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Article

Fermentation of Chenopodium formosanum Leaf Extract with Aspergillus oryzae Significantly Enhanced Its Physiological Activities

by
Yi-Min Lin
1,
Ying-Chien Chung
2,*,
Pei-Yu Chen
2,
Yu-Chi Chang
2 and
Wen-Liang Chen
1,*
1
Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu City 300193, Taiwan
2
Department of Biological Science and Technology, China University of Science and Technology, Taipei City 115311, Taiwan
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(5), 2917; https://doi.org/10.3390/app13052917
Submission received: 11 January 2023 / Revised: 20 February 2023 / Accepted: 22 February 2023 / Published: 24 February 2023
(This article belongs to the Special Issue Characterization of Bioactive Compounds and Antioxidant Activity of Plant)
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Abstract

:
Chenopodium formosanum Koidz (CF) is an indigenous cereal plant of Taiwan. Its high content of secondary metabolites and nutrients has attracted attention for its use in skin care products and functional foods. However, most studies have focused on the extract of CF seeds, which are relatively expensive, and none have investigated the effects of combining extraction and fermentation. In this study, we evaluated the utility of using extracts of different parts of CF, i.e., the leaves, stems, and unhulled and hulled seeds. We first made aqueous, ethanolic, methanolic, and ethyl acetate extracts of the four parts. After assessing their biological activities, we selected only unfermented and fermented CF leaf methanolic extracts for subsequent analysis. None of the concentrations of fermented CF leaf extract (≤400 mg/L) were cytotoxic, and all exhibited antioxidative, anti-inflammatory, antimicrobial, skin-whitening, moisturizing, and antiaging activities. The concentrations of protocatechuic acid, epicatechin, gallic acid, and quercetin increased the most after fermentation. Therefore, they were subjected to a molecular docking analysis, which revealed that quercetin and epicatechin may contribute the most to skin-whitening and antiaging properties, respectively. In conclusion, fermented CF leaf methanolic extracts can be useful as a functional ingredient in health foods, botanical drugs, and cosmetic products.

1. Introduction

Chenopodium formosanum Koidz (CF, also known as “djulis”) is an indigenous cereal plant of Taiwan, an annual fast-growing underutilized cereal, and cultivated in some areas inhabited by aboriginal people [1]. CF leaves are colorful, and its seeds are bright red [2]. The genus of CF is the same as that of quinoa (Chenopodium quinoa), but quinoa seeds are not necessarily red [3]. CF seeds have substantial nutritional value and exhibit biological activities [3]. CF seed extracts possess antioxidative, anti-inflammatory, anticancer, hepatoprotective, antiadipogenic, antihypertensive, repellency, and insecticidal properties [4,5,6,7]. CF is rich in diverse secondary metabolites with substantial pharmacological activities and nutritional value. Thus, CF has received increasing attention for use in skincare products and functional foods [8,9]. CF seed extracts may be beneficial for skin protection and skincare. For example, CF seed extracts could alleviate injury caused by UVB in HaCaT cells and protect the skin from photoaging [3,10]. These benefits may be attributed to its antioxidative and anti-inflammatory properties [11,12]. These biological effects are exerted by bioactive compounds present in various parts of CF [6].
Previous studies have focused on CF seeds, which are relatively expensive; however, the roots, leaves, and stems of CF, which are often wasted, can also be highly beneficial for extract production [3,5,13]. The biological activities of hulled and unhulled seeds differ significantly [14]. Thus, if other parts of CF can be used for their antioxidative, anti-inflammatory, skin-whitening, or antiaging properties, agricultural waste could be minimized and manufacturing costs could be reduced [15].
Fermentation can modify original molecules to enhance the flavor and antioxidative activity of a product [16]. Appropriate microorganisms used for fermentation also increase the multifunctional activities of fermented herbs and reduce their cytotoxicity [17,18]. For example, Magnolia officinalis fermented by Aspergillus niger has higher antityrosinase, antioxidative, antimicrobial, and antiaging activities and lower cytotoxicity [19]. L. acidophilus-fermented CF prevented colon carcinogenesis in rats by increasing superoxide dismutase and catalase activities [20].
To the best of our knowledge, few reports exist on the functional analysis of different CF parts, and none have investigated the effects of combining extraction and fermentation. This study used a novel approach to develop CF leaves as a raw ingredient, which can be environmentally friendly and have economic value.

2. Materials and Methods

2.1. Plant Material, Extract Preparation, and Fermentation

Djulis (Chenopodium formosanum Koidz) cultivated in Taitung County (Taiwan) was supplied by Haugooli. It was identified by Professor Bau-Yuan Hu and a voucher specimen (20210208) was deposited in the herbarium of China University of Science and Technology, Taiwan. It was harvested during the spring of 2021. Leaves, stems, and unhulled and hulled seeds of CF were harvested separately, air-dried in the shade at ambient temperature, powdered using a blender, and passed through a 0.5 mm mesh. For extraction, 10 g of the powder was mixed with 100 mL of distilled water, 95% ethanol, 100% methanol, or 100% ethyl acetate and stirred (150 rpm) at room temperature. After 1 h, the mixture was filtered and the filtrate was evaporated at 60 °C (for the water extract), 50 °C (for the ethanolic extract), 45 °C (for the methanolic extract), or 40 °C (for the ethyl acetate extract) to remove the solvent fraction by using a rotary vacuum evaporator (Panchum Scientific Corp., Kaohsiung City, Taiwan). The residues were frozen and lyophilized using a shelf freeze dryer (Uniss Corp., Taipei City, Taiwan).
Aspergillus oryzae (ATCC 30104) was purchased from the Bioresource Collection and Research Center (BCRC), Taiwan, and was cultured in potato dextrose broth and incubated at 24 °C for 10 days. To obtain the fermented extract, 0.2 g of the freeze-dried extract was first added to 200 mL of phosphate-buffered solution (pH 5.5) containing 4 × 108 spores of A. oryzae and incubated at 24 °C. After 5 days of cultivation, the fermented solution was centrifuged at 9000× g for 6 min and the supernatant was collected. Subsequently, the solution was filtered and the filtrate was evaporated at 60 °C using a rotary vacuum evaporator (Dlab scientific Inc., Riverside, CA, USA). The residue was a fermented CF extract.

