Bryophyllum Tubiflorum Research Articles

In Vitro Organogenesis and Epiphyllous Secondary Buds Formation in Kalanchoe tubiflora (Harvey), Hamet, (Crassulaceae)

ABSTRACT: In-vitro organogenesis and epiphyllous secondary buds production in Kalanchoe tubiflora were carried out using MS medium supplemented with appropriate growth regulators. This protocol can be valuable for the mass propagation of this plant species, which is crucial for effective conservation efforts. The highest yield of whitish green massive callus was obtained in MS medium supplemented with 2.0 mg/l of 2,4-dichlorophenoxy acetic acid and 1.0 mg/l of Indole-3-butyric acid. Rapid shoot formation was achieved in MS medium supplemented with 2.0 mg/l of Benzyl amino purine, 1.0 mg/l of kinetin, and 1.0 mg/l of IBA. Effective root induction was observed in MS medium supplemented with 1.5 mg/l of Indole-3-acetic acid, 1.0 mg/l of BA, and 0.5 mg/l of Naphthalene acetic acid. Epiphyllous secondary bud induction was successfully achieved in Murashige and Skoog medium supplemented with 1.0 mg/l of BAP, 0.5 mg/l of kinetin, 0.5 mg/l of IBA, and 0.5 mg/l of Gibberellic acid 3. The plantlets were successfully acclimatized and thrived well in the greenhouse environment. This protocol has significant potential for large-scale production of K. tubiflora and can contribute to its conservation efforts.

Comparative petiole and stem anatomy of species of the genera Bryophyllum and Kalanchoe in South-western Nigeria

The petiole and the stem anatomy of Kalanchoe and Bryophyllum species were studied to document characters that are of taxonomic importance among the species. The species are Kalanchoe crenata, Kalanchoe lanceolata, Bryophyllum pinnatum, Bryophyllum delagoense and Bryophyllum houghtonii. Transverse sections of the median region of the petioles and the stem of each of the species were made using a Reichert sliding microtome at 8-15μm thickness. Unifying and diagnostic features were also identified. Unifying features such as starch grains present on the petiole, parenchymatous pith in the stem were recorded. Diagnostic features such as biseriate to multi seriate epidermis in the stem of Bryophyllum pinnatum were also recorded. The unifying characters suggest that the species of the two genera should be combined into one genus Kalanchoe with sections under it.

Impact of colchicine on leaf morphology and stomatics of Kalanchoe tubiflora (Harv.) Raym.-Hamet (Crassulaceae)

The demand of medicinal plants for consumption is greatly increasing worldwide. The conventional breeding programs are generally dependent on the environment prone to biotic and abiotic stresses. These added to the low content of secondary metabolites at harvest, bring the need for artificial development of polyploid individuals as an alternative to increase productivity. Consequently, the present study evaluated the effect of different colchicine concentrations and exposure time, on Kalanchoe tubiflora leaf morphology and stomata. Initially, K. tubiflora seedlings were harvested and submitted to colchicine concentrations of 0, 0.025, 0.05 and 0.1 % and at two exposure times (24 and 48 hours). Subsequently, morphological measurements such as plant height, leaf width, leaves number, leaf length, leaf thickness and leaf volume every 15 days were made for 16 weeks after planting. Then, the stomata were characterized, taking into account the width, length, stomatal index and the number of chloroplasts per stoma. A significant increase in leaf morphology was found in colchicine treatments of 0.025 % at 48 h and 0.1 % at 24 h. A significant increase in stomatal morphology with the treatment of 0.025 % at 24 h was also recorded. This shows that the correct application of colchicine in term of quantity and time could produce greater growth in a short period and increase the biomass of K. tubiflora medicinal plant.

Trichoderma viride inoculated in the growing medium for the vitamin C increase in the leaves of Kalanchoe spp. and defense against Pithyum sp.

