Research

, Volume: 19( 1) DOI: 10.37532/0972-768X.2021.12(1).405

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The Synergistic Efficacy of Medemia argun, Arecaceae, Seed's Ethanolic Extract to Insulin as Antidiabetic and Hepatoprotective Agent in Rats

Asmaa Nabil Mohie^1*, Mahmoud Abdel Razik Doheim², Ragab Abdel Fattah El Masry³, and Ayman Mohamed El-Sayed⁴

¹Department of Biochemistry, Faculty of Agriculture, Zagazig University, Egypt

²Department of Biochemistry, Faculty of Agriculture, Zagazig University, Egypt

³Department of Biochemistry, Faculty of Agriculture, Zagazig University, Egypt

⁴Department of Biochemistry, Faculty of Agriculture, Zagazig University, Egypt

*Correspondence:

Mohie AN, Department of Biochemistry, Faculty of Agriculture, Zagazig University, Egypt, E-mail: dr.asmaanabil88@gmail.com

Abstract

 

Diabetes mellitus (DM) is an incurable metabolic disorder, with a global incidence of about 2.8%. Adverse effects of chemical drugs necessitated searching for medicinal plants as alternative therapies. Medemia argun (MA) is an ancient Egyptian palm, rich in proanthocyani-dins (PACs), which are essential constituent of human diet. It was highly valued in ancient Egypt, although the reason for that necessitates further study.

Thirty male rats were allocated into 5 equal groups. Type I diabetes mellitus (DM Type I) was induced by single intraperitoneal (IP) injection of alloxan (120 mg/kg). Animals were then treated with insulin (30 U/kg, daily) +/- MA seed ethanolic extract (100 mg/kg, orally, once daily). Random blood sugar (RBS) level, liver transaminase activity (ALT and AST), albumin, total bilirubin as well as histopathological examination of liver tissues were investigated in all groups.

MA when given solely by oral gavage, significantly lowered RBS level either alone or with insulin. Similar effects were seen on serum ALT and AST activities, without affecting bilirubin, albumin levels significantly. It also preserved hepatic cords, portal triad structures, sinusoids, Von-Kupffer cells and stroma better than insulin alone.

MA seed ethanolic extract potentially improved insulin outcomes in diabetes and restored liver tissue changes when used alone or combined with insulin.

Keywords

Medemia argun; Type I diabetes mellitus; Liver functions;Rats

Introduction

Diabetes mellitus (DM), belongs to metabolic diseases, where the principal symptom of the disease is persistently elevated sugar levels in the blood. It can be classified into two main types, I and II. Diabetic complications possess possible blindness, amputation of the lower limb, renal failure and cardiac arrest (stroke). In developed countries, about 87% to 91% of the diagnosed diabetic people belong to type II diabetes, 7%to 12% have type I diabetes, whiles 1% to 3% to have other types of the disease [1]. Due to the side effects of conventional medicine, many physicians prescribe natural products as an alternative to conventional treatment for the treatment of various diseases, including diabetes [2]. Many herbals have been proved for their potential to protect against hepatotoxicity in many disciplines [3, 4].

Medemia argun (MA) is an ancient palm rich in proanthocyanidins (PACs). These polyphenolic compounds are widely distributed in plants and are an integral part of the human diet [5]. Little studies were published about MA, it is a fan palm, grows at the Nubian Desert Oases of South Egypt and North of Sudan. The fruit has been found in the tombs from the 5th Dynasty (2500 BC) and Roman times (6–7th century AD), especially inside the tomb of Tut Ankh Ammon [6]. The PAC derived from MA nuts is an efficient protector against oxidative/nitrative stress associated with many ailments [5].

The presence of PACs with efficient antioxidant activity may underline the usefulness of argun nuts during ancient times and may explore the potential use of this fruit as a novel rich source of hydrolyzable PACs containing afzelechin, catechin, and gallocatechin [5].

In this study, we tested the possible synergistic potential of MA seed ethanolic extract against experimental type I diabetes, induced by alloxan, as well as, the therapeutic value of the extract on some liver functions, which was documented by histological examination of the liver tissue, whether used alone or as an adjuvant to insulin.

Materials and Methods

Chemicals

Alloxan was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mixtard insulin (soluble insulin 30% and isophane insulin 70%), was obtained from the Egyptian local market, imported from Novo Nordisk, Denmark. All other chemicals used were of analytical grade.

