Novel bioactive extract from yarrow obtained by the supercritical antisolvent-assisted technique inhibits lipid metabolism in colorectal cancer

Background: Altered lipid metabolism in cancer is associated to dissemination and prognosis. Bioactive compounds naturally occurring in Achillea millefolium L. (yarrow) have been reported to exert antitumour activities. Food biotechnology may provide on-demand mixtures of bioactive compounds with complementary activities in cancer treatment. Methods: Supercritical-antisolvent-precipitation (SAS) has been applied to fractionate the bioactive compounds from an Ultrasound-Assisted-Extraction yarrow extract resulting in two extracts with distinct polarity, yarrow-precipitate-(PP) and yarrow-separator-(Sep). Total phenolic content and relevant essential oils have been characterized. Antioxidant, anti-inflammatory and antiproliferative activities have been compared. Moreover, the effect on the inhibition of colorectal cancer cells’ bioenergetics has been evaluated. Results: Yarrow-PP exerted the highest antioxidant activity, even higher than the complete UAE-yarrow extract, meanwhile yarrow-Sep showed the highest anti-inflammatory activity, even higher than the complete UAE-yarrow extract. Interestingly, yarrow-Sep inhibited key lipid metabolic targets in CRC cells extensively shown to be implicated in cancer dissemination and prognosis —SREBF1, FASN, ABCA1 and HMGCR— and epithelial to mesenchymal targets—CDH1, ATP1B1, CDH2 and Vimentin—augmenting cell adhesion. Conclusions: In summary, SAS technology has been applied to provide a novel combination of bioactive compounds, yarrow-Sep, which merits further research to be proposed as a potential complementary nutraceutical in the treatment of CRC.


Introduction
Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Metastasis is the last step of cancer, being the main responsible factor for morbidity and mortality.In fact, more than 90% of mortality associated with CRC is due to metastasis.Currently, the altered lipid metabolism in cancer is a topic of interest due to the prominent role of lipids regulating the progression in various types of tumors.Lipids and lipid-derived molecules have been shown to activate growth regulatory pathways and promote malignancy in several cancers (Aguirre-Portoles et al., 2017;Bouzas et al., 2022).During tumor development, lipid-associated alterations include i) an increase in the de novo lipogenesis driven by the sterol regulatory element-binding factor (SREBF1) and downstream molecular targets, fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD-1); ii) an increase in cholesterogenesis by means of the expression of hydroxy-methylglutaryl-CoA reductase (HMGCR), affecting plasmatic membrane fluidity and remodeling the extracellular tumor microenvironment (Aguirre-Portoles et al., 2018); and iii) an increased uptake of extracellular fatty acids by means of the increased expression of the lipoprotein low-density receptor (LDLR) and cluster differentiation 36 (CD36), which have also been demonstrated to dictate metastatic tropism in several cancers (Montero-Calle et al., 2022).Importantly, many authors have described the link between the altered expression of lipid metabolic genes and development and prognosis in cancer (Sanchez-Martinez et al., 2015;Vargas et al., 2015).
The development of innovative approaches, such as fractionation, precipitation, and nanoencapsulation, may contribute to provide on-demand mixtures of bioactive compounds with complementary and/or synergistic activities (Oskoueian et al., 2020;Shafaei et al., 2020;Zarei et al., 2021).In this regard, supercritical antisolvent (SAS) precipitation has been demonstrated to act as a very powerful technique for the fractionation of plant extracts, providing further combinations of bioactive compounds (Mur et al., 2021).In this process, a polar liquid solution of a plant extract is continuously sprayed in a cocurrent with supercritical CO 2 (SCCO 2 ).Thus, more polar compounds precipitate due to their insolubility in SCCO 2 ; meanwhile, less-polar compounds remain dissolved in SCCO 2 , being recovered downstream by pressure reduction of SCCO 2 (Langa et al., 2019).At the end of the process, two fractions, the precipitate fraction and the separator fraction, are obtained containing compounds of different polarities.This technique can be considered as environmentally sustainable since it does not generate any type of waste (Gimenez-Rota et al., 2019).Moreover, CO 2 creates an inert environment, reducing the degradation of bioactive compounds due to its low critical temperature.Thus, Villalva et al. (2019) used SAS to fractionate the bioactive compounds present in an ethanolic extract of yarrow, obtaining two fractions, one enriched in phenolic compounds and the other enriched in essential oil components (Villalva et al., 2019).
The objective of this work was to apply the SAS technology to fractionate the bioactive compounds of an ultrasound-assisted extraction from yarrow (UAE yarrow), to obtain fractions with potential activity as co-adjuvants in CRC treatment.The chemical composition of fractions obtained was characterized in terms of total phenolic compounds and essential oil composition.The antioxidant, anti-inflammatory, and antiproliferative activities were also determined.In addition, the effect on cell bioenergetics of colorectal cancer cells was evaluated.

UAE yarrow extract
Yarrow inflorescences and upper dried leaves (batch number 395.CC.26101) were supplied by a Spanish company specialized in plant material supplies (Plantafarm, Leon, Spain).Samples were ground using a knife mill (Grindomix GM 200, Retsch, Llanera, Spain), and the particle size was reduced to <500 µm.
UAE was carried out following the method described by Villalva et al. (2021).In brief, a Branson 250 digital device (Branson Ultrasonic, Danbury, United States) with an electric power of 200 W and frequency of 60 Hz was used.Ground plant material (20 g) was extracted with ethanol (ratio of 1:10, plant:solvent) for 20 min and 60% amplitude, using a probe of ½' diameter.Next, the samples were filtered, and ethanol was partially removed under rotary evaporation at 35 °C (IKA RV 10, Madrid, Spain) to achieve a concentrated solution at 17.8 mg/mL.

SAS precipitation
The supercritical antisolvent precipitation was performed using the supercritical technology equipment Thar SF 2000 (Thar Technology, Pittsburgh, PA, United States) at 15 MPa and 40 °C.A comprehensive explanation about the equipment and the process design used can be found elsewhere (Quintana et al., 2019).
Concisely, the experiment started by pumping SCCO 2 (at 60 g/ min flow rate along the experiment) until the pressure and temperature conditions were attained into the precipitation vessel.Then, the UAE yarrow solution (17.8 mg/mL) was pumped into this vessel at 1 mL/min for 45 min while maintaining the SCCO 2 flow rate.After mixing, yarrow extract components not soluble in the SCCO 2 + ethanol supercritical phase were obtained in the precipitation vessel and were collected (precipitate fraction, yarrow PP).Meanwhile, the fraction soluble in the SCCO 2 + ethanol matrix went into the separator vessel, where ethanol and this fraction of phytochemicals were recovered.Next, additional SCCO 2 was pumped during 15 min to remove the residual solvent from the precipitation vessel.
The samples obtained in the separator were rotary evaporated (RV 10 control VWR, IKA, Staufen, Germany) until an oleoresin product was obtained (separator fraction, yarrow Sep).Dry SAS fractions, PP, and Sep were kept in glass vials at −20 °C under darkness until analysis.Prior to closing the vials, a stream of N 2 was passed.
Chromatograms were recorded at 280, 320, 340, and 360 nm.Compounds were identified according to a previous HPLC-PAD-MS/MS study carried out by the research group by comparing their accurate mass spectra and their fragmentation pattern with the literature (Villalva et al., 2021).Analytical standards were used, if available, to confirm the correct identification of each compound regarding its retention time and UV−Vis spectrum.Quantification was performed using calibration curves of analytical standards, except for 6-hydroxyluteolin-7-O-glucoside and luteolin-Cglucoside (luteolin-7-O-glucoside standard was used), schaftoside isomer and apigenin-C-hexoside-C-pentoside (schaftoside), methyl apigenin-glucoside (vitexin), and dihydroxy-trimethoxyflavone, methoxyacacetin, and methoxyflavone (casticin).Ethyl gallate was added as internal standard in each analyzed sample.The results were expressed as mg phenolic compound/g dry sample.

