Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application.

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4-5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell's resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells.

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the upregulation of CFS (cheilanthifoline synthase) to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h respectively, under MJ elicitation. Besides, MJ upregulated the expression of TNMT (tetrahydroprotoberberine N-methyltransferase) to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs' level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Biotechnological Exploration of Transformed Root Culture for Value-Added Products.

Medicinal plants produce valuable secondary metabolites with anticancer, analgesic, anticholinergic or other activities, but low metabolite levels and limited available tissue restrict metabolite yields. Transformed root cultures, also called hairy roots, provide a feasible approach for producing valuable secondary metabolites. Various strategies have been used to enhance secondary metabolite production in hairy roots, including increasing substrate availability, regulating key biosynthetic genes, multigene engineering, combining genetic engineering and elicitation, using transcription factors (TFs), and introducing new genes. In this review, we focus on recent developments in hairy roots from medicinal plants, techniques to boost production of desired secondary metabolites, and the development of new technologies to study these metabolites. We also discuss recent trends, emerging applications, and future perspectives.

Modern Trends in the In Vitro Production and Use of Callus, Suspension Cells and Root Cultures of Medicinal Plants.

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.

Developmental Methylome of the Medicinal Plant Catharanthus roseus Unravels the Tissue-Specific Control of the Monoterpene Indole Alkaloid Pathway by DNA Methylation.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental- and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.

Recent Advances in the Tissue Culture of American Ginseng (Panax quinquefolius).

The in vitro tissue culture of medicinal plants is considered as a potential source for plant-derived bioactive secondary metabolites. The in vitro tissue culture of American ginseng has wide commercial applications in pharmaceutical, nutraceutical, food, and cosmetic fields with regard to the production of bioactive compounds such as ginsenosides and polysaccharides. This review highlights the recent progress made on different types of tissue culture practices with American ginseng, including callus culture, somatic embryo culture, cell suspension culture, hairy root culture, and adventitious root culture. The tissue culture conditions for inducing ginseng callus, somatic embryos, cell suspension, hairy roots, and adventitious roots were analyzed. In addition, the optimized conditions for increasing the production of ginsenosides and polysaccharides were discussed. This review provides references for the use of modern biotechnology to improve the production of bioactive compounds from American ginseng, as well as references for the development and sustainable utilization of American ginseng resources.

Report: Pharmacognostic and physicochemical screening of Euphorbia nivulia Buch.-Ham.

Euphorbia nivulia Buch.-Ham. (Euphorbiaceae) is commonly known as Indian Spurge Tree in English, and "Saj Thor" or "Jhanami booti" in local language. The plant is used traditionally in the treatment of various diseases like inflammation, fever, worm infection, asthma, cough, wounds and diabetes. In current study fresh as well as dried aerial parts of the plant and cut sections were examined, both macroscopically and microscopically. The study also deals with fluorescence analysis and phytochemical characteristics and other WHO recommended methods for standardization. WHO guidelines on quality control for medicinal plants materials were used for pharmacognostical evaluation of E. nivulia, phytochemical screening helps in determining the predominant classes of active constituents responsible for the activity. The present work will be helpful in identification of the fresh and dried samples of aerial parts pharmacognostically and anatomically. These studies will serve as a reference for correct identification and may be helpful in checking any type of adulteration. These observations will also help in differentiating this species from closely related species of the same genus and family.

Implication of palynological techniques for the authentication of adulterated drugs traded with the same name in different herbal markets of district Lahore, Pakistan.

Adulteration in traded medicinal plants is a significant issue nowadays and use of these adulterated medicinal plants can impose harmful impact to end user. However, this problem can be overcome by ensuring the identification of traded medicinal plants which are used in making different herbal medicines. In this regard, palynological markers are considered to be an important taxonomic tool in the identification of original medicinal plant from its adulterant. Hence this study attempted to provide particular reliable palynological markers for distinguishing selected medicinal plants from their adulterants, that is, Cinnamomum verum versus Canella winterana, Cinnamomum tamala versus Cinnamomum obtusifolium, Gymnema sylvestre versus Gymnema lactiferum, Artemisia maritima versus Artemisia absinthium, Achillea millefolium versus Adhatoda vasaka, Sphaeranthus indicus versus Sphaeranthus africanus, Averrhoa carambola versus Butea monosperma, and Morus nigra versus Morus alba. Results demonstrated great variations in multiple palynological characters between original medicinal plant and its adulterant such as in pollen size, shape, colpi length, exine, intine thickness, and fertility. In equatorial view, circular to spheroidal shape of pollen was found in A. millefolium while oblate shape was observed in A. vasaka. Similarly B. monosperma pollen was 34 µm, whereas pollen of its adulterant A. carambola was 21 µm. Moreover, colpi length of A. maritima was 11.8 µm, whereas 4.5 µm in A. absinthium. Hence it can be concluded that palynological characters are commendably helpful in identification of genuine medicinal plant from its adulterant.

Morphological and microscopic identification of three major medicinal Dendrobium species in Ta-pieh Mountains area.

Dendrobium is an important medicinal material in China. It has the effect of nourishing the stomach, nourishing yin and clearing heat. In China, there are many types of Dendrobium, and different Dendrobium species have different efficacy. The present study is aimed at distinguishing three major Dendrobium species from morphological and microscopic identification in Ta-pieh mountains area. In this article, the roots, stems and leaves of Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme are used as materials to compare the differences of tissues of these three Dendrobium species by morphological indexes and microscopic identification of different Dendrobium. The stem morphology of these three Dendrobium species was significantly different except for stem internodes number and the middle part of the stem diameter by measuring the stems of Dendrobium. To ensure the safe use of Dendrobium, we built a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. huoshanense, D. officinale, and D. moniliforme. The microscopic results show that different types of Dendrobium exhibit different states that can be distinguished under normal light normal and fluorescence microscopy. This comparative study of morphology and microscopy contributes to the development of identification and quality evaluation of Dendrobium.

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described.

A new anti-inflammatory ß-carboline alkaloid from the hairy-root cultures of Eurycoma longifolia.

One new ß-carboline alkaloid 7-methoxy-(9H-ß-carbolin-1-il)-(E)-1-propenoic acid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) were isolated from the hairy-root cultures of Eurycoma longifolia. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells were investigated. Compound 1 strongly inhibited the production of NO while 2 and 3 having weak or inactive effect. Consistently, compound 1 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase.

Biofertilization with Azospirillum brasilense improves in vitro culture of Handroanthus ochraceus, a forestry, ornamental and medicinal plant.

Biofertilization with plant growth-promoting rhizobacteria is a potential alternative to plant productivity. Here, in vitro propagation of Handroanthus ochraceus (yellow lapacho), a forest crop with high economic and environmental value, was developed using the Azospirillum brasilense strains Cd and Az39 during rhizogenesis. Epicotiles of in vitro plantlets were multiplied in Woody Plant Medium (WPM). For rooting, elongated shoots were transferred to auxin-free Murashige-Skoog medium with Gamborg's vitamins and WPM, both at half salt concentration (½MSG and ½WPM), and inoculated with Cd or Az39 at the base of each shoot. Anatomical studies were performed using leaves cleared and stained with safranin for optical microscopy and leaves and roots metalized with gold-palladium for scanning electron microscopy (SEM). In ½WPM auxin-free medium, A. brasilense Cd inoculation produced 55% of rooting, increased root fresh and dry weight (45% and 77%, respectively), and led to lower stomata size and density with similar proportion of open and closed stomata. Both strains selectively increased the size or density of glandular trichomes in ½MSG. Moreover, bacteria were detected on the root surface by SEM. In conclusion, the difference in H. ochraceus response to A. brasilense inoculation depends on the strain and the plant culture media. Cd strain enhanced rooting in auxin-free ½WPM and produced plantlets with features similar to those expected in ex vitro plants. This work presents an innovative in vitro approach using beneficial plant-microorganism interaction as an ecologically compatible strategy in plant biotechnology.

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites.

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative "chemical factory". However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.

Propiedades anti-Helicobacter pylori de los extractos de Psidium guajava y Coptis chinensis / Atividade anti-Helicobacter pylori de los extractos de Psidium guajava e Coptis chinensis / Anti-Helicobacter pylori activity of extracts of Psidium guajava and Coptis chinensis

Se postula y fundamenta Ia utilidad de una mezcIa de extractos de plantas medicinales con propiedades sinérgicas compuesta por Psidium guajava L. estandarizado en su contenido de heterósidos flavónicos y Coptis chinensis Franch., estandarizado en su contenido de aIcaIoides benzofenantridínicos, para eI desarroIIo de un fitomedicamento útiI para eI tratamiento y prevención de Ia gastritis crónica provocada por Helícobacter pylori. EI estudio explora eI potencial que tiene Ia mezcIa para inhibir eI crecimiento in vitro de diversas cepas clínicas de H. pylori resistentes a los antibióticos convencionales, así como, su acción protectora deI epiteIio gástrico, al impedir Ia adherencia de Ia bacteria alas celulas AGS en cultivo (AU)

Descreve-se e fundamenta-se a utilidade de uma associagáo de extratos de plantas medicinais com propriedades sinergicas composta por Psidium guajava L., extracto padronizado em glicósidos flavónicos e Coptis chinensis Franch, extracto padronizado em alcalóides benzofenantridínicos, para o desenvolvimento de um medicamento a base de plantas para o tratamento e prevençáo da gastrite crónica provocada por Helicobacter pylori. O estudo explora o potencial que tem esta associaçáo para inibir o crescimento in vitro de diversas estirpes clinicas de H. pylori resistentes aos antibióticos convencionais, assim como, a sua acçáo protectora do epitelio gástrico, ao impedir a aderencia das bacterias ás culturas celulares de AGS (AU)

It is described the synergistic properties of a mixture of Psidium guajava L., extract standardized in its content of flavone glycosides and Coptis chinensis Franch, extract standardized in its content of benzophenantridinic alkaloids, for developing a phytodrug for the treatment and prevention of chronic gastritis induced by Helicobacter pylori. The study explores the properties of this combination of extracts by inhibiting in vitro growth of antibiotic-resistant clinical H. pylori strains and preventing adherence of the bacteria to human AGS cell cultures (AU)

Botanical features for identification of Gymnosporia arenicola dried leaf.

Gymnosporia arenicola Jordaan (Celastraceae) is a shrub or small tree, which naturally occurs in coastal sand dunes of Southern Mozambique and South Africa. Its dried leaf is often used in traditional medicine for the treatment of infectious and inflammatory diseases. Hereby, we present results of studies carried out according to the pharmacopoeia standards for the identification of herbal drugs, in the whole, fragmented, and powdered plant material. These results were complemented with scanning electron microscopy and histochemical techniques. The leaf microscopic analysis revealed a typical dorsiventral mesophyll with a corresponding spongy parenchyma-palisade parenchyma ratio of 0.60, anomocytic and paracytic stomata, papillate cells with a diameter of 4.00 ± 0.40 µm, multicellular uniseriate nonglandular trichomes with a length of 27.00 ± 4.10 µm and cristalliferous idioblasts containing calcium oxalate cluster crystals with a diameter of 23.04 ± 5.84 µm. The present findings demonstrate that the G. arenicola leaf has both nonglandular trichomes and hypoderm, features not previously described in the corresponding botanical section (Gymnosporia sect. Buxifoliae Jordaan). The establishment of these new botanical markers for the identification of G. arenicola leaf is essential for quality, safety and efficacy reasons.

[Clonal micropropagation of a rare species Hedysarum theinum Krasnob (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers].

In the present study, a protocol was developed for the in vitro propagation of a rare medicinal plant, Hedysarum theinum (tea sweetvetch), from axillary buds, and identification of the regenerants was performed with the use of ISSR markers. It was demonstrated that Gamborg and Eveleigh medium supplemented with 5 µM 6-benzylaminopurine was the best for H. theinum for initial multiplication. On the other hand, half-strength Murashige and Skoog (MS) basal medium supplemented with 7 µM α-naphthaleneacetic acid proved to be the best for explant rooting. Molecular genetic analysis of the H. theinum mother plants and the obtained regenerants was performed with six ISSR markers. Depending on the primer, four to ten amplified fragments with sizes ranging from 250 to 3000 bp were identified. Our results confirmed the genetic stability of regenerants obtained in five passages and their identity to the mother plant.

Methyl jasmonate, yeast extract and sucrose stimulate phenolic acids accumulation in Eryngium planum L. shoot cultures.

Eryngium planum L. has been reported as a medicinal plant used in traditional medicine in Europe. The tissue cultures may be an alternative source of the biomass rich in desired bioactive compounds. The purpose of this study was to investigate the influence of the biotechnological techniques on the selected phenolic acids accumulation in the agitated shoot cultures of E. planum. Qualitative and quantitative analyses of those compounds in 50% aqueous - methanolic extracts from the biomass were conducted by applying the HPLC method. Methyl jasmonate (MeJA), yeast extract (YE) and sucrose (Suc) stimulated accumulation of the phenolic acids: rosmarinic (RA), chlorogenic (CGA) and caffeic (CA) in in vitro shoot cultures. Cultivation of shoots in liquid MS media supplemented with 1.0 mg L(-1) 6-benzyladenine and 0.1 mg L(-1) indole-3-acetic acid in the presence of 100 µM MeJA for 48h was an optimum condition of elicitation and resulted in approximately 4.5-fold increased content of RA + CGA + CA in plant material compared to the control (19.795 mg g(-1) DW, 4.36 mg g(-1) DW, respectively). The results provide the first evidence that the selected phenolic acids can be synthesized in elicited shoot cultures of flat sea holly in higher amount than in untreated shoots.

[Microscopic identification of dai medicine alpiniae kwangsiensis rhizoma and its confused species].

OBJECTIVE: To observe microscopic characteristics of rhizome of Alpinia kwangsiensis, Alpinia platychilus, Alpinia blepharocalyx, and to provide basis for their identification. METHODS: Microscopic identification of root transverse section by paraffin tissue section and free-hand section and powder were carried out to distinguish them. RESULTS: The microscopic identification can be made by cell structure of epidermis and endodermis, the existence of nonglandular hair, the lignification degree of vascular bundle fiber, the number and existence style of vascular bundle fiber, the number and size of tube, and so on. The powder identification can be made mainly according to the existence of nonglandular hair and spiral vessel, the shape of starch grain, and so on. CONCLUSION: The microscopic characteristics of rhizome can provide basis for the identification of three Alpinia species.