Hydro-Ethanolic Leaf Extract of Ziziphus Abyssinica Hochst Ex A. Rich

Boakye-Gyasi et al. BMC Complementary and Alternative Medicine (2017) 17:231 DOI 10.1186/s12906-017-1750-z RESEARCH ARTICLE Open Access Hydro-ethanolic leaf extract of Ziziphus abyssinica Hochst Ex A. Rich (Rhamnaceae) exhibits anti-nociceptive effects in murine models Eric Boakye-Gyasi1*, Isaac Tabiri Henneh1,2, Wonder Kofi Mensah Abotsi1, Elvis Ofori Ameyaw3 and Eric Woode1 Abstract Background: Despite substantial advances in pain research and treatment, millions of people continue to suffer from pain and this has been attributed mainly to the unavailability of effective and safer analgesics. The use of plants as medicines is still widespread and plants constitute a large source of novel phytocompounds that might become leads for the discovery of newer, effective and safer alternatives. Various parts of Ziziphus abyssinica have been used in folk medicine in several African countries as painkillers. However, there is no report on the possible anti-nociceptive effects of this plant especially the leaves, hence the need for this current study. Methods: The possible anti-nociceptive activity of hydro-ethanolic leaf extract of Ziziphus abyssinica (EthE) was assessed in rodents using chemical (acetic acid, formalin and glutamate), thermal (tail-immersion test) and mechanical/inflammatory (carrageenan) models of nociception. Results: EthE (30-300 mg/kg, p.o.) dose-dependently and significantly inhibited chemical-induced nociception with a maximum inhibition of 86.29 ± 2.27%, 76.34 ± 5.67%, 84.97 ± 5.35%, and 82.81 ± 5.97% respectively for acetic acid, formalin (phase 1), formalin (phase 2) and glutamate tests at its highest dose. EthE also dose-dependently and significantly increased reaction times in both tail-immersion and carrageenan-induced hypernociceptive tests. The activities of the extract in the various models were comparable with the effect of morphine hydrochloride and diclofenac sodium used as standard analgesic drugs. Conclusion: Oral administration of hydro-ethanolic leaf extract of Ziziphus abyssinica ameliorates nocifensive behaviours associated with chemical-, thermal- and mechanical/inflammatory - induced nociceptive pain. Keywords: Ziziphus Abyssinica, Nociception, Formalin, Acetic acid, Glutamate, Carrageenan, Tail-immersion Background research and treatment, millions of people continue to One of the most common reasons why people seek suffer from pain. This has been attributed to either in- medical care is pain [1]. It is also a major reason for ab- appropriate use of analgesics or unavailability of effective senteeism from work, underemployment and unemploy- and efficacious analgesics thereby increasing cost of pain ment and results in a huge financial loss to individuals treatment. Already, opioids and non-opioids which are and countries as a whole [2]. This is because aside the mostly prescribed for the management of pain have cost of treatment, it also complicates the treatment of limited use because of their numerous side effects [4]. other ailments [3]. Despite substantial advances in pain This underscores the need to search for newer drugs with improved efficacy and safety. The use of plants as medicines is still widespread espe- * Correspondence: [email protected] cially in Africa [5]. Plants also constitute a large source 1Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, of novel phytocompounds that might become leads for Ghana the discovery of new pharmaceutical agents which may Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Boakye-Gyasi et al. BMC Complementary and Alternative Medicine (2017) 17:231 Page 2 of 12 be useful for the management and prevention of diseases a desiccator containing activated silica until it was ready w and ailments [6]. One of such plants of medicinal im- for use. The yield obtained was 10.8% /w. portance is Ziziphus abyssinica (Hochst Ex A. Rich) ‘ ’ which is commonly known as Catch thorn in English Phytochemical screening ‘ ’ and Larukluror among the Sissala people of Ghana [7]. Preliminary qualitative phytochemical screening was The leaves and other parts of the plant have been used conducted on EthE using standard procedures [21]. traditionally to treat pneumonia, tonsillitis, Newcastle disease, snake bite, burns, wounds, tachycardia, pectoral Animals pain, migraines and as pain-killers [7–10]. Extracts from ICR mice (20-25 g) and Sprague-Dawley rats (170-250 g) different parts of the plant have been reported previously of both sexes were bought from Noguchi Memorial In- to have antioxidant [11], antimicrobial [11–13] anti- stitute for Medical Research, University of Ghana, diarrheal [14] molluscicidal [15] antiplasmodial [16] and Legon, Ghana. They were kept in stainless cages anti-ulcerogenic effects [17]. There have also been (34 × 47 × 18 cm) in groups of five at the animal house reports on the anti-nociceptive effects of various parts of facility of FPPS, KNUST, Kumasi. The animals were plants in the Ziziphus genus including Zizyphus spina- given normal commercial diet obtained from Agricare christi [18], Zizyphus oxyphylla [19] and Ziziphus Limited, Kumasi, Ghana and water was given ad libitum. mucronata [20] in animal models. However, there is no They were kept under normal laboratory conditions with report on the possible anti-nociceptive effects of this regards to room temperature and humidity. In all experi- plant especially the leaves, hence the need for this ments, animals were randomly assigned to either treat- current study. Based on the traditional use of the plant ment or control group. All the techniques and protocols in the management of pain and previous published data used in the study were done in accordance with estab- on the anti-nociceptive effects of other species of Zizi- lished public health guidelines in “Guide for Care and phus, the present study examined the anti-nociceptive Use of Laboratory Animals” [22]. Also, all protocols used properties of the hydro-ethanolic leaf extract of Ziziphus in the study were approved by the Departmental Ethics abyssinica in animal models that could predict both Committee. peripheral and central-mediated pain as well as neuro- genic and inflammatory pain. Drugs and chemicals Method The following chemicals and drugs were used in the Plant collection study: carrageenan, formalin and acetic acid (British Fresh leaves of Ziziphus abyssinica were collected from Drug House, Poole, England); morphine sulphate (Duo- Ejura (7°23′00.16″N, 1°22′00.00″W) in the Ejura- pharma (M) Sdn Bhd, Malasia); diclofenac sodium (Denk Sekyedumase Municipal of Ashanti Region in the month of Pharma, Germany) and L-glutamic acid (Sigma-Aldrich October, 2015. It was authenticated by Mr. Clifford Asare Inc., St. Louis, MO, USA). of the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), Kwame Anti-nociceptive activity Nkrumah University of Science and Technology (KNUST), Acetic acid-induced writhing Kumasi, Ghana. A voucher specimen (KNUST/HM/2016/ The test was carried out as described previously [23, 24]. L003)wasdepositedattheDepartmentofHerbalMedi- Mice (n = 5) received either vehicle (10 mL/kg distilled cine’sherbarium. water p.o.), EthE (30-300 mg/kg p.o.) or diclofenac (10- 100 mg/kg i.p.). After 60 min (p.o.) or 30 min (i.p.), they Plant extraction received intraperitoneal injection of acetic acid (10 mL/kg v About 1 kg of fresh matured leaves of Ziziphus abyssi- of 0.6% /v). They were then individually placed in testing nica were air dried for fourteen days in a room and with chambers (a Perspex chamber of 15 cm × 15 cm × 15 cm). the aid of a hammer mill pulverized into fine powder. A mirror placed at 45° to the floor level allowed complete An amount of 600 g of the powdered leaves was ex- view of the animals in the camcorder (Sony-Handycam, v tracted with 4 L of 70% /v ethanol for 48 h period using model: HDRCX675/B, Tokyo, Japan) which was used to a Soxhlet apparatus (Aldrich® Soxhlet Extraction Appar- capture the nociceptive behaviors of the mice following atus, Z556203, St. Louis, MO, USA). The extract acetic acid injection. This was captured for 30 min and obtained was labeled as EthE and subsequently concen- later tracked using a public domain software JWatcher™ trated using a rotary evaporator (Rotavapor R-215 software Version 1.0 developed by University of California, model, BÜCHI Labortechnik AG, Flawil, Switzerland) Los Angeles, USA and Macquarie University, Sydney, under reduced pressure and temperature (70 °C). This Australia. The total number of writhes per every five- was further dried on a water bath and then preserved in minute time bloc was obtained and this was used to plot Boakye-Gyasi et al. BMC Complementary and Alternative Medicine (2017) 17:231 Page 3 of 12 time course curves from which the areas under the curves Glutamate - induced hypernociception (AUCs) were calculated. The test was performed in mice (20 – 25 g) as previously described [29, 30]. Glutamate (20 μL; 10 μmol/paw) was Formalin-induced nociception in mice administered into the ventral surface of rats’ right hind This test was performed as has been described previ- paws (n = 5) after they had been pretreated, 1 h with ously [25, 26]. Seven groups (n = 5) of ICR mice were EthE (30-300 mg/kg, p.o.) or 30 min prior to morphine used for the test. Each mouse was placed in one of (3 mg/kg, i.p.).

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