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International Journal of Medicinal Mushrooms, 20(7):623–636 (2018)
623
1521-9437/18/$35.00 © 2018 Begell House, Inc. www.begellhouse.com
Cosmetic and Skincare Benets of Cultivated
Mycelia from the Chinese Caterpillar Mushroom,
Ophiocordyceps sinensis (Ascomycetes)
Wai-Yin Cheng,1 Xue-Qin Wei,1 Ka-Chai Siu,1,2 Ang-Xin Song,1,2 & Jian-Yong Wu1,2,*
1Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon,
Hong Kong; 2State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), The Hong Kong
Polytechnic University, Shenzhen Research Institute, Shenzhen, People’s Republic of China
*Address all correspondence to: Jian-Yong Wu, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
Hung Hom, Kowloon, Hong Kong; jian-yong.wu@polyu.edu.hk
ABSTRACT: Mushrooms are potential sources of novel natural cosmeceutical ingredients. This study was conducted
to evaluate the cosmetic (skincare) benets of the valuable medicinal species Ophiocordyceps sinensis (=Cordyceps
sinensis). The mycelial extracts of 2 O. sinensis strains, Cs-HK1 and Cs-4, prepared sequentially with ethyl acetate,
ethanol, and hot water were tested with in vitro assays for tyrosinase-, collagenase-, and elastase-inhibitory activity.
The ethyl acetate extracts of both fungal strains showed potent antityrosinase and antielastase activity, with low half-
maximal inhibitory concentrations (0.14–0.47 mg/mL) comparable to those of the respective reference compounds
(arbutin and epigallocatechin gallate). All mycelial extracts exhibited moderate or signicant anticollagenase activity;
most extracts showed a signicant photoprotective effect with a sun protection factor up to 25. The results from this
study show the potential use of O. sinensis as a source of cosmetic ingredients for skincare applications.
KEY WORDS: anticollagenase, antielastase, antioxidant, antityrosinase, Cordyceps sinensis, medicinal mushrooms
and fungi, mycelial extract, Ophiocordyceps sinensis
ABBREVIATIONS: EGCG, epigallocatechin gallate; EtOAc, ethyl acetate; EtOH, ethanol; HW, hot water; IC50, half-maximal
inhibitory concentration; SPF, sun protection factor; TEAC, Trolox-equivalent antioxidant capacity; UVR, ultraviolet radiation
I. INTRODUCTION
Today, “natural” is a hot selling point for many cosmetic products on the market because of public concerns
with the potential harmful effects of synthetic chemicals on the human body and a general belief that natural
products are safe and healthy. Interest is growing worldwide in the cosmetic benets of bioactive natural
products extracted from medicinal plants and herbs. These medicinal natural products can offer protective
and therapeutic functions such as anti-inammatory, antimicrobial, and antioxidant/antiaging effects, but they
also produce direct skincare effects when applied topically, such as antiwrinkle, skin-whitening/lightening,
skin-moisturizing, and skin-toning benets and protection against UV rays.1,2
Skin aging is generally classied as intrinsic aging, caused by natural physiological processes in the
human body, and extrinsic aging such as photoaging, which is mainly attributed to exposure to ultraviolet
radiation (UVR).3 Oxidative stress caused by reactive oxygen species from endogenous and exogenous sources
is regarded as the primary cause for both intrinsic and extrinsic aging.4 Therefore, the use of synthetic or
natural antioxidants is considered to be the most effective strategy to retard skin-aging processes.4 Sunscreens
can protect skin from UVR and delay the onset and progression of skin aging.5 Synthetic sunscreens may,
however, cause adverse effects such as skin irritation, allergy, and endocrine disruption.6,7 Natural products
including avonoids, anthraquinones, and tannins, which have a structure similar to that of synthetic sun-
screens, have been shown to be capable of absorbing UVR.8–10
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Cheng et al.
