and a Silane Reduced Analogue

$QWLR[LGDQW9DOXHDQG$QWLSUROLIHUDWLYH(IÀFDF\RI0LWUDJ\QLQH

Asian Pac J Cancer Prev, 15 (14), 5659-5665

Introduction

Mitragynine dominated M.speciosa Korth (Ketum) leaves has widely recognised in Southeast Asia as an effective analgesic medicinal agents owing to its opioid- like effects (Matsumoto et al., 2004; Adkin et al., 2011).

Mitragynine was used as as an alternative to opium to combat craving and opioid withdrawal symptons (Matsumoto et al., 2004) and for pain management (Nelson et al. 2010; Vicknasingam et al., 2010).

+\GUR[\O ï2+ DQG SHUR[\O UDGLFDOV 522ï have been associated with carcinogenesis, coronary heart disease, and many other health issues related to advanced age (Gupta et al., 2009; Fu-Qiang et al., 2013;

Jing-A et al, 2013; Simay et al., 2013; Wesen et al., 2013; Joanna and Ewa, 2014). Therefore, exogeneous supply of antioxidants is essential as a complimentary and replacement of depleted endogeneous antioxidants due to aging (Sirinya et al., 2014). The antioxidant might be used in prevention of aging-associated diseases, LQFOXGLQJFDQFHUE\VXSSUHVVLQJWKHIRUPDWLRQRI526

and scavenging free radicals (Fu-Qing et al., 2013; Jing-A

1Centre for Drug Research, Universiti Sains Malaysia, Penang, ²School of Medicine, International Medical University, Kuala Lumpur, Malaysia )RUFRUUHVSRQGHQFHVPDKVXÀ#XVPP\JRKWHLNEHQJ#\DKRRFRP

Abstract

Background: To investigate the antioxidant value and anticancer functions of mitragynine (MTG) and its silane-reduced analogues (SRM) in vitro. Materials and Methods: MTG and SRM was analyzed for their reducing power ability, ABTS radical inhibition and 1,1-diphenyl-2-picryl hydrazylfree radicals scavenging activities.

)XUWKHUPRUHWKHDQWLSUROLIHUDWLRQHIÀFDF\ZDVHYDOXDWHGXVLQJ077DVVD\RQ.DQG+&7FDQFHUFHOOOLQHV

YHUVXV1,+7DQG&&'&RQRUPDOFHOOOLQHVUHVSHFWLYHO\Results: SRM and MTG demonstrate moderate DQWLR[LGDQWYDOXHZLWK$%76DVVD\7UROR[HTXLYDOHQWDQWLR[LGDQWFDSDFLW\7($&“PPROWUROR[PPRO

DQG“PPROWUROR[PPROUHVSHFWLYHO\DQG'33+,&_ “PJP/DQG,&_ “PJP/

UHVSHFWLYHO\%RWK07*DQG650GHPRQVWUDWHHTXDOSRWHQF\,&_ “DQG,&_ “UHVSHFWLYHO\ WRZDUGV.FHOOOLQHVFRPSDUDEOHWRFRQWUROEHWXOLQLFDFLG%$,&_“%RWKFRPSRXQGVVKRZHG

FRQFHQWUDWLRQGHSHQGHQWF\WRWRWR[LFLW\HIIHFWVDQGH[HUWSURIRXQGDQWLSUROLIHUDWLYHHIÀFDF\DWFRQFHQWUDWLRQ!

ƫ0WRZDUGV+&7DQG.FDQFHUFHOOOLQHVFRPSDUDEOHWRWKRVHRI%$DQG)8)OXRURXUDFLO

)XUWKHUPRUHERWK07*DQG650H[KLELWKLJKVHOHFWLYLW\WRZDUGV+&7FHOOOLQHVZLWKVHOHFWLYHLQGH[HVRI

DQGUHVSHFWLYHO\FRPSDUHGWR)86, &RQFOXVLRQV:7KHVHÀQGLQJVUHYHDOHGWKDWWKHPHGLFLQDO

and nutitional values of mitragynine obtained from ketum leaves that growth in tropical forest of Southeast Asia and its analogues does not limited to analgesic properties but could be promising antioxidant and anticancer or chemopreventive compounds.

.H\ZRUGV 0LWUDJ\QLQHUHODWHGDQDORJXHVDQWLR[LGDQWYDOXHDQWLSUROLIHUDWLYHHIÀFDF\

5(6($5&+$57,&/(

$QWLR[LGDQWYDOXHDQG$QWLSUROLIHUDWLYH(IÀFDF\RI0LWUDJ\QLQH

and a Silane Reduced Analogue

Teik Beng Goh

.RK5KXQ<LDQ

, Mohd Nizam Mordi

6KDULI0DKVXÀ0DQVRU

et al., 2013; Simay et al., 2013; Joanna and Ewa, 2014).

Antioxidant activity is involved in cancer prevention at the initiation stage (chemopreventive) while antiproliferative activity is targeting cancer cells at the promotion and progression stage (Ahmad and Normaliza, 2013; Sami et al., 2013; Nejib et al., 2013; Eduardo et al., 2013;

papavadee et al., 2013; Puthuparampil et al., 2013; Tarika et al., 2014). Antioxidant activity had been reported for M.

Speciosa extracts (Suhanya et al., 2009) but no research on antioxidant activity of the pure mitragyine had been published.

The natural plant-based polyphenolic antioxidants VXFKDVÁDYDQRLGVÁDYDQRODQWKRF\DQLQVUDQGURVPDULQLF

acid and etcare very common (Supakit et al., 2013; Sami et al.,2013; Nejib et al., 2013; papavadee et al., 2013;

Eduardo et al., 2013; Sadegh et al., 2013). Herraiz et al.

(2003 and 2004) has reported the discovery of non-phenolic DQWLR[LGDQWEDVHGRQơFDUEROLQHDQGLQGROLFVFDIIROGDQG

elaborate in detail the anti-oxidative mechanism. These results suggest that indole alkaloid related analogues in general may conform to a family of radical scavengers and antioxidants other than the polyphenolic group that

$VLDQ3DFLÀF-RXUQDORI&DQFHU3UHYHQWLRQ9RO



might act as potential protectants and free radical sinks at a physiological pH. This has prompted us to search and evaluate pure compounds other than polyphenolic which might have antioxidant value.

Mitragynine was found to be hepatotoxic and this necessitate further evaluation of its toxicology and pharmacological activities such as cytotoxicity.

(Saidin, 2008; Saidin et al, 2008) had discovered that mitragynineexhibited dose-dependent inhibition of cell SUROLIHUDWLRQLQWKHFHOOOLQHV+HS*+(.0&/

F+RODQG6+6<<DWFRQFHQWUDWLRQ!ƫJPOZLWK

VXEVWDQWLDOFHOOGHDWKDWƫJPO

MTT assay was performed on mitragynine analogues in vitro to screen for their potential cytotoxic effect as anticancer drugs that inhibit the proliferation and the JURZWKRIWKHFHOO&\WRVWDWLFOHDGLQJWRWKHSURJUDPPHG

cell death (apoptosis) (Zafer and ayse, 2013). Screening on anti-proliferation functionality of mitragynine related analogues on other cell lines is prudent since MTG demonstrate potent cytotoxicity activities against SH- SYS5 at high dose (Saidin, 2008).

Mitragynine could be promising alternatives for future pain management treatments (Matsumoto et al., 2004).

However the potential cytotoxicity of mitragynine and its derivatives is still not fully documented in literature.

Therefore, this prompted us to evaluate the cytotoxicity of mitragynine and its silane reduced derivative, which could be safer analogues due to its reduced indole double bond, on human cell lines in this study. Antoine et al.

(2008) reported that reduced indole bond (indoline) might help to improve anti-tumour activity. Hence, the other objective is to evaluate whether the reduction of indole GRXEOH ERQG LQ 07* WR LWV DQDORJXH LQGROLQH 650

contribute any further enhancement in cytotoxicity and anti- proliferation functionality to human cancer cell lines as compared to MTG..

Materials and Methods

Materials

MTG was obtained from ketum leaves using the SXEOLVKHGSXULÀFDWLRQSURWRFRO7HLN%HQJHWDO

650ZDVREWDLQHGXVLQJWKHSXEOLVKHGV\QWKHWLFPHWKRG

7HLN%HQJHWDO4XHUFHWLQ%+7WUROR[DQGDOOWKH

other reagents used were of analytical grade and purchased from Sigma-Aldrich.

DPPH radical-scavenging assay

'33+IUHHUDGLFDOVFDYHQJLQJDFWLYLW\RI07*650

TXHUFHWLQDQG%+7ZDVPHDVXUHGXVLQJWKHPHWKRGRI

&KDQJ+DR HW DO  6XSDNLW HW DO 6DPSOH

DQGVWDQGDUGƫ/DWYDULRXVFRQFHQWUDWLRQV

DQGPJP/ZHUHPL[ZLWK

P/RIP0'33+VROXWLRQ$IWHUDPLQLQFXEDWLRQ

DWURRPWHPSHUDWXUHƒ&LQWKHGDUNWKHUHGXFWLRQRI

the DPPH free radical was measured at 517 nm against 2 P/RI'33+VROXWLRQLQƫ/RIPHWKDQRODVFRQWURO

7KH UDGLFDOVFDYHQJLQJ DFWLYLW\ 56$ ZDV FDOFXODWHG

using the following formula:% RSA=[(A0- A1)÷A0] × 100 where A0 is the absorbance of the control; and A1 is the absorbance of samples after 30 min.

ABTS antioxidant assay

7KH7($&ZHUHGHWHUPLQHGE\WKH$%76DQWLR[LGDQW

DVVD\ 3LQFKXN HW DO  &KDQJ +DR HW DO 

$%76ZDVSURGXFHGE\UHDFWLQJP0$%76LQ+₂2

ZLWK  ƫ0 SRWDVVLXP SHUVXOSKDWH VWRUHG LQ GDUN DW

ƒ&IRUKEHIRUHXVH7KH$%76VROXWLRQZDV

diluted to an absorbance of 0.70±0.02 at 734 nm in water.

7KHQ  ƫ/ VWRFN VROXWLRQV RI 07* DQG 650 %+7

quercetin and Trolox at concentration 0.5-4 mM were DGGHGWRP/RI$%76VROXWLRQ7KHDEVRUEDQFHZDV

measured at 734 nm, 6 min after addition of Trolox and VDPSOHV7KH7($&YDOXHVRIWKHVDPSOHVZHUHFDOFXODWHG

by relating the decrease in absorbance to that of the Trolox solution on a molar basis.

Measurement of reducing capacity using the FRAP assay 7KHIHUULFUHGXFLQJDQWLR[LGDQWSRZHUDVVD\)5$3 ZDV HPSOR\HG ZLWK PLQRU PRGLÀFDWLRQ &KDQJ+DR HW

DO%ULHÁ\P/RIÀYHGLIIHUHQWFRQFHQWUDWLRQV

RI 07* 650 TXHUFHWLQ DQG %+7  

DQGPJP/LQPHWKDQROZDVPL[HGZLWK

 P/ RI 3%6 S+  DQG  P/ RI  SRWDVVLXP

hexacyanoferrate (lll).The solution was then incubated IRUPLQLQDƒ&ZDWHUEDWKEHIRUHP/RI

WULFKORURDFHWLFDFLG7&$ZDVDGGHG(DFKVROXWLRQZDV

centrifuged at 3000 rpm for 10 min. The upper layer of WKHVROXWLRQP/ZDVPL[HGZLWKGLVWLOOHGZDWHU

P/DQG)H&O₃P/DQGWKHDEVRUEDQFHZDV

measured at 700 nm.

Cell culture

1RUPDOFRORQÀEUREODVW&&'&RQRUPDOPRXVH

ÀEUREODVW 1,+7 HU\WKURLGOHXNDHPLF .  DQG

FRORQ FDUFLQRPD FHOO OLQHV +&7  ZHUH SXUFKDVHG

IURPWKH$PHULFDQ7\SH&XOWXUH&ROOHFWLRQ5RFNYLOOH

0'DQGZHUHFXOWXUHGLQ'XOEHFFR·VPRGLÀHG(DJOH·V

PHGLXP *LEFR,QYLWURJHQ VXSSOHPHQWHG ZLWK 

IRHWDOERYLQHVHUXPXQLWP/SHQLFLOOLQDQGƫJ

P/VWUHSWRP\FLQDWƒ&XQGHU&2₂.

MTT cell antiproliferation assay

07* 650 EHWXOLQLF DFLG %$ DQG ÁXRURXUDFLO

)8ZHUHGLVVROYHGLQGLPHWK\OVXOSKR[LGH'062WR

IRUPP0VWRFNVROXWLRQDQGZHUHNHSWDWƒ&3ULRUWR

the assay, the stock solutions were diluted in the complete culture medium to produce test solutions (range from 6.5 WRƫ06ROXWLRQVRI'062LQWKHFRPSOHWH

culture medium were used as the vehicle control.To HYDOXDWHZKHWKHUWKHDQWLSUROLIHUDWLYHDFWLRQZDVVSHFLÀF

to tumour cells, these pure compounds were also tested RQQRQWXPRXULJHQLFFHOOOLQHV1,+7DQG&&'&R

%ULHÁ\WKHFHOOVZHUHVHHGHGLQZHOOPLFURWLWUHSODWHV

DQGWUHDWHGZLWKYDULRXVFRQFHQWUDWLRQVRI07*650

)8DQG%$UDQJHIURPƫ07KHSODWHVZHUH

incubated for 24 hbefore the test samples and the vehicle FRQWUROZHUHDGGHGIROORZHGE\LQFXEDWLRQDWƒ&IRU

K6XEVHTXHQWO\ƫORIPJP/077$FURV2UJDQLFV

%HOJLXPZDVDGGHGWRHDFKZHOODQGLQFXEDWHGIRUDQRWKHU

4 h before the media were removed by aspiration.

The formazan product generated from the viable FHOOVZDVGLVVROYHGLQ'062DQGPHDVXUHGDWQP

$QWLR[LGDQW9DOXHDQG$QWLSUROLIHUDWLYH(IÀFDF\RI0LWUDJ\QLQH against the reference wavelength at 650 nm. The growth

inhibitory effect of the test samples was determined by comparing the optical density of the treated sample to the optical density of the control. The results were expressed DVWKHSHUFHQWDJHRIFHOOYLDELOLW\>2'VDPSOH2'FRQWURO

x 100%] (Zafer and Ayse, 2013). A dose-response curve was plotted, and the concentrations of the compounds UHTXLUHGWRLQKLELW,&₅₀) of the cells’ viability was either determined by interpolating the curve or calculated XVLQJ3URELW$QDO\VLV63669HUVLRQ&KLFDJR,/

USA). All the experiments were carried out in triplicate.

Morphological changes of the untreated, cancer and normal cells treated with 6.5, 12.5, 25, 50, 100 and 200 ƫ00,7DQG650IRUDQGKZHUHYLHZHGXQGHU

an inverted light microscope.

Results

Antioxidant activity

DPPH-scavenging activity: The DPPH radical VFDYHQJLQJ DFWLYLW\ IRU 07* DQG 650 LV VKRZQ LQ

Figure 1a. The order of inhibition was as follows:

TXHUFHWLQ!%+7!650!07*7KHDQWLR[LGDQWSRWHQFLHV

DV GHWHUPLQHG E\ WKH ,&₅₀ YDOXH ERWK 07* DQG 650

LQGLFDWHWKHLUJRRGDQWLR[LGDQWDFWLYLW\7KH,&₅₀ values RI 07* DQG 650 ZHUH “ DQG “ PJ

ml, respectively (Table 1). The antioxidant activity of 9$1 ZDV ORZHU UHODWLYH WR TXHUFHWLQ ,&₅₀=0.20±0.00 PJPO DQG %+7 ,&₅₀ “ PJPO 7DEOH 

KRZHYHU WKH DQWLR[LGDQW DFWLYLW\ RI 650 DQG 07*

PHDVXUHG E\ WKH ,&₅₀ value, was higher than those of HGLEOH IUXLWV ZKRVH ,&₅₀ values have been reported by HDUOLHUUHVHDUFKHUVRUDQJH,&₅₀ “PJPODQG

VWDUIUXLW,&₅₀=3.80±2.10) (Pin-Der-Duh, 1998).

Trolox equivalent antioxidant capacity 7KH 7($&

antioxidant capacity values of MTG (1.96±0.02 mmol

WUROR[PPRODQG650“PPROWUROR[PPRO were much higher than the value for Trolox, which was used as a reference, but lower than the values for quercetin “ PPRO WUROR[PPRO DQG %+7 “

PPROWUROR[PPRO)LJXUHE7KXVWKH$%76UDGLFDO

scavenging activities of all the tested samples were in the IROORZLQJRUGHUTXHUFHWLQ!%+7!650!07*

5HGXFLQJFDSDFLW\: Figure 1c compares the reducing FDSDELOLWLHVRI07*DQG650WRWKRVHRITXHUFHWLQDQG

%+77KHUHGXFLQJSRZHUVRIWKHVHPLWUDJ\QLQHUHODWHG

analogues and standard compounds had the following RUGHUTXHUFHWLQ!%+7!650!07*7KHIHUULFUHGXFLQJ

abilities of the compounds were weaker than those of TXHUFHWLQDQG%+7EXWWKHVHUHVXOWVLQGLFDWHGWKDWWKLV

compounds are mild electron and hydrogen donors and might be able to terminate radical chain reactions by converting free radicals to more stable products via donating a hydrogen atom or by electron transfer reactions from indole nucleus (Herraiz et al., 2003; Herraiz et al., 2004).

Antiproliferation activity

$QWLSUROLIHUDWLRQ HIÀFDF\: All of the mitragynine

Figure 1. ^a'33+6FDYHQJLQJ$FWLYLW\9HUVXV&RQFHQWUDWLRQRI07*650%+7DQG4XDUFHWLQ^b7($&

7UROR[(TXLYDOHQWDQWLR[LGDQWFDSDFLW\YDOXHP0RI07*650FRPSDUHGWR%+7DQG4XHUFHWLQ^cReducing 3RZHURI07*DQG650FRPSDUHGWR%+7DQG4XHUFHWLQ

A) B) C)

Figure 2. (IIHFWRI07*650RQ$.%+&7

 RQ +XPDQ 7XPRU &HOO /LQHV ,QKLELWLRQ  DQG & 1,+7 ' &&'  &R 1RUPDO &HOO /LQH

,QKLELWLRQDW&RQFHQWUDWLRQVRI

DQG—0%HWXOLQLFDFLGVHOHFWLYHWROHXNHPLD and 5-Fluorouracil (selective to colon cancer) was used as the UHIHUHQFH FRPSRXQG 7KH 07* DQG 650 VKRZ LQKLELWLRQ

against cells’ proliferation in dosedependent manner. Values are means of three experiments (n=3), error bars equal±SD

Table 1. ,&_ ,QKLELWRU\ &RQFHQWUDWLRQ DW  RI

SXUH 07* DQG 650 9HUVXV 6WDQGDUG$QWLR[LGDQW

%+7 TXHUFHWLQ (DFK 9DOXH LV ([SUHVVHG DV WKH

PHDQ“6'Q 

7\SHRI6WDQGDUG3XUH&RPSRXQG ,&₅₀PJPO ,&₅₀ (µM)

Quercetin 0.20±0.00 0.66

MTG 3.75±0.04 5.72

650 “ 

%+7 “ 

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5662

UHODWHGDQDORJXHV07*DQG650ZHUHDVVD\HGIRUDQWL

proliferative activities against 2 cancer cell lines (K 562 DQG+&7DQGQRUPDOFHOOOLQHV1,+7DQG&&'

&RXVLQJWKH077DVVD\$GLW\DHWDO$OYLQHW

al., 2014) after 48 hours of incubation in comparison with

%$DQG)8,WZDVLQWHUHVWLQJWRQRWHWKDWERWK07*

DQG650H[KLELWHTXLYDOHQWDQGSRWHQWF\WRWR[LFHIIHFWV

RQ WKH /HXNDHPLD FDQFHU . ZLWK ,&₅₀=(25.20±1.53 ƫ0DQG,&₅₀ “ƫ0UHVSHFWLYHO\FRPSDUHG

WR %$ ,&₅₀ “ ƫ0 7DEOH  +RZHYHU WKH

selective indexes is only 1.42 and 1.26 for MTG and 650 UHVSHFWLYHO\ FRPSDUHG WR %$ 6,  ZKLFK LV

two time higher.

%RWK07*DQG650GLVSOD\HGWKHPRGHUDWHSRWHQF\

ZLWK,&₅₀ “ƫPDQG“ƫPUHVSHFWLYHO\

DJDLQVW+&7FHOOOLQHVFRPSDUHGWR)8“ (Table 2). However, from the dose concentration curve

)LJXUHDWFRQFHQWUDWLRQDERYHƫ007*DQG650

demonstrate comparable percentage of inhibition (>80%) RUHIÀFDF\FRPSDUHGWR%$DQG)8,QFRQFOXVLRQ07*

DQG650JHQHUDOO\H[KLELWHGSRWHQWF\WRWR[LFHIIHFWDQG

anti-tumour properties toward the colon cancer cell lines +&7DVZHOODVWKHOHXNDHPLDFDQFHUFHOOOLQH.DW

KLJKGRVH)LJXUH,QWKLVVWXG\07*UHODWHGDQDORJXHV

selectively exhibited more potent cytotoxic activity toward WKH.OHXNLPLDFDQFHUFHOOOLQHVWKDQ+&7FRORQ

cancer cell lines. The leukaemia cell lines were more susceptible and sensitive than colon cell lines toward these 07*DQDORJXHV7KH6,GDWDOLVWHGLQ7DEOHLQGLFDWHERWK

07*DQG650H[KLELWHGKLJKVHOHFWLYLW\WRZDUG+&7

ZLWKUHVSHFWWR&&'&RZLWKVHOHFWLYHLQGH[HVRI

and 2.93 respectively compared to standard anticancer drugs for colon carcinoma, 5-FU, which had selective indexes of 0.60.

7KHLQYLWURDQWLFDQFHUDFWLYLW\RI07*DQG650DW

GLIIHUHQWFRQFHQWUDWLRQVDJDLQVW.+&71,+7

DQG &&'&R DIWHU  K RI WUHDWPHQW ZDV SORWWHG

respectively to obtain a dose response curve as shown LQ)LJXUH,WZDVREVHUYHGWKDWLQFRPSDULVRQWRWKH

SRVLWLYHFRQWURO%$DQG)8ERWKFRPSRXQGVFDXVHG

VLJQLÀFDQWSGRVHGHSHQGHQWFHOOGHDWKWRDVLPLODU

H[WHQW%RWK07*DQG650ZKLFKUHGXFHGFHOOYLDELOLW\

WREHORZRIWKHFRQWUROYDOXHDWƫ0)LJXUH

The present study clearly demonstrates the cytotoxic effect RI07*DQG650WRZDUGV+&7DQG.FHOOVDWKLJK

GRVH!ƫ07KHYLDELOLW\RIFHOOVDVPHDVXUHGE\

WKH077DVVD\ZDVVLJQLÀFDQWO\GHFUHDVHGE\LQFXEDWLRQ

with increasing concentrations of MTG related analogues )LJXUH,QFRQFOXVLRQ07*DQG650H[KLELWHGVLPLODU

HIÀFDF\FRPSDUHGWRWKHSRVLWLYHFRQWUROV%$DQG)8

at high dosage (> 100 um).

&HOOV PRUSKRORJ\ DQG DSRSWRVLV: As shown in the photograph in Figure 3, all of the compounds displayed early prominent biochemical hallmarks of programmed cell death (apoptosis): signs of nucleus shrinkage and compaction, cytoplasmic condensation, blebbing of cells, Table 2. &\WRWR[LFLW\$FWLYLW\ 3UHVHQWHG DV 0HGLDQ

,QKLELWRU\&RQFHQWUDWLRQ,&_LQ—PDQGVHOHFWLYLW\

LQGH[6, ,&_1RUPDO&HOO/LQHV,&_&DQFHURXVFHOO

/LQHV RI 0,7 DQG 650 RQ &DQFHU &HOO /LQHV DQG

1RUPDO &HOO DIWHU  K RI 7UHDWPHQW 5HVXOWV DUH

6KRZQDV$YHUDJH“6'Q 

 ,&₅₀—06,,&₅₀QRUPDOFHOOOLQHV,&₅₀ cancerous cell lines)

 &HOO/LQHV

&RPSRXQGV +&7 &&'&R . 1,+7

MTG 47.10±3.47, 148.00±4.93 25.20±1.53 35.80±1.05

3.14 1.42

650 “ “ “ “

2.93 1.26

%$   “ “

3.82

5-FU 14.00±0.86 7.32±0.63 - -

0.52

*%HWXOLQLF$FLG6HOHFWLYHWR/HXNHPLDDQG)OXRURUDFLO6HOHFWLYHWR&RORQ

cancer) was used as the reference compound. Values are express as mean±standard GHYLDWLRQRIWKUHHLQGHSHQGHQWH[SHULPHQWV67'1,+7DQG&&'&RZDV

XVHGDVQRUPDOFHOOIRUFRPSDULVRQ6HOHFWLYLW\LQGH[6,RI0,7DQG650RQ

FDQFHUFHOOOLQHVDERYHZDVFDOFXODWHGEDVHGRQ,&₅₀RI1,+7,&RI&&'

&RDQG,&₅₀RI6DPSOHVZLWK6,YDOXHJUHDWHUWKDQZHUHFRQVLGHUHGWRKDYH

high selectivity

)LJXUH+XPDQ(HU\WKURP\HOREODVWRLG/HXNHPLD.1,+7FRORQFDQFHUFDUFLQRPD+&7+XPDQ

QRUPDOFRORQLFÀEUREODVWFHOOOLQH&&'&RFHOOLPDJHVZHUHWDNHQXQGHUDQLQYHUWHGSKDVHFRQWUDVWPLFURVFRSHDWð

PDJQLÀFDWLRQZLWKDGLJLWDOFDPHUDDWKDIWHUWUHDWPHQWZLWKWKHVDPSOHVUHVSHFWLYHO\$'LQLQGLFDWHVHIIHFWVRIWZR

FRPSRXQGV07*DQG650RQ.1,+7+&7DQG&&'&RFHOOOLQHVUHVSHFWLYHO\FRPSDUHGWRFRQWURO$DQGVWDQGDUG

anti-cancer drugs, betulinic acid [(1D) and (2D)] and 5-FU [(3D) and (4D)]. Photomicrograph (1A), (2A), (3A) and (4A) depicts the .1,+7+&7DQG&&'&RFHOOVIURPWKHFRQWUROJURXSIRUPHGDFRPSDFWFRQÁXHQWOD\HU$3KRWRPLFURJUDSK

GHSLFWVWKHF\WRWR[LFHIIHFWRIWKHFRPSRXQG07*%,WFDQEHFOHDUO\VHHQWKDWWKHSRSXODWLRQRIFHOOVUHGXFHGPRGHUDWHO\

DIWHUKRIWUHDWPHQW3KRWRGHSLFWVWKHWUHDWPHQWZLWK650GHPRQVWUDWHGVLJQLÀFDQWF\WRWR[LFHIIHFWRQ.FHOOSUROLIHUDWLRQ

&7UHDWPHQWZLWKEHWXOLQLFDFLGVKRZHGPDUNHGLQKLELWLRQLQFHOOSUROLIHUDWLRQ'7KHSLFWXUH'UHYHDOHGWKDWWKH

population of the cells is reduced drastically with 5-FU (3-4D)

$QWLR[LGDQW9DOXHDQG$QWLSUROLIHUDWLYH(IÀFDF\RI0LWUDJ\QLQH apoptotic cell debris separated from mother cells and the

IRUPDWLRQRIYDFXROHVDXWRSKDJ\%RODQGHWDO

7KHUHIRUH07*DQG650DUHDSRSWRVLVLQGXFLQJDJHQWV

with differing potency against colon and leukaemia cancer cell lines.

'LVFXVVLRQ

The DPPH assay is a reliable method to to determine the radical-scavenging activity of antioxidant, which are known to be a major factor in the biological damage caused by oxidative stress (Supakit et al., 2013; Sami et al., 2013; Vazhapilly et al., 2013). The DPPH assay LVEDVHGRQWKHJUDGXDOTXHQFKLQJRIWKHVWDEOH'33+‡

(2,2-diphenyl-1-picrylhydrazyl) free radical by the proton or electron donated from the antioxidant.The radical scavenging activity (Yang et al., 2014) of mitragynine related analogues could be partly attributed to its hydrogen- donating ability (Herraiz et al., 2003; Herraiz et al., 2004), whilst its ß-carboline nuclei are free radical inhibitors that react promptly with DPPH free radicals (Herraiz et al., 2003; Herraiz et al., 2004).

7KHDQWLR[LGDQWFDSDFLW\H[SUHVVHGDVP07($&LV

a simple and reliable measure of the antioxidant activity (Kriangsak et al., 2006; Herraiz et al., 2003; Herraiz et al., 2004). Moreover, the antioxidant activity measured LQWHUPVRIP07($&7UROR[LVPRUHPHDQLQJIXODQG

GHVFULSWLYHWKDQWKH,&₅₀ value (Kriangsak et al., 2006).

Mitragynine related analogues are good antioxidants and radical scavengers,as they exhibited greater reactivity than Trolox. This result was not unexpected because ơFDUEROLQHKDVEHHQUHSRUWHGWRSRVVHVVJUHDWFDSDFLW\

WRTXHQFKIUHHUDGLFDOV$%76‡+HUUDL]HWDO

Herraiz et al., 2004; Sami et al., 2013).

The effectiveness of the quenching is dependent on the molecular weight, number of aromatic rings and nature of the hydroxyl group substitution (Herraiz et al.,

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these compounds showed that all of the compounds with a indolic nuclear moiety exhibited antioxidant activity DJDLQVWWKH$%76‡DWDSK\VLRORJLFDOS+

The reducing power of pure compounds may be XVHG DV D VLJQLÀFDQW LQGLFDWRU WR SUHGLFW WKHLU SRWHQWLDO

antioxidant properties and reducing capability (Siddhuraju DQG%HFNHU9D]KDSLOO\HWDO9DULRXVVWXGLHV

have demonstrated the positive relationships between antioxidant activity and reducing power (George et al., 2012; Sami et al., 2013; Vazhapilly et al., 2013). The absorbance values expresses the reducing power of the sample studied.A stronger absorbance value indicates higher antioxidant activity. Antioxidants are reductants that inactivate oxidants via a redox reaction (Siddhuraju DQG %HFNHU ,Q WKLV DVVD\ WKH )H^IHUULF\DQLGH

complex is used as an indicator of electron-donating activity. The capability of these pure compounds to act as reducing agents caused the reduction of the Fe^ ferricyanide complex to its ferrous form, which was measured at 700 nm.

The result from a single antioxidant experiment must be cautiously interpreted because it merely provides a deductive suggestion and estimation regarding the

antioxidant properties. Therefore, a multiple assay approach is crucial to determine conclusively the antioxidant properties (Herraiz et al., 2003; Herraiz et DO7KH$%76DQG'33+PHWKRGVDUHWKHPRVW

popular spectrophotometric methods for determining the radical-scavenging activity. This study indicated that indole skeleton without phenolic group possess moderate antioxidant capacities and could play a crucial role in providing potential antioxidants and free radical scavengers. These results suggest that indoles, in general, may conform to a family of radical scavengers and antioxidants other than the polyphenolic group that might act as potential protectants and free radical sinks at a physiological pH.

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anti-proliferation at high dosage and merit further LQYHVWLJDWLRQRQRWKHUFHOOOLQHV7KLVUHVXOWYHULÀHGZLWK

what was reported by (Saidin, 2008) that mitragynine exhibit marked cytotoxicity at higher concentration.

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the indole double bond on MTG to indoline does not FRQWULEXWHWRDQ\VLJQLÀFDQWHQKDQFHPHQWLQWKHLQKLELWLRQ

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backbone for chemotherapy against colon cancer, was EHORZDJDLQVWWKH+&7FHOOOLQH6DVLSDZDQ

HW DO  5DPHVK HW DO  :LUDWFKDQHH HW DO

2010). Therefore, it was generally toxic toward cells.

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treatment compared with conventional drugs such as 5-FU since they are relatively safe for non-cancerous KXPDQFHOOVVXFKDV&&'&R2QHLPSRUWDQWFULWHULRQ

for a cancer therapeutic drug is that it must have minimal or no side-effects on normal cells in patients undergoing chemotherapy. Therefore, anticancer drugs should have KLJKVHOHFWLYHLQGH[6DVLSDZDQHWDO5DPHVKHW

DO:LUDWFKDQHHHWDO7KH6,YDOXHGHVFULEHV

the selectivity of the pure compound for cancerous cell lines relative to that for normal cells (Sasipawan et al.,

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2004; Wiratchanee et al., 2010).

The selectively induced apoptosis suggests that these mitragynine related analogues might be potent lead compounds for the development of future cancer therapeutics. Apoptosis is a better method for destroying damaged cells than necrotic cell death; thus, agents that can induce apoptosis are desirable for cancer treatment %RODQGHWDO6DGHJKHWDO1HMLEHWDO

Zafer and Ayse, 2013).

Mitragynine analogues had moderate antioxidant YDOXHV EDVHG RQ WKH '33+ DVVD\ $%76 DVVD\ DQG

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activities were much higher than those of the water-soluble vitamin E analogue Trolox and of edible fruits that are considered to have good antioxidant properties. The MTG DQG 650 H[KLELW SRWHQW DQWLWXPRXU SURSHUWLHV WRZDUG

the leukaemia cell line K 562 but showed only moderate F\WRWR[LF HIIHFW RQ +&7  FHOO OLQHV +RZHYHU ERWK

07* DQG 650 GHPRQVWUDWH PDUNHG DQWLSUROLIHUDWLYH

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in vitro experimental results, we can conclude that both 07*DQG650DUHFRPSDUDEOHLQSRWHQF\DQGHIÀFDF\

towards K 562 cell lines than existing standard drug for OHXNHPLDFDQFHU %$ +RZHYHU %$ DUH PRUH VHOHFWLYH

WKDQ 07* DQG 650 %RWK 07* 6,  DQG 650

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analogues could be a safer option for cancer treatment.

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leukaemia cell lines were more susceptible than colon cell lines toward these mitragynine analogues.

Mitragynine and its analogue exhibit potent and selective cytotoxic effect via apoptotic pathway, and KLJKDQWLSUROLIHUDWLRQHIÀFDF\DWKLJKGRVH!XP WRZDUGV .  DQG +&7  FDQFHU FHOO OLQHV 7KH

results of this study also showed that mitrgynine related analogues exhibited moderate antioxidant and free radical scavenging activities. The hydrogen donating ability of these compounds has been proven through the assessment of reducing power ability and radical scavenging activities.

07* DQG 650 DUH D FODVV RI DQWLR[LGDQW DQG UDGLFDO

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the values of mitragynine analogues as a new source of antioxidant to prevent the progress of various oxidative stress-induced diseases including cancer. Furthermore, WKLV VWXG\ MXVWLÀHV WKH HIÀFDF\ RI PLWUDJ\QLQH UHODWHG

analogues as potential anticancer and chemo-preventive medicine. This study supports the biological relevancy and popular traditional use of mitragynine obtained from 0LWUDJ\QD 6SHFLRVD .RUWK .HWXP RU %LDN%LDN WKDW

growth in tropical forest of Southeast Asia.

Acknowledgements

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Sains Malaysia.

References

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proliferation of cervical and leukemic cancer cells by penicillin G. Asian Pac J Cancer Prev, 14, 2127-30.

$GNLQV-(%R\HU(%0FFXUG\&50LWUDJ\QDVSHFLRVD

a psychoactive tree from Southeast Asia with opioid activity.

Curr Top in Med Chem, 11, 1165-75.

$KPDG)$51RUDPDOL]D01&UXFLIHURXV9HJHWDEOHV

Dietary phytochemicals for cancer prevention. Asian Pac J Cancer Prev, 14, 1565-70.

$OYLQ5$(YDQJHOLQH&$6RQLD'-$SRSWRVLVLQGXFLQJ

DFWLYLW\RI+3/&IUDFWLRQIURPVoacanga globosa%ODQFR 0HUURQKXPDQFRORQFDUFLQRPDFHOOOLQH+&7Asian Pac J Cancer Prev, 15, 617-22.

$QWRLQH '- :LOOLDPV '3 3DUN %.  'UXJLQGXFHG

hepatotoxicity: state of the science. Expert Opin Drug Metab Toxicol, 4, 1415-27.

%RODQG . )ODQDJDQ / 3UHKQ -  3DUDFULQH FRQWURO RI

WLVVXHUHJHQHUDWLRQDQGFHOOSUROLIHUDWLRQE\&DVSDVHCell Death Dis, 4, 725.

)X4LDQJ 6<LQJ / ;LDQJ6KL . HW DO  3URJUHVV RQ

understanding the anticancer mechanisms of medicinal mushroom: Inonotus obliquus. Asian Pac J Cancer Prev, 14, 1571-8.

*HRUJH9&.XPDU'56XUHVK3.HWDO$SRSWRVLV

induced cell death due to oleanolic acid in hacat keratinocyte cells-a proof-of-principle approach for chemopreventive drug development. Asian Pac J Cancer Prev, , 2015-20.

Gupta SK, Selvan VK, Agrawal SS, et al (2009). Advances in pharmacologicalstrategies for the prevention of cataract development. Indian J Ophthalmol, , 175-83.

Herraiz T, Galisteo T (2004). Endogenous and dietary indoles:

DFODVVRIDQWLR[LGDQWVDQGUDGLFDOVFDYHQJHUVLQWKH$%76

assay. Free Radical Res, , 323-31.

+HUUDL]7*DOLVWHR-&KDPRUUR-/7U\SWRSKDQUHDFWV

with naturally occurring and food-occurring phenolic DOGHK\GHVWRJLYHSKHQROLFWHWUDK\GURơFDUEROLQHDONDORLGV

activity ss antioxidants and free radical scavengers. J Agri Food Chem, 51, 2168-73.

.ULHQJVDN78QDURM%.HYLQ&HWDO&RPSDULVRQRI

$%76 '33+ )5$3 DQG 25$& DVVD\V IRU HVWLPDWLQJ

antioxidant activity from guava fruit extracts. J Food Comp Ana, 19, 669-75.

.UXN-'XFKQLN(2[LGDWLYHVWUHVVDQGVNLQGLVHDVHV

possible role of physical activity. Asian Pac J Cancer Prev, 15, 561-8.

/HH-$.R-+-XQJ%*HWDO)HUPHQWHGSUXQXVPXPH

with probiotics inhibits 7,12-Dimethylbenz[a]anthracene DQG 2WHWUDGHFDQR\O SKRUERODFHWDWH LQGXFHG VNLQ

carcinogenesis through alleviation of oxidative stress. Asian Pac J Cancer Prev, 14, 2973-8.

0DGULJDO%XMDLGDU(0DUWLQR5RDUR/*DUFLD$JXLUUH.HWDO

(2013). Grapefruit juice suppresses azoxymethane-induced colon aberrant crypt formation and induces antioxidant capacity in mice. Asian Pac J Cancer Prev, 14, 6851-6.

Mat J, Siti A, Hamid ANA, et al (2013). Apoptosis induction in MV4-11 and K562 human leukemic cells by SHUHVNLDVDFKDURVD &DFWDFHDH OHDI FUXGH H[WUDFW Asian Pac J Cancer Prev, 15, 475-81.

0DWVXPRWR.+RULHV6,VKLNDZD+HWDO$QWLQRFLFHSWLYH

effect of 7-hydroxymitragynine in mice: discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa. Life Science, , 2143-55.

1HMLE * 0RVWDID ,: 9DQGLWD 6 HW DO  1DEDJ

(Zizyphusspina-christi) extract prevents aberrant crypt foci development in colons of azoxymethane-treated rats by abrogating oxidative stress and inducing apoptosis. Asian Pac J Cancer Prev, 14, 5031-5.

1HOVRQ -/ /DSRLQW - +RGJPDQ 0- HW DO  6HL]XUH

and coma following kratom (mitragynina speciosa korth) Exposure. J Med Toxicol, 6, 424-6.

3DSDYDGHH.5RRQJWDZDQ6$WKLNRP6$QWLR[LGDQW

effects of gamma-oryzanol on human prostate cancer cells.

Asian Pac J Cancer Prev, 14, 5421-5.

3LQFKXN , 6KRYDO + 'RWDQ < HW DO  (YDOXDWLRQ RI

antioxidants: scope, limitations and relevance of assays.

ChemPhys Lipids, 165, 638-47.