2.2. Microbial Strains, Cells, and Regents

Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Cutibacterium acnes (ATCC 10723), Candida albicans (ATCC 10231), and A. brasiliensis (ATCC 16404) were used to evaluate the antimicrobial activity of the fermented CF leaf extract. All these microbial strains were purchased from the Bioresource Collection and Research Center (BCRC) in Taiwan. Human epidermal melanocytes (HEMn) were obtained from Cascade Biologics (Portland, OR, USA). The human skin keratinocyte (HaCaT) cell line was kindly provided by Professor GH Wang (Xiamen Medical College, China). RAW 264.7 murine macrophages were obtained from the BCRC.
Mushroom tyrosinase, collagenase, elastase, hyaluronidase, and MMP-1 protein were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemicals used in the study were of analytical grade (purity > 99.2%) and purchased from Sigma-Aldrich. Cultural media used were purchased from DIFCO (Tucker, GA, USA). Proinflammatory cytokines (TNF-α and IL-6) were obtained from Invitrogen (Carlsba, CA, USA).

2.3. Analysis of Antioxidative Activity

The antioxidative potentials of various extracts or fermented extracts were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS). First, a 100 μM DPPH solution was freshly prepared in pure ethanol. Next, extracts at various concentrations (1 mL) were individually added to 1 mL of ethanol and 500 μL of the DPPH solution and maintained in the dark at room temperature. After 30 min, the absorbance was measured at 517 nm by using an Epoch enzyme-linked immunosorbent assay (ELISA) reader (BioTek Instruments, Santa Clara, CA, USA) [19]. BHT was used as a positive control. All experiments were conducted in triplicate. The DPPH scavenging activity of the extract was calculated as follows:
DPPH scavenging activity ( % ) = [ 1 − ( O D 517   o f   s a m p l e O D 517   o f   c o n t r o l ) ] × 100  
The ABTS solution was prepared by mixing 7 mM ABTS with 2.45 mM K2S2O8 (1:1) in the dark, and the ABTS solution was diluted with methanol before use until the absorbance at 734 nm reached 0.7. Next, 50 μL of the sample was mixed with 950 μL of the ABTS solution for 30 min, and the absorbance was measured at 734 nm by using an Epoch ELISA reader [13]. BHT was again used as the positive control. All experiments were conducted in triplicate. The ABTS scavenging activity of the extract was calculated as follows:
ABTS scavenging activity ( % ) = [ 1 − ( O D 734   o f   s a m p l e O D 734   o f   c o n t r o l ) ] × 100
The IC50 value of the ABTS scavenging activity was determined at 50% scavenging activity.

2.4. Total Phenol and Flavonoid Quantification

The total phenolic content (TPC) was estimated as the gallic acid equivalent (GAE) per dried weight of the sample and expressed as the mg-GAE/g-DW, per the method described by Kujala et al. (2000) [21]. A standard gallic acid solution was prepared by dissolving 10 mg of gallic acid in 10 mL of methanol and then diluted to desirable concentrations in methanol. Next, 0.5 mL of gallic acid solution or the fermented extract was mixed with 1 mL of Folin–Ciocalteu phenol reagent and then neutralized with 1 mL of 7.5% Na2CO3 solution for 3 h. After centrifugation, the absorbance of the supernatant in the mixture was measured at 760 nm against a blank solution (reagents except extract or gallic acid) by using a UV–Vis spectrophotometer (UV-2600i, Shimadzu, Japan). The calibration curve of absorbance (y) versus gallic acid concentrate (x) had the equation y = 0.0518x + 0.0684 (R2 = 0.9992).
The total flavonoid content (TFC) was estimated as the quercetin equivalent (QE) per dried weight of the sample and expressed as mg-QE/g-DW in accordance with the method described by Chuang et al. (2018) with little modification [5]. A standard quercetin solution was prepared by dissolving 40 mg of quercetin in 10 mL of methanol and then diluted to desirable concentrations in methanol. Next, 1 mL of quercetin solution or the fermented extract was mixed with 4 mL of distilled water, 0.3 mL of 5% NaNO2, and 0.3 mL of 10% AlCl3 solution. After 10 min of stirring, 2 mL of 1 M NaOH and 2.4 mL of distilled water were added in turn, and the resulting mixture was gently stirred. After 30 min, the absorbance of the mixture was measured at 415 nm against a blank solution (reagents minus the extract or quercetin) by using a UV–Vis spectrophotometer. The calibration curve of absorbance (y) versus the concentration of quercetin (x) had the equation y = 0.0753x + 0.0412 (R2 = 0.9951).

2.5. Analysis of Cell Viability

To evaluate the cytotoxic effects of the unfermented or fermented extract on HEMn, HaCaT, or Raw 264.7 cells, the MTT method was adopted (Wang et al., 2017) [16]. The HEMn cells were cultivated in Medium 254 supplemented with human melanocyte growth supplement. HaCaT cells were cultivated in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. Raw 264.7 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h of incubation, these cells (6 × 105 cells/well) in a 96-well plate were treated with the fresh medium (as the control group) or extract (10–400 mg/L) for 72 h. Thereafter, 20 μL of MTT solution (5 mg/mL) was added to each well and reacted at 37 °C for 4 h under 5% CO2. The solution was removed and 150 μL of DMSO was added to dissolve formazan crystals for 10 min. The absorbance of each well was measured at 570 nm by using an Epoch ELISA reader. Cell viability was calculated using the following equation:
Cell viabillity ( % ) = ( O D 570   o f   s a m p l e O D 570   o f   c o n t r o l ) × 100

2.6. Analysis of Tyrosinase Activity and Melanin Content

The antityrosinase activity of various CF extracts was analyzed using the method reported by Zheng et al. (2012) [22]. These CF extracts were prepared in DMSO solution at different concentrations. Subsequently, 30 μL of the sample was added to 970 μL of 0.05 mM PBS and homogeneously mixed with 2 mL of solution containing L-tyrosine (1 mL, 100 mg/L) and mushroom tyrosinase solution (1 mL, 350 units/mL) in the dark. After a 20 min reaction period, the absorbance of the solution was measured at 490 nm by using a UV–Vis spectrophotometer. Koji acid was used as the positive control. All experiments were conducted in triplicate. The concentration of half the original tyrosinase activity inhibited was defined as the IC50 value. The antityrosinase activity of the extract was calculated as follows:
antityrosinase activity ( % ) = [ ( A − B ) − ( C − D ) ] ( A − B ) × 100
where A is the absorbance without the extract (control), B is the absorbance without the extract and enzyme (blank of A), C is the absorbance with the extract and enzyme, and D is the absorbance without the enzyme (blank of C).
The effect of the fermented extract on the antityrosinase activity and melanin content in HEMn cells was analyzed using the method reported by Wu et al. (2021) with slight modifications [23]. Briefly, 5 × 106 HEMn cells/well were incubated in Medium 254 supplemented with human melanocyte growth supplement at 37 °C. After a 24 h cultivation period, the cells were treated with different final concentrations of the fermented extract (5–50 mg/L) in 24-well plates for 24 h. Subsequently, the cells were collected, washed with PBS, lysed with a cell lysis solution, sonicated at 4 °C for 5 min by using an ultrasonic sonicator (Qsonica, Newtown, CT, USA), and then centrifuged at 6500× g for 20 min by using a micro-ultracentrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were reacted with 2.5 mM L-DOPA in 0.1 M PBS in 96-well plates for 60 min. The absorbance was measured at 475 nm by using an Epoch ELISA reader. To examine the melanin content in HEMn cells, 5 × 106 HEMn cells/well were first incubated for 24 h and then treated with the fermented extract for 24 h. Thereafter, the cells were collected through trypsinization and centrifugation. The cell pellets were suspended in 100 μL of 1 N NaOH containing 10% DMSO and heated at 70 °C for 1.5 h. The absorbance was measured at 405 nm by using an Epoch ELISA reader. The melanin content in the HEMn cells was determined through comparison with a synthetic melanin standard obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.7. Analysis of Antiaging and Aquaporin 3 Activities

The collagenase activity was measured using a modified fluorogenic DQ-gelatin assay using the method reported by Vandooren et al. (2011) [24]. The collagenase activity was determined by measuring the absorbance at an excitation wavelength of 485 nm and an emission wavelength of 528 nm by using a Synergy 2 microplate reader (BioTek Instruments, Santa Clara, CA, USA). The elastase activity was measured using the method reported by Karim et al. (2014), with MeOSuc-Ala-Ala-Pro-Val-pNA as the substrate [25]. The elastase activity was determined by measuring the absorbance at 405 nm by using an Epoch ELISA reader. A hyaluronidase activity assay was conducted using the method reported by Thring et al. (2009), with hyaluronic acid as the substrate [26]. The hyaluronidase activity was determined by measuring the absorbance at 600 nm using an Epoch ELISA reader. The MMP-1 activity was determined as described by Tsai et al. (2014) [27]. A human MMP-1 ELISA kit (R&D, Minneapolis, MN, USA) was used. The MMP-1 activity was determined by measuring the absorbance at 450 nm using an Epoch ELISA reader. The aquaporin 3 (AQP3) activity was analyzed according to the method of Ikarashi et al. (2020) [28] and briefly described as follows. HaCaT cells (3 × 105 cells/well) were first cultured in a 6-well cell culture plate for 48 h and then treated with the fermented CF leaf extract. After 48 h of treatment, the cells were collected and washed twice with PBS solution. The total mRNA in the HaCaT cells was extracted using a commercial RNA extraction kit and reverse transcribed into cDNA. Target gene expression was amplified using the following primer set: forward, 5′-AGACAGCCCCTTCAGGATTT-3′; reverse, 5′-TCCCTTGCCCTGAATATCTG-3′) [29]. The mRNA expression level or AQP3 activity was detected using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), estimated using the delta-delta Ct method, and normalized to that of GAPDH.

2.8. Analysis of Antimicrobial Activity

A dilution tube method was used to determine the minimum inhibitory concentration (MIC) of the fermented CF leaf methanolic extract against E. coli, S. aureus, P. aeruginosa, and C. acnes in accordance with the method reported by Rahman et al. (2013) [29]. Briefly, 2 mL of fermented extracts, 2 mL of TSB, and 1 mL of inoculum (3 × 106 CFU/mL) were mixed in a test tube and aerobically incubated at 37 °C for 18 h. However, reinforced clostridial broth was used for C. acnes and anaerobically cultivated at 37 °C for 48 h. MIC is defined as the lowest concentration of the fermented extract that prevents the visible growth of E. coli, S. aureus, P. aeruginosa, and C. acnes.
To determine the minimum fungicidal concentration (MFC) of the fermented extracts against C. albicans and A. brasiliensis, the standard plate count method was adopted. Briefly, 1 mL of the fermented extract and 100 mL of specific broth (yeast malt broth for C. albicans and potato dextrose broth for A. brasiliensis) containing inoculum (6 × 105 CFU/mL or 6 × 105 spores/mL) were mixed in an Erlenmeyer flask and incubated at 25 °C for 5 days. The lowest concentration of the fermented extract showing no visible growth on subculturing was considered as the MFC.

2.9. Analysis of Anti-Inflammatory Activity

The ROS level in Raw 264.7 cells was measured using a fluorescent dye protocol (the oxidant-sensitive probe DCFH-DA) according to the method reported by Huang et al. (2019) [30]. The cells (6 × 105 cells/mL) were cultured with a mixture containing 0–250 mg/L of fermented extracts and 1 mg/L of lipopolysaccharides (LPS). LPS alone was the positive control. After 24 h of cultivation, the cells were stained with 20 μM DCFH-DA for 15 min, and ELISA was adopted to measure ROS production. To measure the NO level in raw 264.7 cells, the cells were cultured with fermented extracts and LPS for 24 h. Next, the same volume of the cell supernatant was mixed with Griess reagent for 10 min. The amount of NO produced in the cells was measured at 550 nm and estimated from a NaNO2 standard curve. To investigate the anti-inflammatory activity, the levels of the proinflammatory cytokines TNF-α and IL-6 were assessed using commercial ELISA kits. The samples were prepared as described in the previous NO assay. The cytokine levels in Raw 264.7 cells were analyzed at 620 nm in accordance with the manufacturer’s instructions. Data were calculated according to standard curves of each cytokine standard.

2.10. Molecular Docking Analysis

To determine the possible source or reason of the enhanced physiological activity of the fermented CF leaf extract, three-dimensional (3D) crystal structures of tyrosinase, collagenase, elastase, and hyaluronidase were retrieved from the Protein Data Bank database, and the 3D structures of chemical ingredients with significantly increased concentrations in the CF leaf extract after fermentation (i.e., protocatechuic acid, epicatechin, gallic acid, and quercetin) were obtained from the human metabolome database. To explore the binding affinities between receptors and ligands, automated flexible docking of ligands was performed using AutoDock Tools v1.5.7. The two-dimensional diagrams of protein–ligand interactions were visualized using Discovery Studio v3.5 (Biovia, San Diego, CA, USA).

2.11. Statistical Analysis

Data were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. Statistical significance was set at p < 0.05. All statistical analyses were conducted using SPSS (version 20.0; SPSS, Chicago, IL, USA). IC50 values were calculated from an equation for the linear regression curve between probit percent inhibition and log concentration by using Origin software (OriginaLab, Northampton, MA, USA) [31].

3. Results and Discussion

3.1. Physiological Activities of Unfermented Extracts Derived from Various Parts of CF by Using Different Extraction Solvents

The solvents used to obtain CF leaf extracts had different extraction yields, which can be arranged as follows (in descending order): distilled water (34.6%) > 100% methanol (32.1%) > 95% ethanol (30.2%) > 100% ethyl acetate (22.8%). This indicates that the extraction yield of the solvent increased with its polarity. Similar yield trends were noted when these solvents were used to extract other parts of CF.
DPPH and ABTS assays are rapid, straightforward, and inexpensive and are thus widely used to examine antioxidative activity in herbal products [32]. Figure 1 illustrates the physiological activities of the unfermented extracts of various parts of CF obtained using different extraction solvents. Water and 100% methanol were suitable extraction solvents for examining DPPH scavenging activity in leaves, unhulled seeds, and hulled seeds (Figure 1A). In terms of the average DPPH scavenging activity, the order of antioxidative capacity was the highest for unhulled seeds, followed by leaves, hulled seeds, and then stems for various solvents. Leaf and unhulled seeds had higher antioxidative capacities than the other parts (p < 0.05). Wu et al. (2021) indicated that the DPPH radical scavenging activity of the CF leaf extract was higher than that of the unhulled seed extract obtained using 75% ethanol [15]. In our study, the highest DPPH scavenging activity (95.3% ± 1.4%) was observed in the 100% methanolic extract of CF unhulled seeds and the lowest (17.5% ± 1.2%) was noted in the 95% ethanolic extract of CF hulled seeds. The DPPH scavenging activity of the positive control BHT (1000 mg/L) was 93.5% ± 0.8%. Figure 1B presents the ABTS scavenging activity of the unfermented extracts of various parts of CF obtained using different extraction solvents. Water and 100% methanol were the optimal extraction solvents for various parts of CF. CF leaf, unhulled seed, and hulled seed extracts (except those obtained using 100% ethyl acetate as the extraction solvent) exhibited high ABTS scavenging activity. The highest ABTS scavenging activity (96.1% ± 1.3%) was observed for the methanolic CF leaf extract. The lowest activity (17.1% ± 0.2%) was observed for the 100% ethyl acetate extract of CF hulled seeds. The ABTS scavenging activity of the positive control BHT (1000 mg/L) was 95.8% ± 0.7%. These results indicated that CF leaves had higher antioxidative capacity, similar to CF seeds. Different antioxidative activities in various parts (sprout, seeds, bran, pericarp, leaves, stem, and roots) of C. quinoa were also observed by Lim et al. (2020) [33].
Figure 1C presents the antityrosinase activity of the unfermented extracts of various parts of CF obtained using different extraction solvents. Among all solvents, 100% methanol and 95% ethanol were suitable as extraction solvents for various parts of CF. The highest antityrosinase activity (98.3% ± 0.3%) was noted in the CF leaf methanolic extract. A relatively high antityrosinase activity (95.2%–96.8%) was noted in the ethanolic or methanolic extracts of CF unhulled and hulled seeds. The antityrosinase activity of the positive control koji acid (1000 mg/L) was 99.2% ± 0.4%.

