AAVS_13_10_2313-2322

Introduction

Plastic is one of the most prevalent pollutants worldwide (Kumar et al., 2025). Around 400 million tons of plastic waste each year is anticipated to rise significantly in the near future (OECD, 2022). Polystyrene plastic is one of the commercial plastic materials produced to meet human needs (Ho et al., 2018). It is intesively used in the manufacture of food container, utensils, electronics, cosmetics, agriculture, automotive, and building construction materials (Frias and Nash, 2019). Inefficient waste disposal and recycling systems contribute to the accumulation of polystyrene in the environment. Plastic waste is not easily biodegradable and can fragment into microplastics (1–1000 µm) and nanoplastics (1–1000 nm) through mechanical wear, ultraviolet radiation, and biological processes (Ahamed and Akhtar, 2024). Nanoplastics are of particular concern because of their small size, which enables them to enter organisms through ingestion, inhalation, and dermal contact (Prata et al., 2020).

In aquatic ecosystems, nanoplastics disrupt biodiversity by affecting species composition and survival, accumulate in organisms, and act as carriers of toxic pollutants, eventually entering the food chain and posing ecological and health risks (P et al., 2025). At the cellular level, PS-NPs induce oxidative stress, cell apoptosis, DNA damage, cancer, and over-expression of inflammatory cytokine (Banerjee and Shelver, 2021). The induction of apoptosis is mainly mediated by mitochondrial dysfunction and subsequent activation of caspase-dependent pathways (Guerrero et al, 2008). Elevated pro-inflammatory cytokines that are not balanced with anti-inflammatory cytokines will increase cytotoxic Nitric Oxide (NO) levels, thereby exacerbating tissue damage due to nanoplastic exposure (Gopal et al., 2019).

Butterfly pea (Clitoria ternatea L.) is a traditional Asian medicinal plant to treat diarrhea, fever, relieve allergies, and prevent diabetes mellitus (Singh et al., 2018). C. ternatea L. contains tannins, saponins, triterpenoids, phenols, flavonoids, alkaloids, anthocyanins, and other compounds (Jeyaraj et al., 2022). It has been reported to exhibit antioxidant and anti-inflammatory activities, with studies showing protection against carrageenan-induced inflammation and cytokine modulation during SARS-CoV-2 infection (Swathi et al., 2020; Nugraha et al., 2021). These properties suggest that C. ternatea may mitigate oxidative stress and immune dysregulation caused by PS-NPs exposure.

Although the adverse effects of PS-NPs are well documented in aquatic organisms such as fish and mollusks, little is known about their impact on mammalian systems relevant to human health. To the best of our knowledge, no previous study has evaluated the protective effects of C. ternatea extract (CTE) against PS-NPs-induced toxicity in mammals. Therefore, the present study aimed to investigate the anti-inflammatory and immunomodulatory effects of ethanolic CTE in Wistar rats exposed to orally administered PS-NPs and to profile its bioactive compounds using GC-MS analysis to identify potential anti-inflammatory constituents. We hypothesized that CTE would attenuate PS-NPs induced inflammation and immunological alterations in Wistar rats, primarily through its bioactive compounds with potential anti-inflammatory activity.

Materials and Methods

Research period and location

The experimental study was carried out in the Animal Laboratory and Histology Laboratory, Department of Biology, Faculty of Science and Technology, Universitas Airlangga. This research was carried out from June 2024-January 2025.

Chemicals

Butterfly pea flowers (Clitoria ternatea L.) were obtained from local market in Bekasi, Indonesia. Butterfly pea flowers were identified by the Department of Biology, Faculty of Science and Technology, Universitas Airlangga. A voucher of specimen with number 02/06.CT/2025 was deposited at Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia. Polystyrene nanoplastics (100 nm in size) were purchased from Sigma-Aldrich Solution (Merck Millipore, Germany) Product No. 43302, Batch No. BCCK7019). According to the vendor’s certificate of analysis, the particles had a nominal diameter of 0.100 μm, calibrated particle diameter of 0.095 μm, standard deviation of 0.004 μm, and a polydispersity index (PDI) of 4%. IL-1, IL-6 and TNF-α kits were obtained from BT-Lab ELISA kit (Bioassay Technology Laboratory, China). All chemical reagents used in this study were of analytical grade.

