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Ethnopharmacological relevance Tylophora indica (Burm. f.) Merr. commonly known as ananthamool is a climbing perennial plant which is widely used in Indian traditional medicine. T. indica exhibits diverse range of pharmacological activities viz. antiasthmatic, antidiarrheal, anticancer, antiarthritic, antiepileptic, anti-inflammatory etc. Aim of the study Present review aims to grant an up-to-date insight into the botany, ethnopharmacology, phytochemistry, pharmacology and toxicology of T. indica, exploring its future research and opportunities. Material and methods Comprehensive information regarding T. indica was collected using the keywords Tylophora indica or Indian ipecac or ananthamool in various electronic databases ACS, Google Scholar, Pubmed, Science Direct, SciFinder, Web of Science, Springer Link and Wiley. In addition, some books and book chapters were also consulted. Results T. indica has been traditionally used in India, Bangladesh and Sri Lanka in the form of various preparations like powder, decoction, pulp, paste and extract alone or in combination with other herbs against various ailments like skin disorders, inflammation, cough, asthma, diarrhea, cancer, microbial infections etc. In vitro and in vivo pharmacological studies on T. indica revealed its potential as antiasthmatic, antiallergic, anti-inflammatory, anticancer, antimicrobial, antioxidant, antidiarrheal agent etc. A diverse range of phytochemical constituents have been isolated and identified from T. indica namely alkaloids (Tylophorine, Tylophorinine, Tylophorinidine), flavonoids (Kaempferol & Quercetin) terpenoids (α-Amyrin & β-Amyrin) and sterols (β-sitosetrol). Amongst which phenanthroindolizidine alkaloids isolated from roots and leaves are largely explored and considered to be the most active constituent of plant. Conclusion Present review provides an insight into botany, ethnopharmacology, phytochemistry, pharmacology and toxicology of T. indica. As an important traditional Indian medicine, few ethnobotanicals use of T. indica have been supported by modern pharmacological studies, especially in asthma, cancer and inflammation. Among compounds from various phytochemical classes, phenanthoindolizidine alkaloids namely tylophorine and tylophorinidine alkaloids have been considered as bioactive components of the plant and widely investigated. However, further identification, isolation and quantification employing some advanced hyphenated techniques viz. LC-MS/MS, LC-NMR to discover new pharmacologically active phytoconstituents in the management of different diseases. Several investigators have highlighted possible therapeutic roles of T. indica extracts and isolated compounds. Moreover, information about various aspects of T. indica pertaining to phytochemistry, toxicology and quality control are still unresolved. Further in-depth studies are required to discover key features viz. structure activity relationships, mode of action, safety and toxicity and therapeutic potentials T. indica in clinical settings.

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Cancer chemotherapies or antitumor agents mainly remain the backbone of current treatment based on killing the rapidly dividing cancer cell such as tylophora alkaloids and their analogues which have also demonstrated anticancer potential through diverse biological pathways including regulation of the immune system. The introduction of durable clinically effective monoclonal antibodies, however, unmasked a new era of cancer immunotherapies. Therefore, the understanding of cancer pathogenesis will provide new possible treatment options, including cancer immunotherapy and targeted agents. Combining cytotoxic agents and immunotherapies may offer several unique advantages that are complementary to and potentially synergistic with biologic modalities. Herein, we highlight the dynamic mechanism of action of immune modulation in cancer and the immunological aspects of the orally active antitumor agents tylophora alkaloids and their analogues. We also suggest that future cancer treatments will rely on the development of combining tumor-targeted agents and biologic immunotherapies.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T lymphocytes caused by human T-cell lymphotropic virus type 1 (HTLV-1). There are an estimated 5 million to 20 million HTLV-1-infected individuals worldwide; their lifetime risk of developing ATL is 3-5 %, and high HTLV-1 proviral loads have been shown to be an independent risk factor. Although conventional chemotherapeutic regimens used against other malignant lymphomas have been administered to ATL patients, the prognosis is often poor. In previous studies, we screened 459 extracts from 344 plants to isolate components exhibiting antiproliferative activity against HTLV-1-infected T-cell lines (MT-1 and MT-2). In our continuing search for potential anti-HTLV-1 natural products, 15 extracts of Asclepiadaceae plants were further tested against MT-1 and MT-2 cells. The MeOH extract of aerial parts of Tylophora tanakae showed antiproliferative activity. Activity-guided fractionation resulted in the isolation of 6 phenanthroindolizidine alkaloids (including a new compound), and we examined their antiproliferative activity against MT-1 and MT-2 cells. The EC50 value of some of the alkaloids was in the low nanomolar range, comparable to that of the clinically used antineoplastic drug doxorubicin. Structure-activity relationship analyses suggested that a 14β-hydroxy moiety is essential for activity against HTLV-1-infected T cells. In contrast, the presence of a 2-methoxy moiety, a 7-methoxy moiety, or an N-oxide moiety appears to reduce the potency of the antiproliferative activity against HTLV-1-infected T cells.

