Yunnan Baiyao - Above Top Secret

Copyright © 2009 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Biomedical Nanotechnology
Vol. 5, 472–476, 2009
Identification of Nanofibers in the Chinese
Herbal Medicine: Yunnan Baiyao
Scott C. Lenaghan1 , Lijin Xia1 , and Mingjun Zhang2 ∗
1
2
206 Dougherty Hall, University of Tennessee, 1512 Middle Dr, Knoxville, TN 37996, USA
408 Dougherty Hall, University of Tennessee, 1512 Middle Dr, Knoxville, TN 37996, USA
Yunnan Baiyao is a traditional Chinese herbal medicine that has been used to treat wounds for over
100 years. Here, we use Atomic Force Microscopy (AFM) to determine nano-scale structures of the
Yunnan Baiyao. AFM images revealed uniform nanofibers present in relatively high abundance in
a solution of this medicine. Fibers were typically 25.1 nm in diameter and ranged in length from
86–726 nm due to processing. Due to the unique adhesive and structural properties of nanofibers,
we concluded that these fibers may play a role in platelet aggregation, leading to clotting, and the
sealing of wounds.
Keywords: AFM, Yunnan Baiyao, Nanofiber.
RESEARCH ARTICLE
1. INTRODUCTION
Yunnan Baiyao (syn Yunan Bai yao, Yunnan Paiyao) is
a traditional Chinese herbal medicine that has been used
for the treatment of a variety of ailments since its inception in 1902. The formulation of this medicine remains
a closely guarded Chinese secret, and remained unknown
to the western world until the Vietnam War (1959–1975)
when American troops began to notice that the Vietnamese
carried small bottles with them to treat battlefield wounds.
Upon further inquiry, the Americans discovered that the
Vietnamese were using bottles of Yunnan Baiyao, and purchased these bottles to treat their own wounds.1
Claims have been made that Yunnan Baiyao can treat
a wide variety of diseases and ailments. Most commonly
Yunnan Baiyao has been used to reduce bleeding and to
increase clotting and platelet aggregation. It has also been
reported that Yunnan Baiyao can be used as an antibacterial or even applied topically to help to seal wounds.2
Other reports claim that it has cytotoxic effects and can be
used for the treatment of cancer.3–7 Despite its widespread
use, very little clinical evidence exists to substantiate
these claims and the mechanism used to generate these
health benefits remains unknown. Due to the secretive
nature, only some of the components of Yunnan Baiyao
are known (Table I). Isolation of these components from
Yunnan Baiyao has led to the discovery of a large number
of saponins that contribute to the antibacterial and antiinflammatory properties of the drug.8 Other components
have been shown to have both anti-coagulant, and platelet
∗
Author to whom correspondence should be addressed.
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J. Biomed. Nanotechnol. 2009, Vol. 5, No. 5
aggregating roles, which seem to counter one another.9–11
In the early 1970’s, there was a theory stating that Yunnan
Baiyao contained micro to nano-scale particulate matter
that could potentially help to cause platelet aggregation,
although this claim was never proven.2
Recently, however, it has been demonstrated that
nanoparticles and nanotubes can have drastic effects on
the abilities of platelets to activate and respond to injury.
Researchers have found that the effect of platelet aggregation induced by nanoparticles is dependent on the composition of the nanoparticles. Gold nanoshells strongly
increase platelet aggregation and can induce thrombosis,
while nanosilver has anti-coagulatory effects that decrease
the formation of clots.12 13 A more comprehensive study
found that the greatest effects on platelet aggregation were
seen with carbon nanotubes, followed by the mixed carbon
nanoparticles.14 Isolated biological nanoparticles have also
been shown to have increased effects on platelet aggregation, leading to faster wound healing in vitro.15
2. RESULTS
By using AFM, we observed a large number of
micro- and nano-scale particulates from the recommended
concentration of Yunnan Baiyao (10 mg ml−1 ). At this concentration, the density of particulate matter was too great
to accurately image. To reduce the number of large particulate matter, a dilution series was conducted to determine the
optimal imaging conditions. It was determined that a concentration of 1 mg ml−1 in distilled water was optimal for
imaging. To reduce the number of micro-scale particulates,
1550-7033/2009/5/472/005
doi:10.1166/jbn.2009.1056
Lenaghan et al.
