Analyzing of Major Active Luteolin in Vernonia Cinerea

International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
Analyzing of Major Active Luteolin in Vernonia Cinerea
Guan-Rui Lai and Chiao-Song Wu

times higher than that of liquid.
Supercritical fluid extraction is a process with no
pollution, no toxic solvent, no organic residues. Carbon
dioxide can be easily removed in supercritical fluid
extraction.
Abstract—This study investigates the extraction of the
Vernonia cinerea using supercritical carbon dioxide extraction
under the following conditions pressure at 300, 400 and 500
bar, temperatures at 313, 323 and 333 K, CO2 flow rate at 2
and 3 L/min. The best operation of extraction is under 500 bar,
333 K and 3 L/min CO2 flow rate. The reverse-phase high
performance liquid chromatography method was developed
for determining major active components of Vernonia cinerea.
We dissolve the sample with methanol and analyzed by using a
MetaChem C18-A(250 × 4.6m, 5μ) column with a gradient
elution composed of ultra-pure water and acetonitrile. The
wavelength of UV detector was set at 280 nm and the flow-rate
was set at 1 mL/min. Under this condition, the ingredients of
Vernonia cinerea can be detected.
Index Terms—Vernonia cinerea, supercritical carbon
dioxide, luteolin, flavonoids.
I. INTRODUCTION
Organic solvent is usually used to extract product. But
organic solvent must be separated out and therefore the
procedure is quite complicated.
Extraction of biomass by Press method is easy to destroy
the structure and thus affect the quality of the raw materials.
The most commonly used in cellulite fruit extraction of
essential oil, the extraction yield is good, the procedure is
simple, but the extract containing a lot of impurities must be
made through the separation and purification, only relatively
pure product make it suitable for high-value products.
Steam distillation is most commonly used for oil
extraction, the hot steam extract the essential oils of the plant,
but if the essential oil has the thermal unstable property. The
other drawback of steam distillation is that only some of
active composition is extracted. Substance exist as gas,
liquid or solid phase, but when the temperature and pressure
above its critical temperature and critical pressure, it enters
the so-called supercritical fluid state, shown in Fig. 1. When
the temperature is below the critical temperature, It have
obvious interface between the gas-liquid phase, but the
critical point is reached, this interface disappear. The
physical properties of the supercritical fluid are between the
gas and liquid. The density of supercritical fluid is near to
the density of liquid. So that the dissolution power of
supercritical fluid is higher than that of gas. The viscosity of
supercritical fluid is near to that of the gas, so the conveying
power in the supercritical fluid is higher than that in liquid.
The diffusion coefficient of supercritical fluid is 10 to 100
Fig. 1. The phase diagram of the substance [1].
II. VERNONIA CINEREA
Vernonia cinerea is a perennial grass plants. Its stems with
vertical stripes is erected, The height of Vernonia cinerea is
forty to one hundred centimeters. The shape of leaves are
ovate or oval, the leaves have three to seven centimeters
long and one to three centimeters wide.
Vernonia cinerea is distributed in mainland China, Fujian,
Guangdong, Guangxi, Jiangxi, Hunan, Sichuan, Yunnan
and Vietnam, Myanmar, India, the Philippines, Australia,
Africa and other places. It is distributed in Taiwan among
the plains, the waterfront and the low altitude of the
mountain. The stems of Vernonia cinerea are cooked or fried
[2].
A. The Physiologically Active
Vernonia cinerea is picked in late spring to winter and is
collected after the removal of soil and then dried for use.The
whole plant of Vernonia cinerea is used as medicine .Its
flavors are Cool or slightly cold, bitter, slightly sweet
slightly acrid. The effectiveness of Vernonia cinerea is free
to fever, to stop bleeding, the anti-inflammatory,
detoxification, swelling.
Flavonoids categories (Flavonoids) are widely distributed
in nature. It is a type of important natural organic
compounds. The ionization states of Flavonoids exist in
plants. The majority type of Flavonoids is combined with
sugar into glycosides type. The flavonoids categories refer
to the two benzene rings through mutual associates with
Manuscript received January 3, 2013; revised March 26, 2013.
Guan-Rui Lai is with Ming Chi University of Technology, Department
of Chemical Engineering, TW (e-mail: [email protected]).
DOI: 10.7763/IJBBB.2013.V3.233
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
The extraction tube was put into the thermostatic chamber.
The temperature of thermostatic chamber was set at 313, 323
and 333 k.
Carbon dioxide comes from CO2 cylinder. The
temperature of carbon dioxide condenser was set at 1 0C.
Carbon dioxide from condenser was pumped into the
extraction tube by high pressure pump. The pressure of high
pressure pump was set to 300, 400 and 500 bar.
The extraction tube was controlled in constant
temperature and pressure. The time of static extraction was
set at 30 minute. After the static extraction, dynamic
extraction is proceeding. The flow rate of carbon dioxide in
dynamic extraction was set at 2 and 3L/min, and the extract
of herb was collected every thirty minutes and the weight of
extract. The reverse-phase high performance liquid
chromatography was used to determine major active
components of Vernonia cinerea from supercritical carbon
dioxide extraction. We extracted the sample with methanol
and analyzed by using a metachem C18-A(250×4.6mm, 5μ)
column with a gradient Elution composed of ultra-pure
water and acetonitrile. The wavelength of UV detector was
set at 280nm and the flow-rate was set at 1mL/min. Under
this condition, the ingredients of Vernonia cinerea can be
detected [2].
three carbon chain compounds. Natural flavonoid classified
on the mother nucleus of compounds often contain
substituents such as a hydroxyl group, a methoxy group, a
methyl group, an abnormal pentenyl group, etc. According
to their structural characteristics of Flavonoids , can be
divided into the flavonoids (Flavone), flavonols (Flavonol),
Isoflavones (Isoflavone) and flavanones of alcohol
(Flavanol).Flavonoid group of compounds is often exited as
crystalline solid majority or minority (such as flavonoid
glycoside analog) powder. Flavonoid types of six
Compounds of different structure have different
solubility[3]. The flavonoid glycosides types is soluble in
methanol and ethanol polar organic Solvent, due to the
molecules and intermolecular permutation closely. It is
difficult to dissolve in water. Flavonols, such as a flat-type
molecule, due to stuffing closely intermolecular attraction is
large, it is difficult to dissolve in the water. Flavonoid types
of plant ingredients with a wide range of different plant
components may have different physiological activity and
pharmacodynamics role[4]. Currently there are number of
thousands of flavonoid group of compounds is separated,
including domestic and foreign scholars. From Vernonia
cinerea carved from the flavonoid group of compounds
identified active ingredients, such as: apigenin (Apigein),
luteolin (Luteolin), Quercetin (Quercetin) [5]. Luteolin has a
role on the cardiovascular system, central nervous system
function, Enhanced cell immunity,anti-cancer and
anti-tumor function of a variety of pharmacological.
B. Supercritical Extraction Technology
The extraction of Supercritical fluid carbon dioxide is
better than traditional extraction. The component of extract
depends on the temperature and pressure. Changing the
temperature and pressure can vary the component solubility
of extract in supercritical carbon dioxide. The temperature in
the supercritical fluid extraction process is low. The active
ingredient in herb is not destroyed. So the heat sensitive
active ingredient can be extracted.
Supercritical fluid carbon dioxide extraction is
non-polluting, non-toxic, non-organic residues and
environmentally-friendly, Supercritical fluid carbon dioxide
extraction make the process of extraction and solvent
removal into one simplified operation and improve
extraction efficiency. Supercritical carbon dioxide
extraction technology is a room temperature extraction, no
damage to the heat-sensitive substances. Have a great impact
on the destructive effect of high temperature food nutrition,
especially vitamins. Supercritical carbon dioxide extraction
is a process without the participation of oxygen and light and
with stable product quality [6].
Fig. 2. Schematic diagram of the supercritical extraction. 1:CO2 Tank,
2:Control Valve, 3:Cooler,4:Air Compressor, 5:Compressor Pressure
transducer and Controler, 6:Mixed Block, 7:Filter, 8:Extracted Tube,
9:Heater and Thermometer, 10:Sample Collect Bottle, 11:Wet Test Meter,
12:Co-solvent Bottle, 13:Liquid Pump, 14:Micro Valve.
IV. RESULTS AND DISCUSSION
III. EXPERIMENTAL METHOD
Vernonia cinerea is ground by artificial method. The
ground powder size was controlled within 1mm. Then
ground power was placed in an oven at 60 ℃ and was dried
for 3 hours. Water was removed before extraction. Water in
herb will affect the results of the experimental data. After
drying, 10 g of Vernonia cinerea powder was put into the
extraction tube. The volume of extraction tube is 100 mL.
Fig. 3. The extraction curve of Vernonia cinerea at pressure 500 bar,
temperature 333 K, flow 2 L/min under the drying time.
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
dioxide density is decreased and the solute solubility in the
carbon dioxide fluid is reduced. Another factor is that when
the extraction temperature increases, the vapor pressure of
solute increases. The solute dissolving power increased. In
this study, the yield of extraction increase with increasing
temperature as shown in Fig. 4 - Fig. 6. In the fixed
temperature and pressure, it will increase the yield of
extraction with increasing CO2 flow rate as shown in Fig.6.
The mass transfer resistance will decrease with increasing
CO2 flow rate. If we fixed the temperature and the CO2 flow
rate. It will increase the yield of extraction with increasing
extraction pressure as shown in Fig. 7. In high extraction
pressure, the density of supercritical fluid will increase and
the solubility of solute will increase.
Fig. 4. Pressure 300 bar flow 2 L/min and not under the same temperature
extraction curve.
Fig. 7. Pressure 500 bar, temperature of 333 K, not the same flow extraction
curve.
Fig. 5. Pressure 400 bar, the flow 2 L/min different temperature extraction
curve.
Fig. 8. Temperature of 333 K, flow 3 L/min and not under the same pressure
extraction curve.
23'
8
12
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28
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Fig. 9. Same pressure 300 bar and flow rate of 2 ml / min at 313 K.
365
min
49.597'
47.144'
38.923'
35.546'
29.711'
31.065'
32.868'
27.777'
25.258'
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20.863'
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15.312'
10.171'
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2.8
08'
3.7
4.5
61'
6.3
95'98'
If the samples in supercritical fluid extraction process
have moisture, water may form ice to cause instrument
blocked. Thus we remove excess moisture from samples.
The experimental results of Vernonia cinerea extraction
at300 K, 500 bar, 3 L/min CO2 flow rate are shown in Fig. 3.
The drying time of largest yield in extraction is 2 hours.
When the pressure and CO2 flow rate are fixed, we change
the extraction temperature. Two factors will affect the yield
of extraction. When the temperature increase, the carbon
8.328'
67'
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6.6
7.3
mV
Fig. 6. Pressure 500 bar, the extraction flow 2L/min different temperature
curve.
International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
500bar. Fig. 15 is the HPLC figure of standard Luteolin. The
retention time of HPLC column is about 10 minutes.
mV
mV
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45.544'
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35.546'
29.711'
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32.868'
27.777'
25.258'
19.279'
20.863'
22.051'
15.312'
Fig. 14. A temperature of 333 k, flow 3 ml / min the same under 500 bar.
2.808'
3.767'
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6.395' 6.698'
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30.979'
32.732'
32
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29.738'
28
25.303'
24
21.794'
20
19.076'
16
Fig. 10. Same pressure 300 bar and flow rate of 2 ml / min at 323 K.
15.097'
12
400
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2.802'
3.768'
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mV
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3.767'
4.477'
400
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min
Fig. 11. Same pressure 300 bar and flow rate of 2 ml / min at 333 K.
mV
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12
REFERENCES
18.770'
19.811'
16
20
[1]
24
28
32
[2]
36
45.868'
47.388'
37.148'
38.780'
29.293'
30.789'
32.384'
33.893'
25.902'
22.877'
15.332'
17.108'
18.126'
19.355'
20.338'
10.458'
11.747'
13.118'
1.892'
2.846'
3.829'
4.835'
6.107'
6.503' 6.938'
7.594'
8.311'
8
12.539'
mV
400
350
300
250
200
150
100
50
0
-50 4
54.848'
46.182'
47.804'
49.677'
39.201'
37.080'
29.343'
30.818'
32.432'
33.947'
28
[3]
min
Fig. 13. A temperature of 333 k, flow 3 ml / min the same under 400 bar.
[4]
Fig. 9 - Fig. 11 is at the pressure300bar and flow rate 2
mL / min at different temperature were compared, the
retention time of Luteolin in HPLC is about 8.4min. Fig. 9 Fig. 11 shows that the content of Luteolin will be almost
same between 313K and 333K. Fig. 12 - Fig. 14 is at the
temperature 333K and flow rate 3 mL / min at different
pressure were compared, Fig.12 - Fig. 14 shows that in
400bar the content of Luteolin will be larger than 300bar and
[5]
[6]
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54.761'
16
Fig. 12. A temperature of 333 k, flow 3 ml / min the same under 300 bar.
52.123'
12
V. CONCLUSION
The traditional extraction used a large amount of organic
solvent. Organic solvent is pollution liquid. It needs much
energy and money to separated organic solvent from extract.
It is not suitable for heat sensitive herbs. In this study, the
best condition of extraction is under extraction pressure 500
bar, extraction temperature 333 K, the CO2 flow rate 3
L/min.
49.416'
8
Fig. 15. Luteolin component of the standard retention time.
10.483'
11.787'
13.159'
13.809'
15.393'
17.180'
18.103'
19.706'
20.374'
21.883'
23.177'
24.968'
25.998'
2.844'
3.828'
4.898'
6.082' 6.541'
6.963'
7.602'
8.332'
400
350
300
250
200
150
100
50
0
-50 4
International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
Guan-Rui Lai lives in No. 157, Lane 82, Chung
Cheng Road, Cian Jhuang Village, Daliao,
Kaohsiung, Taiwani and he was born on January
30, 1987. He was graduate from Minhsin
University of Science and Technology in
Hsinchu city Taiwan and now is studying in
Ming Chi University of Technology. His
research field is the technology of supercritical
Chiao-Song Wu got his doctor in National Taiwan
University. He worked in The Associate Professor
of Department of Chemical Engineering, Ming Chi
University of Technology.
fluid extraction.
367