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This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L−1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.

Fresh, shade-dried and powdered samples of leaf, stem and root of Chitrak (Plumbago zeylanica L) were subjected to fractional distillation in a Soxhlet apparatus using four organic solvents. Chloroform, acetone and ethanol extracts from root of Plumbago showed higher zone of inhibition of 22.66±1.52, 21.5±1.29 and 16.5±1.29 mm and minimum inhibitory concentration (MIC) of 1.0, 10.0 and 10.0 μg/ml, respectively against Escherichia coli. Root and leaf extracts by ethanol, chloroform and acetone showed higher antibacterial activity against E. coli as compared to standard Kanamycin (MIC-100 μg/ml). The high performance thin layer chromatography (HPTLC) fingerprints were used for the quantitation of two bioactive markers: gibberellic acid and quinol R in the plant powder of different organs. Maximum content of gibberellic acid was found in acetone extract of the root (59.74%, Rf 0.79) followed by methanol root extract (53.01%). HPTLC provides a chromatographic fingerprint of phytochemicals and is suitable for confirming the identity and purity of medicinal plant raw materials. Key words: Plumbago zeylanica, antimicrobial property, bioactive compound, kanamycin, Escherichia coli, high performance thin layer chromatography (HPTLC), phytochemical fingerprint.

The plant species Plumbago zeylanica ( P. zeylanica ) is a multipurpose medicinal herb of family Plumbaginaceae. This plant is a natural gift to mankind credited with potential medicinal properties such as anti-cancer, anti-atherogenic, cardiotonic, hepatoprotective, anti-fungal, diabetes and neuroprotective assets to list of few. In this study, genetic diversity and relationships among various Plumbago accessions, collected from different geographical regions of India, was assessed using simple sequence repeat (SSR), sequence related amplified polymorphism (SRAP) and internal transcribed spacer (ITS) markers. SSR and SRAP markers showed highest values of Nei's genetic diversity and Shannon information index among populations. We also observed statistically significant genetic differentiations among and within populations ( P P. zeylanica populations into similar clusters which corroborate the above analysis to be useful for genetic diversity analysis of this plant. Our data signifies that SSR and SRAP are both reliable and effective tools for analyzing genetic diversity in P. zeylanica . However, based on our chosen dataset, we find ITS marker less significant for the genetic diversity analysis of this multifaceted plant. This information would be useful towards the identification, characterization and conservation of this species at the molecular level.

Agrobacterium rhizogenes mediated hairy root culture of Plumbago rosea L. is an attractive alternative for the production of plumbagin which is the major bioactive compound in P. rosea tuberous roots. The traditional industries form the major consumer of the tuberous roots as these are used in many ayurvedic preparations. The present work investigates the prospects of utilizing hairy roots in the place of tuberous roots based on bacterial survival test of hairy roots and comparison through phytochemical analyses (TLC, Spectrophotometry, HPLC and LC-MS). Since the traditional system of medicine follows stringent curing procedure before incorporation of the roots in medicinal preparations, cured tuberous and hairy roots were also compared. The phytochemical profile of hairy roots was remarkably similar to that of tuberous roots. Curing caused no change in the phytotchemical composition of the roots but only a reduction in the amount of plumbagin and other molecules. Plumbagin was reduced to 0.372 ± 0.026% dry weight (DW) in cured tuberous roots (1.15 ± 0.08% DW in uncured) and 0.061± 0.0043% DW in cured hairy roots (1.32 ± 0.09% DW in uncured). An 11.3 fold increase in root biomass with 1.56% DW plumbagin obtained in bioreactor as against 5.39 fold in shake-flasks (with 1% w/v inoculum over 3 weeks period), adds to the prospects of its applicability in traditional systems. The results suggest a refurbishment of conventional high quantity cured roots in traditional preparations with low quantity uncured roots, irrespective of root types.

The aim of the study was to evaluate the potential anticandidal activity of ethanolic extracts of some ethnobotanically important higher plants against Candida species. Plants crude extracts were prepared and their an- ticandidal activity was evaluated. Minimum inhibitory value was evaluated for the most promising plant extracts and their mode of action was studied using scanning electron microscopy method. The ethanolic extracts of 26 plants were evaluated for anticandidal activity against four human pathogenic yeast species—Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. The potential anticandidal activity was observed in ethanolic extract of Berberis aristata, Costus speciosus and Plumbago zeyla- nica. The SEM analysis showed that, unlike control cells representing normal oval shape with smooth surface, treatment of C. albicans with B. aristata, C. speciosus and P. zeylanica significantly affected the external morphology due to cell deformation, shrinking, cracks on cell surface. These three plants may serve as leads in the development of potential anticandidal agents.