The animals

All experimental protocols were authorized by the Faculty of Dental Medicine (Ethical Clearance Commission), Universitas Airlangga, Indonesia (ethical clearance number 0592/HRECC.FODM/VI2024). Male Wistar rats (150-180 g) were procured from the Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia. These rats were placed under standardized controlled conditions (room temperature, balanced light-dark cycle, free access for rat food and water).

Preparation of plant extract

According to Singh et al. (2018) with modification, the flowers were cut into small pieces and left to air-dried for 7-14 days until they reached a consistent weight. During the air-drying process, the flowers were not exposed to sunlight and oven’s high temperature. Once dried, they were ground into a powder. Then, 1.5 kg of dried material was macerated in 96% ethanol (Merck Millipore, Germany) at a ratio of 1:8 (w/v) for 72 hours, at room temperature (25 oC) with constant stirring (Singh et al., 2018). The resultant mixture was subsequently subjected to evaporation using rotary evaporator. Afterward, the extract underwent lyophilization. Before being administered to the experimental animals, the extract was reconstituted in distilled water daily. The rats received the extract at doses of 100, 200, and 400 mg/kg BW.

GCMS analysis

Bioactive compounds were identified using GC-MS (Agilent Technologies 7890A, United States), with data analysis performed using the GC-MS D5975C software. Helium was used as the carrier gas and the temperature programming was set with initial temperature of 60oC. The final temperature of the oven was 350oC. 2 µL sample was injected with splitless mode. The total running time for a sample is 45 minutes (Deshmukh and Jadhav, 2013). Comparing the average peak area to the total peak areas will provide the relative percentage of each component.

Animal experiment

The animal experiment was conducted based on the protocol described by Safitri et al. (2025) with slight modifications. Following a two-week acclimation period, five treatment groups were determined, including a normal control group (K) without administration of PS-NPs and without administration of Clitoria ternatea L. flower extract (CTE), a negative control group K- (administrated with 10 µL/kg BW of PS-NPs, equivalent to 10.05 mg/kg BW), and three treatment groups receiving different doses of CTE after exposure to PS-NPs. The treatment groups were P1 with the administration of 100 mg/kg BW of CTE; P2 with administration of 200 mg/kg BW of CTE; P3 with administration of 400 mg/kg BW of CTE). The CTE doses were selected according to Singh et al. (2018), where comparable doses produced significant activity in rodents, thereby allowing assessment of dose response effects. CTE was administered approximately within 2 hours after the administration of PS-NPs. The administration of animal treatments was carried out via oral gavage once daily for 35 consecutive days following a 14-day acclimatization period. The 35-day treatment period was chosen to simulate subchronic exposure and allow sufficient time for observing cumulative toxic effects of PS-NPs and the potential protective effects of CTE.

On day 36, the animals were anesthetized intramuscularly with a ketamine/xylazine mixture (90 mg/kg and 10 mg/kg, respectively) and then sacrificed. Blood samples (2-3 mL) were collected using a 23G needle. A portion of the blood was transferred into EDTA tubes for hematological parameter analysis, while the remaining portion was placed into plain tubes (without EDTA) to obtain the serum for further analysis. The kidney tissue was taken from the rats and processed for histological analysis.

Hematological analysis

Hematological parameters were measured within maximum of 3 hours after blood collection. Hematological analysis was conducted according to Pramudya et al. (2025). Coulter counters (Abbot Cell-Dyn 1700 Hematology Analyzer, GMI, USA) were used to calculate: White blood cells (WBC) count (103/mm3), red blood cells (RBC) count (106/mm3), hemoglobin concentration (g/dL), hematocrit percentage (%), and thrombocytes count (103/mm3).

Measurement of IL-1, IL-6 and TNF-α

Serum was obtained by centrifuging whole blood at 3,000 rpm for 10 minutes. IL-1, IL-6, and TNF-α levels were measured by Enzyme Linked Immuno Sorbent Assay (ELISA) method according to kit’s protocol (Bioassay Technology Laboratory, China) (Susilo et al., 2025). The absorbances were read at 450 nm using a microplate reader. IL-1, IL-6, and TNF-α levels were determined using the standard curve.

Histological analysis of kidney

Histological analysis was carried out by fixing kidney samples in 10% neutral buffered formalin for 48 h (Merck Millipore, Germany). Standard operating procedure for histology was used as guidance (Triwahyudi et al., 2023). Tissues were placed in cassettes, washed and processed with serial alcohol and xylol. Then, tissues were embedded in three times paraffin (1 h each). Sections were cut at thickness of 4 μm, stained with hematoxylin and eosin, and examined under light microscope (Olympus Medical Indonesia, Indonesia). Histological assessments included measurements of glomerulus diameter, proximal and distal convoluted tubule diameters (µm), the percentage of necrosis in proximal and distal convoluted tubules, and epithelial thickness of both tubules. All parameters were quantified from multiple randomly selected fields using a digital microscope camera (Optilab Miconos, Indonesia) and compared across the normal control, negative control, and CTE treatment groups.

Statistical analysis

The data were analyzed using the SPSS software v.24 (IBM Corp., USA). Statistical procedures followed the method described by Triwahyudi et al. (2023). One-way analysis of variance (ANOVA) was performed for all parameters, with p-values less than 0.05. Tukey’s post hoc test was applied for multiple comparisons. The results are presented as mean ± standard deviation (SD).

Results

Identification of bioactive compounds of CTE

Table 1 presented the GC-MS results of the CTE. 22 compounds were identified in CTE. The major component with the highest percentage peak area was 1-Methyl-6,7,8,9-tetrahydrol[1,3] thiazepinol[2,3-f]purine-2,4(1H,3H)-dione (67.63%), followed by Dimethylaminodiborane and 3-Amino-4,5-diphenyl-isothiazole with percentage of peak area of 6.01% and 3.56%, respectively. Three main compounds in the extract belong to the alkaloid group. Other compounds were identified belong to alkaloid and flavonoid groups. The GC-MS analysis revealed the presence of compounds from CTE that provide beneficial effects on the body.

CTE affects hematology parameter

As shown in Table 2, The negative control (3.5 x 103 ± 206 cells/mm³) exhibited a significantly lower WBC count compared to the normal control (P=0.011). Furthermore, the WBC count in the CTE 200 (2.9 x 103 ± 102 cells/mm³) and CTE 400 (3.6 x 103± 210 cells/mm³) groups was significantly different compared to the negative control (P<0.001 and P<0.045, respectively). In line with WBC result, percentage of hematocrit also increased significantly in CTE 200 (36.50 ± 0.49%) and CTE 400 (35.50 ± 1.71%) (P=0.046). In hemoglobin measurement, CTE 100 (13.87 ± 0.29 g/dL), and CTE 200 (14.63 ± 0.37 g/dL) displayed significant changes in hemoglobin concentration (P=0.016 and P=0.004) when compared to negative control. There were no significant differences observed in RBC count and thrombocyte count.

CTE suppressed levels of IL-1, IL-6, and TNF-α

According to Figure 1, negative control (7.89 ± 0.87 pg/L) showed a slight increase in IL-1 levels compared to normal control (P=0.937). Levels of IL-1 were significantly decreased in CTE 100 (5.15 ± 0.31 pg/L) (P<0.001), CTE 200 (5.88 ± 0.79 pg/L) (P=0.045), and CTE 400 (5.13 ± 0.67 pg/L) (P<0.001) compared to negative control group and normal control, indicating suppression below baseline levels.

Table 1: Twenty-two compounds were identified from CTE by GC-MS.

