Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Protective Effects of Neem Ethanolic Extract on Streptozotocin-induced Diabetes: Modulation of C-Peptide and Peroxynitrite Levels in Liver Male Rats

B
Bushra R. Ibrahim1,*
L
Luma W. Khaleel1
1Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Iraq.

ABSTRACT

Background: Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia due to insufficient insulin production or the body’s inability to effectively use insulin. Neem extract, derived from the leaves of the Azadirachta indica (Neem), is used in various applications, particularly in traditional medicine and as a natural pesticide. This study aims to evaluate the therapeutic effects of neem ethanolic extract on type 1 diabetes mellitus by determining changes in blood glucose levels, serum C-peptide concentrations and peroxynitrite formation in diabetic rats. Specifically, the study seeks to investigate neem’s potential to improve glycemic control, preserve endogenous insulin secretion as indicated by C-peptide levels and reduce oxidative and nitrosative stress measured through peroxynitrite levels.

Methods: Ninety Male Rats that were 180-200 g in weight were randomly divided in to six groups 15 rats in each one and kept for approximately seventy days, animal groups: G1: (Negative control). G2: (Positive control) will receive a single STZ dose (50 mg/kg B.W. I/P) to induce diabetes. G3: Will daily receive neem ethanolic extract (500 mg/kg) orally. G4: Diabetic animals will daily receive neem ethanolic extract (400 mg/kg B.W) orally. G5: Diabetic animals will daily receive neem ethanolic extract (500 mg/kg B.W) orally. G6: Diabetic Animals will daily receive Insulin (3 I.U) S/c.

Result: Our results showed that the diabetic group showed an increase in Glucose, Peroxynitrite and a decrease in C-peptide and disruption of the hepatic cords in liver tissue when compared to the control group. When diabetic animals were treated with neem extract and insulin, we observed an improvement in both Peroxynitrite, C-peptide, Glucose and a normal liver structure and a normal distribution of central veins and hepatic cords in liver tissue when compared to the positive group.

INTRODUCTION

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from insufficient insulin secretion, impaired insulin action, or both. The global prevalence of DM continues to rise, making its long term complications a major public health concern (Saeedi et al., 2019; Al-Saeedi et al., 2025). Beyond disturbances in glucose metabolism, diabetes is associated with progressive damage to multiple organs, largely driven by oxidative stress and chronic inflammation (Kapucu et al., 2021; Mahmood et al., 2024; Othman et al., 2024; Hasan et al., 2022; Fakri et al., 2023; Balaky et al., 2021; Muhsin et al., 2021).  
       
The liver plays a central role in glucose and lipid homeostasis and is particularly vulnerable to diabetic injury. In diabetes, sustained hyperglycemia enhances the production of reactive oxygen species (ROS) through pathways such as glucose autoxidation, mitochondrial dysfunction and activation of the polyol pathway. Excessive ROS generation overwhelms endogenous antioxidant defenses, leading to oxidative stress, inflammatory responses and structural damage in hepatic tissue. These processes contribute to hepatocellular injury, fibrosis and impaired liver function, which further exacerbate metabolic dysregulation in diabetic individuals (Alsieni et al., 2021; Hasan et al., 2024; Najim et al., 2024).
       
One important mediator of oxidative and nitrosative stress in diabetes is peroxynitrite (ONOO-), a highly reactive nitrogen species formed by the rapid reaction of nitric oxide with superoxide anions. Peroxynitrite induces lipid peroxidation, protein nitration and DNA damage, thereby amplifying inflammation and cellular dysfunction in the liver. Elevated levels of peroxynitrite have been implicated in the progression of diabetic complications, highlighting its relevance as a marker and mechanistic contributor to hepatic oxidative damage (Ghasemi et al., 2023; Pirabbasi et al., 2024; Ahmed et al., 2025).
       
C peptide, a cleavage product released in equimolar amounts with insulin from pancreatic β cells, is increasingly recognized as more than a biologically inert peptide. In diabetes, particularly in insulin deficient states, reduced C peptide levels reflect β cell dysfunction. Emerging evidence suggests that C peptide exerts protective effects on microvascular function, inflammation and oxidative stress. Therefore, assessment of C peptide provides valuable insight into pancreatic function and the severity of diabetic metabolic disturbances (Laila et al., 2022).
       
Experimental models are essential for elucidating the mechanisms underlying diabetic liver injury and for evaluating potential therapeutic interventions. Streptozotocin (STZ) is widely used to induce experimental diabetes due to its selective toxicity toward pancreatic β cells. STZ enters β cells via the glucose transporter GLUT2, causing DNA alkylation, oxidative stress and subsequent insulin deficiency. This model reliably mimics key metabolic and oxidative features of diabetes, including hyperglycemia induced liver damage, making it suitable for investigating hepatoprotective strategies (Al-awadhi et al., 2024; Mahmoud Al-Doori et al., 2025).
       
Current antidiabetic therapies primarily aim to control blood glucose levels but often provide limited protection against oxidative stress mediated organ damage, including hepatic dysfunction. Consequently, there is growing interest in complementary and alternative therapies derived from natural products that can target oxidative stress and inflammation alongside glycemic control (Patil et al., 2021).
       
Azadirachta indica is a medicinal plant extensively used in traditional medicine and has attracted scientific attention for its antidiabetic, antioxidant and anti inflammatory properties. Neem contains a wide range of bioactive compounds, including flavonoids, terpenoids and alkaloids, which have demonstrated free radical scavenging and anti inflammatory activities. These properties suggest that neem extract may mitigate oxidative stress, suppress inflammatory pathways and protect liver tissue against diabetes induced damage (Cardoso et al., 2025).
       
Current therapies often provide limited protection against oxidative stress-mediated organ damage. Consequently, there is interest in natural products like Azadirachta indica, which contains bioactive compounds such as flavonoids and terpenoids with potent insulin-mimetic activity (Panda et al., 2023; Pandita et al., 2022). This study evaluates the protective effects of neem on hepatic dysfunction and oxidative markers in STZ-induced diabetic rats.

MATERIALS AND METHODS

The Fresh leaves of Azadirachta indica were collected from Al-Nahrain University, Jadiriyah, Baghdad, during June 2024. The sample was identified and classified by the University of Baghdad grassland, College of Science, Department of Biology.
 
Preparation of extract
 
The leaves were properly washed and air-dried at room temperature for two weeks. The dried leaves were ground into powder using a corona manual grinding machine. Exactly 100 g of the ground leaves of neem were soaked in 1 liter of ethanol for 24 hrs. It was sieved and filtered using Whatman no1 (125 mm) filter paper. The filtrate was evaporated to dryness using a rotary evaporator and the paste was put in a stoppered universal bottle and stored in the refrigerator until needed. The paste was dissolved in distilled water before use (Obiajulu and Chukwuemeka, 2020).
 
Induction of diabetes in male rats
 
Diabetes was induced by streptozotocin (STZ) (50 mg/kg) diluted in 5 ml citrate buffer having pH 4.5, which was injected intraperitoneally (IP) to 60 rats. fasting condition were preserved for 18 hours to stimulate a diabetic situation. The injection volume of the diluted STZ was determined (Jenna and Wurster, 2021).
 
Experimental design
 
Ninety Male Rats (Rattus norvegicus) weighted 180-200g, were randomly divided into six groups 15 rat in each one and kept for approximately seventy days. The room temperature ranged 22 to 25°C with relative humidity conditions water supply, commercial food and a 12-hour light/12-hour dark cycle for 14 days before the beginning of the experiment. Diabetes was induced in 60 rats for four groups by STZ (50 mg/kg) diluted in 5 ml citrate buffer having pH 4.5 which was injected intraperitoneally (IP) to rat and preserved fasting condition for 18 hours to stimulate diabetic situation.
 
Animals groups
 
G1: (Negative control): The animals received normal saline orally.
G2: (Positive control): The animals received single STZ dose (50 mg/kg B.W I/P) to induced diabetes (Omolaoye et al., 2018).
G3: Healthy animals, received neem ethanolic extract (500 mg/kg B.W) daily and orally.
G4: Diabetic animals received neem ethanolic extract (400 mg/kg B.W) daily and orally (Seriana et al., 2021).
G5: Diabetic animals received neem ethanolic extract (500 mg/kg B.W) daily and orally.
G6: Diabetic Animals received insulin (3 I.U) daily and S/c.
 
Blood and tissue sampling
 
Each animal received a dosage of (≥ 100 mg/kg) of sodium pentobarbital solution as anesthesia IP. Blood samples were taken by cardiac puncture of the rats and centrifuged for 15 min at 4000 rpm to collect serum before being subjected to biochemical and immunological analysis. The liver was cleaned with cold saline after their removal from the animals to be fixed with 10% neutral formalin for histological examination. The samples were kept at -20°C.
 
Biochemical analysis
 
Determination of glucose
 
Serum glucose levels were determined using the modified kinetic glucose oxidase peroxidase (GOD-PAP) method (Trinder, 1969). Measurements were performed using a commercial kit provided by Agappe Diagnostics Ltd, India.
 
