Morphine Withdrawal Attenuating Effect, Toxicity and Alkaloid Composition of Sophora Alopecuroides L. Var. Alopecuroides
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Research Journal of Pharmacognosy (RJP) 4(1), 2017: 59-66 Received: Aug 2016 Accepted: Oct 2016 Original article Morphine withdrawal attenuating effect, toxicity and alkaloid composition of Sophora alopecuroides L. var. alopecuroides S. Kianbakht1*, R. Hajiaghaee1, A. Ramezani Salehabad2 1Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran. 2Behsazan Machinery Manufacturing Company, Rafsanjan, Kerman Province, Iran. Abstract Background and objectives: The seeds of Sophora alopecuroides L. var. alopecuroides may benefit treatment of opioid dependence. Therefore, the plant alkaloid composition, toxicity and effects on morphine withdrawal were studied. Methods: The alkaloid composition was determined by GC and GC/MS analysis. Mice were made dependent by morphine injected 3 times a day for 3 days. The withdrawal jumping and diarrhea were induced by administration of naloxone 2 h after the 10th injection of morphine on the day 4. The ethanol 90% extract (100, 200, 300 mg/kg), alkaloid fraction (5, 10, 20 mg/kg), morphine (50 mg/kg) or saline were injected 30 min before naloxone. All drugs were injected subcutaneously to groups each consisting of 10 mice. To assess toxicity, different doses of the ethanol or aqueous extracts dissolved in normal saline were gavaged once to groups each consisting of 30 mice. Afterward, the numbers of dead animals within 72 h after gavage were counted and LD50 was calculated. Results: Matrine, cytisine, sophoridine, n-methyl cytisine, sophocarpine and sophoramine were the major alkaloids. All doses of the total extract, alkaloid fraction and morphine decreased jumping and diarrhea significantly compared to the saline (p<0.001). The effects of the total extract and alkaloid fraction were not significantly different from morphine (p>0.05). The ethanol and aqueous extracts LD50 were 355 mg/kg and 540 mg/kg, respectively. Conclusion: The plant inhibited opioid withdrawal with efficacy comparable to morphine. The alkaloids may be involved in the effect. The ethanol and aqueous extracts are moderately and slightly orally toxic, respectively. Keywords: addiction, alkaloid, Sophora alopecuroides, toxicity Introduction Opioid dependence is a worldwide health α2-adrenoceptor agonists and adjunct medications problem that has economic, personal and public are used for the opioid detoxification. In health consequences. There are three major antagonist maintenance, naltrexone (an opioid approaches to pharmacologic treatment of opioid antagonist) is used. The pharmacologic agents dependence: opioid detoxification, agonist with proven efficacy in the agonist maintenance maintenance and antagonist maintenance. Opioid are buprenorphine, buprenorphine/naloxone and detoxification is utilized mainly for transition methadone [1,2]. The pharmacologic agents used into or out of a maintenance program over a very in the treatment of opioid dependence have short period of time. Methadone, buprenorphine, important limitations in efficacy and safety. © Available at: http://rjpharmacognosy.ir Copy right 2014 by the Iranian Society of Pharmacognosy *Corresponding author: [email protected], Tel/Fax: +98263-4764010, Fax: +98263-4764021 Kianbakht S. et al. Novel pharmacologic options with more efficacy water for 3 days with three changes of the and better safety profile are needed for opioid solution. The resulting extract was filtered and dependence treatment [3,4]. evaporated under vacuum into a dried powder (30 Plants can be a source of new opioid dependence g, 6% and 13 g, 2.6% for ethanol and aqueous pharmacotherapies [5,6]. Sophora alopecuroides extracts, respectively). L. var. alopecuroides (S. alopecuroides), (Leguminosae) is a perennial herb growing in Extraction of alkaloids Western Asia including Iran and Central Asia. Alkaloid extraction was carried out as described The plant seeds have been traditionally used for by Kamada et al. [12]: 200 mL of CHCl3- Me treatment of many disorders including pain, OH- NH4OH (15: 5: 1) was added to 600 mg of inflammation, diarrhea, fever, bacterial 90% ethanol extract and sonicated for 10 min. infections, chronic liver diseases, heart failure After filtration, the residue was washed with 200 and hypertension [7]. Moreover, decoction of the mL of solution twice. The pooled filtrate was plant seed has been administered orally for evaporated to dryness. Five mL of CHCl3 and 2 detoxification and maintenance treatment of mL of 1 N H2SO4 were added to the residue and opium and heroin addicts in Iran. The plant and the solution was mixed. The CHCl3 phase was removed and the H SO phase was adjusted to pH its alkaloids have been described as poisonous 2 4 10 with 28% NH OH. Alkaloids were extracted [8]. Quinolizidine alkaloids are responsible for 4 once with 2 mL and twice with 1 mL of CHCl the