plants

Article
Bitter Melon (Momordica charantia L.) Fruit Bioactives
Charantin and Vicine Potential for Diabetes Prophylaxis
and Treatment
Mahwish 1 , Farhan Saeed 1 , M. Tauseef Sultan 2 , Ayesha Riaz 3 , Sagheer Ahmed 4 , Nicusor Bigiu 5 ,
Ryszard Amarowicz 6, * and Rosana Manea 5

1 Institute of Home & Food Sciences, Government College University, Faisalabad 38000, Pakistan;
[email protected] (M.); [email protected] (F.S.)
2 Institute of Food Science and Nutrition, BZU, Multan 60800, Pakistan; [email protected]
3 Institute of Home Sciences, University of Agriculture, Faisalabad 38000, Pakistan; [email protected]
4 Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad 44000, Pakistan;
[email protected]
5 Faculty of Medicine, Transilvania University of Brasov, 500019 Brasov, Romania;
[email protected] (N.B.); [email protected] (R.M.)
6 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-748 Olsztyn, Poland
* Correspondence: [email protected]

Abstract: Natural products are gaining clinical significance in modern day health care systems to pre-
vent diseases. Bitter melon, a health promoting vegetable, is traditionally used for medical nutrition



therapy to cure diabetes but to reap maximum health claims, vigilant control of its substances in diet


is crucial as part of curative action for effective diabetes management. In the present research, first
Citation: Mahwish; Saeed, F.; Sultan, phase focused on detection of key bioactive components, i.e., charantin and vicine in different parts
M.T.; Riaz, A.; Ahmed, S.; Bigiu, N.;
of its fruit. In the second phase, normal and hyperglycemic Sprague Dawley rats were fed on skin,
Amarowicz, R.; Manea, R. Bitter
flesh and whole fruit of bitter melon at 150 and 300 mg/kg body weight and assessed for diabetes
Melon (Momordica charantia L.) Fruit
prophylaxis and treatment. The highest amount of charantin (0.16 ± 0.02 mg/g) was recorded in flesh
Bioactives Charantin and Vicine
while vicine was present in abundance in whole fruit (0.21 ± 0.01 µg/100 g). In normal rats, bitter
Potential for Diabetes Prophylaxis
and Treatment. Plants 2021, 10, 730.
melon supplementation was helpful in managing the onset of diabetes. Hyperglycemic rats showed
https://doi.org/10.3390/ diabetic complications including polydipsia, polyuria, glycosuria, renal hypertrophy and increased
plants10040730 glomerular filtration rate. However, bitter melon consumption showed significant improvements in
these parameters. The most potent dose was 300 mg/kg whole fruit that resulted in 31.64% lowering
Academic Editor: Corina Danciu of blood glucose level and 27.35% increase in insulin level in hyperglycemic rats.

Received: 17 March 2021 Keywords: bitter melon; Momordica charantia; diabetes; charantin; vicine; glycemic control; hypoglycemia
Accepted: 5 April 2021
Published: 8 April 2021

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Sedentary lifestyle and poor dietary habits lead towards occurrence of various disor-
published maps and institutional affil-
ders such as diabetes, cardiovascular diseases, hypertension, etc. Plants and their products
iations.
have tendency to ameliorate such disorders and can play a helpful role in preventing
diseases. The dietary components play an integral part in promoting the health due to
presence of phytochemicals and phytonutrients. They are helpful in mitigating serious
disorders such as diabetes, hypercholesterolemia, hyperlipidemia, inflammation, etc.
Copyright: © 2021 by the authors.
Bitter melon (Momordica charantia L.) is considered due to its chemical moieties [1].
Licensee MDPI, Basel, Switzerland.
The bitter melon is a member of family Cucurbitaceae and is also known as balsam pear,
This article is an open access article
bitter gourd and karela in different regional languages. It is consumed as a vegetable
distributed under the terms and
in different tropical and subtropical regions of world, owing to its distinctive taste and
conditions of the Creative Commons
Attribution (CC BY) license (https://
nutritional profile. Green fruits of bitter melon contain vitamin A, C, thiamine, niacin,
creativecommons.org/licenses/by/
riboflavin and minerals [2]. It naturally possesses high phenolic content, i.e., gallic acid,
4.0/).
alkaloids, saponins, flavonoids, etc. Due to this rich phytochemistry, bitter melon exhibits

Plants 2021, 10, 730. https://doi.org/10.3390/plants10040730 https://www.mdpi.com/journal/plants

Plants 2021, 10, 730 2 of 13

highest antioxidant activity among its family. Owing to the presence of various bioactive
components, bitter melon holds some pharmacological properties and exerts various health
enhancing properties such as scavenging of free radicals, hypoglycemic and hypolipidemic
activities [3]. Diabetes mellitus is a growing threat to human health that affects millions
of peoples each year. The dietary patterns significantly influence the pathogenesis of
diabetes mellitus by incorporating healthy foods in normal diets along with physical
activity is an effective strategy. Many studies suggest the effectiveness of bitter melon
against diabetes prophylaxis [4–9]. Many in vivo studies confirmed the hypoglycemic
potential of bitter melon and treatment due to the presence of numerous hypoglycemic
agents such as alkaloids, flavonoids, saponin, catechins, charantin, vicine, and polypeptide-
p fractions [10]. Many experiments supported that bitter gourd proved to be effective in
insulin production and can be hepato-renal protective. However, inadequacies were present
in most of the studies due to poor study design [11]. Furthermore, some hypoglycemic
studies reported contradictory results with negligible positive effects of bitter melon intake
during diabetes [12,13]. In many studies, the data were insufficient and indecisive to
advocate its use in diabetes management [11,14]. Some studies evaluated effectiveness of
only specific part of bitter melon with single dosage for limited time period [12]. In these
studies, no information was provided in relation to role of bioactive molecules in diabetes
management. It was, therefore, important to conduct a more conclusive and systematic
study. Hence, present study was designed to compare different parts of bitter melon fruit for
charantin and vicine contents and subsequent feeding trial to assess individual or combined
role of these natural products in improving diabetes and associated complications.

2. Materials and Methods

2.1. Procurement and Handling of Raw Material
Bitter melon fruits were procured from Vegetable Research Section, Ayub Agriculture
Research Institute, Faisalabad, Pakistan. These fruits were washed carefully to remove ad-
hered unhygienic substances and other contaminants. After washing, the outer warty skin
was peeled and collected separately. After peeling, flesh part was obtained by removing
seeds. Some fruits were cut into small size as a whole. These isolated fruit portions were
kept for few days at room temperature to dry. Fine powder was made of this dried material
by using laboratory grinder (Panasonic, Osaka, Japan, Model MJ-W176P) and absolute
refined powder was collected by passing through a sieve and finally packed separately in
glass bottles for further evaluation.

2.2. Reagents and Standards

HPLC grade reagents and standards were purchased from Sigma-Aldrich (Tokyo,
Japan) and Merck (Darmstadt, Germany). Diagnostic kits were purchased from Cayman
Chemicals (Europe) and Sigma-Aldrich Bioassay (Darmstadt, Germany).

2.3. Quantification of Charantin

The estimation of charantin contents in samples were determined by protocol of
Pitipanapong et al. [15]. Firstly, 1.0 g powder of bitter melon samples was taken to extract
with ethanol and further extracted in methanol by ultra-sonication. In this way, viscous
crude extract of charantin was obtained by filtration of extracts and subsequent evaporation.
Before analyzed by HPLC, crude extract was purified. HPLC analysis was performed with
C-18 Inertsil ODS-3 column (size of particles 5.0 µM, 4.6 mm × 250 mm ID). Methanol-
water (100:2 v/v) was used as mobile phase with 1 mL/min flow rate. The wavelength for
UV detection was 204 nm and volume for sample injection was 200 µL.

2.4. Quantification of Vicine

The extraction of bitter melon powder samples was done with water (10–25 mL) for
ten min by ultra-sonication. The mixture was centrifuged and supernatant was collected
separately in volumetric flask. All the samples were filtered before analysis through HPLC.
Plants 2021, 10, 730 3 of 13

HPLC analysis was performed with C-18 reversed phase column 250 mm × 4.6 mm, 5 µM).
Methanol-phosphate buffer with pH 3.0 (10:90, v/v) was used as mobile phase. Detector
was set at 280 nm [16].

2.5. Efficacy Study

To probe anti-diabetic effect of bitter melon, Sprague Dawley rats were kept in Animal
Room, College of Pharmacy, Government College University, Faisalabad. Prior to feeding
trial, rats were acclimatized by giving basal diet for few days. During eight week feeding
trial, temperature and relative humidity was maintained to 25 ± 2 ◦ C and 55 ± 5%, respec-
tively, with regular alteration in light/dark period consisting of 12 h. Central Government
Ethical Committee Guidelines were strictly followed during the course of study (Approval
no. FSD-16/M-234).

2.6. Diet and Dosage

For control group, the diet is composed of 66% corn starch, 10% corn oil, 10% cellulose,
10% protein contents, 3% minerals and 1% vitamin mixture. For experimental groups, skin,
flesh, whole fruit powder of bitter melon in doses of 150 mg/kg and 300 mg/kg body
weight was added. In this way, seven groups of rats were made consisting of twenty rats
in each group. Each group is further split into normal rats and hyperglycemic rats. The
normal rats were given diet without excessive sucrose while hyperglycemic rats were given
40% sucrose in diet to induce hyperglycemia and diabetes. The rats were given access to
water and feed on ad libitum basis.

2.7. Testing
Each group was observed for water intake, feed intake and weight gain in 24 h period
during the course of the study. The volume of urine was also noted by using metabolic
cages in which rats were placed for 24 h and collected urine under a layer of toluene.
The amount of reducing sugar in urine of rats was determined by 3,5-dintro salicylic
acid method [17]. Creatinine was measured in blood and urine by Owen’s method [18].
Glomerular filtration rate (GFR) was assessed by following formula [19]:

GFR(ml/min)
Urine volume (ml)×Urinary Creatinine (mg/dl)×1000 (g)
= Body weight (g)×Plasma creatinine (mg/dl)×1440 (min)

Blood samples were collected in EDTA coated tubes at 28 and 56 day of trial. Isolation
of serum was done by centrifugation @ 4000 rpm for 6 min in centrifuge machine (Rotrofix
32-A Heltich, Westphalia, Germany). For biochemical evaluation, these samples of sera
were kept through Microlab (Rendox Toerauta RX-Monza, County Monaghan, Republic
of Ireland). The glucose and insulin level was calculated by procedure of Katz et al. [20]
and Ahn et al. [21], respectively. The percent increase or decrease in biochemical traits of
experimental groups was calculated in comparison to control groups. At the end of trial,
the weight of the right and left kidneys were determined separately.

2.8. Statistical Analysis

The data was statistically analyzed using Statistical Package (Microsoft Excel 2016 and
SPSS v20.). Level of significance was determined by ANOVA and LSD for comparison [22].

3. Results
3.1. Quantification of Charantin and Vicine
The charantin and vicine contents (Figure 1) in different bitter melon fruit parts were
determined through HPLC before administration of diet to Sprague Dawley rats. The total
amount of charantin was found high in flesh part (0.16 ± 0.02 mg/g) than whole fruit
(0.11 ± 0.02 mg/g) and skin (0.08 ± 0.01 mg/g). Meanwhile, whole fruit possessed high
Plants 2021, 10, 730 4 of 13

amount of vicine (0.210 ± 0.010 µg/100 µg) than flesh (0.131 ± 0.005 µg/100 µg) and skin
(0.114 ± 0.006 µg/100 µg).

Figure 1. Charantin and vicine contents in skin, flesh and whole fruit of bitter melon. x,y,z indicated
significant differences in charantin contents (p < 0.05); a,b,c indicated significant differences in vicine
contents (p < 0.05).

3.2. Feed and Water Intake

The diet containing bitter melon fruit skin, flesh and whole fruit at the doses of 150 and
300 mg/kg body weight increases the consumption of water in normal rats with highest
increase was observed in rats fed with 300 mg/kg body weight of whole fruit (Table 1).

Table 1. Impact of bitter melon supplementation on water intake of rats.

Water Intake (mL/24 h)

Groups 28th day 56th day
Normal Rats Hyperglycemic Rats Normal Rats Hyperglycemic Rats
d 29.35 ± 2.15 a d 36.65 ± 2.05 a
Control 23.7 ± 1.15 27.9 ± 2.12
SF-L 24.1 ± 1.34 c 25.94 ± 1.49 b 28.1 ± 1.77 c,d 31.02 ± 2.34 b
SF-H 24.3 ± 1.40 c 24.85 ± 1.46 c 28.4 ± 2.15 c 29.01 ± 2.17 d
FF-L 24.4 ± 1.88 b,c 25.03 ± 2.11 b 28.3 ± 1.63 c 30.85 ± 1.69 c
FF-H 24.7 ± 1.35 b 24.07 ± 1.75 d 28.6 ± 2.19 b 28.79 ± 1.56 e
WF-L 24.8 ± 2.01 a,b 24.91 ± 1.68 b 28.7 ± 2.03 b 30.71 ± 2.62 c
WF-H 25.2 ± 1.48 a 23.86 ± 1.33 d 29.00 ± 1.59 a 28.63 ± 2.22 e
SF—Skin fed rats; FF—Flesh fed rats; WF- Whole fruit fed rats; L—Lower amount of bitter melon in experimental diet (150 mg/kg body
weight), H—High amount of bitter melon in experimental diet (300 mg/kg body weight); Values are mean ± SD of ten rats; Superscript
letters within the same column indicate significant (p < 0.05) differences.

In hyperglycemic rats, consumption of water increased in control and significant

reduction in water intake was observed by incorporation of bitter melon in diet in dose
dependent manner. Whole fruit and flesh of bitter melon were found more effective
than skin in preventing polydipsia conditions in hyperglycemic rats. In normal rats,
supplementation of bitter gourd skin, flesh and whole fruit has resulted in reduction of
feed intake compared to control while polyphagia condition was noticed in hyperglycemic
control rats as they consumed an excessive quantity of diet compared to rats fed with bitter
melon (Table 2).
Plants 2021, 10, 730 5 of 13

Table 2. Impact of bitter melon supplementation on feed intake of rats.

Feed Intake (g)

Groups 28th day 56th day
Normal Rats Hyperglycemic Rats Normal Rats Hyperglycemic Rats
Control 15.1 ± 1.12 a 17.03 ± 1.78 a 21.2 ± 2.49 a 23.22 ± 1.33 a
SF-L 14.6 ± 0.76 b 14.96 ± 1.39 b 20.5 ± 2.03 b 20.94 ± 1.11 b
SF-H 14.3 ± 1.38 b 13.80 ± 1.01 c 20.1 ± 1.98 b 19.78 ± 1.04 c
FF-L 14.3 ± 1.22 b 14.94 ± 0.78 b 20.4 ± 1.62 b 20.87 ± 0.92 b
FF-H 14.1 ± 0.94 b,c 13.76 ± 1.39 c 19.8 ± 0.99 b,c 19.45 ± 1.17 c
WF-L 13.9 ± 0.45 c 14.82 ± 1.54 b 19.9 ± 1.07 b,c 20.73 ± 1.43 b
WF-H 13.7 ± 0.88 c 13.64 ± 1.44 c 19.6 ± 0.69 c 19.36 ± 1.36 c
Abbreviations and description as in Table 1.

3.3. Body Weight Gain

Normal control rats gained more weight with the passage of time than bitter melon
fed rats (Table 3). In hyperglycemic control rats, body weight did not increase as much,
while apprehensible body weight increase was observed in hyperglycemic rats fed with
bitter melon. Increase in body weight was more pronounced in hyperglycemic rats fed
with 300 mg/kg body weight of bitter melon than 150 mg/kg body weight.

Table 3. Impact of bitter melon supplementation on body weight of rats.

Body Weight (G)

Groups 28th Day 56th Day
Normal Rats Hyperglycemic Rats Normal Rats Hyperglycemic Rats
Control 158.66 ± 3.01 a 145.55 ± 2.75 c 215.90 ± 4.22 a 181.34 ± 2.61 c
SF-L 155.91 ± 2.77 b 151.18 ± 2.04 b 208.67 ± 4.06 b 203.66 ± 3.59 b
SF-H 154.75 ± 3.52 c 151.66 ± 1.89 a,b 208.40 ± 2.88 b 205.33 ± 3.11 a
FF-L 154.62 ± 1.95 c 151.43 ± 2.09 b 208.33 ± 2.54 b 204.50 ± 3.49 b
FF-H 154.34 ± 2.67 c 152.57 ± 2.67 a 208.13 ± 1.97 b 206.56 ± 3.34 a
WF-L 154.32 ± 3.00 c 151.71 ± 0.95 b 208.26 ± 2.42 b 204.66 ± 2.68 b
WF-H 153.68 ± 2.18 d 152.83 ± 1.90 a 208.01 ± 2.97 b 206.60 ± 2.36 a
Abbreviations and description as in Table 1.