Identification of Nanofibers in the Chinese Herbal Medicine: Yunnan Baiyao
Table I. Known components of Yunnan Baiyao.
Common name
Pseudoginseng,
San qi
Wild yam root
Scientific name
Known functions
Decreased platelet
activation9
Dioscorea hypoglauca Cytotoxicity of
cancer cells4 5
Sweet geranium Erodium stephanianum Antiviral, antioxidative27 28
Gao liang jiang Alpinia officinarum
Antimicrobial8
Borneol
Cytotoxicity,
antimicrobial3 10 25
Dragon’s blood Daemonorops draco
Cytotoxicity, anti-platelet
functions6
Himalayan
Paris polyphylla var.
Cytotoxicity, antimicrobial3–6
trillium
Panax pseudoginseng
chinensis
the solution was passed through a sterile 0.2 m syringe filter. This eliminated the background particles, and provided
a smooth surface for imaging nano-scale structures (Fig. 1).
To ensure that no contamination occurred during sample
preparation, controls were established for distilled water,
syringe filtered distilled water, and the filter itself. The control samples were free from nanoparticles, and imaging
of the filter revealed no damage from use. Non-uniform
nanoparticles were observed in all Yunnan Baiyao samples,
but varied greatly in size and did not possess a definitive
shape. Contrary to the nanoparticle data, a large number
of uniform nanofibers were present in the Yunnan Baiyao
solution (Fig. 1).
Fig. 2. 3-D rendering of nanofibers. Left, top-down view of 3-D rendering demonstrating the uniform height of the nanofibers. Right, tilted angle of
the same nanofibers demonstrating the depth of field, and height of nanofibers relative to the background.
J. Biomed. Nanotechnol. 5, 472–476, 2009
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RESEARCH ARTICLE
Fig. 1. Nanofibers isolated from Yunnan Baiyao solution. Left, 5 m × 5 m scan showing the organization of nanofibers on the surface of the silica
wafer. Right, 3 m × 3 m scan of surface showing nanofibers.
RESEARCH ARTICLE
Identification of Nanofibers in the Chinese Herbal Medicine: Yunnan Baiyao
Lenaghan et al.
Fig. 3. Phase image of nanofibers showing the difference in phase shift compared to background. A shift of ∼11.5 was observed for nanofibers when
the background was subtracted. This shift indicates that the nanofibers have a greater adhesive interaction with the tip than the surrounding material.
Measurements from 100 fibers on three separate samples, revealed an average length of 299.6 nm (Stdev =
1632 nm), with a wide range, from 86–726 nm. On
subsequent scans, long fibers were observed ranging from
1–1.5 m. The large variation in length of the fibers was
due to shearing from the filtration process. The nanofibers
were uniform in diameter, with an average of 25.1 nm
(Stdev = 25 nm) and a range from 20 to 29 nm. Similarly
the height of the fibers was consistent with an average
of 3.93 nm (Stdev = 002 nm) and a range from 3.90 to
3.99 nm. The height uniformity can easily be seen in a
3-D rendering of height (Fig. 2). Most often, the nanofibers were found in bundles, with the fibers overlapping or
in close contact with one another. Phase images obtained
from scans of Yunnan Baiyao indicated that the fibers were
much softer than the surrounding silica surface, and that
the fibers had consistent shifts in phase, ∼11.5 when subtracted from the background, as indicated by the color of
the fibers in Figure 3. From the phase difference between
the nanofibers and the surrounding background material,
we conclude that there is an adhesive interaction between
the tip and the nanofibers. The increased adhesion from
these fibers contributes to their medicinal effects.
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3. DISCUSSION
While there exists some evidence that the medicinal
properties of Yunnan Baiyao can be related to chemical constituents isolated during the compounding process,
saponins and triterpenes alone cannot completely explain
the mechanism of action of this curious drug. The elucidation of nanofibers within a solution of Yunnan Baiyao
leads to the possibility that there are physical factors, in
addition to chemical ones, that lead to increased platelet
aggregation and wound sealing.
