Hepatoprotective potentials of Bryophyllum pinnatum against doxorubicin induced liver damage in Wistar rats

 Corresponding author: Gabriel Otu Ujong Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Hepatoprotective potentials of Bryophyllum pinnatum against doxorubicin induced liver damage in Wistar rats. Gabriel Otu Ujong * Department of Human Physiology, University of Cross River State. Okuku Campus, Cross River State, Nigeria. World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 Publication history: Received on 04 September 2025; revised on 10 October 2025; accepted on 13 October 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.28.1.3510 Abstract Doxorubicin (Dox), an antibiotic used in tumors treatment, has several substantial adverse effects that limit its clinical utilization. Dox administration impairs liver function, noting the vital role of liver in the body, it is pertinent to finding effective protective agents to combat Dox-induced liver damage. This study was therefore set to ascertain the hepatoprotective role of Bryophyllum pinnatum (Crassulacaea) against Dox-induced liver damage in rats. Five groups of 7 rats each were used for the study. The control received normal rat feed and drinking water. Group 2 (Dox only) received 50 mg/kg of Doxorubicin i.p. once daily. Group 3 (Extract group) took 600mg/kg of the ethanolic extract of Bryophyllum pinnatum orally once daily. Group 4 (Dox + extract) had Doxorubicin + extract and group 5 (Dox + Bis) received Doxorubicin + 5mg/kg of Bisoprolol orally once daily. The feeding regimens lasted 28 days. Blood samples and liver tissues were collected under ketamine (60 mg/kg body weight). Our results revealed that administration of induced liver damage in rats with a significant increase in Caspases-3 concentration and decrease in BCL-2 levels. Doxonly group also had significant (p<0.05) elevation of serum hepatic injury biomarkers (ALT, AST, ALP, total and direct bilirubin) as well as significant (p<0.05) reduction in albumin concentration. Furthermore, all the hepatic photomicrographs featured injuries to the hepatocytes. All these anomalies were ameliorated following treatment with ethanolic extract of Bryophyllum pinnatum or Bisoprolol. These findings reveal the hepatoprotective potentials of Bryophyllum pinnatum extract against doxorubicin-induced liver damage in rats. Keywords: Doxorubicin; Bryophyllum pinnatum; Bisoprolol; Hepatoprotection; Caspases; BCl-2 1. Introduction Doxorubicin (DOX) is a popular drug widely used to treat breast cancer, leukemia, bronchogenic carcinoma, gastric carcinoma, sarcomas and thyroid carcinomas as well as hematological malignancies [1]. Although DOX has a prominent antitumor activity, its adverse effects on non-cancerous cells limit its use [2]. It is metabolized by liver microsomal enzymes and cytoplasmic reductases, resulting in the accumulation of toxic and immunogenic intermediates that have been implicated in the induction of liver injury [3]. DOX toxicity is caused by a variety of processes including DNA damage in cancerous, preventing cell proliferation [4]. while in non-cancerous cells, it triggers oxidative stress and impairs the mitochondrial activity by building up reactive oxygen species (ROS) [5]. DOX could stimulate oxidative stress by inhibiting the transcription factor; nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that synchronizes cellular redox homeostasis and controls the antioxidant and detoxification responses [6]. The pathway mediating DOX-induced impairment of redox homeostasis has been reported in numerous organs, like heart, kidneys and liver [7]. “Plants and plant parts are being used to prevent as well as allay symptoms, or revert abnormalities. Most of the pharmaceutical products currently dispensed by physicians have a long history of use as herbal remedies including opium, aspirin, digitalis and quinine” [8]. Additionally, modern medicine utilizes active compounds isolated World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 