Compound Experimental protocol Antitumoral activity and/or mechanism Animal/cell line tested Reference Anaphylaxis model Apoptotic manifestations via phospho-ser 15-p53 into mitochondria Mast cells [11 ] Skin carcinogenesis model Inhibition of the proliferation associated genes c-Myc and H-ras and antiapoptotic gene Bcl2 along with upregulation of proapoptotic genes Bax, p53, and active caspase-3 Mice [12 ] Trypan-blue assays Cytotoxic activity B16-F10, Sbcl2, WM3211, WM98-1 and WM1205Lu, PC-3, human gingival fibroblasts, oral mucosal, neutrophils—male guinea pig, rat hepatocytes cells [14 , 15 , 23 , 32 , 33 , 48 , 49 ] Melanoma cell proliferation Deregulation of the E2F family of transcription factors, transcriptional activity of E2F1 Sbcl2, WM3211, WM98-1, and WM1205Lu cells [15 ] Flow cytometry analysis Cytotoxic activity P-815, K-562, CEM, and MCF-7 cells [13 ] VL irradiation time Antioxidative reactivity HSG, HSC-2, and HL-60 cells [17 ] MTT assay Cytotoxic activity B16-F10, P-815, K-562, CEM, MCF-7, MCF-7 gem, HeLa, DU-145, KB, HSG, human dental pulp, murine peritoneal macrophages HL-60, HepG-2, B16, cells [13 , 19 –22 , 25 –29 , 38 , 45 , 46 , 48 ] DPPH assay Antioxidative activity Caco-2 cells and VH10 fibroblasts [18 ] Flow cytometer analysis Enhanced the accumulation of cells in the S and G2/M phase which may be unable to divide HeLa cells DAPI staining Increase in the number of apoptotic cells In vitro hemolytic activityHemolytic activity Human erythrocytes [19 ] Caspase-3 colorimetric assay Induce caspase 3-mediated apoptosis RT-PCR Anticancer activities via apoptosis induction and anti-inflammatory downregulation of Bcl-2, COX-2, and IL-1β RT-PCR Downregulated the expression of Bcl-2, COX-2, and IL-β HeLa cells [20 ] Flow cytometer analysis Increased population of cells G2/M phase by 4.5-fold
PC-3 cells
[24 ] Western blot and RT-PCR analysis Reduced expression of antiapoptotic protein Bcl-2 and enhanced expression of proapoptotic protein Bax DPPH radical-scavenging activity Formation of dimers HSG cells [25 ] ELISA Reduced the nicotine-induced ROS, NO generation, and iNOSII expression Murine peritoneal macrophages [27 ] Spectrophotometric analysis Increase in LDH release DU-145 and KB cells [28 ] ESR analysis Activity of the production of phenoxyl radicals with most efficiently scavenged reactive oxygen
HSG cells
[29 ] Laser cytometry analysis Production of ROS induced by VL-irradiated is significantly affected by pH Antioxidants production Produced antioxidants in alkaline solutions Human salivary gland and oral squamous cells [30 ] DPPH assay Apoptosis-inducing effect HGF and HSG cells [31 ] TBA analysis lipid oxidation Depleted intracellular glutathione; protect cells from the genetic attack of reactive oxygen species via inhibition of xanthine oxidase activity and lipid peroxidation
Oral mucosal fibroblasts
[32 ] ATP assay Decreased cellular ATP level in a concentration- and time-dependent manner NR assay Intracellular glutathione levels HFF and HepG2 cells [33 ] Dichlorofluorescein assay Reduction in the intracellular level of GSH HSG cells [34 ] CAs assay Induced a dose-dependent increase of aberrant cells V79 cells
[41 ] Topo II activity assay Inhibition of topoisomerase II Croton oil induced skin carcinogenesis Inhibition of the proliferation associated genes c-Myc and H-ras and antiapoptotic gene Bcl2 along with upregulation of proapoptotic genes Bax, p53, and active caspase-3 Swiss mice [36 ] DMBA/TPA-induced carcinogenesis in murine skin Declined of hyperplasia, epidermal ODC activity, and protein expression of iNOS, COX-2, and secretion of proinflammatory cytokines