Gas chromatography-mass spectrometry analysis
Essential oils from the total UAE yarrow extract and its fractions, yarrow PP and yarrow Sep, were identified and quantified by gas chromatography (GC)-mass spectrometry (MS)-FID, following the method developed by Villanueva-Bermejo et al. (Villanueva-Bermejo et al., 2017).For this purpose, an Agilent 7890 A system (Agilent technologies, Santa Clara, CA, United States) equipped with a split/ splitless injector, FID detector, and mass spectrometer detector (5975C triple-axis) was used.The analyses were performed using an Agilent HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm phase thickness) and helium as inert gas, maintaining a flow rate of 1 mL/ min.The chromatographic method started with a temperature of 40 °C, which was increased to 150 °C at 3 °C/min and held at 150 °C for 10 min, then increased from 150 °C to 300 °C with a ramp of 6 °C/min, and finally, maintained at 300 °C for 1 min.Samples (1 µL) were injected in the spitless mode.The temperatures for the injector, mass spectrometer ion source, interface, and quadrupole were 250 °C, 230 °C, 280 °C, and 150 °C, respectively.The mass spectrometer was operated in the electron impact mode with an energy of 70 eV and was used in the total ion current (TIC) mode, and the mass interval scan ranged from 40 to 500 m/z.
Compound identification was carried out by comparing the mass spectral fragmentation patterns with the Wiley 229 mass spectral library, as well as by contrasting their corresponding retention index (RI) with those described in the literature.The results were expressed as the percentage of area contribution of the total amount of identified compounds.

Measurement of the antioxidant activity
Antioxidant activity was determined using ABTS •+ assay, according to the protocol (Re et al., 1999).In brief, for the reaction, 10 µL of the samples were added to 990 µL of diluted ABTS •+ radical solution.Then, the reaction took place in the dark until the absorbance reached a plateau.After that, the absorbance was measured at 734 nm, and the results were expressed as TEAC values (mmol Trolox/g extract).For this purpose, Trolox was used as the reference standard, with the following regression equation used for TEAC determination: Y = 47.88X+ 0.9237 and R 2 = 0.9943, where X and Y represent mM Trolox and the inhibition percentage, respectively.
The cytotoxic effect of the extracts was tested on differentiated macrophages, following the MTT assay by Mosmann (1983).
For the anti-inflammatory assay, differentiated cells were incubated with 0.05 μg/mL lipopolysaccharide (LPS) from E. coli 055:B5 (Sigma-Aldrich, Madrid, Spain) in the presence of the samples for 24 h.Then, the supernatants were collected and kept frozen at −80 °C.The release of cytokines TNF-α, IL-1β, and IL-6 was measured in the supernatants of differentiated cells using an ELISA kit (BD Biosciences, Aalst, Belgium), according to the manufacturer's instructions.

Analysis of the inhibition of CRC cell viability
Colorectal cancer cells DLD1 and SW620 were purchased from the American Type Culture Collection (Manassas, VA, United States) and cultured in DMEM (2 mM glutamine) and 10% fetal bovine serum (LONZA Iberica, S.A) in 5% CO 2 atmosphere at 37 °C and 95% humidity.
DLD1 and SW620 cells were plated in 96-well plates at densities of 8,000 cells/well and 15,000 cells/well, respectively, overnight (o/n) to allow the cells to attach.The next day, the cells were treated for 48 h with UAE yarrow or Sep yarrow extracts dissolved in DMSO diluted in DMEM (Cultek, Madrid, Spain) growth medium at different concentrations.Subsequently, the culture medium was supplemented with 10% sterile filtered MTT at 5 μg/mL.After 3 h, the medium was removed, and the insoluble formazan crystals were dissolved in 100 µL/ well of DMSO.Absorbance was measured at 560 nm in a VICTOR Nivo multimode plate reader (PerkinElmer, Madrid, Spain).The inhibition of growth (mitochondrial function) due to extracts was expressed as a percentage of viable cells in experimental wells relative to control wells to calculate IC 50 values.

