Preparation and characterization of the inclusion complex of Baicalin (BG) with β-CD and HP-β-CD in solution: An antioxidant ability study

Article abstract of DOI:10.1016/j.saa.2009.03.025

The formation of the complexes of BG with β-CD and HP-β-CD was studied by UV-vis absorption spectroscopy, fluorescence spectra, Phase-solubility measurements and nuclear magnetic resonance spectroscopy (NMR) in solution. The formation constants (K) of com

Full text of DOI:10.1016/j.saa.2009.03.025

Spectrochimica Acta Part A 73 (2009) 752–756
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Preparation and characterization of the inclusion complex of Baicalin (BG) with
␤-CD and HP-␤-CD in solution: An antioxidant ability study
Jinxia Lia,b, Min Zhanga, Jianbin Chaoa,∗, Shaomin Shuangb
a The Institute of Applied Chemistry of Shanxi University, Taiyuan 030006, PR China
b College of Chemistry and Chemical Engineering of Shanxi University, Taiyuan 030006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The formation of the complexes of BG with ␤-CD and HP-␤-CD was studied by UV–vis absorption
spectroscopy, fluorescence spectra, Phase-solubility measurements and nuclear magnetic resonance
spectroscopy (NMR) in solution. The formation constants (K) of complexes were determined by fluores-
cence method and Phase-solubility measurements. The results showed that the inclusion ability of ␤-CD
and its derivatives was the order: HP-␤-CD > ␤-CD. In addition, the experimental resulted confirmed the
existence of 1:1 inclusion complex of BG with CDs.
The antioxidant ability studies of BG and CDs complexes were done. The results obtained indicated that
the BG/HP-␤-CD complex was the most reactive form, and then was the BG/␤-CD complex; the last was
BG. Special configuration of complex has been proposed on NMR technique.
Received 22 November 2008
Received in revised form 21 February 2009
Accepted 19 March 2009
Keywords:
Baicalin
Cyclodextrins
Absorption spectroscopy
Fluorescence
NMR
© 2009 Elsevier B.V. All rights reserved.
DPPH•
1. Introduction
several types of cells [12–14] inducing cell death in human hepato-
cellular carcinonia cell [15] and in human promyelocytic leukemia
Investigations of molecular recognition have attracted much
attention in supramolecular chemistry involving natural and
artificial host–guest systems [1]. The inclusion process of phar-
maceutical molecules with CDs usually results in a modulation
of the physicochemical and pharmaceutical properties of guest
molecules, such as increased solubility, improved chemical stabil-
ity and bioavailability, reduced toxicity controlled-rate release and
so on [2–4]. Therefore, it would be of great importance to com-
prehensively understand the inclusion behavior of molecules of
pharmaceutical interests with CDs. Recently, various hydrophilic,
hydrophobic and ionic cyclodextrin derivatives have been utilized
to extend the physicochemical properties and inclusion capacity of
natural cyclodextrin [5,6]. HP-␤-CD is a water-soluble derivative of
␤-CD, which has been widely studied as a complexion agent for
many pharmaceuticals. The ability of CDs to form inclusion com-
plexes is highly affected by size, shape, hydrophobicity and the form
of the guest’s molecular.
HL-60 cells [16] and so on. However, in spite of the wide spectrum
of pharmacological properties, its use in pharmaceutical filed is
limited because of its poor solubility.
When the fluorescent guests are included in the CD cavity, the
non-radiative decay processes of luminophores are significantly
attenuated and hence fluorescence emission increased [17–19]. Due
to its high sensitivity, selectivity and instrumental simplicity, fluo-
rescence method has been used to investigate the phenomena of
inclusion complexes and determine the association constants of
complexes [19–21]. High resolution nuclear magnetic resonance
(NMR) is also a powerful tool for studying CD complexes [22] that
can provide not only quantitative information, but also detailed
information on geometry of the complex.
The present work was designated to study the complexation
of BG utilizing two different cyclodextrins (HP-␤-CD and ␤-CD) to
improve the solubility and to determine the effect of the complex-
ation process on their antioxidant capacity.
