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1.
Am J Chin Med ; 49(8): 1871-1895, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34961421

RESUMO

Shikonin is one of the primary active components extracted from the dried root ofZicao (Lithospermum erythrorhizon, Onosma paniculata, or Arnebia euchroma), a traditional Chinese herbal medicine. Shikonin is known to not only exert anti-proliferative, anti-inflammatory, and anti-angiogenic activities, but also play a crucial role in triggering the production of reactive oxygen species, suppressing the release of exosomes, and inducing apoptosis. Increasing evidence suggests that shikonin has a protective effect against skin diseases, including psoriasis, melanoma, and hypertrophic scars. In order to evaluate the application potential of shikonin in the treatment of skin diseases, this review is the first of its kind to provide comprehensive and up-to-date information regarding the uses of shikonin and its derivatives on skin diseases and its underlying mechanisms. In this review, we have focused on the signaling pathways and cellular targets involved in the anti-dermatosis effects of shikonin to bridge the gaps in the literature, thereby providing scientific support for the research and development of new drugs from a traditional medicinal plant.


Assuntos
Lithospermum , Naftoquinonas , Dermatopatias , Humanos , Inflamação , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Dermatopatias/tratamento farmacológico
2.
J Obstet Gynaecol Res ; 47(11): 3789-3796, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34505328

RESUMO

OBJECTIVE: To investigate the optimal dose of mifepristone and lithospermum combination regimen on medical abortion in early pregnancy rats without increasing side effects. STUDY DESIGN: Sixty sexually mature female Sprague Dawley (SD) rats with early pregnancy were randomly allocated into 10 groups, including a control group (treated with 0.5% CMC-Na) and nine experiments (treated with 1 mg/kg mifepristone, and 90, 180, 270, and 540 mg/kg lithospermum, and 90/180/270/540 mg/kg lithospermum +1 mg/kg mifepristone, respectively). The hormone levels, factors associated with endometrial bleeding, oxidative stress, and apoptotic proteins in the endometrium, were then investigated. RESULTS: The results demonstrated that 540 mg/kg lithospermum plus 1 mg/kg mifepristone treatment significantly improved the abortion rate when compared with the control group. Compared with the 1 mg/kg mifepristone, 540 mg/kg lithospermum plus 1 mg/kg mifepristone treatment did not induce significant increase in factors associated with abnormal endometrial bleeding, such as matrix metalloproteinase-9 (MMP9). However, mifepristone and lithospermum combination regimen promoted the expression level of malondialdehyde (MDA), activated caspase 3, caspase 9 and Bax, meanwhile, reduced the expression of superoxide dismutase (SOD) and Bcl-2. CONCLUSION: These findings provided strong evidence that mifepristone and lithospermum combination regimen can obtain satisfactory abortion effect without increasing the expression level of bleeding-related factors.


Assuntos
Abortivos não Esteroides , Abortivos Esteroides , Aborto Induzido , Lithospermum , Misoprostol , Animais , Mifepristona/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley
3.
Nutrients ; 13(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34579088

RESUMO

The incidence of atopic dermatitis (AD), a disease characterized by an abnormal immune balance and skin barrier function, has increased rapidly in developed countries. This study investigated the anti-atopic effect of Lithospermum erythrorhizon (LE) using NC/Nga mice induced by 2,4-dinitrochlorobenzene. LE reduced AD clinical symptoms, including inflammatory cell infiltration, epidermal thickness, ear thickness, and scratching behavior, in the mice. Additionally, LE reduced serum IgE and histamine levels, and restored the T helper (Th) 1/Th2 immune balance through regulation of the IgG1/IgG2a ratio. LE also reduced the levels of AD-related cytokines and chemokines, including interleukin (IL)-1ß, IL-4, IL-6, tumor necrosis factor-α (TNF-α), thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, regulated on activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 in the serum. Moreover, LE modulated AD-related cytokines and chemokines expressed and secreted by Th1, Th2, Th17, and Th22 cells in the dorsal skin and splenocytes. Furthermore, LE restored skin barrier function by increasing pro-filaggrin gene expression and levels of skin barrier-related proteins filaggrin, involucrin, loricrin, occludin, and zonula occludens-1. These results suggest that LE is a potential therapeutic agent that can alleviate AD by modulating Th1/Th2 immune balance and restoring skin barrier function.


Assuntos
Dermatite Atópica/tratamento farmacológico , Lithospermum/química , Extratos Vegetais/farmacologia , Pele/efeitos dos fármacos , Pele/patologia , Animais , Benzofuranos/química , Benzofuranos/farmacologia , Citocinas/genética , Citocinas/metabolismo , Depsídeos/química , Depsídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Extratos Vegetais/química , Pele/imunologia , Baço/citologia , Equilíbrio Th1-Th2/efeitos dos fármacos
4.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502210

RESUMO

The present study reports a green chemistry approach for the rapid and easy biological synthesis of silver (Ag), gold (Au), and bimetallic Ag/Au nanoparticles using the callus extract of Lithospermum erythrorhizon as a reducing and capping agent. The biosynthesized nanoparticles were characterized with ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD) analysis, and transmission electron microscopy (TEM). Our results showed the formation of crystalline metal nanostructures of both spherical and non-spherical shape. Energy dispersive X-ray (EDX) spectroscopy showed the characteristic peaks in the silver and gold regions, confirming the presence of the corresponding elements in the monometallic particles and both elements in the bimetallic particles. Fourier-transform infrared (FTIR) spectroscopy affirmed the role of polysaccharides and polyphenols of the L. erythrorhizon extract as the major reducing and capping agents for metal ions. In addition, our results showed that the polysaccharide sample and the fraction containing secondary metabolites isolated from L. erythrorhizon were both able to produce large amounts of metallic nanoparticles. The biosynthesized nanoparticles demonstrated cytotoxicity against mouse neuroblastoma and embryonic fibroblast cells, which was considerably higher for Ag nanoparticles and for bimetallic Ag/Au nanoparticles containing a higher molar ratio of silver. However, fibroblast migration was not significantly affected by any of the nanoparticles tested. The obtained results provide a new example of the safe biological production of metallic nanoparticles, but further study is required to uncover the mechanism of their toxicity so that the biomedical potency can be assessed.


Assuntos
Antineoplásicos/farmacologia , Ouro/química , Lithospermum/química , Nanopartículas Metálicas/administração & dosagem , Neuroblastoma/tratamento farmacológico , Extratos Vegetais/farmacologia , Prata/química , Animais , Antineoplásicos/química , Apoptose , Células Cultivadas , Nanopartículas Metálicas/química , Camundongos , Células NIH 3T3 , Neuroblastoma/patologia
5.
DNA Res ; 28(5)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34424327

RESUMO

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contigN50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon's roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT's functional differentiation in evolution.


Assuntos
Lithospermum , Naftoquinonas , Aciltransferases , Lithospermum/genética
6.
Se Pu ; 39(7): 708-714, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34227368

RESUMO

Lithospermum erythrorhizon has the functions of cooling blood, activating blood, as well as detoxifying and penetrating rash. Lithospermum oil extracted from Lithospermum erythrorhizon can prevent and treat diaper rash, skin ulceration, eczema, and other skin diseases. Supercritical fluid extraction is the optimal method for the extraction of active components from lithospermum. In this study, an analytical method was established for simultaneously determination of six active components in lithospermum oil with high performance liquid chromatography (HPLC), and the contents of the active components as the evaluation index were used to investigate several important factors in the preparation of lithospermum oil by supercritical fluid extraction. The optimized HPLC conditions were as follows: separation column, Diamonsil C18 (250 mm×4.6 mm, 5 µm); mobile phases, acetonitrile containing 0.1% (v/v) formic acid-0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate (75∶25, v/v); flow rate, 1 mL/min; injection volume, 15 µL; room temperature; photodiode array detector (PAD); detection wavelength, 275 nm. The supercritical fluid extraction was optimized for ensuring stability of the amounts of effective components and the reliability of the quality of lithospermum oil. This will serve as the basis for preparation and quality control processes. Three factors and three levels orthogonal tests were adopted to investigate the important factors, viz. the pressure, temperature and CO2 flow rate in the preparation of lithospermum oil. The results showed that the developed HPLC-PAD method can be used for the simultaneous determination of shikonin, acetylshikonin, ß-acetoxyisovaleryl akanin, isobutyryl shikonin, ß,ß-dimethylacryl shikonin, and 2-methylbutyryl shikonin in 30 min. The method has good precision, accuracy and repeatability. The contents of the active components were the highest when the extraction pressure, extraction temperature, and CO2 flow rate were 23 MPa, 40 ℃, and 27 L/h, respectively. The optimized conditions are suitable for the preparation and actual production of lithospermum oil. The HPLC-PAD method is simple, feasible, accurate, and reliable. It can be used for the preparation and quality control of lithospermum oil by supercritical fluid extraction. Thus, with this method, the stability of the contents of active ingredients and the reliability of the quality of lithospermum oil can be ensured; moreover, safe and effective drug use can be realized. The established method has obvious advantages over the traditional process and is a good candidate for widespread use.


Assuntos
Lithospermum , Compostos Fitoquímicos/análise , Óleos Vegetais/química , Cromatografia Líquida de Alta Pressão , Cromatografia com Fluido Supercrítico , Lithospermum/química , Reprodutibilidade dos Testes , Temperatura
7.
Phytomedicine ; 82: 153460, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33476976

RESUMO

BACKGROUND: Shikonin, a naphthoquinone compound extracted from the root of Lithospermum erythrorhizon, has been extensively studied for its antitumor activity. However, the systematic pathways involved in Shikonin intervention in human colon cancer has not yet clearly defined. PURPOSE: This study was to evaluate the cytotoxic effects of Shikonin in colon cancer, as well as investigate the potential biomarkers from a global perspective and the possible antitumor mechanisms involved. METHODS: In this work, cell viability, cell cycle and cell apoptosis in human colon cancer cells were assessed to evaluate the antitumor activity of Shikonin. Transcriptomics and metabolomics were integrated to provide the perturbed pathways and explore the potential mechanisms. The crucial proteins and genes involved were further validated by immunohistochemistry and real-time quantitative PCR. RESULTS: Shikonin revealed a remarkable antitumor potency in colon cancer. Cell cycle was significantly arrested at the S phase as well as apoptosis was induced in SW480 cell line. Furthermore, a total of 1642 differentially expressed genes and 40 metabolites were detected after Shikonin intervention. The integrated analysis suggested that the antitumor effect was mainly attributed to purine metabolism, arginine biosynthesis, pyrimidine metabolism, urea cycle and metabolism of amino acids. The up-regulated expression of proteins vital for arginine biosynthesis was subsequently validated by immunohistochemistry in xenograft mice. Notably, supplemental dNTPs and arginine could significantly reverse the cytotoxic effect induced by Shikonin and the genes participating in purine metabolism and arginine biosynthesis were further determined by RT-qPCR. CONCLUSION: Our findings provide a systematic perspective in the therapeutic effect of Shikonin which might lay a foundation for further research on Shikonin in colon cancer.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Naftoquinonas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lithospermum/química , Metabolômica , Camundongos , Naftoquinonas/farmacologia , Raízes de Plantas/química , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Phytother Res ; 35(1): 463-476, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32779300

RESUMO

Triple-negative breast cancer (TNBC) is heterogeneous disease with a poor prognosis. It is therefore important to explore novel therapeutic agents to improve the clinical efficacy for TNBC. The inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is always overexpressed in many types of tumors, including TNBC and regarded as a potential target for cancer therapy. Through screening a library of natural products, we identified shikonin, a natural bioactive component of Lithospermum erythrorhizon, is a novel and selective IMPDH2 inhibitor. Enzymatic analysis using Lineweaver-Burk plot indicates that shikonin is a competitive inhibitor of IMPDH2. The interaction between shikonin and IMDPH2 was further investigated by thermal shift assay, fluorescence quenching, and molecular docking simulation. Shikonin treatment effectively inhibits the growth of human TNBC cell line MDA-MB-231, and murine TNBC cell line, 4T1 in a dose-dependent manner, which is impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. Most importantly, IMPDH2 knockdown significantly reduced cell proliferation and conferred resistance to shikonin in TNBC. Collectively, our findings showed the natural product shikonin as a selective inhibitor of IMPDH2 with anti-TNBC activity, impelling its further study in clinical trials.


Assuntos
Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Naftoquinonas/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lithospermum/química , Camundongos , Simulação de Acoplamento Molecular , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
9.
Exp Eye Res ; 203: 108419, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33383026

RESUMO

Lithospermum erythrorhizon (L. erythrorhizon), used in traditional medicine, is a potent wound healing, anti-inflammatory and antioxidant plant. However, the effects of L. erythrorhizon on retinal degenerative diseases remain unknown. Here, we explored the protective effects of L. erythrorhizon in in vitro and in vivo retinal degeneration. We found that ethanol extract of L. erythrorhizon (EELE) and the dichloromethane fraction of L. erythrorhizon (MCLE) significantly increased cell viability under glutamate/BSO-induced excitotoxicity/oxidative stress in R28 cells. Treatment with EELE and MCLE reduced the intracellular reactive oxygen species (ROS) and the levels of apoptotic proteins, such as cleaved PARP and cleaved caspase-3. Furthermore, oral administration of EELE and MCLE in an in vivo optic nerve crush mouse model decreased RGC cell death and increased retinal thickness. The major compound between EELE and MCLE was found to be lithospermic acid A (LAA), which has been shown to prevent the elevation of ROS in R28. Therefore, EELE and MCLE have protective effects against the death of retinal cells in vitro and in vivo, and the major compound, LAA, has an antioxidant effect on retinal cells, suggesting that EELE and MCLE could be beneficial agents for retinal degenerative diseases, including glaucoma.


Assuntos
Lithospermum/química , Traumatismos do Nervo Óptico/tratamento farmacológico , Fitoterapia/métodos , Extratos Vegetais/uso terapêutico , Raízes de Plantas/química , Degeneração Retiniana/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Benzofuranos/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Depsídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compressão Nervosa , Traumatismos do Nervo Óptico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Tomografia de Coerência Óptica
10.
J Dermatolog Treat ; 32(3): 297-301, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-31424962

RESUMO

BACKGROUND: Herbal extracts with fewer adverse effects can be an alternative to these drugs because they can target various molecular pathways of acne pathogenesis. OBJECTIVES: To evaluate the clinical efficacy of herbal extracts (mangosteen, Lithospermum officinale, Tribulus terrestris L., Houttuynia cordata Thunb) for the treatment of mild to moderate acne vulgaris. METHODS: Sixty patients were randomized in a 1:1 ratio to receive blinded treatment with herbal extracts or vehicle for 8 weeks. Inflammatory and non-inflammatory acne lesion counts, Investigator's Global Assessment, patient's satisfaction and safety profiles were assessed. We also performed skin biopsy at baseline and week 8 to confirm immunological changes with immunohistochemistry staining. RESULTS: By the end of the study period, both inflammatory and non-inflammatory acne lesion counts were significantly decreased in herbal extracts group (p< .05). In immunohistochemistry staining, expressions of IL-1α, IL-8, and keratin 16 were significantly decreased in herbal extracts group compared to vehicle group from baseline to week 8. There was no serious adverse events in both groups. CONCLUSIONS: This herbal extracts can be a new therapeutic option for patients with mild to moderate acne vulgaris who are reluctant to use drugs.


Assuntos
Acne Vulgar/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Acne Vulgar/patologia , Administração Cutânea , Adulto , Método Duplo-Cego , Esquema de Medicação , Feminino , Garcinia mangostana/química , Garcinia mangostana/metabolismo , Humanos , Interleucina-1alfa/metabolismo , Lithospermum/química , Lithospermum/metabolismo , Masculino , Satisfação do Paciente , Extratos Vegetais/química , Índice de Gravidade de Doença , Pele/metabolismo , Pele/patologia , Resultado do Tratamento , Adulto Jovem
11.
Molecules ; 25(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322297

RESUMO

The potential of plant extracts as bioinsecticides has been described as a promising field of agricultural development. In this work, the extracts of Punica granatum (pomegranate), Phytolacca americana (American pokeweed), Glandora prostrata (shrubby gromwell), Ulex europaeus (gorce), Tagetes patula (French marigold), Camellia japonica red (camellia), Ruta graveolens (rue or herb-of-grace) were obtained, purified, and their activity against Spodoptera frugiperda (Sf9) insect cells was investigated. From the pool of over twenty extracts obtained, comprising different polarities and vegetable materials, less polar samples were shown to be more toxic towards the insect cell line Sf9. Among these, a dichloromethane extract of R. graveolens was capable of causing a loss of viability of over 50%, exceeding the effect of the commercial insecticide chlorpyrifos. This extract elicited chromatin condensation and the fragmentation in treated cells. Nanoencapsulation assays of the cytotoxic plant extracts in soybean liposomes and chitosan nanostructures were carried out. The nanosystems exhibited sizes lower or around 200 nm, low polydispersity, and generally high encapsulation efficiencies. Release assays showed that chitosan nanoemulsions provide a fast and total extract release, while liposome-based systems are suitable for a more delayed release. These results represent a proof-of-concept for the future development of bioinsecticide nanoformulations based on the cytotoxic plant extracts.


Assuntos
Agentes de Controle Biológico/química , Praguicidas/química , Extratos Vegetais/química , Folhas de Planta/química , Animais , Camellia , Quitosana/química , Fabaceae , Insetos , Inseticidas/análise , Lipossomos/química , Lithospermum , Nanoestruturas , Phytolacca americana , Romã (Fruta) , Ruta , Solventes , Soja/efeitos dos fármacos , Tagetes
12.
Plant Cell Physiol ; 61(10): 1798-1806, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810231

RESUMO

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeídos/metabolismo , Benzoquinonas/metabolismo , Lithospermum/enzimologia , Naftoquinonas/metabolismo , Terpenos/metabolismo , Lithospermum/metabolismo , Redes e Vias Metabólicas
13.
Sci Rep ; 10(1): 13555, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782359

RESUMO

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.


Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , Lithospermum/genética , Doenças das Plantas/genética , Folhas de Planta/genética , Proteínas de Plantas/antagonistas & inibidores , Plantas Medicinais/genética , Secoviridae/genética , Lithospermum/virologia , Doenças das Plantas/virologia , Folhas de Planta/virologia , Proteínas de Plantas/genética , Plantas Medicinais/virologia , Secoviridae/patogenicidade
14.
Pharmacol Res ; 161: 105123, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32822867

RESUMO

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.


Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/prevenção & controle , Antagonistas do Ácido Fólico/farmacologia , Lithospermum , Receptor ErbB-2/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antraquinonas/isolamento & purificação , Antraquinonas/farmacocinética , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Antagonistas do Ácido Fólico/isolamento & purificação , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Lithospermum/química , Camundongos Transgênicos , Raízes de Plantas , Receptor ErbB-2/genética , Transdução de Sinais , Distribuição Tecidual , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Plant Physiol ; 184(2): 753-761, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32727911

RESUMO

Several Boraginaceae plants produce biologically active red naphthoquinone pigments, derivatives of the enantiomers shikonin and alkannin, which vary in acyl groups on their side chains. Compositions of shikonin/alkannin derivatives vary in plant species, but the mechanisms generating the diversity of shikonin/alkannin derivatives are largely unknown. This study describes the identification and characterization of two BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1), from Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae that primarily produces the shikonin/alkannin derivatives acetylshikonin and ß-hydroxyisovalerylshikonin. Enzyme assays using Escherichia coli showed that the acylation activity of LeSAT1 was specific to shikonin, whereas the acylation activity of LeAAT1 was specific to alkannin. Both enzymes recognized acetyl-CoA, isobutyryl-CoA, and isovaleryl-CoA as acyl donors to produce their corresponding shikonin/alkannin derivatives, with both enzymes showing the highest activity for acetyl-CoA. These findings were consistent with the composition of shikonin/alkannin derivatives in intact L erythrorhizon plants and cell cultures. Genes encoding both enzymes were preferentially expressed in the roots and cell cultures in the dark in pigment production medium M9, conditions associated with shikonin/alkannin production. These results indicated that LeSAT1 and LeAAT1 are enantiomer-specific acyltransferases that generate various shikonin/alkannin derivatives.


Assuntos
Aciltransferases/metabolismo , Lithospermum/enzimologia , Naftoquinonas/metabolismo , Aciltransferases/genética , Escherichia coli , Lithospermum/genética , Especificidade por Substrato
17.
Phytochemistry ; 175: 112375, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32305685

RESUMO

Shikonin is a natural naphthoquinone derivative that specifically occurs in boraginaceous plants, and the major active ingredient of the medicinal plant Lithospermum erythrorhizon. Previously, a cytochrome P450 oxygenase (CYP) CYP76B74 catalyzing 3″-hydroxylation of geranylhydroquinone (GHQ) - a key intermediate of shikonin biosynthesis, was identified from cultured cells of Arnebia euchroma. However, the enzymes catalyzing oxidation of the geranyl side-chain of GHQ from L. erythrorhizon remain unknown. In this study, we performed transcriptome analysis of different tissues (red roots and green leaves/stems) from L. erythrorhizon using RNA sequencing technology. Highly expressed CYP genes found in the roots were then heterologously expressed in Saccharomyces cerevisiae and functionally screened with GHQ as the substrate. As the result, two CYPs of CYP76B subfamily catalyzing the oxidation of GHQ were characterized. CYP76B100 catalyzed the hydroxylation of the geranyl side-chain of GHQ at the C-3″ position to form 3″-hydroxyl geranylhydroquinone (GHQ-3″-OH). The enzyme CYP76B101 carried out oxidation reaction of GHQ at the C-3″ position to produce a 3″-carboxylic acid derivative of GHQ (GHQ-3″-COOH) as well as GHQ-3″-OH. This enzyme-catalyzed oxidation reaction with GHQ as the substrate is reported for the first time. This study implicates CYP76B100 and CYP76B101 as having a potential role in shikonin biosynthesis in L. erythrorhizon.


Assuntos
Lithospermum , Naftoquinonas , Sistema Enzimático do Citocromo P-450 , Terpenos
18.
Drug Des Devel Ther ; 14: 577-589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103900

RESUMO

Background: Shikonin, the main ingredient of Lithospermum erythrorhizon, has been reported to have antitumor effects via multiple targets and signaling pathways. However, the detailed mechanism underlying the effects in cervical cancer still remained unknown. Methods: MTT, wound-healing, transwell assays and flow cytometry experiments were used to measure cell growth, migration, invasion, and cell cycle analysis. Western blot was used to examine protein levels of Snail, Vimentin and E-cadherin. The expression level of miR-183-5p was measured via qRT-PCR. The E-cadherin promoter activity was detected via Secrete-PairTM Dual Luminescence Assay Kit. The transient transfection experiments were used for silencing of E-cadherin and overexpression of Snail genes. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings. Results: We showed that shikonin inhibited cell viability, migration and invasion, and induced cell cycle arrest in a dose-dependent manner in cervical cancer Hela and C33a cells. Mechanistically, we found that shikonin increased miR-183-5p expression and inhibited expression of transcription factor Snail protein. The mimics of miR-183-5p reduced, while the inhibitors of miR-183-5p reversed shikonin-inhibited Snail protein expression. In addition, shikonin decreased Vimentin, increased E-cadherin protein expressions and E-cadherin promoter activity, the latter was reversed in cells transfected with exogenous Snail overexpression vectors. Moreover, silencing of E-cadherin significantly abolished shikonin-inhibited cervical cancer cell growth. Similar findings were also observed in vivo using one xenograft mouse model. Conclusion: Our results show that shikonin inhibits EMT through inhibition of Snail and stimulation of miR-183-5p expressions, which resulted in induction of E-cadherin expression. Thus, blockade of EMT could be a novel mechanism underlying the anti-cervical cancer effects of shikonin.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , MicroRNAs/genética , Naftoquinonas/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antígenos CD/genética , Antineoplásicos Fitogênicos/administração & dosagem , Caderinas/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Lithospermum/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/administração & dosagem , Fatores de Transcrição da Família Snail/genética , Neoplasias do Colo do Útero/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Plant Physiol ; 182(4): 1933-1945, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31974127

RESUMO

Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in Lithospermum erythrorhizon localizes to the cytosol, in which GPP is utilized for the biosynthesis of naphthoquinone pigments, which are shikonin derivatives. This study describes the identification of the cytosol-localized GPPS gene, LeGPPS, through EST- and homology-based approaches followed by functional analyses. The deduced amino acid sequence of the unique LeGPPS showed greater similarity to that of farnesyl diphosphate synthase (FPPS), which generally localizes to the cytosol, than to plastid-localized conventional GPPS. Biochemical characterization revealed that recombinant LeGPPS predominantly produces GPP along with a trace amount of FPP. LeGPPS expression was mainly detected in root bark, in which shikonin derivatives are produced, and in shikonin-producing cultured cells. The GFP fusion protein in onion (Allium cepa) cells localized to the cytosol. Site-directed mutagenesis of LeGPPS and another FPPS homolog identified in this study, LeFPPS1, showed that the His residue at position 100 of LeGPPS, adjacent to the first Asp-rich motif, contributes to substrate preference and product specificity, leading to GPP formation. These results suggest that LeGPPS, which is involved in shikonin biosynthesis, is recruited from cytosolic FPPS and that point mutation(s) result in the acquisition of GPPS activity.


Assuntos
Citosol/metabolismo , Geraniltranstransferase/metabolismo , Lithospermum/metabolismo , Cumarínicos/metabolismo , Geraniltranstransferase/genética , Monoterpenos/metabolismo , Mutagênese Sítio-Dirigida , Naftoquinonas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo
20.
BMC Complement Altern Med ; 19(1): 325, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752825

RESUMO

BACKGROUND: Osteoarthritis (OA) is an age-related joint disease with characteristics that involve the progressive degradation of articular cartilage and resulting chronic pain. Previously, we reported that Astragalus membranaceus and Lithospermum erythrorhizon showed significant anti-inflammatory and anti-osteoarthritis activities. The objective of this study was to examine the protective effects of ALM16, a new herbal mixture (7:3) of ethanol extracts of A. membranaceus and L. erythrorhizon, against OA in in vitro and in vivo models. METHODS: The levels of matrix metalloproteinase (MMP)-1, -3 and - 13 and glycosaminoglycan (GAG) in interleukin (IL)-1ß or ALM16 treated SW1353 cells were determined using an enzyme-linked immunosorbent and quantitative kit, respectively. In vivo, the anti-analgesic and anti-inflammatory activities of ALM16 were assessed via the acetic acid-induced writhing response and in a carrageenan-induced paw edema model in ICR mice, respectively. In addition, the chondroprotective effects of ALM16 were analyzed using a single-intra-articular injection of monosodium iodoacetate (MIA) in the right knee joint of Wister/ST rat. All samples were orally administered daily for 2 weeks starting 1 week after the MIA injection. The paw withdrawal threshold (PWT) in MIA-injected rats was measured by the von Frey test using the up-down method. Histopathological changes of the cartilage in OA rats were analyzed by hematoxylin and eosin (H&E) staining. RESULTS: ALM16 remarkably reduced the GAG degradation and MMP levels in IL-1ß treated SW1353 cells. ALM16 markedly decreased the thickness of the paw edema and writhing response in a dose-dependent manner in mice. In the MIA-induced OA rat model, ALM16 significantly reduced the PWT compared to the control group. In particular, from histological observations, ALM16 showed clear improvement of OA lesions, such as the loss of necrotic chondrocytes and cartilage erosion of more than 200 mg/kg b.w., comparable to or better than a positive drug control (JOINS™, 200 mg/kg) in the cartilage of MIA-OA rats. CONCLUSIONS: Our results demonstrate that ALM16 has a strong chondroprotective effect against the OA model in vitro and in vivo, likely attributed to its anti-inflammatory activity and inhibition of MMP production.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Cartilagem Articular/efeitos dos fármacos , Osteoartrite , Extratos Vegetais/farmacologia , Animais , Astragalus propinquus/química , Cartilagem Articular/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glicosaminoglicanos/análise , Humanos , Ácido Iodoacético/efeitos adversos , Lithospermum/química , Masculino , Metaloproteinases da Matriz/análise , Medicina Tradicional do Leste Asiático , Camundongos Endogâmicos ICR , Osteoartrite/induzido quimicamente , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Substâncias Protetoras/farmacologia , Ratos
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