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Hoveniae Semen Seu Fructus (HSF) is a fruit and seed of Hovenia dulcis Thunb. Here, we first report HSF water extract contains a potent anti-influenza A virus (IAV) effect via inhibition of neuraminidase and hemagglutinin and a direct virus-killing effect. We examined the effect of WHSF on IAV infection using GFP-tagged Influenza A/PR/8/34. FACS analysis and fluorescent microscopy showed a dose-dependent inhibitory effect of WHSF against IAV infection. WHSF also suppressed the cytopathic effects of H1N1 and H3N2 IAV. Immunofluorescence and qPCR analyses confirmed WHSF significantly decreases IAV protein and RNA expressions. Time of addition and plaque assays revealed WHSF affects viral replication in the early stage via modulating hemagglutinin and inducing virucidal effects. Furthermore, WHSF suppressed the neuraminidase activity of H1N1 and H3N2 IAV. Quercetin, not taxifolin in WHSF inhibited IAV infection. Our results suggest WHSF could be developed as a natural antiviral agent to prevent IAV infection.

Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this study, we evaluated the anti-influenza A virus (IAV) activity of TOF by detecting GFP expressed by influenza A virus (A/PR/8/34-GFP) infection. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that TOF potently inhibited IAV infection, dose-dependently. Consistently, immunofluorescence and Q-PCR analysis results confirmed TOF significantly represses IAV protein and RNA expression. TOF inhibited IAV infection at the binding and entry step upon viral infection and interferes with HA protein. Further, TOF exhibited a virucidal effect and inhibited the neuraminidase activity of IAV. Additionally, TOF prevented the cytopathic effect caused by H1N1 and H3N2 IAV infection. Amentoflavone among the constituents in TOF exerted the strongest anti-IAV effect. Myricetin, quercetin, and quercitrin also inhibited IAV infection. However, the potent anti-IAV effect of TOF may be related to the synergistic effect of constituents, not by a single specific compound. Our results suggest TOF exhibits a significant inhibitory effect against IAV infection at multi-stages via the blockage of viral attachment and entry, inhibition of neuraminidase, and induction of virucidal effects.

Lilii Bulbus, the bulb of tiger lily, has anti-oxidant and anti-tumorigenic properties. However, the effects of Lilii Bulbus on learning, memory, and hippocampal neurogenesis remain unknown. This study investigated whether water extract of Lilii Bulbus (WELB) affects memory ability and hippocampal neurogenesis. Behavioral analyses (Morris water maze and passive avoidance test), immunohistochemistry, cell proliferation assay, and immunoblot analysis were performed. WELB (50 and 100 mg/kg; for 14 days) enhanced memory retention and spatial memory in normal mice as well as in scopolamine-treated mice with memory deficits. Furthermore, the administration of WELB significantly increased the number of proliferating cells and surviving newborn cells in the dentate gyrus of the hippocampus in normal mice. We found that WELB has a pro-neurogenic effect by increasing the activation of brain-derived neurotrophic factor (BDNF)/cAMP response element-binding protein (CREB) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) in the hippocampus. Moreover, we confirmed that WELB (100 and 200 μg/ml) significantly increased NE-4 C and primary embryonic NSCs proliferation. Inhibition/knockdown of MEK/ERK blocked WELB-induced MEK/ERK phosphorylation and NSCs proliferation. Hence, MEK/ERK activation was required in WELB-induced NSCs proliferation. Our study demonstrates the first evidence for WELB promoting hippocampal neurogenesis and memory; pro-neurogenic activity may enhance brain plasticity, with implications for treating neurodegenerative diseases.

