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).

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 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.

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.

Cancer chemopreventive effect of bergenin from Peltophorum pterocarpum wood.

The aqueous extract of Peltophorum pterocarpum (Fabaceae) wood exhibited potent inhibitory effects against EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and against melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells, as well as potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity. Two phenolic acid derivatives, bergenin (1) and gallic acid (2), were isolated from the ethyl acetate (AcOEt)-soluble fraction obtained from the extract. Compound 1 exhibited potent inhibitory effect against EBV-EA activation 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. Both compounds 1 and 2 exhibited melanogenesis-inhibitory activities in α-MSH-stimulated B16 melanoma cells, and, in addition, compound 2 showed strong DPPH radical-scavenging activity.

A biological active artificial saliva formulation containing flower mucilage from Ceylon Spinach (Basella alba Linn.).

Ceylon Spinach (Basella albe) is an edible perennial vine found in tropical Asia and Africa, known as vegetables containing mucilage. Its mucilage from flowers was extracted by microwaving and precipitated with 95% ethanol. Five artificial saliva formulations composing of mucilage from Ceylon Spinach, calcium chloride (CaCl2), potassium chloride (KCl) and sodium fluoride (NF) were developed. The best formulation No.5 containing 0.61% of the mucilage with the non-Newtonian pseudoplastic flow (8.9 ± 0.2 cP) and the wetting time (12.50 ± 2.24 min) similar to the normal human saliva was selected. This artificial saliva formulation exhibited biological activities including an antioxidative activity by DPPH free radical scavenging with the SC50 of 14.26 ± 2.00 mg/ml (0.05 folds of ascorbic acid), and the adhesion inhibition of S. mutans on hydroxyapatite beads at 17.01 ± 7.75%, while the natural human saliva exhibited an increase bacterial adhesion of 33.10 ± 9.70%. The safety of this formulation which gave no cytotoxicity on normal human gingival fibroblasts at 99.20 ± 21.09% cell viability was also demonstrated. The results from this study have indicated high biological activity and safety of the developed formulation containing mucilage from Ceylon Spinach which is potential to be used as artificial saliva for xerostomia patients.

Potent in vitro Anti-mouth Cancer (KB) and Immunostimulating Activities of the Job's Tears (Coix lachryma-jobi Linn.) Seed Semi-purified Extract Cocktails Containing Linoleic Acid.

The crude methanolic and hexane extracts of non-cooked, steamed and roasted from three Job's Tears cultivars were prepared and further semi-purified by liquid-liquid extraction techniques and silica gel column. The six single semi-purified extracts (F1-F6) were combined as nine cocktails (CT1, CT6, CT8, CT13, CT14, CT21, CT24, CT25 and CT31) according to the IC50 values from the preliminary study and investigated for anti-proliferative and apoptotic induction on mouth cancer cell line (KB) and immunostimulating as well as antioxidative activities. The highest anti-proliferative activity was observed in CT13 showing the IC50 value of 0.53±0.45 µg/mL which was higher than 5-fluorouracil and doxorubicin of 20.34 and 1.60 times, respectively. CT1 which was the combination of F1-F6 and CT13 which was the combination of F4-F6 exhibited significant strong synergistic activity with the combination index value (CI) of 0.28. CT1 at 200 µg/mL showed the highest percentages of apoptotic cells (40.65±10.97%) with no necrotic cells, but lower than cisplatin (100 µg/mL) of 2.18 times. CT14 gave the highest immunostimulating activity with the phagocytosis percentage of 13.0±1.7%, but lower than lipopolysaccharide of 1.08 times. CT31 gave the highest free radical scavenging and metal chelating activities with the SC50 and MC50 values of 0.73±0.07 and 1.99±0.24 µg/mL, but lower than ascorbic acid and EDTA of 18.25 and 4.33 times, respectively. The linoleic acid contents related to anti-cancer activity were also examined by HPLC. This study has demonstrated that CT1 composing of F1-F6 at the percentage ratio of 0.71:2.06:81.38: 8.47:4.92:2.46 was the potential cocktails of the semi-purified extracts from the Job's Tears which can be further developed as a novel active compound for oral cancer treatment.