The present investigation deals with chemical standardization and aflatoxin inhibitory and antioxidant activity of a herbomineral formulation, sahaj vati prepared in two batches (batch I and II) by mixing shilajeet (thick exudates of the mountain during summer season), guggul (Commiphora mukul (stocks) hook.), haridra (Curcuma longa L) and chitrak (Plumbago zealanica L) with levigation of Agnimantha kwatha (Premna mucronata L). Batch I was prepared by mixing shilajeet, guggul, haridra and chitrak in equal proportion (25% each by weight) while batch II comprised shilajeet (44.7%), guggul (44.7%), haridra (4.8%) and chitrak (5.8%) by weight. Sahaj vati II showed highest (96%) inhibition of aflatoxin production at concentration of 10 000 µg/ml. Suddha guggul exhibited strong antioxidant activity showing lowest IC50 at concentration of 20.56 µg/ml. Based on these findings, the newly formulated herbomineral formulation could be recommended for value addition of nutraceuticals by enhancing their shelf life against lipid per oxidation and aflatoxin contamination.

Summary The genus Plumbago has a concentration of indigenous species in eastern tropical Africa and Madagascar: nine out of a total of between twelve and twenty-five species. In the Flora of Ethiopia and Eritrea, Vol. 5, published in 2006, only two indigenous species were accounted for: the widespread and common P. zeylanica and a new species, P. truncata, restricted to south-western Ethiopia. The name P. truncata was not formally validated. Since then more collections and field observations of Plumbago have been made in Ethiopia: after revision of the entire material it is concluded that P. truncata is conspecific with P. dawei, known from Uganda, Kenya, Tanzania and Madagascar, and that another tropical East African species, P. montis-elgonis, known from Kenya and Tanzania, also occurs in south-western Ethiopia. In Ethiopia the two species are restricted to areas originally covered by moist forest: P. dawei to Transitional Rain Forest and Riverine Forest, while P. montis-elgonis to the lowermost zone of...

Background: Kuth (Saussurea lappa), Nisotha (Operculina turpethum), Akarakara (Anacyclus pyrethrin) and Chitrak (Plumbago zeylanicum) are some common plants used in Ayurvedic system of medicines and herbal drugs. Objective: The objective of the present study is to evaluate the quality of the samples of same plant marketed into different area, on the standardization parameters given in Ayurvedic pharmacopoeia. Methods: Three different marketed samples of these roots were subjected to pharmacognostic, physicochemical and phytochemical analysis and results were compared to the standards given in Ayurvedic pharmacopoeia. Results: Variations were found in physicochemical and phytochemical parameters of two samples in each case of Saussurea lappa, Operculina turpethum, Anacyclus pyrethrin and of one sample in case of Plumbago zeylanicum. Pharmacognostical parameters were found same as given in Ayurvedic pharmacopoeia for all the samples. Conclusions: The outcome of the study suggested that there is a lot of difference in the quality of the same drug marketed in different parts of country which may also cause variation in products prepared from them. These findings may be very useful for the identification of the species which may be useful to pharmaceutical industries for the quality control of the commercial samples.

The efficacy of aqueous leaf extracts of three Plumbago (Plumbago indica, P. capensis, P. zeylanica) species on anthracnose fungus Colletotrichum gloeosporioides were assessed. Out of the extract of three plants screened, that of Plumbago indica showed the best result both in inhibition of conidial germination up to (98.9%), and radial mycelial growth (98.75%) of the pathogen over the control and in the pectinolytic enzyme studies also P. indica showed a minimum activity of Polymethyl esterase (1.91U), Polymethyl galacturonase (endo PMG-2.77%; exo PMG-0.028U) and Pectin trans eliminate; the least amount of sugar (endo PTE-17.14%; exo PTE-11.11U) was released in P. indica. In cellulolytic enzyme studies, the amount of viscosity loss due to the activity of 1,4-β endoglucanase was remarkably reduced (3.57%) in P. indica compared to the other two species. P. capensis showed moderate results; P. zeylanica showed minimum inhibition of the enzyme activity of the pathogen. Results show that among all three plumba...

The genus Meloidogyne includes species of the most common nematodes to affect crops around the world. The species M. enterolobii is notable for affecting and causing serious losses in the production of guava trees and various other economically important crops in Brazil. The aim of this study was to evaluate the susceptibility of 10 plant species to parasitism by M. enterolobii, and the in vitro and in vivo effects of their leaf extracts on the pathogen. Initially seedlings of Solenostemon scutellarioides, Dieffenbachia amoena, Spigelia anthelmia, Plumbago scandens, Ricinus communis, Chenopodium ambrosioides, Azadirachta indica, Morinda citrifolia, Jatropha curcas and Datura stramonium were inoculated with 5,000 eggs of M. enterolobii to evaluate their susceptibility to nematode infection. For the test in vitro, a 5% concentration of the leaf extracts were added to Petri dishes, and 50 J2 of M. enterolobii were placed in each dish. After 48 hours incubation, the juveniles were evaluated for motility and mortality in the extracts. For the test in vivo, leaf extracts were used at the same concentration, however with only the seven most-promising in vitro species. This assay included the following sequence: inoculation of 5,000 eggs in autoclaved and moist soil contained in 1L pots; application of 30 ml of extract to the soil after 24 hours; transplanting of ‘Santa Clara’ tomato seedlings the following day; and reapplying the extract after 7 and 14 days. The results were evaluated 45 days after nematode inoculation. It was seen that the species D. amoena, R. communis, A. indica, M. citrifolia, J. curcas and D. stramonium displayed highly resistant behaviour; S. anthelmia, P. scandens and C. ambrosioides were very resistant, whereas S. scutelarioides was susceptible to the nematode. With the in vitro test, extracts from seven of the 10 species caused 70.4% to 97.4% J2 mortality. Applying the best leaf extracts to the soil was efficient in reducing M. enterolobii infestation in roots of the tomato.

