Dendrobium Densiflorum Research Articles

Chemical composition, antioxidant activity and bioactive constituents of six native endangered medicinal orchid species from north-eastern Himalayan region of India

Orchidaceae is one of the largest and most diversified group of flowering plants within the families of angiosperm. Medicinal properties of orchids are less explored, but it is drawing significant attention of the scientific communities recently. The north eastern Himalayan region of India is well-known orchid hotspot with prevalence of two-third of total orchid species of the country. In the present study, an attempt was made to investigate the phytochemical composition of six native, endangered medicinal orchid species of this region namely Dendrobium nobile, Dendrobium moschatum, Dendrobium densiflorum, Acampe papillosa, Coelogyne nitida and Arundina graminifolia . The nutritional as well as phytochemical composition and antioxidant activity of the extracts were analyzed by spectrophotometric methods. Minerals were estimated by atomic absorption spectroscopy (AAS) and flame photometer. Quantification of phenolics and bioactive metabolites from methanol extracts of the selected orchid species were carried out by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GCMS) methods. Results revealed that Dendrobium moschatum contained highest total carbohydrate (6.49 g 100g −1 ) and protein (11.30 g 100g −1 ). Dendrobium densiflorum was rich in total flavonoid content (35.54 mg quercetin equivalent QE 100g −1 ) and total alkaloid content (11.70 g 100g −1 ). It also showed highest 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity (64.4 µg ml −1 ). Acampe papillosa exhibited maximum total phenol content (4.09 g gallic acid equivalent GAE 100g −1 ), tannin (3.17 g tannic acid equivalent TAE 100g −1 ) and saponin (7.20 g 100g −1 ) content whereas Coelogyne nitida showed 281.7 µg ml −1 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) scavenging activity. Iron, zinc, copper, manganese, sodium, potassium and calcium contents were higher in the Dendrobium species compared to other medicinal orchids under study. Quercetin (4518.3 mg kg −1 in Dendrobium moschatum ) and catechin (1211.1 mg kg −1 in Dendrobium densiflorum ) were the major phenolic compounds present abundantly in these orchid species. Metabolite analysis by GCMS showed presence of total twenty-six major bio-active compounds (like dendroban-12-one, embramine, scoparone etc.) in these six orchid species. The current study presents scientific evidence in support of the traditional therapeutic application of the studied orchid species and proves that these are potential sources of biologically and pharmacologically active ingredients. • Phytochemical composition analysis and bio prospecting of six medicinal orchids were carried out . • Dendrobium leaves are rich in carbohydrate and minerals but stems are rich in protein content. • Dendrobium densiflorum is rich in flavonoid, alkaloid, zinc, bio active compounds and antioxidant activity. • Acampe papillosa is rich in total phenol, total tannin and total saponin content . • Coelogyne nitida exhibits higher antioxidant activity due to higher quercetin content.

Application of DNA barcoding tool for authentication of traditionally important medicinal Dendrobium (Orchidaceae) species from Sikkim Himalaya

Dendrobium, the second largest genus of the Orchidaceae family, is equally important for ornamental value and traditional medicine. Sikkim Himalaya region is rich in traditional knowledge of medicinal plants and their diversity. However, these regions were least explored through modern molecular tools. Reference databases association with DNA Barcoding technique is a new tool for species authentication. Here, we tested the efficiency of DNA barcoding loci (ITS, matK, rbcL, trnH-psbA) based on similarity search, characters based and phylogenetic methods to authenticate the medicinally important Dendrobium species. DNA from young leaves of studied species were isolated, and ITS (∼700 bp), rbcL(∼650 bp), matK (∼ 800 bp), trnH-psbA (∼ 450 bp) were amplified and sequenced for species-level identification. ITS, rbcL and trnH-psbA regions showed easy amplification and sequencing. Among the 54 sequences, 9 sequences were first time submitted to GenBank. In case of similarity search, ITS showed most efficient to identify all Dendrobium species except two species Dendrobium thyrsiflorum and Dendrobium densiflorum. However, the characters-based method easily differentiated between Dendrobium thyrsiflorum and Dendrobium densiflorum. The similarity-based methods and ITS sequence information together may help correct species identification for medicinal plants of Dendrobium.

