Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats

ARTICLE IN PRESS beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

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Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats

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Q1

Pramod Mourya a, Ajay Shukla a, Gopal Rai a, Santram Lodhi b,* a

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b

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Department of Pharmacy, Guru Ramdas Khalsa Institute of Science and Technology, Barela, Jabalpur, M.P. India Department of Pharmacy, Smt. Sharadchandrika Suresh Patil College of Pharmacy, Chopda, Jalgaon (M.S.) 425107, India

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A R T I C L E

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I N F O

A B S T R A C T

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Article history:

The objective of present study was to investigate hypoglycemic and hypolipidemic effect of

Received 6 September 2016

ethanolic and aqueous extracts of Ziziphus oenoplia (L) stem bark against Alloxan induce hy-

Received in revised form 26

perglycemia in rats. Hyperglycemia was induced by an injection of alloxan monohydrate

November 2016

150 mg/kg (i.p.). After 72 hr, the rats having Blood Glucose Level (BGL) above 150 mg/dL were

Accepted 10 December 2016

selected for the investigation. At two different doses (200 mg/kg and 400 mg/kg b.w.) of aqueous

Available online

and ethanolic extracts were observed antidiabetic effect for 12 consecutive days. BGL was

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monitored after 1, 3, 6 and 12 days and compared with Metformin (250 mg/kg). Alpha amylase

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Keywords:

and alpha glucosidase activity of both extracts were also determined. Phytochemical study

Alloxan

revealed the presence of glycosides, flavonoids, alkaloids and terpenoids in ethanol extract

Metformin

and flavonoids, carbohydrates and proteins found in aqueous extract of Z. oenoplia bark. Oral

Diabetes mellitus

administration of both extracts showed significant (P < 0.05) antihyperglycemic activity in

Ziziphus oenoplia

dose dependent manner in alloxan induced diabetic rats. The diabetic rats had significant (P < 0.01) reduction in blood glucose; serum liver enzyme level (AST, ALT, and ALP) and lipid

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profile were compared with normal rats. Significant effects of aqueous and alcoholic extract

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in alpha amylase and alpha glucosidase activity were observed in rats. The ethanolic and

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aqueous extract reveals the reduction in the blood glucose level, inhibition of alpha amylase

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and alpha glucosidase enzymes which support antidiabetic effect (reduce postprandial glucose

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levels) of Z. oenoplia and this may be due to presence of flavonoids constituents.

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© 2016 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is

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an open access article under the CC BY-NC-ND license (http://creativecommons.org/

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licenses/by-nc-nd/4.0/).

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1.

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Introduction

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Diabetes mellitus is a syndrome resulting from a variable interaction of hereditary and environmental factors, characterized

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by damaged of β-cells from pancreas and complication of vascular disease. Diabetes is a major and one of the most common chronic diseases in the world (Vinuthan et al., 2007). It is a group of metabolic disease characterized by hyperglycemia, altered metabolism of lipids, carbohydrate and protein; this may be

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Q2

* Corresponding author. S. Lodhi, Department of Pharmacy, Smt. Sharadchandrika Suresh Patil College of Pharmacy, Chopda, Jalgaon (M.S.) 425107, India. E-mail address: [email protected] (S. Lodhi). http://dx.doi.org/10.1016/j.bjbas.2016.12.002 2314-8535/© 2016 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

