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Abstract
Secondary metabolites from plants are a good source for the NSAID drug development. We studied the analgesic activity ofethanolic extract of Erythrina variegata L. (Fabaceae) followed by molecular docking analysis. The analgesic activity of Erythrinavariegata L. is evaluated by various methods viz., acetic acid-induced writhing test, hot plate and tail immersion test. Subsequently,molecular docking analysis has been performed to identify compounds having activity against COX-1 and COX-2 enzymes byusing GOLD docking fitness. The result of preliminary phytochemical screening revealed that the extract contains alkaloids andflavonoids. In analgesic activity tests, the extract at the doses of 50, 100 and 200 mg/kg body weight (b.w.) produced a increase inpain threshold in a dose dependent manner. In acetic acid induced writhing test, the inhibitory effect was similar to the referencedrug diclofenac sodium. The extract showed 18.89% writhing inhibitory effect at the dose 200 mg/kg b.w., whereas diclofenacsodium showed 79.42% inhibition of writhing at a dose of 10 mg/kg b.w. The results of tail immersion and hot plate test alsoshowed potential analgesic activity of the extract which is also comparable to the standard drug morphine (5 mg/kg b.w.). Dockingstudies shows that phaseollin of Erythrina variegata L. has the best fitness score against the COX-1 which is 56.64 and 59.63 for COX-2 enzyme. Phaseollin of Erythrina variegata L. detected with significant fitness score and hydrogen bonding against COX-1 andCOX-2 is reported for further validation.
Background
Natural products are a major source of drugs and about half ofthe pharmaceuticals in use today are derived from naturalproducts []. Application of advanced drug screening methodsshow plant compounds have variety of structures withbioactivities, including anti-tumor, anti-inflammatory, antiviraland hepatoprotective properties []. Inflammation andpain are major areas of interest for discovery starting with theidentification of morphine form opium [,]. The non-steroidalanti-inflammatory drugs (NSAIDs) are among the most widelyused drugs in the control of postoperative pain but often causea number of side-effects []. Cyclooxygenase (COX), the keyenzyme required for the conversion of arachidonic acid toprostaglandins was first identified over 20 years ago []. Theenzyme exists in at least two isoforms, COX-1 and COX-2.Although both the isoforms catalyze the same biochemicaltransformation, the two isoforms are subject to a differentexpression regulation []. COX-1 is a constitutive enzyme and isresponsible for the supply of prostaglandins which maintainthe integrity of the gastric mucosa and provide adequatevascular homeostasis whereas COX-2 is an inducible enzymeand is expressed only after an inflammatory stimulus [].Literature studies indicate that direct tissue contact of NSAIDsgives the side effects like gastric upset, irritation, and ulceration[], and also confirms that gastrointestinal side effects ofNSAIDs such as irritation and GI bleeding are due to thepresence of a free carboxylic group in the parent drug [,].Thus, developing new agents with minimum or without sideeffects is an extensive research area in the present scenario. Dawnstar 1 042 – air combat game. Thegenus Erythrina comprises of about 110 species of trees andshrubs. The name 'coral tree' is used as a collective term forthese plants. Coral tree is indigenous to the Old World tropics,possibly originally from India to Malaysia, but is native ofancient westward to Zanzibar and eastward to easternPolynesia (the Marquesas). It is typically found on sandy soil inlittoral forest, and sometimes in coastal forest up to 250 m(800ft) in elevation. The coral tree is cultivated particularly asan ornamental tree and as a shade and soil improvement tree (itfixes nitrogen) for other tree crops such as coffee and cacao [,]. Leaves are stomachic, anthelmintic, laxative, diuretic,gatactagogue and emmenagogue; applied externally fordispersing venereal buboes, relieve pain of the joints andinflammations; juice is poured in to the ear to relief earache andis used as an anodyne in toothache. The bark is astringent,febrifuge anti-bilious and anthelmintic; useful in dysentery andas a collyrium in ophthalmia. The roots are emmenagogue [,].Phytochemical studies [] suggest that Erythrina variegataL. contain compounds like Isoquinoline, Isococcolinine,Erythrinin A, Erythrinin C, Erythrinin B, Osajin, AlpinumIsoflavone, Erythrabyssin II, Erycristagallin, 6-Hydroxygenistein, Lupiwighteone, Hypaphorine, Erysovine,Erysopitine, Erysotrine, Erysonine, Erythratidine, Erythrinine,Erythramine, Erythraline, Nororientaline, Erybidine, LReticuline,Coreximine, Euchrenone B10, Eryvarins Q,Abyssinone V, Phaseollin, Campesterol, Scoulerine,Erystagallin A, Orientanol B, Robustone, Stachydrine,Erysovine. Therefore, it is of interest to evaluate the analgesicactivity of the ethanolic bark extract of Erythrina variegata L. andalso the compound for this activity by in silico moleculardocking analysis [].
