Antidiabetic and antihyperlipidemic activity of Dracaena Cinnabari Balf. Resin ethanolic extract of Soqatra Island in Experimental Animals

Background: - Dracaena Cinnabari balf is a species plant in Agavaceae family. It is a tree endemic to the Island of Socotra,Yemen . It was used as a dye and medicine in Socotra and the Mediterranean basin. Some researches demonstrated both hypoglycemic and hypolipidemic of its resin of Dracaena Cochinchinensis , a traditional Chinese tree. Accordingly, this study aimed to evaluate the antidiabetic potential of the ethanolic extract of dracanea cinnabari balf resin (DCBR), endemic in Socotra Island, in Alloxan induced diabetes, and evaluation of possible antihyperlipidemic activity of the same extract in high-cholesterol diet-induced hyperlipidemia in male albino rats. Methods : - Experimental diabetes was induced in rats with intraperitoneal injection a single dose of Alloxan (150 mg/kg). Hyperlipidemia was induced in rats by administration of 1% cholesterol in diet and 0.5 % Hydrogen Peroxide in drinking water for 14 days. Fasting blood glucose levels were measured at 1st day, 7th day and 14th day. Pancreas histopathology was done at the end. Estimation of serum lipid profile and liver histopathology was done at the end of 14 day. The results were expressed as mean ± SD and differences among the groups of animals were compared using one-way ANOVA. Results:- Administration of ethanolic extract of resin of two doses (100mg and 300mg\kg) in alloxan induce diabetic rats resulted in a significant decrease in FBG levels with a recovery in destruction of pancreas cell compared with untreated group .Also the same doses of extract showed significant decrease in triglycerides ,total cholesterol ,low density lipoprotein, very low density lipoprotein and alkaline phospatase in comparing with untreated groups. Conclusion: - From the results of present study we can conclude the ethanolic extract of Dracaena cinnabari Balf . have a hypoglycemic and hypolipidemic activity in experimental animals , these activity are time and dose dependent.

Healthy male albino rats of Wistar strain weighing 150-250g.They were obtained from the Central Animal House, Sana'a University. The present study was approved before start off experiment by the animal Ethics committee, 00243,Goverment of Yemen constituted, Sana'a university. The animals were housed in standard polypropylene cages and maintained within the same room temperature and humidity with 12:12 hour light and dark cycle. All the rats were given a 14-day period of acclimatization before starting the experiment.
 Induction of diabetes : Diabetes was induced by intraperitoneal administration of a single dose of alloxan(150 mg/kg) [20]. Male albino rats were fasted for 18 h. The fasting blood sugar (FBS) levels of the rats were determined with blood from the rats' tail vein using an auto analyzer (AccuCheck Performa Advantage II, Roche).Freshly prepared Alloxan monohydrate dissolved in normal saline (0.9% w/v NaCl) and injected as a single dose of 150 mg/kg intraperitoneally to induce hyperglycemia. To avoid fatal hypoglycemia as a result of massive pancreatic insulin release, rats were Kept for the next 24 h on 5% glucose [21] . After 72 hours, rats with fasting blood glucose Level more than 250 mg/dL were considered to be diabetic and selected for studies [22]. The experiment was approved by the Institutional Ethical Committee, Faculty of Medicine and Health Sciences, Sana`a University. Experimental design: Evaluation of antidiabetic activity of DCBR methanolic extract in alloxan -induced diabetic rats . The albino rats were allocated randomly into five groups, each containing six rats :-Group 1: Normal control, without treatment rats. Group2: Diabetic control, without treatment rats Group3: Diabetic rats received Ethanolic extract (100 mg/kg/day, p.o [23] Group4: Diabetic rats received Ethanolic extract (300 mg/kg/day, p.o.) [23] Group5: Diabetic rats received glibenclamide (2.5 mg/kg/day, p.o.) [24] After 72 hours from administration of alloxan rats with hyperglycemia more than 250 mg\dl were selected and used for experiment . The rats were administered the extract (dissolved in Tween 80) and chemical drug for two weeks orally by using a feeding cannula. The experimental rats were weighed every day to compare the body weight change in different groups through the experimental period. FBG was estimated before the start of treatment ,on 7 th day and 14 th days of treatment . All rats were later sacrificed on 14th days by diethyl ether and the Pancreases were weighted separately then collected in10 % formalin for histopathological examination.

