2 Analgesic Activity of Ethanol Leaf Extract Of

Danraka et al., Nig. Journ. Pharm. Sci., September, 2020, Vol. 19 No.2, 11-24

ANALGESIC ACTIVITY OF ETHANOL LEAF EXTRACT OF HYMENODICTYON FLORIBUNDUM (Hochst. & Steud) B. L. Rob AND IT’S POSSIBLE MECHANISM OF ACTION IN MICE *Danraka, R.N., Maje, I. M. and Ejiofor, J. I Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. *Author for correspondence: [email protected]

ABSTRACT

The plant Hymenodictyon floribundum is a common shrub in Africa that has been reported to be used traditionally in the treatment of pain and inflammatory diseases. In this study, the analgesic activity of the ethanol leaf extract of Hymenodictyon floribundum (HF) in mice was studied after phytochemical screening and LD50 determination. HF (375, 750 and 1500 mg/kg p.o) was evaluated for analgesic activity using acetic acid induced writhing and hot plate methods in mice. The ethanol leaf extract exhibited significant (P < 0.05) and dose-dependent analgesic activity comparable to that of the reference drug morphine (10 mg/kg body weight, p.o) in both acetic acid and hot plate method. The preliminary phytochemical screening of the extract revealed the presence of flavonoids, tannins, saponins, steroids glycosides, anthraquinones and phenols. The oral median lethal dose (LD50) of the extract in mice was found to be greater than 5,000 mg/kg body weight. The pretreatment of mice with prazosin, naloxone, and propranolol significantly (p<0.05) decreased the analgesic effect of the extract, while pretreatment with yohimbine, and glibenclamide, each, had no significant effect on its analgesic activity. The findings suggest that the ethanol extract of HF possesses analgesic activity possibly via opioidergic, α1 and β-adrenergic receptors pathways and supports the ethnomedicinal claims of the plant for the management of pain. Keywords: Hymenodictyon floribundum, analgesic, phytochemical, writhes, acetic acid, hot plate

INTRODUCTION The International Association for the Study of Pain and the World Health Organization Research in phytomedicine is now a define pain as “an unpleasant sensory and common practice in the search for bioactive emotional experience associated with actual substances used as medicines (Sen and or potential tissue damage, or described in Samantha, 2014). The plant Hymenodictyon terms of such damage.” (IASP, 2014.). floribundum belongs to the family of Although the drugs used in the treatment of Rubiaceae with a grey-black stem that is 4 to pain are available, people still complain of 9 meters tall and grows in a rocky terrain. pain. Under-treated or unrelieved pain also (Bridson and Verdcout, 2003). There are places a high financial burden on humans over 30 species of Hymenodictyon and (Stewart et al 2003). Most of the available several ethnobotanical claims have pointed analgesic like the non-steroidal anti- to the use of H. floribundum as an effective inflammatory drugs (NSAIDs) cause anti-inflammatory and analgesic plants. H. gastrointestinal bleeding and excelsum for example has been scientifically immunosuppression (Hougee, 2008), while proven to possess significant anti- the relatively safe cyclooxygenase-inhibitors inflammatory and analgesic properties (Kar such as celecoxib, are often not affordable to et al., 2013). the general populace (Tick et al.,2018). In a

