Cardiac glycosides from the roots of Streblus asper Lour. with activity against Epstein-Barr virus lytic replication.

Cardiac glycosides (CGs) show potential broad-spectrum antiviral activity by targeting cellular host proteins. Herein are reported the isolation of five new (1-5) and eight known (7-13) CGs from the roots of Streblus asper Lour. Of these compounds 1 and 7 exhibited inhibitory action against EBV early antigen (EA) expression, with half-maximal effective concentration values (EC50) being less than 60 nM, and they also showed selectivity, with selectivity index (SI) values being 56.80 and 103.17, respectively. Preliminary structure activity relationships indicated that the C-10 substituent, C-5 hydroxy groups, and C-3 sugar unit play essential roles in the mediation of the inhibitory activity of CGs against EBV. Further enzyme experiments demonstrated that these compounds might inhibit ion pump function and thereby change the intracellular signal transduction pathway by binding to Na+/K+-ATPase, as validated by simulated molecular docking. This study is the first report that CGs can effectively limit EBV lytic replication, and the observations made in this study may be of value for lead compound development.

Melanogenesis-inhibitory activities of limonoids and tricyclic diterpenoids from Azadirachta indica.

The chemical constituents of the roots and bark of Azadirachta indica were investigated, leading to the isolation of six tricyclic diterpenoids and four limonoids including a new compound, azadirachtin J (4). The structures were elucidated on the basis of NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Furthermore, melanogenesis-inhibitory activities of the isolated compounds were evaluated. As a result, compounds 1-3 and 10 exhibited superior inhibitory activities against melanogenesis with no, or almost no, toxicity to the cells (86.5-105.1% cell viability). Western blot analysis showed that compounds 1 and 3 exhibited melanogenesis inhibitory activities in α-MSH-stimulated B16 melanoma cells due to, at least in part, inhibition of the expression of MITF, followed by a decrease in the expression of tyrosinase, TRP-1, and TRP-2. Compounds 1 and 3 exhibited tyrosinase inhibitory activities (IC50 values of 44.86 µM and 69.85 µM respectively). Docking results confirm that the active inhibitors strongly interact with tyrosinase residues.

Limonoids and tricyclic diterpenoids from Azadirachta indica and their antitumor activities.

The chemical constituents of the roots, seeds, and bark of Azadirachta indica var. siamensis were investigated, leading to the isolation of six tricyclic diterpenoids and five limonoids, including two new compounds (2, 5). The structures were elucidated based on NMR spectroscopic techniques, mass spectrometry and single-crystal X-ray diffraction as well as comparison with the literature. Moreover, the cytotoxicity activities of the isolates were evaluated. The results indicated that the compounds 1-3, 5-9 exhibited cytotoxicities against one or more cancer cell lines tested, with IC50 values in the range of 1.7-88.1 µM. The mechanism of action studies indicated that the most active compound, compound 5, could induce the apoptosis of AZ521 cells. Furthermore, the Western blot analysis showed that compound 5 could reduce the expression levels of procaspases-3, -8, -9 and promote the expression of Bid and AIF.

Characterization, quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper.

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na+,K+-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na+,K+-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-ß-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na+,K+-ATPase (IC50 7.55-13.60 µM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na+,K+-ATPase. Compound 4 could reasonably bind to the active sites of Na+,K+-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na+,K+-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na+,K+-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.

Melanogenesis-Inhibitory and Cytotoxic Activities of Triterpene Glycoside Constituents from the Bark of Albizia procera.

Five oleanane-type triterpene glycosides including three new ones, proceraosides E-G (1-3), were isolated from a MeOH-soluble extract of Albizia procera bark. The structures of 1-3 were determined by use of NMR spectra, HRESIMS, and chemical methods. Compounds 1-5 exhibited inhibitory activities against the proliferation of the A549, SKBR3, AZ521, and HL60 human cancer cell lines (IC50 0.28-1.8 µM). Additionally, the apoptosis-inducing activity of compound 2 was evaluated by Hoechst 33342 staining and flow cytometry, while the effects of 2 on the activation of caspases-9, -8, and -3 in HL60 cells were revealed by Western blot analysis.

Melanogenesis-Inhibitory and Cytotoxic Activities of Chemical Constituents from the Leaves of Sauropus androgynus L. Merr. (Euphorbiaceae).

A new steroid, 20-hydroxyisofucosterol (stigmasta-5,24(28)-diene-3ß,20ß-diol) (7), along with six known compounds 1 - 6 were isolated from the MeOH extract of the leaves of Sauropus androgynus L. Merr. (Euphorbiaceae). The structure of new steroid was determined by HR-APCI-MS and various NMR techniques in combination with literature data. Subsequently, their anti-inflammatory, cytotoxic activities against five human cell lines, as well as inhibitory activities against the α-MSH induced melanogenesis on the B16 cell line were evaluated. As the results, steroid compounds, 6 and 7 exhibited moderate cytotoxic to HL60, AZ521, SKBR3, and A549 tumor cell lines (IC50 26.9 - 45.1 µm) with high tumor selectivity for A549 relative to WI38 cell lines (SI 2.6 and 3.0, resp.). And, flavonoid compounds, 4 and 5 exhibited superior inhibitory activities against melanogenesis (67.0 - 94.7% melanin content), even with no or low toxicity to the cells (90.1 - 99.6% cell viability) at the concentrations from 10 to 100 µm. Furthermore, Western blot analysis suggested that compound 5 could inhibit melanogenesis by suppressing the protein expressions of MITF, TRP-1, TRP-2, and tyrosinase.

Chemical Constituents of the Seed Cake of Camellia oleifera and Their Antioxidant and Antimelanogenic Activities.

There is a growing interest in the exploitation of agricultural byproducts. This study explored the potential beneficial health effects from the main biowaste, tea seed pomace of Camellia oleifera Abel (Theaceae), produced when tea seed is processed. Eighteen compounds were isolated from the 70% EtOH extract of the seed cake of C. oleifera. Their structures were determined by ESI-MS, 1 H- and 13 C-NMR together with literature data. All fractions and compounds were evaluated for the antioxidant and melanogenesis inhibitory activities. As the result, AcOEt fraction has the best in vitro antioxidant and antimelanogenesis activities, compounds 7 - 12 and 15 showed remarkable antioxidant activity, compounds 4, 6, 8, and 15 - 17 exhibited superior inhibitory activities against melanogenesis. Furthermore, tyrosinase inhibitory activity assay suggested that compound 8 could suppress melanogenesis by inhibiting the expression of tyrosinase.

Nitric Oxide Production-Inhibitory Activity of Limonoids from Azadirachta indica and Melia azedarach.

Seventy-three limonoids isolated from three Meliaceae plants, Azadirachta indica, A. indica var. siamensis, and Melia azedarach, or semi-synthesized from the Meliaceae limonoids, were evaluated for their inhibitory activity against nitric oxide (NO) production in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), as a primary screening test for anti-inflammatory agents. Of the compounds tested, 21 compounds exhibited inhibitory activity (IC50 4.6 - 58.6 µm) without any significant toxicity (IC50 > 100 µm) which were more potent than l-NMMA (NO-production inhibitory activity, IC50 65.6 µm; cytotoxicity, IC50 > 100 µm), and among which, nine compounds, i.e., 17-hydroxy-15-methoxynimbocinol (6), ohchinin (20), 1-cis-cinnamoyl-1-decinnamoylohchinin (24), salannin (27), methyl nimbidate (32), isosalannin (55), nimbolinin D (58), mesendanin E (69), and 7-deacetylgedunin (73) exhibited potent inhibitory activity (IC50 4.6 - 29.3 µm). In particular, compounds 6 (IC50 7.3 µm), an azadirone-type limonoid, and 73 (IC50 4.6 µm), a gedunin-type limonoid, exhibited remarkable activity. Western blot analysis revealed that 27 and 73 reduced the expression levels of the inducible NO synthase and cyclooxygenase-2 proteins in a concentration-dependent manner. These findings suggest that limonoids of A. indica, A. indica var. siamensis, and M. azedarach, and their semi-synthetic derivatives may be effective against inflammation.

Melanogenesis-Inhibitory and Cytotoxic Activities of Limonoids, Alkaloids, and Phenolic Compounds from Phellodendron amurense Bark.

Four limonoids, 1 - 4, five alkaloids, 5 - 9, and four phenolic compounds, 10 - 13, were isolated from a MeOH extract of the bark of Phellodendron amurense (Rutaceae). Among these, compound 13 was new, and its structure was established as rel-(1R,2R,3R)-5-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1-(methoxycarbonylmethyl)indane-2-carboxylic acid methyl ester (γ-di(methyl ferulate)) based on the spectrometric analysis. Upon evaluation of compounds 1 - 13 against the melanogenesis in the B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), four compounds, limonin (1), noroxyhydrastinine (6), haplopine (7), and 4-methoxy-1-methylquinolin-2(1H)-one (8), exhibited potent melanogenesis-inhibitory activities with almost no toxicity to the cells. Western blot analysis revealed that compound 6 inhibited melanogenesis, at least in part, by inhibiting the expression of protein levels of tyrosinase, TRP-1, and TRP-2 in α-MSH-stimulated B16 melanoma cells. In addition, when compounds 1 - 13 were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), duodenum (AZ521), and breast (SK-BR-3) cancer cell lines, five compounds, berberine (5), 8, canthin-6-one (9), α-di-(methyl ferulate) (12), and 13, exhibited cytotoxicities against one or more cancer cell lines with IC50 values in the range of 2.6 - 90.0 µm. In particular, compound 5 exhibited strong cytotoxicity against AZ521 (IC50 2.6 µm) which was superior to that of the reference cisplatin (IC50 9.5 µm).

Melanogenesis-Inhibitory and Antioxidant Activities of Phenolics from Periploca forrestii.

Two new tetrahydrofuran-type lignans, (-)-gentioluteol 9-O-ß-d-glucopyranoside (1), (-)-berchemol 9-O-ß-d-apiofuranosyl-(1→6)-O-ß-d-glucopyranoside (2), along with sixteen known compounds 3 - 18 were isolated from the 95% EtOH extract of the stems of Periploca forrestii. The structures of the new tetrahydrofuran-type lignans were determined by HR-ESI-MS and various NMR techniques in combination with CD method. Then, their antioxidant abilities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, and ferric-reducing antioxidant power (FRAP) assays. Meanwhile, a similar trend was obtained in tripartite antioxidant assays, which compounds 7 - 9 and 11 exhibited potent abilities. Subsequently, the evaluation of all compounds against the α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis on the B16F10 cell line, compounds 5 - 11, 15, and 16 exhibited inhibitory effects with no or weak toxicity at low concentration. Of these, compound 8 exhibited the strongest inhibition melanogenesis ability. Furthermore, Western blot analysis suggested that compound 8 could inhibit melanogenesis by suppressing the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase.

Biological Activities of Phenolics from the Fruits of Phyllanthus emblica L. (Euphorbiaceae).

Seven phenolic compounds, 1 - 7, including a new organic acid gallate, mucic acid 1-ethyl 6-methyl ester 2-O-gallate (7), were isolated from the MeOH extract of the fruits of Phyllanthus emblica L. (Euphorbiaceae). The structures were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluated for their antioxidant abilities by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. The inhibitory activities against melanogenesis in B16 melanoma cells induced by α-MSH, as well as cytotoxic activities against four human cancer cell lines were also evaluated. All phenolic compounds, 1 - 7, exhibited potent antioxidant abilities (DPPH: IC50 5.6 - 12.9 µm; ABTS: 0.87 - 8.43 µm Trolox/µm; FRAP: 1.01 - 5.79 µm Fe2+ /µm, respectively). Besides, 5 - 7, also exhibited moderate inhibitory activities against melanogenesis (80.7 - 86.8% melanin content), even with no or low toxicity to the cells (93.5 - 101.6% cell viability) at a high concentration of 100 µm. Compounds 1 - 3 exhibited cytotoxic activity against one or more cell lines (IC50 13.9 - 68.4%), and compound 1 with high tumor selectivity for A549 (SI 3.2).

Melanogenesis-Inhibitory Activities of Isomeric C-seco Limonoids and Deesterified Limonoids.

Treatment of eight C-seco limonoids including six of salannin-type, 1 - 6, and two of nimbin-type, 7 and 8, with a combination of BF3 · Et2 O and iodide ion yielded the isomeric C-seco derivatives, i.e., six isosalannins, 1a - 6a, and two isonimbins, 7a and 8a, respectively. Ohchinin (1) was further subjected to LiAlH4 reduction which yielded a deesterified trihydroxy limonoid, nimbidinol (9). In addition, ten limonoids including seven of azadirone-type, 10 - 16, and three of gedunin-type, 17 - 19, all of which possess no ester functionality in the molecule, were obtained from the neutral fraction of Azadirachta indica seed extract after alkaline hydrolysis. Among the above, twelve compounds, i.e., 1a - 4a, 6a, 9, 13 - 16, 18, and 19, were new compounds, and their structures were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluation of all these limonoids for their inhibitory activities against melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), five structurally modified limonoids, 3-deacetyl-28-oxosalannin (6a), 9, 17-epi-17-hydroxynimbocinol (14), 17-epi-17-hydroxy-15-methoxynimbocinol (15), and 7-deacetyl-17-epinimolicinol (18), in addition to a natural limonoid, 1, exhibited potent inhibitory activities with 26 - 66% reduction of melanin content at 100 µm concentration with almost no or low toxicity to the B16 melanoma cells (70 - 99% cell viability at 100 µm).

Cytotoxic and Apoptosis-inducing Activities of Taraxastane-type Triterpenoid Derivatives in Human Cancer Cell Lines.

Twenty-eight taraxastane-type triterpenoid derivatives 4 - 31 were prepared from the naturally occurring triterpenoids faradiol (1) and heliantriol C (3). The cytotoxic activities of these compounds and arnidiol (2) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol 3,16-di-O-l-alaninate (31) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5 - 2.7 µm. In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane-type triterpenoid derivatives might provide useful antitumor agents with apoptosis-inducing activity.

Biological Activities of Triterpenoids and Phenolic Compounds from Myrica cerifera Bark.

Seven triterpenoids, 1 - 7, two diarylheptanoids, 8 and 9, four phenolic compounds, 10 - 13, and three other compounds, 14 - 16, were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin (1), ursolic acid (3), and myricanol (8) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK-BR-3 (breast) human cancer cell lines (IC50 3.1 - 24.2 µm). Compound 8 induced apoptotic cell death in HL60 cells (IC50 5.3 µm) upon evaluation of the apoptosis-inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases-3, -8, and -9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis-inhibitory activity in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), erythrodiol (7), 4-hydroxy-2-methoxyphenyl ß-d-glucopyranoside (13), and butyl quinate (15) exhibited inhibitory effects (65.4 - 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 - 107.4% cell viability) at 100 µm concentration. In addition, 8, myricanone (9), myricitrin (10), protocatechuic acid (11), and gallic acid (12) revealed potent DPPH radical-scavenging activities (IC50 6.9 - 20.5 µm).

Melanogenesis-inhibitory activity and cancer chemopreventive effect of glucosylcucurbic acid from shea (Vitellaria paradoxa) kernels.