The article presents the results of research on succulents such as Kalanchoe pinnata, Kalanchoe tubiflora and Kalanchoe gastonis-bonnieri, aimed at improving plant growth and defense against the pathogenic fungus Pythium sp., through the use of Trichoderma viride inoculated in the medium. Objectives of the work were: 1) use Trichoderma viride to assess whether the use of this endophytic fungus can increase the growth rate of Kalanchoe pinnata, Kalanchoe tubiflora and Kalanchoe gastonis-bonnieri, plants generally slow in their growth cycle; 2) evaluate if using Trichoderma viride can lead to an increase in vitamin C content in the leaves of Kalanchoe spp.; 3) assess and evaluate how the use of Trichoderma viride allows greater protection of the roots from Pithyum sp. which often affects the roots of these succulents. The two experimental groups in cultivation were: i) group without Trichoderma spp., irrigated with water and substrate previously fertilized; ii) group with Trichoderma viride, irrigated with water and substrate previously fertilized. The test showed a significant increase in agronomic parameters analyzed in plants treated with Trichoderma viride. In fact, all plants treated showed a significant increase in height and number of leaves per plant, vegetative and root weight of plants, number and weight of new shoots, total number of germinated seeds/ 100 seeds sown and a significant reduction in average germination time. In addition, plants treated with Trichoderma show an increase in leaf vitamin C content, dry mass and a reduction in plant mortality due to Pythium sp. Tricoderma can also optimize the use of fertilizers and water by facilitating the cultivation of plants in certain locations subject to biotic and abiotic stress.

Three bufadienolides induce cell death in the human lung cancer cell line CL1‐5 mainly through autophagy

The effects of 3 bufadienolides, namely kalantuboside B, kalantuboside A, and bryotoxin C, isolated from Kalanchoe tubiflora (Harvey) were evaluated and characterized in CL1-5 highly metastatic human lung cancer cells. In contrast to their apoptosis-promoting activity in other cancer cells, these bufadienolides only slight or did not induce apoptosis in CL1-5 cancer cells. Instead, they activated an autophagy pathway, as indicated by increased autophagosome formation. Autophagy induced by these bufadienolides was demonstrated to be linked to the down-regulation of p-mTOR and the up-regulation of LC3-II, ATG5, ATG7, and Beclin-1. Our findings revealed an autophagy as the alternative mechanism of drug action by bufadienolides in CL1-5 lung cancer cells and provided evidence that bufadienolides are a potential therapeutic strategy for highly metastatic human lung cancer.

Phenolic profile by HPLC-ESI-MS/MS and enzymatic inhibitory effect of Bryophyllum delagoense

Bryophyllum delagoense (Eckl. & Zeyh.) Druce, native to Madagascar, is popularly known as “abyssian cactus” and popularly used in folk medicine as an analgesic and healing agent. The study methodology was divided into the phytochemical study: fractionation and identification of phenolic compounds by HLPC-ESI-MS/MS from the methanolic extract (ME), and fractions (DCMF and EAF) of leaves of B. delagoense, and biological activity with acetylcholinesterase and α-glucosidase inhibition of extracts and fractions by in vitro enzymatic techniques. Twenty-seven phenolic compounds were identified, being the highest concentration of syringic acid (87.848 mg g−1). The DCMF fraction showed the best biological activity for inhibition of α-glucosidase enzyme (0.125 mg mL−1).

Kalantuboside B induced apoptosis and cytoprotective autophagy in human melanoma A2058 cells: An in vitro and in vivo study

Kalantuboside B (KB), a natural bufadienolide derivative extracted from the succulent plant Kalanchoe tubiflora, is well-known for its cardiotonic, immunomodulatory, and anti-inflammatory properties. In this study, we tested in vitro and in vivo anti-cancer efficacy with low concentrations of KB (5–30 ng/mL; 8.7–52.2 nM) on A2058 melanoma cells; and for the molecular mechanisms that underlie them. KB significantly inhibited the cell viability and colony formation via arresting the cell cycle at G2/M phase. There was an association with a decrease in Cyclin A/B1, Cdc25C, and Cdc2 expressions. Further, this treatment indicated the induction of apoptosis, DNA fragmentation, cytochrome c release, and caspase-3, -8, -9, and -12 activation, and PARP cleavage, which shows that mitochondrial, death-receptor, and ER-stress signaling pathways are involved. KB-induced autophagy was apparent from enhanced LC3-II accumulation, GFP-LC3 puncta, and AVO formation. Surprisingly, KB-mediated cell death was potentiated by 3-MA and CQ to suggest the role of autophagy as a cytoprotective mechanism. Moreover, KB-treated A2058 cells enhanced intracellular ROS generation and antioxidant NAC prevented apoptosis and reversed cytoprotective autophagy. Interestingly, KB-induced apoptosis (PARP cleavage) and cytoprotective autophagy (LC3-II accumulation) were mediated by the up-regulation of the ERK signaling pathway. It was also shown that KB promoted cytoprotective autophagy by a calcium dependent-p53 downregulation pathway. In vivo data showed that KB suppressed tumor growth significantly in A2058-xenografted nude mice. A Western blot indicated cell-cycle inhibition (cyclin A reduction), apoptosis induction (PARP cleavage and Bcl-2 inhibition), and cytoprotective autophagy (LC3-II upregulation and p53 downregulation) in KB-treated A2058-xenografted mice. Our findings suggested that KB-induced ROS pathway plays a role in mediating the apoptosis and cytoprotective autophagy in human melanoma cells. Thus, KB is considered to be a putative anti-tumor agent.

Bryophyllum pinnatum and Related Species Used in Anthroposophic Medicine: Constituents, Pharmacological Activities, and Clinical Efficacy.

Bryophyllum pinnatum (syn. Kalanchoe pinnata) is a succulent perennial plant native to Madagascar that was introduced in anthroposophic medicine in the early 20th century. In recent years, we conducted a large collaborative project to provide reliable data on the chemical composition, pharmacological properties, and clinical efficacy of Bryophyllum. Here, we comprehensively review the phytochemistry, as well as the pharmacological and clinical data. As to the pharmacology, special emphasis is given to properties related to the use in anthroposophic medicine as a treatment for "hyperactivity diseases", such as preterm labor, restlessness, and sleep disorders. Studies suggesting that B. pinnatum may become a new treatment option for overactive bladder syndrome are also reviewed. Tolerability is addressed, and toxicological data are discussed in conjunction with the presence of potentially toxic bufadienolides in Bryophyllum species. The few data available on two related species with medicinal uses, Bryophyllum daigremontianum and Bryophyllum delagoense, have also been included. Taken together, current data support the use of B. pinnatum for the mentioned indications, but further studies are needed to fully understand the modes of action, and to identify the pharmacologically active constituents.

Assessment of a stenophagous weevil, Osphilia tenuipes (Coleoptera: Curculionidae), as a potential biological control agent for weedy Bryophyllum spp. (Crassulaceae) in Australia

Bryophyllum delagoense (Crassulaceae) is a serious weed in Australia and a target for biological control. Following preliminary testing in South Africa, the host range of a Madagascan conderine weevil, Osphilia tenuipes, was determined within a quarantine facility in Queensland, Australia. The primary test consisted of exposing potted plants, selected from an approved host test list, to attack by the weevil in a no-choice design. Tests of oviposition on cut stems and adult preference in a choice design were also undertaken. Osphilia tenuipes used all Bryophyllum spp. found in Australia as hosts and also several other exotic species of Crassulaceae, including Kalanchoe blossfeldiana, Kalanchoe spathulata and Echeveria sp. Native Australian species of Crassulaceae, all Crassula spp., were not attacked, nor were any plant species outside the family Crassulaceae. The preference test showed that the weevil fed on and oviposited in Bryophyllum pinnatum, K. blossfeldiana (two varieties) and Kalanchoe sexangularis in the presence of B. delagoense. The climate matching software CLIMEX was used to develop models that indicated that much of Australia, including southern states, would be climatically suitable for both B. delagoense and O. tenuipes, and that the predicted distribution of both organisms would move southward with climate change. Because the conflicts of interest attributed to the non-target attack are thought to be resolvable, the insect is to be assessed for release under the provisions of Australia’s Biological Control Act.

