Phytochemical Analysis and Antioxidant Property of Rhizome extracts aqueous of Phragmites australis in Alloxan Diabetic Rats

 

Derouiche Samir, Azzi Manel, Hamida Abir

Department of Cellular and Molecular Biology, Faculty of Natural Science and Life,

University of Echahid Hamma Lakhdar -Eloued, El-oued 39000, El-oued, Algeria

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

 

ABSTRACT:

The present study was aimed to investigate the effect of phragmites australis aqueous extracts on lipid peroxidation and antioxidant status in some tissues of alloxan induced diabetic rats. In our experiment study, rats were divided into three groups of five animals in each; Control, diabetic and diabetic + 200 mg/kg extract plant groups. All rats except those in the control group were made diabetic using a single intraperitoneal injection of 150 mg/kg alloxan. Administration of extract began four days after induction of diabetes and lasted for three weeks. The results of the phytochemical analysis revealed that Total phenol and flavonoid content in P. Australis aqueous extract shows highest concentration in aqueous extract of P. australis (34.10mg GA EQ/gm, 3.27mg QEQ/gm). contained various bioactive compounds, including tanin, terpenoids, glycosides and flavonoids. Administration of (200mg/kg body weight/day) extracts in diabetic rats has remarkably improved the elevated levels of fasting blood glucose, triglyceride and LDL concentration. A significant decrease in lipid peroxidation (p<0.001) and small increase in the reduced glutathione (GSH) contents in kidney tissue of diabetic rats were observed. Our studies demonstrate the anti-hyperglycemic and anti-oxidative potential of phragmites australis, which could exert beneficial effects against the diabetes and associated free radicals complications in kidney tissue.

 

KEYWORDS: Phragmites Australis, diabetes, stress oxidative, Alloxan.

 

 

 

 


1. INTRODUCTION:

Diabetes affects more than 6.6% of the world's population according to data from the International Diabetes Federation [1]. it remains a devastating disease, with a huge cost in terms of human suffering and health care expenditures [2]. Diabetes is a heterogeneous group of metabolic diseases that occurs when the pancreas does not produce enough insulin or when the body cannot effectively use insulin or both [3].

 

Many complications of chronic hyperglycemia of diabetes often occur later as damage in kidneys, heart, eyes, nerves and blood vessel [4]. These complications are long supposed to be related to the chronic elevation of glucose which induces oxidative stress by several mechanisms, the glycemic balance therefore plays a very important role in the prooxidant / antioxidant balance [5]. Medicinal plants have therapeutic power through the antioxidant properties against free radicals, such as vitamins, polyphenol and flavonoids and other secondary metabolites [6]. Several plants have been described as treatment for diabetes [7]. Therefore, medicinal plants have hypoglycaemic compounds, which makes it possible to discover new pharmaceutical molecules and food supplements with a therapeutic effect [8]. Medicinal plants have the capacity to produce secondary metabolites which represent a major source of molecules that can be used by humans, in particular in the pharmacological field [9]. Phragmites australis, commonly known as Gueseb, is a weed mainly found in unwooded wetlands such as at the edge of a stream [10]. The basis of our choice for this plant where it is used as an antidiabetic treatment in Sahara of Algeria. The aim of this study was to investigate the effect of aqueous extracts of phragmites australis on stress oxidative and lipid peroxidation in some tissue of alloxan induced diabetic rats.

 

2. MATERIALS AND METHODS:

2.1. Collection and extraction of rhizomes material:

Fresh rhizomes of the plants were collected in October from a village in Touggourt of Ouargla state, Algeria. 10 g of powder of the rhizomes dissolved in 100ml of hot distilled water to be macerated for 24hours at room temperature. After filtration on a muslin cloth, then on filter paper, this filtrate was then evaporated to dryness under reduced pressure at 65°C. using a rotary evaporator.

 

2.2. Animals:

The study is performed on 12 albino Wister rats aged 2-4 months and weighting between 250 and 375g at the beginning of the experiment, these animals brought from the Pastor Institute of Algeria, are launched in faculty of sciences of nature and life, the University of El-Oued,

 

2.3. Treatment of Animals:

After induction of diabetes, diabetic and non-diabetic rats were divided into three groups of four rats each, and kept in the same conditions:

Group 1: control receiving a standard diet with normal drinking water for 21 days.

Group 2: diabetics receiving a standard diet with normal drinking water for 21 days.

Group 3: diabetic + AEPA: diabetic rats treated with aqueous extract of Phragmites australis (200mg /kg b.w.) for 21 days.

 

2.4. Blood collection and preparation of tissue samples:

At the end of 21 days of AEPA treatment, rats were fasted for 16h, decapitated and blood samples were transferred into ice-cold centrifuge tubes. The serum was prepared by centrifugation, for 10min at 3000 revolutions/min and utilized for biochemical assays. Liver, kidney, heart and pancreas from each rat was removed immediately and stord in – 20°C until use for oxidative stress evaluation.

