Comparative Effects of Some Medicinal Plants: Anacardium occidentale, Eucalyptus globulus, Psidium guajava, and Xylopia aethiopica Extracts in Alloxan-Induced Diabetic Male Wistar Albino Rats

Insulin therapy and oral antidiabetic agents/drugs used in the treatment of diabetes mellitus have not sufficiently proven to control hyperlipidemia, which is commonly associated with the diabetes mellitus. Again the hopes that traditional medicine and natural plants seem to trigger researchers in this area is yet to be discovered. This research was designed to compare the biochemical effects of some medicinal plants in alloxan-induced diabetic male Wistar rats using named plants that are best at lowering blood glucose and hyperlipidemia and ameliorating other complications of diabetes mellitus by methods of combined therapy. The results obtained showed 82% decrease in blood glucose concentration after the 10th hour to the fortieth hour. There was significant increase P < 0.05 in the superoxide dismutase activity of the test group administered 100 mg/kg of A. Occidentale. There was no significant difference P > 0.05 recorded in the glutathione peroxidase activity of E. globulus (100 mg/kg) when compared to the test groups of P. guajava (250 mg/kg) and X. aethiopica (250 mg/kg). Catalase activity showed significant increase P < 0.05 in the catalase activity, compared to test groups. While at P > 0.05, there was no significant difference seen between test group and treated groups. Meanwhile, degree of significance was observed in other parameters analysed. The biochemical analysis conducted in this study showed positive result, attesting to facts from previous works. Though these individual plants extracts exhibited significant increase in amelorating diabetes complication and blood glucose control compared to glibenclamide, a synthetic antidiabetic drug. Greater performance was observed in the synergy groups. Therefore, a poly/combined formulation of these plants extracts yielded significant result as well as resolving some other complications associated with diabetics.


Introduction
We envisage that using only one or two of these plants extracts separately to determine its antidiabetic activities may not yield effective result. Therefore we set out to combine the plants extracts to investigate their effect arising from more than two entities that will produce an effect greater than their individual effects.
The effect of applying them synergistically to ameliorate diabetes mellitus and its associate diseases was mainly designed. Insulin therapy and oral antidiabetic agents/drugs used in the treatment of diabetes mellitus have not sufficiently proven to control hyperlipidemia. Also the adverse effect/limitations of both modes of treatments, together with the cost of procuring these drugs, have led to several research works on the efficiency of the alleged hypoglycemic and antidiabetic activity of these plants. Therefore this research work is geared towards the study of the named plants that are best at lowering blood glucose and hyperlipidemia and ameliorating other complications of diabetes mellitus.
Anacardium occidentale L. (see Figure 1(a)) leaves stem and bark extracts are utilized widely for the treatment of diarrhea, dysentery, and colonic pain. It has also been reported to possess antidiabetic, antibacterial, anti-inflammatory, and antiulcerogenic properties [1]. The leaves are also used in Brazil for eczema, psoriasis, scrofula, dyspepsia, genital problems, and venereal diseases, as well as for impotence, bronchitis, cough, intestinal colic, leishmaniasis, and syphilis-related skin disorders. The seed coat and the shell that remains after the extraction of the nut are used as fuel for burning purposes [2]. The use of plants in traditional medicine has been identified as a means of studying the potentiality of future medicines. In the year 2000, over 122 compounds used in conventional medicine were identified by researchers as derivatives of "ethnomedical" plants sources, with 80% of these compounds used for the same or similar traditional ethnomedical [3]. Medicinal plants possess curative properties with secondary metabolites that vary in chemical structures such as saponins, tannins, essential oils, alkaloids, and flavonoids to mention but few [4]. They proved to have contributed to the treatment of diseases such as HIV/AIDS, malaria, diabetes, sickle-cell, anaemia, mental disorders [5], and microbial infections [6,7]. Iwu et al. [6] reported that the main benefits of using plants derived medicine are that they are considerably affordable and safer compared to synthetic options, thus providing intense therapeutic benefits and more affordable treatments.
The major chemical composition of Eucalyptus globulus (see Figure 1(b)) are -pinene, -pinene, terpenes, tannins, and so forth [8]. The blue gum flowers are considered as vital source of nectar and pollen greens for bees, thus yielding honey while their leaves are used for herbal tea and for therapeutic purposes [9]. Studied the antidiabetic effects of this plant in the pancreatic islets of diabetic rats. Upon the administration of aqueous Eucalyptus globulus, a reduction in weight loss and increase in water and food intake compared to the streptozotocin untreated diabetic rats administered with streptozotocin. Psidium guajava is an evergreen small tree or shrub whose origin is America [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Its commonly called in different dialects as follows: English, Guava; Hausa: Gwaaba; Yoruba: Guafa; and Igbo: Ugwoba. Deguchi and Miyazaki [25] studied the effect of aqueous Psidium guajava (guava) leaf extract (see Figure 1(c)) on alpha-glucosidase enzymes (that can digest carbohydrate). Results showed that the extracts were able to inhibit the in vitro activities of maltase, sucrase and alpha-amylase in a dose dependent perspective. In vivo studies showed that aqueous extracts of Psidium guajava leaves inhibit both sucrase and maltase activities in the intestinal mucosa of diabetic mice, with natural inhibition of mixed competitive and noncompetitive inhibition [26] while the antioxidant status was elevated in a dose dependent manner [27]. Xylopia aethiopica is an evergreen aromatic tree that belongs to the Annonaceae plant family, as shown in Figure 1(d). The fruits are said to have consisted of -pinene (19%), -terpinene (14.7%), trans-pinocarveol (8.6%), and P-cymene (7.3%) as essential oil [28]. The extracts of dried Xylopia aethiopica fruit were found to inhibit human cervical cancer cell lines C33A, inducing apoptosis and cell cycle arrest in C33A cells in a dose dependency [29]. Adaramoye et al. [30] study the effect of dried fruit extracts protection on Wistar albino rats from adverse effect of whole body radiation. He reported that a synergic treatment attenuated the adverse effects of irradiation on liver glutathione S-transferase (GSH), catalase activities after a week of exposure.
Diabetes insipidus is a metabolic disorder caused by deficiency of vasopressin (antidiuretic) a pituitary hormone that regulates reabsorption of water by the kidney. It is characterized by the production of large volume of diluted urine and constant thirst, although it is a rare kind of disease. According to [31], diabetes mellitus is a chronic metabolic disorder of carbohydrates, proteins, and fats occurring in the endocrine system [32,33] due to absolute or relative deficiency of insulin secretion with/without varying degree of insulin resistance [34,35]. Thus, a combined therapy of these plants extracts become necessary as a trial version for controlling and treating diabetes and associate diseases.

Methodology
2.1. Methods. The leaves of these plants, namely, Anacardium occidentale, Eucalyptus globulus, Psidium guajava, were collected from the premises of University of Nigeria, Nsukka, while the fruits of Xylopia aethiopica were purchased from a local market in Delta State.

Materials
3.1. Animals. The experimental animals used in this study were male Wistar albino rats obtained from the Faculty of Veterinary Medicine, University of Nigeria, Nsukka. Their age ranged between 12 and 14 weeks old, with average body weight 170-260 g.

Experimental Design.
Sixty male Wistar rats were housed in separate cages, acclimatized for seven days. Thereafter, they were divided into fifteen groups of four rats per cage. They were maintained for 12 hours light and dark cycle at tropical conditions. The rats had free access to water and were fed with normal rat diet ad libitum. The feeds were obtained from Vital Feeds, UAC Nigeria. In the beginning of the experiment, the rats were weighted in order to predetermine dosage. They were grouped into various treatment groups from groups 1 to XV. However, groups 1-XIV were all diabetic; treatment was administered for 40 hours, arranged as follows.
Group I was given Anacardium occidentale 100 mg/kg body weight, orally.
Group II also was administered 250 mg/kg body weight of Anacardium occidentale orally.
Group III received orally 100 mg/kg body weight Eucalyptus globulus.
Group IV was given 250 mg/kg body weight of Eucalyptus globulus orally.
Group V got orally 100 mg/kg body weight of Psidium guajava.
Group VI also was administered orally 250 mg/kg body weight Psidium guajava.
Group VII received 100 mg/kg body weight Xylopia aethiopica orally.
Group VIII was given orally 250 mg/kg body weight Xylopia aethiopica.
Group IX was administered a mixture of 100 mg/kg body weight Anacardium occidentale + Eucalyptus globulus orally.
Group X received 250 mg/kg body weight of the mixture of Anacardium occidentale + Eucalyptus globulus by administration.
Group XI was given the mixture, Psidium guajava + Xylopia aethiopica of 100 mg/kg body weight through oral route of administration.
Group XIII received 5 mg/kg body weight via oral intervention of glibenclamide.
Group XIV diabetic but administered no treatment rather was fed with just feed and water.
Group XV was given 10% dimethyl sulphoxide DMSO to act as positive control.
3.3. Chemicals/Reagents/Samples. Dimethyl sulphoxide (DMSO) was purchased from Serva, Heidelberg, New York, while chloroform (trichloromethane), sulphuric acid and alloxan monohydrate were bought from Sigma Aldrich Chemicals, Germany. Thiobarbituric acid was from Lab Tech chemical, Avishkar, and trichloroacetic acid from Kermel. Sodium chloride, cupric acid were purchase from Mayer and Baker, England. The commercial kits were of Randox laboratories Ltd, Crumlin Co. Antrim, UK.

Induction of Diabetes
A quantity of 180 mg/kg alloxan monohydrate was dissolved in normal saline (0.9% Nacl) and administered intraperitoneally to induce diabetes, after subjecting the animals to fasting for 20 hours with their base lines blood glucose determined. Three days after induction of diabetes, the rats with fasting blood glucose higher than 250 mg/dL were used for the experiments. Blood glucose concentrations were determined every eight hours for 40 hours. Meanwhile, food and water were removed from cages four hours prior to testing. While the administration of the plant extracts was carried out every 12 hours. In the end of 40th hour, cardiac dislocation method was used to sacrifice the animals while blood and liver samples were collected for further biochemical test.

Preparation of Plant Extracts
The leaves of three plants were air dried to constant weight at room temperature and reduced to powder by a commercial miller. Quantities of 600 g of each of plant such as Anacardium occidentale + Eucalyptus globulus, Psidium guajava + Xylopia aethiopica were pulverized, and plants materials were macerated in 2.7 litres of analytic chloroform. After 48 hours, the resulting extracts were filtered with Whatman number 1 filter paper and the filtrates were concentrated with rotary evaporator at minimal pressure; the yield of extracts was calculated using the following (see also  (2), where the volumes given were arrived out as follows: where : dose used (g/kg body weight of test animal), : body weight (g), : concentration (g/mL), and : volume (mL).

Phytochemical Analysis
The crude extracts of each plant were screened/analyzed qualitatively by the method modified by [36] for the presence of alkaloids, saponins, steroids, flavonoids, glycosides, tannin, and oils.
The 5 mg/kg of glibenclamide test group was found to be significantly different < 0.05, compared to groups A. occidentale (100 mg/kg), E. globulus (100 mg/kg) and diabetic untreated group. Figure 5 shows the effect of some variable doses of each extract on triglyceride concentrations of experimental rats, with diabetic untreated group having the highest triglyceride value. In other groups, apart from DMSO, upon administration of these extracts, subsequent decrease in various treatment groups especially 250 mg/kg was seen. Group 7 showed significant decrease < 0.05 in triglyceride level of test group A. occidentale 250 mg/kg and treatment groups given P. guajava (100 mg/kg), X. aethiopica (100 mg/kg), and 5 mg/kg glibenclamide, and diabetic untreated group. However no significant difference > 0.05 was observed in groups A. occidentale (100 mg/kg), E. globulus (100, 250 mg/kg), P. guajava (250 mg/kg), X. aethiopica (100, 250 mg/kg), P. guajava + X. aethiopica (100, 250 mg/kg), and DMSO control. A significant difference < 0.05 was observed in group 5, compared to test groups 2, 10, 11, 12, 14, and 15.
Diabetes mellitus is characterized by distorted lipid metabolism, with increase in blood lipids. Hence Figure 4 showed the effect of varying extracts dose on cholesterol level. A considerable reduction was observed in groups treated with  P. guajava (100, 250 mg/kg) compared to glibenclamide group and thus reductions with other groups when compared with the diabetic untreated group. In Figure 5, it can be deduced that there was a greater decrease in triglyceride level in groups administered with A. occidentale (250 mg/kg), E. globulus (250 mg/kg), A. occidentale + E. globulus (100, 250 mg/kg), and P. guajava + X. aethiopica (100, 250 mg/kg) and slight decrease with groups given A. occidentale (100 mg/kg), X. aethiopica (100 mg/kg), P. guajava (100, 250 mg/kg), and X. aethiopica (100 mg/kg) compared to the diabetic untreated group. Figure 6 shows the activity of aspartate aminotransferase based on the effect of the varying doses of the various plant extracts. From the plots it was deduced that there was more significant difference in AST levels of the groups administered A. occidentale + E. globulus (100, 250 mg/kg), P. guajava + X. aethiopica (100 mg/kg), and P. guajava (100 mg/kg) and a lesser reduction in groups A. occidentale (250 mg/kg), E. globulus (100, 250 mg/kg), and P. guajava + X. aethiopica when compared to glibenclamide group. Phytochemical screening of these plants showed the presences of alkaloids, flavonoids, tannins, saponins, and so forth, which are known to perform specific and various functions and hence exhibit different biochemical and pharmacological actions such as cell toxicity and cell protection [37].
The reductions in ALT and AST levels are said to be caused by the hepatocellular and cardiac protection offered   by these extracts. This is further confirmed by the work of Shen et al., 2008, [38] polyherbal formulation in liver function enzymes in diabetic rats. That upon the administration of herbal formula led to a decrease in ALT and AST levels, as observed in groups treated with different doses of the polyherbal formulation which has X. aethiopica as one of its component. The implication of this is that the extract did not produce toxic effects on both the cardiac and hepatic tissues while in the diabetic untreated group there was a noticeable elevation in these two enzymes, an indication of hepatic and cardiac tissue damage. Components such as flavonoids a polyphenol have potent antioxidant capabilities. In 1986 Torell et al. [39] and Faure et al. [40] showed that they inhibited peroxidation of polyunsaturated fatty acids in cell membranes while [3] reported that flavonoids from Helichrysum genus inhibited the formation of two powerful peroxidation agents, namely, superoxide ions and hydroxyl radicals. However, Figure 7 shows the effect of the plant extracts on alanine aminotransferase (ALT) activity upon the administration of these extracts. A sharp reduction was observed in synergy groups administered A. occidentale + X. aethiopica (100, 250 mg/kg) and P. guajava + X. aethiopica (100, 250 mg/kg) compared to glibenclamide group. The groups treated with extracts P. guajava (250 mg/kg) and X. aethiopica (100, 250 mg/kg) had significant reductions in ALT levels compared to groups A. occidentale (100, 250 mg/kg) and E. globulus (100, 250 mg/kg) compared with the glibenclamide group.
In Figure 8, the effect of varying doses of plant extracts on alkaline phosphatase activity in diabetic rats can be seen. A greater reduction is shown in groups administered A. occidentale (100 mg/kg), P. guajava (250 mg/kg), E. globulus (100 mg/kg), X. aethiopica (100 mg/kg), A. occidentale + E. globulus (100, 250 mg/kg), and P. guajava + X. aethiopica (100, 250 mg/kg). A minimal extent of reduction in the serum ALP levels in groups E. globulus (250 mg/kg) and P. guajava (100, 250 mg/kg) was observed compared to the glibenclamide treated group. A much more significant elevation plasma ALP levels were seen in diabetic untreated group compared to every other groups. The reductions in serum ALP levels are a result of cellular membrane/hepatocellular membrane protective effect of the plants extracts, which is supposedly a factor of phytochemical constituents. In 2010 Uboh et al. [41] showed that the aqueous extracts of P. guajava infer the same hepatocellular protection in rats that is linked to flavonoids, a phytochemical component. Flavonoids have been reported to possess antioxidant activity [42], thus protecting cell membranes from peroxidative actions of free radicals. Hence Figure 9 showed the effect of extracts on levels of malondialdehyde (MDA). A more significant decrease of MDA levels was observed in groups treated with E. globulus (250 mg/kg), X. aethiopica (250 mg/kg), and A. occidentale + E. globulus (250 mg/kg) and a significant reduction was observed in groups A. occidentale (100 mg/kg), P. guajava (250 mg/kg), and P. guajava + X. aethiopica (100, 250 mg/kg) compared to the glibenclamide and the diabetic untreated groups. These biochemical effects are supported by the works of Hsieh et al. [43] who reported that the polyphenolic components of P. guajava extracts have high concentration and its equivalent to gallic acid at the rate of 165.61 mgg −1 . Also, polyphenolics and flavonoids are excellent scavengers of free radicals, ferrous ions chelators [44]. In 2007, Hsieh et al. showed that quercetin, a phytochemical component of gallic acid and ferulic acid in an extract of P. guajava, inhibited the formation of advance glycation and end-products. Quercetin has been shown in vivo and in vitro to possess antiglycative biochemical properties whereby it inhibits diabetic complications. Reference [45] also prevented oxidative -cell damaged in streptozotocin animal subjects in vivo [46]. The work of [47] on the stem-bark extract of A. occidentale in rats fed with high-fructose diet did not cause plasma membrane damage. Eucalyptus globulus leaves contain flavonoids such as quercetin that possess antioxidant properties. Figure 10 shows the effect of these plants extracts on catalase activity; from the plots a significant increase in levels of catalase was recorded in treated groups P. guajava (100, 250 mg/kg), X. aethiopica (100, 250 mg/kg), and P. guajava + X. aethiopica (100, 250 mg/kg). In Figure 11, the levels of vitamin C are depicted by the individual plots, as effects of the various extracts at varying doses with significant elevation recorded in groups E. globulus (250 mg/kg), P. guajava (100, 250 mg/kg), X. aethiopica (250 mg/kg), and A. occidentale + X. aethiopica (250 mg/kg), and fairly increase with groups treated with A. occidentale (250 mg/kg) and P. guajava + X. aethiopica (250 mg/kg). Also with a fair increase in groups administered A. occidentale (100 mg/kg) and A. occidentale + E. globulus (250 mg/kg), compared to the glibenclamide treated. The elevations in the levels of these enzymic and nonenzymic antioxidants after the administration of the various plants extracts indicate the presence of antioxidant components that offers cellular protection from peroxidative actions of free radicals produced during diabetes mellitus. From the various figures and plots of these antioxidants with regard to the effect of the extracts, it can be inferred that the antioxidant levels in the diabetic untreated groups show significant reductions. This implies that peroxidative reactions by free radicals had occurred. Therefor, the marker for this biochemical process "malondialdehyde" is a factor for the determination of this reaction.
