fruits of cv.
Publisher’s Note: MDPI stays neutral
No. 486, whereas the lowest phenol content was detected in the shade-dried samples of semi-ripe
with regard to jurisdictional claims in
fruits of Isfahan. cv. Flavonoid content was higher with the shade-drying method, irrespective of
published maps and institutional affil-
cultivar. In conclusion, considering the fruit yield and active biological compounds in the studied
iations.
cultivars, cv. No. 486 should be grown commercially because of its higher yield and production of
other secondary metabolites.

Keywords: bitter melon; charantin; medicinal plant; diabetes; active ingredients; fruit yield
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
1. Introduction
conditions of the Creative Commons
Attribution (CC BY) license (https:// Diabetes is one of the most common and dangerous chronic diseases in humans. It
creativecommons.org/licenses/by/ has been estimated that, by 2025, more than 75% of people living in developing countries
4.0/). will have diabetes [1]. According to global statistics in 2015, it was estimated that there

Plants 2021, 10, 1370. https://doi.org/10.3390/plants10071370 https://www.mdpi.com/journal/plants

Plants 2021, 10, 1370 2 of 19

were 415 million people with diabetes, and that, by 2040, one in 10 adults (642 million) will
have diabetes [2]. The indicated difference reveals that the proportion of people infected
with the disease in the community will be much higher than expected. The number of
people with diabetes in Iran is higher than the global average [3]. For this reason, the study
of new effective plant sources for curing diabetes is among the research priorities of the
Ministry of Health and Medical Education of Iran.
Bitter melon, a useful medicinal plant, is used in most developing countries, particu-
larly in Asia and Africa, as a herb, medicine, and vegetable [4]. Unripe fruit and seeds of
bitter melon have been the subject of extensive research with respect to phytochemical and
pharmaceutical properties [5]. The medicinal value of this plant, which can be used to treat
many diseases, including diabetes, has attracted the attention of scientists worldwide [6].
All parts of this plant, including the leaves and fruits, are used in traditional medicine in
Asia, Africa, and even Western countries to cure diabetes [7]. It has been reported that leaf
and fruit extracts of bitter melon have antioxidant effects [8]. Fresh fruit or ground dried
fruit is effective at reducing blood glucose levels [9].
Genetic and environmental factors and farming systems have significant effects on
the quantity of bioactive compounds in medicinal plants. Different melon cultivars have
produced types of active ingredients. In this study, bitter melon cultivars from Japan and
the Philippines were evaluated to obtain information about charantin [10]. The charantin
content in Japanese cultivars was higher than that in the Filipino cultivars reported by Kim
et al. (2014). New plant species transferred to other areas express the same characteristics
as those in the place of origin, although, by planting in a new area, changes in some
characteristics, such as the duration of growth periods and phytochemicals, are expected.
Studies have been carried out on the adaptability and yield of bitter melons, where it was
observed that variations are more closely related to growth period duration, flowering
time, fruit ripening, and other morphological and biochemical characteristics [11].
Yield is a complex attribute that is strongly influenced by environmental factors [12].
The yield potential of different varieties of a single plant species varies from one en-
vironment to another, and varieties do not show the same yield potential in different
environments [11]. The content and composition of a plant’s bioactive chemicals differ
from their origin to other locations because of changes in metabolic activity, which is af-
fected by environmental factors [12]. Important environmental factors that affect the quality
and quantity of bioactive compounds are light, temperature, soil, irrigation, and altitude
above the free sea level [13,14]. This plant is located in India, Bangladesh, the Philippines,
Thailand, Malaysia, China, Japan, eastern and southeastern Pakistan, Indonesia, Australia,
the tropical regions of Africa, South America, and the Caribbean [7]. Bitter melons are
grown and harvested in limited areas of Iran. However, the detection of phytochemicals
from different plant parts, such as leaves and fruits, has not yet been reported. This is the
first report comparing different bitter melon cultivars and their adaptability to Karaj envi-
ronmental conditions in Iran, as well as the determination of effective bioactive compounds
in their leaves and fruits.

2. Results
2.1. Variation of Vegetative Traits among the Cultivars
Significant differences were observed among bitter gourd cultivars for vegetative
characteristics (Table 1). The differences in plant height, number of leaves per plant, leaf
area, and plant dry weight were significant at the 1% level of probability, whereas the
number of branches per plant was significant at the 5% level of probability.
Plants 2021, 10, 1370 3 of 19

Table 1. Mean comparison of vegetative traits of six bitter melon cultivars.

Leaf No Leaf Area Plant Plant Height Number of

Cultivar
Per Plant (cm2 /10 Leaf) Dry Weight (g) (cm) Branches/Plant
Iranshahr 1735.33 ± 15 c 64.7 ± 2.5 a 541.00 ± 15 ab 337.00 ± 12 ab 128.00 ± 3.5 b
Hybrid Mestisa 2312.66 ± 21 ab 46.4 ± 1.6 b 433.00 ± 17 abc 311.00 ± 9 ab 170.00 ± 6.7 ab
Hybrid No. 486 2756.33 ± 25 a 60.8 ± 3.2 a 666.00 ± 23 a 355.00 ± 11 a 168.00 ± 3.5 ab
Local Japanese 2162.66 ± 14 bc 58.5 ± 2.1 a 489.00 ± 19 abc 242.00 ± 18 bc 146.00 ± 4.4 b
Isfahan 1646.66 ± 9 c 60.8 ± 2.4 a 381.00 ± 21 bc 332.00 ± 21 ab 152.00 ± 4.6 ab
Ilocano 2184.00 ± 11 ab 15.5 ± 1.6 c 222.00 ± 18 c 175.00 ± 13 c 229.00 ± 5.4 a
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

The maximum and minimum plant heights were 355.33 and 175.33 cm for cv. Hybrid
No. 486 and cv. Ilocano, respectively. This attribute plays an important role in determining
the appropriate distance between rows during commercial field cultivation.
Among the cultivars, the Thai cultivar (Hybrid No. 486) produced the highest number
of leaves/plant (2756), while the lowest number of leaves (1646) was observed in cv. Isfahan
(Table 1). The mean leaf area of 10 leaves per plant was evaluated for each cultivar (Table 1);
these were found to vary from 15.46 cm2 (cv. Ilocano) to 64.73 cm2 (cv. Iranshahr). The
highest plant dry weight was observed in Hybrid No. 486 (665.66 g) and the lowest
was observed in cv. Ilocano (222.33 g) (Table 1). Considering that all parts of the plant,
including the leaves and fruits, are used for pharmaceuticals and traditional medicine,
Hybrid No. 486 showed the best performance in terms of both leaf and fruit dry weight.
The cv. Ilocano, with the lowest plant height, had the highest number of branches per plant
(229.33). The lowest number of branches was observed in cv. Iranshahr (cv. palee) and cv.
Local Japanese (Table 1).

2.2. Variation of Reproductive Traits among the Cultivars

Significant differences (p ≤ 0.01) were observed among the cultivars in terms of their
reproductive characteristics. Male flowers appeared earlier than female flowers in all the
cultivars. However, there was a 1 week difference between the cultivars in days to the
appearance of the first male flower (Table 2). The first male flower appeared in cv. Local
Japanese at 25.33 days after planting, which was the latest among the cultivars. The other
cultivars did not show any significant differences in this regard. The first male flower
appeared in Hybrid Mestisa and Hybrid No. 486 19 days after planting.

Table 2. Mean comparison of reproductive characteristics of six bitter melon cultivars.

Traits
Node Number Days Difference for
Days to First Days to First Male: Female Number of
Cultivar at 1st Female Male and Female
Male Flower Female Flower Flower Ratio Fruits/Plant
Flower Appear Flower Appearance
Iranshahr 21.00 b 29.66 ab 16.00 a 8.66 ab 15.46 bc 33.18 b
Hybrid Mestisa 19.00 b 25.33 c 9.00 b 6.33 b 17.11 ab 33.07 b
Hybrid No. 486 19.33 b 25.00 c 12.6 ab 5.66 b 13.21 c 34.35 b
Local Japanese 25.33 a 31.33 ab 17.00 a 6.00 b 20.06 a 24.51 c
Isfahan 21.00 b 33.00 a 14.33 a 12.00 a 12.20 cd 32.22 b
Ilocano 19.33 b 28.33 bc 13.66 a 9.00 ab 9.03 d 133.33 a
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

The first female flowers appeared 25 days after planting in Hybrid Mestisa and Hybrid
No. 486, and 33 days after planting in cv. Isfahan. For example, the fruit yield of Hybrid
No. 486 and Hybrid Mestisa, which showed shorter differences, resulted in higher fruit
yield (Table 2).
 No. 486
Local Japa-
25.33 a 31.33 ab 17.00 a 6.00 b 20.06 a 24.51 c
nese
Isfahan 21.00 b 33.00 a 14.33 a 12.00 a 12.20 cd 32.22 b
Ilocano 19.33 b 28.33 bc 13.66 a 9.00 ab 9.03 d 133.33 a
Plants 2021, 10, 1370 Means (three replications) in each column, followed by at least one letter in common are not sig- 4 of 19
nificantly different at the 5% probability level using Tukey′s test.

2.2.1. Dry Fruit Yield (g/Plant)

2.2.1. Dryof
Analysis Fruit Yield showed
variance (g/Plant) that the dry fruit yield per plant and its components
Analysis of
were significantly variance
different (p showed that thethe
≤ 0.01) among drybitter
fruit melon
yield per plant and
cultivars. its components
Hybrid No. 486 were
had significantly different
the highest fruit yield (p ≤ plant
per 0.01) among the bitter
(0.42 kg.plant melon cultivars.
−1) followed by Hybrid Hybrid No.(0.38
Mestisa 486 had the
highest fruit yield per plant (0.42 −1 ) followed by Hybrid Mestisa (0.38 kg.plant−1 )
kg.plant −1) (Figure 1). Considering thekg.plant
fresh and dry fruit weight per plant, Hybrid No.
(Figure
486 had 1). Considering
the highest the freshbyand
weight, followed dry fruit
Hybrid Mestisaweight
fromper
theplant, Hybrid
Philippines No. 486
(Figure 1). had the
highest weight, followed by Hybrid Mestisa from the Philippines (Figure 1).

4
Fresh fruit yield
kg/bush

0
Iranshahr Hybrid Hybrid No. Local Japanese Isfahan Ilocano
Plants 2021, 10, x FOR PEER REVIEW
Mestisa 486 5 of 20

Bitter melon cultivars

0.5
0.45
0.4
0.35
Dry fruit yield

0.3
kg/bush

0.25
0.2
0.15
0.1
0.05
0
Iranshahr Hybrid Hybrid No. Local Japanese Isfahan Ilocano
Mestisa 486

Bitter melon cultivars

Figure
Figure 1. Fresh
1. Fresh andfruit
and dry dry fruit yield/plant
yield/plant (Immature
(Immature stage) stage) of sixmelon
of six bitter bitter cultivars
melon cultivars grown
grown in Karaj,inIran.
Karaj, Iran. Columns
Columns with with
the same letter are not significantly different at the 1% probability level using Tukey’s
the same letter are not significantly different at the 1% probability level using Tukey’s test. test.

2.2.2. Fruit Number per Plant

The number of fruits per plant ranged from 24 to 133. The highest fruit number per
plant was observed in cv. Ilocano and could be attributed to the lower ratio of male to
Plants 2021, 10, 1370 5 of 19

2.2.2. Fruit Number per Plant

The number of fruits per plant ranged from 24 to 133. The highest fruit number per
plant was observed in cv. Ilocano and could be attributed to the lower ratio of male to
female flowers (Table 2).

2.2.3. Fruit Characteristics

There were significant (p ≤ 0.01) differences among bitter melon cultivars in terms of
fruit length and fruit diameter at the unripe and semi-ripe stages, as well as a change in
aril color at the ripe stage.

2.2.4. Fruit Length

At the full ripe stage, fruit color changed from green to orange. At this stage, hybrid
No. 486 had the longest fruit (32.50 cm) (Table 3).

Table 3. Mean comparison of the fruit characteristics of six bitter melon cultivars at different developmental stages.

Length of Fruit Diameter of Fruit

(cm) (cm)
Fruit
Aril Color Aril Color
Unripe Semi-Ripe Ripe Unripe Semi-Ripe Density
Change Change
(g/cm3 )
Iranshahr 16.96 c 19.00 c 22.90 c 25.16 b 5.19 a 6.00 a 6.00 b 0.76 bc
Hybrid Mestisa 23 a 28.93 a 30.00 a 32.16 a 4.75 a 5.25 a 6.09 b 0.65 e
Hybrid No. 486 21 ab 25.00 b 30.00 a 32.50 a 5.14 a 5.77 a 6.76 a 0.73 cd
Local Japanese 18.00 bc 20.00 c 24.43 bc 27.16 b 4.81 a 5.72 a 6.33 ab 0.69 de
Isfahan 20.30 ab 23.93 b 27.26 ab 28.50 b 5.06 a 5.77 a 6.43 ab 0.81 ab
Ilocano 6.70 d 8.46 d 9.63 d 11.00 c 3.61 b 4.14 b 4.43 c 0.86 a
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

2.2.5. Fruit Diameter

The diameter of the fruit was measured at the unripe, semi-ripe, and color-changing
stages of the ariels. Measurements at the completely ripe stage were not possible. The
highest fruit diameter (5.19 cm) was observed in the cv. Iranshahr (cv. Palee), and the
lowest (3.61 cm) belonged to cv. Ilocano.

2.2.6. Fruit Density

Assessment of fruit density in different cultivars during unripe to ariel color-changing
stages indicated that, during the development of the fruit, its density decreased consid-
erably during the last stages (Figure 2). The study of the cross-sections of fruits of bitter
melon cultivars in different fruit developmental stages supported this observation. At 50%
of the final fruit volume, cv. Ilocano had the highest fruit density, while hybrid Mestisa
had the lowest (Table 3).

2.2.7. Number of Seeds per Fruit

The total number of seeds/fruit varied from 27 to 39.33 (Table 4). The highest total
seeds/fruit (39.33 seeds) belonged to cv. Iranshahr (cv. palee) and the lowest (27 seeds)
belonged to cv. Ilocano. There was a positive correlation between seed number per fruit and
fruit size in medium-weight medicinal pumpkins. However, there was no such relationship
in the heavier fruits. The redistribution of photosynthetic assimilates from fruits to seeds is
a genetic attribute.
 Assessment of fruit density in different cultivars during unripe to ariel color-chang-
ing stages indicated that, during the development of the fruit, its density decreased con-
siderably during the last stages (Figure 2). The study of the cross-sections of fruits of bitter
Plants 2021, 10, 1370
melon cultivars in different fruit developmental stages supported this observation. At 50%
6 of 19
of the final fruit volume, cv. Ilocano had the highest fruit density, while hybrid Mestisa
had the lowest (Table 3).

0.95

0.9
Fruite density g/cm3

0.85 Iranshahr
Hybrid Mestisa
0.8
Hybrid No. 486
0.75 Local Japanese
Isfahan
0.7
Ilocano
0.65

0.6
Unripe Semi-ripe Ripe
Fruite growth satges
Figure 2. Changes
Figure in the
2. Changes fruit
in the density
fruit densityofofdifferent
different bitter meloncultivars
bitter melon cultivarsduring
during fruit
fruit developmental
developmental stages
stages from from
unripeunripe
to to
ariel ariel
colorcolor
change stages.
change stages.

Table 4. Total seed number

2.2.7. per fruit,
Number 1000-seed
of Seeds perweight,
Fruit and color indices for six bitter melon cultivars.
The total number of seeds/fruit varied from 27 to 39.33 (Table 4). The highest total
Traits
Cultivars seeds/fruit (39.33
Total Number of
seeds) belonged to cv. Iranshahr (cv.
1000-Seed Weight
palee)
Color Indexand the lowest (27 seeds)
belonged to cv. Ilocano.
Seeds/Fruit (g) There was a positive
L* correlation
a betweenb seed number∆L per fruit
and fruit size in medium-weight medicinal pumpkins. However, there was no such rela-
Iranshahr 39.33 ± 3.1 a 179.00 ± 3.5 c 28.64 bc −8.00 ab 13.64 b −65.66 bc
Hybrid Mestisa 30.50 ±tionship
2.6 abc in the heavier
190.50 ±fruits.
2.7 b The redistribution
41.36 a of13.15
− photosynthetic
d 21.53 aassimilates
−52.95from
a fruits
Hybrid No. 486 27.66 to seeds
± 1.7 c is a genetic attribute.
216.16 ± 1.9 a 22.34 c −7.37 a 11.80 b −71.96 c
Local Japanese 29.00 ± 2.4 bc 214.66 ± 4.1 a 27.21 bc −10.85 cd 16.22 ab −67.09 bc
Isfahan 38.00 ± 3.1 ab 157.33 ± 5.7 e 29.36 b −8.06 ab 13.59 b −64.95 b
Ilocano 27.00 ± 4.2 c 168.00 ± 6.2 d 32.67 b −10.07 bc 15.63 b −61.63 b
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test. *CIELAB color space also referred to as L*a*b* and ∆L are color indices defined by the International Commission
on Illumination (abbreviated CIE) in 1976.

2.2.8. Thousand Seed Weight

The 1000-seed weight is an indicator of seed size. The mean comparison of the
1000-seed weight of bitter melon cultivars showed that Hybrid No. 486 had the highest
(216.6 g) 1000-seed weight, followed by cv. Local Japanese (214.66 g) (Table 4).

2.2.9. Fruit Color

Mean comparison of color-related coefficients, including L, a, b, and ∆L, showed that
there were significant differences between bitter melon cultivars for fruit color indices
(Table 4). The highest L (41.36) was recorded for Hybrid Mestisa (in other words, its fruit
color was brightest), while the lowest L (22.34) belonged to Hybrid No. 486 (i.e., the fruit
color of this cultivar was darker than the remaining cultivars). The “L” index determines
the color luminosity, where a higher “L” value is representative of the brighter color of
the fruit. In our study, the lowest fruit brightness was observed in Hybrid No. 486, which
Plants 2021, 10, 1370 7 of 19

was consistent with the high levels of fruit tubercle. The highest luminosity was observed
in Hybrid Mestisa, which had the least tubercle on the fruit surface. Therefore, it can
be expected that cultivars with fewer tubercle fruits would be brighter and have greater
marketability. The mean comparison for “a” index (which indicates the color of the fruit)
indicated a significant difference among the cultivars, with the largest “a” index (7.37) in
Hybrid No. 486, which represents the green color of the fruit (Table 4). The “b” index
determines the brightness or darkness of color; the highest “b” (21.53) belonged to Hybrid
Mestisa, which indicates light green, while the lowest “b” index (11.8) was recorded for
Hybrid No. 486 (Table 4). The low “b” index indicated the dark-green color of the fruit in
this cultivar.

2.3. Phytochemical Characteristics

2.3.1. Total Phenol Content
The total phenol content of fruits (Regardless of the drying method and developmental
stages) was the highest (17.8 mg gallic acid equivalent (GAE) in Hybrid No. 486, followed
by Hybrid Mestisa, cv. “Isfahan,” Local Japanese, and cv. Iranshahr. The Ilocano had the
lowest level (10.42 mg GAE) (Table 5). The highest total phenol content in Hybrid No. 486
was obtained in freeze-dried fruits in the unripe and ripe stages (25.17 and 24.12 mg GAE,
respectively). However, the lowest phenol content (3.57 mg GAE) in cv. Isfahan was in the
semi-ripe stage and shade-dried samples (Tables 5 and 6).

Table 5. Fruit phenol content of six cultivars of bitter melon (mg GA equivalent).

Fruit Phenol Content (mg GA eq.)

Unripe Semi-Ripe Ripe
Shade Freeze Shade Freeze Shade Freeze
Cultivars
Drying Drying Drying Drying Drying Drying
Iranshahr 9.56 ± 1.02 f–k 22.51 ± 1.3 a–c 5.09 ± 0.91 j–k 10.49 ± 1.05 d–k 7.96 ± 1.04 g–k 12.01 ± 1.11 c–k
Hybrid Mestisa 14.92 ± 2.01 a–j 21.30 ± 0.95 a–d 6.76 ± 0.82 i–k 7.62 ± 1.63 h–k 18.85 ± 2.13 a–g 17.34 ± 1.65 a–i
Hybrid No. 486 10.78 ± 1.7 d–k 25.17 ± 1.65 a 9.43 ± 0.56 f–k 16.56 ± 1.87 a–i 20.70 ± 2.16 a–e 24.12 ± 2.11 ab
Local Japanese 8.34 ± 0.85 g–k 17.87 ± 2.01 a–h 8.76 ± 0.47 g–k 15.47 ± 1.54 a–j 15.72 ± 1.84 a–j 13.78 ± 1.63 b–k
Isfahan 12.26 ± 1.13 c–k 20.15 ± 1.94 a–f 3.57 ± 0.21 k 13.99 ± 1.33 b–k 18.42 ± 1.38 a–h 12.22 ± 0.65 c–k
Ilocano 9.64 ± 1.1 e–k 11.54 ± 1.63 c–k 7.62 ± 0.64 h–k 6.65 ± 0.87 i–k 11.84 ± 1.12 c–k 15.21 ± 1.55 a–j
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

Table 6. Cultivar × drying method and cultivar × development stage interaction effects on fruit total phenolic content
(mg GAE).

