anti - aging effect of medicinal plants is as a result of the antioxidant or free radical scavenging properties of their ingredients ( 2 - 4 ) . cinnamon ( cinnamomum zeylanicum ) and clove ( syzygium 
 aromaticum ) are rich in two active ingredients , which are , cinnamaldehyde and eugenol , respectively ( 5 , 6 ) . these two phytochemicals have different biological activities including , cytotoxic and apoptotic effects on the cancer cell lines ( 6 - 8 ) . anti - mutagenicity , anti - oxidant , anti - inflammatory and anti - depressant activities have also been reported for cinnamaldehyde and eugenol ( 5 , 9 - 11 ) . the effect of phytochemicals on telomere length and telomerase interacting proteins of proliferating stem cells have not been reported yet . gene folds and cell characteristic changes could manifest the ultimate effect of any interaction between chemical components and intracellular proteins . recently , we have reported that cinnamaldehyde and eugenol affect the human adipose derived stem cells ( hascs ) viability , doubling time and differentiation ( 12 ) using cheminforma - tics and experimental approaches . in the present work , the effect of cinnamaldehyde and eugenol on hascs telomere length was determined . also , three important telomere/ telomerase interacting proteins ( tips ) including tert , protein kinase b isoform 1 ( akt1 ) and dkc1 gene expressions under cinnamaldehyde and eugenol treatment were assessed . tert and dkc1 gene s products are necessary for whole enzyme telomerase activity ( 13 ) ; protein kinase b phosphorylates and activates the telomerase enzyme ( 14 ) . a probable mechanism of action of herbal ingredients may be the direct binding of phytochemi - cals to the cell signaling molecules ; this has been shown for some phytochemicals , since the last decade ( 15 , 16 ) . further , experimental methods are becoming more complicated , sensitive and accurate , but they are not empirical to study the ligand - protein docking . computational biology tools help in exploring and assessing the probable ligand - protein interactions in a virtual environment . therefore , in the present investigation , using computational biology and docking tools , the probable cinnamaldehyde and eugenol interactions with tips were explored . cinnamaldehyde ( hplc , > 99.9% ) , eugenol ( hplc grade ; > 98% ) , dmso and antibiotics were from sigma - aldrich . the plasmid dna of psicheck-2 , used in telomere length determination , was a gift from dr farideh talebi . human ascs were prepared , after filling a consent form by a 34-year - old woman who was admitted to a hospital for caesarean section . the hascs preparation and stem cell confirmation methods were as described method in our recent publication ( 12 ) . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . we have selected this concentrations according to the cheminformatics and experimental information about the best results of doubling time of hascs denoted in recently published work . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . rna extraction ( geneall ) , complementary dna ( cdna ) synthesis ( viogene ) and total dna extraction ( qiagen ) were performed according to the kit instructions . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . primers and pcr programs are shown in tables 1 and 2 . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length docking study needs 3ds structures of the proteins and ligands . the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . therefore , 3dss were downloaded from pdb or modeled using swissmodel server ( http://swissmodel.expasy.org/ ) ( 19 , 20 ) . table 3 shows the 3ds of modeled tips using swissmodel server . for docking analysis , the mol2 format of cinnamaldehyde and eugenol molegro virtual docker ver.4.2 was used for docking analysis ; this software has about 87% docking accuracy ( 22 ) . the predicted tertiary structures of some tips or unites tr : telomerase enzyme ; rnp : ribonucleoprotein ; tert : telomerase gene the gene folds , absolute quantities of telomere length and docking scores were analyzed by spss software ver.20 . the meansd was compared using analysis of variances ( anova ) and tukey - hsd with a confidence interval of 95% . also , cluster analysis was performed on the docking scores to select the best scores presentation as graphic views of docking . for clustering , the algorithm of nearest neighbor method and squared euclidean distance that was standardized with z score in spss software were used . string database at the url http://string-db.org/ predicts and plots the protein - protein direct or indirect association networks . the string confidence of association net tools was adjusted on the lowest confidence level ( 0.15 ) to cover more associations . cinnamaldehyde ( hplc , > 99.9% ) , eugenol ( hplc grade ; > 98% ) , dmso and antibiotics were from sigma - aldrich . the plasmid dna of psicheck-2 , used in telomere length determination , was a gift from dr farideh talebi . human ascs were prepared , after filling a consent form by a 34-year - old woman who was admitted to a hospital for caesarean section . the hascs