Research on spent coffee grounds: from oil extraction to its potential application in cosmetics

Research on spent coffee grounds: from oil extraction to its potential application in cosmetics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Research on spent coffee grounds: from oil extraction to its potential application in cosmetics Adrianna Maria Piasek, Paula Bardadyn, Zoja Trojan, Karolina Jelonek, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6358386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Sep, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract Coffee is one of the most widely consumed beverages globally, generating significant amounts of waste, including spent coffee grounds (SCG). SCG contains valuable compounds, particularly oil, which constitutes 10–20% of its composition, depending on the plant species. This study developed an optimized method for extracting the lipid fraction efficiently, without requiring extensive time, specialized equipment, or high costs. The most effective extraction was achieved using hexane at its boiling point for 30 minutes, with an SCG-to-solvent ratio of 1:5 (m:v). Given its composition and beneficial properties, SCG oil holds potential for various industrial applications, with this research focusing on its suitability for the cosmetics industry. Notably, SCG oil demonstrated high antioxidant activity compared to commercially available coffee oils and showed no cytotoxic effects on 2D and 3D skin cell cultures. These findings highlight SCG oil as a sustainable, eco-friendly alternative to conventional cosmetic raw materials, contributing to both sustainable development and innovation in the cosmetics sector. spent coffee grounds oil antioxidants cosmetics 2D cell culture 3D skin model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights ● The coffee market generates huge amounts of waste, including tons of SCG. ● One of SCG's most important and valuable fractions is oil (10–20%). ● Optimal parameters of SCG oil extraction were selected and it was efficiently obtained. ● SCG oil was tested for antioxidant activity, which is much higher than that of pressed ones. ● SCG oil is not cytotoxic and reduces ROS production in skin cells. 1. Introduction Every day, people drink about 3.5 billion cups of coffee, making it one of the most popular beverages in the world [ 1 ]. The biggest coffee lovers are Scandinavians – statistically, one Finn drinks 4 cups a day, which gives about 12 kilograms of coffee per year [ 2 ]. Unfortunately, coffee processing generates a huge amount of waste, among which coffee cherry husk, coffee pulp and coffee chaff (coffee silverskin) can be distinguished. These raw materials, managed inadequately, are a heavy burden on the environment. Other well-known coffee by-products are the spent coffee grounds (SCG). Huge amounts of SCG are mainly formed during the production of instant coffee – about 2 kilograms of wet grounds for every kilogram of instant coffee. Important sources are also our households, coffee shops, gas stations, etc. – brewing coffee produces about 0.9 grams of ground per 1 gram of coffee [ 1 ]. It is estimated that both of these phenomena cause the formation of about 6 million tons of spent coffee grounds per year. A small part is further used as fertilizers, but the vast majority ends up in landfills, where the organic mass decomposes into acidic leachate and greenhouse gases [ 3 , 4 ]. The big scale of the SCG problem has attracted the interest of many researchers, who are constantly looking for new methods of dealing with this issue. As a result of numerous studies, it has been proven that what we consider as waste, is in fact rich in various components that can be further handled [ 5 – 7 ]. Many factors contribute to SCG composition, e.g. coffee bean type, brewing time, temperature and pressure. Because of this variability, as well as the overwhelming number of different compounds, it is not possible to fully determine the exact contents of this biomass. However, the analysis of the available data establishes its approximate composition, according to which they consist mainly of polysaccharides (38.6–53.3%), lignin (24.0–33.0%), proteins (6.7–13.6%), antioxidants (1.5–2.5%), minerals (1.0–2.0%) and lipids (10.0–20.0%) [ 8 ]. Individual ingredients can be applied in many different ways. This work focuses on the valorization of the lipid fraction, which has a long list of potential applications, e.g. in the food and energy industries, as well as in the cosmetic market, which is the most important from the point of view of the conducted research. Commercially available coffee oils are produced by cold-pressing the beans. This method, although mostly organic, generates further waste and is not suitable for spent coffee grounds. The most common way of obtaining SCG oil, also used in this work, is the traditional solvent extraction, but new techniques such as supercritical fluid extraction, microwave- or ultrasonic-assisted extraction are also popular [ 9 ]. SCG oil, derived from grounds, is a brown liquid with a distinct coffee aroma. It consists primarily of triacylglycerols (TAG), which account for approximately 85% of its composition. Additionally, it contains smaller amounts of diacylglycerols (DAG), monoacylglycerols (MAG), and free fatty acids (FFAs). The profile of fatty acids building the lipid fraction (esterified and in the free form) depends on many parameters, including the origin of beans and grounds and the method of oil extraction, but it is similar to other vegetable oils, as shown in Fig. 1 . SCG oil mainly consists of linoleic acid (C 18:2 ), palmitic acid (C 16:0 ), stearic acid (C 18:0 ), oleic acid (C 18:1 ) and smaller quantities of arachidic acid (C 20:0 ) as well as linolenic acid (C 18:3 ). Linoleic acid is considered as one of the essential fatty acids and in the appropriate amounts it allows to reduce cholesterol in blood, decrease the risk of atherosclerosis and hypercholesterolemia [ 10 , 11 ]. Similar properties are attributed to oleic acid [ 12 ]. Palmitic acid, despite its bad reputation, in controlled amounts can also be beneficial for our organisms, ensuring the physical properties of cell membranes [ 13 ]. In cosmetics, all three acids are responsible mainly for emulsification and protection against water loss. Stearic acid has a similar function but it is also a gentle surfactant with possible anti-inflammatory properties which can reduce skin redness [ 14 ], whereas linolenic acid is known as a lightening agent in the case of ultraviolet (UV)-induced skin hyperpigmentation [ 15 ]. Except acylglycerols and FFAs, compounds such as sterols, tocopherols and diterpenes, including kahweol and cafestol (known for their ultraviolet protective properties, as well as anti-inflammatory and antioxidant activity) and many others can be found [ 16 – 18 ]. In addition, SCG oil, like its commercial counterparts obtained from coffee beans, turned out to be a substance rich in numerous antioxidants, among which chlorogenic acids and caffeine can be distinguished [ 19 , 20 ]. In this study, the antioxidant activity of SCG oil was tested using chemical colorimetric assays, which require prior extraction of antioxidants to organic solvent miscible with water. This action is commonly used in oil testing and various methods were used to optimize extraction in this work. Finally, when introducing a cosmetic or its ingredient to the market it is crucial to check its influence on the skin. Since animal testing of cosmetic formulations as a whole, as well as of individual ingredients, is against the European Union law, such testing should be carried out on in vitro models. These studies mostly use 2D and 3D models consisting of human skin cells (Piasek et al., 2023). That is why the last part of this study was dedicated to confirming the usefulness of the obtained SCG oil on skin cell models. 2. Materials and methods 2.1. Materials Technical grade n-hexane for extraction was supplied from VWR. Silica gel plates were purchased from Merck or VWR, iodine crystals from WARCHEM and reference lipid mixture (Lipid Standard, Mono-, Di-, & Triglyceride Mix) from Sigma-Aldrich. Analytical grade hexane, diethyl ether, acetic acid, methanol and ethanol were delivered by Avantor Performance Materials Poland, and acetone by Merck. Sodium carbonate, sodium acetate, ethyl acetate, sodium hydroxide, hydrochloric acid and dimethyl sulfoxide (DMSO) were acquired from Chempur. All other reagents used for antioxidant activity and total polyphenol content assays i.e. iron (III) chloride hexahydrate, 2,4,6-tris(2-pirydyl)-s-triazine complex (TPTZ), iron (II) sulfate heptahydrate, ammonium acetate, neocuproine, copper (II) chloride dihydrate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 3,4,5-trimethoxybenzoic acid (gallic acid), ammonium persulfate (APS), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocâlteu reagent were supplied by Sigma-Aldrich. HaCaT keratinocytes were purchased from the German Cancer Research Center - Deutsches Krebsforschungszentrum. Dulbecco’s Modifieds Eagle Medium (DMEM), 0.25% trypsin-EDTA, staurosporine, glutathione (GSH), fluorescein diacetate (FDA), propidium iodide (PI), crystal violet (CV), paraformaldehyde and 2′,7′- dichlorodihydrofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and penicillin/streptomycin mix (10.000 U/ml pen and 10 mg/ml strep) were obtained from Gibco, phosphate-saline buffer (PBS) from VWR, tetrazolium salt (MTT) from Thermo Fisher Scientific, hydrogen peroxide (H 2 O 2 ) from Avantor Performance Materials Poland, and Pluronic F127 from BASF Polska. For comparison, green coffee oil (CO1, Zrób Sobie Krem) and two roasted coffee oils (CO2, Etja and CO3, Manufaktura Natura) were used. 2.1. Oil extraction Spent coffee grounds (SCG), obtained from the local coffee shop (Warsaw, Poland), were subjected to drying for 24 hours in order to decrease the water content to about 5–6%. Dried SCG were sifted through a sieve to separate impurities and homogenize the material. The appropriate mass of coffee grounds was weighed and placed in a round bottom flask in which coffee oil was extracted with hexane at 68°C (hexane boiling point) for a specified period - individual process parameters were tested to select the best ones. During optimization, extractions were performed in 500 ml, 1000 ml and 2000 ml round bottom flasks to examine process scalability. Different extraction times, i.e. 30 min, 45 min and 60 min, were also evaluated. Another tested parameter was the ratio of SCG-to-solvent, 5 variants were checked − 1:2.5, 1:3.75, 1:5, 1:6.25, 1:7.5. Last but not least, the multiplicity of extraction was also analyzed. After extraction, the solid phase was separated from the liquid – the grounds, without the lipid fraction, were dried overnight and weighed. The yield of oil extraction was determined by sample mass loss according to the following equation: $$\:\%\:extraction\:yield=\frac{{SCG}_{c}-{SCG}_{x}}{{SCG}_{c}}\times\:100\%$$ where SCG c – the weight of the sample before extraction, SCG x – the weight of the sample after extraction. To recover the solvent for the next extractions and separate crude SCG oil, the liquid phase was evaporated on a vacuum evaporator. 2.2. Thin layer chromatography (TLC) The composition of the main lipid classes in SCG oil was determined by thin layer chromatography, performed on silica gel plates. Before separation, the chamber was saturated with vapors of mobile phase, which was a mixture of hexane, diethyl ether and acetic acid (40:10:1, v:v:v). Aliquots of 1 µl of SCG oil and three commercially available coffee oils were applied on TLC plates. The lipid composition was identified by the comparison with the reference lipid mixture, which was also applied on the plate. After developing and drying the plates, they were observed under UV light at a wavelength of 254 nm and then stained with iodine vapors. 2.3. Extraction of antioxidants from the oil To determine the antioxidant activity of SCG oil, it was necessary to extract antioxidants with an organic solvent miscible with water. Based on the information presented in literature [ 21 – 24 ] and our research, methanol and ethanol solutions were selected for this purpose. These included methanol concentrations (40%, 60% and 80%) and 48% ethanol. 1 ml of oil (extracted from SCG or purchased on the market) was added to 20 ml of the solvent receiving a 1:20 oil-to-solvent (v:) ratio. During optimization, tests were also carried out in 1:1, 1:2, 1:4, and 1:10 oil-to-solvent (v:v) ratios. Mixtures were shaken on a laboratory shaker at 250 rpm for a selected period. During these tests, extraction times such as 10 min, 30 min, 1 h, 4 h and 24 h were investigated. Extraction of each sample was carried out three times. 2.4. Antioxidant activity and total phenolic content Antioxidant activity of SCG oil and commercial coffee oils was determined by chemical tests (FRAP, CUPRAC, DPPH and ABTS), and total phenolic content (TPC) was evaluated by the Folin-Ciocâlteu method – these techniques are based on the measurement of the absorbance change of the tested solutions caused by the presence of compounds with specific properties. The results are presented as concentrations to different standards (depending on the test) or a percentage of SCG oil antioxidant activity (control). 