B16 Murine Cells Research Articles

Culturable Diversity and Biological Properties of Bacterial Endophytes Associated with the Medicinal Plants of Vernonia anthelmintica (L.) Willd

As one of the important traditional medicinal plants listed in the Chinese pharmacopoeia, Vernonia anthelmintica (L.) Willd has been shown to possess various biological activities. In this study, we characterized culturable endophytic bacteria associated with the medicinal plant V. anthelmintica collected from Hotan within the Xinjiang autonomous region of China. Bacterial endophytes were identified via 16S rRNA gene sequence analysis and compared to similar sequences from the GenBank. Isolated strains exhibited 99.08–100% similarity to Bacillus haynesii XJB-5, Bacillus proteolyticus XJB-16, Bacillus halotolerans XJB-35, Bacillus safensis XJB-71, Pseudomonas punonensis XJB-7, Lysinibacillus fusiformis XJB-17, Streptococcus lutetiensis XJB-66, Leclercia adecarboxylata XJB-12, Paenibacillus alvei XJB-14, and Pantoea agglomerans XJB-62. The ethyl acetate extracts of the bacterial endophytes demonstrated various pharmacological properties, such as antimicrobial, cytotoxic, antidiabetic, and antioxidant activity, according to a melanin content assay and have shown tyrosinase activity in murine B16 cells. A crude extract of B. halotolerans XJB-35 displayed more powerful biological activities than other bacterial endophytes; therefore, this strain was studied further in order to select the optimized parameters for enhancing the synthesis of bioactive compounds. The optimal culture medium was found to be nutrient broth (NB) medium, using peptone as its carbon source and yeast extract as its nitrogen source. A 24 h incubation time produced the optimal conditions for the maximum growth of B. halotolerans XJB-35 and the production of bioactive compounds. Moreover, we investigated the volatile chemical component of the dichloromethane fraction using GC-MS analysis. Our findings provide valuable information regarding the synthesize of bioactive natural products by B. halotolerans XJB-35 for use by the medicinal and pharmaceutical industries.

Diversity and Biological Activities of Endophytic Fungi from the Flowers of the Medicinal Plant Vernonia anthelmintica.

Secondary metabolites produced by endophytic fungi are an important source of biologically active compounds. The current research was focused on the biological activities of ethyl acetate extracts of fungi, isolated and identified from Vernonia anthelmintica flowers for the first time. In addition, an investigation of the non-polar chemical composition of dichloromethane-ethyl acetate extracts of the most active fungal strain was carried out. The isolates included Ovatospora senegalensis NR-03, Chaetomium globosum NR-04, Thielavia subthermophila NR-06, Aspergillus calidoustus NR-10, Aspergillus keveii XJF-23 and Aspergillus terreus XJF-3 species. Strains were identified by 18S rRNA gene sequencing methods and were registered in GenBank. Crude extracts of the fungi displayed in vitro biological activities, including antimicrobial and cytotoxic activities. A melanin content assay was performed on murine B16 cells. An ethyl acetate extract of O. senegalensis NR-03 showed high anticancer and antimicrobial activity; therefore, we also studied the non-polar chemical composition of the dichloromethane-ethyl acetate fraction and identified 52 non-polar compounds with the different medium. This investigation discovered that the secondary metabolites of the total extract of endophytic fungi could be a potential source of alternative natural antimicrobial, cytotoxic and melanin synthesis activity in their host plant, and the isolation of bioactive metabolites may provide a lead to new compounds of pharmaceutical significance.

Secondary Metabolites and their Biological Activities from Endophytic Fungal Strain Aspergillus terreus XJA8 Associated with Vernonia anthelmintica

Two isocoumarins (6-methoxymellein and 6-hydroxymellein), one pyrrole compound (minaline), one lactone derivative (butyrolactone II), five phenolic acids (phenylacetic, veratric (3,4-dimethoxybenzoic), 4-hydroxyphenylacetic, 4-hydroxybenzoic and protocatechuic acid), and two phenolic acid methyl esters ((2-hydroxyphenyl) acetic acid methyl ester and methyl 4-hydroxyphenylacetate), along with 1,3-bis(4-hydroxyphenyl) propan-2-one were isolated from the fungal strain Aspergillus terreus XJA8. The fungal strain was isolated from the root of the medicinal plant Vernonia anthelmintica for first time. All isolated secondary metabolites were evaluated for their antimicrobial activity and melanin synthesis in murine B16 cells.

