Comparative Study of the Antioxidant Constituents, Activities and the GC-MS Quantification and Identification of Fatty Acids of Four Selected Helichrysum Species

Helichrysum Mill. (Asteraceae) is a plant genus comprising distinctively of aromatic plants of about 500–600 species. Since most of these plants have not been previously studied, extensive profiling helps to validate their folkloric uses and determine their potential value as sources of plant-derived drug candidates. This study, therefore, aims to investigate the antioxidant activity (DPPH, NO, FRAP); total antioxidant capacity, total phenolic, total flavonoid, and fatty acid compositions of the aqueous acetone extracts from four Helichrysum plants namely, Helichrysum pandurifolium, Helichrysum foetidum, Helichrysum petiolare, and Helichrysum cymocum. The results obtained showed that the H. cymocum extract had the best DPPH radical scavenging activity (IC50 = 11.85 ± 3.20 µg/mL) and H. petiolare extract had the best nitric oxide scavenging activity (IC50 = 20.81 ± 3.73 µg/mL), while H. pandurifolium Schrank extract (0.636 ± 0.005 µg/mL) demonstrated the best ferrous reducing power, all of which are comparable with results from ascorbic acid used as the standard. The IC50 values of the radical scavenging activity ranged from 11.85–41.13 µg/mL (DPPH), 20.81–36.19 µg/mL (NO), and 0.505–0.636 µg/mL (FRAP), for all the plants studied. The H. petiolare has the highest total antioxidant capacity (48.50 ± 1.55 mg/g), highest total phenolic content (54.69 ± 0.23 mg/g), and highest total flavonoid content (56.19 ± 1.01 mg/g) compared with other species. The fatty acid methyl esters were analysed using gas chromatography-mass spectrometry (GC-MS). The results obtained showed variations in the fatty acid composition of the plant extracts, with H. petiolare having the highest saturated fatty acid (SFA) content (7184 µg/g) and polyunsaturated fatty acid (PUFA) content (7005.5 µg/g). In addition, H. foetidum had the highest monounsaturated fatty acid (MUFA) content (1150.3 µg/g), while H. cymocum had the highest PUFA:SFA ratio of 1.202. In conclusion, the findings from this study revealed that H. pandurifolium Schrank, H. foetidum, H. petiolare, and H. cymocum are repositories of natural bioactive compounds with potential health-promoting benefits that need to be investigated, for both their antioxidant activity in a number of disease conditions and for further exploration in drug discovery and development projects.


Plant Extraction
The leaves of the plants were cleaned and air-dried to a constant weight and the dried samples were pulverised using an electronic blender, grounded, and weighed. The powdered plant materials in conical flasks were soaked and subjected to intermittent stirring in 90% aqueous acetone and warmed in the water bath at 60 • C for 2 h with slight modification [22]. The mixture was filtered with Whatman cellulose filter paper under pressure using a pump and the plant material was subjected to a second extraction after soaking overnight and the filtrate pooled before rotary evaporation. The final residue or extract obtained was allowed to dry in the fume cupboard and stored at −20 • C until required for use. (1 mg extract dissolved in 1 ml acetone is used in subsequent analysis).

In Vitro Evaluation DPPH Radical Scavenging Activity Assay
The DPPH radical scavenging activity assay was performed as previously described [23]. Briefly, a 2000 µL stock concentration of DPPH (0.004 g in 100 mL methanol) was added to aliquots of 500 µL plant extracts at different concentrations (10-250 µg/mL) and the reaction mixture was shaken in the dark for 30 min at room temperature. The controls contained the DPPH solution without the plant extract, while methanol was used as the blank. A decrease in absorbance of the test mixture read at 517 nm will result from quenching of DPPH free radicals after the exposure time interval. The following formula is used to determine the scavenging effects of the plant extracts: where A 0 is the absorbance of the blank and A 1 is the absorbance of the extract.

Nitric Oxide Scavenging Activity Assay
Sodium nitroprusside generates Nitric oxide (NO) in aqueous physiological pH, measured in the Greiss reaction that produces nitrite ions as previously described [24]. Briefly, 4000 µL of the plant extract or standard solution at different concentrations (10-250 µg/mL) was added to 1000 µL of Sodium nitroprusside solution and 2000 µL of the mixture was added to 1200 µL of the Griess reagent containing 1% sulphanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 2% H 3 PO 4. The absorbance of the chromophore formed during diazotisation of nitrite with sulphanilamide, and its subsequent coupling with naphthyl ethylenediamine dihydrochloride, was measured at 550 nm. The percentage (%) inhibition activity was calculated from the following equation, with ascorbic acid as the standard: where A 0 is the absorbance of the control while A 1 is the absorbance of the extract or standard.

Reducing Power Assay
Different concentrations of plant extracts (10-250 µg/mL) and corresponding concentrations of standard ascorbic acid were added to 2500 µL and 2500 µL of phosphate buffer (pH 6.6) and 1% potassium ferricyanide, respectively. Incubation of the mixture was done at 50 • C for 20 min after which, 2500 µL of 10% trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. Thereafter, 2500 µL of the supernatant was added to 2500 µL distilled water and 500 µL of freshly prepared 0.1% ferric chloride solution [25], and absorbance was read at 700 nm. Ascorbic acid was used as the standard at the various concentrations.

Estimation of Total Phenolic Compounds
The Folin-Ciocalteu reagent method was used to determine the phenolic content as previously described [26]. Briefly, 500 µL of the plant extracts and 100 µL of Folin-Ciocalteu reagent (0.5 N) were added and incubated for 15 min at room temperature after which 2500 µL of sodium carbonate (7.5% w/v) was added to the mixture (plant extract + Folin-Ciocalteu) and incubated for 30 min at room temperature, and absorbance read at 760 nm. Phenolic concentration was expressed as gallic acid equivalent (GAE) (mg/g of dry mass) as the reference value.

Total Flavonoid Content Estimation
Aluminium chloride solution was used to determine the flavonoid content as previously described, with quercetin as the standard [27]. Briefly, 1000 µL of a 100 µg/mL extract stock solution was added to 3000 µL of methanol and mixed with 200 µL of 10% aluminium chloride, 200 µL of 1 M potassium acetate, and 5600 µL of distilled water. The mixture was incubated at room temperature for 30 min, and absorbance read at 415 nm. The calibration curve was prepared from quercetin solutions in methanol, at the various concentrations.

Determination of Total Antioxidant Capacity
The plant extract (3000 µL) was added to 3000 µL of the reagent solution containing 0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate as previously described [28]. The tubes containing the mixture were capped and incubated in the water bath at 95 • C for 90 min and allowed to cool at room temperature, followed by an absorbance reading at 695 nm against the blank. The extraction of 100 mg plant extracts was done for 3 h at 60 • C using 1 ml of 70% methanol, and 130 µL of the extract was freeze-dried and derivatised using 30 µL N, O-Bis (trimethylsilyl)trifluoroacetamide (BSTFA), and 100 µL acetonitrile at 60 • C for 30 min. The sample was then transferred into a 2 mL GC vial, and 1 µL was injected onto the GC-MS/MS in splitless mode.

Chromatographic Separation
Helium gas at a flow rate of 1 mL/min, injector temperature maintained at 250 • C, and separation of the analytes was performed on a non-polar Rxi-5Sil MS (30 m, 0.25 mm ID, 0.25 µm film thickness) (instrument type, Trace 1300, Thermo Scientific, Waltham, MA, USA) coupled to triple quadrupole mass spectrometer (TSQ 8000, Thermo Scientific). The oven temperature was programmed as follows: 100 • C for 4 min, then ramped to 180 • C at 10 • C/min rate and held for 2 min before finally ramped at 20 • C/min until 320 • C and held for 5 min. The mass spectrometer detector (MSD) operated in tandem mass spectrometry (MS/MS) mode, the source, and quad temperature were maintained at 250 • C and 150 • C, respectively. The transfer temperature was maintained at 250 • C. Briefly, 100 mg of the plant extract was vortexed and sonicated at room temperature for 30 min in a mixture of 1 m of chloroform and 1 mL of methanol. This was centrifuged at 3000 rpm for 1 min after which 500 µL of the chloroform fraction (bottom layer) was completely dried with a gentle stream of nitrogen, reconstituted and vortexed with 500 µL of methyl tert-butyl ether (MTBE), and 100 µL was derivatised with 30 µL of trimethyl sulfonium hydroxide (TMSH). Thereafter, 1 µL of the derivatised sample was injected into the GC-MS, in a 5:1 split ratio.

Chromatographic Separation
Helium gas at a flow rate of 1.2 mL/min, injector temperature maintained at 240 • C and separation of the FAMEs was performed on a polar RT-2560 (100 m, 0.25 mm ID, 0.20 µm film thickness) capillary column (instrument type, 6890 N, Agilent technologies network) coupled to Agilent technologies inert XL EI/CI Mass Selective Detector (MSD) (5975, Agilent Technologies Inc., Palo Alto, CA, USA). The oven temperature was programmed as follows: 100 • C for 4 min, then ramped to 240 • C at 3 • C/min rate and held for 10 min. The mass spectrometer detector (MSD) operated in scan mode, and the source and quad temperature were maintained at 250 • C and 150 • C, respectively. The transfer temperature was maintained at 250 • C. The mass spectrometer was operated under electron impact (EI) mode at ionisation energy of 70 Ev, scanning from 40 to 650 m/z.

Statistical Analysis
The mean ± SD from three experimental observations in triplicates of data were used for statistical analysis. The in vitro antioxidant assays were analysed using the ANOVA test, followed by Tukey's test, with statistical significance at (p < 0.05).

In Vitro Antioxidant Capacities and Profiles of the Four Helichrysum Species
The antioxidant capacity of the aqueous acetone extracts of four Helichrysum species, namely H. pandurifolium, H. foetidum H. petiolare, and H. cymocum, were investigated.

DPPH Scavenging Activity
The radical inhibitory or scavenging activity of the selected aqueous acetone extracts of the Helichrysum species (10-250 µg/mL) was concentration-dependent. Results showed that the extracts caused increased activity with increasing concentrations as shown in Table 1, which is in tandem with results from previous studies [6,29]. Figure 1 shows IC 50 values of 14.17 ± 1.77 µg/mL (H. pandurifolium), 41.13 ± 3.62 µg/mL (H. foetidum), 23.57 ± 2.59 µg/mL (H. petiolare), and 11.85 ± 3.20 µg/mL (H. cymocum) respectively. Hence, the ranking order for the scavenging free radical activity could be represented as H. cymocum >H. pandurifolium > H. petiolare >H. foetidum for these extracts, with ascorbic acid used as the standard, showing the best radical scavenging activity and an IC 50 value of 2.66 µg/mL compared with all four selected Helichrysum species.

Reducing Power Activity
The reducing power dose-response curves of all extracts (10-250 μg/mL) of the selected Helichrysum species are concentration-dependent, as shown in Table 3. The ranking order for the reducing power at the highest concentration of 250 μg/mL indicates that H. cymocum ˃ H. foetidum ˃ H. pandurifolium ˃ H. petiolare of 0.636 μg/mL, 0.619 μg/mL, 0.602 μg/mL and 0.505 μg/mL, respectively ( Figure 3). The ascorbic acid has the highest value of 0.853 μg/mL at the highest concentration of 250 μg/mL.

Reducing Power Activity
The reducing power dose-response curves of all extracts (10-250 µg/mL) of the selected Helichrysum species are concentration-dependent, as shown in Table 3. The ranking order for the reducing power at the highest concentration of 250 µg/mL indicates that H. cymocum > H. foetidum > H. pandurifolium > H. petiolare of 0.636 µg/mL, 0.619 µg/mL, 0.602 µg/mL and 0.505 µg/mL, respectively (Figure 3). The ascorbic acid has the highest value of 0.853 µg/mL at the highest concentration of 250 µg/mL.    On the other hand, the phenolic content was 53.11 ± 0.47 mg/g for H. pandurifolium, 42.14 ± 0. 50 mg/g for H. foetidum, 54.69 ± 0.23 mg/g for H. petiolare, and 47.93 ± 0.57 mg/g for H. cymocum acetone extracts, respectively. Overall, H. petiolare has the best antioxidant capacity,total flavonoids, and total phenolics compared with other species (Figure 4). Previous studies have shown that many factors, including genetic diversity, biological, environmental, seasonal variations as well as the harvesting period, may account for any differences or similarities seen in the results of TF, TP, and TAC of plant extracts for the same plant species [30][31][32].

Total Phenolic Acid and Phenolic Aldehyde Composition
In table 4, the composition of the phenolics and their aldehyde content in different species vary. The vanillin (78.5 μg/g), protocatechuic acid (297.5 μg/g), aldhyde (13.5 μg/g), and caffeic acid (1424.3 μg/g) were more in the aqueous ace tract of H. petiolare in comparison with that of H. pandurifolium, H. foetidum, an mocum. It was also observed that syringaldehyde (23.1 μg/g), m-coumaric aci μg/g), and ferulic acid (144.3 μg/g) were higher in H. pandurifolium compared wit H. petiolare, H. foetidum, and H. cymocum. Additionally, vanillic acid (43.5 μg/g) an maric acid (0.122 μg/g) showed a higher concentration in H. foetidum compared other species, meanwhile, syringic acid (13.7 μg/g) and gallic acid (685.7 μg/g) wer in the aqueous acetone extract of H. cymocum in comparison with H. pandurifolium iolare, and H. foetidum as showed in Table 4. Hence, it is important to know that ea ponent was determined using an external standard calibration by GC-MS/MS.

Total Phenolic Acid and Phenolic Aldehyde Composition
In Table 4, the composition of the phenolics and their aldehyde content in the four different species vary. The vanillin (78.5 µg/g), protocatechuic acid (297.5 µg/g), coniferaldhyde (13.5 µg/g), and caffeic acid (1424.3 µg/g) were more in the aqueous acetone extract of H. petiolare in comparison with that of H. pandurifolium, H. foetidum, and H. cymocum. It was also observed that syringaldehyde (23.1 µg/g), m-coumaric acid (0.392 µg/g), and ferulic acid (144.3 µg/g) were higher in H. pandurifolium compared with that of H. petiolare, H. foetidum, and H. cymocum. Additionally, vanillic acid (43.5 µg/g) and p-coumaric acid (0.122 µg/g) showed a higher concentration in H. foetidum compared with the other species, meanwhile, syringic acid (13.7 µg/g) and gallic acid (685.7 µg/g) were higher in the aqueous acetone extract of H. cymocum in comparison with H. pandurifolium, H. petiolare, and H. foetidum as showed in Table 4. Hence, it is important to know that each component was determined using an external standard calibration by GC-MS/MS.  Table 5 shows the amount (µg/g) of the individual fatty acid and classes of fatty acids of the aqueous acetone extract of H. pandurifolium, H. foetidum, H. petiolare, and H. cymocum. Eleven saturated fatty acids (C 12 -C 24 ), two monounsaturated fatty acids (C 16:1 , C 18:1 n 9 (cis) ), and two polyunsaturated fatty acids (C 18:2 n 6 (cis) , C18:3n3) were identified in the extracts of the four plants. The amount of the fatty acids varied widely, viz, 3.1 to 728.3 µg/g (for saturated fatty acids), 76.7 to 1057.9 µg/g (for monounsaturated fatty acids), and 624.9 to 4688.6 µg/g (for polyunsaturated fatty acids), respectively. The PUFA:SFA ratios are 0.604 (H. pandurifolium), 0.726 (H. foetidum), 0.975 (H. petiolare), and 1.202 (H. cymocum) with attributable health benefits. Table 6 depicts the GC-MS chromatograms of the fatty acids of aqueous acetone extract of H.pandurifolium, H.foetidum H.petiolare, and H.cymocum while Figure 5 shows the area of the peaks, ratio area, and the retention time (R, time) of aqueous acetone extract of H. pandurifolium, H. foetidum H. petiolare, and H. cymocum.  The peaks, ratio area, and the retention time obtained from gas chromatography-mass spectrometry (GC-MS) are described by the National Institute of Standards and Technology (NIST) library to that of a known compound. This is depicted in the different compositions of fatty acids in the above table.

Composition of Saturated Fatty Acids
FOR PEER REVIEW

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, antitumoural, 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 [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.