Hepatotoxicity is the main secondary effect caused by methotrexate, it is the drug widely used to treat autoimmune diseases, malignant tumors, cancer, psoriasis, rheumatoid arthritis (RA), lupus, ectopic pregnancy, leukemia, and other ailments.

In recent years, in vivo and in vitro research has been carried out focused on the search for hepatoprotective agents that help reduce the damage caused by this drug. This work aims to describe the hepatoprotector effect of natural bioactive compounds (carvacrol, phloridzin, berberine, pentoxifylline, chlorogenic acid, gallic acid, resveratrol, rutin, quercetin, thymoquinone, lycopene, alpha lipoic acid, ozone and melatonin), isolated from medicinal plants as well as from organic extracts of medicinal plants such as Curcuma longa, Sphaeralcea angustifolia, Balanites aegyptica, Morus nigra, Spinacea oleracea, mixture from four medicinal plants (Boerhaavia diffusa, Cratoera nurvala, Nelumbo nucifera and Rheum emodi) and pollen) that prevent and/or protect the liver from damage caused by methotrexate. The majority of these evaluations have been performed in in vivo models.

• Hepatotoxicity

• Methotrexate

• Rheumatoid arthritis

• Medicinal plants

• Natural bioactive compounds

• Hepatoprotection

Jiménez-Arellanes MA (2024) Medicinal Plants Extracts and Pure Secondary Metabolites with Hepatoprotective Effect against Damage Caused by Methotrexate: A Review. JSM Clin Case Rep 12(3): 1241.

RA: Rheumatoid Arthritis; MP: Medicinal Plants; NBC: Natural Bioactive Compounds.

Pharmacological effect of methotrexate and rheumatoid arthritis

Methotrexate (MTX, 1) is an anti-metabolite of folic acid (2) that is used for the treatment and prophylaxis of some ailments, such as autoimmune diseases, malignant tumors, cancer, psoriasis, rheumatoid arthritis (RA), lupus, ectopic pregnancy, leukemia, and neoplasic diseases, among other illnesses [1,2].

It is a main drug known as primordial Disease Modifying Antirheumatic Drug (DMARD) use in the treatment of RA. MTX has been used for more than 20 years due to its effectiveness, tolerability, rapid action and its ability to stop the progression of the disease, added to its low cost. In addition, can be used as monotherapy or in combination with other drug, including the biological therapy [3,4].

RA develops with permanent pain, burning, itching, hypersensitivity, redness, loss of function and mobility of the 

upper and then lower articulations; it also causes chronic inflammation of the articulations, affecting above all the sinovial tissue, and causing damage at the bone level, tendons and ligaments, deforming the joints. It has a multifunctional, systemic, autoimmune etiology, characterized mainly by an episodic symmetrical polyarthritis, chronic and deforming, and by producing joint disability over the long term [5]. Its diagnosis is made by interpreting clinical signs and symptoms of the disease [6,7]. Its prevalence fluctuates between 0.5 and 2% in the population of industrialized countries, with an incidence of 200 cases per 100,000 inhabitants, being more frequent in women than men (relation 3:1), and is presented between 30 and 55 years of age, although it can occur at any age, and there are currently cases of RA in young people (<25 years).

RA is a public health problem throughout the world, due to its great economic and social impact due to its prevalence and complications, especially due to prolonged treatment and the disability it generates. In Mexico, it affects 1.6% of the population, with higher incidence in women, and is the first cause of rheumatology care [8], generating mainly handicap and disability, reducing the quality of life of the patient, and impacting the economy of the country, the patient, and his family [9].

The treatment of the RA is based mainly on the use of DMARD drugs (which include MTX, azathioprine, sulfasalazine, leflunomide, and D-penicillamine) and biological therapy, with the object of reducing the symptoms, preventing structural damage and disability. These drugs cause severe secondary effects, such as hepatotoxicity (HPT), leucopenia, myelosuppression, pneumonitis and an elevated risk of bacterial and viral infections, such as osteoporosis [1,6,10].

The dose of MTX should be > 10 mg/week, administered orally, although it can be administered parenterally, depending on the patient’s conditions. It is recommended to start with 7.5 and increase to 10 mg/week in a single dose for 4 weeks, together with folic acid (5 to 10 mg) one day after administering MTX.

Subsequently, a progressive increase of between 2.5 to 5 mg/ week is carried out until reaching 20-30 mg/week within the first 6 months, depending on the clinical response and tolerance of the patient. It is advisable to monitor the response-efficacy of the drug with clinical and radiological studies for four weeks to determine its effectiveness [3,11,12].

Mechanism of action of the MTX in RA

This drug is used at low doses in chronic inflammatory processes such as RA, due to its dual action (immunosuppressant and anti-inflammatory), since it has the ability to block cellular metabolism and inhibits cell division, because it inhibits the proliferation and induces apoptosis of activated T lymphocytes by reducing purine metabolism and causes extracellular release of adenosine. In addition, it acts as an anti-inflammatory through specific receptors (Type A2 and A3).

