Identification of two polysaccharides from Prunella vulgaris L. and evaluation on their anti-lung adenocarcinoma activity

Article abstract of DOI:10.3390/molecules15085093

Prunella vulgaris L. (PV) has been used for tumor therapy in Traditional Chinese Medicine for centuries, however, systematic research on extracted PV polysaccharides believed to possess various biological activities, as well as their preventive and anti-t

Full text of DOI:10.3390/molecules15085093

Molecules 2010, 15, 5093-5103; doi:10.3390/molecules15085093
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molecules
www.mdpi.com/journal/molecules
ISSN 1420-3049
Article
Identification of Two Polysaccharides from Prunella vulgaris L.
and Evaluation on Their Anti-Lung Adenocarcinoma Activity
Liang Feng 1,2, Xiao-Bin Jia 1,*, Feng Shi 1 and Yan Chen 1
1
Key Laboratory of Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of
Chinese Medicine, Jiangsu, Nanjing, 210028, China; E-Mails: shifeng_1985_wcl@163.com (F.S.);
ychen202@yahoo.com.cn (Y.C.)
2
Biotechnology Labortory of Chinese Medicine, Macau University of Science and Technology,
Macau, China; E-Mail: wenmoxiushi@163.com (L.F.)
* Author to whom correspondence should be addressed; E-Mail: jxiaobin2005@hotmail.com;
Tel.: 86-25-856378091; Fax: 86-25-85637809.
Received: 12 May 2010; in revised form: 13 July 2010 / Accepted: 26 July 2010 /
Published: 27 July 2010
Abstract: Prunella vulgaris L. (PV) has been used for tumor therapy in Traditional
Chinese Medicine for centuries, however, systematic research on extracted PV
polysaccharides believed to possess various biological activities, as well as their preventive
and anti-tumor effects on lung cancer has not been reported. In this study, two
polysaccharides (P31 and P32) were isolated from the aqueous extract of PV and purified
through ethanol precipitation, followed by deproteination using DEAE-52 gel-filtration
chromatography. The main monosaccharide composition of polysaccharide P32 was
analyzed by GC. It was found that polysaccharide P32 consisted of rhamnose, arabinose,
xylose, mannose, glucose and galactose in a molar ratio of 3.46:49.32:58.91:0.43:2.64:
3.11, respectively. In order to evaluate polysaccharide P32’s anti-lung adenocarcinoma
activities and immunomodulation effects, a C57BL/6 mouse-Lewis lung carcinoma (LLC)
model was established and investigated. Our results showed that polysaccharides of PV
had anti-lung cancer activity and could increase the thymus index and the spleen index in
tumor-bearing mice, suggesting possible immunomodulation effects.

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Keywords: Prunella vulgaris L.; polysaccharide; GC; anti-lung adenocarcinoma; immune
function
1. Introduction
Prunella vulgaris L. (PV) belongs to the Labiatae family of perennial plants and is widely
distributed in Asia and Europe [1,2]; its dried spikes are often utilized in Traditional Chinese Medicine.
In the Chinese Pharmacopoeia, PV is commonly used to treat headaches, high blood pressure, diseases
of the lymphatic system, goiter and tuberculosis. It has long been used clinically in China for the
prevention and therapy of non small-cell lung cancer. Currently, PV is considered more compatible
with other anti-tumor herbs in lung cancer treatment. It contains components such as terpenoids,
flavonoids, polyphenols [3] and polysaccharides that are known to be associated with tumor inhibitory
effects. Polysaccharides is some of its main bioactive compounds that possesses immunoregulatory
[4,5], anti-inflammatory [6,7], anti-virus [8] and antioxidant [9] activities. In general, qualitative and
quantitative analyses of the polysaccharide contents in PV are performed using methods such as
colorimetry and high performance liquid chromatography (HPLC). Gas chromatography (GC) is a
convenient method also widely employed for the identification of polysaccharide compositions [10,11].
In this study, the compositions of purified polysaccharides extracted from PV were analyzed by GC.
Moreover, the effects of these polysaccharides on lung adenocarcinoma and on immune regulation
were investigated in vivo using the C57BL/6 mouse-Lewis lung carcinoma (LLC) model.
2. Results and Discussion
2.1. Isolation of polysaccharide from PV
Proteins were removed using the trichloroacetic acid method and the Sevag method (applied once)
[12,13]. Samples were scanned at a wavelength of 190–600 nm. As shown in Figure 1, no presence of
proteins and nucleic acids were detected after protein removal, as indicated by the disappearance of the
absorption at 260–280 nm.
Figure 1. The UV absorbance spectra of polysaccharide before and after deproteinization.
A
B
proteins and
nucleic acids
The UV absorption wavelength of proteins and nucleic acids is from 260–280 nm so this absorption
peak was used to determine if the polysaccharide solution contained proteins and nucleic acids (A.
Polysaccharide before protein removal; B. Polysaccharide after protein removal).

