Potential Therapeutic Activity of Mango (Mangifera indica L.) Leaves on Excess Consumption of Fructose in Rats

Background and Objective: High fructose consumption has increased worldwide. It causes various metabolic, genetic and histologic alterations. Alternative medicine, primarily herbal plants, has been proposed to alleviate the negative effects of high fructose consumption. The main objective of this study was to explore the efficacy of supplementation or treatment with mango leaves against high fructose induced alterations in male rats. Methodology: Mango leaves nutritional and active components were determined. A total of sixty male adult rats were used in this study. Fifteen rats were kept as healthy (negative control group; rats fed on balanced diet) while in others metabolic alterations were induced by consumption of high fructose diet ad libitum. Rats fed on high fructose diet were splited into 3 groups (15 rats in each), one group set as positive control group; rats fed on high fructose diet only and the other 2 groups; mango treated group; rats fed on high fructose diet until induction of hyperglycemia (one month and half) then fed on high fructose diet with replacement of fiber with 5% mango leaves and mango supplemented group; rats fed on high fructose diet with 5% mango leaves replacing fiber. Results: Mango leaves contain significant amounts of crude protein, crude fat, carbohydrates, crude fiber, ash, total flavonoids and polyphenols that controlled and corrected the following high fructose consumption results. Consumption of high fructose diet significantly (p≤0.05) increased final body weight (FBW), body weight gain (BWG), abdominal circumference (AC), Lee index and Original Research Article Gabal et al.; JPRI, 33(64B): 192-204, 2021; Article no.JPRI.78565 193 body mass index (BMI). High fructose also significantly (p≤0.05) increased levels of systolic blood pressure (SBP), serum triacylglycerol (TAG), total cholesterol (TC), fasting blood glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), serum tumor necrosis factor-α (TNF-α), leptin, malondialdehyde (MDA), advanced glycation end products (AGEs) and adipocyte size as well as blood histone deacetylase (HDAC) enzyme activity. High fructose consumption contrarily caused significant decrease (p≤0.05) in levels of quantitative insulin check index of insulin sensitivity (QUICKI), adiponectin, muscular insulin receptor substrate-1 (IRS-1) and glucose transporter-4 (GLUT-4) gene expression as well as blood reduced glutathione (GSH). Furthermore, microscopic examinations of the pancreatic and adipose tissues corroborated the biochemical findings. Conclusion: Mango leaves are a cheap source of macro and micronutrients as well as active constituents. By limiting metabolic and genetic abnormalities caused by high fructose consumption, either mango leaf supplementation or therapy improved and ameliorated all biochemical and microscopic data. The mango leaves supplemented group showed the most improvement.


INTRODUCTION
Fructose is a simple sugar that is found naturally in honey and fruits. It is utilized as artificial sweetener. Sugar-sweetened beverages and processed foods are the main source of fructose in diet. Consumption of these products became higher especially in children and adolescents [1].
Chronic consumption of high fructose in diet was found to be linked with increased risk of different diseases [2]. A recent epidemiological analysis in humans also found an association between metabolic syndrome (MS) prevalence and sugar availability. MS is a cluster of hyperglycemia, dyslipidemia, insulin resistance, elevated blood pressure and abdominal obesity [3].
Metabolic abnormalities due to high fructose consumption for a long time results in hyperglycemia that explained by the defect in glucose uptake, insulin receptors, signaling and action that may be due to genetic and epigenetic dysregulation [4,5].
In the last few years there has been an unusual growth in the scope of herbal medicine. The usage of these natural drugs increased worldwide because of their low cost with minimal side effects. Mango (Mangifera indica L.) is a succulent stone fruit belongs to Anacardiaceae family. Mango is cultivated in different parts of the world, particularly in tropical countries. It has been well authenticated that mango leaves are a remarkable source of both macro and micro nutrients [6].
Mango leaves provide energy from macronutrients including carbohydrates, proteins and fats. Leaves also contain dietary fiber, phenolic compounds, vitamins and other phytochemicals that are vital to normal human development, growth and health. Mango leaves have varied health promoting chemical constituents including mangiferin. Mangiferin is a natural polyphenol of C-glycosylxanthone structure, with various pharmacological/ physiological activities. Mangiferin promotes gut regularity and health. It also have antioxidant and anti-inflammatory benefits [7].
The purpose of this study was to see if mango leaves might compete with high fructose diet-induced genetic, metabolic, oxidative, and inflammatory alterations in male rats.

