Agrocybe cylindracea, an edible mushroom, is widely cultivated for its abundance of nutrients and flavor, and many of its metabolites are reported to have beneficial roles, such as medicinal effects on tumors and chronical illnesses. However, the lack of genomic information has hindered further molecular studies on this fungus. Here, we present a genome assembly of A. cylindracea together with comparative genomics and pathway analyses of Agaricales species. The draft, generated from both next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing platforms to overcome high genetic heterozygosity, is composed of a 56.5 Mb sequence and 15,384 predicted genes. This mushroom possesses a complex reproductive system, including tetrapolar heterothallic and secondary homothallic mechanisms, and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources. Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances, including B vitamins, unsaturated fatty acids, and N-acetylglucosamine. RNA-seq data show that A. cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage, whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis. These results reflect diverse functions of the two anatomical structures of the fruiting body. Our comprehensive genomic and transcriptomic data, as well as preliminary comparative analyses, provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components. Agrocybe cylindracea, belonging to the Agrocybe genus, is an edible mushroom that is very popular for the unique flavor and high nutritional content of its fruiting body. A. cylindracea is also regarded as a multipurpose food supplement due to the high levels of nutrients and bioactive compounds present in this species. Some studies have shown that active extracts of A. cylindracea have effects on various human diseases Agaricus bisporus, Laccaria bicolorCoprinopsis cinereaAgrocybe aegerita (A. aegerita), which belongs to the same genus as A. cylindracea, are rich in carbohydrate hydrolases and have the ability to degrade lignocellulosic biomass. Sequencing of A. aegerita also led to analyses of the carbohydrate-active genes and fruiting-related genes A. cylindracea is grown in many countries, including China, and has marked economic benefits for farmers. However, molecular research on A. cylindracea has been limited to lectins and active polysaccharide extracts.Climatic and geographical diversity have resulted in the availability of a large variety of edible fungal species for domestication worldwide. As the largest mushroom clade, many Agaricales, such as A. cylindracea and a preliminary analysis of the complex reproductive system of this organism. Comparative analysis was conducted between the genome of A. cylindracea and the published genomes of 16 Agaricales fungi Agaricus bisporus and poisonous mushrooms such as Galerina marginata, to analyze the phylogeny of Agaricales species. We also attempted to construct a molecular framework of nutrient homeostasis, from element absorption to biosynthesis. Our work provides insights into the molecular details of A. cylindracea as both a food and potential medicine.Here, we present a genome assembly of A. cylindracea genome is estimated to be ~58.2 Mb (Based on the k-mer (17-mer) distribution, the size of our assembled ~58.2 Mb . The two~58.2 Mb . Assembl~58.2 Mb . Our corA. cylindracea genome assembly , including 747,935 single-nucleotide variants (SNVs) (1.32% of the genome) and 124,042 indels and substitutions. Based on structural annotation of the genes, 269,636 SNVs in genes are predicted, and among these SNVs, 165,407 exonic SNVs are synonymous, whereas 104,229 are nonsynonymous, and others are outside the protein-coding regions , with a mitochondrial intermediate peptidase (MIP) nearby. This species has three pheromone-coding genes and five pheromone receptor-coding genes in the MAT B locus, which are clustered on contig394  and heterothallic (self-sterile) mechanisms . A majorontig394 A. These ontig394 B.Figure Saccharomyces cerevisiae and Chromocrea spinulosa, exhibit MAT switching behavior, termed secondary homothallism, changing the original MAT and producing fertile offspring A. cylindracea. It is also supported by the prediction of five silent information regulator (Sir)-coding sequences that participate in the silencing of the other MAT that is located near the expressed MAT locus , followed by G. marginata, Hebeloma cylindrosporum (H. cylindrosporum), and Hypholoma sublateritium (H. sublateritium). There are 732 orthologous blocks, containing 56.61% of the A. cylindracea genes (8709) and 60.36% of the A. aegerita genes (8520). The species-specific A. cylindracea genes, compared with the A. aegerita genes, are enriched in ion binding, protein binding, nitrogen compound metabolic process, and primary metabolic process  of the A. cylindracea genes are homologues to the rest  in G. marginata is absent in A. cylindracea and A. aegerita. This gene is located on highly conserved blocks among the three species  superfamily members, 88 auxiliary activity (AA) enzymes, 30 carbohydrate esterases (CEs), 65 glycosyltransferases (GTs), 8 polysaccharide lyases (PLs), and 56 carbohydrate-binding module (CBM) superfamily members. Interestingly, 254 (58%) of these proteins are membrane-bound or secreted and possess a signal peptide domain , GH9 (1 copy), GH44 (1 copy), GH45 (1 copy), and lytic polysaccharide monooxygenase (LPMO) (17 copies) families, which are associated with cellulose deconstruction indracea . A. cyliA. cylindracea degrades all polysaccharides of plant cell walls and lignin in addition to cellulose A. cylindracea, four of which are manganese peroxidases. The AA2 family includes class II lignin-modifying peroxidases such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP), which are major enzymes for lignin degradation Similar to other white rot fungi, A. cylindracea degrades crude proteins from cultivation substrates as nitrogen source. Using the MEROPS database as a reference, we identified 2177 peptidases, including 256 proteins with a signal peptide domain. The most abundant peptidases are prolyl oligopeptidases (family S09/218 genes), from serine peptidases; Copia transposon peptidases (A11/133), from aspartic peptidases; and prolyl aminopeptidases (S33/130). The high abundance of peptidases suggests effective utilization of external nitrogen resources and high protein content.A. cylindracea possesses a reliable transport system for absorbing nutrients, as supported by the fact that 359 nutrient transport-associated proteins have been identified based on Pfam annotation, including ABC and MFS family transporters that are responsible for transporting oligopeptides, amino acid sugars, and metal ions.In addition, A. cylindracea can utilize carbon sources efficiently to provide energy and materials for the biosynthesis of other nutrients, such as amino acids, B vitamins, polysaccharides, and unsaturated fatty acids.To elucidate the molecular mechanism of nutrient metabolism, we constructed the metabolic processing networks, including the carbon cycle and biosynthetic pathways of amino acids and other nutrients . Almost A. cylindracea, in which all the 1,3-β-glucan synthesis-associated enzymes were identified, together with ten predicted beta-1,6-glucan biosynthesis-associated proteins, KRE6 and SKN1  are both highly expressed and expanded. Moreover, chitinase, which hydrolyzes chitin to produce N-acetylglucosamine (GlcNAc), is expressed at high levels in the A. cylindracea fruiting bodies. These expanded and highly expressed genes indicate the ability of A. cylindracea to produce functionally active carbohydrate.Beta-glucans, especially beta-1,3-glucans, exhibit high biological activity in immunomodulatory, anti-inflammatory, and antitumor processes and SKN1  [25]; HoA. cylindracea is also an ideal source of amino acids, unsaturated fatty acids, and B vitamins. The nutrients in A. cylindracea are diverse and highly active  and isopentenyl diphosphate (IPP) A. cylindracea to synthesize ubiquinone  clusters containing 24 related genes. Polyketide synthases (PKSs) are known to be responsible for both aromatic and highly reduced polyketide metabolites A. cylindracea. A total of 28 secondary metabolite gene clusters were detected | > 1), including 1744 upregulated genes in pilei and 1153 in stipes  synthesis, namely, delta-9-desaturase (EC1.14.19.1) and delta-12-desaturase (EC1.14.19.-), are also highly expressed in pilei. Interestingly, many annotated carbohydrate hydrolases, including beta-1,3-glucan, maltose, glycogen, trehalose, and dextrin hydrolases, are significantly upregulated in pilei.Our transcriptomic study yielded 2897 differentially expressed genes (DEGs) in the fruiting bodies  A. cylindracea genome is highly heterozygous, most likely due to outcrossing during cultivation and the complex reproductive system of this organism. During heterothallic sexual reproduction . The highly expressed glutamine synthetase in A. cylindracea is a central enzyme in nitrogen assimilation and biosynthesis of glutamine A. cylindracea, can increase the linoleic acid content in vitro and in vivo experiments. Genome sequences and transcriptomic analyses make breeding for high-quality strains and improved yields increasingly realistic and provide information regarding the sequences and expression characteristics of the key genes for research on biological synthesis.In this study, we describe an assembly of the A. cylindracea genome assembly and transcriptomic analysis provide further information for basic research on cultivation and drug development. A further refined A. cylindracea reference genome will be an important genomic resource for studies on sexual reproduction systems, genetic diversity, and domestication trait selection. Genomic analyses will allow researchers to identify the related genes that affect the quality, nutrient content, and biological efficiency of A. cylindracea. With more sequencing of different strains, DNA barcoding could be exploited for screening the excellent or specific strains rapidly and stably.The A. cylindracea AC9 strain for sequencing and analysis. The AC9 strain was derived from wild strains picked by Dengxue Lu in Chaoyang village, Xingcun town, Wuyishan city  in 1999 and cultivated by the Gansu Provincial Academy of Sciences Institute of Biology in China. The wild strains grew on the rotten roots of Camellia oleifera trees. We isolated the strains at the top of the stipes by tissue isolation. After years of breeding and selection, the biological efficiency reached 75%, with good taste and appearance.We chose the A. cylindracea mycelia with different insert sizes were constructed and sequenced with the Illumina HiSeq 2000 and PacBio RS II platforms in Beijing Institute of Genomics, including three paired-end libraries , two mate-pair libraries (8 kb and 10 kb), and one PacBio library , and PCR duplications of the raw data using in-house Perl scripts A. cylindracea: M = N × (L − K + 1)/L and G = T/N  Jellyfish (version 2.1.3) de novo assembler Platanus High-quality paired-end reads were assembled into contigs by the de novo-assembled transcripts to the assembly.To evaluate the genome assembly, three approaches were used in our study: (1) raw data mapping, where we calculated mapping rates by mapping paired-end reads back to the assembly; (2) core gene mapping, where we used CEGMA http://picard.sourceforge.net). Heterozygous variations (SNVs and indels) were detected with SAMtools (version 1.3.1) and GATK (version 3.5) The high-quality reads were mapped to the assembly by BWA (version 0.7.15-r1140), and duplicate reads were removed using MarkDuplicates from PICARD tools (version 1.119) , which employs two programs, namely, RECON and RepeatScout, was applied with default settings to construct a de novo repetitive library from the assembly, which was further annotated.RepeatModeler  Three strategies were used for gene prediction: (1) −5. InterProScan http://www.kegg.jp/kegg/). Carbohydrate-active and peptidase enzymes were classified by aligning predicted protein sequences against the CAZy and MEROPS databases with an E-value cutoff of 10−5. Pheromone precursors were predicted by TransDecoder (https://transdecoder.github.io/) and the Pfam database in the flanking regions of pheromone receptors.Predicted genes were annotated by a BLAST search against NCBI databases with an E-value cutoff of 10A. cylindracea AC9 mycelia, constructed from pileus and stipe RNAs, were sequenced on the Illumina HiSeq 2000 platform  was used to obtain improved alignment results. FPKM was calculated using Cufflinks (version 2.0.2) P < 0.05, |fold change| > 2). Triple duplication was used to reduce sampling bias . Gene families were constructed using the OrthoMCL (v2.0.9) The protein sequences of Serpula lacrymans as the outgroup. Orthologous gene blocks between A. cylindracea and other fungi were identified using MCscanX Single-copy gene families were extracted for phylogenetic analysis. Protein alignments of each family were performed with MUSCLE A. cylindracea enzymes onto metabolic pathways using the “basic pathway mapping tool” from the KEGG website. We selected the most important nutrient metabolic pathways, including the carbon cycle and B vitamin and polysaccharide metabolic pathways, for further analysis. In addition, we checked the A. cylindracea-specific pathways via manual inspection and constructed a network of energy and nutrient pathways.We mapped annotated http://www.accc.org.cn/, and the China General Microbiological Culture Collection Center (ID: CGMCC5.2198), which is accessible at http://www.cgmcc.net/.The strain has been submitted to the Agricultural Culture Collection of China (ID: ACCC 53291), which is accessible at http://bigd.big.ac.cn/gsa. The data analyzed during this study are deposited at NCBI under BioProject PRJNA353318 (GenBank: MPNV00000000). Phylogenetic data have been deposited in the TreeBASE repository (ID: 22964), which is accessible at http://www.treebase.org/.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive Yuan Liang: Formal analysis, Visualization, Writing-original draft, Writing-review & editing. Dengxue Lu: Resources, Conceptualization, Writing-review & editing. Sen Wang: Formal analysis, Visualization, Writing-original draft. Yuhui Zhao: Resources. Shenghan Gao: Formal analysis. Rongbin Han: Resources. Jun Yu: Writing-review & editing. Weili Zheng: Resources, Supervision. Jianing Geng: Supervision, Writing-review & editing. Songnian Hu: Conceptualization, Supervision. All the authors read and approved the final manuscript.The authors declare that they have no competing interests.


Mycobacterium abscessus is an extensively drug–resistant pathogen that causes pulmonary disease, particularly in cystic fibrosis (CF) patients. Identifying direct patient-to-patient transmission of M. abscessus is critically important in directing an infection control policy for the management of risk in CF patients. A variety of clinical labs have used molecular epidemiology to investigate transmission. However, there is still conflicting evidence as to how M. abscessus is acquired and whether cross-transmission occurs. Recently, labs have applied whole-genome sequencing (WGS) to investigate this further and, in this study, we investigated whether WGS can reliably identify cross-transmission in M. abscessus.M. abscessus isolates from 62 patients, seen at 4 hospitals in 2 countries over 16 years.We retrospectively sequenced the whole genomes of 145 M. abscessus but does provide enough information to replace multiple existing molecular assays. We detected 1 episode of possible direct patient-to-patient transmission in a sibling pair. We found that patients acquired unique M. abscessus strains even after spending considerable time on the same wards with other M. abscessus–positive patients.We have shown that a comparison of a fixed number of core single nucleotide variants alone cannot be used to infer cross-transmission in M. abscessus through direct patient-to-patient transmission or a common reservoir. Tracking transmission using WGS will only realize its full potential with proper environmental screening, as well as patient sampling.This novel analysis has demonstrated that the majority of patients in this study have not acquired  Mycobacterium abscessus is not common. Environmental screening may provide a better understanding of the acquisition of M. abscessus infections.Whole-genome sequencing should replace the current molecular typing used routinely in clinical microbiology laboratories. The patient-to-patient spread of  Mycobacterium abscessus (recently renamed as Mycobacteroides abscessus) [M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii [Mycobacterium abscessus is an important pathogen that has emerged in the CF patient population and that has been associated with poor clinical outcomes, especially following lung transplantation [M. abscessus are frequently not listed for transplant; therefore, the acquisition of this pathogen is considered to be a serious complication in this group.scessus) , is a grbolletii , 2. Thesantation . This isantation . CF patiM. abscessus strains has been studied using Variable Nucleotide Tandem Repeats (VNTR) and Multi Locus Sequence Typing (MLST) [M. abscessus remains contested [Mycobacterium abscessus is ubiquitous in the environment, with its niche hypothesized to be free-living amoeba [The epidemiology of g (MLST) . The clug (MLST) , 9. A doontested , 11, witontested , 12–14. g amoeba , 16, butM. abscessus isolates. We also wanted to utilize the data to investigate the scale of patient-to-patient transmission and whether identification of single nucleotide variants (SNVs) by WGS can confirm transmission. To do this, we sequenced the genomes of 145 M. abscessus clinical isolates from a well-characterized cohort of 62 patients from 4 hospitals in 2 countries over 16 years.In this retrospective study, we assessed the utility of using WGS to characterize subspecies, antimicrobial resistance (AMR) profiles, and typing of M. abscessus isolates from 30 patients at Hospital de la Santa Creu I Sant Pau (bcn_hsp), Hospital Clínic (bcn_hcl), and Hospital Vall d’Hebron (bcn_hvh) in Barcelona, Spain, and 112 isolates from 32 patients from Great Ormond Street Hospital (GOSH) in London, United Kingdom . SNVs were called against the reference genome using freebayes v1.2.0 [Sequenced reads for all samples were first mapped to s v1.2.0 , and varPotential regions of recombination were identified from the consensus genome sequences using Gubbins v2.3.1 . RegionsM. abscessus subsp. massiliense sequences, as well as for M. abscessus subsp. abscessus ST-1 and ST-26 sequences. All samples in each subtree were mapped against a suitable reference. Mycobacterium abscessus subsp. massiliense str. GO 06 was used as the reference sequencing for study massiliense sequences, and the de novo assembly of the earliest ST-26 study sequence (ldn_gos_2_520) was used as a reference for other ST-26 samples. Mycobacterium abscessus subsp. abscessus ATCC 19977 was again used as the reference for ST-1 sequences, as it is the same sequence type. All subtrees were generated using the same method outlined above, apart from the ST-26 subtree, which did not use Gubbins: instead, variants were filtered if 3 SNVs were found within a 100 bp window.Separate subtrees were also inferred for Sequence clusters to infer possible transmission were generated using 3 different methods on each subtree. First, we used an SNV threshold that was based on the upper bounds of all within-patient diversity and was applied to complete linkage hierarchical clustering based on a pairwise SNV matrix. Secondly, we assigned clusters using the R package rPinecone, as it incorporates SNV thresholds and root-to-tip distances and has been useful when applied to clonal populations . Lastly,de novo assembly of bacterial genomes using St. Petersburg genome Assembler (SPAdes) and pilon, wrapped in the Unicycler v0.4.4 package [All samples underwent  package . Assembl package  and comp package . To geneM. abscessus isolates from 62 patients. There were 33 M. abscessus isolates from Barcelona, subdivided into 24 M. abscessus subsp. abscessus, 2 M. abscessus subsp. bolletii, and 7 M. abscessus subsp. massiliense. There were 112 M. abscessus isolates from the United Kingdom, subdivided into 78 M. abscessus subsp. abscessus, 1 M. abscessus subsp. bolletii, and 33 M. abscessus subsp. massiliense. Sample MLST definitions, VNTR definitions, and AMR-associated mutations are shown in We obtained WGSs for 145 M. abscessus subsp. massiliense isolates between patients. We used an SNV matrix from mapping against a reference (M. abscessus subsp. abscessus ATCC19977), as well as hierBAPS and rPinecone, to predict sequence clusters. The sequence clusters generated from the single-reference SNV matrix provided no further information than the MLST profiles and, in many cases, provided spurious findings with large groups of isolates clustered with no epidemiological link , compared to 70 545 (IQR 59 947–71 891) across all isolates from the subspecies.Mapping to a single reference genome led to the inability of a single SNV cut-off, or model, to exclude unrelated isolates from sequence clusters, because the number of pairwise SNV distances varied greatly between both subspecies and specific lineages . For exaM. abscessus subsp. abscessus and M. abscessus subsp. massiliense isolates, as well as further subtrees for ST-1 (VNTR II), ST-26 (VNTR I), and ST-23/ST-48 (VNTR III) isolates. We also integrated the presence of accessory genes when interrogating possible sequence clusters for transmission  were identified , compared to 18% (IQR 12–37%) for isolates not in the same sequence cluster.M. abscessus after their first visit to the clinic  that had attended a lung function laboratory on 3 occasions within a month of each other. In this case, the only way transmission could have occurred would be if ldn_gos_7, who was already infected, contaminated the environment and the infection was then transmitted to ldn_gos_46. The predominant view [M. abscessus remains present but undetectable by conventional methods for this time period or, intriguingly, could cause a latent infection, like what occurs with Mycobacterium tuberculosis. To the best of our knowledge, this has never been a demonstrated part of the pathogenesis of M. abscessus infection, and may be worthy of further investigation.In addition to conducting the core genome SNV analysis, we also found that the integration of accessory genome information is a useful indicator of relatedness within eviously  that theant view  that humM. abscessus–dominant clones [M. abscessus will only realize its full potential with the proper screening of environmental sources, alongside longitudinal patient sampling.In agreement with previous studies, we found an international distribution of t clones . We founClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz526_suppl_Supplementary_Figure_S1Click here for additional data file.ciz526_suppl_Supplementary_Figure_S2Click here for additional data file.ciz526_suppl_Supplementary_LegendsClick here for additional data file.ciz526_suppl_Supplementary_MethodsClick here for additional data file.ciz526_suppl_Supplementary_Table_1Click here for additional data file.


Marrubium vulgare is a plant with high bioactive potential. It contains marrubiin, a labdane diterpene that is characteristic for this genus, as well as a complex mixture of phenolic compounds. According to numerous studies, M. vulgare acts as a good antioxidant agent, and due to this, it could potentially be useful in treatments of cancer, diabetes mellitus, and liver diseases. In addition, its anti-inflammatory, wound-healing, antihypertensive, hypolipidemic, and sedative potential are discussed. Apart from that, its antimicrobial activity, especially against Gram+ bacteria, fungi, herpes simplex virus, and parasites such as Toxoplasma gondii, Trichomonas vaginalis, and Plasmodium berghei-berghei was recorded. Additionally, it could be used as a chicken lice repellent, herbicide, and natural insecticide against mosquito larvae and natural molluscicide. In veterinary medicine, M. vulgare can be used as an anthelmintic against the eggs and larvae of bovine strongyles parasites, and as an antibiotic against bovine mastitis caused by resistant bacterial strains. Due to the mentioned benefits, there is a tendency for the cultivation of M. vulgare in order to ensure high-quality raw material, but more firm scientific evidence and well-designed clinical trials are necessary for the well-established use of M. vulgare herb and its preparations. Marrubium vulgare L. (M. vulgare), originated in the region between the Mediterranean Sea and Central Asia, has become a widespread species, currently inhabiting all continents [Marrubium derives from the Hebrew word marrob, meaning bitter juice, while vulgare means ‘common’ or ‘well known’. In the English language, the name ‘horehound’ comes from the Old English words har and hune, meaning downy plant. The most common folk name in the Serbian language is očajnica meaning a ’desperate woman’, because tea from this herb was typically used as a bitter remedy by women who were unable to conceive . Nevertheless, the most prevailing traditional application of M. vulgare worldwide is for the treatment of gastrointestinal and respiratory disorders.ntinents . The LatM. vulgare along with highlighting the interest in this medicinal plant as a source of herbal remedies. This is supported by the traditional use of M. vulgare preparations in medicine as stated by the European Medicines Agency (EMA) commission [This review article is aimed at comprehensively describing the botanical features, phytochemicals, and pharmacological properties of mmission . It is bmmission ,4,5.M. vulgare is an annual or perennial herb from Lamiaceae family, with a tough, woody, branched taproot or numerous fibrous lateral roots and numerous stems, which are quadrangular, erect, very downy, and from 20 to 100 cm high. Leaves are roundish, ovate, usually toothed, petiolate, veined, and hoary on the surface, and they are arranged in opposite pairs on a long stem. Inflorescences are formed in axils of upper leaves, with white flowers in crowded axillary whorls. The calyx is tubular, lobed, and 10-toothed, each tooth with a small hooked spine/bristle. Corolla is white to pale lavender, tubular, and bilabiate; the upper lip is 2-lobed, bifid, and erect, while the lower lip is 3-lobed with a larger middle lobe. Corolla tube includes style, stamens, and anthers with divergent sacs [M. vulgare generally appear in the early spring, and they are regularly visited by nectar-gathering bees [ent sacs . The shaing bees . Seeds lM. vulgare vegetative and generative organs is densely covered with glandular and non-glandular trichomes. There are two types of glandular trichomes: peltate and capitate. Peltate trichomes consist of a short stalk cell and a large head with secretory cells arranged in a circle. The substance produced by secretory cells passes through apical walls and accumulates within a space between the cuticle and the cell wall layer. The plant has a capitate, long trichomes, with basal cells, a long stalk neck cell, and a unicellular head. There are two types of short capitate trichomes: those with a bicellular head and those with a unicellular stalk. As for non-glandular trichomes, they can be multicellular uniserate or multicellular branched [The surface of branched .M. vulgare. In order to secure the supply of good quality raw material with a high content of marrubiin and other diterpenes, as well as phenolics, the cultivation of this plant is becoming increasingly common. In addition to choosing the assortment and agricultural practices appropriate for specific agro-ecological conditions, the postharvest processing of raw material plays a key role.Bearing in mind its vast potential for medicinal use, as well as the continuous discovery of its alternative beneficial properties, lately there has been a growing interest and demand for M. vulgare is reproduced exclusively by seeds, through direct sowing or seedlings production. However, after the collection of mature seeds, its germination rate is low (35%). Following one month storage, germination increases to 78–80% [o 78–80% . Seeds sM. vulgare grown from direct sowing in the field and seedlings production was assessed in the experiments conducted in Poland. It was established that the seed sowing term significantly affected M. vulgare cropping. Actually, a much higher fresh herb yield was obtained from the plantation established in the spring than from that established in autumn [M. vulgare cropping in the second year.The cropping of n autumn . HarvestM. vulgare plants with nitrogen biofertilizer (Biofert), potassium biofertilizer (Available K), or with a combination of the two showed that when applied abundantly, the fertilizers enable the M. vulgare plant to reach its maximum height, and also enhance fresh and dry herb weights and essential oil production [2/s lead to significant linear, curvilinear, and quadratic effects on nitrogen, boron, and manganese, respectively, in aerial parts [The biofertilization of oduction . Physiolal parts .M. vulgare prefers alkaline soils [2+, K+, and Ca2+ in a Cu2+ concentration-dependent manner in the aboveground part of the plant. Treatment with copper stimulates antioxidant enzymes activities (superoxide dismutase (SOD) and catalase (CAT) enzymes) and increases the total phenolics (flavonoid) content. These findings lead the authors to conclude that M. vulgare possesses an intrinsic capability to cope with the Cu stress by the activation of enzymatic and non-enzymatic antioxidant systems.ne soils . Amri etne soils  reportedM. vulgare, which appears to be highly sensitive to salinity, especially the content of the main bioactive compound marrubiin, which decreased with higher NaCl concentration.Other results  showed tM. vulgare plant specimens . In addition, soil samples were analyzed for boron content via the atomic absorption spectrophotometer technique. Their report confirmed that compared to the boron concentration in soil, M. vulgare samples accumulated boron levels that were 4 times higher in flowers, more than 4 times higher in leaves, 3 times higher in stem, and about 3 times higher in root, which showed that the members of the natural populations of M. vulgare can tolerate high boron stress.Boron plays a very important role in sugar synthesis in cell walls, nucleic acid, phenolics, hormone, carbohydrate and protein metabolism, cell elongation and pollen tube formation in plants. However, the deficiency and toxicity levels of boron are quite close when it comes to plants, which need only trace amounts of boron for vital activities. Ardic et al.  used a cMarrubii herba (flowering aboveground parts) are harvested just before obtaining a full green color. M. vulgare has a musky odor that changes by drying into a pungent yet pleasant odor and bitter, aromatic taste [Marrubii herba (dried to 17.20% of fresh herba weight) has a total fiber content of 9.50%, total ash content of 10.70%, acid insoluble ash content of 1.73%, and water-soluble ash content of 8.90%. They also reported that the alcohol soluble extractive value is 8.66%, which suggested that most of the constituents of the plants were soluble in alcohol. Furthermore, the water-soluble extractive value is approximately 5.90%, while the petroleum ether soluble extractive value is 2.77%.ic taste . Mittal ic taste  reportedM. vulgare produces trace amounts of essential oil, usually between 0.03% and 0.06% [O-α-l-arabinopyranosyl-β-d-glucopyranoside) as identified compounds [M. vulgare extracts [M. vulgare accumulates diterpenes of labdane type as principle bitter components, up to 3 mg/g of fresh weight [S)-hydroxymarrubiin, 11-oxomarrubiin, 3-deoxo-15(S)-methoxyvelutine, marrubenol, marruliba-acetal, cyllenil A, polyodonine, and preleosibirin [M. vulgare is 156 mg/g [Marrubii herba is presented in nd 0.06% ,8 with mnd 0.06% . Non-volompounds ,16. Sesqextracts ,18. M. vh weight , with maosibirin ,15,20,21osibirin ,23,24. T156 mg/g . The revMarrubiin attributes include a low turnover, high stability, and little catabolism, which are core characteristics required for therapeutic compounds and nutraceuticals of economic importance. It is responsible for the therapeutic properties such as antinociceptive, antioxidant, antigentoxic, cardioprotective, vasorelaxant, gastroprotective, antispasmodic, immunomodulating, antioedematogenic, analgesic, and antidiabetic properties. In addition, marrubiin is considered a potential substrate for potent active compounds, particularly marrubiinic acid and marrubenol. Furthermore, marrubiinic acid possesses antinociceptive activity, while marrubenol is vasorelaxant .M. vulgare is an abundant source of various phenolic compounds, major classes being phenolic acids, phenylpropanoid (cinnamic) acids, and esters and flavonoids. The amount of total cinnamic acid derivatives is estimated to 14.09 mg/100 mg of dry material [M. vulgare contain tannins (up to 7%) where the estimated amount of condensed tannins is 16.55 mg catechin/100 g of dry material [trans-cinnamic, ferulic, o-coumarinic, p-coumarinic and sinapic acids are determined. Chologenic acid and other cinnamic acid esters were quantified. It was considered that as a part of the Lamioideae subfamily, M. vulgare is characterised by bitter components, a low amount of essential oil, and an absence of rosmarinic acid [M. vulgare extracts [M. vulgare extracts. The list of individual bioactive phenolic acids and esters is given in Furthermore, material . M. vulgmaterial ,26. Detanic acid . Howeverextracts ,22,39. HM. vulgare extracts [In flavonoid fractions, flavone aglycones such as apigenin, luteolin, chrysoeriol, and diosmetin; flavone glycosides such as vicenin II and vitexin; and other flavone derivatives such as lactoylflavones, luteolin-7-lactate, and apigenin7-lactate can be found . Among fextracts ,22.M. vulgare are various nitrogen-containing compounds such as choline and betonicine, amino acids and alkaloid stachydrine, polysaccharides, as well as minerals, in particular potassium salts [Other compounds determined in the herbal material of um salts ,17,42. TMarrubiin attributes include a low turnover, high stability, and low catabolism, which are core characteristics required for therapeutic compounds and nutraceuticals of economic importance. In addition, marrubiin is considered to be a potential substrate for potent active compounds, such as marrubinic acid and marrubenol .The marrubiin and related diterpenoid metabolites predominantly accumulate in leaves and leaf trichomes. The central diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) turns into peregrinol diphosphate, which is a bicyclic prenyl diphosphate intermediate that features a hydroxyl group at C-9, which is characteristically present in marrubiin and related metabolites .The accumulation of furanic labdane diterpenes in plantlets seemingly depends on the developmental stage. No furanic labdane diterpenes were detected in plantlets during the first four to five weeks following germination. At this time, the leaves become more differentiated, and the number of trichomes on leaves is increasing. Young leaves and buds contain the highest content of furanic labdane diterpenes, which are mostly stored in peltate glandular trichomes .Marrubium vulgare can be confused with Ballota nigra L., Nepeta cataria L., Stachy germanicus L., Marrubium incantum DESR, or Marrubium remotum KIT [In order to ensure the health benefits of herbal drugs and to avoid potential health hazards, it is necessary to provide plant material of satisfactory quality. The first step in the quality control of herbal drugs is to identify the herb. otum KIT . For thaotum KIT .The source and quality of raw materials largely depends on good agricultural practices and manufacturing processes. These are certainly essential steps for the quality assurance of herbal medicines and play the key role in guaranteeing the quality and stability of herbal preparations. All of the above-mentioned factors affect the safety and efficacy of herbal drugs .3/h) shows an inverse effect. M. vulgare leaves dried at 80 °C and 300 m3/h contain relatively high amounts of total phenols in comparison with other drying temperatures [Drying is used to dehydrate plants by reducing the moisture content that encourages biochemical deterioration, as well as the growth of microorganisms. The drying air temperature is a determining factor in drying kinetics. The drying rate decreases in low drying temperature. Any increment in the drying air temperature  increases the energy efficiency, while any increment in the air flow rate , the best results in terms of marrubiin extraction yield were obtained by extracting samples at 120 °C with 100% ethanol for 15 min (3 × 5 min microwave cycles) [Advanced control and optimization of extraction are very important in the field of chemical engineering for the development of processes that will result in high yield with minimal time and energy costs . The con cycles) .M. vulgare extraction can be used as a method of choice to obtain high-quality powders that preserves the constant amorphous structure after 6 months storage time. Furthermore, recoveries of both phenolic acids and flavonoids were distinctly higher when 10% maltodextrin was added as a carrier, which suggested that maltodextrin addition protects bioactive compounds from thermal degradation. This is particularly emphasized in the case of rutin content, which was 4-fold higher when a carrier was included [M. vulgare, can be successfully separated from complex mixtures by chitin, which is an abundant natural polymer [Subcritical included . It is r polymer .M. vulgare with water and various concentrations (20–80%) of methanol, ethanol, and acetone, as well as at various temperatures (20–60 °C) and extraction times (30–450 min). They concluded that the highest amount of phenolics (293.34 mg GAE/g DW) was obtained with 60% methanol solution at 25 °C for 180 min. The total flavonoids content was the highest (79.52 mg CE/g DW) with 80% methanol solution at 20 °C for 450 min, and condensed tannins (28.15 mg CE/g DW) with 60% acetone solutions at 50 °C and for 180 min.Bouterfas et al.  tried toM. vulgare than from the fresh plant. The proportion of water pretreatment is also relevant and affects the yield extraction [Optimal conditions for obtaining essential oil are a 3% mass ratio, extraction time of 180 min, powder particles with 0.1–0.63 mm in diameter, and an extraction temperature of 120 °C. A much higher yield of essential oil is obtained from the dry plant material of traction . The temtraction  reportedMarrubium vulgare herb tea is used as a cough suppressant, and expel catarrh. ‘Materia Medica Vegetabilis’ gave directions for the preparation of M. vulgare decoction with honey against bronchitis and coughs [M. vulgare herbal medicinal products are used as an expectorant in cough associated with cold, for symptomatic treatment of mild dyspeptic complaints, such as bloating flatulence, and in temporary loss of appetite [M. vulgare has a bitter value of 3.0, acting as the bitter gustative receptores (on the base of the tongue) stimulator. This effect leads to the increase in gastric and biliar secretion and the stimulation of appetite [M. vulgare is one of the most popular herbal pectoral remedies in traditional medicine, and it is used as a bitter tonic, expectorant, and diuretic [M. vulgare includes treatment for jaundice, painful menstruation, and as a laxative in higher doses [M. vulgare [Marrubii herba is traditionally used in a number of countries outside Europe [d coughs . The herappetite ,57. M. vappetite . M. vulger doses ,60. Exteer doses . The imp vulgare ,61. Litee Europe . Generale Europe .M. vulgare can be applied as herbal tea for oral use , powdered herbal substance , expressed juice  and liquid extract . The use in children under 12 years of age is not recommended. Safety during pregnancy and lactation has not been established [M. vulgare  and pregnant rats treated with the extract exhibited a significant decrease in hematological parameters: red blood cells, hematocrit, hemoglobin, and mean corpuscular volume. The extract of M. vulgare caused a significant decrease on the mean implantations of fetuses and their size. As for the macroscopic and histological appearance of uterus; these data showed no change in normal treated rats. Contrary to these, the treated pregnant rats showed a severe histological change characterized by interrupted gestation, as well as the lysing of placental and embryo tissue in the uterus. All these results support the hypothesis of an abortifacient effect of M. vulgare [Leonorus cardiaca, which is a plant that is known for its emenagogue activity and contraindicated in pregnancy [Marrubum vulgare on hormonal parameters in a female rat model (receiving 500 mg/kg and 1000 mg/kg of M. vulgare extract for 21 days) of polycystic ovarian syndrome were studied. Luteinizing hormone (LH) hormone significantly decreased at a dose of 1000 mg/kg, and estradiol and progesterone decreased at doses of 500 mg/kg and 1000 mg/kg, while testosterone decreased at a dose of 1000 mg/kg. These findings contribute to the potential influence of examined extract on female hormones, thus questioning their safe usage in pregnancy [M. vulgare is mentioned in certain literature as a potentially nephrotoxic plant [ vulgare . Possiblregnancy . Additioregnancy . In addiic plant . HoweverM. vulgare extracts have been studied. As a good antioxidant agent, M. vulgare proved to be very useful in treatments of cancer, diabetes mellitus, and liver diseases. In addition, many studies indicated that this plant possesses anti-inflammatory and hemostatic effects, as well as antihypertensive, sedative potential, and antimicrobial properties.In recent years, numerous pharmacological effects of The imbalance in homeostatic processes between oxidants and antioxidants in the body, which is caused by free radicals, leads to oxidative stress. Oxidative stress is considered to be the primary cause of aging and a wide variety of human diseases, such as cancers, diabetes, neurodegenerative disorders, rheumatoid arthritis, etc. Antioxidants are substances that significantly delay, prevent, or inhibit oxidative damage to target molecules .M. vulgare methanol extracts were determined using DPPH  free radical scavenging assay and the results revealed a strong activity with the half maximal inhibitory concentration (IC50) value of 8.24–12.42 μg/mL [M. vulgare essential oil exhibits IC50 value of 153.84 μg/mL, which is about two times higher than a synthetic antioxidant (butylated hydroxytoluene or BHT) [M. vulgare extracts (261.41 and 272.90 μmol TE/g respectively), while the lower activity was observed when essential oil and isolated marrubin were investigated [M. vulgare leaves were tested for their antioxidant activity in rapeseed oil at 80 °C. The effect of the extracts on the edible oil stability was assessed by measuring weight gain, peroxide value, and UV absorption [M. vulgare was associated with the presence of marrubiin, along with phenolics and flavonoids exerting a synergistic effect.The in vitro antioxidant properties of 42 μg/mL ,31. The  or BHT) . Photochstigated . Djeridastigated  determinsorption . Acetonesorption , the higM. vulgare methanolic extracts showed a dose-dependent ferric reducing capacity  with a value equal to 50.01 μg AAE/g of extract when compared to ascorbic acid  [M. vulgare  showed IC50 of 64.07 mg AAE/g of dry extract and IC50 for the neutralization of DPPH, hydroxyl (OH), and nitroso (NO) radicals were 13.41, 63.99, and 64.86 μg/mL, respectively. Furthermore, inhibition on lipid peroxidation was examined in vitro [M. vulgare extracts.In addition, valents) . Other avalents) ,28 reporM. vulgare extracts on DPPH radical but concluded that this effect varied significantly depending on the type of the organic solvent used for extraction and sampling location.Bouterfas et al. 2016  also detM. vulgare extracts using different in vitro tests, but research should be directed toward in vivo experiments in order to elucidate their full antioxidant capacity.Lastly, all these finding indicate the antiradical potential of v/v) and petroleum ether extracts on CCl4-induced liver cell toxicity in mice showed that liver and kidney function parameters remained in the normal levels in groups treated with M. vulgare extracts. The administration of M. vulgare ethanolic extracts significantly enhanced SOD (superoxide dismutase) and CAT  activity, as well as total antioxidant capacity, with significant reduction in lipid peroxide concentration when extracts were used as protective or therapeutic agents [v/v) of M. vulgare in different concentrations  was assessed by measuring lipid profile parameters such as AST (aspartate aminotransferase), ALT , ALP , GSH (reduced gluthation), SOD, and malondialdehyde (MDA), as well as by histopathological examination of CCl4-induced liver damage in rats. Different concentrations of extracts showed a significant antihepatotoxic effect by reducing the levels of AST and ALT significantly, whereas the ALP level was insignificantly decreased. Regarding the antioxidant activity, these extracts exhibited a significant decrease in SOD and contents of GSH and MDA (a biomarker of membrane lipids peroxidation). These findings showed that different concentrations of M. vulgare extract protect liver against CCl4-induced hepatotoxicity, and the effect may be attributed to its antioxidant activity [Investigation of the hepatoprotective and therapeutic effect of ethanol–water extract , GR (glutathione reductase), and GST (glutathione transferase) activities in rat liver tissue. In addition, it increased the GSH content and decreased the production of MDA level, adding to alleviated histopathological changes in rats’ livers treated with CCl4 [Methanol extracts of ith CCl4 .In another animal study, marrubic acid exhibited a significant antihepatotoxic activity by reducing the elevated levels of serum enzymes such as serum glutamate oxaloacetate transaminase (SGOT) by 40.16%, serum glutamate pyruvate oxaloacetate transaminase (SGPT) by 35.06%, and alkaline phosphatase (ALP) by 30.51% [M. vulgare were tested on CCl4-induced acute liver injury in rats through the in silico method. All compounds showed expected and similar bioactivity, especially when it comes to liver-associated enzymes inhibition [The hepatoprotective potential of 12 pure compounds  characteristic for hibition .M. vulgare are often used traditionally in cancer treatment [reatment ,76, but M. vulgare essential oil to inhibit the proliferation of cervical cancer (HeLa) cell lines with an IC50 value of 0.258 μg/mL. M. vulgare ethanol–water extracts  reduced the viability of melanoma (B16) and glioma (U251) in a dose-dependent manner. By demonstrating the ability of M. vulgare extracts to inhibit proliferation, induce apoptosis, and cytoprotective autophagy, the results suggested that this plant could be a good candidate for anti-melanoma and anti-glioma therapy [M. vulgare was evaluated for its in vitro cytotoxic activity by measuring the percentage of viable cells’ glioblastoma multiforme cell lines  using a luminescence system. After evaluating the cytotoxicity impact that M. vulgare has on U87 (IC50: 270.3 μM), LN229 (IC50: 343 μM), and T98G (IC50: 336.6 μM) from glioblastoma multiforme cell lines, it was concluded that it is the most efficient in two cell lines, U87 (69.9%) and LN229 (71%) [Zarai et al.  reported therapy . In addiracts 70:, v/v redM. vulgare ethanol–water extracts  and six pure compounds  were also tested against Ehrlich tumor cell lines, human tumor cell lines U251 (brain tumor) and MCF7 (breast cell lines) [M. vulgare displayed a moderate effect on human myeloid leukemia (K562) and imatinib-resistant human myeloid leukemia (K562R) cells, as well as on human B cell precursor leukemia cell lines (697). The authors of this study suggested that these results provide a common natural source for the hemi-synthesis of future ladanein-derived flavones and the study of their antileukemic activity. Tlili et al. [M. vulgare and other Tunisian plant extracts on leukemia and colon cancer cell lines . Subsequently, the anti-inflammatory activity was assessed, and the results showed that white horehound possesses the highest activity in the group of analyzed plants.The in vitro anticancer activity of l lines) . Alcoholl lines)  showed ti et al.  examine M. vulgare against a human melanoma cancer cell line (A375) and normal human skin fibroblasts (BJ) using a 3--2,5-diphenyl-tetrazolium bromide test, cell cycle analysis, and real-time monitoring of cell viability. Surprisingly, examined fractions demonstrated a low total phenolic content and did not show significant antioxidative properties, but the nonhydrolyzed PhA fraction exhibited cytotoxic activity against a human melanoma cancer cell line, without affecting normal fibroblasts. Both acidic and alkaline hydrolysis abolished this activity, indicating that the esterified forms of phenolic compounds exhibit the observed cytotoxic effects. Since M. vulgare is abundant in phenylpropanoid (cinnamic) esters and phenylethanoid glycosides, further investigation of these compounds may provide an insight into the exact anticancer mechanism of action and use as a model for cancer treatment drug development.Kozyra et al.  investigM. vulgare possessed the largest number of plant–constituent–target gene interactions indicating cell cycle arrest and apoptosis in addition to the inhibition of cellular proliferation as possible mechanisms and marked this plant as a potential source for the supportive treatment of cancer.Moreover, Shawky  used theM. vulgare on isoproterenol-induced myocardial infarction in a rat model showed that serum creatinine kinase-MB was subsided by 52.2–69.0% (depending on the dose of M. vulgare extract). In addition, the treatment with extracts significantly reduced myocardial myeloperoxidase activity in myocardial infarction [M. vulgare compared to control. The results of this study demonstrated that M. vulgare extracts have strong protective effects against isoproterenol-induced myocardial infarction, and it seems possible that this protection is a result of its anti-inflammatory properties. Furthermore, the 11-oxomarrubiin, vulgarcoside A, and 3-hydroxyapigenin-4′-O-(6′′-O-p-coumaroyl)-β-d-glucopyranoside from the methanolic extract of M. vulgare exhibited moderate to low levels of inhibition on NO production, while vulgarcoside A also showed a moderate inhibition effect on pro-inflammatory cytokinine TNF-α [M. vulgare showed inhibitory activity toward the cyclooxygenase (COX) enzyme, which plays a key role in the transformation of arachidonic acid into pro-inflammatory prostaglandins and is associated with inflammation [Investigation of the anti-inflammatory effect of the methanolic extracts of farction ,82. Levene TNF-α . Glycosiammation .M. vulgare at a dose of 200 mg/kg in rats treated with carrageenin causes a significant decrease (87.30%) of inflammation compared to standard positive control (diclofenac), which showed 85.52% protection in this test [M. vulgare exhibits significant and dose-related antioedematogenic effects. The treatment of mice with marrubiin caused a dose-dependent inhibition of carregeenan, bradykinin, and histamine-induced extravasation of Evans blue in mice ears, with maximal inhibitions of 63.0%, 70.0%, and 73.7%, respectively. The other phlogistic agonists, such as prostaglandin E2, caused an inhibition of less than 50%. In addition, marrubiin significantly inhibited the ovalbumin-induced allergic edema in actively sensitized animals. These results demonstrate that the systemic administration of marrubiin exerts a non-specific inhibitory effect on pro-inflammatory agent-induced microvascular extravasation of Evans blue in mice ears [p-benzoquinone-induced abdominal constriction test showed that methanolic extracts of M. vulgare have a similar effect as reference drugs indomethacin and acetylsalicylic acid [The assessment of anti-inflammatory activity showed that the oral administration of methanolic extract of his test . In a moice ears . The evalic acid .M. vulgare  have on COX showed that these compounds inhibited the formation of hormones, such as prostaglandins and peroxasalandine, which contribute to the production of inflammatory intermediators [An in vitro investigation of the anti-inflammatory effect that six compounds from ediators .M. vulgare aqueous extracts  was evaluated as a cure agent in mice previously infected with Salmonella typhimurium and as a protective agent in mice infested with S. typhimurium [M. vulgare (100 mg/kg body weight) showed high immunomodulatory effect in the level of double positive T cells, interleukin (17AIL-17) and interferon-gamma (IFN-γ).The immunomodulatory effect of different concentrations of himurium . AccordiM. vulgare in different doses . It was concluded that all doses reduced the physical activity of mice. They also induced muscle relaxation [The potential to reduce morphine withdrawal signs in animals had been studied using aqueous and ethanolic extracts of laxation .Marrubium vulgare has an ethnomedical record as an antidiabetic agent [M. vulgare 80% ethanol extracts exerted a moderate effect (IC50 = 12.66 μg/mL). Moreover, a series of in vivo experiments were carried out on alloxan-induced diabetes during which the experimental animals were treated twice a day with aqueous extract of M. vulgare for 15 days [M. vulgare aqueous extract induced significant dose-dependent antidiabetic and antihyperlipidemic effects. A dose of 100 mg/kg reduced blood glucose by 50%, whereas doses of 200 and 300 mg/kg showed more than 60% reduction of the same parameter. A decrease of total lipids, triglycerides, and cholesterol levels were observed in animals treated with M. vulgare when compared to the diabetic control group. Glibenclamide was used as a reference and showed similar effects, and the authors hypothesized that flavonoids and verbascoside derivatives present in examine extract caused the observed effects [ic agent ,86. Certic agent . Hellal ic agent  performe 15 days . Oral ad effects .M. vulgare methanolic extracts resulted in a significant lowering of blood glucose level, serum urea, uric acid, and creatinine, as well as a correction of lipid profiles when compared to streptozotocin-induced diabetic rats. These methanolic extracts significantly increased the glucose uptake of liver and skeletal muscles. Contrary to this, they reduced the glucose absorption in the everted rat jejunum. These results suggest that the effect of M. vulgare extract may be due to extrapancreatic mechanisms. This antidiabetic effect is an outcome of the modulation of glycogen synthesis and the inhibition of intestinal glucose absorption.Chakir et al.  showed tM. vulgare extracts  on autoimmune diabetes mellitus induced by cyclosporine and streptozotocin. When compared to diabetic mice, the animals from the group treated with extracts of M. vulgare showed a decrease in blood glucose levels, pancreatic levels of interferon gamma (IFN-γ), and NO. M. vulgare extracts also caused a significant decrease in total cholesterol, low-density lipoprotein (LDL) cholesterol, very-low-density lipoprotein (VLDL) cholesterol, and triglycerides. Additionally, the serum insulin levels were significantly increased after treatment with M. vulgare.Another study  showed tM. vulgare extract showed a neutral effect on blood glucose levels.In addition, Alkofahi et al.  screenedM. vulgare on the contractile reactivity of isolated aorta were analyzed in an experimental model of streptozotocin-induced diabetic rats after two months of oral administration of M. vulgare [M. vulgare. In addition, M. vulgare-treated rats showed a lower concentration of KCl and noradrenaline in comparison to the control group. Based on these results, it was concluded that the oral administration of M. vulgare during 2 months could attenuate the contractile responsiveness of the vascular system, which may prevent the development of hypertension in diabetic rats.The incidence of atherosclerosis and cardiovascular diseases increases in diabetes mellitus patients. Therefore, the effects of  vulgare . The resM. vulgare extract on induced diabetes in experimental animals, the duration of these studies were short, and also another model of type 2 diabetes studies would be interesting to elucidate the exact mechanisms of action of this plant. In addition, there has been one clinical trial that contradicted findings from animal studies. Namely, aqueous extract (infusion of dried M. vulgare leaves) was tested to evaluate a clinical effect in 22 type 2 diabetic patients that had poor response to conventional treatment. The results showed that infusion reduced glucose levels by only 0.64%, cholesterol by 4.16%, and triglycerides by 5.78% [Although there is an evident antihyperglycemic effect of by 5.78% . FinallyM. vulgare is widely used as an antihypertensive treatment in folk medicine. Crude extracts of the aerial parts of M. vulgare show a potent in vitro inhibition of KCl-induced contraction of rat aorta. Bioguided fractionations, spectroscopic analysis, and chemical derivatization revealed furanic labdane diterpenes, marrubenol, and marrubiin as the most active compounds [M. vulgare water extract had on the systolic blood pressure, cardiovascular remodeling, and vascular relaxation in spontaneously hypertensive rats, it was observed that treatment with M. vulgare produced a decrease in systolic blood pressure. In addition, it had significant antihypertrophic effect in aorta and it improved acetylcholine (ACh)-induced relaxation of mesenteric artery. These results demonstrated that in addition to its antihypertensive effect, M. vulgare water extract improved the impaired endothelial function in spontaneously hypertensive rats [The water extract of ompounds ,37. By aive rats .M. vulgare herb extracts  using Triton WR-1339-induced hyperlipidemia in mice. Extracts were applied using 0.1 and 0.25 LD50 concentrations. After 7 h and 24 h of treatment, the intragastric administration of all extracts caused a significant decrease of plasma total cholesterol; LDL cholesterol and triglyceride levels were also significantly lowered by all extracts. Additionally, more polar extracts (methanol and ethyl acetate) showed a significant ameliorative action on elevated atherogenic index (AI) and LDL/HDL-C ratios [One study evaluated the hypocholesterolemic and hypotriglyceridemic activities of four C ratios . These fC ratios .Marrbium vulgare herb extract, it would be useful to evaluate its possible usage as a part of nutrcaceuticals approach to this indication [Moreover, since metabolic syndrome as a cluster of conditions includes increased blood pressure, high blood sugar, and abnormal cholesterol or triglyceride levels, all of which are influenced by dication .M. vulgare extract was applied [M. vulgare extract. For indomethacin-induced ulcers, the percentages of ulcer inhibition were 50.32%, 66.24%, and 82.17% for the groups treated with 25, 50, and 100 mg/kg of M. vulgare. In both models, the marrubiin (25 mg/kg) produced a significant reduction in all parameters compared to the control group. There was also a significant increase in pH and mucus production in groups treated with M. vulgare extracts and marrubiin. The results demonstrated that the gastroprotective effect induced by these extracts and marrubiin is related to the activity of NO and endogenous sulfhydryls, which are important gastroprotective factors.In the model of ethanol-induced ulcers, a significant reduction in all analyzed parameters was observed when  applied . The curM. vulgare extracts  was tested on four strains of bacteria: Staphylococcus aureus, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa. The antibacterial activity of different fractions performed on solid agar medium showed little or no effect. This implies that the antibacterial activity proved with crude extract of M. vulgare was likely induced by synergistic action in chemical ingredients present in the extracts [M. vulgare showed a significant antimicrobial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Proteus vulgaris, and Candida albicans [The antibacterial potential of different extracts . The metalbicans .M. vulgare against two fungal strains; Aspergillus niger ATCC 16,404 and Candida albicans ATCC 10,231 using the solid medium diffusion method. The minimum inhibitory concentrations (MICs) obtained range between 6.25 and 100 μg/mL and led to experiencing strong antifungal inhibition, which often exceeded the effect of marketed antifungals  that marked M. vulgare flavonoids as potentially powerful antifungal agents [M. vulgare and marrubiin can be used as antifungal agents for the treatment of skin dermatophyte infections. They examined the effect of acetone and methanol extracts, essential oil, and marrubin  against the dermatophytes fungi Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum, and against two fungi strains .Moreover, one study was undertaken to determine the antifungal activity of flavonoids (flavans and flavanols) extracted from the leaves of l agents . In addil agents  concludeM. vulgare essential oil has a significant effect on microorganisms, especially Gram+ bacteria with inhibition zones and MIC values in the range of 6.6–25.2 mm and 1120–2600 μg/mL, respectively, whereas Gram– bacteria exhibited higher resistance. When it comes to antifungal activity, among the four strains tested, Botrytis cinerea exhibited the strongest response to M. vulgare essential oil, with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum, and Aspergillus niger were less sensitive to this essential oil [tial oil .M. vulgare in wound reparation demonstrated that this extract, which was rich in poliphenolic compounds  and marrubiin (6.62%), showed antioxidant and wound-healing properties by promoting cell migration and the proliferation of fibrosis [M. vulgare [The study of use of methanolic extract of fibrosis . The ass vulgare . A positM. vulgare showed antiviral activity with selectivity indices of 3.11, 2.8, and 1.28, respectively. This study revealed that the hexane fraction disrupts the early steps of cyclic replication, including HSV-1 attachment, in a dose-dependent manner [Acute and recurrent herpes simplex virus type 1 (HSV-1) infections remain an important problem due to the emergence of acyclovir resistant virus. As a result of that, the search for novel antiviral bioactive compounds from plants has intensified in recent years. The antiviral activity of methanol, hexane, and chloroform extract of t manner .Toxoplasma gondii is an intracellular parasite that causes many symptoms, such as encephalitis and congenital disorders. Using MTT -2–5-diphenyltetrazolium bromide) cell-proliferation assay in vitro, it was concluded that M. vulgare could be very useful against this parasite [parasite .M. vulgare essential oil and extracts  have potent antiprotozoal activity against Trichomonas vaginalis. After 48 h of exposure, the essential oil with a minimal inhibitory concentration value of 291 μg/mL showed the highest antiprotozoal activity, followed by ethyl acetate (541 μg/mL), methanol (1000 μg/mL), and n-hexane (1500 μg/mL). According to the findings of this study, the compounds of M. vulgare have a significant effect on T. vaginalis [aginalis .M. vulgare was evaluated in mice infected with chlorquine-sensitive Plasmodium berghei-berghei using curative, suppressive, and prophylactic experimental animal models. Preliminary phytochemical screening and intraperitoneal LD50  estimation of the extract were carried out. In all doses tested, the extract produced significant curative and suppressive effects with minimal prophylactic effect. The extract also significantly prolonged the survival time of treated mice (up to 22 d), compared to the negative control group (11 d). The results of this study suggest that the ethanol extract of M. vulgare possesses curative and suppressive antiplasmodial activity in mice at all tested doses [The antiplasmodial activity of ethanolic extract of ed doses .M. vulgare were evaluated using the egg hatch assay and larval mortality assay. After a 24 h exposure period at a concentration of 50 mg/mL, the mortality rate was 45.8% for the aqueous extract and 51% for the ethanolic extract. These findings showed that M. vulgare extracts have potential anthelmintic effect on eggs and larvae of bovine strongyles parasites in vitro [The anthelmintic activities of ethanolic and aqueous extract of in vitro .M. vulgare shows good antibacterial activity against methicillin-resistant staphylococci and multi-resistant Escherichia coli strains isolated from animals with mastitis manifestation [Bovine mastitis is the most serious diary problem in terms of economic losses to the dairy industry. With new generations of virulence and resistant bacteria, finding alternative treatments with medicinal plants to control these pathogenic strains is very popular. Among others, the ethanol extract of estation .M. vulgare has been popularly used on chicken farms to prevent lice and frequent scratching of animals, which intensified its planting on farms [M. vulgare was tested against fourth instar larvae of the mosquito Culex pipiens. The obtained results indicated a sensitivity of C. pipiens larvae that was even higher when the exposure time of larvae to the insecticide is extended. The greatest mortality rate (94%) was achieved with 900 mg/L and a 72 h exposure to M. vulgare extract, whereas a 59% mortality rate was achieved with 900 mg/L and a 72 h exposure period. These results may provide an opportunity to develop alternatives to environmentally hazardous chemicals using some readily available, affordable plants which are mostly harmless to different living organisms [M. vulgare has a remarkable toxicity on the snails of both Schistosoma mansoni and S. haematobium species [M. vulgare leaf extract and rizosphere soil extract significantly influence the seed germination and seedling growth of Sinapis arvensis and Latuca sativa in laboratory conditions. However, the allelopatic effects depend on target species. These extracts can be used as an important source of natural herbicides to control weeds in crop fields [In Spain, on farms . Moreoverganisms . Further species . In addip fields .M. vulgare produce structurally highly diverse groups of secondary metabolites, thus representing the valuable source of bioactive compounds and preparations with health-promoting effects: antioxidant, hepatoprotective, antiproliferative, anti-inflammatory, antidiabetic, and antimicrobial being the most investigated. Although these effects of M. vulgare extracts, essential oil, marrubiin, flavan and flavonol type of flavonoids, and phenylethyl esters were studied, the pharmacokinetics of these compounds and the concentrations of extracts, essential oil, and isolated compounds needed for the pharmacological effect have yet to be established. White horehound is a part of traditional medicine systems worldwide; it is generally recognized as safe, but well designed clinical trials are needed in order to shift from traditional to a well-established use of M. vulgare herb preparations for the prevention and treatment of various ailments.


