Antinociceptive Ecdysteroids and Other Constituents of Palisota hirsuta K . Schum ( Commelinaceae )

1 Department of Pharmaceutical Sciences, Faculty of Health Sciences, Kumasi Polytechnic, Kumasi, Ghana. 2 Department of Biomedical Sciences, Faculty of Allied Sciences, College of Health and Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana. 3 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. 4 Department of Chemistry, College of Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.


INTRODUCTION
Pain is a normal manifestation in everyday life and serves as a vital defensive function.However, uncontrolled pain can dramatically diminish quality of life (Page et al., 1997).It is an unpleasant sensation that can be either acute or chronic and is as a consequence of complex neurochemical process in the peripheral and central nervous system (Richard et al., 2012).Pain and inflammation is the most common manifestation of diseases that affect millions of people worldwide (Bhatia et al., 2013).According to a WHO report, about 70-80% of the world's populations rely on non-conventional medicines mainly from herbal sources in their primary health care.Plants are still the reservoirs for obtaining drugs and drug leads (Newman and .Cragg, 2007) and are also a good source of anti-nociceptive compounds; morphine from Papaver somniferum L. (Papaveraceae).
Palisota hirsuta is used traditionally for its pain-relieving and antiseptic effects.The plant juice and crushed pieces of twigs are used as a poultice for fractures, bruises, joint pains, nail ulcers and lymph gland inflammation.The whole plant decoction is used for urethral problems.Leaf preparations are used against lice (Bellomaria and Kacou, 1995).Boakye-Gyasi et al., (2014), demonstrated that oral administration of an ethanolic leaf extract of Palisota hirsuta attenuates pain-related behavior in vincristineinduced neuropathic pain model.Sarpong et al., (2013) also showed that the roots of P. hirsuta and its constituents, mainly 20hydroxyecdysone, show anti-inflammatory and antioxidant activities.However, the root of Palisota hirsuta and its compounds have not being thoroughly evaluated for analgesic activity as suggested by folklore medicine.Therefore, the present study aimed at evaluating the possible anti-nociceptive activity of the .hydroethanolic root extracts, isolate and characterise the antinociceptive constituents.

Plant collection and processing
Palisota hirsuta roots were collected from Esaase-Bontefufuom in the Amansie West district of Ashanti Region of Ghana, in May 2014.The plant was authenticated by Dr George Henry Sam of the Department of Herbal Medicine, Kwame Nkrumah University of Science and Technology; a voucher specimen was kept in the Faculty of Pharmacy and Pharmaceutical Sciences' Herbarium (No. FP 1008).The roots were washed and sun-dried for seven (7) days and then milled into coarse powder.

Extraction
The powdered roots of P. hirsuta (2 kg) was then soxhlet extracted with 4 litres of 70% ethanol.The crude extract (cristened, PHC), was then concentrated with a rotary evaporator (R-114, Buchi, Switzerland) at a temperature of 55 • C under vacuum to give a yield of 200 g (11.69%.). 100 g of the crude extract (PHC) was dissolved in 80% methanol and defatted with Petroleum ether (BDH) 40-60 °C to afford an extract, PHE and the remaining methanol soluble extract as PHM.

Experimental
NMR and MS were employed as the techniques to identify the structure and identity of compounds in this project/study. 1 H NMR, 13 C NMR and 2D NMR (COSY, NOESY, HMQC and HMBC) spectra were obtained on a Bruker 400 MHz instrument.One bond 1 H-13C connectivities were determined with HSQC while two-and three-bond 1 H-13C connectivities were determined by HMBC experiments.Chemical shifts were reported in δ (ppm) using the solvent (DMSO).GC-MS was also utilized to resolve the compounds which were mixtures of fatty acids and for that matter difficult to separate with silica gel.

Anti-Nociceptive Activity of Extracts and Compounds
Drugs: Formalin was obtained from BDH, Poole, England and Morphine hydrochloride was purchased from Phyto-Riker Pharmaceuticals, Accra, Ghana.
Animals: Mice [Imprinting Control Region (ICR)] (30-40g) were purchased from Centre for Scientific Research into Plant Medicine Akwapim Mampong, Ghana and housed in the animal facility of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology (KNUST).The animals were housed in groups of six in stainless steel cages (34×47×18 cm) with soft wood shavings as bedding, fed with normal commercial pellet diet (GAFCO, Tema), given water ad libitum and maintained under laboratory conditions (temperature 24-28 C, relative humidity 60-70%, and 12 hour light-dark cycle).All procedures and techniques used in these studies were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH, Department of Health and Human Services publication no.85 -23, revised 1985).All protocols used were approved by the Department of Pharmacology Ethics Committee, KNUST, Kumasi.

