Young and mature leaves of Azadirachta indica (neem) display different antidiabetic and antioxidative effects

ABSTRACT Azadirachta indica (AI), popularly known as neem in India, has been used in the Indian Ayurvedic medicinal system for thousands of years in the management of varied ailments. The present study was undertaken to evaluate the antioxidant, antidiabetic, and anti-inflammatory activity of the crude ethanolic extract of the young and mature leaves of Azadirachta indica. We used FTIR to analyze the functional groups present in ethanolic AI extracts. Extracts of the young (YLE) and mature leaf (MLE) were administered to streptozotocin-induced diabetic rats. YLE and MLE reduced blood glucose, restored weight and maintained insulin and lipid levels, highlighting the antidiabetic action of both extracts. Significantly, the effect observed with YLE was greater as compared to MLE. The oxidative stress biomarkers were also normalized with respect to type 1 diabetic control. FTIR analysis showed several peaks present in the YLE 30 which were absent in the MLE. We found a decrease in the levels of Q3 inflammatory cytokines (IL-6 and TNF-alpha) in type 1 diabetic rats supplemented with YLE. YLE displays better anti-diabetic activity in comparison to MLE. Our results lend credence to the use of YLE of AI leaves as a useful supplementary candidate for future diabetes management therapy.


Introduction
It is estimated that there are about 419 million adults aged between 20 and 79 years old worldwide who are currently living with diabetes; this situation is alarming and it is projected to reach 642 million in the year 2040, an increase of 55% worldwide [1].A more worrying and disturbing situation is that over 80% of those afflicted live in medium-and low-income countries where access to modern drugs is limited.Due to the lack of a known cure for diabetes mellitus and the attendant toxicity of the existing drugs used in the management of DM, the World Health Organization encourages the scientific community to explore the possibilities of using plants and plant products in the search for an intervention [2,3].
Spurred by the history and success of the drug artemisinin from Artemisia annua and metformin from Galega officinalis plants, we are encouraged to revisit Azadirachta indica (AI) to search for antioxidant and antidiabetic activity.Azadirachta indica A. Juss is commonly referred to as the neem tree, belonging to the Meliaceae family.
All over the world, neem is widely used as an alternative therapy in the treatment, management and control of various ailments [4,5].An earlier report supported the role neem and its ingredients play in preventing the pathogenesis of disease and scavenging free radical production.Studies using animal models have shown that neem and its main components are essential for managing cancer through the modulation of several molecular pathways, including p53, pTEN, NF-B, PI3K/Akt, Bcl-2, and VEGF.It is regarded as a safe medicinal herb that modifies a wide range of biological functions without causing any negative side effects [6,7].Although many studies have been conducted on neem, to the best of our knowledge there has been none comparing the antidiabetic activity of the ethanolic extract of the young and matured leaf.There are reports that different parts of a plant have different activity and that there is a difference in the chemical composition of the young and mature leaves of the same plant; young parts of the plant contain a higher amount of phytochemicals, ascorbic acid and chlorophyll when compared to the matured leaves [8,9].
The present investigation is carried out to evaluate the elemental constituents of young and mature leaf extract of Azadirachta indica exploring the antidiabetic activity of the young and mature leaf extract of AI by assessing the antioxidant, oxidative stress indicators, and the plasma membrane redox system after the induction of diabetes in experimental rats.

Plant material
Azadirachta Indica leaves were collected from the Science Faculty Campus, University of Allahabad, India, in March 2017 (the voucher specimen was confirmed and deposited in the Herbarium of the Botany Department, Faculty of Science, University of Allahabad).

Extract preparation
The leaves were washed under running tap water and then with distilled water, blotted dry with tissue paper, air-dried under shade (25-29°C) and milled into a coarse powder.Four hundred grams (400 g) of the powder was added to 800 ml of absolute ethanol and homogenized.The resultant homogenate was kept in the refrigerator (4°C) for 48 h and thereafter filtered using a cheese cloth followed by Whatman filter paper no. 1.The filtrate was then lyophilized to complete dryness [10].