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UVR causes melanocytes in the basal layer of the epidermis to produce melanin, a natural pigment that
determines skin color and protects skin against UVR.11,12 Tyrosinase is the enzyme that catalyzes the rst 2
rate-limiting steps of melanin synthesis, namely the hydroxylation of l-tyrosine and the oxidation of l-dopa
to dopaquinone.13 However, abnormal accumulation of melanin leads to skin hyperpigmentation problems
such as freckles, senile lentigines, and melisma.14 To meet the cosmetic need for skin whitening, tyrosinase
inhibitors such as arbutin, hydroquinone, and kojic acid are now widely applied in skin-whitening products
in the cosmetic industry.14 Collagenase is a metalloproteinase responsible for the degradation of collagen,
an important protein for maintaining skin strength and elasticity.15 Elastase is a protease responsible for
the breakdown of elastin, which is an important protein found within the extracellular matrix and is vital
for giving elasticity to skin.16–19 In addition to elastin, elastase can cleave collagen, bronectin, and other
proteins in the extracellular matrix.18,19 Because collagen and elastin are the main structural components of
skin, inhibitors of collagenase and elastase can prevent or slow the formation of skin wrinkles and sagging.
Although most of the natural ingredients in cosmetic and cosmeceutical products are from plants
and animals, edible and medicinal mushrooms have been recognized as a new and promising source of
natural cosmetic ingredients.20,21 Edible and medicinal mushrooms provide a diverse array of bioactive
compounds, including polysaccharides and secondary metabolites, with many health benets such as
antitumor, immunomodulatory, antioxidative, antimicrobial, and anti-inammatory effects.22,23 These fungi
or their constituents have found wide application in functional foods and nutraceutical products. To date,
however, only a relatively small number of medicinal fungi have been applied in commercial cosmetic
products for topical skincare and haircare. Much more research is needed to explore and assess the cosmetic
functions of fungi and their active components for better use of valuable healthful and cosmetic products.
Ophiocordyceps sinensis (Berk.) Sung et al. (=Cordyceps sinensis; Ophiocordicypetaceae,
Ascomycetes), generally known as the Chinese caterpillar mushroom, or Dong-Chong-Xia-Cao in Chinese,
is a medicinal species with numerous of health-promoting and therapeutic effects such as anticancer,
immunomodulatory, antioxidative, antiaging, neuroprotective, and hepatoprotective actions.24 Because
the natural O. sinensis caterpillar mushroom, which is formed of a fruiting body on an insect larva, is
rare and very expensive, mycelial fermentation is widely applied for commercial production of the fungal
components. Although O. sinensis has been applied in some commercial cosmetic products,20,21 the cos-
metic benets of O. sinensis are still not well documented in the literature. Cs-HK1 is a Tolypocladium
fungus isolated from the wild C. sinensis fruiting body. Its mycelial biomass and exopolysaccharides from
mycelial fermentation have shown signicant health benets.25 Cs-4 (Paecilomyces hepiali) is a fungus
ofcially approved by Chinese authorities for use in the commercial production of O. sinensis health foods.
This study was conducted to assess the cosmetic potential of Cs-HK1 and Cs-4 fungi by measuring
their antityrosinase, anticollagenase, antielastase, antioxidant, and sunscreen properties. The mycelial
biomass produced by liquid fermentation was extracted with solvents of different polarities (ethyl acetate,
ethanol, and water) in order to compare the activities of the different extracts.
II. MATERIALS AND METHODS
A. Materials
Cs-4 mycelial powder was provided by Jiangxi Guoyao Ltd. (Nanchang, Jiangxi, China). Mushroom
tyrosinase, l-tyrosine, 4-hydroxyphenyl-β-d-glucopyranoside (arbutin), porcine pancreatic elastase,
N-succinyl-Ala-Ala-Ala-p-nitroanilide, collagenase, N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA),
epigallocatechin gallate (EGCG), and edetate disodium were purchased from Sigma-Aldrich; homosalate,
from Macklin Biochemical (Shanghai, China); and catechin, from Yuanye Biology Ltd. (Shanghai, China).
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Cosmetic Benets of O. sinensis Mycelia 625
The Cs-HK1 mycelial biomass was prepared by mycelial liquid fermentation in our laboratory, as
reported previously.25 Cs-HK1 mycelial biomass maintained on potato dextrose agar medium was inocu-
lated into 50 mL liquid medium in a 250-mL Erlenmeyer ask and incubated as the starter culture for
liquid fermentation. The starter culture broth was transferred into 1-L Erlenmeyer asks, each lled with
250 mL liquid medium (at a 4% v/v inoculation ratio), to start the mycelial fermentation. The liquid medium
comprised 40 g/L glucose, 10 g/L yeast extract, 5 g/L peptone, 1 g/L KH2PO4, and 0.5 g/L MgSO4·7H2O.
The liquid culture or mycelial fermentation occurred at 20°C and 150 rpm on a shaker for 7 days. The
mycelial biomass was recovered from the liquid broth by centrifugation (6000 rpm for 15 minutes)
and freeze-dried.
B. Preparation of Mycelial Extracts
The dry mycelial powders of Cs-HK1 and Cs-4 were sequentially extracted with 3 solvents (from low to
high polarity): absolute ethyl acetate (EtOAc), absolute ethanol (EtOH), and water. The solid-to-solvent
ratio was xed at 1:10 for EtOAc and EtOH extraction and at 1:15 for water extraction; the mixture of solid
and solvent was placed in Erlenmeyer asks. For EtOAc and EtOH extraction, the solid-liquid mixture
was stirred magnetically at 150 rpm and 30°C for 24 hours. The liquid extract was separated from the solid
residues by vacuum ltration, then concentrated by vacuum evaporation, and nally dried completely in
an oven at 55°C, yielding the EtOAc and EtOH extracts. For hot water (HW) extraction, the mixture was
stirred on a hot plate at 97°C for 3 hours, and the liquid extract was collected after centrifugation and
freeze-dried, yielding the HW extract.26,27
C. Analysis of Chemical Components of Mycelial Extracts
The HW extracts of Cs-HK1 and Cs-4 mycelia were dissolved in distilled water for all analyses; the
EtOAc and EtOH extracts were dissolved in ethanol in order to analyze total phenolics and avonoids,
and in distilled water to analyze total carbohydrate and (water-soluble) protein content. All the mixtures
were stirred overnight to achieve complete dissolution. Total carbohydrate content was determined with
the anthrone test, using glucose as the standard,28 and the protein content was determined with the Lowry
method, using bovine serum albumin as the standard.29 Total phenolic content was determined with the
Folin-Ciocalteu assay using gallic acid as the standard.26 Total avonoid content was determined with the
aluminium chloride colorimetric assay using catechin as the standard.30
D. Enzyme Activity Assays of Mycelial Extracts
For the enzymatic inhibition assays, the HW extract samples were dissolved in the buffer solutions; the
EtOAc and EtOH extracts were dissolved in dimethyl sulfoxide at a 2% (w/v) nal concentration for the
tyrosinase inhibition assay and at 1% for both the anticollagenase and the antielastase assays. All assays
were performed in triplicate, and the results were averaged. The absorbance of the enzyme-substrate
reaction solution with the mycelial extracts was recorded at room temperature with a UV-visible spectro-
photometer. The enzyme inhibition activity for all assays was calculated with Eq. (1) for tyrosinase and
elastase, or Eq. (2) for collagenase:
(1)
International Journal of Medicinal Mushrooms
Cheng et al.
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(2)
where AC is the absorbance of the negative control and AS is that of the samples or the positive control.
1. Anti–Tyrosinase Activity Assay
The antityrosinase assay was evaluated on the basis of the inhibition of mushroom tyrosinase activity by
the sample, with l-tyrosine as the substrate, according to a protocol described previously.31 Tyrosinase
solution (700 units/mL, 50 μL) was mixed with 250 μL extract solution and 200 μL 0.2 M phosphate buffer
(pH 6.5), and maintained at room temperature under gentle agitation for 90 minutes. Immediately after
500 μL 0.03% l-tyrosine was added, the absorbance was recorded at 475 nm with a spectrophotometer.
Arbutin was included as an antityrosinase reference for the positive control.
2. Anti–Collagenase Activity Assay
As described previously by Van Wart and Steinbrink,32 the anticollagenase assay was performed using col-
lagenase from Clostridium histolyticum (EC.3.4.23.3) and the synthetic substrate, FALGPA. Collagenase
solution (0.83 units/mL, 80 μL) was mixed with an equal volume of the sample solution and 50 mmol/L
tricine buffer (10 mmol/L CaCl2 and 400 mmol/L NaCl; pH 7.5), and maintained at room temperature
for 15 minutes. Immediately after 160 μL 2 mmol/L FALGPA was added, the absorbance was recorded
at 335 nm with a spectrophotometer for 20 minutes. EGCG was included as the positive control for the
anticollagenase assay.
3. Anti–Elastase Activity Assay
The antielastase assay was conducted as reported by Thring et al.15; we used porcine pancreatic elastase
(E.C.3.4.21.36) and, as the substrate, N-succinyl-Ala-Ala-Ala-p-nitroanilide. Elastase solution (24 μg/mL,
25 μL) was mixed with 250 μL test extracts and 25 μL 0.2 M Tris-HCl buffer (pH 8.0), and maintained
at room temperature for 15 minutes. Immediately after 300 μL substrate (1.6 mmol/L) was added, the
absorbance was recorded at 410 nm with a spectrophotometer for 20 minutes. EGCG was used as the
positive control for the anti–elastase activity assay.
E. Determination of Potential Sunscreen Activity
The extract samples were dissolved to a nal concentration of 0.2 mg/mL with absolute EtOH (analytical
grade) for the EtOAc and EtOH extracts or with distilled water for the HW extracts. All the sample solu-
tions were prepared through the use of constant stirring overnight at room temperature and then ltration
through lter paper. The absorbance of the sample solution was scanned from 200 to 400 nm (at 5-nm
intervals) in a 1-cm quartz cuvette on a UV-visible spectrophotometer. The solvent for the particular extract
(ethanol or water) was used as a blank for the absorbance measurement. The sun protection factor (SPF)
was calculated with the equation developed by Mansur et al.33:
(3)
where CF represents the correction factor; EE(λ), the erythemal efciency spectrum; I(λ), the solar intensity
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Cosmetic Benets of O. sinensis Mycelia 627
spectrum; and A(λ), the absorbance of the sample. Homosalate was used as the reference sunscreen and
was prepared at 8% to determine the correction factor so that the formulation has a SPF value of 4, accord-
ing to a method from the US Food and Drug Administration. The normalized product function EE(λ)
I(λ), which represents the relation between the erythemogenic effect and the solar intensity between 290
and 320 nm (in 5-nm increments), was determined according to the method described by Sayre et al.,34
as shown in Table 1. The SPF of different extracts can be estimated with the Mansur equation and the
normalized product function.
F. Determination of Antioxidant Activities
1. DPPH Radical Scavenging Activity
The DPPH radical scavenging assay was modied from a procedure reported by Zhan et al.35 The HW
extract was dissolved in 0.05 M acetate buffer (pH 5.5), and the EtOAc and EtOH extracts were dissolved
in absolute ethanol. An 1-mL aliquot of the sample solution was mixed with 0.25 mL 0.50 mmol/L DPPH
in ethanol and incubated at room temperature in the dark for 30 minutes, after which absorbance was
measured at 517 nm against the solvent blank. Trolox in ethanol and 0.05 M acetate buffer (pH 5.5) with
5% ethanol was used as the positive control; the extract solution without DPPH was used as the sample
blank. The scavenging activity was calculated with Eq. (4):
(4)
where Ab, As, and Asb represent the absorbance of the blank, the extract or positive control, and the sample
blank, respectively. The half-maximal effective concentration—the extract concentration at which 50%
of DPPH radicals are scavenged—was obtained from the concentration-response curve.
2. Trolox-Equivalent Antioxidant Capacity Assay
Trolox-equivalent antioxidant capacity (TEAC) for scavenging ABTS radical cations was determined as
reported previously.36 Trolox was used as the antioxidant reference, and the activity was expressed as
TABLE 1: Normalized Product Function Used to
Calculate Sun Protection Factors34,a
Wavelength (nm) EE × I (Normalized)
290 0.0150
295 0.0817
300 0.2874
305 0.3278
310 0.1864
315 0.0839
320 0.0180
Total 1
EE, erythemogenic effect; I, solar intensity.
aNo unit.
International Journal of Medicinal Mushrooms
Cheng et al.
628
micromoles of Trolox per gram of sample. ABTS radical cations were generated through the reaction
of ABTS with potassium persulfate at room temperature in the dark. The extract sample solution was
mixed with an equal volume (500 μL) of the ABTS radical cation solution for 20 minutes, after which
absorbance was measured at 734 nm. The radical scavenging activity was presented as the percentage
radical reduction, as for the aforementioned DPPH scavenging activity (Eq. 4).