It is known that nanoparticles and nanotubes play a significant role in platelet activation, and that the amount of
activation and subsequent beneficial or detrimental effects
can be related to the composition of the nanoparticles.12–15
In a similar manner, we believe that the nanofibers discovered in Yunnan Baiyao play a role in the activation of
platelets, which may help to explain some of its beneficial effects. The nanofibers are most likely carbon based
due to their extraction from a variety of plants, and carbon nanotubes have been demonstrated to have the greatest
effects on increasing platelet activation. Increased platelet
activation by the nanofibers would lead to an increased
clotting rate that would reduce bleeding, one of the most
J. Biomed. Nanotechnol. 5, 472–476, 2009
Lenaghan et al.
Identification of Nanofibers in the Chinese Herbal Medicine: Yunnan Baiyao
J. Biomed. Nanotechnol. 5, 472–476, 2009
created from a variety of different components. The other
is through small plant fibers, such as lignin, that are naturally derived from the crushed herbs. Nano-scale imaging
of herbal medicines is not commonly practiced, however,
AFM studies of herbal remedies may help to shed light on
how some of these mysterious medicines function.
EXPERIMENTAL DETAILS
Yunnan Baiyao. Yunnan Baiyao was purchased from a Chinese Grocery Store, Ranch 99, Milpitas, CA. The powder
was solubilized in distilled water at varying concentrations
from 10 mg/ml to 1 mg/ml. The powder was vortexed until
a uniform distribution was achieved and placed at 4 C
until use.
Sample Preparation. To reduce the number of large particulates, the solution containing the Yunnan Baiyao powder was filtered through a 0.4 m and 0.2 m sterile
syringe filter. After filtration, the solution was again vortexed, and then approximately 20 l was spotted onto a
silica wafer that had been previously cleaned, sonicated,
and dusted with a compressed air canister. Controls using
distilled water and a bare silica wafer were prepared alongside the experimental samples. All samples were allowed
to dry overnight at 23 C under a laminar flow hood.
Experiments were conducted in triplicate to ensure that the
results were reproducible.
AFM Imaging. All imaging was performed with an Agilent 5500 AFM system. Samples were scanned under both
contact and AC mode to determine the best imaging conditions for the samples. A Budget Sensors® Tap300Al
cantilever (Resonant freq. 300kHz, force constant 40 N/m)
was used for optimal imaging. Image processing was
performed using the PicoImage (Agilent Technologies)
software package. Samples were scanned by two independent researchers to verify the identity of the fibers, and
ensure that no artifacts were introduced from a single user.
Random fibers (100) were chosen for analysis of length,
height, and diameter from three separate preparations. All
measurements were made using PicoImage.
References and Notes
1. J. Polesuk, J. M. Amodeo, and T. S. Ma, Microchemical investigation
of medicinal plants. X. Analysis of the Chinese herbal drug Yunnan
Bai Yao. Mikrochim Acta. 507 (1973).
2. C. W. Ogle, S. Dai, and J. C. Ma, The haemostatic effects of the
Chinese herbal drug Yunnan Bai Yao: A pilot study. Am. J. Chin
Med. Gard City NY 4, 147 (1976).
3. E. Horvathova, D. Slamenova, L. Marsalkova, M. Sramkova, and
L. Wsolova, Effects of borneol on the level of DNA damage induced
in primary rat hepatocytes and testicular cells by hydrogen peroxide.
Food Chem. Toxicol. 47, 1318 (2009).
4. K. Hu and X. Yao, The cytotoxicity of protoneodioscin (NSC698789), a furostanol saponin from the rhizomes of Dioscorea
collettii var. hypoglauca, against human cancer cells in vitro. Phytomedicine 9, 560 (2002).
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RESEARCH ARTICLE
common uses of Yunnan Baiyao. Chemical components
of Yunnan Baiyao have been shown to both increase, and
decrease platelet activation and coagulation (Table I), and
thus the presence of nanofibers would further aid in allowing rapid clot formation and the reduction of bleeding
times.
Another potential benefit from the nanofibers would be
to facilitate wound sealing to prevent infection. It has been
well-established in the literature that nanofibers have very
strong adhesive benefits.16–18 As in the case of the gecko,
it allows the gecko to easily climb surfaces using bundles of nanofibers on the surface of its feet.19–21 In addition to the adhesive properties, nanofibers have also been
used as scaffolding to initiate the repair of damaged tissue. Schneider et al., found that the combination of a
nanofiber scaffold with epidermal growth factor resulted
in the regeneration of new skin 5 times faster than without the scaffold.22 Furthermore, Long et al. concluded
that nanofiber scaffolding was effective in wound healing of rabbits with full-thickness skin defects.23 In this
same manner, it is likely that the nanofibers found in
Yunnan Baiyao act as scaffolding to initiate the repair of
wounds. This scaffolding would allow wounds to repair
more rapidly, substantiating the claims that Yunnan Baiyao
effectively aids in sealing wounds. In effect, the nanofibers
may act as an adhesive bandage that allows a wound to
seal by adhesion of the skin layers. In all samples tested, it
was observed that nanofibers tended to be found in clumps,
and tightly associated with one another, even after being
passed through the filter. In highly concentrated samples,
there was too much background for the AFM to achieve
a contrast that would have allowed visualization of the
fibers, thus a series of dilutions was used to optimize the
imaging and visualize individual nanofibers. In a concentrated form, as in the recommended doses for treatment of
wounds externally (250 mg), the number of fibers would
be greatly increased over our experimental samples. This
would lead to a network of nanofibers that would be able
to bind to one another and form scaffolding that may lead
to increased cell growth. The true length of the fibers
could not be elucidated in this study due to shearing, however fibers of several micrometers were observed in some
experiments.
The discovery of consistent and uniform nanofibers in
Yunnan Baiyao helps to elucidate the mechanism of action
of this Chinese herbal medicine. By applying similar nanofibers to other drugs, it may be possible to increase their
effectiveness, specifically in the area of wound healing and
blood clotting. Recently there has been a resurgence in the
study of herbal remedies for the treatment of a variety of
ailments, including cancer.24 In fact, recent studies have
included Yunnan Baiyao and clinical data could dramatically increase the use of this drug.25 26 There are in general
two possible ways for nanofiber formulation. The first is
through the compounding process that these nanofibers are
RESEARCH ARTICLE
Identification of Nanofibers in the Chinese Herbal Medicine: Yunnan Baiyao
5. K. Hu and X. Yao, The cytotoxicity of methyl protoneogracillin
(NSC-698793) and gracillin (NSC-698787), two steroidal saponins
from the rhizomes of Dioscorea collettii var. hypoglauca, against
human cancer cells in vitro. Phytother Res. 17, 620 (2003).
6. C. C. Shen, S. Y. Tsai, S. L. Wei, S. T. Wang, B. J. Shieh, and
C. C. Chen, Flavonoids isolated from Draconis Resina. Nat. Prod.
Res. 21, 377 (2007).
7. Y. Wang, Y. J. Zhang, W. Y. Gao, and L. L. Yan, Anti-tumor constituents from Paris polyphylla var. yunnanensis. Zhongguo Zhong
Yao Za Zhi 32, 1425 (2007).
8. H. Huang, D. Wu, W. X. Tian, X. F. Ma, and X. D. Wu, Antimicrobial effect by extracts of rhizome of Alpinia officinarum Hance may
relate to its inhibition of beta-ketoacyl-ACP reductase. J. Enzyme
Inhib Med Chem. 23, 362 (2008).
9. X. Cui, T. Sakaguchi, Y. Shirai, and K. Hatakeyama, Orally administered Panax ginseng extract decreases platelet adhesiveness in 66%
hepatectomized rats. Am. J. Chin Med. 27, 251 (1999).
10. Y. H. Li, X. P. Sun, Y. Q. Zhang, and N. S. Wang, The antithrombotic
effect of borneol related to its anticoagulant property. Am. J. Chin
Med. 36, 719 (2008).
11. W. J. Tsai, H. T. Hsieh, C. C. Chen, Y. C. Kuo, and C. F.
Chen, Characterization of the antiplatelet effects of (2S)-5-methoxy6-methylflavan-7-ol from Draconis Resina. Eur. J. Pharmacol.
346, 103 (1998).
12. T. Akchurin, T. Aissiou, N. Kemeny, E. Prosk, N. Nigam, and S. V.
Komarova, Complex dynamics of osteoclast formation and death in
long-term cultures. PLoS ONE 3, e2104 (2008).
