1. Introduction
Momordica charantia Linn. (Cucurbitaceae) is referred to as bitter melon or bitter gourd and has recently attracted considerable attention for its various physiological activities, such as its antitumor [
1–
3], anti-inflammatory [
4], antioxidant [
5,
6], antibacterial [
7,
8], hypoglycemic [
9–
11], hypocholesterolemic [
12], hypotriglyceridemic [
13], and immunostimulating activities [
14]. Previous investigations have shown that the fruits and leaves of
M. charantia had rich phenolics and exhibited a high antioxidant activity [
5]. Nowadays, it has been used as a traditional antidiabetic remedy in eastern countries and areas for many years [
11,
15]. Fresh bitter melon is also used as a nourishing food, as it contains: 93.8% water, 0.9% protein, 0.1% lipid, 3.3% dietary fiber, 20 kJ energy per 100 g, 0.6% ash, and a small quantity, 0.05%, of vitamin C [
16].
The antidiabetic evaluation of bitter melon has been well investigated in streptozocin- or alloxan-induced diabetic rats, mice and rabbits, high-fat diet-induced obesity mice, as well as in humans with type 2 diabetes [
9,
13,
17–
20]. The hypoglycemic potential components in bitter melon have been identified as glycosides, saponins, alkaloids, triterpenes, polysaccharides, proteins, and steroids [
14,
21]. Although several pure chemicals were isolated from bitter melon and applied for investigating their antidiabetic mechanisms, the mixture of these hypoglycemic chemicals such as saponins or charantins seemed to present a significantly higher bioactivity. For example, the hypoglycemic chemicals of bitter melon are proven as a mixture of steroidal saponins known as charantins and alkaloids [
21]. The antidiabetic mechanisms of bitter melon also have been proposed. Bitter melon has shown to stimulate glycogen storage by liver and insulin secretion by islets of Langerhans [
13,
22]. Bitter melon suppresses weight gain and has the potential to reduce adiposity [
23]. Moreover, bitter melon supplementation lowered serum and hepatic triglyceride in normal rats [
24]. Bitter melon may possess insulin-like properties, preserved pancreatic islet beta cells [
23,
25]. A recent study proved that bitter melon could upregulate the significance of glucose transporter 4 (GLUT-4), peroxisome proliferator-activated receptor γ (PPARγ) and phosphatidylinositol 3 kinase (PI3K) by augmenting the glucose uptake and homeostasis [
26]. Bitter melon can also improve insulin sensitivity by increasing insulin-stimulated insulin receptor substrate-1 (IRS1) tyrosine phosphorylation in high-fat diet-fed mice/rats [
27,
28]. Hence, the synergic effect of these bioactive components would possibly make the contribution. Therefore, the question is: “Do we need to evaluate the antidiabetic activity of bitter melon by using purified samples?”
Superfine grinding technology is a new technology and a useful tool for preparing superfine powder [
29]. Compared with other samples ground with traditional mechanical methods, superfine powder bears good physical properties like dispersibility and solubility. To date, the superfine grinding technology has also been applied in biotechnology and foodstuffs and shown a high potential for many other commercial applications [
30]. For example, the
Astragalus membranaceus powder obtained with superfine grinding had high water-holding capacity, high fluidity, high water solubility index and high protein solubility [
31]. Now, Chinese markets sell the bitter melon powder processed by hot drying and milling which might result in the inactivation of components in the samples. Nevertheless, lyophilized bitter melon had also previously been superfine-ground with its particle size less than 50 μm by our group. Similar to other foodstuffs, superfine grinding bitter melon powder after the lyophilization process retains the whole chemical compositions of fresh samples, such as proteins, polysaccharides, glycosides, saponins, alkaloids, and triterpenes, while its physical properties received significant changes after the grinding process. However, few references are presented for evaluating the antidiabetic activity of superfine grinding bitter melon powder containing a total of bioactive compositions.
The objectives of this study were: (1) to obtain the bitter melon lyophilized superfine grinding powder (BLSP) and bitter melon hot air drying superfine grinding powder (BAP); (2) to compare their differences in physical/chemical properties, antidiabetic activity and their mechanisms in vivo; and, (3) to conclude the processing effect on the bioactivity of bitter melon.
3. Experimental Section
3.1. Plant Material
The fresh fruits of wild bitter melon were obtained from Lvjian Agricultural Station (Yangzhong City, China) and were authenticated by Jiangsu Academy of Agricultural Science. The bitter melon selected for the present study had a 20–25 mm diameter and green appearance.
Bitter melon lyophilized superfine grinding powder (BLSP) was prepared by washing the unripe bitter melons with tap water, removing the seeds, lyophilizing the remaining portion with the pressure of 30 Pa for 24 h at 5 °C, and superfinely ground with a HSF high-speed hammer mill (National Special Superfine Powder Engineering Research Center of China, Nanjing, China). The preparation methods of bitter melon hot air drying superfine grinding powder (BAP) was similar to those of the BLSP, except the 60 °C hot air procedure (24 h) and grounding with a QE-100g mill (Zhejiang Yili Co., Jinghua, China). BAP and BLSP were filtered through a 150 μm screen before further experiments. The obtained samples were stored at 4 °C for further use.
3.2. Determination of Physical Properties of BLSP and BAP
The Morphological characterization of BLSP and BAP was analyzed with a Field Emission Scanning Electron Microscope (FE-SEM: Jeol JSM-7001F: with Au-coated, operated at 10 kW). Particle size distribution of BLSP and BAP were measured by a laser diffraction instrument (Mastersizer 2000, UK). The moisture contents were determined by using a HB43-S moisture analyzer (Mettler Toledo, Switzerland). The water activity (Aw) values were determined by using AQUA Lab (Decagon Devices, Inc., USA). The water solubility index of 20 min was determined by using the method described by Zhao
et al.[
55].
