SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF
MITRAGYNA SPECIOSA
Norsita Tohar1, Khalijah Awang2
1
Centre for Foundation Studies in Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia
2
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia
Email : khalijah@um.edu.my
ABSTRACT
Mitragyna speciosa is a narcotic plant but with specific medicinal
importance. The main components of the leaves of M. speciosa are indole alkaloids.
Previous scientific researches have shown that the alkaloidal extracts possess very potent
anti-inflammatory, analgesic and opioid properties. The three most abundant indoles are
mitragynine, paynanthine and speciogynine, the first two of which appear to be unique to
this species. All these compounds were obtained from classical organic solvent (ethyl
acetate, dichloromethane or methanol) extraction. CO2 extract of M. speciosa has never
been studied yet. Hence, in this present investigation, supercritical fluid extraction (SFE)
of M. speciosa was performed. Comparison of the TLC of SFE extracts with that of the
ethyl acetate extract from this preliminary study shows no alkaloid was extracted out with
100% CO2. Alkaloids were only extracted with eluent system using ethanol as co-solvent
and the optimal conditions was CO2-ethanol (80% EtOH), 40 oC and 5000psi. CO2 was
chosen as the media of extraction because it is non-toxic and environmentally safe to
prepare and use.
ABSTRAK
Mitragyna
speciosa
merupakan
tumbuhan
narkotik
tetapi
mempunyai kepentingan perubatan yang tertentu. Indol alkaloid adalah komponan utama
daun M. speciosa. Kajian saintifik terdahulu menunjukkan bahawa ekstrak alkaloidnya
mempunyai ciri-ciri anti-radang, analgesik dan ’opioid’. ’Mitragynine’, ’paynanthine’
dan ’speciogynine’ merupakan tiga indol utama dengan ’mitragynine’ dan ’paynanthine’
adalah unik bagi spesis ini. Kesemua sebatian tersebut telah diperolehi cecara
pengekstrakan klasik dengan menggunakan pelarut organik (etil asetat, diklorometana
dan metanol). Belum ada kajian yang dilakukan untuk ekstrak CO2 M. Speciosa maka
pengekstrakan ’supercritical fluid’ telah dijalankan dalam penyelidikan ini. Dari kajian
awal ini, perbandingan TLC di antara ekstrak SFE dengan ekstrak etil asetat
menunjukkan tiada alkaloid yang terekstrak keluar melalui penggunakan 100% CO2.
Alkaloid hanya diperolehi dengan sistem pelarut yang menggunakan etanol sebagai
pelarut bersama dan keadaan optima ialah CO2-etanol ( 80% EtOH), 40 oC dan 5000 psi.
CO2 telah dipilih sebagai media pengekstrakan kerana ia tidak toksik dan mesra alam
untuk disediakan dan digunakan.
Keywords: Mitragyna speciosa, mitragynine, SFE, CO2
INTRODUCTION
In view of increasing environmental and health concern about the use of organic solvents
in the extraction of natural products, there has been growing interest in using supercritical
fluids. The supercritical extraction using carbon dioxide in the supercritical state, i. e.
pressure > 73.8 bar and temperature > 31.06 oC, allows extracts to be obtained that are of
better quality than those extracted with organic solvents, whose residues, even if present
as traces, contaminate the extract. The technique permits the process to be performed at
low temperatures, avoiding the degradation of termolabile compounds and limiting
hydrolytic phenomena that lead to loss of compounds soluble in water. CO2 is a non-toxic
solvent and can be easily and completely removed from products; moreover it possesses
unusual properties such as high compressibility, liquid-like density, high diffusivity, low
viscosity and low surface tension under supercritical condition. So, supercritical fluid
shows a greater ability to diffuse into the ultrafine matrix than the conventional organic
solvents, thus improving extraction yield of desired materials from complex matrices [1].
Mitragyna belongs to the family Rubiacea and is found in tropical and subtropical
regions of Asia and Africa. Mitragyna speciosa is found growing in Thailand and
Malaysia. In Malaysia it is found in the northern states of Kedah and Perlis as well as in
the East coast states and is commonly known as Ketum or Biak by the local Malays.
Mitragyna speciosa has many applications in traditional use. There are descriptions of its
use as a tonic, cure for fever, treatment for diarrhea and as morphine substitute [2,3]. In
Thailand, leaf of this plant has been consumed for its opium-like effect and its coca-like
stimulant ability to combat fatigue and enhance tolerence to hard work under a scoring
sun [2,4,5]. Due to its unique medicinal properties, a number of chemical,
pharmacological, principle and the mechanisms underlying the biological activities
studies have been carried out over the last forty years [6-12].
In the present work we show the results concerning the extraction of crude from the
leaves of M. speciosa, by means of supercritical carbon dioxide, in a single extraction
step. To best of our knowledge, this is the first report on CO2 supercritical fluid extract of
M. speciosa.
MATERIALS AND METHODS
Plant Material
Fresh leaves and barks of M. speciosa were collected from the forest in Sg.Setong,
Jelawang, Kelantan on the 5th of November 2006. The voucher specimens KL 5321 were
deposited at the Herbarium of the Department of Chemistry,University of Malaya.
Supercritical extraction with carbon dioxide
SFE without co-solvent :
In this typical experimental study, about 25 g dried and ground leaves were placed in 100
mL extraction vessel of the analytical-scale SFT 100 XW SFE system (illustrated in Fig.
