<strong>Research Progress on Mechanisms Underlying Aconitines Analgesia</strong>

Abstract

Abstract:Aconitum has abundant resources and has been prescribed in China and other eastern Asia countries for analgesic，anti-rheumatic and neurological indications for centuries. As the principal active constituents present in Aconitum，diterpenoid alkaloids possess therapeutic activities against inflammation，nociception and tumors，and can be divided into four skeletal categories：C18-，C19-，C20- and bis-diterpenoid alkaloid. In this review，we summarized the research progress on the analgesic effects and the underlying mechanisms of several clinically effective diterpenoid alkaloids in Aconitum including bulleyaconitine （C19-diterpenoid alkaloid），bullatine A （C20-diterpenoid alkaloid） and lappaconitine （C18-diterpenoid alkaloid），as well as aconitine （C19-diterpenoid alkaloid）. We also explored the scientific rationale for the Aconitum processing.Bulleyaconitine，bullatine A，lappaconitine and aconitine exhibit potent analgesic effects in a variety of animal models of pain hypersensitivity as well as in clinic. Their antinociception is
entirely mediated by stimulation of spinal microglial dynorphin A expression via a Gs/cAMP/ PKA/p38β/CREB signal transduction pathway，which then activates the post-synaptic neuronal κ-opioid receptors. Processing of aconitine induces the increase of benzoylaconine，which has lower toxicity and higher therapeutic index. In addition，aconitines or the Aconitum extracts produce antinociception without induction of tolerance and inhibit morphine antinociceptive tolerance，which is also through stimulation expression of spinal dynorphin A.

Key words:conitines,bulleyaconitine,bullatine A,lappaconitine,analgesia,dynorphin A,microglia

CLC Number:

R964

HUANG Qian，SUN Ming-li，LI Teng-fei，WANG Yong-xiang.Research Progress on Mechanisms Underlying Aconitines Analgesia[J]. Acta Neuropharmacologica, 2017, 7(3): 21-32.

References

[1] Chi Jung Tai, Mohamed El-Shazly, Wu Tung-ying, et al. Clinical aspects of aconitum preparations [J]. Planta Med, 2015, 81 (12-13):1017-1028.

[2] 安婧娴, 刘芳, 刘芳, 等. 近年来乌头属植物二萜生物碱化学成分及其镇痛活性研究进展 [J]. 中南药学, 2016, 14 (5):521-5.

[3] 吴超. 我国乌头属植物研究进展 [J]. 新疆医科大学学报, 2011, 34 (10):1153-7.

[4] Xiao Pei-gen, Wang Feng-peng, Gao Feng, et al. A pharmacophylogenetic study of Aconitum L. (Ranunculaceae) from China [J]. Acta Phytotaxono Sin, 2006, 44 (1):1-46.

[5] 蔡超群, 杨春华, 梁敬钰, 等. 乌头属二萜生物碱成分构效关系研究进展 [J]. 海峡药学, 2013, 25 (3):1-5.

[6] Tang Xi-can, Liu Xue-jun, Lu Wei-hua, et al. Studies on the analgesic action and physical dependence of bulleyaconitine A [J]. Yao Xue Xue Bao, 1986, 21 (12):886-891.

[7] Li Teng-fei, Fan Hui, Wang Yong-xiang. Aconitum-derived bulleyaconitine a exhibits antihypersensitivity through direct stimulating dynorphin a expression in spinal microglia [J]. J Pain, 2016, 17 (5):530-548.

[8] 杨杏林, 杨大男, 杨清锐, 等. 草乌甲素的研究进展 [J]. 世界临床药物 2013, 34 (2):80-83.

[9] 唐希灿. 镇痛抗炎新药滇西嘟拉碱甲 [J]. 新药与临床, 1986, 5 (2):120-1.

[10] 龙丽, 赵金霞, 贾汝琳, 等. 草乌甲素对大鼠佐剂性关节炎的影响 [J]. 临床药物治疗杂志, 2013, 11 (1):16-18.

[11] Zhu He-quan, Xu Jing, Shen Kai-feng, et al. Bulleyaconitine a depresses neuropathic pain and potentiation at C-fiber synapses in spinal dorsal horn induced by paclitaxel in rats [J]. Exp Neurol, 2015, 273:263-272.

[12] 刘延青, 丁晓宁, 王应德. 草乌甲素片治疗常见慢性疼痛的临床研究 [J]. 中国疼痛医学杂志, 2011, 17 (5):314-315.

[13] 吐尔逊江. 草乌甲素穴位注射治疗坐骨神经痛49例疗效观察 [J]. 新疆中医药, 1999, 17 (3):32-33.

[14] 郑兴华. 草乌甲素和大剂量丹参注射液缓解晚期癌性疼痛16例初探 [J]. 临床医学, 2005, 25 (2):65-6.

[15] Wang Chi-fei, Peter Gerner, Wang Sho-yao, et al. Bulleyaconitine a isolated from aconitum plant displays long-acting local anesthetic properties in vitro and in vivo [J]. Anesthesiology, 2007, 107 (1):82-90.

[16] Wang Chi-fei, Peter Gerner, Birgitta Schmidt, et al. Use of bulleyaconitine A as an adjuvant for prolonged cutaneous analgesia in the rat [J]. Anesth Analg, 2008, 107 (4):1397-1405.

[17] Zhu Da-yuan, Bai Dong-lu, Tang Xi-can. Recent studies on traditional Chinese medicinal plants [J]. Drug Dev Res, 1996, 39 (2):147-57.

[18] Mitsuo Murayama, Tomoko Ito, Chohachi Konno, et al. Mechanism of analgesic action of mesaconitine. I. Relationship between analgesic effect and central monoamines or opiate receptors [J]. Eur J Pharmacol, 1984, 101 (1-2):29-36.

[19] Mitsuo Murayama, Hiroshi Hikino H. Effect of cyclic AMP on mesaconitine-induced analgesia in mice [J]. Eur J Pharmacol, 1985, 108 (1):19-23.

[20]Tomoko Isono, Tsutomu Oyama, Akitoshi Asami, et al. The analgesic mechanism of processed Aconiti tuber: the involvement of descending inhibitory system [J]. Am J Chin Med, 1994, 22 (1):83-94.

[21] Yasuyuki Suzuki, Kazuhiro Goto, Atsushi Ishige, et al. Antinociceptive effect of Gosha-jinki-gan, a Kampo medicine, in streptozotocin-induced diabetic mice [J]. Jpn J Pharmacol, 1999, 79 (2):169-175.

[22] Omiya Y, Goto K, Suzuki Y, et al. Analgesia-producing mechanism of processed Aconiti tuber: role of dynorphin, an endogenous kappa-opioid ligand, in the rodent spinal cord [J]. Jpn J Pharmacol, 1999, 79 (3):295-301.

[23] Xu Hong-meng, Hideko Arita, Masakazu Hayashida, et al. Pain-relieving effects of processed Aconiti tuber in CCI-neuropathic rats [J]. J Ethnopharmacol, 2006, 103 (3):392-397.

[24] Wang Chao, Sun Danni, Liu Chun-fang, et al. Mother root of Aconitum carmichaelii Debeaux exerts antinociceptive effect in Complet Freund's Adjuvant-induced mice: roles of dynorpin/kappa-opioid system and transient receptor potential vanilloid type-1 ion channel [J]. J Transl Med, 2015, 13: 284.

[25] China Pharmacopoeia Committee. Pharmacopoeia of the People?s Republic of China 2005 [M]. Beijing, People?s Medical Publishing House, 2005.

[26] Angela Ameri. The effects of Aconitum alkaloids on the central nervous system [J]. Prog Neurobiol, 1998, 56 (2):211-235.

[27] Chodoeva A, Bosc J-J, Robert J. Aconitum alkaloids and biological activities [M]. In Natural Products. Springer, 2013: 1503-1523.

[28] Jutta Friese, Johannes Gleitz, Ulrike T Gutser, et al. Aconitum sp. alkaloids: the modulation of voltage-dependent Na+ channels, toxicity and antinociceptive properties [J]. Eur J Pharmacol, 1997, 337 (2-3):165-174.

[29] China Pharmacopoeia Committee. Pharmacopoeia of China [M]. Beijing, People's Medical Publishing House, 1977.

[30] Li GN: Yunnan Zhiwu Zhi .Chinese Medicine Record of Yunnan[M]. Yunnan: Yunnan Science and Technology Press; 1990.

[31] 张红宇, 王璐. 雪上一枝蒿片的药效学研究 [J]. 中华临床医学杂志, 2005, 6 (3):3-4.

[32] Teng Shi-yong, Zhang Si-xi, Niu Kai, et al. Development and validation of an LC-MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study [J]. Biomed Chromatogr, 2015, 29 (12):1798-1804.

[33] Judith Singhuber, Zhu Ming, Sonja Prinz, et al. Aconitum in Traditional Chinese Medicine-A valuable drug or an unpredictable risk? [J]. J Ethnopharmacol, 2009, 126 (1):18-30.

[34] Ren Wei, Yuan Lin, Li Jun, et al. Ethanolic extract of aconiti brachypodi radix attenuates nociceptive pain probably via inhibition of voltage-dependent Na(+) channel [J]. Afr J Tradit Complement Altern Med, 2012, 9 (4):574-583.

[35] Tang Xi-can, Wang Tsu-wu, Hsu Bin. The analgesic actions of Bullatine A, delavaconitinum and isoaconitine [J]. Acta Pharmaceu Sin, 1966, 13 (3):227-228.

[36] Huang Qian, Mao Xiao-fang, Wu Hai-yun, et al. Bullatine a stimulates spinal microglial dynorphin A expression to produce anti-hypersensitivity in a variety of rat pain models [J]. J Neuroinflammation, 2016, 13 (1):214.

[37] Li Jun, Ren Wei, Huang Xian-ju, et al. Bullatine A, a diterpenoid alkaloid of the genus Aconitum, could attenuate ATP-induced BV-2 microglia death/apoptosis via P2X receptor pathways [J]. Brain Res Bull, 2013, 97:81-85.

[38] 李芸, 胡昌江, 徐婷, 等. 高乌头炮制前后高乌甲素含量测定及小鼠急性毒性实验 [J]. 中成药, 2016(1): 179-181.

[39] 徐斌, 张琴, 郭夫江, 等. 高乌头的研究进展 [J]. 中成药, 2016(4): 882-890.

[40] 谈永霞, 王鸣刚, 张继, 等. 乌头属植物研究进展 [J]. 安徽农业科学, 2011(24): 14644-14646.

[41] Ono M, Satoh T. Pharmacological studies of lappaconitine. Analgesic activities [J]. Arzneimittelforschung, 1988, 38 (7):892-895.

[42] Tang X C, Zhu M Y, Feng J, et al. Pharmacologic actions of lappaconitine hydrobromide[J]. Yao Xue Xue Bao, 1983, 18 (8):579-84.

[43] Mamoru Ono, Tetsuo Satoh. Pharmacological studies on lappaconitine: antinociception and inhibition of the spinal action of substance P and somatostatin [J]. Jpn J Pharmacol, 1991, 55 (4):523-530.

[44] Ou Shan, Zhao Yan-dong, Xiao Zhi, et al. Effect of lappaconitine on neuropathic pain mediated by P2X3 receptor in rat dorsal root ganglion [J]. Neurochem Int, 2011, 58 (5):564-573.

[45] Zhu Xiao-cui, Ge Chen-tao, Wang Pan, et al. Analgesic effects of lappaconitine in leukemia bone pain in a mouse model [J]. Peer J, 2015, 3:e936.

[46] 孙明丽, 黄茜, 王永祥. 高乌甲素的镇痛作用及其机制的研究 [J]. 中国药理学通报, 2015, 31:81.

[47] 安一明, 戴建平, 陈力, 等. 高乌甲素贴片治疗癌症疼痛的临床研究 [J]. 中国肿瘤临床与康复, 1997(2):86-7.

[48] 阙铁生, 钟进才, 周晓敏. 高乌甲素透皮贴剂治疗中重度癌痛 [J]. 中国临床康复, 2005(2): 211.

[49] 晓敏, 钟进才, 阙铁生, 等. 高乌甲素贴片治疗肝癌疼痛的疗效 [J]. 广西医学, 2005(10): 1528-1530.

[50] 高丽丽, 康爽. 高乌甲素用于甲状腺术后镇痛的临床观察 [J]. 黑龙江医学, 2015(07):771-773.

[51] Gong QA, Li M. Effect of lappaconitine on postoperative pain and serum complement 3 and 4 levels of cancer patients undergoing rectum surgery[J]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 2015, 35 (6):668-672.

[52] 王井泉. 高乌甲素治疗56例胆道术后病人疼痛的临床疗效分析 [J]. 中国临床药理学与治疗学, 2010(5): 551-554.

[53] Sterling N Wright. Irreversible block of human heart (hH1) sodium channels by the plant alkaloid lappaconitine [J]. Mol Pharmacol, 2001, 59 (2):183-192.

[54] Guo Xin, Tang Xi-can. [Effects of reserpine and 5-HT on analgesia induced by lappaconitine and N-deacetyllappaconitine] [J]. Acta Pharmacologica Sinica, 1990, 11 (1):14-18.

[55] Ono M, Satoh T. Pharmacological studies on lappaconitine: possible interaction with endogenous noradrenergic and serotonergic pathways to induce antinociception [J]. Jpn J Pharmacol, 1992, 58 (3):251-257.

[56] Ulrike Seitz, Angela Ameri A. Different effects on [3H]noradrenaline uptake of the Aconitum alkaloids aconitine, 3-acetylaconitine, lappaconitine, and N-desacetyllappaconitine in rat hippocampus [J]. Biochem Pharmacol, 1998, 55 (6):883-888.

[57] Maria Luisa Rojo, Antonio Rodriguez-Gaztelumendi, Angel Pazos, et al. Differential adaptive changes on serotonin and noradrenaline transporters in a rat model of peripheral neuropathic pain [J]. Neurosci Lett, 2012, 515 (2):181-186.

[58] Lu Guang-hua, Dong Zheng-qi, Wang Qing, et al. Toxicity assessment of nine types of decoction pieces from the daughter root of Aconitum carmichaeli (Fuzi) based on the chemical analysis of their diester diterpenoid alkaloids [J]. Planta Med, 2010, 76 (8):825-830.

[59] Wen Rui-qing, Li Dong-hui, Zhao Xin, et al. [Rationality of the processing methods of aconiti lateralis radix (Fuzi) based on chemical analysis] [J]. Yao Xue Xue Bao, 2013, 48 (2):286-290.

[60] Karoline Peter, Johann Schinnerl, Susanne Felsinger, et al. A novel concept for detoxification: complexation between aconitine and liquiritin in a Chinese herbal formula ('Sini Tang') [J]. J Ethnopharmacol, 2013, 149 (2):562-569.

[61] Sun Bo, Li Ling, Wu Sheng-ming, et al. Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry [J]. Anal Biochem, 2009, 395 (2):125-133.

[62] Huang Qin-an, Zhang Yin-mei, He Yi, et al. Studies on hydrolysis of aconitine [J]. Zhongguo Zhong yao za zhi, 2007, 32 (20):2143-2145.

[63] Kentaro Wada, Makoto Nihira, Hideyuki Hayakawa, et al. Effects of long-term administrations of aconitine on electrocardiogram and tissue concentrations of aconitine and its metabolites in mice [J]. Forensic Sci Int, 2005, 148 (1):21-29.

[64] Wang Yu-guang, Wang Sheng-qi, Liu Yong-xue, et al. Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2006, 844 (2):292-300.

[65] Zhang Hong-gui, Sun Ying, Duan Ming-yu, et al. Separation and identification of Aconitum alkaloids and their metabolites in human urine [J]. Toxicon, 2005, 46 (5):500-506.

[66] Zhang Min, Peng Chong-sheng, Li Xiao-bo. In vivo and in vitro metabolites from the main diester and monoester diterpenoid alkaloids in a traditional chinese herb, the aconitum species [J]. Evid Based Complement Alternat Med, 2015, 2015:252434.

[67] Li Teng-fei, Gong Nian, Wang Yong-xiang. Ester hydrolysis differentially reduces aconitine anti-hypersensitivity mediated by spinal microglial dynorphin A expression and neurotoxicity: Implications for the Aconitum processing [J]. Front Pharmacol, 2016, 7:367.

[68] Yang Chang-lin, Huang Zhi-fang, Zhang Yi-han, et al. Effects of steaming and baking on content of alkaloids in Aconite Lateralis Radix (Fuzi) [J]. China J Chin Materia Medica, 2014, 39 (24):4798-803.

[69] Reisine T, Pasternak G: Opioid analgesics and antagonists. In Goodman and Gilman's the Pharmacological Basis of Therapeutics[M]. 9th edition. Edited by JG H, LE L, PB M, et al. New York: McGraw-Hill; 1996: 521-577

[70] Gong Nian, Wang Yan-chao, Wang Hhi-li, et al. Interactions of the potent D-amino acid oxidase inhibitor CBIO with morphine in pain and tolerance to analgesia [J]. Neuropharmacology, 2012, 63 (3):460-468.

[71] Michael H Bekhit. Opioid-induced hyperalgesia and tolerance [J]. Am J Ther, 2010, 17 (5):498-510.

[72] Shu Hai-hua, Masakazu Hayashida, Huang Wen-qi, et al. The comparison of effects of processed Aconiti tuber, U50488H and MK-801 on the antinociceptive tolerance to morphine [J]. J Ethnopharmacol, 2008, 117 (1):158-165.

[73] Shogo Tokuyama, Ryuji Nagae, Emiko Mashida, et al. Involvement of kappa opioid receptors in formalin-induced inhibition of analgesic tolerance to morphine in mice [J]. J Pharm Pharmacol, 2007, 59 (8):1109-1115.

[74] Masakatsu Takahashi, Toshihiko Senda, Hiroshi Kaneto. Role of spinal kappa opioid receptors in the blockade of the development of antinociceptive tolerance to morphine [J]. Eur J Pharmacol, 1991, 200 (2-3):293-297.

[75] Huang Qian, Sun Ming-li, Chen Yuan, et al. Concurrent bullatine A enhances morphine antinociception and inhibits morphine antinociceptive tolerance by indirect activation of spinal κ-opioid receptors [J]. J Ethnopharmacol, 2016, 2017:151-159.

[76] Shu Hai-hua, Hideko Arita, Masakazu Hayashida, et al. Effects of processed Aconiti tuber and its ingredient alkaloids on the development of antinociceptive tolerance to morphine [J]. J Ethnopharmacol, 2006, 103 (3):398-405.

[77] 李荣, 吕洪伟: 吗啡与吗啡加高乌甲素用于妇科手术后PCIA效果的临床研究. 山东省第十六次麻醉学学术会议; 中国山东济宁. 2013: 5.

[78] 陈新建, 陈彦青. 高乌甲素复合舒芬太尼用于老年患者胃癌术后静脉自控镇痛 [J]. 临床麻醉学杂志, 2011(10):989-990.

[79] Shu Hai-hua, Masakazu Hayashida, Shunsuke Chiba, et al. Inhibitory effect of processed Aconiti tuber on the development of antinociceptive tolerance to morphine: evaluation with a thermal assay [J]. J Ethnopharmacol, 2007, 113 (3):560-563.

[80] Shu Hai-hua, Hideko Arita, Masakazu Hayashida, et al. Inhibition of morphine tolerance by processed Aconiti tuber is mediated by kappa-opioid receptors [J]. J Ethnopharmacol, 2006, 106 (2):263-271.