A series of tylophora alkaloids and their derivatives (1-38) were synthesized and systematically bioassayed for their anti-HIV activity for the first time. The results revealed that most of these alkaloids possess good HIV inhibitory effect. Especially, compounds 1, 4 and 25 displayed low nanomolar levels of anti-HIV activ- ity (inhibitory effect at 50 nM: 1: 71%; 4: 60%; 25: 64%), which is about similar to that of the first HIV inhibitor AZT (inhibitory effect at 50 nM: 70%) and slightly lower than that of the first HIV integrase inhibitor MK-0518 (inhibitory ef- fect at 50 nM: 82%). Present studies provide fundamental support for the development of tylophora alkaloids as novel HIV inhibitors.

Diversities in the pollinia morphology of the family Asclepiadaceae in respect to pollinium shape, size, color, length and breadth of pollinial sac, length and breadth of corpusculum and length and breadth of caudicle were studied in 13 species belonging to eight genera (Cynanchum L., Calotropis R.Br., Glossonema Decaisne., Leptadenia R.Br., Pergularia L., Sarcostemma R.Br., Tylophora R.Br and Vincetoxicum N. M. von Wolf.) Calotropis procera (Ait.) Ait. f. yielded largest pollinia sac. (906.4 4.4.7 ?m) whilst the smaller one (109.8 × 48.0 ?m) was represented by Cynanchum pollens Hemsley and Lace. In respect of the length and breadth of the corspuseulem, C. Procera yielded the lowest one bt Leptodaenia pyrotechnium yielded the smallest (65.2 × 41.2 ?m). DOI: http://dx.doi.org/10.3329/bjb.v43i3.21590 Bangladesh J. Bot. 43(3): 249-253, 2014 (December)

The alkaloids of Tylophora have been shown to have an anti-asthmatic and anti-inflammatory response. The leaves of the plant are used in Ayurvedic medicine for the treatment of asthma, bronchitis, and arthritis. In the cur-rent study, the phyto-constituents and major secondary metabolites found in Tylophora indica leaves and stem were isolated and extracted using differ-ent solvents and analysed by FT-IR spectroscopy to have a semi qualitative analysis by comparing the structure of tylophorine (major secondary metab-olite found in Tylophora leaves having anti-asthamatic activity) with the ex-tracted product. The chloroformic leaf extract (200μl) of T. indica was inves-tigated for altered immunologic response in mice. In response to plant ex-tract, lymphocyte count increased in mice representing the immuno-stimulatory effect of plant extract.

Aims: The present studies were initiated to develop a cost effective protocol for micropropagation,as a mean for conservation of medicinal plant -Tylophora indica(Burm f.)Merill. The plant is threatened andneeds immediate conservation, therefore, the study was undertaken with following objective: •In vitromultiplication of Tylophora indicausing nodal axillary bud proliferation and through organogenesis of callus. Study Design:For all experiments ten repli cates were used per treatment and all the experiments were repeated three times. Data have been presented as Mean ± Standard deviation.

Eleven new alkaloids (1–11), together with eleven known phenanthroindolizidine alkaloids (12–22) were isolated from the whole plants of Tylophora atrofolliculata (Asclepiadaceae). Their structures were characterized by means of NMR spectroscopies including HSQC, HMBC, 1H–1H COSY and NOESY experiments, assisted by high-resolution MS and CD spectral analyses. HIF-1-mediated reporter gene assay in T47D cells was used to evaluate the inhibitory effects of isolated alkaloids on HIF-1 activation. Most alkaloids exhibited extremely potent inhibitory effects with IC50 values in the low nanomolar range. The potency of 14 and 17 are comparable to manassantin B, one of the most potent HIF-1 inhibitors identified so far. Structure–activity relationship (SAR) analyses showed the necessities for high activity, i.e. non-planarity at indolizidine moiety, substitution types and patterns on the phenanthrene and indolizidine moieties. This is the first report on HIF-1 inhibitory activity of phenanthroindolizidine alkaloids, providing a new insight into their underlying anti-cancer mechanism.