3.4. Estimation of Urine and Reducing Sugar Excretion

The hyperglycemic rats produced more urine than the normal rats. This polyuria
condition prevailed in hyperglycemic control group (Table 4). The dietary supplementation
of bitter melon unveiled substantial decrease in urine excretion.

Table 4. Impact of bitter melon supplementation on urine volume of rats.

Urine Volume (mL/24 h)

Groups 28th Day 56th Day
Normal Rats Hyperglycemic Rats Normal Rats Hyperglycemic Rats
Control 14.3 ± 2.23 a 20.1 ± 1.01 a 18.3 ± 1.13 a 26.8 ± 2.85 a
SF-L 13.7 ± 1.72 b 16.5 ± 2.19 b 17.6 ± 1.31 b 21.3 ± 3.03 b
SF-H 13.6 ± 1.63 b 15.8 ± 1.43 b 17.5 ± 0.99 b 20.65 ± 1.26 b
FF-L 13.3 ± 0.76 b,c 15.7 ± 1.29 b 17.3 ± 1.84 b 20.4 ± 1.64 b
FF-H 13.3 ± 1.33 b,c 14.9 ± 2.07 b 16.9 ± 1.22 b,c 20.2 ± 2.13 b,c
WF-L 13.2 ± 0.89 c 15.2 ± 0.96 c 16.8 ± 1.71 c 19.9 ± 0.78 c
WF-H 13.0 ± 2.13 c 14.7 ± 1.56 d 16.7 ± 2.17 c 19.7 ± 1.16 c
Abbreviations and description as in Table 1.
Plants 2021, 10, 730 6 of 13

The amount of reducing sugar in excreted urine was negligible (in milligrams) in
normal control and experimental rats (Table 5). The urine samples of hyperglycemic control
rats showed large quantity of reducing sugar and decreased considerably in rats fed with
diet containing skin, flesh and whole fruit of bitter melon. The most potent dose was
300 mg/kg body weight of the whole fruit of bitter melon in reducing glycosuria condition
followed by flesh and skin.

Table 5. Impact of bitter melon supplementation on reducing sugar in urine.

Reducing Sugar in Urine (G)

Groups 28th Day 56th Day
Normal Rats Hyperglycemic Rats Normal Rats Hyperglycemic Rats
Control 0.02 ± 0.00 a 2.91 ± 0.32 a 0.02 ± 0.01 a 4.32 ± 0.75 a
SF-L 0.03 ± 0.02 a 2.16 ± 0.11 b 0.03 ± 0.02 a 2.18 ± 0.42 b
SF-H 0.02 ± 0.01 a 1.86 ± 0.21 c 0.02 ± 0.01 a 1.96 ± 0.38 b
FF-L 0.05 ± 0.02 a 1.99 ± 0.36 c 0.04 ± 0.02 a 2.02 ± 0.33 b
FF-H 0.02 ± 0.00 a 1.58 ± 0.58 c 0.02 ± 0.01 a 1.74 ± 0.27 c
WF-L 0.02 ± 0.01 a 1.87 ± 0.13 c 0.03 ± 0.00 a 1.89 ± 0.14 b
WF-H 0.03 ± 0.01 a 1.43 ± 0.19 d 0.02 ± 0.00 a 1.63 ± 0.36 c
Abbreviations and description as in Table 1.

3.5. Estimation of Kidney Weight

The assessment of renal hypertrophy was determined by observing the kidney weights
in relation to 100 g body weight (Table 6). Normal rats showed no abnormal increase in
kidney weight. During hyperglycemic conditions, increase in kidney weight is more
obvious in control rats specifying renal hypertrophy. Bitter melon dietary supplementation
significantly reduced this condition.

Table 6. Impact of bitter melon supplementation on kidney weight.

Kidney Weight (G) Kidney Weight (G)

in Normal Rats in Hyperglycemic Rats
Groups
Right Left Right Left
Control 0.69 ± 0.04 a 0.68 ± 0.01 a 0.89 ± 0.06 a 0.87 ± 0.03 a
SF-L 0.64 ± 0.03 b 0.63 ± 0.02 b 0.78 ± 0.03 b 0.78 ± 0.02 b
SF-H 0.63 ± 0.02 b 0.62 ± 0.01 b 0.76 ± 0.04 c 0.77 ± 0.02 b
FF-L 0.64 ± 0.02 b 0.63 ± 0.03 b 0.76 ± 0.03 c 0.75 ± 0.04 c
FF-H 0.62 ± 0.01 c 0.62 ± 0.04 b 0.74 ± 0.03 d 0.72 ± 0.03 d
WF-L 0.64 ± 0.03 b 0.62 ± 0.02 b 0.69 ± 0.02 e 0.70 ± 0.01 d,e
WF-H 0.62 ± 0.02 c 0.62 ± 0.02 b 0.68 ± 0.02 e 0.68 ± 0.02 e
Abbreviations and description as in Table 1.

