|Year : 2020 | Volume
| Issue : 1 | Page : 20-25
Effect of Cymbopogon citratus aqueous extract on Areca catechu-Induced lesions in the liver of adult wistar rats
Gerald Ikechi Eze, Kevin Aiwanfoh Akonoafua
Department of Anatomy, University of Benin, Benin City, Edo State, Nigeria
|Date of Submission||13-May-2020|
|Date of Acceptance||15-May-2020|
|Date of Web Publication||31-Jul-2020|
Mr. Kevin Aiwanfoh Akonoafua
P. O. Box 10095, Ugbowo Headquarters, Benin City, Edo State
Source of Support: None, Conflict of Interest: None
Background: Cymbopogon citratus has been reported to possess antimicrobial, antioxidant, antimutagenic, and anticarcinogenic properties, as well as the ability to modify gene transcription while the seeds of Areca catechu are known to contain carcinogenic compounds. Aim: This study evaluated the effects of aqueous extract of C. citratus on A. catechu-induced liver injury in adult Wistar rats. Materials and Methods: Thirty rats weighing between 180 g and 250 g were obtained from the laboratory animal facility of the Department of Anatomy, University of Benin. The animals were randomly divided into six groups of five animals each. Group A served as control and received distilled water only, Group B received A. catechu only, Group C received C. citratus only, Group D received A. catechu and low dose C. citratus, Group E received A. catechu and moderate dose C. citratus, while Group F received A. catechu and high dose C. citratus. The experimental animals were acclimatizing for 2 weeks, before the administration of extracts, which was through oral gavage, for 4 weeks. The rats were sacrificed after the 4th week. Liver function tests and appropriate histological procedures were carried out. Results: Significant increases (P < 0.05) of aspartate transaminase and alanine aminotransferase levels were recorded in the group that received A. catechu only, whereas the administration of C. citratus in various doses inhibited the rise in their levels. Conclusion: Histological analysis showed that A. catechu caused inflammation and distortion of the histoarchitecture of the liver, while C. citratus attenuated these effects.
Keywords: Areca catechu, Cymbopogon citratus, hepatitis, medicinal plants, wistar rats
|How to cite this article:|
Eze GI, Akonoafua KA. Effect of Cymbopogon citratus aqueous extract on Areca catechu-Induced lesions in the liver of adult wistar rats. Niger J Exp Clin Biosci 2020;8:20-5
|How to cite this URL:|
Eze GI, Akonoafua KA. Effect of Cymbopogon citratus aqueous extract on Areca catechu-Induced lesions in the liver of adult wistar rats. Niger J Exp Clin Biosci [serial online] 2020 [cited 2020 Aug 8];8:20-5. Available from: http://www.njecbonline.org/text.asp?2020/8/1/20/291192
| Introduction|| |
Medicinal plants have been identified and used throughout the human history. From time immemorial, the use of herbs to treat diseases has been paramount in the universe, especially before the modern medicine. Medicinal plants are the various types of plants used in herbalism, and some of these plants have pharmacological activities. These plants are considered as the rich resources of ingredients which can be used in the drug development and synthesis. Some of these plants are the important source of nutrition and as a result of that they are recommended for their therapeutic values. Besides that, these plants play a critical role in the development of human cultures around the whole world.
A large amount of archaeological evidence exists which indicates that humans were using medicinal plants during the Paleolithic. The World Health Organization estimates that 80% of some Asian and African countries presently use herbal medicine for some aspects of primary health care. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions and to defend against attack from the predators such as insect and mammals., In 2001, researchers identified 122 compounds used in the modern medicine which were derived from ethnomedical plant sources.
Since medicinal plants are still majorly used in third world countries, there is a need to study their quality, safety, and efficacy. Investigations in the chemical and biological activities of plants during the past two centuries have yielded components for the development of modern synthetic organic chemistry and the emergence of medicinal chemistry as a major route for the discovery of novel and more effective therapeutic agents. Plant foods, especially vegetables contribute to both local diets and ethno medicine in developing countries especially Nigeria.