[81] Takemori A E, Loh H H, Lee N M. Suppression by dynorphin A and [des-Tyr1]dynorphin A peptides of the expression of opiate withdrawal and tolerance in morphine-dependent mice [J]. J Pharmacol Exp Ther, 1993, 266 (1):121-124.

[82] Sourisak Sounvoravong, Masakatsu Takahashi, Mihoko N Nakashima, et al. Disability of development of tolerance to morphine and U-50,488H, a selective kappa-opioid receptor agonist, in neuropathic pain model mice [J]. J Pharmacol Sci, 2004, 94 (3):305-312.

[83] Kenzie L Preston, Annie Umbricht, J R Schroeder, et al. Cyclazocine: comparison to hydromorphone and interaction with cocaine [J]. Behav Pharmacol, 2004, 15 (2):91-102.

[84] Sharon L Walsh, Eric C Strain, Mary E Abreu, et al. Enadoline, a selective kappa opioid agonist: comparison with butorphanol and hydromorphone in humans [J]. Psychopharmacology (Berl), 2001, 157 (2):151-162.

[85] Pfeiffer A, Brantl V, Herz A, et al. Psychotomimesis mediated by kappa opiate receptors [J]. Science, 1986, 233 (4765):774-776.

[86] Li TF, Wu HY, Wang YR, et al. Signaling mechanisms underlying bullyaconitine A-induced dynorphin A expression and mechanical antiallodynia in neuropathy [J]. Sci Rep, 2017, in press.

[87] Wu Hai-yun, Mao Xiao-fang, Fan Hui, et al. p38β MAPK signaling mediates exenatide-stimulated microglial β-endorphin expression [J]. Mol Pharmacol, 2017, DOI: https://doi.org/10.1124/mol.116.107102.

[88] Jia Yu, Gong Nian, Li Teng-fei, et al. Peptidic exenatide and herbal catalpol mediate neuroprotection via the hippocampal GLP-1 receptor/beta-endorphin pathway [J]. Pharmacol Res, 2015, 102: 276-285.

[89] Fan H, Gong N, Li TF, et al. The non-peptide GLP-1 receptor agonist WB4-24 blocks inflammatory nociception by stimulating beta-endorphin release from spinal microglia [J]. Br J Pharmacol, 2015, 172 (1):64-79.

[90] Gong Nian, Xiao Qi, Zhu Bin, et al. Activation of spinal glucagon-like peptide-1 receptors specifically suppresses pain hypersensitivity [J]. J Neurosci, 2014, 34 (15):5322-5334.

[91] Fan Hui, Li Teng-fei, Gong Nian, et al. Shanzhiside methylester, the principle effective iridoid glycoside from the analgesic herb Lamiophlomis rotata, reduces neuropathic pain by stimulatine spinal microglia beta-endorphin expression [J]. Neuropharmacology, 2016, 101: 98-109.

[92] Zhu Bin, Gong Nian, Fan Hui, et al. Lamiophlomis rotata, an orally available Tibetan herbal painkiller, specifically reduces pain hypersensitivity states through the activation of spinal glucagon-like peptide-1 receptors [J]. Anesthesiology, 2014, 121 (4):835-851.

[93] Gong Nian, Fan Hui, Ma Ai-niu, et al. Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors [J]. Neuropharmacology, 2014, 84:31-45.

[94] Xu Meng, Wu Hai-yun, Liu Hao, et al. Morroniside, a secoiridoid glycoside from Cornus officinalis, attenuates neuropathic pain by activation of spinal glucagon-like peptide-1 receptors [J] Br J Pharmacol, DOI: 10.1111/bph.13720.

[95] Huang Qian, Mao Xiao-fang, Wu Hai-yun, et al. Cynandione A attenuates neuropathic pain through p38β MAPK-mediated spinal microglial expression of β-endorphin [J]. Brain Behav Immun, 2017, DOI: 10.1016/j.bbi.2017.02.005.

Research Progress on Mechanisms Underlying Aconitines Analgesia

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