Antidiabetic efficacy of Methanol extract Brown Alga Lobophora variegata on Alloxan stimulated Hyperglycemic Wistar Albino Rats

 

Sathyaseelan Thennarasan1, Subbiah Murugesan1, Vajiravelu Sivamurugan2*

1Division of Algal Biotechnology and Bionano Technology, Post Graduate and Research Department of Botany, Pachaiyappa’s College, Chennai – 600 030, India.

2Post Graduate and Research Department of Chemistry, Pachaiyappa’s College, Chennai – 600 030, India.

*Corresponding Author E-mail: sivaatnus@gmail.com

 

ABSTRACT:

To investigate the effect of methanol extract of Lobophora variegata for its antihyperglycemic activity against alloxan induced diabetic rats. The methanol residue of Lobophora variegata was given orally at a dosage of     10 mg/kg weight to diabetic animals for about 28 days. The outcome of algae residue feeding was estimated by various biochemical and haematological parameters such as RBC, WBC, HB and platelets and lipid profile. Histopathological evaluation was made in the pancreas. After feeding of methanol extract in diabetic rats for     28 days, the blood glucose has significantly decreased, while the increase in liver glycogen level was observed. In addition, the regeneration of the pancreas of the treated animals was also noticed. The methanol residues of the Lobophora variegata possess very effective anti-hyperglycemic activity on the diabetic rats as compared to Glipizide.

 

KEY WORDS: Lobophora variegata, Alloxan monohydrate, Carbohydrate metabolism, Diabetes mellitus, Brown algae.


 

 

INTRODUCTION:

Diabetes mellitus is a metabolic disorder of multiple   etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both.1 The pharmacological drugs are either too expensive or have undesirable side effects or contraindications.2 Throughout the world, many traditional treatments for diabetes exist and therein lies a hidden wealth of potentially useful natural products for the control of diabetes.3,4

 

Macroalgae are included as part of a healthy lifestyle in different countries and are consumed entirely or are used as extracts or food additives.5 Remarkable resources of marine bioactives are from species like sponges (31%), red algae (4%), brown algae (5%), green algae (1%), microorganisms (15%), coral (24%), ascidians (6%), mollusks (6%), others (8%). Several red, brown and green macroalgae have shown antidiabetic properties (e.g., Rhodomelacon fervoides, Ecklonia cava, Palmaria, Alaria and Ascophyllum). A bromophenol isolated from the red alga Rhodomelacon fervoides have potent PTP1B inhibitory action in vitro.6,7 HPN also significantly decreased plasma glucose, serum triglycerides and total cholesterol in a mouse model.7 Two other bromophenols, 2,4,6-tribromophenol and 2,4-dibromophenol, isolated from the red alga Grateloupia elliptica showed inhibition against Saccharomyces cerevisiae α-glucosidase and against Bacillus stearothermophilus α-glucosidase.8 Besides inhibiting against PTP1B and α-glucosidase, some bromophenols also inhibit aldose reductase (Suzen and Buyukbingol), the first enzyme of the polyol pathway responsible for the fructose formation from glucose. Subbiah Murugesan et al., (2016) reported antidiabetic efficacy of red algae Portieria hornemannii and Spyridia fusiformis on alloxan stimulated hyperglycemic activity in Wistar albino rats.9

 

Regarding brown algae, methanol extracts of the brown algae Pelvetia siliquosa, Ecklonia cava and                   E. stolonifera reduced plasma glucose levels in diabetic rats.10,11 Phenolic extracts of the brown algae Alaria and Ascophyllum exhibited inhibitory effects on α-amylase activity, with Ascophyllum also inhibiting α-glucosidase.12 The phlorotannin Phlorofucofuroeckol-A isolated from Ecklonia stolonifera showed significant inhibitory effects against AGEs.13 The phlorotannins Dieckol and Eckol, isolated from Eisenia bicyclis, successfully inhibited α-amylase, while Diphloretho hydroxycarmalol, a phlorotannin isolated from the brown alga Ishigeo kamurae, showed inhibitory effects against both α-glucosidase and α-amylase.14

 

The polyphenol-rich extracts from Ulva rigida reduced plasma glucose levels in diabetic rats.15 Popov and Krivoshapko (2013) studied a total mixture of polar lipids from Sargassum pallidum, Ulva fenestrata, Zostera marina under conditions of impairments of carbohydrate and lipid metabolism in mouse models.16 Laminaria angustata Kjellman var. longissima, able to reduce blood glucose levels in the Wistar rat model.17 Sharifuddin et al.,(2015) reviewed beneficial roles of seaweeds for diabetes prevention and management. They highlighted the healthy nutritional composition that may benefit diabetic patients.5 Based on the evidences, we attempted to find novel antidiabetic drugs from the marine brown alga Lobophora variegata and evaluate the antihyperglycemic activity against Wistar albino rats.