The seeds of Medemia argun, Arecaceae (Palmae) were separated from their fruits, put in a dry and clean place for 5 days, crushed into small particles by a grinder. The particles of the powdered seeds were kept for 2 days in conical flask containing 70% ethanol. The mouth of the conical flask was covered with aluminum foil and kept in a shaker for 24 hrs. for continuous agitation at 150 rev/min for thorough mixing and also complete elucidation of active materials to dissolve in the respective solvent. Then, extract was filtered by using filter paper. The solvent from the extract was removed by using rotary vacuum evaporator with the water bath temperature of 45 ºC. Finally, the crude extract were collected in petri dishes, kept in a dry and clean place for 2 days. Then crude extract was ready to use for the experiment by dispensing in sterile normal saline for intraperitoneal[7, 8].

Animals and experimental design

The procedures of the experiment followed International Standards of Animal Ethics for Animal Care and Use. Thirty adult male Wistar albino rats weighing100-120 g were randomly allocated for the study. The animals were housed in polyethylene cages in a moderately humid room under a controlled 12 hours light/dark cycle and left under constant environmental and nutritional conditions with free access to standard food and water ad libitum. Animals were kept 2 weeks for acclimatization before starting the experiment, then, randomly classified into 5 groups, as per Table 1. DM was confirmed 1 week after alloxan injection by measuring blood glucose levels; only animals with blood glucose more than 200 mg/dl were considered diabetic[9]. The treatment continued for one week, then, animals were sacrificed. The setting of the experiment was in the animal house of Faculty of Medicine, Zagazig University, Egypt.

TABLE 1. Experimental groups

Blood samples collection

At the end of the experiment, rats were sacrificed. Blood samples were obtained directly after the decapitation of animals, centrifuged at 2500 rpm for 10 min after standing for 45 minutes at 4ºC. The obtained sera were kept at -80ºC right biochemical investigations.

Liver tissue sampling

Liver tissues were dissected out, washed by cold saline, blotted by tissue paper, kept in formol saline solution (10% v/v) for histological examination.

Serum biochemical investigations

Random blood glucose levels [12], alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities [13], albumin [14], and total bilirubin [15] levels were spectrophotometrically analyzed.

Histopathological work up

Fixation and tissue processing: Formalin preserved hepatic tissues were processed in an automated tissue processor. The process consisted of an initial 2 steps fixation and dehydration. Fixation comprising tissue immersion in 10% buffered formalin solution for 48 hours, followed by removal of fixative in distilled water for 30 minutes. Dehydration was then carried out by running the tissues through a graded series of alcohol (70%, 90%, and 100%). The tissue was initially exposed to 70% alcohol for 120 minutes, followed by 90% alcohol for 90 minutes and then two cycles of absolute alcohol, each for one hour. Dehydration was then followed by clearing the samples in several changes of xylene. It consisted of tissue immersion for an hour in a mixture comprising 50% alcohol and 50% xylene, followed by pure xylene for one and a half hours. Samples were then impregnated with molten paraffin wax, then embedded and blocked out. Paraffin sections (4–5 um) were stained with hematoxylin and eosin [16]. Stained sections were examined for inflammation, degenerations, necrosis, and other pathological changes.

Statistical analysis

Data were statistically analyzed using SPSS, USA, all of the data of control and treated groups were expressed as (Mean ± SE). Two-Tail ANOVA and unpaired t-test were carried out to find if there was any significant difference between control and treated groups. P values <0.05, were considered significant.

Results and Discussion

The study revealed that, alloxan administration to rats, induced diabetes mellitus (DM), within 10 days, manifested by a significant increase in random blood glucose levels. The activity of both ALT and AST were also significantly elevated. Treatment of diabetic animals with MA seed extract alone, could keep these parameters near normalcy. Induction of DM didn’t significantly change both albumin and total bilirubin, nor, MA action was also significant. Insulin treatment of diabetic rats significantly restored RBS, ALT, AST activities, albumin and total bilirubin values, compared to diabetic ones. Co treatment of MA with insulin for diabetic rats significantly lowered RBS than insulin alone, although didn’t add a value to insulin on other parameters (Table 2).