G. Statistical Analysis
All enzyme assays and tests were conducted in triplicate and the results expressed as the mean ± standard
deviation and calculated with SPSS data analysis software. The statistical signicance of treatment effects
was determined by 1-way analysis of variance with the least signicant difference post hoc test (for dif-
ferences among the tested samples), or by the Student t test at P < 0.05 (for differences between tested
samples and the positive control).
III. RESULTS AND DISCUSSION
A. Yield and Chemical Composition of Cs-HK1 and Cs-4 Mycelial Extracts
Table 2 shows the yield and chemical composition of various mycelial extracts from Cs-HK1 and Cs-4.
The HW extract was most abundant from both Cs-HK1 (248.4 mg/g) and Cs-4 (144.3 mg/g), with much
higher yields than those of the EtOH and EtOAc extracts. For Cs-HK1, the yield of the EtOH extract
(43.3 mg/g) was slightly more than that of the EtOAc extract (26.6 mg/g). The relative yields of the EtOAc,
EtOH, and HW extracts from Cs-HK1 were consistent with those reported by Wu et al.27 For Cs-4, The
yield of EtOAc extract (103.4 mg/g) was nearly double that of the EtOH extract (50.8 mg/g).
Among the major components of the Cs-HK1 and Cs-4 extracts (except for the Cs-4 EtOAc extract),
proteins generally accounted for the largest proportion, followed by carbohydrates; the HW extracts had
the highest protein and carbohydrate contents. No proteins or carbohydrates were detected in the EtOAc
extract of Cs-4, but in most cases the EtOAc extracts had relatively high phenolic and avonoid contents.
The HW extract yields were much higher than organic solvent extract yields, implying that water-soluble
TABLE 2: Yield and Composition of Cs-HK1 and Cs-4 Mycelial Extracts with Different Solvents
Extracts Yield
(mg/g)
Carbohydrates
(wt%)
Proteins
(wt%)
Phenolics
(mg GAE/g)
Flavonoids
(mg CE/g)
Strain Cs-HK1
EtOAc 26.6 2.9 ± 0.32 9.4 ± 0.21 27.1 ± 1.00 46.8 ± 6.15
EtOH 43.3 2.7 ± 0.09 11.5 ± 0.74 15 ± 0.91 32.9 ± 3.09
HW248.4 10.5 ± 1.08 40.5 ± 1.42 28.3 ± 1.07 2.87 ± 0.17
Strain Cs-4
EtOAc 103.4 n.d. n.d. 58.5 ± 1.49 173.3 ± 6.42
EtOH 50.8 1.4 ± 0.05 20.3 ± 0.10 11.4 ± 0.69 27.2 ± 0.84
HW 144.3 23.5 ± 1.31 33.3 ± 0.81 10.8 ± 0.97 4.48 ± 0.51
Yield and contents are based on the total mass of dry mycelia. CE, catechin equivalent; EtOAc, ethyl acetate; EtOH, ethanol;
GAE, gallic acid equivalent; HW, hot water; n.d., not detected.
Volume 20, Issue 7, 2018
Cosmetic Benets of O. sinensis Mycelia 629
components, including total carbohydrate and protein, were most abundant. The extraction yield of a
component depends not only on the actual content but also on the polarity and other molecular properties
of the component in the raw material.
B. Inhibitory Effects of Mycelial Extracts on Tyrosinase, Collagenase, and Elastase
Figure 1 shows the activities of 3 enzymes—tyrosinase, collagenase, and elastase—in the presence of
Cs-HK1 and Cs-4 mycelial extracts at various concentrations. The activities of tyrosinase and collagenase
were inhibited to different degrees by all 3 solvent extracts (EtOAc, EtOH, and HW). Elastase activity
FIG. 1: The inhibitory effects of various concentrations of different mycelial extracts from Ophiocordyceps sinensis
strains Cs-HK1 and Cs-4 on tyrosinase activity (arbutin as the positive control) (A), collagenase activity (EGCG as
the positive control) (B), and elastase activity (EGCG as the positive control) (C). EGCG, epigallocatechin gallate;
EtOAc, ethyl acetate; EtOH, ethanol; HWE, hot water extract.
A
B
C
International Journal of Medicinal Mushrooms
Cheng et al.
630
was inhibited by the EtOAc and EtOH extracts of the Cs-HK1 fungus and the EtOAc extract of the Cs-4
fungus, but not by the HW extracts of the 2 fungi nor by the EtOH extract of Cs-4. All the inhibitory
effects followed a concentration-dependent trend. Table 3 presents the half-maximal inhibitory concen-
trations (IC50) derived from the concentration curves. Overall, the EtOAc extracts of both fungi had the
most potent, and the HW extract had relatively weak, inhibitory activity against the 3 enzymes. In most
cases, the EtOAc and EtOH extracts of Cs-HK1 showed more signicant activity against the 3 enzymes
than those of Cs-4. Comparison of IC50 showed that the EtOAc extract of Cs-HK1 is even more potent
than the positive control against tyrosinase and elastase activity.
As shown in Fig. 1A and Table 3, the potency of both fungal extracts against tyrosinase was in the
following rank order: EtOAc > EtOH > HW. The antityrosinase activity of the HW extract of Cs-HK1 was
signicantly lower than that of the EtOAc and EtOH extracts, whereas the activity of the HW extract of
Cs-4 was about 50% less than that of the EtOAc and EtOH extracts. The results are comparable with those
in the literature on the antityrosinase effects of mushroom extracts. Chien et al.37 evaluated the antityrosi-
nase activities of several mushrooms including Agaricus brasiliensis, Antrodia camphorata, Cordyceps
militaris, and Ganoderma lucidum; G. lucidum was the most potent, with an IC50 of 0.32 mg/mL. Their
study also showed a similar trend for the different solvent extracts, with the EtOH extract being more
potent than the HW extract. Xu et al.31 evaluated the tyrosinase activity of polysaccharides isolated from
14 wild mushrooms, among which the most active were from Chroogomphus rutilus (IC50 = 0.46 mg/mL)
and Handkea utriformis (IC50 = 0.78 mg/mL); these IC50 were comparable to that of the Cs-4 HW extract.
It has been suggested that phenolic compounds including avonoids, phenolic acids, and stilbenes
from natural sources are attributable to tyrosinase inhibition.38–40 Comparison of the results showed a
rough correlation between avonoid content (Table 2) and the antityrosinase IC50 (Table 3), as both are
in the rank order EtOAc > EtOH > HW. In addition to the contributions of individual components, the
TABLE 3: IC50 of Different Mycelial Extracts of Cs-HK1 and Cs-4 (Final Concentration 0.2 mg/mL) on the
Basis of Tyrosinase, Collagenase, and Elastase Activity, and Sun Protection Factor
Extract IC50 (mg/mL) Sun Protection
Factor
Tyrosinase Collagenase Elastase
Cs-HK1
EtOAc 0.28 ± 0.02 0.29 ± 0.03 0.14 ± 0.01 16.2 ± 1.6
EtOH 0.53 ± 0.02 0.14 ± 0.01 0.46 ± 0.04 11.3 ± 2.7
HW 2.49 ± 0.01 0.60 ± 0.01 n.d. 10.2 ± 0.5
Cs-4
EtOAc 0.33 ± 0.02 0.47 ± 0.01 0.37 ± 0.01 3.1 ± 0.2
EtOH 0.36 ± 0.02 0.20 ± 0.01 n.d. 25.4 ± 0.3
HW 0.71 ± 0.05 0.87 ± 0.12 n.d. 18.5 ± 1.8
Positive controls
Arbutin 0.64 ± 0.03 n.d. n.d. n.d.
EGCG n.d. 0.07 ± 0.01 0.19 ± 0.01 n.d.
Homosalate n.d. n.d. n.d. 4.0 ± 0.3
EGCG, epigallocatechin gallate; EtOAc, ethyl acetate; EtOH, ethanol; HW, hot water; IC50, half-maximal inhibitory concen-
tration; n.d., not determined.
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Cosmetic Benets of O. sinensis Mycelia 631
complex extracts of Cs-HK1 and Cs-4 mycelia can perform biological functions synergistically or through
the cooperative actions of different compounds.41
As shown in Fig. 1B and Table 3, the EtOH extracts of both fungi exhibited the most potent anti-
collagenase activity and HW extracts, the lowest activity. The EtOH extract of Cs-HK1 showed the highest
potency, with an IC50 of 0.14 mg/mL, which was not signicantly different from that of the positive control,
EGCG (IC50 = 0.07 mg/mL; P > 0.05). Similar to our results, German-Baez et al.42 reported the IC50 of
EGCG against collagenase to be 0.05 mg/mL. Collagenase is a zinc-containing proteinase that requires
Ca2+ for activity, and its inhibitors generally are chelating compounds such as EDTA, carboxylate, hydroxa-
mate, phosphonate/phosphate, and thiol, which have binding afnity to Zn2+or Ca2+.32,43 Components of
O. sinensis—such as amino acids, which are capable of binding with heavy metal ions—may contribute to
the anticollagenase activity.44,45 Previous studies have suggested that polyphenols and phenolic acids have
collagenase-inhibitory activity.46–48 Some polysaccharides may also contribute to anticollagenase activity.21
As shown in Fig. 1C and Table 3, the elastase activity was inhibited by only 3 of the mycelial extracts:
the EtOAc extracts of both fungi and the EtOH extract of Cs-HK1. The EtOAc extract of Cs-HK1 had
the most potent inhibitory effect, with a low IC50 of 0.14 mg/mL, which was similar to that of the positive
control, EGCG (IC50 = 0.19 mg/mL; P > 0.05, no signicant difference). The results are comparable with
those in the literature on the antielastase effects of medicinal plant extracts. Lee et al.49 reported the anti-
elastase activities of 150 medicinal plants, most of which had IC50 > 0.2 mg/mL, except the Areca catechu
palm, which had an IC50 of 0.04 mg/mL. Similar to the results of the present study, German-Baez et al.42
showed that the less polar EtOAc extracts of several plants (tea) had stronger elastase-inhibitory activity
than the more polar methanol extracts. In another study, 23 extracts from 21 plant species were tested
against porcine elastase, and all of these extracts caused less inhibition than EGCG.15 In comparison with
the results from the literature, the antielastase activity of the Cs-HK1 EtOAc extract was among the highest.
Flavonoids such as kaempferol, quercetin, and myricetin have signicant antielastase activities.50 The
avonoid 3′-hydroxyfarrerol, which has a chemical structure similar to that of the well-known isocoumarins
(serine proteinase inhibitors), may act as a reversible, noncompetitive inhibitor of neutrophil elastase.51,52
In this study, the avonoid content and antielastase activity showed the similar trend of EtOAc > EtOH >
HW, which may suggest the possible contribution of avonoid compounds to elastase inhibition. However,
the relation requires verication through more vigorous assessment of avonoid components and their
effects on elastase activity.
C. Sunscreen Effects of Different Mycelial Extracts
The SPF of mycelial extracts are shown in Table 3. The Cs-4 EtOH extract exhibited the highest SPF
(25.4), followed by several other extracts, including the EtOAc, EtOH, and HW extracts of Cs-HK1 and
the HW extract of Cs-4; these had similar SPFs in the range of 10.2 to 18.5. The Cs-4 EtOAc extract
had a relatively low SPF value (3.1). According to the US Food and Drug Administration,53 3 types of
sunscreens are available: products with an SPF of 2–12 that provide minimal sun protection, those with
an SPF of 12–30 that provide moderate sun protection, and those with an SPF ≥ 30 that impart high sun
protection . Therefore, most of the mycelial extracts impart moderate or minimal sun protection. However,
other components in the formulation, such as esters, emulsiers, and emollients, can interact with the
active sunscreen ingredients to increase or decrease the actual SPF.54
Sunscreen products usually provide photoprotective effects either by reecting and scattering UVR
or by directly absorbing the light rays, thereby reducing the amount or intensity of harmful UVR reach-
ing the skin.55 The Cs-HK1 and Cs-4 extracts may contain molecules with structures similar to those of
chemical sunscreens and that can act as natural photoprotective agents.56 In addition, polysaccharides
International Journal of Medicinal Mushrooms
Cheng et al.
632
within the mycelial extracts may also offer photoprotective effects.57 These effects may be attributed to
the combined or cooperative action of different components of the extracts. Cs-HK1 and Cs-4 extracts
are generally regarded as safe and nontoxic to humans.57,58
D. Antioxidant Activities of Cs-HK1 and Cs-4 Mycelial Extracts
As shown in Fig. 2, all mycelial extracts of the Cs-HK1 and Cs-4 fungi exhibited a concentration-dependent
scavenging effect on DPPH radicals (Fig. 2A) and ABTS radical cations (Fig. 2B) at different levels.
Table 4 presents the corresponding half-maximal effective concentrations for scavenging DPPH radicals
and the TEAC values for scavenging ABTS radical cations. The EtOH extracts of both fungi showed the
highest activity, the lowest half-maximal effective concentration for scavenging DPPH radicals, and the
highest TEAC for scavenging ABTS radical cations. Overall, all the Cs-HK1 extracts exhibited stronger
radical scavenging activities than the Cs-4 extracts with the same solvents.
Two previous studies also showed that EtOH extracts of various medicinal mushrooms were more
active than HW extracts in scavenging free radicals.59,60 Although phenolics and avonoids are generally
recognized as major antioxidant components, the activity data in Table 4 do not imply a consistent trend
with the phenolic and avonoid contents shown in Table 2. In addition to the total amount of a compo-
nent, its structure and property can signicantly inuence its bioactivity.61–63 Other components such as
FIG. 2: Antioxidant activities of different mycelial extracts of Ophiocordyceps sinensis strains Cs-HK1 and Cs-4 in
scavenging DPPH radicals (A) and ABTS radical cations (B) (Trolox-equivalent antioxidant capacity assay). Error
bars represent the standard deviation (n = 3). EtOAc, ethyl acetate; EtOH, ethanol; HW, hot water.
A
B
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Cosmetic Benets of O. sinensis Mycelia 633
polysaccharides, peptides, and proteins also play a signicant role in radical scavenging.64–66 Many pre-
vious studies have linked antityrosinase (whitening), anticollagenase, antielastase, and photoprotective
properties with the antioxidant activities of plant and fungal extracts.67–69 In the present study, however, a
direct and quantitative correlation was not established between these skincare benets and the antioxidant
activities or the amounts of antioxidants.
IV. CONCLUSIONS
This study systematically assessed the potential cosmetic benets of 2 strains of O. sinensis through
in vitro enzyme assays of the mycelial extracts. Some of the mycelial extracts showed potent antityrosi-
nase, anticollagenase, and antielastase activity comparable to or more favorable than that of the reference
compounds. In most cases, the least polar EtOAc extract was more active than the most polar HW extract.
However, no clear or consistent correlation was found between the enzyme-inhibitory activity and the
antioxidant activity or the major chemical components. The results substantiate the cosmetic benets,
encouraging the use of O. sinensis extracts for skincare and cosmeceutical applications with skin-whitening,
antiwrinkle, antioxidative, and sunscreen functions. Further studies should be conducted to identify the
active components and to investigate the underlying mechanisms of action and the structure-function
relations of active ingredients.
ACKNOWLEDGMENTS
This work was supported by the Shenzhen Basic Research Program Project (Grant No. JCYJ20160531184200806),
a Research Grant Council Collaborative Research Fund equipment grant (Grant No. C5031-14E), and the
Hong Kong Polytechnic University.
TABLE 4: Antioxidant Activities of Different Mycelial Extracts from
Cs-HK1 and Cs-4 Measured by DPPH Radical Scavenging Activity and
Trolox-Equivalent Antioxidant Capacity Assay
Extract DPPH (EC50, mg/mL) TEAC (µmol Trolox/g)
Cs-HK1
EtOAc 1.38 ± 0.1 111.0 ± 15.8
EtOH 1.08 ± 0.1 148.4 ± 29.0
HW 3.20 ± 0.2 100.3 ± 11.3
Cs-4
EtOAc 9.21 ± 0.5 74.5 ± 26.3
EtOH 1.12 ± 0.1 131.8 ± 22.3
HW 5.68 ± 0.3 90.35 ± 3.9
Positive controls
Trolox in EtOH 0.01 ± 0.0 n.d.
Trolox in buffer 2.06 ± 0.1 n.d.
Differences were signicant between data within the same column (P < 0.05). EC50,
half-maximal effective concentration; EtOAc, ethyl acetate; EtOH, ethanol; HW, hot
water; n.d., not determined; TEAC, Trolox-equivalent antioxidant capacity.
International Journal of Medicinal Mushrooms
Cheng et al.
634
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