Antihyperglycemic and Antihyperlipidemic Effect of Persea americana in High Fat Diet and Low Dose Streptozotocin Induced T2DM Male Albino Wistar Rats

Background: Type II diabetes is a major health problem worldwide, and is increasing in an alarming rate globally and in Ethiopia due to change in dietary habits and sedentary life style. Even though there is no effective cure for diabetes, early control of blood glucose signi�cantly reduces the risk of diabetic complications. Different types of ingredients present in medicinal plants that act on a variety of targets by various modes and mechanisms are used to treat diabetes with minimum cost and side effect. Therefore, the objective of the present study was to investigate the antidiabetic effect of Persea americana mill fruit juice in high fat diet (HFD) and low dose Streptozotocin (STZ) induced type 2 diabetic (T2DM) male albino Wistar rats. Methods: Thirty six male albino Wistar rats weighing form 150-200g were divided in into six different groups: group I (normal control); Group II (diabetic control); Group III (metformin control) and Group IV – Group VI (treatment groups). Group I was fed on standard pellet and group II – group VI were fed on HFD for 4 weeks to induce pre-diabetes and insulin resistance followed by low dose STZ injection to induce T2DM. The treatment groups (group IV, V and VI) were given 632 mg/Kg, 1264 mg/Kg and 1896 mg/Kg/day of Persea americana fruit juice for six weeks, respectively to compare with normal, diabetic and 7mg/Kg metformin treated groups. After forty-ve days of treatment, the rats were fasted overnight (12 to 14 hours), anaesthetized and blood sample was collected by cardiac puncture for biochemical tests (fasting blood glucose (FBG), lipid pro�le, total protein and creatinine). The results were analyzed using SPSS version 22.0. One way ANOVA followed by Post hoc Tukey’s multiple comparisons were done to compare the mean differences among the experimental groups, and p-values < 0.05 were considered statistically signi�cant. Results: In high dose (1896 mg/Kg/day) Persea americana mill fruit juice treated group, food consumption, body weight, FBG, and LDL-C were signi�cantly reduced and HDL-C was signi�cantly increased (p < 0.005) compared with diabetic control group. Moderate dose (1264mg/Kg/day) treated group showed a decrease in FBG on 6 th week and improve HDL-C levels. Treating the rats with Persea americana fruit juice changed TG, total protein and creatinine levels although not signi�cant. Oral antidiabetes drug (metformin) signi�cantly reduced pellet consumption, body weight, FBG and lipid pro�le. Conclusion: Overall, Persea americana mill fruit juice showed antihyperglycemic and antihyperlipidemic effect particularly through reduction of fasting blood glucose, LDL-C and increasing HDL-C in T2DM induced


