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Full Research Article

Defensive Influence of Ginkgo Biloba Extract on Toxicity Induced by Isotretinoin in Swiss Albino Mice

D
Doaa M. Elnagar1,*
W
Wejdan S. Alqahtani1
W
Waad S. Alsubaei1
S
Shahad A. Shikh Alghannameh1
A
Aisha H Alqarni1
D
Danah A. Alturbak2
S
Sohailah M. AlNefaie1
N
Norah M. Alqahtani1
A
Albandry H. Alrajeh1
K
Khalid E. Ibrahim1
1Department of Zoology, College of Science , King Saud University, Riyadh, 12372, Saudi Arabia.
2Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, 12372, Saudi Arabia.

ABSTRACT

Background: Isotretinoin is a systemic retinoid derived from vitamin A that is widely used for the treatment of severe recalcitrant acne. 

Methods: Forty male albino mice were segregated into four groups, the first group was the control, the second group was treated with an oral administration of Ginkgo biloba extract (GBE) 120 mg/kg, the third group was treated with an oral administration of isotretinoin (ISO) 40 mg/kg and the fourth group was pre-treated with GBE one hour prior to the ISO administration. Liver enzymes, lipid profile and pro-inflammatory mediators histamine (HA) and prostaglandin (PG) were estimated. Histopathological and histochemical examination for liver and spleen were also performed.

Result: Liver and spleen indices, liver enzymes and lipid profile were significantly higher due to treatment of ISO (p≤0.05) compared to control, which decreased significantly in the group treated with GBE and ISO compared to the third group. In contrast, pro-inflammatory mediators showed no alterations among groups. Additionally, histopathological examination revealed severe hepatocytic degeneration and massive inflammation in ISO-treated group besides great edema and hemorrhage in spleen. GBE pre-treatment to ISO resulted in the decline of the pathological signs in liver and spleen. Intense TNF-α expression was expressed in liver and spleen of animals ISO-treated that was significantly down regulated in animals GBE pre-treated to ISO. The findings demonstrated that isotretinoin (ISO) caused marked adverse physiological and histopathological changes in hepatic and splenic tissues, while Ginkgo biloba extract (GBE) exerted a protective effect against liver and spleen injury.

INTRODUCTION

Isotretinoin (13-cis-retinoic acid) ISO is one of the most effective retinoids used for many dermal diseases such as acne vulgaris, pimples, black heads, healing of oral premalignant diseases and reduction of skin tumors (Raleigh et al., 2000). Retinoids are vitamin A-derived signaling molecules. Vitamin A is synthesized in mammals and also obtained from plant sources such as vegetables and fruits, as well as animal sources like butter and eggs (Bushue and Wan, 2010). Furthermore, retinoids saved in the body as retinyl esters in liver stellate cells (Blaner et al., 2009). ISO controls the acne by inhibiting sebaceous glands’ proliferation, hair follicles keratinization and inflammatory cytokines activity (Kumaş et al., 2018).
       
Retinoid therapy is generally linked to notable toxicity, including hyperostosis, headaches, hypercalcemia and hepatic damage (Hunsu et al., 2021; Sayed and Bekhet, 2020). Long term ISO use has been reported to cause renal and hepatic disorders, elevate oxidative stress and even contribute to depressive symptoms in some patients (Erturan et al., 2012; Ozkol et al., 2015). Isotretinoin induced a hazardous teratogenicity like other retinoid compounds such as craniofacial malformations and other serious birth defects (Balon and Riba, 2016; Pinheiro et al., 2013). Recent findings of psychotic disorders such as neurodevelopmental disorders like autism, psychosis and intellectual disability were reported (Suuberg, 2019).
       
Ginkgo biloba, or maidenhair tree, is an ancient East Asian species known from fossils. It typically grows 20-35 m tall, with some Chinese trees reaching 50 m. Its leaf extracts contain key bioactive compounds, including ginkgolides (terpenes), flavonoid glycosides such as quercetin, myricetin, kaempferol and isorhamnetin, proanthocyanidins, bilobalides and characteristic ginkgo alkylphenols and flavones (van Beek, 2002; van Beek and Montoro, 2009; Wang et al., 2018). Ginkgo biloba extracts possesses marked pharmacological impacts, such as protecting nerves, preventing platelet aggregation, antioxidation and scavenging free radicals and oxidative stress inhibitor (Aziz et al., 2018; Pu et al., 2024; Sugavasi et al., 2019).
       
The present study was performed to evaluate the impact of Ginkgo biloba extract to reduce the toxicity of isotretinoin in Swiss albino mice.

MATERIALS AND METHODS

Isotretinoin was obtained from United Pharmaceutical Company-Saudi Arabia. Ginkgo biloba was obtained from Now food Company, USA.
 
Experimental animals
 
Adult male Swiss albino mice weighing approximately 25-30 g and aged about 14 weeks were provided for the present study. The experiment were performed in Central Research Laboratory, King Saud University, Riyhad and housed under standard laboratory conditions.The mice were provided with a standard pellet diet and water ad libitum throughout the experimental period.
 
Experimental design
 
Forty male Swiss albino mice were segregated into four groups, the first group was the untreated control, the second group was treated with a daily oral administration of GBE 120 mg/kg, the third group was treated with a daily oral administration of ISO 40 mg/kg and the fourth group was pre-treated with GBE one hour prior to the ISO administration as previous regime, the duration of experiment was 28 days.  

Collection of samples
 
At the end of experiment animals were under euthanized using CO2 flow then weighed and dissected using sterile sharp tools. Blood samples were collected from the heart and underwent a CBC test using a hematology analyzer (Mindray - China). Liver and spleen samples were collected, weighed and cut into 2 portions, first one was saved at -20°C for homogenizing and the second portion was fixed in 10% formalin.
 
Liver and spleen indices
 
The animals were weighed to get the total body weight, then liver and spleen samples were weighed. Index was calculated according to the following formula:

 
Liver enzymes estimation
 
Liver samples stored at -20°C were homogenized in cold PBS with ratio 1:5 for 3 min, then centrifuged twice for 20 min at 4°C and supernatants were filtered. Supernatants were used for liver enzymes (ALT, AST and ALP) detection using commercial kits (Spectrum-Egypt).
 
Lipid profile demonstration
 
Supernatants were used for demonstration of lipid profile parameters (TG, TC, HDL and LDL), using commercial kits (Spectrum-Egypt).

Inflammatory mediators’ determination
 
Histamine and prostaglandin inflammatory mediators were measured using ELISA technique (E-EL-0032 and E-EL- 0034) Elab Science-China.
 
Histopathological analysis
 
Liver and spleen pieces were fixed in 10% formalin, then dehydrated through ascending alcohol grades and cleared in xylene. Samples were embedded, sectioned and stained with H andE and PAS. Sections were imaged using a Nikon light microscope (Japan). Image analysis was performed using Image J and Fiji softwares. A histopathological scoring system was applied for liver evaluation (Kleiner et al., 2005), according to the following profile: hepatocellular ballooning, inflammation, steatosis, edema, hemorrhage which graded as, 0=none, 1=mild, 2=moderate, 3=severe. Histopathological scoring system of spleen (Wang et al., 2021) was performed according to the following criteria: White pulp depletion, lymphoid follicle disorganization, red pulp congestion, hemorrhage, hyaline degeneration and inflammatory cell infiltration, it was graded as 0 = Normal histological architecture, 1 = Minimal changes (<10% of tissue affected), 2 = Mild changes (10-25%), 3 = Moderate changes (26-50%), 4 = Severe changes (>50% of tissue affected).
 
Immunohistochemical study
 
Paraffin sections were deparaffinized, rehydrated and subjected to antigen retrieval in EDTA buffer (pH/ 9) using a microwave for 7 min. Sections were incubated overnight at 4°C with primary antibodies: TNF α (E AB 33121) for liver and spleen and P53 (E AB 33472, Elabscience China) for spleen. After PBS washing, sections were incubated with secondary antibody at 37°C for 2 hr, followed by Avidin-Biotin complex for 1 hr at 37°C, then DAB for 30 min. Sections were dehydrated and mounted. Imaging was performed using a Nikon light microscope (Japan) and Fiji software was used to quantify expression distribution and optical density.
 
