VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

DETECTION AND QUANTIFICATION OF L-DOPA IN

SELECT SPECIES OF SLIME MOLDS AND PUFFBALLS

A thesis submitted to

The School of Biotechnology, International University

In partial fulfillment of the requirements for the degree of

B.S in 2019-2020

Student name: Đỗ Xuân Anh Kiệt – BTBTIU16085

Supervisor: Assoc. Prof. Trần Thị Mỹ Hạnh

9/2020
ACKNOWLEDGEMENT
I would like to express my very great appreciation to Assoc. Prof. Dr. Tran Thi My
Hanh, my thesis advisor, for her help and support. It’s thanks to her conscientious
academic support that I’ve earned much experience, knowledge, and improved
myself. I deeply esteem how she put her enthusiasm, patience, and thoughtfulness in
treating me and other students.

I would like to offer my special thanks to Prof. Stephenson (Universe of Arkansas,

Arkansas, USA) and Dr. Edward Grand (Mahidol University, Nakhon Pathom, Thailand)
for generously giving me the puffball samples for my research.

I am particularly grateful for the assistance given by the marine, central, algae lab
technicians: Ms. Chau, Ms. Lan, and Mr. Long for providing me knowledge about the
lab instruments and their kindness.

Assistance provided by my seniors: MSc. Tuyen, Mr. Quan, Ms. Quyen, Ms. Nhi was
greatly appreciated. I would also like to thank my labmates, Ms. Nguyen, Ms. Ha,
Nam, Ms. Vy, Ms.Yen for their great help, the positive psychological atmosphere they
provided, and an especial thank to Ms. Nhu who shared her experience, knowledge
and supported the chemicals for my TLC test. My grateful thanks are also extended to
my volunteer - Truc for being energetic, enthusiastic, and willing to help.

I would also like to extend my thanks to the technicians of the laboratory B16 in
VNUHCM-University of Science for being open-hearted and sharing their knowledge
about HPLC to me.

Finally, I wish to thank my parents for their support and encouragement throughout
my study.
DETECTION AND QUANTIFICATION OF L-DOPA IN
SELECT SPECIES OF SLIME MOLDS AND PUFFBALLS
Kiet X.A Do1, Hanh T.M Tran2
1
School of Biotechnology, International University, Vietnam National University in
HCMC.
2
Corresponding author’s email address: ttmhanh@hcmiu.edu.vn
 Contents

Abstract..................................................................................................................1
I. Introduction........................................................................................................2
II. Materials and methods.......................................................................................9
1. Materials:........................................................................................9
2. Method...........................................................................................10
2.1. P. polycephalum culture..........................................................10
2.2. P. polycephalum collection......................................................10
2.3. L-DOPA extraction for TLC and HPLC.......................................10
III. Results and discussions..................................................................................12
1. Detection of L-DOPA in the samples by TLC...................................12
2. High performance liquid chromatography......................................14
IV. Conclusions and recommendations..................................................................18
V. References....................................................................................................................................19
Abstract
L-DOPA is an amino acid which is clinically used for Parkinson's deasease’s
treatment.Even though synthetic L-DOPA is effective and in high demand, but some
severe side effects have been reported from patients on long term L-DOPA medication.
The objectives of this study were to detect the presence and determine L-DOPA
contents in slime molds (Physarum. polycephalum), and puffballs (Calvatia
craniiformis, and Lycoperdon pyriforme) by thin-layers chromatography (TLC) and
high-performance liquid chromatography (HPLC).

To detect the presence of L-DOPA, TLC was applied using homogenate of formic acid
2% and samples, silica gel plate 60 F254 (Merck PGaA, Germany) as stationary phase,
and the solvent mixture of isopropanol: ethyl acetate: water: glacial acetic acid
(29:19:10:1) as mobile phase. The results suggested the presence of L-DOPA in both
the slime mold and puffball samples.

HPLC-UV quantification found that the L-DOPA content in P. polycephalum plasmodia

(solid culture) was 2.07 (mg/g), the microplasmodia (liquid culture was 2.49 (mg/g)
and that of immature fruiting body of C. craniiformis was 2.22 (mg/g), mature fruiting
body was 2.28 (mg/g), and young fruiting body of L. pyriforme was 2.14 (mg/g).
Generally, the L-DOPA contents between these species are very similar to each other,
but significantly lower than that of Mucuna pruriens (a common source of plants for L-
DOPA). The future research should be conducted to optimize the biomass along with
L-DOPA contents of potential slime mold and puffball species.