 

MATERIALS AND METHODS:

Collection of Seaweed:

In the present study Lobophora variegata J. V. Lamouroux) Womersley ex E.C. Oliveira was collected from coastal regions of Mandapam, South India. The freshly collected seaweed samples were rinsed with sterilized sea water and shade dried until constant dry weight obtained. The methanol residue of the above alga was obtained by soaking 1g of the dried algae powder in 100 mL of methanol overnight at 60°C the solvent extract was filtered followed by removal of solvent using rotary evaporator yielded 250 mg of extract residue.

 

Animal and Experimental design:

The Wistar strain of male albino rats having 180 and  200 g body weight was used in the current study. After keeping the animals in the laboratory condition for a week for acclimatization, the experiment was initiated. The study was conducted after obtaining institutional animal ethical committee clearance and reference number is (IAEC/KMCP/152/FT/4379/2013-2014). Chemical stimulation of diabetes in Wistar rats diabetes was stimulated by freshly prepared solution of monohydrate of Alloxan was injected intrapertioneally at 150 mg. kg-1 of body weight prepared in physiological saline after fasting overnight for 12 hrs.18 The elevated glucose level in plasma was determined at 72 hrs and then on the 7thday, after injection, this data confirmed hyperglycemia. To diagnose the diabetes, the threshold value of fasting plasma glucose level was taken as >126 mg/dL.

 

Experimental design:

In the experiment, a total of 24 rats were divided into 4 groups (18 diabetic surviving rats and 6 normal rats), each group with 6 rats was used. Diabetes was induced in rats 3 days before starting the experiment.

 

Group I: Normal control rats, received 0.9% saline at a dose of 10 mL/kg/body weight/rat/day for 28 days.

Group II:  Alloxan induced diabetic rats, received 10 mg/kg body weight/rat/day of Alloxan monohydrate through Intra peritoneally injection for 28 days.

Group III: Diabetic rats treated with Glipizide (5 mg/kg/body weigh/rat/day) dissolved in aqueous solution and given orally for 28 days.

Group IV: Diabetic rats treated with methanol extract of L. variegata (10 mg/kg/body weight/rat/day) dissolved in aqueous solution and given orally for 28 days.

 

Sample collection:

The treatment with algal residue was continued for 28 days. After that, the body weight and blood glucose level were monitored. The blood was collected retro-orbitally under light ether anesthesia using capillary tubes. The blood sample was collected in new ampoules containing anticoagulant agents, EDTA and plasma was separated from the blood in a T8 electric centrifuge at a rotation of 2000 rpm for about 2 mins. The liver and pancreas were immediately removed by dissection from the animals after the sacrifice and organs were washed in ice-cold saline and pancreas was subjected to histopathological studies and the enzyme activities are estimated from the liver.

 

Biochemical analysis:

Estimation of blood glucose:

Glucose assay kit (One Touch Ultra) provides direct measurement of glucose in various biological samples (Trinder, 1969).19

 

 

Hepatic glucokinase and hexokinase activity:

The small portion of liver was washed in ice cold 150 mM solution of potassium chloride containing 1 mM of EDTA and homogenized using ice cold buffer (10 mM of cysteine and 1 mM of EDTA in 0.1 mL of Tris-HCl at pH 7.4) and centrifuged for 20 mins at 4 °C. Glucose phosphorylation was assayed by means of Glucose 6 phosphate dependent spectrophotometric method.20

 

Glucose-6-phosphatase assay:

The small portion of the liver was homogenized using 40 times of its weight of the ice-cold buffer (0.1 Citrate/KOH, pH 6.5) and cheese cloth used for filtration. Glucose-6-phosphatase activity was determined by phosphate release method and concentration of H3PO4 was determined in the assay by colorimetric method.21

 

Glycogen content:

The tissue sample was washed using hot concentrated KOH (30%) and followed by anthrone reagent. Glycogen content was measured using a colorimetric method.22

 

Determination of haematological parameters:

The blood samples were evaluated for HB, WBC, RBC and Platelets by using an auto analyzer (MISPA-EXCEL, Japan).

