846 J. Agric. Food Chem.

2002, 50, 846−849

Extraction of Pharmaceutical Components from Ginkgo biloba

Leaves Using Supercritical Carbon Dioxide

CHUN YANG,* YAN-RONG XU, AND WEI-XI YAO

Laboratory of Environmental-Analytical Chemistry and Ecological Toxicology, Research Center for

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

Ginkgo biloba extract (GBE) has many remarkable pharmacological and clinical effects, and it is the
most frequently used product as a phytomedicine in many countries. The combination of primary
extraction with 70% ethanol followed by extraction using supercritical carbon dioxide provides an
efficient and economical means for obtaining flavonoids and terpenoids from Ginkgo biloba leaves.
The supercritical fluid extraction (SFE) is affected by pressure, temperature, and the concentration
of modifier in the extractant. At the most favorable experimental conditions of 300 MPa, 60 °C, and
carbon dioxide containing 5% ethanol as modifier, the yield of GBE powder is 2.1% (based on the
air-dry weight of Ginkgo biloba leaves) compared to a yield of only 1.8% by conventional solvent
extraction. The contents of flavonoids and terpenoids in SFE products are 35.9% and 7.3%,
respectively, which are significantly higher than the general standards of 24% and 6%, respectively.

KEYWORDS: Supercritical fluid extraction; carbon dioxide; Ginkgo biloba leaves; GBE

INTRODUCTION and determination of effective components present in Ginkgo

biloba (8-11). However, even though SFE extraction of Ginkgo
Ancient Ginkgo biloba survived from the era when dinosaurs biloba has many advantages, research and development in this
became extinct over 200 million years ago. Its amazing vitality field is still not sufficient. Actually, so far, there are very few
has attracted an increasing exploration into its potential ap- references describing the process in detail, possibly due to the
plication in health foods and supplements. It is proved that lack of technical confidence. In this paper SC CO2 was
Ginkgo biloba leaves contain a large number of active com- tentatively applied to extract pharmaceutical components from
pounds, the most important of which are flavonol glycosides Ginkgo biloba leaves and raw GBE, and the variables such as
(flavonoids) and terpenoids. These effective pharmaceutical pressure, temperature, and modifier were optimized in order to
substances, in particular the terpenoids, regulate blood flow to achieve satisfactory results.
the brain, thus helping to counteract memory loss, depression,
and lack of alertness which may develop in old age. The major MATERIALS AND METHODS
therapeutic uses of Ginkgo biloba extract (GBE) are in many
areas such as vascular insufficiency and hearing disorders (1- General Experimental Procedures. Green Ginkgo biloba leaves
4). were collected in autumn in Nankou near Beijing. The leaves were
rinsed with distilled water and air-dried in the laboratory. The dried
As is well-known, conventional solvent extraction is rather leaves were pulverized to pass No. 5 mesh screen (5-mm openings)
tedious, and it inevitably introduces hazardous residual of using a FZ 103 plant pulverizer (Qijiawu Plant of Scientific Instruments,
organic solvent into the products. To get high-quality GBE, Heibei, China).
supercritical carbon dioxide (SC CO2) is regarded as an ideal In this study, extraction with ethanol was considered as the primary
substitute. SC CO2 has unique physicochemical properties such treatment prior to the SFE extraction. Pulverized Ginkgo biloba leaves
as high diffusivity, lower viscosity, and lower surface tension, (500 g) were extracted with 70% ethanol (1 L × 2 times) for 2 h under
which make it an ideal extractant for the extraction of target reflux. More extraction steps are often needed in order to completely
components from the hosting matrixes. Once extracted and extract the effective components from Ginkgo biloba leaves. After
fractionated into its different components, the products are filtration, the aqueous solution was distilled to remove extractant, where
the raw product denoted as GBERaw contains approximately 4% of total
completely free of residual solvent. Nowadays, supercritical fluid
flavonoids (abbreviated as TFL) and less than 1% of total terpenoids
extraction (SFE) technology has been widely used for the (TTE). The yield of GBERaw was approximately 16% based on the air-
analysis of natural products and pharmaceuticals (5-7), and dry weight of Ginkgo biloba leaves. In the conventional solvent
this technology is receiving increased attention for the extraction extraction, further refinement of extractives with organic solvents such
as chloroform and acetone was followed by a series of purifications
* Address correspondence to this author at the Division of Chemistry, with columns filled with polycaprolactam, alumina oxide, and resins
Nanyang Technological University, 1 Nanyang Walk, Singapore 637616 such as XAD and GDX. These steps can lead to generally satisfactory
(telephone 65-790-3928; fax 65-896-9434; e-mail cyang@nie.edu.sg). GBE product (GBESE).

