Issues in Biological Sciences and Pharmaceutical Research Vol.7(5), pp.82-90, December 2019 Available online at https://www.journalissues.org/IBSPR/ https://doi.org/10.15739/ibspr.19.011 Copyright © 2019 Author(s) retain the copyright of this article ISSN 2350-1588

Original Research Article

Histological study on the effect of aqueous extract of leaf on pancreas of hyperglycemic wistar rats

Received 26 September, 2019 Revised 15 November, 2019 Accepted 22 November, 2019 Published 10 December, 2019

Uthman Ademola Yusuf1*, The present study was carried out to assess some effects of medica Gibson Sijumbila1, leaf extract treatment on the following parameters in hyperglycemic Samuel Bolaji Mesole2, Wistar rats: diabetic hyperglycaemia and microanatomy of pancreas. The John Amos Mulemena1, male adult Wistar rats (40) weighing between 160-200g were indiscriminately classified into four consisting of 10 rat namely: (A) normal Kingsley Kamvuma1, control, (B) hyperglycemic only, (C) hyperglycemic treated with citron and 3 Olugbenga Olawale Eweoya (D) citron only. Aqueous leaf extracts of citron were administered orally at and 400mg/kg body weight daily for 6 weeks. After 6weeks extract of Thomas Kehinde Adenowo4 administration, animals were sacrificed by cervical dislocation. Organ

1Department of Anatomy, School of weight was also taken while the pancreas was processed for light Medicine and Health Sciences, microscopy. Data were analysed using one way ANOVA. p<0.05 was Mulungushi University, Livingstone considered significant. After 6 weeks the glucose level of Campus, . hyperglycemic+Citron and Citron only groups significantly lowered, 2Department of Anatomy, Texila increase in body weight and relative pancreatic weight were observed American University, Lusaka, Zambia. relative to the hyperglycemic group. The histological examination of the 3Department of Anatomical Sciences, pancreas of hyperglycaemic rats showed reduction Islet of Langerhans with University of . . 4Anatomy Department, Faculty of fibrotic tissue while hyperglycemic+Citron rats was similar to control rats. Basic Medical Sciences, College of Aqueous leaf extract of citronhas the ability to ameliorate the damage Health Sciences, Olabisi Onabanjo initiated by hyperglycemia on the pancreas of adult male Wistar rats. University, Sagamu Campus, Nigeria Key words: Pancreas, streptozotocin, Citrus medica, hyperglycemia, Wistar rat. *Corresponding Author Email: [email protected]

INTRODUCTION

In adult humans, the pancreas weighs about 80g and lies artery which have the corresponding veins that drain into retroperitonealy in the transpyloric plane (Banks et al., the portal system of veins (Verspohl, 2009). In view of its 2010). The second and third portions of the duodenum dual blood supply, ischemia to the pancreas from vascular curve around the head of the pancreas (Verspohl, 2009). obstruction is uncommon (Banks et al., 2010). The pancreas The spleen is adjacent to the pancreatic tail. The regions of is innervated by both the parasympathetic and sympathetic the pancreas are the head, neck, body and tail (Banks et al. nerve fibers (Rayan et al., 2014). The efferent 2010). The distal end of the common bile duct passes parasympathetic fibers are contained within the branches through the head of the pancreas and joins the pancreatic of the vagus nerve that originates in the dorsal vagal duct entering the duodenum (Barnby, 2015). The arterial complex (tenth cranial nerve nucleus) in the medulla of the blood supply to the pancreas are from the splenic and brain. The terminal branches of the vagus nerve synapse pancreaticoduodenal arteries branches of gastroduodenal with intrapancreatic ganglia while the sympathetic Issues Biol. Sci. Pharma. Res. 83

