Paolo Robert P.

Bueno
New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Soybean
Glycine max (L.) Merr., Fabaceae
major source of healthy food in many Asian
countries

In China, green vegetable soybeans maodou

are vegetable-type fruits of soybean plants that
are harvested at the green stage for use as a
vegetable before they mature and dry

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Green vegetable soybeans
Rich in protein, -3 fatty acids, carbohydrates,
fiber
Micronutrients: folic acid, manganese,
vitamin K, potassium, and zinc
Isoflavones, saponins, and phenolic
compounds

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Green vegetable soybeans
Exhibited various degrees of
antioxidative and anti-
inflammatory activities
Insert journals here

to date, have not been reported to exhibit

immunosuppression activity

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Objectives
To isolate and identify compounds that has inhibitory
activities on the proliferation of concanavlin A (Con A)-
activated lymphocytes

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Research Scheme
Fresh Soy fruit (5.0 kg) Dried Soy fruit (1.2 kg)
(Whole pod of green vegetable soybeans) sun-dried
MeOH extraction under reflux (2x)
Concentrated under reduced pressure

Crude MeOH extract (225g)

Dissolution in H2O; Solvent Fractionation

Pet Ether Fraction CHCl3 Fraction EtOAc Fraction (32.5

kg)
Vacuum chromatography (60-100 mesh);
Gradient Elution (CHCl3:MeOH); each 6.0L

90:10 Eluate (380mg) 70:30 Eluate (3.2g) 60:40 Eluate (5.2g)

Medium-pressure Liquid Chromatography Vacuum chromatography Medium-pressure

Elution with 95:5, 90:10, 85:15, 80:20 Liquid
(CHCl3:MeOH); 500mL Chromatography
F1 F2 F3 F4 F5
(30mg)
F1 F2 F3 F4 F5
(7.5mg)
Reversed-Phase HPLC
Elution with MeOH:H2O (80:20)
Sephadex LH-20 gel;
Elution with pure MeOH

compd. 1 compd. 2 compd. 3 compd. 4 compd. 5

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Con A-activated Lymphocyte
Inhibition ICR mice
Sacrificed by cervical dislocation

Spleen Sample

ICR mice of similar age and weight

Placed in cold Hanks solution & torn apart;
Pass thru a mesh (200-mesh)

(2025g) Hemolyzed (1mmol/L tris-HCl and 1% NH4Cl)

Suspended to RPMI 1640 culture medium

from the Experimental Animal Center of Cultured lymphocytes

Soochow University (1 x 104 cells/well)
animal room condition: temperature
(22 2 C), light (12 h light/dark
2 set-up:
1) Seeded cultured lymphocytes

cycle), and humidity (50 10%) 2) Activated Con-A set-up

Animal study proposal was approved Spontaneous Control Cyclosporine A Treatment groups
by the Institutional Animal Care and (Lymphocyte alone) ( Positive Control: 1M) ( 0, 0.25, 0.5, 1.0 & 2.0 g/mL)
Use Committee of Soochow University
(Permit IACUC2007-13) Incubated for 24 hrs

+10L CCK8; incubated 3h

for 370C

Read @ A450nm

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids
Four known ones:
Compound 3: Ginsenine
Compound 4: (1S,3S)-1-methyl-1,2,3,4-
tetrahydro--carboline-3-carboxylic acid
Compound 5: (1R,3S)-1-methyl-1,2,3,4-
tetrahydro--carboline-3-carboxylic acid, 5,7
and
Compound 6: indole-3-carboxylic acid

yield two new alkaloids, 1 and 2

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
COMPOUND 1
Yellowish powder
M.W.: C13H10O2N2
Indicating 10 degrees of
unsaturation

COMPOUND 2
White amorphous powder
M.W.: C13H14O2N2
Indicating 8 degrees of
unsaturation
HR-ESI-MS spectrum of compounds 1 and 2
New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids
Infrared Spectra Absorption bands
3369, 1718, and 1610 cm-1 3356, 3288, and 1645 cm-1

COMPOUND
COMPOUND 1
Secondary amine N-H Stretch
2
O-H Stretch
Hydroxy
,-unsaturated lactone C=O Stretch N-H Stretch
Secondary amine
,-unsaturated imide C=N Stretch N-H Bend
Secondary amine

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

1H NMR spectra of compound 1

One NH group
Four aromatic protons

Presence of an indole skeleton of an unsubstituted moiety in the

aromatic ring
New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

1H 1H COSY spectra of compound 1

four aromatic protons located at C-6, C-7, C-8, and C-9

two methylene groups at C-2 and C-3
methine group was established at C-3a

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

13C NMR spectra of compound 1

Resonance for 13 C atoms

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

HSQC spectra of compound 1

Two CH2
Five CH
Six quaternary C atoms

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

HMBC spectra of compound 1

correlations between H 4.85, 4.33 (H-2) and C 155.2 (C-

1a) and between H 3.19, 2.75 (H-3) and C 155.2 (C-1a)
indicated that the CN double bond was established at C-1a
and C-1
,-unsaturated lactone group was established at C-3a and
C-5 on the basis of the HMBC correlation between H 4.34
(H-3a) and C 174.4 (C-5)

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

compound 1: 3,3a-dihydro-2H-pyrrolo[2,3:5,6]-
pyrano[4,3-b]indol-5(10H)-one

Named: soyalkaloid A

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

1H NMR spectra of compound 2

One doublet methyl signal

Four aromatic protons
Two hetero-substituted methine
one methylene signal

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

13C NMR spectra of compound 2

similar to those of ginsenine, except for the chemical

shift changes for C-3 (+3.0 ppm) at 172.3, C-4 (4.0
ppm) at 53.6 and for C-10a (2.0 ppm) at 130.3

suggesting that compound 2 has -OH

instead of the -OH in ginsenine

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
Isolation and Identification of Two New
Alkaloids

compound 2: 4-hydroxy-1-methyl-1,2,4,5-
tetrahydroazepino[3,4-b]indol-5-(10H)-one
Named: isoginsenine

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
 Inhibitory activities on the proliferation of
concanavlin A (Con A)-activated lymphocytes

ONLY compound 1 at concentrations from 0.5 to 2.0 mol/mL

significant decreases Con A-activated lymphocytes
proliferation of lymphocytes; however, it could dramatically reduce the proliferation of activated lymphocytes
induced by Con A

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
 Conclusion
Six alkaloid known ones:
Formerly described;
Compound 3: Ginsenine
Compound 4: (1S,3S)-1-methyl-1,2,3,4-tetrahydro--carboline-3-
carboxylic acid
Compound 5: (1R,3S)-1-methyl-1,2,3,4-tetrahydro--carboline-3-
carboxylic acid, 5,7 and
Compound 6: indole-3-carboxylic acid

Two new alkaloids

Compound 1: 1: 3,3a-dihydro-2H-pyrrolo[2,3:5,6]-
pyrano[4,3-b]indol-5(10H)-one (soyalkaloid A)
Compound 2: 4-hydroxy-1-methyl-1,2,4,5-
tetrahydroazepino[3,4-b]indol-5-(10H)-one (isoginsenine)

Soyalkaloid A could dramatically reduce the

proliferation of activated lymphocytes induced by
Con A

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes
THANK YOU!

Paolo Robert P. Bueno

New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes