Hypoglycemic and hypolipidemic effects of different parts and formulations of bitter gourd (Momordica Charantia).

BACKGROUND: Cardiovascular diseases and diabetes are responsible for large number of deaths throughout the globe. Bitter gourd has the potential to become a component of the diet or a dietary supplement for diabetic and pre-diabetic patients owing to the presence of insulin like molecules. Recent investigations have suggested that bitter gourd extracts may ameliorate high fat diet induced obesity and hyperlipidemia in animal models. Moreover, its supplements in food result in lowering weight gain and visceral fat mass.
METHODS: The current study was designed to investigate the nutraceutical potential of skin, flesh and whole fruit of bitter gourd cultivars against hyperglycemia and hyperlipidemia. For the purpose, various bitter gourd cultivars were procured from local market. Bio-evaluation studies were carried out on biochemical parameters using rodent experiment model.
RESULTS: From results, it was revealed that maximum reduction in blood glucose skin 1.06%, flesh 2.65%, whole fruit 4.29%, total cholesterol skin 6.60%, flesh 6.04%, whole fruit 6.70%, low density lipoprotein skin 5.55%, flesh 6.81%, whole fruit 6.60%, and triglycerides skin 0.04%, flesh 3.38%, whole fruit 2.02%, were observed. Moreover, insulin skin 2.14%, flesh 3.52%, whole fruit 2.73%, production was slightly enhanced with improved levels of high density lipoprotein in whole fruit of bitter gourd.
CONCLUSION: Overwhelmingly, it may be inferred here that bitter gourd holds the potential to significantly improve diabetic conditions and associated late complications with no ill effects on body organs.

Background

Incidence of chronic diseases is a rising apprehension globally with ever escalating reported cases of physiological syndromes. Besides, continued oxidative stress and oxidative damage lead to chronic inflammation and other physiological abnormalities [1]. Among these chronic diseases, diabetes and cardiovascular (CVD) are responsible for large number of deaths in the world [2]. Diabetes mellitus is an emerging global health perspective that is prevalent in more than 285 million people worldwide. It has been anticipated that diabetes affected people will be around 439 million by 2030. Furthermore, it has been projected that about 75% of the affected people will be from developing countries [3]. As far as the hyperlipidemia is concerned, it is lipid metabolism disorder and major risk factor for the development of cardiovascular aberration. It is prevalent among 7% of the adult population with an estimated 25 million people affected [4, 5].

Diabetes mellitus treatment often results in some late complex abnormalities including nephropathy, neuropathy and retinopathy etc. Due to adverse responses and rely upon low cost therapeutic ways, about 30% of diabetic patients use alternative therapeutic ways [6]. Various plant based remedial strategies are being utilized worldwide to cope with the chronic diseases and infections as preventive and curative measure. According to the statistics of WHO plant based medicines are being used by nearly 80% of the people for their primary healthcare worldwide. This analeptic potential of plant based medication is ratified to an array of valued phytochemicals present predominantly in their waste products. Among these, bitter gourd and its various components and formulations can be used due to their sugar lowering effects via biochemical, pharmacological and physiological modes [7, 8].

Bitter gourd (Momordica charantia L.) is a climbing perennial, tendril-bearing vine belongs to family cucurbitaceae. In the past, it was frequently used as antidote for diabetes, stomach pain, wounds, tumors, malaria, rheumatism, colic, inflammation, measles and fevers [9, 10]. Owing to the presence of insulin like molecules, bitter gourd has the potential to become a component of the diet or a dietary supplement for diabetic and pre-diabetic patients [11]. Recently, many researchers evaluated the role of bitter gourd in lowering blood glucose level [12, 13], cholesterol [13, 14] and visceral fat mass [15].

Recent researches also suggested that bitter gourd extracts may ameliorate high fat diet induced obesity and hyperlipidemia in animal models. Bitter gourd supplements in food result in lowering weight gain and visceral fat mass. This might be due to increase in the level of oxidation of fatty acid and ultimately reduction in weight and peritoneal fat deposition [15]. Most of these studies have been conducted with fruit pulps only. Very little information is available to compare the different parts and formulations of the plant in parallel experiments.

The present study was planned with the objectives to determine hypoglycemic and hypolipidemic effect of bitter gourd on normal, hyperglycemic and hyperlipidemic rats and to identify the part of the plant where the hypoglycemic and hypolipidemic principle is concentrated. Moreover, assessment of different formulations was also undertaken to find the suitable dose under these conditions.

Methods

Procurement of raw material

Fruits of bitter gourd were procured from Vegetable Research Section, Ayub Agriculture Research Institute, Faisalabad. These fruits were washed thoroughly under running tap water to remove adhered dirt, dust and other foreign debris.

Biological assay

To evaluate the hypoglycemic and hypolipidemic properties of skin, flesh and whole fruit powder of bitter gourd, an efficacy trial was planned. For the purpose, male Sprague Dawley rats were procured from the national institute of health, Islamabad. Initially, the rats were acclimatized by feeding basal diet for 1 week period. The environmental conditions were controlled throughout the trial like temperature (23 ± 2 °C) and relative humidity (55 ± 5%) along with 12 h light-dark period. At the initiation of study, some rats were dissected to establish the baseline trend. During efficacy trial, three types of studies were conducted independently by involving normal, hyperglycemic and hyperlipidemic. In Study I, rats were fed on normal diet whereas in study II and study III, high sucrose and high fat diets were administrated, respectively. In the animal modeling, seven groups of rats were formed in three different studies assigning 10 rats in each group. During the entire trial, bitter gourd formulations based feed was given to the respective groups.

Feed plans for experimental rats

For control group, experimental diet was prepared by using corn oil (10%), corn starch (66%), protein (10%), cellulose (10%), mineral (3%) and vitamin mixture (1%). In experimental groups, bitter gourd was added in the aforementioned diet (Table 1).

Table 1

Diet plan used in the studies

	Study I (Normal diet)							Study II (High sucrose diet)							Study III (High cholesterol diet)
Groups	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
Diet	D0	D1	D2	D3	D4	D5	D6	D0	D1	D2	D3	D4	D5	D6	D0	D1	D2	D3	D4	D5	D6

D0: Control

D1: Diet containing bitter gourd skin powder 150 mg / kg of body weight

D2: Diet containing bitter gourd skin powder 300 mg / kg of body weight

D3: Diet containing bitter gourd flesh powder 150 mg / kg of body weight

D4: Diet containing bitter gourd flesh powder 300 mg / kg of body weight

D5: Diet containing bitter gourd whole fruit powder 150 mg / kg of body weight

D6: Diet containing bitter gourd whole fruit powder 300 mg / kg of body weight

Feed and water intake

The gross feed intake of each group was calculated every day, excluding the spilled diet throughout the study period. The net water intake was also recorded on daily basis by measuring the difference in graduated bottles.

