Identification of Tibetan medicine Dida based on DNA barcoding.

The purpose of this study was to test the ability of DNA barcoding to identify the herbal raw trade of Tibetan medicine Dida in China. A reference database for plant-material DNA barcodes was successfully constructed and used to identify 36 commercially samples of Dida collected from Southwest China. The ITS sequence was amplified from these samples and the efficiency of the PCR amplification of ITS was 100%. The DNA sequencing results revealed that 3 samples (8.3%) were authenticated as Swertia chirayita, 2 sequences (5.6%) were authenticated as Swertia mussotii, 3 sequences (8.3%) were authenticated as Swertia ciliata, as recorded in the Tibetan Pharmacopeia. The other samples were authenticated as adulterants and all of them originated from common plants belonging to Saxifraga, Swertia and Halenia. This result indicates Dida pieces that are available in the market have complex origins and may indicate a potential safety issue and DNA barcoding is a convenient tool for market supervision.

[Analysis and Discrimination of the Medicinal Plants Swertia Davidi Franch Based on Infrared Spectroscopy].

Fourier-transform infrared spectroscopy combined with partial least squares discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) were used to rapidly discriminate the Swertia davidi Franch which collected from different origins. The original infrared spectra data of different parts of all the 70 samples which collected from four different regions were preprocessed by automatic calibration, automatic smoothing, the first derivative and the,second derivative. Then the processed data were imported into OMNIC 8.2 and the absorption peaks were compared; PLS-DA was performed by SIMCA-P⁺ 10.0 and the effect of discrimination of different origins was compared by 3D score plot of the first three principal components; the infrared spectral data were imported into SPSS 19. 0 for HCA to compare classification results of different parts by the dendrogram. The results showed that: (1) There were differences among the spectra of the roots of different origins in the spectral peaks in 1,739, 1,647, 1,614, 1,503, 1,271, 1,243, 1,072 cm⁻¹. The spectra of the stems of different origins showed differentiation in the wavelength in 1 503, 1 270, 1 246 cm⁻¹; (2) The characteristic peaks of different parts of the same origin were different; (3) PLS-DA indicated that the data which were processed by automatic correction, automatic smoothing and second derivative have showed the best classification. In addition, the discrimination of roots which collected from different origins could be the best; (4) Tree diagram of HCA showed that the accuracy rate of cluster in roots, stems and leaves were 83%, 56%, and 70%, respectively. In conclusion: FTIR combined with PLS-DA and HCA can rapidly and accurately differentiate S. davidi that collected from different origins, the origin discrimination effect of different parts was clearly different that the classification of roots is the best, the second derivative could enhance the specificity of the samples, the classification in 3D score plot could be visualized and obvious.

RP-HPLC analysis of seco-iridoid glycoside swertiamarin from different Swertia species.

Genus Swertia is valued for its great medicinal potential; mainly Swertia chirayita (Roxb. ex Fleming) H. Karst. is used in traditional medicine for a wide range of diseases. Seco-iridoid glycosides like swertiamarin is referred with enormous pharmacological potentials. The aim of the study was to identify a suitable substitute to S. chirayita by quantifying seco-iridoid swertiamarin from five different Swertia species endemic to the Western Ghats. The reverse-phase high-performance liquid chromatography diode array detector analyses were performed and chromatographic separation was achieved on a Lichrospher 100, C18e (5 µm) column (250-4.6 mm). A mobile phase consisting of acetonitrile and water (25:75) was used for separation. Results indicated that the concentration of the marker compound has been found to vary largely between and within the species from different localities. The content of swertiamarin was the highest in S. chirayita compared to the other species studied herein, advocating the use of Swertia minor as an alternate source to S. chirayita.

Phytochemical investigation of crude methanol extracts of different species of Swertia from Nepal.

BACKGROUND: The genus Swertia is reported to contain potent bitter compounds like iridoids, xanthones and c-glucoflavones that are known to heal many human disorders. In contrast to high ethnomedicinally valued Swertia chirayita, its other species have not been studied extensively, in spite of their common use in traditional medicinal system in Nepalese communities. So, the present study attempts to investigate the content of total polyphenols, flavonoids, antioxidant activity and estimate the rough content of amarogentin, swertiamarin and mangiferin from different species of Swertia from Nepalese Himalayas. METHODS: Whole plant parts of S. chirayita (SCH), S. angustifolia (SAN), S. paniculata (SPA), S. racemosa (SRA), S. nervosa (SNE), S. ciliata (SCI) and S. dilatata (SDI) were collected; total phenolic and flavonoid contents were quantified spectrophotometrically and in vitro DPPH free radical scavenging assay was measured. Thin layer chromatography was performed on TLC aluminium plates pre-coated with silica gel for identification of swertiamarin, amarogentin and mangiferin from those species and semi quantitative estimation was done using GelQuant.NET software using their standard compounds. RESULTS: The phenolic content was highest in the methanol extract of SCH (67.49 ± 0.5 mg GAE/g) followed by SDI, SRA, SNE, SCI, SPA and SAN. The contents of flavonoids were found in the order of SCH, SPA, SRA, SNE, SDI, SCI and SAN. Promising concentration of phenolics and flavonoids produced promising DPPH free radical scavenging values. The IC50 values for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test was lowest in SCH (23.35 ± 0.6 µg/ml), even lower than the standard ascorbic acid among the seven studied species. A significant correlation of 0.977 was observed between the polyphenol content and antioxidant values. The TLC profile showed the presence of all three major phytochemicals; amarogentin, swertiamarin and mangiferin in all of the plant samples. CONCLUSION: Among the seven studied species, SCH showed anticipating results in total phenol content, flavonoid content and DPPH radical scavenging test. The less considered species of Swertia can be a potential source of bioactive amarogentin, and other useful therapeutic compounds in the alarming status of Swertia chirayita as shown by the phytochemical analysis.

Quantitative determination of three pentacyclic triterpenes from five Swertia L. species endemic to Western Ghats, India, using RP-HPLC analysis.

Aim of this study was to identify pentacyclic triterpenoids betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) from five Swertia species endemic to Western Ghats, which are used as substitutes as well as adulterants to Swertia chirayita. Our results indicate that the concentration of active compound vary largely among and within the species from different localities. OA was found in a higher amount in all species compared to BA and UA. From the results, it was clear that BA, OA and UA are present in the endemic species collected from Western Ghats, thus advocating the use of these species as alternate sources to S. chirayita. This in due course may release pressure of exploitation from natural resources of S. chirayita and help to bring it out from an endangered category from conservation point of view.

[Studies on genetic diversity of three Tibetan herbs].

The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.

Phylogenetic examination of crude drugs derived from Yunnanese Swertia plants.

Aiming to examine whether the genetic background of the crude drugs derived from four Yunnanese Swertia plants and their chemical constituent profiles correlate, we analyzed the nucleotide sequences of their nuclear ribosomal DNA regions including ITS1, 5.8S ribosomal RNA gene, and ITS2, together with those of Japanese S. japonica and S. pseudochinensis from Hebei Province. The result that two of the Yunnanese Swertia plants, S. binchuanensis and S. punicea, were genetically similar may explain their similarity in chemical constituent profiles. On the other hand, in spite of differences in chemical profile, S. decora and S. pseudochinensis were genetically close. The other Yunnanese Swertia plants, S. delavayi, and S. japonica, stood at intermediate positions between these two genetically similar pairs. The result suggests that although genetic background would have an influence, environmental factors, e.g., soil and weather conditions, might be critical for their production of secondary metabolites.

Application of microscopy for discrimination of eight Swertia species utilized in the traditional Chinese medicine "Qingyedan".

Many Swertia species are utilized as a traditional medicine under the name "Qingyedan" in China, but are easily confused with one another. To distinguish eight Swertia species (S. mileensis, S. cincta, S. patens, S. punicea, S. delavayi, S. nervosa, S. macrosperma, and S. yunnanensis) and to ensure their safety and efficacy, the microscopic and macroscopic characteristics of the roots, stems, leaves, and flowers of them were examined. The results showed that microscopic and macroscopic features helpful for authentication of the eight species were the sinuosity of the anticlinal walls of epidermal cells and presence or absence of hairs on the leaf lamina; presence or absence of V-shaped fibers and fibers with sinuous abaxial wall in the sepals; shape of epidermal cells and pattern of papillae on hairs on the margin of corolla nectary; distribution of stomata in leaf and sepal epidermises, stone cells in cortex and phloem of roots and in cortex and pith of stems, crystals in parenchymatous cells of mesophyll and stem, stomata size, stem diameter, and 4- or 5-merous flowers, and so on. Two keys to the eight Swertia species based on macroscopic and microscopic characteristics are presented. The study indicates that microscopy and related techniques are convenient, practicable, and can be unambiguously applied for authentication of Swertia species.

Effect of storage conditions and storage periods on seed germination in eleven populations of Swertia chirayita: a critically endangered medicinal herb in Himalaya.

Effect of different storage conditions (room temperature, 4°C, and -15°C) and different storage periods over 24 months on seed germination in Swertia chirayita collected from different altitudes in Sikkim Himalaya was determined. Multivariate ANOVA revealed significant (P < 0.0001) effect of storage condition and storage period on seed germination and mean germination time. Seed germination percentage significantly (P < 0.01) varied between 87.78% (Sc5) and 100% (Sc2) during initial testing. Comparatively, high seed germination, low mean germination time, and low rate of fall in seed germination percentage in seeds stored at 4°C over different storage period were recorded. In addition, above 50% seed germination in majority of the populations even after 24 months of storage suggests 4°C as the most appropriate storage condition for long-term storage of seeds of S. chirayita.

AFLP markers for identification of Swertia species (Gentianaceae).

The genus Swertia is well known for its medicinal properties, as described in the Indian pharmacopoeia. Different members of this genus, although somewhat similar in morphology, differ widely in their pharmacological and therapeutic properties. The most important species of this genus, with maximal therapeutic properties, is S. chirayita, which is often adulterated with other less-potent Swertia spp. There is an existing demand in the herbal drug industry for an authentication system for Swertia spp, in order to enable their commercial use as genuine phytoceuticals. To this end, we used amplified fragment length polymorphism (AFLP) to produce DNA fingerprints for six Swertia species. Nineteen accessions (2 of S. chirayita, 3 of S. angustifolia, 2 of S. bimaculata, 5 of S. ciliata, 5 of S. cordata, and 2 of S. alata) were used in the study, which employed 64 AFLP selective primer pairs. Only 46 selective primer pairs were found to be useful for all the accessions. A total of 5312 fragments were produced by these 46 primer pairs. Species-specific markers were identified for all six Swertia species (131 for S. chirayita, 19 for S. angustifolia, 181 for S. bimaculata, 47 for S. ciliata, 94 for S. cordata, and 272 for S. alata). These AFLP fingerprints of the Swertia species could be used to authenticate drugs made with Swertia spp and to resolve adulteration-related problems faced by the commercial users of these herbs.

[Investigation on medicinal plant resources of Swertia in Yunnan province].

OBJECTIVE: To explore the species and distribution of medicinal plants Swertia in Yunnan province, and provide scientific basis for sustainable utilization. METHOD: Field investigation was carried out, specimens were collected, and literature and data were consulted. RESULT: There were 35 species and 2 varieties (including 1 newly recorded species), most of them were used as medicinal plants, and an identification index was established. CONCLUSION: The results provide reliable foundation for comprehensive utilization and in-depth study of Swertia in Yunnan province.

Molecular profiling of "Chirayat" complex using Inter Simple Sequence Repeat (ISSR) markers.

"Chirayat" represents a group in which different species of Swertia and some other non-Swertia species are used as local substitutes/adulterants for the Swertia chirayita. Twelve different taxa recognized under "Chirayat" complex were characterized using ISSR markers. On the basis of preliminary screening, 16 informative primers were selected for molecular profiling. All the ISSR primers generated distinct DNA amplification profiles, which could be used for identification of an individual taxon in the group. Primers with anchors at 5' end generated the most polymorphic profiles. Based on their ability to distinguish between the taxa, expressed as resolving power (Rp), primer 885 showed the highest resolving power and 855 the least. A total of 332 reproducible bands were scored and all of them were polymorphic. In the cluster analysis of Swertia SPP., based on the band data, two main clusters were observed. The first consisted of S. angustifolia and S. cordata; and the other of the remaining species of Swertia. Based on the similarity coefficient, S. lurida was closest to S. chirata. The utility of ISSR markers for the authentication of commercial crude drug samples is also demonstrated. The results show that ISSR is an efficient and reliable marker system for the molecular authentication of medicinal plants as well as commercial crude drug samples.

Identification of Swertia mussotii and its adulterant Swertia species by 5S rRNA gene spacer.

OBJECTIVE: This research focused on analyzing the differences of 5S rRNA gene spacer sequences on Swertia mussotii and its commonly used adulterants, including S. franchetiana, S. wolfangiana and S. chirayita. METHOD: DNA was extracted from the collected Swertia samples. 5S rRNA intergenic spacers were amplified by PCR, sequenced and analyzed. RESULT: 5S rRNA gene spacer sequences were different between S. mussotii and its other three adulterants. Sequence divergence among species ranged from 30.6% to 65.0%. CONCLUSION: 5S rRNA spacers may be used as molecular authentication markers to differentiate S. mussotii and other commonly used Swertia adulterants. This result provides reliable and simple reference for the authentication of Swertia genus species.

Two-dimensional LC-MS analysis of components in Swertia franchetiana Smith.

An off-line 2-D-LC approach was developed for the separation and identification of components in extracts of Swertia franchetiana Smith, which is a traditional Chinese medicine. An octadecylsilica column was used for further separation of fractions from a cyano column with UV and MS detection. In this method, a longer column was used in the second dimension to improve the separation ability, and "on-column focusing" was adopted to reduce sample loss and pollution on concentrating the fractions from the first dimension. More than 118 and 575 components were detected in the chloroform and n-butanol extracts by this method, respectively. In contrast, only 20 and 95 peaks were detected by the one-dimensional method. Most of the additionally detected compounds were of low-abundance components. Many compounds can be preliminarily identified according to their UV and mass spectra in the two-dimensional method. The obtained results not only demonstrated the powerful resolution of 2-D-LC, but also led to in-depth comprehension of the UV and mass spectra of complex samples.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii.

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA RPL16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA RPL16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

[Determination of xanthones in Swertia mussotii and Swertia franchetiana by high performance liquid chromatography].

A simple and rapid method for simultaneous separation and determination of xanthones in Swertia mussotii and Swertia franchetiana by high performance liquid chromatography has been established. The analysis was performed on a Kromasil C18 column (250 mm x 4.60 mm i.d., 5 microm) (at 20 degrees C) eluted with methanol and 0.1% aqueous phosphoric acid (73: 27 in volume ratio) as mobile phase at a flow rate of 1.0 mL/min and with UV detection at 260 nm. Results showed that xanthones were separated successfully from each other and from other interfering components. There was a good linear relationship between the content of component and its peak area for each xanthone, with the correlation coefficients of 0.999 2 - 0.999 9. The convenient method can be used for quantitative analysis of xanthones.

Morphological and ultrastructural diversity of orbicules in Gentianaceae.

Minute granules of sporopollenin, called orbicules, can be observed on the innermost tangential and/or radial walls of secretory tapetum cells. Orbicules were investigated in 53 species of 34 Gentianaceae genera using light microscopy, scanning electron microscopy and transmission electron microscopy. This selection covered all different tribes and subtribes recognized in Gentianaceae (87 genera, +/-1650 species). Orbicules were found in 38 species (23 genera) distributed among the six tribes recognized in Gentianaceae. The orbicule typology is based on those described previously in Rubiaceae. Of the six orbicule types described previously, Type II orbicules are lacking. Type III orbicules are most common (17 species). Hockinia Gardner is the only representative with Type I orbicules. The number of representatives with orbicules belonging to the other orbicule types are equally distributed among the species studied: seven species possess Type IV orbicules, six species Type V and six species Type VI. The systematic usefulness of this typology is discussed in comparison with the latest systematic insights within the family, and palynological trends in Gentianaceae. Orbicule data have proven to be useful for evaluating tribal delimitation within Rubiaceae and Loganiaceae s.l.; however, they seem not to be useful for tribal delimitation in Gentianaceae. In the tribes Potalieae and Gentianeae orbicule data may be useful at subtribal level.

[A comparison of the its sequences of the Tibetan medicine "zang yin chen"--Swertia mussotti and its adulterant species].

AIM: To identify the medicine and search for new medicine resource at the molecular level, the ITS of "Zang Yin Chen"--Swertia mussotti and its adulterant species were sequenced. METHODS: The double-stranded DNA was amplified using PCR systems 9,600 kits and sequenced on an ABI 377 automated sequencer from both directions. RESULTS: The ITS sequences of S. mussotti in different populations showed no variation. It has the unique ITS sequence and shows distinct difference from its adulterant species. In the phylogenetic tree based on the ITS data of S. mussotti and all vicarious species constructed by Paup, S. franchetiana and S. mussotti clustered together with high bootstrap support. CONCLUSION: ITS sequences can be used for the molecular authentication between the S. mussotti and its adulterant species. S. franchetiana can be regarded as a new medicine resource of "Zhang Yin Chen".

[Comparing Swerita daridi with Swertia mileencis on pharmacologic action].

The article compared Swertia daridi with Swertia mileencis about chemical composition and pharmacological action. The study showed two species drug's bitter taste chemical composition and pharmacological action are approximately equal. It can be tried to replace Swertia daridi with Swertia mileencis.