Antioxidant Compounds of Gayam Seed (Inocarpus fagiferus Fosb) to Prevent Atherosclerosis through Increases of SOD Activity and Improvement of Lipid Profile on Wistar Rat.

Ant ioxidant Compounds of Gayam Seed

( I nocarpus fagiferus Fosb) to Prevent

At herosclerosis t hrough I ncreases of SOD Act ivity

and I mprovement of Lipid Profile on W istar Rat

By

I. M ade Sukadana, Ida Bagus Putra M anuaba, I. W ayan W ita,

I. W ayan Putu Sutirta Yasa and Sri Rahayu Santi

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J. Biol. Chem. Research

Volume 32 ( 1) 2015 Pages No. 28 -35

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An I nternational Journal of Life Sciences and Chemistry

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J. Biol. Chem. Research. Vol. 3 2, No. 1: 28- 35, 2015

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RESEARCH PAPER Received: 20/ 11/ 2014 Revised: 11/ 12/ 2014 Accepted: 01/ 01/ 2015

Antioxidant Compounds of Gayam Seed ( I nocarpus

fagiferus Fosb) to Prevent At herosclerosis through

I ncreases of SOD Act ivity and I mprovement of Lipid

Profile on Wistar Rat

I. M ade Sukadana

*

, Ida Bagus Putra M anuaba

*

, I. W ayan W ita

* *

,

I. W ayan Putu Sutirta Yasa

* *

and Sri Rahayu Santi

*

* Chemistry Department, Faculty of M athematic and Natural Sciences, Udayana University, Indonesia

* * Faculty of M edicinal, Udayana University, Indonesia

ABSTRACT

This study aims to prove the potency of ethanol extract of gayam seed to prevent atherosclerosis through increase of SOD activity a nd improvement of lipid profile plasma blood of W istar ra t for 4 months. This wa s experimental study with randomized posttest only control group design. The samples w ere 25 W istar rat, randomized into 5 groups: group K1 (nega tive control), group K2 (positive control, feed high fat diet), group P1(high

fat diet + ethanol extract dose 50 mg/ kgbw), group P2 (high fat diet + ethanol extract dose

100 mg/ kgbw), and group P3 (high fa t diet + ethanol extract dose 150 mg/ kgbw ). This

paper also describes the determination of the antioxidant a ctivity a nd identification of the active compounds using gas chromatography-ma ss spectrometry (GCM S) a nalysis.

From the results it is evident tha t ethanol extra cts in va ried doses decrease the level of lipidsof tota l cholesterol, triglyceride, and LDL cholesterol, but do not affect the level of HDL cholesterol. They a lso increase SOD a ctivity in blood plasm of W istar rat. Thus the extract potential to prevent atherosclerosis disease. The etha nol extract w hich a ctive as an antioxidant w ith IC50= 280 ppm with contains thirteen compounds, i.e., one fla vonoid

compound, eight fa tty acid and fa tty acid ester compounds, and four unknow n compounds according to W iley 229 Library.

Antioxidant……….……….Wistar Rat Sukadana et al., 20 15

INTRODUCTION

Screening of bioactive com pounds from plant can be conduct ed w it h the phyt opharm acologic approaches and phyt ochem ical screening approaches. One of t he phyt opharm acologic approaches relies on et nobot any, i.e., screening bioact ive com pound of plant based on it s use as t radit ional m edicine by cert ain societ y (Farnsw ort h, 1996). Tradit ional m edicine represent s one of Indonesia’ s cultural asset s and has been em pirically proven from generat ion t o generat ion (Kardinan and Taryono, 2003).

A plant which can be exploit ed as t raditional medicine is gayam (Inocarpus fagiferus Fosb). The seed of this plant is applicable t o heal t he at herosclerosis and ischem ic heart disease and pot ential as antioxidant agent based on em pirical st udies in Fiji Island (Sotheesw aran and Sharif, 1994; Segat ri, 1995). This is possibly because of t he chem ical com pounds cont ent of the gayam seed in Fiji, especially those of secondary m et abolic compounds such as phyt ost erol, diacylgliserol,



-t ocopherol, linolenic acid, and linoleic acid as t he m ajor com pounds (Sotheesw aran and Sharif, 1994). Nevert heless t here is no research explaining t he correlat ion bet ween t he com pounds in the seed with the t radit ional healing, although m any people have proven t he effect and benefit of gayam seed, let alone scient ific evidences of gayam seed in Bali. Only t he st at em ent of Agest ia and Sugrani (2009) which explains that t here is correlat ion bet w een t he st ructure of fat t y acid or flavonoid and t heir possibilit y as antioxidant activit y. Cert ain fat ty acids like PUFA (polyunsat urat ed fatt y Acids) have ant ioxidant activit y as exogen ant ioxidant w hich can st imulate endogen antioxidant such as SOD (Superoxide Dismust ase).

SOD is a cat alysis react ion t o convert anion superoxide (O2

-) t o hydrogen peroxide and oxygen t hat are not react ive. Expression and act ivit y of SOD effect and response at vascular cells w it h oxidative st ress (Chauhan et al., 2003; Faraci, 2003; Gunnet t et al., 2003).Therefore, act ivit y of SOD can be used as a biomarker of at heroscl erosis disease (Didion et al., 2002). The ot her SOD activit y, t he level of lipid profile such as tot al cholest erol, t riglyceride, HDL-cholest erol, and LDL-cholest erol in serum or plasm a can also is used t o det erm ine dam ages of blood vessel like in atherosclerosis (Budiana, 2008). So that bot h SOD and levels of lipid profile a biomarkers t o invest igat e t he at herosclerosis disease, and the ext ract of gayam seed is exp ect ed t o cont ain antioxidant compounds (Sukadana, 2012).

Therefore it is crucial to invest igat e t he chem ical cont ent s of t he gayam seed and their bio-act ivit y as an antioxidant agent also to invest igat e w het her et hanol ext rbio-act of gayam seed can prevent at herosclerosis t hrough increases SOD activit y and improvem ent of lipid profile. In this paper t he ext ract ion process of t he identificat ion antioxidant compounds from the seed and the det erm inat ion of secondary met abolit e. The capacit y ant ioxidant t ow ard DPPH and it s effect of at herosclerosis t hrough measurem ent of SOD and level of lipid profileare described.

M ATERIAL AND M ETHODS

M at erials used in this research are: gayam (Inocarpus fagiferus Fosb) seed obtained from Tabanan Bali and t axonomically ident ified by LIPI’s Kebun Raya " Eka Karya" Bali. Chemicals used are et hanol (p.a and t echnical), aquadest , DPPH (diphenylphicrylhydrazil), and phyt ochemical reagent .

Antioxidant……….………. Wistar Rat Sukadana et al., 2 0 15

Anim al used is Wist ar rat and with ethical clearance No. 0144/ KE-PH/ IX/ 2013 dat ed: 4 Sept em ber 2013. Chem icals t hat used t o analysis lipid profile were provided by UPT. Balai Laboratorium Kesehat an Provinsi Bali. Ot her mat erial w ere What m an No.1, ket am ine and xylazine for anest hesia, blood plasm , st andard feed BR1 CP511B, high fat diet feed consist of m ixt ure BR1 CP511B and ot her mat erials with high cholest erol.

Equipments

Equipm ent s used include a set of glass w ares, ext ract or, analyt ical balance, blender, knife, rot ary vacuum evaporat or, desicat ors, and t est t ubes, t est ing dishes, volumet ric pipet t es, rat cage, syringe, m icro haematocrit tubes (Cat . 7493 21), EDTA Eppendorf t ubes, UV-vis spect rophotomet er, and gas chrom atographs-mass spect romet ers (GCM S).

Procedure

Extraction of compounds from gayam seed (Inocarpus fagiferus Fosb).

About 5.85 kg dried powder of gayam (Inocarpus fagiferus Fosb) seed w as macerat ed using 21.5 L et hanol 96% (Et OH). M acerat ion process w as conduct ed 5 tim es at 24 hours each. The et hanol ext ract w as evaporat ed using rot ary vacuum evaporat or t o obt ain a concent rat ed Et OH ext ract . It w as t est ed for antioxidant activit y with t he DPPH t est and it w as com pared with Vit amin E. Furthermore t he concent rat ed Et OH ext ract w as analyzed using Gas Chrom atographs-M ass Spect rom et ers (GCM S) t o det erm ine it s com ponent s. These Et OH ext ract w as t hen was applied t o anim al model on W ist ar rat 20 g/ rat / days t o prove t he pot ency of concent rat ed Et OH ext ract of gayam seed t o prevent atherosclerosis t hrough increase of SOD act ivit y and improvement of lipid profile using t o randomized post t est only cont rol group design with 5 groups.