Methodology
Plant material collection and identification:
The whole plant was collected from Chittagong Universityhilly forest, Bangladesh on March 2012. A voucher specimenfor this plant has been maintained in Bangladesh NationalHerbarium, Dhaka, Bangladesh (Accession No. 36148).
Preparation of plant material:
Barks were sundried for 7 days and later dried in drier at 40°Cfor about an hour. The dried bark were then ground intopowder using high capacity grinding machine and stored inairtight plastic container with necessary markings foridentification and kept in cool, dark and dry place for theinvestigation. The bark of the plant material was extracted withethanol. After completion of the extraction, the liquid wasfiltered using a sterilized cotton filter []. It was thenevaporated by rotary evaporator for about 1 hr. Further, it waskept for drying till it solidifies from liquid form. Then solventwas completely removed and obtained 6 g (yield 1.5%) driedcrude extract which was used for preliminary phytochemicalgroup tests and then subjected to for rest of the experiments.
Preparing animals:
For the experiment Adult Swiss albino mice (BALB/c)weighing between (12-300) g of either sex were collected fromanimal sources department of ICDDRB, Dhaka. The animalswere maintained under normal laboratory condition and keptin standard polypropylene cages at room temperature of (30 ±2)°C and 60% to 65% relative humidity and provided withstandard diet and water. The institutional animal ethicalcommittee approved all protocols for animal experiment. Eachgroup consists of five mice and to denote individual animal,they were marked as group I, II, III, for test samples at thedoses of 50, 100 and 200 mg/kg body weight and a control andpositive control group was also maintained for every tests.
Preliminary phyto-chemical screening:
One gram of the ethanol extract of E. variegata was dissolved inethanol and was subjected to preliminary phytochemicalscreenings for determining nature of phytoconstituents [].
Active Dock 1 1 1938
Acetic acid-induced writhing test:
The anti-nociceptive activity of the extract was studied usingacetic acid-induced writhing model in mice []. The animalswere divided into control, positive control and test groups withfive mice in each group. The animals of test groups receivedtest samples at the doses of 50, 100 and 200 mg/kg bodyweight. Positive control group received standard drugdiclofenac sodium at the dose of 10 mg/kg body weight. Testsamples were administered orally 30 min before intraperitonealadministration of 0.6% acetic acid but diclofenacsodium was administered 15 min before injection of acetic acid.After an interval of 5 min, the mice were observed for specificcontraction of body referred to as ‘writhing' for the next 30 min[].
Hot plate method:
The paws of mice are very sensitive to temperature at (55 ±0.5)°C, which are not damaging to the skin. The animals wereplaced on Eddy׳s hot plate kept at a temperature of (55 ± 0.5)°C.A cut off period of 30 sec [], was observed to avoid damage tothe paw. Reaction time was recorded when animals licked theirfore or hind paws, or jumped at 0, 30, 60 90 and 120 min afteroral administration of the samples []. The animals of testgroups received test samples at the doses of 50, 100 and 200mg/kg body weight. Positive control group received standarddrug diclofenac sodium at the dose of 10 mg/kg b.w. and salinewater.
