Acta Medica Philippina

The Effect of Swietenia Mahogani (Mahogany)Seed Extract On Indomethacin-Inducd Gastric

Ulcers In Female Sprague-Dawley Rats.

Bacsal, K; Chavez, L;Diaz I; Espina, S; Javillo, J; Manzanilla, H.; Motalban' J.;Panganiban, C.; Rodriguez, A.; Sumpaico C.; Talip, B.; Yap, S.

UPCM, Deparunent of Phamacology

ABSTRACT

Swietenia mahogani (mahogany) has beenreported to have medicinal uses, suchas teatment

for hypertensio n, cancer, anaebiasis, che st painsand intestinal parasitism. This study examinedthe effect of mahogany seed extacts onindome thacin-induced gas tric ul ce r s in femaleSpragrc-Dawley rats. Fortyfive 2-3 nnntholdrats wei ghing I 50-200 g were randomly assignedinto 5 trestment groups, namely: [1] 59opolyvinylpyrrolidone (PVP), [2] misoprostol( 1 44 ue I kS BW ), [ 3 ] 0. I a g mala gany se ed extractlkg BW, t4l 0.28 g mahogany seed extract I kgBW, and tsl 0.57 g nahogany seed extractlkgBW. After three days of administration oftreatncnls using gavage tnethod, the rats weresacrificed, and their stomachs graded grosslyusing Besl's Ulcer Staging Index.H i s to pat lw lo gi c gr adi ng w as al so c o nduc te d. Attlu doses utilized in this study, nnhogany seedextraclwasfound to have a potential effect on thehealing of gastric ulcers. It is speculated thatthis effect can be auributed to the plwspholipidand long chain unsatwatedfatty acid content ofmahogany seeds. Further studies arerecomnended to verifi these ftndings.

INTRODUCTION

ThePhilippines is faced with countless challcngesin its quest for attaining a Newly IndustrializedCountry status (NIC) by thc ycar2000. For thesereasons, we have opened our doors to numerous

multinational companies which have beenbauling out for market share in various industries,one of which is the drug indusry.

Filipinos encountera numberof problems whenthe need !o use drugs arises such as: theiravailability, accessibility, and affordability.Because of the fierce competition among thevarious drug companies, the prices ofdrugs usedto treat common illnesses have escalated. Thus,most impoverished Filipinos have troubleprocuring these essential drugs that would meettheir health needs. Since most drug companiesare more or less located in the major cities,people from far-flungareas in the Philippines areleftwith nootherchoicebuttobuy theseexpensivedrugs or improvise with the use of herbalmedicine.

Our country, blessed with a vast number ofindigenous resources, has turned to herbalmedicine as an alternative to commercially-produced drugs. They are cheaper and easier !oobtain, thus, they can be used by more Filipinos.In this light, there has been an upsurge in herbalmedicine research in order to investigate andmaximize fully the potential therapeutic use ofplants. Pharmacological and oxicological studiaswill prevent the indiscriminate use of plants withboth therapeutic uses and toxic side effects andinsure the safcty and efficacy of these test species.Many of our countrymen suffer from pepticulcers, the major causes of which are excessivealcohol and/oTNSAID intake, smoking and stres.There are a number of medicines which act

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Acta Medica Philip,pina

through different mechanisms used for thetreatment of ulcers. Investigations are cunentlybeing undertaken to discover medicinal plantswith anti-ulcer functions. The discovery of aplant with anti-ulcer properties would becontribuory o the growing list of medicinalplants currently being ,used in the country,alleviating some of the problems of coat andavailability of commercially-produced drugs.

Swictenia nwhogani (mahogany) seeds havebeen reported to have medicinal value fortrsatment of hypertension, cancer, amoebiasis,coughs, chest pains and intestinal parasitism.The biologically active ingredienrs,tetranortriterpenoids and fatty acids areconsidered o be responsible for the.se therapeuticeffects. It is this vast range of uses of mahoganyseeds that has peaked our interest to investigatethe potential anti-ulcer activity of S. mahoganiseed extract.

Research Question

Among female twoto three month-oldSprague-Dawley ras with gasric ulcers induced usingindomethacin at30mglkg?t4 hours before, howwill the daily oral administration of Swietenianahogani (mahogany) seed extracts at0.l4 gkg, 0.28 g/kg and 0.57 gftg affect the gastricmucosa, measured by the size and number ofulcers using the Ulcer Staging Index, comparedto those of female sprague-Dawley rats withsimilarly induced gastric ulcers and administeredwith misoprostol or polyvinylpyrrolidonesolution?

Objectives

General Objective:

To determine the effects of Swietenia mahogani(mahogany) sced extracts on indomethacin-induced gastric ulcers in femalc Sprague-Dawleyrats.

Specific Objectives:

To determine tlre size and number of gastricmucosal lessions in female Spnague-Dawley ras!o which 0.14 g/kg body weight,0.28 /kg bodyweight and 0.57 gAg body weight mahoganyseed exnact were administered, as measured bythe Ulcer Staging Index.

To compare the scorBs of the gastric mucosae oftterats ineach of the following treatmentgroups:O.la g,ftg body weight mahqgnny segd extract;0.28 gkg body weight mahqgpny seed extract;0.57 gkg mahogany seed exract; misoprostrolsolution; and polyvinylpynolidone solurion.

