Matrix metalloproteinase-1 inhibitory activities of Morinda citrifolia seed extract and its constituents in UVA-irradiated human dermal fibroblasts.

The objective of this study was to examine whether a 50% ethanolic extract (MCS-ext) of the seeds of Morinda citrifolia (noni) and its constituents have matrix metalloproteinase-1 (MMP-1) inhibitory activity in UVA-irradiated normal human dermal fibroblasts (NHDFs). The MCS-ext (10 µg/mL) inhibited MMP-1 secretion from UVA-irradiated NHDFs, without cytotoxic effects, at 48 h after UV exposure. The ethyl acetate-soluble fraction of MCS-ext was the most potent inhibitor of MMP-1 secretion. Among the constituents of the fraction, a lignan, 3,3'-bisdemethylpinoresinol (1), inhibited the MMP-1 secretion at a concentration of 0.3 µM without cytotoxic effects. Furthermore, 1 (0.3 µM) reduced the level of intracellular MMP-1 expression. Other constituents, namely americanin A (2), quercetin (3) and ursolic acid (4), were inactive. To elucidate inhibition mechanisms of MMP-1 expression and secretion, the effect of 1 on mitogen-activated protein kinases (MAPKs) phosphorylation was examined. Western blot analysis revealed that 1 (0.3 µM) reduced the phosphorylations of p38 and c-Jun-N-terminal kinase (JNK). These results suggested that 1 suppresses intracellular MMP-1 expression, and consequent secretion from UVA-irradiated NHDFs, by down-regulation of MAPKs phosphorylation.

Inhibitory effects of Morinda citrifolia extract and its constituents on melanogenesis in murine B16 melanoma cells.

The objective of this study was to examine the effects of Morinda citrifolia (noni) extract and its constituents on α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in cultured murine B16 melanoma cells (B16 cells). A 50% ethanolic extract of noni seeds (MCS-ext) showed significant inhibition of melanogenesis with no effect on cell proliferation. MCS-ext was more active than noni leaf and fruit flesh extracts. Activity guided fractionation of MCS-ext led to the isolation of two lignans, 3,3'-bisdemethylpinoresinol (1) and americanin A (2), as active constituents. To elucidate the mechanism of melanogenesis inhibition by the lignans, α-MSH-stimulated B16 cells were treated with 1 (5 µM) and 2 (200 µM). Time-dependent increases of intracellular melanin content and tyrosinase activity, during 24 to 72 h, were inhibited significantly by treatment with the lignans. The activity of 1 was greater than that of 2. Western blot analysis suggested that the lignans inhibited melanogenesis by down regulation of the levels of phosphorylation of p38 mitogen-activated protein kinase, resulting in suppression of tyrosinase expression.

Effects of unripe Citrus hassaku fruits extract and its flavanone glycosides on blood fluidity.

The enhancement of blood fluidity may lead to improvements in skin problems resulting from unsmooth circulation or blood stagnation. Since a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits may be a useful ingredient in skin-whitening cosmetics, the present study was designed to examine the effect of CH-ext on blood fluidity. CH-ext concentration-dependently inhibited in vitro collagen-induced rabbit platelet aggregation and in vitro polybrene-induced rat erythrocyte aggregation. The CH-ext showed in vitro fibrinolysis activity in fibrin plate assay. Activity-guided fractionation of the CH-ext using antiplatelet activity, inhibitory activity of erythrocyte aggregation, and fibrinolysis activity revealed that these activities of CH-ext were attributable to naringenin-7-glycoside (prunin). Successive oral administration of CH-ext to rats inhibited the lipopolysaccharide (LPS)-induced decrease of blood platelets and fibrinogen, and LPS-induced increase of fibrin degradation products (FDP) in LPS-induced disseminated intravascular coagulation (DIC) model rats. Effects of CH-ext on blood fluidity were analyzed by a micro channel array flow analyzer (MC-FAN). Preventive oral administration of CH-ext to rats showed dose-dependent reduction of the passage time of whole blood flow of the DIC model rats in comparison with that of the vehicle control rats. These results imply that CH-ext may have effects which improve effects on blood fluidity.

Inhibitory effects of devil's claw (secondary root of Harpagophytum procumbens) extract and harpagoside on cytokine production in mouse macrophages.

Successive oral administration (50 mg/kg) of a 50% ethanolic extract (HP-ext) of devil's claw, the secondary root of Harpagophytum procumbens, showed a significant anti-inflammatory effect in the rat adjuvant-induced chronic arthritis model. HP-ext dose-dependently suppressed the lipopolysaccharide (LPS)-induced production of inflammatory cytokines [interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha)] in mouse macrophage cells (RAW 264.7). Harpagoside, a major iridoid glycoside present in devil's claw, was found to be one of the active agents in HP-ext and inhibited the production of IL-1beta, IL-6, and TNF-alpha by RAW 264.7.

Antiallergic activity of unripe Citrus hassaku fruits extract and its flavanone glycosides on chemical substance-induced dermatitis in mice.

Oral administration of a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits collected in July exhibited a potent dose-dependent inhibition of IgE (immunoglobulin E)-mediated triphasic cutaneous reaction at 1 h [immediate phase response (IPR)], 24 h [late phase response (LPR)] and 8 days [very late phase response (vLPR)] after dinitrofluorobenzene challenge in mice. Naringin, a major flavanone glycoside component of CH-ext, showed a potent dose-dependent inhibition against IPR, LPR and vLPR. Neohesperidin, another major glycoside component of CH-ext, showed an inhibition against vLPR. The effect of CH-ext on type IV allergic reaction was examined by determining inhibitory activity against ear swelling in mice by using the picryl chloride-induced contact dermatitis (PC-CD) model. Oral administration (p.o.) of CH-ext and subcutaneous administration (s.c.) of prednisolone inhibited ear swelling during the induction phase of PC-CD. The inhibitory activities of combinations of CH-ext (p.o.) and prednisolone (s.c.) against PC-CD in mice were more potent than those of CH-ext alone and prednisolone alone, without enhancing the adverse effects. Other combinations of prednisolone (s.c.) and flavanone glycoside (p.o.) components of CH-ext, i.e. naringin and neohesperidin, exerted similar synergistic effects.

Inhibitory effects of Citrus hassaku extract and its flavanone glycosides on melanogenesis.

The 50% ethanolic extract (CH-ext) obtained from the unripe fruit of Citrus hassaku exhibited significant tyrosinase inhibitory activity. The CH-ext showed antioxidant activity, such as superoxide dismutase (SOD)-like activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. Activity-guided fractionation of the CH-ext indicated that flavanone glycoside-rich fractions showed potent tyrosinase inhibitory activity. Further examination revealed that the tyrosinase inhibitory activity and antioxidant activity of the CH-ext were attributable to naringin and neohesperidin, respectively. The CH-ext showed inhibition of melanogenesis without any effects on cell proliferation in cultured murine B16 melanoma cells after glucosamine exposure. The topical application of the CH-ext to the dorsal skin of brownish guinea pigs showed in vivo preventive effects against UVB-induced pigmentation.

Inhibitory effects of constituents of Morinda citrifolia seeds on elastase and tyrosinase.

A 50% ethanolic extract (MCS-ext) from seeds of Morinda citrifolia ("noni" seeds) showed more potent in vitro inhibition of elastase and tyrosinase, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than extracts of M. citrifolia leaves or flesh. Activity-guided fractionation of MCS-ext using in vitro assays led to the isolation of ursolic acid as an active constituent of elastase inhibitory activity. 3,3'-Bisdemethylpinoresinol, americanin A, and quercetin were isolated as active constituents having both tyrosinase inhibitory and radical scavenging activities. Americanin A and quercetin also showed superoxide dismutase (SOD)-like activity. These active compounds were isolated from noni seeds for the first time.

Histamine release inhibitory activity of Piper nigrum leaf.

Oral administration of a methanolic extract of Piper nigrum leaf (PN-ext, 50, 200 and 500 mg/kg) showed a potent dose-dependent inhibition of dinitrofluorobenzene (DNFB)-induced cutaneous reaction at 1 h [immediate phase response (IPR)] after and 24 h [late phase response (LPR)] after DNFB challenge in mice which were passively sensitized with anti-dinitrophenyl (DNP) IgE antibody. Ear swelling inhibitory effect of PN-ext (50, 200 and 500 mg/kg, per os (p.o.)) on very late phase response (vLPR) in the model mice was significant but weaker than that on IPR. Oral administration of PN-ext (50, 200 and 500 mg/kg for 7 d) inhibited picryl chloride (PC)-induced ear swelling in PC sensitized mice. PN-ext exhibited in vitro inhibitory effect on compound 48/80-induced histamine release from rat peritoneal mast cells. Two lignans of PN-ext, (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), were identified as major active principles having histamine release inhibitory activity.

Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.

Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice.

Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

(-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

Melanogenesis stimulation in murine B16 melanoma cells by Kava (Piper methysticum) rhizome extract and kavalactones.

Melanogenesis stimulation activity of aqueous ethanolic extracts obtained from several different parts of five Piper species, namely Piper longum, P. kadsura, P. methysticum, P. betle, and P. cubeba, were examined by using cultured murine B16 melanoma cells. Among them, the extract of P. methysticum rhizome (Kava) showed potent stimulatory effect on melanogenesis as well as P. nigrum leaf extract. Activity-guided fractionation of Kava extract led to the isolation of two active kavalactones, yangonin (2) and 7,8-epoxyyangonin (5), along with three inactive kavalactones, 5,6-dehydrokawain (1), (+)-kawain (3) and (+)-methysticin (4), and a glucosylsterol, daucosterin (6). 7,8-Epoxyyangonin (5) showed a significant stimulatory effect on melanogenesis in B16 melanoma cells. Yangonin (2) exhibited a weak melanogenesis stimulation activity.

Melanogenesis stimulation in murine b16 melanoma cells by umberiferae plant extracts and their coumarin constituents.

Melanogenesis stimulation activities of seven ethanolic extracts obtained from Umbelliferae plants used as Chinese crude drugs, namely the roots of Angelica dahurica BENTH. et HOOK., A. biserrata SHEN et YUAN, Notopterygium incisum TING, Heracleum lanatum MICHX., and H. candicans WALL., and the fruits of Cinidium monnieri (L.) CUSSON and C. formosanum YABE, were examined by using cultured murine B16 melanoma cells. Among them, the extract (5, 25 microg/ml) of H. lanatum showed a potent stimulatory effect on melanogenesis with significant enhancement of cell proliferation in a dose-dependent manner. The melanogenesis stimulatory effects of sixteen coumarins (1-16) isolated from the seven Umbelliferae crude drugs were also examined. Among them, linear-furocoumarins [psoralen (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4)] and angular-furocoumarin [sphondin (13)] exhibited potent melanogenesis stimulation activity. From the view point of structure-activity relationships, it may be assumed that a linear-furocoumarin ring having a hydrogen and/or methoxyl group at 5 and 8 positions such as 1, 2, 3 and 4 was preferable for the melanogenesis stimulation activity. The introduction of a prenyl group into the furocoumarin ring was disadvantageous. Coumarin derivatives having a simple coumarin ring were inactive.

Studies on palauan medicinal herbs. II. Activation of mouse macrophages RAW 264.7 by Ongael, leaves of Phaleria cumingii (Meisn.) F. Vill. and its acylglucosylsterols.

The extract of Ongael [leaves of Phaleria cumingii (MEISN.) F. VILL.], a Palauan medicinal herb, enhanced an in vitro phagocytic activity of mouse macrophages RAW 264.7 cells (RAW 264.7). Activity-guided fractionation of the Ongael extract by the in vitro phagocytosis assay using RAW 264.7 led to the isolation of a mixture of acylglucosylsterols (1) as an active constituent along with other inactive constituents, tetracosanol and mangiferin. On the basis of chemical modifications and spectral analyses, the compound 1 was deduced to be a mixture of the known 3-O-(6-O-acyl-beta-D-glucosyl)-beta-sitosterols, the acyl moiety being mainly palmitoyl (57%), oleoyl (12%) and alpha-linolenoyl (12%) with small amount of stearoyl (7%) and linoleoyl (4%).

Melanogenesis stimulation in murine B16 melanoma cells by Piper nigrum leaf extract and its lignan constituents.

A methanolic extract from the leaves of Piper nigrum L. showed a significant stimulatory effect on melanogenesis in cultured murine B16 melanoma cells. Activity-guided fractionation of the methanolic extract led to the isolation of two known lignans, (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), together with a new lignan, (-)-3-desmethoxycubebinin (3). Among these lignans, 1 and 2 showed a significant stimulatory activity of melanogenesis without any significant effects on cell proliferation.

Antipruritic effect of Cnidii Monnieri Fructus (fruits of Cnidium monnieri CUSSON).

Antipruritic effects of 70% ethanol extract (CM-ext) of Cnidii Monnieri Fructus (dried fruits of Cnidium monnieri CUSSON, Umberifferae) were investigated. In mice, an oral administration of CM-ext (200 and 500 mg/kg) inhibited compound 48/80-induced scratching behavior without influence on spontaneous locomotion. Isopimpinellin (3) and osthol (1), coumarin derivatives isolated from CM-ext, showed an inhibitory effect on compound 48/80-induced scratching behavior.