Background
Diabetes mellitus (DM) is a metabolic disorder, which either results from de ciency in insulin production by the pancreas or inability of the insulin produced to bind effectively to its receptor on the cell surface.Either of these conditions leads to accumulation of glucose in the blood and results in chronic hyperglycaemia (1,2).Untreated chronic hyperglycaemia can in turn lead to long-term complications, including micro-vascular and macro-vascular problems that cause disturbances of carbohydrate, fat and protein metabolism, and it covers a wide range of heterogeneous diseases (3).World health organization (WHO) estimated that globally, 425 million adults aged over 18 years were living with diabetes in 2017 and this will increase to 629 million by 2045 (4).The diabetes prevalence in sub-Saharan Africa, which was 12.1 million in 2016 is expected to rise to 23.9 million by 2030 (5).
Diabetes mellitus could be categorized into several groups: type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), gestational diabetes, and other speci c types, but the vast majority cases of diabetes fall into T1DM and T2DM broad categories (6).Type 1 diabetes is arisen from absolute de ciency of insulin secretion while the more prevalent category, T2DM, is due to combination of resistance to insulin action and an inadequate compensatory insulin secretory response (7).Both T1DM and T2DM are characterized by hyperglycaemia, excessive urine production, compensatory thirst, increased uid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism (8).
Type 2 DM is the most common type of diabetes and accounts for 90-95% of all diabetic cases, and mostly prevalent in people older than 45 years who are overweight (9).It is characterized by a signi cant insulin production ranging from less than normal to above normal, but the body is unable to utilize it e ciently (9,10).
The change in dietary habits and a sedentary lifestyle are the two main causes responsible for the development of T2DM when insulin resistance is established as a precondition (1,12).
Yet, there is no effective cure for diabetes, and the oral antihperglycemic drugs and insulin currently used in managing the disease are associated with several undesirable side effects.The use of oral antidiabetic drugs is limited due to their adverse side effects such as the haematological, cutaneous and gastrointestinal reactions, hypoglycaemic coma and impairment of liver and kidney functions.In addition, they are not suitable to use during pregnancy (2,8).Moreover, providing a modern medical healthcare across the world is still a far-off goal due to economic constraints (13).
Apart from the currently available therapeutic options, many herbal medicines have been recommended for the treatment of diabetes.A variety of ingredients present in medicinal plants are thought to act on a variety of diabetic targets by various modes and mechanisms (14).Many important drugs used in medicine today are directly or indirectly derived from plants due to its bioactive constituents such as; alkaloids, steroids, tannins and some plant phytochemicals, including cafestol, avonoids and carotenoids (15).Persea americana is the tree originated from Mexico and nowadays cultivated in different parts of the world, including Ethiopia (8,13).The phytochemical constituents of Persea americana have been studied and analysis had shown the presence of polyphenols that has one to two times more protein than any other fruit, contains low amount of simple sugar and appreciable amount of dietary ber, high in manganese, phosphorous, iron and potassium, but low in sodium, vitamin E, vitamin C, carotene, thiamin, ribo avin, nicotinic acid and folate (16).Persia ameriana fruit pulp has a various bene cial health effects such as anti-obesity, hepatoprotective, antiosteoarthritis, anti-carcinogenic effects, suppressing liver injury, wound healing activity and chemo-protective activities (17)(18)(19)(20)(21).It has also been reported to exhibit several pharmacological effects in ethnomedicine, including treatment for diarrhoea, intestinal parasites, skin and seed oil for weight loss (22,23).Thus, the present study was aimed to investigate the antidiabetic effects of Persia ameriana fruit pulp juice in high fat diet and low dose SZT induced diabetic male albino Wistar rats.

Methods And Materials
Study Design and Period An experimental study was conducted at animal laboratory of Biochemistry Department, College of Health Sciences, Addis Ababa University, Etiopia from December 2018 to May 2019 to investigate the antidiabetic effect of Persea americana fruit pulp juice in male albino Wistar rats.

Ethical Consideration
The study was conducted after the proposal was evaluated and approved by Department of Biochemistry Research and Ethical Review Committee (DRERC) by protocol no.M.Sc.13/17.All experimental activities conducted in the laboratory was in accordance to ethical declaration of national and international standards for experimental animals, which protect the right of experimental animal and minimize suffer, hunger, pain, thirst, injury, discomfort and fear to the best minimal level.

Sample Size Determination
An appropriate sample size determination is important since too small size misses the real effect in an experiment while a sample size larger than necessary will lead to wasting resources and ethical issues on sacri ced animals (24).Number of animals was calculated according to Federer rule :(k-1) (n-1) ≥15 where k, is the number of groups and n, number of subjects per group; and drop-out size (do) was estimated 10%, the minimal size of sample was determined as: ndo= 5/(1-do) 2 = 5/(1-0.1) 2= 5/0.81= 6.2≈6where ndo is minimal sample size (25).The size of sample in each group was at 6 rats.The study was conducted on six groups, so the total sample size was 6 groups × 6 rats = 36 rats.

Experimental Animals and Study Protocol
Male albino Wistar rats (4-5 weeks) weighing 150-200 g were obtained from Department of Pharmacology, College of Health Sciences, Addis Ababa University.The animals were housed in polypropylene plastic cages and maintained under standard laboratory conditions of room/optimum temperature and 12 hour light/dark cycle at Biochemistry animal laboratory and fed on a standard commercial rat pellet and water ad libitum.At 9 -10 weeks of age, the rats were randomly assigned into six groups.Throughout the experiment, each rat in the assigned group was identi ed by giving a speci c number on its tail by permanent marker.The allocation and treatment of the rats were as follows: Group I: Normal control rats injected citrate buffer and administered 3.5 ml/Kg of distilled water orally Group II: HFD and STZ induced diabetic rats that served as diabetic control and were given distilled water daily.
Group III: HFD and STZ induced diabetic rats treated with 7 mg/Kg of metformin orally Group IV: HFD and STZ induced diabetic rats treated with 632 mg/Kg of Persea americana fruit juice.Group V: HFD and STZ induced diabetic rats treated with 1264 mg/Kg of Persea Americana fruit juice.
Group VI: HFD and STZ induced diabetic rats treated with 1896 mg/Kg-BW/day of Persea Americana fruit juice.

High Fat Diet (HFD) Preparation
Many experiments have used rats fed with commercial lard (animal fat) as an obesity model (26).However, since commercial lard is not available in Ethiopia, the HFD used in this experiment was prepared from locally available bovine fat.To prepare the HFD, 40% puri ed fat was mixed with 60% of pellet (40%/60% w/w).Since the standard pellet powder consists of 20% fat, 60% carbohydrate and 20% protein (27), the nal percentage of fat is 52% (i.e., 52%/100% W/W).Hence, the term, 'high fat diet' used in this study refers to a diet containing 52% fat, 36% carbohydrate and 12% of protein.

Induction of Type 2 Diabetes Mellitus
There are different methods to induce diabetes mellitus in animal model (28).In our study, high fat diet (HFD) and low dose streptozotocin (STZ) induced diabetic rat model was used.This model involves a combination of a high fat diet to bring about hyperinsulinemia, insulin resistance and glucose intolerance followed by treatment with the β cell toxin STZ, which results in a severe reduction in functional β cell mass.These two stressors were designed to induce the features of human T2DM pathology to the rats within a shorter period of time scale.

Streptozotocin Injection
After 4 weeks of dietary manipulation, the rats were fasted overnight and injected intraperitoneally with freshly prepared STZ at a concentration of 35 mg/Kg body weight in 0.1M citrate buffer (pH = 4.5).The rats were given 5% glucose solution from 4 to 8 hours to reduce STZ induced hypoglycemia (29), and fasting blood glucose was determined after 72hrs by glucometer.The rats with blood glucose level above 200 mg/dL were considered as diabetic and those with normal blood glucose level were re-injected STZ to induce T2DM.

Preparation of Persea americana Fruit Juice and Dosage Calculation
Persea americana mill fruit was purchased from Jimma town, 350 Km South West of Addis Ababa, Ethiopia and identi ed by taxonomists in national herbarium, Addis Ababa University.The ripen fruit was washed carefully with distilled water and cut off by spoon, peeled off and the seed removed.Then the edible part of fruit was mixed by blender to prepare a juice as consumed by humans.In our study, Persea americana juice was used to treat diabetic induced male albino Wistar rats.The juice was prepared by adding water to 80gm of wet fruit pulp up to 200ml mark.Then the juice was dried to know the weight of the fruit powder that is found in juice.The dry mass of the fruit in 200ml juice was found to be 44.5gm.The volume of juice given to rats was also extrapolated from volume of human daily consumption, which is (200ml of juice /day in 70Kg).In this study the average body weight (BW) of rats were 250gm.Therefore, the volume of juice given to rats was 0.71ml.The approximate amount of Persea americana fruit pulp powder in 0.71ml of juice is approximately158mg.Accordingly, 158mg, 316mg and 474mg /250gm BW/day was considered as a low, moderate and high dose of Persea americana fruit pulp juice, respectively.Similarly, 0.7, 1.4 and 2.1ml/250gm BW/day were considered as low, moderate and higher volume that is given to rats daily.The doses (158mg, 316mg and 474mg /250gm BW/day) were converted to standard unit (mg/Kg) as 632, 1264, and 1896mg/Kg/day, respectively and used in this thesis document.The Persea Americana fruit juice was administered for six weeks orally from 9:30-10:30 AM.

Extrapolation of Metformin Dose
Safe and effective drug dosing is necessary regardless of its purpose of administration.There are several instances where in the initial dose of a particular drug is unavailable in a speci c species.Therefore, choosing starting dose of such drugs for research, experiments, or clinical trials in animals and humans is a concern.The human dose of metformin drug was extrapolated to animal dose as follows (30).

Blood Sample Collection, Serum Preparation and Storage
At the end of the experiment, the rats were fasted overnight and euthanized by anesthetizing with diethyl ether and then blood was collected by direct cardiac puncture to serum separator tube.Then, the blood was left at room temperature for 30 minutes to coagulate and centrifuged at 3000 rpm for 10 minutes.The serum sample was transferred to necked tube and stored in deep freezer at -80 °C until the biochemical analyses were done.

Biochemical Tests
Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), total protein (TP) and creatinine were determined with chemistry analyzer.Low density lipoprotein cholesterol (LDL-C) level was calculated using Frieldwald's formulae (31).Overnight fasting blood glucose was collected from the tail of the rats, and measured with One Touch GlucoSure on 0, 14 th , 28 th and 42 th days of the experiment.

Statistical Data Analysis
The statistical data were analyzed using SPSS version 22. 00.The results of food consumption, body weight and biochemical parameters were expressed as mean ± SEM.The statistical differences between the means were examined using analysis of variance (ANOVA) followed by Post Hoc Tukey's multiple comparisons.The p-values < 0.05 were declared as statistically signi cant.

Effect of Persea americana on Food Intake
The food intake of diabetic control group rats was signi cantly increased (p = 0.001) when compared with normal control group.In high dose treated group, the mean pellet consumption was signi cantly reduced when compared with diabetic control group (p = 0.004).However, no signi cant difference was observed in moderate and high dose treated group compared to the normal control group (Table 1).

Effect of Persea americana on Body Weight
Before the induction of T2DM, there was no signi cant difference in the mean values of body weight among all groups of the rats.However, at the end of experiment, the mean body weight of diabetic control, low dose Persea americana and metformin treated groups signi cantly decreased compared to body weight of normal control group (p = 0.001; 0.007 and 0.044), respectively.Interestingly, the mean body weight of high dose treated group signi cantly increased compared with that of diabetic control group (p = 0.002).There was no signi cant difference in body weight between normal control and high dose treated groups (Figure 1).

Effects of Persea americana on Fasting Serum Glucose Level
Before treatment, the fasting serum glucose levels were signi cantly increased in all diabetic induced rats compared with normal control (p < 0.05).The mean value of fasting serum glucose was signi cantly reduced in rats treated with high dose of Persea americana on 4 th (p = 0.036) and 6 th week (p =0.012) compared with diabetic control group.Statistically signi cant decrease in serum glucose was also observed in moderate dose Persea americana juice treated group after the sixth week of treatment as compared with diabetic control group (p = 0.02).
Metformin treated group showed signi cant reduction in fasting serum glucose on the 2 nd (p = 0.044), 4 th (p = 0.021) and 6th (p = 0.013) weeks of treatment, respectively compared with diabetic control group (Figure 2).

Effects of Persea americana on Lipid Pro le
After the induction of diabetes and subsequent treatment with Persea americana juice, the mean values of TC (p = 0.006) and LDL-C (p = 0.007) in high dose treated diabetic induced rats were signi cantly reduced when compared with the mean values in the diabetic control group.In metformin treated group, the mean values of serum TC (p = 0.003) and LDL-C (p = 0.006) were signi cantly decreased compared with that of the diabetic control group.The mean values of TC (p = 0.002) and LDL-C (p = 0.004) in diabetic control group were signi cantly increased compared with that of the normal control group.Interestingly, there was no signi cant difference in mean values of TC and LDL-C in moderate and high dose Persea americana juice, and metformin treated groups when compared to normal control group.The mean value of fasting serum HDL-C was decreased in diabetic control rats, compared to normal control group (p = 0.002).On the other hand, the mean value of HDL-C was signi cantly increased in moderate (p = 0.046) and high (p = 0.034) doses of Persea americana and metformin (p = 0.014) treated groups as compared with diabetic control group.No signi cant difference was found in mean HDL-C level among the normal control, metformin control and high dose treated groups.The mean value of TG did not show any signi cant difference among all the study groups (p > 0.05), (Table 2).

Effects of Persea americana on Total Protein and Creatinine
Regarding serum total and creatinine levels, no signi cant difference was observed among the study groups (Table 3).
Table 3.The effects of Persea americana fruit juice on total protein and creatinine of diabetic rats The results are expressed as mean ± SEM (n=6); TP -Total Protein; NC -normal control; DC -diabetic control; Mtmetformin control; A -low dose treated group; B -moderate dose treated group; C -high dose treated group

Discussion
Persea americana is a fruit used as human food and in treating different diseases.In the present study, Persea americana juice was used to treat high fat diet and low dose STZ induced T2DM in male albino Wistar rats for six weeks.The effect of Persea americana on food consumption, body weight, fasting blood glucose, lipid pro le, total protein and creatinine was investigated in the diabetic rats.
In this study, food intake was higher in diabetic control group compared with in normal control group.It was also observed that the food consumption in high dose and moderate dose Persea americana juice treated groups of rats was lower than in diabetic control group.In addition, the pellet intake was reduced in the high dose treated group when compared with pellet intake in metformin treated group.The appetite decreasing of effect of Persea americana fruit juice is due to the presence of dietary ber and fruit oil matrix, which acts by slowing gastric emptying and increasing early satiety in the rats (32,33).
Slight body weight loss was observed in HFD and low dose STZ-induced diabetic rats following oral administration of both Persea americana fruit juice and metformin.In diabetic rats, decreased body weight may be due to an excessive breakdown of tissue proteins and lipid to provide energy as the body cannot use carbohydrate due to insulin insu ciency (34).The improvement in body weight seen in diabetic rats treated with Persea americana juice is most probably due to an improved metabolic activity that makes the body system more capable of maintaining blood glucose homeostasis (35).Similarly, 7mg/Kg of metformin treated rats gained body weight when compared with the diabetic group after six weeks of the treatment.However, the effect of metformin was less compared with that of Persea americana juice in normalizing body weight.The study done by Duerta et al. also reported that the β-sitosterol found in Persea americana showed body weight loss activity by reducing compulsive eating and fat accumulation in the abdominal region (36).The bioactive compounds found in Persea americana fruit juice may help in suppressing free radicals generation due to hyperglycemia and control muscle over wasting that resulted from glycemic control in treated diabetic rats, and ultimately lead to normalize the level of body weight (37).
In the rst week of the experiment, fasting blood glucose concentration was elevated in all diabetic induced rats compared with normal control rats.This increase in fasting blood glucose concentration is an important characteristic feature of T2DM.However, for diabetic rats that received Persea americana juice, their fasting blood glucose was non-signi cantly reduced in the second week of the experiment.The fasting blood glucose level was signi cantly decreased on fourth and sixth weeks of the experiment in rats treated with moderate and high dose of Persea americana pulp juice when compared with glucose level of diabetic control rats.These results agree with Rao and Adinew's results that reported albino Wistar rats received ethanolic extract of Persea americana fruit 300 mg/Kg/day, orally for 4 weeks, showed a decrease in the fasting blood glucose level, glycosylated hemoglobin, blood urea, and serum creatinine (35).Similar nding was also reported by Thenmozhi et al. in which treatment of Sprague Dawley rats with n-hexane fraction from hydromethanolic (2:3) extract of Persea americana fruit 30mg/Kg orally for 8 weeks showed decrease in fasting blood glucose level (38).A randomized clinical trials study done by Sabate et al. on 26 healthy overweight individuals also revealed that consumption of half avocado signi cantly reduced the blood insulin and glucagon-like peptide-1 levels (39).Moreover, the results of Wien et al. investigation on healthy overweight adults showed that avocado in lunch meal attenuated rise in postprandial blood insulin levels 30 min after start of the lunch meal and diminished the desire to eat compared with the avocado-free control group (33).On the other hand, low dose Persea americana juice did not cause signi cant change in fasting blood glucose throughout six weeks of the treatment, but decreased the levels to some extent.
The antihyperglycemic effect of avocado fruits may be related to insulin mimetic or stimulatory effect and its ability to stimulate the remaining pancreatic βcells in animal models, making them able to secrete more insulin (35).The phytochemicals like avonoids, saponins, tannins and alkaloids found in avocado act as anti-oxidants and contain insulin stimulating substances such as insulin receptor substrate, glycogen synthase and glucose dependent insulotropic polypeptide.Persea americana could decrease glucose level by increasing insulin secretion or peripheral glucose utilization in the gut of normal treated rats (40).The D -manno heptulose found in Persea americana may also be responsible for hypoglycemic effect by decreasing the rate of glycolysis via hexokinase inhibition and weight control via appetite reduction.
In metformin treated group, the fasting blood glucose is signi cantly reduced starting from second week of the experiment when compared with diabetic control group.Metformin reduces hyperglycemia by reducing gluconeogenesis, increasing insulin receptor sensitivity, especially in muscle cells, and decreasing glucose uptake in the intestine (41)(42)(43).On the sixth week, no signi cant difference was found in fasting glucose levels among rats treated with moderate and high dose of Persea americana juice and metformin.
