Invest Clin 67(1): 92 - 107, 2026 https://doi.org/10.54817/IC.v67n1a07

Corresponding author: Yingtao Zhang. Department of Nephrology, Yan’an People’s Hospital, Yan’an, 716000, China.

E-mail: snkzyt@sina.com

Phyllanthin identified from Phyllanthus

amarus attenuates arsenite-induced liver

and kidney damage: Role of NF-kB pathway

inhibition.

Jiarui Xu1, Smeeta Sadar2, Hemant Kamble3 and Yingtao Zhang4

1Department of Poisoning and Occupational Diseases, Emergency Center, Shandong

Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.

2Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune, Maharashtra.

3Shri Wagheshwar Gramvikas Pratishthan’s Pharate Patil College of Pharmacy,

Mandavgan Pharata, Maharashtra, India.

4Department of Nephrology, Yan’an People’s Hospital, Yan’an, China.

Keywords: Antioxidant; Hepatotoxicity; Nephrotoxicity; NF-κb; Oxidative stress;

Phyllanthus amarus; Sodium arsenite.

Abstract. Sodium arsenite is a common and highly toxic inorganic arse-

nic compound that causes liver and kidney damage. Phyllanthus amarus is well

known for its protective effects on these organs. This study aimed to identify

the active phytoconstituents of the methanolic extract of P. amarus (PAME) and

to explore their effects on arsenite-induced liver and kidney toxicity in experi-

mental rats. The standardization of P. amarus extract was performed using high-

performance liquid chromatography (HPLC). Male Wistar rats developed liver

and kidney toxicity after daily oral administration of sodium arsenite (5 mg/

kg) for 4 weeks. The rats were simultaneously given coenzyme Q10 (CoQ10; 10

mg/kg) or PAME (50, 100, and 200 mg/kg). Results showed that HPLC analy-

sis detected phyllanthin at a retention time of 25.41 minutes with an area of

71.84%. Arsenite treatment caused a significant (p<0.001) increase in hepatic

enzymes (ALT, AST, and ALP), renal markers (BUN, uric acid, and creatinine),

and direct and total bilirubin in the serum. It also significantly increased hepatic

and renal levels of malondialdehyde, nitric oxide, NF-κB p65, interleukins (ILs),

and TNF-α (p<0.001), while decreasing hepatic antioxidant enzymes (GSH and

SOD) and overall hepatic antioxidant capacity. Notably, P. amarus extract (200

mg/kg) markedly (p<0.001) mitigated arsenite-induced changes in these serum

markers, oxidative stress indicators, NF-kB p65, and inflammatory cytokines. It

also improved the structure of liver and kidney tissues, maintained cellular ar-

chitecture, and reduced necrosis and inflammation. In conclusion, these results

suggest that phyllanthin from P. amarus protects against arsenite-induced liver

and kidney damage by inhibiting NF-κB activation, reducing inflammatory cyto-

kine release, and decreasing oxidative and nitrosative stress, thereby enhancing

overall antioxidant capacity. Therefore, P. amarus extract may be a promising

treatment for pesticide-related liver and kidney injuries in rats.

Phyllanthin protected arsenite-induced toxicity 93

Vol. 67(1): 92 - 107, 2026

La filantina identificada en Phyllanthus amarus atenúa

el daño hepático y renal inducido por arsenito: papel

de la inhibición de la vía NF-Kb.

Invest Clin 2026; 67 (1): 92 – 107

Palabras clave: Antioxidante; Hepatotoxicidad; Nefrotoxicidad; NF-κb; Estrés oxidativo;

Phyllanthus amarus; Arsenito de sodio.

Resumen. El arsenito de sodio es un compuesto inorgánico de arsénico,

de prevalencia elevada y altamente tóxico, que causa toxicidad hepática y

renal. Phyllanthus amarus está bien documentado por sus efectos hepato-

protectores y nefroprotectores. Este estudio tuvo como objetivo examinar los

fitoconstituyentes activos del extracto metanólico de P. amarus (PAME) y sus

mecanismos de acción sugeridos contra la toxicidad hepática y renal inducida

por el arsenito en ratas experimentales. La estandarización del extracto de P.

amarus se realizó mediante cromatografía líquida de alta resolución (HPLC).

Ratas Wistar machos desarrollaron toxicidad hepática y renal tras la adminis-

tración oral continua de arsenito de sodio (5 mg/kg) durante 4 semanas. A

las ratas se les administró por vía oral coenzima Q10 (CoQ10; 10 mg/kg) o

PAME (50, 100 y 200 mg/kg) de forma concomitante. En los resultados, el

análisis de HPLC mostró la presencia de filantina con un tiempo de retención

de 25,41 min y un área de 71,84%. La administración de arsenito dio lugar

a un aumento significativo (p<0,001) de las enzimas hepáticas (ALT-alanina

aminotransferasa), AST (aspartato aminotransferasa) y ALP (fosfatasa alcali-

na), de las enzimas renales (BUN (nitrógeno ureico en sangre), ácido úrico

y creatinina) y de la bilirrubina directa y total en el suero. También elevó

efectivamente (p<0,001) los niveles hepáticos y renales de malondialdehído,

óxido nítrico, NF-κB (factor nuclear kappa de la cadena ligera de las células

B activadas) p65, IL (interleucinas) y TNF-α (factor de necrosis tumoral alfa),

y disminuyó las enzimas antioxidantes GSH (glutatión) y SOD (superóxido

dismutasa), así como la capacidad antioxidante total hepática. Sin embar-

go, el extracto de P. amarus (200 mg/kg) atenuó notablemente (p<0,001)

las alteraciones inducidas por el arsenito en estos marcadores séricos, los

parámetros de estrés oxidativo, NF-κB p65 y los niveles de citoquinas infla-

matorias. También mejoró la histología hepática y renal, preservó la arquitec-

tura celular y redujo la necrosis e inflamación. En conclusión, estos hallazgos

sugieren que la filantina de P. amarus ejerce efectos protectores contra la

hepatotoxicidad y la nefrotoxicidad inducidas por el arsenito al inhibir la ac-

tivación de NF-κB y disminuir la liberación de citoquinas inflamatorias y el

estrés oxidativo-nitrosativo, mejorando así la capacidad antioxidante general.