Significance of the Study

This research will make a valuable contribution!o the developmentof anti-ulcer drugs ttr,at utitzemedicinal plants, in order to more efficiently andappropriately respond to the healtlr needs of theordinary Filipino.

Review of Related Literature

A peptic ulcer is a breach in the mucosa of theduodenum and stomach, which extends throughthe muscularis mucos:l into the submucosa ordeeper. Ulcers are mone commonly found in theduodenum than in the antrum of the stomach.They are chronic, most often solitary which tendto be less than 4 cm in diameter (1).

Normally, the gastric mucosil is protected by acompacdy ananged epithelium and thick layerof mucus. Other suggested protective factors arethe regenerative capacity of tle mucosal cells,alkalinity ofpancreatic and intestinal juices whichneutralize gastric contents and an adequatevascular supply (2). All these are aimed atpreventing digestion of the somach by pure acidgasric juice.

However, resistance of the stomach to digestiondoes fail. The following facors have beenpostulated to cause ulcer.

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Gastric Mucosal Injury

Peptic ulcers are produced by an imbalancebetween thd gasroduodenal mucosal defensiveand agressive forces. Agressive forces involve(l) acid secretion; (2) peptic activity derivedfrom pepsinogen; (3) Helicobacter pyloriinfection of the gastric antrum ; (4) use of NSAIDS(5) impaired inhibition of stimulatorymechanisms such as gastrin relcase (6) increasedparietal cell mass, (7) increased sensitivity tosecreOry stimuli (8) ircreased basal acid secretorydrive (l).

In vitro experiments have shown that digestionof the said mucus by the gasnic acids, especiallypepsin I, predisposes mucosal barrier to erosionwhich eventually results in the development ofulcers. (l) However, there are certainmechanismsfactors within the gasrointestinaltract that protects the stomach from theseaggressive factors. These defensive forcesirclude the following l) secretion of surfacemucus layer by epithelial cells. The nature andadherent characteristics of the mucous, aglycoprotein get secreted by surface epitheliumover the gastric wall, is responsible in protectingthe underlying cell fiom the damage caused bythe mechanical forces of digestion. Likewise, itlubricates the mucosa, assisting the movement offood over the surface and retains water within itsgelatinous matrix; (2) secretion of bicarbonateinto the mucus layer, thus producing a pH gradientfrom the highly acidic gastric lumen to the almostneutral mucosal surface (3) protection againstdiffusion of H+ ions into the mucosa by thespecialized apical surface of the gastric mucosalcells (4) remartable regenerative capacity ofmucosal epithelial cells for rapid repair of injuries(5) mucosal elaboration of prostaglandins havingcytoprotective activity, possibly by maintainingadequate mucosal blood flow and by stimulatingsecretion of mucus and bicarbonate. Possiblecauses include increased parietal cell mass,increase sensitivity to secretory drive andimpaired inhibition of stimulatory mechanismslike gastrin release (l).

Acta Medica Philippina

Mucosal Blood flow. A decreased in bloodsupply to 0re lesser curvature of the stomach hasbeen suggested to play a role in the developmentofchronic ulceration on the affected part. Focalischemia and consequent necrosis of certainsegments of the gasric wall also lead to ulcers.

Environmental Factors. Some commonexogenous agents such as NSAIDS, alcohol,smoking and dietary salts are thought to damagethe mucosa.

Symptomatology. A symptom complex refenedto as "ulcer pain" is fle usual manifestation of anactive ulcer. Two factors have been proposed Loexplain ulcer pain. These are (l) the action ofacid gasricjuice on the raw surface of the ulcer,and (2) cause of muscular nature unconnectedwith the action of tlre acid juice on the mucus.The inflammatory foci in fte muscularis mucosagive rise to contractions in the neighborhood ofthe ulcer which increases the inragastric pressureand the tension of muscle fibers, thus producinga sensation of pain.

Ulcerpain may range from the classical burningsensation tovaguecomplaints such as asensationof "being hungry". The pain is steady, mild tomoderate, well-localized in the epigastrum andoften relieved by food, water or antacid. It isusually periodic.

Complicat ions. Complicat ions includeperforations, hemonhage and pyloric obsnucdon.Perforations are usually located at the anteriorwall of the duodenum, less commonly in thestomach. A free perforation usually presents anacute abdominal emergency. The patientexperiences sudden intense steady pain in theepigastrum. The patient usually lies still becauseany movement exacerbated the pain. TheaMomen is tender, the abdominal muscles arerigidand bowel sounds arediminished orabsent.Peri toni t is may subsequent ly develop.Hemonhage is another common complication.In cases of massive bleeding, it may lead tohypovolemic shock.

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Acta Medica Philippina

Pyloric obsfuction may be due to several causes.It may be due to duodenitis and antral gastritisimpairing the propulsive motility of the antrumand active relaxation of the pyloric sphincter. Itmay also be due to scarring and relief ofobsrucdon. (2)

Ulcerogenic Agents

Indomethacin, an indole acetic acid derivative,was the product of a laboraory search for drugswirh anti-inflammatory properties. It wasintroduced in 1963 for the treatmentof rheumatoidarthritis and related disorders.