$QWLR[LGDQW9DOXHDQG$QWLSUROLIHUDWLYH(IÀFDF\RI0LWUDJ\QLQH Pin-Der D (1998). Antioxidant activity of burdock (Arctium

lappa linne); its scavenging active oxygen. J Am Oil Chem Soc, , 455-61.

Puthuparampil NA, Santhibhavan PP, Anand N, et al (2013).

&KHPRSUHYHQWLYHHIIHFWRIAmorphophallus campanulatus (roxb) blume tuber against aberrant crypt foci and cell proliferation in 1, 2-dimethylhydrazine induced colon carcinogenesis. Asian Pac J Cancer Prev, 14, 5331-9.

5DPHVK %% 6HOLQD )' 3DWULFN - HW DO  6HOHFWLYH

cytotoxic activities of two novel synthetic drugs on human EUHDVWFDUFLQRPD0&)FHOOVAnti cancer Research, 29, 2993-6.

Sadegh F, Ali M, Ardekani E, Zabihi ZA, et al (2013).

Antioxidant and apoptotic effects of an aqueous extract of XUWLFDGLRLFDRQ WKH 0&) KXPDQ EUHDVW FDQFHU FHOO OLQH

Asian Pac J Cancer Prev, 14, 5317-23.

6DLGLQ1$&\WRWR[LFLW\RIH[WUDFWRI0DODVLDQPLWUDJ\QD

speciosa korth and its dominant alkaloid mitragynine Ph D.

7KHVLV,PSHULDO&ROOHJH

6DLGLQ 1$ 5DQGDOO 7 7DND\DPD + HW DO  0DOD\VLDQ

kratom, a phyto-pharmaceutical of abuse: studies on the mechanism of cytotoxicity. Toxicology, , 19-20.

6DPL5&KXQ-XDQ/<DQ=HWDO&DEEDJHEUDVVLFD

ROHUDFHD/ YDU FDSLWDWD SK\WRFKHPLFDOV ZLWK DQWLR[LGDQW

DQGDQWLLQÁDPPDWRU\SRWHQWLDOAsian Pac J Cancer Prev, 14, 6657-62.

6DVLSDZDQ01DWKLGGD:6DKDSDW%HWDO&\WRWR[LF

and apoptotic effects of six herbal plants against the human hepatocarcinoma(HepG2) cell line. Chinese Med, 6, 1-8.

6LGGKXUDMX3%HFNHU.7KHDQWLR[LGDQWDQGIUHHUDGLFDO

scavenging activities of processed cowpea (Vignaunguiculata /Seed Extracts Food Chem, , 10-9.

Simay G, Ercan K, Nese K (2014). Preventive effects of resveratrol against azoxymethane induced damage in rat liver. Asian Pac J Cancer Prev, 14, 2367-70.

6LULQ\D 7 &KDUDWGD 3 :DQLGD , HW DO  &OHLVWRFDO\[

nervosum Extract Ameliorates chemical-induced oxidative stress in early stages of rat hepatocarcinogenesis G. Asian Pac J Cancer Prev, 15, 2825-30.

6XKDQ\D 3 -X]DLOL $ 6XUDVK 5 HW DO  (YDOXDWLRQ

of antioxidant and antibacterial activities of aqueous, methanolic and alkaloid extracts from mitragyna speciosa 5XELDFHDHIDPLO\/HDYHVMolecules, 14, 3964-74.

6XSDNLW .$ .KDMRUQVDN 7 3DQXZDQ & HW DO 

Antioxidant and anti-cancer cell proliferation activity of propolis extracts from two extraction methods. Asian Pac J Cancer Prev, 14, 6991-5.

7DULND 7 6LULQ\D 7 &KDUDWGD 3 HW DO  &DQFHU

chemopreventive effect of Spirogyra neglecta (Hassall) kützing on diethylnitrosamine-induced hepatocarcinogenesis in rats. Asian Pac J Cancer Prev, 15, 1611-6.

7HLN%HQJ * 6LGGLTXL - 5D]DN + HW DO $ VLPSOH

and costeffective isolation and purification protocol of mitragynine from mitragynaspeciosa korth leaves.

Malaysian J Anal Sci, 15, 54-60.

7HLN%HQJP 00 HW DO  8QH[SHFWHG UHGXFWLRQ RI WKH

indole double bond in mitragynine using n-butylsilane and FDWDO\WLF WULV SHQWDÁXRURSKHQ\O ERUDQHV Synth Comm, 44, 1291-5.

9LFNQDVLQJDP%1DUD\DQDQ6%HQJ*70DQVRU60

The informal use of ketum (mitragyna speciosa) for opioid withdrawal in the northern states of peninsular Malaysia and implications for drug substitution therapy. Int J Drug Policy, 21, 283-8.

9D]KDSLOO\ &* 'HYDQJD 51. 3DODPDGDL .6 HW DO 

&RPSDUDWLYH VWXGLHV WR HYDOXDWH UHODWLYH In vitro potency of luteolin in inducing cell cycle arrvest and apoptosis in

+D&D7DQG$FHOOVAsian Pac J Cancer Prev, 14, 631-7.

:HVHQ$0 -ZDQ$=$WKHHU$0  ,QYHVWLJDWLRQ RI

antioxidant status in multiple myeloma patients: effects of therapy. Asian Pac J Cancer Prev, 14, 3663-7.

:LUDWFKDQHH09LWKRRQ9:DQQD&HWDO&\WRWR[LF

activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells In vitro. BMC Complementary and Alternative Med, , 1-8.

<DQJ/:HQ-XQ0%DR.XQ4HWDO%OHQGLQJRIVR\

EHDQRLOZLWKVHOHFWHGYHJHWDEOHRLOV,PSDFWRQR[LGDWLYH

stability and radical scavenging activity. Asian Pac J Cancer Prev, 15, 2883-9.

=DIHU$$\VH,*$QWLR[LGDQWVPD\SURWHFWFDQFHUFHOOV

from apoptosis signals and enhance cell viability. Asian Pac J Cancer Prev, 14, 4611-4.