PILOT PRODUCTION OF 5-HTP FROM THE SEEDS OF GRIFFONIA SIMPLICIFOLIA

Abstract

The seeds of Griffonia simplicifolia contain substantial quantities of 5-Hydroxytryptophan (5-HTP). The effect of factors such as particle size, temperature and volume of solvent were quantified. Using two-way ANOVA, the percentage contribution to the total variation by the individual factors observed were: volume, 69.26%; temperature, 15.55% and particle size, 11.94%. The total contribution from the three factors was 96.75%. The remaining 3.25% can be attributed to interaction of the above factors. This confirms that the combined effect of these factors is synergistic. The best solvent system for the recrystallization of the crude extract was water/ethanol 20:80. Recrystallized 5-HTP which was obtained from seeds of Griffonia was assayed monthly over three months. Conditions employed were refrigerator (approx. 5°C), room temperature (approx. 30°C), 40°C and 60°C. The amount of 5-HTP present in the seeds was determined by High Performance Liquid Chromatography, using the HPLC method earlier developed for the direct assay of 5-HTP from the seeds of Griffonia 1. The purified 5-HTP crystals were subjected to stability studies, assuming first order kinetics. The rate

Full text

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PILOT PRODUCTION OF 5-HTP FROM THE SEEDS OF GRIFFONIA
SIMPLICIFOLIA
Addotey J. N. A.* and Adosraku R. K.
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical
Sciences, College of Health Sciences, KNUST, Ghana.
ABSTRACT
The seeds of Griffonia simplicifolia contain substantial quantities of 5-
Hydroxytryptophan (5-HTP). The effect of factors such as particle
size, temperature and volume of solvent were quantified. Using two-
way ANOVA, the percentage contribution to the total variation by the
individual factors observed were: volume, 69.26%; temperature,
15.55% and particle size, 11.94%. The total contribution from the three
factors was 96.75%. The remaining 3.25% can be attributed to
interaction of the above factors. This confirms that the combined effect
of these factors is synergistic. The best solvent system for the
recrystallization of the crude extract was water/ethanol 20:80.
Recrystallized 5-HTP which was obtained from seeds of Griffonia was
assayed monthly over three months. Conditions employed were
refrigerator (approx. 5°C), room temperature (approx. 30°C), 40°C and
60°C. The amount of 5-HTP present in the seeds was determined by
High Performance Liquid Chromatography, using the HPLC method
earlier developed for the direct assay of 5-HTP from the seeds of Griffonia1. The purified 5-
HTP crystals were subjected to stability studies, assuming first order kinetics. The rate
constant for the degradation was 1.059 E-03 day-1 at refrigerator temperature (approx. 5°C) as
compared to 1.195 E-03 day-1 for that kept at room temperature. The t90 values for
refrigerator temperature and room temperature were 99.62 days and 88.28 days respectively.
The difference in shelf life was thus 11 days. This is considered economically not significant
considering the cost of refrigeration.
KEYWORDS: Griffonia simplicifolia, recrystallization, stability studies, pilot production.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.041
Volume 5, Issue 6, 204-221 Research Article ISSN 2278 – 4357
*Corresponding Author
Addotey J. N. A.
Department of
Pharmaceutical
Chemistry, Faculty of
Pharmacy and
Pharmaceutical Sciences,
College of Health
Sciences, KNUST, Ghana.
Sciences, College of
Health Sciences, KNUST,
Ghana.
Article Received on
11 April 2016,
Revised on 01 May 2016,
Accepted on 21 May 2016
DOI: 10.20959/wjpps20166-6982

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1.0 INTRODUCTION
In recent years, there has been a renewed interest in obtaining biologically active compounds
from natural sources. Contributing to this worldwide attention towards formulations based on
natural products are their low toxicity, their complete biodegradability, their availability from
renewable sources and in most cases, their low-cost compared with those of compounds
obtained by total chemical synthesis. Owing to this renewed attention to pharmaceuticals,
agrochemicals and nutraceuticals (functional foods) obtained from natural sources, the study
of bioactive secondary metabolites, traditionally carried out mainly by chemists, has
increasingly attracted the attention of pharmacologists, biologists, botanists, agronomists, etc.
stimulating co-operative work.
Griffonia simplicifolia is a stout, woody, climbing shrub growing to about 3m with greenish
flowers and inflated black pods. The seeds contain substantial quantities of the amino acid, 5-
hydroxytryptophan (5-HTP). Today, the seed of this climbing shrub is used in the treatment
of fibromyalgia, headaches, insomnia, depression and stomach problems.
Griffonia seeds continue to be exported from Ghana. Therefore it was considered prudent to
develop a simple method of production of 5-HTP from the seeds of Griffonia in reasonable
purity prior to export. This will add value to the export. Stability studies carried out on the
seeds showed that 5-HTP content decreased at rates of 4.62% and 6.77% at room and
refrigerator temperatures respectively.[2] In order to reap economic benefits, 5-HTP of
reasonable purity will have to be extracted from the seeds. There is however no information
on the stability of 5-HTP which had been extracted from the seeds. The kinetics of the
degradation of extracted 5-HTP were thus studied in order to predict most stable conditions
of storage prior to export.
1.1 OBJECTIVES
 To investigate the factors affecting the extraction of 5-HTP from the seeds of Griffonia
simplicifolia using various solvents. (I.e. effect of factors such as temperature, time and
volume of solvent on amount of 5-HTP extracted)
 To determine the most effective recrystallization conditions for the purification of the
extracted 5-HTP.
 To carry out stability studies on the purified 5-HTP
 To estimate the shelf life of the 5-HTP powder obtained.

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2.0 MATERIALS AND METHODS
2.1 Materials
Thermostatically controlled Gallenkamp, Ovens, Buchi rotary evaporator, Buchi recirculator
chiller, Buchi water bath, Stuart Scientific Flask shaker, Ceramic mortar and pestle
Analytical Balance (Adams Instrument), HPLC Apparatus- LC-10AT Shimadzu pump with
programmable absorbance detector (783A Applied Biosystems), Integrator- shimadzu CR
501 chromatopac, Column -phenomenex hypersil 5micron C18 250 × 4.60 mm, Blender,
Refrigerator Melting point apparatus.
2.2 Reagents
Methanol, Distilled water, Ethanol (96%).
2.3 Extraction of 5-HTP prior to assay
Approximately 1.0000g of Griffonia seeds was accurately weighed. This was transferred into
a porcelain mortar and triturated with a 1:1 mixture of water and methanol (solvent) into a
smooth paste. The paste was quantitatively transferred into a 100 ml volumetric flask using
solvent and additional solvent was added to the mark. The mixture was shaken with a shaker
for 15 min. The mixture was filtered under pressure using a Buchner funnel and Whatman
No.1 filter paper discarding the first 10 ml. The filtrate was conveniently diluted and
subjected to analysis by HPLC. An appropriate concentration of pure 5-HTP was used as
reference.
2.4 HPLC analysis of crude extract using pure 5-HTP
The complete chromatograph consisted of LC-10AT Shimadzu pump with programmable
absorbance detector (783A Applied Biosystems) and shimadzu CR 501 chromatopac
Integrator. Column used was phenomenex hypersil 5micron C18 250 × 4.60 mm. the mobile
phase consisted of 3% methanol in 0.05M KH2PO4 buffer at pH 4.8 eluted isocratically at
1.5ml/min. 20ul portions of a suitable concentration of pure 5-HTP as well as seed extract
were loaded and injected in turn unto the column. The eluent was monitored at 275 nm and
A.U.F. of 0.200. The peak areas were estimated from the chromatogram and used as a
measure of concentration.
2.5 Preparation of mobile phase (1L)
100 ml of 0.5M potassium dihydrogen orthophosphate was diluted to 800 ml with distilled
water; the pH was adjusted to pH 4.8 with 0.1M sodium hydroxide and the resulting solution