2.4.1. FRAP method The principle of the FRAP method is based on the ability to reduce iron (from Fe 3+ to Fe 2+ ). Before performing an assay, the FRAP reagent was prepared by mixing an acetate buffer (pH 3.6, prepared from sodium acetate, Milli-Q water and acetic acid, pH was adjusted with sodium hydroxide and hydrochloric acid), 20 mM iron (III) chloride hexahydrate and 10 mM 2,4,6-tripyridyl-s-triazine complex (TPTZ) in the ratio of 10:1:1 (v:v:v). Aliquots of 5 µl of samples were added to wells, each sample was run in triplicate. Subsequently, 145 µl of the FRAP reagent was added to each sample. After 30 minutes of incubation at room temperature and without access to light the absorbance of the samples was measured at a wavelength of 593 nm. Antioxidant activity was determined against a standard curve of iron (II) sulfate heptahydrate. 2.4.2. CUPRAC method The mechanism of the CUPRAC assay is similar to the FRAP method and it is based on the antioxidants' ability to reduce copper (from Cu 2+ to Cu + ). Before the analysis, the CUPRAC reagent consisting of 10 mM ammonium acetate (pH 7.0, prepared from ammonium acetate and Milli-Q water, pH was adjusted with sodium hydroxide and hydrochloric acid), 7.5 mM neocuproine in methanol, 10 mM copper (II) chloride dihydrate and Milli-Q water in a ratio of 1:1:1:0.6 (v:v:v:v), was prepared. Aliquots of 20 µl of samples in triplicates were added to wells, then 145 µl of the CUPRAC reagent was also added. The plate was incubated for 30 minutes at room temperature and without access to light. Later the absorbance of samples was measured at a wavelength of 450 nm. Antioxidant capacity was evaluated based on the standard curve of trolox. 2.4.3. DPPH assay The DPPH assay is based on the measurement of the scavenging capacity of the tested sample towards the stable free radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Before performing the test, a 0.25 mM methanolic solution of DPPH was prepared and its absorbance at a wavelength of 520 nm was checked - if the absorbance is not 0.30 +/- 0.02, the solution should be adjusted to this value by adding DPPH or methanol. Aliquots of 25 µl of samples and 75 µl of prepared solution were added to wells, each sample was tested in three repetitions. The plate was incubated at room temperature for 15 minutes without access to light and then the absorbance at 520 nm was measured. The concentration of antioxidants was calculated by using a standard curve of trolox. 2.4.4. ABTS method The ABTS assay is based on the ability to scavenge the ABTS •+ radical cation, which needs to be pre-generated 12–16 hours before the test, by preparing a 2.45 mM solution of ammonium persulfate (APS) in 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Aliquots of 25 µl of samples and 75 µl of ABTS solution were added to wells, each sample was tested in triplicate. The plate was incubated for 30 minutes at room temperature and without access to light. Next, the time absorbance of samples was measured at a wavelength of 734 nm. Antioxidant concentration was determined against a standard curve of trolox. 2.4.5. Total polyphenol content The total polyphenol content (TPC) was measured using the Folin-Ciocâlteu method. A volume of 10 µl of samples was added to wells in triplicate. Subsequently, 50 µl of the Folin-Ciocâlteu reagent was added to each sample. Following 3 minutes of incubation, 140 µl of 5% sodium carbonate was also added. After 90 minutes of incubation without access to light, the absorbance of each sample was measured at a wavelength of 765 nm. TPC was determined based on the standard curve of gallic acid. 2.5. Cell culture HaCaT keratinocytes, purchased from the DKFZ institute, were cultivated in a DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin mixture at 37°C under a 5% CO 2 atmosphere. The cell line was routinely cultured into a 75 cm 2 culture flask. When the confluency was about 80%, keratinocytes were harvested with 0.25% trypsin and seeded at a density of 1 x 10 4 cells/ml into 96-well plates. For 2D culture non-treated flat-bottom plates were used and cells were then incubated for 24 hours (37°C, 5% CO 2 ), whereas for the spheroid culture, cells were seeded into pretreated (a 1% of Pluronic F127 incubated overnight, and PBS washed) round-bottomed well plates and incubated for 48 hours (37°C, 5% CO 2 )(limiting cell adhesion method [ 25 ]). 2.6. Preparation of oil samples and cell treatment Before the cells were treated, oil samples were dissolved in a selected organic solvent, i.e. acetone (Ac) or ethyl acetate (EA). First, the oil was diluted in a solvent at concentrations ranging from 3.13 to 50%, and then the mixtures were added to the DMEM medium at a concentration of 0.5% (2D culture experiments) or 1% (comparative study between 2D and 3D culture). The final oil content ranged from 0.016 to 0.250% for 2D culture studies and from 0.016 to 1.000% for 2D and 3D culture comparative experiments. DMEM was supplemented with 1% FBS (for FDA, PI and MTT assays) or without FBS (during ROS and crystal violet assays) and a standard 1% penicillin/streptomycin mixture. 24 hours (2D culture) or 48 hours (spheroids) after cells were seeded into plates, they were treated with SCG oil and 0.5% (2D) or 1.0% (3D) solvent solution, which was used as a control. Additionally, keratinocytes were treated with 2 µM staurosporine (positive control in FDA, PI and MTT assays) and with 0.5% H 2 O 2 (positive control in ROS and CV assays) and glutathione (GSH; negative control in ROS assay). 2.7. Determination of cytotoxicity 2.7.1. FDA/PI assay FDA/PI assay can be used to evaluate the viability of the cells. Fluorescein diacetate (FDA) and propidium iodide (PI) are two fluorescent dyes, which stain living and dead cells, respectively [ 26 ]. Cells were treated with oil dissolved in Ac/EA and a pure solvent as a control. After 24 hours of incubation, 10 µl of FDA and PI mixture (prepared in pure DMEM medium at a concentration of 80 µg/ml for both dyes) was added to each well. The plate was incubated for 10 minutes in the dark and fluorescence of each well was measured at the wavelength of 490/526 ± 9 nm (for FDA) and 534/635 ± 9 nm (for PI). The number of living and dead cells was determined against cells treated with a pure solvent solution and described as a percentage of control. 2.7.2. MTT assay MTT colorimetric assay is based on the activity of mitochondrial dehydrogenase, involved in the reduction of yellow tetrazolium salt to purple formazan, which is insoluble in water [ 27 ]. Following the FDA/PI assay, the medium was removed from each well and replaced with 50 µl of 0.5 mg/ml MTT in the pure DMEM medium. Then the plates were incubated for 2 hours at 37°C under a 5% CO 2 atmosphere. Next, to dissolve the obtained formazan, the medium was once again removed and replaced with 100 µl of DMSO. The absorbance of each well was measured at a wavelength of 570 nm. Cell viability was determined against cells treated with a solvent and described as a percentage of control. 2.7.3. CV assay Crystal violet stains all acidic (negatively charged) components of cells ( i.e. DNA, RNA and proteins), which is why it may be used to determine the total number of cells in a well [ 26 ]. After 24 hours of incubation with the added substances, the medium was removed, and cells were washed with a PBS buffer to remove dead-floating cells. Subsequently, keratinocytes were fixed with a 3.7% solution of paraformaldehyde in PBS for 10 minutes. After that time 0.05% methanolic solution of crystal violet was added and then the plate was incubated for 30 minutes. CV was then removed, and cells were washed with Milli-Q water three times. Plates were dried overnight at room temperature and next 50 µl of methanol was added to dissolve CV. The absorbance of each well was measured at a wavelength of 540 nm and the number of cells after treatment was determined against a control (cells treated with a pure solvent). 2.8. ROS assay ROS assay is used to determine the amount of reactive oxygen species produced by cells [ 27 ] . Before the addition of SCG oil, the medium was replaced by 50 µl of 10 µM 2',7′-dichlorofluorescein diacetate (DCF-DA) in PBS. The plate was incubated for 30 minutes, then DCF-DA was removed, and oil samples were added. The fluorescence of each well was measured at a wavelength of 485/525 ± 9 nm twice – after 1 and 24 hours of incubation. The ROS amount was determined in relation to the control i.e. cells treated with a solvent. 2.9. Statistical analysis All experiments were carried out in triplicates. Numerical results are presented in figures as a mean ± standard deviation (SD). The received data were analyzed using appropriate tests to assess differences between groups. One-way ANOVA was applied to compare the means across multiple independent groups, determining whether there were statistically significant differences among them. Independent samples were used to compare the means between the two groups by the t-test to evaluate if the observed differences were statistically significant. A significant level of α = 0.05 was set for all statistical tests and is marked by (*) in each graph. 3. Results The results presented in this research were obtained within an advanced biorefinery concept enabling the efficient valorization of spent coffee grounds (SCG) through a carefully designed sequence of processes. This approach allows for the extraction of coffee oil, antioxidants, lignin and sugars. The idea was undertaken by the Polish company EcoBean in collaboration with the Warsaw University of Technology and has progressed to the stage of a fully developed conceptual design, protected under patent application numbers P.447416 and PCT/PL2024/050107. This article specifically focuses on refining and optimizing the SCG oil extraction process as part of the broader strategy for comprehensive SCG utilization, as well as checking their antioxidant properties and cytotoxicity toward skin cells. 3.1. Optimization of oil extraction from SCG Optimization of valuable compound extraction is a key stage of each research. In the case of SCG, this element affects not only the quality of the obtained oil but also the required time and operational costs. Moreover, when choosing the best extraction parameters, environmental aspects also cannot be ignored. That is why, we have identified key parameters of oil extraction from SCG which are: the time of extraction (30, 45 and 60 min), SCG-to-hexane ratio (1:7.5; 1:6.25; 1:5; 1:3.75 and 1:2.5) and extraction multiplicity (1, 2 and 3). Each extraction process was carried out as described in the methods (subsection 2.1 .) taking into account the parameter under study. The results are shown in Fig. 2 . The results presented in Fig. 2 A, show that an extension of the extraction time beyond 30 minutes does not lead to a much higher efficiency. The short extraction time is advantageous in terms of process energy costs. Figure 2 B demonstrates that efficiency improves with the increase in the amount of solvent used while considering the ratio of SCG-to-hexane. It is worth noting that the same oil extraction efficiency was obtained in a 500 ml flask with a ratio of 1:7.5 and a 4 times larger scale (2000 ml) with a grounds-to-solvent ratio of 1:3.75. Consequently, as the scale of the process increases, there is a possibility to use less solvent to obtain satisfactory process yields. This is a very optimistic prognosis for the implementation of this extraction method in industry. Last but not least, the multiplicity of the process was checked. As shown in Fig. 2 C, a single extraction allows to obtain most of the oil, whereas the second and third extractions performed on the same SCG did not increase the yield significantly. Based on all the presented results, a single 30-minute oil extraction from SCG in a ratio of 1:5 was selected as optimal. 3.2. Oil composition determined by TLC analysis The TLC profiles of SCG oil and commercial coffee oils (CO1-CO3) are shown in Fig. 3 . The main lipid class detected in all tested oils was triacylglycerols. Other classes were spotted in smaller quantities and different proportions. SCG oil contains significantly more 1,3-diacylglycerols than its counterparts obtained from coffee beans and smaller amounts of monoacylglycerols. In the case of 1,2-diacylglycerols, they were not detected in any of the samples. Usually, fats and lipids are not detected under UV light due to the lack of fluorophores - therefore standards were not viewable (Fig. 3 A). However, lipids present in SCG oil and commercial coffee oils were visible, meaning that their acylglycerols may contain fluorescent compounds. 3.3. Optimization of antioxidant extraction from SCG oil For testing the antioxidant properties, the literature proposes various methods including FRAP, CUPRAC or DPPH assays. Thus, an essential step for investigating the antioxidant properties of the SCG oil was the extraction of compounds with such properties. Unfortunately, the described methods did not give satisfactory results. Based on the available sources, several experiments were carried out to select appropriate extraction parameters, i.e. type of solvent (methanol or ethanol), solvent concentration (40%, 48%, 60% or 80%), extraction time (10 min, 30 min, 1 h, 4 h and 24 h) and oil-to-solvent ratio (1:1, 1:2, 1:4, 1:10 and 1:20). After the extraction, chemical test for antioxidant properties were performed to evaluate which process was the most efficient at extracting antioxidants from SCG oil. The results presented in Fig. 4 depict the most promising variants from all of the performed tests, but the initial trials are described in the attached Supplementary materials (Table S1, Figure S1 and S2) . Due to the received effects (Figure S2) , it was decided to significantly increase the amount of solvent used (1:10 and 1:20 oil-to-solvent ratio (v:v)) and the extraction time (4 h and 24 h). In addition, due to lower cytotoxicity, the possibility of using ethyl alcohol was also checked. All extractions were performed three times on the same SCG oil. The first extraction of antioxidants from the SCG oil is the most efficient one, for the repeated extractions (2nd and 3rd) relatively low concentrations of antioxidants were obtained. In the FRAP assay (Fig. 4 A), two variants were the most promising and both were using an 80% methanol, however, they differ in the length of the extraction run (24 h and 4 h) and the ratio of oil-to-solvent (1:10 and 1:20). From an economic point of view, a single extraction of antioxidants from SCG oil is much more cost-effective, that is why, we compared how the amount of extracted antioxidants differs between the 1st and 2nd extractions. This analysis revealed that more antioxidants were obtained in the sample once extracted with 80% methanol for 4 h and at a ratio of 1:20 SCG oil-to-solvent. 3.4. Comparison of the antioxidant effect of SCG oil and commercial oils Coffee oil pressed from beans is well-known in the cosmetic industry, mostly for its antioxidant activity. During this research, we have compared the properties of commercial products and the oil extracted from coffee waste. In each case, antioxidants were extracted from the oil with the optimized methodology presented in subsection 2.3. Next, chemical tests for antioxidant properties (like ABTS, CUPRAC, FRAP) and TPC assay were carried out. These results are presented in Fig. 5 . All conducted studies have shown that SCG oil has the highest antioxidant properties compared to other oils, for which the results obtained are much lower. In the CUPRAC test (Fig. 5 B), there is about a 10-fold difference between SCG oil and CO1. This fact is very promising in terms of possible applications of SCG oil in cosmetics. 3.5. Effects of SCG oil on human skin cells in 2D and 3D models Due to its interesting composition and antioxidative properties, SCG oil may have a valuable effect on skin cells, which was presented in Section 3.4. Studying this effect was problematic because of the very limited solubility of the oil in the medium used for cell culture. It was necessary to use an additional solvent to enable the distribution of oil in various concentrations in the culture medium. This solvent should have several properties, including having the least possible impact on the cells and not affecting the influence of the oil. We examined the use of three solvents: acetone, ethyl acetate and DMSO. Despite the initial good results for DMSO, it was eliminated from the tests due to the observed precipitation of oil in DMSO with longer storage, as well as the problem of sample freezing while storing the oil in DMSO at low temperatures (5°C). While testing SCG oil on the viability of skin cells in a monolayer (2D), the solvent concentration was maintained at a constant level of 0.5% for acetone and ethyl acetate, which does not cause a threat to the cells. Different viability test results for SCG oil-acetone or SCG oil-ethyl acetate addition to HaCaT keratinocytes are shown in Fig. 6 . These include conducted measurements like the MTT test, FDA/PI assay and CV staining. The decrease in values at MTT, FDA and CV assays is typically connected with harmful effects of the investigated compounds, whereas in PI assay it is shown by the increase in values. Studies indicating the metabolic activity of cells (MTT and FDA) showed a similar tendency (Fig. 6 A and B). In oil concentrations up to 0.150%, there are no significant differences between the used solvents. The results achieved for oil dissolved in ethyl acetate during the FDA test presented lower values ​​(around 70% of control), which indicates a lower activity of cytoplasmic esterases. However, in the case of oil dissolved in acetone, there was a visible difference at the highest oil concentration (0.250%), where the cell metabolic activity was significantly lower (by about 30%) suggesting partial cell death. These observations confirm the results obtained using propidium iodide staining (Fig. 6 C). In the case of the highest concentration, cell membrane damage was significantly higher for cells treated with oil dissolved in acetone. Crystal violet staining carries information about the number of remaining cells in the well, as well as the number of negatively charged elements in the cells (proteins, DNA) can be deduced. This staining revealed interesting results and differences within the tested solvents (Fig. 6 D). Treatment of cells with oil dissolved in both tested solvents does not result in detaching cells from the culture base. Moreover, in the case of cells with the addition of oil dissolved in ethyl acetate, we noticed a positive change - an increased number of negatively charged elements in the cells and/or an increase in the number of cells. We then examined if SCG oils dissolved in acetone or ethyl acetate are capable of reducing the reactive oxygen species production in keratinocytes. The results were gathered in Fig. 7 . As mentioned earlier, we were seeking a solvent for SCG oil that should not interfere with the action of the active compound (Fig. 7 ). When SCG oil dissolved in acetone was added to the cells (Fig. 7 A), we observed a rise in free radicals’ release by keratinocytes as the oil concentration decreased and the solvent proportion increased. Perhaps, this would not be surprising if it were not for the comparison to the results obtained for SCG oil dissolved in ethyl acetate (Fig. 7 B), where the same antioxidant properties were maintained for all concentrations of the added oil. Moreover, a longer-lasting effect of these properties for oil dissolved in ethyl acetate is visible when comparing the results obtained for treating cells for 1 h and 24 h. In the case of treating cells with oil dissolved in acetone after 24 h, the results are almost as high as for the positive control, proving the increased release of free radicals by the cells takes place. To better predict the potential effect of SCG oil on the human skin, we compared its influence between keratinocytes seeded as monolayer (2D) as well as in spheroids (3D) prepared by the limiting-cell adhesion method. Spheroids are a more complex model than 2D culture and provide a more accurate imitation of real cell interactions [ 25 ]. Considering the results obtained in the ROS assay, ethyl acetate was from now on used as an oil solvent for further studies on human skin cells. For this comparative experiment, a wider range of oil concentrations was used (up to 1%). The SCG oil cytotoxicity was assessed by the MTT as well as FDA/PI assays in both 2D and 3D models, and the results are presented in Fig. 8 . This study showed that SCG oil in the tested concentrations does not adversely affect cell viability (Fig. 8 A and B) and their metabolic activity (Fig. 8 C) (all results are above 90%). Furthermore, there is no significant difference between the results obtained in 2D and 3D cultures. Higher oil concentrations (1% v/v) even had a more favorable effect on the metabolic activity of keratinocytes in the spheroids than in a monolayer (Fig. 8 C). 4. Discussion In this research, we focused on the extraction of oil from spent coffee grounds which can be used as a valuable cosmetic ingredient. The optimization of oil extraction from SCG allowed for the selection of appropriate process parameters. A 30-minute extraction and a 1:5 (SCG:hexane) ratio is sufficient to extract most of the oil. The described data show the relationship between the process efficiency and the amount of solvent used - the higher the ratio of hexane to grounds, the greater the efficiency. These results are similar to the ones present in the literature [ 28 , 29 ]. In both cited research these oils were extracted using soxhlet apparatus, which is difficult to implement in the industry. Higher extraction yields with the growth of the process scale (using the same SCG:solvent ratio) allow us to have optimistic hopes for introducing the process on an industrial scale. Due to the wide variety of raw materials, the extracted oil requires characterization. One of the simplest methods that make it possible is TLC. The obtained results suggest the presence of triacylglycerols and 1,3-diacylglycerols, with the simultaneous absence of 1,2-diacylglycerols and monoacylglycerols in our SCG oil. In addition, there were also other signals visible on the TLC plates, most likely coming from glycerol, sterols, free fatty acids, ceramides, and cholesterol, as these were observed in other studies [ 30 – 32 ]. So far, their specific identification in SCG oil has failed but will be continued in the future. Apart from its composition, the antioxidant properties of the SCG oil were also checked. These tests required prior extraction of antioxidants to the organic solvent. Despite many described methods of antioxidant extraction from various types of oils, no satisfactory ready-to-use method for SCG oil was found in literature [ 23 , 33 – 38 ]. That is why, it was decided to optimize this process within this study. The comparison of the selected conditions with those reported in the literature shows some similarities. When it comes to the time required for the process, the 4-hour extraction is the longest described one. Considering the oil-to-solvent ratio, the closest reported is the ratio 1:10 (m:v) used by [ 24 , 35 ]. The best extractant turned out to be 80% methanol. The same solvent and concentration were used by Khemakhem [ 23 ] and Marfil [ 34 ], respectively. The obtained methanol extracts with antioxidants from the SCG oil were tested for antioxidant properties (ABTS, CUPRAC, FRAP tests) and for the total polyphenol content. A high antioxidant content of SCG oil was demonstrated compared to the tested commercial coffee oils, which is consistent with the expectations and information presented in the literature [ 35 , 39 ]. This fact is very promising in terms of the possible application of SCG oil in cosmetics. The antioxidant properties of green coffee oil and cosmetics containing it were checked by Wagemaker [ 37 ]. Despite the low antioxidant properties of the oil, its presence in cosmetic formulations improved its total antioxidant activity which grew with the increase of oil concentration. All described results indicate that oil extracted from coffee waste can be environmentally friendly and a better alternative to currently used products in terms of antioxidant properties. Before using SCG oil in the in vitro studies performed on cells, it was necessary to select an organic solvent that could mix well with the oil. Interestingly, none of the other works concerning coffee oil [ 40 – 42 ] mention this important fact. It seems that the others were applying coffee oil directly to the cell culture medium. Our observations of SCG oil show that it did not mix well with the culture medium and remained as the upper layer of the two-phase solution. Therefore, it was decided to choose an organic solvent that would facilitate its dispersion. We have selected polar acetone and slightly less polar ethyl acetate. The outcomes obtained for both solvents differed from each other, which may result from their different effects on human skin cells. Due to relatively low toxicity and good miscibility with many substances, acetone is one of the most commonly used solvents in cell culture studies [ 43 ]. Research conducted by Farkas [ 44 ] has shown that it enhanced the proliferation of HaCaT cells and increased the level of transcripts characteristic for proliferating keratinocytes (α5 integrin, KGFR and cyclin D1). The second tested solvent, ethyl acetate, although less commonly used in the in vitro studies, was found to be less cytotoxic than acetone, as reported by Koc [ 45 ]. In our study, ethyl acetate also turned out to be a better solvent for SCG oil, especially while testing the possibility of ROS production in cells. Our study highlights an important problem often encountered in biological research, where the solvent itself may not induce a cytotoxic effect but may inhibit the health-promoting effects of the compound being tested. To be able to use coffee oil in cosmetics, it is necessary to check its cytotoxic properties against cells. MTT, FDA, PI and crystal violet tests were performed on skin cell cultures - keratinocytes. The presented values ​​indicate low toxicity of SCG oil toward the investigated skin cells, which was also demonstrated by Kanlayavattanakul on mouse melanoma cells B16-F10 with concentrations ranging from 0.1 to 10 µg/ml of the coffee oil [ 40 ]. Cubas et al. compared the effects of untreated coffee waste oil and oil treated with non-thermal plasma on fibroblast cells BALB/3T3 clone A31. They showed less toxicity of the oil after treatment with non-thermal plasma toward cells. Particularly, in the concentration range of 0.031–0.125 mg/ml [ 41 ]. In addition to extensive research on SCG oil, numerous studies on green coffee oil are also relevant to the findings presented in this paper. For instance, cytotoxicity tests on keratinocytes revealed that green coffee oil exhibited no harmful effects at the tested concentrations [ 42 , 46 ], similarly to the results of our study. Green coffee oil was also tested by Oliveira et al. on female rats. Among other things, the tests for acute toxicity showed no changes in all organs assessed [ 47 ]. Interestingly, we are the first to carry out tests on the 3D skin model, which enabled checking the effect of substances on cells in an environment more similar to the body thanks to intercellular interactions and the results confirm that SCG oil was safe for use. Furthermore, there were no significant differences between the results obtained in 2D and 3D culture, both results stated that there is no cytotoxicity of the SCG oil toward skin cell models. 