FIELD OF INVENTION:
The present invention relates to method development for the production of HPLC grade 40% Ldopa
from the seeds of Mucuna pruriens by using non-hazardous solvent system. The present
invention relates to method for extraction of Mucuna pruriens having 40% natural L-Dopa
content for the treatment of neurological and physiological disorders.
DESCRIPTION OF THE RELATED ART:
The methodology adopted in this study was to search through various Patent databases using
keywords and international patent classifications. Technical publications were obtained by
sea'rching through various technical literature sources using different keywords. Relevant
patent and technical documents were then short listed.
Reference may be made to European Patent Application Number: 20030809721, titled, 'Mucuna
pruriens and extracts thereof for the treatment of neurological diseases', Publication Date:
04/23/2008, Filing Date: 10/02/2003, where Mucuna pruriens seeds or seed powder is
extracted. The methodology is as follows:
1. Hexane extract: 200 gm pulverized seed material of Mucuna pruriens were shaken at room
temperature in 200 ml n-hexane for 18 hrs. After filtration, the material was further washed
with 100 ml n-hexane and filtered. The filtrates were collected and solvent distilled off to obtain
a yellowish oily liquid (5.5 g) in a yield of 2.75% (wfw). The 50 mg extract gave clear solution in 1
ml DMSO (5% v/v).
2. Acetone extract: The residue of the n-hexane extraction was shaken for 18 hrs. at room
temperature in acetone (200 ml) and filtered. The residue was extracted once more with 200 ml
acetone by further shaking for 18 hrs. The residue was washed with acetone (100 ml) and
filtered. After pooling, the filtrates were evaporated by distillation under reduced pressure
yielding an yellowish mass (2.02 g) to a yield of 1.0% (wfw).
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3. Water-Ethanol (1:l) extract: The residue (ca. 96 g) was shaken for 18 hrs. at room
temperature in 500 ml of a mixture of water, ethanol, 1:l with 0.5% ascorbic acid. The solvent
was filtered and concentrated under reduced pressure at a temperature of 35" C.
Above extraction procedure was repeated four times. After concentration, the filtrates were
collected and the solvent reduced to one tenth and kept at 2-4" C. for 24 hour. The crystallized
matter was filtered and filtrate was kept for another 24 hours at 24" C. The crystallized matter
was again filtered and taken together to get 1.75 g crystals in a yield of 1.75%. The filtrate was
evaporated to dryness to get a solid mass (22.51 g) with 1.78 g as crude L-DOPA obtained after
crystallization.
4. Chloroform extract: 100 g of the pulverised seeds of M. pruriens were shaken for 18 hours at
room temperature in 1.7% ammoniated chloroform (300 ml). The extract was filtered and the
extraction was repeated three times. The concentrated extract was washed with water (100 ml)
and further concentrated to afford 4.0 g extract in a yield of 4.0 (w/w).
5. Ethanol extract: 20 g of the pulverized seeds of M. pruriens were shaken for 18 hrs at room
temperature in EtOH (100 ml). After filtration, the residue was again shaken for 18 hours and
filtered. The process was repeated for a total of four extractions. The filtrates were
concentrated and pooled together to get 1.34 g extract. The extract was tested for the presence
of L-DOPA by TLC, positively.
6. Aqueous extract: The residue obtained from the ethanol extraction, as stated above, was
further shaken for 18 hrs. at room temperature in demineralised water and filtered. The
extraction was repeated for three times more. The filtrates were pooled together and water
distilled off, after passing SO2 to prevent the oxidation of L-DOPA. The extract (4.68 g) was
tested for the presence of L-DOPA.
7. Acetone extract: 10 g of the pulverised seeds of M. pruriens were shaken for 18 hours at room
temperature in acetone (50 ml). After filtration, the residue was again extracted with acetone.
The extraction was repeated three times more. The filtrates were pooled together and the
solvent distilled off. The extract (0.37g) was used for the presence of phosphatides.
DRAWBACKS OF THE PRIOR ART:
1. The technical problem underlying in the manufacturing process of extract is use of acetone,
dimethylformamide and hexane as its possible presence as residual solvent in the extract, to be
used for treating neurological diseases including Parkinson's disease, is fatal.
2. The extraction parameters are not standardized for the repeatability and reproducibility of
the process to be called as 'Standard Operating Procedure'.
3. The manufacturing process for the said extract is multi-step, resulting high cost of the
product.
4. Solvents used in the process are hazardous in nature for Operators also.
5. The process is silent about the content of the L- Dopa in the extracted produced by using the
method.
Therefore, in view of cited prior arts, the present invention provides an economical process for
production of Mucuna pruriens having 40% natural L-Dopa content.
OBJECTS OF THE INVENTION:
The main object of the present invention is to develop a process for the production of M.
pruriens seeds extract, as economic process.