Anticancer activity of Kalanchoe tubiflora extract against human lung cancer cells in vitro and in vivo.

Uncontrolled cell proliferation is a common feature of human cancer. Some of herbal extract or plant-derived medicine had been shown as an important source of effective anticancer agents. We previously reported that an n-BuOH-soluble fraction of Kalanchoe tubiflora has antiproliferative activity by inducing mitotic catastrophe. In this study, we showed that the H2 O-soluble fraction of Kalanchoe tubiflora (KT-W) caused cell cycle arrest, and senescence-inducing activities in A549 cells. We used 2 dimensional PAGE to analyze the protein expression levels after KT-W treatment, and identified that the energy metabolism-related proteins and senescence-related proteins were disturbed. In vivo experiments showed that the tumor growths in A549-xenografted nude mice were effectively inhibited by KT-W. Our findings implied that KT-W is a putative antitumor agent by inducing cell cycle arrest and senescence. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1663-1673, 2016.

Quantification of Bufadienolides in Bryophyllum pinnatum Leaves and Manufactured Products by UHPLC-ESIMS/MS.

A quantitative assay for determination of the main bufadienolides bersaldegenin-1-acetate (1), bersaldegenin-3-acetate (2), bryophyllin A (3), and bersaldegenin-1,3,5-orthoacetate (4) in Bryophyllum pinnatum leaves and manufactured products was developed and validated. The assay involved extraction by pressurised liquid extraction, followed by quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy. The ultrahigh performance liquid chromatography-tandem mass spectroscopy method was applied to various batches of leaves harvested on several dates from plants grown at two locations (Brazil and Germany). In addition, press juices prepared from plants cultivated in Germany and Brazil were analysed. The total bufadienolide content ranged from 16.28 to 40.50 mg/100 g dry weight in leaves from plants grown in Brazil. The total content of these four bufadienolides was significantly lower in plants cultivated in Germany (3.78-12.49 mg/100 g dry weight, resp.). The total amounts of bufadienolides were 0.091-0.163 mg/100 mL and 0.89-1.16 mg/100 mL in press juices obtained from plants cultivated in Germany and Brazil, respectively. When analysing single leaves from individual plants, the content of bufadienolides was markedly higher in young leaves. For comparative purposes, the content of these bufadienolides was also determined in Bryophyllum daigremontianum and Bryophyllum tubiflorum. Bersaldegenin-1,3,5-orthoacetate (4) was predominant in the leaves of B. daigremontianum and in the stems of B. tubiflorum, while the leaves of B. tubiflorum contained very low amounts of 1-4.

Water loss prevention plays a greater role than ROS scavenging in dehydration tolerance of Kalanchoe tubiflora epiphyllous buds

Different plants and plant organs have various strategies to cope with drought stress.Kalanchoe tubifloraplants have a strong ability to prevent water loss and to scavenge reactive oxygen species. The epiphyllous bud of the plant serves as a reproduction unit and is able to generate plantlets even under conditions of extreme drought. The aim of this project was to study the antioxidant-stress response and osmoregulation ofK. tubifloraepiphyllous buds to dehydration and to compare the drought-tolerance mechanisms of the plant body and the epiphyllous buds. With the progression of bud dehydration, relative water content, seedling survival rate and dry weight per seedling decreased, and electrolyte leakage, hydrogen peroxide and malondialdehyde content and superoxide anion production rate increased. The activities of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased under dehydration. The content of proline, soluble sugars and soluble protein increased in dehydrated buds proportionally with the extent of water loss. These data indicate that the drought-tolerance mechanism of theK. tubiflorabud was different from that of its mother plant. The buds invest more energy to prevent water loss during drought stress, and their antioxidant defense weakens.