Determination of C-peptide
 
The kits were purchased from (SunLong Biotech) under kits numbered (ABIN6963725 C-Peptide) and this test was performed using the (ELISA Kit) technique.
 
Determination of peroxynitrite
 
The kits were purchased from (Sunlong Biotech) under kits numbered (Rat Peroxynitrite Anion (ONOO-)) and this test was performed using the (ELISA Kit) technique.
 
Histopathological investigations
 
After taken from rats following scarification, liver tissue samples were fixed in 10% neutralized formaldehyde, embedded in paraffin wax and then stained with hematoxylin and eosin. were used to stain tissues and to demonstrate fatty changes, respectively according to (Al-Khuzaay et al., 2024; Yahya et al., 2024).
 
Ethical approval
 
Ethical approval was obtained for conducting experiments on laboratory animals under the approval obtained from the University of Baghdad - College of Veterinary Medicine -Animal Care and Use Committee, according to approval number P.G/1200 issued on 23/6/2024.
 
Statistical analysis
 
The Statistical Packages of Social Sciences-SPSS (2019) program was used to detect the effect of difference groups and period in study parameters. LSD-Least significant difference (two way) was used to significant compare between means in this study.

RESULTS AND DISCUSSION

The results of the biochemical analysis are summarized in the following Tables. As shown in Table 1, STZ- induced diabetic rats exhibited a significant increase in glucose levels, which was markedly reduced by neem extract treatment (p≤0.05). Changes in serum C-peptide concentration across the experimental groups are detailed in Table 2, showing partial restoration in treated groups. Furthermore, the levels of peroxynitrite in tissue were significantly attenuated following neem administration, as presented in Table 3.

Table 1: Effect of different doses at three periods of neem leaves extract and insulin treatment on serum glucose concentration (mg/dl) in STZ-induced diabetic male rats.



Table 2: Effect of different doses at three periods of neem leaves extract and insulin treatment on serum C-peptide concentration (pg/ml) in STZ-induced diabetic male rats.



Table 3: Effect of different doses at three periods of neem leaves extract and insulin treatment in lung tissue peroxynitrite(ng/L) in STZ-induced diabetic male rats.


 
Histopathological results of liver
 
Histological examination of the liver (summarized in Fig 1) showed that diabetic rats (G2) suffered from coagulative necrosis and fatty droplets (Fig1 B). In contrast, rats treated with neem ethanolic extract 500 mg/kg(G5) showed near-normal liver architecture (Fig1 E), comparable to the negative control (Fig 1 A).

Fig 1: A Histological section of the liver of rats of negative control group shows normal liver architecture that characterized by normal central veins (V) with hepatic cords (Black arrow), H and E 40X. B: Histological section of the liver of rats of positive control group shows area of coagulative necrosis that characterized by hypereosinophilic pyknotic and shrunken hepatocytes, disturbing of hepatic cords (Black arrow) with variably sized clear intracytoplasmic vacuoles (Fatty droplets, blue arrow), H and E, 40X. C: Histological section of the liver of rats that received Neem only shows normal liver architecture that characterized by normal central veins (V) and hepatic cords (Black arrow) distribution with no clear pathological changes, H and E 40X. D: Histological section of the liver of DM rats that treated with 400mg/kg Neem shows moderate multifocal of variably sized periportal granulomas (Black arrow) that replaced individual necrotic hepatocytes. Granulomatous foci characterized by aggregation of mononuclear cells (lymphocytes and macrophages), H and E, 40X. E: Histological section of the liver of DM rats treated with 500 mg/kg. Neem shows normal liver architecture characterized by normal central veins (V) and hepatic cords distribution with some individual apoptotic/necrotic hepatocytes (Black arrow), H and E, 40X. F: Histological section of the liver of diabetic rats that treated with insulin shows normal liver architecture characterized by normal central veins (V) and hepatic cords distribution. However, some individual apoptotic and necrotic hepatocytes and mononuclear cells aggregated around central veins were seen (Black arrow), H and E, 40X.


       
The present study demonstrates that ethanolic Azadirachta indica leaf extract exerts significant antidiabetic and hepatoprotective effects in streptozotocin (STZ)-induced diabetic rats. Treatment with neem extract, particularly at 500 mg/kg, resulted in a significant reduction in blood glucose levels. This glucose-lowering effect may be linked to improved peripheral glucose utilization and GLUT4 translocation, comparable to the efficacy of insulin treatments noted by Patel et al., (2018). As shown in Table 1, restoration of C-peptide concentrations, attenuation of oxidative stress as indicated by reduced peroxynitrite levels and marked improvement in liver histoarchitecture. These effects were time-and dose-dependent and, in several parameters, comparable to insulin treatment.
       
STZ-induced diabetic rats exhibited persistent hyperglycemia throughout the experimental period, confirming successful induction of diabetes and reflecting impaired insulin secretion and utilization. In contrast, normal control animals and non-diabetic rats treated with neem extract (500 mg/kg) maintained normoglycemia, indicating that the extract did not induce hypoglycemia under physiological conditions According to (Nagatani et al., 1996; Wang et al., 2010; Guerre-Millo et al., 1985).
       
Treatment with neem extract at both 400 and 500 mg/kg significantly reduced blood glucose levels compared with diabetic controls. The glucose-lowering effect was more pronounced at 500 mg/kg, with a statistically significant decrease observed at day 60 (p≤0.05), indicating a clear dose- and time-dependent response. However, glucose levels remained higher than those of normal controls, suggesting partial rather than complete glycemic normalization. Insulin-treated rats showed a marked reduction in glucose levels, though values remained slightly elevated relative to non-diabetic controls (Patel et al., 2018).
       
The hypoglycemic effect of neem extract may be attributed to improved peripheral glucose utilization. A plausible mechanism supported by previous studies is the upregulation of insulin signaling and increased GLUT4 translocation in skeletal muscle, enhancing glucose uptake and improving glycemic control. This mechanism provides a single, coherent explanation for the observed glucose reduction and avoids redundant or unsupported pathways (Morris et al., 2019).
       
C-peptide levels remained stable in normal controls and non-diabetic rats treated with neem extract, confirming preserved endogenous insulin secretion. In contrast, diabetic control rats showed a significant reduction in C-peptide levels (p≤0.05), reflecting STZ-induced cytotoxic damage to pancreatic β-cells and impaired insulin synthesis.        

Neem extract treatment partially restored C-peptide levels in diabetic rats, with the 500 mg/kg dose showing superior efficacy compared to 400 mg/kg. The extract at 400 mg/kg maintained C-peptide levels up to day 60, followed by a significant decline after stop of treatment (70 day), whereas the higher dose sustained improved levels for a longer duration. Insulin-treated rats also demonstrated a significant increase in C-peptide at day 60, likely due to reduced glucotoxic stress on surviving β-cells rather than direct stimulation of insulin synthesis (El-Beltagy et al., 2021; Abduallah et al., 2023; McCalla et al., 2024; Dholi et al., 2011).
       
As shown in Table 2. These findings indicate that neem extract may preserve residual β-cell function by mitigating STZ-induced cytotoxicity, rather than by modulating autoimmune mechanisms, which are not relevant to this experimental model (Dholi et al., 2011; Boldison et al., 2019). This is supported by Gabr et al., (2023), who noted the impact of antioxidants on preventing pancreatic B-cell damage.
       
Peroxynitrite levels remained within normal ranges in negative controls and non-diabetic rats treated with neem extract. Diabetic control rats exhibited a significant elevation in peroxynitrite levels (p≤0.05), consistent with enhanced oxidative and nitrosative stress associated with diabetes and increased nitric oxide synthase activity (Ghorbani et al., 2011; Saravanan et al., 2006; Wilhelm et al., 2008).
       
Treatment with neem extract at both doses significantly reduced peroxynitrite levels compared with diabetic controls, with the 500 mg/kg dose producing greater normalization. This reduction suggests an attenuation of oxidative stress, which may contribute to improved pancreatic and hepatic function. Insulin-treated rats showed an initial increase in peroxynitrite at day 60, followed by a significant decrease at day 70 (p≤0.05) after stop of treatment (70 day), indicating delayed oxidative stress modulation according to (Alfaris et al., 2021).
       
These results support the role of neem extract in reducing diabetes-associated oxidative stress in vivo.  The bioactive potential of neem in combating systemic disturbances is consistent with the findings of (Veerendrakumar et al., 2023 and Kumar et al., 2021). Who emphasized the role of traditional plants in biochemical restoration. As shown in Table 3. Histological examination of liver tissue from diabetic control rats revealed marked pathological alterations, including coagulative necrosis, hepatocellular shrinkage, pyknotic nuclei and disruption of hepatic cords, consistent with diabetes-induced hepatic injury (Chen et al., 2022; Fakhri et al., 2021; Huang et al., 2024).                           