innervation originates in the lateral grey matter of the Diabetes mellitus is a disorder of glucose metabolism thoracic and lumbar segment of the spinal cord (Frank et whereby the body is not properly making use of glucose in al., 2003). The bodies of the postganglionic sympathetic the blood stream, therefore compromising a necessary neurons are located in the hepatic and celiac plexuses of function for cell nutrition and function (Faramarz et al., nerve (Ryan et al., 2014). The postganglionic nerve fibers 2012). Type 1 diabetesis also called insulin-dependent innervate the blood vessels of the pancreas (Ryan et al., diabetes (Baynes et al., 2009). It used to be called juvenile- 2014). onset diabetes, because it often begins in childhood and The endocrine pancreas refers to those cells within the accounts for about 10% of all diabetic cases, affecting pancreas that synthesize and secrete hormones. It takes the approximately 20 million people Worldwide (Baynes et al., form of many small clusters of cells called the islets of 2009). Type 2 diabetes is also known as adult-onset Langerhans or, more simply, islets (Franks et al., 2003). diabetes, accounting for about 95% of the cases of Diabetes Humans have roughly one million islets (Burns and Edil, mellitus in adults (Kia Soo et al., 2012). Gestational diabetes 2012). In standard histological sections of the pancreas, is known to be Diabetes triggered by pregnancywhich islets are seen as relatively pale-staining groups of cells account for about 2% to 10% of Diabetes mellitus embedded in a sea of darker-staining exocrine tissue pregnancies (Kia Soo et al., 2012). (Burns and Edil, 2012). Diabetes mellitus had been connected with disabling and The pancreatic islets house three major cell types, each of life threatening complications such as retinopathy, which produces a different endocrine product: neuropathy, nephropathy, dermopathy, cardiomyopathy and hepatopathy (Faramarz et al., 2012). a. Alpha cells (cells) which secrete the hormone After the classic work of Banting and Best, (1922) on glucagon. insulin, indefinite number of findings had accumulated over b. Beta cells ( cells) which produce insulin and are the years. Currently, botanicals are being screened for their the most abundant of the islet cells. efficacy and safety in the management of Diabetes mellitus c. Delta cells (cells) which secrete the hormone and its complications (Shibibet al., 1993; Choudhury et al., somatostatin, which is also produced by a number of other 2017). In this regard, there is laboratory-based evidence endocrine cells in the body. that the fruit juice of Momordica charantia reverses The different cell types within an islet are not randomly hyperglycemia in rats by decreasing gluconeogenesis and distributed - beta cells occupy the central portion of the increasing insulin secretion (Shibib et al., 1993; Choudhury islet and are surrounded by a "rind" of alpha and delta cells et al., 2017). Additional potential herbal sources of new (Ryan et al., 2014). Aside from the insulin, glucagon and chemical entities for the management of Diabetes mellitus somatostatin, a number of other "minor" hormones have include Coccina indica (Kimble et al., 1996), Gymnema been identified as products of pancreatic islet cells (Frank sylvestre (Baskaran et al., 1990) and Panax quinquefolius et al., 2003). Islets are richly vascularized, allowing their (ginseng) (Sievenpiperet al., 2004). Others include Annona secreted hormones ready access to the blood circulation. muricata (Adewole and Caxton-Martins, 2006), Hypoxis Although the islets comprise only about 1-2% of the mass hemerocallidi (Ojewole, 2006), Berberis lyceum (Gulfraet al., of the pancreas, they receive about 10 to 15% of the 2007) Aloe vera (Noor et al., 2008), Trichosanthes pancreatic blood flow (Frank et al., 2003). Additionally, cucumerina (Adeeyo et al., 2011) and Allium cepa (Yusuf et they are innervated by parasympathetic and sympathetic al., 2012). neurons, and nervous signals clearly modulate the Chronic hyperglycemic oxidative stress is implicated in secretion of insulin and glucagon (Burns and Edil, 2012). the pathogenesis of these complications (Jeanette et al., Hyperglycemia is the technical term for high blood 2005). Some reports have shown that antioxidants may glucose or blood sugar. High blood glucose happens when protect people from the disabling effects of Diabetes the body has too little insulin or when the body cannot use mellitus by mopping up free oxygen and superoxide radicals insulin properly (Pais et al., 2007), other conditions that (Jeanette et al., 2005). It has also been shown by Opara can cause hperglycemia are pancreatitis, cushing’s (2004) that depletion of antioxidant appears to be a major syndrome, unusual hormone-screating tumors, pancreatic risk factor for developing complications, and that cancer, certain medications and severe illnesses antioxidant supplements lowered the risk. Besides, (Sommerfield et al., 2004). Temporary hyperglycemia is impaired insulin levels or action in Diabetes mellitus often benign and asymptomatic in nature. Blood glucose predisposes to dyslipidemia and increased risk of levels can rise well above the normal values for significant atherosclerosis (Rossetti and Goldberg, 2002).In view of the periods without producing any permanent effects or fact that emphasis is now on herbal therapy, searchlight is symptoms (Sommerfield et al., 2004). However, chronic on herbs that are edible of which Citron is qualified. The hyperglycemia at levels more than slightly above the Citron is a large fragrant citrus fruit with a thick rind, normal values can produce a very wide variety of serious botanically classified as Citrus medica (Kalpesh et al., 2012). complications over a period of years, chronic It is one of the four original citrus fruits from which most hyperglycemia that persists even in fasting states is most other citrus types developed through natural hybrid commonly caused by Diabetes mellitus (Sommerfield et al., speciation or artificial hybridization (Taha et al., 2012). A 2004). source of confusion is that citron or similar words in Uthman et al. 84

