Using Micropropagation to Develop Medicinal Plants into Crops.

Medicinal plants are still the major source of therapies for several illnesses and only part of the herbal products originates from cultivated biomass. Wild harvests represent the major supply for therapies, and such practices threaten species diversity as well as the quality and safety of the final products. This work intends to show the relevance of developing medicinal plants into crops and the use of micropropagation as technique to mass produce high-demand biomass, thus solving the supply issues of therapeutic natural substances. Herein, the review includes examples of in vitro procedures and their role in the crop development of pharmaceuticals, phytomedicinals, and functional foods. Additionally, it describes the production of high-yielding genotypes, uniform clones from highly heterozygous plants, and the identification of elite phenotypes using bioassays as a selection tool. Finally, we explore the significance of micropropagation techniques for the following: a) pharmaceutical crops for production of small therapeutic molecules (STM), b) phytomedicinal crops for production of standardized therapeutic natural products, and c) the micropropagation of plants for the production of large therapeutic molecules (LTM) including fructooligosaccharides classified as prebiotic and functional food crops.

Plant microbiome-dependent immune enhancing action of Echinacea purpurea is enhanced by soil organic matter content.

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p < 0.0001). A change in soil organic matter content from 5.6% to 67.4% led to a 4.2-fold increase in the macrophage activation potential of extracts from E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p < 0.001). These results indicate that the level of soil organic matter is an agricultural factor that can alter the bacterial microbiome, and thereby the activity, of E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

Activities and Prevalence of Proteobacteria Members Colonizing Echinacea purpurea Fully Account for Macrophage Activation Exhibited by Extracts of This Botanical.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Molecular Phylogeny, Diversity, and Bioprospecting of Endophytic Fungi Associated with wild Ethnomedicinal North American Plant Echinacea purpurea (Asteraceae).

The endophytic fungal community associated with the ethnomedicinal plant Echinacea purpurea was investigated as well as its potential for providing antifungal compounds against plant pathogenic fungi. A total of 233 endophytic fungal isolates were obtained and classified into 42 different taxa of 16 genera, of which Alternaria alternata, Colletotrichum dematium, and Stagonosporopsis sp. 2 are the most frequent colonizers. The extracts of 29 endophytic fungi displayed activities against important phytopathogenic fungi. Eight antifungal extracts were selected for chemical analysis. Forty fatty acids were identified by gas chromatography-flame-ionization detection (GC-FID) analysis. The compounds (-)-5-methylmellein and (-)-(3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin were isolated from Biscogniauxia mediterraneaEPU38CA crude extract. (-)-5-Methylmellein showed weak activity against Phomopsis obscurans, P. viticola, and Fusarium oxysporum, and caused growth stimulation of C. fragariae, C. acutatum, C. gloeosporioides, and Botrytis cinerea. (-)-(3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin appeared slightly more active in the microtiter environment than 5-methylmellein. Our results indicate that E. purpurea lives symbiotically with different endophytic fungi, which are able to produce bioactive fatty acids and aromatic compounds active against important phytopathogenic fungi. The detection of the different fatty acids and aromatic compounds produced by the endophytic community associated with wild E. purpurea suggests that it may have intrinsic mutualistic resistance against phytopathogen attacks in its natural environment.

Diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus Opuntia humifusa (Cactaceae) from the United States.

The endophytic fungal community associated with the native cactus Opuntia humifusa in the United States was investigated and its potential for providing antifungal compounds. A hundred-eight endophytic fungal isolates were obtained and identified by molecular methods into 17 different taxa of the genera Alternaria, Aureobasidium, Biscogniauxia, Cladosporium, Cryptococcus, Curvularia, Diaporthe, Epicoccum, Paraconiothyrium, Pestalotiopsis and Phoma. The most frequent species associated with O. humifusa were Alternaria sp. 3, Aureobasidium pullulans and Diaporthe sp. The fungal community of O. humifusa had a high richness and diversity; additionally, the species richness obtained indicates that the sample effort was enough to recover the diversity pattern obtained. Six extracts of endophytes showed antifungal properties and (1)H NMR analyses of the extracts of Alternaria sp. 5 Ohu 8B2, Alternaria sp. 3 Ohu 30A, Cladosporium funiculosum Ohu 17C1 and Paraconiothyrium sp. Ohu 17A indicated the presence of functional groups associated with unsaturated fatty-acid olefinic protons and fatty acid methylene and methyl protons. GC-FID analysis of these extracts confirmed the presence of a mixture of different fatty acids. The (1)H NMR analyses of Biscogniauxia mediterranea Ohu 19B extracts showed the presence of aromatic compounds. From the extract of B. mediterranea we isolated the compound 5-methylmellein that displayed moderate antifungal activity against the phytopathogenic fungi Phomopsis obscurans. Our results suggest that native medicinal cacti of the United States can live symbiotically with rich and diverse endophytic communities and may be a source of bioactive molecules, including those able to inhibit or control plant disease pathogens.

