A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Antimicrobial activities and phytochemicals of Murraya paniculata L. flowers, leaves and bark crude extracts

Aims@#Murraya paniculata (L.) has been widely employed in medicine, has also been modified to serve as an ingredient in health foods and found application in cosmetics. This study was aimed to assess the biological activities of M. paniculata by analyzing the chemical compositions of its flowers, leaves and bark.@*Methodology and results@#Crude extracts drawn from the flowers, leaves and bark of M. paniculata underwent testing to determine the antibacterial properties in terms of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), as well as the overall chemical composition, total phenolic content, flavonoids and antioxidant activity. Crude extract of leaves exhibited the most potent antibacterial activity when tested against Staphylococcus aureus TISTR 1466, Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 27853. The crude extract from bark delivered the most significant antibacterial activity when tested against Micrococcus luteus TISTR 9341, Escherichia coli ATCC 1261, Pseudomonas sp., Streptococcus sp. and Methicilin resistant S. aureus (MRSA). For all crude extracts, the MIC value against M. luteus TISTR 9341 was 12.5 mg/mL. Meanwhile, the MBC value for the crude extract of leaves against B. subtilis ATCC 6633 was 12.5 mg/mL, whereas, for flower and bark crude extracts, the MBC value against S. aureus TISTR 1466 was 25 mg/mL. Antioxidant activity was at its highest for the crude extract from bark (IC50 = 1.36 mg/mL). The highest phenolic content was recorded for the crude extract from bark, while the highest flavonoid content came from the crude extract of leaves (70.81 ± 0.31 mgGAE/g extract and 115.73 ± 1.18 mgQE/g extract, respectively).@*Conclusion, significance and impact of study@#The research findings suggest that the crude extracts of M. paniculata leaves and bark show greater significant levels of bioactivity than was the case for crude extracts from flowers. The research findings could help in exploring the possibilities of using M. paniculata for pharmaceutical purposes and in aquaculture.

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction（PCR） method to accurately discriminate the crude materials of Murrayae Folium et Cacumen， Murraya exotica and M. paniculata. MethodBased on the difference in chloroplast genome sequences of M. exotica and M. paniculata， species-specific identification primers P03 and P04 of M. exotica and M. paniculata were designed according to single nucleotide polymorphism （SNP） on the chloroplast genome. A multiplex allele-specific PCR identification method was established for the identification of M. exotica and M. paniculata following the optimization of annealing temperature， number of cycles， and primer concentration ratio. The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model. ResultIn this multiplex allele-specific PCR identification method， about 330 and 230 bp of specific fragments were amplified from DNA templates of M. exotica and M. paniculata， respectively， under the following conditions：cycle number of 31， annealing temperature of 60 ℃， and primer concentration ratio of P03 and P04 of 1∶2. Consistent results were obtained for samples from different sources. ConclusionThe multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen， which can be used for the simultaneous identification of M. exotica and M. paniculata by the length of fragments in a single identification assay.

Comparison of Murraya exotica and Murraya paniculata by fingerprint analysis coupled with chemometrics and network pharmacology methods / 中国天然药物

There are two source plants for the traditional Chinese medicine Murrayae Folium et Cacumen (MFC) in Chinese Pharmacopoeia, i.e. Murraya exotica L. and M. paniculata (L.) Jack. Herein, a chemical comparison of M. exotica and M. paniculata by high performance liquid chromatography (HPLC) fingerprint analysis coupled with chemometrics and network pharmacology was performed. The main peaks in the fingerprints were identified by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) and authenticated by references. The chemometrics results showed that the HPLC fingerprints of these two species were clearly divided into two categories using hierarchical cluster analysis (HCA) and principal component analysis (PCA), and a total of 13 significantly differentiated markers were screened out by orthogonal partial least squares-discriminant analysis (OPLS-DA). However, the following network pharmacology analysis showed that these discriminated markers were found to act via many common targets and metabolic pathways, indicating the possibly similar pharmacological effects and mechanisms for M. exotica and M. paniculata. The above results provide valuable evidence for the equivalent use of these two plants in clinical settings. Moreover, the chromatographic fingerprint analysis coupled with chemometrics and network pharmacology supplies an efficient approach for the comparative analysis of multi-source TCMs like MFC.

