Indian Journal of Biochemistry & Biophysics
Vol. 44, February 2007, pp. 56-60
NOTE
Extraction of bioactive principles from
Mucuna pruriens seeds
Laxminarain Misra* and Hildebert Wagner$
*Central Institute of Medicinal and Aromatic Plants,
Lucknow 226015, India
$
Department of Pharmacy, Center for Pharma Research,
Butenandtstr. 5-7, University of Munich,
81377 Munich, Germany
Received 5 May 2006; revised 22 December 2006
Mucuna pruriens (L.) DC. Syn. M. prurita Hook. (Papiliona-
ceae) is used in male impotency, as aphrodisiac, in sexual debility,
and as nervine tonic. It also possesses anti-parkinson property,
possibly due to the presence of L-DOPA. In the present study,
attempts were made to develop the suitable method(s) for extrac-
tion of L-DOPA/other active components from the seeds using
different solvents. The various extracts were also screened for
their neuroprotective and antioxidant activities. In addition, TLC
and HPLC fingerprinting of the extracts for amino acid compo-
nents were also developed for preliminary and sophisticated
analysis. The L-DOPA could be obtained in good yield on extrac-
tion with EtOH-H2O mixture (1:1) using ascorbic acid as protec-
tor. Interestingly, n-propanol extract, which contained negligible
amount of L-DOPA, had shown significant neuroprotective activ-
ity, suggesting that some components, other than L-DOPA, might
also be responsible for anti-Parkinson property of seeds. The ex-
tract (MW-0100) containing mainly amino acids and water-
ethanol extract (1:1) (MWEL-1299) showed promising antioxi-
dant activity (EC50 = 2.5 µg) against DPPH radicals. MWEL-1299
also exhibited encouraging results against 1-methyl-4-
phenylpyridinium ion (MPP+) toxicity. The TLC fingerprinting
may be used to authenticate the plant material in herbal industry.
Keywords: Mucuna pruriens (L.) DC. Syn. M. prurita Hook.,
Papilionaceae, L-DOPA, Extraction procedures, Anti-
oxidant activity, Neuroprotective activity, Anti-
Parkinson Activity.
Mucuna pruriens (L.) DC. syn. M. prurita Hook.
(Papilionaceae) is an annual twining herb, found in
bushes and hedges at damp places, ravines and scrub
jungles throughout the plains of India. It is cultivated
for its pods as vegetable and young leaves as fodder.
Seeds are consumed as meal after removing anti-
nutritional factors1. The pods are anthelmintic and
________
*Corresponding author
E mail: laxmisra@hotmail.com
Tel: +91-0522-2717529; Fax: +91-0522-2342666
seeds exhibit anti-inflammatory and aphrodisiac prop-
erty2. In India, seeds are used as a tonic and for male
vitality in traditional medicine. They have also shown
anti-Parkinson property, probably due to the presence
of L-DOPA (a precursor of neurotransmitter
dopamine). The content of dopamine in the brain
tissue is reduced due to the blockade of conversion of
tyrosine to L-DOPA. It is resumed, when L-DOPA is
given externally, resulting in relief in the Parkinson’s
disease3.
The bioassay based extraction and fractionation of
M. pruriens seeds for neuroprotective and antioxidant
activities has not been reported, so far. In the present
study, attempts have been made to develop the
suitable method(s) using different solvents for
extraction of L-DOPA/other active components from
the seeds. The extracts have also been tested for their
neuroprotective and antioxidant activities. TLC and
HPLC fingerprinting of the seed extracts have also
been developed for preliminary and sophisticated
analysis.
Materials and Methods
The seeds of Mucuna pruriens were purchased
from the local market of Lucknow, India and
identified and preserved as described4.
Extraction of L-DOPA and polar components
(i) With water under SO2 protection (MW-1299)
The dried milled seeds (100 g) were defatted with
acetone (300 ml) by shaking for 48 h at room tem-
perature (RT) and defatted material was extracted
with water (3 × 500 ml) by shaking overnight. The
residual material was removed by filtration and
filtrates were pooled and concentrated. SO2 was
passed through the concentrate (2 bubbles/s for
10 min) until the saturation and decolourized by shak-
ing with activated charcoal. Crude L-DOPA was crys-
tallized at 5°C and recrystallized in water at 0°C
(yield 0.98%) and confirmed by comparison with au-
thentic sample and spectral data4,5.
(ii) With water-ethanol (1:1) under ascorbic acid protection
(MWEL-1299)
The dried, milled seeds (100 g) were defatted with
acetone (300 ml) by shaking for 48 h at RT and
defatted material was extracted with water-ethanol
 NOTES
(1:1) with 0.1% ascorbic acid (3 × 500 ml) by shaking
overnight. The residue was removed by filtration and
filtrates were pooled and concentrated. The concen-
trate after crystallization yielded crude L-DOPA,
which on further recrystallization in hot water gave
pure crystals (yield 1.78%).