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diluted to 1000 ml with distilled water. 970 ml of this was added to 30 ml of methanol to
produce 1000 ml of the mobile phase.
2.6 Preparation of 0.5 M Potassium dihydrogen orthophosphate
Approximately 6.8050 g of potassium dihydrogen orthophosphate was weighed and dissolved
with distilled water to 100 ml
2.7 Determination of optimum solvent composition for recrystallization of 5-HTP
About 0.1g of the powdered substance was placed in a small test tube (75 × 11 mm) and the
solvent was added a drop at a time with continuous shaking of the test tube. After about 1 ml
of the solvent had been added, the mixture was heated to boiling. If the sample dissolved in 1
ml of cold solvent or upon gentle warming, the solvent was considered unsuitable. If the
entire solid did not dissolve, more solvent was added in 0.5 ml portions and again heated to
boiling after each addition. If 3 ml of solvent was added and the substance did not dissolve on
heating, the substance was regarded as sparingly soluble in that solvent and another solvent
was sought. If the compound dissolved (or almost completely dissolved) in the hot solvent,
the tube was cooled to determine whether crystallization occurs. If crystals separate, the
amount was noted. The process was repeated with other possible solvents, using a fresh test
tube for each experiment, until the best solvent was found; the approximate proportions of the
solute and solvent giving the most satisfactory results were recorded.
2.8 Recrystallization of crude 5-HTP
A specific quantity of crude 5-HTP was weighed and placed in a round bottomed flask of
suitable size fitted with a reflux condenser. Slightly less than the required quantity of solvent
was added together with a few pieces of porous porcelain to prevent ‘bumping’. The mixture
was heated to boiling point on a water bath, and more solvent was added down the condenser
until a clear solution, apart from insoluble impurities, was produced. The hot solution was
rapidly filtered through a fluted filter paper supported in a relatively large funnel with a short
wide stem. The funnel was warmed in an electric oven before filtration was started, and
supported in a conical flask of sufficient size to hold all the solution. The conical flask was
stood on a steam bath and the filtrate was kept boiling gently so that the warm solvent
vapours maintained the temperature of the solution undergoing filtration. This was to prevent
premature crystallization. The filtered solution was covered with a watch glass and then set
aside to cool undisturbed.

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2.9 Stability studies on recrystallized 5-HTP from Griffonia seeds
Recrystallized 5-HTP obtained from seeds of Griffonia was assayed monthly over three
months. Conditions employed were refrigerator (approx. 5°C), room temperature (approx.
30°C) and 40°C and 60°C.
2.10 Assay of recrystallized 5-HTP crystals
An amount of purified 5-HTP (0.0200 - 0.0500 g) was weighed accurately. This was
dissolved in 1:1 water-methanol to form 50.00 ml solution. 0.50 ml of this solution was
diluted to 25.00 ml. The resultant solution was analysed by HPLC using pure 5-HTP as
reference.
3.0 RESULTS
3.1 Quantification of the contribution of factors affecting extraction
Percentage yield of 5-HTP obtained per seed weight was estimated. The contribution of a
particular factor was estimated by calculating the difference between the percentage yield
obtained in the absence and presence of that factor.
Table Error! No text of specified style in document.-1 Contribution of particle size (100 ml
solvent) 30ºC
Peak area
%yield
Powdered
160.00
4.16
Triturated
244.73
6.37
Difference
2.21
Table Error! No text of specified style in document.-2 Contribution of Particle size (20 ml
solvent) 30ºC
Peak area
%yield
Powdered
51.10
1.33
Triturated
72.99
1.90
Difference
0.57
Table Error! No text of specified style in document.-3 Contribution of particle size (100 ml
solvent) 80ºC
Peak area
%yield
Powdered
250.11
6.51
Triturated
345.00
8.98
Difference
2.47

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Table Error! No text of specified style in document.-4 Contribution of Particle size (20 ml
solvent) 80ºC
Peak area
%yield
Powdered
86.82
2.26
Triturated
150.22
3.91
Difference
1.75
powdered
paste
0
2
4
6
8
10 20 ml at room temp
100 ml at room temp
20ml at 80 deg
100ml at 80 deg
particle size
% yield
Figure Error! No text of specified style in document..1 Graphs showing the effect of
particle size on yield of 5-HTP extracted
 %yield = weight of 5-HTP /weight of seed taken*100%
 20ml at room temperature refers to yield obtained from 1g of powdered seeds which was
extracted with 20ml of solvent at room temperature
 100ml at room temperature refers to yield obtained from 1g of powdered seeds which was
extracted with 100ml of solvent at room temperature
 20ml at 80 deg refers to yield obtained from 1g of powdered seeds which was extracted
with 20ml of solvent at 80°C
 100ml at 80 deg refers to yield obtained from 1g of powdered seeds which was extracted
with 100ml of solvent at 80°C
Table Error! No text of specified style in document.-5 Contribution of Temperature (for
powdered seeds) 100 ml
Temperature/ºC
Peak area
%yield
Room temp (30)
160.00
4.16
80
250.11
6.51
Difference
1.35