Intracellular MTX, in the form of polyglutamate, inhibits purine synthesis by blocking 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) and dihydrofolate reductase; also inhibits pyrimidine synthesis by blocking thymidylate synthase [Figure 1] [4,13].

Figure 1: Mechanism of anti-inflammatory action of the MTX (taken from Goicoechea-García) [14]

On the other hand, MTX reduces the production of proinflammatory cytokines, such as interleukin (IL) 12A among others, interferon gamma (INF-γ), and increases the codification of genes that code for anti-inflammatory proteins (IL-4 and IL- 10). This causes an accumulation of intracellular AMPc and expels its outside the cell, where it is transformed into adenosine and joins to the A2a receptor of the lymphocyte (ADORA2A), favoring the synthesis of protein-kinase A (pKA), blocking the formation of interferons and, in consequence, inhibiting the synthesis of inflammatory cytokines [5,14]. MTX reduces the production of other pro-inflammatory agents, such as prostaglandins and leukotrienes, as well as some proteolytic enzymes [5].

Toxicity of methotrexate

MTX at low doses is a safe and tolerable drug, but it has been reported that it causes various secondary effects, depending on the folate (mucosis or medular toxicity) or independent of the folate, as idiosyncratic reactions at the pulmonary level, hepatic toxicity and effects at the neurological level [5]. It is estimated that 50% of patients treated with MTX have the probability of developing cancer: three times more of developing melanomas or lung cancer and five times more of showing lymphoma [6]. Adverse effects occur in up to 80% of patients, this causes more than 35% to abandon treatment; effect that affects the progression of RA and complication in its treatment. Other effects that MTX causes are HPT, nephrotoxicity, bone marrow toxicity, pulmonary fibrosis and gastrointestinal toxicity [13,15]. Nephrotoxicity is the result of crystallization of the renal tubular lumen, this being one of the main reasons for abandonment of the treatment, since more than 90% of MTX is excreted by urinary means, increasing serum creatinine and urea, causing uremia, hematuria and renal failure [13,16,17]. Approx. between 10 and 20 % of MXT is metabolized in the liver, producing 7-hydroxy-MXT (active form) and approximately 5% of this drug is metabolized in the intestine (by intestinal flora), forming 4-amoni-desoxyN-10-methylpteroic acid. In addition, between 5 to 20% of the MTX and between 1 and 5% of its metabolite (7-hydroxyMXT) is eliminated by bile, which means it is recommended to periodically review the parameters of hepatic and renal function [14]. The HPT is the most common and serious side effect of the prolonged treatment with MTX; it induces histological changes in the liver, including steatosis, hypertrophy of stellate cells, anisonucleosis and hepatic fibrosis. This HPT appears to increase with the total accumulated dose [18], through an imbalance in the endogenous antioxidant system, due to the fact that it promotes oxidative stress in the hepatic cells, unleashing peroxidation of lipids, reduction in glutation and oxidative stress, which causes deterioration in mitochondrial function [19].

The main risk factors for developing hepatopathy from MTX are: alcohol abuse, the existence of previous hepatic diseases (hepatitis B and C), age, nutritional status, obesity and diabetes, among others [20]. Upon initiating treatment with MTX and increasing the dose, it is recommended to analyze hepatic enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphate (AP)], creatinine and hemogram every month, due to the fact that high levels of AST are related with higher incidence of HPT [21]. MTX should be suspended when AST surpasses three times the upper limit for normality; however, it can be reintroduced at low dose once the level has normalized. Alterations in ALT are frequent but transitory, and upon observing a permanent increase in its concentration, hepatic biopsies are recommended to discard other causes for the increase, such as the use of AINEs, obesity, and/or chronic alcohol consumption [12]. Taking into account the severe damage caused by MTX in the liver, it is necessary to explore the kinds of substances (plant extracts and/or natural or synthetic compounds) used to counteract this damage.

Hepatoprotector effect in vivo of the polar extracts from medicinal plants against the damage caused by MTX

Moghadam et al. [19], evaluated the hepatoprotector effect in vivo of the ethanolic extract (EtOH) from Curcuma longa (C. longa) in a murine model (albino Wistar rats) with hepatic damage caused by MTX. The author used the following treatment program: Group 1: control (SSI/intragastric route -i.g.-); Groups 2 and 3: received extract of C. longa (100 and 200 mg/ kg/i.g., respectively) over 30days; Group 4: MTX (20 mg/kg), administered by intraperitoneal route (i.p.) in single dose at day 30; and Groups 5 and 6 received only the extract of C. longa (and 200 mg/kg/i.g.) over 30 days, respectively, plus MTX (20 mg/ kg/i.p.) in a single dose at day 30. At the end, the animals were sacrificed 4 days after administration of MTX and blood levels of ALT and AST (indicators of hepatic damage), AP and bilirubin (markers of biliary function), albumin (Alb, hepatic function) were determined, as well as levels of superoxide dismutase (SOD), catalase (CAT), glutation peroxidase (GSH-Px) and oxidized lipids (Lpx) in hepatic tissue. In addition, hepatic damage was evaluated at the histological level. MTX caused severe hepatic damage with a confidence interval of p <0.05, since levels of AST, AP, ALT increased and antioxidant capacity (SOD, CAT, GSH-Px) decreased; histological analysis of the hepatic tissue of the animals showed severe centrilobular and periportal degeneration, hyperemia in the vena porta, increased infiltration of arterial inflammatory cells and necrosis; on the contrary, these histopathological alterations were lower in the group that received C. longa extract (200 mg/kg), where hyperemia and necrosis were also reduced. The extract showed its hepatoprotective effects through the regulation of the antioxidant levels (SOD, CAT, GSH-Px) and the regulation of hepatic markers AST, ALT and AP, causing an improvement in bile and liver synthesis. The authors concluded that the extract of C. longa reduces the HPT of MTX because it contains antioxidant and anti-inflammatory compounds.

Montaser et al. [21], reported the effect of aqueous extract of Balanites aegyptiaca (BA), melatonin (MEL, 3), and ursodesoxycolic acid (UDCA) against HPT caused by MTX in male rats (Sprague Dawely). They used the following treatments: Group I: control [0.5 mL carboxymethylcellulose (CMC) plus 1 mLde SSI], Group II: MTX (13.4 mg/kg/i.p., dissolved in SSI) on day 30, Group III-V: MEL (10 mg/kg), BA (100 mg/kg) and UDCA (20 mg/kg), respectively, administered by i.g. during 30 days plus a single dose of MTX (13.4 mg/kg/i.p., single dosis) on day 30. At the end of the treatment, AST, ALT and AP were quantified, and the parameters of oxidative stress (SOD, CAT, GST, GR and GPx) were determined in hepatic tissue, together with the total antioxidant effect, quantification of tumor necrosis factor (TNF-α) and histological analysis. The results showed a significant increase in ALT, AST, AP, gamma glutamyl transferase (GGT), total and direct bilirubin, as well as levels of TNF-α, oxydized glutation (GSSG), Lpx and nitric oxide (NO) in the MTX group and the group of MTX + UDCA; these levels were lower, while levels of PT, Alb, total antioxidant activity, GSH, GSH-Px, glutation reductase (GR), GST and SOD had a significant reduction in the groups treated with MTX and MTX + UDCA compared with the control group. It was noted that the damage caused by MTX diminished upon administering MEL and BA, since there was an increase in serum levels of PT and Alb, but reduced AST, ALT, AP levels; there was also a significant decrease in Lpx, NO and GSSG; therefore, it was concluded that BA and MEL helped reduce hepatic damage caused by MTX due to their antioxidant activity. In addition, they recommended not administering UDCA together with MTX, because the author observed an increase in inflammation and in liver size.

In another investigation, the protector effect of EtOH extract from Morus nigra (MUL) leave was evaluated against the damage caused by MTX in male albino rats. The animals were divided into four groups: Group I: healthy control, Group II: MUL extract (500 mg/kg/i.g.) administered during 14 days, Group III: MTX (20 mg/ kg/i.p.) on the third day of the experiment, and Group IV: MUL extract (at 500 mg/kg/i.g.) administered during 14 days + MTX (20 mg/kg), one single dose on the third day of the experiment. On day 15, serum and hepatic tissue samples were taken to determine markers of hepatic function (AST, high density lipids -HDL, AP, total proteins -TP- and Alb) and for histological analysis. The results indicated that MTX caused a significant increase on ALT, AST, AP and HDL levels compared to the healthy controls, while the animals administered with MUL + MTX showed a reduction in these parameters compared with MTX (p <0.05); histological analysis of the livers of the group that received MTX showed changes in the hepatic architecture, such as centrilobular hepatic necrosis, cellular infiltration with fibrosis, and group IV, which received MTX plus MUL extract, did not show alteration. The authors concluded that Morus nigra extract protects against hepatic damage caused by MTX [22].

Additionally, the aqueous extract from Spinacea oleracea L. leave was evaluated against the damage caused by MTX in albino rats. In this study, they used three groups of animals: Group I: healthy control, Group II: MTX (20 mg/kg/i.p., single dose at the beginning of treatment) plus SSI during 5 days, and Group III: MTX + aqueous extract of S. oleracea (200 mg/kg/i.g.) administered during 7 days before and 5 days after administration of MTX (20 mg/kg/i.p.). At the end of the treatment, the animals were sacrificed to take samples of blood and hepatic tissue to determine AST, ALT, AP and Bil), oxidative stress (Lpx, GHS) and histological analysis. The results obtained show that the administration of MTX caused an increase in Lpx and reduction in levels of GSH, AP, while these changes were inverted in the group treated with the S. oleracea extract. At the histological analysis, less damage was observed in the group treated with MTX plus S. oleracea extract. It was concluded that the protector effect of the aqueous extract of Spinacea oleracea against the HPT caused by MTX could be attributed to the combined effects of quercetin (QE, 4) and kaempferol (5); these two flavones were detected in the extract by HPLC analysis [23].