Formalin induced nociception
The formalin test first described by Dubuisson and Dennis, (1977) was carried out as described by Malmberg and Yaksh, (1995) with a few modifications.Each animal was assigned and acclimatized to one of 20 formalin test chambers (a Perspex chamber 15 × 15 × 15 cm) for thirty minutes before formalin injection (Wilson et al., 2002).The mice were then pretreated with the test drugs (30 min for i.p. route and 1 hr for oral route) before intraplantar injection of 10 l of 5 % formalin.The animals were immediately returned individually into the testing chamber.A mirror angled at 45˚ below the floor of the chamber allowed a complete view of the paws.The behaviour of the animal was then captured over a 60 minute period for analysis by a camcorder (Everio™ model GZ-MG1300, JVC, Tokyo, Japan) placed in front of the mirror.Pain response was scored for 60 min, starting immediately after formalin injection.
The first phase of the formalin test was defined conservatively as 0-10 minutes and the second phase 10-60 minutes post formalin injection (Wilson et al., 2002).Nociceptive behaviour was quantified by counting the incidents of spontaneous biting/licking of the injected paw (Zochodne et al., 2001) using the public domain software JWatcher™ Version 1.0 (University of California, Los Angeles, USA and Macquarie University, Sydney, Australia available at http://www.jwatcher.ucla.edu/).Nociceptive score was determined for each 5-mins time block in each phase by measuring the amount of time spent biting/licking the injected paw.The product of the frequency and duration of biting/licking was used as nociceptive score.Mice were randomly selected for one of the following study groups: Group I: Morphine (1, 3, and 10 mg kg -1 ), Group II: P. hirsuta extract, PHC (30, 100 and 300 mg kg -1 ), Group III: Vehicle treated control, Group IV: Methanol fraction, PHM (30, 100 and 300 mg kg -1 ), Group V: Petroleum Ether Fraction, PHE (30, 100 and 300 mg kg -1 ), Group VI: PH IV (1, 10 and 30 mg kg -1 ), Group VII: PH V (1, 10 and 30 mg kg -1 ).

Statistical Analysis
The statistical analyses were performed by ANOVA, followed by Newman-Keuls post hoc tests using GraphPad Prism for Windows version 5 (GraphPad Software, San Diego, USA).The results were expressed as the mean + S.E.M to show variation in groups.Differences are considered significant when P<0.05.

Formalin-induced nociception
Formalin administration produced a typical pattern of flinching and licking behaviour (Figures 1 & 2), produced a marked and dose-related inhibition of both phases of formalininduced nociception first phase (P < 0.0001) and second phase (P < 0.0001).The highest dose produced maximal inhibitions of 40.89±24.10%and 66.25±32.08% for the early and late phases of the crude extract PHC.For the pet-ether fraction, PHE the highest dose caused maximal nociceptive inhibition of 45.02±29.81%and 80.50±4.628%.The maximal inhibitions caused by the highest dose of the methanol fraction PHM was 31.72±15.29%and 61.0±41.97%for the two phases.
Considering the isolates, the pattern of inhibition was not regular for the isolated compounds.For instance, with PH I, the maximal inhibitions caused by the doses (phase 1: 10 mgkg -1 and phase 2: 30 mgkg -1 ) were 55.08±14.90%and 63.57±25.91%.The highest dose (30 mgkg -1 ) caused maximal inhibitions of nociception of 71.39±9.192%and 89.19±3.816%for PH IV and 61.34±11.07%and 82.89±3.978%and for PH V (Figure 2).Similarly, morphine (1-10 mg kg -1 , i.p) produced inhibition of both the neurogenic (P<0.0001) and inflammatory (P < 0.0001) pain phases (Figure 1a-b).Morphine, reduced the duration of formalin evoked nociceptive behavior by a maximum percentage of 92.49 ±4.679% in the early phase and 95.16±5.497% in the late phase of the formalin test (Figure 1a-b).Examining the results obtained in the formalin test, it was deduced that; PHC, PHE, PHM, PH I, IV, V and morphine significantly reduced the time spent in licking the injured paw, an indication that these compounds possess strong anti-nociceptive or analgesic properties.

Identification of the compounds
The fatty acid mixture PH-1 was subjected to GC/MS.Compounds were identified by their mass spectra, interpretation of their fragmentation patterns and comparing retention times of their peaks to those of standard compounds analysed previously in literature (National Institute of Standards and Technology (NIST), 2011).The fatty acid components were identified as hexanoic acid, stearic acid and eicosanoic acid as previously reported by Sarpong et al., (2013).
PH IV was isolated as an off white amorphous solid with a melting point of 240-242°C [Literature: 237.5-239.5°C](Hikino and Hikino, 1990).It showed positive Liebermann's test.The Electron Spray Ionization Mass Spectrometer(ESI-MS) revealed a molecular ion peak [M-H]+ at m/z of 479 which corresponded to the molecular formular, indicating six degrees of unsaturation, ascribed to α, βunsaturated ketone and four steroid rings (Yun-.Song et al., 2007).

DISCUSSION
In the formalin test, the early phase is considered to be due to direct activation of nociception neurons by formalin, whereas the late phase reflects pain generated in acutely injured tissue (Hunskaar and Hole, 1987;Tang et al, 2007).The licking response induced by formalin, results from a combination of peripheral input and spinal cord sensitization (Tjolsen et al., 1992).The intraplantar injection of formalin, releases prostaglandin 2(PGE 2 ), nitrous oxide ( NO) and kinins in the spinal cord (Tjolsen et al., 1992).Taking this into account, the anti-nociception of Palisota hirsuta could be dependent on either peripheral or central sites of action or both.Centrally acting drugs, such as opioids, inhibit both phases of pain by equally inhibiting the effect produced by prostaglandins released at this level in response to inflammation (Ferreira, 1981;Hunskaar and Hole, 1987;Shibata et al, 1989) and by endogenous opioids through their action on the central nervous system.It has been demonstrated (Tjolsen et al., 1992) that the late phase in formalin test depends on an inflammatory reaction in peripheral tissue.Peripheral acting drugs such as diclofenac (Rosland et al., 1990) which block prostaglandin synthesis, reduce nociception mostly in the late phase but can also affect the early stage (Ortiz et al., 2008).In fact, the anti-nociceptive effects of the extracts and compounds, exhibited in the formalin test, suggest an involvement at both central and peripheral levels, which further implies that the extract and the isolates possess not only anti-nociceptive but also antiinflammatory activity.This result therefore agrees with the antiinflammatory activity of P. hirsuta established by Sarpong et al., (2013).
From Figure 1a-f and 2a-f, there is the indication of maximal inhibitions or reductions of the licking time in the early and late phases, respectively.Similarly, morphine (1-10 mg kg -1 , i.p.) produced marked inhibition of both the neurogenic (P<0.0001) and inflammatory (P<0.0001)pain phases (Figure 1ab).Morphine, reduced the duration of formalin evoked nociceptive behavior by a maximum percentage of 92.49±4.67% in the early phase and 95.16±5.497% in the late phase of the formalin test (Figure 1a and b).The analgesic activity of 20-hydroxyecdysone (PH IV) was very much comparable to the standard drug morphine.The anti-nociceptive actions of the fractions and various isolates of the roots of Palisota hirsuta are being reported for the first time.From the results obtained, the anti-nociceptive properties of Palisota hirsuta are not in doubt due to the steroidal nature of the compounds found in the plant.Thus this research has shown that the root extract and its steroidal compounds have profound analgesic effects.However, the anti-nociceptive action is not dose dependent for the extract, fractions and the isolates used for the study.Woode et al., (2009), previously demonstrated the antinociceptive effect of P. hirsuta leaf extract.They highlighted that the analgesic effect of P. hirsuta might partially or wholly be due to the stimulation of peripheral opioid receptors through the activation of the nitric oxide-cyclic GMP-ATP-sensitive K + (NO/cGMP/K + ATP)-channel pathway without tolerance induction after chronic administration.This gives scientific credence to the use of the whole plant for treatment of pain and inflammatory conditions in folklore medicine.
Phytoecdysteroids have received attention as anabolic steroids.Chermnykh et al., (1988) compared the anabolic activities of 20-hydroxyecdysone and that of metandrostenolone and found the former to increase protein and carbohydrate metabolism and was non-toxic to the reproductive organs.Another ecdysteroid, ecdysterone, has also been reported to exhibit potent oxytocic activity in the guinea pig uterus assay besides inhibiting nicotine and serotonin-induced contractions during guinea pig ileum assay (Parameswaran et al, 2001) and may have some implication in the folkloric use to treat infertility.There is ample evidence of the varied biological activities of the ecdysteroids especially 20-hydroxyecdystone, however this is the first time the anti-nociceptive activity has been established.