Experimental animals
The experimental rats (36 male Wistar rats, 4 months old) were allowed to stabilize in the laboratory environment for 1 week, and then randomly assigned equally into six groups: Group I -Normal control (NC), receiving no treatment at all Group II -Type 1 Diabetic control (DC) Group III -Type 1 Diabetic + YLE, diabetic treated with young leaf extract (YLE) 200 mg/ kg body weight.
Group IV -Type 1 Diabetic + MLE, diabetic rats treated with mature leaf extract (MLE) 200 mg/kg body weight Group V -Normal + YLE, normal rats received 200 mg/kg body weight Group VI -Normal + MLE, normal rats received 200 mg/kg body weight Animals were kept in a temperaturecontrolled room (25 ± 5°C) with 12-h lightdark cycles.All rats had free access to drinking water and were fed a normal laboratory diet.The protocol of this study conforms to the guidelines of the Animal Care and Ethics Committee of the University of Allahabad.

Induction of diabetes with Streptozotocin (STZ)
The method of [11] was used to induce type-1 diabetes and briefly described; diabetes was induced in overnight fasted experimental rats with STZ (45 mg/kg body weight) dissolved in freshly prepared citrate buffer (0.1 M, pH 4.5).STZinjected animals were provided with a 20% glucose solution to drink overnight to overcome the initial drug-induced hypoglycemic mortality.After 4 days, rats with fasting blood glucose ≥250 mg/ dl were considered Type 1 diabetic and included in the study.

Extract administration
Azadirachta indica young and matured leaf extract was dissolved in distilled water (vehicle) and administered orally via gastric intubation at a dose of 200 mg/kg body weight.

Collection of blood, isolation of red blood cells and plasma
At the end of the experimental period (28 days), blood samples were collected by cardiac puncture into 10 unit/ml heparin anticoagulant syringes, and were then processed to obtain plasma and packed red blood cells (PRBC).The red blood cells were pelleted by centrifugation at 800 × g for 10 min at 4°C.The plasma was removed and immediately frozen at −80°C until used for assays; the buffy coat and the RBCs were washed thrice with cold phosphate-buffered saline (PBS) (0.9% NaCl and 10 mmol L −1 Na 2 HPO 4 ; pH 7.4) and then used for the experiment.Blood glucose values were determined using an Accu-Chek Active Glucometer (Roche Diagnostics, Mannheim, Germany).
Determination of serum triglyceride and total cholesterol was performed using reagent kits from Span diagnostic and ERBA diagnostics and measurements were made on an Erba Mannheim Chem.-7 analyzer.Fasting insulin level was measured by the ELISA kit supplied by Krishgen Biosystems, India.

FTIR spectroscopic analysis
Fourier transform infrared spectrophotometer (FTIR) was used for identifying the types of chemical bonds (functional groups) present in compounds.The FTIR spectra were obtained using an FTIR-8400S Fourier transform infrared spectrophotometer (Germany).The spectra were recorded in transmission mode from 4,000 to 500 cm −1 (mid-infrared region) at a resolution of 0.44 cm −1 .

Erythrocyte reduced-glutathione (GSH) determination
The method of Beutler et al. [12] was used to determine the erythrocyte GSH.The method is based on the ability of the -SH group to reduce 5,5 0-dithiobis, 2-nitrobenzoic acid (DTNB) and form a yellow-colored anionic product whose optical density is measured at 412 nm.The concentration of GSH expressed in mg mL −1 PRBC sand was determined from the standard plot.

Erythrocyte PMRS activity assay
Erythrocyte PMRS activity was measured by the reduction of ferricyanide as described earlier [13].Briefly, 0.2 ml of packed RBC was suspended in PBS containing 5 mM glucose and 1 mM freshly prepared potassium ferricyanide to a final volume of 2.0 ml.The suspensions were incubated for 30 min at 37°C and then centrifuged at 800 × g at 4°C.The supernatant was collected and assayed for ferrocyanide content using 4,7-diphenyl-1,10-phenanthroline disulfonic acid disodium salt, and absorption was recorded at 535 nm (0 = 20,500 M −1 cm −1 ).The results are expressed in mmol ferrocyanide/ml PRBC/30 min.