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2.2. Molecular weight determination of polysaccharide
A standard curve was generated based on the Mw, Ve and Kav of dextran standards with various
molecular weights (Table 1) as follows: Y = -3.4754X + 6.5431 (r = 0.9991, where Y is the logarithm
of molecular weight, and X is the Kav value. The standard curve is shown in Figure 2.
Table1. The computed value of dextrans,blue dextran and glucose Mw,Ve and Kav.
Dextran
T-10
Mw
10,000
LgMw
4
Ve ( min)
12.643
11.344
10.895
9.325
Kav=(Ve-Vo)/(Vt-Vo)
0.7353
T-40
40,000
4.602
4.845
5.699
6.301
0.5494
T-70
70,000
0.4852
T-500
T-2000
500,000
2,000,000
0.2606
7.930
0.061
Note: Ve was retention time of dextrans; Vo was retention time of blue dextran; Vt was retention
time of glucose at 14.495.
Figure 2. The standard curve of Mw and Kav.
6.5
6.0
5.5
5.0
4.5
4.0
0.0
0 .1
0.2
0.3
0.4
0.5
0.6
0 .7
0 .8
Three polysaccharide components (P31, P32, P33) were observed in the HPLC chromatograms
(Figure 3) with molecular weights of 242,641, 58,060 and 4,980, respectively (Table 2).
Figure 3. HPLC chromatograms of glucose (A), T-10 (B), T-40 (C), T-70 (D), T-500 (E),
T-2000 (F), blue dextran (G), PV (H). T-10, T-40, T-70, T-500 and T-2000 correspond to
different molecular weight dextran standards listed in Table 1 with Mw = 10,000, 40,000,
70,000, 500,000 and 2,000,000, respectively.
A
B

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Figure 3. Cont.
C
D
E
F
P32
G
0.004
0.002
0.000
H
P31
P33
0
5
10
Minutes
15
20
Table 2. The retention time and molecular weight of polysaccharides from PV.
Components
Retention time(min)
Molecular weight
P31
P32
P33
9.833
11.082
13.227
242,641
58,060
4,980
In the purification of polysaccharides, DEAE-52 cellulose was used in order to obtain the final
polysaccharide fractions (P31, P32). The elution curve (Figure 4) has a symmetrical peak shape,
indicating good separation. The results indicate that polysaccharide P32 (between 150 -200 tubes) was
the main component; therefore, it was selected for further characterization.

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Figure 4. Absorbance by tube number.
2.3. Monosaccharide composition of polysaccharide and its molar ratio
Polysaccharide P32 was analyzed by GC and the results are shown in Figure 5. Each
monosaccharide peak in the order of increasing retention time was identified as rhamnose, arabinose,
xylose, mannose, glucose, galactose and myo-inositol hexaacetate. The corresponding retention times
were 16.88, 17.38, 17.82, 23.57, 23.89, 24.48 and 27.28 min, respectively. The molar ratio of
rhamnose: arabinose: xylose: mannose: glucose: galactose was 3.46: 49.32: 58.91: 0.43: 2.64: 3.11.
Figure 5. The gas chromatogram of mixed substances and polysaccharide P32 of PV.
uV(x100,000)
C
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
95.0
90.0
85.0
80.0
75.0
70.0
65.0
60.0
55.0
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
Ara
Glu
Gal
A
myo-Inositol
Hexaacetate
Man
Rha
Xyl
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
min
B
The GC conditions as follows: gas flow rate: nitrogen carrier gas for the 50 mL/min, hydrogen 37 mL/min,
air as 370 mL/min; column temperature: The temperature-program 130 ºC (5 min) → 4 ºC /min → 240 ºC
(5min); detector temperature of 300 ºC; gasification chamber temperature of 280 ºC.( A. mixed substances;
B. polysaccharide P32 sample)