RNA isolation and gene expression analysis
CRC cells were plated and, after an overnight incubation, were treated with two different concentrations of each extract based on the previously determined concentrations, inhibiting 50% of cell viability (IC 50 ).Non-treated cells were kept as controls.Total RNA was isolated from each condition with the RNeasy Mini Kit (QIAGEN Iberica, Madrid, Spain), following the manufacturer´s instructions.RNA quality and quantity were checked by UV spectroscopy (NanoDrop 2000 Spectrophotometer, Thermo Scientific, Waltham, Massachusetts, United States).
RNA extracted was subsequently used for real-time quantitative polymerase chain reaction (qPCR).In brief, 1 µg of total RNA was reverse-transcribed with the High-Capacity RNA-to-cDNA Master Mix system (Life Technologies, Madrid, Spain).qPCR was performed in the Quant Studio PCR System (Life Technologies) using the Veri Quest SYBR Green qPCR Master Mix, and specific oligos complementary to the genes of interest, including lipid metabolism-related genes-SREBF1, FASN, ABCA1, and SCD1-and epithelial-to-mesenchymal transition (EMT) genes-CDH1, ATP1B1, CDH2, and Vimentin-are indicated in Supplementary Table S1.The relative quantification (RQ) of gene expression for each gene was determined following the 2 −ΔΔCt Livak method (Livak and Schmittgen, 2001).RQ Manager Software and ExpressionSuite Software (Applied Biosystems, Madrid, Spain) were applied to calculate the relative expression of each gene.

Analysis of mitochondrial respiration by extracellular flux analysis of the oxygen consumption rate
To analyze the effect of the extracts on mitochondrial respiration, we monitored the extracellular flux of the oxygen consumption rate (OCR) (Cell Mito Stress Test), after the injection of several modulators of the electron transport chain, with the XFe96 Cell Bioanalyzer (Seahorse Biosciences-XFe96, Madrid, Spain).Optimal cell density and drug titration were previously determined.Prior to the experiments, the cells were pre-treated with the indicated doses of the extracts for 48 h.Non-treated cells were kept as controls.
In brief, for the Mito Stress assay, CRC cells were seeded onto 96-well XFe96 cell culture microplates at a density of 8,000 cells/well.The next day, the medium was changed to substrate-limited DMEM without glucose, glutamine, and sodium pyruvate (Life Technologies, Madrid, Spain), and then, it was supplemented with 0.5 mM glucose, 1.0 mM GlutaMAX (Life Technologies, Madrid, Spain), 0.5 mM carnitine (Sigma-Aldrich, Madrid, Spain), and 10% FBS fetal bovine serum for 6-8 h.Then, media were changed to XFe DMEM supplemented with 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES and adjusted to pH 7.4.
Cells were incubated for 45 min-1 h at 37 °C without CO 2 .Three different modulators of mitochondrial respiration were sequentially injected.After basal OCR determination, oligomycin (1.5 µM), which inhibits ATPase, was injected to determine the amount of oxygen dedicated to ATP production by mitochondria.To determine the maximal respiration rate or spare respiratory capacity, FCCP (carbonyl cyanide-4-(trifluoro-methoxy) phenylhydrazone) was injected (0.8 µM) to free the gradient of H + from the mitochondrial intermembrane space and, thus, to activate maximal respiration.Finally, antimycin A and rotenone (0.5 µM) were added to completely inhibit the mitochondrial respiration.

Lipid accumulation: oil red O staining
CRC cells treated for 48 h with the extracts were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 30 min.After washing twice with PBS and with 60% isopropanol, cells were stained with 3 g/L oil red O (in 60% isopropanol) at RT for 30 min.Next, cells were washed thrice with PBS and left to dry at RT for 10 min for visualizing using a microscope (Leica DM 2000 LED, Madrid, Spain).Finally, cells were resuspended in isopropanol, and absorbance was measured at 510 nm in a VICTOR Nivo multimode plate reader (PerkinElmer, Madrid, Spain).