Body weight gain

The gain in body weight for each group of rats was monitored on weekly basis to estimate any suppressing effect of bitter gourd formulations.

Hypoglycemic perspectives

In each group, at respective intervals (4th and 8th week) glucose concentration was estimated by GOD-PAP method as described by Katz et al. [16], whereas, insulin level was estimated by following the instructions of Ahn et al. [17].

Serum lipid profile

Serum cholesterol level was determined using CHOD–PAP method [18], low density lipoproteins (LDL) by following the procedure of according to the guidelines of Kim et al. [18], high density lipoprotein (HDL) by HDL Cholesterol Precipitant method [19] and triglycerides level by liquid triglycerides (GPO–PAP) method as described by Kim et al. [18].

Liver functioning tests

For liver soundness, alanine transferase (ALT), aspartate transferase (AST) and alkaline phosphatase (ALP) were estimated [20]. The ALT and AST levels were measured by dinitrophenylhydrazene (DNPH) through Sigma Kits 58–50 and 59–50, respectively whereas; Alkaline Phosphatase-DGKC was used for ALP assessment.

Kidney functioning tests

The serum samples were analyzed for urea by GLDH-method, whilst creatinine by Jaffe procedure via commercial kits to evaluate the kidney functioning [21, 22].

Weight of body organ

After the trial period, the mice were dissected and body organs like heart, lungs, kidney, liver pancreas and spleen were collected and weighed.

Statistical analysis

The generated data was being applied by completely randomized design (CRD) and further subjected to statistical analysis using Statistical Package (Microsoft Excel 2010 and Statistix 8.1). Analysis of variance technique (ANOVA) was used to determine the level of significance [23].

Results

Neither feeding with different parts of bitter gourd fruit nor the variations in concentration of bitter gourd in diet influenced food intake during the experimental period. However, this trait affected significantly with time intervals (weeks) in all studies (Fig. 1). The non-substantial effect of addition of bitter gourd in diet on feed intake is in harmony with the findings of Klomann et al. [24]. The different parts and variation in amount of bitter gourd in diet imparted significant effect on water intake in all the studies. The maximum water intake was observed in control group than other groups fed with diet containing bitter gourd (Fig. 2). Shetty et al. [25] reported that water intake increase in diabetic group but the supplementation of bitter gourd in diet significantly decreases the consumption of water. The excessive water intake is a characteristic sign of diabetes. Parmar et al. [26] found that there was an increase in intake of water in diabetic rats as compared to rats of control groups.

Fig. 1

Feed intake (g/rat/day) in different studies with different diets

Fig. 2

Water intake (mL/rat/day) in different studies with different diets

Body weight

Body weight affected substantially with diets containing different parts and concentrations with the passage of time (Fig. 3). It was noted that weight is reduced slightly by giving bitter gourd in rats fed with normal diet while in sucrose and cholesterol fed rats, weight is increased considerably in experimental groups in comparison to their respective control groups. Shetty et al. [25] also found a marginal increase in body weight of diabetic rats fed with diet containing bitter gourd. Similar findings by Hossain et al. [27] also strengthen the current results that body weight of diabetic groups treated with bitter gourd was higher than the untreated diabetic group.

Fig. 3

Body weight gain (g/rat/day) in different studies with different diets

Effect on studied blood parameters

The results of various blood parameters depicted encouraging effect of utilization of bitter gourd in study I, study II and study III (Table 2, 3 and 4).

Table 2

Effects of bitter gourd on blood parameters in comparison to control in normal rats (Study I)

		Control diet (D0)	Skin		Flesh		Whole fruit
			150 mg/kg (D1)	300 mg/kg (D2)	150 mg/kg (D3)	300 mg/kg (D4)	150 mg/kg (D5)	300 mg/kg (D6)
Glucose (mg/dL)	0 day	88.23 ± 1.14abc	89.67 ± 2.18ab	88.97 ± 3.29abc	90.97 ± 2.17a	89.03 ± 1.85abc	89.17 ± 2.40abc	89.93 ± 2.49ab
	28 day	89.90 ± 2.55ab	88.73 ± 1.27abc	88.03 ± 1.36abc	89.63 ± 2.85ab	88.10 ± 1.97abc	88.00 ± 4.23abc	87.80 ± 2.08abc
	56 day	90.37 ± 1.22a	88.60 ± 2.25abc	88.03 ± 0.51abc	87.47 ± 1.21abc	86.73 ± 1.86bc	86.73 ± 1.65bc	86.07 ± 1.23c
Insulin (μIU/mL)	0 day	9.67 ± 0.32abc	9.37 ± 0.21abcde	9.53 ± 0.25abcde	9.57 ± 0.23abcde	9.70 ± 0.35ab	9.30 ± 0.10abcde	9.60 ± 0.36abcd
	28 day	9.17 ± 0.06bcde	9.17 ± 0.61bcde	9.20 ± 0.56bcde	9.50 ± 0.10abcde	9.30 ± 0.40abcde	9.00 ± 0.36e	9.37 ± 0.31abcde
	56 day	9.03 ± 0.31de	9.10 ± 0.36cde	9.33 ± 0.32abcde	9.07 ± 0.35de	9.37 ± 0.15abcde	9.13 ± 0.25bcde	9.87 ± 0.76a
Cholesterol (mg/dL)	0 day	78.63 ± 1.95bcd	79.70 ± 1.42bc	79.53 ± 1.88bc	79.60 ± 3.24bc	78.47 ± 1.48bcd	80.27 ± 0.95b	79.60 ± 2.34bc
	28 day	84.13 ± 0.67a	78.13 ± 0.70bcd	76.37 ± 0.87defg	77.27 ± 1.96cdef	75.17 ± 1.98efg	78.37 ± 2.12bcd	75.93 ± 1.70defg
	56 day	85.83 ± 0.35a	77.13 ± 1.19cdef	74.60 ± 0.98fg	75.17 ± 2.22efg	74.00 ± 0.95g	75.90 ± 2.56defg	74.67 ± 1.58fg
LDL (mg/dL)	0 day	29.87 ± 2.06ab	29.93 ± 2.28ab	29.63 ± 3.14ab	28.63 ± 1.21ab	29.80 ± 2.09ab	31.03 ± 2.56a	29.07 ± 2.15ab
	28 day	29.97 ± 2.39ab	29.50 ± 2.46ab	29.77 ± 1.40ab	29.33 ± 1.79ab	28.60 ± 1.47ab	27.60 ± 1.15bc	27.27 ± 1.30bc
	56 day	29.10 ± 1.51ab	29.00 ± 1.81ab	28.07 ± 3.07ab	28.77 ± 2.06ab	27.90 ± 1.92abc	27.97 ± 1.72ab	27.93 ± 1.55abc
HDL (mg/dL)	0 day	33.83 ± 1.80g	34.07 ± 0.71fg	35.23 ± 2.32efg	34.77 ± 1.71efg	35.03 ± 1.64efg	33.43 ± 1.17g	34.20 ± 1.93efg
	28 day	37.30 ± 2.29def	37.53 ± 1.27cde	39.07 ± 2.05bcd	40.87 ± 1.56abc	41.73 ± 1.75ab	40.53 ± 2.18abcd	41.13 ± 2.04ab
	56 day	38.80 ± 3.17bcd	40.23 ± 1.65abcd	41.90 ± 2.76ab	41.23 ± 2.90ab	42.63 ± 1.55a	42.73 ± 1.57a	42.77 ± 2.70a
Triglycerides (mg/dL)	0 day	68.43 ± 0.71	67.77 ± 2.29	67.60 ± 0.89	67.80 ± 2.00	68.37 ± 1.65	67.87 ± 1.19	67.37 ± 1.50
	28 day	68.20 ± 1.40	67.37 ± 1.43	66.60 ± 1.07	67.53 ± 0.87	67.50 ± 2.12	67.43 ± 3.31	67.03 ± 3.25
	56 day	68.33 ± 3.26	68.10 ± 1.37	67.63 ± 1.59	67.23 ± 1.71	66.13 ± 2.64	66.73 ± 2.84	66.03 ± 3.16
ALP (IU/L)	0 day	165.20 ± 3.73	165.77 ± 6.55	164.47 ± 3.84	164.57 ± 7.40	164.83 ± 3.45	164.83 ± 2.60	163.77 ± 7.40
	28 day	163.83 ± 2.76	162.73 ± 1.94	161.67 ± 2.31	163.10 ± 6.58	162.30 ± 1.61	163.27 ± 3.48	162.10 ± 2.76
	56 day	164.87 ± 3.35	163.17 ± 4.43	163.33 ± 2.08	163.47 ± 3.09	162.10 ± 2.31	163.13 ± 4.44	162. 43 ± 1.46
ALT (IU/L)	0 day	42.53 ± 1.90ab	42.90 ± 1.35a	41.37 ± 0.93abcde	42.00 ± 1.15abcd	42.07 ± 1.23abc	42.93 ± 2.38a	42.60 ± 1.21ab
	28 day	42.90 ± 1.37a	41.47 ± 0.49abcde	41.40 ± 0.87abcde	41.37 ± 0.93abcde	41.40 ± 2.18abcde	40.87 ± 0.85abcde	39.83 ± 0.81cde
	56 day	42.70 ± 2.23ab	41.37 ± 0.99abcde	41.27 ± 0.91abcde	40.20 ± 1.06bcde	39.87 ± 0.38cde	39.60 ± 2.16de	39.37 ± 2.40e
AST (IU/L)	0 day	136.63 ± 2.87abc	137.40 ± 0.89abc	137.57 ± 1.70ab	136.37 ± 2.08abc	136.30 ± 1.35abc	136.30 ± 2.38abc	135.77 ± 1.42abc
	28 day	136.40 ± 2.26abc	135.43 ± 1.89abc	135.37 ± 0.85abc	135.20 ± 1.54abc	135.17 ± 0.40bc	135.20 ± 1.00abc	135.13 ± 1.07bc
	56 day	137.67 ± 1.20a	136.40 ± 0.56abc	135.13 ± 1.24bc	135.10 ± 1.54bc	135.08 ± 1.03c	135.10 ± 1.13bc	135.07 ± 1.95c
Serum creatinine (mg/dL)	0 day	0.70 ± 0.02	0.72 ± 0.02	0.70 ± 0.02	0.71 ± 0.02	0.70 ± 0.03	0.72 ± 0.02	0.71 ± 0.02
	28 day	0.73 ± 0.05	0.71 ± 0.02	0.70 ± 0.03	0.70 ± 0.02	0.71 ± 0.02	0.70 ± 0.04	0.69 ± 0.02
	56 day	0.74 ± 0.03	0.69 ± 0.01	0.69 ± 0.01	0.69 ± 0.02	0.68 ± 0.03	0.69 ± 0.01	0.69 ± 0.01
Serum urea (mg/dL)	0 day	26.47 ± 0.60abc	27.33 ± 1.00ab	27.63 ± 0.81a	26.10 ± 0.62bcd	26.57 ± 0.57abc	26.53 ± 0.87abc	27.53 ± 0.49a
	28 day	27.50 ± 1.06a	25.71 ± 0.45cde	25.71 ± 0.45cdef	24.94 ± 0.87defg	24.44 ± 0.55efg	25.04 ± 0.22defg	24.21 ± 0.86fghi
	56 day	27.57 ± 1.12a	24.67 ± 1.45efg	23.04 ± 0.82i	24.08 ± 0.78ghi	24.04 ± 0.70ghi	23.24 ± 0.41hi	23.00 ± 0.38i

Values are Mean ± SD of 3 independent determinations; different letters in a row represent significant differences (p < 0.05)

Table 3

Effects of bitter gourd on blood parameters in comparison to control in hyperglycemic rats (Study II)

		Control diet (D0)	Skin		Flesh		Whole fruit
			150 mg/kg (D1)	300 mg/kg (D2)	150 mg/kg (D3)	300 mg/kg (D4)	150 mg/kg (D5)	300 mg/kg (D6)
Glucose (mg/dL)	0 day	88.17 ± 0.99i	88.80 ± 1.61i	89.00 ± 3.00i	89.10 ± 1.41i	88.03 ± 0.91i	89.83 ± 2.27i	90.40 ± 1.93i
	28 day	113.80 ± 2.03c	102.00 ± 2.26def	100.33 ± 1.93efg	98.13 ± 1.91fgh	97.00 ± 1.35gh	97.50 ± 1.57gh	94.47 ± 3.70h
	56 day	142.93 ± 2.70a	117.83 ± 3.07b	112.23 ± 2.46c	110.80 ± 3.36c	104.53 ± 4.23d	103.57 ± 3.00de	97.70 ± 2.17gh
Insulin (μIU/mL)	0 day	9.40 ± 0.26g	9.47 ± 0.23g	9.60 ± 0.36g	9.63 ± 0.25g	9.83 ± 0.51g	9.23 ± 0.12g	9.50 ± 0.10g
	28 day	10.80 ± 0.46f	13.83 ± 0.76d	14.00 ± 0.44cd	13.90 ± 0.72d	14.50 ± 0.10bcd	14.40 ± 0.17bcd	14.97 ± 0.35ab
	56 day	12.23 ± 0.74e	14.77 ± 0.81abc	14.93 ± 0.75ab	14.80 ± 0.79abc	15.13 ± 0.49ab	15.10 ± 0.30ab	15.33 ± 0.25a
Cholesterol (mg/dL)	0 day	79.50 ± 1.13e	79.55 ± 0.92e	80.45 ± 1.77e	79.75 ± 0.64e	79.65 ± 0.78e	78.00 ± 1.84e	79.80 ± 2.12e
	28 day	98.27 ± 1.12c	91.87 ± 1.34d	90.50 ± 1.44d	90.40 ± 2.98d	89.87 ± 1.00d	90.63 ± 2.45d	90.20 ± 3.38d
	56 day	129.07 ± 1.25a	103.07 ± 1.83b	100.80 ± 1.30bc	99.50 ± 1.13bc	97.97 ± 1.77c	98.37 ± 1.52c	98.00 ± 3.22c
LDL (mg/dL)	0 day	29.53 ± 3.23hi	28.87 ± 1.37hi	29.30 ± 2.57hi	28.17 ± 1.45i	28.93 ± 2.66hi	28.70 ± 1.55hi	28.07 ± 1.46i
	28 day	38.93 ± 1.55f	33.63 ± 1.45g	32.10 ± 2.11gh	31.80 ± 1.95gh	29.77 ± 2.49hi	31.40 ± 3.02ghi	29.17 ± 1.85hi
	56 day	63.85 ± 2.47a	55.80 ± 1.87b	51.07 ± 1.77c	49.67 ± 2.40cd	46.33 ± 2.80de	49.57 ± 1.55cd	45.63 ± 2.51e
HDL (mg/dL)	0 day	34.93 ± 2.37de	35.20 ± 1.55de	34.90 ± 2.23de	35.10 ± 2.66de	34.50 ± 1.57de	34.77 ± 0.99de	33.53 ± 0.93e
	28 day	35.93 ± 2.27de	37.23 ± 0.95d	41.73 ± 2.24c	41.60 ± 1.51c	41.73 ± 1.75c	40.87 ± 1.99c	42.00 ± 1.90c
	56 day	35.97 ± 2.25de	42.23 ± 1.19c	43.23 ± 1.17bc	42.90 ± 1.61bc	43.30 ± 0.98ab	45.63 ± 2.80ab	47.43 ± 1.12a
Triglycerides (mg/dL)	0 day	67.43 ± 0.76g	68.30 ± 1.21g	68.40 ± 2.11g	68.03 ± 2.31g	68.13 ± 1.98g	68.93 ± 1.32g	67.53 ± 0.78g
	28 day	81.43 ± 2.70de	78.37 ± 1.56ef	77.37 ± 2.43f	77.33 ± 1.53f	76.53 ± 2.15f	77.30 ± 1.70f	76.30 ± 2.80f
	56 day	92.87 ± 3.80a	87.00 ± 2.66b	85.43 ± 1.01bc	85.53 ± 1.60bc	85.10 ± 1.64bc	84.90 ± 2.76bc	82.37 ± 2.44cd
ALP (IU/L)	0 day	165.33 ± 4.52e	164.10 ± 5.85e	164.90 ± 1.51e	164.53 ± 2.01e	165.63 ± 4.87e	164.20 ± 3.28e	164.73 ± 3.54e
	28 day	193.43 ± 2.54b	189.80 ± 6.12bcd	187.47 ± 4.66bcd	185.63 ± 4.57cd	184.33 ± 2.06d	184.50 ± 3.68d	184.33 ± 5.66d
	56 day	203.40 ± 2.46a	193.67 ± 3.91b	193.60 ± 3.05b	191.53 ± 3.51bc	191.20 ± 2.65bc	191.47 ± 4.28bc	187.83 ± 1.89bcd
ALT (IU/L)	0 day	42.63 ± 0.90bcd	42.20 ± 1.31bcd	42.83 ± 1.75bcd	41.17 ± 1.08d	42.17 ± 0.90bcd	42.00 ± 2.39cd	41.03 ± 2.18d
	28 day	44.23 ± 1.88bc	42.47 ± 0.93bcd	42.23 ± 1.70bcd	42.13 ± 1.10bcd	41.30 ± 0.87d	41.93 ± 0.47cd	41.83 ± 2.76cd
	56 day	49.37 ± 0.70a	44.57 ± 0.83b	43.93 ± 1.56bc	43.87 ± 1.63bc	43.30 ± 1.11bcd	43.37 ± 1.16bcd	43.20 ± 1.42bcd
AST (IU/L)	0 day	135.27 ± 2.55g	136.90 ± 0.44g	136.97 ± 1.67g	136.70 ± 1.00g	137.10 ± 0.62g	136.13 ± 0.67g	136.43 ± 0.93g
	28 day	146.67 ± 2.29b	142.57 ± 1.96cdef	142.37 ± 2.06ef	142.50 ± 1.64def	142.27 ± 0.90ef	142.07 ± 1.12f	141.90 ± 0.85f
	56 day	151.93 ± 2.10a	145.40 ± 1.35b	145.13 ± 1.24b	145.20 ± 1.54b	144.83 ± 1.42bcd	145.00 ± 1.41bc	144.60 ± 1.31bcde
Serum creatinine (mg/dL)	0 day	0.74 ± 0.04de	0.74 ± 0.05de	0.73 ± 0.01e	0.72 ± 0.02e	0.74 ± 0.01de	0.73 ± 0.03e	0.73 ± 0.02e
	28 day	0.91 ± 0.03a	0.87 ± 0.02b	0.86 ± 0.02b	0.86 ± 0.02b	0.85 ± 0.01b	0.85 ± 0.01b	0.84 ± 0.01b
	56 day	0.91 ± 0.03a	0.80 ± 0.02c	0.79 ± 0.03c	0.79 ± 0.03c	0.78 ± 0.02c	0.78 ± 0.02c	0.77 ± 0.01cd
Serum urea (mg/dL)	0 day	27.21 ± 1.03bcd	27.31 ± 0.75bcd	27.90 ± 2.11bcd	27.20 ± 0.40bcd	27.80 ± 1.49bcd	25.90 ± 1.44d	27.57 ± 1.01bcd
	28 day	30.90 ± 1.56a	28.67 ± 0.87b	28.30 ± 1.40bc	27.60 ± 1.93bcd	27.43 ± 1.10bcd	26.93 ± 0.81bcd	26.67 ± 1.03bcd
	56 day	32.48 ± 1.13a	27.75 ± 0.90bcd	27.47 ± 1.14bcd	27.17 ± 2.05bcd	27.10 ± 1.51bcd	26.73 ± 1.15bcd	26.21 ± 1.58cd

