Biological and toxicological evaluation of aerial parts extracts of locally grown Cleome austroarabica

2.1 Chemicals and materials

The standards and chemicals such as chloroform, dimethyl sulphoxide (DMSO, purity 99%) and methanol were used in this study obtained from Fisher Chemical Company, UK. Hexane was collected from Daejung, Korea. Ethyl acetate was collected from Carbon Group, Ireland. Filter papers were purchased from Whatman, UK. The control ciprofloxacin antibiotic, silica gel and TLC plate were obtained from E. Merck, Germany. Brine shrimp eggs was purchased from the USA. Sea water was collected from Wave beach, Muscat, Sultanate of Oman.

2.2 Microorganism

The clinically isolated selected microorganism such as Escherichia coli (E. coli), Haemophilus influenza (H. influenza), Enterococcus faecalis (E. faecalis), and Staphylococcus aureus (S. aureus) were obtained from one of local hospitals in Nizwa, Sultanate of Oman.

2.3 Sample collection

The aerial parts of C. austroarabica were collected from the initial campus of the University of Nizwa, Birkat Al Mouz, Nizwa, Oman, in November 2016. The morphological authentication was done using the microscopic method by Dr. Syed Abdullah Gilani, Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa. The collected aerial parts were cleaned and dried at ambient temperature.

2.4 Extraction

The dried plant was ground into coarse powder by using blender machines and the yield was around 414.1 gm. The powdered samples (400 gm) were macerated in about 1 L of methanol solvent for one week (Hossain et al., 2013). The extracts were filtered by using a Buchner funnel and concentrated at 40 °C using a rotary evaporator to give yield 37.35 gm. Then the residue (35 gm) was suspended in ethanol and water mixture (1:1) and finally fractioned with hexane, chloroform, and ethyl acetate consecutively to give the corresponding extracts. The mother solvent was evaporated completely and the weight of each residue was recorded.

2.5 Antibacterial activity

The antibacterial activity of each polarity aerial extract of C. austroarabica was evaluated by using the disc diffusion method (Abdullah et al., 2016; Matani et al., 2015). In this experiment, four concentrations of each polarity extract (2000, 1000, 500, 250 µg/ml) were prepared by the dilution method using dimethyl sulphoxide (DMSO). Filter paper discs of 5 mm diameter size were dipped in each concentration. The discs were put on agar plates that were inoculated with the clinically isolated Gram (+) positive and Gram (−) bacteria. The solvent DMSO as a negative control and ciprofloxacin (250 µg/ml) as a positive control were used. After inoculated, the plates were kept in the incubator for 24 h at 37 °C. An antibacterial activity of each concentration of each polarity extract was evaluated manually by measuring the diameter of the inhibited zone.

2.6 Cytotoxic activity

The toxicity of each prepared aerial extract of C. austroarabica was done by using shrimp assay, which is usually called as sea monkeys. Shrimp invertebrate organisms about 1 mm in size with brownish colour was used for the calculation of cytotoxic activity (Lieberman, 1999).

2.7 Fractionation of lowest LC₅₀ by column chromatography

The chloroform aerial extract gave lowest LC₅₀ value_. The chloroform extract (5 gm) was subjected to column chromatography over silica gel (25 × 75 cm, 100 gm) by using n-hexane, n-hexane: chloroform, chloroform, chloroform: ethyl acetate, ethyl acetate, ethyl acetate: methanol mixtures as a mobile phase (Sohail et al., 2017). All collected fractions were on TLC and those with similar pattern were combined and screened for their cytotoxic activity.

3.1 Yield of extracts

The aerial course powdered of C. austroarabica were extracted directly with methanol by maceration method. The residue was reextracted by different solvents with increasing polarity. The approximate yields and their percentage of yield of each individual extract are given in Table 1. The highest percentage of the yield of methanol extract was about 10.6% obtained from 414.1 gm of aerial parts of coarse powder of C. austroarabica (Table 1). After fractionation, the amount and yield of hexane and chloroform was higher than the ethyl acetate and hydro alcoholic extracts (Table 1). Therefore, the aerial parts of C. austroarabica contained relatively higher amounts of nonpolar ingredients compared to the polar fractions.