Another object of the present invention is to produce an extract of the defined content of LDopa
by using the aforesaid process.
Another object of the present invention is to avoid human exposure with hazardous solvents
and /- as residual solvents.
Yet another object of this invention is to reduce the long operation span.
Still another object of the present invention is development of the commercially economic art.
SUMMARY OF THE INVENTION:
The present invention provides a commercially economical process a with repeatable,
standardized method for the production of Mucuna pruriens seeds extract containing 40% LDopa
by HPLC which avoids human exposure to hazardous solvents.
In a preferred embodiment of the present invention, the method for production of M. pruriens
seed extract having atleast 40 % L-Dopa by HPLC comprises the steps of: grinding 1.0 Kg Mucuna
pruriens (Kaunch) seeds and extracting with 4.0 liter of 45 % alcohol having water 55 % (pH 3.8-
5.5), with rotation at 45-60°C for three hours; filtering and allowing it to settle for 3 hours;
performing two more extractions each with 3.0 liter solvent of 45 % alcohol; concentrating the
extracts obtained by three extractions at 45-55" C and high vacuum to about 1.0 liter (total
volume) and allowing it to settle for 10 hours, at about 10%; filtering the whole mass; mixing
the solid mass obtained from above step with 50 ml water followed by filtering; dyring the solid
mass dried under vacuum, at 60-65"c for 7 hours; grinding and sieving the mass through 60
mesh to obtain free flowing yellow brown powder of 40 % L-Dopa.
BRIEF DESCRIPTION OF THE DRAWINGS:
It is to be noted, however, that the appended drawings illustrate only typical embodiments of
this invention and are therefore not to be considered for limiting of its scope, for the
invention may admit to other equally effective embodiments.
Fig. 1 illustrates thin layer chromatograph of Mucuna pruriens seeds (raw material) & Mucuna
pruriens extract.
Fig. 2 illustrates HPLC chromatogram I of Powder of Mucuna Pruriens extract.
Fig. 3 illustrates HPLC chromatogram Il of Powder of Mucuna Pruriens extract.
Fig. 4 illustrates HPLC chromatogram I of Mucuna Pruriens extract (Standard Sample).
Fig. 5 illustrates. HPLC chromatogram II of Mucuna Pruriens extract (Standard Sample).
Fig.6 illustrates. HPLC chromatogram Ill of Mucuna Pruriens extract (Standard Sample).
DESCRIPTION OF THE PREFERRED EMBODIMENTS:
Accordingly, the present invention provides a commercially economical process with repeatable
standardized method for the production of Mucuna pruriens seeds extract containing atleast
40% L-Dopa by HPLC using non-hazardous solvent system.
Mucuna pruriens L. (Leguminosae), commonly known as kaunch in India, is a climbing legume
endemic in India and in other parts of the tropics including Central and South America. In
Ayurvedic system of medicine, Mucuna seed preparations are in contemporary use for the
treatment of Parkinson's disease (PD), described in Ayurvedic text as Kampavata. Different
preparations from the seed of Mucuna are used for the management of ageing, rheumatoid
arthritis, diabetes, male infertility and nervous disorders. L-Dopa from M. pruriens is 2-3 times
more effective than synthetic without side effect. It is used to make dopamine, an important
brain chemical involved in mood, sexuality and movement. It is converted into dopamine after it
enters the blood stream. The blood carries the dopamine into the brain, where it naturally
increases HGH production from the pituitary gland.
For the production of M. pruriens seed extract having L-dopa NLT 40 % by HPLC, Mucuna
pruriens (Kaunch) seeds are grinded and extracted with 45% alcohol having water 55% (pH 3.9),
with rotational at 45-60°C for three hours. The solution is filtered and allowed to settle for 3
hours. Similarly, two more extractions each with 45 % alcohol + 55 % R.0 Water (pH 3.96
maintained by using Glacial acetic Acid 0.300 liter), at 45-60°c, for 3 hours are done. The extracts
are concentrated at 45-55°C on high vacuum and allowed to settle for 10 hours, at about 10°C.
The whole mass is filtered and then mixed with about 50 ml water and again filtered. The solid
mass is dried under vacuum at 60-65°C for 16 hours, grinded and sieved through 60 mesh to
obtain free flowing yellow.brown powder NLT 40 % L-Dopa. Yield obtained is approximately 2.17
%.
The invention is described in detail with reference to the examples given below. The examples
are provided just to illustrate the invention and therefore, should not be construed to limit
the scope of the invention.