Baicalin (BG), a flavonoid present in the root of Scutellaria
baicalensis Georgi (Fig. 1), has attracted considerable attention
because of the activities, such as antibacterial [7], anti-HIV activ-
ity [8], attenuating oxidative stress [9–11], inhibiting the growth of
2. Experimental
2.1. Apparatus and materials
UV-757CRT spectrophotometer (Shanghai Precision and Sci-
entific Instrument Co. Ltd.); Fluorescence measurements were
performed by F-2500 FL spectrofluoremeter (Hitachi) using 1 cm
quartz cell and both the slits were set at 20 nm with the excitation
∗
Corresponding author at: The Institute of Applied Chemistry of Shanxi Univer-
sity, Taiyuan 030006, PR China. Tel.: +86 3517017838.
E-mail address: chao@sxu.edu.cn (J. Chao).
1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2009.03.025

J. Li et al. / Spectrochimica Acta Part A 73 (2009) 752–756
753
Fig. 1. The chemical structure of Baicalin.
wavelength at 270 nm and the emission at about 350 nm. All the
NMR date was obtained on Bruker Avance DRX 300MHZ NMR spec-
trometer.
A stock solution of 1.0 × 10−4 mol/l BG (provided by Dr. Zhang
and was purified by recrystallization) was prepared by dissolving
and diluting its crystals in water. CDs and DPPH• were purchased
from Sigma–Aldrich, Inc., St. Louis, MO. All other reagents were
of analytical-reagent grade and were used without purification.
Doubly distilled water was used throughout. All experiments were
carried out at room temperature.
Fig. 2. The absorption spectra of 1.0 × 10−6 mol/LBG in the present of ␤-CD and the
concentration of ␤-CD is 0–5 × 10−3 M.
2.5. Determination of antioxidant activity by the scavenging of
the stable radical DPPH•
The antioxidant activity was measured, wherein the bleaching
rate of a stable free radical, DPPH• is monitored at a characteristic
wavelength in the presence of the sample. In its radical form, DPPH•
absorbs at 517–520 nm, but upon reduction by an antioxidant or a
radical species its absorption decreases.
2.2. Procedure
A 0.1 or 1 ml aliquot of the stock solution of BG was transferred
into a 10 ml volumetric burette, and then an appropriate amount
of 1.0 × 10−2 mol/l CDs (␤-CD and HP-␤-CD) was added. The solu-
tion was diluted to a final volume of 10 ml with distilled water. The
final mixture solution was dissolved thoroughly under ultrasonic
for 30 min, and then equilibrated for 30 min at 20 1 ◦C. The work-
ing solution was transferred into a 1 cm × 1 cm quartz cell to record
absorption and fluorescence spectra. All measurements of absorp-
tion and fluorescence were made against a blank solution treated
in the same way but without BG in a 1.0 cm quartz cell.
A volume of 2 ml of 1.0 × 10−5 M DPPH• was used. Furthermore,
DPPH• is insoluble in aqueous solution the scavenging study was
performed in mixture of ethanol–water (20:80).
The reaction was started by addition of 1 ml of BG (1.0 × 10−5 M),
BG/␤-CD, and BG/HP-␤-CD complex samples, which correspond
to the 3 mM cyclodextrin concentration from the Phase-solubility
studies. All the solution were balanced for 5 min in room tempera-
ture, then the bleaching of DPPH• was followed at 520 nm.
The decrease in absorbance at 520 nm was measured against a
blank of ethanol–water (20:80) 1 and 2 ml 1.0 × 10−5 M DPPH• to
estimate the radical scavenging capacity of each antioxidant sam-
ple. The results were expressed as percentage DPPH• elimination
calculated according to the following equation [24]:
2.3. NMR measurements
1 × 10−4 mol/l BG and 1 × 10−4 mol/l CDs (HP-␤-CD and ␤-CD)
solutions with a volume ratio of 1:1 were mixed thoroughly. With
D2O as solvent, 1H NMR spectra was obtained at 300.13 MHz with
10 ␮s as 90◦ pulse width. All experiments were performed at
20 1 ◦C.
ꢀ
ꢁ
1 − As
AU =
100,
(2)
A0
where AU is radical-scavenging activity, As is absorbance of sample
and A0 absorbance of blank sample.
2.4. Phase-solubility study
Solubility measurements were based on the Phase-solubility
technique [23]. Namely, excess amount of solid BG (8 mg) was
added to a series of 10 ml stopper burette that contained increasing
amount of CDs (1.0 × 10−2 mol/l, 0–9 ml, including ␤-CD and HP-␤-
CD). These obtained suspensions were shaken by ultrasonic method
for 3 h at room temperature, and then were filtered after being
placed for 7 days. The filtrate was diluted and analyzed through
UV method. Phase-solubility profile was obtained by plotting the
solubility of BG versus the concentration of CDs.
3. Results and discussion
3.1. UV spectroscopy
Fig. 2 shows the absorption spectra of BG in the absence and
presence of CDs at room temperature. The absorption of BG varied
significantly with the addition of ␤-CD. BG alone in water exhibited
two absorption peaks at 275 and 314 nm, respectively. The increase
in ␤-CD concentration from 1 to 5 m mol/l resulted in an increase in
the absorption of BG. Simultaneously, as the ␤-CD increase a weak
red shift of absorption peak at 275 nm and a weak blue shift of peak
at 314 nm was observed. These might be partly attributed to the
change of chromophore groups in BG molecular due to the complex
formation between BG and ␤-CD through hydrophobic interaction,
and suggested that the likely formation of an inclusion complex
between BG and ␤-CD. Similar phenomena were observed for the
HP-␤-CD.
The apparent stability constant (Ks) of the complexes were cal-
culated according to the following equation
slope
S0(1 − slope)
Ks =
(1)
where S0 is the solubility of BG at room temperature in absence
of CDs and slope means the corresponding slope of the Phase-
solubility diagrams.

754
J. Li et al. / Spectrochimica Acta Part A 73 (2009) 752–756
Fig. 4. Double reciprocal plots for BG complexes to ␤-CD or HP-␤-CD. (ꢀ): ␤-CD;
(ꢀ): HP-␤-CD.
Fig. 5. Phase-solubility diagram of BG and CDs. (ꢀ) ␤-CD, (ꢀ) HP-␤-CD.
3.3. Phase-solubility measurements
Fig. 5 showed that CDs enhanced the poor aqueous solubility
of BG, thus proving a certain degree of its inclusion complexation
in aqueous solutions, the results observed showed a linear behav-
ior for ␤-CD (r2 = 0.9927) and HP-␤-CD (r2 = 0.9842), and consistent
with 1:1 molecular complex formation for CDs and BG. The binding
constant (Ks) of the complexes were shown in Table 1. As shown in
Table 1, the binding constant and solubility of BG determined with
CDs followed the rank order HP-␤-CD > ␤-CD. The results were as
the same as fluorescence results.
Fig. 3. Fluorescence emission spectra of 1.0 × 10−6 mol/l BG in CDs. a: ␤-CD
(0–6.0 mM); b: HP-␤-CD (0–6.0 mM).
3.4. NMR measurements
To ascertain the structure of the inclusion complexes between
BG and CDs, 1H NMR spectroscopy studies of free drug and inclusion
complexes were therefore undertaken. Figs. 6 and 7 illustrated the
change of hydrogen atom of BG and CDs before and after forming
the inclusion complexes. The difference in hydrogen chemical shift
values between BG in the free and complexed state were presented
3.2. Fluorescence study
Fig. 3 showed that adding CDs (including ␤-CD and HP-␤-CD)
to BG solution resulted in a significant enhancement of the flu-
orescence signal. The excitation wavelength was at 270 nm, the
maximum emission wavelength at 358 and 349 nm, respectively.
With the increasing of CDs, the emission wavelength appeared blue
shift, and a new increasing emission wavelength was observed with
the concentration increasing of HP-␤-CD at about 430 nm. These
suggested that the inclusion complexes were likely formed between
BG and CDs. The CD cavity provided an apolar environment for the
BG molecule and the motion of the BG molecule in the cavity was
largely confined. Thus, the enhanced rigidity of the BG molecule
resulted in an increase of its fluorescence quantum yield.
Table 1
Apparent stability constant (Ks) of BG inclusion.
CDs complex
Linear equation
Ks (M−1
)
r2
␤-CD
HP-␤-CD
y = 0.0843x + 1.0864
y = 0.1186x + 1.2338
864
1143
0.9927
0.9842
The inclusion formation constant (K) is a measure of the com-
plexing power of CD. The formation constant and ratio of the
complex were obtained from fluorescence data using the modified
Benesi–Hildebrand equation [25]
1
1
1
˛
=
+
(3)
(F − F0)
([CDs] K˛)
where, F and F0 represent the fluorescence intensity of BG in the
presence and absence of CDs, respectively; K is a forming constant;
˛ is a constant. Fig. 4 shows the double reciprocal plots of 1/(F − F0)
versus 1/[CD]. The good linear relationship obtained when 1/(F − F0)
were plotted against 1/[CDs] supports the existence of a 1:1 com-
plex. These data suggested the inclusion ability of HP-␤-CD was
bigger than ␤-CD.
Fig. 6. 1H NMR spectra of BG and inclusion complexes: the order were BG, BG/␤-CD
and BG/HP-␤-CD from the below to the up.

J. Li et al. / Spectrochimica Acta Part A 73 (2009) 752–756
755
Fig. 7. 1H NMR spectra of ␤-CD and BG/␤-CD inclusion complex from the below to
the up.
Table 2
The 1H NMR chemical shifts corresponding to BG in the absence and presence of CDs
in D2O.
BG (H)
BG (ı0)
BG/␤-CD (ı1)
ꢀı1
BG/HP-␤-CD (ı2)
ꢀı2
H-8
H-3
6.364
6.573
7.218
7.552
6.313
6.554
7.179
7.502
−0.051
−0.019
−0.039
−0.040
6.636
6.719
7.341
7.676
0.272
0.146
0.123
0.124
H-3ꢀ4ꢀ5ꢀ
H-2ꢀ6ꢀ
in Tables 2 and 3 showed the hydrogen chemical shift change values
of CDs after forming the complexes.
Fig. 8. The structure of inclusion complexes between BG and CDs. a: BG/HP-␤-CD;
b: BG/␤-CD.
It can be seen from the figures that the H-8, H-3, H-3ꢀ, H-4ꢀ, H-5ꢀ
and H-2ꢀ, H-6ꢀ of BG exhibited larger chemical shifts, namely, the A,
B and C ring of BG were all entered into the cavity of ␤-CD and HP-
␤-CD, because of the diminished freedom of rotation caused by the
penetration of BG molecule into the CDs cavity. And the seam time,
the H-5 of ␤-CD experienced larger chemical shift than H-3, which
illustrated that the molecular of BG entered into the cavity of ␤-CD
from the small ring-edge side of ␤-CD; while The H-3 of HP-␤-CD
experienced larger chemical shift than H-5, which illustrated that
the molecular of BG entered into the cavity of HP-␤-CD from the
large port.
From all the above, the mechanism of complex between BG and
CDs were shown as follow (Fig. 8).
3.5. Scavenging study of DPPH• by free or complexed-BG
Fig. 9. The consume percentage of DPPH• in presence of BG free and complexes
DPPH• is a stable free radical generating a deep violet solution in
organic solvents. Its progressive discoloration when in the presence
of BG indicated that it is acting as an antioxidant.
forms.
The scavenging ability was measured as a relative scavenging in
presence of free or complex BG. Fig. 9 was in according with scav-
enging ability is related with enhanced solubility of BG. Also theses
results indicated that the complexes formed maintained the BG
antioxidant activity.
The antioxidant activity of phenolic compounds depends on the
position and degree of hydroxylation, as well as the nature of rad-
icals of the ring structure. Anti-oxidative activity is intensified by
Furthermore, since the mechanism of DPPH• reduction is
known, the amount remaining of both reagents may be determined.
The rate of the DPPH•-scavenging reaction was measured by
monitoring the decrease in absorbance at 520 nm due to DPPH•.
Fig. 9 showed the consumption of DPPH• which indicates that the
complexed BG with CDs were more effective than free BG, with the
HP-␤-CD complex (77.78) > ␤-CD complex (72.22) > free BG (38.89).
Table 3
1H NMR chemical shifts CDs and the inclusion complexes in D2O.