No.	Retention time	Area (%)	Compound name	Molecular formula
1	3.507	67.63	1-Methyl-6,7,8,9-tetrahydrol[1,3]thiazepinol[2,3-f]purine-2,4(1H,3H)-dione*	C10H12N4O2S
2	4.102	6.01	Dimethylaminodiborane	C2H6B2N
3	3.753	3.56	3-Amino-4,5-diphenyl-isothiazole	C15H12N2S
4	5.346	2.47	3’H-Cycloprop(1,2)-5-cholest-1-en-3-one, 1’-carboethoxy-1’-cyano-1,2-dihydro-	C32H49NO3
5	4.734	2.10	Propargyl alcohol, heptafluorobutyrate	C7H3F7O2
6	3.901	2.10	Propanenitrile, 3-chloro-	C3H4ClN
7	4.017	1.75	Nickel, 2,6, 10-dodecatriendi-1, 12-diyl-	C12H18Ni
8	4.435	1.65	Methyl 8-bromo-3-oxooctanoate	C9H15BrO3
9	4.877	1.91	1H-Benzotriazole-1-methanamine, N-phenyl-	C13H12N4
10	4.655	1.27	1-(2,5-Dimethoxyphenyl)-3-nitro-1,2,4-triazole	C10H10N4O4
11	4.535	1.20	N-[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]guanidine	C10H9ClN4S
12	5.095	1.03	3-Methyl-3,5--(cyanoethyl)tetrahydro-4-thiopyranone	C12H16N2OS
13	5.177	0.95	Molybdenum, dicarbonylbis(.eta.-4-2-methylenecycloheptanone)-	C18H26MoO4
14	6.070	0.86	Phosphoramidous dichloride, dimethyl-	C2H6Cl2NP
15	5.002	0.85	2,4-Dinitrobenzoic acid	C7H4N2O6
16	4.614	0.85	3-Bromo-2-naphthoic Acid	C11H7BrO2
17	5.270	0.81	2-Pyridinecarbonitrile, 6-chloro-	C6H3ClN2
18	5.724	0.68	Phosphinodithioic acid, diphenyl-	C12H11PS2
19	5.965	0.63	N-chloro-2-propyn-1-amine	C3H4ClN
20	5.823	0.60	[1,2,4]Triazolo[1,5-a]pyrimidine, 2-ethylsulfanyl-5,7-dimethyl-	C9H12N4S
21	3.250	0.55	Pteridine-8-oxide, 6-aldoximino-2-amino-4(3H)-oxo-	C7H6N6O3
22	6.306	0.53	1,2,3,4,9,10-Hexahydro-9,10-exo-epoxy-4a,9a-exo-o-benzeno-1,4-exo-methanoanthracene	C21H18O

*Tentative identification based on NIST/Wiley GC-MS library; not confirmed with authentic standard.

Table 2: The CTE influenced WBC count, RBC count, hemoglobin concentration, hematocrit percentage, and number of thrombocytes after NP exposure. Values are represented as mean ± SD (n = 5). Asterisks denote statistical significance. (*): significant difference compared to negative control group. (**): significant difference compared to both normal and negative control group.

Parameter	Groups					P value
Parameter	Normal control	Negative control	CTE 100 mg/kg BW	CTE 200 mg/kg BW	CTE 400 mg/kg BW	P value
WBC (cell/mm3)	4.1 x 103 ± 224	3.5 x 103 ± 206*	3.7 x 103 ± 544	2.9 x 103 ± 102**	3.6 x 103 ± 210*	<0.001
RBC (cell/mm3)	6.6x106±1.24x105	6.7x106±1.4x105	7.3x106±9.4x104	7.4x106±8.1x104	7.5x106±4.7x104	0.1
Hemoglobin (g/dL)	10.63 ± 0.45	11.53 ± 0.53	13.87 ± 0.29*	14.63 ± 0.37**	12.97 ± 1.16	0.003
Hematocrit (%)	28.87 ± 4.47	35.03 ± 2.02	35.40 ± 1.20	36.50 ± 0.49**	36.50 ± 0.49**	0.036
Thrombocyte (103/mm3)	293x103±6.5x103	302x103±46x103	295x103±13x103	321x103±36x103	290x103±12x103	0.802

Figure 2 showed that CTE 100 with administration of 100 mg/kg BW C. ternatea extract had the highest level of IL-6 of all groups (6.12 ± 1.05 ng/L). CTE 100 demonstrated a significant increase (P<0.001) compared to negative control and normal control. In contrast, compared to negative control group, CTE 200 group (3.34 ± 0.48 ng/L) lowered IL-6 levels significantly (P=0.027). Meanwhile, CTE 400 group (4.34 ± 0.39 ng/L) with higher doses had no significant difference compared to negative control group (P=0.920).

Serum level of TNF-α also increased in negative control group (175.49 ± 7.20 ng/L) (Figure 3). Compared to negative control group, CTE 200 (171.95 ± 13.35 ng/L) showed no significant difference in TNF-α level (P=0.991). On the other hand, TNF-α levels in CTE 100 (141.20 ± 14.95 ng/L) and CTE 400 (105.71 ± 11.61 ng/L) were declined significantly (P=0.002 and P<0.001). These results indicated that PS-NPs exposure could elevate the level of pro-inflammation cytokines and CTE administration could lower pro-inflammation cytokines levels.

CTE improves kidney structural changes induced by nanoplastics or CTE ameliorates nanoplastics-induced kidney damage

Histopathological examination revealed characteristic features of tubular necrosis in the negative control group, including altered cytoplasmic eosinophilia, swelling, loss of the brush border, nuclear fragmentation, and detachment of necrotic cells into the tubular lumen (Figure 4). Quantitative analysis showed that PS-NPs exposure reduced glomerular diameter compared with the normal control, although this difference was not statistically significant. Administration of CTE, particularly at 200 mg/kg BW, significantly increased glomerular diameter relative to the negative control (P= 0.03). CTE treatment at all doses markedly reduced the percentage of necrotic proximal tubules, with the strongest effect observed at 200 mg/kg BW (P= 0.006). Similarly, necrosis in distal tubules was significantly lower in the 200 and 400 mg/kg BW groups (P= 0.03 and P < 0.001, respectively). Regarding epithelial thickness, only CTE 400 mg/kg BW significantly increased the proximal tubular epithelium compared with the negative control (P = 0.04). No significant differences were observed for the diameters of proximal and distal tubules or the epithelial thickness of distal tubules (Table 3).

Discussion

The findings of this study support the potential therapeutic use of CTE in mitigating immune dysregulation and organ damage caused by PS-NPs. This study demonstrated that CTE has significant anti-inflammatory and immunomodulatory effects against nanoplastic-induced toxicity in rats. CTE administration significantly increased RBC count and hemoglobin levels, and decreased the levels of key pro-inflammatory cytokines including IL-1, IL-6, and TNF-α. In addition, CTE treatment improved kidney histology by increasing glomerulus diameter and reducing the percentage of necrosis in convoluted tubules.

Table 3: Administration of CTE affected histology of kidney after NP exposure. Values are represented as mean ± SD (n = 5). Asterisks denote statistical significance. (*): significant difference compared to negative control group. (**): significant difference compared to both normal and negative control group.

Kidney structure	Groups					P value
Kidney structure	Normal control	Negative control	CTE 100 mg/kg BW	CTE 200 mg/kg BW	CTE 400 mg/kg BW	P value
Diameter of glomerulus (µm)	73.15±1.46	68.88 ± 2.27	69.14 ± 4.27	75.97±1.88*	66.85±4.38	0.003
Diameter of proximal convoluted tubules (µm)	31.01±1.47	31.69 ± 2.56	30.99 ± 0.43	32.09 ± 1.39	31.94±1.04	0.775
Diameter of distal convoluted tubules (µm)	20.54±1.67	20.96 ± 1.91	21.98 ± 2.30	21.06 ± 1.44	21.04±1.70	0.851
Necrosis of proximal convoluted tubules (%)	43.71±3.59*	65.92 ± 7.00	52.19±8.34*	48.94±4.03*	51.31±5.94*	0.001
Necrosis of distal convoluted tubules (%)	37.92±3.29	70.94 ± 2.75	61.16 ± 6.84	55.47±5.53**	52.25±5.51**	<0.001
Epithelial thickness of proximal convoluted tubules (µm)	9.69±0.47	9.67 ± 0.50	10.08 ± 0.52	10.33 ± 0.61	10.90±0.66*	0.031
Epithelial thickness of distal convoluted tubules (µm)	5.25±0.33	5.23 ± 0.58	5.41 ± 0.48	5.51 ± 0.42	5.36 ± 0.23	0.876

These findings are consistent with Maneesai et al. (2022) reported that C. ternatea extract reduced oxidative stress markers and improved vascular structure in hypertensive rats. However, our study extends this knowledge by demonstrating its efficacy specifically against nanoplastic-induced damage, which had not been previously reported. While, Swathi et al. (2021) demonstrated anti-inflammatory activity of C. ternatea in carrageenan-induced arthritis, our study uniquely shows its immunomodulatory effect in an environmental toxicology model, thus broadening the application of this plant to novel contexts of pollutant-induced immunotoxicity. One of the strengths of this study is the use of a controlled oral nanoplastic exposure model in rats, which closely mimics real-world exposure routes. Additionally, the dose-dependent design allows for a more comprehensive understanding of the extract’s biological effects and potential therapeutic window.

In recent years, PS-NPs pollution issue has intensified, garnering significant attention worldwide. Previous research reported that hazardous substances in the environment, including PS-NPs, can induce inflammatory responses (Hayati et al., 2023). PS-NPs are consumed by animals via the food chain, resulting in direct organ damage and potentially activating immune responses that lead to inflammation and disease. In line with that, our study showed that PS-NPs exposure could increase the levels of pro-inflammation cytokines significantly compared to normal control. Administration of CTE could lower the levels of pro-inflammatory cytokines. Many diseases development are related with over-production of TNF-α (Wahyuningsih et al., 2018). Thus, we found this result beneficial. The modulation in IL-1 and IL-6 levels following the administration CTE indicates that the extract may regulate immune function by suppressing cytokine levels, aiding in the control of pro-inflammatory cytokines. Reducing pro-inflammatory cytokine levels may help protect healthy cells from damage. Excessive inflammation was induced by over-expression of pro-inflammatory cytokines (Wahyuningsih et al., 2018).

Although reductions in cytokine levels (IL-1, IL-6, and TNF-α) were observed, the effects were not strictly dose-dependent. Notably, IL-6 levels were significantly elevated at the lowest dose (100 mg/kg), whereas suppression was observed at 200 mg/kg, and no significant change occurred at 400 mg/kg. TNF-α levels, on the other hand, showed modest reductions across the CTE groups without a clear linear pattern. This suggests a possible biphasic or hormetic response, in which lower doses may transiently stimulate cytokine expression, while moderate doses suppress it. Such non-linear responses can be explained by receptor saturation, feedback inhibition, and complex interactions among CTE phytochemicals (Maxwell, 2020; Saini et al., 2023). While such non-linear responses are sometimes reported in phytochemical studies, the precise mechanisms remain unclear. The present findings should therefore be interpreted as evidence of immunomodulatory activity rather than a consistent therapeutic effect, and further mechanistic studies are needed to clarify the basis of this dose–response pattern. These findings indicate that CTE possesses immunomodulatory activity, although its effects may vary depending on the administered dose.

The increase in free radicals due to PS-NPs can impact the health of blood cells. Our study showed that PS-NPs exposure could alter the WBC. Administration of CTE at the lower dose (100 mg/kg BW) increased the WBC count. These findings suggest that PS-NPs exert immunotoxic effects characterized by leukopenia alongside increased pro-inflammatory cytokines. CTE did not restore WBC counts to baseline but ameliorated systemic inflammation and tissue damage, indicating an immunomodulatory rather than leukocyte-restorative effect. In line with our study, Xu et al. (2023) reported that exposure to micro/-nanoplastics could significantly decreased the WBC in mice. In RBC and hemoglobin measurements, the PS-NP group showed lower values compared to the CTE-treated groups. CTE administration was associated with a modest but statistically significant increase in hemoglobin. PS-NPs are known to exert suppressive effects on RBC through disruptions in lipid membrane composition, leading to RBC damage and reduced hemoglobin and hematocrit levels. Therefore, the observed changes may indicate a mild protective effect of CTE on erythrocytes, although the biological significance of these small increases remains uncertain. The presence of oxidative stress induced by PS-NPs leads to an imbalance between oxidant and antioxidant systems within cells. This condition triggers a series of reactions that damage lipid membranes, proteins, and DNA, contributing to the death of red blood cells, thus affecting RBC (Wang and Zennadi, 2021). This situation indicates that PS-NPs capable of crossing the blood vessel barrier can lead to pathophysiological events in blood cells.

Polystyrene nanoplastics contribute significantly to environmental pollution due to its non-biodegradable condition (Yan et al., 2023). Food or water contamined with PS-NPs could enter the gastrointestinal system. Once absorbed, they enter the bloodstream and accumulate in important organs, including kidneys, where they interfere with normal function and cause toxicity. The kidneys are frequently targeted by nanoscale materials due to their role in toxin elimination (Li et al., 2023). Study conducted by Lu and Wei (2024) reported that PS-NPs caused inflammatory response in porcine kidney. Other studies show that PS-NPs can also damage animal’s kidney (Hu and Palic, 2020; Li et al., 2023). Our study revealed that nanoplastic could affect diameter of glomerulus and percentage of necrosis in proximal and distal convoluted tubules. The non-linear pattern observed in glomerular diameter, where only the 200 mg/kg dose showed improvement while the 400 mg/kg dose resulted in further reduction, suggests a possible biphasic effect. This finding emphasizes the complexity of CTE’s biological activity and warrants further investigation.

On the other hand, there have been many explorations to reduce the toxicity of PS-NPs by administration of exogenous material especially from plants. Indonesia is one of the countries with a wealth of flora in the world and has various kinds of plants that have potential for health, one of which is Clitoria ternatea. Clitoria ternatea or butterfly pea traditionally referred to as ‘bunga telang,’ in Indonesia. This plant is a perennial, twining herba belongs to the Fabaceae family. This flower is widely distributed around tropical area. The CTE contains bioactive compounds namely, phenols, flavonoids, anthocyanin, kaempferol, tannins, myricetin glycosides, alkaloids, proteins, and carbohydrates (Jeyaraj et al., 2022; Indrianingsih et al., 2024) Based on our GC-MS result, the major peak (67.63%) was tentatively annotated as 1-Methyl-6,7,8,9-tetrahydrol[1,3]thiazepinol[2,3-f]purine-2,4(1H,3H)-dione, a compound analogous to buthiopurine, according to the NIST/Wiley library match. Although this annotation remains tentative and requires confirmation with authentic standards, buthiopurine is classified as a purine alkaloid. Purine alkaloids have been reported to exhibit antioxidant, anti-inflammatory, cytotoxic, antimicrobial, immunomodulatory, and hepatoprotective activities (Letchuman et al., 2024). Buthiopurine is also known as a derivative of mercaptopurine (Comparative Toxicogenomic Database, 2024), a compound with immunosuppressive properties through inhibition of purine metabolism. Therefore, if present, this tentative compound in CTE might contribute to similar biological activities. In our study, the protective effect of CTE against PS-NP-induced oxidative stress and kidney tissue damage suggests that its bioactive compounds, including flavonoids, anthocyanins, and possibly purine alkaloids, may underlie the observed antioxidant and immunomodulatory effects. Nevertheless, further structural elucidation using complementary techniques such as LC-MS/MS or NMR spectroscopy will be required to validate the identity of this compound.

Despite the promising findings, this study has some limitations. The duration of PS-NPs exposure and CTE treatment was relatively short. Moreover, mechanistic insights at the molecular level, such as signaling pathways or gene expression, were not investigated. While general clinical observations (body weight, activity, and appearance) did not reveal overt abnormalities among groups, a systematic scoring of clinical signs was not performed. Similarly, although all rats underwent necropsy, no remarkable gross post-mortem abnormalities were observed beyond the histological alterations already reported; however, detailed documentation of post-mortem changes was not included, as the main focus of this study was on hematological and histological outcomes.

Future research should explore the molecular mechanisms underlying the observed effects, including the modulation of ROS and oxidative stress markers, cytokine signaling pathways (e.g., NF-αB, Nrf2), and gene expression profiling. It is also recommended to evaluate long-term effects and conduct studies involving both male and female animals to confirm sex-specific responses to CTE treatment.

Conclusion

We concluded that CTE could modulate immune response and exhibit anti-inflammation properties by decreasing IL-1, IL-6, TNF-α levels to prevent over-production of pro-inflammatory cytokines after nanoplastics exposure, decreasing number of WBC, and increasing hemoglobin. CTE also has antioxidant activity and could repair the histology of kidney (diameter of glomerulus, percentage of necrosis in proximal and distal convoluted tubules, and epithelial thickness of proximal convoluted tubules) after exposed to 10 µL/kg BW of PS-NPs. Thus, the study suggests that C. ternatea L. flower, cultivated in Indonesia, may serve as an alternative compound to enhance immune response and mitigate the toxicity of PS-NPs entering the body.

Acknowledgment

The authors thank the Universitas Airlangga for funding this research through Penelitian Dasar Unggulan (PDU) in the fiscal year 2024. No. 1667/UN3. FST/PT.01.02/2024

Novelty Statement

The novelty of our study lies in being the first to investigate the immunomodulatory and anti-inflammatory potential of Clitoria ternatea extract against polystyrene nanoplastic-induced toxicity in a mammalian model (rats), whereas most previous nanoplastic studies have focused primarily on aquatic organisms such as fish and mollusks. Furthermore, this work integrates toxicological, phytochemical, immunological, and histopathological assessments within a single experimental framework, providing a holistic understanding of the protective mechanisms involved. In addition, buthiopurine was identified as a major bioactive compound with anti-inflammatory activity through GC–MS profiling, contributing novel insights into the pharmacological potential of C. ternatea.

Author’s Contribution

MP: Conceptualization, study design, funding acquisition, investigation, data analysis, drafting and reviewing the manuscript.

GAK, ZSM, KCL, AOP, NIZ: Investigation, data collection, data analysis.

AH: Study design, reviewing the manuscript.

ESS: Reviewing the manuscript.

Data availability

All data supporting the findings of this study are included in the article.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI or AI-assisted technologies were used in the preparation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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