2013

http://informahealthcare.com/arpISSN: 1381-3455 (print), 1744-4160 (electronic)

Arch Physiol Biochem, 2013; 119(2): 81–87! 2013 Informa UK Ltd. DOI: 10.3109/13813455.2013.775159

ORIGINAL ARTICLE

Inhibitory activities of Ulva lactuca polysaccharides on digestiveenzymes related to diabetes and obesity

Sahla BelHadj1, Olfa Hentati2, Abdelfattah Elfeki1, and Khaled Hamden2

1Laboratory of Animal Ecophysiology, University of Sfax, Faculty of Sciences of Sfax, PO Box 95, Sfax, Tunisia and 2High School of Biotechnology of

Sfax (ISBS), Road of Soukra Km 4, PO Box 1175, Sfax, Tunisia

Abstract

The aim of this study was to evaluate the effect of alga Ulva lactuca polysaccharides (ULPS) onkey enzymes related to diabetes and obesity. This marine natural product, ULPS, exertedpotential inhibition on key enzymes related to starch digestion and absorption in both plasmaand small intestine mainly a-amylase by 53% and 34% and maltase by 97 and 164%respectively, leading to a significant decrease in blood glucose rate by 297%. Moreover, ULPSpotentially inhibited key enzymes of lipid metabolism and absorption as lipase activity in bothplasma and small intestine by 235 and 287% respectively, which led to a notable decreaseof blood LDL-cholesterol and triglycerides levels, and in the counterpart an increase inHDL-cholesterol level in surviving diabetic rats. Additively, ULPS significantly protected theliver-kidney functions, by decreasing of aspartate transaminase (AST), alanine transaminase(ALT) and gamma-glytamyl transpeptidase (GGT) activities and creatinine, urea and albuminrates in plasma.

Keywords

a-amylase, kidney toxicity, lipase, liverdysfunction, maltase

History

Received 7 November 2012Revised 14 January 2013Accepted 7 February 2013Published online 17 April 2013

Introduction

Diabetes mellitus is a major and growing public health

problem throughout the world, with an estimated worldwide

prevalence in 2008 of more than of 347 million people, and is

a heterogeneous disorder with varying prevalence among

different ethnic groups and reported to constitute the 16th

leading cause of global mortality (Danaei et al., 2011). It is

generally recognized that patients with diabetes are at risk for

numerous severe complications, including diabetic hyperlip-

idemia, liver-kidney complications and hypertension

(Hamden et al., 2010a,b; Hamden et al., 2011a,b). Dietary

carbohydrates and fatty acids represent some of the major

nutrients needed to maintain human health, and disacchar-

idases and lipases play important roles in the digestion of food

and absorption of sugars and fatty acids. Several enzymes

secreted by the small intestine, namely maltase, lactase,

sucrase, and lipase, are generally known to break down those

polysaccharides and lipids into monosaccharides and free

fatty acids (Hamden et al., 2010). One of the therapeutic

approaches for decreasing postprandial hyperglycaemia is to

retard absorption of glucose by the inhibition of carbohydrate-

hydrolysing enzymes such as a-amylase and a-glucosidase, in

the digestive organs (Hamden et al., 2011b; Liu et al., 2011a).

Much effort has been extended in search of effective

a-amylase and a-glucosidase inhibitors from the natural

resources in order to develop physiologically functional food

or to introduce a natural antibiotic agent (Akkarachiyasit

et al., 2011; Jo et al., 2011). However, the continuous use of

those synthetic agents should be limited because those agents

may cause side effects such as flatulence, abdominal cramp,

vomiting, and diarrhoea (Kast, 2002). Therefore, numerous

studies have been carried out to find out natural agents to

inhibit a-amylase and a-glucosidase from natural products

which do not show any side effects (Hamden et al., 2011b).

In recent years, many marine resources have attracted

attention in the search for bioactive compounds to develop

new drugs and health foods (Liu et al., 2011b; Sun et al.,

2011). Marine algae have been identified as an under-

exploited plant resource and a source of functional food

(Senni et al., 2011). The isolation of compounds have shown

that they have a variety of biological activities such as anti-

oxidant, anti-coagulant, anti-hypertension, anti-bacterial, and

anti-tumour activities (Chiu et al., 2012; Go et al., 2011; Oh

et al., 2011; Senni et al., 2011). Algal polysaccharides have

been demonstrated to play an important role as free-radical

scavengers in vitro and anti-oxidants for the prevention of

oxidative damage in living organisms (Anastyuk et al., 2009;

El Gamal et al., 2012; Zhang et al., 2010; Wijesekara et al.,

2011). The structure and mechanisms of the pharmaceutical

effects of bioactive polysaccharides on diseases have

been extensively studied, and more natural polysaccharides

with different curative effects have been tested and even

applied in therapies.

