INTRODUCTION

Medicinal plants are the richest alternative bio-resources to synthetic drugs (^{Obi et al., 2012}). Presently, there is a revitalization of interest in the use of medicinal plants by over 80% of the world population (^{Ogbonnia et al., 2011}). In African countries, a large proportion of the population depends solely on herbal medicines for their primary health care needs. This is because medicinal plants are accessible, acceptable and affordable among the populace when compared to synthetic drugs (Obi et al., 2012).

Dryopteris filix-mas, Dryoperidaceae is a deciduous evergreen perennial herb growing up to 60 -150 cm tall, with rhizomes. It is an effective ground cover plant that is native to Europe, Asia, and North America. It has a large light-green triangular frond (^{Sekendar et al., 2012}). It is commonly found in streams, shady places and wetland areas. It is commonly referred to as male fern, Aspidium, water loving fern, worm fern and shield fern (^{Uwumarongie, Enike, Bafor, 2016}). Its vernacular names include; fougère mâle (local French), sarkhas; shurud (Arabic), Eraketa (Urhobo), and Akpaka or Akolor (Igbo). The edible parts of Dryopteris filix-mas include; the leaves and root. The rhizomes can be eaten raw or cooked as part of a regimen for losing weight. In traditional medicine, the leaves and rhizome are used in the treatment of rheumatoid arthritis, inflammation, malaria, worm infestation, internal haemorrhage, uterine bleeding, fever, mumps, carbuncles and sores (Sekendar et al., 2012). The leaves, roots and rhizomes are used to rinse hair and to treat dandruffs (Sekendar et al., 2012; ^{Soare et al., 2012}). Literature had reported its anti-diarrheal activity (Uwumarongie, Enike, Bafor, 2016), antioxidant and cytotoxic activities (Sekendar et al., 2012), anti-helmintic activity (^{Urban et al., 2014}) and anti-microbial activity (Soare et al., 2012).

Following the ethnomedicinal values of Dryopteris filix-mas in Nigeria, there are no scientific literatures to validate its safety profile due to its repeated use overtime in the treatment and management of various diseases. The main purpose of this study is to evaluate the sub-chronic toxicities of Dryopteris filix-mas leaf extract on some toxicological parameters.

MATERIAL AND METHODS

RESULTS AND DISCUSSION

This study evaluated the sub-chronic toxicity profile of Dryopteris filix-mas in albino rats. From LD₅₀ study, there were no signs of toxicity or death recorded at various doses (100, 1000, 2000, 3000 and 5000 mg/kg) of the extract after 24 hours and subsequently for 14-days when compared to control group. This suggests that the extract is not toxic on short term exposure in this experimental condition. LD₅₀ value above 5000 mg/kg is classified as non-toxic (^{Muhammad et al., 2015}). Phytochemical screening revealed the presence of tannins, flavonoids, saponins, steroids, alkaloids, terpenoids and reducing sugars in the leaf extract of Dryopteris filix-mas (Table I). These secondary metabolites have been shown to exert various toxicological effects apart from their benefits (^{Aksel, 2010}; ^{Yadav, Agarwala, 2011}). In support of this finding, Uwumarongie and co-workers in the study of the phytochemical constituents of Dryopteris filix mas leaf also reported the presence of glycosides, tannins, flavonoids, steroids and other nutrients (Uwumarongie, Enike, Bafor, 2016).

Hematological parameters assessment is vital among various toxicity biomarkers to know whether test substances affect the hematopoitic system (^{Bashir et al., 2015}). From Table II, there was significant (*p<0.05) increase in PCV, RBC and hemoglobin on 31^st day following the administration of 250 and 500 mg/kg of Dryopteris filix-mas leaf extract to albino rats. This suggests that Dryopteris filix-mas could promote red blood cell production at 250 and 500 mg/kg doses after 30 days of its exposure to animals. Plant secondary metabolites such as flavonoids and terpenoids have been reported to promote erythropoisis (^{Osano et al., 2016}) and presence of these metabolites in the extract (Table I) may account for increase in PCV, RBC and hemoglobin on day 31^st. Non-significant changes in PCV, hemoglobin and RBC levels on 61 and 91^st days suggest that the extract does not promote red blood cell production at 250 and 500 mg/kg after longer duration of exposure. Long term intake of medicinal plants rich in saponins had been reported to cause hemolysis of red blood cells (^{Ekpenyong, Akpan, Udoh, 2012}). Study by ^{Kumar, Karthik, Rao (2011}) also revealed that presence of saponins in extracts of some Indian medicinal plants resulted in hemolytic effects due to alteration in the erythrocyte membrane. Thus, presence of saponins in the extract may account for the reversibility in high PCV, RBC and hemoglobin levels of 31^st day on 61 and 91^st days.

Values are presented as mean ± Standard error of mean (n =5). *p<0.05: Statistically significantly different from control group.

From Table III, there was significant (*p<0.05) decrease in WBC and lymphocyte counts and significant (*p<0.05) increase in granulocyte and medium size cells count on 91^st day following the administration of 250 and 500 mg/kg of Dryopteris filix-mas leaf extract to albino rats. This suggests that bioaccumulation of the extract due to its 90 days exposure to animals could disrupt bone marrow function resulting to insufficient production of leukocytes. ^{Debelo et al. (2016}) stated that reduction in WBC level is associated with immune suppression. Studies by ^{Unakalamba, Ozougwu, Ejere (2013}) revealed that saponins in medicinal plants could cause decrease production in WBC. Usually, reduction in total leukocyte and lymphocyte count correlates with increase in MID and granulocyte count (Yadav et al., 2010). Non significant change in these parameters following 28 days recovery studies suggests that leukocyte suppression effect of the extract was reversible.

Values are presented as mean ± Standard error of mean (n = 5). *p<0.05: Statistically significantly different from control group.

From Table IV, there was no significant difference (p>0.05) in PCT, MPV, MCV, MCH, and MCHC levels on 31, 61 and 91^st days in animals treated with various doses (62.5, 125, 250 and 500 mg/kg) of Dryopteris filix-mas leaf extract. This indicates that components of these blood parameters may not be deleteriously affected by the extract.

Values are presented as mean ± Standard error of mean (n =5). p>0.05: Not statistically significantly different from control group.

Increase in cholesterol and triglyceride levels could be associated with cardiovascular and coronary heart disease (^{Kayode et al., 2016}). From this study, significant reduction in total cholesterol and triglyceride levels at 250 mg/kg dose on day 61 (Table V) suggests that 250 mg/kg dose could promote lipid lowering. Presence of saponins and flavonoids in medicinal plants have been reported to play significant roles in cholesterol and lipid lowering (^{Yadav, Agarwala, 2011}; ^{Builders, Isichie, Aguiyi, 2012}).

Values are presented as mean ± Standard error of mean (n =5). *p<0.05: Statistically significantly different from control group.

Liver enzymes (AST, ALT, ALP), total protein and albumin concentrations are considered when assessing liver function. Therefore, alteration in the level of these biomarkers could be an indication of liver injury. When the liver is exposed to toxicants, damage to its membrane may occur, thereby causing leakages and elevation in the level of these enzymes in the blood circulation. On the other hand, whenever the liver is protected, its membrane remains intact thereby causing non-alteration or reduction in its enzyme levels (^{Otunola, Afolayan, 2017}).

From Table VI, significant increase in liver enzymes (ALT, AST and ALP) in 250 and 500 mg/kg groups on 91^st day suggests that the extract could cause hepatic injury when used for long duration. These biochemical changes were corroborated by the liver histopathology results (Figure 1) characterized by congestion of the hepatic triad leading to displacement of red blood cells from the intravascular space to the loose connective tissues in dose dependent manner. In recovery studies, significant increase in liver enzymes, as well as histopathological changes observed on 91^st day were not observed (Table VI and Figure 2), suggesting that the injuries attributed to long term exposure to the extract for 90 days was reversible.

Values are presented as mean ± Standard error of mean (n =5). *p<0.05: Statistically significantly different from control group.

Electrolytes (sodium, potassium, chloride and calcium), urea and creatinine are put into consideration when assessing kidney function. Increase in renal biomarkers as well as alteration in kidney cytoarchitecture is an indication of compromised renal functions (^{Ogbonnaya, Uadia, 2016}).