Histopathological examination of Pancreas:
For histological study, specimens of pancreases were immersed in neutral buffered formalin (10%) for 48 hours. Then tissue dehydration was done using ascending grades of alcohol (ethanol), followed by tissue clearing using xylene. The tissues were transferred to molten paraffin for impregnation and embedded in paraffin blocks. After fine sectioning, staining was done using hematoxylin and eosin (H and E) stain and examined under a microscope [25]. All the sections were examined under a light microscope under X400 magnifications. Photomicrographs of lesions were taken for observations and documentation of histopathological lesions.
 Induction of hyperlipidemia: Hyperlipidemia was induced in rats by administration of 1% cholesterol in diet and 0.5 % Hydrogen Peroxide in drinking water for 14 days [26]. Experimental design : Evaluation of antihyperlipidemic activity of DCBR methanolic extract in experimental rats. The albino rats were allocated randomly into five groups, each containing six rats :-After experimental period, and after an overnight fasting, animals in different groups were anesthetic under mild diethyl ether and the blood was withdrawn by retro-orbital method to determine serum lipid profile. Serum obtained by immediate centrifugation of blood samples using centrifuge at 3000 rpm for 15 min. at room temperature. The concentration of total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) was determined by using enzymatic commercial kits marketed by QCA Ltd, Spain. While VLDL and atherogenic index in plasma was calculated as per Friedewald estimation [28] . Then, the animals were sacrificed to take the liver and aorta specimens for histopathological examination.

Histopathological examination of liver and Aorta:
Specimens of liver and aorta were immersed in neutral buffered formalin (10%) for 48 hours. Then tissue dehydration was done using ascending grades of alcohol (ethanol), followed by tissue clearing using xylene. The tissues were transferred to molten paraffin for impregnation and embedded in paraffin blocks. After fine sectioning, staining was done using hematoxylin and eosin (H and E) stain and examined under a microscope [25]. All the sections were examined under a light microscope under X400 magnifications. Photomicrographs of lesions were taken for observation and documentation of histopathological lesions.

Statistical analysis:
The result were expressed as mean ± SD and differences among the groups of animals were compared using one-way ANOVA with post-hoc LSD's test. Statistical significance was set at P ≤ 0.05. Statistical analysis was performed using Microsoft excel, and SPSS software version 20.

RESULTS:
Result of antidiabetic activity of DCBR methanolic extract in alloxan -induced diabetic rats.  Relative change in body weight: Measurement of the weight of all animals from the first day to the last day of the experiment showed an irregular change and differences in the groups as shown in Table (1). The weight of group 1 increased significantly from 1st day to 14th day in opposite to group 2. While, the groups that treated with 100 mg/kg (group 3) and 300 mg/kg DCBR extract (group 4) showed noticeable improvement to normal weight during treatment period whereas the weight were dropped dramatically because of Alloxan injection. Group 5 that was treated with 2.5 mg/kg Glibenclamide kept the same weight level during treatment period.

 Relative change in weight of pancreas:
As shown in table (2), after 14 day of treatment, there was a significant decrease in pancreas weight in group 2, in comparison to group 1 (p-value≤ 0.05). Group 3 which was treated with 100 mg/kg DCBR extract showed an increase in pancreas weight. We got the same result with group 5 which was treated with 2.5 mg/kg Glibenclamide, whereas group 4 which was treated with 300 mg/kg DCBR extract, showed a significant increase in the pancreas weight in comparison to group 2

Table (2):
The effect of DCBR extract on change in the pancreas weight (g) of Alloxaninduced diabetic rats; n=6