Plant
The mango leaves were obtained from the Ministry of Agriculture in Giza, Egypt, and botanists verified their authenticity (Botany Department, Women Faculty, Ain Shams University). The leaves were rinsed in water and dried entirely in well-ventilated stores under proper circumstances before being processed into powder with an electric grinder [8].

Chemicals
Fructose and other chemicals were obtained from Cairo, Egypt-EL-Gomhouria Company for Trading Chemicals.

Animals
Sixty male adult Sprague-Dawley strains weighing (100 ± 5 g) were kept in wire cages for acclimation (7 days) in the well-ventilated animal house of El-Azhar University's Faculty of Pharmacy till the end of the trial period (12weeks). They were given a 12 hour light/dark cycle and a standard diet as well as unlimited tap water. All animal experiments were approved by the Institutional Animal Ethics Committee, which followed the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals.

Diet
A balanced diet was created in accordance with the American Institute of Nutrition's AIN-93M and amended by Reeves et al. [9]. According to Faure et al. [10], a high fructose diet was produced.

Determination of mango leaves nutritional and active composition
Mango leaves crude protein, crude fat, carbohydrates, crude fiber and ash were determined following AOAC technique [11]. Folin-Ciocalteu reagent was used to measure total flavonoids and polyphenols, as described by Arnous et al. [12].

Experimental design
The rats were divided into four groups, each with 15 rats, and given the following treatments: Group I: healthy negative control group of rats that have been fed a well-balanced diet. Group II: Positive control group; rats fed on high fructose diet. Group III: Mango treated group; rats fed on high fructose diet until induction of hyperglycemia (one month and half) then fed on high fructose diet with replacement of fiber with 5% mango leaves [8]. Group IV: Mango supplemented group; rats fed on high fructose diet with 5% mango leaves replacing fiber.

Anthropometric measurements  Body weight gain (BWG), abdominal circumference (AC), Lee index and body mass index (BMI)
Animals were weighed twice a week using electrical balance. All rats were fasted overnight at the termination of the experiment (90 days), final body weight (FBW) were measured using electrical balance. The abdominal circumference and body length (nose-to-anus length) were measured using non-stretchable tape for calculation of lee index and body mass index (BMI) under deep anesthesia. Lee index is calculated as cubic route of final body weight (g) divided by the length (cm); where > 0.30 considered obese. The BMI was calculated as final body weight (g) divided by the square of the anal-nasal length (cm); where > 0.68 considered obese [13,14].

Systolic blood pressure (SBP)
Systolic blood pressure was measured under anesthesia the day of sacrifice, using a blood pressure tail cuff system (Model 29 pulse amplifier with adapted rodent restrainer and tail cuff sensor, iitc incorporated).Three readings were taken consecutively to calculate the average as a final reading for SBP [15].

Handling of blood and organs samples
Rats were anaesthetized before being sacrificed. Blood was drawn from the hepatic portal vein and separated into two tubes, one for blood and the other to separate serum for biochemical analysis. The pancreatic and adipose tissues were extracted to be analyzed microscopically, while thigh muscle tissues were separated to evaluate gene expression.

Biochemical determination
Blood fasting glucose concentration and insulin level were determined by glucometer and enzyme immunoassay test kit [16,17]. Blood histone deacetylase (HDAC) enzyme activity was measured using a colorimetric assay kit (BioVision, CA, USA) following Strahl and Allis method [18] while blood reduced glutathione level was determined calorimetrically according to Beutler et al. [19]. Homeostasis model assessment-insulin resistance (HOMA-IR), and the quantitative insulin check index of insulin sensitivity (QUICKI) were calculated according to the following equations [20]: Serum malondialdehyde (MDA) and advanced glycation end products (AGEs) levels were determined using standard determination methods [21,22]. Serum total cholesterol (TC) and total triacylglycerol (TAG) were measured using Biodignostic kits (Giza, Egypt).

