2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of extracts from Aloiampelos striatula

Hexane, chloroform, acetone, methanolic and water extracts from leaves and stem-bark of Aloiampelos striatula were evaluated for their antioxidant activity by 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay. The hexane, chloroform, acetone, methanolic and water extracts from leaves of A. striatula showed scavenging activity ranging from 10.30±0.41 30.14±1.4, 30.49±0.85 45.95±0.22, 38.45±0.49 51.65±0.95, 34.83±1.53 64.98±0.45 and 54.37±1.24 66.74±0.89%, respectively, at various concentrations. The hexane, chloroform, acetone, methanolic and water extracts from stem -bark of A. striatula showed scavenging activity ranging from 12.53±3.07 29.81±2.43, 17.80±5.17 37.20±2.13, 6.15±0.24 62.79±3.82, 38.23±1.18 67.32±0.55 and 3.16±0.40 70.66±0.18%, respectively, at various concentrations. Additionally, the IC50 values of these extracts were also determined and were found to be in the range of <200 to >3000 μg/mL. The positive control, vitamin C, showed an IC50 value of <200 μg/mL. The therapeutic applications of A. striatula are often in traditional Basotho medicine in the Kingdom of Lesotho, which include treating skin problems, high blood pressure, sexually transmitted illnesses, common colds, blood cleansing, stomach ailments and dressing of wounds. Further studies on this plant are required to explore this plant for its commercial applications.


Introduction
Aloiampelos striatula belongs to the Asphodelaceae family of the genus Aloiampelos (Reynolds, 1950;Van Wyk and Smith, 1996;Seleteng-Kose et al., 2015). The vernacular names of this plant are mohalakane and seholobe (Seleteng-Kose et al., 2015). A. striatula is also known by other names such as Aloe striatula, hardy aloe and striped -stemmed aloe. A. striatula is distributed throughout the Kingdom of Lesotho and South Africa (Reynolds, 1950). The western region of the Kingdom of Lesotho and Eastern Cape and Free State provinces of South Africa are rich in A. striatula (Reynolds, 1950). A. striatula has a slender semi-woody stem, grows into a large shrub and reaches up to 2 -meter height. A. striatula produces reddish-orange or yellowish flowers (Smith and Van Wyk, 2008). A. striatula has dark green curved thin leaves, which are much alike aloe vera leaves. The therapeutic applications of A. striatula in the traditional Basotho medicine in the Kingdom of Lesotho (Seleteng-Kose et al., 2015) includes treating skin problems, high blood pressure, sexually transmitted illnesses, common colds, blood cleansing, stomach ailments and dressing of wounds (Van Wyk and Gericke, 2000;Moffett, 2010;Seleteng-Kose et al., 2015). Particularly, the leaves of A. striatula reported having most of the therapeutic potentials (Seleteng-Kose et al., 2015). The leaves are soaked in water and consumed by the Basotho for various ailments (Moteetee and Van Wyk, 2011;Moteetee et al., 2017). Our literature search showed that this plant has not been studied extensively both phytochemically and pharmacologically. To the best of our knowledge, the antioxidant activity of A. striatula has not been reported previously. Therefore, the aim of this study was to evaluate the antioxidant activity of hexane, chloroform acetone, methanolic and water extracts obtained from the leaves and stem-bark of A. striatula by DPPH radical scavenging assay. Additionally, the IC 50 values of these extracts were also determined. This is the first report of this kind from this species gathered from the Kingdom of Lesotho.

Plant materials
The leaves and stem-bark of A. striatula were collected at Mafikeng village, Roma, Maseru district, The Kingdom of Lesotho, Southern Africa. The plant material was identified by Dr Lerato Seleteng-Kose. Voucher specimen for leaves (AALS) and stem-bark (AASB) were kept in the Organic Chemistry Research Laboratory, Department of Chemistry and Chemical Technology, Faculty of Science and Technology, The National University of Lesotho, Roma campus, The Kingdom of Lesotho.

Preparation of plant extracts
Approximately, 10kg of leaves and 6kg of stem-bark of A. striatula were collected, allowed to air -dry at room temperature separately for six weeks and then ground into fine powder using a laboratory blender (Waring Blender, Blender 80119, Model HGB2WT93, 240V AC, 3.5 AMPs, Laboratory and Analytical Supplies). For the current study, a mass of 90. 65, 101.55, 203.11, 85.65 and 263.96 g of powdered leaves was taken separately in RB-flasks and was extracted as per the procedure given in the literature (Pillai et al., 2019;Matamane et al., 2020) with slight modification as detailed below. Approximately, 1 L of hexane, chloroform, acetone, methanol and water were added separately into each RB-flask. The mixtures were macerated for 2 weeks with occasional shaking. The resulting solutions were filtered off separately and the solvents were removed by simple distillation. A mass of 2.24, 10.63, 29.38 and 26.32 g of hexane, chloroform, acetone and methanolic extracts, respectively, were obtained after removal of solvents. However, in the case of water extract, the solvent was removed as much as possible by vacuo and the resulting mass was left to stand for two weeks in a fume hood. A mass of 55.47 g of high viscous water extract was obtained and was used for further analysis. Using similar extraction procedures, 1.10, 0.89, 0.75, 0.09 and 2.10 g of hexane, chloroform, acetone, methanolic and water extracts were obtained, respectively from 99.98, 101.34, 104.22, 100.00 and 98.99 g of powdered stem-bark.

DPPH radical scavenging assay and determination of IC 50 values
DPPH radical scavenging assay was carried out using a method as described in the literature (Kim et al., 2002;Dontha, 2016;Pillai et al., 2019;Matamane et al., 2020). Briefly, a stock solution of methanolic extract was prepared at a concentration of 3.0 mg of extract in 1 mL of 50% methanol (v/v). Serial dilutions were made from this stock solution to obtain solutions with concentrations of 3000, 2000, 1500, 1000, 800, 500 and 200 μg/mL. Solutions without extract concentration served as a negative control (Pillai et al., 2019). A solution of 3.94 mg of DPPH in 100 mL of methanol served as oxidant, which was prepared just before use and stored in dark to minimize degradation (Pillai et al., 2019). A volume of 0.1 mL sample of plant extract solution was mixed with 1.0 mL of 0.1 mM DPPH solution and 0.45 mL of 50 mM Tris -HCL buffer (pH = 7.40) (Pillai et al., 2019). Similarly, stock solutions of hexane, chloroform, acetone and water extracts were prepared at a concentration of 3.0 mg of extract in 1 mL of 50% methanol (v/v). Further dilutions were made from these stock solutions to obtain solutions with concentrations of 3000, 2000, 1500, 1000, 800, 500 and 200 μg/mL (Pillai et al., 2019). A volume of 0.1 mL each of this extract was mixed separately with 1.0 mL of 0.1 mM DPPH solution and 0.45 mL of 50 mM Tris -HCL buffer (pH = 7.40). A stock solution of vitamin C (0.3 g) in 50% methanol (v/v) was prepared and serial dilutions were made as previously, which served as a positive control. A volume of 0.1 mL of this solution was mixed with 1.0 mL of 0.1 mM DPPH solution and 0.45mL of 50 mM Tris -HCL buffer (pH = 7.40). The mixtures were incubated for 30 min and the absorbance (optical density) of the mixture was measured at 517 nm using an MRC spectrophotometer (Mode Spectro UV -11 S/N: UEB 1704020). The following equation was used to calculate the percentage of DPPH radical scavenging activity of extracts.
Where A test = Absorbance in the presence of extract or positive control and A cont = Absorbance of negative control (i.e. without extract).
The IC 50 value is defined as the concentration of extract that inhibits the formation of DPPH radical by 50% (Moyo et al., 2013;Ndhlala et al., 2013). A lower value of IC 50 represents higher antioxidant activity and vice versa. The IC 50 values were calculated using Microsoft Excel by plotting extract concentration versus percentage inhibition of DPPH radical. Each experiment was carried out in triplicate and the average of the three values was used to calculate IC 50 value for each extract. FULL PAPER Standard deviation was calculated for each concentration from the three values of the experiment.

Results and discussion
The water extract from leaves of A. striatula (E5) was the most potent among all ten extracts. The extracts E2, E3, E4 and E9 showed significant scavenging activity at all concentrations. The extracts E8 and E10 showed lower scavenging activity at lower concentrations but at higher concentrations, both they exhibited significant activities. As stated previously, at higher concentrations, E8 and E10 might have contained scavenging rich phytochemicals such as polyphenols, phenolics, flavonoids etc. in sufficient quantities. The extracts E1, E6 and E7 showed weak to moderate scavenging activity at all concentrations and showed relatively lower scavenging activity among all ten extracts. The leaves of A. striatula reported having many therapeutic applications (Van Wyk and Gericke, 2000;Moffett, 2010;Seleteng-Kose et al., 2015). However, the therapeutic applications of stem-bark and root of A. striatula have not been reported, previously. This study also confirmed that the extracts from the leaves of A. striatula showed higher scavenging activity than the extracts from stem-bark. Particularly, the water extract from leaves of A. striatula (E5) showed the highest scavenging activity among all ten extracts and identified as the potent extract.

Conclusion
In this study, hexane, chloroform, acetone, methanolic and water extracts from leaves and stem-bark of A. striatula were evaluated for their antioxidant activity by DPPH radical scavenging assay. Their radical scavenging activity at various concentrations was found to be in the range of 6.15±0.24 to 70.66±0.18%. Additionally, the IC 50 values of these extracts were also determined and were found to be in the range of <200 to >3000 µg/mL. The positive control, vitamin C, showed an IC 50 value of <200 µg/mL. The water extract from leaves of A. striatula showed similar IC 50 value as that of positive control. i.e. <200 µg/mL. This water extract from leaves of A. striatula finds therapeutic applications in the traditional Basotho medicine in the Kingdom of Lesotho. Our study also confirmed this fact that the water extract from leaves of A. striatula showed the highest scavenging potential and identified as the most potent among all ten extracts. This is the first report of this kind from this species collected from the Kingdom of Lesotho. Further studies on this plant will be useful to explore for any commercial applications.