Quantitative Classification of Murraya paniculata Leaf Traits and Endogenous Hormone Contents Analysis / 中国实验方剂学杂志

Objective:To carry out germplasm resource evaluation and new variety breeding of <italic>Murraya paniculata</italic> and improve the germplasm quality， so as to ensure the demand， yield and quality of medicinal materials. Method:Following resource investigation and collection， 17 traits of 107 <italic>M. paniculata</italic> germplasm samples， like plant type， basal diameter， leaf shape， leaf length， and leaf width were determined and then subjected to principal component analysis and factor analysis for screening the principal component factors. Nine primary traits were selected as variables for further cluster analysis using Ward's method and Euclidean distance. According to the characteristics of medicinal parts， the core germplasms were screened out. Then the contents of auxin， zeatin， zeatin nucleoside， isopentenyl adenine， isopentenyl adenine riboside， dihydrozeatin， and dihydrozeatinriboside in the leaves were measured by ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）， followed by their correlation analysis with agronomic trait. Result:The variation coefficients of petiole length， branching number， and basal diameter were large. The nine main factors could be classified into four categories， with a contribution rate of 72.822%. The cluster analysis with Ward's method and Euclidean distance showed that 107 germplasm samples were clustered into six clusters and 61 core germplasms were identified. Such traits as leaf length， leaf width， petiole length， leaf surface， and petiole color were found to play an important role in the classification of <italic>M. paniculata</italic> germplasms. The content of zeatin nucleoside exhibited significant positive correlations with leaf length （<italic>P</italic><0.01）， petiole length （<italic>P</italic><0.01）， and leaf width （<italic>P</italic><0.05）. Conclusion:These results have laid the foundation for further selection and breeding of <italic>M. paniculata</italic> new varieties.

Chemical composition and in vitro antibacterial activity of essential oils from Murraya paniculata (L. ) Jack (Rutaceae) ripe and unripe fruits against bacterial genera Mycobacterium and Streptococcus

This study aims to investigate chemical composition of essential oils from Murraya paniculata (L.) Jack (Rutaceae) ripe and unripe fruits and determine their in vitro antibacterial activity. Essential oils were extracted by hydrodistillation from Murraya paniculata (L.) Jack ripe and unripe fruits collected in the Cerrado, in Rio Verde, southwestern Goiás, Brazil. They were analyzed by gas chromatography with flame ionization detector (GC-FID) and by gas chromatography-mass spectrometry (GC-MS). Sesquiterpenes, which represent the most abundant class of compounds in oils, predominated in both ripe and unripe fruits. Major constituents of essential oils extracted from ripe fruits (RF-EO) were (-caryophyllene (21.3%), (-ylangene (13.3%), germacrene-D (10.9%) and (-zingiberene (9.7%) whereas the ones of unripe fruits (UF-EO) were sesquithujene (25.0%), (-zingiberene (18.2%), germacrene-D (13.1%) and (-copaene (12.7%). In vitro antibacterial activity of essential oils was evaluated in terms of its minimum inhibitory concentration (MIC) values by the broth microdilution method in 96-well microplates. Both essential oils under investigation showed moderate anti-streptococcal activity against the following bacteria: Streptococcus mutans, S. mitis, S. sanguinis, S. sobrinus and S. salivarius. MIC values ranged between 100 and 400 µg/mL. Regarding the antimycobacterial activity, essential oils from M. paniculata (L.) Jack unripe and ripe fruits were active against Mycobacterium kansasii (MIC = 250 µg/mL), moderately active against M. tuberculosis (MIC = 500 µg/mL) and inactive against M. avium (MIC = 2000 µg/mL). This study was pioneer in revealing similar chemical profiles of both essential oils extracted from Murraya paniculata (L.) Jack unripe and ripe fruits, besides describing their in vitro anti-streptococcal and antimycobacterial activities.

Evaluación de la calidad, seguridad y capacidad antioxidante de Murraya paniculata L y su tintura / Evaluation of the quality, safety and antioxidant capacity of Murraya paniculata L and its tincture

INTRODUCCIÓN: Murraya paniculata (L.) Jack (Rutaceae) es empleada tradicionalmente en nuestro país para el alivio del dolor y la inflamación asociados a enfermedades osteomioarticulares, pero no se reportan estudios que profundicen en elementos tan importantes como lo constituyen su calidad y seguridad. En el presente trabajo se evalúa su potencialidad para ser empleado como alternativa terapéutica. MÉTODOS: la droga vegetal (hojas frescas de M. paniculata), y su derivado galénico (tintura), fueron sometidas a un estudio farmacognóstico que incluyó la evaluación macromorfológica de las hojas de la especie, así como la determinación de sus índices numéricos de calidad y el tamizaje fitoquímico. Asimismo la tintura obtenida fue sometida a la evaluación de sus requisitos organolépticos, índices numéricos de calidad, análisis capilar, tamizaje fitoquímico y cromatografía en capa delgada; desde el punto de vista preclínico, fue evaluada su seguridad a través de un estudio de toxicidad aguda dérmica y su capacidad antioxidante mediante las técnicas FRAP y DPPH. RESULTADOS: los parámetros de calidad establecidos fueron incluidos en la Guía de fitofármacos y apifármacos. Los resultados preclínicos obtenidos en la evaluación de la tintura fueron satisfactorios, ya que no generó efectos tóxicos, el análisis cualitativo DPPH evidenció su capacidad antioxidante y el análisis por FRAP mostró un poder antioxidante similar al de la vitamina C. CONCLUSIONES: los resultados alcanzados permiten garantizar que M. paniculata sea una alternativa segura y eficaz para el uso en la terapéutica, como parte del arsenal de nuestro sistema de salud en Medicina Natural y Tradicional.

INTRODUCTION: Murraya paniculata (L.) Jack (Rutaceae) has been traditionally used in our country to treat pain and inflammation caused by osteoarticular disorders. However, there are no reports of studies about such important topics as its quality and safety. The present paper evaluates the potential use of M. paniculata as a therapeutic alternative. METHODS: a pharmacognostic study was conducted of the plant drug (fresh leaves of M. paniculata) and its galenic derivative (tincture) which included a macromorphological evaluation of the leaves, determination of numerical indices of quality and phytochemical screening. The tincture obtained was evaluated for organoleptic requirements and numerical indices of quality, and underwent capillary analysis, phytochemical screening and thin-layer chromatography. Preclinical safety was assessed in an acute dermal toxicity study. Antioxidant capacity was examined by FRAP and DPPH. RESULTS: the quality parameters obtained were included in the Guide of Phytomedicines and Apimedicines. Preclinical evaluation of the tincture was satisfactory, since no toxic effects were found. Qualitative DPPH analysis revealed antioxidant capacity and FRAP found an antioxidant power similar to that of vitamin C. CONCLUSIONS: results confirm the status of M. paniculata as a safe, effective therapeutic alternative within the natural and traditional medicine stock of our country's health system.

Validación de un método por CLAR para la cuantificación de L-prolina en la tintura al 20 por ciento de Murraya paniculata L. Jack / Validation of a high performance liquid chromatography method for quantitation of L-proline in 20 percent tincture from Murraya paniculata L. Jack

INTRODUCCIÓN: la búsqueda de técnicas analíticas para el control de la calidad de los medicamentos constituye un aspecto de gran interés en el campo farmacéutico, más si van dirigidas al estudio del o los marcadores químicos de las plantas medicinales, sus extractos y fitomedicamentos. OBJETIVO: validar un método de cromatografía líquida de alta resolución (CLAR) para la determinación cuantitativa del aminoácido L-prolina como sustancia marcador en la tintura de Murraya paniculata L. Jack. MÉTODOS: en el método por CLAR, la separación se realizó en una columna C-18 (UP5ODB-150/046), se utilizó como fase móvil una mezcla de solución buffer fosfato, pH ajustado a 2,4 y acetonitrilo (70:30 v/v), con una velocidad de flujo de 0,6 mL/min, modo isocrático, con detección ultravioleta a 440 nm. El volumen de inyección de la muestra fue de 20 µL. El método fue validado según la categoría I, siguiendo las exigencias internacionales. RESULTADOS: la curva de calibración fue lineal en el rango de concentraciones ensayadas (30 a 375 µg/mL), se observó una buena precisión con coeficientes de variación menores del 2 por ciento. Los valores de recobrado estuvieron dentro de los límites establecidos para los métodos cromatográficos (98-102 por ciento). Se demostró la especificidad del método, al no presentarse interferencias de picos adicionales en la zona de elusión del compuesto de interés (L-prolina). CONCLUSIONES: el método analítico por CLAR, validado para la cuantificación del aminoácido L-prolina en la tintura de M. paniculata, demostró ser lineal, preciso, exacto y específico bajo las condiciones de estudio(AU)

INTRODUCTION: the search for analytical methods that may monitor the quality of drugs is an issue of great interest in the pharmaceutical field, even more if they are directed to studying chemical markers of medicinal plants, their extracts and phytomedicines. OBJECTIVE: to validate a high-resolution liquid chromatography (HPLC) method for the quantitative determination of the L-proline amino acid as a marker substance in Murraya paniculata L. Jack tincture. METHODS: in the HPLC, the separation was performed on a C-18 (UP5ODB-150/046) column, with a mixture of phosphate buffer solution, pH adjusted to 2.4 and acetonitrile (70:30 v/v) used as mobile phase, the flow rate was 0.6 mL/min, isocratic mode with UV detection set at 440 nm. The injection volume of the sample was 20 µL. The method was validated according to category I, following international requirements. RESULTS: the calibration curve was linear over the concentration range tested (30-375 mg/mL), good precision was observed with a variation coefficient less than 2 percent. Recovery values were within the limits for chromatographic methods (98-102 percent). The method was specific since there was no-interference by additional peaks in the elution zone of the compound in question (L-proline). CONCLUSIONS: the HPLC analytical method, validated for the quantification of L-proline amino acid in M. paniculata tincture, proved to be linear, precise, accurate and specific under the study conditions(AU)

Acercamiento al género Murraya (Rutaceae) y a la especie Murraya paniculata (L) Jack / Approach to Murraya (Rutaceae) genus and Murraya paniculata (L) Jack species

Introducción: Murraya paniculata (L) Jack (Rutaceae) es empleada tradicionalmente en algunas provincias de Cuba para el alivio del dolor y la inflamación asociados a enfermedades osteomioarticulares. No se reportan estudios en el país que profundicen en la composición química de esta especie o del género Murraya, ni tampoco relacionados con información etnomédica y actividades biológicas comprobadas. Objetivos: reunir y analizar información científica actualizada referente al género, y a esta especie en particular, como fuente natural de compuestos biológicamente activos que determinan sus potencialidades medicinales. Métodos: se incluyeron en el análisis artículos científicos y libros relacionados con los temas de composición química, usos tradicionales y evaluaciones farmacológicas, así como otros elementos de interés de plantas medicinales. Resultados: la información analizada puede servir de base para el desarrollo de nuevas investigaciones que avalen el empleo en la terapéutica de productos fitoterápicos de elevada eficacia, seguridad y calidad. La revisión de los resultados de otros grupos de investigación permite establecer estrategias racionales de investigación científica, que contribuyan al uso racional de los recursos que se encuentran en universidades y centros de investigación y salud. Conclusiones: los elementos encontrados en la bibliografía consultada permiten asegurar que la especie Murraya paniculata puede ser potencialmente empleada en fitoterapia, debido entre otras cosas, al importante número de metabolítos secundarios identificados con actividad farmacológica reconocida

Introduction: Murraya paniculata (L) Jacq (Rutaceae) is traditionally used in some Cuban provinces for pain relief and inflammation associated to osteomyoarticulary diseases. There has been no reported studied at domestic level, which delves into neither the chemical composition of this species or of Murraya genus nor the confirmation of etnomedical information and biological actions. Objectives: to gather and analyze updated scientific information about this genus, particularly this species, as likely natural source of biologically active compounds responsible for their medicinal potentialities. Methods: the analysis comprised scientific articles and books on chemical composition, traditional uses and pharmacological assessment, and other interesting elements of medicinal plants. Results: the analyzed information can provide the basis for the development of new research studies that will support the use of highly effective, safe and quality phytotherapeutic products. The review of the results achieved by other research groups allows drawing reasonable scientific research strategies to contribute to the rational use of resources by universities and research and health institutions. Conclusions: the elements found in the literature review allow assuring that Murraya paniculata species may be potentially used in phytotherapy because of the significant numbers of detected secondary metabolites with recognized pharmacological action

Antinociceptive and bioactivity of leaves of Murraya paniculata (L. ) Jack, Rutaceae / Antinociceptiva e bioatividade das folhas de Murraya paniculata (L. ) Jack, Rutaceae

The results of antinociceptive and toxicological studies on the ethanol extract of the leaves of Murraya paniculata (L.) Jack, Rutaceae, are reported. The extract (250 and 500 mg/kg dosages) was found to produce a profound antinociceptive activity in a dose dependent manner. The extract showed considerable brine shrimp toxicity (LD50 = 32 μg/mL).

Os resultados dos estudos de atividade antinociceptiva e toxicológicos do extrato etanólico de Murraya paniculata (L.) Jack, Rutaceae são relatados. O extrato (nas doses de 250 e 500 mg/kg) produziu um alta atividade antinociceptiva na forma dose-dependente. O extrato também mostrou considerável toxicidade no teste de Artemia salina (DL50 = 32 μg/mL).

Constituintes químicos das folhas de Murraya paniculata (Rutaceae) / Chemical constituents from leaves of Murraya paniculata (Rutaceae)

A separação cromatográfica do extrato hexânico e da fase em CH2Cl2 do extrato etanólico das folhas de Murraya paniculata resultou no isolamento de um triterpeno (24-metileno-cicloartan-3β-ol), um fenilpropanóide (cafeato de metila) e sete cumarinas preniladas [isomeranzina, acetato de murranganona, murrayatina, murrangatina, hidrato de meranzina, febalosina e murranganona]. Dentre as substâncias isoladas, as cumarinas foram detectadas anteriormente em M. paniculata ao passo que 24-metileno-cicloartan-3β-ol e cafeato de metila estão sendo descritos pela primeira vez no gênero Murraya. Os extratos e frações além das substâncias puras foram submetidos à avaliação do potencial antimicrobiano frente à Staphylococcus aureus e Escherichia coli indicando que somente a cumarina hidrato de meranzina mostrou fraca atividade.

Chromatographic separation of the hexane extract and the CH2Cl2 phase from the ethanol extract from leaves of Murraya paniculata yielded one triterpenoid (24-methylene-cycloartan-3β-ol), one phenylpropanoid (methyl caffeate) and seven coumarins [isomeranzine, murranganone acetate, murrayatine, murrangatine, meranzine hydrate, phebalosine and murranganone]. All the isolated coumarins were previously obtained from M. paniculata while 24-methylene-cycloartan-3β-ol and methyl caffeate have been described for first time in the Murraya genus. The crude extracts, fractions and pure substances were submitted to evaluation of antimicrobial potential against Staphylococcus aureus and Escherichia coli which indicated that the coumarin meranzine hydrate showed weak activity.