The alkaloid (-)-tylophorine was isolated from a sample of Tylophora indica, and the crude extract was analyzed by HPLC/MS(n) and chiral HPLC/MS. While the literature states that the naturally occurring form of this alkaloid is the R-enantiomer and that its S-antipode is usually not found in nature, we confirmed the hypothesis of Govindachari and Nagarajan that natural levorotatory tylophorine is indeed a nearly racemic mixture with a slight excess of the R-enantiomer.

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Tylophora mayottae W.D. Stevens, Labat & Barthelat, spec. nova (Fig. 3, 4). Typus: M AYOTTE: Grande Terre, Mamoudzou, r��serve foresti��re de Majimbini, La Convalescence, 5.IX.2001, Barthelat et al. 483 (holo-: P [P00229464]! iso-: BR!, K!, MAO!, MO!). Tylophora mayottae W.D. Stevens, Labat & Barthelat is similar to T. coriacea Marais, but with smaller flowers which are rotate and a shorter corolla tube (0.2-0.5 mm) and with guide rails which are shorter (0.15-0.2 mm). Twining and trailing vine with white latex. Stems rooting at nodes, woody and corky below, with hirsutulous indument present at the nodes and sometimes extending in a continuous line along the internodes; internodes 3-14 cm long. Leaves opposite; blades 6.7-13.5 �� 4-8.8 cm, ovate to elliptic, apex acute to acuminate, base obtuse to truncate or shallowly lobed with a sinus up to 7 mm deep, both surfaces glabrous, glossy, succulent, margin thickened, lateral veins 4 to 6, colleters 2 to 10, sometimes raised on a fleshy pad; petioles 0.7-3.4 cm, glabrous or hirsutulous on the upper side. Inflorescence 4.5- 6.0 cm long, with hispidulous indument forming a continuous line on the axis; peduncle 1.7-2.2 cm when in flower, up to 5.3 cm long when in fruit. Flowers borne on pedicels 0.5- 0.6 cm long; bracts 2.2 �� 0.3 mm, linear to lanceolate; calyx apparently without colleters, lobes 1.3-1.3 �� 0.5 mm, lanceolate, apex acute, glabrous or with a few hairs on midrib and margin; corolla pale pink, glabrous outside, densely white-hispid inside with hairs 0.15 mm long, tube 0.2���0.5 mm, lobes 2-2.5 �� 1-1.2 mm, lanceolate with acute apex; corona pale yellow 1.0 mm high, 1.5-1.6 mm in diam., 5-lobed; lobes free, erect, more or less reaching to the base of anthers, apices rounded; c o r p u s c u l u m n a r ro w l y e l l i p s o i d, 0.2 -0.2 2 �� 0.0 7 - 0.08 mm, translators 0.09 mm long, pollinia ellipsoid, 0.18-0.2 �� 0.14-0.16 mm; ovaries glabrous. Follicles 8.2 �� 1.1 cm, narrowly fusiform, glabrous, smooth, green; seeds 10-11 �� 5.5-6.5 mm, dark brown, margin 0.8 mm wide, irregularly toothed on distal third, surface smooth, finely hispidulose on both sides; coma 2-2.5 cm long, white. Distribution, habitat and phenology. ��� Tylophora mayottae is only known from Mayotte, it has been recorded in sub-humid to humid forest between 200 to 400 m in the Majimbini and Combani forest reserves on Grande Terre and in the coastal forests at Saziley on Grande Terre and at Moya on Petite Terre. It has been recorded in flower in October and November, and in mature fruit in September, but flowering and fruiting probably spans at least from August to December. Vernacular names and local use. ��� The new species is known from data recorded by Barthelat & Boullet (2005) or on herbarium labels as ���Vahy rountou���, ���Vahy rotono be��� or ���Pamba suisui be��� in Shibushi dialect, and ���Ouvamba suisui bole��� in Shimaore dialect. The plant is used in the preparation of magic potions known as ���grigris��� (M. M���changama pers. comm.). Conservation status. ��� Tylophora mayottae is rare and clearly has a restricted distribution. It is known from only three recent collections from three separate protected areas, and three collections from the XIXth century, two of which have no locality details other than ��� Mayotte ���. The preliminary risk of extinction of Tylophora mayottae is therefore assessed as ��� Vulnerable��� [VU D2] following the IUCN Red List Categories and Criteria (IUCN, 2012). Further information on the extant population is needed to be able to provide a more reliable assessment of the conservation status of the new species. Notes. ��� Tylophora mayottae is vegetatively similar to T. coriacea, which is restricted to Mauritius, Reunion and the Seychelles, but differs from it by its smaller flowers which are rotate (vs. subcampanulate), with a shorter corolla tube (0.2-0.5 vs. 1.7-2 mm) and shorter guide rails (0.15-0.2 vs. 0.6-0.8 mm). In a recent molecular study (Liede-Schumann et al., 2012), Tylophora and a variety of other tropical genera were placed in synonymy with the north-temperate genus Vincetoxicum Wolf. However the authors are not convinced of the merits of this view, and prefer to maintain the genus Tylophora as distinct until more compelling evidence is available. Paratypi. ��� M AYOTTE: Grande Terre, Bandr�����, Saziley, La Convalescence, 30.IX.2003, Barthelat et al. 1242 (P); Grande Terre, Bandr�����, Saziley, La Convalescence, 29.XI.2005, Barthelat et al. 1526 ���); sine loc., Boivin s.n. (P); sine loc., XI.1850, Boivin 3210 (K, P); for��t de Combani, 21.X.1884, Humblot 1336 (K, P)., Published as part of Stevens, W. Douglas, Labat, Jean-No��l & Barthelat, Fabien, 2016, Two new species of Apocynaceae, Asclepiadoideae from Mayotte, pp. 127-134 in Candollea 71 (1) on pages 131-132, DOI: 10.15553/c2016v711a15, http://zenodo.org/record/5721433, {"references":["Barthelat, F. & V. Boullet (2005). Index de la flore vasculaire de Mayotte - version 2005 - 1. In: Rolland, R., V. Boullet & J. - P. Quod (ed.), Mayotte: Biodiversite et Evaluation Patrimoniale: 115 - 187. DAF de Mayotte & CBN Mascarin.","Liede-Schumann, S., H. Kong, U. Meve & M. Thiv (2012). Vincetoxicum and Tylophora (Apocynaceae: Asclepiadoideae: Asclepiadeae) - two sides of the same medal: Independent shifts from tropical to temperate habitats. Taxon 61: 803 - 825."]}

Tylophora lui Y.H. Tseng & C.T. Chao, a new species of Apocynaceae from southern Taiwan, is described and illustrated. It resembles T. ovata in the shape and size of the leaves and flowers, but differs by the indumentum of stems, the number of flowers, and by the glabrous and slender inflorescences. An identification key to the Taiwanese Tylophora species is provided.

Biochemical changes in respect to Peroxidase, Polyphenol oxidase, IAA oxidase, Total amino acid, Reduc- ing sugar and Protein content were studied during the process of shoot regeneration in the callus culture of Tylophora indica (Burm. F.) Merrill. The following stages: dedifferentiated callus, differentiating green callus, differentiating callus with shoot primordial, differentiating callus with well-developed multiple shoots were selected for present study. Mature leaf was used as control. From the present investigation, it was proved that the process of differentiation had high metabolic activity. High peroxidase and polyphenol oxidase and IAA oxidase activities were recorded in differentiating green callus. Protein content along with reducing sugar and total amino acid contents were decreased significantly during differentiation as compared to control. Increased metabolic rate during in vitro organogenesis help Tylophora for rapid growth and multiplication.

Background: Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity. The specific biomolecular targets of these alkaloids have not yet been clearly identified. (+)-(13aS)-deoxytylophorinine is a new phenanthroindolizidine alkaloid originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata in our institute. (+)-(13aS)-deoxytylophorinine exerts both in vitro and in vivoanticancer activities. Methods: The in vivo anticancer effects and toxicity of this compound were investigated in mice, and interactions between this compound and double-helical DNA sequences were studied in detail with circular dichroic spectroscopy and fluorescence spectroscopy. Viscosity measurements were applied to check the interactive mode between this compound and DNA. Results: Potent anticancer effects were observed in vivo. Also, concentration-dependent interactions were observed and this compound seemed to interact in a sequence-specific manner with AT-repeated sequences of double-helical DNA. Such interactions were proved to be intercalating by viscosity measurements. Conclusions: Anticancer alkaloid (+)-(13aS)-deoxytylophorinine can have sequence-specific interactions with DNA in an intercalating manner.