Statistical analysis
 
Statistical analyses were performed using GraphPad Prism version/ 10 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean±SEM. Group differences were evaluated using one way ANOVA followed by appropriate post hoc. For experiments with two independent variables, two way ANOVA was applied.  P value≤0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Modulation of hematological indices in response to treatment
 
ISO exposure caused inflammatory shift, with significant increases in total WBCs, lymphocytes, monocytes and granulocytes. GBE alone kept leukocyte counts normal and GBE pre treatment partially reduced ISO induced leukocytosis. RBCs and hemoglobin remained stable across all groups, while ISO significantly lowered MCV, a change reversed by GBE. Platelet counts rose sharply with ISO but were largely normalized by GBE (Fig 1).

Fig 1: Hematological parameters across experiment.


 
Assessment of liver and spleen organ indices
 
ISO administration significantly increased both liver and spleen indices compared with controls, indicating organ enlargement. Pre treatment with GBE before ISO markedly significantly reduced liver index and non-significant decrease of spleen indexes relative to ISO (Fig 2).

Fig 2: Effects of GBE and ISO on liver and spleen indices in mice.


 
Biochemical assessment of liver function enzymes
 
ISO administration caused hepatic dysfunction, with significant increases in ALT, AST and ALP confirming hepatocellular and cholestatic injury. GBE pre treatment before ISO significantly reduced ALT and ALP and non significant decline in AST. ISO also induced dyslipidemia, elevating triglycerides, total cholesterol and LDL. GBE pre treatment lowered triglycerides and LDL, while HDL remained unchanged across all groups (Fig 3).

Fig 3: Liver enzymes and lipid profile analysis across experimental groups.


 
Quantification of inflammatory mediator levels
 
Measurements of histamine and prostaglandin showed no treatment related changes across all experimental groups (Fig 4).

Fig 4: Effects of GBE and ISO on histamine and prostaglandin concentrations.


 
Liver histopathology analysis
 
General histopathological changes of liver
 
Microscopic examination showed that both the control liver (Fig 5A) and the GBE only group (Fig 5B) maintained normal hepatic. ISO treatment (Fig 5C) caused severe hepatocellular injury, including heavy inflammatory infiltration, widespread cytoplasmic degeneration and marked parenchymal damage. In the GBE+ISO group (Fig 5D), these toxic changes were visibly reduced, with less leukocyte infiltration and diminished cytoplasmic vacuolization.

Fig 5: Photomicrographs of liver stained routinely.


 
Histochemical evaluation of hepatic glycogen content
 
PAS stained liver sections showed differences among the groups. The control and GBE liver (Fig 6A,B) displayed dense, uniform PAS positive glycogen. ISO treatment (Fig  6C) caused marked glycogen depletion, with weak PAS staining consistent with ballooning degeneration and metabolic stress. In the GBE+ISO group (Fig 6D), glycogen stores were noticeably restored.

Fig 6: Photomicrographs of liver stained histochemically with PAS.


 
Comparative assessment of hepatic histopathological scoring system
 
The liver histopathological scores showed clear group differences. Both the control and GBE only groups displayed normal hepatic architecture with a total score of 0. ISO treated animals exhibited severe pathological signs, reaching the highest score of 12. Pre treatment with GBE before ISO markedly reduced these lesions, lowering the total score to 6 (Table 1).

Table 1: Liver histopathological scoring system across experimental animals.


 
Evaluation of TNF-α mediated inflammation expression in liver
 
The immunohistochemical localization of TNF α showed no detectable TNF α expression with minimal distribution and optical density values in control and GBE treated group (Fig  7A,B) similarly showed absent immunoreactivity. However, ISO treated animals (Fig 7C) exhibited strong TNF α upregulation, with markedly increased distribution and optical density percentages. Pre treatment with GBE before ISO (Fig 7D) visibly reduced TNF α staining distribution area % (Fig 7E) and optical density (Fig 7F).

Fig 7: Photomicrographs of liver stained immunohistochemically for TNF-α mediated inflammation expression.



General microscopic investigation of spleen and quantitative morphometric analysis of splenic lymphoid nodes
 
The control and GBE only groups showed normal splenic architecture, with well defined white pulp nodules and distinct red pulp regions (Fig 8A,B). ISO treatment caused marked splenic injury, including lymphoid depletion, reduced immune cell density, hemorrhage and edema and significant decline in lymph node area (Fig 8C). In the GBE+ISO group, partial restoration was observed (Fig 8D); although some inflammatory features persisted, such as giant macrophages, residual eosinophilic deposits and white pulp fusion into red pulp, with an insignificant increase in lymph node area (Fig 8E).

Fig 8: Histopathological examination of splenic tissue.


       
Analysis of splenic histopathological scoring system
 
The histopathological score of splenic tissues revealed that both the control and GBE treated animals revealed normal splenic architecture registered total score of zero. Contrastingly, ISO treated animals showed severe pathological changes reaching the highest cumulative score (19). Pre administration of GBE to ISO markedly attenuated these alterations, as reflected by reduction of total score to be (11), indicating the impact of GBE to decline pathology of splenic toxicity ISO-induced (Table 2).

Table 2: Splenic histopathological scoring system across experimental animals.


 
Assessment of TNF-α mediated inflammation expression in spleen
 
Immunohistochemical assessment of splenic TNF α showed weak reactivity in the control (Fig 9A) and GBE treated groups (Fig 9B). ISO administration (Fig 9C) produced a marked upregulation of TNF α. This elevation was reduced in the GBE+ISO group (Fig 9D). Quantitatively, TNF α distribution and optical density (Fig 9E,F) increased significantly in the ISO group versus control, but both parameters declined markedly in the GBE pre treated group compared with ISO alone.

Fig 9: Photomicrographs of spleen stained immunohistochemically for TNF-α expression.


 
Determination of P53 mediated apoptosis expression in spleen
 
Immunohistochemical evaluation of splenic P53 showed basal reactivity in the control (Fig 10A) and GBE treated groups (Fig 10B). ISO treatment (Fig 10C) produced a marked rise in P53 positive nuclei. This elevation was declined in the GBE pre treated group (Fig 10D). Quantitatively, P53 distribution and optical density (Fig 10E,F) were significantly increased in the ISO group versus control, but both parameters declined markedly in the GBE+ISO group compared with ISO alone.

Fig 10: Photomicrographs of spleen stained immunohistochemically for P53 expression.


       
ISO induced abundant leukocytosis, with marked increase in total WBCs and all major leukocyte subsets. ISO can trigger systemic inflammation through toll like receptor activation and elevated pro inflammatory cytokines, which stimulate bone marrow myelopoiesis (Dessinioti et al., 2020). GBE alone maintained normal leukocyte counts and its marked ability to reduce ISO induced leukocytosis identified it as a potential biological response modifier. This protective action stems from the combined effects of ginkgolides and flavonoids, which inhibit key pro inflammatory cytokines (Tabassum et al., 2022).
       
ISO caused selective hematological disruption, with RBCs and hemoglobin remaining stable but MCV significantly decreased and platelet counts markedly elevated. These alterations were consistent with ISO related bone marrow effects. The reduced MCV reflected a shift toward microcytosis without loss of red cell mass (Alyasi et al., 2021; Ataseven and Ugur Bilgin, 2014). GBE pre administration corrected ISO induced hematological changes, restoring MCV and normalizing platelet counts (Omidkhoda et al., 2019; Wendo et al., 2024).
       
ISO administration significantly increased liver and spleen indices, indicating systemic organ enlargement. The hepatomegaly corresponds to ISO’s extensive hepatic metabolism. The rise in spleen index reflected activation of systemic inflammatory resulted in splenomegaly (Ataseven and Ugur Bilgin, 2014; Tawanwongsri et al., 2025). GBE pre treatment significantly lowered the liver index in ISO treated animals as evidence for antioxidant and anti inflammatory actions. However, its influence on the spleen index was non significant (Abd-Ellah and Mariee, 2007; Ren et al., 2019).
       
ISO caused significant hepatocellular damage, reflected by elevated ALT, AST and ALP markers of membrane leakage. These findings align with clinical reports showing liver enzyme elevations in a subset of retinoid treated patients. GBE pre treatment markedly decreased enzyme activity. This improvement revealed that GBE flavonoids, including quercetin and kaempferol, stabilize hepatocyte membranes and prevent cytosolic enzyme leakage (He et al., 2006; Tawanwongsri et al., 2025).
       