Extraction with chloroform in basic medium for maximum
extraction of non-polar nitrogenous substances (MCH-1299)
The dried milled seeds (100 g) were defatted with
acetone (300 ml) by shaking for 48 h at RT and
defatted material was extracted with chloroform in
1.7% 15 N ammonia (3 × 300 ml) by shaking over-
night. The residual material was removed by filtration
and filtrates were pooled and concentrated. The
concentrate was washed with water several times to
bring down the pH to 7.0. After removal of solvent,
4.0 g crude extract was obtained.
Extraction of lesser polar constituents (with limited concen-
tration of L-DOPA)
The dried milled seeds (20 g each) were extracted
with the following solvents (4 × 100 ml) by shaking
overnight and concentrated to obtain crude extracts:
(i) n-butanol (MBL-0100), (ii) n-propanol
(MPL-0100), (iii) ethanol (MEL-0100) and (iv)
methanol (MML-0100). The non-polar constituents
were earlier identified by us6.
Extraction of alkaloids (MPL-0100ALK)
The dried milled seeds (500 g) were defatted with
acetone (1.25 L) by shaking for 48 h at RT. The
defatted material was extracted with n-propanol
(3 × 1.25 L) by shaking overnight to obtain 8.84 g
extract as described4,7.
Extraction of amino acids (MW-0100)
As amino acids constituted the main active compo-
nents of seeds, attempts were made to develop an
efficient procedure for their extraction. The dried
milled seeds (20 g) were defatted with acetone
(50 ml) by shaking for 48 h at RT and defatted
material was extracted with water (3 × 100 ml) by
shaking overnight. The residual material was removed
by filtration and filtrates were pooled and concen-
trated. The extract was treated with activated charcoal
to remove the polymerized L-DOPA and obtain
mainly amino acids (2.5 g) containing little L-DOPA.
o-Phthaldialdehyde (OPA) preparation for HPLC
The extract MWEL-1299 (5 mg) because of its
value in the herbal industry was chosen for further
study. It was dissolved in water (25 µl) and methanol
57
(250 µl) and to it OPA (250 µl) was added, and used
as ready solution for HPLC analysis. Similarly, OPA
solutions of about 3-dozen reference samples (Merck)
of various amino acids (0.5 mg was dissolved in
400 µl water and 100 µl OPA was added) were pre-
pared for the HPLC analysis. The HPLC was done in
HP 1090A instrument with Lichrospher RP18, 5 µm
(Merck) as stationary phase and the following mobile
phases: A: 0.05 M sodium acetate-methanol-THF
(180:19:1); and B: methanol with gradient as: 15%
(4 min), 20% (11 min), 30% (10 min), 50% (7 min),
86% (1 min) at 40°C with a flow rate 1.0 ml/ min;
injection volume 20 µl for extracts and 5 µl for
references; UV detection at 337 nm.
TLC fingerprinting of extract/isolates of MWEL-1299
TLC plates were run in n-butanol-n-propanol-
water-acetic acid (3:3:2:1) and spots were visualized
by ninhydrin reagent (300 mg dissolved in 100 ml
n-butanol and 3 ml acetic acid). About 3-dozen refer-
ence samples (Merck) of various amino acids were
run as co-TLC with the most appropriate extract
(MWEL-1299) to ascertain the presence of the fol-
lowing amino acids (Rf in parenthesis): L-histidine
(0.01), L-lysine (0.02), L-cystine (tentative) (0.12),
glutathione (0.12), L-serine (0.14), glycine (0.16),
L-glutamine (0.17), L-threonine (0.18), L-alanine
(0.20), β-alanine (0.22), 5-methoxy-N,N-dimethyl-
tryptamine (0.26), L-proline (0.26), L-methionine
(0.28), 4-amino butyric acid (0.29), L-DOPA (0.32),
L-tyrosine (0.48), 5-methoxytryptamine (0.50), hy-
droxytryptamine (0.59) and tryptamine (0.60).
Antioxidant and neuroprotective activity tests
Antioxidant activity of the extracts was tested by
scavenging of DPPH radicals8. For testing of neuro-
protective activity, effect of extracts on growth and
survival of dopamine (DA) neurons in culture was
determined9. The enhancement of growth after 1 week
of treatment with varied concentrations of each ex-
tract (0.00, 0.05, 0.50, 5.0, and 50.0 µg/m) was meas-
ured by tritiating DA uptake Promising extracts were
also investigated for DA uptake after 1-methyl-4-
phenylpyridinium ion (MPP+) toxicity.
Results and Discussion
Extraction of L-DOPA
The yield of crude extracts obtained with different
solvents is given in Table 1. Extraction with ethanol-
water (1:1) under ascorbic acid protection gave the
58 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, FEBRUARY 2007

higher yield of L-DOPA (1.78%), as compared to ex- Extraction of alkaloidal components

traction with water using SO2 as protective agent Among various extracts, n-propanol contained the
against oxidation (L-DOPA 0.98%). Extraction of maximum concentration of isoquinoline alkaloids4
seeds with chloroform in basic medium afforded and was almost devoid of amino acids and their
crude extract in 4% yield, whereas different alcohols derivatives. Ethanol extract contained the lesser
yielded crude extracts ranging from 5.0-9.7% concentration of these alkaloids. Earlier, we isolated
(the lowest and highest yield was obtained with four tetrahydro-isoquinolines and identified by NMR
n-propanol and methanol respectively). TLC indicated spectra from MWEL-1299 extract of M. pruriens
the presence of negligible amount of L-DOPA in seeds4,7.
n-propanol extract, while n-butanol, ethanol and
methanol extracts showed brighter spots of L-DOPA Extraction and analysis of amino acids
and other amino acids (Table 1). Extraction of seeds with water (MW-1299) gave
extract (30.5%) and after defatting with acetone and
Among amino acids, L-DOPA was the major com- extraction with ethanol (MEL-W-0100) afforded
ponent (~50%), even after its removal by crystalliza- sticky solid (26%) containing mainly amino acids and
tion from the extract (MWEL-1299). Although most polysaccharides The extraction with water-ethanol
of the extracts contained similar classes of com- (1:1) yielded 22.5% non-sticky solid matter (Table 2).
pounds, MWEL-1299 is preferred by the industry, as Although amino acids were analyzed earlier by
the extraction method is simple, extract is non-sticky HPLC10, but for quick analysis from the extract,
and contains mainly desirable secondary metabolites.
Table 2—Analysis of amino acids of MWEL-1299 extract
As MW-1299 and MEL-W-0100 showed the presence of M. pruriens seeds by HPLC
of similar constituents, further defatting the acetone
defatted material with ethanol was not desirable. Amino acids Retention time Content (%)
Ascorbic acid (added)* 1.0 -
Glutathione 1.2 3.33
Table 1—Results of extraction for polar and lesser polar u.i. 1.5 6.66
constituents from M. pruriens seeds (defatted with acetone) Glutamic acid 2.1 5.99
u.i. 2.5 1.00
Extraction for polar constituents u.i. 3.0 3.33
Extract Solvent Crude extract TLC identification by u.i. 3.9 0.83
no. yield (%) reference compounds L-Serine 5.0 0.33
u.i. 5.8 0.17
MW- H2O (+SO2) 30.5%, L-DOPA, amino acids, L-Histidine 6.2 0.27
1299 Sticky solid polysaccharides, etc. u.i. 7.5 0.17
L-Threonine 8.2 1.50
*MEL- H2O (+SO2) 26.0%, L-DOPA, amino acids, u.i. 9.0 0.10
W-0100 Sticky solid polysaccharides, etc. L-DOPA** 10.0 48.91
MWEL H2O-EtOH, 22.5%, solid L-DOPA, amino acids, β-Alanine/taurine/L-arginine 10.5 18.60
-1299 1:1 (+ascorbic alkaloids, lipids etc. L-Alanine 12.5 1.00
acid) 4-Aminobutyric acid 13.5 2.00
L-Tyrosine 14.4 0.67
Extraction with alcohols for lesser polar constituents u.i. 20.0 1.00
MBL- n-Butanol 7.6 Amino acids, L-DOPA Methionine 22.1 0.17
0100 (minor) L-Valinine 22.8 0.67
L-Phenylalanine 24.2 0.67
MPL- n-Propanol 5.0 Isoquinoline alkaloids, u.i. 25.8 0.17
0100 L-isoleucine Isoleucine 26.3 0.50
L-Leucine 27.1 0.33
MEL- Ethanol 6.7 Amino acids, L-
u.i. 29.1 0.10
0100 DOPA, Isoquinoline
L-Lysine 29.6 1.33
alkaloids
5-Methyltryptamine 30.1 0.10
MML- Methanol 9.7 Amino acids, L-DOPA u.i 32.0 0.10
0100 (major)
*Ascorbic acid was added during the extraction to protect from
*After defatting by acetone, the residual material was double- polymerization of L-DOPA in aqueous medium. **L-DOPA
defatted by ethanol for extraction by water to obtain mainly the (1.78%) was crystallized from the extract before HPLC. u.i. =
most polar constituents Unidentified
 NOTES 59

simpler HPLC and TLC methods were developed. (Table 2). The TLC was also useful for quick moni-
The HPLC (MWEL-1299) indicated the presence of toring of the authenticity of M. pruriens, as it tenta-
several amino acids, of which 19 could be identified. tively gave the amino acids composition in the extract
However, L-DOPA was the major amino acid (Table 2). TLC analysis using reference compounds
(>50%), even after crystallizing out the maximum also confirmed the presence of all these amino acids.
amount from the extract before HPLC measurement.
Other major amino acids identified were Antioxidant and neuroprotective activity
β-alanine/taurine/L-arginine (18.6%) present in com- The extracts MW-0100 and MWEL1299 showed
bination or individually as they had the same retention promising antioxidant activity (EC50 = 2.5 µg),
time, glutamic acid (5.99%) and glutathione (3.33%) whereas MEL-0100, MPL-0100 and MCH-1299
exhibited insignificant (EC50 = 72, 90 and 300 µg,
Table 3—Effect of M. pruriens extracts on growth and respectively), indicating that the activity had some
survival of dopamine (DA) neurons in culture relation with polar components of the seeds. The
extract MPL-0100 (n-propanol extract) gave highest
Extract no. Concentration DA uptake
(% control)
response in neuroprotective testing on the growth and
(µg/ml)
survival of DA neurons in culture, followed by
*MHX-1299 0.05 123 MEL-0100 (ethanol extract) and MBL-0100
0.50 105 (n-butanol extract). These extracts had dopamine
5.00 92 uptake of 205, 185, and 139% respectively (Table 3).
50.00 100
Other extracts did not yield any promising results
*MAC-1299 0.05 115 These findings suggested that neuroprotective activity
0.50 95 might be attributed to the lesser polar constituents.
5.00 92
50.00 102 Protective effect against MPP+ toxicity
*MBL-0100 0.05 139 The protective effect of some of the extracts
0.50 107 against MPP+ toxicity using dopamine uptake after
5.00 88 MPP+ treatment is given in Table 4. MWEL-1299
50.00 125
(water-ethanol extract, 1:1, concentration 50 µg/ml)
*MPL-0100 0.05 205 showed encouraging results of DA uptake (22 and 14
0.50 185
5.00 170
DPM x 1000 respectively with no and 5 µM MPP+).
50.00 190
Table 4—Protective effect against MPP+ toxicity
*MEL-0100 0.05 185 of M. pruriens extract
0.50 145
5.00 130 Extract no. Concentra- DA uptake (DPM × 1000) DA uptake
50.00 140 tion No MPP+ 5 µM MPP+ (% of no
*MML-0100 0.05 123 (µg/ml) MPP+)
0.50 119
5.00 115 MWEL-1299 0.0 20.0 5.0 25
50.00 105 0.5 20.5 6.5 31
5.0 18.0 6.0 33
*MCH-1299 0.05 128 50.0 22.0 14.0 62
0.50 118
5.00 105 MW-0100 0.0 12.0 4.0 39
50.00 102 0.5 9.5 5.0 52
5.0 10.5 5.5 50
*MWEL-1299 0.05 125 50.0 14.5 8.0 60
0.50 112
5.00 108 MCH-1299 0.0 13.0 4.0 31
50.00 100 0.5 12.0 3.5 32
5.0 15.5 4.5 29
*MW-0100 0.05 121 50.0 17.5 6.0 34
0.50 102
5.00 103 MHX-1299 0.0 17.0 4.0 26
50.00 85 0.5 16.5 4.5 28
5.0 17.5 5.0 30
*0.00 µg/ml was taken as control (100%) 50.0 19.5 5.5 29
60 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, FEBRUARY 2007
Similarly, the percent of no MPP+ DA uptake was
significant in MWEL-1299 (62%), followed by
MW0100 (60%), whereas MCH-1299 and
MHX-1299 did not give any significant activity.
These results suggested the extract with non-polar
constituents gave almost negligible effect.
In conclusion, the present study demonstrated that
L-DOPA could be extracted in good yield from
M. pruriens seeds using EtOH-H2O (1:1) using ascor-
bic acid as protector. Interestingly, n-propanol extract,
which contained negligible amount of L-DOPA had
shown significant neuroprotective activity, suggesting
that the whole extract of M. pruriens seeds could be
superior to pure L-DOPA in treatment of Parkinson-
ism. The TLC/HPLC fingerprinting of extract may be
used to authenticate the plant material in herbal prepa-
rations/formulations industry.
Acknowledgement
The authors acknowledge CMI GMbH, Munich for
a research grant and Profs C W Olanow and C
Mytilineou (Mount Sinai Medical School, New York)
and R Gebhardt (Institute of Biochemistry, University
Hospital, Leipzig, Germany) for testing the neuropro-
tective and antioxidant activities. Director, CIMAP is
acknowledged for granting leave to LM.
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