Antioxidant Activity of Artocarpus heterophyllus Lam. (Jack Fruit) Leaf Extracts: Remarkable Attenuations of Hyperglycemia and Hyperlipidemia in Streptozotocin-Diabetic Rats

The present study examines the antioxidative, hypoglycemic, and hypolipidemic activities of Artocarpus heterophyllus (jack fruit) leaf extracts (JFEs). The 70% ethanol (JFEE), n-butanol (JFBE), water (JFWE), chloroform (JFCE), and ethyl acetate (JFEAE) extracts were obtained. Both JFEE and JFBE markedly scavenge diphenylpicrylhydrazyl radical and chelate Fe+2in vitro. A compound was isolated from JFBE and identified using 1D and 2D 1H- and 13C-NMR. The administration of JFEE or JFBE to streptozotocin (STZ)-diabetic rats significantly reduced fasting blood glucose (FBG) from 200 to 56 and 79 mg%, respectively; elevated insulin from 10.8 to 19.5 and 15.1 μU/ml, respectively; decreased lipid peroxides from 7.3 to 5.4 and 5.91 nmol/ml, respectively; decreased %glycosylated hemoglobin A1C (%HbA1C) from 6.8 to 4.5 and 5.0%, respectively; and increased total protein content from 2.5 to 6.3 and 5.7 mg%, respectively. Triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), VLDL-C, and LDL/HDL ratio significantly declined by -37, -19, -23, -37, and -39%, respectively, in the case of JFEE; and by -31, -14, -17, -31, and -25%, respectively, in the case of JFBE; as compared to diabetic rats. HDL-C increased by +37% (JFEE) and by +11% (JFBE). Both JFEE and JFBE have shown appreciable results in decreasing FBG, lipid peroxides, %HbA1C, TC, LDL-C, and TG levels, and increasing insulin, HDL-C, and protein content. The spectrometric analysis confirmed that the flavonoid isolated from JFBE was isoquercitrin. We can conclude from this study that JFEE and JFBE exert hypoglycemic and hypolipidemic effects in STZ-diabetic rats through an antioxidative pathway that might be referred to their flavonoid contents.


INTRODUCTION Preparation of Plant Extracts
The air-dried, powdered jack fruit leaves were subjected to preliminary phytochemical screening. The results revealed the presence of carbohydrates, flavanoids, glycosides, sterols, and tannins; however, steam volatiles and saponins were absent.

Spectrometric Analysis
Jack fruit leaves (750 g) were extracted repeatedly with 70% ethanol at room temperature. The ethanolic crude extract (JFEE) was concentrated under vacuum to yield a residue of 74 g (9.8% w/w). The extract was suspended in water and partitioned with petroleum ether, chloroform, ethyl acetate, and n-butanol. The JFBE (20 g, 27.02% w/w) was applied to Di-anion column (Ø 1 m × 5 cm, 200 g) and eluted with 500 mL methanol:water (50:50) to give three fractions. The third fraction yielded 35 subfractions. Fraction numbers 30-35 (0.12 g, 6% w/w) were applied on silica gel column (110 g, Ø 55 × 11 cm) using ethyl acetate:formic acid:acetic acid:water 100:11:11:2, 100:11:11:4, 100:11:11:6, 100:11:11:8, and 100:11:11:10 to give five fractions that were pooled together, concentrated under reduced pressure, and purified on a Sephadex LH-20 column using 90% methanol as eluent, followed by crystallization, to give 40 mg of a yellow powder that elicited a brown color at 254 nm and produced a bright yellow color upon spraying with AlCl 3 , which was identified spectroscopically using both 1D and 2D 1 H-and 13 C-NMR techniques. The 1 H-and 13 C-NMR spectra of this compound were recorded at Taibah University, Saudi Arabia, on an Avance II Bruker FT-NMR spectrometer 400 (400 MHz) using CD 3 OD or DMSO-d6 as solvents and TMS as an internal standard. Chemical shifts are expressed as δ ppm units.

DPPH · Assay
The hydrogen atom or electron donation ability of the JFEs was measured as a result of the bleaching of purple-colored methanol solution of DPPH · . Briefly, each of the JFEs (JFEE, JFBE, JFWE, JFCE, and JFEAE) were used in this experiment at concentrations of 0.2, 0.4, and 0.6 mg/mL for each of the mentioned extracts in triplicate; 50 µL of each concentration was added to 5 mL of 0.004% methanol solution of DPPH · . After a 30-min incubation at room temperature, the absorbance was recorded against blank at 517 nm [18]. Vitamin E was used as a reference antioxidant. The percentage of inhibition of DPPH · free radical scavenging activity = [A c -A s /A c ]  100, where, A c : absorbance of control and A s : absorbance of sample.

