Evaluation of analgesic activity and toxicity of alkaloids in Myristica fragrans seeds in mice

Journal of Pain Research Dovepress open access to scientific and medical research O ri g inal R esearch Open Access Full Text Article Journal of Pain Research downloaded from https://www.dovepress.com/ by 37.59.46.207 on 13-Jul-2018 For personal use only. Evaluation of analgesic activity and toxicity of alkaloids in Myristica fragrans seeds in mice This article was published in the following Dove Press journal: Journal of Pain Research 1 August 2013 Number of times this article has been viewed A Al-Shammary Hayfaa 1 AA Malik Al-Saadi Sahar 2 M Al-Saeidy Awatif 3 College of Science, Department of Medical Analysis, Thi-Qar University, Thi-Qar, Iraq; 2College of Science, Biology Department, University of Basrah, Basrah, Iraq; 3College of Science, Biology Department, Thi-Qar University, Thi-Qar, Iraq 1 Introduction Correspondence: A Al-Shammary Hayfaa College of Science, Department of Medical Analysis, Thi-Qar University, Al-mtanzah Street, Nassriya, Thi-Qar, Iraq Email submit your manuscript | www.dovepress.com Dovepress http://dx.doi.org/10.2147/JPR.S45591 Powered by TCPDF (www.tcpdf.org) Aim: To examine the analgesic effect of alkaloids in Myristica fragrans seed in a mouse model of acetic acid-induced visceral pain. Methods: Alkaloids were extracted from ground nutmeg seed kernels with 10% acetic acid in 95% ethyl alcohol. Visceral pain was induced in male and female BALB/c mice by intraperitoneal injection of 0.6% acetic acid. Analgesic effect of alkaloids (0.5 gram or 1 gram per kilogram [g/kg], by mouth) was assessed by evaluating writhing response. Acute toxicity was tested in response to 2, 3, 4, 5, or 6 g/kg of alkaloid extract; the median lethal dose (LD50) was determined by probit analysis. Results: Alkaloid extract at a dose of 1 g/kg significantly reduced the number of writhing responses in female, but not male mice; 0.5 g/kg of alkaloid extract had no effect in either sex. The LD50 was 5.1 g/kg. Signs of abnormal behavior, including hypoactivity, unstable gait, and dizziness were seen in animals given a dose of 4 g/kg or higher; abnormal behavior lasted for several hours after administration of the alkaloids. Conclusion: According to the classification of Loomis and Hayes, M. fragrans seed alkaloids have analgesic activity and are slightly toxic. Keywords: analgesic, mice, LD50, acetic acid, visceral pain, nutmeg Myristica fragrans Houtt (nutmeg) is an aromatic evergreen tree of the plant family Myristicaceae.1 Nutmeg, the actual seed of the tree, is important in folk medicine, where it is used to treat colds, fever, catarrh, general respiratory ailments, and skin diseases like scabies. It is also used as an appetite stimulant, carminative, antiemetic, and abortifacient.2,3 In controlled laboratory studies, M. fragrans has been shown to possess insulin-like,4 insecticidal,5–8 antibacterial,9–12 and antioxidant activities.13 However, prolonged use of nutmeg can cause degenerative changes in the kidney, spleen, liver, heart, medial geniculate body, and superior colliculus.13–16 Alkaloids are any of a class of naturally occurring, organic nitrogen-containing bases. Traditionally isolated from plants, alkaloids have been increasingly found in animals, insects, marine invertebrates, and microorganisms.17–19 Plant-derived alkaloids elicit many biological effects, including analgesia. Previous studies have demonstrated that an acetone-soluble substance within the n-hexane extract of M. fragrans exerts analgesic activity;20,21 however, the identity of the active constituents responsible for the analgesic activity remains unknown. Thus, the present study was designed to study the analgesic effect of alkaloids extracted from M. fragrans seeds in mice subjected Journal of Pain Research 2013:6 611–615 611 © 2013 Hayfaa et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. Dovepress Hayfaa et al to acetic acid-induced visceral pain and to assess the acute toxicity of these alkaloids. Journal of Pain Research downloaded from https://www.dovepress.com/ by 37.59.46.207 on 13-Jul-2018 For personal use only. Materials and methods Plant materials Dried M. fragrans seeds were collected from Iraqi markets in Thi-Qar City, Iraq and authenticated as M. fragrans seeds by AA Malik Al-Saadi Sahar. Only the seed kernels (nutmeg) were used in this study. Detection of alkaloids Seed kernels were ground to a fine powder. Fifty mL of 4% HCl was added to 10 grams of nutmeg powder, heated to a boil, cooled, and filtered. Three drops each of Mayer’s reagent/ picric acid/Dragendorff’s reagent were added to 0.5 mL of filtrate. The presence of alkaloid was indicated as a white precipitate (Mayer’s reagent), a yellow precipitate (picric acid), or an orange precipitate (Dragendorff’s reagent).22 Alkaloid extraction Crude alkaloid compounds were extracted according to the modified method of Harborne.22 Crushed seed kernels (20 grams) were suspended in 200 mL of hexane, and lipid soluble impurities were removed by continuous extraction using a Quickfit® Soxhlet apparatus (Sigma-Aldrich, St Louis, MO, USA). Kernels were dried under laboratory conditions and subjected to an extraction process with 200 mL of 10% acetic acid in 95% ethyl alcohol for 24 hours in the Soxhlet apparatus. The extract was filtered through No 1 Whatman filter paper (BDH Pharmaceuticals, London, UK) and concentrated by a rotary evaporator (Bibby Scientific Ltd, Staffordshire, UK) at 45°C to 20 mL. The pH was adjusted to 9 by adding concentrated ammonium hydroxide solution, and the solution was partitioned three times with 50 mL of chloroform in a separation funnel, which was shaken vigorously and left to stand each time. The extract separated into two layers. The lower (chloroform) layer contained the alkaloids, which was confirmed with Mayer’s reagent, picric acid, or Dragendorff’s reagent. The chloroform layer was concentrated using a rotary evaporator and left to dry under laboratory conditions. Dried alkaloids were stored in a clean, dark vial at 4°C. 20°C ± 2°C with a 12-hour day/night cycle and access to food and water ad libitum. Animals were treated in accordance with the Ethical Guidelines for the Investigation of Experimental Pain in Conscious Animals issued by the International Association for the Study of Pain, and were approved by the local animal care ethics committee.23 Acute toxicity study Male and female mice were divided into six groups of twelve mice each (six males and six females). Animals were matched for weight and size and allowed to acclimate for 3 days. Groups were given vehicle or 2, 3, 4, 5, or 6 g/kg of nutmeg alkaloids suspended in 0.4 mL of 70% ethanol:distilled water (1:3 by volume) by oral gavage. Animals were observed for 72 hours for behavioral changes or mortality. The median lethal dose (LD50) was determined by probit analysis. Analgesic activi (...truncated)