Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww ANALGESIC EFFECT OF VANADYLE SULFATE AND METHYLENE BLUE ON EXPERIMENTALLY INDUCED PAIN IN MICE Khudhair Abd Al-Rahman1 Muhmood Abed Al-Ani Mustafa Ahmed Jasim Muna Muhammed Ismael Physiology and Pharmacology Dept., College of Vet. Med., Diyala University- Iraq 1Corresponding author: dr.khudhairalani@gmail.com ABSTRACT The present study was designed to determine the potential analgesic effects of both Vanadyl sulfate and Methylene blue simultaneously and in combination using isobolographic analysis, to determine the sort of interaction of Vanadyl sulfate and Methylene blue by up and down method and investigate the analgesic activity of Vanadyl sulfate and Methylene blue in mice. The calculated median lethal dose (LD50) was (456.08 mg Kg -1 B.W) of vanadyl sulfate and (1878 mg Kg-1 B.W) of methylene blue. The analgesic activity of Vanadyl sulfate at the dose (152 mg kg-1) and Methylene blue at the dose (626 mg kg-1) by using acetic acid induces writhing and hot plate methods to induced pain in albino male mice. The test and standard drugs (Ibuprofen 100 mg kg-1) significantly (p<0.05) reduced the number of abdominal constriction and stretching of hind limb induced by acetic acid injection. The analgesic activity results by acetic acid showed a significant decrease in the numbers of writhing in (30) minutes of test-1 (Vanadyl Sulfate) and test-2 (Methylene blue) at a level of (P<0.05) in comparison with control group while analgesic activity results by hot plate method that showed a significant increase in reaction time at (30, 60,120 and 180) minutes of: Test -1 (Vanadyl Sulfate) and: Test -2 (Methylene blue) at a level of (P<0.05) in comparison with control group. Key words: Pain, Analgesic, Ibuprofen, Methylene blue, Vanadyl sulfate, Acetic acid. INTRODUCTION Pain is that the commonest reason for physician consultation in most developed countries (Debono et al. 2013). It's a serious symptom in several medical conditions and can interfere with a human quality of life and general functioning (Moore et al. 2015). Hypnotic suggestion, social support, distraction or excitement its psychological factors may be considerable affect pain's unpleasantness or intensity (Weyers, 2006). In some arguments place fourth in assisted killing or suicide debates, has been used the pain as an argument to permit people who are terminally sick to lives end (IASP, 1979). As a result of it's a complex, subjective phenomenon, process pain has been a Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww challenge. The International Association for the study of Pain's widely used definition states: "Pain is an emotional experience and unpleasant sensory associated with potential or actual tissues harm, or described in terms of such harm" (Bonica, 1979). Methylene blue, is known as methylthioninium chloride, is a dye and medication. It is used mainly in treatment methemoglobinemia (BNF, 2015). Specifically, used to treatment methemoglobinemia levels when are more than 30% and in which there are symptoms despite oxygen therapy (Alan, 1984). Methylene blue, previously been used for treating the urinary tract infections and cyanide poisoning but this use is no longer recommended. It's given by injection into a vein (BNF, 2015). Use often turns the sweat, stool and urine blue to green in color. The uses of methylene blue during the pregnancy maybe harm the baby and not given in methemoglobinemia is likely very dangerous (Alan, 1984). Methylene blue is a thiazine dye (BNF, 2015). It works by converting the ferric iron (Fe3+) to ferrous iron (Fe2+) in hemoglobin (Alan, 1984). Vanadyl (IV) sulfate, VOSO4, is an inorganic compound of vanadium. It's one of the most common sources of vanadium in the laboratory and it's very hygroscopic blue solid, reflective its high stability. The vanadyl ion features, it (VO2+), that has been referred to as "most stable diatomic ion" (Bauer et al. 2005). Vanadyl sulfate is an intermediate during extraction of vanadium from petroleum residues, the vanadium is a major commercial source (Crans et al. 2011). Some experimental drugs and food supplements are components from vanadyl sulfate. Vanadyl sulfate work as insulin-like effects (Krivovichev et al. 2007). The most water-soluble sulfates, the vanadyl sulfate are rare in nature. Pauflerite are an anhydrous form (Hawthorne et al. 2001), a mineral of fumarolic origin. Hydrated forms, also rare, include trihydrate, hexahydrate, and pentahydrates (minasragrite, orthominasragrite, (Schindler et al. 2003) and anorthominasragrite (Berneth and Azine, 2008). The main aim of present study was to investigate the analgesic effects of both vanadyl sulfate and methylene blue on pain induced by acetic acid and hot plate methods in albino mice. MATERIALS AND METHODS Experimental Animals Twenty- four Wister albino male mice, the body weight ranged between 25- 30 g and about 3 months of age were used in this experiment. Placed the mice in a special housing room in College of Veterinary Medicine/ Department of Physiology and Pharmacology for 2 weeks for acclimation. Standard rodent diet Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww and tap water were freely available. Housing conditions were maintained at 20- 25 oC in the air-conditioned room, the light/ dark cycle was 14/10 in housing place, while the air of the room was changed continuously by using ventilation vacuum. LD50 measurement of Vanadyl sulfate and Methylene blue Determination of acute toxic median lethal dose (LD50) of both Vanadyl sulfate and Methylene blue in albino male mice was done by up and down method (Dixon, 1980), and it was calculated according to the following equation: LD50 = xf + kd LD50 = Median lethal dose xf = Last dose used in the experiment d = Difference between doses k = Factor of change from Acetic acid induced writhing The analgesic activity evaluation by writhing method induced by acetic acid. The acetic acid solution in concentration (1% v/v) prepared with distilled water. Ibuprofen solution used as (Reference drug) (dose-100 mg kg-1 10 ml-1) was prepared with normal saline. Each of test -1: A solution of vanadyl sulfate (152 mg kg-1) according to LD50 result, and test -2: A solution methylene blue (626 mg kg-1) according to LD50 result, was prepared in (10) ml of normal saline. The mice were divided into (4) groups (6) mice in each group. Food was withdrawn before about (12) hours of administration of Ibuprofen, vanadyl sulfate and methylene blue. The Ibuprofen, vanadyl sulfate and methylene blue were given orally and after (60) minutes writhing were induced by (ip) injection of (1%) of acetic acid as (0.1 ml 10 g-1) of body weight. The writhing were recorded for (30) minutes (Shanmugasundaram and Venkataraman, 2005). Hot plate method The hot plate method applies by used hot plate apparatus, consists of an electrically heated surface. The temperature is between 55-56 °C. The mice are placed on the hot plate and the time recorded by a stop-watch until either jumping or licking occurs. Each of test -1: A solution of vanadyl sulfate (152 mg kg-1) according to LD50 result, and test -2: A solution methylene blue (626 mg kg-1) according to LD50 result was prepared. The mice were divided into (4) groups (6) mice in each group, the mice were marked individually. Food was withdrawn before about 12 hours of administration of Ibuprofen, vanadyl sulfate and methylene blue. The Ibuprofen, vanadyl sulfate and methylene blue were Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww given orally and after 60 minutes placed the animals on the hot plate and record the observations at the time (0, 30, 60, 120 and 180) minutes (Oz et al., 2011). Statistical Analysis The values are expressed as the mean ± the standard error (M±SEM). The values were analyzed