5. Conclusion Given the vast amount of spent coffee grounds generated daily worldwide, finding sustainable ways to repurpose this waste is essential. Oil extraction is one of the most well-established approaches for utilizing SCG. In this study, we propose a simple, efficient extraction method that requires no specialized equipment or excessive time while ensuring high yield. Due to its unique composition, SCG oil holds potential for various industries, with our research specifically exploring its application in cosmetics. Additionally, this study introduces an innovative protocol for extracting antioxidants from SCG oil. The extracted oil demonstrated superior antioxidant properties compared to commercially available green and roasted coffee oils and exhibited no cytotoxicity toward keratinocyte cells in both 2D and 3D models. These findings highlight SCG oil as a promising, sustainable ingredient for the cosmetics industry. Declarations Competing Interests The entire research was carried out within an advanced biorefinery concept enabling the efficient valorization of spent coffee grounds (SCG) through a carefully designed sequence of processes by the Polish company EcoBean in collaboration with the Warsaw University of Technology. This approach allows for the extraction of coffee oil, antioxidants, lignin, and sugars, and is currently covered by patent application numbers P.447416 and PCT/PL2024/050107. The authors declare that they have disclosed all relevant affiliations and potential conflicts of interest related to this study. Funding The research was supported by the UE framework program Horizon Europe (ID project: 27170) ‘Upcycling coffee waste into useful raw materials and green products’ and the Ministry of Science and Higher Education program ‘Implementation PhD’. Author contributions Conceptualization: Adrianna Maria Piasek and Anna Sobiepanek; Formal analysis: Adrianna Maria Piasek, Paula Bardadyn and Anna Sobiepanek; Funding acquisition: Adrianna Maria Piasek, Łukasz Wysocki, Tomasz Kobiela and Anna Sobiepanek; Investigation: Adrianna Maria Piasek, Paula Bardadyn, Zoja Trojan and Karolina Jelonek; Methodology: Adrianna Maria Piasek and Anna Sobiepanek; Project administration: Adrianna Maria Piasek, Paula Bardadyn and Anna Sobiepanek; Resources: Adrianna Maria Piasek, Łukasz Wysocki and Anna Sobiepanek; Supervision: Adrianna Maria Piasek and Anna Sobiepanek; Visualization: Adrianna Maria Piasek, Paula Bardadyn and Zoja Trojan; Writing – original draft: Adrianna Maria Piasek and Paula Bardadyn; Writing – review and editing: Adrianna Maria Piasek, Paula Bardadyn, Zoja Trojan, Karolina Jelonek, Łukasz Wysocki, Tomasz Kobiela and Anna Sobiepanek. Ethical Approval Not applicable. This research does not need any ethical approval. Availability of data and materials Data and materials are available on request from the corresponding author Dr. Anna Sobiepanek ( [email protected] ). Acknowledgments Pluronic F127 was a kind gift from Dr. Michał Stepulak from BASF Polska. Moreover, we would like to express our sincere gratitude to Professor Andrzej Chwojnowski from the Nałęcz Institute of Biocybernetics and Biomedical Engineering PAS for his invaluable guidance and insightful discussions throughout the development of this work. References Blinová, L.; Sirotiak, M.; Bartošová, A.; Soldán, M. Review: Utilization of Waste From Coffee Production. Research Papers Faculty of Materials Science and Technology Slovak University of Technology 2017 , 25 , 91–101, doi:10.1515/rput-2017-0011. Kate MacDonnell Coffee Consumption by Country in 2024: Top 10 Countries. Corner Coffee STORE 2024. Forcina, A.; Petrillo, A.; Travaglioni, M.; Di Chiara, S.; De Felice, F. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6358386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444180926,"identity":"c2fcad61-ba07-4441-bcc7-f491e5005760","order_by":0,"name":"Adrianna Maria Piasek","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Adrianna","middleName":"Maria","lastName":"Piasek","suffix":""},{"id":444180927,"identity":"48cd798c-646e-4efc-90bf-837dc436aa04","order_by":1,"name":"Paula Bardadyn","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Bardadyn","suffix":""},{"id":444180928,"identity":"8369a510-6c7f-47b7-9bf5-19472368962b","order_by":2,"name":"Zoja Trojan","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Zoja","middleName":"","lastName":"Trojan","suffix":""},{"id":444180929,"identity":"66a0b6d8-1efe-4bbe-a17b-326f6b0d2525","order_by":3,"name":"Karolina Jelonek","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Jelonek","suffix":""},{"id":444180930,"identity":"8b7daf73-d7e0-4970-b1f9-ac4e77d3ff55","order_by":4,"name":"Łukasz Wysocki","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Łukasz","middleName":"","lastName":"Wysocki","suffix":""},{"id":444180931,"identity":"f05e81b5-b1a5-4621-8617-82d151750c22","order_by":5,"name":"Tomasz Kobiela","email":"","orcid":"","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Kobiela","suffix":""},{"id":444180932,"identity":"a16b5e16-8f35-44ea-9d6e-2030c761c819","order_by":6,"name":"Anna Sobiepanek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBCDBDYoLQemeICYj1gtxnAtbIS0wOjEBkJa+Gf3HvzA8Mcmj4+99+Dngoq09O0SCYwP3rYx5OHSInHnXLIEY1taMRvPuWTpGWdycnfOSGA2nNvGUIzTYTdyDCQYGw4ntknkGEjztlXkbridwAZkMCS24dAhfyPH+AfDH6AW+TfGv3n/VaQb3E5g/41Pi8GNHDMJBjaQLTxm0rwNOQlALWzM+LQYArVYJIL9kpdmzXMszXDn/IfNknPOSeD0ixzQYTc+AENMvv3s4ds8Ncny5jyHD354U2aTx4/L+yCQACZ5oE5lYGwAUhIJ+HRAAVwLsjmjYBSMglEwChgYAIRNVRT7v63qAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3186-7582","institution":"Warsaw University of Technology: Politechnika Warszawska","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Sobiepanek","suffix":""}],"badges":[],"createdAt":"2025-04-02 06:58:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6358386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6358386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12649-025-03284-2","type":"published","date":"2025-09-20T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81615798,"identity":"eea28490-a425-479d-bffe-8a50d328d259","added_by":"auto","created_at":"2025-04-29 08:09:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":769082,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of fatty acid composition of SCG oil and other vegetable oils. Created in BioRender. Sobiepanek, A. (2025) https://BioRender.com/m45w351\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/d99cbf94f84bcfc260c32746.png"},{"id":81613240,"identity":"5f06411e-f96c-4cb3-9074-6406d96ecd62","added_by":"auto","created_at":"2025-04-29 07:44:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86328,"visible":true,"origin":"","legend":"\u003cp\u003eEfficiency of oil extraction; optimization of time (A), SCG:hex ratio (B) and extraction multiplicity (C).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/780e5e9f960887c0e70dfc9c.png"},{"id":81613242,"identity":"3f3ac7cd-a081-447a-a4f3-44f12d3a7822","added_by":"auto","created_at":"2025-04-29 07:44:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303464,"visible":true,"origin":"","legend":"\u003cp\u003eTLC profiles of coffee oils (commercial (CO1, CO2, CO3) and extracted from SCG) with standard (S) visualized under UV light (A) and stained with iodine vapors (B). TLC plates show the separation of acylglycerols.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/df26928a6098f8f3fcb5395f.png"},{"id":81614897,"identity":"8637fd02-e5e8-4fe1-9a0f-b52a123caf53","added_by":"auto","created_at":"2025-04-29 08:00:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105186,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of various samples obtained under different conditions during optimization of extraction parameters; results of FRAP [A] and DPPH [B] assays in relation to standard (FeSO\u003csub\u003e4\u003c/sub\u003e x 7H\u003csub\u003e2\u003c/sub\u003eO and trolox, respectively). Statistical significance was checked within each extraction group and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 is marked on graphs with (*).\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/38baa314d5cb27d6a4d5da97.png"},{"id":81614894,"identity":"57c6f79b-3952-4f0a-ba1a-964e11e0d399","added_by":"auto","created_at":"2025-04-29 08:00:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118786,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the antioxidant properties (based on ABTS [A], CUPRAC [B], FRAP [C], and total polyphenol content [D] tests) of coffee oil extracted from spent coffee grounds and commercial products available on the market in relation to control, i.e. SCG oil from the first extraction. Statistical significance was checked within each extraction group and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 is marked on graphs with (*).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/0182fc9fb8fe023db1dd1553.png"},{"id":81614895,"identity":"265edffc-bd34-4c88-a95c-d8459b458978","added_by":"auto","created_at":"2025-04-29 08:00:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":151431,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of SCG oil on human skin cells in 2D culture. Acetone and ethyl acetate were used as solvents for SCG oil. [A] MTT assay - cell viability assessment, [B, C] FDA/PI assay - amount of living and dead cells, [D] CV staining - cell quantity assessment.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/7f2d3fbcfddafa6ecc4893f8.png"},{"id":81613692,"identity":"ae17964c-0424-4b58-8eb9-26de9a51bed8","added_by":"auto","created_at":"2025-04-29 07:52:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81844,"visible":true,"origin":"","legend":"\u003cp\u003eProduction of reactive oxygen species (ROS) by cells treated with SGC oil dissolved in acetone [A] or ethyl acetate [B]. H2O2 and GSH were used as positive and negative controls, respectively. Statistical significance with \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 is marked on graphs with (*).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/4280cec863e835df2a8b1f7b.png"},{"id":81615734,"identity":"597565ad-ef17-4240-b438-fae2302a7cb5","added_by":"auto","created_at":"2025-04-29 08:08:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":102882,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the effect of oil extracted from spent coffee grounds and dissolved in ethyl acetate on human skin cells in 2D and 3D cultures.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/f07fb180a053f2798bc78f28.png"},{"id":91890004,"identity":"03773e82-7ae5-42d2-a94b-85ec1aea0c73","added_by":"auto","created_at":"2025-09-22 16:03:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2850551,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/88c0bf33-89f6-4936-a9cf-e16ef3f357cf.pdf"},{"id":81613254,"identity":"a626be77-5a70-468e-9350-5b165ee606ee","added_by":"auto","created_at":"2025-04-29 07:44:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":257427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCreated in BioRender. Sobiepanek, A. (2025) https://BioRender.com/t67d586\u003c/p\u003e","description":"","filename":"PublicationLicenseMar302025Graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/b98c928fcd287cbcf0e635a2.pdf"},{"id":81613682,"identity":"5a3c7a0f-2598-4e14-956e-1374157e8f0d","added_by":"auto","created_at":"2025-04-29 07:52:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":195270,"visible":true,"origin":"","legend":"","description":"","filename":"Piaseketal.2025Supplementfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6358386/v1/d1550dd3e9317c0e9b08b24f.pdf"}],"financialInterests":"","formattedTitle":"Research on spent coffee grounds: from oil extraction to its potential application in cosmetics","fulltext":[{"header":"Highlights","content":"\u003cp\u003e● The coffee market generates huge amounts of waste, including tons of SCG.