 

 

 

2.5. Determination of the total Phenol content:

The determination of the total polyphenols was carried out according to the Folin-Ciocalteu (FC) method [11]: 100μl of Phragmites australisextract are mixed with 500 μl of the FC reagent and 400μl of Na 2 CO 3 at 7.5% (w /v). The mixture is stirred and incubated in the dark and at room temperature for ten minutes and the absorbance is measured at 760nm by a UV spectrophotometer. The results are expressed in mg gallic acid equivalent/g of dry vegetable material with reference to the calibration curve of gallic acid.

 

2.6. Determination of the total flavonoid content:

The determination of total flavonoids was carried out according to the method described by Dehpeur et al. [12]: 500μl of each extract to be analyzed are added to 1500μl of 95% methanol, 100μl of 10% (m/v) AlCl 3, 100μl of 1M sodium acetate and 2.8ml of distilled water. The mixture is stirred and then incubated in the dark and at room temperature for 30minutes. The blank is made by replacing the extract with 95% methanol and the absorbance is measured at 415nm using a UV spectrophotometer. The results are expressed in mg equivalent quercetin/g of dry vegetable material with reference to the standard curve of quercetin.

 

2.7. Estimation of oxidative stress markers:

In our study, the determination of MDA concentration, carbonyl compounds such as malondialdehyde react with thiobarbituric acid (TBA) to yield absorbent pink chromophores at 532 nm according to the method of YAGI., 1976 [13]. The glutathione was determined using a colorimetric method of (Weckbercker and Cory., 1988) [14] by a spectrophotometer, the measurement of optical density results from the formation of 2-nitro-5-mercocapturic acid from the reduction of dithio-bis-2-nitrobenzoic acid which is called Ellman's reagent with SH groups exist in GSH. The enzymatic activity of glutathione peroxidase (GPx) was measured by the method of (FLOHE and GUNZLER 1984) [15]. This method is based on the reduction of hydrogen peroxide (H2O2) in the presence of reduced glutathione (GSH), the latter is transformed into (GSSG) under the influence of the GPx.

 

2.8. Statistic study:

Data were reported as mean± SEM. Results comparisons were carried out by using 1-way analysis of variance followed by the Student t test to compare means among the groups. Differences were considered statically significant at p<0.05.

 

3. RESULTS:

3.1. Phenolic and flavonoid compounds:

The total phenolic and flavonoids compounds was expressed in terms of Gallic acid equivalents (mg GA eq/gm dry wt) and of Quercetin equivalents (mg QE/gm dry wt) respectively, using the following equation based on the calibration curve: Y = 0.0094x - 0.0905 R2 = 0.997 for phenolic compounds and Y = 0.040x + 0.291 R2 = 0.995 for flavonoids compounds. Total phenolic and flavonoids contents are represented in Table 1.

 

Table 1: Total phenol and flavonoids contents in aqueous extract of rhizome Phragmites australis.

Plant

Total phenol content

mg GA eq/gm dry wt

Flavonoid content

mg Quer eq/gm dry wt

Phragmites Australis

15.75±0,775

4.85±0,154

 

3.2. Liver oxidative stress markers:

Our results (table 2) show a significant increase (p<0.05) in lipid peroxidation (MDA) and a significant decrease (p<0.001) in GSH levels and no change in GPx activity in diabetic rats as compared with control. The treatment of diabetic rats with aqueous extract of Phragmites australis induces a significant decrease (p<0.05) in liver MDA level and an improvement in liver GSH and GPx activity in rat compared to diabetic rats.

 

Table 2: Oxidative stress parameters in liver of control and experimental groups

Parameters

MDA

(nmol/gtissus)

GSH

(nmol/g tissus)

GPx

(UI/mg Pr)

Control

1.386±0.257

2.532±0.535

1.57+0.03

Diabetic

1.529±0.139*

1.548±0.140***

1.25+0.17

Diabetic + AEPA

1.481±0.350 a

1.828 ±0.350a

6.35+0.82*a

Data are expressed as mean± SD (n=4).

*p<0.05, **p<0.01, ***p<0.001: significantly different from control, ap<0.05, b p<0.01, c p<0.001: significantly different from diabetic,

 

3.3. Heart oxidative stress markers:

Our results show that cardiac GSH concentration is significantly decreased (p<0.01) and there is no significant variation in the level of MDA and GPx activity in diabetic rats compared with control. Treatment with the extract of Phragmites australis increased the level of GSH (p<0.01) and improvement of MDA level and GPx activity in the heart of rats in comparison with diabetic rats (table 3).

 

Table 3: Oxidative stress parameters in heart of control and experimental groups

Parameters

MDA

(nmol/g tissus)

GSH

(nmol/g tissus)

GPx

(UI/mg Pr)

Control

1.647±0.392

1.956±0.249

0.96+0.28

Diabetic

1.621±0.407

1.589±0.043**

1.40+0.02

Diabetic + AEPA

0.902±0.170**b

2.191±0.284b

6.65+1.12***c

Data are expressed as mean± SD (n=4).

