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Introduction
According to the United States Drug Administration (FDA), dietary supplements are those products that are ingested orally and contain a dietary ingredient. These may include vitamins, minerals, amino acids, herbal, and botanical extracts among other compounds that can supplement the daily diet (FDA, 2022a). The demand for these products intensified considerably due to the arrival of SARS-CoV-2. Hamulka et al. (2021) point out that during the COVID-19 outbreak in 2020, the population's interest in inquiring about dietary supplements that help improve the immune system increased. The main compounds searched on the web were vitamins (C and D) and medicinal plant extracts (garlic, ginger, and turmeric). Dietary supplements can help strengthen the immune system and treat and prevent the main non-communicable diseases in the world, such as cardiovascular and respiratory diseases, cancer, diabetes, and obesity, among others (Bruins et al., 2019; OMS, 2022).
Likewise, the appearance of chronic-degenerative diseases is closely associated with oxidative stress (O S), which can be defined as an imbalance between antioxidants and oxidants such as free radicals (FR) and reactive oxygen species (ROS), which cause damage to macromolecules, and trigger a cascade of signaling involved with pro-inflammatory factors, which subsequently give rise to a wide variety of non-communicable diseases (Byrne et al., 2021; Charlton et al., 2021; Pizzino et al., 2017; Ramachandra et al., 2021; Sabbatino et al., 2021; Sies, 2020). Therefore, a balanced diet, rich in phytochemicals and nutraceuticals, can minimize the impact of OS (Prior & Cao, 2000), since it has been observed that they have the capacity to provide antioxidant activity against FR and ROS, in addition to exerting anti-inflammatory (Leyva-López et al., 2016), anticancer (Criollo-Mendoza et al., 2022), hypoglycemic (Gutiérrez-Grijalva et al., 2022), and hypocholesterolemic activities (Das et al.,2022), among others.
The dietary supplement industry and market are constantly changing as consumers increasingly demand products that meet quality and safety standards and also satisfy the nutritional and functional needs sought. The dietary supplements with the greatest demand are those that contain vitamins, minerals, amino acids, enzymes, and phytochemicals. These are presented individually or as mixtures, which can come from herbal extracts from chemical synthesis or by isolating and purifying the compounds of interest (Hassan et al., 2020; Lordan, 2021).
The mixture of phytochemicals and extracts from plants, medicinal fruits, and other nutraceuticals can enhance its biological activity, creating a synergy between the compounds. However, on some occasions, they are made with ethnopharmacological knowledge, lacking tests that guarantee their functionality and safety, so it is important to inquire about these products. According to Brzezicha et al. (2021), it is estimated that more than 80 % of the world's population uses some type of dietary supplement or herbal remedy, so it is necessary to study its safety and effectiveness. For this, study models have been proposed in cell lines, microorganisms, and living organisms. In the present work, the use of the biological model of Artemia salina was suggested since it has been widely used to evaluate the toxicity of natural compounds. It is economical and reproducible and allows the possible toxicity of the compounds to be elucidated before an evaluation in cell lines or murine models (Karchesy et al., 2016; Wanyoike et al., 2004). For this study, dietary supplements made from extracts of botanical origin, such as medicinal plants and edible fruits abundant in phytochemicals such as flavonoids, phenolic acids, terpenes, phytosterols, fatty acids, vitamins, and minerals with antioxidant, anti-inflammatory, antidiabetic, and antihypercholesterolemic therapeutic indications, were selected, so this research aimed to evaluate the antioxidant, biofunctional and toxicological potential of dietary supplements marketed in Mexico, made from medicinal plants, botanical extracts and edible fruits.
Materials and Methods
Ten dietary supplements based on medicinal plants, natural extracts, and edible fruits of different chemical nature were analyzed (Table 1). They were purchased in stores that commercializes dietary supplements in Culiacan, Sinaloa, Mexico. Green tea leaves (Camellia sinensis L.) were used as a reference for the antioxidant and toxicological assays.
Table 1 Characteristics of the dietary supplements analyzed.
| Code | Ingredients | Therapeutic indication** | Serving/day | Weight (mg)* | Dose/day (mg) | Origin |
|---|---|---|---|---|---|---|
| Suppl.1 | Bioflavonoid extract from Citrus limon, C. sinensis, C. paradisi, C. reticulata, C. aurantifolia, and Citrus sinensis peel hesperidin extract. | Antioxidant | 1-3 capsules | 650 | 1,950 | Canada |
| Suppl.2 | Standardized concentrate of Vaccinium corymbosum 36:1 and anthocyanins. | Antioxidant | 1-3 capsules | 512.5 | 1,537.5 | Canada |
| Suppl.3 | Dry extract of: C. longa, C. sinensis, Arthrospira platensis, L. ododes, R. officinalis, S. hispanica, T. officinale, R. rosea, A. muricata, E. oleracea, M. citrifolia, G. mangostana, A. propinquus, Aphanizomenon flos-aquae, seed of V. vinifera, P. africana. Mix of amino acids, carotenoids, vitamins, and minerals. | Antioxidant | 1 tablet | 1,200 | 1,200 | Mexico |
| Suppl.4 | Dry extract of G. biloba leaves. Vitamins and minerals. | Mental focus | 1-2 tablets | 445 | 890 | Mexico |
| Suppl.5 | M. charantia extract, of which 10% are bitter phytochemicals such as charantin | Glycemic control | 1 capsule | 500 | 500 | EUA |
| Suppl.6 | A mixture of standardized phytosterols (β-sitosterol, campesterol, stigmasterol). G. monogyna extract, A. hippocastanum, V. vinifera, V. myrtillus, A. sativum, C. annuum, C. sinensis, P. cuspidatum root resveratrol. Mix of vitamins and minerals. | Cardiovascular health | 3 tablets | 1,003.5 | 3,010.5 | EUA |
| Suppl.7 | Powder of O. ficcus, O. joconostle, A. sativum, C. sinensis, C. longa, C. limon, C. cyminum, I. sonorae. | Glycemic control | 6 capsules | 400 | 2,400 | Mexico |
| Suppl.8 | J. communis berries, P. alba leaves, and P. silvestris leaves. | Anti-inflammatory | 6 capsules | 300 | 1,800 | Mexico |
| Suppl.9 | Extract of C. vulgaris, C. sempervirens, A. uva-ursi, C. pubescens. | Anti-inflammatory and antioxidant | 2 tablets | 500 | 1,000 | Mexico |
| Suppl.10 | Dried fruit of organic L. barbarum | Antioxidant | 3 capsules | 500 | 1,500 | Mexico |
| Control | C. sinensis leaves | Antioxidant | N/D | N/D | N/D | N/D |
Note: Suppl. (Supplement); USA (United States of America); N/D (Not declared). *Weight in mg of each tablet/capsule. **Noted by the manufacturer.