CRC cell adhesion
The effect of the extracts on cell adhesion was analyzed after plating the cells on a Matrigel matrix (0.4 μg/mm 2 ) (Sigma-Aldrich, Madrid, Spain).Afterward, 96-well plates (Corning, Somerville, Massachusetts, United States) were coated with 100 µL Matrigel matrix (diluted in 0.1 M NaHCO 3 ) and incubated at 4 °C O/N.Then, 96-well plates were blocked with 200 µL of sterile adhesion medium (DMEM 0.5% BSA) during 2 h at 37 °C, and CRC cells were fluorescently stained with 10 µL of 1 mg/mL BCEBF (Sigma, Madrid, Spain) per 1 mL of DMEM during 30 min at 37 °C and 5% CO 2 .Then, cells were harvested with 4 mM EDTA-PBS, and 2.5 × 10 5 cells were resuspended in 50 µL of the sterile adhesion medium and transferred to each pre-coated well, previously removing the adhesion medium.At this point, cells were treated with the extracts at the indicated concentrations in quadruplicates.Subsequently, cells were incubated during 2 h at 37 °C and 5% CO 2 , and after that, non-attached cells were removed by decantation.Wells were washed twice with 100 µL PBS 1X to properly eliminate all non-attached cells, and finally, attached cells were lysed with 50 µL of 10% SDS in PBS.Plates were incubated on a shaker for 20 min at room temperature and in darkness.The fluorescence signal was read with the Spark multimode microplate (TECAN, Chapel Hill, North Carolina, United States) at 436 nm-535 nm excitation-emission, respectively.

Statistical analysis
Statistical analysis of the data was carried out with Statgraphics v. Centurion XVI for Windows software (StatPoint Inc., Warranton, VA, United States).Data were reported as the mean ± standard deviation.A one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test at p < 0.05 was used to look for differences between means.All analyses were conducted in triplicate.
RT-qPCR results were analyzed using two-way ANOVA (Bonferroni post hoc test).Statistical differences were considered significant when p-value was <0.05.

Results and discussion
3.1 SAS fractionation of the UAE yarrow extract and biochemical characterization of the resulting fractions SAS fractionation of the UAE yarrow extract was carried out at 15 MPa and 40 °C.Previous studies performed by the research group indicated that these were the most suitable conditions to obtain a selective fractionation of the different compounds in the extract (Villalva et al., 2019).After the SAS process, two main fractions were obtained: precipitate (yarrow PP) and separator (yarrow Sep) fractions.
As several biological activities of yarrow extracts, including antitumor activity, have been related to phenolic compounds, an extensive characterization of these compounds recovered in the precipitate and separator fractions was carried out by HPLC-PAD.The chromatogram of phenolic compounds of the UAE yarrow extract is shown in Supplementary Figure S1.As depicted in Table 1, most of the phenolic compounds were recovered in the precipitate fraction (yarrow PP), except for the less-polar ones consisting of three methoxylated flavone structures.Particularly, 3,5-DCQA, the most abundant compound in the UAE yarrow extract, showed an enrichment of approximately 2.1 times in the precipitate fraction.Moreover, other abundant phenolics in the extract, such as vicenin II, schaftoside, and luteolin 7-O-glucoside, also showed a 1.9-fold enrichment in the precipitate fraction (Yarrow PP).These results are consistent with the lower solubility of the polar phenolic compounds in the SCCO 2 -ethanol mixture, most of them being precipitated and recovered in the precipitate fraction.Giménez-Rota et al. ( 2019) also reported a lower solubility of the polar phenolic compounds, such as rosmarinic acid, in the SCCO 2 -ethanol mixture (Giménez-Rota et al., 2019).
Considering that the main biological activities of yarrow extracts have also been attributed to the components of the essential oil, yarrow PP and yarrow Sep fractions were also analyzed by GC-MS.Table 2 illustrates that essential oil components were mostly recovered in the separator fraction (yarrow Sep); meanwhile, these compounds were not found in the precipitate fraction (yarrow PP).As explained before, this result is in accordance with the higher solubility of essential oil components in the SCCO 2 -ethanol mixture, due to its low polarity.The most abundant identified bioactive compounds were terpenes and terpenoids such as borneol and grandisol, followed by caryophyllene oxide.Although the UAE yarrow extract also presented a similar GM-MS profile, the total chromatographic area (expressed as AUC) was higher for the separator fraction.

Antioxidant activity of the UAE yarrow extract and its derived SAS fractions
The antioxidant activity of precipitate (yarrow PP) and separator (yarrow Sep) fractions was determined using the ABTS radical method (TEAC value).As it can be observed in Table 3, the precipitate fraction (yarrow PP) showed the highest antioxidant activity; meanwhile, the separator fraction (yarrow Sep) presented the lowest one.These results are in concordance with the enrichment in phenolic compounds found in the precipitate fraction as the antioxidant activity is generally attributed to the presence of these compounds.Thus, Tajner-Czopek et al. ( 2020) described high antioxidant activity of dicaffeoylquinic acids (Tajner-Czopek et al., 2020) and reported the antioxidant activity of luteolin and its glucosides (Choi et al., 2014).

Anti-inflammatory activity of the UAE yarrow extract and its SAS fractions
In the first step, the cytotoxicity of the UAE extract and SAS fractions on THP-1/M cells was evaluated using the MTT method.The results indicated that at 30 μg/mL (concentration used to carry out the anti-inflammatory assays), the cell viability was ≥95% for all samples.
As shown in Figure 1, THP-1/M cells activated with LPS (positive control) presented an increase in the secretion of all pro-inflammatory cytokines studied (TNF-α, IL-1β, and IL-6) compared to non-activated controls (negative control).When the activation of THP-1/M was carried out in the presence of 30 μg/mL of the UAE extract and SAS fractions, a decrease in TNF-α secreted level was observed, compared with levels obtained for positive control.However, while the separator fraction inhibited the secretion up to the levels of the negative control, the precipitate fraction only decreased the secretion by 40%.The UAE yarrow extract showed a TFN-α inhibition value of 80%.Regarding IL-1β, the UAE extract and separator fraction presented a decrease in the secretion of this cytokine, although the greatest decrease was achieved in the separator fraction (80% decreased).The results obtained for the IL-6 release in the presence of samples were similar to those obtained for TNF-α, the separator fraction being the most active in terms of reducing the IL-6 release to basal levels.
Although both SAS fractions presented anti-inflammatory activity, the separator fraction was much more active than the precipitate one and the complete UAE yarrow extract.The antiinflammatory activity of yarrow PP may be related to its content in phenolic compounds, more specifically with dicaffeoylquinic acids, luteolin, apigenin, and their glycosides.In this regard, Park et al. (2022) indicated that 3,5-DCQA (the main DCQA in the precipitate fraction) treatment reduced LPS-induced pro-inflammatory cytokine expression in microglial cells (Park et al., 2022).Moreover, Caporali et al. (2022) reviewed the anti-inflammatory activity of luteolin and luteolin 7-O-glucoside (Caporali et al., 2022).They concluded that these components demonstrated an interesting anti-inflammatory action, both in vitro and in vivo, mainly in the interaction with JAK/STAT3 and NF-kB.On the other hand, although yarrow Sep contained a high quantity of methoxylated flavones that have been reported to possess anti-inflammatory activity (Mu et al., 2019), these fractions were mainly enriched in essential oil components, such as borneol, grandisol, and caryophyllene oxide.The antiinflammatory activity of these terpenes and terpenoids is well known and has been summarized by Kim et al. (2020).Thus, the anti-inflammatory activity found in this fraction may be associated with the presence of several components including phenolic compounds-such as methoxylated flavones-and terpenes and terpenoids-such as borneol, grandisol, and caryophyllene oxide.

Yarrow Sep diminished the cell viability of colorectal cancer cells in a higher manner than UAE yarrow
Next, the effect of the extracts on cell viability of colorectal cancer cells was evaluated.With this purpose, DLD1 colorectal cancer cells were treated for 48 h with the extracts at different concentrations.Meanwhile yarrow Sep compromised DLD1 cell viability higher than UAE yarrow, the IC 50 values-concentration that inhibits 50% of cell viability-being 37.6 ± 4.8 and 78.6 ± 4.5 for yarrow Sep and UAE yarrow, respectively; yarrow PP did not compromise the cell's viability at the doses tested (IC 50 > 150 μg/ mL) (Figure 2).
The higher inhibition of the CRC cell viability of yarrow Sep may be related to its higher enrichment in essential oil components, mainly terpenes and terpenoids, compared to the complete UAE yarrow.In this regard, casticin, a methoxylated flavone, and additional essential oil components have been proposed to inhibit the proliferation of several cancer cells, including colorectal cancer cells (Chan et al., 2018;Masyita et al., 2022).
These results are interesting as there is no correlation between the antioxidant and the antiproliferative activities.The antioxidant activity of bioactive compounds is controversial in cancer treatment due to its dual effect, protecting against oxidative stress and DNA damage previous to tumor development but enhancing the redox balance once tumor is established, facilitating cancer cell dissemination.Thus, yarrow Sep inhibited cancer cell proliferation, this activity being independent of its antioxidant activity.

Yarrow Sep inhibited lipid metabolism related targets in CRC cells higher than UAE yarrow
Yarrow Sep, being rich in volatile compounds-such as borneol, caryophyllene oxide, eucalyptol, sabinene, and terpineol, as identified by GC-MS-is also present in a supercritical fluid extract from yarrow (SFE yarrow) with effects on the inhibition of lipid metabolism in vitro and in preclinical models of high-fat diet-induced obesity and pancreatic cancer xenografts (Mouhid et al., 2019a;Mouhid et al., 2019b).Next, the effect of yarrow Sep on key lipid metabolism targets implicated in colorectal cancer progression and dissemination was evaluated.A supercritical extract from rosemary (SFE-RE), previously shown to inhibit lipid metabolism in CRC, was used for comparison purposes (Gomez de Cedron et al., 2019b;Bouzas et al., 2022).
As shown in Figure 3A, yarrow Sep significantly inhibited the expression of main drivers of the de novo lipogenesis and cholesteroigenesis, SREBF1, FASN, ABCA1, and HMGCR.On the contrary, an increase in the expression of ApoA1, previously described to counteract the pro-tumoral effect of ABCA1 in CRC, was found (Aguirre-Portoles et al., 2017).This was also corroborated by the in vitro staining of neutral lipids (Figure 3B), where yarrow Sep significantly reduced the neutral lipid content compared to nontreated cells.
These results were also validated in the metastatic CRC cell line SW620, where only yarrow Sep significantly reduced the expression   S2).These results suggest that volatile bioactive compounds in yarrow Sep seem to be the main drivers of the inhibition of lipid metabolism targets implicated in the inhibition of CRC cell viability.

Yarrow Sep extract inhibited CRC cell bioenergetics higher than UAE yarrow
As yarrow Sep reduced the intracellular lipid content, next it was investigated if this may be translated into a reduction of cell    bioenergetics.Thus, the OCR of CRC cells previously pre-treated with the extracts, UAE yarrow or yarrow Sep, at two different doses, ½ × IC 50 and 1 × IC 50 , for each extract was evaluated.After 48 h of treatment, the same number of cells were plated in the absence of the extracts to eliminate the effects due to the inhibition of cell viability.Then, cells were exposed to a substrate-limited medium with reduced availability of extracellular fatty acids (FAs) (1% FBS) and glucose (0.5 mM glucose).Then, 0.5 mM carnitine was added to favor the use of intracellular FAs for fatty acid oxidation (FAO).
As shown in Figure 4, yarrow Sep inhibited basal respiration rate (BRR) and maximal respiration rates (MRR) and the calculated ATP levels in a dose-dependent manner.Thus, yarrow Sep compromises the oxidative phosphorylation when there is reduced availability of FAs in line with the inhibition of lipid metabolism targets by the extract.These results were also validated in the metastatic CRC cell line SW620, where only yarrow Sep significantly reduced the respiration rates, both in basal and stressed conditions (Supplementary Figure S3).
As far as our knowledge, no previous studies have evaluated the effects of yarrow-derived extracts on the inhibition of CRC cell bioenergetics.

Yarrow Sep extract inhibited the EMT process in a higher manner than UAE yarrow
Finally, due to metastasis being the last step of cancer and the described role of lipid metabolism in the promotion of cancer dissemination (Chaffer and Weinberg, 2011), the effect of UAE yarrow and yarrow Sep on the key targets of EMT was explored.
As shown in Figure 5A, yarrow Sep significantly inhibited the expression of the mesenchymal markers, vimentin and N-cadherin, and upregulated the expression of the epithelial marker E-cadherin compared to non-treated cells.The in vitro analysis of cell adhesion functional assay also showed the effect of yarrow Sep significantly augmenting the cell adhesion capacity of CRC cells compared to control non-treated cells (Figure 5B).These results were also observed in the metastatic CRC cell line SW620, where only yarrow Sep significantly augmented the expression of E-cadherin, although no effects were found on the inhibition of mesenchymal markers.(Supplementary Figure S4).
These results suggest that yarrow Sep inhibits the EMT process at least partially mediated by the effects on the inhibition of key lipid metabolism targets implicated in cancer dissemination.