*p<0.05, **p<0.01, ***p<0.001: significantly different from control, ap<0.05, b p<0.01, c p<0.001: significantly different from diabetic,

 

3.4. Kidney oxidative stress markers:

Obtained results (table 4) show an increase in MDA level (p<0.05) and no change in GSH concentration and in GPx activity in kidney of diabetic rats as compared with control. The treatment of diabetic rats with aqueous extract of Phragmites australis induces a decrease in liver MDA level (p<0.001) and an improvement in kidney GPx activity as compared to the diabetic rats.

 

Table 4: Oxidative stress parameters in kidney of control and experimental groups

Parameters

MDA

(nmol/g tissus)

GSH

(nmol/g tissus)

GPx (UI/mg Pr)

Control

2.27±1.36

2.42±0.40

1.10+0.06

Diabetic

3.55±0.69*

2.069±0.13

1.45+0.3

Diabetic + AEPA

2.30±±0.079c

2.2±0.1

8.54+0.7***c

Data are expressed as mean± SD (n=4).

*p<0.05, **p<0.01, ***p<0.001: significantly different from control, ap<0.05, b p<0.01, c p<0.001: significantly different from diabetic

 

3.5. Pancreas oxidative stress markers:

Our results show that MDA level is significantly decreased (p<0.001) and GSH concentration (p<0.001) and GPx activity (p<0.05) are significantly decreased in pancreas of diabetic rats compared with control. Treatment with the extract of Phragmites australis increased the level of GSH (p<0.01) and improvement GPx activity and no change of MDA level in the pancreas of rats in comparison with diabetic rats (table 5).

 

Table 5: Oxidative stress parameters in pancreas of control and experimental groups

Parameters

MDA

(nmol/g tissus)

GSH

(nmol/g tissus)

GPx

(UI/mg Pr)

Control

1.60±0.099

4.31±1.54

1.73+0.26

Diabetic

1.80±0.013***

1.5±0.029***

1.03+0.28*

Diabetic + AEPA

1.37±0.496 a

3.033±0.70b

4.46+4.41***c

Data are expressed as mean± SD (n=4).

*p<0.05, **p<0.01, ***p<0.001: significantly different from control, ap<0.05, b p<0.01, c p<0.001: significantly different from diabetic

 

4. DISCUSSION:

The results of the quantitative phytochemical analysis carried out on the aqueous root extract of Phragmites australis showed the presence of certain bioactive compounds such as flavonoids and polyphenols with high concentration. Flavonoids are naturally occurring compounds derived from plants with variable phenolic structures [16]. The flavonoids act on the complications of diabetes by their antioxidant and anti-enzymatic power, neutralizing the effect of free radicals and limiting the inflammatory reaction in the different tissues [17]. Our result shows an increase in MDA lipid peroxidation marker in kidneys and pancreas in diabetic rats. The oxidation of lipids makes the membranes more rigid and therefore less fluid [18]. The increase of MDA plays a very important role in the alteration of γ-glutamylcysteinesynthetas (γ-GCS), the enzyme responsible for glutathione synthesis [19] in this context our results show a decrease of GSH in different tissues. Explained this result by the state of diabetes induced by Alloxan, glucose is used by the polyol route and turns into sorbitol, this reaction consumes NADPH, the latter is essential for the regeneration of glutathione molecules by the enzyme glutathione reductase which also explains the decrease in glutathione in diabetics [20]. Increase of MDA could be the result of a significant modification of the cellular redox status in the pancreas in favor of prooxidants, since Alloxanhas been found to generate free radicals which, by their oxidizing power, are at the origin of oxidation DNA, lipids and carbohydrates leading to the death of β cells and the onset of diabetes [21]. On the other hand Alloxan rats treated with the aqueous extract of the Phragmites australis which may contains cellular chemoprotective compounds provided with an antiperoxidant power playing an effect against the deleterious effect of the free radicals, which explains the maintenance of MDA and GSH at its levels in the organs studied (liver, heart, kidney and pancreas).polyphenols and flavonoids, have been studied as a nutrient well known for their anti-inflammatory and antioxidant effects [22]. Also, there is a significant increase in GPx and tissue SOD activity, therefore, a large enzymatic improvement in oxidative stress in treated versus untreated diabetic rats. On the other hand, according to the study by Shahid et al (2014) [23]. Phragmites australis contains Mn, Zn and Cu, these are essential metals in defense against oxidative stress. the richness of Phragmit australis by phenol and flavonoids gave it an antioxidant character.

 

5. CONCLUSION:

The evidences suggest that Phragmites australis have a hypoglycemia and beneficial effect for the protection against metabolic disturbed and diabetic complications.

 

6. CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

 

7. ACKNOWLEDGEMENTS:

we would like to thank the Faculty of Sciences of Nature and Life, University of El Oued, Algeria for the permission to utilize the facilities to make this work. Also We thank members of Algiers Pasteur Institute for providing the rats.

 

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Received on 23.09.2019            Accepted on 17.10.2019           

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2019; 9(4):249-252.

DOI: 10.5958/2231-5713.2019.00041.2