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Effects of Cymodocea nodosa extract on metabolic disorders and oxidative stress in alloxan-diabetic rats
Biomedicine & Pharmacotherapy 89 (2017) 257–267
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Original article
Effects of Cymodocea nodosa extract on metabolic disorders and oxidative stress in alloxan-diabetic rats Rihab Ben Abdallah Kolsia,* , Hichem Ben Salahb , Neila Jardakc , Rim Chaabend, Abdelfattah El Fekie, Tarak Rebaic , Kamel Jamoussid , Noureddine Alloucheb , Hafedh Belghithf , Karima Belghitha a
Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, 3038 Sfax, Tunisia Laboratory of Chemistry of Natural Substances, Faculty of Sciences of Sfax, PB 802, 3018 Sfax, Tunisia c Histology, Orthopaedic and Traumatology Laboratory Sfax Faculty of Medicine, Sfax, Tunisia d Biochemistry Laboratory, CHU Hedi Chaker, Sfax, Tunisia e Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, Tunisia, f Enzyme and Bioconversion Unit, Biotechnology Center of Sfax, Tunisia b
A R T I C L E I N F O
Article history: Received 29 November 2016 Received in revised form 30 January 2017 Accepted 10 February 2017 Keywords: Cymodoces nodosa LC–ESI–MS/MS a-Amylase Diabetes complication
A B S T R A C T
This new study aimed to evaluate for the first time the effect of Cymodocea nodosa extract (CNE) on a-amylase activity, hyperglycemia and diabetes complications in the alloxan-induced diabetic rats. The in vitro evaluation and oral administration of CNE to surviving diabetic rats inhibited key enzyme related to hyperglycemia as a-amylase, helped to protect the b cells of the rats from death and damage confirmed by oral glucose test tolerance (OGTT), which leads to decrease in blood glucose level by 49% as compared to untreated diabetic rats. The CNE also decreased the triglyceride, low density lipoprotein (LDL) cholesterol and total cholesterol rates in the plasma of diabetic rats by 46%, 35%, and 21%, respectively, and increased the high density lipoprotein (HDL) cholesterol level by 36%, which helped maintain the homeostasis of blood lipid. When compared to those of the untreated diabetic rats, the superoxide dismutase, catalase, and glutathione peroxidase levels in the pancreas, liver and kidney of the rats treated with this supplement were also enhanced significantly. Moreover, a significant decrease was observed in the lipid peroxidation level in the tested organs of diabetic rats after CNE administration. This positive effect of CNE was confirmed by histological study. Overall, the findings presented in this study demonstrate that CNE has both a promising potential with a valuable hypoglycemic and hypolipidemic functions. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a chronic and complex metabolic disorder characterized by hyperglycemia, insulin resistance, and relative insulin deficiency [1]. It represents one of the leading causes of mortality and morbidity worldwide. It is a major and increasing health problem that affects millions of people from all gender and age groups worldwide. Chronic elevation of blood glucose will eventually lead to tissue damage which can be found in many organ and systems [2,3]. The generation of free radical and oxidative stress play great role in the pathogenesis of diabetes and its late complications [4].
* Corresponding author. E-mail address: [email protected] (R. Ben Abdallah Kolsi). http://dx.doi.org/10.1016/j.biopha.2017.02.032 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Free radicals can be formed in diabetic animals, and induce oxidative stress, which may impair function of liver and kidney, in which a significant decrease in antioxidant enzyme activities (GSH-Px, SOD, CAT) and antioxidant GSH level, and lipid peroxidation level is increased [5]. Renewed attention in recent decades to alternative medicines and natural therapies has stimulated a new wave of interest in ethnomedicine. The plant kingdom has become a target for the search for new drugs and biologically active compounds [6] because both insulin and oral hypoglycemic drugs possess undesirable side effects [7]. Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes [8,9], but only a few have received scientific scrutiny. To our knowledge, so far no other investigations has been carried out on Cymodocea nodosa (C. nodosa) regarding to its use in traditional medicine.
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This marine plant is phanerogam in Mediterranean and Aegean Sea with their foremost role in marine ecosystem dynamics such as providing food and shelter source for several species, while stabilizing the sea floor [10]. In the present study, we have investigated the effect of Cymodocea nodosa extract (CNE) on hyperglycemia and diabetes complications, considering its antioxidant properties as well as pancreatic islet architecture and its effects on liver-kidney functions in alloxan-induced diabetes rats. 2. Materials and methods 2.1. Plant material and preparation of C. nodosa extract C. nodosa sample were collected from Sfax-Chebba coastal area (southeast of Tunisia) in spring 2014, washed thoroughly with sea water and then with fresh water in order to remove the sand particles and epiphytes, milled in a mechanical grinder for 5 min, to obtain a fine and homogeneous powder and then were stored in hermetic bags at room temperature (25 C). The extraction was carried out using a mixture of ethanol and water (200 ml, 4:1 v/v) was added to the 50 g of C. nodosa powder and kept under agitation for 24 h. Subsequently, the solution was filtered using Whatman No 3 filter paper. The extract was concentrated by evaporation to dryness at 40 C, and the residue obtained was stored in glass vials, at 4 C in the dark until further use. 2.2. Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) analysis A reverse-phase high-performance liquid chromatography technique was developed to identify and quantify the major compounds contained in the CNE. Concentrations were calculated based on peak areas compared to those of external standards. The HPLC chromatograph was a Schimadzu apparatus equipped with a LC-10ATvp pump and a SPD-10Avp detector. The column was (4.6 mm 250 mm) (Shim-pack, VP-ODS) and the temperature was maintained at 40 C. The flow rate was 0.3 ml/min. The mobile phase used was 0.1% phosphoric acid in water (A) versus 70% acetonitrile in water (B) for a total running time of 40 min, and the gradient changed as follows: solvent B started at 20% and increased immediately to 50% in 30 min. After that, elution was conducted in the isocratic mode with 50% solvent B within 5 min. Finally, solvent B decreased to 20% until the end of running time. 2.3. In vitro a-amylase inhibitory assay The assay mixture containing 200 ml of 0.02 M sodium phosphate buffer, 20 ml of a porcine pancreatic a-amylase enzyme and the CNE in concentration range 25, 50, 100 and 200 mg/ml were incubated for 10 min at room temperature followed by addition of 200 ml of starch in all test tubes. The reaction was terminated with the addition of 400 ml DNS reagent and placed in boiling water bath for 5 min, cooled and diluted with 15 ml of distilled water and absorbance was measured at 540 nm. The control sample was prepared without any extract. The% inhibition was calculated according to the formula of Ben Abdallah Kolsi et al. [11] Inhibitionð%Þ ¼
Abs540ðControlÞAbs540ðExtractÞ 100 Abs540ðControlÞ
The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by no linear regression analysis from the mean inhibitory values.
Acarbose was used as specific inhibitor of a-amylase. All tests were performed in triplicate. 2.4. Acute toxicity study The diabetic rats were divided into six groups of seven animals each. Group 1 served as control, received a volume of 10 ml/kg of body weight (bw) of NaCl 0.9% orally. Groups 2, 3, 4, 5 and 6 were administered respectively at the doses of 100, 200, 500, 1000 and 2000 mg/kg bw orally in a volume of 10 ml/kg. After the oral administration of CNE, animals were observed individually at least once every 30 min during the first 48 h, periodically. 2.5. Animals Male Wistar rats, with body weight of 180–200 g and bred in the Central Animal House and obtained from the Central Pharmacy, Tunisia, were used in this study. The animals were maintained in a controlled environment and under standard conditions of temperature and humidity with an alternating light-dark cycle. The handling of the animals was approved by the Tunisian Ethical Committee for the care and use of laboratory animals. 2.6. Induction of diabetes in experimental animals Rats were injected intraperitoneally with a freshly prepared solution of alloxan monohydrate dissolved in a normal saline solution of NaCl 0.9% pH 4 at a dose of 150 mg/kg bw [11]. This injection can provoke fatal hypoglycemia as a result of reactive massive release of pancreatic insulin, rats were also given orally 5– 10 ml of a 20% glucose solution after 6 h. Rats were then kept for the next 24 h on a 5% glucose solution as beverage to prevent too severe hypoglycaemia [12]. After 2 weeks, rats displaying glycosuria and hyperglycemia (blood glucose levels 2 g/l) were retained for the experiments. 2.7. Experimental procedure, body weight and blood glucose measurement Four groups of rats, seven in each received the following treatment schedule. Group I:(Control) Normal rats. Group II: (Diab) Alloxan-diabetic rats. Group III: (Diab + CNE) Alloxan-diabetic rats received 200 mg/ kg bw of CNE [13]. Group IV: (Diab + Acar) Alloxan diabetic rats treated with 5 mg/ kg bw of (acarbose) [13]. The fasting body weight and blood glucose levels were estimated on 1, 7, 14, 21 and 28 days periodically. 2.8. Glucose tolerance test Three days before decapitation of rats, the oral glucose tolerance test (OGTT) was measured; all groups were fasted overnight. Control animals were given 1 ml of distilled water orally, all other groups were administrated glucose 2 g/kg/bw orally by gastric gavages route. Blood glucose levels were determined at 0, 30, 60, 120 and 240 min subsequent to received glucose and fasting glucose was measured. Blood samples were collected from a small cut in the tail vein and blood glucose levels were measured automatically with the aid of a one touch glucometer (Bionime, Pharmatec, Tunisia) by glucose oxidase-peroxidase method using strips [7]. Results achieved were taken for evaluating short- and long-term effects of CNE on diabetic rats.