Tail immersion test:
The procedure is based on the observation that morphine likedrugs selectively prolongs the reaction time of the typical tailwithdrawal reflex in mice animals of the control, positivecontrol and test groups were treated with diclofenac sodium(10 mg/kg body weight), saline water (10 ml/kg body weight)and test samples at the doses of 50, 100 and 200 mg/kg bodyweight respectively. 1 to 2 cm of the tail of mice was immersedin warm water kept constant at 55°C. The reaction time was thetime taken by the mice to deflect their tails. The first readingwas discarded and the reaction time was recorded as a mean ofthe next three readings. A latency period of 30 sec was definedas complete analgesia and the measurement was stopped whenthe latency period exceeded to avoid injury to mice. The latentperiod of the tail-flick response was taken as the index of antinociceptionand was determined at 0, 30, 60, 90 and 120 minafter the administration of the test drugs and standard [].Definition of groups and their treatments were as some of thehot plate test. The hot plate latencies and tail flick weresequentially measured at 0, 30, 60, 90 and 120 min with thesame cut off time of 30 sec for the safety of animals.
Statistical analysis:
The results of statistical analysis for animal experiment wereexpressed as mean ± SEM. Data were analyzed by pairedsample t test. The results obtained were compared with thecontrol group. The criterion for statistical significance was ***p<0.01 and *p< 0.05. All the statistical tests were carried out usingSPSS statistical software.
In silico molecular docking analysis:
For docking analysis Gold 4.12 is used to predict the potentactive compound E. variegata against the active site of COX-1and COX-2 enzymes where compounds are collected from theliterature review.
Ligands preparation:
From the literature review, all compounds-Isoquinoline,Isococcolinine, Erythrinin A, Erythrinin C, Erythrinin B, Osajin,Alpinum Isoflavone, Erythrabyssin II, Erycristagallin, 6-Hydroxygenistein, Epilupeol, Lupiwighteone, Hypaphorine,Erysovine, Erysopitine, Erysotrine, Erysonine, Erythratidine,Erythrinine, Erythramine, Erythraline, Nororientaline,Erybidine, L-Reticuline, Coreximine, Euchrenone B10,Eryvarins Q, Abyssinone V, Phaseollin, Scoulerine, ErystagallinA, Orientanol B, Robustone, Stachydrine, Erysovine are drawnin Symyx Draw 4.0 and then prepared for docking using theSybyl 7.3 Molecular Modeling Suite of Tripos, Inc. 3Dconformations were generated using Concord 4.0 [],hydrogen atoms were added and charges were loaded usingthe Gasteiger and Marsili charge calculation method []. Basicamines were protonated and acidic carboxyl groups were deprotonatedprior to charge calculation. The AMPPD ligand wasminimized with the Tripos Force Field prior to docking usingthe Powell method with an initial Simplex [] optimizationand 1000 iterations or gradient termination at 0.01kcal/(mol*A). Input ligand file format was mol2 for all dockingprograms investigated.
Protein preparation and active site determination:
The crystal structure COX-1 and COX-2 enzymes are collectedprotein data bank [] pdb id: 2OYE (COX-1) and 6 COX (COX-2). Two enzymes are prepared according to the dockingprotocol of Gold. Iconbox 2 1 1 ubkg download free. The active site of these enzyme identifiedaccording to the giving information by Harman et al. 2007 []for COX-1 and Kurumbail et al. 1996 [] for COX-2.