Rats treated with neem extract showed dose-dependent histological improvement. The 400 mg/kg dose resulted in moderate restoration with residual periportal granulomatous changes, whereas the 500 mg/kg dose exhibited near-normal liver architecture, characterized by intact central veins and well-organized hepatic cords. These findings indicate superior hepatoprotective efficacy at the higher dose. Insulin-treated rats also demonstrated near-normal liver morphology (Mahata et al., 2021; Alrefaei et al., 2025; El-Megharbel et al., 2022).                    
       
Overall, neem extract at 500 mg/kg provided the most effective protection against diabetes-induced hepatic damage, likely through combined glycemic control and oxidative stress reduction (Husen et al., 2021).

CONCLUSION

In conclusion, this study demonstrates that ethanolic extract of Azadirachta indica leaves possesses potent antidiabetic and hepatoprotective properties in STZ-induced diabetic rats. The extract, particularly at a dose of 500 mg/kg, significantly improved glycemic control and restored serum C-peptide levels while reducing nitrosative stress via the modulation of peroxynitrite. Furthermore, neem treatment successfully attenuated hepatic structural damage, promoting the restoration of normal liver architecture. These findings suggest that neem extract could serve as a valuable natural therapeutic agent for managing type 1 diabetes and its associated hepatic complications. Future research should focus on identifying the specific bioactive compounds responsible for these protective effects.

ACKNOWLEDGEMENT

This study did not receive any grant from any funding agency.
 
Funding
 
No financial support for our research.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES


  1. Abduallah, A.M., Ahmed, A.E., Bajaber, M.A. and Alalwiat, A.A. (2023). Evaluation of the Antidiabetic Effects of methanolic extracts of neem (Azadirachta indica) seeds on the streptozotocin- induced wistar rats. Pakistan Veterinary Journal. 43(4): 792-798.

  2. Ahmed, R.M., Abdullah, A.R., Majeed, A.K. and Hasan, A.F. (2025). The effect of clove oil on the expression of xly gene in tomato infected with Fusarium oxysporum. Agricultural Science Digest. 45: 91-97. doi: 10.18805/ag.DF-794.

  3. Al-awadhi, R.M., Abdelrazek, H.M.A., Abouelhassan, E.M., Kamel, M.S., Attia, N. A. and Awad, A.A. (2024). Neem leaves extract reduces sex steroids and gonadal function in female wistar albino rats. Biomedical and Pharmacology Journal. 17(3): 1471-1481.

  4. Alfaris, N.A., Alshammari, G.M., Altamimi, J.Z., Aljabryn, D.H., Alagal, R.I., Aldera, H. and Yahya, M.A. (2021). Ellagic acid prevents streptozotocin-induced hippocampal damage and memory loss in rats by stimulating Nrf2 and nuclear factor-êB and activating insulin receptor substrate/PI3K/ Akt axis. Journal of Physiology and Pharmacology. 72(4). doi: 10.26402/jpp.2021.4.02. 

  5. Al-Khuzaay, H.M., Al-Juraisy, Y.H., Hasan, A.F. and Tousson, E. (2024). Antitumor activity of β-glucan isolated àrom date fruits ùn cancer cells in vivo. Opera Medica et Physiologica. 11(3): 41-48.

  6. Alrefaei, A.F. and Elbeeh, M.E. (2025). Hepatoprotective effects of Glycyrrhiza glabra in diabetic male rats: Addressing liver function, oxidative stress and histopathological changes. Biology. 14(3): 307.

  7. Al-Saeedi, R.F.H., Mwafaq, R.K. and Jasim, N.A. (2025). Nephrotoxicity of iron oxide nanoparticles in male mice. Agricultural Science Digest. 46(1): 38-43. doi: 10.18805/ag.DF-751.

  8. Alsieni, M.A., El Rabey, H.A., Al-Sieni, A.I. and Al-Seeni, M.N. (2021). Comparison between the antioxidant and antidiabetic activity of fenugreek and buckthorn in streptozotocin induced diabetic male rats. BioMed Research International. 2021(1): 7202447.

  9. Balaky, H.M. and Kakey, I.S. (2021). Indications of liver and kidney functions in non-insulin dependent diabetic patients. Iraqi Journal of Science. pp 769-778.

  10. Boldison, J., Da Rosa, L.C., Davies, J., Wen, L. and Wong, F.S. (2019). Dendritic cells license regulatory B cells to produce IL-10 and mediate suppression of antigen- specific CD8 T cells. Cellular and Molecular Immunology. 17(8): 843-855. https://doi.org/10.1038/s41423-019- 0324-z.

  11. Cardoso, C.A., Parca, A.V., Facciotti, P.R., Machado, L.C., de Oliveira, V.C., de Campos, S.A.G.D. and dos Santos, M.D. (2025). Natural pesticide, Azadirachta indica A. juss. (Neem), disrupted reproductive parameters of male rats. Journal of Ethnopharmacology. 337: 118927.

  12. Chen, H., Luo, Y., Tsoi, B., Gu, B., Qi, S. and Shen, J. (2022). Angong niuhuang wan reduces hemorrhagic transformation and mortality in ischemic stroke rats with delayed thrombolysis: Involvement of Peroxynitrite-mediated MMP-9 activation. Chinese Medicine. 17(1): 51.

  13. Christian, E.O. and Chukwuemeka, E.F. (2020). Antidiabetic and modulatory effect of ethanol extract of neem leaf on some essential biochemical parameters of streptozotocin- induced diabetic rats. International Journal of Biochemistry Research and Review. 28(4): 1-11.

  14. Dholi, S.K., Raparla, R., Kanaki, S.K., Bhaskar, P. and Priyanka, S. (2011). Comparative evaluation of antidiabetic activity of Cinnamomum tamala Linn. leaf extracts in STZ induced diabetic rats. International Journal of Pharmaceutical Sciences and Drug Research. 3(3): 221-224.

  15. El-Beltagy, A.E.F.B., Saleh, A.M., Attaallah, A. and Gahnem, R.A. (2021). Therapeutic role of Azadirachta indica leaves ethanolic extract against diabetic nephropathy in rats neonatally induced by streptozotocin. Ultrastructural Pathology. 45(6): 391-406.

  16. El-Megharbel, S.M., Al-Thubaiti, E.H., Qahl, S.H., Al-Eisa, R.A. and Hamza, R.Z. (2022). Synthesis and spectroscopic characterization of dapagliflozin/Zn (II), cr (III) and Se (IV) novel complexes that ameliorate hepatic damage, hyperglycemia and oxidative injury induced by streptozotocin- induced diabetic male rats and their antibacterial activity. Crystals. 12(3): 304.

  17. Fakhri, S., Patra, J.K., Das, S.K., Das, G., Majnooni, M.B. and Farzaei, M.H. (2021). Ginger and heart health: From mechanisms to therapeutics. Current Molecular Pharmacology. 14(6): 943-959.

  18. Fakri, Y.A. and Al-Jowari, S.A.K. (2023). Antithyroid peroxidase and thyroid hormones in a sample of Iraqi patients with type 2 diabetes mellitus. Iraqi Journal of Science. pp 1618-1624.

  19. Gabr, N.M., Elbayoumy, F.M.A.E., Omar, A.E.D.S.E.S., El-Lateef, A.E.L.S.A., Eissa, A.A.M.A., Younes, A.M. and Abbas, M.A.M. (2023). Impact of antioxidants on pancreatic B- cell damage caused by Streptozotocin in adult male albino rats. International Journal of Medical Arts. 5(1): 2944- 2950.

  20. Ghasemi, A. and Jeddi, S. (2023). Streptozotocin as a tool for induction of rat models of diabetes: A practical guide. EXCLI Journal. 22: 274.

  21. Ghorbani, A., Omrani, G.R., Hadjzadeh, M.A.R. and Varedi, M. (2011). Effects of rat C-peptide-II on lipolysis and glucose consumption in cultured rat adipose tissue. Experimental and Clinical Endocrinology and Diabetes. 119(6): 343-347.

  22. Guerre-Millo, M., Leturque, A., Girard, J. and Lavau, M. (1985). Increased insulin sensitivity and responsiveness of glucose metabolism in adipocytes from female versus male rats. The Journal of Clinical Investigation. 76(1): 109-116.

  23. Hasan, N.T. and Ali, S. (2024). Impact of obesity on serum levels of neuropeptide-Y in type 2 diabetics in relation to glycemic status. Iraqi Journal of Pharmaceutical Sciences. 33(4SI): 282-289.

  24. Hasan, Z.Y.M. and Abdullah, J.M. (2022). Effects of gel extract from Aloe vera cultivated in Iraq on blood glucose level and lipid profile in induced diabetic mice. Iraqi Journal of Science. 63(1): 124-130.

  25. Huang, W., Zheng, J., Wang, M., Du, L. Y., Bai, L. and Tang, H. (2024). The potential therapeutic role of melatonin in organ fibrosis: A comprehensive review. Frontiers in Medicine. 11: 1502368.

