Antihyperglycemic, Antihyperlipidemic and Antiapoptotic Activities of Micromelum Minutum Seeds in Diabetic Rats

Khaled MM Koriem1,2*, Muhammad E Aminuddin3, Azham S Kader3 and Noruddin R Sheikh4 1Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia (USM), Malaysia 2Medical Physiology Department, National Research Centre, Dokki, Giza, Egypt 3Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia 4Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Malaysia


Introduction
Diabetes mellitus is one of the most common chronic diseases in the whole world. It is a complex, multi-factorial disease which affects the quality, quantity, and style of an individual's life [1]. Diabetes mellitus is a chronic metabolic disorder, mainly characterized by disruption in carbohydrates, protein, and fat metabolism caused by the complete or relative insufficiency of insulin action [2]. When the amount of blood glucose in the blood increases, for example, after a meal, it triggers the release of the hormone insulin from the pancreas. Insulin stimulates muscle and fat cells to remove glucose from the blood and stimulates the liver to convert glucose to glycogen, causing the blood sugar level to decrease to the normal level and consequently stimulate pancreas to secret glucagon hormone to increase blood glucose level where glucagon causes the liver to convert stored glycogen into glucose and high amount of glucose is circulating in the blood (hyperglycemia). To keep the normal level of glucose in blood, the kidney removes the extra sugar from the blood and excretes it in the urine. Hyperglycemia can be handed initially with oral synthetic agent and insulin therapy. But these synthetic agents produce some serious side effects and are relatively expensive for developing countries [3,4].
The research for a new and natural source to be used in the treatment of diabetes was increased in the last decade. The medicinal plants provide a new, avaliable and cheap source for developing new drugs nowadays. Natural products account for more than 40% of all pharmaceuticals on the market today, where from 1941 to 2002, over 50% of all the drugs, or new drug entities, available for cancer treatment were derived from natural resources [5]. The dependence of large rural population on medicinal plants for treatment of diabetes is because of its availability and affordability [6]. Additionally, after the approbation made by WHO on diabetes mellitus, exploration on hyperglycemic agents from medicinal plants has become more significant [7].
The Micromelum minutum (Family Rutaceae) is a shrub that reaches up to 3 m in height, growing widely in Southeast Asia and the Pacific Islands. Its synonym is Micromelum pubescens Blume [8] and is known in Malaysia as chemomar, cherek or kematu. The leaves are used traditionally as a febrifuge, the stems as a carminative, and the flowers and fruits as an expectorant and a purgative, respectively [9]. The genus Micromelum species are known to contain 6-and 8-prenylated coumarins [10][11][12]. Micromelum minutum seeds was chosen for current study because the seeds are rich source of coumarins. Previous investigation of the leaves of the plant led to the isolation of bioactive coumarin derivatives such as microminutin [13], flindersine, 7,8-dioxygenated coumarins, and triterpenes [13][14][15], as well as a pyranoquinoline alkaloid [13]. Lekphrom et al. [16] isolated a new 7-oxygenated coumarin, 7-demethylmurralonginol isovalerate, and murralonginol, together with seven known coumarins (7-oxygenated coumarins, murralonginol isovalerate, murralongin, micromelin, scopoletin, microminutin, murrangatin, and minumicrolin, from the fruits of Micromelum minutum. The methanolic extract of Micromelum minutum exhibit the highest antimutagenic activity in Mutagens, Trp-P-1 among 118 samples (108 species) of edible thailand plants was examined by the Ames Test [17]. Micromelum minutum leaves showed cytotoxic activity against leishmania major and cancer cells (lung adenocarcinoma and leukaemia) [18].
This study was conducted to investigate the anti-hyperglycemic, anti-hyperlipidemic and anti-apoptotic activities of Micromelum minutum seeds ethanolic extract in diabetic rats. Preparation of the ethanolic extract: Micromelum minutum seeds (1.5 kg) were air-dried in an oven at 40°C for 4 days and then the dry plant was cut and pulverized. Dried Micromelum minutum seeds (500 g) were placed in 1000 ml of distilled boiling water and kept at room temperature for 15 min. The dried powdered plant material was macerated for 7 days using 70% ethanol as a solvent. The solvent was then eliminated by a rotary vacuum evaporator under reduced pressure and the subsequent extract lyophilized, representing a yield of 15% of the dry material extracted. The extract was evaporated to dryness to give dried ethanolic extract (150 g) according to the method of Chopra et al. [19]. The extraction process was taken one month from collection of the plant seeds until final ethanolic extract was obtained.

