α-Glucosidase inhibitory constituents from Acanthopanax senticosus Harm leaves

Article abstract of DOI:10.3390/molecules17066277

A new triterpene glycoside, 3-O-[(α-L-rhamnopyranosyl)(1→2)]- [β-Dglucuronopyranosyl-6-O-methyl ester]-olean-12-ene-28-olic acid (1) and a new indole alkaloid, 5-methoxy-2-oxoindolin-3-acetic acid methyl ester (5) were isolated from the leaves of Acanthopanax senticosus Harms along with six known compounds. The structures of the new compounds were determined by means of 2D-NMR experiments and chemical methods. All the isolated compounds were evaluated for their glycosidase inhibition activities and compound 6 showed significant α-glucosidase inhibition activity.

Full text of DOI:10.3390/molecules17066277

Molecules 2012, 17, 6277-6289; doi:10.3390/molecules17066277
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Scutellaria baicalensis Alleviates Cantharidin-Induced Rat
Hemorrhagic Cystitis through Inhibition of Cyclooxygenase-2
Overexpression
Steven Kuan-Hua Huan 1,2, Kun-Teng Wang 3,4, Shauh-Der Yeh 2, Chia-Jung Lee 3, Li-Chun Lin 3,
Der-Zen Liu 5 and Ching-Chiung Wang 3,*
1
Division of Urology, Department of Surgery, Chi Mei Medical Center, No.21, Taikang,
Liuying Dist., Tainan City 73657, Taiwan; E-Mail: skhsteven@yahoo.com.tw
2
Graduate Institute of Clinical Medical Research, Taipei Medical University, 250 Wu-Hsing Street,
Taipei City 11031, Taiwan; E-Mail: d8602003@tmu.edu.tw
3
School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street,
Taipei City 11031, Taiwan; E-Mails: b8706014@tmu.edu.tw (K.-T.W.);
m303092003@tmu.edu.tw (C.-J.L.); snoopy0615@hotmail.com (L.-C.L.)
4
Section of Biologics & Advanced Therapeutic Product Analysis, Division of Research and Analysis,
Food and Drug Administration, No.161-2, Kunyang St, Nangang District, Taipei City 11561, Taiwan
5
Graduate Institute of Biomedical Materials, Taipei Medical University, 250 Wu-Hsing Street,
Taipei City 11031, Taiwan; E-Mail: tonyliu@tmu.edu.tw
* Author to whom correspondence should be addressed; E-Mail: crystal@tmu.edu.tw;
Tel.: +886-2-2736-1661 (ext. 6161); Fax: +886-2-2732-9368.
Received: 12 April 2012; in revised form: 13 May 2012 / Accepted: 15 May 2012 /
Published: 25 May 2012
Abstract: Cantharidin, an active component in mylabris, is used in traditional Chinese
medicine (TCM) to treat scabies and hepatoma, but accompanied by hemorrhagic cystitis.
Evidence shows that cantharidin induces human bladder carcinoma cell death through
COX-2 overexpression in vitro. In TCM, Scutellaria baicalensis is usually used to cure
mylabris-induced hematuria. This work was undertaken to determine the mechanisms of
cantharidin-induced rat hemorrhagic cystitis and explore the uroprotective effect of
S. baicalensis. In vitro results showed cantharidin could induce cytotoxicity through
prostaglandin (PG)E2 overproduction of T24 cells. Boiling-water extract of S. baicalensis
(SB-WE) could significantly inhibit PGE2 production and COX-2 expression in
lipo-polysaccharide-induced RAW 264.7 cells, indicating obvious anti-inflammatory
abilities. In vivo results indicated that cantharidin caused rat hemorrhagic cystitis with

