Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.


Introduction
Natural medicines, extracted from herbs and other living sources such as serpent venoms, have been used by humans since the earliest times. In contrast, the use of mineral substances as medicines was an innovation of Paracelsus introduced as recently as the These can be grouped in subfamilies based on evolutionary relationships (different colors of α subunits), ligand specificity and, in the case of β2 and β7 integrins, restricted expression on white blood cells. Integrins can be grouped into two larger classes that bind to cell surface cell adhesion molecules (CAMs) and ECM ligands. They can be further classified as collagen-binding integrins (α1β1, α2β1, α10β1, and α11β1), RGD-recognizing integrins (α5β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3), laminin-binding integrins (α3β1, α6β1, α7β1, and α6β4), and leukocyte integrins (αLβ2, αMβ2, αXβ2, and αDβ2). The β2 integrin subunit (CD18) can pair with one of the four α subunits (αL-CD11a, αM-CD11b, αX-CD11c, and αD-CD11d) [16]. The α4β1 and α9β1 integrins recognize fibronectin and VCAM-1. The β2 and β7 integrins are restrictedly expressed by leukocytes). Asterisks show the alternatively spliced cytoplasmic domains. This figure is based on the study of Hynes [10] and Yue et al. [17]. (RGD, Arg-Gly-Asp sequence; VCAM-1, a vascular cellular adhesion molecule-11).

Prestimulation with Cytokines
To demonstrate the inhibitory effect of a natural compound on cellular adhesion, cells, typically from the human umbilical vein endothelial cell line (HUVEC), are usually first treated with certain cytokines to stimulate the expression of CAM ( Figure 2). In vivo, lipopolysaccharide (LPS, from Gram-negative bacteria) stimulates the immune response by interacting with its leukocyte membrane receptor, the Pattern Recognition Receptor (PRR) CD14 (with TLR4-MD2), to induce the generation of cytokines such as tumour necrosis factor α (TNF-α), interleukin-1 and -6 (IL-1, IL-6) ( Figure 3). TNF-α is also involved in systemic inflammation [2,18]. It is primarily produced by activated monocytes or macrophages [19]. Note, cytokine generation, increase of expression of cytokines can also be stimulated by certain plant extracts (for example, among others, garlic (Allium sativum) decreases the level of IL-1α, IL-2, IL-6, IL-12, TNF-α and IFN-γ, however, increases IL-10), as summarized by Spelman, et al. [20]. After stimulation of the endothelial cells, the plant (or venom) extract is then added to them. The natural compound may downregulate expression of the adhesion molecules, resulting in the diminution of cell adhesion, and the compound has an antiinflammatory effect in consequence. The ICAMs and VCAMs are the most researched adhesion molecules [5]. We elaborate on this in the next section.
Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 54 increases IL-10), as summarized by Spelman, et al. [20]. After stimulation of the endothelial cells, the plant (or venom) extract is then added to them. The natural compound may downregulate expression of the adhesion molecules, resulting in the diminution of cell adhesion, and the compound has an antiinflammatory effect in consequence. The ICAMs and VCAMs are the most researched adhesion molecules [5]. We elaborate on this in the next section.

Inhibition of CAMs by Suppression of Their Expression (Downregulation)
The phytochemicals of olive oil (from Olea europaea) and red wine (from Vitis vinifera), oleuropein (monoterpene, seco-iridoid glucoside), hydroxytyrosol (phenylethanoid), tyrosol (phenylethanoid), elenolic acid (monoterpene, seco-iridoid) and resveratrol (stilbene) at nutritionally relevant concentrations have been shown to inhibit endothelial adhesion molecule expression ( Figure 2). This provides a strongly suggestive basis for the atheroprotective property of the so-called "Mediterranean diet" [21]. increases IL-10), as summarized by Spelman, et al. [20]. After stimulation of the endothelial cells, the plant (or venom) extract is then added to them. The natural compound may downregulate expression of the adhesion molecules, resulting in the diminution of cell adhesion, and the compound has an antiinflammatory effect in consequence. The ICAMs and VCAMs are the most researched adhesion molecules [5]. We elaborate on this in the next section.
Walnut (Juglans regia) extract and its principal active component ellagic acid decreased the stimulated endothelial expression of ICAM-1 and VCAM-1, indicating a mechanism for the known antiatherogenic and osteoblastic activity of the substance [18]. A walnutenriched diet may therefore indeed be cardioprotective and inhibit osteoporosis [18].
Curcumin (diphenylheptanoid) from the Curcuma longa rhizome also downregulated the expression of adhesion molecules and, hence, monocyte adhesion [13]. Saponin (triterpene) astagaloside IV from Mongolian milkvetch (Astagalus membranaceus) decreased the LPS-induced expression of VCAM-1 and E-selectin on the surface of HUVEC, hence this Chinese traditional medicinal herb is predicted to have anti-inflammatory efficacy; however, ICAM-1 was not affected [22].
Tripertygium wilfordii is a vine-like plant that grows in south China, and in the Chinese pharmacopoeia the extract from its root is prescribed for treating long-term rheumatoid arthritis and systemic lupus erytnematosus [7,8]. Chang et al., applied IL-1α to stimulate HUVEC cells; treatment with a high concentration (50 ng/mL) of the herb extract (containing wilforonide, alkaloids, diterpenes, triterpenes, b-sitosterol, daucosterol, dulcitol and glycosides [7,23]) had a significant inhibitory effect on both the expression and secretion of the cellular adhesion molecules, and thus may be a potential therapeutic agent for the treatment of inflammatory diseases [7].

Mechanism of Downregulation
The downregulation of CAMs by natural products is achieved by inhibiting their gene expression.
The molecular details involve the uptake of the natural product by the cytoplasm followed by interaction between the compound and transcription factors for adhesion molecule genes. Activation of the transcription factor nuclear factor κB (NF-κB), is mediated by the proinflammatory cytokines mentioned above, for instance TNF-α, and triggers gene expression of adhesion molecules ( Figure 4). NF-κB binding sites are found in the promoter region of E-selectin, ICAM-1 and VCAM-1 [13,[24][25][26][27]. NF-κB is in the cytoplasm in inactive form, complexed to IκB "nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor" [13]. When cells are stimulated with TNF-α, the IκB is phosphorylated, ubiquitinated, and degraded. The thereby activated NF-κB translocates to the nucleus and transcriptionally up-regulates cytokine receptors as well as the adhesion molecules [13,26,28,29].
Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 54 Walnut (Juglans regia) extract and its principal active component ellagic acid decreased the stimulated endothelial expression of ICAM-1 and VCAM-1, indicating a mechanism for the known antiatherogenic and osteoblastic activity of the substance [18]. A walnut-enriched diet may therefore indeed be cardioprotective and inhibit osteoporosis [18].
Curcumin (diphenylheptanoid) from the Curcuma longa rhizome also downregulated the expression of adhesion molecules and, hence, monocyte adhesion [13]. Saponin (triterpene) astagaloside IV from Mongolian milkvetch (Astagalus membranaceus) decreased the LPS-induced expression of VCAM-1 and E-selectin on the surface of HUVEC, hence this Chinese traditional medicinal herb is predicted to have antiinflammatory efficacy; however, ICAM-1 was not affected [22].
Tripertygium wilfordii is a vine-like plant that grows in south China, and in the Chinese pharmacopoeia the extract from its root is prescribed for treating long-term rheumatoid arthritis and systemic lupus erytnematosus [7,8]. Chang et al., applied IL-1α to stimulate HUVEC cells; treatment with a high concentration (50 ng/mL) of the herb extract (containing wilforonide, alkaloids, diterpenes, triterpenes, b-sitosterol, daucosterol, dulcitol and glycosides [7,23]) had a significant inhibitory effect on both the expression and secretion of the cellular adhesion molecules, and thus may be a potential therapeutic agent for the treatment of inflammatory diseases [7].

Mechanism of Downregulation
The downregulation of CAMs by natural products is achieved by inhibiting their gene expression.
The molecular details involve the uptake of the natural product by the cytoplasm followed by interaction between the compound and transcription factors for adhesion molecule genes. Activation of the transcription factor nuclear factor κB (NF-κB), is mediated by the proinflammatory cytokines mentioned above, for instance TNF-α, and triggers gene expression of adhesion molecules ( Figure 4). NF-κB binding sites are found in the promoter region of E-selectin, ICAM-1 and VCAM-1 [13,[24][25][26][27]. NF-κB is in the cytoplasm in inactive form, complexed to IκB "nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor" [13]. When cells are stimulated with TNF-α, the IκB is phosphorylated, ubiquitinated, and degraded. The thereby activated NF-κB translocates to the nucleus and transcriptionally up-regulates cytokine receptors as well as the adhesion molecules [13,26,28,29].  The expression and function of integrins on various immune cells are summarized in Table 1. Table 1. Ligands and functions of different integrins of human leukocytes (RGD, Arg-Gly-Asp sequence; VCAM-1, vascular cellular adhesion molecule-1; ICAM-1, Intercellular adhesion molecule-1) [10,[30][31][32][33]. various inflammatory diseases. Both compounds inhibited the TNF-α-induced expression of ICAM-1 [46].
Ganoderma lucidum, a medicinal mushroom, has been used in traditional Chinese medicine to prevent and treat various diseases, for example cancer [47]. A polysaccharide derived from the fungus interacted with cell surface proteins and β1-integrin expression was diminished, while β-actin expression was not affected [47].
We summarize the different modes of pathway intervention involving gene expression in Table 2. Table 2. Different modes of pathway intervention involving gene expression by natural compounds.

P1
Inhibiting the dephosphorylation of I κB and, hence, the activation of NF-κB P2 Inhibiting translocation of activated NF-κB into the nucleus P3 Inhibition of binding of activated NF-κB to promoter sites for CAM expression

Intervention at the ECM
Flavonoid baicalein, derived from the root of Scutelaria baicalensis, a widely used Chinese herbal medicine that has been used in anti-cancer and anti-inflammatory therapy [48], has an inhibitory effect on the expression of matrix metalloproteinases (MMPs) [48], which are involved in the degradation of the extracellular matrix. Destruction of basement membranes and stromal extracellular matrix is critical for favoring metastasis and invasion of malignant cells [48]. The MMPs have therefore a role in promoting tumour growth, invasion and metastasis. Treatment of human breast carcinoma (MDA-MB-231) cells with baicalein inhibited the expression of MMP-2/9, which is a result of the mitogen-activated protein kinase (MAPK) signaling pathway [48] (Figure 5). adhesion molecule 1 (ICAM-1) expression and inhibiting endothelial lipase (EL) expression in TNF-α-treated HUVECs and macrophages by blocking the NF-kB pathway [45].
Ganoderma lucidum, a medicinal mushroom, has been used in traditional Chinese medicine to prevent and treat various diseases, for example cancer [47]. A polysaccharide derived from the fungus interacted with cell surface proteins and β1-integrin expression was diminished, while β-actin expression was not affected [47].
We summarize the different modes of pathway intervention involving gene expression in Table 2.

Mode
Action P1 Inhibiting the dephosphorylation of I κB and, hence, the activation of NF-κB P2 Inhibiting translocation of activated NF-κB into the nucleus P3 Inhibition of binding of activated NF-κB to promoter sites for CAM expression

Intervention at the ECM
Flavonoid baicalein, derived from the root of Scutelaria baicalensis, a widely used Chinese herbal medicine that has been used in anti-cancer and anti-inflammatory therapy [48], has an inhibitory effect on the expression of matrix metalloproteinases (MMPs) [48], which are involved in the degradation of the extracellular matrix. Destruction of basement membranes and stromal extracellular matrix is critical for favoring metastasis and invasion of malignant cells [48]. The MMPs have therefore a role in promoting tumour growth, invasion and metastasis. Treatment of human breast carcinoma (MDA-MB-231) cells with baicalein inhibited the expression of MMP-2/9, which is a result of the mitogenactivated protein kinase (MAPK) signaling pathway [48] (Figure 5). Flavonoid (chalcone) butein (3,3,2 ,4 -tetrahydroxychalcone) is an active substance found in several plants, such as Semecarpus anacardium, Dalbergia odorifera, Caragana jubata and Rhus verniciflua [49]. It has been demonstrated that it decreased leukocyte adhesion to A549 cells through the inhibition of TNF-α-induced ICAM-1 and VCAM-1 expression by inhibiting the NF-κB/MAPK/Akt signaling pathway. Butein also inhibits ROS generation [49], and may prevent TNF-α-induced airway inflammation [49] (Figure 4).

Inhibition of CAM Binding by Blocking Specific Cell-Surface Receptor Sites
We can distinguish three types of mechanisms inhibiting cell adhesion by blocking (B) specific receptor sites (Table 3). Table 3. Three types of mechanisms inhibiting cell adhesion by blocking specific receptor sites.