preparation and stem cell confirmation methods were as described method in our recent publication ( 12 ) . four groups of hascs studied were treated for 48 hr : 2.5 m / ml cinnamaldehyde treated , 0.1 g / ml eugenol treated , 0.01% dmso treated and untreated hascs . cinnamaldehyde and eugenol were primarily dissolved in 0.01% dmso in phosphate buffer , which is the solvent . we have selected this concentrations according to the cheminformatics and experimental information about the best results of doubling time of hascs denoted in recently published work . according to mentioned study , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol exert better cell viability and doubling time on hascs ( 12 ) . treated and untreated hascs were assessed for the telomere length and the expression of tert , akt1 and dkc1 genes . rna extraction ( geneall ) , complementary dna ( cdna ) synthesis ( viogene ) and total dna extraction ( qiagen ) were performed according to the kit instructions . semi - quantitative pcr was done for tert , akt1 , dkc1 and actin - beta gene expression , and quantitative pcr ( qpcr ) was done for telomere length determination . primers and pcr programs are shown in tables 1 and 2 . for absolute telomere length by diploid genome measurement using qpcr , primer pairs used for expression analysis of human tert , akt1 , dkc1 and housekeeping actin - beta genes oligomers used for absolute quantification of hascs telomere length the telomerase interacting proteins ( tips ) were selected according to the telomerase database ( http://telomerase.asu.edu/ ) ( 18 ) . therefore , 3dss were downloaded from pdb or modeled using swissmodel server ( http://swissmodel.expasy.org/ ) ( 19 , 20 ) . table 3 shows the 3ds of modeled tips using swissmodel server . for docking analysis , the mol2 format of cinnamaldehyde and eugenol were prepared from zinc database ( http://zinc.docking.org ) ( 21 ) . molegro virtual docker ver.4.2 was used for docking analysis ; this software has about 87% docking accuracy ( 22 ) . the predicted tertiary structures of some tips or unites tr : telomerase enzyme ; rnp : ribonucleoprotein ; tert : telomerase gene the gene folds , absolute quantities of telomere length and docking scores were analyzed by spss software ver.20 . the meansd was compared using analysis of variances ( anova ) and tukey - hsd with a confidence interval of 95% . also , cluster analysis was performed on the docking scores to select the best scores presentation as graphic views of docking . for clustering , the algorithm of nearest neighbor method and squared euclidean distance that was standardized with z score in spss software were used . string database at the url http://string-db.org/ predicts and plots the protein - protein direct or indirect association networks . the string confidence of association net tools was adjusted on the lowest confidence level ( 0.15 ) to cover more associations . untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . k562 cells , telomerase positive cell lines , were used to confirm whether the designed primers are competent for amplification of detectable tert in qpcr reaction . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . the 3ds prediction was done for human gar1p , nhp2 , nop10 , pinx1 , stau2 , tert and dyskerin that had no 3ds on the protein data bank . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . clustering for docking scores performed by the nearest neighbor method and the measure of squared euclidian distance . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . cinnamaldehyde tend to inhabit the hydrophobic parts of the proteins , although in some cases , it formed hydrogen bonds with hydrophilic domains of tips . according to the cluster analysis , the ligand - protein docking scores were divided into 8 distinct clusters . the dendrogram of cluster analysis represents calculated distances between the best phytochemicals - tips docking scores ( figure 4 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . analysis of variance ( anova ) shows there is a significant difference between all scores ( ci= 0.95 ; p= 0.000 ) there was a significant difference between docking scores of tips - phytochemicals ( ci= 0.95 ; p - value= 0.000 ) . as an example of docking results and according to the cluster analysis , only the best interactions from each category are presented in this article ( figure 6 - 13 ) . an interesting result was obtained for eugenol and telomeric dna binding domain of hnrnpa1 protein ( pdb i d : 1pgz ) . the eugenol was exactly docked in the binding site of the telomeric dna purines of hnrnpa1 ( figure 14 ) . docking result of rpl22 ( pdb i d : 3j3a ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 6 of clustering docking result of hsp90aa1 ( pdb i d : 3q6n ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 3 of clustering docking result of ku70-ku80 hetero - complex ( pdb i d : 1jeq ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 4 of clustering docking result of 14 - 3 - 3 ( pdb i d : 2br9 ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 1 of clustering docking result of kip ( pdb i d : 1y1a ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . this score belongs to category 8 of clustering docking result of smn1 ( pdb i d : 1g5v ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bound of ligand - protein interaction ( striated line ) in binding site pocket . this score belongs to category 7 of clustering docking result of hnrnpa1 ( pdb i d : 1pgz ) protein with eugenol ; left : binding site of eugenol ( green ) on the protein ; right : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . the molecule eugenol locates in a deep binding pocket of protein figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . was selected for drawing the association net ; apart from the stau2 protein , all the 30 proteins had at least a weak association . according to the string - db biological function analyzer ( 23 ) , the most important biological pathways , the tips , play roles include : intrinsic apoptosis pathway , intracellular transport , cellular localization , membrane organization , cell organelle organization , protein localization , etc . also , molecular functions affected by tips include : the telomeric dna binding , poly(a ) rna binding , telomerase activity , rna binding , rna - directed dna polymerase activity and telomeric rna binding . besides , protein kinase c inhibitory activity , ion channel binding , enzyme binding and nitric oxide synthase regulatory activity . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 untreated hascs were compared with 0.01% dmso , 2.5 m / ml cinnamaldehyde or 0.1 g / ml eugenol treated ones . k562 cells , telomerase positive cell lines , were used to confirm whether the designed primers are competent for amplification of detectable tert in qpcr reaction . 0.01% dmso and 2.5 m / ml cinnamaldehyde treated hascs had higher akt1 folds ( p<0.05 ) when compared with the untreated cells . but the akt1 folds did not change in the 0.1 g / ml eugenol treated cells as compared to the untreated hascs ( p>0.05 ) ( figure 1 ) . the 0.01% dmso and 2.5 m / ml cinnamaldehyde significantly increased the dkc1 gene folds in hascs ( p<0.05 ) . but 0.1 g / ml eugenol decreased the dkc1 folds ( p<0.05 ) compared with the untreated cells ( figure 2 ) . untreated hascs has the longest telomere length as compared to 0.01% dmso , 2.5 m / ml cinnamal - dehyde or 0.1 g / ml eugenol treated hascs ( figure 3 ) . there was no statistical difference among 0.01% dmso and untreated or phytochemicals treated cells ( p>0.05 ) . generally , the phytochemicals treated hascs had shortened telomeres as compared to the untreated cells ( p<0.05 ) . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the akt1 expression folds in hascs . nevertheless , the 0.1 g / ml eugenol did not affect akt1 expression significantly tukey - hsd and anova analysis for dkc1 folds comparison . the figure shows the 0.01% dmso or 2.5 m / ml cinnamaldehyde increase the dkc1 expression folds in hascs . but the 0.1 g / ml eugenol decreased dkc1 expression significantly telomere length comparison between examined groups . the control hascs , 2.5 m / ml cinnamaldehyde and 0.1 g / ml eugenol significantly decreased the telomere length ( p - value < 0.05 ) but not in comparison with 0.01% dmso group ( p - value > 0.05 ) figure 4 shows the gene name , number of docking sites and the best minimum docking scores of cinnamaldehyde and eugenol with 22 tips . the 3ds prediction was done for human gar1p , nhp2 , nop10 , pinx1 , stau2 , tert and dyskerin that had no 3ds on the protein data bank . dendrogram of cluster analysis for 22 tips plus telomerase ( tert ) ; left table represents number of binding sites on each protein for both cinnamaldehyde and eugenol and minimum docking scores . clustering for docking scores performed by the nearest neighbor method and the measure of squared euclidian distance . some important docking results are shown in figure 6 - 14 docking scores of both cinnamaldehyde and eugenol were analyzed using cluster analysis ( figure 4 ) and one - way anova ( figure 5 ) . cinnamaldehyde tend to inhabit the hydrophobic parts of the proteins , although in some cases , it formed hydrogen bonds with hydrophilic domains of tips . according to the cluster analysis , the ligand - protein docking scores were divided into 8 distinct clusters . the dendrogram of cluster analysis represents calculated distances between the best phytochemicals - tips docking scores ( figure 4 ) . the cinnamaldehyde and eugenol docked with tips in molegro virtual docker ( mvd ) software . analysis of variance ( anova ) shows there is a significant difference between all scores ( ci= 0.95 ; p= 0.000 ) there was a significant difference between docking scores of tips - phytochemicals ( ci= 0.95 ; p - value= 0.000 ) . as an example of docking results and according to the cluster analysis , only the best interactions from each category are presented in this article ( figure 6 - 13 ) . an interesting result was obtained for eugenol and telomeric dna binding domain of hnrnpa1 protein ( pdb i d : 1pgz ) . the eugenol was exactly docked in the binding site of the telomeric dna purines of hnrnpa1 ( figure 14 ) . docking