Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
ISSN 0976 – 044X
Research Article
Antioxidant, Cytotoxic and Apoptotic Activities of Crude Extract of Alpinia purpurata on
Cervical Cancer Cell Line
1
2
2
Enock Kiage Oirere , Palanirajan Anusooriya , Deivasigamani M alarvizhi , Chinthamony Arul Raj², Velliyur Kanniappan Gopalakrishnan
1
1,2*
Depart m ent of Bioinf ormat ics, Karpagam Universit y, Coim bat ore, Tam il Nadu, India.
Depart m ent of Biochemist ry, Karpagam Universit y, Coimbat ore, Tamil Nadu, India.
2
* Corresponding author’s E-mail: vk.gopalakrishnan@karpagam.ac.in
Accepted on: 02-12-2015; Finalized on: 31-01-2016.
ABSTRACT
Alpinia purpurat a (Vieill.) K. Schum belongs in the Zingiberaceae family and is a very popular garden plant in India, it possesses
moderate antibacterial and anticancer act ivities. To st udy t he ant ioxidant , cytot oxic and apopt ot ic activit ies of n-hexane leaf ext ract
of A. purpurata ; antioxidant act ivit y was det ermined by measuring (i) the scavenging effect of plant ext ract against 2, 2-diphenyl-1picryl hydrazyl (DPPH) and 2, 2′-azinobis-3-ethylbenzot hiazoline-6-sulfonat e (ABTS), hydroxyl radical scavenging, superoxide radical
scavenging, nit ric oxide radical scavenging, hydrogen peroxide radical scavenging, met al chelat ing act ivit y and (ii) reducing power
capacit y and ferric reducing power (FRAP). Cyt ot oxicit y was det ermined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt et razolium
bromide (M TT assay) and acridine orange/ et hidium bromide st aining t o assess t he ant icancer act ivit y of t he crude ext ract on HeLa
cells. The ant ioxidant activit y of t he plant ext ract was found t o be close t o t hat of st andard ascorbic acid. DPPH radical scavenging
act ivit y IC50 value was 495 ± 1.43µg/ ml and 440± 1.39µg/ ml for ext ract and ascorbic acid respect ively. ABTS radical scavenging
act ivit y IC50 value was 420± 2.26µg/ ml and 400 ± 2.10µg/ ml for extract and ascorbic acid respect ively. The reducing power and ferric
reducing activity of t he ext ract increased wit h the increase of t heir concent rations. In M TT assay, leaf ext ract inhibit ed HeLa cells in a
dose-dependent manner, showing cyt otoxicit y wit h IC50 of 41.25µg/ ml. M orphological changes observed by fluorescent miscroscopy
on the leaf ext ract t reat ed cells st ained wit h acridine orange/ ethidium bromide suggest ed an apopt ot ic activit y. This st udy clearly
demonst rated t hat n-hexane leaf ext ract of Alpinia purpurata is a good ant ioxidant and induces apopt osis in HeLa cells.
Keywords: Alpinia purpurata , n-hexane, HeLa cell line, ant ioxidant , cyt ot oxicit y, apopt osis.
INTRODUCTION
T
he oxidat ive st ress is one of t he m ajor causat ive
fact ors in t he induction of various chronic and
1
degenerat ive ailm ent s w hich includes cancer. Folk
m edicine is used in m any places and plant s cont ain a rich
source of nat ural antioxidant s t hat m ost ly serve as
2
candidates for t he developm ent of novel drugs. Am ong
t he
count less
nat urally
occurring
antioxidant s
carot enoids, ascorbic acid and phenolic com pounds have
3
t he best perform ance. They inhibit lipid peroxidat ion,
scavenge free radicals and act ive oxygen species by
proliferat ing a react ion cycle and chelating heavy met al
4
ions. In t he search for sources of nat ural antioxidant s
and com pounds w it h radical scavenging act ivit y during
t he recent years, som e have been found such as
5
6
echinacoside in Echinaceae root , ant hocyanin, phenolic
7
8
com pounds, w at er ext ract s of roast ed Cassia t ora , w hey
9
10
prot eins, and t hioredoxin h prot ein from sweet pot at o.