10.1021/jf010945f CCC: $22.00 © 2002 American Chemical Society

Published on Web 01/16/2002
Supercritical CO2 Extraction of Ginkgo biloba J. Agric. Food Chem., Vol. 50, No. 4, 2002 847

Table 1. L4(23) Tests and Results for Supercritical CO2 Extraction of

Ginkgo biloba
variables
T P ethanol GBE products
test no. (°C) (MPa) (%) W (g) color
1 50 10.0 1.0 0.17 ± 0.01 light yellow
2 50 30.0 5.0 0.48 ± 0.02 orange yellow
3 80 10.0 5.0 0.44 ± 0.06 orange yellow
4 80 30.0 1.0 0.38 ± 0.02 yellow
level I (g) a 0.33 0.31 0.28
level II (g) 0.41 0.43 0.46
∆W2 (g2)b 0.0064 0.0144 0.0324
Figure 1. Schematic diagram of extraction of Ginkgo biloba leaves using
supercritical CO2: (1) CO2 tank; (2) reservoir for modifier; (3) SFE pump; a The values for level I and level II are the averages of the weight of GBE
(4) cooling jacket; (5) modifier pump; (6) mixer; (7) SFE-931 extractor; products at lower values and higher values of the variable, respectively. b ∆W2
(8) preheater; (9) extraction chamber; (10) heater; (11) collection vessel; represents the square of the difference between the values of levels I and II.
and (12) valves.
The schematic process of SC CO2 extraction of GBE is depicted in leaves using SC CO2. The product appears viscous and dark
Figure 1. The laboratory-scale SFE-931 processing unit was designed green, which demonstrates that the contents of flavonoids and
and constructed in-house by the Research Center for Eco-Environmental terpenoids were both very low, whereas chlorophyll and glucides
Sciences, Chinese Academy of Sciences, Beijing, China. The 316 remained in the products. Efforts including changing the
stainless steel chamber with an inner diameter of 10 mm, a length of
operating temperature and pressure, as well as adding modifier,
50 mm, and a wall thickness of 4 mm, was designed to withstand high
temperatures and high pressures (cf. 40.0 MPa). A known amount of were applied to improve the process; unfortunately, none were
sample (typically 1.0 to 5.0 g) was placed in the extraction chamber, noticably effective. This demonstrates that it is not feasible to
and silica gel was used to eliminate the dead volume. Liquid CO2 and obtain high-quality GBE via a single-step direct SFE extraction
absolute ethanol were separately introduced to a mixer at a predeter- from Ginkgo biloba leaves. On the other hand, the contents of
mined volumetric ratio by two pumps. Liquid CO2 into the SFE pump TFL and TTE are only around 1.2 and 0.4 g, respectively, in
was supplied by means of condensation or by placing the tank in a 100 g of dry Ginkgo biloba leaves (16). The use of direct SFE
position reverse to that used for direct liquid CO2 transfer. Extractant extraction from Ginkgo biloba leaves would involve a greater
from the mixer was transferred to the extraction chamber. In the first CO2 consumption, thereby it is not an economical means. In
operating mode, enough extractant was introduced to allow for a 5-min this case, a pretreatment step is essential, resulting in significant
static conditioning for sufficient contact between matrixes and extrac-
reductions in the volume and mass of material and the
tant. The second operating mode was followed by a steady flow of SC
CO2 under the dynamic extraction condition. Certain temperature and elimination of many unwanted substances, by which the SFE
pressure were maintained to allow CO2 in the supercritical state. of the effective contents is more cost-effective and practicable.
Dynamic extraction lasted 30 min to 1.0 h with an approximate flowrate Optimization of Experimental Conditions. Before the SC
of 1.0 mL/min. Products from the chamber were collected in a vessel CO2 extraction of GBERaw, experimental conditions were
containing 10 mL of 70% ethanol solution. The solvent was vaporized, optimized first. Three variables, including pressure (P), tem-
and the GBE was obtained for subsequent qualitative and quantitative perature (T), and modifier volume, were selected as possible
analysis by high-performance liquid chromatography (HPLC). factors which influence the SFE extraction process. The
Measurement of Composition of GBEs by HPLC. According to experimental design by orthogonal method [L4(23)] focused on
related literature (12-15) pretreatment of GBE samples was essential the three variables, each chosen at two levels (Table 1), and
in order to obtain accurate results. GBE powder (0.1 g) was weighed
all experiments were performed in triplicate using 3.5 g of
and dissolved into a methanol/water solution. With respect to the
measurement of the total flavonoids in samples, hydrolysis of GBE GBERaw powder. In this work, the experimental results collected
was conducted in 2% HCl aqueous solution under 90 °C before HPLC- are weight (W) of GBE and the product coloration, because they
UV analysis. To determine the total terpenoids, including ginkgolides could vary with the experimental conditions and could reflect
and bliobalide, GBE aqueous solution was loaded on an acidic alumina the overall product quality. In general, yellow GBE product
column and eluted with methanol. The methanol in the eluate was contains more flavonoids than orange or brown GBE products.
vaporized under a nitrogen gas stream to a predetermined volume for To determine the effect of the variability in the experimental
assaying by HPLC method using a refractive index (RI) detector. system caused by variables, the two averages of the weight of
Separation and assaying of components of GBE were conducted on GBE products for level I and level II are examined according
an HPLC (LC-1050, Hewlett-Packard Co., Palo Alto, CA) equipped
to the results. The squares of the differences between the two
with an UV detector and a refractive index detector (LC-25 Perkin-
Elmer Co., Wellesley, MA). The separation was performed on a C18
levels (∆W2) are then computed from the averages. As can be
column (4.0 mm i.d. × 150 mm length, particle size 5.0 µm). The noted, the largest difference is obtained from the results for the
mobile phase was a methanol/water/phosphoric acid (58:41.5:0.5, v/v) variable of modifier, which indicates that the modifier present
mixture under the optimal flow rate for the individual analyte to achieve in SC CO2 affects extraction of GBE in a most significant way.
complete analytical separation with a short analysis time. The concen- Thereby, it can be concluded that the most important influential
trations of the analytes were determined with external standards for factor is the modifier, followed by pressure and temperature.
calibration. The method detection limits are 1 ng level for three Moreover, in view of the results summarized in Table 1, the
flavonoids: quercetin (QU), kaempferol (KA), and isorhamnetin (iso- optimized conditions are higher content of modifier, higher
RH) by HPLC/UV, and 1 µg level for ginkgolides A, B, and C (GA, temperature, and higher operating pressure.
GB, and GC) and bilobalide (BB) by HPLC-RI (14, 15).
Although increasing pressure and temperature can enhance
the extracting efficiency of SC CO2 and improve the product
RESULTS AND DISCUSSION
quality of active compounds from Ginkgo biloba leaves, because
Direct SFE from Ginkgo biloba Leaves. This study at- carbon dioxide is a nonpolar extractant, the polarity change by
tempted to directly extract effective contents from Ginkgo biloba means of altering temperature and pressure has limited effect
848 J. Agric. Food Chem., Vol. 50, No. 4, 2002 Yang et al.

Table 2. Influence of Modifier on the Extraction of GBE with Table 4. Comparison of GBEs Produced by Conventional Solvent
Supercritical CO2 at 30.0 MPa and 60 °C Extraction and by Supercritical CO2 Extraction
ethanol (%) yield (%) color TFL (%) TTE (%) flavonoids (%) terpenoids (%)
0 0.3 light yellow 15.3 ± 0.5 1.2 ± 0.1 GBE yielda (%) QU KA iso-RH GA GB GC BB
1.0 1.6 light yellow 23.4 ± 1.1 4.8 ± 0.3
GBESE 1.8 ± 0.2 20.6 6.2 5.0 0.5 0.3 0.2 1.7
5.0 2.1 golden yellow 35.9 ± 1.3 7.3 ± 0.6
GBESFE 2.1 ± 0.1 19.6 8.8 7.5 1.7 1.0 0.4 4.2
10.0 2.2 brown 29.4 ± 1.2 5.8 ± 0.6
a Calculated from the weight of final GBE products and the feed of dry Ginkgo

Table 3. Effects of Pressure and Temperature on the Extraction of biloba leaves.

GBE with Supercritical CO2 Containing 5% Ethanol
position of thermally liable active ingredients present in the
experimental conditions GBE products finished products. Therefore, in general, it is recommended that
P (MPa) T (°C) yield (%) color operation be maintained at appropriate pressure and medium
8.0 60 0.8 light yellow temperature, say 30.0 MPa and 60 °C.
15.0 60 1.4 light yellow Extraction of Active Components from Raw GBE using
25.0 60 1.8 yellow Supercritical CO2. According to the results mentioned in the
30.0 60 2.1 golden yellow previous sections, the SFE of GBERaw took 30 min with SC
30.0 40 1.7 light yellow
30.0 80 2.1 orange yellow CO2 containing 5 vol % of ethanol as modifier, at 30.0 MPa
30.0 100 2.3 brown and 60 °C. The product of GBESFE obtained under these SFE
conditions appeared as golden-yellow powder with bitter
taste and is completely soluble in ethanol and 50% methanol
on the extraction of more polar substances, such as flavonoids in water.
and terpenoids. To increase the solubility of the moderately polar Table 4 presents the comparison of GBEs extracted from
solutes and improve their isolation selectivity, small amounts GBERaw with conventional organic solvents and SF CO2. The
of polar cosolvent can be used to modify the polarity and 2.1% yield of GBESFE is 15% greater than the 1.8% yield of
selectivity of SC CO2. Ethanol is by far the most common GBESE by conventional solvent extraction. The contents of total
solvent used to enhance the supercritical extraction for food or flavonoids and total terpenoids in GBESFE are both higher than
medical processes because of its low toxicity. the generally recognized critical values of 24% and 6%,
The data in Table 2 show that the addition of ethanol in SC respectively. The 7.3% of terpenoid contents is 2.7 times higher
CO2 can substantially improve the extraction efficiency of than the value of 2.7% obtained from GBESE. In addition,
flavonoids and terpenoids. This demonstrates that a minor although the whole process of GBESFE involves ethanol used
fluctuation in the volume composition of modifier has a rather in the primary step and SFE step, in fact, ethanol is often
considerable impact on the polarity of extractant, and subse- selected as an extractant or an additive in food and drug
quently on the overall extraction efficiency of GBE. It was also processing. Therefore, the presence of a trace amount of ethanol
found that the contents of GBEs in terms of flavonoids and has no or little negative influence on the GBE quality.
terpenoids vary with the volume composition of modifier in
SC CO2. It is noted that the TFL and TTE in GBE for SC CO2
ACKNOWLEDGMENT
extraction modified with 10% ethanol are both lower than those
in GBE for 5% ethanol modifier. One possible way to explain
We thank W. E. Liu, T. M. Liu, and S. G. Chu for their help
the decrease of the purity is that more polar substances were
and support.
extracted together with active compounds because the addition
of modifier also increases their solubility in the extractant. On
the other hand, as the modifier in the extractant increases to a LITERATURE CITED
certain content, a higher temperature is required to reach the
increased critical temperature (Tc). Therefore, the content of (1) Braquet, P. The effects of PAF-acether on the cardiovascular
ethanol should be in a range of 5-10 vol %. system and their inhibition by a new highly specific PAF-acether
On the basis of the results from the orthogonal experiments, receptor antagonist BN52021. Pharm. Res. Commun. 1986, 8,
pressure and temperature have effect on the extraction of GBE 717-722.
(2) Kleijnen, J.; Knipschild, P. Ginkgo biloba for cerebral insuf-
using SC CO2. The influences of the two variables were further
ficiency. Br. J. Clin. Pharm. 1992, 34, 352-358.
investigated in order to achieve the best results. Table 3 shows (3) Vesper, J.; Hansgen, K. D. Efficacy of Ginkgo biloba in 90
the experimental conditions and the yield and color of GBE outpatients with cerebral insufficiency caused by old age.
products. As can be seen, while the temperature was set at 60 Phytomedicine 1994, 1, 9-16.
°C and with a volume of 5% of ethanol as modifier, the yield (4) Hofferberth, B. The efficacy of EGB 761 in patients with senile
of GBE increases from 0.8 to 2.1% with an increase of pressure dementia of the Alzheimer type, a double-blind, placebo-
from 8.0 to 30.0 MPa. This is explained by the fact that the controlled study on different levels of investigation. Hum.
density and viscosity of supercritical carbon dioxide change and, Psychopharm. 1994, 9, 215-222.
therefore, its extracting power increases. It is also worth (5) Ge, F. H.; Li, J.; Wang, H. B.; Hui, G. Progress of the application
mentioning that high pressure is bound to result in greater cost of supercritical CO2 technology in the natural product and
pharmaceutical analysis. Zhongyaocai 1995, 18, 316-319.
for the extraction operating system, as well as stringent operation
(6) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Supercritical fluid
design and increased energy demand. Likewise, increased chromatography and extraction. Anal. Chem. 1996, 68, 487-
temperatures have a favorable effect on the extraction efficiency. 514R.
However, when the temperature reaches 80 °C, the effective (7) Yuan, Y. F., Zhou, J., Zheng, X. M. Studies on volatile oil in
contents possibly begin to decrease according to the color of Ligusticum chuanxiong by supercritical fluid extraction.
GBEs. Moreover, elevated temperature could lead to decom- Zhongguo Yaoxue Zazhi 2000, 35, 84-87.
Supercritical CO2 Extraction of Ginkgo biloba J. Agric. Food Chem., Vol. 50, No. 4, 2002 849