Therapeutic Himalayan herbs: Folklore uses, bioactive phytochemicals, and biological activities of medicinal orchids used by Nomads

Natural products have bioactive properties which have been exploited for human and animal use for hundreds of years. Orchids are one of the largest, diverse, and most evolved groups of plants within monocot angiosperms. Different species under this category have been recognized as the most threatened plants due to poor seed germination, the need for endophytic mycorrhizal partners, and habitat destructions coupled with climate change. The majority of orchid taxa are known to have therapeutic potential due to the presence of bioactive substances, such as alkaloids, bibenzyls, phenanthrenes, phenanthrenequinones, glycosides, carbohydrates, flavonoids, and stilbenoids. In this study, twenty-eight indigenous Himalayan orchids were documented from six Himalayan states and one union territory of India viz ., Acampe ochracea, Aerides odorata, A. multiflora, Anoectochilus setaceus , Arundina graminifolia , Bulbophyllum odoratissimum , Coelogyne corymbosa , C. punctulata , Crepidium acuminatum , Cymbidium aloifolium , C. elegans , Dendrobium densiflorum , D. fimbriatum , D. fugax , D. nobile , Eulophia graminea , Habenaria dentata , H. marginata , Herminium lanceum , Liparis nervosa , Papilionanthe teres , Phaius tankervilleae , Pholidota imbricata , P. pallida , Porpax muscicola , Rhynchostylis retusa , Vanda coerulea and V. cristata familiar to nomadic tribes for use as medicine in their daily life. Data collected on species identity and nomenclature, local names, uses, bioactive constituents, pharmacological bioactivities, and phenology are presented. These findings will help in preserving Himalayan traditional knowledge, conservation of endangered plants, and research and development associated with wild orchids or similar taxa elsewhere in the globe.

Scoparone Induces Expression of Pluripotency Transcription Factors SOX2 and NANOG in Dermal Papilla Cells.

Dermal papilla cells (DPCs) regulate hair follicle development. We aimed to investigate the effect of scoparone from Dendrobium densiflorum on DPCs in the induction of stem cell properties and pluripotency-related proteins. DPC viability was evaluated by the MTT assay. Apoptosis or necrosis of DPCs was determined by Hoecsht33342/PI nuclear staining analysis. Expression of OCT4, NANOG and SOX2 genes was determined using Real-Time Polymerase Chain Reaction (PCR). Immunocytochemistry and western blot analysis were performed to determine pluripotency related proteins. Scoparone increased the expression of pluripotency related transcription factors SOX2 and NANOG, while it had minimal effects on OCT4 levels. Scoparone exerted its stemness-enhancing activity through the up-regulation of Akt-dependent inhibition of GSK3β, resulting in increased cellular levels of β-catenin. Our results show a potential novel activity and mechanism of action of scoparone on human DPCs that could facilitate the development of hair enrichment approaches.

The complete chloroplast genome sequence of Dendrobium densiflorum and its phylogenetic implications

Dendrobium is one of the largest genera in Orchidaceae with approximately 1500 species. Many of them have been used as Traditional Chinese Medicine (TCM) for hundreds of years. Here, the first complete chloroplast genome sequence of D. densiflorum was reported and characterized. The complete cpDNA of D. densiflorum is a circular molecule of 153,122 bp, which contains 76 protein-coding genes, 30 tRNA genes, and four rRNA genes. Our phylogenetic analysis indicated that the newly sequenced cpDNA of D. densiflorum could be used for the phylogenetic study of Dendrobium species.

Abstract 2687: Cypripedin inhibits non-small cell lung cancer migration through suppression of epithelial-to-mesenchymal transition

Abstract Lung cancer is one of the most common cause of cancer-related death worldwide because of high metastasis rate. Cancer migration is a prerequisite event among multi-complex processes of metastasis, being one of the interested target for cancer therapy. Accumulative evidence emphasizes that epithelial-to-mesenchymal transition (EMT) plays an important behavior exerting cell motility and survival, and relevant to cancer metastasize. Even the therapeutic approaches have been developed, lung cancer metastasize remains challenge for drug discovery and development. Recent studies highlight the potent anticancer properties of the compounds derived from Orchid. However, the underlying mechanism was unrevealed. This study demonstrated that cypripedin, a phenanthrenequinone extracted from Dendrobium densiflorum, possess an ability to inhibit in vitro cell migration in non-small cell lung cancer H460 and H23 cells. The number of motile cells, examined by transwell migration and wound healing assay, were gradually reduced in a dose-dependent concentration of cypripedin, along with the reduction of colony number and size formed under an anchorage-independent growth. Mechanistic investigation shows that the mesenchymal markers including N-Cadherin, Vimentin and Slug were significantly down-regulated following cypripedin treatment. Our finding suggests that cypripedin potentiates anti-migrative activity in non-small cell lung cancer through the attenuation of EMT process, which support further extensive studies against lung cancer. Citation Format: Surassawadee Treesuwan, Varisa Pongrakhananon. Cypripedin inhibits non-small cell lung cancer migration through suppression of epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2687.

Cypripedin diminishes an epithelial-to-mesenchymal transition in non-small cell lung cancer cells through suppression of Akt/GSK-3\u03b2 signalling

Lung cancer appears to have the highest rate of mortality among cancers due to its metastasis capability. To achieve metastasis, cancer cells acquire the ability to undergo a switch from epithelial to mesenchymal behaviour, termed the epithelial-to-mesenchymal transition (EMT), which is associated with poor clinical outcomes. Drug discovery attempts have been made to find potent compounds that will suppress EMT. Cypripedin, a phenanthrenequinone isolated from Thai orchid, Dendrobium densiflorum, exhibits diverse pharmacological activities. In this study, we found that cypripedin attenuated typical mesenchymal phenotypes, including migratory behaviour, of non-small cell lung cancer H460 cells, with a significant reduction of actin stress fibres and focal adhesion and with weakened anchorage-independent growth. Western blot analysis revealed that the negative activity of this compound on EMT was a result of the down-regulation of the EMT markers Slug, N-Cadherin and Vimentin, which was due to ATP-dependent tyrosine kinase (Akt) inactivation. As a consequence, the increase in the Slug degradation rate via a ubiquitin-proteasomal mechanism was encouraged. The observation in another lung cancer H23 cell line also supported this finding, indicating that cypripedin exhibits a promising pharmacological action on lung cancer metastasis that could provide scientific evidence for the further development of this compound.

Correction to: Cypripedin, a phenanthrenequinone from Dendrobium densiflorum, sensitizes non-small cell lung cancer H460 cells to cisplatin-mediated apoptosis.

In original publication of the article, unfortunately one of the author names was published incorrectly as "Boonchu Sritularuk". The correct name is "Boonchoo Sritularak".

Cypripedin, a phenanthrenequinone from Dendrobium densiflorum, sensitizes non-small cell lung cancer H460 cells to cisplatin-mediated apoptosis.

The life-threatening potential of lung cancer has increased over the years due to its acquisition of chemotherapeutic resistance, especially to cisplatin, a first-line therapy. In response to this development, researchers have turned their attention to several compounds derived from natural origins, including cypripedin (CYP), a phenanthrenequinone substance extracted from Dendrobium densiflorum. The aim of the present study was to investigate the ability of CYP to induce apoptosis and enhance cisplatin-mediated death of human lung cancer NCI-H460 cells using cell viability and apoptosis assays. The induction of apoptosis by CYP was observed at a concentration of > 50μM with the appearance of morphological changes, including DNA condensation and chromatin fragmentation. Together with, CYP was able to activate caspase-3 and downregulate the anti-apoptotic proteins Bcl-2 and Bcl-xL. Also, a non-cytotoxic dose of CYP synergistically potentiated the effect of cisplatin in non-small cell lung cancer line H460 cells, which clearly exhibited the apoptotic phenotype. Western blot analysis revealed that the underlying mechanism involved the downregulation of anti-apoptotic Bcl-xL, whereas the levels of other apoptotic regulatory proteins were not altered. This study provides interesting information on the potent effect of CYP as a chemotherapeutic sensitizer that could be further developed to improve the clinical outcomes of lung cancer patients.

Photosynthetic Characteristics of Four Wild Dendrobium Species in China

Photosynthetic physiology of Dendrobium nobile, Dendrobium pendulum, Dendrobium chrysotoxum, and Dendrobium densiflorum was studied. A bimodal diurnal variation of the net photosynthetic rate (Pn) was observed in the four Dendrobium species with the first peak [5.09 to 6.06 μmol (CO2) per m−2·s−1] ≈1100 hr and the second peak [3.83 to 4.58 μmol (CO2) per m−2·s−1] at 1500 hr. No CO2 fixation was observed at night. For all four Dendrobium species, the light compensation point (LCP) was 5 to 10 μmol·m−2·s−1, light saturation point (LSP) ranged from 800 to 1000 μmol·m−2·s−1, apparent quantum yield (AQY) was 0.02, and CO2 compensation points (CCP) and saturation point (CSP) were 60 to 85 μmol·mol−1 and 800 to 1000 μmol·mol−1, respectively. Carboxylation efficiency (CE) values ranged from 0.011 to 0.020. The optimum temperature for photosynthesis was between 26 and 30 °C. The measurement of Pn seasonal variation indicated that July to August had the higher Pn for Dendrobium species. Additionally, the chlorophyll a/b (Chl a/b) ratios of the leaves were 2.77 to 2.89. Measurement of key enzymes in the photosynthetic pathway indicated relatively high Ribulose-1,5-bisphosphate carboxylase (RuBPCase) and glycolate oxidase (GO) activities but very low phosphoenolpyruvate carboxylase (PEPCase) activities. It suggested that these four Dendrobium species are typical semishade C3 plants.

Effects of NPK and 6-Benzylaminopurine on Growth and Flowering of Two Orchid Genera

The rising demand for herbal medicine and environmental degradation necessitates the development of more productive management practices for many valuable orchid species. The nitrogen, phosphorous and potassium (NPK) fertilisation and 6-benzylaminopurine (BAP) application has been advised as a practice to address these trends. However, it is not known that at what rate the plant would efficiently endorse the growth. Performance of three NPK ratios, viz., (10:10:10), (20:10:15) and (30:10:15), with three dosages, i.e. 1, 2 and 3 g/l, for each ratio and nine concentrations, i.e. 15, 25, 50, 75, 100, 125, 150, 175 and 200 mg/l, of BAP supplement on two rare orchids Dendrobium densiflorum and Phaius tankervilleae was evaluated in green house conditions. At 1 g/l of (30:10:15) ratio NPK application, both the species showed significantly highest plant height, leaf length, leaf width and number of leaves. Shortest time interval from inflorescence emergence to first flower opening was observed when 200 mg/l of BAP was applied. The results indicate that NPK with high N and K promote vegetative growth and the cytokinin (BAP), at a concentration of 200 mg/l, has a great potential for speeding up flowering process in both the genera.

Performance of Orchid Species in Shevaroy Hills of Eastern Ghats

Performance of 24 orchid species was assessed in the net-house of Horticultural Research Station, Tamil Nadu Agricultural University, Yercaud. Among the species studied, plant height was significantly high (105cm) in Spathoglotts plicata. At the end of the vegetative stage, number of leaves was maximum (30) in Aerides. Leaf was longest (49.8cm) and widest (17.6cm) in Rhyncostylis retusa. Paphiopedilum insigne produced largest flower size (14.4cm across). Pedicel length was highest in Phaius tankervillae (10.3cm) and Renanthera inshootiana (7.1cm). Aerides multiflorum recorded lowest pedicel length (1.24cm). Epidendrum radicans and Spathoglottis plicata produced flowers round the year. Lucia virides recorded maximum life (29 days) on plant, followed by Arachnanthe clarkei (18 days), Dendrobium densiflorum (18 days) and Oncidium flexosum (16 days), while, minimum (6 days) was observed in two orchid species, Aerides crispum and Cattleya sp. Maximum (23 days) vase life was recorded in Lucia virides. Results of the investigation thus reveal that among the 24 orchid species studied, Epidendrum radicans, Lucia virides and Paphiopediluminsigne performed better in terms of vegetative and floral characters under Shevaroy hill condition.

Retraction Note: Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Artificial seeds were obtained through encapsulation of protocorm-like bodies (PLBs) of Dendrobium densiflorum in calcium alginate beads. This paper demonstrates the alginate-encapsulation and conversion (complete plantlet regeneration) from PLBs, the effect of storage conditions (at different temperature; 4, 8, 16 °C, RT and duration; 15, 30, 45, 60, 75, 90 days) on viability of encapsulated plant materials as well as the assessment of genetic fidelity of the regenerants. Individual PLBs were encapsulated in calcium alginate beads for mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3 % sodium alginate and 100 mM calcium chloride (CaCl2·2H2O). The highest percentage of conversion (100 %) of encapsulated PLBs (capsules) was obtained on MS2 medium (MS medium + 2 mg/l BAP). Capsules were successfully stored till 60 days at 8 °C with conversion frequency of 95.5 %. Plantlets regenerated from encapsulated beads were acclimatized successfully with 95 % survival rate. A total of 40 primers were screened, out of which 10 primers successfully generated 39 scorable bands, ranging from 0.2 to 1.3 kb amplicons. The uniform RAPD banding profile among the plantlets derived from encapsulated PLBs following 60 days of storage confirmed genetic fidelity.

Erratum to: Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Erratum to: Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

RETRACTED ARTICLE: Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

RETRACTED ARTICLE: Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Isolation and structural characterization of a neutral polysaccharide from the stems of Dendrobium densiflorum

A novel neutral heteropolysaccharide (DDP-1-D) was purified from hot water extracts of dried stem of Dendrobium densiflorum by DEAE-52 and Sephacryl S-200 High-Resolution Chromatography. The heteropolysaccharide had an average molecular weight about 9440Da. It was composed mainly of glucose and mannose in the ratio of 3.01:1. Structural features of DDP-1-D were elucidated by a combination of chemical and instrumental techniques, including FT-IR, GC–MS, periodate oxidation–Smith degradation, 1H and 13C NMR spectroscopies (including COSY, TOCSY, HSQC, and HMBC spectra). The results indicated that DDP-1-D is a mannoglucan and has a backbone consisting of (1→4)-linked α-d-Glcp, (1→6)-linked α-d-Glcp, (1→2)-linked α-d-Manp and (1→4)-linked β-Manp. This is the first study to provide clear evidence for the structure of the polysaccharide in D. densiflorum.

<i>In vitro</i> seed germination in <i>Cymbidium elegans</i> Lindl. and <i>Dendrobium densiflorum</i> Lindl. ex Wall. (Orchidaceae)

A comparative study of in vitro seed germination of two endangered orchid species, viz. Cymbidium elegans Lindl. and Dendrobium densiflorum Lindl. ex Wall., was carried out on Murashige and Skoog's (MS) medium, supplemented with different concentrations and combination of 6-benzylaminopurine (BAP) and á-Naphthalene acetic acid (NAA). The hormone-free MS medium and MS medium supplemented with various growth hormones were found effective for in vitro seed germination of both species. However, the seeds of these two species showed variation in their germination behavior. Hormone-free MS basal medium was found most effective for seed germination of D. densiflorum; whereas, basal medium supplemented with BAP (1mg/l) was effective for C. elegans. The seeds of D. densiflorum showed quick response in earlier germination, protocorm formation and further development into seedlings in comparison to C. elegans. In C. elegans, germination of immature seeds started after nine weeks of inoculation; whereas in D. densiflorum, the initiation of germination started after five weeks of culture. The variations in seed germination, protocorm formation and seedling differentiation in the two orchid species might be due to the differences in their genetic constitution and the presence of different endogenous growth stimulating substances present in their seeds. The present study has provided useful information for in vitro clonal mass multiplication of these commercially important orchid species. Key-words: growth hormone; in vitro study; orchid.DOI: 10.3126/botor.v6i0.2917 Botanica Orientalis - Journal of Plant Science (2009) 6: 100-102

Genetic characterization of the nine medicinal Dendrobium species using RAPD

The nine species of Dendrobium were distinguished from each other by the banding pattern generated by the sixteen 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. Distinctive bands generated from the nine species were observed. RAPD analysis was also applied to estimate the genetic relationship among the nine species. A dendrogram was constructed based on a data matrix of 323 polymorphic bands originated by the sixteen random primers. Four groups were identified, one consisting of Dendrobium huoshanense and Dendrobium loddigesii, the second consisting of Dendrobium bellatulum and Dendrobium fimbriatum. The third cluster contained Dendrobium candidum, Dendrobium densiflorum and Dendrobium exile. The rest were grouped in the fourth cluster. The principal coordinate analysis (PCA), that is, the plot drawn on the basis of the RAPD data, clearly distinguished the nine species into four groups, which also support the notion in the dendrogram described above.

Changes in endogenous hormone levels and redox status during enhanced adventitious rooting by rare earth element neodymium of Dendrobium densiflorum shoot cuttings

The effects of neodymium nitrate (Nd 3+) on the adventitious rooting of Dendrobium densiflorum shoot cuttings were studied. The addition of Nd 3+ (5 μmol/L) to culture medium significantly increased rooting frequency. Histological investigation showed that Nd 3+ did not change the process of root initiation. Nd 3+ did not influence total endogenous cytokinin levels, but significantly increased the level of endogenous indole-3-acetic acid (IAA) in the base of shoot cuttings. Compared to the control, the ratio of IAA/cytokinins was very high in the Nd 3+ treatment. These results suggested that the enhanced rooting frequency may be related to the increase in endogenous IAA level in Nd 3+ treatment. Analysis of enzyme activities showed that the enhanced accumulation of the endogenous IAA by Nd 3+ should not be attributed to inhibition of IAA decomposition by IAA oxidase or promotion of cytokinin decomposition by cytokinin oxidase. Besides, Nd 3+ increased the ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the process of adventitious rooting while the ratio of ascorbate (ASC) to dehydroascorbate (DHA) was not affected.

Micropropagation of Dendrobium densiflorum Lindl. ex Wall. through protocorm-like bodies: effects of plant growth regulators and lanthanoids

An efficient microprogation protocol has been developed for Dendrobiumdensiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l−1 BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l−1 BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l−1 neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.