3.6. Estimation of Glomerular Filtration Rate

Significantly higher glomerular filtration rate (Table 7) was noticed in hyperglycemic
control rats and regular bitter gourd consumption in these rats markedly influenced this
trait to normalization.
Plants 2021, 10, 730 7 of 13

Table 7. Impact of bitter melon supplementation on glomerular filtration rate of rats.

Glomerular Filtration Rate (mL/min)

Groups
Normal Rats Hyperglycemic Rats
Control 1.13 ± 0.13 a 3.32 ± 0.33 a
SF-L 1.06 ± 0.09 b 2.76 ± 0.27 b
SF-H 1.04 ± 0.11 b 2.70 ± 0.28 b
FF-L 1.04 ± 0.16 b 2.75 ± 0.22 b
FF-H 1.03 ± 0.07 b,c 2.63 ± 0.31 b
WF-L 1.04 ± 0.09 b 2.45 ± 0.21 c
WF-H 1.01 ± 0.14 c 2.33 ± 0.14 c
Abbreviations and description as in Table 1.

3.7. Estimation of Blood Glucose

In normal rats, due to feeding of bitter melon, blood sugar level was maintained in
experimental group of rats. In hyperglycemic rats, blood sugar level reduced significantly
in experimental groups. The maximum reduction was observed in rats fed with whole
fruit of bitter gourd (300 mg/kg body weight) in both 28th (Figure 2) and 56th (Figure 3)
day samples.

Figure 2. Percent decrease in glucose level after feeding bitter gourd on 28th day. Bars that do not share similar letters
denote statistical significance (p < 0.05).
Plants 2021, 10, 730 8 of 13

Figure 3. Percent decrease in glucose level after feeding bitter gourd on 56th day. Bars that do not share similar letters
denote statistical significance (p < 0.05).

3.8. Estimation of Insulin Concentration

Skin, flesh and whole fruit of bitter melon in diet induced a significant increase in
insulin release from the pancreas of diabetic rats at both low (150 mg/kg body weight) and
high (300 mg/kg body weight) concentrations in a dose dependent manner as compared
to control values in normal and hyperglycemic rats. Skin fed rats showed a lesser effect
on insulin release compared to flesh and whole fruit. Treatments of hyperglycemic rats
with bitter melon for 4 and 8 weeks resulted in significant increase in insulin production
(Figures 4 and 5). This section may be divided by subheadings. It should provide a concise
and precise description of the experimental results and their interpretation, as well as the
experimental conclusions that can be drawn.
Plants 2021, 10, 730 9 of 13

Figure 4. Percent increase in insulin level after feeding bitter gourd on 28th day. Bars that do not share similar letters denote
statistical significance (p < 0.05).

Figure 5. Percent increase in insulin level after feeding bitter gourd on 56th day. Bars that do not share similar letters denote
statistical significance (p < 0.05).
Plants 2021, 10, 730 10 of 13

4. Discussion
There is an erratic approach in the literature regarding effectiveness of bitter melon
on glucose control to make a decisive conclusion. The present, detailed study highlighted
the effectiveness of skin, flesh and whole fruit containing seed as a dietary approach
against development of diabetes and its treatment. The present results clearly revealed
that bitter melon given at 300 mg/kg body weight improved the diabetic status to a
reasonable extent. Bitter melon possessed high concentration of hypoglycemic agents like
charantin and vicine, which are the mainstays in treatment of diabetes. Charantin and
vicine contents were first observed in skin, flesh and whole fruit containing seeds before
bio-evaluation. Pitipanapong et al. [15], Ham and Wang [23] and Desai et al. [24] reported
variable concentration of charantin in this plant. Similarly, a higher amount of vicine was
assessed in seeds than fruit and leaves by Zhang et al. [16]. The skin, flesh and whole fruit
of bitter melon with known quantities of charantin and vicine were administered in daily
diet of experimental rats.
Improvements in diabetic conditions were observed after giving bitter melon. The in-
crease in water intake in experimental groups of normal rats is due to increase in metabolic
rate and fatty acid metabolism after incorporation of bitter melon in diet. The excessive
water consumption by hyperglycemic rats is primarily associated with pre-diabetic state
and characteristic sign of onset of diabetes. This notion is favored by the findings of Parmar
et al. [25], who reported high intake of water in hyperglycemic rats. In diabetic rats, intake
of water gradually reduced after giving bitter melon [25].
Feed intake reduced to certain degree due to incorporation of bitter melon in diet.
Chen et al. [26], Reyes et al. [27] and Huang et al. [28] also observed lesser feed intake
in rats after adding bitter melon, but this impact is trivial to be considered or even in
some cases have no impact on feed intake [29]. The gradual increase in body weight was
observed in normal control, whereas that increase was lesser in diabetic control rats. The
other diabetic groups of rat that fed on bitter melon, improvement in body weight was
observed [30]. Bitter melon inclusions in the diet prevent the polyuria condition associated
with diabetes. In hyperglycemic rats, urine sugar was very high and in agreement with
previous studies [31]. Dietary supplementation of bitter melon minimizes sugar excretion
in urine during diabetes. Increase in kidney size and malfunctioning is generally associated
with diabetes. This increase is mainly due to increase in diameter and length of capillaries.
In current study, bitter gourd supplementation has resulted in partial but significant
decrease in ratio of kidney to body weight. Increase in glomerular filtration rate in hyper-
functional kidney mostly occurs during the initial diabetic stage [32] and metabolic control
for a long period is helpful in reducing kidney filtration during diabetes [33]. In our
study, the glomerular filtration rate increased considerably in hyperglycemic control. The
feeding of bitter melon to hyperglycemic rats showed significant reduction in glomerular
filtration rate. The hypoglycemic ability of bitter gourd is discussed in different scientific
explorations, advocating its use in various forms. The results regarding reduction in
blood glucose level by consuming bitter melon are in accordance with the findings of Jafri
et al. [30]. In their study, hyperglycemic rats showed a substantial decrease in glucose when
fed with bitter melon powder. In another study, acetone extract of whole bitter melon fruit
powder was used in doses of 25, 50 and 75 mg/100 g body weight and was observed to have
significant blood glucose lowering ability (13.30 to 50%) in alloxan diabetic albino rats [34].
On the other hand, some studies reported contradictory results of bitter melon intake with
no positive effect in lowering blood glucose level and diabetic conditions [12,13].
The hypoglycemic ability of bitter melon is due to presence of bioactive molecules that
play a pivotal role in many physiological, pharmacological and biochemical processes [35].
The possible factors involved in reducing blood glucose level by bitter melon consumption
include skeletal and peripheral muscles stimulation to enhance glucose utilization [36,37],
preventing uptake of glucose from intestine [38–40], maintaining enzymatic activities re-
lated to glucose metabolism [41], restoring stability of pancreatic β-cells and increasing
their functionality [42]. Bitter melon contains charantin and vicine, which are collectively
Plants 2021, 10, 730 11 of 13

found more effective in diabetes management as charantin is a typical cucurbitane-type

triterpenoid in M. charantia and is a potential substance with antidiabetic properties. Cha-
rantin could be used to treat diabetes and can potentially replace treatment. It is a mixture
of two compounds, namely, sitosteryl glucoside and stigmasteryl glucoside [43].
In the present study, bitter melon supplemented feed had a positive effect on pancreatic
cells to enhance insulin secretion and maintenance of serum insulin level [44,45]. This
increase in insulin production might be due to β-cells structural and functional stability
after consuming diet supplemented with bitter melon [34,43] or increasing the number
of β-cells in Islets of Langerhans [44]. According to Xiang et al. [46], certain chemical
constituents in bitter melon operated as a growth factor for pancreatic β-cells. The presence
of bioactive moieties that act as hypoglycemic agents including charantin and vicine are
spontaneously involved in insulin production or possess insulin-like activity. The chemical
constituents present in bitter melon help in minimizing oxidative damage by neutralization
of free radicals activity and performing prompt actions to control the death of β-cells. From
the above discussion, it is deduced that all parts of fruits of bitter melon are significantly
effectual against diabetes and 300 mg/kg body weight of whole fruit was the most potent
dose and can be preferred for use in hyperglycemic conditions and diabetes prophylaxis
and treatment.

5. Conclusions
It is concluded that charantin is present in a large quantity in flesh parts and vicine
is mainly concentrated in the whole fruit containing seeds. Current research intervention
suggested that individual phytochemical like charantin or vicine is less effective in diabetes
management. The complex interaction of these hypoglycemic agents of bitter melon can
play a more operative role in delaying the pathogenesis of diabetes mellitus. However, the
clinical studies following the double blind randomized controlled trials on human subjects
must be conducted to warrant their use in humans.

Author Contributions: Conceptualization, M. and F.S.; investigation, M.; writing—original draft

preparation, M.T.S.; S.A., and R.A., writing—review and editing, N.B.; R.M. and A.R. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Higher Education Commission, Islamabad, Pakistan, grant
number 2AV2–124.
Institutional Review Board Statement: Central Government Ethical Committee Guidelines were
strictly followed during the course of study (Approval no. FSD-16/M-234).
Informed Consent Statement: Not applicable.
Data Availability Statement: All the data produced here is available and can produced when required.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Grover, J.K.; Yadav, S.; Vats, V. Medicinal plants of India with anti-diabetic potential. J. Ethnopharmacol. 2002, 81, 81–100. [CrossRef]
2. Gupta, M.; Sharma, S.; Gautam, A.; Bhadauria, R. Momordica charantia Linn. (Karela): Nature’s silent healer. Int. J. Pharm. Sci. Rev.
Res. 2011, 11, 32–37.
3. Virdi, J.; Sivakami, S.; Shahani, S.; Suthar, A.C.; Banavalikar, M.M.; Biyani, M.K. Antihyperglycemic effects of three extracts from
Momordica charantia. J. Ethnopharmacol. 2003, 88, 107–111. [CrossRef]
4. Kulkarni, P.; Lohidasan, S.; Mahadik, K. Isolation, characterisation and investigation of in vitro antidiabetic and antioxidant
activity of phytoconstituents from fruit of Momordica charantia Linn. Nat. Prod. Res. 2019, 1–3. [CrossRef]
5. Kim, S.K.; Jung, J.; Jung, J.H.; Yoon, N.; Kang, S.S.; Roh, G.S.; Hahm, J.R. Hypoglycemic efficacy and safety of Momordica charantia
(bitter melon) in patients with type 2 diabetes mellitus. Complement. Ther. Med. 2020, 52, 102524. [CrossRef] [PubMed]
6. Cortez-Navarrete, M.; Martínez-Abundis, E.; Pérez-Rubio, K.G.; González-Ortiz, M.; Méndez-Del Villar, M. Momordica charantia
Administration Improves Insulin Secretion in Type 2 Diabetes Mellitus. J. Med. Food 2018, 21, 672–677. [CrossRef]
7. Thent, Z.C.; Das, S.; Zaidun, N.H. Emerging Trends On Drug Delivery Strategy of Momordica charantia against Diabetes and its
Complications. Curr. Drug Deliv. 2018, 15, 453–460. [CrossRef]
Plants 2021, 10, 730 12 of 13

8. Xu, X.; Shan, B.; Liao, C.H.; Xie, J.H.; Wen, P.W.; Shi, J.Y. Anti-diabetic properties of Momordica charantia L. polysaccharide in
alloxan-induced diabetic mice. Int. J. Biol. Macromol. 2015, 81, 538–543. [CrossRef]
9. Jia, S.; Shen, M.; Zhang, F.; Xie, J. Recent Advances in Momordica charantia: Functional Components and Biological Activities. Int.
J. Mol. Sci. 2017, 18, 2555. [CrossRef]
10. Tan, S.P.; Kha, T.C.; Parks, S.E.; Roach, P.D. Bitter melon (Momordica charantia L.) bioactive composition and health benefits: A
review. Food. Rev. Int. 2016, 32, 181–202. [CrossRef]
11. Ooi, C.P.; Yassin, Z.; Hamid, T.A. Momordica charantia for type 2 diabetes mellitus. Cochrane Database Syst. Rev. 2010. [CrossRef]
12. Kasbia, G.S.; Arnason, J.T.; Imbeault, P. No effect of acute, single dose oral administration of Momordica charantia Linn., on
glycemia, energy expenditure and appetite: A pilot study in non-diabetic overweight men. J. Ethnopharmacol. 2009, 126, 127–133.
[CrossRef] [PubMed]
13. Yin, R.V.; Lee, N.C.; Hirpara, H.; Phung, O.J. The effect of bitter melon (Mormordica charantia) in patients with diabetes mellitus: A
systematic review and meta-analysis. Nutr. Diabetes 2014, 4, e145. [CrossRef]
14. Peter, E.L.; Kasali, F.M.; Deyno, S.; Mtewa, A.; Nagendrappa, P.B.; Tolo, C.U.; Ogwang, P.E.; Sesaazi, D. Momordica charantia L.
lowers elevated glycaemia in type 2 diabetes mellitus patients: Systematic review and meta-analysis. J. Ethnopharmacol. 2019, 231,
311–324. [CrossRef]
15. Pitipanapong, J.; Chitprasert, S.; Goto, M.; Jiratchariyakul, W.; Sasaki, M.; Shotipruk, A. New approach for extraction of charantin
from Momordica charantia with pressurized liquid extraction. Sep. Purif. Technol. 2007, 52, 416–422. [CrossRef]
16. Zhang, H.; Wang, Y.; Zhang, X.; Liu, M.; Hu, Z. Analysis of vicine in bitter melon with high performance liquid chromatography.
Anal. Lett. 2003, 36, 1597–1605. [CrossRef]
17. Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. [CrossRef]
18. Owen, J.A.; Iggo, B.; Scandrett, F.J.; Stewart, C.P. The determination of creatinine in plasma or serum, and in urine; a critical
examination. Biochem. J. 1954, 58, 426–437. [CrossRef] [PubMed]
19. Yokozawa, T.; Chung, H.Y.; He, L.Q.; Oura, H. Effectiveness of green tea tannin on rats with chronic renal failure. Biosci. Biotechnol.
Biochem. 1996, 60, 1000–1005. [CrossRef]
20. Katz, A.; Nambi, S.S.; Mather, K.; Baron, A.D.; Follmann, D.A.; Sullivan, G.; Quon, M.J. Quantitative insulin sensitivity check index:
A simple, accurate method for assessing insulin sensitivity in humans. J. Clin. Endocrinol. Metab. 2000, 85, 2402–2410. [CrossRef]
21. Ahn, J.; Choi, W.; Kim, S.; Ha, T. Anti-diabetic effect of watermelon (Citrullus vulgaris Schrad) on Streptozotocin-induced diabetic
mice. Food Sci. Biotechnol. 2011, 20, 251–254. [CrossRef]
22. Steel, R.G.; Torrie, J.H. Principles and Procedures of Statistics: A Biometrical Approach; McGraw-Hill: New York, NY, USA, 1986.
23. Han, C.; Wang, J. Optimization of conditions for charantin extraction in PEG/Salt aqueous two-phase systems using response
surface methodology. Open Complemen. Med. J. 2009, 1, 46–50. [CrossRef]
24. Desai, S.; Tatke, P.; Mane, T.; Gabhe, S. Isolation, characterization and quantitative HPLC-DAD analysis of components of
charantin from fruits of Momordica charantia. Food Chem. 2020, 345, 128717. [CrossRef]
25. Parmar, K.; Patel, S.; Patel, J.; Patel, B.; Patel, M. Effects of bittergourd (Momordica charantia) fruit juice on glucose tolerance and
lipid profile in type-II diabetic rats. Int. J. Drug Dev. Res. 2011, 3, 139–146.
26. Chen, Q.; Chan, L.L.; Li, E.T. Bitter melon (Momordica charantia) reduces adiposity, lowers serum insulin and normalizes glucose
tolerance in rats fed a high fat diet. J. Nutr. 2003, 133, 1088–1093. [CrossRef] [PubMed]
27. Reyes, B.A.; Bautista, N.D.; Tanquilut, N.C.; Anunciado, R.V.; Leung, A.B.; Sanchez, G.C.; Magtoto, R.L.; Castronuevo, P.;
Tsukamura, H.; Maeda, K.I. Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous
cyclicity of alloxan-induced diabetic rats. J. Ethnopharmacol. 2006, 105, 196–200. [CrossRef]
28. Huang, H.L.; Hong, Y.W.; Wong, Y.H.; Chen, Y.N.; Chyuan, J.H.; Huang, C.J.; Chao, P.M. Bitter melon (Momordica charantia L.)
inhibits adipocyte hypertrophy and down regulates lipogenic gene expression in adipose tissue of diet-induced obese rats. Br. J.
Nutr. 2008, 99, 230–239. [CrossRef]
29. Klomann, S.D.; Mueller, A.S.; Pallauf, J.; Krawinkel, M.B. Antidiabetic effects of bitter gourd extracts in insulin-resistant db/db
mice. Br. J. Nutr. 2010, 104, 1613–1620. [CrossRef]
30. Jafri, S.; Ismail, M.; Zaman, G. Effect of Momordica charantia (karela) in alloxan induced diabetic rats. Pak. J. Sci. 2009, 61, 220.
31. Nandini, C.D.; Sambaiah, K.; Salimath, P.V. Dietary fibres ameliorate decreased synthesis of heparan sulphate in streptozotocin
induced diabetic rats. J. Nutr. Biochem. 2003, 14, 203–210. [CrossRef]
32. Christiansen, J.S.; Gammelgaard, J.; Frandsen, M.; Parving, H.H. Increased kidney size, glomerular filtration rate and renal
plasma flow in short-term insulin-dependent diabetics. Diabetologia 1981, 20, 451–456. [CrossRef]
33. Feldt-Rasmussen, B.; Hegedüs, L.; Mathiesen, E.; Deckert, T. Kidney volume in type 1 (insulin-dependent) diabetic patients with
normal or increased urinary albumin excretion: Effect of long-term improved metabolic control. Scand. J. Clin. Lab. Investig. 1991,
51, 31–36. [CrossRef]
34. Singh, N.; Gupta, M. Regeneration of beta cells in islets of Langerhans of pancreas of alloxan diabetic rats by acetone extract of
Momordica charantia (Linn.) (bitter gourd) fruits. Ind. J. Exp. Biol. 2007, 45, 1055–1062. [PubMed]
35. Bhushan, M.S.; Rao, C.; Ojha, S.; Vijayakumar, M.; Verma, A. An analytical review of plants for anti diabetic activity with their
phytoconstituent and mechanism of action. Int. J. Pharm. Sci. Res. 2010, 1, 29–46. [CrossRef]
36. Cummings, D.E.; Overduin, J. Gastrointestinal regulation of food intake. J. Clin. Investig. 2007, 117, 13–23. [CrossRef]
Plants 2021, 10, 730 13 of 13

37. Akhtar, N.; Khan, B.A.; Majid, A.; Khan, H.M.; Mahmood, T.; Gulfishan; Saeed, T. Pharmaceutical and biopharmaceutical
evaluation of extracts from different plant parts of indigenous origin for their hypoglycemic responses in rabbits. Acta Pol. Pharm.
2011, 68, 919–925. [PubMed]
38. Uebanso, T.; Arai, H.; Taketani, Y.; Fukaya, M.; Yamamoto, H.; Mizuno, A.; Uryu, K.; Hada, T.; Takeda, E. Extracts of Momordica
charantia suppress postprandial hyperglycemia in rats. J. Nutr. Sci. Vitaminol. 2007, 53, 482–488. [CrossRef] [PubMed]
39. Jeong, J.-H.; Lee, S.-H.; Hue, J.-J.; Lee, K.-N.; Nam, S.Y.; Yun, Y.W.; Jeong, S.-w.; Lee, Y.H.; Lee, B.J. Effect of bitter melon (Momordica
charantia) on anti-diabetic activity in C57BLI/6J db/db mice. Korean J. Vet. Res. 2008, 48, 327–336.
40. Abdollah, M.; Zuki, A.; Goh, Y.; Rezaeizadeh, A.; Noordin, M. The effects of Momordica charantia on the liver in streptozotocin-
induced diabetes in neonatal rats. Afr. J. Biotechnol. 2010, 9, 5004–5012.
41. Shibib, B.A.; Khan, L.A.; Rahman, R. Hypoglycaemic activity of Coccinia indica and Momordica charantia in diabetic rats: Depression
of the hepatic gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase and elevation of both liver and
red-cell shunt enzyme glucose-6-phosphate dehydrogenase. Biochem. J. 1993, 292, 267–270. [CrossRef]
42. Gadang, V.; Gilbert, W.; Hettiararchchy, N.; Horax, R.; Katwa, L.; Devareddy, L. Dietary bitter melon seed increases peroxisome
proliferator-activated receptor-γ gene expression in adipose tissue, down-regulates the nuclear factor-κB expression, and alleviates
the symptoms associated with metabolic syndrome. J. Med. Food 2011, 14, 86–93. [CrossRef]
43. Joseph, B.; Jini, D. Antidiabetic effects of Momordica charantia (bitter melon) and its medicinal potency. Asian Pac. J. Trop. Dis.
2013, 3, 93–102. [CrossRef]
44. Fernandes, N.P.; Lagishetty, C.V.; Panda, V.S.; Naik, S.R. An experimental evaluation of the antidiabetic and antilipidemic
properties of a standardized Momordica charantia fruit extract. BMC Complement. Altern. Med. 2007, 7, 29. [CrossRef]
45. Mohammady, I.; Elattar, S.; Mohammed, S.; Ewais, M. An evaluation of anti-diabetic and anti-lipidemic properties of Momordica
charantia (bitter melon) fruit extract in experimentally induced diabetes. Life Sci. J. 2012, 9, 363–374.
46. Xiang, L.; Huang, X.; Chen, L.; Rao, P.; Ke, L. The reparative effects of Momordica charantia Linn. extract on HIT-T15 pancreatic
beta-cells. Asia Pac. J. Clin. Nutr. 2007, 16 (Suppl. 1), 249–252. [PubMed]