Badr [24], described the protector effect of the aqueous extract from pollen (PAM, rich source of flavonoids with antioxidant activity) against the HPT caused by MTX in male albino mice, with the following treatment programs: Group I: control; Group II: MTX (single dose 20 mg/kg/i.p.) on day 7 of the study; Group III: administered for 7 days with PAM (100 mg/kg/ i.g.), Group IV: PAM (100 mg/kg/i.g.) before the administration of MTX (20 mg/kg/i.p.) administered during 7 days,; and Group V: PAM (100 mg/kg/i.g.) after the administration of MTX (20 mg/ kg/i.p.) over 7 days. At the end of the study, the animals were sacrificed to take samples such as serum and hepatic tissue to determine AST, ALT, AP, glutamate transferase (γ-GT), SOD and CAT. In addition, histological liver analysis and quantification of interleukins (IL-1β, IL-6, IL-4 and IL-10) values were performed. The results obtained indicated that the MTX group showed an increase in levels of ALT, AST, AP and γ-GT compared with controls. On the other hand, the PAM group showed a reduction in these hepatic markers (p< 0.05) compared with the group of MTX plus PAM; therefore, the administration of PAM before MTX showed a better hepatoprotector effect. In addition, the PAM group showed improved antioxidant effect and immune response compared with the p PAM/MTX group due to the increase in the production of SOD and CAT. The results showed significant elevation of IL-4, IL-10 and reduction in IL1β and IL-6 in all the groups treated with pollen compared with the MTX group, which suggests that PAM contains immunomodulator substances and that its administration prior to or after the use of MTX reduces hepatic damage.

Another work described the protector effect of pollen administered over 35 days against the alterations caused by MTX in hepatic and renal tissues. This study was performed in rabbits: Group I: control (SSI), Group II: MTX (0.25 mg/kg/i.p., single dose administered during 35 days), Group III: PAM (50 mg/kg/i.g.) + MTX (0.25 mg/kg/i.p.) and Group IV: PAM (50 mg/kg/day). At the end of the study (day 36), the animals were sacrificed and blood samples, liver and kidney tissue were taken for histological analysis. The results obtained showed that MTX caused hydropic degeneration, picnosis, sinusoidal dilation and hyperplasia of the bile conduit, together with renal tubular degeneration, glomerular contraction and a hialine precipitation, while the pollen (PAM) group showed a lower degree of these morphometric alterations. However, glomerular contraction and degeneration of the renal tubules was partially protected in animals that received MTX plus pollen, and in biochemical results there was a significant reduction in AST and ALT in the group that received pollen + MTX. It was therefore concluded that treatment with pollen has little protector effect against the toxicity caused by MTX [25].

Recently, Sharma et al. [26], described the protector effect of the EtOH extract (70%) of a mixture of 4 medicinal plants (Boerhaavia diffusa, Cratoera nurvala, Nelumbo nucifera and Rheum emodi) against the nephrotoxic damage caused by MTX. This mixture was tested at doses of 200 and 300 mg/kg, in male Wistar rats with the following program: Group I: control, Group II: MTX (7 mg/kg/day) administered by i.p. from day 5 to 8, Groups III and IV: MTX (7 mg/kg/day) from day 5 to 8 plus the mixture of 4 plants at doses of 200 and 300 mg/kg, respectively, for 8 days. The animals were sacrificed one day later and serum was obtained to determine parameters of renal function (levels of urea, uric acid, creatinine, protein, globulin and albumin) and markers of oxidative stress (AST, ALT, CAT, Lpx, AP); these markers were also determined in the kidney. In addition, histological analysis was performed. The results generally showed that both doses (200 and 300 mg/kg) of the polyherbal preparation decreased the urea, uric acid, creatinine, proteins, globulins and albumin levels in serum and also in the kidney compared to the MTX group, without reaching the healthy control levels. The parameters of oxidative stress were also reduced in the MTX groups plus extract from the 4 plants at the doses tested without reaching the levels of controls. It should be noted that they also tested a mixture from 3 plants (B. diffusa, C. nurvala, and R. emodi) at doses of 150 and 250 mg/kg; however, this mixture was less active than the 4-plant mixture. Both mixtures (4 and 3 species) protected against renal damage caused by MTX; these mixtures showed good antioxidant activity in vitro (> 45%) with respect to positive control (gallic acid, 83.42%); also, their flavonoid content was high and showed a good percentage (>35%) of inhibition of xantin oxidase (trial in vitro).

Hepatoprotector effect of pure compounds (natural and synthetic) against damage caused by MTX

Carvacrol (CAR, 6) showed a protector effect against the hepatic toxicity caused by MTX, by being administered for 7 days in Wistar rats (225 ± 30 g). This trial was performed as follows: Group I (control); Group II (MTX); and Group III, (CAR + MTX). From day 1, Group III received, for 7 days, CAR (73 mg/kg/i.p.), and from day 2, Groups II and III received a single dose of MTX (20 mg/kg/i.p.). On day eight, a blood sample and hepatic tissue were obtained to quantify ALT, AST, AP, Lpx, CAT, total oxidant state (TOS) and histological analysis. The results obtained showed that levels of Lpx, ALT, AST and AP increased significantly in the group that only received MTX compared with the control group. On the other hand, the results of the group given CAR + MTX showed a good effect against exposure to MTX, due to the observed significant reduction in Lpx and increase in AST. This was confirmed with the histological analysis, where significant hepatic lesion was observed in the MTX group compared with the CAR + MTX group; it was concluded that pretreatment with CAR notably reduces the hepatic damage caused by MTX.

Dalaklioglu et al. [28], reported the protector effect of resveratrol (RVT, 7) against the HPT caused by MTX over a period of 3 months. The study was performed in Wistar rats under the following treatment: Group I: Control, Group II: MTX (7 mg/kg/ day/i.p.), once a day for 3 consecutive days, Group III: MTX (7 mg/kg/day/i.p.) + RVT (20 mg/kg/day/i.p.) and Group IV: RVT (20 mg/kg/day/i.p.). The first dose of RVT was administered 3 days before the injection of MTX and continued over 3 days. After 3 months, the animals were anesthetized to take a blood sample and sacrificed by cervical dislocation for taking hepatic tissue. The parameters evaluated were: liver histology, quantification of AST, ALT, AP; determination of the parameters of oxidative stress [substances reactive to thiobarbiturate acid (TBARS), glutation-S-transferase (GST) and CAT]. The results showed that the administration of MTX significantly increased levels of ALT, AST and AP compared to the control group, and a reduction was observed in TBARS, CAT and GST values in the group receiving MTX + RVT; these markers (TBARS, CAT and GST) also increased in the livers of animals that received only MTX respect to controls. Treatment with RVT significantly reduced the HPT caused by MTX. These results demonstrated that RVT has a protector effect against the HPT caused by MTX, by inhibiting the peroxidation of lipids by oxidative stress. It was therefore concluded that treatment with RVT could be a promising strategy to reduce the HPT caused by MTX.

Chlorogenic acid (CGA, 8) has also shown hepatoprotector effect against the toxicity caused by MTX in rats. Group I: control (SSI), Group II: received a single dose of MTX administered by i.p. on day 18. Groups III and IV were pretreated with CGA at 50 and 100 mg/kg, respectively, for 20 days and a single dose of MTX (on day 18). On day 21, the animals were sacrificed by cervical dislocation and liver samples were taken. The results showed that the MTX group had a significant increase in the markers of toxicity, histological changes, reduced activity of SOD, CAT, and GPx, while the CGA group showed a reduction of the liver damage; also, there was a reduction in the levels of markers of oxidative stress. These findings show the hepatoprotective effect of CGA, because reduced of the proinflammatory cytokines and apoptotics effect, in addition, it stimulated the antioxidant system in liver tissue. Therefore, the administration of CGA helps to reduce the toxicity of MTX by being a substance with antioxidant activity [29].

In another work, the beneficial effect of lycopene (Lyc, 9) against the hepatic toxicity caused by MTX in Sprague-Dawley rats was studied, using the following scheme: Group I: Lyc (10 mg/ kg/i.g. dissolved in corn oil) administered during 10 days; Group II: MTX (20 mg/kg/i.p., single dose) dissolved in corn oil on day 1; Group III: MTX (20 mg/kg/i.p., single dose) plus Lyc (10 mg/kg/i.g.) administered during 10 days after the MTX administration. Upon ending the experiment, ALT, AP, AST and parameters of oxidative stress [CAT, IL-1β, total hepatic antioxidant capacity (TAC) and total oxidative state (TOS)] were quantified and histological analysis performed. The results indicated severe tissue damage, sinusoidal dilation and infiltration, congestion, degeneration and increase in levels of TNF-α, IL-1β, TOS and TAC in hepatic tissue on the MTX group respect to controls. On the other hand, a significant reduction in AST, ALT and pro-inflammatory cytokines (TNF-α, IL-1β) in the MTX plus Lyc group respect to MTX group was observed, suggesting that lycopene reduces hepatic damage, oxidative stress and inflammation. The authors concluded that compound 9 is efficient in reducing HPT caused by MTX through the reduction of levels of pro-inflammatory cytokines, but not of TOS, at a dose of 10 mg/kg [30].

Phloridzin (PHL, 10) has a protector effect against the HPT caused by MTX compared with positive control N-acetylcysteine (NAC), for which male rats were used: Group I: healthy control, Group II: PHL (40 mg/day/i.g./10 days), Group III: MTX (20 mg/kg/i.p., single dose) administered on day 3, and Groups IV, V and VI received NAC (150 mg/kg/day), PHL (40 mg/kg/day) and PHL (80 mg/kg/day), respectively, administered during 10 consecutive days; the three latter groups received MTX (20 mg/kg/i.p., single dose) on the third day after initiating treatment. Upon ending the administration, ALT, AST, LDH, TNF-α, cyclooxygenase-II (COX-2), TAC, TBARS, GSH, nitrite (NO -), CAT, GST and SOD were quantified as biomarkers of oxidative stress; expression of hepatic caspase-3 was also evaluated. In the animals treated with PHL, hepatic lesion was significantly reduced, due to reduced levels of ALT, AST and LDH, TNF-α and levels of COX- 2; also, significant reduction was observed on NO - and TBARS levels, with significant increases in TAC, GSH, GST, CAT and SOD respect to MTX group. PHL protects against hepatic lesions in rats by reducing oxidative stress, inflammation and apoptosis in the liver, and may be promising to alleviate and/or prevent HPT caused by MTX [1].

