INTRODUCTION:

Antioxidants are substances that when present in low concentrations, compared to those of an oxidizable substrate significantly delay or prevent oxidation of that substance¹.

Antioxidants are believed to play a role in preventing the development of some chronic diseases such as diabetes². When the concentration of ROS exceeds those of antioxidant neutralizing species, a condition known as oxidative stress occurs. Oxidative stress has been implicated in several diseases including atherosclerosis, cancer, malaria, rheumatoid arthritis, chronic fatigue syndrome, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Evidence via monitoring biomarkers such as the presence of ROS and RNS as well as antioxidant defense has indicated oxidative damage may be implicated in the pathogenesis of these diseases³. Antioxidants both synthetic and natural antioxidants have been used to ameliorate the actions of free radicals in biological systems. Several in vitro and in vivo studies have further indicated a positive correlation in the use of antioxidants to mitigate free radical related pathologies^4-6. Prolong use of synthetic antioxidants revealed that they have several side⁷. Natural antioxidants are believed to be safer than the synthetic ones⁵. Natural antioxidants are also more accessible and affordable. Therefore there is need to search for medicinal plants with antioxidant potentials and to isolate compounds responsible for the activity. Medicinal plants with antioxidant activity have continuously been utilized both in traditional and contemporary medicine for management of free radical related diseases and other disease conditions because plants contain numerous phytochemical constituents and many of them are known to exhibit a wide range of biological activity⁸.

Aubrevillea kerstingii is a deciduous tree belonging to the family fabaceae. It is native to West Africa⁹. Various parts of the plant are used in treatment of oedema, kidney diseases and are used as diuretics, laxatives and for treatment of gout (Burkill, 1985). Aubrevillea kerstingii is being used traditionally for the treatment of various ailments associated with oxidative stress with little scientific evidence. This study was carried out in order to isolate antioxidant compound from the stem bark of Aubrevillea kerstingii.

MATERIALS AND METHODS:

Plant Collection, Identification and Preparation:

The stem bark of Aubrevillea kerstingii was collected from DajinDala, unguwarDala, along old Birnin Gwari Road, Gwargwaji Zaria Local Government Area of Kaduna State. It was identified at the Herbarium Unit of the Department of Botany, Ahmadu Bello University Zaria, Nigeria and was given a voucher number. The fresh stem bark of Aubrevillea kerstingii was air dried under shade at room temperature, pulverized and stored in an airtight container for further use.

Extraction:

The powdered stem bark of Aubrevillea kerstingii (1.5kg) was extracted with n-hexane, ethyl acetate and methanol using sequential maceration in increasing order of polarity. The extracts were concentrated on rotary evaporator. The extracts (n-hexane, ethyl acetate and Methanol) were kept in desiccators for further use¹⁰.

Antioxidant activity:

Qualitative Screening for Antioxidant Activity:

This was carried out using TLC autographic screening on pre-coated silica plate. The TLC profile of each of the extracts was developed in a suitable solvent system and sprayed with 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) as detecting reagent. A yellow spot against a purple background suggest presence of free radical scavenging compounds¹¹.

Quantitative screening:

The determination of DPPH radical scavenging activity of the 3 stem bark extracts was carried out according to the method described by standard method¹². Sample stock solution of each extract (0.1mg/ml) was diluted with methanol into various concentrations: 12.5, 25 50, 100, and 200µg/ml respectively. To each sample, 2.5ml (of 12.5, 25 50, 100, and 200µg/ml in methanol) was added to 1ml of DPPH solution (0.3mM in methanol). After 30minutes of reaction at room temperature, the absorbance of the solution was measured at 518nm. The free radical scavenging activity of the extract was determined by comparing its absorbance with that of a control (DPPH with no sample). The ability to scavenge the DPPH radical was calculated using the following equation;

Control - Sample

Percentage inhibition of absorbance = ------------------- x 100

Control

Ascorbic acid of different concentration was used as standard.

Statisticalanalysis:

All the data obtained from the studies were expressed as Mean±Standard Error of Mean (SEM). Data was statistically evaluated using one-way ANOVA, followed by Dunnett test. The values were considered significant when p < 0.05¹⁹.

Isolation of antioxidant compound from ethyl acetate extract of Aubrevilleakerstingii:

The ethyl acetate extract (5g) was pre-adsorbed on silica gel and loaded into a column pre packed with 100g silica gel (60-120 Merck). The extract was eluted with n-hexane: ethyl acetate in a gradient elution starting from n-hexane (100%)²⁰. The eluates were collected in 30ml fractions. A total of 250 fractions were collected. The fractions were combined on the basis of their TLC profiles using DPPH and sulphuric acid as detecting reagents to give 24 sub-fractions which were labeled E1-E24.

Fraction E21 was re-chromatographed on a silica gel packed column (50cm x 1cm) then eluted with chloroform: methanol 95:5 to 60:40. A total of 37 fractions were collected and pooled together based on their TLC profile and labeled A₃₇– A_41.A single homogenous spot was obtained on TLC with two different solvent systems chloroform: methanol (9:1). The compound appeared as white needles and was labeled as A37.

Identification and Structural Elucidation of the Active Compound (s):

Melting Point:

This was done by using Toshniwal melting point apparatus. Thin walled capillary tubes were used to hold melting point samples which was sealed at one end and placed in the melting point apparatus. The substance was heated at a slow heating rate and the temperature was recorded using a thermometer from where the sample started melting to where it has melted.²²

Nuclear Magnetic Resonance (NMR):

The isolated compound was identified by using NMR spectroscopy (Indian Institute of Technology HauzKhas, New Delhi, India). The ¹H-NMR and ¹³C-NMR spectra were obtained in CDCl₃ with NMR spectrometer (BrukerAvance III 400MHZ). The chemical shifts (δ) were reported in parts per million (ppm)²¹.

RESULTS:

Antioxidant Assay:

Qualitative Screening for Antioxidant Activity:

The ethyl acetate extract developed in suitable solvent system and sprayed with 0.3mM DPPH showed yellow spots against purple background confirming the presence of antioxidant compounds. The n-hexane extract did not show yellow or white spots when sprayed with DPPH solution (Figure 1).

Figure 1: Chromatogram of ethyl acetate extract of Aubrevillea kerstingii stem bark using n-hexane: ethyl acetate sprayed with 0.3mM DPPH solution

Quantitative Antioxidant Studies of Aubrevillea kerstingii Stem bark Extracts:

DPPH scavenging activity of the extracts revealed a concentration dependent increase in free radical scavenging activity. The n-hexane extract showed percentage inhibition of extract of 2.43, 4.30, 16.87, 19.70 and 20.98% at concentrations of 12.5, 25, 50, 100 and 200µg/ml respectively. The ethyl acetate extract showed percentage inhibition of extract of 20.60, 49.67, 60.55, 69.19 and 73.10% at concentrations of 12.5, 25, 50, 100 and 200µg/ml respectively while the Methanol extract showed percentage inhibition of extract of 14.92, 45.58, 59.54, 62.96 and 69.75% at concentrations of 12.5, 25, 50, 100 and 200µg/ml respectively (Figure 2). The n-hexane ethyl acetate and methanol showed IC₅₀ values of 98.58μg/ml, 36.79μg/ml and 49.27μg/ml respectively compared while ascorbic acid has IC₅₀of 17.89μg/ml (Table 1).

Figure 2: Antioxidant activity of n-hexane, ethyl acetate and methanol extracts of Aubrevillea kerstingii Stem bark and Ascorbic acid in different concentrations

Table 1: IC₅₀for Antioxidant activity of Aubrevillea kerstingii Stem bark extracts

Extracts	IC₅₀(μg/ml)
n-hexane	98.58
Ethyl acetate	36.79
Methanol	49.27
Ascorbic acid	17.89

Compound A37

A white needle like substance that appears as a single homogenous spot on TLC with Rf value of 0.63 was obtained after E21 was re-chromatographed at solvent system of chloroform: methanol (95:5) (Figure 3).

Figure 3:Chromatogram of Compound A37 developed in Chloroform: methanol (9:1) sprayed with 10% sulphuric acid heated at 105°C for 2 minutes

NMR analysis of Compound A37:

Compound A37 appeared as a white needle like substance with melting point of 293-298°C. The ¹HNMR spectrum of A37(400 MHz, CDCl₃) revealed the following chemical shift values at 3.47(d), 5.29(d), 2.35 (m), 2.22(m), 1.63(m), 1.32 (m), 1.01(s), 0.83 (s), 0.84 (s), 1.18(s), 0.91(s), 0.94(s). The ¹³C NMR (200 MHz, CDCl₃) showed signals at δ 180.10, 145.00, 122.70, 77.52, 54.30, 48.37, 46.60, 46.51, 42.30, 42.10, 39.99, 38.14, 37.85, 37.71, 34.25, 33.20, 33.16, 33.10, 31.05, 28.79, 28.52, 26.76, 24.62, 24.21, 24.00, 23.97, 18.58, 17.70, 16.99, 15.32 (Table 2).

Table 2: ¹H and ¹³C NMR data of A37 Compared with Literature

Carbon position	¹H NMR	¹³C NMR
1		37.84
2		26.76
3	3.47 (d)	77.52
4		38.14
5		54.30
6	1.52 (m)	18.58
7	1.48 (m)	33.10
8		39.99
9	1.57 (m)	48.37
10		37.71
11		23.97
12	5.29 (d)	122.70
13		145.00
14		42.30
15		28.52
16		24.00
17		46.60
18	2.35 (m)	42.10
19	2.22 (m)	46.51
20		31.05
21	1.63(m)	34.25
22	1.32 (m)	33.16
23	1.01 (s)	28.79
24	0.83 (s)	16.99
25		15.32
26	0.84 (s)	17.70
27	1.18 (s)	24.62
28		180.10
29	0.91 (s)	33.18
30	0.94 (s)	24.21

Figure 4: Proposed Structure of Compound A37

DISCUSSION:

The DPPH test provides information on the reactivity of compounds with a stable free radical DPPH that gives a strong absorption band at 517nm in visible region. When the odd electron becomes paired off in the presence of a free radical scavenger the absorption reduces and the DPPH solution is decolorized as the colour changes from deep violet to light yellow. The degree of reduction in absorbance is reflective of the antioxidant potency of the extract¹⁴. Methanol and Ethyl acetate extracts developed in suitable solvent system and sprayed with 0.3mM DPPH showed yellow spots against purple background confirming the presence of antioxidant compounds. The n-hexane extract did not show yellow or white spots when sprayed with DPPH Solution. DPPH scavenging activity of the extracts revealed a concentration dependent increase in free radical scavenging activity. The decrease in absorbance by the DPPH radical with increase in concentration of the extracts which manifested in the rapid discolouration of the purple DPPH, suggest that the ethyl acetate and methanol extracts of Aubrevillea kerstingii stem bark has antioxidant activity due to its proton donating ability. The extracts were found to highly scavenge free radicals when compared to standard antioxidants. The reducing capacity of compounds could serve as indicator of potential antioxidant properties¹⁵.

Fraction E21 subjected to further column chromatography using chloroform: methanol (95:5) yielded a compound that was homogenous on TLC. It was labeled as compound A37.The 1H NMR showed 48 proton signals. The signals between δ-0.84 and δ-1.98 may be due to the presence of triterpenes skeleton while a signal at δ-3.47 may be due to a proton attached to oxygen. The ¹³C NMR consists of 30 carbons peaks. The peak at δ-180.10 may be due to the presence of carbonyl group. The two peaks at δ-122.70 and δ 145.00 may be due to the presence of a pair of sp2 hybridized carbons. The peaks at δ-26.76., 15.52, 16.99, 17.70, 24.61, 33.18 and 24.21 are likely due to methyl substituent as reported by Naveen and Jangwan, 2012¹⁶, 2013. The spectral data of A37 is in agreement with that of oleanolic acid reported by Martins et al., 2013¹³. The oleanolic acid isolated appeared as white crystal and showed an R_fvalue of 0.63 when developed on TLC. This was not in agreement with that reported by Vyas and Argal 2014¹⁶, who reported oleanolic acid from Lantana camarahas an R_fvalue of 0.50. The melting point of oleanolic acid in this study was 293-298°C. This is similar to that of oleanolic acid reported by Naveen and Jangwan, 2012¹⁶. The compound isolated in this study was reported in various literatures to possess antioxidant activity. Antioxidant properties of the oleanolic acid isolated from Cassia auriculatawas reported by Senthilkumaret al., 2014¹⁸.

CONCLUSION:

The methanol extract and ethyl acetate extracts showed significant antioxidant activity while the n-hexane extract showed no significant antioxidant activity in vitro using DPPH assay. The presence of oleanolic acid may be responsible for the antioxidant activity of stem bark of Aubrevillea kerstingii. This plant may therefore serve as potential antioxidant agent.

ACKNOWLEDGEMENTS:

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

CONFLICT OF INTEREST STATEMENT:

The authors declare that there is no conflict of interest.

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Received on 14.05.2021 Modified on 24.12.2021

Asian J. Pharm. Tech. 2022; 12(4):285-289.

DOI: 10.52711/2231-5713.2022.00046