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: jayashreedr2012@gmail.com

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.