Drying Method Development Stage

Cultivars Shade Drying Lyophilized Unripe Semi-Ripe Ripe
Iranshahr 7.54 ± 1.60 d 15.00 ± 0.50 bc 16.04 ± 1.27 abc 7.79 ± 1.32 ef 9.98 ± 1.15 cdef
Hybrid Mestisa 13.51 ± 1.35 bc 15.42 ± 1.10 b 18.11 ± 1.15 ab 7.19 ± 1.28 f 18.09 ± 0.95 ab
Hybrid No. 486 13.64 ± 0.95 bc 21.95 ± 1.25 a 17.98 ± 1.50 ab 13.00 ± 0.80 bcdef 22.41 ± 1.34 a
Local Japanese 10.94 ± 1.30 bcd 15.71 ± 1.30 b 13.10 ± 1.85 bcdef 12.11 ± 0.125 bcdef 14.75 ± 1.27 bcde
Isfahan 11.42 ± 2.10 bcd 15.45 ± 0.95 b 16.21 ± 1.12 abc 8.78 ± 1.55 def 15.32 ± 1.15 bcd
Ilocano 9.70 ± 1.12 cd 11.14 ± 1.12 bcd 10.59 ± 0.85 cdef 7.13 ± 1.35 f 13.53 ± 1.40 bcdef
Mean 11.12 15.78 15.34 9.33 15.68
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

2.3.2. Total Flavonoid Content

Investigating the amounts of flavonoids in different stages of fruit development
(regardless of cultivar and drying method) showed that, with increasing developmental
stages of fruit from the unripe stage, the flavonoid content decreased significantly (p ≤ 0.01).
The highest flavonoid content in the unripe stage was 15.31 mg quercetin equivalent
(mg QE) and the lowest was equal to 6.57 mg QE in the ripe fruit (Table 7). The dual
Plants 2021, 10, 1370 8 of 19

interaction effects of cultivar × drying method and cultivar × development stage on fruit
total flavonoid content are shown in Table 7. The results showed that the highest total
flavonoid content (19.42 mg QE) was in Hybrid No. 486 in the unripe fruit stage in shade-
dried samples. The lowest flavonoid content was observed in the freeze-dried cv. Local
Japanese and Hybrid Mestisa in semi-ripe fruit and ripe fruit. However, the flavonoid
content was lower in the ripe fruits of cv. Iranshahr (cv. Palee) for both drying methods
(Tables 7 and 8).

Table 7. Fruit flavonoid content of six cultivars of bitter melon (mg quercetin equivalent).

Fruit Flavonoid Content (mg Quercetin eq.)

Unripe Semi-Ripe Ripe
Cultivars Shade-Drying Freeze-Drying Shade-Drying Freeze-Drying Shade-Drying Freeze-Drying
Iranshahr 15.63 ± 3.06 a–g 18.44 ± 1.85 ab 8.70 ± 0.88 d–k 5.77 ± 0.55 i–k 4.37 ± 0.35 k 4.08 ± 0.34 k
Hybrid Mestisa 18.08 ± 1.65 abc 11.05 ± 1.66 a–k 9.49 ± 0.65 d–k 3.60 ± 0.61 k 7.40 ± 0.62 g–k 3.58 ± 0.61 k
Hybrid No. 486 19.42 ± 1.71 a 16.07 ± 1.23 a–f 11.94 ± 1.04 a–k 14.07 ± 0.85 a–i 9.61 ± 0.88 c–k 9.63 ± 0.55 c–k
Local Japanese 16.93 ± 1.35 a–d 13.45 ± 1.45 a–j 7.80 ± 1.32 f–k 6.17 ± 0.55 h–k 4.28 ± 0.55 k 8.13 ± 0.81 e–k
Isfahan 16.47 ± 1.45 a–e 14.50 ± 1.66 a–h 9.11 ± 1.12 d–k 7.76 ± 0.77 f–k 5.03 ± 0.41 jk 6.46 ± 0.68 h–k
Ilocano 15.21 ± 1.66 a–g 8.51 ± 0.95 d–k 7.94 ± 0.88 f–k 11.08 ± 0.86 a–k 10.37 ± 0.87 b–k 5.86 ± 0.88 i–k
Means (three replications), in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

Table 8. Cultivar × drying method and cultivar × development stage interaction effects on fruit flavonoid content (mg QE).

Drying Method Development Stage

Cultivars Shade Drying Lyophilized Unripe Semi-Ripped Ripe
Iranshahr 9.57 ± 0.85 abcd 9.43 ± 0.45 bcd 17.04 ± 1.60 ab 7.24 ± 1.12 ef 4.23 ± 0.50 f
Hybrid Mestisa 11.66 ± 0.50 abc 6.08 ± 1.20 d 14.57 ± 1.40 abc 6.55 ± 0.55 ef 5.49 ± 0.25 f
Hybrid No. 486 13.66 ± 1.10 a 13.26 ± 0.75 ab 17.75 ± 1.35 a 13.00 ± 1.50 abcd 9.62 ± 0.30 cdef
Local Japanese 9.67 ± 1.25 abcd 9.25 ± 1.10 bcd 15.19 ± 1.12 ab 6.99 ± 0.55 ef 6.21 ± 0.10 f
Isfahan 10.20 ± 1.40 abcd 9.57 ± 0.80 abcd 15.49 ± 1.50 ab 8.43 ± 0.65 def 5.75 ± 0.70 f
Ilocano 11.18 ± 1.35 abc 8.48 ± 1.15 cd 11.86 ± 0.50 bcde 9.51 ± 0.80 cdef 8.12 ± 0.35 def
Mean 10.99 9.35 15.31 8.62 6.57
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

2.3.3. Fruit Charantin Content

There was a significant difference (p ≤ 0.01) among the bitter melon cultivars for
fruit charantin content. The dual interaction effects of cultivar × drying method and
cultivar × development stage also had a significant (p ≤ 0.01) effect on fruit charantin
content. The mean comparison of fruit charantin content is presented in Table 9. Fruit cha-
rantin content in different stages of fruit development indicated that the highest charantin
was 13.84 µg/g in semi-ripe stage, while it was 12.46 µg/g in unripe fruits, and the lowest
in ripe fruits. The fruit charantin content in different bitter melon cultivars subjected to
different drying methods in the three fruit development stages is presented in Figure 3. The
fruit charantin content in the lyophilized samples was higher than that in the shade-dried
samples, irrespective of cultivar. In both methods, the highest fruit charantin content was
obtained in the semi-ripe fruit (Figure 3).
 Plants 2021, 10, 1370 9 of 19

Table 9. Cultivar × drying method and cultivar × development stage interaction effects on fruit charantin content (µg/g).

Drying Method Development Stage

Cultivars
Shade-Drying Lyophilized Unripe Semi-Ripe Ripe Mean
Iranshahr 7.87 ± 0.91 def 17.93 ± 1.37 ab 15.25 ± 1.51 bcd 15.55 ± 1.02 bc 7.91 ± 0.90 hij 12.9 ± 1.14
Hybrid Mestisa 7.09 ± 0.63 ef 17.39 ± 0.31 abc 15.21 ± 0.54 bcd 16.44 ± 0.58 b 5.06 ± 0.30 k 12.2 ± 0.47
Hybrid No. 486 9.06 ± 1.15 def 21.79 ± 1.41 a 13.72 ± 0.58 de 18.54 ± 1.36 a 14.01 ± 0.90 cde 15.4 ± 0.94
Local Japanese 6.37 ± 0.41 f 10.65 ± 0.3 def 9.07 ± 0.33 hi 9.28 ± 0.35 gh 7.18 ± 0.75 j 8.5 ± 0.47
Isfahan 5.60 ± 0.51 f 12.89 ± 0.83 bcd 8.93 ± 0.42 hij 10.97 ± 0.96 fg 7.84 ± 0.65 hij 9.2 ± 0.67
Plants 2021, 10, x FOR PEER REVIEW 10 of 20
Ilocano 9.15 ± 0.56 def 12.36 ± 0.66 cde 12.61 ± 0.85 ef 12.26 ± 0.40 ef 7.38 ± 0.60 ij 10.8 ± 0.61
Mean 7.52 15.50 12.46 13.84 8.23 11.5 ± 0.71
Means (three replications) in each column, followed by at least one letter in common are not significantly different at the 5% probability
level using Tukey’s test.

