Zanthoxylum Species: A Comprehensive Review of Traditional Uses, Phytochemistry, Pharmacological and Nutraceutical Applications.

Zanthoxylum species (Syn. Fagara species) of the Rutaceae family are widely used in many countries as food and in trado-medicinal practice due to their wide geographical distribution and medicinal properties. Peer reviewed journal articles and ethnobotanical records that reported the traditional knowledge, phytoconstituents, biological activities and toxicological profiles of Z. species with a focus on metabolic and neuronal health were reviewed. It was observed that many of the plant species are used as food ingredients and in treating inflammation, pain, hypertension and brain diseases. Over 500 compounds have been isolated from Z. species, and the biological activities of both the plant extracts and their phytoconstituents, including their mechanisms of action, are discussed. The phytochemicals responsible for the biological activities of some of the species are yet to be identified. Similarly, biological activities of some isolated compounds remain unknown. Taken together, the Z. species extracts and compounds possess promising biological activities and should be further explored as potential sources of new nutraceuticals and drugs.

Development of conventional PCR and real-time PCR assays to discriminate the origins of Chinese pepper oil and herbal materials from Zanthoxylum.

BACKGROUND: To ensure the safety, quality and therapeutic efficacy of processed foods and herbal medicines, it is important to identify and discriminate economically motivated adulterants. Zanthoxylum schinifolium is sold at a higher price than other Zanthoxylum species and is frequently adulterated with closely related Zanthoxylum species because of its high demand as a Korean food ingredient and medicinal material in markets. In addition, the pericarps of three Zanthoxylum species (Z. schinifolium, Z. bungeanum and Z. piperitum) are defined as herbal medicine Zanthoxyli Pericarpium in Korean pharmacopoeias, but not Z. piperitum in Chinese pharmacopoeias. Further confusion arises in the morphological similarity between Z. armatum (adulterant) and Z. bungeanum. Therefore, the aim of this study was to develop a sequence characterized amplified region (SCAR) marker for discrimination of four Zanthoxylum species. RESULTS: With the goal of developing rapid and reliable tools for genetic discrimination of authentic Zanthoxyli Pericarpium, we designed species-specific SCAR markers, based on ITS2 sequences, that generate amplicons of less than 200 bp. Using these markers, we established both conventional and real-time PCR assay methods capable of differentiating samples at the species level. We validated the ability of SCAR markers to authenticate edible oil and herbal medicine, and confirmed that some herbal medicines contaminated with Z. armatum are being distributed as Zanthoxyli Pericarpium in Korean and Chinese markets. CONCLUSIONS: The SCAR markers and PCR methods described represent powerful tools for protecting against adulteration and ensuring standardization of processed foods and herbal medicine. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Phylogeny and biogeography of the pantropical genus Zanthoxylum and its closest relatives in the proto-Rutaceae group (Rutaceae).

Zanthoxylum L. (prickly ash) is the only genus in the Citrus L. family (Rutaceae) with a pantropical distribution. We present the first detailed phylogenetic and biogeographic study of the genus and its close relatives in the proto-Rutaceae group. Our phylogenetic analyses based on two plastid and two nuclear markers show that the genus Toddalia Juss. is nested within Zanthoxylum, that earlier generic and intrageneric classifications need revision, and that the homochlamydeous flowers of the temperate species of Zanthoxylum are the result of a reduction from heterochlamydeous flowers. The biogeographic analyses reveal a Eurasian origin of Zanthoxylum in the Paleocene or Eocene with successive intercontinental or long-range migrations. Zanthoxylum likely crossed the North Atlantic Land Bridges to colonize the Americas in the Eocene, and migrated back to the Old World probably via the Bering Land Bridge in the Oligocene or Miocene. Zanthoxylum also colonized several Pacific Islands and the Hawaiian clade shows phylogenetic incongruence between the plastid and nuclear datasets, suggesting hybridization. The Hawaiian species are one of the rare examples of endemic Hawaiian lineages that are older than the current main islands.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge.

Rapid Determination of the Geographical Origin of Chinese Red Peppers (Zanthoxylum Bungeanum Maxim.) Based on Sensory Characteristics and Chemometric Techniques.

In this paper, principal component analysis (PCA), linear discriminant analysis (LDAp, artificial neural networks (ANN), and support vector machine (SVM) were applied to discriminate the geographical origin of Chinese red peppers (Zanthoxylum bungeanum Maxim.). The models based on color, smell and taste may discriminate quickly and effectively the geographical origin of Chinese red peppers from different regions, but the successful identification rates may vary with different kinds of parameters and chemometric methods. Among them, all models based on taste indexes showed an excellent ability to discriminate the geographical origin of Chinese red peppers with correct classifications of 100% for the training set and the 100% for test set. The present study provided a simple, efficient, inexpensive, practical and fast method to discriminate the geographical origin of Chinese red peppers from different regions, which was of great importance for both consumers and producers.

Comparative study on alkaloids and their anti-proliferative activities from three Zanthoxylum species.

BACKGROUND: Alkaloids have been considered as the most promising bioactive ingredients in plant species from the genus Zanthoxylum. This study reports on the compositions and contents of the Zanthoxylum alkaloids (ZAs) from three Zanthoxylum species, and their potential anti-proliferation activities. METHODS: An HPLC-UV/ESI-MS/MS method was established and employed to analyze the alkaloids in different Zanthoxylum extracts. The common and unique peaks and their relative contents were summarized and compared to evaluate the similarity and dissimilarity of the three Zanthoxylum species. Meanwhile, inhibitory activity tests to four carcinoma cell lines, i.e., stomach tumor cells (SGC-7901), cervical tumor cells (Hela), colon tumor cells (HT-29) and Hepatic tumor cells (Hep G2), were carried out in vitro to evaluate the bioactivities of the ZAs. RESULTS: Seventy peaks were detected in the crude total alkaloid samples, and 58 of them were identified. As a result, 13 common peaks were found in the extracts of all the three Zanthoxylum species, while some unique peaks were also observed in specific species, with 17 peaks in Z. simulans, 15 peaks in Z. ailanthoides and 11 peaks in Z. chalybeum, respectively. The comparison of the composition and relative contents indicated that alkaloids of benzophenanthridine type commonly present in all the three Zanthoxylum species with high relative contents among the others, which are 60.52% in Z. ailanthoides, 30.52% in Z. simulans and 13.84% in Z. chalybeum, respectively. In terms of activity test, Most of the crude alkaloids extracts showed remarkable inhibitory activities against various tumor cells, and the inhibitory rates ranged from 60.71 to 93.63% at a concentration of 200 µg/mL. However, SGC-7901 cells seemed to be more sensitive to the ZAs than the other three cancer cells. CONCLUSION: The alkaloid profiles detected in this work revealed significant differences in both structures and contents among Zanthoxylum species. The inhibitory rates for different cancer cells in this study indicated that the potential anti-cancer activity should be attributed to quaternary alkaloids in these three species, which will provide great guidance for further exploring this traditional medicinal resource as new healthcare products.

[Comparative study of HPLC fingerprint between Zanthoxylum bungeanum and Zanthoxylum schinifolium].

OBJECTIVE: To establish the HPLC fingerprint of Zanthoxylum bungeanum and Zanthoxylum schinifolium for finding the difference. METHODS: Samples were extracted with 50% methanol 25 mL by ultrasonic wave and then separated on Hypersil BDS C18 (250 mm x 4.6 mm, 5 microm) column. Gradient elution was carried out with a mobile phase of methanol-water. The detection wavelength was 268 nm, the column temperature was set at 35 degrees C, the flow rate was 1.0 mL/min and the analytic time was 130 min. The software "Similarity Evaluation System for Chromatographic Fingerprint of TCMs" (Version 2004A) was employed to generate the mean chromatogram and carry out the similarity analysis of the samples. SPSS 17.0 was employed to carry out the cluster analysis. RESULTS: Similarity of Z. bungeanum was 0.909 - 0.992 and that of Z. schinifolium was 0.930 - 0.999. There were 27 common peaks in HPLC Fingerprints of Z. bungeanum and 24 in that of Z. schinifolium. Their HPLC standard fingerprints were obvious difference. They belonged to different categories in cluster analysis. CONCLUSION: The method is simple, accurate and rapid. It could obviously distinguish Z. bungeanum from Z. schinifolium. So it suggests that HPLC fingerprints should be one of the quality control indexes of Zanthoxyli Pericarpium.

[Application of two FTIR pattern recognition methods to the Zanthoxylum nitidum geographical origins determination].

In the present work, the authors explored a rapid method of the Zanthoxylum nitidum geographical origins determination. Based on Fourier transform infrared spectroscopy (FTIR) technology, the band of 1 800-400 cm(-1) which is the IR fingerprint of Zanthoxylum nitidum, the Fisher ratio and the soft independent modeling of class analogies (SIMCA) were used to build a classification model. Respectively, four kinds of Zanthoxylum nitidum in the Guangxi region were detected by the model, and the model was verified by calculating their recognition rate and rejection rate. The results show that the authors can accurately extract the overall information of Chinese herbal medicines by using the FTIR, also established a pattern recognition model to predict unknown samples, and obtained satisfactory recognition rate and rejection rate, indicating that the model has stronger ability of identification. The detection on real time was carried out rapidly with the Fisher model, suggesting that the model has more practical value.

Elucidation of Japanese pepper (Zanthoxylum piperitum De Candolle) domestication using RAD-Seq.

Japanese pepper, Zanthoxylum piperitum, is native to Japan and has four well-known lineages (Asakura, Takahara, Budou, and Arima), which are named after their production area or morphology. Restriction-site associated DNA sequencing (RAD-Seq) was used to analyse 93 accessions from various areas, including these four lineages. Single nucleotide variant analysis was used to classify the plants into eight groups: the Asakura and Arima lineages each had two groups, the Takahara and Budou lineages each had one group, and two additional groups were present. In one Asakura group and two Arima groups, the plants were present in agricultural fields and mountains, thus representing the early stage of domestication of the Japanese pepper. The second Asakura lineage group was closely related to plants present in various areas, and this represents the second stage of domestication of this plant because, after early domestication, genetically related lineages with desirable traits spread to the periphery. These results demonstrate that domestication of Japanese pepper is ongoing. In addition, this study shows that spineless plants are polyphyletic, despite the spineless lineage being considered a subspecies of Japanese pepper.

Quality evaluation of different varieties of Zanthoxylum bungeanum Maxim. peels based on phenolic profiles, bioactivity, and HPLC fingerprint.

This work aims to evaluate the quality of seven varieties of Zanthoxylum bungeanum peels based on phenolic compounds, bioactivity, and HPLC fingerprint combined with chemometrics analysis, and pick out the key chemical compounds. As a result, S2 (Fengxian Dahongpao) showed the highest content of total phenolic and rutin, as well as the strongest antioxidant activity, followed by S6 (Hancheng Shizitou). From HPLC fingerprint, eight common characteristic peaks were selected and proved to be effective in controlling the quality of Z. bungeanum peels with the total contribution of 92.775%. The similarity values of each sample ranged between 0.696 and 0.970. In addition, hyperoside was identified as another key chemical indicator for quality evaluation of Z. bungeanum peels by PCA. Accordingly, seven varieties of Z. bungeanum were classified into four groups, among which group 4 (Fengxian Dahongpao, S2) was considered as an outstanding variety, followed by group 1 (S5, S6, and S7) and group 3 (S3 and S4), and group 2 (Fugu late-maturing, S1) was the last one. These results will be helpful to establish an effective and comprehensive evaluation system of Z. bungeanum. PRACTICAL APPLICATION: This study not only provides another index for quality evaluation of Z. bungeanum peels, but it also expects to be a theoretical basis for high-quality germplasm cultivation and the development of Z. bungeanum peels as food additives, pharmaceutical, or health care products.

Genome survey of Zanthoxylum bungeanum and development of genomic-SSR markers in congeneric species.

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum.

Identification of different species of Zanthoxyli Pericarpium based on convolution neural network.

Zanthoxyli Pericarpium (ZP) are the dried ripe peel of Zanthoxylum schinifolium Sieb. et Zucc (ZC) or Zanthoxylum bungeanum Maxim (ZB). It has wide range of uses both medicine and food, and favorable market value. The diverse specifications of components of ZP is exceptional, and the common aims of adulteration for economic profit is conducted. In this work, a novel method for the identification different species of ZP is proposed using convolutional neural networks (CNNs). The data used for the experiment is 5 classes obtained from camera and mobile phones. Firstly, the data considering 2 categories are trained to detect the labels by YOLO. Then, the multiple deep learning including VGG, ResNet, Inception v4, and DenseNet are introduced to identify the different species of ZP (HZB, DZB, OZB, ZA and JZC). In order to assess the performance of CNNs, compared with two traditional identification models including Support Vector Machines (SVM) and Back Propagation (BP). The experimental results demonstrate that the CNN model have a better performance to identify different species of ZP and the highest identification accuracy is 99.35%. The present study is proved to be a useful strategy for the discrimination of different traditional Chinese medicines (TCMs).

Comparative Transcriptome Analysis and Expression of Genes Reveal the Biosynthesis and Accumulation Patterns of Key Flavonoids in Different Varieties of Zanthoxylum bungeanum Leaves.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. However, the transcriptional sequencing has not been investigated, and the molecular basis for the flavonoid biosynthesis remains unclear in this plant. This paper, the key flavonoids (epicatechin, rutin, hyperoside, trifolin, quercitrin, and afzelin) contents were determined in the leaves of 10 Z. bungeanum varieties from a common garden. Results show the leaves of Z. bungeanum mainly contained hyperoside (11.410-21.721 mg/g) and quercitrin (9.401-18.016 mg/g). The total content of these key components was the highest in Fengxian Dahongpao (66.012 mg/g) and the lowest in Fugu (32.223 mg/g). Three varieties (Hancheng stingless, Fugu, and Fengxian Dahongpao) with significant differences in the total content of key flavonoids were selected for transcriptome analysis to obtain flavonoid biosynthesis-related genes. In total, 83 522 unigenes were obtained, 40 668 (48.69%) unigenes were annotated, and 6656 differentially expressed genes (DEGs) were identified. Comparison of the other two varieties, Fugu had many differentially expressed genes indicating the particularity of its variety. Flavonoid-related DEGs of 22 structural genes, including three PALs, one CYP73A, three 4CLs, six CHSs, one CHI, one F3H, one DFR, two ANSs, one ANR, one FLS, and two CYP75B1s, as well as nine MYBs were obtained. These structural genes had different expression patterns in different Z. bungeanum varieties. It is worth noting that the genes expressing the flavonoid 3'5' hydroxylase are absent in Z. bungeanum. Furthermore, quantitative real-time PCR experiment showed consistent results in transcriptome analysis. The RNA-Seq data set of this study sheds lights on the molecular mechanism of flavonoid biosynthesis in Z. bungeanum, provides valuable information for the metabolic regulation of flavonoids, and may serve as a guide for future breeding programs.

Authentication of Zanthoxylum Species Based on Integrated Analysis of Complete Chloroplast Genome Sequences and Metabolite Profiles.

We performed chloroplast genome sequencing and comparative analysis of two Rutaceae species, Zanthoxylum schinifolium (Korean pepper tree) and Z. piperitum (Japanese pepper tree), which are medicinal and culinary crops in Asia. We identified more than 837 single nucleotide polymorphisms and 103 insertions/deletions (InDels) based on a comparison of the two chloroplast genomes and developed seven DNA markers derived from five tandem repeats and two InDel variations that discriminated between Korean Zanthoxylum species. Metabolite profile analysis pointed to three metabolic groups, one with Korean Z. piperitum samples, one with Korean Z. schinifolium samples, and the last containing all the tested Chinese Zanthoxylum species samples, which are considered to be Z. bungeanum based on our results. Two markers were capable of distinguishing among these three groups. The chloroplast genome sequences identified in this study represent a valuable genomics resource for exploring diversity in Rutaceae, and the molecular markers will be useful for authenticating dried Zanthoxylum berries in the marketplace.

De novo transcriptome assembly of Zanthoxylum bungeanum using Illumina sequencing for evolutionary analysis and simple sequence repeat marker development.

Zanthoxylum, an ancient economic crop in Asia, has a satisfying aromatic taste and immense medicinal values. A lack of genomic information and genetic markers has limited the evolutionary analysis and genetic improvement of Zanthoxylum species and their close relatives. To better understand the evolution, domestication, and divergence of Zanthoxylum, we present a de novo transcriptome analysis of an elite cultivar of Z. bungeanum using Illumina sequencing; we then developed simple sequence repeat markers for identification of Zanthoxylum. In total, we predicted 45,057 unigenes and 22,212 protein coding sequences, approximately 90% of which showed significant similarities to known proteins in databases. Phylogenetic analysis indicated that Zanthoxylum is relatively recent and estimated to have diverged from Citrus ca. 36.5-37.7 million years ago. We also detected a whole-genome duplication event in Zanthoxylum that occurred 14 million years ago. We found no protein coding sequences that were significantly under positive selection by Ka/Ks. Simple sequence repeat analysis divided 31 Zanthoxylum cultivars and landraces into three major groups. This Zanthoxylum reference transcriptome provides crucial information for the evolutionary study of the Zanthoxylum genus and the Rutaceae family, and facilitates the establishment of more effective Zanthoxylum breeding programs.

[Authentication of Zanthexylum bungeanum Maxin population and adulterants by analysis of rDNA ITS sequences].

AIM: To study the difference of rDNA ITS sequences between Zanthexylum bungeanum populations and their adulterants in main habitants of China so as to provide molecular markers for identifying Zanthexylum bungeanum populations against adulterants. METHODS: rDNA ITS regions (including ITS-1, 5.8S and ITS-2) of 7 populations of Zanthexylum bungeanum which are separate located in Gansu, Shanxi, Sichuan, Hebei provinces, and 3 adulterants were sequenced by PCR products sequencing method or clone sequencing method. RESULTS: The sequences of rDNA ITS region of Zanthexylum bungeanum were reported for the first time, and the sequences of ITS region ranged from 619 to 620 bp, and the length difference amoung Zanthexylum bungeanum and their adulterants is 4 bp. There are 15 variable sites, 12 informative sites and 3 authenticable sites among Zanthexylum bungeanum populations. The difference of rDNA ITS regions amoung Zanthexylum bungeanum and their adulterants is obvious, the number of variable sites is 71. CONCLUSION: The difference of rDNA ITS sequences can be used to authenticate accurately the populations of Zanthexylum bungeanum and their adulterants. These populations of Z. bungeanum which have close relationship always distribute in near geographic areas. The characteristics of rDNA ITS sequence can be used as good markers for authenticating Zanthexylum bungeanum populations form their adulterants.

[Study of chemical constituents of the essential oil form Pericarpium Zanthoxyli by GC-MS].

The chemical components of the essential oil from Pericarpium Zanthoxyli collected from Jinan city of Shandong province and Hengshui city of Henan province were analyzed by GC-MS. 36 components from Pericarpium Zanthoxyli in Jinan city of Shandong province that composed of about 87% of the total essential oil were separated and identified. 20 components from Pericarpium Zanthoxyli in Hengshui city of Henan province that composed of about 74% of total essential oil were separated and identified.

Searching for novel cancer chemopreventive plants and their products: the genus Zanthoxylum.

The genus Zanthoxylum (Rutaceae) comprises about 250 species, of which many are used as food, often as condiments, substituting pepper due to the pungent taste of fruits, seeds, leaves, and bark, and therapeutic remedies especially in Eastern Asian countries and in Central America. The whole plant is also consumed as an ingredient of soups and salads. The aim of this review is to examine in detail from a phytochemical and pharmacological point of view what is reported in the current literature about the anti-cancer and chemopreventive properties of phytopreparations or individual active compounds obtained from edible plants belonging to this genus.

Leaf essential oil composition of five Zanthoxylum species from Monteverde, Costa Rica.

The leaf essential oils from five species of Zanthoxylum (Rutaceae) from Monteverde, Costa Rica, have been obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The species examined include Z. fagara, Z. acuminatum, Z. melanostictum, Z. monophyllum, and an undescribed species. The most abundant classes of compounds found in Zanthoxylum leaf oils are acyclic and menthane monoterpenoids as well as simple alcohols, aldehydes, and ketones. In terms of molecular diversity, menthane and acyclic monoterpenoids, cadinane and mesocyclic sesquiterpenoids, and simple alcohols, aldehydes, and ketones dominate the essential oils of Zanthoxylum species. Monoterpenoids make up the majority of the mass of the leaf oils of Z. monophyllum, Z. acuminatum, Z. fagara, and Zanthoxylum sp. nov. Linalool, 4-terpineol, alpha-terpineol, and trans-2-hexenol, are found in all of the Zanthoxylum species examined in this study.