Evaluating the oestrogenic activities of aqueous root extract of Asparagus africanus Lam in female Sprague-Dawley rats and its phytochemical screening using Gas Chromatography-Mass Spectrometry (GC/MS)

Chemicals and drugs

Meloxicam was obtained from Bal Pharma, India. Fulvestrant (ICI 182, 870) and 17α-ethinyl estradiol were obtained from Sigma-Aldrich, Inc. St Louis. RNase plus mini, and RNase-Free DNase kits were purchased from Qiagen, Germany. All chemicals are of analytical grade.

Plant material collection and extraction

The sample of the A. Africanus plant was collected in Damaturu metropolitan, Yobe State, Nigeria. The plant was authenticated by plant Taxonomist (Dr. Kien-Thai, Yong) and the sampled plant was assigned a reference number (KLU 48696) and deposited in the Herbarium, University of Malaya. The root plant part was processed into a fine powder under laboratory conditions, and 50 g of fine powder was extracted in 500 mL of distilled water for 24 hr (Dunn, Turnbull & Sharon, 2004). The extract was filtered using Whatman filter paper No.4. and the filtrate was kept in the freezer at −20 °C before being evaporated to dryness in a vacuum using freeze dryer (Eyela FDU-1200; Tokyo, Japan) maintained at −50 °C for three days. The obtained yield of crude extract (7.0%) was kept in a fridge at 4 °C for further analyses.

Evaluation of the oestrogenic activity of A. africanus

All experimental procedures were carried out according to the approval of the ethics committee for animal experimentation, Faculty of Medicine, University of Malaya, Malaysia (Ethics Reference no.:2015-180505/PHAR/AEI). The female Sprague-Dawley rats were obtained from the animal experimentation unit, Faculty of Medicine, University of Malaya. The animals were housed in polycarbonate cages in a controlled environment (temperature, 23 °C ± 2 °C; relative humidity, 50 ± 10%; frequent ventilation; and an illumination schedule of 12-h light/12-h dark). The experimental animals were allowed free access to phytoestrogen-free diet (Altromin 1324 FORTI) and tap water. Thirty immature bilaterally ovariectomised female Sprague-Dawley rats with body weights ranging from 160–180 g were used for this study. The rats were randomly divided into five groups comprising six each (n = 6). Three groups of animals were administered a subcutaneous (SC) injection of A. africanus extract dissolved in distilled water (DH₂O) at three different doses (50, 200 and 800 mg/kgBW) twice daily for a total doses of 100, 400, and 1,600 mg/kg per day for three consecutive days from day 14 to day 16 post ovariectomy (Bo-Mi et al., 2009). Corn oil (5 ml/kg BW) and 17α-Ethinyl estradiol (EE: 1 mg/kg BW) were administered SC and served as negative and positive control, respectively. The animals were monitored for any clinical signs and abnormal behaviours daily throughout the experimental period (Jain et al., 2016).

In the second batch of the experiment, another 30 rats (n = 6) were pre-treated with SC injection of 1 mg/kg BW fulvestrant (ICI 182, 870), which is an oestrogen receptor antagonist (ORA), 30 min before each treatments. All animals were observed for vaginal cornification and early vaginal opening (Marcondes, Bianchi & Tanno, 2002; Srivastava et al., 2007) and were euthanised 24 h after the last dose of treatment using an intraperitoneal overdose of 150 mg/kg ketamine and 15 mg/kg xylazine. The body weights of the animals were recorded, and the uteri tissues were harvested for gene expression analyses (Bo-Mi et al., 2009; Deepak, Rema & Roy, 2015). The blood was collected, and the separated serum was used for hormonal analysis (Yoshinaka et al., 2009).

