Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3627

Full Length Research Paper

DPPH radical scavenging activity of extracts from Rhamnus prinoides

Manoharan Karuppiah Pillai
  • Manoharan Karuppiah Pillai
  • Department of Chemistry and Chemical Technology, Faculty of Science and Technology, National University of Lesotho, Roma Campus, P. O. Roma 180, Kingdom of Lesotho, South Africa.
  • Google Scholar
Lehlohonolo Isaac Santi
  • Lehlohonolo Isaac Santi
  • Department of Chemistry and Chemical Technology, Faculty of Science and Technology, National University of Lesotho, Roma Campus, P. O. Roma 180, Kingdom of Lesotho, South Africa.
  • Google Scholar
Sibusisiwe Magama
  • Sibusisiwe Magama
  • Department of Biology, Faculty of Science and Technology, National University of Lesotho, Roma Campus, P. O. Roma 180, Kingdom of Lesotho, South Africa.
  • Google Scholar


  •  Received: 24 May 2019
  •  Accepted: 05 July 2019
  •  Published: 31 August 2019

 ABSTRACT

Hexane, chloroform, ethyl acetate and methanolic extracts from leaves and stem-bark of Rhamnus prinoides were evaluated for their antioxidant activity by DPPH radical scavenging assay. The leaves extracts showed scavenging activity ranging from 03.33±0.89 to 55.03±3.40 µg mL-1 while the stem-bark extracts showed relatively strong scavenging activity ranging from 03.65±1.02 to 59.55±2.27 µg mL-1. The IC50 values of R. prinoides hexane leaves extract (RPHELS), R. prinoides chloroform leaves extract (RPCHLS), R. prinoides ethyl acetate leaves extract (RPEALS), R. prinoides methanolic leaves extract (RPMELS), R. prinoides hexane stem-bark extract (RPHESB), R. prinoides chloroform stem-bark extract (RPCHSB), R. prinoides ethyl acetate stem-bark extract (RPEASB) and R. prinoides methanolic stem-bark extract (RPMESB) were found to be >3000, >3000, >3000, 950.42, ~1500, 710.50, ~1000 and 902.78 µg mL-1, respectively. The positive control ascorbic acid showed an IC50 value of <200 µg mL-1. From this study, we concluded that the extracts from R. prinoides showed promising antioxidant activity. R. prinoides finds therapeutic applications in the traditional medicine. Further research is required to commercialize products from this plant.

Key words: Antioxidant, ascorbic acid, Rhamnus prinoides, radical scavenging assay, methanolic extract, chloroform extract, hexane extract, ethyl acetate extract.

 


 INTRODUCTION

Rhamnus prinoides belongs to the Rhamnaceae family (Dale, 2000; Dlamini and Turner, 2002). R. prinoides is also known by other names such as African Dogwood, Glossy-leaf and mififi. R. prinoides is widely distributed in East and South African countries (Alemu et al., 2007; Abegaz et al., 1999) which include Ethiopia, Botswana, Eritrea,  Lesotho,   Namibia,   South    Africa,   Swaziland,  Uganda and Kenya (Ashine, 2015). R. prinoides grows up to 4.5-m height and found in evergreen forests, in the wild and along water streams. Although, R. prinoides is a slow growing plant in low rainfall areas, it can grow 1 m per annum in wet areas (Ferede et al., 2018). The leaves begin with pale green and turn to dark and shiny on maturation. The  roundish  red  berries  attract  bees  and domestic fowl. R. prinoides casts a very deep shade such that it will not allow other plants to grow around it. R. prinoides flowers towards the end of the year and fruiting occurs at the beginning of the year. R. prinoides finds therapeutic applications in the traditional medicine. The decoction of roots has been used to treat pulmonary tuberculosis, pneumonia, bladder and kidney problems (Maliehe, 1997; Van Wyk and Moteetee, 2011). The bark has been used to induce vomiting. An extract of the root together with the bark of Erythrina tomentosa has been used to relief colic. The leaves have been applied as a liniment to simple sprains. R. prinoides has been used to provide a special aroma and flavor (Shale and Gashe, 1991; Abegaz et al., 1999). R. prinoides has also been used in the beer industry as a hopping agent. Geshoidin, a naphathalenic glycoside, present in the stem-bark is responsible for providing bitterness in alcoholic beverages (Nindi et al., 1999). The antioxidant activity of 97% ethanolic extracts from leaves of R. prinoides has been reported previously (Ashine, 2015). Methanolic and aqueous extracts from roots of R. prinoides have also evaluated for their DPPH radical scavenging activity (Kimondo et al., 2019). However, to the best of our knowledge, the scavenging activity of hexane, chloroform, ethyl acetate and methanolic extracts of the leaves and stem-bark of R. prinoides has not been reported previously, particularly the plant species gathered from the Kingdom of Lesotho. The aim of the present study was to evaluate the antioxidant activity of these extracts by DPPH radical scavenging assay and to determine their IC50 values. The results obtained are communicated in this article.

 


 MATERIALS AND METHODS

Plant materials

The leaves and stem-bark of R. prinoides were collected from the foothills of Popa and Popanyane Mountains at Mokhokhong village, Roma, Maseru district, the Kingdom of Lesotho, Southern Africa in January 2019. A voucher specimen viz. Santi/RPLS/2019 for leaves and Santi/RPSB/2019 for stem-bark were kept separately in the Organic Research Laboratory, Department of Chemistry and Chemical Technology, Faculty of Science and Technology, National University of Lesotho, Roma Campus, Maseru district, Kingdom of Lesotho, Southern Africa.

Processing of materials

The leaves were allowed to air dry at room temperature for two weeks and then ground into powder using a commercial blender (Waring Blender, Blender 80119, Model HGB2WT93, 240V AC, 50-80 Hz, 3.6AMPs, Laboratory and Analytical Supplies). The chopped stem-barks were allowed to air dry at room temperature for two weeks and ground into powder using the blender.

Preparation of plant extracts

The powdered leaves (300.043 g)  of  R.  prinoides  were  extracted with methanol under cold conditions for 3 days. The solution was filtered using a filter paper (Boeco, Germany). The solvent was removed by vacuo and the same procedure was repeated once again. Finally, the plant material was extracted with hot methanol. 40.1858 g of combined methanol extract was obtained after removal of solvent. The same extraction procedure was followed to get hexane (3.2274 g), chloroform (10.6285 g) and ethyl acetate (11.4763 g) extracts from 300.254, 300.131 and 299.921 g of powdered leaves, respectively. The powdered stem-bark (299.530 g) of R. prinoides was extracted first with methanol at room temperature for 3 days followed by a reflux condition for 6 h. 32.2047 g of combined crude methanol extract was obtained after removal of solvent. The same extraction procedures were followed to get hexane (2.5895 g), chloroform (5.4327 g) and ethyl acetate (8.1493 g) extracts from 300.014, 300.157, and 300.422 g of powdered stem-bark, respectively.

Chemicals and solvents used

Ascorbic acid, DPPH, hexane (AR grade, 99.5%), chloroform (AR grade, 99.5%), ethyl acetate (AR grade, 99.5%) and methanol (AR grade, 99.5%) were all purchased from Sigma-Aldrich.

Antioxidant activity

The antioxidant activity of the extracts was carried out using 1,1-diphenyl-2-picrylhydrazyl (DPPH) as described in literature (Kim et al., 2002). Briefly, stock solution of the 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-1. Solutions without extract concentration served as negative control. 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. 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 (p H7.40). Similarly, stock solutions of hexane, chloroform and ethyl acetate 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, 200 and 0 µg mL-1. 0.1 mL each of extract was mixed separately with 1.0 mL of 0.1 mM DPPH solution and 0.45 mL of 50 mM Tris-HCL buffer (pH7.40). A stock solution of ascorbic acid (0.3 g) in 50% methanol (v/v) was prepared and serial dilutions were made as previously and served as positive control. 0.1 mL 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). The mixtures were incubated for 30 min and their optical density was measured at 517 nm. Percentage inhibition of DPPH free radical was calculated using the equation:

DPPH Scavenged (%) = [(Acont - Atest)/Acont] × 100

where Atest = Absorbance in the presence of extract or positive control and Acont = Absorbance of negative control.

The IC50 value is defined as the concentration (in µg mL-1) of extract that inhibits the formation of DPPH radical by 50% (Moyo et al., 2013; Ndhlala et al., 2013). A lower value of IC50 represents higher antioxidant activity. The IC50 values were calculated from graphs by plotting extract concentrations vs. percentage inhibition of DPPH radical using Microsoft Excel. Each experiment was carried out in triplicate and the averages of the three values were used to calculate IC50 values. Standard deviation was calculated for each concentration from the three values of the experiment.

Statistical analysis

Results were expressed as means of three determinations. One-way analysis of variance (ANOVA) was used to compare means at the significance level p < 0.05. All analysis was performed by Microsoft Excel software.

 


 RESULTS AND DISCUSSION

Table 1 summarizes the DPPH radical scavenging activity of hexane, chloroform, ethyl acetate and methanolic extracts of the leaves and stem-bark of R. prinoides. R. prinoides hexane leaves extract (RPHELS) showed 3.33±0.89, 3.80±2.60, 8.61±1.39, 10.12±0.84, 13.39±2.94, 20.10±3.23 and 34.56±6.51% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. The positive control, ascorbic acid, showed 53.01±3.98, 53.46±0.14, 53.51±0.77, 53.82±0.54, 54.12±1.64, 54.34±0.92 and 56.45±5.45% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. These results revealed that RPHELS showed very weak radical scavenging activity relative to positive control at all concentrations. R. prinoides chloroform leaves extracts (RPCHLS) showed 23.01±3.44. 27.15±5.18, 30.35±1.02, 31.38±0.11, 47.37±4.14, 48.38±4.15 and 48.49±3.17% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. This result showed that RPCHLS has lower activity than positive control at all concentrations. R. prinoides ethyl acetate leaves extract (RPEALS) showed 17.71±1.02, 17.96±0.82, 19.48±1.50, 28.51±0.62, 36.05±6.26, 38.94±5.75 and 42.33±5.88% of scavenging activity at 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. This result revealed that RPEALS exhibited weak activity at low concentrations relative to positive control. However, at high concentration of 3000 µg/mL, it showed higher scavenging activity of 42.33±5.88%. R. prinoides methanolic leaves extract (RPMELS) showed 21.01±3.80, 33.74±1.42, 41.50±2.73, 50.93±5.27, 52.93±2.88, 54.34±2.70 and 55.03±3.40% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. This result revealed that RPMELS exhibited weak activity at low concentrations relative to positive control. However, RPMELS has comparable activity as that of positive control at high concentrations.

 

 

R. prinoides hexane stem-bark extract (RPHESB) showed 12.39±3.19, 18.93±1.04, 30.47±0.82, 42.15±4.06, 50.24±1.50, 51.52±2.47 and 52.15±1.06% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. This result revealed that RPHESB exhibited weak activity at low concentrations relative to positive control. However, at higher concentrations such as 1500, 2000 and 3000 µg/mL, RPHESB exhibited comparable activity as that of positive control. R. prinoides chloroform stem-bark extract (RPCHSB) showed 42.37±5.65, 43.30±2.98, 52.23±2.46, 53.64±4.42, 54.06±1.41, 54.41±2.17 and 55.22±2.48% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. Thus, RPCHSB exhibited comparable activity as that of positive control at all concentrations except at concentrations of 200 and 500 µg mL-1. RPCHSB showed 42.37±5.65 and 43.30±2.98% of scavenging at concentrations 200 and 500 µg/mL, respectively, while the positive control showed 53.01±3.98 and 53.46±0.14% of scavenging activity at concentrations 200 and 500 µg mL-1, respectively. R. prinoides ethyl acetate stem-bark extract (RPEASB) showed 3.65±1.02, 40.04±1.50, 47.09±4.36, 50.31±7.80, 51.61±2.27, 54.23±1.83 and 57.43±3.28% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg/mL, respectively. Thus, RPEASB exhibited very weak activity relative to positive control at low concentration of 200 µg mL-1. R. prinoides methanolic stem-bark extract (RPMESB) showed 30.09±5.26, 41.69±2.27, 45.80±2.54, 52.78±6.43, 55.37±3.90, 56.19±3.58 and 59.55±2.27% of scavenging activity at concentrations 200, 500, 800, 1000, 1500, 2000 and 3000 µg mL-1, respectively. This result revealed that RPMESB exhibited weak activity at low concentrations relative to positive control. However, at high concentrations such as 2000 and 3000 µg/mL, it showed higher scavenging activity of 56.19±3.58 and 59.55±2.27%, respectively. Among the extracts (RPHELS, RPCHLS, RPEALS, RPMELS, RPHESB, RPCHSB, RPEASB and RPMESB) from R. prinoides, RPMESB showed the highest scavenging activity (Table 1). For comparison and clarity, the percentage of scavenging activity of these extracts at various concentrationsare is as shown in Figures 1 and 2. The IC50 values of hexane, chloroform, ethyl acetate and methanolic extracts of the leaves and stem-bark of R. prinoides are shown in Table 2. RPHELS, RPCHLS, RPEALS, RPMELS, RPHESB, RPCHSB, RPEASB and RPMESB exhibited IC50 values of >3000, >3000, >3000, 950.42, ~1500, 710.50~1000 and 902.78 µg mL-1, respectively. RPCHSB is the most potent with IC50 value of 710.50 µg mL-1. The positive control ascorbic acid showed an IC50 value of <200 µg mL-1.