(8) Yao, W. X.; Chen, X. M.; Zhang, Y. Q. Determination of (14) Yao, W. X.; Zhang, Z.; Su, Z. Y.; Yao, Y. Y. Ecological
flavonoid compounds in Ginkgo biloba leaves by supercritical evolution of ginkgo flavonoids and its determination using
fluid extraction and high performance liquid chromatography. hydrolysis and HPLC. Yaowu Fenxi Zazhi (Supplement) 1995,
China Chem. Lett. 1995, 6, 589-592. 15, 381-383.
(9) van Beek, T. A.; Taylor, L. T. Sample preparation of standardized (15) Yao, W. X.; Yang, C.; Tian, Y.; Liu, T. M.; Xu, Y. R. Rapid
extracts of Ginkgo biloba by supercritical fluid extraction. determination of ginkgolides and bilobalides in leaves of Ginkgo
Phytochem. Anal. 1996, 7, 185-191. biloba Lour and its extracts by HPLC. Yaowu Fenxi Zazhi 1999,
(10) Deng, Q. H.; Gao, Y. A. study on effective components from
1, 38-40.
ginkgo leaves by second supercritical fluid extraction.
(16) Yu, Q.; Shen, Z. B.; Chen, X.; Tan, W. H.; Wang, C. Z. Study
Zhongcaoyao 1999, 30, 419-422.
of the factors affecting the effective components in ginkgo leaves.
(11) Shen, G.; Yao, W. X. Determination of ginkgolic acid using
supercritical fluid extraction and high performance liquid chro- The Proceedings of Chinese Conference on Ginkgo biloba.
matography. Fenxi Huaxue 2000, 28, 985-988. Shanghai, China, 1997; pp 78-83.
(12) Hasler, A.; Sticher, O. Identification and determination of
flavonoid from Ginkgo biloba by high performance liquid
chromatography. J. Chromatogr. 1992, 605, 41-48. Received for review July 20, 2001. Revised manuscript received
(13) van Beek, T. A.; Scheeren, H. A.; Rantio, T.; Melger, W. C.; November 15, 2001. Accepted November 16, 2001.
Lelyveld, G. P. Determination of ginkgolides and bilobalide in
Ginkgo biloba leaves and phytopharmaceuticals. J. Chromatogr.
1991, 543, 375-387. JF010945F