Molecules 2012, 17
hematuria via c-Fos and COX-2 overexpression. SB-WE was given orally to
6278
cantharidin-treated rats, whereby hematuria level, elevated PGE2 and COX-2 protein
overexpression were significantly and dose-dependently inhibited by SB-WE. The
anti-inflammatory components of SB-WE are baicalin and wogonin, whose contents were
200.95 ± 2.00 and 31.93 ± 0.26 μg/mg, respectively. In conclusion, cantharidin induces rat
cystitis through c-Fos and COX-2 over-expression and S. baicalensis can prevent the
resulting hematuria because of its anti-inflammatory effects.
Keywords: mylabris; cantharidin; Scutellaria baicalensis; hemorrhagic cystitis;
cyclooxygenase-2; hematuria; c-Fos
1. Introduction
Cantharidin is a major component of mylabris (Mylabris phalerata Pallas), commonly known as
Spanish Fly. In Europe and India, mylabris is used as an aphrodisiac. In traditional Chinese medicine
(TCM), people use it to treat scabies and hepatoma [1,2]. However, people who take an overdose of
mylabris will be poisoned by cantharidin. According to clinical case reports, cantharidin poisoning
results in burning of the mouth, hematemesis, hepatomegaly, irritation of the genitourinary tract,
hematuria, and dysuria [1,3,4]. Moreover, in a previous study we found that cantharidin induced
secondary necrosis and cyclooxygenase (COX)-2 overexpression in bladder epithelial cells [5].
In TCM, doctors use Scutellaria baicalensis Georgi (Labitae) to cure mylabris-induced hematuria [6].
S. baicalensis is recorded for its traditional heat-clearing and damp-drying properties and used to treat
respiratory tract infections, vigorous fever, diarrhea, spontaneous external bleeding, jaundice, and
hepatitis. In addition, S. baicalensis also plays a therapeutic role in anti-pyretic and detoxification
treatments [7–12]. In terms of its chemical components, S. baicalensis is rich in flavonoids, including
chrysin, baicalein, baicalin, 7-methylbaicalein, norwogonin, oroxylin A, scutellarein, wogonin, and
wogonoside [13,14]. Among the various components in S. baicalensis, baicalin and wogonin are the
two major ones and are reported to have anti-inflammatory properties. In Lin’s study, baicalin showed
significant anti-inflammatory effects on LPS-induced raw paw edema. Wogonin also displayed PGE2
down-regulative effects in interleukin-1 beta or tumor necrosis factor-alpha-induced NIH/3T3 cell
lines [15–17]. Furthermore, baicalin and wogonin were well-documented to promote urination in
anesthetized dogs, rabbits, and mice [18].
Nowadays, few study discuss rat hemorrhagic cystitis. Researchers usually use cyclophosphamide
and ifosfamide, which are DNA-alkylating agents used in cancer therapy, as animal cystitis inducers.
Metabolites of cyclophosphamide and ifosfamide can induce urinary bladder irritation and hematuria
through COX-2 and inducible nitric oxide synthase (iNOS) overexpression [19,20]. Based on the
above reasons, we assumed that cantharidin might cause bladder inflammation and then induce
hematuria, via a mechanism which possibly the same as that for cyclophosphamide and ifosfamide.
In this study, we tried to establish a cantharidin-induced rat hematuria model by directly injecting
cantharidin into the bladder of female Wistar rats and discuss the mechanism of its irritation. The
boiling-water extract of S. baicalensis (SB-WE) was also used to treat cantharidin-induced rat

Molecules 2012, 17
6279
hematuria and we explored its uroprotective mechanism. In vitro nitric oxide (NO) and prostaglandin
(PG)E2 inhibition abilities and in vivo immunohistochemistry, hematuria level and COX-2 expression
of rat bladder were used to evaluate the uroprotection of SB-WE. Besides, the quality of the SB-WE
was monitored by the levels of two substance markers, baicalin and wogonin, with a high-performance
liquid chromatographic (HPLC) method.
2. Results
2.1. Cantharidin Induced T24 Cell Death
The structure of cantharidin (2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid
anhydride, C10H12O4, m.w. 196.2) is displayed in Figure 1. It is an odorless and colorless powder.
Figure 1. Structure of cantharidin.
O
CH3
O
O
CH3
O
Figure 2A shows that cantharidin significantly induced T24 cell necrosis at 10 μM. To explore
whether cantharidin resulted in cell death through COX-2 expression and PGE2 production, NS398, a
cyclooxygenase-2-specific inhibitor, was used.
Figure 2. NS398 significantly inhibited cantharidin-induced (A) cytotoxicity and (B) PGE2
production in T24 cells. NS398: A selective COX-2 inhibitor. ** p < 0.001. n = 3.
180
160
(A)
140
120
**
100
80
60
40
20
0
Cantharidin (10 M)
-
+
+
+
+
NS398 (M)
25
50
100
100
-

Molecules 2012, 17
6280
Figure 2. Cont.
40
30
20
10
0
(B)
**
**
Cantharidin (10 M)
-
-
+
-
+
+
NS398 (50 M)
-
+
As shown in Figure 2A/2B, NS398 significantly ameliorated the cytotoxicity at 100 μM and
decreased PGE2 production in cantharidin-treated T24 cells at 50 μM. These results suggest that the
cantharidin-induced cell death could be attenuated through treatment with anti-inflammatory agents.
2.2. Cantharidin Induced Rat Hemorrhagic Cystitis Through Initiating Urinary Bladder Inflammation
In an in vivo assay, cantharidin (0.5 mg/kg) was directly injected into the bladder of female rats,
where it induced severe hematuria and dysuria after 16 h (Figure 3).
Figure 3. The boiling-water extract of S. baicalensis (SB-WE) dose-dependently alleviated
cantharidin-induced hematuria and dysuria in Wistar rats.
Cantharidin (0.5 mg/kg)
SB-WE (mg/kg)
200
100
0
Blank
Hematuria level
Urine volume
1+
2+
3+
0+
7.5 mL
7.5 mL
4.0 mL
8.5 mL
The pathology examination showed that cantharidin induced colliquative necrosis of the urinary
bladder epithelium and induction of neutrophil infiltration in cantharidin-treated Wistar rats (Figure 4A).
Besides, the immunohistochemistry results showed that c-Fos, an inflammation-related transcription
factor, was significantly increased in rat bladder after cantharidin injection (Figure 4B). The urinary
bladders were homogenized and protein extracted for the Western blot analysis.

Molecules 2012, 17
Figure 4. (A) Hematoxylin and eosin (H&E) and (B) immunohistochemical staining of
6281
c-Fos to elucidate the uroprotective effects of the boiling-water extract of S. baicalensis
(SB-WE) on cantharidin-induced rat cystitis. The solid black triangle pointed out the
neutrophil infiltrations and significant c-Fos signal.
Figure 5 shows that the COX-2 expression level in the cantharidin-treated group was 1.5 fold higher
than in the blank group.
Figure 5. Effects of the boiling-water extract of S. baicalensis (SB-WE) against
cantharidin-induced rat cystitis and correlated with (A) Western blot and (B) quantitative
analysis (B) of COX-2 protein expression. B, blank; D, DMSO. * p < 0.05, ** p < 0.001, n = 5.
Cantharidin (0.5 mg/kg)
_
(A)
SB-WE (mg/kg)
COX-2
B
D
50
100
200
400
α- Tubulin
2.0
1.5
1.0
0.5
0.0
(B)
*
*
*
*
**
_
SB-WE (mg/kg)
400
H O
DMSO
100
50
200
2
Cantharidin (0.5 mg/kg)

Molecules 2012, 17
6282
2.3. Anti-Inflammatory Effects of Boiling-Water Extracts of S. baicalensis (SB-WE)
To investigate the anti-inflammatory effects of SB-WE, a LPS-induced RAW 264.7 cell model was
used. Results showed that NO and PGE2 production by LPS-induced RAW 264.7 cells was
significantly inhibited by the SB-WE in a dose-dependent manner, and the respective 50% inhibitory
concentration (IC50) values were 79.86 and 35.02 μg/mL (Figure 6).
Figure 6. Anti-inflammatory effects of the boiling-water extract of S. baicalensis (SB-WE)
on (A) NO and (B) PGE2 expressions in LPS-induced RAW 264.7 cells. n = 3.
80
(A)
60
40
20
0
12.5
25
50
100
SB-WE (g/mL)
120
100
80
60
40
20
0
(B)
12.5
25
50
100
SB-WE (g/mL)
2.4. Uroprotective Effects of Boiling-Water Extracts of S. baicalensis (SB-WE)
As shown in Figure 3, the SB-WE dose-dependently alleviated the hematuria and increased the
urine volume, indicating that the toxicity of cantharidin was reduced by S. baicalensis. The hematuria
level was reduced from 3+ to 1+ in cantharidin-treated group after oral administration of SB-WE
(200 mg/kg). Urine volume in the SB-WE group (200 mg/kg) was about 1.5 fold higher than the
cantharidin-treated group (4.0 mL in cantharidin-treated group and 7.5 mL in SB-WE group). In
histological analysis, the colliquative necrosis of the urinary bladder epithelium and neutrophil
infiltration were attenuated in a dose-dependent manner by administration of SB-WE (Figure 4A).

