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Supercritical CO2 extraction of nimbin from

neem seeds?An experimental study
Article in Journal of Food Engineering March 2001
DOI: 10.1016/S0260-8774(00)00131-X

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Journal of Food Engineering 47 (2001) 289293

www.elsevier.com/locate/jfoodeng

Supercritical CO2 extraction of nimbin from neem seeds an

experimental study
Pathumthip Tonthubthimthong a, Supaporn Chuaprasert a, Peter Douglas b,*,
Wilai Luewisutthichat a
a

Department of Chemical Engineering, King Mongkut's University of Technology, Thonburi, Bangkok 10140, Thailand
b
Department of Chemical Engineering, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
Received 7 January 2000; accepted 26 July 2000

Abstract
Nimbin is one of the many substances found in neem seeds and is reported to have several medicinal properties and uses. For
example, it is an anti-pyretic, can be used to treat arthritis, hypoglycaemia, peptic ulcers, anti-secretory activity, and it can also be
used as an antibiotic. In this paper, we present the results of a preliminary experimental study to extract nimbin from neem seeds
using CO2 supercritical uid extraction (SFE). The operating pressure in the extraction was varied from 10 to 26 MPa, the temperature was varied from 308 to 333 K and the ow rate was varied from 0.24 to 1.24 ml/min. An optimum extraction rate was
observed at a pressure of 23 MPa when operating at 308 K. Best extraction conditions occurred at 23 MPa, 308 K and a ow rate of
1.24 ml/min for a 2 g sample of neem. The measured extraction rate was found to be about 0.18 mg of nimbin/g neem seed per hour
of operation which is equivalent to about 0.35 kg nimbin extracted per kg nimbin present in neem seeds. The future work needs to
focus on the interaction between the various operating parameters such as temperature, pressure and ow rate of supercritical
carbon dioxide. In addition physical properties i.e., particle size, porosity need to be determined in order that a model can be
developed and tested. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Supercritical uid extraction; Carbon dioxide; Nimbin; Neem

1. Introduction
Azadirachta indica A. Juss is a medicinal plant,
commonly known as neem, which has long been used in
agriculture and medicine. According to folklore, the
bark, leaves and fruit have been used as medicines for
the treatment of a variety of human ailments, particularly against diseases of bacterial and fungal origin
(Chawla, Kumar, & Bansal, 1994). Neem has recognised
insecticidal activity as well as anti-inammatory, analgesic, anti-pyretic, and anti-malarial activities (Dhawan
& Patnaik, 1993). In addition, neem oil is reported to
have contraceptive ecacy (Khare, Srivastava, Sharma,
& Tewari, 1984; Riar et al., 1991; Garg, Talwar, &
Upadhyay, 1994). Chemical and therapeutic studies
have been studied since the beginning of century. Two
crystalline bitter principles, nimbin and nimbinin were

Corresponding author. Tel.: +1-519-888-4567 ext. 2913; fax: +1519-746-4979.

E-mail address: pdouglas@cape.uwaterloo.ca (P. Douglas).

rst isolated from the oil along with amorphous bitter

principle nimbidin.
Conventional production methods such as solvent
extraction and soxhlet, although eective for extraction,
can lead to degradation of heat sensitive compounds as
well as leave traces of toxic solvents in the solute. This is
a concern for food and medicinal extracts. Supercritical
uid technology may be a viable alternative to solvent
extraction methods. The phenomenon of supercritical
uid technology extraction was observed over 100 years
ago but has been slow to nd commercial applications,
due in part to the sophisticated and expensive high
pressure equipment and technology required (Jarvis &
Morgan, 1997).
Carbon dioxide is generally the most desirable solvent
for supercritical uid extraction (SFE). The critical
temperature of carbon dioxide is only 304 K, which
makes it attractive for the extraction of heat sensitive
compounds. In addition it is an inert, non-ammable,
non-explosive, inexpensive, odourless, colourless, clean
solvent that leaves no solvent residue in the product, it is
also non-toxic and is generally accepted as a harmless

0260-8774/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 0 - 8 7 7 4 ( 0 0 ) 0 0 1 3 1 - X

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P. Tonthubthimthong et al. / Journal of Food Engineering 47 (2001) 289293

ingredient in pharmaceuticals and food. In addition,

carbon dioxide has a low surface tension and viscosity
and high diusivity which make it attractive as a supercritical solvent (Roy, Goto, Kodama, & Hirose,
1996). The diusivity of supercritical carbon dioxide is
one to two orders of magnitude higher than for other
uids, which permits rapid mass transfer, resulting in a
larger extraction rate than that obtained by conventional liquid extraction.
Although much has been written in the last 10 years
about SFE very little has been written about supercritical extraction of nimbin from neem seeds. Only two
papers have been found on the supercritical extraction
of nimbin from neem (D'Andrea, Ferri, Maccioni, van
der Esch, & Vitali, 1994 and Johnson & Morgan, 1997).
Johnson and Morgan (1997) report on the use of CO2 to
extract nimbin as well as using CO2 and a methanol cosolvent in an extractor made from a piece of high
pressure HPLC tubing. They investigated the eect of
pressure and methanol co-solvent but did not study the
eect of temperature, CO2 ow rate, or sample size.
Their results indicated that nimbin extraction yield increased continuously with pressure and reached a maximum yield of about 40% at 34 MPa, the maximum
pressure tested. When 6% of a methanol co-solvent was
used the extraction rate increased to 70%.
Since nimbin is a medical substance and the use of a
co-solvent in the extraction process might aect the
medicinal properties of the extracted nimbin and might
hinder future FDA approval for such a commercial
process we have focussed on the use of CO2 only as a
solvent.
2. Experimental
In this experimental study, nimbin was extracted
from neem seed kernels using supercritical carbon dioxide. The following extractor operating conditions
were studied:
CO2 ow rate;
CO2 pressure;
extractor temperature and
weight of neem sample.
The experiments were carried out in an ISCO Model
SFX 2-10 SFE unit shown in Fig. 1. Dried neem seeds
were obtained locally and stored in a sealed glass jar at
ambient conditions. Without any pre-treatment the
neem seeds were ground into a powder. The particle size
was measured and found to be about 600 lm. Approximately 2 g of the ground neem powder was placed
in the 10 ml sample unit, glass wool was placed on top of
the sample to ll the remaining volume in the unit. The
sample unit was placed in the ISCO unit and allowed to
equilibrate to the pre-set extraction temperature. CO2
was then charged into the high pressure pump from the

Fig. 1. Schematic diagram of supercritical extraction unit.

CO2 cylinder. The high pressure pump further compressed the CO2 to the desired pressure. Then the valve
between the pump and the extractor was opened and the
CO2 was allowed to ow through the sample. A back
pressure valve at the outlet was adjusted to maintain a
constant volumetric throughout the period of the experiment. However, it should be noted that it was difcult to maintain a constant ow rate at the beginning
and the end of the experimental run. The product gas,
CO2 plus nimbin, was bubbled into the collecting tube
lled with 10 ml of methanol. Although Johnson and
Morgan (1997) used a 5050 mixture of toluene and
ethanol methanol has also been recommended as a solvent for nimbin. Siddiqui and Mitra (1942) recommended methanol and, in fact, Johnson and Morgan
(1997) also used methanol as a co-solvent with CO2 and
also to dissolve the trapped nimbin prior to analysis by
GPLC. The collection tube with methanol was replaced
every 10 min with a new tube lled with 10 ml of
methanol so that a dynamic extraction curve could be
obtained. The collection tubes were wrapped with aluminium foil to prevent photo degradation of the nimbin.
The nimbinmethanol sample was later analysed for
nimbin in a Waters HPLC spherisorb ODS2 column
(250 mm  4:6 mm ID) lled with 5 lm particles. The
analysis was performed under isocratic conditions at a
ow rate of 1 ml/min using mobile phase water: acetonitrile (40:60), at the wavelength of 217 nm. A Waters 600
controller/pump, Waters 486 tuneable adsorbance detector and a Waters 717 plus autosampler were also used.
3. Results and discussion
The extracted material collected in the collection
tubes formed a two-phase liquid mixture with methanol

P. Tonthubthimthong et al. / Journal of Food Engineering 47 (2001) 289293

291

in the collection tubes. Neem oil was yellowish in colour

and collected at the bottom of the collection tube. The
continuous methanol phase was analysed for nimbin.
The maximum yield obtained was about 0.35 mg of
nimbin per 2 g sample of neem seeds. This is equivalent
to 0.35 kg of nimbin per kg of nimbin in neem seeds
since the amount of nimbin in neem seeds is about 0.5 mg/g
of seeds (Chinnasamy, Harishankar, Uday Kumar, &
Rukmini, 1993). Johnson and Morgan (1997) reported a
maximum extraction of approximately 0.45 kg of nimbin per kg of nimbin in neem seeds. However, it is
somewhat dicult to compare their results with ours
since they used a dierent experimental set-up and
experimental conditions. They used a piece of HPLC
high pressure tubing rather than a commercial supercritical extractor. They operated at a constant ow rate
of 1 ml/min, a temperature of 328 K, a maximum
pressure of 34 MPa and used a 5050 tolueneethanol
solvent in the collector tube.