Values are Mean ± SD of 3 independent determinations; different letters in a row represent significant differences (p < 0.05)

Table 4

Effects of bitter gourd on blood parameters in comparison to control in hyperlipidemic rats (Study III)

		Control diet (D0)	Skin		Flesh		Whole fruit
			150 mg/kg (D1)	300 mg/kg (D2)	150 mg/kg (D3)	300 mg/kg (D4)	150 mg/kg (D5)	300 mg/kg (D6)
Glucose (mg/dL)	0 day	87.50 ± 0.80h	87.97 ± 0.67h	88.63 ± 1.38h	89.50 ± 1.57h	88.80 ± 1.31h	88.50 ± 2.08h	87.83 ± 2.00h
	28 day	98.43 ± 1.21bcde	96.77 ± 1.25def	96.40 ± 1.23def	96.10 ± 2.13defg	95.40 ± 2.39fg	95.70 ± 1.84efg	94.63 ± 1.72fg
	56 day	106.33 ± 2.52a	100.33 ± 1.53b	99.87 ± 1.86bc	98.57 ± 1.03bcd	97.30 ± 2.56cdef	95.27 ± 1.99fg	93.50 ± 0.60g
Insulin (μIU/mL)	0 day	9.53 ± 0.15h	9.57 ± 0.12h	9.66 ± 0.23h	9.36 ± 0.27h	9.68 ± 0.91h	9.86 ± 0.19h	9.57 ± 0.74h
	28 day	10.67 ± 0.75g	10.87 ± 0.74g	11.07 ± 0.67fg	10.77 ± 0.46g	11.27 ± 0.32efg	11.20 ± 0.46efg	11.83 ± 0.64cdef
	56 day	11.37 ± 0.96defg	12.00 ± 0.62cde	12.93 ± 0.38ab	12.17 ± 0.32bcd	12.97 ± 0.67ab	12.57 ± 0.47abc	13.10 ± 0.20a
Cholesterol (mg/dL)	0 day	80.77 ± 3.15h	80.47 ± 3.72h	79.90 ± 2.01h	79.47 ± 3.07h	80.47 ± 2.34h	79.53 ± 3.43h	78.67 ± 3.06h
	28 day	128.00 ± 4.36bc	114.87 ± 3.82ef	109.33 ± 2.42g	110.67 ± 1.31fg	107.40 ± 2.26g	107.33 ± 1.53g	109.33 ± 4.86g
	56 day	160.67 ± 4.16a	129.33 ± 2.52b	125.30 ± 3.08bc	123.37 ± 3.28cd	116.70 ± 2.54e	124.70 ± 2.86bc	118.83 ± 1.91de
LDL (mg/dL)	0 day	29.73 ± 3.87i	28.63 ± 1.08i	29.03 ± 2.75i	28.67 ± 2.15i	27.90 ± 0.89i	28.30 ± 2.10i	28.73 ± 1.50i
	28 day	49.40 ± 1.35f	40.57 ± 1.63g	40.17 ± 1.27g	39.57 ± 1.53 gh	39.00 ± 0.26gh	37.37 ± 2.30gh	36.13 ± 1.63h
	56 day	74.33 ± 3.31a	63.20 ± 3.51b	60.23 ± 3.40bc	57.00 ± 1.28cd	54.67 ± 2.40de	54.57 ± 1.55de	53.20 ± 2.54e
HDL (mg/dL)	0 day	34.57 ± 2.12h	34.27 ± 1.06h	34.83 ± 2.80h	32.23 ± 1.68h	34.30 ± 1.45h	34.83 ± 1.75h	33.40 ± 1.35h
	28 day	35.93 ± 1.46gh	39.13 ± 1.24fg	42.40 ± 3.20ef	43.07 ± 3.82def	43.33 ± 2.69de	45.73 ± 2.74bcde	47.70 ± 1.31abc
	56 day	35.97 ± 1.72gh	44.90 ± 2.41cde	47.03 ± 2.85bcd	46.00 ± 2.69bcde	49.30 ± 1.97ab	49.20 ± 4.68ab	51.63 ± 4.07a
Triglycerides (mg/dL)	0 day	67.70 ± 2.12e	68.27 ± 1.06e	67.63 ± 2.80e	68.80 ± 1.68e	69.17 ± 1.45e	68.70 ± 1.75e	67.37 ± 1.35e
	28 day	96.67 ± 4.09c	81.67 ± 2.18d	80.10 ± 2.46d	81.03 ± 3.10d	79.43 ± 1.63d	78.57 ± 3.19d	77.03 ± 2.75d
	56 day	124.67 ± 3.06a	103.57 ± 3.37b	99.70 ± 1.25bc	99.27 ± 4.34bc	99.23 ± 5.19bc	98.67 ± 2.34c	98.23 ± 4.47c
ALP (IU/L)	0 day	163.93 ± 3.04f	162.27 ± 4.73f	165.07 ± 3.21f	164.93 ± 4.46f	165.37 ± 3.75f	163.87 ± 3.21f	164.40 ± 1.66f
	28 day	196.13 ± 1.70b	190.37 ± 5.93bcde	187.53 ± 5.15de	186.43 ± 3.57de	187.00 ± 3.65de	186.17 ± 2.27de	185.00 ± 3.60e
	56 day	210.43 ± 4.04a	194.67 ± 2.28bc	194.57 ± 3.44bc	192.03 ± 4.24bcd	191.53 ± 3.21bcd	191.30 ± 4.55bcd	189.03 ± 3.48cde
ALT (IU/L)	0 day	42.27 ± 1.25fg	42.37 ± 0.80fg	41.17 ± 0.40g	42.03 ± 1.46fg	41.20 ± 1.49g	42.00 ± 1.55fg	41.43 ± 1.33g
	28 day	47.80 ± 1.23bc	43.40 ± 0.56efg	43.30 ± 1.92efg	43.40 ± 0.82efg	43.03 ± 1.26fg	43.27 ± 1.04fg	43.07 ± 1.00fg
	56 day	53.77 ± 1.76a	48.77 ± 1.50b	47.27 ± 0.91bc	46.50 ± 3.18bcd	45.87 ± 1.53cde	44.57 ± 3.10def	44.17 ± 1.70def
AST (IU/L)	0 day	136.03 ± 1.22e	137.00 ± 0.78e	136.63 ± 0.76e	136.47 ± 0.93e	136.43 ± 0.85e	137.23 ± 0.55e	135.73 ± 1.11e
	28 day	156.00 ± 1.73c	148.07 ± 0.71d	147.70 ± 2.02d	148.07 ± 1.12d	147.27 ± 1.10d	147.47 ± 1.52d	147.83 ± 1.05d
	56 day	172.67 ± 2.11a	162.07 ± 1.99b	161.67 ± 1.46b	161.87 ± 2.03b	161.50 ± 1.47b	161.13 ± 1.79b	161.53 ± 1.70b
Serum creatinine (mg/dL)	0 day	0.73 ± 0.02f	0.75 ± 0.02f	0.74 ± 0.01f	0.73 ± 0.02f	0.75 ± 0.01f	0.72 ± 0.02f	0.73 ± 0.02f
	28 day	0.98 ± 0.98b	0.91 ± 0.91c	0.89 ± 0.89c	0.91 ± 0.91c	0.89 ± 0.89c	0.89 ± 0.89c	0.88 ± 0.88cd
	56 day	1.17 ± 0.05a	0.84 ± 0.03de	0.83 ± 0.04e	0.84 ± 0.02de	0.82 ± 0.02e	0.82 ± 0.01e	0.81 ± 0.01e
Serum urea (mg/dL)	0 day	26.23 ± 0.29f	26.73 ± 0.84ef	27.07 ± 1.25ef	26.67 ± 0.68ef	27.16 ± 1.01ef	27.25 ± 0.27ef	28.05 ± 0.68e
	28 day	35.07 ± 2.65b	32.83 ± 1.63cd	32.53 ± 0.83cd	32.43 ± 1.20cd	32.29 ± 0.52cd	31.57 ± 0.96d	31.40 ± 0.89d
	56 day	37.47 ± 1.12a	34.00 ± 0.75bc	33.13 ± 0.29cd	33.07 ± 1.31cd	32.50 ± 0.52cd	33.00 ± 0.61cd	32.23 ± 1.15d

