Browsing by Author "Asha Sankar, M"
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ThesisItem Open Access Exploitation of invitro cultures of Indian Madder(Rubia cordifolia.Linn) for anticancerous compounds(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Labade, Dinesh Sitaram; KAU; Asha Sankar, MThe present investigation on “Exploitation of in vitro cultures of Indian Madder (Rubia cordifolia L.) for anticancerous compounds” was carried out at the Plant Tissue Culture Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara and Amala Cancer Research Centre, Thrissur during the period 2006-2008. The study was undertaken with the objective to standardize the in vitro techniques for initiation and proliferation of static and suspension cultures of Rubia cordifolia and to screen the in vitro cultures for synthesis of naphthoquinone and quantify it. It was also envisaged to enhance the level of product synthesis in in vitro cultures and to assess the anticancerous activity of in vitro and in vivo extracts in terms of cytotoxicity, antioxidant and prooxidant activities in vitro. Leaf, nodal and root derived callus cultures of Rubia cordifolia were established in vitro. Explants were pre treated with the fungicide, Bavistin 2.5 per cent for 15 minutes. Surface sterilization with mercuric chloride (HgCl2) at 0.1 per cent for 1 min and 30 sec was effective for yielding healthy, contamination free cultures from nodal segments and leaves, respectively. MS medium at full strength, supplemented with NAA at 2 mg l-1 along with BA at 0.5 mg l-1 was observed ideal for initiation and proliferation of calli. The auxin synergist phloroglucinol, when supplemented to the medium, did not not yield encouraging results, with respect to callusing in the experimental species. Root derived cultures were inferior with respect to callus initiation and proliferation, registering low values for all the parameters studied. Incubating in vitro cultures under illuminated condition at 26 ± 2 C was superior to dark incubation, with respect to callus initiation and proliferation. Chloroform – methanol at 8.5 :1.5 ratio was indentified as the appropriate solvent system for detection of naphthoquinone on thin layer chromatograms in the test extracts of the experimental species, with alcoholic KOH (10 per cent) as the spray reagent. Ms medium at full strength, fortified with NAA and BA at 2.0 mg l-1 and 0.5 mg l-1 respectively, which recorded maximum naphthoquinone synthesis, was standardized as the production medium. Enhancing concentration of sucrose to 5 per cent in the production medium, did not elicit a positive response on naphthoquinone production in vitro. Reducing nitrate concentration of the production medium, to half and one fourth the original concentration, resulted in enhanced in vitro synthesis of the target compound. Supplementing the production medium with yeast extract (1 per cent and 2 per cent) as well as precursor feeding with phenyl alanine and tyrosine each at levels of 50 mg l-1, 100 mg l-1 and 150 mg l-1 exerted a favourable influence on synthesis of naphthoquonines, in vitro. Incubation in dark resulted in marginal increase in in vitro production of naphthoquinones. Incorporation of autoclaved mycelia of Pythium aphanidermeatum at levels of 2.0 per cent and 5.0 per cent resulted in enhanced in vitro production of naphthoquinone. The abiotic elicitor, salicylic acid at concentration of 10 μM and 100 μM resulted in maximum synthesis of naphthoquinones in in vitro root cultures (8.76 units g -1 calli) of Rubia cordifolia. Immobilization of test calli with sodium alginate – calcium chloride complex as well as subjecting the in vitro cultures to stress conditions, as imposed by sorbitol failed to bring about an enhancement in the in vitro production of naphthoquinones. None of the explants employed in the study induced hairy roots, when co- cultured with the Agrobacterium rhizogenes strains, MTCC 2364 and MTCC 532. Based on cell count, subculturing intervals of leafs, nodal and root derived suspension were fixed as 24, 27 and 27 days respectively with the respective packed cell volume as 0.93 per cent, 0.83 per cent and 0.80 per cent. Naphthoquinone was detected, in ex vitro and in vitro test extracts at all levels of maturity tested. Both ex vitro and in vitro root extracts exihibited maximum cytotoxicity, as revealed by the percentage of cell death on DLA and EAC cell lines as well as their IC50 values. As compared to whole plant extract, in vitro systems of the experimental species exhibited least antioxidant action. Extent of pro-oxidant activity was higher in in vitro root extract of the experimental species.ArticleItem Open Access Hydration-dehydration technique, a mid-storage correction to prolong viability of rice seeds(Kerala Agricultural University, 1996) Girija, T; Sukumara Dev, V P; Asha Sankar, M; Rajappan Nair, N; KAUThesisItem Open Access In vivo and in vitro screening of sida spp. for ephedrine content(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1998) Asha Sankar, M; KAU; Sreekandan Nair, GThe present investigations on in vivo and in vitro screening of Sida spp. For ephedrine content were carried out at the experiment field and Plant Tissue Culture Laboratory of the Department of plantation Crops and Spices and Biochemistry laboratory of All India Co-ordinated Research Project on Medicinal and Aromatic Plants, College of Horticulture, Kerala Agricultural university, Vellanikkara, Thrissur, from September 1993 to march, 1998. The objectives of the study were to screen four commonly occurring Sida spp. Viz S. rhombifolia ssp. Retusa, S. acuta, S. rhombifolia ssp. Rhombifolia and S. cordifolia for ephedrine under in vivo and in vitro conditions and to explore the possibilities of upgrading the content of this alkaloid in vitro. Leaf, stem and root callus cultures of the four experimental species were established in vitro. Half strength MS medium supplemented with 2, 4-D 1 mg 1-1 was observed ideal for initiation and proliferation of calli. Kinetin at 0.3 and 1.0 mg 1-1 enhanced the callus inducing property of NAA. Among the species tested, S. acuta recorded significantly superior performance registering high callus index values of 319.60 and 311.19 respectively for leaf and stem cultures on half MS medium incorporated with 2, 4-D 1.0 mg 1-1 . The auxin synergist, phloroglucinol at levels of 100.0 mg 1-1 and 125.0 mg 1-1 was singularly effective in promoting callusing in Sida spp. Incubating leaf and stem cultures under illuminated conditions at 27 + 10C was significantly superior to incubation in dark or at 10-110C. Root explants were inferior to leaf and stem explants in inducing and proliferating calli. Successful regeneration of roots from leaf and stem calli of the experiment species was achieved with NAA, 1.0 mg 1- . Stimulatory effects of the growth factor combination, NAA and kinetin at 1.0 mg 1-1 and 0.3 mg 1-1 were reflected in number of roots regenerated. The promotive effects of 2 ip in root regeneration were more evident in stem cultures. NAA and kinetin at 0.5 mg 1-1 and 1.0 mg 1-1 respectively initiated shoots in leaf (20.10 per cent) and stem (26.93 per cent) callus cultures. Substituting sucrose with maltose in proportions of 1:2 in half strength MS media fortified with NAA and kinetin each at 1.0 mg 1-1 initiated embryoids in S. rhombifolia spp rhombifolia and S. cordifolia. Half MS media supplemented with NAA and kinetin, each at 1.0 mg 1-1 was standardised as the production medium which recorded positive response in leaf calli of S. cordifolia with respect to synthesis of ephedrine in qualitative and chromatographic tests. Butanol – glacial acetic acid – water at 4:1:1 was identified as the appropriate solvent system with ninhydrin as the localizing spray. Incorporation of yeast extract at 2.0g 1-1 and the precursor phenyl alanine at 50.0 mg 1-1 and 100.0 mg 1-1 elicited synthesis of ephedrine in leaf and stem calli of S. cordifolia. Methionine, another precursor failed to elicit synthesis of alkaloid in callus cultures of Sida spp. Addition of osmoregulant, polyethylene glycol at 2.0 per cent exerted a favourable influence on synthesis of ephedrine in leaf and stem calli of S. cordifolia. Definite presence of ephedrine in in vitro cultures of S. cordifolia was confirmed by eliciting the cultures with autoclaved mycelia of Pythium aphanidermatum at 500.0mg 1-1, 2.0g 1-1 and 5.0 g 1-1. Supplementing elicitation with precursor feeding was particularly beneficial to synthesis of ephedrine, wherein apart from S. cordifolia leaf callus cultures of S. rhombifolia ssp. Rhombifolia synthesised ephedrine. Immobilization or irradiation of calli failed to produce the alkaloid. Success in establishment of hairy root cultures from in vitro calli of S. cordifolia was dependant on the efficiency of the strain of Agrobacterium rhizogenes employed Strain A4 induced hairy roots in 50 per cent cultures each. In leaf and stem calli of S. cordifolia and 16.67 per cent cultures in root calli of the same species. Sucessful liquid suspensions of the experimental species could be established with a critical cell density of 2 g calli in 50 ml culture media, subcultured at an interval of 17 days with an inoculum ratio of 1:4. S. cordifolia was most effective with respect to proliferation in liquid suspensions registering an increase in packed cell volume of 200 per cent and 150 per cent respectively in leaf and stem calli, at 17 days subculture. Estimation of content of ephedrine in positively responding in vitro systems revealed that elicitation coupled with precursor feeding produced highest content of 0.0208 per cent and 0.0107 per cent respectively in leaf and stem calli of S. cordifolia. Barring cultures fed with phenyl alanine, static cultures synthesized higher amounts of ephedrine as compared to suspensions. Total free amino acid content of alkaloid producing fresh calli exceeded that of unproductive fresh and aged calli while total phenol content registered low values in alkaloid producing calli. Studies on yield parameters of field grown Sida spp. Revealed that total yield per plant and mean root yield varied significantly with stages of harvest, the maximum values being obtained at harvest at 9 months after planting. However stages of harvest did not influence the content of crude extractables of the experimental species significantly. S. rhombifolia ssp. Retusa ranked superior with respect to total biological yield and shoot yield per plant. Leaf extracts of S. cordifolia recorded comparatively higher content of ephedrine (0.0089) when harvested at 7 months after planting.ThesisItem Open Access Standardization of good agricultural practices (GAP)in Kacholam (Kaempferia galanga L)for yield and quality(Department of Plantation crops and spices,College of Horticulture, Vellanikkara, 2011) Chandana, R; KAU; Asha Sankar, MAn investigation on “Standardization of good agricultural practices (GAP) in kacholam (Kaempferia galanga L.) for yield and quality”, was carried out at the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, during 2010-2011 to standardize organic resource management and post harvest handling practices in kacholam for optimizing yield and quality, leading to formulation of good agricultural practices in the crop. Results of the study revealed that the treatments which included FYM along with biofertilizers proved superior in terms of earliness in germination whereas FYM supplemented with Kalanchoe pinnata recorded the maximum germination percentage of 98.61 and all other treatments were on par. Plants which receive FYM along with biofertilizers recorded the maximum leaf number (30.67) at 6 MAP. Vermicompost supplemented with kalanchoe and FYM enriched with biofertilizers recorded the highest values for foliage spread at later stages of crop growth. In the present study, the superiority registered by the control plots receiving inorganic fertilizers with respect to vegetative growth parameters at early stages of growth was not evident at later stages. Experimental plots applied with FYM enriched with biofertilizers recorded the highest value for fresh rhizome yield per plant (80.30 g) and fresh (9.93 t ha-1) as well as dry (3.19 t ha-1) rhizome yields per hectare, which were on par with the rest of the treatments. Plots incorporated with FYM and kalanchoe, recorded significantly higher values for dry recovery (36.4 %). Control plots applied with inorganic fertilizers recorded the lowest dry recovery percentage (31.42 %) as well as dry rhizome yield (2.32 t ha-1). Essential oil content was significantly higher in plots applied with FYM supplemented with kalanchoe (1.47 %) and oleoresin content recorded a significantly higher recovery percentage of 3.42 per cent in plots applied with FYM supplemented with kalanchoe and biofertilizers. Control plots which received inorganic fertilizers recorded the least recovery of essential oil (0.87 %). FYM supplemented with kalanchoe, singly and along with biofertilizers and chromolaena mulch, recorded highest available P (92.48 kg ha-1) and N (503.8 kg ha-1) content in soil respectively. Vermicompost supplemented with kalanchoe recorded high content of soil K and all other treatments were on par. Control plots registered the lowest soil K content. Higher plant uptake of major nutrients was observed in vermicompost treated plots. Enhanced population of soil microbes was recorded by the use of organic nutrients and biofertilizers. FYM supplemented with biofertilizers and vermicompost applied singly and along with kalanchoe and biofertilizers recorded the maximum bacterial, fungal and actinomycetes population. During the course of experiment, maximum incidence of bacterial wilt was observed in FYM manure received plots. Composite samples of kacholam registered no significant variation in dry recovery during sun drying, shade drying and oven drying. Maximum quality constituents were retained in sun dried samples of the crude drug, recording 1.2 per cent essential oil and 3.4 per cent oleoresin though the variations were not significant. Storage studies revealed the least percentage loss in weight (0.98 %) and residual moisture content (6.78 %) in samples stored in polyethyleneterephthalate (PET) bottles, at 6 months after storage. Essential oil and oleoresin contents recorded the maximum values during storage in PET bottles and polyethylene bags respectively. Samples stored in polyethylene bags and PET bottles did not record any insect infestation and had registered minimum microbial infection. Percentage loss of essential oil and oleoresin ranged from 5-30 per cent and 22-55 percent depending on method of storage.ThesisItem Open Access Survey, collection and characterization of 'Kizharnelli' (Phyllanthus spp.) of Kerala(Department of plantation crops and spices, College of horticulture, Vellanikkara, 2015) Shafna Kalarikkal; KAU; Asha Sankar, MAn investigation on “Survey, collection and characterization of „Kizharnelli‟ (Phyllanthus spp.) of Kerala”, was carried out at the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, and CARe KERALAM, Koratty, Thrissur during 2013-2014, with the objective of morphological and phytochemical characterization of Phyllanthus accessions collected after surveying select locations of Kerala and assessment of quality of traded crude drug of Phyllanthus by detecting species admixtures and estimating phytochemical constituents. Genus Phyllanthus belonging to the family Phyllanthaceae, consists of about 833 species and is chiefly distributed in moist humid tropics. The most wide spread species of this genus, P. amarus, reputed for its hepatoprotective activity which contain phyllanthin and hypophyllanthin and is used in traditional medicine against jaundice. Other commonly ocuuring herbaceous Phyllanthus spp. of Kerala are P. virgatus var. virgatus, P.virgatus var. gardnerianus, P. rheedei, P. airy-shawii, P. maderaspatensis and P. urinaria. Taxomonic confusion exists in identification of these herbaceous Phyllanthus spp., mainly due to their similarity in gross morphology, close proximity in growth habitat as well as referring them with a common vernacular name, „Kizharnelli‟. Preponderance of other Phyllanthus spp. often leads to ignorant as well as deliberate adulteration/substitution in its raw drugs, resulting in lowering the efficacy of the medication. Other Phyllanthus species have not been subjected to in-depth phytochemical and clinical investigations. A total of fourty seven Phyllanthus accessions were collected from coastal regions, plains, midlands and high ranges of southern, central and northern zones of Kerala, of which, seventeen accessions were from southern zone, fourteen from central and sixteen from northern zones. The collected Phyllanthus accessions were decoded into respective species based on the key characters of herbaceous Phyllanthus spp. described in Flora of Madras Presidency. Out of the fifteen qualitative characters observed, no notable variability was observed for six qualitative characters viz., growth habit, branching pattern, leaf margin, flower colour, capsule colour and capsule shape. Erect growth habit, spreading branching pattern, entire leaf margin, depressed globose capsule shape, pale green flower colour and yellowish green capsule colour were noticed in all the accessions. Stem colour, leaflet colour, rachis colour, leaflet shape, leaflet apex, leaflet base and peduncle colour were highly varying among the accessions. The lone accession of P. maderaspatensis had obcordate leaf apex. P. virgatus var. gardnerianus and P. virgatus var. virgatus were observed to have longest pedicel length (1.0 cm), while rest of the Phyllanthus spp. had a pedicel length of 0.1 cm. P.amarus had five sepals and rest of the species, six. Highest plant height (90.1 cm), fresh weight (16.21 g) and dry weight (13.81 g) were observed for P. virgatus var. gardnerianus. Broadest leaves were observed in P. rheedei, and longest leaflets (2.01 – 2.21 cm) in P. virgatus var. gardnerianus and P. virgatus var. virgatus. The accessions of P. urinaria (22.1 – 28.8 cm) registered shortest stems length. P. amarus was distributed equally in the three zones surveyed and P. urinaria was predominantly observed in southern and central zones. Lone accession of P. maderaspatensis was observed in southern zone. P. virgatus var. virgatus was not represented at all in southern zone. The northern zone had representations of all herbaceous species of Phyllanthus under study, except P. maderaspatensis. P. amarus was equally distributed in coastal regions, plains, midlands and high ranges. P. virgatus var. gardnerianus had representation only in high ranges, while P. maderaspatensis was represented only in coastal regions. Coastal regions represented fewer species of Phyllanthus, while, high ranges registered maximum representation of herbaceous Phyllanthus species. Clustering of Phyllanthus accessions based on morphological parameters revealed that P. urinaria, P. airy-shawii, and P. amarus occurred in more than one cluster which indicates the presence of morphovariants in them. P. maderapatensis and P. rheedei formed single separate clusters indicating their individual morphological identity. Altitude wise clustering based on morphological parameters also presented a similar clustering pattern. In species wise assessment of growth and yield parameters of collected accessions during pot culture, P. amarus and P. urinaria, recorded significant differences, only with respect to plant height, wherein, accessions from coastal and midlands were rated superior in P. amarus. With respect to number of leaflets, P. virgatus var. virgatus from midlands of central zone was significantly superior. Biochemical characterization of Phyllanthus accessions revealed highest contents of total extractives (0.55 g to 0.61g) and phyllanthin (0.32 - 0.46 %) in P. amarus. Phyllanthin was absent in P. urinaria. Maximum content of phenol was recorded in P. airy-shawii (232.1 -252.1 mg g-1) followed by P. urinaria (196.2 -221.2 mg g-1). P. airy-shawii recorded lowest EC50 value (211.3 to 222.3 μg ml-1), indicating highest antioxidant capacity. A positive correlation noticed between total phenol content and antioxidant capacity. Clustering of Phyllanthus spp. in central zone based on biochemical parameters grouped, P. rheedei and P. virgatus var. virgatus in a single cluster while, they existed in separate clusters during clustering based on morphological parameters. Thus, morphologically dissimilar Phyllanthus spp. possess comparable contents of active ingredients. Hence, from the therapuetic point of view, substitution is possible between the species that are clustered together based on contents of active ingredients. Clustering based on temporal sites revealed that altitude can influence the content of certain active ingredients of Phyllanthus spp. During organoleptic evaluation of raw drug samples RD-1 and RD-2, the raw drug sample RD-1, was superior, devoid of any species admixtures. In the raw drug sample RD-2, though the predominant species was P. amarus, presence of P. airy-shawii was detected. The biochemical parameters of raw samples of Phyllanthus from RD-1 and RD-2 did not register any appreciable difference with the reference sample, with respect to all biochemical parameters studied.ThesisItem Open Access Utilisation of in vitro cultures of Tinospora cordifolia Miers (chittamrithu) for berberine(Department of Plantation Crops, College of Horticulture, Vellanikkara, 2002) Kalimuthu, M; KAU; Asha Sankar, MThe present investigation on "Utilisation of in vitro cultures of Tinospora cordifolia Miers. (Chittamrithu) for berberine" was carried out in the Plant Tissue Culture and Biochemistry Laboratories, College of Horticulture, Vellanikkara, Thrissur during the period 1999-2001. The study was undertaken with the objective to standardise the in vitro techniques for initiation and proliferation of static and suspension cultures of T cordifolia and to screen the in vitro cultures for synthesis of berberine and quantify it. It was also envisaged to enhance the level of product synthesis in in vitro cultures. Leaf, petiole and stem derived callus cultures of Vellanikkara and Madurai ecotypes were established in vitro. Surface sterilisation with mercuric chloride (HgCh) at 0.1 per cent for 8 min was most effective in all the explants. MS medium at full strength supplemented with NAA at 4 mg r ' was observed ideal for initiation and proliferation of calli. Kinetin at 3 mg r' and BA at 4 mg r' enhanced the callus inducing property of NAA. Both the ecotypes responded equally for most of the parameters observed, with respect to callusing. The auxin synergist, phloroglucinol at levels of 100.0 mg r' and 125 mg r' and casein hyrdolysate at 100, 200 and 300 mg r' registered favourable influence on callusing. Incubating leaf and stem cultures under illuminated conditions at 26±1 QC was significantly superior to incubation in dark. Successful regeneration of roots from leaf and stem calli of the experimental ecotypes was achieved on MS medium at half strength supplemented with NAA or lAA each at 2 mg r'. Calli derived from Madurai ecotype performed better with respect to root regeneration. None of the treatments tried resulted in a positive response with respect to shoot initiation from callus cultures of both the ecotypes. Substituting sucrose with lactose in proportions of 2: 1 in MS medium at full strength fortified with NAA at 1 mg r ' and Kin at 4 mg r' initiated embryoids in both the ecotypes. MS media at full strength supplemented with NAA and BA or NAA and Kin each at 2 mg r', was standardised as the production medium, which recorded maximum berberine synthesis. Butanol-glacial acetic acid-water at 7:1:2 was identified as the appropriate solvent system for detecting the alkaloid with Dragendorff's reagent as the localizing spray. Substituting sucrose with lactose maintaining a proportion of 2: 1 and reducing the phosphorus level in basal medium to half the original strength resulted in increased levels of berberine synthesis. The precursor phenyl alanine at 100, 150 and 200 mg r' elicited synthesis of berberine. Addition of osmoregulants, polyethylene glycol at 2.0 and 3.0 per cent and mannitol at 1.5 per cent exerted a favourable influence on synthesis of berberine in Tinospora. Incorporation of autoclaved mycelia of Pythium aphanidermatum at 0.5, 1.0 and 1.5 g r' and immobilisation of calli with sodium alginate-calcium chloride complex revealed a positive influence on synthesis of berberine. Liquid suspensions of Vellanikkara and Madurai ecotypes registered 0.92 and 0.87 per cent of packed cell volume. Based on critical cell density, the liquid suspension were subcultured at 16 days interval. As compared to static cultures, suspensions synthesized lesser quantity of berberine. Berberine was detected only in stem extracts of ex vitro plants. When compared to ex vitro samples, in vitro cultures yielded higher quantities of berberine. The highest berberine yield (23.176 ug/g of callus) was obtained from stem cultures maintained in solid MS media supplemented with NAA 2 mg r' + BA 2 mg r ' and autoclaved mycelia of P. aphanidermatum at 0.5 g r'. Madurai ecotype performed better with respect to berberine synthesis with a mean value of 17.565 ug of berberine/gram of callus whereas Vellanikkara ecotype synthesized 16.051 ug/g of callus under positively responding treatments.