Group K1: negat ive cont rol (rat group with feed st andard)

Group K2: posit ive cont rol (rat group with feed high fat diet )

Group P1: rat group with feed high fat diet + et hanol ext ract dose 50 m g/ kg bw

Group P2: rat group with feed high fat diet + et hanol ext ract dose 100 mg/ kg bw

Group P3: rat group with feed high fat diet + et hanol ext ract dose 150 mg/ kg bw

The experiment w as conduct ed for four mont hs t hen the blood of each rat groups w ere analyzed for t heir SOD and lipid profile as t ot al cholest erol, t riglyceride, HDL cholest erol, and LDL cholest erol. These dat a w ere analyzed using one w ay Anova (SPSS version 19.0 for Window s).

RESULTS AND DISCUSSION

Antioxidant activity of ethanol extract from gayam seed (Inocarpus fagiferus Fosb).

The result of macerat ion of about 5.85 kg dried powder of gayam (Inocarpus fagiferus Fosb) seed using 21.5 L ethanol was about 87.272 g brow n concent rat ed et hanol ext ract .

Antioxidant activit y t est s using DPPH show ed that t here w as t he et hanol ext ract w as more act ive t han the vit am in E (as ant ioxidant st andard (see Figure 1). The value of IC50which w as

less t han IC50 =1000 m eans that the ext ract is potent active as antioxidant .

[image:5.612.117.519.91.222.2]

Antioxidant……….………. Wistar Rat Sukadana et al., 2 0 15

Figure 1. Antioxidant activity of ethanol extract.

Lipid Profile and SOD Activity of W istar Rat Plasma

In t he lipid profile of t he rat plasma for 4t h m ont h experim ent shown in Figure 2.The lipid profile and the one w ay Anova analysis show t hat all of the variable have normal dist ribut ion and homogenit y (p>0.05) except SOD act ivit y (p<0.05). The highest level of t ot al cholest erol in shown by rat groups w ith high fat diet (K2) w it h significant difference

(p<0.05) if compared w ith ot her rat groups. M ean of t he t ot al cholest erol level for K1i s

50.00



4.42 m g/ dl, K2is 82.60



5.73 mg/ dl, P1is 66.20



7.29 m g/ dl, P2is 70.80



8.11 m g/ dl,

and P3 is 70.40



6.87 m g/ dl.

Figure 2. Lipid profile of W istar rat blood plasma.

Significant decrease of t otal cholest erol level occured aft er t he rat groups w ere given high fat diet and et hanol ext ract of gayam seed of 50 mg/ kg bw (P1, p<0.05). higher doses like

in groups P2 and P3do not give significant difference in levels of t ot al cholest erol com pared

t o K2 (p<0.05) but it s level increase compared t o P1, while t he level of t ot al cholest erol P2is

not significantly different com pared w it h P3 (p>0.05). This is because by t he compound

com ponent s in gayam seed like sat urat ed fat t y acid in higher dose w ill affect t he level of t ot al cholest erol.The level of t riglyceride decreases very significant ly (p<0.05) aft er rat w it h high fat diet w ere given et hanol ext ract dose of 50 m g/ kg bw (P1, mean: 101.2



7.623

m g/ dl)) compared t o K2 (m ean. 154,0



37.236 m g/ dl), while higher ext ract doses like in

t reat m ent group P2 and P3do not increase t he level of t riglyceride significantlly (p>0.05), but

if t he t w o groups w ere com pared (P2; mean. 128.4



32.447 m g.dl and P3; m ean. 113.2



33.094 m g/ dl), they are not significantlly different (p>0.05).

J . Biol. Chem. Resear ch 3 1 Vol. 3 2 , ( 1) : 2 8 - 3 5 (2 0 15 )

25

280

0 100 200 300

Vit am in E Et hanol ext ract IC50

(ppm)

0 50 100 150 200

K1 K2 P1 P2 P3

50

82.6

66.2 70.8 70.4

47.2 154

101.2

128.4

113.2

30.18 39 33.54 35.7 32.94

9.46 12.8 12.42 9.42 14.82

Concentration (mg/ dl)

[image:5.612.151.462.371.507.2]

Antioxidant……….……….Wistar Rat Sukadana et al., 2 0 15

Theorit ically, level of HDL cholest erol should increases if ant ioxidant agent w as given in higher dose, but t he result of these experiment show that t he level of HDL cholest erol t ends t o unchange (t heir m eans are not significantly different, p>0.05). The m ean of HDL cholest erol for all of rat groups, K1, K2, P1, P2, and P3are 30.18



2.57 mg/ dl, 39.00



5.41

m g/ dl, 33.54



2.39 m g/ dl, 35.70



3.84 mg/ dl and 32.94



4.37 mg/ dl respect ively. The result of mult iple comparisons t est show t hat K2 vs K1 (p<0.05), P1 vs K2 (p<0.05), P2 vs K2

(p>0.05), P3 vs K2 (p<0.05), P2 vs P1 (p>0.05), P3 vs P1 (p>0.05), and P3 vs P2 (p>0.05). M ixture

of m any compounds t hat w ere cont ained in t he et hanol ext ract of gayam seed possibly causes t hese cont radict ion.

LDL cholest erol is a bad cholest erol. Figure 2. show s t hat m ean level of LDL cholest erol in all rat groups, K1, K2, P1, P2, and P3are 9.46



4.593 m g/ dl, 12.68



7.203 m g/ dl, 9.42



6.062

m g/ dl, and 14.82



7.626 mg/ dl respect ively. It m eans t hat all of them are not significantly different (p>0.05).

The result of analysis of SOD activit y show s that concent rat ed et hanol ext ract in varied doses can increase act ivit y of ant ioxidant endogen SOD. This m eans t he com pounds in et hanol ext ract can be expect ed t o prevent at herosclerosis disease. In the normal rat (K1)

t he percent age of rat e inhibition (48.746



5.303%) higher t han the percent age of rat e inhibition of high fat diet rat group (K2), 17.254



4.373 % (p<0.05). In hypercholest erolem ia

rat group (K2) plak at herosclerosis occurs t hat decreasest he SOD act ivit y. Futhermore, if t o

hypercholest erolem ia condition rat ext ract w as given in varied doses t he compounds in the ext ract w ill increase t he endogen SOD act ivit y as show n in Figure 3.

Figure 3. SOD activity of W istar rat blood plasma.

In t reat m ent group P1, where rat w it h high fat diet a given et hanol ext ract at 50 m g/ kg bw ,

t here is increase in percentage of rat e inhibition significant lly (p<0.05) compared t o K2, and

w hen rat s are given higher doses like in P2, it drast ically increase (p<0.05). Nevert heless, if

rat s w ere given et hanol ext ract w ith dose of 150 m g/ kg bw (P3) higher t han that of P2,

percent age of rat e inhibition is not significant ly different (p>0.05), possibilit y because the com pounds in gayam seed do not act as ant ioxidant s.

J . Biol. Chem. Resear ch 3 2 Vol. 3 2 , ( 1) : 2 8 - 3 5 (2 0 15 )

48.746

17.254

37.085

84.339 86.881

0 10 20 30 40 50 60 70 80 90 100

K1 K2 P1 P2 P3

Rate Inhibition

Antioxidant……….……….Wistar Rat Sukadana et al., 20 15

Identification of ethanol extract

[image:7.612.117.492.236.384.2]

The result of phytochemical t est indicat es t hat the et hanol ext ract cont ains t rit erpenoids, st eroids, flavonoids and phenolat e compounds. Chromatograph of GCM S analysis indicat es t hat the et hanol ext ract cont ains t hirt een component s (Figure 4). The WILEY229.LIB dat a base approach suggests t hat t he ext ract contains one flavonoid compound (homopt erocarpine, Rt . 28.042), eight fatt y acids and fat t y acid est er com pounds (palm itic acid, Rt. 23.157; et hyl palm itic, Rt . 23.488; linoleic acid, Rt. 24.831; et hyl linoleic, Rt . 25.120; et hyl oleic, Rt. 25.181; et hyl st earic, Rt . 25.426; et hyl eicosanoic, Rt. 27.212;and et hyl docosanoic, Rt. 28.877) and four unknown com pounds with ret ent ion t im es of23.319, 24.892, 25.317, and 30.647 m inutes (see Figure 4).

Figure 4. Chromatograph of ethanol extract.

The st ruct ures of all compounds are shown in Figure 5, w hile t he unknown compounds are not shown.

Figure 5.The structures of the compounds in the ethanol extract.

