key: cord-0801001-dp7n26qv authors: Poletto, Patrícia; Alvarez-Rivera, Gerardo; Torres, Talyta M.S.; Mendiola, Jose A.; Ibañez, Elena; Cifuentes, Alejandro title: Compressed fluids and phytochemical profiling tools to obtain and characterize antiviral and anti-inflammatory compounds from natural sources date: 2020-06-07 journal: Trends Analyt Chem DOI: 10.1016/j.trac.2020.115942 sha: c79afd37ccadf49c714aeb503d92f44ac159d895 doc_id: 801001 cord_uid: dp7n26qv Many natural compounds, found mainly in plants, are associated with the treatment of various diseases. The search for natural therapeutic agents includes compounds with antiviral and anti-inflammatory activities. Among the many steps involved in bioprospection, extraction is the first and most critical step for obtaining bioactive compounds. One of the main advantages of using compressed fluids extraction is the high quality of the final product obtained due to the use of green solvents, while the selectivity towards target compounds can be tuned by adjusting the process parameters, especially pressure, temperature and solvent characteristics. In this review, a discussion is provided on the power of compressed fluids, such as supercritical fluid extraction (SFE), pressurized liquid extraction (PLE) and subcritical water extraction (SWE) to obtain antiviral and anti-inflammatory compounds from natural sources. In addition, an adequate knowledge about the identity and quantity of the compounds present in the extract is essential to correlate biological activity with chemical composition. Phytochemical profiling tools used for identification and quantification of these bioactive natural compound are also discussed. It can be anticipated that after the current SARS-COV-2 pandemic, the search of new natural compounds with antiviral and anti-inflammatory activity will be a hot research topic, so, this review provides an overview on the technologies currently used that could help this research. In nature, there is a wide variety of plant-derived compounds with different biological 44 activities, including antiviral and anti-inflammatory. Bioprospection can be defined as "the 45 methodical search for novel pharmaceutical (and other) products from living beings". The 46 search for natural compounds against viral infections is a very hot topic due to the large number 47 of related diseases and the ease of viral spread [1] [2] [3] . This problem is even more evident now 48 due to the SARS-CoV-2 pandemic. On the other hand, the search for natural anti-inflammatory 49 compounds is also an important issue, since inflammation is suggested to play a crucial role in 50 many diseases, including viral infections [4, 5] ; in fact, the infection mediated by SARS-CoV-2 51 has strongly been associated with several inflammatory processes. 52 Polyphenols, terpenes, fatty acids and polysaccharides are some types of natural 53 compounds that have been tested as antiviral and anti-inflammatory agents. These compounds 54 are usually present in spices, flowers and leaves of many plants and herbal species used in 55 traditional medicine in many countries. In addition, interesting compounds can be obtained from 56 food, agricultural by-products and marine sources (e.g., seaweeds and microalgae) [6] . The 57 evaluation of natural compounds as therapeutic agents is not intended to replace synthetic drugs, 58 but to alleviate their use and attenuate the toxic side-effects and to help to develop new 59 candidates of future pharmaceuticals [1, 7, 8] . However, extraction, identification and in vitro or 60 in vivo confirmation of their activity are challenges that need to be overcome for the 61 development and use of these natural bioactive compounds. 62 The first question raised is the technology needed to obtain the compounds, standardize 63 the product and make the process scalable. In this sense, it is important to consider the actual 64 need for using sustainable techniques and solvents, able to comply with the Green Chemistry and 65 Green Engineering principles [9] . Among the different techniques that can be employed, those 66 based on compressed fluids fulfill these requirements and have already been used to extract 67 target bioactive compounds from different biomasses [6, 10] . Techniques such as supercritical 68 fluid extraction (SFE), pressurized liquid extraction (PLE) and subcritical water extraction 69 (SWE), stand out from conventional techniques due to the use of non-toxic solvents (or 70 minimizing its amount), short extraction times and highly tunable selectivity, improving the 71 quality of extracts and valuing applications as therapeutic agents. 