 

Estimation of lipid profile:

Sample collection and analysis were made to fast overnight at least for a minimum of 8 hrs. 5 mL of fasting venous blood was collected from the antecubital vein under aseptic precaution from each subject in plain bottles. After collection of blood sample, the blood was allowed to clot (for 10 min) and serum was separated by centrifugation at 2500 rpm for 20 min and the serum removed and stored at a 4°C pending assay for lipid profile. Each serum sample from different groups was evaluated for the following parameters using on Glaxo kits ERBA Chem-5 semi auto analyzer.

1.      Total cholesterol (mg/dl)

2.      Triglyceride (mg/dl)

3.      HDL-cholesterol (mg/dl) Additional two parameters were calculated using Friedewald formula.

4.      LDL-cholesterol (mg/dl)

 

Estimation of phospholipids:

An aliquot 0.1 mL of lipid extract was taken into microkjeldahl flask and 0.4 mL of 60% perchlororic acid solution was added. It was digested directly on a sand bath for 20 minutes. Glass beads (2-3) were added to each flask to avoid bumping during digestion. The 0.1 mL of digested solution was taken in a tube and was added to 8 ml water. It was then mixed with 2.0 mL colouring reagent (1 part of 10% ascorbic acid and 6 part of 0.42%) was added. It was incubated in a heated water bath at 37°C for 1 hr. After mixing absorbance was taken at 660 nm in colorimeter against H2O.

 

Histopathological examination:

The small portion of pancreas from each animal was removed after sacrificing the animal and was collected in 10% formalin solution, and immediately processed by the paraffin technique. Haematoxylin-eosin was used to stain the paraffin section.23Each pancreas sample was seen at 400 X using microscope and evaluated based on the injuries.24

 

Statistical analysis:

The biochemical parameters and haematological were subjected to statistical analysis by one-way Analysis of Variance to determine the significant difference between the groups was done with Graph pad Prism software. All Pairwise Multiple Comparison Procedures by Student-Newman-Keuls Method. If the statistical significant difference was p < 0.05 data accepted for assessment.

 

RESULTS:

Effect on body weight of normal and experimental animals:

Table.1 illustrates the values of body weight of normal and experimental animals in each group. The final body weight of the normal control animals at the end of the experimental period (28 days) was 230.40 g and the growth in terms of body weight during this period was 3.5% (Table 1). Diabetic induced rats registered lesser body weight during the entire experimental period as compared to normal animals. The initial body weight of the diabetic rats was 11% less as compared to control animals and this increased to record a 45% lesser body weight than the control at the end of the experimental period. The positive controls, i.e. drug (Glipizide 5 mg/kg body weight) treated animals showed a recovery from body weight loss. Treatment with methanol extract residue of L. variegate at a dose of 10 mg/kg body weight, exhibited an excellent recovery to keep the body weight comparable to that of control animals.

 

Effect on blood glucose levels:

Non-diabetic control (normal) animals remained steadily euglycemic throughout the course of study (Table 2). Alloxan induced diabetic animals recorded more than twofold higher glucose as compared to normal animals and on the 28thday, the diabetic induced animals had 154% more glucose than the normal animals (Table 2). Neither drug treatment (positive control) (Glipizide 5 mg/kg body weight) nor the treatment with the methanol extract residue of L. variegata at a dose of 10 mg/kg body weight could bring back the glucose levels to normal levels (Table 2). The level of glucose observed on zero day in animals treated with the algal extract was 196.26 ± 5.56%) decreased to 153.92 ± 3.90% in                4 weeks. However, as compared to control animals, the values were 69% higher (Table 2). The anti-hyperglycemic effect observed in diabetic induced animals in response to treatment with an algal extract was not sufficient enough to bring the glucose levels back to normal. Nevertheless, the observed anti-hyperglycemic effect is comparable to that noted for drug (Glipizide) treated animals.