  26. Husen, F., Hernayanti, H., Ekowati, N., Sukmawati, D. and Ratnaningtyas, N.I. (2021). Antidiabetic effects and antioxidant properties of the saggy ink cap medicinal mushroom, coprinus comatus (Agaricomycetes), in streptozotocin-induced hyperglycemic rats. International Journal of Medicinal Mushrooms. 23(10): 9-21.

  27. Jenna, I., Wurster, L., Rachel and Brown, E.P. et al. (2021). Streptozotocin induced hyperglycemia alters the cecal metabolome and exacerbates antibiotic induced dysbiosis. Cell Repo. 37(11): 110113.

  28. Kapucu, A. (2021). Crocin ameliorates oxidative stress and suppresses renal damage in streptozotocin induced diabetic male rats. Biotechnic and Histochemistry. 96(2): 153-160.

  29. Kumar, S., Prashant, M., Ram, B. and Dwivedi, K.N. (2021). Role of katankateryadi kwatha in insulin secretion and restoration of biochemical changes in streptozotocin-nicotinamide induced diabetes mellitus type 2 in rats. International Journal of Applied Biology and Pharmaceutical Technology.  12(3): 362-379.

  30. Laila, N., Sitasiwi, A.J. and Isdadiyanto, S. (2022). Effect of neem leaf extract on reproductive potential of male rats fed high-fat diet. Biosaintifika: Journal of Biology and Biology Education. 14(1): 135-146.

  31. Mahata, L.E., Ali, H. and Murni, A.W. (2021). Effect of streptozotocin on liver histology damage in rats’ model of gestational diabetes mellitus. Int. J. Res. Rev. 8: 18-22.

  32. Mahmood, N.M.S., Mahmud, A.M. and Maulood, I.M. (2024). Role of melatonin in attenuation of vascular angiotensin 1-7 reactivity via calcium channels and endothelial derived relaxing factors in induced diabetic aortic rats. Iraqi Journal of Science. pp 4256-4270.þ

  33. Mahmoud Al-Doori, M.A., Farhan Al-Halbosiy, M.M., Mohammed, H.Z.Y., Hussein, S. M., Hasan, A.F. and El-Wahsh, H.M. (2025). The antileshmaniasis effect of Brassica juncea seeds fatty acids on the growth of leishmania tropica promastigotes isolated culture in vitro. Asian Journal of Dairy and Food Research. 44: 90-98. doi: 10.18805/ajdfr.DRF-553.

  34. McCalla, G.A. and Brown, P.D. (2024). Suitability of the neonatal streptozotocin diabetes model and folklore therapeutic use of low-dose neem leaf extract to treat hyperglycemia associated with type 2 diabetes mellitus. Tropical Journal of Natural Product Research. 8(1): 5925.

  35. Minokoshi, Y., Kahn, C.R. and Kahn, B.B. (2003). Tissue-specific ablation of the GLUT4 glucose transporter or the insulin receptor challenges assumptions about insulin action and glucose homeostasis. Journal of Biological Chemistry.  278(36): 33609-33612.

  36. Morris, J.C.B. Gonzales, J.J., De La, C. et al. (2019). The highly pure neem leaf extract, scne, inhibits tumorigenesis in oral squamous cell carcinoma via disruption of pro-tumor inflammatory cytokines and cell signaling. Front Oncol. 13(9): 890.

  37. Muhsin, S.N. and Faris, H.N. (2021). Quality of life of patients with type 2 diabetes mellitus in Thi-Qar-Iraq. International Journal of Pharmaceutical Research. 13(1): 5438.

  38. Nagatani, S.H.O.J.I., Bucholtz, D.C., Murahashi, K.U.M.I.K.O., Estacio, M.A., Tsukamura, H.I.R.O.K.O., Foster, D.L. and Maeda, K.I. (1996). Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats. Endocrinology. 137(4): 1166-1170.

  39. Najim, H.D., Mohammed, M.M. and Rahmah, A.M. (2024). Influence of adding empagliflozin to conventional anti-diabetic therapy on quality-of-life scale for type 2 diabetic patients.  Iraqi Journal of Pharmaceutical Sciences. 33(3): 142- 149.

  40. Obiajulu, C.E., Francis, C.E., Chukwudi, O.O., Daniel and Onwusulu, N.  (2020). Aqueous extract of azadirachta indica leaves favorably alters the course of streptozotocin-induced diabetes in rats: A comparative prospective cohort study. Biomedical Research and Therapy. 7(7): 3877-388.

  41. Obiajulu, A.O. and Chukwuemeka, E.E.O. (2020). E-human resource management and organizational performance of selected federal agencies in Nigeria. International Journal of Academic Research in Business and Social Sciences. 10(12): 1-15. https://doi.org/10.6007/IJARBSS/v10-i12/ 8212.

  42. Omolaoye, T.S., Skosana, B.T. and du Plessis, S.S. (2018). Diabetes mellitus-induction: Effect of different streptozotocin doses on male reproductive parameters. Acta Histochemica. 120(2): 103-109.

  43. Othman, H.K., Mahmud, A.M. and Maulood, I.M. (2024). The roles of potassium channels and nitric oxide in the modulation of apelin induced-relaxation in isolated diabetic rat aorta. Iraqi Journal of Science. pp 644-658.

  44. Panda, C., Sharma, P., Dixit, U.S. and Pandey, L.M. (2023). Potential and prospective of traditional Indian medicinal plants for the treatment of diabetes. Journal of Biologically Active Products from Nature. 13(4): 316-360.

  45. Pandita, D., Pandita, A. and Bhanu, C. (2022). Antidiabetic Plants for Drug Discovery: Pharmacology, Secondary Metabolite Profiling and Ingredients with Insulin Mimetic Activity. CRC Press.

  46. Patel, M.J.S., Tripathy, K.D.M. et al. (2018). A supercritical CO2 extract of neem leaf (A. indica) and its bioactive liminoid, nimbolide, suppresses colon cancer in preclinical models by modulating pro-inflammatory pathways. Mol Carcinog.  57(9): 1156-1165. 

  47. Patil, S.M., Shirahatti, P.S., VB, C.K., Ramu, R. and Prasad, N. (2021). Azadirachta indica A. Juss (neem) as a contraceptive: An evidence-based review on its pharmacological efficiency. Phytomedicine. 88: 153596.

  48. Pirabbasi, E., Zangeneh, M.M., Zangeneh, A., Moradi, R. and Kalantar, M. (2024). Chemical characterization and effect of Ziziphora clinopodioides green synthesized silver nanoparticles on cytotoxicity, antioxidant and antidiabetic activities in streptozotocin induced hepatotoxicity in wistar diabetic male rats. Food Science and Nutrition. 12(5): 3443-3451.

  49. Saeedi, P., Petersohn, I., Salpea, P., Malanda, B., Karuranga, S., Unwin, N. and Committee, I.D.F.D.A.  (2019). Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the international diabetes federation diabetes atlas. Diabetes Research and Clinical Practice. 157: 107843.

  50. Saravanan, G. and Leelavinothan, P. (2006). Effects of syzygium cumini bark on blood glucose, plasma insulin and C-peptide in streptozotocin induced diabetic rats. Int. J. Endocrinol. Metab. 4: 96-105. 

  51. Satyanarayana, K., Sravanthi, K., Shaker, I.A. and Ponnulakshmi, R. (2015). Molecular approach to identify antidiabetic potential of Azadirachta indica. J. Ayurveda Integr Med. 6: 165-174.

  52. Seriana, I., Akmal, M., Darusman, D., Wahyuni, S., Khairan, K. and Sugito, S. (2021). Neem leaf (Azadirachta indica A. Juss) ethanolic extract on the liver and kidney function of rats. The Scientific World Journal. 2021(1): 7970424.

  53. Trinder, P. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochen. 6: 24-27.

  54. Veerendrakumar, P., Neralla, M., Satheesh, T. and Venkidasamy, B. (2023). Comparative extraction and bioactive potential of the leaf extracts of Azadirachta indica for combatting postoperative head and neck infections: An in vitro study. Cureus. 15(12): e51303.

  55. Wang, Z., Yang, Y., Xiang, X., Zhu, Y., Men, J. and He, M. (2010). Estimation of the normal range of blood glucose in rats. Wei Sheng Yan Jiu=Journal of Hygiene Research. 39(2): 133-137.

  56. Wilhelm, B., Kann, P. and Pfützner, A. (2008). Influence of C peptide on glucose utilization. Journal of Diabetes Research. 2008(1): 769483.

  57. Yahya, A., Adil, O.W., Mohammed, H.O., and Hasan, A.F. (2024). Histopathological and immunohistochemical studies on the effects of silver oxide nanoparticles (AgNPs) on male rats’ liver. Journal of Bioscience and Applied Research. 10(3): 392-398.
Published In
Asian Journal of Dairy and Food Research
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Protective Effects of Neem Ethanolic Extract on Streptozotocin-induced Diabetes: Modulation of C-Peptide and Peroxynitrite Levels in Liver Male Rats

    B
    Bushra R. Ibrahim1,*
    L
    Luma W. Khaleel1
    1Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Iraq.

    ABSTRACT

    Background: Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia due to insufficient insulin production or the body’s inability to effectively use insulin. Neem extract, derived from the leaves of the Azadirachta indica (Neem), is used in various applications, particularly in traditional medicine and as a natural pesticide. This study aims to evaluate the therapeutic effects of neem ethanolic extract on type 1 diabetes mellitus by determining changes in blood glucose levels, serum C-peptide concentrations and peroxynitrite formation in diabetic rats. Specifically, the study seeks to investigate neem’s potential to improve glycemic control, preserve endogenous insulin secretion as indicated by C-peptide levels and reduce oxidative and nitrosative stress measured through peroxynitrite levels.

    Methods: Ninety Male Rats that were 180-200 g in weight were randomly divided in to six groups 15 rats in each one and kept for approximately seventy days, animal groups: G1: (Negative control). G2: (Positive control) will receive a single STZ dose (50 mg/kg B.W. I/P) to induce diabetes. G3: Will daily receive neem ethanolic extract (500 mg/kg) orally. G4: Diabetic animals will daily receive neem ethanolic extract (400 mg/kg B.W) orally. G5: Diabetic animals will daily receive neem ethanolic extract (500 mg/kg B.W) orally. G6: Diabetic Animals will daily receive Insulin (3 I.U) S/c.

    Result: Our results showed that the diabetic group showed an increase in Glucose, Peroxynitrite and a decrease in C-peptide and disruption of the hepatic cords in liver tissue when compared to the control group. When diabetic animals were treated with neem extract and insulin, we observed an improvement in both Peroxynitrite, C-peptide, Glucose and a normal liver structure and a normal distribution of central veins and hepatic cords in liver tissue when compared to the positive group.

    INTRODUCTION

    Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from insufficient insulin secretion, impaired insulin action, or both. The global prevalence of DM continues to rise, making its long term complications a major public health concern (Saeedi et al., 2019; Al-Saeedi et al., 2025). Beyond disturbances in glucose metabolism, diabetes is associated with progressive damage to multiple organs, largely driven by oxidative stress and chronic inflammation (Kapucu et al., 2021; Mahmood et al., 2024; Othman et al., 2024; Hasan et al., 2022; Fakri et al., 2023; Balaky et al., 2021; Muhsin et al., 2021).  
           
    The liver plays a central role in glucose and lipid homeostasis and is particularly vulnerable to diabetic injury. In diabetes, sustained hyperglycemia enhances the production of reactive oxygen species (ROS) through pathways such as glucose autoxidation, mitochondrial dysfunction and activation of the polyol pathway. Excessive ROS generation overwhelms endogenous antioxidant defenses, leading to oxidative stress, inflammatory responses and structural damage in hepatic tissue. These processes contribute to hepatocellular injury, fibrosis and impaired liver function, which further exacerbate metabolic dysregulation in diabetic individuals (Alsieni et al., 2021; Hasan et al., 2024; Najim et al., 2024).
           
    One important mediator of oxidative and nitrosative stress in diabetes is peroxynitrite (ONOO-), a highly reactive nitrogen species formed by the rapid reaction of nitric oxide with superoxide anions. Peroxynitrite induces lipid peroxidation, protein nitration and DNA damage, thereby amplifying inflammation and cellular dysfunction in the liver. Elevated levels of peroxynitrite have been implicated in the progression of diabetic complications, highlighting its relevance as a marker and mechanistic contributor to hepatic oxidative damage (Ghasemi et al., 2023; Pirabbasi et al., 2024; Ahmed et al., 2025).
           
    C peptide, a cleavage product released in equimolar amounts with insulin from pancreatic β cells, is increasingly recognized as more than a biologically inert peptide. In diabetes, particularly in insulin deficient states, reduced C peptide levels reflect β cell dysfunction. Emerging evidence suggests that C peptide exerts protective effects on microvascular function, inflammation and oxidative stress. Therefore, assessment of C peptide provides valuable insight into pancreatic function and the severity of diabetic metabolic disturbances (Laila et al., 2022).
           
    Experimental models are essential for elucidating the mechanisms underlying diabetic liver injury and for evaluating potential therapeutic interventions. Streptozotocin (STZ) is widely used to induce experimental diabetes due to its selective toxicity toward pancreatic β cells. STZ enters β cells via the glucose transporter GLUT2, causing DNA alkylation, oxidative stress and subsequent insulin deficiency. This model reliably mimics key metabolic and oxidative features of diabetes, including hyperglycemia induced liver damage, making it suitable for investigating hepatoprotective strategies (Al-awadhi et al., 2024; Mahmoud Al-Doori et al., 2025).
           
    Current antidiabetic therapies primarily aim to control blood glucose levels but often provide limited protection against oxidative stress mediated organ damage, including hepatic dysfunction. Consequently, there is growing interest in complementary and alternative therapies derived from natural products that can target oxidative stress and inflammation alongside glycemic control (Patil et al., 2021).
           
    Azadirachta indica is a medicinal plant extensively used in traditional medicine and has attracted scientific attention for its antidiabetic, antioxidant and anti inflammatory properties. Neem contains a wide range of bioactive compounds, including flavonoids, terpenoids and alkaloids, which have demonstrated free radical scavenging and anti inflammatory activities. These properties suggest that neem extract may mitigate oxidative stress, suppress inflammatory pathways and protect liver tissue against diabetes induced damage (Cardoso et al., 2025).
           
    Current therapies often provide limited protection against oxidative stress-mediated organ damage. Consequently, there is interest in natural products like Azadirachta indica, which contains bioactive compounds such as flavonoids and terpenoids with potent insulin-mimetic activity (Panda et al., 2023; Pandita et al., 2022). This study evaluates the protective effects of neem on hepatic dysfunction and oxidative markers in STZ-induced diabetic rats.

    MATERIALS AND METHODS

    The Fresh leaves of Azadirachta indica were collected from Al-Nahrain University, Jadiriyah, Baghdad, during June 2024. The sample was identified and classified by the University of Baghdad grassland, College of Science, Department of Biology.
     
    Preparation of extract
     
    The leaves were properly washed and air-dried at room temperature for two weeks. The dried leaves were ground into powder using a corona manual grinding machine. Exactly 100 g of the ground leaves of neem were soaked in 1 liter of ethanol for 24 hrs. It was sieved and filtered using Whatman no1 (125 mm) filter paper. The filtrate was evaporated to dryness using a rotary evaporator and the paste was put in a stoppered universal bottle and stored in the refrigerator until needed. The paste was dissolved in distilled water before use (Obiajulu and Chukwuemeka, 2020).
     
    Induction of diabetes in male rats
     
    Diabetes was induced by streptozotocin (STZ) (50 mg/kg) diluted in 5 ml citrate buffer having pH 4.5, which was injected intraperitoneally (IP) to 60 rats. fasting condition were preserved for 18 hours to stimulate a diabetic situation. The injection volume of the diluted STZ was determined (Jenna and Wurster, 2021).
     
    Experimental design
     
    Ninety Male Rats (Rattus norvegicus) weighted 180-200g, were randomly divided into six groups 15 rat in each one and kept for approximately seventy days. The room temperature ranged 22 to 25°C with relative humidity conditions water supply, commercial food and a 12-hour light/12-hour dark cycle for 14 days before the beginning of the experiment. Diabetes was induced in 60 rats for four groups by STZ (50 mg/kg) diluted in 5 ml citrate buffer having pH 4.5 which was injected intraperitoneally (IP) to rat and preserved fasting condition for 18 hours to stimulate diabetic situation.
     
    Animals groups
     
    G1: (Negative control): The animals received normal saline orally.
    G2: (Positive control): The animals received single STZ dose (50 mg/kg B.W I/P) to induced diabetes (Omolaoye et al., 2018).
    G3: Healthy animals, received neem ethanolic extract (500 mg/kg B.W) daily and orally.
    G4: Diabetic animals received neem ethanolic extract (400 mg/kg B.W) daily and orally (Seriana et al., 2021).
    G5: Diabetic animals received neem ethanolic extract (500 mg/kg B.W) daily and orally.
    G6: Diabetic Animals received insulin (3 I.U) daily and S/c.
     
    Blood and tissue sampling
     
    Each animal received a dosage of (≥ 100 mg/kg) of sodium pentobarbital solution as anesthesia IP. Blood samples were taken by cardiac puncture of the rats and centrifuged for 15 min at 4000 rpm to collect serum before being subjected to biochemical and immunological analysis. The liver was cleaned with cold saline after their removal from the animals to be fixed with 10% neutral formalin for histological examination. The samples were kept at -20°C.
     
    Biochemical analysis
     
    Determination of glucose
     
    Serum glucose levels were determined using the modified kinetic glucose oxidase peroxidase (GOD-PAP) method (Trinder, 1969). Measurements were performed using a commercial kit provided by Agappe Diagnostics Ltd, India.
     