therapeutic and adverse effects of S. 3 from the solution. The combined extracts were alopecuroides. The alkaloids are very soluble in filtered after adding anhydrous Na2SO4 and were ethanol and water. The alkaloids have shown a evaporated to dryness at 40 °C (265 mg). variety of pharmacological effects on the immune, nervous, gastrointestinal and GC (gas chromatography) and GC/MS (gas cardiovascular systems and also anticancer and chromatography-mass spectrometry) analyses antimicrobial activities [9,10]. One of the The extraction of alkaloids was analyzed on a alkaloids called cytisine and its analog Younglin Acm 600 instrument with an FID varenicline have been used to treat nicotine detector operated with a split/splitless injector addiction [11]. There has been no study on the (Younglin, Korea) and DB-5 capillary column, phytochemical characteristics of the S. 30 m×0.25 mm i.d., 0.25 μm film thickness alopecuroides from Iran. Moreover, the toxicity (Agilent, USA). Carrier gas: helium, linear and effects of S. alopecuroides on the opioid velocity: 30 cm/sec, flow: 0.8 mL/min. Injection withdrawal have not been evaluated so far. temperature: 290 oC. Injection volume: 1.0 μL. Therefore, the present study was undertaken to Injection mode: split (1:50). Temperature examine these subjects. program: 50 oC for 5 min, rising at 3 oC/min to o o o 240 C, then rising at 15 C/min to 300 C, held o o Experimental at 300 C for 3 min. FID (290 C): H2 flow: 50 Plant material mL/min; air flow: 400 mL/min. GC/MS analysis The seeds of S. alopecuroides were collected was performed on an Agilent 6890/5973 N from Kerman Province, Iran at the fruiting stage instrument and DB-5 capillary column (30 (November, 2015). A voucher specimen of the m×0.25 mm i.d., 0.25 μm film thickness). Carrier gas: helium, Linear velocity: 32.4 cm/sec, flow: plant was deposited at the Herbarium of the o Institute of Medicinal Plants, ACECR, Karaj, 0.8 mL/min; injection temperature: 290 C; Iran. injection volume: 1.0 μL. Injection mode: split (1:10); temperature program: 50 oC, for 5 min, o o Extraction rising at 3 C/min to 240 C, then rising at 15 oC/min to 300 oC, held at 300 oC for 3 min; MS The plant seeds were dried, powdered (500 g) o and macerated with 90% ethanol solution or interface temperature: 290 C, MS mode: EI, 60 RJP 4(1), 2017: 59-66 Sophora alopecuroides attenuates morphine withdrawal Ionization voltage: 70 eV; mass range: 40-500 u; of 30 mice (15 males and 15 females) in a scan speed: 3.18 scans/sec; interval: 0.50 sec (2 volume of 5 mL/kg. Afterward, the numbers of Hz). Data handling was conducted using a Chem. dead animals within 72 h after gavage were Station (Agilent, USA). counted and LD50 was calculated by the graphical method of Miller and Tainter as described Identification of the components previously [17,18]. The administered doses were The compounds from the alkaloids extract were 12.5, 25, 50, 100, 200, 300, 400, 500, 750 and identified by comparison of their retention 1000 mg/kg. indices which were calculated by using the retention times of injected n-alkanes (C8–C28) Effects on opioid withdrawal in mice (obtained from Fluka, USA) with the same The effects on morphine withdrawal were chromatographic conditions, along with the evaluated using a method described previously fragmentation patterns of the mass spectra with [19]. To induce morphine dependence in the those reported in the literatures and the published mice, morphine was given with the following mass spectra or WILEY library (13). The dosage schedule. Morphine was injected thrice a percentage of the identified compounds was day at 9:30 am, 1:30 pm and 5:30 pm, using the calculated based on GC peak areas without any doses 50, 50 and 75 mg/kg respectively for 3 correction factors as described previously [13- days. The higher afternoon dose was aimed to 16]. minimize overnight withdrawal. Moreover, a 50 mg/kg dose of morphine was given in morning of Drugs the 4th day (2 h before naloxone injection). Morphine sulfate was purchased from the Darou Hyperactivity and Straub tail effect were noted Pakhsh pharmaceutical company (Iran). after morphine injection. Naloxone hydrochloride was obtained from the Naloxone (2 mg/kg) was given 2 h after the last Sigma-Aldrich company (USA). All drugs and injection of morphine in the 4th day. extracts were dissolved in normal saline. The Subsequently, the animals were placed singly on drugs and extracts were prepared immediately a piece of blotting paper in a cylindrical glass (25 prior use and injected subcutaneously in a cm in diameter, 40 cm height) for 30 min. volume of 5 mL/kg. The doses of total extract, Naloxone immediately caused morphine alkaloid fraction and morphine were as follows. withdrawal signs as jumping and diarrhea. The Total extract: 100, 200 and 300 mg/kg; alkaloid number of jumps and feces weight during the 30 fraction: 5, 10 and 20 mg/kg; morphine: 50 min period was recorded for each animal. The mg/kg.