Figure 3.Figure Fruits charantin

Fruits3.charantin contentcontent in six bitter
in six bitter melonmelon cultivars
cultivars during
during different
different developmental
developmental stage
stage andand drying
drying condition
condition
(A): shade-dried,
(A): shade-dried, (B): freeze-dried.
(B): freeze-dried.

Table 9. Cultivar × drying method and cultivar × development stage interaction effects on fruit charantin content (μg/g).

Drying Method Development Stage

Cultivars
Shade-Drying Lyophilized Unripe Semi-Ripe Ripe Mean
Plants 2021, 10, 1370 10 of 19

The highest charantin content was detected in Hybrid no. 486 in semi-ripe fruit using
the freeze-drying method. Since this cultivar had the highest fruit yield, it is suitable
for cultivation under Karaj conditions. The lowest charantin content was detected in cv.
Isfahan in an unripe stage in shade-dried samples and Hybrid Mestisa in the fully ripe
Plants 2021, 10, x FOR PEER REVIEWstage using freeze-dried samples. It can be concluded that Hybrid no. 486 had high11fruit
of 20
yield and the highest fruit charantin content in the semi-ripe stage using freeze-dried
samples; thus, it can be recommended for growth in Karaj conditions.
2.3.4. Leaf
2.3.4. Leaf Charantin Content
Charantin Content
Mean leaf
Mean leafcharantin
charantincontent
contentin in
sixsix bitter
bitter melon
melon cultivars
cultivars is presented
is presented in Figure
in Figure 4.
4. There
There were significant differences between the cultivars; cv. Iranshahr with
were significant differences between the cultivars; cv. Iranshahr with 11.08 µg/g had the11.08 μg/g
had the leaf
highest highest leaf charantin
charantin content, content,
while cv.while
Localcv. Local Japanese
Japanese had the(4.83).
had the lowest lowest (4.83).

20.00
Charantin (μg/g)

15.00

10.00

5.00

0.00
Iranshahr Hybrid Hybrid Local Isfahan Ilocano
Mestisa No. 486 Japanese

Figure 4. Leaf charantin content in six bitter melon cultivars grown under Karaj conditions.

3. Discussion
Figure 4. Leaf charantin content in six bitter melon cultivars grown under Karaj conditions.
The perusal of mean performance of the bitter gourd cultivars revealed that plant
height, number of leaves per plant, leaf area, and plant dry weight were significant at the
3. Discussion
1% level of probability, whereas the number of branches per plant was significant at the 5%
level The perusal ofSignificant
of probability. mean performance
differencesofinthe bitter
plant heightgourd cultivarsbitter
of different revealed
gourdthat plant
cultivars
height, number
reportedly of leaves
previously per plant,
ranged fromleaf area,
98 cm and
[15] toplant
575 cm dry[16].
weightIt haswere significant
been reportedatthat the
1% level of probability, whereas the number of branches per
plant leaf numbers vary with cultivars and other management practices [16–20]. Other plant was significant at the
5% level of also
researchers probability.
showed Significant
significant differences
differences in plant height
between cultivarsof different bitter
for leaf area, andgourd
our
cultivars reportedly previously ranged from 98 cm [15] to 575 cm
findings are in agreement with their results [21,22]. Moreover, the high herb-producing [16]. It has been reported
that plant
cultivar leaf numbers
(Hybrid No. 486) vary
hadwith
morecultivars and other
green leaves management
and higher mean leaf practices [16–20].
area (Table 1),
Other researchers
resulting in a higher alsoamount
showedof significant differences
photosynthesis, thusbetween cultivars
representing for leafsource
a strong area, andfor
our findings
producing are in agreement
secondary withDifferences
metabolites. their results in [21,22].
the numberMoreover, the high
of branches herb-produc-
among cultivars
ing cultivar
did not follow (Hybrid
the sameNo. 486)
trendhad more green
as biomass leaves andIslam
production. higheret mean leaf area
al. (2014), who(Table
studied 1),
resulting in a higher amount of photosynthesis, thus representing
15 indigenous bitter melon landraces with small fruits, reported that the population with a strong source for pro-
ducingand
small secondary metabolites.
round fruits Differences
had a short main branchin theandnumber of branches
numerous lateral among
branches cultivars
compared did
to
notthose
follow plants bearing
the same trendrectangular
as biomassfruits [22]. This
production. observation
Islam et al. (2014), is in
whoagreement
studied 15 with
in-
our findings,
digenous bitterparticularly
melon landraces for cv.with
Ilocano. Although
small fruits, the Ilocano
reported that thecultivar
population produced
with small the
highest
and round numberfruitsofhad
branches/plant
a short main (Tablebranch4),andplant height was
numerous the branches
lateral lowest in compared
this cultivar, to
which negatively
those plants bearingaffected the leaf fruits
rectangular area. Iranshahr
[22]. This cultivars
observation produced the lowestwith
is in agreement number our
of branches/plant
findings, particularly butforthe
cv.larger
Ilocano.leaf. The number
Although of branches
the Ilocano cultivarisproduced
the resultthe ofhighest
apical
dominance, and it is important to determine the optimum plant
number of branches/plant (Table 4), plant height was the lowest in this cultivar, whichdistance on the row during
cultivation.
negatively affectedA plant’s thecompact
leaf area.canopy affects
Iranshahr light absorption
cultivars producedand the carbon
lowest acquisition
number of
efficiency
branches/plant for photosynthesis
but the larger and leaf.vegetative
The number growth by changing
of branches leaf area
is the result index (LAI),
of apical domi-
which is a special genetic trait of plant cultivars, also influenced
nance, and it is important to determine the optimum plant distance on the row during by cultural practices [20].
The presence of highly significant correlations among different plant
cultivation. A plant’s compact canopy affects light absorption and carbon acquisition ef- cultivar traits indicated
dependency of one characterand
ficiency for photosynthesis on other, and there
vegetative growthwas by a negative
changing correlation
leaf area of number
index (LAI),of
branches/plant with plant height and leaf size in another report [23].
which is a special genetic trait of plant cultivars, also influenced by cultural practices [20].
The presence of highly significant correlations among different plant cultivar traits indi-
cated dependency of one character on other, and there was a negative correlation of num-
ber of branches/plant with plant height and leaf size in another report [23].
The results showed that male flowers appeared earlier than female flowers in all the
cultivars. The difference in the appearance of the first female flower was much higher than
Plants 2021, 10, 1370 11 of 19

The results showed that male flowers appeared earlier than female flowers in all the
cultivars. The difference in the appearance of the first female flower was much higher than
that of male flowers among the studied cultivars. Therefore, cv. Local Japanese and cv.
Isfahan had comparatively late flowering among the other cultivars. Fruits of the early
flowering cultivars reached the harvesting stage faster. Fruit harvesting stimulated the
appearance of more female flowers in all of the cultivars. The appearance of the male flower
before the female flower plays a special role in pollination and fruit formation [24]. Rathod
et al. (2008) reported that the number of days from direct sowing until the appearance of
the first male flower varied between 37.25% and 40.23% [25]. The timing of male and female
flower appearance depends on the genetic and environmental factors that are related to the
adaptation of the cultivars to different regions, and some researchers have reported that
this can vary from 24 to 41 days [15,21,22,26–30]. This is in agreement with the results of
the present study, which showed 12 days difference in the appearance of male and female
flowers. The time between the appearance of the first male and female flowers among the
studied cultivars varied between 5.66 and 12 days after transplantation. Our observations
showed that a shorter difference between the appearance of the first male and female
flowers led to a higher fruit yield.
On average, the first female flower appeared at nodes 9–17 (in Philippine and Japanese
cultivars, respectively) (Table 2); which agrees with Dalamu et al. (2012) who reported
that the first flower appeared at the ninth node [31]. It can, thus, be concluded that the
earlier appearance of female flowers at the lower nodes is strongly correlated with early
ripening and fresh and dry fruit yield. Gupta et al. (2015) reported that the number of
days to the appearance of the first female flower had a positive and significant correlation
with some traits, including fruit yield [11]. Other researchers have also shown that male
flowers appear at lower nodes compared to female flowers [14,19,20,27,31]. The number
of fruits and the fruit yield/plant in bitter melon are highly dependent on the presence
of male and female flowers (sex ratio). The sex ratio ranged from nine (cv. Ilocano) to 20
(cv. Local Japanese) in the different cultivars. Sex ratio strongly affected pollination and
fruit formation. A greater sex ratio can provide sufficient pollen for a longer period to
fertilize female flowers. Other researchers have also reported significant differences in sex
ratios among different bitter melon cultivars, for example, from 6.85 to 27.29 [25,32], 7.90 to
9.77 [33], 19 to 20 [29,30], 17.99 to 33.50 [15], 10 to 30 [28], and 17.17 to 22.18 [27].
The highest fresh fruit yield (5.22 kg/plant) was obtained from Hybrid No. 486, while
cv. Ilocano produced the lowest yield (2.98 kg/plant) (Figure 2). Although it produced the
highest fruit number per plant (133.33), cv. Ilocano had a lower fresh fruit yield because of
its small fruit size. This finding is consistent with that of Dey et al. (2006), who reported
that genotypes with smaller fruits had lower fresh fruit yields [33]. The fresh fruit yield of
bitter melon is economically important and depends on the variety and crop management
practices. The average economic yield was 8000–10,000 kg·ha−1 . Some cultivars may
produce up to 20,000–40,000 kg·ha−1 [5,6,33] and even higher (25 to 80 tons) [34]. These
variations can be attributed to climatic conditions, soil fertility, and the cultivar used.
In Taiwan, a yield of 61–108 tons·ha−1 was obtained under greenhouse conditions from
grafted seedlings [35], and, in India, yields from 21 to 44 tons·ha−1 were reported [6]. The
lower yield of our research is related to the shorter growing period in Karaj, Iran, than in
tropical regions such as India and the Philippines.
Our results revealed that the dry fruit yield per plant and yield attributing traits were
significantly different among the bitter melon cultivars. The differences in the order of
cultivars with respect to fruit yield on the basis of fresh or dry fruit yield was related to
the moisture percentage of each cultivar. Several factors affect fruit water content such as
fertilization [18], cultivar, fruit density, time of harvest, maturity, and texture, and it varied
between 91% and 93% [36].
The current results showed that the highest fruit number per plant was observed in cv.
Ilocano and could be attributed to the lower ratio of male to female flowers. No significant
differences were observed among the remaining cultivars in this trait. The fruit number per
Plants 2021, 10, 1370 12 of 19

plant depends on the plant’s genetics, growth conditions, and crop management practices,
as reported by several studies [24,32,37,38]. Our findings are consistent with those of these
researchers, except for cv. Ilocano. However, a fruit number per plant of 6.6 to 68 has been
reported [3,19,26,27].
According to our results, the observed differences in fruit length can be attributed
to the different stages at which the fruit length was measured. Fruit length was also
reported by different researchers as 15.82–38.83 cm [27], 8.78–33 [4], 9.23–24.47 cm [15,32],
and 2.91–11.60 cm [37]. Dudhat and Patel showed that nutrient management affects fruit
length [38].
Our results showed that the highest fruit diameter was observed in the cv. Iranshahr
(cv. Palee), and the lowest belonged to the cv. Ilocano. Other researchers reported fruit
diameters of 2.2 to 7.4 cm [19,32,39], which is consistent with our findings at unripe and
semi-ripe stages.
In this study, the highest and the lowest numbers of total seeds/fruit belonged to cv.
Iranshahr (cv. palee) and cv. Ilocano, respectively. Moreover, there was a positive correla-
tion between seed number per fruit and fruit size in medium-weight medicinal pumpkins.
Hand pollination in Momordica charantia produced heavier fruit, and open pollination and
fruit size were related to the number of seeds/fruit [40]. Total seeds/fruit had a wide
range reported by several researchers, i.e., 7.4–28.43 [25], 7.4–28.43 [37], 15.65–33 [27], and
10.45–26.04 [22].
According to our results, the mean comparison of the 1000-seed weight of bitter melon
cultivars showed that Hybrid No. 486 had the highest 1000-seed weight, followed by cv.
Local Japanese. It was previously reported by many researchers that the 1000-seed weight
varied among different bitter melon genotypes, that is, 58 g [40], 166 g [41], and 21.4 g [37].
Our findings are in agreement with some of these reports.
Our results of the phytochemical assay showed that the total phenol content of fruits
was the highest in Hybrid No. 486, followed by Hybrid Mestisa, cv. “Isfahan,” Local
Japanese, cv. Iranshahr (ee), and cv. Ilocano. A comparison of the effect of different
drying methods on the phenolic content of fruit in different cultivars indicated that the
freeze-drying method was superior to shade-drying. Horax et al. (2005) reported the
phenolic compounds in four types of bitter melons in both oven-dried and freeze-dried
samples as being between 4.55 and 82.4 mg/g dry weight [42]. They also reported the
phenolic compounds in the seeds as being from 4.61 to 40.08 mg/g dry weight. They
concluded that the oven-drying method was superior to freeze-drying. In another study,
Horax et al. (2010) investigated the phenolic content of bitter melon pericarps and seeds.
The results showed that the total phenol content in bitter melon seeds was higher than that
in fruit pericarps [43]. Kubola and Siriamornpun (2008) evaluated phenolic compounds in
different organs of bitter melon (leaves, stems, unripe, and ripe fruits) and showed that the
leaves contained the highest total phenol content, and unripe fruit contained higher total
phenol content than the ripe fruit [44]. Tan et al. (2014) grew six varieties of bitter melon
in greenhouse conditions and compared the total phenol content of the fruit, showing
that this trait varied from 1.5 to 7.9 mg GAE [45]. The phenolic contents measured in the
present study were higher than those reported by Tan et al. (2014). The higher levels of
phenolic compounds may be due to genotypic differences, climatic conditions, and crop
management practices. As mentioned earlier, although secondary metabolite production is
under genetic control, environmental factors also have significant effects.
According to our results, the highest total flavonoid content was obtained in Hybrid
No. 486 in the unripe fruit stage in shade-dried samples. The lowest flavonoid content
was observed in the freeze-dried cv. Local Japanese and Hybrid Mestisa in semi-ripe
fruit and ripe fruit. However, the flavonoid content was lower in the ripe fruits of cv.
Iranshahr (cv. Palee) for both drying methods. Tan et al. (2014) investigated the extraction
efficiency of flavonoids from bitter melon fruits and showed that the solvent type had a
significant effect on flavonoid extraction efficiency. The lowest extraction efficiency (<2 mg
of retinol per gram of fruit dry weight) was obtained, while the highest flavonoid content
Plants 2021, 10, 1370 13 of 19

(approximately 25 mg/g equivalent to retinol) was obtained when acetone was used. They
finally suggested that the acetone solvent acted to extract the maximum flavonoids of bitter
melon [45]. Therefore, the variations in the results obtained by different researchers are
primarily due to the climatic conditions and secondarily due to the solvent used in the
extraction of flavonoids.
The current results showed that the fruit charantin content at different stages of
fruit development was highest at the semi-ripe stage, followed by unripe and ripe fruits.
Secondary metabolite production is controlled by different plant pathways such as malonic
acid, mevalonic acid, and shikimic acid pathways. During plant developmental stages,
some genes are activated and some genes are silenced, resulting in the different secondary
metabolite composition of medicinal plants [46]. Another important factor that affects
secondary metabolite content and composition is the post-harvest technology, especially
the drying condition and methods. During the drying period, transformation of some
primary compounds to others takes place under the control of some enzymes [47]. In
our research, charantin content for different drying methods indicated the superiority of
lyophilized compared to shade-dried. Zhang et al. (2009) also examined the effects of the
drying method on the phytochemical characteristics of bitter melons and showed that the
freeze-drying method improved the phytochemical composition of the plant [48].
Kim et al. (2014) compared the charantin content of 10 Japanese and Filipino bitter
melon cultivars and concluded that Japanese cultivars were superior to the Filipino culti-
vars. Among the cultivars studied in their research, “Peacock” (Japanese cultivar) had the
highest fruit charantin content (711 µg/g), while “Trident” 357 (Filipino cultivar) had the
lowest (29.8 µg/g) [48]. The findings of our study are consistent with those reported by
Lee et al. (2015) and Goo et al. (2016) [49,50]. These differences in fruit charantin content
in different studies are due to differences in genotypes and growing conditions [50]. The
fruit total phenol, flavonoid, and charantin content, and ripe fruit color of the six bitter
melon cultivars studied here indicated that cultivars with darker fruit color contained more
secondary metabolites than the cultivars with brighter fruits. Habicht et al. (2011) also
showed that the cultivars with white fruit had the lowest amount of saponin in comparison
to the darker cultivars. Therefore, the results of this study are consistent with the findings
of Habicht et al. (2011) [51].
Our results showed that cv. Iranshahr had the highest leaf charantin content, while
cv. Local Japanese had the lowest. Lee et al. (2015) examined the charantin content
of the Korean native population of Momordica charantia, “Erabu”, and Japanese Dragon
cultivars and reported that the mean leaf charantin content was 144 µg/g, while the average
fruit charantin was 55.27 µg/g. They stated that improved Japanese cultivars are more
expensive than the native Korean population [49]. The difference between the findings of
the present study and those of Lee et al. (2015) is mainly due to differences in cultivars
and climatic conditions. Goo et al. (2016) showed significant differences among eight
indigenous populations of bitter melons (two from Indonesia and six from India) for leaf
charantin content. They reported the leaf charantin content of three Indian populations
as KSI3 (5 µg/g), KSI7 (5.9 µg/g), and KSI8 (5.8 µg/g). They also confirmed that leaf
charantin content was greater than that of fruits [50].

4. Materials and Methods

4.1. Plant Materials
This study was conducted in the greenhouse and research field of Seed and Plant
Improvement Institute in Karaj, Iran, with geographical coordinates latitude 35◦ 480 N and
longitude 50◦ 580 E, and an elevation of 1292 m above free sea level (Figure S1, Supple-
mentary Materials). Six bitter melon cultivars, namely, Hybrid No. 486 (from Thailand),
Hybrid Mestisa (from the Philippines), Iranshahr (cv.Palee from India), Ilocano (from the
Philippines), Isfahan, and Local Japanese, collected from different regions in Iran and other
countries, were used in this study (Figure S2, Supplementary Materials).
Plants 2021, 10, 1370 14 of 19

4.2. Plant Establishment in the Field

Seeds were sown in small pots (200 cm3 ) for seedling production. The medium was
prepared using a mixture of perlite, peat moss, and soil (1:1:1). Irrigation was regularly
applied to the bottom of the pots. The greenhouse temperature during the seed germination
process was 20 ± 2 ◦ C, and the relative humidity was adjusted to 55%. Seedlings were
transplanted into the field at the 4–5-leaf stage. Each experimental plot consisted of one
row, with a 200 cm distance between rows and 50 cm between two plants within rows.
A tape irrigation system was used to irrigate the plots. Soil samples were taken from a
depth of 0–30 cm before seedling planting (three samples). The results of the soil analyses
are presented in Table 10. Considering the results of soil analysis (Table 10), 75 kg·ha−1 of
urea, 150 kg·ha−1 of triple-super phosphate, and 100 kg·ha−1 of potassium sulfate were
applied before planting as basal fertilizers. Humic acid (Humax) was applied 3 weeks after
transplanting the seedlings in the field. Two top dressings of urea, once at the beginning of
flowering (75 kg·ha−1 ) and another at the fruit setting (75 kg·ha−1 ) stages, were applied.
Zinc sulfate (40 kg·ha−1 ) was applied at the beginning of the flowering stage.

Table 10. Physical and chemical properties of soil in experimental field at Karaj, Iran.

EC N P K
Clay Silt Sand Texture OC% pH Fe Zn Cu Mn B
(Ds/m) (total) (ava) (ava)
mg/kg
Clay- 0.63 ± 1.82 ± 7.56 ± 27.6 ± 7.97 ± 0.90 ± 2.22 ± 16.5 ± 0.44 ±
27 40 33 600 ± 15 295 ± 6.3
loam 0.02 0.21 0.23 2.6 2.1 0.03 0.14 0.3 0.06

OC: Organic Carbon; EC: Electrical Conductivity, B: Boron.

4.3. Experimental Design and Recorded Parameters

This study was designed and implemented in two separate experiments.
Experiment 1: In the first experiment, the vegetative, reproductive, and fruit traits of
bitter melon cultivars were evaluated using a randomized complete block design with three
replications. Parameters at vegetative stage, i.e., plant height, number of leaves/plant, leaf
area, leaf fresh weight/plant and leaf dry weight/plant, above-ground dry weight/plant,
number of branches, and stem moisture content were recorded. Reproductive traits in-
cluded days to the appearance of the first male and female flowers, the node number
on which the first male or female flower appeared, male/female flower ratio, number
of fruits/plant, total number of seeds/fruit, 1000-seed weight, fruit color-related indices
(using KONICA MINOLTA’s Hunter-Leaf machine, Chiyoda City, Tokyo, Japan) at 2 cm
below the fruit’s tail), fresh/unripe fruit weight, ripe fruit weight, length, diameter, weight,
density, and volume of fruit were measured and evaluated. Fresh fruit yield per plant was
measured using the technique described by Palada and Chang (2003) [6].
Experiment 2: In the second experiment, the effect of the time of harvesting (unripe,
semi-ripe, and ripe) and drying methods (shade-dried and freeze-dried) on the phytochem-
ical attributes of the fruit, i.e., the total phenol, flavonoids, and charantin contents were
examined using factorial arrangements in a randomized block design with three replica-
tions. All means then compare statistically by Tukey’s test. Fruits of each cultivar were
sampled at three different stages, unripe, semi-ripe, and ripe, following the methodology
reported by Horax et al. 2010. Unripe is described as when the pericarp is still green and
the seed is not fully grown yet (about 2 weeks after flowering when it is 25% of the full
size) [43,52]. Semi-ripe is described as when the green pericarp and seed are fully grown
(approximately 3–4 weeks after flowering when it is 50% of full size). Ripe is described as
when the pericarp is yellow, and the seed arils and internal tissues are red (approximately
4–5 weeks after flowering and 100% fruit size). In addition, fruit growth parameters were
evaluated when the green pericarp and seed were fully grown, the seed arils turned pink,
and fruits were 80% of full size. Following sampling, the internal texture of the fruit and
the development stage of the seeds were examined and recorded upon cutting the fruit
 and the seed is not fully grown yet (about 2 weeks after flowering when it is 25% of the
full size) [43,52]. Semi-ripe is described as when the green pericarp and seed are fully
grown (approximately 3–4 weeks after flowering when it is 50% of full size). Ripe is de-
scribed as when the pericarp is yellow, and the seed arils and internal tissues are red (ap-
Plants 2021, 10, 1370 proximately 4–5 weeks after flowering and 100% fruit size). In addition, fruit growth 15 ofpa-
19
rameters were evaluated when the green pericarp and seed were fully grown, the seed
arils turned pink, and fruits were 80% of full size. Following sampling, the internal texture
of the fruit and the development stage of the seeds were examined and recorded upon
(Figure 5). fruit
cutting the The freeze-drying
(Figure 5). Theconditions used
freeze-drying for drying
conditions thefor
used fruit samples
drying are listed
the fruit samples in
Table 11.
are listed in Table 11.