3.3. Analysis of Chemical Components of BLSP and BAP
Two grams of powders were extracted with 100 mL distilled water at 100 °C for 30 min, filtering the infusions to obtain the resulting supernatant. After cooling and filtering, the volume was made up to 100 mL with distilled water. Water-soluble nitrogen content was analyzed with the Bradford procedure based on an established calibration curve. The results were expressed as BSA (Bovine serum albumin) equivalents in mg/g dry material. Additionally, water-soluble sugar content was analyzed by using the anthranone reagent and the results were expressed as d-glucose equivalents percent. The total of polyphenols was analyzed by the Folin-Ciocalteu method. Briefly, this solution (0.5 mL) was mixed with 2.5 mL of distilled water, 1.5 mL of 7.5% sodium carbonate (Na2CO3), and 0.5 mL of Folin-Ciocalteau reagent. After incubation at 45 °C for 30 min, the absorbance of the reaction mixture absorbance was measured at 750 nm, and the content was expressed as gallic acid equivalents in mg/g dry material.
The total flavonoid content was determined according to the aluminum chloride colorimetric method [
6]. The total flavonoid content was expressed in milligrams of rutin equivalents per gram of dry material. The total saponin content was determined used the method described by Xu & Dong [
56] and expressed as Ginsenoside Rg1 equivalents percent.
3.4. Animals
Sprague–Dawley male rats weighing 200 ± 20 g were obtained from the Laboratory Animal Research Center of Jiangsu University (LARC, Zhenjiang, China) with the license number SCXK (SU) 2009–0002 and SYXK (SU) 2008–0024. The rats were caged individually in LARC at temperature of 22 ± 2 °C and a relative humidity of 40%–60%, and artificially illuminated on an approximate 12 hrlight/dark cycle. The air exchange was about 18 times/h. All the rats were provided food and filtered tap water ad libitum.
3.5. Induction of Diabetes
Rats were randomly separated into two groups: normal control (NC) and diabetic groups. The NC group was fed with a basic diet and experimental animals were fed with a high-fat and high-sucrose diet (containing of 7% lard, 15% sucrose and 78% basic diet) for 4 weeks. After feeding the rats with these diets for 4 weeks, the rats were then made to fast overnight before treatment. Type 2 diabetic rats were induced by a single intraperitoneal injection of STZ (Sigma Chemical Co., St. Louis, MO, USA) freshly dissolved in a 0.1 mol/L citrate buffer (pH 4.5) at a dosage of 35 mg/kg body weight. The NC group was administered with citrate buffer (pH 4.5). Three days later, the rats were confirmed as the diabetic model when their fasting plasma glucose levels exceeded 11.1 mmol/L. Diabetes were stabilized in these STZ-treated rats over a period of 7 days before the experiment.
3.6. Experimental Design
Experimental rats with 12 of normal model and 30 of STZ-diabetic model were divided into 7 groups of 6 each. Group 1 (NC + water) consisted of normal rats treated with distilled water (4 mL/kg body weight); Group 2 (NC + BLSP 400) consisted of normal rats treated with BLSP (400 mg/kg bw); Group 3 (DC (diabetic control) + water) consisted of diabetic rats treated with distilled water (4 mL/kg bw); Group 4 (BLSP 400) consisted of diabetic rats treated with BLSP (400 mg/kg bw); Group 5 (BLSP 800) consisted of diabetic rats treated with BLSP (800 mg/kg bw); Group 6 (BAP 400) consisted of diabetic rats treated with BAP (400 mg/kg bw); Group 7 (Metformin 400) consisted of diabetic rats treated with 400 mg/kg bw of metformin (Beijing Jingfeng Pharmaceutical CO., LTD, Beijing, China).
During the five-week treatment, the body weight of each rat and the food/water intake volumes were measured weekly, then the animals were anesthetized with chloral hydrate. Blood samples were collected via abdominal aorta puncture. The blood samples were centrifuged at 3500 rpm for 10 min to obtain the serum, which was kept at −20 °C for further analysis.
3.7. Blood Biochemical Assays
Fasting blood glucose was measured using OneTouch Ultra Blood Glucose Meter (Johnson & Johnson Medical (China) Ltd., Shanghai, China).
The sera were assayed for triglycerides (TG), cholesterol (CHOL), high-density lipoproteins cholesterol (HDLC) and low-density lipoproteins cholesterol (LDLC) levels with an Olympus AU2700 Clinical Chemistry Analyzer (Olympus Inc., Japan). The insulin levels were determined using commercial rat insulin ELISA kits (R & D Systems China Co. Ltd., Shanghai, China).
3.8. Oxidative Stress Markers and Antioxidant Enzymes
The end product of liver tissue lipid peroxidation quantity was expressed by the content of the malondialdehyde (MDA). The MDA content was determined using commercially available kits (Nanjing Jiancheng Bio CO., Nanjing, China). While the levels of SOD and glutathione peroxidase (GPx) of the liver tissue were also determined using commercially available kits (Nanjing Jiancheng Bio CO., Nanjing, China).
3.9. Histopathologic Procedures
Pancreatic tissues were harvested from the sacrificed rats. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, cut to approximately 4 μm sections, and stained with hematoxylin and eosin (H&E). The slides were viewed on a Zeiss Axiovert 40 Microscope (Carl Zeiss, Oberkochen, Germany).
3.10. Statistical Analyses
The data were presented as group mean values ± SD (standard deviation) and were analyzed by one-way analysis of variance (ANOVA). All the statistical analyses were performed using SPSS v14.0 (SPSS Inc., Chicago, IL, USA). p values <0.05 were considered as significant.