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Table Error! No text of specified style in document.-6 Contribution of Temperature (for
triturated seeds) 100 ml
Temperature/ºC
Peak area
%yield
30
244.73
6.37
80
345.00
8.98
Difference
2.61
Table Error! No text of specified style in document.-7 Contribution of Temperature (for
powdered seeds) 20 ml
Temperature/ºC
Peak area
%yield
30
51.10
1.33
80
86.82
2.26
Difference
0.93
Table Error! No text of specified style in document.-8 Contribution of Temperature (for
triturated seeds) 20ml
Temperature/ºC
Peak area
%yield
30
72.99
1.90
80
150.22
3.91
Difference
2.01
ROOM TEMP
80 DEG
0
2
4
6
8
10 Powdered at 20ml
Powdered at 100ml
Paste at 20ml
Paste at 100ml
temperature
% yield
3.2 Graphs showing the effect of temperature on yield of 5-HTP extracted
 %yield = weight of 5-HTP /weight of seed taken*100%

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 Powdered at 20ml refers to yield obtained from 1g of powdered seeds which was
extracted with 20ml of solvent
 Powdered at 100ml refers to yield obtained from 1g of powdered seeds which was
extracted with 100ml of solvent
 Paste at 20ml refers to yield obtained from 1g of triturated seeds which was extracted
with 20ml of solvent
 Paste at 100ml refers to yield obtained from 1g of triturated seeds which was extracted
with 100ml of solvent
Table Error! No text of specified style in document.-9 Contribution of volume (for
powdered seeds)rt
Volume/ml
Peak area
%yield
20
51.10
1.33
100
160.00
4.16
Difference
2.83
Table Error! No text of specified style in document.-10 Contribution of volume (for
triturated seeds)rt
Volume/ml
Peak area
%yield
20
72.99
1.90
100
244.73
6.37
Difference
4.47
Table Error! No text of specified style in document.-11 Contribution of volume (for
powdered seeds) 80 ºC
Volume/ml
Peak area
%yield
20
86.82
2.26
100
250.11
6.51
Difference
4.25
Table Error! No text of specified style in document.-12 Contribution of volume (for
triturated seeds) 80 ºC
Volume/ml
Peak area
%yield
20
150.22
3.91
100
345.00
8.98
Difference
5.07

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20 ml
100 ml
0
2
4
6
8
10 Powdered at room temp
powdered at 80 deg
paste at room temp
paste at 80 deg
volume
% yield
Figure Error! No text of specified style in document..3 Graphs showing the effect of
volume of solvent on yield of 5-HTP extracted
 %yield = weight of 5-HTP /weight of seed taken*100%
 Powdered at room temp refers to yield obtained from 1g of powdered seeds which was
extracted with solvent at room temperature
 Powdered at 80 deg refers to yield obtained from 1g of powdered seeds which was
extracted with solvent at 80°C
 Paste at room temp refers to yield obtained from 1g of triturated seeds which was
extracted with solvent at room temperature
 Paste at 80 deg refers to yield obtained from 1g of triturated seeds which was extracted
with solvent at 80°C.
Source of Variation
% of total variation
P value
other conditions
85.93
0.0064
particle size
11.94
0.0263
Source of Variation
P value summary
Significant?
other conditions
**
Yes
particle size
*
Yes

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powdered
paste
0
2
4
6
8
particle size
% yield
Figure Error! No text of specified style in document..4 ANOVA results between effect of
particle size and other factors
ROOM TEMP
80 DEG
0
2
4
6
8
temperature
% yield
Figure Error! No text of specified style in document..5 ANOVA results between effect of
temperature and other factors
Source of
Variation
% of total
variation
P value
other conditions
82.89
0.0042
temperature
15.55
0.0120
Source of Variation
P value summary
Significant?
other conditions
**
Yes
temperature
*
Yes

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20 ml
100 ml
0
2
4
6
8
volume
% yield
Figure Error! No text of specified style in document..6 ANOVA results between effect of
volume and other factors
Table Error! No text of specified style in document.-13Determination of Suitable Solvent
Composition for Recrystallization Of 5-Htp
Solvent
Cold
Hot
Methanol
Soluble
very soluble
Water
Partially soluble
very soluble
Ethanol
Insoluble
Sparingly soluble
Water :ethanol 50:50
Insoluble
very soluble
Water :ethanol 25:75
Insoluble
Soluble
Water :ethanol 20:80
Insoluble
Soluble
Water :ethanol 15:85
Insoluble
Soluble
Water :ethanol 10:90
Insoluble
Sparingly soluble
Table Error! No text of specified style in document.-14 Table showing change in purity of
5-HTP during purification
Weight of seeds/g
Amount of 5-
Weight of extract
%purity of
Source of Variation
% of total variation
P value
other conditions
28.03
0.0432
volume
69.26
0.0031
Source of Variation
P value summary
Significant?
other conditions
*
Yes
volume
**
Yes

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HTP expected/g
extract
104.6240
9.3950
25.3754
45.27
100.3131
9.0081
22.2062
42.88
weight of crystals
formed after
washing and drying
% purity of
crystals
weight of crystals
formed after
recrystallisation
% purity of
crystals
8.964
78.34
6.4326
90.33
8.570
72.87
5.1520
92.54
a) b)
Figure Error! No text of specified style in document..7 HPLC Chromatogram for water-
methanol extract a) before and b) after recrystallization

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Figure Error! No text of specified style in document..1 HPLC Chromatogram for Pure 5-
HTP
Figure Error! No text of specified style in document..8 HPLC Chromatogram for 5-HTP
crystals after stability studies a) after 30days and b) after 90days
3.2 Stability studies
Table Error! No text of specified style in document.-15 results for stability test at 5ºC
Time/days
Concentration(%w/w)
log C
log C - log Co
0
90.96
1.65782
0
45
85.80
1.632457
-0.02536
90
83.14
1.61878
-0.03904
Table Error! No text of specified style in document.-16 Results for stability test at 30ºC
Time/days
Concentration(%w/w)
log C
log C - log Co
0
90.96
1.65782
0
45
84.54
1.626032
-0.03179
90
82.48
1.615319
-0.0425
Table Error! No text of specified style in document.-17 Results for stability test at 45ºC
Time/days
Concentration
log C
log C - log Co
0
90.96
1.65782
0
45
84.38
1.62521
-0.03261
90
80.96
1.607241
-0.05058
Table Error! No text of specified style in document.-18 Results for stability test at 60ºC
Time/days
Concentration
log C
log C - log Co

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0
90.96
1.65782
0
45
83.12
1.618676
-0.03914
90
80.10
1.602603
-0.05522
Table Error! No text of specified style in document.-19
TIME/days
5ºC
30ºC
40ºC
60ºC
0.
0.000000
0.000000
0.000000
0.000000
45.
-0.02536271
-0.03178775
-0.03261048
-0.03914446
90.
-0.03903997
-0.04250134
-0.0505795
-0.05521748
Graphs of logC- logCo versus time were plotted for all temperatures and these were used to
estimate the rate constants at the respective temperatures.
020 40 60 80 100
-0.08
-0.06
-0.04
-0.02
0.00 5
30
40
60
TIME/DAYS
LOG C-LOG Co
Figure Error! No text of specified style in document..9 Plot of log C- log C0 versus time
Table Error! No text of specified style in document.-20 Table for the Arrhenius Plot
SLOPE(S)
K(-2.303 × S)/day-1
log k
t/ ºC
T/K
1/T
-0.00046
0.001059
-2.97523
5
278
0.003597
-0.000519
0.001195
-2.92249
30
303
0.0033
-0.000595
0.001369
-2.86356
40
313
0.003195
-0.000665
0.001531
-2.81502
60
333
0.003003

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0.0028 0.0030 0.0032 0.0034 0.0036 0.0038
-3.2
-3.1
-3.0
-2.9
-2.8
-2.7
-2.6
1/T
log k
Figure Error! No text of specified style in document..10 Arrhenius Plot of experimental
results
3.3 Sample calculation of shelf life
At 30C, From graph of (log C – log C0) against time, slope = -k/2.303
Therefore k = -2.303  slope
k = -2.303  (-0.000519) = 1.1954  10-3 day-1
Shelf life, t90
From the relation, log (C0/C) = kt / 2.303
Where C0 = initial concentration
C = concentration at time, t
K = rate of decomposition
T = [2.303/k] log (C0/C)
T90 = [2.303/k] log (100/90)
= [2.303/1.1954  10-3]  0.0458
T = 88.24 days.
T = 2.94 months
Table Error! No text of specified style in document.-21 Shelf lives calculated at the various
temperatures
TEMPERATURE (ºC)
K (day-1)
t 90 in days (months)
5
1.059 E-03
99.62 (3.32 )
30
1.195 E-03
88.28 (2.94 )

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40
1.369 E-03
77.06 (2.57 )
60
1.531 E-03
68.91 (2.30 )
4.0 DISCUSSION
4.1Factors affecting extraction
The assay results carried out under the various conditions were analyzed statistically using
two-way ANOVA and the percentage contribution to the total variation by the individual
factors observed were: volume, 69.26%; temperature, 15.55% and particle size, 11.94%. The
total contribution from the three factors was 96.75%. The remaining 3.25% can be attributed
to interaction of the above factors. This confirms that the combined effect of these factors is
synergistic.
4.2 Determination of solvent for recrystallization
The primary property which was taken into account in the selection of solvent for
recrystallization was safety. This is because, the 5-HTP produced was to be used orally and
hence issues of toxicity had to be carefully considered. Based on availability, probable
solvents were; water, methanol, ethanol (95%) and ether. 5-HTP was found to be insoluble in
ether and since methanol toxic, the list of suitable solvents reduces to water and ethanol
(95%).
Experimentally, 5-HTP was found to be too soluble in water and insoluble in ethanol (95%).
Thus, neither of the two could be used solely as a solvent for recrystallization. It was
therefore imperative that a mixture of the two solvents be used.
The most suitable combination was found to be water/ethanol 20:80. This combination
 Had a high solvent power for 5-HTP at elevated temperature and a comparatively low
solvent power at the laboratory temperature.
 Dissolved the impurities to a very small extent. (The HPLC chromatograms for the crude
and purified extracts clearly depicts this).
 Readily yielded well-formed crystals of the purified compound.
Was easily removed from the crystals of the purified compound i.e. possess a relatively low
boiling point. (This was possible due to the larger composition of ethanol as compared to
water. Ethanol has a lower boiling point than water
4.3 Stability studies

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The straight-line curve with negative slope obtained for the plot of log C- log C0 versus time
suggests first order kinetics for the degradation. This implies that the rate of the degradation
depends essentially on the concentration of 5-HTP present at each point in time.
The shelf life (t90) of a pharmaceutical product is the length of time the product may safely be
stored on the dispensary shelf before significant decomposition occurs3. This is important
since, drugs may decompose to inactive products or toxic compounds. The t90 values for
refrigerator and room temperatures were 99.62 days and 88.28 days respectively. The
difference in shelf life was thus approximately 11 days. This may not be considered
economically significant considering the cost of refrigeration.
Projecting from the rate of degradation of 5-HTP in the seeds, the amount which would have
been lost after 3 months would be 13.86% and 20.31% after storage at room temperature and
refrigeration respectively. This is greater than the 10% loss after storage of the extracted 5-
HTP in 100 days.
5.0 CONCLUSION
Generally it was observed that an increase in the temperature and volume of solvent provided
an increase in yield of 5-HTP extracted. A reduction in particle size of the powdered seeds by
crushing the seeds, also increases the amount of 5-HTP extracted. A combination of the
effects of the above factors was found to yield the highest amount of 5-HTP.
The degradation of 5-HTP extracted from the seeds of Griffonia simplicifolia obtained in
Ghana follows first order kinetics and is temperature dependent. The 5-HTP produced if
stored under refrigerator conditions will remain within specifications for approximately 100
days. Extracting 5-HTP from the seeds of Griffonia simplicifolia reduces the degradation in
the seeds.
REFERENCES
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