result of rpl22 ( pdb i d : 3j3a ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . it is the best score of dockings and belong to the category 5 of clustering docking result of tert ( modeled structure ) protein with eugenol ; above : binding site of the eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 6 of clustering docking result of hsp90aa1 ( pdb i d : 3q6n ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 3 of clustering docking result of ku70-ku80 hetero - complex ( pdb i d : 1jeq ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 4 of clustering docking result of 14 - 3 - 3 ( pdb i d : 2br9 ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 1 of clustering docking result of kip ( pdb i d : 1y1a ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . this score belongs to category 2 of clustering docking result of rnpc2 ( pdb i d : 2cq4 ) protein with cinnamaldehyde ; above : binding site of cinnamaldehyde ( green ) on the protein ; below : binding pocket has not hydrogen bounds . this score belongs to category 8 of clustering docking result of smn1 ( pdb i d : 1g5v ) protein with eugenol ; above : binding site of eugenol ( green ) on the protein ; below : hydrogen bound of ligand - protein interaction ( striated line ) in binding site pocket . this score belongs to category 7 of clustering docking result of hnrnpa1 ( pdb i d : 1pgz ) protein with eugenol ; left : binding site of eugenol ( green ) on the protein ; right : hydrogen bounds of ligand - protein interaction ( striated lines ) in binding site pocket . figure 15 shows an association network between tips and telomerase ; this is plotted using string data base ( see materials and methods ) . was selected for drawing the association net ; apart from the stau2 protein , all the 30 proteins had at least a weak association . according to the string - db biological function analyzer ( 23 ) , the most important biological pathways , the tips , play roles include : intrinsic apoptosis pathway , intracellular transport , cellular localization , membrane organization , cell organelle organization , protein localization , etc . also , molecular functions affected by tips include : the telomeric dna binding , poly(a ) rna binding , telomerase activity , rna binding , rna - directed dna polymerase activity and telomeric rna binding . besides , protein kinase c inhibitory activity , ion channel binding , enzyme binding and nitric oxide synthase regulatory activity . some abbreviations in the string - db plotted net were not mentioned in the text ; they include : rbm38 equals rnpc1 ; rbm39 equals rnpc2 ; xrcc6 equals ku70 ; xrcc5 equals ku80 ; ncl equals nucleolin ; la equals ssb ; cib1 equals kip ; ywhaq equals 14 - 3 - 3 ; tmed10 equals p23 recently , we have reported that cinnamaldehyde and eugenol affect the hascs doubling time and differentiation ( 12 ) . in the current study , cinnamalde - hyde and eugenol did not increase the tert expression to a detectable level in hascs . however , human mesenchymal stem cells had been previously reported to be telomerase negative ( 24 ) . we have used k562 cell lines , as a positive cell line model for tert gene expression during real - time pcr method validation . as far as we know , there is not known chemical material with a definite positive effect on the telomerase expression in adscs . eugenol decreased the akt1 expression but cinnamaldehyde increased or decreased it as compared to the untreated or dmso treated cells , respectively . when 0.01% dmso is also present in the cinnamal - dehyde and eugenol groups , as the solvent of phytochemicals ( see materials and methods ) , the ultimate effect of both phytochemicals on the akt1 gene expression will decrease . this evidence confirms more potent effect of eugenol on the decrease of the akt1 expression . cinnamaldehyde or dmso - treated hascs increased the dkc1 gene expression folds but the eugenol decreased it severely as compared to the untreated cells . these findings suggest that the final targets of cinnamaldehyde and eugenol are different in the intracellular environment . the effect of cinnamal - dehyde and eugenol on some tips or cell aging was reported previously . for example , cinnamaldehyde up - regulates the stau2 expression in mutz-3 cell line and human peripheral blood monocyte - derived dendritic cells ( 25 ) . according to the comparative toxicogenomics database ( http://ctdbase.org/ ) , king and colleagues reported that cinnamaldehyde induces the dna damage in hct116 , a human colon cancer cell line ( 9 ) . the result of the present study on telomere length showed that damage such as telomere shortening may be obvious ( figure 3 ) . the hallmark of some publications is the insignificance of the effect of solvents on the gene expression of treated cells . king and coworkers did not report the effect of dmso on the hct116 cell line . in the current study , it was shown that a low concentration of dmso increases both akt1 and dkc1 gene folds when compared with the untreated cells . the findings of this study infer the importance of the solving agent ( dmso ) effects on the gene expression and cell