Pharmacological activities of cilantro's aliphatic aldehydes against Leishmania donovani.

Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as a potential source of new drugs against leishmaniasis. In this study, we investigated the antileishmanial activity of cilantro (Coriandrum sativum) essential oil and its main components: (E)-2-undecenal, (E)-2-decenal, (E)-2-dodecenal, decanal, dodecanal, and tetradecanal. The essential oil of C. sativum leaves inhibits growth of Leishmani donovani promastigotes in culture with an IC50 of 26.58 ± 6.11 µg/mL. The aliphatic aldehydes (E)-2-decenal (7.85 ± 0.28 µg/mL), (E)-2-undecenal (2.81 ± 0.21 µg/mL), and (E)-2-dodecenal (4.35 ± 0.15 µg/mL), all isolated from C. sativum essential oil, are effective inhibitors of in vitro cultures of L. donovani promastigotes. Aldehydes (E)-2-decenal, (E)-2-undecenal, and (E)-2-dodecenal were also evaluated against axenic amastigotes and IC50 values were determined to be 2.47 ± 0.25 µg/mL, 1.25 ± 0.11 µg/mL, and 4.78 ± 1.12 µg/mL, respectively. (E)-2-Undecenal and (E)-2-dodecenal demonstrated IC50 values of 5.65 ± 0.19 µg/mL and 9.60 ± 0.89 µg/mL, respectively, against macrophage amastigotes. These cilantro compounds showed no cytotoxicity against THP-1 macrophages.

Antifungal activity of extracts from endophytic fungi associated with Smallanthus maintained in vitro as autotrophic cultures and as pot plants in the greenhouse.

The endophytic fungal assemblages associated with Smallanthus sonchifolius (Poepp.) H. Rob. and Smallanthus uvedalius (L.) Mack. ex Small growing in vitro autotrophic cultures and in the greenhouse were identified and evaluated for their ability to produce bioactive compounds. A total of 25 isolates were recovered that were genetically closely related to species of the genera Bionectria , Cladosporium , Colletotrichum , Fusarium , Gibberella , Hypocrea , Lecythophora , Nigrospora , Plectosphaerella , and Trichoderma . The endophytic assemblages of S. sonchifolius presented a greater diversity than the group isolated from S. uvedalius and demonstrated the presence of dominant generalist fungi. Extracts of all fungi were screened against the fungal plant pathogens. Ten extracts (41.6%) displayed antifungal activities; some of them had a broad antifungal activity. The phylotypes Lecythophora sp. 1, Lecythophora sp. 2, and Fusarium oxysporum were isolated from in vitro autotrophic cultures and displayed antifungal activity. The presence of bioactive endophytic fungi within S. sonchifolius and S. uvedalius suggests an ecological advantage against pathogenic attacks. This study revealed reduced numbers of endophytes in association with both Smallanthus species in controlled cultivation conditions compared with the endophytic communities of hosts collected in the wild environments. Even as reduced endophytic communities, these fungi continue to provide chemical protection for the host.

Bioassay-directed isolation and identification of phytotoxic and fungitoxic acetylenes from Conyza canadensis.

Conyza canadensis (L.) Cronquist syn. (horseweed) is a problematic and invasive weed with reported allelopathic properties. To identify the phytotoxic constituents of the aerial parts, a systematic bioactivity-guided fractionation of the dichloromethane extract was performed. Three active enyne derivatives, (2Z,8Z)-matricaria acid methyl ester, (4Z,8Z)-matricaria lactone, and (4Z)-lachnophyllum lactone, were identified. The lactones inhibited growth of the monocot Agrostis stolonifera (bentgrass) and the dicot Lactuca sativa (lettuce) at 1 mg mL(-1), while the (2Z,8Z)-matricaria acid methyl ester was less active. In a dose-response screening of the lactones for growth inhibitory activity against Lemna paucicostata , (4Z)-lachnophyllum lactone was the most active with an IC50 of 104 µM, while the (4Z,8Z)-matricaria lactone was less active (IC50 of 220 µM). In a fungal direct bioautography assay, the two lactones at 10 and 100 µg/spot inhibited growth of the plant pathogenic fungi Colletotrichum acutatum , Colletotrichum fragariae , and Colletotrichum gloeosporioides . In a dose-response screening of the lactones against six different plant pathogenic fungi, (4Z,8Z)-matricaria lactone was more active than the commercial fungicide azoxystrobin on Col. acutatum , Col. fragariae , and Col. gloeosporioides at 30 µM and about as active as the commercial fungicide captan against Col. gloeosporioides , while (4Z)-lachnophyllum lactone was less active.