Background:Leishmaniasis is an endemic tropical disease caused byLeishmaniaparasites, transmitted mainly by phlebotomine sandflies, impacting both health and socioeconomic wellbeing. Currently there are inadequate therapeutic measures to manage the disease thus indicating the need for the development of affordable and effective therapeutic interventions from herbal plants as alternative medicine. This study investigated theinvitroantileishmanial effects of blends of crude extracts ofSolanum nigrumandPlumbago capensisagainstLeishmania major.Methods:The promastigote parasites ofLeishmania majorwere cultured and grown for 3 days in different concentrations of the individual extracts to determine minimum inhibitory concentrations (MIC). Thein vitroantileishmanial efficacy was determined by exposing promastigotes and macrophages infected withL. majorto the blends of extracts in ratios of 2000:250, 1000:500, 500:1000 and 250:2000. Finally, nitric oxide released byL. majorinfected macrophages that were treated with the plant extracts at ratio of 125:125 was quantified using a standard nitrite curve.Results:The individual methanol extracts were most effective in inhibiting the growth of promastigotes with MIC values between 0.25 mg/ml and 1.0 mg/ml as compared to aqueous extracts. The most active ratios for the blends were 250:2000 and 2000:250 for methanolic and aqueous blends respectively. The infection rates and multiplication indices associated with all the combined extracts were significantly different (P< 0.05) from those of pentostam and amphotericin B at all the concentrations studied. The OD for the combined test extracts ranged between 0.034 and 0.041 and these corresponded to < 5 µM of NO.Conclusion:Findings from this study demonstrate that combination therapy usingS. nigrumandP. capensisextracts is effective in treatingLeishmania major infection. Based on our findings we recommendin vivostudies to be conducted to determine the efficacy of these combined therapies againstLeishmaniamajor.

A bioassay-guided fractionation and chemical examination of chloroform extract of Plumbago capensis roots resulted in isolation and characterization of two new napthaquinone derivatives (4, 8) along with six known compounds (1-3, 5-7). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested for their mosquito larvicidal activity against fourth instar larvae of Aedes aegypti, and compared with that of rotenone. Among the tested compounds, isoshinanolone (3) and plumbagin (1) showed excellent toxicity with LC(50) values of 1.26 and 5.43 microg/mL. New compound (8) displayed moderate toxicity against the tested mosquito species.

Males of the predaceous stink bug Podisus maculiventris (Say) (Heteroptera: Pentatomidae: Asopinae) emit low frequency tremulatory signals. Laser vibrometry was used to record and analyze naturally emitted signals, focusing on variation in signal velocity and frequency during transmission through plants (Phaseolus vulgaris L. and Plumbago auriculata Lam.) as a function of distance from the vibrational source. Signal velocity varied individually between 2 and 15 mm/s recorded on a plant close to the calling male and decreased by 0.3 to 1.5 dB/cm on bean and 0.3 to 0.9 dB/cm on plumbago. The dominant frequency of signals was variable at frequencies below 50 Hz. On bean frequencies centered around 10 Hz or 20 Hz were dominant for signals recorded at the source. Transmission through bean resulted in an increase in the 20 Hz peak relative to other frequencies in the signal. Variation of the dominant frequencies of signals transmitted through plumbago stems were more predictable, showing typical changes in amplitude relative to the distance from the source. The regular variation of the dominant frequency along the stem with linear increase of signal velocity at decreasing distance from the source may provide plant-dwelling insects with information about the distance to the calling individual.

In the present study, Plumbago rosea Linn. (Plumbaginaceae), one of the folk medicinal plants commonly used as an antifertility agent, was evaluated for its antifertility effect. Five successive so...

In this contribution, new data concerning the Italian distribution of alien vascular flora are presented. It includes new records, exclusions and confirmations for Italy or for Italian administrative regions for taxa in the genera Ageratum, Aster, Buddleja, Cedrus, Centranthus, Cephalotaxus, Clerodendrum, Cotoneaster, Cyperus, Honorius, Lantana, Ligustrum, Morus, Muscari, Oenothera, Opuntia, Platycladus, Plumbago, Pseudotsuga, Sedum, Sporobolus, Stachys, Ulmus and Yucca. A nomen novum, Stachys talbotii, is proposed as a replacement name for Sideritis purpurea.

Traditionally Plumbago rosea L. is used as an abortifacient in the Southeast Asian region. Methanolic root extract of a local species of Plumbago rosea L. was studied to evaluate its traditional antifertility claim. Interestingly, it was found to possess dose related inhibitory effect on uterine contractile responses elicited by oxytocic agents on isolated uteri of pregnant and pseudo-pregnant rats. Furthermore, it was found to possess significant (p < 0.05) fetotoxic activity along with mild abortive potential in pregnant mice when given orally at high doses (400 and 800 mg kg(-1)) once daily for ten days starting from day 10 of gestation. The results derived indicated possible presence of utero-active compound (s) in this plant that inhibited oxytocic agents induced uterine motility. Moreover, pronounced fetotoxic and mild abortifacient potentials observed at higher doses in pregnant mice might support its accredited traditional use to avoid unwanted pregnancy.

The present study was undertaken to determine the antibacterial potential of Plumbago species viz., Plumbago zeylanica Linn., Plumbago auriculata Lam. and Plumbago rosea Linn. collected from various localities of South India. For the bio-efficacy studies six different extracts of Plumbago species with various concentrations viz., 20, 40, 60, 80 and 100µg/ml against Gram positive and Gram negative bacterial pathogens viz., Staphylococcus aureus (MTCC 737), Streptococcus pyogenes (MTCC 1928), Bacillus subtilis (MTCC 441), Klebsiella pneumoniae (MTCC 109), Morganella morganii (MTCC 662) and Pseudomonas aeruginosa (MTCC 1688) using well diffusion method. Among the eighteen different extracts of three different Plumbago species, highest frequency of antibacterial activity (54%) was recorded in P. rosea. The antibacterial activity of various extracts in different concentration of the selected Plumbago species are as follows: P. rosea (54%) > P. zeylanica (49%) > P. auriculata (40%). The ethanolic extract of all the Plumbago species revealed superior bactericidal activity compared to other tested extracts. The ethanolic extracts of P. zeylanica, P. auriculata and P. rosea showed 91%, 50% and 99% of activity against Gram positive pathogens and 66%, 26% and 89% of activity against Gram negative pathogens. The bioactive compound plumbagin and extract of aerial parts of Plumbago species show a wide spectrum of antibacterial activity. The compound shows promise as a new drug for various bacterial infectious diseases. Hence, this study offers a base of using Plumbago species as herbal alternative for the synthesis of antimicrobial agents.

Plumbagin, (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural substance found in the roots of plant species in the genus Plumbago, has been used as a traditional medicine against many diseases. In this study, Caenorhabditis elegans was used as a model for testing the anthelmintic effect of plumbagin. The compound exhibited a nematicidal effect against all stages of C. elegans: L4 was least susceptible, while L1 was most susceptible to plumbagin with an LC50 of 220 and 156 μM, respectively. Plumbagin inhibited C. elegans development from L1 to adult stages with an IC50 of 235 μM, and body length was also reduced at concentrations of 25 and 50 μg/ml. Brood sizes decreased from 203±6 to 43±6 and 18±3 eggs per hatch in plumbagin-treated worms at 10, 25, 50 μg/ml, respectively. Furthermore, plumbagin was lethal to strains resistant to the nematicides levamisole, albendazole, and ivermectin, indicating that it possesses a strong and unique nematicidal action. Plumbagin decreased the number of mitochondria in hypodermal and intestinal cells and body wall muscles and damaged the ultrastructure of these tissues. Taken together, plumbagin may be a new drug against parasitic nematodes.

Knowledge of the natural genetic variation and structure in a species is important for developing appropriate conservation strategies. We aimed to examine (i) the patterns and levels of morphological and genetic variability within/among populations of Plumbago zeylanica and ascertain whether these variations are dependent on geographical conditions; and (ii) evaluated genetic differentiation and population structure within the species. ISSR and RAPD markers were used to study the genetic structure and variation within and among populations collected from 13 widespread regions in India. High levels of genetic diversity and significantly high genetic differentiation were revealed by both the markers among all studied populations., Knowledge of the natural genetic variation and structure in a species is important for developing appropriate conservation strategies. As genetic diversity analysis among and within populations of Plumbago zeylanica remains unknown, we aimed (i) to examine the patterns and levels of morphological and genetic variability within/among populations and ascertain whether these variations are dependent on geographical conditions; and (ii) to evaluate genetic differentiation and population structure within the species. A total of 130 individuals from 13 populations of P. zeylanica were collected, covering the entire distribution area of species across India. The genetic structure and variation within and among populations were evaluated using inter-simple sequence repeat (ISSR) and randomly amplified DNA polymorphism (RAPD) markers. High levels of genetic diversity and significantly high genetic differentiation were revealed by both the markers among all studied populations. High values of among-population genetic diversity were found, which accounted for 60 % of the total genetic variance. The estimators of genetic diversity were higher in northern and eastern populations than in southern and western populations indicating the possible loss of genetic diversity during the spread of this species to Southern India. Bayesian analysis, unweighted pair group method with arithmetic average cluster analysis and principal coordinates analysis all showed similar results. A significant isolation-by-distance pattern was revealed in P. zeylanica by ISSR (r = 0.413, P = 0.05) and RAPD (r = 0.279, P = 0.05) analysis. The results obtained suggest an urgent need for conservation of existing natural populations along with extensive domestication of this species for commercial purpose.

The effect of drought on the growth and gas exchange of six bedding plant species—agastache [Agastache urticifolia (Benth.) O. Kuntze `Honeybee Blue'], dusty miller (Cineraria maritima L. `Silverdusty'), petunia (Petunia ×hybrida `Wave Purple'), plumbago (Plumbago auriculata Lam. `Escapade'), ornamental pepper (Capsicum annuum L. `Black Pearl'), and vinca [Catharanthus roseus (L.) G. Don `Titan']—was quantified under greenhouse conditions. Seeds were sown in January and seedlings were grown in the greenhouse until 18 Apr., when two irrigation treatments—drought (D, ≈18% volumetric moisture content at reirrigation) and control (C, ≈25% volumetric moisture content at reirrigation)—were initiated. Leaf net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration (E) were determined in response to a range of substrate moisture content (from ≈5% to 30% by volume) and temperature (from 20 °C to 40 °C). Dry weight of agastache, ornamental pepper, and vinca was unaffected by drought, whereas that of other species was reduced. Leaf area of plumbago and height of plumbago and vinca were reduced by drought. As substrate moisture content decreased from 25% to 10%, Pn, E, and gs decreased linearly in all species except petunia and plumbago. Leaf net photosynthetic rate of all species declined as leaf temperature increased from 20 °C to 40 °C. In contrast, E of all species, except petunia, increased as temperature increased. Transpiration rate of petunia increased as temperature increased from 20 °C to 30 °C, and then decreased between 30 °C and 40 °C. Although petunia had the highest Pn among the tested species, its Pn and gs declined more rapidly compared with the other species as temperature increased from 20 °C to 40 °C or as substrate moisture content decreased, indicating that petunia was most sensitive to high temperature and drought.

The solubility of 1,4-naphthoquinone, plumbagin, lawsone, and juglone in supercritical carbon dioxide was determined spectroscopically at 40 degrees C, and in the pressure range 8-18 MPa. Their solubilities at 12 MPa were between 0.3 and 10 g L(-1). Plumbagin from Plumbago scandens L. roots was extracted at 40 degrees C and 20 MPa. The extracted plumbagin mass fraction was up to 0.2% in fresh roots but down to about 0.006% in aged roots. n-Hexane and chloroform extraction of such aged roots indicates that the older and dryer the roots are, the stronger they bind plumbagin. Reversed-phase HPLC indicated a relatively pure plumbagin extract with supercritical carbon dioxide.

A combination of spices (Piper nigrum, Piper longum and Zingiber officinale), herbs (Cyperus rotundus and Plumbago zeylanica) and salts make up Amrita Bindu. The study was focused to evaluate the antioxidant property of individual ingredients in Amrita Bindu against the free radical 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The analysis revealed the antioxidant potential of the ingredients in the following order: Piper nigrum > Piper longum > Cyperus rotundus > Plumbago zeylanca > Zingiber officinale. Two different experiments were designed. In experiment I, rats were fed with normal diet whereas in experiment II rats were given feed mixed with Amrita Bindu for 3 weeks (4 g/kg of feed). Rats from both experimental groups were challenged against a single intraperitonial injection of phenylhydrazine (PHZ) (7.5 mg/kg body weight). At the end of 24 and 72 h, blood was analysed for free radicals and antioxidant levels. It was interesting to note that rats with Amrita Bindu pretreatment showed significantly lower levels of free radicals, lipid peroxidation and protein carbonyls along with significantly higher levels of antioxidants when compared with rats without Amrita Bindu pretreatment on PHZ administration. These results reveal that Amrita Bindu, a salt-spice-herbal mixture exerts a promising antioxidant potential against free radical induced oxidative damage.

Centrifugal liquid-liquid partition chromatography presents significant advantages for the separation and purification of plant metabolites owing to the short operational time of the process and the elimination of possible irreversible adsorption of compounds. The crude chloroform extract from roots of Plumbago auriculata was analysed by countercurrent chromatography using hexane:ethyl acetate:methanol:water (40:10:10:2, v/v) as solvent system. The isolation of the naphthoquinones plumbagin and epi-isoshinanolone, the steroids sitosterol and 3-O-glucosylsitosterol, plumbagic and palmitic acids was easily achieved. Naphthoquinones are typical components of Plumbago species and they show interesting biological activities.