Indomethacin has prominent anti-inflammatory,analgesic and antipyrctic properties similar tothose of salicylates. Indomethacin is a potentinhibitor of the prostaglandin cyclooxygenase.Italso inhibis the motility of polymorphonuclearleucocytes. Like many other aspirin-like drugs,indomethacin increases oxidat ivephosphorylation in supratherapeuticconcentrations and expresses thebiosynthesis ofmucopolysachharides. In addition, it increasesintracellular concentations, of cyclic AMPinhibiting phosphodiesterase (3).

It is rapidly and completcly absorbed from thegastrointestinal tract, with peak plasmaconcentations attaincd from I to 2 hours. Thehalf-life is about4.5 hours. Indomethacin is907oprotein-bound and is biotransformed bydemethylation and deacylation. Both the parentdrug and the biotransformation products undergoenterohepatic circulation. Severe side-effectsinclude headache, verl.igo, diarrhea and pepticulceration with chronic usc (4).

Non-stcroidal anti-inflammatory drugs such asaspirin and indomcthacin, have been associatedwith gastric and duodenal ulcers. Sevcral theorieshave bcen proposed regarding the mechanismsby which thesc drugs act.

It has bcen postulatcd that the mucosal injuryproduccd by the aforementioned drugs ismultifactorial. Howcver, indomethacin in

particular is thought o promote peptic ulcerationvia itsinhibition ofclyclooxygenase tlut is neededin the formation of endoperoxides, of whichprosaglandins @2) is an example. The duodenalmucosa produces prostaglandin E2 andbicarbonate which are needed for the maintenarrceof is integrity.

A study of indomethacin-induced ulcers in themouse showed that two doses of indomethacin,administered subcutaneously at 85 mg/kg bodyweight, induced well-defined gastrointestinalulcers accompanied by inflammatory andvascular changes in the stomach and smallintestine. Maximal damage was observed 20hours after the second dose of indomethacin.Morphometric analysis identified changes in allcompdtments of the small intestine. There wasa marked reduction in the length of the smallintestine, intestinal dilation, a significantdecreasein villous height, with the formation ofsubepithelial blisters or blebs within villi, andsubmucosal vascular dilatation. There was nochange in the number of villi or submucosalarterioles or in the loial amountof musclepresentin the wall of the intestine.

Indomethacin has also been found to increasegastric acid secretion due to the loss ofhydrogenion-back-diffusion through a damaged mucosalbarrier, and not due to a direct reduction inparietal cell secretion which explains itsulcerogenic action.

Anti-Ulcer Agents

Misoprostol

Misoprostol (Cybtec), a l5-deoxy- 15-hydroxy-l6methyl analogue of prostaglandin E, has amolecular weight of 382. It has been proven bythe FDA effective for ttre treatment of pepticulcer disease. The mechanism involves themaintenance of mucosal integrity which isfacilitated by an increase in mucosal blood flow.This resuls in an increase in n utdents and oxygenthat may be delivered to the cell. At the sametime, toxic substances may be removed more

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efficiently from the tissucs. (5) Specifically,misoprostol acts by inhibiting thc secretion ofgastric acid, both basally :rnd in response to food,histamine, pentagastrin, and coffee, by directaction on parietal cells (6, 7, 8). Thiscytoprotective property of Misoprostol can beattributed o its ability to stimulate increasedmucous secretion and bicarbonate secretion bythe gastric mucosa. In an experimentcomparingthe mucosal protective propcrties of MisoprosLol,Cimetidine and S ucralfatc in rats, Misoprostol attwo doses of 100 and 200 ug/kg reduced themean number of gastric lesions by 587o and,7$Voof control, respectively. Gastric ulcers wereinduced by aspirin, indomethacin, stress, sodium,taurocholate and ethanol. In all 5 experimentalulcer models the gastric mucosal protectiveactivity of misoprostol was not rclatcd to itsantisecretory effccts as the doses used did nothave significant antisccrctory activity in the rat.It was concludcd ftat misoprostol providcdmucosal protcction by a nlcchanism that docs notrequire a reduction of gastric acid sccretion forits protective effcct. (9)

Misoprostol is absorbed rapidly after oraladministration and is rnetabolizcd to the free acidform. The plasma concentrations of Misoprostolare highest approximately 30 minutes afteradministrations of the drug. Plasma binding isnot extensive, and plasma drug concentrationsare not substantially affected by age. Renalexcretion is minor, and the dose docs not. nrcdadjustment in patients with renal impairment.(10)

Misoprostol is generaly well tolerated, and it hasno majorcentral nervous system orcardiovasculareffects. The most frequently reported adverseevent is diarrhea which can be so severe as towarrant discontinuation of trcatmcnt. The otherimportant side effcct of Misoprostol is increaseduterine contractility which can provokc abortion.Misoprostol is contraindicatcd in pregnantwomen and has bcen sold as an abortifacient inBrazil. (ll)

Acta Medica Philippina

Mahogany ( Swietenia mahogani)

Swietenia mahogani, otherwise known asmahogany or West Indian mahogany, belongs tothe family Meliaceae (Mahogany family), a grcupof plants known to have insecticidal activities aswell as medicinal uses. (12)