Biochemical investigation (FBG):
The groups 2, 3, 4 which were injected with 150mg/kg alloxan showed a significant increase in the FBG values more than 200mg/dl (p-value ≤ 0.05) in comparison to group1. After 7days of treatment , the dose of 100 mg/kg DCBR extract(group 3) , 300 mg/kg DCBR extract(group 4) and 2.5 mg/kg Glibenclamide (group 5) showed a significant change in comparison to group 1 and group 2 (p-value ≥ 0.05) , as well as after 14 days of treatment. By the way, group 4 did not show a significant change in FBG in comparable to group 1,

Histopathological examination of pancreas:
 Macroscopic observations : The pancreas was subjected to macroscopic and microscopic investigation to detect irregularities and abnormalities of the structure .Macroscopic analysis of the control group demonstrated normal pinkish appearance (Fig.1a) in comparison to the pancreas of untreated diabetic, depletion large area of the pancreas as a result of alloxan injection 150 mg/kg, (Fig.5c). Treated groups with 300 mg/kg extract , 100 mg/kg extract ,and 2.5 mg/kg Glibenclamide showed clear preserved tissue (Fig. 1e, 1g, 1i) respectively).

 Microscopic observations :
The microscoic appearance of the control group islet cells (group 1) was normal (Fig.5b). Untreated diabetic group (group2) revealed a breakdown of micro-anatomical features including necrosis as in (Fig. 1d). The comparison of the untreated diabetic group to normal group, there was a destruction in the islet cells with irregular shape and atrophy (Fig. 1d). (Fig. 1f, 1h, 1j) figures showed treated diabetic rats with 100 mg/kg extract (group 3), 300 mg/kg extract (group 4) and 2.5 mg/kg Glibenclamide (group 5) respectively. The comparison of these three groups with the untreated diabetic rats, there was an evidence of recovery of langerhans islets, more obvious islet pattern with well outlined boundaries, vacuolation was reduced or absent in many islets.

Result of antihyperlipidemic activity of DCBR methanolic extract in experimental rats.  Relative change in body weight:
Based on the result reflected on table (4), we could say that the rats of groups 2,3,5 (hyperlipidemic without treatment, 100 mg/kg DCBR extract, 10 mg/kg Atorvastatin respectively, showed an increased growing in body weight throughout the entire period of   Relative change in weight of liver: As shown in table (5), after 14 day of treatment ,there was a significant change in the liver weight of group 2 in comparison to group 1 (p-value≤ 0.05). also, other three groups of treatment with 100mg/ kg DCBR extract (group 3), 300 mg/ kg DCBR extract (group 4)and 10 mg/kg Atorvastatin (group 5) showed a significant change and decrease the liver weight in comparison to group2 (p-value≤ 0.05)

 Biochemical parameters: TC, TG ,HDL,LDL, VLDL, and AI:
As in table (6) the rats when fed with high-fat diet (1% cholesterol) showed a remarkable hyperlipidemia. For the whole group, there was a significant increase in TC, TG, LDL, VLDL Al, and a decrease in HDL in comparison to group1 (p-value≤ 0.05). When we compare the treated groups with group 2, we noticed that: the treated group with 100 mg/kg DCBR extract(group 3) showed a significant decrease in TC, TG, LDL,VLDL and AI and a significant increase in HDL (p-value≤ 0.05), (where the change in TC,TG,LDL was insignificant, and VLDL was significant) .The higher dose of 300 mg/kg DCBR extract (group 4)showed a significant effect in decreasing TC, TG , LDL , and AI. In addition, there is no effect on HDL. The last group which was treated with 10 mg /kg Atorvastatin showed no effect on TC, TG. But, also showed a significant increase in HDL and no significant effect on VLDL and AI. Histopathological examination of liver and Aorta  Macroscopic observations : The liver was subjected to macroscopic and microscopic investigation to detect irregularities and abnormalities of the structure. Macroscopic analysis of the liver in hyperlipidemic group (group 2) demonstrated major light brown color changes of the liver lobes (Fig.3a, 3b). While the liver of the normal group (group 1) looks in a normal appearance i.e deep brown color with smooth surface (Fig.2a). The hyperlipidemic groups treated with 100mg/kg DC extract (group 3), 300 mg/kg DC extract (group 4), and 10 mg/kg Atorvastatin (group 5) shown pale brown coloration of the hepatic soft tissue (Fig. 4a,4d,4g). In addition, inducing high fat diet shows a clear accumulation of fat in the adipose tissue inside the rat abdomen. Which is clear in group 2 (Fig3b &3c). The treated group with 100mg/kg DC extract shows no big difference in the amount of the accumulation of the fat in adipose tissue (Fig.4b). Whereas other doses of 300 mg/kg DC extract and 10 mg/kg Atorvastatin shows a notable decrease Fig. 4.