Extraction
500 mg of each dietary supplement was weighed and dissolved with 10 mL of 80 % ethanol (EtOH) assisted by ultrasound, with a power of 50 Hz and a frequency of 40 KHz (Cole Parmer Ultrasonic Bath EW-08895-15) at 45 °C, for 2 h. After time, the extracts were centrifuged at 10,000 rpm, at 4°C for 15 min, and the supernatant was recovered and used for the following determinations. For the determination of volatile organic compounds (VOCs), hexane and methanolic extracts of supplement 8 and methanolic extracts of supplement 3 were obtained. These solvents were used to extract the hydrophilic and hydrophobic compounds. The same extraction conditions mentioned above were used.
Determination of Total Reducing Capacity (TRC) and Total Flavonoids (TF)
For the determination of TRC, the Folin-Ciocalteu method described by Swain & Hillis (1959), was used with slight modifications. A calibration curve of gallic acid (0-0.4 mg/mL) was carried out, which was used as a standard. 80 % EtOH was used as blank. In a 96-well Costar® microplate, 10 μL of the extract was mixed with 230 μL of distilled water and 10 μL of the 2N Folin-Ciocalteu reagent and incubated for 3 min, then 25 μL of Na2CO3 was added and kept 2 h in darkness. The absorbance was read at 725 nm in a microplate reader Synergy HT (BioTek, Inc, EUA). The results were expressed in mg equivalents of gallic acid (GAE) per gram.
The TF was determined following the method of Ebrahimzadeh et al. (2018), which consisted of adding 10 μL of the extracts to a 96-well microplate, followed by 250 μL of distilled water, 10 μL of 10% AlCl3 and 10 μL of 1M CH3CO2K. It was allowed to incubate for 30 min in the darkness, and the absorbance was read at 415 nm using a Synergy HT microplate reader (Biotek, Inc, USA). 80% EtOH was used as blank, and a quercetin standard curve from 0 to 1.0 mg/mL was constructed. The results were expressed in mg quercetin equivalents (EQ) per gram.
Antioxidant activity (AOX)
Oxygen Radical Absorbance Capacity (ORAC)
It was carried out following the method described by Huang et al. (2002) with slight modifications. A 75 mM phosphate buffer adjusted to pH 7.4 was prepared, a 22.5 mg/mL fluorescein solution in phosphate buffer (stock solution), of which 100 μL was taken and made up to 10 mL with the phosphate buffer (intermediate solution). From this last solution, 400 μL was taken and volumetric to 25 mL with phosphate buffer (working solution). A solution of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) 2.6 mg/mL was prepared with phosphate buffer and a calibration curve of 400 μM Trolox in phosphate buffer.
In a 96-well microplate, 230 μL of distilled water was deposited in the outer rows and columns to maintain the working wells at a stable temperature (37 °C). The working solution of fluorescein and AAPH was deposited in the microplate reader (Synergy HT, BioTek, Inc, USA) to be automatically dispensed. 25 μL of blank (phosphate buffer), calibration curve (Trolox), and samples were deposited, and the microplate was placed inside the reader at a temperature of 37 °C, and the experiment was carried out. The microplate reader automatically dispensed 150 μL fluorescein and 50 μL AAPH and took readings for 70 min at 60 s intervals with an excitation wavelength of 485 nm and emission at a wavelength of 580 nm. The results were expressed as equivalent µmol of Trolox per gram.
ABTS radical scavenging capacity
It was carried out using the method proposed by Thaipong et al. (2006), with slight modifications. A 7.4 mM ABTS (2,2'-Azinobis-3-ethyl-benzo-thiazoline-6-sulfonic acid depletion) stock solution and a 2.6 mM potassium persulfate stock solution were prepared; both were mixed in equal volumes, and They were left to react, protected from light, at room temperature for 16 h. From this mixture, the working solution was prepared, which consisted of diluting it with absolute EtOH until obtaining an absorbance of 0.7 at 734 nm. A 1 mM Trolox stock solution was prepared, with a calibration curve from 0.1 to 1.0 mM. For the assay, 10 μL of the blank (80 % EtOH), calibration curve, and sample were deposited in a 96-well microplate, followed by 190 μL of the ABTS radical, and allowed to react for 2 h at room temperature, in the darkness. Once the time had elapsed, the absorbance was read at 734 nm in a Synergy HT microplate reader (BioTek, Inc., USA). The results were expressed as equivalent mmol of Trolox per gram.
