Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Withania somnifera and Artemisia annua. The over-expression and inhibition dual strategy revealed that the additional OkHMGR in-planta could afford endogenous flow of isoprenoid units towards synthesis of terpenoids. The present study provides in-depth insight of OkHMGR in regulation of biosynthesis of non-plastidal isoprenoids. This is first report on any gene of MVA/isoprenoid pathway from under-explored Camphor Tulsi belonging to genus Ocimum. Studies also suggested that OkHMGR could be a potential tool for attempting metabolic engineering for enhancing medicinally important terpenoidal metabolites in plants. Ocimum (Tulsi), an important member of Lamiaceae family comprises of about 150 species that are the inhabitants of tropical and warm temperate regions of Asia, Central and South Africa1. The Tulsi has been used as a source of medicinal preparations since ages, mentioned in various ancient literatures such as Charak Samhita, Rigveda etc. In Sanskrit the meaning of Tulsi is “one that is incomparable”. It is classified as a “Rasayana,” the herb which improves the health. The prominently used species of genus Ocimum are O. basilicum, O. sanctum, whereas O. gratissimum and O. kilimandscharicum are less explored2. O. kilimandscharicum is known for camphor as major constituent in its essential oil and is a perennial herb, native of East Africa and is also found in India, Turkey and Thailand4. The plant is rich in aromatic essential oil that makes it economically and medically important. O. kilimandscharicum contains pool of various secondary metabolites with a major proportion of monoterpenoids (≥ 90.0%) out of which oxygenated monoterpenoids may constitute upto 70% while hydrocarbons may constitute about upto 25%5. It has been reported that the essential oil content in Camphor Tulsi varied between 0.70–2.0% on dry weight basis6. Seed and leaf essential oil of O. kilimandscharicum also contains minor proportions of other monoterpenoids such as -bisabolene, terpineol, linalool, (Z)-cinnamic acid methyl ester, camphor etc are reported to possess various antimicrobial, antispasmodic, bactericidal, carminative, anti-helminthic, hepatoprotective, antiviral and larvicidal activities8. Camphor is the most distinct compound of the plant which may serve various medicinal and industrial purposes8. The terpenoid metabolites are produced by the plant through MEP (2C-methyl-D-erythritol 4-phosphate) and MVA  pathway in plastids and cytosol, respectively9. Both the pathways are actively involved in the biosynthesis of isoprenoid moieties operative from their respective compartments11. Mono, di-, tetra- terpenoids are mainly biosynthesized by MEP pathway whereas MVA pathway is reported for the biosynthesis of sesquiterpenoids and triterpenoids11. Our earlier studies on W. somnifera showed that the mevalonate pathway contributes dominantly to the synthesis of IPP than non-mevalonate pathway and HMG-CoA to mevalonic acid via HMGR enzyme is the rate-determining step13. Undoubtedly, HMGR gene governs the MVA pathway derived isoprenoids and plays a key role in isoprenoid biosynthesis. HMGR gene has been isolated from number of plant species including W. somnifera, S. miltiorrhiza, A. indica and other plants20. Different isoforms of HMGR have also been reported in cotton and tomato, which might have different roles to play under the influence of different environmental and developmental conditions16. HMGR is responsible for regulating the production of diverse terpenoids virtually in all plants and holds central regulatory position in terpenoid bearing plants  followed by Andrographis paniculata (86%), Picrorhiza kurrooa (86%), Panax ginseng (83%), W. somnifera (78%), Arabidopsis thaliana (78%) and Nicotiana tabacum (75%). Plants HMGR possess HMG-CoA binding motifs (EMPIGYVQIP) & (TTEGCLVA) and NADPH-binding motifs (DAMGMNM) & (GTVGGGT) which were also found in the OkHMGR  exhibits closeness to S. miltiorrhiza and the functional motifs were very similar to other plant HMGRs  introduced in the enzyme assay influenced the catalytic activity of the enzyme. It was observed that addition of K+ increased the activity of enzyme  and the maximum reaction velocity (Vmax) were determined , 0.425µmole min−1 mg−1 enzyme protein, 14.53 s−1 and 1.6 × 104 M−1 s−1 respectively.Full length ned Fig. . The Km,OkHMGR transcripts in O. kilimandscharicum was found to be highest in the flower followed by other tissues  and salicylic acid (SA) as exhibited by the exogenous supplementation studies. A substantial increase in transcript abundance was observed with MeJA  hormone for 3 h, elevated the expression of OkHMGR transcripts , caryophyllene (37.87%) and germacrene D (19.1%) were observed  reduction in total withanolide content and 39–43% reduction in total sterol content compared to untreated untransformed tissues. Over-expressed lines treated with inhibitors showed lowering in gene expression levels, withanolide and sterol content to varying extents in comparison to untreated but transformed tissues . Among various species, we primarily focused on O. kilimandscharicum as it contains more terpenoids than any other Ocimum species, which are known for the presence of phenylpropanoids. The isolated OkHMGR with a molecular mass of 60 kDa (Supplementary Table S. miltiorrhiza with highest similarity. The characterstic motifs such as HMG-CoA binding motif (TTEGCLVA and EMPVGYVQIP) and NADPH binding motifs (DAMGMNM and GTVGGGT) in OkHMGR sequence revealed that OkHMGR shares the conserved motifs with other plant species  and (EMPVGYVQIP) while the S-domain (DAMGMNM), forms the binding site for NADPH9.HMGRs have been broadly classified under two categories: class I and class II. Class I are found in eukaryotes and, are membrane bound while class II in prokaryotes and are soluble in naturem, Vmax, optimum pH, optimum temperature etc. Variation in the catalytic activity due to change in pH is a consequence in pattern of ionic bonding between the substrate and enzyme under acidic or alkaline conditions  than NADPH (903 µM) showed that the enzyme has more affinity for its substrate rather than its cofactor. Other reports on HMGR where the enzyme had higher affinity for HMG-CoA than NADPH such as in A. thaliana, Raphanus raphanistrum, Solanum tuberosum with lower Km for HMG-CoA as compared to Km for co-factor NADPH17. This clearly suggests that the availability of the substrate could be a rate controlling factor.Biochemical characterization of the HMGR through its catalytic properties provided a detailed account of the kinetic nature of the protein as revealed by KO. kilimandscharicum, HMGR exhibited differential pattern of gene expression in different tissues of the plant. Similarly, in other plants such as in C. arachnoids, highest expression in stem tissues and lowest in leaves was recorded27. In S. miltiorrhiza, HMGR expression was detected in roots, stems and leaves but highest expression was found in roots, followed by stem and leaf tissues28. In, this study, high expression levels in flower tissues indicate that OkHMGR may have role in growth and development of flower or metabolite accumulation in inflorescence29  and epicedrol synthase (ECS) of A. annua in N. benthamiana, intermediate enzymes of artemisinin biosynthesis pathway was reported to show remarkable increase in the artemisinin production41. The percentage of the essential oil constituents in over expressed tissues was higher42 along with 5 folds higher transcripts in over-expressed tissues. Another report from Parthenium argentatum (guayule) also showed that the over-expression of HMGR led to 65% increase in polyisoprenoids rubber43. Interestingly, when change in individual constituents was being studied we noted that not only sesquiterpenes like caryophyllene, germacrene D but also some of the monoterpenes such as beta-myrcene, limonene, beta ocimene, camphene also increased30. The stable transformed plants of O. kilimandscharicum confirm the above results (data not shown). The moderate enhancement in monoterpenoid may account probably to the observations that an immediate conversion of the C5 moieties might have been influenced, although major impact was shed on MVA dependent metabolites. To further affirm our findings, the gene was transformed in different species of Ocimum namely, O. basilicum, O. sanctum and O. gratissimum, which represent another class in terms of phytochemical moieties. In all the three species over-expression led to the increase in total essential oil content, individual essential oil constituents and mRNA transcript levels. It is reported earlier that the monoterpenoid essential oils specifically ocimene are derived from MEP pathway as confirmed by 13C labelling and inhibitor treatment studies45. Interestingly, it was reported that in ripen strawberry, both monoterpenoid  and sesquiterpenoid (nerolidol) are biosynthesized in the cytosol as the gene FaNES1 which is capable of synthesizing both  and (nerolidol) depending upon the presence of its precursors GPP and FPP lacks plastid targeting peptide sequence at N-terminal. The observations were further confirmed by feeding studies where labeling of mevalonic acid led to labeling of linalool46. It was shown that the two isoforms of A. thaliana GPP synthase are involved in organelle specific manner, one being targeted to the plastids while the other was functionally linked to cytosol47. Shikonin, a monoterpene derivative from L. erythrorhizon is also produced by MVA pathway and negatively regulated by mevalonin treatment48. The reason which can be anticipated for this is loss of plastid targeting peptide due to some splicing event. Another reason is presence of dual isoforms of a synthase targeted to different cellular compartments which arises due to some mutation or difference in selection of first or second methionine group for initiation of protein synthesis or other post translational modifications49. The importance of HMGR is not restricted to triterpenoids only but carotenoids were also affected by its expression. Over-expression of HMGR increased the total carotenoids by up to 43%  HMGR could drive the artemisinin content50. These findings further proved that HMGR is involved in synthesis of sesqui-terpenes, especially artemisinin. Similarly, W. somnifera contains withanolides and sterols as an important phytoconstituents and HMGR is known to be involved in the production of various triterpenoids and sterols. In W. somnifera, over-expression of OkHMGR, transiently led to increase in mRNA transcripts and hence withanolides and sterols as well.Results in HMGR transcripts as well as terpenoid production. In transformed lines, over-expression and inhibition both the phenomenon were acting together as inhibition in transformed lines was less than that of control lines. These results were common in all the plants studied thereby confirming that HMGR has a major function in the synthesis of terpenoids. However, in case of W. somnifera, inhibitor treatment was more pronounced than over-expression while in some change in withanolide content was more than that of sterol content. The variation may arise due to the fact that triterpenoids and sterols are not the immediate products of HMGR. They are intervened by a series of enzymes and their intermediates which are finally converted to product. The plant tissues are acted upon by different downstream enzymes which drive the metabolite pool differentially towards the biosynthesis of terpenoids depending upon the conditions such as over-expression, silencing, inhibitor treatments etc. It has already been established that different tissues and chemotypes of W. somnifera have different withanolide content which to a great extent is governed by HMGR thereby imparting its role in withanogenesis51. Increased HMGR expression with diversion in pathway it selects i.e. either withanolides or sterols may also be due to post-transcriptional modifications of HMGR. Involvement of HMGR in triterpenoid synthesis was earlier reported when over-expression of P. ginseng HMGR in A. thaliana increased the content of sterols and triterpenoids, and in P. ginseng increased ginsenosides52. Although MVA dependent pathway has major participation in sterol and triterpenoid biosynthesis52, yet MEP pathway too has a role with extent, depending on the class of terpenoids51. Our earlier report on W. somnifera has established that major contribution was from MVA pathway rather than MEP pathway25.The application of inhibitor strategy onto untransformed control, vector control and over-expressed lines exhibited decrease in HMGR from less explored O. kilimandscharicum, is reported which has active participation and regulatory role in MVA derived phytomolecules not only in native plant which has terpenoid rich essential oil but in other medicinally important plants such as W. somnifera and A. annua. This is the first report of transformation of a gene in O. kilimandscharicum and hence opens-up new avenues for determination of function of more pathway genes downstream HMGR thereby providing a better understanding of biosynthetic pathway. The study also provides a novel OkHMGR which can be used as a powerful sequence to drive MVA led isoprenoids in medicinally and aromatically important plants.In conclusion, a novel regulatory O. kilimandscharicum plants growing at CSIR-Central Institute of Medicinal and Aromatic Plant, Lucknow53. RNA was analyzed quantitatively and qualitatively and used for cDNA preparation using RevertAid First Strand cDNA Synthesis Kit  according to manufacturer’s protocol. Degenerate primer HMGRDGF and HMGRDGR were designed . Partial OkHMGR sequence was used to design 3′ and 5′ RACE primers. To amplify putative downstream region, PCR reaction was performed with 3′ RACE primer pair of OkHMGRDF1 and oligodT (for primary reaction) and OkHMGRDF2 and oligodT (for nested PCR reaction) under similar conditions as used for partial fragment with change in annealing temperature i.e. 52 °C. For amplification of upstream region, 5′ RACE primer pairs OkHMGRUR1 and AAP (for primary reaction) and OkHMGRUR2 and AUAP (for nested PCR reaction) were used with difference in annealing conditions i.e. 58 °C for 45 s. 50 µl reaction volume was set up for PCR reactions containing 100 ng template, 20 picomole of primers, 25 µl master mix and remaining volume was maintained using nuclease free water. Amplified fragments of both upstream and downstream regions were analyzed, eluted from gel, cloned in pJET1.2 cloning vector and transformed in DH5α E. coli cells. Transformed colonies were screened via colony PCR, plasmids were sequenced and confirmed as part of HMGR by BLAST analysis.Total RNA was extracted from OkHMGR. Using this assembled full length sequence forward OkHMGRFLF and reverse OkHMGRFLR primers were designed containing SalI and HindIII restrictions sites respectively for full length amplification of OkHMGR. The PCR conditions were as follows: 94 °C for 3.0 min, 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 2.0 min followed by a final extension of 72 °C for 7 min. The resultant product was cloned in pJET1.2 cloning vector and transformed into DH5α competent cells. By colony PCR the positive clones of OkHMGR cDNA were screened. Full length OkHMGR was further confirmed by restriction digestion with SalI and HindIII restriction enzymes.All the three sequences were assembled to make full length sequence of OkHMGR were taken and multiple sequence alignment was done by the Mega 6.0654. Motifs and domains were assigned by the online web databases as MEME server55. Physiochemical properties were carried out, by using protpram tool from expasy server (https://web.expasy.org/protparam/). Secondary structure prediction was carried by using GORIV sever, widely used for modeling purposes (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page = /NPSA/npsa_gor4.html).A total of 108 HMGR sequences from 55 plants along with OkHMGR and pET28a plasmid were digested by SalI and HindIII restriction enzymes and both were ligated by T4 DNA ligase. Ligated product, pET-OkHMGR, was transformed in BL21 (DE3) competent cells and confirmed by colony PCR and restriction digestion. For over-expression of pET-OkHMGR in E. coli (BL21) the cells were induced by 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to grow at 18 °C for overnight. The cell lysate was prepared by sonication and analysed by12% SDS gel. For purification of recombinant OkHMGR enzyme, one liter recombinant E. coli BL21 (DE3) culture was induced and purified through Ni-NTA affinity column chromatography. Fractions obtained after elution were screened for protein concentration (A280) as well as for its catalytic activity.Plasmid DNA of pJET cloned full length via HMGR enzyme was carried out at 30 °C using spectrophotometer at 340 nm essentially by using the method of Dale et al.17. Assay mixture consisted of 0.05 mM HMG-CoA, 3.0 mM NADPH, 7.5 mM dithiothreitol (DTT) and 150 mM potassium phosphate buffer (pH 7.0). Absorption coefficient of 6220 M−1 cm−1 at 340 nm for NADPH was used for computation of activity. Further, various catalytic properties of OkHMGR enzyme were estimated by altering required parameters.HMG-CoA-dependent oxidation of NADPH OkHMGR transcripts was determined in six different tissues of the plant i.e. young leaf, middle/mature leaf, large/fully mature leaf, stem, bud and flower as described earlier56. Gene specific qRT  primers were designed using Beacon Designer Software  values. For different stresses and elicitor treatments leaves were treated for 4 hours. In case of wounding the leaves were injured with scalpel blade at different sites and treated samples were then collected after 0.5 h, 3 h and 24 h. Heat and cold stress were given at 55 °C and 4 °C respectively for different time durations i.e. 0.5, 1, 2, 4 hrs. Leaves were also treated with NaCl , salicylic acid and methyl jasmonate , and gibberellic acid  for 3 hrs. All the tissues were subjected to RNA isolation, cDNA preparation and transcript profiling.Relative level of OkHMGR in plants, the coding sequence of gene was cloned in plant binary vector pCAMBIA1303. Coding sequence of OkHMGR cloned in pJET 1.2 cloning vector, was digested with BglII enzyme. The resulting fragment was cloned at BglII site of pCAMBIA1303 under the control of CaMV35S promoter. After confirmation of ligation event through colony PCR and digestion, the resulting pCAMBIA:OkHMGR construct was transformed in A. tumefaciens strain GV3010 via freeze thaw method57. Transformation of pCAMBIA:OkHMGR construct in A. tumefaciens was confirmed by colony PCR with nptII primers  was used.Supplementry FiguresSupplementary Tables


Hypericum perforatum L.) herbal products, there is limited oversight, frequent off-label use and insufficient monitoring of adverse drug reactions. In this study, we use amplicon metabarcoding (AMB) to authenticate 78 H. perforatum herbal products and evaluate its ability to detect substitution compared to standard methods using thin-layer chromatography (TLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Hypericum perforatum was detected in 68% of the products using AMB. Furthermore, AMB detected incongruence between constituent species and those listed on the label in all products. Neither TLC nor HPLC-MS could be used to unambiguously identify H. perforatum. They are accurate methods for authenticating presence of the target compounds, but have limited efficiency in detecting infrageneric substitution and do not yield any information on other plant ingredients in the products. Random post-marketing AMB of herbal products by regulatory agencies could raise awareness among consumers of substitution and would provide an incentive to manufacturers to increase quality control from raw ingredients to commercialized products.Many herbal products have a long history of use, but there are increasing concerns over product efficacy, safety and quality in the wake of recent cases exposing discrepancies between labeling and constituents. When it comes to St. John’s wort ( Hypericum perforatum L.) herbal products are popular in complementary and alternative medicine, and are widely used to treat mild to moderate depression but have a much broader traditional use1. These products play an important role in primary healthcare, and their popularity is determined by consumer health concerns, cultural habits and by the belief that they are natural and thus safe2. Hypericum perforatum is among the top-selling herbs and is sold as over-the-counter (OTC) products in pharmacies, supermarkets, health shops and through e-commerce3. Products are typically labeled as natural foods or dietary supplements, and claims regarding their possible health benefits appear on labels and in associated advertising. In 2011, the global market for herbal products was estimated to be US$83 billion4 with Europe being the largest market.St. John’s wort  or combine different approaches into a single procedure 13. Herbal products are usually highly processed and have numerous ingredients, and applying these methods might not enable the accurate identification of all plant ingredients, especially if target species are admixed with other species within the same genera. To complement traditional identification methods, the EMA, as well as the United States Food and Drug Administration (FDA), support the use of innovative analytical technologies such as DNA barcoding.The European Medicines Agency (EMA) is the European Union agency that is responsible for the evaluation of medicinal products. However, differences in herbal medicine classification exist between EU/EEA member states and this complicates quality monitoring of these products14. Sanger sequencing based DNA barcoding studies have revealed widespread levels of substitution: 6% in saw palmetto herbal dietary supplements15, 16% in ginkgo products16, 25% in black cohosh17, 33% in herbal teas18, and 50% in ginseng19. A blind test of 44 herbal products sold in North America using DNA barcoding7 found that 59% contained species not listed on the labels, and only two out of twelve screened companies had products free of substitution, contamination or unreported fillers7. High-throughput sequencing based amplicon metabarcoding (AMB)20 studies can provide insights into species composition of complex mixtures of DNA such as processed herbal products. For example, in a study by Coghlan et al.5, the species composition of 15 highly processed traditional Chinese medicines (TCM) were evaluated using high-throughput sequencing and found that these contained species and genera included on CITES appendices I and II. Other AMB studies have shown similar concerns of varying quality and product label-content fidelity. For instance, Ivanova et al.21 found that 15 tested herbal supplements contained non-listed, non-filler plant DNA and Cheng et al.22 showed that the quality of 27 tested herbal preparations was highly affected by the presence of contaminants.DNA barcoding is a validated molecular identification method that can provide species-level resolution that is commonly used in authentication of taxonomic provenance of herbal products(Hypericum perforatum), marketed in the EU/EEA both as herbal food supplement and herbal drug. St. John’s wort has traditional indications in nervous system, psychiatric, gastrointestinal, hepatobiliary, renal and urinary, respiratory, thoracic, endocrine, musculoskeletal, metabolism and nutritional disorders, as well as in infections and infestations23. The mode of action of the major responsible bioactive compounds of H. perforatum is still not completely known, but it seems that they act in a synergetic manner to achieve the clinical effectiveness24. However, several studies showed that the antidepressant activity is associated mainly to the phloroglucinol derivative hyperforin25 and the naphthodianthrones hypericin and pseudohypericin26. The quantity and quality of active constituents in Hypericum herbal medicines are highly affected by the manufacturing process1. Treatments involving St. John’s wort are generally safe, but several studies show that use in combination with other drugs can cause potentially life-threatening adverse drug reactions due to pharmacokinetic interactions3. These adverse interactions are still not fully understood despite extensive studies in its mechanisms. Hypericum extracts can cause serious side effects when administrated simultaneously with some antidepressants27. Other studies reported adverse reactions due to high doses of hypericin in Hypericum products leading to a phototoxic effect that may induce photodermatitis and can also decrease or nullify the effect of other drugs when administrated simultaneously28. Hall et al.29 reported exacerbated vaginal breakthrough bleeding, and Murphy et al.30 observed evidence of follicle growth and probable ovulation when simultaneously administrating H. perforatum with low-dose oral contraceptives.In this study, we investigated complex herbal products containing St. John’s wort Hypericum herbal products compared with standard identification approaches suggested by the European Pharmacopoeia and the European Medicine Agency, such as HPLC-MS and TLC.The aim of this study was to evaluate the ability of using AMB to detect substitution in single and multi-ingredient 31, and includes a TLC based identification assay intended to distinguish H. perforatum from other species, including other species within the same genus. The European Pharmacopoeia 8.031Hyperici herba monograph TLC test yields four zones, corresponding to rutin, hyperoside, pseudohypericin and hypericin, calibrated by two reference solution compounds, rutin and hyperoside  hyperoside, hypericin, and pseudohypericin present, but rutin in low or undetectable concentrations. This made presence of H. perforatum or other Hypericum species with indistinguishable chromatograms unlikely, but could indicate presence of H. maculatum or other Hypericum species with indistinguishable chromatograms, for example H. elegans; (3) rutin detected or not, and hyperoside, pseudohypericin and hypericin not detected. This ruled out presence of Hypericum species. Fifty-five out of 74 (74%) samples contained H. perforatum or an indistinguishable adulterant, or a mixture of Hypericum species including H. perforatum or an indistinguishable adulterant. Nine out of 74 (12%) samples contained Hypericum species other than H. perforatum or indistinguishable adulterants, or a mixture of those other Hypericum species. Ten out of 74 (14%) samples did not contain Hypericum species in detectable amounts  out of 78 failed. The test results could be grouped into three categories: (1) rutin, hyperoside, pseudohypericin and hypericin present. This could indicate presence of nts Fig. .Figure 2H. perforatum using HPLC-MS is based on the presence of the two main bioactive compounds in Hyperici herba, hyperforin and hypericin. The Hyperici herba monograph in the European Pharmacopoeia 8.031 states that the dried drug should have a minimum content of total hypericins, expressed as hypericin, corresponding to 0.08% to verify the presence of H. perforatum. A literature review of 88 taxa of Hypericum with qualitative (74 taxa32) and quantitative (27 taxa33) measures of hyperforin and hypericin, together with the six test control species measured in this study  samples hyperoside, hypericin, and pseudohypericin were present, but rutin was present in low or undetectable concentrations, which made presence of H. perforatum or other Hypericum species with indistinguishable chromatograms unlikely, but could indicate presence of H. maculatum or other Hypericum species with indistinguishable chromatograms, such as H. elegans, or a mixture of Hypericum species. In ten out 74 (14%) samples rutin was detected or not, and hyperoside, pseudohypericin and hypericin were not detected, which ruled out presence of Hypericum species. The test results could further be grouped into three categories: (1) hyperforin content higher than hypericin content. This could indicate presence of H. perforatum or other Hypericum species with high levels of hyperforin, e.g. H. elegans, H. olympicum or H. polyphyllum; (2) hyperforin content equal to or lower than hypericin content. This could indicate presence of H. maculatum or other species with low levels of hyperforin, such as H. barbatum Jacq., H. hirsutum L., H. humifusum L., H. linarioides Bosse, H. richeri Vill., H. rumeliacum Boiss. or H. tetrapterum Fr.; (3) no hyperforin or hypericin detected. This indicates that material from Hypericum species is absent or that these compounds are present in undetectable amounts. It should be noted that low levels of hyperforin could also indicate low content of H. perforatum in the tested product. Sixty-one out of 74 samples (82%) had hyperforin content higher than hypericin content; eight out of 74 (11%) had hyperforin content equal to or lower than hypericin content; and five out of 74 (7%) had no detectable hyperforin or hypericin . The tested products showed presence of rutin, hyperoside, pseudohypericin and hypericin in 55 out of 74 (74%) samples, which could indicate cin Fig. .The DNA extracted from 78 samples was highly variable in quantity and quality. Fragment Analyzer measurements gave results for 47 (60%) samples, with DNA concentration ranging from 0.01 to 140 ng/μl. Thirty-one samples (40%) did not contain measurable DNA concentrations: 11 capsules, eight tablets, eight herbal teas and four extracts. PCR amplification reactions were performed for all 78 samples and amplicons were obtained from 76% of the samples for nrITS1 and 73% for nrITS2. The highest amplification rate was obtained for herbal teas (97% for nrITS1 and 88% for nrITS2), followed by capsules (62% for nrITS1 and 62% for nrITS2), tablets (43% for nrITS1 and 45% for nrITS2) and extracts (27% for nrITS1 and 40% for nrITS2). There was no significant correlation between sample total DNA concentration and nrITS amplicon concentration or between amplicon concentration and sequenced reads or bases  for nrITS1 and 47% (37/78 samples) for nrITS2. A dataset consisting of 1,511,356 reads, fulfilling our trimming and filtering quality criteria, was obtained, including 737,010 nrITS1 and 774,346 nrITS2 reads . Forty samples out of 78 samples (51%) yielded no molecular operational taxonomic units (MOTUs) for either nrITS1 or nrITS2 and are excluded from the results and discussion . These included 21 herbal teas, eight capsules, nine tablets and two extracts , and for multi ingredient products, 62% (13 out of 21). The overall ingredient fidelity  for multi ingredient products was 41% and for all products 57%. The following four species were found in more than 50% of the samples: Hypericum perforatum (68%), Convolvulus arvensis L. (63%), Achillea millefolium L. (53%) and Urtica dioica L. (50%). Plant taxa present in more than 20% of the samples are listed in Table Hypericum perforatum was found in all four products, and in addition an average of 20.3 other species.The number of species detected per sample ranged from one to 57, with an average of 18.7 species per sample. Out of the 17 successfully analyzed single ingredient samples, those containing only H. perforatum, that was detected in 68% (26) of the MOTU yielding samples, several other Hypericum species were also detected: H. humifusum in 21% (8), H. tetrapterum in 13% (5) and H. hirsutum in 3% (1). Other Hypericum species were never detected without H. perforatum suggesting that adulteration by admixture is more widespread than complete substitution. The five most common species detected with AMB, but which were not present on the label of the products were: Agrostis gigantea Roth (Poaceae) detected in 24%, Centaurea cyanus L. (Compositae) in 21%, Vaccinium myrtillus L. (Ericaceae) in 21%, Lolium perenne L. (Poaceae) in 18%, and Apera spica-venti (L.) P. Beauv. (Poaceae) in 16% of the samples. A total of 34 anemophilous (wind-pollinated) species were detected, including 31 grasses and the woody species Fraxinus excelsior L., Humulus lupulus L., and Juglans regia L. , and HPLC-MS could be used to detect higher hyperforin than hypericin content in 23 samples (88%). All three methods were in agreement for 19 samples (73%). For the 12 samples that yielded MOTUs using AMB but in which H. perforatum was not detected, TLC could be used to detect rutin, hyperoside, hypericin and pseudohypericin in six samples (50%), and HPLC-MS could be used to detect higher hyperforin than hypericin content in six samples (50%). For five samples (42%), the results from TLC and HPLC-MS were in agreement.In Fig. H. perforatum and/or adulteration of products containing H. perforatum with other Hypericum species, and the common and hyperforin-less H. maculatum in particular, requires the use of accurate analytical methods for the quality control of herbal products of H. perforatum. Identification of Hypericum species is feasible using taxonomic identification keys, but recognition of species in the field is challenging and many Hypericum species have superficially similar morphology. Positive identification requires the researchers to study flower, leaf and stem morphology including diagnostic characters that might be absent early or late in the flowering season. Identification is further complicated as H. perforatum may occasionally hybridize with other species in the genus, resulting in hybrids that have intermediate morphology and secondary metabolite spectra35. Eastern Europe is a significant source of Hypericum material for European herbal products36, and here material is mostly wild-harvested and several different species co-occur, which could easily lead to intentional and/or accidental picking of the different species.Misidentification of H. perforatum  have spectra typical of H. perforatum but also several other species. The 74 analyzed samples had hypericin contents ranging from 0–0.03%, and none passed the minimum threshold set by the European Pharmacopoeia  have no detectable hyperforin or hypericin, eight out of 74 (11%) have low content or absence of 20 for identifying plant species diversity in a range of products47. Comparative identification of processed food and pharmaceutical products is challenging as compared to substrates that can be used for morphological identification, such as pollen clumps48 and pollen in honey45. The advantage of pharmaceutical products and traditional and complementary alternative medicines is that these have their putative contents printed on the package22. Galimberti et al.48, Richardson et al.46, and Hawkins et al.45 used rbcL and trnH-psbA, and nrITS2 and rbcL, respectively, to analyze DNA from pollen in pollen grains and honey to investigate honey bee foraging preferences. Cheng et al.22 used amplicon metabarcoding to analyze nine traditional Chinese medicines (TCMs) and detected on average 4.8 species using nrITS2 and 2.8 using trnL. Coghlan et al.6 analyzed TCMs for presence of both animal and plant ingredients and found over 68 plant families and eight vertebrate genera in these products. Ivanova et al.21 used universal nrITS primers and found a host of plant species in eight herbal supplements, as well as many fungi due to specificity of these primers in amplifying fungal nrITS. In this study, plant specific nrITS primers were used to amplify nrITS1 and nrITS2, and an average of 18.7 species were detected per sample. In addition, the presence of H. perforatum was detected in 26 out of 38 sequenced samples (68%). These findings corroborate previous results that amplicon metabarcoding is an effective way to investigate species composition in products that contain a mixture of DNA from different species. Looking at the subset of registered herbal medicines that yielded AMB data, all four were found to contain H. perforatum, and this was supported by TLC results for all four, and by HPLC results in only two cases.Several studies have shown the resolution and efficacy of DNA metabarcodinget al.21 reported a slightly higher success rate with eight out of 15 samples (53%) for nrITS2 AMB of herbal products from Echinacea, Gingko, Hypericum, Trigonella, and Valeriana. Cheng et al.22 reported a 100% success rate for 30 individual samples of TCM, all of which were unprocessed crude drugs. The varying degrees of success probably reflect the quality and type raw material, but also the many details in the analysis that can be varied to optimize the results, roughly in order of significance: extraction procedures and purification, primers, markers, identification approach, clustering, MOTU thresholds, sequencing platform, filtering, quality thresholds and chimera removal, library preparation, and amplification protocols.The relatively low success rate (49%) after applying strict read quality and filtering criteria makes this method challenging to use for routine screening at this time. Ivanova 49. Secondly, AMB is a PCR-based method, and variation in nrITS copy number, primer annealing, and amplification bias all influence the number of taxon-specific reads50. Thirdly, incomplete reference databases and sequences with incorrect species names, can render taxonomic identifications prone to uncertainties.Quantifying contamination is important when focusing on the tolerated levels of foreign matter in herbal pharmaceuticals. Quantifying relative species abundances based on sequence read numbers from samples with unknown ingredients is hampered by several factors. Firstly, AMB relies on the availability of DNA, but plant DNA can be removed or highly degraded during the harvesting, drying, storage, transportation, and processing H. perforatum, in 68% of the samples, but in addition other Hypericum species were detected, H. humifusum in 21% (8), H. tetrapterum in 13% (5) and H. hirsutum in 3% (1) of the samples. Hypericum tetrapterum belongs to the same taxonomic section as H. perforatum but is more closely related to H. maculatum, whereas H. humifusum and H. hirsutum belong to sections Oligostema and Hirtella s.l., respectively52. These species can co-occur in natural habitats from which H. perforatum is wild crafted. These other Hypericum species were never detected without H. perforatum suggesting that adulteration by admixture is more widespread than complete substitution.AMB detected the target species, 53. The detection of additional plant species, other that the ones from the label or those that can be expected as substitutes, contaminants or fillers, may be explained by (1) amplified PCR chimeras; (2) false-positive BLAST identifications due to incomplete or error-prone reference databases; or (3) presence of pollen from anemophilous (wind-pollinated) species. Thirty-four of the MOTUs belong to anemophilous species, including 31 grasses of which Agrostis gigantea Roth (Poaceae) was detected in 24% of samples, Lolium perenne L. (Poaceae) in 18%, and Apera spica-venti (L.) P.Beauv. (Poaceae) in 16%. DNA from pollen from these species can end up in the products through co-occurrence with the collected material in natural habitats as well as during other steps in the process chain.The overlooked species diversity through poor primer fit and amplification bias is difficult to quantify but some diversity is likely missedH. perforatum and simultaneously to detect substitution, adulteration and/or admixture of other species. These results corroborate with previous results that show the usefulness of metabarcoding for use in complementing traditional methods of quality control for consumer safety22. It should be emphasized however that the relatively low success rate of generating sequence reads per product makes this method challenging to use for routine screening, as only 49% of the tested products yielded sequences as compared to 95% of products that could be used for TLC and HPLC-MS analyses. Moreover, the high sensitivity of AMB in detecting everything from grass pollen on field-collected plants to plant dust left in production equipment requires a careful consideration of the concept of contamination. AMB results for herbal pharmaceutical authentication should be interpreted with a focus on presence and or absence of target species, i.e. the labeled ingredients, but alarms need not be raised over trace contaminations from species plausibly present in the cultivation, transport or production chain. However, the TLC and HPLC-MS results show that these methods are of limited applicability with regard to detecting species substitution, but may be used efficiently to detect target compounds. The clear advantage of HPLC-MS over TLC is the ability to quantify constituents and to screen for a vastly larger number of compounds. If product safety relies on threshold levels of specific bioactive compounds, absence of toxins, allergens and admixed pharmaceuticals, then chemical analysis methods are more relevant than DNA based composition analysis, but if product fidelity, species substitution or adulteration is suspected then the latter method outperforms in terms of resolution. Development of novel molecular markers and approaches for genomic barcoding are likely to increase the resolution of DNA barcoding in species-level identification. Several others have advocated the use of DNA barcoding and metabarcoding in herbal product authentication and herbal pharmacovigilance56 and adoption of standards for quality control by regulatory agencies could raise product quality and increase consumer confidence.The metabarcoding results confirm that AMB can be used to test for the presence of Hypericum perforatum according to the label were randomly purchased in European countries (and Turkey), including Romania (51), Germany (5), Poland (4), Turkey (4), Slovakia (3), Spain (2), UK (2), Austria (2), Czech Republic (1), France (1), Italy (1), Sweden (1) and the Netherlands (1). The samples were bought from pharmacies (44), herbal shops (25), super markets (2) or via e-commerce (7), and were sold as herbal teas (44), capsules (15), tablets (14) and extracts (5). According to the label information, the products included 38 single ingredient products, 33 products contained between two and ten ingredients and seven products contained more than ten ingredients. These medicinal products for scientific analyses were imported into Norway under Norwegian Medicines Agency license no. 16/04551–2. An overview of the samples including label information, but not the producer/importer name, lot number, expiration date or any other information that could lead to the identification of that specific product can be found in Supplementary Table Seventy-eight herbal products that included H. elegans (voucher ARNHM01He), H. maculatum (ARNHM01Hm), H. maculatum (92151 UMF CLUJ), H. olympicum (ARNHM01Ho), H. patulum (ARNHM01Hpa), H. perforatum (92141 UMF CLUJ) and H. polyphyllum (ARNHM01Hpo) were used as references for the identification and quantification of the main compounds. The reference species were selected to include the main adulterant, H. maculatum , species with known high levels of hyperforin and hypericin, H. polyphyllum and H. olympicum, as well as random species collected during fieldwork in Romania, H. patulum and H. elegans. All voucher specimens are deposited in the Herbarium of the Alexander Borza Botanical Garden (CL) of Babes-Bolyai University, Cluj-Napoca, Romania.For the phytochemical analysis, aerial parts of Hyperici herba monograph in the European Pharmacopoeia 8.031. Control solutions were prepared by mixing 500 mg of ground identified and vouchered material of selected Hypericum species with 10 ml of methanol  for 10 min at 60 °C. After cooling, the obtained solution was filtered. The reference solution was obtained by dissolving 5 mg of hyperoside  and 5 mg of rutin  in 5 ml of methanol. The test solutions of the Hypericum products were prepared in the same way as the control solution. Herbal products were processed depending on the pharmaceutical formulation and following the principles of the same extraction procedure, which were adapted to each pharmaceutical formulation. The analysis of the samples was performed in triplicate on SilicaGel plates , in twin bands of 10 mm, consisting of a 10 µl test sample and a 5 µl reference solution. As a mobile phase, a mixture of formic acid :distilled water:ethyl acetate  (6:9:90 V/V/V) was used. After migration of the principal components, the plates were dried at 100–105 °C for 10 min. Detection was performed in UV light, at 365 nm, after spraying with a mixture of 1% methanolic diphenylboryloxyetylamine (Sigma-Aldrich) and 5% methanolic polyethylene glycol 400 (Sigma-Aldrich) and 30 min incubation.Samples were processed according to the Hypericum extracts were prepared by adding 750 mg of powdered plant material of Hypericum species, H. perforatum and H. maculatum, to 15 ml methanol in a glass tube57. The tubes were capped and agitated in the dark at 25 °C for 3 hours on a digital ceramic hotplate stirrer (Arec. X. Velp Scientifica). The extracts were filtered and diluted (1:100) in the mobile phase consisting of a mixture of 1 mM acetonitrile (Merck)/ammonium acetate (Merck) 45/50 (V/V) in double distilled, deionised water (Infusion Solution Laboratory of the University of Medicine and Pharmacy Cluj-Napoca - Romania), and 1 µl of the mixture was injected into the HPLC chromatographic system. Quantities of the herbal product test samples were individually adapted in order to identify and quantify the reference compounds and the extraction procedure was subsequently followed as described above for the references. The HPLC system used was an 1100 series Agilent Technologies model  consisting of a G1312A binary pump, an in-line G1379A degasser, a G1329A autosampler, a G1316A column thermostat and an Agilent Ion Trap Detector 1100 SL. Chromatographic separation was performed on a Zorbax SB-C18  column (Agilent Technologies) equipped with a Zorbax SB-C18 precolumn with the mobile phase above, at 45 °C with a flow rate of 0.6 ml/min. The detection of analytes was performed in triplicate in non-reactive MS2 mode for the quantification of hypericin  or in reactive MS2 mode for hyperforin (Sigma), negative ion ionisation, using an ion trap mass spectrometer equipped with an electrospray ionisation ion source (ESI): capillary +2500 V, nebulizer 40 psi (nitrogen), dry gas nitrogen at 8 l/min, dry gas temperature 350 °C.2 > 0.998) were obtained. Positive identification of the target compounds was performed by mass-spectrometry, and quantification of hypericin and hyperforin was based on peak area  in comparison with the standard curves.Standard calibration curves were obtained by plotting the peak areas of standard concentrations of hypericin  and hyperforin  against their nominal concentrations. Two linear regression equations . The final elution volume was 100 μl, and extracted DNA was quantified using a Fragment Analyzer™  and a DNF-488-33 HS Genomic DNA Reagent Kit .Total DNA was extracted using a modified CTAB extraction method from small amounts of each herbal product (about 300 mg)59. The forward primers were labeled with unique 10 bp multiplex identifier (MID) tags and the reverse primers with uniform truncated P1 (trP1) tags. Thermal cycling was carried out in 25 μl reaction volumes, and each reaction contained 5 μl 5X Q5 reaction buffer , 1.5 μl 10 μM of each primer , 0.5 μl 10 mM dNTPs, 0.25 μl 20 U/μl Q5 High-Fidelity DNA Polymerase , 5 μl 5X Q5 High GC enhancer, 10.75 μl of Milli-Q ultrapure water and 0.5 μl of template DNA. The following thermocycling protocol was used: 30 s of initial denaturation at 98 °C, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 30 s, and elongation at 72 °C for 30 s, followed by a final elongation step at 72 °C for 2 min. The annealing temperature was 56 °C for nrITS1, and 71 °C for nrITS2.All amplicon libraries were prepared using fusion PCR based on two nuclear ribosomal target sequences, internal transcribed spacers nrITS1 and nrITS2. PGM fusion primers were based on 17SE and 5.8 I1, and 5.8 I2 and 26 SE, respectivelyThe size, purity and the molar concentration (nmol/l) of each amplicon library was measured using a Fragment Analyzer™  and a DNF-910 dsDNA Reagent Kit . An equimolar pool (2 ng/μl/library) was prepared from the amplicon libraries using the Biomek 4000 Laboratory Automation Workstation . Agencourt AMPure XP  was used for removal of unincorporated primers and nucleotides using the manufacturer’s instructions (Agencourt AMPure XP v. B37419AA). The total concentration of the purified pooled amplicon library stock and three serial dilutions  were analyzed using the Fragment Analyzer™  and DNF-488 High Sensitivity Genomic DNA Analysis Kit in order to identify the optimum concentration range for the template preparation.An Ion Chef (Life Technologies (LT), Thermo-Fisher Scientific, USA) was used to prepare pooled Ion AmpliSeq libraries (LT) for emulsion PCR and to load the sequencing chips. The input DNA template concentration was adjusted to the number of Ion Sphere Particles (ISPs) and added to the emulsion PCR master mix. The emulsion PCR was done using the Ion Chef, and template-positive ISPs were enriched and loaded on an Ion 318 v2 Chip (LT) and sequenced on an Ion Torrent Personal Genome Machine (LT) using an Ion PGM Sequencing 400 kit (LT). Sequencing read data was analyzed and demultiplexed into FASTQ files per sample using Torrent Suite version 5.0.4 (LT).60 available as a Galaxy pipeline at the Naturalis Biodiversity Center (http://145.136.240.164:8080/). Using the HTS pipeline, nrITS1 and nrITS2 primer sequences were used to demultiplex the sequencing reads per sample and to filter out reads that did not match any of the primers. PRINSEQ61 was used to determine filtering and trimming values based on read lengths and Phred read quality62. All reads with a mean Phred quality score of less than 26 were filtered out, as well as reads with a length of less than 300 bp. Remaining reads were trimmed to a maximum length of 440 bp for nrITS1 and 350 bp for nrITS2. CD-HIT-EST63 was used to cluster reads into molecular operational taxonomic units (MOTUs) defined by a sequence similarity of >99% and a minimum number of ten reads. The consensus sequences of non-singleton MOTUs were queried using BLAST64 against a reference nucleotide sequence database, with a maximum e-value of 0.05, a minimum hit length of 100 bp and sequence identity of >97%. The number of reads per MOTU, as well as the BLAST results per MOTU, were compiled using custom scripts from the HTS Barcode Checker pipeline60. The reference sequence database consisted of a local copy of the NCBI/GenBank nucleotide database.FASTQ read files were processed using the HTS-barcode-checker pipelineIon-Torrent amplicon read data is deposited in DRYAD: doi:10.5061/dryad.32j7r.Supplementary DataSupplementary Data S7


Angelica sinensis, a nucleotide signature consisting of 37-bp (5′-aatccgcgtc atcttagtga gctcaaggac ccttagg-3′) was developed. It is highly conserved and specific within Angelica sinensis while divergent among other species. Then, we designed primers (DG01F/DG01R) to amplify the nucleotide signature region from processed materials. 15 samples procured online were analysed. By seeking the signature, we found that 7 of them were counterfeits. 28 batches of Chinese patent medicines containing Danggui were amplified. 19 of them were found to contain the signature, and adulterants such as Ligusticum sinense, Notopterygium incisum, Angelica decursiva and Angelica gigas were detected in other batches. Thus, this nucleotide signature, with only 37-bp, will broaden the application of DNA barcoding to identify the components in decoctions, Chinese patent medicines and other products with degraded DNA.It is very difficult to identify Angelicae sinensis radix (Danggui) when it is processed into Chinese patent medicines. The proposed internal transcribed spacer 2 (ITS2) is not sufficient to resolve heavily processed materials. Therefore, a short barcode for the identification of processed materials is urgently needed. In this study, 265 samples of Angelicae sinensis radix and adulterants were collected. The ITS2 region was sequenced, and based on one single nucleotide polymorphism(SNP) site unique to  Angelica sinensis (Oliv.) Diels, is a well-known Pharmacopoeia-recorded material medica in China456Angelica biserrata, whose synonym is Angelica pubescens f. biserrata; Ligustici rhizoma et radix (Gaoben), derived from Ligusticum sinense or Ligusticum jeholense; Peucedani decursivi radix (Zihuaqianhu), derived from Angelica decursiva, whose synonym is Peucedanum decursivum; Notopterygii rhizoma et radix (Qianghuo), derived from Notopterygium incisum or Notopterygium franchetii; the root of Levisticum officinale, A. gigas, A. magaphylla, A. valida, A. acutiloba also can be adulterants with Angelicae sinensis radix. Among these, A. gigas is used as “Danggui” in Korea and Yanbian, Jilin Province; A. acutiloba is also a statutory “Danggui” species recorded in the Japanese Pharmacopoeia. Because it has morphological characters that are similar to those of its adulterants, taxonomic identification based on morphology is very difficult, especially after it has been processed into Chinese patent medicines. Previous studies have reported that ferulic acid can be used as a chemical marker to detect Angelicae sinensis radix in Chinese herbal decoctions via HPLC910Angelicae sinensis radix (Danggui) derived from et al. used ITS2 to distinguish commercial Rhodiola products successfully and found that 60% contained adulterantset al. identified Acanthopanacis cortex and its adulterants with ITS2et al. designed specific ITS primers to identify A. sinensis and its adulterantsA. sinensis from Chinese patent medicines or decoctions. Meusnier et al. proposed a “mini-barcode” sequence to overcome the amplification problem of degraded DNAet al. successfully obtained an 88-bp fragment from TCM decoction, but the amplification of the longer fragment was unsuccessfulet al. designed six mini-barcodes (127~314-bp) to detect processed fish products. While the 650-bp length (CO1) could distinguish only 20.5% of the species tested, mini-barcoding achieved 88.6% successful identification, indicating that shorter fragment lengths are more likely to be amplified from degraded DNAAglaia stellatopilosaet al. successfully developed a 23-bp signature for identification of ginseng products and found some adulterants in Chinese patent medicinesDNA barcoding technology uses a standard gene fragment for rapid identification of unknown specimens to species level. Its accuracy and repeatability have made it successful in species identification13A. sinensis species. The method was also used for analysis of Chinese patent medicines containing Angelicae sinensis radix.In this study, we aimed to develop a short gene identifier for the identification of Angelicae sinensis radix and its Chinese patent medicines. A 37-bp (5′-aatccgcgtc atcttagtga gctcaaggac ccttagg-3′) nucleotide signature was found. This exclusive nucleotide signature of Angelicae sinensis radix is completely conserved within the A. sinensis and closely related species from experimental materials and our previous study were aligned, and the aligned length was 454-bp long. The results showed that 126 sequences of the A. sinensis species were highly conserved. Simultaneously, a SNP at position 193 was found to be unique to A. sinensis. Based on this SNP site, we developed a 37-bp nucleotide signature (5′-aatccgcgtc atcttagtga gctcaaggac ccttagg-3′) exclusively specific for A. sinensis.A total of 265 ITS2 sequences of Angelica species from GenBank were annotated to this 37-bp nucleotide signature region and were aligned using MEGA 5.0  was amplified and sequenced using the universal primer pair ITS2F/3R. In addition to “Danggui extract powder” and the decoctions, 13 other products were successfully amplified and sequenced. We designed a specific primer pair to amplify the signature from the degraded DNA. The results showed that the shorter barcode can still be amplified and that the length of amplification is 167-bp . The PCRThere are many types of Chinese patent medicines containing Angelicae sinensis radix. Most Chinese patent medicines contain different species, and we selected Huoxue Zhitong Capsule for the preliminary experiment. Huoxue Zhitong Capsule contains Angelicae sinensis radix and 5 other ingredients as follows: Notoginseng Radix et Rhizoma (Sanqi), Olibanum (Ruxiang), Borneolum Syntheticum (Bingpian), Eupolyphaga Steleophaga (Tubiechong) and Pyritum (Zirantong). We tested the availability of the specific designed primer pair DG01F/DG01R to amplify the nucleotide signature region of Angelicae sinensis radix from this Chinese patent medicine. The results showed that the targeted nucleotide signature of Angelicae sinensis radix was successfully amplified and sequenced from the 2 batches of Huoxue Zhitong Capsules.The other 26 batches of Chinese patent medicines containing Angelicae sinensis radix were also tested, including Wuji Baifeng Pills, Deida Pills, Renshen Jianpi Pills. All the Chinese patent medicines contained 8 to 56 ingredients. Details regarding the Chinese patent medicines are shown in A. gigas was found in 1 batch of Guipi Pill and 1 batch of Duhuo Jisheng Pill. Other adulterants such as Ligustici rhizoma et radix (Gaoben), Peucedani decursivi radix (Zihuaqianhu) and A. acutiloba, were also detected. Another type of adulterant Carum carvi, was also found in 2 batches of Niuhuang Qingxin Pills  was detected in 1 batch of Dingkun Pill and 1 batch of Wuji Baifeng Pill, and in Pills . These rDue to the fact that Chinese patent medicine ingredients are extremely complex, no standardization for Chinese patent medicines has been established. Additionally, because market supervision is not strict and given the lack of regulations in this field, the composition and quality of these medicines and healthcare products are not guaranteedet al. used it to identify Angelica species and demonstrated that the ITS region is the most suitable DNA barcode for identification of this genus, with 100% PCR and sequencing successet al.Molecular identification can be a powerful complement to traditional identification methods in identifying Chinese patent medicines. Yuan A. sinensis signature. Via Basic Local Alignment Search Tool (BLAST) analysis, we found that the nucleotide signature was not present in any other species. Thus, the nucleotide signature developed in this study can be used as a “species-specific marker” for Angelicae sinensis radix.The ideal nucleotide signature should be completely conserved within a specific species. In this region, even the closest related species has one locus that differs from the As the main adulterant on the market, Angelicae pubescentis radix is another type of Pharmacopoeia-recorded material medica, and its price is lower than that of Angelicae sinensis radix. The two medicinal components share similar morphological characteristics but have different pharmacological properties and medicinal values. Angelicae sinensis radix is mainly used to enrich the blood and promote blood circulation as a dietary therapy, while Angelicae pubescentis radix is known to rheumatism and relieve pain in waist and knee. They should not be used as substitute for the medicine labelled in the patent medicine.Carum carvi, which is not mentioned as a labelled component, was also amplified (169-bp) from 2 batches of Niuhuang Qingxin Pills. This result indicates that Carum carvi adulterated with target species Saposhnikoviae radix (Fangfeng) is a very common phenomenon in Chinese patent medicines. In this study, the 167-bp fragment containing the nucleotide signature was successfully amplified from powder, slices, extract powder, decoctions and Chinese patent medicines using the new primer DG01F/DG01R. Samples with the nucleotide signature were successfully identified as Angelicae sinensis radix. This nucleotide signature method is a type of absolute identification, and it will broaden the applications of DNA barcoding for market supervision.By seeking the nucleotide signature, the percentage of adulterated samples was 26.92% in Chinses patent medicine and 77.78% in “Danggui powder”. In addition to Angelicae sinensis radix and its adulterants, et al. reported a novel method for detecting Hg2+ using double-stranded DNA-carrying gold nanoparticles (dsDNA-GNP) within 1 minute with the naked eyeet al. developed a SNP microarray with capture and detection probes to simply, rapidly, and sensitively assay the genotyping of the samplesBecause Angelicae sinensis radix can be identified very accurately and rapidly using the 37-bp nucleotide signature, this method can be developed to provide on-site and real-time detection technology. In recent years, using gold nanoparticles (GNPs) for on-site detection has become a rapidly developing research field. Naoki In the future, we can combine GNPs and this 37-bp nucleotide signature method to develop an on-site and real-time detection technology. With this technology, specific detection probes, complementary target sequences and oligonucleotides with this 37-bp nucleotide signature sequence can be designed. Due to the high sensitivity of this technology, its use, with species-specific probes, can be extended to identify degraded DNA samples. Futhermore, we also aimed to develop an inexpensive and user-friendly biological sensor to detect Angelicae sinensis radix with a low limit of detection enabling Chinese patent medicines to be identified in a fast, effective and stable manner.In conclusion, the nucleotide signature developed in this study provides users with an easy and absolute authentication method and may make a major contribution to the detection of counterfeit products in the Chinese patent medicine market.A. sinensis and its adulterants were collected from China and Japan. The details regarding these samples are shown in Angelica genus were downloaded from GenBank to verify their nucleotide signatures. A total of 429 complete ITS2 sequences representing 72 species of Angelica were obtained for further study  was boiled in 300 mL of double-distilled water for 120 min, 180 min and 240 min, and the decoctions were used for DNA extraction.Specimens: The samples were ground into fine powder using a Retsch MM400 laboratory mixer mill  at a frequency of 30 Hz. Then genomic DNA was extracted using a Plant Universal Genomic DNA Kit , according to the manufacturer’s instructions. ITS2 was amplified using universal primersg for 3 min to obtain the precipitate for DNA extraction. Chinese patent medicines were subjected to cryogenic grinding in liquid nitrogen using a mortar and pestle. After adding 700 μL of pre-wash buffer  to wash the Chinese patent medicine powder several times until the supernatant was clear and colourless, the mixture was centrifuged at 7500× g for 5 min at room temperature. Then, genomic DNA was extracted from the precipitate according to the protocol of the Plant Universal Genomic DNA Kit (Tiangen).Decoctions and Chinese patent medicines: Decoctions were centrifuged at 12000× TM, Bio-Rad, Hercules, CA, USA) using the following thermal program: 94 °C for 5 min, followed by 40 cycles of denaturation (94 °C for 30 s), annealing (53 °C for 30 s), elongation (72 °C for 45 s), and a final 10 min extension at 72 °C. The PCR products were examined using 1.5% agarose gel electrophoresis and purified for bidirectional sequencing using an ABI 3730XL sequencer  based on the Sanger sequencing method, at the National Key Facilities for Crop Genetic Resources and Improvement, the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.A new primer pair, DG01F/DG01R, for amplification of the degraded DNA was designed using Primer Premier 6.0 software . PCR was performed in a 25-μL reaction system containing 12.5 μL of 2× PCR Master Mix , 2.0 μL of each primer (2.5 μM), and 1 μL (about 50 ng) of DNA templates and filled with double-distilled water. The reactions were performed with a thermal cycler  in a pMDTM19-T Vector , according to the manufacturer’s protocol. Ampicillin-resistant single colonies on Luria-Bertani medium were confirmed and selected by PCR with primers DG01F/DG01R. The above cultured clones were used for sequencing at the National Key Facilities for Crop Genetic Resources and Improvement, the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.TA cloning experiments: PCR products of Chinese patent medicines were tested via agarose gel electrophoresis, and the desired bands were excised and recovered using a TIANgel Midi Purification Kit (Tiangen). Angelica species were selected and aligned using MEGA 5.0 softwareSequences were edited and assembled manually using CodonCode Aligner 5.1.4 . ITS sequences from GenBank were annotated using the Hidden Markov model (HMM) to obtain ITS2 sequencesHow to cite this article: Wang, X. et al. A Nucleotide Signature for the Identification of Angelicae Sinensis Radix (Danggui) and Its Products. Sci. Rep.6, 34940; doi: 10.1038/srep34940 (2016).


Necrotizing enterocolitis (NEC) continues to be a major cause of neonatal morbidity and mortality. We describe the added value of therapeutic drug monitoring by presenting the case of a preterm infant with severe NEC treated with meropenem. Dosing strategy will achieve adequate patient outcome when treating pathogens with elevated MIC. As safe as meropenem is, there are not enough data for 40 mg/kg, every 8 h infused over 4 h; accordingly, strict monitoring of blood levels is mandatory. Based on our findings, a 4 h prolonged infusion of 40 mg/kg meropenem, every 8 h, will achieve an adequate patient outcome. Necrotizing enterocolitis (NEC), one of the most common gastrointestinal emergencies in preterm infants, continues to be a major cause of neonatal morbidity and mortality. Early recognition and aggressive management have improved clinical outcome, although the exact etiology remains unknown. Given the polymicrobial intra-abdominal nature of this infection, broad-spectrum or combination antimicrobial agents are most often initiated . Meropen9/L, normal range: 150–450.10 E9/L). He was started on IV cefotaxime, vancomycin, and metronidazole, serial abdominal exams were performed, and the pediatric surgical team was consulted. Blood cultures remained negative. Seven days after initiation of medical therapy (DOL 36), the surgical team reevaluated the infant. It was decided to proceed with an exploratory laparotomy given the progressive clinical decline and respiratory alerts. Intraoperative findings included malodorous and purulent free fluid present in abdomen, clear necrotizing enterocolitis (NEC grade IIIb) affecting the whole colon (with 4 covered perforations) and the distal small bowel, with intra-abdominal leakage of feces and excessive bleeding leading to a complete colectomy until the sigmoid, while creating a jejunostomy and small fistula. On postoperative day 6, a focal fluid collection was detected by ultrasonography. Peritoneal fluid culture was positive for Enterobacter cloacae complex, while peripheral blood cultures remained negative. The antimicrobial therapy was changed to IV meropenem, amikacin, and fluconazole based on E. cloacae complex susceptibilities : 1 μg/mL (E-test)). Based on the severity of the situation, appropriate meropenem dosage was optimized by means of real-time TDM monitoring. Serum samples were analysed using a validated High-Performance Liquid Chromatography method with Diode-Array Detection. Meropenem was initially administered as a 30-minute infusion of 20 mg/kg, every 8 h. TDM monitoring, conducted 1 h after the infusion of the fourth dose, revealed the complete absence of meropenem. The optimal transport conditions  were respected. Therefore the dose was increased to 30 mg/kg . Based on the infected intra-abdominal collection and increase of inflammatory parameters  a new laparotomy was performed on DOL 52 for drainage of it. Intraoperatively the infected collection and the existing but necrotic jejunostomy were removed. The frailty of the bowel was too high to create a new stoma, so a first Petzer sonde was placed in the terminal ileum and sorted cutaneously, and a second Petzer sonde was placed for a more proximal perforation. In the light of the infectious problem, very difficult wound healing and complete dehiscence of the wound suture were observed, followed by slow granulation over the following weeks. Peritoneal fluid cultures remained positive for E. cloacae complex with a gradual increase of MIC until an intermediate susceptibility value, 6 μg/mL (EUCAST), but with negative molecular carbapenemase producing Enterobacteriaceae screening. On DOL 59, after complete eradication of E. cloacae complex (repeated negative cultures), the antibiotic regimen was switched to IV ciprofloxacin and metronidazole for a 14-day course, after 18 days of extended meropenem infusion. A fourth laparotomy followed aiming to create a distal stoma, while during the same intervention the proximal stoma was closed. The patient received 5 weeks of broad-spectrum antibiotics in total. He was discharged in a stable clinical condition at 36 weeks of postmenstrual age weighing 4,370 grams.A preterm male infant was born by cesarean section at 30 and 5/7 weeks' gestation weighing 1,140 grams, of which the monochorionic diamniotic twin pregnancy was complicated by twin-to-twin transfusion syndrome (grade I) and severe intrauterine growth restriction. Maternal antibiotics and betamethasone were administered prior to delivery. Apgar scores of 7 and 9 were assigned at 1 and 5 minutes of life. He knew a good start with a spontaneous heart activity and received 30% oxygen and PEEP by Neopuff, which was replaced by nasal continuous positive pressure ventilation (CPAP), which persisted during his stable transport to the neonatal intensive care unit (NICU) and continued until day 4. He was started on caffeine for prevention of apnea and bradycardia, and it could be stopped on day of life (DOL) 16. Due to immaturity of preterm intestinal mucosa and risk for development of NEC minimal enteral feeds were initiated on DOL 2 and only on DOL 7 the quantity was prudently increased, reaching full enteral feeds by DOL 17. On DOL 29 he had an acute onset of progressive abdominal distention and general malaise, clinical and radiologic compatible with NEC stage IIb (modified Bell's staging). Abdominal RX findings included explicit signs of pneumatosis intestinalis at the right hypochondrium and the left flank and residual air at the level of the vena porta bifurcation. Enteral feeds were stopped, and gastric decompression with continuous suctioning and a sepsis workup was initiated. A complete blood count, metabolic profile, blood gas, and blood culture were drawn, which revealed anemia  and thrombocytopenia  as premature infants could be thought of as immune compromised for whom a target of 40–50% T > MIC may be insufficient. These targets were defined based on the Clinical and Laboratory Standards Institute (CLSI) recommended MIC breakpoints of meropenem for Pseudomonas aeruginosa (susceptible: <2 μg/mL and resistant: >4 μg/mL) which closely correspond to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (susceptible: ≤2 μg/mL and resistant: >8 μg/mL) [μg/mL) [ E. cloacae complex (2 μg/mL) and PK-PD target of 75% of the dosing interval > MIC with the assumption that neonates are immune-compromised host. In general, for intermediate or resistant microorganisms (with meropenem MICs ≥ 2 μg/mL) better PK/PD target attainment is obtained with prolonged 4 h infusion [ E. cloacae complex with T > MIC: 50% of time > 4 μg/mL and 75% of time > 2 μg/mL.Severe NEC is a life-threatening condition requiring prompt intravenous therapy with broad-spectrum antimicrobial agents . Meropen8 μg/mL) . Limitedinfusion . A poteninfusion . In conc


Perfluorooctyl bromide (PFOB) enclosed nanoparticles (NPs) as ultrasonic contrasts have shown promising results in the recent years. However, NPs display poor contrast enhancement in vivo. In this work, we used the copolymers poly(lactide-co-glycolide) carboxylic acid (PLGA-COOH) and poly(lactide-co- glycolide) poly(ethylene glycol) carboxylic acid (PLGA-PEG-COOH) as a shell to encapsulate PFOB to prepare a nanoultrasonic contrast agent. The NPs were small and uniform (210.6 ± 2.9 nm with a polydispersity index of 0.129 ± 0.016) with a complete shell nuclear structure under the transmission electron microscopy (TEM). In vitro, when concentration of NPs was ≥10 mg/ml and clinical diagnostic frequency was ≥9 MHz, NPs produced intensive enhancement of ultrasonic gray-scale signals. NPs could produce stable and obvious gray enhancement with high mechanical index (MI) (MI > 0.6). In vivo, the NPs offered good ultrasound enhancement in tumor after more than 24 h and optical imaging also indicated that NPs were mainly located at tumor site. Subsequent analysis confirmed that large accumulation of fluorescence was observed in the frozen section of the tumor tissue. All these results caused the conclusion that NPs encapsulated PFOB has achieved tumor-selective imaging in vivo. In recent years, ultrasonic molecular imaging has become a promising method for cancer diagnostics because it can image an intact living body at cellular and subcellular level with high spatial and temporal resolution, low cost, portability, and lack of ionizing irradiation . UltrasoNanobubbles were used in most studies related to nanoscale UCAs because they produced good sound reflection –8. HowevHere, we would use hybrid membrane materials of PLGA-PEG-COOH and PLGA-COOH packaged PFOB to prepare nanoscale UCAs, which all had complete core-shell structure with a good gray enhancement in the aqueous solution in vitro. In in vivo experiments, the enhancement of ultrasound gray scales at the tumor site was analyzed after tail intravenous injection. Finally, tumor-selective imaging of NPs was assessed by both small animal optical imaging and fluorescence microscope.2-PEG-COOH (Mw = 3400) was purchased from Beijing Kaizheng Biotech Co., Ltd. . Polyvinyl alcohol , 4′-6-diamidino-2-phenylindole (DAPI), and coumarin-6 were provided by Sigma-Aldrich . Perfluorooctyl bromide (PFOB) was purchased from Aladdin Industrial Corporation . HepG2 cells (human liver hepatocellular carcinoma cell line) were purchased from the Cell Institute of the Chinese Academy of Sciences . All other reagents were of analytical pure grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. .Poly (50/50) with terminal carboxylate groups  was purchased from Jinan Daigang Biotech Co., Ltd. . NH2 to PLGA-COOH according to a carbodiimide–N- hydroxysuccinimide (EDC/NHS)-mediated chemistry [3 and characterized by 1H NMR at 400 Hz  to determine the modification ratio of PEG on PLGA.Carboxylate-functionalized copolymer PLGA-PEG was synthesized by conjugating COOH-PEG-NHhemistry   aqueous solution to form a preemulsion. The preemulsion was sonicated at 300 W for 2 min over ice. Organic solvents were then evaporated for 3 h in a thermostatic bath (30°C) to remove the methylene chloride. To acquire microparticles encapsulated PFOB (MPs) as contrast, the organic solvents were evaporated immediately after the preemulsion's formation. NPs/MPs labeled coumarin-6/DiR were prepared by adding 50 μl coumarin-6/DiR to the organic solution prior to emulsification to label the polymer phase.PFOB was encapsulated in nanoparticles by modifying the emulsion solvent evaporation method  Figure . BrieflyParticle diameter, size distribution, and zeta potential of the NPs/MPs were measured by a dynamic light-scattering system  at 25°C. The structure of the NPs was examined by transmission electron microscopy . The morphology of microparticles was observed by optical microscope .A weighed amount of NPs was dissolved into methanol following 5 min ultrasonic treatment and then centrifuged for 10 min. PFOB concentration of supernatant liquor was measured by gas chromatograph (GC) at 300°C, using flame ionization detector (FID). Percentages of encapsulation efficiency (% EE) were calculated based on the following equations:Each NP/MP suspension sample was filled in an Eppendorf tube. Ultrasound images were obtained in a nonlinear mode with a commercial ultrasound imaging system . All images were acquired using the same instrument parameters: frame rate (FR) 26, brightness (BG) 20, and dynamic range (DR) 65 db.7 HepG2 cells were inoculated subcutaneously into the right hind legs of the mice. All in vivo experiments began when the diameter of tumors reached 0.8–1.2 cm. The laboratory animal management committee and ethics committee at the Third Xiangya Hospital of Central South University approved all animal experiments.Nude male BALB/c mice (age of 5 weeks) were obtained from Hunan Slake Jingda Experimental Animal Co., Ltd. . Approximately 1.0 × 10Tumor-bearing nude mice were divided into two groups at random: the control group (three mice injected with MPs) and the experimental group (six mice injected with NPs). No animal deaths occurred in the experimental process.0 referred to image gray-scale intensity before the injection, and RGVx referred to image gray-scale intensity after the injection [Mice were anesthetized with 1% pentobarbital sodium by abdominal injection (1 mg/100 g); 0.3 ml samples were injected into the body via the tail vein. Images were collected before and after injection: 0.5 h, 2 h, 12 h, 24 h, and 48 h, using a L74M transducer with 10 MHz and MI of 1.0. No instrument parameters changed during this experiment. All data and images were stored for offline analysis. Because of individual differences and tumor heterogeneity, gray-scale images were quite different. We defined the quantitative gray scale as follows.njection .IVIS Lumina II  was used for in vivo optical imaging. NPs and MPs containing DiR were injected into 2 groups of mice (3 mice per group). Then mice were put into an opaque black box. The excitation filter of DiR was 745 nm and emission filter of it was 810–875 nm. Images were obtained by the CCD camera at 0 h, 0.5 h, 2 h, 12 h, 24 h, and 48 h. All the data were analyzed by Living Image® Software 4.0 .μm slices. Frozen sections were stained with DAPI for labeling the nuclei of tumor cells. Images were obtained by using a fluorescence microscope (Olympus). DAPI and coumarin-6 were excited at 340 and 466 nm, respectively, and the emission was recorded at 488 and 504 nm, respectively.NPs or MPs labeled coumarin-6 was injected into two groups of mice (3 mice per group). After being injected for 24 h, tumor-bearing mice were killed, and collected tumors were sectioned into 5 t test.All experiments were conducted in triplicate. All data were expressed as the mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) and 1H-NMR (3), methane , and methine  proton of the PLGA segment, respectively. The peak at 3.7 ppm was associated with the methene  proton of the PEG chain. By using the relative molecular weights and the integration of characteristic peaks at 5.20 and 3.7 ppm, the conjugation efficiency of NH2-PEG-COOH to PLGA-COOH was estimated to be 12.5%.The chemical composition of the synthesized product was confirmed by 1H-NMR . The chaThe size distribution and zeta-potential of the NPs and MPs were assessed. The mean diameter of the NPs was 212.7 ± 2.76 nm  with a pTo compare the ultrasonic reflectance ability of NPs with that of MPs, in vitro ultrasound imaging was acquired by using diagnostic high-frequency ultrasound (10 MHz) . The resWe also studied the influence of concentration and probe frequency on ultrasonic signals produced by NPs. Enriched NPs were diluted with degassing deionized water . We also separately studied the influence of mechanical index (MI) on NPs echo reflection. We selected a 10 mg/ml sample as the research object. P = 0.001).Tissue harmonic imaging- (THI-) mode imaging was carried out on two groups of tumor-bearing mice. The tumor images of the contrast enhancement were provided by NPs  and MPs In addition, the images of liver and kidney were also analyzed before and after injection of NPs. The results showed that there was no significant gray contrast enhancement in liver and spleen after injection of NPs (data not shown).We conducted small animals living optical imaging to further confirm that the NPs could gather in the tumor tissue. In NP group , the redTo further confirm the microscopic localization of NPs, the nude mice were sacrificed and frozen sections of the tumors were examined by fluorescence microscope. Tumor tissues of the nucleus were dyed blue by DAPI. NPs with green fluorescence appeared in areas beyond the nucleus in tumor tissue, but MPs with green fluorescence were not in tumor tissues .The purpose of this study is to use PFOB packaged PLGA-PEG-COOH and PLGA-COOH to prepare nanoscale UCA, which can generate contrast enhancement in vitro and in vivo. Regardless of the changes of the ratio of PLGA-PEG-COOH/PLGA-COOH, it is possible for a nanoparticle to maintain a complete shell structure by using such a mixture of membrane materials to package PFOB . When thPEG molecular weight of 2000 or more can avoid the RES . A PEG-3μl, the T/R was minimal. Therefore, when PFOB was >60 μl, free PFOB droplets appeared [In the preparation of nanoscale UCA, particle size and size distribution are important parameters that determine the fate of UCA in vivo studies. To get through the tumor's endothelial pore  and escape from the RES trapping effect , the optimal diameter of NPs for clinical employment should be less than 300 nm . In the appeared .Ultrasound contrast agents have the ability to enhance echogenicity. Higher concentration could produce more echo reflection and stronger echo signal. The intensity of echo reflection was directly proportional to the concentration of the particles . AccordiWe separately studied the effect of the MI on echo reflection. The MI is a measure of the power of an ultrasound beam. The higher the MI was valued, the greater the energy of ultrasonic emission was and the greater echo reflection results were . EchogenThe ability of NPs to reflect ultrasound was almost the same as that of MPs but their time of contrast enhancement was longer than that of MPs in vivo due to the small size of NPs. The time of the contrast enhancement process was longer than 24 h, similar to Rapoport's research . The timTarget, distribution, and metabolism of contrast agents in vivo can be observed by small animals living optical imaging . The tenHistofluorescence imaging revealed the location of the NPs after intravenous injection. The tumors are heterogeneous. Endothelial monolayers and the cells lining of the tumor vessel are defective. This presented intercellular openings, transcellular holes, and endothelial fenestrae. The functional pore size of different tumors varied ranging from 200 nm to 2000 nm . In the PFOB, with good echo enhancement ability, is the candidate of nanoultrasonic contrast agents. But PFOB is soluble neither in water nor in oil and it cannot be injected directly into the body, so usually it needs to be wrapped in a shell. The membrane materials that make up the shell should be able to completely package PFOB within the nanometer range, and the generated NPs have a good echo enhancement in vitro. They also enable NPs to escape the removal of RES in vivo and image after gathering in tumor tissues.In this work, we used membrane materials, PLGA-COOH and PLGA-PEG-COOH packaged PFOB, to produce nanometer UCA. Nanoparticles were all shell-core structures. We evaluated their echogenic ability in vitro and in vivo. In vivo fluorescence imaging and frozen section further confirmed that the NPs could accumulate in tumor tissues. Their characteristics suggested that NPs may be applicable to ultrasonic molecular imaging and targeting therapy/drug/gene delivery to tumor.


Hypovitaminosis D and secondary hyperparathyroidism are frequent among HIV-infected patients. As there are no data about the best supplementation therapy both in treatment and in maintenance, we conducted an observational study of 300 HIV-infected patients for whom vitamin D and parathormone (PTH) had been measured in order to validate a protocol of vitamin D supplementation in patients with HIV-infection. Patients with vitamin D deficiency (defined as 25(OH)D < 10 ng/mL), insufficiency (defined as 25(OH)D < 20 ng/mL), or hyperparathyroidism (PTH > 65 pg/mL) were supplemented with cholecalciferol 16.000IU (0.266 mg) weekly (if deficiency) or fortnightly (if insufficiency or high PTH levels). Rates of normalization of 25(OH)D (levels above 20 ng/mL) and PTH levels (<65 pg/mL) were analyzed. Multivariate analysis of factors related to normalization was carried out. With a median follow-up of 2 years, 82.1% of patients with deficiency and 83.9% of cases with insufficiency reached levels above 20 ng/mL. However, only 67.2% of individuals with hyperparathyroidism at baseline reached target levels (<65 pg/mL). Independent factors for not achieving PTH objective were tenofovir (TDF) and protease inhibitors use. In HIV-infected patients with hypovitaminosis, the protocol of cholecalciferol supplementation normalized vitamin D levels regardless of antiretroviral regimen in a high proportion of patients but it was less effective to correct hyperparathyroidism. In recent years, a growing number of studies have reported high prevalence rates of vitamin D deficiency among HIV-infected patients . The preThe relevance of the high prevalence of hypovitaminosis D among HIV patients is that vitamin D is not only a well-established factor for bone disease –11, but Risk factors for hypovitaminosis D are female sex, dark skin pigmentation, low dietary intake, seasonality, insufficient sun exposure, and high body mass index –19. ReceThe systematic screening for vitamin D deficiency is controversial. The most recent EACS guidelines  suggest Arguments against the widespread screening for vitamin D deficiency in HIV-infected patients include the unclear benefit of vitamin D replacement for nonmusculoskeletal outcomes, the cost of laboratory testing and treatment, and the potential toxicities of some supplementation approaches . FurtherTherefore, accepting the evidence that adequate vitamin D levels are beneficial for HIV patients we implemented a protocol for vitamin D supplementation and analyzed the factors influencing the achievement of these target levels in a clinical population where a protocol for vitamin D supplementation has been implemented. Moreover, we also analyzed the relationship between vitamin D and PTH levels.An observational study was conducted at the Department of Infectious Diseases at Hospital del Mar , analyzing 300 HIV-infected out-patients who were naïve or on stable HAART  for whom vitamin D levels had been measured (fasting status). Patients were followed up in our department and visited between June 2010 and October 2013. The study was approved by the hospital ethical committee and all subjects provided written informed consent. All patients' data were anonymized for data management and statistical analysis. As an observational study, there was no control arm for comparison and hence no randomization.Vitamin D  status was categorized as insufficient when <20 ng/mL and deficient when <10 ng/mL and hyperparathyroidism as PTH levels  >65 pg/mL. Secondary laboratory variables included serum levels of calcium (8.5–10.5 mg/dL), phosphate (2.5–4.8 mg/dL), alkaline phosphatase (40–129 IU/L), CD4 and CD8 lymphocytes counts, and HIV viral load .The following demographic and clinical data were assessed: gender, age, race, risk factors for HIV-infection, HAART, and hepatitis C and B coinfection. For the analysis were excluded patients with renal insufficiency  equation), hepatic insufficiency (defined as Child Pugh stage C), patients under chemotherapy, and those already receiving vitamin D, bisphosphonates, and calcium supplements .Subjects were supplemented with 16.000 IU or 0.266 mg of vitamin D3  weekly, fortnightly, or monthly according to vitamin D baseline and PTH concentrations and according to the supplementation protocol designed by the Department of Infectious Diseases at Hospital del Mar . Those pUnivariate analyses were performed with chi-square test and ORs and its 95% confidence interval was estimated by logistic regression.Data of 300 participants from the 3 follow-up moments were pooled resulting in 798 registers. With the data arranged in this manner a Linear Mixed Model was performed to analyse the relationship between PTH and vitamin D, adjusted by TDF. In this model the individual (the identifier) was included as a random effect.To analyse the factors associated to achieve the objective of 25(OH)D level above 20 ng/mL and PTH level below 65 pg/mL a chi-square analysis was done for the univariate analysis and a logistic regression model for the multivariate analysis; both analyses were done in patients with baseline 25(OH)D < 20 ng/mL and baseline 25(OH)D < 10 ng/mL.Statistical analysis was performed with R Statistical Package .The characteristics of the 300 patients included in the study are presented in The protocol described in At baseline, 84 patients were vitamin D deficient and 186 insufficient. All patients, both deficient and insufficient, were offered supplementation but all those who declined this option were not supplemented. A total amount of 22 patients of those with vitamin D deficiency and 58 of those with insufficiency declined supplementation.p < 0.0001). Vitamin D objective was influenced by neither TDF exposure (p: 0.65) nor nonnucleoside exposure (p: 0.23). A high proportion of patients on PI therapy achieved 25(OH)D concentration >20 ng/mL ; however, the difference did not reach statistical significance. At the multivariate analysis, the only significant factor associated with achieving the objective of 25(OH)D concentration >20 ng/mL was cholecalciferol supplementation (p < 0.0001).62 patients with vitamin D deficiency (73.8%) were supplemented . After cp = 0.0001). The results were similar in those subjects with vitamin D insufficiency. The proportion of participants with vitamin D <20 ng/mL was 62% (186 patients) of whom 128 subjects were supplemented with cholecalciferol. More patients in the supplemented regimen achieved vitamin D concentration >20 ng/mL after treatment than those not supplemented . Moreover, PTH values were significantly higher, 10.643 pg/mL, in those patients exposed to TDF (p < 0.001)  .No changes on HAART due to vitamin D for PTH levels were made. Those HAART switches during supplementation were made for either virologic failure or adverse events related to antiretroviral therapy.There were no differences between patients supplemented and those not with respect to the frequencies of possible adverse events including infectious diseases, fractures, cancer, and cardiovascular diseases, so we considered there were no events attributable to vitamin D replacement. During the course of the study no patient in replacement group presented toxicities . The mean viral load, CD4 number and CD4%, calcium, phosphate, and alkaline phosphatase measurements did not differ between baseline and the end of the study.Considering that hypovitaminosis is associated with unfavorable outcomes of some chronic infections such as HIV  and the The results of this study demonstrate that supplementation of HIV-infected individuals with vitamin D is well tolerated, safe, and effective, as more than 95% of the patients achieved vitamin D levels above 20 ng/mL, with the proposed schedule of administration.In the current study, as in others, supplementation did not significantly affect calcium, phosphorus, and alkaline phosphatase during the period of observation. In addition, no differences were observed in the mean viral load, CD4 number, and CD4% between baseline and the end of the study. Moreover, in contrast to other studies, neither baseline concentration of vitamin D nor vitamin objectives were influenced by the cART use , 22, 40.On the other hand, PTH elevation plays a direct role over bone disease among HIV-infected patients, as it acts on bone to release calcium and persistent PTH elevations are associated with bone loss across a range of clinical conditions –43. StudBecause low vitamin D was associated with PTH increases, we hypothesized that vitamin D repletion may reduce the risk of PTH abnormalities. Many studies have shown that serum PTH is inversely related to serum 25(OH)D, and that with the increase in serum 25(OH)D there is a decrease in serum PTH that usually reaches a plateau. In the Endocrine Society guidelines , the staIn different studies vitamin D has been associated with TDF-linked hyperparathyroidism emphasizing that both, TDF use and vitamin D status, influence in PTH values. Although the mechanism by which TDF produces hyperparathyroidism is unclear, a previous study suggests that the increased hydroxylation rates and tubular phosphate losses, which drive calcium preservation and possibly altered bone metabolism, are dependent on vitamin D status . MoreoveLimitations of the present study are mainly related to the observational design nature of the current analyses. In this respect, the results reported herein are only associations from which no conclusions regarding causality can be drawn. The 25(OH)D measurements were done along the whole year, independently of seasonality. On the other hand we did not examine sun exposure, nor intakes of dietary calcium, although dietary calcium deficiency is rare in our population due to Mediterranean diet, and serum calcium and phosphorus were normal during the complete follow-up. Calcium urinary excretion was not measured.In conclusion we demonstrate that the proposed scheme for vitamin D supplementation in HIV-infected patients is safe and valid for correcting vitamin D abnormalities and to improve raised PTH levels, but not enough for normalizing them, especially in patients exposed to tenofovir or protease inhibitors.