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Angel of human health: Current research updates in toad medicine
- March 2015
- American Journal of Translational Research 7(1):1-14
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Abstract and Figures
There are currently 34 genera and 410 species of toads in the world. The medicinal parts of toads mainly include their venom, skin, and clothing. The toad's venom and skin possess the same chemical components, mainly the toad venom lactone class, and their pharmacological effects primarily include the maintenance of strong heart, antitumor, antivirus, anti-infection, and analgesic effects. So far, the produces from the medicinal raw materials of the toad are widely used clinically around the world, especially in China, Japan, and South Korea. About 50 varieties of medicines are used in the clinical treatment of various complicated diseases in China, such as "Liushen pills" which was popular in the whole world. Toads are mainly used in treating malignant tumors (e.g., liver cancer, gastric cancer, esophageal cancer, colon cancer, cervical cancer, among others), and some major diseases such as hepatitis B. Despite the therapeutic effects of toad-derived medicines on human health, there is insufficient research and development of toad-derived medicines by leading drug companies. In order to harness the beneficial effects of the resources of the toad species, it is the responsibility of global pharmaceutical researchers to develop and generate economically feasible toad-derived therapeutic products, while promoting maximum protection to the resources of the toad species.
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Am J Transl Res 2015;7(1):1-14
www.ajtr.org /ISSN:1943-8141/AJTR0003150
Review Article
Angel of human health: current research updates in
toad medicine
Qian Yang1,2*, Xuanxuan Zhou1,2*, Meng Zhang1,2*, Linlin Bi1,2, Shan Miao1,2, Wei Cao1,2, Yanhua Xie1,2, Jiyuan
Sun1,2, Haifeng Tang1,2, Ying Li1,2, Qing Miao1,2, Siwang Wang1,2
1Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, China; 2The
Cultivation Project of Collaborative Innovation Center for Chinese Medicine in QinBa Mountains, Xi’an 710032,
China. *Equal contributors.
Received October 14, 2014; Accepted November 25, 2014; Epub January 15, 2015; Published January 30, 2015
Abstract: There are currently 34 genera and 410 species of toads in the world. The medicinal parts of toads mainly
include their venom, skin, and clothing. The toad’s venom and skin possess the same chemical components, mainly
the toad venom lactone class, and their pharmacological effects primarily include the maintenance of strong heart,
antitumor, antivirus, anti-infection, and analgesic effects. So far, the produces from the medicinal raw materials of
the toad are widely used clinically around the world, especially in China, Japan, and South Korea. About 50 varieties
of medicines are used in the clinical treatment of various complicated diseases in China, such as “Liushen pills”
which was popular in the whole world. Toads are mainly used in treating malignant tumors (e.g., liver cancer, gastric
cancer, esophageal cancer, colon cancer, cervical cancer, among others), and some major diseases such as hepati-
tis B. Despite the therapeutic effects of toad-derived medicines on human health, there is insufcient research and
development of toad-derived medicines by leading drug companies. In order to harness the benecial effects of the
resources of the toad species, it is the responsibility of global pharmaceutical researchers to develop and generate
economically feasible toad-derived therapeutic products, while promoting maximum protection to the resources of
the toad species.
Keywords: Toad, cancer, hepatitis B
Introduction
As a creature, the toad has made outstanding
contributions to human health. There are 34
general and 410 species of toads all over the
world that includes Australia as well as Mada-
gascar [1-3]. Toads are probably the most com-
mon amphibians in China. Within the Bufonidae
family, more than half of all species belong to
the same genus, namely the toad. Bufo mem-
bers of this genus are the most predominant
species.
There are 2 general, 17 species or subspecies
of toads in China [4]. The three main varieties
are: 1) the Bufo bufo gargarizans, 2) the Bufo
raddei, and 3) the Bufo melanostictus. Toads
have a bloated shape, and their bodies are full
of bumpy warts, hence they are known as
Laihama, and are hailed as the world’s ugliest
animals. However, they have an indelible func-
tion in medical history, and have been recorded
in China’s Zhang Zhong jing (Classical Presc-
ription), Sun Simiao’s “Valuable Prescriptions
for Emergencies”, Hua Tuo’s “Hua Tuo Reen-
gineering”, and Li Shizhen’s “Compendium of
Materia Medica”. Today, the toad continues to
be essential for the health of mankind. Toads
have been recorded as an invaluable source of
medicine for humans in ancient and modern
Chinese medicine, as well as in modern scien-
tic research.
Modern research in toad medicines
At present, the components of toads that can
be directly used as medicine are mainly toad
venom, toad skin, and toad clothing. The mainly
composition were approximately 103 toad
venom ligand compounds and 12 indole alka-
loids as chemical constituents in toad [5]. The
main composition of dry toad, toad skin and
Toad medicine
2 Am J Transl Res 2015;7(1):1-14
toad clothing compared to toad venom is basi-
cally the same; however, the difference in the
concentration of the components is large. Thus,
the differences in pharmacological functions
and clinical applications of the varied toad
medicines are very distinct.
The chemical composition of toad venom
Toad venom is the most complex composition
of toads. The difference in chemical composi-
tion varies greatly based on the variety of the
toad venom, area of production, differences in
the methods of collecting and processing. Toad
venom has both fat soluble and water-soluble
chemical components, based on the active
Figure 1. Structure of main chemical ingredients in the toad venom. A. Cinobufagin (CB). B. Resibufogenin (RBG). C.
Bufalin (BL). D. Cinobufotalin (CBTL). E. Bufotenine (BT). F. Bufotenidine (BTD). G. Bufobutanoicacid; H. Serotonin. I.
Bufotenine N-oxide. J. N-methyl serotonin.
Figure 2. Chemical construction of Bufotaline.
Toad medicine
3 Am J Transl Res 2015;7(1):1-14
ingredient that inuences the dissolving prop-
erties. In order to determine the composition of
the chemical structure of the fat-soluble class
in toad venom, it has been observed that cino-
bufagin (C26 H34O6, CBG, Figure 1A), resibufo-
genin (C24 H32O4, RBG, Figure 1B), and bufalin
(C24H34O4, BL, Figure 1C). These three chemi-
cals account for about 10% of the dry weight
(CBG or RBG content in a small number of sam-
ples of toad venom can be as high as 7 to 8%)
[6]. Cinobufotalin (C24 H34O6, CBT, Figure 1D),
BL, CBG, and RBG content in the range of 0.05-
5.12% (w/w), are derived from Bufo bufo gar-
garizans of toad venom, and the concentration
of of RBG, CBG, BL, CBT are in the range of
0.94-2.15%, 1.19-3.03%, 0.46-1.07% and
0.29-0.87%, respectively. The concentrations
in toad venom from Bufo melanostictus are in
the range of 0.12-3.69%, 0.19-5.12%, 0.08-
1.71% and 0.05-1.14%, respectively [7]. Water-
soluble ingredients include bufotoxins such as
the toad diene class, the cardiac glycosides
ene toad venom, the indole alkaloids (Figure
1E-J), the alcohols and polysaccharides, the
amino acids, the peptides, adrenaline, toad
venom tryptamine like serotonin, toad venom
tryptamine, toad venom quaternary ammoni-
um, toads and ning, and dehydrogenation toad
venom tryptamine, toad venom tryptamine
hydrobromide, and others [8].
Toad toxins and toad color amine content have
the strongest pharmacological effects in toad
venom. Bufotalin (C26H36O6, BTL, and Figure 2)
is one of the most important active compounds.
Yang et al [9] separated toad venom spirit,
3-butyl-2-acyl arginine acetate, from the toad
skin. Shimada, et al [10] extracted spirit-3-sym-
plectic-2-acyl-L-histidine, 1-methyl-3-methyl his-
tidine, histidine ester and ve other toad toxins
from the toad venom and the skin of toads, in
Taiwan and Bufo melanostictus.
Cardiac steroid compounds, can be divided into
free-type and combination-type compounds.
The free-type compounds are called bufogenin,
such as BL, RBG, and others. Combination-type
compounds are called bufotoxin, such as fat
toad venom ligands, 3-sulfuric acid esters.
These compounds are toad diene hydroxyl acid
lactone compounds (Figure 3) [1 1] .
Figure 3. Toad alkadiene acid lactone compounds. A-C. Showed kinds of the mother structures.
Figure 4. The class indole alkaloids. A. Toad thiamethoxam nowadays. B. Sterol compounds. C. Chemical structure
of the mother structures.
Toad medicine
4 Am J Transl Res 2015;7(1):1-14
Toad venom lubricious amine compounds
belong to the class of indole alkaloids. Li Weixi
[12] isolated toad thiamethoxam from the dry
toad (Figure 4A). The chemical composition
content of toad thiamethoxam is high in the dry
toad and is low in the toad venom. The dry toad
also contains cholesterol, beta sitosterol, and
palmitic acid cholesterol ester (Figure 4B, 4C).
Such compounds are divided into compounds
containing lactone structure, such as the toad
ring amide B (C8H12N2O4, bufo gargarizans-B,
BGZ-B, Figure 5A) [13], the toad ring amide D
(C8H12N2O3, bufo gargarizaine-D, BGZ-D, Figure
5B) [14], and exclude lactone structure com-
pounds, such as toad ring amide C (C16H26N4O9,
bufo gargarizans-C, BGZ-C, Figure 5C) [13].
It has been reported that the water-soluble
components that have been separated from
the dry toad, contain ingredients like dipeptide,
pyrimidine, and adenosine [14]. In addition,
they also contain photopigments [15] and all
kinds of inorganic elements [16].
The chemical composition of toad skin
The moisture content of toad skin was 10.99%
on an average, the total ash content on an aver-
age was 13.21%, the acid insoluble ash con-
tent was 4.58% on an average, the CB and RBG
contents were 0.13% and 0.03%, respectively
[17]. Yan Ziping [18] performed 10 times the
amount of 80% ethanol reux extraction of toad
skin, the average concentration of CBG and
RBG, in the preparation of the extract, was
0.20% and 0.11%, respectively. There is a
major impact on the toad skin when using dif-
ferent extraction methods to extract ingredi-
ents like toad venom lactone. Cao Xutao et al
[19] were able to extract RBG, CBG, BL, telo-
cinobufagin (C24H34O5, TCBG, Figure 5D), BTL,
desacetylcinobufotalin (C24 H34O6, DTCBT, Fig-
ure 5E), hellebrigenin (HBG), arenobufagin (AB),
gamabufotalin (GBTL), 11β-hydroxylresibufo-
genin (11β-HRB), CBT, and other 11 kinds of
bufogenin components from the toad skin.
Zhao Dazhou et al [20] did a comparative study
on toad diene lactone compounds and their
concentration in the toad venom and the toad
skin, and found that both contain the same
diene lactone composition that mainly includes
four indole alkaloids. The concentration of
dehydrogenation toad tryptamine is higher in
the toad skin, while it is low in toad venom.
Sterols in the toad venom mainly consist of
sitosterol, while in toad skin; the sterols are
mainly cholesterol and cholesteryl palmitate.
The skins of the Taiwan toad (Bufo vulgaris for-
Figure 5. Toad ring amide compounds structures. A. Bufogargarizaine B. B. Bufogargarizaine D. C. Bufogargarizaine
C. D. Telocinobufagin. E. Desacetylcinobufotalin. F. Hellebrigenin.
Toad medicine
5 Am J Transl Res 2015;7(1):1-14
mosus Boulenger), green toad (B. viridis Laur?)
from Japan, Bangkok toad (B. bankorensis
Borbour) from Taiwan and Bufo Bufo gargari-
zans (B. Bufo gargarizan Cantor) from China
were analyzed and found to possess succinic
acid, adipic acid and pimelic acid instead of
symplectic 2 acyl arginine ester compounds,
and sulfuric acid ester compounds. The skins
of the toad (B. americanus) from North America
and Bufo melanostictus (B. melanostictus
Schneider) from Taiwan were analyzed and
shown to contain I-histidine, L-1-methyl histi-
dine, and L-part 3 instead of arginine-methyl
histidine-bufatoxin compounds [21]. Therefore,
the chemical composition and the concentra-
tions in toad skin from different regions in the
world display a large difference. Xu Naiyu et al
[16] analyzed and determined the chemical ele-
ments in the toad skin of Bufo gargarizans from
China, and showed that the toad skin contains
calcium, magnesium, sodium, manganese,
iron, zinc, copper, phosphorus, silicon, and the
silver elements. The calcium content is the
highest, followed by iron and magnesium.
Chemical composition of toad clothing
Chinese scholars [16] rst analyzed the chemi-
cal composition of toad clothing from Bufo gar-
garizans and identied eight compounds: 1)
hexadecanoic acid cholesterol ester, 2) choles-
terol, 3) 5-alpha, 8-alpha-epidioxycholesta-
6-en-3 beta-ol, 4) cholesteric-5-ene-3 beta,7
beta- diol, 5) cholesteric-7-ene-3 beta, 5 alpha,
6 beta-three glycol, 6) 3-18 alkoxy-1, 2-propyl-
ene glycol, 7) delta4,5(E), delta9,10(Z)-sheath
amino alcohol (fteen carbonic acid amides),
and 8) toad thiamethoxam. The compounds (3),
(5)-(7) were obtained from Bufo gargarizans
and the pseudopelade at the rst time. In addi-
tion, other reports have documented that toad
clothing contains RBG in the range of 31.3-80
μg/g [22-24].
Pharmacological research
Cardio tonic effect
The effect of RBG, in toad venom, on cardiac
function is stronger, followed by BL and CBG.
Studies have shown that the impact of CBG on
anesthesia, blood pressure, and normal cardi-
ac function is not obvious; however, there is a
positive effect on the articial blood loss-
induced hypotension of the drug. There is an
increase in cardiac output and an increase in
arterial pressure, but the impact on the heart
rate is not obvious [25]. The inhibitory effect of
the toad venom on Na+-K+ ATPase in red blood
cells is very strong, and leads to an increased
myocardial intracellular Na+ concentration. The
Ca2+ levels in the myocardial cells get excited
and reinforce a myocardial contraction [26].
Toad venom can prolong the brinogen coagu-
lation time; its anticoagulant effect is similar to
urokinase, by making the brin dissolve acti-
vated enzymes that increase the coronary
artery perfusion ow and the myocardial nutri-
tional blood ow to improve microcirculation,
and thus increases the myocardial oxygen sup-
ply. In addition, the toad venom can cause
peripheral vascular contraction thereby mim-
icking the effect of adrenaline. Xie Jingtian [27]
conrmed RBG can decrease membrane reac-
tion in Pu Ken wild ber of dog and sheep, slow
conduction and generate excitement anti-
arrhythmic drugs.
Anti-tumor effect
Antitumor activity is one of the most important
pharmacological effects of toad venom. The
IC50 for toad steroid alkene compounds on
tumor cells is in the range of 1 to 10 nM. The
antitumor mechanism involves inducing tumor
cell apoptosis, promoting tumor cell differentia-
tion, increasing immunity, and inhibiting prolif-
eration and angiogenesis of tumor endothelial
cells [28]. Kamano [29] tested 80 types of nat-
ural toad steroid materials and its chemical
derivatives on liver cancer cells. The in vitro
inhibitory effect of PLC/PRF/5 showed that the
C-17 alpha-pyrone ring, 3 beta-OH, 14 beta-OH
(or 14 beta, 15 beta-ternary ring oxygen), C/D
ring cis-fused groups, and other similar struc-
tures are necessary for the antitumor activity.
When 19-methyl oxidized into aldehyde group
the activity was enhanced, and when the 14-is
a beta-OH of toad steroid ene role the activity
was stronger than 14 beta and 15 beta with
ternary oxygen ring structure, and HBG, BL in all
subjects the strongest active compounds (IC50
were 1.6×10-4 μg/mL and 2.8×10-4 μg/mL). All
the toad steroids contain 14 beta and 15 beta
ternary ring oxygen compounds, where the
activities are the strongest (IC50 = 7.4×10-4 μg/
mL). In such compounds 19-oxidation of alde-
hyde group activity was enhanced, while the
Toad medicine
6 Am J Transl Res 2015;7(1):1-14
hydroxymethyl activity was weakened; the
activity of 16-bit acetoxyl group was signicant-
ly enhanced, while introducing the hydroxyl or
other ester base was reduced [30]. Chen Xiaoyi
et al [31] found that BL inhibits the growth of
the poorly differentiated gastric cancer, MGC-
803, and the IC50 is about 0.1 μmoL/L. Yin
Peihao et al [32] observed that BL signicantly
inhibits BxPC-3 pancreatic cancer cell growth.
There is a positive correlation between inhibi-
tion, drug concentration, and the time of action.
Su Yonghua [33] showed that the different con-
centrations of BL damage the human liver cell
membrane. BL, rather than CBG and RBG, has
a pronounced inhibitory effect on SMMC-7721
and BEL-7402, and that the same effect on
growth inhibition was lower than that of mito-
mycin. The team of Qu Junle [34] found that BL
signicantly inhibits the growth of ASTC-a-1
cancer cells from human lung adenocarcinoma
in the form of concentration dependence. Jiang
[35] veried that BL has an obvious pharmaco-
logical activity on the human non-small cell
lung cancer cell line A549, and this inhibition
activity was positively correlated with the time
of administration and the dose of the drug. The
IC50 of BTL on human Hela cells, PLC/PRF/5
cells, human nasopharyngeal carcinoma Kb
cells, and human granulocyte leukemia HL60
cells are 0.11 μΜ [36], 3.4×10-4 μg/mL [29],
0.19 μg/mL/mL [37], and 0.01 μg/mL [38],
respectively. Su [38] found that IC50 for BTL-
treated human hepatoma Hep3B, human
colorectal cancer HT-29, and human breast
cancer MCF7 cells, after 6 days of differentia-
tion, were 0.24 ± 0.02 mΜ, 0.234 ± 0.03 μM,
and 0.15 ± 0.02 μM, respectively. The above
results show that the BTL antitumor effect is
not only signicant, but BTL has a wide antitu-
mor spectrum. Tian [39] detected the antineo-
plastic activity of 10 kinds of bufadienolide
compositions on HepG2 and A549 cells, and
found that the IC50 of BL were 0.6 ± 0.0 μM and
0.6 ± 0.1 μM, and the IC50 of BTL were 0.5 ± 0.1
μM and 0.9 ± 0.2 μM, respectively, compared
with that of adriamycin (0.3 ± 0.1 μM and 0.1 ±
0.0 μM) that showed no signicant difference.
Belgian scholar Moreno [40] performed a com-
parative study of the antitumor effects of 27
kinds of bufadienolides in 6 strains of anthro-
pogenic tumor cells, Hs683, MCF-7, PC-3,
A549, U373, SKMEL-28, and tumor cells of 2
strains of the rat, CT26 and WT B16F10. The
conrmed average IC50 of the gamabufotalin
rhamnoside, bufotalin, hellebrin and argentino-
genin were low, ie, 50 ± 11 nM, and the IC50 of
the gamabufotalin rhamnoside on 6 kinds of
anthropogenic tumor cells were low, ie, 3.0 ±
0.0 nM. The antitumor mechanism of bufadi-
enolides may either involve inhibition of the
Na+-K+ ATPase activity of tumor cells or ClC-3Cl-
is catalyzed to start inhibiting the P13K/Akt/
mTOR signaling pathways, which have an anti-
tumor role [41].
Induction of tumor cell differentiation
BL is the main component in inducing tumor
cell differentiation [42]. The main mechanism
is to inhibit topoisomerase II and the activities
of protein kinase A and protein kinase C. By
lowering WT1 gene expression, BL induces dif-
ferentiation of K562 cells. When BL is in the
range of 0.01 moL/L-0.026 moL/L, there is
only cell differentiation, and when BL is in the
range of 0.026 μmoL/L-0.05 μmoL/L, WT1 pro-
tein and mRNA levels will be lower. It has been
shown that decreased WTl gene expression
can promote differentiation and apoptosis of
K562 cells [43]. Research by Takai [44, 45]
showed that endometrial cancer cells (HHUA,
HEC-1B), ovarian cancer (SKOV3, OMC-3), pla-
cental villi cancer cells BeWo, and gynecologi-
cal malignant tumor cells are extremely sensi-
tive to BL, and the drug mainly inhibits tumor
cell differentiation in the G0/G1 phase.
Induction of apoptosis of tumor cells
Huang [46] found that BL, at a concentration of
0.01 μmoL/L, inhibits the growth of human le-
ukemia cells HL60 and apoptosis occurs after
24 h in the cells. Bone sarcoma cells, U2OS,
treated with BL for 48 h, display condensed
chromatin and have the typical apoptotic body
that increases with the increase in the concen-
tration of BL. This indicates the cytotoxicity of
BL and its effect on promoting apoptosis [47].
BL has an effect on target-induced tumor cell
apoptosis, for example, BL can increase the
expression of the Bax protein and lower the
Bel-2 protein levels, to induce the apoptosis of
tumor cells (eg, U2OS, HL60, HepG2, A549)
[48]. Sun [49] studied and found that when BL
acts on human lung adenocarcinoma ASTC-a-1
cells, it can produce a large number of reactive
oxygen species, strengthen caspase 3 activity
at the same time, induce the transfer of the Bax
apoptosis protein from the cytoplasm to the
Toad medicine
7 Am J Transl Res 2015;7(1):1-14
mitochondria, thereby inducing apoptosis in
ASTC-a-1. Kurosawa [50] found that specic
inhibitors of protein kinase c (protein kinaze c,
PKC) can signicantly decrease the leukemia
expression of BL-inducing IL-1 beta gene of the
THP-1 human mononuclear cells, and thus
explain that BL may induce differentiation and
apoptosis of the tumor cells through the PKC
pathway. Recent studies suggest that the mem-
brane Na+-K+ ATPase is the main target of BL
and has an anti-tumor effect, through specic
inhibition of Na+-K+ ATPases, and by launching a
series of activated downstream proteins relat-
ed to apoptosis signaling pathways, thus, even-
tually leading to apoptosis [51, 52].
Autophagy induced by tumor cells
Our researches as well as previous reports [53]
have conrmed that BL can signicantly cause
apoptosis and autophagy of human hepatocel-
lular carcinoma HepG2 cells [54]. It has also
been conrmed that BL causes cell death in
human colon cancer cell lines, HT-29 and Caco-
2, by inducing autophagy and not by inducing
apoptosis. The mechanism of autophagy invo-
lves BL to induce cancer cells to produce a lot
of reactive oxygen species (ROS). In turn, ROS
activates the JNK pathway-raised autophagy
related genes ATG5 and BECN1 expression and
initiates the autophagy of tumor cells.
Anti-angiogenesis
Studies by Lee [55] conrm that 5, 10 and 20
nmol/L of BL have signicant inhibitory effects
on angiogenesis, and the inhibition rates are
45.3%, 62.8%, and 75.6%, respectively. Wang
Nayao [56] found that Huachansu injection
contained the BL component in the chicken
embryo allantois membrane and had an inhibi-
tory effect on angiogenesis.
The analgesic effect of anesthesia
Zhang Wei [57] conrmed that toad venom has
an analgesic action with 6 kinds of fat-soluble
components, of which 1.3 mg/kg RBG and 1.3
mg/kg CBG display the most signicant analge-
sic effects. The analgesic effect of 0.8 mg/kg
BL is relatively stable, and the analgesic effect
of the South American toad toxins, BL and BTL,
are weaker than the CBG. The local anesthesia
effect of BL is 30 to 60 times more than
cocaine, 300 times more than procaine, and
the anesthesia time is long. There are no cen-
tral poisoning symptoms and local stimulation,
and the mechanism may be associated with
the slow release of acetylcholine of muscle
cells [58].
Other activity
Toad venom exerts rapid and signicant anti-
bacterial effects on Staphylococcus aureus
and alpha hemolytic Streptococcus. Toad
venom also displays therapeutic effects on
purulent disease induced by some antibiotics,
but also has the capacity to increase blood cap-
illary permeability during inhibition, reduces
medicinal overow, and is benecial in eliminat-
ing swelling, and so on. CBG activates the mice
abdominal cavity macrophages, improves
phagocytosis, kills bacteria and inhibits the
growth of bacteria [59]. Studies conrm that
the toad clothing extract can inhibit Na+-K+
ATPase activity [60], reduce blood sugar [61],
inhibit HIV-1 [62], and inhibit mice S180 sarco-
ma, H22 liver cancer, Lewis lung cancer, liver
ascites, and HCA growth pharmacological
effects, among others [63].
Toxicity
Toad venom
The oral LD50 of toad venom is 0.36 mg/kg, and
the common adult clinical oral dose is 3-5 mg/
day (the maximum dose cannot exceed 135
mg/day) [64]. He Shilin [65] conrmed the LD50
of toad venom extracts by different fabrication
processes. Intravenous injection of mice pro-
duced a rough extract of 0.04 g/kg of toad
venom, an alcohol extract of 0.21 g/kg, and a
water extract of 0.9 g/kg of toad venom. Toad
venom, produced by intravenous injection, of
75% ethanol extract in mice for 14 days showed
an LD50 of 0.60 g/kg [66]. The intravenous LD50
of toad venom was 41 mg/kg, LD50 of toad
venom by subcutaneous injection was 96.60
mg/kg, and that by intraperitoneal injection
was 36.24 mg/kg [67].
Toad skin
A research of Jin Qiquan [68] shown that a mix-
ture of toad skin water and fat displayed an
intravenous LD50 of 3.81 ± 0.22 mg/kg in mice,
and an intraperitoneal injection showed an
LD50 of 26.27 ± 0.30 mg/kg. It was observed
that the original toxic drug ingredients are
Toad medicine
8 Am J Transl Res 2015;7(1):1-14
mainly toad toxins and ester soluble cons-
tituents.
Lactone class ingredients of toad venom
The LD50, by intraperitoneal injection of BL and
CBG were 2.22 mg/kg and 4.38 mg /kg, respec-
tively. The LD50 of toad aglucone by rapid intra-
venous injection was 4.25 mg/kg, by slow intra-
venous injection was 15 mg/kg, the LD50 by
intraperitoneal injection was 14 mg/kg, the
LD50 by subcutaneous injection was 124.50
mg/kg, and orally, the LD50 was 64 mg/kg. The
LD50 of toad turney intravenous injection was
1.30 mg/kg. The intravenous LD50 of BTL in
dogs is 0.36 mg/kg, with an oral minimal lethal
dose of 0.98 mg/kg. The LD50 of BL mice tail
intravenous injection was 2.26 mg/kg [69]. The
LD50 of BTL tail intravenous injection in mice
was 4.13 mg/kg [70].
Secretio bufonis injection
The LD50 of rat peritoneal injection was 102.65
mg/kg [71]. The main adverse reactions of the
toad venom injection in clinical use are phlebi-
tis, rare allergic reactions and arrhythmia, but
most of the symptoms can be controlled by
regulating the dripping speed, preventing leak-
age, and careful nursing that are effective to
prevent these side reactions. Other general
side effects of oral preparations are lighter and
can be effectively prevented through a control
dose [72].
Cinobufacini injection
Our study conrmed that the LD50 of the intra-
venous Cinobufacini injection in the rat was
312.95 mg/kg. Clinical side effects mainly are
high-risk vascular irritation reaction (about
70.63%), followed by drug fever (10.71%), and
allergic reactions (9.13%) [73].
To sum up, the toad lactone class is the main
medicinal ingredient. The toad lactone is a lip
soluble constituent, where the most important
toxic target organ is the heart. The intensity of
toxicity decreased in the order of AB>BL>TCBG
>HBG>GBTL>CBT>BTL>RBG>DTCBT [74].
Drug research
Toad venom as raw materials for drugs
In China, toad venom has been made into a
variety of drugs, such as injections, oral liquids,
lm agents, pills, the transdermal drug delivery
system, among others, and is mainly used for
clinical treatment of multiple premature beats,
increase in eosinophils, leukemia, skin cancer,
neurodermatitis, tuberculous stula, tooth-
ache, and local anesthesia [75, 76]. In recent
years, some scholars have been conducting
research in new dosage forms, mainly lipo-
somes, microspheres, beta entrapped cyclo-
dextrin complex, albumin nanoparticles, aero-
sols, Papua agents, the microemulsion com-
bine, and others [77, 78]. For example, injec-
tions made by Chinese Anhui Keyuan pharma-
ceutical group Co. Ltd. (approval number:
Z34020604) and the Chinese Jiangsu Ange
pharmaceutical Co. Ltd. (approval number:
Z32020694), with main ingredients called
indole alkaloid derivatives, indoles, total alka-
loid in serotonin (C10H12ON2) per milliliter meter
>18 μg, have effects in heat-clearing and detox-
ication; Liushen pills (approval number:
Z32020481), the compound toad venom mas-
tic (approval number: Z20063321), Tianchan
capsule (approval number: Z20020056), the
toad venom Jiuxin pill (approval number:
Z22022831), Xinli pill (approval number:
Z44021844), Japan’s approval and production
of KYUSHIN (approval number: C310621) these
medicines are widely used in kinds of cancer
(digestive, respire atory, urinary and reproduc-
tive system, skin and head and neck cancer
patients), a variety of diseases such as asthma,
acute and chronic cardiac failure.
Toad skin as raw materials for drugs
Toad skin also has been made in kinds of medi-
cal preparations. Such as Cinobufacini injec-
tion which contains ingredients like toad venom
ligands, with the concentration of the ingredi-
ents in the range of 4.62 to 5.80 mg/L [79],
and shows detoxication, detumescence
effects, pain relief, antiviral effects, promotes
bone marrow proliferation, and enhances
immunity effect, among other functions. The
injection can be used in the clinical treatment
of advanced tumors and chronic hepatitis B
[80-82]. Its antitumor mechanisms include the
drug-regulated immune function [83, 84], inhi-
bition of tumor cell proliferation [85], induction
of tumor cell apoptosis [86] and differentiation
[87], inhibition of tumor angiogenesis [88], and
reversal of multi-drug resistance [47]. The main
representative products as Cinobufacini tab-
lets (Z34020272), Cinobufacini oral liquid
Toad medicine
9 Am J Transl Res 2015;7(1):1-14
(Z34020644), Cinobufacini capsule (Z2009-
0944), Antike capsule (Z10960071), contains
indole alkaloids, reducing sugars, amino acids,
and toad toxins such as the toad tryptamine
[89], widely used in detoxication, tumescence,
pain relief, and the treatment of medium and
advanced cancer, chronic hepatitis B, among
other diseases.
Treatment of malignant tumors in clinical ap-
plication
Cao Jie [90] conrmed that the toad venom
injection in combination with the chemical ther-
apy treatment of 60 cases of late malignant
tumors, can signicantly improve the body’s
immune function, improve the quality of life of
patients, signicantly increase white blood
cells, and reduce the toxic reaction caused by
chemotherapy. Zhao Jianqing [91] performed a
comparative study of toad venom injection with
chemotherapy and chemotherapy alone to ana-
lyze the effect on the treatment of advanced
non-small cell lung cancer, in terms of toxicity
and immune function. Radiotherapy with toad
venom injection of 31 cases of patients with
advanced esophageal cancer [92] and chemo-
therapy with toad venom injection of 34 cases
of non-small cell lung cancer [93] conrmed
that toad venom injection can signicantly
enhance the curative effect, relieve local pain,
reduce fever, increase the appetite, improve
the immune function and mental health, and
other qualities of life. Huang Zhifen’s [94] clini-
cal observation conrmed that chemotherapy
with toad venom injection in the treatment of
advanced gastric cancer can act synergistical-
ly. Qiu Bingli [95] treated 60 cases of advanced
malignant tumors with the Cinobufacini injec-
tion (34 cases of lung cancer, 16 cases of
digestive tract tumor, 9 cases of primary liver
cancer and l case of neurogenic tumor), and the
total effective rate (PR+SD) was 68.33%. Wang
Changjun [96] reports two methods, percuta-
neous left subclavian artery perfusion
Cinobufacini injection and hepatic artery che-
moembolization (HAC), to treat all the 30
patients with inoperable advanced liver cancer.
The total effective rate of the Cinobufacini
group was 70%, and signicantly improved liver
function and the prevention and treatment of
liver brosis. The total effective rate of the HAC
group was 76.7%. Ma Jinli [97] studied 109
patients with NSCLC (gemcitabine + cisplatin +
Cinobufacini injection) and a control group of
108 cases (gemcitabine + cisplatin). The effec-
tiveness of treatment group and control group
were 55.96% and 37.96%, respectively.
Leukopenia, abnormal renal function, and gas-
trointestinal reaction in the treatment group
were signicantly lower than the control group.
Liu Xiaohong [98] reported that 42 cases of
advanced primary liver cancer patients (HAC +
Cinobufacini injection) compared with and a B
group (pure HAC) with conventional treatment.
The results showed the total effective rate of
the A group was 83.3%, which is signicantly
higher than 57.1% of group B (P<0.01). The T
cell subgroup number and NK cell activity of
group A is signicantly higher than group B.
Cinobufacini injection and radiation combined
treatment can obviously reduce the adverse
reactions of tumor radiotherapy, improve the
patients’ tolerance.
Zhang et al [99] conrmed that Cinobufacini
injection static drops companied with gamma
knife treatment made side effects of radiother-
apy and pain obviously lower than the control
group without Cinobufacini injection static
drops, and 1-year survival rate of the joint group
was 73.33% higher than control group of
43.33% (P<0.01). Toad venom “Jiening’s” elec-
tuary treatment of advanced carcinoma of
severe pain has the characteristics of quick
effect, reduced time with pain, and less adverse
reactions. The joint use of 20 tablets of mor-
phine can increase the analgesic action [100].
The total effective rate of complex toad venom
powder treatment of cancer pain was 93.3%,
and the average duration of pain relief was
16.5 h. The improvement in the quality of life
and the stability factor is 90% [101]. Liu et al
[102] reported that the toad electuary was
mainly used to treat various kinds of cancerous
pain in 332 cases. The total effective rate was
92.65%. With Chanwu Cataplasm (the prescrip-
tion is mainly composed of toad venom, radix
aconiti, among others) treatment of 120 cases
of patients with advanced lung cancer pain, 45
cases were greatly improved (37.5%), 60 cases
were markedly improved (50%), 15 cases were
invalid (12.50%), and 120 cases of patients
had a pain duration of an average of 11.5 h
[103]. Gong Zipeng et al [104, 105] observed
that the analgesic effect of the Cinobufacini
injection is mainly mediated by peripheral opi-
oid receptors, and has nothing to do with the
central opioid receptor. The analgesic action
Toad medicine
10 Am J Transl Res 2015;7(1):1-14
may not produce similar effects as the central
opioid receptor in blocking drug addiction and
the withdrawal syndrome caused by side
effects, but whether it is addiction needs to be
further analyzed.
The toad venom injection, 10-20 ml + 5% glu-
cose liquid, in a 500 ml intravenous drip was
used to treat 50 patients of suspected inuen-
za a (H1N1). The signicant efciency reached
90%. The comprehensive curative effect is
superior to the Antondine and the radix bupleu-
ri injection. Researchers [106] have observed
this curative effect may be associated with the
toad venom injection and displays antiviral and
antibacterial effects, enhances immunity,
relieves cough, and eliminates phlegm.
Cinobufacini injection suppresses HBV replica-
tion. The total effective rate was 55%, which
was signicantly higher than the control group
(20%). The signicant difference was found in
the two groups (p<0.05) [107]. Pan Yiren [108]
conducted the treatment of 50 cases of abnor-
mal alpha-fetoprotein (AFP) in patients with
chronic hepatitis B. one group was treated with
30 ml Ganlixin injection + 20 ml Cinobufacini
injection and the control group with 30 ml
Ganlixin injection. Results from the treatment
group AFP showed a rapid decrease (including
18 cases dropped to normal, rate of 62.4%)
compared with the control group (P<0.005).
The normal control group showed no signicant
AFP, and the percentage is only 22.7%. In one
year liver cancer was detected in only 2 cases
(9.1%). Liu Hui [109] reported that HBeAg nega-
tive conversion ratios for the treatment group
(add Cinobufacini injection) at the end of the
treatment, or treatment after six months, were
62.90% (22/35) and 65.70% (23/35), respec-
tively, while those of the control group were
37.90% (11/29) and 41.40% (12/29), respec-
tively. HBV-DNA negative conversion ratios in
the treatment group at the end of the treatment
and 6 months after the treatment were 65.70%
(23/35) and 68.60% (24/35), respectively
while the control group was 41.40% (12/29)
and 41.40% (12/29), respectively. This sug-
gests that Cinobufacini injection can obviously
improve the Ara-Amp negative conversion ratio
of HbeAg and HBV-DNA, enhance the body’s
immune function, and improve the antiviral
effect.
Epilogue
To sum up, toad, as an important member in
the biological world, not only maintains the bio-
logical chain of balance and evolution, but also
plays an important role in the protection of
human health. Toads and humans live together
on earth. Human beings should rationally pro-
tect the toad species in the earth’s biosphere
and promote the breeding of toads. Therefore,
there should be more scientic and economic
value for toad medicines that will guarantee the
prosperity of the biosphere on earth, and
hence, safeguard the health of humans.
Disclosure of conict of interest
None.
Address correspondence to: Siwang Wang, Institute
of Materia Medica, School of Pharmacy, Fourth
Military Medical University, 169 West Changle Road,
Xi’an 710032, Shanxi, China. Tel: 0086 29
84772519; Fax: 0086 29 83224790; E-mail: wang-
siwang_wsw@163.com; wangsiw@fmmu.edu.cn
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... The skin secretions and venom of amphibians are rich sources of bioactive compounds, such as peptides, bufadienolides, alkaloids, proteins and biogenic amines, which have contributed meaningfully to human health and most pharmaceutical researches [26,27]. Yang et al. [27] maintained that the toad toxins from the parotoid or skin glands have significant therapeutic value for a good number of diseases. ...
... The skin secretions and venom of amphibians are rich sources of bioactive compounds, such as peptides, bufadienolides, alkaloids, proteins and biogenic amines, which have contributed meaningfully to human health and most pharmaceutical researches [26,27]. Yang et al. [27] maintained that the toad toxins from the parotoid or skin glands have significant therapeutic value for a good number of diseases. Against this backdrop, this study screened toad venoms for its antiplasmodial potency in Swiss albino mice infected with P. berghei. ...
Article
Full-text available
- Mar 2021
... For these reasons, these compounds may serve as leads for developing novel heart medications and analgesics. Furthermore, preparations from anuran venoms including toad cake showed encouraging in vitro antitumor activity that might be associated with the inhibition of cell proliferation, the promotion of angiogenesis of tumor endothelial cells, the induction of apoptosis, the promotion of differentiation, and the stimulation of tumor-directed immune responses [72][73][74]. Comparable results were obtained with various isolated bufadienolides [72][73][74]. Thus, ingredients of anuran skin secretions may also turn out useful against cancer. ...
... Furthermore, preparations from anuran venoms including toad cake showed encouraging in vitro antitumor activity that might be associated with the inhibition of cell proliferation, the promotion of angiogenesis of tumor endothelial cells, the induction of apoptosis, the promotion of differentiation, and the stimulation of tumor-directed immune responses [72][73][74]. Comparable results were obtained with various isolated bufadienolides [72][73][74]. Thus, ingredients of anuran skin secretions may also turn out useful against cancer. ...
... In ancient times, secretions from the paratoid glands of toads were used for hunting and to execute prisoners (Pinto, 1995;Dewick, 1997), but also medicinally as diuretic, cardiac stimulant, expectorant, analgesic and anti-inflammatory (Chen et al., 2018;Draasch-Fernandes et al., 2019). Additional investigations indicated that compounds present in the secretion of the paratoid gland of toads also display cytotoxic, antimalarial, antifungal, antiviral, antitrypanosomal, antileishmania and antibacterial activities (Tempone et al., 2008;Ferreira et al., 2013;Yang et al., 2015;Oliveira et al., 2019). ...
Article
- May 2022
- TOXICON
... A family of bufadienolide glycosides (bufadienolides) has been identified as the pharmacological compounds responsible for the antitumor activity of cinobufacini (Huachansu) [34,35]. Cinobufagin (CBG) and bufalin are the two major bufadienolides in cinobufacini (Huachansu) and other forms of toad extracts, and both have demonstrated potent antitumor efficacy in vitro and in vivo [27,36]; however, the underlying molecular mechanisms remain poorly understood. Several studies reported that ROS levels were markedly increased in CBG-treated cancer cell lines [37,38], but the role of ROS in the induction of cancer cell toxicity by CBG has been unclear. ...
... When harassed, viscous white fluid containing BDs is secreted from these glands ( Fig. 1a) (Barbosa et al. 2009;Hutchinson and Savitzky 2004;Lichtstein et al. 1992). BDs function by inhibiting the activity of Na + /K + ATPase, similar to other cardiotonic steroids (Yang et al. 2015). Due to this toxicity, there are few natural enemies of toads (Licht and Low 1968;Mohammadi et al. 2018;Shimada et al. 1977;Toledo and Jared 1995;). ...
... The main compounds that can be found are biogenic amines, bufadienolides, peptides and proteins and alkaloids [17,18]. These compounds are responsible for a large panel of potential therapeutic indications such as cardiotonic/anti-arrhythmic, antidiabetic, immunomodulatory, antibacterial/antifungal, antiprotozoal, antiviral, antineoplastic, sleep inducing, analgesic, contraceptive, endocrine activity, behavioral changes or wound healing [19,20]. Among antiproliferative activities, some specific ones have already been highlighted on melanoma cells [21][22][23]. ...
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