Determination of erythrocyte MDA content
Erythrocyte MDA was measured according to the method of Esterbauer and Cheeseman [14] with slight modification.0.2 ml of Packed RBC was suspended in 3 ml GPBS containing 0.5 mM glucose, pH 7.4.0.2 ml of the suspension was then added to 1 ml of 10% TCA and 2 ml of 0.67% thiobarbituric acid (TBA), boiled for 20 min at 90-100°C, and then cooled.This resultant mixture was then centrifuged at 1000 × g for 5 min, and the absorbance of the supernatant was read at 532 nm.The concentration of MDA in erythrocytes was calculated using extinction coefficient (e = 31,500) and is expressed as nmol mL −1 of packed erythrocytes

Determination of plasma sialic acid
The method proposed by [15] was used and described briefly; To 500 μl of sample solution diluted (20 times), 0.10 ml of 0.04 M periodic acid was added.The solution was thoroughly mixed, and the resultant mixture was kept in an ice bath for 30 min.Thereafter, 1.25 ml of resorcinol working solution brought to a final volume of 50 ml with 10 M HCl was added, mixed, and incubated in the water bath at 98°C for 5 min.Lastly, 3.25 ml of n-butanol was added.The resultant solution was mixed vigorously, and the tubes were placed in a water bath at 37°C for 3 min for stabilization of color.The absorbance was measured immediately at 625 nm.

Total antioxidant activity by FRAP method
The total antioxidant potential of the plasma was determined using a modification of the ferric reducing ability of plasma (FRAP) assay as reported by Benzie and Strain [38].FRAP reagent was prepared from 300 mmol L −1 acetate buffer, pH 3.6, 20 mmol L −1 ferric chlorides and 10 mmol L −1 2,4,6-tripyridyl-s-triazine made up of 40 mmol L −1 hydrochloric acids.All three solutions were mixed in the ratio of 10:1:1 (v/v/v), respectively, and 3 ml of FRAP reagent was then thoroughly mixed with 100 ml of plasma.The absorbance was read at 593 nm at 30-s intervals for 4 min.FRAP values are expressed as mmol Fe(II)/L plasma.

Advanced Oxidation Protein Products (AOPP) assay
The assay of AOPP levels was performed by modification of the method of Witko-Sarsat et al., [16].Two milliliters of plasma was diluted with 1:5 in PBS, and 0.1 ml of 1.16 M potassium iodide was then added to each tube, followed by 0.2 ml acetic acid after 2 min.The absorbance of the reaction mixture was immediately read at 340 nm against a blank containing 2 ml of PBS, 0.1 ml of KI and 0.2 ml of acetic acid.The chloramine-T absorbance at 340 nm was linear within the range of 10-100 mmol L −1 , and AOPP concentrations were expressed as mmol L −1 chloramine-T equivalents.

Determination of membrane protein carbonyls
The method of [17] was used to measure the erythrocyte membrane protein carbonyls.Erythrocyte membrane samples (0.2 mL) in PBS/ 0.4 mL plasma were taken in 2 tubes as test and control samples.A total of 4.0 mL of 10 mmol L −1 2,4-DNPH, prepared in 2 mmol L −1 HCl, was added to the test sample, and 4.0 mL of 2 mmol L −1 HCl alone was added to the control sample.The contents were mixed thoroughly and incubated for 1 h in the dark at 37°C.Twenty percent trichloroacetic acid (TCA) (w/v) was added to both tubes, and the mixture was left on ice for 10 min.The tubes were then centrifuged at 850 × g for 20 min to obtain protein pellets.Finally, the protein pellets were dissolved in 6 mmol L −1 guanidine hydrochloride and incubated for 10 min at 37°C.Spectra of the supernatant were taken at 370 nm.The carbonyl content was calculated using an absorption coefficient of 22,000 mol L −1 cm −1 , and data were expressed in nmol mg −1 protein.

Measurement of cytokines levels in the serum
Cytokine levels were measured according to the instructions provided by the company (Krishgen Biosystems-India) and following the previously published paper by [18].The plate was read at 450 nm by using an ELISA microplate reader (Spectrostar Nano-BMG LabTech, Germany).The results are reported in Pg/mL.

Statistical analysis
Statistical analyses for the experiments were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA.Results are expressed as the mean ±SD for statistical analysis of the data group means and were compared by one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test.P < 0.05 was considered to be statistically significant.