13. S. Shrivastava, T. Bera, S. K. Singh, G. Singh, P. Ramachandrarao,
and D. Dash, Characterization of antiplatelet properties of silver
nanoparticles. ACS Nano. 3, 1357 (2009).
14. A. Radomski, P. Jurasz, D. Alonso-Escolano, M. Drews,
M. Morandi, T. Malinski, and M. W. Radomski, Nanoparticleinduced platelet aggregation and vascular thrombosis. Br. J. Pharmacol. 146, 882 (2005).
15. K. Chu, V. Kaul, W. G. Owen, and M. Jayachandran, Biological nanoparticles and platelet activation. FASEB J. 22, 924
(2008).
16. R. L. Price, M. C. Waid, K. M. Haberstroh, and T. J. Webster, Selective bone cell adhesion on formulations containing carbon nanofibers. Biomaterials 24, 1877 (2003).
17. A. Salehi-Khojin, J. J. Stone, and W.-H. Zhong, Improvement of
interfacial adhesion between UHMWPE fiber and epoxy matrix
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Lenaghan et al.
using functionalized graphitic nanofibers. J. Compos. Mater. 41,
1163 (2007).
L. R. Xu, L. Li, C. M. Lukehart, and H. Kuai, Mechanical characterization of nanofiber-reinforced composite adhesives. J. Nanosci.
Nanotechnol. 7, 2546 (2007).
A. Mahdavi, L. Ferreira, C. Sundback, J. W. Nichol, E. P. Chan, D. J.
Carter, C. J. Bettinger, S. Patanavanich, L. Chignozha, E. Ben-Joseph,
A. Galakatos, H. Pryor, I. Pomerantseva, P. T. Masiakos, W. Faquin,
A. Zumbuehl, S. Hong, J. Borenstein, J. Vacanti, R. Langer, and J. M.
Karp, A biodegradable and biocompatible gecko-inspired tissue adhesive. Proc. Natl. Acad. Sci. USA 105, 2307 (2008).
N. W. Rizzo, K. H. Gardner, D. J. Walls, N. M. Keiper-Hrynko,
T. S. Ganzke, and D. L. Hallahan, Characterization of the structure
and composition of gecko adhesive setae. J. R. Soc. Interface 3, 441
(2006).
B. Zhao, N. Pesika, K. Rosenberg, Y. Tian, H. Zeng, P. McGuiggan,
K. Autumn, and J. Israelachvili, Adhesion and friction force coupling
of gecko setal arrays: Implications for structured adhesive surfaces.
Langmuir 24, 1517 (2008).
A. Schneider, J. A. Garlick, and C. Egles, Self-assembling peptide
nanofiber scaffolds accelerate wound healing. PLoS ONE 3, e1410
(2008).
J. H. Long, W. Y. Tan, R. W. Jiang, and Y. Z. Zhang, [Experimental study on gelatin/polycaprolactam composite nanofiber scaffold in
wound healing]. Zhonghua Shao Shang Za Zhi. 24, 42 (2008).
W.-j. Ruan, M.-d. Lai, and J.-g. Zhou, Anticancer effects of Chinese
herbal medicine, science or myth? Journal of Zhejiang UniversityScience B 7, 1006 (2006).
Z. Cai, S. Hou, Y. Li, B. Zhao, Z. Yang, S. Xu, and J. Pu, Effect of
borneol on the distribution of gastrodin to the brain in mice via oral
administration. J. Drug Target 16, 178 (2008).
T. Okuda, K. Mori, and N. Hayashi, Constituents of Geranium thunbergii Sieb. et Zucc. III. Application of determination methods of
tannin activity with hemoglobin and of ellagitannin with nitrous acid
(author’s transl). Yakugaku Zasshi 96, 1143 (1976).
Z. Sroka, H. Rzadkowska-Bodalska, and I. Mazol, Antioxidative
effect of extracts from Erodium cicutarium L. Z Naturforsch C
49, 881 (1994).
J. Zielinska-Jenczylik, A. Sypula, E. Budko, and H. RzadkowskaBodalska, Interferonogenic and antiviral effect of extracts from
Erodium cicutarium. II. Modulatory activity of Erodium cicutarium
extracts. Arch Immunol Ther Exp. Warsz 36, 527 (1988).
Received: 13 August 2009. Accepted: 11 September 2009.
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