Rapid analysis of lignans from leaves of Podophyllum peltatum L. samples using UPLC-UV-MS.

A new rapid UPLC-UV-MS method has been developed that permits the analysis of four lignans (4'-O-demethylpodophyllotoxin, podophyllotoxin, α-peltatin and ß-peltatin) in P. peltatum L. Podophyllotoxin is a natural lignan that is being used as a precursor for the semi-synthetic anti-cancer drugs etoposide, teniposide and etopophos. The chromatographic separation was achieved using a reversed-phase C18 column with a mobile phase of water and acetonitrile, both containing 0.05% formic acid. Analyses of P. peltatum leaves collected from different colonies within a single site indicated a significant variation in 4'-O-demethylpodophyllotoxin, α-peltatin, podophyllotoxin and ß-peltatin content. Within 3.0 min four main lignans could be separated with detection limits of 0.1, 0.3, 0.3 and 0.2 µg/mL, respectively. 4'-O-demethylpodophyllotoxin and α-peltatin appeared most prominently among the lignans obtained. The podophyllotoxin content was found in the range of 0.004-0.77% from 16 samples collected from 6 colonies within the same site. The content of podophyllotoxin is directly proportional to the content of 4'-O-demethylpodophyllotoxin and inversely proportional to α-peltatin and ß-peltatin content. LC-mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of four lignans in various populations of plant samples. By applying principal component analysis and hierarchical cluster analysis, Podophyllum samples collected from various colonies within a location were distinguished.

The role of biotechnology in the production of the anticancer compound podophyllotoxin.

Podophyllotoxin is a plant-derived compound found in Podophyllum sp. that is used to produce semi-synthetic anticancer pharmaceuticals such as etoposide, teniposide, and etoposide phosphate. This chapter describes the role of biotechnology to produce podophyllotoxin and our attempts to domesticate Podophyllum peltatum L., also known as the American mayapple. The domestication research on mayapple included surveys of the natural population, identification of high yielding genotypes, propagation, cultivation, sustainable harvest procedures and the development of protocols for in vitro germplasm bank.

Frequency and timing of leaf removal affect growth and podophyllotoxin content of Podophyllum peltatum in full sun.

Podophyllotoxin is a pharmaceutical compound found in leaves and rhizomes of American mayapple (P. peltatum L.), a species being investigated as an alternative to that of the Indian mayapple (P. emodi). Leaves alone can serve as a renewable source of podophyllotoxin (and other lignans) leaving rhizomes undisturbed to produce leaf biomass in subsequent years. It is not known, however, how frequently or severely plants can be defoliated without adversely affecting future plant growth, lignan content, or podophyllotoxin yield (g.m(-2)). This study compared harvest strategies that were mild to severe in frequency and timing of leaf removal. A wild population in full sun was subjected to leaf removal treatments of varying frequency (every year, every 2nd or 3rd year) and timing (early or late). Control plots not previously harvested were included every year. Plots were 1.0 m2 and established during spring of 2001. Duration of the study was four years. P. peltatum plants did not tolerate the most severe harvest treatment: annual harvest frequency in combination with early harvest time. Early annual harvests reduced total leaf dry mass and total leaf area in a consistent and linear manner. In contrast, plants tolerated annual harvests when conducted late in the growing season and tolerated early harvests when conducted every 2nd or 3rd year. The number of sexual shoots was reduced to zero by early annual harvests. Podophyllotoxin content was 2.7 to 6.5 times greater in leaves harvested early compared to those harvested late, though content was significantly greater in only two out of four years. In conclusion, we can recommend leaf removal every year from well-established P. peltatum populations grown in full sun if harvests are conducted late in the growing season. This harvest strategy ensures maximum podophyllotoxin yield without jeopardizing future leaf biomass yield. Leaves harvested early appear to have greater podophyllotoxin content, but we discourage early harvest every year. Instead, our results indicate that leaves can be harvested early every other year without reducing long-term performance of P. peltatum populations.

Geographic information system method for assessing chemo-diversity in medicinal plants.

The spatial distribution of wild germplasm of Podophyllum peltatum L. (American mayapple) has been analyzed using the Geographic Information System (GIS) with the objective to develop a method and a database for evaluation of biotic and abiotic factors influencing drug yield, and to map elite genotypes for propagation and improvement. The field assessment followed a standard procedure including geographical coordinates of each accession, leaf biomass randomly harvested, identification of associate species, collection of herbarium specimen, soil sample and digital pictures of the site. By overlaying morphological and chemical data with geomorphic information, a thematic map was created locating the podophyllotoxin-rich accessions and the uniqueness of each site was recorded for post-collection analysis. This work has enabled the establishment of a database of P. peltatum germplasm in Mississippi with drug yield linked to spatial locations for rational utilization of our natural resources. While this method integrates information of well-characterized diverse in situ P. peltatum germplasm, it might become a strategy for curators to reduce cost for establishing and maintaining ex situ collections since the genetic material is geo-referenced.

Micropropagation of Anemopaegma arvense: conservation of an endangered medicinal plant.

Anemopaegma arvense (Vell.) Stellfeld ex de Souza is a medicinal species also known as Catuaba. All commercially available formulations of Catuaba have been produced from wild crafting, the gathering of plant biomass from its native habitat. Nodal segments were used as explants and 4.8 new buds per explant were induced on MS media supplemented with 4.4 microM of kinetin in 30 days. The percentage of in vitro rooting was low, although acclimatization of unrooted plants into soil was successfully achieved. For germplasm maintenance, cultures of A. arvense were stored on 4 % (w/v) sorbitol and maintained viable at low growth rate without subcultures for six months.

Variation of podophyllotoxin in leaves of Eastern Red Cedar (Juniperus virginiana).

Leaves of Eastern red cedar (Juniperus virginiana L. Cupressaceae) have been reported to contain podophyllotoxin, a pharmaceutical compound used to manufacture drugs for treatment of cancer, rheumatoid arthritis, genital warts, psoriasis, and multiple sclerosis. Podophyllotoxin content of leaves of immature, mature male, and mature female plants (approximately 1.45 mg x g -1) was significantly higher than that of leaves of juvenile plants (0.60 mg x g -1). Sampling date also affected podophyllotoxin content. Leaves harvested in January and April exhibited higher podophyllotoxin contents (1.56 and 1.45 mg x g -1, respectively) than leaves harvested in February and June (1.06 and 1.08 mg x g -1, respectively). There was no obvious pattern or trend in the data due to sampling date. There was no significant interaction between plant type and sampling date. These results indicate that foliage of mature Eastern red cedar, a waste product of the lumber industry, could be a low-yielding, but relatively stable, source of podophyllotoxin.

Podophyllum peltatum possesses a beta-glucosidase with high substrate specificity for the aryltetralin lignan podophyllotoxin.

A beta-glucosidase with high specificity for podophyllotoxin-4-O-beta-D-glucopyranoside was purified from the leaves of Podophyllum peltatum. The 65-kDa polypeptide had optimum activity at pH 5.0 and was essentially inactive at pH 6.5 or above. Maximum catalytic activity of this glucosidase was obtained at 45 degrees C, but the enzyme was not heat stable. This beta-glucosidase displayed higher substrate specificity for podophyllotoxin-4-O-beta-D-glucopyranoside than for the other lignans tested, and for the (1-->3) linkage of laminaribiose than for other glucosidic linkages.

Micropropagation of Hydrastis canadensis: Goldenseal a North American endangered species.

An in vitro propagation protocol for rapidly producing Hydrastis canadensis L., Goldenseal, plantlets from disk tissue of young leaves was developed. Leaf explants were inoculated on MS medium supplemented with various concentrations of NAA and TDZ for production of callus. Two-month-old calli were sub-cultured on MS media containing cytokinins (BA, kinetin, TDZ) in different concentrations for shoot initiation. The optimum level of callus induction and maintenance was in 5.3 microM NAA in combination with 2.2 microM of TDZ. Shoot multiplication was achieved on MS medium with 2.2 microM TDZ in combination with 0.5 microM NAA. The alkaloid profile of micropropagated plantlets was similar to the profile of the mother plants. These results suggest that our in vitro propagation protocol will produce a positive impact in the conservation of H. canadensis.

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa.

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.

Evaluation of Podophyllum peltatum accessions for podophyllotoxin production.

In an effort to develop a sustainable source of podophyllotoxin for the production of anticancer drugs such as etoposide, teniposide and etopophos, Podophyllum peltatum accessions with podophyllotoxin-rich leaf biomass were identified and transplanted to different growing conditions by vegetative cuttings. Results indicate that the lignan profile in leaves does not change over time or due to environment conditions. Podophyllotoxin and alpha-peltatin content in the blades seems to be stable with an inverse relationship of concentration between these compounds. A podophyllotoxin-rich leaf accession showed low biosynthetic capability to synthesize alpha- and beta-peltatin and the converse was also true, indicating that selection and cultivation of high-yielding podophyllotoxin leaf biomass may reduce production costs.