3.2 Antimicrobial activity

The activity of methanol extract and its derived different polarities aerial fractions of C. austroarabica were determined against clinically isolated Gram (+and −) bacterial strains (Najwa and Hossain, 2018). In our current experiment, two Gram (+) bacteria: Staphylococcus aurous (S. aurous), Enterococcus faecalis (E. aurous) and two Gram (−) bacteria: Haemophilus Influenza (H. Influenza), and Escherichia coli (E. coli) were used against various polarities aerial extracts at different concentrations. Unfortunately, the experimental results showed that there was no growth inhibition obtained against the tested Gram (+and −) bacteria at any concentrations (Table 2). However, the ethanol extract of leaves and flowers of other species of C. viscosa belongs to this family showed activity against E. coli, P. vulgaris and P. aeruginosa (Abreu et al., 2013). Another study conducted by Muhaidat et al on the essential oils of C. droserifolia and C. trinervia species belong to the same family and showed that it gave a significant inhibition against different pathogen bacterial strains (Muhaidat et al., 2015). But, the selected plant species which were grown in Oman did not give any activity, it could be due to the sensitivity of applied bacterial strains or the dose of the extract samples (Najwa and Hossain, 2018). There is no reports and data available on antimicrobial studies of the selected species in Oman. Therefore, we are unable to compare our results with the reported values.

3.3 Cytotoxic activity

The cytotoxicity of the prepared aerial extracts of C. austroarabica was evaluated by brine shrimp lethality (BSL) assays reported by several authors (Matani et al., 2015; Rehab and Hossain, 2016). All the prepared aerial extracts at different concentrations have shown activity against the brine shrimp larvae. Among the five prepared different polarities aerial extracts, the hexane, chloroform, ethyl acetate and hydro alcoholic extracts have displayed significant activity against the brine shrimp larvae. The mortality percentages of the shrimp larvae exposed to different aerial extracts of C. austroarabica are shown in Table 3. The order of activity was chloroform > methanol > ethyl acetate > hexane > hydro alcoholic extract. Moreover, there is an increase in the mean percentage of mortality with increase in concentration of aerial extract. Both the hexane and hydro alcoholic extracts gave the highest LC₅₀ value 640.26 µg/ml (Table 3). According to the definition of LC₅₀, the high LC₅₀ value means it has less toxicity. That means, both the hexane and hydro alcoholic extracts contained less toxic compounds (high LC₅₀ value means less toxic). The minimum LC₅₀ value was obtained from the chloroform extracts compared to positive control KMNO₄ (Fig. 2). In our present experiment, the chloroform extract showed the lowest LC₅₀ value. Therefore, the chloroform extract is the most potent toxic aerial extract among the five prepared extracts from the C. austroarabica.

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Fig. 2

Comparison of LC₅₀ values of methanol (MeOH), hexane, chloroform (CHCl₃), ethyl acetate (EtOAc), hydro alcoholic (Hydro) and potassium permanganate (KMNO₄).

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According to the LC₅₀ value, chloroform extract was selected for the separation of toxic ingredients. The column chromatography method was used to separate the ingredients from the chloroform extract. After fractionation of the chloroform extract by column chromatography, seven fractions (Fraction 1 (0.45gm); Fraction 2 (0.15gm), Fraction 3 (0.14gm), Fraction 4 (0.47gm), Fraction 5 (0.42gm), Fraction 6 (0.48gm) and Fraction 7 (0.2 gm) were obtained by using the mobile phase e.g., n-hexane, n-hexane: chloroform, chloroform, chloroform: ethyl acetate, ethyl acetate, and ethyl acetate: methanol mixtures. All the isolated fractions from chloroform aerial extract were tested for toxicity in the same way mentioned by using brine shrimp napulii. All chloroform fractions gave cytotoxic activity at very high concentrations as compared to the original chloroform extract (Fig. 3). Some cytotoxic activity was done by the authors of the other species such as C. droserifolia, C. viscosa and C. burmanni belongs to the selected family. They reported that the C. droserifolia plant species showed significant activity against breast cancer cell (MGF7) and colon adenocarcinoma (HCT116) (Ezzat and Adel Motaal, 2012). Another study conducted on cytotoxic activity on the C. viscosa and C. burmanni species by Pillali and Nair. They mentioned that methanol extracts of both the plant species exhibited significant toxicity against the shrimp nauplii when compared to standard potassium permanganate (positive control) and thus is possibly a good indicator of toxicity (Pillai and Nair, 2011). There are no reports and data available on the selected species. Therefore, we are unable to compare our results with the reported data due to the lack of literature on the toxicological activity of C. austroarabica.

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Fig. 3

LC₅₀ values of different fractions of chloroform extract.

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