Lotus (Nelumbo nucifera Gaertn.) is a perennial aquatic plant, that has been used in folk to treat various diseases. Here, we first demonstrate the water extract of lotus leaf (WLL) has a strong inhibitory effect on influenza A virus (IAV) infection by inhibiting neuraminidase (NA) and hemagglutinin (HA). To investigate the effect of WLL on IAV infection, we used green fluorescent protein (GFP)-tagged Influenza A/PR/8/34, fluorescence-activated cell sorting (FACS) analysis, and plaque reduction assay. WLL significantly repressed viral infection, dose-dependently. Immunofluorescence (IF) analysis confirmed that WLL reduces influenza HA, NA, M2, and NP viral proteins expression. WLL strongly inhibited HA and reduced NA activity of IAV. Among six components in WLL, isoquercitrin exerted potent antiviral efficacy. Additionally, WLL potently inhibited the cytopathic effect caused by H1N1 or H3N2 IAV. In conclusion, our results suggest that WLL could be used as a natural viral inhibitor for H1N1 and H3N2 influenza viral infection.

Epimedium koreanum Nakai (EKN) is a popular plant in Korean and Chinese medicine for treating a variety of ailments. The aqueous extract of EKN has a significant inhibitory impact on influenza A virus (IAV) infection by directly blocking viral attachment and having a virucidal effect, according to this study. Using fluorescent microscopy and fluorescence-activated cell sorting (FACS) with a green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus, we examined the effect of EKN on viral infection. By viral infection, EKN strongly suppresses GFP expression, and at a dosage of 100 µg/mL, EKN decreased GFP expression by up to 90% of the untreated infected control. Immunofluorescence and Western blot analyses against influenza viral proteins revealed that EKN decreased influenza viral protein expression in a dose-dependent manner. EKN inhibited the H1N1 influenza virus's hemagglutinin (HA) and neuraminidase (NA), preventing viral attachment to cells. Furthermore, EKN had a virucidal impact and inhibited the cytopathic effects of H1N1, H3N2 and influenza B virus infection. Finally, our findings show that EKN has the potential to be developed as a natural viral inhibitor against influenza virus infection.

Because tyrosinase catalyzes essential steps in melanin synthesis, tyrosinase inhibitors are widely used for skin–lightening. To reduce the side effects of synthetic compounds, safer tyrosinase inhibitors isorated from natural products have gained attention. Here, we examined the anti-melanogenic effect of the seed of Ziziphus jujuba and identified eight major compounds, of which spinosin exhibited the strongest tyrosinase inhibitory activity (IC50 = 47 µM). When further examined, spinosin suppressed αMSH- or UVB-induced melanogenesis in B16F10 cells without cytotoxicity. The anti-melanogenic effect was also shown in a human skin model. As an underlying mechanism, in silico analysis showed that spinosin may bind to and suppress tyrosinase activity by forming multiple hydrogen bonds and hydrophobic interactions with the binding pocket of tyrosinase. Lineweaver-Burk and Dixon plots further revealed that spinosin acts as a competitive inhibitor of tyrosinase (Vmax, 11.61 × 103, 11.63 × 103, and 11.62 × 103, Km, 0.139, 0.185, and 0.239 for the con, 10 µM, 50 µM of spinosin, respectively). In conclusion, spinosin, a major compound in the seed of Ziziphus jujuba, could be a novel additive for treating pigmentation disorders or skin-lightening cosmetics.

The biological effect of lappaol A, a lignan, is derived from Arctium lappa, and is relatively unknown; however, lignans have various beneficial properties. Therefore, we investigated the effects of lappaol A on the immunoglobulin E/antigen (IgE/Ag)-mediated allergic response in mast cells and mice. Lappaol A significantly reduced the release of β-hexosaminidase, IL-4, TNF-α, PGD2 and LTC4 with IC50 values of 131.3 μM, 11.58 μM, 59.50 μM, 17.85 μM and 10.99 μM, respectively, in IgE/Ag-activated RBL-2H3 cells. Moreover, lappaol A suppressed the activation of the arachidonate and FcεRI signaling cascades in the cells. Furthermore, lappaol A suppressed the IgE/Ag-mediated passive cutaneous anaphylaxis reaction, ear edema and mast cell infiltration in the ear tissues of IgE/Ag-exposed mice. In conclusion, these findings suggest that lappaol A exhibits anti-allergic activity by inhibiting the activation of the arachidonate and FcεRI signaling cascades in IgE/Ag-activated mast cells. Therefore, this information may help to elucidate the anti-allergic action of lappaol A and Arctium lappa L.

Background: The leaf of Pinus densiflora known as pine needles has been used to treat vascular disease, gastrointestinal diseases, and urinary diseases in traditional medicine. We evaluated anti-osteoporotic effect of water extract of Pinus densiflora (WEPN) on acute bone loss and osteoclastogenesis induced by receptor activator for nuclear factor-κB ligand (RANKL). Methods: After oral administration of WEPN (0.25 g/kg) for 5 days, femora were collected, and bone parameter [trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular number (Tb. N), and bone mineral density (BMD)] were analyzed by micro-CT analysis. Anti-osteoclastic effect of WEPN was examined using tartrate-resistant acid phosphatase activity and activation of RANKL signaling pathway. Results: We found that WEPN significantly attenuated RANKL-induced decrease of BV/TV, Tb.Th., Tb.N, and BMD but increase of Tb. Sp in femora. WEPN dose-dependently decreased osteoclastogenesis accompanied by inhibiting the activation of RANKL signaling components (JNK, p38, and p65) and mRNA expression level of osteoclast specific genes (NFATc1, c-Fos, TRAP, cathepsin K, DC-STAMP, and carbonic anhydrate). Conclusion: WEPN inhibition on osteoclastogenesis could contribute to attenuate RANKL-induced trabecular bone loss in vivo. Therefore, it might suggest that WEPN could be prescribed in traditional medicine or used in health functional food to prevent or treat osteoporotic bone diseases. Keywords: Pinus densiflora, Osteoclast, Receptor activator for nuclear factor-κB ligand, Nuclear factor of activated T cells cytoplasmic 1

Background: Traditional medicine and herbal prescriptions are becoming more popular, and they account for a large share of the worldâs healthcare research studies, developments, and market demands. Increasing scientific evidence of the substantive efficacies such as preventive health keeping pharmaceutical materials and dietary supplements can be found elsewhere. Above all, safety should be the critical premise for considering developmental materials such as pharmaceuticals without side effects and toxicity. Methods: The authors formulated KIOM2012H (K2H) using four herbs that were reported to have medicinal effectsâincluding anticancer, antiaging, antimicrobial, inflammation, and neuroprotective properties. In order to examine the toxicity, single and repeated dose toxicity, and genotoxicities of bacterial mutation, micronucleus, and chromosomal aberration assays were conducted. Results: All experimental observations and results showed normal findings. Toxicities or abnormal signs were not observed in all experimental assays, including oral administration, animal behavior, clinical findings, and changes in body weight in vivo. In vitro bacterial cultures produced no revertant colonies, and no increased numbers of structural or numerical aberrant metaphases were found in the metaphase chromosomes examined. Moreover, no significant increased frequency of micronucleus was observed in any of the doses used. Overall, no acute toxicity or genotoxicity was found in all analysis parameters in all the assays conducted. Conclusion: Reviewing the results as a whole, K2H extract was regarded as a safe material with no toxicity, and can be applied for the research and development of complementary and alternative medicines with improved efficacy in current therapeutic healthcare, based on traditional medicine and herb resources. Keywords: genotoxicity, herb, toxicity, traditional medicine

Background: The hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. Methods: Tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. Results: We found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. Conclusion: The present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis. Keywords: Uncaria sinensis, Osteoclasts, Receptor activator for nuclear factor-κB ligand, Nuclear factor of activated T-cells, Cytoplasmic 1