 

 

 

 

The DPPH radical scavenging activity of 97% ethanolic extract from leaves of R. prinoides has previously been reported and its maximum radical scavenging was found to be 81.148% at a concentration of 24 mg mL-1 (Ashine, 2015) and the IC50 value was determined to be 5.2 mg mL-1. The positive control, ascorbic acid, showed 93.77% scavenging activity with an IC50 value of 0.24 mg mL-1 in the same assay (Ashine, 2015). Therefore, when compared with the present study on the hexane, chloroform, ethyl acetate and methanolic extracts from leaves of R. prinoides, this 97% ethanolic extract from leaves of R. prinoides showed higher radical scavenging activity and lower IC50 value. This 97% ethanol might have more  extractive  power of  active  constituents  than the solvents used in the present study. Additionally, the collection of plant materials at different geographic locations  will  also  play  a  vital  role  in  determining  the active constituents of extracts. Methanolic and aqueous roots extracts from R. prinoides have also been screened for   their  DPPH  radical  scavenging  activity.  Their  IC50  values were found to be 377.27 and ~250 µg mL-1, respectively (Kimondo et al., 2019). The positive control, ascorbic acid, showed an IC50 value 50.32 µg mL-1 in the same assay (Kimondo et al., 2019). Additionally, the kinetics of acetylcholinesterase (AChE) inhibitory activity of aqueous extract from R. prinoides has previously been reported (Catherine and Edward, 2009). The IC50 value for R. prinoides was found to be 0.201 mg mL-1. The AChE inhibitory activity of R. prinoides was found to be higher than that of some Portugese and Danish medicinal plants (Ferreira et al., 2006; Adersen et al., 2006). Biologically important secondary metabolites such as emodin,  physcion,  prinoidin,  rhamnazin,  geshodin  and many other emodin-derived compounds have been reported from R. prinoides (Van Staden and Drewes, 1994; Abegaz and Kebete, 1995). Alkaloids, flavonoids, terpenoids, anthraquinones, saponins, polyphenols, etc., classes of compounds have also been reported from various extracts of R. prinoides (Molla et al., 2016).

 

 

 

 

 


 CONCLUSION

DPPH radical scavenging activity of hexane, chloroform, ethyl acetate and methanolic extracts from leaves and stem-bark  of  R.  prinoides collected from the Kingdom of Lesotho have been evaluated. The leaves extracts showed scavenging activity ranging from 03.33±0.89 to 55.03±3.40 µg mL-1 while the stem-bark extracts showed relatively strong scavenging activity ranging from 03.65±1.02 to 59.55±2.27 µg mL-1. The IC50 values of these extracts were also determined and were found to be in the range of 710.50 to >3000 µg mL-1. R. prinoides finds therapeutic applications in the traditional medicine and showed promising antioxidant activity. Therefore, further studies will be useful to commercialize products from this plant. 

 


 CONFLICT OF INTERESTS

The authors declared no conflict of interests.

 


 ACKNOWLEDGEMENTS

The authors acknowledge the National University of Lesotho for its overall support.

 



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