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,India
e-mail: jayashreedr2012@gmail.com
Introduction
 Plant 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, antiinflammatory properties, anti-malarial activities, and also
an immuno-modulators.
Selected Medicinal Plant Species ,
Weeds – Natural Habitats.
Eclipta alba(Gutter/Pit)
Leucas aspera (Construction Site)
Euphorbia hirta(Open Field)
Achyranthes aspera(Wasteland)
Objectives:
 To
investigate effects of arbuscular
inoculums in selected medicinal plants
mycorrhizal
 To enhance the growth & yield of plant biomass
 To encourage and promote these wild crafted herbs for its
nutritional values
 Global priorities in developing new drugs from principle
biochemical components with good economic returns for
the farmers.
Materials and Methods
•
Green house test:
Sl.
No
.
AM Fungi
Control
Propogules Dry wt.
(g)
-
-
1
•
•
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).
2
Acaulospora
bireticulata
2.8 x 106
0.4
A. lacunose
2.8 x 107
0.4
A. laevis
2.8 x 105
0.4
Glomus mosseae
1.4 x 105
0.8
G. aggregatum
2.1 x 107
0.5
G. geosporum
3.5 x 105
0.3
G. fasciculatum
3.5 x 105
0.3
3
4
5
AM fungi inoculum load used
based on its propagules number
(g) for selected medicinal plants
6
7
8
Mycorrhizal Parameters
%Root Colonization
Spore Count
Physiological Parameters
Analysis 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 foilAtomic 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
Euphorbia hirta
Eclipta alba
Leucas aspera
Whole plant- AMF inocuilated Selected Medicinal Plant
(90 DAI)
Achyranthes aspera
Euphorbia hirta
Eclipta alba
Leucas aspera
Mycorrhizal Parameters
% Root Colonization and Spore Count
Number of Vesicles
Number of Arbuscules
Hyphae & Mycelium-Vesicles
Spore Count & Structures
Spore count G.aggregatum
Aculospora
Chlmydomonas Shape - Glomus
Physiological Parameters
Quantitavive Analysis
Primary and Secondary Metabolites
Carbohydrate contents
8
A.aspera
6
E.alba
4
E.hirta
2
0
L.aspera
6
A.aspera
6
E.alba
4
E.hirta
2
L.aspera
0
12
10
A.aspera
E.alba
4
E.hirta
2
L.aspera
0
8
Phenol contents
Aminoacid contents
10
8
10
Carbohydrates (%)
10
12
Amino acid (%)
Protein Contents
Phenols (%)
Proteint (%)
12
8
A.aspera
6
E.alba
4
2
0
E.hirta
L.aspera
Quantitavive Analysis
Primary and Secondary Metabolites
12
A.aspera
25
20
E.alba
15
E.hirta
10
L.aspera
5
0
Alkaloids (%)
30
Alkaloid contents
10
8
A.aspera
6
E.alba
4
E.hirta
2
L.aspera
0
Terpenoid contents
6
Terpenoids (%)
Flavonoids (%)
Flavonoid contents
35
5
A.aspera
4
3
2
1
0
E.alba
E.hirta
L.aspera
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 Sodium (ppm/g)
Estimation of Magnesium (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
•
•
•
•
•
•
•
•
•
•
L.aspera –Leucosterol & Nicotine
E.hirta-Quercitine
Control 0.33mg/g
G.mosseae 0.98mg/g
G.fasciculatum 0.80mg/g
G.bireticulata 0.59mg/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
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.