ABSTRACT

Title: Coordinated Anti-angiogenic, Analgesic Potential and Phytochemical !aluation of "numa#i $Podocarpus macrophyllus% and Chico $Achras zapota% Lea!es &tract Pro'onents: d(in Ar)ae Alconcel, Paul *enedict A'ostol, Darrell d Tua+on School: ,eneral Santos -o'e Christian School *loc# . Dadiangas-eights, Sal!ani St., ,eneral Santos City, Phili''ines Inumaki is a small to medium size evergreen tree, reaching 2 m tall. !he leaves are stra"#sha"ed, $#%2 cm long, and about % cm broad, &ith a central midrib. !he cones are borne on a short stem, and have 2#' cm. scales. !he treatment o( and anti#angiogenesis is common health "roblem in our generation today. !here(ore this study aims to (ind a chea"er alternative agent in treating angiogenesis, (ever and body "ains through anti#angiogenic, and anti"yretic testing o( the combined extracts o( Inumaki )Podocarpus macrophyllus) and Chico )Achras zapota) is undertaken. Chico )Achras zapota) Chico leaves are one o( the most ignored yet has signi(icant value in the (ield o( medicine. !he "ro"onents (urther investigate the uses o( both "lant and (ound that its leaves have medicinal used, one o( those is anti#carcinogenic "ro"erty conducted by the researchers using Chorioallantoic *embrane )C+*, +ssay. !he "ro"onents bought - .uck eggs (rom a "ro(essional breeder, /uickly a(ter, the duck eggs &ere incubated (or % days. 0hile the "ro"onents &ere &aiting (or the eggs to develo" blood vessel, they "re"ared the inumaki and Chico leaves then soaked it in *ethanol to get the extract. !he extracts &ere obtained &ith the use o( a 1otary 2va"orator. !hen, the "ro"onents made (ive treatments, inumaki extract, Chico extract, combined extract, "ositive control merca"tu"urine and a negative control )&ater,. !hen, (ilter "a"ers &ere soaked on each treatment and "laced on the C+* layer o( the egg. !he eggs &ere then /uickly sealed and incubated (or another '3 hours. +(ter the incubation "rocess, the blood vessels o( the treated eggs &ere observed under a microsco"e and com"ared to the control unit. !he anti#angiogenic screening o( inumaki extract, Chico 2xtract and the combined extract sho&ed a signi(icant result in decreasing the number o( blood vessels in the embryo o( the duck egg based on the re4ection o( the null hy"othesis in the analysis o( variance. 0hen tested (or analgesic "otential, Inumaki sho&ed a sustained resistance to "ain &ith a mean resistance o( '%.$5 seconds (ollo&ed by chico &ith --.'5 seconds. !he "hytochemical screening sho&ed that the Inumaki is "ositive (or +lkaloids, 6lavoniods, 7eucoanthocyanins and !annins8 on the other hand, the extract is negative (or 9nsaturated :teroids and :a"onin. 6or Chico, it is "ositive (or +lkaloids, 9nsaturated :terols, 6lavonoids, 7eucoanthocyanin and !annins8 only negative to :a"onins. !hus, the "ro"onents concluded that the methanol extract o( Inumaki and Chico,and as &ell as the combined, have antibacterial, anti#angiogenic "ro"erties, analgesic "otential and contains secondary metabolites8 there(ore can be used as a much chea"er alternative to commercial medicine.

