The toxicity study of Aquilaria malaccensis (agarwood) leaves aqueous extract on male reproductive system in Sprague Dawley rats

Abstract

Aquilaria malaccensis (AM) or locally known as ‘gaharu’ (agarwood) is a species of Aquilaria genus and belongs to the Thymelaeaceae family. It is widely distributed in Malaysia, Indonesia, and the Borneo Islands. Traditionally, its leaves were used to relieve bruises and studies have shown that they function as an antioxidant, aphrodisiac, and tranquilizer. Despite its proven beneficial medicinal properties, information regarding its toxicity is limited. Therefore, this study was conducted to investigate the male reproductive toxicity of AM. Prior to the toxicity study, the quality and antioxidant property of standardized A. malaccensis leaves aqueous extract (AMLAE) was determined by a set of phytochemical screening, High-Performance Liquid Chromatography (HPLC) analysis, heavy metal, microbial contamination and in vitro antioxidant tests. The general toxicity of AMLAE was evaluated based on acute and sub-acute oral administration in Sprague Dawley (SD) rats according to the Organization for Economic Cooperation and Development (OECD) Guideline 420 and 407 respectively. The OECD Guideline 421 was selected to evaluate the male reproductive toxicity analysis which comprised of control group and three AMLAE-treated groups (100, 300 and 500mg/kg) respectively. In total of 63 days of oral administration was carried out prior to the one-to-one mating activity with female. Male necropsy was conducted upon sperm-positive vaginal smear to evaluate the male reproductive parameters. Pregnant female rats were necropsied on day 21 to evaluate the reproductive outcomes via caesarean hysterectomy. The extraction protocol successfully yielded 17.64% powder extract. Phytochemical analyses revealed the presence of saponins, phenolics, tannins, flavonoids and aromatic compounds. No microbial and heavy metal contamination was detected. HPLC analysis of the AMLAE revealed that it contained mangiferin (31.08mg/g) as one of its major constituents. AMLAE indicated strong cupric ion reducing power and potent scavenging activity with 740.83mmol Trolox equivalent/g and 1.24 ± 0.27μg/ml respectively. The assessment of acute toxicity revealed that AMLAE did not influence mortality, clinical behaviours, body weight gain, or necropsy findings at a dose of 2000mg/kg body weight. In the sub-acute toxicity, both male and female rats had shown abnormalities in the liver and kidney histology at the dose of 2000mg/kg. No significant findings were recorded in male reproductive parameters and reproductive outcomes on pregnant rats except significant elevations in the in vivo antioxidant activity, hormonal concentration, testicular histology, protamination level and protamine 1 gene expression. Data from present results revealed that AMLAE did not exhibit toxicity on male reproductive system and the no observed adverse effect level for male reproductive toxicity was ˃500mg/kg via oral route.