 

Table 1: Efficiency of methanol extract of L. variegata on body weight of normal and experimental animals

Groups

Initial body weight

Final body weight

Normal control (G I)

222.48 ± 3.28

230.40 ± 4.32

Negative control  (Alloxon monohydrate (G II))

199.65 ± 3.05

157.45 ± 2.35(*a)

Positive control (Glipizide (G III))

214.45 ± 3.60

217.22 ± 3.30(*b)

Treatment control (Diabetic+Methanolic extract of L. variegata (G IV))

221.60 ± 3.88

229.45 ± 3.48(*b)

Units: gram

Values are expressed as mean ± SEM.

No. of animals in each group (n) = 6

Values were found out by using One Way ANOVA followed by Newman Keul’s multiple range test.

(a*)  values were significantly different from Initial Body Weight of GI at (p<0.01).

 

Table 2: Efficiency of 4 weeks treatment with methanol extract of L. variegata on glucose levels in alloxan diabetic rats.

Groups

0-DAY

14th DAY

28th  DAY

Normal control(G I)

87.15 ± 3.60

92.20 ± 3.85

91.00  ± 3.82

Negative control (Alloxon monohydrate (G II))

161.80 ± 5.75

189.20 ± 5.96(*a)

231.30 ± 6.30(*a)

Positive control (Glipizide (G III))

183.76 ± 5.65

153.40 ± 4.60(*b)

141.20 ± 3.42(*b)

Treatment control (Diabetic+Methanolic extract of L. variegata (G IV))

196.26 ± 5.56

161.24 ± 4.65(*b)

153.92 ± 3.90(*b)

Unit: mg%

N = 6 in each group, Values are expressed as Mean ± SEM.

Values were found out by using One Way ANOVA followed by Newman Keul’s Multiple range tests.

(*a) values were significantly different from normal control (GI) at (p<0.001).

(*b) values were significantly different from diabetic control (GII) at (p<0.001).

 

 

Effect on glycogen content:

Glycogen content of liver tissue was estimated on the 28th day in all the four groups of experimental animals [non-diabetic control, diabetic control, positive control (drug treated) and algal extract treated groups] as shown in Table 3. In diabetic induced animals (Group II), liver glycogen content decreased significantly by 72.96% as compared to that in non-diabetic control animals. Treatment with Glipizide increased this liver glycogen level by 180%, while the methanol extract residue treatment by 143% (Table 3). However, these values are less than that of normal (Group I) animals by 29% and 34% respectively.

 

Table 3: Efficiency of administration of methanol extract of L. variegata on glycogen content of liver tissue of rats

Groups

Liver tissue glycogen content

Normal control (G I)

48.90 ± 1.45

Negative control (Alloxon monohydrate (G II))

13.22 ± 0.35(*a)

Positive control (Glipizide (G III))

37.90 ± 1.23(*b)

Treatment control (Diabetic+Extract of L. variegata (G IV))

32.25 ± 1.05(*b)

Unit: (mg/g tissue)

 

Effect of hepatic enzymes:

Hepatic enzymes Hexokinase, Glucokinase and Glucose-6-phosphate were estimated in normal control (Group I), diabetic control (Group II) (positive control (Group III) and treatment control (Group IV) animals, 28 days after induction of diabetes. The results are given in Table 4.

 

Non-diabetic control animals recorded 0.232 ± 0.004, 32.60 ± 0.80 and 0.4145 ± 0.006 for the enzyme hexokinase, glucokinase and glucose–6–phosphate. Diabetic induced animals showed reduced levels of these enzymes in their liver tissue. Hexokinase registered nearly 50% reduction while glucokinase and glucose-6-phosphate exhibited 187% and 400% decreases respectively as compared to non-diabetic controls. On treatment with Glipizide (5 mg/kg body weight) or the algal drug (10 mg/kg body weight), the values showed improvements. The observed levels between these two groups of animals are comparable with each other (Table 4). However, they could not be restored to levels observed in non-diabetic controls. Nevertheless, the algal drug was able to induce a recovery in the levels of the enzymes as compared to the control animals.

 


Table 4: Efficiency of administration of methanolic extract of L. variegata on hepatic enzymes involved in carbohydrate metabolism in rats.

Groups

Hexokinase

Glucose 6-Phosphate

Glucokinase

Normal control (G I)

0.232 ± 0.004

0.145 ± 0.006

32.60  ± 0.80

Negative control (Alloxon monohydrate (G II))

0.106 ± 0.002*a

0.147 ± 0.002*a

6.35 ± 0.12*a

Positive control (Glipizide (G III))

0.169 ± 0.003*b

0.335 ± 0.005*b

20.69 ± 0.45*b

Treatment control (Diabetic+Extract of  L. variegata (G IV))

0.149 ± 0.002*b

0.274 ± 0.004*b

18.25 ± 0.35*b

Unit = one unit is as 50% inhibition of NBT/mg protein. ; Unit = μmoles of H2O2 utilized/min/mg protein.; Unit = μg/100 mg tissue.

N = 6 in each group, values are expressed as Mean ± SEM; Values were found out by using One Way ANOVA followed by Newman Keul’s multiple range tests.; *a values were significantly different from normal control (GI) at (p<0.001); *b values were significantly different from diabetic control (GII) at (p<0.001); Values with different superscripts in the same row differ significantly at the 5%  level (p< 0.05).


Effect on haematological parameters:

The effects of methanol extract residue of L. variegata on haematological parameters in diabetic induced rats are shown in Table 5. At the end of the study period 28 days, statistically significant differences could not be seen in the mean values for WBC and RBC counts, HB level and platelet values as compared to the non-diabetic animals.

 

Effect on lipid profile:

Table 6 shows the levels of serum total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and phospholipids of the experimental animals in each group. Total cholesterol, triglycerides, high density lipoprotein, low density lipoprotein (LDL) and phospholipids levels were significantly increased, whereas HDL level was increased in alloxan induced diabetic rats as compared to normal rats. Treatment of normal and alloxan induced diabetic rats with L. variegata at a dose of
10 mg/kg body weight for 28 days resulted in a marked decrease in the levels of total cholesterol, triglycerides, HDL, LDL which are nearly equal to that of Glipizide treating animals. The observed levels exhibit an excellent recovery in the levels of lipids caused by the induction of diabetes.

 

Table 5: Efficiency of administration of methanol extract of L. variegata on haematological parameters

Groups

WBC 

RBC

HB

Platelets

Normal control

(G I)

9.65 ± 0.75

5.47 ± 0.35

11.65 ± 0.68

328.45 ± 36.15

Negative control
(Alloxon monohydrate (G II))

9.25 ± 0.68

5.46 ± 0.32

12.04 ± 0.75

316.50 ± 30.15

Positive control
(Glipizide (G III))

8.87 ± 0.60

5.37 ± 0.30

15.08 ± 0.42

293.45 ± 28.45

Treatment control
(Diabetic+Extract of  L. variegata

(G IV))

8.66 ± 0.56

5.06 ± 0.28

13.26 ± 0.40

297.75 ± 29.20

Unit: For WBC -103/µL, RBC-106/µL, HB-gm/dL, Platelet-103/mL

 

 


 

Table 6: Efficiency of administration of methanolic extract of L. variegata on total cholesterol, triglyceride, HDL-C, LDL-C and phospholipids.

Groups

Total Cholesterol

 Triglyceride

HDL-C

LDL              

Phospholipids

Control (GI)

83.90 ± 3.52

87.50 ± 2.55

49.28 ± 1.85

15.85 ± 1.55

123.65 ± 2.50

Diabetic control (GII)

230.20 ± 6.92**(a)

157.60 ± 4.54**(a)

30.60 ± 1.12**(a)

37.85 ±  2.31**(a)

204.30 ± 6.30**(a)

Diabetic+Glipzide (GIII)

104.85 ± 3.26**(b)

93.80 ± 2.55**(b)

43.98 ± 1.43**(b)

20.35 ± 1.86**(b)

147.42 ± 3.80**(b)

Treatment control
(Diabetic+ Methanolic extract of                  L .variegata (G IV)

123.60 ± 3.58**(b)

106.70 ± 2.80**(b)

39.44 ± 1.37**(b)

28.52 ± 1.92**(b)

161.65 ± 4.07**(b)

Units: mg/dl

Values are expressed as mean ± SEM.

Values were compared by using analysis of variance (ANOVA) followed by Newman-Keul's multiple range tests. 