CD
␤-CD
(ı0)
BG/␤-CD
(ı1)
HP-␤-CD
BG/HP-␤-CD
(ı2)
ꢀ
(H)
ꢀı1
0.003
−0.001
−0.069
−0.014
−0.013
(ı0
)
ꢀı2
H-4
H-2
H-5
H-6
H-3
3.376
3.413
3.633
3.633
3.728
3.379
3.412
3.584
3.619
3.715
3.260
3.346
3.461
3.615
3.749
3.259
3.344
3.466
3.616
3.764
−0.001
−0.002
0.005
0.001
0.015

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J. Li et al. / Spectrochimica Acta Part A 73 (2009) 752–756
the presence of a second hydroxy group, through the formation of
an intramolecular hydrogen bond [26]. It might be that that the
–OH positions of BG molecules is close enough to secondary –OH
groups of CDs to form hydrogen bonds and contribute to antioxidant
activity [27]. Therefore the formation of an “intramolecular” hydro-
gen bond of the inclusion complex is possible and consequently an
increase of antioxidant capacity is expected.
[8] J.A. Wu, A.S. Attele, L. Zhang, Ch.S. Yuan, Anti-HIV activity of medicinal herbs:
usage and potential development, Am. J. Chin. Med. 29 (2001) 69–81.
[9] Z.H. Shao, C.Q. Li, T.L. Vanden Hoek, L.B. Becker, P.T. Schumacker, J.A. Wu, A.S.
Attele, C.S. Yuan, Extract from Scutellaria baicalensis Georgi attenuates oxidant
stress in cardiomyocytes, J. Mol. Cell. Cardiol. 31 (1999) 1885–1895.
[10] Z.H. Shao, T.L. Vanden Hoek, Y. Qin, L.B. Becker, P.T. Schumacker, C.Q. Li, L. Dey,
E. Barth, H. Halpern, G.M. Rosen, C.S. Yuan, Baicalein attenuates oxidant stress
in cardiomyocytes, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) 999–1006.
[11] Z. Gao, K. Huang, H. Xu, Protective effects of flavonoids in the roots of Scutellaria
baicalensis Georgi against hydrogen peroxideinduced oxidative stress in HS-
SY5Y cells, Pharmacol. Res. 43 (2001) 173–178.
[12] L. Qain, K. Okita, T. Murakami, M. Takahashi, Inhibitory effect of baicalein on the
growth of cultured hepatoma cells (HUH-7), Biotherapy 4 (1990) 1664–1670.
[13] H.C. Huang, H.R. Wang, L.M. Hsieh, Antiproliferative effect of baicalein, a
flavonoid from a Chinese herb, on vascular smooth muscle cell, Eur. J. Pharmacol.
251 (1994) 91–93.
[14] T. Inoue, E.K. Jackson, Strong antiproliferative effects of baicalein in cultured rat
hepatic stellate cells, Eur. J. Pharmacol. 378 (1999) 129–135.
[15] Y. Matsuzaki, N. Kurokawa, S. Terai, Y. Matsumura, N. Kobayashi, K. Okita, Cell
death induced by baicalein in human hepatocellular carcinoma cell lines, Jpn.
J. Cancer Res. 87 (1996) 170–177.
[16] Y.C. Li, Y.S. Tyan, H.M. Kuo, W.C. Chang, T.C. Hsia, J.G. Chung, Baicalein induced
in vitro apoptosis undergo caspases activity in human promyelocytic leukemia
HL-60 cells, Food Chem. Toxicol. 42 (2004) 37–43.
[17] K. George, V. Willingham, H. Wu, D. Gridley, G. Nelson, F.A. Cucinotta, Chromo-
some aberrations in human lymphocytes induced by 250 MeV protons: effects
of dose, dose rate and shielding, Adv. Space Res. 30 (4) (2002) 891–899.
[18] L.A. Blyshak, M. Rollie-Taylor, D.W. Sylvester, A.L. Underwood, G. Patonay, I.M.
Warner, Characterization of naphthoate surfactants in normal and reverse
micellar systems via luminescence spectroscopy, J. Colloid Interf. Sci. 136 (2
(May)) (1990) 509–518.
4. Conclusion
The present study has demonstrated the inclusion complex
interaction between BG with ␤-CD and HP-␤-CD in the solution.
Among CDs, the inclusion ability of HP-␤-CD was stronger than
that of ␤-CD. And the activity of eliminating free radical DPPH•
were HP-␤-CD inclusion complex > ␤-CD inclusion complex > free
BG. In addition, the fluorescence spectroscopy and Phase-solubility
measurements data showed the formation of 1:1 stoichiometric
complex of BG with ␤-CD and HP-␤-CD over the concentration
range evaluated. Moreover, the study demonstrated that CDs sev-
ered as drugs carrier system in a dosage-controlled manner and can
increase the solubility, stability and antioxidant activity of guest
molecular. A mechanism was set up to expound the structure of
the inclusion complexes.
[19] J.A. Arancibia, G.M. Escandar, Two different strategies for the fluorimetric deter-
mination of piroxicam in serum, Talanta 60 (6 (August)) (2003) 1113–1121.
[20] G.M. Escandar, D. González Gómez, A. Espinosa Mansilla, A. Mun˜oz de la Pen˜a,
H.C. Goicoechea, Determination of carbamazepine in serum and pharmaceu-
tical preparations using immobilization on a nylon support and fluorescence
detection, Anal. Chim. Acta 506 (2 (March)) (2004) 161–170.
Acknowledgement
This work was supported by the national Natural Science Foun-
dation of Shanxi Province (No. 2006011017).
[21] T.A. Betts, G.C. Catena, J. Huang, K.S. Litwiler, J. Zhang, J. Zagrobelny, F.V. Bright,
Fiber-optic-based immunosensors for haptens, Anal. Chim. Acta 246 (1 (May))
(1991) 55–63.
References
[22] J.B. Chao, D.P. Meng, J.S. Li, H. Xu, Sh.P. Huang, Preparation and study on the novel
solid inclusion complex of ciprofloxacin with HP-␤-cyclodextrin, Spectrochim.
Acta A 60 (2004) 729–734.
[1] A.D. Hamilton, Molecular recognition (Tetrahedron symposia No.56), Tetrahe-
dron 51 (1995) 343.
[2] J. Szejtli, Introduction and general overview of cyclodextrin chemistry, Chem.
Rev. 98 (1998) 1743–1754.
[3] K. Uekama, F. Hirayama, T. Irie, Cyclodextrin drug carrier systems, Chem. Rev.
98 (1998) 2045–2076.
[4] M.E. Cortes, R.D. Sinisterra, M.J. Avilacampos, N. Tortamano, R.G. Rocha, The
chlorhexidine: ␤-cyclodextrin inclusion compound: preparation, characteriza-
tionandmicrobiologicalevaluation, J. Incl. Phenom. Macrocycl. Chem. 40 (2001)
297–302.
[23] T. Higuchi, K.A. Connors, Phase solubility techniques, Adv. Anal. Chem. Instrum.
4 (1965) 117–212.
ˇ
[24] M. Strazˇisˇar, S. Andrensˇek, A. Smidovnik, Effect of ␤-cyclodextrin
on antioxidant activity of coumaric acids, Food Chem. (2008),
doi:10.1016/j.foodchem.2008.02.051.
[25] K.A. Connors, Binding Constants. The Measurement of Molecular Complex Sta-
bility, Wiley, New York, 1987.
[26] F. Natella, M. Nardini, M. Di Felice, C. Scaccini, Benzoic and cinnamic acid deriva-
tives as antioxidants: structure-activity relation, J. Agric. Food Chem. 47 (1999)
1453–1459.
[27] A. Kokkinou, S. Makedonopoulou, D. Mantzafos, The crystal structure of the
1:1 complex of ␤-cyclodextrin with trans-cinnamic acid, Carbohydr. Res. 328
(2000) 135–140.
[5] F. Hirayama, K. Uekama, Cyclodextrin-based controlled drug release system,
Adv. Drug Deliv. Rev. 36 (1999) 125–141.
[6] N. Ono, H. Arima, F. Hirayama, K. Uekama,
A moderate interaction of
maltosyl-alpha-cyclodextrin with Caco-2 cells in comparison with the parent
cyclodextrin, Biol. Pharm. Bull. 24 (2001) 395–402.
[7] M. Kubo, Y. Kimura, T. Odani, T. Tani, K. Namba, Studies on Scutellaria radix. Part
2: the antibacterial substance, Planta Med. 43 (1981) 194–201.