French, Lithuanian, Hungarian, Finnish,Latvian, the West was soaked in 4000 mls of distilled water for 24 hours in a Slavic languages, and all Germanic languagesbut English are measuring cylinder. The mixture was homogenised in false friends, as they refer to the (Sharangouda and Explosion Proof Blender for two minutes, then filtered Patil, 2010). Indeed, into the 16th Century, the English name using a whatman filter (Grade 1circles, diameter 15 mm, Citron included the lemon and perhaps the as well. In Z274844). The filterate in the round bottom flask was put most Arabic languages it is called Turunj, or similar, but in on a heating mantle (Barnstead/ eletrothermal) at 100°C Syria and many other Islamic countries it is called Kabbad; for 7 hours. The concentrate formed was taken to an oven in Japanese it is called Bushukan(Klein, 2014). In Nigeria at 50°C for 1hour; the final residue of about 80 g was a dark Citrus medica is called several names in different languages green mass which was stored at room temperature of 25°C such as ‘Osan jaganhin’ (Aiyeloja and Bello, 2006), ‘Oronbo until reconstituted for uses. nla’ (Adeogun et al., 2014) in Yoruba, also in Igbo it is called ‘Oloma-oyinbo’ and ‘Babban lemu’ in Hausa (Aiyeloja and Animals and animal management Bello, 2006). From ancient through medieval times, the citron was A total number of Forty (40) adult presumably healthy male used mainly for medicinal purposes: to combat sea- Wistar rats (Rattus norvegicus) were used for this sickness, pulmonary troubles, intestinal ailments, scurvy study,weighing about 160-200g of 8 to 10weeks old. and other disorders. Citron juice with wine was considered Animals were kept in four cages (10 rats per cage) and an effective antidote to poison, as Theophrastus reported. housed in the animal holdings of the Department of In the Ayurvedic system of medicine, the juice is still used Anatomy, Faculty of Basic Medical Sciences, Animal House, for treating conditions like nausea, vomiting, and excessive Olabisi Onabanjo University Ikenne. They were maintained thirst (Bairagi et al., 2011).Citron has been used in the on standard animal feeds (Wealth-gate pelletized feeds) treatment and management of many diseases for many and allowed access to clean water and feeds freely (ad years, both in traditional and complementary medicine in libitum). Asia, Europe and some parts of Africa (Kalpesh et al., 2012). It has also been reported to be effective in the management Induction of hyperglycemia in rats of chronic metabolic disorders such as Diabetes mellitus (Archana et al., 2011). A number of biological effects, such The animals were acclimatised for four weeks and blood as anticancer, antimycobacterial, antiviral, apoptosis- glucose level was monitored before induction of inducing and immunomodulatory activities have been hyperglycaemia. Hyperglycaemia was induced in twenty reported for Citrus medica (Kabra et al., 2012). The major overnight-fasted indiscriminately designated rats by a constituents of Citrus medica are the lecitins, polypeptides single intraperitoneal administration of Streptozotocin at known as viscotoxins and a number of phenolic compounds 70 mg/kg bw (Lal et al., 2000). Streptozotocin (STZ) was (e.g., digallic acid, O-coumaric acid) found in their free state dissolved in citrate buffer (1ml, pH 4.5) just prior to or as glycosides (Bhmya and Begum, 2009). Phytochemical injection. Hyperglycemia was allowed to develop for properties of fruit decoction, peels (rind of fruit) and leaves 72hours (Lenzen, 2008). Rats with Fasting Blood Glucose ≥ of Citrus medica Linn had been found to have 250 mg/dl were considered hyperglycemic (Tende, 2011). alkaloids,flavonoids, phenols, carbohydrates and mucilage; Control and citron only rats received a single the peels had alkaloids, flavonoids, steroids, phenols and intraperitoneal injection of 1ml citrate buffer (1ml/kg bw; carbohydrates; the leaves contained alkaloids, flavanoids, pH 4.5). vitamins C, steroids and glycosides (Taha et al., 2012). The flavonoids reported from the fruits are hesperidin: 3,5,6- Experimental design trihydroxyl -4,7 -di methoxy flavone; 3,5,6-trihydroxy- 3’,4’,7- trimethoxy flavones (Bhmya and Begum, 2009). The Forty (40) animals were divided into four groups of ten Citronleaf has abundance of antioxidants (Taha et al., (10) animals each. The group A is the control group were 2012).The study is therefore aimed at investigating some normoglycemic animals that received neither STZ nor ameliorative effects of aqueous extract of citron leaf on the citron leaf extract, Group B was hyperglycemic rats that pancreatic histo-architecture of hyperglycemic wistar rats. received distilled water solely, Group C was hyperglycemic rats that received citron extract only and Group D was normoglycemic group that received solely citron extracts. MATERIALS AND METHODS Citron (citrus medica) mode of administration The authentication of Citron leaves was done at Forestry Research Institute of Nigeria (FRIN), Jericho hill, Ibadan The dose of the aqueous extracts of citron employed in and voucher number (No. FHI. 110913) was assigned. these studies was adopted from the report of Taha et Citron aqueous leaf extracts were prepared as reported by al.(2012). Physiological saline was used in dissolving citron Taha et al., (2012). Fresh leaves of citron were air-dried daily and was administered orally with use of oro-gastric (under shade). The leaves were ground to coarse powder cannula to Group C rats (n=10) at 400 mg/kg bw (at 9.00 – using an electric blender. The powdered sample of 400 g 10.00 a.m. every day) for a maximum period of 6 weeks, Issues Biol. Sci. Pharma. Res. 85

Group D rats (n=10) were administered 400 mg/kg bw of RESULTS citronextracts. average body weights of the rats (g). Measurement of blood glucose Figure 1 showed the weekly changes in body weight of rats The blood glucose was evaluated in nightlong fasted rats at in various groups. The body weight of rats in numerous 9:00 – 10:00 hours using Glucose oxidase method of one groups throughout the 4 weeks of acclimatisation were touch ultra 2 glucometer (Accu-Chek Compact Plus). Blood normal with no significant difference when compared to was obtained from the median caudal vein of the tail by Group A (control) (P>0.05). At the second week, there was snipping the tip of the tail. The blood glucose level was a rise of 8.3% in the average body weight of the rats in monitored from twenty-eight (28) days (acclimatisation Group B of hyperglycemic only (174.16± 3.21g), period) before the induction of hyperglycaemia and for 42 howeveronce the second of treatment, their average body days of treatment (Adewole and Caxton-Martins, 2006). weight started decreasing and by the sixth week (166.07± 3.28g) there was a decrease of 3.7% compared to the initial Measurement of the body weight (g) average body weight at week 0 (170.64±3.14g) with significant difference when compared to Group A P<0.05. In Body weights (g) of the rats were recorded for four weeks Group C of hyperglycemic+Citrus medica, there was a rise (acclimatisation period) before induction of of 7.9% at second week of treatment (173.06± 3.79g), hyperglycaemia and on a weekly basis throughout the however at sixth week (178.95±3.37g) the rise was 12.1% experimental treatment for a period of 6 weeks. Body when compared with their weight at week 0 (169.92±3.88 Weight was taken with a weighing scale (Venus VT 30 SL); g) with no significant difference when compared to Group (Adeeyo et al., 2011). A(P>0.05).