ISO exposure produced dyslipidemic profile, with significant elevations in major lipid fractions an expected effect of synthetic retinoids. This disturbance likely reflected ISO induced activation of hepatic cytochrome P450 enzymes and reduced lipoprotein lipase activity. GBE pre treatment modified these abnormalities, markedly lowering lipid levels. This improvement is attributed to its bilobalides and ginkgolides, which enhance fatty acid oxidation through PPAR α upregulation (Alrasheed et al., 2024; Czauderna et al., 2018).
       
ISO caused marked hepatocellular injury, with leukocyte infiltration and cytoplasmic degeneration. These lesions reflected ISO induced lipid peroxidation and membrane destabilization. GBE pre treatment countered these effects, reducing inflammatory cell recruitment and cytoplasmic vacuolization through free radical scavenging and lysosomal stabilization that may be attributed to high content of flavonoids (Mahadevan and Park, 2008; Taziki et al., 2022).
       
The immunohistochemical assessment showed that ISO markedly increased hepatic TNF-α expression, indicating a strong pro inflammatory state (Alresheedi et al., 2025). Pre treatment with GBE significantly decreased TNF-α expression, that GBE interfered with NF-κB signaling, the main transcriptional driver of TNF-α expression (Wang et al., 2015).       
       
ISO caused splenic injury, with lymphoid depletion, hemorrhage, edema and disruption of white  and red pulp architecture. GBE pre treatment produced only partial improvement; despite some architectural restoration, persistent features such as giant macrophages and white pulp fusion into red pulp indicate incomplete recovery (Daye et al., 2020; Saipriya, 2025). ISO exposure sharply increased splenic TNF-α and p53 expressions, indicating an intensified inflammatory and apoptotic response within the splenic parenchyma. Pre treatment with GBE markedly reduced TNF-α and p53 expressions demonstrating strong immunomodulatory activity (Li et al., 2020; Mahmoud et al., 2019; Vousden and Prives, 2009; Di Meo et al., 2020).

CONCLUSION

ISO exposure caused broad systemic toxicity, including hematological disturbances, dyslipidemia and structural injury in both liver and spleen. These effects were associated with heightened inflammation and apoptosis. GBE provided strong protective effects, improving liver function and structure through antioxidant and anti inflammatory actions. GBE still downregulated key inflammatory and apoptotic markers in both organs, demonstrating a solid modulatory role against ISO induced toxicity.

ACKNOWLEDGEMENT

Authors appreciate (Ongoing Research Funding program, ORF-2026-759) for funding this work, King Saud University, Riyadh, Saudi Arabia.
 
Funding
 
The project was funded by Ongoing Research Funding program, ORF-2026-759, King Saud University, Riyadh, Saudi Arabia.
 
Statements
 
Ethical approval
 
The experiment was carried out according to the Institutional Review Board (IRB), King Saud University, Riyadh, Saudi Arabia, the ethics reference No. KSU-SE-24-63.
 
Data Availability
 
Data are available from the corresponding author.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES


  1. Abd-Ellah, M.F. and Mariee, A.D. (2007). Ginkgo biloba leaf extract (EGb 761) diminishes adriamycin-induced hyperlipidaemic nephrotoxicity in rats: association with nitric oxide production. Biotechnology and Applied Biochemistry. 46(Pt 1): 35-40. https://doi.org/10.1042/BA20060085.

  2. Alrasheed, A.A., Alsadhan, K.F., Alfawzan, N.F., AbuDujain, N.M., Alnasser, A.H. and Almousa, H. (2024). Impact of isotretinoin on blood lipids and liver enzymes: A retrospective cohort  study in Saudi Arabia. Therapeutics and Clinical Risk Management. 20: 567-575. https://doi.org/10.2147/TCRM. S466113.

  3. Alresheedi, N.H., Samaha, M.M., Ibrahim, T.M. and Abu-Elsaad, N.M. (2025). Eupatilin protects against isotretinoin induced hepatotoxicity through immunomodulation of TLR4/MyD88/TRAF6 and NF-κB/NrF2 pathways. European Journal of Pharmacology. 1003: 177920. https://doi.org/ https://doi.org/10.1016/j.ejphar.2025.177920.

  4. Alyasi, A., Al Hawsawi, K., Malebari, B.A., Mandili, R. and Alqasim, D. (2021). Isotretinoin-Induced Thrombocytosis in a Patient With Acne Vulgaris: A Case Report. Cureus. 13(7): e16716. https://doi.org/10.7759/cureus.16716.

  5. Ataseven, A. and Ugur Bilgin, A. (2014). Effects of Isotretinoin on the Platelet Counts and the Mean Platelet Volume in Patients with Acne Vulgaris. The Scientific World Journal. 2014(1): 156464. https://doi.org/https://doi.org/10.1155/ 2014/156464.

  6. Aziz, T.A., Hussain, S.A., Mahwi, T.O. and Ahmed, Z.A. (2018). Efficacy and safety of Ginkgo biloba extract as an “add- on” treatment to  metformin for patients with metabolic syndrome: A pilot clinical study. Therapeutics and Clinical Risk Management. 14: 1219-1226. https://doi.org/10.214 7/TCRM.S169503.

  7. Balon, R. and Riba, M. (2016). Should women of childbearing potential be prescribed valproate? a call to action. The Journal of Clinical Psychiatry. 77(4): 525-526. https:// doi.org/10.4088/JCP.15com09958.

  8. Blaner, W.S., O’Byrne, S.M., Wongsiriroj, N., Kluwe, J., D’Ambrosio, D.M., Jiang, H., Schwabe, R.F., Hillman, E.M.C., Piantedosi, R. and Libien, J. (2009). Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid  storage. Biochimica et Biophysica Acta. 1791(6): 467-473. https://doi.org/10. 1016/j.bbalip.2008.11.001.

  9. Bushue, N. and Wan, Y.J.Y. (2010). Retinoid pathway and cancer therapeutics. Advanced Drug Delivery Reviews. 62(13): 1285-1298. https://doi.org/10.1016/j.addr.2010.07.003.

  10. Czauderna, C., Palestino-Dominguez, M., Castven, D., Becker, D., Zanon-Rodriguez, L., Hajduk, J., Mahn, F.L., Herr, M., Strand, D., Strand, S., Heilmann-Heimbach, S., Gomez- Quiroz, L.E., Wörns, M.A., Galle, P.R. and Marquardt, J.U. (2018). Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver. PloS One. 13(12): e0209067. https://doi.org/10.1371/journal. pone. 0209067.

  11. Daye, M., Belviranli, M., Okudan, N., Mevlitoglu, I. and Oz, M. (2020). The effect of isotretinoin therapy on oxidative damage in rats. Dermatologic Therapy. 33(6): e14111. https://doi.org/10.1111/dth.14111.

  12. Dessinioti, C., Zouboulis, C.C., Bettoli, V. and Rigopoulos, D. (2020). Comparison of guidelines and consensus articles on the management of patients with acne with oral isotretinoin. Journal of the European Academy of Dermatology and Venereology/ : JEADV. 34(10): 2229- 2240. https://doi.org/10.1111/jdv.16430.

  13. Di Meo, F., Cuciniello, R., Margarucci, S., Bergamo, P., Petillo, O., Peluso, G., Filosa, S. and  Crispi, S. (2020). Ginkgo biloba Prevents Oxidative Stress-Induced Apoptosis Blocking p53 Activation in Neuroblastoma Cells. Antioxidants (Basel, Switzerland). 9(4): 279. https://doi.org/10.3390/ antiox9040279.

  14. Erturan, İ., Naziroðlu, M. and Akkaya, V.B. (2012). Isotretinoin treatment induces oxidative toxicity in blood of patients with acne vulgaris: A clinical pilot study. Cell Biochemistry and Function. 30(7): 552-557. https://doi.org/10.1002/ cbf.2830.

  15. He, S.X., Luo, J.Y., Wang, Y.P., Wang, Y.L., Fu, H., Xu, J.L., Zhao, G. and Liu, E.Q. (2006). Effects of extract from Ginkgo biloba on carbon tetrachloride-induced liver  injury in rats. World Journal of Gastroenterology. 12(24): 3924-3928. https://doi.org/10.3748/wjg.v12.i24.3924.

  16. Hunsu, V.O., Facey, C.O.B., Fields, J.Z. and Boman, B.M. (2021). Retinoids as Chemo-Preventive and Molecular-Targeted Anti-Cancer Therapies. International Journal of Molecular Sciences. 22(14). https://doi.org/10.3390/ijms22147731.

  17. Kleiner, D.E., Brunt, E.M., Van Natta, M., Behling, C., Contos, M.J., Cummings, O.W., Ferrell, L.D., Liu, Y., Torbenson, M.S., Unalp Arida, A., Yeh, M., McCullough, A.J., Sanyal, A.J. and Network, N.S.C.R. (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 41(6): 1313. https://journals.lww. com/hep/fulltext/2005/06000/design_and_validation_ of_a_histological_scoring.15.aspx.

  18. Kumaş, M., Eþrefoğlu, M. and Güler, E.M. (2018). Protective effects of silymarin against isotretinoin induced liver and kidney injury in mice. Indian Journal of Experimental Biology. 56(3): 158-163.

  19. Li, C., Liu, K., Liu, S., Aerqin, Q. and Wu, X. (2020). Role of Ginkgolides in the Inflammatory Immune Response of Neurological Diseases: A Review of Current Literatures. Frontiers in Systems Neuroscience. 14: 45. https://doi. org/10.3389/fnsys.2020.00045.

  20. Mahadevan, S. and Park, Y. (2008). Multifaceted therapeutic benefits of Ginkgo biloba L.: chemistry, efficacy,  safety and uses. Journal of Food Science. 73(1): R14-9. https://doi.org/ 10.1111/j.1750-3841.2007.00597.x

  21. Mahmoud, A.M., Wilkinson, F.L., Sandhu, M.A., Dos Santos, J.M. and Alexander, M.Y. (2019). Modulating oxidative stress in drug-induced injury and metabolic disorders: The role of natural and synthetic antioxidants. In Oxidative medicine and cellular longevity (Vol. 2019, p. 3206401). https://doi.org/10.1155/2019/3206401.

  22. Omidkhoda, S.F., Razavi, B.M. and Hosseinzadeh, H. (2019). Protective effects of Ginkgo biloba L. against natural toxins, chemical toxicities and radiation: A comprehensive review. Phytotherapy Research. 33(11): 2821-2840. https://doi.org/https://doi.org/10.1002/ptr.6469.

  23. Ozkol, H.U., Ozkol, H., Karadag, A.S., Bilgili, S.G., Tuluce, Y. and Calka, O. (2015). Oral isotretinoin therapy of acne patients decreases serum paraoxonase-1 activity  through increasing oxidative stress. Drug and Chemical Toxicology. 38(1): 63-66. https://doi.org/10.3109/01480545.2014.905590.

  24. Pinheiro, S.P., Kang, E.M., Kim, C.Y., Governale, L.A., Zhou, E.H. and Hammad, T.A. (2013). Concomitant use of isotretinoin and contraceptives before and after iPledge in  the United States. Pharmacoepidemiology and Drug Safety. 22(12): 1251-1257. https://doi.org/10.1002/pds.3481.

  25. Pu, X., Fu, Y., Yang, Y. and Xu, G. (2024). Ginkgo biloba extract alleviates CCl4-induced acute liver injury by regulating PI3K/AKT signaling pathway. Heliyon. 10(4): e26093. https://doi.org/https://doi.org/10.1016/j.heliyon.2024. e26093.

  26. Raleigh, S., Verschoyle, R., Bowskill, C., Pastorino, U., Staniforth, J., Steele, F., Dinsdale, D., Carthew, P., Lim, C., Silvester, J. and Gescher, A. (2000). Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol. British Journal of Cancer. 83: 935-940. https://doi.org/ 10.1054/bjoc.2000.1421.

  27. Ren, M., Zhang, Y., Li, R. and Li, Q. (2019). Effect of ginkgo biloba L. extract on oxidative stress and activity of protein kinase C in spleen tissue of mice with irradiation damage. Journal of Jilin University Medicine Edition. 45: 223-227. https://doi.org/10.13481/j.1671-587x.20190202.

  28. Saipriya, S. (2025). Drug-Induced Alterations in Splenic Structure and Function. International Journal of Pharmaceutical Research and Applications. 10(3): 903-914. https://doi. org/10.35629/4494-1003903914.

  29. Sayed, A. and Bekhet, G. (2020). Impact of Vitamin A on the expression profile of development-controlling genes in chick embryos. Indian Journal of Animal Research. 1-8. doi: 10.18805/ijar.B-1256.

  30. Sugavasi, R., Sivanesan, S., Gudemalla, K., Mundugaru, R. and Swaminathan, M. (2019). Effect of Ginkgo biloba extract on hematological and biochemical alterations in fluoride intoxicated wistar rats. Research Journal of Pharmacy and Technology. 12: 3839. https://doi.org/10.5958/0974- 360X.2019.00659.0.

  31. Suuberg, A. (2019). Psychiatric and developmental effects of isotretinoin (Retinoid) treatment for  acne vulgaris. Current Therapeutic Research, Clinical and Experimental. 90: 27-31. https://doi.org/10.1016/j.curtheres.2019. 01.008.

  32. Tabassum, N., Das, R., Lami, M.S., Chakraborty, A.J., Mitra, S., Tallei, T.E., Idroes, R., Mohamed, A.A.R., Hossain, M.J., Dhama, K., Mostafa-Hedeab, G. and Emran, T. Bin. (2022). Ginkgo biloba: A Treasure of Functional Phytochemicals with Multimedicinal Applications. Evidence-Based Complementary and Alternative Medicine: ECAM, 2022, 8288818. https://doi.org/10.1155/2022/8288818.

  33. Tawanwongsri, W., Kanchanasuwan, T. and Eden, C. (2025). Isotretinoin and Hepatotoxicity in Patients with Acne: A Narrative Review. Cosmetics. 12(1): 17. https://doi.org/ 10.3390/cosmetics12010017.

  34. Taziki, S., Gholamzadeh, F. and Hosseini, R. (2022). The hepatoprotective effects of taurine against oxidative stress induced by  isotretinoin in rats. Journal of Biochemical and Molecular Toxicology. 36(11): e23178. https://doi.org/10.1002/jbt. 23178.

  35. van Beek, T.A. (2002). Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatography A. 967(1): 21-55. https://doi.org/10.1016/s0021-9673(02)00172-3.

  36. van Beek, T.A. and Montoro, P. (2009). Chemical analysis and quality control of Ginkgo biloba leaves, extracts and  phytopharmaceuticals. Journal of Chromatography A. 1216(11): 2002-2032. https://doi.org/10.1016/j.chroma. 2009.01.013.

  37. Vousden, K.H. and Prives, C. (2009). Blinded by the Light: The Growing Complexity of p53. Cell. 137(3): 413-431. https:/ /doi.org/10.1016/j.cell.2009.04.037.

  38. Wang, H., Li, S., Cui, Z., Qin, T., Shi, H., Ma, J., Li, L., Yu, G., Jiang, T. and Li, C. (2021). Analysis of spleen histopathology, splenocyte composition and haematological parameters in four strains of mice infected with Plasmodium berghei K173. Malaria Journal. 20(1): 249. https://doi.org/10. 1186/s12936-021-03786-z.

  39. Wang, J., Mao, S., Wang, J., Jiao, Y., Zhang, X., Zhang, Y., Wang, Z., Zhang, Q. and Li, S. (2018). Effect of Ginkgo biloba extract on cognitive function and neurotransmitter levels in rats with vascular dementia. Indian Journal of Animal Research. 52(8): 1141-1145. doi: 10.18805/ijar.v0iOF.8467.

  40. Wang, Y., Wang, R., Wang, Y., Peng, R., Wu, Y. and Yuan, Y. (2015). Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38  MAPK, NF-κB/IkBα and Bcl- 2/Bax signaling. Drug Design, Development and Therapy. 9: 6303-6317. https://doi.org/10.2147/DDDT.S93732.

  41. Wendo, J.K., Mbaria, J.M., Nyariki, J.N. and Isaac, A.O. (2024). Ginkgo biloba attenuated detrimental inflammatory and oxidative events due to Trypanosoma brucei rhodesiense in mice treated with melarsoprol. PLOS Neglected Tropical Diseases. 18(4): 1-26. https://doi.org/10.1371/ journal.pntd.0012103.
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    Full Research Article

    Defensive Influence of Ginkgo Biloba Extract on Toxicity Induced by Isotretinoin in Swiss Albino Mice

    D
    Doaa M. Elnagar1,*
    W
    Wejdan S. Alqahtani1
    W
    Waad S. Alsubaei1
    S
    Shahad A. Shikh Alghannameh1
    A
    Aisha H Alqarni1
    D
    Danah A. Alturbak2
    S
    Sohailah M. AlNefaie1
    N
    Norah M. Alqahtani1
    A
    Albandry H. Alrajeh1
    K
    Khalid E. Ibrahim1
    1Department of Zoology, College of Science , King Saud University, Riyadh, 12372, Saudi Arabia.
    2Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, 12372, Saudi Arabia.

