Effect of Arbuscular mycorrhizal symbiosis on biosynthesis of active ingredients in selected...
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Transcript of Effect of Arbuscular mycorrhizal symbiosis on biosynthesis of active ingredients in selected...
Effect of Arbuscular mycorrhizal symbiosis on biosynthesis of active ingredients in
selected Medicinal plants
Tejavathi D.H. Jayashree D.R. *
*D.R. Jayashree ,Research Scholar, Department of Botany, Jnanabharathi,Bangalore University,Bangalore-560 056,Karnataka,Indiae-mail: [email protected]
IntroductionPlant Metabolites & Mineral Nutrients – "SPARK PLUGS OF LIFE"Vision of Health and Agriculture.Eco-friendly farming system for sustainable
Agriculture.Biofertilizers-Arbuscular Mycorrhizal Fungi (AMF), symbiotic Plant-fungus association. (Lewis, 1985; Paracer and Ahmadjian, 2000)Arbusclar systemic classification Morton & Benny, 1990; Phylum: Glomeromycota Order: Glomales & Endogonales Families: Glomaceae, Acaulosporaceae Genera: Glomus (60sp.), Sclerocystis(1sp.), Acaulospora(14sp.), Entrophospor(3sp.)
Plant Fungal Association
Cost-benefit model (Broucher, 1985; Lewis, 1985 &Tuomi et. al., 2001).
Non-Conventional Green Leafy Vegetables
- a wild crafted medicinal herbs, for study
- Achyranthes aspera L. (Amaranthaceae),
Achyranthine. - Leucas aspera (Willd.)Spreng
(Lamiaceae), • Leucasperol A,B &
Leucolactone. - Eclipta alba (L.)Hassk , (Asteraceae), Wedelolactone & ecliptine. - Euphorbia hirta L. (Euphorbiaceae), Euphorbin & quercitin.
Phytochemical Metabolites & Mineral Nutrients
Primary and secondary Metabolites
Protein, Carbohydrates and Aminoacids• Phenols,Flavonoids,Alkaloids and Terpenoids • Common Nutromineral Element Constituents:• NPK, Primary macronutrients• Ca & Mg, Secondary macronutrients• Na&Fe,micronutrients/traceelements
Medicinal Value:• Antitumor,antidiabetic,antiasthmatic,antimicrobial,
anti-inflammatory properties, anti-malarial activities, and also an immuno-modulators.
Selected Medicinal Plant Species , Weeds – Natural Habitats.
Euphorbia hirta(Open Field) Eclipta alba(Gutter/Pit)
Leucas aspera (Construction Site) Achyranthes aspera(Wasteland)
ObjectivesObjectives:: To investigate effects of arbuscular mycorrhizal To investigate effects of arbuscular mycorrhizal
inoculums in selected medicinal plantsinoculums in selected medicinal plants
To enhance the growth & yield of plant biomassTo enhance the growth & yield of plant biomass
To encourage and promote these wild crafted To encourage and promote these wild crafted herbs for its nutritional values herbs for its nutritional values
Global priorities in developing new drugs from Global priorities in developing new drugs from principle biochemical components with good principle biochemical components with good economic returns for the farmers. economic returns for the farmers.
Materials and MethodsMaterials and Methods
• Green house test:
• A completely randomized pot experiment 3 replicates & 8 treatments for each of 4 Medicinal plant species, maintained for 30, 60 & 90 DOI (Days of Inoculum).
• AM fungi inoculum load used based on its propagules number (g) for selected medicinal plants
Sl. No.
AM Fungi
Propogules
Dry wt. (g)
1
Control - -
2
Acaulospora bireticulata
2.8 x 106 0.4
3
A. lacunose 2.8 x 107 0.4
4
A. laevis 2.8 x 105 0.4
5
Glomus mosseae
1.4 x 105 0.8
6
G. aggregatum
2.1 x 107 0.5
7
G. geosporum 3.5 x 105 0.3
8
G. fasciculatum
3.5 x 105 0.3
Mycorrhizal Parameters%Root Colonization
Spore Count
Physiological ParametersAnalysis of Plant Metabolites and
Mineral Contents • Plant collection: Whole plant species were
harvested after 90 days of inoculation & dried in hot air oven at 600celcius for 12-24 hours, powdered with Mortar & Pestle.
• Extract preparation: By Wet Digestion Method• 1g dried plant sample +
HNO3:HCLO3+H2SO4(10:4:1)• heated at 200oC ; reduced to 1 ml ( clear soln.),
diluted to 100ml(DW) & quantitatively estimated.
Quantitative Analysis Colorimetric/Sphectrophotometric
Plant Metabolites
Primary metabolites:Total proteins: Lowry’s Method, BSA, 660 nm. Total Carbohydrates: Anthrone Method, Glucose, 630nm. Total Amino acids: Ninhydrin Method, Tyrosine, 570nm. Secondary metabolites: Total Phenols: Folic-Ciocalteu , pyrocatechol, 760 nm.Total Flavonoids: Aluminium Chloride, Quercitine, 415 nm.Total Alkaloids: BromoCresol Green, Atropine, 470 nm.Total Tepenoids:
Quantitative Analysis Colorimetric/Sphectrophotometric
Method• NPK (Jackson, 1973)• Nitrogen - Microkjeldahl method, Na2CO3 (0.1N).• Phosphorous - Vanadomolybdate phosphoric method,
Phosphate (490nm)• Potassium - Flame photometer, K .• Calcium - Raghuramulu et al,., 2003 , CaCl2.
• Magnesium - Barton, 1948 & Michelson,1957), Mg foil- Atomic absorption spectrophotometer 248nm.
• Sodium - Jones Method, 2001, NaCl• Iron - Wongs Method ,1928 Ferrous Ammonium
Sulphate.
HPLC Analysis – Principle Bioactive components of selected medicinal Herbs with
Mycorrhizal inocultaions Sample Preparation: 1 g powered plant sample-5ml
Methanol;grinded, centrifuged-10,000rpm,5 mins. Supernatent,used for HPLC analysis
Standard Preparations: 10 mg of each standards – 25ml (Mobile
Phase) Methanolic Water(70:30) :High gradient
grade volumetric flask,Class A. Filtered standard of 20µl loop was injected using Rhedoyne injector.
HPLC Analysis
Sample analysis: RPHPLC (Merck)-Mobile phase The analytes were resolved using C18
(4.6 mm x 250mm particle size 5mm) at 1600 psi.
Methanolic extract –filtered 0.22 µm & injected, HPLC instrument (Dionex Sunnyvate CA, USA); delivery system (Ultimate 3000 pump LPG-3400 A); dectector (UVD-3000).
Calculations: Amount of principle biochemical Component
Results – GrowthParameters(90 DAI)
Achyranthes aspera Eclipta alba
Euphorbia hirta Leucas aspera
Whole plant- AMF inocuilated Selected Medicinal Plant(90 DAI)
Achyranthes aspera Eclipta alba
Euphorbia hirta Leucas aspera
Mycorrhizal Parameters% Root Colonization and Spore Count
Number of Arbuscules Number of Vesicles
Hyphae & Mycelium-Vesicles
Spore Count & Structures
Spore count G.aggregatum
Chlmydomonas Shape - Glomus
Aculospora
Physiological ParametersQuantitavive Analysis
Primary and Secondary Metabolites
Quantitavive AnalysisPrimary and Secondary Metabolites
Plant Mineral Nutrients Primary Macroelements - NPK
Estimation of Nitrogen (%) Estimation of Phosphorous (%)
Estimation of Potassium (%)
Mineral elements Analysis Secondary Macronutrients -Ca & Mg Micronutrients - Na & Fe
Estimation of Calcium (ppm/g) Estimation of Magnesium (ppm/g)
Estimation of Sodium (ppm/g) Estimation of Iron (ppm/g)
HPLC Chromagram – AMF inoculated medicinal plants
Effects of AMF in the selected Medicinal herbs
Quantified values – Biochemical compound in the Medicinal plants inoculated with different species of
AMF
A.aspera –Betaine
Control 0.16 mg/g
G.fasciculatum 0.69 mg/g
G.mosseae 0.20 mg/g
A.laevis 0.23 mg/g
• E.alba-Wedelolactone• Control 0.8 mg/g• G.aggregatum 4.002 mg/g• G.mosseae 1.5mg/g• G.fasciculatum 1.4mg/g
• E.hirta-Quercitine• Control 0.33mg/g• G.mosseae 0.98mg/g• G.fasciculatum 0.80mg/g• G.bireticulata 0.59mg/g
L.aspera –Leucosterol & Nicotine
Showed non-significance results
CONCLUSION
Present analysis clearly indicates that the selected wildcraft herb considered as weeds have the potential to accumulate high levels of Primary, Mineral nutrients with AMF inoculants.
Mycorrhizal fungi are able to enhance the absorption of nutrients from the soil by mass flow through roots and diffused slowly thus, increasing in biomass as well as Princeple biochemical compontnts
OUT LOOK ON NEW FIELDS FOR STANDARDISATION
• Cultivating such wild herbs can fetch pocket money to the poor farmers, as well provide nutrient dietary food rather a medicine.
• Isolating and standardizing the principle bioactive compounds from such plants are remarkable accounts for the Pharmaceutical industries.