Example 1:
Extraction of L-Dopa in Pilot Plant
15.0 Kg Mucuna pruriens Seeds
(Cross grinded)
1st extraction in vertical extractor with 60 liter of 45 % (27 liter) alcohol + 55 % (33 liter) R.0
Water (pH 3.89 maintained by using Glacial acetic Acid 0.400 liter), at 45-60°c, for 3 hours
Fraction transferred in storage vessel I
2nd extraction in vertical extractor with 45 liter of 45 % (20.25 liter) alcohol + 55 % (24.75 liter)
R.0 Water (pH 3.96 maintained by using Glacial acetic Acid 0.300 liter), at 45-60°c, for 3 hours
Fraction transferred in storage vessel I1
3rd extraction in vertical extractor with 45 liter of 45 % (20.25 liter) alcohol + 55 % (24.75 liter)
R.0 Water (pH 3.98 maintained by using Glacial acetic Acid 0.300 liter), at 45-60°C, for 3 hours
Fraction transferred in storage vessel Ill
The content of storage vessel I, II & Ill retransferred into vertical extractor leaving behind the
settled residue.
The contents I, II & Ill mixed by circulation (using circulation pump) in vertical extractor
Half of the content transferred from vertical extractor to storage vessel
(To have the optimum distillation)
Distillation of Mucuna pruriens extract under vacuum at 68e C, with intermittent transfer of
fraction from storage vessel, time taken for this process (31 burs)
Concentrated extract 9.5 liter to Recovered Solvent 96 Liter
separate storage vessel transferred (methanol +water)
The concentrated extract transferred from storage vessel to plastic can of 10 liter capacity each
(2 cans) 1
Contents crystallized at 100 C for 10 hours,
The whole mass filtered by using filter cloth (300 mesh) under vacuum
Solid mass again mixed with R.0 water
Filtered again by using filter cloth (300 mesh) under vacuum
Obtained wet solid mass 92.58 gm
dried under vacuum, for 16 hours at 40-45cC.
Filtrate
Grinded & passed through sieve (60 mesh) 9.0 liter filtrate stored in storage vessel
1 326 gm Mucuna extract powder obtained The 100 ML filtrate
(Part-I) concentrated on
Water bath to make thick paste
(Part-ll)
Analysis of Mucuna extracts powder by Analysis of 97.73 content
HPLC For L-Dopa - 79 .11% gm by HPLC for
L-Dopa content- 2.39 %
Table-1: shows comparative summary of the trials for extracting L-Dopa
Trial - I with 5 kg Mucuna seed.
Trial - II with 37.5 kg Mucuna seed
Trial - Ill with 15 kg Mucuna seed
S. No Parameter
1. Extraction solvent:
1st Extract (3 hrs)
2nd & 3rd Extract
(each 3 hrs)
pH 3.9
2. Extraction temp
3. Settling duration
4. Distilation temp
5. Conc. volume
6. Ppt. duration
Trial-ll Trial-Ill
45% alcohol with similar similar
0.66% acidic water
(Acetic acid) pH 3.8
45% alcohol with similar similar
0.59 % Acidic water
50°C
5 hours
68" C
3.6 L
10 hours
7. Ppt temp 10°C
8. Washing with water 0.4 L
50" C 50" C
5 hours 5 hours
68" C 68" C
27 L 9.5 L
10 hours 10 hours
10" C 10°C
3 L 1.2 L
-11- 0 4 AU6 2014
9. Drying temp 45" C 45°C 45°C
10. Drying duration 16 hours 16 hours 16 hours
11. Yield
12. Assay HPLC
Table 2: shows solvent selection for 40% L-Dopa extract preparation
S. No Solvent Observation Remarks
1. Water at room temp. Traces of L-Dopa Not useful
2. Water at 40°C and 65°C Traces of L-Dopa Conc. increased as
compared to s.no.1
3. Pure alcohol at room temp. Traces of L-Dopa Not useful
4. 20% alcohol with water Seeds powder could Not useful
not exhaust even
after 6th extraction.
5. 20% alcohol with acidic
water (1% glacial acetic
acid at 40°c)
6. 45% alcohol with acidic
water (0.035% HCL (v/v))
at 40°c
7. 45% alcohol with acidic
water 2% (glacial acetic
After 8 consecutive Improvements
extraction exhaust required
for L-Dopa
Drug exhaust for Final product dark
L-Dopa after brown, improvement
3 extractions requires
Traces of L-dopa Standardization
after 3 extraction requires, final product
0 4 AUG 2014
acid at 45"~) as yellow brown.
8. 45% alcohol with acidic Traces of L-dopa Process has
water (1.21% glacial acetic after 3 extraction repeatability
acid at 50°c) and reproducibility
Example 2:
Thin Layer chromatography of raw Mucuna pruriens seeds and Mucuna pruriens extract
containing L-dopa content.
Test solution-Raw Material: 10 mesh powder of 1.0 gm, Mucuna pruriens seeds were extracted
with 20 ml alcohol at ambient temperature on water bath for 15 minutes. The extract was
cooled and filtered and concentrated to 10 ml.
Test solution-Extract: 60 mesh powder of 0.4 gm Mucuna pruriens seed extract was extracted
with 20 ml alcohol at ambient temperature on water bath for 15 minutes. The extract was
cooled and filtered and concentrate to 10 ml.
Solvent system:
N-Butanol: Acetic acid: Water (4:l:l)
Procedure:
10 pI each of raw material test solution and extract test solution was applied on precoated silica
gel 60 F254 TLC plate (E. Merck) of uniform thickness (0.2 mm). The plate in the solvent system
was developed to a distance of 6.2 cm.
Observations:
In visible light one spot showed at Rf 0.38. Under (UV) four spots at Rf 0.22,0.33,0.38 & 0.61 (all
blue). On exposure to iodine vapor five spot appeared at Rf 0.22 (yellow), 0.27 (yellow), 0.38
(brown), 0.53 (light brown) and 0.6l(light yellow). On spraying 10 % sulphuric acid and heating
the plate at 105"c for ten minutes seven spot appeared at Rf 0.12 (pink), 0.20 (pink), 0.29 (pink),
0.35 (pink), 0.38 (pink), 0.56 (yellow) and 0.61 (yellowish).
The observations showed that the Rf value of sample of raw material complied with the sample
of Mucuna pruriens extract.
Table 3: shows standardized extract NLT 40 % L-Dopa from Mucuna pruriens (Kaunch) seeds
S.No. Parameter Limits
1. Assay, L-Dopa content (By HPLC) NLT 40 % w/w
2. Description
3. Extractive value- In water
4. pH (1 % w/v aqueous solution)
Free flowing powder
NLT 80 %W/W
5.5-7.0
5. Loss on drying (at 1050 c) NMT 8 %W/W
6. Ash content
7. Acid insoluble ash
NMT 10 % w/w
NMT 1.0 % w/w
8. Aflatoxins As per IP
9. Water content (By K.F. Method) NMT 5 % w/w
10. Sieve analysis - 20 to 60 mesh As per customer
Specification (Physical
11. Heavy metals
12. Total microbial count
property)
As per IP (NMT 20 ppm)
NMT 1x105 cfu/ gm
13. Yeast & Mould count NMT 100 cfu/gm 0 4 AUF 2014
Table 4: shows the stability of Mucuno pruriens extract during storage
TEST Initial After After After After
Month 1 Month 2 Month 3 Month 4 month
Physical OK OK OK OK OK
Appearance
Texture OK OK
L-Dopa 45.85%
Content
Color
Odour
Brown Not altered Not altered Not altered Not
altered
Characteristic Not altered Not altered Not altered Not
altered
6.09 6.13
4.0% 4.0%
pH (~%w/v) 6.04 . 6.10
Loss on 3.8 % 3.8%
Drying (at 105°C)
Ash Content 2.48 % 2.51 %
Acid 0.019% 0.021%
Insoluble Ash
Water soluble 52.61 % 52.81%
Extractive value
Microbial 9600cfu/ 9614cfu/ 9622cfu/ 9626cfu/ 9632cfu/
Count gm gm gm gm gm
Numerous modifications and adaptations of the system of the present invention will be
apparent to those skilled in the art, and thus it is intended by the appended claims to cover all
such modifications and adaptations which fall within the true spirit and scope of this
invention.

WE CLAIM:
1. A method for the production of Mucuna pruriens seeds extracts containing not less than
40% L-Dopa by HPLC comprising the steps of:
a. grinding 5.0 -15 Kg Mucuna pruriens (Kaunch) seeds and extracting with 45 %
alcohol having water 55 % (pH 3.8-5.5), with rotation at 45-60°C for three hours;
b. filtering and allowing the above solution to settle for 3 hours;
c. performing two more extractions each with 45 % alcohol;
d. concentrating the extracts obtained by three extractions at 45-55" C and high
vacuum to about 1.0 liter (total volume) and allowing it to settle for 10 hours, at
about 10K;
e. filtering the whole mass; mixing the solid mass obtained from above step with
50 ml water followed by filtering;
f. dyring the solid mass under vacuum, at 60-65"c for 7 hours;
g. grinding and sieving the mass through 60 mesh to obtain free flowing yellow
brown powder of 40 % L-Dopa.
2. The method for the production of Mucuna pruriens seeds extracts containing not less
than 40% L-Dopa by HPLC as claimed in claim 1, wherein second and third extraction is
performed with 45% alcohol with acidic water (1.21% glacial acetic acid at 50°c).
3. The method for the production of Mucuna pruriens seeds extracts containing not less
than 40% L-Dopa by HPLC as claimed in claim 1, wherein the extract is stable upto four
months.