Results of Figure 12 show the effect of plants extracts on blood glucose concentration, which records glucose level relative to time and dose as seen in the trend of decline. Sokeng et al. [48] showed that the methanolic extract of Anacardium occidentale effectively reduced the blood glucose level in streptozotocin-diabetic rats; this reduction was shown to be more effective with subsequent fractions of the methanolic extract. The mechanism of these extracts supposedly is attributed to the direct stimulation of insulin in the remaining pancreatic cells. In other words the extracts are involved in an insulin-like extrapancreatic stimulation, such as the stimulation of glucose utilization and reduction of hepatic gluconeogenesis [49]. Repeated administration of methanolic extracts and its fractions culminated in a decrease in blood and urine glucose levels [48]. The hypoglycemic effects of these plants are further attributed to their phytochemical constituent. Soussi et al. [50] examined the antihyperglycemic/hypoglycemic effect of Eucalyptus globulus in diabetic mice, whereby dietary administration of these plants ameliorated loss of body weight, polydipsia, and blood glucose level in streptozotocin-diabetic mice. This effect is linked to the protection or regeneration of pancreaticcell following the exposure to the diabetogen-streptozotocin and its action by modulation of insulin secretion. Also in the works of Soussi et al. [50] the pancreatic effects of these plants and their blood glucose lowering ability were reported. The antidiabetic effect of Psidium guajava leaves is connected to their phytochemical constituent such as the various flavonoids, terpenoids, and glycosides as reported by [7,51,52]. The leaves of these plants are used in the reduction of postprandial blood glucose elevation, improvement of hyperinsulinemia in marine models [53]. Also, Pinto et al. [54] in his experiment tested a group of diabetic rats by the administration of some herbal plants that had P. guajava as one of them. Upon the oral administration of aqueous extracts of the leaves at a dose 500 mg/kg body weight for 15 days on, recorded a significant reduction of blood glucose as 43.59% compared to 47 and 74% of glibenclamide.
The fruits of Xylopia aethiopica have been reported to possess hypoglycemia ability as well as other biochemical activities, thus confirming its usage as an antidiabetic agent [55]. This was further supported by the polyherbal formula of Shen et al., [38] which composed of X. aethiopica as one of its component, which showed a significant reduction in plasma blood glucose upon the administration of alcoholic extracts of polyherbal formula. This was found to have done better than glibenclamide. Also, in 2008 Shen et al. showed a significant decrease in plasma glucose of diabetic Wister rats upon the administration/treatment with plant extracts where X. aethiopica was one of the extracts. The reduction in blood glucose was found to be dose dependent. Figure 13 showed greater reduction in plasma blood glucose with synergic formulations of extracts A. occidentale + E. globulus compared to that of P. guajava + X. aethiopica and glibenclamide. This is largely attributed to the plants extracts combined formulation, which is gotten from the individual hypoglycemic activity shown in Figure 13. This is further buttressed by the synergetic works of Shen et al., 2008, on the polyherbal formulation that showed effective reductions in plasma glucose concentrations of diabetic rats than that of glibenclamide. A factor responsible for this could be the synergistic interaction of some phytochemical components such as polyphenols and tannins.
Glucose-6-phosphatase is a key enzyme in the homeostasis of blood glucose through the catalysis of terminal step both in gluconeogenesis and glycogenolysis. Fructose-1, 6bisphosphatase is one of the key enzymes of gluconeogenic pathway [57].
Hepatic, cardiac tissues release aspartate aminotransferase and alanine aminotransferase; therefore the elevation of plasma concentrations of these enzymes is an indicator of hepatic and cardiac damage [58], as in the case of complications in diabetes mellitus while alkaline phosphatase function as a biochemical marker enzyme for maintaining membrane integrity [59]. The increase in plasma levels of this enzyme suggests peroxidation of cell membrane, which occurs during diabetes.

Conclusion
These individual plants extracts exhibited significant increase in ameliorating diabetes complication and blood glucose control compared to glibenclamide, a synthetic antidiabetic drug. Greater performance was observed in the synergy groups. Therefore, a poly/combined formulation of these plants extracts will give considerable effects as well as yielding significant result and may resolved some other complications associated with diabetics.