Recent Patents on Biotechnology (v.8, #1)

Preface by Rongling Wu (1-1).

Calanolides are naturally occurring pyranocoumarins found particularly in Calophyllum species (family Clusiaceae/Guttiferae), and are well known for their potent anti-human immunodeficiency virus (HIV) activity. Various preclinicaland clinical studies with this class of compounds are going on, and the National Cancer Institute (NCI) has beenplaying an active and supportive role in this regard. The present review covers an up-to-date literature of naturally occurringcalanolides in view of their anti-HIV potential and total syntheses including information on related patents.

The number of botanical dietary supplements in the market has recently increased primarily due to increasedhealth awareness. Standardization and quality control of the constituents of these plant extracts is an important topic, particularlywhen such ingredients are used long term as dietary supplements, or in cases where higher doses are marketed asdrugs. The development of fast, comprehensive, and effective untargeted analytical methods for plant extracts is of highinterest. Nuclear magnetic resonance spectroscopy and mass spectrometry are the most informative tools, each of whichenables high-throughput and global analysis of hundreds of metabolites in a single step. Although only one of the twotechniques is utilized in the majority of plant metabolomics applications, there is a growing interest in combining the datafrom both platforms to effectively unravel the complexity of plant samples. The application of combined MS and NMR inthe quality control of nutraceuticals forms the major part of this review. Finally I will look at the future developments andperspectives of these two technologies for the quality control of herbal materials.

Withanolides from Withania somnifera Dunal: Development of Cellular Technology and their Production by Neelam S. Sangwan, Farzana Sabir, Smrati Mishra, Shilpi Bansal, Rajender S. Sangwan (25-35).
Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous system ofmedicine in India for over thousands of years. In view of its varied therapeutic potential, the plant has also been the subjectof considerable scientific attention. The major chemical constituents of the Withania genus, the withanolides, are agroup of naturally occurring C28-steroidal lactones built on an intact or rearranged ergostane framework, in which C22and C26 are oxidized to form a six-member lactone ring. In recent years, numerous pharmacological investigations havebeen carried out utilizing W. somnifera extracts and several patents have been filed on pharmacological and medicinal importanceof withanolides and extracts of W. somnifera, individually or in combination. Considering the immense importanceof withanolides for medicinal purposes, the establishment of strategies to improve withanolides yield are highly desirable.Under natural conditions, W. somnifera possesses restricted levels of withanolides then, alternatives for obtainingwithanolides in better yields are imperative. In vitro approaches followed by metabolic engineering could be attractivetools to achieve this goal. Therefore, we present here an overview of the development of various protocols for in vitro tissueregeneration from W. somnifera and in vitro secondary metabolite production as well. The review also gives an accountof selected patents on various important activities of phytochemicals and extracts of W. somnifera.

The use of plants for healing diseases is one of the oldest medical practices and there are several studies showingthat botany and medicine are related. Recent researches have shown that around 25% of new chemical entities and42% of anticancer drugs marketed worldwide from 1981 to 2006 are obtained from natural products and their derivatives.One-third of the botanical biodiversity of the planet is in South American tropical ecosystems (Neotropical). Over the centuries,plants have been used by indigenous people for curing diseases. This strong tradition has been a great challenge forthe scientific community in order to validate the folkloric medicinal use of herbs. In this way, a very few Neotropicalpharmaceutical products have reached the market in industrialized countries, even though they have a considerable plantdiversity. Plants synthesize several organic compounds which are not related to their growth and development and arecalled secondary metabolites or natural products that are derived from central or primary metabolism. Because plants aresessile organisms, they have to respond quickly to environmental changes in order to escape and survive under unfavorableconditions. Drought is one of the most worldwide serious impediments for crop yields producing adverse negative effectson plant growth, by impacting leaves and roots growth, stomatal conductance, photosynthetic rate and biomass gain.The three major classes of secondary metabolites are produced from pathways of different primary metabolites, includingglycolysis, tricarboxilic acid cycle, aliphatic amino acids, pentose phosphate pathway, shikimate pathway and aromaticamino acids. This review compiles the metabolic changes occurring at primary metabolite level and total biosynthesis ofnatural products with potential for the development of new drugs in response to drought.