1
I. Introduction
L-3,4-dihydroxyphenylalanine (L-DOPA) is an amino acid derived from the
hydroxylation of the amino acid L-tyrosine (Slominski et al., 2012). L-DOPA was first
isolated from Vicia faba by Marcus Guggenheim from 1913 and has always been the
first drug of choice in the treatment of Parkinson’s disease since discovered in the
1960s. Parkinson’s disease is a long-term degenerative disorder. It’s caused by the
brain cell’s death in a region of the midbrain called substantia nigra. This leads to the
deficiency of dopamine in this region that affects the motor system. L-DOPA, which
able to cross the blood-brain barrier whereas dopamine cannot (Hardebo et al., 1980),
is converted by decarboxylase into dopamine, a neurotransmitter that responsible for
neural communication that controls body movement and a key factor in the treatment
of the disease. For more than 50 years since the clinical use of L-DOPA was
discovered, L-DOPA has still been mentioned as the gold standard for Parkinson’s
disease treatment (Tambasco et al., 2018).

Figure 1.1. Dopa decarboxylase reaction (Raboni, 2010)

Therefore, many approaches have been made to meet the high demand for L-DOPA.
L-DOPA is currently synthesized chemically for pharmaceutical use by an asymmetric
synthesis method called the Monsanto process (Knowles et al., 1986). On the other
hand, L-Tyrosine, which is a precursor of L-DOPA is used for biotechnologically
synthesis of L-DOPA by a one-step process of oxidation catalyzed by tyrosinase
(Algieri et al., 2012). In fact, L-DOPA is an intermediate in melanin biosynthesis. Thus,
many species that have black pigment melanin are selected to research as a possible
source of L-DOPA. Conventional production of L-DOPA involves the extraction of L-
DOPA from Mucuna pruriens seeds and it is marketed as tablets under various brand
names (Ali & Haq, 2006a). Thanks to the characteristics such as, simple cultivation

2
process, low risk of contamination Mucuna pruriens bean is very common as a L-DOPA
source though the L-DOPA

Figure 1.2. Myxomycetes life cycle (Parker et al., 2016)

content is not too high and requires long duration cultivation at least 45 days. The
following sources share many characteristics such as, requiring extraction step,
contamination risk, and requiring induced factor or L-tyrosine as a precursor. Though,
they take less time in cultivation. The other plant sources mainly from Mucuna
pruriens cell suspension (Chattopadhyay et al., 1994), callus cultures of banana
(Bapat et al., 2000) and Portulaca grandiflora (Rani et al., 2007). L-DOPA is also

3
produced from Aspergillus oryzae, fungal biomass in buffer containing L-tyrosine (Ali &
Haq, 2006a) and Acremonium rutilum produced in a potato dextrose broth
(Krishnaveni et al., 2009). The yeast (Yarrowia lipolytica) biomass is used to produce
the L-DOPA in a buffer containing L-tyrosine provided with enhancing material
diatomite (Ali et al., 2007). The bacterial species reported to synthesize L-DOPA are
Escherichia intermedia cells immobilized in polyacrylamide gel using pyrocatechol
(Para & Baratti, 1988) and Erwinia herbicola using catechol as a substrate (Koyanagi
et al., 2005). It is also produced from mutant strains of actinomycetes in a broth
containing L-tyrosine (Sukumaram et al., 1979; Surwase and Javhab., 2010).

Although tissue cultures of Mucuna species have been recommended as possible

sources of L-DOPA, its commercial production has faced the problems of large-scale
cultivation (Loganathan, 1998). Under the effect of induced factor L-tyrosine, the
callus tissue was cultured and has 9.47 grams L-DOPA over 100 grams dry sample
which is the highest value in the study after 15 days of cultivation (Raghavendra,
2011). The most common source is from Mucuna seeds. Based on the research of Y.A.
Adebowale et al., 2005, L-DOPA mass extracted from seeds of 6 species of Mucuna
genus is around 3.87 to 7.12 grams over 100 grams dried sample. Among Mucuna
species, M. pruriens is the most common name in the PD treatment. Since in short-
term they have the same effect with the synthesized L-DOPA treatment, the long-term
treatment of M. pruriens still has side effect. M. pruriens seed contains polyphenols
which can bind to proteins that reduce their digestibility and inhibit hydrolytic enzyme
activity which are digestive enzymes (Lampariello et al., 2012). This leads to the
intestinal problem of the patients who use M. pruriens seed in long term.

Myxomycetes known as slime molds were confused as a part of the fungi kingdom.
They are now classified as Protista. Slime molds have the appearance of gelatinous
slime. The slime molds are single-cell organisms but can congregate together to move
as a single body to seek for a food source and produce fruiting bodies to release
spore.

Plasmodium is the vegetative stage of myxomycetes. This stage can be described as a

mass of protoplasm with varies in size and morphology in different species. The
organisms are very mobile to seek for food in the

plasmodium stage. Myxomycetes feed by sweeping over and cover all the bacteria and
organic material in their path (Tran et al., 2015). Myxomycetes grow very fast at this
stage. Especially, it takes a short time to collect sample in just around 4-7 days of

4
laboratory cultivation. Plasmodia can be cultured in solid medium and liquid medium.
In liquid medium, small plasmodia can be fragmented into microplasmodia, which can
be subcultured repeatedly to yield large quantities of microplasmodia. Both plasmodia
and microplasmodia share the same characteristics of rapid growth rate and absence
of cell wall which are suitable for cultivation and L-DOPA extraction (Daniel & Rusch,
1961; Tran et al., 2015). One group of myxomycetes that has been successfully
cultured is the order Physarum (Gray and Alexopoulos et al., 1968, Lado et al., 2007).
Physarum polycephalum belong to this group and has been used as a model organism
in many studies. It was considered as a potential source for biodiesel due to the rapid
rate of growth and produce a considerable amount of lipids (Poulos & Thompson,
1971; Tran et al., 2012). The optimization of biomass and lipid of it is also evaluated
(Tran et al., 2015).

Figure 1.3. P. Polycephalum Figure 1.4. P. Polycephalum

plasmodia microplasmodia

Stemontitis is a common genus that characterized by the tall brown sporangia.

Stemontitis herbatica belongs to this group. Loganathan, 1998 has been succeeded in
L-DOPA determination at the sporangia development process of this species. He
successfully identified the presence of L-DOPA with TLC, FTIR, and mass spectral
analysis. By the column chromatography purification, he isolated L-DOPA and
evaluated the L-DOPA quantity that is around 50 mg/1g fresh biomass. The isolated L-
DOPA went through FTIR and mass spectral analysis along with standard L-DOPA to
examine the purification of L-DOPA.

There are some unpublished articles from our group about the presence of L-DOPA in
the other species. Le, et al., 2017, has determined the amount of L-DOPA is 2.5165
mg/g dried biomass in Phy. oblonga and 2.2138 mg/g dried biomass in Ph.

5
polycephalum using HPLC. Nguyen, et al., 2019 showed evidence that incubation time,
UV light and light can affect the L-DOPA production in Ph. polycephalum by using
HPLC. The data showed that L-DOPA content in the incubation condition increased in
the first 2 days then started to decrease. The data also showed that the L-DOPA
content decreased in light and UV light exposure conditions. But for the sample
cultured in liquid medium in light exposure condition, L-DOPA content increased in the
first 4 hours then started to decrease. On the other hand, Tran et al., 2020 concluded
optimum Hematin concentration in the culture medium is 2.5 µg/ml to optimize L-
DOPA content in Ph. polycephalum.

For not being intergrated as plasmodium form, the microplasmodia can asynchronous
divide their cells in different microplasmodium to grow. That results in slime molds
being incubated as microplasmodia has the higher biomass/time than slime molds
being incubated as plasmodia (Dove and Rusch, 1980). Therefore, to optimize all of
the slime molds cultivation, the goal is to transfer them to their consistent liquid
medium.

Puffball is the name of the mushrooms that release clouds of visible spore when their
fruiting bodies burst. Puffballs were classified as the same group of taxonomy in the
past but at the present, they are separated into 3 genera Calvatia,
Calbovista, and Lycoperdon (Freedman, 1987). Stalk-visible puffballs are classified as
false puffballs. All of the false puffballs are inedible while most of the true puffballs are
edible at their immature stage because their inner white flesh hasn’t been
differentiated to gleba – the spore-bearing inner mass (Miller, 1977). Their fruiting
bodies are also visible to search for and durable. As puffballs have these properties,
they have advantages over the slime molds because the slime molds fruiting bodies
are very small, ephemeral, and soft (Rikkinen, 2019). Since myxomycetes haven’t
been reported about their toxicity, they are not commonly used as food because they
mostly settle on unsanitary spots due to their feeding behavior.

6
 Figure 1.5. Calvatia craniiformis samples

There are some bioactive compounds that have been found from Lycoperdon
pyriforme: 4-methoxy-benzene- 1-azoformamide (1) and 4-methoxybenzene-1-0"-
azoxyformamide (2) and the chlorinated derivative 3.5-dichloro-4- methoxybenzene-
1-ONN-azoxyfonnamide (3). Compounds (1) and (2) are active against the plant
parasitic nematode Meloidogyne incognita but not active against the saprophytic
nematode Caenorhabditis elegans, exhibit weak antimicrobial effect against Nadsonia
fulvescens and Penicillium notatum. Compound (3), which is less active towards
nematodes but more cytotoxic compared to (1) and (2) (B. Köpcke et al., 2006; Z. M.
Thu et al., 2020). In another study, β-Glucosinase has been characterized and isolated
successfully from  L. pyriforme (M.Y. Akatin, 2011).

From the appearance the fruiting bodies, we can see the puffballs has the color of dark
pigments (black, brown) which can be the evidence of melanin presence. Especially,
the name puffball was given based on their cloud of brown spores when they burst. It
is known that the inner white flesh turns into dark color due to the differentiation of
the inner white flesh into spore with brown color. Therefore, there’s must be a
relationship between the spore forming process with the melanin synthesis. Since L-
DOPA is one of the precursors of melanin synthesis pathway, puffballs have the strong
evidences of the L-DOPA presence.

The objectives of this research were to detect the presence of L-DOPA in slime molds
(P. polycephalum), and puffballs (C. craniiformis, and L. pyriforme) by thin-layers

7
chromatography (TLC) and then quantify the L-DOPA contents of the sample using
HPLC. This project provides information on L-DOPA production in slime molds and
puffballs, which can be used to clarify the potential of these species for Parkinson’s
disease treatment.

8
 II. Materials and methods.
1. Materials:
P. polycephalum was obtained from the myxomycetes culture collection of the Applied
Microbiology Laboratory (School of Biotechnology, Ho Chi Minh International University, Ho
Chi Minh City, Vietnam). P. polycephalum was cultured in both solid and liquid medium. The
solid medium had the addition of 1.5% agar and the pH adjusted to 5.6 while liquid pH was
adjusted to 4.6. The other ingredients followed the optimal medium recipe:

Table 2.1.1. Optimal medium compositions (Truong et al., 2019)

Optimal medium

Citric acid •H2O 40.4 g

FeCl2 •4H2O 0.6 g

MgSO4 •7H2O 6.0 g

CaCl2 •2H2O 6.0 g

ZnSO4 •7H2O 0.34 g

Tryptone 6.59 g

Dextrose 20.0 g

Yeast Extract 3.00 g

KH2PO4 20.0 g

Distilled water 1.00 L

9
C. craniiformis puffball fruiting bodies were kindly collected in July from the lawn of
the University of Mahidol in Thailand and provided by Dr. Edward Grand (Mahidol
University, Nakhon Pathom, Thailand).

L. pyriforme puffball fruiting bodies were kindly collected and provided by Prof.
Stephenson (University of Arkansas, Northwest Arkansas, USA).

The puffballs samples were dried and grounded into fine powder using herbal grinder
machine.

