Hispanamide: An Antioxidant Phenolic Propanamide from the Leaves of Acalypha Hispida (Burn.F)

Acalypha hispida (family Euphorbiaceae) is widely known for its medicinal applications in ethno-medicine. The most active antioxidant fraction of the leaf methanol extract was investigated with the aim of isolating and elucidating the active constituents. Bioassay guided isolation and purification was achieved using standard chromatographic techniques and invitro evaluation of ability of fractions, isolates and standards to scavenge 2, 2-Diphenyl-1-picrylhydrazyl radical (DPPH). The concentration of the pure isolate and standards required to scavenge 50% of the hydroxyl radical (IC 50 ) was carried out to assess their antioxidant effectiveness. The lower the IC 50 value, the greater the antioxidant effectiveness of the compound. Various spectroscopic techniques were used in the structural elucidation. The active isolate was identified as 4-hydroxy-N-[1-(hydroxymethyl)-2-phenylethyl-benzenepropanamide and named Hispanamide. Hispanamide exhibited a higher antioxidant effectiveness (IC 50 of 0.339 mg/ml) than the antioxidant standards Ascorbic acid (IC 50 of 0.373 mg/ml) and α-Tocopherol (IC 50 of 0.482 mg/ml) used for the assay. The order of decreasing antioxidant effectiveness is hispanamide > Ascorbic acid > α-Tocopherol. This is the first report of the isolation of this phenyl propanamide from plant source and its occurrence in the plant validates the ethnomedicinal uses of Acalypha hispida .


Introduction
The role of free radicals in many diseased conditions has been established. Various biochemical reactions in our body generate reactive oxygen species which are capable of damaging important bio-molecules and if not effectively scavenged by cellular constituents, constitute serious health hazards to man since they lead to degenerate diseases such as cancer, premature aging, diabetes, tumors, cirrhosis, cardiovascular, nervous, rheumatic and pulmonary disorders, cardiovascular diseases, cataract and macular degeneration (Halliwell & Gutteridge, 1990;Halliwell et al, 1992). Continuous intake of antioxidants is said to be necessary and important in reducing cellular damage and ageing process (Potterat, 1997).
Different parts of the plant is used in ethno-medicine for the treatment of kidney ailments, leprosy, gonorrhea, asthma, as a laxative, expectorant and diuretic (Iwu et al,1999;Kafaru, 2000;Sofowora, 2008). Due to the detection of many bioactive compounds in plants with possible antioxidant activity, there has been increased interest in the relationship between antioxidants and disease risks (Nilsson, 2006). There is therefore need for discovery of more plants and compounds with antioxidant activities.
The presence of phenolics, flavonoids, glycosides, steroids, saponins, phlobatannins, and hydroxyanthraquinones had been detected in a previous phytochemical screening of the aqueous and leaf methanolic extracts of A. hispida (Iniaghe et al, 2010;Okorondu et al, 2009). Antifungal properties of extracts of leaves of A. hispida have also been reported (Ejechi and Soucey, 1999). In addition, alcoholic extracts of A. hispida have been reported to be biologically active against P. aeruginosa, E. coli as well as S. aureus and S. typhi (Okorondu et al, 2009). Gallic acid, corilagin, cycloartane-type triterpenoids, flavonoids like quercetin and kaempferol derivatives have been isolated from the plant (Adesina et al, 2000;Bergitte et al, 2003;Gutierrez-Lugo et al, 2002). We had earlier reported the toxicity and antioxidant activity of fractions obtained from chromatographic separation of leaf methanolic extract of A. hispida (Onocha et al, 2011).
This present research report is a bioassay guided isolation, purification and characterization of the pure isolate from the most active antioxidant fraction obtained from the previous work (Onocha et al, 2011). In continuation of our research work on bioactive components and derivatives from Nigerian medicinal Euphorbiaceae plants and search for source of new antioxidants and therapeutic drugs from plant source (Onocha et al, 2004;Oloyede et al, 2010a,b ;Onocha and Ali, 2010;Onocha and Olusanya, 2010;2011;Oloyede et al., 2011a,b), we now report for the first time from plant source, the isolation of Hispanamide: an Antioxidant Phenolic Propanamide from the leaves of Acalypha hispida. It had earlier been synthesized from Phenyl Alaninol, 4hydroxy hydrocinnamic acid and triethylamine as one of the N-substituted benzenepropanamides invented, synthesizesd and patented by Kaplan and Tiqwa (2014) as nonsteroidal drugs for use in the treatment of pain and inflammation.

Fractionation Procedure
In the previous study (Onocha et al, 2011), of the 236 fractions collected (with 14 pooled fractions B1 -B14), fraction B9 (139-150 -eluted with 30% MeOH in EtOAc) interestingly, was found to exhibit a higher significant activity (99.7 %) at the concentration of 0.1 mg/ml than all the fractions and standards used in scavenging hydroxyl radical. It was also found to be the most active of all the fractions at scavenging free radicals at concentrations ranging from 0.0625 mg/ml to 1.0 mg/ml with the following percentage inhibition: 91.6, 92.9, 93.8, 94.1 and 94.9 (at 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively). At the same concentration ranges, it exhibited higher activity than the standards Ascorbic Acid (with percentage inhibition: 44.3, 65.4, 67.8, 68.7 and 90.9) and α -Tocopherol (with percentage inhibition: 10.4, 12.1, 12.4, 12.4 and 15.4) while it was comparable in activity to that of the standard butylated hydroxylanisole (with percentage inhibition: 91.9, 93.9, 94.0, 94.3 and 95.4) used for the assay.
2.4 Scavenging Effect on 2, 2-diphenyl-1-picryhydrazyl (DPPH) The ability of the extracts to scavenge DPPH radical was determined according to Oloyede et al., 2010b;Onocha et al., 2011. 2, 2-diphenyl-1-picryhydrazyl radical (3.94 mg), a stable radical was dissolved in methanol (100ml) to give a 100 µm solution. To 3.0 ml of the methanolic solutions of DPPH was added 0.5 ml of each of the bulked fractions with doses ranging from 0.0625 mg/ml to 1.0mg/ml. The decrease in absorption at 517 nm of DPPH using UV-Visible spectrophotometer was measured 10 minutes later. The actual decrease in absorption was measured against that of the control (Table 1) and the percentage inhibition was also calculated using the formula: I (%) = Acontrol -Asample / Acontrol X 100. The same experiment was carried out on ascorbic acid and α-tocopherol which are known antioxidants. All test and analysis were run in triplicates and the results obtained were averaged. In the DPPH scavenging radical method, the percentage inhibition of the B9YB isolate was the highest at all concentrations (( Figure 3). The IC50 (the concentration of the samples required to scavenge 50 % of the DPPH radicals) was used to examine the antioxidant effectiveness of sample. The lower the IC50, the greater the overall effectiveness of the suspected antioxidant sample. The results obtained revealed that B9YB had the best antioxidant effectiveness with IC50 of 0.339mg/ml followed by Ascorbic acid with IC50 of 0.373mg/ml and the synthetic antioxidant standard α-Tocopherol at IC50 of 0.482 mg/ml.

Conclusion
A number of N-substituted benzenepropanamides have been invented, synthesizesd and used alone as nonsteroidal drugs in the treatment of pain and inflammation or in combination with other analgesic. The isolation and elucidation of Hispanamide, one of such derivatives for the first time from natural sources (plants) and the establishment of its antioxidant effectiveness enhances its anti-inflammatory properties and could be useful for the treatment of ailments resulting from oxidative stress. Futhermore, its presence in the plant justifies the ethnomedicinal uses of Acalypha hispida.