Category Pick only one box and mark an X on it. ;iochemistry ;otany Chemistry Com"uter :cience 2arth and :"ace :cience 2ngineering 2nvironmental :cience *athematics *edicine and <ealth *icrobiology Physics =oology >erontology

I/We hereby certify that the above is the result of work done in my/our research . ______________________ Signature of Team Leader ___________________ Date

Chapter 1 THE PROBLEM AND ITS SETTING The cha'ter 'ro!ides the readers of the nature, sco'e, 'ro/lem, and its historical and theoretical /ac#ground. "t includes the follo(ing: /ac#ground of the study, statement of the 'ro/lem, hy'othesis, significance of the study, re!ie(s of related literature, o'erational definition of terms and sco'e and limitation. 0.0 *ac#ground of the Study Angiogenesis is a normal 'rocess in gro(th and de!elo'ment of the em/ryo and fetus, as (ell as in (ound healing. -o(e!er, this is also a fundamental ste' in the transition of tumors from a dormant state to a malignant state. 1ith this 'rocess (e can sim'ly find out if an o/)ect can cure or lessen the effects of certain cancers, /y the means of /lood !essel count. This is done due to the fact that (ithout the /lood !essels, the cancer tissues (hich are 'resent in our /ody cannot and (ill not e&ist, these /lood !essels su''ly the needed nutrients for the cancer tissues. 2urthermore, angiogenesis cannot /e done (ith non-fertile eggs /ecause (ith these #inds of eggs, there is no s'erm cell and egg cell formation. The main significance is on the anti-angiogenic 'ro'erty of this lea!es, as (ell as the anti-/acterial, and the secondary 'lant meta/olites. These secondary 'lant meta/olites include the follo(ing: Al#aloids, Sa'onins, 3nsaturated Sterols, Tannins, 2la!onoids, and others. 0.2 Statement of the Pro/lem

S'ecifically, this study (as conducted to ans(er the follo(ing 4uestions: a. Does the "numa#i $Podocarpus macrophyllus) and Chico (Achras zapota% lea!es e&tract e&hi/it antiangiogenic 'ro'erty, and analgesic 'otential5 1hat are the secondary meta/olites 'resent in the e&tract of "numa#i $ Podocarpus macrophyllus) and Chico (Achras zapota% lea!es5

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0.6

,eneral 7/)ecti!es

The o/)ecti!e of this study is to e!aluate the 'hytochemical constituents, analgesic 'otential and antiangiogenic 'ro'erty of the crude methanol e&tract of "numa#i $ Podocarpus macrophyllus) and Chico (Achras zapota% lea!es 0.8 -y'otheses ALT 9:AT"; : The e&tract of "numa#i $ Podocarpus macrophyllus ) and Chico (Achras zapota% lea!es ha!e anti-angiogenic, 'ro'erties, analgesic 'otential and contains essential secondary meta/olites. The e&tract of "numa#i $Podocarpus macrophyllus ) and Chico (Achras zapota% lea!es ha!e no anti-angiogenic, analgesic 'otential and does not contain essential secondary meta/olite.

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Significance of the Study

This study determines the secondary meta/olites 'resent in "numa#i $ Podocarpus macrophyllus) and Chico (Achras zapota% lea!es e&tract. The com'letion of this study (ould 'ro!ide information on the efficacy of "numa#i $Podocarpus macrophyllus) and Chico (Achras zapota% lea!es, (ith its cou'ling e&tract efficacy as an anti/acterial, and anti-angiogenic agent (ith analgesic 'otential.

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Sco'e and Limitations

This study is only limited to e!aluate the cou'ling anti-angiogenic, analgesic 'otential and 'hytochemical constituents of "numa#i $Podocarpus macrophyllus) and Chico (Achras zapota% lea!es e&tract. The anti-angiogenic 'ro'erty can /e studied through the use of merca'tu'urine as the 'ositi!e control, (ater $negati!e control%, inuma#i e&tract, Chico, and the com/ined e&tract of the said 'lants and /y solely de'ending on the /lood !essel formation of the medium, (hile the 'hytochemical constituents (ill undergo a series of tests. The said e&'eriments (ere conducted from August to 7cto/er 2>00 in the Science La/oratory of ,eneral Santos -o'e Christian School 0.? 9e!ie( of 9elated Literature Anti-Angiogenic

Anti-angiogenic is a 'ro'erty 're!enting the 'hysiological 'rocess in!ol!ing the gro(th of ne( /lood !essels from 're-e&isting !essels. $(((.(i#i'edia.org@(i#i@Angiogenesis%