Materials
Ethanolic extract purity, quality and stability methods: Purity tests (Microbiological, Pesticide residues, Heavy metals, Radioactive residues, Chemical, Foreign organic matter and Sulfated ash) were performed in accordance with Malaysian accepted protocol requirements and accredited to ISO/IEC 17025 consult the WHO guidelines on stability and quality controls methods for medicinal plants [20,21]. The plant extract was stored in a tightly cooling (-4°C) sealed container away from heat and light that prevent any loss of extract solvent and entry of water; where plant extract dissolved in 1ml distilled water and given orally to individual rat.
Determination of lethal dose fifty (LD 50 ) of Micromelum minutum extract: Forty adult albino rats were used in this study and divided into 5 equal groups each of 8 rats. The LD 50 of the extract was determined in mg/kg body weight [22], where LD 50 = DM-ΣZ.d/m, DM= the highest dose used, Z= the number of dead rats of two successive doses divided by two, d=difference between two successive doses, m= the number of rats in each group. Administrations with oral 10% of the LD 50 of the extract were found to be safe [23].
Thin layer chromatography (TLC) for coumarins isolation: TLC was used to resolve and isolate the coumarins constituents from the plant extract. Dried seeds of the plant were extracted with acetone at room temperature. The ethanol extract (2.36 g) was subjected to silicagel column chromatography eluting with hexane and hexane-acetone (7:3, 3:2, 1:1, 1:4), successively, to give 6 fractions. Each fraction was further subjected to silica-gel column chromatography and preparative TLC with appropriate combinations of hexane, CH 2 Cl 2 , iso-Pr 2 O, benzene, CHCl 3 , EtOAc, acetone, and MeOH as developing solvents to give another six coumarins as described in the results below. c) Kits reagents obtained from Biomerieux Company through local supplier for measuring serum glucose, triglycerides and total cholesterol fractions. Doses of drugs used were calculated according to Paget and Barnes [24] administered orally by gastric tube. The extract is also administered orally by a gastric tube.

Methods
Induction of diabetes: Hyperglycemia was induced by injecting streptozotocin (STZ) at a dose of 150 mg/kg intraperitoneall (ip) and the animals were kept under observation. After 48 hrs, the animals were tested for blood glucose using enzymatic colorimetrical method [25]. Twelve days after the STZ injection, rats with fasting blood glucose levels greater than 200 mg/dl were considered diabetic.

Determination of anti-hyperglycemic and anti-hyperlipidemic activities:
Five groups each of 8 diabetic rats were used for both anti-hyperglycemic and anti-hyperlipidemic activities. In antihyperglycemic group, the first group was kept as negative control administered saline orally. The second group is diabetic rats. The 3 rd group; diabetic rats treated orally with Glibenclamide (2.5 mg/kg) was used as reference positive control drug suspended in saline [26]. The 4 th group; diabetic rats treated daily with oral dose of 100 mg/kg bwt of the ethanolic extract of Micromelum minutum seeds; while the 5 th group; diabetic rats received daily an oral dose of 25 mg/kg bwt of Microminutinin coumarin for 15 days and 30 days, respectively. At zero time, 15 & 30 days after administration of negative and positive controls as well the extract and coumarin, blood samples were collected from the retro-orbital plexus through the canthus of the anesthetized rats after an overnight fasting and serum was isolated by centrifugation and total blood glucose was determined.
In anti-hyperlipidemic group the animals were divided also into collected for Immunohistochemical (IHC) staining techniques allow for the visualization of antigens via sequential application of a specific antibody to the antigen (primary antibody), a secondary antibody to the primary antibody and an enzyme complex with a chromogenic substrate with interposed washing steps. The enzymatic activation of the chromogen results in a visible reaction product at the antigen site. The specimen may then be counterstained and overslipped. Results are interpreted using a light microscope [27,28] Staining protocol: Staining procedure for p53 and bcl-2 was performed according to Hsu and Raine [29] and Elias et al. [30].
Histopathological studies: At the end of experimental study; the animals were sacrificed by mild ether anesthesia. The pancreas tissues were collected, excised and rinsed in ice-cold 0.9% saline solution. They were blotted dry and fixed in 10% formalin for 48 hours. Then, they were subject to dehydration with acetone of strength 70, 80 and 100% respectively each for 1hour and embedded in paraffin wax. 7.0 μm thick paraffin sections of the tissue samples from control and treated animals were stained with haematoxylin-eosin for photomicroscopic observations [31].

Statistical analysis:
The results are expressed as mean ± standard error (SE). Statistical significance was determined through twoway analysis of variancs (ANOVA), followed by Student's t -test. P values less than 0.05 were considered statistically significant *P ≤ 0.05 significant difference compared to control (-ve control), **P ≤ 0.01 highly significant differences compared to control (-ve control). a P ≤ 0.05 significant difference compared to diabetic (+ve control) group. b P ≤ 0.01 highly significant difference compared to diabetic (+ve control) group.

Results
TLC isolates twelve's coumarins depending on the eluate used as follow; the hexane-acetone (7:3) eluate gave micromarin-A (1) (57.9mg), micromelin (7) (28.8mg), murralonginol isovalerate (8) (0.4mg), microminutinin (9) (471.9mg), 6-methoxymicrominutinin (10) (9.7mg), micromarin-F (4) (10.7mg), and micromarin-G (5) (1.1mg). The hexane-acetone (1:1) eluate gave microminutin (11) (105.1mg), micromarin-H (6) (1.0mg), micromarin-C (3) (1.2mg), and murrangatin (12) (0.5mg). The hexane-acetone (1:4) eluate gave micromarin-B (2) (7.5mg). The structures of these twelve's coumarins are illustrated in Figure 1. Microminutinin coumarin (9) was used in this study due to the majority of this compound in the plant seeds. Table 1 revealed the effect of Micromelum minutum seeds ethanolic extract and Microminutinin coumarin on serum glucose levels in diabetic rats. It is clear that there is a significant decrease in serum glucose levels after 15 and 30 days of administration of both extract and coumarin. The Micromelum minutum seeds ethanolic extract (100 mg/kg bwt) was more effective in lowering serum glucose level than Microminutinin coumarin (25mg/kg bwt). Furthermore, the dose of the standard drug Glibenclamide (2.5mg/kg bwt) was more potent in lowering effect of diabetic rats' levels than both doses of the extract and coumarin, respectively.  Table 2 illustrated the effect of Micromelum minutum seeds ethanolic extract and Microminutinin coumarin on serum cholesterol, triglycerides, HDL and LDL levels in diabetic rats. The data show that there was a significant decrease in the cholesterol, triglycerides and LDL levels, while an increase in the HDL level was recorded. Administration with 100 mg/kg bwt of Micromelum minutum seeds ethanolic extract exhibited a higher increase in the HDL than coumarin in dose of 25 mg/ kg bwt. Moreover, the dose of the standard drug Atorvastatin (1mg/ kg bwt) was more potent in lowering effect of hyperlipidemic rats' levels than the doses of Micromelum minutum extract and coumarin, respectively. Table 3 exhibited the change in body weight in diabetic rats compared to control. It is clear that there is an increase in body weight in diabetic rats compared to control; while the administration of Atorvastatin, Micromelum minutum extract and coumarin, respectively at zero time, 15 days, 1 and 2 months decreased the body weight where 100 mg/kg bwt of Micromelum minutum seeds ethanolic extract exhibited a higher decrease in body weight than coumarin in dose of 25 mg/kg bwt. Moreover, the dose of Atorvastatin (1mg/kg bwt) was more potent in lowering effect of hyperlipidemic rats' levels than the dose of plant extract and coumarin, respectively. Figure 2 showed the photomicrographs of immunohistochemically stained pancreas sections. The saline administration showed normal amount of expressed p53 (Figure 2 A). On the other hand, the amount of p53 was potentially decreased in diabetic rats. (Figure 2 B) compared to control rats. The treatment of diabetic rats with Micromelum minutum extract and Microminutinin coumarin increased the amount of expressed p53 (Figure 2 C and D) compared to diabetic rats. Histopathological investigation of the pancreas of control and diabetic rats are shown in Figure 4, A-D. Pancreas of normal control animals showed normal islets, whereas that of diabetic animals showed atrophy of β-cells and vascular degenerative changes in the islets. Micromelum minutum seeds ethanolic extract (100 mg/kg) and Microminutinin coumarin (25mg/kg bwt) treatment increased the number of islets as compared to that of diabetic animals. Results expressed as mean ± SE (n=8), *Significantly difference from control at P ≤ 0.05,** Highly significantly difference from control at P ≤ 0.01, a Significantly difference from diabetes at P ≤ 0.05, b Highly significantly difference from diabetes at P ≤ 0.01, Saline, Glibenclamide, plant seeds extract and Microminutinin were given in the same volume (1ml). Serum glucose was measured at Zero, 15 & 30 days of Saline, Glibenclamide, Extract and Microminutinin administration. Results expressed as mean ± SE (n=8), **Significantly difference from control at P ≤ 0.01, b significantly difference from diabetes at P ≤ 0.01, Saline, Atorvastatin, plant seeds extract and Microminutinin were given in the same volume (1ml). Lipid profile was measured at Zero, 1 & 2 months of Saline, Atorvastatin, Extract and Microminutinin administration.

Discussion
Pharmacological studies on the Micromelum minutum seeds (Family Rutaceae) grown in Malaysia are limited. The selection of the plant Micromelum minutum due to it's widely distribution in Southeast Asia and the Pacific Islands where the leaves are used traditionally as a febrifuge, the stems as a carminative, and the flowers and fruits as an expectorant and a purgative, respectively [9]. In this study the evaluation of antihyperglycemic, antihyperlipidemic and antiapoptotic Activities of Micromelum minutum in diabetic rats was done.
In the present study, the effects of the Micromelum minutum seeds ethanolic extract and Microminutinin coumarin on serum glucose levels in diabetic rats were done. The result revealed that there is a significant decrease in glucose levels after extract (100 mg/kg bwt) and coumarin (25mg/kg bwt) administration. Coumarins are reported to have antioxidant activity and preserve the levels of other antioxidants in human plasma [32,33]. Coumarins also inhibited lipid peroxidation and increased the activity of antioxidants, superoxide dismutase and catalase [34]. Micromelum minutum seeds extract significantly reduced the blood glucose levels of diabetic rats indicating that the mechanism of action may be due to coumarin ingredient; where coumarin inhibits hyperglycemia [34,35]. Hyperglycemia inhibits antioxidants and its cellular transport. Since the chemical structure of antioxidants is similar to that of glucose, it shares the membrane transport system with glucose and hence competes with it for its transport, where glucose-lysine mixtures (GL) at 150°C increased the ability to reduce lipid peroxidation and decreased the free radical scavenging activity [36]. Thus the elevation in glucose concentration may depress natural antioxidants inside the body. Therefore when coumarin is supplied from exogenous source as from Micromelum minutum seeds, it is able to do its antioxidant duty and protect the pancreas and decrease serum glucose. These observations were supported by Von Stebut et al. [37] who succeeded to obtain a successful treatment of adult multisystemic langerhans cell histiocytosis from coumarin.
In this study, the effects of the Micromelum minutum seeds extract and Microminutinin coumarin on serum cholesterol, triglycerides, HDL and LDL levels in diabetic rats were done. The result revealed that there is a significant decrease in the cholesterol, triglycerides and LDL; while an increase in HDL concentrations after extract and coumarin administration. Administration with 25mg/kg bwt of coumarin Data represents mean ± SE (n=8), ** highly significantly difference from control at P≤ 0.01; a Significantly difference from diabetes at P≤ 0.05, b Highly significantly difference from diabetes at P≤ 0.01, Saline, Atorvastatin, plant seeds extract and Microminutinin were given in the same volume (1ml). Body weight was measured at Zero, 15 days, 1 & 2 months of Saline, Atorvastatin, Extract and coumarin administration   exhibited a significant increase in HDL value, while 100 mg/ kg bwt of the plant extract also showed a significant increase in HDL value but higher; which is considered a beneficiary effect in the treatment of dyslipidemia condition. The HDL mediates the reverse transport of cholesterol from peripheral tissues to the liver for disposal by excretion into bile. This process will disallow the slow accumulation of lipids in artery walls. This effect may be attributed to the coumarin contents of the plant seeds which was reported to exhibit lipid lowering effect and significantly decrease total cholesterol level [38][39][40].
In this study, increase in body weight in diabetic rats was observed. However, obesity, which represents one of the main features of the metabolic syndrome, is commonly associated with diabetes syndrome [41]. The administration of both Micromelum minutum seeds extract and Microminutinin coumarin Micromelum minutum seeds extract and Microminutinin coumarin to diabetic rats decrease body weight. These results are in agreement with that of Pari and Rajarajeswari [42] and Guerrero-Analco et al. [35] who reported that the administration of coumarin to diabetic rats resulted in alterations in the metabolism of glucose with subsequent reduction in plasma glucose levels and body weight.
In the current study, anti-apoptotic activity of Micromelum minutum seeds extract and Microminutinin coumarin were recorded. p53 and bcl-2 are closely related to the majority of human toxicity and cancer [43]. Apoptotic marker, p53 is a critical regulator of apoptosis in many cells. It stimulates a wide network of signals that act through either extrinsic or intrinsic pathways of apoptosis [44] by activating the transcription of downstream genes such as p21 and Bax to induce apoptotic process which inhibiting the growth of cells with damaged DNA. On the other hand, bcl-2 has been reported to function primarily by blocking the apoptosis pathway [45]. Bcl-2 gene product is a negative regulator of apoptosis, which forms a heterodimer complex with Bax and neutralizes the effect of pro-apoptosis [46]. Our results indicated that diabetes decreased p53 expression while increased bcl-2 expression. On the other side, the treated with Micromelum minutum seeds extract and its coumarin ingredient to diabetic rats increased p53 expression while decreased bcl-2 expression. These results were in agreement with that of Mulware [47] who reported that diabetes cause induction of oxidative-induced DNA damage by ROS may lead to isolated base lesions or single-strand breaks, complex lesions like double-strand breaks, and some oxidative generated clustered DNA lesions (OCDLs) which are linked to cell apoptosis and mutagenesis. On the other side, coumarin has anti-apoptotic activity, an inhibitor of p53 abrogated glucolipotoxicity-induced ROS generation and p53 expression [48]. The antiapoptotic activity of Micromelum minutum may be related to its mahanine ingredient (a carbazole alkaloid occurs in Micromelum minutum; where concentration of 10 µM mahanine caused a complete inhibition of cell proliferation and the induction of apoptosis in a time dependent manner. Mahanine-induced cell death was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase like activities, poly (ADPribose) polymerase cleavage, release of cytochrome c into cytosol and stimulation of reactive oxygen species generation. The mahanine activated various caspases such as caspase-3, -6, -8 and -9 (like) activities but not caspase-1 like activity. More than 70% cell survival was observed in the presence of a caspase-3 inhibitor. The overall results suggested that mahanine down regulates cell survival factors by activation of caspase-3 through mitochondrial dependent pathway, and disrupts cell cycle progression [49,50]. High blood glucose levelsinduced apoptosis by regulating the gene expression of the bcl-2 and p53; where bcl-2 expression stimulated by high glucose level [51].
Histopathological studies of pancreas of control and diabetic rats of both Micromelum minutum seeds ethanolic extract-and Microminutinin coumarin-treated groups indicate that the plant ethanolic extract has cytoprotective properties.
In this study; we find out that these major amounts of coumarins in Micromelum minutum seeds ethanolic extract are responsible for all plant seeds activities; and the plant ethanolic extract was more potent in its activities than its Microminutinin coumarin. This may be related to synergistic effects among different coumarins contents of the plant seeds ethanolic extract.
Micromelum minutum seeds are used in Malaysia as a house remedy and people are used to drink it without a known acceptable dose. From this study, we found that 100mg/kg bt Micromelum minutum seeds ethanolic extract was more potent in all activities studied; so if human standard weight was 70kg; then 7g of seeds ethanolic extract was recommended for human daily consumption. In this study we collected 1.5 kg of the plant seeds to obtain finally 150g of Micromelum minutum seeds ethanolic extract; so 70g of Micromelum minutum seeds in divided doses was the equivalence of recommended daily amount for human consumption of raw herb (seeds of Micromelum minutum) used for this study and the clinical treatment requires management by a health-care provider.

Conclusion
According to the previous data, it could be concluded that Micromelum minutum seeds extract could be used for the treatment of hyperglycemia, hyperlipidemia and apoptosis. Coumarins contents are responsible for all these activities of plant seeds ethanolic extract. However, further clinical studies are warranted to establish it's effectiveness in humans and it will be interesting to see whether Micromelum minutum seeds can reverses existing complications associated with diabetes, like in real case scenarios.