** (a) Values are significantly different from normal control GI at p<0.001.

** (b) Values are significantly different from toxic control control GII at p<0.01.


 

Histopathological studies:

In histopathological study, the Fig.1a showed normal acini and normal cellular population in the islets of Langerhans in the pancreas of normal diabetic rats (Group-I). Fig.1b showed extensive damage and reduced number of islets of Langerhans in the pancreas of diabetic rats (Group-II). Fig.1c showed restoration of the normal cellular population size of islets with hyperplasia by Glipizide (Group-III). Fig.1d showed partial restoration of normal cellular population and enlarged the size of β-cells with hyperplasia in the methanol extract residue of L. variegata treated rats (Group IV).

 

Figure 1: Histopathological study of pancreas of rats:

a) Section shows Structure of Pancreas section of GP1 (Normal control); b) Pancreas section of GP2 (Toxic control-(Alloxan monohydrate); c) Pancreas section of GP3 (Standard control-(Alloxan monohydrate+Glipizide) and d) Pancreas section of GP4 (methanol extract of L. vareigata10 mg/kg/rat)

 

DISCUSSION:

The present study was undertaken to assess anti-hyperglycemic property of methanol extract of                   L. variegata in rats as well as to provide an basic approach for the evaluation of its traditional preparation in order to scientifically validate the therapeutic preparation in the control of diabetes. To the best of our knowledge, this is the first report that analyzes the antidiabetic potential of L. variegata in experimental diabetes. Management of diabetes without any side effects is still a challenge to the modern medicine. This leads to increasing the demand for searching for new drugs from natural origin no or less side effects.

 

The body weight got decreased in alloxan-induced diabetic rats.25 The administration of methanol extract of L. variegata at a dose of 10 mg/kg the final body weight increased, whereas the same decreased in alloxan-induced diabetic rats. The ability of these algal extracts to protect massive body weight loss seems to be due to its ability to reduce hyperglycemia.

 

In the current study diabetes mellitus was induced by alloxan monohydrate at a dose of 150 mg/kg body weight i.p. Alloxan causes enormous reduction in insulin release through the destruction of β cells of the islets of Langerhans. In the present study, we have observed that a significant increase in plasma glucose level in alloxan induced diabetic rats, whereas treatment with Glipizide (5 mg/kg body weight), the methanol extract of               L. variegata at a dose of 10 mg/kg body weight showed significant antihyperglycemic activity in mild degree of hyperglycemia. In mild diabetes, the maximum percent reduction in glucose level was seen in groups receiving 10 mg/kg body weight per day of methanol extract of            L. variegata.

 

As reported earlier, the liver glycogen content was reduced significantly in diabetic control as compared to non-diabetic control in the present study.26 Treatment with the methanol extract of L. variegata at a dose of          10 mg/kg body weight prevented this alteration in glycogen content of liver tissue, but could not normalize the content of glycogen of the non-diabetic control. This prevention or depletion of glycogen in the liver is possibly due to either stimulation of insulin release from β-cells.27In uncontrolled or poorly controlled diabetes, there is an increased glycosylation of a number of proteins, including.28 During diabetes the excess glucose present in the blood reacts with haemoglobin. Therefore, the total level decreases in alloxan diabetic rats.29 The significant and consistent antidiabetic effect of methanol extracts of L. variegata in alloxan-induced diabetic rats in also is due to enhanced glucose utilization by peripheral tissues.

 

The alloxan treated animals exhibited a decrease in hepatic glycogen content which may be due to an increase in glucose-6-phosphatase activity and a low level of hexokinase activity.30 An increase in hepatic glycogen content in algal extracts administered animals suggests that the activation of glycogen synthase for which the substrate glucose-6-phosphate could have been readily provided by increased hexokinase activity.31,32 These observations clearly indicate the potential of these algal extracts to reduce gluconeogenesis both alone and in combination. Thus,  L. variegata, reduced blood glucose levels and increased glycogenesis and glycolysis, reduced gluconeogenesis and brought the glucose metabolism towards normal levels in diabetic rats. Moreover, the effect of                       L. variegata on the carbohydrate metabolism in diabetic rats is found to be similar to that of Glipizide.