TABLE 2. Variations in serum random blood glucose, ALT and AST activities, total bilirubin and albumin levels, among experimental groups, (Values are expressed as Mean ± SEM, n=6)

N.B: The P value represents the difference between all groups, Pa represents differences from the negative control group, Pb represents differences from the diabetic control group. P values <0.05, were considered significant. Results were computed by Two-tail ANOVA

Examination of liver tissue (Figure 1) showed that induction of DM showed moderate inflammatory infiltration in the portal area with predominance of eosinophil and neutrophil infiltrations in the portal triads (Plate 2), in comparison to control ones, that showed normal hepatic parenchyma with preserved hepatic cords, portal triad structures, sinusoids, Von-Kupffer cells and stroma (Plate 1). Treatment of DM rats with MA showed normal hepatic parenchyma, although some hepatocytes exhibited hydropic degeneration (Plate 3). Insulin treatment to DM group showed mild to moderate congestion of the hepatic and portal blood vessels, lymphatic dilatation, mild cholangitis and degenerative changes, few cloudy swelling and hydropic changes (Plate 4). Co treatment of MA to insulin for diabetic rats showed normal hepatic parenchyma with preserved hepatic cords, portal triad structures, sinusoids, Von-Kupffer cells and stroma. Mild biliary proliferation was seen in few sections, the Von-kupffer cells in such sections appeared hypertrophied (Plate 5).

Figure 1: It shows histopathologic findings among studied groups (all were stained with H&E):

The words “Diabetes” denotes “a passer through a siphon” while “Mellitus” means “sweet”, both are derived from the Greek words. It is believed that Greeks used these terms in such a way, due to the extensive urine excretions produced by diabetic patients that attracted some insects [17, 18]. It is caused by deficiency or ineffectiveness of insulin, resulting in an increase in glucose levels in the blood. It leads to damage of many body systems, particularly blood vessels, eyes, kidney, heart and nerves[19]. DM has been classified into two common types i.e. insulin dependent diabetes mellitus (IDDM, Type I) and non-insulin dependent diabetes mellitus (NIDDM, Type II). Type I diabetes is believed to be an autoimmune disease characterized by a local inflammatory reaction in and around islets that is followed by the destruction of B cells, whereas Type II diabetes is characterized by peripheral insulin resistance and impaired insulin secretion[20]. In the present work, alloxan induced DM in the treated rats, this is well known reported model[21]. It also significantly up regulated enzyme activities of ALT and AST, without significant effect on both serum albumin and total bilirubin. This action was registered before as well [22]. It showed moderate inflammatory infiltration in the portal area with predominance of eosinophil and neutrophil infiltrations in the portal triads of liver tissue. This tissue change was also noticed before by other researchers, who reported a hepatic sinusoidal enlargement, macrovesicular hepatocyte fatty degeneration along with structure loss, steatohepatitis, and peri-portal fibrosis [22].

In this study, treatment with MA for diabetic rats significantly lowered blood glucose levels. This extract wasn’t reported to have anti diabetic activity up to our knowledge, but, the main component of the extract, PACs, was reported to lower blood glucose levels significantly, which is in agreement with our results[23].MA treatment also, significantly down regulated liver enzyme activity of both ALT and AST. PACs were reported to have the same effect [24]. The same authors also published in the same work a prominent hepatoprotective efficacy of PACs on liver tissue morphology. Insulin therapy, significantly lowered blood glucose, liver ALT and AST activities and albumin and total bilirubin in diabetic rats. Similarly, it was reported that insulin significantly lowered liver ALT and AST activities in diabetic rats[25]. It also showed mild to moderate congestion of the hepatic and portal blood vessels, lymphatic dilatation, mild cholangitis and degenerative changes, meaning that it failed to restore alloxan-induced liver tissue changes [26]. The addition of MA extract to insulin, significantly improved the insulin effect on blood sugar, although this short treatment period didn’t significantly change insulin effect on liver functions of the diabetic group. The effect of PACs, combined with insulin was reported elsewhere to have better insulinomimic activity on insulin-sensitive cell line [27] and on diabetic mice, through improving insulin sensitivity [28].

Conclusion

The combination of insulin and MA extract rendered normal hepatic parenchyma and preserved hepatic cords, portal triad structures, sinusoids, Von-Kupffer cells and stroma in diabetic rats. Diabetes mellitus has many complications on the liver and other vital tissues. The addition of MA seed extract, which is rich in PACs, can potentiate the insulin-like response among diabetic subjects, through the restoration of blood sugar and maintaining liver enzyme activities. The addition of MA also keeps better liver tissue architecture and minimizes inflammatory response due to DM.