    Determination of C-peptide
     
    The kits were purchased from (SunLong Biotech) under kits numbered (ABIN6963725 C-Peptide) and this test was performed using the (ELISA Kit) technique.
     
    Determination of peroxynitrite
     
    The kits were purchased from (Sunlong Biotech) under kits numbered (Rat Peroxynitrite Anion (ONOO-)) and this test was performed using the (ELISA Kit) technique.
     
    Histopathological investigations
     
    After taken from rats following scarification, liver tissue samples were fixed in 10% neutralized formaldehyde, embedded in paraffin wax and then stained with hematoxylin and eosin. were used to stain tissues and to demonstrate fatty changes, respectively according to (Al-Khuzaay et al., 2024; Yahya et al., 2024).
     
    Ethical approval
     
    Ethical approval was obtained for conducting experiments on laboratory animals under the approval obtained from the University of Baghdad - College of Veterinary Medicine -Animal Care and Use Committee, according to approval number P.G/1200 issued on 23/6/2024.
     
    Statistical analysis
     
    The Statistical Packages of Social Sciences-SPSS (2019) program was used to detect the effect of difference groups and period in study parameters. LSD-Least significant difference (two way) was used to significant compare between means in this study.

    RESULTS AND DISCUSSION

    The results of the biochemical analysis are summarized in the following Tables. As shown in Table 1, STZ- induced diabetic rats exhibited a significant increase in glucose levels, which was markedly reduced by neem extract treatment (p≤0.05). Changes in serum C-peptide concentration across the experimental groups are detailed in Table 2, showing partial restoration in treated groups. Furthermore, the levels of peroxynitrite in tissue were significantly attenuated following neem administration, as presented in Table 3.

    Table 1: Effect of different doses at three periods of neem leaves extract and insulin treatment on serum glucose concentration (mg/dl) in STZ-induced diabetic male rats.



    Table 2: Effect of different doses at three periods of neem leaves extract and insulin treatment on serum C-peptide concentration (pg/ml) in STZ-induced diabetic male rats.



    Table 3: Effect of different doses at three periods of neem leaves extract and insulin treatment in lung tissue peroxynitrite(ng/L) in STZ-induced diabetic male rats.


     
    Histopathological results of liver
     
    Histological examination of the liver (summarized in Fig 1) showed that diabetic rats (G2) suffered from coagulative necrosis and fatty droplets (Fig1 B). In contrast, rats treated with neem ethanolic extract 500 mg/kg(G5) showed near-normal liver architecture (Fig1 E), comparable to the negative control (Fig 1 A).

    Fig 1: A Histological section of the liver of rats of negative control group shows normal liver architecture that characterized by normal central veins (V) with hepatic cords (Black arrow), H and E 40X. B: Histological section of the liver of rats of positive control group shows area of coagulative necrosis that characterized by hypereosinophilic pyknotic and shrunken hepatocytes, disturbing of hepatic cords (Black arrow) with variably sized clear intracytoplasmic vacuoles (Fatty droplets, blue arrow), H and E, 40X. C: Histological section of the liver of rats that received Neem only shows normal liver architecture that characterized by normal central veins (V) and hepatic cords (Black arrow) distribution with no clear pathological changes, H and E 40X. D: Histological section of the liver of DM rats that treated with 400mg/kg Neem shows moderate multifocal of variably sized periportal granulomas (Black arrow) that replaced individual necrotic hepatocytes. Granulomatous foci characterized by aggregation of mononuclear cells (lymphocytes and macrophages), H and E, 40X. E: Histological section of the liver of DM rats treated with 500 mg/kg. Neem shows normal liver architecture characterized by normal central veins (V) and hepatic cords distribution with some individual apoptotic/necrotic hepatocytes (Black arrow), H and E, 40X. F: Histological section of the liver of diabetic rats that treated with insulin shows normal liver architecture characterized by normal central veins (V) and hepatic cords distribution. However, some individual apoptotic and necrotic hepatocytes and mononuclear cells aggregated around central veins were seen (Black arrow), H and E, 40X.


           
    The present study demonstrates that ethanolic Azadirachta indica leaf extract exerts significant antidiabetic and hepatoprotective effects in streptozotocin (STZ)-induced diabetic rats. Treatment with neem extract, particularly at 500 mg/kg, resulted in a significant reduction in blood glucose levels. This glucose-lowering effect may be linked to improved peripheral glucose utilization and GLUT4 translocation, comparable to the efficacy of insulin treatments noted by Patel et al., (2018). As shown in Table 1, restoration of C-peptide concentrations, attenuation of oxidative stress as indicated by reduced peroxynitrite levels and marked improvement in liver histoarchitecture. These effects were time-and dose-dependent and, in several parameters, comparable to insulin treatment.
           
    STZ-induced diabetic rats exhibited persistent hyperglycemia throughout the experimental period, confirming successful induction of diabetes and reflecting impaired insulin secretion and utilization. In contrast, normal control animals and non-diabetic rats treated with neem extract (500 mg/kg) maintained normoglycemia, indicating that the extract did not induce hypoglycemia under physiological conditions According to (Nagatani et al., 1996; Wang et al., 2010; Guerre-Millo et al., 1985).
           
    Treatment with neem extract at both 400 and 500 mg/kg significantly reduced blood glucose levels compared with diabetic controls. The glucose-lowering effect was more pronounced at 500 mg/kg, with a statistically significant decrease observed at day 60 (p≤0.05), indicating a clear dose- and time-dependent response. However, glucose levels remained higher than those of normal controls, suggesting partial rather than complete glycemic normalization. Insulin-treated rats showed a marked reduction in glucose levels, though values remained slightly elevated relative to non-diabetic controls (Patel et al., 2018).
           
    The hypoglycemic effect of neem extract may be attributed to improved peripheral glucose utilization. A plausible mechanism supported by previous studies is the upregulation of insulin signaling and increased GLUT4 translocation in skeletal muscle, enhancing glucose uptake and improving glycemic control. This mechanism provides a single, coherent explanation for the observed glucose reduction and avoids redundant or unsupported pathways (Morris et al., 2019).
           
    C-peptide levels remained stable in normal controls and non-diabetic rats treated with neem extract, confirming preserved endogenous insulin secretion. In contrast, diabetic control rats showed a significant reduction in C-peptide levels (p≤0.05), reflecting STZ-induced cytotoxic damage to pancreatic β-cells and impaired insulin synthesis.        

    Neem extract treatment partially restored C-peptide levels in diabetic rats, with the 500 mg/kg dose showing superior efficacy compared to 400 mg/kg. The extract at 400 mg/kg maintained C-peptide levels up to day 60, followed by a significant decline after stop of treatment (70 day), whereas the higher dose sustained improved levels for a longer duration. Insulin-treated rats also demonstrated a significant increase in C-peptide at day 60, likely due to reduced glucotoxic stress on surviving β-cells rather than direct stimulation of insulin synthesis (El-Beltagy et al., 2021; Abduallah et al., 2023; McCalla et al., 2024; Dholi et al., 2011).
           
    As shown in Table 2. These findings indicate that neem extract may preserve residual β-cell function by mitigating STZ-induced cytotoxicity, rather than by modulating autoimmune mechanisms, which are not relevant to this experimental model (Dholi et al., 2011; Boldison et al., 2019). This is supported by Gabr et al., (2023), who noted the impact of antioxidants on preventing pancreatic B-cell damage.
           
    Peroxynitrite levels remained within normal ranges in negative controls and non-diabetic rats treated with neem extract. Diabetic control rats exhibited a significant elevation in peroxynitrite levels (p≤0.05), consistent with enhanced oxidative and nitrosative stress associated with diabetes and increased nitric oxide synthase activity (Ghorbani et al., 2011; Saravanan et al., 2006; Wilhelm et al., 2008).
           
    Treatment with neem extract at both doses significantly reduced peroxynitrite levels compared with diabetic controls, with the 500 mg/kg dose producing greater normalization. This reduction suggests an attenuation of oxidative stress, which may contribute to improved pancreatic and hepatic function. Insulin-treated rats showed an initial increase in peroxynitrite at day 60, followed by a significant decrease at day 70 (p≤0.05) after stop of treatment (70 day), indicating delayed oxidative stress modulation according to (Alfaris et al., 2021).
           
    These results support the role of neem extract in reducing diabetes-associated oxidative stress in vivo.  The bioactive potential of neem in combating systemic disturbances is consistent with the findings of (Veerendrakumar et al., 2023 and Kumar et al., 2021). Who emphasized the role of traditional plants in biochemical restoration. As shown in Table 3. Histological examination of liver tissue from diabetic control rats revealed marked pathological alterations, including coagulative necrosis, hepatocellular shrinkage, pyknotic nuclei and disruption of hepatic cords, consistent with diabetes-induced hepatic injury (Chen et al., 2022; Fakhri et al., 2021; Huang et al., 2024).                           

    Rats treated with neem extract showed dose-dependent histological improvement. The 400 mg/kg dose resulted in moderate restoration with residual periportal granulomatous changes, whereas the 500 mg/kg dose exhibited near-normal liver architecture, characterized by intact central veins and well-organized hepatic cords. These findings indicate superior hepatoprotective efficacy at the higher dose. Insulin-treated rats also demonstrated near-normal liver morphology (Mahata et al., 2021; Alrefaei et al., 2025; El-Megharbel et al., 2022).                    
           
    Overall, neem extract at 500 mg/kg provided the most effective protection against diabetes-induced hepatic damage, likely through combined glycemic control and oxidative stress reduction (Husen et al., 2021).

    CONCLUSION

    In conclusion, this study demonstrates that ethanolic extract of Azadirachta indica leaves possesses potent antidiabetic and hepatoprotective properties in STZ-induced diabetic rats. The extract, particularly at a dose of 500 mg/kg, significantly improved glycemic control and restored serum C-peptide levels while reducing nitrosative stress via the modulation of peroxynitrite. Furthermore, neem treatment successfully attenuated hepatic structural damage, promoting the restoration of normal liver architecture. These findings suggest that neem extract could serve as a valuable natural therapeutic agent for managing type 1 diabetes and its associated hepatic complications. Future research should focus on identifying the specific bioactive compounds responsible for these protective effects.

    ACKNOWLEDGEMENT

    This study did not receive any grant from any funding agency.
     
    Funding
     
    No financial support for our research.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

    REFERENCES


    1. Abduallah, A.M., Ahmed, A.E., Bajaber, M.A. and Alalwiat, A.A. (2023). Evaluation of the Antidiabetic Effects of methanolic extracts of neem (Azadirachta indica) seeds on the streptozotocin- induced wistar rats. Pakistan Veterinary Journal. 43(4): 792-798.

    2. Ahmed, R.M., Abdullah, A.R., Majeed, A.K. and Hasan, A.F. (2025). The effect of clove oil on the expression of xly gene in tomato infected with Fusarium oxysporum. Agricultural Science Digest. 45: 91-97. doi: 10.18805/ag.DF-794.

    3. Al-awadhi, R.M., Abdelrazek, H.M.A., Abouelhassan, E.M., Kamel, M.S., Attia, N. A. and Awad, A.A. (2024). Neem leaves extract reduces sex steroids and gonadal function in female wistar albino rats. Biomedical and Pharmacology Journal. 17(3): 1471-1481.

    4. Alfaris, N.A., Alshammari, G.M., Altamimi, J.Z., Aljabryn, D.H., Alagal, R.I., Aldera, H. and Yahya, M.A. (2021). Ellagic acid prevents streptozotocin-induced hippocampal damage and memory loss in rats by stimulating Nrf2 and nuclear factor-êB and activating insulin receptor substrate/PI3K/ Akt axis. Journal of Physiology and Pharmacology. 72(4). doi: 10.26402/jpp.2021.4.02. 

    5. Al-Khuzaay, H.M., Al-Juraisy, Y.H., Hasan, A.F. and Tousson, E. (2024). Antitumor activity of β-glucan isolated àrom date fruits ùn cancer cells in vivo. Opera Medica et Physiologica. 11(3): 41-48.

    6. Alrefaei, A.F. and Elbeeh, M.E. (2025). Hepatoprotective effects of Glycyrrhiza glabra in diabetic male rats: Addressing liver function, oxidative stress and histopathological changes. Biology. 14(3): 307.

    7. Al-Saeedi, R.F.H., Mwafaq, R.K. and Jasim, N.A. (2025). Nephrotoxicity of iron oxide nanoparticles in male mice. Agricultural Science Digest. 46(1): 38-43. doi: 10.18805/ag.DF-751.

    8. Alsieni, M.A., El Rabey, H.A., Al-Sieni, A.I. and Al-Seeni, M.N. (2021). Comparison between the antioxidant and antidiabetic activity of fenugreek and buckthorn in streptozotocin induced diabetic male rats. BioMed Research International. 2021(1): 7202447.

    9. Balaky, H.M. and Kakey, I.S. (2021). Indications of liver and kidney functions in non-insulin dependent diabetic patients. Iraqi Journal of Science. pp 769-778.

    10. Boldison, J., Da Rosa, L.C., Davies, J., Wen, L. and Wong, F.S. (2019). Dendritic cells license regulatory B cells to produce IL-10 and mediate suppression of antigen- specific CD8 T cells. Cellular and Molecular Immunology. 17(8): 843-855. https://doi.org/10.1038/s41423-019- 0324-z.

    11. Cardoso, C.A., Parca, A.V., Facciotti, P.R., Machado, L.C., de Oliveira, V.C., de Campos, S.A.G.D. and dos Santos, M.D. (2025). Natural pesticide, Azadirachta indica A. juss. (Neem), disrupted reproductive parameters of male rats. Journal of Ethnopharmacology. 337: 118927.

    12. Chen, H., Luo, Y., Tsoi, B., Gu, B., Qi, S. and Shen, J. (2022). Angong niuhuang wan reduces hemorrhagic transformation and mortality in ischemic stroke rats with delayed thrombolysis: Involvement of Peroxynitrite-mediated MMP-9 activation. Chinese Medicine. 17(1): 51.

    13. Christian, E.O. and Chukwuemeka, E.F. (2020). Antidiabetic and modulatory effect of ethanol extract of neem leaf on some essential biochemical parameters of streptozotocin- induced diabetic rats. International Journal of Biochemistry Research and Review. 28(4): 1-11.

    14. Dholi, S.K., Raparla, R., Kanaki, S.K., Bhaskar, P. and Priyanka, S. (2011). Comparative evaluation of antidiabetic activity of Cinnamomum tamala Linn. leaf extracts in STZ induced diabetic rats. International Journal of Pharmaceutical Sciences and Drug Research. 3(3): 221-224.

    15. El-Beltagy, A.E.F.B., Saleh, A.M., Attaallah, A. and Gahnem, R.A. (2021). Therapeutic role of Azadirachta indica leaves ethanolic extract against diabetic nephropathy in rats neonatally induced by streptozotocin. Ultrastructural Pathology. 45(6): 391-406.

    16. El-Megharbel, S.M., Al-Thubaiti, E.H., Qahl, S.H., Al-Eisa, R.A. and Hamza, R.Z. (2022). Synthesis and spectroscopic characterization of dapagliflozin/Zn (II), cr (III) and Se (IV) novel complexes that ameliorate hepatic damage, hyperglycemia and oxidative injury induced by streptozotocin- induced diabetic male rats and their antibacterial activity. Crystals. 12(3): 304.

    17. Fakhri, S., Patra, J.K., Das, S.K., Das, G., Majnooni, M.B. and Farzaei, M.H. (2021). Ginger and heart health: From mechanisms to therapeutics. Current Molecular Pharmacology. 14(6): 943-959.

    18. Fakri, Y.A. and Al-Jowari, S.A.K. (2023). Antithyroid peroxidase and thyroid hormones in a sample of Iraqi patients with type 2 diabetes mellitus. Iraqi Journal of Science. pp 1618-1624.

    19. Gabr, N.M., Elbayoumy, F.M.A.E., Omar, A.E.D.S.E.S., El-Lateef, A.E.L.S.A., Eissa, A.A.M.A., Younes, A.M. and Abbas, M.A.M. (2023). Impact of antioxidants on pancreatic B- cell damage caused by Streptozotocin in adult male albino rats. International Journal of Medical Arts. 5(1): 2944- 2950.

    20. Ghasemi, A. and Jeddi, S. (2023). Streptozotocin as a tool for induction of rat models of diabetes: A practical guide. EXCLI Journal. 22: 274.

    21. Ghorbani, A., Omrani, G.R., Hadjzadeh, M.A.R. and Varedi, M. (2011). Effects of rat C-peptide-II on lipolysis and glucose consumption in cultured rat adipose tissue. Experimental and Clinical Endocrinology and Diabetes. 119(6): 343-347.

    22. Guerre-Millo, M., Leturque, A., Girard, J. and Lavau, M. (1985). Increased insulin sensitivity and responsiveness of glucose metabolism in adipocytes from female versus male rats. The Journal of Clinical Investigation. 76(1): 109-116.

    23. Hasan, N.T. and Ali, S. (2024). Impact of obesity on serum levels of neuropeptide-Y in type 2 diabetics in relation to glycemic status. Iraqi Journal of Pharmaceutical Sciences. 33(4SI): 282-289.

    24. Hasan, Z.Y.M. and Abdullah, J.M. (2022). Effects of gel extract from Aloe vera cultivated in Iraq on blood glucose level and lipid profile in induced diabetic mice. Iraqi Journal of Science. 63(1): 124-130.

    25. Huang, W., Zheng, J., Wang, M., Du, L. Y., Bai, L. and Tang, H. (2024). The potential therapeutic role of melatonin in organ fibrosis: A comprehensive review. Frontiers in Medicine. 11: 1502368.

    26. Husen, F., Hernayanti, H., Ekowati, N., Sukmawati, D. and Ratnaningtyas, N.I. (2021). Antidiabetic effects and antioxidant properties of the saggy ink cap medicinal mushroom, coprinus comatus (Agaricomycetes), in streptozotocin-induced hyperglycemic rats. International Journal of Medicinal Mushrooms. 23(10): 9-21.

    27. Jenna, I., Wurster, L., Rachel and Brown, E.P. et al. (2021). Streptozotocin induced hyperglycemia alters the cecal metabolome and exacerbates antibiotic induced dysbiosis. Cell Repo. 37(11): 110113.

    28. Kapucu, A. (2021). Crocin ameliorates oxidative stress and suppresses renal damage in streptozotocin induced diabetic male rats. Biotechnic and Histochemistry. 96(2): 153-160.

    29. Kumar, S., Prashant, M., Ram, B. and Dwivedi, K.N. (2021). Role of katankateryadi kwatha in insulin secretion and restoration of biochemical changes in streptozotocin-nicotinamide induced diabetes mellitus type 2 in rats. International Journal of Applied Biology and Pharmaceutical Technology.  12(3): 362-379.

    30. Laila, N., Sitasiwi, A.J. and Isdadiyanto, S. (2022). Effect of neem leaf extract on reproductive potential of male rats fed high-fat diet. Biosaintifika: Journal of Biology and Biology Education. 14(1): 135-146.

    31. Mahata, L.E., Ali, H. and Murni, A.W. (2021). Effect of streptozotocin on liver histology damage in rats’ model of gestational diabetes mellitus. Int. J. Res. Rev. 8: 18-22.

    32. Mahmood, N.M.S., Mahmud, A.M. and Maulood, I.M. (2024). Role of melatonin in attenuation of vascular angiotensin 1-7 reactivity via calcium channels and endothelial derived relaxing factors in induced diabetic aortic rats. Iraqi Journal of Science. pp 4256-4270.þ

    33. Mahmoud Al-Doori, M.A., Farhan Al-Halbosiy, M.M., Mohammed, H.Z.Y., Hussein, S. M., Hasan, A.F. and El-Wahsh, H.M. (2025). The antileshmaniasis effect of Brassica juncea seeds fatty acids on the growth of leishmania tropica promastigotes isolated culture in vitro. Asian Journal of Dairy and Food Research. 44: 90-98. doi: 10.18805/ajdfr.DRF-553.

    34. McCalla, G.A. and Brown, P.D. (2024). Suitability of the neonatal streptozotocin diabetes model and folklore therapeutic use of low-dose neem leaf extract to treat hyperglycemia associated with type 2 diabetes mellitus. Tropical Journal of Natural Product Research. 8(1): 5925.

    35. Minokoshi, Y., Kahn, C.R. and Kahn, B.B. (2003). Tissue-specific ablation of the GLUT4 glucose transporter or the insulin receptor challenges assumptions about insulin action and glucose homeostasis. Journal of Biological Chemistry.  278(36): 33609-33612.

    36. Morris, J.C.B. Gonzales, J.J., De La, C. et al. (2019). The highly pure neem leaf extract, scne, inhibits tumorigenesis in oral squamous cell carcinoma via disruption of pro-tumor inflammatory cytokines and cell signaling. Front Oncol. 13(9): 890.

    37. Muhsin, S.N. and Faris, H.N. (2021). Quality of life of patients with type 2 diabetes mellitus in Thi-Qar-Iraq. International Journal of Pharmaceutical Research. 13(1): 5438.

    38. Nagatani, S.H.O.J.I., Bucholtz, D.C., Murahashi, K.U.M.I.K.O., Estacio, M.A., Tsukamura, H.I.R.O.K.O., Foster, D.L. and Maeda, K.I. (1996). Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats. Endocrinology. 137(4): 1166-1170.

    39. Najim, H.D., Mohammed, M.M. and Rahmah, A.M. (2024). Influence of adding empagliflozin to conventional anti-diabetic therapy on quality-of-life scale for type 2 diabetic patients.  Iraqi Journal of Pharmaceutical Sciences. 33(3): 142- 149.

    40. Obiajulu, C.E., Francis, C.E., Chukwudi, O.O., Daniel and Onwusulu, N.  (2020). Aqueous extract of azadirachta indica leaves favorably alters the course of streptozotocin-induced diabetes in rats: A comparative prospective cohort study. Biomedical Research and Therapy. 7(7): 3877-388.

    41. Obiajulu, A.O. and Chukwuemeka, E.E.O. (2020). E-human resource management and organizational performance of selected federal agencies in Nigeria. International Journal of Academic Research in Business and Social Sciences. 10(12): 1-15. https://doi.org/10.6007/IJARBSS/v10-i12/ 8212.

    42. Omolaoye, T.S., Skosana, B.T. and du Plessis, S.S. (2018). Diabetes mellitus-induction: Effect of different streptozotocin doses on male reproductive parameters. Acta Histochemica. 120(2): 103-109.

    43. Othman, H.K., Mahmud, A.M. and Maulood, I.M. (2024). The roles of potassium channels and nitric oxide in the modulation of apelin induced-relaxation in isolated diabetic rat aorta. Iraqi Journal of Science. pp 644-658.

    44. Panda, C., Sharma, P., Dixit, U.S. and Pandey, L.M. (2023). Potential and prospective of traditional Indian medicinal plants for the treatment of diabetes. Journal of Biologically Active Products from Nature. 13(4): 316-360.

    45. Pandita, D., Pandita, A. and Bhanu, C. (2022). Antidiabetic Plants for Drug Discovery: Pharmacology, Secondary Metabolite Profiling and Ingredients with Insulin Mimetic Activity. CRC Press.

    46. Patel, M.J.S., Tripathy, K.D.M. et al. (2018). A supercritical CO2 extract of neem leaf (A. indica) and its bioactive liminoid, nimbolide, suppresses colon cancer in preclinical models by modulating pro-inflammatory pathways. Mol Carcinog.  57(9): 1156-1165. 

    47. Patil, S.M., Shirahatti, P.S., VB, C.K., Ramu, R. and Prasad, N. (2021). Azadirachta indica A. Juss (neem) as a contraceptive: An evidence-based review on its pharmacological efficiency. Phytomedicine. 88: 153596.

    48. Pirabbasi, E., Zangeneh, M.M., Zangeneh, A., Moradi, R. and Kalantar, M. (2024). Chemical characterization and effect of Ziziphora clinopodioides green synthesized silver nanoparticles on cytotoxicity, antioxidant and antidiabetic activities in streptozotocin induced hepatotoxicity in wistar diabetic male rats. Food Science and Nutrition. 12(5): 3443-3451.

    49. Saeedi, P., Petersohn, I., Salpea, P., Malanda, B., Karuranga, S., Unwin, N. and Committee, I.D.F.D.A.  (2019). Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the international diabetes federation diabetes atlas. Diabetes Research and Clinical Practice. 157: 107843.

    50. Saravanan, G. and Leelavinothan, P. (2006). Effects of syzygium cumini bark on blood glucose, plasma insulin and C-peptide in streptozotocin induced diabetic rats. Int. J. Endocrinol. Metab. 4: 96-105. 

    51. Satyanarayana, K., Sravanthi, K., Shaker, I.A. and Ponnulakshmi, R. (2015). Molecular approach to identify antidiabetic potential of Azadirachta indica. J. Ayurveda Integr Med. 6: 165-174.

    52. Seriana, I., Akmal, M., Darusman, D., Wahyuni, S., Khairan, K. and Sugito, S. (2021). Neem leaf (Azadirachta indica A. Juss) ethanolic extract on the liver and kidney function of rats. The Scientific World Journal. 2021(1): 7970424.

    53. Trinder, P. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochen. 6: 24-27.

    54. Veerendrakumar, P., Neralla, M., Satheesh, T. and Venkidasamy, B. (2023). Comparative extraction and bioactive potential of the leaf extracts of Azadirachta indica for combatting postoperative head and neck infections: An in vitro study. Cureus. 15(12): e51303.

    55. Wang, Z., Yang, Y., Xiang, X., Zhu, Y., Men, J. and He, M. (2010). Estimation of the normal range of blood glucose in rats. Wei Sheng Yan Jiu=Journal of Hygiene Research. 39(2): 133-137.

    56. Wilhelm, B., Kann, P. and Pfützner, A. (2008). Influence of C peptide on glucose utilization. Journal of Diabetes Research. 2008(1): 769483.

    57. Yahya, A., Adil, O.W., Mohammed, H.O., and Hasan, A.F. (2024). Histopathological and immunohistochemical studies on the effects of silver oxide nanoparticles (AgNPs) on male rats’ liver. Journal of Bioscience and Applied Research. 10(3): 392-398.
    In this Article
      Contact us
      Follow us
      Published In
      Asian Journal of Dairy and Food Research

      Editorial Board

      View all (0)