 

Lipid peroxidation is one of the characteristic features of chronic diabetes and its mediated tissue damage has been observed in diabetic conditions.33 Hyperglycemia generates reactive oxygen species (ROS), which in turn cause lipid peroxidation and membrane damage.34 Increased concentrations of lipid peroxides in the liver are reported to decrease cytochrome P450 and cytochrome b5 activities, which may affect the drugs metabolizing activity in chronic diabetes.35 The diabetic animals in the present study registered lowered levels of GSH reflecting its increased utilization owing to oxidative stress; while a significant elevation of GSH levels in L. variegata treated diabetic rats coincided with a significant decline in lipid peroxidation. It appears that the effect of the methanol extract of L. variegata on GSH could be at two levels, either through increasing the biosynthesis of GSH or through inhibiting its utilization by reducing oxidative stress.

 

The antioxidant enzymes SOD (superoxide dismutase) and CAT (catalase) play an important role in reducing cellular stress. SOD scavenges the superoxide radical by converting it to hydrogen peroxide and molecular oxygen while CAT brings about the reduction of hydrogen peroxides and protects higher tissues from the highly reactive hydroxyl radicals (Brioukhanov and Netrusov, 2004). In the present investigation, both these enzymes registered low levels of activity in diabetic controls indicating diabetes-induced stress. Such a decline in these enzyme activities has also been reported earlier.36,37 The L. variegata, when administered to the diabetic animals improved both SOD and CAT activities substantially, reflecting the antioxidant potency of these algal extracts. The effects of L. variegata alone on antioxidants (GSH, SOD, CAT and LPO) were found to be better than those of Glipizide administered diabetic animals.

Decreased enzymatic activity of hexokinase, glucokinase and substrate glucose-6- phosphate has been reported in diabetic animals resulting in depletion of liver and muscle glycogen.38 Treatment with methanol extract of L. variegata has significantly increased the hexokinase, glucokinase activity and glucose-6-phosphate level in the liver, indicating an overall increase in glucose influx and thus the methanol extract of L. variegata seems to have an overall effect of an increase in glucose utilization. These studies also establish that algal extract showed no adverse effect on haematological parameters including WBC, RBC counts, Hb and platelets. Thus, the methanol extract of L. variegata can be presumed to be free from toxicological effects.

 

The level of serum lipids is usually elevated in diabetes mellitus, and such an elevation represents the risk of coronary heart disease (CHD). A lowering of serum lipid concentration through diet or drug therapy seems to be associated with a decrease in the risk of vascular disease. The abnormally high concentration of serum lipids in diabetic subject is mainly due to increased mobilization of free fatty acids from the peripheral fat depots, since insulin inhibits the hormone sensitive lipase. However, glucagon, catecholamines and other hormones enhance lipolysis. The marked hyperlipidaemia that characterized the diabetic state may therefore be regarded as a consequence of the uninhibited actions of lipolytic hormones on the fat depots.

 

In the alloxan-induced diabetes mellitus, the rise in blood glucose is accompanied by an increase in serum cholesterol and triglycerides. The levels of cholesterol and triglycerides and low-density lipoprotein (LDL) levels were brought to near normal by the treatment with L. variegata at a dose of 10 mg/kg body weight and in alloxan induced diabetic rats.

 

In the alloxan-induced diabetes mellitus, the rise in blood glucose is accompanied by an increase in serum cholesterol and triglycerides. This might suggest that the effect may be due to extra intestinal action or probably by an insulin releasing mechanism of the tested drug.39,40

 

A large amount of glycogen was also observed after treating diabetic rats with the methanol extract of                 L. variegata, suggesting the possibility of increased glycogen formation through enhanced glycogen synthase enzyme activity, and a probable mechanism of its hypoglycemic effect. The blood glucose data obtained clearly indicate that the methanol extract of L. variegata produced significant and consistent anti–hyperglycemic effect in alloxan induced diabetic rats. Continuous treatment with methanol extract of L. variegata for a period of 28 days produced a significant decrease in the blood glucose levels of diabetic rats.

In the present study, elevated levels of serum TC (total cholesterol), TG (triglyceride) and LDL-C (Low density lipoprotein cholesterol) decreased  HDL-C (High density lipoprotein cholesterol)  levels in alloxan-induced diabetic rats.41 On the other hand, the induction of diabetes by alloxan caused decrease in body weight in the diabetic control rats. This was in accordance with (Li and Kim, 2011) who suggests that this may due to alloxin inducing a catabolic effect on protein metabolism. It may be achieved by retarding protein synthesis and stimulating protein degradation. Continuous administration of methanol extract of          L. variegata had decreased the blood glucose, total cholesterol and triglycerides significantly, while protein levels increased significantly. The results of serum glucose level, total cholesterol, triglycerides and protein are consistent with the finding of earlier reports in rats.42,43 The plasma insulin level was decreased in diabetic control when compared with normal control rats. After 28 days treatment with methanol extract of          L. variegata, the level of plasma insulin was significantly increased when compared with diabetic control. Diabetic rats treated with 10 mg/kg body weight of the methanol extract of L. variegata and rats treated with glipizide alone showed a progressive decline in level towards normal while diabetic rats treated with 10 mg/kg body weight showed only moderate decline. Our findings of elevated values of cholesterol and triglycerides recorded in diabetic rats were indicative of abnormal fat metabolism that is commonly encountered in diabetes mellitus. In diabetic patients, glucose metabolism progressively diminishes and the metabolism rapidly turns to utilization of fatty acids for energy purposes. Diabetic rats showed a progressive decrease in HDL levels and a corresponding increase in LDL levels when compared to the normal rats indicating abnormal fat metabolism encountered in diabetes mellitus. Diabetic rats treated with 10 mg/kg body weight of extract as well as diabetic rats treated with Glipizide alone maintained their values within normal limits. However, rats treated with 10 mg/kg body weight showed only a moderate response. The levels of cholesterol and triglycerides and Low density lipoprotein (LDL) levels were brought to near normal by the treatment with L. variegata at a dose of 10 mg/kg body weight in alloxan induced diabetic rats.

 

The effect of L. variegata at a dose of 10 mg/kg body weight on diabetic hypertriglyceridemia could be through its control of hyperglycaemia. This is in agreement with the facts that:

1.      The level of glycemic control is the major determinant of total and very low-density lipoprotein (VLDL), triglyceride concentrations.44

2.      Improved glycemic control following sulfonylurea therapy decreases the levels of serum VLDL and total triglycerides.45

 

The main ‘anti-atherogenic’ lipoprotein (HDL) is involved in the transport of cholesterol from peripheral tissues in the liver (Segal et al., 1984)46 and thereby it acts as a protective factor against coronary heart disease (CHD).47

 

The level of HDL-cholesterol was decreased in diabetic rats when compared with normal rats.48 Our results clearly show that the level of HDL-cholesterol was increased in alloxan induced diabetic rats when treated with L. variegata at a dose of 10 mg/kg body weight. These results suggest that L. variegata at a dose of               10 mg/kg body weight has protective effect against alloxan-induced diabetes and its complications.

 

Histopathology:

The histopathological studies also showed regeneration of β-cells of the pancreas and so might be of value in diabetes treatment. Histopathological studies also revealed that L. variegata and Glipizide have significantly improved the histological architecture of the islets of Langerhans. The groups treated with                   L. variegata (10 mg/kg body weight) and Glipizide (5 mg/kg body weight) showed greater persistence of Islets of Langerhans and lesser degree of necrotic changes as compared to the untreated alloxan-induced diabetic rats. Similar kind of work was reported by Menelo Ciudadano Hongayo (2011) in Cystoseir amoniliformis. This would explain further as this histology result showed normal cells like elongated and rounded islets of Langerhans. However, abnormalities can still be observed.

 

CONCLUSION:

In conclusion, the methanol extract of L. variegata at a dose of (5 mg/kg body weight) exhibited significant anti-hyperglycemic activity in normal and alloxan-diabetic rats. The extract also showed improvement in the body weight, liver glycogen content and carbohydrate metabolizing enzymes as well as regeneration of β-cells of the pancreas. The histopathological results showed some signs of regeneration in the cells of the pancreas. Therefore, based on the present investigation, it is concluded that the methanol extract of L. variegata could be a good candidate for developing novel pharmaceutics. However, further pharmacological and biochemical investigations are necessary to clearly elucidate the mechanism of action and will be helpful in projecting this alga as a therapeutic target in the diabetes research.

 

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

ACKNOWLEDGEMENTS:

We are grateful to thank Dr. N. Chidambaranathan, Vice Principal and HOD of Pharmacology, K.M College of Pharmacy, Madurai, India, for his help and support extended by providing the experimental animal facilities.

 

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Received on 23.05.2017       Accepted on 28.07.2017     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2017; 7 (3): 157-165.

DOI: 10.5958/2231-5713.2017.00026.5