Background: Black ginseng has a more potent biological activity than non-steamed ginseng. We investigated the effects of long-term intake of dietary black ginseng extract (BG) on antioxidant activity in aged mice. We also compared the effects of BG on cognitive deficits with those of white ginseng extract (WG) and red ginseng extract (RG). Methods: Ten-month-old mice were fed an AIN-93G-based diet containing 10 g/kg (low dose, L) or 30 g/kg (high dose, H) WG powder, RG powder, or BG powder for 24 wk. We measured serum lipids, the activities of antioxidant enzymes, and malondialdehyde levels. Additionally, the protein expression levels of choline acetyltransferase and vesicular acetylcholine transporter, which are presynaptic cholinergic markers in the cortex and hippocampus of the brain, were measured by western blotting. Results: Triglyceride levels were reduced in all the extract-treated mice, except those in the LBG group. High-density lipoprotein cholesterol levels in the HBG group were higher than those in the control group. Total cholesterol levels were reduced in the LBG group. Additionally, glucose levels in the HBG group were significantly reduced by 41.2%. There were lower levels of malondialdehyde in the LBG group than in the control group. Furthermore, glutathione reductase activity increased in the HWG group and the HRG group. The protein expression levels of choline acetyltransferase and vesicular acetylcholine transporter significantly increased in all the ginseng-treated groups. Conclusion: The results suggest that supplementation with the tested ginseng extracts may suppress the cognitive decline associated with aging, via regulation of the cholinergic and antioxidant defense systems.

Background: The traditional medicine oyaksungi-san (OY) has been prescribed in East Asia for hundreds of years for the treatment of stroke, paralysis, and ataxia. OY also has therapeutic effects on arthralgia, myalgia, and rheumatoid arthritis, and recent studies have shown its protective effects against apoptosis of hippocampal cells and its anti-inflammatory effects on the peripheral blood cells of patient with cerebral infarction. Many studies have explored the use of traditional medicine and herb materials in the development of safe, novel, and effective pharmaceuticals with fewer side effects. These efforts commonly adopt a bioconversion tool for fermentation with beneficial microbes. However, only pharmaceuticals with high levels of safety and low levels of toxicity can be used in healthcare system. Methods: OY water extract was fermented with Lactobacillus and assayed for acute toxicity and genotoxicity. Single dose acute toxicity, bacterial reverse mutation, chromosome aberrations, and micronucleus were observed and assayed in rats, histidine/tryptophan auxotrophic bacteria, Chinese hamster ovary fibroblast cells, and mice bone marrow cells, respectively. Results: All the experimental animals showed no abnormal behavior, clinical signs, body weight increases, or mortality. In the bacterial cultures, no revertant colonies were observed. Morphological and numerical chromosomal aberrations were not found in all metaphases examined. Frequency of induced micronuclei was not significantly increased in all doses applied. Conclusion: As a whole, no acute toxicity or genotoxicity were observed in all the assays examined. Therefore, fermented OY is considered to be a safe material that can be used for development of complementary and alternative medicine using bioconversion. Keywords: fermentation, genotoxicity, oyaksungi-san, traditional medicine, toxicity

Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (A. rugosa), commonly known as Korean mint, has traditionally been used for both food and medicine, depending on which part of the plant is used. We investigated the anti-proliferative activities of A. rugosa extract in vascular smooth muscle cells (VSMCs) and analysed several molecular pathways to determine the active component of the extract. A. rugosa extract inhibited PDGF-BB-stimulated VSMC proliferation and DNA synthesis. A. rugosa extract caused arrest of the cell cycle at the G0/G1 transition by up-regulating the levels of p21WAF1/CIP1 and p27KIP1. Among the components of the A. rugosa extract reported in a previous study, rosmarinic acid (RA) only inhibited DNA synthesis and dose-dependently suppressed PCNA expression. These results indicate that A. rugosa extract, which includes the active component RA, demonstrates anti-proliferative activity by arresting the cell cycle at the G0/G1 phase through the upregulation of p21WAF1/CIP1 and p27Kip1 expression.

Background: Galla Rhois has been considered to have medicinal properties against diarrhea, excessive sweating, bleeding, and chronic cough in Asian countries. Gallotannins, which are Galla Rhois-derived tannins, have been reported to possess biological and pharmacological activities, especially anticancer activity. In this study, we evaluated the effect of steaming at a temperature over 120 °C on the chemical constituents and biological activities of the water extract of Galla Rhois (GRE). Methods: GRE was steamed at a temperature over 120 °C (AGRE), and its specific constituents were analyzed; the results were validated using a high-performance liquid chromatography–diode array detector system. To evaluate the anticancer effect of GRE and AGRE, cell viability assay, cell cycle analysis, and Western blot analysis were performed in HCT116 human colon cancer cells. Results: Steaming markedly increased the contents of gallic acid and ellagic acid in GRE, and GRE or AGRE treatment reduced the viability of HCT116 cells. Notably, the steaming process enhanced the growth inhibitory effect of GRE in cancer cells. AGRE induced apoptosis through the activation of caspase-3, caspase-8, and caspase-9. Additionally, AGRE regulated the activation of mitogen-activated protein kinases including extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase, whereas GRE did not. However, both GRE and AGRE inhibited the activation of AKT. Conclusion: Compared with GRE, AGRE is more potent in its ability to induce apoptosis in HCT116 cells; therefore, we suggest that the steaming process may be useful as a feasible method for improving the anticancer effect of GRE.

Background: Yijin-tang (YJ) has been used traditionally for the treatment of cardiovascular conditions, nausea, vomiting, gastroduodenal ulcers, and chronic gastritis. In this study, a simple and sensitive high-performance liquid chromatography (HPLC) method was developed for the quantitation of nine bioactive compounds in YJ: homogentisic acid, liquiritin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, 6-gingerol, and pachymic acid. Methods: Chromatographic separation of the analytes was achieved on an RS Tech C18 column (4.6 mm × 250 mm, 5 μm) using a mobile phase composed of water containing 0.1% (v/v) trifluoroacetic acid (TFA) and acetonitrile with a gradient elution at a flow rate of 1.0 mL/min. Results: Calibration curves for all analytes showed good linearity (R2 ≥ 0.9995). Lower limits of detection and lower limits of quantification were in the ranges of 0.03–0.17 μg/mL and 0.09–0.43 μg/mL, respectively. Relative standard deviations (RSDs; %) for intra- and interday assays were < 3%. The recovery of components ranged from 98.09% to 103.78%, with RSDs (%) values ranging from 0.10% to 2.59%. Conclusion: This validated HPLC method was applied to qualitative and quantitative analyses of nine bioactive compounds in YJ and fermented YJ, and may be a useful tool for the quality control of YJ.

Aloe has long been used in food products, beverages and cosmetics, and as a traditional medicine to treat various diseases in many countries. In the present study, a new chromone, aloe resin E (1), and a new pyrone, aloenin C (11), together with thirteen known compounds were isolated from aqueous dissolved Aloe exudates and their structures were identified by spectroscopic analysis. Nuclear factor kappa B (NF-κB) inhibitory activity of the isolated compounds was evaluated using an NF-κB luciferase assay in HepG2 cells. Among them, 7-hydroxy-5-(hydroxymethyl)-2-methylchromone (4), 5-((S)-2′-oxo-4′-hydroxypentyl)-2-hydroxymethylchromone (9), and aloenin aglycone (13) showed significant inhibitory effects against TNFα-induced NF-κB transcriptional activity in a dose-dependent manner with IC50 values ranging from 14.92 to 18.70 µM. Furthermore, the transcriptional inhibition of compounds 4, 9, and 13 was confirmed by a decrease in the expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) genes in HepG2 cells.

Background: Magnolia officinalis cortex has been traditionally used to treat stomach and intestine diseases in traditional Korean medicine. In this study, we investigated the effect of water extract of M. officinalis cortex (WEMC) on osteoclast differentiation and function. Methods: Phytochemical characterization of WEMC was performed by high-performance liquid chromatography analysis. Osteoclast differentiation of bone marrow-derived macrophages was determined by tartrate-resistant acid phosphatase activity assay. Receptor activator of nuclear factor-κB ligand (RANKL) signaling factors and transcription factors regulating osteoclast differentiation were analyzed by Western blot and real-time polymerase chain reaction. Bone resorption function of mature osteoclasts was examined by using culture plate coated with inorganic crystalline calcium phosphate. Furthermore, the in vivo effect of WEMC on osteoporosis was examined using RANKL-induced bone loss model, characterized by micro-computed tomography and bone metabolism marker analysis. Results: WEMC inhibited RANKL-induced osteoclast differentiation and the bone resorbing activity of mature osteoclasts. WEMC contains gallic acid and honokiol as active constituents contributing to the inhibitory effect of WEMC on osteoclast differentiation. Further, WEMC suppressed RANKL-induced activation of p38 and nuclear factor-κB pathways and expression of osteoclastogenic transcription factors such as c-Fos for AP-1 and nuclear factor of activated T cells cytoplasmic 1. Ectopic overexpression of a constitutive active form of nuclear factor of activated T cells cytoplasmic 1 rescued the antiosteoclastogenic effect of WEMC. Consistent with the in vitro results, WEMC suppressed RANKL-induced trabecular bone loss in mice. Conclusion: WEMC might have a therapeutic potential to treat pathological bone diseases due to increased osteoclast differentiation and function.

Background: Dental caries is considered to be a preventable disease, and various antimicrobial agents have been developed for the prevention of dental diseases; however, many bacteria show resistance to existing agents. In this study, 14 medicinal herbs were evaluated for antimicrobial activity against five common oral bacteria as a screen for potential candidates for the development of natural antibiotics. Methods: Aqueous extracts of medicinal herbs were tested for activity against Enterococcus faecalis, Actinomyces viscosus, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sanguis grown in brain heart infusion (BHI) broth. A broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A disk diffusion assay was performed by inoculating bacterial cultures on BHI agar plates with paper disks soaked in each of the medicinal herb extracts. Inhibition of the synthesis of water-insoluble glucans by S. mutans was also investigated. Results: The aqueous extracts of many of the 14 medicinal herbs demonstrated antimicrobial activity against the five types of pathogenic oral bacteria. The extracts of Sappan Lignum, Coptidis Rhizoma, and Psoraleae Semen effectively inhibited the growth of oral bacteria and showed distinct bactericidal activity. The extracts of Notoginseng Radix, Perillae Herba, and Psoraleae Semen decreased the synthesis of water-insoluble glucans by the S. mutans enzyme glucosyltransferase (GTase). The present study is the first to confirm the antimicrobial activity of the extract of Sappan Lignum against all five species of oral bacteria strains. Conclusion: These results suggest that certain herbal medicines with proven antimicrobial effects, such as Sappan Lignum and Psoraleae Semen, may be useful for the treatment of dental diseases.

Many medicinal plants such as a Panax ginseng and Morus alba (mulberry tree) have been widely used as depigmenting agents in Asia. To maximize their synergistic effects on melanogenesis, new herbal decoctions were created by mixing Ginseng Radix Alba (GR) and Mori Radicis Cortex (MC) at a ratio of 3:2 which called GMC decoction. A decoction of GR and Mori Ramulus (MR), which called GMR, was also formulated in order to compare the anti-melanogenic capacity. Combined decoctions, GMC and GMR, significantly decreased mushroom tyrosinase activity in vitro; however, single extracts, including MC and MR, showed weaker inhibitory activity. Melanin content assay and Fontana–Masson staining confirmed that two decoctions showed stronger inhibitory effects on the forskolin-induced melanin level in B16 cells, without cytotoxicity. Our findings suggest that ginseng in combination with mulberry tree enhances the anti-melanogenic effect in vitro.