    ABSTRACT

    Background: Isotretinoin is a systemic retinoid derived from vitamin A that is widely used for the treatment of severe recalcitrant acne. 

    Methods: Forty male albino mice were segregated into four groups, the first group was the control, the second group was treated with an oral administration of Ginkgo biloba extract (GBE) 120 mg/kg, the third group was treated with an oral administration of isotretinoin (ISO) 40 mg/kg and the fourth group was pre-treated with GBE one hour prior to the ISO administration. Liver enzymes, lipid profile and pro-inflammatory mediators histamine (HA) and prostaglandin (PG) were estimated. Histopathological and histochemical examination for liver and spleen were also performed.

    Result: Liver and spleen indices, liver enzymes and lipid profile were significantly higher due to treatment of ISO (p≤0.05) compared to control, which decreased significantly in the group treated with GBE and ISO compared to the third group. In contrast, pro-inflammatory mediators showed no alterations among groups. Additionally, histopathological examination revealed severe hepatocytic degeneration and massive inflammation in ISO-treated group besides great edema and hemorrhage in spleen. GBE pre-treatment to ISO resulted in the decline of the pathological signs in liver and spleen. Intense TNF-α expression was expressed in liver and spleen of animals ISO-treated that was significantly down regulated in animals GBE pre-treated to ISO. The findings demonstrated that isotretinoin (ISO) caused marked adverse physiological and histopathological changes in hepatic and splenic tissues, while Ginkgo biloba extract (GBE) exerted a protective effect against liver and spleen injury.

    INTRODUCTION

    Isotretinoin (13-cis-retinoic acid) ISO is one of the most effective retinoids used for many dermal diseases such as acne vulgaris, pimples, black heads, healing of oral premalignant diseases and reduction of skin tumors (Raleigh et al., 2000). Retinoids are vitamin A-derived signaling molecules. Vitamin A is synthesized in mammals and also obtained from plant sources such as vegetables and fruits, as well as animal sources like butter and eggs (Bushue and Wan, 2010). Furthermore, retinoids saved in the body as retinyl esters in liver stellate cells (Blaner et al., 2009). ISO controls the acne by inhibiting sebaceous glands’ proliferation, hair follicles keratinization and inflammatory cytokines activity (Kumaş et al., 2018).
           
    Retinoid therapy is generally linked to notable toxicity, including hyperostosis, headaches, hypercalcemia and hepatic damage (Hunsu et al., 2021; Sayed and Bekhet, 2020). Long term ISO use has been reported to cause renal and hepatic disorders, elevate oxidative stress and even contribute to depressive symptoms in some patients (Erturan et al., 2012; Ozkol et al., 2015). Isotretinoin induced a hazardous teratogenicity like other retinoid compounds such as craniofacial malformations and other serious birth defects (Balon and Riba, 2016; Pinheiro et al., 2013). Recent findings of psychotic disorders such as neurodevelopmental disorders like autism, psychosis and intellectual disability were reported (Suuberg, 2019).
           
    Ginkgo biloba, or maidenhair tree, is an ancient East Asian species known from fossils. It typically grows 20-35 m tall, with some Chinese trees reaching 50 m. Its leaf extracts contain key bioactive compounds, including ginkgolides (terpenes), flavonoid glycosides such as quercetin, myricetin, kaempferol and isorhamnetin, proanthocyanidins, bilobalides and characteristic ginkgo alkylphenols and flavones (van Beek, 2002; van Beek and Montoro, 2009; Wang et al., 2018). Ginkgo biloba extracts possesses marked pharmacological impacts, such as protecting nerves, preventing platelet aggregation, antioxidation and scavenging free radicals and oxidative stress inhibitor (Aziz et al., 2018; Pu et al., 2024; Sugavasi et al., 2019).
           
    The present study was performed to evaluate the impact of Ginkgo biloba extract to reduce the toxicity of isotretinoin in Swiss albino mice.

    MATERIALS AND METHODS

    Isotretinoin was obtained from United Pharmaceutical Company-Saudi Arabia. Ginkgo biloba was obtained from Now food Company, USA.
     
    Experimental animals
     
    Adult male Swiss albino mice weighing approximately 25-30 g and aged about 14 weeks were provided for the present study. The experiment were performed in Central Research Laboratory, King Saud University, Riyhad and housed under standard laboratory conditions.The mice were provided with a standard pellet diet and water ad libitum throughout the experimental period.
     
    Experimental design
     
    Forty male Swiss albino mice were segregated into four groups, the first group was the untreated control, the second group was treated with a daily oral administration of GBE 120 mg/kg, the third group was treated with a daily oral administration of ISO 40 mg/kg and the fourth group was pre-treated with GBE one hour prior to the ISO administration as previous regime, the duration of experiment was 28 days.  

    Collection of samples
     
    At the end of experiment animals were under euthanized using CO2 flow then weighed and dissected using sterile sharp tools. Blood samples were collected from the heart and underwent a CBC test using a hematology analyzer (Mindray - China). Liver and spleen samples were collected, weighed and cut into 2 portions, first one was saved at -20°C for homogenizing and the second portion was fixed in 10% formalin.
     
    Liver and spleen indices
     
    The animals were weighed to get the total body weight, then liver and spleen samples were weighed. Index was calculated according to the following formula:

     
    Liver enzymes estimation
     
    Liver samples stored at -20°C were homogenized in cold PBS with ratio 1:5 for 3 min, then centrifuged twice for 20 min at 4°C and supernatants were filtered. Supernatants were used for liver enzymes (ALT, AST and ALP) detection using commercial kits (Spectrum-Egypt).
     
    Lipid profile demonstration
     
    Supernatants were used for demonstration of lipid profile parameters (TG, TC, HDL and LDL), using commercial kits (Spectrum-Egypt).

    Inflammatory mediators’ determination
     
    Histamine and prostaglandin inflammatory mediators were measured using ELISA technique (E-EL-0032 and E-EL- 0034) Elab Science-China.
     
    Histopathological analysis
     
    Liver and spleen pieces were fixed in 10% formalin, then dehydrated through ascending alcohol grades and cleared in xylene. Samples were embedded, sectioned and stained with H andE and PAS. Sections were imaged using a Nikon light microscope (Japan). Image analysis was performed using Image J and Fiji softwares. A histopathological scoring system was applied for liver evaluation (Kleiner et al., 2005), according to the following profile: hepatocellular ballooning, inflammation, steatosis, edema, hemorrhage which graded as, 0=none, 1=mild, 2=moderate, 3=severe. Histopathological scoring system of spleen (Wang et al., 2021) was performed according to the following criteria: White pulp depletion, lymphoid follicle disorganization, red pulp congestion, hemorrhage, hyaline degeneration and inflammatory cell infiltration, it was graded as 0 = Normal histological architecture, 1 = Minimal changes (<10% of tissue affected), 2 = Mild changes (10-25%), 3 = Moderate changes (26-50%), 4 = Severe changes (>50% of tissue affected).
     
    Immunohistochemical study
     
    Paraffin sections were deparaffinized, rehydrated and subjected to antigen retrieval in EDTA buffer (pH/ 9) using a microwave for 7 min. Sections were incubated overnight at 4°C with primary antibodies: TNF α (E AB 33121) for liver and spleen and P53 (E AB 33472, Elabscience China) for spleen. After PBS washing, sections were incubated with secondary antibody at 37°C for 2 hr, followed by Avidin-Biotin complex for 1 hr at 37°C, then DAB for 30 min. Sections were dehydrated and mounted. Imaging was performed using a Nikon light microscope (Japan) and Fiji software was used to quantify expression distribution and optical density.
     
    Statistical analysis
     
    Statistical analyses were performed using GraphPad Prism version/ 10 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean±SEM. Group differences were evaluated using one way ANOVA followed by appropriate post hoc. For experiments with two independent variables, two way ANOVA was applied.  P value≤0.05 was considered statistically significant.

    RESULTS AND DISCUSSION

    Modulation of hematological indices in response to treatment
     
    ISO exposure caused inflammatory shift, with significant increases in total WBCs, lymphocytes, monocytes and granulocytes. GBE alone kept leukocyte counts normal and GBE pre treatment partially reduced ISO induced leukocytosis. RBCs and hemoglobin remained stable across all groups, while ISO significantly lowered MCV, a change reversed by GBE. Platelet counts rose sharply with ISO but were largely normalized by GBE (Fig 1).

    Fig 1: Hematological parameters across experiment.


     
    Assessment of liver and spleen organ indices
     
    ISO administration significantly increased both liver and spleen indices compared with controls, indicating organ enlargement. Pre treatment with GBE before ISO markedly significantly reduced liver index and non-significant decrease of spleen indexes relative to ISO (Fig 2).

    Fig 2: Effects of GBE and ISO on liver and spleen indices in mice.


     
    Biochemical assessment of liver function enzymes
     
    ISO administration caused hepatic dysfunction, with significant increases in ALT, AST and ALP confirming hepatocellular and cholestatic injury. GBE pre treatment before ISO significantly reduced ALT and ALP and non significant decline in AST. ISO also induced dyslipidemia, elevating triglycerides, total cholesterol and LDL. GBE pre treatment lowered triglycerides and LDL, while HDL remained unchanged across all groups (Fig 3).

    Fig 3: Liver enzymes and lipid profile analysis across experimental groups.


     
    Quantification of inflammatory mediator levels
     
    Measurements of histamine and prostaglandin showed no treatment related changes across all experimental groups (Fig 4).

    Fig 4: Effects of GBE and ISO on histamine and prostaglandin concentrations.


     
    Liver histopathology analysis
     
    General histopathological changes of liver
     
    Microscopic examination showed that both the control liver (Fig 5A) and the GBE only group (Fig 5B) maintained normal hepatic. ISO treatment (Fig 5C) caused severe hepatocellular injury, including heavy inflammatory infiltration, widespread cytoplasmic degeneration and marked parenchymal damage. In the GBE+ISO group (Fig 5D), these toxic changes were visibly reduced, with less leukocyte infiltration and diminished cytoplasmic vacuolization.

    Fig 5: Photomicrographs of liver stained routinely.


     
    Histochemical evaluation of hepatic glycogen content
     
    PAS stained liver sections showed differences among the groups. The control and GBE liver (Fig 6A,B) displayed dense, uniform PAS positive glycogen. ISO treatment (Fig  6C) caused marked glycogen depletion, with weak PAS staining consistent with ballooning degeneration and metabolic stress. In the GBE+ISO group (Fig 6D), glycogen stores were noticeably restored.

    Fig 6: Photomicrographs of liver stained histochemically with PAS.


     
    Comparative assessment of hepatic histopathological scoring system
     
    The liver histopathological scores showed clear group differences. Both the control and GBE only groups displayed normal hepatic architecture with a total score of 0. ISO treated animals exhibited severe pathological signs, reaching the highest score of 12. Pre treatment with GBE before ISO markedly reduced these lesions, lowering the total score to 6 (Table 1).

    Table 1: Liver histopathological scoring system across experimental animals.


     
    Evaluation of TNF-α mediated inflammation expression in liver
     
    The immunohistochemical localization of TNF α showed no detectable TNF α expression with minimal distribution and optical density values in control and GBE treated group (Fig  7A,B) similarly showed absent immunoreactivity. However, ISO treated animals (Fig 7C) exhibited strong TNF α upregulation, with markedly increased distribution and optical density percentages. Pre treatment with GBE before ISO (Fig 7D) visibly reduced TNF α staining distribution area % (Fig 7E) and optical density (Fig 7F).

    Fig 7: Photomicrographs of liver stained immunohistochemically for TNF-α mediated inflammation expression.



    General microscopic investigation of spleen and quantitative morphometric analysis of splenic lymphoid nodes
     
    The control and GBE only groups showed normal splenic architecture, with well defined white pulp nodules and distinct red pulp regions (Fig 8A,B). ISO treatment caused marked splenic injury, including lymphoid depletion, reduced immune cell density, hemorrhage and edema and significant decline in lymph node area (Fig 8C). In the GBE+ISO group, partial restoration was observed (Fig 8D); although some inflammatory features persisted, such as giant macrophages, residual eosinophilic deposits and white pulp fusion into red pulp, with an insignificant increase in lymph node area (Fig 8E).

    Fig 8: Histopathological examination of splenic tissue.


           
    Analysis of splenic histopathological scoring system
     
    The histopathological score of splenic tissues revealed that both the control and GBE treated animals revealed normal splenic architecture registered total score of zero. Contrastingly, ISO treated animals showed severe pathological changes reaching the highest cumulative score (19). Pre administration of GBE to ISO markedly attenuated these alterations, as reflected by reduction of total score to be (11), indicating the impact of GBE to decline pathology of splenic toxicity ISO-induced (Table 2).

    Table 2: Splenic histopathological scoring system across experimental animals.


     
    Assessment of TNF-α mediated inflammation expression in spleen
     
    Immunohistochemical assessment of splenic TNF α showed weak reactivity in the control (Fig 9A) and GBE treated groups (Fig 9B). ISO administration (Fig 9C) produced a marked upregulation of TNF α. This elevation was reduced in the GBE+ISO group (Fig 9D). Quantitatively, TNF α distribution and optical density (Fig 9E,F) increased significantly in the ISO group versus control, but both parameters declined markedly in the GBE pre treated group compared with ISO alone.

    Fig 9: Photomicrographs of spleen stained immunohistochemically for TNF-α expression.


     
    Determination of P53 mediated apoptosis expression in spleen
     
    Immunohistochemical evaluation of splenic P53 showed basal reactivity in the control (Fig 10A) and GBE treated groups (Fig 10B). ISO treatment (Fig 10C) produced a marked rise in P53 positive nuclei. This elevation was declined in the GBE pre treated group (Fig 10D). Quantitatively, P53 distribution and optical density (Fig 10E,F) were significantly increased in the ISO group versus control, but both parameters declined markedly in the GBE+ISO group compared with ISO alone.

    Fig 10: Photomicrographs of spleen stained immunohistochemically for P53 expression.


           
    ISO induced abundant leukocytosis, with marked increase in total WBCs and all major leukocyte subsets. ISO can trigger systemic inflammation through toll like receptor activation and elevated pro inflammatory cytokines, which stimulate bone marrow myelopoiesis (Dessinioti et al., 2020). GBE alone maintained normal leukocyte counts and its marked ability to reduce ISO induced leukocytosis identified it as a potential biological response modifier. This protective action stems from the combined effects of ginkgolides and flavonoids, which inhibit key pro inflammatory cytokines (Tabassum et al., 2022).
           
    ISO caused selective hematological disruption, with RBCs and hemoglobin remaining stable but MCV significantly decreased and platelet counts markedly elevated. These alterations were consistent with ISO related bone marrow effects. The reduced MCV reflected a shift toward microcytosis without loss of red cell mass (Alyasi et al., 2021; Ataseven and Ugur Bilgin, 2014). GBE pre administration corrected ISO induced hematological changes, restoring MCV and normalizing platelet counts (Omidkhoda et al., 2019; Wendo et al., 2024).
           
    ISO administration significantly increased liver and spleen indices, indicating systemic organ enlargement. The hepatomegaly corresponds to ISO’s extensive hepatic metabolism. The rise in spleen index reflected activation of systemic inflammatory resulted in splenomegaly (Ataseven and Ugur Bilgin, 2014; Tawanwongsri et al., 2025). GBE pre treatment significantly lowered the liver index in ISO treated animals as evidence for antioxidant and anti inflammatory actions. However, its influence on the spleen index was non significant (Abd-Ellah and Mariee, 2007; Ren et al., 2019).
           
    ISO caused significant hepatocellular damage, reflected by elevated ALT, AST and ALP markers of membrane leakage. These findings align with clinical reports showing liver enzyme elevations in a subset of retinoid treated patients. GBE pre treatment markedly decreased enzyme activity. This improvement revealed that GBE flavonoids, including quercetin and kaempferol, stabilize hepatocyte membranes and prevent cytosolic enzyme leakage (He et al., 2006; Tawanwongsri et al., 2025).
           
    ISO exposure produced dyslipidemic profile, with significant elevations in major lipid fractions an expected effect of synthetic retinoids. This disturbance likely reflected ISO induced activation of hepatic cytochrome P450 enzymes and reduced lipoprotein lipase activity. GBE pre treatment modified these abnormalities, markedly lowering lipid levels. This improvement is attributed to its bilobalides and ginkgolides, which enhance fatty acid oxidation through PPAR α upregulation (Alrasheed et al., 2024; Czauderna et al., 2018).
           
    ISO caused marked hepatocellular injury, with leukocyte infiltration and cytoplasmic degeneration. These lesions reflected ISO induced lipid peroxidation and membrane destabilization. GBE pre treatment countered these effects, reducing inflammatory cell recruitment and cytoplasmic vacuolization through free radical scavenging and lysosomal stabilization that may be attributed to high content of flavonoids (Mahadevan and Park, 2008; Taziki et al., 2022).
           
    The immunohistochemical assessment showed that ISO markedly increased hepatic TNF-α expression, indicating a strong pro inflammatory state (Alresheedi et al., 2025). Pre treatment with GBE significantly decreased TNF-α expression, that GBE interfered with NF-κB signaling, the main transcriptional driver of TNF-α expression (Wang et al., 2015).       
           
    ISO caused splenic injury, with lymphoid depletion, hemorrhage, edema and disruption of white  and red pulp architecture. GBE pre treatment produced only partial improvement; despite some architectural restoration, persistent features such as giant macrophages and white pulp fusion into red pulp indicate incomplete recovery (Daye et al., 2020; Saipriya, 2025). ISO exposure sharply increased splenic TNF-α and p53 expressions, indicating an intensified inflammatory and apoptotic response within the splenic parenchyma. Pre treatment with GBE markedly reduced TNF-α and p53 expressions demonstrating strong immunomodulatory activity (Li et al., 2020; Mahmoud et al., 2019; Vousden and Prives, 2009; Di Meo et al., 2020).

    CONCLUSION

    ISO exposure caused broad systemic toxicity, including hematological disturbances, dyslipidemia and structural injury in both liver and spleen. These effects were associated with heightened inflammation and apoptosis. GBE provided strong protective effects, improving liver function and structure through antioxidant and anti inflammatory actions. GBE still downregulated key inflammatory and apoptotic markers in both organs, demonstrating a solid modulatory role against ISO induced toxicity.

    ACKNOWLEDGEMENT

    Authors appreciate (Ongoing Research Funding program, ORF-2026-759) for funding this work, King Saud University, Riyadh, Saudi Arabia.
     
    Funding
     
    The project was funded by Ongoing Research Funding program, ORF-2026-759, King Saud University, Riyadh, Saudi Arabia.
     
    Statements
     
    Ethical approval
     
    The experiment was carried out according to the Institutional Review Board (IRB), King Saud University, Riyadh, Saudi Arabia, the ethics reference No. KSU-SE-24-63.
     
    Data Availability
     
    Data are available from the corresponding author.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

    REFERENCES


    1. Abd-Ellah, M.F. and Mariee, A.D. (2007). Ginkgo biloba leaf extract (EGb 761) diminishes adriamycin-induced hyperlipidaemic nephrotoxicity in rats: association with nitric oxide production. Biotechnology and Applied Biochemistry. 46(Pt 1): 35-40. https://doi.org/10.1042/BA20060085.

    2. Alrasheed, A.A., Alsadhan, K.F., Alfawzan, N.F., AbuDujain, N.M., Alnasser, A.H. and Almousa, H. (2024). Impact of isotretinoin on blood lipids and liver enzymes: A retrospective cohort  study in Saudi Arabia. Therapeutics and Clinical Risk Management. 20: 567-575. https://doi.org/10.2147/TCRM. S466113.

    3. Alresheedi, N.H., Samaha, M.M., Ibrahim, T.M. and Abu-Elsaad, N.M. (2025). Eupatilin protects against isotretinoin induced hepatotoxicity through immunomodulation of TLR4/MyD88/TRAF6 and NF-κB/NrF2 pathways. European Journal of Pharmacology. 1003: 177920. https://doi.org/ https://doi.org/10.1016/j.ejphar.2025.177920.

    4. Alyasi, A., Al Hawsawi, K., Malebari, B.A., Mandili, R. and Alqasim, D. (2021). Isotretinoin-Induced Thrombocytosis in a Patient With Acne Vulgaris: A Case Report. Cureus. 13(7): e16716. https://doi.org/10.7759/cureus.16716.

    5. Ataseven, A. and Ugur Bilgin, A. (2014). Effects of Isotretinoin on the Platelet Counts and the Mean Platelet Volume in Patients with Acne Vulgaris. The Scientific World Journal. 2014(1): 156464. https://doi.org/https://doi.org/10.1155/ 2014/156464.

    6. Aziz, T.A., Hussain, S.A., Mahwi, T.O. and Ahmed, Z.A. (2018). Efficacy and safety of Ginkgo biloba extract as an “add- on” treatment to  metformin for patients with metabolic syndrome: A pilot clinical study. Therapeutics and Clinical Risk Management. 14: 1219-1226. https://doi.org/10.214 7/TCRM.S169503.

    7. Balon, R. and Riba, M. (2016). Should women of childbearing potential be prescribed valproate? a call to action. The Journal of Clinical Psychiatry. 77(4): 525-526. https:// doi.org/10.4088/JCP.15com09958.

    8. Blaner, W.S., O’Byrne, S.M., Wongsiriroj, N., Kluwe, J., D’Ambrosio, D.M., Jiang, H., Schwabe, R.F., Hillman, E.M.C., Piantedosi, R. and Libien, J. (2009). Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid  storage. Biochimica et Biophysica Acta. 1791(6): 467-473. https://doi.org/10. 1016/j.bbalip.2008.11.001.

    9. Bushue, N. and Wan, Y.J.Y. (2010). Retinoid pathway and cancer therapeutics. Advanced Drug Delivery Reviews. 62(13): 1285-1298. https://doi.org/10.1016/j.addr.2010.07.003.

    10. Czauderna, C., Palestino-Dominguez, M., Castven, D., Becker, D., Zanon-Rodriguez, L., Hajduk, J., Mahn, F.L., Herr, M., Strand, D., Strand, S., Heilmann-Heimbach, S., Gomez- Quiroz, L.E., Wörns, M.A., Galle, P.R. and Marquardt, J.U. (2018). Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver. PloS One. 13(12): e0209067. https://doi.org/10.1371/journal. pone. 0209067.

    11. Daye, M., Belviranli, M., Okudan, N., Mevlitoglu, I. and Oz, M. (2020). The effect of isotretinoin therapy on oxidative damage in rats. Dermatologic Therapy. 33(6): e14111. https://doi.org/10.1111/dth.14111.

    12. Dessinioti, C., Zouboulis, C.C., Bettoli, V. and Rigopoulos, D. (2020). Comparison of guidelines and consensus articles on the management of patients with acne with oral isotretinoin. Journal of the European Academy of Dermatology and Venereology/ : JEADV. 34(10): 2229- 2240. https://doi.org/10.1111/jdv.16430.

    13. Di Meo, F., Cuciniello, R., Margarucci, S., Bergamo, P., Petillo, O., Peluso, G., Filosa, S. and  Crispi, S. (2020). Ginkgo biloba Prevents Oxidative Stress-Induced Apoptosis Blocking p53 Activation in Neuroblastoma Cells. Antioxidants (Basel, Switzerland). 9(4): 279. https://doi.org/10.3390/ antiox9040279.

    14. Erturan, İ., Naziroðlu, M. and Akkaya, V.B. (2012). Isotretinoin treatment induces oxidative toxicity in blood of patients with acne vulgaris: A clinical pilot study. Cell Biochemistry and Function. 30(7): 552-557. https://doi.org/10.1002/ cbf.2830.

    15. He, S.X., Luo, J.Y., Wang, Y.P., Wang, Y.L., Fu, H., Xu, J.L., Zhao, G. and Liu, E.Q. (2006). Effects of extract from Ginkgo biloba on carbon tetrachloride-induced liver  injury in rats. World Journal of Gastroenterology. 12(24): 3924-3928. https://doi.org/10.3748/wjg.v12.i24.3924.

    16. Hunsu, V.O., Facey, C.O.B., Fields, J.Z. and Boman, B.M. (2021). Retinoids as Chemo-Preventive and Molecular-Targeted Anti-Cancer Therapies. International Journal of Molecular Sciences. 22(14). https://doi.org/10.3390/ijms22147731.

    17. Kleiner, D.E., Brunt, E.M., Van Natta, M., Behling, C., Contos, M.J., Cummings, O.W., Ferrell, L.D., Liu, Y., Torbenson, M.S., Unalp Arida, A., Yeh, M., McCullough, A.J., Sanyal, A.J. and Network, N.S.C.R. (2005). Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 41(6): 1313. https://journals.lww. com/hep/fulltext/2005/06000/design_and_validation_ of_a_histological_scoring.15.aspx.

    18. Kumaş, M., Eþrefoğlu, M. and Güler, E.M. (2018). Protective effects of silymarin against isotretinoin induced liver and kidney injury in mice. Indian Journal of Experimental Biology. 56(3): 158-163.

    19. Li, C., Liu, K., Liu, S., Aerqin, Q. and Wu, X. (2020). Role of Ginkgolides in the Inflammatory Immune Response of Neurological Diseases: A Review of Current Literatures. Frontiers in Systems Neuroscience. 14: 45. https://doi. org/10.3389/fnsys.2020.00045.

    20. Mahadevan, S. and Park, Y. (2008). Multifaceted therapeutic benefits of Ginkgo biloba L.: chemistry, efficacy,  safety and uses. Journal of Food Science. 73(1): R14-9. https://doi.org/ 10.1111/j.1750-3841.2007.00597.x

    21. Mahmoud, A.M., Wilkinson, F.L., Sandhu, M.A., Dos Santos, J.M. and Alexander, M.Y. (2019). Modulating oxidative stress in drug-induced injury and metabolic disorders: The role of natural and synthetic antioxidants. In Oxidative medicine and cellular longevity (Vol. 2019, p. 3206401). https://doi.org/10.1155/2019/3206401.

    22. Omidkhoda, S.F., Razavi, B.M. and Hosseinzadeh, H. (2019). Protective effects of Ginkgo biloba L. against natural toxins, chemical toxicities and radiation: A comprehensive review. Phytotherapy Research. 33(11): 2821-2840. https://doi.org/https://doi.org/10.1002/ptr.6469.

    23. Ozkol, H.U., Ozkol, H., Karadag, A.S., Bilgili, S.G., Tuluce, Y. and Calka, O. (2015). Oral isotretinoin therapy of acne patients decreases serum paraoxonase-1 activity  through increasing oxidative stress. Drug and Chemical Toxicology. 38(1): 63-66. https://doi.org/10.3109/01480545.2014.905590.

    24. Pinheiro, S.P., Kang, E.M., Kim, C.Y., Governale, L.A., Zhou, E.H. and Hammad, T.A. (2013). Concomitant use of isotretinoin and contraceptives before and after iPledge in  the United States. Pharmacoepidemiology and Drug Safety. 22(12): 1251-1257. https://doi.org/10.1002/pds.3481.

    25. Pu, X., Fu, Y., Yang, Y. and Xu, G. (2024). Ginkgo biloba extract alleviates CCl4-induced acute liver injury by regulating PI3K/AKT signaling pathway. Heliyon. 10(4): e26093. https://doi.org/https://doi.org/10.1016/j.heliyon.2024. e26093.

    26. Raleigh, S., Verschoyle, R., Bowskill, C., Pastorino, U., Staniforth, J., Steele, F., Dinsdale, D., Carthew, P., Lim, C., Silvester, J. and Gescher, A. (2000). Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol. British Journal of Cancer. 83: 935-940. https://doi.org/ 10.1054/bjoc.2000.1421.

    27. Ren, M., Zhang, Y., Li, R. and Li, Q. (2019). Effect of ginkgo biloba L. extract on oxidative stress and activity of protein kinase C in spleen tissue of mice with irradiation damage. Journal of Jilin University Medicine Edition. 45: 223-227. https://doi.org/10.13481/j.1671-587x.20190202.

    28. Saipriya, S. (2025). Drug-Induced Alterations in Splenic Structure and Function. International Journal of Pharmaceutical Research and Applications. 10(3): 903-914. https://doi. org/10.35629/4494-1003903914.

    29. Sayed, A. and Bekhet, G. (2020). Impact of Vitamin A on the expression profile of development-controlling genes in chick embryos. Indian Journal of Animal Research. 1-8. doi: 10.18805/ijar.B-1256.

    30. Sugavasi, R., Sivanesan, S., Gudemalla, K., Mundugaru, R. and Swaminathan, M. (2019). Effect of Ginkgo biloba extract on hematological and biochemical alterations in fluoride intoxicated wistar rats. Research Journal of Pharmacy and Technology. 12: 3839. https://doi.org/10.5958/0974- 360X.2019.00659.0.

    31. Suuberg, A. (2019). Psychiatric and developmental effects of isotretinoin (Retinoid) treatment for  acne vulgaris. Current Therapeutic Research, Clinical and Experimental. 90: 27-31. https://doi.org/10.1016/j.curtheres.2019. 01.008.

    32. Tabassum, N., Das, R., Lami, M.S., Chakraborty, A.J., Mitra, S., Tallei, T.E., Idroes, R., Mohamed, A.A.R., Hossain, M.J., Dhama, K., Mostafa-Hedeab, G. and Emran, T. Bin. (2022). Ginkgo biloba: A Treasure of Functional Phytochemicals with Multimedicinal Applications. Evidence-Based Complementary and Alternative Medicine: ECAM, 2022, 8288818. https://doi.org/10.1155/2022/8288818.

    33. Tawanwongsri, W., Kanchanasuwan, T. and Eden, C. (2025). Isotretinoin and Hepatotoxicity in Patients with Acne: A Narrative Review. Cosmetics. 12(1): 17. https://doi.org/ 10.3390/cosmetics12010017.

    34. Taziki, S., Gholamzadeh, F. and Hosseini, R. (2022). The hepatoprotective effects of taurine against oxidative stress induced by  isotretinoin in rats. Journal of Biochemical and Molecular Toxicology. 36(11): e23178. https://doi.org/10.1002/jbt. 23178.

    35. van Beek, T.A. (2002). Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatography A. 967(1): 21-55. https://doi.org/10.1016/s0021-9673(02)00172-3.

    36. van Beek, T.A. and Montoro, P. (2009). Chemical analysis and quality control of Ginkgo biloba leaves, extracts and  phytopharmaceuticals. Journal of Chromatography A. 1216(11): 2002-2032. https://doi.org/10.1016/j.chroma. 2009.01.013.

    37. Vousden, K.H. and Prives, C. (2009). Blinded by the Light: The Growing Complexity of p53. Cell. 137(3): 413-431. https:/ /doi.org/10.1016/j.cell.2009.04.037.

    38. Wang, H., Li, S., Cui, Z., Qin, T., Shi, H., Ma, J., Li, L., Yu, G., Jiang, T. and Li, C. (2021). Analysis of spleen histopathology, splenocyte composition and haematological parameters in four strains of mice infected with Plasmodium berghei K173. Malaria Journal. 20(1): 249. https://doi.org/10. 1186/s12936-021-03786-z.

    39. Wang, J., Mao, S., Wang, J., Jiao, Y., Zhang, X., Zhang, Y., Wang, Z., Zhang, Q. and Li, S. (2018). Effect of Ginkgo biloba extract on cognitive function and neurotransmitter levels in rats with vascular dementia. Indian Journal of Animal Research. 52(8): 1141-1145. doi: 10.18805/ijar.v0iOF.8467.

    40. Wang, Y., Wang, R., Wang, Y., Peng, R., Wu, Y. and Yuan, Y. (2015). Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38  MAPK, NF-κB/IkBα and Bcl- 2/Bax signaling. Drug Design, Development and Therapy. 9: 6303-6317. https://doi.org/10.2147/DDDT.S93732.

    41. Wendo, J.K., Mbaria, J.M., Nyariki, J.N. and Isaac, A.O. (2024). Ginkgo biloba attenuated detrimental inflammatory and oxidative events due to Trypanosoma brucei rhodesiense in mice treated with melarsoprol. PLOS Neglected Tropical Diseases. 18(4): 1-26. https://doi.org/10.1371/ journal.pntd.0012103.
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