Ferrous Ion (Fe ++ ) Chelating Activity
The Fe ++ chelating ability of JFEs was monitored by the absorbance of Fe ++ -ferrozine complex at 562 nm. Briefly, JFEE, JFBE, JFWE, JFCE, and JFEAE were used at concentrations of 0.2, 0.4, and 0.6 mg/mL in triplicate. A volume of 0.4 mL of each extract was added to 0.2 mL 2 mM FeCl 2 . The reaction was initiated by addition of 0.4 mL 5 mM ferrozine. The total volume was adjusted to 4 mL with ethanol. Then, the mixture was shaken vigorously and left at room temperature for 10 min, followed by measuring absorbance at 562 nm [19]. Absorbance of the solution was then measured spectrophotometrically at 562 nm [19]. Vitamin E was used as a reference antioxidant. The percentage of inhibition of Fe ++ -ferrozine complex formation was calculated by using the equation: Fe ++ chelating effect percentage = [1 -A s /A c ]  100, where A C : absorbance of control and A S : absorbance of sample.

Experimental Animals
Male Wistar rats weighing 170-250 g were used in this study. Animals were maintained under standard conditions of temperature (24 ± 5 o C) and relative humidity (55 ± 5%), with a regular 12 h light:12 h dark cycle, and allowed free access to standard laboratory food and water 7 days before starting the experiment and during the whole period of the experiment. All animals were fed with common pellet diets and water ad libitum. All animals were treated humanely in accordance with the guideline for care of animals as set by the World Health Organization.

Induction of Diabetes Mellitus
Diabetes was induced in the rats by a single intraperitoneal (i.p.) injection of freshly prepared STZ (60 mg/kg b.w.) in normal saline. Two days after STZ administration, blood samples were obtained from the tips of the rat's tail and the fasting blood glucose (FBG) levels determined using a OneTouch® Ultra® glucometer (LifeScan, U.S.A.) to confirm diabetes. The diabetic rats exhibiting blood glucose levels above 190 mg% were included in this study [20]. The biochemical effects of JFEE and JFBE were compared to GLB (a reference hypoglycemic drug).

Experimental Design and Treatment Regimen
Rats were subdivided randomly into five groups (eight rats/group) and treated as follows:- Group I: Normal control rats received 0.1 mL DMSO and 0.5 mL 5% Tween 80 for 10 days by oral lavage.  Group II: STZ-diabetic rats received 0.1 mL DMSO and 0.5 mL 5% Tween 80 for 10 days by oral lavage.  Group III: Diabetic rats orally received GLB (600 g kg -1 day -1 ) in 0.1 mL DMSO and 0.5 mL 5% Tween 80 for 10 days by oral lavage [21].  Group IV: STZ-diabetic rats orally received JFEE dissolved in 0.1 mL DMSO and 0.5 mL 5% Tween 80 at a dose of 200 mg kg -1 day -1 for 10 days by oral lavage [22].  Group V: STZ-diabetic rats orally received JFBE dissolved in 0.1 mL DMSO and 0.5 mL 5% Tween 80 at a dose of 200 mg kg -1 day -1 for 10 days by oral lavage [22].
On the 11th day, the fasting rats were subjected to light ether anesthesia and killed by cervical dislocation. Trunk blood was collected into heparinized chilled tubes containing sodium fluoride (to inhibit glycolysis). Serum was separated by centrifugation at 4 o C and stored at -20 o C until further use.

Measurements of Biochemical Parameters
Sera were assayed for FBG, total protein content, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) using kits purchased from Spinreact, Spain. The very-low-density lipoprotein cholesterol (VLDL-C) was calculated by dividing the values of TG by 5 [23]. The LDL/HDL ratio was also calculated. The lipid peroxides (expressed as thiobarbituric acid reactive substance, TBARS) was measured in serum [24]. Serum insulin level was estimated using an enzyme immunoassay kit purchased from SPI-BIO (Society of Pharmacology and Immunology-BIO), France. The whole blood with EDTA was used to estimate glycosylated hemoglobin percentage (%HbA1C) using a diagnostic kit purchased from Beckman Coulter, U.K.