Polymethoxy-substituted phenanthrene-9-carboxylic acids or their methylate are key intermediates for the synthesis of tylophora alkaloids and their analogs. An intramolecular oxidative coupling reaction of unfunctionalized 2,3-disubstituted phenyl acrylic acids and derivatives promoted by di-tert-butylperoxide gave above intermediates in high yields. The mild reaction conditions and easy purification procedures of this method provide a new approach for the synthesis of phenanthrenes.

All 301 names in Tylophora and the small genera in Asclepiadeae-Tylophorinae now considered synonymous with Vincetoxicum, namely Amblyoglossum, Belostemma, Biondia, Blyttia, Diplostigma, Goydera, Hoyopsis, Hybanthera, Iphisia, Ischnostemma, Merrillanthus, Microstephanus, Nanostelma, Neohenrya, Oncostemma, Pentabothra, Pentastelma, Pleurostelma, Podostelma, Pycnostelma, Rhyncharrhena, Sphaerocodon, Spirella, Tylophoropsis and Vincetoxicopsis, are listed. A total of 107 new combinations are made (104 in Vincetoxicum, one in Dregea, one in Marsdenia, and one in Lygisma), 25 nomina nova have been coined, 141 typifications are undertaken and 10 new synonyms are proposed.

Tylophora indica is very popularly used for the treatment of asthma based on its traditional use for asthma. Tylophora is perennial climbing plant native to the plains, forests, and hills of southern and eastern India. A method has been developed for different extracts of Tylophora indica for HPTLC fingerprinting analysis for identification and quantification of marker compound. For chloroform extract-Chloroform(90): Methanol (5) : Ethyl acetate (5) v/v, Methanol Extract-Toluene(5): Chloroform(90), Ethyl acetate(5) v/v and for Petroleum ether extract-Hexane(40) : Ethyl acetate (60) v/v. The HPTLC fingerprinting profile developed for different extracts of Tylophora indica will help in proper identification and quantification of marker compound.

Vincetoxicum xinpingense, a new species from the Ailao Mountains in Yunnan Province, China, is described and illustrated. It is morphologically similar to V. sublanceolatum, Tylophora forrestii, and T. chingtungensis, but can be distinguished by several characters: shape and size of leaves, size of corolla lobes, length and shape of corona, attachment position of corona to the anthers, and orientation of pollinia. In addition, conservation status and other relevant notes are provided.

A rapid synthetic approach to the tylophora alkaloids antofine and 13aα-secoantofine is presented that makes use of a multicatalytic oxidative aminochlorocarbonylation/Friedel–Crafts reaction as the key step. This reaction, along with a one-pot, three-step telescoped process offers a three or four-pot sequence to access the title compounds in high overall yield.

A new species of Tylophora, T. corollae Bullock ex Meve & Liede (Asclepiadaceae), is described from Ethiopia. This delicate twiner is characterized by yellow, shield-shaped corona lobes with apical appendages.The non-twining shrub up to lm high, T. fleckii (Schltr.) N.E. Br., so far known to be endemic to Namibia, is for the first time recorded from NE Africa. The interesting biogeographical implications resulting from the extraordinary gap of 4500km in its distribution are discussed.

A concise, efficient and modular route involving Parham-type cycloacylation as the key step has been used to synthesize six enantiopure phenanthro-indolizidine alkaloids 1a-c. The preparation of enantiomerically pure tylophora alkaloids and their seco analogues on a large-scale is now feasible. The alcohol intermediates 8a-c, which are difficult to prepare by other synthetic methodologies, have been synthesized by a metallation-cyclization-reduction sequence in excellent yields.

A new secoiridoid, secamonoide A (1), and a new xanthone glycoside, secamonoide B (2), together with nine known compounds, were isolated from the aerial parts of Tylophora secamonoides Tsiang. Their structures were elucidated on the basis of spectroscopic methods. An antimicrobial bioassay showed that secamonoides A and B exhibited weak activities (MIC values greater than 100 mg/ml) against some hospital bacteria in vitro.

Pollinators of two Cynanchum, five Tylophora, and 16 Vincetoxicum species were observed in 26 populations in Japan. The following pollination systems were observed in 18 species: moth pollination, generalized insect pollination, wasp pollination, dipteran pollination, both dipteran and moth pollination, and autogamy. Principal component analysis based on 13 floral characters indicated that the size of the pollinator tended to increase with sizes of all characters measured. Furthermore, species that have developed interstaminal parts of the corona and concealed stigmatic chambers tend to be pollinated by long-tongued insects. The phylogenetic distribution of pollinator types showed that species belonging to Clade I are pollinated exclusively by Diptera, whereas those of Clade II are pollinated by four insect orders. The most prominent pollinator transition in the Tylophora–Vincetoxicum complex is dipteran to moth pollination. The most common morphological change of the plants from dipteran pollination to moth pollination, or vice versa, is modification of the corolla. In the Tylophora–Vincetoxicum complex, pollination mode shifts are generally accompanied by modifications of the corona and the structure of gynostegium. One hypothesis for the rapid radiation observed in Clade II is that a widely distributed species may have partitioned its distribution in the relatively near past and adapted to various environments, in which the dominant pollinators were different, and that the local races may then have diverged from each other after they were isolated. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 325–341.

A concise, efficient and modular approach to the tylophora alkaloids is described, a family of potent cytotoxic agents that are equally effective against drug sensitive and multidrug resistant cancer cell lines. The advantages of the chosen route are illustrated by the total syntheses of the phenanthroquinolizidine cryptopleurine (1) and the phenanthroindolizidines (-)-antorine (2), (-)-tylophorine (3), and their only recently isolated congener (-)-ficuseptine C (4). The key steps consist in a Suzuki cross-coupling between a (commercial) boronic acid and a simple aryl-1,2-dihalide followed by elaboration of the resulting products into the corresponding 2-alkynyl-biphenyl derivatives 27, 33, 41 and 46. The latter undergo PtCl 2 -catalyzed cycloisomerizations with formation of the functionalized phenanthrenes 28, 34, 42 and 47, which were transformed into the targeted alkaloids by a deprotection/Pictet-Spengler annulation tandem. Due to the flexibility and robust character of this approach, it might enable a systematic exploration of the pharmacological profile of this promising class of bioactive natural products.

The purpose of this study was to develop a new micropropagation system for Tylophora indica, an important medicinal plant in India, using root explants as starting material. Root explants cultured on MS medium supplemented with 6-benzyladenine (BA) or 2-isopentyladenine (2iP) produced organogenic nodular meristemoids (NMs) within 4 weeks. NMs induced from the cut ends of root segments showed two types of organogenic response--direct shoot bud formation and somatic embryogenesis--when maintained on induction medium. In 42% of the explants, NMs developed shoot buds directly in the presence of 10.72-26.80 microM BA. On average, 18.5 +/- 0.7 shoots per gram of NM tissue were obtained after each 4-week subculture. Elongation of microshoots and root initiation were correlated with the auxin used, with the optimal response occurring in the presence of 28.54 microM indole-3-acetic acid. In 39% of the explants, NMs dedifferentiated into friable embryogenic callus (FEC) in the presence of BA or 2iP after 12 weeks of culture. Of the different treatments, MS medium supplemented with 10.72 microM BA was the most effective in inducing FEC and somatic embryogenesis: at this concentration 64% of the cultured NMs developed FEC and, on the same medium, 89% of the FEC produced globular somatic embryos (SEs). FEC biomass increased nearly five-fold with every 4-week subculture, and about 30 SEs were recovered per gram of FEC during this period. The best conversion of mature SEs to complete plantlets was obtained on basal MS medium-42%. Plantlets derived via somatic embryogenesis and shoot organogenesis were successfully hardened (88-96%) and transferred to the field.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC/MSn) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MSn, LC/UV-MS and LC/MSn techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS). Copyright © 2003 John Wiley & Sons, Ltd.

A metal-free intramolecular oxidative coupling of 2,3-disubstituted phenylacrylic acids furnishing polymethoxy-substituted phenanthrene-9-carboxylic acids (II), key intermediates for the synthesis of tylophora alkaloids, is developed.

Summary Our preliminary studies with tylophora alkaloids had shown that they inhibit cellular immune responses like contact sensitivity to dinitro-flurobenzene and delayed hypersensitivity to sheep red blood cells, in vivo. Investigations were hence carried out to determine the cellular targets of tylophora alkaloids in in vitro systems. Con A induced proliferation of splenocytes was used as a model system to study the effect of the alkaloids on cellular immune responses. The alkaloid mixture was found to inhibit proliferation of splenocytes at higher concentrations and augment the same at lower concentrations. Both macrophages and T cells were found to be vulnerable to tylophora alkaloids. The alkaloid mixture suppressed IL-2 production in Con A stimulated splenocytes at the inhibitory or higher concentrations and enhanced production at the lower concentrations. IL-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentrations of tylophora. These studies indicate that tylophora alkaloids have a concentration dependent biphasic effect on Con A induced mitogenesis. At lower concentrations they augment Con A induced lymphoproliferation by enhancing IL-2 production. Inhibition of proliferation at higher concentrations of the alkaloid is due to inhibition of IL-2 production and activation of macrophages, which have a cytostatic effect.

Tylophora alkaloids have been shown to have antiasthmatic, antiinflammatory and antianaphylactic properties. Since all these disorders are a consequence of altered immunological status, the effect of these alkaloids on model immune reactions were studied. Crude extract of the leaves of Tylophora indica inhibited delayed hypersensitivity reaction to sheep red blood cells in rats when the alkaloid mixture was administered before and after immunization with these cells. The alkaloid mixture also inhibited contact sensitivity to dinitro-fluorobenzene in mice when given prior to or after contact sensitization. Lymphocytes taken from contact sensitized mice, when treated with tylophora alkaloid in vitro and transferred into naive syngeneic hosts, could suppress the transfer of delayed type hypersensitivity (DTH) response. However, the tylophora alkaloids could not suppress primary humoral (IgM) immune response to SRBC in mice at the same dose. These studies suggest that tylophora alkaloids suppress cellular immune responses when administered at any stage during the immune response.

This paper gives an account of two new black mildews, namely, Asterina prataparajii and Meliola mahamulkarii, infected the leaves of Tylophora dalzelli and Osyris arborea, respectively, were found new and are described and illustrated here in detail.

Oncostemma cuspidatum K. Schumdescribed from the West African island of Sao Tome, is recognized as being synonymous with Tylophora oblonga N.E. Bra widespread and variable species of mainland African rainforests. The monotypic genus Oncostemma K. Schum. is reduced to synonymy with Tylophora R. Br. (Asclepiadaceae–Asclepiadeae–Astephaninae).

Tylophora tridactylata Goyder (Apocynaceae: Asclepiadoideae) is described from the Eastern Arc mountains of southern Tanzania and illustrated. Its conservation status is assessed.

A modified classification of the family Asclepiadaceae R. Br.s.s. into three tribes — Secamoneae, Asclepiadeaes.l. and Stapelieaes.l. — is proposed. The position of attachment of the caudicles to the pollinia is suggested as a criterion for defining the erect and pendulous stature of the pollinaria. The concept of the transverse stature of pollinaria has been abandoned. In addition to the stature of the pollinaria, the morphology of the anther sacs (whether or not embedded in the tissue of the anther wings) and the position of anther wings with respect to the anther sacs are suggested as supplementary characters for tribal classification of the family. The characters of the gynoecium, particularly the presence or absence of true styles and the sharp constriction between stigma-head and ovaries (i.e. clavuncular morphology) have also been suggested as useful in differentiating Asclepiadeaes.l. and Stapelieaes.l., along with the stature of the pollinaria. The circumscription of Asclepiadeae is emended to accommodate taxa of the former tribe Gonolobeae as a subtribe. The circumscription of Stapelieae has been retained in a wider sense, as suggested by Decaisne (1844). The tribes Fockeeae Kunze, Liede & Meve (1994), Marsdenieae Benth. (1876), Ceropegieae Benth. (1876), and Stapelieaes.s.sensuBenth. (1876; non Decne., 1844) have been relegated to subtribe status in the tribe Stapelieae Decne. (1844). Homology of the different parts of the gynoecium in the Asclepiadeae (s.l.) with those in the Stapelieae (s.l.) has been drawn. Segments of the style have been distinguished into ‘true style’ and ‘pseudostyle’, the former as parts of the ovary segment in development, the latter as stigma segment in development. The genusTylophoraR. Br. which was formerly treated under Stapelieae Decne. has been transferred to Asclepiadeae based on the morphology of the pollinaria, gynoecium and seed coat architecture.

The antimicrobial efficacy of two phenanthroindolizidine alkaloids, tylophorinidine hydrochloride (TdnH) and tylophorinine hydrochloride (TnnH), isolated from the plant Tylophora indica (local name, Antamul) was evaluated. These were screened for in vitro antifungal and antibacterial activities. Both compounds exhibited potent antifungal activity displaying minimum inhibitory concentrations (MIC) in the range of 2-4 microg/mL for TdnH and 0.6-2.5 microg/mL for TnnH against Candida species.

An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC₅₀ values ranging from 84 nM to 20.6 μM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI₅₀ values ranging from 4 nM to 24.2 μM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure–activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5–9).

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity as well as other effects like antiamebicidal, antiviral, and anti-inflammatory activities. The specific biomolecular targets of these compounds have not yet been clearly identified. S-(+)-Deoxytylophorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata. Potent anticancer activity was observed in vitro and in vivo. Neurotoxicity of CAT was also studied and it was far less serious than that of vinblastine. Interactions between this compound and DNA had been studied in detail in our laboratory previously, and we further studied its interactions with RNA.

1. Acetyltylophoroside (AcT) and tylogenin inhibit Na+/K+-ATPase in spite of having structures very different from cardiac glycosides (CGs). 2. Calculation of the lowest energy conformations of AcT and tylogenin and superpositions of these with the X-ray conformations of CGs and chlormadinone acetate led to a model for the interaction of these different types of Na+/K+-ATPase inhibitors with the receptor.

The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC₅₀) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure-activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC₅₀ values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.

Phenanthroindolizidine-based tylophora alkaloids have been reported to have potential antitumor, anti-immuno and, anti-inflammatory activity. The structure-activity relationships of a series of tylophora alkaloids were studied to guide future drug design. Our results indicate that although these compounds are structural analogs, their potency of cytotoxicity, selectivity against NF-kappaB signaling pathway, and their inhibitory effects against protein and nucleic acid synthesis are different. Because they do not have an identical spectrum of targets, the studied compounds are structural, but may not be functional analogs.

Tylophorine (1) and related phenanthroindolizidine alkaloids, also known as Tyophora alkaloids, have been isolated principally from plants of the family Asclepiadaceae , including members of the genus Tylophora . Because of their profound cytotoxicity, these compounds have been targets of synthetic modification. Evaluation of (+)-( S )-tylophorine and its analogs in the National Cancer Institute's antitumor screen showed a uniform and potent inhibitory effect on the cell growth (G1 50 ≅ 10 -8 M) against all 60 cancer cell lines, with notable selectivity toward several refractory cell lines, including melanoma and lung tumor cell lines. However, to date, this compound class has not been successfully developed for clinical use in cancer due to the CNS toxicity of tylocrebrine reported in 1966. Most recently, tylophorine analogs have shown significant inhibitory effects on NF-κB mediated transcription, a unique mode of antitumor action that differs from those of the current known antitumor drugs. This recent discovery points to a great potential for developing tylophorine derivatives as a new antitumor drug class. Motivation is high in many research laboratories and a renewed and expedited exploration of tylophora alkaloids is anticipated. Therefore, this article contains a comprehensive review of isolation, total synthesis, antitumor activity and structure-activity relationship (SAR) correlations of tylophorine-related alkaloids.

The methanolic extract of Tylophora asthamatica (Asclepiadaceae) leaves was bioassayed by a leaf disc method for feeding deterrence using Spilosoma obliqua Walker as the test insect. Tylophorine, isolated from the extract was evaluated under laboratory and greenhouse conditions for its feeding deterrence, persistence and systemic properties by using the natural host plant (Ricinus communis) of the test insect. The antifeedant compound azadirachtin and crude neem extract were used as standards. Tylophorine produced complete inhibition of feeding under laboratory conditions and was persistent in field trials for two days as determined by the damage rating score. However, both tylophorine and the crude neem extract were shown to be nonsystemic when tested in the Hoagland culture media.

Five phenanthroindolizidine alkaloids (PA) were chemically synthesized and seven were isolated from Tylophora atrofolliculata. To facilitate future drug design of phenanthroindolizidine alkaloids as potential antitumor agents, we have explored the structure-activity relationships (SAR) of this class of compounds. We demonstrated that DCB-3503 and tylophorinidine (PA-7) were among the most active compounds against tumor growth both in vitro and in vivo. In the hepatocellular carcinoma cell line HepG2, the GI(50)s of DCB-3503 and PA-7 were 35+/-5 nM and 11+/-5 nM, respectively. DCB-3503 and PA-7 significantly inhibited HepG2 tumor growth in nude mice at a dose of 9 mg/kg given by intraperitoneal (ip) injections twice a day every third day for a total of four cycles (P