of the curve, also increased. These results are expected

because each experiment was conducted using essentially the same amount of CO2 only the ow rate or
velocity was varied. Therefore, during a given interval of
time more CO2 passes through the sample when the ow
rate is high thereby extracting more nimbin in a given
time interval. This is shown more clearly when the extraction yield is plotted versus the amount of CO2
consumed in Fig. 2(b). The extraction rate is not aected
by ow rate to the same degree but is only a function of
the amount of CO2 that passes through the sample. In
fact for the two highest ow rates, the curves lie on the
same line indicating that the exit concentration of solute
is independent of ow rate and therefore equilibrium is
considered to exist. If intraparticle diusion resistance is
important then the extraction rate will be a function of
the CO2 consumed and the higher the ow rate the lower
the slope. Others have observed this phenomenon
(Goto, Sato, & Hirose, 1993).

3.1. Eect of solvent ow rate

3.2. Eect of solvent pressure

The rst set of extraction experiments was carried out

at a constant pressure of 20 MPa and an oven temperature of 328 K using 2 g of ground neem seeds and pure
carbon dioxide. The CO2 feed ow rate was varied from
0.241.24 ml/min. Fig. 2 shows the eect of CO2 ow
rate on the extraction of nimbin as a function of time in
Fig. 2(a) and as a function of the amount of carbon
dioxide consumed in Fig. 2(b). The maximum amount
of nimbin extracted is constant at about 0.35 kg nimbin
extracted/kg nimbin available in the sample. At 328 K
and 20 MPa and at a CO2 ow rate of 1 ml/min Johnson
and Morgan (1997) obtained a maximum extraction
yield of about 0.04 kg nimbin extracted/kg nimbin
available in the sample after an extraction for 30 min.
From our experiments at a ow rate of 1.24 ml/min we
obtained about 0.25 kg/kg and at a ow rate of 0.62 ml/
min we obtained about 0.10 kg/kg; interpolating to a
ow rate of 1 ml/min yields 0.19 kg nimbin/kg nimbin in
neem seeds.
From Fig. 2(a) it was found that when the ow rate
was increased, the extraction rate, indicated by the slope

The second set of SFE experiments was carried out at

a constant temperature of 328 K using 2 g of ground
neem seeds and using supercritical carbon dioxide at
ow rate of 0.62 ml/min and pressures varying from 10
to 26 MPa. The results, shown in Fig. 3, indicated that
as the pressure is increased the extraction rate also increases. This phenomenon is well known and explained
by the fact as the pressure increases the density of the
supercritical uid approaches that of the liquid resulting
in an increase in the solvating power. Johnson and
Morgan (1997) also studied the eect of pressure on the
extraction rate at a constant temperature of 328 K and
ow rate of 1 ml/min. Their results show that as pressure
was increased the extraction also increased; the maximum extraction that they obtained was 0.45 kg nimbin/
kg nimbin in neem seeds at 34.4 MPa.
Fig. 3 shows that once the pressure was increased
from 23 to 26 MPa the extraction rate decreased, indicating an optimum extraction pressure exists in the
range of 23 MPa. This is a phenomenon that has been
observed in other systems (Chuaprasert, Boon-

Fig. 2. (a) Eect of ow rate on total yield at 328 K and 20 MPa, (b) eect of ow rate on total yield at 328 K and 20 MPa.

292

P. Tonthubthimthong et al. / Journal of Food Engineering 47 (2001) 289293

Fig. 3. Eect of pressure on total yield at 328 K and 0.62 ml/min.

