The preparation and microbiological evaluation of the inhibitory effects of some acrylic acid derivatives

The following acrylic acid derivatives have been prepared and microbiologically evaluated as possible inhibitors of the growth of lactobacilli; indoleacrylic acid, β-(2-quinolyl)-, β-(3-quinolyl)-, β-(4-quinolyl) acrylic acids, cinnamic acid, p-hydroxycinnamic acid, p-dimethylaminocinnamic acid, p-diethylaminocinnamic acid, thienylacrylic acid, furylacrylic acid, and α-ethylacrylic acid.The utilization of tryptophan by Leuconostoc mesenteroides P-60 and Lactobacillus arabinosus was inhibited by the isomeric quinolylacrylic acid derivatives as well as by indoleacrylic acid. With this latter compound and the β-(3-quinolyl)acrylic acid, competitive inhibition was shown.p-Hydroxycinnamic acid inhibited the utilization of phenylalanine and tyrosine by all the organisms tested. At similar concentrations neither cinnamic acid nor phenol exerted any inhibitory effect.The effects of all inhibitors could be at least partially reversed by the addition of larger quantities of the corresponding amino acids.     

Role de la phenylalanine dans la biosynthese des flavonoides et acides “cinnamique” dans les tiges volubiles de Periploca Graeca

Phenylalanine is the precursor of the cinnamic acids and coumarins in the stems and leafs of P. graeca L. Esterification of p-coumaric acid by quinic acid is required before oxidation to chlorogenic acid. In our experiments, we did not obtain radioactive flavonols from ¹⁴C phenylalanine. PAL activity varies as a result of light and temperature in the same manner as the level of flavonoids (especially the phenolic acids). This enzyme, therefore, plays a regulatory role in the synthesis of these phenolic substances. The variation in PAL activity during illumination does not follow the same course as described for other plants.     

Bacterial Anabaena variabilis phenylalanine ammonia lyase: A biocatalyst with broad substrate specificity

Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids.     

The effect of herbicides,plant growth regulators and other compounds on phenylalanine ammonia-lyase activity

The in vitro and in vivo effects of a number of herbicides and plant growth regulators on phenylalanine ammonia-lyase (PAL) activity were investigated. The most elective in vitro inhibitors were product analogs, t-cinnamic and p-coumaric acids, and carbonyl reagents, hydroxylamine and nitromethane. Application of the herbicides diuron, dalapon, amiben, and chloropropham, to plants resulted in a decrease in the intracellular concn of PAL. The inhibitory effect of alachlor was found to be dose-responsive and somewhat specific. A correlation between PAL inhibition and herbicidal activity was observed for hydroxylamine. The cytokinin, pyranyl benzyladenine, (PBA) increased PAL activity in pigweed. The possibility of developing herbicides acting through PAL inhibition is discussed.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.     

Metabolic profiling, free-radical scavenging and tyrosinase inhibitory activities of Lemna minor whole plants cultivated in various concentrations of proline and sucrose

Metabolic profiling of Lemna minor whole plants cultivated in proline and sucrose at various concentrations was performed using gas chromatography-mass spectrometry coupled with multivariate statistical analysis. In total, 46 compounds including alkaloids, amino acids, fatty acids, organic acids, phenolics, phytosterols, purines, and sugars were assigned, and the relative levels of these metabolites were compared in the L. minor whole plant samples. Free-radical scavenging and tyrosinase inhibitory activities were also investigated in L. minor whole plants cultivated in proline and sucrose at various concentrations. The whole plants cultivated in 3% sucrose and 0.5 mM proline for 42 days exhibited the highest antioxidative and tyrosinase inhibitory activities, and biomass accumulation. Total phenolic content and cinnamic acid content were also highest under those conditions, which might have contributed to the free-radical scavenging and tyrosinase inhibitory activities. There were strong correlations between free-radical scavenging activity and total phenolic content, and between tyrosinase inhibition activity and cinnamic acid content in L. minor whole plants cultivated under various conditions.     

Phenolcarboxylic acids from medicinal herbs exert anticancer effects through disruption of COX-2 activity

Integrated research of herbs and formulas characterized by functions of promoting blood circulation and removing blood stasis is one of the most active fields in traditional Chinese medicine. This paper strives to demonstrate the roles of a homologous series of phenolcarboxylic acids from these medicinal herbs in cancer treatment via targeting cyclooxygenase-2 (COX-2), a well-recognized mediator in tumorigenesis. We selected thirteen typical phenolcarboxylic acids (benzoic acid derivatives, cinnamic acid derivatives and their dehydration-condensation products), and found gallic acid, caffeic acid, danshensu, rosmarinic acid and salvianolic acid B showed 50% inhibitory effects on hCOX-2 activity and A549 cells proliferation. 2D-quantitative method was introduced to describe the potential structural features that contributed to certain bioactivities. We also found these compounds underwent responsible hydrogen bonding to Arg120 and Ser353 in COX-2 active site residues. We further extensively focused on danshensu [d-(+)-β-(3,4-dihydoxy-phenylalanine)] or DSS, which exerted COX-2 dependent anticancer manner. Both genetic and pharmacological inhibition of COX-2 could enhance the ability of DSS inhibiting A549 cells growth. Additionally, COX-2/PGE₂/ERK signaling axis was essential for the anticancer effect of DSS. Furthermore, combined treatment with DSS and celecoxib could produce stronger anticancer effects in experimental lung metastasis of A549 cells in vivo. All these findings indicated that phenolcarboxylic acids might possess anticancer effects through jointly targeting COX-2 activity in cancer cells and provided strong evidence in cancer prevention and therapy for the herbs characterized by blood-activating and stasis-resolving functions in clinic.     

Carbonic anhydrase inhibitors. Inhibition of mammalian isoforms I–XIV with a series of natural product polyphenols and phenolic acids

A series of phenolic acids and phenol natural products, such as p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, ferulic acid, gallic acid, syringic acid, quercetin, and ellagic acid, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). All mammalian isozymes of human (h) or murine (m) origin hCA I–hCA XII, mCA XIII and hCA XIV were inhibited in the low micromolar or submicromolar range by these (poly)phenols (K_Is in the range of 0.87–7.79 μM). p-Hydroxybenzoic acid was the best inhibitor of all isozymes (K_Is of 0.87–35.4 μM) and the different isozymes showed very variable inhibition profiles with these derivatives. Phenols like the ones investigated here possess a CA inhibition mechanism distinct of that of the sulfonamides/sulfamates used clinically or the coumarins. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule and bind more externally within the active site cavity, making contacts with various amino acid residues. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs.     

Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots

Phenylalanine ammonia-lyase (PAL) activity, 11 phenolic acids and lignin accumulation in Matricaria chamomilla roots exposed to low (3 μM) and high (60 and 120 μM) levels of cadmium (Cd) or copper (Cu) for 7 days were investigated. Five derivatives of cinnamic acid (chlorogenic, p-coumaric, caffeic, ferulic and sinapic acids) and six derivatives of benzoic acid (protocatechuic, vanillic, syringic, p-hydroxybenzoic, salicylic acids and protocatechuic aldehyde) were detected. Accumulation of glycoside-bound phenolics (revealed by acid hydrolysis) was enhanced mainly towards the end of the experiment, being more expressive in Cu-treated roots. Interestingly, chlorogenic acid was extremely elevated by the highest Cu dose (21-fold higher than control) suggesting its involvement in antioxidative protection. All compounds, with the exception of chlorogenic acid, were detected in the cell wall bound fraction, but only benzoic acids were found in the ester-bound fraction (revealed by alkaline hydrolysis). Soluble phenolics were present in substantially higher amounts in Cu-treated roots and more Cu was retained there in comparison to Cd. Cu strongly elevated PAL activity (by 5.4- and 12.1-fold in 60 and 120 μM treatment, respectively) and lignin content (by 71 and 148%, respectively) after one day of treatment, indicating formation of a barrier against metal entrance. Cd had slighter effects, supporting its non-redox active properties. Taken together, different forms of phenolic metabolites play an important role in chamomile tolerance to metal excess and participate in active antioxidative protection.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Some allelochemicals of Pteridium aquilinum and their involvement in resistance to Pieris brassicae

A number of polar and semi-polar compounds from bracken, Pteridium aquilinum L. Kühn, were screened for feeding deterrent activity to Pieris brassicae L. At natural concentrations, 2 out of 4 flavonoids tested were stimulatory and one was inhibitory; while 2 out of 4 benzoic, and 4 out of 5 cinnamic acid derivatives were inhibitory. Prunasin and its enzymatic hydrolysates had no effect on feeding. It was concluded that braken flavonoids and cyanogenic glycoside were unlikely to be involved in resistance of polar bracken extracts to P. brassicae, while benzoic and cinnamic acids and condensed tannins were probably involved. A number of other benzoic and cinnamic acid derivatives not occurring in bracken were screened, and distinct relationships between chemical structure and feeding deterrent activity to P. brassicae were demonstrated.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

Structure–activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid,sorbic acid and hexanoic acid

In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d–6g, 10d–12g, 16d–18g and 22d–24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC₅₀ value: 3.64?µmol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Alterations in peroxidase activity and phenylpropanoid metabolism induced by Nacobbus aberrans Thorne and Allen, 1944 in chilli (Capsicum annuum L.) CM334 resistant to Phytophthora capsici Leo.

Chilli CM334 (Capsicum annuum L.) is resistant to Phytophthora capsici Leonian (Pc), but Nacobbus aberrans Thorne and Allen, 1944 (Na) broke down its resistance in plants previously infected by the nematode. Peroxidase (POD) and L-phenylalanine ammonia-lyase (PAL) activity, total soluble phenols (TSP) and chlorogenic acid concentration in CM334 plants inoculated with either or both pathogens (Na-Pc) were compared; also, the toxic effect of some phenolic acids on Na was tested in vitro. The highest POD activity (5.3 μM tetraguaiacol mg^?1 protein min^?1) was registered in plants inoculated only with Pc, while those inoculated only with Na showed the lowest (3.3 μM) (P?≤?0.05). PAL activity was 39.9 nM trans-cinnamic acid μg^?1 protein min^?1 in plants inoculated only with Pc, and it was lower (19.3 nM) and similar in non-inoculated plants or those with Na and with Na-Pc (P?≤?0.05). Usually, plants inoculated with Pc alone had higher contents of TSP (P?≤?0.05) (1.9 mg tannic acid g^?1 dry matter) and plants inoculated with Na or Na-Pc had lower levels (0.8 and 0.9 mg) than those non-inoculated (1.3 mg). CM334 plants inoculated with Na showed a significant reduction (10–37% and 12–17%, in roots and leaves) in the concentration of chlorogenic acid as compared to the non-inoculated. Vanillic, trans-cinnamic, p-coumaric and syringic acids had greater nematicidal effects (P?≤?0.05) than chlorogenic acid in vitro. Apparently Na modified the defence responses in CM334 plants as POD and PAL activities and TSP and chlorogenic acid concentrations were reduced.     

Effect of a novel aromatic cytokinin derivative on phytochemical levels and antioxidant potential in greenhouse grown Merwilla plumbea

In an attempt to elucidate the carry-over effect of cytokinins (CKs) on phytochemical and antioxidant activity of acclimatized plants, tissue culture-derived Merwilla plumbea supplemented with three CK types at four (0.25, 0.5, 1 and 2 µM) concentrations were grown for 6 months ex vitro. Phenolic acids including the hydroxybenzoic and hydroxycinnamic acid derivatives in M. plumbea were quantified using ultra performance liquid chromatography while the antioxidant activity was evaluated using oxygen radical absorbance capacity (ORAC). Different concentrations of gallic acid, protocatechuic acid, p-hydroxybenzoic acid and salicylic acid were observed with all the treatments with the exception of non-treated plants. Most phytochemicals (for example, gallic acid, ferulic acid protocatechuic acid and caffeic acid) were highest in plants obtained from 0.25 µM meta-topolin riboside (mTR). Likewise, plants derived from 2 µM mTR had the highest ORAC (684 µmol g^?1 trolox equivalents) activity. Bearing in mind that therapeutic effects of medicinal plants are often associated to their phytochemical content, the current results are an indication on how the intricate in vitro environment (CK type and concentration in this case) affects the growth and general physiology of micropropagated plants especially after acclimatization.     

Comparative metabolic activity related to flavonoid synthesis in leaves and flowers of Chrysanthemum morifolium in response to K deficiency

K limitation could decrease the flavonoids content in many Chinese traditional herbs. These results may be caused by the influence of K deficiency on the synthesis pathway of flavonoids. In this paper, we aim to study the influence of K deficiency on secondary metabolites and activities of phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H) enzymes in the process of flavonoid synthesis in Chrysanthemum morifolium Ramat. The results show that K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium while the same effect was obtained on C4H and PAL. Total flavonoids content increased with the content of cinnamic acid and p-coumaric acid in the plant under K deficiency, respectively. The regression equations between content of flavonoids and cinnamic acid/ p-coumaric acid should be expressed in term of linear effects (R²?=?0.9809, P?<?0.001 for cinnamic acid and R²?=?0.9929, P?<?0.0001 for p-coumaric acid, respectively). But the effect of phenylalanine on flavonoids should be expressed in term of quadratic pattern (R²?=?0.9375, P?<?0.05). There were two kinds principal components from chorismate to coumaryl CoA synthesis process, principal 1 was the substrate (phenylalanine, cinnamic acid, p-coumaric acid and PAL) and principal 2 was the enzyme (4CL, C4H), the principal 1 was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.17% and 9.64%), however, the factor loading (correlation coefficient of measured targets and the corresponding principal component) in principal 1 was opposite under different K application.     

A series of cinnamic acid derivatives and their inhibitory activity on intestinal α-glucosidase

Inhibition of α-glucosidase and α-amylase delays the digestion of starch and disaccharides to absorbable monosaccharides, resulting in a reduction of postprandial hyperglycemia. Finding effective mammalian α-glucosidase inhibitors from natural sources can be beneficial in the prevention and treatment of diabetes mellitus. We investigated the inhibitory activity of cinnamic acid derivatives against rat intestinal α-glucosidase and porcine pancreatic α-amylase in vitro. Among 11 cinnamic acid derivatives, caffeic acid, ferulic acid, and isoferulic acid were the most potent inhibitors against intestinal maltase with IC₅₀ values of 0.74?±?0.01, 0.79?±?0.04, and 0.76?±?0.03?mM, respectively, whereas ferulic acid (IC₅₀?=?0.45?±?0.01?mM) and isoferulic acid (IC₅₀?=?0.45?±?0.01?mM) were effective intestinal sucrase inhibitors. However, all cinnamic acid derivatives were found to be inactive in pancreatic α-amylase inhibition. Kinetic analysis revealed that intestinal maltase was inhibited by caffeic acid, ferulic acid, and isoferulic acid in a mixed-inhibition manner. In addition, ferulic acid and isoferulic acid inhibited intestinal sucrase in a mixed type manner, whereas caffeic acid was a non-competitive inhibitor. The combination of isoferulic acid and acarbose showed an additive inhibition on intestinal sucrase. This study could provide a new insight into naturally occurring intestinal α-glucosidase inhibitors that could be useful for treatment of diabetes and its complications.     

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.