B1
Blocking adhesion receptors on the surface of mobile cells (e.g., leukocytes)

B2
Blocking adhesion receptors on the surface of tissue cells (e.g., of the endothelium)

B3
Blocking adhesion motifs in the extracellular matrix (ECM) In contrast to intervention at the level of protein expression, natural compounds can also specifically block cell recognition motifs, such as the amino acid triplet RGD. For example, the polyphenol EGCG from green tea has been shown to block RGD motifs (B) (Arg-Gly-Asp) and, hence, inhibit adhesion [50].
We recall that integrins are transmembrane heterodimers, a family of plasma membrane receptors that mediate adhesion of leukocytes to ECM [51]. Integrins are also involved in pathophysiological processes as well, such as genetic and autoimmune diseases, metastasis and thrombosis and, thus, integrins are important therapeutic target structures [51,52]. Blocking or disruption of binding to integrin receptors is therefore an important topic in industrial drug discovery [51,53]. Serpent venom disintegrins, a family of low molecular weight proteins, typically contain the RGD motif, and are known to block integrin activities by binding with high affinity to the integrins [51,54]. For example, rhodostomin, a snake venom from Calloselasma rhodostoma, blocked the integrin ανβ3 and also affected pp125 FAK phosphorylation and the actin cytoskeleton [55]. Rhodostomin contains an RGD motif that specifically inhibits the integrin-binding function [56]. It can be produced in Pichia pastoris (methylotrophic yeast) as well and it inhibits platelet aggregation with a K(I) of 78 nM as potent as native rhodostomin [56]. However, its D51E mutant blocks platelet aggregation with a K(I) of 49 mM [56]. Structural analysis of rhodostomin and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets [56]. Two minor differences between them were inferred from their backbone dynamics and 3D structures [56]. The docking of rhodostomin into integrin αIIbβ3 showed that between the backbone amide and carbonyl groups of the D51 residue were formed hydrogen bonds with the integrin residues R216 and R214, respectively [56]. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex [56].
Not only snake venom affects integrin-mediated adhesion, but herbs as well. Epigallocatechin-gallate (EGCG) (flavonoid ester) is the main polyphenol of green tea (Camellia sinensis). Many studies have shown its beneficial effect on human health [1]. The majority of them demonstrated direct effects on cell adhesion and movement. In a previous study we showed that EGCG indirectly affects HeLa cell adhesion: the cells cannot adhere onto EGCG-pre-treated model ECM coatings [50]. The polyphenol formed multilayers in poly-L-lysine polyethylene-glycol-RGD (PLL-g-PEG-RGD) chains, and blocked the RGD motifs [50]. EGCG alters the properties of mucin as well; EGCG-mucin mixtures showed that discrete particles are formed and their size increases with the ratio of EGCG to mucin [57]. Another natural compound, cistifolin (benzofuran derivative) from the rhizome of the gravel root (Eupatorium purpureum), known as an anti-rheumatic herbal drug, was identified as a potent inhibitor of β1 and β2 integrin-mediated cell adhesion and, thus, has therapeutic potential for diseases where integrin adhesion molecules play a significant role [58].

Classical Techniques for Measuring Cell Viability
Experimental natural compounds are added to stimulated cell cultures to induce a CAM response, which is commonly measured by labeling methods, mainly the enzymelinked immunosorbent assay (ELISA), Western blot, and flow cytometry. ELISA uses antibodies linked to enzymes that create a color change (e.g., by altering a dye) to identify the examined substances [13]. The Western blot is a widely used technique in biology to detect specific proteins in a sample by letting animal-derived or synthetic antibodies bind to them [13]. ELISA and Western blot techniques cannot be used for measuring cell viability directly; however, for example, Western blot can be applied as a complementary method to study the mechanism of cell death (apoptosis, autophagy markers, etc.), thus it provides a lot of information about the mechanism. Flow cytometry is usually a laser-based technique for cell sorting and counting. A wide range of fluorophores can be used in flow cytometry measurements. Fluorophores are fluorescent labels that can attach to the antibody that recognizes the target molecule of the cell [2,59]. However, impedance-based flow cytometers also exist, known as Coulter counters, which are well established label-free methods for sizing and counting cells and particles [60].
The terms "cell viability" or "compound cytotoxicity" have broad meanings in drug discovery. For in vitro monolayer cell cultures, a compound is considered to be cytotoxic if the compound interferes with cellular attachment, or significantly alters cellular morphology, cell growth and cell viability. A variety of assay methods can be used to estimate the number of viable eukaryotic cells after exposure of the investigated compounds. These cell-based assays are often used for screening collections of compounds to determine if the compounds have effects on cell proliferation or show cytotoxic and cytostatic effects. Cell-based assays are also widely used for monitoring organelle function. These screening methods have been devised to examine a broad variety of parameters associated with biochemical events necessary for sustaining viability, especially as evinced by membrane integrity. The quantities emerging from metabolism (especially ATP-based viability) assays are proportional to viable cell number. Cytotoxicity assays determine parameters proportional to the degree of cell death. The fundamental difference between the approaches depends on the length of exposure to the compound (short-term exposures (4 h or less) may adversely affect metabolism markers or ATP content before measurable membrane integrity changes, and long-term exposures (24 h or more), particularly after early primary necrosis, may lead to underestimation of cytotoxicity owing to degradation of marker enzyme activity after its release into the extracellular environment [62][63][64][65].
Most cell viability and cytotoxicity assays can be divided into three categories: those that (i) exploit the loss of membrane integrity; (ii) directly measure metabolic markers or ATP content; and (iii) assess metabolic activity. Other forms of detection exist. Crystal violet staining can reveal the adherence of cells and thus be used to measure the viability of adherent cells [66][67][68]. Determination of the loss of membrane integrity: these assays rely on the breakdown/disintegration of the cell membrane to allow different molecules to enter the cell, or allow intracellular compounds to be secreted to the extracellular area. Living cells are not permeable to it, but dead cells are (hence acquire dark coloring) and can be detected in the population after exposure treatment of the cells. (B) The basis of the MTT assay is that the yellow, water-soluble tetrazole becomes purple, insoluble formazan by the action of mitochondrial dehydrogenase of living cells. Cytotoxic and cytostatic activities can be determined from the optical density of the control and treated cells (ICM: incomplete medium, CM: complete medium). The formazan can be conveniently extracted by DMSO for colorimetric measurement.
The terms "cell viability" or "compound cytotoxicity" have broad meanings in drug discovery. For in vitro monolayer cell cultures, a compound is considered to be cytotoxic if the compound interferes with cellular attachment, or significantly alters cellular morphology, cell growth and cell viability. A variety of assay methods can be used to estimate the number of viable eukaryotic cells after exposure of the investigated compounds. These cell-based assays are often used for screening collections of Living cells are not permeable to it, but dead cells are (hence acquire dark coloring) and can be detected in the population after exposure treatment of the cells. (B) The basis of the MTT assay is that the yellow, water-soluble tetrazole becomes purple, insoluble formazan by the action of mitochondrial dehydrogenase of living cells. Cytotoxic and cytostatic activities can be determined from the optical density of the control and treated cells (ICM: incomplete medium, CM: complete medium). The formazan can be conveniently extracted by DMSO for colorimetric measurement.
Metabolic assays primarily focus on measuring ATP levels, or the reduction of tetrazolium salts, or resazurin dyes inside living cells.
Cellular proliferation causes a change in the ratios of certain metabolites e.g., NADPH/ NADP, FADH/FAD, FMNH/FMN, and NADH/NAD. These metabolic intermediates are then capable of reducing tetrazolium salts to formazan product, which can be detected. Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a non-fluorescent redox dye, which when reduced to resorufin becomes a red compound, so the color change can be detected.
The most common method for assaying live cell proliferation is measuring the amount of DNA synthesis, which is done by adding a labeled DNA analog called BrdU (5-bromo-2deoxyuridine (BrdU), which is incorporated into the DNA instead of thymidine. To assess the incorporation of BrdU into the DNA colorimetric ELISA or immunohistochemical staining methods are used. A newer approach is to detect the incorporation of the alkyne containing thymidine analog EdU. The incorporation can be detected by a copper catalyzed azide-alkyne cycloaddition.

Limitations and Considerations When Using Membrane Integrity or Metabolic Assays
The listed substrates (Tables 4-7) all have distinct advantages and disadvantages when compared to each other. Assay sensitivity, noise-to-signal ratio, ease of use, and also reagent stability are all factors that have to be considered. Metabolic assays also need to consider that the reduction of said substrates are impacted by other intracellular metabolic activity, and have no direct effect on the cells viability or cytotoxicity of a studied compound.
In fixed samples such as animal tissues and cell population, proliferation can still be measured, but only by immunostaining for specific proliferative markers. Ki-67 is a nuclear protein that is associated with cell proliferation and ribosomal RNA transcription. Traditional antibodies for Ki-67 can only be used to stain frozen (not paraffin embedded) samples. MIB-1 antibodies however target a different epitope of Ki-67, and thus can be used to stain formalin and paraffin fixed samples, making the use of Ki-67 as a proliferative marker easier. Another commonly used marker is the proliferating cell nuclear antigen. This protein expedites DNA synthesis by holding the polymerase to the DNA, so it is expressed widely in the nucleus during DNA synthesis, making it an effective marker of cell proliferation. Other markers that can be used include MCM2 also. It has to be noted that all such assays measuring DNA synthesis directly or indirectly are sensitive to the stage of the cell in the cell cycle at the time the measurement is carried out.  1 Trypan Blue: Staining with trypan blue is one of the oldest viability assays. In a viable cell, the intact membrane will prevent trypan blue from entering cells. In dead or dying cells, trypan blue will enter the cell, staining it blue. This method was traditionally quantified manually using microscopes and hemocytometers, making it very labor-intensive. However, the recent availability of affordable automated cell counters makes this assay less time consuming and more accurate. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) is a tetrazolium salt that gets reduced by both mitochondrial and extra-mitochondrial dehydrogenases to form insoluble blue formazan crystals, meaning a solubilization step is required before the assay can be read. MTS/XTT: MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) substrates are similar to MTT. However, one advantage is that the reactions are carried out intracellularly in the presence of the intermediate electron acceptor phenazine methosulfate (PMS), which enhances their sensitivity. In addition, the reduced formazan product is soluble and gets released to the culture media, removing the need for the extra solubility step that is required with MTT. However, phenol red in cell culture media, fatty acids, and serum albumin have all been reported to distort data obtained from MTS, XTT, and WST assays over prolonged incubation periods. WST: Water-soluble tetrazolium salts (WSTs) are cell-impermeable tetrazolium dyes that get reduced extracellularly via plasma membrane electron transport, and combined with the electron acceptor PMS to generate water-soluble formazan dyes. LDH assay: Lactate dehydrogenase is a ubiquitous, stable cytoplasmic enzyme that converts lactate to pyruvate. If the cell membrane has been damaged, LDH, and therefore, its enzymatic activity is released from cells and can be detected in cell culture media.  [112] 1 Alamar Blue: is a resazurin compound that gets reduced to resorufin and dihydroresorufin in viable cells. It can enter live cells so does not require cell lysis, and is stable in culture media. This assay has the added advantage that it can be measured in both fluorimetric and colorimetric plate readers. Pace et al., reported incubation time dependent cytotoxic effect [104]. Calcein-AM: Calcein-acetoxymethylester is a non-fluorescent dye that is used in both cell viability and apoptosis assays and its lipophilic, allowing easy passage through the cell membrane. Once inside the viable cell, intracellular esterases cleave the ester bonds of the acetomethoxy group, resulting in the formation of a fluorescent anionic and hydrophilic calcein dye. Non-viable cells do not contain active esterases. Also need to consider that: Cu 2+ , Co 2+ , Fe 3+ , Mn 2+ , and Ni 2+ quench the fluorescent signal from calcein at physiological pH, which means care must be taken to select the appropriate cell culture media.     The most frequently used techniques in cellular adhesion experiments with natural compounds are based on dyes (absorbing or fluorescing) as labels. These methods have several drawbacks; however, (i) in general they are not high-throughput techniques, since the procedures are time-consuming and complicated, (ii) many dyes are cytotoxic and therefore disturb the normal physiological activity of the cells; furthermore their structures are often quite comparable to those of the natural compounds under investigation, leading to interference, (iii) typically only end-point data is gathered; i.e., no information on the kinetics of the processes is obtained, (iv) many natural compounds investigated for possible therapeutic benefits have low molecular weight, and frequently are environmentally sensitive (e.g., to oxidation) and thermally or photolytically unstable.
A good example of this instability is the green tea polyphenol EGCG [1,50]. Investigating the physiological effects of such compounds by labeling techniques exacerbates the difficulties.
Recent developments, such as the use of quantum dots (semiconductor nanoparticles) and gold nanoparticles instead of organic dyes as labels do little to alleviate these disadvantages.

Measurement Techniques for Monitoring Cellular Movement and Adhesion
Cell migration takes place inside or on the extracellular matrix surface [157]. The lamellae of the cell have to adhere to the matrix components to generate traction forces for cell movement [157]. The actin filaments of the cytoskeleton are linked through the cell membrane to the substrate at focal adhesion points, which are connected by actin filaments such as to promote the direction of movement and are located under the entire body of the cell [157,158]. Hence, adhesion and migration are in close relation to each other; adhesion is essential to achieve cellular movement. Thus, measuring cell migration with migration assays provides information about cell adhesion as well [157]. In Table 8, we summarize the advantages and disadvantages of migration assays. Spreading and adhesion play crucial roles in development and maintenance of cell homeostasis and in many further complex functional processes [159,160]. Many types of assays have been developed to measure the adhesion strengths (forces) and can be categorized into single cell and population cell studies [158,161,162]. Cell adhesion detachment events for single cell studies can be initiated via the breakage of molecular bonds (e.g., optical tweezer techniques and micropipette aspiration), cell population studies via static adhesion (centrifugation technique), and cell population studies via dynamic adhesion (e.g., microfluidic techniques, spinning disk, and flow chamber) [158,161,162]. The measurement of migration and adhesion under experimental in vitro conditions can provide important information about natural compounds, yielding quantitative data about cellular movement and adhesion albeit in a drastically simplified environment compared to the in vivo situation [50].

Label-Free Biosensors
The concept of labeling is rooted in the poor sensitivity of many traditional biological molecular detection techniques. Biomolecules are mostly composed of light elements with small masses and optical polarizabilities and are only rarely fluorescent. The techniques based on electrons, X-rays, visible light and mass spectrometry, which have been so successful in physics and chemistry, become much less potent when moved to the world of biology. By artificially coupling a biomolecule to an artificial entity with an electron density, optical polarizability or mass that may be orders of magnitude greater than that of the biomolecule, the latter suddenly becomes "visible" to the technique being employed to detect it. The euphoria that followed this sudden visibility tended to completely overwhelm continuing appraisal of whether the labeling itself might mask the biological effect that was the primary object of investigation [189].
The advent of new technologies sufficiently sensitive to remove the need to label the biomolecules to make them visible opened up a new world of label-free detection [190][191][192]. Natural compounds usually have small molecular weight where labeling can be problematic or even impossible, especially if their binding pocket is small or embedded. Label-free biosensors are emerging tools to investigate the mode of action of small molecules as well. They eliminate all of the disadvantages of the classical techniques listed above, because these methods do not require labels or dyes, which may disturb the samples. Furthermore, in general, the measurement procedures are not difficult, relatively cheap, and not timeconsuming. In the field of natural compound research (for example active compounds in traditional Chinese medicines), novel methods, for example, resonant waveguide grating (RWG) [50,193,194] and holographic microscopy can be applied without using any dyes or other labels [195].
These techniques, particularly the highly sensitive waveguide-based methods [57,194,[196][197][198][199][200][201][202], constitute highly promising novel phenotypic assays for drug discovery [203] owing to their capacity to reveal the complexity of drug actions and interactions and provide a holistic view of receptor-ligand interactions in living cells. Waveguide sensors are based on the phenomenon of the evanescent field generated by waveguided (usually visible) light being modulated by the presence of drugs, proteins and other biomolecules and living cells [204,205]. An adlayer, which may be constituted from a phospholipid membrane, extracellular matrix proteins or tissue is firstly placed onto the planar waveguide, where it is in contact with physiological medium containing the analyte of interest, such as the natural compound [204,205]. Molecular processes taking place in or on this adlayer modify the parameters of light propagating in the waveguide, any of which can serve as signal transducers [205,206].
Label-free techniques are especially useful for kinetic monitoring of biological events [207][208][209], even at single-cell level [210]. So they have become more and more popular in the field of drug discovery and in other areas focusing on the biological roles of small molecules [1,203]. Furthermore, the same techniques can be readily adapted to measure the dynamics of cell shape changes [211]. Cell adhesion assays and cell migration tests can be achieved in a completely label-free way, with in situ exposure to the natural compounds and, indeed, any drug. For example, a recent study of Peter et al. applied holographic microscopy to quantitatively show that EGCG reduced the motility speed and migration of HeLa cells [195]. Holographic microscopy creates 3D images of cells and the changes in cell morphology due to EGCG could be observed in real time [195].
These techniques are more and more important to systematically study the effects of active substances on cell behavior, especially cell adhesion, and to investigate different biological materials (such as membranes and matrices) and living cells in a high-throughput format [50]. In a recent experiment, Peter et al. showed that EGCG can block the RGD (Arg-Gly-Asp) motif, an important cell adhesion ligand and directly affect matrix properties by hydrogen bonding (Figure 7) [50]. The other novelty of this study is that the polymer coating fabrication, its treatment with the small EGCG molecule (a green tea polyphenol), and the observation of cell adhesion could be all studied online using a high-throughput RWG biosensor, with different EGCG and oxidized EGCG concentrations [50]. This and similar methodologies should be applicable to other extracellular matrix interactions with small natural compounds ( Figure 7).
Recently, it was concluded that the shape of the kinetic curves obtainable with the label-free biosensor can be used to quantify in vitro cell viability in a fast and highly sensitive manner [59].
Natural compounds are generally small molecules that can self-associate into colloidal aggregates in aqueous buffer [212]. In the early stage of drug discovery, this phenomena is the principal cause of false results [212]. Wang et al. reported RWG-based This and similar methodologies should be applicable to other extracellular matrix interactions with small natural compounds ( Figure 7).
Recently, it was concluded that the shape of the kinetic curves obtainable with the label-free biosensor can be used to quantify in vitro cell viability in a fast and highly sensitive manner [59].
Natural compounds are generally small molecules that can self-associate into colloidal aggregates in aqueous buffer [212]. In the early stage of drug discovery, this phenomena is the principal cause of false results [212]. Wang et al. reported RWG-based assays to identify natural compound aggregation and characterize its influence on membrane receptors in living cells [212]. They showed that the colloidal aggregates may cause false activity in DMR desensitization assays [212]. A series of RWG-based assays for colloidal aggregate detection and characterization of their promiscuity were developed [212]. RWG-based assays can be applied as practical tools to distinguish between real and false responses, providing useful reliable results in the early stages of drug discovery [212].

Living Cell Movements-Holographic Microscopy
The realization of in situ monitoring of living cell movements (i.e., crawling along the endothelium) is a very important advance [195,213]. There are anyway few techniques to study cell movements; classical ones are mostly directed at migration studies and they also have their drawbacks [195]. For instance, filter assays (Zigmond and Dunn chambers, Boyden chamber) measure cell migration over a membrane in response to chemoattractant compounds [195]. These assays are very specialized, requiring cells to migrate through both a matrix and the pores of a filter [213]; however, very few cell lines can migrate through both of them [195]. Single cell movements can be investigated by using time-lapse imaging, usually requiring fluorescent labels [195,213]. As discussed above, fluorescent imaging may disturb the cells and the imaging time is limited by bleaching of the fluorescent marker [195,213]. In contrast to fluorescent imaging, holographic microscopy is a label-free technique [195,214]. It is usually critical to observe and quantitatively record live cell behavior, especially directional movement (migration) and random movement (motility), and also the shape changes to understand the behavior of the cells in such environments and to be able to make inferences regarding further therapeutic possibilities [195]. Although traditional herbal extracts have become more and more popular for treating illnesses, the literature of systematic quantitative studies is still limited [1].

Preparation of Natural Compounds
Natural products with their broad chemical diversity and bioactivity spectrum are sought after by the pharmaceutical industry and they continue to provide new structures with promising effects and to offer templates for the development of scaffolds of novel drug candidates. However, natural product discovery programs have been abandoned over the past 30 years by many pharmaceutical companies [215][216][217].
Natural products present their own challenges for drug discovery, including screening, isolation, characterization and optimization, which doubtless contributed to the decline in their pursuit by the pharmaceutical industry. In recent years, several technological and scientific developments-such as improved analytical tools, genome mining, engineering strategies, and microbial culturing-are addressing these challenges, preparing the way for new opportunities and interest in natural compounds [218,219].
Here, we briefly summarize recent technological developments that are facilitating natural product-based drug discovery, highlight selected applications and discuss key opportunities. Innovative compound identification from natural products requires a multidisciplinary approach utilizing numerous technologies [220].
Isolation and purification of natural compounds are the most important and difficult steps for molecular structure identification, in vitro testing, quantity control and further industrial production. The natural compounds are usually in complex matrix material and the active moieties are present in low concentration [220] which are often accompanied by co-occurring irrelevant common metabolites accumulated in high amounts. Selection of appropriate techniques and strategies is essential for getting the target compounds at high yield. Over the past decades, many types of new extraction, isolation and purification techniques (classical solvent extraction procedures, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), extraction with ionic liquids, accelerated (pressurized) solvent extraction (ASE), supercritical fluid extraction (SFE), extraction on solid phases, distillation methods, membrane filtration, preparative high-performance liquid chromatography (HPLC), counter-current chromatography (CCC), supercritical fluid chromatography (SFC), etc.) have been applied. With developments in separation science and microplatebased in vitro high-throughput screening (HTS) assays, natural compound research gained remarkable momentum recently. Nevertheless, despite these developments of extraction, separation, analytical and structural elucidation techniques, isolation of natural compounds from plants, marine organisms or microorganisms is challenging, with problems including post-harvest changes in material quality [221,222]. Extraction is the first step to separate most natural products from the raw materials. The method progresses through the following stages: (i) solvent penetration into the matrix; (ii) the components dissolve in the solvent; (iii) the components diffuse out of the matrix. A brief but excellent summary of the various extraction methods used for natural products is presented by Zhang and co-workers [223]. A more detailed description is presented in a book chapter by Cavalcanti et al. [224]. The analyses of extracts, fractions, isolates, compounds from different natural matrices, studies on chemotaxonomy, chemical fingerprinting, metabolome elucidation, and structural evaluation have become easier because of the availability of a number of instrumental analytical techniques, e.g., GC-MS, LC-PDA, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, and CE-MS, NMR, NIR, FT-IR.
Atanasov and co-workers [219] have detailed the traditional bioactivity-guided isolation process and its limitations, and also ideas on addressing them. The general process begins with the extraction steps and the method applied determines which type of compounds will be present in the particular extract (solvents with hydrophilic chemical character will result in a higher abundance of hydrophilic compounds). Following the identification of an extract with promising bioactivity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the individual active compound or compounds are isolated. To isolate such new antiproliferative/antimicrobial compounds from a series of extracts, highthroughput label-free activity screening by label-free biosensors could be applied. The identities of the pure compounds responsible for an extract's activity can be determined by bioactivity-guided fractionation using novel label-free biosensor systems [1,[204][205][206][207][208][209][210][211][212][213][214].
A systems biology-guided approach coupled with application of commensurate technologies (genomics, proteomics, metabolomics, in silico modeling, etc.) should provide new opportunities for identifying new and better drug candidates. Molecular libraries of lead compounds from natural products will serve as sources of lead extracts/compounds for innovative drugs ( Figure 8).

Natural Compounds of Plant, Animal and Fungal Origin
We summarize and categorize those natural compounds found in the literature that affect cellular adhesion and migration. The sources of the active compounds are divided into three groups; plants (Table 9), animals (Table 10), or fungi (Table 11). The compounds produced by these organisms include primary and secondary metabolites. They show structural and functional diversities, which are the result of biosynthetic processes modulated by natural selection. Defense molecules-antimicrobial peptides (AMPs)-are commonly produced to resist stresses. These oligo-and polypeptides are synthesized by ribosomes or via non-ribosomal peptide synthetases. Many AMPs exhibit broad-spectrum antimicrobial and anticancer activity. Plants are a remarkably promising source of these molecules and they are also considered to affect plant growth and development [225]. AMPs can be classified into cationic or anionic compounds according to their net charge. Their function, isolation and other aspects have been reviewed [225][226][227][228].
In the cases of plant or fungal origin, it has been well known for centuries that active substances can be extracted from individual plant organs (i.e., leaves, flowers, rhizomes, fruits, seeds, roots, stems, bark and xylem). These parts can be consumed as hot water infusions (e.g., green tea, herbal infusions), as spices (e.g., pepper, clove), as raw fruits or extruded/fermented products (e.g., red wine, olive oil). Such natural compounds are consumed day-by-day as food, but their ability to cure diverse diseases has been known for thousands of years (e.g., in Chinese traditional medicine).
In our tables, if the literature information is too scanty to allow us to be more specific, we simply write P (pathway intervention involving gene expression) or B (cell adhesion inhibition by blocking specific receptor sites) to indicate the mechanism.

Natural Compounds of Plant, Animal and Fungal Origin
We summarize and categorize those natural compounds found in the literature that affect cellular adhesion and migration. The sources of the active compounds are divided into three groups; plants (Table 9), animals (Table 10), or fungi (Table 11). The compounds produced by these organisms include primary and secondary metabolites. They show structural and functional diversities, which are the result of biosynthetic processes modulated by natural selection. Defense molecules-antimicrobial peptides (AMPs)-are commonly produced to resist stresses. These oligo-and polypeptides are synthesized by ribosomes or via non-ribosomal peptide synthetases. Many AMPs exhibit broad-spectrum antimicrobial and anticancer activity. Plants are a remarkably promising source of these molecules and they are also considered to affect plant growth and development [225]. AMPs can be classified into cationic or anionic compounds according to their net charge. Their function, isolation and other aspects have been reviewed [225][226][227][228].   Ellagic acid HPLC, Cell-enzyme- Eupatorium purpureum (rhizome) Giardia activity 30957]) assay, Transmission and scanning electron microscopy, MTT assay [230] Elenolic acid, Hydroxytyrosol,             In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238][239][240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion ( Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides Disintegrin In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238][239][240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion ( Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides produced by several fungal strains, for example Microsphaeropsis sp. [241]. These compounds and metabolites from the peribysin group are also detailed in Table 11.  In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238][239][240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion ( Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides produced by several fungal strains, for example Microsphaeropsis sp. [241]. These  In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238][239][240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion ( Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides produced by several fungal strains, for example Microsphaeropsis sp. [241]. These compounds and metabolites from the peribysin group are also detailed in Table 11.

Echis carinatus
(venom) In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.
Statins, cholesterin-lowering active ingredients, are among the most widely prescribed medicines. Naturally occurring statins (e.g., lovastatin and mevastatin) are produced by different fungi, a synthetic derivate (simvastatin) of a fungal fermentation product is found in pharmacopeias alongside synthetic statins (e.g., atorvastatin, fluvastatin). In Table 11, the synthetic derivatives are labeled (syn). Lovastatin is naturally produced by e.g., Pleurotus ostreatus (oyster mushroom) [236] and mevastatin was first isolated from the Penicillium citrinum [237]. Several pleiotropic effects of statins have been revealed, including enigmatic effects on cancer [238][239][240]. Naturally occurring statins can suppress cell migration, invasion, and cell adhesion ( Table 8). The effects of such suppression on cancer metastasis would appear to be obvious. We focus on studies in which cell migration, invasion and adhesion have been studied in vitro. Another metabolite type, the macrosphelides (MSs; A, B, C, D, E, F, G, H, etc.), are macrolides produced by several fungal strains, for example Microsphaeropsis sp. [241]. These compounds and metabolites from the peribysin group are also detailed in Table 11. In the cases of plant or fungal origin, it has been well known for centuries that active substances can be extracted from individual plant organs (i.e., leaves, flowers, rhizomes, fruits, seeds, roots, stems, bark and xylem). These parts can be consumed as hot water infusions (e.g., green tea, herbal infusions), as spices (e.g., pepper, clove), as raw fruits or extruded/fermented products (e.g., red wine, olive oil). Such natural compounds are consumed day-by-day as food, but their ability to cure diverse diseases has been known for thousands of years (e.g., in Chinese traditional medicine).
In our tables, if the literature information is too scanty to allow us to be more specific, we simply write P (pathway intervention involving gene expression) or B (cell adhesion inhibition by blocking specific receptor sites) to indicate the mechanism.
In case of animal origin, the most popular category of substance is serpent venom (Table 10). However, a recent study of Mattia et al. showed that edible insects and invertebrates can be a source of useful polyphenols as well [235]. For example, black ants (Lasius niger), mealworms (Tenebrio molitor) and grasshoppers contain the highest levels of total polyphenols [235]. More experiments are needed to understand whether eating insects and other invertebrates might be beneficial to humans [235], although we note that some cultures have, for example, regarded locusts as comestible for millennia.

SARS-CoV-2 and Possible Treatments with Herbal Extracts
A novel coronavirus (nCoV) started infecting humans since late 2019. The pathogen is called "Severe Acute Respiratory Syndrome-related Coronavirus 2" (SARS-CoV-2). It can cause a fatal respiratory disease, called Coronavirus disease 2019 (COVID- 19), and acute respiratory distress syndrome (ARDS) as well [271]. The COVID-19 pandemic spread quickly inducing a worldwide problem, because the virus is highly contagious; transmission occurs presumably mainly via airborne droplets [271]. SARS-CoV-2 belongs to the genus Betacoronavirus of the large family of Coronaviridae [271]. Externally, the virus displays a corona-shaped layer of spikes that play a significant role in the infection process [272]. The virions are able to recover from the drastic mechanical perturbation imposed by atomic force microscopy [272]. The global structure is temperature-resistant, but the virion surface is denuded of spikes if heated [272]. These properties obviously influence the infectivity of the virus. The spike protein is a large glycoprotein trimer, which contributes to host receptor binding, cell tropism and pathogenesis [271]. A recent study showed that the surface of the native virion displays a dynamic brush owing to the rapid motions and flexibility of the spikes [272]. By binding host receptors, the virus genome penetrates into the cytoplasm of the host cell [271]. The angiotensin-converting enzyme II (ACE2) is a receptor for SARS-CoV-2, but it is suggested that the virus may use integrins too as receptors, binding to them through the conserved RGD motif [50,51,273]) present on the spike protein [271]. Members of the integrin family are commonly used as receptors by many other human viruses as well, and RGD is the minimal sequence required for binding [271]. Integrins have high expression in lungs and other vital organs whereas ACE2 is found to have negligible occurrence in the lungs [274]. Expression of integrins is high in lung cells (especially αVβ6, αVβ8, α5β1) and the ICAM-1. Thus, the high infectivity of the virus may be at least partly due to the RGD-integrin-mediated cell-adhesive property [274]. Phosphorylation sites on the spike protein induce Tyr, PKC and cAMP signaling pathways, which activate calcium ion channels or get activated by calcium [274]. Thus the RGD-integrin interaction clearly occurs in a calcium-dependent manner [274]. This interaction may then unleash a "cytokine storm" due to TNFα and IL-6 activation [274]. The lowering of divalent ion concentrations in the lungs by pulmonary EDTA chelation therapy may inhibit virus-host attachment [274]. Glycans may have multiple roles during viral entry [275]. Inhibition of N-glycan biosynthesis was shown to enhance spike protein proteolysis, leading to a decrease of receptor-binding domain presentation on the virus [275]. Thus, another idea for treatment is to administer chemical inhibitors of glycosylation [275]. Hence, although vaccines have already been developed and authorized for emergency use, extensive research work and brainstorming continue for the development of future drugs against SARS-CoV-2 ( Figure 9).
Already-existing synthetic drugs (favipiravir, ivermectin, remdesivir, among others) have been shown to be effective for treatment, but the application of these drugs may have direct or indirect side effects (for example, pain, liver problems, allergy, etc.) [276]. Plants are an abundant source of natural antiviral compounds that may be an effective option, because most of them are safer compared to synthetic drugs, although there are exceptions [276]. Some of these metabolites have protective effects against different microbes; however, the role of most metabolites still remains unknown for us. Bhuiyan et al. created a large collection of antiviral compounds from 219 medicinal plants [276], from which, it can be inferred that polyphenols work against coronaviruses by actuating or inhibiting cellular signaling pathways or inhibiting 3-chymotripsin-like protease (3CL pro ) and papainlike protease (PL pro ) [276]. Polyphenol compounds from Broussonetia papyrifera, Sambucus and Pelargonium, and flavonoid-type compounds (quercetin, apigenin) showed activity against human coronavirus [276,277]. Different types of alkaloids (for example tylophorine, 7-methoxy cryptopleurine, etc.) have anti-SARS activity by inhibiting protease and RNA and protein synthesis, and chloroquine has been reported to have anti-SARS-CoV-2 activity [276]. Saponins (amphipatic triterpenes) showed antiviral effects against a lot of viruses (Influenza virus, Dengue virus, rotaviruses, among others) [276,278]. Among triterpenes, ginkgolide A can strongly inhibit the SARS-CoV-2 protease [276], and glycyrrhizin was found to be effective against influenza [278]. In the case of SARS-CoV-2, glycyrrhizin was found to have the highest binding affinity with the viral S protein [276,279], but curcumin, apigenin and chrisophanol also bind to this part of the virus according to in silico molecular docking [279]. In a recent study, EGCG from green tea beverage was shown to inhibit infection of live virus and its variants by inhibiting spike binding to ACE2 receptor [280]. Virus can attach to ACE2 receptor or integrins of the host cell. RGD-integrin interaction occurs in calcium-dependent manner [274]. As the result of the process, the virus penetrates into the cell and starts to copy itself. (B) Several compounds bind to the spike protein or even may alter it [275] and prevent virus-receptor attachment. (C) Lowering divalent ion concentrations in the lungs with pulmonary EDTA chelation therapy may inhibit virus-host interaction [274].
Already-existing synthetic drugs (favipiravir, ivermectin, remdesivir, among others) have been shown to be effective for treatment, but the application of these drugs may have direct or indirect side effects (for example, pain, liver problems, allergy, etc.) [276]. Plants are an abundant source of natural antiviral compounds that may be an effective option, because most of them are safer compared to synthetic drugs, although there are exceptions [276]. Some of these metabolites have protective effects against different microbes; however, the role of most metabolites still remains unknown for us. Bhuiyan et al. created a large collection of antiviral compounds from 219 medicinal plants [276], from which, it can be inferred that polyphenols work against coronaviruses by actuating or inhibiting cellular signaling pathways or inhibiting 3-chymotripsin-like protease (3CL pro ) and papain-like protease (PL pro ) [276]. Polyphenol compounds from Broussonetia papyrifera, Sambucus and Pelargonium, and flavonoid-type compounds (quercetin, apigenin) showed activity against human coronavirus [276,277]. Different types of alkaloids (for example tylophorine, 7-methoxy cryptopleurine, etc.) have anti-SARS activity by inhibiting protease and RNA and protein synthesis, and chloroquine has been reported to have anti-SARS-CoV-2 activity [276]. Saponins (amphipatic triterpenes) showed antiviral effects against a lot of viruses (Influenza virus, Dengue virus, rotaviruses, among others) [276,278]. Among triterpenes, ginkgolide A can strongly inhibit the SARS-CoV-2 protease [276], and glycyrrhizin was found to be effective against influenza [278]. In the case of SARS-CoV-2, glycyrrhizin was found to have the highest binding affinity with the viral S protein [276,279], but curcumin, apigenin and chrisophanol also bind to this part of the virus according to in silico molecular docking [279]. In a recent study, EGCG from green tea beverage was shown to inhibit infection of live virus and its variants by inhibiting spike binding to ACE2 receptor [280]. (A) Virus can attach to ACE2 receptor or integrins of the host cell. RGD-integrin interaction occurs in calcium-dependent manner [274]. As the result of the process, the virus penetrates into the cell and starts to copy itself. (B) Several compounds bind to the spike protein or even may alter it [275] and prevent virus-receptor attachment. (C) Lowering divalent ion concentrations in the lungs with pulmonary EDTA chelation therapy may inhibit virus-host interaction [274].
It should be noted that the risk factors for a severe course of COVID-19 in intensive care unit patients are chronic obstructive pulmonary disease, renal dysfunction, hypertension, diabetes mellitus and coronary heart disease. Elderly adults and patients with chronic illnesses and obesity are vulnerable [278]. There is preliminary evidence that nutrientrelated disorders are associated with greater susceptibility to infection [278].
Herbal remedies have a potentially preventive effect, mainly acting through supporting the immune system, for example Astragalus membranaceus or Echinacea purpurea [277]. Clinical studies showed that extracts from Pelargonium sidoides, Sambucus nigra and Cistus incanus are effective treatments of infectious respiratory illnesses [277,278]. Kalus et al., a decade before the COVID-19 pandemic, showed that Cistus incanus extract significantly decreased the symptoms of 160 patients with infections of the upper respiratory tract (caused by bacteria, influenza, and other viruses) and the level of C-reactive protein inflammatory marker was also decreased [281]. EGCG green tea polyphenol was shown to bind to the hemagglutinin of influenza virus [281] as well, implying that regular consumption of green tea should decrease the influenza infection rate, too [278]. In summary, consuming extracts of herbs, vegetables and fruits improve overall health due to their phytochemicals and nutrients, and these compounds may prevent or attenuate the symptoms of COVID-19.

Conclusions
Most investigations into the effect of natural products on cellular adhesion are focused on the adhesion molecules belonging to the immunoglobulin superfamily of CAMs (ICAM-1 and VCAM-1), and selectins and integrins [5]. These molecules are implicated in several widespread diseases, such as various types of inflammation, rheumatism, atherosclerosis and cancer. Metabolites obtained from plants, fungi and venoms may offer therapeutic potential by regulating adhesion molecules, generally down-regulation [5]. We summarized natural metabolites having diverse structures, which influence cellular adhesion and migration by modulation of adhesion molecules. The reported effects of natural products, either presented to model cell systems as complex extracts or as purified active principles, generally manifest themselves as: (i) inhibition of cell-cell adhesion (i.e., circulating to endothelial cells); and (ii) a general anti-inflammatory effect. The mechanism of (i) is either the blocking of specific adhesion sites (such as the RGD motif and/or its binding complement) on the cell surface (the group of mechanisms labeled B-blocking adhesion receptors on the surface of immune or tissue cells, or motifs in the ECM), or the downregulation of the cell adhesion molecules (the group of mechanisms labeled P-inhibiting the dephosphorylation, translocation or binding of certain factors). The latter group is often effected by inhibition of signaling pathways involving NF-κB. The mechanism of (ii) is by suppressing ROS, achieved by the sacrificial oxidation of the natural product. To map the properties and effects of natural compounds, "classical" labeling techniques have been applied to monitor cellular adhesion and movement. New label-free methods can provide more information (especially kinetic information) about the effects of extracts more sensitively and conveniently without using any cell physiology-altering dyes or other labels. In this review, the preparation method of natural compounds was summarized and the natural product banks were mentioned as well. We systematized recent studies about natural compounds referring to their effects on cell adhesion and movement; the active substances were categorized based on their origin (floral, faunal or fungal). From these collected results it can be clearly seen that applying natural compounds can be a cure against tumors and inflammatory diseases, but more clinical tests would be desirable. Synthetically modified versions of the substances can be also used to cure illness. Finding new, still unknown natural compounds and to map the exact effects of already known extracts from traditional medicine are both important to create new, better, and more effective therapeutic drugs. Some plant extracts showed antiviral activity on SARS-CoV-2 as well, thus natural compounds may be used to attenuate or even prevent the symptoms of COVID-19 as well.
Our review aimed at collecting evidence in order to answer a specific research question [282], namely the role of natural compounds in cellular adhesion and migration, including the characterization methods of these bioactive compounds. We have elucidated the main points of interest (definition of natural compounds and their isolation, adhesion and migration process, effects on cell viability, invasive and non-invasive assays, label free techniques) with inclusion and exclusion criteria. We have made a careful and systematic search of the literature, using the following keywords: natural compound of plant, fungi and other origin, cell adhesion, migration, motility, movement, CAM, integrin, cancer cell, stimulation, inflammation, viability, cytotoxicity, flow cytometry, dyes, label-free, biosensors, preparation, isolation, intracellular pathogens SARS-CoV-2. Approximately 280 relevant paper wereselected. Very recent (2021) and old references (articles from the 1970s and even from 1962) were evaluated. Our criteria were that the compound must be a natural one (i.e., natural origin), with effect on cell adhesion and/or migration.