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  • Research ArticleEffects of Salvadora persica Extract on the Hematological andBiochemical Alterations against Immobilization-Induced Rats

    Kholoud S. Ramadan and Salha A. Alshamrani

    Department of Biochemistry, Faculty of Girls Science, King Abdulaziz University, Jeddah, Saudi Arabia

    Correspondence should be addressed to Kholoud S. Ramadan; [email protected]

    Received 28 May 2015; Revised 10 June 2015; Accepted 14 June 2015

    Academic Editor: Gary Lopaschuk

    Copyright 2015 K. S. Ramadan and S. A. Alshamrani. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

    A total of 24 rats were divided into 4 groups: control, stress, extract alone, and stress + extract ( = 6 each), for total 21 days oftreatment.The immobilization stress was induced in rats by putting them in 20 cm 7 cm plastic tubes for 2 h/day for 21 days. Ratswere postorally treated with Salvadora persica at a dose of 900mg/kg body weight via intragastric intubations. At the end of thetest period, hematological and biochemical parameters were determined in blood and serum samples with determination of vitalorgans weights.The vital organ weights were not significantly affected in stressed rats as compared to control rats. Compared to thecontrol group, the stress treated group showed significances in several hematological parameters, including decreases inWBC,RBC,and PLT counts. Furthermore, in comparison to the control group, the stress group showed significantly increased blood glucose,serum total cholesterol, LDL-cholesterol, and triacylglycerols levels and decreased HDL-cholesterol level. The hematological andbiochemical parameters in the stress + extract treated group were approximately similar to control group. The SP extract restoredthe changes observed following stress treatment.

    1. Introduction

    Stress can be considered as an important main cause of manydiseases like coronary artery diseases, cancer, and diabetesmellitus [1]. Stress has become an increasingly popular andwidely applied term in our everyday languages. People havewitnessed the physical damage of stress that can work on thebody [2].

    Immobilization is a suitable and easy method to causephysical stress which leads to restrictingmobility and aggres-sion in animal model [3]. However, raising evidence hasimplied that the production of free radicals plays an impor-tant role in this process.

    Focusing on the usage of natural antioxidants is anew strategy for mitigating oxidative damage. Many ofthe negative effects of oxidative stress are decreased aftersupplementation with certain dietary antioxidants [4]. Thereis an increasing interest in total medicinal plant extracts,the largest value of which may be due to their constituentsthat subscribe to the modulation of the oxidative balance

    in vivo. Additionally, the special advantage of total plantextracts is that they are easily accessible products, withoutpurification, to apply them in possible prevention of diseases[5].

    The medicinally important species of Salvadora persicaL. (SP), also known as Miswak, mustard tree, and tooth-brush tree, distributed mainly in tropical and subtropicalAsia. Miswak belongs to the family of Salvadoraceae andevery part of the plant is used as a medicinal purposeamong global Muslim community. Various phytochemicalstudies on Salvadora persica reported the presence of alka-loids, salvadorine, flavonoids, steroids, trimethylaine, andsalvadoricine. Various ingredients of Salvadora persica havevaluable important biological properties, including criticalantibacterial, antifungal activity and antidiabetic [6, 7].

    Many studies reported that oral ingestion of medicinaldrugs can alter the hematological parameters ranges to eitherpositive or negative [8]. Many of these therapeutic effectshave been confirmed by contemporary scientific researchand their antistress effects have not been well researched.

    Hindawi Publishing CorporationScientificaVolume 2015, Article ID 253195, 5 pages

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    Therefore, this study was planned to investigate the anti-stress effects of administration of Salvadora persica extracton hematological and biochemical alterations induced byimmobilization stress in rats.

    2. Materials and Methods

    2.1. Collection and Authentication of Plant. Salvadora persicaL. (SP) was purchased from local market in Jeddah, Kingdomof Saudi Arabia, and authenticated by Herbarium, KingAbdulaziz University.

    2.2. Preparation of Aqueous Extract. The fresh Miswak rootsticks were cut into small pieces and allowed to dry atroom temperature for one week. Then they were groundin grinding machine to fine powder, mixed with distilledwater, and extracted for 24 h at 150 rpm at 25C in a shaker.The mixture was then centrifuged at 3000 rpm for 20min.The supernatants were subsequently filtered through What-man No. 1 filter paper and the filtrate was concentrated inrotary evaporator (Buchi Rotavapor R-200) at 70C and waslyophilized.The resulting powder was packed in a glass bottleand stored at 4C until needed. It was dissolved in distilledwater to prepare the exact aqueous dose (900mgKg1 bodyweight) for intragastrical injection [9].

    2.3. Animals and Experimental Design. Adult male albinorats (weighing 120160 g)were obtained fromCentral AnimalHouse in Jeddah, Saudi Arabia. The animals were housed inacrylic cages in standard conditions of temperature beforestarting the experiments to adapt to the laboratory conditionand fed with commercial diet and water ad libitum. Theexperimental protocol was approved by the InstitutionalAnimal Ethical Committee (IAEC) of Saudi Arabia, Jeddah.

    The immobilization stress was induced in rats by puttingthem in 20 cm 7 cm plastic tubes for 2 h/day for 21 days[10, 11]. There are several 3mm holes at the far end of thetubes for breathing that allows ample air but animals willbe unable to move. Twenty-four rats were randomly dividedinto four groups of six animals each. Experimental groupswere designed as follows: control group that received distilledwater; stressed group; Salvadora persica (SP) that receiveddaily extract (900mg/Kg bw) intragastrically using animalfeeding intubation needles at the same time of day, for 21days; stress + extract group. Rats were subjected to 2 h stressonce a day for a period of 21 days except the nonstressgroup. Following stress session, rats were returned to homecages and were able to access food and water freely for theremainder of the day. Rats in different groups were weighedon days 0, 7, 14, and 21. They were closely observed forbehavioral and general morphological changes.

    2.4. Vital Organs Weights. At the termination of treatment,21 days, vital organs (heart, brain, liver, and kidneys) wereharvested from scarified rats.Theywere washed with ice-coldsaline solution (0.9%w/v), blotted, and weighted.The weightof each organ was standardized to 100 g body weight of eachanimal.








    0 1 2 3



    ght (





    Figure 1: Effects of SP extract (900mg/kg bw) on body weight incontrol and immobilization-induced stress rats (21 days).The resultsare expressed as the mean SD for 6 animals in each group.

    2.5. Hematological Assay. Blood samples were collected fromrats into heparinized tubes under light ether anesthesia.White blood cells (WBC), lymphocyte and monocyte ratio,red blood cells (RBC), hematocrit (Hct), hemoglobin (Hb),mean cell volume (MCV), mean cell hemoglobin (MCH),mean cell hemoglobin concentration (MCHC), and plateletcount (PLT) were measured on Hematology Analyzer (Aba-cus Junior Vet 5, Austria).

    2.6. Biochemical Analysis. At the end of the experimentalperiod, animals were fasted for 812 h before blood collectionin order to cause no interference in the analysis of blood glu-cose and serum lipid profile. Blood samples were withdrawnby end tail vein cuttingmethod fromovernight fasted animalsand blood glucose was measured by one touch electronicglucometer ACU check. Other blood samples were collectedand allowed to coagulate at room temperature for 30min andwere subsequently centrifuged at 3000 g for 10min. Serumwas removed and stored at 80C until analysis.

    Estimation of biochemical parameters such as serum totalcholesterol [12], triacylglycerols [13], and HDL-cholesterol[14] wasmeasured.The LDL andVLDL levels were calculatedusing the formulae [15].

    2.7. Statistical Analysis. Data were analyzed by one-wayanalysis of variance (ANOVA) followed by Students -testsusing a commercially available statistics software package(SPSS forWindows, V. 15.0) program. Results were presentedas means SD. values

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    Table 1: Effects of SP extract on rat organs weights (per 100 g body weight) in control and immobilization-induced stress rats.

    Groups Relative organ weightLiver Kidney Brain Heart

    Control 3.43 0.2 0.76 0.06 0.69 0.03a 0.3 0.03Stress 3.45 0.5 0.77 0.07 0.8 0.08b 0.32 0.04SP extract 3.46 0.32 0.78 0.1 0.69 0.02a 0.32 0.001Stress + extract 3.33 0.4 0.62 0.2 0.73 0.04a 0.33 0.03All the values are expressed as mean SD ( = 6 for each group). Values not sharing a common alphabet as superscripts are significantly different from eachother at the level of < 0.05.

    Table 2: Hematological parameters in control and experimental groups.

    Control Stress SP extract Stress + extractWBC (103/mm3) 8.48 0.3a 5.2 0.03b 11.2 0.36c 10.3 0.8c

    Lymphocyte (%) 85.33 2.8 90.6 2.2 89.9 3.1 89.8 3.2Monocytes (%) 11.2 2.6 7.23 0.4 9.45 2.2 7.73 1.5RBC (106/mm3) 7.33 0.35a 6.8 0.25b 7.6 0.36a 7.9