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After 30 days of oral treatment once a day, all animals were sacrificed by decapitation in order to minimize the handling stress and the trunk blood collected. Pancreas, liver and kidney were rapidly removed and cleaned of fat; all these samples were stored at (80 C) until used. 2.9. Biochemical assays The analyses of serum lipase were performed to determine the serum level of a specific protein (enzyme) involved in digestion, and lipids levels of triglycerides (TG), total cholesterol (T-Ch), low density lipoprotein cholesterol (LDL-Ch), high density lipoprotein cholesterol (HDL-Ch) were also explored. Serum LDL-cholesterol
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concentration was determined according to formula Shertzer et al. [14]: LDL cholesterol = total cholesterol– (triglycerides/5) –HDL cholesterol. The levels of glucose and a-amylase activity in the serum were measured using the commercial kits (Biolabo, France) on an automatic biochemistry analyzer (BS 300, China) at the biochemical laboratory of Hedi Chaker Hospital of Sfax. Serum activity of aspartate and alanine aminotransferase (AST, ALT), lactate dehydrogenase (LDH), creatinine, albumin and urea rates were measured by standardized enzymatic procedures using commercial kits from (Biolabo, Lyon, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) at the biochemical
Fig. 1. HPLC–MS chromatogram at 250 nm and chemical structures of the flavonoids identified from ethanolic extract of C. nodosa .: catechin (3), quercetin-3-O-rutinoside (6), quercetin-3-O-glucoside (7), isorhamnetin-3-O-rutinoside (8).
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laboratory of Hedi Chaker Hospital of Sfax. The antioxidant activities were measured, after the homogenization of the pancreas, liver and kidney in a phosphate buffer. The level of total protein was determined by the method of Lowry et al. [15], using bovine serum albumin as the standard at 660 nm. The lipid peroxidation was assessed in the sera of various organs of rats experimentally used as control and all treated groups of animals by the quantification of thiobarbituric acid reactive substances (TBARS) using the method of Yoshioka et al. [16]. The activity of superoxide dismutase (SOD) was assayed by the spectrophotometric method of Marklund and Marklund [17] and expressed as U/ mg protein. The glutathione peroxidase (GPX) activity was measured by the method described by Paglia et al. [18] and expressed as mmoles GSS/(min mg protein). The catalase (CAT) activity was determined by adopting the method of Mueller et al. [19] at 240 nm and expressed as mmoles of H2O2 consumed/ (min mg protein).
Compound 3 displayed UV maximum absorption at 276 nm and gave [MH] ion at m/z 289. It was identified as catechin [20,21]. Compounds 4 (tR 4.3 min; lmax 323 nm) and 5 (tR 5.2 min; lmax 276 nm) were tentatively assigned as ferulic and cinnamic acid derivatives, respectively according to their [MH] ions (m/z 385 and 293, respectively). They produced characteristic MS2 ions at m/ z 193 [ferulic acid-H] and at m/z 147 [cinnamic acid-H]. Compounds 6 (tR 8.3 min) and 7 (tR 10.2 min) displayed two maximum UV absorptions at 254 and 354 nm and exhibited the same MS2 ion at m/z 301 which corresponds to quercetin derivatives. Moreover, these two compounds showed parent ions [MH] at m/z 609 and at m/z 463. Thus, they were identified as quercetin-3-O-rutinoside and quercetin-3-O-glucoside [21]. Compound 8 released MS2 ions at m/z 315 and 300 assigned to isorhamnetin derivative. Furthermore, this compound showed molecular ion [MH] at m/z 623, therefore, it was characterized as isorhamnetin-3-O-rutinoside [21].
2.10. Histological analyses
3.2. In vitro a-amylase inhibitory assay
Classical procedure was used for histology. After fixation in Bouin solution, pieces of fixed tissue were embedded into paraffin, cut into 5 mm slices and colored with hematoxyline-eosine.
One of the therapeutic approaches to decrease hyperglycemia is to delay the absorption of glucose by inhibiting the hydrolysis of carbohydrates such as amylolytic enzymes (a-amylase), in the gastrointestinal tract [22]. It is generally recognized that carbohydrates are digested into oligosaccharides by a-amylase and then digested by gluco-amylase (alpha-dextrinases) to maltose and maltotriose. Our in vitro studies demonstrated an appreciable a-amylase inhibitory activity of CNE was concentration dependent with a maximum of 73.03% (Table 2). The IC50 values show that the tested extract (65.22 mg/ml) has a lower potency over a-amylase compared to the acarbose specific inhibitor (19.40 mg/ml). Therefore, partial but significant inhibition of a-amylase by the CNE could help to modulate the release rate of glucose from starch, whereas the inhibition of a-amylase plays a crucial role in the stage of starch digestion resulting in decreasing the release of
2.11. Statistical analysis Data are presented as means standard deviation (SD). Statistical significance was assessed by the Fisher’s test. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) analysis of CNE The phenolic constituents of the CNE were analyzed for the first time by HPLC-DAD-ESI/MS2. Their retention time, absorbance spectrum and MS/MS spectra were compared with those of candidate phenolic compounds reported in previous literature. Eight phenolic compounds were detected and tentatively identified. The HPLC-UV/DADchromatogram at 254 nm is illustrated in Fig. 1. The retention times (tR), UV values (lmax) and the molecular ions of the phenolic compounds are summarized in Table 1. In this study, four flavonoids (3, 6, 7 and 8) and three phenolic acid derivatives (2, 4 and 5) were characterized from the CNE. Compound 1 (tR 3.2 min; lmax 330 nm) gave a molecular ion [MH] at m/z 391. Its MS2 spectrum presented two fragment ions at m/z 216 and m/z198, however, the compound 1 could not be identified. Compound 2 (tR 3.6 min; lmax 314 nm) was tentatively identified as sinapic acid derivative, whose identities were assigned based on their pseudo molecular ions at m/z 223 [sinapic acid-H] and at m/z 179 [sinapic acid-H-CO2].
Table 2 In vitro a-amylase inhibition of Cymodocea nodosa extract and acarbose commercial drug. Concentration(mg/ml)
% of inhibition
CNE
25 50 100 200
32.38 0.11 40.16 0.01 52.29 0.09 73.03 0.08
Acarbose
5 10 20 40
13.27 0.06 29.80 0.08 51.52 0.02 86.62 0.03
IC50 value (mg/ml) 65.22 mg/ml
19.40 mg/ml
Values are expressed as mean S.D. for three independent determinants.
