The chromosome-level genome of double-petal phenotype jasmine provides insights into the biosynthesis of floral scent

Jasmine(Jasminum sambac Aiton)is a well-known cultivated plant species for its fragrant flowers used in the perfume industry and cosmetics.However,the genetic basis of its floral scent is largely unknown.In this study,using PacBio,Illumina,10×Genomics and highthroughput chromosome conformation capture(Hi-C)sequencing technologies,a high-quality chromosome-level reference genome for J.sambac was obtained,exploiting a double-petal phenotype cultivar‘Shuangbanmoli’(JSSB).The results showed that the final assembled genome of JSSB is 580.33 Mb in size(contig N50=1.05 Mb;scaffold N50=45.07 Mb)with a total of 39618 predicted protein-coding genes.Our analyses revealed that the JSSB genome has undergone an ancient whole-genome duplication(WGD)event at 91.68 million years ago(Mya).It was estimated that J.sambac diverged from the lineage leading to Olea europaea and Osmanthus fragrans about 28.8 Mya.On the basis of a combination of genomic,transcriptomic and metabolomic analyses,a range of floral scent volatiles and genes were identified involved in the benzenoid/phenylpropanoid and terpenoid biosynthesis pathways.The results provide new insights into the molecular mechanism of its fragrance biosynthesis in jasmine.

High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometric Analysis of Bilobalide and Ginkgolides in Ginkgo biloba L. Leaves

The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Ginkgo biloba L. by high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) was carried out. The separation was performed on Inertsil ODS3 column with methanol-water (36:64) as mobile phase, with 1 mL·min -1 of flow rate at 35℃. Then the mass spectrum analysis was conducted by ZMD micromass electrospray ionization (ESI)-mass spectrometer (MS). The HPLC total ion chromatogram and selected ion chromatogram (with 325, 407, 423, 439 of m/z) of the sample and ESI-/MS mass spectra of the peaks in the chromatograms were obtained. So bilobalide, ginkgolide A, B, C and J in Ginkgo biloba L. leaves were identified. The method is easy and rapid, with a good accuracy.

Natural products:Perspectives in the pharmacological treatment of gastrointestinal anisakiasis

Objective: To evaluate the activity of different natural products against L3 larvae of Anisakis type I. Methods: Information on investigations into the activity of natural products against the L3 larvae of Anisakis simplex was collected from scientific journals, books, theses and reports via a library and electronic search(using Pubmed, Scopus, Medline, Web of Science and Science Direct). The search terms included: natural products, medicinal plants, essential oils, terpenic derivatives, Anisakis, antinematodal activity. Results: In the literature reviewed numerous papers were found concerning the in vitro and in vivo activity against Anisakis type I of various natural products(plant extracts, essential oils and their major components). Analysis of the results showed that in vitro tests the Melaleuca alternifolia essential oil and the ar-turmerone isolated from Curcuma longa displayed the greatest activity. In vivo, the most active compound was perillaldehyde, the main component of Perilla frutescens essential oil. Conclusions: This study shows that some natural products exhibited promising antianisakis properties.

The use of environmentally sustainable bio-derived solvents in solvent extraction applications——A review

Replacement of volatile organic compounds （VOCs） by greener or more environmentally sustainable solvents is becoming increasingly important due to the increasing health and environmental concerns as well as economic pressures associated with VOCs. Solvents that are derived from biomass, namely bio-derived solvents, are a type of green solvent that have attracted intensive investigations in recent years because of their advantages over con- ventional VOCs, such as low toxicity, biodegradability and renewability. This review aims to summarize the use of bio-derived solvents in solvent extraction applications, with special emphasis given to utilization of biodiesels and terpenes. Compared with the conventional VOCs, the overall performance of these bio-derived solvents is comparable in terms of extraction yields and selectivity for natural product extraction and no difference was found for metal extraction. To date most researchers have focused on laboratory scale thermodynamics studies. Future work is required to develop and test new bio-derived solvents and understand the kinetic performance as well as solvent extraction nilnt nlant studies.

Analyzing Method on Biogenic Volatile Organic Compounds

In order to analyze biogenic volatile organic compounds in the atmosphere, an automated gas chromatography is developed and employed at the laboratory of National Center for Atmospheric Research (NCAR) during January to July, 2000. A small refrigerator was used so as to remove water in the air sample from gas line, and get accurate concentrations of volatile organic compounds. At 5°C, good water removing efficiency can be obtained at controlled flow rate. Air samples were collected around the building of Mesa Lab. of NCAR and analyzed by this gas chromatography system. This paper reports this gas chromatography system and results of air samples. The experimental results show that this gas chromatography system has a good reproducibility and stability, and main interesting volatile organic compounds such as isoprene, monoterpenes have an evident diurnal variation.

A systematic summary of natural compounds in Radix Glycyrrhizae

Objective： To accumulate data from studies on the compounds identified from Gancao （Radix Glycyrrhizae, GC）, andthen systematically summarize and classify these compounds according to their structural characteristics. Methods： Fivedatabases （CNKI, VIP, Wanfang data, CBM, and Pubmed） were used to search for studies on the chemical structure ofcompounds from GC. The retrieval time of the respective databases was from their inception to March 2016. Accordingto the inclusion and exclusion criteria, the papers were carefully screened, and the data were extracted. Results： A totalof 653 compounds from GC were collated from 252 articles, including flavonols （n = 201） belonging to 10 subgroups,terpenes and saponins （n = 167）, coumarins （n = 30） belonging to 3 subgroups, aliphatics （n = 206）, aromatics （n = 35）,and others （n = 14）. The flavones, represented by liquiritin and liquiritigenin, were the most reported compounds isolatedfrom GC, followed by terpenes and saponins such as glycyrrhizic acid and glycyrrhetinic acid. Conclusion： The morethan 600 natural compounds in GC may be responsible for GC’s anti-inflammatory, phlegm-expelling, cough- andpain-relieving activities.

Role of essential oils in food safety: Antimicrobial and antioxidant applications

Essential oils(EOs)are more complex and comprise a number of volatile and natural bioactive compounds,which often used in food industries as the best alternatives.This review focuses on the impact of EOs and the roles of their major components in food manufacturing as natural preservatives with the related mechanisms of action.In addition,the major bioactive molecules of different types of EOs and their pharmacological activities such as antioxidant,antifungal and antimicrobial effects on crop protection were also discussed.The major compounds of EOs represent potential antioxidant,antimicrobial and antifungal activities through various mechanisms.Different types of EOs such as tea tree oil,lemon oil,clove oil,cinnamon oil and thyme oil from various traditional plants,have significantly showed better antimicrobial and antioxidant activities,and also effectively increased the shelf lives of the cereal products and increased the quality of food safety.The major groups of EOs such as terpenes and aromatic volatile compounds,play a key role in food safety without affecting the quality.Due to their various activities including antioxidant and antimicrobial activities,EOs could be used as alternative preservatives to increase the shelf lives of cereals and crops.

Analyses of BioVOCs variation related to vegetation predominance in the Natural Park of Ampezzo Dolomites, UNESCO world heritage area of Dolomites

The determination of emitted Biogenic Volatile Compounds （BioVOCs） profile via Field Air Analysis （FAA） - Solid Phase Micro Extraction （75μm Carboxen/PDMS portable field sampler） was performed using GC-MS. The six sampling sites located in the Natural Park of Ampezzo Dolomites, part of the UNESCO＇s World Heritage List, are characterized by different predominating vegetation. The ubiquitous compound hexanal was chosen as internal standard, in order to compare the amounts of the different compound in the different sites. The main terpenes identified were α-pinene, 13-pinene, 6-3-carene and d-limonene, while 1,8 cineole was the highest abundant oxygenated terpene. In the site next the＇national road a higher amount of benzene derivatives （ben- zene, toluene, o-xylene, p-xylene and ethylbenzene） were observed. In the site relatively close to the road a marked decrease of benzene deriva- tives was observed. In the sites close to conifers （i.e. Fir, Pine and Larch） the terpenic content observed was higher than in the site close to the mixed forest （mainly large-leaf and some conifers）. Some terpenes were observed also in the high-quote meadow site, although of lower abun- dance than in the other sites. The highest abundance of terpenes was ob- served in the Pinus cerebra （Swiss Pine） site.

Naturally Occurring Terpenes:A Promising Class of Organic Molecules to Address Influenza Pandemics

Since the olden times,infectious diseases have largely affected human existence.The newly emerged infections are excessively caused by viruses that are largely associated with mammal reservoirs.The casualties of these emergencies are significantly influenced by the way human beings interact with the reservoirs,especially the animal ones.In our review we will consider the evolutionary and the ecological scales of such infections and their consequences on the public health,with a focus on the pathogenic influenza A virus.The nutraceutical properties of fungal and plant terpene-like molecules will be linked to their ability to lessen the symptoms of viral infections and shed light on their potential use in the development of new drugs.New challenging methods in antiviral discovery will also be discussed in this review.The authors believe that pharmacognosy is the“wave of future pharmaceuticals”,as it can be continually produced and scaled up under ecofriendly requirements.Further diagnostic methods and strategies however are required to standardise those naturally occurring resources.

Evaluation of Terpenes’Degradation Rates by Rumen Fluid of Adapted and Non-adapted Animals

The aim of the present study was to evaluate terpenes degradation rate in the rumen fluid from adapted and non-adapted animals.Four castrated healthy animals,two rams and two bucks,were used.Animals were daily orally dosed for 2 weeks with 1 g of each of the following terpenes,α-pinene,limonene andβ-caryophyllene.At the end of each week,rumen fluid(RF)samples were assayed in vitro for their potential to degrade terpenes over time.For each animal,a 10 mL reaction medium(RM)at a ratio 1:9(v/v)was prepared and a terpenes solution at a concentration of 100μg/ml each,was added in each RM tube.Tubes were incubated at 39℃ under anaerobic conditions and their contents sampled at 0,2,4,8,21 and 24 h.RF could degrade terpenes as it was shown by the significantly(P<0.05)higher overall degradation rates.Individual terpene degradation rates,were significantly(P<0.05)higher in week 5 for limonene and marginally(P=0.083)higher also in week 5 forα-pinene.In conclusion,the findings of the present preliminary study suggest that terpenes can be degraded in the rumen fluid.

A likely autotetraploidization event shaped the Chinese mahogany(Toona sinensis)genome

Chinese mahogany(Toona sinensis) is of considerable medical and economic importance, and its genome has been deciphered. However, the process underlying its polyploidy is unclear, and the chromosomal evolutionary trajectory is poorly understood. Here, by reanalysing the T.sinensis genome, we found evidence of a tetraploidization event(T. sinensis special tetraploidization, TST) that occurred approximately 15-17 million years ago(MYA) after the core eudicot-common hexaploidization(ECH or gamma) event. We characterized the synonymous nucleotide substitution rates(Ks values) of collinear genes and found that T. sinensis genes affected by the TST evolve at a slower rate than Acer yangbiense genes. Furthermore, we identified homologous genes related to polyploidization and speciation and constructed multiple alignments with different reference genomes. Notably, the significant balance of gene retention and loss characterized in the two TST-derived subgenomes suggests an autopolyploid nature of the TST. Moreover, we deduced the chromosomal karyotypes of the two subgenomes and identified 7chromosomal fusions that have shaped the T. sinensis genome;more information is available on a newly constructed karyotype platform(http://www.cgrpoee.top/Toona_sinensis/index.html). The T. sinensis genome preserves the ancestral chromosome structure of dicotyledons well and could serve as a good reference for understanding genomic changes in other Meliaceae and Sapindales plants. In addition, we verified that tandem duplication and the ECH have promoted the expansion of terpene synthase(TPS) genes;conversely, the TST seems to have inhibited expansion of these genes. This present effort has clarified the polyploidy events of the T. sinensis genome, filled gaps in the history of karyotype evolution, and laid a solid foundation for further genomic studies in the Meliaceae research community and beyond.

Functional characterization of(E)-β-caryophyllene synthase from lima bean and its up-regulation by spider mites and alamethicin

（E）-β-Caryophyllene is a sesquiterpene compound widely distributed in plants and functions in plant defence. However, little is known about the sequence and function of （E）-β-caryophyllene synthase in lima bean （Phaseolus lunatus）. Here, we report a new full-length cDNA （PICAHS） encoding （E）-β-caryophyllene synthase, a possible key enzyme of plant defence. The cDNA of PICAHS contains an open reading frame of 1 761 bp, encoding a protein of 586 amino acids with a predicted mass of 67.95 kDa. The deduced amino acid sequence shows 52% identity with sesquiterpene synthase MtCAHS of Med- icago truncatula. Based on phylogenetic analysis, PICAHS is classified as the terpene synthases （TPS）-a subfamily. The recombinant enzyme, expressed in Escherichia coil, catalysed the formation of a major product （E）-β-caryophyllene （82%） and a minor product a-humulene （18%） from farnesyl dJphosphate. Real-time quantitative PCR （qRT-PCR） analysis found that the PICAHS transcript was significantly up-regulated in leaves after treatment with spider mites and alamethicin （ALA）, suggesting its ecological function in plant defence.

Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium‘Siberia’

Terpenoids are the main components contributing to the fragrance of Lilium‘Siberia’,and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids.Although the major terpene synthases in Lilium‘Siberia’have been identified,how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower.This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium,and disclose the related underlying transcriptional regulation mechanism.In this study,we identified three R2R3-MYB TFs—LiMYB1,LiMYB305 and LiMYB330,which were involved in regulating the biosynthesis of terpenes in Lilium‘Siberia’.Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds.When LiMYB1,LiMYB305 and LiMYB330were overexpressed in flowers,the release of some main monoterpenes,such as linalool and ocimene,as well as the expression of TPS genes,especially for LiTPS2,were enhanced.A virus-induced gene silencing(VIGS)assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes.The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2.Moreover,the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330,indicating their synergistic roles in the regulation of floral terpene biosynthesis.In general,these results indicated that LiMYB1,LiMYB305,and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.

The chromosome-scale genome assembly of Jasminum sambac var. unifoliatum provides insights into the formation of floral fragrance

Jasmine [Jasminum sambac(L.) Ait.], a tropical and subtropical plant emits a sweet, heady fragrance during flower opening. However, the molecular mechanisms underlying this phenomenon remain largely unknown. In the present study, integrated Illumina sequencing, Pacbio sequencing, and high-throughput chromatin conformation capture(Hi-C) scaffolding was used to generate a 495.60 Mb genome assembly of J.sambac var. unifoliatum cultivar ‘Fuzhou Single-petal’(JSU-FSP), with contig N50 of 16.88 Mb;96.23% of the assembly was assigned to 13 pseudochromosomes. The genome harbors 30 989 protein-coding genes, and 49.47% of the assembled sequences are repetitive sequences. The analysis of duplication modes showed that 51% of genes were duplicated through dispersed duplication, and expanded gene families are mainly involved in photosynthesis, which may be responsible for the light-loving characteristic specific to jasmine. Transcriptome analysis revealed that at least 35 structural genes involved in the biosynthesis of volatile terpenes(VTs), volatile phenylpropanoid/benzenoids(VPBs),fatty acid-derived volatiles(FADVs), and indole were highly expressed in the flower-opening stage, both preharvest and postharvest, and are proposed to be important in endowing flower aroma. Additionally, at least 28 heat shock protein(HSP) and 11 β-glucosidase(BGLU) genes may be involved in the formation of floral fragrance. These findings provide insights into the formation of the floral fragrance of jasmine and will promote germplasm utilization for breeding improved jasmine varieties.

Molecular Regulation of the Metabolic Pathways of the Medicinal Plants: <i>Phyla dulcis</i>

Phyla (Lippia) dulcis contains hernundulcin sesquiterpene zero-caloric sweetener that is about a thousand times sweeter than sucrose, and also bitter constituents including camphor and limonene. There is yet no simple method to remove the undesirable constituents. The yield of sweetener hernundulcin is very low, and there is no simple method to maximize its composition. The aim of the project was to characterize the mRNA targets that regulate the primary and terpenoid metabolic enzymes of P. dulcis. Restriction fragment differential display polymerase chain reaction of P. dulcis glutamate dehydrogenase-synthesized RNA showed that many mRNAs encoding β-caryophyllene, (+)-epi-α-bisabolol, bicyclogermacrene, bifunctional sesquiterpene, and geraniol synthases shared sequence homologies with ribulose-1,5-bisphophatase carboxylase, granule-bound starch synthase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and phosphoenol pyruvate carboxylase. Sequence similarities between mRNAs encoding primary metabolic enzymes and terpene synthases suggested that photosynthesis could regulate terpenoid metabolism in order to increase the yield of sweetener hernundulcin.

Plant Senescence: The Role of Volatile Terpene Compounds (VTCs)

Senescence is a natural, energy-dependent, physiological, developmental and an ecological process that is controlled by the plant’s own genetic program, allowing maximum recovery of nutrients from older organs for the survival of the plant, as such;it is classified as essential component of the growth and development of plants. In some cases, under one or many environmental stresses, senescence is triggered in plants. Despite many studies in the area, less consideration has been given to plant secondary metabolites, especially the role of VTCs on plant senescence. This review seeks to capture the biosynthesis and signal transduction of VTCs, the physiology of VTCs in plant development and how that is linked to some phytohormones to induce senescence. Much progress has been made in the elucidation of metabolic pathways leading to the biosynthesis of VTCs. In addition to the classical cytosolic mevalonic acid (MVA) pathway from acetyl-CoA, the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, originating from glyceraldehyde-3-phosphate (GAP) and pyruvate, leads to the biosynthesis of isoprenoid precursors, isopentenyl diphosphate and dimethyl allyl diphosphate. VTCs synthesis and emission are believed to be tightly regulated by photosynthetic carbon supply into MEP pathway. Thus, under abiotic stresses such as drought, high salinity, high and low temperature, and low CO2 that directly affect stomatal conductance and ultimately biochemical limitation to photosynthesis, there has been observed induction of VTC synthesis and emissions, reflecting the elicitation of MEP pathway. This reveals the possibility of important function(s) of VTCs in plant defense against stress by mobilizing resources from components of plants and therefore, senescence. Our current understanding of the relationship between environmental responses and senescence mostly comes from the study of senescence response to phytohormones such as abscisic acid, jasmonic acid, ethylene and salicylic acid, which are extensively involved in response to various abiotic and biotic stresses. These stresses affect synthesis and/or signaling pathways of phytohormones to eventually trigger expression of stress-responsive genes, which in turn appears to affect leaf senescence. Comparison of plant response to stresses in relation to patterns of VTCs and phytohormones biosynthesis indicates a considerable crosstalk between these metabolic processes and their signal to plant senescence.

Frankincense derived heavy terpene cocktail boosting breast cancer cell(MDA-MB-231) death in vitro

Objective: To investigate the anti-cancer effect of frankincense derived heavy oil obtained by Soxhlet extraction method on breast cancer cells(MDA-MB-231), and to study its chemical profile using gas chromatography mass spectrometry analysis.Methods: Hexane was used to extract heavy oil from frankincense resin. Chemical profiling of heavy oil was done using Perkin Elmer Clarus GC system with mass spectrometer. MDA-MB-231 cells were treated with different dilutions(1:1 000, 1:1 500,1:1 750, 1:2 000, 1:2 250, 1:2 500, 1:2 750, 1:3 000, 1:3 250) of heavy oil for 24 h. The cells were observed by using light microscopy. Cell viability was measured by MTT assay.Results: Gas chromatography mass spectrometry chemical profiling of frankincense derived heavy oil revealed the presence of terpenes such as a-pinene(61.56%), a-amyrin(20.6%), b-amyrin(8.1%), b-phellandrene(1.47%) and camphene(1.04%). Heavy terpene cocktail induced significant MDA-MB-231 cell death at each concentration tested. Noticeably, very low concentration of Soxhlet derived heavy terpenes elicits considerable cytotoxicity on MDA-MB-231 cells compared to hydro distillated essential oil derived from frankincense resin.Conclusions: Extracting anti-cancer active principle cocktail by simple Soxhlet method is cost effective and less time consuming. Our in vitro anti-cancer data forms the rationale for us to test heavy terpene complex in breast cancer xenograft model in vivo. Furthermore, fractionation and developing frankincense heavy terpene based breast cancer drug is the major goal of our laboratory.

Simple Method for Simultaneous Quantitation and Fingerprint Analysis of Ginkgo biloba Tablets by High Performance Liquid Chromatography-UV-Evaporative Light Scattering Detector

By optimizing the separation and analytical conditions, a reliable, simple and accurate high-performance liquid chromatography（HPLC） method coupled with ultraviolet（UV） and evaporative light scattering detector（ELSD） was developed for the simultaneous determination of lactones and flavonoid, that is, bilobalide, ginkgolide A, ginkgolide B, and rutin, in more than 10 batches of Ginkgo biloba tablets from different pharmaceutical companies. The method could also be applied to fingerprint research, for a more general evaluation of the quality of this preparation. The separation of the components was achieved on a Hanbon C18 column with gradient elution using water and methanol containing 0.1% trifluoroacetic acid（TFA）. The column temperature was 30 ℃ and the flow-rate of the mobile phase was 0.6 mL/min. The drift tube temperature of the ELSD was set at 100 ℃, and the nitrogen flow-rate was 2 L/min. Good linear relationships were shown with correlation coefficients for analytes exceeding 0.9913. The limits of detection（LODs, S/N=3） and the limits of quantitation（LOQs, S/N=10） were 0.00887-0.0508 μg/μL and 0.0171- 0.0636 μg/μL, respectively, on the column. The relative standard deviations（RSD） of the analytes were less than 3.61% for intraday and 3.70% for interday, respectively. The average recovery rates obtained were in the range of （97.3±4.3）% to （101.9±3.1）% for all the compounds. The results of quantitative and fingerprint analysis proved that the contents of the components were totally similar in the preparation of Ginkgo biloba tablets from the same pharmaceutical company; whereas, they varied significantly in the preparations of Ginkgo biloba tablets from different companies.

Fractions of a chloroform extract of ajenjo leaves (Artemisiamendozana DC. var. mendozana) inhibit the proliferation,viability and clonogenicity of B16-F0 melanoma cells

The ajenjo,Artemisia mendozana DC.var.mendozana(Asteraceae),grows in the Andean foothills of Mendoza and San Juan,Argentina,and is used as a medicinal plant for its antispasmodic and antifungal properties.The aim of this work was to obtain fractions of a chloroform extract of ajenjo leaves and to evaluate the in vitro effects on proliferation,viability and clonogenicity of B16-F0 melanoma cells.Using a silica gel chromatography column,120 fractions were collected and grouped according to the chromatographic profile in 9 main fractions(F1–F9).Their major compounds identified were:terpenes(F1),terpenes and sesquiterpene lactones(F2–F3),sesquiterpenes(F4–F6)and phenols and sesquiterpenes(F7-9).B16-F0 cells were incubated for 72 h with DMSO(vehicle)or 0.1 mg/ml F1–F9.At 72 h of culture,F1 decreased both the growing index(GI)and cell viability.F2 and F3 both decreased GI and only F3 decreased clonogenic activity.F4 and F5 both decreased GI.Only F5 decreased cell viability and F4 decreased clonogenicity.Consequently,fractions F6–F8 did not affect any of the cell parameters assayed,while F9 decreased cell viability and inhibited clonogenicity.

Effects of combined cannabidiol(CBD)and hops(Humulus lupulus)terpene extract treatment on RAW 264.7 macrophage viability and inflammatory markers

This study investigates the potential of cannabidiol(CBD),one major cannabinoid of the plant Cannabis sativa,alone and in combination with a terpene-enriched extract from Humulus lupulus(“Hops 1”),on the LPS-response of RAW 264.7 macrophages as an established in vitro model of inflammation.With the present study,we could support earlier findings of the anti-inflammatory potential of CBD,which showed a dose-dependent[0-5μM]reduction in nitric oxide and tumor necrosis factor-alpha(TNF-α)released by LPS-stimulated RAW 264.7 macrophages.Moreover,we observed an additive anti-inflammatory effect after combined CBD[5μM]and hops extract[40μg/mL]treatment.The combination of CBD and Hops 1 showed effects in LPS-stimulated RAW 264.7 cells superior to the single sub-stance treatments and akin to the control hydrocortisone.Furthermore,cellular CBD uptake increased dose-depend-ently in the presence of terpenes from Hops 1 extract.The anti-inflammatory effect of CBD and its cellular uptake positively correlated with terpene concentration,as indicated by comparison with a hemp extract containing both CBD and terpenes.These findings may contribute to the postulations for the so-called“entourage effect”between cannabinoids and terpenes and support the potential of CBD combined with phytomolecules from a non-cannabi-noid source,such as hops,for the treatment of inflammatory diseases.