Non-significant change in electrolytes and total protein (Table VII) suggests that the physiological function of these biomarkers may not be hampered following the intake of the extract for 90 days.

Values are presented as mean ± Standard error of mean (n =5). p>0.05: Not statistically significantly different from control group.

However, significant increase in kidney function parameters, urea and creatinine (Table VIII) on the 91^st day suggests that the extract could be associated with kidney injury due to its longer duration of exposure. These changes were corroborated by the kidney histopathology result which is characterized by predominant glumeruli proliferation with loose Bowmans’ space in extensive areas of the kidney in dose dependent manner (Figure 3). Also, this suggests that doses below 250 mg/kg may be tolerated and safe even beyond 90 days exposure. Significant increase in kidney function parameters and kidney histopathological changes observed on the 91^st day were not observed in the recovery studies (Table VIII and Figure 4), suggesting that the injuries attributed to long term exposure of the extract for 90 days was not permanent.

Values are presented as mean ± Standard error of mean (n =5). *p<0.05: Statistically significantly different from control group.

Liver and kidney toxicities observed on 91^st day of this study could be attributed to the effects of some secondary metabolites present in the extract of Dryopteris filix-mas as revealed in the phytochemistry result (Table I). Studies have revealed that exposure to high levels of plant secondary metabolites such as tannins, saponins, glycosides and alkaloids could cause hepatorenal toxicity (^{Netala et al., 2014}; ^{Louis et al., 2014}; ^{Mariangela et al., 2016}). Report had also shown that long term consumption of medicinal plant rich in flavonoids could result to auto-oxidation of reactive oxygen species thereby causing liver and kidney toxicities (^{Namjoo et al., 2013}). Long term intake of cardiac glycosides and diterpenoid glycosides had been reported to cause renal proximal tubule necrosis as well as centrilobular hepatic necrosis in animals (^{Chikezie, Ibegbulem, Mbagwu, 2015}). Nearly all alkaloids from plants are believed to be responsible for nitrogen secretion (like urea and uric acid in animals) and could be toxic when ingested in large quantity (^{Olivoto et al., 2017}). ^{Builders, Isichie, Aguiyi (2012}) also revealed that presence of tannins in stem bark extract of Parkia biglobosa may be responsible for its liver and kidney damage. ^{Osano et al. (2016}) also revealed that saponins and tannins in methanol leaf extracts of Prosopis juliflora may be associated with its hepetorenal toxicity. From this study, presence of tannins, saponins, glycosides, flavonoids and alkaloids in Dryopteris filix-mas leaf extract may be responsible for the liver and kidney toxicities observed on 91^st day.

Non-significant difference (p>0.05) in body weight gain of animals treated with various doses of the extract for 90 days (Table IX) suggests that sub-chronic administration of the extract does not contribute to body weight reduction. ^{Ekpenyong, Akpan, Udoh (2012}) reported that high level of hydrolysable tannins (non-digestable form) from phytochemicals could cause appetite suppression and body weight reduction when compared to condensed tannins.

Values are presented as mean ± Standard error of mean (n =5). p>0.05: Not statistically significantly different from control group.

Non-significant changes in organs weights on 91^st day and in recovery studies (Tables X) indicate that the extract may not be associated with hypertrophy or hyperplasia. Studies by ^{Otunola, Afolayan (2017}) revealed that increase or decrease in organ weights may be associated with marked organ toxicity.

Values are presented as mean ± Standard error of mean (n = 5). p>0.05: Statistically significantly different from control group

Studies by ^{Moke, Ilodigwe, Erhirhie (2015}) revealed that evaluation of ulcerogenic properties of medicinal plants could aid in validating their safety profile relating to long term use. Absence of ulcerations in the stomach mucosa of rats that were exposed to the extract for 90 days (Table XI) suggest that the extract is not be ulcerogenic in the stomach mucosa on long term use.

CONCLUSIONS

Acute toxicity study revealed that ethanol leaf extract of Dryopteris filix-mas may be safe on single dose exposure. However, 90 days repeated exposure of animals to 250 and 500 mg/kg doses of this extract may cause reduction in leukocyte counts and hepatorenal injuries, which may be reversible.

Thus, caution should be exercised by consumers who use high doses of the extract over a long period of time.