Tibetan Medicinal Plant Research Articles

Chemical Analysis of Lancea tibetica using UPLC-UV/MS and ESI-MSn Methods

Lancea tibetica Hook. F. et Thoms (Family: Scrophulariaceae) is a Tibetan medicinal plant used for the prevention and treatment of leukemia, heart disease and cough in traditional Chinese medicine. The pharmacological and antioxidant effects of L. tibetica are mainly attributed to the content of phenylpropanoid glycosides and lignans [1,2]. As part of a program to address the issues of authenticity of material source, safety, quality assurance, and quality control of botanical and dietary supplements, nine furofurano lignans along with verbascoside, isoverbascoside, β-sitosterol and oleanolic acid were identified from L. tibetica in our research group. In the present work, both UPLC-UV/MS and ESI-MS/MS methods were applied for the quantitative and qualitative determination of lignans, verbascoside and isobascoside in L. tibetica. The reference standards include verbascoside (1), isoverbascoside (2), lantibeside (3), phillyrin (4), lantibeside B (5), lantibeside C (6), tibeticoside A (7), styraxjaponoside C (8), sylvatesmin (9), (+)-piperitol (10) and horsfieldin (11). The separation of standard compounds was achieved by using C-18 column material in UPLC method coupled with PDA and SQD detectors; a mixture of water and acetonitrile, both containing 0.05% formic acid, has been selected as the mobile phase. The column temperature was maintained at 40°C. Structures of reference standards were further characterized by ESI-MS/MS method, and discussed the proposed fragmentation pathway. The developed UPLC-UV/MS and MS/MS methods will be useful for the determination of lignans, verbascoside and isobascoside in L. tibetica, and in future biological studies related to L. tibetica.

Quantitative Determination of 10 Phenylpropanoid and Lignan Compounds inLancea tibeticaby High-Performance Liquid Chromatography with UV Detection

An HPLC method was developed for simultaneous determination of one phenylpropanoid glycoside, verbascoside (1), and nine lignans, including lantibeside (2), phillyrin (3), lantibeside B (4), lantibeside C (5), tibeticoside A (6), styraxjaponoside C (7), sylvatesmin (8), (+)-piperitol (9), and horsfieldin (10), from the Tibetan medicinal plant Lancea tibetica Hook. F. et Thoms. The analysis was performed within 45 min. The extraction method was optimized with different solvent systems. The HPLC method was validated for linearity, repeatability, accuracy, limits of detection, and limits of quantification. The limits of detection and limits of quantification of 10 analytes were found to be less than 0.1 and 0.5 µg/mL, respectively. The RSD for intra- and inter-day analyses was less than 4.2 %, and the recovery efficiency was 90-105 %. The method was used to analyze different populations of L. tibetica collected in China. HPLC profiles showed that the concentrations of analytes were different in samples collected from different areas of China. Verbascoside was the dominant component in three out of five plant samples; compounds 2, 3, 6, and 8 accounted for over 62 % yields in total lignan contents. The method is useful for identification, quality assurance, and quality control of L. tibetica and its related products.

Diterpenoid alkaloids from a Tibetan medicinal plant Aconitum richardsonianum var. pseudosessiliflorum and their cytotoxic activity

The chemical constituents from Aconitum richardsonianum var. pseudosessiliflorum were investigated. The roots of this plant were extracted three times with 90% EtOH at the room temperature. The ethanol extracts were combined and concentrated under reduced pressure to yield residue, which was suspended in water and successively partitioned with chloroform. The chloroform extraction was isolated and purified by silica gel and Sephadex LH-20 column chromatography. Six compounds were isolated and elucidated as delelatine (1), isodelpheline (2), 3-acetylaconitine (3), isoatisine (4), nordhagenine A (5) and yunaconitine (6). Compounds 1–5 were obtained from Aconitum Brunneum for the first time. Compound (1) showed significant cytotoxic activities (IC50=4.36μM) against the human tumor cell line P388.

Hispidulin, a small flavonoid molecule, suppresses the angiogenesis and growth of human pancreatic cancer by targeting vascular endothelial growth factor receptor 2‐mediated PI3K/Akt/mTOR signaling pathway

Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti-inflammatory and anti-oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs in a dose-dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF-induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice. To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF-triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal-regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis.

Simultaneous separation and analysis of two bioactive xanthones in the Tibetan medicinal plantGentianopsis paludosa(Hook. f.) Ma by micellar electrokinetic capillary chromatography

Summary 1,7-Dihydroxy-3,8-dimethoxyxanthone (X1) and 1,8-dihydroxy-3,7-dimethoxyxanthone (X2) are two important xanthones of the Tibetan medicinal plant Gentianopsis paludosa (Hook. f.) Ma. They are very similar in structure, the only difference being exchange of OH and OCH3 at the 7 and 8 positions. By calculations based on the geometry of the molecules using the MM+ force field, the different distances between the hydroxyl groups of the two xanthones were obtained (4.64774 Å for X2 and 7.19412 Å for X1), therefore, the two hydroxyl groups of X1 should freely interact with more water molecules than those of X2 in aqueous solution. In other words, X2 is more hydrophobic than X1. Micellar electrokinetic capillary chromatography (MEKC) was therefore chosen for separation of the compounds. The optimum separation conditions were: 20 mm borate + 20 mm SDS (pH 9.8) as running buffer, 17.5 kV applied potential, and detection wavelength 260 nm. The two xanthones were well separated in 9.0 min, with Gaussian peak shapes. The repeatability of the MEKC method (expressed as RSD) for X1 and X2 was 0.9 and 1.1%, respectively, for migration time, and 3.1 and 1.4% for peak area. The limits of detection (S/N = 3) were 0.41 μg mL−1 for X1 and 0.82 μg mL−1 for X2. The recovery of the MEKC method for the two xanthones was also satisfactory.

Application of HPLC-Tandem-MS for authentication of the traditional Tibetan medicinal plant Rhodiola rosea

Rhodiola rosea, a high-altitude plant, is extensively being used in traditional folk medicine in Tibet to treat fatigue, asthma, haemorrhage, depression, anaemia, impotence, gastrointenstinal ailments, infections, and nervous system disorders [1]. Some other Rhodiola species, in particular, R. alterna, R. brevipetiolata, R. crenulata, R. kirilowii, and R. sachalinensis are often marketed as R. rosea, and thus, the therapeutic effects of R. rosea are not achieved. In an attempt to develop a mass spectrometry method for discriminating among these species, peptides from the six Rhodiola species were analyzed and isolated by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-Tandem-MS). On the basis of the mass spectral fingerprints, 27 Rhodiola samples were successfully differentiated by principal component analysis (PCA) of the mass spectral raw data. The PCA results were also validated with cluster analysis and supervised PCA analysis. Using these fingerprints, some R. rosea-specific peptides were detected. This method provides effective and accurate identification of Rhodiola rosea.

Metabolotaxonomy of Tibetan medicinal plant Halenia elliptica with HPLC

Metabolotaxonomy of Tibetan medicinal plant Halenia elliptica with HPLC

From Valley to Alpine Mountain: Diversity and Utilization of Tibetan Medicinal Plants in Deqin

A field and interdisciplinary survey of Tibetan medicinal plant resources was undertaken in Deqin Country of Northwest Yunnan in 2008. Three typical habitats,river valley,mid-mountain and alpine regions were selected to document the use knowledge and species diversity used locally,and to evaluate the importance and vulnerability of medicinal plant species. 144 medicinal plant species of 63 families and 126 genera were recorded in this paper. Most of them (64%) are used through collecting wild plants,and the other 36% are supplied by pilot cultivation or conventional plantation. 26% of total 144 species are being threatened because of commercial harvesting and limited resources. Among the three surveyed plots along altitude gradients,the river valley has the least vulnerable plant species,while alpine region has the most plant species which are being threatened by overharvesting,limited by slow growth rate,and exacerbated by the difficulty of man-made cultivation. And reasonable strategies should be developed for plants resources in this and other special habitats.

Marker Compounds from the Herb Lancea tibetica and Identification by HPTLC

Lancea tibetica Hook. F. et Thoms, a Tibetan medicinal plant, is traditionally used for the prevention and treatment of leukemia, intestinal angina, heart disease and cough by the Zang people [1]. Sylvatesmin has been reported to have anti-tumor and anti-oxidant activity in vitro [2,3]. A phytochemical investigation led us to isolate and identify 15 compounds from this plant, including furofuranoliganans, phenylpropanoid glycosides, triterpenes and iridoid glycosides. Eleven compounds, including 7 furofuranoliganans, verbascoside, isoverbascoside, β-sitosterol and oleanolic acid were used as the marker compounds to identify this plant by HPTLC method. Three of these furofuranoliganans (shown in Fig.1) (tibeticoside, lantibeside, and lantibeside C) have only been reported from this plant. A fast and reliable HPTLC method was developed for the quality analysis of this widely used Tibetan medicine. The HPTLC profiles are shown in Fig.2 and Fig.3. The developed method was successfully applied for the identification of 5 Lancea tibetica samples collected from different regions in China. The developed method was validated for specificity, stability, repeatability, and robustness.

Comparative Analysis of the Major Constituents in the Traditional Tibetan Medicinal Plants Saussurea laniceps and S. medusa by LC–DAD–MS

Liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry was developed for the qualitative and quantitative comparison of the main constituents in Saussurea laniceps (SL) and S. medusa (SM), two species of plants used under the name “Xuelianhua” in traditional Tibetan medicine. A method validation including linearity, limit of detection, precision and recovery was performed. The results showed that a good linearity with R2 > 0.99 was achieved, and the limit of detection of the quantified constituents was reported to be between 0.8 and 3.3 ng. The relative standard deviation value was below 3.82% for repeatability, and recovery studies for the quantified compounds were found to be within the range 90.92–103.12%. The unique properties of the present method were evaluated by analyzing twelve related herbal samples including five S. laniceps samples and seven S. medusa samples. Twenty-two compounds including phenolic acids, cumarins, lignanoids and flavonoids were identified by online ESI–MS and by comparison with literature data and standard compounds, and seven of them were quantified by LC–DAD simultaneously. The results demonstrated that the common constituents in the two herbs were protocatechuic acid, syringoside, chlorogenic acid, isoquercitroside, 1,5-dicaffeoylquinic acid, apigenin 7-O-β-d-glucoside, chrysoeriol 7-O-β-d-glucoside, acacetin 7-O-β-d-glucoside, apigenin and chrysoeriol. In the present study, it was found that the characteristic constituents were umbelliferone, scopoletin and their glucosides in S. laniceps, as well as arctiin and arctigenin in S. medusa. It was feasible to choose these characteristic compounds for the quality evaluation as well as chemical authentication of the two related herbs. The results also support discrimination between the two species when using them in folk medicine.

Chemical constituents investigation of Daphne tangutica

A phytochemical study of an ethanol-soluble extract from the root barks of Daphne tangutica Maxim., a traditional Tibetan herb medicine, led to the isolation of 30 compounds, including eight daphnane diterpenes, nine coumarines, six lignans, five phenylpropanoid derivatives, β-sitosterol and p-hydroxy benzonate. Two compounds out of these isolates are new daphne diterpene analogs, and their structures were established as 1,2α-dihydro-5β-hydroxy-6α,7α-epoxy-resiniferonol-14-benzonate, and 1,2β-dihydro-5β-hydroxy-6α,7α-epoxy-resiniferonol-14-benzonate, respectively, on the basis of spectroscopic methods. Additionally, this is the first time that 13 known compounds have been isolated and identified from this traditional Tibetan medicinal plant.

Allele-specific primers for diagnostic PCR authentication of Halenia elliptica, a traditional Tibetan medicinal plant

Allele-specific primers for diagnostic PCR authentication of Halenia elliptica, a traditional Tibetan medicinal plant

In vivo and in vitro antiviral activity of five Tibetan medicinal plant extracts against herpes simplex virus type 2 infection

Extracts from five Tibetan medicinal plants collected from the Tibetan Plateau were evaluated for antiviral activity against herpes simplex virus type 2 (HSV-2) in vitro and in vivo. Viral plaque reduction assays showed that extracts from four out of five plants inhibited HSV-2 infection significantly with 50% effective concentrations (EC50) values ranging from 0.35 ± 0.11 to 1.83 ± 0.21 mg/mL. The other plant, Swertia mussotii Franch. (Gentianaceae), exhibited activity in inhibiting the viral biosynthesis. In the attachment assay, two plants, Dracocephalum heterophyllum Benth. (Lamiaceae) and Dracocephalum tanguticum Maxim. (Lamiaceae) reduced the attachment of HSV-2 to cell surface. Interestingly, all of the extracts showed virucidal activity. Analyzed by real-time PCR, three extracts showed strong inhibition of HSV DNA replication with Dracocephalum heterophyllum and Dracocephalum tanguticum at the concentration of 4 mg/mL and Lagotis brevituba Maxim. (Scrophulariaceae) at 1 mg/mL. BALB/c mice were used for determining in vivo efficacy. Mice encephalitis herpes models were established by infection with HSV-2. The extracts of Dracocephalum heterophyllum, Dracocephalum tanguticum, and Swertia mussotii at a dose of 1 g/kg per day significantly prolonged the mean survival times and reduced the mortality of HSV-2 infected mice compared with control group (P < 0.05). Taken together, we conclude that the antiviral mechanisms of these plants involve various stages of virus replication. Extracts from three of these plants, Dracocephalum heterophyllum, Dracocephalum tanguticum, and Swertia mussotii, may be possible candidates in developing anti-HSV-2 medicine.

Medicinal plants used by Tibetans in Shangri-la, Yunnan, China

BackgroundMedicinal plants used by the local people in Xizang (Tibet) have been investigated since the 1960s. The others out of Xizang, however, have been less understood, although they may be easily and strongly influenced by the various local herbal practices, diverse environments, local religious beliefs and different prevalent types of diseases. In 2006, two ethnobotanical surveys were organized in the county of Shangri-la, Yunnan Province, SW China, to document the traditional medicinal plants used by the Tibetan people.MethodsAfter literature surveying, four local townships were selected to carry out the field investigation. Three local healers were interviewed as key informants. The methods of ethnobotany, anthropology and participatory rural appraisal (PRA) were used in the field surveys. Plant taxonomic approach was adopted for voucher specimen identification.ResultsSixty-eight medicinal plant species in 64 genera of 40 families were recorded and collected. Among them, 23 species were found to have medicinal values that have not been recorded in any existing Tibetan literatures before, and 31 species were recorded to have traditional prescriptions. Moreover, the traditional preparations of each species and some folk medicinal knowledge were recorded and analyzed. These traditional prescriptions, preparations, new medicinal plants and folk medicinal knowledge and principles were discovered and summarized by local traditional Tibetan healers through times of treatment practices, and were passed down from generation to generation.ConclusionAs a part of the cultural diversity of Tibetan community, these traditional medicinal knowledge and experiences may provide data and information basis for the sustainable utilization and development of Tibetan medicine, and may contribute to the local economic development. However, for many reasons, they are disappearing gradually as time goes by. Our study showed that there were abundant traditional Tibetan medicinal prescriptions and using methods. It implies that more Tibetan medicinal plants and traditional knowledge can be discovered. Further research should be done to save the wealth of these traditional medicinal knowledge and experiences before they are dying out.

Eastern Himalayan alpine plant ecology, Tibetan ethnobotany, and climate change

Tibetan culture and livelihoods depend on native plants for medicine, food, grazing, wood, as well as cash from market sales. The Medicine Mountains (part of the Hengduan Mountains) of the eastern Himalayas, with tremendous plant diversity derived from steep gradients of both elevation and precipitation, have traditionally been an important source of Tibetan medicinal plants. We examine climate change in this area and vegetation patterns influenced by biogeography, precipitation and elevation (NMS and CCA ordinations of GLORIA plots). The Alpine environment has the highest plant diversity and most useful plants and is the most susceptible to climate change with impacts on traditional Tibetan culture and livelihoods—particularly Tibetan medicine and herding.

Herbs and Traders in Transit: Border Regimes and the Contemporary Trans-Himalayan Trade in Tibetan Medicinal Plants

This article discusses the contemporary cross-border trade in medicinal plants between Nepal and Tibet. As Tibetan pharmacy extensively relies on raw materials not native to Tibet, long-distance trade in medicinal materials is not a new phenomenon. However, with the recent creation of a Tibetan medicine industry in the People’s Republic of China (PRC) and the increasing demand for herbs from India and Nepal, the contemporary herb trade is facing new challenges. Surging trade volumes, notions of patient safety, growing ecological concerns, and the current political situation in Tibet have led to increased efforts at rendering legible and controlling the transit of traders and herbs across the border. The ethnographic account of a Tibetan plant trader’s business trip to Nepal serves as a starting point for a discussion of these efforts and the traders’ tactical manoeuvres to deal with them. The notion of ‘border regimes’ is introduced to analyse the regulations, their implementation and side-effects that condition the current situation.

Conserving Tibetan Medicinal Plants in the Khawakarpo Region

To local Tibetans, Mt. Khawakarpo is not only an important sacred mountain but also a major storehouse of medicinal plants, which they use. Recent field surveys show that Mt. Khawakarpo and adjacent areas (‘the Khawakarpo region’) are home to 144 species of household medicinal plants from 126 genera of 63 families, which are used in both Chinese and Tibetan medicine. These plants include eight Class I and Class II nationally protected species (four being CITES—Convention on International Trade in Endangered Species of Wild Flora and Fauna—listed); of these, 37 species have been recognized by local practitioners of Tibetan medicine as plants in need of conservation. Results from fieldwork indicates that initiatives to conserve plants used in Tibetan medicine should consider not only their biological properties but also their cultural values to local communities; in order for these efforts to be sustainable, the communities must be brought in as key players. The fieldwork described here comprised a participatory resources inventory, the creation of community-based medicinal plants reserves, cultivation trials, and training opportunities for medicinal practitioners to share indigenous knowledge and acquire skills in plant conservation and harvesting on a sustainable basis.

Applied Vulnerability Assessment of Useful Plants: A case study of Tibetan Medicinal Plants from Nepal

Wagner, A., Kriechbaum, M. & Koch, M. A.: Applied Vulnerability Assessment of Useful Plants: A case study of TIbetan Medicinal Plan~s from Nepal. - Bot. jahrb. Syst. 127: 01lO-OO0. 2008. - ISSN 0006·8152. The growing demand for medicinal and aromatic plants (MAPs) that are harvested from the wild exerts an increasing pressure on these important natural resources of the Himala­ yas. In the present study, 126 species of higher plants from the Muktinath Valley, Kali Gandkhi., in Central Nepal are documented. Endangerment was assessed by adapting the Rapid Vulnerability Approach (RVA). The RVA-tesr shows rhar seven of these species have a high potential endangerment, 102 species a moderate potential and 17 species no potential For valuation of the reliability of the RVA-test, a statistical analysis was conducted. The six indicators used in thisstudy do not show any or only minor inter-correlations, and resulting threat values are independent from each other. We can thus show that each individual indieator has a significant effect on the assessment of endangerment. As a result, the RVA as used herein is suirable for an approximate assessment of endangerment of MAPs.

Development of an HPLC-DAD–ESI-MS n method for quantitative analysis of Saussurea tridactyla

An improved HPLC-DAD–ESI-MS n method has been developed to simultaneously quantify eight major compounds in Saussurea tridactyla Sch.-Bip. ex Hook. f. which has long been used as a traditional Tibetan medicine. This method was validated to be sensitive, precise and accurate with the LODs of 0.11–5.01 μg/ml, the overall intra-day and inter-day variations less than 2.70%, and the overall recovery over 98.0%, respectively. The correlation coefficients ( r 2) of the calibration curves were higher than 0.991. This newly established method was successfully applied to reveal the difference in the chemical profiles and contents of these analyses in S. tridactyla from different localities. In addition, by comparison UV and MS spectra with those of authentic compounds and literatures, a total of fourteen peaks were identified. It can be concluded that this method was effective to ensure the safety and efficacy consistency of S. tridactyla, and can be applied to other traditional Tibetan medicinal plants from different resources in Tibet.

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita

Aim of the study Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita. Materials and methods A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively. Results The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin ( 1), jaceosidin ( 2), cirsilineol ( 3), cirsimaritin ( 4), hispidulin ( 5), quercetin ( 6), 6-methoxytricin ( 7), acacetin ( 8), and apigenin ( 9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol ( 3), 6-methoxytricin ( 7) and apigenin ( 9) significantly inhibited T cell proliferation and activation in the bioassays. Conclusion The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.