Evaluation of the therapeutic potential of vomit seed ( Strychnos nux-vomica ) homeopathic treatment in acute acetaminophen-induced toxicity

Purpose: To investigate the effect of vomit seed homeopathic drugs (14CH and 30CH) on paracetamol-induced toxicity in rats. Methods: Thirty-two male Wistar rats were divided into 4 groups of 8 animals each (n = 8) and treated for 12 days. During the treatment period, group (G) one (G1) which constitutes the control group received filtered water, whereas groups two (G2), three (G3), and four (G4) were gavaged with a toxic dose of paracetamol 1/4 of LD 50 . Next, Strychnos nux-vomica (0.5 mg/kg) homeopathic dilutions of 14CH and 30CH were administered to G3 and G4, respectively. Hematological, biochemical, and antioxidant markers were assessed. Results: Homeopathic drug groups, namely G3 and G4, showed significant recovery in some hematological (red blood cells count and hemoglobin concentration; p < 0.01) and biochemical parameters (urea, creatinine, glutamic transaminase, and alkaline phosphatase; p < 0.001). The level of malondialdehyde significantly decreased while glutathione level significantly increased in treated rats. Conclusion: The antioxidant and anti-hemolytic properties of vomit seeds protect against paracetamol-induced acute toxicity in rats by reducing oxidative stress, hepatotoxicity, and nephrotoxicity in rats.


INTRODUCTION
Acetaminophen, (paracetamol) is a popular analgesic and antipyretic that is often prescribed and used as a primary treatment. It is widely known that excessive intake of paracetamol may result in acute liver failure due to the generation of reactive oxygen species and formation of toxic metabolite N-acetyl-benzoquinone imines [1]. Administering high doses of paracetamol may result in severe renal failure which is frequently accompanied or preceded by substantial hepatic dysfunction [2].
Strychnos nux-vomica (Loganiaceae) is an evergreen tree native to South-East Asia and India. It is a medium-sized tree that grows in largely open environments [3]. There are at least 84 compounds known to exist in Nux vomica, including alkaloids, glycosides, flavonoids, terpenoids, steroids, and organic acids, according to previous characterization studies [4]. Seeds of Strychnos nux-vomica L are used in traditional medicine to treat a variety of health conditions including anemia, lumbago, asthma, pneumonia, constipation, hyperglycemia, malaria, skin problems, paralysis, muscle spasms, and decreased appetite [5]. Before taking it orally, Nux vomica is processed by extraction and standardization with other suitable excipients. Its dosage is usually carefully monitored, and it is not taken frequently.
Homeopathy is a well-regarded complementary medicine, particularly in Europe and India. In homeopathy, organic and inorganic natural substances are used to restore "vital power," alleviate illnesses and restore health [6]. Few research has been published on the use of homeopathy against the toxicity of paracetamol. The present study attempts to examine the effectiveness of the microdoses (14CH) and (30CH) of Strychnos nux-vomica as a homeopathic drug against paracetamol poisoning.

EXPERIMENTAL Plant material
Seeds of Strychnos nux-vomica were identified and sorted after rigorous quality control. The seeds were washed and air-dried. Then, they were crushed and stored in glass vials until use.

Animals
Male Wister rats (n = 42) weighing between 165 and 247 g, were acquired from the Animal Laboratory of Natural and Life Science Faculty of El Oued University. Animal study protocol was approved by the Ethics Committee of the University of El Oued, El Oued, Algeria (approval no. 42/EC/DCMB/FLNS/UE2020). All experiments were carried out in accordance with the guidelines for Animal Research: Reporting in vivo experiments 2.0 [7].

Homeopathic strain preparation
Using the Strychnos nux-vomica seeds, a homeopathic strain remedy was prepared. The parent solution is a basic component that serves as a starting point for manufacturing homeopathic medicine. The preparation of the mother tincture was made by maceration in a water/ethanol mixture at 80 % v/v.

Preparations for centesimal Hahnemannian (CH) dilutions
The CH dilution equation is presented in Eq 1.
x CH = 10 -2x …….. (1) One volume of the mother tincture was added to 99 volumes of solvent 80 % v/v ethanol to obtain a CH dilution. The dilution was vigorously agitated as a result of dynamization action. This allows for creation of the first level of dilution, or 1 CH = 1 % = 0.01. Repeating the process, one volume of 1 CH solution was made up with 99 volumes of solvent. Subsequently, the solution was electrified to produce 2 CH. This process was continued until appropriate dilution in CH was achieved. There are two steps in the formulation stage of homeopathic remedies: dilution and potency. The solution was shocked repeatedly and strongly agitated after each dilution. This dynamization would discharge therapeutic substances by causing the formation of nanoparticles that keep onto the imprint of original substances and the information it contains. The solvent then grasps this information during subsequent dilutions [8].

Study design
Rats were acclimatized for two weeks, during which they were fed standard food and kept at room temperature, constant humidity, and a 12hour photoperiod. The animals were equally divided into one control group (G1) and three experimental groups (G2, G3, and G4). The control group received filtered water, whereas the experimental groups were provided with a toxic dose of paracetamol equivalent to 1/4 of the LD50 (485 mg/kg) by gavages. In addition to the toxic dose of paracetamol, G3, and G4 were treated with the homeopathic drug (14CH and 30CH) of Strychnos nux-vomica (0.5 mg/kg), respectively.

Blood and tissue samples
To conduct biochemical analysis, blood samples in heparinized tubes were drawn from the jugular vein of rats. After dissection, the liver, heart, and kidneys were separated, washed with physiological water, weighed, and fixed in formalin to use in the histopathological study. One gram of tissue (heart, liver, kidney) from each rat was ground and homogenized in TBS and then centrifuged at 3000 rpm for 15 min. The supernatant was filtered and used to determine oxidative stress parameters.

Antioxidant properties
The stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity was used to measure the antioxidant activity of plant extracts and the standard Butylated hydroxytoluene (BHT). The DPPH radicalscavenging assay was carried out according to the method described by Mubarak et al [9] with some modifications. Eq 2 was used to compute the radical scavenging activity. RS (%) = ((Ab-As)/Ab)100 ……….. (2) where RS is DPPH radical scavenging, Ab is the absorbance of blank, and As is the absorbance of sample.

Hemolysis assay
Hemolysis assay was done as described by Henkelman et al [10]. Blood (5 ml) was collected from healthy volunteers in tubes containing 5.4 mg of EDTA to prevent coagulation and centrifuged at 1000 rpm for 10 min at 4 o C. The erythrocytes were washed three times with PBS. The washed RBCs were diluted to a hemoglobin concentration of 10 g/L with ultra-pure water, which resulted in complete lysis, and aliquots were stored at −80 °C. Fifty microliters of ten dilutions of erythrocytes suspension were mixed with 100 µL of test sample (S) (Strychnos nuxvomica), and the reaction mixture was incubated at 37 ºC water bath for 60 min. The absorption of hemoglobin was measured at 450 nm. Percentage hemolysis (H) was calculated using Eq 3.
Where C is the control

Serum biochemical parameters
A type auto-analyzer (BIOLIS 24j) with an appropriate reagent package for each parameter was used to determine blood glucose, urea, creatinine, alanine aminotransferase, and aspartate aminotransferase using the colorimetric method.

Oxidative stress parameters
The thiobarbituric acid (TBA) method was used to determine Malondialdehyde (MDA) level. The MDA reacts with TBA to give pink-absorbing chromophores at 532 nm. Glutathione was determined by a spectrophotometer using the colorimetric method [11].

Statistical analysis
One-way analysis of variance was used to statistically assess the impact of homeopathic medicine. A Duncan's multiple comparison post hoc test was used to detect whether any statistically significant difference exists between the obtained means. Differences were considered significant at p < 0.05.

Antioxidant property
The researchers first assessed the antioxidant capacity of the homeopathic strain (hydroalcoholic extract of Vomit seeds). Figure 1 demonstrates the DPPH radical-scavenging activity of plant extract and Butylated hydroxytoluene (BHT) at various doses. The S. nux-vomica seed extract showed the highest inhibitory action, attaining a peak of 97.35 % at a dose of 0.5 mg/mL, while BHT showed a maximum inhibitory activity of 77.63 % at the same concentration. The extract of seeds has an IC50 of 6.86 g/mL. With respect to the common BHT, IC50 equals 257 g/mL.

Hematological parameters
The effects of homeopathic formulations 14CH and 30CH on hematological parameters in control and acetaminophen-intoxicated rats are presented in Table 1. The red blood cell (RBC) count and hemoglobin (Hg) concentration significantly decreased after the paracetamol intake in experimental groups compared to control group. Table 2 shows the effect of Strychnos-nuxvomica homeopathic drugs on serum biochemical markers in control and paracetamolintoxicated rats. The latter showed a significant increase in the serum levels of urea, creatinine, glutamic transaminase (GTO and GTP), and alkaline phosphatase compared to their counterparts in control group. The kidney and liver injuries were reduced significantly after the concurrent intake of 14CH and 30CH S. nux Vomica extracts.

Oxidative stress markers
The obtained results are presented in Table 3. Paracetamol induced significant increases in liver and kidney malondialdehyde (MDA) levels against a significant decrease in glutathione (GSH) levels of the experimental groups compared to control group. Unlike paracetamolintoxicated rats, the 14CH and 30CH homeopathic treated groups significantly alleviated disharmony and restored normal secretion of oxidative stress markers.    13.6±0.4 c *P < 0.05, **p < 0.01, ***p < 0.001 compared to control group; a p < 0.01, b p < 0.01, c p < 0.001, compared to paracetamol group

DISCUSSION
The concept of "let like be healed by like" underlies one of the most significant applications of alternative and integrative medicine (homeopathy). Homeopathy treats a variety of illnesses by using highly diluted medicines [12]. The goal of current investigation was to assess the biological characteristics and protective effect of homeopathic remedies prepared from Vomit seeds against paracetamol-induced toxicity in rats. The damaging effects of oxidative stress are inhibited or avoided thanks to natural antioxidants found in plants. It is widely demonstrated in literature that free radical scavengers such as polyphenols, flavonoids, glycosides, steroids, tannins, alkaloids, and phenolic substances are present in S. nuxvomica extract [5]. Hemolysis of human RBCs is a highly useful model to examine the effects of free radicals on membrane oxidative damage and assess the antioxidant activity of novel drugs. The erythrocyte membrane is prone to damage, and it may lose its integrity as a result of lipid peroxidation, releasing hemoglobin (hemolysis) and intracellular K + ions [13].
The results showed that plants, particularly phenolic compounds which are responsible for the protection of red blood cells against lipid peroxidation, are rich in antioxidants [5]. Highdose paracetamol destroyed RBCs and also produced thrombocytopenia and hemolytic anemia [14]. Paracetamol may stop the kidneys from releasing erythropoietin. The number of RBC and Hb, values significantly decreased after paracetamol overdose, but the number of WBC significantly rose. Paracetamol toxicity is substantially correlated with the development of inflammatory reactions and immune cell dysfunction [15]. The co-administration of homeopathic treatments 14CH and 30CH reduced considerably the toxic effects of paracetamol and reversed these alterations. According to Patel et al [3], Strychnos-nuxvomica has bioactive compounds such as alkaloids, saponins, tannins, flavonoids, and glycosides. This explains their therapeutic actions. The results of this study revealed that kidney damage that was caused by paracetamol overdose was characterized by a significant increase of creatinine and blood urea in the paracetamol-intoxicated rats' group compared to control group. The obtained findings also showed that an overdose of paracetamol caused severe hepatotoxicity in rats as explained by an increase in hepatic enzymes GOT, GPT, and PAL when compared to control group.
Numerous harmful substances are known to build up in the liver, where they are detoxified. It was reported that a high intake of paracetamol damages the kidneys and liver by producing reactive oxygen species, increasing lipid peroxidation, depleting antioxidant enzymes, and activating cytokines that cause cell death and tissue damage [16]. The harmful effects of paracetamol on the kidneys and liver were significantly reduced after administering the homeopathic S. nux vomica extract.
According to the findings, treatment with paracetamol induced a considerable increase in MDA levels while concurrently inhibiting the level of GSH in the liver and kidney of rats [17]. As a result of toxicity, there is a fairly significant association between the rise in lipid peroxidation (LPO) and the fall in reduced glutathione (GSH). Malonaldehyde (MDA), a byproduct of the degradation of lipids in LPO, damages the structure of membranes as a result of a series of peroxidative processes [18]. Cytochrome P450 transforms paracetamol into a toxic metabolite (N-acetyl-p-benzoquinone imine). The glutathione detoxifies this metabolite, forming an acetaminophen-glutathione conjugate (APAP-SG). Increased APAP-SG synthesis reduces GSH levels, which results in cell death [19]. In the event of hepatotoxicity, free radicals are produced. Glutathione has antioxidant properties manifested through scavenging of free radicals. Thus, the S. nux-vomica homeopathic drug has antioxidant properties that prevent lipid peroxidation through decreasing MDA.

CONCLUSION
The study reveals the presence of antioxidant and anti-hemolytic principles in vomit seeds. Homeopathic treatment demonstrates superior recovery in renal parameters and liver enzyme levels. The results suggest that acetaminophen toxicity is effectively reversed with Strychnos nux-vomica 14CH and 30CH homeopathic treatment. Strategies to examine the findings of this study in humans should be explored.

Ethical approval
Approval for this work was obtained the Ethics Committee of the University of El Oued, El Oued, Algeria (approval no. 42/EC/DCMB/FLNS/ UE2020).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.