Values are Mean ± SD of 3 independent determinations; different letters in a row represent significant differences (p < 0.05)

Glucose and insulin

The study intervals led to an enhancement in the glucose level of control groups. However, bitter gourd enriched diets substantially suppressed this trait with passage of time and the lowest glucose concentration was observed in groups of rats fed with 300 mg/kg body weight of whole fruit of bitter gourd in all the studies (Table 2, 3 and 4). Means concerning insulin were 9.03 ± 0.31, 9.10 ± 0.36, 9.33 ± 0.32, 9.07 ± 0.35, 9.37 ± 0.15, 9.13 ± 0.25 and 9.87 ± 0.76 μIU/mL in study I, 12.23 ± 0.74, 14.77 ± 0.81, 14.93 ± 0.75, 14.80 ± 0.79, 15.13 ± 0.49, 15.10 ± 0.30 and 15.33 ± 0.25 μIU/mL in study II, 11.37 ± 0.96, 12.00 ± 0.62, 12.93 ± 0.38, 12.17 ± 0.32, 12.97 ± 0.67, 12.57 ± 0.47 and 13.10 ± 0.20 μIU/mL in study III for D0, D1, D2, D3, D4, D5 and D6 respectively which clearly indicated that increase in amount of bitter gourd in diet has resulted in production of more insulin.

Cholesterol

Means concerning cholesterol revealed that the highest cholesterol (85.83 ± 0.35 mg/dL) was in D0 that reduced to 77.13 ± 1.19 (D1), 75.90 ± 2.56 mg/dL (D5), 75.17 ± 2.22 mg/dL (D3), 74.67 ± 1.58 mg/dL (D6), 74.60 ± 0.98 mg/dL (D2), 74.00 ± 0.95 mg/dL (D4) in study I. The study interval of 0, 28, 56 day explicated an obvious decrease in cholesterol level from commencement till termination of this study in groups fed with diet containing bitter gourd powder. The cholesterol in diabetic control group D0 (Study II) was 129.07 ± 1.25 mg/dL that substantially reduced to 103.07 ± 1.83, 100.80 ± 1.30, 99.50 ± 1.13, 97.97 ± 1.77, 98.37 ± 1.52 and 98.00 ± 3.22 mg/dL in D1, D2, D3, D4, D5 and D6 respectively. In Hypercholesterolemic rats (Study III), enormous increase in cholesterol was observed in control group D0 (160.67 ± 4.16 mg/dL) while experimental groups fed with bitter gourd showed reduction in the level of cholesterol as 129.33 ± 2.52, 125.30 ± 3.08, 123.37 ± 3.28, 116.70 ± 2.54, 124.70 ± 2.86 and 118.83 ± 1.91 mg/dL in D1, D2, D3, D4, D5 and D6, respectively. The study interval of 0, 28, 56 day explicated an obvious decrease in cholesterol level from commencement till termination of the study in groups fed with diet containing bitter gourd powder.

LDL

LDL level was varied insignificantly from initiation to termination of trial (0, 28th & 56th day) in study I. In this study, maximum LDL level was observed in D0 as 29.10 ± 1.51 mg/dL that substantially reduced in D1 (29.00 ± 1.81 mg/dL) trailed by D3 (28.77 ± 2.06 mg/dL), D2 (28.07 ± 3.072 mg/dL), D5 (27.97 ± 1.72 mg/dL), D6 (27.93 ± 1.55 mg/dL) and D4 (27.90 ± 1.92 mg/dL). In study II, the recorded values showed a diminishing trend in LDL level with values of 55.80 ± 1.87, 51.07 ± 1.77, 49.67 ± 2.40, 46.33 ± 2.80, 49.57 ± 1.55, 45.63 ± 2.51 mg/dL in D1, D2, D3, D4, D5 and D6, respectively compared to control group D0 (63.85 ± 2.47 mg/dL). Similarly, the highest value of LDL was in control group (74.33 ± 3.31 mg/dL) followed by D1 (63.20 ± 3.51 mg/dL), D2 (60.23 ± 3.40 mg/dL), D3 (57.00 ± 1.28 mg/dL), D4 (54.67 ± 2.40 mg/dL), D5 (54.57 ± 1.55 mg/dL) and D6 (53.20 ± 2.54 mg/dL) indicated that bitter gourd powder in diet is helpful in lowering LDL level. The low density lipoproteins are the major carrier for cholesterol in blood [28]. Bitter gourd has the ability to reduce this ‘bad cholesterol’ from the blood. Temitope et al. [29] orally administered aqueous extract of bitter gourd at dose of 80, 100, 120, 140 mg/kg body weight for 14 days. Significant decline in LDL in experimental groups were observed compared to rats fed with normal diet.

HDL

HDL values increased after consumption of bitter gourd in diet. The study interval influenced significantly on the increase in HDL level and the highest increase was observed in D6 in all the studies. The higher amount of HDL in blood is considered valuable because it is found good for health and mostly designated as ‘good cholesterol’. The decline in HDL concentration in blood leads to wide ranging cardiovascular complications. Bitter gourd in diet is helpful in increasing the level of HDL in blood. Bano et al. [30] revealed that increased the serum HDL level rise up to 45% by administering aqueous extract of bitter gourd for 5 weeks. Temitope et al. [29] examined different doses of bitter gourd to analyze HDL level in blood and found diets containing 100 mg/kg and 140 mg/kg body weight as suitable dietary approaches and resulted in marked increase in the HDL level than the rest of the treatments.

Triglycerides

The values for triglycerides rise gradually in control group while reduction was observed by supplementation of bitter gourd in diet. Consistent with current results, Jayasooria et al. [31] determined the effect of bitter gourd on lipid metabolism and found significant reduction of 39.2 and 40.5% in hepatic triglycerides in cholesterol rich and cholesterol free diets, respectively. The present results also supported by the findings of Bano et al. [30] determined 20% decline in serum triglyceride level due to dietary intake of bitter gourd in diabetic rats. Hossain et al. [27] observed significant elevation in triglycerides level (47.02%) in diabetic control rat groups. However, bitter gourd at the rate of 250, 500, 750 mg reduced the triglyceride level to 30.30%, 33.84 and 37.43%, respectively. The findings indicated a decline of triglycerides in a dose dependent manner. They observed effect of diet containing bitter gourd on triglyceride level during study period of 90 days. They obtained blood samples at every 15 days interval and observed continuous reduction in triglycerides compared to control group. Recently,

Liver function test

The reduction in ALP, ALT and AST level was recorded after feeding of diet containing bitter gourd compared to control groups. Serum creatinine and urea concentration was also remained in normal ranges due to feeding of bitter gourd in diet. The current data is comparable to the earlier investigations of Hossain et al. [27] confirmed a reduction in serum ALP, ALT and AST of rats treated with bitter gourd extract. In another study noted that bitter gourd is helpful in reducing the amount of ALT and AST. Moreover, Sathishsekar and Subramamian [32] also reported hepato-protective role of bitter gourd. The present data was comparable with the findings of Nagy et al. [33] showing decline in serum creatinine and urea level after bitter gourd administration.

Effect on different organs

Means for weight of different organs (Table 5) depicted non-significant impact of diet on various organs except for kidney and liver weight that were found higher in diabetic control rat as compared to rats fed with bitter gourd supplemented food. Liver weight was also noted slightly higher in control groups of hypercholesterolemic rats. Platel et al. [34] confirms the current results showed bitter gourd had no adverse effect on major organs of the body. A considerable elevation in kidney weight of diabetic rats might be due to hyperplasia and hypertrophy of tubular and mesangial cells of the kidney [35]. Treating the diabetic rats with bitter gourd caused a decrease in the weight of kidney and liver up to normal ranges.