Apopt osis or programm ed cell deat h is a vast ly organized
process w hich leads t o m orphological and biochem ical
changes including nuclear pycnosis and cyt oplasm
condensat ion t o produce m em brane-bound apopt otic
bodies t hat are phagocytosed by m acrophages or
adjacent cells. Com pounds t hat suppress or inhibit t he
proliferat ion of t um or cells by inducing apopt osis have
11
pot ent ial as ant it um or agent s. Am ong m ost comm on
neoplast ic diseases affect ing w omen, cervical carcinom ais
one of t hem , com es second aft er breast cancer w hich is
t he m ost prevalent , each year t here is approxim at ely half
12
a m illion new cases w orldw ide. Hence, t he developm ent
of chem ot herapeutic or chem oprevent ive agent s against
cervical cancer is very vit al t o m inim ize t he cases caused
by t his disease.
The plant s belonging t o Zingiberaceae are know n t o
13,14
cont ain m edicinal propert ies.
A range of essent ial oils
15
is found in t he species of Zingiberaceae.
Rhizom e
ext ract s of som e species of the m edicinal Zingiberales are
16
broadly used as food and t radit ionally as rem edy.
Leaves of various Zingiberaceae are also used in food
17
flavoring and as indigenous m edicine. Alpinia species
are w ell know n m edicinal herbs t hat have been proven by
previous researches t o have several effect s, nam ely ant i18,19
20,21
inflamm at ory,
ant ioxidant ,
ant im icrobial,
22,23
24
ant iderm at ophyt ic,
ant inociceptive,
25
26
hepat oprotect ive,
imm unost im ulat ory,
and
27,28
ant icancer
act ivities. Alpinia is t he principal genus in
ginger fam ily in which Alpinia purpurata (Vieill.) K. Schum
29
is a very popular garden plant in India. The sharp odour
of rhizom e could enhance appet it e, t ast e and voice. It is
also used for treatm ent of renal diseases, rheum at ism ,
30
sore t hroat and headache.
The M ABA (M icroplat e
Alam ar Blue Assay) assay st udy of t he crude et hanolic
ext ract of t he various part s of A. purpurat a depict ed t he
leaf ext ract t o possess t he highest act ivit y, followed by
31
t he rhizome and flow er ext ract s.
The plant has
m oderat e ant ibact erial and ant icancer act ivit ies w hich
m ay be due t o t he rich phyt ochemicals in t he leaves of A.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
28
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
32
purpurata . The object ive of t he present st udy w as t o
assess t he cyt ot oxic and apopt ot ic effect of n-hexane leaf
ext ract of Alpinia purpurata on cervical cancer HeLa cell
line and ant ioxidant activit y of t he plant .
M ATERIALS AND M ETHODS
ISSN 0976 – 044X
purpurata w as estim ated by t he met hod described by
39
Dinis. The Reducing pow er capacity w as evaluat ed by
40
t he m odified m et hod of Oyaizu. FRAP assay w as used t o
est im ate t he reducing capacit y of leave ext ract , according
41
t o t he met hod of Benzie and St rain.
Cytotoxicity Assay
General Experimental Procedures
The plant pow der w as exhaust ively extracted using
soxhlet apparat us and t he extract w as evaporat ed t o
dryness using rot ary flash evaporat or (Buchi t ype).
Hum an cervical cancer cell line (HeLa) w as purchased
from Nat ional Centre for Cell Science (NCCS, Pune, India)
and w ere m aint ained in Dulbecco’s m odified eagles
m edia
(HIM EDIA).
3-(4,5-dimet hylthiazol-2-yl)-2,5diphenyl t et razolium bromide (M TT), t rypsin-EDTA
(HIM EDIA) and Fet al bovine serum (FBS) w ere purchased
from Invitrogen (USA). Organic solvent s, silica gels and
CO2 incubat or w ere obt ained from NEW BRUNSWICK
SCIENTIFIC (NBS), EPPENDORF, GERM ANY. DNA-binding
dyes Acridine Orange and Et hidium Brom ide (Sigm a,
USA). Optical densit y w as read using a micro plate reader
(ELISASCAN, ERBA). The st ained cells w ere observed by a
fluorescent m icroscope (Olym pus CKX41 w it h Opt ika Pro5
cam era).
Plant collection and extraction
The leaves of t he plant A. purpurat a were collected from
t he nat ural habit at s of Kanyakum ari district , Tamil Nadu,
India. The plant specim en w as aut hent icat ed by Dr. G.V.S
M urt hy, Bot anical Survey of India, Coim bat ore, TNAU
Cam pus, India. A voucher specimen w as deposit ed in t he
laborat ory for fut ure reference (BSI/ SC/ 5/ 23/ 1033
11/ Tech). The voucher specim en w as deposit ed at t he
herbarium of Karpagam Universit y, Coim bat ore. The
leaves of A. Purpurat a w ere w ashed t horoughly in t ap
w at er, shade dried and pow dered. The pow der (100g)
w as exhaust ively extracted wit h n-hexane in t he ratio of
1:5 (w / v) for 24 hr by using soxhlet apparat us. The extract
w as evaporat ed t o dryness using rot ary flash evaporat or
(Buchi type).
Antioxidant Activity
The scavenging activit y DPPH free radicals of t he nhexane leave ext ract of Alpinia purpurata w as rest rained
33
according t o t he procedure described by Blois. The
Nit ric oxide w as generat ed by sodium nit roprusside and
m easured by t he GriessIllosvoy reaction by t he met hod of
34
Green. The superoxide scavenging activit y of t he nhexane ext ract of Alpinia purpurata w as m easured by
reduction of nit robluetet razolium (NBT) met hod of
35
Font ana. The hydroxyl radical scavenging act ivit y w as
m easured w it h a slight m odificat ion of Elizabet h and
36
Rao. The abilit y of t he n-hexane leave extract of Alpinia
purpurata t o scavenge hydrogen peroxide w as
37
det erm ined according t o t he met hod of Ruch. The ABTS
radical cation scavenging activit y w as performed w it h
38
slight m odificat ions described by Re. The chelat ing of
ferrous ions by t he n-hexane leave ext ract of Alpinia
The
M TT
[3-(4,5-dimet hylthiazol-2-yl)-2,5-diphenylt et razolium bromide] colorimet ric assay w as used t o
evaluat e t he anti-proliferative act ivit y of n-hexane crude
ext ract. This assay is based on t he m et abolic reduct ion of
soluble M TT by mit ochondrial enzym e act ivit y of viable
t um or cells int o an insoluble (dark purple) color form azan
product , w hich can be m easured spect rophot om et rically
aft er dissolving in dim et hylsulfoxide (DM SO). Cellular
t oxicit y of t he crude hexane ext ract from A. purpurat a
leaves on cult ured cells w as measured using t his
42
4
m et hod. Briefly, 200 µl of cells (5×10 cells/ m l) w ere
seeded in 96 w ell m icroplat es and incubated for 24 hr
(37°C, 5% CO2 air hum idified). Then 20 µl of prepared
concentrat ions of 6.25, 12.5, 25, 50 and 100µg/ml of t he
sam ple w as added and t he cells divided int o 6 groups.
The sam ples w ere initially dissolved in DM SO before
being added t o t he culture media. Cont rol group
cont ained DM SO at t he sam e concent rat ion [0.5% (v/ v)]
of t he treat ed group. Aft er 24hr of incubat ion, 20 µl of
M TT solut ion (5 m g/ m l in phosphat e buffer solut ion) w as
added and the plat es incubated for anot her 3 hr. 150 µl
of m edium cont aining M TT w ere t hen gent ly replaced by
DM SO and pipet t ed t o dissolve any form azan cryst als
form ed. Optical densit y w as read at 540 nm using DM SO
as blank in a m icro plat e reader (ELISASCAN, ERBA) and
t he cell percent viability w as calculated by t he form ula [%
viabilit y = (OD of Test / OD of Control) X 100]. The 50%
reduction in cell num ber relat ive t o t he control or IC50
w as est ablished by extrapolation from linear regression of
experiment al dat a.
Apoptosis studies with AO/ EB staining method
DNA-binding dyes Acridine Orange (AO) and Et hidium
Brom ide (EB) (Sigm a, USA) w ere used for t he
43
m orphological det ect ion of apopt otic and necrotic cells.
AO is t aken up by bot h viable and non-viable cells and
em it s green fluorescence if int ercalat ed into double
st randed nucleic acid (DNA). EB is t aken up only by nonviable cells and emit s red fluorescence by int ercalat ion
int o DNA. HeLa Cells were added t o a final concentrat ion
4
of 5×10 / ml t hen 0.6ml w ere seeded at t he 6-w ell plat es
and incubat ed for overnight . Cells w ere left untreat ed or
t reat ed w ith IC50 concent ration of 41.25 µg/ m l of plant
ext ract.
Aft er being cult ured for 24 hrs and 48 hrs, t he cells w ere
w ashed by cold PBS and t hen st ained wit h a m ixt ure of
AO (100 µg/ m l) and EB (100 µg/ m l) at room t em perat ure
for 10m in.
The st ained cells w ere w ashed tw ice w it h 1X PBS and
observed by a fluorescence microscope in blue filt er of
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
29
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
ISSN 0976 – 044X
fluorescent m icroscope (Olym pus CKX41 w it h Opt ika Pro5
cam era).
act ivit y also increases w it h
concentrat ion of t he extract.
Statistical analysis
The reducing abilit y of a com pound generally depends on
t he presence of reduct ant s w hich have exhibit ant ioxidat ive pot ent ial by breaking t he free radical chain,
49
donat ing a hydrogen at om . The plant ext ract reduces
t he m ost Fe3+ ions in a concentrat ion dependent m anner. The high reducing pow er is t he high absorbance
at 700 nm . The reducing power of t he ext ract is
com pared w it h Ascorbic acid (Figure 3A). From previous
research it w as found t hat plant -derived ext ract s
cont aining ant ioxidant principles has also cyt ot oxicit y
50
act ivit y t ow ard t um or cells.
All experiment s w ere conduct ed in triplicat e and t he dat a
obt ained w ere represent ed as mean ± st andard
deviations t hat w ere int erpret ed using M icrosoft Office
Excel 2007®.
RESULTS AND DISCUSSION
Free radical
properties
scavenging potential
and
antioxidant
Nat ural product s play vit al role in chem ot herapy, having
cont ribut ed
a
great er
percent age
in
cancer
44,45
chemot herapeut ic pharm aceuticals.
Agent s w it h
capabilit y of inhibiting cell proliferation, inducing
apopt osis or m odulating signal t ransduction are current ly
46
used for t he treatm ent of cancer. Flavonoids are pot ent
w at er-soluble ant ioxidant s and free radical scavengers
w hich prevent oxidat ive cell dam age and have st rong
ant icancer activity. Som e of t he research evidence
suggest t hat t he free radicals induce oxidat ive dam age t o
biom olecules (lipids, prot eins and nucleic acids) and t he
dam age result t o various diseases in hum ans for inst ance
diabet es m ellitus, at herosclerosis, ageing, inflamm at ion,
47
cancer, and AIDS.
In t his research, t he antioxidant and anticancer activity of
A. purpurata w as invest igat ed. There are various m et hods
t o det ermine t he ant ioxidant capacit ies w hich differ in
t erm s of t heir determ inat ion principles and experiment al
48
conditions.
For m easuring ant ioxidant act ivit y, t he
paramet ers such as DPPH radical scavenging activity,
ABTS radical scavenging act ivit y, hydroxyl radical
scavenging act ivit y, superoxide radical scavenging
act ivit y, nit ric oxide radical scavenging act ivit y, hydrogen
peroxide radical scavenging act ivit y, met al chelating
act ivit y, reducing pow er capacit y and FRAP assay are
used. In radical scavenging activit y, all t he above nine
ant ioxidant s show
good scavenging activity in
concentrat ion dependent m anner. The scavenging
t he
increase
in
t he
Ant ioxidant properties, specifically radical scavenging
act ivit y, is very crucial due t o t he deplet ion and dam aging
effect t o free radicals in living organism s.
The DPPH radical scavenging (%) activit y of n-hexane
leave ext ract of Alpinia purpurata , com pared t o st andard
ascorbic acid, is show n in Figure 1A. The IC50 value of nhexane leave extract of Alpinia purpurat a and ascorbic
acid w ere found t o be 495 ± 1.43µg/ m l and 440±
1.39µg/ ml respect ively.
The plant ext ract exhibited pot ent ABTS radical cat ion
scavenging act ivit y in concentrat ion dependent m anner
w it h t he IC50 being 420 ± 2.26µg/ ml and t he IC50 of t he
st andard ascorbic acid w as found t o be 400 ± 2.10µg/ m l
(Figure 1B).
The nit ric oxide radical scavenging activity of leaf extract
of Alpinia purpurata , IC50 value w as 250 ± 1.83µg/ ml and
for ascorbic acid w as 160 ± 1.45 µg/ m l (Figure 1C). The
superoxide scavenging act ivit y of n-hexane leave extract
of t he Alpinia purpurat a w as increased m arkedly w it h t he
increase of concentrat ions (Figure 1D). The half inhibit ion
concentrat ion (IC50) of leave ext ract w as 400 ± 2.88µg/ m l
and ascorbic acid w as 300 ± 1.85µg/ ml.
Figure 1: (A) DPPH radical scavenging assay; (B) ABTS radical scavenging act ivit y; (C) Nit ric oxide scavenging act ivit y; (D)
Superoxide radical scavenging act ivit y.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
30
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
ISSN 0976 – 044X
Figure 2: (A) Hydrogen peroxide radical scavenging act ivit y; (B) M et al chelat ing act ivit y; (C) Hydroxyl radical scavenging
act ivit y.
Figure 3: (A) Reducing pow er activit y; (B) Ferric reducing antioxidant pow er assay.
Hydrogen peroxide radical scavenging act ivit y of nhexane leaf ext ract of Alpinia purpurata increased in dose
dependent m anner in com parison w it h t he st andard
ascorbic acid. The IC50 value of leaf ext ract and ascorbic
acid w ere found t o be 450± 2.86µg/ m l and 350 ±
2.27µg/ ml respect ively (Figure 2A). The met al chelating
act ivit y is show n in figure 2B. The result s are expressed as
percent age m et al chelat ing activity. Ext ract exhibit ed
dose dependent m et al chelating activit y w ith an IC50
value of 355 ± 2.81µg/ml and t he IC50 of st andard
ascorbic acid w as found t o be 265 ± 1.92µg/ml. The
hydroxyl radical scavenging act ivit y of t he n-hexane leaf
ext ract of Alpinia purpurat a w as found t o be effective
w it h t he IC50 of 410 ± 2.87µg/ml and t he st andard
ascorbic acid w as found t o be 285± 2.16µg/ml (Figure 2C).
The reducing power pot entials of t he n-hexane leave
ext ract of Alpinia purpurat a in comparison w it h t he
st andard ascorbic acid at 700 nm is explained in figure 3A.
The t endency for ferric ion reducing act ivit ies of Alpinia
purpurata and ascorbic acid is show n in figure 3B. The
result indicat e that t he reducing power and ferric
reducing act ivit y of t he leaf ext ract increased wit h t he
increase in their concent rations.
Cytotoxicity and Apoptosis study
Apopt osis induct ion is t he preferred approach in t he
developm ent of anticancer drugs as it is t he m ost
effect ive w ay of treat ing cancer. In t his st udy, t he aim
w as t o invest igate t he cyt ot ot oxic effect s and if t he
apopt ot ic m echanism on HeLa cells is induced by Alpinia
purpurata extract. The cyt ot oxicit y of Alpinia purpurat a
ext ract on HeLa w as evaluated by M TT assay based on
percent age of cell viabilit y. N-Hexane-leaf ext ract w as
found t o have cyt ot oxic effect against HeLa, show ing cell
proliferat ion inhibition in a concentrat ion-dependent
m anner. The IC50 for A. purpurata crude ext ract w as
found t o be 41.25µg/mL (Figure 4).
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
31
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
The result s obt ained from t he present st udy show ed t hat
A. purpurata has an anticancer activit y. Apparent
hallm ark m orphologies of apopt osis w ere observed in
t reat ed HeLa cells in dual acridine orange and et hidium
bromide st aining (Figure 5). Cont rol HeLa cells w hich
w ere elongated and spindle in shape becam e disfigured
aft er Alpinia purpurat a extract t reat ment . Cell shrinkage
w it h irregular shape, cyt oplasm ic condensat ion, nuclear
pycnosis and blebbing followed by appearance of
apopt ot ic bodies led t o a decrease of the num ber of
viable cells aft er t reat ment wit h t he extract for 24 and 48
hrs respect ively. Nutrient depletion in grow t h media or
cont act inhibit ion result ing t o nat ural deat h of cells m ay
be t he cause for t he appearance of few apopt ot ic cells in
t he cont rol HeLa.
ISSN 0976 – 044X
CONCLUSION
In conclusion, t his st udy clearly dem onst rat ed t hat nhexane-leaf extract of Alpinia purpurat a is a good
ant ioxidant and induces apopt osis in HeLa cells. These
result s suggest t hat Alpinia purpurata t hat act as an
apopt ot ic inducer could become a potent ial anticancer
agent in developm ent of t he drug. Fut ure prospect s of
t his st udy involve ident ificat ion and isolat ion of cyt ot oxic
and apopt ot ic inducer bioact ive com pounds from the leaf
ext ract of t his plant w it h furt her extrapolat ion on t heir
m olecular struct ures and mechanism of act ion. M ore
st udies can be carried out t o determ ine t he cyt ot oxic and
apopt ot ic effect s on several cell lines follow ed by in vivo
st udies.
Conflict of interest
The aut hors declare that t here is no conflict of int erest s
regarding t he publicat ion of t his paper.
Acknowledgement: The aut hors are t hankful t o t he
Chancellor, t he Chief Executive Officer, t he Vice
Chancellor, and t he Regist rar of Karpagam Universit y for
providing facilit ies and encouragem ent.
REFERENCES
Figure 4: M TT analysis, show s n-hexane-leaf extract
inhibit ed HeLa cells in a dose-dependent m anner, w it h
cyt ot oxicit y of IC50 (41.25µg/ ml).
Figure 5: AO/ EB St aining. For HeLa cells A. Show s t he
cont rol. B. Treat ed w it h ext ract (41.25µg/ ml) for 24hr C.
Treat ed w it h extract (41.25µg/ ml) for 48hr.
1.
Young IS, Woodside JV, Ant ioxidant s in healt h and disease,
J ClinPat hol, 54, 2001, 176-186.
2.
Lin CC, Huang PC, Ant ioxidant and hepat oprot ect ive effect s
of Acathopanax senticosus, Phyt ot her Res, 14, 2002, 48994.
3.
Duh PD, Tu YY, Yen GC, Antioxidants act ivit y of aqueous
ext ract of Harnjyur ( Chrysant hemum morifolium Ramat),
Lebensmwiss Technol, 32, 1999, 269-77.
4.
Sundararajan R, Haja NA, Venkat esan K, M ukherjee K, Saha
BP, Bandyopadhyay A, M ukherjee PK, Cyt isuss coparius
Link-A natural ant ioxidant , BM C Complement Altern M ed,
6, 2006, 1-7.
5.
Hu C, Kitt s DD, St udies on t he antioxidant act ivit y of
Echinaceae root ext ract , J Agric Food Chem, 48, 2000,
1466-72.
6.
Espin JC, Soler-Rivas C, Wichers HJ, Viguera-Garcia C,
Ant hocyanin-based nat ural colorants: a new source of
ant iradical activit y for foodst uff, J Agric Food Chem, 48,
2000, 1588-92.
7.
Rice-Evans CA, M iller N, Paganga G, Ant ioxidant properties
of phenolic compounds, Trends Plant Sci, 2, 1997, 152-59.
8.
Yen GC, Chuang DY, Antioxidant properties of wat er
ext ract s from Cassia tora L. in relat ion to t he degree of
roast ing, J Agric Food Chem, 48, 2000, 2760-65.
9.
Tong LM , Sasaki S, M c Clements DJ, Decker EA,
M echanisms of the antioxidant act ivit y of a high molecular
weight fract ion of whey, J Agric Food Chem, 48, 2000,
1473-78.
10. Huang DJ, Chen HJ, Hou WC, Lin CD, Lin YH, Active
recombinant t hioredoxin h prot ein wit h antioxidant
act ivit ies from sweet pot at o ( Ipomoea bat atas[L.] Lam
‘Tainong 57’) storage root s, J Agric Food Chem, 52, 2004,
4720-24.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
32
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
11. Frankfurt OS, Krishna A, Apopt osis-based drug screening
and detection of select ive t oxicit y t o cancer cells,
Ant icancer Drugs, 14, 2003, 555-61.
ISSN 0976 – 044X
and Thailand used t radit ionally to
Et hnopharmacol, 100, 2005, 237–43.
t reat
cancer, J
12. Franco EL, Schlecht NF, Saslow D, The epidemiology of
cervical cancer, Cancer J, 9, 2003, 348-59.
28. An N, Zou ZM , Tian Z, Luo XZ, Yang SL, Xu LZ,
Diarylhept anoids from the rhizomes of Alpinia officinarum
and t heir ant icancer activit y, Fitot erapia, 79, 2008, 27–31.
13. Kumar VP, Chauhan NS, Padh H, Rajani M , Search for
ant ibact erial and ant ifungal agents from select ed Indian
medicinal plants, J Et hnopharmacol, 107, 2006, 182-88.
29. Sabu M , Zingiberaceae and Cost aceae of sout h India, India:
Indian Associat ion for Angiosperm Taxonomy, Calicut
Universit y, 2006.
14. Venkat esan G, Ganesan S, Kesavan L, Banumat hy N,
Et hnobot any of
Et hnic group Thott ianaickan of
Tiruchirapalli Dist rict , TamilNadu, India. In: Prajapat hi ND,
Prajapathi T, Jajpura S, Advances in M edicinal Plants, Vol. I.
(eds) Asian M edicinal Plants and Healt h Care Trust ,
Jodhpur, Rajast han, 2005, 59–75.
30. Prajapathi ND, Purohit SS, Arun KS, Kumar T, A Handbook
of medicinal plant s, A complet e source Book, India:
Agrobios; 2003.
15. Ibrahim H, Zakaria M , Essent ial oils from three M alaysian
Zingiberaceae species, M alays J Sci, 9, 1987, 73-76.
16. Ibrahim H, Khalid N, Hussin K, Cult ivat ed gingers of
peninsular
M alaya:
ut ilizat ion,
profiles
and
micropropagation, Garden’s Bull Singap, 59, 2007, 71-88.
17. Larsen K, Ibrahim H, Khaw SH, Saw LG, Gingers of
Peninsular M alaysia and Singapore, Nat ural Hist ory
Publicat ions (Borneo): Kot a Kinabalu Sabah, 1999, 135.
18. Zoghbi M GB, Andrade EHA, M aia JGS, The essential oil of
Vit exagnus-castus L. growing in t he Amazon region, Flavour
Fragrance J, 14, 1999, 211–13.
19.
Israf DA, Khaizurin TA, Syahida A, Lajis NH, Khozirah S,
Cardamonin inhibits COX and iNOS expression via inhibition
of
p65NF- κB
nuclear
t ranslocat ion
and
Iκ-B
phosphorylat ion in RAW 264.7 macrophage cells, M ol
Immunol, 44, 2007, 673–79.
20. Habsah M , Amran M , M ackeen M M , Lajis NH, Kikuzaki H,
Nakat ani N, Rahman AA, Ghafar, Ali AM , Screening of
Zingiberaceae ext ract s for ant imicrobial and antioxidant
act ivit ies, J Et hnopharmacol, 72, 2000, 403–10.
21. Chen IN, Chang CC, Ng CC, Wang CY, Shyu YT, Chang TL,
Ant ioxidant and ant imicrobial act ivit y of Zingiberaceae
plants in Taiwan, Plant Foods Hum Nut r, 63, 2008, 15–20.
22. Trakranrungsie N, Chat chawanchonteera A, Khunkitt i W,
Et hnovet erinary st udy for ant idermatophyt ic act ivit y of
Piper betle, Alpinia galanga and Allium ascalonicum
ext ract s in vit ro , Res Vet Sci, 84, 2008, 80–4.
23. Gibbs RD, Chemot axonomy of Flowering Plants, 1
M cGill Queen Universit y Press, M ont real; 1974.
st
ed.
31. Villaflores OB, M acabeo APG, Gehle D, KrohnK, Franzblau
SG, Anguinaldo AM . Phyt oconstit uent s from Alpinia
purpurata and their in vit ro inhibit ory act ivit y against
M ycobact erium tuberculosis, Pharmacogn M ag, 6, 2010,
339-44.
32. Raj CA, Sophia D, Ragavendran P, St arlin T, Rathi M A,
Gopalakrishnan VK, Leaf ext ract of Alpinia purpurata (Vieill)
K. Schum screened for it s phyt ochemical const ituent s and
ant ibact erial and ant icancer act ivit ies, Chin J Integr M ed,
10, 2012, 1460-64.
33. Blois M , Ant ioxidant determinat ions by the use of a st able
free radical, Nature, 181, 1958, 1199-1200.
34. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS,
Tannenbaum SR, Analysis of nit rate, nit rite and 15 N in
Biological fluids, Anal Biochem, 126, 1982, 131-36.
35. Font ana M , M osca L, Rosei M A, Int eract ion of Enkephalines
wit h oxyradicals, BiochemPharmacol, 61, 2001, 1253-57.
36. Elizabet h K, Rao M NA, Oxygen radical scavenging act ivit y of
curcumin, Int J Pharm, 58, 1990, 237-40.
37. Ruch RJ, Cheng SJ, Klaunig JE, Prevent ion of cyt ot oxicit y
and inhibit ion of int racellular communication by
ant ioxidant catechins isolated from Chinese green tea.
Carcinogenesis, 10, 1989, 1003-1008.
38. Re R, Pellegrini N, Prot eggente A, Pannala A, Yang M , RiceEvance C, Ant ioxidant activit y applying an improved ABTS
radical cation decolorization assay, Free Radic Biol M ed, 26,
1999, 231-237.
39. Dinis TCP, M adeira VM C, Almeida LM , Act ion of phenolic
derivat ives (Acet o aminophen, Salycilat e and 5aminosalycilate) as inhibitors of membrane lipid
peroxidat ion and as peroxyl radical scavengers, Arch
Biochem Biophys, 315, 1994, 161-169.
24. Arambewela LSR, Arawwawala LDAM , Rat nasooriya WD,
Ant inocicept ive effect s of aqueous and ethanolic ext ract s
of Alpinia calcarata rhizomes in rat s, J Et hnopharmacol, 95,
2004, 311–16.
40. Oyaizu M , St udies on product s of browning react ions:
ant ioxidant activit ies of product s of browning reaction
prepared from glucoseamine. Jpn J Nut r, 44, 1986, 307315.
25. Kadot a S, Tezuka Y, Prasain JK, Ali M S, Banskot a AH, Novel
diarylheptanoids of Alpinia blepharocalyx, Curr Topics M ed
Chem, 3, 2003, 203–25.
41. Benzie IFF, St rain JJ, The ferric reducing abilit y of plasma
(FRAP) as a measure of “ Ant ioxidant power” The FRAP
assay, Anal Biochem, 239, 1996, 70-76.
26. Bendjeddou D, Lalaoui K, Sat t a D, Immunost imulating
act ivit y of t he hot wat er-soluble polysaccharide ext ract s of
Anacyclus pyrethrum , Alpinia galangal and Citrullus
colocynthis, J Et hnopharmacol, 88, 2003, 155–60.
42. Arung ET, Wicaksono BD, Handoko YA, Kusuma IW, Yulia D,
Sandra F, Ant i- cancer propert ies of Diet hylet her Ext ract of
Wood from Sukun (Ant ocarpusalt ilis) in Human Breast
Cancer (T47D) cells), Trop J Pharm Res, 8, 2009, 217-324.
27. Lee CC, Hought on P, Cytot oxicit y of plant s from M alaysia
43. Zhang JH, YU J, Li WX, Cheng CP, Evaluation of M n
2+
st imulated and Zn inhibit ed apopt osis in rat corpus luteal
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
2+
33
Int. J. Pharm. Sci. Rev. Res., 36(2), January – February 2016; Art icle No. 06, Pages: 28-34
ISSN 0976 – 044X
cells by flow cyt omet ry and fluorochromes st aining, Chin J
Physiol, 41, 1998, 121-26.
curiosit y, cause and consequence, Lancet , 344, 1994, 72124.
44. Chen C, Chia-Li W, Shin H, Lin JK, Cyt ot oxic prenyflavanones
from Taiwanese propolis [J]. J Nat Prod, 66, 2003, 503-6.
48. Cao G, Prior RL, Comparision of different analytical
met hods for assessing t ot al antioxidant capacit y of human
serum, ClinChem, 44, 1998, 1309-15.
45. Suzuki I, Hyashi I, Takaki T, Groveman DS, Fujimiya Y,
Ant it umor and ant icyt openic effect s of aqueous ext ract s of
propolis in combinat ion with chemotherapeutic agents,
Can Biot her Radiopharm, 17, 2002, 553-62.
46. De Flora S, Ferguson LR, Overview of mechanisms of cancer
chemoprevent ive agent s, J M ut at Res, 591, 2005, 8-15.
47. Halliwell B, Free radicals, ant ioxidant s and human disease:
49. Sinnathambi A, M azumder PM , Ashok P, Narayanan LS. In
vit ro antioxidant and free radical scavenging activit y of
Alst onia scholaris Linn. R.Br, Iran J Pharmacol Therapeut , 6,
2008, 191-96.
50. Gordon M H, The mechanism of t he antioxidant act ion in
vit ro , In: Hudson BJF, Food Ant ioxidant s.[M ]. London:
Elsevier Aplied Sci, 1990, 1-18.
Source of Support: Nil, Conflict of Interest: None.
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
© Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed.
34