research sponsored by World Health floribundum were shade-dried until constant Organization to study the global burden of weight was obtained and then reduced into disease, it was found that conditions fine powder with the aid of a mortar and characterized by presence of pain (low back pestle. The powdered leaf material (700 g) pain, neck pain, other musculoskeletal was extracted exhaustively with aqueous w disorders, migraine, and falls) accounted for ethanol (70% /v) using continuous Soxhlet 5 of the top 10 conditions. (Vos et al., apparatus for 72 hours. The extract was 2017). Low back pain was responsible for concentrated to dryness on a water-bath set 83 million disability and was the greatest at 50°C, subsequently packed in an amber contributor of years lived with disability bottle and stored in a desiccator prior until (Hartvigsen et al., 2018). Since traditional use. The extract was referred to as ethanol medicine has been the major source of most leaf extract of Hymenodictyon floribundum drugs and has better acceptability because of (EEHF). Solutions of the extract were perceived safety (wang et al.,2014), it safe prepared freshly with Normal Saline for to say that there is a possibility of more each study. The percentage yield of the discovery through traditional medicine. extract was calculated using the formula: Though not documented, the leaves of Hymenodictyon floribundum like H. excelsum, is claimed to possess analgesic and anti-inflammatory properties by the Qualitative Phytochemical Analysis ethnic people of kuregu, Zaria Local Government Area, Kaduna state and hence The extract was subjected to phytochemical the need to validate this claim screening in accordance with standard protocol as described by Evans (2002). The 2.0 MATERIALS AND METHODS extract was screened by adopting simple chemical tests to detect the presence of Plant Collection and Identification alkaloids, flavonoids, saponins, cardiac glycosides, tannins, anthraquinones, The whole plant consisting of the stem (with carbohydrates, steroids and triterpenes. the leaves attached) and the root was collected from Kargi Hill along Birnin Animals Gwari road, Zaria Local Government Area, Kaduna state in October 2019. The collected Adult male Swiss Albino mice (18-25g) plant was identified and authenticated, by were obtained from the Animal House comparing with an existing specimen by a Facility of the Department of Pharmacology taxonomist, Malam Sanusi Namadi of the and Therapeutics, Ahmadu Bello University, Department of Botany, Faculty of Life Zaria, Nigeria. The animals were housed in Sciences, Ahmadu Bello University, Zaria. a well-ventilated room in their cages and A voucher specimen number of ABU900124 provided with normal rodent feed and water was assigned to the plant by comparing with ad libitum until the end of the study. They an existing voucher specimen. were maintained under normal 12 hours light/dark cycle. All experiments were Extraction of plant material conducted in accordance with the guideline of National Institute of Health (NIH, 1998). Plant extraction was carried out according to the method described by Kupchan et al. Ethical approval for the use of animals was (1973). The leaves of Hymenodictyon sought from the Ahmadu Bello University

Committee on Animal Use and Care At the end of this test, mice that survived (ABUCAUC) and an approval number of were euthanized with chloroform and ABUCAUC/2020/011 was given. subsequently incinerated 2.4 Drugs/chemicals Evaluation of Analgesic Activities in Mice Glacial acetic acid (May and Baker limited, Acetic acid induced writhing in mice Dagenham, England), ethanol (Sigma Aldrich, St. Louis Mo, USA), morphine The method previously described by Koster sulphate (Martindale Pharmaceuticals, UK), et al (1959) was adopted for this study. hydrochloric acid (May and Baker, UK), Thirty (30) male Swiss albino mice were sulphuric acid (May and Baker, UK), ferric divided into 5 groups of six (6) mice. Mice chloride anhydrous (Avishkar, India), in group I were administered with Normal ammonia (Lobachemie, India), naloxone Saline (10 ml/kg, p.o) and served as a hydrochloride (Abcam Plc, Cambridge, negative control, while mice in group V UK), prazosin hydrochloride (Abcam Plc, received morphine (10 mg/kg, p.o.) and Cambridge, UK), glibenclamide (Abcam served as a positive control. Group II, III Plc, Cambridge, UK), yohimbine and IV received graded doses of EEHF (375, hydrochloride (Abcam Plc, Cambridge, 750 and 1500 mg/kg respectively) via oral UK), propranolol hydrochloride (Abcam Plc, route. Sixty (60) minutes after oral Cambridge, UK). All the reagents were of administration, acetic acid 0.6% v/v (10 analytical grade. ml/kg) was administered to each mouse via intraperitoneal route and was placed in an Acute Toxicity Studies observation cage. Five (5) minutes after acetic acid injection, the number of writhes Median Lethal Dose (LD50) determination was counted for each mouse for a period of was conducted using Organization for 10 minutes. A reduction in the number of Economic Co-operation and Development writhes as compared to the vehicle treated (OECD 423) guidelines in mice. In this animals was considered as evidence for the method, two groups of three (3) animals presence of analgesia and expressed as each were fasted prior to dosing (food but percent inhibition of writhes. not water was withheld for 3 hours). The fasted body weight was determined for each animal and the dose was then calculated according to the body weight. Food was further withheld for 1-2 hours in mice after Mc = Mean number of writhes (control) the extract has been administered. In the first Mt = Mean number of writhes (test) phase, 2000 mg/kg of the test substance was administered to each mouse in a single oral Hot Plate test in Mice dose using oral canula and the mice were observed for 48 hours for clinical sign and The method of Eddy and Leimbach (1953) symptoms of toxicity including death. The was adopted. The hot plate was provided by second phase was conducted in a similar Ugo Basile hot plate (model 7280, manner to the first but with dose of 5000 Germany) at the laboratory of the mg/kg. Department of Pharmacology, Faculty of Pharmacy, Bayero University Kano, Nigeria. Mice were individually placed on a