Table 5

Means for organ weight in different studies

Organs	Studies	Group of rats fed with different diets
		D0	D1	D2	D3	D4	D5	D6
Heart	Study I	0.95 ± 0.03	0.93 ± 0.02	0.93 ± 0.02	0.92 ± 0.02	0.91 ± 0.03	0.92 ± 0.02	0.90 ± 0.03
	Study II	0.95 ± 0.02	0.94 ± 0.01	0.93 ± 0.02	0.93 ± 0.01	0.92 ± 0.01	0.93 ± 0.03	0.92 ± 0.02
	Study III	0.95 ± 0.03	0.95 ± 0.01	0.93 ± 0.01	0.94 ± 0.02	0.93 ± 0.01	0.93 ± 0.03	0.93 ± 0.03
Lung	Study I	1.59 ± 0.06	1.60 ± 0.06	1.62 ± 0.09	1.57 ± 0.11	1.56 ± 0.07	1.60 ± 0.11	1.60 ± 0.02
	Study II	1.59 ± 0.03	1.62 ± 0.05	1.62 ± 0.04	1.58 ± 0.09	1.63 ± 0.03	1.58 ± 0.09	1.59 ± 0.06
	Study III	1.61 ± 0.08	1.63 ± 0.04	1.62 ± 0.07	1.60 ± 0.08	1.65 ± 0.04	1.61 ± 0.06	1.61 ± 0.03
kidney	Study I	1.94 ± 0.02	1.90 ± 0.03	1.88 ± 0.03	1.89 ± 0.07	1.87 ± 0.05	1.89 ± 0.07	1.88 ± 0.04
	Study II	2.01 ± 0.07	1.91 ± 0.04	1.87 ± 0.04	1.90 ± 0.06	1.88 ± 0.04	1.89 ± 0.03	1.87 ± 0.05
	Study III	1.96 ± 0.04	1.91 ± 0.04	1.89 ± 0.05	1.90 ± 0.06	1.88 ± 0.03	1.90 ± 0.03	1.90 ± 0.06
Liver	Study I	6.72 ± 0.27	6.60 ± 0.10	6.55 ± 0.22	6.59 ± 0.23	6.51 ± 0.20	6.56 ± 0.12	6.53 ± 0.19
	Study II	7.23 ± 0.37	6.69 ± 0.24	6.66 ± 0.12	6.69 ± 0.10	6.60 ± 0.14	6.63 ± 0.17	6.59 ± 0.24
	Study III	7.58 ± 0.21	7.04 ± 0.20	6.85 ± 0.16	6.96 ± 0.17	6.84 ± 0.09	6.93 ± 0.26	6.77 ± 0.07
Pancreas	Study I	2.02 ± 0.11	2.03 ± 0.20	2.05 ± 0.11	2.02 ± 0.03	2.02 ± 0.13	2.04 ± 0.06	2.03 ± 0.07
	Study II	1.87 ± 0.06	2.01 ± 0.09	2.01 ± 0.07	2.03 ± 0.04	2.04 ± 0.10	2.04 ± 0.05	2.05 ± 0.07
	Study III	1.95 ± 0.11	2.04 ± 0.10	2.02 ± 0.03	2.04 ± 0.04	2.01 ± 0.13	2.03 ± 0.04	2.02 ± 0.03
Spleen	Study I	0.49 ± 0.04	0.51 ± 0.06	0.49 ± 0.02	0.49 ± 0.01	0.48 ± 0.03	0.49 ± 0.05	0.48 ± 0.03
	Study II	0.41 ± 0.04	0.51 ± 0.04	0.50 ± 0.04	0.50 ± 0.05	0.49 ± 0.04	0.50 ± 0.01	0.48 ± 0.03
	Study III	0.48 ± 0.03	0.51 ± 0.05	0.51 ± 0.02	0.51 ± 0.06	0.50 ± 0.03	0.49 ± 0.05	0.49 ± 0.02

Discussions

The findings of Jafri et al. [36] are in harmony with the current study, they reported substantial decrease in glucose of hyperglycemic rats after consuming bitter gourd powder. Singh and Gupta [37] also confirmed antihyperglycemic property of this plant in diabetic condition in both animals as well as in humans. Virdi et al. [38] also observed antihyperglycemic property of bitter gourd. In another study, Jayasooriya et al. [31] observed effects of bitter gourd powder in cholesterol free and cholesterol enriched diets fed rats and noted that there was a continuous decline in glucose level in groups of rat fed with cholesterol free diet while no significant influence was noted in rats fed with cholesterol enriched diets. Clouatre et al. [39] revealed reduction in blood glucose level by giving bitter gourd extracts at 50 mg/kg of body weight in normal rats.

The results for insulin in current study are in accordance with Mohammady et al. [40] noted significant increase in insulin in diabetic rats treated with bitter gourd as compared to diabetic control group. Similarly, Fernandes et al. [41] observed positive effect of bitter gourd extract on serum insulin level. The increase in insulin level in the diabetic rats after giving diet supplemented with bitter gourd might be due to recovery of beta cells of Langerhans [37, 41]. Other studies indicated that addition of bitter gourd in diet enhance the number of beta cells [40]. However, researchers reported that bitter gourd did not involve in restoring of these cells rather it enhance the activity of beta cells.

The current investigation has shown bitter gourd effectiveness against cholesterol synthesis which is in accordance with the work of Jayasooria et al. [31] who used freeze-dried powder of bitter gourd to note its effect on lipid parameters in rats fed with diet supplemented with and without cholesterol. They observed 32.0 and 22.4% decline in total cholesterol level in the absence and presence of dietary cholesterol, respectively. Abas et al. [42] revealed that total cholesterol was significant increase in diabetic rats compared to control. Consumption of bitter gourd for 28 days showed significant reduction in cholesterol compared to diabetic control group. In an experiment on Wistar rats, decrease in cholesterol was noticed after consuming bitter gourd fruit extract [41]. Similarly, Bano et al. [30] observed significant reduction (21%) in cholesterol after oral administration of aqueous extract of bitter gourd for a period of 5 weeks. Later, in an antidiabetic study by Wehash et al. (2012) on male Sprague-Dawley rats indicated lipid lowering effect of bitter gourd. Hossain et al. (2012) investigated antihyperglycemic and antihyperlipidemic effect of aqueous extract of bitter gourd at daily dose of 250, 500 and 750 mg/kg body weight. They observed 12.88, 14.44 and 17.21% reduction in serum cholesterol, respectively.

Wehash et al. [43] observed significant reduction in LDL level from 141.51 to 31.18 mg/dL in diabetic group fed with control diet and bitter gourd supplemented diet, respectively.another researcher noted increase in LDL in STZ induced diabetic control and decline in all the experimental groups at the end of 28 day study period. Similarly, Chaturvedi et al. [44] also found reduction in amount of low density lipoproteins in blood by administering bitter gourd extracts. The lowering of LDL by consuming bitter gourd in diet might be due to secretion of Apolipoprotein-B by the liver.

Conclusions

It is evident from the present research findings that conspicuous from the contemporary endeavor that among various parts of bitter gourd, whole fruit part in a dose of 300 mg/kg body weight found to be suitable in lowering blood glucose level, increasing serum insulin level, reduction in cholesterol, increase in HDL and minimize the amount of low density lipoprotein and triglycerides. Moreover, level of ALP, ALT, AST, serum creatinine and urea reduced to normal ranges by consumption of bitter gourd. Being veracious and concise, it is executed that bitter gourd be endowed with vivid approaching to improve the effect of oxidative stress, thus stopping the chain reactions implicated in the onset of chronic diseases. The outcomes of current project found bitter gourd most effective against obesity and chronic aberrations such as hyperglycemia and hyperlipidemia. It is vigorously suggested to devise diet based modules to use bitter gourd against lifestyle related disorders.