J . Biol. Chem. Resear ch 3 3 Vol. 3 2 , ( 1) : 2 8 - 3 5 (2 0 15 )

O

OH

O

O CH2CH3

Palmitic acid E thyl palm itic

O

OH

O

O CH2CH3

O

O CH2CH3

Linoleic acid Ethyl linoleic

Ethyl oleic

O

O CH2CH3

Ethyl stearic

O

O CH2CH3

Ethyl eicosanoic

O

O CH2CH3

E thyl docosanoic

O

O

O

O

[image:7.612.109.506.432.663.2]

Antioxidant……….………. Wistar Rat Sukadana et al., 2 0 15

CONCLUSIONS

Conclusions of t hese research es are:

1. The et hanol ext ract s in varied doses decrease t he levels of lipid profile i.e., tot al cholest erol, t riglyceride, and LDL cholest erol, except the HDL cholest erol.

2. Concent rat ed et hanol ext ract increases SOD act ivit y in blood plasma of Wist ar rat so it is pot ent ial to prevent atherosclerosis disease.

3. The et hanol ext ract is identified as ant ioxidant compounds with IC50 = 280 ppm . It

contains t hirt een component s; one flavonoids com pound (homopt erocarpine), eight fat t y acids and fat t y acid est er com pounds (palmitic acid, et hyl palmitic, linoleic acid, et hyl linoleic et hyl oleic, et hyl st earic, et hyleicosanoic and et hyl docosanoic) and four unknown com pounds.

Further research

In vivo experiment of et hanol ext ract t o obt ain it s pot ential as anti atherosclerosis w it h relevant variables including ICAM -1, VCAM -1, SOD-2, SOD-3, M DA, et c.

ACKNOW LEDGEM ENTS

We w ould like t o express our grat it ude t o Dikt ithat gave fund through Hibah Dokt or and Udayana Universit y t hat facilit at es our research.

REFERENCES

Agest ia, R. and Dan Sugrani, A., 2009. M akalah Kimia Organik Bahan Alam Flavonoid (Quercet in), Program S2 Kimia, Universit as Hasanuddin, Sumat ra.

Budiana, 2008, M emahami Dampak Kolest erol, Available from : URL:

ht tp:/ / w ww .dew anfam ily mult iply.com, accest 12 December 2011.

Chauhan, S.D., Seggara, G., Vo, P.A., M acallist er, R.J., Hobbs, A.J. and Ahluw alia, A. 2003. Prot ect ion against Lipopolysaccharide-induced Endot helial Dysfunction in Resist ance and Conduit Vasculature of iNOS Knockout M ice. FASEB J, 17: 773–5.

Didion, S.P., Ryan, M .J., Didion, L.A., Fegan, P.E., Sigmund, C.D. and Faraci, F.M . 2002. Increased Superoxide and Vascular Dysfunct ion in Cu Zn SOD-deficient M ice. Circ

Res, 91: 938 –44.

Faraci, F.M . 2003. Hyperhomocyst einem ia: a M illion W ays to Lose Cont rol. Art erioscler

Thromb Vasc Biol, 23: 371-3.

Farnsw ort h, N.R., 1996. Biological and Phyt ochemical Screening of Plant, J. Pharm. Science,

55, 3, 225-276.

Gunnet t , C.A., Heist ad, D.D. and Faraci, F.M . 2003. Gene-t arget ed M ice Reveal a Crit ical Role for Inducible Nit ric Oxide Synt hase in Vascular Dysfunct ion during Diabet es.

St roke.34: 2970 –4.

Kardinan, A. and danTaryono, 2003. Tanam an Obat Penggem pur Kanker, PT Agro M edia Pust aka, Jakart a.

Segat ri, P., 1995.Taru Prem ana Khasiat Tanam-t anamanuntuk Obat Tradisional, Upadasast ra, Denpasar: 20.

Antioxidant……….……….Wistar Rat Sukadana et al., 2 0 15

Sotheesw aran, S. and Sharif, M .R. 1994.Lipids from t he Seeds of Seven Fijian Plant Species.

Food Chemist ry, 49: 11-3.

Sukadana, I.M . 2012.Phyt ochemical and Antioxidant Activit y Test of Et hanol Ext ract Gayam Seed (Inocarpus fagiferus Fosb).Unpublish:1-5.

Corresponding author: Dr. I. M ade Sukadana, Chemist ry Depart m ent , Facult y of M at hem at ics and Nat ural Sciences, Udayana Universit y, Indonesia.

Email:imsukadana@yahoo.com