This plant is a more or less decidous, erect treegrowing up to l0 meters or more in height, witha heavy, dark green, dense crown. The trunk ismore or less butlressed. The bark is dark grayand ridged. S. mahogani yields a highly prizedreddish brown wood. This species is similar tothe broad-leaved Mahogany (Swieteniamacrophylla king), but it differs in its smallerleaves, leafles, and fruis. (13)

In the Philippines, mahogany is considered asone of the most important group of trecs used fordecorative purposes (14) but it has also beenrcported to have medicinal values, namcly,troaLment for hypertension, cancer, amocbiasis,chest pains and intestinal parasitism. Relatedspecies have been known to be effectiveantihelminthics and anti-cancer agents. (15)

Isolation of chemical components from seedsusing differenttechniques showed high saturatedand unsaturated fatty acid content. It was alsoreported to be positive for the presence ofphospholipids, neutml lipids and glycolipids frommahogany seed extracts ( 16).Phosphatidylcholine is the mostabundant. Otherstudies revealed the presence oftetranorri Frpenoids (specifically swietenoloidsand swietenines, diacetate (12, 14,15) resins andphenolic compounds (14). Oral administrationof triterpenoid saponins has been reported toprotect rats from developing gastric ulcersinduced by various experimental models:NSAIDS, stress and Shay rat method (17).

Polyvinylpyrrolidone (PVP), thc control

Polyvinylpyrrol idone (PVP), now morecommonly known as providone is a mixture ofessent ial ly l inear synthet ic polymers of

l 3 l

Acta Medica Philippina

vinylpynolidone. Physically it is described as afine whiteor very slightly cream colored, odorlesspowder. It is hygroscopic, soluble in lvater,alcohol and chloroform but insoluble in ether.As aSVo solution in water, it has a pH varyingfrom 3-7 (18).

It is mostly neutral and its toxicity is very low,butat thesame time it is said to havea detoxicanteffect. It also has no antigen cffcct (19). Thercmporary accepable daily intake sel by theFAQ ilHO in 1983 is 25mg/kg. The acute oraltoxicity in rats and guinea pigs is above lOOg/kg.

Ljses of PVP in Medicine

In extemal applications, it serves to increase lhesolubility of substances which are difficult todissolve in water. It is also of importance asdispersing agent (up to 57o solution), bindingagent, and lubricant. In opthalmology it is usedas an admixture in eye droPs.

PVP can be taken orally. It is used as anadmixture in tablets either as a binder or as coat(0.5-57o solution). It is not absorbed in themucous membrances of the gastrointestinal tract(if so, in negligible amounts) (19).

Effects of PVP

Wesscl states thatdamagc from contactwith skinor mucous mcmbrancs by oral intakc o[ PVP hasnot becn known of sincc, as statcd earlicr, ils

absorption in these situations can be ignored. Itis only with parcntal administration that someeffects are noted.

PVPretention orelimination behavesreciprocallywith the molecular weight of the compound, asthose with molecular weighs of 20,000 andabove are eliminated aflet a delay and partlyretained for wceks or months.

After l0 years of administration of long termadministration of PVP, accumulation occurs in

$ereticulo-entothelial system' These sometimes

manifest as joint pains, hepatesplenomcgalyand dyspnea. This is called the PVP disease.

All authors agee that the storage of highmolecular PVP in the liver, lungs, secreloryorgans and the granulation tissues has nounfavorable influence on the functioning of theorgans or lhe tendcncy of the tissues to hcal.

PVP has bcen studicd for possible carcinogcniceffecs and none have becn noted.

In the pathophysiology of peptic ulcers, it isknown thatboth acidand pepsin are importantinits occurrence (l).

Helicobacter Pylori

Phospholipase activity has been implicated inthe mucosal damage caused by H. pylori-inducedulceration. According to Berstad, the organismpossibly produces or induces the host to releasephospholipases that metabolize ttre protectivelayer of phospholipids (20). Studies byYamashiro on H. pylori colonized the mucouslayer tlrereby covering normal gastric epithelium;(2) lay atop inegular microvilli as well as amongthe intercellular junctions of damaged cells; and(3) adhered firmly by specific junction zone andlosely by filamentous appendages to thc epithelialsurfaces of cells which lacked evidence of aprolective mucous laYer (21).

METHODOLOGY

Study Design. This experiment utilized arandomized controlled parallel design. Thetreatment groups were as follows:

Group treatment No. of rats

A GFontrol (PVP solution) l0B (+)misoprostol (l44ug/kg) l0C mahogany extrrct (0.lag7kg)-dose I l0D mahogany extract (0.28glkg)-dose 2 l0E mahogany extract (0.57 gAg)'dose 3 l0

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Experimental Animals. Forty-five femalesprague-Dawley rats weighing between 150-200grams and aged 2-3 montts were used in thisstudy. Two rats were kept in every cage.

Preparation of the Treatment Solutions(Details are shown in Appendices l and 2)

Mahogany. The three dosesof mahoganyexuact,namely, O.l4Zgftg, 0.2839kg, and 0.57 2gKg,wer€ computed based on the initial findings ofacute loxicity experiments in mice.

During each expcrimental session, a stock ofmahogany seed extract. rvith a concentration of0.1316 extracU ml solution, was prcparcd. Thiswas dissolved in l5Vo polyvinylpynolidone

@VP) solution.