Swiss mice
[42 ] TUNEL assay Upregulation of p53 expression with a concomitant increase in p21WAF1 levels in epidermal cells indicating induction of damage to the DNA Flow cytometric analysis cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors WM1205Lu cells
[24 ] TUNEL assay Induces apoptosis in melanoma tumors WM1205Lu cells DPPH assay Antioxidative properties HL-60 and HepG-2 cells [48 ] Sulforhodamine B assay Cytotoxic activity SK-OV-3, XF-498, and HCT-15 cells [76 ] Murine Ehrlich ascites and solid carcinoma models Inhibit the growth of Ehrlich ascites BALB/c mice [44 ] DPPH assay Antioxidation activity HepG2 cells [22 ] Western blot analysis Decreased the protein expression of BSP in a concentration-dependent manner Human dental pulp cells [35 ] DPPH assay Antioxidant effect Raw 264.7 cells [43 ] VL irradiation/MTT assay Generation of eugenol radicals HSG and HGF cells
[36 ] Laser cytometer Generation of ROS ESR analysis Produced phenoxyl radicals HSG and HGF cells [37 ] Superoxide generation/spectrophotometer Stimulation the production of superoxide ( Neutrophils—male guinea pig [40 ] DPPH assay Antioxidative properties HL-60 and HepG-2 cells [48 ] UDS assay Cytotoxicity and genotoxicity effects B6C3F1 mouse hepatocytes
[47 ] F-344 rat hepatocytes L-Lactate assay Cytotoxic effect B6C3F1 mouse hepatocytes F-344 rat hepatocytes MTT assay Cytotoxic activity HL-60, HepG-2, WM266-4, SK-Mel-28, LCP-Mel, LCM-Mel, PNP-Mel, CN-MelA, and GR-Mel cells [16 , 48 ] WST assay Cytotoxic and genotoxic properties V79 cells [49 ] Corn oil gavage Carcinogenic activity is based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) F344/N rats and B6C3F1 mice [50 ] Trypan-blue exclusion assay Cytotoxic activity Rat hepatocytes [55 ]
HSG cells
[29 ] DPPH radical-scavenging activity Cormation of dimers Dichlorofluorescein assay Reduction in the intracellular level of GSH
[39 ] MTT assay Inhibition of cell proliferation WM266-4, SK-Mel-28, LCP-Mel, LCM-Mel, PNP-Mel, CN-MelA, and GR-Mel cells [16 ] WST assay Cytotoxic and genotoxic properties V79 cells [49 ] L-Lactate assay Cytotoxic effect B6C3F1 mouse hepatocytes
[47 ] F-344 rat hepatocytes UDS assay Cytotoxicity and genotoxicity effects B6C3F1 mouse hepatocytes F-344 rat hepatocytes Trypan-blue exclusion assay Potential cytotoxic effects Rat hepatocytes and SCC-4 cells [47 , 51 , 54 ] Flow cytometric assay Induction of apoptosis of cells by involvement of mitochondria- and caspase-dependent signal pathway
SCC-4 cells
[51 ] Western blotting analysis Upregulation of the protein expression of Bax and Bid and downregulation of the protein levels of Bcl-2 (upregulation of the ratio of Bax/Bcl-2), resulting in cytochrome c release, promoted Apaf-1 level, and sequential activation of caspase-9 and caspase-3 in a time-dependent manner Real-time PCR mRNA expressions of caspases 3, 8, and 9 MTT assay Cytotoxic effect
Human BMFs
[52 ] Western blot analysis Activate NF-κ B expression that may be involved in the pathogenesis of OSF and mediated by ERK activation and COX-2 signal transduction pathway Fura-2 as a probe assay Induced a 2+ 2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca2+ influx PC3 cells [53 ] Comet assay/(DAPI) staining Induced apoptosis (chromatin condensation) and DNA damage HL-60 cells