3.2. Comparison of Physiological Activities of Unfermented and Fermented Methanolic Extracts of CF Leaves and Unhulled Seeds

On the basis of the results presented in the preceding section, we used only the methanolic extracts of CF leaves and unhulled seeds for our subsequent fermentation experiments. Figure 2 illustrates the physiological activities of the unfermented and fermented methanolic extracts of CF leaves and unhulled seeds. Compared with the DPPH scavenging activity assay, ABTS is more suitable because this assay can be used for measuring the antioxidative capacity of hydrophilic and hydrophobic compounds [34]. Thus, an ABTS scavenging activity assay was conducted. Figure 2A presents that the A. oryzae-fermented methanolic CF extract was more effective compared with the unfermented CF extract (methanolic extract). The IC50 values of the unfermented leaf extract, fermented leaf extract, unfermented unhulled seed extract, and fermented unhulled seed extract were 293.6, 22.9, 362.7, and 63.5 mg/L, respectively. The ABTS scavenging activities of the fermented leaf extract and seed extract were higher than those of the unfermented leaf extract and unfermented unhulled seed extract by 12.8 and 5.7 times, respectively. The IC50 value (22.9 mg/L) of the fermented leaf extract for scavenging ABTS was superior to that (100 mg/L) of the ethanolic extract of CF hull and that (2350 mg/L) of the CF product fermented by L. plantarum [30,35]. The improvement in the ABTS scavenging activity of the A. oryzae-fermented CF leaf extract was more pronounced compared with that of the seed extract.
Figure 2B presents the antityrosinase activity of the unfermented and fermented extracts of CF leaves and unhulled seeds. After fermentation, the antityrosinase activity of the extracts was enhanced. The IC50 values of the unfermented and fermented leaf extracts and unfermented and fermented unhulled seed extracts were 226.5, 14.1, 282.8, and 29.6 mg/L, respectively. The antityrosinase activities of the fermented leaf and unhulled seed extracts were 16.1 and 9.6 times higher than those of the corresponding unfermented extracts, and the effect of fermentation was significant on the CF leaf extract. The antityrosinase activities of extracts after fermentation were considerably higher than those of the CF hull extract after high pressure extraction [30]. Thus, the fermented extracts were more active than the unfermented extracts. CF leaves are typically discarded as waste; therefore, the CF leaf methanolic extract was selected for fermentation in subsequent experiments.

3.3. TPC, TFC, Antiaging Activity, and Antimicrobial Activity of Fermented CF Leaf Methanolic Extract

Although some studies have evaluated the antioxidative activity of CF seeds, few have focused on the TPC, TFC, and antiaging activity (especially its IC50 value) of CF leaves. Collagen and elastin constitute the major components of the extracellular matrix (ECM) of the dermis, which are broken down by collagenase and elastase activities, which are the characteristic biomarkers of skin aging [36]. MMPs can cleave the dermal ECM, thereby damaging skin integrity [3]. Therefore, inhibiting the activities of these degradation enzymes can prevent or retard skin aging.
Table 1 presents the TPC, TFC, and antiaging activities of the methanolic extract of CF leaves fermented by A. oryzae. The results indicated that the TPC and TFC of the fermented leaf extract were 162.5 ± 6.7 mg-GAE/g-DW and 87.1 ± 2.8 mg-QE/g-DW, respectively. The TPC of the fermented leaf extract was higher than that in previous reports: 97.19 mg-GAE/g-DW (leaf extract), 26.11 mg-GAE/g-DW (seed extract), 3.24 mg-GAE/g-DW (seed extract), 11.861 mg-GAE/g-DW (seed extract), and 28.97 mg-GAE/g-DW (product of seed fermented by L. plantarum) [5,8,9,15,37]. The TFC of the fermented leaf extract was less than that of the 75% ethanolic leaf extract (156.15 mg-QE/g-DW) but higher than that of the seed extracts (2.55 and 3.52 mg-QE/g-DW) [5,15,37]. Moreover, collagenase, elastase, hyaluronidase, and MMP-1 activities (IC50) of the fermented leaf extract were 113.5 ± 9.2, 137.4 ± 16.2, 106 ± 7.6, and 176.4 ± 13.8 mg/L, respectively. As a result of the high correlation between aging and oxidative stress, antioxidants may help retard aging [38]. Hong et al. (2016) indicated that CF can inhibit MMP-1 expression in cellular/animal models [3]. Lin et al. (2021) reported that CF water extracts (0.5%) exhibited high antiaging activity by regulating the expression of skin-related and collagen-related genes [12]. Our study is the first to report the antiaging activity (i.e., anti-collagenase, anti-elastase, anti-hyaluronidase, and anti-MMP-1) of the fermented CF leaf extract, and even a low concentration (100–200 mg/L) was highly effective (Table 1), which may be because of its high TPC, as supported by previous reports [39].
Excessive antibiotic use has markedly increased antibiotic resistance and adverse effects, compelling researchers to explore safe and effective remedies [40]. Plant-derived products have unmatched chemical diversity and provide abundant opportunities for new antimicrobial drug development [41]. Table 2 lists the antimicrobial activity of the A. oryzae-fermented methanolic extract of CF leaves. To apply the fermented CF leaf extract in the cosmetic industry, its antimicrobial activity against five strains (S. aureus, E. coli, P. aeruginosa, C. albicans, and A. brasiliensis) mandatorily used for a USP 51 antimicrobial effectiveness test as well as C. acnes was evaluated. The results indicated that the MIC values of E. coli, S. aureus, P. aeruginosa, and C. acnes were 150 ± 12, 180 ± 14, 140 ± 9, and 120 ± 5 mg/L, respectively. The MFC values of C. albicans and A. brasiliensis were 230 ± 26 and 280 ± 21 mg/L, respectively. The fermented CF leaf extract was active against these tested strains. A. brasiliensis was a less sensitive strain, possibly due to the use of the same fungal genus (A. oryzae) as the fermenting strain. C. acnes was the most sensitive strain among tested bacterial strains, indicating that the fermented leaf extract was a potential ingredient for skin treatment. The antimicrobial activity (MIC value) of CF is less documented; therefore, we compared that of C. quinoa instead. Park et al. (2017) reported that the ethanolic extract of C. quinoa seed did not exhibit strong antimicrobial activity against these strains [42]. Pereira et al. (2020) indicated that the MIC values of E. coli and S. aureus were 270 mg/L and 482 mg/L and the MFC value of A. brasiliensis was 352 mg/L [43]. The high antimicrobial activity of the fermented leaf extract may be attributed to the presence of rich phenolic compounds (Table 1) [44], thus indicating its potential use for medicinal purposes.

3.4. Cytotoxicity of Fermented CF Leaf Methanolic Extract

An increasing CF extract concentration may produce toxic components (e.g., saponin, alkaloids, or phytic acid) and cause cytotoxicity [12]. Regardless of the potency of the physiological activity of the fermented CF leaf extract, it cannot be used if it causes cytotoxicity. Therefore, we examined the cytotoxic effect of the fermented CF leaf extract on HaCaT and Raw 264.7 cells as well as the effects of both unfermented and fermented extracts on HEMn cell survival. Figure 3 presents the cytotoxic effects of these extracts on HEMn, HaCaT, and Raw 264.7 cells. All concentrations of the fermented CF leaf extract were nontoxic (viability ≥ 92.7%) for all tested cells. The viability of HaCaT cells was >94.6% ± 0.5% compared with that of control cells when the concentration of the fermented extract was ≤400 mg/L, implying that it is potentially safe for skin treatment. Hong et al. (2016) reported that the CF seed extract at concentrations of ≤150 mg/L did not affect HaCaT cell viability [3]. Moreover, the viability of LPS-stimulated RAW 264.7 cells was 92.7% ± 1.6% at ≤400 mg/L. This suggests that a dose of ≤400 mg/L of the fermented CF leaf extract is suitable for anti-inflammatory studies. The results also revealed that the fermented CF leaf extract had a considerably lower cytotoxicity in HEMn cells, whereas the unfermented extract, even at 250 mg/L, exhibited significant cytotoxicity (p < 0.05) compared with the control. Moreover, the viability of HEMn cells at 400 mg/L of unfermented and fermented extracts was 78.2% ± 1.4% and 92.8% ± 2.5%, respectively. Thus, the fermented extract at 400 mg/L was considered safe for all the three cell lines and may therefore be safe and effective for use in antioxidative, antiaging, antimicrobial, and skin-whitening products (Table 1 and Table 2).

3.5. Effect of Fermented CF Leaf Methanolic Extract on Skin Hydration

The CF extract exerts moisturizing effects; thus, we explored the role of AQP3, a protein implicated in skin hydration, which is crucial to skin aging [12]. AQP3, which is encoded by the AQP3 gene, functions as a water channel in the basal layer keratinocytes of the epidermis and regulates the water content in the stratum corneum. A decreased water content in the skin results in wrinkles and loss of elasticity [45,46]. Given that the fermented CF leaf extract exhibited antioxidative, skin-whitening, antiaging, and antimicrobial activities in this study, we believed that it is also effective in moisturizing. Figure 4 displays the effects of the fermented CF leaf extract at different concentrations on the relative mRNA expression (AQP3) in HaCaT cells, which increased with the increase in extract concentration. AQP3 expression was 2.43 ± 0.08 and 4.18 ± 0.31 times higher than that in the control group at concentrations of 100 and 250 mg/L, respectively. These findings indicate that the fermented CF leaf extract significantly improved skin hydration and may thus help prevent wrinkles and maintain skin elasticity [11].

3.6. Anti-Inflammatory Effects of Fermented CF Leaf Methanolic Extract

IL-6, TNF-𝛼, NO, and ROS play crucial roles in skin abnormalities, including the infiltration of inflammatory cells, redness, swelling, and uncomfortable feelings [3]. Therefore, the effects of the fermented leaf extract on their production/activity were analyzed. Figure 5 indicates that the fermented CF leaf extract reduced NO, IL-6, and TNF-α production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. NO production decreased from 31.6 ± 2.1 μM (control, without fermented extract) to 11.9 ± 0.8 μM (at 250 mg/L), a 62.3% reduction, which was more effective than that of the CF hull extract obtained through high-pressure extraction [30]. IL-6 production decreased from 282 ± 2.6 μg/L (control) to 218 ± 2.5 μg/L (at 250 mg/L), a 22.7% reduction. TNF-α production decreased from 126 ± 6.6 μg/L (control) to 47.2 ± 1.5 μg/L (at 250 mg/L), a 62.5% reduction. Furthermore, ROS production in LPS-stimulated RAW 264.7 cells was reduced by 46.7% when 250 mg/L of the fermented extract was applied. A study indicated that phenolic acids and flavonoids in the water extract of CF seeds exerted antioxidative and anti-inflammatory effects in cellular/animal models [3]. Chu et al. (2022) reported that the water extract of hulled CF seeds reduced ROS generation [9]. These results of anti-inflammatory (Figure 5) and high polyphenol contents (Table 1) indicated that the fermented CF leaf extract can reduce oxidative stress and increase anti-inflammatory activity.

3.7. In Vivo Skin Whitening Activity of Fermented CF Leaf Methanolic Extract

The tyrosinase structure differs among mushrooms, mice, and humans; thus, the results of antityrosinase activity obtained from the nonhuman sources of tyrosinase may not reflect the real effects. Figure 6 displays the effects of the fermented extract of CF leaves on antityrosinase activity and melanin production in HEMn cells. The IC50 value of in vivo antityrosinase activity calculated from the regression equation between antityrosinase activity and the extract concentration was 33.2 mg/L, which is different from the IC50 value (14.1 mg/L) examined for mushroom tyrosinase (Figure 2B). The antityrosinase activity of the fermented extract was 33.6% ± 1.5% at 5 mg/L and 58.3% ± 2.4% at 50 mg/L (1.74 times higher). Similarly, the melanin production in HEMn cells was 63.5% ± 2.1% and 32.8% ± 0.6% (1.93 times lower), respectively. The curve of actual melanin production was inconsistent with that of theoretical melanin production (Figure 6). The higher inhibitory effect of melanin production indicated the involvement of additional mechanisms other than antityrosinase activity in the skin-whitening activity. Lin et al. (2021) reported that CF water extracts at 0.5% reduced melanin by approximately 30% compared with the control group [12]. Together, the findings exhibited excellent skin-whitening activity for the methanolic leaf extract of CF fermented by A. oryzae.
Polyphenols, such as phenolic acids and flavonoids, have high antioxidative activities due to the presence of several hydroxyl groups in their ring structures [30,41]. The fermented CF leaf extract was abundant in phenolic compounds and flavonoids (Table 1), which likely led to its high antityrosinase activity. De Freitas et al. (2016) indicated that rutin (also present in high concentrations in the fermented CF leaf extract) can effectively inhibit tyrosinase production and block melanin precipitation [47]. Together, these findings indicate a relationship between skin whitening activity and fermented CF leaf extract, likely due to its rutin content.

3.8. Molecular Docking Analysis

The concentrations of quercetin, gallic acid, protocatechuic acid, and epicatechin considerably increased after fermentation; therefore, they were subjected to a molecular docking analysis to evaluate their roles in skin-whitening and antiaging mechanisms. Table 3 lists the docking results of protocatechuic acid, epicatechin, gallic acid, and quercetin. The results revealed various degrees of theoretical binding affinities against the same enzyme. Quercetin exhibited the best binding energy (−6.8 kcal/mol) against tyrosinase. Epicatechin exhibited the best binding energy against antiaging enzymes (collagenase, elastase, and hyaluronidase). Protocatechuic acid exhibited relatively low binding energies against the four types of tested enzymes compared with other phytochemical compounds. The docking interactions and docking complex of optimal phytochemical compounds against tested enzymes are presented in Figure 7. Quercetin and epicatechin formed various molecular forces when they bound to tested enzymes, which facilitated protein–ligand complex formation. Among the compounds, quercetin exhibited the best docking interactions against tyrosinase due to the formation of H bonds with Ser274 and Pro271, π-cation bonds with Lys463 and Lys239, and π-alkyl bonds with Pro488 and Leu268 (Figure 7A). Epicatechin exhibited the best docking interactions against collagenase, which were favored by the formation of H bonds with Asn287 and Tyr345, π-cation and π-anion bonds with Asp 340 and Lys291, and π-π interactions between Try337 and the aromatic ring of epicatechin (Figure 7B). Epicatechin exhibited the best docking interactions against elastase, which were favored by the formation of H bonds with Ser214 and π interactions with His57 (π-cation), Phe215 (π-π), Leu998 (π-sigma), and Val216 (π-alkyl) (Figure 7C). Epicatechin exhibited the best docking interactions against hyaluronidase, which were favored by the formation of H bonds with Gly187 and Asp297 and π-π and π-alkyl interactions with Tyr189 (Figure 7D). On the basis of these results, we believe that the significantly increased concentrations of quercetin and epicatechin after fermentation may contribute the most to the antityrosinase and antiaging activities of the CF leaf extract.

4. Conclusions

Our findings reveal that the combination of extraction and fermentation significantly enhanced the biological activities of CF leaves, including antioxidative, anti-inflammatory, antimicrobial, skin-whitening, antiaging, and moisturizing activities. In particular, the CF leaf extract exhibited high biological activities even at low concentrations (e.g., 50 mg/L). The CF leaf extract revealed a better performance and a significantly lower cytotoxicity than the CF seed extract. Given that CF leaves are typically thrown away, their use can reduce agricultural waste and production costs. Moreover, a molecular docking analysis of the CF leaf extract indicated that quercetin had the highest skin-whitening effects and epicatechin had the highest antiaging effect. In conclusion, the fermented CF leaf extract is promising as a functional ingredient in health foods, botanical drugs, and cosmetic products.

Author Contributions

All authors collaborated to carry out the work presented here. Y.-M.L., P.-Y.C. and Y.-C.C. (Yu-Chi Chang) performed the experiments; Y.-C.C. (Ying-Chien Chung) and W.-L.C. conceived and designed the experiments; Y.-M.L. and Y.-C.C. (Ying-Chien Chung) wrote the paper; and W.-L.C. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Science and Technology of Taiwan, grant numbers NSTC 111-2622-E-157-002- and MOST 110-2313-B-157-001-MY2.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Physiological activities of unfermented extracts (1000 mg/L) of various parts of C. formosanum obtained using different extraction solvents. (A) DPPH scavenging activity, (B) ABTS scavenging activity, and (C) Antityrosinase activity. BHT was the positive control for antioxidant activity. Koji acid was the positive control for antityrosinase activity. Data are expressed as the means and standard deviations of three independent experiments. Significant difference was expressed by * p < 0.05.
Figure 1. Physiological activities of unfermented extracts (1000 mg/L) of various parts of C. formosanum obtained using different extraction solvents. (A) DPPH scavenging activity, (B) ABTS scavenging activity, and (C) Antityrosinase activity. BHT was the positive control for antioxidant activity. Koji acid was the positive control for antityrosinase activity. Data are expressed as the means and standard deviations of three independent experiments. Significant difference was expressed by * p < 0.05.
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Figure 2. Physiological activities of unfermented and fermented methanolic extracts of C. formosanum leaves and unhulled seeds. (A) ABTS scavenging activity and (B) antityrosinase activity. Data are expressed as the means and standard deviations of three independent experiments.
Figure 2. Physiological activities of unfermented and fermented methanolic extracts of C. formosanum leaves and unhulled seeds. (A) ABTS scavenging activity and (B) antityrosinase activity. Data are expressed as the means and standard deviations of three independent experiments.
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Figure 3. Cytotoxic effects of C. formosanum leaf extracts on HEMn, HaCaT, and Raw 264.7 cells. Fresh medium was used as a control. Data are expressed as the means and standard deviations of three independent experiments (* p < 0.05 vs. blank control).
Figure 3. Cytotoxic effects of C. formosanum leaf extracts on HEMn, HaCaT, and Raw 264.7 cells. Fresh medium was used as a control. Data are expressed as the means and standard deviations of three independent experiments (* p < 0.05 vs. blank control).
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Figure 4. Effect of fermented C. formosanum leaf extract on relative mRNA expression (AQP3) in HaCaT cells. Cells were treated without extract (control). Data are expressed as the means and standard deviations of three independent experiments.
Figure 4. Effect of fermented C. formosanum leaf extract on relative mRNA expression (AQP3) in HaCaT cells. Cells were treated without extract (control). Data are expressed as the means and standard deviations of three independent experiments.
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Figure 5. Effects of fermented C. formosanum leaf extract on NO, IL-6, and TNF-α production in LPS-stimulated RAW 264.7 cells. LPS alone was positive control. Data are expressed as the means and standard deviations of three independent experiments.
Figure 5. Effects of fermented C. formosanum leaf extract on NO, IL-6, and TNF-α production in LPS-stimulated RAW 264.7 cells. LPS alone was positive control. Data are expressed as the means and standard deviations of three independent experiments.
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Figure 6. Effects of fermented C. formosanum leaf extract on antityrosinase activity and melanin production in HEMn cells. Data are expressed as the means and standard deviations of three independent experiments.
Figure 6. Effects of fermented C. formosanum leaf extract on antityrosinase activity and melanin production in HEMn cells. Data are expressed as the means and standard deviations of three independent experiments.
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Figure 7. Docking interactions and docking complex of the optimal phytochemical compounds against tested enzymes. (A) Molecular docking of the interactions between quercetin and tyrosinase. (B) Molecular docking of the interactions between epicatechin and collagenase. (C) Molecular docking of the interactions between epicatechin and elastase. (D) Molecular docking of the interactions between epicatechin and hyaluronidase.
Figure 7. Docking interactions and docking complex of the optimal phytochemical compounds against tested enzymes. (A) Molecular docking of the interactions between quercetin and tyrosinase. (B) Molecular docking of the interactions between epicatechin and collagenase. (C) Molecular docking of the interactions between epicatechin and elastase. (D) Molecular docking of the interactions between epicatechin and hyaluronidase.
Applsci 13 02917 g007aApplsci 13 02917 g007b
Table
Table 1. TPC, TFC, and antiaging activity of the methanolic extract of C. formosanum leaves fermented with A. oryzae.
Table 1. TPC, TFC, and antiaging activity of the methanolic extract of C. formosanum leaves fermented with A. oryzae.
Phenolic YieldAntiaging Activity (IC50, mg/L)
TPC (mg-GAE/g-DW)TFC (mg-QE/g-DW)Collagenase ActivityElastase ActivityHyaluronidase ActivityMMP-1 Activity
Leaf extract162.5 ± 6.787.1 ± 2.8113.5 ± 9.2137.4 ± 16.2106 ± 7.6176.4 ± 13.8
Table
Table 2. Antimicrobial activity of the methanolic extract of C. formosanum leaves fermented by A. oryzae.
Table 2. Antimicrobial activity of the methanolic extract of C. formosanum leaves fermented by A. oryzae.
Antimicrobial Activity
MIC (mg/L)MFC (mg/mL)
E. coliS. aureusP. aeruginosaC. acnesC. albicansA. brasiliensis
Leaf extract150 ± 12180 ± 14140 ± 9120 ± 5230 ± 26280 ± 21
Table
Table 3. Results of the molecular docking analysis of protocatechuic acid, epicatechin, gallic acid, and quercetin.
Table 3. Results of the molecular docking analysis of protocatechuic acid, epicatechin, gallic acid, and quercetin.
Protocatechuic AcidEpicatechinGallic AcidQuercetin
Docking Affinity Score (kcal/mol)
Tyrosinase –−2.5−5.9−6.8
Collagenase−5.1−7.0−4.9−6.3
Elastase−4.8−6.7−4.9−6.5
Hyaluronidase−6.2−8.2−6.4−7.4
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Lin, Y.-M.; Chung, Y.-C.; Chen, P.-Y.; Chang, Y.-C.; Chen, W.-L. Fermentation of Chenopodium formosanum Leaf Extract with Aspergillus oryzae Significantly Enhanced Its Physiological Activities. Appl. Sci. 2023, 13, 2917. https://doi.org/10.3390/app13052917

AMA Style

Lin Y-M, Chung Y-C, Chen P-Y, Chang Y-C, Chen W-L. Fermentation of Chenopodium formosanum Leaf Extract with Aspergillus oryzae Significantly Enhanced Its Physiological Activities. Applied Sciences. 2023; 13(5):2917. https://doi.org/10.3390/app13052917

Chicago/Turabian Style

Lin, Yi-Min, Ying-Chien Chung, Pei-Yu Chen, Yu-Chi Chang, and Wen-Liang Chen. 2023. "Fermentation of Chenopodium formosanum Leaf Extract with Aspergillus oryzae Significantly Enhanced Its Physiological Activities" Applied Sciences 13, no. 5: 2917. https://doi.org/10.3390/app13052917

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