2. Method
2.1. P. polycephalum culture
P. polycephalum in liquid culture took 4 days of incubation at room temperature. Each
sample was incubated in a 100-ml volumetric flask covered with a black plastic bag to
prevent light in the orbital shaker. Each flask of medium received 10 ml of the liquid
sample and 100 µl of hematin (Daniel et al., 1962).
P. polycephalum in solid culture took 2 days of incubation at room temperature. Each
sample was incubated in a 200mm petri dish and contained in a carton box to prevent
light. Each petri dish received a portion of plasmodium around 1.5 cm2 and 100 µl of
hematin (Daniel et al., 1962).

2.2. P. polycephalum collection

P. polycephalum microplasmodia in liquid medium: After incubation, the flask content
was transfer to falcons to be centrifuged 6000 rpm in 20 min. The supernatant was
collected and maintained at -20oC. The samples were freeze-dried later.
P. polycephalum plasmodia in solid medium: After incubation, plasmodia were scraped
to collect without damaging the surface of solid medium. The sample were maintained
at -20oC and freeze-dried later.

2.3. L-DOPA extraction for TLC and HPLC

2.3.1 Thin-layers chromatography.
Standard L-DOPA was diluted into 2% formic acid at the concentration of 1 mg/ml.

P. polycephalum: 1g of dried sample was homogenized with 10 ml formic acid 2%

(w/v) (Loganathan, 1998). The homogenate was centrifuged at 6000 rpm for 25

10
minutes. The supernatant was filtered through nylon syringe filter (0.45 µm, Φ 15
mm). The supernatant was collected to be used in the TLC process.
Puffballs need the cell-wall disruption treatment before going through the TLC test. 1g
of the sample was suspended in 15 ml cold buffer (Tris 50 mmol/L, pH 7.5). Mortar
and pestle were used to demolish the samples. The supernatant is collected by
applying centrifugation on homogenate (6.000 rpm, 20 minutes, 4°C). The
supernatant was filtered through nylon syringe filter (0.45 µm, Φ 15 mm). The
supernatant was mixed well with formic acid in a 1:1 ratio.
The thin-layers chromatography uses the system of silica gel plate C60 F254 (Merck
KGaA, Germany) as stationary phase and isopropanol: ethyl acetate: water: glacial
acetic acid (29:19:10:1) (Loganathan, 1998) as mobile phase. After the TLC plate had
been developed, it was dyed by ninhydrin dye. The ingredient of ninhydrin dye is 1.5g
ninhydrin, 100mL n-butanol, and 3 mL glacial acetic acid (Cai, 2008).

2.3.2. High performance liquid chromatography

Dried samples were homogenized with ACN to extract the organic compounds and
several chemicals with the purpose to improve the polarity and solubility. The
homogenate was extracted by the QuEchERS method. The products of the QuEchERS
method added Dansyl groups to become Dansyl derivatives to increase their
nonpolarity (Takeuchi, 2005).

2.4. HPLC analysis

The Dansyl derivatives were analyzed by HPLC-UV (Agilent HPLC 1200 series) at the
wavelength of 250 nm with the system of column ZORBAX Extend-C18, 80, 5 µm, 4,6
x 250 mm as stationary phase and methanol: water (7:3) as mobile phase. The
concentrations were determined by their peak areas. The standard L-DOPA was used
for determining the relationship between peak area and concentration and build a
linear equation y= ax+b. Base on the linear equation, the sample concentration was
estimated with provided peak areas (Plenis et al., 2019).

11
III. Results and discussions
1. Detection of L-DOPA in the samples by TLC
Capillary tubes were used to for doting, thus the volume of each drops are not
accurately equal to each other. Samples were maintained in ice bucket to prevent
protein denaturation. After doting 1 spot, the capillary tube was washed by acetone
before taking new sample. The TLC plate for slime molds samples (Figure
3.1.1.1) (75 mm) was larger than the TLC plate for puffball (Figure 3.1.1.2) (50 mm).
Though the larger TLC plate took longer retention time - 45 min to develop and the
smaller one took 15 min to develop, the separation wasn’t improved much - the L-
DOPA spot of L-DOPA standard on the larger plate still had a long smear.

Rentention factor (Rf) value of L-DOPA was calculated by

distance spot moved

Rf =
distance solvent moved

54 mm
The Rf of L-DOPA in the larger TLC plate (Figure 3.1.1.1) is R f 1= =0.831
65 mm

35 mm
The Rf of L-DOPA in the smaller TLC plate (Figure 3.1.1.2) is R f 2= =0.833
42mm

12

Figure 3.1.1. TLC plate of standard

L-DOPA and P. polycephalum
samples
(1) 1 drop of 1 mg/ml standard L-DOPA.
Figure 3.1.2. TLC plate of standard
(2) 1 drop of P. polycephalum in solid L-DOPA and C. craniiformis samples
medium homogenate

(3) 2 drops of P. polycephalum in solid medium homogenate

(4) 1 drop of P. polycephalum in liquid medium homogenate

(5) 2 drops of P. polycephalum in liquid medium homogenate

(6) 1 drop of 1 mg/ml standard L-DOPA

(7) 4 drops of 1 mg/ml of C. craniiformis with immature spore homogenate

(8) 4 drops of 1 mg/ml of C. craniiformis with mature spore homogenate

The TLC process didn’t go through any treatment to separate amino acids and
proteins. Therefore, ninhydrin could have dyed the both types of compounds and the
bands at L-DOPA position can be the mixture of L-DOPA and several amino acids
which have the similar polarity. Even though, the presence of these bands raise the
possibility of the L-DOPA presence and be the basis for HPLC analysis.
The result of TLC was not very clear as it couldn’t form bands separately. Even
though, we can still see the blur bands are present at the same distance of standard
L-DOPA spot movement in the samples. Besides, the bands of L-DOPA of samples
were expected to have light color because 1 mg dried biomass of slime molds was
diluted into 10 ml formic acid solution and 1 mg fresh biomass of puffballs was diluted
into 30 ml including Tris buffer and formic acid solution. Since the dilution factor of
puffballs is doubled in comparison with slime molds, the amount of puffball
homogenate in the TLC test was also double.
The TLC plate for the slime mold test (7.5 cm) was longer than the puffball’s one
because it was supposed to separate the bands properly with the longer retention
time. But still, the bands could not separate as well as expected so the puffball
samples use the normal length TLC plate (5 cm). The method still needs improvement
for the proper separation since the L-DOPA standard still has a long smear below the
band.

13
The method of extraction for puffballs was not fit for extracting L. pyriforme sample.
The maximum rpm was 6000 which was not enough to separate the supernatant from
the residue.

14
2. High performance liquid chromatography
All the HPLC results have the presence of the peaks at the same retention time in
HPLC result of L-DOPA standard which can be considered as L-DOPA’s peaks. L-DOPA
from all of the samples were extracted effective since all their L-DOPA expected peaks
base on L-DOPA standard (Figure 3.1.2.1) are separated from the other components’
peaks reliably. The shapes of the peaks are also reliable in height and width (the
height is not low and width is not large).

Figure 3.2.1. HPLC result of fruiting bodies of standard L-dopa

Figure 3.2.2. HPLC result of fuirting bodies of L. pyriforme

Table 3.2.1 L-DOPA content of L. pyriforme

Samples L-DOPA content (mg/g)

L. pyriforme 2.14

15
In TLC results, bands of microplasmodia of P. Polycephalum in solid medium had
denser color. Base on the HPLC, the TLC result is explainable since in microplasmodia
result, there are presence of more compounds that are nearly similar to L-DOPA. On
the other hand, the L-DOPA content in microplasmodia is also much higher than
plasmodia. There would be a prediction that protein content in microplasmodia is
higher than plasmodia

Figure 3.2.3. HPLC result of P. polycephalum plasmodia in sold medium

Figure 3.2.4. HPLC result of P. polycephalum microplasmodia in liquid medium

Table 3.2.2. L-DOPA content of P. polycephalum

Samples L-DOPA content (mg/g)

Plasmodia of P. polycephalum 2.07
Microplasmodia of P. polycephalum 2.49

16
The TLC results of C. craniiformis don’t have much different between bands in sample
with immature spores and sample with mature spores’ ones. Besides, their L-DOPA are
also not much different from each other. The TLC result is quite explainable base on
the HPLC result since they also didn’t have many differences in the L-DOPA-like
components.

Figure 3.2.5. HPLC result of C. craniiformis with immature spores

Figure 3.2.6. HPLC result of C. craniiformis with mature spores

Table 3.2.3. L-DOPA content of C. craniiformis

Samples L-DOPA content (mg/g)

C. craniiformis with immature spores 2.22
C. craniiformis with mature spores 2.28
3

2.49
2.5
2.22 2.28
2.07 2.14
2
L-DOPA conc. (mg/g)

1.5

0.5

0
Solid medium P. Liquid medium P. Immature spore Mature spore C. L. pyriforme
polycephalum polycephalum C. craniformis craniformis

17
 Figure 3.2.7. L-DOPA content of slime molds and puffballs

 Comparison among the slime mold samples

The microplasmodia in liquid medium has a higher biomass growth rate in comparison
with plasmodia in solid medium (Dove and Rusch, 1980). On the other hand,
according to this HPLC result, the liquid medium can be considered to be the more
effective L-DOPA bioproduction culture for P. polycephalum (Table 3.1.2.1). 
 Comparison among the puffball samples
Among 3 samples, the C. cranifomis with mature spore has the highest L-DOPA
content (Table 3.2.2.1). Base on this result, the spore production can be a factor that
affects the L-DOPA production. On the other hand, both 2 samples of C.
cranifomis have higher L-DOPA content than L. pyriforme. Therefore, L. pyriforme’s L-
DOPA content can be considered as lower than C. cranifomis. 
 Comparison of all the samples
The P. polycephalum in liquid medium has the highest L-DOPA content while P.
polycephalum in solid medium has the lowest L-DOPA content (Figure 3.2.2.1). It can
be concluded that the liquid medium improved the biosynthesis of the L-DOPA of P.
polycephalum effectively. Therefore, the liquid medium may be the ideal condition
for P. polycephalum to synthesize L-DOPA.
 Comparison between the samples with M. pruriens - the most common
source of L-DOPA bioproduction.
The highest L-DOPA content sample in the project - P. polycephalum in liquid medium
(2.49 mg/g) has L-DOPA which is lower than L-DOPA content in M. pruriens L-DOPA
content which varies between 5.8 mg/g to 64.2 mg/g (Pulikkalpura et al., 2015).
Since the L-DOPA content is lower, microplasmodia of slime molds has very short
cultivation time and their L-DOPA extraction process of it is less complicated because
microplasmodia of slime molds don’t have cell wall like M. Prurien. Puffballs also have
advantage of their edibility. While the M. pruriens seeds are hard to eat as well as
their polyphenols hinder the disgestion process (Lampariello et al., 2012), the puffballs
are easier material to process as food since the inner white flesh has attractive flavor
and texture.
 About L-DOPA and HPLC
This HPLC method still can’t distinguish between L-DOPA and it’s isomer – D-DOPA.
Therefore, the L-DOPA contents which are determined in the project are still
questionable as to if it’s L-DOPA or not. To confirm this, NMR is needed in future

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research. There’s an available HPLC method to separate L-DOPA and D-DOPA by
forming the L-Leucine derivatives of DOPA (Hermansson & Wiese, 1981).

IV. Conclusions and recommendations

For the purpose to detect L-DOPA in slime mold, puffball samples, the TLC system
need improvement for the proper performance. The bands should be separated more
effective and have no smear. There are some suggestion to solve the problem: the
sample should be diluted more to prevent the overload of solvent or the ratio of acetic
acid in solvent should be increased to prevent the formation of hydrogen bonds
between organic compounds and silica gel. Even though, the bands are still acceptable
since all the samples had their L-DOPA content detected by HPLC analysis.
The HPLC-UV using dansyl technique has been proven as a consistent technique for L-
DOPA detection in slime molds and puffballs. The HPLC results show the similarity of
L-DOPA content between the targeted species: P. polycephalum in solid medium (2.07
mg/g), P. polycephalum in liquid medium (2.49 mg/g), C. craniiformis with immature
spore (2.22 mg/g), C. craniiformis with mature spore (2.28 mg/g), L. pyriforme (2.14
mg/g). The HPLC results still require the confirmation that the detected L-DOPA is not
it’s isomer – D-DOPA.

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