[51 ] Flow cytometric analysis Increased the production of reactive oxygen species (ROS) and Ca2+ and reduced the mitochondrial membrane potential Western blotting analysis/confocal laser microscopy Promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), and activating transcription factor 6α (ATF-6α ) Flow cytometric analysis Promoted the levels of CD11b and Mac-3 that might be the reason for promoting the activity of phagocytosis; NK cells [58 ] Ames test Mutagenicity activity Salmonella TA 98[59 ]
[56 ] Comet assay Significant dose-dependent increase in the degree of DNA (strand breaks) Cytotoxic or genotoxic effect in vivo —i.p./Comet assay Increase in mean Comet tail moment in peripheral blood leukocytes and in the frequency of micronucleated reticulocytes HepG2 cells TUNEL assay Activity of caspases 3, 8, and 9 A549 cells [58 ] Western blot assay Cleavages of PARP, accompanied by an accumulation of cytochrome c and by the activation of caspase-3 SK-N-SH cells [60 ] 61 ] Trypan-blue exclusion assay Cytotoxic activity Rat hepatocytes [55 ] Trypan-blue assay Cytotoxic activity HeLa, rat hepatocytes cell [21 , 23 , 55 , 64 ] MTT assay Cytotoxic activity HT-1080, ML1-a cells [63 ] Boyden-chamber assay Reduced 40 and 85%
HT-1080 cells
[62 ] Gelatin zymography and RT-PCR analyses Inhibitory effect of MMP-2 and MMP-9 and downregulate the expression of matrix metalloproteinases (MMPs) 2 and 9 and upregulate the gene expression of tissue inhibitor of metalloproteinase- (TIMP-) 1 Expression of MMPs, TIMPs, and uPA assays Decreased mRNA expression of urokinase plasminogen activator (uPA) Western blot analysis Suppressed the phosphorylation of AKT, extracellular signal-regulated kinase (ERK), p38, and nuclear transcription factor kappa B (NF-κ B) Fluorometric assay Increases in the levels of ADP and AMP Rat hepatocytes
[62 ] CCK-8 assayEstrogenic effect based on the concentrations of the hydroxylated intermediate, 4OHPB MCF-7 cells Western blot analysis Suppressed TNF-induced activation of the transcription factor AP-1, c-jun N-terminal kinase, and MAPK-kinase ML1-a cells [63 ] Colorimetric e fluorometric assays Reduced the levels of nucleic acids and MDA, and increased NP-SH concentrations EAT cells in the paw of Swiss mice [65 ] Salmonella tester strains Salmonella typhimurium strains TA1535, TA100, and TA98 Induction of hepatic tumors Carcinogenic in the induction of hepatomas B6C3F1 mice [67 ] Induction of skin papillomas Carcinogenic in the induction of skin papillomas CD-1 mice SRB assay Cytotoxic activity A549, SK-OV-3, SK-MEL-2, and HCT15 cells [70 ] Salmonella tester strains Salmonella typhimurium strains TA1535, TA100, and TA98 Induction of hepatic tumors Carcinogenic in the induction of hepatomas B6C3F1 mice [67 ] Induction of skin papillomas Carcinogenic in the induction of skin papillomas CD-1 mice MTT assay Cytotoxic activity A375, HCT 116, MCF-7, P388, L-1210, 3LL, SNU-C5, HL-60, U-937, HCT 116, L1210 mouse, and Syrian hamster embryo cells [71 , 77 , 78 , 80 , 84 , 89 ] TRPA1 and TRPM8 gene expression Reduce the proliferation of melanoma cells; this effect is independent of an activation of TRPA1 channels A375, G361, SK-Mel-19, SK-Mel-23, and SK-Mel-28 cells [77 ] Sulforhodamine B assay Cytotoxic activity HeLa, A549, SK-OV-3, SK-MEL-2, XF-498, and HCT-15 cells
[76 ] Ames test Not mutagenic Strains (TA 98, TA 100, TA 1535, and TA 1537) of Salmonella typhimurium DTNB assay TrxR inactivation Recombinant rat TrxR
[78 ] Western blot analysis Induced an adaptive antioxidant response through Nrf2-mediated upregulation of phase II enzymes, including TrxR induction HCT 116 cells XTT assay Inhibitory effects on the growth of cells
Hep G2 cells
[80 ] Western blot analysis Increase in the CD95 (APO-1/CD95) protein expression in Hep G2 cells Inhibited the expression of Bax, p53, and CD95, as well as the cleavage of PARP. This pretreatment also prevented the downregulation of Bcl-XL in cells Trypan-blue assay Inhibited the proliferation of cells PLC/PRF/5 cells [81 ] Flow cytometer analysis Activation of proapoptotic PLC/PRF/5 cells
[83 ] Western immunoblot analysis Prevented the phosphorylation of JNK and p38 proteins DAPI/Fluorometric method Induced apoptosis in cells
P388, L-1210, 3LL, SNU-C5, HL-60, U-937, and HepG2 cells
[71 ] Flow cytometry analysis Induces the ROS-mediated mitochondrial permeability transition and resultant cytochrome c release cis -DDP-inducedPotentiated the inactivating effect of cis -DDP in all phases of the cell cycle NHIK 3025 cells [82 ] NRU assay Induced the fragmentation of nuclei (Plate 2), which Hep-2 cells
[87 ] Genotoxicity assays—DNA repair test Involve DNA damage as one of the factors involved in the mammalian cytotoxicity LDH-cytotoxicity assay Potent inhibitory effect against human hepatoma cell growth
HepG2 and Hep3B cells
[88 ] Western blot analysis JAK2-STAT3/STAT5 pathway may be important targets Decreased the protein levels of cyclin D1 and proliferative cell nuclear antigen (PCNA) but increased the protein levels of p27Kip1 and p21Waf1/Cip1 Flow cytometry analysis Inducing apoptosis and synergizing the cytotoxicity of CIK cells K562 cells [92 ] Spectral analysis Induced an adaptive antioxidant response through Nrf2-mediated upregulation of phase II enzymes, including TrxR induction S180 in mice [89 ] MTT assay Cytotoxic activity NIH/3T3 cells [90 ] Lymphoproliferation—Con A, LPS, or PMA plus ionomycin Inhibit the lymphoproliferation and induce a T-cell differentiation from CD4CD8 double positive cells to CD4 or CD8 single positive cells Mice splenocytes
[74 ] Flow cytometry analysis Capability to block the cell growth and stimulate a differentiation to mature cell IgM-secreting B cells to SRBC Decreased level of IgM to be due to the lower level of B-cell proliferation Balb/c mice ELISA Inhibits proliferation and DNA synthesis Caco-2 cells
[79 ] Radioimmunoassay Decreased intracellular cAMP levels Flow cytometry analysis Influence on the tumor cell cycle: G2-M period shortened, cell cycle lengthened, and cell proliferation inhibited U14 cells [92 ] cis -DDP-inducedPotentiated the inactivating effect of cis -DDP in all phases of the cell cycle NHIK 3025 cells [82 ] Trypan-blue assay Anticancer activity HL-60, A549, PC3, Du145, LN-CaP, A172, U251, SKMEL28, and A375 cells [93 , 94 ] Flow cytometry analysis Inhibition and induced-differentiation on human osteogenic sarcoma cells Human osteogenic sarcoma cells [95 ] MTT assay Cytotoxic activity HepG2 cells
[97 ] Spectrophotometer Higher antioxidant capacity NRU assay Cytotoxic activity Mac Coy cells [96 ] MTT assay Antiviral activity EHV-1 [98 ]
Rat hepatocytes
[54 ] Waters chromatograph Decrease in cell viability, accompanied by losses of ATP, GSH; 100 ] Trypan-blue exclusion assay Cytotoxic activity RPMI8226, U266, and IM-9 cells
[99 ] Flow cytometry Induced caspases 3, 9, and 8 activities RPMI8226 cells Western blot analysis TNF-α -induced apoptosis ELISA Downregulation of NF-κ B activity TNF-α -induced apoptosis In vivo assayAnticancer effects with no toxic effects NOD/SCID mouse