hot plate (55±1°C), before drug treatment so various pain pathways involved in the that each animal serves as its own control. analgesic activity of H. floribundum. The The time taken for the animal to either pathways investigated and the standard licked the paw, withdrawal of the paws or antagonists/blockers used for the studies jumped was taken as reaction time and were as follows: recorded. A cut-off time of 30 sec was used to avoid damage to the paw. Male Swiss a) opioidergic, using naloxone (a albino mice (30) were divided into 5 groups nonselective opioid receptor antagonist, 2 of six (6) mice each. Mice in group I were mg/kg, i.p), administered Normal Saline (10 ml/kg, p.o) b) α1-adrenergic, using prazosin (an α1- and served as a negative control, while adrenoceptor antagonist, 1 mg/kg, i.p), group V mice received morphine (10 mg/kg, i.p) and served as a positive control. Group c) α2-adrenergic, using yohimbine (an α2- II, III and IV received graded doses of adrenoceptor antagonist, 1 mg/kg, i.p), EEHF (375, 750 and 1500 mg/kg respectively) via oral route. The latency was d) β-adrenergic, using propranolol (a non- observed and recorded after 0, 60, 90, 120 selective β-adrenergic blocker, 20 mg/kg, and 150 minutes. The prolongation of the i.p) latency times was taken as an analgesic response (percentage maximum possible e) potassium ATP, using glibenclamide (a effect MPE). K-ATP channel blocker, 5 mg/kg, i.p), Mice were randomly divided into 6 groups of six (6) mice each for the pathways. All the groups were treated as follows: Where; Group I: Normal Saline (10 ml/kg, p.o) Test = latency to respond alone after treatment Group II: EEHF (1,500 mg/kg, p.o) alone Baseline = latency to respond Group III: morphine (10 mg/kg, p.o) alone prior to treatment and Group IV: receptor antagonist/blocker (as Cut-off (30 secs) = preset time at which mentioned). the test was ended in the absence of a response Groups V: pretreated with receptor antagonist/blocker 30 minutes before Mechanistic Studies administering The involvement of opioidergic, adrenergic, EEHF (1500 mg/kg, oral). cholinergic and K+- channel pathways in the elucidation of analgesic activities of the Group VI: pretreated with receptor extract was investigated using mouse model antagonist/blocker 30 minutes before of acetic acid induced writhing by Rangel et administering morphine (10 mg/kg, oral). al. (2012) as previously described. Sixty (60) minutes post treatment (30 The most active dose of Hymenodictyon minutes for groups IV, VII and VIII), mice floribundum extract was subjected to were challenged with acetic acid to induce mechanistic studies to investigate the

writhing by adopting the method of Koster steroids/triterpenes, terpenoids, et al. (1959) as previously described. carbohydrates and anthraquinones (Table 1).

Statistical Analysis Estimation of Median Lethal Dose (LD50) of Ethanol Leaf Extract of leaf of Data obtained were analyzed using one-way Hymenodictyon floribundum analysis of variance (ANOVA) and Mixed design ANOVA in SPSS version 25. Results The LD50 of the ethanol leaf extract of H. are presented as tables and graph. floribundum was estimated to be greater than 5000 mg/kg body weight and there RESULTS were no signs of toxicity such as salivation, sleep, tremor, lacrimation, convulsion, Percentage Yield of the Ethanol Leaf diarrhea, lethargy, respiration and change in Extract of leaf of Hymenodictyon behavioural pattern and mortality. floribundum Effect of Ethanol Extract of H. The percentage yield of the dark brown floribundum on Acetic-acid induced ethanol leaf extract of Hymenodictyon Writhes in Mice floribundum was 10.4%. The extract was able to alleviate pain by Preliminary Phytochemical constituents reducing the number of writhes observed in The preliminary phytochemical screening of the animals. The decrease was statistically the ethanol leaf extract of H. floribundum significant (p < 0.05) compared to Normal revealed the presence of alkaloids, cardiac Saline and was dose-dependent (Figure 1). glycosides, saponin, tannins, flavonoids,

Table 1: Phytochemical constituents present in the ethanol leaf extract of leaf of Hymenodictyon floribundum Phytochemical constituents Inference Alkaloids present Cardiac glycosides present Saponins present Tannins present Triterpenes/Steroids present Terpenoids present Carbohydrates present Anthraquinones present

Figure 1: Effect of Ethanol Extract of H. floribundum on Acetic-acid induced Writhes in Mice

Data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett Post-hoc Test.* p< 0.05 compared to Normal Saline, ** p < 0.01 compared to Normal Saline. HF = Ethanol Extract of Hymenodictyon floribundum; S.E.M. = Standard Error of Mean Effect of Ethanol Extract of H. floribundum on Reaction time in Hot-plate induced Pain in Mice Compared to the baseline pain threshold, the only 1500mg/kg of the extract significantly extract at all doses was able to increase pain (p< 0.05) increased pain threshold when threshold over time and displayed a dose- compared to normal saline; the increase in dependent increase in latency to reaction in latency to pain was comparable to morphine the positive direction. At all-time points, at the highest dose of the extract (Figure 2). .

Figure 2: Effect of Ethanol Extract of H. floribundum on Reaction time in Hot-plate induced Pain in Mice

Data was analyzed using Mixed-Design analysis of variance (ANOVA) followed by Dunnett Post-hoc Test.* p< 0.05 compared to T0, # p< 0.05 compared to Normal Saline. HF = Ethanol Extract of Hymenodictyon floribundum; S.E.M. = Standard Error of Mean. T= Test time where T0 is baseline, T1, T2, T3 and T4 represent 60 min, 90 min, 120 min and 150 min after treatment administration.

Result of Mechanistic Study significantly different from the extract (p<0.01). The analgesic activity of the The mean number of writhes produced by extract in the presence of yohimbine was mice treated with glibenclamide was significantly reduced (p= 0.002) compared significantly higher than that of the extract to when the extract was administered alone at the highest dose (p< 0.05) but was (Figure 4). comparable to that produced by normal saline treated group (p > 0.05). However, The effect of prazosin was comparable to the analgesic activity of the extract as well that of normal saline (p>0.05). The as that of morphine were unaffected in the analgesic activity of the extract and presence a K+-Channel blocker, morphine were unaltered in the presence of glibenclamide (p > 0.05) (Figure 3). prazosin (p>0.05) (Figure 5). The effect of yohimbine was comparable to that of normal saline (p>0.05) but was

Figure 3: Effect of Ethanol Extract of K+-Channel

Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Bonferroni Post-hoc test. * is p< 0.002 compared to EEHF1500, # is p < 0.05 compared to Morphine. N/Saline = Normal Saline; EEHF = ethanol Extract of Hymenodictyon floribundum; GLB = Glibenclamide. Propranolol, given alone, produced activity Opioid Pathway- Naloxone significantly that was similar to that produced by normal increased the number of writhes when given saline (p>0.05) but significantly reduced the alone and when administered before the analgesic activity of the extract (p<0.01). extract. The analgesic activity of the extract The analgesic activity of was unaltered in and morphine were significantly reduced (p the presence of propranolol (Figure 6). >0.05) in the presence of naloxone (Figure 7).

Figure 4.0: Effect of Ethanol Extract of H. floribundum on α2-receptor

Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Bonferroni Post-hoc test. * is p< 0.05 compared to EEHF1500, # is p < 0.05 compared to YHB. N/Saline = Normal Saline; EEHF = Ethanol Extract of Hymenodictyon floribundum; YHB = Yohimbine.

Figure 5.0: Effect of Ethanol Extract of H. floribundum on α1-receptor

Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Bonferroni Post-hoc test. * is p< 0.05 compared to EEHF1500, # is p < 0.05 compared to PRZ. N/Saline = Normal Saline; EEHF = Ethanol Extract of Hymenodictyon floribundum; PRZ = Prazosin.

Figure 6.0: Effect of Ethanol Extract of H. floribundum on β-receptor

Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Bonferroni Post-hoc test. * is p< 0.05 compared to EEHF1500. N/Saline = Normal Saline; EEHF =ethanol Extract of Hymenodictyon floribundum; PRN = Propranolol.

Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Bonferroni Post-hoc test. * is p< 0.05 compared to EEHF1500, # is p < 0.05 compared to Morphine. N/Saline = Normal Saline; EEHF = ethanol Extract of Hymenodictyon floribundum; NLX = Naloxone.

DISCUSSION factors; the extract is greatly metabolized before it gets to the central compartment Acetic-acid causes pain through peripheral leading to insufficient amount in the CNS nociceptive pathway (Ma and Zhang, 2011). with lower doses; and secondly, there is a This method is highly responsive and quick high possibility that receptor occupancy in blocking the detection of painful effects plays a key role in the analgesic activity of of compound(s) at dose level that may the extract (Nick, 2018). appear inactive in other procedures like tail- flick test (Usman et al., 2008; Yerima et al., Several phytochemicals such as saponins, 2009). The irritant indirectly activates the tannins, and cardiac glycosides which are release of nociceptive endogenous mediators present in the ethanol leaf extract of (bradykinin, serotonin, and prostaglandin) Hymenodictyon floribundum are known to and pro-inflammatory cytokines (TNF-α and have analgesic activity (Calixto et al., 2000). IL-1β) to bring about painful sensation There are also reports on the analgesic (Kakoti et al., 2013). The abdominal effects of alkaloids, and saponins (Choi et constriction produced by acetic acid can be al., 2005; Reanmongkol et al., 2005). used to evaluate peripherally acting analgesics like non-steroidal anti- Majority of analgesic agents are known to inflammatory drugs (NSAIDs) (Aiyelero et block several nociceptive pathways in al., 2009; Mishra et al., 2011; Kakoti et al., addition to the inhibition of prostaglandin + 2013) and centrally acting analgesics like synthesis. These pathways include; the K - morphine (Donkor et al., 2013; Kakoti et al., Channel (Tsantoulas and McMahon, 2014), 2013). Acetyl salicylic acid (ASA) and adrenergic α1, α2 (Daniel et al., 2009) and β NSAIDs decrease writhes caused by acetic receptors (Elif et al., 2010) as well as acid by interfering with cyclo-oxygenase opioidergic pathway (Machelska and Melih, (COX) in peripheral tissues by inhibiting the 2018). All these pathways have known release and/or synthesis of inflammatory antagonists which have been employed in mediators (Donkor et al., 2013) this study to determine the possible mechanism of action of the extract. The K+- The ability of the extract to dose- channel is blocked by sulphonamides such dependently reduce pain caused by acetic as glibenclamide (Kenia et al., 2006); α1 acid is an indication of its peripheral-acting and α2 adrenoceptors are blocked by analgesic potential. In the hotplate test, a prazosin (Mycek, 1997) and yohimbine well-established centrally-mediated pain (Katzung and Masters, 2013) respectively; pathway, the extract was also significantly propranolol is a non-selective β- able to increase pain threshold, although the adrenoceptor blocker (Perez, 2006) while analgesic (Bhalke and Pal, 2012). The effect the opioid receptors situated at the spinal was more pronounced at the highest dose of and supraspinal area of central nervous the extract after 2 hours of administration. system are blocked by naloxone (Mary- This time-dependent effect is an indication Jeanne et al., 1983). This study revealed of slow and possibly a complete absorption that glibenclamide, yohimbine, prazosin, across the blood brain barrier (Nick, 2018) propranolol and naloxone are all devoid of since the analgesic effect started few hours analgesic activity since the mean number of after drug administration and lasted several writhes produced by mice treated with these hours before wearing off. The fact that the antagonists/blockers were significantly analgesia was more pronounced at the higher than that produced by highest dose of highest dose is an indication of several