944 from plants and about 80% of these active ingredients indicate a positive correlation between their modern therapeutic use and the traditional use. Bryophyllum pinnatum is a succulent, perennial plant about 1.0 m tall, with a fleshy cylindrical stem, a reddish colour (for the youngest), and potential health benefits [9]. Common names of Bryophyllum pinnatum include “life plant”, “air plant”, “African never die” and “Litana wa” in Nigeria, Bryophyllum pinnatum has been used to treat hypertension [10], rheumatism, body pain, arthritis, heartburn, skin ulcers, peptic ulcers, diabetes mellitus, microbial infections. Others include immune-modulatory, CNS depressant, analgesic, anti-inflammatory, antiulcer, insecticidal, anti-diabetic, anticonvulsant and antioxidant properties. Caspases (cysteine aspartases, or cysteine-dependent aspartate-directed proteases, or cysteine-aspartic proteases), belonging to the family of proteases, are significant in the regulation of apoptosis and inflammatory processes and in development [11], as the intrinsic or extrinsic pathways of apoptosis both induce cell death by activating caspases [12]. It has been shown in several studies that BIS offers cardio-protective effects against apoptosis, autophagy, and oxidative stress through modulation of immune and inflammatory mediators. The BCl-2 family is a group of structurally related proteins that participate in the regulation of apoptosis, some as inhibitors and others as activators of this process [13]. Bcl-2, the first member of this family to be discovered, is an antagonist of apoptosis [14]. Additionally, BIS demonstrated a protective effect against myocardial ischaemia/reperfusion injury by reducing the size of an infarct. Bisoprolol was found to decrease serum levels of creatinine kinase and lactate dehydrogenase, lower the apoptotic index, and suppress tumour necrosis factor alpha (TNF-α) and interleukin 6 mediators, thus down regulating the caspase 3 and caspase 12 signaling pathways. When drugs are taken over long periods or in high doses could cause hepatotoxicity. This could be prevented through early intervention for better outcomes [15]. Despite the clinical efficacy of doxorubicin in cancer treatment, its use is often associated with cardiac toxicity, leading to adverse effects such as hepatotoxicity characterized by elevated bilirubin levels and disruption of hepatic cytoarchitecture. Recent research suggests potential therapeutic benefits of ethanolic leaf extract of Bryophyllum pinnatum (Crassulaceae) and bisoprolol in mitigating cardiac toxicity, hepatic functions and tissue integrity in doxorubicin-induced toxicity remain inadequately understood. Therefore, this research tends to evaluate the effect of ethanolic leaf extract of Bryophyllum pinnatum and bisoprolol on some liver function indices in doxorubicin induced liver damage in rats. 2. Materials and method 2.1. Collection and preparation of plant materials The plant Bryophyllum pinnatum (Ccrassulaceae) (Africa never die) was obtained from Ugep local market, in Yakurr Local Government Area of Cross River State, on March, 2024. It was identified and authenticated in the Department of Botany, University of Calabar. Its ethanolic leaf extract was prepared in University of Cross River State (UNICROSS), Okuku Campus. 2.2. Preparation of Crassulaceae (africa never die) extract Bryophyllum pinnatum (African never die) leaves were cleaned, washed to remove debris and dried. They were then grinded into powder using Allyson blender (model FY-999), after grinding. It was soaked in 98% ethanol solution (BDH chemical Ltd Poole England) for 24 hours and was then filtered, after 24 hours the supernatant was extracted, first using second time using white cellulose material, and then by Whatman number 1 filter paper, oven dried at 45oC into paste form and stored at normal room temperature. The extraction gave a percentage yield of about 5.3%. The extract was stored at 4oC till further use. 2.3. Experimental animal Thirty-five (35) Wistar rats weighing between 150-250 were obtained from the animal house of physiology Department, faculty of Basic Medical Sciences, University of Cross River State (UNICROSS) Okuku campus. The animals were allowed to acclimatize for one week in the medical Physiology Department where the experiment was carried out. The animals were housed in well-ventilated cages. The beddings, feed and water were replaced every day, and kept under controlled environmental conditions (room temperature of about 27oC and 12-hour light/dark cycle). The animals were given feed and water freely. 2.4. Experimental design The animals were group into five group containing 7 rats in each group. World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 945 • Group 1 (Control): received normal rat chow and drinking water • Group 2 (Extract fed): took 600 mg/kg body weight of Crassuaceae extract orally • Group 3 (Dox only): Received 50 mg/kg body weight of doxorubicin • Group 4 (Dox + Extract): took 50 mg/kg body weight of doxorubicin + 600 mg/kg body weight of Crassuaceae extract orally • Group 5 (Dox + Bis): took 50 mg/kg body weight of doxorubicin + 5 mg/kg body weight of bisoprolol orally Animals were also fed with normal salt and water ad libitum. Treatment lasted for 28 days. 2.5. Collection of blood samples After 28 days of experimental regimens, the animals were sacrifice under 60 mg/kg body weight ketamine intraperitoneally. Blood was collected via cardiac puncture using a 5 ml syringe into plain capped sample bottles, which was then centrifuged at 3000 RPM for 10 minutes). Serum was extracted from the supernatant and taken for laboratory assay. 2.6. Harvesting of liver The liver was harvested from each animal and placed in capped sample bottles containing 10 % formaldehyde for preservation, which would later be taken for further analysis to determine the cardiac biomarkers and histological study. 2.7. Determination of biochemical parameters 2.7.1. Determination Caspase-3 Caspase-3 and its inhibition can be determined by using the fluorescence correlation spectroscopy (FCS) technique. FCS is a highly sensitive method based on measuring the fluorescence fluctuations in a laser highly focused detection volume of less than 1.0 fL (10-15 L). This method is widely used for studying molecular diffusion and concentration, chemical kinetics, enzyme activity assay, and molecular interaction such as protein−protein interaction drug−protein in vitro and in vivo. However, FCS is limited for application in enzyme activity and molecule interactions since the difference in the molecule weight of the un-cleaved substrate and cleaved substrate should be more than 8 times. FCS to determine caspase-3 and matrix metalloproteinase-9 activities by increasing the molecular weight of the substrates, Fluorescence cross-correlation spectroscopy (FCCS) is not dependent on molecular size changes, and applied FCCS to determine protease activity of caspase-3 in live cells. 2.7.2. Determination of BCl-2 The expression of BCl-2 has been detected in different types of hepatocytes in monoand binucleated hepatocytes during chronic hepatitis B. In animal cells, BCl-2 proteins have been found on the outer mitochondrial membrane, which is supposed to form a complex with the voltage-gated anion channel porin (VDAC). Interaction between BCl-2 with VDAC3or VDAC1-derived peptides has been shown to protect against cell death by inhibiting cytochrome c release. Bcl-2 family proteins have also been detected in the perinuclear envelope and found to be broadly located in many tissues. The ability of the BCl-2 family proteins to form oligomeric pores in artificial lipid bilayers has been documented. When liberated from mitochondria, apoptogenic factors (Smac/Diablo homologue, cytochrome c homologue, etc.) activate caspases, which, in turn, induce programmed cell death. 2.7.3. Determination of conjugated bilirubin Conjugated bilirubin, in the presence of a solubilizing agent, is coupled with 3,5-dichlorophenyl diazonium in a strongly acidic medium. The intensity of the red azo dye formed is directly proportional to the total bilirubin and can be determined photometrically (546 nm). 2.7.4. Determination of unconjugated bilirubin A 200-μl reaction mixture containing 150 μl UnaG solution (400 pmol) and 50 μl diluted bilirubin solution or serum (for a final UnaG concentration of 2 μM) was prepared, with 5 μl artificial unconjugated bilirubin solution or newborn serum added to 995 μl of 0.1 M phosphate-buffered saline (PBS; 200-fold dilution) as we have previously shown. A microplate reader SH-9000; Corona Electric Co., was used at 37 °C with fluorescence filters for excitation and emission wavelengths of 498 and 527 nm, respectively. Bilirubin concentrations ranging from 0.0 to 66.8 mg/dl were prepared using artificial World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 946 bilirubin standard solutions including albumin (Arrows Co., Osaka, Japan) and were measured to generate a standard calibration curve. Serum samples were diluted 200-fold with PBS and fluorescence intensity was measured. Serum concentrations of unconjugated bilirubin were extrapolated from the standard curve. 2.7.5. Determination of alanine aminotransferase (ALT) Kinetic method according to the International Federation of Clinical Chemistry [16]. Assay Principle: The series of the reaction involved in the assay system is as follows: • The amino group is enzymatically transferred by ALT present in the sample from alanine to the carbon atom of 2-oxoglutarate yielding pyruvate and L-glutamate. • Pyruvate is reduced to lactate by lactate-dehydrogenase (LDH) present in the reagent with the simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD). The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. • Endogenous sample pyruvate is rapidly and completely reduced by LDH during the initial incubation period so that it does not interfere with the assay. Procedure: Macro Semi-micro Reagent R 1.0mL 500 µL Specimen 100µL 50 µL It was mixed and initial absorbance read after 60 seconds and then the timer was started simultaneously. It was read again after 60, 120 and 180 seconds. The mean absorbance change per minute (∆A/min) was thereafter determined. Calculation: The ALT activity formula: U/l = 1746 x ∆A 340 nm /min 2.7.6. Determination of alkaline phosphatase (ALP) ALP – by Colorimetric method, [17]. Assay Principle Colorimetric determination of alkaline Phosphatase activity according to the following reaction: Phenyl phosphate Alkaline phosphatase Phenol + Phosphate (at pH 10) Phenol liberated is measured in the presence of 4-aminoantipyrine and Potassium ferricyanide. The presence of sodium arsenate in the reagent stops the enzymatic reaction. Calculation: Optical density of serum sample – Optical density of serum blank x (n) Optical density of Standard n = 20 (Kind and King IU/100 ml) n = 142 (IU/L) World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 947 2.7.7. Determination of aspartate aminotransferase (AST) This was done by colorimetric method [18]. Principle: The reaction involved in the assay system is as follows: The amino group is enzymatically transferred by AST present in the sample from L-aspartate to the carbon atom of 2oxoglutarate yielding oxaloacetate and L-glutamate. L-Aspartate + 2-Oxoglutarate AST Oxaloacetate + L-Glutamate AST activity is measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4dinitrophenylhydrazine. Calculation: AST activity was obtained from the formula table in U/L 2.8. Measurement of total plasma proteins The Biuret method was employed [19] based on the principle that the Cupric ions in alkaline solution react with peptide bonds in proteins producing a violet colour which is proportionate to the amount of protein present (Biuret reaction). 