Molecules 2012, 17
6283
Besides, the signal of c-Fos was also reduced by the SB-WE (Figure 4B, solid black triangles). As
shown in Figure 5A, the cantharidin-induced COX-2 overexpression was alleviated by treatment with
different dosages of the SB-WE (Figure 5). The above results indicated that cantharidin-induced
hematuria and dysuria might be correlated with an inflammatory pathway. The SB-WE displayed
significant anti-inflammatory abilities and contributed to prevent cantharidin-induced hematuria in
Wistar rats.
2.5. Quality Control of the Boiling-Water Extract of Scutellaria baicalensis (SB-WE)
The HPLC chromatogram profile of the SB-WE is shown in Figure 7. The respective retention
times of baicalin and wogonin were 16.5 and 40.9 min. The respective contents of baicalin and
wogonin in the SB-WE were 200.95 ± 2.00 and 31.93 ± 0.26 μg/mg.
Figure 7. HPLC chromatogram of baicalin and wogonin in the boiling-water extract of
S. baicalensis (SB-WE). The respective retention times of baicalin and wogonin were 16.5
and 40.9 min.
3. Discussion
Cantharidin is a sesquiterpenoid toxicant and can be extracted from many species of blister beetle.
In China, mylabris (Mylabris phalerata Pallas) is a kind of blister beetle used for anti-cancer or
anti-arthritic drugs [21]. However, the therapeutic threshold of mylabris is very narrow. Many adverse
reactions of mylabris were reported in clinical use [22–24]. According to case reports of cantharidin
poisoning, cantharidin induces fever and hematuria in intoxicated patients [25]. However, the
mechanisms of cantharidin-induced fever and hematuria are not clear. As shown in Figure 2,
cantharidin induced PGE2 production and necrosis in T24 cells which were alleviated by NS398, a
selective inhibitor of COX-2. Evidence showed that COX-2-dependent cell death was also found in
apoptosis-resistant cancer cells treated with an endogenous cannabinoid and anandamide [26]. Hence,
we suggest that the possible mechanism of fever and hematuria induced by cantharidin is through
induction of COX-2 overexpression by bladder epithelial cells.
In the discovery of uroprotective agents, cyclophosphamide and ifosfamide, two well-known
DNA-alkylating agents, are commonly used as hematuria inducers. Metabolites of these two

Molecules 2012, 17
6284
anti-cancer agents, acrolein and 4-hydroxyifosfamide, had been confirmed to be a potential inducers of
urothelial irritation. Evidence has shown that hemorrhagic cystitis occurred by directly damaging with
these two metabolized products, while lead to the ulceration, edema, neovascularization and necrosis
of the bladder [27,28]. Evidence indicates that it takes 12–24 h to induce hemorrhagic cystitis after
treatment with cyclophosphamide and ifosfamide [19,20]. In addition, cantharidin could rapidly induce
blisters on the skin with topical exposure [29]. Cantharidin is also used as an inflammatory inducer to
establish a new animal inflammation model [30], but cantharidin has a high systemic toxicity and
causes diarrhea, congestion of the gastrointestinal tract and kidneys in vivo [31,32]. In this study,
cantharidin was used to induce bladder inflammation and hematuria via direct injection into rat
bladder without causing any systemic toxicity within only one day (Figures 3 and 4). After that, the
uroprotective effects of S. baicalensis were evaluated on the basis of this acute rat hemorrhagic cystitis
model. Firstly, results showed that SB-WE inhibited NO and PGE2 production by LPS-induced RAW
264.7 cells, indicating that SB-WE displayed strong anti-inflammatory abilities in vitro (Figure 6).
Secondly, SB-WE also could reduce cantharidin-induced inflammation and hematuria through
inhibition of c-Fos and COX-2 expressions in an in vivo assay. In TCM, S. baicalensis is a medicine
widely used as an anti-inflammatory therapy and the characteristic of S. baicalensis is to clear internal
heat. In addition, S. baicalensis is widely used as an anti-pyretic and detoxifying herb and is reported
to have anti-cancer, anti-oxidative, and anti-inflammatory effects [14,17,21,33]. A study by Zhang et al.
showed that S. baicalensis could inhibit tumor cell proliferation via suppression of COX-2 expression [21].
Our results show that the obvious COX-2 inhibition abilities of S. baicalensis contributed to the
uroprotective effects against cantharidin-induced hemorrhagic cystitis. Baicalin and wogonin, the
principal compounds in root of S. baicalensis, display significantly anti-inflammatory effects and have
been used as the quality control indicators of the boiling-water extract.
Taken together, we first established a pharmacological animal model for discovering uroprotective
agents. The mechanism of cantharidin-induced rat hemorrhagic cystitis was through overexpression of
inflammatory-related mediators, c-Fos and COX-2. S. baicalensis, a traditional anti-inflammatory herb,
displayed significant uroprotective effects against cantharidin-induced hematuria via inhibition of
c-Fos and COX-2 overexpression.
4. Experimental
4.1. Chemicals
Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT), N-(2-cyclohexyloxy-4-nitrophenyl) methane sulfonamide (NS398) and other chemicals were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM),
fetal bovine serum (FBS), antibiotics, glutamine, and trypsin-EDTA were obtained from Gibco (Grand
Island, NY, USA). COX-2 (clone C-20), α-tubulin (clone TU-02), and c-Fos (clone 4) antibodies for
Western blotting and immunohistochemistry were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Acetonitrile and trifluoroacetic acid (TFA) for the HPLC analysis were
chromatographic grade and were obtained from Merck (Darmstadt, Germany). Purified deionized
water was prepared using the Millipore Milli-RO system (Milli-QRG, Billerica, MA, USA).

Molecules 2012, 17
6285
4.2. Cell Culture
RAW 264.7 (murine macrophage) and T24 (human bladder carcinoma) cell lines were maintained
in DMEM, with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, adjusted to contain 3.7 g/L
sodium bicarbonate, and were maintained at 37 °C with 5% CO2.
4.3. Animals
Female Wistar rats (200~250 g) were bought from BioLASCO Taiwan (Yilan County, Taiwan), and
maintained at 21 ± 2 °C with food and water ad libitum, and kept on a 12-h light/12-h dark cycle. All
Wistar rats used in this experiment were cared for according to the Ethical Regulations on Animal
Research of Taipei Medical University (approval No.: LAC-98-0050).
4.4. Isolation of Cantharidin
Dried mylabris (Chinese blister beetles) was bought from a traditional Chinese medicine store in
Taipei, and voucher specimens (MP-002) were deposited in the Graduate Institute of Pharmacognosy
Science, Taipei Medical University. The isolation procedure was the same as in our previous study [5].
The structure of cantharidin is shown in Figure 1.
4.5. Preparation and Quality Control of the Boiling Water Extract of S. baicalensis (SB-WE)
Dried roots of S. baicalensis were purchased from a traditional Chinese medicine store in Taipei
and identified by Dr. Hsien-Chang Chang. Voucher specimens (SB-001) were deposited in the
Graduate Institute of Pharmacognosy Science, Taipei Medical University. The preparation method was
modified from our previous study [34]. Radices of S. baicalensis were immersed in purified deionized
water and boiled for at least 30 min until half of the original amount was left. Aqueous solutions were
then filtered, and freeze-dried under vacuum.
Two substance markers, baicalin and wogonin, were used to evaluate the quality of the boiling
water extract of S. baicalensis (SB-WE). The HPLC equipment comprised an SCL-10Avp System
Controller, an SPD-M10A Diode Array Detector, an LC-10ATvp Liquid Chromatograph Pump, an
SIL-10Avp Auto Injector, FCV-10Avp Flow-Channel Selection Valves (Shimadzu, Tokyo, Japan),
and an ERC-3415 Degasser (ERC, Altegolfsheim, Regensburg, Germany). The stationary phase
consisted of a Purospher® STAR RP-18e reversed-phase column (5 μm, 4 mm i.d. × 250 mm, Merck).
Acetonitrile-water was used as the mobile phase in the gradient mode as follows: Acetonitrile: 0~17 min,
40%, 17~45 min, 40% to 100%. The flow rate was 1 mL/min, and the oven temperature was
maintained at 40 °C. We used a UV wavelength of 280 nm to detect the baicalin and wogonin.
4.6. Cytotoxicity, PGE2 and NO Inhibitory Assay
The protocols of the cytotoxicity, PGE2, and NO inhibitory assays were modified from our previous
study [35]. RAW 2624.7 cells (4 × 105 cells/mL) were seeded on 96-well plates and co-treated with
LPS (500 ng/mL) and the SB-WE. T24 cells (1 × 105 cells/mL) were seeded on 96-well plates and
co-treated with cantharidin and NS398. After 18 h incubation, cytotoxicity was examined by an MTT

Molecules 2012, 17
6286
assay, and the cell culture supernatant was collected for PGE2 detection using a commercial assay kit
(Assay Designs, Farmingdale, NY, USA). NO was measured as nitrite production in the medium after
24 h of incubation with or without the SB-WE, Briefly, nitrate in the medium was converted to nitrite
and measured spectrophotometrically at 540 nm after the Griess reaction.
4.7. Cantharidin Induced Hemorrhagic Cystitis in Wistar Rats
Wistar rats were starved for 24 h with free access to water and anesthetized with Zoletil® (Virbac,
Carros cedex, France). The urinary bladders of the rats were instilled with cantharidin (0.5 mg/kg) via
a transurethral injection with a 4-Fr. ureteral catheter (BARD, Murray Hill, NJ, USA). After 1 h,
cantharidin-treated rats were orally administrated SB-WE (50, 100, 200, and 400 mg/kg). Each group
had five test rats. Urine was continuously collected for 16 h after administration of the SB-WE, and
then the rats were sacrificed to obtain the urinary bladders. Hematuria levels were analyzed by a
PocketChemTMUA analyzer (Arkray, Tokyo, Japan). According to the operation manual, hematuria
levels could be divided into four levels: 0+, 1+, 2+ and 3+ depending on the hemoglobulin content in
urine. Urinary bladders were fixed in 10% paraformaldehyde, gradually dehydrated in ethanol, and
embedded in paraffin. Immunohistochemical staining for c-Fos was performed on formalin-fixed,
paraffin-embedded tissue sections. Staining was done using conditions recommended by the vendor.
The protocol of COX-2 protein expression was modified from our previous study [5]. COX-2 Protein
expression levels were analyzed with the AlphaImager Imaging System.
4.8. Statistical Analysis
Data are presented as the mean and standard deviation (SD) and were analyzed with Student’s t-test
and One-Way ANOVA by SPSS software. The in vitro and in vivo assays were performed by 3 and 5
independent trials, respectively. The significance level used for statistic analysis displayed as: * p < 0.05,
** p < 0.001.
5. Conclusions
In summary, we found that cantharidin-induced rat hemorrhagic cystitis through up-regulating
expression of COX-2 and the inflammation-related transcription factor, c-Fos. In addition,
S. baicalensis extracts could reduce the cantharidin-induced cytotoxicity, COX-2, PGE2 and c-Fos
expression in vitro and in vivo. Our results could be used as a drug screening platform to discover
novel uroprotective drugs.
Acknowledgments
The authors would like to express their gratitude for the grants from Chi-Mei Medical Center and
Taipei Medical University (96CM-TMU-10).

Molecules 2012, 17
6287
References and Notes
1. Hundt, H.K.; Steyn, J.M.; Wagner, L. Post-mortem serum concentration of cantharidin in a fatal
case of cantharides poisoning. Hum. Exp. Toxicol. 1990, 9, 35–40.
2. Liu, D.; Chen, Z. The effects of cantharidin and cantharidin derivates on tumour cells. Anticancer
Agents Med. Chem. 2009, 9, 392–396.
3. Graziano, M.J.; Waterhouse, A.L.; Casida, J.E. Cantharidin poisoning associated with specific
binding site in liver. Biochem. Biophys. Res. Commun. 1987, 149, 79–85.
4. Karras, D.J.; Farrell, S.E.; Harrigan, R.A.; Henretig, F.M.; Gealt, L. Poisoning from “Spanish fly”
(cantharidin). Am. J. Emerg. Med. 1996, 14, 478–483.
5. Huan, S.K.; Lee, H.H.; Liu, D.Z.; Wu, C.C.; Wang, C.C. Cantharidin-induced cytotoxicity and
cyclooxygenase 2 expression in human bladder carcinoma cell line. Toxicology 2006, 223,
136–143.
6. Wang, G.S. Medical uses of mylabris in ancient China and recent studies. J. Ethnopharmacol.
1989, 26, 147–162.
7. Yin, J.; Wennberg, R.P.; Miller, M. Induction of hepatic bilirubin and drug metabolizing enzymes
by individual herbs present in the traditional Chinese medicine, yin zhi huang. Dev. Pharmacol. Ther.
1993, 20, 186–194.
8. Lam, T.L.; Lam, M.L.; Au, T.K.; Ip, D.T.; Ng, T.B.; Fong, W.P.; Wan, D.C. A comparison of
human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol
extracts of Chinese medicinal herbs. Life Sci. 2000, 67, 2889–2896.
9. Hong, T.; Jin, G.B.; Cho, S.; Cyong, J.C. Evaluation of the anti-inflammatory effect of baicalein
on dextran sulfate sodium-induced colitis in mice. Planta Med. 2002, 68, 268–271.
10. Park, S.; Hahm, K.B.; Oh, T.Y.; Jin, J.H.; Choue, R. Preventive effect of the flavonoid, wogonin,
against ethanol-induced gastric mucosal damage in rats. Dig. Dis. Sci. 2004, 49, 384–394.
11. Di, B.; Feng, N.; Liu, W. Pharmacokinetic comparisons of Shuang-Huang-Lian with the different
combinations of its constitutional herbs. J. Ethnopharmacol. 2006, 107, 401–405.
12. Lu, Y.; Joerger, R.; Wu, C. Study of the chemical composition and antimicrobial activities of
ethanolic extracts from roots of Scutellaria baicalensis Georgi. J. Agric. Food Chem. 2011, 59,
10934–10942.
13. Kimura, Y.; Yokoi, K.; Matsushita, N.; Okuda, H. Effects of flavonoids isolated from scutellariae
radix on the production of tissue-type plasminogen activator and plasminogen activator
inhibitor-1 induced by thrombin and thrombin receptor agonist peptide in cultured human
umbilical vein endothelial cells. J. Pharm. Pharmacol. 1997, 49, 816–822.
14. Huang, W.H.; Chien, P.Y.; Yang, C.H.; Lee, A.R. Novel synthesis of flavonoids of Scutellaria
baicalensis Georgi. Chem. Pharm. Bull. (Tokyo) 2003, 51, 339–340.
15. Lin, C.C.; Shieh, D.E. The anti-inflammatory activity of Scutellaria rivularis extracts and its
active components, baicalin, baicalein and wogonin. Am. J. Chin. Med. 1996, 24, 31–36.
16. Li, H.B.; Jiang, Y.; Chen, F. Separation methods used for Scutellaria baicalensis active
components. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2004, 812, 277–290.
17. Chi, Y.S.; Kim, H.P. Suppression of cyclooxygenase-2 expression of skin fibroblasts by wogonin, a
plant flavone from Scutellaria radix. Prostaglandins Leukot. Essent. Fatty Acids 2005, 72, 59–66.

Molecules 2012, 17
6288
18. Yang, X.; Han, F.M.; Cheng, Z.Y.; Chen, Y. Determination of baicalin and chlorogenic acid in
yinhuang granule by capillary electrophoresis. Se Pu 1990, 17, 573–575.
19. Cho, K.H.; Hyun, J.H.; Chang, Y.S.; Na, Y.G.; Shin, J.H.; Song, K.H. Expression of nitric oxide
synthase and aquaporin-3 in cyclophosphamide treated rat bladder. Int. Neurourol. J. 2010, 14,
149–156.
20. Macedo, F.Y.; Mourao, L.T.; Palheta, R.C., Jr.; Juca, D.M.; Lima, R.C., Jr.; Neto Jde, S.;
Magalhaes, P.J.; Santos, A.A.; Souza, M.H.; Brito, G.A.; et al. Cyclooxygenase-2 contributes to
functional changes seen on experimental hemorrhagic cystitis induced by ifosfamide in rat urinary
bladder. Cancer Chemother. Pharmacol. 2011, 67, 935–943.
21. Zhang, D.Y.; Wu, J.; Ye, F.; Xue, L.; Jiang, S.; Yi, J.; Zhang, W.; Wei, H.; Sung, M.; Wang, W.; et al.
Inhibition of cancer cell proliferation and prostaglandin E2 synthesis by Scutellaria baicalensis.
Cancer Res. 2003, 63, 4037–4043.
22. Worthley, L.I. Clinical toxicology: Part II. Diagnosis and management of uncommon poisonings.
Crit. Care Resusc. 2002, 4, 216–230.
23. Silverberg, N. Pediatric molluscum contagiosum: Optimal treatment strategies. Paediatr. Drugs
2003, 5, 505–512.
24. Al-Binali, A.M.; Shabana, M.; Al-Fifi, S.; Dawood, S.; Shehri, A.A.; Al-Barki, A. Cantharidin
Poisoning due to Blister Beetle Ingestion in Children: Two case reports and a review of clinical
presentations. Sultan Qaboos Univ. Med. J. 2010, 10, 258–261.
25. Mallari, R.Q.; Saif, M.; Elbualy, M.S.; Sapru, A. Ingestion of a blister beetle (Mecoidae family).
Pediatrics 1996, 98, 458–459.
26. Patsos, H.A.; Greenhough, A.; Hicks, D.J.; Al Kharusi, M.; Collard, T.J.; Lane, J.D.; Paraskeva, C.;
Williams, A.C. The endogenous cannabinoid, anandamide, induces COX-2-dependent cell death
in apoptosis-resistant colon cancer cells. Int. J. Oncol. 2010, 37, 187–193.
27. Kanat, O.; Kurt, E.; Yalcinkaya, U.; Evrensel, T.; Manavoglu, O. Comparison of uroprotective
efficacy of mesna and amifostine in Cyclophosphamide-induced hemorrhagic cystitis in rats.
Indian J. Cancer 2006, 43, 12–15.
28. Kurowski, V.; Wagner, T. Urinary excretion of ifosfamide, 4-hydroxyifosfamide, 3- and
2-dechloroethylifosfamide, mesna, and dimesna in patients on fractionated intravenous ifosfamide
and concomitant mesna therapy. Cancer Chemother. Pharmacol. 1997, 39, 431–439.
29. Maglio, D.; Nightingale, C.H.; Nicolau, D.P. Production and resolution of cantharidin-induced
inflammatory blisters. Int. J. Antimicrob. Agents 2003, 22, 77–80.
30. Clementi, G.; Amico-Roxas, M.; Caruso, A.; Catena Cutuli, V.M.; Prato, A.; Maugeri, S.;
de Bernardis, E.; Scapagnini, U. Effects of CGRP in different models of mouse ear inflammation.
Life Sci. 1994, 54, PL119–124.
31. Chang, C.C.; Liu, D.Z.; Lin, S.Y.; Liang, H.J.; Hou, W.C.; Huang, W.J.; Chang, C.H.; Ho, F.M.;
Liang, Y.C. Liposome encapsulation reduces cantharidin toxicity. Food Chem. Toxicol. 2008, 46,
3116–3121.
32. Sánchez-Barbudo, I.S.; Camarero, P.R.; García-Montijano, M.; Mateo, R. Possible cantharidin
poisoning of a great bustard (Otis tarda). Toxicon 2012, 59, 100–103.

Molecules 2012, 17
6289
33. Huang, W.H.; Lee, A.R.; Yang, C.H. Antioxidative and anti-inflammatory activities of
polyhydroxyflavonoids of Scutellaria baicalensis GEORGI. Biosci. Biotechnol. Biochem. 2006,
70, 2371–2380.
34. Wang, K.T.; Chen, L.G.; Chou, D.S.; Liang, W.L.; Wang, C.C. Anti-oxidative abilities of
essential oils from Atractylodes ovata rhizome. Evid. Based Complement. Alternat. Med. 2011,
doi:10.1093/ecam/neq006.
35. Wang, K.T.; Chen, L.G.; Tseng, S.H.; Huang, J.S.; Hsieh, M.S.; Wang, C.C. Anti-inflammatory
effects of resveratrol and oligostilbenes from Vitis thunbergii var. taiwaniana against
lipopolysaccharide-induced arthritis. J. Agric. Food Chem. 2011, 59, 3649–3656.
Sample Availability: Samples of the cantharidin are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).