Hormonal assay

ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA) were used to measure oestrogen, progesterone and luteinising hormone (LH) levels. Briefly, the plate was washed manually with 350 µl washing buffer and soaked for two minutes, followed by aspiration of the content from the plate. The process was repeated three times. 50 µl of standard or sample was added to each well and 50 µl of antibody was added immediately to each well and incubated for 45 min at 37 °C. The plate was washed three times and 100 µl HRP-Streptavidin Conjugate (SABC) working solution was added to each well and further incubated for 30 min at 37 °C. Aspiration was continued, and washing procedures were repeated five times. Then, 90 µl of tetramethylbenzidine (TMB) substrate solution was added and the content incubated for 15–20 min at 37 °C. Finally, 50 µl of stop solution was added, and the absorbance was measured at 450 nm immediately. The result was extrapolated from the plotted standard curve (US Food and Drug Administration, FDA, 2016).

Gene expression assay

Quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to identify the expression levels of oestrogen receptor alpha (ERα) and calbindin-D9K (Calb3) genes in treated and untreated rats. Total RNA was extracted from uterus tissues using RNase plus mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.

The concentration and purity of extracted RNA samples were determined by NanoDrop spectrophotometer 2000c (Thermo Fisher Scientific, Waltham, MA, USA), while the quality of RNA was further determined by Bioanalyzer Agilent RNA 6000 Nano Kit (Agilent, USA). One microgram of total RNA was reverse transcribed to cDNA using high capacity RNA-to cDNA Master Mix supplied by Applied Biosystems (Foster City, CA, USA). The obtained cDNA was stored at −20 °C until further used. For RT-PCR, ERα (Rn01640372_m1) and calbindin (Rn00560940_m1) assay genes were supplied from TaqMan (Applied Biosystems). A volume of 1 µl complementary cDNA was used for qPCR which was performed using standard conditions. The amplification and quantification reactions were performed using the StepOne Plus Real-Time PCR System (Applied Biosystems, USA) according to TaqMan gene expression assay protocol. GAPDH (Rn0177563_g1) and HPRT-1 (Rn01527840_m1) were used as the endogenous reference genes. Cycling kinetics was performed in 40 cycles, to ensure linearity of PCR product (Thuy & Eui-Bae, 2009). The real-time PCR reaction was performed in triplicate, and the averages of the obtained threshold cycle (C_t) values were processed for further calculations according to the comparative C_t method.

Gene expression values were calculated according to the 2^−ΔΔCtmethod (Livak & Schmittgen, 2001). The ΔC_t value of each sample was determined by subtracting the average C_t value of the endogenous reference genes from the average C_t value of the target gene. The ΔΔC_t value was then calculated by subtracting the ΔC_t value of the treated sample from the ΔC_t value of the untreated control.

Finally, the gene expression levels were calculated as 2^−ΔΔCt giving the final value that was normalised to the reference genes and relative to the control sample values of the studied genes. GenEx Enterprise software (MultiD Analyses, Göteborg, Sweden) for quantitative real-time PCR (qRT-PCR) expression profiling, was used to analyse and normalise the qRT-PCR data (Kubista & Sindelka, 2007).

Phytochemical screening using GC/MS

A total of 5 mg of aqueous root extracts was re-dissolved in 5 mL of deionised water and filtered using polytetrafluoroethylene (PTFE) 0.2 µm pore size. With the aid of a vial, 1.5 mL filtrate was placed in the autosampler. Phytochemicals were screened using GC/MS -QP2010 ULTRA (Shimadzu Corp., Kyoto, Japan) with electron impact ionisation. The column used was RTX (fused silica) column with the dimension of length 30.0 m_x thickness 0.25 mm, and diameter 0.25 µm (Srivastava, Mukerjee & Verma, 2015).

The GC/MS analysis was carried out under the following conditions; sample injection was performed using a 10 µL syringe with an injection volume of 1 µL. Helium gas was used as a carrier gas with a flow rate of 4.8 mL/min with a split ratio of 1:10. The temperature setting for the column was 40 °C, which was later increased at 15 min of the start-up by 10 °C per minute to 300 °C. This temperature was maintained for 30 min. The total run time/post run time was 48 min. The injector was set at room temperature, and detector voltage was 0.86 kV. The mass spectrometer scan ranges were from 28–500 amu at 1 spectra/sec, with ionising voltage and ionisation current of 70 eV (Yuet et al., 2013; Srinivasa, Ammani & Rose, 2015).