72 Once the natural extract is obtained using the mentioned green extraction processes and 73 its bioactivity confirmed using in vitro or in vivo assays, an in depth study of its chemical 74 composition is mandatory and frequently it faces the challenge of identifying and elucidating the 75 structure of compounds many times new and unknown. For this reason, the analytical 76 identification and quantification of compounds are important tasks that complement the 77 extraction step. Chromatographic techniques coupled to (tandem) high resolution mass 78 spectrometry are widely employed due to its capacity to identify the structure, composition and 79 concentration of compounds in a very fast and sensitive way [11] . Interestingly, once the 80 phytochemical profile of the extracts is obtained, the analytical tool selected can assist in 81 improving the extraction focusing on the target bioactive compound(s). 82 In this review, we discuss the applicability and highlight the power of compressed fluids 83 as an alternative method to classical extraction methods to obtain natural compounds against 84 viral and inflammatory diseases. Besides, the main analytical techniques used for their adequate 85 phytochemical profiling are also discussed (see Figure 1 ). 86 through an in silico study, reported 13 natural compounds, found in 26 Chinese herbs commonly 93 used to treat viral respiratory infections, as potentially active to treat Covid-19; these compounds 94 have been confirmed to directly inhibit important proteins in SARS (Severe Acute Respiratory 95 Syndrome) and MERS (Middle East Respiratory Syndrome) and, considering the genetic 96 similarities between SARS and MERS coronavirus and the new SARS-CoV-2, it is expected that 97 they could be also effective against the new coronavirus. This type of studies opens a horizon for 98 technologies capable of selectively extracting these compounds, such as compressed fluids 99 techniques. It is expected that, in addition to the domestic use of medicinal herbs, obtaining the 100 compounds through a standardized process will facilitate the development of vaccines, adjuvants 101 and/or drugs. In this section we present the studies on the antiviral activity of compounds 102 obtained by compressed fluids, found in literature. An overview of the natural matrices used as 103 sources of compounds, the class of compounds obtained, the extraction technique and conditions 104 applied, as well as the analytical technique used for the phytochemical characterization is 105 provided in Table 1 . All these studies employed in vitro approaches to confirm the antiviral 106 bioactivity. The antiviral biological mechanisms of these compounds are out of the scope of this 107 work. 108 Herpes simplex virus (HSV) affects adults and children, there is no cure or vaccine and, 109 currently, there is an urgent need to develop new treatments due to the appearance of strains 110 resistant to acyclovir, the drug most frequently used to reduce the infection time and pain 111 produced by HSV [2] . According to data in Table 1 Polysaccharides are another class of natural compounds, which have been highlighted for 119 their effective antiviral activity against various viruses. In Table 1 , we can see that algae and 120 microalgae are the most common sources of antiviral polysaccharides [17] [18] [19] , and HSV has 121 been the target virus in these studies. However, a common conclusion in all these studies was 122 that the antiviral activity was higher when the host cells were pretreated with polysaccharides 123 before the virus infection. In this sense, including sub-and supercritical extracts in normal diets 124 may allow to boost the immune response. Therefore, the effects of future viral infections may be 125 lower, reducing the spread of the outbreak by considering these polysaccharides pretreatment. 126 One of the main diseases triggered by a virus is acute respiratory infection, especially 127 influenza viruses. Langeder et al. [1] reported that flavonoids represent the most important class Terpenes, fatty acids and phenolics represent the classes of compounds extracted by 141 compressed fluids already tested for anti-inflammatory disorders. In general, the anti-142 inflammatory activity of these compounds is related to the inhibition of pro-inflammatory 143 pathways associated to many diseases. The overview of anti-inflammatory compounds extracted 144 by SFE is provided in Table 2 . The compounds extracted by PLE and SWE with anti-145 inflammatory activity are presented in Table 3 . The data provide information on the natural 146 source and the class of compounds obtained responsible of the anti-inflammatory activity. The 147 anti-inflammatory biological mechanisms of these compounds are out of the scope of this work. 148 Terpenes are the compounds most frequently studied in extracts from compressed fluids 149 with anti-inflammatory activity. These compounds are widely distributed in many plants, mainly 150 in aerial parts (leaves and stems) and underground parts (roots). The anti-inflammatory potential 151 of terpenes were described by Hortelano et al. [23] , revealing that these compounds can be 152 promising natural therapeutic agents. As can be seen in Table 2 such as Z-ligustilide (phtalide) and 6-gingerol (polyphenol) extracted from a mixture of ginseng 169 and ginger (Angelica sinensis and Zingiber officinal) also relieved intestinal inflammation in rat 170 models [31] . Finally, the in vitro gastrointestinal release of γ-oryzanol, a terpene obtained from 171 rice bran, exhibited a potent anti-inflammatory activity, which helped to prevent colorectal 172 cancer [32] . 173 Among the phenolic compounds, those belonging to the groups of catechol derivatives, 174 coumarins, and flavonoids were considered the best anti-inflammatory compounds, based on 175 their structure, molecular weight, polarity, among other physicochemical properties [7] . suggested that these compounds could be used as a potential vaccine adjuvant. 192 Collectively, these findings corroborate the antiviral and anti-inflammatory potential of 193 bioactive compounds obtained from different natural sources extracted using compressed fluids ' 194 techniques. In the following section, we will discuss the applicability of compressed fluids in the 195 extraction of the above mentioned compounds. 196 197 Green extraction is a term derived from Green Chemistry, whose 12 principles were 200 suggested by Anastas and Warner [9] . In summary, the principles set out the ways in which 201 chemical products and processes can be more sustainable. In this context, Chemat et al. that allows extracting at low temperatures. Other benefits associated to the use of CO 2 are its low 213 cost, non-flammability and GRAS (Generally Recognized As Safe) status. Moreover, after 214 extraction CO 2 is released as gas (which can be recycled) and therefore, no solvent residues are 215 found in the extract or in the unextracted matrix that can be further used or processed. Co-216 solvents can be used to assist in the extraction of compounds with low affinity for CO 2 217 (compounds of medium and high polarity). If the amount of co-solvent is high enough to form 218 two phases or one phase above the bubble point curve, but below the critical composition, the 219 extracting solvent is called gas-expanded liquid (GXL) and mainly uses carbon dioxide to 220 expand the liquid [41] . In fact, by changing only the amount of CO 2 in the solvent, the same 221 system can highly modify its physicochemical properties (such as dielectric constant, transport 222 properties, hydrogen-bonding ability, miscibility and acidity), behaving as switchable solvents at 223 medium-high pressure, as shown in Figure 2 . For further reading on this topic, see Herrero et al. 224 [41] . 225 SFE has some advantages compared to conventional techniques. One of the most 226 interesting is related to the tunability of the solvent. Above the critical conditions of temperature 227 and pressure, the change of the binomial T and P can generate a "new solvent" with different 228 values of solubility, diffusivity and viscosity, modifying its extraction power [42] . PLE (also 229 called Accelerated Solvent Extraction, ASE ® , or Pressurized Solvent Extraction) and SWE 230 operate under subcritical conditions, at temperatures above the atmospheric boiling point of the 231 solvent, usually ethanol and/or water, and below its critical temperature (and applying a pressure 232 high enough to maintain the solvent in liquid state). In general, non-polar compounds, such as 233 terpenes, fatty acids and sterols are extracted by SFE due to the solvent characteristics, as can be 234 seen in Table 2 , and also described in Figure 3 ; while polar or semi-polar compounds are 235 preferentially extracted by GXL, PLE and SWE (Table 3) . PLE and SWE processes, generally 236 use higher temperatures, unlikely SFE and GXL. The binomial T and P allows the solvent to 237 remain in a liquid state while varying its physicochemical