To combat the resistance, synergist piperonyl butoxide (PBO) incorporated LLINs is one of the available options. In the current phase II hut trial, the efficacy of Veeralin®LN was compared with MAGNet®LN and untreated net in reducing the entry, induced exit, mortality and blood feeding inhibition of target vector species.The performance of VeeralinAn. culicifacies.The performance of Veeralin was equal to MAGNet in terms of reducing hut entry, inhibiting blood feeding and inducing exophily, and with regard to causing mortality Veeralin was better than MAGNet. When compared to untreated net, a significant reduction in hut entry and blood feeding and an increase in exophily and mortality were observed with Veeralin. In cone bioassays, unwashed Veeralin caused > 80% mortality of Veeralin performed equal to  or better than  MAGNet and could be an effective tool against pyrethroid resistant malaria vectors. Controlling the malaria vectors with indoor residual spraying (IRS) of insecticides and/or long-lasting insecticidal nets (LLINs) is the major approach used for preventing malaria transmission . Recent Anopheles in experimental hut trials [®LN conducted in a setting (an area) where the malaria vector, An. culicifacies was resistant to synthetic pyrethroids [An. culicifacies in terms of preventing hut entry, inducing exit, inhibiting blood-feeding and causing mortality in a tribal district of Odisha State, India.Current interest of the manufacturers is to focus on developing innovative control strategies that can combat pyrethroid resistance , 11. Fort trials . As thert trials , 14 pre-t trials . Two PBOt trials . The curethroids . The perethroids   and cooler months (November–December). The two vector species that transmit malaria in this area were Anopheles fluviatilis and An. culicifacies. Anopheles fluviatilis was highly susceptible to synthetic pyrethroids whereas, An. culicifacies was found to be resistant [The current Phase II trial was carried out in experimental huts located at the village, Kandhaguda of Malkangiri District (17° 45′ N to 18° 40′ N and 81° 10′ E to 82° 00′ E) in Odisha State. The major human malaria parasite species prevailing in this area was esistant  with mod®LN, manufactured by VKA Polymers, Tamil Nadu, India, the pyrethroid insecticide, alpha-cypermethrin was integrated into monofilament polyethylene fibres of 130 denier at a target dose of 6.0 g AI/kg, equivalent to 216 mg AI/m2 of insecticide, and 2.2 g/kg of PBO, equivalent to 79.2 mg/m2. In the current trial, six arms viz., Veeralin unwashed and washed 20 times, MAGNet unwashed and 20 times washed (positive control), untreated net (negative control) and 25 times washed Veeralin®LN were compared. All the trial nets (size 180 × 160 × 150 cms) were sponsored by VKA Polymers. Each arm had six replicate nets and every night different replicate nets of the same arm were tested in each hut for each week. Chemical analysis and cone bioassays before any wash and after 20 or 25 washes were carried out on additional nets.In the test LLIN, VeeralinAnopheles stephensi mosquitoes were used as An. culicifacies, the target test mosquito species was not available at the rearing facility of Indian Council of Medical Research (ICMR)—Vector Control Research Centre (VCRC), Puducherry, where washing of nets was carried out. Bioassays were also carried out at the field site i.e., after the evaluation in the experimental huts, exposing wild caught pyrethroid resistant An. culicifacies and pyrethroid susceptible Anopheles jeyporiensis on one net selected randomly from the six replicate nets of each arm used in the experimental huts. On each net, bioassays were performed on all the five panels (one roof and four side panels). Two cone tests were carried out on each panel, releasing five mosquitoes per cone test. Thus, a total of 50 mosquitoes each of the susceptible and resistant species were tested per net. The mosquitoes were exposed for 3 min and after the exposure, they were held for 24 h at temperature 27 ± 2 °C and humidity 75 ± 10%. During the holding time, the mosquitoes were provided with sugar solution for feeding. The mortality was recorded after 24 h.Nets were washed according to the WHO washing protocol . Cone biTo determine the alpha-cypermethrin and PBO content in the treatment arms, chemical analysis of LLIN samples was done, prior to and after washing 20 or 25 times and after hut evaluation. Prior to wash, 5 pieces, one (30 × 30 cm) from each of the 5 positions, were cut and removed from one of the three additional nets of each arm. For chemical analysis, net samples were also obtained from 20 times washed  and 25 times washed net , and similarly from unwashed arms. After experimental hut study, from each arm, one of the six replicate nets used in the huts was randomly chosen for the analysis. The samples, after packing in aluminium foil, were labelled and stored at + 4 °C and sent to the WHO Collaborating Centre, Gembloux, Belgium, for analysing the active ingredient content of alpha-cypermethrin and PBO.For sleeping in experimental huts, 12 adult volunteers, two (preferably husband and wife) for each hut, were selected from the village through organizing a meeting.To engage human participants in the hut evaluation, clearance was obtained from the ICMR-VCRC Human Ethics Committee. Prior written consent was obtained from the volunteers for their participation. The volunteers were told to sleep under the net using the bed materials that were allotted to them from 19.00 to 05.30 h with a small interval for dinner and not to make any fire or smoke inside the hut.Before starting the evaluation, in each of the six replicate nets of all the arms, a total of six holes (each one: 4 cm × 4 cm) were made to give an effect of a torn net. Six teams, each with two sleepers, were formed. The nets and the sleepers were rotated as per the Latin square design to reduce the differences due to attractiveness of huts and volunteers. One treatment arm was tested for one week in each hut with one replicate net each day. The sleepers were rotated among the huts daily, whereas, the arms were rotated weekly. The collection and processing of mosquitoes have been described elsewhere .An. culicifacies in each arm was recorded. As the variance was higher than the mean; negative binomial regression analysis  was done by taking the number collected as dependent variable and the experimental arms as independent variable. The number of An. culicifacies exited, blood fed and dead after 24 h  day-wise and arm-wise were analysed using logistic regression (using SPSS 16.0) by taking mosquitoes exited, fed and dead  as dependent variables and arms as categorical covariates. For overall comparison, the negative control (untreated net) was kept as reference category. Further comparisons between the treated arms were made by removing negative control from the grouped data and holding the positive control (unwashed MAGNet) as reference category. The mean alpha-cypermethrin and PBO content in the net samples prior to any wash, after washing and after hut evaluation were compared using one-way ANOVA followed by a rank (LSD) test.The number of mosquitoes entered, exited, dead inside the hut and the number that succeeded to feed on volunteers were calculated for each arm by compiling the data collected over 12 weeks. The day-wise entry of An. culicifacies formed 69.4%, An. fluviatilis 2.3%, and Anopheles subpictus and Anopheles vagus (non-vectors) together formed 19.5%, and 8.8% was culicines. As the trial was carried out at the end of the rainy season, the number of An. fluviatilis collected was very small. Therefore, further analysis was restricted to An. culicifacies, which was resistant to alpha-cypermethrin.In total, 72 collections, completing two rotations (six weeks to complete one rotation) across all treatments, were done for each of the six arms in the experimental huts. A total of 650 mosquitoes were collected. Among them, An. culicifacies that entered the huts (entry) with unwashed Veeralin, 20 times washed Veeralin, unwashed MAGNet, 20 times washed MAGNet, untreated nets and Veeralin washed 25 times was 0.47 ± 0.11, 0.56 ± 0.08, 0.51 ± 0.10, 0.46 ± 0.08, 3.61 ± 0.28, and 0.65 ± 0.11, respectively from 72 collections during 12 weeks  than the untreated one. It was noted from the incidence rate ratio (IRR) that, among the treated arms, the hut entry was the minimum with MAGNet washed 20 times followed by unwashed Veeralin. However, no significant difference was seen in the entry between the five treated arms indicating a similar deterrent effect of all the treated arms  followed by unwashed Veeralin (52.9%). However, the 95% CI for the odds ratios showed no significant difference in the exit rate between the treated arms .The five treated arms induced significantly a higher exophily (p < 0.05) than the untreated arm. The exit rate of ms Table . By taki2 = 89.435, df = 5, p < 0.05). Compared to untreated arm, all treated arms recorded a significantly reduced feeding rate. Between the treatment arms, no significant difference was found, as shown by 95% CI for the Odds ratios. Personal protection determines the level of protection offered by each treatment arm. Among the five treatment arms, the personal protective effect was the maximum with MAGNet washed 20 times (92.3%) followed by unwashed MAGNet (91.5%), Veeralin washed 20 times (91.1%), unwashed Veeralin (90.7%) and Veeralin washed 25 times (88.2%).The lowest blood feeding (i.e. the highest feeding inhibition) was observed with Veeralin washed 20 times (55.0%) followed by unwashed MAGNet (56.8%) Table. Overall2 = 18.979, df = 4, p = 0.001) , the maximum being with unwashed Veeralin 94.1%)  content of alpha-cypermethrin in the net samples of the three Veeralin arms viz., unwashed Veeralin, Veeralin washed 20 times and 25 times was 5.61 ± 0.12 (SD), 5.61 ± 0.08 and 5.63 ± 0.06 g/kg, and the nets complied with the target dose of 6.0 g/kg ± 25% (4.5 g/kg-7.5 g/kg). The alpha-cypermethrin content after 20 and 25 washes was 5.44 ± 0.07 g/kg and 5.49 ± 0.11 g/kg, corresponding to an overall alpha-cypermethrin retention of 97% and 98%, respectively. After hut evaluation, the alpha-cypermethrin content was 5.59 ± 0.06, 5.52 ± 0.05 and 5.44 ± 0.12 g/kg in Veeralin unwashed and washed 20 times and 25 times, respectively. After washing (20 or 25 times), there was a decline in alpha-cypermethrin content (p < 0.05 by One-way ANOVA) compared to the base-line concentration, but, within the specifications of ± 25%. No further diminishing of active ingredient content was observed after the hut trial (p > 0.05).In the two MAGNet arms, unwashed and 20 times washed, the initial alpha-cypermethrin content was 5.82 ± 0.05 and 5.80 ± 0.05 g/kg and that met comply with the target dose of 5.8 g/kg ± 25% (4.4–7.3 g/kg). Compared to the initial level, the active ingredient concentration reduced to 5.46 ± g/kg after 20 washes (p < 0.05), but within the accepted margin of ± 25%. The corresponding active ingredient retention was 94%. After the hut evaluation, alpha-cypermethrin content did not decrease significantly (p > 0.05), as it was 5.59 ± 0.08 for MAGNet unwashed and 5.36 ± 0.13 g/kg for MAGNet washed 20 times.The mean base-line PBO content in the three Veeralin arms  were within the acceptable range of the target dose of 2.2 g/kg ± 25% (1.7–2.8 g/kg). After 20 and 25 washes, there was a decrease in PBO content to 2.01 ± 0.02 g/kg and 2.01 ± 0.06 g/kg (p < 0.05 by one-way ANOVA); however, the content was within the specification of ± 25%. The overall retention of PBO content after washing was 82% and 83%, respectively. After hut trial, while no further decline in PBO content (2.02 ± 0.03 g/kg) was noticed in Veeralin washed 20 times (p > 0.05), there was a decrease in 25 times washed Veeralin (1.94 ± 0.04 g/kg)(p < 0.05). In unwashed Veeralin, after hut trial, PBO content declined to 2.31 ± 0.03 g/kg compared to the base-line (p < 0.05). However, in all cases, the PBO content was within the acceptable range of ± 25% (1.7–2.8 g/kg).An. culicifacies to alpha-cypermethrin + PBO nets in comparison to only alpha-cypermethrin nets in reducing the entry and blood-feeding and in inducing exophily and mortality in experimental huts. There was no entry of An. culicifacies into the huts on many occasions when the volunteers slept under the treated nets (of the five treatment arms), signifying a higher deterrent effect of the pyrethroid insecticide, alpha-cypermethrin used in the treated nets. In spite of the holes made, the deterrent effect was also realized from the 10 alive An. culicifacies collected inside the untreated nets whereas, only one alive and one dead were found under the nets of the five treated arms. Thus, all the treated arms significantly reduced the entry of An. culicifacies in to the huts compared to the untreated arm. Further, the washed treatment arms did not differ significantly from the unwashed with regard to hut entry indicating no impact of washing of Veeralin LN/MAGNet LN on their deterrent effect.The current study assessed the response of An. culicifacies (25%) was also noticed from the huts with untreated net (negative control), which might be due to its innate behaviour. An experimental hut study conducted recently in the same area showed that the exit rate (induced exophily) of An. culicifacies in the untreated arm was 23.9% [Considerable exit of as 23.9% . HoweverAmong the treated arms, Veeralin washed 20 times (41.9%) effected the maximum inhibition of blood feeding followed by MAGNet unwashed (40.0%). The feeding rate was significantly lower in all the treatment arms than the untreated arm. When compared to the positive control, the blood feeding inhibition in the treatment arms did not differ significantly.An. culicifacies in the five treated arms varied from 55.3 to 94.1%, the maximum was by unwashed Veeralin and that was much higher than the mortality caused by the positive control. Further, the cone-bioassay results showed that only unwashed Veeralin caused > 80% mortality of An. culicifacies (pyrethroid resistant) at the end of hut evaluation. However, the enhanced mortality produced by alpha-cypermethrin + PBO LLINs was not sustained after 20 washes. Similar results were obtained in experimental hut trials in Benin, Burkina Faso and Cameroon, where deltamethrin-PBO LLINs produced significantly a higher mortality of An. gambiae sensu lato (s.l.) (pyrethroid resistant) compared to non-PBOLLINs, with higher mortality observed with unwashed LLINs [The total mortality of ed LLINs , 13.An. culicifacies after hut evaluation showed that unwashed Veeralin met the efficacy criteria of > 80% mortality [An. stephensi and An. jeyporiensis) were exposed to all the five treatment arms, 100% mortality was observed. It is known that against susceptible mosquito populations, pyrethroid-PBO nets may not have any effect [An. culicifacies in experimental huts. The chemical analysis though, indicated a loss of insecticide and PBO after 20 or 25 washes, their content, even after the loss, remained within the accepted range of ± 25%. Therefore, the marked increase in mortality noticed with Veeralin LN was probably due to the addition of the synergist, PBO, which is known to inhibit the activity of the enzymes that are responsible detoxification of pyrethroids [An. culicifacies with Veeralin in experimental huts and this could be due to the presence of different resistance mechanisms that are not affected by the synergist, PBO [The cone-bioassays conducted during the current trial with pyrethroid resistant ortality . When pyy effect , but comy effect . Overallethroids . Neverthist, PBO .Phase II LLIN evaluations mainly focus on two entomological parameters, blood feeding inhibition and mortality, which are crucial for obtaining interim recommendation/pre-qualification . In the An. culicifacies.Experimental hut studies conducted in West and Central Africa, Tanzania and Togo with high pyrethroid resistance showed that pyrethroid-PBO nets performed better than standard-LLINs , 10, 25,In the current study, Veeralin LN performed equal to  or better than  the reference LN (MAGNet) and fulfilled the WHO-PQ criteria of Phase II evaluation of LLINs. In view of increasing development of pyrethroid resistance in malaria vectors, PBO incorporated Veeralin LN could be an option to be considered for malaria control after fully qualified as a vector control product by the WHO.


Erigeron breviscapus is an important medicinal plant in Compositae and the first species to realize the whole process from the decoding of the draft genome sequence to scutellarin biosynthesis in yeast. However, the previous low‐quality genome assembly has hindered the optimization of candidate genes involved in scutellarin synthesis and the development of molecular‐assisted breeding based on the genome. Here, the E. breviscapus genome was updated using PacBio RSII sequencing data and Hi‐C data, and increased in size from 1.2 Gb to 1.43 Gb, with a scaffold N50 of 156.82 Mb and contig N50 of 140.95 kb, and a total of 43,514 protein‐coding genes were obtained and oriented onto nine pseudo‐chromosomes, thus becoming the third plant species assembled to chromosome level after sunflower and lettuce in Compositae. Fourteen genes with evidence for positive selection were identified and found to be related to leaf morphology, flowering and secondary metabolism. The number of genes in some gene families involved in flavonoid biosynthesis in E. breviscapus have been significantly expanded. In particular, additional candidate genes involved in scutellarin biosynthesis, such as flavonoid‐7‐O‐glucuronosyltransferase genes (F7GATs) were identified using updated genome. In addition, three candidate genes encoding indole‐3‐pyruvate monooxygenase YUCCA2 (YUC2), serine carboxypeptidase‐like 18 (SCPL18), and F‐box protein (FBP), respectively, were identified to be probably related to leaf development and flowering by resequencing 99 individuals. These results provided a substantial genetic basis for improving agronomic and quality traits of E. breviscapus, and provided a platform for improving other draft genome assemblies to chromosome‐level. Erigeron breviscapus is a medicinal plant with great potential in Compositae. Scutellarin, a major active component of flavonoids abundant in its leaves, is widely used as a prescription drug in treating cardiovascular and cerebrovascular diseases  currently prepared from E. breviscapus extracts  by genomic analysis and synthetic biology , globe artichoke (Cynara cardunculus var. scolymus), safflower (Carthamus tinctorius L.), fleabanes (E. breviscapus), lettuce (Lactuca sativa), sunflower (Helianthus annuus L.), sweet wormwood (A. annua), chrysanthemum (C. nankingense and C. seticuspe), cup plant (Silphium perfoliatum) and milk thistle . Among them, only sunflower and lettuce in Compositae have reached the chromosome level  and candidate genes controlling late flowering and number of leaves, re‐captured candidate genes involved in major flavonoids biosynthesis, and established a stable genetic transformation system. The results will provid valuable technical support and genetic resources for genetic improvement and optimization of scutellarin biosynthesis in E. breviscapus in the future, and also provide a template for improvement of other draft genomes to chromosome‐level.In this study, we used the third generation of genome sequencing technology to update the genome assembly of 22.1E. breviscapus genomic sequencing was provided by Longjin Pharmaceutical Co., Ltd, and planted in the greenhouse at Yunnan Agricultural University. Total genomic DNA was extracted from fresh leaves of 60‐day‐old plants using the GenElute Plant Genomic DNA Miniprep Kit (Sigma‐Aldrich). 40 μg of sheared DNA was used to construct SMRT Cell libraries with an insert size of 17 kb. These libraries were sequenced in SMRT DNA sequencing cells with P6/C4 chemistry. Together with the long reads obtained from the previous study . Illumina reads were trimmed by software trimmomatic‐0.36 with default parameters and were used to correct base‐calling by proovread with the default parameters  integrated in HiC‐Pro , salicylic acid (SA) and gibberellin (GA) hormones on gene expression, 60‐day‐old plants were treated with 200 μmol/l ABA, SA and GA by foliar spray, and then leaves harvested at 4, 12, and 24 hr after treatment, respectively. Five to eight leaves from different plants were mixed and represent a biological repeat, and three biological replicates were performed. All collected samples were immediately frozen in liquid nitrogen and stored at −80°C until use. Total RNA was extracted using Qiagen RNeasy Plant Mini Kits. According to the manufacturer's instructions, total RNA‐seq libraries were prepared using TruSeq RNA Library Preparation Kit, v. 2 (Illumina), and were subsequently pair‐end sequenced with a read length of 150 bp on the HiSeq 4000 platform. In total, about 688.9 billion RNA‐seq reads were obtained, representing about 103.3 Gb of raw data. According to the following metrics: (a) to remove adaptors; (b) to trim 3′ end bases with a quality score below 20; and (c) to remove low‐quality reads with base Ns or more than 10% of bases with a quality score below 20, raw RNA‐seq reads were trimmed and filtered by Trimmomatic .The quality‐filtered short fragments from the Illumina platform were subjected to 2.5For repeat annotation, Tandem Repeat Finder  and were subjected to TBLASTN analysis to the E. breviscapu assembled genome with a cutoff E‐value of 1e−5  against several databases including the nr  was applied to identify the potential orthologous gene families among E. breviscapus, A. annua, C. cardunculus, H. annuus, L. sativa, A. thaliana, N. tabacum, O. sativa and V. vinifera. To construct the phylogenetic tree, MUSCLE  was provided for calibration and the program MCMCtree from the PAML package  libraries of V. vinifera, L. sativa, H. annuus, E. breviscapus, A. annua, A. thaliana, and O. sativa) were identified using the HMMER3 (http://hmmer.janelia.org/) software and the Pfam‐A data sets was downloaded from PFAM (http://pfam.janelia.org/). The seed file for CHI domain (PF02431) was obtained, and Pfam‐A data sets for six other types, namely PAL, CHS, 4Cl, F6H, FSII, and F7GAT were transferred from fasta sequences downloaded from NCBI to stockholm file by perl script “fasta2sto.pl”. The domain file was used as the first template to scan the gene family whereby the output genes were filtered out with an E‐value below 1e‐10. The filtered genes were used as second templates for a second round of scanning of the target gene families. Similarly, the second phase of output genes was filtered out with an E‐value of 1e‐10. The putative genes from the gene family were identified. Finally, the gene families of all seven species were filtered by blast with the downloaded sequence with cutoff: identity ≥ 70%.The specific gene families of seven species  and 50 with high leaf number (>120), were selected from the greenhouse of Longjin Pharmaceutical Co., Ltd. For phenotypic evaluation, three traits including branch number, leaf number and plant weight were counted. Young leaves collected from single individuals were immediately frozen in liquid nitrogen. Total DNA was extracted with the DNAsecure plant kit and at least 2 µg of genomic DNA for each sample was used to construct a sequencing library. 72‐ single‐end 100bp  and 27 paired‐end 150 bp  sequencing libraries with an insert size of approximately 400 bp were sequenced on BGI‐500 and Illumina HiSeq 4000 sequencer, respectively  was used to colour the phylogenetic tree. Principal component analysis (PCA) was performed with the Genome‐wide Complex Trait Analysis  software  phylogenetic tree with 100 bootstrap using SNPhylo  for three traits, respectively. GWAS was performed with a linear mixed model (LMM) in genome‐wide efficient mixed model association  software  was calculated by R package qqman  from Pfam  genes in the 2.12PAL, C4H, 4Cl, CHS, CHI, and FSⅡ were cloned from leaves of E. breviscapus. The target fragments and pCambia1301‐35SN were cut by the restriction enzymes as shown in Table Agrobacterium tumefaciens strain EHA105 by electroporation. E. breviscapus transgenic plants were generated as described previously  of 2.13E. breviscapus using the RNAprep pure Plant Kit (Tiangen) and reverse transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa). PCR amplification was performed in a Roche LightCycler 96 (Roche) using the SYBR Green qRT‐PCR Master Mix (TaKaRa) according to the manufacturer's instructions. The thermal profile for SYBR Green qRT‐PCR was 95°C for 2 min, followed by 40 cycles of 95°C for 20 s, 58°C for 20 s, and 72°C for 20 s. The actin gene was chosen as a reference gene to control for normalization. Samples for high‐performance liquid chromatography (HPLC) analysis were prepared as described previously . Total RNA was extracted from leaves of transgenic 33.117‐mer curves, the heterozygosity of E. breviscapus was approximately 2.04%  relative to synonymous substitution (Ks) within certain genes may account for the adaptive evolution of organisms at the molecular level . A total of 14 genes were present in both lists of genes under positive selection , cinnamate‐4‐hydroxylase (C4H), 4‐coumaroyl‐CoA‐ligase (4Cl), chalcone synthase (CHS), chalcone isomerase (CHI), and flavone synthase II (FS II) to form apigenin, which is the precursor for breviscapine biosynthesis  and hydroxycinnamoyl‐CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT)  belong to the UGT88 gene family  were clustered together into the UGT88X branch (evm.ugt.ctg3868.3 was the same with previously published genome), while another gene (evm.ugt.ctg3868.3) belongs to the UGT88F subfamily , 8,346,064 bp on chromosome 5  and 105,039,479 bp on chromosome 6 . While for leaf number and plant weight, the strongest peak were identified on 135,967,818 bp on chromosome 5  and 135,881,643 on chromosome 3 , respectively. By looking deep into genes close to those peaks, six candidate genes were identified, which were evm.model.ctg208.1, evm.model.ctg2378.1, evm.model.ctg2956.4, evm.model.ctg566.11, evm.model.ctg566.17, and evm.model.ctg2581.6 , serine carboxypeptidase‐like 18 (SCPL18), serine/threonine‐protein phosphatase (PP1), late embryogenesis abundant protein (LEA), and F‐box protein (FBP), respectively  for a long time, which shows morphological adaptation of rosette leaves and the accumulation of flavonoids in leaves. Previous studies found that the content of total flavonoids in E. breviscapus was positively correlated with altitude, and the correlation coefficient was 0.91999  Active ingredients have been decided, and the curative effects are clear. (b) There is an efficient transgenic system which provides a good technical platform for functional gene validation and gene editing. (c) De novo biosynthesis of scutellarin in engineered yeast has been accomplished (Liu et al., E. breviscapus such as luteolin, kaempferol, quercetin and hesperetin (Chu et al., E. breviscapus is an ideal model medicinal plant in traditional Chinese medicine research, and updated genome assembly and identified candidate genes are definitely helpful for further improvement and utilization of this medicinal herb.In conclusion, among 10,000 medicinal plants, S.Y., and Y.D. conceived the study. M.C., and M.Y. collected and grew the plant material. R.L., Y.Z., D.L., J.Y., and G.C. performed experiments. S.D., Y.L., W.F., and H.S. assembled the genome. X.D., S.H., G.Z., G.L., Y.Z., W.C., and Y.D. annotated and analysed the genome. S.H., and X.D. wrote the manuscript. All authors read and approved the final manuscript.Table of ContentsClick here for additional data file.Fig S1‐S8Click here for additional data file.Table S1‐S20Click here for additional data file.


Plasmodium vivax malaria is poorly understood. The objective of this study was to describe the PK/PD relationship of chloroquine and its major metabolite, desethylchloroquine, in a P. vivax volunteer infection study. We analyzed data from 24 healthy subjects who were inoculated with blood‐stage P. vivax malaria and administered a standard treatment course of chloroquine. The PK of chloroquine and desethylchloroquine was described by a two‐compartment model with first‐order absorption and elimination. The relationship between plasma and whole blood concentrations of chloroquine and P. vivax parasitemia was characterized by a PK/PD delayed response model, where the equilibration half‐lives were 32.7 hours  27.4–40.5) for plasma data and 24.1 hours (95% CI 19.0–32.7) for whole blood data. The estimated parasite multiplication rate was 17 folds per 48 hours (95% CI 14–20) and maximum parasite killing rate by chloroquine was 0.213 hour−1 (95% CI 0.196–0.230), translating to a parasite clearance half‐life of 4.5 hours (95% CI 4.1–5.0) and a parasite reduction ratio of 400 every 48 hours (95% CI 320–500). This is the first study that characterized the PK/PD relationship between chloroquine plasma and whole blood concentrations and P. vivax clearance using a semimechanistic population PK/PD modeling. This PK/PD model can be used to optimize dosing scenarios and to identify optimal dosing regimens for chloroquine where resistance to chloroquine is increasing.Chloroquine has been used for the treatment of malaria for > 70 years; however, chloroquine pharmacokinetic (PK) and pharmacodynamic (PD) profile in  WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?WHAT QUESTION DID THIS STUDY ADDRESS?☑ Chloroquine has been extensively used for > 70 years; however, the relationship between chloroquine pharmacokinetic (PK) and parasite killing has not been quantified using semimechanistic population PK/pharmacodynamic (PD) modeling.P. vivax killing effect in a volunteer infection study using an induced blood‐stage malaria model.WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?☑ This study investigated the PK/PD relationship between chloroquine plasma and whole blood concentrations and P. vivax parasites. This study demonstrates the importance of biological matrices in chloroquine PK monitoring and found a delayed effect of chloroquine on killing of P. vivax parasites.HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?☑ This is the first study characterizing the PK/PD relationship of chloroquine plasma and whole blood concentrations on clearance of in silico prediction tool for determining dosing strategies that reduce the development of resistance and ensure chloroquine therapeutic effect against P. vivax with decreased sensitivity to chloroquine.☑ The developed PK/PD model provides a valuable Plasmodium vivax.P. falciparum malaria in almost all regions of the world. However, chloroquine remains a valuable antimicrobial agent for the treatment of P. vivax malaria in most countries because of its low cost and well‐understood safety profile.Globally, 225 million cases of malaria have been reported with 7.5 million cases due to P. vivax emerged in Papua New Guinea in the late 1980s,P. vivax malaria is difficult due to lack of a continuous in vitro P. vivax culture system, and by the inability of molecular genotyping to discriminate between recrudescence , relapse, and new infections.Chloroquine resistance to P. vivax malaria have been characterized in only a few studies.P. vivax, would provide a valuable tool for clinical prediction of the efficacy of various dosing regimens, and for optimizing the current dosing regimen, particularly to combat rising resistance to chloroquine.Inadequate chloroquine dosing leads to subtherapeutic exposure and increased risk of treatment failure and resistance development. The pharmacokinetic (PK) and pharmacodynamic (PD) properties of chloroquine and its active metabolite, desethylchloroquine, in P. vivax induced blood‐stage malaria model.P. vivax malaria field studies.In this study, we investigated the PK/PD relationship of chloroquine and desethylchloroquine by analyzing data from a volunteer infection study (VIS) using the P. vivax‐infected erythrocytes administered intravenously on day 0. Chloroquine was administered as chloroquine phosphate tablet (Avloclor). Subjects received a total chloroquine dose of 1.55 g base for ≥ 60 kg adults or 25 mg base/kg for < 60 kg adults orally over 3 days on day 8, 9, or 10. The study was approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee and the Australian Defence Human Research Ethics Committee. All subjects provided written informed consent before participating.Study design, procedure, and main results were described in detail elsewhere.P. vivax counts were collected before inoculation, daily from day 4 until parasitemia was detected, twice‐daily thereafter until treatment day, before first dose on treatment day, and at 2, 4, 8, 12, 16, 24, 30, 36, 48, 60, 72, 96, 120, 168, 240, and 480 hours after the first dose.Samples for measuring chloroquine and desethylchloroquine concentrations in plasma and whole blood were collected before chloroquine administration and at 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, 168, 240, and 480 hours after first dose administration. Blood samples for P. vivax parasitemia was measured using quantitative real‐time polymerase chain reaction targeting the 18S rRNA gene.Chloroquine and desethylchloroquine concentrations were measured using liquid chromatography tandem mass spectrometry. The lower limit of quantification (LLOQ) was 1.35 µg/L for chloroquine plasma, 1.0 µg/L for whole blood chloroquine, 1.3 µg/L for plasma desethylchloroquine, and 1.75 µg/L for whole blood desethylchloroquine concentrations. P. vivax parasitemia measurements. Of these measurements, 8.9% of chloroquine plasma, 11.8% of desethylchloroquine plasma, 8.9% of chloroquine whole blood, and 10% of desethylchloroquine whole blood concentrations were below the LLOQ. Chloroquine and desethylchloroquine concentrations in erythrocytes were calculated and correlations between drug concentrations in whole blood and erythrocytes were determined  and 619 Nonlinear mixed effects modeling was performed using Monolix version 4.3.3 . For data programming, preprocessing and postprocessing of the modeling, and simulations MATLAB R2015b  and the MATLAB toolbox IQMtools version 1.2.2.2  was used. The graphic processing of the Monolix output for the final model was performed with R (version 3.5.1)FigureP. falciparum strains.P. vivax, desethylchloroquine PK data were incorporated into the PK model with the aim to be used in the PK/PD model. Data below the LLOQ were treated using the left‐censoring method, by maximizing the likelihood of the data being below LLOQ.Chloroquine and desethylchloroquine plasma and whole blood concentrations were converted into molar units using the molecular masses of 319.8 and 291.8 g/mol, respectively. The chloroquine phosphate doses were converted to the equivalent (base) chloroquine dose by multiplying chloroquine phosphate by 0.62 and converted into molar units. Correlations between chloroquine (Spearman’s rho = 0.99) and desethylchloroquine (Spearman’s rho = 0.98) whole blood and erythrocyte concentrations were high FigureS1. GivFrel was tested for the second, third, and fourth doses vs. the first dose. Additive, proportional, and combined error models were tested to explain random unexplained variability  and elimination  models were tested. Interindividual variability (IIV) was assumed to be log‐normally distributed. Correlations between PK parameters were evaluated by estimating the off‐diagonal components of the variance‐covariance matrix. Interoccasion variability related toP < 0.05) and backward‐deletion (P < 0.01) approach. Continuous covariates were modeled as power function and categorical covariates as linear function  is independent of whether plasma or whole blood concentrations were measured in describing the parasite killing effect, a commonEmaxCQ parameter was used to fit the two models simultaneously. To technically enable the simultaneous fit, the observed parasitemia levels were duplicated to link each set separately to the empirical Bayes estimates of the PK parameters for plasma or whole blood concentrations. We tested several PD models, including a direct effect (Emax), a turnover, and an effect compartment model , parasite reduction ratio over 48 hours (PRR48), parasite clearance half‐life (PCt1/2), and time above chloroquine plasma and whole blood concentration at half of maximum effect  were estimated using individual PD parameters obtained via empirical Bayes estimates . A VPC was performed by simulating 1,000 observations at each time point and 95% CI of the 5th, 50th, and 95th simulated percentiles were plotted against observations. In addition, successful convergence of the minimization routine, reasonable physiological plausibility, and precision of parameter estimates were also taken into account during the model development.EmaxCQ,EC50CQ plasma, andEC50CQ whole blood values  were included in the analysis.TableFigureAll subjects from the previously reported clinical trial .Individual empirical Bayesian estimates of PK parameters from plasma and whole blood data were used simultaneously to describe the drug concentration‐effect relationship. Parasite killing was described by a delayed effect of chloroquine concentration on the parasite. The equilibration half‐life for chloroquine in plasma was 32.7 hours (95% CI 27.4–40.5) and in whole blood 24.1 hours (95% CI 19.0–32.7), indicating that chloroquine concentration in the effect compartment takes some time to reach equilibrium with levels measured in plasma and whole blood. The PD parameters and their IIVs were estimated with a reasonable precision Table3. TheEC50CQ plasma,EC50CQ whole blood; transfer rate constant of chloroquine plasma, and whole blood concentration from and to the central compartment and an effect compartment . These parameters were difficult to estimate because none of the subjects had recrudescence. As a result, we fixedEC50CQ whole blood to 0.28 μmol/L, a value previously reported in the literature based on relapse data.EC50CQ plasma is six folds lower thanEC50CQ whole blood, based on the whole blood‐to‐plasma ratio of chloroquine concentrations in this study. The geometric mean of estimatedPMR48 was 17.4‐fold per 48 hours (95% C: 14.3–20.4). The arithmetic mean ofPRR48 was the same between plasma and whole blood , corresponding to a PRR of 400 every 48 hours (95% CI 320–500). The geometric mean of estimatedPCt1/2 was 4.5 hours (95% CI 4.1–5.0). The geometric mean of estimated time aboveEC50CQ plasma was 14.3 days (95% CI 13.5–15.1) and time aboveEC50CQ whole blood was 18.3 days (95% CI 17.0–19.6).Sensitivity analysis revealed that the estimates of some parameters changed substantially with different initial condition values:FigureEmax CQ for this subject is higher than the rest of subjects (0.365 hour−1 vs. others: 0.172–0.292 hour−1). The model predictive performance was confirmed with VPC plots  was required when using chloroquine plasma data, and of ≤ 146 µg/L (0.456 µmol/L) when using chloroquine whole blood data.Two scenarios of chloroquine resistance were considered. In the first scenario, simulations were performed by varying theP. vivax parasites using a semimechanistic population PK/PD modeling. The final population PK model for plasma and whole blood samples was a two‐compartment model for both chloroquine and desethylchloroquine, with first‐order absorption and elimination. A delayed effect model was used to describe the effect of chloroquine on the P. vivax kinetics with equilibration half‐life of at least 24 hour. Given chloroquine’s long half‐life, this delayed effect is not clinically relevant in uncomplicated malaria. However, a delayed effect is undesirable for property of drugs used for treating severe malaria or for antimalarials with short half‐lives.This is the first study characterizing the PK/PD relationship of chloroquine plasma and whole blood concentrations on clearance of Vc CQF) values reported in the published studies varied from 6.7–60.3 L/kg for chloroquine, and from 0.03–1.3 L/kg for desethylchloroquine . These values were comparable to the values estimated in this study . The apparent clearance of chloroquine(CLCQF) estimated in this study was slightly higher at 0.78 L/h/kg (95% CI 0.71–0.85 L/h/kg) L/h/kg than the published values . The apparent clearance of desethylchloroquine (CLDCQF) value from literature ranged between 0.03 and 0.65 L/h/kg, which is within the range of theCLDCQF value estimated in this study . This discrepancy may be attributable to different study populations, extensive PK sampling time points, differences in parasitemia levels, and use of concomitant antimalarial therapy. Published data on the population PK of hydroxychloroquine ) are limited, with two studies reporting a one‐compartment PK model, probably due to limited samplingThe structural PK model described in this study was similar to two published studies.The analysis presented in this report represents the first attempt to develop a PK model of chloroquine and desethylchloroquine using whole blood concentrations. PK parameter estimates of whole blood samples were generally lower than that of plasma samples. This is due to differences in the partitioning of chloroquine and desethylchloroquine between erythrocytes and plasma. In this study, chloroquine and desethylchloroquine whole blood concentrations were higher than in plasma, which is in accordance with the results of other studies.P. falciparum malaria in patients receiving oral artesunate therapy.P. vivax trophozoites and schizonts are insensitive to chloroquine.EC50 difficult. ChloroquineEC50 for whole blood was fixed to a value previously reported in the literature based on the assumption that the drug sensitivity of parasites at the time of recrudescence is similar to that calculated from relapse data.EC50CQ plasma was assumed to be six folds lower than theEC50CQ whole blood based on whole blood to plasma ratio of chloroquine concentrations. Despite these limitations, this is the first study to characterize PK/PD relationship of chloroquine plasma and whole blood concentrations on clearance of P. vivax parasites. This is also the first time that the parasite killing effect of chloroquine was investigated in a P. vivax induced blood stage VIS.The delay between appearance of chloroquine in plasma and whole blood and parasite killing was described using an effect compartment model. The utility of this delayed response model has been reported in modeling of EC50CQ biological matrix was used as a PK determinant of chloroquine therapeutic outcome. With a log10 PRR48 of 2.6 (95% CI 2.5–2.7), 10 days are required for chloroquine to clear parasites in a hypothetical patient with a baseline parasitemia of 10.EC50 for 14–18 days, this ensures that all asexual parasites are removed from circulation, thereby curing the blood‐stage infection. The estimated log10 PRR48 in this study is similar to the reported values of chloroquine‐sensitive isolates (range 2.6–3.6),10 PRR48 estimated for chloroquine is lower than artesunate (3.2),P. vivax malaria. Simulations showed that chloroquine efficacy was sensitive to changes inEmaxCQ. AnEmaxCQ reduction of 14% for plasma samples and 25% for whole blood samples resulted in treatment success rate of < 90%. In other reported in silico simulations, changes inEmaxCQ were also found to have the largest effect on treatment outcome.The time aboveP. vivax induced blood‐stage malaria model. This model improves our understanding of the concentration‐effect relationship of chloroquine in P. vivax malaria and can be applied to optimize chloroquine dosing regimens. The PK/PD model of chloroquine developed, which includes estimation of parasite growth from samples collected pretreatment, can be used within a simulation framework to explore a range of scenarios. These include varying dosing schemes for chloroquine resistant P. vivax infection by varying theEmax orEC50, the effect of incomplete adherence to the standard 3‐day dosing regimen on outcome, or varying the dosage schedule so as to minimize drug toxicity, which is primarily related to peak plasma concentration (Cmax) could be simulated. Another model applicability, particularly the PK model, is the prediction of chloroquine dosing regimens for treatment of COVID‐19. Simulations of various dosing schemes using the developed PK model to achieve exposure above in vitro inhibitionEC50 of 1.13–7.36 µMin vivoEC50 of 6.78–44.16 µM (based on whole blood‐to‐plasma ratio of 6:1 in this study), as well as prediction of chloroquine concentrations in the lungs . MMV is funded by a number of donors including USAID, Bill and Melinda Gates Foundation, UK Department for International Development, Norwegian Agency for Development Cooperation, Irish Aid, Newcrest Mining Limited, Australian Aid, Swiss Agency For Development and Co‐Operation, and Wellcome Trust. These funders had no role in the design, conduct, or analysis of the trial. J.S.M. was supported by an Australian National Health and Medical Research Foundation Practitioner Fellowship. J.A.S. is funded by an Australian National Health and Medical Research Council Senior Research Fellowship 1104975. This work was supported in part by the Australian Centre for Research Excellence in Malaria Elimination, funded by the NHMRC (1134989).N.G., S.C., and J.J.M. are employees of Medicines for Malaria Venture (MMV); all other authors declared no competing interests for this work.A.N.A.R., J.A.S., and J.S.M. wrote the manuscript. S.C., J.J.M., and J.S.M. designed the research. J.S.M. performed the research. A.N.A.R., L.M., N.G., and A.K. analyzed the data.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.Fig S6Click here for additional data file.Fig S7Click here for additional data file.Supplementary MaterialClick here for additional data file.


The limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.5 to 2.5 pg/mg and from 2 to 5 pg/mg, respectively. The intraday and interday precisions were within 13.32% at LOQ, low, medium, and high levels. The accuracies were within the range of 85.63–116.1%. The extraction recoveries were in the range of 89.42–119.68%, and the matrix effects were within the range of 44.81–119.77%. Furthermore, the method was successfully applied to the detection and quantification of fentanyl and sufentanil in hair samples from two authentic cases. Thus, this method has great potential for detecting fentanyl analogues and novel synthetic opioids in forensic work.The recent emergence of new fentanyl analogues and synthetic opioids on the drug market poses a global public health threat. However, these compounds cannot typically be identified using existing analytical methods. In this study, we aimed to develop and validate a rapid and sensitive method based on ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 37 fentanyl analogues and novel synthetic opioids in hair samples. Hair samples (20 mg) were extracted by cryogenic grinding in an extraction medium of methanol, acetonitrile, and 2 mmol/L ammonium acetate (pH 5.3). Following centrifugation of the samples, the analytes were separated using a WATERS Acquity UPLC HSS T Although many countries are trying to curb this trend, e.g., more than 170 NPSs are currently controlled in China, NPSs still pose a threat to public health. Most NPSs are synthetic cannabinoids and designer cathinones, but there has been a sharp increase in the consumption of novel synthetic opioids, particularly fentanyl and its analogues3. Fentanyl, a μ-opioid receptor agonist, is used as an analgesic and anesthesia adjuvant with a 50–100 times higher potency than morphine6. However, numerous illicit fentanyl substances have been used to adulterate other abuse drugs such as heroin, cocaine, and methamphetamine. Fentanyl and its analogues, which have high potency and numerous side effects, e.g., respiratory depression, have caused many fatalities7. This problem is further aggravated by the easy availability of these substances via complex international networks and their use with other drugs9. Daniulaityte et al.10 reported that in Montgomery County (located in southeast Ohio), the number of unintentional overdose death cases in which positive tests for fentanyl analogues were obtained increased by 337% between the second half of 2015 and the first half of 2017.Recently, new psychoactive substances (NPSs) have emerged in illicit markets13, urine14, saliva17, and dried blood spots19. However, it has been reported that the stability of some fentanyl analogues in whole blood is one month or less21. In other words, fentanyl analogues have a short period of storage time in such samples. Moreover, fentanyl is metabolized quickly in vivo, as the half-life of fentanyl in adults is 3.7 h and in infants is 5.3–21.1 h22. Therefore, to supplement these methods, it is necessary to develop methods for analyzing hair samples.As a consequence of increasing fatalities and the emergence of new fentanyl analogues and novel synthetic opioids, forensic laboratories must update their analytical methods for the identification and quantification of these drugs in various biological matrices. Some methods have been developed for the detection of these substances in whole blood24. To the best of our knowledge, only a few comprehensive methods using LC–MS/MS have been reported for analyzing fentanyl analogues and novel synthetic opioids in hair25. However, only a limited number of compounds were included in these methods.Hair, as an alternative or complementary matrix to conventional matrices, is a research hotspot in forensic science. Hair analysis can provide a longer detection window because substances may remain in hair for a long time without significant degradation. Furthermore, hair is easy to access, transport, and store26. Moreover, as fentanyl analogues typically have low active concentration27, higher sensitivity is required for the analysis of fentanyl analogues in hair. Compared with other methods, such as gas chromatography with nitrogen–phosphorous detection28 and gas chromatography–mass spectrometry29, liquid chromatography–tandem mass spectrometry31 is promising for analyzing fentanyl and its analogues in hair owing to its short analysis time, selectivity, and sensitivity.Despite the advantages of hair analysis, the drug concentration in hair after a single use is usually on the pg/mg levelIn this study, an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the identification and quantification of 31 fentanyl analogues and 6 novel synthetic opioids in hair. Furthermore, the developed method was successfully applied to authentic cases.N-desmethyl U-47700, U-48800, U-50488, and W-18 (containing 1 mg/mL free base); 4-anilino-N-phenethylpiperidine (4-ANPP), norcarfentanil, acryl fentanyl, butyryl fentanyl, isobutyryl fentanyl, para/ortho-fluorofentanyl, para-fluorobutyryl fentanyl (PFBF), 4-fluoroisobutyryl fentanyl, cis-3-methylfentanyl, β-hydroxythiofentanyl, valeryl fentanyl, ocfentanil, furanyl fentanyl, sufentanil, remifentanil, carfentanil, cyclopropylfentanyl, and methoxyacetylfentanyl (containing 100 μg/mL free base); and acetyl fentanyl (containing 50 μg/mL free base) were obtained as methanol or acetonitrile solutions from CERILLIANT . 3-Methylthiofentanyl, trans-3-methylfentanyl, α-methylfentanyl, β-hydroxyfentanyl, β-hydroxy-3-methylfentanyl, thiofentanyl, and tetrahydrofuranyl fentanyl (THF-F) (1 mg/mL) were synthesized by the Criminal Investigation Department of the Shanghai Public Security Bureau . The deuterated internal standards (ISs) of fentanyl-d5 , norfentanyl-d5 , and U-47700-d3 (used for the novel synthetic opioids) were purchased from Cerilliant.Standards of fentanyl, norfentanyl, alfentanil, acetyl norfentanyl, U-47700, High-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were obtained from SIGMA-SLDRICH . Formic acid (98%), ammonium acetate (98%), and ammonium formate (98%) were obtained from FLUKA . Ultrapure water was produced using a Milli-Q system .Working solutions were obtained by diluting their single solutions in one methanol mixture, and further dilutions of this mixture in methanol. Working solutions were prepared at 10 different concentration levels .d5, norfentanyl-d5, and U-47700-d332.An IS mixture (10 ng/mL) was obtained by spiking the extraction medium (EM), which consisted of methanol, acetonitrile, and 2 mmol/L ammonium formate  with 250 μL of a mixture (100 ng/mL) of fentanyl-Blank hair samples, provided voluntarily by the laboratory staff, were used for spiked calibration standards and quality control (QC) samples. The real hair samples used were from suspected users who were arrested and investigated by police. All the samples were stored at room temperature until analysis. All participants provided written informed consent and all study protocols were approved by the Ethics Committee of Academy of Forensic Science, Shanghai, China.g and filtered (pore size 0.22 μm). Finally, 200 μL of filtrate was transferred into an autosampler vial.To remove contaminants, the samples were washed with water two times and acetone three times, and then air-dried at room temperature. The washed samples were cut into 2–3 mm pieces and weighed (20 mg) in 2 mL tubes. Then, ceramic beads and 1 mL of the EM (containing 10 ng/mL IS) were added to tube. Subsequently, the hair samples were extracted by cryogenic grinding using a Bead Ruptor system  at a speed of 6 m/s for 20 s and then allowed to cool for 40 s. This process was repeated 10 times. The pulverized samples were centrifuged for 3 min at 14,000 × 3 column  fitted with a 1.8 μm HSS T3 guard column. The mobile phase was composed of 20 mmol/L ammonium acetate solution containing 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). The temperature of the autosampler was set at 4 °C and the injected volume was 5 μL. The gradient elution procedure is shown in Table The UHPLC-MS/MS analysis was performed on an Acquity UPLC system  coupled to a QTRAP 6500 PLUS triple quadruple linear ion trap mass spectrometer . Sample separation was performed using on a WATERS Acquity UPLC HSS TThe mass spectrometer was equipped with an electrospray interface operating in positive ionization mode. The source temperature and ion spray voltage were set to 500 °C and 5,500 V, respectively. The gas parameters were set as follows: collision-activated dissociation (CAD) gas, medium; curtain gas (CUR), 30 psi; nebulizing gas, 40 psi; and heater gas, 40 psi. Detection was performed using multiple reaction monitoring (MRM) with two transitions for each analyte and IS. The first transition was used for quantification and the second for qualification. The MRM transitions and optimized mass spectrometric parameters for each compound are listed in Table 33 guidelines and several recent criteria36 for method validation. Furthermore, the recovery and matrix effect (ME) were evaluated as described by Matuszewski et al.37.The method was developed according to the Society of Hair Testing (SoHT)38.The method selectivity was assessed using eight different sources of blank hair and spiking with the IS (10 ng/mL) to evaluate potential interference. Moreover, interference from possible coadministered medications was investigated according to our previous procedureTo determine the LODs and LOQs, blank hair samples were spiked with analyte concentrations of 5.0, 2.5, 1.0, and 0.5 pg/mg, and three replicates of each concentration were analyzed. The concentration that gave a signal-to-noise (S/N) ratio greater than 3 for both the MRM transitions was chosen as the LOD. The LOQ was defined as the lowest calibration point with a coefficient of variation (CV) of less than 20% for the precision and accuracy in the range of 80–120%.Calibration standards  were prepared by adding the working solutions to 20 mg of blank hair. In addition, QC samples were prepared at four concentration levels: LOQ (2 and 5 pg/mg), low (10 pg/mg), medium (500 pg/mg), and high . To determine linearity, seven sets of calibrators (two replicates for each set) were analyzed. The calibration curves were constructed by plotting the peak area ratios between each analyte and IS versus the concentration using 1/x weighting.n = 6) and over four days (n = 24), respectively. The CV for the precision should not exceed 15% for the low, medium, and high samples, whereas that for the LOQ sample should not exceed 20%. The accuracy was determined as the percentage ratio of the measured and theoretical values.The method precision and accuracy were assessed by analyzing spiked blank hair samples at four QC levels . The precision was expressed as the CV. The intraday and interday precision were determined by analyzing six replicates on one day , medium (500 pg/mg), and high (2000 pg/mg) levels. Hair samples from six drug-free individuals were used. For each level, the samples were divided into three groups . Set 1 consisted of neat standard solutions containing all the analytes in the EM. Set 2 was obtained by extracting the blank hair samples of six individuals and then spiking with the analytes. Set 3 was obtained by extracting the spiked hair samples using the method described in Sect. Sample extraction. The extraction recovery was calculated as the percentage ratio of the peak area of set 3 to the peak area of the set 2. The ME was defined as the percentage ratio of the peak area of set 2 to the peak area of set 1.According to the method recommended by Matuszewski et al.n = 6) after storage in the autosampler at 4 °C for 24 h.The stability of each analyte in hair was determined by injecting the extracted samples at three levels . In our study, 31 fentanyl analogues including 5 pairs of isomers were analyzed. To separate the isomers, we optimized the gradient elution based on previous studies13. First, we compared analyte separation using a WATERS T3 column and a RESTEK PPFP column  and found that better separation was achieved using the former column  of the EM was investigated. The recoveries of all compounds were in the range of 84.34–96.08%, 89.10–108.63%, and 83.27–104.15% with volumes of 500, 800, and 1,000 µL, respectively, with ME values in the range of 358.40–504.59%, 79.49–115.06%, and 77.92–104.30%, respectively. The EM volume had a great impact on the ME. In particular, when the analytes were extracted with 500 μL of the EM, the ME value increased significantly. However, for the recovery, the effect of the EM volume was not significant. Finally, the extraction solution was EM and the volume was 1,000 μL.No interfering signals were observed at the retention times of the analytes and ISs. Furthermore, there was no interference in blank hair spiked with common drugs of abuse and pharmaceuticals. The retention times are summarized in Table 2 value obtained for each analyte. To the best of our knowledge, this is the first method for quantifying 31 fentanyl analytes and 6 novel synthetic opioids. The LODs for all the compounds ranged from 0.5 to 2.5 pg/mg, and the LOQs ranged from 2 to 5 pg/mg. Busardò et al.25 reported a method to quantify 22 fentanyl analogues in hair with LODs of 3–7 pg/g and LOQs of 11–21 pg/g. The calibration curves of all the analytes were established in different concentration range, but with acceptable correlation coefficients (R2 > 0.99). According to previous reports44, the concentrations of fentanyl analogues in hair samples are typically in the following ranges: 3–2,800 pg/mg for fentanyl, 15.1–149 pg/mg for norfentanyl, and 3–104 pg/mg for 4-ANPP. In addition, a concentration of 44 pg/mg has been reported for furanyl fentanyl41. Therefore, the linearity ranges obtained for these compounds in our study cover the ranges observed in authentic cases.Table 36.The precision and accuracy obtained for each analyte are listed in Table The extraction recovery and ME data are summarized in Table The stability results for each analyte are shown in Table Following validation, the developed method was applied to the determination of fentanyl and its analogues in hair from authentic cases. The MRM chromatograms of cases 1 and 2 are shown in Fig. 44.reported a case in which a patient with a chronic and heavy toothache was treated with a fentanyl patch for 22 consecutive days. For a 5 cm hair sample cut into segments 0–1 cm, 1–2 cm, 3–4 cm, and 4–5 cm, the fentanyl concentration in all the segments was in the range of 60 pg/mg (LOQ) to 480 pg/mg. Compared with multiple doses, the concentration of fentanyl in hair was lower after a single dose, which may be why norfentanyl was not detected in our real case.A 35-year-old female patient without a history of drug abuse underwent surgery for thyroid disease. Anesthesia induction was performed by endotracheal intubation during surgery. One month after the operation, the patient's hair was collected and then the 0–3 cm segment of the hair sample was analyzed. In this case, fentanyl was detected at a concentration of 8.02 pg/mg. Schneider et al.A 51-year-old man was reported to police by his colleague. According to the informant, the man may have been using drugs for a long time. After hair collection, the hair sample was cut into three segments . Then, these samples were analyzed using our proposed method. Sufentanil was detected in the hair sample at concentrations of 183.91, 131.68, and 31.48 pg/mg for S1, S2, and S3, respectively. However, no metabolites were detected in the hair sample owing to the parent drugs being largely incorporated inside the keratin matrix from sweat, the bloodstream, and the sebum before metabolization. In this case, the observed concentration of sufentanil in hair will provide a reference for future forensic work.In this study, a sensitive, simple, rapid, and robust UHPLC-MS/MS was developed and validated for determination of 31 fentanyl analogues and 6 novel synthetic opioids in hair samples. This method covers fentanyl analogues and novel synthetic opioids that are common or new to the drug market. Furthermore, the developed method was successfully applied to authentic cases.


Within the next 24 h, the levels of inflammatory factors, correlation between active ingredients and inflammatory cytokines, the pathological changes of lung tissue, and protein expression of the JAK1/STAT3 signaling pathways were analyzed one by one. Finally, the concentration order of components absorbed in rat serum was berberine > palmatine > jatrorrhizine > coptisine > evodin > chlorogenic acid > evodiamine. Compared with the MOD group, the TNF-α, IL-6, and IFN-γ in the ZJF-H group were significantly reduced (p < 0.05). Moreover, the TNF-α decreased significantly accompanied by the increase of berberine, chlorogenic acid, jatrorrhizine, palmatine, evodin, and evodiamine in serum . Compared with the MOD, the area of lung injury, the expressions of JAK1, p-JAK1, STAT3, and p-STAT3 were significantly decreased under the treatment of ZJF (p < 0.05). Therefore, downregulating the JAK1/STAT3 signaling pathways was a potential avenue of ZJF in reversing lung injury induced by sepsis.Lung injury was the common and serious complication of sepsis, a systemic inflammatory response syndrome caused by severe infections. Chinese medicine had unique advantages in attenuating inflammatory response, such as Zuojinfang (ZJF). ZJF was a classical compound herb formula composed of Coptidis Rhizoma and Euodiae Fructus in a ratio of 6 : 1. In this paper, 15 ingredients in ZJF were identified and 8 of them absorbed into rat's serum were quantified by HPLC-MS/MS. Subsequently, sepsis-induced lung injury model was replicated in rats by cecal ligation and puncture. 60 SD rats were randomly divided into 6 groups ( Sepsis was defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection . At pres1 [Fortunately, it was found that some Chinese herbs had unique advantages in reducing the production of drug-resistant bacteria and inhibiting the generation of proinflammatory factors. Moreover, active ingredients obtained from Chinese herbs or compound preparation of Chinese medicine like emodin , ginseno1 , glycyrr1 , and Xue1  usually κB signaling pathway [β, IL-6, and TNF-α [Zuojinfang (ZJF) was a classical formula composed of Coptidis Rhizoma (CR) and Euodiae Fructus (EF) in a ratio of 6 : 1. ZJF was always applied to treat gastric ulcer, gastroesophageal reflux, gastritis, and pyloric obstruction , 25. The pathway . In addind TNF-α . Based oThe purpose of this study was to verify the efficacy and mechanism of ZJF in reversing lung injury induced by sepsis. Firstly, HPLC-MS/MS was applied for the qualitative and quantitative analysis of ZJF. And then the 8 ingredients absorbed into the rat's serum were also determined by HPLC-MS/MS. The inflammatory factor levels were tested by enzyme-linked immunosorbent assay (ELISA). Pathologic changes in lung tissues were evaluated via pathological section with hematoxylin and eosin (HE) staining. The expressions of JAK, p-JAK1, STAT3, and p-STAT3 were determined by western blot assay. Our study not only evaluated the therapeutic effect of ZJF on lung injury induced by sepsis but also provided a new perspective for resolving the mechanism of ZJF in regulating the JAK1/STAT3 signaling pathway.Animal experiments were carried out in accordance with the Guidelines of Animal Experimentation in Jiangsu University , and the protocol was also approved by its Animal Ethics Committee.Coptidis Rhizoma Franch and Evodiarutaecar pa (Juss.) Benth. var. officinalis (Dode) Huang, respectively. ZJF was obtained by mixing CR (120 g) and EF (20 g), crushing, and passing through 60 molybdenum sieves.Chlorogenic acid, jatrorrhizine, palmatine, berberine, evodin, evodiamine, and rutaecarpine (purity > 98.0%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . Coptisine (purity > 98.5%) was purchased from Prof. Hao Zhang . CR was provided by Nanjing Pharmacy . EF was provided by Evodiae Planting Base . CR and EF were identified by Prof. Chengying Wu  as n = 10): control group , sham operation group , model group , ZJF low-dose group , ZJF high-dose group , and positive control prednisolone group .Cecal ligation and puncture –33 was aμg/mL), jatrorrhizine (35.0 μg/mL), coptisine (18.0 μg/mL), palmatine (13.0 μg/mL), berberine (43.0 μg/mL), evodin (35.0 μg/mL), evodiamine (42.0 μg/mL), and rutaecarpine (32.0 μg/mL) was prepared in methanol. The solutions were stored in refrigerator at 4°C and kept in darkness. The mixed standard stock solution was diluted into a series of standard working solution by methanol.Mixed standard stock solution containing chlorogenic acid  was extracted twice with 65% ethanol by refluxing for 90 min each time . Two-parμL of supernatant was taken and frozen-spare.After intragastric administration of ZJF extracts (freeze-dried powder dispersed in 0.5% CMC-Na), the rats were subjected to blood collection from orbit at 6 h, 12 h, and 24 h, respectively. 0.2 mL of plasma was taken into the heparinized centrifuge tube, centrifuged at 4000 rpm for 10 min, and 50 μL of the serum, vortexed for 2 min, and centrifuged for 10 min to obtain 400 μL of the supernatant. It was vacuum-dried at 40°C until ethyl acetate was evaporated, reconstituted by adding 100 μL of methanol, ultrasonically mixed for 5 min, and centrifuged for 10 min to obtain 80 μL of the supernatant for chromatographic analysis.0.5 mL of ethyl acetate was added to 50 18 column  was used for the chromatographic separation under gradient elution with acetonitrile -0.3% glacial acetic acid  at a flow rate 0.4 mL/min. The column injection volume was 5 μL, and column temperature was 20°C. MS acquisition was carried out in the ESI-positive ionization mode. Detection mode was set to multiple reaction monitoring , and nebulizer pressure 25 psi.ZORBAX XDC-Cing MRM, . The conr2 > 0.9997) in a relatively wide concentration range  was precisely removed into 0.1 mL blank plasma samples and their peak areas were recorded (A) by HPLC-MS/MS after the same pretreatment as described in μL mixed standard solution (10 ppm) was added into 0.1 mL methanol with the same operation and their areas were measured as “B.” Their recovery was calculated by “(A/B) × 100%”. The recovery rate of 8 active ingredients in serum showed stable recovery between 83.7% and 89.5%.10 μL standard solution of series concentrations and 10 μL sample solution were prepared; then, the diluent was added (40 μL). Standard and sample wells were added with horseradish peroxidase- (HRP-) labelled antibody (100 μL) to detect the antibody. The reaction wells were sealed with the sealing plate membrane and kept in 37°C water bath for 60 min. Discard the liquid, dry the plates, fill them up with washing fluid for 1 min, shake off the washing liquid, dry the plates, and repeat the washing process 5 times. The substrates were added (50 μL) to all the wells and incubated for 15 min at 37°C. 15 min later, the OD value of each well was measured at 450 nm wavelength. The standard curve was obtained with the linear regression of standard concentration and OD value, and the corresponding concentration of the OD value of the sample was found on this curve. The concentration of samples could be found from the standard curve according to their OD values.Detection was performed according to the manufacturer's instructions for the enzyme-linked immunosorbent assay kits. 50 μm serial sections. Then, the sections were gradually dewaxed, dehydrated, stained with HE, immersed in xylene and alcohol, stained with hematoxylin for 5 min, stained with eosin for 3 min, and reimmersed in alcohol and xylene. Finally, the morphological changes [Collected lung tissues were fixed in 10% formalin for 48 h. The right upper lobe and hilar organizations were routinely washed, dehydrated, embedded in paraffin, and cut into 5  changes , 36 of tβ-actin as the control.Protein samples of lung tissue were prepared and subjected to protein gel electrophoresis (Bio-Rad). The gel was transferred to a membrane, blocked, and incubated with a primary antibody: the JAK1 , p-JAK1 , STAT3 , and p-STAT3  diluted 1 : 1000. The diluted primary antibody was incubated at room temperature for 2 h. Secondary antibody was incubated for 30-60 min. Membranes were visualized with enhanced chemiluminescence. ImageJ software  was used for analyzing protein bands. The relative density of each band was calculated to p < 0.05 were considered as significant.All data were presented as the mean ± SD. Statistical analysis was performed using GraphPad Prism 5 . Differences were analyzed by one-way analysis of variance . Differences tR) and corresponding parent ion (m/z) produced by HPLC-MS/MS (The extracted ion chromatogram (EIC) of 15 components  was retrLC-MS/MS . They weLC-MS/MS .The contents of the above 8 components in serum at 6 h, 12 h, and 24 h after oral administration of ZJF at high and low doses were quantitatively analyzed. Finally, only the rutaecarpine was not detected, and the other 7 components were successfully quantified. Results showed that the content of Coptis alkaloids in serum was always the highest, including berberine (100.28 ± 2.31 ng/mL), jatrorrhizine (11.42 ± 0.71 ng/mL), coptisine (9.33 ± 0.43 ng/mL), and palmatine (20.73 ± 1.55 ng/mL), especially the berberine. Besides, the contents of evodin (2.64 ± 0.44 ng/mL) and chlorogenic acid (3.85 ± 0.78 ng/mL) obtained from Euodiae Fructus were also prominent in serum. Furthermore, the content of these active ingredients in serum decreased significantly from 6 h to 24 h after administration .α, IFN-γ, and IL-6 were the inflammatory factors, and SOD was an antioxidant index, which were related to inflammation. Compared with the sham group, the levels of TNF-α, IFN-γ, and IL-6 at 6 h, 12 h, and 24 h in the MOD group were all significantly increased (p < 0.05) along with the significant decrease of SOD (p < 0.05). After the intervention of ZJF and PNSL, the expressions of TNF-α, IFN-γ, and IL-6 were reduced and the SOD level was increased  and the level of SOD was significantly increased (p < 0.01). Therefore, ZJF significantly inhibited the inflammatory expression in septic rats.TNF-ncreased . More spα, IL-6, and IFN-γ) at same time points . The levels of inflammatory cytokines were regarded as the ordinate, and the contents of active ingredients were the abscissa for linear regression (α was negatively correlated with berberine (p < 0.0001), chlorogenic acid (p < 0.01), jatrorrhizine (p < 0.01), palmatine (p < 0.01), evodin (p < 0.05), and evodiamine (p < 0.05). Moreover, there was no significant correlation between the other inflammatory factors (IL-6 and IFN-γ) and the active ingredients.The correlation analysis was conducted between the components and the inflammatory factors (TNF-gression . The resp < 0.01). After treatment with ZJF and PNSL, the area of lung injury decreased significantly compared with the MOD . Compared with the MOD, the PNSL also significantly downregulated the protein expression of JAK1, p-JAK1, STAT3, and p-STAT3 (p < 0.01). Similar to the PNSL group, the expression of JAK1, p-JAK1, STAT3, and p-STAT3 was also downregulated in the ZJF-H group (p < 0.01). Therefore, ZJF reversed sepsis caused lung injury by downregulating the expression of JAK1/STAT3-related proteins  and ultimately reduced the level of inflammatory mediators (p < 0.05). Therefore, this study confirmed that ZJF could reverse sepsis-induced lung injury by inhibiting the JAK1-STAT3 signal pathway and reducing the expression of inflammatory factors.Undoubtedly, excessive inflammation response was the major contributing factor for sepsis-induced organ injury . This imα, IFN-γ, and IL-6) in serum were analyzed according to the data collected at 6 h, 12 h, and 24 h after oral administration of ZJF extract. Fortunately, the concentration of TNF-α decreased significantly accompanied by the increase of various components, including berberine (p < 0.0001), chlorogenic acid (p < 0.01), jatrorrhizine (p < 0.01), palmatine (p < 0.01), evodin (p < 0.05), and evodiamine (p < 0.05). However, this notable negative correlation did not exist in other inflammatory factors and ingredients. This study suggested that berberine, chlorogenic acid, jarrorhizine, palmatine, evodin, and evodiamine had inhibitory effects on sepsis-induced inflammatory response, especially the TNF-α. Through literature review, other laboratory also found that berberine [In order to explore the intrinsic communication of components and inflammation, the correlations between 8 components and 3 inflammatory factors  can also be regulated through other signaling pathways, such as JAK-STAT [α levels. Therefore, downregulating the level of inflammatory factors by inhibiting the JAK-STAT signaling pathway was one of the mechanisms of ZJF for the treatment of sepsis. ZJF reversed the lung injury induced by sepsis was related to the inhibition of inflammatory response under the synergistic effect of multicomponents, such as chlorogenic acid, jatrorrhizine, palmatine, berberine, evodiamine, and rutaecarpine. In the subsequent study, the mechanism of these components in treating lung injury induced by sepsis will be continued to explore.There are few studies on antisepsis of ZJF, Coptis chinensis (CR), Evodia rutaecarpa (EF), and their main components (berberine and rutaecarpine). In the existing reports, it was found that ZJF, CR, and EF downregulated the expression of inflammatory factors, such as TNF- pathway , 43. HowJAK-STAT –46. TherZJF reversed the lung injury caused by sepsis through inhibiting JAK1/STAT3 expression and subsequently reducing inflammatory mediator production. More importantly, berberine, chlorogenic acid, jarrorhizine, palmatine, evodin, and evodiamine were important potential substances of ZJF in treating lung injury induced by sepsis.


VS-5584 is a small-molecular compound that showed equivalent activity against mTOR and all class I PI3K isoforms and demonstrated preclinical activity in diverse cancer cell lines and xenograft tumor model, and rational combination of VS-5584 and other target therapies achieved promising outcomes in oncology. In the present study, we established and validated a simple and sensitive UPLC-MS/MS method for the determination of VS-5584 in plasma samples. VS-5584 was separated via an Acquity UPLC BEH C18 column, with a mobile phase composed of acetonitrile and 0.2% formic acid in water (40 : 60). The calibration curve displayed a good linearity in the range of 1.0–1000 ng/mL, with satisfactory accuracy (−13.6% < RE% < 8.8%) and precision . The validated method was then applied to a pharmacokinetic study in rats. After administration of 10 mg/kg, VS-5584 was absorbed quickly and reached a peak concentration of 473.2 ± 72.0 ng/mL after 20 min. The established method allows for the quantification of VS-5584 in rat plasma in detail and can be utilized to successfully describe the pharmacokinetic profile of VS-5584. The increased activity of the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signalling pathway has been associated with increased growth, survival, and proliferation in many human cancers . Thus, tin vitro, and further suppress A375 melanoma xenograft growth in nude mice after oral administration +, and an m/z of 312.73 for VS-5584 and 366.65 for IS were defined as product ions, as these were the most abundant product ions in the MS2 scan spectra, respectively. Additionally, the ionization of m/z 354.78 → 312.73 for VS-5584 and 410.64 → 366.65 for IS was stable and reproducible. The MRM reaction of IS was consistent with previous reports [To get a favorable mass response, VS-5584 and IS were infused directly into the mass spectrometer with a syringe pump, respectively. Better responses were achieved in positive ionization mode as VS-5584 and IS are nitrogenous compounds. Ion  reports , 19.To obtain suitable retention time and high detection sensitivity, the mobile phase system was determined in acetonitrile, methanol, and proportions of acid in water. As the organic phase in the mobile phase, acetonitrile achieved a smoother baseline in the IS channel. Then, 40% acetonitrile was chosen, compared to 35% and 30% acetonitrile in the mobile phase, resulting in a total run time of 3.0 min. Subsequently, a mixture of acetonitrile and water containing 0.2% formic acid (40 : 60) at a flow rate of 0.20 mL/min was chosen as the mobile phase system.To simplify the sample treatment, plasma proteins were precipitated prior to treatment. Acetonitrile and methanol were tested as extraction solvents. Satisfactory recoveries were acquired with both of the two solvents, but acetonitrile yielded a thinner IS peak shape.Blank samples were free of endogenous interfering substances at the retention times of VS-5584 (2.32 min) and IS (1.45 min), indicating sufficient selectivity of this method. Representative MRM chromatograms of blank plasma, VS-5584, and the IS in rat plasma are presented in y = 0.03554x + 0.00632, r = 0.9977). The LLOQ was determined to be 1.0 ng/mL.In rat plasma, the calibration curve of VS-5584 demonstrated good linearity in the range of 1.0–1000 ng/mL (typical equation: After injection of ULOQ, no obvious chromatographic peak was detected in blank samples at the retention times of VS-5584 and the IS; thus, no carryover was found in this method.As shown in The percentage recoveries of VS-5584 and IS spiked in rat plasma were found to be greater than 89.3%. t1/2 of 3.28 ± 1.37 h at the terminal phase. AUC0 ⟶ 12 h, AUC0 ⟶ ∞ were found to be 865.2 ± 192.1 and 928.8 ± 228.0 h ng/mL, respectively. Vz/F and CL/F were found to be 54.1 ± 29.6 L/kg and 11.3 ± 2.5 L/h/kg, respectively.The established method was successfully used for the first time to study the pharmacokinetic behaviour of VS-5584 in rats after an oral administration of 10 mg/kg. The time course of plasma VS-5584 concentrations is shown in To our knowledge, we established and validated for the first time a simple and sensitive UPLC-MS/MS method for the determination of VS-5584 in rat plasma. The established analytical method was successfully applied to describe the pharmacokinetic profile of VS-5584 after oral administration in rats. The established method is suitable for future preclinical and clinical studies such as pharmacokinetic studies and drug-drug interaction studies. Additionally, the established method can be used as a reference for developing a high-throughput bioassay and for therapeutic monitoring of VS-5584 in human plasma.


Alstonia scholaris (L.) R. Br. (Apocynaceae) is an evergreen tree that has been used to treat lung diseases. In this study, the toxicity profile of indole alkaloids from leaves of A. scholaris was investigated. In acute toxicity tests, mice were administered total alkaloids (TA) and five indole alkaloids. In a chronic toxicity test, rats were continuously administered TA  for 13 weeks, followed by a 4-week recovery. A single administration of TA affected the behavior of mice, and at 12.8 g/kg bw, prone position, shortness of breath, wheezing, and convulsion were observed. The half-lethal dose (LD50) in mice was 5.48 g/kg bw, almost 2740 times the clinical dose in humans. Among the five indole alkaloids, the maximum tolerance dose in mice ranged from 0.75 to 4 g/kg bw. The TA-treated rats did not die and showed no adverse effects or dose-dependent changes in weight or food and water consumption, despite fluctuations in hematological and biochemical parameters compared with historical data. Furthermore, both gross and histopathological observations revealed no abnormalities in any organ. With daily oral administration to rats, the non-observed-adverse-effect-level of TA was 100 mg/kg bw. The results indicate that TA is safe for clinical use.The online version of this article (10.1007/s13659-020-00237-1) contains supplementary material, which is available to authorized users. Aristolochia and Asarumcan, is well known to cause kidney failure and cancer, particularly of the urinary tract in humans. Such adverse effects have aroused concern about the safety of herbal medicines  GPT3-1) issued by the China Food and Drug Administration on March 2005, the period for testing drug administration in animals should be based on the expected period of clinical use of that substance in humans. The repeated oral administration of a test substance for 1–3 months in animals is thought to be comparable to the administration for less than 1 month in humans. The objective of chronic toxicity studies is to determine the possible clinical adverse reactions caused by the substance, including the nature and degree of harm, the dose–response and time–response relationships, the effects on target organs or tissues, and the reversibility, and then predict the starting dose in clinical trials and the safe dose range for repeated drug use. The doses of 50, 100, and 300 mg/kg bw TA used in this study were equivalent to 25, 50, and 150 times, respectively, the usual dose in humans.In this study, both male and female rats treated with doses of 50, 100, and 300 mg/kg bw TA once daily showed no signs of clinical toxicity or mortality. Changes in food and water ingestion are generally used as indicators of the harmful effects of drugs and chemicals . In the The liver and kidneys are frequent targets of drug action, because the liver is the primary organ for drug biotransformation, and the kidneys are the primary organs for drug excretion. Organ weight is one of the main parameters used to evaluate the effects of test substances in toxicity studies . In the The hematopoietic system, which is highly sensitive to toxic substances, can be altered when poisonous plants are ingested . And chaHistopathological examination is a fundamental step in preclinical toxicology research that is used to further validate whether tissues or internal organs are damaged . Accordi50) (administered via gavage) in mice of 5.48 g/kg bw was approximately 2740 times the recommended dose for patients. In addition, acute toxicity testing demonstrated that picrinine, vallesamine, sholaricine, 19-epischolaricine, and echitamine were safe for mice when used alone. Moreover, no lethality, significant alterations in hematological and serum biochemical indices, or adverse histopathological effects were evident in the chronic toxicity study (13 weeks) with rats using doses 25, 50, and 150 times the clinical dose. On the basis of this result, the TA should be considered safe for use in clinics and deserve further development as a pharmaceutical product.In summary, at the tested oral doses ranging from 2.2 to 12.8 g/kg bw, TA produced some signs of acute toxicity in mice in a dose-dependent manner, which might be related to toxicity to the neuromuscular, central nervous system and autonomic nervous system. The half-lethal dose . The plants were collected in 2006 in Pu’er City, Yunnan Province, People’s Republic of China. Dr. Xiao–Dong Luo of the Kunming Institute of Botany, Chinese Academy of Sciences, identified the plants, and the plant name was checked against https://www.theplantlist.org. A voucher specimen (no. Luo20060407) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Chinese Academy of Sciences.Leaves of A. scholaris were extracted with 90% ethyl alcohol under reflux conditions (3 h × 4 times) at room temperature, and the solvent was evaporated in vacuo to obtain the ethanolic extract. The ethanolic extract was dissolved in 0.3% aqueous HCl solution and filtered; the residue was the nonalkaloid fraction. Then, the acidic solution, adjusted to pH 9–10 with 10% aqueous ammonia, was extracted with ethyl acetate to obtain the total alkaloids (TA) fraction (batch no. 20070512). In addition, the picrinine, vallesamine, sholaricine, 19-epischolaricine, and echitamine used in this study were isolated from the leaves or barks of A. scholaris  rats (60–110 g) and Institute of Cancer Researcch (ICR) mice (18–20 g) were purchased from Kunming Medical University, Kunming City, Yunnan Province, People’s Republic of China (license no. SCXK (Dian) 2005-0008). Animals were housed in a room at 24 ± 1 °C and 50–60% relative humidity under a 12 h light–dark cycle and maintained with lights on at 9:00 and off at 21:00. The animals were acclimatized to the laboratory environment for 7 days and allowed free access to water and a standard diet before the initiation of treatment. All animals were fasted but provided water for 10–12 h before the start of the experiment. Animals were euthanized using pentobarbital sodium  followed terminal heart stick and cervical dislocation or induction of bilateral pneumothoraces as secondary means of assuring euthanasia. There are no deviations from the standards and regulations promulgated under the Animal Welfare Act.Acute toxicity was evaluated in mice according to the testing guidelines for the safety evaluation of drugs (Notification [Z] GPT2-1) issued by the China Food and Drug Administration in March 2005. In the experiment, the dose of all test substances were set according to the pretest results (Data not shown). Fasted mice were randomly divided into different groups of 10 each . The groups were the following.Total indole alkaloids (TA): The mice were divided into seven groups and treated with TA orally at doses of 0, 2.2, 3.1, 4.4, 6.3, 9.0, and 12.8 g/kg bw. The treated groups were administered TA at the volume of 30 mL/kg, whereas the control group (0 g/kg bw) received an equal volume of 1% carboxymethylcellulose.A. scholaris at the volume of 20 mL/kg, whereas the control group received an equal volume of 1% carboxymethylcellulose.Alkaloid compounds: Five groups were intragastrically administered picrinine (Pic) at 2.0 g/kg bw, vallesamine  at 4.0 g/kg bw, scholaricine (Sch) at 0.75 g/kg bw, 19-epischolaricine (Epi) at 2.0 g/kg bw, or echitamine (Ech) at 2.0 g/kg bw. The treated groups were administered the different compounds from The symptoms of toxicity and mortality were recorded systematically at 1, 2, 4, and 6 h after administration of the test substances. The number of live mice was noted after 24 h, and once-daily observations were performed for a further 14 days. The animals were intraperitoneally injected with pentobarbital sodium and sacrificed by cervical dislocation to gross observation.A. scholaris adverse effects according to the testing guidelines for the safety evaluation of drugs (Notification [Z] GPT3-1) issued by the China Food and Drug Administration on March 2005. Treatments were administered for 13 weeks, followed by 4 weeks of recovery. In this test, 160 rats were divided into four groups of 40 rats  that were treated orally with 0 (control), 50, 100, or 300 mg/kg bw TA in a volume of 10 mL/kg daily. Rats in the control group were given an equal volume of 1% carboxymethylcellulose. Daily observations were conducted for toxicological signs, mortality, and physiological and behavioral changes. Bodyweights were recorded on day one of the study and twice weekly thereafter. Body alterations were recorded, and the dose was adjusted in accordance with body weight to maintain the target dose level in all animals. Food consumption and water intake were also measured each week. Rats were anesthetized and blood samples were collected from the common carotid artery with EDTA-2K, sodium citrate (1:9), vacuum blood collection tubes. The hematological, coagulative, and biochemical indices were evaluated (see methodology below) after TA treatment for 7 and 13 weeks and after the 4-week recovery period. Following blood collection, gross observation of the major organs was carefully performed, and the absolute and relative (organ to body weight ratio) weights of organs and tissues, including the brain, heart, liver, spleen, lung, kidney, adrenal gland, thymus, testis, epididymis, uterus, and ovary, were measured. All organs were weighed directly after dissection to avoid mechanical injury and stored in 10% neutral buffered formalin.A chronic toxicity test was performed on rats in order to evaluate the target organs and the reversibility of Blood samples were treated with EDTA-2K to perform hematological assessments. A HEMAVET 950  automated hematology analyzer was used to measure the white blood cell (WBC) count, neutrophils (NE), lymphocytes (LY), monocytes (MO), red blood cell (RBC) count, hemoglobin (Hb), red blood cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocytes (RET), and platelets (PLT). Blood was treated with sodium citrate before prothrombin time (PT) was measured.+), potassium ions (K+), chloride ions (Cl−), ionized calcium (iCa), total calcium (TCa), and potential of hydrogen (pH) value were measured using a Beckman AU680 automatic biochemistry analyzer .Serum was obtained after centrifugation of blood at 3000 revolutions for 10 min and collected in 1.5 mL centrifuge tubes. In the serum, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), triglycerides (TG), total cholesterol (TC), sodium ions  from the control and 300 mg/kg bw groups at 7 and 13 weeks and after the recovery period were douched with running tap water for 4 h, dehydrated via a graded ethanol series, dewaxed in xylene, embedded in paraffin, cut into 5 µM sections using a microtome, and stained with hematoxylin and eosin (H&E). Moreover, both small intestine and colon were observed by H&E staining at the end of the week 7 treatment. The examination of additional organs such as the small intestine, colon, brain, adrenal gland, testis, ovary, and uterus was performed at the end of the week 13 administration. Pathological sections of tissues were examined under a Leica DM6000B light microscope and photomicrographs  obtained. When lesions were observed in the 300 mg/kg bw group, the diseased organs were also examined in the 100 mg/kg.bw group.Data are presented as the mean ± standard deviation, and statistical analyses were performed using SPSS 17.0 software . The variance in the data of all parameters was checked for homogeneity by using Bartlett’s procedure. When the data were homogeneous, one-way ANOVA was used. In the cases of heterogeneous data, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the LSD and Dunnett’s T3 multiple tests were employed for comparisons between control and treated groups. A nonparametric test was selected for the ranked data. Results were considered significant at p < 0.05.Supplementary file1 (PDF 582 kb)Below is the link to the electronic supplementary material.