Correspondence: Olfa Hentati, High School of Biotechnology of Sfax(ISBS), Road of Soukra Km 4.5, PO Box 1175, Sfax 3038, Tunisia. Tel:+216 74 677 637/74 674 354. Fax: +216 74 674 364. E-mail:olfa_hentati@yahoo.fr

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The current study attempts to contribute to this line of

research by investigating the therapeutic action of polysac-

charides from Ulva lactuca on diabetic status as well as the

activities of a-amylase, maltase and lipase with respect to

small intestinal and liver-kidney toxicity in alloxan-induced

diabetic rats.

Materials and methods

Sample collection and extraction

Ulva lactuca was collected from Sfax province, White Sea

coast, Tunisia (at 34� 31N and 10� 33E) in October 2011.

The collected sample was washed with seawater many times

and further washed two times with distilled water to remove

epiphytes, salts and sands and contamination from other

algae. They were air dried in shade and ground by a blender to

give small size pieces (2 mm) then stored in plastic bags at

room temperature in a dry dark place before use. Sulphated

polysaccharide extraction procedure by Hamden et al. (2010a)

was followed with some modifications. Briefly, 500 g (n¼ 5)

of air shade dried algae were roughly cut and autoclaved

in 5 L of water at 100 �C for 3 h. The slurry was separated

by gauze and filtered. The filtrate was dialysed against tap

water for 48 h, and then concentrated to about 1000 mL

under reduced pressure and then 95% ethanol (5 L) was

added. The mixture was allowed to stand for overnight at

room temperature. The precipitate was collected and washed

twice with absolute ethanol, then dried at 50 �C. The crude

polysaccharide extract was stored at 4 �C and used for animal

experiments.

Animals and treatments

The assays of the present study were conducted on adult male

Wistar rats, weighing 212� 19 g, which were obtained from

the local Central Pharmacy, Tunisia. All rats were kept in an

environmentally controlled breeding room (temperature:

20� 2 �C, humidity: 60� 5%, 12–12 h dark/light cycle)

where they had standard diets and free access to tap water.

The experimental protocols were conducted in accordance

with the guide for the care and use of laboratory animals

issued by the University of Sfax, Tunisia, and approved by the

Committee of Animal Ethics.

Diabetes was induced in rats by a single intraperitoneal

injection of freshly prepared alloxan solution in normal

saline at a dose of 150 mg/Kg body weight (Hamden et al.,

2009).

One week later, the blood glucose level was measured by a

glucometer in tail and only rats with a higher rate of glucose

2 g/L are used for experimentation. After injection of alloxan,

the percentage of the viability of rats was 71%. Before the

treatment, the rats were divided into five groups of eight

animals each as follows:

Group 1: The day of the experiments, eight surviving diabetic

rats were scarified before treatment as referent surviving

diabetic rats [Diab0].

Group 2: Diabetic control rats at day 30 [Diab30]

Group 3: Diabetic rats treated with ULPS by gastric gavage

route food (180 mg/kg of body weight/day during 30 days)

(Hassan et al., 2011) and termed [DiabþULPS].

Group 4: Normal rats were used as controls, were scarified at

day 30, considered as referent non-diabetic rats at day 30

[Con30].

Group 5: Normal rats treated with ULPS by gastric gavage

route food (50 mg/kg of body weight/day during 30 days) and

termed [ConþULPS].

One month later, the rats were weighed and sacrificed by

decapitation, and their trunk blood collected. The serum was

prepared by centrifugation (1500� g, 15 min, 4 �C).

Biochemical analysis

Alloxan, maltose, sucrose, and lactose were purchased from

Sigma-Aldrich (St. Louis, MO, USA), the glucose; HDL, TC,

TG, AST, ALT, GGT and LDH were from Biomaghreb

analyticals (Tunis, Tunisia). All other chemicals used were of

analytical grade. The mucosal small intestine of each rat was

excised and the lumen was flushed out several times with

0.9% NaCl. The mucosal washing and the scraped mucosa

were pooled, homogenized, and centrifuged (5000� g,

15 min). The supernatant was frozen and stored at �80 �Cfor further use in subsequent enzymatic assays. The activities

of a-amylase, and maltase activities were obtained by

measuring the amount of glucose released from various

substrates (Dahlqvist, 1968; Maeda et al., 1985). For oral

sugar tolerance test, the carbohydrates loaded were as follows:

glucose (2 g/Kg), maltose (2 g/Kg), starch (1 g/Kg) by gastric

gavage route. For oral lipid tolerance test, oil was admini-

strated by gastric gavage (2 g/kg). After oil administration,

blood samples were collected from the tail vein at 3, 6, 9 and

12 h and TG level was measured. For histochemical proced-

ures, tissue specimens of the liver and kidney were obtained

and fixed with 10% buffered formalin, and subsequently

embedded in paraffin. After that, the paraffin-embedded

samples were cut in sections (thickness, 5 mm) and then

stained with hematoxylin-eosin. The samples were then

examined using an Olympus CX41 light microscope.

Statistical analysis

Data are presented as means� SD. Determinations were

performed from eight animals per group and differences were

examined by a one-way analysis of variance (ANOVA)

followed by the Fisher test (Stat View). The significance was

accepted at p50.05.

Results

a-amylase and maltase activities in small intestine ofcontrol and treated diabetic rats

The results revealed that diabetes induced a considerable

increase in the a-amylase, and maltase activities in the plasma

by 53 and 80% respectively and in mucosal small intestine of

the untreated rats by 80 and 164%, respectively, which

increases the glucose rate by 297% in the plasma of diabetic

rats. In ULPS treated diabetic rats, the activities of those

enzymes underwent considerable improvement. In fact, the

administration of ULPS to diabetic rats at a dose of 180 mg/kg

body weight was observed to reduce the activities of all those

enzymes both in the plasma and in the intestine of surviving

diabetic rats. The results indicate that, when compared with

82 S. BelHadj et al. Arch Physiol Biochem, 2013; 119(2): 81–87

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that of the untreated diabetic rats, the inhibitory effect of

a-amylase and maltase activities decreased and the glucose

concentration in the plasma underwent a reduction by 43%

after the administration of ULPS to surviving diabetic rats.

In non-diabetic rats, the administration of ULPS has no side

effects and values of all indices are identical to those of

control rats (Figure 1).

Effect of ULPS on blood glucose level and oralcarbohydrate tolerance test (OCTT) in diabetic rats

With the intent to assess the effect of orally administered

ULPS on systemic glucose homeostasis and to confirm the

potential inhibitory action of key mucosal intestinal enzymes

on carbohydrate digestion, we performed an oral glucose,

starch and maltose tolerance test in conscious fasted rats after

ULPS administration. These results clearly showed that acute

oral administration of ULPS reduced significantly peak

glucose concentration 60 min after glucose, starch and

maltose administration, as compared with untreated diabetic

rats (Figure 2).

Effect of ULPS on Lipase activity in plasma andintestine; and oral lipid tolerance test in diabetic rats

Figure 3 indicates that compared with the control, there was a

significant increase in the activity of the lipase plasma and

mucosal small intestine of diabetic rats. However, after the

administration of ULPS to surviving diabetic rats, a consid-

erable reduction in plasma and intestinal lipase activity by 51

and 48% was observed, which leads to a significant decrease

in TC, LDL-C and TG by 30, 51 and 33% and increase of

HDL-C by 48%. In non-diabetic rats treated with ULPS,

lipase activity is similar to that of control.

An OLTT was performed in all rats at the end of the

nutritional intervention period. Rats were fasted for 12 h

Figure 1. Effect ULPS on small intestine and plasma a-amylase and maltase activities of control and experimental groups of rats. Values arestatistically presented as follows: *p50.05 significant differences compared to controls. @p50.05 significant differences compared to diabetic rats day0. &p50.05 significant differences compared to diabetic rats day 30. $p50.05 significant differences compared to diabetic rats day 30 treated withULPS.

DOI: 10.3109/13813455.2013.775159 Therapeutic effect of Ulva lactuca polysaccharides on diabetes 83

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before the test and blood samples were taken from the tail

vein for the determination of TG level. Then, an oral oil

challenge (2 g/kg body weight) was given by gavage and

plasma samples were taken at 3, 6, 9 and 12 h after oil

administration. The plasma TG level was increased 3 and 6 h

after oral administration of the ULPS, this being followed by a

decrease 6 h after the administration. The addition of 50 mg/

kg of ULPS significantly suppressed the elevation of plasma

TG level for 3 h after oil gavage, its effect was the strongest at

9 h after oil administration.

Liver functions of control and treated diabetic rats

Table 1 demonstrates that, compared with non-diabetic rats,

the diabetic rats undertook an increase in terms of the AST,

ALT, LDH and GGT activities by 88, 83, 63 and 44%

respectively in plasma (Table 1). Interestingly, the adminis-

tration of ULPS to surviving diabetic rats seems to have

reverted back this increase and ameliorated all indices related

to liver dysfunction induced by diabetes. Findings from

further histological analyses were found to confirm the

positive effect of ULPS. In fact, no histological injuries were

evidenced in the liver of normal rats at both the beginning

(Figure 4B) and the end of experimentation (Figure 4C), as

compared to normal rats (Figure 4A). As shown in

Figure 4(C), fatty cysts, indicated by arrow, appeared in the

hepatic tissues of diabetic rats. However, the administration of

ULPS to surviving diabetic rats protects liver tissues

(Figure 4D).

Kidney functions of control and treated diabetic rats

Table 1 evidenced that the administration of ULPS to

surviving diabetic rats seems to have reverted back the

increase of plasma creatinine and urea and the decrease of

albumin levels as compared to untreated diabetic rats.

Figure 2. Blood glucose level and oral glucose, starch and maltose tolerance test on control and experimental groups of rats. Statistical analyses asgiven in caption to Figure 1.

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Figure 3. Activities of lipase in small intestine and plasma and oral oil tolerance test of control and experimental groups of rats. Statistical analyses asgiven in caption to Figure 1.

Table 1. Effect of ULPS on liver dysfunction indices (AST, ALT and LDH activities in plasma) and metabolic disorders (T-Ch, TG and LDH-Ch inplasma) in diabetic rats.

Diab0 Con30 Diab30 DiabþULPS ULPS

Liver dysfunction indicesAST (U/L) 160� 21* 129.6� 13 179� 16*# 130.8� 8�# 125.9� 14ALT (U/L) 78.8� 9* 47.8� 2.8 88.1� 14* 49.3� 7.3�# 41.8� 3.6LDH (U/L) 1085� 78* 876� 65 1435� 221*# 942� 75�# 789� 54GGT (U/L) 7.09� 1.78 6.76� 0.78 9.76� 3.91 6.21� 1.43 5.81� 0.67

Kidney toxicity indicesCreatinine (mg/L) 25.7� 2.1* 20.6� 1.3 34.1� 2.6*# 26.8� 1.8�# 17.6� 2.1Urea (g/L) 1.13� 0.09* 0.84� 0.03 1.61� 0.26*# 1.03� 0.09�# 0.67� 0.06Albumin (g/L) 31.7� 2.4* 47.1� 2.1 24.3� 2.6*# 39.8� 3.4�# 51.1� 3.4

Lipid profileT-Ch (g/L) 1.47� 0.09* 1.23� 0.12 2.30� 0.12*# 1.61� 0.12*� 1.56� 0.09HDL-Ch (g/L) 0.61� 0.06* 0.68� 0.06 0.49� 0.06*# 0.73� 0.05*�# 0.95� 0.08LDL-Ch (g/L) 0.86� 0.09* 0.55� 0.15 1.81� 0.12*# 0.88� 0.12� 0.61� 0.11TG (g/L) 1.22� 0.19* 0.91� 0.17 1.59� 0.26*# 1.04� 0.17*�# 0.87� 0.08

Values are statistically presented as follows: *p50.05 significant differences compared with controls.�p50.05 significant differences compared with diabetic rats day 0.#p50.05 significant differences compared with diabetic rats day 30.

DOI: 10.3109/13813455.2013.775159 Therapeutic effect of Ulva lactuca polysaccharides on diabetes 85

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The positive effect of ULPS was confirmed by histological

analyses. In fact (see Figure 5B), a diabetic rat at day 0 and

(Figure 5C) a diabetic rat at day 30 showed histopathological

changes (e.g. capsular space shrinkage and glomerular

hypertrophy) as compared with a control rat (Figure 5A);

however after ULPS administration to surviving diabetic rats

(Figure 5D) a potential protective action was shown.