Determination of insulin receptor
substrate-1 (IRS-1) and glucose transporter-4 (GLUT-4) genes expressions using real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted by Trisol reagent (Invitrogen, CA) from anterior thigh muscle tissue using total RNA purification kit. RNA and cDNA concentration were quantified using Nanodrop method to make sure they are pure and suitable to conduct real time PCR [23]. IRS-1 and GLUT-4 genes expressions were measured in three replicates using 18S rRNA expression level as an internal reference. Data were normalized using the 2 −ΔΔCt method [24].

Microscopic examination
Pancreas as well as white adipose tissues from all rats were dissected out and fixed instantaneously in formalin (10%) solution for 24 h. Paraffin blocks were prepared and 5μm thick sections were subjected to microscopic examination. Hematoxylin and Eosin stains (H & E) were used with magnification power 200 and 400 [25]. Image-Pro plus Version 5.0 (Media Cyberetics) was used to measure adipocyte average diameters as described by Chen and Farese [26] at the Cairo University's Veterinary Medical Laboratory.

Statistical analysis
The SPSS software was used to analyze the results (version 16). The findings were expressed as mean ± standard deviation (ANOVA; F-test) and the least significant difference (L.S.D) was calculated following to Levesque [27].

Nutritional and Active Composition of Mango Leaves
From analysis data of mango leaves Fig. [1], it was found that leaves contain significant amounts of crude protein, crude fat, carbohydrates, crude fiber and ash. This explain the high nutritive value of mango leaves with low caloric content on comparison. Leaves also were found to contain valuable amounts of polyphenols and flavonoids with many health benefits.

Effect of mango leaves on the anthropometric measurements of male rats fed on high fructose diet
Current research results revealed that there was significant increase (p≤0.05) in the final body weight (FBW),body weight gain (BWG), abdominal circumference (AC), lee index, and body mass index (BMI) in positive control group with respect to the healthy negative control group leading to obesity in rats fed with high fructose diet .On contrary, there was significant decrease (p≤0.05) in FBW,BWG,AC, lee index, and BMI in mango supplemented and treated groups and that mango leaves oral supplementation to rats fed on high fructose diet from the beginning prevented and improved these values with respect to the positive control group as presented in Table 2.

Effect of mango leaves on systolic blood pressure and serum lipids fractions levels in male rats fed on high fructose diet
It is obvious from current results that, high fructose intake significantly increased (p≤0.05) systolic blood pressure (SBP), serum triacylglycerol (TAG) and total cholesterol (TC) levels in comparison with healthy negative control group as shown in table (3) leading to hypertension and hyperlipidemia . The increase in these parameters was inhibited by oral mango leaves administration. The mango supplemented group showed the most significant improvements.

Effect of mango leaves on fasting blood glucose level, insulin level, resistance and sensitivity in male rats fed on high fructose diet
Compared with the healthy negative control rats, high fructose intake caused significant (P≤0.05) increase in the blood levels of fasting glucose, insulin, homeostasis model assessmentinsulin resistance (HOMA-IR), with significant decrement in the quantitative insulin check index of insulin sensitivity (QUICKI) index. As compared to the positive control group, mango leaves treatment of high fructose fed rats resulted in a significant (P≤0.05) reduction in insulin, fasting blood glucose, HOMA-IR, and an increase in the QUICKI index, lowering the likelihood of developing diabetes mellitus (Table  4). Oral supplementation with mango leaves prevented diabetes mellitus and improved all of the tested parameters.   In comparison to healthy negative control rats fed a balanced diet, serum levels of tumor necrosis factor-α (TNF-α)and leptin hormone were found to be significantly (P≤0.05) increased in positive control rats fed a high fructose diet, accompanied by a significant reduction in serum adiponectin hormone level (P≤0.05). On comparison to the positive control group, oral administration of mango leaves demonstrated a significant (P≤0.05) improvement in TNF-α, leptin resistance status, with a significant (P≤0.05) decrease in serum leptin levels and an increase in serum adiponectin levels ( Table 5). The mango supplemented group showed the greatest improvement.

Effect of Mango Leaves on Blood Histone Deacetylase (HDAC) Enzyme Activity, Muscular Insulin Receptor Substrate-1 (IRS-1) and Glucose Transporter-4 (GLUT-4) Gene Expression in Male Rats Fed on High Fructose Diet
Histone deacetylase enzyme activity in blood (HDAC) was significantly (P≤0.05) increased accompanied with significant (P≤0.05) decrease in gene expression of IRS-1 and GLUT-4 in positive control rats fed with high fructose diet in comparison with the healthy negative control rats fed on the balanced diet. Whereas oral administration of mango leaves showed significant (P≤0.05) amelioration in the tested parameters as compared with positive control group ( Table 6). The most improvements were recorded in mango supplemented group.

Effect of Mango Leaves on Oxidative Status in Male Rats Fed on High Fructose Diet
Oxidative biomarkers measured in serum that illustrated in Table (7) showed that MDA and AGEs contents were significantly increased (p≤0.05) accompanied with significant reduction in blood antioxidant GSH level in positive control rats fed with high fructose diet as compared to healthy negative control rats composing a state of oxidative stress. Remarkably, mango leaves treatment and supplementation was linked with a significant increase in antioxidant GSH level and decrease in oxidative stress indicators MDA as well as AGEs content in comparison with positive control group.

Effect of Mango Leaves on Adipocyte Size in Male Rats Fed on High Fructose Diet
The size of adipocytes as presented in table (8) is significantly increased (p≤0.05) in positive control group on comparison with healthy negative control group, while mango treated and supplemented groups showed significant decrease (p≤0.05) in visceral fat thickness and mean adipocyte diameter as compared to positive control group .Protective effect of mango leaves against expansion of adipose tissue by high fructose diet was observed. The outcomes of biochemical analysis were supported by microscopic examination of adipose tissue.

Effect of Mango Leaves on Microscopic Examination of Pancreatic and White Adipose Tissues in Male Rats Fed on High Fructose Diet
Microscopic examination of pancreatic and white adipose tissues (Fig. 2 and 3) showed the degenerative alterations caused by high fructose consumption, as well as the protective action of mango leaves, supplied biochemical analysis with support. Microscopic examination of the negative control group pancreas showed normal parenchyma with normal pancreatic acini, duct, and islets (Fig. 2a), while that of positive control group revealed acute pancreatitis with congested blood vessels and mononuclear cells infiltrations (Fig. 2b). Mango treated group reported normal pancreatic tissue with slight congestion in the blood vessels (Fig. 2c) while mango supplemented group denoted normal histological pancreatic tissues the same as control negative group (Fig. 2d), The adverse effect of high fructose intake has been ameliorated in mango treated group and counteracted in mango supplemented group.

Table 5. Effect of mango leaves on serum tumor necrosis factor-α (TNF-α), leptin and adiponectin levels in male rats fed on high fructose diet
There was no significant difference between means with the typical alphabetical superscripts in the same column, significant difference (p≤ 0.05). All values are presented mean ±SD, n=10 Table 6.   Microscopic examination of the negative control group adipose tissues displayed normal adipocytes distribution with regular sizes (Fig.  3a), while that of positive control group revealed increased size with abnormal distribution (Fig. 3b). Mango treated group reported slight increased size with normal distribution of white adipose tissues (Fig. 3c) while mango supplemented group denoted normal histological adipose tissues the same as control negative group (Fig. 3d). The adverse effect of high fructose intake has been ameliorated in mango treated group and counteracted in mango supplemented group.

DISCUSSION
Mangifera indica L. is an important fruit cultivated in various countries all over the world. Mango leaves have been studied for their beneficial health effects. Mango leaves contain significant amounts of crude protein, crude fat, carbohydrates, crude fiber and ash as well as a plethora of phytochemicals including polyphenols and flavonoids. Phytochemicals include mangiferin, benzophenones and phenolic acids. Mango leaves also contain antioxidants such as ascorbic acid, flavonoids, tocopherols and carotenoids. Mango leaves extracts have been studied for their different biological activities and various bioactivities. Mango leaves can be used as a potential ingredient for the development of functional foods and pharmaceutical drugs considering their nutritional and phytochemical profile and beneficial effects [28,29]. Through this study the role of feeding mango leaves in association with high fructose diet was evaluated.
Fructose has a chemical formula as glucose (C 6 H 12 O 6 ). Fructose metabolism differs from that of glucose. Fructose is completely extracted by hepatic tissues and rapidly converted into glucose, lactate, glycogen, and fat [30]. Metabolic syndrome develops as a result of a high-fructose diet, which includes symptoms such as a high body mass index (BMI), hypertension, dyslipidemia, hyperglycemia, and insulin resistance. This combination of variables can lead to obesity and type 2 diabetes [31].
It was found that rats fed on high fructose diet gained weight and became obese with high BMI, Lee index associated with accumulation of fat in the abdomen as indicated by increased abdominal circumference. These observations were accompanied by increased systolic blood pressure and increased fat synthesis as indicated by increased triacylglycerol and cholesterol levels in serum. These metabolic effects is due to the nature of fructose metabolism.
Although fructose is a monosaccharide, it is not directly metabolized to provide energy in most metabolic activities (ATP). Fructose, on the other hand, aids glycogen formation in the liver via a metabolic pathway that overlaps with gluconeogenesis. Fructose promotes the creation of triacylglycerol (TAG) from glycerol and the synthesis of fatty acids. TAG is then stored as fat until the deposited glucose is depleted, triggering a negative feedback loop. However, because glucose levels are rarely reduced, digested fructose is considered excessive and is primarily stored as fat. In this setting, increased fructose consumption is linked to the occurrence of metabolic syndrome criteria [32].
On the other hand mango leaves supplementation and treating effects were obvious on experimental rats affected by consumption of high fructose diet. Mango leaves active constituents regulated fat metabolic pathways through controlling of different genes expression in rats causing an amelioration in fat deposition in the rat's body and correction of obesity markers and body weight. This is due to mango leaves fiber and polyphenolic active components that modulate the expression of transcriptional factors and enzymes related to adipogenesis [33][34][35].
High fructose consumption for a time causes a state of hyperglycemia, hyperinsulinemia, insulin resistance and finally induction of type-2 diabetes. Fructose consumption causes a decreased glycemic index and does not need insulin hormone to be trans-located into cells. While glucose needs insulin. After fructose sucking, it is imparted to the liver. Fructose can be metabolized in the liver to produce glucose (and glycogen), lactic acid, or de novo lipogenesis. Gluconeogenesis converts around half of the fructose consumed as an oral dosage (30-70g) to glucose after an overnight fast. The other fructose metabolic cascade that produces lactic acid happens only when fructose ingestion is substantial. Lactic acid is known to produce muscle pain and soreness. Excessive fructose consumption has been shown to enhance lipogenesis. The conversion of fructose to glucose raises the risk of type 2 diabetes. In addition, fructose can disrupt blood glucose balance by inducing insulin resistance in the muscles and liver. In human experiments, fructose has been reported to produce insulin resistance [36,37].On contrary administration of mango leaves to rats normalized blood glucose level, insulin level and sensitivity. These results was confirmed by previous studies that examined the effect of leaves powder, water extract and ethanolic extract on diabetic rats. The main reason that mango leaves have anti-diabetic properties is their active constituents principally mangiferin in addition to other constituents as anthocyanidins including cyanidin, delphinidin and peonidin, leucoanthocyanins, gallic tannins, catechin and flavonoids as quercetin [38][39][40][41].
Study results revealed that high fructose administration was accompanied by an inflammatory status increasing levels of inflammatory markers including TNF-α and leptin with decreasing anti-inflammatory adiponectin level. Excess adipogenesis and lipogenesis would occur as a consequence of high fructose intake, resulting in elevated adipocytokines like leptin. Despite the fact that leptin maintains energy homeostasis by balancing hunger, but excessive production leads to leptin resistance. Hyperphagia, obesity, and an increased percentage of fat mass occur as a consequence [42]. Because it produces and secretes various immune-modulatory substances known as adipokines, adipose tissue may be considered an endocrine organ. Proinflammatory adipokines (such as TNF-α) and anti-inflammatory adipokines (i.e., adiponectin) are two types of cytokines. Adipocyte function is disrupted in obesity, with increased expression of proinflammatory adipokines and decreased expression of anti-inflammatory adipokines, resulting in a chronic, low-grade inflammatory state [35].
The fluctuating production of these metabolites can lead to inflammation, hypertension and insulin resistance [43][44][45]. Individually, each of these adipo-secretomes exerts mild effects, but may collectively result in one or more systemic complications [32]. Mango leaves supplemented and treated rats showed decreased levels of pro-inflammatory cytokines as TNF-α and leptin with increasing anti-inflammatory adiponectin. This is attributed to anti-inflammatory characteristics of mango leaves active components including polyphenols and flavonoids making mango leaves as a good functional food for obese and metabolic syndrome individuals [35,29].
Mango leaves addition to rat's diet attenuated the genetic and epigenetic modifications caused by high fructose consumption. It was noticed that high fructose intake is linked with initiation of type 2 diabetes in rats with increased HDAC activity and decreased IRS-1 and GLUT-4 gene expression. Diabetes mellitus is characterized by persistent hyperglycemia. Hyperglycemia is due to the metabolic abnormalities. These abnormalities are explained by the defects in glucose uptake and insulin action. Defective regulation of glucose transporter-4 (GLUT-4) protein occurs due to the inhibition of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) followed by initiation of downstream signaling events that prevents downstream signaling and insulin action [46].
The role of epigenetic histone modifications in diabetes is under investigation. HDACs are group of enzymes that remove the acetyl group from histone proteins on DNA, making DNA less accessible to transcription factors. HDACs have been revealed to play a significant role in the regulation of several key genes linked to diabetes by regulating the acetylation of histone and transcriptional factors that re-control glucose homeostasis. As a result, HDACs play an important role in glucose metabolism and physiological insulin signaling. Thus, HDAC inhibition increase GLUT-4 translocation and insulin-induced glucose uptake in skeletal muscle [47].
High fructose diet detrimental effects are associated with the oxidative stress and that was confirmed by increased oxidative stress parameters in positive control rats. High fructose diet caused increased MDA and AGEs levels and decreased GSH level. High fructose diet increased lipid peroxidation leading to more formation of MDA. It also caused a state of hyperglycemia that initiate the formation of AGEs in tested rats. Opposing to this state of oxidative stress; antioxidants were depleted including GSH level [32,48,49]. Mango leaves attenuated oxidative stress due to their antioxidant capacity as they contain flavonoids and polyphenols [50].
The microscopic examination of pancreatic and adipose tissue supported the biochemical analysis results. Pancreas tissue examination of rats fed on high fructose diet revealed acute pancreatitis with congested blood vessels and mononuclear cells infiltrations thus may affect the function of β cells badly over time causing progression of type2 diabetes. While mango leaves active components protected pancreatic tissues from inflammation and consequent degeneration in supplemented and protected groups. Also, the microscopic examination of adipose tissue revealed increased size with abnormal distribution [49]. This is due to obesity caused by high fructose intake and confirmed by adipocyte size measurement. On the other hand addition of mango leaves powder to rat's diet controlled obesity and abnormalities in adipose tissues [32].

CONCLUSION
Mango leaves contain beneficial nutrients and active constituents with health benefits. By limiting metabolic and genetic abnormalities caused by high fructose consumption, either mango leaf supplementation or therapy improved and ameliorated all biochemical and microscopic data. The mango leaves supplemented group showed the most improvement. Mango leaves should be added to the human diet as a condiment to maintain health.

DISCLAIMER
The products applied in this research are frequently and most often used in our field of study and country. There is no conflict of interest between the authors and producers of the products because we do not plan to use them as a means of pursuing legal action, but rather to further knowledge. Furthermore, the research was not supported by the production firm, but rather by the authors' own efforts.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All authors hereby declare that they followed the "Principles of laboratory animal care" (NIH publication No. 85-23, updated 1985) as well as any applicable country laws. All animal experiments were carried out according to a procedure authorized by Ain Shams University's Local Institutional Animal Ethics Committee.

NOTE
The study emphasises the efficacy of "herbal medicine," an ancient practice still practiced in some parts of India. This ancient concept should be carefully reviewed in light of modern medical research, and if proven to be suitable, it can be used in part.