Analge ic A drug or medicine gi!en to reduce 'ain (ithout resulting in loss of consciousness. Analgesics are sometimes referred to as 'ain#iller medications

In!"a#i Podocarpus macrophyllus $Ausama#i or "numa#i% is a conifer in the genus Podocarpus, family Podocar'aceae. "t is the northernmost s'ecies of the genus, nati!e to southern Ba'an and southern China. Ausama#i and "numa#i are Ba'anese names for this tree, and Ausama#i is increasingly /eing used as the nglish name as (ell, re'lacing the old, /otanically inaccurate names C/uddhist 'ineC and Cfern 'ineC. (http://en wikipedia or!/wiki/Podocarpus"macrophyllus) Chico Lea$e Are different in si+es. "t is irregular in sha'e and similar in si+e. They are mostly green yet turn /ro(n (hen they (ilt. They are highly ornamental and are seldom used. $(((.(i#i'ili'inas.org@chico% Chorioallantoic "e"%rane Chorioallantoic mem/rane are also #no(n as C/reathing mem/ranesC. "t is made u' of the chorion and the allantois (hich /egin to fuse at a/out day <. The chorion lines the inside surface of the 'orous eggshell (here it can e&change o&ygen and car/on dio&ide freely. The /lood !essels of the allantois 'ic# u' o&ygen and ta#e it to the em/ryo. They also /ring (aste 'roducts such as car/on dio&ide and urine from the em/ryo to release into the chorioallantoic ca!ity.

$(((.(i#i'edia.org@(i#i@Chorioallantoic_mem/rane%

Chapter & Material an' Metho' &(1 Material an' E)!ip"ent This su/-section 'resents the ra( materials and la/oratory e4ui'ment to /e used in the e&'erimental setu'. These (ere categori+ed /ased on 're'aration of e&tract, 're'aration of 'a'er disc, anti/acterial susce'ti/ility test, 'hytochemical screening, analgesic screening and anti-angiogenic testing. Daterials and "nstruments used for anti-angiogenic screening Ta%le *(1 Material ! e' +or Anti-Angiogenic Screening La/oratory 4ui'ments 0. Petri Dish 2. 2ilter Pa'er 6. *ea#er 8. Scal'el ?. 2orce's <. Dicrosco'e =. ,lass Slide .. Co!er Sli' E. Alcohol Lam' 0>. -ot Plate 00. Digital *alance 02. 0>>>mL rlenmeyer 2las# 06. "ncu/ator

Primary Daterials 0. Duc# gg 2. *agras lea!es e&tract 6. Chico lea!es e&tract

Material an' In tr!"ent ! e' +or Ph,toche"ical creening

The said study used !arious instruments and e4ui'ments to 'erform the 'hytochemical screening. Ta/le 6.< lists the materials needed for the e&'eriment. Ta%le *(* Material ! e' +or Ph,toche"ical An' Analge ic Screening Primary Daterials 0. *agras and Chico lea!es e&tract 2. 2 D -ydrochloric acid $-Cl% 6. Po(dered Sodium Chloride $:aCl% 8. DayerFs reagent ?. Petroleum ther <. Chloroform =. thanol .. -ydrochloric acid $-Cl% E. Dagnesium Stri's 0>. Sodium Chloride 00. Anhydrous Sodium Sulfate *(E.peri"entation an' General Proce'!re La/oratory 4ui'ments 0. Test Tu/e 2. Test Tu/e 9ac# 6. *ea#er 8. -ot Plate ?. !a'orating Dish <. Stirring 9od =. Sto''er .. Distilled 1ater E. Cruci/le Tongs 0>. ,raduated Cylinder