Mehrzadi et al. [31], described the protector effect of berberine (BBR, 11) in male Wistar rats. The animals were treated as follows: Group I: Control (SSI/10 days), Group II: MTX (20 mg/kg/i.p.) administered on day 9; Group III: BBR (100 mg/kg) administered during 10 days and at day 9 a single dose of MTX (20 mg/kg/i.p.) and Group IV: BBR (100 mg/kg/i.g.) administered during 10 days.

On day 11, blood samples were taken to determine ALT, AST and AP levels, and later the liver was extracted for histological analysis and to determine parameters of oxidative stress, such as Lpx, GSH, oxidated protein (OP), nitric oxide (NO), CAT, SOD, and GSH-Px. In addition, expression of SOD and GSH-Px were determined by real- time PCR. The results showed that MTX significantly increased levels of AST, ALT and AP (p< 0.001) compared with healthy controls; it also increased levels of Lpx, PO, NO - and increased the activity of myeloperoxidase -MPO- (with p< 0.001, p< 0.01, p< 0.05 and p< 0.01, respectively). In addition, MTX also reduced the activity of GSH, SOD, GSH-Px and CAT (p< 0.001) respect to control. By administering BBR for 10 days, a reduction in AST and ALT (p< 0.001), of Lpx (p< 0.001) and of GSH were observed and an increase in the activity of GSH-Px (p< 0.05). Therefore, it was concluded that BBR is useful to prevent the HPT caused by MTX by exercising a beneficial effect on oxidative stress.

The protector effect has also been reported for pentoxifylline (PTX, 12) and alpha lipoic acid (ALA, 13) against the HPT and nephrotoxicity caused by MTX in male Sprague-Dawley rats. The study included Group I: control, Group II: MTX (20 mg/kg)

administered in day one, Group III: MTX + PTX (20 + 50 mg/kg) and Group IV: MTX + ALA (20 + 100 mg/kg). PTX and ALA were administered daily for 10 days. Upon ending the experiment, GSH-Px, SOD, CAT, Lpx, NO and xanthine oxidase (XO) levels were determined in the liver and kidney tissue. In serum, gamma glutamyl transferase (GGT), direct bilirubin (DBil) and urea were determined. The results indicated a significant reduction in the damage caused by MTX in the groups treated with PTX and ALA, compared to the group that only received MTX, the protector effect was better in the group that received ALA that PTX. Increases were also found in GGT, urea and levels of CAT, Lpx, NO and XO in both groups (PTX and ALA), while GSH-Px only increased in the liver. ALA and PTX protect against the toxic effect caused by MTX in the liver and kidneys, being more active ALA [17].

Another work has reported the preventive effect of gallic acid (GA, 14) on the oxidative stress caused by MTX in rat liver. They used four groups: Group I: control, Group II: MTX (20 mg/ kg/i.p.) administered on day 9, Group III: MTX (day 9) + GA (30 mg/kg/day/i.g.) and Group IV: GA (30 mg/kg/day/i.g.), GA was administered during 10 days. On day 11, the blood sample and liver tissue were taken. The biochemical markers (AST, ALT and AP, Lpx and GSH levels) of hepatic lesion were also quantified, as well as the CAT, SOD, GPx activity and the expression of SOD2 and GPx1 genes by RT-PCR, as well as the histological analysis were made. The results showed that GA reduced the AST, ALT and AP levels, and also GSH, GPx, CAT, and SOD activity were reduced respect to MTX group. Likewise, the expression of GPx1 and SOD2 was reduced. In addition, the histological results showed that MTX caused hepatic damage and that GA improved the histological changes, indicating that GA protects against the damage caused by MTX due to antioxidant efect [32].

The protector effect of quercetin (QE, 4) on renal lesions caused by MTX in male Sprague Dawley rats has been reported. In this case, they used three groups: Group I, control (SSI); Group II, MTX (20 mg/kg/i.p., single dose) administered on day one and Group III, MTX (20 mg/kg/i.p.), administered on day one + QE (5 mg/kg/i.g.). QE was administered 30 minutes before MTX on the first day and this was administered for four more days. At the end of the experiment, the kidneys were extracted for histopathological analysis and for analysis of oxidative stress. MTX group showed alteration in the renal structure, with tubular degeneration and dilation and detachment of epithelial cells. Fewer degenerative changes were observed in the QE group, with a histological appearance similar to the control group. Furthermore, QE reduced the number of apoptotic cells. Lpx levels were elevated in the MTX group respect to controls and MTX + QE groups; a reduction was also observed in SOD, GSH-Px and CAT activity in the MTX/QE group. The authors concluded that QE reduces the toxic effects caused by MTX [33].

Another study reported the evaluation of QE (4) against toxicity caused by MTX in male Sprague-Dawley rats using the following regimen: Group I: healthy control, Groups II and III: MTX (0.25 and 0.125 mg/kg), Group IV: MTX (0.125 mg/kg)+ QE (500 mg/kg), Group V: MTX (0.25 mg/kg) + QE (500 mg/ kg); these treatment were administered by oral route during 14 days. At the end of treatment, blood sample was taken and the brain, lungs, heart, liver, kidney, spleen, stomach, jejune and ileon were extracted for histological analysis. The results showed that QE had an important protector effect against damage caused by MTX on the respiratory, hepatic and renal tissue. The MTX groups (at 0.25 and 0.125 mg/kg) showed significant corporal weight loss and increased of AST, ALT, AP and PT levels compared with healthy control, while the MTX/QE group showed a significant reduction respect to MTX group. Likewise, the urea and creatinine levels decreased in MTX/QE mice respect to MTX group. On the other hand, the animals with MTX at 0.125 and 0.25 mg/kg showed severe histological changes in lung, liver, kidney and spleen, showing alterations such as: sinusoidal dilation and slight congestion of the hepatic parenchyma, congested septal cappilaries, abnormal alveola, compared with the healthy group, while the MTX + QE group showed less damage on these organs (liver, lung and kidney). The author concluded that QE has a good protector effect, attenuating the toxicity caused by MTX in the liver, kidney and respiratory apparatus [34]. In another study, also reporte the protector effect of QE against the renal toxicity cause by MTX through biochemical and histopathological analyses, where QE was administered for 9 days; this assay was performed in adult male Wistar rats divided into: control group (SSI i.p.), MTX group (20 mg/kg/i.p., single dose on the third day), MTX group (20 mg/kg/i.p., a single dose on the day three) plus QE (50 mg/kg). QE was administered by i.g. 2 days before MTX and for 6 consecutive days after the MTX administration. At end of the experiment, blood sample and renal tissue were taken to determine SOD and Lpx, along with histological analysis and apoptotic changes through an assay of terminal marking of deoxynocliutyl-tranferase (dUTP) and expression of caspase-3. The results indicated nephrotoxic tissue damage, increased apoptotic index and increase in the expression of caspase-3 (p<0.05) in the MTX group compared with the control. In the MTX/QE group the histopathological damage was low and the expression of the apoptotic index, and caspase-3 diminished respect to MTX group. The Lpx level increased in the MTX group, and in MTX/QE group this value was significantly reduced. Furthermore, the SOD level was higher in the MTX group compared to the MTX/QE mice. Administration of MTX caused oxidative stress and structural and functional damage in renal tissue, and the continuous administration of QE reduced oxidative stress through its antioxidant properties, making QE promising to alleviate the renal toxicity caused by MTX [13].

Another flavone investigated for its potential HPP effect in female Wistar rats (200-250 g) was rutin (15); for this study, three groups were used: Group I: control (0,5 ml SSI/i.p.), Group II: MTX (20 mg/kg/i.p., administered on first day) and Group III: MTX (20 mg/kg/i.p. on first day) + rutin (100 mg/kg/i.p.) administered during 10 days. From blood sample and hepatic tissue some parameters of oxidative stress (Lpx, GSH-Px and SOD) were determined, as well as markers of hepatic damage (AST and ALT) and histological analysis of the liver. The results indicated that the MTX + rutin group showed less histological lesion compared with the group that only received MTX. In addition, a significant increase in Lpx and ALT levels was observed in MTX group, while SOD and GSH-Px values diminished in this group respect to control groups. It was concluded that rutin may be an adjuvant to reduce the side effects generated by MTX during therapy [35]. The hepatoprotective effects of the flavonoids maybe is due to their anti-inflammatory and antioxidant activities.

Another compound that has been investigated as a HPP agent against the damage caused by MTX is thymoquinone (16, isolated from Nigella sativa). This study was carried out in Wistar rats as follows: Group I: thymoquinone (10 mg/kg/day/) administered for 10 days, Group II: MTX (20 mg/kg/i.p.), a single dose on the third day of treatment, Group III: MTX (20 mg/kg/i.p.), a single dose on day 3 plus thymoquinone (10 mg/kg/day/i.g.) for 10 days, and Group IV: control (vehicle). On day 11, the animals were sacrificed and blood (serum) samples taken to determine urea, creatinine, ALT, AST, GSH, CAT, iNOS, Lpx, TNF-α, NF-κB/p65, COX- 2 and caspase-3. In addition, the liver and kidney were extracted for histological analysis. Thymoquinone favored corporal weight gain and reduced levels of the renal (urea and creatinine) and hepatic (ALT and AST) markers respect to MTX group, improving the effect on hepatic and renal function. GSH and CAT levels determined in liver and kidney was reduced in the MTX/ thymoquinone group compared with the MTX group. Values of GSH, Lpx and the nitrite/nitrate ratio in the kidney were similar to the control and thymoquinone groups; however, this change was not observed in the liver. Levels of iNOS and TNF-α increased in the MTX group and diminished in the MTX/thymoquinone group. At the histological level, liver and kidney architecture of the MTX/thymoquinone group was similar to controls, while the kidney from the MTX group showed glomerular atrophy, dilated renal tubule, and in the liver TNF-α and the expression of NF-kB and COX-2, while in the thymoquinone group reduced these parameters. The authors concluded that thymoquinone has antioxidant, antinitrosative, anti-inflammatory and antiapoptotic properties, so it can be used as a hepato- and nephroprotective agent to prevent and/or reduce the damage caused by MTX [36,37].