characteristics (lower viscosity, higher 238 transport properties) yielding a more powerful extraction. High temperatures combined with high 239 pressures may modify the dielectric constant of the solvent and, therefore, the selectivity of the Regarding the applicability of compressed fluids for extraction of antiviral and anti-246 inflammatory compounds, the major inputs come from SFE, being terpenes the main target 247 family. According to Table 1 , the pressures and temperatures used to extract antiviral 248 compounds varied between 20-30 MPa and 40-50 °C, respectively. Table 2 shows that 249 depending on the matrix, anti-inflammatory terpenes were recovered at SFE conditions ranging 250 from 9 to 40 MPa and temperatures from 30 to 65 °C, with different CO 2 ethanolic extract presented more efficient inhibition than hexane and water extracts against HSV. 279 The major compounds were fucosterol and palmitic acid, whose presence was higher in the most resulted in a highly anti-inflammatory extract, whose power was increased by the use of alum as 305 Compounds like carotenoids, phenolics and flavonoids were extracted from different 307 natural sources using PLE and SWE. For instance, a mixture of water and ethanol (50:50) at 308 80ºC was efficient to recover phenolic compounds from spinach leaves with good anti-309 inflammatory activity. However, low amounts of carotenoids were found in this extract 310 compared to SFE, presenting both extracts an important anti-inflammatory activity [36] . SWE extract showed higher antiinflammatory response compared to hot water and ethanol extractions (10 mg/mL) [48] Natural 476 products against acute respiratory infections: Strategies and lessons learned Natural 479 products-derived chemicals: Breaking barriers to novel anti-HSV drug development, 480 Viruses Plant Derived Antivirals: A 482 Potential Source of Drug Development 485 Inflammatory responses and inflammation-associated diseases in organs Angelica sinensis and Zingiber officinale roscoe ameliorates TNBS-induced colitis in rats Development of colorectal-targeted dietary supplement 586 tablets containing natural purple rice bran oil as a colorectal chemopreventive Phenolic 589 compounds: Natural alternative in inflammation treatment Assessment of the bioactive capacity of extracts 592 from Leptocarpha rivularis stalks using ethanol-modified supercritical CO2 Antioxidant and anti-inflammatory activities of Lonicera 596 japonica Thunb. var. sempervillosa Hayata flower bud extracts prepared by water, ethanol 597 and supercritical fluid extraction techniques Extraction of functional ingredients from spinach (Spinacia oleracea L.) using 601 liquid solvent and supercritical CO2 extraction Winemaking by-products as anti-604 inflammatory food ingredients Morus alba and active compound oxyresveratrol exert 608 anti-inflammatory activity via inhibition of leukocyte migration involving MEK/ERK 609 signaling Augmentation of humoral and cellular 613 immunity in response to Tetanus and Diphtheria toxoids by supercritical carbon dioxide 614 extracts of Hippophae rhamnoides L. leaves Green extraction of natural products: Concept and 617 principles Gas expanded liquids and switchable solvents Experimental design of supercritical fluid extraction -A review Compressed fluids (SFE, PLE 625 and SWE) for the extraction of bioactive compounds Supercritical anti-solvent fractionation for improving antioxidant and anti-629 inflammatory activities of an Achillea millefolium L. extract Anti-infammatory, 632 antinociceptive activity of an essential oil recipe consisting of the supercritical fluid CO2 633 extract of white pepper, long pepper, cinnamon, saffron and myrrh in vivo Effect of different non-conventional extraction methods on the 637 antibacterial and antiviral activity of fucoidans extracted from Nizamuddinia zanardinii Utilization of plant-based agricultural waste by subcritical water 641 treatment Antioxidative and anti-inflammatory activities of Citrus unshiu peel extracts using a 644 combined process of subcritical water extraction and acid hydrolysis Supercritical fluid extraction of heather (Calluna vulgaris) and evaluation of anti-648 hepatitis C virus activity of the extracts Supercritical CO2 extraction of Aloysia gratissima leaves and evaluation of anti-653 inflammatory activity Inhibitory Effect of Supercritical Extracts from Arctium lappa L. on the Lectin Pathway of 657 the Complement System Protective Effect of SFE-CO2 of Ligusticum chuanxiong Hort 661 Against d-Galactose-Induced Injury in the Mouse Liver and Kidney Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary 665 extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: 666 The influence of vehicle type Supercritical fluid 669 extraction as an alternative process to obtain essential oils with anti-inflammatory 670 properties from marjoram and sweet basil Supercritical sage extracts as anti-inflammatory food ingredients Supercritical CO2 extract and essential oil of aerial part of Ledum palustre L. 677 -Chemical composition and anti-inflammatory activity Activity and Chemical Composition of Essential Oil Extracted with Supercritical CO2 681 from the Brown Seaweed Undaria pinnatifida Extraction of the volatile oil from Carum carvi of Tunisia and Lithuania by 685 supercritical carbon dioxide: Chemical composition and antiulcerogenic activity Green extraction of oil from Carum carvi seeds 689 using bio-based solvent and supercritical fluid: Evaluation of its antioxidant and anti-690 inflammatory activities Reinforcement of barrier function and 693 scalp homeostasis by Senkyunolide A to fight against dandruff Comparison of the anti-696 inflammatory activities of supercritical carbon dioxide versus ethanol extracts from leaves 697 of Perilla frutescens Britt. Radiation mutant Anti-inflammatory activities of the products of supercritical fluid extraction from 701 Litsea japonica fruit in RAW 264.7 cells Supercritical extract of Seabuckthorn 704 Leaves (SCE200ET) inhibited endotoxemia by reducing inflammatory cytokines and 705 nitric oxide synthase 2 expression A supercritical CO 2 extract from seabuckthorn leaves inhibits pro-inflammatory 709 mediators via inhibition of mitogen activated protein kinase p38 and transcription factor 710 nuclear factor-κB Chemical 714 composition, antioxidant activity, neuroprotective and anti-inflammatory effects of cipó-715 pucá (Cissus sicyoides L.) extracts obtained from supercritical extraction Standardized supercritical Co2 extract of 718 acanthus ilicifolius (Linn.) leaves inhibits the pro-inflammatory cytokine tumor necrosis 719 factor-Α in lipopolysaccharide-activated murine raw 264.7 macrophage cells Anti-inflammatory and antitumour activity of 722 various extracts and compounds from the fruits of Piper longum L Anti-inflammatory effect of supercritical-carbon dioxide fluid extract from flowers 726 and buds of Chrysanthemum indicum Linnén, Evidence-Based Complement Green processes and sustainability: An overview on the extraction 729 of high added-value products from seaweeds and microalgae A supercritical CO 2 extract of neem leaf (A. indica) and its bioactive 733 liminoid, nimbolide, suppresses colon cancer in preclinical models by modulating pro-734 inflammatory pathways The Highly Pure Neem Leaf Extract, SCNE, Inhibits 738 Tumorigenesis in Oral Squamous Cell Carcinoma via Disruption of Probing the therapeutical potential of 742 conventional and supercritical fluid extract of Zingiber officinale to mitigate ulcer, 743 inflammation, hepatotoxicity and nephron toxicity Enhanced extraction of oleoresin from Piper nigrum by 746 supercritical carbon dioxide using ethanol as a co-solvent and its bioactivity profile A valepotriate-enriched fraction from Valeriana glechomifolia 750 Meyer inhibits leukocytes migration and nociception in formalin test in rodents, Brazilian 751 Pinus densiflora needle supercritical fluid 753 extract suppresses the expression of pro-inflammatory mediators iNOS, IL-6 and IL-1β, 754 and activation of inflammatory STAT1 and STAT3 signaling proteins in bacterial 755 lipopolysaccharide-challenged murine macrophag, DARU Anti-inflammatory effect of supercritical extract 759 and its constituents from Ishige Okamurae Evaluation of wound healing 762 activity of Thunbergia laurifolia supercritical carbon dioxide extract in rats with second-763 degree burn wounds Copaíba (Copaifera sp.) leaf extracts obtained by 767 CO2 supercritical fluid extraction: Isotherms of global yield, kinetics data, antioxidant 768 activity and neuroprotective effects Experimental 771 study on anti-inflammatory activity of a TCM recipe consisting of the supercritical fluid 772 CO 2 extract of Chrysanthemum indicum, Patchouli Oil and Zedoary Turmeric Oil in 773 vivo Hydrothermal processing of β-775 glucan from Aureobasidium pullulans produces a low molecular weight reagent that 776 regulates inflammatory responses induced by TLR ligands The authors are grateful to CAPES-PRINT, project number 88887.310560/2018-00 471 (Brazil) and Project AGL2017-89417-R (MINECO, Spain). 472The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: