Makoto Fukatsu

Biological Activities of Phenolic Compounds and Triterpenoids from the Galls of Terminalia chebula

Nine phenolic compounds, including two phenolic carboxylic acids, 1 and 2, seven hydrolyzable tannins, 3–9, eight triterpenoids, including four oleanane‐type triterpene acids, 10–13, and four of their glucosides, 14–17, isolated from a MeOH extract of the gall of Terminalia chebula Retz. (myrobalan tree; Combretaceae), were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells induced by α‐melanocyte‐stimulating hormone (α‐MSH), against the EpsteinBarr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, and against TPA‐induced inflammation in mice. Their 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activities and cytotoxic activities against four human cancer cell lines were also evaluated. Compounds 6–9 and 12 exhibited potent inhibitory activities against melanogenesis (39.3–66.3% melanin content) with low toxicity to the cells (74.5–105.9% cell viability) at a concentration of 10 μM. Western‐blot analysis revealed that isoterchebulin (8) reduced the protein levels of MITF (=microphtalmia‐associated transcription factor), tyrosinase, and TRP‐1 (=tyrosine‐related protein 1), mostly in a concentration‐dependent manner. Eight triterpenoids, 10–17, showed potent inhibitory effects on EBV‐EA induction with the IC50 values in the range of 269–363 mol ratio/32 pmol TPA, while these compounds exhibited no DPPH scavenging activities (IC50>100 μM). On the other hand, the nine phenolic compounds, 1–9, exhibited potent radical‐scavenging activities (IC50 1.4–10.9 μM) with weak inhibitory effects on EBV‐EA induction (IC50 460–518 mol ratio/32 pmol TPA). The tannin 6 and seven triterpenoids, 10–16, have been shown to inhibit TPA‐induced inflammation (1 μg/ear) in mice with the ID50 values in the range of 0.06–0.33 μmol/ear. Arjungenin (10) exhibited inhibitory effect on skin‐tumor promotion in an in vivo two‐stage mouse‐skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter. Compounds 1, 2, 4, 5, 7–9, 12, and 13, against HL60 cell line, compounds 1 and 4, against AZ521 cell line, and compounds 1, 11, and 12, against SK‐BR‐3 cell line, showed moderate cytotoxic activities (IC50 13.9–73.2 μM).

Antioxidative and Melanogenesis‐Inhibitory Activities of Caffeoylquinic Acids and Other Compounds from Moxa

The MeOH extract of moxa, the processed leaves of Artemisia princeps Pamp. (Asteraceae), exhibited potent 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity and melanogenesis‐inhibitory activity in α‐melanocyte‐stimulating hormone (α‐MSH)‐stimulated B16 melanoma cells. Eight caffeoylquinic acids, 1 and 6–12, five flavonoids, 13–17, two benzoic acid derivatives, 18 and 19, three coumarin derivatives, 20–22, four steroids, 23–26, and six triterpenoids, 27–32, were isolated from the MeOH extract. Upon evaluation of compounds 1, 6–23, and four semisynthetic caffeoylquinic acid esters, 2–5, for their DPPH radical‐scavenging activity, 15 compounds, 1–13, 17, and 19, showed potent activities (IC50 3.1–16.8 μM). The 15 compounds exhibited, moreover, potent inhibitory activities (51.1–92.5% inhibition) against peroxidation of linoleic acid emulsion at 10 μg/ml concentration. In addition, when 27 compounds, 1–8, 10, 12, 13, 15–18, 20–25, and 27–32, were evaluated for their inhibitory activity against melanogenesis in α‐MSH‐stimulated B16 melanoma cells, five caffeoylquinic acids, i.e., chlorogenic acid (1), ethyl chlorogenate (3), propyl chlorogenate (4), isopropyl chlorogenate (5), and butyl chlorogenate (6), along with homoorientin (17) and vanillic acid (18), exhibited inhibitory activities with 33–62% reduction of melanin content at 100 μM concentration with no or almost no toxicity to the cells (89–114% of cell viability at 100 μM). Western blot analysis showed that compound 6 reduced the protein levels of microphtalmia‐associated transcription factor (MITF), tyrosinase, tyrosine‐related protein 1 (TRP‐1), and TRP‐2 mostly in a concentration‐dependent manner, suggesting that this compound inhibits melanogenesis on α‐MSH‐stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP‐1, and TRP‐2. Furthermore, four compounds, 13, 15, 16, and 30, exhibited cytotoxicities against HL60 human leukemia cell line (IC50 7.0–11.1 μM), and nine compounds, 14–16, 23, 26–28, 31, and 32, showed inhibitory effects (IC50 272–382 mol ratio/32 pmol 12‐O‐tetradecanoylphohrbol‐13‐acetate (TPA)) against EpsteinBarr virus early antigen (EBV‐EA) activation induced by TPA in Raji cells.

Cytotoxic and Apoptosis‐Inducing Activities of Steviol and Isosteviol Derivatives against Human Cancer Cell Lines

Seventeen steviol derivatives, i.e., 2–18, and 19 isosteviol derivatives, i.e., 19–37, were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines, nine steviol derivatives, i.e., 5–9 and 11–14, and five isosteviol derivatives, i.e., 28–32, exhibited activities with single‐digit micromolar IC50 values against one or more cell lines. All of these active compounds possess C(19)‐O‐acyl group, and among which, ent‐kaur‐16‐ene‐13,19‐diol 19‐O‐4′,4′,4′‐trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC50 values in the range of 1.2–4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis. These results suggested that acylation of the 19‐OH group of kaurane‐ and beyerane‐type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis‐inducing activity.

Cytotoxic Activities and Anti-Tumor-Promoting Effects of Microbial Transformation Products of Prenylated Chalcones from Angelica keiskei

Three prenylated chalcones, 4‐hydroxyderricin (1), xanthoangelol (2), and xanthoangelol F (3), isolated from Angelica keiskei, were transformed by the fungus Aspergillus saitoi. These chalcones were converted to flavanones (i.e., 4, 8, and 12), and prenyl‐chain‐hydrated (i.e., 5, 7, 9–11, and 13) and ring‐B‐hydroxylated (i.e., 6) chalcones. The structures of three new metabolites, 7, 9, and 13, were established as 2″,3″‐dihydro‐4,3″‐dihydroxyderricin, 6″,7″‐dihydro‐7″‐hydroxyxanthoangelol, and 6″,7″‐dihydro‐7″‐hydroxyxanthoangelol F, respectively. Upon evaluation of cytotoxic activities of compounds 1–13, the metabolite 7 exhibited potent cytotoxicity against HL60 cells, and this cell death was revealed to be mostly due to apoptosis. In addition, compounds 1–4, 7–10, 12, and 13 were examined for their inhibitory effects on the induction of EpsteinBarr virus early antigen (EBV‐EA) by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells. All compounds tested showed inhibitory effects against EBV‐EA activation with potencies higher than that of β‐carotene. Furthermore, the metabolite 13 exhibited inhibitory effect on skin tumor promotion in an in vivo two‐stage mouse skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.

Melanogenesis-Inhibitory Saccharide Fatty Acid Esters and Other Constituents of the Fruits of Morinda citrifolia (Noni)

Five new saccharide fatty acid esters, named nonioside P (3), nonioside Q (4), nonioside R (8), nonioside S (10), and nonioside T (14), and one new succinic acid ester, butyl 2‐hydroxysuccinate (=4‐butoxy‐3‐hydroxy‐4‐oxobutanoic acid) (31), were isolated, along with 26 known compounds, including eight saccharide fatty acid esters, 1, 2, 5, 6, 7, 9, 12, and 13, three hemiterpene glycosides, 15, 17, and 18, six iridoid glycosides, 21–25, and 27, and nine other compounds, 20, 28, 29, and 32–37, from a MeOH extract of the fruit of Morinda citrifolia (noni). Upon evaluation of these and five other glycosidic compounds, 11, 16, 19, 26, and 30, from M. citrifolia fruit extract for their inhibitory activities against melanogenesis in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), most of the saccharide fatty acid esters, hemiterpene glycosides, and iridoid glycosides showed inhibitory effects with no or almost no toxicity to the cells. These compounds were further evaluated with respect to their cytotoxic activities against two human cancer cell lines (HL‐60 and AZ521) and their inhibitory effects on EpsteinBarr virus early antigen (EBV‐EA) activation induced with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.

Melanogenesis-Inhibitory and Cytotoxic Activities of Diarylheptanoids from Acer nikoense Bark and Their Derivatives

Nine cyclic diarylheptanoids, 1–9, including two new compounds, i.e., 9‐oxoacerogenin A (8) and 9‐O‐β‐D‐glucopyranosylacerogenin K (9), along with three acyclic diarylheptanoids, 10–12, and four phenolic compounds, 13–16, were isolated from a MeOH extract of the bark of Acer nikoense (Aceraceae). Acid hydrolysis of 9 yielded acerogenin K (17) and D‐glucose. Two of the cyclic diarylheptanoids, acerogenin A (1) and (R)‐acerogenin B (5), were converted to their ether and ester derivatives, 18–24 and 27–33, respectively, and to the dehydrated derivatives, 25, 26, 34, and 35. Upon evaluation of compounds 1–16 and 18–35 for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with α‐melanocyte‐stimulating hormone (α‐MSH), eight natural glycosides, i.e., six diarylheptanoid glycosides, 2–4, 6, 9, and 12, and two phenolic glycosides, 15 and 16, exhibited inhibitory activities with 24–61% reduction of melanin content at 100 μM concentration with no or almost no toxicity to the cells (88–106% of cell viability at 100 μM). In addition, when compounds 1–16 and 18–35 were evaluated for cytotoxic activity against human cancer cell lines, two natural acyclic diarylheptanoids, 10 and 11, ten ether and ester derivatives, 18–22 and 27–31, and two dehydrated derivatives, 34 and 35, exhibited potent cytotoxicities against HL60 human leukemia cell line (IC50 8.1–19.3 μM), and five compounds, 10, 11, 20, 29, and 30, against CRL1579 human melanoma cell line (IC50 10.1–18.4 μM).

Glycosidic Inhibitors of Melanogenesis from Leaves of Passiflora edulis

A new flavonoid glycoside, chrysin 6‐C‐β‐rutinoside (chrysin α‐L‐rhamnopyranosyl‐(1→6)‐C‐β‐glucopyranoside; 2), and two new triterpene glycosides, (31R)‐31‐O‐methylpassiflorine (7) and (31S)‐31‐O‐methylpassiflorine (8), along with 14 known glycosides, including three flavonoid glycosides, 1, 3, and 4, six triterpene glycosides, 5, 6, and 9–12, three cyano glycosides, 13–15, and two other glycosides, 16 and 17, were isolated from a MeOH extract of the leaves of Passiflora edulis (passion flower; Passifloraceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluation of compounds 1–17 against the melanogenesis in the B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), three compounds, isoorientin (1), 2, and (6S,9R)‐roseoside (17), exhibited inhibitory effects with 37.3–47.2% reduction of melanin content with no, or almost no, toxicity to the cells (90.8–100.2% cell viability) at 100 μM. Western blot analysis showed that compound 2 reduced the protein levels of MITF, TRP‐1, and tyrosinase, in a concentration‐dependent manner while exerted almost no influence on the level of TRP‐2, suggesting that this compound inhibits melanogenesis on the α‐MSH‐stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of TRP‐1 and tyrosinase. In addition, compounds 1–17 were evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.

Glycosidic inhibitors of melanogenesis from leaves of Momordica charantia.

Eight glycosidic compounds, 1–8, including two new compounds, (4ξ)‐α‐terpineol 8‐O‐[α‐L‐arabinopyranosyl‐(1→6)‐β‐D‐glucopyranoside] (5) and myrtenol 10‐O‐[β‐D‐apiofuranosyl‐(1→6)‐β‐D‐glucopyranoside] (7), were isolated from the BuOH‐soluble fraction of a MeOH extract of Momordica charantia leaves. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1–8 on the melanogenesis in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), these compounds were found to exhibit inhibitory activities with 7.1–27.0% and 23.6–46.4% reduction of melanin content at 30 μM and 100 μM, respectively, with no or almost no toxicity to the cells (80.0–103.5% of cell viability at 100 μM). Western blot analysis showed that compound 7 reduced the protein levels of MITF, tyrosinase, TRP‐1, and TRP‐2 mostly in a concentration‐dependent manner, suggesting that this compound inhibits melanogenesis on the α‐MSH‐stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP‐1, and TRP‐2.

Melanogenesis Inhibitory Activity of Sesquiterpenes from Canarium ovatum Resin in Mouse B16 Melanoma Cells

Four known sesquiterpene alcohols, i.e., 1–4, ten triterpene alcohols, i.e., 5–14, and four triterpene acids, i.e., 15–18, were isolated from the MeOH extract of Canarium ovatum resin (elemi resin). Upon evaluation of the previously described compounds 1–18 on the melanogenesis in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), three sesquiterpene alcohols, i.e., cryptomeridiol (1), 4‐epicryptomeridiol (2), and cadin‐1(14)‐ene‐7α,11‐diol (4), exhibited inhibitory effects with 27.4–34.1 and 39.0–56.9% reduction of melanin content at 50 and 100 μM, respectively, with no or very low toxicity to the cells (80.9–103.9% of cell viability at 100 μM). Western‐blot analysis revealed that compounds 1 and 2 reduced the protein levels of MITF (=microphtalmia‐associated transcription factor), tyrosinase, and TRP‐2 (=tyrosine‐related protein 2), mostly in a concentration‐dependent manner, suggesting that these compounds exhibit melanogenesis inhibitory activity on α‐MSH‐stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase and TRP‐2. Three sesquiterpene alcohols, i.e., 1, 2, and 4, are, therefore, considered to be valuable as potential skin‐whitening agents.

Cytotoxic activities of amino acid-conjugate derivatives of abietane-type diterpenoids against human cancer cell lines.

Nine amino acid conjugate derivatives, each 2–10 and 12–20, were prepared from abietic acid (1) and dehydroabietic acid (11), respectively, and they were evaluated for their cytotoxicities against four human cancer cell lines, i.e., leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3). All compounds showed cytotoxicities against HL60 with IC50 values in the range of 2.3–37.3 μM. In addition, most of the derivatives exhibited moderate cytotoxicities against the other cancer cell lines. Among the derivatives, methyl N‐[18‐oxoabieta‐8,11,13‐trien‐18‐yl]‐L‐tyrosinate (19) exhibited potent cytotoxic activities against four cancer cell lines with IC50 values of 2.3 (HL60), 7.1 (A549), 3.9 (AZ521), and 8.1 μM (SK‐BR‐3). Furthermore, all derivatives were shown to possess high selective cytotoxic activities for leukemia cells, since they exhibited only weak cytotoxicities against normal lymphocyte cell line RPMI1788.