relative pancreatic weight(%) Animal sacrifice

The relative weight of the pancreases in various groups After 42 days of treatment the animals were sacrificed by were shown in Figure 2. There was a significant decrease in cervical dislocation. Laparotomy was performed, the pancreatic weight in Group B (0.538±0.032) compared to pancreas was harvested, rinsed in phosphate buffered Group A (0.916±0.072) (P<0.05). In Group C (0.810±0.045) solution and fixed in 10% formol saline for histological the relative pancreatic weight was not significantly procedures namely; Haematoxylin and Eosin stain and different from Group A(P>0.05) and there was no Gomori stain(Bancroft and Steven, 1992). significant different between Group D (0.981±0.064) and Group A when compared. The relative pancreatic weight (%) blood glucose levels on weekly basis (mg/dl) The relative pancreatic weight of the rat was evaluated as the ratio of respective weight of the pancreas and the Figure 3, the levelsof glucose of different groups of rats on terminal body weight of the same rat, the unit was recorded weekly basis. The blood glucose level of rats in numerous as percentage (%) using sensitive weighing balance groups throughout 4 weeks of acclimatisation were (SonyF3G brand); (Adeeyo et al., 2011). normoglycemic in nature with no significant difference when compared with rats in Group A (P>0.05). The Group B Photomicrography rats were hyperglycaemic at week 0 (253.16±5.77 mg/dl) and remained therefore till the end of the sixth week of Photomicrography of histological sections of the Pancreas treatment (367.53±7.11 mg/dl). The worth was were taken with an Olympus Microscope (New York, United considerably significantly increase from Group A (74.65± State of America) coupled with camera at Department of 3.38 mg/dl) (P<0.05). Whereas the Group C (265.15±6.05 Human Anatomy, Kampala International University, Gongo mg/dl) rats had high blood glucose level at week zero up to La mboto, Dar es Salaam, . week three that was considerably significantly increased compared with Group A rats at P<0.05. From the fourth Statistical analysis week the blood glucose levels of Group C (98.17± 3.98 mg/dl) were similar to Group A (74.65± 3.38 mg/dl) that Data were analysed using Excel 2016 (Microsoft weren’t significantly different at P>0.05. Corporation, U.S.A). Data were expressed as mean ± standard error of the mean (mean±SEM). Mean values were compared using one way ANOVA. P values less than 0.05 HISTOLOGICAL FINDINGS (P<0.05) were taken to be statistically significant. All graphs were drawn with Excel 2016 (Microsoft Corporation Using Haematoxylin and eosin (H&E) and Gomori stains to U.S.A). study the histology of pancreas in the various grouped rats Uthman et al. 86

Figure 1: Histogram chart of Average Body weight on weekly basis. Data were expressed as Mean±SEM. P<0.05*

Figure 2: Bar chart of Relative pancreatic weight in Percentage (%). Data were expressed as Mean±SEM. P<0.05*

LEGEND: NC: Normal Control HG: Hyperglycemic HGC: Hyperglycemic+Citrus medica CM: Citrus medica only Issues Biol. Sci. Pharma. Res. 87

Figure 3: Histogram chart of Blood Glucose on weekly basis. Data were expressed as Mean±SEM (P<0.05*).

a a a a

a a a

* a A a B

a a

a * a

a *

a a a a

C a D

Figure 4: Photomicrograph of the Pancreas showing the pancreatic islet at day 42. (Arrow black: Islet of langerhans; a: Acini, *: blood vessel). A normal control, B hyperglycemic, C hyperglycemic+Citrus medica and D Citrus medica only. H&E stain. Mag. X400

The control group pancreatic islets appeared normal with by other acidophilic cells. The acini were also well normal cytoarchitectures showing lots of basophilic cells distributed. (Figure 4A and 5A). In the hyperglycemic group occupying the central portions of the islets and surrounded (B) of rats, the pancreatic islets appeared smaller with few Uthman et al. 88

a a a a * a a a a

A B a a a a a a a * C D

Figure 5: Photomicrograph of the Pancreas showing the pancreatic islet at day 42. (Arrow black: Islet of langerhans; a: Acini, *: blood vessel). A normal control, B hyperglycemic, C hyperglycemic+Citrus medica and D Citrus medica only. Gomori stain. Mag. X400

cellularity.There was also some degree of fibrosis in this through accentuation of release of insulin from B cells of group (Figure 4B and 5B); The hyperglycemic+ Citrus islets of Langerhans of pancreas, prevention of uptake of medica group pancreatic islet appeared a bit small with glucose from gastrointestinal tract as seen in alpha- cellularity and the acini were well distributed. (Figure 4C gluconidase or pancreatic amylase enzyme inhibitors, and 5C) and Citrus medica only group (D) of rats appeared prevention of gluconeogenesis and glucogenolysis normal with numerous cells present in the pancreatic islet (Adewole et al., 2007). Citrus medica antihyperglycemic and the acini were well distributed (Figure 4D and 5D). activities had been reported to be due to the presence of alpha gluconidase (Ahmad et al., 2008) and pancreatic amylase enzyme inhibitors (Nizam et al., 2014). These DISCUSSION enzymes inhibit the digestion of glucose into an absorbable product, hence the inability of blood glucose to increase The present study evaluated the possible ameliorative after glucose intake. The presence of these inhibitors was effects of citron on the hyperglycemia effects of STZ on the reported in like Morus alba, which was able to pancreas, treatment of STZ induced hyperglycaemia rats exhibit antihyperglycaemic activity (Sudha et al., 2011). with aqueous leaf extract of Citrus medica, at a dose of Also the antihyperglyceamic activity of Citrus medica leaf 400mg/kg/d, produced normoglycaemia in 83.2% of rats extract was due to the presence of antioxidants like by the end of the third week of treatment and all the flavonoids, phenol in it (Taha et al., 2012). Duong et al. animals had become normoglycaemic by the end of the (2003) reported that flavonoids and phenols were powerful forth week. Normoglycemia was maintained in these rats hydrosoluble antioxidants in biological fluids. The from fourth week to the end of experiment. antioxidants were able to prevent further destruction of Antihyperglycemic activities of Citrus medica leaf extract beta cells in the pancreatic islets. The declining of the body had been reported earlier by Taha et al.(2012), which was weight of the rats in hyperglycemic group after second an agreement with the results of this study. week might due to the low level of insulin which had been Antihyperglycemic activities are usually achieved reported by Raheleh et al., 2016 that low level of insulin Issues Biol. Sci. Pharma. Res. 89

leads to inability of the body to breakdown glycogen for Adewole SO, Ojewole JA, Caxton-Martins EA (2007). energy and the body begins to breakdown its own stored Protective effect of quercetin on the morphology of fat foe energy, this causes rapid weight loss. In pancreatic beta cells of streptotozocin treated diabetic hyperglycemic+Citrus medica group there was an increase rats. Afri. J. Trad. Comple. Alternat. Med.4:64-74.Crossref in the body weight from the second week of the treatment Ahmad G, Hossan F, Fariba S, Mansour M (2008). The this might due to the presence of antioxidant in the Citrus inhibitory effect of some Iranian plants extracts on the medica which had been reported by Rahelehet al., 2016 that alpha glucosidase. Iranian, J Basic Med. Sci. 11(1):1-9. is capable of reducing blood glucose level which was an Aiyeloja AA, Bello OA (2006). Ethinobotanical potentials of agreement with this study. common herbs in Nigeria. A case study of Enugu state. The histological findings showed that hyperglycemic Acad. J. 1(1):16-22. group (Figure 4B and 5B) islet reduced with paucity of cells Archana N, Amit J, Vijay J, Tiralh K (2011). Antidiabetic and and presence of fibrotic tissue due to the STZ given to these Hypolidemic activity of Citrus medica Linn seed extract in group which was taken up by β cells, the STZ caused the streptozotocin induced diabetic rats. Pharmaco. J. death of these cells by DNA fragmentation (Suwattanee et 23(12):407-415. al., 2014). In the hyperglycemic+ Citrus medica group Bairagi GB, Kabra AO, Mandate RJ (2011). Anthelmintic (Figure 4C and 5C) also there is reduction in the islet with activity of Citrus medica L. Leaves in Indian adult numerous and viable cells present. In this group, citron earthworm. Internat. J. Pharma. Techno. Res. 3(2):664- treated, early reversal of hyperglycaemia might have 667. brought about increase in G-6-PDH levels with associated Bancroft JD, Steven A (1992). 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