ISOLATION, IDENTIFICATION AND BIOLOGICAL ACTIVITY OF ENDOPHYTIC BACTERIA BACILLUS HAYNESII XJB-6 ASSOCIATED WITH MEDICINAL PLANT VERNONIA ANTHELMINTICA STE

The present study aimed to the isolation, molecular identification and biological properties of endophytic bacterium Bacillus haynesii XJB-6 associated with the medicinal plant Vernonia anthelmintica stem. Isolated bacterial strain identification was based on 16S rrna gene sequence analysis and comparison with similar sequences from genbank. The 16S rrna gene sequence similarities 99.26% of endophytic bacterium isolated from the stem of V. Anthelmintica showed that the Bacillus haynesii. Crude ethyl acetate extract of B. Haynesii XJB-6 increased melanin synthesis in murine B16 cells in a dose-dependent manner.

Mechanisms of 4-Dimethylamino-4'-Methoxy Chalcone in Promoting Melanin Synthesis

Background and aims: Vitiligo, a pigmentation-associated disease, affects 1–2% of the global population andis difficult to treat. The pathogenetic mechanism of vitiligo remains unclear. Vernonia anthelmintica (L.) Willd. extract for vitiligo treatment was initially recorded 300 years ago. Itschalcone compounds are believed to play essential roles in this treatment. In a previous study, chalcones were shown to enhance melanin production and tyrosinase activity inmouse B16 cells. Materials and methods: In this study, the effects were investigated of 4-dimethylamino-4'-methoxy chalcone (DMC) on theexpression of tyrosinase (TYR), tyrosinase-related protein (TRP)-1, tyrosinase-relatedprotein (TRP)-2, and microphthalmia-associated transcription factor (MITF)on murine B16 cells. Moreover, the signaling pathways of melanogenesis regulation,and the effects of DMCon the AC/cAMP/PKA/CREB (CREB and p-CREB), p38mitogen-activated protein kinase (MAPK) (MAPK, p-p38 MAPK, ERK andp-ERK), Wnt/β-catenin (β-catenin), and SWI/SNF pathways (SOX-10) proteinexpression levels were examined by Western blot. Results: The data showed that DMCcould promote melanin production by upregulating the p-CREB, p-p38,p-ERK and β-catenin proteins.

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.

Digital holographic microscopy evaluation of dynamic cell response to electroporation.

Phase-derived parameters and time autocorrelation functions were used to analyze the behavior of murine B16 cells exposed to different amplitudes of electroporation pulses. Cells were observed using an off-axis digital holographic microscope equipped with a fast camera. Series of quantitative phase images of cells were reconstructed and further processed using MATLAB codes. Projected area, dry mass density, and entropy proved to be predictors for permeabilized cells that swell or collapse. Autocorrelation functions of phase fluctuations in different regions of the cell showed a good correlation with the local effectiveness of permeabilization.

Abstract 2665: Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models

Abstract Major therapeutic advances in the field of tumor immunotherapy are hindered by the ability to overcome the immunosuppressive tumor microenvironment (TME), which prevents the immune system from mounting an efficient anti-tumor response. Pulsed focused ultrasound (pFUS) studies has shown some promise as an immunomodulatory strategy for altering TME. This study evaluated the temporal changes following non-ablative pFUS on TME immune cell profiles in spleen (Sp), lymph nodes (LN), and tumors (B16 melanoma and 4T1 mammary carcinoma). Murine B16 and 4T1 cells were subcutaneously, bilaterally implanted into C57BL/6J and BALB/c mice (n=10 tumors/time-point), respectively. Ultrasound guided pFUS (VIFU 2000, Alpinion) was administered at 1 MHz, at 6 MPa PNP to 5 mm tumors. Entire tumor volume was sonicated with a 2 mm spacing between points using a 10 ms pulse length, 10% duty cycle and a pulse repetition frequency of 10 Hz. At days 1, 3, and 5 post-sonication, Sp, LN and tumors were harvested, fixed and processed for flow cytometry (FACS) analysis of immune cell populations [cytotoxic (Tcyt), helper (Th), and regulatory (Treg) T cells, natural killer (NK), dendritic cells (DC), F4/80+ macrophages (M1 and M2), myeloid-derived suppressor cells (MDSC)] and immune checkpoint markers (CTLA-4, PD-1 and PD-L1). The immune response to pFUS differed between the B16 and 4T1 tumor bearing mice. B16 mice at days 1, 3 and 5 post-sonication revealed a 3-4 fold increase in Tcyt, Th, NK and M1 macrophage activity in tumor, which peaked at day 3, and decreased by day 5, compared to control tumors. At day 5, a shift of cell populations toward LN, with increased Treg, Tcyt, NK, F4/80, M1 and M2 activity was observed. 4T1 mice showed high levels of Tcyt in Sp on day 1, and NK cells on day 3 in LN. Interestingly, these cell populations decreased in Sp and LN by day 5, with 3-4 fold increase in 4T1 tumors, along with increasing macrophage and DC activity. Despite the differences in both tumor types, results suggest that pFUS altered the TME by inducing anti-tumor responses by activating both innate (macrophages, NK cells) and adaptive (Tcyt, Th cells) arms of immunity. This study reveals the differences in immune cell profiles following sonication between the two tumor types and underscores the importance of not generalizing the effect of pFUS on the TME. This analysis also suggests that the global changes to the immune cell profile represent a shift from anti-inflammatory, immunosuppressive TME towards an anti-tumor TME. Optimizing pFUS parameters to enhance immune cell infiltration, would presumably lead to slowing tumor growth rates and when coupled with immune checkpoint inhibits could serve as neoadjuvant approach in treating malignancy. Citation Format: Parwathy Chandran, Gadi Cohen, Scott R. Burks, Joseph A. Frank. Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2665.

Secondary metabolites produced by endophytic Pantoea ananatis derived from roots of Baccharoides anthelmintica and their effect on melanin synthesis in murine B16 cells

Five indole derivatives, 1H-indol-7-ol (1), tryptophol (2), 3-indolepropionic acid (3), tryptophan (4), 3,3-di(1H-indol-3-yl)propane-1,2-diol (5) and two diketopiperazines, cyclo(L-Pro-L-Tyr) (6), cyclo[L-(4-hydroxyprolinyl)-L-leucine (7) along with one dihydrocinnamic acid (8) were isolated from Pantoea ananatis VERA8, that endophytic bacteria derived from Baccharoides anthelmintica roots. This is a first report towards an isolation of endophytic strains (funji or bacteria) from the B. anthelmintica herb. The synergetic properties of the total extract compositions, as well as effects of the pure isolated secondary metabolites evaluated on their melanin synthesis in murine B16 cells towards for vitiligo treatment.

Design, synthesis, and toward a side-ring optimization of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones and their effect on melanin synthesis in murine B16 cells

Herein, we report the synthesis of 48 novel 3-sulfonylamides containing a tricyclic thieno[2,3-d]pyrimidin-4(3H)-one moiety, and their influence on melanin synthesis in murine B16 cells. All target sulfonylamides were synthesized through key intermediate 3-nitro-thieno[2,3-d]pyrimidin-4(3H)-ones using three types of ipso-nitration reactions. In this case, we converted the pyrido[1,2-a]- fragment of the thieno[2,3-d]pyrimidine moiety to pyrrolo[1,2-a]- and azepino[1,2-a]- side-rings in order to evaluate the bioactivities of the synthesized derivatives for a structure activity-relationships point of view. The obtained results suggest that some of the selected compounds revealed a promising influence on melanin synthesis in murine B16 cells and may serve as lead compounds for further drug discovery and development.

Corrigendum to: Diversity-oriented synthesis of amide derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones and evaluation of their influence on melanin synthesis in murine B16 cells

Corrigendum to: Diversity-oriented synthesis of amide derivatives of tricyclic thieno[2,3-<i>d</i>]pyrimidin-4(3<i>H</i>)-ones and evaluation of their influence on melanin synthesis in murine B16 cells

Design, Synthesis and Biological Activity of Novel Furocoumarin Derivatives as Stimulators of Melanogenesis and Tyrosinase in B16 Cells

Sixteen novel and four known(4a, 4d, 4e, 4h) amine derivatives of furocoumarin were synthesized, then submitted to evaluation as stimulators of melanogenesis and tyrosinase in B16 murine cells. Among them, five compounds(4g, 4j―4m) showed potent activating effect on both melanogenesis and tyrosinase in vitro compared with positive control(8-MOP), the most widely used drugs for vitiligo in clinic. Noticeably, compounds 4h and 4j, which contain morpholine and piperazine, were recognized as the most effective stimulator of melanogenesis and tyrosianse in B16 cells separately. These derivatives may serve as lead compounds for further drug discovery for the treatment of vitiligo.

Novel Furocoumarin Derivatives Stimulate Melanogenesis in B16 Melanoma Cells by Up-Regulation of MITF and TYR Family via Akt/GSK3β/β-Catenin Signaling Pathways.

The extracts of Ficuscarica L. and Psoralen corylifolia L. are traditional Uygur medicines for the treatment of vitiligo, and its active ingredients furocoumarins, were are found to be the most effective agents against this skin disorder nowadays. Therefore, a series of novel easter derivatives (8a–8p) of furocoumarin were designed and synthesized based on our previous research to improve this activity in the present study. The synthesized derivatives were biologically evaluated for melanin synthesis in murine B16 cells and the SAR (structure-activity relationship) was summarized. Eight derivatives were more potent than positive control (8-MOP, 8-methoxypsoralan), especially compounds 8n (200%) and 8o (197%), which were nearly 1.5-fold potency when compared with 8-MOP (136%). Furthermore, the signaling pathway by which 8n activates the melanin biosynthesis was defined. Our results showed that it not only elevated the melanin content, but also stimulated the activity of tyrosinasein a concentration-dependent manner. Increasing of phosphorylation of Akt (also named PKB, protein kinase B) and non-activated GSK3β (glycogen synthase kinase 3 beta), which inhibited the degradation of β-catenin were observed through Western blot analysis. The accumulation of β-catenin probably led to the activation of transcription of MITF (microphthalmia-associated transcription factor) and TYR (tyrosinase) family, as well as the subsequent induction of melanin synthesis.

Sensitivity Of Murine Melanoma Cells B16 To Plasma Needle Treatment

Melanoma is the most dangerous form of skin cancer originated from the pigment-producing melanocytes in the basal layer of the epidermis. Its capability to produce metastasis, along with the development of quimio or radioresistance makes this kind of cancer very dangerous and hard to heal, so is important an early detection as well as the search of new treatments. B16 murine cell line has been used extensively as a model for the study of this type of cancer. Non thermal plasma needle has been effectively used in the inactivation of microorganisms, in the disinfection of several types of tissues and to accelerate the wound healing processes. It also has been tested as an inductor of apoptosis in HepG2 cancer cell line. The observed effects of plasma upon cells has been attributed to the generation of reactive oxygen/nitrogen species (RONS), which can disrupt the plasmatic membrane and react with several biomolecules including DNA. The aim of the present work is to evaluate the sensitivity of the murine melanoma cell line B16 to a helium-generated plasma needle exposure. B16 cells were grown at 37°C and 5% CO2 atmosphere, in minimal essential medium with 10% FBS. Cells were harvested by tripsinization, washed twice with Hanks balanced saline solution and further incubated for at least one hour in MEM at 37°C to recover. Blood samples were obtained from healthy donors by venopunction and lymphocytes were separated by using the Ficoll Hypaque technique. Nucleated cells were collected, washed twice with HBSS, resuspended in RPMI-1640 with 10% FBS and kept at 37°C for at least one hour. Both cell types were resuspended in HBSS and then 200 µl aliquots were distributed in a microwell plate and exposed to plasma generated by a flow of 0.7 LPM of helium through a 13.56 MHz radiofrequency generator at a power of 5 W. The needle was kept at a distance of 2 mm. Cell death was evaluated by the fluorescein diacetate technique. After treatments, cells were stained with a 1:1 fluorescein diacetate (80 µg/ml) and ethidium bromide (50 µg/ml) solution and observed with an epifluorescence microscope. Living cells are stained in green while dead cells are stained in red. Genotoxicity was assessed by means of the comet assay. After treatments, cells were mixed with an equal volume of 1% low melting point agarose, poured on top of fully frosted microscope slides and immerse in cold lysis solution for an hour. Slides were then transferred to an electrophoresis cell, covered with the electrophoresis solution (0.3M NaHO, 0.1 mM Na2EDTA) for 20 minutes to allow DNA unwinding and then a current was applied (20 V, 300 mA, 20 minutes). Slides were stained with 60 µl of a 20 µg/ml ethidium bromide solution and observed under an epifluorescence microscope. Comets were scored by means of the Comet Assay IV Analyzer (Perceptive Instruments Inc.). The results show that the exposure to plasma produced cytotoxicity and genotoxicity on B16 cells in a dose-dependent manner. It was observed that the effect of non-thermal plasma was higher in cells of melanoma of mouse comparing to the effect observed in the lymphocytes.

Diversity-oriented synthesis of amide derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones and evaluation of their influence on melanin synthesis in murine B16 cells

Abstract A diversity-oriented synthesis of amide-containing thieno[2,3-d]pyrimidin-4(3H)-ones is reported. All compounds were tested for their influence on melanin synthesis in murine B16 cells. The azepine fragment in thieno[2,3-d]pyrimidin-4(3H)-one skeleton significantly increases the melanin content.

Synthesis and biological evaluation of novel sulfonamide derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones on melanin synthesis in murine B16 cells

Phototherapy with psoralens as photosensitizers is the commonly used method for the treatment of vitiligo. Considering the structure similarity between psoralens and thieno[2,3-d]pyrimidinones, a series of novel sulfonamide derivatives containing tricyclic thieno[2,3-d]pyrimidinone were synthesized and evaluated for melanin synthesis in murine B16 cells. All new compounds were characterized by 1H NMR, 13C NMR, IR and HRMS (ESI). Among them, 6 compounds demonstrated excellent activity than positive control (8-methoxylpsoralen, 8-MOP) with more than 1.5-fold potency. Compound 11w with dichloro substitution at meta-positions in the benzenesulfonyl moiety was the most potent one (658.3 ± 8.7%), exhibiting 5.0-fold stronger activity than 8-MOP (130.9 ± 8.6%). The difluoro analog compound 11o increased melanin synthesis in murine B16 cells with a 4.35-fold potency as compared to 8-MOP. These compounds may serve as lead compounds for further drug discoveries for the treatment of vitiligo.

Synthesis and in vitro biological evaluation of novel coumarin derivatives containing isoxazole moieties on melanin synthesis in B16 cells and inhibition on bacteria

A novel series of coumarin derivatives 6a–o, bearing isoxazole moieties were designed and synthesized. After that, they were evaluated for melanin synthesis in murine B16 cells and inhibitory effect on the growth of CA (Candida albicans), EC (Escherichia coli), SA (Staphylococcus aureus). It was found that eleven compounds (6b–f, 6j–o) showed a better activity on melanin synthesis than positive control (8-MOP). Among them, compounds 6d (242%) and 6f (390%), with nearly 1.6 and 2.6-fold potency compared with 8-MOP (149%) respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.Seven halogen substituted compounds exhibited moderate antimicrobial activity against CA. It is interesting that 6e–f and 6l–m, which had two halogens on the benzene showed a comparable activity with Amphotericin B against CA.The evaluation of melanin synthesis in B16 cells and inhibitory effect on bacteria of above structurally diverse derivatives had also led to an outline of structure-activity relationship.

Design, synthesis and bioactivity of chalcones and its analogues

The Vernohia anthelmintica L.’s extract is one of the most popular Uygur medicines used for vitiligo. It is believed that the chalcone compounds of the plant play an important role in the treatment since they may activate tyrosinase and improve melanin production. In this study, twenty-one chalcones and nine analogues were synthesized in view of three different components of chalcone (A, B ring and α, β-unsaturated carbonyl). After biological evaluation of their activity on tyrosinase in cell-free systems, the result showed that most compounds (except polyhydroxy chalcones) possess activator effect on the tyrosinase, especially for 13a–15a, 20a and 1b, which bearing a comparable activity to the positive control 8-MOP. SAR of these tyrosinase activator was summed up for the first time as well. Finally, compound 13a was found to increase melanin contents and tyrosinase activity 1.75 and 1.3 fold, respectively, compared with that of untreated murine B16 cells at the concentration of 40μg/mL.

The depigmenting effect of natural resorcinol type polyphenols Kuwanon O and Sanggenon T from the roots of morus australis

Ethnopharmacological relevanceMorus australis, one of the major Morus species growing in East Asia, is rich in phenolic compounds. The extract of M. australis has been used as skin whitening components for a long period. The action mechanisms of its principal constituents are still unclear. This study aims to evaluate the skin lightening effects of phenolic compounds extracted from the root of M. australis in different melanocyte systems and artificial skin models. Materials and methodsThe depigmenting effect of resorcinol type polyphenols (RTPs) from the root extract of M. australis was evaluated in murine b16 and melan-a cell lines using a combined sulforhodamine B assay. Tyrosinase activity and the expression of melanogenesis proteins were evaluated for the mechanism study. The artificial skin model is used as a replacement of the animal test. ResultsOnly Kuwanon O and Sanggenon T were found to have significant depigmenting effects in both murine b16 and melan-a cell lines. Their depigmenting mechanisms are slightly different in the two cell systems. In b16 cells, Kuwanon O and Sanggenon T, together with the other two RTPs, induced post-transcriptional degradations of MITF without suppressing its mRNA expression, leading to significant decreases of TRP-1 and TRP-2 production. While in melan-a cells, the levels of tyrosinase families were suppressed via MITF downregulation at both transcription and translation level by RTPs, with Kuwanon O inducing the greatest suppression. Further evaluations in artificial skin model demonstrated the outstanding depigmenting effects of Kuwanon O and Sanggenon T. ConclusionsKuwanon O and Sanggenon T from M.australis root extract are two potential skin whitening ingredients. To screen resorcinol flavonone derivatives with an isoprenyl group in the Diels-Alder substituent might be an option for the search of potent hypopigmenting agents from plants.