Pseudomonas aeruginosa is one of the most prominent opportunistic bacteria in airways of cystic fibrosis patients and in immunocompromised patients. These bacteria share the same polymicrobial niche with other microbes, such as the opportunistic fungus Aspergillus fumigatus. Their inter-kingdom interactions and diverse exchange of secreted metabolites are responsible for how they both fare in competition for ecological niches. The outcomes of their contests likely determine persistent damage and degeneration of lung function. With a myriad of virulence factors and metabolites of promising antifungal activity, P. aeruginosa products or their derivatives may prove useful in prophylaxis and therapy against A. fumigatus. Quorum sensing underlies the primary virulence strategy of P. aeruginosa, which serves as cell–cell communication and ultimately leads to the production of multiple virulence factors. Understanding the quorum-sensing-related pathogenic mechanisms of P. aeruginosa is a first step for understanding intermicrobial competition. In this review, we provide a basic overview of some of the central virulence factors of P. aeruginosa that are regulated by quorum-sensing response pathways and briefly discuss the hitherto known antifungal properties of these virulence factors. This review also addresses the role of the bacterial secretion machinery regarding virulence factor secretion and maintenance of cell–cell communication. Pseudomonas aeruginosa and Aspergillus fumigatus, two ubiquitous microbes, occurs in environmental niches, such as soil and water. Researchers are most interested in how their interactions in the human biosphere may affect the propensity for disease, particularly in the immunocompromised host and in persons with cystic fibrosis . C. CP. aerentified ,132.P. aeruginosa phenazines, has been identified in cystic fibrosis patient sputum at concentrations up to 135 µM [P. aeruginosa phenazines are reported to exert fungicidal effects on A. fumigatus. Phenazines can diffuse across the cell membranes and act as reducing agents, causing uncoupling of oxidative phosphorylation and generating toxic intracellular ROS that are harmful to the fungus [P. aeruginosa and A. fumigatus at the molecular level [P. aeruginosa, PCA, was converted into 1-HP, which was further transformed into phenazine-1-sulphate (PS) and 1-methoxyphenazine (1MP) [A. fumigatus also mediated the conversion of PCA into pyocyanin and promoted the production of mixed phenazine dimers [P. aeruginosa [P. aeruginosa phenazines  on A. fumigatus mitochondrial architecture and hyphal growth where the primary mode of inhibition was ROS–RNS-mediated oxidative stress [A. fumigatus; whereas low-to-moderate levels of ROS can act as a sporulation signal in fungal development, higher levels of ROS can act as a toxic substance [A. fumigatus development switches from vegetative growth to conidiation in response to the phenazine gradient in the co-culture medium, correlating with levels of phenazine radicals produced in the phenazine-mediated redox cycles [P. aeruginosa that enables the bacteria to switch from iron-denial-based (pyoverdine-based) to toxin-based (pyocyanin-based) antifungal activity in response to environmental iron concentrations [P. aeruginosa exerted phenazine pyocyanin-based antifungal mechanisms to compete with A. fumigatus [3 or blood) led to the reduction of pyoverdine content while increasing the phenazine content in bacterial supernatants. Subsequent experiments revealed strong antifungal activity of these supernatants against A. fumigatus biofilms. Similarly, P. aeruginosa supernatants not containing pyocyanin were significantly less antifungal toward A. fumigatus biofilms under high-iron conditions. We also found that pure pyocyanin at concentrations over 125 µM interfered with fungal metabolism [Pyocyanin, one of the o 135 µM . High pyo 135 µM . P. aerue fungus . A remarar level . They dene (1MP) . These pne (1MP) . The exae dimers . The exaruginosa . Briard e stress . This ste stress . Interese stress ,137. Zhex cycles . Our rectrations . We founumigatus . In thistabolism . TherefoP. aeruginosa possesses an array of secretion systems (Types I–VI), which are distinguished by the characteristics of components forming the secretion machinery [P. aeruginosa, important for iron acquisition, is an example of a protein secreted through the Type I Secretion System (T1SS) [P. aeruginosa that contributes to cytotoxicity and acute infections [P. aeruginosa identified with T3SS are ExoS, ExoT, ExoU, and ExoY [P. aeruginosa contains one or more genomic island-associated Type IV secretion systems (T4SS) that play a crucial role in horizontal gene transfer of integrative and conjugative elements [P. aeruginosa is EstA protein, which has esterase activity and is involved in the production of rhamnolipids, cell motility, and biofilm formation [P. aeruginosa harbors three Type VI secretion (T6S) loci, hemolysin co-regulated protein secretion island-I, II, and III . Two major known substrates of T6SS in P. aeruginosa are hemolysin-co-regulated protein (Hcp) and valine–glycine repeat protein G (VgrGs) [P. aeruginosa can perceive T6SS-mediated aggression of neighboring cells [P. aeruginosa were originally identified in the PAO1 strain and later studied in the reference strain PA14 [P. aeruginosa-mediated pathogenesis in progressive lung diseases [Serratia marcescens [Candida albicans, Candida glabrata, and Saccharomyces cerevisiae [P. aeruginosa T6SS protein Tse2, which showed toxic effects and inhibition of growth in the yeast Saccharomyces cerevisiae [P. aeruginosa [Pseudomonas T6SS as a potential antifungal secretion machinery and a target for future therapeutic studies. Secretory systems of P. aeruginosa could also be a target of therapeutic approaches to bacterial control [Like other Gram-negative bacteria, achinery . These sm (T1SS) ,142. Secm (T1SS) . The Typfections ,143,144,and ExoY . Among tand ExoY . P. aeruelements , and theormation . The Typ (VgrGs) . While q (VgrGs) . Another (VgrGs) . It is r (VgrGs) ,150. Galng cells  and recong cells . The recain PA14 . Studiesain PA14 . The HSIdiseases ; HSI-IIIdiseases . Recent rcescens ,155. Therevisiae ,155. Effrevisiae . To evalruginosa . These s control . The bacP. aeruginosa anti-infectives that can inhibit the growth and/or reduce the virulence of the pathogen [P. aeruginosa biofilms to restore susceptibility toward antibiotics [Pseudomonas pathogenicity and thus infection [Pseudomonas infections have not been very successful, owing to such factors as development of resistance to antimicrobials, adverse drug effects on the host, or undesirable drug–drug interactions, combination therapy (including antibiotics and antivirulence drugs) and alternative approaches have been proposed [P. aeruginosa pathogenesis in the mammalian host, should be explored to examine effects on P. aeruginosa intermicrobial interactions, in order to evaluate the roles of these secretions systems as the as yet unquantified components of P. aeruginosa antifungal weaponry.Numerous research approaches are being pursued to develop pathogen . The stribiotics . Targetinfection ,161. As proposed ,162. Theproposed  and T2SSproposed . The appproposed ,166, assproposed , proteinproposed ,169,170,proposed . Blockagproposed  or throuproposed  targetinPseudomonas–Aspergillus interactions we have detailed must be translated into the in vivo effects. As our review here focuses on the in vitro and molecular mechanisms of Pseudomonas pathogenicity, a more extensive review of the in vivo effects is required and awaited. Animal models would suggest co-infection in the lungs with P. aeruginosa and A. fumigatus in leukopenic mice is a deadly combination [P. aeruginosa and A. fumigatus has received scant attention in animal models that mimic cystic fibrosis, where the effects of P. aeruginosa infection alone have received much attention [P. aeruginosa infection has preceded A. fumigatus infection, or A. fumigatus is commonly seen with P. aeruginosa, which might suggest that P. aeruginosa predisposes to A. fumigatus establishing itself as an infection, or that the pathologic processes in cystic fibrosis, having advanced as a result of repeated insults, provides a welcome milieu for A. fumigatus infection. Studies have indicated eradicating P. aeruginosa from cystic fibrosis lungs is associated with increased risk of acquiring A. fumigatus [A. fumigatus isolates in cystic fibrosis do not differ in their susceptibility to P. aeruginosa inhibitory factors compared to other isolates. [P. aeruginosa more commonly preceding A. fumigatus, studies have shown that once A. fumigatus infection has become established, more episodes of P. aeruginosa infection will follow than would otherwise be the case [The effect of the bination . The intttention ,190,191.ttention ,193,194,ttention , P. aeruumigatus ,197, altumigatus . We havesolates. . Howeverthe case . Again, the case .P. aeruginosa and A. fumigatus interactions play a crucial role in the pathology of cystic fibrosis and less defined roles in immunocompromised patients. Such interactions alter the shared environmental niche and can be beneficial or inhibitory for both the species and can result in undesired effects on the mammalian host. Intermicrobial competitions and metabolite exchange between P. aeruginosa and A. fumigatus have long been studied. Stronger emphasis has been placed on co-culture experiments of Pseudomonas and Aspergillus species, which resulted in discoveries with potential therapeutic significance. It is our belief that Pseudomonas–Aspergillus interaction continues to be a promising field for mycology research and that more mycologists will be eventually attracted to these studies. This review revisits the quorum-sensing-regulated virulence factors of P. aeruginosa and describes how these factors affect and interact with the opportunistic fungi A. fumigatus. We hope that this review will be helpful to re-emphasize the complex and sophisticated nature of the Pseudomonas–Aspergillus interaction and indicate new avenues for future mycology studies. Better understanding of this interaction could suggest avenues for therapeutic intervention, for the benefit of the host.


Small bowel obstruction secondary to endometriosis is extremely rare. This etiology is reported in 0.10% of cases and diagnosed incidentally.32-year old female, critically ill patient was diagnosed with small bowel obstruction. After medical stabilization of multiple comorbidities, diagnostic laparoscopy was done and converted to open right ileocolectomy with ileo-transverse anastomosis. Definite diagnosis was ileal obstruction caused by transmural endometriosis. The patient's recovery was uneventful. At one-year follow-up, she remains asymptomatic.We present the uncommon case of ileal obstruction due to transmural endometriosis that was treated surgically. We also present current literature review focusing on diagnostic and treatment methods of this rare disease.Endometriosis should be included in the differential diagnosis of small bowel obstruction. The treatment of choice is bowel resection. This etiology is reported in 0.10% of cases and diagnosed incidentally. Using the Surgical Case Report (SCARE) Guidelines [2A 32-year-old nulliparous woman presented into Emergency Room with colicky abdominal pain that lasted 48 h. Additionally, she had diarrhea, abdominal distension, nausea, vomiting, dry cough, nasal congestion, headache, and fever. She was taking antibiotics for H. pylori gastritis. Her medical history included endometriosis. She was taking oral contraceptives intermittently. She had right oophorectomy at age 22 due to endometrioma, and right salpingectomy at age 25 due to ectopic pregnancy. In-vitro fertilization and embryo transfer failed at age 29.2/FiO2 ratio 370. Abdominal CT scan with IV contrast showed small bowel diameter of 5 cm with air-fluid levels and transition point at 5 cm from the ileocecal valve  on postoperative day (POD) 0. On POD 1, she had bowel sounds and slowly recovered her bowel function. On POD 4, she was stared on liquid diet. Her diet was slowly progressed and the TPN weaned. By POD 8, she was tolerating soft diet, having bowel function, and no signs of active infection. She was discharged home.The macroscopic pathological examination consisted of a 20 cm long ileum segment and a 6 cm cecum and colon segment. The external surface was light brown, with congestive areas. The luminal surface had hematic material and showed a variable diameter secondary to stenotic areas, with purplish areas of hemorrhagic appearance. Cross sections showed small dilated cystic areas in relation to fibrosis and mural bleeding.Microscopically, we found multiple foci along the enterocolonic wall of glandular and tubular structures, lined by epithelial cuboidal-columnar cells without atypia, surrounded by endometrial stroma with lymphoplasmacytic inflammatory infiltrate, focal hemorrhage, and hemosiderin-laden macrophages. There was important vascular proliferation as well as extensive subserosal fibrous bands. Immunostains for Estrogen Receptor and PAX8 were positive, which confirmed the endometrial nature of the lesions. The diagnosis of enterocolonic transmural endometriosis with serosal adhesions was established .Fig. 4PaDefinite diagnosis was ileal obstruction secondary to transmural endometriosis. At one-year follow-up, patient remains asymptomatic. She has been referred to Gynecology for further treatment of endometriosis.3Endometriosis is defined by ectopic endometrium . 10% of From the patients with endometriosis, 10% have enterocolonic endometriosis . From thOnly 0.10% of women with ileal endometriosis develop ileal obstruction . PhysiopIleal obstruction caused by endometriosis is diagnosed incidentally . LuminalIleal endometriosis is mostly identified in sections with hematoxylin and eosin staining. Immunohistochemical analysis is also useful. Immunostains most used are Estrogen Receptor (ER), Progesterone Receptor (PR), Pair-Box 8 (PAX8), and Cluster of Differentiation 10 (CD10) . In our Bowel resection is the treatment of choice in patients with ileal obstruction due to endometriosis . SurgicaOur case is unique. Ileal obstruction was secondary to transmural endometriosis. Additionally, our patient had C. difficile colitis and Influenza AH1N1 at the same time.4Ileal obstruction due to endometriosis is extremely rare and diagnosed incidentally. However, it should be contemplated as possibility in the differential diagnosis of women with small bowel obstruction. Definite diagnosis is established through pathological examination and immunohistochemical analysis. The treatment of choice is bowel resection.None.Sources of funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Ethical approval: Exception: Case report of interesting case encountered during normal medical practice.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Adriana Santos-Manzur: treating physician, concept and design, data collection, data analysis, writing.David Ricardo Valdez-Bocanegra: concept and design, data collection, data analysis, writing.María Cristina Ornelas-Flores: data collection, data analysis.Janet Pineda-Díaz: data collection, data analysis.Enrique Stoopen-Margain: data collection, data analysis.Not applicable.Adriana Santos-Manzur.Not commissioned, externally peer reviewed.


Cutaneous leishmaniasis has been endemic since decades. Millions of cases are reported worldwide specially in developing and underdeveloped countries. There are 2 major types of cutaneous leishmaniasis based on the causating species found in different regions of the world. These include New and Old World cutaneous leishmaniasis, which are self-healing, but if not treated, these may cause severe scars and many other complications like mucosal involvement. The conventional gold standard treatment for both types is mainly intralesional or parenteral administration of antimonial. Lately, a great deal of research has been done on development of topical treatment based on single agent or combination therapy. This review summarizes the current state of literature regarding therapeutic outcome of topical treatment against cutaneous leishmaniasis caused by different species in different regions. Leishmania is an intracellular parasite from trypanosomatidae family among protozoa; consisting of almost 20 species which are transmitted by female sandflies (90 species), causing leishmaniasis. There are 3 main types of leishmaniasis; visceral or kala-azar leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis. VL is the deadliest form, while CL the most common form. Throughout the world, about 1.5 to 2 million new cases occur per year, among which 500,000 cases consist of VL and 1 to 1.5 million cases of CL. In 2018, a report showed that over 85% of new CL cases has occurred in Afghanistan, Algeria, Bolivia, Brazil, Colombia, Iran, Iraq, Pakistan, the Syrian Arab Republic, and Tunisia ..L. braziImiquimod (IMQ) is a member from the class of drugs known as imidazo-quinolines with the ability to modulate the immune response by up-regulating different inflammatory mediators, i.e., cytokines like tumor necrosis factor alpha , interleukin 12 (IL-12), interferon alpha , and interferon beta (IFN beta) ,17. PrevL. tropica). Ali Raza and colleagues also determined that the response of systemically administered MA (20 mg/kg/day) was not affected by topical application of IMQ 5% cream in OWCL caused by L. tropica ..StreptomL. major was achieved with ointment containing 15% PA and 12%/5% MBCl. Variation in MBCl concentration showed no significant differences in clinical outcome ..50].L. major model by decreasing parasite burden and delaying lesion progression. When early and late treatment initiation has been compared, more notable results were obtained for the group where treatment was started after 3 days of onset of infection. Common adverse effect reported was irritation. These studies showed a greater potential in BPQ and its pro-drugs to be used in future topical treatments of CL [Buparvaquone (BPQ) is hydroxyl naphthoquinone studied for having antiprotozoal activity since long . Its antts of CL  in additts of CL ,57.L. major species; it showed poor penetration of the drug with least improvement of the lesion size and parasite load along with intense skin irritations after 5 days of treatment [L. mexicana and L. major CL when treated with topical 6% MT [L. (Viannia) braziliensis and L. (Viannia) panamensis. These discrepancy in results might be due to variation in causating species, treatment duration, and formulation components [Miltefosine (MT), also known as Hexadecylphosphocholine, is a phospholipid derivative having potent anticancer and in vitro leishmanicidal activity. It is approved for oral administration in breast cancer and VL ,59; howereatment . Contraral 6% MT . Similarmponents .L. major species [Najafian and colleagues have recently reported that nanoliposomal MT has had greater therapeutic outcome as compared to conventional formulation when studied in vitro in dermal macrophages of BALB/c mice infected with Iranian strain of  species . Similar species . Both thL. major when morphine ointment was applied topically as compared to the placebo used [Morphine is an alkaloid acting as an immunomodulatory agent. It has been found to be able to improve the healing of CL wounds  when appebo used . This efL. major [L. infantum [L. major [Artemether, a free radical producing antiparasitic drug, also showed promising antileishmanial effects against various leishmania species, i.e., L. major ,69 and Linfantum  both in L. major . This maL. tropica-infected BALB/c mice. The SSG-NDL gel was reported to greatly enhance the effect of topical formulation via significantly reducing the lesion size (4-fold increase in activity) and also reducing the IC50 value from 1.65 mg/ml of plain SSG ointment to 1.3 mg/ml of SSG-NDL gel. This report is also an example of how novel DDS can greatly enhance the therapeutic outcome of the conventionally used drugs [Recently, SSG has been loaded a nanodeformable liposomal (NDL) DDS for topical application among ed drugs .L. maxicana. However, it needs further investigation for effective regimens to be used in the topical treatment of CL [Azole antifungals are used for a wide range of fungal infections including yeast infections of various mucocutaneous surfaces like mouth, throat, esophagus, and genital organs. Additionally, azole antifungals have also shown antileishmanial properties in combination with pentavalent antimonial drugs. Researchers investigated 1% fluconazole cream twice daily for approximately 6 weeks in combination with once weekly IL glucantime. It was found that fluconazole cream potentiated the effect of glucantime (64% CR) and can be used in treating CL lesion . Anothernt of CL .leishmania species in different regions has different therapeutic outcome. Topical treatment combined with parenteral administration has shown higher therapeutic efficacy. Several new and old drug candidates with modified dosage forms are in pipeline which will increase topical therapeutic options for treatment of CL. In the future, pharmacodynamic and pharmacokinetic analysis of drugs and new dosage forms used in the topical treatment of CL will be discussed in detail.In developing and third world countries, CL is still endemic because of limited number of drugs available to patients. Moreover, most of these treatment regimens require parenteral administration which poses serious discomfort for patients, poor patient compliance, higher cost of therapy, and lower therapeutic outcome among patients. Various topical treatments are being studied since long to overcome these problems since it is more convenient dosage forms for CL that provide high concentration of drug at the site of infection. This review has highlighted that variation in the Treatment of both types of cutaneous leishmaniasis has greatly been advanced from parenteral to topical treatment options with significant therapeutic outcomes.Leishmania species. Variation in therapeutic outcome has been reported for different species in different regions.Several combination treatments comprising of topical and parenteral modalities are developed and studied in different regions reporting different There are many findings which showed that the modifications in the drug delivery systems from conventional to novel dosage forms can greatly enhance the therapeutic outcome of many topically used drugs; it therefore emphasizes to do more future research on improving the dosage forms to get optimum therapeutic outcome and to counter the resistance toward these agents.Salah AB, Buffet PA, Morizot G, Massoud NB, Zâatour A, Alaya NB, et al. WR279, 396, a third generation aminoglycoside ointment for the treatment of Leishmania major cutaneous leishmaniasis: a phase 2, randomized, double blind, placebo controlled study. 2009;3(5):e432.Ben Salah A, Ben Messaoud N, Guedri E, Zaatour A, Ben Alaya N, Bettaieb J, et al. Topical paromomycin with or without gentamicin for cutaneous leishmaniasis. N Engl J Med. 2013;368(6):524–32.Arevalo I, Tulliano G, Quispe A, Spaeth G, Matlashewski G, Llanos-Cuentas A, et al. Role of imiquimod and parenteral meglumine antimoniate in the initial treatment of cutaneous leishmaniasis. 2007;44(12):1549–54.Layegh P, Rajabi O, Jafari MR, Emamgholi Tabar Malekshah P, Moghiman T, Ashraf H, et al. Efficacy of Topical Liposomal Amphotericin B versus Intralesional Meglumine Antimoniate (Glucantime) in the Treatment of Cutaneous Leishmaniasis. J Parasitol Res. 2011:656523.Carneiro G, Aguiar MG, Fernandes AP, Ferreira LAM. Drug delivery systems for the topical treatment of cutaneous leishmaniasis. Expert Opin Drug Deliv. 2012;9;1083–1097.