Docking using GOLD (Genetic Optimization for Ligand Docking):
GOLD utilizes genetic algorithm to explore the rotationalflexibility of receptor hydrogens and ligand conformationalflexibility []. In GOLD docking was carried out using thewizard with default parameters population size (100); selectionpressure (1.1); number of operations (10,0 00); number ofislands (1); niche size (2); and operator weights for migrate (0),mutate (100) , and crossover (100) were applied. The active sitewith a 10 Å radius sphere was defined by selecting an activesite residue of protein. Default Genetic Algorithm settings wereused for all calculation s and a set of 10 solutions were savedfor each ligand. GOLD was used by a GoldScore fitnessfunction. GoldScore is a molecular mechanism like function andhas been optimized for the calculation of binding positions ofligand. It takes into account four terms:
Fitness = S(hb_ext) + 1.3750*S(vdw_ext) + S(hb_int) + 1.0000*S(int)S(int) = S(vdw_int) + S(tors)
Where, Shb_ext is the protein-ligand hydrogen bonding andsvdw_ext are the vanderwaals interactions between protein andligand. Shb_int are the intramolecular hydrophobic interactionswhereas Svdw_ int is the contribution due to intra molecularstrain in the ligand.
Phytochemical screening:
Preliminary phytochemical screening of the crude ethanolicextracts of the barks of E. variegata revealed the presence ofalkaloid and flavonoids Table 1 (see supplementary material).
Acetic acid-induced abdominal writhing test:
The analgesic effect ethanolic extracts of E. variegata at doselevel of 50, 100 and 200 mg/kg body weight on acetic acidinduced writhing in mice was exhibited. Doses of the extractinhibited writhing response induced by acetic acid in a dosedependent manner in which group III (200 mg/kg b.w.)exhibits the highest 18.89% of inhibition and is comparable tothe reference drug diclofenac sodium (79.42%) Table 2 (seesupplementary material).
Hot plate test:
The tail withdrawal reflex time following administration of theethanolic extracts of E. variegata at dose level of 50, 100 and 200mg/kg b.w. was found almost remain the same consistencywith increasing dose of the sample which is comparable tothe reference drug Table 3 (see supplementary material).
Tail immersion test:
The tail withdrawal reflex time following administration of theethanolic extracts of E. variegata at dose level of 50, 100 and 200mg/kg b.w. was found almost remain the same consistencywith increasing dose of the sample which is comparable to thereference drug Table 4 (see supplementary material).
Docking analysis:
The compounds of E. variegata mentioned above were subjectedto dock in the active site of COX-1 and COX-2 enzyme by golddocking method. The results of docking analysis of COX-1 andCOX-2 enzyme are listed in Table 5 (see supplementarymaterial). After docking the ligand protein complex was savedin pdb format then subjected for analysis in the AccelrysDiscovery Studio Visualizer. Docking studies showed thatphaseollin has the best gold fitness score against the COX-1which is 56.64 and 59.63 for COX-2 enzyme. Molecular analysisshowed that phaseollin form two hydrogen bonds withresidues of the active site of COX-1 enzyme. It is that phaseollinmade bonds with TYR354 with a distance 2.98478 Å between 4no oxygen of phaseollin with hydrogen of Tyr354. Anotherhydrogen bond was formed between 1 no hydrogen ofphaseollin and O of SER499 where bonding distance is 1.86273Å. Similarly, In COX-2 enzyme two hydrogen bonds areformed between the oxygen of SER499 and hydrogen ofTYR354 with the hydrogen at 1st and oxygen at 4th position ofphaseollin where bonding distance is 1.48447 Å and 1.54784 Årespectively. Interaction between the phaseollin with the COX-1and COX-2 are represented in Figure 1.
Interaction of phaseollin with a) COX-1 and b) COX-2.
Discussion
The present study has established analgesic potential of E.variegata using acetic acid induced writhing test for visceralpain and tail immersion and hot plate tests for pain mediatedby central activity. Acetic acid induced writhing in mice is amodel of visceral pain, which is highly sensitive and useful forscreening peripherally acting analgesic drugs. E. variegata plantextracts caused dose dependent anti-nociception againstchemical induced pain in mice. Ethanolic extracts of the bark ofE. variegata were treated in test animals at a dose of 50, 100 and200 mg/kg b.w. The ethanolic extracts of E. variegata at the doseof 200 mg/kg b.w. were found to exhibit the highest 18.89%writhing inhibitory response, where the reference drugdiclofenac sodium showed about 79.42% writhing inhibitoryresponse at a dose of 10 mg/kg b.w.