the extract but were comparable to that These suggest the non-involvement of the produced by normal saline treated group. α2 adrenoceptor antagonist and potassium channel blocker pathways in the analgesic The extract most probably does not act via activity of the extract. But pretreatment of + ATP gated K - Channel pathway since animals with naloxone, prazosin and blockade of this channel did not negatively propranolol significantly reversed the impair its analgesic activity. Yohimbine, an analgesic action of the extract. These α1- adrenoceptor antagonist was able to suggest the involvement of the opioidergic, impair the analgesic activity of the extract α1 and β-adrenergic pathways in the by significantly increasing the average analgesic activity of the extract. writhes number in mice treated with the extract following pretreatment of the The ability of prazosin to potentiate antagonist- this is an indication that the morphine analgesic has been reported extract most likely acts via the activation of (Ozdogan et al., 2003; Ozdogan et al., α1- receptor in the sympathetic pathway. It 2004). The analgesic potential of is unlikely that the extract acts via α2- propranolol has been reported (Sadrabadi et adrenoceptor pathway since even in the al., 2011). The therapeutic uses of presence of a known α1-adrenoceptor propranolol include; the management of blocker, prazosin, the analgesic activity of migraine headache prophylaxis; and several the extract remained unaltered. Propranolol clinical studies in adults have found that is a non-selective β-adrenoceptor antagonist. chronic therapy with propranolol reduces the In the presence of this blocker, the analgesic frequency and severity of migraine in 60 to activity of the extract at the highest dose was 80% of patients (Caruso et al., 2000; significantly altered, an indication that the Sadrabadi et al., 2011). extract owes it analgesic activity partly to the activation of β-receptor. The analgesic Conclusion activity of the extract might also be The result of the study suggests that the attributed to the activation of some or all of ethanolic leaf extract of Hymenodictyon the opioid receptors since its analgesic floribundum may contain bioactive activity was greatly impaired by the constituents with analgesic activity mediated administration of an opioid antagonist prior through the central and peripheral to its own administration. mechanism and may involve interaction It makes sense that the extract may likely which are related to opioidergic, α1 and β- owe its analgesic activity to the activation of adrenergic receptors pathways and further α1, β adrenoceptor and opioidergic support the ethnomedical claim of the use of pathways. The results obtained in the the plant in the management of pain. present work suggest the involvement of Acknowledgement three main systems in the analgesic activity of the extract in the acetic acid induced Authors wish to thank the technical staff of writhing test in mice. The analgesic activity the Department of Pharmacology and of the extract was found to be related Therapeutics for their contributions and opioidergic, α1 and β-adrenergic receptors. expertise during the laboratory stage of this research. In this study, pretreatment of mice with yohimbine and glibenclamide both failed to reverse the analgesic activity of the extract.

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