1. Introduction
The discovery of natural, safe, and very effective antioxidants has highlighted the need to address health-related problems in recent years. The effectiveness and safety of antioxidant use and the integrity of the body’s antioxidant system are linked to healthy living and the prevention of both life and non-life-threatening diseases. Exogenous or dietary antioxidants work in tandem with the body’s antioxidant system to protect against or combat reactive oxygen species (ROS), also known as free radicals, which cause oxidative stress [
1,
2]. The overproduction of ROS, such as superoxide anion (O
2−), per hydroxy radical (HOO
.), hydroxyl radical (HO
.) singlet oxygen (
1O
2), and hydrogen peroxide (H
2O
2), involve consistent or persistent electron reductive pathways to molecular oxygen [
3,
4]. As a result, chain reactions or processes are created, which cause lipid peroxidation, leading to damage to cell membrane phospholipids, DNA, and protein molecules which are often implicated as oxidative stress inducers in cancer, diabetes mellitus, inflammation, stroke, immunosuppression, anaemia, and neurodegenerative diseases [
5].
Antioxidants are chemicals that counteract the imbalances caused by oxidative processes, triggering a defence mechanism against the overproduction of free radicals. Natural, safe, and potent antioxidants that provide defence against the harmful effects or actions of free radicals are gaining immense interest in medical research, as they offer protection against free radicals. Synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butyl hydroquinone (TBHQ), are linked to high levels of toxicity in humans and are generally expensive [
6,
7,
8,
9]. For instance, BHA, a synthetic phenolic antioxidant widely utilised in various sectors, affects endocrine functions, causing significant alterations in oestrogen secretion and steroid hormone homeostasis [
10,
11,
12]. Hence, antioxidants in plant or other natural sources, with few or no adverse effects, are preferred alternatives to synthetic antioxidants, especially because they are affordable and readily available. Fatty acids (FAs) are essential chemical constituents in the cells, which serve as fuel for many biological and metabolic activities, including muscular contraction, and have both nutritional and medicinal values. Medicinal plants are excellent sources of fatty acids in nature and occur in different forms like saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) [
13]. Most of the components of medicinal plants, including fatty acids, terpenes, alkaloids, tannins, terpenoids, saponins, have been shown in various studies to prevent and treat many oxidative stress-related disease conditions [
14,
15].
The genus
Helichrysum Mill is a distinctively aromatic medicinal plant of the family Asteraceae, well-distributed in many countries worldwide, including South Africa.
Helichrysum Mill consists of approximately 600 plant species with at least one-third (245 species) available in South Africa. Many of these species differ in morphology and are therefore classified into 30 different groups. Plants in this genus have been traditionally used for the treatment of such human ailments as cold, cough, skin infections, inflammation, insomnia, cystitis, jaundice, stomach pain, menstrual pain, asthma, arthritis disorders, diabetes mellitus wound healing, etc. The in vitro antioxidant, antifungal, anti-inflammatory, anti-bacterial, hepatoprotective, anti-proliferative, and anti-diabetic properties of some species in this genus have been previously studied [
16,
17], however the therapeutic potential of
Helichrysum petiolare & B. L Burtt,
Helichrysum cymocum (L) D. Don,
Helichrysum foetidum (L). Moench,
Helichrysum pandurifolium Schrank as sources of antioxidants, fatty acids, and other constituents has not been fully explored [
18]. Only the antioxidant activities of the acetone and methanol extracts of
H. petiolare, the essential oils from the leaves of
H. cymocum, and the methanolic extract of
H. foetidum have been reported in the literature [
19,
20,
21]. Therefore, in this study, GC-MS analysis of the extracts of four selected
Helichrysum species was done to identify and quantify total phenolic content (TPC), total flavonoids content (TFC), antioxidant activity as well as total fatty acids and lipids.
4. Discussion
Antioxidants, fatty acids, and other constituents of medicinal plants have been reported to be beneficial for preventing, alleviating, or treating, oxidative stress-induced diseases [
33,
34]. Antioxidants are known to be involved in halting redox imbalances by activating the antioxidant defence system to scavenge free radicals through a number of mechanisms, including increased chain-breaking antioxidant activity (synergistic effect), conversion of unstable hydroperoxides in a non–radical pathway to stable components (reducing effect), singlet oxygen (quencher), conversion of pro-oxidant metal derivatives to stable products (metallic chelation), inactivation of pro-oxidant enzymes, decreased activity of free radical oxidation reactions, and inactivation of autoxidation of chain reactions [
35,
36].
Antioxidants have great therapeutic value as anti-viral, anti-fungal, anti-bacterial, anti-tumoural, anti-cancer, anti-angiogenic, anti-inflammatory, anti-allergic, anti-diabetic, and neuroprotective actions [
37,
38,
39,
40]. Flavonoids and phenolics are the main phytocompounds present in most medicinal plants, with more than 4500 flavonoid compounds having been identified [
41] and over 8000 phenolic compounds reported [
42,
43]. Flavonoids and phenolics with potent antioxidant activities have been shown to effectively modulate oxidative stress-related diseases through clearly-defined mechanisms of action [
6]. Apart from their medicinal use, antioxidants are also used in the food industry to preserve and improve the shelf-lives of most foods [
44].
The determination of the antioxidant potential of plant extracts is dependent on the different methods used and their underlying mechanisms, which explains the multiplicity of techniques in most related studies [
45,
46]. Therefore, DPPH scavenging activity, nitric oxide scavenging activity, and reducing power activity were used in this study to investigate the antioxidant activities of the aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum. The phenolic antioxidants have been shown to disrupt the formation of ROS and other free radicals by the transfer of hydrogen atoms from its hydroxyl group [
47] while the antioxidant flavonoids are known to stabilise ROS via their scavenging actions through the oxidation of free radicals into more stable but less active or reactive radicals [
47]. In this study, the aqueous acetone extracts of
H. pandurifolium, H. foetidum, H. petiolare, and H. cymocum produced radical decolourisation of the DPPH solution because of the high free radical scavenging activity of the plant extracts [
5,
48].
The results reveal that the extracts tested have a dose-dependent activity. In fact, at the concentration of 250 μg/mL, the aqueous acetone extracts tested reduce the DPPH radical with an excellent percentage of 90.36 ± 1.00%, 85.28 ± 1.34%, 89.18 ± 0.59%, 89.55 ± 1.22%, 89.55 ± 1.22% for aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum, respectively. Additionally, the IC
50 is inversely proportional to the antioxidant capacity of a compound. However, the lowest value of IC
50 indicates a strong antioxidant capacity of a compound.
H. Cymocum showed the lowest IC
50 values of 11.85 ± 3.20 µg/mL which had better antioxidant activity compared with
H. pandurifolium, H. foetidum, and
H. petiolare, (
Figure 1). The antioxidant power of the aqueous acetone extracts could be explained by the presence of phenolic compounds including flavonoids present in the species of
Helichrysum studied and which are known as antioxidant substances with the ability to trap radical species and reactive forms of oxygen. (
Figure 1).
The results of the IC
50 DPPH assay of the methanolic extracts of similar species namely
H. dasyanthum, H. excisum, and
H. felinum were 12.33, 13.67, and 20.71 μg/mL, respectively, which were within the range of the IC
50 obtained in our study, as reported by Lourens et al. [
21]. However, only the
H. pandurufolium of IC
50 (41.13 ± 3.62 µg/mL) is similar and in agreement with those reported [
49] with the species name,
H. chionophilum, H. plicatum subsp. plicatum, and H. arenarium subsp. Aucheri having IC
50 of 40.5, 48.0, and 47.6 μg/mL, respectively. From literature, the flavonoids are the main compounds in the helichrysum genus with remarkable antioxidant activity, as reported [
49,
50].
The reaction of sodium nitroprusside with oxygen produces nitric oxide and nitrite that scavenge free radicals via diazotisation with a sulphanilamide acid coupled reaction, producing a pink colour [
51]. The antioxidant activities in NO assay involve the donation of protons to the nitrite radicals that show decreased absorbance. In line with the antioxidant activity, the nitric oxide scavenging revealed dose-dependent activity. It is worth mentioning here that all the doses are highly significant among the groups. Although
H. petiolare (20.81 ± 3.73 µg/mL) with the lowest IC
50 indicates the best nitric oxide scavenging effect and good antioxidant compared with the IC
50 of
H. pandurifolium (36.19 ± 2.08 µg/mL),
H. foetidum (24.31 ± 3.67 µg/mL), and
H. cymocum (24.68 ± 4.78 µg/mL). At the concentration of 250 μg/mL, the aqueous acetone extracts tested have NO scavenging activity with an excellent percentage of 78.64 ± 0.38%, 80.34 ± 0.38%, 82.67 ± 0.58%, 81.98 ± 0.48%, and 89.55 ± 1.22% for aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum, respectively. The difference in the antioxidant of aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum could be attributed to the variation in the chemical composition. Indeed, several types of bioactive compounds known for their antioxidant activity [
52,
53] are identified in
H. petiolare with high levels of some compounds, including phenolic (caffeic acid, coniferaldehyde, protocatechuic acid, vanillin) compared to the other species (
Table 4). The reducing power of natural products or plant extracts indicates their potential to transfer electrons from Fe
3+ to Fe
2+, which is synonymous with the antioxidant activity and is linked to reductones that donate a hydrogen atom to break the free radical chain, thus preventing peroxide formation [
54]. The colour change from yellow to various shades of green and blue following treatment is dependent on the reducing power of the plant extract, with the blue colour indicating the highest reducing power. Thus, with increasing concentration of the aqueous acetone extract of
H. pandurifolium, H. foetidum, H. petiolare, and H. cymocum, the observed blue colour indicates greater reducing power, which is similar to results in previous studies [
55,
56].
Consequently, the decrease in absorbance observed is an indication of the extent of nitrite radical scavenging potentials [
57] and this could be attributed to components such as flavonoids, as reported in previous studies [
58,
59]. Similarly, the aqueous acetone extracts of
H. pandurifolium,
H. foetidum H. petiolare, and
H. cymocum can act as natural antioxidants with relative activities scavenging free radical species. The reducing ability or potential is synonymous with the free radical scavenging activity of the plant extracts which is attributable to different amounts of the plant’s phytochemicals constituents [
6] Overall, the antioxidant activities of these plant extracts are attributed to the constituents of total phenolic, total flavonoid, and total antioxidant capacity.
The fatty acid and lipid composition of the aqueous acetone extracts of
H. pandurifolium, H. foetidum, H. petiolare, and H. cymocum were determined by fatty acid methyl esters (FAMEs) analysis involving the derivatisation, which was analysed by gas chromatography [
60]. Previous studies have shown that geographical location, plant species, and seasonal changes could influence the fatty acid content of plants [
61,
62]. Unsaturated (monounsaturated and polyunsaturated) fatty acids have been reported to ameliorate cardiovascular diseases, modulate inflammation and support the immune system against cancer, diabetes mellitus, neurodegenerative diseases, etc. [
63,
64]. This study has shown that the aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum contain various amounts of fatty acids with different compositions, as previously reported [
61,
65]. Our results showed two monounsaturated (MUFA) and two polyunsaturated fatty acids (PUFA), most of which cannot be synthesised by the human body and are only available in dietary sources, making them of great nutritional health benefit [
65,
66]. Stearic acid (C18:0), oleic acid (C18:1n9 (cis)), and linoleic acid (C18:2n9 (cis)), with known health benefits, were high in the aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and
H. cymocum as revealed in
Table 5, which is similar to findings from previous studies that involved different extractants, different parts, and different
Helichrysum species, e.g.,
H. chionophilum and
H. plicatum subsp. [
65]. The high dietary fatty acid ratio of PUFA:SFA are implicated in oxidative stress and are prone to lipid peroxidation because PUFA is highly susceptible, however, raising the PUFA/SFA ratio in the body helps to prevent cardiovascular disease (CVD) and conditions [
67]. The PUFA/SFA varied considerably in the aqueous acetone extracts of
H. pandurifolium, H. foetidum H. petiolare, and H. cymocum having 0.604, 0.726, 0.975, and 1.202 (for PUFA/SFA), respectively in our study, and these were seen to be comparable with the values in some seaweed plants considered to be of great health benefits in literature [
13]. To the best of our knowledge, no study has reported the comparative study of antioxidant activities, constituents, and fatty acid compositions of four selected aqueous acetone extracts of the
Helichrysum species. However, few studies have investigated the antioxidant activity of one species of this plant [
18]. The many folkloric benefits of the plants in the
Helichrysum species are under-explored in scientific investigations [
18]. Natural, plant-based fatty acids are considered to be the best sources of dietary fatty acids because it has been recommended to prevent cardiovascular (CVD) and other disease conditions [
67]. Thus, they could serve as potential sources of effective nutraceutical compounds for the prevention of various disease conditions.
In conclusion, our work provides relevant information on the phenolic, flavonoid, antioxidant capacity, and fatty acid profiles of the aqueous acetone extracts of H. pandurifolium, H. foetidum H. petiolare, and H. cymocum which demonstrate significant antioxidant activities. Since these constituents have been reported in previous studies to be effective in the prevention and treatment of various diseases, further research leading to possible drug discovery and development from these four Helichrysum species, especially for diabetes and its related cognitive decline conditions, is encouraged.