\u003c/p\u003e\u003cp\u003e● One of SCG's most important and valuable fractions is oil (10\u0026ndash;20%).\u003c/p\u003e\u003cp\u003e● Optimal parameters of SCG oil extraction were selected and it was efficiently obtained.\u003c/p\u003e\u003cp\u003e● SCG oil was tested for antioxidant activity, which is much higher than that of pressed ones.\u003c/p\u003e\u003cp\u003e● SCG oil is not cytotoxic and reduces ROS production in skin cells.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eEvery day, people drink about 3.5\u0026nbsp;billion cups of coffee, making it one of the most popular beverages in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The biggest coffee lovers are Scandinavians \u0026ndash; statistically, one Finn drinks 4 cups a day, which gives about 12 kilograms of coffee per year [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Unfortunately, coffee processing generates a huge amount of waste, among which coffee cherry husk, coffee pulp and coffee chaff (coffee silverskin) can be distinguished. These raw materials, managed inadequately, are a heavy burden on the environment. Other well-known coffee by-products are the spent coffee grounds (SCG). Huge amounts of SCG are mainly formed during the production of instant coffee \u0026ndash; about 2 kilograms of wet grounds for every kilogram of instant coffee. Important sources are also our households, coffee shops, gas stations, etc. \u0026ndash; brewing coffee produces about 0.9 grams of ground per 1 gram of coffee [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is estimated that both of these phenomena cause the formation of about 6\u0026nbsp;million tons of spent coffee grounds per year. A small part is further used as fertilizers, but the vast majority ends up in landfills, where the organic mass decomposes into acidic leachate and greenhouse gases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The big scale of the SCG problem has attracted the interest of many researchers, who are constantly looking for new methods of dealing with this issue. As a result of numerous studies, it has been proven that what we consider as waste, is in fact rich in various components that can be further handled [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany factors contribute to SCG composition, e.g. coffee bean type, brewing time, temperature and pressure. Because of this variability, as well as the overwhelming number of different compounds, it is not possible to fully determine the exact contents of this biomass. However, the analysis of the available data establishes its approximate composition, according to which they consist mainly of polysaccharides (38.6\u0026ndash;53.3%), lignin (24.0\u0026ndash;33.0%), proteins (6.7\u0026ndash;13.6%), antioxidants (1.5\u0026ndash;2.5%), minerals (1.0\u0026ndash;2.0%) and lipids (10.0\u0026ndash;20.0%) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Individual ingredients can be applied in many different ways. This work focuses on the valorization of the lipid fraction, which has a long list of potential applications, e.g. in the food and energy industries, as well as in the cosmetic market, which is the most important from the point of view of the conducted research.\u003c/p\u003e \u003cp\u003eCommercially available coffee oils are produced by cold-pressing the beans. This method, although mostly organic, generates further waste and is not suitable for spent coffee grounds. The most common way of obtaining SCG oil, also used in this work, is the traditional solvent extraction, but new techniques such as supercritical fluid extraction, microwave- or ultrasonic-assisted extraction are also popular [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSCG oil, derived from grounds, is a brown liquid with a distinct coffee aroma. It consists primarily of triacylglycerols (TAG), which account for approximately 85% of its composition. Additionally, it contains smaller amounts of diacylglycerols (DAG), monoacylglycerols (MAG), and free fatty acids (FFAs). The profile of fatty acids building the lipid fraction (esterified and in the free form) depends on many parameters, including the origin of beans and grounds and the method of oil extraction, but it is similar to other vegetable oils, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. SCG oil mainly consists of linoleic acid (C\u003csub\u003e18:2\u003c/sub\u003e), palmitic acid (C\u003csub\u003e16:0\u003c/sub\u003e), stearic acid (C\u003csub\u003e18:0\u003c/sub\u003e), oleic acid (C\u003csub\u003e18:1\u003c/sub\u003e) and smaller quantities of arachidic acid (C\u003csub\u003e20:0\u003c/sub\u003e) as well as linolenic acid (C\u003csub\u003e18:3\u003c/sub\u003e). Linoleic acid is considered as one of the essential fatty acids and in the appropriate amounts it allows to reduce cholesterol in blood, decrease the risk of atherosclerosis and hypercholesterolemia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similar properties are attributed to oleic acid [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Palmitic acid, despite its bad reputation, in controlled amounts can also be beneficial for our organisms, ensuring the physical properties of cell membranes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In cosmetics, all three acids are responsible mainly for emulsification and protection against water loss. Stearic acid has a similar function but it is also a gentle surfactant with possible anti-inflammatory properties which can reduce skin redness [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], whereas linolenic acid is known as a lightening agent in the case of ultraviolet (UV)-induced skin hyperpigmentation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExcept acylglycerols and FFAs, compounds such as sterols, tocopherols and diterpenes, including kahweol and cafestol (known for their ultraviolet protective properties, as well as anti-inflammatory and antioxidant activity) and many others can be found [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, SCG oil, like its commercial counterparts obtained from coffee beans, turned out to be a substance rich in numerous antioxidants, among which chlorogenic acids and caffeine can be distinguished [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, the antioxidant activity of SCG oil was tested using chemical colorimetric assays, which require prior extraction of antioxidants to organic solvent miscible with water. This action is commonly used in oil testing and various methods were used to optimize extraction in this work.\u003c/p\u003e \u003cp\u003eFinally, when introducing a cosmetic or its ingredient to the market it is crucial to check its influence on the skin. Since animal testing of cosmetic formulations as a whole, as well as of individual ingredients, is against the European Union law, such testing should be carried out on \u003cem\u003ein vitro\u003c/em\u003e models. These studies mostly use 2D and 3D models consisting of human skin cells (Piasek et al., 2023). That is why the last part of this study was dedicated to confirming the usefulness of the obtained SCG oil on skin cell models.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eTechnical grade n-hexane for extraction was supplied from VWR. Silica gel plates were purchased from Merck or VWR, iodine crystals from WARCHEM and reference lipid mixture (Lipid Standard, Mono-, Di-, \u0026amp; Triglyceride Mix) from Sigma-Aldrich. Analytical grade hexane, diethyl ether, acetic acid, methanol and ethanol were delivered by Avantor Performance Materials Poland, and acetone by Merck. Sodium carbonate, sodium acetate, ethyl acetate, sodium hydroxide, hydrochloric acid and dimethyl sulfoxide (DMSO) were acquired from Chempur. All other reagents used for antioxidant activity and total polyphenol content assays i.e. iron (III) chloride hexahydrate, 2,4,6-tris(2-pirydyl)-s-triazine complex (TPTZ), iron (II) sulfate heptahydrate, ammonium acetate, neocuproine, copper (II) chloride dihydrate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 3,4,5-trimethoxybenzoic acid (gallic acid), ammonium persulfate (APS), 2,2\u0026rsquo;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Cioc\u0026acirc;lteu reagent were supplied by Sigma-Aldrich. HaCaT keratinocytes were purchased from the German Cancer Research Center - Deutsches Krebsforschungszentrum. Dulbecco\u0026rsquo;s Modifieds Eagle Medium (DMEM), 0.25% trypsin-EDTA, staurosporine, glutathione (GSH), fluorescein diacetate (FDA), propidium iodide (PI), crystal violet (CV), paraformaldehyde and 2\u0026prime;,7\u0026prime;- dichlorodihydrofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and penicillin/streptomycin mix (10.000 U/ml pen and 10 mg/ml strep) were obtained from Gibco, phosphate-saline buffer (PBS) from VWR, tetrazolium salt (MTT) from Thermo Fisher Scientific, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) from Avantor Performance Materials Poland, and Pluronic F127 from BASF Polska. For comparison, green coffee oil (CO1, Zr\u0026oacute;b Sobie Krem) and two roasted coffee oils (CO2, Etja and CO3, Manufaktura Natura) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Oil extraction\u003c/h2\u003e \u003cp\u003eSpent coffee grounds (SCG), obtained from the local coffee shop (Warsaw, Poland), were subjected to drying for 24 hours in order to decrease the water content to about 5\u0026ndash;6%. Dried SCG were sifted through a sieve to separate impurities and homogenize the material. The appropriate mass of coffee grounds was weighed and placed in a round bottom flask in which coffee oil was extracted with hexane at 68\u0026deg;C (hexane boiling point) for a specified period - individual process parameters were tested to select the best ones.\u003c/p\u003e \u003cp\u003eDuring optimization, extractions were performed in 500 ml, 1000 ml and 2000 ml round bottom flasks to examine process scalability. Different extraction times, i.e. 30 min, 45 min and 60 min, were also evaluated. Another tested parameter was the ratio of SCG-to-solvent, 5 variants were checked \u0026minus;\u0026thinsp;1:2.5, 1:3.75, 1:5, 1:6.25, 1:7.5. Last but not least, the multiplicity of extraction was also analyzed.\u003c/p\u003e \u003cp\u003eAfter extraction, the solid phase was separated from the liquid \u0026ndash; the grounds, without the lipid fraction, were dried overnight and weighed. The yield of oil extraction was determined by sample mass loss according to the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:extraction\\:yield=\\frac{{SCG}_{c}-{SCG}_{x}}{{SCG}_{c}}\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere SCG\u003csub\u003ec\u003c/sub\u003e \u0026ndash; the weight of the sample before extraction, SCG\u003csub\u003ex\u003c/sub\u003e \u0026ndash; the weight of the sample after extraction.\u003c/p\u003e \u003cp\u003eTo recover the solvent for the next extractions and separate crude SCG oil, the liquid phase was evaporated on a vacuum evaporator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Thin layer chromatography (TLC)\u003c/h2\u003e \u003cp\u003eThe composition of the main lipid classes in SCG oil was determined by thin layer chromatography, performed on silica gel plates. Before separation, the chamber was saturated with vapors of mobile phase, which was a mixture of hexane, diethyl ether and acetic acid (40:10:1, v:v:v). Aliquots of 1 \u0026micro;l of SCG oil and three commercially available coffee oils were applied on TLC plates. The lipid composition was identified by the comparison with the reference lipid mixture, which was also applied on the plate. After developing and drying the plates, they were observed under UV light at a wavelength of 254 nm and then stained with iodine vapors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction of antioxidants from the oil\u003c/h2\u003e \u003cp\u003eTo determine the antioxidant activity of SCG oil, it was necessary to extract antioxidants with an organic solvent miscible with water. Based on the information presented in literature [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and our research, methanol and ethanol solutions were selected for this purpose. These included methanol concentrations (40%, 60% and 80%) and 48% ethanol. 1 ml of oil (extracted from SCG or purchased on the market) was added to 20 ml of the solvent receiving a 1:20 oil-to-solvent (v:) ratio. During optimization, tests were also carried out in 1:1, 1:2, 1:4, and 1:10 oil-to-solvent (v:v) ratios. Mixtures were shaken on a laboratory shaker at 250 rpm for a selected period. During these tests, extraction times such as 10 min, 30 min, 1 h, 4 h and 24 h were investigated. Extraction of each sample was carried out three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antioxidant activity and total phenolic content\u003c/h2\u003e \u003cp\u003eAntioxidant activity of SCG oil and commercial coffee oils was determined by chemical tests (FRAP, CUPRAC, DPPH and ABTS), and total phenolic content (TPC) was evaluated by the Folin-Cioc\u0026acirc;lteu method \u0026ndash; these techniques are based on the measurement of the absorbance change of the tested solutions caused by the presence of compounds with specific properties. The results are presented as concentrations to different standards (depending on the test) or a percentage of SCG oil antioxidant activity (control).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. FRAP method\u003c/h2\u003e \u003cp\u003eThe principle of the FRAP method is based on the ability to reduce iron (from Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e). Before performing an assay, the FRAP reagent was prepared by mixing an acetate buffer (pH 3.6, prepared from sodium acetate, Milli-Q water and acetic acid, pH was adjusted with sodium hydroxide and hydrochloric acid), 20 mM iron (III) chloride hexahydrate and 10 mM 2,4,6-tripyridyl-s-triazine complex (TPTZ) in the ratio of 10:1:1 (v:v:v). Aliquots of 5 \u0026micro;l of samples were added to wells, each sample was run in triplicate. Subsequently, 145 \u0026micro;l of the FRAP reagent was added to each sample. After 30 minutes of incubation at room temperature and without access to light the absorbance of the samples was measured at a wavelength of 593 nm. Antioxidant activity was determined against a standard curve of iron (II) sulfate heptahydrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. CUPRAC method\u003c/h2\u003e \u003cp\u003eThe mechanism of the CUPRAC assay is similar to the FRAP method and it is based on the antioxidants' ability to reduce copper (from Cu\u003csup\u003e2+\u003c/sup\u003e to Cu\u003csup\u003e+\u003c/sup\u003e). Before the analysis, the CUPRAC reagent consisting of 10 mM ammonium acetate (pH 7.0, prepared from ammonium acetate and Milli-Q water, pH was adjusted with sodium hydroxide and hydrochloric acid), 7.5 mM neocuproine in methanol, 10 mM copper (II) chloride dihydrate and Milli-Q water in a ratio of 1:1:1:0.6 (v:v:v:v), was prepared. Aliquots of 20 \u0026micro;l of samples in triplicates were added to wells, then 145 \u0026micro;l of the CUPRAC reagent was also added. The plate was incubated for 30 minutes at room temperature and without access to light. Later the absorbance of samples was measured at a wavelength of 450 nm. Antioxidant capacity was evaluated based on the standard curve of trolox.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. DPPH assay\u003c/h2\u003e \u003cp\u003eThe DPPH assay is based on the measurement of the scavenging capacity of the tested sample towards the stable free radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Before performing the test, a 0.25 mM methanolic solution of DPPH was prepared and its absorbance at a wavelength of 520 nm was checked - if the absorbance is not 0.30 +/- 0.02, the solution should be adjusted to this value by adding DPPH or methanol. Aliquots of 25 \u0026micro;l of samples and 75 \u0026micro;l of prepared solution were added to wells, each sample was tested in three repetitions. The plate was incubated at room temperature for 15 minutes without access to light and then the absorbance at 520 nm was measured. The concentration of antioxidants was calculated by using a standard curve of trolox.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. ABTS method\u003c/h2\u003e \u003cp\u003eThe ABTS assay is based on the ability to scavenge the ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e radical cation, which needs to be pre-generated 12\u0026ndash;16 hours before the test, by preparing a 2.45 mM solution of ammonium persulfate (APS) in 2\u0026prime;-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Aliquots of 25 \u0026micro;l of samples and 75 \u0026micro;l of ABTS solution were added to wells, each sample was tested in triplicate. The plate was incubated for 30 minutes at room temperature and without access to light. Next, the time absorbance of samples was measured at a wavelength of 734 nm. Antioxidant concentration was determined against a standard curve of trolox.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5. Total polyphenol content\u003c/h2\u003e \u003cp\u003eThe total polyphenol content (TPC) was measured using the Folin-Cioc\u0026acirc;lteu method. A volume of 10 \u0026micro;l of samples was added to wells in triplicate. Subsequently, 50 \u0026micro;l of the Folin-Cioc\u0026acirc;lteu reagent was added to each sample. Following 3 minutes of incubation, 140 \u0026micro;l of 5% sodium carbonate was also added. After 90 minutes of incubation without access to light, the absorbance of each sample was measured at a wavelength of 765 nm. TPC was determined based on the standard curve of gallic acid.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell culture\u003c/h2\u003e \u003cp\u003eHaCaT keratinocytes, purchased from the DKFZ institute, were cultivated in a DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin mixture at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The cell line was routinely cultured into a 75 cm\u003csup\u003e2\u003c/sup\u003e culture flask. When the confluency was about 80%, keratinocytes were harvested with 0.25% trypsin and seeded at a density of 1 x 10\u003csup\u003e4\u003c/sup\u003e cells/ml into 96-well plates. For 2D culture non-treated flat-bottom plates were used and cells were then incubated for 24 hours (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e), whereas for the spheroid culture, cells were seeded into pretreated (a 1% of Pluronic F127 incubated overnight, and PBS washed) round-bottomed well plates and incubated for 48 hours (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e)(limiting cell adhesion method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Preparation of oil samples and cell treatment\u003c/h2\u003e \u003cp\u003eBefore the cells were treated, oil samples were dissolved in a selected organic solvent, i.e. acetone (Ac) or ethyl acetate (EA). First, the oil was diluted in a solvent at concentrations ranging from 3.13 to 50%, and then the mixtures were added to the DMEM medium at a concentration of 0.5% (2D culture experiments) or 1% (comparative study between 2D and 3D culture). The final oil content ranged from 0.016 to 0.250% for 2D culture studies and from 0.016 to 1.000% for 2D and 3D culture comparative experiments. DMEM was supplemented with 1% FBS (for FDA, PI and MTT assays) or without FBS (during ROS and crystal violet assays) and a standard 1% penicillin/streptomycin mixture.\u003c/p\u003e \u003cp\u003e24 hours (2D culture) or 48 hours (spheroids) after cells were seeded into plates, they were treated with SCG oil and 0.5% (2D) or 1.0% (3D) solvent solution, which was used as a control. Additionally, keratinocytes were treated with 2 \u0026micro;M staurosporine (positive control in FDA, PI and MTT assays) and with 0.5% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (positive control in ROS and CV assays) and glutathione (GSH; negative control in ROS assay).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of cytotoxicity\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. FDA/PI assay\u003c/h2\u003e \u003cp\u003eFDA/PI assay can be used to evaluate the viability of the cells. Fluorescein diacetate (FDA) and propidium iodide (PI) are two fluorescent dyes, which stain living and dead cells, respectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Cells were treated with oil dissolved in Ac/EA and a pure solvent as a control. After 24 hours of incubation, 10 \u0026micro;l of FDA and PI mixture (prepared in pure DMEM medium at a concentration of 80 \u0026micro;g/ml for both dyes) was added to each well. The plate was incubated for 10 minutes in the dark and fluorescence of each well was measured at the wavelength of 490/526\u0026thinsp;\u0026plusmn;\u0026thinsp;9 nm (for FDA) and 534/635\u0026thinsp;\u0026plusmn;\u0026thinsp;9 nm (for PI). The number of living and dead cells was determined against cells treated with a pure solvent solution and described as a percentage of control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. MTT assay\u003c/h2\u003e \u003cp\u003eMTT colorimetric assay is based on the activity of mitochondrial dehydrogenase, involved in the reduction of yellow tetrazolium salt to purple formazan, which is insoluble in water [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Following the FDA/PI assay, the medium was removed from each well and replaced with 50 \u0026micro;l of 0.5 mg/ml MTT in the pure DMEM medium. Then the plates were incubated for 2 hours at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Next, to dissolve the obtained formazan, the medium was once again removed and replaced with 100 \u0026micro;l of DMSO. The absorbance of each well was measured at a wavelength of 570 nm. Cell viability was determined against cells treated with a solvent and described as a percentage of control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. CV assay\u003c/h2\u003e \u003cp\u003eCrystal violet stains all acidic (negatively charged) components of cells (\u003cem\u003ei.e.\u003c/em\u003e DNA, RNA and proteins), which is why it may be used to determine the total number of cells in a well [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. After 24 hours of incubation with the added substances, the medium was removed, and cells were washed with a PBS buffer to remove dead-floating cells. Subsequently, keratinocytes were fixed with a 3.7% solution of paraformaldehyde in PBS for 10 minutes. After that time 0.05% methanolic solution of crystal violet was added and then the plate was incubated for 30 minutes. CV was then removed, and cells were washed with Milli-Q water three times. Plates were dried overnight at room temperature and next 50 \u0026micro;l of methanol was added to dissolve CV. The absorbance of each well was measured at a wavelength of 540 nm and the number of cells after treatment was determined against a control (cells treated with a pure solvent).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ROS assay\u003c/h2\u003e \u003cp\u003eROS assay is used to determine the amount of reactive oxygen species produced by cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e. Before the addition of SCG oil, the medium was replaced by 50 \u0026micro;l of 10 \u0026micro;M 2',7\u0026prime;-dichlorofluorescein diacetate (DCF-DA) in PBS. The plate was incubated for 30 minutes, then DCF-DA was removed, and oil samples were added. The fluorescence of each well was measured at a wavelength of 485/525\u0026thinsp;\u0026plusmn;\u0026thinsp;9 nm twice \u0026ndash; after 1 and 24 hours of incubation. The ROS amount was determined in relation to the control \u003cem\u003ei.e.\u003c/em\u003e cells treated with a solvent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were carried out in triplicates. Numerical results are presented in figures as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The received data were analyzed using appropriate tests to assess differences between groups. One-way ANOVA was applied to compare the means across multiple independent groups, determining whether there were statistically significant differences among them. Independent samples were used to compare the means between the two groups by the t-test to evaluate if the observed differences were statistically significant. A significant level of α\u0026thinsp;=\u0026thinsp;0.05 was set for all statistical tests and is marked by (*) in each graph.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe results presented in this research were obtained within an advanced biorefinery concept enabling the efficient valorization of spent coffee grounds (SCG) through a carefully designed sequence of processes. This approach allows for the extraction of coffee oil, antioxidants, lignin and sugars. The idea was undertaken by the Polish company EcoBean in collaboration with the Warsaw University of Technology and has progressed to the stage of a fully developed conceptual design, protected under patent application numbers P.447416 and PCT/PL2024/050107. This article specifically focuses on refining and optimizing the SCG oil extraction process as part of the broader strategy for comprehensive SCG utilization, as well as checking their antioxidant properties and cytotoxicity toward skin cells.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Optimization of oil extraction from SCG\u003c/h2\u003e \u003cp\u003eOptimization of valuable compound extraction is a key stage of each research. In the case of SCG, this element affects not only the quality of the obtained oil but also the required time and operational costs. Moreover, when choosing the best extraction parameters, environmental aspects also cannot be ignored. That is why, we have identified key parameters of oil extraction from SCG which are: the time of extraction (30, 45 and 60 min), SCG-to-hexane ratio (1:7.5; 1:6.25; 1:5; 1:3.75 and 1:2.5) and extraction multiplicity (1, 2 and 3). Each extraction process was carried out as described in the methods (subsection \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e.) taking into account the parameter under study. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, show that an extension of the extraction time beyond 30 minutes does not lead to a much higher efficiency. The short extraction time is advantageous in terms of process energy costs. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB demonstrates that efficiency improves with the increase in the amount of solvent used while considering the ratio of SCG-to-hexane. It is worth noting that the same oil extraction efficiency was obtained in a 500 ml flask with a ratio of 1:7.5 and a 4 times larger scale (2000 ml) with a grounds-to-solvent ratio of 1:3.75. Consequently, as the scale of the process increases, there is a possibility to use less solvent to obtain satisfactory process yields. This is a very optimistic prognosis for the implementation of this extraction method in industry. Last but not least, the multiplicity of the process was checked. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, a single extraction allows to obtain most of the oil, whereas the second and third extractions performed on the same SCG did not increase the yield significantly. Based on all the presented results, a single 30-minute oil extraction from SCG in a ratio of 1:5 was selected as optimal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Oil composition determined by TLC analysis\u003c/h2\u003e \u003cp\u003eThe TLC profiles of SCG oil and commercial coffee oils (CO1-CO3) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The main lipid class detected in all tested oils was triacylglycerols. Other classes were spotted in smaller quantities and different proportions. SCG oil contains significantly more 1,3-diacylglycerols than its counterparts obtained from coffee beans and smaller amounts of monoacylglycerols. In the case of 1,2-diacylglycerols, they were not detected in any of the samples.\u003c/p\u003e \u003cp\u003eUsually, fats and lipids are not detected under UV light due to the lack of fluorophores - therefore standards were not viewable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, lipids present in SCG oil and commercial coffee oils were visible, meaning that their acylglycerols may contain fluorescent compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Optimization of antioxidant extraction from SCG oil\u003c/h2\u003e \u003cp\u003eFor testing the antioxidant properties, the literature proposes various methods including FRAP, CUPRAC or DPPH assays. Thus, an essential step for investigating the antioxidant properties of the SCG oil was the extraction of compounds with such properties. Unfortunately, the described methods did not give satisfactory results. Based on the available sources, several experiments were carried out to select appropriate extraction parameters, i.e. type of solvent (methanol or ethanol), solvent concentration (40%, 48%, 60% or 80%), extraction time (10 min, 30 min, 1 h, 4 h and 24 h) and oil-to-solvent ratio (1:1, 1:2, 1:4, 1:10 and 1:20). After the extraction, chemical test for antioxidant properties were performed to evaluate which process was the most efficient at extracting antioxidants from SCG oil. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depict the most promising variants from all of the performed tests, but the initial trials are described in the attached \u003cem\u003eSupplementary materials (Table S1, Figure S1 and S2)\u003c/em\u003e. Due to the received effects \u003cem\u003e(Figure S2)\u003c/em\u003e, it was decided to significantly increase the amount of solvent used (1:10 and 1:20 oil-to-solvent ratio (v:v)) and the extraction time (4 h and 24 h). In addition, due to lower cytotoxicity, the possibility of using ethyl alcohol was also checked. All extractions were performed three times on the same SCG oil.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe first extraction of antioxidants from the SCG oil is the most efficient one, for the repeated extractions (2nd and 3rd) relatively low concentrations of antioxidants were obtained. In the FRAP assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), two variants were the most promising and both were using an 80% methanol, however, they differ in the length of the extraction run (24 h and 4 h) and the ratio of oil-to-solvent (1:10 and 1:20). From an economic point of view, a single extraction of antioxidants from SCG oil is much more cost-effective, that is why, we compared how the amount of extracted antioxidants differs between the 1st and 2nd extractions. This analysis revealed that more antioxidants were obtained in the sample once extracted with 80% methanol for 4 h and at a ratio of 1:20 SCG oil-to-solvent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Comparison of the antioxidant effect of SCG oil and commercial oils\u003c/h2\u003e \u003cp\u003eCoffee oil pressed from beans is well-known in the cosmetic industry, mostly for its antioxidant activity. During this research, we have compared the properties of commercial products and the oil extracted from coffee waste. In each case, antioxidants were extracted from the oil with the optimized methodology presented in subsection 2.3. Next, chemical tests for antioxidant properties (like ABTS, CUPRAC, FRAP) and TPC assay were carried out. These results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAll conducted studies have shown that SCG oil has the highest antioxidant properties compared to other oils, for which the results obtained are much lower. In the CUPRAC test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), there is about a 10-fold difference between SCG oil and CO1. This fact is very promising in terms of possible applications of SCG oil in cosmetics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Effects of SCG oil on human skin cells in 2D and 3D models\u003c/h2\u003e \u003cp\u003eDue to its interesting composition and antioxidative properties, SCG oil may have a valuable effect on skin cells, which was presented in Section 3.4. Studying this effect was problematic because of the very limited solubility of the oil in the medium used for cell culture. It was necessary to use an additional solvent to enable the distribution of oil in various concentrations in the culture medium. This solvent should have several properties, including having the least possible impact on the cells and not affecting the influence of the oil. We examined the use of three solvents: acetone, ethyl acetate and DMSO. Despite the initial good results for DMSO, it was eliminated from the tests due to the observed precipitation of oil in DMSO with longer storage, as well as the problem of sample freezing while storing the oil in DMSO at low temperatures (5\u0026deg;C). While testing SCG oil on the viability of skin cells in a monolayer (2D), the solvent concentration was maintained at a constant level of 0.5% for acetone and ethyl acetate, which does not cause a threat to the cells. Different viability test results for SCG oil-acetone or SCG oil-ethyl acetate addition to HaCaT keratinocytes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. These include conducted measurements like the MTT test, FDA/PI assay and CV staining. The decrease in values at MTT, FDA and CV assays is typically connected with harmful effects of the investigated compounds, whereas in PI assay it is shown by the increase in values.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStudies indicating the metabolic activity of cells (MTT and FDA) showed a similar tendency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). In oil concentrations up to 0.150%, there are no significant differences between the used solvents. The results achieved for oil dissolved in ethyl acetate during the FDA test presented lower values ​​(around 70% of control), which indicates a lower activity of cytoplasmic esterases. However, in the case of oil dissolved in acetone, there was a visible difference at the highest oil concentration (0.250%), where the cell metabolic activity was significantly lower (by about 30%) suggesting partial cell death. These observations confirm the results obtained using propidium iodide staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In the case of the highest concentration, cell membrane damage was significantly higher for cells treated with oil dissolved in acetone.\u003c/p\u003e \u003cp\u003eCrystal violet staining carries information about the number of remaining cells in the well, as well as the number of negatively charged elements in the cells (proteins, DNA) can be deduced. This staining revealed interesting results and differences within the tested solvents (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Treatment of cells with oil dissolved in both tested solvents does not result in detaching cells from the culture base. Moreover, in the case of cells with the addition of oil dissolved in ethyl acetate, we noticed a positive change - an increased number of negatively charged elements in the cells and/or an increase in the number of cells.\u003c/p\u003e \u003cp\u003eWe then examined if SCG oils dissolved in acetone or ethyl acetate are capable of reducing the reactive oxygen species production in keratinocytes. The results were gathered in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs mentioned earlier, we were seeking a solvent for SCG oil that should not interfere with the action of the active compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). When SCG oil dissolved in acetone was added to the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), we observed a rise in free radicals\u0026rsquo; release by keratinocytes as the oil concentration decreased and the solvent proportion increased. Perhaps, this would not be surprising if it were not for the comparison to the results obtained for SCG oil dissolved in ethyl acetate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), where the same antioxidant properties were maintained for all concentrations of the added oil. Moreover, a longer-lasting effect of these properties for oil dissolved in ethyl acetate is visible when comparing the results obtained for treating cells for 1 h and 24 h. In the case of treating cells with oil dissolved in acetone after 24 h, the results are almost as high as for the positive control, proving the increased release of free radicals by the cells takes place.\u003c/p\u003e \u003cp\u003eTo better predict the potential effect of SCG oil on the human skin, we compared its influence between keratinocytes seeded as monolayer (2D) as well as in spheroids (3D) prepared by the limiting-cell adhesion method. Spheroids are a more complex model than 2D culture and provide a more accurate imitation of real cell interactions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Considering the results obtained in the ROS assay, ethyl acetate was from now on used as an oil solvent for further studies on human skin cells. For this comparative experiment, a wider range of oil concentrations was used (up to 1%). The SCG oil cytotoxicity was assessed by the MTT as well as FDA/PI assays in both 2D and 3D models, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. This study showed that SCG oil in the tested concentrations does not adversely affect cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B) and their metabolic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) (all results are above 90%). Furthermore, there is no significant difference between the results obtained in 2D and 3D cultures. Higher oil concentrations (1% v/v) even had a more favorable effect on the metabolic activity of keratinocytes in the spheroids than in a monolayer (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this research, we focused on the extraction of oil from spent coffee grounds which can be used as a valuable cosmetic ingredient. The optimization of oil extraction from SCG allowed for the selection of appropriate process parameters. A 30-minute extraction and a 1:5 (SCG:hexane) ratio is sufficient to extract most of the oil. The described data show the relationship between the process efficiency and the amount of solvent used - the higher the ratio of hexane to grounds, the greater the efficiency. These results are similar to the ones present in the literature [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In both cited research these oils were extracted using soxhlet apparatus, which is difficult to implement in the industry. Higher extraction yields with the growth of the process scale (using the same SCG:solvent ratio) allow us to have optimistic hopes for introducing the process on an industrial scale.