Phytolacca Dodecandra (L’ Herit) (Phytolaccaceae) Methanol Root Extract Protects Liver from Acetaminophen-Induced Injury in Rats

Abstract

Phytolacca dodecandra (L’ Herit), or ‘Endod’, is one of the widely known medicinal plants in Ethiopia. Berries of the endod have been used as a detergent for centuries. The present study was aimed to test the hepatoprotective effects of the plant against acetaminophen (APAP)-induced liver injury in rats. Mice of either sex were used for oral acute toxicity tests and APAP-induced lethality tests. Hepatoprotective experiments were done on male rats using 2 g/kg of APAP to induce liver damage. Liver enzymes, total bilirubin (TB), and lipid profile were determined. Liver tissues were also examined histopathologically to see a morphologic change in the control and experiment groups. The protective effect of the plant extract was also tested through sodium pentobarbital (SPB)-induced sleeping time. A significant increase in serum levels of liver enzymes, TB, low-density lipoprotein (LDL), and triglycerides (TGs) was seen from oral administration of 2 g/kg APAP. Total cholesterol (TC) and high-density lipoprotein (HDL) levels were decreased. Serum levels of all parameters were reversed to normal after administration of silymarin 100 mg/kg and, 100, 200, and 400 mg/kg doses of the extract. A significant dose-dependent hepatoprotective effect of Phytolacca dodecandra Methanol Root Extract (PDME) was seen in terms of LDL. Histopathological investigations and SPB-induced sleeping time confirmed the findings of biochemical analysis. The findings of the present study indicate that PDME protected the liver from APAP injury.

Background

Plants have been used since ancient times and a myriad of pharmaceutically active compounds have been isolated from plants. ¹ Several plant metabolites demonstrated diverse biological activity. Flavonoids have shown remarkable antioxidant, anti-inflammatory, antibacterial, antifungal, and antitumor activity. ² Diallyl Disulfide, a bioactive compound from garlic, has demonstrated anti-tumor activity against many types of tumor cells. ³ Furthermore, Vitamin E long-chain metabolite ameliorates asthma ⁴ and Camellia tea improved glycemic control in type II diabetic patients. ⁵

Traditional knowledge and medicinal plants play a major role in safeguarding both human and animal health in developing countries. ⁶ Similarly, medicinal plants are widely used by the Ethiopian population. ⁷ Phytolacca dodecandra (L’ Herit), commonly known as Endod in Amharic, belongs to the genus Phytolaccaceae and it is widely known for its use as a detergent. ⁸ P. dodecandra has long been known for its molluscicidal effects, ⁹ and recent studies revealed several additional medicinal uses of the plant in Ethiopia. ¹⁰

The most commonly documented traditional uses of P.dodecandra is for the treatment of skin itching, gonorrhea, leeches, intestinal worms, anthrax, malaria, abdominal pain, bloating, wound, and rabies.^11–13 In addition, it has an abortifacient activity, and the plant is traditionally used as contraception. ¹⁴ It is also used in combination with plants such as; Momordica foetida, Justicia schimperiana, and Croton macrostachyus for liver disorders traditionally diagnosed by yellowish discoloration of the eye.^15–18

Moreover, most of P.dodecandra's traditional uses have been confirmed by invitro and invivo experimental models. Aqueous leaf extracts of P. dodecandra have shown abortifacient effects in rats. ¹⁹ Additionally, antimalarial, antirabies, spermicidal, and larvicidal activities of P.dodecandra have been confirmed experimentally.^14,20–24 The plant has also shown in vitro antihelmintic and anti-microbial activities.^25–29 Because of its cytotoxic activity, p.dodecandra is considered a potential plant pesticides.^30,31 Several secondary metabolites including phenolics, terpenoids, anthraquinones, alkaloids, and flavonoids were found in extracts of different parts of p.dodecandra. These secondary metabolites might attribute to the observed activities of the plant. ²⁹

Free radicals are implicated as a cause of many diseases. ³² Different products having anti-oxidant effects could prevent the ailments that would happen as a result of reactive free radicals exposure. Accordingly, the antioxidant effect of P.dodecandra has been studied and the plant has shown promising free radical scavenging activities. The methanol and aqueous extracts of the plant have shown antioxidant activity against both ABTS (2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid) and DPPH ((2,2 DiPhenyl-1-PicrylHydrazyl) radical scavenging tests. ³³ These findings suggest the potential of P.dodecandra free radical scavenging and organ protection from free radicals. The aim of the present study was, therefore, to test the hepatoprotective effects of P.dodecandra in rats by using APAP to induce liver injury.

Methods

Equipment

Mechanical grinder, volumetric flasks, funnel, gauze (Nylon clothes), filter paper (Whatman number one), plastic sample holder, labeler, deep freezer, rotavapor (BÜCHI Rotavapor R-200), freeze-drier (OPERON, Made in Korea), heating bath (BÜCHI Heating Bath B- 490), aluminum foil, mortar and pestle, spoon, beakers, measuring cylinders, sensitive digital weighing balance, disposable glove, mice cages, rat cages, oral gavages, syringe, serum separator tubes (SST) (Vacuum Blood Collection Tube Gel & Clot Activators, 5 ml, Henso Medical (Hangzhou) Co., Ltd, China), centrifuge machine (Eppendorf Centrifuge 5804R), fully automated serum analyzer (CobasR 6000), desiccators, surgical glove, surgical blades, scissors, forceps, refrigerator, tissue cassettes, frost-ended slides, heater, tissue processor (Tissue Tek II Rotary Sekura TissueProcessor), cool plate, water bath, rotary microtome, autostainer and microscope (Leica DM750) with ICC50 HD camera were used.

Chemicals and Reagents

Silymarin tablets (LiverubinR Cure Quick Herbals Alchem international Pvt. Ltd), methanol (CARLO ERBA Reagent), APAP tablets (Para-Denk 500 Denk Pharma), Tween-80, distilled water, normal saline (0.9% sodium chloride solution), paraffin, xylene, hematoxylin solution, Formaldehyde 35% (SIGMA-ALDRICH Co.), 2% eosin solution, closed reagent cassettes of Roche Company for all alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP),total bilirubin (TB), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglycerides (TGs) (Roche Diagnostic, Indiapolis) were used. All chemicals and reagents were bought from Germany.

Plant Materials

The root of P. dodecandra was collected from Saden Sodo area, South West Shoa Zone, Oromia, 110 kilometers south of Addis Ababa, Ethiopia. Plant identification and authentication were done at the Ethiopian Public Health Institute (EPHI), Addis Ababa, Ethiopia. The voucher number (GD-002) was given to the specimen and it was kept in the herbarium of the EPHI for future reference.

Preparation of Phytolacca dodecandra Methanol Root Extract

The root of P. dodecandra was washed with tape water and kept under a shed without direct exposure to the sunlight for two weeks. The dried part was chopped, and grinded by a mechanical grinder into a coarse powder. The course powder (400 g) was macerated in 80% methanol for 72 h with occasional shaking. The method of extraction was in line with the way the plant is used traditionally. The filtrate obtained using gauze, and filter paper was concentrated by rotavapor in a heating bath. Lyophilizer was used to remove the aqueous part of the solvent and an 8.60% yield was finally obtained. (Figure 1)

Figure 1.

Schematic diagram showing how Phytolacca odecandra methanol 80% root extract was prepared.

Animals

The animals used were mice of either sex and male rats weighing 30–35 g and 160–182 g, respectively. All experimental methods were carried out in compliance with the requirements outlined in the Committee for the Purpose and Control of Supervision of Experiments on Animals’ guidelines for the care and use of experimental animals. ³⁴ Accordingly, a letter of ethical approval (Ref. No. ERB/SOP/368AB/13/2021) was obtained from Addis Ababa University Ethical Review Board. To reduce the impacts of environmental change and travel-related stress, animals were adapted to animal house conditions for seven days before experimentation. They were then divided randomly into different groups and kept at room temperature in polypropylene cages that were lined with soft wood flakes as bedding. Except for during fasting periods, animals were given free access to ordinary pellets and conventional tap water.

The standard drug, plant extract, and vehicles were calculated as per their body weight and provided using oral gavages. On the prepared checklist, every administration was documented to avoid any skipped or repeated administrations. Animals were treated with intra-nasal chloroform during the procedures to reduce suffering, and they were ultimately sacrificed by cervical dislocation.

Oral Acute Toxicity Test

Based on the limit test guidelines of OECD guideline-425, an acute toxicity test of the root 80% methanol extract of P. dodecandra was conducted on a group containing five mice of either sex. ³⁵ Prior to the experiment, mice were fasted for four hours, and while they were fasting, they had unlimited access to free water. Each animal received orally a single dose of PDME, 2000 mg/kg, diluted in 2% Tween-80 in distilled water. Mice were fasted for two hours and then allowed unlimited access to food and water. Within 12 h, the animals were again monitored for any physical or behavioral changes, with the first 4 h receiving particular focus. The animals were kept under close observation for such serious toxicological symptoms as breathing difficulties, loss of appetite, overall weakness, agitation, writhing, lack of motor coordination, muscular relaxation, drowsiness, and profound sleep. They were also monitored daily for the following 14 days for mortality, and the test was repeated using 3000 mg/kg of extracts.

Acetaminophen-Induced Lethality Test

The acetaminophen (APAP) lethality test was performed as described previously. ³⁶ Animals were divided into two groups of ten mice each, fasted for twelve hours with free access to water before extract administration, and fasted for an additional hour following APAP administration. Group I, a control group, received the vehicle, 2% Tween-80 dissolved in distilled water whereas group II was given 400 mg/kg of PDME dissolved in 2% Tween-80. Because this test was used as a preliminary, variations in the number of death among the two groups was supposed to attribute to the test substance. Accordingly, all animals were given a fatal dose of APAP (1 g/kg) an hour later. The number of deaths occurring within 24 h was counted, and the following formula was used to compute the percentage of protection:

$$\mathrm{Percent}\mathrm{protection}(\text{\%})={\displaystyle \frac{a}{b}\times 100,}$$ (1)

where a represents the number of animals still alive 24 h after the lethal dose of APAP was administered, and b represents the total number of animals in the group at the start.

Acetaminophen Dose Selection

Based on the results of our prior study, the dose that causes a significant biochemical change without causing any physical harm was chosen. Accordingly, liver damage was caused by giving a single dose of APAP (2 g/kg).

Hepatoprotective Activity Against Acetaminophen-Induced Liver Damage

For the hepatoprotective testing of the Phytolacca dodecandra Methanol Root Extract (PDME), there were six groups with five rats each. This was aimed to see the dose dependency in hepatoprotective effects of the PDME. The six groups included the PDME group, which received 100, 200, or 400 mg/kg of the extract; the silymarin group, which received 100 mg/kg of the standard drug in 2% Tween-80; the normal and negative control groups, which both given 2% Tween-80. The standard medication and test chemicals were freshly made by dissolving in 2% Tween-80 and administered every day for 10 days. On the tenth day, one hour following the last dose administration, all animals except those in the normal control group received 2 g/kg APAP in NS; while animals in the normal control group were given the vehicle, NS.

Biochemical Analysis

The levels of TB and the liver enzymes ALT, AST, and ALP were measured. The TC, TGs, HDL, and LDL were also assessed using the cobas^R 6000, a fully automated machine. The following formula was used to calculate the extract's hepatoprotective activity, which was expressed as a percent protection of the extract through biochemical parameters:

$$\text{\%}\mathrm{Protection}=[(a-b)÷(a-c)]\times 100,$$ (2)

where a is the average value of the marker generated by the hepatotoxin (APAP), b is the average value of the marker created by the hepatotoxin combined with the test chemicals, and c is the average value of the biomarker generated by the vehicle. P-values of less than 0.05 were regarded as significant. Every biomarker was evaluated following Maqsoodet al. ³⁷

Histopathological Analysis

The left lateral lobe, right medial lobe, and the caudate lobe of the liver gave three gross slices, which were then stored in a tissue cassette with their identifiers and fixed in 10% buffered neutral formalin solution until processing. Each tissue block's sections were cut at a thickness of 4 µm, stained with hematoxylin for 10 min, and then counterstained for 20 s with 2% eosin solution. DPX (Dibutylphthalate Polystyrene Xylene) mounting medium was used to cover-slip the stained slides. Histomorphological defects were graded depending on the kind and severity of morphologic changes following other previous studies. ³⁸ A Leica DM750 microscope equipped with an ICC50 HD camera was used to take selected photos of the sections at a 10x magnification.

Sodium Pentobarbitone-Induced Sleeping Time

Sodium pentobarbitone (SPB) was used to induce sleep and the test was done according to our previous study. ³⁶ Sleep duration (the time gap between sleep onset, loss of writhing reflex, and time of awakening, gain of the reflex) in minutes was recorded.

Statistical Analysis

Statistical analysis was done using SPSS (Statistical Package for Social Sciences) version 25 software. Results were presented as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) was done to see the differences in the means of the groups. Significant differences were determined using the Tukey posthoc test with multiple comparisons. A p-value of less than 0.05 was considered statistically significant. ³⁶

Results

Oral Acute Toxicity Test

Following administration of P. dodecandra 80% methanol root extracts at doses of 2000 and 3000 mg/kg, no mortality was noted over 14 days. Additionally, no toxicity indicators were seen during the monitoring time. Therefore, it is expected that the extract's oral LD50 s are more than 3000 mg/kg.

APAP-Induced Lethality Test

Only two of the mice who were given 400 mg/kg PDME per kilogram died within 24 h after receiving 1 g/kg APAP, indicating 80% protection, while all of those who were not given PDME died (Figure 2). Additionally, these observations were held for one week, beyond which no additional observations were taken.

Figure 2.

Effects of PDME on APAP-induced mortality. (APAP-acetaminophen, PDME-80% Methanol root extract of P. dodecandra).

Biochemical Analysis

Effects of PDME on Liver Enzymes and Total Bilirubin

The serum levels of all indicators were increased significantly (p < 0.05) after 2 g/kg of APAP was administered as shown in Table 1. However, administration of various PDME doses reduced the elevated levels of the biomarkers.

Table 1.

Effects of PDME on Serum Levels of Liver Enzymes and TB of Rats Orally Administered 2 g/kg APAP, (Mean ± SEM).

Group	ALT	AST	ALP	TB
Normal control	56.85 ± 0.21	205.95 ± 14.07	146.00 ± 49.15	0.09 ± 0.01
Negative control	183.45 ± 175.61a	375.55 ± 186.25a	235.50 ± 30.96a	0.18 ± 0.04a
100 mg/kg, PDME	69.30 ± 15.89be (124.5)	206.850 ± 45.59bde (99.4)	145.75 ± 34.86bd (100.3)	0.13 ± 0.01bde (55.6)
200 mg/kg, PDME	56.93 ± 18.82 be (99.94)	241.80 ± 66.61b (78.9)	178.66 ± 96.52 b (63.5)	0.16 ± 0.06 b (22.2)
400 mg/kg, PDME	78.50 ± 7.47 b (82.9)	277.80 ± 106.68 b (57.6)	165.25 ± 55.79 b (78.5)	0.15 ± 0.01 b (33.3)
100 mg/kg, Silymarin	84.25 ± 6.66 b (78.4)	270.70 ± 66.67 b (61.8)	201.00 ± 50.61 b (38.5)	0.12 ± 0.01 b (66.7)

Compared with: ^anormal control, ^bnegative control, ^cPDME100, ^dPDME200, ^ePDME400, P < 0.05. Numbers in bracket show percent protection.

(ALP-alkaline phosphatase, ALT-alanine aminotransferase, AST-aspartate aminotransferase, mg/dl-milligram per deciliter, mg/kg- milligram per kilogram, PDME-80% Methanol root extract of P. dodecandra, TB-total bilirubin, U/L-units per liter).

Effects of PDME on Lipid Profiles

Following the administration of APAP, a significant rise in the blood levels of LDL, and TGs and a fall in TC and HDL levels were seen (Table 2). However, administration of different doses of PDME caused those readings to decrease to normal, and increment of HDL and TC toward normal levels.

Table 2.

Effects of PDME on Lipid Profiles of Rats Orally Administered 2 g/kg APAP, (Mean ± SEM).

Group	HDL (mg/dl)	LDL (mg/dl)	TC(mg/dl)	TG (mg/dl)
Normal control	22.33 ± 1.55	9.65 ± 0.63	47.20 ± 6.17	73.00 ± 30.86
Negative control	18.90 ± 5.72a	12.10 ± 2.07a	44.77 ± 1.69a	89.52 ± 28.78a
100 mg/kg, PDME	25.05 ± 6.05b (179.30)	10.20 ± 4.27bde (77.55)	50.07 ± 6.99 b (218.1)	67.10 ± 0.53bde (135.7)
200 mg/kg, PDME	39.20 ± 14.55 b (591.8)	9.00 ± 1.41bce (126.5)	69.96 ± 20.40 b (1036.6)	81.73 ± 21.79bce (47.15)
400 mg/kg, PDME	32.85 ± 8.97 b (406.7)	7.57 ± 1.47bcd (184.9)	52.27 ± 5.94 b (308.64)	63.70 ± 10.17bcd (156.3)
100 mg/kg, Silymarin	23.42 ± 4.57 b (131.7)	9.92 ± 1.06 b (88.9)	45.15 ± 4.65 b (15.64)	49.57 ± 7.67 b (241.8)

Compared with: ^anormal control, ^bnegative control, ^cPDME100, ^dPDME200, ^ePDME400, P < 0.05. Numbers in bracket show percent protection. (HDL, high-density lipoprotein; LDL, low-density lipoprotein; mg/dl, milligram per deciliter; mg/kg, milligram per kilogram; PDME, 80% Methanol root extract of P. dodecandra; TC, total cholesterol; TGs, triglycerides).

Histopathological Analysis

Histopathological study showed a significant alteration of the normal architecture of the liver in the negative control group which received 2 g/kg APAP (Figure 3). However, pre-administration of PDME resulted in various degrees of tissue protection, as shown in Table 3.

Figure 3.

Images of the liver sections obtained from different groups.

(I) Normal control groups: rats received 2% Tween-80 and then NS, (II) Negative control groups: rats given only APAP, (III) Rats given PDME at 100 mg/kg, (IV) Rats given PDME at 200 mg/kg, (V) Rats given PDME at 400 mg/kg, (VI) Positive control group: rats given silymarin at 100 mg/kg. Areas of tissue necrosis are indicated by black arrows, while areas of varied degrees of vacuolar degeneration are indicated by red arrows. APAP-acetaminophen, g/kg-gram per kilogram, mg/kg- milligram per kilogram, NS- normal saline, PDME-Methanol 80% root extract of P. dodecandra.

Table 3.

Effects of PDME at Different Doses on APAP-Induced Liver Injury.

Group	Liver tissue necrosis	Vacuolar degeneration
Normal control, NS	−	−
Negative control, APAP alone	+++	++
100 mg/kg, PDME	−	+
200 mg/kg, PDME	−	−
400 mg/kg, PDME	−	+
100 mg/kg, Silymarin	−	+

−, normal; +, mild effect; ++, moderate effect; +++, severe effect. (APAP-acetaminophen, mg/kg- milligram per kilogram, NS- normal saline, PDME-80% Methanol root extract of P. dodecandra).

Effects of PDME on SPB-Induced Sleeping Time

APAP 2 g/kg resulted in an increment of SPB-induced sleeping time. Administration of the PDME at a high dose decreased the sleeping time to normal. The extract alone showed no change in sleeping time compared to the normal control (Table 4).

Table 4.

Effects of PDME on SPB-Induced Sleeping Time in Rats Orally Administered 2 g/kg APAP.

Group	Sleep duration (in minutes)
Normal control, vehicle + SPB	82.32 ± 4.33
Negative control, APAP + SPB	131.01 ± 32.21a
PDME + SPB	77.23 ± 8.09a
PDME + APAP + SPB	106.66 ± 65.80b
Silymarin100 mg/kg + APAP + SPB	90.10 ± 5.11bc

Compared with: ^anormal control, ^bnegative control, ^cPDME400 mg/kg, P < 0.05. (APAP, acetaminophen; mg/kg, milligram per kilogram; PDME, 80% Methanol root extract of P. dodecandra; SPB, Sodium pentobarbitone 150 mg/kg).

Discussion

Phytolacca dodecandra, also known as the African soap berry plant, is found in different regions of the world with variable morphology. ³⁹ The species is widely distributed over various parts of Asia, South America, and Sub-Saharan Africa, including Ethiopia.^40,41 Ethiopians have traditionally been using berries of P. dodecandra as soap for washing clothes. ⁸

Traditionally, P.dodecandra has broad medicinal use for many diseases. For example, either the powdered root or leaf of the plant drunk in a mix with water is used for the treatment of liver diseases.^16–18 The whole part of the plant is also used traditionally for abdominal pain and abortion. ¹⁰ Most of the traditional claims have attracted scientific investigation. It had been long since Aklilu Lemma showed molluscicidal activity of the plant and it has triggered a lot of interest in the plant among scientists.^42,43 However, there are scarce data on the effect of the plant on liver histopathology and biochemical parameters. We investigated the hepatoprotective effect of P. dodecandra in the rat model. P. dodecandra has a hepatoprotective effect in acetaminophen-induced liver injury in rat models.

Anti-oxidant and hepatoprotective effects of the plant had previously been revealed. ⁴⁴ The previous study was done in mice models and used a carbon tetrachloride to induce liver injury. The rationale for conducting the present study was to see the effect of the plant on acetaminophen-induced liver injury in rats. Hepatotoxicity potential and its easy accessibility made acetaminophen a major cause of drug-induced acute liver injury. APAP is metabolized to N-acetyl-p-benzoquinoneimine (NAPQI) which causes oxidative stress, depletes glutathione, and ultimately causes liver damage. ⁴⁵ In this regard, it is worth using this model than the rarely used drugs or toxins. Additionally, in the present study, male rats were used for hepatoprotective tests. Rats are preferred to mice and feasible for the hepatoprotective study in animal model.^46,47

In the present study, administration of the plant extract reduced acetaminophen-induced lethality in mice. The antioxidant activity of the plant might be responsible for this biological effect of the plant extract. ³³ Moreover, the plant's anti-inflammatory effect also might have contributed to the decline in death. ⁴⁸ Consequently, the plant was further tested for hepatoprotective activity in rat models. The liver injury markers such as liver enzymes; ALT, AST, ALP, lipid profiles; HDL, LDL, TC, TGs, and serum bilirubin levels were measured, and liver histopathology was investigated.

Administration of APAP has increased the serum level of all the biomarkers, except HDL and TC, and altered the normal architecture of the liver. The damage caused by NAPBQI on the hepatocytes might have altered cell membrane integrity and resulted in the increment in the extracellular level of the enzymes. ⁴⁹ The toxin might have contributed to the inability of the liver to regulate metabolism. The observed abnormal serum levels of lipid profiles and total bilirubin in the experimental group compared to the control might be attributed to a failure in metabolism regulation.^49,50

Change in the serum levels of the liver biomarkers depends on the doses of PDME administrated. The maximum change in AST, ALP, TB, and HDL was observed at 100 mg/kg of PDME while 200 mg/kg and 400 mg/kg doses of PDME showed maximum protection in terms of ALT and TGs, respectively. This might be attributed to different phytoconstituents in the extract possessing protective effects with their different characteristics of concentration-effect relationship. Additionally, PDME has shown a dose-dependent change in plasma LDL levels. An increase in the active constituents as the dose increases may explain the observed activity.

On another hand, PDME administration increased the level of TC at different doses. A similar result was observed in the previous study done on the plant. ⁴⁴ Liver is the principal site for cholesterol endogenous biosynthesis and homeostasis maintenance. ⁵¹ However, overall statistically non significant liver protection was observed for PDME compared to silymarin. Moreover, the results of the biochemical analysis were confirmed through histopathological examination. Morphological alterations observed in the negative control were not observed in animals that received PDME. PDME at 100 mg/kg resulted in a visible decrement in tissue damage, which is inline with the findings of biochemical analysis.

In a normal liver the metabolism of SPB is carried out timely. But liver injury impairs SPB metabolism. As a result, significant effect of SPB is observed in terms of prolonged sleeping time.^52,53 We used SPB as an additional indicator of liver-protecting effect of the extract. The results of the test, SPB-induced prolonged sleeping time, is also in agreement with the biochemical and morphological studies. Extract was found not to have sleep-inducing property as no significant difference was seen between the two groups; negative controls and those which received the extract alone. Therefore, higher sleeping time in negative control compared to the normal was due to liver injury caused by the NAPBQI. This implies that the liver did not metabolize SPB as timely as normal. However, the extract has reversed the prolonged sleeping time by protecting the liver from damage.

Schistosomiasis affects the liver and is known to cause changes in the lipid profile and liver enzymes. Additionally, fibrosis and enlargement of the liver are clinical symptoms of schistosomiasis. ⁵⁴ Liver damage is caused by eggs of Schistosoma mansoni, the causative parasite of schistosomiasis, which is lodged into the liver and evokes the immune system leading to hepatic granuloma and fibrosis. ⁵⁵ Both Schistosoma mansoni and acetaminophen cause liver injury in part with similar mechanisms. This implies that p. dodecandra could have a protective effect against schistosomiasis induced liver injury. The presence of secondary metabolites such as phenolics, terpenoids, anthraquinones, alkaloids, and flavonoids, which had shown promising hepatoprotective activity, support the plant's traditional use for liver disease and schistosomiasis. ²⁹ These pieces of evidence and the antioxidant activities of different parts of the plant ²⁷ might have contributed to the liver-protecting activity of P. dodecandra observed in the current study.

Silymarin is known to reverse all toxicities caused by high doses of acetaminophen.^56,57 Similarly, the present study, has shown the greatest protection in terms of all biomarkers, histopathological examination, and SPB-induced sleeping time. Generally, this experimental study justifies the traditional use of P. dodecandra for liver diseases and, more specifically, indicates that APAP-induced liver injury can be prevented by the hepatoprotective effect of the plant.

Limitations of the Study

The present study tested the protective effect of the plant extract. However, the plant was not studied for reversing APAP-induced liver damage. Moreover, the study did not show the effects of solvent fractions of the extract which could be the goal of further research in the future.

Conclusion

The present study shows that 80% methanol root extract of P.dodecandra significantly decreased the serum levels of liver enzymes, low-density lipoprotein, and triglycerides, and increased total cholesterol and high-density lipoprotein levels. There was also histopathological improvement after plant extract administration in the rat model. This finding suggests 80% methanol root extract of P.dodecandra has a hepatoprotective effect. However, further investigation is required to identify the potential active constituent responsible for the observed biological effect of the plant extract. Nevertheless, the finding augments the traditional uses of the plant.

Abbreviations

ALP

Alkaline Phosphatase

ALT

Alanine Amino Transferase

APAP

Acetaminophen (N-acetyl-p-aminophenol)

AST

Aspartate Amino Transferase

HDL

High Density Lipoprotein

IP

Intra-peritoneal

LD₅₀

Median Lethal Dose

LDL

Low Density Lipoprotein

NAPQI

N-acetyl-p-benzoquinone imine

PDME

Phytolacca Dodecandra Methanol Root Extract

SEM

Standard Error of Mean

SPB

Sodium Pentobarbital

TB

Total Bilirubin

TC

Total Cholesterol

TGs

Triglycerides