Results
General biological parameters of experimental rats (body weight, fasting insulin, triglyceride, and total cholesterol level) are mentioned in Table 1.Body weight and fasting insulin level are significantly (P < 0.05) decreased in the diabetic group (DC) when compared with the control group (NC), while the YLE-supplemented group shows a significant (P < 0.05) increase in both values.The MLE-supplemented rats also show a decreased value of both parameters.Triglyceride and total cholesterol levels significantly increased (P < 0.05) in DC groups of rats, while the YLE-supplemented group showed significantly decreased (P < 0.05) the level of triglyceride and total cholesterol.
The blood glucose level of Type 1 diabetic rats was significantly higher when compared to the normal control rats as shown in Figure 1a.The oral administration of the YLE and MLE at a dose of 200 mg/kg body weight to the experimental rats lowered the blood glucose level as compared to the untreated diabetic rats (P < 0.05).The dose of 200 mg/kg body weight each of YLE and MLE restored blood glucose to normal levels, but YLE showed better action when compared to the untreated group and MLE supplemented group, which support our FT-IR spectrum of active component found greater content in YLE with respect to MLE.
FT-IR spectrum analysis: The FTIR spectrum was used for the identification of the functional groups of the active component in the plant based on the values of the peak in the region of the infrared radiation.The young and matured leaf extract of AI was passed into the FTIR, and the separation of the functional groups of the components was based on the peak ratio.FT-IR spectroscopic analysis revealed the presence of alcohols, phenols, alkanes, alkynes, alkyl halides,  [39,4].
The result of this study showed that glutathione (GSH) values in Figure 3a of diabetic rats were significantly (P < 0.05) decreased when compared to the rats in the control group.Treatment with young and matured leaf ethanolic extract of AI (200 mg/kg body weight) caused a significant (P < 0.05) increase in the GSH level compared to diabetic control groups.The results also support our FT-IR spectrum data that YLE contains greater active phenolic content with respect to MLE, so it has better radical saving activity when compared with MLE.Plasma membrane redox system (PMRS) activity in erythrocytes of the experimental rats is shown in Figure 3b.It was observed that there was a significant (P < 0.05) increase in the erythrocytes PMRS activity in the diabetic control group when compared to the normal control group.The administration of both AI extracts (200 mg/kg body weight) caused a significant (P < 0.05) reduction in the PMRS activity.
Lipid peroxidation measured in terms of malonaldehyde (MDA) is presented in Figure 3c.It was observed that the MDA level in erythrocytes was significantly increased in diabetic rats.The administration of young extract of AI shows a graphical representation of the effect of STZ administration on the blood glucose levels of experimental rats and the resultant effect on the rats when the YLE and MLE 200 mg/kg was administered.There was a significant increase (P < 0.05) in the blood glucose levels of the diabetic control when compared to normal, and there was a significant decrease (P < 0.05) in blood sugar level of the treated groups when compared to the diabetic group.The values are given as mean ± standard deviation of replicate determinations (n = 6).STZ-induced diabetes resulted in significantly (P < 0.05) increase level of PMRS compared to normal control groups.The oral administration of AI extract (200 mg/kg body weight) up to 28 days per day significantly (P < 0.05) decreased level of PMRS in STZ-induced diabetic rats.Values are expressed as mean ±SD for 6 rats in each group.c) Lipid peroxidation as malonaldehyde (MDA) levels in normal and experimental groups.The MDA levels of STZ-induced diabetic rats were significantly higher (P < 0.05) when compared with the normal control rats, however.Oral administration of AI extract offered significantly (P < 0.05) decreased in MDA level in diabetic rats.para.d) Total antioxidant capacity of plasma measured in terms of FRAP value the experimental rats.FRAP value is expressed as µmol Fe(II) per l of plasma.Data are represented as mean ± SD (n = 6).Significant (P < 0.05) difference was obtained between the normal and STZ-induced diabetic rats.(200 mg/kg body weight) to diabetic rats induced a significant (P < 0.05) decrease in MDA levels in erythrocytes when compared to the diabetic control group, which confers its antioxidant property.MLE-supplemented group also shows a decrease in the MDA level, but YLE provides better results because it contains a greater amount of active components with respect to MLE which was already reported in our FI-TR spectrum result.
Animals of the diabetic group showed a significant (P < 0.05) decrease in plasma FRAP value when compared to the normal control groups, Figure 3d.Treatment of STZinduced diabetic animals with both extracts of AI (200 mg/kg body weight) caused a significant (P < 0.05) higher FRAP activity.But YLE provides better changes due to its antioxidant property.
Figure 4a shows the level of plasma sialic acid after streptozotocin treatment and the treatment with AI extract at 200 mg/kg body weight.Streptozotocin-induced diabetic rats showed a significant increase in the levels of plasma sialic acid.The ethanolic AI extracts showed a mild sialic acid lowering action in the diabetic-treated group of rats as well as in the normal rats, and a reduction close to nondiabetic levels.
The level of the advanced oxidation protein product (AOPP) in plasma observed in this work was significantly (P < 0.05) increased in streptozotocin-induced diabetic rats when compared to rats in the normal group.The treatment with YLE AI extracts resulted in a significant reduction in the level of AOPP level in diabetic rats, YLE supplemented group showed better action when compared to the untreated group and The level of sialic acid was significantly higher in diabetic rats in comparison with normal control.Significant (P < 0.05) difference was obtained between normal control and diabetic groups.b) Plasma AOPPs level measured as free radical mediated protein oxidation during rat aging.Concentration of AOPP is expressed as mmol/L of chloramine-T equivalents.Significant (P < 0.05) difference was obtained normal and control group (one way ANOVA post hoc Bonferroni test).c) Protein carbonyl (PCO) content in plasma of normal and experimental rats groups.PCO level in STZ-induced diabetic rats significantly (#P < 0.05) increased compared to control rats.Oral supplementation of YLE and MLE 200 mg/kg body weight for 28 days treatment significantly (*P < 0.05) decreased the PCO level in plasma compared to diabetic control rats.PCO content is expressed in nmol/mg protein.Values are expressed as mean ± SD for 6 rats in each group.
the MLE-supplemented group because YLE contains a greater amount of active components found greater content with respect to MLE as can be seen in Figure 4b.
The results of protein carbonyl formation in the erythrocyte membrane in the experimental rats are given in Figure 4c.There is a significant increase (p < 0.05) in protein oxidation in the diabetic rats when compared to the control group.The administration of the young and matured ethanolic leaf extract of 200 mg/kg body weight to the diabetic rats led to a significant reduction (p < 0.05) in the protein carbonyl.YLE supplementation provides better results because it contains a greater amount of active components with respect to MLE which was already reported in our FI-TR spectrum result.
The results of inflammatory cytokine levels (IL-6 and TNF-alpha) are represented in Figure 5 (a,b), respectively.Our finding shows that significantly (P < .05)increased both inflammatory cytokine levels in the diabetic group (DC) when compared with the control group (NC), while YLE-supplemented group shows a significant (P < .05)decrease in both values.