2.9. Measurement of serum albumin Serum albumin was measured based on the principle that albumin binds with bromocresol green (BCG) at pH 4.2 causing a slight change in absorbance of the yellow BCG dye. The blue-green colour formed is proportional to the concentration of albumin present when measured photometrically between 580 and 630 nm with maximum absorbance at 625 nm [20, 21]. Calculation: Albumin (g/dl) = Absorbance of test × conc. of standard (g/dL) Absorbance of standard (g/dL) 3. Results 3.1. Serum BCL-2 level in the different experimental groups Result of the serum BCL-2 levels of control, Doxorubicin (Dox), Extract, Dox + Extract and Dox + Bisoprolol were 6.70 ±0.33, 1.40 ±0.15, 7.40 ±0.10, 5.50 ±0.12 and 4.50 ±0.14 ng/mL respectively. The result was significant lower (p<0.01) in Dox only when compared with the control, Dox + Extract and Dox + Bis groups. Values obtained for Dox + Extract and Dox + Biso were significantly (p<0.05) lower compared with the extract group, Figure 1. 3.2. Serum caspase-3 level in the different experimental groups The result of the serum caspase-3 levels in of control, Doxorubicin (Dox), Extract, Dox + Extract and Dox + Bisoprolol were 2.50 ±0.13, 16.0 ±0.23, 3.50 ±0.07, 2.20 ±0.14 and 1.80 ±0.14 ng/mL respectively. The result showed a significantly higher (p˂0.01) values in Dox only group when compared with the control. However, values obtained for other groups were significantly (p<0.05) lower compared with Dox only group, Figure 2. 3.3. Serum total proteins, albumin and globulin levels in the different experimental groups The serum total proteins concentration (g/L) for the different experimental groups were: control (63.00 ±0.71), Dox (66.00 ±0.51), Extract (58.00 ±0.37), Dox + Extract (61.00 ±0.71) and Dox + Bis (68.00 ±0.68). It was significantly (p<0.05) higher in Dox only group compared with control. Values obtained for extract only group and Dox + Extract group were significantly (p<0.05) lower compared with control, Dox only and Dox + Bis groups, Table 1. World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 948 The serum albumin concentration (g/L) for the different experimental groups were: control (39.00 ±0.51), Dox (33.00 ±0.71), Extract (31.00 ±0.51), Dox + Extract (25.00 ±0.58) and Dox + Bis (30.00 ±0.81). It was significantly (p<0.05) lower in other experimental groups compared with control, Table 1. The serum globulin concentration (g/L) for the different experimental groups were: control (24.00 ±0.45), Dox (33.00 ±0.68), Extract (27.00 ±0.37), Dox + Extract (35.00 ±0.66) and Dox + Bis (39.00 ±1.10). It was significantly (p<0.05) higher in Dox only, Dox + Extract and Dox + Bis groups compared with control and extract groups, Table 1. 3.4. Serum liver enzymes (AST, ALT and ALP) levels in the different experimental groups Result of the serum AST levels (IU/L) of control, Doxorubicin (Dox), Extract, Dox + Extract and Dox + Bisoprolol were 22.00 ±0.71, 40.00 ±1.50, 26.00 ±0.71, 26.00 ±0.71 and 29.00 ±0.51 ng/mL respectively. It was significantly (p<0.05) higher in Dox only group compared with control and other experimental groups, Table 1. The ALT concentration (IU/L) in the control group was (11.00 ±0.58), mean value obtained for Dox only group (23.00 ±0.97) was significantly (p<0.05) higher compared with control. The Extract only, Dox + Extract, and Dox + Bis groups had significantly (p<0.05) lower ALT levels compared with Dox only group, Table 1. The ALP concentration (IU/L) in the Dox only group was significantly (p<0.05) higher compared with control group (that is 171.00 ±1.30 and 105.00 ±1.40 respectively). The mean values of ALP concentration for the Extract only, Dox + Extract, and Dox + Bis groups were significantly (p<0.05) lower compared with Dox only group, Table 1. 3.5. Serum total bilirubin level in the different experimental groups The result of the serum total bilirubin levels (µmol/L) for the different groups were: control (5.10 ±0.33), Dox (9.90 ±0.59), Extract (3.5±0.093), Dox + Extract (8.0±0.60) and Dox + Bis (7.5±0.18). The result showed a significantly (p<0.05) higher level in Dox only group when compared with the control group. Values obtained for Dox + Extract and Dox + Bis groups were significantly (p<0.05) higher compared with other groups. The extract only groups had the lowest total bilirubin level compared with other groups, Table 2. 3.6. Serum conjugated and unconjugated bilirubin levels in the different experimental groups The result of the serum conjugated bilirubin levels (µmol/L) were: control (2.4±0.071), Dox (5.0±0.071), extract (2.2±0.10), Dox + Extract (4.3±0.17) and Dox = Bis (3.7±0.14). Serum conjugated bilirubin levels were also significantly (p<0.05) higher in Dox only groups compared with control and other experimental groups. Values obtained for the extract only group was also significantly (p<0.05) lower compared with other groups. While values obtained for Dox + Extract and Dox + Bis groups were significantly (p<0.05) higher compared with other groups, Table 2. Serum unconjugated bilirubin levels were also significantly (p<0.05) higher in Dox only, Dox + Extract and Dox + Bis groups compared with control and extract only groups. Values obtained for the extract only group was also significantly (p<0.05) lower compared with other groups, Table 2. 3.7. Histological study of the Liver As shown in figures 3 to 6, the photomicrograph of the liver tissue in the control shows a central vein surrounded by hepatocytes (dark dotted cells) appearing normal. In the Dox group, the photomicrograph of the liver tissue shows a central vein surrounded by hepatocytes (dark dotted cells) marked alterations in the liver cytoarchitecture. Photomicrograph of the liver tissue in the Dox + Extract group shows a central vein surrounded by hepatocytes (dark dotted cells) appearing normal. In the Dox + Bis group, the photomicrograph of the liver tissue shows a central vein surrounded by hepatocytes (dark dotted cells) appearing normal. World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 949 Values are expressed as mean ±SEM, n = 7l * = p<0.01 compared with control; # = p<0.01 versus other treated groups; + = p<0.05 compared with Dox + Extract group; a =p<0.05 compared with Extract group Figure 1 Comparison of BCL-2 concentration in the different experimental groups Values are expressed as mean ±SEM, n = 7; ** = p<0.01 compared with control; # = p<0.01 versus other treated groups; + = p<0.05 compared with DOX+CRA group, a =p<0.05 compared with DOX+CRA group Figure 2 Comparison of Caspases concentration in the different experimental groups World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 950 Table 1 Serum proteins and liver enzymes concentration in the different experimental groups Total protein (g/L) Albumin (g/L) Globulin (g/L) AST (IU/L) ALT (IU/L) ALP (IU/L) Control 63.00 39.00 24.00 22.00 11.00 105.00 ±0.71 ±0.51 ±0.45 ±0.71 ±0.58 ±1.40 Dox 66.00 33.00 33.00 40.00 23.00 171.00 ±0.51* ±0.71* ±0.68* ±1.50* ±0.97* ±1.30* Extract 58.00 31.00 27.00 26.00 17.00 89.00 ±0.37*,a ±0.51* ±0.37a ±0.71*,a ±0.51*,a ±0.86*,a Dox+ Extract 61.00 25.00 35.00 26.00 21.00 143.00 ±0.71a ±0.588*,a ±0.66*,b ±0.71*,a ±0.68*,b ±0.75*,a,b Dox + Bis 68.00 30.00 39.00 29.00 19.00 164.00 ±0.68b ±0.81*,a,c ±1.10*,a,b,c ±0.51*,a ±0.86*,a ±0.71*,a,b,c Values are expressed as mean ±SEM, n = 7. * = p<0.05 vs control; a = p<0.05 vs Dox; b = p<0.05 vs Extract; c = p<0.05vs Dox + Extract Table 2 Serum bilirubin concentrations and platelet count in the different experimental groups Total bilirubin (µmol/L) Conjugated bilirubin (µmol/L) Unconjugated bilirubin (µmol/L) Control 5.10 2.40 2.70 ±0.33 ±0.07 ±0.20 Dox 9.90 5.00 4.90 ±0.598 ±0.07* ±0.33* Extract 3.50 2.20 1.30 ±0.09*,a ±0.10a ±0.10*,a Dox+ Extract 14.00 9.10 4.90 ±0.60*,a,b ±0.17*,a,b ±0.39*,b Dox + Bis 14.00 8.70 5.30 ±0.18*,a,b ±0.14*,a,b ±0.16*,b Values are expressed as mean ±SEM, n = 7. * = p<0.05 vs control; a = p<0.05 vs Dox; b = p<0.05 vs Extract; c = p<0.05vs Dox + Extract World Journal of Advanced Research and Reviews, 2025, 28(01), 943-956 951 H & E. x 300 Figure 3 Control group H & E stain x 300 Figure 4 Dox only group