Table 1 Identification of phenolic compounds of Cymodocea nodosa extract by HPLC-DAD-ESI/MS2 Peak
tR (min)
HPLC-DAD lmax (nm)
[MH] (m/z)
Main fragment ions MS2 (m/z)
Molecular formula
Tentative identification
1 2 3 4 5 6 7 8
3.2 3.6 3.8 4.7 8.3 9.3 12.0 12.7
330 314 278 323 276 255; 356 254; 354 255; 355
391 391 289 385 293 609 463 623
198; 216; 276 223; 179 245; 205 193; 176; 240 147 301; 300; 271 301; 343 315; 300; 271
– – C15H14O6 – – C27H30O16 C21H19O12 C28H32O16
Unknown Snapinic acid derivative Catechin Ferrilic acid derivative Cinnamic acid derivative Quercetin-3-O-rutinoside Quercetin-3-O-glucoside Isorhamnetin-3-O-rutinoside
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glucose, but his complete inhibition is undesirable because it could cause intestinal disorders [23]. The appreciable a-amylase inhibitory activity of the CNE could be associated with the presence of phenolic compounds specially flavonoids. Several reports revealed that the activity of a –amylase is effectively inhibited by flavonoids, such as naringenin, quercetin-3-O-rutinoside, luteolin, catechin, quercetin-3-O-glucoside [24,25], indicating that these phenolic compounds are able to inhibit the activities of carbohydrate-hydrolysing enzymes, due to their ability to bind with proteins [26]. Therefore, our extract can be considered a new natural source possessing properties for the fight against type 2 diabetes. To show equal preference for a-amylase enzyme it is always necessary to do the corresponding in vivo activity. Thus proof of concept needs to be demonstrated in preclinical animal studies, it was essential to confirm the in vivo experiments action following oral administration to live animals. 3.3. Effect of CNE on body weight and blood glucose levels The results presented in Table 3 indicated that diabetes induced a 52% loss of body weight compared with the control group. The oral administration of CNE and acarbose induced a significant (P < 0.05) increase in body weight by 69.45% and 73.91%, respectively, accompanied by a significant (P < 0.05) decrease of the blood glucose levels by 49.64% and 54.22% respectively after 4 weeks of treatment compared with the rats from the untreated diabetic group (Table 3). This reduction on body weight is related to continues lipolysis by a lack of insulin due to the installation of the diabetes and the degradation of structural proteins by disruption of the metabolism of carbohydrates [27]. While the moderation is due to the control of hyperglycemia and activation of the synthesis of structural proteins [28], where blood glucose levels became normal and stable, what shows the positive effect of treatment. 3.4. Effect of CNE on OGTT The periodic time course of changes observed in blood glucose levels during the oral glucose tolerance test (OGTT) (0–240 min) shows that the blood glucose levels of normal rats were noted to increase to a maximum of 1.30 g/l after 60 min of oral glucose challenge and to return to normal levels after 240 min (Fig. 2). The administration of 2 g/ml glucose to untreated diabetic rats induced an increase in their blood glucose levels, with peak of 3.25 g/l. The administration of CNE and acarbose, on the other hand, decreased the glucose peak in blood glucose levels to 1.67 and 1.38 g/l, respectively, significantly (P < 0.05) compared to diabetic rats. Glucose was probably attached to the OH functional groups of the phenolic compounds of CNE and the standard drug to reduce the glucose levels. In this study, the results suggest the usefulness of C. nodosa for further development as a therapeutic agent for impaired glucose tolerance and diabetes mellitus.
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The same effect has been shown by Posidonia oceanica is a widely allocated phanerogam in Mediterranean and Aegean Sea [7]. This which clearly shows a very active use of glucose by peripheral tissues, explained by an increase in glucose tolerance in these tissues when animals are treated with some marine plants. 3.5. Effect of CNE on a-amylase activity, serum glucose levels and pancreas architecture Type 1 diabetes is a progressive auto-immune disease wherein the b-cells producing insulin are destroyed by the immune system. It is characterized by the accumulation of lymphocytes, which infiltrate and destroy pancreatic islets, thereby causing carbohydrate metabolism disorders associated with high serum glucose levels or hyperglycaemia [5]. The results of the present study indicated that CNE administration to surviving diabetic rats induced considerable reductions in the plasma and intestine a-amylase activity by 26.74% and 47.11%, respectively, as compared to the untreated diabetic rats (Fig. 3). The oral treatment of surviving diabetic rats by CNE stimulated the b-cells regeneration in the pancreas, which led to an increase in insulin secretion. Both a-amylase activity inhibition and pancreatic b-cells protection from damage and death (Fig. 4C) induced a decrease in the blood glucose level by 45.66% as compared to the untreated group of diabetic rats (Fig. 5). One of the therapeutic strategies commonly used for the decrease of postprandial hyperglycemia in non insulin dependent diabetic topics is retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing the amylolytic enzymes (a-amylase), in the digestive tract [22]. Moreover, the CNE inhibited this key enzyme responsible for the conversion of starch to simple sugars absorbable by the small intestine by completely blocking access to the active site of the a-amylase. Increasing the activity of this enzyme observed at different levels leads necessarily to increase the intestinal absorption of glucose and accumulation in the bloodstream. It has been shown that treatment with the CNE and acarbose possess the ability to entrap glucose molecules in the blood and slow down their absorption, which leads to the hypoglycemic effect (Fig. 5) that could be explained by three reasons: (1) protection of pancreatic cells from progressive damage enhanced by alloxan, (2) the stimulation of Langerhans islets and (3) improvement of peripheral sensitivity to remnant insulin and antioxidant properties of C. nodoa. The results of the present study indicated also that the injection of alloxan to rats induced b-cell damage, with depleted islet cells of Langerhans (atrophy), cellular degeneration, pancreatic injury, and a clear decline in the area occupied by b-cells. The administration of CNE to surviving diabetic rats was noted to protect the pancreatic b-cells from death and damage and to induce more insulin secretion in the blood, which enhanced metabolism and body weight.
Table 3 Effect of Cymodocea nodosa extract on body weight and blood glucose in Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Body weight
Control Diab Diab + CNE Diab + Med
Blood glucose
Before treatment
After treatment
Before treatment
After treatment
167.35 4.13 170.23 3.80 159.12 4.51 162.09 3.67
249.52 6.19 119.68 5.33 202.80 2.77 208.14 5.06
0.79 1.58 2.79 2.81 2.64 0.77 2.55 2.25
0.84 0.91 2.84 3.16 1.43 0.89 1.30 4.02
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Glucose level in serum (g/l)
Cont
Diab
Diab+CNE
Diab+Med
4 3.5 3 2.5 2 1.5 1 0.5 0
0
30
60
120
240
Time (min) Fig. 2. Effects of Cymodocea nodosa extract on glucose tolerance. Glucose levels obtained from Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Acar: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05.
Fig. 3. Inhibitory effects of Cymodocea nodosa extract on alpha-amylase activity in the serum (A) and intestine (B) in Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Acar: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. aP < 0.05 compared with normal control rats; bP < 0.05, compared with diabetic rats and cP < 0.05 compared with treated rats.
Furthermore, some marine plants extracts are known for their hypoglycemic ability, the properties of marine origin have been extensively investigated in the literature [7,29]. The data in Table 4 show a significant increase in serum indices indicating liver dysfunction in untreated diabetic rats namely activities of AST, ALT and LDH by 21.14%, 28.65% and 7.89%, respectively, compared with the normal rats (P < 0.05). Generally, diabetes mellitus is associated with hyperlipidaemia. For this reason, we have evaluated the lipid parameters which is of crucial importance through its use in the treatment of several cardiovascular diseases and diabetes control [30]. We found a significant increase in total cholesterol (T-Ch), cholesterol low density lipoprotein (LDL-Ch) and triglycerides (TG) by 16.79%, 34.54% and 78.88%, respectively associated with a significantly lower rate of high density lipoproteins (HDL-Ch) in serum (87.5%) of the untreated diabetic rats compared with controls (Table 4). This observed hyperlipidemia can be explained by the degradation of the lipid compounds or fatty tissue by insulin deficiency that inhibits 3-hydroxy 3-methyl glutamyl coenzyme A reductase
(HMG-CoA reductase) involved in the biosynthesis of cholesterol [31]. The administration of CNE was also noted to decrease hepatic toxicity and to regulate the lipid profile as evidenced by the decrease observed for the AST, ALT, LDH, LDL-C, T-Ch and TG levels and the increase recorded for HDL-C (Table 4). This action of CNE supports its lipid lowering activity in diabetic condition and therefore it helps to prevent diabetic associated complication. These results are in agreement with previous findings in the literature, concerning marines phanerogams distributed in the Mediterrenean Sea, was found to decrease this biochemicals parameters levels and prevent its oxidation in vivo [7,29]. Hyperglycemia causes non-enzymatic glycation of proteins following the Maillard reaction and alters energy metabolism, which may lead to high rates ROS and the development of diabetes complications [32]. The oxidative stress for diabetes also includes redox imbalance from altered metabolism of lipids, carbohydrates and decrease of antioxidants defense. The results revealed that the hepatic tissues of diabetic rats underwent a decrease in SOD, CAT and GPX activities and an
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Fig. 4. Histological study of pancreas from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats, massive destruction of b-cells; (C) alloxan-diabetic rats,treated with CNE; (D) alloxan-diabetic rats treated with acarbose medicament, a partial protector effect of b-cells from pancreas was observed (H&E 100).
increase in TBARS levels. The rate of reactive substances with thiobarbituric acid (TBARS) was determined at the level of the organ tested because they are used to indicate the presence of a lipid peroxidation resulting from the establishment of an oxidative stress. This perturbations were restored significantly (p < 0.05) by the administration of CNE and acarbose (Table 5), which shows the beneficial effect of treatment able to mitigate oxidative stress and enhance antioxidant defenses in diabetic rats. In fact, the histological analysis of liver sections taken from diabetic rats showed lipid droplets in the cytoplasm of hepatic tissues due to the deficiency in insulin, responsible for metabolizing fats (Fig. 6B). These results are consistent with several other reports on experimentally induced diabetes in animal models [33,34]. The positive effects of CNE and acarbose were further evidenced by the decreased prevalence and accumulation of fat cells in the liver tissues (Fig. 6C, D). 3.6. Effect of CNE on renal dysfunction parameters and architecture The variations of parameters of kidney toxicity in experimental rats were highlighted by the study of biochemical and oxidative stress parameters (Table 4 and 5) and confirmed by histological study (Fig. 7). Hyperglycaemia induced an increase in albumin,
creatinine and urea contents in blood plasma by 21.59%, 37.39% and 104.23%, respectively and a decrease of SOD, CAT and GPX activities in the kidney of diabetic rats by 61.92%, 34.74% and 73.45%, respectively compared with the control rats. An increase of 362.79% in TBARS levels was also observed. Furthermore, a marked decrease in kidney toxicity was observed following CNE treatment, which was evidenced by 14.48%, 27.95% and 31.61% decrease in the biochemical parameters (Table 4), however, noted to increase the SOD, CAT and GPX activities in the kidney by 109.95%, 48.33% and 211.65% respectively and to decrease the TBARS level by 71.35% compared with untreated diabetic rats (Table 5). The significant increase in the concentration of biochemical indices in the serum of untreated diabetic rats compared to control rats. It can be explained by the accelerated protein degradation liver and plasma [35] or to the degradation of some compounds protein of the body due to the administration of alloxan or compounds made by food, which can be degraded into amino acids and urea. Kidney is the most important organ, which play a pivotal role in regulating various physiological processes in the body. Damage to the kidney inflicted by nephrotoxin agents is of grave consequences [5]. In diabetic animals, free radicals can rapidly accumulated and form oxidative stress, which may impair function of kidney, in which significantly decreased antioxidant activities. Persistent
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Fig. 5. Effects of Cymodocea nodosa extract on serum glucose levels in Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. aP < 0.05 compared with normal control rats; bP < 0.05, compared with diabetic rats and cP < 0.05 compared with treated rats.
Table 4 Effects of administration of Cymodocea nodosa extract on liver-kidney toxicity and lipid profile in blood of Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Control
Diab
Diab + CNE
Diab + Med
Liver-toxicity AST (UI/l) ALT (UI/l) LDH (UI/l)
155.42 46.93b 50.85 6.69b 676.42 172.48b
188.28 23.38a,c 65.42 11.75a,c 729.85 185.55a,c
166.57 55.34b 59.00 11.71a,b 649.28 163.55a,b
161.42 31.06b 58.14 6.12a,b 662.42 108.61a,b
Kidney-toxicity Albumin (g/l) Urea (mmol/l) Creatinine (mmol/l)
32.42 5.22b 5.90 0.48b 25.70 3.90b
39.42 2.65a,c 12.05 1.75a,b 35.31 5.93a,c
33.71 5.46b 8.24 1.03a,b 25.44 4.01b
32.57 2.81b 8.30 1.45a,b 15.82 5.04b
Lipid profile T G (mmol/l) T-Ch (mmol/l) LDL-Ch (mmol/l) HDL-Ch (mmol/l)
0,90 0,29b 1.31 0.21b 0.55 0.18b 0.32 0.06b
1.61 0.40a 1.53 0.34a,c 0.74 0.23a,c 0.60 0.11a,c
1.31 0.50a,b 1.34 0.21b 0.5 0.08b 0.40 0.05a,b
1.39 0.11b 1.35 0.1a,b 0.42 0.10a,b 0.39 0.08a,b
Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. a P < 0.05 compared with normal control rats. b P < 0.05, compared with diabetic rats and. c P < 0.05 compared with traited rats.
hyperglycaemia leads to increased production of free radicals [36]. In our study, the activities of SOD, CAT and GPX decreased in diabetic rats as reported earlier which could be due to inactivation caused by alloxan-generated ROS. Several reports indicate that the compounds responsible for antioxidative activity of marine plants are mainly phenolic diterpenes such as carnosoic acid, carnosol, rosmanol [37], and other phenolic acids, such as rosmarinic and caffeic acids [38]. It is possible that the C. nodosa extract due to its presence of several bioactive antioxidant principles and their synergistic properties may be caused an improving effect in antioxidant status of diabetic rats. The negative action of hyperglycaemia on the kidney functions is found in perfect correlation with histological study showed an increase in Bowman’s space and glomerular atrophy in the kidneys of diabetic rats (Fig. 7B). On the other hand the administration of
CNE and acarbose to diabetic rats also regulated the Bowman’s space size and prevented glomerular atrophies and cortical structure but with some alterations relatively less significant to the diabetic rats (Fig. 7C, D). 4. Conclusion The present work is the first investigation dealing with the hypoglycemic and protective effects of CNE in alleviating oxidative stress and free radicals as well as in enhancing enzymatic defenses in diabetic rats. The results from the present study confirm the possibility that the major function of the CNE is on the protection of vital tissues pancreas, kidney and liver and reducing the complications usually resulting from oxidative stress in diabetes mellitus.
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Table 5 Effects of Cymodocea nodosa extract administration on SOD, CAT, GPX and TBARS levels in liver, pancreas and kidney in Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Groups Liver Control Diab Diab + CNE Diab + Med Pancreas Control Diab Diab + CNE Diab + Med Kidney Control Diab Diab + CNE Diab + Med
SOD (U/mg protein)
CAT (mmole H2O2/min/mg protein)
GPX (mmol GSH/min/mg protein)
TBARS (nmol/mg protein)
22.16 0.16b 11.02 0.39a,c 20.08 0.01b 19.45 1.09b
42.15 0.64b 23.31 0.49a,c 44.07 0.64b 39.09 0.47a,b
4.20 0.02b 0.96 0.01a,c 3.72 0.02a,b 3.19 0.08a,b
0.84 0.01b 2.39 0.01a,c 0.94 0.27b 0.90 0.48b
19.45 0.02b 9.95 0.95a,c 18.41 0.03b 17.58 0.03a,b
32.02 0.04b 19.71 0.05a 26.43 0.03a,b 28.69 0.02a,b
2.15 0.03b 0.54 0.01a,c 1.94 0.11a,b 2.04 0.03b
0.65 0.01b 2.74 0.02a,c 0.59 1.16b 0.50 0.01a,b
12.66 0.02b 4.82 0.03a,c 10.12 0.61a,b 10.26 0.02a,b
50.22 0.03b 32.77 0.02a,c 48.61 0.61b 49.41 0.41b
6.14 0.02b 1.63 0.01a,c 5.08 0.02a,b 5.55 0.02a,b
0.43 0.01b 1.99 0.02a,c 0.57 0.01a,b 0.62 0.02a,b
Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. a P < 0.05 compared with normal control rats. b P < 0.05, compared with diabetic rats and. c P < 0.05 compared with traited rats.
Fig. 6. Histological comparison of liver from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats, appearance of lipid accumulation in liver cells; (C) alloxan-diabetic rats treated with CNE and (D) alloxan-diabetic rats treated with acarbose medicament, a decrease in the abundance of lipid accumulation in liver cells was showed (H&E 100).
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Fig. 7. Histological comparison of kidneys from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats showed Bowman’s space size and atrophy of glomerulus; (C) alloxan-diabetic rats treated with CNE and (D) alloxan-diabetic rats treated with acarbose medicament, a protector effect was showed (H&E 100).
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Original article
Effects of Cymodocea nodosa extract on metabolic disorders and oxidative stress in alloxan-diabetic rats Rihab Ben Abdallah Kolsia,* , Hichem Ben Salahb , Neila Jardakc , Rim Chaabend, Abdelfattah El Fekie, Tarak Rebaic , Kamel Jamoussid , Noureddine Alloucheb , Hafedh Belghithf , Karima Belghitha a
Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, 3038 Sfax, Tunisia Laboratory of Chemistry of Natural Substances, Faculty of Sciences of Sfax, PB 802, 3018 Sfax, Tunisia c Histology, Orthopaedic and Traumatology Laboratory Sfax Faculty of Medicine, Sfax, Tunisia d Biochemistry Laboratory, CHU Hedi Chaker, Sfax, Tunisia e Laboratory of Animal Ecophysiology, Faculty of Sciences of Sfax, Tunisia, f Enzyme and Bioconversion Unit, Biotechnology Center of Sfax, Tunisia b
A R T I C L E I N F O
Article history: Received 29 November 2016 Received in revised form 30 January 2017 Accepted 10 February 2017 Keywords: Cymodoces nodosa LC–ESI–MS/MS a-Amylase Diabetes complication
A B S T R A C T
This new study aimed to evaluate for the first time the effect of Cymodocea nodosa extract (CNE) on a-amylase activity, hyperglycemia and diabetes complications in the alloxan-induced diabetic rats. The in vitro evaluation and oral administration of CNE to surviving diabetic rats inhibited key enzyme related to hyperglycemia as a-amylase, helped to protect the b cells of the rats from death and damage confirmed by oral glucose test tolerance (OGTT), which leads to decrease in blood glucose level by 49% as compared to untreated diabetic rats. The CNE also decreased the triglyceride, low density lipoprotein (LDL) cholesterol and total cholesterol rates in the plasma of diabetic rats by 46%, 35%, and 21%, respectively, and increased the high density lipoprotein (HDL) cholesterol level by 36%, which helped maintain the homeostasis of blood lipid. When compared to those of the untreated diabetic rats, the superoxide dismutase, catalase, and glutathione peroxidase levels in the pancreas, liver and kidney of the rats treated with this supplement were also enhanced significantly. Moreover, a significant decrease was observed in the lipid peroxidation level in the tested organs of diabetic rats after CNE administration. This positive effect of CNE was confirmed by histological study. Overall, the findings presented in this study demonstrate that CNE has both a promising potential with a valuable hypoglycemic and hypolipidemic functions. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is a chronic and complex metabolic disorder characterized by hyperglycemia, insulin resistance, and relative insulin deficiency [1]. It represents one of the leading causes of mortality and morbidity worldwide. It is a major and increasing health problem that affects millions of people from all gender and age groups worldwide. Chronic elevation of blood glucose will eventually lead to tissue damage which can be found in many organ and systems [2,3]. The generation of free radical and oxidative stress play great role in the pathogenesis of diabetes and its late complications [4].
* Corresponding author. E-mail address: [email protected] (R. Ben Abdallah Kolsi). http://dx.doi.org/10.1016/j.biopha.2017.02.032 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Free radicals can be formed in diabetic animals, and induce oxidative stress, which may impair function of liver and kidney, in which a significant decrease in antioxidant enzyme activities (GSH-Px, SOD, CAT) and antioxidant GSH level, and lipid peroxidation level is increased [5]. Renewed attention in recent decades to alternative medicines and natural therapies has stimulated a new wave of interest in ethnomedicine. The plant kingdom has become a target for the search for new drugs and biologically active compounds [6] because both insulin and oral hypoglycemic drugs possess undesirable side effects [7]. Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes [8,9], but only a few have received scientific scrutiny. To our knowledge, so far no other investigations has been carried out on Cymodocea nodosa (C. nodosa) regarding to its use in traditional medicine.
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This marine plant is phanerogam in Mediterranean and Aegean Sea with their foremost role in marine ecosystem dynamics such as providing food and shelter source for several species, while stabilizing the sea floor [10]. In the present study, we have investigated the effect of Cymodocea nodosa extract (CNE) on hyperglycemia and diabetes complications, considering its antioxidant properties as well as pancreatic islet architecture and its effects on liver-kidney functions in alloxan-induced diabetes rats. 2. Materials and methods 2.1. Plant material and preparation of C. nodosa extract C. nodosa sample were collected from Sfax-Chebba coastal area (southeast of Tunisia) in spring 2014, washed thoroughly with sea water and then with fresh water in order to remove the sand particles and epiphytes, milled in a mechanical grinder for 5 min, to obtain a fine and homogeneous powder and then were stored in hermetic bags at room temperature (25 C). The extraction was carried out using a mixture of ethanol and water (200 ml, 4:1 v/v) was added to the 50 g of C. nodosa powder and kept under agitation for 24 h. Subsequently, the solution was filtered using Whatman No 3 filter paper. The extract was concentrated by evaporation to dryness at 40 C, and the residue obtained was stored in glass vials, at 4 C in the dark until further use. 2.2. Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) analysis A reverse-phase high-performance liquid chromatography technique was developed to identify and quantify the major compounds contained in the CNE. Concentrations were calculated based on peak areas compared to those of external standards. The HPLC chromatograph was a Schimadzu apparatus equipped with a LC-10ATvp pump and a SPD-10Avp detector. The column was (4.6 mm 250 mm) (Shim-pack, VP-ODS) and the temperature was maintained at 40 C. The flow rate was 0.3 ml/min. The mobile phase used was 0.1% phosphoric acid in water (A) versus 70% acetonitrile in water (B) for a total running time of 40 min, and the gradient changed as follows: solvent B started at 20% and increased immediately to 50% in 30 min. After that, elution was conducted in the isocratic mode with 50% solvent B within 5 min. Finally, solvent B decreased to 20% until the end of running time. 2.3. In vitro a-amylase inhibitory assay The assay mixture containing 200 ml of 0.02 M sodium phosphate buffer, 20 ml of a porcine pancreatic a-amylase enzyme and the CNE in concentration range 25, 50, 100 and 200 mg/ml were incubated for 10 min at room temperature followed by addition of 200 ml of starch in all test tubes. The reaction was terminated with the addition of 400 ml DNS reagent and placed in boiling water bath for 5 min, cooled and diluted with 15 ml of distilled water and absorbance was measured at 540 nm. The control sample was prepared without any extract. The% inhibition was calculated according to the formula of Ben Abdallah Kolsi et al. [11] Inhibitionð%Þ ¼
Abs540ðControlÞAbs540ðExtractÞ 100 Abs540ðControlÞ
The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by no linear regression analysis from the mean inhibitory values.
Acarbose was used as specific inhibitor of a-amylase. All tests were performed in triplicate. 2.4. Acute toxicity study The diabetic rats were divided into six groups of seven animals each. Group 1 served as control, received a volume of 10 ml/kg of body weight (bw) of NaCl 0.9% orally. Groups 2, 3, 4, 5 and 6 were administered respectively at the doses of 100, 200, 500, 1000 and 2000 mg/kg bw orally in a volume of 10 ml/kg. After the oral administration of CNE, animals were observed individually at least once every 30 min during the first 48 h, periodically. 2.5. Animals Male Wistar rats, with body weight of 180–200 g and bred in the Central Animal House and obtained from the Central Pharmacy, Tunisia, were used in this study. The animals were maintained in a controlled environment and under standard conditions of temperature and humidity with an alternating light-dark cycle. The handling of the animals was approved by the Tunisian Ethical Committee for the care and use of laboratory animals. 2.6. Induction of diabetes in experimental animals Rats were injected intraperitoneally with a freshly prepared solution of alloxan monohydrate dissolved in a normal saline solution of NaCl 0.9% pH 4 at a dose of 150 mg/kg bw [11]. This injection can provoke fatal hypoglycemia as a result of reactive massive release of pancreatic insulin, rats were also given orally 5– 10 ml of a 20% glucose solution after 6 h. Rats were then kept for the next 24 h on a 5% glucose solution as beverage to prevent too severe hypoglycaemia [12]. After 2 weeks, rats displaying glycosuria and hyperglycemia (blood glucose levels 2 g/l) were retained for the experiments. 2.7. Experimental procedure, body weight and blood glucose measurement Four groups of rats, seven in each received the following treatment schedule. Group I:(Control) Normal rats. Group II: (Diab) Alloxan-diabetic rats. Group III: (Diab + CNE) Alloxan-diabetic rats received 200 mg/ kg bw of CNE [13]. Group IV: (Diab + Acar) Alloxan diabetic rats treated with 5 mg/ kg bw of (acarbose) [13]. The fasting body weight and blood glucose levels were estimated on 1, 7, 14, 21 and 28 days periodically. 2.8. Glucose tolerance test Three days before decapitation of rats, the oral glucose tolerance test (OGTT) was measured; all groups were fasted overnight. Control animals were given 1 ml of distilled water orally, all other groups were administrated glucose 2 g/kg/bw orally by gastric gavages route. Blood glucose levels were determined at 0, 30, 60, 120 and 240 min subsequent to received glucose and fasting glucose was measured. Blood samples were collected from a small cut in the tail vein and blood glucose levels were measured automatically with the aid of a one touch glucometer (Bionime, Pharmatec, Tunisia) by glucose oxidase-peroxidase method using strips [7]. Results achieved were taken for evaluating short- and long-term effects of CNE on diabetic rats.
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After 30 days of oral treatment once a day, all animals were sacrificed by decapitation in order to minimize the handling stress and the trunk blood collected. Pancreas, liver and kidney were rapidly removed and cleaned of fat; all these samples were stored at (80 C) until used. 2.9. Biochemical assays The analyses of serum lipase were performed to determine the serum level of a specific protein (enzyme) involved in digestion, and lipids levels of triglycerides (TG), total cholesterol (T-Ch), low density lipoprotein cholesterol (LDL-Ch), high density lipoprotein cholesterol (HDL-Ch) were also explored. Serum LDL-cholesterol
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concentration was determined according to formula Shertzer et al. [14]: LDL cholesterol = total cholesterol– (triglycerides/5) –HDL cholesterol. The levels of glucose and a-amylase activity in the serum were measured using the commercial kits (Biolabo, France) on an automatic biochemistry analyzer (BS 300, China) at the biochemical laboratory of Hedi Chaker Hospital of Sfax. Serum activity of aspartate and alanine aminotransferase (AST, ALT), lactate dehydrogenase (LDH), creatinine, albumin and urea rates were measured by standardized enzymatic procedures using commercial kits from (Biolabo, Lyon, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) at the biochemical
Fig. 1. HPLC–MS chromatogram at 250 nm and chemical structures of the flavonoids identified from ethanolic extract of C. nodosa .: catechin (3), quercetin-3-O-rutinoside (6), quercetin-3-O-glucoside (7), isorhamnetin-3-O-rutinoside (8).
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laboratory of Hedi Chaker Hospital of Sfax. The antioxidant activities were measured, after the homogenization of the pancreas, liver and kidney in a phosphate buffer. The level of total protein was determined by the method of Lowry et al. [15], using bovine serum albumin as the standard at 660 nm. The lipid peroxidation was assessed in the sera of various organs of rats experimentally used as control and all treated groups of animals by the quantification of thiobarbituric acid reactive substances (TBARS) using the method of Yoshioka et al. [16]. The activity of superoxide dismutase (SOD) was assayed by the spectrophotometric method of Marklund and Marklund [17] and expressed as U/ mg protein. The glutathione peroxidase (GPX) activity was measured by the method described by Paglia et al. [18] and expressed as mmoles GSS/(min mg protein). The catalase (CAT) activity was determined by adopting the method of Mueller et al. [19] at 240 nm and expressed as mmoles of H2O2 consumed/ (min mg protein).
Compound 3 displayed UV maximum absorption at 276 nm and gave [MH] ion at m/z 289. It was identified as catechin [20,21]. Compounds 4 (tR 4.3 min; lmax 323 nm) and 5 (tR 5.2 min; lmax 276 nm) were tentatively assigned as ferulic and cinnamic acid derivatives, respectively according to their [MH] ions (m/z 385 and 293, respectively). They produced characteristic MS2 ions at m/ z 193 [ferulic acid-H] and at m/z 147 [cinnamic acid-H]. Compounds 6 (tR 8.3 min) and 7 (tR 10.2 min) displayed two maximum UV absorptions at 254 and 354 nm and exhibited the same MS2 ion at m/z 301 which corresponds to quercetin derivatives. Moreover, these two compounds showed parent ions [MH] at m/z 609 and at m/z 463. Thus, they were identified as quercetin-3-O-rutinoside and quercetin-3-O-glucoside [21]. Compound 8 released MS2 ions at m/z 315 and 300 assigned to isorhamnetin derivative. Furthermore, this compound showed molecular ion [MH] at m/z 623, therefore, it was characterized as isorhamnetin-3-O-rutinoside [21].
2.10. Histological analyses
3.2. In vitro a-amylase inhibitory assay
Classical procedure was used for histology. After fixation in Bouin solution, pieces of fixed tissue were embedded into paraffin, cut into 5 mm slices and colored with hematoxyline-eosine.
One of the therapeutic approaches to decrease hyperglycemia is to delay the absorption of glucose by inhibiting the hydrolysis of carbohydrates such as amylolytic enzymes (a-amylase), in the gastrointestinal tract [22]. It is generally recognized that carbohydrates are digested into oligosaccharides by a-amylase and then digested by gluco-amylase (alpha-dextrinases) to maltose and maltotriose. Our in vitro studies demonstrated an appreciable a-amylase inhibitory activity of CNE was concentration dependent with a maximum of 73.03% (Table 2). The IC50 values show that the tested extract (65.22 mg/ml) has a lower potency over a-amylase compared to the acarbose specific inhibitor (19.40 mg/ml). Therefore, partial but significant inhibition of a-amylase by the CNE could help to modulate the release rate of glucose from starch, whereas the inhibition of a-amylase plays a crucial role in the stage of starch digestion resulting in decreasing the release of
2.11. Statistical analysis Data are presented as means standard deviation (SD). Statistical significance was assessed by the Fisher’s test. P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) analysis of CNE The phenolic constituents of the CNE were analyzed for the first time by HPLC-DAD-ESI/MS2. Their retention time, absorbance spectrum and MS/MS spectra were compared with those of candidate phenolic compounds reported in previous literature. Eight phenolic compounds were detected and tentatively identified. The HPLC-UV/DADchromatogram at 254 nm is illustrated in Fig. 1. The retention times (tR), UV values (lmax) and the molecular ions of the phenolic compounds are summarized in Table 1. In this study, four flavonoids (3, 6, 7 and 8) and three phenolic acid derivatives (2, 4 and 5) were characterized from the CNE. Compound 1 (tR 3.2 min; lmax 330 nm) gave a molecular ion [MH] at m/z 391. Its MS2 spectrum presented two fragment ions at m/z 216 and m/z198, however, the compound 1 could not be identified. Compound 2 (tR 3.6 min; lmax 314 nm) was tentatively identified as sinapic acid derivative, whose identities were assigned based on their pseudo molecular ions at m/z 223 [sinapic acid-H] and at m/z 179 [sinapic acid-H-CO2].
Table 2 In vitro a-amylase inhibition of Cymodocea nodosa extract and acarbose commercial drug. Concentration(mg/ml)
% of inhibition
CNE
25 50 100 200
32.38 0.11 40.16 0.01 52.29 0.09 73.03 0.08
Acarbose
5 10 20 40
13.27 0.06 29.80 0.08 51.52 0.02 86.62 0.03
IC50 value (mg/ml) 65.22 mg/ml
19.40 mg/ml
Values are expressed as mean S.D. for three independent determinants.
Table 1 Identification of phenolic compounds of Cymodocea nodosa extract by HPLC-DAD-ESI/MS2 Peak
tR (min)
HPLC-DAD lmax (nm)
[MH] (m/z)
Main fragment ions MS2 (m/z)
Molecular formula
Tentative identification
1 2 3 4 5 6 7 8
3.2 3.6 3.8 4.7 8.3 9.3 12.0 12.7
330 314 278 323 276 255; 356 254; 354 255; 355
391 391 289 385 293 609 463 623
198; 216; 276 223; 179 245; 205 193; 176; 240 147 301; 300; 271 301; 343 315; 300; 271
– – C15H14O6 – – C27H30O16 C21H19O12 C28H32O16
Unknown Snapinic acid derivative Catechin Ferrilic acid derivative Cinnamic acid derivative Quercetin-3-O-rutinoside Quercetin-3-O-glucoside Isorhamnetin-3-O-rutinoside
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glucose, but his complete inhibition is undesirable because it could cause intestinal disorders [23]. The appreciable a-amylase inhibitory activity of the CNE could be associated with the presence of phenolic compounds specially flavonoids. Several reports revealed that the activity of a –amylase is effectively inhibited by flavonoids, such as naringenin, quercetin-3-O-rutinoside, luteolin, catechin, quercetin-3-O-glucoside [24,25], indicating that these phenolic compounds are able to inhibit the activities of carbohydrate-hydrolysing enzymes, due to their ability to bind with proteins [26]. Therefore, our extract can be considered a new natural source possessing properties for the fight against type 2 diabetes. To show equal preference for a-amylase enzyme it is always necessary to do the corresponding in vivo activity. Thus proof of concept needs to be demonstrated in preclinical animal studies, it was essential to confirm the in vivo experiments action following oral administration to live animals. 3.3. Effect of CNE on body weight and blood glucose levels The results presented in Table 3 indicated that diabetes induced a 52% loss of body weight compared with the control group. The oral administration of CNE and acarbose induced a significant (P < 0.05) increase in body weight by 69.45% and 73.91%, respectively, accompanied by a significant (P < 0.05) decrease of the blood glucose levels by 49.64% and 54.22% respectively after 4 weeks of treatment compared with the rats from the untreated diabetic group (Table 3). This reduction on body weight is related to continues lipolysis by a lack of insulin due to the installation of the diabetes and the degradation of structural proteins by disruption of the metabolism of carbohydrates [27]. While the moderation is due to the control of hyperglycemia and activation of the synthesis of structural proteins [28], where blood glucose levels became normal and stable, what shows the positive effect of treatment. 3.4. Effect of CNE on OGTT The periodic time course of changes observed in blood glucose levels during the oral glucose tolerance test (OGTT) (0–240 min) shows that the blood glucose levels of normal rats were noted to increase to a maximum of 1.30 g/l after 60 min of oral glucose challenge and to return to normal levels after 240 min (Fig. 2). The administration of 2 g/ml glucose to untreated diabetic rats induced an increase in their blood glucose levels, with peak of 3.25 g/l. The administration of CNE and acarbose, on the other hand, decreased the glucose peak in blood glucose levels to 1.67 and 1.38 g/l, respectively, significantly (P < 0.05) compared to diabetic rats. Glucose was probably attached to the OH functional groups of the phenolic compounds of CNE and the standard drug to reduce the glucose levels. In this study, the results suggest the usefulness of C. nodosa for further development as a therapeutic agent for impaired glucose tolerance and diabetes mellitus.
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The same effect has been shown by Posidonia oceanica is a widely allocated phanerogam in Mediterranean and Aegean Sea [7]. This which clearly shows a very active use of glucose by peripheral tissues, explained by an increase in glucose tolerance in these tissues when animals are treated with some marine plants. 3.5. Effect of CNE on a-amylase activity, serum glucose levels and pancreas architecture Type 1 diabetes is a progressive auto-immune disease wherein the b-cells producing insulin are destroyed by the immune system. It is characterized by the accumulation of lymphocytes, which infiltrate and destroy pancreatic islets, thereby causing carbohydrate metabolism disorders associated with high serum glucose levels or hyperglycaemia [5]. The results of the present study indicated that CNE administration to surviving diabetic rats induced considerable reductions in the plasma and intestine a-amylase activity by 26.74% and 47.11%, respectively, as compared to the untreated diabetic rats (Fig. 3). The oral treatment of surviving diabetic rats by CNE stimulated the b-cells regeneration in the pancreas, which led to an increase in insulin secretion. Both a-amylase activity inhibition and pancreatic b-cells protection from damage and death (Fig. 4C) induced a decrease in the blood glucose level by 45.66% as compared to the untreated group of diabetic rats (Fig. 5). One of the therapeutic strategies commonly used for the decrease of postprandial hyperglycemia in non insulin dependent diabetic topics is retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing the amylolytic enzymes (a-amylase), in the digestive tract [22]. Moreover, the CNE inhibited this key enzyme responsible for the conversion of starch to simple sugars absorbable by the small intestine by completely blocking access to the active site of the a-amylase. Increasing the activity of this enzyme observed at different levels leads necessarily to increase the intestinal absorption of glucose and accumulation in the bloodstream. It has been shown that treatment with the CNE and acarbose possess the ability to entrap glucose molecules in the blood and slow down their absorption, which leads to the hypoglycemic effect (Fig. 5) that could be explained by three reasons: (1) protection of pancreatic cells from progressive damage enhanced by alloxan, (2) the stimulation of Langerhans islets and (3) improvement of peripheral sensitivity to remnant insulin and antioxidant properties of C. nodoa. The results of the present study indicated also that the injection of alloxan to rats induced b-cell damage, with depleted islet cells of Langerhans (atrophy), cellular degeneration, pancreatic injury, and a clear decline in the area occupied by b-cells. The administration of CNE to surviving diabetic rats was noted to protect the pancreatic b-cells from death and damage and to induce more insulin secretion in the blood, which enhanced metabolism and body weight.
Table 3 Effect of Cymodocea nodosa extract on body weight and blood glucose in Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Body weight
Control Diab Diab + CNE Diab + Med
Blood glucose
Before treatment
After treatment
Before treatment
After treatment
167.35 4.13 170.23 3.80 159.12 4.51 162.09 3.67
249.52 6.19 119.68 5.33 202.80 2.77 208.14 5.06
0.79 1.58 2.79 2.81 2.64 0.77 2.55 2.25
0.84 0.91 2.84 3.16 1.43 0.89 1.30 4.02
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Glucose level in serum (g/l)
Cont
Diab
Diab+CNE
Diab+Med
4 3.5 3 2.5 2 1.5 1 0.5 0
0
30
60
120
240
Time (min) Fig. 2. Effects of Cymodocea nodosa extract on glucose tolerance. Glucose levels obtained from Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Acar: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05.
Fig. 3. Inhibitory effects of Cymodocea nodosa extract on alpha-amylase activity in the serum (A) and intestine (B) in Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Acar: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. aP < 0.05 compared with normal control rats; bP < 0.05, compared with diabetic rats and cP < 0.05 compared with treated rats.
Furthermore, some marine plants extracts are known for their hypoglycemic ability, the properties of marine origin have been extensively investigated in the literature [7,29]. The data in Table 4 show a significant increase in serum indices indicating liver dysfunction in untreated diabetic rats namely activities of AST, ALT and LDH by 21.14%, 28.65% and 7.89%, respectively, compared with the normal rats (P < 0.05). Generally, diabetes mellitus is associated with hyperlipidaemia. For this reason, we have evaluated the lipid parameters which is of crucial importance through its use in the treatment of several cardiovascular diseases and diabetes control [30]. We found a significant increase in total cholesterol (T-Ch), cholesterol low density lipoprotein (LDL-Ch) and triglycerides (TG) by 16.79%, 34.54% and 78.88%, respectively associated with a significantly lower rate of high density lipoproteins (HDL-Ch) in serum (87.5%) of the untreated diabetic rats compared with controls (Table 4). This observed hyperlipidemia can be explained by the degradation of the lipid compounds or fatty tissue by insulin deficiency that inhibits 3-hydroxy 3-methyl glutamyl coenzyme A reductase
(HMG-CoA reductase) involved in the biosynthesis of cholesterol [31]. The administration of CNE was also noted to decrease hepatic toxicity and to regulate the lipid profile as evidenced by the decrease observed for the AST, ALT, LDH, LDL-C, T-Ch and TG levels and the increase recorded for HDL-C (Table 4). This action of CNE supports its lipid lowering activity in diabetic condition and therefore it helps to prevent diabetic associated complication. These results are in agreement with previous findings in the literature, concerning marines phanerogams distributed in the Mediterrenean Sea, was found to decrease this biochemicals parameters levels and prevent its oxidation in vivo [7,29]. Hyperglycemia causes non-enzymatic glycation of proteins following the Maillard reaction and alters energy metabolism, which may lead to high rates ROS and the development of diabetes complications [32]. The oxidative stress for diabetes also includes redox imbalance from altered metabolism of lipids, carbohydrates and decrease of antioxidants defense. The results revealed that the hepatic tissues of diabetic rats underwent a decrease in SOD, CAT and GPX activities and an
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Fig. 4. Histological study of pancreas from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats, massive destruction of b-cells; (C) alloxan-diabetic rats,treated with CNE; (D) alloxan-diabetic rats treated with acarbose medicament, a partial protector effect of b-cells from pancreas was observed (H&E 100).
increase in TBARS levels. The rate of reactive substances with thiobarbituric acid (TBARS) was determined at the level of the organ tested because they are used to indicate the presence of a lipid peroxidation resulting from the establishment of an oxidative stress. This perturbations were restored significantly (p < 0.05) by the administration of CNE and acarbose (Table 5), which shows the beneficial effect of treatment able to mitigate oxidative stress and enhance antioxidant defenses in diabetic rats. In fact, the histological analysis of liver sections taken from diabetic rats showed lipid droplets in the cytoplasm of hepatic tissues due to the deficiency in insulin, responsible for metabolizing fats (Fig. 6B). These results are consistent with several other reports on experimentally induced diabetes in animal models [33,34]. The positive effects of CNE and acarbose were further evidenced by the decreased prevalence and accumulation of fat cells in the liver tissues (Fig. 6C, D). 3.6. Effect of CNE on renal dysfunction parameters and architecture The variations of parameters of kidney toxicity in experimental rats were highlighted by the study of biochemical and oxidative stress parameters (Table 4 and 5) and confirmed by histological study (Fig. 7). Hyperglycaemia induced an increase in albumin,
creatinine and urea contents in blood plasma by 21.59%, 37.39% and 104.23%, respectively and a decrease of SOD, CAT and GPX activities in the kidney of diabetic rats by 61.92%, 34.74% and 73.45%, respectively compared with the control rats. An increase of 362.79% in TBARS levels was also observed. Furthermore, a marked decrease in kidney toxicity was observed following CNE treatment, which was evidenced by 14.48%, 27.95% and 31.61% decrease in the biochemical parameters (Table 4), however, noted to increase the SOD, CAT and GPX activities in the kidney by 109.95%, 48.33% and 211.65% respectively and to decrease the TBARS level by 71.35% compared with untreated diabetic rats (Table 5). The significant increase in the concentration of biochemical indices in the serum of untreated diabetic rats compared to control rats. It can be explained by the accelerated protein degradation liver and plasma [35] or to the degradation of some compounds protein of the body due to the administration of alloxan or compounds made by food, which can be degraded into amino acids and urea. Kidney is the most important organ, which play a pivotal role in regulating various physiological processes in the body. Damage to the kidney inflicted by nephrotoxin agents is of grave consequences [5]. In diabetic animals, free radicals can rapidly accumulated and form oxidative stress, which may impair function of kidney, in which significantly decreased antioxidant activities. Persistent
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Fig. 5. Effects of Cymodocea nodosa extract on serum glucose levels in Cont: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. aP < 0.05 compared with normal control rats; bP < 0.05, compared with diabetic rats and cP < 0.05 compared with treated rats.
Table 4 Effects of administration of Cymodocea nodosa extract on liver-kidney toxicity and lipid profile in blood of Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Control
Diab
Diab + CNE
Diab + Med
Liver-toxicity AST (UI/l) ALT (UI/l) LDH (UI/l)
155.42 46.93b 50.85 6.69b 676.42 172.48b
188.28 23.38a,c 65.42 11.75a,c 729.85 185.55a,c
166.57 55.34b 59.00 11.71a,b 649.28 163.55a,b
161.42 31.06b 58.14 6.12a,b 662.42 108.61a,b
Kidney-toxicity Albumin (g/l) Urea (mmol/l) Creatinine (mmol/l)
32.42 5.22b 5.90 0.48b 25.70 3.90b
39.42 2.65a,c 12.05 1.75a,b 35.31 5.93a,c
33.71 5.46b 8.24 1.03a,b 25.44 4.01b
32.57 2.81b 8.30 1.45a,b 15.82 5.04b
Lipid profile T G (mmol/l) T-Ch (mmol/l) LDL-Ch (mmol/l) HDL-Ch (mmol/l)
0,90 0,29b 1.31 0.21b 0.55 0.18b 0.32 0.06b
1.61 0.40a 1.53 0.34a,c 0.74 0.23a,c 0.60 0.11a,c
1.31 0.50a,b 1.34 0.21b 0.5 0.08b 0.40 0.05a,b
1.39 0.11b 1.35 0.1a,b 0.42 0.10a,b 0.39 0.08a,b
Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. a P < 0.05 compared with normal control rats. b P < 0.05, compared with diabetic rats and. c P < 0.05 compared with traited rats.
hyperglycaemia leads to increased production of free radicals [36]. In our study, the activities of SOD, CAT and GPX decreased in diabetic rats as reported earlier which could be due to inactivation caused by alloxan-generated ROS. Several reports indicate that the compounds responsible for antioxidative activity of marine plants are mainly phenolic diterpenes such as carnosoic acid, carnosol, rosmanol [37], and other phenolic acids, such as rosmarinic and caffeic acids [38]. It is possible that the C. nodosa extract due to its presence of several bioactive antioxidant principles and their synergistic properties may be caused an improving effect in antioxidant status of diabetic rats. The negative action of hyperglycaemia on the kidney functions is found in perfect correlation with histological study showed an increase in Bowman’s space and glomerular atrophy in the kidneys of diabetic rats (Fig. 7B). On the other hand the administration of
CNE and acarbose to diabetic rats also regulated the Bowman’s space size and prevented glomerular atrophies and cortical structure but with some alterations relatively less significant to the diabetic rats (Fig. 7C, D). 4. Conclusion The present work is the first investigation dealing with the hypoglycemic and protective effects of CNE in alleviating oxidative stress and free radicals as well as in enhancing enzymatic defenses in diabetic rats. The results from the present study confirm the possibility that the major function of the CNE is on the protection of vital tissues pancreas, kidney and liver and reducing the complications usually resulting from oxidative stress in diabetes mellitus.
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Table 5 Effects of Cymodocea nodosa extract administration on SOD, CAT, GPX and TBARS levels in liver, pancreas and kidney in Control: normal rats; Diab: alloxan-diabetic rats; Diab + CNE: alloxan-diabetic rats treated with CNE; Diab + Med: alloxan-diabetic rats treated with acarbose medicament. Groups Liver Control Diab Diab + CNE Diab + Med Pancreas Control Diab Diab + CNE Diab + Med Kidney Control Diab Diab + CNE Diab + Med
SOD (U/mg protein)
CAT (mmole H2O2/min/mg protein)
GPX (mmol GSH/min/mg protein)
TBARS (nmol/mg protein)
22.16 0.16b 11.02 0.39a,c 20.08 0.01b 19.45 1.09b
42.15 0.64b 23.31 0.49a,c 44.07 0.64b 39.09 0.47a,b
4.20 0.02b 0.96 0.01a,c 3.72 0.02a,b 3.19 0.08a,b
0.84 0.01b 2.39 0.01a,c 0.94 0.27b 0.90 0.48b
19.45 0.02b 9.95 0.95a,c 18.41 0.03b 17.58 0.03a,b
32.02 0.04b 19.71 0.05a 26.43 0.03a,b 28.69 0.02a,b
2.15 0.03b 0.54 0.01a,c 1.94 0.11a,b 2.04 0.03b
0.65 0.01b 2.74 0.02a,c 0.59 1.16b 0.50 0.01a,b
12.66 0.02b 4.82 0.03a,c 10.12 0.61a,b 10.26 0.02a,b
50.22 0.03b 32.77 0.02a,c 48.61 0.61b 49.41 0.41b
6.14 0.02b 1.63 0.01a,c 5.08 0.02a,b 5.55 0.02a,b
0.43 0.01b 1.99 0.02a,c 0.57 0.01a,b 0.62 0.02a,b
Data represent mean SD (n = 7 for each group). Values differ significantly at P < 0.05. a P < 0.05 compared with normal control rats. b P < 0.05, compared with diabetic rats and. c P < 0.05 compared with traited rats.
Fig. 6. Histological comparison of liver from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats, appearance of lipid accumulation in liver cells; (C) alloxan-diabetic rats treated with CNE and (D) alloxan-diabetic rats treated with acarbose medicament, a decrease in the abundance of lipid accumulation in liver cells was showed (H&E 100).
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Fig. 7. Histological comparison of kidneys from (A) Control: normal rats showing normal architecture; (B) alloxan-diabetic rats showed Bowman’s space size and atrophy of glomerulus; (C) alloxan-diabetic rats treated with CNE and (D) alloxan-diabetic rats treated with acarbose medicament, a protector effect was showed (H&E 100).
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