![Experimental and literature values [27] of 13 C NMR shifts (ppm) of faurinone.](https://www.researchgate.net/publication/364298576/figure/tbl4/AS:11431281089122001@1665415734970/Experimental-and-literature-values-27-of-13-C-NMR-shifts-ppm-of-faurinone_Q320.jpg)
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Citation: Adewinogo, S.O.; Sharma,
R.; Africa, C.W.J.; Marnewick, J.L.;
Hussein, A.A. Chemical Study and
Comparison of the Biological
Activities of the Essential Oils of
Helichrysum petiolare,H. cymosum, and
H. odoratissimum.Plants 2022,11,
2606. https://doi.org/10.3390/
plants11192606
Academic Editor: Stefania Garzoli
Received: 24 August 2022
Accepted: 30 September 2022
Published: 3 October 2022
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plants
Article
Chemical Study and Comparison of the Biological Activities
of the Essential Oils of Helichrysum petiolare,H. cymosum,
and H. odoratissimum
Selena O. Adewinogo 1, Rajan Sharma 1, * , Charlene W. J. Africa 2, Jeanine L. Marnewick 3
and Ahmed A. Hussein 1, *
1Chemistry Department, Cape Peninsula University of Technology, Bellville Campus, Symphony Road,
Bellville 7535, South Africa
2Medical Biosciences, University of the Western Cape, Bellville 7535, South Africa
3Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology,
Symphony Rd., Bellville 7535, South Africa
*Correspondence: sharmar@cput.ac.za (R.S.); mohammedam@cput.ac.za (A.A.H.)
Abstract:
Helichrysum species are prominent South African medicinal plants. From the essential
oils (EOs) of three Helichrysum species, H.petiolare,H.odoratissimum, and H.cymosum, sixty-three
constituent components were identified, with hydrocarbons and oxygenated monoterpenes and
sesquiterpenes as major components. The compounds were analyzed by gas chromatography-
mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. In H.petiolare
EO, the major components were faurinone (20.66%) and (E)-
β
-ocimene (17.21%). Faurinone was
isolated from this EO for the first time. In H.odoratissimum, 1,8-cineole (17.44%) and
α
-pinene, and
γ
-curcumene (15.76%) were the major components whereas, in H.cymosum,
α
-pinene (29.82%) and
(E)-caryophyllene (19.20%) were the major components. In the antibacterial activity study, the EOs
were tested against Staphylococcus aureus,Escherichia coli, and Pseudomonas aeruginosa. The EOs were
found to possess low antibacterial, anti-tyrosinase, and photoprotection activities and moderate
antioxidant capacities, thus establishing these Helichrysum EOs as valuable antioxidant agents.
Keywords:
essential oils; Helichrysum;H. petiolare;H. odoratissimum;H. cymosum; antioxidant; antibac-
terial; tyrosinase inhibition; sun protection factor
1. Introduction
Helichrysum Mill. is a large genus comprising over 600 species spread throughout
Africa, Europe, North America, and Australia. Out of these, nearly 244–250 Helichrysum
species occur in Southern Africa (including Namibia) with extensively varied morpholo-
gies [
1
,
2
]. Helichrysum species are popular materials in the traditional medicines of Europe,
Asia, and Africa, where their herbal teas are used to treat fever, cough respiratory problems,
digestive disorders, skin inflammation, and wounds [3–5].
Essential oils (EOs) and their volatile constituents have been important materials for
preventing and treating human diseases for a long time [
6
]. Helichrysum EOs are well
studied in the literature [
7
–
10
] and show promising biological potencies. H. italicum EO,
also called “immortelle”, is already a renowned ingredient of cosmetics. It is said to
promote blood flow in the skin, regenerate it, and help attenuate signs of aging such as fine
lines and wrinkles [5].
As the ethnomedicinal records propose Helichrysum species as a skin remedy in the
quest to explore the South African flora for novel cosmeceutical ingredients, H. petiolare,H.
odoratissimum, and H. cymosum EOs were selected for phytochemical investigation. Table 1
summarizes the studies related to the EO’s of these Helichrysum species with regard to
chemical composition and biological activity.
Plants 2022,11, 2606. https://doi.org/10.3390/plants11192606 https://www.mdpi.com/journal/plants
Plants 2022,11, 2606 2 of 16
Table 1.
Review of previous studies on essential oils of the selected Helichrysum species under focus.
Name Locality
Studies on Essential Oils
References
Analysis
Method Major Components Biological Tests
Helichrysum
petiolare Hilliard
and B.L.Burtt
SA GC-MS
1,8-Cineole (22.4%),
(E)-caryophyllene (14.0%),
p-cymene (9.8%)
Antimicrobial,
antioxidant, and
anti-inflammatory
Lourens et al. [1]
SA GC-MS
Caryophyllenyl alcohol
(36.42–45.26%),
β-hydroagarofuran
(19.45–25.64%), δ-cadinene
(3.39–4.76%)
None Giovanelli et al.
[5].
Helichrysum
cymosum (L.)
D.Don subsp.
cymosum
Tanzania GC-MS
(E)-Caryophyllene (27.02%),
caryophyllene oxide (7.65%),
p-cymene (7.55%).
Antimicrobial Bougatsos et al.
[8].
SA GC-MS
1,8-Cineole (20.4%), α-pinene
(12.4%), (E)-caryophyllene
(10.8%)
Antimicrobial and
antimalarial and
cytotoxic
Van Vuuren et al.
[11].
SA TLC and
GC-MS
1,8-Cineole (20.4–34.6%),
(E)-caryophyllene (8.4–10.8%),
α-pinene (3.6–12.4%).
Antimicrobial Reddy [12].
Cameroon GC-FID and
GC-MS
δ-3-Carene (16.1%),
(E)-caryophyllene (12.0%),
camphene (7.4%).
Radical scavenging
and antifungal
Tchoumbougnang
et al. [13].
SA GC-MS
(Z)-
β
-Ocimene (35.61–50.44%),
(E)-caryophyllene
(15.03–16.62%), α-humulene
(5.28–8.68%).
None Giovanelli et al.
[5].
Helichrysum
odoratissimum (L.)
Sweet
Zimbabwe GC-MS
α-Pinene (15.0%),
α-humulene (13.0%),
(E)-caryophyllene (9.6%).
None Gundidza and
Zwaving [14].
SA GC-MS
p-Menthone 35.4%, pelugone
34.2%, 1,8-cineole 13.0% (fresh
plant material).
None Asekun et al. [15].
SA TLC and
GC-MS
(E)-Caryophyllene
(9.3–25.2%), limonene
(11.6–19.6%), and 1,8-cineole
(11.2–17.1%).
Antimicrobial Reddy [12].
SA GC-MS
Limonene (14.55%),
1.8-cineole (6.56%), α-pinene
(4.20%).
Repellent and
fumigation against
maize weevil
Odeyemi et al.
[16].
SA GC-MS β-Pinene (51.6%), limonene
(16.9%), α-humulene (5.6%)
Antimicrobial and
cytotoxic Lawal et al. [17].
Uganda GC-MS
Palmitic acid (27.1%),
humulene (14.1%),
(E)-caryophyllene (12.6%).
Antimicrobial Ocheng et al. [18].
SA GC-MS
α-Pinene (4.11–18.39%),
(E)-caryophyllene
(9.67–15.85%), 1,8-cineole
(2.74–13.35%).
None Giovanelli et al.
[5].
Additionally, as per the literature, these EOs have not been evaluated before for their antityrosinase activity
(Table 1). Therefore, the present research aimed to elucidate the chemical composition of the essential oils of
these three selected Helichrysum species, and biologically evaluate them for their antimicrobial, antioxidant,
antityrosinase, and photoprotective activity.
Plants 2022,11, 2606 3 of 16
2. Results and Discussion
2.1. Yield and Chemical Composition of the Helichrysum Essential Oils
The hydrodistilled plant materials of H.petiolare and H.cymosum were isolated as pale
green and clear essential oils, respectively. H.petiolare had a higher essential oil yield of
0.25% (v/w) against 0.15% (v/w) for H.cymosum.
The Helichrysum essential oils were found to collectively contain high amounts of
α
-pinene up to 29.82% as in H.cymosum EO and 1,8-cineole up to 17.44% as in H.odoratissi-
mum EO, as shown in Table 2. These compounds have previously been reported as major
constituents in the GC-MS analyses of these Helichrysum species.
α
-Pinene has been re-
ported in African H. odoratissimum EOs in amounts as high as 43.4% and
40.6–47.1% [19,20]
.
Lourens et al. [
21
] have reported 1,8-cineole (22.4%) as the major compound in H.petiolare
from the plant collected in Pretoria. Reddy [
12
] found 1,8-cineole as the major compound
in H.cymosum EO as 20.4–34.6%. In H.odoratissimum EO, 1,8-cineole compound has been
found between 6.56 and 17.1% [5,12,15,16].
Table 2. GC-MS analysis of the Helichrysum essential oils.
Mass Spectral Matching Composition (%) Experimental
RI
Literature
RI Identification
H. petiolare H. odoratissimum H. cymosum
α-Pinene 7.49 15.76 29.82 938 939 ARI, MS
Camphene - 0.32 0.44 951 950 BRI, MS
β-Pinene 10.54 5.18 2.56 981 979 ARI, MS
Myrcene 0.50 0.41 0.78 993 990 ARI, MS
α-Terpinene - 1.51 1.83 1017 1017 BRI, MS
1,8-Cineole 9.87 17.44 15.13 1035 1032 BRI, MS
(E)-β-ocimene 17.21 0.42 8.24 1051 1050 ARI, MS
β-Ocimene (undefined
isomer) 3.79 - 3.26 1057 - Wb MS
γ-Terpinene 0.73 0.82 2.50 1063 1060 BRI, MS
allo-Ocimene 6.66 - 3.01 1136 1132 ARI, MS
Borneol - - 0.45 1164 1166 BRI, MS
Terpinen-4-ol 0.57 0.63 2.18 1176 1177 BRI, MS
α-Terpineol - 5.51 0.82 1193 1190 BRI, MS
Lavandulyl acetate 0.99 - - 1294 1290 ARI, MS
Myrtenyl acetate 0.41 - - 1325 1326 ARI, MS
α-Copaene 0.65 - - 1372 1376 BRI, MS
Unknown - 1.13 - - - -
Lavandulyl propionate 0.41 - - 1384 - Match
Italicene - 3.24 - 1409 1402 BRI, MS
(E)-Caryophyllene - 7.30 19.20 14.22 1420 BRI, MS
α-Humulene 3.01 2.06 0.83 1450 1453 BRI, MS
Unknown - - 0.36 1486 - -
γ-Curcumene - 15.76 - 1487 1481 BRI, MS
Phenyl ethyl
2-methylbutanoate 0.90 - - 1488 1487 ARI, MS
Ar-Curcumene - 7.63 -
Unknown 5.29 3.06 - 1499 - -
7-epi-α-Selinene - - 0.60 1510 1517 BRI, MS
Sesquicineole - 2.75 - 1514 1516 ARI, MS
Lavandulyl isovalerate 1.28 - - 1514 1509 ARI, MS
δ-Cadinene 2.05 1.13 - 1522 1523 BRI, MS
Unknown - 0.54 - 1531 - -
α-Calacorene 0.68 0.40 - 1539 1540 BRI, MS
Faurinone 20.66 - - 1568 - MS, NMR
Caryophyllene oxide - 1.66 2.65 1578 1580 BRI, MS
Plants 2022,11, 2606 4 of 16
Table 2. Cont.
Mass Spectral Matching Composition (%) Experimental
RI
Literature
RI Identification
Viridiflorol - 0.45 - 1585 1591 BRI, MS
Unknown 0.43 - - 1602 - -
Junenol - 0.59 - 1610 1618 ARI, MS
Unknown 1.93 - - 1642 - -
Unknown 0.62 - - 1649 - -
Valeranone 1.07 - - 1666 1672 BRI, MS
Monoterpene
hydrocarbons: 46.92 24.42 53.24
Oxygenated
monoterpenes: 12.25 23.58 18.58
Total monoterpenoids: 59.17 48.00 71.82
Sesquiterpene
hydrocarbons: 6.39 37.52 20.63
Oxygenated
sesquiterpenes: 23.01 5.99 2.65
Total sesquiterpenoids: 29.40 43.51 23.28
Diterpene hydrocarbons: 0.60 0.72 3.09
Phenylpropanoids: 0.90 0.00 0.00
Total identified: 90.07 92.23 98.19
Unidentified: 8.27 4.73 1.82
Total 98.34 96.96 100.01
A
= Adams [
22
].
B
= Babushok et al. [
23
]. Wb = NIST Chemistry WebBook [
24
]. MS = In addition to RI, the MS of
the analyzed compound matched with the MS of the compound in [
22
] and/or NIST Chemistry WebBook [
24
].
Wb MS = The MS of the analyzed compound matched with the compound listed in [
24
]. Match = no RI or MS
available in the literature. The compound was reported solely based on the mass spectral match with NIST14
libraries reported by MassHunter software (Agilent Technologies, Inc., Santa Clara, CA, USA) (Probability < 0.04).
Unknown = The MS of the compound could not be matched with the available literature data. U = Undefined.
Higher n-paraffin needed.
H.odoratissimum and H.cymosum EOs both featured a high content of (E)-caryophyllene
at 7.30% and 19.20%, respectively, as was previously identified in previous reports by
Reddy [
12
] (9.3–25.2%), and Bougatsos et al. [
8
] (27.04%). (E)-
β
-ocimene found in high
content in H.petiolare EO (17.21%) and H.cymosum EO (8.24%) was not identified in
previous analysis reports of these two essential oils. In this study,
γ
-Curcumene was found
to be present as a prominent constituent (15.76%) in H.odoratissimum EO, which is much
higher than the percentage composition (2.15%) reported in an earlier report [
16
]; however,
ar-curcumene, another dominant compound in this EO (7.63%), has also been reported in
the Cameroonian H.odoratissimum EO in a higher content of 20.3% [19].