Two jasmonate derivatives, glucosylcucurbic acid (1) and methyl glucosylcucurbate (2), were isolated from the MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels. These and their deglucosylated derivatives, cucurbic acid (3) and methyl cucurbate (4), were evaluated for their melanogenesis-inhibitory and cancer chemopreventive potencies. Compounds 1, 3, and 4 exhibited potent melanogenesis-inhibitory activities in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Western-blot analysis revealed that compounds 1 and 3 reduced the protein levels of MITF (=microphthalmia-associated transcription factor), tyrosinase, TRP-1 (=tyrosine-related protein 1), and TRP-2 mostly in a concentration-dependent manner. In addition, compound 1 exhibited inhibitory effects against Epstein-Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, against TPA-induced inflammation in mice, and against 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.

Potent anti-proliferative effects against oral and cervical cancers of Thai medicinal plants selected from the Thai/Lanna medicinal plant recipe database "MANOSROI III".

CONTEXT: Thai/Lanna medicinal plant recipes have been used for the treatment of several diseases including oral and cervical cancers. OBJECTIVE: To investigate anti-proliferative activity on human cervical (HeLa) and oral (KB) cancer cell lines of medicinal plants selected from Thai/Lanna medicinal plant recipe database "MANOSROI III". MATERIALS AND METHODS: Twenty-three methanolic plant crude extracts were tested for phytochemicals and anti-proliferative activity on HeLa and KB cell lines for 24 h by the sulforhodamine B (SRB) assay at the doses of 1 × 10(1)-1 × 10(-6 )mg/ml. The nine extracts with the concentrations giving 50% growth inhibition (GI50) lower than 100 µg/ml were further semi-purified by liquid/liquid partition in order to evaluate and enhance the anti-proliferative potency. RESULTS: All extracts contained steroids/triterpenoids, but not xanthones. The methanolic extracts of Gloriosa superba L. (Colchinaceae) root and Albizia chinensis (Osbeck) Merr. (Leguminosae-Mimosoideae) wood gave the highest anti-proliferative activity on HeLa and KB cell lines with the GI50 values of 0.91 (6.0- and 0.31-fold of cisplatin and doxorubicin) and 0.16 µg/ml (28.78- and 82.29-fold of cisplatin and doxorubicin), respectively. Hexane and methanol-water fractions of G. superba exhibited the highest anti-proliferative activity on HeLa and KB cell lines with the GI50 values of 0.15 (37- and 1.9-fold of cisplatin and doxorubicin) and 0.058 µg/ml (77.45- and 221.46-fold of cisplatin and doxorubicin), respectively. DISCUSSION AND CONCLUSION: This study has demonstrated the potential of plants selected from MANOSROI III database especially G. superba and A. chinensis for further development as anti-oral and cervical cancer agents.

Limonoids and flavonoids from the flowers of Azadirachta indica var. siamensis, and their melanogenesis-inhibitory and cytotoxic activities.

A new limonoid, 7-O-acetyl-7-O-debenzoyl-22-hydroxy-21-methoxylimocinin (2), and two new flavonoids, 3'-(3-hydroxy-3-methylbutyl)naringenin (7) and 4'-O-methyllespedezaflavanone C (9), along with nine known compounds, including two limonoids, 1 and 3, and seven flavonoids, 4-6, 8, and 10-12, were isolated from a MeOH extract of the flowers of Azadirachta indica A.Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their melanogenesis-inhibitory activities in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH). Compound 2 (16.9% melanin content at 30 µM), 6-deacetylnimbin (3; 49.6% melanin content at 100 µM), and kaempferide (10; 41.7% melanin content at 10 µM) exhibited inhibitory effects with no, or almost no, toxicity to the cells (81.0-111.7% cell viability). In addition, evaluation of their cytotoxic activities against HL60, A549, AZ521, and SK-BR-3 human cancer cell lines, isoazadironolide (1), 4'-O-methyl-8-prenylnaringenin (5), euchrestaflavanone A (8), 9, and 3-methoxy-3'-prenylnaringenin (12) revealed potent cytotoxicities against one or more cell lines with IC50 values in the range of 4.5-9.9 µM.

Three new and other limonoids from the hexane extract of Melia azedarach fruits and their cytotoxic activities.

A defatted fraction obtained from the hexane extract of the fruits of Melia azedarach L. (chinaberry tree; Meliaceae) exhibited cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines with IC50 values in the range of 2.9-21.9 µg/ml. Three new limonoids, 3-deacetyl-4'-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), along with 16 known limonoids, 1-4, 6-13, 15, 16, 18, and 19, and one known triterpenoid, 20, were isolated from the fraction. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. These compounds were evaluated for their cytotoxic activities against the four cancer cell lines mentioned above. 3-Deacetyl-4'-demethyl-28-oxosalannin (16), which exhibited potent cytotoxicity against AZ521 (IC50 3.2 µM) cells, induced typical apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometry. This work provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica.

Cytotoxic and apoptosis-inducing activities, and anti-tumor-promoting effects of cyanogenated and oxygenated triterpenes.

Two of each semisynthetic lanostane- and cycloartane-type triterpenes with a cyano-enone functionality, i.e., 13 and 18, and 23 and 28, respectively, sixteen of their synthetic intermediates, 9-12, 14-17, 19-22, and 24-27, along with seven semisynthetic oxygenated triterpene acetates, 29-35, and eight natural hydroxy triterpenes, 1-8, were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. One natural triterpene, 8, and ten semisynthetic triterpenes, 9, 13, 15, 18, 23, 25, 28, 29, 32, and 33, exhibited potent cytotoxicities against one or more cell lines with IC50 values in the range of 1.4-9.9 µM. Two lanostane-type triterpenes with a cyano-enone functionality, 3-oxolanosta-1,8,24-triene-2-carbonitrile (13) and 3-oxolanosta-1,8-diene-2-carbonitrile (18), induced apoptosis in HL60 cells, as observed by membrane phospholipid exposure in flow cytometry. Western blot analysis showed that 13 and 18 significantly reduced procaspases-3, -8, and -9, and increased cleaved caspases-3, -8, and -9. These findings indicated that compounds 13 and 18 induced apoptosis in HL60 cells via both the mitochondrial and the death receptor-mediated pathways. In addition, upon evaluation of the inhibitory effects on EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, seven natural triterpenes, 1-6 and 8, and ten semisynthetic triterpenes, 9, 10, 14, 15, 19, 20, 24, 25, 29, and 30, exhibited inhibitory effects which were higher than that of ß-carotene, a vitamin A precursor studied widely in cancer-chemoprevention animal models.

Limonoids from Azadirachta indica var. siamensis extracts and their cytotoxic and melanogenesis-inhibitory activities.

Six new limonoids, 7-benzoyl-17-epinimbocinol (5), 3-acetyl-7-tigloylnimbidinin (8), 1-isovaleroyl-1-detigloylsalanninolide (15), 2,3-dihydro-3α-methoxynimbolide (16), deacetyl-20,21-epoxy-20,22-dihydro-21-deoxyisonimbinolide (26), and deacetyl-20,21,22,23-tetrahydro-20,22-dihydroxy-21,23-dimethoxynimbin (27), along with 28 known limonoids, 1-4, 6, 7, 9-14, 17-25, and 28-34, and two known flavonoids, 35 and 36, have been isolated from the extracts of bark, leaves, roots, and seeds of Azadirachta indica A. Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. Eleven compounds, 1, 2, 4-7, 13, 16, 17, 29, and 30, exhibited potent cytotoxicities against one or more cell lines with IC50 values in the range of 0.1-9.3 µM. Compound 16 induced apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis on AZ521 cells revealed that compound 16 activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that 16 induced apoptosis via both mitochondrial and death receptor pathways in AZ521. In addition, upon evaluation of all compounds against the melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), 20 limonoids, i.e., 1-3, 6, 9-11, 18, 19, 21-29, 32, and 34, and two flavonoids, 35 and 36, exhibited melanogenesis-inhibitory activities, with no, or almost no, toxicities to the cells at lower and/or higher concentrations, which were more potent than the reference arbutin, a known melanogenesis inhibitor. Western blot analysis showed that nimbin (18) 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, indicating that 18 inhibits melanogenesis on a 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.