For each test group, the volume of stock solutionadministered to the rats was computed based onthe body weight of each rat. To ensure that thevolumes are administered equally among all theras in the different treatmcnt groups, an additionalvolume of PVP solution was added to make I mlof mahogany extract solution.

Misoprostol (Cytotec). For each experimentsession, two tablets of Cytotec containing 200 ugof misoprostol werc crushcd using a mortar andpestle, and dissolve in l0 ml of NSS. Thisyielded a solution with a concentration of 40 ug/ml. Computations werc made in order todetermine, the amount of solution to beadministered to each rat, based on the dose ofl44ugkgbody weight.

Indomethacin. For each scssion, four 25gcapsules of indomcthacin wcrc dissolved in l0ml o f NSS, y ie ld ing a so lu t ion w i th aconcentration of IOg/mi. Thc amount of sol utiongiven to each rat was computcd based on the doseof 30mg/kg body weight. (23)

Ulcer- induct ion. Aftcr one week ofacclimatization, the rats were subjectcd to a24-hour fast. On the lirst day of experimentation,

Acta Medica Philippina

the rats were numbered, weighed and randomlyassigned o the five treatrnent groups as describedabove. Indomethacin was computed based onthe weight of erch rat, w.ls administered viaintragasric gavage.

Administration of Treatments. After 24 hours,initial adminisration of the different treatments@VP solution, misoprostol solution, mahoganydose 1, mahogany dose 2, and mahogany dose 3)for day I were done also by intragasric gavage.

The treatment wereadministered thrice, 24 hounaparl During all the reatment days, the rats werekept in their cages with food provided for 24hours until tre next trealrnent schedule.

Six hours after the ttrird treatment(3), the subjectswere sacrificed by chloroform inhalation in anair-tight jar. The stomach, were surgicallyremoved by cuuing from the esophageal andpyloric ends. The isolated stomach were openedand cut along the greater curvature. The contentswere removed, and the stomach cleaned, usingnormal saline solution. Care was exercised so asto keep the mucosal blood vessels intact. Thestomach was spread over a dissecting pan and theperipheral borders securely pinned.

Outcome Evaluation

Gross Analysis. Grading of the ulcers wasconducted by the same observer who was unawareof the treatment given to the rats. The stomachswere examined under a dissection microscope.The diameten of the lesions were measuredusing a ruler. G.rading was based on Best's Ulcerstaging Index shown below (22).

[.esion Score

Deep linear ulcer > l0 mm longDeep linear ulcer < l0 mm longCircular ulcer I -3 mmCircularulcer< I mmFraction of stomach showingevidence of hemonhage

42I0.52

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Histopathologic Analysis. After grading theulcers from thegross specimens, all the slomachswere cut into three segments, namely: fundus,body and pylorus; and then brought to UPDepartment of Pathology for histopatologicanalysis. Since thcre is no established criterionfor quantitative evaluation of ulcers, the resultswere graded based on the number of ulcers,presence of necrosis, and degree of infl ammationas evidenced by cellular proliferation. Thecategories utilized in this study are as follows:

l. Number of ulcers

No. ScoreNormal / 0 0

t - 2 I3 - 4 2> 4 3

Absence of ulcers and "hcalcd" ulcers weregiven a score of 0. An ulccr is considercd"healed" if there is evidence of capillary andfibroblastic proliferation and granulation tissueformation.

2. Presence of necrosis

( + ) o( - ) I

Necrotic tissues in mild forms of ulcers areusually confined at the superficial areas of theulcer. The specimen is graded positive if thenecrotic tissue extends tc the submucosa,muscular, or decper layers of the stomach.

3. Degree of iflammation

No cellular infiltratcs 0Acute inflammatory cclls ILymphocytic/

Eosinophil inliltration 2

The degree of inflammation is graded accordingto the presence and type of inflammatory cell

infiltration. Acute inflammatory cells, which areobserved beneath the ulcerative lesions, arecomposed of polymorphonuclear cellswhilelymphoid aggregates can be observed also in themucosa extending to the submucosa.

Statistical Analysis. Since tlre data were obtainedusing subjective methods, the scores wereanalyzed using the Kruskal-Wallis one wayanalysis of variance, a non-parametric procedure.This is used to t€st the null hypothesis (H) thatthe samples are from identical populations (24).I-evel of significance was set at alpha = 0.05.

Separate analyses were done for scores obrainedfrom gross examination and histopathologicgrading of ulcers.

RESI.JLTS

Forty-five (45) female Sprague-Dawley rats wererandomly assigned to five treatment goupsnamely: three doses of Mahogany extracts (doseI = 0.148/kB body weight; dose 2 = 0.28 g/kgbody weight; dose 3=0.5'l gke body weight)with Misoprostol (M) and PVP as positive andnegative control, respectively. Nine rats wererandomly assigned to each teatment group.

Prior to thecompletion of the treatmentregimen,rats died. PVP, Misoprostol and Mahogany doseI groups had two deaths each, while Mahoganydose 3 incurred one death. These deaths werepostulated to be caused by gastrointestinalbleeding, profuse diarrhea and severe tapeworminfection.

Analysis of Gross Specimens

Thc scores of the 5 troatmcnt groups obtaincdfrom Best's Ulcer Staging Index were compared.The mean ranks of the 5 treatrnent groups aresummarized in Table l. Kruskall Wallis one wayANOVA showed a statistically signifi cant resultat alpha=0.05.

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Table 1. Mean ranksof Ulcers AmongTreatmentGroups Using tllcer Staging Index

TREATMENTCROUP MEANRANK N

PVP 32.2rMISOPROSTOL I8.I4

DOSE l 17.14DOSE 2 19.17DOSE 3 12.00

77798

Kruskall Wallis H = 13.23 (Critical value atdf4,9.49)

Histopathologic Results

Upon viewing under the microscope, the slidescontaining the sections of the rat stomach, thepresence of ulcers, was observed in all of theEeatment grcups. However, these were presentin varying degrees. Ulceration reached thesubmucosa in majority of the specimens, whileonly the mucosa was injured in a few.

There was also evidence of necrosis, andinflammatory cell infi lration basically involvingpolymorpho-nuclear cells, particularly,neurophils. Early granulation tissue was likewisefound, indicating eady stages of regeneration.Mean ranks for statistical analysis werecomputedfor each of the treatment groups (Table 2).

Table 2. Mean Ranks of Ulcers AmongTreatment Groups Using HistopathologicAnalysis

TREATMENTGROUP MEANBANK n

PVP 31.00 7MrsoPRosToL 18.93 7

DOSE l 18.50 7DOSE 2 16.22 9DOSE 3 14.50 8

Kruskall Wallis H = 9.97, significant atalpha=0.05

Acta Medica Philippina

Differences among the 5 treatment groups werefound o be statistically significantatalpha=O.05).

DISCUSSION

The desruction of the gasric mucosal barrierconsisting of tlre surface epithelium and mucouscoatis the main causeof ulcer (l). Thii destructionmay be due to, among other factors, an increasein gastric acid secretion, decrease in mucusproduction or decrease in mucosal blood flow.

This study investigated the effecs of mahoganyseed extract on gastric ulcers compared toMisoprostol, a drug whose ulcer-healing effectshave been extensively studied, andpolyvinylpyrrol idone, an inert solvent.Mahogany seed extract was found to have aneffect on the gastric mucosal healing similar tothat of Misoprostol. Mean ranks for Misoprostoland Mahogany doses 1,2 and 3 for both. the grossand histopathologic fi ndings were very near eachother. All the doses had lower mean ranks thanmisoprostol. Further, Misoprosol and the threedoses of Mahogany extract were all found tohave lower mean rank than the PVP, the negativecontrol. This suggest that mahogany indeed hasa potential effect.

How is this healing achieved? Based on studies,Misoprostol is said to make healing possiblethrough several mechanisms. It decreases gastricsecretion (9) without decreasing the blood supplythat allows more nutrients and oxygen to bedelivered to the cells. It also removes toxicsubstances efficiently; and increases mucus andbicarbonate secretion which renders the mucosaless acidic and protects it from further injury (5).

On theotherhand,research on mahogany has yetto reach its zenith; therefore is properties havenot been extensively studied. Nevertheless,several mechanisms may be postulated to play arole in protecting or healing the gastric mucos:r.

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The mucus laycr on tlrc gastric cpitltclium mayrender some protection !o thc mucosa, but this isnot sufficicnt Anothcrphysical barricrof surface-active phospholipid dircctly absorbed ino theluminal lining of thc stomach has bccn proposcd.The finding that intact mucosa is hydrcphobicsupports $is hypothcsis (25,26).

One such phospholipid is phosphatidylcholine.At the molecular lcvel, the dimensions of fullysaturated phosphatidylcholine are ideal for closepacking to form a barrier bccause of the qualityof the cross-sectional arca of thc polar and non-polar moieties (27).

It is intcresting to note that thc packed fatty acidbarrier already cxists in the mucosa, but anadditional absorbcd monolaycr of phospholipidwill also coat and protcct thc mucosa from acid(26\.

Mahogany sccd extract is high in lipids,particularly neutral lipids, glycolipids andphospholipids, thc most abundant of which isphophat idylchol ine (28). Based on thehypothcsized mcchanism, above, therefore,mahogany secd extracl may provide a protectivephospholipid coat on the injurcd mucosa suchthat progression of ulcer into a more severe statemay be inhibited, and thcrcforc allow healing toproceed.

Another possible mechanism could involve theanti-bacterial property of mahogany seed extracL

Helicobacter pylori has becn found to play amajor role in inducing or aggravating ulceration(29). Although thcse bactcria do not normallysurvive in rats, thcy havc bccn found to occurnaturally in humans (30).

H. pylori f,osscsscs thc ability to dcstroy thephospholipid cpithclial laycr of thc mucosa attdbecausc of its sphingomyclinasc activity, itdcstroys sphingomyclin neccssary lorccll to celladhcsion (12). In addition H. pylori reduceshydrophobicity, thcrcby rcsulting in phospholysisof the mucus layer.

Mahogany sced extractconhins high amounts oflong chain unsaturated fatty acids, includingl8:3, l8:2 and l8:l (28). Studieson long chainfatty acids have found anti-ulcer properties, themechanism of which is postulated to be associatedwith the inhibition ofgastric bacterial metabolism.This anti-bacterial property increases withincreasing saturation on Cl8 (29).

S ince mahogany seed has a h igh fatty component,it has a potential anti-bactcrial action. Thistheory, if proven effective especially against H.pylori, would provide apromising addition tn thevast array of ulcer treatment mcthods.

The three doses of mahogany secd extract wereall found to be better than the negative control.Even at the lowcst dosc, some anti-ulcer activilywas obscrved. Bascd on initial toxicity studies,mahogany was found to haveavcry low toxicity.This implies a high therapeutic index formahogany, which is idcal for any drug. Ergo,mahogany is a relatively safe therapcutic agenl

CONCLUSION &RECOMMENDATIONS

Mahogany seed extract (doses 1,2 and 3) werefound to have a better effect on the healing ofgilstric ulcers than the negtive control, PVP.The observed healing effect is similar to that ofmisoprostol but this needs to be furtherinvestigated.

A multiple comparisons procedure should bedone o pinpoint which of the groups differ fromeach other.

REFERENCES

l. Abd El-Dayem AM, El-Againy MA.Non-wood forest products,physiochemical characteristics andfatty-acid composition of three seedoils.Acta horticulturac 1993: 333:287 -293.

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t Acepcion VFB, ct. al. A comparativestudy of misoprostol and grcen bananaon thcir protcctivc and hcaling cffcctsagainst indomcthacin-induced gastriculccrs in rats. Rcscarch papcrsubmittcdtothc Dcpartmcnt of Pharmacology, UPCollcge of Mcdicinc, 14 Oct. 1994.Aguwa-CN and Okunj i -CO.Gastrrointcstinal studics of Pyrcnacanthasraudtii lcaf extracts. J Ethnopharmacol.1986 Jan; 15 (l):45:55.Akdamar K, Agrawal N. Ertan A.Inhibition of noctumal gastric secrctionin normal human voluntecrs bymisoprostol: a synthctic prostaglandinEl methyl estcr analog. Am JCastrocnterol 1982: 7 7 :9O2-9M.A l Somal , N . Suscept ib i l i t y o fHclicobactcr activity of manuka honcy.J. of Rovaf Soc. Of Med Jan. 1994;87'9-12.Asante, MA et al. Casric MucxosalHydrophobicity and H. pylori infectiondcnsity: a mcchanism for pcptic ulccrpathogenesis? Gut 1995; 36(suppl l).Ballingr A. Cytoprotection withmisoprostol: usc in thc treatmcnt andprevention of ulcers. Dig. Dis 1994Jan-Feb: l2 (l):37-45.BauerRf, et. al. Comparative mucosalprotcctive propcrtics of misoprostol,cimetidine and sucralfate. DigDis Sci1986; suppl 8ls-85s.Berstad K, et al. Phospholipase A2activity in gasuic juice from patientswith active and H. pylori+radicatedhealcd duodenal ulcer. Aliment-Pha.acol-Thcr. 1994 Apr; 8(2):175-180.Best RB, lrwis DA, Nasser, N. Theanti-ulccrogcnic activity of thcunripeplantain banana (Musa species). BrJPharmac 1984; 82: 107-l 16.Bhalare HA, et al. Lipid Compositionof some secds of central India J FoodSci Tcch 1993; 30:54-55.Buencamino RB ct al. A comparativestudy on the cffcctivity of commcrcial

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antacid and diffcrent conccntrations ofeggshell suspension in the short-tcrmtre:ttment of ethanol-induccd gastriculccr. Rcscarch papcr submittcd lo theDcpartment of Physiology, UP Collegeof Mcdicine, 1992.Chan KC, Tang TS, Toh HT. Isolationof swietcnolidc diacctate from swicteniamacrophylla. Phytochem 197 6; | 5:429 -430.Corless, et al. Survival of Hclicobacterby pylori in animal models. Gut.1995;36 (suppl l). Davies GR, et al.Hclicobactcr pylori stimulates antralrnucosal rcactive oxygen metabolitcproduction in vivo. Gut 1994;35:179-185.Davis GA, Fordtran JS, Dajani EZ.Dosc-rcsponsc, mcal-stimutarcd gastricanticcscrctory study of proslaglandinEl analog, misoprostal, in man. Dig DisSci 1988;33:298-302.Hills BA and LichtcnbergcrlM. Gastricmucosal barricr: hydrophobicity ofstrcrchcd stomach lining. Am J. Physiol.1985:244:G&3-7.Hills BA, Butlcr BD and LichtcnbcrgcrLM. Gastr ic mucosal barr icr:hydrophobic lining to thc lumen of thestomach. Am J Physiol. 1983: 7;G-56r-8.Hopkins RJ and Morr is JG.Hclicobactcrpylori: thc missing link inpcrspcctive. Am J Med 1994 Sep; 97(3):265-277.lcung FW. Disscociatcd effecs ofmisoprostol on gastric acid secretionand mucosal blood flow. Dig Dis Sci;1986;31 : suppl 86s-90s.Mata R. Segura-Correa R. Newtetranortriterpenoids from swietcniahumilis. J Nat Prod 1993:56: 1567-t574.Rosulta ct al. Drug theraphy in pcpticulcer discase. Department ofPharmacology, UP Col lege ofMcdicinc. No date.Salud EC. Extract ivcs of four philippine

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17.7.

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2r .

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10.

l l .

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t37

22.

23.

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mahogany species 1. Benzene-solublefraction. Forpide Dig 1977 ;6:24-27 .Thompson L, et al. Inhibitory effect ofpolysaturated fatty acidson the growthof Helicobacter pylori: a possibleexplanation of the effect of diet onpeptic ulceration. Gut 1994; 35:1557 -

1561.U. P. College of Forestry Compilationpp.62-63.Veldhuyzen van Zanten, S. J. O.;S herman , P. M. Indications for treatmentof Helicobacter pylori infection: asystcmatic overview. Can Med AssocJ 1994:150 (2): 189-198.Walt R. R. Misoprostrol for the treatmentof pcptic ulcer and antiinflammatorydrug-induced gastroduodenalulceration. New England Joumal ofMedicine 1992. 327 (22): 157 5-1580.Wesscl, W.; School; M; Winkler, E.Polyvinylpyrrol idone (PVP), I t 'sDiagnostic, Thcrapcutic and TechnicalApplication and Consequences Thereof.Arzneim.-Forsch. (Drug Research).1971;21(10): 1468-1482 Wilson D.Therapeut ic aspects of prostaglandinsin the treatment of pcptic ulcer disease.Dig Dis. Sci. 1986;31: suppl42s-46s.Wood AJJ. Misoprostol for thetreatment of pept ic ulcer andant i in f lam m a tory -drug- in d ucedgastroduodenal ulceration. NE J Med1992:321:1575-158Yamashiro Y, etal. Helicobacterpyloricolonization inchildren with gastritisand peptic ulcer. II. Ultrastructuralchange of the gastric mucosa. ActaPaediatr Jpn. 1994 Apr; 36(2\:17l-r75.

APPENDIX 1PREPARATION OF SOLUTIONS

Preparation of Mahogany SolutionA. Seed Extraction

I.

24.

25.

The mahogany seeds were dried andcoarsely ground. 1.Ot kg of the groundplant material was covered with 3.0 Ldistilled water in a glass vessel andboiled for 30 minutes, colled to roomtemperature and filtered in vacuothrough cheesecloth. The plant marcafter filtration of the first extract wasthen boiled again in 3.0 L distilled water,this time for two hours and filtered. Theextract was allowed to stland overnightand decanted using f,rlter paper. 3.0 L ofthe combined extract was ro0a-vaporizedto yield34.7 Vo decoction, equivalent tol32.8gof the concentrated extracl

Storage

The concentrated extract was keptrefrigerated in a vial wrpped inaluminum foil.

Solutions from the concentrated extractwere made fresh everyday.

Preparation of PVP

A 5Vo PYP l0 solution was used assolvent for the mahogany extracts. 5.0gof PVP powder were dissolvedthoroughly in 50 mll- of distilled wateruntil a clear liquid was obtained. Moredistilled water was added o make 100mL. The pH of the solution was noted.

Preparation of Mahogany StockSolution

Twenty mL of the 0.13 16 g extracVmlstock solution was prepared per day.2.362 g of the mahogany extract wasweighed using the analytical balance.

26.

27.

B.

e.28.

29.

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It was dissolved in l0ml- of distilled 1.599 seed xwater. More distilled water was added _= _; x=0.0318g seedp0g mouseto make the solution 20 ml. The pH of 10009/kg m 20 mousethe solution was also taken.

0.03128g1kg x7 =A.22269 seed/200 g ratII. Preparation of Misoprostol Solution (0.22269f2Wra0(l0OA0= Ll13gseed/kgrat

Two 200 ug tablets of misoprostol were Conversion of g seed to g extract:ground finely and dissolved in l0ml- 1.113x0.1277=0.142 g extract/kg ratnormal saline solution. Theresulting4Oug/ml suspension wascontinuously Dose 2: 0.283 g extract/kg rat (equivalent tomixed to present the particles from 3.17 g seed/kg mouse)settling. The dose administered to theratswas l44ttglkg' 3'lTgseed ^,*=0.*rosseed20/smouse

III. Preparation of Indomethacin Solution 10009/kg m 205

The resulting l0 mg/ml solution was 0.0634x7=0.4438 g seed/200 g ratadministered to the rats ata dose of 30 (0.4438g2$grarx1000gAg=2.2t9gs,eAkgntmg/kg body weight. 2.219x0.1277--0.292g extracr/kg rat

APPENDIX 2. Dose 3:0.59 g extract/kg (equivalent to 6.4 gseed/kg mouse)

COMPUTATIONS6.49 seed x

Based on the pre-test results of acute toxicity -=-;x=0.l28gseed2Ogmouseresults on mahogany seed, the no effect dose 10009/kg m 20s(NED) is at 6.4 seed/kg mouse. Doses for ratswere computed using a log dose interval of 0.3 0.128x7=0'896 gseedl}00gtat

srarring airhis NED. (0.896g/200g)(10009/kg)={.48g seed/kg rat4.48x0.127 7 =0.59 gkg rat

Dose 1: 0.142 g extract/kg rat (equivalent to1.S9g seed/kg mouse) Stock solution: 0.1316 g extract/ml solution

Conversion of dosage in mouse to rat based on 0'572 g extract x

surface area ratios (Conversion Factor=7) = -; x=0.13169 extracV2309rat1000grat 230e

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