The tail immersion test is considered to be selective to examinecompounds acting through non-opoid receptor; the extractincreased mean basal latency, which indicates that it may actvia centrally, mediated analgesic mechanism. Narcoticanalgesics inhibit both peripheral and central mechanism ofpain, while non-steroidal anti-inflammatory drugs inhibit onlyperipheral pain []. The extract inhibited both mechanisms ofpain, suggesting that the plant extract may act as a narcoticanalgesic.
The hot plate and tail immersion tests are widely used forassessing central anti-nociceptive activities. Opioid agentsexhibit their analgesic effects both via supra-spinal and spinalreceptors []. The present experiments, ethanolic extracts of E.variegata exhibited a statistically significant. It seems quitepossible that the lower doses of the extract have more potentcentral anti-nociceptive effect. It has been suggested that theopioid mechanisms mediate anti-nociceptive effect of ethanolicextracts of E. variegata.
Ethanolic extract of E. variegata produced a dose-dependentanti-nociceptive effect on the glutamate-induced paw lickingresponse. Recently, found that the nociceptive responseinduced by glutamate appears to involve peripheral, spinal andsupraspinal sites of action and is greatly mediated by bothNMDA and non- NMDA receptors as well as by the release ofnitric oxide or by some nitric oxide-related substance. Hence,an effect of the plant extract directly on the receptors or secondmessengers related to these transmitters could avoid thenociceptive response. The effect of E. variegata againstnociception induced by glutamate is of great interest sinceglutamate plays a significant role in nociceptive processing inboth central and peripheral nervous systems [].
Advances in computational techniques have enabled virtualscreening to have a positive impact on the discovery process.Virtual screening utilizes docking and scoring of eachcompound from a dataset and the technique used is based onpredicting the binding modes and binding affinities of eachcompound in the dataset by means of docking to an X-raycrystallographic structure []. Some recent studies havefocused on certain factors such as the size and diversity of theligand dataset, wide range of targets and the evaluation ofdocking programs []. In our present studies, by means ofgold docking, we docked 33 compounds of E. variegata into theactive site of the COX-1 and COX-2 enzymes. In view of theabove, fitness score values were measured using GOLD 4.12showed that phaseollin has the highest fitness score of 56.64was noticed with COX-1 and fitness score of 59.63 wasobserved for COX-2, suggesting that more interaction ofphaseollin has more in COX-1 and COX-2 than the othercompounds.
Conclusion
Results show that plant extract of E. variegata possessesmoderate analgesic potential. Though the involvement ofopioid receptor has been determined using naloxone, furtherstudies are needed using different agonists (such as adrenergic,serotonergic etc.) to completely understand the exactmechanisms of its anti-nociceptive activity. It seems possiblethat E. variegata contains chemical constituents with analgesicproperty for consideration in drug development. In the presentstudy the analgesic activity of the ethanolic bark extract of E.variegata was done and its compound was successfully dockedonto the both COX-1 and COX-2. Thus, phaseollin could beconsidered as a potent analgesic molecule against COX-1 andCOX-2 for further validation.
Competing interests
All authors declare that they have no competing interests.
Authors׳ contributions
MMNU has designed the study, performed data analysis andinterpretation and wrote the manuscript. TBE has wrote themanuscript and also modify the in silico parts. MMRM hasprovided assistance in taxonomical identification andcollections of voucher specimen׳s numbers for the plants andmodify the in silico parts. RD has performed data analysis andwrote the in silico parts.
Supplementary material
Footnotes
Citation:Uddin et al, Bioinformation 10(10): 630-636 (2014)
References
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