The major compound in H.petiolare essential oil (compound
1
; RI = 1568) identified
by GC-MS analysis was structurally elucidated as faurinone (Figure 1) through NMR and
MS analyses. This sesquiterpene ketone has never been reported before in the essential
oil of H.petiolare. The structural elucidation of compound
2
(RI = 1499) is also discussed
further below.
Plants 2022, 11, x FOR PEER REVIEW 5 of 16
of H. petiolare. The structural elucidation of compound 2 (RI = 1499) is also discussed fur-
ther below.
2.2. Structural Elucidation of Compound 1
Compound 1 (5 mg) was identified as faurinone (Figure 1) by MS, 1H-NMR, and 13C-
NMR spectroscopic techniques, and the spectroscopic data were compared with the pre-
viously published literature, as presented in the following sections.
Figure 1. Chemical structure of faurinone.
2.2.1. Mass Spectrometry
The ion fragment peaks obtained in the MS spectrum (Figure 2) were compared to
the data first reported by Hikino et al. [25]. The molecular ion [M+] at m/z = 222.3 suggested
the molecular formula to be C15H26O, which translates to the three degrees of unsaturation
as it is for faurinone. A base peak was found at m/z = 123.2, which is typical of isomers of
faurinone as described by Weyerstahl et al. [26].
Figure 2. Mass spectrum of faurinone.
2.2.2. 1H NMR Spectroscopy
The 1H NMR spectrum showed four methyl signals, and two of them appear at 0.74
ppm and 0.80 ppm (d, 6.2 Hz, Me-12, Me-13), and the other two methyl groups appear as
singlets at 1.02 (Me-10) and 2.19 (Me-15); the latter is adjacent to a carbonyl group (C-14)
(Figure 1). The spectrum also showed a proton resonating at 2.31 (ddd, 3.6, 10.7, 14.4 Hz)
and assigned to H-4, and another proton at 1.95 (dd, 5.0, 10.7 Hz) and assigned to H-5, in
addition to a cluster of protons between 1.92 and 0.80 ppm. The proton signals are sum-
marized in Table 3.
Table 3. Summary of identified protons in the 1H spectrum of faurinone.
Title 1 δ (ppm) Multiplicity (J = Hz)
Experimental Reported
H-4 2.31 ddd (3.6, 10.7, 14.4) tt * (10.4, 3.8) [27]
H-5 1.95 dd (10.7, 5.01) dd * (10.4, 4.8) [27]
H-10 (Me) 1.02 s 1.03 s [25]
H-(12,13) (Me) 0.74 d (6.12)
0.80 d (6.12)
0.74 d (5)
0.81 d (5) [25]
H-15 (Me) 2.19 s 2.18 s [25]
* chemical shifts were not reported.
Figure 1. Chemical structure of faurinone.
Plants 2022,11, 2606 5 of 16
2.2. Structural Elucidation of Compound 1
Compound
1
(5 mg) was identified as faurinone (Figure 1) by MS,
1
H-NMR, and
13
C-NMR spectroscopic techniques, and the spectroscopic data were compared with the
previously published literature, as presented in the following sections.
2.2.1. Mass Spectrometry
The ion fragment peaks obtained in the MS spectrum (Figure 2) were compared to the
data first reported by Hikino et al. [
25
]. The molecular ion [M
+
] at m/z= 222.3 suggested
the molecular formula to be C
15
H
26
O, which translates to the three degrees of unsaturation
as it is for faurinone. A base peak was found at m/z= 123.2, which is typical of isomers of
faurinone as described by Weyerstahl et al. [26].
Plants 2022, 11, x FOR PEER REVIEW 5 of 16
of H. petiolare. The structural elucidation of compound 2 (RI = 1499) is also discussed fur-
ther below.
2.2. Structural Elucidation of Compound 1
Compound 1 (5 mg) was identified as faurinone (Figure 1) by MS, 1H-NMR, and 13C-
NMR spectroscopic techniques, and the spectroscopic data were compared with the pre-
viously published literature, as presented in the following sections.
Figure 1. Chemical structure of faurinone.
2.2.1. Mass Spectrometry
The ion fragment peaks obtained in the MS spectrum (Figure 2) were compared to
the data first reported by Hikino et al. [25]. The molecular ion [M+] at m/z = 222.3 suggested
the molecular formula to be C15H26O, which translates to the three degrees of unsaturation
as it is for faurinone. A base peak was found at m/z = 123.2, which is typical of isomers of
faurinone as described by Weyerstahl et al. [26].
Figure 2. Mass spectrum of faurinone.
2.2.2. 1H NMR Spectroscopy
The 1H NMR spectrum showed four methyl signals, and two of them appear at 0.74
ppm and 0.80 ppm (d, 6.2 Hz, Me-12, Me-13), and the other two methyl groups appear as
singlets at 1.02 (Me-10) and 2.19 (Me-15); the latter is adjacent to a carbonyl group (C-14)
(Figure 1). The spectrum also showed a proton resonating at 2.31 (ddd, 3.6, 10.7, 14.4 Hz)
and assigned to H-4, and another proton at 1.95 (dd, 5.0, 10.7 Hz) and assigned to H-5, in
addition to a cluster of protons between 1.92 and 0.80 ppm. The proton signals are sum-
marized in Table 3.
Table 3. Summary of identified protons in the 1H spectrum of faurinone.
Title 1 δ (ppm) Multiplicity (J = Hz)
Experimental Reported
H-4 2.31 ddd (3.6, 10.7, 14.4) tt * (10.4, 3.8) [27]
H-5 1.95 dd (10.7, 5.01) dd * (10.4, 4.8) [27]
H-10 (Me) 1.02 s 1.03 s [25]
H-(12,13) (Me) 0.74 d (6.12)
0.80 d (6.12)
0.74 d (5)
0.81 d (5) [25]
H-15 (Me) 2.19 s 2.18 s [25]
* chemical shifts were not reported.
Figure 2. Mass spectrum of faurinone.
2.2.2. 1H NMR Spectroscopy
The
1
H NMR spectrum showed four methyl signals, and two of them appear at
0.74 ppm and 0.80 ppm (d, 6.2 Hz, Me-12, Me-13), and the other two methyl groups appear
as singlets at 1.02 (Me-10) and 2.19 (Me-15); the latter is adjacent to a carbonyl group (C-14)
(Figure 1). The spectrum also showed a proton resonating at 2.31 (ddd, 3.6, 10.7, 14.4 Hz)
and assigned to H-4, and another proton at 1.95 (dd, 5.0, 10.7 Hz) and assigned to H-5,
in addition to a cluster of protons between 1.92 and 0.80 ppm. The proton signals are
summarized in Table 3.
Table 3. Summary of identified protons in the 1H spectrum of faurinone.
Title 1
δ(ppm) Multiplicity (J= Hz)
Experimental Reported
H-4 2.31 ddd (3.6, 10.7, 14.4) tt * (10.4, 3.8) [27]
H-5 1.95 dd (10.7, 5.01) dd * (10.4, 4.8) [27]
H-10 (Me) 1.02 s1.03 s[25]
H-(12,13) (Me) 0.74 d(6.12)
0.80 d(6.12)
0.74 d(5)
0.81 d(5) [25]
H-15 (Me) 2.19 s2.18 s[25]
* chemical shifts were not reported.
2.2.3. 13C NMR Spectroscopy
The
13
C NMR spectrum revealed fifteen carbons that were classified according to
DEPT-135 into four methyls, five methylene, four methines, one fully substituted carbon,
and a carbonyl group. The peak at
δ
212.1 ppm indicated the presence of the carbonyl
carbon (C-14) previously reported at
δ
211.8 ppm [
27
]. The remaining peaks from
δ
51.9 to
21.4 ppm were representative of typical saturated (sp
3
hybridized) carbons unaffected by
electronegative atoms, as found in the structure of faurinone. Besides the carbonyl carbon,
the absent peak at
δ
41.6 ppm in the DEPT-135 confirmed the presence of a quaternary
carbon at position 1, as reported at the same ppm value by Bos et al. [
27
] and Weyer-
stahl et al. [
26
]. The typical shifts in the saturated carbons in the
13
C NMR of compound
1
obtained experimentally were in close agreement with the reported values of faurinone [
26
].
Plants 2022,11, 2606 6 of 16
The summary of the comparison with the literature on chemical shifts is presented in
Table 4.
Table 4. Experimental and literature values [27] of 13C NMR shifts (ppm) of faurinone.
Carbon * Multiplicity Compound 1 Faurinone
CH2t21.38 21.4
C-12 q22.23 22.2
C-13 q23.05 23.0
CH2t26.54 26.5
C-11 d29.04 29.0
C-15 q29.34 29.2
CH2t30.88 30.8
CH2t32.35 32.4
C-10 q36.67 36.6
C-1 s41.60 41.6
C-4 d47.31 47.3
C-5 d49.31 49.3
C-6 d50.94 50.9
C-14 s212.10 211.8
* The methylene groups could not be assigned in this work.
2.3. Structural Elucidation of Compound 2
Compound
2
(RI = 1499) could not be fully identified. Weak signals obtained in
NMR analysis did not permit complete analysis. However, its mass spectrum showed
similarities with the published data. In the literature, compound
2
was identified as
β
-
dihydroagarofuran (RI = 1499) in the essential oil of H. petiolare from South Africa as one
of the major compounds (19.45–25.65%) by Giovanelli et al. [
5
]. The mass spectrum of
compound
2
(Figure 3) shows similarity to that of
β
-dihydroagarofuran [
22
] with the
molecular ion [M
+
] = 222.3, the base peak m/z= 207.3, and other peaks m/z= 189.2 and,
m/z= 149.2.
Plants 2022, 11, x FOR PEER REVIEW 6 of 16
2.2.3. 13C NMR Spectroscopy
The 13C NMR spectrum revealed fifteen carbons that were classified according to
DEPT-135 into four methyls, five methylene, four methines, one fully substituted carbon,
and a carbonyl group. The peak at δ 212.1 ppm indicated the presence of the carbonyl
carbon (C-14) previously reported at δ 211.8 ppm [27]. The remaining peaks from δ 51.9
to 21.4 ppm were representative of typical saturated (sp3 hybridized) carbons unaffected
by electronegative atoms, as found in the structure of faurinone. Besides the carbonyl car-
bon, the absent peak at δ 41.6 ppm in the DEPT-135 confirmed the presence of a quater-
nary carbon at position 1, as reported at the same ppm value by Bos et al. [27] and
Weyerstahl et al. [26]. The typical shifts in the saturated carbons in the 13C NMR of com-
pound 1 obtained experimentally were in close agreement with the reported values of
faurinone [26]. The summary of the comparison with the literature on chemical shifts is
presented in Table 4.
Table 4. Experimental and literature values [27] of 13C NMR shifts (ppm) of faurinone.
Carbon* Multiplicity Compound 1 Faurinone
CH2 t 21.38 21.4
C-12 q 22.23 22.2
C-13 q 23.05 23.0
CH2 t 26.54 26.5
C-11 d 29.04 29.0
C-15 q 29.34 29.2
CH2 t 30.88 30.8
CH2 t 32.35 32.4
C-10 q 36.67 36.6
C-1 s 41.60 41.6
C-4 d 47.31 47.3
C-5 d 49.31 49.3
C-6 d 50.94 50.9
C-14 s 212.10 211.8
* The methylene groups could not be assigned in this work.
2.3. Structural Elucidation of Compound 2
Compound 2 (RI = 1499) could not be fully identified. Weak signals obtained in NMR
analysis did not permit complete analysis. However, its mass spectrum showed similari-
ties with the published data. In the literature, compound 2 was identified as β-dihy-
droagarofuran (RI = 1499) in the essential oil of H. petiolare from South Africa as one of the
major compounds (19.45–25.65%) by Giovanelli et al. [5]. The mass spectrum of compound
2 (Figure 3) shows similarity to that of β-dihydroagarofuran [22] with the molecular ion
[M+] = 222.3, the base peak m/z = 207.3, and other peaks m/z = 189.2 and, m/z = 149.2.
Figure 3. Mass spectrum of compound 2.
Figure 3. Mass spectrum of compound 2.
2.4. Antibacterial Activity: Minimum Inhibitory Concentration (MIC) Using the Broth
Microdilution Method
Cutaneous infections pose global health problems [
28
]. Preventing and treating bac-
terial skin infections can necessitate topical antimicrobials, which may be an antibiotic.
Theoretically, a topical antibiotic presents advantages over systemic administration such as
delivering high concentrations of active ingredients to the affected site and less systemic
toxicity [
29
]. The Helichrysum EOs were tested against three skin pathogenic bacteria,
S. aureus,E. coli, and P. aeruginosa. The MICs of the EOs were taken as the lowest con-
centration inhibiting visible bacterial growth of strains tested, as detected by the INT
(p-iodonitrotetrazolium chloride) reagent and expressed in mg/mL (presented in Table 5).
Plants 2022,11, 2606 7 of 16
Table 5. MICs (mg/mL) of Helichrysum EOs and control.
Sample Micro-Organisms
S. aureus E. coli P. aeruginosa
H. petiolare >25.6 12.8 12.8
H. odoratissimum 12.8 12.8 12.8
H. cymosum >25.6 12.8 12.8
Ampicillin <0.2 <0.2 R *
* R = resistant.
The MICs of the Helichrysum EOs were found between 12.8 and 25.6 mg/mL, whereas
the positive control ampicillin exhibited MIC values lower than 0.2 mg/mL (P.aeruginosa
is resistant to ampicillin). Compared to the threshold MIC value of 2 mg/mL for EOs [
30
],
the results indicate that the EOs possess poor antibacterial activities.
2.5. Antioxidant Capacities
The magnitude of antioxidative capacities of the Helichrysum essential oils was evalu-
ated by four
in vitro
antioxidant capacity assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH),
2,2
0
-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant
power (FRAP), and oxygen radical absorbance capacity (ORAC) assays. The results are
summarized in Table 6.
Table 6. Antioxidant capacities of Helichrysum EOs in the DPPH, ABTS, FRAP, and ORAC assays.
Sample
DPPH * ABTS * FRAP * ORAC *
mg/mL % RSA6
min ±SD
% RSA6
min ±SD
TEAC (µmol
TE/L ±SD) mg/mL FRAP (µmol
AAE/L ±SD)
ORAC (µmol
TE/L ±SD)
H. petiolare
2 14.41 ±0.51 84.42 ±0.43 9131.4 ±45.5
2−750.5 ±11.5 6587.3 ±126.3
1 8.98 ±0.40 77.96 ±0.71 8445.9 ±76.1
0.5 5.29 ±0.20 67.08 ±0.76 7281.7 ±81.5
H. odoratissimum
2 4.09 ±0.95 60.74 ±1.24 6603.8 ±132.6
23026.6 ±184.6 6624.8 ±10.8
1 1.27 ±0.43 46.72 ±0.96 5103.8 ±102.7
0.5 −0.57 ±0.03 28.16 ±0.84 3117.5 ±89.5
H. cymosum
2 5.58 ±0.61 40.26 ±0.33 4412.2 ±35.7
2897.4 ±173.1 6549.7 ±99.9
1 3.14 ±0.00 23.69 ±0.70 2639.6 ±75.3
0.5 1.58 ±0.51 10.70 ±0.22 1250.1 ±23.9
Trolox®
2 94.94 ±0.02
- - - - -
1 94.78 ±0.06
0.5 94.45 ±0.04
Gallic acid
2
–
97.97 ±0.13 605,840 ±27811.3
2635,500 ±
4070.9 –
1 97.96 ±0.16 355,740 ±7127.6
0.5 98.05 ±0.03 195,220 ±6241.5
EGCG ** – – – – 2 – 26,904 ±328.2
* Average values of triplicate measurements (n = 3); RSA: radical scavenging activity; SD = standard devi-
ation; RSD = relative standard deviation; TE: Trolox
®
equivalent; AAE: ascorbic acid equivalent. ** EGCG:
(-)-epigallocatechin gallate.
In the DPPH assay, the EOs were found to possess a very low percentage of radi-
cal scavenging activities (% RSA). The highest % RSA was exhibited by H.petiolare EO
as 14.41
±
0.51% at 2 mg/mL against 94.94
±
0.02% for Trolox
®
positive control at the
same concentration. In the ABTS assay, H.petiolare EO exhibited the highest antioxidant
capacity with 84.42
±
0.43% and 9131.4
±
45.5
µ
mol TE/L at 2 mg/mL. The % RSA was
close to that of the gallic acid positive control, found as 97.97
±
0.13% at the same con-
centration. In the FRAP and ORAC assays, H.odoratissimum EO was found to exhibit
the highest antioxidant capacities at 2 mg/mL with 3026.6
±
184.6
µ
mol AAE/L and
Plants 2022,11, 2606 8 of 16
6624.8
±
10.8
µ
mol TE/L, respectively. In these two assays, the positive controls exhibited
10- and 100-fold higher capacities than the highest-performing essential oil. Addition-
ally, H.petiolare EO was found inactive in the FRAP assay with an equivalence value of
−750.5 ±11.5 µmol AAE/L
, whereas H.cymosum EO was the second-best performing EO
in the assay (
897.4 ±173.1 µmol AAE/L
). Overall, using the reference controls as bench-
marks, the results suggest that the EOs possess low-to-moderate antioxidant capacities.
2.6. Tyrosinase Inhibition
Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase, is a copper-containing
enzyme that has a central role in the production of melanin, the pigment responsible
for the color of the skin. It catalyzes the first two steps of the multiphase process of
melanogenesis, the biosynthesis of melanin. As tyrosinase inhibitors are increasingly
prevalent in cosmeceuticals aiming to reduce hyperpigmentation [
31
], it is important to
investigate EOs as natural options in this regard. In the present work, the selected essential
oils were tested in the tyrosinase inhibition assay exploring the monophenolase activity
of the enzyme by monitoring the absorbance of L-DOPA (
λ490
nm) using L-tyrosine as a
substrate. The essential oils were tested at 200
µ
g/mL and 50
µ
g/mL and compared to
kojic acid, a standard tyrosinase inhibitor used in cosmetics, at the same concentrations.
The results were obtained, as presented in Table 7.
Table 7.
Summary of the tyrosinase inhibition assay results for the Helichrysum EOs at 200
µ
g/mL
and 50 µg/mL.
Samples Tyrosinase Inhibition (%)
at 200 µg/mL at 50 µg/mL
H. petiolare 62.66 ±11.96 22.22 ±1.46
H. odoratissimum 63.30 ±2.35 28.62 ±0.30
H. cymosum 61.59 ±10.45 25.42 ±1.80
Kojic acid 96.24 ±3.62 98.34 ±0.80
Overall, the EOs exhibited significantly lower tyrosinase inhibition values than kojic
acid at 200 and 50
µ
g/mL. At both concentrations, the EOs performed near equally in the
range of 61.59
±
10.45 to 63.30
±
2.35% at 200
µ
g/mL and 22.22
±
1.46 to 28.62
±
0.30%
at 50
µ
g/mL, whereas kojic acid was found as 96.24
±
3.62% and 98.34
±
0.80%
µ
g/mL
at respective concentrations. Since the enzyme inhibition is concentration dependent,
the values obtained indicate that collectively the Helichrysum EOs are relatively weak
tyrosinase inhibitors.
2.7. Sun Protection Factor (SPF)
Solar UV rays are the protagonists in external cutaneous aging in humans and provoke
a myriad of dermatological complications including skin cancer [
32
–
35
]. Herein, the SPF
values of the Helichrysum essential oils were determined by measuring the absorbance of
dilute hydroalcoholic solutions of EOs (0.1% v/v) at 290–320 nm at 5 nm intervals then
calculated using the equation given by Mansur et al. [
36
]. The results are presented in
Table 8.
Plants 2022,11, 2606 9 of 16
Table 8.
Spectrophotometric absorbances of hydroalcoholic aliquots of the Helichrysum essential oils
and their calculated SPF.
Wavelength (nm) EE(λ)×I(λ) ** Employed Absorbance *
H. petiolare EO H. odoratissimum EO H. cymosum EO
290 0.0150 0.2999 ±0.0060 0.0632 ±0.0020 0.2955 ±0.0054
295 0.0817 0.2813 ±0.0079 0.0436 ±0.0048 0.2244 ±0.0085
300 0.2874 0.2129 ±0.0165 0.0354 ±0.0024 0.1259 ±0.0063
305 0.3278 0.1290 ±0.0112 0.0283 ±0.0011 0.0746 ±0.0038
310 0.1864 0.0796 ±0.0070 0.0250 ±0.0015 0.0478 ±0.0024
315 0.0837 0.0548 ±0.0057 0.0235 ±0.0005 0.0342 ±0.0015
320 0.0180 0.0384 ±0.0036 0.0208 ±0.0010 0.0254 ±0.0010
Calculated SPF 1.511 0.309 0.956
* Values represent mean absorbance values
±
standard deviation of triplicate measurements, n = 3; ** constant
values of erythemogenic effect (EE) of radiation with wavelength
λ×
solar intensity (I) at wavelength
λ
determined
by Sayre et al. [37].
According to the study, the SPF of the essential oils was found to be 1.511, 0.956, and
0.309, for H.petiolare,H.cymosum, and H.odoratissimum Eos, respectively. As compared to
the previously reported threshold SPF value of 2 [
38
,
39
], the results may not establish these
Helichrysum EOs noteworthy for sunscreen formulations.
3. Materials and Methods
3.1. Plant Material
Two out of the three species studied, H.petiolare (6.0 kg) and H.cymosum (3.5 kg), were
wildly harvested from the University of the Western Cape campus in December 2018. Their
voucher specimens were authenticated by Hlokane Mabela and deposited at the Horticul-
tural Sciences Department of the Cape Peninsula University of Technology. The essential
oil of H.odoratissimum was purchased directly from a local South African establishment
(Pure Indigenous [Indigo Trading] African Helichrysum, 100% Organic Essential Oil).
3.2. Extraction of Essential Oil
The fresh aerial parts (leaves, stems, and flowers) of the plants were subjected to
hydrodistillation using the Clevenger-type apparatus for 3 h as per the European Pharma-
copeia guidelines [
40
]. The essential oil was recovered by decantation in glass vials and
stored in the dark at 4
◦
C until further use. The oil yield was expressed as the average
percentage of volume in mL per weight in g (% v/w) of triplicate analyses.
3.3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The GC-MS analyses were carried out according to the procedure previously reported
by Kuiate et al. [
19
] with some adjustments. The instrument consisted of an Agilent
GC-7820A fitted with an HP-5MS fused silica column (30 m
×
0.25 mm i.d.
×
0.25
µ
m
film thickness) and coupled with an Agilent 5977E mass selective compartment (Agilent
Technologies, Inc. USA). The oven temperature was programmed at 50
◦
C for 5 min,
50–220 ◦C
at a rate of 2
◦
C
·
min
−1
then 220
◦
C temperature hold for 5 min for the first ramp.
For the second ramp, the temperature was set to 300
◦
C at a rate of 25
◦
C
·
min
−1
. Helium
was used as a carrier gas at 1 mL.min
−1
flow rate and pressure of 7.6522 psi. Sample
injection of 1
µ
L of 1% (v/v) solution diluted in n-hexane was splitless and operated at 250
◦
C. A reference standard of homologous n-paraffin series of C
8
-C
20
(Sigma-Aldrich
®
, Cat
no. 04070) was prepared and co-injected under identical experimental conditions as those
for samples for the determination of retention indices (RIs). The MS spectra were obtained
on electron impact at 70 eV scanning from 30.0 to 650 m/z.
The identification of the constituents was achieved by computerized matching (MassHunter
software, Agilent Technologies, Inc., Santa Clara, USA) of each mass spectrum generated
with those stored in the instrument’s built-in mass spectral libraries (National Institute of
Plants 2022,11, 2606 10 of 16
Standards and Technologies, NIST), then by comparing the experimental RIs [
41
] and gener-
ated mass spectra with those of the NIST online data collection [
24
] and the literature [
22
,
23
].
The relative amounts of individual constituents were calculated automatically based on the
total ion count detected by the GC-MS and expressed as percentage composition.
3.4. Isolation and Purification of H. Petiolare Essential Oil’s Components
A silica slurry of 3.547 g of H.petiolare EO was packed in a silica gel column (
40 cm ×4 cm
).
The separation was performed using a gradient elution of hexane: ethyl acetate (Hex: EA) in
order of increasing polarity from 100:0 to 94:6 (Hex: EA). The separation yielded 53 fractions
(20–50 mL) labeled as 1–53 which were further concentrated at 45 ◦C.
Fraction 29 was subjected to preparative thin-layer chromatography (prep TLC) to
purify compound
1
. Fraction 29 (20 mg) was dissolved in 750
µ
L of hexane then 250
µ
L
was loaded on three individual silica gel 60 F254 TLC plates (20 cm
×
10 cm; Merck,
Germany). Subsequently, the plates were developed at 97:3 hexane: ethyl acetate (double
run). Compound
1
was marked under
λ254
nm, scrapped off, and eluted with hexane.
Compound
2
was purified from fraction 31 by prep TLC at 92:8 hexane: ethyl acetate
(double run) as previously described.
The MS spectra of compound
1
and compound
2
were obtained by dissolving 0.5 mg
in 300
µ
L of hexane and analyzing the samples by the method previously described. Their
1
H NMR and
1
3C NMR spectra were recorded at 20
◦
C using deuterated chloroform on a
Bruker Avance
™
400 MHz spectrometer (Germany). The chemical shifts of
1
H and
13
C in
ppm (δ) were determined with tetramethylsilane (TMS) used as an internal reference.
3.5. Antibacterial Assay
3.5.1. Micro-Organisms
The essential oils were tested against three skin pathogenic bacterial strains. These
were one Gram-positive strain, wild-type (WT) S.aureus, and two Gram-negative strains,
wild-type (WT) E.coli and wild-type (WT) P.aeruginosa.
3.5.2. Preparation of Media
The bacterial species were resuscitated by inoculation into brain heart infusion (BHI)
broth (Oxoid UK, Cat. no. CM1135) and incubated at 37
◦
C for 24 h, after which, each
strain was streaked aseptically onto tryptone soya agar for a single colony formation
and incubated at 37
◦
C for 24 h. The cell suspensions were performed in sterile saline,
standardized at 0.5 McFarland standard (Remel
™
, Kansas, Cat. no. R20410) at 1.5
×
10
8
colony forming units (CFU)/mL. Then, the working suspensions were obtained by a second
1:100 dilution onto BHI to approximately 106 CFU/mL.
3.5.3. Broth Microdilution Susceptibility Assay
The broth microdilution test was performed, as previously described by Lourens et al. [
21
]
and Sartoratto et al. [
42
] with slight adjustments. An EO stock solution of 51.2 mg/mL was
prepared with a BHI:dimethyl sulphoxide (DMSO) (1:1) solution. In a 96-well plate, 100
µ
L
of BHI was added to the experimental wells in triplicate except in well 1. Then, 200
µ
L
of EO stock solution was added to well 1, from which a serial dilution was performed to
the last experimental well. Subsequently, 100
µ
L of cell suspension was added to establish
the two-fold 25.6–0.2 mg/mL sample concentration range and a bacterial cell suspension
of approximately 5
×
10
5
CFU/mL. The plate was incubated at 37
◦
C for 20 h. After
incubation, the antimicrobial activity was detected by adding 40
µ
L of 0.2 mg/mL INT
(Sigma-Aldrich
®
, Cat no. I10406) aqueous solution. The plates were incubated at 37
◦
C
for 1 h. The MICs were defined as the lowest concentration of essential oil that inhibited
visible growth, as indicated by the color change of INT. Ampicillin (Sigma-Aldrich
®
, Cat
No. A9393) was used as a positive control.
Plants 2022,11, 2606 11 of 16
3.6. Antioxidant Capacity Assays
3.6.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay
The DPPH assay was performed according to the method previously described by
Bondet et al. [
43
] with slight modifications. In a clear 96-well plate, 275
µ
L of DPPH reagent
(Sigma-Aldrich
®
, Cat no. D9132) (absorbance of 2.0
±
0.1 at 517 nm) was added to 25
µ
L
of EO sample and Trolox
®
(Sigma-Aldrich
®
, Cat no. 238831) positive control (2.0, 1.0, and
0.5 mg/mL). For the blank, ethanol was added instead of the sample. The total volume
of the assay was 300
µ
L. The absorbance was read at 517 nm and 37
◦
C at the 6 min time
point. The EO/Trolox
®
sample was read in triplicate (n = 3). The % RSA of the samples
was calculated using Equation (1).
% RSA6 min =1−Asam ple
Ablank
(1)
where A
sample
is the absorbance signal of the EO sample and A
blank
is the absorbance signal
of the DPPH solution (ethanol in place of the sample) at 517 nm and 6 min. The results were
expressed as the mean percentage of triplicate measurements (±standard deviation, SD).
3.6.2. 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) Assay
The ABTS assay was performed according to Re et al. [
44
] with slight modifications.
The ABTS radical cation (ABTS
•
+) (Sigma-Aldrich
®
, Cat no. A1888) stock reagent was
produced by reacting 5 mL of freshly prepared 7 mM ABTS solution with 88
µ
L of a freshly
prepared 140
µ
M K
2
S
2
O
8
(Merck, Cat no. 105091) then allowing the mixture to sit overnight
for 16 h in the dark at room temperature. In a clear 96-well plate, 275
µ
L of ABTS
•
+ reagent
(absorbance of 2.0
±
0.1 at 734 nm) was added to 25
µ
L of each ethanolic Trolox
®
working
standard (50
µ
M, 100
µ
M, 150
µ
M, 250
µ
M, and 500
µ
M) and EO sample (2.0, 1.0, and
0.5 mg/mL). Gallic acid (Sigma-Aldrich
®
, Cat No. G7384) was used as a positive control.
For the blank, ethanol was added instead of the sample. The total volume of the assay was
300
µ
L. The absorbance was read at 734 nm and 37
◦
C at the 6 min time point. The EO
sample, working standard, and gallic acid sample were read in triplicate (n = 3). The %
RSA of each EO or positive control working solution was calculated using Equation (1),
where A
sample
is the absorbance signal of the EO sample/positive control and A
blank
is the
absorbance signal of the ABTS
•
+ solution (ethanol in place of the sample) at 734 nm. The
results were expressed as the mean percentage of triplicate measurements (
±
standard
deviation, SD). The Trolox
®
equivalent capacity assay (TEAC) values were reduced from
the linear regression (R
2
= 0.9980) of Trolox
®
concentrations (
µ
M) and the absorbance
readings at 734 nm at 6 min and expressed as mean (
±
SD) of triplicate measurements in
µmol Trolox®equivalents per litre of the sample tested (µmol TE/L).
3.6.3. Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC assay was performed according to the method described by Prior et al. [
45
]
with slight modifications. In a black 96-well plate, 12
µ
L of the Trolox
®
working solutions
(83
µ
M, 167
µ
M, 250
µ
M, 333
µ
M, and 417
µ
M were prepared with phosphate buffer at pH
7.4) and EO sample (2.0 mg/mL) were added in triplicate (n = 3). Subsequently, 138
µ
L
of fluorescein solution was added followed by 50
µ
L of freshly prepared by dissolving
2,2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) (Sigma-Aldrich
®
, Cat no.
440914) in phosphate buffer (150 mg of AAPH in 6 mL buffer). (-)-Epigallocatechin gallate
(EGCG) (Sigma-Aldrich
®
, Cat no. E4143) was used as a positive control. For the blank,
the phosphate buffer was added in place of the sample. The total volume of the assay
was 200
µ
L and the temperature was set at 37
◦
C. Readings of the EO/EGCG samples
(2.0 mg/mL) and Trolox
®
working standard solutions were taken using the excitation
wavelength set at 485 nm and the emission wavelength at 530 nm for 2 h at 1 min reading
interval. After analysis, the data points of the blank, EO sample, EGCG sample, and
Trolox
®
working standards were summed up over time to obtain the area under the fluo-
Plants 2022,11, 2606 12 of 16
rescence decay curve (AUC). The ORAC values were calculated using the linear regression
(R
2
= 0.9861) equation (Y = aX + c) between Trolox
®
concentration (Y) (
µ
M) and the net
area (blank-corrected) under the fluorescence decay curve (X). The results were expressed
as the mean (
±
SD) of triplicate measurements in
µ
mol of Trolox
®
equivalents per litre of
the sample tested (µmol TE/L).
3.6.4. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was conducted as recommended by Benzie and Strain [
46
] with slight
adjustments. Firstly, the fresh blue FRAP reagent was prepared by mixing 30 mL of acetate
buffer, 3 mL of 2,4,6-tris [2-pyridyl]-s-triazine (TPTZ) (Merck, Cat no. T1253) with 3 mL
of FeCl3solution and 6.6 mL of distilled water. Then, an L-ascorbic acid (Sigma-Aldrich®,
Cat no. A5960) standard series of 50
µ
M, 100
µ
M, 200
µ
M, 500
µ
M, and 1000
µ
M was
prepared from a 1 mM of L-ascorbic acid stock solution in distilled water. Lastly, in a clear
96-well plate, 300
µ
L of the FRAP reagent was added to 10
µ
L of L-ascorbic acid working
standard solutions and EO sample (2.0 mg/mL) in triplicate (n = 3). Gallic acid was used
as a positive control. For the blank, the phosphate buffer (pH 3.6) was added instead of
the sample. The total volume of the assay was 310
µ
L. The absorbance of TPTZ-Fe (II) in
the samples was read at 593 nm at 37 ◦C for 30 min. The results were calculated using the
linear regression (R
2
= 0.9965) of the L-ascorbic acid (AA) standard series concentrations
(
µ
M) and absorbance signals expressed as mean (
±
SD) of triplicate measurements in
µ
mol
L-ascorbic acid equivalents per litre of the sample tested (µmol AAE/L).
3.7. Anti-Tyrosinase Assays
3.7.1. Essential Oil Samples and Positive Control Preparation
EO working solution (10 mg/mL) was prepared with a DMSO: Tween
®
20 (1:1) solution
to facilitate dispersion of the oils which was further diluted to 1 mg/mL working solution
with methanol. A 10 mg/mL kojic acid working solution was made up of 100% DMSO and
then diluted to 1 mg/mL with methanol.
3.7.2. Tyrosinase Inhibition Assays
The tyrosinase inhibition assay was performed as described previously by Popoola et al. [
47
]
and Cui et al. [
48
] with slight modifications. The concentrations of the EO sample and
kojic acid chosen, 200
µ
g/mL and 50
µ
g/mL, respectively, were achieved by setting up
the 96-well plate in the following order: 70
µ
L of the sample (1 mg/mL) then 30
µ
L of
tyrosinase enzyme (500 U/mL). Each concentration of the sample and positive control
was set up in two different wells whereby, one of the wells received enzyme and the other
well had no enzyme volume added. All volume deficits were compensated by adding
excess buffer. The negative controls, 10% v/v of 1:1 DMSO: Tween
®
20 in methanol for
the EO and 10% v/v DMSO in methanol for kojic acid were treated the same way. The
plate was incubated at 37
◦
C (
±
2.0
◦
C) for 5 min. Thereafter, the reaction was initiated by
adding 110
µ
L of L-tyrosine (2 mM) and subsequently incubated at 37
◦
C (
±
2.0
◦
C) for
30 min. The absorbance of L-DOPA was read at 490 nm on a Multiskan
™
spectrum plate
reader (Thermo Fisher Scientific, Waltham, MA, USA). Two independent experiments were
carried out in triplicate and the percentage of tyrosinase inhibition was calculated using
Equation (2).
Tyrosinase inhibition (%) = (A−B)−(C−D)
(A−B)×100 (2)
where Ais the negative control with an enzyme, Bis the negative control without enzyme,
Cis the EO sample or kojic acid with enzyme and Dis the EO sample or kojic acid without
enzyme. The inhibition percentages were expressed as the mean (
±
standard deviation) of
duplicate measurements. One-way ANOVA was used to compare the absorbance values of
the two groups (p< 0.05).
Plants 2022,11, 2606 13 of 16
3.8. Sun Protection Factor (SPF)
The protocol used for this assay was conducted as per Kaur and Saraf [
49
]. The
solubility of the EO in different ratios of ethanol and water was tested by taking 10% to
50% of ethanol in distilled water. The maximum solubility was detected at ethanol: water
in a 40:60 ratio, above which turbidity developed. Thereafter, an initial stock solution of 1%
v/v was prepared by making up 10
µ
L of the EO to 1 mL of ethanol:water (40:60). Then, out
of this stock, 0.1% v/v in 40:60 ethanol: water was prepared. Subsequently, 100
µ
L of the
EO aliquot and the blank (ethanol: water, 40:60) were injected into the 96-well plate and
read in triplicate (n = 3) over the 290 nm-320 nm range at a 5 nm interval. The SPF value of
the essential oil was calculated following the method by Mansur et al. [
36
]. The mean of
the observed absorbance values was multiplied by their respective erythemogenic effect
(EE) times solar intensity at wavelength
λ
values, EE (
λ
)
×
I (
λ
), then their summation was
obtained and multiplied with the correction factor (=10). The calculation is described as
Equation (3).
SPFspectrophotometric =CF ×
320
∑
290
EE (λ)×I(λ)×Abs (λ)(3)
where CF is the correction factor (=10), EE (
λ
) is the erythemogenic effect of radiation
at wavelength
λ
, I (
λ
) is the solar intensity at wavelength
λ
, and Abs (
λ
) represents the
spectrometric absorbance value at wavelength
λ
. The values of EE (
λ
)
×
I (
λ
) are constant
values that were determined by Sayre et al. [37], as shown in Table 9.
Table 9. Relationship between erythemogenic effect and radiation intensity.
Wavelength (nm) EE ×I (Normalized)
290 0.0150
295 0.0817
300 0.2874
305 0.3278
310 0.1864
315 0.0837
320 0.0180
Total 1
4. Conclusions
The present work aimed to investigate the chemical composition of three South African
Helichrysum essential oils and to explore their biological activities in the quest to find
medicated fragrant ingredients to be used in cosmetic formulations. The GC-MS and
NMR analyses revealed that their major constituents were hydrocarbons and oxygenated
monoterpenes (
α
-pinene, 29.82% in H. cymosum; 1,8-cineole, 17.44% in H. odoratissimum)
and oxygenated sesquiterpenes (faurinone, 20.66% in H. petiolare). This is the first report
elucidating faurinone in the essential oil of H. petiolare. The EOs of all the three reported
species of Helichrysum in this study had
α
-pinene and 1,8-cineole in common and as one of
the major phytoconstituents.
α
-pinene and 1,8-cineole are associated with pharmacological
activities such as antimicrobial, antioxidant, and antitumor effects. However, the biological
evaluation of these EOs did not correspond to the reported pharamacological activities of
these phytoconstituents. This investigation reiterates the fact that the bioactive functional
property of a natural product such as essential oil cannot be linked to a single compound or
a group of compounds; rather, it can be a result of the concerted effect of many secondary
metabolites. Among the
in vitro
biological activities, this study is the first to report tyrosi-
nase inhibition and sun protection factor of these Helichrysum essential oils. According to
the results obtained, the essential oils possessed low antibacterial, anti-tyrosinase activities
and photoprotection but moderately promising antioxidant capacities. This study estab-
Plants 2022,11, 2606 14 of 16
lishes that H. petiolare,H. odoratissimum, and H. cymosum essential oils have great potential
to complement antioxidant formulations.
Author Contributions:
All the authors have participated and contributed substantially to this
manuscript. S.O.A.: methodology, investigation, data curation, writing the original draft. R.S.:
supervision, validation, Writing—Review and editing. C.W.J.A.: methodology, validation, resources-
biology-antioxidants. J.L.M.: methodology, validation, resources-biology-antioxidants. A.A.H.:
conceptualization, methodology, supervision, resources, Writing—Review and editing. All authors
have read and agreed to the published version of the manuscript.
Funding:
This study was supported by National Research Foundation, South Africa (grant number
106055), and “The APC was funded by the Cape Peninsula University of Technology”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data supporting reported results can be found at https://etd.cput.
ac.za/handle/20.500.11838/3340?mode=simple (accessed on 30 September 2022).
Acknowledgments:
Thanks to Hlokane Mabela of the Horticultural Sciences Department of the Cape
Peninsula University of Technology for identification of plants.
Conflicts of Interest: The authors declare no conflict of interest.
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