Highlights and conclusion
SAS precipitation was successfully applied to fractionate UAE yarrow into two extracts with different polarities: the yarrow precipitate (yarrow PP), rich in polar phenolic bioactive compounds, and yarrow separator (yarrow Sep), rich in essential oil components.
Most of the UAE yarrow phenolic compounds were recovered in the precipitate fraction yarrow PP, highlighting 3,5-DCQA.The yarrow Sep fraction was enriched in essential oil components, such as borneol, grandisol, and caryophyllene oxide.
Yarrow PP exerted the highest antioxidant activity, even higher than the complete UAE yarrow; meanwhile, yarrow Sep showed the highest anti-inflammatory activity, even higher than the complete UAE yarrow.
Yarrow Sep inhibited key lipid metabolic targets shown to be implicated in CRC dissemination and poorer prognosis.More specifically, yarrow Sep significantly inhibited the expression of the main drivers of the de novo lipogenesis and cholesterogenesis-SREBF1, FASN, ABCA1, and HMGCR-in line with the reduction of the neutral lipid content quantified in yarrow Sep-treated cells compared to non-treated cells.
These results suggest that volatile bioactive compounds present in the yarrow Sep fraction may be responsible for the inhibition of lipid metabolism in CRC.
In summary, SAS technology was applied to provide a novel combination of bioactive compounds, yarrow Sep, which merits further investigation to be proposed as a potential complementary nutraceutical in the treatment of CRC.

FIGURE 1
FIGURE 1Levels of TNF-α, IL-1β, and IL-6 secreted by THP-1/M activated with LPS in the presence of extract and SAS fractions.a-d Different letters indicate statistical differences between samples for each interleukin.Significance level at p < 0.05 according to Fisher's least significant difference (LSD) test.

FIGURE 2
FIGURE 2 Effect of the yarrow extracts on cell viability (MTT assay) of DLD1 colorectal cancer cells.Dose-response curves of the cell proliferation assay after 48 h of treatment with increasing concentrations of UAE yarrow and yarrow Sep extracts.Data represent the mean ± SEM of three independent experiments, each performed in triplicate.
FIGURE 3 (A) Effect of UAE yarrow and yarrow Sep extracts in the expression of lipid metabolic targets in the DLD1 CRC cells.(B) Red Oil O staining quantification after treatment with the extracts for 48 h.Asterisks indicate significant statistic differences *p-value <0.05; **p-value <0.01 (three to four replicates, three independent experiments).

FIGURE 4
FIGURE 4 Effect of yarrow UAE and yarrow Sep in the mitochondrial oxidative phosphorylation of CRC cancer cells.Mitochondrial respiration quantification by flux analysis of the oxygen consumption rate (OCR) of DLD1 cells previously pre-treated with yarrow UAE and yarrow Sep extracts for 48 h.The basal respiration rate, spare respiratory capacity, ATP production, and proton leak of 8,000 cells per condition are compared.Data represent the mean ± SEM of four to six replicates.Asterisks indicate statistically significant differences *p < 0.05; **p < 0.01; ***p < 0.005; and ****p < 0.001 relative to the control non-treated cells.
FIGURE 5 (A) Effect of yarrow UAE and yarrow Sep extracts in the expression of EMT targets in the DLD1 CRC cells.(B) In vitro functional cell-based assay for the cell adhesion capacity of colorectal cancer cells.Asterisks indicate statistically significant differences *p < 0.05; **p < 0.01; ***p < 0.005; and ****p < 0.001 relative to the control non-treated cells (three replicates, three independent experiments).

TABLE 2
Essential oil compounds identified by GC-MS.RI and peak area contribution (%) of the original UAE extract and the separator fraction.

TABLE 3
of the master regulator transcription factor SREBF1 gene expression (p < 0.045) (Supplementary Figure