Por lo tanto, el extracto de P. amarus podría constituir un tratamiento eficaz

para el daño hepático y renal inducido por pesticidas en ratas.

Received: 20-10-2025 Accepted: 12-11-2025

94 Xu et al.

Investigación Clínica 67(1): 2026

INTRODUCTION

Sodium arsenite is a common, highly

toxic inorganic arsenic compound. Environ-

mental exposure to arsenic through drink-

ing arsenic-contaminated groundwater can

cause several health hazards, particularly af-

fecting the liver and kidneys 1. In more than

70 countries, approximately 140 million

people drink water with arsenic concentra-

tions exceeding the WHO provisional limit

of 10 μg/L 2. According to models, approxi-

mately 94–220 million people are at risk of

exposure to high arsenic levels in groundwa-

ter3. When exposed, sodium arsenite induces

inflammation and oxidative stress, primarily

in the kidneys and liver, thereby disrupting

homeostasis.

Disruption of this balance, along with

the reactive nitrogen species (RNS) and

reactive oxygen species (ROS)-mediated

induction of oxidative stress, triggers he-

patocellular injury 4. Additionally, arsenite-

induced hepatotoxicity and nephrotoxicity

are characterized by mitochondrial damage

and ATP depletion, which in turn lead to oxi-

dative stress. This is evidenced by increased

oxidative markers, such as lipid peroxida-

tion (malondialdehyde, MDA) and nitrite/

nitrate levels, as well as decreased antioxi-

dant defenses, including glutathione (GSH),

catalase, and superoxide dismutase (SOD) 5.

Sodium arsenite enhances pro-inflammatory

signaling pathways and induces apoptosis in

renal and hepatic tissues by upregulating

proteins such as caspase-3 and tumor necro-

sis factor-alpha (TNF-α) 5,6. Consequently, re-

searchers aim to strengthen the antioxidant

defense system to reduce the production of

free radicals induced by arsenite toxicity.

Current therapeutic strategies for

arsenite-induced toxicity involve chelation

therapy and antioxidant treatment. Che-

lation therapy is a standard method, with

2,3-dimercaptosuccinic acid (DMSA) being

the most commonly used chelating agent,

which helps bind and remove arsenic from

the bloodstream. However, DMSA alone is

not enough to eliminate arsenic from in-

tracellular compartments, leading to toxic-

ity and cell damage. As a result, combined

therapies are being investigated to improve

their effectiveness 7. A new approach is to

add antioxidants alongside chelators. Coen-

zyme Q10 (CoQ10), which has antioxidant

properties, protects against arsenic-induced

intracellular damage and, when used with

DMSA, offers enhanced protection against

arsenite toxicity 7. Dual therapy not only

promotes the removal of arsenic from the

extracellular space but also protects against

intracellular arsenic toxicity, demonstrating

broader pharmacological benefits in arsenic

poisoning. However, these treatments are

very costly, necessitating affordable alterna-

tives. Plant-derived medicinal compounds

present a promising, low-cost option for ad-

dressing arsenite-induced toxicity.

To counteract sodium arsenite toxicity,

many studies have examined the protective

effects of both natural and synthetic mol-

ecules. For example, naringin, hesperidin,

and lipoic acid have been shown to reduce

arsenic toxicity by restoring biochemical pa-

rameters, decreasing oxidative stress, and

inhibiting inflammatory and apoptotic cas-

cades8,9. These findings support the potential

of such molecules to lessen sodium arsenite-

induced hepatic and renal damage through

their antioxidant and anti-inflammatory

properties. Phyllanthus amarus Schum. and

Thonn., also known as Bhuia amla, is a me-

dicinal herb of great importance in the sci-

entific field of Ayurvedic medicine 10. It has

been traditionally used for over 2000 years

to treat secondary hepatitis and various liver

injuries 10. It has numerous traditional uses

and is commonly employed to treat condi-

tions such as jaundice, gonorrhea, heavy

menstruation, and diabetes 11. Qualitative

analyses of the phytochemical composition

of P. amarus have identified a wide range of

compounds, including lignans (phyllanthin

and hypophyllanthin), alkaloids, and biofla-

vonoids (e.g., quercetin). While it remains to

be confirmed which of these possesses anti-

Phyllanthin protected arsenite-induced toxicity 95

Vol. 67(1): 92 - 107, 2026

oxidant properties, scientific reports indicate

that the herb exerts maximal effects on the

liver and kidneys 10,12-14. Such liver specificity

is rooted in its traditional use for jaundice,

as evidenced by the report by Santos et al. 15.

Studies have demonstrated the antioxidant

effect of the ethanolic extract of P. amarus,

indicating its protective role in experimen-

tal models of kidney and liver damage 11-14.

However, the exact mechanisms underlying

the hepatoprotective and nephroprotective

effects of these compounds against arsenite-

mediated hepatic and renal toxicities still re-

main unknown. Therefore, this study aimed

to investigate the biochemical mechanisms

and phytoconstituents responsible for the

hepatoprotective effects of P. amarus in an

experimental model of arsenite-induced liver

and kidney damage.

MATERIALS AND METHODS

P. amarus methanolic extract -

preparation and identification

Air-dried powder from P. amarus aeri-

al parts underwent maceration at ambient

temperature using methanol (distilled).

This process involved soaking and occasional

agitation for 7 days, followed by filtration.

The filtrate was dried in a tray dryer at 40°C,

yielding a semi-solid P. amarus methano-

lic extract (PAME). Subsequently, colloidal

silicon dioxide was incorporated, and the

mixture was dried in a vacuum tube. Phyto-

chemical analysis of PAME was conducted

using high-performance liquid chromatogra-

phy (HPLC) to quantify phyllanthin content.

Analyses were conducted using an HPLC sys-

tem (reverse-phase C18 column, 250 × 4.6

mm, flow rate 1.5 mL/min). For isolation

and detection, a mobile phase comprising

acetonitrile and buffer in a 40:60 volume ra-

tio was employed. The buffer was prepared

by dissolving potassium hydrogen phosphate

(0.136 g) in o-phosphoric acid (0.5 mL). The

optimal injection volume was 20 μL, and the

detector wavelength was set to 230 nm. The

autosampler temperature was maintained at

10°C, and the system operated at 1000 psi 16.

Animals

White male Wistar rats aged 8–10

weeks were obtained from the animal facil-

ity at Shandong First Medical University.

The rats were kept in an environment with

controlled temperature (24 ± 1°C), humid-

ity (45-55%), and a normal light-dark cycle.

During the study, the rats had free access to

standard pellet feed and water. The Zhinan-

zhen Biology Ethics Committee approved

the research protocol (approval number:

A2024000414).

Experimental design

The rats were divided into six groups of

15 animals each and received the following

treatments: Group 1: gum acacia (1% sus-

pension, 10 mg/kg; Normal group), Group

2: gum acacia (1% suspension, 10 mg/kg) +

Sodium arsenite (5 mg/kg) (vehicle control

group), Group 3: Coenzyme Q10 (10 mg/

kg, 1% suspension in gum acacia) + sodium

arsenite (5 mg/kg) (CoQ10-treated group),

and Groups 4 to 6: standardized extract of P.

amarus (50 mg/kg, 100 mg/kg, or 200 mg/

kg, 1% suspension in gum acacia) + sodium

arsenite (5 mg/kg) (PA-treated groups), all

administered orally for 28 days.

On the final day of the experiment

(day 28), blood samples were collected from

anesthetized rats via retro-orbital puncture,

stored in glass tubes, and centrifuged for 10

minutes at 2,000 × g at 4°C. Serum levels

of albumin, ALT (alanine transaminase),

AST (aspartate transaminase), ALP (alkaline

phosphatase), bilirubin (direct and total),

BUN (Blood Urea Nitrogen), cholesterol,

creatinine, LDL (Low-Density Lipoprotein),

HDL (High-Density Lipoprotein), LDH (Lac-

tate Dehydrogenase), triglycerides, and

uric acid were measured using reagent kits

according to the provided procedure (Ac-

curex Biomedical Pvt. Ltd., Mumbai, India).

The animals were then euthanized, and the

96 Xu et al.

Investigación Clínica 67(1): 2026

liver and kidneys were quickly removed and

weighed with a balance at temperatures be-

low 4°C. The tissues were divided into three

sections and stored at -80°C. One section

was used to assess oxidative and nitrosative

stress markers (MDA (malondialdehyde),

GSH (reduced glutathione), NO (nitric ox-

ide), SOD (superoxide dismutase) activity)

and total antioxidant capacity (TAC) fol-

lowing previously reported methods 16-22.

Another portion was analyzed to determine

the concentrations of pro-inflammatory cy-

tokines (IL-6, IL-1β, and TNF-α) and NF-κB

p65 using a commercially available ELISA

kit (Thermo Fisher Scientific, USA). The re-

maining tissue was examined histologically

using hematoxylin and eosin (H&E) stain-

ing. Changes observed in the histological

characteristics were classified according to

a previously established grading system 23.

Statistical analysis

GraphPad Prism software (version 5.0;

GraphPad, San Diego, USA) was used for

statistical analysis. One-way analysis of vari-

ance (ANOVA) followed by Dunnett’s post

hoc test was performed. A two-sided Fisher’s

exact test was used to calculate the correla-

tion coefficients. The results are presented

as mean ± SEM, with statistical significance

set at p<0.05.

RESULTS

Phyllanthin - Isolation and identification

PAME had a 59.12% w/w yield and con-

tained glycosides, lignans, steroids, tannins,

and phenols. HPLC column analysis lasted

40 minutes, during which phyllanthin was

detected at 25.41 minutes, with a peak area

of 71.84% (Fig. 1).

Body, liver, kidney, and spleen weights

Body weight was effectively decreased

(p<0.001), while a significant (p<0.001)

increase in spleen, kidney, and liver weights

(both absolute and relative) was observed

in vehicle control rats compared to nor-

mal rats. Rats treated with PA (200 mg/kg)

showed a significant (p<0.001) reduction in

the elevated weights of the spleen, kidney,

and liver, along with a marked (p<0.001)

increase in body weight compared to vehi-

cle control rats. However, PA treatment at

doses of 50 and 100 mg/kg did not produce

any notable changes in the absolute or rela-

tive weights of the liver, spleen, and kidneys,

nor in body weight, compared to the vehicle

control group. CoQ (10 mg/kg) treatment

effectively decreased spleen, kidney, and

liver weights (p<0.001) and increased body

weight (p<0.001) compared to the vehicle

control group (Table 1).

Fig. 1. HPLC chromatogram of the standardized P. amarus extract showing a phyllanthin peak at RT = 25.41

min. mAU, milli-absorbance units.

Phyllanthin protected arsenite-induced toxicity 97

Vol. 67(1): 92 - 107, 2026

Serum parameters

Compared to the normal group, serum

levels of BUN, uric acid, creatinine, direct

and total bilirubin, LDH, ALP, AST, and ALT

were significantly (p<0.001) elevated, while

albumin levels were notably (p<0.001) de-

creased in the vehicle control group. These

changes suggest substantial hepatic and

renal injury caused by sodium arsenite, as

these parameters are closely linked to liver

and kidney functions. Treatment with PA

(200 mg/kg) led to significant improve-

ments, evidenced by a marked (p<0.001) re-

duction in serum BUN, ALT, AST, creatinine,

uric acid, direct bilirubin, total bilirubin,

LDH, and ALP levels, along with a significant

(p<0.001) increase in albumin compared to

the vehicle control group. Additionally, CoQ

(10 mg/kg) significantly (p<0.001) inhib-

ited arsenite-induced changes in serum cre-

atinine, BUN, uric acid, albumin, bilirubin

(direct and total), LDH, AST, ALT, and ALP

levels compared to the control (Tabla 2).

Lipid profile

Following chronic sodium arsenite

administration, a significant reduction

(p<0.001) in serum HDL and a notable

(p<0.001) increase in serum cholesterol,

LDL, and triglyceride levels were observed

in vehicle control rats compared to normal

rats. These decreases in serum HDL and the

elevations in cholesterol, LDL, and triglyc-

eride levels were clearly (p<0.001) reduced

with PA (200 mg/kg) treatment. Similarly,

CoQ (10 mg/kg) administration result-

ed in significant improvements, increas-

ing (p<0.001) HDL levels and decreasing

(p<0.001) LDL, cholesterol, and triglycer-

ide levels in the serum compared to vehicle

control rats. However, no significant chang-

es in serum cholesterol, HDL, LDL, or tri-

glyceride levels were seen following PA (50

and 100 mg/kg) treatments (Table 3).

Hepatic and renal antioxidant parameters

Compared with normal rats, sodium ar-

senite administration significantly reduced

hepatic total antioxidant capacity (TAC) in

the vehicle control group. However, PA (200

mg/kg) effectively increased hepatic TAC

(p<0.001) compared to the vehicle control

group. Notably, CoQ (10 mg/kg) also sig-

nificantly increased hepatic TAC (p<0.01)

relative to the vehicle control group (Fig.

2A). Compared to the normal group, sodi-

um arsenite markedly affected hepatic and

renal antioxidant levels, as indicated by a

significant (p<0.001) decrease in hepatic

and renal GSH and SOD levels, followed by

Table 1. Effect of P. amarus on body weight and organ weights.

Treatment Normal Vehicle control CoQ (10) PA (50) PA (100) PA (200)

Body weight (g) 239.70 ± 3.81 213.20 ± 3.47### 234.70 ± 4.17*** 211.70 ± 2.94 218.00 ± 1.39 223.50 ± 2.35***

Liver weight (g) 5.48 ± 0.22 7.34 ± 0.29### 5.52 ± 0.33*** 7.23 ± 0.30 7.31 ± 0.15 5.73 ± 0.29***

Liver weight /

Body weight

22.90 ± 1.11

34.47 ± 1.54###

23.65 ± 1.79***

34.20 ± 1.57

33.51 ± 0.60

25.69 ± 1.43***

Kidney weight (g) 1.20 ± 0.01 1.81 ± 0.01### 1.29 ± 0.01*** 1.77 ± 0.01 1.79 ± 0.01 1.30 ± 0.01***

Kidney weight /

Body weight

5.01 ± 0.08

8.49 ± 0.16###

5.50 ± 0.14***

8.39 ± 0.13

8.21 ± 0.06

5.81 ± 0.09***

Spleen weight (g) 0.19 ± 0.02 0.75 ± 0.02### 0.36 ± 0.02*** 0.74 ± 0.03 0.73 ± 0.02 0.52 ± 0.03**

Spleen weight

/ Body weight

(x10–3)

0.79 ± 0.08

3.53 ± 0.12###

1.54 ± 0.11***

3.52 ± 0.19

3.33 ± 0.07

2.31 ± 0.15**

The results are presented as mean ± SEM, based on a sample size of 6. A one-way analysis of variance (ANOVA) was

used for statistical analysis, and Dunnett’s test was applied to each parameter individually. **p<0.01, ***p<0.001:

vehicle control group, and ###p<0.001: normal group. CoQ (10), Coenzyme Q (10 mg/kg); PA, Phyllanthus amarus.

98 Xu et al.

Investigación Clínica 67(1): 2026

a substantial (p<0.001) increase in nitric

oxide and MDA levels in the hepatic and re-

nal tissues of the vehicle control group. PA

(200 mg/kg) effectively (p<0.001) restored

GSH and SOD levels and significantly low-

ered MDA (p<0.001) and nitric oxide levels

in hepatic and renal tissues compared to the

vehicle control group. CoQ (10 mg/kg) ad-

ministration also demonstrated strong hepa-

toprotective and nephroprotective effects by

effectively increasing (p<0.001) hepatic and

renal GSH and SOD levels and markedly re-

ducing (p<0.001) hepatic and renal nitric

oxide and MDA levels relative to the vehicle

control group (Fig. 2B-2E).

Table 2. Effect of P. amarus on serum hepatic and renal biomarker levels.

Treatment Normal Vehicle control CoQ (10) PA (50) PA (100) PA (200)

BUN (mg/dL) 25.85 ± 1.47 50.72 ± 1.29### 31.48 ± 1.26*** 47.85 ± 0.89 46.91 ± 1.38 34.38 ± 1.26***

Creatinine

(mg/dL)

0.63 ± 0.06

2.07 ± 0.11###

0.92 ± 0.07***

2.12 ± 0.13

2.08 ± 0.12

1.57 ± 0.09***

Uric acid

(mg/dL)

1.90 ± 0.12

4.41 ± 0.12###

2.50 ± 0.06***

4.36 ± 0.11

4.40 ± 0.14

3.26 ± 0.13***

Albumin

(mg %)

6.75 ± 0.56

2.39 ± 0.51###

6.04 ± 0.41***

2.43 ± 0.50

2.61 ± 0.43

4.46 ± 0.40***

Direct bilirubin

(mg %)

0.20 ± 0.01

0.67 ± 0.01###

0.29 ± 0.02***

0.66 ± 0.02

0.62 ± 0.01

0.42 ± 0.01***

Total bilirubin

(mg %)

0.12 ± 0.01

0.31 ± 0.02###

0.16 ± 0.01***

0.31 ± 0.02

0.31 ± 0.01

0.23 ± 0.01***

ALP (IU/L) 50.47 ± 3.77 392.80 ± 3.81### 94.35 ± 3.69*** 383.80 ± 6.32 367.80 ± 6.12 257.90 ± 3.71***

AST (IU/L) 63.92 ± 13.33 284.30 ± 14.17### 102.00 ± 11.65*** 289.50 ± 12.34 264.50 ± 10.44 144.00 ± 11.42***

ALT (IU/L) 28.32 ± 7.65 146.50 ± 6.27### 42.75 ± 10.28*** 145.00 ± 9.97 140.00 ± 6.73 63.36 ± 8.47***

LDH (mg %) 482.00 ± 119.90 3418.00 ± 212.80### 682.30 ± 152.20*** 3399.00 ± 405.50 3239.00 ± 406.40 1456.00 ± 131.40***

The results are presented as mean ± SEM, based on a sample size of 6. A one-way analysis of variance (ANOVA) was

performed for statistical analysis, followed by Dunnett’s test applied individually to each parameter. ***p<0.001:

vehicle control group and ###p<0.001: normal group. AST, Aspartate transaminase; ALP, Alkaline phosphatase; ALT,

alanine transaminase; BUN, Blood Urea Nitrogen; CoQ (10), Coenzyme Q (10 mg/kg); LDH, Lactate Dehydroge-

nase; PA, Phyllanthus amarus.

Table 3. Effect of P. amarus on serum lipid profile.

Treatment Normal Vehicle control CoQ (10) PA (50) PA (100) PA (200)

Cholesterol

(mg %)

18.71 ± 3.14

65.06 ± 8.72###

35.70 ± 2.42***

61.54 ± 4.62

60.73 ± 5.07

29.74 ± 4.02***

HDL

(mg %)

60.85 ± 1.09

22.26 ± 3.42###

56.31 ± 3.68***

23.99 ± 2.07

21.94 ± 4.74

56.53 ± 3.15***

LDL

(mg %)

2.10 ± 0.35

5.02 ± 0.51###

2.12 ± 0.45***

5.14 ± 0.47

5.07 ± 0.47

2.34 ± 0.35***

Triglyceride

(mg %)

60.33 ± 14.89

185.30 ± 19.41###

72.25 ± 9.03***

167.30 ± 17.33

156.90 ± 13.88

122.4 ± 10.23***

The results are presented as mean ± SEM, based on a sample size of 6. A one-way analysis of variance (ANOVA)

was conducted for statistical analysis, and Dunnett’s test was subsequently applied to each parameter individually.

***p<0.001: vehicle control group and ###p<0.001: normal group. CoQ (10), Coenzyme Q (10 mg/kg); HDL, high-

density lipoprotein; LDL, low-density lipoprotein; PA, Phyllanthus amarus.

Phyllanthin protected arsenite-induced toxicity 99

Vol. 67(1): 92 - 107, 2026

Protein expressions of hepatic and renal

ILs, TNF-α, and NF-kB p65

Compared to the normal group, hepatic

and renal ILs (IL-6 and IL-1β), TNF-α, and NF-

kB p65 protein expression were significantly

increased (p<0.001) in the vehicle control

group. Administration of PA (200 mg/kg) effec-

tively reduced hepatic and renal ILs, TNF-α, and

NF-kB p65 protein levels (p<0.001) compared

with the vehicle control group. Treatment with

CoQ (10 mg/kg) demonstrated a substantial

(p<0.001) protective effect against arsenite-

induced liver and kidney damage, as shown by

decreased hepatic and renal ILs, TNF-α, and

NF-kB p65 protein expression compared to the

vehicle control group (Table 4).

There was a strong, positive, and statis-

tically significant correlation between serum

ALT levels and hepatic TNF-α (R2=0.737

and p<0.05), IL-1β (R2=0.8578 and

p<0.01), IL-6 (R2=0.7493 and p<0.05),

and NF-κB p65 (R2=0.8692 and p<0.01)

(Fig. 3A-3D). Serum levels of BUN were posi-

tively and significantly correlated with re-

nal TNF-α (R2=0.7693 and p<0.05), IL-1β

(R2=0.7315 and p<0.05), IL-6 (R2=0.8323

and p<0.01), and NF-κB p65 (R2=0.8927

and p<0.01) (Fig. 3E-3H).

Hepatic histopathology

The liver sections from the normal

group showed well-preserved architectural

features, including hepatocytes arranged

in orderly cords radiating from the central

vein, with uniform cellular dimensions and

transparent cytoplasm. The nuclei were cen-

trally positioned and exhibited normal mor-

phology. No necrosis was observed; however,

mild inflammation was noted (Fig. 4A). The

histology of hepatic tissue from the vehicle

control group showed histopathological

changes, including significant (p<0.001)

necrosis, distorted hepatocyte arrangement,

and infiltration of inflammatory cells (Fig.

4B). Histological examination of liver sec-

tions from rats treated with CoQ (10 mg/kg)

revealed a notable (p<0.001) improvement

in morphology compared to the vehicle con-

trol group. Hepatocytes mostly maintained

their normal structure, with mild necrosis

Fig. 2. P. amarus effects on hepatic and renal oxidative stress. A quantitative chart of the total antioxidant

capacity of the liver (A). Quantitative measurements of SOD (B), GSH (C), MDA (D), and nitric oxide

(E) levels in hepatic and renal tissues. Results are presented as mean values with SEM, based on a

sample size of 6. One-way ANOVA was used for statistical analysis, with Dunnett’s test applied to each

parameter separately. ***p<0.001: vehicle control group and ###p<0.001: normal group. CoQ (10),

Coenzyme Q (10 mg/kg); GSH, glutathione peroxidase; NO, nitric oxide; MDA, malondialdehyde; PA,

Phyllanthus amarus; SOD, superoxide dismutase.

100 Xu et al.

Investigación Clínica 67(1): 2026

and inflammation, and their cytoplasm was

less eosinophilic than in the vehicle control

group (Fig. 4C). Histological analysis of liv-

er tissue from the PA (50 and 100 mg/kg)-

treated groups showed infiltration of inflam-

matory cells, vacuolation, and interstitial

edema (Fig. 4D and 4E). Liver sections from

the PA (200 mg/kg)-treated group showed

mainly preserved hepatocytes, as evidenced

by well-maintained cellular architecture and

minimal necrotic changes. The cytoplasm

showed reduced vacuolation, and inflam-

matory cell infiltration was significantly de-

creased (p<0.001) compared to the vehicle

control group (Fig. 4F, Fig. 4M).

Renal histopathology

The renal sections of the normal group

showed well-preserved kidney architecture.

The glomeruli and renal tubules were clearly

Fig. 3. Simple regression analysis of hepatic TNF-α (A), IL-1β (B), IL-6 (C), and NF-kB p65 (D) with ALT le-

vels, and renal TNF-α (E), IL-1β (F), IL-6 (G), and NF-kB p65 (H) with BUN levels. A two-sided Fisher’s

test was used to calculate the correlation coefficients. ALT, alanine transaminase; BUN, blood urea

nitrogen; ILs, interleukins; NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor-alpha.

Table 4. Effect of P. amarus treatment in hepatic and renal Interleukins

and NFkB-P65 levels.

Treatment Normal Vehicle control CoQ (10) PA (50) PA (100) PA (200)

Hepatic TNF-α

(pg/mg)

11.23 ± 1.57

56.96 ± 2.54###

22.17 ± 3.18***

52.83 ± 4.32

51.09 ± 4.76

26.16 ± 4.50***

Hepatic IL-1β

(pg/mg)

8.61 ± 0.88

111.50 ± 2.67###

24.17 ± 2.71***

109.00 ± 2.80

98.19 ± 5.61

55.69 ± 4.80***

Hepatic IL-6

(pg/mg)

29.19 ± 5.06

169.30 ± 8.19###

81.67 ± 7.37***

152.20 ± 6.82

152.20 ± 10.47

100.60 ± 9.31***

Hepatic NF-kB p65

(pg/mg)

141.50 ± 14.43

653.30 ± 7.00###

168.10 ± 8.93***

646.20 ± 13.25

559.30 ± 20.79

274.60 ± 16.08***

Renal TNF-α

(pg/mg)

3.12 ± 0.67

14.13 ± 1.23###

4.42 ± 0.54***

12.46 ± 0.76

11.88 ± 1.00

5.58 ± 0.85***

Renal IL-1β

(pg/mg)

11.39 ± 2.17

79.58 ± 5.70###

37.92 ± 4.17***

74.58 ± 4.82

71.94 ± 5.08

48.33 ± 3.71***

Renal IL-6 (pg/mg) 9.09 ± 1.56 97.02 ± 6.43### 22.78 ± 3.20*** 94.39 ± 5.91 94.90 ± 1.97 24.70 ± 6.04***

Renal NF-kB p65

(pg/mg)

78.84 ± 1.69

214.70 ± 2.14###

98.41 ± 2.86***

213.10 ± 2.79

203.10 ± 2.48

119.90 ± 2.29***

The results are expressed as mean ± SEM, based on a sample size of 6. A one-way analysis of variance (ANO-

VA) was used for statistical analysis, and Dunnett’s test was subsequently applied to each parameter individually.

***p<0.001: vehicle control group and ###p<0.001: normal group. CoQ (10), Coenzyme Q (10 mg/kg); ILs, Inter-

leukins; NF-κB, nuclear factor kappa B; PA, Phyllanthus amarus; TNF-α, tumor necrosis factor-alpha.

Phyllanthin protected arsenite-induced toxicity 101

Vol. 67(1): 92 - 107, 2026

Fig. 4. P. amarus on rat hepatic and renal pathology.

