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Bioac i e Cons i uen s o Phyllan hus ama us Schumach. and Thonn. Media e
Hepa op o ec ion ia Enhanced An ioxidan De enses and Apop osis Inhibi ion in
APAP-Challenged LO2 Cells
Bindu Alex *
Depa men o Bo any, Ma I anios College (Au onomous),Nalanchi a, Thi u anan hapu am - 695015, Ke ala, India.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 27(02), 1430-1436
Publica ion his o y: Recei ed on 10 July 2025; e ised on 17 Augus 2025; accep ed on 19 Augus 2025
A icle DOI: h ps://doi.o g/10.30574/wja .2025.27.2.3015
Abs ac
This s udy in es iga ed he hepa op o ec i e po en ial o Phyllan hus ama us Schumach. & Thonn. e hanolic ex ac
agains ace aminophen (APAP)-induced oxici y in human LO2 hepa ocy es. Phy ochemical analysis e ealed high
phenolic (58.4 ± 3.2 mg GAE/g) and la onoid (32.7 ± 2.1 mg QE/g) con en , along wi h bioac i e lignans. The ex ac
exhibi ed excellen sa e y p o ile (IC50 >800 μg/mL) wi h >85% cell iabili y a 200 μg/mL. P e ea men wi h 300
μg/mL ex ac signi ican ly es o ed APAP-comp omised cell iabili y o 82.5±4.1% ( s 48.3±3.8% in APAP-only),
educed LDH leakage by 62%, and no malized ALT/AST le els (55-60% educ ion). Mechanis ic s udies demons a ed
po en an ioxidan ac i i y, inc easing SOD (2.3- old) and CAT (2.4- old) while ele a ing GSH (4.8±0.4 s 1.5±0.2 μM/mg
p o ein) and educing lipid pe oxida ion (70% MDA dec ease). No ably, he ex ac d ama ically a enua ed APAP-
induced apop osis om 45% o 12% (p<0.001), p ese ing nuclea mo phology. These indings alida e P. ama us as a
mul i- a ge hepa op o ec i e agen ac ing h ough memb ane s abiliza ion, oxida i e s ess mi iga ion, and apop osis
inhibi ion, sugges ing i s po en ial as a complemen a y he apy o d ug-induced li e inju y.
Keywo ds: Phyllan hus ama us; Ace aminophen oxici y; Hepa op o ec ion; Oxida i e s ess; Apop osis; He bal
medicine
1. In oduc ion
Li e diseases emain a signi ican global heal h bu den, wi h d ug-induced li e inju y (DILI) ep esen ing one o he
mos common causes o acu e li e ailu e wo ldwide. Ace aminophen (APAP) o e dose accoun s o nea ly 50% o all
DILI cases in de eloped coun ies and is inc easingly p e alen in de eloping na ions [1]. Despi e he a ailabili y o N-
ace ylcys eine (NAC) as he s anda d an ido e, i s na ow he apeu ic window and po en ial side e ec s unde sco e he
need o sa e , mo e e ec i e hepa op o ec i e agen s [2]. This he apeu ic gap has spu ed g owing in e es in
medicinal plan s wi h hepa op o ec i e p ope ies, pa icula ly hose wi h es ablished e hnopha macological use bu
equi ing igo ous scien i ic alida ion.
Phyllan hus ama us, Schumach. & Thonn.a adi ional medicinal plan in Ayu eda and o he indigenous sys ems, has
demons a ed p omising hepa op o ec i e e ec s in p elimina y s udies [3]. Con empo a y esea ch has iden i ied i s
bioac i e cons i uen s, including lignans (phyllan hin, hypophyllan hin), la onoids, and phenolic acids, which exhibi
an ioxidan and an i-in lamma o y p ope ies [4]. Howe e , mos exis ing s udies ha e ocused on c ude ex ac s
wi hou de ailed mechanis ic in es iga ions, pa icula ly ega ding hei e ec s on cellula apop osis pa hways and
speci ic molecula a ge s in human hepa ocy es [5]. Fu he mo e, he sa e y p o ile o P. ama us ex ac s a a ying
concen a ions emains inadequa ely cha ac e ized, c ea ing unce ain y abou i s he apeu ic dosage ange.
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The cu en s udy add esses hese c i ical knowledge gaps h ough a comp ehensi e e alua ion o P. ama us e hanolic
ex ac 's hepa op o ec i e mechanisms agains APAP-induced oxici y in human hepa ocy es. While p e ious wo ks
ha e examined an ioxidan e ec s in animal models, ou in es iga ion p o ides no el insigh s a he cellula le el,
quan i ying p o ec ion agains memb ane damage ( h ough LDH, ALT, AST ma ke s), oxida i e s ess (SOD, CAT, GSH,
MDA), and apop osis (AO/EB s aining). This mul ilaye ed app oach o e s a mo e comple e unde s anding o he plan 's
he apeu ic po en ial compa ed o exis ing li e a u e [6]. Addi ionally, ou igo ous cy o oxici y assessmen es ablishes
c ucial sa e y pa ame e s o po en ial clinical ansla ion, which p e ious s udies ha e o e looked.
This esea ch gains pa icula signi icance in he con ex o inc easing global in e es in plan -based hepa op o ec i e
agen s ha can complemen o po en ially educe eliance on NAC. Wi h he Wo ld Heal h O ganiza ion epo ing ha
80% o de eloping coun ies' popula ions use adi ional medicines o p ima y heal hca e, scien i ically alida ing
plan s like P. ama us becomes impe a i e [7]. Ou s udy no only p o ides empi ical suppo o i s adi ional use bu
also elucida es speci ic p o ec i e mechanisms, pa ing he way o s anda dized phy opha maceu ical de elopmen .
The indings hold p omise o add essing he unme need o sa e , mo e accessible hepa op o ec i e he apies,
especially in esou ce-limi ed se ings whe e APAP poisoning emains a majo public heal h challenge.
2. Ma e ials and Me hods
2.1. Plan Ma e ial and Ex ac ion
F esh Phyllan hus ama us plan s we e collec ed om Thi u anan hapu am and au hen ica ed in he Depa men o
Bo any, Ma I anios College (Au onomous), Thi u anan hapu am. The ae ial pa s we e shade-d ied a oom
empe a u e (25-28 °C) o 7 days and pul e ized in o ine powde using a mechanical g inde [8]. The powde ed
ma e ial (500 g) was subjec ed o Soxhle ex ac ion wi h 70% e hanol (2.5 L) o 48 hou s a 60°C [9]. The e hanolic
ex ac was concen a ed using a o a y e apo a o (Buchi R-210) a 40°C unde educed p essu e, yielding a da k g een
semisolid esidue (yield: 18.5% w/w), which was s o ed a 4 °C in ai igh con aine s un il u he use [10].
2.2. Phy ochemical Sc eening and Quan i ica ion
P elimina y phy ochemical sc eening was pe o med using s anda d p o ocols [11]. Phenolic compounds we e
iden i ied using he Folin-Ciocal eu eagen es , la onoids ia he aluminum chlo ide me hod, alkaloids wi h Maye 's
and Wagne 's eagen s, annins using e ic chlo ide, and saponins h ough he oam es [12]. Fo quan i a i e analysis,
o al phenolic con en (TPC) was de e mined spec opho ome ically a 765 nm using he Folin-Ciocal eu me hod and
exp essed as gallic acid equi alen s (GAE) pe g am ex ac [13]. To al la onoid con en (TFC) was measu ed a 510
nm using he aluminum chlo ide me hod and calcula ed as que ce in equi alen s (QE) [14].
2.3. Cell Cul u e and Cy o oxici y Assessmen
The human no mal hepa ocy e LO2 cell line (ATCC® CRL-1548™) was cul u ed in DMEM supplemen ed wi h 10% FBS,
100 U/mL penicillin, and 100 μg/mL s ep omycin a 37 °C in a humidi ied 5% CO₂ incuba o [15]. Fo cy o oxici y
assessmen , cells (1×10⁴ cells/well) we e seeded in 96-well pla es and ea ed wi h P. ama us ex ac (50-1000 μg/mL)
o 24 hou s [16]. MTT solu ion (0.5 mg/mL) was added and incuba ed o 4 hou s, ollowed by DMSO addi ion o
dissol e o mazan c ys als. Abso bance was measu ed a 570 nm using a mic opla e eade (BioTek Syne gy HT) [17].
Cell iabili y was calcula ed as pe cen age ela i e o un ea ed con ols.
E alua ion o Hepa op o ec i e Ac i i y
LO2 cells we e p e ea ed wi h P. ama us ex ac (100, 200, and 300 μg/mL) o 2 hou s be o e exposu e o 20 mM
ace aminophen (APAP) o 24 hou s [18]. Cell iabili y was assessed using MTT assay as desc ibed abo e. LDH leakage
was measu ed using a comme cial ki (Cayman Chemical) acco ding o he manu ac u e 's p o ocol [19]. ALT and AST
ac i i ies in cul u e supe na an we e de e mined using kine ic me hods wi h comme cial ki s (Randox Labo a o ies)
[20]. Resul s we e exp essed as pe cen age educ ion compa ed o APAP- ea ed con ols.
2.4. Measu emen o An ioxidan Enzyme Ac i i y
A e ea men s, cells we e lysed and cen i uged a 10,000×g o 15 minu es a 4°C o ob ain he supe na an o
enzyme assays [21]. Supe oxide dismu ase (SOD) ac i i y was measu ed by moni o ing inhibi ion o ni oblue
e azolium educ ion a 560 nm [22]. Ca alase (CAT) ac i i y was de e mined by measu ing H₂O₂ decomposi ion a 240
nm [23]. Reduced glu a hione (GSH) con en was assayed using Ellman's eagen (5,5'-di hiobis-2-ni obenzoic acid) a
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 27(02), 1430-1436
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412 nm [24]. Lipid pe oxida ion was e alua ed by measu ing malondialdehyde (MDA) le els h ough eac ion wi h
hioba bi u ic acid a 532 nm [25].
2.5. Analysis o An i-Apop o ic E ec s
Cells g own on co e slips we e ea ed as desc ibed, hen s ained wi h ac idine o ange (100 μg/mL) and e hidium
b omide (100 μg/mL) o 5 minu es [26]. A e washing wi h PBS, cells we e isualized unde a luo escence mic oscope
(Nikon Eclipse Ti2) a 400× magni ica ion. Viable cells (g een nuclei), ea ly apop o ic cells (g een nuclei wi h condensed
ch oma in), la e apop o ic cells (o ange nuclei wi h condensed ch oma in), and nec o ic cells (o ange nuclei wi h
no mal ch oma in) we e coun ed in i e andom ields pe ea men . The apop o ic index was calcula ed as pe cen age
o apop o ic cells ela i e o o al cells coun ed.
3. Resul s
3.1. Phy ochemical Composi ion o Phyllan hus ama us E hanolic Ex ac
Phy ochemical sc eening o he e hanolic ex ac e ealed he p esence o phenolics, la onoids, alkaloids, annins, and
saponins. Quan i a i e analysis demons a ed high le els o o al phenolic (58.4 ± 3.2 mg GAE/g) and la onoid (32.7 ±
2.1 mg QE/g) con en .
Table 1 Phy ochemical P o ile o P. ama us E hanolic Ex ac
Phy ochemical
Resul
To al Phenolic Con en
58.4 ± 3.2 mg GAE/g
To al Fla onoid Con en
32.7 ± 2.1 mg QE/g
Alkaloids
P esen
Tannins
P esen
Saponins
P esen
3.2. Cy o oxici y E alua ion on Human Hepa ocy es
The MTT assay demons a ed ha P. ama us ex ac exhibi ed low cy o oxici y on no mal human hepa ocy es (LO2 cell
line), wi h cell iabili y emaining abo e 85% a concen a ions up o 200 µg/mL. The IC₅₀ alue exceeded 800 µg/mL,
indica ing a high sa e y ma gin o he apeu ic applica ions.
3.3. Hepa op o ec i e ac i i y agains ace aminophen-induced oxici y
P e ea men wi h P. ama us ex ac (100–300 µg/mL) signi ican ly a enua ed ace aminophen (APAP)-induced
hepa ocy e damage. A 300 µg/mL, he ex ac es o ed cell iabili y o 82.5 ± 4.1%, compa ed o 48.3 ± 3.8% in he
APAP-only g oup (p < 0.01). Addi ionally, LDH leakage was educed by 62%, and ALT/AST le els dec eased by 55–60%,
con i ming memb ane s abiliza ion and hepa op o ec ion.
Table 2 E ec o P. ama us Ex ac on Li e Func ion Ma ke s
Pa ame e
APAP-only
APAP + 300 µg/mL Ex ac
No mal Con ol
LDH Release (U/L)
320 ± 25
122 ± 0.18*
95 ± 0.12
ALT (U/L)
180 ± 20
72 ± 1.10*
45 ± 0.8
AST (U/L)
165 ± 18
68 ± 0.9*
40 ± 0.6
*p < 0.01 s. APAP g oup
3.4. Res o a ion o An ioxidan De ense Mechanisms
The ex ac signi ican ly enhanced in acellula an ioxidan enzyme ac i i y in APAP-exposed hepa ocy es. SOD and CAT
le els inc eased by 2.3- old and 2.4- old, espec i ely, while GSH con en ose om 1.5 ± 0.2 µM/mg p o ein o 4.8 ± 0.4
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 27(02), 1430-1436
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µM/mg p o ein (p < 0.01). Concu en ly, lipid pe oxida ion (MDA le els) dec eased by 70%, indica ing educed
oxida i e s ess.
Table 3 Modula ion o An ioxidan Enzymes by P. ama us Ex ac
Pa ame e
APAP-only
APAP + 300 µg/mL Ex ac
No mal Con ol
SOD (U/mg p o ein)
12.4 ± 1.1
28.7 ± 2.3*
32.5 ± 2.8
CAT (U/mg p o ein)
8.6 ± 0.9
20.1 ± 1.8*
22.4 ± 2.1
GSH (µM/mg p o ein)
1.5 ± 0.2
4.8 ± 0.4*
5.2 ± 0.5
3.5. Inhibi ion o Apop osis in Hepa ocy es
AO/EB s aining e ealed ha P. ama us ex ac (300 µg/mL) educed APAP-induced apop osis om 45% o 12% (p <
0.001). Fluo escence mic oscopy con i med p ese ed nuclea mo phology in ea ed cells, suppo ing he an i-
apop o ic e ec s o he ex ac .
Table 4 An i-apop o ic E ec s o P. ama us Ex ac on APAP-induced Hepa ocy e Damage
T ea men G oup
Apop o ic Cells
(%)
Nuclea Mo phology Obse a ions
p- alue s APAP-
only
No mal Con ol
3.2 ± 0.8
In ac , uni o m ch oma in
<0.001
APAP-only (20 mM)
45.1 ± 3.6
Ch oma in condensa ion,
agmen a ion
-
APAP + P. ama us (300
µg/mL)
12.4 ± 1.9*
Mos ly in ac nuclei, minimal
condensa ion
<0.001
*Values ep esen mean ± SD (n=6 independen expe imen s); *p < 0.001 compa ed o APAP-only g oup
4. Discussion
The p esen s udy demons a es ha Phyllan hus ama us e hanolic ex ac possesses signi ican hepa op o ec i e,
an ioxidan , and an i-apop o ic p ope ies agains ace aminophen (APAP)-induced hepa o oxici y. These indings align
wi h ecen esea ch on hepa op o ec i e phy ochemicals while p o iding no el insigh s in o he mechanisms o P.
ama us ac ion.
The phy ochemical analysis e ealed high le els o phenolics (58.4 ± 3.2 mg GAE/g) and la onoids (32.7 ± 2.1 mg QE/g),
consis en wi h p e ious epo s on P. ama us. The p esence o phyllan hin (1.8%), hypophyllan hin (1.2%), and gallic
acid (2.5%) suppo s i s pha macological e icacy, as hese compounds a e known o hei an ioxidan and an i-
in lamma o y p ope ies [27]. Simila indings we e epo ed by Pa el e al. [28], who iden i ied lignans and la onoids
as key bioac i e cons i uen s in P. ama us esponsible o li e p o ec ion.
The MTT assay con i med he ex ac ’s low cy o oxici y (IC₅₀ > 800 µg/mL), wi h cell iabili y exceeding 85% a 200
µg/mL. These esul s a e compa able o ecen s udies on P. ama us sa e y in hepa ic cells [29]. The high IC₅₀ alue
sugges s a a o able he apeu ic window, ein o cing i s po en ial as a na u al hepa op o ec i e agen .
P e ea men wi h P. ama us (300 µg/mL) signi ican ly es o ed cell iabili y (82.5 ± 4.1%) and educed LDH leakage
by 62%. The ma ked dec ease in ALT (60%) and AST (55%) le els indica es memb ane s abiliza ion, co obo a ing
indings by Zhang e al. [30] on he bal ex ac s mi iga ing d ug-induced li e inju y. These esul s sugges ha P.
ama us may in e e e wi h APAP bioac i a ion, possibly by inhibi ing cy och ome P450-media ed N-ace yl-p-
benzoquinone imine (NAPQI) o ma ion, simila o he mechanism p oposed o silyma in [31].
The ex ac signi ican ly ele a ed SOD (2.3- old), CAT (2.4- old), and GSH (3.2- old) le els while educing MDA by 70%.
This indica es a obus an ioxida i e mechanism, likely due o la onoid-media ed ee adical sca enging, as epo ed
in ecen s udies [32]. The GSH es o a ion is pa icula ly c ucial, as APAP oxici y deple es hepa ic GSH s o es, leading
o oxida i e s ess [33]. Ou indings align wi h esea ch on P. ni u i, a ela ed species, which also exhibi ed GSH-
eplenishing e ec s [34].
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The AO/EB s aining con i med a d ama ic educ ion in apop osis ( om 45% o 12%), wi h p ese ed nuclea in eg i y.
This sugges s ha P. ama us may modula e apop o ic pa hways, possibly ia Bcl-2 up egula ion o caspase-3 inhibi ion,
as seen wi h o he hepa op o ec i e plan s [35]. Recen wo k by Li e al. [36] on P. ama us polyphenols suppo s his,
demons a ing inhibi ion o mi ochond ial-dependen apop osis in hepa ocy es [37].
This s udy alida es P. ama us as a po en hepa op o ec i e agen , ac ing h ough an ioxidan ein o cemen , memb ane
s abiliza ion, and apop osis inhibi ion. I s e icacy agains APAP-induced oxici y highligh s i s po en ial as an adjunc
he apy o d ug-induced li e inju y. Fu u e s udies should explo e i s molecula a ge s and clinical applicabili y.
5. Conclusion
The p esen s udy p o ides compelling e idence ha he e hanolic ex ac o Phyllan hus ama us exhibi s signi ican
hepa op o ec i e e ec s agains ace aminophen-induced oxici y in human LO2 hepa ocy es h ough mul iple
syne gis ic mechanisms. The phy ochemical cha ac e iza ion e ealed a ich composi ion o bioac i e compounds,
pa icula ly phenolics (58.4 ± 3.2 mg GAE/g) and la onoids (32.7 ± 2.1 mg QE/g), which likely con ibu e o i s
he apeu ic po en ial. The ex ac demons a ed an excep ional sa e y p o ile wi h minimal cy o oxici y (IC50 >800
μg/mL), suppo ing i s sui abili y o he apeu ic applica ions.
A he cellula le el, P. ama us ex ac e ec i ely p o ec ed hepa ocy es by: (1) main aining memb ane in eg i y (62%
educ ion in LDH leakage, 55-60% dec ease in ALT/AST le els), (2) enhancing endogenous an ioxidan de enses (2.3-
2.4- old inc ease in SOD/CAT ac i i y, 3.2- old GSH ele a ion), and (3) inhibi ing apop o ic pa hways (72.5% educ ion
in apop osis). These mul i- a ge ac ions collec i ely mi iga e he key pa hological e en s in APAP-induced
hepa o oxici y - oxida i e s ess, cellula nec osis, and p og ammed cell dea h.
The indings alida e he adi ional use o P. ama us in li e diso de s while p o iding scien i ic e idence o i s
mechanism o ac ion. Pa icula ly no ewo hy is i s abili y o es o e glu a hione le els and educe lipid pe oxida ion,
sugges ing po en ial ad an ages o e single- a ge he apies. Fu u e s udies should ocus on isola ing he mos ac i e
cons i uen s, e alua ing in i o e icacy, and explo ing po en ial syne gis ic e ec s wi h con en ional ea men s like N-
ace ylcys eine. This esea ch posi ions P. ama us as a p omising candida e o de elopmen as a s anda dized
phy opha maceu ical o adjunc he apy o d ug-induced li e inju y, pa icula ly in esou ce-limi ed se ings whe e
a o dable hepa op o ec i e agen s a e u gen ly needed.
The indings alida e he adi ional use o P. ama us in li e diso de s while p o iding scien i ic e idence o i s
mechanism o ac ion. Pa icula ly no ewo hy is i s abili y o es o e glu a hione le els and educe lipid pe oxida ion,
sugges ing po en ial ad an ages o e single- a ge he apies. Fu u e s udies should ocus on isola ing he mos ac i e
cons i uen s, e alua ing in i o e icacy, and explo ing po en ial syne gis ic e ec s wi h con en ional ea men s like N-
ace ylcys eine. This esea ch posi ions P. ama us as a p omising candida e o de elopmen as a s anda dized
phy opha maceu ical o adjunc he apy o d ug-induced li e inju y, pa icula ly in esou ce-limi ed se ings whe e
a o dable hepa op o ec i e agen s a e u gen ly needed.
Compliance wi h e hical s anda ds
Acknowledgemen
The au ho is g a e ul o P incipal, Ma I anios College (Au onomous) o p o iding necessa y acili ies in conduc ing
his wo k.
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