\u003c/p\u003e \u003cp\u003eDue to the wide variety of raw materials, the extracted oil requires characterization. One of the simplest methods that make it possible is TLC. The obtained results suggest the presence of triacylglycerols and 1,3-diacylglycerols, with the simultaneous absence of 1,2-diacylglycerols and monoacylglycerols in our SCG oil. In addition, there were also other signals visible on the TLC plates, most likely coming from glycerol, sterols, free fatty acids, ceramides, and cholesterol, as these were observed in other studies [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. So far, their specific identification in SCG oil has failed but will be continued in the future.\u003c/p\u003e \u003cp\u003eApart from its composition, the antioxidant properties of the SCG oil were also checked. These tests required prior extraction of antioxidants to the organic solvent. Despite many described methods of antioxidant extraction from various types of oils, no satisfactory ready-to-use method for SCG oil was found in literature [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. That is why, it was decided to optimize this process within this study. The comparison of the selected conditions with those reported in the literature shows some similarities. When it comes to the time required for the process, the 4-hour extraction is the longest described one. Considering the oil-to-solvent ratio, the closest reported is the ratio 1:10 (m:v) used by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The best extractant turned out to be 80% methanol. The same solvent and concentration were used by Khemakhem [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Marfil [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], respectively. The obtained methanol extracts with antioxidants from the SCG oil were tested for antioxidant properties (ABTS, CUPRAC, FRAP tests) and for the total polyphenol content. A high antioxidant content of SCG oil was demonstrated compared to the tested commercial coffee oils, which is consistent with the expectations and information presented in the literature [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This fact is very promising in terms of the possible application of SCG oil in cosmetics. The antioxidant properties of green coffee oil and cosmetics containing it were checked by Wagemaker [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Despite the low antioxidant properties of the oil, its presence in cosmetic formulations improved its total antioxidant activity which grew with the increase of oil concentration. All described results indicate that oil extracted from coffee waste can be environmentally friendly and a better alternative to currently used products in terms of antioxidant properties.\u003c/p\u003e \u003cp\u003eBefore using SCG oil in the \u003cem\u003ein vitro\u003c/em\u003e studies performed on cells, it was necessary to select an organic solvent that could mix well with the oil. Interestingly, none of the other works concerning coffee oil [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] mention this important fact. It seems that the others were applying coffee oil directly to the cell culture medium. Our observations of SCG oil show that it did not mix well with the culture medium and remained as the upper layer of the two-phase solution. Therefore, it was decided to choose an organic solvent that would facilitate its dispersion. We have selected polar acetone and slightly less polar ethyl acetate. The outcomes obtained for both solvents differed from each other, which may result from their different effects on human skin cells. Due to relatively low toxicity and good miscibility with many substances, acetone is one of the most commonly used solvents in cell culture studies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Research conducted by Farkas [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] has shown that it enhanced the proliferation of HaCaT cells and increased the level of transcripts characteristic for proliferating keratinocytes (α5 integrin, KGFR and cyclin D1). The second tested solvent, ethyl acetate, although less commonly used in the \u003cem\u003ein vitro\u003c/em\u003e studies, was found to be less cytotoxic than acetone, as reported by Koc [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In our study, ethyl acetate also turned out to be a better solvent for SCG oil, especially while testing the possibility of ROS production in cells. Our study highlights an important problem often encountered in biological research, where the solvent itself may not induce a cytotoxic effect but may inhibit the health-promoting effects of the compound being tested.\u003c/p\u003e \u003cp\u003eTo be able to use coffee oil in cosmetics, it is necessary to check its cytotoxic properties against cells. MTT, FDA, PI and crystal violet tests were performed on skin cell cultures - keratinocytes. The presented values ​​indicate low toxicity of SCG oil toward the investigated skin cells, which was also demonstrated by Kanlayavattanakul on mouse melanoma cells B16-F10 with concentrations ranging from 0.1 to 10 \u0026micro;g/ml of the coffee oil [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Cubas \u003cem\u003eet al.\u003c/em\u003e compared the effects of untreated coffee waste oil and oil treated with non-thermal plasma on fibroblast cells BALB/3T3 clone A31. They showed less toxicity of the oil after treatment with non-thermal plasma toward cells. Particularly, in the concentration range of 0.031\u0026ndash;0.125 mg/ml [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition to extensive research on SCG oil, numerous studies on green coffee oil are also relevant to the findings presented in this paper. For instance, cytotoxicity tests on keratinocytes revealed that green coffee oil exhibited no harmful effects at the tested concentrations [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], similarly to the results of our study. Green coffee oil was also tested by Oliveira \u003cem\u003eet al.\u003c/em\u003e on female rats. Among other things, the tests for acute toxicity showed no changes in all organs assessed [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Interestingly, we are the first to carry out tests on the 3D skin model, which enabled checking the effect of substances on cells in an environment more similar to the body thanks to intercellular interactions and the results confirm that SCG oil was safe for use. Furthermore, there were no significant differences between the results obtained in 2D and 3D culture, both results stated that there is no cytotoxicity of the SCG oil toward skin cell models.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eGiven the vast amount of spent coffee grounds generated daily worldwide, finding sustainable ways to repurpose this waste is essential. Oil extraction is one of the most well-established approaches for utilizing SCG. In this study, we propose a simple, efficient extraction method that requires no specialized equipment or excessive time while ensuring high yield. Due to its unique composition, SCG oil holds potential for various industries, with our research specifically exploring its application in cosmetics. Additionally, this study introduces an innovative protocol for extracting antioxidants from SCG oil. The extracted oil demonstrated superior antioxidant properties compared to commercially available green and roasted coffee oils and exhibited no cytotoxicity toward keratinocyte cells in both 2D and 3D models. These findings highlight SCG oil as a promising, sustainable ingredient for the cosmetics industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe entire research was carried out within an advanced biorefinery concept enabling the efficient valorization of spent coffee grounds (SCG) through a carefully designed sequence of processes by the Polish company EcoBean in collaboration with the Warsaw University of Technology. This approach allows for the extraction of coffee oil, antioxidants, lignin, and sugars, and is currently covered by patent application numbers P.447416 and PCT/PL2024/050107. The authors declare that they have disclosed all relevant affiliations and potential conflicts of interest related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the UE framework program Horizon Europe (ID project: 27170) \u0026lsquo;Upcycling coffee waste into useful raw materials and green products\u0026rsquo; and the Ministry of Science and Higher Education program \u0026lsquo;Implementation PhD\u0026rsquo;.\u003c/p\u003e\n\u003ch5\u003eAuthor contributions\u003c/h5\u003e\n\u003cp\u003eConceptualization: Adrianna Maria Piasek and Anna Sobiepanek; Formal analysis: Adrianna Maria Piasek, Paula Bardadyn and Anna Sobiepanek; Funding acquisition: Adrianna Maria Piasek, Łukasz Wysocki, Tomasz Kobiela and Anna Sobiepanek; Investigation: Adrianna Maria Piasek, Paula Bardadyn, Zoja Trojan and Karolina Jelonek; Methodology: Adrianna Maria Piasek and Anna Sobiepanek; Project administration: \u0026nbsp;Adrianna Maria Piasek, Paula Bardadyn and Anna Sobiepanek; Resources: Adrianna Maria Piasek, Łukasz Wysocki and Anna Sobiepanek; Supervision: Adrianna Maria Piasek and Anna Sobiepanek; Visualization: Adrianna Maria Piasek, Paula Bardadyn and Zoja Trojan; Writing \u0026ndash; original draft: Adrianna Maria Piasek and Paula Bardadyn; Writing \u0026ndash; review and editing: Adrianna Maria Piasek, Paula Bardadyn, Zoja Trojan, Karolina Jelonek, Łukasz Wysocki, Tomasz Kobiela and Anna Sobiepanek.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This research does not need any ethical approval.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and materials are available on request from the corresponding author Dr. Anna Sobiepanek ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePluronic F127 was a kind gift from Dr. Michał Stepulak from BASF Polska. Moreover, we would like to express our sincere gratitude to Professor Andrzej Chwojnowski from the Nałęcz Institute of Biocybernetics and Biomedical Engineering PAS for his invaluable guidance and insightful discussions throughout the development of this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlinov\u0026aacute;, L.; Sirotiak, M.; Barto\u0026scaron;ov\u0026aacute;, A.; Sold\u0026aacute;n, M. 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Acute and Subacute (28 Days) Toxicity of Green Coffee Oil Enriched with Diterpenes Cafestol and Kahweol in Rats. \u003cem\u003eRegulatory Toxicology and Pharmacology\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e110\u003c/em\u003e, 104517, doi:10.1016/j.yrtph.2019.104517.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"spent coffee grounds, oil, antioxidants, cosmetics, 2D cell culture, 3D skin model","lastPublishedDoi":"10.21203/rs.3.rs-6358386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6358386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoffee is one of the most widely consumed beverages globally, generating significant amounts of waste, including spent coffee grounds (SCG). SCG contains valuable compounds, particularly oil, which constitutes 10–20% of its composition, depending on the plant species. This study developed an optimized method for extracting the lipid fraction efficiently, without requiring extensive time, specialized equipment, or high costs. The most effective extraction was achieved using hexane at its boiling point for 30 minutes, with an SCG-to-solvent ratio of 1:5 (m:v). Given its composition and beneficial properties, SCG oil holds potential for various industrial applications, with this research focusing on its suitability for the cosmetics industry. Notably, SCG oil demonstrated high antioxidant activity compared to commercially available coffee oils and showed no cytotoxic effects on 2D and 3D skin cell cultures. These findings highlight SCG oil as a sustainable, eco-friendly alternative to conventional cosmetic raw materials, contributing to both sustainable development and innovation in the cosmetics sector.\u003c/p\u003e","manuscriptTitle":"Research on spent coffee grounds: from oil extraction to its potential application in cosmetics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 07:44:43","doi":"10.21203/rs.3.rs-6358386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-18T11:23:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T06:05:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-04-13T20:23:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T07:09:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-04-02T02:58:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2df317c8-33be-49a9-a07f-70f02c56d84a","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T16:02:53+00:00","versionOfRecord":{"articleIdentity":"rs-6358386","link":"https://doi.org/10.1007/s12649-025-03284-2","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-09-20 15:57:17","publishedOnDateReadable":"September 20th, 2025"},"versionCreatedAt":"2025-04-29 07:44:43","video":"","vorDoi":"10.1007/s12649-025-03284-2","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03284-2","workflowStages":[]},"version":"v1","identity":"rs-6358386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6358386","identity":"rs-6358386","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}