Figure 5.
Figure 5. Exterior
Exteriorand
andinterior
interiorfruit
fruitfeatures
featuresatatdifferent
different developmental
developmental fruit
fruit stages
stages (Hybrid
(Hybrid No.No.
486486 from
from Thailand).
Thailand). Un-
Unripe
ripe stage: left column (A) and (B); semi-ripe stage: Left column (C) and (D); aril color changing stage: right coumn (E)
stage: left column (A,B); semi-ripe stage: Left column (C,D); aril color changing stage: right coumn (E,F); ripe stage: right
and (F); ripe stage: right column (G), growth habit (H).
column (G), growth habit (H).

Table 11. Freeze-drying conditions.

Factor Level
1 Loading temperature Room Temperature
2 Freezing rate 0.5 ◦ C/min
3 Freezing temperature −40 ◦ C
4 Complete freezing duration 2h
5 Temperature ramp rate during the primary drying 8 ◦ C/h

4.4. Phytochemical Analysis

The phytochemical characteristics of dried samples, including total phenol and
flavonoid content, were measured at the Ferdowsi University of Mashhad. Freeze-drying
of samples was performed in the lyophilization section of the Razi Vaccine and Serum
Research Institute in Karaj. Charantin determination was performed using HPLC at Chung-
nam National University, Daejeon, South Korea.
Plants 2021, 10, 1370 16 of 19

4.5. Sample Preparation

Fruits with pulp were cut into pieces (0.5 cm) and dried in the shade at room tempera-
ture (25 ± 3 ◦ C). Aril’s sugar percentage: After mixing the arils with a milling machine, the
pulp was used for soluble solid content evaluation using a refractometer (Digital Palette
PR-32 Atago, Tokyo, Japan).

4.6. Determination of Total Phenol Content

Extraction and determination of total phenol content were performed following the
method of Horax et al. (2005) [42]. Briefly, 1 g of dried fruit was pulverized using a mortar
and pestle. Then, 20 mL of methanol was added and extracted using a water bath at
65 ◦ C for two h. The extracts were filtered using a Buchner funnel and vacuum pump,
and centrifuged at 4750 rpm for 15 min. One milliliter of extract was added to 1 mL of
Folin–Ciocâlteu (0.25 N), 1 mL of sodium carbonate (1 N), and 7 mL of distilled water. For
the blank sample, methanol was added instead of the extract. After vortexing for 1 min, the
samples were incubated for 2 h at room temperature in the dark. Finally, the absorbance
of the resulting solution was measured using a spectrophotometer (Shimadzu PC-1650,
Kyoto, Japan) at 726 nm.

4.7. Determination of Total Flavonoid Content

The total flavonoid content of the fruit was determined according to the method of
Pekal and Pyrzynska (2014). One milliliter of extract was mixed with 0.5 mL of aluminum
chloride, 0.5 mL of water, 0.5 mL of HCl, and 0.5 mL of sodium acetate (1 N). The samples
were then incubated at room temperature for 10 min. Finally, the absorbance of the solution
was measured at 425 nm using a spectrophotometer (Shimadzu PC-1650, Kyoto, Japan) [46].

4.8. Determination of Charantin

Extracting and measuring of charantin was done at Chungnam National University,
Daejeon, South Korea following the method of Kim et al. 2014. One gram of leaf or fruit
sample was mixed with 20 mL of hexane to remove the sample fats. The sample was
then placed under a normal hood to evaporate the hexane. This procedure was repeated
twice. Samples were then extracted with 10 mL of pure methanol using a sonicator
(Branson Ultrasonic Co., Danbury, CT, USA). The extract was filtered through filter paper
and dried using a vacuum evaporator (rotary evaporator) at 40 ◦ C. Finally, the dried
extract was dissolved again in 1 mL of HPLC-grade methanol. Charantin content was
measured using an HPLC (NS-4000 model, Daejeon, South Korea) with a UV detector at a
wavelength of 204 nm. The column used for separation was an Optimapak C-18 column
(4.6 mm × 250 mm, RStech, Daejeon, South Korea) with a flow rate of 0.8 mL·min−1 . A
mobile phase of MeOH/water (98:2 v/v) was used for separation, and the injected volume
of the sample was 20 µL. The identification and quantification of compounds were done by
comparing peak area and the retention times with the external standard (Charantin (92.2%
purity), ChromaDexInc., Irvine, CA, USA). The quantification and analysis of each sample
were performed in triplicate [10].

5. Conclusions
According to all the results of this study, which was conducted for the first time in Iran,
it can be stated that there is potential for economic production of this plant in Karaj climatic
conditions, with cv. No. 486 being the best choice among evaluated cultivars in terms
of vegetative traits and valuable medicinal compounds. Regarding drying of harvested
fruits, although valuable medicinal compounds were better preserved in the freeze-dried
samples, but due to the cost of this method and lack of access to this equipment for farmers,
shade-drying is recommended. In the future, further studies are required to analyze the
individual phenolic compounds and secondary metabolites found in each organ of cv. 486
by using HPLC and GC–TOFMS analysis. This study might help to provide knowledge
on the utilization of strategies to increase the level of different medicinal compounds in
Plants 2021, 10, 1370 17 of 19

cv. No. 486. Moreover, analyzing the gene expression of phenylpropanoid and flavonoid
biosynthetic pathways will help to increase the secondary metabolite production in cv. No.
486 via the bioengineering approach.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/plants10071370/s1, Figure S1: Geographical map of the investigation cite (Karaj) and other
Iranian Cite that the seeds were harvested. Figure S2: Bitter gourd cultivars studied in the research.
Author Contributions: Conceptualization, M.A., S.U.P. and Y.F.; methodology, M.A. and S.U.P.;
validation, M.A. and Y.F.; formal analysis, A.K.; investigation, A.V. and M.A.; resources, M.A., S.U.P.
and Y.F.; data curation, A.V. and M.A.; writing—original draft preparation, A.V. and M.A.; writing—
review and editing, S.U.P., T.M., A.S. and R.S.; visualization, M.A. and A.V.; supervision, M.A.; project
administration, M.A. and Y.F.; funding acquisition, M.A., Y.F. All authors have read and agreed to the
published version of the manuscript.
Funding: The research was supported by Islamic Azad University of Karaj. The authors also thank
the Department of Crop Science, Chungnam National University, Republic of Korea for the sample
analysis. This work was also supported by JST CREST Grant Number JPMJCR17O2.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data is contained within the article or supplementary file.
Conflicts of Interest: On behalf of all authors, the corresponding author states that there are no
conflicts of interest.

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