by using one-way analysis of variance ANOVA, and then the test of the least significant difference LSD applied to find the significant differences (Steel and Torrie, 1984). The significant level of the test was P<0.05. RESULTS Acute Toxicity Study (LD50) of Methylene Blue The value results of oral LD50 of Methylene blue were 1878 mg kg -1 B. W. as in table 1. Table 1. Calculate of LD50 for Methylene blue by up and down method Range of doses BW 1-Kg 1600 mg-3200 Decrease or increase in dose BW 1-Kg 400 mg Death or survival of animal after 24 hours. XXXXOOXO Value of (K) table -0.305 Last used dose (xf) BW 1-Kg 2000 mg 50Value of LD BW 1-Kg 1878 mg O: survival, X: death Acute Toxicity Study (LD50) of Vanadyl sulfate The value results of oral LD50 of Vanadyl sulfate were 456.08 mg kg -1 B.W. as in table 2. Table 2. Calculate of LD50 for Vanadyl sulfate by up and down method Range of doses BW 1-Kg 400 mg – 160 Decrease or increase in dose BW 1-Kg 80 mg Death or survival of animal after 24 hours. OOOOXOXO Value of (K) table 0.701 Last used dose (xf) BW 1-Kg 400 mg 50Value of LD BW 1-Kg 456.08 mg O: survival, X: death Study the interaction between methylene blue & vanadyl sulfate toxic doses The result of isobolographic analysis by using the LD50 dose of both methylene blue and vanadyl sulfate alone and in combination are listed in table 3 and figure 1. Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww Table 3. Isobolograph analysis of both methylene blue &vanadyl sulfate LD50 alone and in combination Parameters Groups Range of doses mg kg-1 B.W. Initial dose mg kg-1 B.W. Final dose mg kg-1 B.W No. of animal Result after 24 hours LD50 mg kg-1 Methylene blue 3200-1600 3200 2000 8 XXXXOOXO 1878 Vanadyl sulfate 160 – 400 160 400 8 OOOOXOXO 456.08 Methylene blue + Vanadyl sulfate 2441.4- 1502.4 + 594.2-364.8 2441.4 + 594.2 1502.4 + 364.8 8+8 XXXXOXXX 1472.91 + 357.49 O: survival, X: death Methylene Blue mg/Kg V a n a d y l S u lf a te m g /K g Isobolographic Analysis in combination alone Figure 1. Isobolograph analysis of both methylene blue and vanadyl sulfate LD50 alone and in combination Writhing Test The results of writhing test induced by acetic acid in mice showed a significant decrease in the numbers of writhing in (30) minutes of both test -1 (Vanadyl Sulfate) and test -2 (Methylene blue) at a level of (P<0.05) in comparison with control group, as in table 4. Table 4. Analgesic Activity of Writhing Test Induced by Acetic Acid Group Treatment Dose (mg kg-1) Writhes numbers in 30 min. (mean ± sem) Inhibition (%) Control Distal Water --------- 57.4 ± 4.645 --------- Standard Ibuprofen 100 34.2 ± 2.753* 40.4 Test - 1 Vanadyl Sulfate 152 37.2 ± 3.965* 35.1 Test - 2 Methylene blue 626 46.4 ± 1.725* 19.1 The values are expressed in Mean ± SE; * Significance at P< 0.05. Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 11 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww Hot Plate Test The results of analgesic activity by hot plate method in mice that showed a significant increase in the reaction time at (30, 60, 120 and 180) minutes of both test -1 (Vanadyl Sulfate) and test-2 (Methylene blue) at a level of (P<0.05) in comparison with control group, as in table 5. Table 5. Analgesic Activity by Hot Plate Method Group Treatment Dose (mg kg-1) Reaction time in seconds at the time (min.) (mean ± sem) 0 30 60 120 180 Control Distal Water ----- 5±0.317 5.4±0.245 5±0.317 5.2±0.375 5.8±0.375 Standard Ibuprofen 100 6±0.317 9.8±0.584* 10. ±0.511* 12.1±1.051* 15.2±0.584* Test - 1 Vanadyl Sulfate 152 5.6±0.245 8.2±0.862* 9.2±0.375* 10.2±0.665* 14±0.838* Test - 2 Methylene blue 626 5.8±0.375 8.6±0.601* 9.8±0.919* 11.2±0.736* 15.4±1.169* The values are expressed in Mean ± SE; * Significance at P < 0.05 DISCUSSION The study indicated that administration of vanadyl sulfate and methylene blue can be suppressed the pain that induced by writhing and hot plate methods in male mice. Local injection of methylene blue has been evaluated in treatments for various pain diseases and has shown a remarkable long-acting analgesic effect (Peng et al. 2007). Methylene blue is a nontoxic dye agent that inhibits the soluble guanylate cyclase and nitric oxide synthase. Nitric oxide regulates physiological functions such as pain and analgesia by activating soluble guanylate cyclase to increase intracellular cyclic guanosine monophosphate (Bhattacharyya and Tracey, 2001). Methylene blue, as an oxidizing-reducing agent, demonstrates a strong affinity to nerve tissues when applied locally, which can directly block the electrical conductivity of nerve fibers, thereby affecting the neural excitability and impulse conductivity. Recent studies have shown that a low dose (0.5% or 1%) of methylene blue may block peripheral nerve fibers at the incision (Kaneria et al. 2007). Hyperglycemia leads to the toxicity of neurons due to increased glucose oxidation, lead to increased reactive oxygen species that may be controlled by the treatment with antioxidant (Andrade et al. 2012). Various studies have suggested the capacity of vanadium to enhance the blood glucose control of diabetics and also to enhance the negative side effects associated with diabetes. Therefore, the vanadyl sulfate may be given the chance to decrease the glucose in blood that leads to decrease in the pain. Diyala Journal of Agricultural Sciences, 10(Special Issue), 61-69, 2018 Al-Rahman et al. 16 1st Scientific Conf., College of Vet. Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww CONCLUSION From the previous result, it is concluded that vanadyl sulfate and methylene blue have a good analgesic effect due to directly block the electrical conductivity of nerve fibers, thereby affecting the neural excitability and impulse conductivity. REFERENCES Andrade, P., V. Visser-Vandewalle, J. S. Rosario. 2012. The thalidomide analgesic effect is associated with differential TNT-a receptor expression in the dorsal horn of the spinal cord as studied in a rat modal of neuropathic pain. Brain Res. 1450: 24-32. Berneth, H. 2008. Azine dyes. In: Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, pp. 475-514. 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Med., Diyala Univ., 2018 w.agriculmag.uodiyala.edu.iq/http://ww األلم المستحدث تجريبياً في الفئران كبريتات الفناديل والمثيلين األزرق في سكن لكل منالتأثير الم منى محمد اسماعيل مصطفى أحمد جاسم محمود عبد العاني 1خضير عبد الرحمن قسم الفسلجة والفارماكولوجي، كلية الطب البيطري، جامعة ديالى، العراق dr.khudhairalani@gmail.comالمسؤول عن النشر: 6 مستخلصال صممت الدراسة الحالية لغرض تقييم قوة التأثيرات المسكنة لكل من الفيندايل سلفيت والمثيلين بلو تحليل باأليزوبولكراف من اجل تقييم قوة التداخل للفيندايل طة استخدام طريقة السامزدوج بوبشكل منفرد و سلفيت والمثيلين بلو عن طريق استخدام طريقة الصعود والنزول، والكشف عن الفعالية المسكنة للفيندايل ملغم 661.56للفيندايل سلفيت LD)50(كانت الجرعة النصف قاتلة و ،في الفئران سلفيت والمثيلين بلو اجريت لغرض فمن وزن الجسم للمثيلين بلو. اما التجربة الثانية 6-كغم ملغم 6666الجسم ومن وزن 6-كغم 6-كغم ملغم 161 ، والمثيلين بلو بجرعة6-كغم ملغم 666لفيندايل سلفيت بجرعة تقييم التأثيرات المسكنة ل . اءطة اعطاء حامض الخليك وطريقة الصفيحة الساخنة الستحداث االلم في ذكور الفئران البيضابوس طة حامض اسبو ةتمدد األطراف الخلفية المستحدثنقباضات البطنية والأظهرت النتائج انخفاض في عدد ا بمستوى 6-كغم ملغم 655فين بجرعة الخليك لكل من الفيندايل سلفيت والمثيلين بلو ودواء األيبوبرو اطة حامض الخليك أظهرت نقصان في عدد س. نتائج الفعالية المسكنة المستحدثة بو(p<0.05)معنوية بالمقارنة (P<0.05)دقيقة لكل من الفيندايل سلفيت والمثيلين بلو عند مستوى معنوية 65االلتواءات لمدة زيادة معنوية في الصحيفة الساخنة اطة استخدام طريقة سمع مجموعة السيطرة، بينما أظهرت النتائج بو دقيقة لكل من الفيندايل سلفيت والمثيلين بلو عند مستوى معنوية (30 ,60 ,120 ,180)فترة الوقت عند (P<0.05) .بالمقارنة مع مجموعة السيطرة أيبوبروفين، مثيلين بلو، فيندايل سلفيت، حامض الخليك. ،مسكن ،ألمالكلمات المفتاحية: