Oligochaeta ramosa (Roxb.) Extract Regulates Lipid Metabolism and Exerts Hepatoprotective Effects in Cadmium-Induced Hepatic Injury in Rats

Environmental pollutants present a potential source of toxicity when exposed to humans. The study was aimed at investigating the potential of Oligochaeta ramosa (Roxb.) as a hepatoprotective agent in cadmium-induced hepatotoxicity causing lipid profile disturbance. The aqueous methanolic (30 : 70 v/v) extract of O. ramosa Roxb. (AME.Or) was subjected to preliminary phytochemical analysis, whereas the antioxidant activity of its constituents was investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The hepatoprotective and antihyperlipidemic effects of AME.Or was investigated by dividing animals into five groups (A–E). Animals were either treated with normal saline or CdCl2 (6.5 mg/kg, intraperitoneally) followed by treatment with silymarin (100 mg/kg), or AME.Or (200 mg/kg) and AME.Or (400 mg/kg) for consecutive three weeks. Blood samples were collected, and the serum lipid profile was assessed on the 11th and 21st day of treatment. Histopathological analysis was performed after euthanization. In vitro analysis of AME.Or revealed 64% inhibition as free radicals scavenging potential during DPPH, total phenolic content (TPC) (79.92 mgGAE/g), and total flavonoids content (TFC) (38.75 mgRE/g). The group intoxicated with CdCl2 showed significantly high (p ≤ 0.05) levels of the liver function indicators and lipid profile than in the control group. The higher dose of AME.Or (400 mg/kg) significantly decreased the aspartate aminotransferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), total bilirubin (p ≤ 0.001), decreased total cholesterol and triglycerides (p ≤ 0.01) while significantly increased high density lipoprotein (HDL; p ≤ 0.01) as compared to the intoxicated group. The histopathological analysis of the liver revealed signs of necrosis in the intoxicated group, while AME.Or treated groups showed marked improvement. The findings accentuate the therapeutic importance of O. ramosa (Roxb.) as a hepatoprotective remedy.

Te folkloric uses include antiemetic, antimicrobial, astringent, purgative, resolvent, antidote, laxative, and antipyretic properties [7,8]. In trivial medicines, the herb has been used for the treatment of skin irritation, cough, external swelling, wounds, and hepatic ailments [9]. Fresh juice of O. ramosa Roxb. is used with Piper nigrum for blood purifcation [10]. Moreover, its aerial parts have been used for the treatment of cough, fever, and diarrhea as a bactericidal against Helicobacter pylori [11].
Te route of cadmium (Cd) exposure plays a key role in determining the extent of cadmium absorption. Most of the Cd is exposed to the body via air, skin, and intestine. Factors including age, diet, lifestyle, and gender also play an important role [12]. Besides, intestinal absorption of Cd is afected by iron, vitamin D, and high dietary fber (Flanagan et al., 1978. Around 5-9% of the total digested Cd is absorbed from the intestine where the iron defciency causes an increase in absorption of Cd by promoting the production of divalent metal ion transport protein in a concentration dependant manner [12][13][14][15]. Te skin absorbs 0.5-0.6% of the Cd in its aqueous solution. Low-cost jewelry might be a source of Cd that is absorbed through the skin [16].
Cd exposure is proven, both experimentally and epidemiologically, to cause hepatic toxicity and lipid imbalance by afecting biological processes via membrane damage, genetic alterations, enzyme inhibition, and electron transport disruption [17]. Cd poisoning occurs as a consequence of industrialized use as well as the presence of the element in inorganic fertilizers. Cd contamination is a major international issue since the metal is known to permeate the food chain and can be bio-accumulated, putting human lives at risk [18].
Since the liver is the key regulator of Cd metabolism and accumulation/deposition, hepatic tissues are more vulnerable to Cd-induced necrosis. It accumulates predominantly in soft tissues (particularly the liver and kidneys), due to the long biological half-life (about 20-30 years in humans) and limited rate of elimination from the body and excessive generation of ROS are the possible mechanisms of Cdtoxicity [19].
Furthermore, oxidative stress, alterations in gene expression and suppression of damaged DNA repair, interference with apoptosis and autophagy, and interaction with bio elements all are mechanisms of Cd toxicity. In hepatocytes, Cd causes oxidative stress, which leads to a variety of pathologies and consequences. Cd toxicity is linked to free radicals-induced lipid peroxidation, which causes a substantial rise in thiobarbituric acid reactive substances (TBARS) in the liver [20].
Limited data is available on the nutritional and compositional status and bioactive potential of O. ramosa Roxb. However, the plant is not much known for its biological role concerning its hepatotonic properties. So, this research aimed to investigate the hepatoprotective potential of the aqueous methanolic extract of O. ramosa Roxb. in Wistar albino rats intoxicated with CdCl 2 .

Collection of Plant Material.
Te dried whole plant of O. ramosa Roxb. was procured from a local market in Bahawalpur, Pakistan. Te plant was authenticated by Te Department of Botany, Islamia University Bahawalpur (IUB), and a specimen was submitted to the herbarium (voucher numbers OR-WP-06-21-185) for future reference.

Preparation of Aqueous Methanolic
Extract of O. ramosa (Roxb.) AME.Or. Te dried whole plant of O. ramosa (Roxb.) was ground to a coarse powder with the commercial grinder and soaked in an aqueous methanolic (30 : 70 v/v) solution. Te soaked material was fltered frstly with muslin cloth and then with Watmann's flter paper to get fltrate. Tis procedure was repeated thrice. Ten the whole fltrate was subjected to rotary evaporation at low temperature (30-45°C) under reduced pressure . A lesser viscous paste of extract was obtained from rotary evaporation, further drying was carried out in the hot air oven. A thick viscous paste of Aqueous methanolic extract of O. ramose (AME.Or) was obtained. At last, it was weighed for the purpose to calculate the percentage yield and stored in an air-tight container with proper labeling at −20°C.

Antioxidant
Potential of AME.Or. Te potential of AME.Or to scavenge oxide free radicals was evaluated by the DPPH method [23] and total phenolic content (TPC) and total favonoid content (TFC) by gallic acid equivalent and rutin equivalent method, respectively [24].

Experimental Animals.
Male Wistar albino rats (n � 30) of either sex (180-220 g of body weight) were caged in the animal house of the Pharmacology research laboratory at the Department of Pharmacology, Faculty of Pharmacy, IUB. All of the animals used for the research were kept in polycarbonate cages of 4(7 × 34 × 18) cm 3 dimensions, with a maximum of six animals per cage, under normal conditions of temperature (25 ± 2°C) and humidity (50-55%), as well as a 12 : 12 hour light and dark cycle. Animals were fed with a standard rodent diet and water ad libitum. Te experiment's protocol was approved by the Institutional Animal Ethics Committee, Department of Pharmacology, Faculty of Pharmacy, IUB, and it was carried out in accordance with the committee's guidelines for the control and monitoring of animal studies under the AEC File No. PAEC/ 21/47.

In Vivo Study
Design. Rats (n � 30) weighing 200 ± 20 g were divided into 5 groups (n � 6). All the rats were acclimatized for 2 weeks before the commencement of the experiments. Te following treatments were provided to each of the six rats in each group over three weeks. Group A rats (normal control) received normal saline (5 ml/kg) through oral gavage for 3 weeks. Group B (intoxicated group) received CdCl 2 prepared in normal saline (6.5 mg/ kg) via an intraperitoneal route on the day 1 st of the study [25]. Group C rats (standard group) were administered with CdCl 2 on day 1, followed by a treatment of silymarin (100 mg/kg via oral gavage) for 3 successive weeks [26]. Group D rats (treatment group 01) were administered orally with AME.Or at a dose of 200 mg/kg for 3 consecutive weeks and CdCl 2 as in group B. Likewise, group E (treatment group 02) received an oral treatment of AME.Or (400 mg/kg) for 3 successive weeks and CdCl 2 as in group B. Doses were carefully selected based on a previous study [27] at the same institute and previous lab experience with this plant.
On the 11 th day, the rats of all the groups were anesthetized using ketamine and xylaxine (10 : 1) at a 2 ml/kg dose, and their blood samples were collected by retro-orbital route for hematological analysis into labeled EDTA bottles to prevent clotting. Serum was collected into plain bottles and allowed to clot after which it was centrifuged at 4000 rpm for 15 minutes. Te serum obtained was stored in a refrigerator at −4°C. On day 21, all the animals were fasted with a free supply of water for 24 h and sacrifced by the cervical dislocation method and serum was collected for hematological analysis. Te liver was dissected from each rat and was kept in 10% formalin solution after weighing followed by the histopathological analysis.

Statistical Analysis.
All the results were expressed as mean ± SEM of n � 6 in each group and statistically evaluated through Graphpad Prism 9.0.1 (La Jolla, CA, USA) to analyze the results by one-way analysis of variance (one-way ANOVA). Bonferroni's post hoc test was conducted to fnd the signifcance among various groups by setting p < 0.05.

Phytochemical Screening of AME.Or.
Phytochemical analysis revealed the presence of alkaloids, tannins, phenols, favonoids, saponins, cardiac glycosides, and carbohydrates (Table 1). AMR.Or inhibited the DPPH free radicals scavenging up to 62.43 ± 2.72%. Te TPC of the extract was found to be 79.92 ± 1.23 mg GAE/g. Likewise, the TFC of AMR.Or was 38.75 ± 0.94 mg RE/g.

Te Efect of AME.Or on Liver Weight and Hepatic
Functions. In the present study, the hepatic parameters, i.e., aspartate aminotransferase/serum glutamic-oxaloacetic transaminase (AST/SGOT), alanine aminotransferase/serum glutamic-pyruvic transaminase (ALT/SGPT), alkaline phosphatase (ALP) and total bilirubin were determined on the 11 th and 21 st day. Te efect of diferent concentrations of AME.Or, i.e., 200 and 400 mg/kg, on the body and the liver weight is given in Table 2, whereas the hepatic parameters of the treated animals are shown in Figure 1. Te Intoxicated group treated with CdCl 2 showed a signifcant increase in hepatic parameters and AME.Or showed remarkable dose-dependent efects, i.e., normalization of hepatic function at a dose of 400 mg/kg p.o. And the results of treatment groups are almost comparable to the standard treatment (silymarin) group.

Lipid Profle (Cholesterol, Triglycerides, HDL, LDL, VLDL, AI).
In the present study, the lipid profle i.e. the total cholesterol, triglycerides, high-density lipoproteins (HDL), low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), and atherogenic index were estimated on the 11 th and 21 st day. Te efect of diferent concentrations of AME.Or; i.e. 200 and 400 mg/kg, on diferent lipid parameters are shown in Table 3. Te intoxicated group treated with CdCl 2 showed marked imbalance (p ≤ 0.05) in lipids levels and AME.Or showed highly signifcant and dosedependent efects, i.e., normalization of lipid profle at dose 400 mg/kg p.o., and the results of treatment groups were almost comparable with the silymarin-treated group.

Te Efect of Various Treatments on the Liver
Histopathology. Te integrity of hepatocytes and the histopathologic alterations after the 21-day treatment with AME. Or are presented in Figure 2. Te intoxicated group showed prominent epithelial damage, enlarged endothelial spaces, disarrangement, and loss of characteristic integrity of the liver. Mild necrosis was observed across the lobules. AME.Or treatment prevented Cd-induced necrotic lesions in a dose-dependent manner. Te hepatic tissue in the treatment groups appeared normal except few scattered necrotic areas.

Discussion
Cd is a toxic heavy metal listed on the priority list of hazardous wastes by USEPA (the United States Environmental Protection Agency) [28]. Because of industrial and agricultural activities, the Cd level in the environment is on a consistent rise [29]. Te Evidence-Based Complementary and Alternative Medicine present study evaluated diferent hepatic serum enzymes (AST/ SGOT, ALT/SGPT, ALP, and TB), considering their levels as a measure of hepatotoxicity [30]. Our fndings afrmed that the hepatic cell integrity is disrupted as a result of Cd exposure with subsequent hepatic failure. Several animal investigations have also found elevated levels of serum hepatic enzymes in rats when administered CdCl 2 , confrming the current fndings and emphasizing the impact of Cd exposure [26,[31][32][33]. Indeed, Cd-intoxication therapy has been investigating some natural phytoconstituents with hepatotonic efects [34]. In this research, O. ramosa (Roxb.) was studied to investigate its hepatotonic efects against Cd-induced hepatic injury with lipids imbalance.
In this study, the intoxicated group showed a signifcant decrease in body weight and an increase in liver weight. Te alteration in body weight may be due to the dysfunction of the glucocorticoid system with impaired glucocorticoid hormones which are implicated in lipids metabolism [34,35]. Another reason for weight loss may be an accumulation of Cd in the liver and other vital organs leading to functional damage and interstitial fbrosis [36]. In contrast to the intoxicated group, AME.Or treatment mitigated the Cd-induced weight loss and fatty liver. Although reported data on traditional medicinal activity data on O. ramosa (Roxb.) is scarce, few ethnobotanical studies highlight it in jaundice and gastrointestinal ailments [11,37].
Hepatic biomarker enzyme levels, cholesterol, triglycerides, and lipoproteins levels are indicators of hepatotoxicity [21,26,35,38]. Te observed signifcant increase (p < 0.001) in the levels of biomarkers, i.e., AST/SGOT, ALT/SGPT, ALP, total cholesterol, LDL, VLDL, triglycerides, total bilirubin, and atherogenic index [39][40][41] and a signifcant decrease (p < 0.001) of HDL level in serum of Cd treated rats is the evidence of hepatotoxicity and hyperlipidemia [42]. Te release of SGOT/AST and ALT/ SGPT into the systemic circulation is triggered by hepatic damage [43] and remains key indicators of hepatotoxicity [44,45]. Cd alters the cellular structure and subsequent Table 2: Te efect of Cdcl 2 on the Body and the Liver Weight. Cdcl 2 was administered via the intraperitoneal route on the 1 st day of the study followed by AME.Or doses at 200 mg/kg and 400 mg/kg. Each group data is presented as g ± SEM of n � 6 rats. When comparing the cadmium-intoxicated group to the control group, the results are deemed insignifcant (ns) if p > 0.05, signifcant ( * ) if p < 0.05, more signifcant ( * * ) if p < 0.01, very signifcant ( * * * ) if p < 0.001, and highly signifcant ( * * * * ) if p < 0.0001. When the control group is compared with the intoxicated group, the signifcance of the results is denoted with ( # ).

Treatment Group
Weight of animals (g) Liver weight (g)   [21]. Current research fndings revealed a signifcant reversal of serum biomarkers to normal in all treatment groups in a dose-dependent manner after administration of AME.Or. AME.Or administered at 400 mg/kg body weight showed signifcant improvement in liver functionality and moderates the damage caused by Cd-intoxication. Indeed the plant is traditionally used in liver diseases for centuries [46] and our fndings correspond to this reputation. Te efect of AME.Or treatment on serum biomarkers can be traced to its antioxidant potential which was investigated by DPPH free radical scavenging potential, TPC, and TFC of the extract [47]. Te antioxidative potential of plants containing favonoids also afects lipid regulation and related immune regulations [48]. Correspondingly, our study showed antioxidative activity along with lipid control and protection against liver enzymes. Te phytochemical analysis also revealed the presence of favonoids among others that forms the basis for antioxidative activities [49] and lipid regulation for future studies. In addition to favonoids, the phytochemical analysis also revealed the presence of alkaloids, favonoids, and phenols that cause to mitigate the cytotoxic efects of intoxication-derived free radicals. Tese fndings are in line with the previously published data on the plant [27].
O. ramosa (Roxb.) extract also possesses lipid-lowering and hepatotonic potential due to its phytochemical

Conclusion
Te present study demonstrated the hepatoprotective potential of AME.Or against Cd-induced hepatotoxicity. Phytochemical screening of aqueous methanolic extract revealed the presence of alkaloids, tannins, phenols, favonoids, saponins, and carbohydrates. Antioxidant potential of AME.Or was also investigated. AME.Or showed signifcant restoration of hepatic biomarkers and lipid profle indicators towards normal dose-dependently (200 and 400 mg/kg). Tus, the study suggested that O. ramosa (Roxb.) possesses hepatotonic potential which is due to its antioxidant efects. Te study was conducted on the rat model of human exposure revealing that AME.Or infuences metabolism and   Or, 400 mg/kg. ( ) Enlarged interstitial spaces of hepatic epithelium. Cdcl 2 was administered via the intraperitoneal route on the 1 st day of study followed by AME.Or doses at 200 mg/kg and 400 mg/kg. Liver sections were processed for histopathological analysis after euthanization. 6 Evidence-Based Complementary and Alternative Medicine prevents abnormalities in hepatic and lipid markers. Terefore, it refects the scientifc ground for folkloric uses of the O. ramosa (Roxb.). Further investigation is needed to clarify the underlying protective mechanisms and active components of AME.Or.

Data Availability
Te data can be obtained from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Saira Aslam planned the study and performed the experiment. Fiaz ud Din Ahmad and Imran Mukhtar contributed to the drafting of the manuscript. Waseem Hassan analyzed the data and prepared the frst draft of the manuscript. Hammad Ahmed performed the experiment. All authors approved the manuscript after complete reading.