Isolation and analgesic property of lupeol from Diospyros mespiliformis stem bark

Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria. Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Kaduna State University (KASU), Kaduna, Nigeria. Department of Medicinal Chemistry and Quality Control, NIPRD, Abuja, Nigeria. Natural Products Laboratories, Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS), University of Strathclyde, Glasgow, UK.

Despite some advantages in the medical use of plant extracts over isolated entities, there is a need to identify the component which is responsible for the observed beneficial effects.This study sequentially extracted and fractionated the stem bark of D. mespiliformis in a bioassay-guided manner using analgesic activity in rats as bioactivity marker in order to identify its active component.A voucher specimen (#5120) was deposited at the herbarium of the Institute.D. mespiliformis is not an endangered plant species, and therefore its collection for purposes of use and scientific study does not require prior authorization.

Extraction, fractionation and compound isolation
Stem bark of the plant was collected, cleaned, dried under shade and grinded into powder.The powdered material (500 g) was sequentially extracted with hexane (DM-1), chloroform (DM-2) and methanol (DM-3) to yield 0.97, 1.23 and 7.16% of the extracts, respectively.The three extracts (DM-1, 2, 3) were preliminary tested for analgesic potency in a pilot experiment (data not shown) using the formalin test (described below); being a model in which both peripheral and centrally mediated pain relief could be measured.The chloroform extract (DM-2) was found to be the most active amongst DM-1, 2, and 3; and was subjected to column chromatography (Still et al., 1978) using silica gel 230 to 400 mesh (Sigma-Aldrich Co., St. Louis, MO, USA).The column was eluted first with hexane, followed by mixtures (100 ml) of hexane: ethyl acetate; and ethyl acetate: methanol in increasing polarity gradient.Fifty ml of the eluates were individually collected, monitored with analytical TLC on precoated silica gel adsorption plates with 250 micron layer thickness (Whatman K5 150 A, Waltham, MA, USA) and visualized under Ultraviolet (UV) light (254/365 Eagle Scientific Ltd, UK).Eluates which were found to have the same thin-layer chromatography (TLC) profile were combined together.A fraction (initially denoted DM-2B) was eluted with mixture of hexane and ethyl acetate (50:50%), and on drying yielded a compound.

Structural elucidation
The isolated compound was identified using calorimetric, colorimetric, chromatographic and spectroscopic techniques.Differential scanning calorimeter (DSC) (NETZSCH DSC 204F1, Netzsch-Gerätebau GmbH, Selb, Germany) was used for thermochemical analysis.UV-visible spectra were recorded on UV-160A instrument (Shimadzu Corporation, Kyoto, Japan) by recording the absorption of 1 mg of the isolated compound in 10 ml ethanol (99%).IR spectra were taken in KBr pellets (FTIR-8400 S (CE), Shimadzu, Japan).The 13 C-and 1 H NMR spectra including 2dimensional 1 H-13 C and 1 H-1 H correlation spectroscopy (COSY) were recorded on Bruker DRX 500 NMR (Bruker BioSpin, Rheinstetten, Germany) equipped with 5-mm QNP probe, 2H lock switch box and BVT 2000 heater.CDCl 3 was used as solvent and Tetramethylsilane (TMS) as internal reference.The chemical shifts were recorded in δ (ppm) and coupling constant in Hz.Distortion enhancement by polarisation transfer (DEPT) analysis was performed for proton attachment, heteronuclear multiple bond correlation (HMBC) for proton-carbon ( 1 H-13 C) 1 H-1 H coupling and correlation spectroscopy (COSY) coupling constants.

Animals
Wistar rats of both sexes obtained from Animal Facility Centre, NIPRD, Abuja, Nigeria, were used for the study.The animals were kept in propylene cages with saw-dust as bedding, and maintained on standard laboratory feeds with water ad libitum.They were used in accordance with Ethical Guidelines for Investigation of Experimental Pain in Conscious Animal (Zimmermann, 1983), in line with NIPRD's standard procedures on laboratory animal usage (NIPRD QMS/SOP no.05:3:06).

Analgesy (Randall-Selitto test)
This test was performed using the modified Randall-Selitto (1957) test with Ugo Basile Analgesy-Meter (No. 7200, Italy).In the test, a meter exerts force at a constantly increased rate on rat paw monitored by a pointer moving along a linear scale.Twenty five rats were grouped into five groups (n = 5) and treated p.o. with vehicle (distilled water; 10 mL/kg), lupeol (25, 50 and 100 mg/kg), or acetylsalicylic acid (ASA) (100 mg/kg).The rat paw was gently placed between the plinth and plunger of the instrument and increased pressure (exerted by 20 g) applied to the middle dorsum of the rat's left hind paw.Stimulus was terminated and force threshold readings taken as soon as nociceptive response were elicited by the rats.Readings were taken pretreatment and at 15, 30 and 60 min after treatment.

Formalin test
The method described by Dubuission and Dennis (1977) was adopted for this assay, with little modification (Adzu et al., 2014).Briefly, the animals were treated p.o. with water (10 mL/kg), lupeol (25, 50 and 100 mg/kg), or acetylsalicylic acid (ASA) (100 mg/kg).They were then injected s.c. with 50 µL solution 2.5% formalin into the sub-plantar surface of rat left hind paw, 30 min after the treatment.Severity of pain was rated in two distinct phases for 60 min: the first phase (0 to 10 min) taken every 2 min and late phase (15 to 60 min) every 5 min using 3 pain-induced behaviour in the following scoring manner: 0 -normal weight baring on the injected paw; 1 -light resting on the paw on the floor; 2-elevation of the injected paw and 3 -for licking, biting or grooming of the injected paw.The mean (±SEM) of the readings was recorded as the pain score, after which the left paw oedema volume of each rat was measured and compared with that of the right hind paw using a digital plethysmometer (LE 7500, LETICA, Spain) 1 h after the formalin injection.

Data analysis
The results were expressed as mean ± standard error of mean.Parametric one way-analysis of variance (ANOVA) was used to analyse the data followed by the Student-Newman-Keuls test for multiple comparison using GraphPad Prism Version 5.01 for Windows (GraphPad Prism Software, San Diego California, USA).
The analgesic potency of the isolated compound was investigated using rats.Such animal models generate reliable data that gives high predictive value in humans (Normandin, 2007); by identifying target and provide proof of efficacy (Hart et al., 2004).In some instances, these in vivo models have advantages over vitro techniques (Houghton et al., 2007).The compound was first tested on mechanical model using analgesy-meter.The test is based on the principle that inflammation increases the sensitivity to nociception and this sensitivity is susceptible to modification by analgesics.The average pre-treatment response of the rats to the model was 4 min, which was maintained throughout the 60 min duration of the experiment by the vehicle control groups.Lupeol and the standard drug (ASA) alleviated the induced pain by prolonging the rats' responses significantly (p< 0.05; Table 1).The fact that lupeol increased the threshold of the intact paw suggests analgesic effect involvement of both peripheral and centrally mediated activity (Vongtau et al., 2004).
The compound was also evaluated against chemically induced pain using formalin test.The test is biphasic, and measures pain of both neurological (first phase) and inflammatory origin (second phase).The test is recommended as a basic pain research for studying the Chemical shifts (δ ppm)  mechanisms of analgesic agents because of its connection to tissue injury (Tjolsen et al., 1992).Lupeol exhibited significant analgesic activity on both phases of the formalin test; with maximal % inhibition of 60% in the first phase, and 31% at the second phase (Table 2).The formalin test model is accompanied by the development Chemical shifts (δ ppm)  of oedema in the injected left paw due to release of inflammatory mediators, and the oedema volume after the assay was taken to evaluate the action of lupeol against this process.Suppressing the induced pain and oedema by lupeol in this study reaffirmed its pain relief effect.
Lupeol, a safe and pharmacologically active triterpenoid is widely distributed in plant kingdom, but less applied in therapy (Gallo and Sarachine, 2009;Siddique and Saleem, 2011).It is known to elicit its activity mainly via the inhibition of tissue response to the induced nociception (Geetha and Varalakshmi, 2001;Chen et al., 2012), especially through the involvement of cytokines (De Lima et al., 2013).

Conclusion
This is neither the first time lupeol is isolated from D. mespiliformis nor its bioactivity being demonstrated.However, this study is unique because it linked the analgesic effect of the plant material to the isolate lupeol; acting alone or synergistically with other phytochemical constituents.This might stimulate more interest in this compound since analgesics prototype like salicylic acid and morphine, and several other bioactive agents in modern pharmacopoeia were derived from products initially used in traditional medicine.

Figure 1 .
Figure 1.DSC spectra of lupeol isolated from the CHCl 3 extract of Diospyros mespiliformis stem bark

Figure 2 .
Figure 2. The 1 H NMR spectra of lupeol isolated from the CHCl 3 extract of Diospyros mespiliformis stem bark.

Figure 3 .
Figure 3.The 13 C NMR spectra of lupeol isolated from the CHCl 3 extract of Diospyros mespiliformis stem bark.

Table 1 .
Inhibition of analgesy in rats by lupeol isolated from the CHCl 3 extract of D. mespiliformis stem bark.

Table 2 .
Inhibition formalin induced noxious stimulus test in rats by lupeol isolated from the CHCl 3 extract of Diospyros mespiliformis stem bark.