Abstract: To investigate the plant growth-promoting effect and stimulation of lignan biosynthesis, the effect of culture filtrates/live co-culture of two arbuscular mycorrhizae-like fungi, Piriformospora indica and Sebacina vermifera, on growth of Linum album cells and on production of podophyllotoxin (PT) and 6-methoxypodophyllotoxin (6-MPT) was studied. For elicitation studies, different volumes of culture filtrates (autoclaved and membrane-filtered) of P. indica/S. vermifera were added to suspension cultures of L. album. The culture filtrates of both the fungi exhibited a positive effect on product formation. For co-culture experiments, both fungi were individually co-cultivated at different concentrations with L. album in suspension cultures for different time periods. This resulted in significant enhancement of PT and 6-MPT content in the plant cells. The activity of phenylalanine ammonia lyase (PAL) was observed to be related to the lignan accumulation, indicating its role as the key enzyme of the phenylpropanoid pathway. The study resulted in total lignan (PT and 6-MPT) production of 745.6 mg/l with a very high PT productivity of 52.4 mg/(l.d).
Abstract: The process of drug discovery is very complex and requires an interdisciplinary effort to design
effective and commercially feasible drugs. The objective of drug design is to find a chemical
compound that can fit to a specific cavity on a protein target both geometrically and chemically.
After passing the animal tests and human clinical trials, this compound becomes a drug available
to patients. The conventional drug design methods include random screening of chemicals
found in nature or synthesized in laboratories. The problems with this method are long design
cycle and high cost. Modern approach including structure-based drug design with the help
of informatic technologies and computational methods has speeded up the drug discovery
process in an efficient manner. Remarkable progress has been made during the past five years
in almost all the areas concerned with drug design and discovery. An improved generation of
softwares with easy operation and superior computational tools to generate chemically stable
and worthy compounds with refinement capability has been developed. These tools can tap into
cheminformation to shorten the cycle of drug discovery, and thus make drug discovery more
cost-effective. A complete overview of drug discovery process with comparison of conventional
approaches of drug discovery is discussed here. Special emphasis is given on computational
approaches for drug discovery along with salient features and applications of the softwares
used in de novo drug designing.
Abstract: Withania somnifera (Solanaceae), commonly
known as ashwagandha, is known to have antiinflammatory,
antitumor, anticonvulsive and
immunosuppressive properties. In the present
study, antioxidant potential of in vitro cultured
cells and roots of W. somnifera was evaluated
within the concentration range of 5-100 μg/ml
using in vitro studies viz. free radical scavenging
capacity on 1, 1-diphenyl,- 2-picrylhydrazyl
(DPPH) radical, 2, 2-azinobis-(3-
ethylbenzothiazoline-6-sulphonate) (ABTS)
radical cation decolourization assay, scavenging
of nitric oxide radical and total antioxidant
capacity. Ascorbic acid was used as standard
compound. Among antioxidant screening models
tested, ethanolic extracts of W. somnifera cells
from transformed callus cultures had shown better
antioxidant potential in comparison to that of roots
and ascorbic acid. Therefore the results justify
the therapeutic application of W. somnifera as
an antioxidant in the indigenous system of
medicine.
Abstract: Pharmacogenomics is derived from integration of genomics, medical, pharmacological and biotechnological principles to develop ideal, safe, efficacious drugs with personalized approach based on the genetic variations that exist in all human beings. It plays an important role in design and discovery of âfuture pharmaceuticalsâ through understanding of newer and specific target identification, better clinical trials, and individualized consideration of drug response and their genetic interactions. The present article will provide insights on pharmacogenomics and its role in development of target oriented personalized drugs based on genetic polymorphism.
Abstract: Erectile dysfunction or impotence is emerging as one of the mostserious life style and stress related disease. Over exertion, physiologicaldisturbances, lowered level of hormones and strained relationship with partner arethe main causes for this disease. Apart from drug therapy and diet control regime,Ayurveda offers natural drugs with proven efficacy for treatment of erectiledysfunction. Ancient medical literature, especially Ayurveda, mentioned severalnatural drugs, alone or in combination, under the chapter of âRasayanaâ andâVajikaranâ to be used in this type of disorders. Garlic is one of them usedtraditionally to enhance sexual power. Psychotherapy alongwith meditation andself belief may also prove very useful approaches to treat this disease. The latestresearch in modern medical science yielded several purified compounds to dealwith bodily disorders. Sildenafil citrate USP and related compounds are the latestaddition in the weaponry of the modern medicinal system used for the treatmentof erectile dysfunction. In present article, two alike drugs of different systems andproperties- Garlic â a traditional drug and Sildenafil citrate â a synthetic drug arecompared. Special emphasis is given to establish the correlation in mechanism ofaction used for similar therapeutic effect i.e. erectile dysfunction along with acomparison on pharmacological and social basis. The results had shown thatgarlic has more or less same therapeutic effect and mechanism of action as thatof Sildenafil citrate. The only step that differs is that Sildenafil citrate increasesblood GMP level by inhibiting its degrading enzyme phosphodiesterase-5 (PDE 5)while garlic acts by activation of guanalyl cyclase enzyme and thus by increasingproduction of GMP in body. Garlic was observed to have lesser side effects andtoxicity, no abuse potential and contraindications in comparison to Viagra. The useof garlic is also justified from social aspect and may be a drug of choice incomparison to Viagra. There is a possibility that an odorless garlic preparationmay have potential to treat erectile dysfunction. Therefore further research workin this direction may be worthwhile.
Abstract: Cell suspension cultures of Linum album were developed from internode portions of in vitro germinated plant in Gamborg's B5 medium supplemented with 0.4 mg naphthalene acetic acid/l. The highest biomass was 8.5 g/l with podophyllotoxin and 6-methoxypodophyllotoxin at 29 and 1.9 mg/l, respectively after 12 d cultivation. Co-cultures of L. album cells with axenically cultivable arbuscular mycorrhiza-like fungi, Piriformospora indica and Sebacina vermifera, were established for the first time. These enhanced podophyllotoxin and 6-methoxypodophyllotoxin production by about four- and eight-fold, respectively, along with a 20% increase in biomass compared to the control cultures.
Abstract: Various cell and hairy root cultures of L. album were developed and analyzed for podophyllotoxin content. Transformed callus and hairy root cultures developed from infection of stem portions of in vitro-germinated L. album plant with Agrobacterium rhizogenes NCIM 5140 strain were selected on the basis of high podophyllotoxin content and growth. Based on the integration of Ri T(L)-DNA and T(R)-DNA, integration of only the ags and not the rol gene in transformed cell culture indicated fragmented integration pattern. The effect of different cultivation media and carbon source on growth and podophyllotoxin production were studied in shake-flask suspension cultures. Detailed batch growth and production kinetics with sugar consumption profile were also established. Maximum volumetric productivity of 4.40 and 2.75 mg/L per day was obtained in cell suspension and hairy root cultures, respectively.
Abstract: The effects of aeration within the range of 0.2-0.5 vvm on transformed and high yielding cell cultures of Linum album were investigated in a 5-L stirred tank bioreactor equipped with low shear Setric impeller. The kinetics of cell growth, substrate utilization, and production of lignans, namely, podophyllotoxin and 6-methoxypodophyllotoxin, were established. Maximum biomass of 23.2 g/L and lignan accumulation levels of 176.3 mg/L podophyllotoxin and 10.86 mg/L 6-methoxypodophyllotoxin were obtained with initial air flow rate of 0.3 vvm. Specified oxygen demand of cells was estimated to be 1.35 g O(2)/g biomass. The optimum oxygen transfer coefficient was found to be 16.7 h(-1) (,) which corresponded to aeration rate of 0.3 vvm. The effect of minimum dissolved oxygen (DO) concentration was investigated with respect to biomass and lignan production by comparing identically aerated and agitated bioreactor cultivations at dissolved oxygen concentrations of 10%, 30%, and 50%. Cell growth and podophyllotoxin accumulation were not affected significantly at these DO levels, but 6-methoxypodophyllotoxin production was enhanced when cells were cultivated at 30% DO level. The maximum volumetric productivities of 18.2 mg/L day and 3.2 mg/L day for podophyllotoxin and 6-methoxypodophyllotoxin, respectively, were obtained. These results establish the key role of oxygen on mass scale production of anticancer lignans by cell cultures of L. album. It may serve as a suitable parameter for scale-up.
Abstract: High yielding transformed callus culture of W. somnifera was established by infecting hypocotyls with Agrobacterium tumefaciens MTCC-2250. Maximum withaferin A content of 0.0875 mg/g dry cell weight and transformation efficiency of 80% were obtained. Confirmation of transformation was done on the basis of the presence of the ags gene by using polymerase chain reaction. Various abiotic elicitors (arachidonic acid, methyl jasmonate, calcium chloride, and copper sulfate) and biotic elicitors (cell extracts and culture filtrates of Alternia alternata, Fusarium solani, and Verticilium dahaliae) were tested at different concentrations to enhance withaferin A production in suspension culture of transformed cells. Maximum enhancements of 5.4 times and 9.7 times, respectively, were obtained when copper sulfate (100 microM) and the cell extract of V. dahaliae (5% v/v) were added separately to suspension cultures. The dual elicitation strategy by the combined addition of these two elicitors resulted in 13.8-fold enhancement of withaferin A content in comparison to control cultures (2.65 mg/L). The present study indicates the potential of this biotechnology-based methodology for the large-scale production of withaferin A.
Abstract: Artemisinin, isolated from the shrub-Artemisia annua, is a sesquiterpene lactone used to treat multi-drug resistant strains of falciparum malaria. It is also effective against a wide variety of cancers such as leukemia and colon cancer. To counter the present low content in leaves and uneconomical chemical synthesis, alternate ways to produce artemisinin have been sought. But this compound remains elusive in cell cultures of A. annua despite the extensive studies undertaken. This work reports the first successful approach for production of artemisinin by cell cultures of Indian variety of A. annua. In the present study, an integrated yield enhancement strategy, developed by addition of selected precursor (mevalonic acid lactone) and elicitor (methyl jasmonate) at optimized concentrations, resulted in 15.2g/l biomass and 110.2mg/l artemisinin, which was 5.93 times higher in productivity in comparison to control cultures.
Abstract: Artemisinin, an endoperoxide containing
sesquiterpene lactone from Artemisia annua, has
proven very effective in treating drug resistant
cases of malaria and cancer. To counter the
present low content in leaves and uneconomical
chemical synthesis, alternate ways to produce
artemisinin have been sought. Inspite of
extensive work in this area, artemisinin remains
elusive in dedifferentiated and differentiated
cultures of A. annua. This work reports the first
successful approach for production of artemisinin
by cell cultures of A. annua cultures of Indian
variety of A. annua. Various precursors of
terpenoid biosynthesis by isoprenoid pathway
were incorporated to study their influence on
artemisinin biosynthesis. Artemisinin content
was maximally increased by 2.0 times, in
comparison to control, when mevalonic acid (50
mg/L) was added as precursor. Various biotic and
abiotic elicitors were also tested at different
concentration. A maximum increase of 3.47 times
in artemisinin accumulation was attained when
methyl jasmonate (5 mg/L) was added. Based
on these results, an integrated bioprocess for
productivity enhancement of artemisinin was
developed. A maximum artemisinin
accumulation of 96.8 mg/L artemisinin was
produced on supplementation of mevalonic acid
and methyl jasmonate as selected precursor and
elicitor respectively, which was 4.79 times higher
in productivity than control callus cultures (20.2
mg/L).
Abstract: Artemisinin, an endoperoxide containing sesquiterpene lactone from Artemisia annua, has proven very effective in treating drug resistant cases of malaria. Due to limited availability and variation in artemisinin content from plant, use of high yielding in-vitro cultures would prove useful in meeting the demand for this important therapeutics. Inspite of extensive work in this area, artemisinin remains elusive in dedifferentiated and differentiated cultures of A. annua. So present study was undertaken to develop Agrobacterium mediated transformed root cultures of A. annua and to combine product enhancement strategies, viz. high yielding root line selection, optimization of media and culture condition, precursor addition and effect of elicitors, to synergistically increase the artemisinin productivity in hairy roots.
This is very first successful tissue culture approach for production of artemisinin by hairy root cultures of Indian variety of A. annua. In the present work, a high yielding transformed root line was selected by Agrobacterium rhizogenes mediated infection of aseptically germinated seedlings. Confirmation of transformation was done by using Polymerase Chain Reaction. To determine optimum media composition and culture condition to maximize biomass and artemisinin production, single parameter optimization method was used. The highest artemisinin content, as determined by HPLC after sodium derivatization, found was 2.9 mg/L. Culture media formulation of half strength of Murashige and Skoog medium with 20 g/L sucrose, 19.5 mM nitrate and 0.5 mM phosphate and culture conditions of 25ï°C temperature, 5.7 pH and 16h/8h light dark cycle were found optimum. Under these optimized culture conditions, various precursors of terpenoid biosynthesis by isoprenoid pathway were incorporated to study their influence on artemisinin biosynthesis. Direct precursors like sodium acetate and mevalonic acid lactone and indirect precursor like casein acid hydrolysate and cholesterol were tested at different concentrations. Artemisinin content was increased by 2.22 times, in comparison to control, when mevalonic acid lactone (20 mg/L) was added. Various biotic (chitosan and yeast extract) and abiotic elicitors (gibberlic acid, calcium chloride, salicylic acid, acetyl salicylic acid, methyl jasmonate) were also tested at different concentration. A maximum increase of 3.88 times in artemisinin accumulation was attained when acetyl salicylic acid (30 mg/L) was added. Based on these results, an integrated bioprocess for synergistic enhancement of artemisinin was developed. A maximum of 9.6 g/L biomass and 15.27 mg/L artemisinin was produced on supplementation of mevalonic acid lactone and acetyl salicylic acid as selected precursor and elicitor respectively, which was 10.9 times higher in productivity than selected hairy roots culture.
Abstract: Biotechnological production of plant-based secondary metabolites by a cell culture-based bioprocess generally suffers from an inherent disadvantage of low yield. In order to overcome this, elicitation is one of the most promising yield-enhancement strategies. Biotic elicitors, especially those of a fungal origin, are known to increase the production of secondary metabolites in plant cell suspension cultures. So far, mostly pathogenic fungi-based elicitor preparations have been widely used for this purpose. This chapter provides details of the application of a symbiotic arbuscular mycorrhiza-like fungus, Piriformospora indica, as an effective elicitor to optimize the concentration of different elicitor preparations and the time of incubation for enhanced production of the anticancer drug podophyllotoxin by cell suspension cultures of Linum album.
Abstract: Inability of arbuscular mycorrhizal fungi to grow in pure cultures without living host roots is one of the most challenging hurdles to studying their symbiotic relationship with plants and their effect on cell growth and secondary metabolite production. In this regard, Piriformospora indica, an arbuscular mycorrhiza-like fungus, can be used as a very promising tool, due to its phytopromotional effect and ability to enhance production of desired phytochemicals. This chapter gives a detailed procedure for developing a co-culture system between plant cells of Linum album and fungal cells of P. indica in suspension cultures, for enhanced production of the anticancer drug podophyllotoxin.
Abstract: Natural products from plants are providing better templates for design of potential chemotherapeutic agents than synthetic drugs. Taxol, podophyllotoxin and camptothecin are some lead molecules, which has proved to be natureâs boon in the treatment of cancer. To meet their ever-increasing demands, biotechnological methods offer an excellent alternative but the economy of such a production is the major hurdle to overcome. The successful industrial application of plant cell cultivation for production of taxol will trigger further research on the other promising plant based chemotherapeutics. As the initial hurdles for large scale cultivation of plant cells have been overcome, the areas of concern now to produce desired products are synergistic product enhancement strategies along with in-depth knowledge of the biosynthetic pathway. Complete pathway for biosynthesis of podophyllotoxin is already established. But alternative pathways of different metabolic fates of lignans, precise sequence of the later steps in taxol biosynthesis and iridoid part of camptothecin biosynthesis are yet to be established.
Understanding of biochemistry, enzymology, physiology, bioreactor design and application of proteomics and genomics are other areas to focus. Several points in a given metabolic pathway can be controlled simultaneously either by over expressing and/or suppression of several enzymes or through the use of transcriptional regulators to control several endogenous genes. So multipoint metabolic engineering offers new perspective for improvement of production of plant based chemotherapeutics
An attempt has been made to provide an overview of research on key anticancer drugs in order to elucidate the biotechnological approaches for their production in cell cultures with special emphasis on the biosynthetic pathway mapping and metabolic engineering.
Abstract: This invention provides a process for enhanced production of bioactive compound using co-culturing of biological materials complementary to each other and that grow in synergism. Particularly the invention illustrates a high yielding process for podophyllotoxin, comprising inoculating sterile Linum album plant cells into liquid plant cell culture media to generate suspension culture and coculturing these plant cells with live fungal cells having phytopromotional activities, preferably Piriformospora indica I Sebacina vermifera. Preferably, the cell culture is of Linum album.