amnauyvitaya, Pengsopar, & Pienprasuh, 1997; Baysal

& Starmans, 1999). Although the existence of an optima
extraction yield as a function of pressure is not discussed
in the literature we feel that its existence is real and can
be explained qualitatively. An increase in pressure results in an increase in the density of the supercritical
uid and a subsequent increase in its solvating power.
However, as the pressure is increased the diusivity decreases and the molecules have more diculty diusing
into the pores to dissolve solute. In addition, an increase
in pressure causes the solid matrix to become more
packed and the void fraction decreases also reducing the
extraction. The solubility of the solute in CO2 is a nonuniform function of temperature and pressure as discussed below. Finally, the selectivity of solute extraction
is a function of pressure, i.e., as the pressure is increased
dierent solutes are preferentially extracted (Brogle,
1982; D'Andrea et al., 1994). Therefore, an increase in
pressure results in opposing forces and it is reasonable
to expect that an optimum pressure may exist. In the
other reported work on the extraction of nimbin Johnson and Morgan (1997) found that increasing pressure
led to higher extraction yields without any indication of
an optimum; they studied pressures between 6.9 and
34.4 MPa. D'Andrea et al. (1994) extracted nimbin,
azadirachtin and 3-tigloylazadirachtol from neem seeds.
They found that nimbin was preferentially extracted at a
CO2 density of 0.8 g/ml; this corresponds to a pressure
of approximately 25 MPa at 328 K; we found an optimum extraction yield at 23 MPa at 328 K.
3.3. Eect of temperature
The third set of SFE experiments was designed to
investigate the eect of extraction temperature on the
extraction rate. Extractions were carried out at a constant pressure of 20 MPa, using 2 g of ground neem
seeds and supercritical carbon dioxide at a ow rate of
0.62 ml/min over a temperature range from 308 to 333
K. The results are presented in Fig. 4. Fig. 4 shows a
general trend of decreasing extraction yield with increasing temperature. In particular we found the best

Fig. 4. Eect of temperature on total yield at 20 MPa and 0.62 ml/min.

temperature was 308 K, the lowest temperature tested.

Most work reported in the literature shows that extraction increases with increasing temperature (Brogle,
1982; Peker, Srinivasan, Smith, & McCoy, 1992; Spanos, Chen, & Schwartz, 1993). However, Goto et al.
(1993) and Stastova, Jez, Bartlova, and Sovova (1996)
present experimental results that do not show any clear
trend as far as temperature is concerned. Gomez, Lopez,
and de la Ossa (1996) present results that imply an optimum temperature. They show that the extraction yield
is inversely proportional to temperature at pressures less
than 15 MPa. They also show that for pressures greater
than 20 MPa a temperature of 313 K results in an extraction yield which is greater than the yield at 283 and
333 K, thereby indicating an optimum in temperature.
McHugh and Krukonis (1994) present solubility data
for naphthalene in CO2 as a function of temperature at
various pressures. The results are by no means uniform;
depending on the pressure the eect of temperature on
solubility can be quite dierent. For pressures less than
15 MPa, an increase in temperature results in a decrease
in the solubility and for higher pressures (1530 MPa)
increasing temperature increases the solubility. It is clear
that the eects of increases in temperature and pressure
on the solubility and extraction yield are not uniform for
all solutes and could lead to the formation of an optimum in both temperature and pressure.
3.4. Eect of sample size
The nal set of experiments was carried out at a
constant pressure of 20 MPa, a temperature of 328 K,
using supercritical carbon dioxide at a ow rate of 0.62
ml/min and neem samples varying in weight from 1 to
2.5 g as shown in Fig. 5. From Fig. 5, we can see that an
increase in sample size has little eect on the specic
extraction yield. The variation in results is felt to be due
to experimental errors. However, it is felt that if the
sample weight is increased beyond some reasonable
value then the extraction rate will decrease because the
porosity of the sample will decrease and the CO2 molecules will not be able to contact the nimbin molecules
easily; we have not reached this point yet. Fig. 5 can be

P. Tonthubthimthong et al. / Journal of Food Engineering 47 (2001) 289293

Fig. 5. Eect of sample size on total yield at 328 K, 20 MPa and

0.62 ml/min.

used as a rough guide to scale-up SFE units if it is assumed that the specic extraction yield remains approximately invariant with increases in the size of the
extractor.
4. Conclusions
1. Nimbin extraction yields of approximately 0.35 kg
nimbin/kg of nimbin in seeds using only supercritical
CO2 extraction were obtained. The extraction yield
was approximately 0.175 mg nimbin/g of neem seeds
and the neem seeds were assumed to contain about
0.5 mg of nimbin/g of neem seeds.
2. The best extraction conditions from experiments were
found to be at: 308 K, 23 MPa and a ow rate of 1.24
ml/min for a 2 g sample of neem.
3. An optimum pressure of extraction appears to exist
at about 23 MPa at a temperature of 328 K.
4. The specic extraction rate was not found to be a function of sample size for the range of samples studied.
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