Discussion

Interestingly, the present study showed that the administration

of an ULPS supplement to surviving diabetic rats potentially

inhibited a-amylase in small intestine and plasma, which is

the key enzyme responsible of the breakdown of starch into

oligosaccharides by catalysing hydrolysis of a-1,4-glucosidic

linkages. These ULPS also inhibited disaccharidases respon-

sible of the breakdown of the disaccharides into simple

sugars, readily available for intestinal absorption. Moreover,

the findings indicate that the administration of ULPS to

surviving diabetic rats significantly decreased the maltase and

sucrase activities in both plasma and intestine, present as key

enzymes and involved in the conversion of oligosaccharides

non absorbable into monosaccharides absorbable in the small

intestine. The inhibitory effects of a-amylase, maltase and

sucrase seem to have limited the process of carbohydrate

hydrolysis and absorption in the intestine (Kasabri et al.,

2010; Wang et al., 2010; Ye et al., 2010). These results are

confirmed by oral tolerance test, which reported that ULPS

ameliorated oral glucose, starch and maltose tolerance test

and established the inhibitory effect of ULPS on key enzymes

related to hyperglycaemia (Goda et al., 2007). In fact in this

investigation, ULPS administration to surviving diabetic rats

suppressed the elevation of blood glucose after oral starch,

glucose, maltose and sucrose ingestion in diabetic rats

suggesting that glucose absorption in small intestine is

delayed.

In addition, the administration of ULPS to surviving

diabetic rats also exerted in vivo inhibitory effects on the key

enzymes of lipid digestion and absorption. In fact, it has been

reported that inhibition of intestinal lipase activity exerts a

therapeutic action on hyperlipidemia, obesity and heart

diseases (Birari & Bhutani, 2007; Sheng et al., 2006). The

enzyme is secreted by the pancreas, transported to small

intestine and hydrolyses non absorbable triglycerides into

simple glycerol and absorbable fatty acids by the small

intestine (Halpern & Mancini, 2003). Therefore, intestine

lipase inhibitors are considered to be a valuable therapeutic

reagent for treating diet-induced obesity in humans (Weigle,

2003). The reduction of lipase activity in pancreas is related

to the amelioration of oral lipid tolerance test. In fact, it has

been reported that alimentary complex lipids are then

converted into triglycerides and free fatty acid by lipase

located in the small intestine and the lipase inhibitors may

reduce postprandial plasma lipid level via retarding the

liberation of TG and free fatty acids and their absorption

(Cairns, 2005; Srivastava & Srivastava, 2004). The thera-

peutic action of the ULPS is confirmed by lower level of TG

and total-cholesterol and higher rate of HDL-cholesterol in

plasma of diabetic rats. Actually, pancreatic lipase inhibition

is one of the most widely studied mechanisms used to

determine the potential efficacy of natural products as

hypolipidemic and anti-cardiovascular diseases agents.

The anti-diabetic and anti-hyperlipidemia actions of ULPS

prevent liver-kidney dysfunctions and showed a decrease of

the plasma indices of hepatic and renal toxicities such as AST,

ALT, LDH, creatinine and urea. Moreover, the ameliorative

action of ULPS in liver-kidney functions was confirmed by

reverting back the appearance of fatty cysts in liver and fatty

Figure 5. Histopathological studies of kidney in the control andexperimental groups of rats. Section of the kidney from (A) a controlrat showing normal architecture; (B) a diabetic rat at day 0 and (C)diabetic rat at day 30 showing histopathological changes (e.g. capsularspace shrinkage and glomerular hypertrophy); (D) diabetic rat treatedwith ULPS, potential protective action was shown.

Figure 4. Histopathological studies of liver in the control and experi-mental groups of rats. Section of the liver from: (A) a control rat showingnormal architecture; (B) a diabetic rat at day 0 and (C) diabetic rat at day30 showing fatty cysts apparition in liver tissues; (D) diabetic rat treatedwith ULPS, potential protective action was shown.

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infiltration in kidney of surviving diabetic rats (Figures 4D

and 5D).

Conclusion

The present study has demonstrated that ULPS significantly

improved glucose and lipid homeostasis in diabetes by

delaying carbohydrate and lipid digestion and absorption.

ULPS therefore represents a potentially useful dietary adjunct

for the treatment of diabetes, obesity and a potential source

for the discovery of new orally active anti-diabetic agent(s).

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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DOI: 10.3109/13813455.2013.775159 Therapeutic effect of Ulva lactuca polysaccharides on diabetes 87

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