Tremorine-induced tremors model is used to evaluate antiparkinsonian drugs because rest tremor is a sign that distinguishes Parkinson's disease (PD) from other diseases. The effects of crude ethanolic extract (CEE) and total acetate fraction (TAF) of Plumbago scandens were investigated at several doses. These extracts at doses of 125 and 250 mg/kg i.p. failed to reduce tremors in tremorine-treated mice. TAF showed significant effects only at a dose of 500 mg/kg. Both CEE and TAF at doses of 1000 and 2000 mg/kg i.p. suppressed the tremors in a dose-dependent fashion for 60 min. Biperiden, an anticholinergic drug, was used as standard at a dose of 3 mg/kg i.p. This study suggests that P. scandens is a plant with possible therapeutic value for PD.

The Plumbago genus belongs to the Plumbaginaceae family and it is known due to its variety of biological uses, most of them attributed to the presence of naphthoquinones. Plumbagin is a naturally occurring naphthoquinone that can be obtained from roots of Plumbago scandens L. In order to find out the better technique for plumbagin extraction, were applied: static maceration, dynamic maceration, with assistance of ultrasonic waves and in Soxhlet apparatus. Four compounds were qualitatively detected in all extracts: the naphthoquinones plumbagin and epi-isoshinanolone, palmitic acid and sitosterol. Plumbagin was always the major component in all analyzed extracts and it was quantitatively determined by gas chromatograph coupled with mass spectrometer. Soxhlet was the most efficient extraction technique however, prolonged heating time promoted plumbagin degradation.O gênero Plumbago pertence à família Plumbaginaceae e apresenta uma variedade de atividades biológicas, a maioria atribuída à presença de naftoquinonas. Plumbagina é uma naftoquinona natural que pode ser obtida a partir de raízes de Plumbago scandens L. Na tentativa de descobrir a melhor técnica de extração dessa substância foram usadas, maceração estática, maceração dinâmica, extração com auxílio de ultrassom e extração em Soxhlet. Quatro substâncias foram detectadas qualitativamente: as naftoquinonas plumbagina e epi-isoshinanolona, o ácido palmítico e o sitosterol. Plumbagina foi sempre o componente majoritário em todos os extratos analisados e sua determinação quantitativa foi realizada através de cromatografia com fase gasosa acoplada à espectrometria de massas. A extração em Soxhlet foi a técnica mais eficiente para obtenção de plumbagina, entretanto a longa exposição a temperaturas elevadas favoreceu a degradação da plumbagina.

Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog's medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM), but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.

Mass multiplication of Plumbago rosea was achieved by indirect organogenesis in young stem, leaf and root explant cultures of 6-9-month-old plants. All the explants responded similarly in a hormonal regime of 2.5 mg/L BA and 1.5 mg/L NAA with the formation of nodular callus in 4 weeks; the callus was divided and subcultured at 4-week intervals in the presence of 3.0 mg/L BA to produce up to 23.5 +/- 1.6 shoots in 18 weeks and then at 2.0 mg/L BA to produce up to 79.6 +/- 1.5 shoots in 23 weeks. The shoots of 2.0-3.5 cm length were rooted easily in half-strength MS agar medium supplemented with 0.1 mg/L IBA and rooted plants established within 4 weeks at a 95-98 % rate without hardening. Eight weeks after establishment, the micropropagated plants were transferred to experimental plots and cultivated for 10 months to obtain a significantly higher number (18.0 +/- 0.5) of larger tuberous roots (137.4 +/- 3.4 g fw/plant) compared to conventional rooted cuttings (14.0 +/- 1.7, 47.9 +/- 1.6 g fw/plant). During this period, the concentration of the root-specific compound, plumbagin recorded per g dw (1.5 %), was higher than that of conventionally propagated plants (0.9-1.0 %). The early formation of plumbagin-rich tuberous roots holds significant potential for the commercial cultivation of the micropropagated plants.

We examined the distribution of DNA-containing organelles (plastids or mitochondria) in pollen from 115 angiosperm species (104 genera, 56 families) to determine whether there was polarized distribution of sperm cells (SC) and generative cells (GC). Using an improved method of fixation and squashing, we found polarization in the GC or SC pairs in mature pollen (obtained from the flower at anthesis) from five species (Abelia spathulata, Campsis grandiflora, Erythrina variegata, Limonium sinuatum, and Tecoma capensis). Including Plumbago, polarization is now known to occur in six genera representing four unrelated families. The results show that the production of pairs of dimorphic SC occurs in species other than those in the genus Plumbago, in which this phenomenon has already been well characterized. These results suggest that differentiation of SC and preferential double fertilization are not restricted to Plumbago and may occur in a variety of angiosperms. In species with significant polarization in GC or SC pairs, polarization of GC occurs at an especially early stage when GC are freed and become spherical, or just after microspore mitosis. This suggests that the distribution of DNA-containing organelles is strictly controlled during the early development of the GC.

Cell cultures of Plumbago roseaL. (synonymous P. indica) were treated with the elicitors prepared from the fungi ( Aspergillus nigerand Rhizopus oryzae), bacteria (Bacillus subtilis and Pseudomonas aeruginosa ), yeast extract and chitosan to induce and enhance the synthesis of plumbagin. Elicitation of plumbagin production in chitosan treated cells was 6.71-fold higher compared to control cells. The treatment of cells with A. niger, R. oryzae and yeast elicitors resulted in two- to three-fold more plumbagin over control cells. Bacterial elicitors did not show much (

In vitro plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige & Skoog (1962) medium supplemented with 1.5 mg litre -1 6-benzylaminopurine, 0.25 mg litre -1 indole-3-acetic acid, 50 mg litre -1 adenine sulfate and 3% (w/v) sucrose. The shoot initials developed within 2-3 wk on the leaf margin as well as from the wounds of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 wk of culture. Shoots rooted easily on medium having half-strength basal Murashige & Skoog (1962) medium supplemented with 0.25 mg litre -1 indole-3-butyric acid and 2% (w/v) sucrose; 84-92% of the in vitro rooted plantlets survived in the greenhouse. The regenerated plantlets appeared morphologically similar to the mother plants. No variation was detected among the regenerated plants by the use of Randomly Amplified Polymorphic DNA (RAPD) markers. This method might be useful for assessing plant improvement programmes.

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm−3 kinetin and 2.0 mg dm−3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm−3 kinetic (Kn), 0.5 mg dm−3 gibberellic acid (GA3) and 0.1 mg dm−3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm−3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.

The genus Plumbago belongs to the family Plumbaginaceae, order Plumbaginales. Comparative chemical profile of P. scandens (native) and P. auriculata (cultivated) was obtained by normal and reversed-phase high performance liquid chromatography with photodiode array detector. Comparison of the ultraviolet espectra and the retention times for the compounds allowed to find similar metabolic patterns in roots, stems and leaves. Four flavonoids, one phenolic acid or derivative and the naphtoquinone plumbagin were comparatively identified to standards.

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 μM 6-benzylaminopurine (BA), 1.4 μM indole-3-acetic acid (IAA), 370 μM adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 μM indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.

Three locally available Plumbago species (Plumbago auriculata, Plumbago rosea and Plumbago zeylanica) have been quantitatively screened for the bioactive marker plumbagin by high performance thin layer chromatography. In general, the root parts were found to be the rich sources for plumbagin. Most significantly, P. rosea was found to accumulate maximum plumbagin in the roots. In contrast, P. auriculata and P. zeylanica are high yielding species for the leaf and stem parts, respectively. Studies have also been extended to evaluate some P. rosea samples collected from southern India and a traditional medicine, Chitrakadi vati, which contains P. zeylanica as the major constituents.

Background: Plumbago rosea is used in traditional systems of medicine for the preparation of formulations used for treating inflammations, cough, bronchitis, and gastrointestinal disorders, and also in conjunction with cancer chemotherapy. In the present study, the cytotoxic and anti-proliferative effects of plumbagin, and the ethanolic root extract of P. rosea (ETPR) was evaluated on SK-MEL 28 melanoma cell lines and human lymphocytes. Materials and Methods: MTT and apoptotic assays were used for the evaluation of cytotoxic and anti-proliferative effects, respectively. In addition, the effect of Plumbagin and ETPR in down regulation of BCL-2 expression is investigated using RT-PCR analysis. Results: Both plumbagin and ETPR dose-dependently decreased the cell viability more potently in melanoma cell lines. P. rosea extract demonstrated significant synergy in inhibiting BCL-2 expression than plumbagin. Moreover plumbagin showed more toxicity in human lymphocytes. Conclusion: Plumbagin has anti-cancer potential, but the side effects limits its use; yet plumbagin, in combination with other ingredients in Plumbago rosea extract, displays significant synergy leading to a stronger anticancer effect with significantly less toxicity.

The chemosterilant activity of two naturally occurring napthaquinones, plumbagin from Plumbago sp. and juglone from Juglans regia has been evaluated using the red cotton bug Dysdercus koenigii. Their activity were compared with synthetic napthaquinones, menadione, two benzoquinones, 2,6-dimethyl and 2,3,6-trimethylbenzoquinone and a hydroquinone 2,6-dimethylhydroquinone. As far as the authors are aware, the present investigation is the first systematic attempt to investigate the effects of quinones on different aspects of reproduction, namely mating behaviour, fecundity and fertility. All the above types of quinones have revealed adverse effects on the reproduction of D. koenigii, which were topically treated as 1-day-old adults with different doses of the compounds depending on their 50% lethality (LD 50 ) values. Analysis of the data revealed a highly statistically significant reduction in the number of eggs, their hatching and further development up to the final instar stage. The sterility index increased with the increase in the dose of any of the above compounds and was highest when both sexes were treated. Among the natural products, juglone induced more sterilizing effects than plumbagin at 5-10 μg/insect. Menadione caused sterility at a very low dose such as 0.5 μg. Of the two benzoquinones, the dimethylbenzoquinone acted at a much lower dose.