aging . cinnamaldehyde and eugenol reduced the telomere lengths as compared to the untreated cells notably , but insignificantly when compared with dmso treated hascs . then , a low concentration of dmso had a negative effect on the hascs telomeres , although not significantly ( figure 3 , p - value= 0.110 ) . cinnamaldehyde and eugenol treated hascs telomeres were not meaningfully different from dmso treated cells ( figure 3 , p > 0.05 ) , although the telomere lengths were shortened . it is assumed that the broad ranges of telomere lengths in the studied groups ( see standard deviations ) , are responsible for the results of telomere length comparisons insignificance . absolute quantification of the telo - mere length using quantitative polymerase chain reaction ( pcr ) showed high coefficient of variations percent ( cv % ) . aviv and colleagues described this fact previously when comparing southern blot and qpcr techniques ( 26 ) . however , the qpcr is more rapid , lesser time consuming , with lower costs , without radioactive hazards and acceptable regression with southern blot ( 17 ) . the decreased telomere length , in the presence of phytochemicals , in the present work could be logical when considering the docking results . the docking results showed that cinnamaldehyde and eugenol attached well within the binding pockets on the tips . some herbal ingredients change the gene expression folds of molecular chaperons such as heat shock proteins ; therefore , medicinal plants ingredients are proposed as chaperon - based medications ( 27 ) . based on the docking results , it is assumed that cinnamaldehyde and eugenol interact with chaperons , such hsp90aa1 , or chaperon inducers ; then they change the behaviors of tips . grover and coworkers , using a virtual study , reported that withaferin a , a phytochemical , targets the hsp90/cdc37-chaperone or co - chaperone complex . they proposed that the withaferin a is a hsp90/cdc37 inhibitor and a potent anticancer agent ( 28 ) . in addition , the findings of the present work proposed that cinnamaldehyde and eugenol might exert similar effects on the telomerase and tips . the molecular effects of cinnamaldehyde and eugenol on the hascs were telomere shortening and the gene expression fold changes . logically , such changes did not occur without involving regulatory events of the cell aging . researchers have shown that the active ingredients of some herbs inhibit the telomerase enzyme dose - dependently ( 29 , 30 ) but others increase its gene expression ( 31 , 32 ) . the role of herbal ingredients on the telomere or telomerase dependent anti - aging or aging induction is not well known . the study on direct interaction of ligands - proteins or biomolecules needs certain tools and professional operators . nevertheless , computational biology tools and analysis , such as docking , helps in predicting the probable direct bindings . intracellular environment has dynamic properties ( 33 ) ; therefore , the chemicals interact with biomolecules in a dynamic way . this infers that the best docking scores are of interest , especially if the dynamic binding site conformations change whenever it is necessary . elizabeth h. blackburn is a noble prizewinner for her works on the telomere and telomerase enzyme . she believes the telomerase and the telomere interacting enzymes exert four distinct biochemical effects on the telomeric dna in vivo :  1 . in fusion with another telomere or dna end , it has been shown that using computational tools , the cinnamaldehyde and eugenol may target tips efficiently . after cinnamaldehyde and eugenol docking with tips or regulating proteins , three effects are probable : telomere shortening , lengthening or no length change . figure 16 proposes a subjective map for the in silico and experimental results of this study . according to the map , the probable effects of the cinnamaldehyde and eugenol on the senescence and expected events after the cell treatment . the cinnamaldehyde and eugenol may bind to tips or act on their gene expression and impel the signal transduction pathways . the left gray box shows the gene names of proteins , which have positive effect on the telomere or telomerase . the left gray box shows two proteins with negative effects on the telomere or telomerase . in our work then , the red lines show the probable occurred events in the current work the hnrnpa1 activates the telomerase ( 35 ) , but as shown in figure 14 , it also interacts with telomeric repeats ( 36 ) . the eugenol docking with hnrnpa1 is the most significant result of docking , which occurred exactly in the telomeric dna binding pocket . such result may suggest a competitive inhibition between telomeric repeats of the dna and the eugenol for binding to the hnrnpa1 . same result was derived for cinnamaldehyde and hnrnpa1 docking and exactly in the binding pocket ( the result is not shown ) . they believed that eugenol metabolism products exert toxic effect on the rat hepatocytes dose - dependently ( 37 ) . also , eugenol inhibits the monoamine oxidase a ( maoa ) , a metabolic enzyme , through the covalent binding ( 11 ) . all