Micropropagation of Kalanchoe tubiflora (Harvey) Hamet

Summary. Kalanchoe tubiflora (Harvey) Hamet is a succulent plant native to Madagascar. Due to its unique morphology, it is a popular ornamental species. However, owing to some valuable metabolites it can also be used in tumor suppression. The present work describes a simple propagation method of K. tubiflora. The stem fragments of K. tubiflora were disinfected with the following sodium hypochlorite concentrations: 0.2% (for 10 or 20 min), 0.4% (10 or 20 min) and 0.6% (10 or 20 min). Next the single-node explants were inoculated on a hormone-free MS medium. The produced plants were transferred to soil and grown in a greenhouse. The applied disinfection procedures were successful. The regeneration of propagules was observed. The average number of new microshoots produced from one explant varied from about 20 to even 52. All microshoots regenerated adventitious roots without transferring them to a rooting medium. The plantlets were successfully acclimatized and continued their growth in the greenhouse.

IDENTIFICATION AND GENETIC DIVERSITY OF PECTOLYTIC PHYTOPATHOGENIC BACTERIA OF MONO- AND DICOTYLEDONOUS ORNAMENTAL PLANTS IN IRAN

Bacterial soft rot can be a destructive disease of ornamental plants. To identify the pathogenic soft rot bacteria occurring in ornamental plants in Tehran and Markazi provinces (Iran), 57 isolates were obtained from 12 different mono- and dicotyledonous hosts and investigated with regard to phenotypic, genotypic and pathogenicity features. Based on phenotypic characteristics, the bacterial strains were identified as Pectobacterium carotovorum subsp. carotovorum. This was confirmed by subspecies-specific primers using PCR. Inoculation of all isolates into Aglaonema leaves confirmed the pathogenecity of the isolated strains. To assess the genetic diversity within Pectobacterium carotovorum subsp. carotovorum populations by rep-PCR, 26 isolates were selected according to their host and geographic distribution as well as two strains from Guilan province. Cluster analysis was conducted using the UPGMA method and revealed a possible close relationship between DNA fingerprints and geographical origins of isolates. Our results showed significant genetic variation among the populations of this pathogen. To the best of our knowledge, this is the first report of Pectobacterium carotovorum subsp. carotovorum on Peperomia obtusifolia, P. caperata, Pilea cadierei, Plectranthus australis, Saintpaulia ionantha and Kalanchoe tubiflora in Iran.

Photoperiodic and thermal requirements for the flowering of Bryo phyllum daigremontianum (Hamet. et Perr.) Berg. and Bryophyllum tubiflorum Harv.

Marked differences were found between the photoperiodic responses of <em>Bryophyllum daigremontianum</em> and <em>Bryophyllum tubiflorum</em>, both species being classified as long-short-day plants (LSDP). The flowering of <em>B. daigremontianum</em> was observed under several photoperiodic conditions, among others under continuous exposure to long day (LD) or after transferring plants from a short-day (SD) to a long-day exposure: both conditions were regarded previously as non-inductive. It is argued that the flowering of <em>B. daigremontianum</em> is conditioned by sufficiently long LD exposure followed by one of two factors: the shortening of the day or the lowering of the temperature. The flowering of <em>B. tubiflorum</em> was observed only under more specific photoperiadic conditions, it seems to be conditioned by several successive changes of the length of the photoperiod.

Species limits, quarantine risk and the intrigue of a polyphagous invasive pest with highly restricted host relationships in its area of invasion

Scirtothrips aurantii is a generalist horticultural pest in its native African range and recently established quite widely in Australia on the invasive succulent weed Bryophyllum delagoense. Paradoxically, this thrips is not polyphagous in its incursive range. The issue is principally one of quarantine. Will the thrips in Australia shift, perhaps adaptively, to citrus, and should the primary focus be on containment around Australian citrus, or does the real quarantine risk exist offshore with thrips present on citrus in Africa? We examined the phylogenetic relationships between Bryophyllum-associated thrips populations in Australia and populations sampled from various host plant species in South Africa (including Bryophyllum) using both CO1 and 28s markers. Eight variable microsatellite markers were developed to assess the extent of gene flow between the thrips on different hosts in South Africa. The COI phylogeny resolved S. aurantii into three distinct clades with samples collected from B. delagoense in South Africa and Australia representing a single clade, a second clade associated with Gloriosa lilies and the third with horticultural hosts. The microsatellite analysis confirmed that the populations associated with citrus and Bryophyllum do not hybridize with one another in sympatry. We conclude that the citrus-damaging thrips are not currently present in Australia and remain a serious quarantine concern in relation to Australian horticulture.

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English

Cardenolides and Bufadienolide Glycosides from Kalanchoe tubiflora and Evaluation of Cytotoxicity

Two new cardenolides, kalantubolide A (1) and kalantubolide B (2), and two bufadienolide glycosides, kalantuboside A (3) and kalantuboside B (4), as well as eleven known compounds were isolated and characterized from the EtOH extract of Kalanchoe tubiflora. The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses including HMQC, HMBC, and NOESY. Biological evaluation indicated that cardenolides (1-2) and bufadienolide glycosides (3-7) showed strong cytotoxicity against four human tumor cell lines (A549, Cal-27, A2058, and HL-60) with IC50 values ranging from 0.01 µM to 10.66 µM. Cardenolides (1-2) also displayed significant cytotoxicity toward HL-60 tumor cell line. In addition, compounds 3, 4, 5, 6, and 7 blocked the cell cycle in the G2/M-phase and induced apoptosis in HL-60 cells.

A new megastigmane from Kalanchoe tubiflora (Harvey) Hamet

One new megastigmane, (6S,7R,8R,9S)-6-oxaspiro-7,8-dihydroxymegastigman-4-en-3-one (1) (tubiflorone, 1), and ten known compounds were isolated and characterized from the EtOH extract of Kalanchoe tubiflora (Harvey) Hamet. Structures of these isolates were assigned based on spectroscopic analyses that included 1D and 2D NMR techniques, such as HMQC, HMBC, and NOESY. The anti-inflammatory activities of selected isolated compounds (1–6 and 9–11) were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compounds 1–4, 6, 9, and 11 possessed nitric oxide inhibitory activity with IC50 values ranging from 15.1±0.9 to 98.9±1.3μM.

Polyphagy in the Australian population of South African citrus thrips (Scirtothrips aurantii Faure)

The South African citrus thrips, Scirtothrips aurantii Faure, 1929, is highly polyphagous in its country of origin. Introduced to Australia in about 2002, populations of this thrips have been found only on the pasture weed Mother of Millions, Bryophyllum delagoense (Eckl. & Zeyh.) 1900. To evaluate the potential risk by these populations to plants of economic significance, under laboratory conditions, whether S. aurantii collected from Mother of Millions in Queensland can survive, reproduce and maintain populations over multiple generations on non-target hosts was tested. That this thrips can form self-sustaining populations on mango, grape, chilli, pea, green bean and blueberry was showed. Extended adult lifespan and some juvenile production were found on lemon and orange, but populations did not persist on these hosts. Furthermore, female thrips accept mango and grape for oviposition as readily as Mother of Millions. This degree of polyphagy under laboratory conditions suggests that caution is needed before promoting this thrips as a biocontrol agent against the pasture weed.