 Microscopic observations :
The normal liver tissue section of group 1 shows normal structure i.e portal vein, hepatic cord, and hepatic sinuside (Fig. 2d). Whereas the hyperlipidemic rat liver tissue section (group 2) shows distortion in the arrangement of cells around the central vein, inflammation Fig. 3g, infiltration Fig3i, fatty changes Fig. 3d, hemorrhage Fig. 3e, hydropic change Fig. 3f, vasodilation Fig.3h. The hyperlipidemic groups treated with 100 mg/kg DC extract (group3), 300 mg/kg DC extract (group4) and 10 mg/kg Atorvastatin ( Group5) brought back the cellular arrangement around the vein in some parts of the tissue that lead to bring the blood vessels to the normal conditions (Fig. 4c, 4f, 4i respectively). Histopathological picture of Aorta all groups does not show any abnormalities Fig. (5).

DISCUSSION:
Alloxan can induce diabetes by destruction of the beta cells of the islets of Langerhans of the pancreas [29]. This results in a decrease of endogenous insulin secretion which paves way for the decreased utilization of glucose by body tissues and consequently elevation of blood glucose level [30]. In current study and after alloxan injection, elevation of blood glucose was observed in group 2,3,4 and 5.
When treated them with the ethanolic extract of D Cinnabari it was indicated that all of them reduced the blood glucose level to an extent. The dose 100 mg/kg decreased FBG significantly (p≤ 0.05) up to 24 % and 53 % after 7, 14 day respectively. Also, the dose 300 mg/kg showed more decrease in FBG significantly (p≤ 0.05) up to 60.5 % and79 % after 7 and 14 days respectively. These results were compatible with [31] [23].
Chen et.al., 2013 demonstrated hypoglycemic effect of total flavonoids of Dracaena cochinchinensis in type 2 diabetes mellitus rats [31]. Also, Gu et al., 2009 showed an antidiabetic effect of Dracaena Cochinchinensis by inhibition on alpha-glucosidase activity and suppressing intestinal carbohydrate absorption and thereby reducing the postprandial increase of blood glucose [23]. In a vitro study reported the ethyl acetate extract of Dragon cinnabari resin has antidiabetic properties with standard Glucose uptake procedure against MCF-7 cell line [19].
The mechanism of antidiabetic properties of the extract is not well known. But, it is returned likely to the phytochemical constituents including flavonoids, alkaloids, tannins, and others. Theses constitutes also obtained from various plant sources and they have been reported to be potent hypoglycemic agents [32].
Flavonoids are the main chemical constituents of dracaena species [33][31] [34]. Numerous studies have been approved the potential role of flavonoids as antidiabetic, antihyperlipidemic and antioxidant [35] [36] [37]. Dracaena cochinchinensis extract, a Chinese traditional dragon blood plant, has shown to possess antidiabetic as well as antihyperlipidemic properties in earlier studies [34] Flavonoids derivative from the methanoilc extract of Origanum majorana L.(family: Lamiaceae) leaves have α-glucosidase enzyme inhibitory activity [38]. Another flavonoids from Cecropia obtusifolia (family: Ceropiaceae) exhibited potent hypoglycemic activity comparable to that of glibenclamide at a dose of 3 mg/kg in diabetic rats [39]. Other flavonoids were isolated from the dried leaves of Myrcia multiflora DC. (family: Myrtaceae) inhibited the activity of the rat lens aldose reductase enzyme so decrease diabetic complications [40].
Flavonoids can decrease the blood glucose by pancreatic and extra pancreatic mechanisms. They can act as insulin secretagogues which increase insulin secretion in vitro and in vivo systems [43] [38]. in similar to Sulfonylureas such as glibenclamide [44] which was used as the reference drug in this study. They also can inhibit α-glucosidase activity in the intestine and reduce glucose absorption from intestine [23].
There are convincing experimental and clinical evidence that the generation of ROS is increased in both Type 1 and Type 2 diabetes and that the onset of diabetes is closely associated with oxidative stress [45]. Flavonoids are rich in phenol groups which work as antioxidant which may have slowed or terminated the production of ROS thereby reversing the diabetic condition [17] [46][47] [48].
In the histopathology, the degeneration observed in the pancreas of alloxan-induced diabetic rats may be due to necrotic action of alloxan on the beta cells [49] [50]. The degenerative changes induced by alloxan in rats pancreas were recovered by DCBR extract and glibenclamide which was followed by regeneration of the islet cells and increased tissue density with attendant improved insulin production and reduction of FBG in rats. This effect was more pronounced at 300 mg/kg DCBR extract. It has been demonstrated that beta cells can regenerate from stem cells located in pancreatic ducts or from progenitor cells residing inside murine islets [51]. Regeneration of the islet cells by DCBR extract may involve promotion of the above mechanisms. Also, Flavonoids are reported to regenerate the damaged pancreatic β -cells in diabetic animals which were acting as antioxidant and reducing the cells damage by alloxan [52].
Furthermore, the current study showed antihyperlipidemic effect of DCBR extract which is agreed with Fan et al. 2014 [34]. High fat diet administered to rats raised the lipid profile and with treatment by extract, lipids are decreased by 16%, 31% TC.21%,29% TG. 8%, 22% LDL. 21%, 29% VLDL with 100 mg/kg and 300 mg/kg respectively. and decrease athereogenic index which is an indicator to decrease the atherosclerosis risks. Increase HDL to 25% with 100 mg/kg dose with no effect with the dose 300 mg/kg. 10 mg/kg of atorvastatin which was used as a reference drug did not show effect on TC and TG. atorvastatin works by inhibiting HMG-CoA reductase, an enzyme found in liver tissue that plays a key role in production of cholesterol in the body. While the outer source of lipid was continuous in food, may this interpret the lack effect of atorvastatin.
Various mechanisms can be explained how flavonoids, which is the main component of Dracaena cinnabari, decreased the lipid profile. Hyperlipidemia related to increased oxidative stress causing significant production of oxygen free radicals, which may lead to oxidative modifications in low-density lipoproteins, which present a significant function in the initiation and progression of atherosclerosis and associated cardiovascular diseases [5].Flavonoids can act directly as scavenging some radical species, thus acting as antioxidants and inhibit the progression of atherosclerosis [47].
Flavonoids can bind with bile acids. since these resin are not absorbed , bile acids are excreted. This results in a decreasing bile acid uptake by the liver, and therefore in a higher conversion of cholesterol to bile acids in the liver [53].Also, flavonoids can Inhibit Pancreatic cholesterol esterase enzyme ,therefore limit the absorption of dietary cholesterol [54][55] [53] 4-then reduce the solubility of cholesterol in micelles which may result in delayed cholesterol absorption [56] [54]. The histopathlogical finding of liver showed an enhancement in some parts of the tissue, because of the effect of the extract in reducing blood lipid. By the way, long-term treatment is required for regeneration the whole tissue.
In conclusion, ethanolic DCBR extract has demonstrated significant antidiabetic and antihyperlipidemic activity. This effect is dose and time dependent. This effect is may be due to the presence of flavonoids in the whole extract, which are working by different mechanisms. However, further studies are recommended to isolate and elucidate the bioactive compound(s) responsible for its antidiabetic and antihyperlipidemic activity and its molecular mechanism of action.