This section sho(s a ste'-/y-ste' 'rocedure on ho( the e&'eriment (as thoroughly done. "t tells us ho( the 'rocedures (ere done and (hat e4ui'ments (ere used. Proce'!re o+ e.tracting the !% tance +ro" In!"a#i /Podocarpus macrophyllus0 an' Chico (Achras zapota) "numa#i $Podocarpus macrophyllus) and Chico (Achras zapota) leaves were collected regardless of their sha'e and si+e. The lea!es (ere cleaned and then soa#ed in methanol for 28 hours. The e&traction 'rocess (as finished /y using 9otary !a'orator. Ph,toche"ical Screening1 2or the 'hytochemical screenings, the 'ro'onents used different #inds of tests in order to determine (hether the e&tracts ha!e secondary meta/olites. 7nly tests that determined the 'resence of al#aloids, sa'onins, leucoanthocyanins, fla!onoids, tanins and 3nsaturated steroids Anti-Angiogenic Screening The anti-angiogenic screening (as done /y means of ma#e a small (hole on the /lunt edge of the egg, then adding 6-? dro's of each e&tract, negati!e and 'ositi!e controls, and the mi&ture of /oth e&tracts. After (hich, the 'ro'onents sealed the eggs then incu/ated for another 8. hours. After the 8. hours, the 'ro'onents /ro#e the eggs and 'laced each of the contents in indi!idual 'etri dishes, then they o/ser!ed the /lood !essels of each egg under a microsco'e.

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Analge ic Screening The mice (ere in)ected (ith >.0 ml of Chi!es, "numa#i and a com/ination of Chi!es and "numa#i e&tracts. After 6> minutes, the researchers conducted the tail flic# method in (hich ? cm of the miceFs tails (ere di''ed in a ?> to ?? degree Celsius of hot (ater. The researchers then too# note of the res'onse time of the mice to(ards the hot (ater. Then after another 6> minutes the researcher again too# do(n notes of the reaction time of the mice. Then finally after an hour the researchers too# the res'onse time of the mice for the last time

Chapter * PRESENTATION2 ANAL3SIS AND INTERPRETATION O4 DATA This cha'ter 'resents the data gathered through actual o/ser!ation and the changes that ha''ened to the /lood !essels of the Duc# eggs incu/ated for 0> days. 5(1 Re !lt o+ the Ph,toche"ical Screening

The results o/tained from the Phytochemical screening sho(s the "numa#i and Chico lea!esF secondary meta/olites. Ta%le 5(1 Secon'ar, Meta%olite o+ the E.tract. Secon'ar, Meta%olite Al#aloi' Saponin 4la$onoi' 7n at!rate' Sterol Le!coanthoc,anin Tannin In!"a#i 66 6 6 6 6 Chico 6 6 6 6 6

The ta/le sho(s the 'ositi!e meta/olites as (ell as negati!e meta/olites in the "numa#i 'lant and the Chico Plant. Al#aloids, Sa'onins, Leucoanthocyanins, and Tannins are all 'ositi!e meta/olites of "numa#i e&tractG (hile 2la!onoids and 3nsaturated Sterols are the negati!e meta/olites of the said 'lant. The Chico e&tracts are 'ositi!e for Al#aloids, Leucoanthocyanins, Tannins, 2la!onoids and 3nsaturated Sterols. 7n the hand, the e&tract is negati!e to Sa'onins. 5(& Re !lt o+ the Anti-Angiogenic Screening 9 PL"CAT"7:S Treatment Chico "numa#i Com/ined Derca'to'urine 1ater 0 0. 20 0= 08 60 2 20 0E 0. 0< 60 6 0= 0= 0< 0= 60 T7TAL ?< ?= ?0 8= E2 D A: 0..<<<= 0E.>>>> 0=.>>>> 0?.<<<= 6>.<<<=