Statistical Analysis
Data were expressed as means ± SEM. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA), followed by the Tukey-Kramer multiple comparison test, to judge the difference between various groups. Significance was accepted at p < 0.05.  (Table 1). The remaining eight protons were attached to oxygen atoms. This suggested the presence of at least nine oxygen atoms in the molecular structure, as we have eight hydroxyl groups and a carbonyl group. Indeed, we incorporated nine oxygen atoms into the partial molecular formula that was derived earlier to get a molecular formula of C 21 H 20 O 9 (mass of 416). The missing mass of 48 corresponds to three oxygen atoms, suggesting that the structure possesses three nonprotonated oxygen atoms, part of a ring system or connecting rings together. In accordance with this analysis, a molecular formula of C 21 H 20 O 12 (mass of 464) was derived. The 1 H-NMR revealed the presence of 12 protons, which corroborated DEPT data. The remaining eight hydroxyl protons exchanged rapidly and therefore exhibited no absorption signals. The five aromatic protons were assigned to two aromatic rings [δ 6. The 13 C chemical shifts that were correlated via HSQC analysis (δ 116.1, 123.0, and 117.8, respectively) suggested that the upfield absorption of the first and third carbon stems from the presence of two adjacent hydroxyl groups in the 3 and 4 aromatic positions, which separated the two spin systems. It is noted that all these protons have been correlated by HSQC to six carbons, which were connected to oxygen atoms as their carbon chemical shifts range from δ 62.0 to 105.4. The most characteristic proton appeared as doublet at δ 5.16 (d, J = 7.9 Hz) and was correlated to the carbon absorption at δ 105.4. This corresponds to the anomeric carbon of the glycosidic bond. The remaining alkyl protons absorbed from δ 3.87 to 3.44 and have been attributed to the cyclic protons of glucose and its alkyl CH 2 group (Fig.  1). According to the analysis presented, we propose that the unknown is 2-(3,4-dihydroxyphenyl) -3-(β-D-  glucofuranosyloxy) -5,7-dihydroxy-4H-1-benzopyran-4-one, also named as quercetine-3-O-βglucopyranoside or isoquercitrin (Fig. 2). The stereochemistry of the glycosidic bond of the anomeric carbon has been assigned as β based on the coupling constant of 1" CH (J = 7.9 Hz) since the α-anomer exhibited a smaller coupling constant for the 1" CH (J = 1.0 Hz). The 1 H-and 13 C-NMR data of isoquercitrin are presented in Table 1 and are as follows: 1

Effect of JFEE and JFBE on Serum Proteins of Diabetic Rats
Injection of STZ into rats produced a significant decline in serum protein content by -64%, p < 0.05, as compared to normal rats ( Table 2). Administration of GLB, JFEE, or JFBE to STZ-diabetic rats elicited a significant elevation in serum protein content by +104, +152, and +128%, respectively, p < 0.05, as compared to STZ-diabetic rats ( Table 2).

) for 10 Days to STZ-Diabetic Rats on Serum Protein Content, FBG, Lipid Peroxides Expressed as TBARS, %HbA1C, and Insulin
Values are expressed as means ± SEM (n = 8). † Significant differences p < 0.05, as compared with normal control animals (group I). * Significant differences p < 0.05, as compared with STZ-treated animals (group II).

Hypoglycemic and Lipid Peroxide Inhibitory Effects of JFEE and JFBE
Injection of STZ into rats exhibited significant elevations in FBG, %HbA1C, and TBARS levels by +264, +100 and +43%, respectively, p < 0.05, as compared to normal control rats (Table 2). These changes were concomitant with a significant decline in serum insulin level by -49%, p < 0.05, as compared to normal rats. Administration of GLB to STZ-diabetic rats produced significant declines in FBG, %HbA1C, and TBARS levels by -29, -15, and -16%, respectively, p < 0.05, associated with a significant increase in serum insulin level by +26%, as compared to STZ-diabetic rats. Administration of JFEE to STZ-diabetic rats elicited a significant increase in serum insulin by +81%, joined with a significant decrease in FBG, %HbA1C, and TBARS levels by -72, -34, and -26%, respectively, p < 0.05, as compared to STZ-diabetic rats. On the other hand, administration of JFBE to STZ-diabetic rats elicited lesser changes than in the case of JFEE intake. These changes involved a significant increase in serum insulin by +40%, associated with a significant decrease in FBG, %HbA1C, and TBARS levels by -61, -26, and -19%, respectively, p < 0.05, as compared to STZ-diabetic rats ( Table 2).

DISCUSSION
During the last 2 decades, traditional medicines have become a topic of interest [26]. The present study was carried out to investigate hypoglycemic and hypolipidemic effects of JFEs in STZ-diabetic rats, compared to the reference hypoglycemic drug GLB. Five different JFEs were obtained and evaluated for their antioxidative activity in vitro using two different models: the DPPH · free radical scavenging assay and the Fe ++ chelating activity assay. The results revealed that the best two fractions that exhibited pronounced in vitro antioxidant activity were JFEE and JFBE and, in turn, both were chosen for subsequent in vivo experiments. This antioxidant activity may be attributed to the high phenolic content [14,27], as evident from the results obtained from the spectrometric analysis that showed the existence of isoquercitrin flavonoid in JFBE. The experimental animal model used in this study was type II diabetes mellitus since a low single dose of STZ can destroy half of pancreatic  cells [28]. The mechanism by which STZ brings about its diabetic state includes pancreatic -cell destruction, which make cells less active [6]. The significant high levels of serum FBG and %HbA1C in STZ-induced diabetic rats were lowered after the oral intake of either JFEE or JFBE. These changes were associated with a significant elevation of serum insulin level. The order of the hypoglycemic activity was JFEE > JFBE > GLB. The present results do not distinguish whether the effects observed are mediated at the level of alterations in fuel metabolism within the intestinal lumen via -amylase inhibitory effects, -cell function including regulation of insulin synthesis and secretion, or insulin action at target tissues [16,29,30]. Oral administration of JFEE or JFBE attenuated hyperglycemia and subsequently %HbA1C was declined due to the powerful hypoglycemic role of JFEE and JFBE [31]. The significant reduction of serum proteins was observed in STZ-diabetic