1). CO2 (99.8 % purity, SFE grade) was purchased from MOX ( Petaling Jaya, Selangor)
and was delivered by SFT-10 Constant Pressure pump. The system conditions studied
were at 40oC and 5000 psi. One-step extractions were performed by feeding CO2 into the
system until the pressure of 5000 psi was attained and the system temperature was set to
40 oC. After 30 minutes of static extraction (no liquid flow), the sample material was
subjected to dynamic extraction by flowing liquid CO2 at a rate of 3 ml/min. During the
extraction, 2 to 4 crude fractions were collected in the vial. Each fraction took between 47 minutes to extract. The extraction of the crude was terminated when it was visually
observed that no extract was being collected in the vial and is termed as exhaustive
extraction. The extracts obtained were concentrated under reduced pressure, yielding dark
green residue.
SFE with ethanol co-solvent :
The supercritical fluid extraction with CO2 and ethanol co-solvent was performed at 5000
psi and 40 oC. The apparatus was equipped with an additional High Performance Liquid
Chromatography (HPLC) pump (Series II Isocratic HPLC pump) for the ethanol. The
CO2 and ethanol were pumped simultaneously. The extracts, containing ethanol, were
evaporated and the final mass of extract weighed.
Conventional extraction
Extraction was carried out by cold percolation. The plant material (1 kg) was first
sprinkled with 25% ammonia (NH3) solution and left to soak for an hour. Later it was
extracted with ethyl acetate for four days at room temperature then filtered. Thereafter,
the solvent was evaporated to dryness under reduced pressure to afford a dark green
coloured sludge.
All crude extracts were sent for 1H NMR experiment and the composition of individual
chromatographic was checked by thin layer chromatographic (TLC, Figure 2).
RESULTS AND DISCUSSION
Preliminary experiments were carried out in order to select relevant factors as well as
their experimental domain to obtain the highest alkaloid crude extraction recovery. The
yield extracted by pure supercritical carbon dioxide was not satisfactory. From the TLC
profile, extraction with 100% CO2 did not furnish any alkaloids. Addition of ethanol
resulted in the extraction of alkaloidal components. Ethanol (95%) was chosen as
modifier in this study and the concentrations used were 20, 50 and 80% (v/v), (in liquid
carbon dioxide). The higher the percentages of modifier, the higher was the extraction
yield achieved. The amounts of crude extracts obtained by SFE method using various
ratio of modifier and by percolation were listed in Table 1.
Alkaloid compound with the Rf value of 0.44 was detectable in each extract with TLC (
developed in dichloromethane-methanol, 95:5, v/v) except in the SFE crude using only
CO2 (Figure 2). Using ethanol as modifier, the TLC patterns indicated the presence of
similar compounds and the concentration of the alkaloidal zone was proportional to the
concentration of the modifier. Only alkolaidal bands were significantly shown by the
crude from material that has been saturated with NH3. This can be attributed to the fact
that salts of alkaloids in plant tissues might be converted to free bases by NH3. This
change can enchance the solubility of a compound in CO2 as well as the degree of
desorption from the plant matrix. As for the percolation extract, different type of alkaloid
and other compounds have been extracted out which were not detected from the TLC of
SFE crude. Interestingly, the major alkaloid found in the ethyl acetate extract,
mitragynine, was not extracted in all the SFE extracts.
In this study, all crude extracts showed the similar patterns of NMR spectrum except for
SFE crude using 100% CO2 .
CONCLUSIONS
Neat carbon dioxide was not sufficient to extract alkaloidal compounds or even any other
compound significantly from M. speciosa at 40 oC and 5000 psi. Increasing the polarity
of the extraction solvent by addition of ethanol had overcome the low yield problem. This
could be explained by the fact that polar constituents in the plant would be easier to
extract with a more polar solvent. Whereby, ethanol as the modifier helps to reduce
matrix interactions and increase supercritical fluid polarity. The extraction yield would
increase much if ethanol is added more as perfectly defined by the highest extraction
yield found with extraction by 80% ethanol. However, in comparison with TLC pattern
of ethyl acetate crude, SFE method using 40 oC and 5000 psi with 80% ethanol was not
capable of extracting more highly polar alkaloid compounds and showed no effect on the
yield of less polar alkaloids even mitragynine, the most abundant component in the plant.
Therefore, optimal conditions for the extraction of those compounds require further
investigation. This study also indicated that SFE method can be used as a means of
fractionation under specific parameters.
ACKNOWLEDGEMENTS
This project was supported by University of Malaya-PJP (FS140/2008C).
The authors would also wish to thank Mr. Marshall Victor from Intran Technologies
Sdn.Bhd. for his assistance with the SFE equipment.
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Table 1.
Percentage of yields by different methods and conditions of extraction
Method
Factors
Yield
Pressure
Temperature
Static
Modifier
(psi)
(oC)
extraction
(ethanol %)a
(%)b
(minute)
SFE
(25.0977 g dried
5000
40
30
0
0.17
5000
40
30
20
1.06
5000
40
30
20
4.05
5000
40
30
50
3.54
5000
40
30
80
8.92
-
-
-
-
6.63
material)
SFE
(25.2715 g dried
material)
SFE
(25.1647 g material
wetted with NH3
SFE
(25.0209 g dried
material)
SFE
(25.0287 g dried
material)
Conventional
extraction
( 1000 g material
percolated with ethyl
acetate
a
Modifier ( ethanol %) = volume of added ethanol (ml)/volume of extraction vessel (100 ml)
b
Extraction yield (%) = (the amount of crude extract/sample mass) x 100
Figure 1.
Flowsheet of the analytical-scale SFT 100 XW SFE system
Compound X
Figure 2.
TLC profile of the crude extracts