1. Microscopic images of liver (A-F) and kidney (G-L) cross-sections from various rat groups, including normal

(A and G), vehicle control (B and H), CoQ (10) treatment (C and I), PA treatment (50 mg/kg) (D and J),

PA treatment (100 mg/kg) (E and K), and PA treatment (200 mg/kg) (F and L). H&E staining at 40X and

100X (inset). Quantitative data showing the effect of P. amarus treatment on rat hepatic and renal patholo-

gies (M). The results are expressed as mean ± SEM, based on a sample size of 6. One-way ANOVA was used

for statistical analysis, followed by Dunnett’s test for each parameter. ***p<0.001: vehicle control group;

###p<0.001: normal group. CoQ (10), Coenzyme Q (10 mg/kg); PA, Phyllanthus amarus. Inflammatory

infiltration (yellow arrow), cellular infiltration (green arrow), and necrosis (blue arrow) are indicated.

102 Xu et al.

Investigación Clínica 67(1): 2026

defined. No signs of inflammation, necro-

sis, or other cellular damage were observed

(Fig. 4G). In contrast, renal tissue from the

vehicle control group exhibited severe kid-

ney damage, with prominent (p<0.001)

tubular necrosis and infiltration of inflam-

matory cells (Fig. 4H). Kidney tissue from

the CoQ10 (10) treated group demonstrat-

ed a significantly preserved renal architec-

ture compared to the vehicle control group

(p<0.001). Although mild inflammation was

present, there was a notable reduction in tu-

bular necrosis and overall structural damage

(Fig. 4I). Groups treated with PA (50 and

100 mg/kg) showed extensive renal tissue

damage similar to that in the vehicle control

group, with clear evidence of inflammation

and tubular necrosis (Fig. 4J and 4K). Treat-

ment with PA (200 mg/kg) showed a marked

(p<0.001) protective effect, comparable to

the vehicle control, with kidney architecture

largely preserved and only minimal signs of

inflammation and reduced tubular damage

(Fig. 4L and 4M).

DISCUSSION

Sodium arsenite causes significant tox-

icity in various biological systems, with ef-

fects ranging from genetic damage to organ

harm. Studies have shown that the liver and

kidneys are especially susceptible to arse-

nic-related toxicity because of their roles

in detoxification and excretion 24,25. Sodium

arsenite triggers notable oxidative stress

in both liver and kidney tissues, leading to

apoptosis and inflammatory responses that

impair their functions. As research advanc-

es, the use of antioxidants and other protec-

tive agents, such as hesperidin, lycopene,

and bosentan, has demonstrated potential

in reducing these toxic effects by lowering

oxidative stress and inflammation and boost-

ing cellular antioxidant defenses 9,26. The

current study examined the possible mech-

anisms by which P. amarus methanolic ex-

tract may protect against arsenite-induced

liver and kidney damage in rats.

Chronic administration of sodium arse-

nite causes acute liver failure and hepatotox-

icity, leading to fatal outcomes 25. Research-

ers have noted that significant increases in

AST, ALT, and ALP levels during arsenite- in-

duced hepatotoxicity serve as indicators of

liver function, along with histological chang-

es 25. Sodium arsenite is absorbed from the

gut and detoxified through oxidative meth-

ylation in the liver. This process, driven by

hepatic enzymes, converts inorganic arsenic

into organic forms like monomethylarsonic

acid and dimethylarsinic acid 27. Paradoxi-

cally, this detoxification pathway depletes

S- Adenosyl methionine and produces triva-

lent methylated metabolites that are more

toxic than the original compound, sodium

arsenite, causing hepatocellular damage25.

Elevated ALT levels are key indicators of

the severity of hepatocellular damage. Simi-

lar to ALT, AST also increases markedly in

arsenite-induced liver injury; however, AST

is less specific to the liver. In severe cases,

AST levels can match or surpass ALT levels,

especially in the later stages of liver necro-

sis. Therefore, increased ALT and AST levels,

along with histological abnormalities during

chronic sodium arsenite exposure, confirm

its hepatotoxic effects 25. Additionally, long-

term exposure to sodium arsenite results in

significant changes in serum biomarkers,

such as BUN, uric acid, and creatinine, in-

dicating renal dysfunction 28. In this study,

elevated levels of ALT, AST, ALP, uric acid,

BUN, and creatinine were observed follow-

ing arsenite administration. These increases

in serum markers correlate with histological

damage in the liver and kidneys, reflecting

the hepatotoxic and nephrotoxic effects of

sodium arsenite. Further histological analy-

sis supported the protective potential of ar-

senite against arsenite-induced structural

damage in hepatocytes, including irregular

and indistinct central veins, cellular dam-

age, tubular necrosis, and increased inflam-

matory cells, which were alleviated after

treatment with P. amarus. Previous studies

have also documented the hepatoprotective

Phyllanthin protected arsenite-induced toxicity 103

Vol. 67(1): 92 - 107, 2026

and nephroprotective effects of P. amarus

through the inhibition of carbon tetrachlo-

ride-induced elevation of hepatic biomark-

ers and high-salt diet-induced increases in

kidney function markers 13,29. The findings

of this study reinforce those from earlier re-

search 13, 29.

Reactive oxygen species (ROS), cy-

tokines, chemokines, and hepatic macro-

phages are key contributors to liver and kid-

ney damage 30. The family of transcription

factors called nuclear factor-κB (NF-κB) is

evolutionarily conserved and remains inac-

tive in the cytoplasm of various cell types.

When activated, NF-κB translocates to the

nucleus, where it plays a critical role in in-

flammatory processes, immune responses,

and programmed cell death. ROS- induced

inflammation is crucial for arsenite- related

liver and kidney injury. Excessive production

of free radicals triggers NF-κB activation at

the inflammation site, leading to the expres-

sion of pro-inflammatory genes, including

TNF-α and interleukins, ultimately raising

cytokine levels. During the acute-phase re-

sponse, pro-inflammatory cytokines are vi-

tal31,32. Elevated levels of TNF-α and IL-1β in

the liver and kidneys serve as important indi-

cators of hepatic and renal damage in rats 33.

Therefore, the transcriptional regulation of

certain inducible inflammatory mediators is

significantly affected by NF- κB 34. Afolabi et

al. reported that intestinal ischemia-reperfu-

sion injury caused a significant increase in

intestinal and hepatic IL-1β and TNF-α lev-

els compared to the sham group 31. However,

administering the methanolic extract of P.

amarus to rats with ischemia-reperfusion in-

jury significantly inhibited hepatic IL-1β and

TNF-α levels 31. Furthermore, previous re-

search demonstrated that P. amarus ethano-

lic extract suppresses NF-κB, a major regula-

tor of inflammation, in RAW 264.7 cells 35.

Additionally, Phyllanthin from P. amarus has

been shown to reduce elevated pro- inflam-

matory cytokine levels by inhibiting NF- κB

activation in high- fat diet- induced fatty liv-

er36. In this study, Phyllanthin from P. amarus

also reduced arsenite-induced increases in

pro-inflammatory cytokine production by in-

hibiting NF-κB. Therefore, the protective ef-

fects of phyllanthin from P. amarus, through

its anti-inflammatory properties, align with

earlier research 31. Moreover, this investiga-

tion consistently showed that the extent of

organ damage, as measured by serum mark-

ers ALT for the liver and BUN for the kid-

ney, was strongly and significantly correlat-

ed with the local inflammatory response in

these organs. Higher levels of organ injury

were associated with increased tissue con-

centrations of proinflammatory cytokines

and transcription factors. The high correla-

tion coefficients (R2> 0.7 for all plots) and

statistical significance (p<0.05) across all

analyses provide strong evidence supporting

this relationship.

P. amarus has been extensively studied

for treating chronic Hepatitis B infection. A

randomized trial involving chronic Hepatitis

B patients (n=60) who received P. amarus

extract for 12 weeks showed a significant

reduction in Hepatitis B virus (HBV) DNA

levels 37,38. Its antiviral effectiveness inhib-

its viral replication and raises liver enzymes

(ALT and AST) 37,38. Patients with chronic

Hepatitis B (n=123) treated with P. amarus

for 6 months experienced decreased HBV

surface antigen (HBsAg) levels, supporting

its antiviral and liver-protective properties39.

Additionally, patients with type 2 diabetes

treated with P. amarus for 10 weeks had sig-

nificant reductions in fasting blood glucose

and glycosylated hemoglobin levels 40. More-

over, in individuals with non-alcoholic fatty

liver disease, P. niruri supplementation low-

ered elevated oxidative stress markers such

as malondialdehyde (MDA), resulting in an

increased overall antioxidant capacity41.

Safety assessments have also shown that P.

amarus is well tolerated. Therefore, P. ama-

rus appears to be a promising herbal treat-

ment for pesticide-induced liver and kidney

damage. However, further research is need-

ed to determine its clinical effectiveness in

these conditions.

104 Xu et al.

Investigación Clínica 67(1): 2026

CONCLUSION

The results of this study showed that P.

amarus methanolic extract effectively pro-

tects against sodium arsenite-induced liver

and kidney damage in rats. The protective

effect of P. amarus is likely due to the pres-

ence of phyllanthin, which reduces NF-κB

activation, thereby decreasing inflammation

and oxidative-nitrosative stress, and boost-

ing overall antioxidant capacity. Clinically,

P. amarus standardized capsules or liquid

extracts can be taken orally, with dosing

carefully controlled based on safety and ef-

fectiveness data from preclinical studies.

The dose should consider factors such as the

severity of arsenic poisoning, the patient’s

body weight, and how long the exposure

lasts, with treatment lasting long enough

for detoxification and tissue healing. Using

P. amarus extract as an additional therapy

alongside traditional chelation could im-

prove patient outcomes by lowering arsenic

levels and reducing biochemical problems

caused by arsenic toxicity.

Acknowledgments

Medical writing support for this manu-

script, under the authors’ guidance, was pro-

vided by Yonnova Scientific Consultancy Pvt.

Ltd. in accordance with Good Publication

Practice Guidelines.

Funding

This study did not receive any funding.

Conflict of interest

No conflict of interest.

Data availability

The raw data supporting this article

will be provided to the corresponding author

upon reasonable request.

Ethical statements

The research protocol was approved

by the Institutional Animal Ethics Com-

mittee (IAEC) of the Zhinanzhen Biol-

ogy Ethics Committee (approval number:

A2024000414). This study was conducted

following the National Institutes of Health

Guide for the Care and Use of Laboratory

Animals.

ORCID numbers auhors

• Jiarui Xu (JK):

0000-0003-4925-2770

• Smeeta Sadar (SS):

0000-0001-8682-303X

• Hemant Kamble (HK):

0009-0003-2865-5172

• Yingtao Zhang (YZ):

0009-0003-9376-7381

Authors contribution

Each author has contributed signifi-

cantly to the development of this manu-

script. JX and HK: conceived and designed

the evaluation, performed parts of the sta-

tistical analysis, and drafted the manuscript;

SS and YZ: conducted data collection and

drafted the manuscript. All authors have

read and approved the final version of the

manuscript.

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