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2.4. In vivo anti-lung adenocarcinoma activity of PV polysaccharide
As can be seen in Figure 6, both the polysaccharide P32 and Prunella extract high-dose groups
showed significant tumor inhibiting effects in adenocarcinoma tumor-bearing C57BL/6 mice,
compared with the saline group (p ≤ 0.05).
Figure 6. Tumor inhibitory rate of polysaccharide P32 in the C57BL/6 mice.
Tumor control rate of polysaccharides in vivo
A
high-dose
low-dose
Cg
p
pe
Tumor weight in C57BL/6 mice
B
high-dose
low-dose
Sg
Cg
p
pe
A. Tumor inhibitory rate; B. Tumor weight.*compared with the Saline group (*p ≤ 0.05);
Sg = saline group; Cg = cyclophosphamide group; p= polysaccharide P32; pe = Prunella extract;
the following expression as the same. High-dose was 10 g crude drug/kg; Low-dose was 5 g crude
drug/kg.
As shown in Table 3, the high-dose polysaccharide P32 group significantly enhanced the
thymus index (p ≤ 0.01) in tumor-bearing mice as compared to saline group. A similar effect of
polysaccharide P32 on spleen index regulation was observed (p≤ 0.01), as shown in Table 4. The
spleen index of the high-dose polysaccharide P32 group was considerably higher compared with the
low-dose group (p ≤ 0.01) and the cyclophosphamide group (Cg) (p ≤ 0.01). Our results indicate that
polysaccharide P32 of PV could enhance the immune response in tumor-bearing mice, which could
contribute to the overall anti-tumor effects of the polysaccharides.

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Table 3. Evaluation of polysaccharide on thymus index in tumor-bearing mice.
Group
Thymus (mg)
Weight (g)
Thymus index (mg/g)
Sg
35.71 7.87
21.43 1.72
1.67 0.28
Cg
30.00 8.16
46.25 7.44
38.57 3.78
20.86 2.54
19.88 2.36
20.43 1.72
1.43 0.30
2.33 0.22**
1.89 0.16
10 g crude drug/kg of p
5 g crude drug/kg of p
10 g crude drug/kg of pe
5 g crude drug/kg of pe
41.25 6.40
34.29 7.87
21.63 2.83
19.43 3.15
1.92 0.26*
1.76 0.25
*compared with the saline group and Cg (*p ≤ 0.05;**p ≤ 0.01;). p= polysaccharide P32; pe = Prunella extract.
Table 4. Evaluation of polysaccharide on spleen index in tumor-bearing mice.
Group
Spleen(mg)
Weight (g)
Spleen index (mg/g)
7.26 0.31
Sg
Cg
155.71 16.18
105.71 16.18
192.50 26.05
150.00 24.49
21.43 1.72
20.86 2.54
19.88 2.36
5.06 0.25
9.71 0.90**
7.32 0.80
10 g crude drug/kg of p
5 g crude drug/kg of p
20.43 1.72
10 g crude drug/kg of pe
5 g crude drug/kg of pe
168.75 34.82
155.71 28.78
21.63 2.83
19.43 3.15
7.79 1.19
8.00 0.52
*compared with the saline group and Cg (*p ≤ 0.05;**p ≤ 0.01;), p= polysaccharide P32; pe = Prunella extract.
The spleen and thymus are two main immune organs that play an important role in anti-tumor
activity [15]. In the tumor immune system, CD4+ T cell activation and proliferation are related to
NF-kappaB pathways. Choi et al [16] demonstrated that the aqueous extract of PV has anti-tumor
activity through the suppression of NF-κB activation. We speculate that polysaccharide P32 can
down-regulate expression of NF-κB and inhibit the proliferation of tumor cells.
3. Experimental
3.1. General
The following instruments were used in this study: Millipore ultrafiltration instrument
(XX814V230), DZF-6051 vacuum oven (Shanghai Jing Wang Experimental Equipment Co., Ltd.),
Model 6820 gas chromatograph system (Agilent Inc.), RTX-5 Sil MS GC capillary column, TSK-GEL
G4000PW (7.5 × 300 mm) gel filtration column (TOSOH Corporation), UV-2450 UV-visible
spectrophotometer (Shimadzu Corporation), ALPHA2-4 freeze-drying equipment (CHRIST
Companies, Germany). Sodium hydroxide, hydrochloric acid, chloroform, butanol, acetone, diethyl
ether, trichloroacetic acid, and ethanol were purchased from Nanjing Chemical Reagent Co., Ltd.
DEAE-52 was purchased from Whatman Company. Glucose, rhamnose, xylose, arabinose, galactose,
mannose were purchased from Sinopharm Chemical Reagent Co., Ltd. Remaining reagents were
AR grade.