due to defects in insulin secretion, insulin action, or both

Q4 (Mourya et al., 2014). Type 2 diabetes mellitus is by far the commonest form of the diseases globally, with developing countries being at the forefront as far as the epidemic is concerned (Pepato et al., 2005; Tiwari and Rao, 2002). There are different types of oral hypoglycemic agents existing along with insulin for the treatment of diabetes mellitus, but due to some complications there is an increasing demand by the patients to use natural products for lowering blood glucose level (Hara and Honda, 1990). Lower level of insulin in diabetic patients leads to increased amino acid level in the blood. It results to increase in transaminase activity (increased level of AST and ALT) that will result in ketogenesis and gluconeogenesis (Amraie et al., 2015). In type 2 diabetes, postprandial hyperglycemia plays an important role in developing stage. There are two major carbohydrate hydrolyzing enzymes i.e. α-amylase and α-glucosidase which are responsible for postprandial hyperglycemia. Alpha-amylase begins the carbohydrate metabolism by hydrolysis of 1, 4-glycosidic linkages of polysaccharides to disaccharides and alpha-glucosidase catalyzes the reaction in which disaccharides convert into monosaccharides, which promote to postprandial hyperglycemia (Shukla et al., 2011). The use of herbal medicines has a long tradition for the management of blood glucose abnormalities. Therefore the researchers continue looking for more effective and safer hypoglycemic agents from natural source (Bhati et al., 2014). Ziziphus oenoplia (L) Mill is also known as Rhamnus oenoplia or Jackal Jujube (family Rhamnacae) distributed throughout the tropical region of India, and Australia. A straggling shrub often semi scan dent by its prickles, and young branches are rusty. Leaves numerous, 2.5–6.5 by 2–2.5 cm, ovate, acute to mentose tips, galabarous, densely silky with hair, petioles 6–8 mm long flowers 12–20 sub sessile calyx hairy outside, petals obviate. Fruits are edible. The bark, fruits, leaves and stems of plant are extensively used in the rural area for stomach, hypotensive, diuretic, wound healing, antibacterial, anti-inflammatory and analgesic (Kirtikar and Basu, 2005). The stem bark of Z. oenoplia Q5 was reported for antioxidant activity (Sameera et al., 2015). Z. oenoplia reveals the presence of alkaloids, flavonoids, phenolic compounds and terpenoids which may be responsible for its medicinal efficacy. Chemical investigation of Z. oenoplia roots has shown the presence of cyclopeptide alkaloids such as Q6 Ziziphine (Suksmrarn et al., 2005; Shukla et al., 2016). Bark and fruits of Z. oenoplia have been used traditionally for antidiabetes mellitus (Prabhavathi and Vijayalakshmi, 2015; Subramoniam, 2016). It is reported that a formulation containing Z. oenoplia along with other species is used for treatment of diabetes and its complications (Krishnan, 2011). The aim of the present study was to investigate the effects of Z. oenoplia stem barks in type 2 diabetes model against alloxan induced diabetic rats to ascertain the traditional claim of plant.

of ABS botanical garden, Salem, Tamil Nadu. Voucher samples were deposited in the herbarium for reference (ZE/221). The fresh stem barks were dried under shade sliced into small pieces and pulverized into coarse powder with mechanical grinder. The powder was passed through sieve no.10 and kept in polythene bags at room temperature for extraction.

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2.2.

Extraction of plant material

The dried coarse powder of Z. oenoplia (L) mill stem barks were defatted with petroleum ether (60–80 °C) in a Soxhlet apparatus by continuous hot extraction. The defatted powder material (marc) thus obtained was further extracted with ethanol (95% v/v) with same method and fresh powder was used with chloroform water extraction by cold maceration method up to complete extraction for 24 hrs. The solvent was removed by distillation under low pressure and evaporation. The resulting semisolid mass was vacuum dried by using rotary flash evaporator. Qualitative analysis of different extracts was carried out to find out the presence of various phytoconstituents (Kokate et al., 2010).

2.3.

Determination of total phenolic content

The total phenolic content in aqueous and ethanolic extracts was determined by colorimetric method with Folin–Ciocaletu reagent (Bozin et al., 2008). A reaction mixture contain 500 μL of 0.1% aqueous dilution of both extracts, 2.5 mL of freshly prepared 0.2M FC reagent and 2 mL of sodium carbonate solution. The mixture was kept in the dark under ambient conditions for 30 min to completion of reaction. Absorbance of the resulting solution was measured at 760 nm in a UV–vis spectrophotometer (Shimadzu, USA). The total phenolic content was expressed as mg of gallic acid equivalents per gram of extracts, using a standard curve of gallic acid (Sigma Aldrich Chemicals Pvt Ltd, Mumbai, India).

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2.

Material and methods

2.1.

Plant collection and preparation

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The plants Z. oenoplia (L) mill were collected from Salem district, Tamil Nadu, India. The plant was identified and authenticated by the botanist Mr. Balsubramanyam, Director

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2.4.

Total flavonoids content

Total flavonoids content from ethanol and aqueous extract was determined by aluminum chloride colorimetric assay method (Park et al., 2008). A test tube containing 0.3 mL of extract, 3.4 mL of 30% methanol, 0.15 mL of NaNO2 (0.5 M) and 0.15 mL of AlCl3.6H2O (0.3 M) was shake up to complete mixing. One milliliter of NaOH (1 M) was added after 5 min, with mixing well and the absorbance was measured at 510 nm. The standard curve of quercetin (Sigma Aldrich Chemicals Pvt Ltd) was made and the total flavonoids content was expressed as milligrams of quercetin equivalents per 100 gm of dried extract.

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Acute toxicity study

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2.5.

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Healthy Wistar albino rats of either sex were divided into two groups, each consisting of five rats. The animals were orally fed extracts in increasing dose levels of 100 to 4000 mg/kg body weight. The study was carried out according to OECD guidelines– 423. All animals were observed continuously for behavior changes up to 2 h (Bhandarkar and Jain, 2015; OECD Guideline, 2001).

Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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2.6.

Animals and experimental design

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Wistar albino rats (150–200 g) of either sex procured from Sri Venkateswara Enterprises, Bangalore, India were used for the experiment. The animals were fed with standard pellet diet (Hindustan lever Ltd. Bangalore) and they were kept under standard environmental conditions of laboratory temperature and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of darkness and light at 12 hours. They were acclimatized to the laboratory condition for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The experimental protocols were approved by Institutional Animal Ethics Committee (IAEC No. P. Col./16/2006). They were divided into seven groups of six animals in each group. Normal control group (I) received only normal saline while standard group (II) received Metformin hydrochloride (Sigma Aldrich Chemicals Pvt Ltd; 250 mg/kg bw) in alloxan (Sigma Aldrich Chemicals Pvt Ltd) treated rats. Diabetic control group (III) received only alloxan (150 mg/kg i.p.). Test group (IV–V) received aqueous extract of Z. oenoplia stem bark (200 and 400 mg/kg bw) in alloxan treated rats. Another test groups (VI–VII) received ethanolic extract of Z. oenoplia stem bark (200 and 400 mg/kg bw) in alloxan treated rats.

3

of the experiment, rats were anesthetized and sacrificed by decapitation. Samples of the free running blood were collected for measurement of lipid profile. TG was determined by using the glyceryl phosphate oxidase method (Chan et al., 2002). Take 0.01 mL of the serum, 1 mL of auto reagent was added and incubated at 37 °C for 5 min. Absorbance was measured at 500 nm. The content was expressed as mg/dL. Total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL) were determined by standard method. AST, ALT and ALP were estimated by using standard kits (Reitman and Frankel, 1957; Singh et al., 2009). For the study of kidney functions, serum urea concentration was determined according to the method of Fawcett and Scott (1960), while serum creatinine concentration was determined by method of Bartels et al. (1972).

2.9.

Histopathological studies

The microscopic examinations of organs (kidneys and pancreas) were collected from different animal groups and store in 10% formalin. After usual processing 6 μm thick sections were cut and stained with hematoxylin and eosin (Luna, 1996). Sections were qualitatively observed under light microscope.

2.10.

Alpha amylase activity

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2.7.

Alloxan induced diabetes

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Alloxan monohydrate (Sigma Chem. Co., St. Louis, MO, USA; 150 mg/kg i.p.) was dissolved in normal saline and injected i.p. after 18 hr fasting of animals to induce hyperglycemia (Alam et al., 2014). After 1 hr of alloxan administration, the animals were fed on standard pellets and water ad libitum. The blood glucose level was monitored, samples collected under mild anesthesia and blood sugar level was measured with an autoanalyzer by using Accu Check advantage II glucose kit. Surviving rats after 72 hr, the rats having Blood Glucose Level (BGL) above 150 mg/dL were selected for the investigation and divided into 7 groups (I–VII) of 6 rats each (Pillai and Santhakumari, 1981; Kumar et al., 2015). The vehicle and reference drug metformin (250 mg/kg) were administered orally (Bailey et al., 1996; Tadakawa et al., 2015) to animals of groups I, II respectively and III group representing the control receiving only alloxan. Group Nos. IV, V, VI and VII received the aqueous and ethanolic extracts at a dose of 200 mg/kg and 400 mg/kg respectively, for 12 consecutive days and BGL was monitored after 1, 3, 6 and 12 days. The effects of alcoholic extracts in normal and diabetic rats were observed by measuring blood glucose level, serum lipid profile and changes in body weight. Serum lipid profile was determined on day 15 after the animals were sacrificed by decapitation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined by Reitman and Frankel method (Bessey et al., 1946; Gutmann and Bergmeyer, 1974; Hyder, 1957).

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2.8.

Biochemical estimation in blood samples

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Blood samples from each group were collected by amputation of the tail tip of rats. Blood glucose level was measured using a glucometer (AccuChek Active, Germany). At the end

Alpha-amylase inhibitory activity of ethanolic and aqueous extracts was carried out according to the standard method (Matsui et al., 2007). In a 96-well plate, reaction mixture containing 50 μL phosphate buffer (100 mM, pH = 6.8), 10 μL α-amylase (2 U/ mL), and the plant extracts in concentration range 20–100 μg/ mL was pre-incubated at 37 °C for 20 min. Then, the 20 μL of 1% soluble starch (100 mM phosphate buffer pH 6.8) was added as a substrate and incubated at 37 °C for 30 min; 100 μL of the DNS color reagent was then added and boiled for 10 min. The absorbance of the mixture was measured at 540 nm using Multiplate Reader (Multiska thermo scientific, version 1.00.40). Acarbose was used as a standard at concentrations range of 20–100 μg/mL. Each experiment was performed in triplicate manner. The results were expressed as percentage inhibition, which was calculated using the formula:

% α-Amylase Inhibition Absorbance of control − Absorbance of test = × 100 Absorbance of standard

2.11.

Alpha glucosidase activity

Alpha-glucosidase inhibitory activity of ethanol and aqueous extracts was carried out according to the standard method (Ademiluyi and Oboh, 2013). In a 96-well plate, reaction mixture containing 50 μL phosphate buffer (100 mM, pH = 6. 8), 10 μL alpha-glucosidase (1 U/mL), and the both extracts in concentration range 20–100 μg/mL was pre incubated at 37 °C for 15 min. Then, 20 μL P-NPG (5 mM) was added as a substrate and further incubated at 37 °C for 20 min. The reaction was finished by adding 50 μL Na2 CO3 (0.1 M). The absorbance of the released p-nitrophenol was measured at 405 nm using Multiplate Reader. Acarbose as standard at same concentration was included. The results were expressed as percentage inhibition, which was calculated as above mentioned equation.

Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

Table 1 – Preliminary phytochemical investigation of plant extracts of Z. oenoplia (L) Mill. Phytochemical constituents

Ethanol extract

Aqueous extract

3.3. + + + + + + − + + + − +

− − + + + − − + − + + +

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Alkaloids Glycosides Flavonoids Carbohydrate Saponins Triterpens Phytosterols Tannins Fixed oil and fat Phenolic compounds Gun and mucilage Proteins and amino acids

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+ ive (positive) = Present; − ive (negative) = Absent.

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3.4.

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2.12.

Statistical analysis

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Data were obtained and subjected to one way analysis of variance followed by Dunnett’s test to determine the statistical significance change in blood glucose level. P < 0.05 was considered significant.

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3.

Results

3.1.

Phytochemical studies

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The extracts of dried stem bark were subjected for phytochemical screening, which reveals the presence of different compounds in plant extract such as alkaloid, glycoside, flavonoid, saponin, carbohydrate, fixed oil and fat, and tannins (Table 1). The percent yields of ethanol and aqueous extracts were 7.4 and 5.8% w/w, respectively.

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3.2.

Total phenolic and flavonoids content

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Acute toxicity study

Acute toxicity study revealed the non-toxic nature of both extracts. There was no lethality or any toxic reactions found at any selected doses at the end of the study period. Based on these results the doses of 200 and 400 mg/kg were selected for the antihyperglycemic activity.

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flavonoid content was found to be 105.5 ± 3.86 and 124.4 ± 5.37 mg (quercetin equivalent) per 100 g of ethanol and aqueous extract.

The total phenolic content in ethanolic and aqueous extracts was found to be 1.24 ± 0.73 mg gallic acid equivalent/g and 0.85 ± 0.59 mg gallic acid equivalent/g respectively. The total

In vivo studies

The present study was aimed preliminary on assessment of the ethanolic and aqueous extracts of the antidiabetic activity of Z. oenoplia bark for antidiabetic study. The extracts showed a dose-dependent decreases in blood glucose level in alloxan induced diabetic rats. As a result, the dose at 200 and 400 mg/ kg bw was selectively treated to diabetic rats for reducing blood glucose level. The blood glucose levels were determined at the time interval for 9 h after single oral administration with ethanolic and aqueous extracts, respectively (Table 2). There was a significant elevation in the blood glucose levels during the experimental time period in diabetic rats, when compared to normal rats. The administration of ethanolic extract significantly (P < 0.05) reduced the blood glucose levels of diabetic rats at 3rd and 6th days after given alloxan. The sharp reduction in glucose levels after oral administration of the ethanolic extract was showed in a time-dependent manner. Results of lipid profile of rat’s blood confirmed that diabetic rats have recorded higher HDL and LDL level than intact control group (Table 3). Rats that were treated with 200 mg/ kg dose of aqueous and alcoholic extract showed decreased both HDL and LDL levels in comparison to untreated diabetic control. Increase in doses of extract shows also significant decrease in lipid components of rat’s blood. Both extracts also found improvement in the conditions of diabetes as supported by decreasing lipid parameters like total cholesterol and triglycerides. The 200 mg/kg and 400 mg/kg extracts showed lowered significantly lipid profile when compared to diabetic control group. Liver enzymes (AST, ALT and ALP) in blood serum were evaluated for increasing level in liver because these enzyme

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Table 2 – Effect of stem bark of Z. oenoplia (L) Mill extracts on blood glucose level of normal and experimental animals during 12 days treatment. Animal groups

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Dose mg/kg

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Normal control Standard treated metformin + alloxan Diabetic control alloxan only Aqueous extract + alloxan Aqueous extract + alloxan Alcoholic extract + alloxan Alcoholic extract + alloxan

_ 250 + 150 150 200 + 150 400 + 150 200 + 150 400 + 150

Blood glucose level (mg/dL) Initial

1st day

3rd day

6th day

12th day

79.6 ± 5.57 288.5 ± 6.84 281.83 ± 10.68 289.66 ± 6.21 292.66 ± 9.09 290.16 ± 9.51 287.33 ± 5.82

78.83 ± 6.40 264.5 ± 6.97 285.16 ± 10.43 246.66 ± 10.62 241.33 ± 8.93* 243.33 ± 9.35* 239.66 ± 6.12*

77.00 ± 4.83 188.66 ± 7.76* 286.83 ± 10.62 175.16 ± 6.01* 171.5 ± 5.35* 160.50 ± 5.50* 154.83 ± 6.08*

77.83 ± 4.83 132.83 ± 6.89* 290.16 ± 10.16 138.00 ± 9.12* 133.83 ± 8.30* 129.33 ± 5.61* 126.66 ± 5.00*

79.00 ± 5.47 125.83 ± 9.80** 294.66 ± 9.16 130.66 ± 10.23* 128.33 ± 15.14** 123.66 ± 5.70* 119.66 ± 6.60**

Values are expressed in mean ± S.D; each group contains 6 animals (n = 6). * P < 0.05, compared with diabetic control values. ** P < 0.01, compared with diabetic control values.

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Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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ARTICLE IN PRESS 5

beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

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Table 3 – Effect of alcoholic and aqueous extract of Z. oenoplia on lipid profile of different treatment groups of rats. Animal groups

Dose

Total cholesterol (mg/ dL)

Triglyceride (mg/dL)

HDL (mg/dL)

LDL (mg/dL)

_ 150 250 + 150 200 + 150 400 + 150 200 + 150 400 + 150

86.42 ± 3.47 125.27 ± 6.51 85.27 ± 5.34* 94.12 ± 4.28 88.62 ± 4.83* 97.27 ± 4.59* 99.43 ± 3.72*

81.37 ± 4.17 133.84 ± 6.42 78.26 ± 5.27* 90.38 ± 4.69 84.67 ± 4.29* 94.84 ± 4.14* 98.28 ± 3.62*

42.05 ± 2.46 68.61 ± 3.27 57.23 ± 3.13 43.28 ± 3.57 40.26 ± 3.61* 52.20 ± 3.29 48.08 ± 2.16*

38.29 ± 2.35 59.72 ± 3.42 48.64 ± 3.28* 37.51 ± 2.16 33.40 ± 2.47* 47.81 ± 2.08 41.28 ± 2.11*

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Normal control Diabetic control alloxan only Standard treated metformin + alloxan Aqueous extract + alloxan Aqueous extract + alloxan Alcoholic extract + alloxan Alcoholic extract + alloxan

412 413 414

Values are expressed in mean ± S.D; each group contains 6 animals (n = 6). * P < 0.05, compared with diabetic control values.

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levels are increased due to liver cells dysfunction and leakage in liver cells membrane. Results showed a significant increase in these enzyme level of serum in alloxan induced diabetes (Table 4). By the treatment of diabetic rats with aqueous and alcoholic extract the enzyme levels were significantly decreased to nearly normal condition comparable to normal rats. Kidney function profile of different groups is shown in Table 5. Diabetic rats treated with aqueous and alcoholic extract in both doses 200 and 400 mg/kg produce significant decreases in serum urea and creatinine concentration that comparable to standard group. Urea measurements have come to be accepted as giving a means of renal function since 50% or greater of urea filtered to the glamorous which is passively reabsorbed through the tubules. After treatment with aqueous extract with 200 and 400 mg/kg doses highly significant decreases were shown in

serum urea from 85.42 ± 6.62 mg/dL to 68.52 ± 5.70 mg/dL and 55.48 ± 4.27 mg/dL, respectively.The creatinine concentration decreases from 3.5 ± 0.34 to 2.3 ± 0.64 and 1.4 ± 0.07 mg/dL, respectively. Treatment with alcoholic extract (200 and 400 mg/ kg dose) decreases significantly serum urea from 85.42 ± 6.62 mg/ dL to 48.49 ± 4.61 mg/dL and 47.81 ± 4.95, respectively. The creatinine concentration decreased from 3.5 ± 0.34 mg/dL to 1.6 ± 0.09 mg/dL and 1.3 ± 0.05 mg/dL, respectively.

3.5.

Histopathological study

The histopathological examination of kidney and pancreas of alloxan induced diabetic rats has revealed destruction of beta cells and reticular changes of islets as evidence of fibrosis. After few days of treatment with Z. oenoplia extracts at a dose of

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Table 4 – Effect of alcoholic and aqueous extract of Z. oenoplia on liver enzymes in blood serum collected from different treatment groups of rats. Animal groups Normal control Diabetic control (alloxan only) Standard treated metformin + alloxan Aqueous extract + alloxan Aqueous extract + alloxan Alcoholic extract + alloxan Alcoholic extract + alloxan

Doses

AST (U/L)

ALT (U/L)

ALP (U/L)

_ 150 250 + 150 200 + 150 400 + 150 200 + 150 400 + 150

36.51 ± 2.45 127.42 ± 10.27 52.63 ± 4.58** 67.25 ± 7.34* 51.92 ± 7.26** 65.28 ± 6.52* 57.42 ± 5.13**

37.28 ± 2.63 124.27 ± 11.64 58.64 ± 4.27** 71.46 ± 7.54* 52.24 ± 6.53** 66.61 ± 6.24** 48.71 ± 5.76**

43.28 ± 3.59 134.65 ± 12.60 62.41 ± 5.38** 78.51 ± 7.53* 64.01 ± 6.42** 71.29 ± 5.89** 65.21 ± 6.37**

Values are expressed in mean ± S.D.; each group contains 6 animals (n = 6). * P < 0.01, compared with diabetic control values. ** P < 0.01, compared with diabetic control values.

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Table 5 – Effect of alcoholic and aqueous extract of Z. oenoplia on kidney function profile of different treatment groups of rats. Animal groups Normal control Diabetic control alloxan only Standard treated metformin + alloxan Aqueous extract + alloxan Aqueous extract + alloxan Alcoholic extract + alloxan Alcoholic extract + alloxan

Doses

Urea (mg/dL)

Creatinine (mg/dL)

_

45.26 ± 3.17 85.42 ± 6.62 43.67 ± 3.81** 68.52 ± 5.70** 55.48 ± 4.27* 48.49 ± 4.61** 47.81 ± 4.95**

0.7 ± 0.04 3.5 ± 0.34 1.7 ± 0.52** 2.3 ± 0.64* 1.4 ± 0.07** 1.6 ± 0.09** 1.3 ± 0.05**

150 250 + 150 200 + 150 400 + 150 200 + 150 400 + 150

Values are expressed in mean ± S.D.; each group contain 6 animals (n = 6). * P < 0.05, compared with diabetic control values. ** P < 0.01, compared with diabetic control values.

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Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

Fig. 1 – Effect of different extracts on histopathological features of kidney: (A) normal control group; (B) standard treated group; (C) diabetic control group; (D) aqueous extract (200 mg/kg) group; (E) aqueous extract (400 mg/kg) group; (F) ethanolic extract (200 mg/kg) group; (G) ethanolic extract (400 mg/kg) group.

Fig. 2 – Effect of different extracts on histopathological features of pancreases: (A) normal control group; (B) standard treated group; (C) diabetic control group; (D) aqueous extract (200 mg/kg) group; (E) aqueous extract (400 mg/kg) group; (F) ethanolic extract (200 mg/kg) group; (G) ethanolic extract (400 mg/kg) group.

200 mg/kg and 400 mg/kg bw, islets of langerhans showed improvement and restoration of normal cellular population size. The histopathological finding of diabetic kidney and pancreas treated with aqueous and ethanolic extract of Z. oenoplia showed marked improvement after 12 days and minimal pathological changes. The activities of these extracts have triggered the beta cells to increase insulin production which promotes glucose uptake and utilization by other tissue. Histopathology of kidney sections is showed in Figure 1. The control group showed the glomeruli appear normal. The tubules are normal and lined by a single layer of cuboidal cells. The standard group showed that glomeruli show mesangial hypercellularity and focal glomerulosclerosis. The tubules are normal. The stroma shows a diffuse infiltrate of lymphocytes. Diabetic control group was observed with focal segmental glomerulosclerosis in glomeruli. The tubules are normal and lined by a single layer of cuboidal cells. The stroma shows a mild chronic inflammatory cell infiltrate of lymphocytes. As a result of treatment with aqueous and ethanolic extract at

200 mg/kg dose, the glomerulus shows mild mesangial hypercellularity. The tubules and stroma both were normal. Group treated with dose of 400 mg/kg aqueous and ethanolic extract represents the glomeruli and tubules were normal. The stroma shows small focal hemorrhages. The histological observations of pancreas sections were shown in Figure 2. Normal control group shows normal structure of islets and architecture is preserved. The acini are lined by round to oval cells with moderate cytoplasm and small round to oval nuclei. Standard group also showed approx resemble to the normal section. But in case of diabetic control group there is a mild infiltrate of lymphocytes at some foci.The ethanolic extract (400 mg/ kg) treated group showed a dense and diffuse infiltrate of lymphocytes within the stroma. The acini cells are normal.

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Alpha-amylase and alpha-glucosidase activity

Ethanolic and aqueous extracts of Z. oenoplia showed IC50 value of 110.26 ± 5.75, and 118.88 ± 5.14 μg/mL respectively in the alpha

Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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3.6.

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Table 6 – α-Amylase inhibitory effects of different extracts of Z. oenoplia. Extracts

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Aqueous extract Alcoholic extract Acarbose

% Inhibition at different concentrations (μg/mL)

IC50 (μg/mL)

20

40

60

80

100

8.35 ± 0.85 12.42 ± 1.40* 14.73 ± 1.86

28.16 ± 1.87* 30.87 ± 2.62* 29.67 ± 2.46

32.46 ± 2.74* 35.18 ± 2.86* 35.72 ± 2.49

36.34 ± 2.84* 38.18 ± 2.95* 39.64 ± 2.89

40.75 ± 3.14* 45.26 ± 3.5*7 45.81 ± 3.86

118.88 ± 5.14 110.26 ± 5.75 –

Values are expressed in mean ± S.D.; each group contains 6 animals (n = 6). * P < 0.05, compared with reference group.

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Table 7 – α-Glucosidase inhibitory effects of different extracts of Z. oenoplia. Extracts

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Aqueous extract Alcoholic extract Acarbose

% Inhibition at different concentrations (μg/mL)

IC50 (μg/mL)

20

40

60

80

100

23.42 ± 1.87* 26.82 ± 1.79* 27.43 ± 1.29

31.29 ± 2.43* 35.43 ± 2.86* 37.16 ± 2.78

38.71 ± 3.17* 41.10 ± 3.85* 45.08 ± 3.79

46.43 ± 3.73* 49.37 ± 3.82* 49.28 ± 3.74

49.34 ± 3.84* 52.71 ± 3.92* 53.04 ± 4.05

105.28 ± 5.26 93.42 ± 4.85 58.87 ± 3.46

Values are expressed in mean ± S.D.; each group contains 6 animals (n = 6). * P < 0.05, compared with reference group.

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amylase inhibition assay. Our findings also revealed that the ethanolic and aqueous extracts of Z. oenoplia efficiently inhibited alpha-glucosidase enzyme in vitro. The ethanolic and aqueous extracts of Z. oenoplia showed IC50 values of 93.42 ± 4.85 and 105.28 ± 5.26 μg/mL respectively in alpha-glucosidase inhibition assay (Table 6 and 7). The present study indicated that Z. oenoplia could be useful in management of postprandial hyperglycemia.

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4.

Discussion

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The phytochemical screening of extracts reveals the presence of different compounds in plant such as glycosides, flavonoids, alkaloids, carbohydrates, terpenoids and proteins. Alloxan is a beta cytotoxin agent which destroys β-cell of islet of langerhans of pancreas of animal resulting in the reduction of release of insulin which leads to increase in blood glucose level (Dhanabal et al., 2007). There were significant decreases in blood glucose level in alloxan induced diabetic rats treated with Z. oenoplia (L) mill extract for entire period of the experiment. The both alcoholic and aqueous extracts of plant were administered at a dose of 200 mg/kg and 400 mg/kg. After 12 days of treatment with alcoholic extract at dose of 400 mg/ kg, the Blood Glucose Level (BGL) decreases from 287.33 to 119.66 mg/dL. The reduction in BGL by aqueous extract at dose 400 mg/kg from 292.66 to 128.33 mg/dL was observed. The results in decrease in BGL comparable with standard drug metformin which decrease BGL from 288.5 to 125.83 mg/dL. However, the blood glucose levels of diabetic rats treated with the ethanol extract were similar or slightly lower than those of the standard treated group rats, suggesting that hypoglycemic components in the plant are greater solubility in ethanol than aqueous. The difference may be attributed to two reasons. One is the nature of biologically active components that are stable in the ethanol. Second possible reason may be the stron-

ger extraction capacity of ethanol which could have produced a greater number of active components responsible for blood glucose-lowering activity. In the present study, the ethanolic and aqueous extracts from Z. oenoplia showed a significant hypoglycemic effect in the alloxan-induced diabetic rats. Therefore, the mechanism of the both extracts is possibly an insulin-independent mechanism. Alloxan induces diabetes by pancreatic cell damage mediated through generation of oxygen free radicals. The primary target of these radicals is the DNA of pancreatic cells causing DNA fragmentation (Shankar et al., 2007). The histological examination of kidney and pancreas of alloxan induced diabetic rats’ revealed destruction of beta cells and reticular changes of islets as evidence of fibrosis. After few days of treatment with Z. oenoplia (L) mill extracts at a dose of 200 mg/ kg and 400 mg/kg, islets of langerhans showed improvement and restoration of normal cellular population size. The histopathological finding of diabetic kidney and pancreas treated with both extracts of Z. oenoplia Mill showed marked improvement after 12 days and minimal pathological changes.The activities of these plants have triggered the beta cells to increase insulin production which promotes glucose uptake and utilization by other tissue (Sepha and Bose, 1956; Shrotri et al., 1963). Results of present studies confirmed that the insulin deficiency as well as increase in blood glucose level will increase cholesterol and triglyceride levels due to fat storage into the liver. Treatment with aqueous and alcoholic extract may improved insulin level by unknown mechanism that will reduces the fat storage in liver. The liver is major organ that is affected by diabetes. If the liver enzyme activity increased, it may result to liver damage (Amraie et al., 2015). Liver enzymes are good indicators for liver functioning. Diabetic conditions also caused secretion and release of liver enzymes to blood circulation due to destruction of cells by change in membrane structure (Udayakumar et al., 2009). Oral administration of aqueous and alcoholic extracts in 200 and 400 mg/kg doses significantly reduces the enzyme level in blood.

Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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beni-suef university journal of basic and applied sciences ■■ (2016) ■■–■■

Diabetes mellitus is a chronic metabolic disorder, characterized by mainly hyperglycemia and disturbances in carbohydrate, protein, and fat metabolism. It causes fading of insulin production or insulin action. Type 2 diabetes is a disease caused by disturbances between blood sugar absorption and insulin production. Postprandial hyperglycemia plays an important role in the development of Type 2 diabetes (Baron, 1998). Control on plasma glucose level is very important for preventing Type 2 diabetes. A drug or diet can delay the production or absorption of glucose by inhibiting carbohydrate hydrolyzing enzymes such as α-amylase and α-glucosidase. This is an important therapeutic approach for diminishing postprandial hyperglycemia (Tiwari and Rao, 2002). Presently, acarbose is an important carbohydrate metabolic enzyme inhibitor in the gastrointestinal tract, but it has some side effects such as diarrhea and intestinal disturbances. Therefore, natural plant originated inhibitors have usual much attention. Alpha-amylase is responsible to begin the carbohydrate digestion process by hydrolysis of 1, 4-glycosidic linkages of polysaccharides to disaccharides and α-glucosidase catalyzes the disaccharides to monosaccharides, which support to postprandial hyperglycemia (Hara and Honda, 1990; Matsui et al., 2007). Hence, these enzyme inhibitors are useful to control the hyperglycemia as well as they delay carbohydrate digestion, which reduce the postprandial plasma glucose level. There are no reports available in the literature about α-amylase and α-glucosidase inhibitory activity of this plant. Hence we aimed to evaluate α-amylase and α-glucosidase inhibitory activity of ethanol and aqueous extracts of Z. oenoplia bark. The results showed that ethanol extract has highest inhibitory potential than aqueous extract. The presence of flavonoids and phenolics in both extracts might have attributed to the potential enzyme inhibition activity of plant. These results can support the possible mechanism followed by flavonoid compounds to control blood glucose levels by the inhibition of α-amylase and α-glucosidase activity in the intestine. Above results can be correlated with significant enzyme inhibitory activity of ethanol and aqueous extract may interfere or delay the absorption of dietary carbohydrate support to the suppression of diet induced plasma glucose level in the small intestine.

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5.

Conclusion

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In conclusion, ethanol extract was found to have a better hypoglycemic effect from barks of Ziziphus oenoplia, because it was more effective than aqueous extract in lowering the blood glucose level of diabetic rats. Aqueous extract has greater hypolipidemic effect than alcoholic extract. We assume that some bioactive components of any medicinal plant may differ in their solubility depending on the extractive solvents used. Since repeated administration of both ethanolic and aqueous extracts did not show any changes in the autonomic and behavioral responses in rats during the experimental period, Z. oenoplia may be considered as an alternative treatment for diabetes. Produced significant reduction in blood glucose level especially to reduce the postprandial glucose levels, liver enzymes level and lipid components, it is further required to

investigate the responsible principle compounds for the inhibitory action of α-amylase and α-glucosidase. This may be beneficial for the development of new antidiabetic agents from native plant resources; hence, a further investigation was needed to explore the possible mechanism of action.

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Acknowledgment

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The authors are grateful to Mr. Balsubramanyam director ABS botanical garden, Salem, Tamil Nadu for providing the plant identification facility. Conflict of Interest The authors have declared that there is no conflict of interest.

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Uncited references

Q7

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Siddiqui, Patil, 2015, Telagari, Hullatti, 2015

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Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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Please cite this article in press as: Pramod Mourya, Ajay Shukla, Gopal Rai, Santram Lodhi, Hypoglycemic and hypolipidemic effects of ethanolic and aqueous extracts from Ziziphus oenoplia (L) Mill on alloxan-induced diabetic rats, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.12.002

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