We characterized the behavior of plastid (pt) and mitochondrial (mt) nucleoids during male gametogenesis in Plumbago auriculata in three dimensions. The behavior of pt-nucleoids and mt-nucleoids differed throughout male gametogenesis. Pt-nucleoids were distributed in a characteristic manner in three stages: in the early microspore, pt-nucleoids assemble around cell nucleus; in the mid-generative cell, pt-nucleoids gather at the internal side of the pollen; in the late-generative cell, pt-nucleoids aggregation turns its pole to the external side of the pollen. We also studied organelle nucleoids in the egg and the central cell by a method in which semi-thick sections of resin-embedded anthers and ovaries were observed by confocal laser scanning microscopy. The number of pt-nucleoids in the sperm cell did not differ significantly from that in the egg. These results suggest that the behavior of DNA-containing organelles is regulated strictly during male gametogenesis in P. auriculata, and that a biparental inheritance of plastids in the Plumbago embryo is more favored than was previously thought.

Plumbagin, a naphthoquinone occurring in the plants belonging to Plumbago sp. is known to have insect growth disrupting activities. In the present study, a comparative evaluation of bioactivities of related naphthoquinones, juglone and menadione and benzoquinones, 2,6-dimethylbenzoquinone and 2,3,6-trimethylbenzoquinone along with 2,6-dimethylhydroquinone have been undertaken using Dysdercus koenigii. The LD50 values of the compounds showed wide variation. Although all the test compounds disrupted the normal growth at sublethal doses, 2,6-dimethylbenzo- quinone and menadione showed activity at low doses.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by 1H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g−1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 μm in diam, produced 8.2 g dry cell wt l−1, but larger aggregates (above 500 μm) favored plumbagin accumulation with an yield of 4.5 mg g−1 dry cell wt.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell.

The aqueous extracts (15 micrograms ml-1 medium) of 22 plants used in folkloric medicine in Palestine were investigated for their antifungal activity and minimum inhibitory concentrations (MICs) against nine isolates of Microsporum canis, Trichophyton mentagrophytes and Trichophyton violaceum. The extract of the different plant species reduced colony growth of the three dermatophytes by 36 to 100% compared with the control treatment. Antimycotic activity of the extract against the three dermatophytes varied significantly (P < 0.05) between test plants. Extracts of Capparis spinosa and Juglans regia completely prevented growth of M. canis and T. violaceum. The most active extracts (90-100% inhibition) were those of Anagallis arvensis, C. spinosa, J. regia, Pistacia lentiscus and Ruta chalapensis against M. canis; Inula viscosa, J. regia and P. lentiscus against T. mentagrophytes; and Asphodelus luteus, A. arvensis, C. spinosa, Clematis cirrhosa, I. viscosa, J. regia, P. lentiscus, Plumbago europea, Ruscus aculeatus, Retema raetam and Salvia fruticosa against T. violaceum. The MICs of these most active plants ranged from 0.6 to 40 micrograms ml-1. The three dermatophytes differed significantly with regard to their susceptibility to plant extracts. Trichophyton violaceum was the most susceptible being completely inhibited by 50% of the extracts followed by M. canis and T. mentagrophytes which were completely inhibited by only 23 and 14% of the extracts, respectively.

Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm.

Withaferin A (WA), a steroidal lactone, and Plumbagin (Pl), a naphthoquinone, from the roots of Withania somnifera and Plumbago rosea, respectively, have been shown to possess growth inhibitory and radiosensitizing effects on experimental mouse tumours. An aqueous extract of the leaves of Ocimum sanctum (OE) was found to protect mice against radiation lethality. Therefore, the radiomodifying effects of the above plant products on the bone marrow of the adult Swiss mouse was studied. Single doses of WA (30 mg kg-1) or Pl (5 mg kg-1) were injected intraperitoneally (ip) and OE (10 mg kg-1) was injected ip once daily for five consecutive days. Administration of extracts was followed by 2 Gy whole body gamma irradiation. Bone marrow stem cell survival was studied by an exogenous spleen colony unit (CFU-S) assay. The effects of WA and Pl were compared with that of cyclophosphamide (CP) and radioprotection by OE was compared with that of WR-2721 (WR). Radiation reduced the CFU-S to less than 50% of normal. WA, CP and Pl significantly enhanced this effect and reduced the CFU-S to almost the same extent (to < 20% of normal), although individually WA and Pl were less cytotoxic than CP. These results indicate that radiosensitization by WA and Pl is not tumour specific. OE significantly increased CFU-S compared with radiotherapy (RT) alone. OE+RT gave a higher stem cell survival (p < 0.05) than that produced by WR+RT. While WR alone had a toxic effect, OE treatment showed no such effect, suggesting that the latter may have an advantage over WR in clinical application.