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3.2. Plant material
Dried spikes of PV was purchased from Medicinal Corporation of Bozhou City, Anhui Province
(batch number 0711128) and authenticated by Professor D.K. Wu from the College of Pharmacy,
Nanjing University of Traditional Chinese Medicine.
3.3. Extraction of crude polysaccharides
Dried PV spikes (200.0 g) were accurately weighed and then extracted twice with distilled water
(2.0 L) under reflux for 2 h. The extracts were combined and centrifuged (4,000 rpm, 30 min). To the
precipitate was added a solution of 85% ethanol (v/v) and the mixture was left standing 12 h and then
filtered. The precipitate was washed successively with 100 mL each of 95% ethanol, anhydrous ethanol,
acetone, diethyl ether (twice), then dried at 40 ºC to obtain the crude polysaccharide P1 (38.7 g).
3.4. Deproteination of crude polysaccharides
Trichloroacetic acid method: Crude polysaccharide solution (100 mL) was added slowly into a 15%
trichloroacetic acid solution (v/v) chilled in an ice bath and stirred constantly until the solution was
without turbidity. The mixture was kept at 4 ºC for 4 h, and then centrifuged to remove the precipitate.
The final pH of the solution was adjusted to neutral by addition of 10% NaOH (5.5 mL). The result
was liquid 1.
Sevag method: To an aliquot of liquid 1 (100 mL) was added chloroform (20 mL), followed by
n-butanol (4 mL). The mixture was shaken by hand for 20 minutes and poured into a separatory funnel.
The denatured proteins were removed at the junction of the water and solvent layer. The extraction was
repeated two times and the extracts were combined to give liquid 2 (P2).
3.5. Molecular weight determination
Polysaccharide and Dextran T standards samples were analyzed on a Shimadzu LC-10AVP HPLC
system equipped with a TSK-GEL G4000PW column (7.5 × 300 mm) and a Shimadzu RID-10A
detector. The mobile phase used consisted of 3.0 mM sodium acetate solution and the flow rate was
1.0 mL/min. The column temperature was maintained at 25 ºC. Polysaccharide solution was filtered
through a 0.45 µm membrane before injection and the injection volume was 10 µL. The HPLC profiles
of polysaccharide sample solutions are shown in Figure 3. The retention times and molecular weights
of the polysaccharide components are summarized in Table 2. The relative molecular mass was
determined according to the standard curve.
3.6. Gel column purification of protein-free polysaccharide
Liquid 2 (P2) was pretreated with DEAE-52 chromatography post-column by eluting with distilled
water, 0.05, 0.10, 0.15, 0.20, 0.50, 1.00, 1.50, and 2.00 mol/L NaCl solution using a DHL-A constant
flow pump at a flow rate of 1 mL/min. An automatic collection device was used to collect the fractions
of 6 mL per tube. The phenol-sulfuric acid method was used and the eluate was monitored at 490 nm
until there was no detection of sugar. The combined eluates were filtered through a Millipore
ultrafiltration system to remove small molecules, and then vacuum concentrated and freeze-dried to

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Molecules 2010, 15
obtain crude polysaccharide. Pretreated P2 was dissolved in distilled water (10 mL) and passed
through a DEAE-52 gel-filtration column. The sample was initially eluted with phosphate buffer
(pH = 7.2) at a flow rate of 6 mL/min for 100 min (tubes 1–100, fractions were collected at 6 mL per
tube), followed by a gradient of 10 mM, l.0 mM, 10 mM NaCl solution (tubes 101-300 at 6 mL per
tube). The fractions were monitored using the phenol-sulfuric acid method and the data was plotted.
Fractions corresponding to the absorbance peaks were combined, concentrated and lyophilized to yield
the final polysaccharide fractions P31 and P32 (total yield: 29.6 g). [14]
3.7. Preparation of myo-inositol hexaacetate as the internal standard
A mixture of inositol (3.0 g), hydroxylamine hydrochloride (4.5 g), acetic anhydride (45 mL) and
pyridine (3 mL) was stirred for 2 h at 90 ºC in a water bath. The reaction mixture was cooled to room
temperature and then poured into ice water (50 mL) to precipitate phytic acid ester. The solid was
filtered and washed with distilled water (20 mL), then dried in oven at 100 ºC and stored for further
use. The final product was analyzed and confirmed by GC as myo-inositol hexaacetate.
3.8. Hydrolysis and derivatization of monosaccharide and polysaccharide
To a test tube was added monosaccharide (5.0 mg), hydroxylamine hydrochloride (5.0 mg),
myo-inositol hexaacetate (5.0 mg) and pyridine (0.5 mL). The reaction mixture was heated for 30 min
at 90 ºC in a water bath with vibration. After the reaction cooled to room temperature, acetic acid
anhydride (0.5 mL) was added and the reaction was heated at 90 ºC for another 30 min to continue the
acetylation reaction to obtain the monosaccharide samples. Polysaccharide P32 sample (5.0 mg) was
dissolved in sulfuric acid solution (10 mL, 2 mol/L) and transferred to an ampoule bottle, then the
bottle was sealed and heated at 105 ºC in the oven for 8 h. After hydrolysis, the solution was
derivatized using the above method described for monosaccharide. The derivatized samples were
injected directly into the GC and analyzed.
3.9. Anti-lung adenocarcinoma activity of polysaccharide in C57BL/6 mice
Male C57BL/6 mice, 6-8 weeks of age and weighing approximately 18–20 g, were obtained from
Shanghai SLAC Laboratory Animal Co., Ltd. and were maintained on a standard environmental
condition. They were fed with standard diet and water ad libitum. Primary tumors were induced by
subcutaneous (s.c.) injection of 106 Lewis cells in 200 µL PBS in the right anterior limb. The mice
were intragastrically administered with 0.4 mL of PV extracted polysaccharide P32 solution (high dose:
75 mg/mL; low dose: 37.5 mg/mL), which was equivalent to 5–10 g crude drug. The Prunella extract
group was intragastrically administered aqueous extract of PV (high dose: 10 g crude drug/kg; low
dose: 5 g crude drug/kg). The drugs were administered every day for 14 consecutive days. Positive
control mice were injected with 0.2 mL/day of cyclophosphamide (20 mg/kg), while blank control
mice received 0.4 mL/day of 0.9% sodium chloride. Mice were weighed before sacrifice, and the
tumor, thymus and spleen were gently extracted. The tumor inhibition rate, thymus index and spleen
index were then calculated. Tissue samples were stored at -20 ºC for future study.

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3.10. Statistical analysis
All data were expressed as mean SD and analyzed by one-way ANOVA with the spss16.0
software. The significant differences within and between groups were investigated.
4. Conclusions
Two polysaccharides extracted from PV (P31 and P32) were successfully separated and purified.
The main polysaccharide P32 was found to consist of rhamnose, arabinose, xylose, mannose, glucose
and galactose. In addition, polysaccharide P32 was found to exhibit anti-lung cancer activity and could
increase the thymus index and spleen index in tumor-bearing mice. The immunomodulation effect may
be one of the mechanisms contributing to the observed anti-lung adenocarcinoma activity.
Acknowledgements
This work was supported by grants from Jiangsu Chinese Medicine Leading Talent (2006) and
Jiangsu Science Foundation (BK2006155). We are pleased to thank Shu Luan for the English
language polishing.
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