Rauvolfia Tetraphylla Research Articles

Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl(2). The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.

An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

Evaluation of in-vitro antibacterial activity and anti-inflammatory activity for different extracts of Rauvolfia tetraphylla L. root bark

ObjectiveTo assess the in-vitro antibacterial activity and anti-inflammatory activity of orally administered different extracts (Hydro-alcoholic, methanolic, ethyl acetate and hexane) of Rauvolfia tetraphylla (R. tetraphylla) root bark in Carrageenan induced acute inflammation in rats. MethodsIn-vitro antibacterial activity was evaluated for extracts against four Gram positive and four Gram negative bacteria by using cylinder plate assay. Hydro-alcoholic extract (70% v/v ethanol) at 200, 400 and 800 mg/kg doses and methanolic, ethyl acetate and hexane extracts at doses 100, 200 and 400 mg/kg were tested for anti-inflammatory activity in Carrageenan induced rat paw oedema model and paw thickness was measured every one hour up to 6 hrs. ResultsAll extracts of R. tetraphylla root bark showed good zone of inhibition against tested bacterial strains. In Carrageenan induced inflammation model, hydro-alcoholic and methanolic extract of R. tetraphylla root bark at three different doses produced significant (P<0.001) reduction when compared to vehicle treated control group and hexane, ethyl acetate extracts. ConclusionIn the present study extracts of R. tetraphylla root bark shows good in-vitro antibacterial activity and in-vivo anti-inflammatory activity in rats.

Rauvotetraphyllines A-E, new indole alkaloids from Rauvolfia tetraphylla

Five new indole alkaloids rauvotetraphyllines A–E (1–5), together with eight known analogues, were isolated from the aerial parts of Rauvolfia tetraphylla. The structures were established by means of spectroscopic methods. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s13659-012-0012-5 and is accessible for authorized users.

Studies on methanolic extract of Rauvolfia species from Southern Western Ghats of India –In vitro antioxidant properties, characterisation of nutrients and phytochemicals

Rauvolfia has long been regarded as medicinal plant. Leaves of all the reported five species of Rauvolfia from southern Western Ghats were chosen to investigate their antioxidant activities, phytochemicals and nutrient composition. The five species were then screened for their antioxidant potentials using various in vitro models such as total antioxidant capacity, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, reducing power and superoxide anion scavenging activity at various concentrations. Nutrients and phytochemical characterisation included determination of tocopherols by HPLC-fluorescence, phenolics, flavonoids, carotenoids, ascorbic acid and pigment composition by spectrophotometric techniques. Rauvolfia serpentina exhibited the highest total phenolic content while Rauvolfia tetraphylla had highest flavonoid content among the five species. R. serpentina showed the highest DPPH radical scavenging activity and also highest pigment composition and vitamin E content, while Rauvolfia densiflora showed highest level of vitamin C content and metal chelating activity among the five species. R. tetraphylla revealed the highest concentration of β carotene. Lycopene was found in very low amounts while comparing with other nutrient compositions and the maximum amount was in R. tetraphylla and least amount was in Rauvolfia beddomei. The results of this study provided an alternative of utilising Rauvolfia leaf as readily accessible source of natural antioxidant in food cosmetic and pharmaceutical industry.

Encapsulation of Rauvolfia tetraphylla microshoots as artificial seeds and evaluation of genetic fidelity using RAPD and ISSR markers

Synseeds of Rauvolfia tetraphylla were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Woody Plant Medium (WPM) (Lloyd and McCown, 1981) and 100 mM calcium chloride. Effect of different medium on in vitromorphogenic response of synseed was evaluated. The maximum frequency (90.3%) of conversion of encapsulated beads into plantlets was achieved on WPM containing 7.5 μM 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. Encapsulated nodal segments stored at 4°C for 1 to 8 weeks also showed successful conversion, followed by development into complete plantlets when returned to regeneration medium. Healthy root formation was achieved in plantlets following 4 weeks of their transfer to Murashige and Skoog medium containing 0.5 μM indole-3-butyric acid (IBA). Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. Genetic stability of synseed-derived plantlets was assessed and compared with mother plant using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. No changes in RAPD and ISSR profiles were found among the regenerated plantlets. This synseed protocol could be useful for conservation, short-term storage and exchange of germplasm of this endangered medicinal plant between national as well as international laboratories. Key words: Encapsulation, germplasm storage, regeneration, synthetic seeds, random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR).

A New Labdane Diterpene from Rauvolfia Tetraphylla Linn. (Apocynaceae)

The air-dried stems and branches of Rauvolfia tetraphylla afforded a new labdane diterpene characterised as 3β-hydroxy-labda-8(17),13(14)-dien-12(15)-olide on the basis of spectroscopic studies including UV, IR, MS and NMR (1H, 13C and HMQC); this is the first report of a new terpenoid constituent from R. tetraphylla, an important medicinal plant widely known for the source of a variety of alkaloids.

Changes in photosynthetic activity, pigment composition, electrolyte leakage, lipid peroxidation, and antioxidant enzymes during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets

The effects of photosynthetic photon flux density (PPFD) on antioxidant metabolism and photosynthetic properties in leaves during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets were investigated. In vitro-propagated plantlets were acclimatized at either 50 (Low-light = LL) or 300 (High-light = HL) μmol m−2s−1 photosynthetic PPFD for 4 weeks under controlled conditions. Increases in chlorophyll (Chl) a, b and carotenoid levels were observed in plantlets acclimatized at both light intensities. At transplantation, micropropagated plantlets were not photosynthetically active, but the net photosynthetic rate increased in newly formed leaves over time during acclimatization. The observed differences in pigment contents and photosynthetic rates suggested adaptation of plantlets from heterotrophic to autotrophic mode of nutrition during acclimatization. Changes in activities of antioxidant enzymes were also observed during acclimatization. Superoxide dismutase activity increased in plantlets acclimatized at HL intensities. Likewise, changes in activity of catalase and ascorbate peroxidase were also detected. These observed changes reflected the ability of plants in developing an antioxidant enzymatic defense system aiding in survival against oxidative stress and in reducing release of free radicals.