DPPH radical scavenging capacity
It was carried out according to the methodology proposed by Karadag et al. (2009), with slight modifications. A 100 μM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution and a 1 mM Trolox stock solution were prepared. A calibration curve was prepared from 0.1 to 1.0 mM Trolox. For the assay, 10 μL of the blank (80 % EtOH), calibration curve, and sample were deposited in a 96-well microplate, followed by 190 μL of the DPPH radical, and allowed to react for 30 min at room temperature, in the darkness. Once the time had elapsed, the absorbance was read at 540 nm in a Synergy HT microplate reader (BioTek, Inc., USA). The results were expressed as equivalent mmol of Trolox per gram.
Determination of volatile organic compounds (VOCs) by GC-IT-MS/MS
The VOCs present in the methanolic and hexanic extracts of supplement No. 8 and methanolic extracts of supplement No. 3 were identified. Identification was performed on an Agilent 7890B gas chromatograph with an ion trap tandem mass spectrometry detector (CG-IT-MS Agilent 240). A VF-5 MS column, 30 m x 0.25 mm x 0.25 µm, was used. The mass spectra were compared to the NIST Mass Spectral Library using the NIST MS search or probability-based match search format as part of Agilent Technologies Workstation MS Software Version 7.0.1; those compounds with a similarity percentage greater than 80 were considered present in the extracts.
Toxicity test in Artemia salina model
It was carried out following the method described by Meyer et al. (1982). A. salina eggs were obtained from a local aquarium in Culiacan, Sinaloa. 3 g of eggs were placed in one L of brine adjusted to pH 7 with 1N sodium hydroxide. They were allowed to hatch for 12 h at 28 °C, with oxygenation and incandescent light. Once the eggs hatched, 10 nauplii were transferred to 6-well microplates. For the bioassay, the nauplii were placed in contact with the evaluated concentrations (100, 200, 300, 400, 500, 1,000, 1,500, 2,000, and 2,500 μg/mL) (only from supplement 8). After 24 h, the surviving crustaceans were counted with a stereoscope. 99 % caffeine obtained from Sigma Merck® was used as a positive control and distilled water as a negative control.
Bioinformatic study
The SuperPred platform (https://prediction.charite.de/) was used to predict the biofunctional potential that the metabolites identified in the extracts of supplements 3 and 8 could potentially have. Targets related to oxidative stress pathologies were obtained from the Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/).
Statistical analysis
All assays were performed by triplicate. The results were expressed as means and standard deviation. A completely randomized one-way ANOVA was performed for the AOX, TRC, and TF analyses, and the means were contrasted using the Tukey test with an α ≤ 0.05. The Minitab18 program (Minitab Inc. State College, Pa., USA) was used for this. For the toxicity study, the IC50 of extract No. 8 was determined by linear regression, represented by the logarithm of the concentration against the lethality percentage.
Results and discussion
Determination of Total Reducing Capacity (TRC) and Total Flavonoids (TF)
Highlights the TRC of supplements 3 and 8 (Table 2). Likewise, the TF content was higher in supplement 3, followed by supplements 4, 5, and 2 (Table 2). In contrast, the TF content in supplement 8 was 3.15 ± 0.31 mg EQ/g, so its TRC may be due to other phytochemicals and compounds present, such as phenolic acids, fatty acids, terpenes, saponin sugars, among others. The Folin-Ciocalteu method is frequently used to estimate the total content of phenolic compounds. However, it has been shown that other compounds, such as terpenes, sterols, vitamins, sugars, alkaloids, ketones, aldehydes, and fatty acids with TRC can interfere with the assay (Magalhães et al., 2010). Therefore, various investigations indicate that it is also a method to estimate the AOX of natural extracts due to the electron transfer that the TRC of an antioxidant compound measures. Additionally, it correlates with other electron transfer assays such as DPPH and ABTS methods (Everette et al., 2010; Lamuela-Raventós, 2018; Magalhães et al., 2006). Also, the molecular structure of phenols can interfere with the test, mainly in the amount and position of the hydroxyl groups present (Magalhães et al., 2010; Platzer et al., 2021). This may explain the very low TF content of supplement 8, which has the highest TRC.
TRC correlates closely with phytochemical content. The composition of supplement 8 stands out for its majority content of Juniperus communis fruits, Pinus sylvestris leaves, and Populus alba leaves. In the fruits of J. communis, a higher content of monoterpenes, diterpenes, and flavonoids has been observed (Ben Mrid et al., 2019; Falasca et al., 2014; Jegal et al., 2017). While in P. sylvestris, mainly monoterpenes, sesquiterpenes, diterpenes, flavan-3-ol type flavonoids, acetylated and glycosylated flavonoids, neolignans, and condensed tannins have been identified (Allenspach et al., 2020; Tegelberg et al., 2018). Likewise, the bark has significant phenolic compounds (Pap et al., 2021). The content of total phenolic compounds in P. sylvestris leaf extracts has also been reported, providing 0.19 mg GAE/g and 51.09 mg QE/g of total flavonoids (Fierascu et al. 2018).
The presence of phenolic acids, lignans, aglycone, and glycosylated flavonoids has been demonstrated in P. alba leaves (Danise et al., 2021; Elsbaey et al., 2019; Tawfeek et al., 2019). Likewise, P. alba leaf extracts contain total phenolic compounds (139.55 ± 8.81 mg GAE/g) and TF (46.12 ± 1.19 mg QE/g) (Elsbaey et al., 2019).
On the other hand, supplement 3 was the one that presented the greatest amount of TF (Table 2). These results are due to the great diversity of phytochemicals present in the product, which have been shown to exert a powerful AOX (Memarzia et al., 2021), with Curcuma longa extracts standing out for their higher concentration in the product followed by extract of Camelia sinensis (Zhang et al., 2019), Arthrospira platensis (Braune et al., 2021) and Rosmarinus officinalis (Ali et al., 2019). It is worth mentioning that this product is a mixture of at least 16 herbal extracts, amino acids, vitamins, minerals, and carotenoids, which also provide TRC and AOX.
Supplement 4 was the third with the highest TRC and TF (Table 2). This product is mainly composed of dry extract of Gingko biloba leaves. It is well known that G. biloba is a good source of phytochemicals, particularly glycosylated flavonoids derived from quercetin, kaempferol, and isorhamnetin (Liu et al., 2015). In contrast, supplements 1 and 2, containing flavonoid extracts, such as hesperidin (flavanone) (Supplement 1), and blueberry concentrate (Vaccinium corymbosum) (Supplement 2), which is abundant in anthocyanins, phenolic acids, and flavonoids (Sun et al., 2018). Manufacturers recommend a daily serving of 1,950 and 1,535.5 mg, respectively. According to our results, they only provide 2.70 and 5.98 mg EQ/g of total flavonoids. These results, lower than those reported by the manufacturers, may be due to the type of solvent used for the extraction (EtOH 80%) and the extraction method (ultrasound), in addition to the reference standard (quercetin), which is a flavonol. At the same time, hesperidin is a flavanone, and the compounds in blueberry are anthocyanins and phenolic acids mainly. Likewise, there may be irregularities in the production processes of this type of product (Wolsko et al., 2005).
Supplement 9 presented a low content of TRC and TF (Table 2). Its main ingredient is Chlorella vulgaris, followed by Cupressus sempervirens, Arctostaphylos uva-ursi, and Capsicum pubescens. C. vulgaris is characterized by presenting phenolic acids, flavonoids, tannins, and triterpenoids, among other compounds (Habashy et al., 2018). It also stands out as a source of hydrophobic compounds, so the solvent used for the extraction probably could not extract them (Ho & Redan, 2022). The leaves of C. sempervirens contain flavonoids and phenolic acids with reducing capacity (Ibrahim et al., 2007). Likewise, the leaves of A. uva-ursi contain arbutin, gallic acid, gallotannins, quercetin glycosides, kaempferol, and myricetin, which have been shown to TRC (Panusa et al., 2015).
Similarly, supplement 10, composed of goji berries, did not present a significant content of TRC and TF (Table 2). This may be because it mainly comprises hydrophobic metabolites such as carotenoids and fatty acids; However, flavonoids and phenolic acids have also been identified (Amagase & Farnsworth, 2011). These significant differences may be due to growing conditions, harvest, variety, origin, biotic and abiotic stress, climate, and handling (Figueiredo et al., 2008). However, it leaves a lack of credibility for the manufacturer since, according to their ingredients, they should have a higher TRC and TF content. Due to the above, it is correct to mention that a specialist should always recommend this type of products, monitor their consumption, and purchase from companies registered with the corresponding health commissions.
AOX in vitro
The AOX results are shown in Table 2. Supplement 3 was the one that showed the highest AOX in the ABTS and DPPH methods, showing significant differences from the other supplements. Phenolic compounds and terpenes are the two phytochemicals that contribute majorly to the AOX of vegetables and medicinal plants (Zhang et al., 2015). In supplement 3, the main ingredient is the rhizome of C. longa, which is abundant in phenolic compounds and terpenes, which is why it exerts a powerful AOX and considerable antiradical capacity in vitro (Altir et al., 2021).
Among the other components of supplement 3 is green tea (C. sinensis), which is recognized for its ability to capture free radicals and reduce biomarkers of oxidative stress (Thitimuta et al., 2017). It also contains Arthrospira plantensis, whose protective properties against oxidative damage have been previously reported (Gutiérrez-Rebolledo et al., 2015). Finamore et al. (2017), reported the antioxidant effects of A. platensis, relating them mainly to its content of phenolic compounds, phycocyanins, and polysaccharides. Regarding the results of the ORAC method, supplement 6 was the one that showed the highest AOX, followed by supplement 4 and 1 (Table 2).
Table 2 Total reducing capacity, total flavonoids, and antioxidant activity of hydroethanolic extracts of food supplements.
| AOX (μmol ET/g) | |||||
|---|---|---|---|---|---|
| Sample | TRC (mg GAE/g) | TF (mg EQ/g) | ABTS | DPPH | ORAC |
| Suppl.1 | 7.47 ± 0.48D,E,F | 2.70 ± 0.02E,F | 0.9 ± 0.00E,F | 0.07 ± 0.01C | 311,720 ± 16,840C,D |
| Suppl.2 | 5.03 ± 0.38E,F | 5.98 ± 0.05C,D,E | 0.074 ± 0.00E,F,G | 0.18 ± 0.00D | 124,980 ± 2,230E |
| Suppl.3 | 28.82 ± 2.09C,D | 11.82 ± 0.83A,B | 4.02 ± 0.04A | 1.91 ± 0.12A | 263,430 ± 24,220 D |
| Suppl.4 | 18.50 ± 2.05C | 9.66 ± 1.14A,B,C | 0.22 ± 0.01D | 0.16 ± 0.01D | 333,770 ± 11,160B,C |
| Suppl.5 | 8.19 ± 0.54D,E,F | 7.53 ± 0.50B,C,D | 0.12 ± 0.01E | 0.03 ± 0.01 D | 45,900 ± 4,840F |
| Suppl.6 | 11.82 ± 0.75C,D,E | 1.61 ± 0.24E,F | 0.26 ± 0.03D | 0.05 ± 0.00 D | 384,220 ± 9,340B |
| Suppl.7 | 7.70 ± 0.75D,E,F | 1.56 ± 0.06E,F | 0.08 ± 0.01E,F,G | 0.5 ± 0.01 D | 150,010 ± 680E |
| Suppl.8 | 43.08 ± 3.13B | 3.15 ± 0.31D,E,F | 2.43 ± 0.02C | 1.28 ± 0.00B | 308,140 ± 10,040C,D |
| Suppl.9 | 0.62 ± 0.16F | 0.13 ± 0.01F | 0.009 ± 0.00G | 0.03 ± 0.02 D | 25,560 ± 3,210F |
| Suppl.10 | 1.32 ± 0.17E,F | 0.12 ± 0.01F | 0.01 ± 0.00F,G | 0.02 ± 0.00 D | 12,810 ± 640F |
| Control* | 78 ± 10.92A | 14.64 ± 2.87A | 3.57 ± 0.04B | 1.62 ± 0.55B | 681,210 ± 50,710A |
Note: *C. sinensis leaves. The results are the means and standard deviation of three replicates. GAE (gallic acid equivalent); EQ (quercetin equivalent); TRC (Total Reducing Capacity); TF (total flavonoids); ET (Trolox equivalent). Different letters in columns indicate statistical differences according to Tukey's test at α ≤ 0.05.
Supplement 6 is a mixture of phytosterols, which have a high radical scavenging capacity and act mainly by protecting cell membranes (Vezza et al., 2020; Yoshida & Niki, 2003). On the other hand, supplement 4 is made from G. biloba, which has AOX, which is related to its content of phenolic compounds (Liu et al., 2007). Supplement 1 is made from plants abundant in flavonoids, whose pharmacological properties are attributed to the inhibition of enzymes that participate in the production of free radicals and their capture capacity, as well as iron chelation (Russo et al., 2000). The results obtained are supported by what was obtained by Ferretti et al. (2010), who previously reported the ability of plant phytosterols to reduce lipid peroxidation of LDL cholesterol, demonstrating their antioxidant activity. The differences in AOX obtained with the different methods could be because the ABTS method allows measuring the antioxidant activity of compounds with a hydrophilic and lipophilic nature. At the same time, DPPH can only dissolve in organic media (Kuskoski et al., 2005), and the ORAC method measures the oxygen radical absorbance capacity, making it one of the methods that provides the most information on the antioxidant potential in biological systems (Zapata et al., 2014).
Free radicals have been related to the incidence and development of chronic diseases such as cardiovascular diseases, various types of cancer, and neurodegenerative diseases, among others (Ginter et al., 2014). This is why products rich in antioxidants are highly attractive to consumers since they consider that consuming this type of supplement does not cause any harm to health (Schroder & Navarro, 2006). However, due to its high doses of phytochemicals, it is necessary to continue evaluating the doses and periods in which this supplementation is appropriate and safe so that possible adverse effects and health damage can be avoided when consumed.
Determination of Volatile Organic Compounds (VOCs)
Only supplements 3 and 8 were selected to determine VOCs because they had the highest nutraceutical content (Table 2). Fatty acids, terpenoids, and phytosterols were identified (Table 3). In supplement 3, aromatic terpenes derived from turmeric stand out. While in supplement 8 a wide variety of terpenes with biological activities were identified.
Table 3 Volatile organic compounds identified in hexanic and methanolic extracts of supplements 3 and 8.
| Supplement | Extract | Compound | Retention time | # CAS | % Similarity |
|---|---|---|---|---|---|
| Suppl. 3 | MeOH | o-Ethylhydroxylamine | 2.41 | 624-86-2 | 91.1 |
| Methenamine, N-hydroxy-N-methyl- | 2.44 | 5725-96-2 | 95.9 | ||
| N-allyl-N, N-dimethylamine | 2.47 | 2155-94-4 | 90.6 | ||
| 3(2H)-Pydazinone | 3.04 | 504-30-3 | 86.0 | ||
| 1H-Tetrazole | 3.15 | 288-94-8 | 81.3 | ||
| 2(5H)-furanone | 3.16 | 597-23-4 | 88.0 | ||
| 1,2-Cyclopentanedione | 3.21 | 3008-40-0 | 94.05 | ||
| Methanamine, N-methoxy- | 3.54 | 1117-97-1 | 93.9 | ||
| 2-pyrrolidinone | 3.71 | 616-45-5 | 92.8 | ||
| 2,4,5-trihydroxypyrimidine | 3.90 | 496-76-4 | 82.5 | ||
| Benzoic acid hydrazide | 3.97 | 613-94-5 | 86.8 | ||
| 1,azabicycle[3.1.0] hexane | 3.99 | 285-76-7 | 84.3 | ||
| 1, methyl-5-fluoruracil | 4.22 | 1000427-92-0 | 86.3 | ||
| Camphor | 4.24 | 76-22-2 | 91.5 | ||
| Catechol | 4.40 | 120-80-9 | 85.6 | ||
| 4-vinylphenol | 4.48 | 2628-17-3 | 96.0 | ||
| 4-Chloro-1-azabicyclo[2.2.2] octane | 4.76 | 5960-95-2 | 92.1 | ||
| 1,2-benzenediol, 3-methoxy- | 4.76 | 934-00-9 | 91.2 | ||
| 2-propen-1-ol, 3-phenyl- | 4.96 | 104-54-1 | 93.1 | ||
| 2-methoxy-4-vinylphenol | 5.00 | 7786-61-0 | 95.1 | ||
| Phenol, 2, 6-dimethoxy- | 5.17 | 91-10-1 | 85.1 | ||
| Benzaldehyde, 2,4-dihydroxy-6-methyl- | 5.48 | 487-69-4 | 85.1 | ||
| Isobisabolene | 6.10 | 1000-424-85-5 | 80.1 | ||
| Pentanoic acid | 6.70 | 109-52-4 | 80.5 | ||
| aR-Turmerone | 6.92 | 5-32-65-0 | 82.0 | ||
| Turmerone | 7.26 | 180315-67-7 | 95.8 | ||
| Heptadecane | 7.33 | 629-78-7 | 96.4 | ||
| (E)-atlantone | 8.08 | 108645-54-1 | 90.1 | ||
| Neophytadiene | 8.46 | 504-96-1 | 80.4 | ||
| Caffeine | 8.78 | 58-08-2 | 84.9 | ||
| Hexadecanoic acid, methyl ester | 9.18 | 112-39-0 | 91.0 | ||
| Turmeronol A | 9.38 | 13165-37-1 | 87.1 | ||
| n-hexadecanoic acid | 9.49 | 57-10-3 | 89.0 | ||
| Hexadecanoic acid, ethyl ester | 9.75 | 628-97-7 | 90.8 | ||
| Methyl.gamma.-linolenate | 10.53 | 16326-32-2 | 95.4 | ||
| 9,12-Octadecanoic acid(Z,Z)-,methyl ester | 10.65 | 112-63-0 | 94.3 | ||
| 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z)- | 10.72 | 301-00-8 | 82.2 | ||
| Phytol | 10.80 | 150-86-7 | 94.9 | ||
| Methyl stearate | 10.89 | 112-61-8 | 91.1 | ||
| Linoleic acid, ethyl ester | 11.22 | 544-35-4 | 94.0 | ||
| Octadecanoic acid, ethyl ester | 11.45 | 111-61-5 | 87.2 | ||
| Curlone | 13.06 | 90.9 | |||
| Campesterol | 19.45 | 474-62-4 | 83.0 | ||
| Stigmasterol | 19.72 | 83-48-7 | 86.5 | ||
| Gamma-sitosterol | 20.26 | 83-47-6 | 83.9 | ||
| Suppl. 8 | MeOH | o-ethylhydroxylamine | 2.41 | 624-86-2 | 89.9 |
| Hydrazine, propyl- | 2.53 | 5039-61-2 | 83.8 | ||
| 1H-imidazole,1-methyl- | 2.90 | 616-47-7 | 88.0 | ||
| 1H-pyrazole,1 methyl- | 2.90 | 930-36-9 | 81.8 | ||
| Methanamide, N-hydroxy-N-methyl- | 3.04 | 5725-96-2 | 83.6 | ||
| 3-amino-1,2,4-triazine | 3.05 | 1120-99-6 | 80.7 | ||
| 1H-tetrazole | 3.16 | 288-94-8 | 85.2 | ||
| 1,2-cyclopentanedione | 3.19 | 3008-40-0 | 95.0 | ||
| N-methoxy-N-methylacetamide | 3.45 | 78191-00-1 | 84.6 | ||
| Thymine | 3.85 | 65-71-4 | 85.8 | ||
| Dihydro-3-methylene-5-methyl-2-furanone | 3.88 | 62873-16-9 | 91.0 | ||
| Phenol, 2-methoxy- | 3.94 | 90-05-1 | 91.9 | ||
| Benzoic acid, methyl ester | 3.96 | 93-58-3 | 83.5 | ||
| Ethanone, 1-(2-hydroxy-6-methoxyphenyl)- | 4.11 | 703-23-1 | 80.8 | ||
| Catechol | 4.38 | 120-809 | 95.1 | ||
| Beta.-D-glucopyranoside-methyl | 6.82 | 709-50-2 | 80.2 | ||
| Neophytadiene | 8.46 | 504-96-1 | 85.9 | ||
| Hexadecanoic acid, methyl ester | 9.17 | 112-39-0 | 92.6 | ||
| n-hexadecanoic acid | 9.47 | 57-10-3 | 91.2 | ||
| 9-octadecanoic acid, methyl ester, (E)- | 10.69 | 1937-62-8 | 82.6 | ||
| Phytol | 10.80 | 150-86-7 | 87.1 | ||
| Octadecanoic acid | 11.17 | 57-11-4 | 80.9 | ||
| Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester | 14.02 | 23470-00-0 | 80.4 | ||
| Squalene | 16.37 | 111-02-4 | 92.9 | ||
| Hentriacontano | 16.79 | 630-04-6 | 88.8 | ||
| Stigmasterol | 19.72 | 83-48-7 | 86.6 | ||
| γ-sitosterol | 20.26 | 83-47-6 | 84.1 | ||
| Olean-12-en-3-ol, acetate, (3.beta.)- | 20.71 | 1616-93-9 | |||
| α-amyrin | 44.74 | 83.5 | |||
| β-amyrin | 20.72 | 559-70-6 | 84.9 | ||
| D-friedoolean-14-en-3-ol | 20.55 | 81654-73-1 | 80.5 | ||
| Hexane | 2-pyrrolidinone | 2.11 | 616-45-5 | 82.2 | |
| (-)-spathulenol | 11.70 | - | 84.5 | ||
| Cubenol | 12.21 | - | 80.2 | ||
| 7-epi-cis-sesquisabinene hyd | 12.39 | - | 74.3 | ||
| Tau-muurolol | 12.54 | - | 79.9 | ||
| Cis-verbenol | 7.63 | - | 84.3 | ||
| (-)-myrtenol | 8.27 | - | 82.1 | ||
| 4-epi-cubedol | 11.09 | - | 82.2 | ||
| Trans-calameno | 11.14 | - | 81.6 | ||
| 1,3-propanediamine | 2.72 | 109-76-2 | 86.6 | ||
| Neophytadiene | 8.45 | 504-96-1 | 94.9 | ||
| 2-pentadecanone, 6,10,14-trimethyl- | 8.51 | 502-69-2 | 89.8 | ||
| n- hexadecanoic acid | 9.44 | 57-10-3 | 82.4 | ||
| Phytol | 10.79 | 150-86-7 | 90.1 | ||
| Heneicosan | 13.89 | 629-94-7 | 83.5 | ||
| Hentriacontano | 15.38 | 630-04-6 | 90.8 | ||
| Eicosan | 15.38 | 112-95-8 | 90.7 | ||
| Squalene | 16.37 | 111-02-4 | 94.3 | ||
| Triacontane | 19.66 | 638-68-6 | 81.5 | ||
| Stigmasterol | 19.71 | 83-48-7 | 81.6 | ||
| γ-sitosterol | 20.25 | 83-47-6 | 83.3 | ||
| D-Friedoolean-14-en-3-ol | 20.53 | 81654-73-1 | 80.5 | ||
| β-amyrin | 20.71 | 559-70-6 | 87.8 |
The compounds identified in supplement No. 3 aR-turmerone, turmerone, (E)-atlantone, turmeronol A, and curlone, are curcuminoids and sesquiterpenes derived from turmeric, the main ingredient of the product (Salem et al., 2022). These metabolites suggest presenting biological activity, mainly anti-inflammatory, anti-cancer, and antioxidant (Jayaprakasha et al., 2005). Likewise, phytosterols identified in C. longa, such as campesterol, stigmasterol, and gamma-sitosterol, have been shown to reduce cholesterol levels in individuals with hypercholesterolemia (Ferguson et al., 2018).
The hexane and methanolic extract of supplement No. 8 extracted a great diversity of metabolites due to the different solvents' polarities. The predominant compounds extracted were terpenes and triterpenes, such as squalene, hentriacontane, α-amyrin, and β-amyrin. Within the product's composition, the content of white poplar leaves, scots pine leaves, and juniper berries stand out, which are abundant in these compounds. In addition, terpenes are the secondary metabolites with the greatest presence and distribution in plants. They stand out for their antioxidant, anti-inflammatory, anticancer, and antibacterial activity (Bajac et al., 2023; Guleria et al., 2021; Ji & Ji, 2021).
Cytotoxicity assays
Extracts with an IC50 less than 1,000 μg/mL were considered toxic, while an IC50 greater than 1,000 μg/mL was considered non-toxic (Meyer et al., 1982). Our results indicated that extract number 8 had an IC50 of 1,562.5 µg/mL, which suggests that it is not toxic for the study model used. The IC50 of the positive control (caffeine) was 800 µg/mL, which is why it is considered slightly toxic. Acute toxicity was evaluated with the A. salina model because it is practical, easy, and economical and provides quick guidance on the toxic potential of organic samples (Aydιn et al., 2016).
As previously mentioned, extract 8 is composed mainly of J. communis berries. Various investigations corroborate our results since the safety of extracts from different species of Juniperus has been demonstrated (Miceli et al., 2020; Taviano et al., 2011). Schneider et al. (2004), reported IC50 values lower than those reported in our research; however, it may be due to the type of extract used (methylene chloride and ethyl acetate); both solvents are extremely toxic for living organisms (Kimura et al., 1971). To our knowledge, there are no investigations on the toxicity of extracts of P. alba leaves and P. silvestris leaves (complementary ingredients of supplement 8); however, a slight cytotoxicity was observed in the latter in NIH 3T3 fibroblasts (Smirnova et al., 2020). The manufacturer of supplement 8 recommends a daily intake of 1,800 mg, so according to our results, caution is advised in its consumption since, although it was not toxic at the concentrations evaluated, this recommendation exceeds them, so manufacturers must assess the recommended doses in vivo models.
Bioinformatic study
The biofunctional potential of the phytochemicals present in supplements No. 3 and 8 was elucidated in Table 4. Only the identified medicinally relevant compounds were selected. In supplement 3, fatty acids and terpenoids derived from turmeric (aR-turmerone, turmerone, (E)-atlantone, turmeronol A, and curlone) were identified, which, according to the bioinformatic analysis, mostly share the targets related to cancer, inflammation and attention deficit hyperactivity. Likewise, phytosterols (campesterol, stigmasterol, and γ-sitosterol) were identified, which are associated with preventing cardiovascular diseases. Regarding the phytochemicals of supplement 8, the terpenes and triterpenes stand out (squalene, hentriacontane, α-amyrin, β-amyrin, D-friedoolean-14-en-3-ol, cubenol, tau-muurolol, cis-verbenol, (-)-myrtenol and 4-epi-cubedol).
According to the analysis carried out in the CTD database (Therapeutic Toxicogenomics Database), ar-turmerone is mainly associated with diabetes involving the genes BAX, CASP3, CYP1A1, PPARG, and TP53. Squalene is associated as a therapeutic agent against coronary conditions (BAX, BCL2, CASP3, CAT, MMP2, and TNF), renal carcinoma (EPAS1, RELA), and diabetes (BAX, BCL2, CASP3, CAT, MMP2, RELA, and TNF). (-)-Myrtenol is associated as a therapeutic agent to treat liver damage, edema, hypocholesterolemia, hyperglycemia, infertility in women, and pancreatic diseases. α-amyrin is a terpene identified in both supplements, associated with treating hyperalgesia, pain, and inflammation. γ-sitosterol is used to treat hypercholesterolemia and liver damage (Davis et al., 2021). The results obtained from the SuperPred database (Table 5) indicate that the compounds exert activity on pathologies related to inflammation, oxidative stress, and the cardiovascular system. According to the present results, the possible mechanisms of action associated with the consumption of these compounds can be elucidated, in addition to providing a broader overview of the use and safety of the use of dietary supplements since these are consumed simultaneously with medications, can cause drug interactions (FDA, 2022b).
Table 4 Molecular targets and therapeutic indications predicted by Comparative Toxicogenomics Database (CTD) of the phytochemicals present in the extracts.
| Compound | Interacting genes | Disease | Inference score |
|---|---|---|---|
| Ar-Turmerone | ABCB1, AHR, BAX, CASP3, CYP1A1, PPARG, TP53 | Diabetes Mellitus (Experimental) | 25.62 |
| Infertility (Male) | 20.89 | ||
| Breast neoplasms | 18.78 | ||
| Colorectal neoplasms | 17.23 | ||
| Esophageal Neoplasms | 14.98 | ||
| α-Amyrin | TACR1 | Bronchial diseases | 6.28 |
| Neurogenic inflammation | 5.76 | ||
| Alcoholism | 5.45 | ||
| Attention deficit disorder with hyperactivity | 5.31 | ||
| γ-Sitosterol | AKT1, APOA1, APOB, BAX, BCL2, BIRC2, CASP3, CASP9, CAT, CDH1, CLEC4E, CYCS, EEIG1, EGFR, ESR1, ESR2, IL10, IL1B, IL6, LDLR, PARP1, TNF, VEGFA | Breast neoplasms | 45.18 |
| Prostatic neoplasms | 37.74 | ||
| Adenocarcinoma | 34.89 | ||
| Brain ischemia | 33.81 | ||
| Reperfusion injury | 32.02 | ||
| Diabetes Mellitus | 31.83 |
Table 5 Molecular targets and therapeutic indications predicted by Superpred of the phytochemicals present in the extracts.
| Compound | Predicted Target | TTD ID | Indication of predicted targets | Probability (%) | Model accuracy (%) |
|---|---|---|---|---|---|
| Ar-Turmerone | Cathepsin D | T67102 | Hypertension | 95.31 | 98.95 |
| T67102 | Multiple sclerosis | 95.31 | 98.95 | ||
| Pregnane receptor X | T82702 | Arteriosclerosis | 93.29 | 94.73 | |
| ADN- liase | T13348 | Glioma | 88.97 | 91.11 | |
| Melanoma | 88.97 | 91.11 | |||
| Eye cancer | 88.97 | 91.11 | |||
| Solid tumor/cancer | 88.97 | 91.11 | |||
| G protein-coupled to receptor 55 | T87670 | Attention deficit hyperactivity | 84.19 | 78.15 | |
| Formyl peptide receptor 1 | T87831 | Inflammation | 83.01 | 93.56 | |
| Curlone | DNA liase | T13348 | Glioma | 93.48 | 91.11 |
| Melanoma | 93.48 | 91.11 | |||
| Eye cancer | 93.48 | 91.11 | |||
| Solid tumor/cancer | 93.48 | 91.11 | |||
| Cathepsin D | T67102 | Hypertension | 91.48 | 98.95 | |
| Multiple sclerosis | 91.48 | 98.95 | 91.11 | ||
| Formyl peptide receptor 1 | T87831 | Inflammation | 83.31 | 93.56 | |
| Peptic ulcer | 83.31 | 93.56 | |||
| G protein-coupled to receptor 55 | T87670 | Attention deficit hyperactivity | 81.9 | 78.15 | |
| Squalane | Cathepsin D | T67102 | Hypertension | 82.96 | 98.95 |
| Multiple sclerosis | 82.96 | 98.95 | |||
| Pregnane X receptor | T82702 | Arteriosclerosis | 82.68 | 94.73 | |
| ADN liase | T13348 | Glioma | 80.29 | 91.11 | |
| Melanoma | 80.29 | 91.11 | |||
| Eye cancer | 80.29 | 91.11 | |||
| Solid tumor/cancer | 80.29 | 91.11 |
Note: TTD (Therapeutic Target Database ID).
Conclusions
The dietary supplements analyzed were shown to contain flavonoids and exert antioxidant activity. The metabolites identified were mostly triterpenes, terpenes, sterols, and fatty acids, which were shown to be related to biomarkers of oxidative stress and inflammation, so their consumption could help minimize the appearance of chronic degenerative diseases. It was possible to elucidate the biofunctional potential of the dietary supplements evaluated, according to the bioinformatic analysis, the phytochemicals present can potentially exert activity in the cardiovascular system and protection against different types of cancer and type 2 diabetes. However, it is necessary to carry out in vivo studies to determine its clinical functionality. Supplement 8 did not show toxicity in the A. salina model; despite this, it is recommended to evaluate acute and chronic toxicity in other biological models since the doses recommended by the manufacturers may present side effects to the consumer's health. The consumption of this type of product is recommended to be supervised by a health specialist, taking extreme precautions if you are undergoing pharmacological treatment since, according to our results, supplements can exert more than one bioactivity due to the diversity of ingredients and compounds present interacting with different molecular targets.