Kelleni et al. [18], reported the protector role of captopril (17, inhibitor of the enzyme convertor of angiotensin) and telmisartan, blocker of the angiotensin II receptor with agonism of the gamma peroxisome proliferator-activated receptor (PPARγ-)] against the HPT caused by MTX in Wistar rats. The animals was divided in: Group I: healthy control; Group II: MTX (20 mg/kg/ i.p.) administered on the fifth day) + captopril (100 mg/kg, for seven days); Group III: MTX (20 mg/kg/i.p.) administered on the fifth day + telmisartan (10 mg/kg, for seven days) and Group IV: MTX (20 mg/kg/ip.r on the fifth day). On the eighth day, samples of blood and hepatic tissue were taken to determine hepatic enzymes (AST, ALT, AP) and parameters of oxidative stress (Lpx, SOD, CAT and NO). The results of this study revealed that the MTX group showed elevated levels of AST, ALT, and AP, and histological alteration. Pretreatment with captopril or telmisartan induced significant liver protection, since it significantly reduced (p<0.05) the serum levels of ALT, AST, AP, and the concentrations of Lpx and NOx, as well as a significant increase in the SOD activity. Furthermore, at a histological level, no kidney or liver damage was observed and a significant reduction was observed in the expression of the enzymes COX-2, iNOS and caspase-3 compared to the MTX group. The authors recommend using captopril or telmisartan in patients that receive MTX; however, its use should be under strict medical supervision.

The hepatoprotector effect has also been reported for pretreatment with ozone (O3) against HPT caused by MTX (administered by i.p.) in male Wistar rats. In this assay the animals were grouped: Group I: control (SSI), Group II: MTX (a single dose, 20 mg/kg/i.p.) administered on fifth day and Group III was pretreated with 5 mL of ozone for 15 days plus a single dose of MTX (20 mg/kg) on fifth day of treatment. On day 16, samples were taken of blood and hepatic tissue to measure levels of ALT, AST, Lpx, cytokines TNF-α, IL-1β, GSH and MPO, and to perform histological examination. The results showed that MTX caused an increase in ALT and AST levels, as well as in Lpx and 

MPO levels, and reduced the concentration of GSH. In the group pretreated with ozone, a significant reduction was observed in these biochemical parameters, along with a reduction in hepatic damage caused by MTX. These authors concluded that ozone helped reduce the HPT caused by MTX in rats [38]. It is important to mention that the scientific literature includes various articles that describe the anti-arthritic activity of medicinal plants and/ or pure compounds and use MTX as positive control, but they do not report the protector effect against the damage cause by MTX [10,39-43].

For this work, scientific articles published in the last 10 years in indexed journal were reviewed, which included descriptions of the hepatoprotector effect from organic medicinal plant extracts and natural or synthetic substances against the hepatic damage caused by MTX. The search tools used were: PubMed, Web of Science, Scopus, Academic Search Complete and Google Scholar. The terms used for the search were: hepatotoxicity, methotrexate, rheumatoid arthritis, medicinal plants, pure natural compounds, and hepatoprotection. A total of 23 articles were found; seven described the HPP effect of medicinal plant extracts and 16 were for natural or synthetic compounds, which were saved and analyzed.

RA is a chronic progressive disease that causes systemic damage and that may present itself at any age. It causes disability and deterioration in the patient; its treatment is based mainly on the use of anti-rheumatics that modify the disease, MTX being the most widely used, in spite of the existence of other alternatives for treatment but with higher cost. However, continuous, indiscriminate use of this drug causes several side effects (nephrotoxicity, hepatotoxicity, myelosuppression, and an elevated risk of contracting bacterial, parasitic and viral infections), which compromise the patient’s health.

Therefore, it is necessary to contribute to the search for treatment alternatives that reduce and/or protect against the side effects caused by MTX and help improve the patient’s quality of life. To date, the polar extracts of only five plant species (C. longa, Balanites aegyptica, Spinacea oleracea, Morus nigra) and pollen have a good hepatoprotector effect against the damage caused by MTX. In addition, only some natural and synthetic compounds (resveratrol, lycopene, chlorogenic acid, gallic acid, carvacrol, alpha lipoic acid, phloridzin, berberine, ozone, pentoxifylline, melatonin, quercetin, rutin, thymoquinone, telmisartan and captopril) have been evaluated in vivo assays (in rats), and showed a good hepatoprotective effect. Polyphenols such as quercetin, resveratrol, rutin, carvacrol chlorogenic acid or gallic acid are the compounds that have shown the best activity presumably due to their anti-inflammatory and antioxidant activities, so these compounds are potential candidates to be evaluated in clinical studies.

This work is a bibliographical review and no experiments were performed with animals or humans, nor is patient data described.

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