mentioned studies show negative effect of eugenol on the cellular proteins and are in accordance with the experimental and virtual findings of the present study . weibel and hansen have shown that the cinnamaldehyde binds to the nucleophilic groups of proteins ( 38 ) . python and coworkers evaluated the effect of cinnamaldehyde on 80 gene expression profiles in dendritic cells and mutz-3 cell line . using dna microarray and qpcr , they showed that some genes were down - regulated but others were up - regulated ( 25 ) . all the mentioned studies confirm the present study results of cinnamaldehyde effect on the gene expression changes . in this work , however , in comparison with the only dmso treated hascs , cinnamaldehyde decreased akt1 and increased dkc1 folds . these findings are in accordance with python and coworkers results on the discrepant behavior of cinnamaldehyde in the gene expression folds . a single ligand - protein docking , as cinnamaldehyde or eugenol to any tip , is not enough for interpreting experimental results . in fact , other protein - protein interrelations are important for their impacts on the special pathway members . the ligand - protein interaction may exert direct or indirect effect on protein - protein interactions in a pathway . tips are good examples of such interactions because of the different roles they play in the cell signaling ( 33 , 39 ) . the network shows that the protein - protein interactions may be weak or strong ; therefore , the docking affects the quantities of signaling changes . mount and pandey reported on the importance of the systems biology application for new drug finding . they emphasized the inter - relations between the different biomolecules in an event , such as cancer development ( 40 ) . in fact , such interrelationships between members of a biological network make sequential steps logical . curcumin , the active ingredient of turmeric , makes sequentially , activation or inactivation in the signaling pathways ( 16 ) . according to the in silico analysis of the present study , the cumulative effects of cinnamaldehyde or eugenol therefore , any prediction or estimation on phytochemical targets is not true without considering pre or post events in a signaling pathway . curcumin down - regulates the cell division , apoptosis and metastatic genes in nf - kappa b signaling in a sequential order ( 16 ) . cinnamaldehyde is proposed as a chemopreventive agent and has toxic effect on cancer cell lines ( 8 , 41 ) . altogether , cinnamaldehyde and eugenol proposed as anticancer agents which affect the human cells through the telomere shortening ; they change the gene folds and have many targets , of which some were checked virtually and 3 , experimentally in the present study . combining the in silico and experiments offer a novel view of the phytochemicals effects on the stem cell aging . this study is a novel one as it focused on the phytochemicals effects on hascs . the results of this study revealed that cinnamaldehyde and eugenol might be useful in cancer therapy or chemoprevention because of the induction of stem cell aging characteristics . in silico predictions , confirmed the results of ex - vivo interventional results . the aims of this study were to explore the stem cell anti - aging properties of the phytochemicals but surprisingly , the adverse results were obtained . the cinnamaldehyde and eugenol could be considered as the potential chemothera - peutic agents for cancer prevention via telomere shortening or telomerase blockade in proliferating cells such as cancer stem cells . however , the field is so large for complementary works on the shelterins , regulatory and epigenetics agents . the authors declare that there is no conflict or competing interest for the publication of the present data .
objective(s):to investigate the effect of cinnamaldehyde and eugenol on the telomere - dependent senescence of stem cells . 
 in addition , to search the probable targets of mentioned phytochemicals between human telomere interacting proteins ( tips ) using in silico studies.materials and methods : human adipose derived stem cells ( hascs ) were studied under treatments with 2.5 m / ml cinnamaldehyde , 0.1 g / ml eugenol , 0.01% dmso or any additive . 
 the expression of tert , akt1 and dkc1 genes and the telomere length were assessed over 48-hr treatment . 
 in addition , docking study was conducted to show probable ways through which phytochemicals interact with tips.results:treated and untreated hascs had undetectable tert expression , but they had different akt1 and dkc1 expression levels ( ci=0.95 ; p<0.05 ) . 
 the telomere lengths were reduced in phytochemicals treated with hascs when compared with the untreated cells ( p<0.05 ) . 
 docking results showed that the tips might be the proper targets for cinnamaldehyde and eugenol . 
 data mining showed there are many targets for cinnamaldehyde and eugenol in the intracellular environment.conclusion:the general effect of cinnamaldehyde and eugenol is their induction of stem cell senescence . 
 therefore , they could be applicable as chemo - preventive or antineoplastic agents .