Discussion
Classically, reductions in blood sugar level, triglyceride and cholesterol level and increase in the level of insulin are the targets for the management and treatment of all types of diabetes; therefore, the ability of the potential hypoglycemic plant to lower the blood sugar level in vivo is the premise that the leaf extract of AI has a potential antidiabetic clinical efficacy.The YLE-supplemented groups provide a better result with respect to the MLE group because the YLE contains a greater amount of phenolic content with respect to MLE which has already been studied with our FI-TR experiments.Reduced glutathione is a ubiquitous low molecular weight and most abundant among endogenous antioxidants that act as a redox buffer protecting against oxidative stress.The reduction in GSH as noticed in the diabetic group indicated that oxidative stress is high within the diabetic rat model as compared to the normal control group.The administration of 200 mg per kg body weight of YLE and MLE AI caused a significant increase in the GSH levels (P < .05)and YLE-supplemented groups provide a better result (14.2% increase) with respect to the MLE.Researchers have reported lower levels of GSH and an increase in oxidative stress in diabetes and related complications [19].The ability of the extracts to act as an antioxidant and their potential use in diseases related to oxidative stress has been scientifically proven [6,20].Advanced oxidation protein products (AOPP) are an established method for measuring shortterm changes in oxidative stress because it rapidly responds to changes [21]; they are the products formed when there is the oxidationmodification of albumin, fibrinogen, and lipoproteins and oxidative stress is said to be greatly involved in this process [22]).When amino acid residues such as tyrosine undergo oxidation, it leads to the formation of dityrosine, aggregation of the protein, crosslinking, and fragmentation occur when ROS-mediated protein damage takes place in vitro [16].The plasma levels of AOPP are elevated in patients with diabetes mellitus and other diseases that involve oxidative stress [23,24].
AOPP are significantly higher in many pathological conditions including diabetes, and they are not only a marker for oxidative stress but a class of renal pathogenic mediators [25], linked to vascular lesions in diabetes, chronic renal insufficiency, and atherosclerosis [26,27].From the present work, it was observed that the AOPP of the diabetic group was significantly higher than that of the control.This is a result of increased glycol-oxidation, oxidantantioxidant imbalance, and coexisting inflammation.. Polyphenols have been demonstrated to possess beneficial levels of antioxidant markers and health-promoting effects in studies [28,29].MLE-supplemented group also shows a decrease in the AOPP level, but YLE provides better results because it contains a greater amount of active components with respect to MLE which is already reported in our FITR spectrum result.
Lipid peroxidation destroys bio-membranes altering their integrity, fluidity and permeability and losing their functionality, leading to the generation of toxic products [30].Lipid peroxides formed by the action of free radicals play a very important role in diabetes and other degenerative diseases (Pandey et al., [31]).The positive result obtained from the young and matured leaf extracts of AI in this work could be a result of the ability of the polyphenols present in them to prevent lipid peroxidation; another reason could be a result of the ability of polyphenols to form adducts with aldehydes like between polyphenols and methyl glyoxal.The better result in YLE groups (7.61% decreased) is due to a large amount of phenolic and active components with respect to MLE.Located in the plasma membrane is the important plasma membrane redox system (PMRS) which has been shown to play a very essential role in the growth, control and defense mechanism of cells.Erythrocyte does not have mitochondria, and PMRS is the only mechanism for trans-plasma membrane electron transport.The results of this work showed that PMRS is significantly increased in the diabetic group when compared with the control group as has been earlier shown by Rizvi and Srivastava [32] who found out that PMRS activity is elevated in diabetes conditions and the reason proffered was that it could be as a result of compensatory mechanisms to mitigate the increase in oxidative stress which occurs in the diabetic rat group.The administration of the ethanolic extracts of young and matured leaf of AI at 200 mg/kg body weight resulted in decreasing the PMRS to near normal values.The mechanism through which this happened might be that it activates the erythrocyte PMRS and AFR reductase activity and through this action, the reducing equivalents are transferred to the extracellular compartments.
The cell surface sialic acids are very crucial in mediating a variety of physiological and pathological processes in the body [33].The results of this work indicated that plasma sialic acid levels were elevated in a diabetic group of rats as compared to the control group as also shown by [34].This elevated level is an indication of vascular damage in the diabetic group.The administration of young and matured AI leaf extracts restores the plasma sialic acid values close to normal values.This sialic acid-lowering action of AI extracts might be attributed to their flavonoid contents as flavonoids are absorbed into the cells within several hours [35].
The use of protein carbonyls as an index of oxidative stress has a great advantage because it is relatively formed in the early formation and it is relatively stable when compared to other carbonylated proteins [36].There is always a relationship between the structure and function of protein in the biological system, therefore oxidative damage to the structure may lead to a loss in specific protein function.Because proteins have a specific and unique biological function, there will be unique functional consequences resulting from their modification in vivo.
Protein carbonyls are oxidation products formed from amino acids such as proline, lysine, threonine and arginine.The formation of protein carbonyl is an indicator of severe damage resulting from oxidation, and it is accompanied by dysfunction of protein, associated with higher hydrophobicity and thermo sensitivity [37].These reports indicate an increase in protein carbonyl formation during oxidative stress [31].An increase in protein carbonyl has also been reported earlier in both type-I and type-II diabetes [36].YLE provides better results because it contains a greater amount of active component with respect to MLE which was already reported in our FI-TR spectrum result, and also supports our hypothesis that it has radical scavenging and antioxidant properties.
Altered levels of metabolites, such as glucose, lipids, fatty acids, and various cytokines from the adipose tissue, activate monocytes and increase the secretion of inflammatory cytokines [40].The findings from this work supported earlier work and the hypothesis in this regard that there was an elevation in the cytokine levels in plasma for rats in the diabetic group.The supplementation with the ethanolic extract of YLE and MLE resulted in a significant (P < .05)reduction of both cytokine levels close to normal, while YLE provides an 8.6% decreased inflammation with respect to MLE.
The outcome of this work indicated that reduced glutathione, PMRS, MDA, sialic acid, and AOPP are increased in STZ-induced diabetic rats, while the ferric-reducing ability of plasma is reduced in the same group of rats.It is also observed that YLE of AI possesses greater antidiabetic and antioxidant activities and that the extracts can increase antioxidant power, reduce oxidative stress, or both in comparison to mature leaves.These results lead credence to the use of these plants by the traditional systems of medicine and may indicate the usefulness of this therapy in tackling the negative effects of ROS and may be used as a part of the management process for diabetics.

Figure 1 .
Figure 1.Panel (a)shows a graphical representation of the effect of STZ administration on the blood glucose levels of experimental rats and the resultant effect on the rats when the YLE and MLE 200 mg/kg was administered.There was a significant increase (P < 0.05) in the blood glucose levels of the diabetic control when compared to normal, and there was a significant decrease (P < 0.05) in blood sugar level of the treated groups when compared to the diabetic group.The values are given as mean ± standard deviation of replicate determinations (n = 6).

Figure 3 .
Figure 3. Panel (a) represents the results of the reduced glutathione (GSH) level in normal, experimental groups of Wistar rats.The GSH level was significantly (P < 0.05) increased in STZ-induced diabetic group of rats in comparison to the control group.The oral administration of the AI extract at a dose of 200 mg/kg body weight up to 28 days significantly (P < 0.05) decreased GSH level compared to diabetic control group.Values are given as mean ± SD for 6 rats in each group.b) Plasma membrane redox system values in normal and experimental groups.STZ-induced diabetes resulted in significantly (P < 0.05) increase level of PMRS compared to normal control groups.The oral administration of AI extract (200 mg/kg body weight) up to 28 days per day significantly (P < 0.05) decreased level of PMRS in STZ-induced diabetic rats.Values are expressed as mean ±SD for 6 rats in each group.c) Lipid peroxidation as malonaldehyde (MDA) levels in normal and experimental groups.The MDA levels of STZ-induced diabetic rats were significantly higher (P < 0.05) when compared with the normal control rats, however.Oral administration of AI extract offered significantly (P < 0.05) decreased in MDA level in diabetic rats.para.d) Total antioxidant capacity of plasma measured in terms of FRAP value the experimental rats.FRAP value is expressed as µmol Fe(II) per l of plasma.Data are represented as mean ± SD (n = 6).Significant (P < 0.05) difference was obtained between the normal and STZ-induced diabetic rats.

Figure 2 .
Figure 2. FTIR spectra of ethanolic extract of Azadirachta indica leaf, showing different spectra and each peak corresponding to a particular functional group.

Figure 4 .
Figure 4. a) Sialic acid content in plasma in ethanolic AI leaf extracts treated STZ-induced diabetic rats.The level of sialic acid was significantly higher in diabetic rats in comparison with normal control.Significant (P < 0.05) difference was obtained between normal control and diabetic groups.b) Plasma AOPPs level measured as free radical mediated protein oxidation during rat aging.Concentration of AOPP is expressed as mmol/L of chloramine-T equivalents.Significant (P < 0.05) difference was obtained normal and control group (one way ANOVA post hoc Bonferroni test).c) Protein carbonyl (PCO) content in plasma of normal and experimental rats groups.PCO level in STZ-induced diabetic rats significantly (#P < 0.05) increased compared to control rats.Oral supplementation of YLE and MLE 200 mg/kg body weight for 28 days treatment significantly (*P < 0.05) decreased the PCO level in plasma compared to diabetic control rats.PCO content is expressed in nmol/mg protein.Values are expressed as mean ± SD for 6 rats in each group.

Figure 5 .
Figure 5. a,b) Inflammatory cytokine level (IL-6 and TNF-alpha) of normal and experimental rats groups.Cytokine level in STZ-induced diabetic rats significantly (#P < 0.05) increased compared to control rats.Oral supplementation of YLE and MLE 200 mg/kg body weight for 28 days of treatment significantly (*P < 0.05) decreased the cytokine level in compared to diabetic control rats.Cytokine content is expressed in pg/ml.Values are expressed as mean ± SD for 6 rats in each group.