Potential cancer chemopreventive and anticancer constituents from the fruits of Ficus hispida L.f. (Moraceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus hispida L.f. (Moraceae) has been used as alternative for traditional medicine in the treatment of various ailments including cancer-cure. The aim of this study was to evaluate the cancer chemopreventive and anticancer activities of crude extracts of F. hispida, with the objective to screen the inhibition of Epstein-Barr virus early antigen, and cytotoxic active components, and provide foundation for potential applications of this promising medical plant. MATERIALS AND METHODS: Compounds were isolated from the MeOH extract of F. hispida fruits, and their structure elucidation was performed on the basis of extensive spectroscopic analysis. The isolated compounds were evaluated for their inhibitory activities against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells, and cytotoxic activities against human cancer cell lines (HL60, A549, SKBR3, KB, Hela, HT29, and HepG2) and a normal cell (LO2) using MTT method. For the compound with potent cytotoxic activity, its apoptosis inducing activity was evaluated by the observation of ROS generation level expression, and membrane phospholipid exposure and DNA fragmentation in flow cytometry. The mechanisms of the apoptosis induction were analyzed by Western blotting. RESULTS: Nineteen compounds, 1-19, including two new isoflavones, 3'-formyl-5,7-dihydroxy-4'-methoxyisoflavone (2) and 5,7-dihydroxy-4'-methoxy-3'- (3-methyl-2-hydroxybuten-3-yl)isoflavone (3), were isolated from the MeOH extract of F. hispida fruits. Five compounds, isowigtheone hydrate (1), 2, 3, 9, and 19, showed potent inhibitory effects on EBV-EA induction with IC50 values in the range of 271-340 molar ratio 32 pmol-1 TPA. In addition, five phenolic compounds, 1-3, 10, and 13, exhibited cytotoxic activity against two or more cell lines (IC50 2.5-95.8µM), as well as compounds 1 and 3 were also displayed high selectivity for LO2/HepG2 (SI 23.5 and 11.8, respectively), while the compound 1-induced ROS generation leads to activated caspases-3, -8, and -9 apoptotic process in HL60 cells. CONCLUSION: This study has established that the MeOH extract of F. hispida fruits contains isoflavones, coumarins, caffeoylquinic acids, along with other compounds including phenolics and steroid glucoside as active principles, and has demonstrated that the chemical constituents of F. hispida may be valuable as potential chemopreventive and anticancer agents.

Potent Phosphodiesterase Inhibition and Nitric Oxide Release Stimulation of Anti-Impotence Thai Medicinal Plants from "MANOSROI III" Database.

Seven plants in the top rank were selected from the "MANOSROI III" database using the two Thai keywords which meant impotence and sexual tonic. Boesenbergia rotunda (L.) Mansf. extract [EDP1-001(1)] gave the highest PDE inhibition activity of 4.36-fold sildenafil, a standard anti-impotence drug. Plumbago indica Linn. extract [EDP2-001(1)] exhibited the highest NO release stimulation activity of 666.85% which was 1.50-fold acetylcholine, a standard drug. Most selected plant extracts were nontoxic to EA.hy926 cells at 1.0 mg/mL. EDP1-001(1) exhibited the LD50 value of acute oral toxicity in male ICR mice of over 5,000 mg/kg body weight. EDP1-001(1) also indicated the improvement of sexual behaviors in the paroxetine-induced sexual dysfunction male mice with the evaluation of number of courtships (NC), mount frequency (MF), intromission frequency (IF), and ejaculatory frequency (EF) at 87.67 ± 6.17, 121.00 ± 23.50, 36.00 ± 3.21, and 13.67 ± 2.96 which were 2.63-, 1.27-, 0.53-, and 0.62-fold sildenafil-treated mice at day 14 of the treatments, respectively. The present study has not only confirmed the traditional use of Thai plants for the treatment of ED but also indicated the potential and application of the "MANOSROI III" database for Thai plant selection to be developed as ED food supplements.

In vitro anti-proliferative activity on colon cancer cell line (HT-29) of Thai medicinal plants selected from Thai/Lanna medicinal plant recipe database "MANOSROI III".

ETHNOPHARMACOLOGICAL RELEVANCE: Thai/Lanna region has its own folklore wisdoms including the traditional medicinal plant recipes. Thai/Lanna medicinal plant recipe database "MANOSROI III" has been developed by Prof. Dr. Jiradej Manosroi. It consists of over 200,000 recipes for all diseases including cancer. To investigate the anti-proliferative and apoptotic activities on human colon cancer cell line (HT-29) as well as the cancer cell selectivity of the methanolic extracts (MEs) and fractions of the 23 selected plants from the "MANOSROI III" database. MATERIALS AND METHODS: The 23 selected plants were extracted with methanol under reflux and evaluated for their anti-proliferative activity by sulforhodamine B assay. The 5 plants (Gloriosa superba, Caesalpinia sappan, Fibraurea tinctoria, Ventilago denticulata and Psophocarpus tetragonolobus) with potent anti-proliferative activity were fractionated by liquid-liquid partition to give 4 fractions including each hexane (HF), methanol-water (MF), n-butanol (BF) and water (WF) fractions. They were tested for anti-proliferative activity and cancer cell selectivity. The ME and fractions of G. superba which showed potent anti-proliferative activity were further examined for morphological changes and apoptotic activities by acridine orange (AO)/ethidium bromide (EB) staining. RESULTS: The ME of G. superba root showed active with the highest anti-proliferative activity at 9.17 and 1.58 folds of cisplatin and doxorubicin, respectively. After liquid-liquid partition, HF of V. denticulata, MFs of F. tinctoria, V. denticulata and BF of P. tetragonolobus showed higher anti-proliferative activities than their MEs. The MF of G. superba indicated the highest anti-proliferative activity at 7.73 and 1.34 folds of cisplatin and doxorubicin, respectively, but only 0.86 fold of its ME. The ME and HF, MF and BF of G. superba and MF of F. tinctoria demonstrated high cancer cell selectivity. At 50 µg/ml, ME, HF, MF and BF of G. superba demonstrated higher apoptotic activities than the two standard drugs. CONCLUSIONS: This present study has not only confirmed the traditional use of the Thai/Lanna medicinal plant recipes for cancer treatments, but also the potential of the selected plant, G. superba for the further development as a modern anti-cancer drug.

Potent Antiproliferative Effect on Liver Cancer of Medicinal Plants Selected from the Thai/Lanna Medicinal Plant Recipe Database "MANOSROI III".

Thai/Lanna medicinal plant recipes have been used for the treatment of several diseases including liver cancer. In this study, methanolic extracts (MEs) of 23 plants were tested for antiproliferative activity on human hepatoma cell line (Hep G2) by the sulforhodamine B (SRB) assay. Nine MEs with potent antiproliferative activity (IC50 < 100 µg/mL) were obtained and further semipurified by liquid/liquid partition extraction. The semipurified fractions were tested for the antiproliferative and antioxidative activities. ME of Stemona collinsae and the semipurified extract and methanol-water fraction (MF) of Gloriosa superba gave the highest antiproliferative activity on HepG2 which were 4.79- and 50.07-fold cisplatin, respectively. The semipurified fractions showed an increased antiproliferative activity. MF of Caesalpinia sappan and HF of Senna alata showed the highest free radical scavenging and metal chelating activities, respectively. The compound in n-hexane fraction (HF) of Ventilago denticulata which showed an increase in antiproliferative activity comparing to its ME was isolated and identified as emodin. This study has demonstrated the potential of the ME from S. collinsae, MF from G. superba, and emodin isolated from V. denticulata, for further development as an antiliver cancer agent.

Potent anti-cervical cancer activity: synergistic effects of Thai medicinal plants in recipe N040 selected from the MANOSROI III database.

ETHNOPHARMACOLOGICAL RELEVANCES: One of the prestigious Thai/Lanna folklore wisdoms is the medicinal plant recipes. Thai/Lanna medicinal plant recipe database MANOSROI III has been developed by Prof. Dr. Jiradej Manosroi. It consists of over 200,000 recipes covering all diseases including cancer. AIM OF THIS STUDY: To investigate the in vitro and in vivo anti-cervical cancer activity and the active constituents of the Thai medicinal plant recipe N040 selected from the MANOSROI III database. MATERIALS AND METHODS: The extracts of recipe N040 and single medicinal plants in the recipe were prepared by hot water and methanol extraction, respectively. The n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH) and water fractions of Caesalpinia sappan, the plant which showed the highest anti-proliferative activity were prepared by liquid-liquid partition extraction. The fraction which showed the highest anti-proliferative activity was further isolated for active constituents. Anti-proliferative activity of recipe N040, methanolic extracts, fractions of Caesalpinia sappan and brazilin, an active constituent on HeLa cell were investigated using sulforhodamine B (SRB) assay. Anti-oxidative activities including free radical scavenging and metal ion-chelating activities, as well as the phenolic and flavonoid contents of these fractions were also determined. The in vivo anti-cancer activity of recipe N040 on HeLa cell xenograft and the subchronic toxicity were performed in nude mice and rats, respectively. RESULTS AND CONCLUSIONS: N040 showed the potent in vitro anti-proliferative activity on HeLa cell with the IC50 value of 0.11 µg/ml. Phytochemicals detected in the plants were steroids/triterpenoids, tannins, flavonoids, saponins and alkaloids. For the single plant, methanolic extract of Caesalpinia sappan gave the highest anti-proliferative activity with the IC50 of 33.46 µg/ml. EtOAc fraction of Caesalpinia sappan showed the highest anti-proliferative and free radical scavenging activities with the IC50 and SC50 of 17.81 and 21.95 µg/ml which were 1.88 and 0.83 folds of its methanolic extract and ascorbic acid, respectively. Poor metal chelating activity (MC50>500 µg/ml) was observed in methanolic extract and all fractions. The highest phenolic and flavonoid contents were observed in the methanolic extract. Brazilin, the known compound isolated from the EtOAc fraction exhibited potent anti-proliferative activity with the IC50 of 0.28 µg/ml which was higher than its methanolic extract and EtOAc fraction of 119.50 and 63.61 folds, respectively, but only 0.39 fold of the recipe extract N040. The tumor size of the HeLa cell xenograft nude mice treated with the recipe N040 at the dose of 44.50mg/kg body weight per day was significantly smaller (p<0.05) than that of the control with the relative tumor weight inhibition of 57.23% which was 0.65 fold of cisplatin. In the subchronic toxicity study, N040 given orally at the dose of 1000 mg/kg body weight per day for 90 days showed no alteration in body weight gain, hematology [except the increase mean corpuscular hemoglobin (MCH) in the treated male rats] and clinical blood chemistry (except the increase blood glucose in the treated male rats) both in female and male rats. Only minor lesions of the organs including lung, liver, kidney and small intestine were observed in both sexes. This study has demonstrated the synergistic effect of the plants composed in the recipe which resulted in the potent anti-cancer activity and confirmed the traditionally use of the recipe N040. In addition, this study has also suggested the compound brazilin isolated from Caesalpinia sappan for its high potential to be further investigated as a novel anti-cervical cancer drug.