Statistical analysis

The results were expressed as a mean ± standard error of the mean (SEM). One way analysis of variance (ANOVA) was employed for data comparison. GenEx statistical analysis was used for the gene expression, and analysis was performed using ANOVA followed by Tukey’s post hoc test. A p-value <0.05 was considered indicative of a statistically significant difference.

Evaluation of the oestrogenic activity of A. africanus

The ethinyl estradiol (EE) treated group showed a significantly reduction in weight gain as compared to control for both with ORA (p < 0.005) and without ORA (p < 0.005). Figure 1 shows that the weight gains of groups treated with AEAA without ORA were 25.8, 40.5 and 57.6 g for the doses 50, 200 and 800 mg/kg, respectively. The differences were not significant as compared to control group (57.0 g). On the other hand, all AEAA treatment groups pre-treated with ORA showed significant (p < 0.005) reduction in weight gain versus control (Fig. 1). The weight gain reduction in the groups pre-treated with the ORA was in a dose-dependent manner, which could be due to a positive synergy between AEAA and ORA.

The concentration of oestrogen (Fig. 2) also depicted a dose-dependent increased pattern with a significant increase in the treated animals without antagonist as compared to control. The mean oestrogen levels of rats treated with AEAA in the absence of ORA were 94.0 (p < 0.05), 106.3 (p < 0.005) and 106.8 (p < 0.005) pmol/L for the doses 50, 200 and 800 mg/kg, respectively, which were significantly higher than control group (57.5 pmol/L). Administration of ORA prior to the treatment reduced the levels of oestrogen as compared to the same group without the antagonist. However, the level of oestrogen in all treated groups was significantly (p < 0.005) higher as compared to the control rats pre-treated with the antagonist.

The mean progesterone levels of rats treated with AEAA in the absence of ORA were 5.05, 5.53 and 3.68 nmol/L for the doses 50, 200 and 800 mg/kg, respectively, which were significantly (p < 0.005) lower than control group (13.75 nmol/L). However, with the administration of ORA, the concentration of progesterone increased significantly (p < 0.005) in 800 mg/kg A. africanus treated rats (Fig. 3). Figure 4 shows the result of luteinising hormone (LH) for treated and untreated SD rats and the results were statistically not significant between groups.

Gene expression

Figures 5 and 6 show the genes expression levels of rats treated without and with ORA, respectively. Treated rats with 200 mg/kgBW A. africanus showed significant (p < 0.005) upregulation of ER α with 7.47 folds as compared to calibrator (Fig. 5). While for the treated animals with ORA, the Calb3 of ethynyl estradiol-treated group was significantly (p < 0.005) overexpressed with 7.64 folds as compared to the calibrator (Fig. 6).

GC/MS chromatogram analysis

GC/MS chromatogram of the aqueous root extract of A. africanus (Fig. 7) shows nine identifiable peaks indicating the presence of nine phytochemical compounds. Chromatograms and spectrums obtained were identified using National Institute of Standards and Technology Library (NIST). Mass Spectrum Search Interpreter version 11 database was used for possible chemical identification by comparing the spectrum and the mass to charge ratio (m/z) of the identified peaks. The details of the compounds are presented in Table 1. The findings revealed two phytoconstituents with steroidal nucleus namely stigmasterol and sarsasapogenin that are structurally related to hormone backbones. Furthermore, other non-steroidal compounds such as; 2(3H)-furanose, dihydro-3-hydroxy-4,4-dimethyl; pyrazinetetramethyl; 1,2-benzenedicarboxylic acid; 7,9-Die-tertt-butyl-1-oxospiro (4,5) deca-6,9-diene-2,8-dione; n-Hexadecanoic acid; butyl citrate; and 3-Dehydro-des-N-26-methyl-dihydro-pseudotomatidine were also detected. The mass spectrum of the identified phytochemicals of aqueous root extract of A. africanus are attached as supplementary materials and labelled as Figs. S1–S9.