The ta/le sho(s the num/er of /lood !essels in the three re'lications of each treatment. Derca'to'urine sho(ed the least mean (ith 0?.<<<= /lood !essels and the negati!e control sho(s the greatest mean (ith 6>.<<<= /lood !essels. The com/ined e&tract of "numa#i and Chico sho(ed a mean /lood !essel formation of 0=.>>. Chico alone sho(ed a mean dlood !essel formation of 0..<<<= (hile for "numa#i it recorded a mean /llod !essels formation of 0E.>>. Ta/le sho(ed that the com/ined e&tract of "numa#i and Chico a''roaches the efficacy of merca'to'urine in reducing the /lood !essels formation, thus reducing the ris# of cancer cell formation /y de'ri!ing the said cancer cell of their nutrition through the /lood !essels. Tu#ey LSD Contrast Derca'to'urine !s com/ines Derca'to'urine !s. chico Derca'to'urine !s. "numa#i Derca'to'urine !s (ater Chico !s "numa#i Difference 0.6 6.> 6.6 -0?.6 >.666 E? H Confidence "nter!al -2.. to ?.8 -0.0 to =.0 ->.. to =.8 -0E.8 to -00.2 -0.2 to =.? 9emar#

significant

Analysis of ;ariance re!ealed that the 2 statistics of 8?.>8 (hich is greater than the 'ooled !alue of 2.6 & 0>-< dictated for the re)ection of the null hy'othesisG that the mean difference /et(een treatments has significant differences (ith each other. Doreo!er, the Tu#ey Least Significance Difference Test sho(ed that the a!erage num/er of mem/rane formations in each treatment significantly differ only (hen merca'to'urine is com'ared to (ater. !idently the treated grou' sho(ed no differences in the mean mem/rane formation (hen com'ared to the 'ositi!e control merca'to'urine. This means, that "numa#i and Chico e&tract or its com/ination e&hi/ited e4ual

effect on the mem/rane formation in the e&'erimental duc# egg. Thus, "numa#i and Chico or its com/ined e&tract has anti-angiogenic 'ro'erty. ANALGESIC SCREENING1 Ta%le 5(- Analge ic re !lt A( Control 9e'lication 1 & * MEAN Thirty minutes after in)ecting e&tract 8 1:inety minutes after in)ecting e&tract 11 1: &:(Sum 19 1*-(Dean 9(;(1;(; 11(-;

Ta/le a/o!e sho(ed that it too# a mean 00.?= seconds for the mice to res'ond to the change in tem'erature (hen it is treated (ith (ater. B( Chico 9e'lication 1 & * MEAN 6> minutes after in)ecting e&tract 1: 5; &:(0 hour and 6> mins after in)ecting e&tract &8(5*(-1(55 Sum *8(9:(;1(9& Dean 19(&5-(&; *-(9< **(59

Ta/le a/o!e sho(ed that it too# a mean 66.8E seconds for the mice to res'ond to the change in tem'erature (hen it is treated (ith chico e&tract.

C( In!"a#i 9e'lication 1 & * MEAN 6> minutes after in)ecting e&tract *8(-: 59(11 8(-: 0 hour and 6> mins after in)ecting e&tract 55(5: -&(;: --(9: Sum 8&(9: 1:1(8: <5(5: Dean 51(9-:(91 *&(&: 51(<9

Ta/le a/o!e sho(ed that it too# a mean 80.<E seconds for the mice to res'ond to the change in tem'erature (hen it is treated (ith "numa#i e&tract.

D( Co"%ine' e.tract 9e'lication 1 & * MEAN 6> minutes after in)ecting e&tract ;(:: *-(9: 5(9: 0 hour and 6> mins after in)ecting e&tract 1;(99 *<(9: 8(:: Sum 5&(: *;(: 58(: Dean 15(:: 1&(*: 1<(:: 15(1:

The data a/o!e for the analgesic screening of "numa#i, Chico and its com/ined e&tracts sho(ed that "numa#i e&tract has the highest analgesic res'onse of 80.<E seconds in ??>C (ater.