Arabidopsis thaliana is the first model plant, the genome of which has been sequenced. In general, intensivestudies on this model plant over the past nearly 30 years have led to many new revolutionary understandings in every singleaspect of plant biology. Here, we review the current understanding of anthocyanin biosynthesis in this model plant.Although the investigation of anthocyanin structures in this model plant was not performed until 2002, numerous studiesover the past three decades have been conducted to understand the biosynthesis of anthocyanins. To date, it appears thatall pathway genes of anthocyanins have been molecularly, genetically and biochemically characterized in this plant. Thesefundamental accomplishments have made Arabidopsis an ideal model to understand the regulatory mechanisms of anthocyaninpathway. Several studies have revealed that the biosynthesis of anthocyanins is controlled by WD40-bHLH-MYB(WBM) transcription factor complexes under lighting conditions. However, how different regulatory complexes coordinatelyand specifically regulate the pathway genes of anthocyanins remains unclear. In this review, we discuss currentprogresses and findings including structural diversity, regulatory properties and metabolic engineering of anthocyanins inArabidopsis thaliana.

Plants are capable of producing a myriad of chemical compounds. While these compounds serve specific functionsin the plant, many have surprising effects on the human body, often with positive action against diseases. Thesecompounds are often difficult to synthesize ex vivo and require the coordinated and compartmentalized action of enzymesin living organisms. However, the amounts produced in whole plants are often small and restricted to single tissues of theplant or even cellular organelles, making their extraction an expensive process. Since most natural products used in therapeuticsare specialized, secondary plant metabolites, we provide here an overview of the classification of the main classesof these compounds, with its biochemical pathways and how this information can be used to create efficient in and explanta production pipelines to generate highly valuable compounds. Metabolic genetic engineering is introduced in lightof physiological and genetic methods to enhance production of high-value plant secondary metabolites.

From Nature to Market: Examples of Natural Products that Became Drugs by Angelo de Fatima, Bruna Silva Terra, Cleiton Moreira da Silva, Daniel Leite da Silva, Debora Pereira Araujo, Leonardo da Silva Neto, Roney Anderson Nascimento de Aquino (76-88).
Nature is an irrefutable source of inspiration for the modern man in many aspects. The observation and understandingof nature have allowed the development of new materials, new sources of energies, new drugs etc. Specifically,natural products provide a great contribution to the development of new agents for the treatment of infections and antitumoragents. However, obtaining natural products directly from animals, fungi, bacteria, plants etc has been considered notenough to attend the high demand by pharmaceutical industries. In this regard, various strategies based on biotechnologicalprocesses or synthetic approaches have been developed. In this scenario the total synthesis can be undoubtedly a usefuland powerful tool for obtaining higher amounts of natural products and/or structural modifications thereof. Herein, weemphasize successful examples of total synthesis of galanthamine, morphine, paclitaxel and podophyllotoxin - naturalproducts approved as pharmaceuticals.

Stressing Conditions as Tools to Boost the Biosynthesis of Valuable Plant Natural Products by Fernanda Gomes da Silva, Lívia Pereira Horta, Raquel de Oliveira Faria, Joao Renato Stehmann, Luzia Valentina Modolo (89-101).
There is a consensus that plants are great sources of metabolites with a broad variety of functions. This is particularlyimportant because plants cannot run away from environmental conditions that can threat their existence. The numerousbiological activities exhibited by plant natural products prompted humanity to use such substances or their derivativesfor the treatment and/or prevention of diseases. The more we know the flora around the world the higher is the chanceto find new lead compounds for the design of more potent drugs or nutraceuticals. This review first deals with Brazilianflora, contextualizing the most studied medicinal species and related patents. It also describes a compilation of relevantworks based on the use of stress conditions to enhance the biosynthesis of valuable metabolites in cell cultures, tissue cultures(hairy roots) and whole plants by using native or crop plants around the world.

Efficient Method for Agrobacterium Mediated Transformation of Artemisia annua L. by Pravej Alam, Anis Mohammad, M.M. Ahmad, Mather Ali Khan, Mohd. Nadeem, Riyazuddeen Khan, Mohd. Akmal, Seema Ahlawat, M.Z. Abdin (102-107).
Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patentshave been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidencesof drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generatetransgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laboriousand delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediatedtransformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performanceof T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times andco-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 underdesiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated thatthe optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

Patent Selections (108-108).