Cymbopogon citratus of the Poaceae family is a tall aromatic coarse grass of 1.5 m high. It is a monocotyledonous hypogeal perennial plant with slender sharp-edged green leaves that has a pointed apex. The stem is reddish brown in color, and it is attached to the bulb by stalk. The entire plant is attached to the soil by fibrous root.C. citratus stalks are commonly used in the cuisines of Africa, the Middle East, and Southeast Asia. It is native to Sri Lanka and South India and is now widely cultivated in the tropical areas of America and Asia. Its oil is used as a culinary flavoring, a scent, and medicine. A tea made from the leaves of West Indian C. citratus has been used to treat fevers, colds, and stomach upset.
C. citratus is believed to have anxiolytic, hypnotic, and anticonvulsant properties, and also cytoprotective, antioxidant, and anti-inflammatory properties.,, However, Leite et al. reported that this same herb had no effect on humans. Its citronellol (an essential oil) constituents from the C. citratus were thought to possess antihypertensive properties. Citronellol has been shown to lower blood pressure, in rats, by a direct effect on the vascular smooth muscle leading to vasodilation.C. citratus is also a folk remedy for cough, elephantiasis, flu, gingivitis, headache, leprosy, malaria, ophthalmia, pneumonia, and vascular disorders. C. citratus is principally taken as a tea to remedy digestive problems, diarrhea, and stomach ache. As a medicinal plant, C. citratus has been considered a carminative and insect repellent. Traditional and alternative medicine is extensively practiced in the prevention, diagnosis, and treatment of various diseases. Although plants has been used for treating several illness, very little is known about their toxicity.
Areca nut (Areca catechu L., Palmaceae) is one of popular traditional herbal medicines used in Indonesia. It is called bijipinang. A. catechu can be chewed, and it is a common masticatory in tropical and subtropical countries.A. catechu has been found to contain mineral, fiber, 50%–60% sugars, 15% lipids (glyceride of lauric, myristic, and oleic acid), 15% condensed tannins (phlobatannin and catechin), polyphenolics (flavonoids and tannin), and 0.2%–0.3% alkaloids (arecoline, arecaidine, guvacine, and guvacoline).
In spite of numerous literature showing that A. catechu has a strong antioxidant activity, this nut is also known as an etiologic association of oral cancer. Suri et al. reported that multiple applications of a dimethylsulfoxide extract of areca nut to hamster cheek pouch resulted in the development of leukoplakia followed by squamous cell carcinoma. Subsequently, Dunham et al. reported that the application of acroline to the cheek pouch of syrian hamsters results in the induction of squamous cell carcinoma. Proliferation of basal cell in the esophageal epithelium and esophageal papilloma was observed. The total tumor response in animal feed areca nut was significantly different from that in animals fed normal diet.
In another study, Rao and Das investigated the carcinogenic potential of areca nut given diet for 12 months at concentrations ranging from 0.25% to 1% and oral feeding of areca nut paste to Swiss mice. Survival rate and body weight of experimental animals were found to be slightly lower than that of the controls. In animals fed areca nut diet for 12 months, hyperplastic changes were observed in oral, esophageal, and stomach epithelium while esophageal carcinoma developed in a limited number of animals.
| Materials and Methods|| |
A. catechu nuts were collected in the University of Benin, whereas the plant C. citratus was collected from Botanical Garden, Sapele road, Benin City and subsequently identified by a curator in the Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Benin City, Edo State.
The result of phytochemical screening of aqueous extract of C. Citratus revealed the presence of tannins, saponins, reducing sugars, flavonoids, and other phenolics compounds, while the preliminary photochemical screening of the aqueous extraction of betel nut showed a positive result for the presence of alkaloids, saponins, phenol, reducing sugar, triterpinoids, and glycosides. However, flavonoids, protein, coumarin, tannins, phytosterols, anthroquinone, anthrocyanides, phlobatannins, and acids substances were absent.
Extracts [Table 1]
|Table 1: Amount of plant material and volume of solvent for the preparation of extracts.|
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After the collection of the plants, they were air-dried for 2 weeks, pulverized, and the aqueous extract of both plants was obtained according to the standard methods.
A total of thirty adult Wistar rats of both sexes were used for the experiment. The animals were purchased from the Animal house of the Department of Anatomy, University of Benin, Benin city, Edo state. The experimental protocols were carried out to conform to the acceptable guidelines on the ethical and humane use of animals in research.
Experimental design [Table 2]
The Wistar rats were randomly selected into six groups of five animals each. Food and water were provided across all groups, ad libitum.
After 28 days of the commencement of the administration of C. citratus and A. catechu, the rats were sacrificed via chloroform anesthesia method. Blood samples were collected in plain bottles, and serum was separated at 2500 rpm for 15 min and biochemical analysis carried out. The liver tissues were harvested, weighed, and promptly fixed.
The liver tissues were excised and stored in bottles containing buffered formal saline, ready for tissue processing. After fixation, the liver tissues were dehydrated in the ascending grades of alcohol, cleared in xylene, and embedded in paraffin wax. The deparaffinized sections were stained routinely with Hematoxylin and Eosin. Photomicrographic plates of the desired sections were taken, using a standard photomicrography setup, namely Olympus XSZ-107BN microscope and an attached Eakins 1080P microscopic camera.
Data were analyzed by the analysis of variance using the Statistical Package for the Social Sciences, IBM SPSS Statistics (version 25.0), New York, United States. The level of significance was set at P < 0.05. The analyzed data were represented in tables.
| Results|| |
The photomicrographs of the control group [Figure 1] showed normal hepatocytes, sinusoids, bile ducts, hepatic artery, and portal vein. [Figure 2] (C. citratus only) shows normal liver architecture and moderate Kupffer cell activation. However, [Figure 3]a-e (A. catechu only) showed varying lesions in the architecture of the liver, after the administration of A. catechu only, including the features of periportal inflammation (hepatitis), Kupffer cell activation, and biliary duct proliferation, whereas [Figure 4], [Figure 5], [Figure 6] showed restoration of the liver architecture, as well as vasoactive effects of vasodilatation and active congestion after the administration of varying dosages of C. citratus. However, the low dose achieved the most remarkable effect, while the high dose had the least effect.
|Figure 1: Control; Rat liver composed of A: hepatocytes, B: sinusoids, C: bile ducts, D: hepatic artery and E: portal vein (H and E, ×100)|
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|Figure 2: Rat liver given Cymbopogon citratus only showing A: normal liver architecture and B: moderate Kupffer cell activation (H and E, ×100)|
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|Figure 3: (a) Rat liver given Areca catechu only showing A: mild vascular congestion, B: mild ductal proliferation, C: moderate periportal infiltrates of inflammatory cells (hepatitis) and D: marked Kupffer cell activation (H and E, ×100). (b) A: mild ductal proliferation, B: periportal infiltrates of inflammatory cells and C: Kupffer activation (H and E, ×400). (c) A: Kupffer cell activation, B: periportal infiltratres of inflammatory cells, C: vascular congestion and D: ductal proliferation (H and E, ×400). (d) A: vascular congestion, B: ductal proliferation, C: periportal infiltrates of inflammatory cells and D: Kupffer cell activation (H and E, ×400). (e) A: vascular congestion, B: periportal infiltrates of inflammatory cells and C: Kupffer cell activation (H and E, ×400)|
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|Figure 4: (a) Rat liver given Areca catechu and low dose Cymbopogon citratus showing A: portal vascular dilatation, B: moderate Kupffer cell activation, C: normal ductal architecture and D: normal hepatocyte architecture (H and E, ×100). (b) A: vasodilatation, B: congestion, C: Kupffer cell activation, D: normal hepatocyte architecture and E: normal ductal architecture (H and E, ×400)|
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|Figure 5:</Figures Bold> (a) Rat liver given Areca catechu and moderate dose Cymbopogon citratus showing A: vasodilatation and congestion, B: moderate Kupffer cell activation, C: normal ductal architecture and D: normal hepatocyte architecture (H and E, ×100). (b) A: normal hepatocyte and B: normal ductal architecture and C: Kupffer cell activation (H and E, ×400)|
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|Figure 6: (a) Rat liver given Areca catechu and high dose Cymbopogon citratus showing A: moderate Kupffer cell activation, B: normal hepatocyte architecture and C: normal ductal architecture (H and E, ×100). (b) A: Kupffer cell activation, B: normal hepatocyte architecture and C: normal ductal architecture (H and E, ×400)|
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| Discussion|| |
This study was carried out to ascertain the effects of A. catechu and C. citratus, singly and in combination, on the liver architecture and enzymes. It is approximated that there are about 600 million areca nut users in the world, and it is the fourth most widely used psychoactive substance (following caffeine, alcohol, and nicotine). Often times, it is used as an ingredient in betel quid and its use is rife, from countries of South Asia to the Pacific Islands of Saipan and Guam. A range of health complications have been identified and linked to areca nut, and systemic adverse effects have been reviewed. Specifically, the evidence for carcinogenicity of areca nut among humans has been evaluated as “sufficient.” On the other hand, C. citratus has been traditionally used to treat gastrointestinal disorders due to its antioxidant and chemo-protective properties.
In the present study, C. citratus was found to be rather innocuous at a dose of 1500 mg/kg body weight, going by the histological result of Group C, where the histoarchitecture of the liver remained normal [Figure 2]. Fandohan et al. reported that no abnormalities in the general behavior or death were observed on day 1 in the group of the rats that were given C. citratus oil at the dose levels of 5–1500 mg/kg body weight. Costa et al. also reported that male mice exposed to a single dose (5–1500 mg/kg b.w.) of C. citratus presented no alterations in general behavior when compared to the controls. Also, the results of the biochemical assays showed that the values gotten from Group C are comparable with that of the control group.
The results from this study revealed that A. catechu- treated Wistar rats had a significantly increased level of aspartate transaminase (AST) and alanine aminotransferase (ALT) [Table 3]. This is in agreement with a study by Dasgupta et al., who reported that arecoline (the active component of A. catechu) (5, 10, and 20 mg/kg, i.p., for 14 days) induces hepatotoxicity, damages the hepatocyte ultrastructure, and significantly increases the serum levels of AST and ALT in a dose-dependent manner.
|Table 3: Table showing the effect of Cymbopogon citratus and Areca catechu on liver function test parameters|
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Chou et al. also indicated that arecoline (0.1–1 mM) is cytotoxic to normal hepatocytes (Clone-9 cells), inducing apoptosis and G0/G1 arrest of the cell cycle. In addition, the water extracts of areca nut (5 and 20 mg/kg) and arecoline (5 and 20 mg/kg, p. o.) also increase the serum levels of ALT, AST, and alkaline phosphatase in mice. Previous investigations by Chang et al. showed that arecoline at concentrations higher than 0.4 mM significantly increases apoptosis and decreases the viability of C2C12 myoblast cells by reducing the activation of signal transducer and activator of transcription-3 (STAT3).
A. catechu was found to be injurious to the liver in this work, as demonstrated by the development of necroinflammatory lesions of portal hepatitis and ductal hyperplasia, in the liver of A. catechu-treated group (Group B). Inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases. Free radicals attack hepatocytes and cause necrosis of parenchymal cells, which promotes inflammatory responses in the liver.
It is, however, worth mentioning that the majority of our biochemical assays indicated no significant difference between the administration of C. citratus at 1000, 2000, and 3000 mg/kg in the protection of the liver from injury. However, the results of the histology suggest that it more potent in the lower and moderate doses. In the present study, we demonstrated that C. citratus significantly reduced the pathological indexes for hepatocytic death and hepatic injury caused by A. catechu in the rat model. In addition, C. citratus attenuated and dramatically suppressed inflammation initiated by A. catechu.
| Conclusion|| |
The results from this study demonstrated that C. citratus protected the rat liver from A. catechu-induced liver lesions by suppressing increases in the biochemical parameters and attenuating hepatic inflammation.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]