Chapter 5 S7MMAR3 O4 4INDINGS2 CONCL7SIONS AND RECOMMENDATION This cha'ter 'resents the summary of findings and conclusions made from the study and recommendations gi!en /y the researchers. 5(1 4in'ing The researchers started (ith the collection of the sam'les 'articularly Podocarpus macrophyllus and Achras zapota in the certain area The collected sam'les (ere then soa#ed (ith Dethanol in rlenmeyer flas#s in the science la/oratory of -o'e Christian School. The 'hytochemical screening (as conducted in the science la/oratory. After each screening, results (ere again recorded des'ite the 'ositi!e or negati!e outcomes. The results on some of the screening (ere 'ositi!e and some are negati!e. The Analgesic screening of the e&tract (as also conducted and the result sho(ed that "numa#i gi!e the longest 'ain resistance among the e&tracts (ith a mean of 80.<E seconds, it e!en e&ceeded the analgesic effect of the com/ined e&tract (ith 08.0> second. 5(& Concl! ion

0>

*ased on the e&'erimentation, results and data gathered, the researchers (ere a/le to formulate the follo(ing conclusions: 0. 2. 6. 8. Dethanol e&tract of "numa#i $Podocarpus macrophyllus% has 'hytochemical 'ro'erties such as Sa'onins, Al#aloids, Leucoanthocyanins, 2la!onoids and Tannins (hile Chico $ Achras zapota% contains 2la!onoids, 3nsaturated sterols, Leucoanthocyanins, al#aloids and Tannins. Com/ined methanol e&tract of "numa#i $Podocarpus macrophyllus) and Chico $Achras zapota% has anti/acterial 'ro'erties that inhi/it the gro(th of /acteria. Com/ined methanol e&tract of *agras $#ucalyptus de!lupta% and Chico $Achras zapota% has antiangiogenic 'ro'erties. "numa#i $Podocarpus macrophyllus% has analgesic 'otential.

5(* Reco""en'ation 2rom the findings of the study, it can /e o/ser!ed that there (ere research ga's. The follo(ing (ould ser!e as research agenda and methodological suggestions in future studies. 0. 2. 6. 8. 2urther studies to /e conducted (ith more /acterial cultures to 'ro!e the !alidity of the inhi/itory effect. Testing (ith other commercially a!aila/le anti/iotics /esides the ones used in this study. 2urther e&'erimentation of the "numa#i and Chico 'lants in terms of its anti-angiogenic 'ro'erty for !alidity. To include anti-inflammatory and anti'yretic 'ro'erty aside from the analgesic 'otential.

Bi%liograph, "nternet: htt':@@(((.'hytochemicals.info@ htt':@@(((.healthline.com@galecontent@/lood-!essels (((.fi.edu.@learn@heart@!essels@!essels.html htt':@@(((.cancer.go!@cancerto'ics htt':@@(((./iology-online.org (((.(i#i'edia.org@(i#i@Assay htt':@@dictionary.reference.com@/ro(se@assay htt':@@dictionary.reference.com@/ro(se@chorioallantoic htt':@@(((.encyclo'edia.com@to'ic@mem/rane.as'& htt':@@(((.medterms.com@scri't@main@art.as'5article#eyI0>20? htt':@@en.(i#i'edia.org@(i#i@2la!onoid en.(i#i'edia.org@(i#i@Air/y-*auer_anti/iotic_testing htt':@@en.(i#i'edia.org@(i#i@Proanthocyanidin (((.scientificcommons.org@statin@anti-angiogenic htt':@@(((.(ordi4.com@definition@Synergy *oo#s: Al/erts, *ruce $2>>2%. C0>. Dem/rane StructureC :e( Jor#: ,arland Science Cal!i L, D(alongo ,CB, D(ingira *A, 9iedl *, Shields BA $0EE?%. CCharacteri+ation of 1attle-Tannin*ased Adhesi!es for Tan+aniaC. $olzforchun!8E

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-ostettmann, A.G A. Darston $0EE?%. %aponins. Cam/ridge: Cam/ridge 3ni!ersity Press 9ogers D2, 1in# D. $0EE.%. Alkaloids: biochemistry& ecolo!y& and medicinal applications. Plenum Press

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