Characterization and Bioactive Potential of Secondary Metabolites Isolated from Piper sarmentosum Roxb.

Piper sarmentosum Roxb. (Piperaceae) is a traditional medicinal plant in South-East Asian countries. The chemical investigation of leaves from this species resulted in the isolation of three previously not described compounds, namely 4″-(3-hydroxy-3-methylglutaroyl)-2″-β-D-glucopyranosyl vitexin (1), kadukoside (2), and 6-O-trans-p-coumaroyl-D-glucono-1,4-lactone (3), together with 31 known compounds. Of these known compounds, 21 compounds were isolated for the first time from P. sarmentosum. The structures were established by 1D and 2D NMR techniques and HR-ESI-MS analyses. The compounds were evaluated for their anthelmintic (Caenorhabditis elegans), antifungal (Botrytis cinerea, Septoria tritici and Phytophthora infestans), antibacterial (Aliivibrio fischeri) and cytotoxic (PC-3 and HT-29 human cancer cells lines) activities. Methyl-3-(4-methoxyphenyl)propionate (8), isoasarone (12), and trans-asarone (15) demonstrated anthelmintic activity with IC50 values between 0.9 and 2.04 mM. Kadukoside (2) was most active against S. tritici with IC50 at 5.0 µM and also induced 94% inhibition of P. infestans growth at 125 µM. Trans-asarone (15), piperolactam A (23), and dehydroformouregine (24) displayed a dose-dependent effect against B. cinerea from 1.5 to 125 µM up to more than 80% inhibition. Paprazine (19), cepharadione A (21) and piperolactam A (23) inhibited bacterial growth by more than 85% at 100 µM. Only mild cytotoxic effects were observed.


Introduction
The Piperaceae family comprises numerous medicinal plants widely used in tropical and subtropical regions around the world. It consists of five genera namely Verhuellia, Zippelia, Manekia, Piper and Peperomia [1]. The most frequently described genera are Piper and Peperomia [1][2][3]. The Piper genus contains about 1000-2000 species with dominant species in their native habitat [4]. Many species of Piper have been used as traditional medicine to treat toothache, fever, chest, pain, cough, asthma, etc. [2]. Previous phytochemicals studies of the Piper genus resulted in the isolation of amide alkaloids, lignans, neolignans and phenylpropanoids as major constituents [2,5]. These isolated compounds displayed a wide range of biological effects including antifungal, antitumor, anti-inflammatory and antioxidant activities [2,[6][7][8].
Piper sarmentosum Roxb. (Piperaceae) is a creeping plant whose vernacular name varies from country to country. It is known as Kaduk and Pokok Kadok in Malaysia, Chaplu in Thailand, Sirih duduk, Akar buguor or Mengkadak in Indonesia, Bolalot in Vietnam and Jiaju, Gelou, Jialou and Shanlou in China [9][10][11]. This species is widely distributed in tropical regions in Northeast India, Southeast Asia and parts of China and has been commonly used in traditional medicine and also as food flavoring agents [12,13]. In Malaysia, the plant is also eaten raw as vegetable and the leaves are boiled in water and taken to relieve fever in malaria and treat coughs, flu, and rheumatism [14]. Furthermore, the whole plant, roots, leaves and fruits of P. sarmentosum have been used for the treatment of colds, gastritis, rheumatoid joint pain, abdominal pain, toothache, diabetes mellitus, worm infections and other diseases for many decades [15,16].
Modern pharmacological studies have shown that crude extracts of P. sarmentosum possess a wide range of biological activities such as antibacterial [17], anti-fungal [18], antiosteoporosis [19,20], anti-depression and neuroprotective [21,22], anti-inflammatory [23,24], anti-cancer [25], hypoglycemic [26], insecticidal [27,28], and antihypertensive activities [29,30]. A variety of chemical constituents, including essential oils, alkaloids, flavonoids, lignans and steroids, have been isolated mostly from the leaves and aerial parts of P. sarmentosum [15]. However, although a large number of chemical components have been isolated and identified from this species, only a few pure compounds have been studied with respect to their biological activity.
In the present study, we report the isolation, structure elucidation and biological effects of the previously undescribed compounds 1-3 along with 31 known compounds from the methanolic leaf extract of P. sarmentosum.

Biological Assays of Isolated Compounds
Earlier review studies of P. sarmentosum have reported diverse pharmacological activities, either as an extract or for some pure compounds [15]. Therefore, the compounds isolated from this species were tested for their anthelmintic, antifungal, antibacterial and cytotoxic properties. These biological examinations were conducted by using established model organisms that are non-pathogenic to humans and selected human cancer cell lines (Biosafety level-1) suitable for rapid screening assays.
The anthelmintic activity was evaluated against Caenorhabditis elegans. This biological screening demonstrated that three phenylpropanoids, methyl 3-(4-methoxyphenyl)-propionate (8), isoasarone (12), and trans-asarone (15) ( 1 H NMR spectra in Figures S27-S29), show anthelmintic activity against C. elegans with 100.0 ± 0.0%, 73.0 ± 1.7%, and 97.4 ± 0.9% percentage mortality, respectively, at a test concentration of 500 ppm ( Figure 5A). These promising compounds 8, 12, and 15 were re-tested against C. elegans with different concentrations ranging from 500 ppm to 100 ppm in order to determine the concentration that kills 50% (LC 50 ) of the nematodes. As shown in Figure 5B, the LC 50 values were calculated (by in-house macro program in Microsoft Excel 2013) to be 174.6 ppm corresponding to 0.9 mM, 425.4 ppm/2.0 mM, and 341.9 ppm/1.6 mM, for compounds 8, 12, and 15, respectively. All three compounds are very unpolar constituents without free hydroxyl functions and accordingly were obtained from the n-hexane fraction. High lipophilicity is a prerequisite for transtegumental diffusion of anthelmintics [74].
The activity of trans-asarone (15) is in accordance with data published by McGaw et al. [75] on the anthelmintic activity against C. elegans of its isomer β-asarone isolated from Acorus species (Acoraceae). To the best of our knowledge, there are no reports on the anthelmintic activity of isoasarone (12) and methyl 3-(4-methoxyphenyl)propionate (8). However, isoasarone (12) has been reported to be toxic against the mosquitos Aedes aegypti, Aedes albopictus and Culex quinquefasciatus [40], while compound 8 exhibited strong antifeedant activity [76]. The obtained results provides scientific evidence of the anthelmintic activity of P. sarmentosum leaves, which are traditionally used to treat worm infections [16]. An earlier study indicated that P. sarmentosum extracts have antifungal properties [18]. However, there is lack of information on compounds responsible for that activity. Therefore, all isolated compounds were tested for their antifungal effects against the phytopathogenic ascomycetes Botrytis cinerea Pars, and Septoria triciti Desm. and the oomycete Phytophthora infestans (Mont.). Briefly, the isolated compounds were tested at a highest concentration of 125 µM, while the commercially available fungicides epoxiconazole and terbinafine at the same concentration as tested samples served as positive controls ( Table 4). As shown in Table 4, seven compounds including the four phenylpropanoids derivatives kadukoside (2), methyl 3-(4-methoxyphenyl)propionate (8), isoasarone (12) and trans-asarone (15), the two alkaloids piperolactam A (23) and dehydroformouregine (24), and the flavonoid 5-hydroxy-7,4 -dimethoxyflavone (4) exhibited activity against the fungi B. cinerea, S. tritici or P. infestans with inhibition rates of about 40% at a concentration of 125 µM after seven days after inoculation. From these compounds, 2, 12 and 15 possess an asarone skeleton. Based on the first hits determined at 125 µM concentration in the initial rapid-screening and sample availability, five compounds were subjected to dose-dependency studies against the fungi S. tritici, B. cinerea and P. infestans. The pathogens were treated with compound concentrations ranging from 1.5 to 125 µM, followed by assay read-out and data analyses. As displayed in Figure 6A, compound 2 was the most active one against S. tritici with inhibition rates of more than 75% at the concentrations of 14 µM and higher, and 48% inhibition at 5 µM. Thus, the IC 50 of 2 calculated by SigmaPlot software was 5.0 ± 0.02 µM. Kadukoside (2) also displayed the highest inhibition activity against the oomycete P. infestans when treated at a concentration of 125 µM ( Figure 6C). As depicted in Figure 6B, the remaining three compounds (15, 23, and 24) exhibited significant activity against B. cinerea. with a dose-dependent effect from 1.5 to 125 µM. The results of compounds 23 and 24 suggest that the alkaloid scaffold is responsible for the antifungal activity. Similar antifungal activity was reported for structurally related piperolactam D and stigmalactam isolated from the aerial parts of Piper parviflorum C. DC. [77]. The substituents and their positions were suggested to be relevant for the distinct antifungal bioactivity [78]. Moreover, isoasarone (12) and trans-asarone (15) have been previously found in P. sarmentosun and other antifungal species such as Boesenbergia pulcherrima (Zingiberaceae) [79] and Acorus species (Acoraceae) [80]. Both of these compounds have been reported to possess antifungal activity against Candida albicans [81]. Surprisingly, in an earlier study trans-asarone did not show in vivo growth inhibition against B. cinerea at a concentration of 125 and even 1000 ppm [82].
Furthermore, all compounds 1-34 were screened for their antibacterial activity against the Gram-negative bacterium Aliivibrio fischeri at 1, 10 and 100 µM ( Table 4). As indicated in Figure 7, five alkaloid compounds namely, trans-N-feruloyltyramine (18), paprazine (19), cepharadione A (21), piperolactam A (23) and dehydroformouregine (24), induced over 40% inhibition of bacterial growth at the highest concentration of 100 µM. Lower concentrations showed no inhibition or even promoted the bacterial growth. In general, alkaloids are nitrogen-containing organic compounds with often significant biological activities. They exist widely in the plant world [5]. The antibacterial activity of compound 19 with 85% inhibition at a concentration of 100 µM was about two-fold better than that of the structurally related compound 18 (41% inhibition). The observed differences in inhibition between the two compounds may be due to the presence of different functional groups at C-3 of the p-coumaroyl moiety. Our results of these two compounds were similar to those reported by Mata et al., which indicate that an extra methoxy group in the p-coumaroyl unit lowers the antibacterial activity [83]. Furthermore, compound 19 which was isolated previously from Cannabis sativa (Cannabaceae) roots has been reported to possess antibacterial activity against a different Gram-negative bacterium, Escherichia coli with an IC 50 value of 0.8 µg/mL [84].
Moreover, the aporphines cepharadione A (21) and piperolactam A (23) almost completely inhibited the Gram-negative bacterium A. fischeri at the highest concentration of 100 µM. Contrary to our results, compound 21 which was isolated recently from the aerial parts of Piper wallichii (Miq.) Hand.-Mazz. showed a different trend in antibacterial activity. This compound was only active against the Gram-positive bacteria Bacillus cereus, Bacillus subtilis and Staphylococcus aureus; however, it was inactive against the three tested pathogenic Gram-negative bacteria E. coli, Pseudomonas aeruginosa and Shigella sonnei [85]. Thus, the compound may exhibit selective antibacterial activity against different Grampositive and Gram-negative species. With regard to compound 23, similar antibacterial properties have been described. For example, in an antimycobacterial bioassay-guided chromatographic study on Piper sanctum (Miq.) Schl. leaves performed by Mata et al. [83], compound 23 displayed good growth inhibition against Mycobacterium tuberculosis with an MIC value of 8 µg/mL. Compound 24 (dehydroformouregine) showed moderate antibacterial activity with 41% inhibition against the Gram-negative bacterium A. fischeri at 100 µM. Despite this compound having been previously isolated from Guatteria ouregou [68], there is no pharmacological activity reported, specifically no antibacterial activity; thus to the best of our knowledge, this is the first report of the antibacterial activity of compound 24.
The cytotoxicity and impact of most isolated compounds on the metabolic cell viability at a reasonable concentration of 10 nM and 10 µM were exemplarily evaluated using HT-29 (human colorectal adenocarcinoma) and PC-3 (human prostate adenocarcinoma) cancer cell lines. The effect on the cancer cell viability was determined by conducting an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The reduction of the tetrazolium dye is assumed to depend on NAD(P)H-dependent mitochondrial oxidoreductases and reflects the metabolic activity of the cells. A high metabolic activity is connected to high proliferation. General cytotoxic effects were determined by using a Crystal Violet (CV) assay. CV is applied to stain adherent intact cells and thus indirectly indicates cell death. Both complementary assays were performed after 48 h treatment with the compounds under investigation. A very potent permeabilizer of cell membranes, digitonin (125 µM), was used as a positive control compromising the cells to the point of 0% of cell viability after 48 h.
As demonstrated in Figure S30 of the supplementary data, most of the compounds tested did not reduce the cell viability below 80% even at the highest concentration of 10 µM. This indicates that compounds are very weakly or inactive against the specific cell lines. Meanwhile, compounds with cell viability below 80% at a concentration of 10 µM could be considered potentially cytotoxic and need further investigation. Briefly, five compounds including two alkaloids, aristolactam BII (22) and dehydroformouregine (24), and three neolignans, magnosalicin (30), andamanicin (31), and magnosalin (32), met this criterion for the HT-29 cell line either in MTT or CV assay at 10 µM. The observed cell viabilities in the MTT assay were 68.4 ± 1.5%, 76.4 ± 3.5% and 62.5 ± 4.2% for compounds 22, 31 and 32, respectively. While in the CV assay the cell viability was 52.9 ± 2.2%, 80.9 ± 5.9%, 71.7 ± 3.3%, 67.8 ± 2.2% and 61.7 ± 3.4% for compounds 22, 24, 30, 31 and 32, respectively. Interestingly, there were no significant variances in cytotoxicity between compounds 31 and 32, which possess stereochemical differences in the position of methyl groups at C-1 and C-2. In addition, three alkaloid compounds, cepharadione A (21), aristolactam BII (22), and piperolactam A (23) reduced cell viability of the PC-3 cell line below 80% at the highest concentration of 10 µM. The IC 50 values of all compounds can be estimated to be above 10 µM.
Out of these seven isolated compounds, only compounds 21, 22 and 23 were previously tested against HT-29 cell lines. Compound 22 previously demonstrated cytotoxic effects with an IC 50 value of 26 µg/mL [86], whereas for compounds 21 and 23 no activity was reported against HT-29 cell lines [87]. Furthermore, no data on 21, 22 and 23 against the human prostate (PC-3) cancer cell line has been published.
In summary, it is remarkable to note that the cell toxicity of the constituents is absent or low. This supports a safe usage of the species P. sarmentosum as food and medicinal plant. In accordance with the traditional application of this species, some isolated compounds from P. sarmentosum possess mild anthelmintic, antifungal, antibacterial or cytotoxic activities. It should be noted that impurities present in the isolated compounds may contribute to the observed effects. Nevertheless, the knowledge gained in this study on the molecular basis of P. sarmentosum will enable the future development of specific extracts and applications not only for human health but also for potential use in agriculture. For those constituents with a stronger effect (IC 50 < 10 µM), a detailed mode of action for the specific biological activity should be addressed in future investigations.

General Methods
The following instruments were used for obtaining physical and spectroscopic data: Column chromatography was performed on silica gel (400-630 mesh, Merck, Germany), Sephadex LH-20 (Fluka, Steinheim, Germany) and Diaion HP20 (Supelco, Bellefonte, PA, USA). Fractions and substances were monitored by TLC. TLC was conducted on precoated Kieselgel 60 F 254 plates (Merck, Darmstadt, Germany) and the spots were detected either by examining the plates under an UV lamp at 254 and 366 nm or by treating the plates with vanillin or natural product reagents. The UV spectra were recorded on a Jasco V-770 UV-Vis/NIR spectrophotometer (Jasco, Pfungstadt, Germany), meanwhile specific rotation was measured with a Jasco P-2000 digital polarimeter (Jasco, Pfungstadt, Germany).
NMR spectra were obtained with an Agilent DD2 400 system at +25 • C (Varian, Palo Alto, CA, USA) using a 5 mm inverse detection cryoprobe. The compounds were dissolved in CD 3 OD (99.8% D) or CDCl 3 (99.8% D), and the spectra were recorded at 399.915 MHz ( 1 H) and 100.569 MHz ( 13 C). 1D ( 1 H, 13 C, and TOCSY) and 2D ( 1 H, 13  The high-resolution mass spectra in both positive and negative ion modes were acquired using either an Orbitrap Elite Mass spectrometer or API 3200 Triple Quadrupole System. The Orbitrap Elite Mass spectrometer (Thermofisher Scientific, Bremen, Germany) was equipped with an HRESI electrospray ion source (spray voltage 4.0 kV, capillary temperature 275 • C, source heater temperature 80 • C, FTMS resolution 100.000), whereas API 3200 Triple Quadrupole System (Sciex, Framingham, MA, USA) was equipped with a turbo ion spray source, which performs ionization with an ion spray voltage on 70 eV. During the measurement, the mass/charge range from 50 to 2000 was scanned.

Plant Material
The dried powdered leaves of Piper sarmentosum Roxb. were supplied by the Institute of Bioproduct Development, Universiti Teknologi Malaysia. The plant material was collected in January 2019 from Negeri Sembilan, Malaysia. A voucher was authenticated (Number: MFI 0039/19) by Dr. Mohd Firdaus Ismail, a botanist at the Institute of Biosciences, Universiti Putra Malaysia. A duplicate of the Herbarium specimen is kept at the Bioorganic Chemistry Department of the Leibniz Institute of Plant Biochemistry, Germany.

Extraction and Isolation
The dried leaf powder (400 g) was extracted five times (1.2 L/each) with 80% aqueous MeOH at room temperature. The combined extracts were concentrated under reduced pressure to obtain a crude MeOH extract (107 g). The extract was suspended in distilled H 2 O (250 mL) and partitioned with n-hexane, EtOAc and n-BuOH, yielding 18, 9 and 17 g of residue, respectively. Further fractionation of the remained aqueous fraction was not extended due to unpromising biological activity. The EtOAc-soluble fraction (9 g) was subjected to a silica gel column (Length, L = 40 cm; diameter, d = 5.5 cm) and eluted with a stepwise gradient of DCM:EtOAc:MeOH (1:0:0 to 0:0:1) to yield ten fractions (E1-E10).   Figure S25).

Anthelmintic Assay
The anthelmintic bioassay was conducted using the Bristol N2 wild type strain of the model organism Caenorhabditis elegans, which was previously demonstrated to correlate with anthelmintic activity against parasitic trematodes [90]. The nematodes were cultured on NGM (Nematode Growth Media) Petri plates using the uracil auxotroph E. coli strain OP50 as food source according to the methods described by Stiernagle [91]. The anthelmintic bioassay was performed according to method developed by Thomsen et al. [90]. The solvent DMSO (2%) and the standard anthelmintic drug ivermectin (10 µg/mL) were used as negative and positive controls in all the assays, respectively. All assays were performed in triplicate.

Antifungal Assay
The antifungal activity was performed against phytopathogenic ascomycetes Botrytis cinerea Pars, and Septoria triciti Desm. and the oomycete Phytophthora infestans (Mont.) de Bary in 96-well microtiter plate assays with minor modification as described by Otto et al. [92]. In brief, the isolated compounds were tested at a highest concentration of 125 µM, while solvent DMSO was serving as negative control (max. concentration 2.5%), and the commercially available fungicides epoxiconazole and terbinafine (Sigma-Aldrich, Damstadt, Germany) served as reference compounds. The pathogen growth was assessed seven days after inoculation by the optical density (OD) at L 405 nm measurement with a TecanGENios Pro microplate reader (five measurements per well using multiple reads in 3 × 3 square). Each experiment was performed in triplicates.

Antibacterial Assay against Aliivibrio fischeri
The isolated compounds were tested at concentrations of 1 and 100 µM against the Gram-negative Aliivibrio fischeri test strain DSM507 (batch no. 1209), with chloramphenicol (100 µM) serving as a positive control as previously reported [3,93].
In brief, 25 mL BOSS medium containing fresh glycerol was incubated at 100 rpm and 23 • C for 16 to 18 h before being diluted with fresh BOSS medium to an appropriate cell number (luminescence value between 30,000 and 50,000 RLU). The assay was carried out in 96-well black flat-bottomed plates (Brand cell GradeTM premium, STERILE R) with a final volume of 200 µL of BOSS medium containing 1% DMSO per well (100 µL of diluted bacterial solution and 100 µL of test solution). The plates were incubated in the dark for 24 h without a lid and without shaking at a temperature of 23 • C and a humidity of 100 percent. The bioluminescence (measured in relative luminescence units, RLU) is proportional to cell density and was calculated after 24 h using the TecanGeniosPro microplate reader. As a result, the entire 1000 ms wavelength range was detected without any preliminary shaking to avoid secondary oxygen effects. The results (mean standard deviation value, n = 6) are given as relative values (percent inhibition) to the negative control (bacterial growth, 1 % DMSO without test compound). Negative values indicate that bacterial growth is accelerating or that luminescence is increasing.

Cytotoxicity Assay
The cytotoxicity and impact on the metabolic cell viability of isolated compounds at 10 nM and 10 µM was evaluated against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cancer cell lines. Both cell lines were purchased from ATCC (Manassas, VA, USA). The cell culture medium RPMI 1640, the supplements FCS and L-glutamine, as well as PBS and trypsin/EDTA were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Culture flasks, multi-well plates and further cell culture plastics were from Greiner Bio-One GmbH (Frickenhausen, Germany) and TPP (Trasadingen, Switzerland), respectively. PC-3 and HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine and 1% penicillin/streptomycin, and in a humidified atmosphere with 5% CO 2 at 37 • C. Routinely, cells were cultured in T-75 flasks until reaching subconfluency (~80%), subsequently cells were harvested by washing with PBS and detached by using trypsin/EDTA (0.05% in PBS) prior to cell passaging and seeding for sub-culturing and assays in 96-well plates [94].
The cell handling and assay techniques were in accordance to the method with minor modification as described by Khan et al. [94]. In brief, anti-proliferative and cytotoxic effects of the compounds were investigated by performing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and CV (crystal violet)based cell viability assays (Sigma-Aldrich, Taufkirchen, Germany), respectively. For this purpose, cells were seeded in low densities in 96-well plates using the aforementioned cell culture medium. The cells were allowed to adhere for 24 h, followed by the 48 h compound treatment. Based on 20 mM DMSO stock solutions, the compounds were diluted in standard growth media to reach final concentrations of 10 nM and 10 µM for cell treatment. For control measures, cells were treated in parallel with 125 µM digitonin (positive control, for data normalization set to 0% cell viability). Each data point was determined in technical quadruplicates and two independent biological replicates. As soon as the 48 h incubation was finished, cell viability was measured.
For the MTT assay, cells were washed once with PBS, followed by incubation with MTT working solution (0.5 mg/mL MTT in culture medium) for 1 h under standard growth conditions. After discarding the MTT solution, DMSO was added in order to dissolve the formed formazan, followed by measuring formazan absorbance at 570 nm, and additionally at the reference/background wavelength of 670 nm, by using a SpectraMax M5 multi-well plate reader (Molecular Devices, San Jose, CA, USA).
For the CV assay, cells were washed once with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). After discarding the PFA solution, the cells were left to dry for 10 min and then stained with 1% crystal violet solution for 15 min at RT. The cells were washed with water and were dried overnight at RT. Afterwards, acetic acid (33% in ultrapure water) was added to the stained cells and absorbance was measured at 570 nm and 670 nm (reference wavelength) using a SpectraMax

Conclusions
This study represents the most comprehensive phytochemical characterization of P. sarmentosum which is used as medicinal and food plant in Asian countries. Investigation of P. sarmentosum leaves yielded three new compounds (1-3) and 31 known ones. Interestingly, 21 of these known compounds were isolated from this species for the first time and thus were not described in previous studies of P. sarmentosum. The structures of all compounds were confirmed by several spectroscopic techniques, i.e 1 H-NMR, 13 C-NMR, 2D NMR, and HRMS. For the first time all isolated compounds were evaluated for their anthelmintic, antifungal, antibacterial and cytotoxic activities to extend the knowledge of their biological effects. It is noteworthy that only very few compounds showed cytotoxic effects, and only at high concentration implying a safe consumption of this species. Some isolated compounds showed anthelmintic, antifungal and antibacterial potential in accordance with the traditional application of the plant species. Our finding suggests that P. sarmentosum can be used as an important source of mild health-promoting effects.  The funder had no role in study design, decision to publish, or manuscript preparation.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.