References

1. Okur ME, Karantas ID, Siafaka PIJAPS. Diabetes Mellitus: A review on pathophysiology, current status of oral pathophysiology, current status of oral medications and future perspectives. Acta Pharm Sci. 2017;55(1): 61-82.
2. Preedy VR. Diabetes: Oxidative stress and dietary antioxidants. Academic Press. 2020.
3. Abass SA. Chemosensitizing effect of Alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma. Biomed Pharmacother. 2018;101:710-718.
4. Abdel-Hamid NM. Herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors. Biomed Pharmacother. 2018;107:1246-1258.
5. Hamed AI. Profiles analysis of proanthocyanidins in the argun nut (Medemia argun-an ancient Egyptian palm) by LC-ESI-MS/MS. J Mass Spectrom, 2014;49(4):306-315.
6. Masullo M. Phenolic compounds from the fruits of medemia argun, a food and medicinal plant of ancient Egypt. Nat Prod Commun. 2016;11(3) 279-282.
7. Al-Hamimi ST. Fast and green extraction method for berry seed lipid extraction using CO₂ expanded ethanol combined with sonication. J European Lipid Science Technology. 2020;122(4):1900283.
8. Nagappan R. Evaluation of aqueous and ethanol extract of bioactive medicinal plant, Cassia didymobotrya (Fresenius) Irwin and Barneby against immature stages of filarial vector, Culex quinquefasciatus Say (Diptera: Culicidae). Asian Pac J Trop Biomed. 2012;2(9):707-11.
9. Spadella CT. Long-term effects of insulin therapy, islet transplantation, and pancreas transplantation in the prevention of glomerular changes in kidneys of alloxan-induced diabetic rats. Transplant Proc. 2005;37(8): 3468-71.
10. Yadav JP. Hypoglycemic and hypolipidemic activity of ethanolic extract of Salvadora oleoides in normal and alloxan-induced diabetic rats. Indian J Pharmacol, 2008;40(1): 23-27.
11. Abd Allah SG, Hassan MK, Abdel Hamid NM, AhmedA. Evaluation of the acute hepatotoxicity of medemia argun seed's extract by determining of ld50 value and dose response curve alfarama. J Basic and Applied Sci. 2020;1(2):90-98.
12. Thompson RH. Colorimetric glucose oxidase method for blood glucose. Clin Chim Acta. 1966;13(1):133-5.
13. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28(1): 56-63.
14. Hinton RJ. Colorimetric determination of human albumin. J Clin Pathol. 1990;43(7):610.
15. Rutkowski RBDEL. An ultramicro colorimetric method for determination of total and direct serum bilirubin. Clin Chem. 1966;12(7): 432-438.
16. Suvarna KLC, Bancroft. Bancroft's Theory and Practice of Histological Techniques 8th Edition ed. 2018: Elsevier. 672.
17. Piero MN. Diabetes Mellitus: A Devastating Metabolic Disorder. Asian J. Biomed. Pharm 2015;4(40): p. 1-7.
18. Patlak M. New weapons to combat an ancient disease: treating diabetes. FASEB J. 2002;16(14):1853.
19. Ismail MY. Clinical evaluation of antidiabetic activity of Trigonella seeds and Aegle marmelos Leaves. Worl Appl Scien J. 2009;70(10):1231-1234.
20. Arora S, Ojha SK, Vohora D. Characterisation of Streptozotocin induced diabetes mellitus in Swiss Albino mice Glo J of Pharmacol. 2009;3(2):81-84
21. Lee MGC, YH, Lee I. Effects of diabetes mellitus induced by alloxan on the pharmacokinetics of metformin in rats: restoration of pharmacokinetic parameters to the control state by insulin treatment. J Pharm Pharm Sci, 2008;11(1):88-103.
22. Lucchesi AN, Cassettari LL, Spadella CT. Alloxan-induced diabetes causes morphological and ultrastructural changes in rat liver that resemble the natural history of chronic fatty liver disease in humans. J Diabetes Res. 2015;494578.
23. Salahuddin AKM. Effects of grape seed proanthocyanidin extract in attenuating diabetic complications in streptozotocin-induced diabetic rats. J Appl Pharm. 2017;9:2-7.
24. Mansouri EL, Khorsandi, Abedi HA. Antioxidant effects of proanthocyanidin from grape seed on hepatic tissue injury in diabetic rats. Iran J Basic Med Sci, 2014;17(6): 460-4.
25. Nolasco EL. Insulin Modulates Liver Function in a Type I Diabetes Rat Model. Cell Physiol Biochem, 2015; 36(4):1467-79.
26. Dal S. Featured Article: Oxidative stress status and liver tissue defenses in diabetic rats during intensive subcutaneous insulin therapy. Exp Biol Med (Maywood). 2016;241(2): 184-92.
27. Pinent M. Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines. Endocrinology. 2004;145(11):4985-90.
28. Zhang HJ. A combination of grape seed-derived procyanidins and gypenosides alleviates insulin resistance in mice and HepG2 cells. J Food Sci. 2009;74(1):H1-7.