Influence of Auxins Combinations on Accumulation of Reserpine in the Callus of Rauvolfia tetraphylla L.

Reserpine is a monoterpene indole alkaloid used to treat hypertension because of its hypotensive property and psychiatric disorders because of its tranquilizing effect. Protocol has been standardized to enhance the synthesis of reserpine in leaf derived calli of Rauvolfia tetraphylla L. by adjusting the auxins combinations in the medium consisting of MS nutrient salts and B5 vitamins. Auxins such as naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were used in 1-5 microM concentration along with 9 microM concentration of 2,4 dichlorophenoxy acetic acid (2,4-D), which was found suitable for callus induction. The combination of (2,4-D) with NAA had been proved to accumulate maximum amount of reserpine followed by 2,4-D with IBA. The IAA with 2,4-D combination yielded very less amount of reserpine than the other combinations and 9 microM 2,4-D alone. The results suggest that there may be synergetic effect of NAA with 2,4-D and IBA with 2,4-D for increase in the biomass and reserpine accumulation and antagonistic effect of IAA with 2,4-D for the above said factors in the callus.

Genetic Diversity in Rauvolfia tetraphylla L.f using RAPD Markers

The present study is the first report of molecular variations in different accessions of Rauvolfia tetraphylla L.f, a medicinally important plant collected from seven locations of Andhra Pradesh, India. Molecular analysis was carried out using RAPD markers. Out of the 40 primers screened from OPA and OPC Kts, a total of 205 scorable polymorphic markers out of 397 total markers were generated. Polymorphism of 51.6% was found with 3 unique markers. Levels of genetic diversity within accessions i.e., the genetic distance ranged from 0.816-0.932. Cluster analysis based on Dice coefficient showed two major groups indicating that mostly in cross-pollinated plants, high levels of differentiation among accessions exists independent of geographical distance. Hence the results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of this species. Understanding such variation would also facilitate their use in various conservational management practices, rootstock breeding and hybridisation programmes.

Antimicrobial activity of Rauvolfia tetraphylla and Physalis minima leaf and callus extracts

The in vitro antimicrobial activity of Rauvolfia tetraphylla and Physalis minima leaf and callus extracts were studied against selected pathogenic fungi and bacteria, following broth dilution assay. Leaves and calli were extracted using absolute alcohol, benzene, chloroform, methanol and petroleum ether. Among the five solvents used, leaf and callus extracted in chloroform of both the plants were found to be more effective against pathogenic bacteria and fungi, where the minimum inhibitory concentration (MIC) ranged between 0.25 to 6 mg/ml. Absolute alcohol extracts showed MIC of 0.25 to 4 mg/ml for bacteria, whereas for fungi it ranged from 0.25 to 100 mg/ml. Extracts of benzene and petroleum ether were ineffective in inhibiting the bacterial and fungal growth or showed poor inhibition. Methanol extract showed MIC of 0.25 to 100 mg/ml against bacterial pathogens and 0.5 to 100 mg/ml against fungal pathogens. The antimicrobial activities of these two indigenous medicinal plants were discussed in the present paper.

Stimulation of reserpine biosynthesis in the callus of Rauvolfia tetraphylla L. by precursor feeding

Reserpine is an important indole alkaloid that is used to treat hypertension and various psychiatric diseases by acting as a tranquilizing agent. In pharmaceutical industries, reserpine is in great demand. Chemical synthesis of reserpine is costlier than extracting it from natural resources. So enhancing this alkaloid in the already available system is a beneficial approach. Tryptophan is the starting material in the biosynthesis of reserpine. Callus was induced from leaf explants of Rauvolfia tetraphylla L. on MS medium supplemented with the combination of 9 μM 2,4-D and 25, 50, 75 and 100 mg/l tryptophan. An increase in the reserpine content was observed at 50 mg/l tryptophan than in other concentrations. Key words: Callus, MS medium, Rauvolfia tetraphylla L., reserpine, tryptophan.

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10μM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

A checklist of birds of Calicut University campus, Kerala

Malappuram District of Kerala. The area is a flat topped laterite hillock, having a campus of about 25 sq. km. The topography of the campus is comparatively plain supporting scrub jungles and cashew plantations in the slopes. The vegetation is dominated by the bushes of Calicopteris flouribunda, Lantana camara, Rauvolfia tetraphylla, Eupatorium odoratum, Clerodendrum infortunatum, etc, interspersed with tress like Mangifera indica, Macaranga peltata, Ficus benghalensis, Ficus religiosa, etc. The campus also has a very large Botanical Garden which attracts many birds.

Screening of 17 Guatemalan medicinal plants for platelet antiaggregant activity

The platelet antiaggregant activity of 17 aqueous extracts of plants traditionally used in Guatemala for the treatment of 'blood disorders' and parasitic infections were assayed. The aggregation of washed human platelets induced by thrombin (0.075 U/mL) was inhibited by extracts of Tridax procumbens, Tagetes lucida, Annona reticulata, Neurolaena lobata, Bixa orellana, Rauvolfia tetraphylla, Petiveria alliacea and Tecoma stans. The other extracts did not have clear inhibitory effects, and indeed caused some platelet aggregation themselves, in the absence of thrombin.