In addition to its anti-hyperglycemic effect, Persea americana fruit juice also showed the ability to alter lipid pro le (TC, LDL-C -C, TG, and HDL-C -C) levels in diabetic albino Wistar rats.The abnormally high concentration of serum lipids in diabetes is mainly due to the increase in the mobilization of free fatty acids from the peripheral depots as insulin inhibits the hormone sensitive lipase.In our study, there was a signi cant increase in the serum TC and LDL-C levels in diabetic rats as compared with normal control rats.The elevated TG level in diabetic rats might be due to the consequence of increased synthesis of triglyceride rich lipoprotein particles (VLDL-C) in the liver and diminished catabolism.Since insulin has a potent inhibitory effect on lipolysis in the adipocytes, insulin de ciency is associated with excess lipolysis and increased in ux of free fatty acids to the liver (44).The increase in LDL-C and VLDL-C levels in the HFD/low dose STZ induced diabetic rats might also be due to over production of LDL-C and VLDL-C by the liver through stimulation of hepatic triglyceride synthesis as a result of free fatty acid in ux.
The increased pool of triacylglycerol-rich lipoproteins, mainly VLDL-C1 observed in type 2 diabetes promotes CETPmediated triacylglycerol enrichment of HDL-C particles, and as a consequence enhances HDL-C catabolism, while depleting them from cholestryl esters, thus decreasing HDL-C cholesterol level (45).
Administration of high dose of Persea americana fruit juice decreased the mean serum TC and LDL-C levels in HFD and low STZ induced diabetic rats compared with diabetic control group.Our results agree with of Pahua-Ramos et al.'s results in which administration of reduced-calorie avocado paste 2 g/Kg/day for 7 weeks showed a signi cant reduction in TC, LDL-C-C and increased insulin sensitivity in high cholesterol and fructose diet fed rats (46).Similar in a study done by Elbadrawy and Shelbaya, administration of hydro-alcoholic extract of avocado130 and 150 mg/Kg/day administrated via stomach tube for 8 weeks showed a signi cant reduction in serum cholesterol, LDL-C-C, and VLDL-C-C, while, the serum level of HDL-C-C was enhanced in HCD rats (47).In addition, in study done by Al-Dosari, oral administration of Persea americana pulp 1 and 2 ml/rat/day for 10 weeks showed a signi cant decrease in serum cholesterol, LDL-C-C, VLDL-C-C and TG levels (48).The LDL-C and TC reducing effect of Persea americana fruit pulp juice is due to its phytosterols, phytostanols and dietary ber, which help reduce cholesterol reabsorption in the intestine and promoting fecal cholesterol excretion, which in turn reduces the level of LDL-C in plasma, increasing the amount of cholesterol excreted from the body and decrease in hepatic cholesterol synthesis (49,50).
In addition, the soluble dietary ber found in avocado juice pulp may decrease the LDL-C and TC levels by binding to bile acid and alters micelle formation, and results in decreased micelle absorption in the small intestine.Moreover, the extract of avocado fruit inhibits the action of acetyl-CoA carboxylase, a key enzyme catalyzing the committed in fatty acid synthesis (51).Treating diabetic rats with metformin decreased the serum TC and LDL-C at the end of the experiment (6 th weeks).Metformin increases insulin sensitivity by decreasing the rate of lipolysis there by slowing the conversion of FFA to lipoprotein precursors in the liver.
Interestingly, the mean value of HDL-C was signi cantly increased in in diabetic rats treated with double and triple doses of Perseaamericana fruit juice when compared with diabetic control rats.This result is in line with the result of Elbadrawy and Shelbaya, in which administration of hydroalcolic extract of avocado pulp 150 mg/Kg for 8 weeks increased HDL-C in HCD rats (47).The possible mechanism by which avocado enhances HDL-C serum level may be by regulating the hydrolysis of certain lipoproteins and their selective uptake, and metabolism by different tissues due to presence of tocopherols, phytosterols and polyphenols in the fruit.Avocado also modi es the structure of the HDL-C lipoprotein by increasing paraoxonase-1 enzyme activity, which is responsible for the hydrolysis of lipid hydroperoxides (52).In the same way, the HDL-C cholesterol level was signi cantly increased in diabetic rats treated with metformin drug compared with in diabetic control rats.
On the other hand, no dose of avocado showed signi cant effect on serum TG level in all treatment groups compared to diabetic control group (p > 0.05).However, there was a decrease in mean value of serum TG after the treatment.This result is in agreement with Pieterse's, in which 200g/day of avocado substituted for 30 g of other mixed dietary fats for 6 weeks didn't signi cantly change the TG, HDL-C, LDL-C and TC levels in energy-restricteddiet volunteers (53).On the contrary to our study, study done by Shehata and Soltan revealed treatment of hypercholesteromic rat with 30% avocado fruit for 4-weeks reduced TG levels signi cantly (54).This difference might be due to the difference in extraction method used.
In T2DM, insulin resistance does not alter only glucose and lipid metabolism, but also protein metabolism.In the present study, the total protein level was reduced in diabetic rats relative to in normal control rats.Diabetes mellitus reduces total protein level by increasing muscle proteolysis, reducing protein synthesis and stimulating hepatic gluconeogenesis.Although the differences were not signi cant, treating diabetic rats with different doses of avocado fruit juice was found to improve the total protein concentration.The metformin treated group also did not show signi cant difference in protein level compared to diabetic control group.In diabetic rats, breaking down of liver and plasma protein enhances serum creatinine level.The administration of Persea americana fruit juice and metformin to diabetic rats decreased serum creatinine level insigni cantly.This indicates that the effect of Persea americana in normalizing protein metabolism disturbance is comparable to that of metformin.

Conclusion
The present study revealed that the antihyperglycemic and antihperlipidemic effect of Persea americana fruit juice in HFD/low dose STZ induced T2DM albino Wistar rats is a dose dependent.Particularly, high dose (1896 mg/Kg/day) of Persea americana juice signi cantly reduced pellet consumption than metformin.Similarly, Persea americana friut juice showed the effect of normalizing body weight and reducing fasting blood glucose level.
Treating diabetic rats with Persea americana juice (1264 mg/Kg, 1896mg/Kg/day) showed signi cant fasting blood glucose reduction on the 4 th and 6 th weeks.It was also found that Persea americana fruit pulp also has a potential reducing effect on LDL-C, TC and increasing effect on HDL-C in STZ and HFD induced diabetic albino rats.Overall, the ndings of our study showed that Persea americana fruit pulp juice has the effect of normalizing metabolic disturbances of carbohydrate and lipid caused by T2DM and HFD, hence it may be helpful in reducing the risk of diabetic complications.

Table 1 .
The effects of Persea americana mill fruit juice on food consumption signi cant difference between all groups as tested by one way ANOVA.The superscript letters "a" and "b" indicate the signi cant differences compared to normal control and diabetic control groups, respectively.
The values are expressed as mean ±SEM.FIT-food intake; NC (I) -normal control; DC (II) -diabetic control; M -(III)metformin control; A (IV) -low dose treated group; B (V) -moderate dose treated group; C (VI) -high dose treated group; * -

Table 2 .
Effects of Persea americana fruit juice on lipid pro le in diabetic rats The results are expressed as mean ± SEM (n=6); NC -normal control; DC-diabetic control; Mt -metformin control; A -low dose treated group; B -moderate dose treated group; C -high dose treated group; TG -Triglyceride; TC-Total Cholesterol; HDL-C, High Density Lipoprotein Cholesterol; LDL-C, Low Density Lipoprotein Cholesterol; a,bstatistically signi cant (p < 0.05) compared with normal and diabetic control, respectively.