I

Asiatic Herpetological Research

February 1992

Evaluation of Snake

Vol. 4, pp. 58-61

Venoms among Agkistrodon Species in China

1Yuancong Chen Dawei Zhang 2 Kexian Jiang 2 and zho-nghui Wang2
1,2,^Shanghai Institute of Biochemistry, Shanghai, China
Shanghai Institute of Biological Products, Shanghai, China

Abstract. -Venom toxicity and enzymatic activities were examined in six species of Chinese
Agkistrodon and Deinagkistrodon acutus. The venom toxicity of A. inlermedius is the strongest, tentimes that of Deinagkistrodon acutus.
The venoms of A. blomhoffii brevicaudus, A. blomhoffii
ussuriensis, and A. shedaoensis are the next strongest. Agkistrodon saxatilis venom had a similar toxicity
as A. strauchii venom and they are more similar to Deinagkistrodon acutus in toxicity.

Key words: Reptilia, Serpentes, Viperidae, Agkistrodon, Deinagkistrodon, China, venom, toxicity.
better than that from A. b. brevicaudus.

Introduction
Agkistrodon snakes are widespread andabundant in China. According to Zhao et(1981) and Chen et al. (1984) theyshould be classified as: (1) Agkistrodonal.

blomhoffii brevicaudus Stejneger, (2) A. b.
ussuriensis Emelianov, (3) A. intermedius(Strauch), (4) A. saxatilis Emelianov, (5)A. shedaoensis Zhao, (6) A. strauchii

Bedriaga (Plate 1), (7) A. monticola
Werner, and (8) Deinagkistrodon acutus(Gloyd, 1979). The later was formerly
regarded as Agkistrodon acutus.

Later, two products, one named Qin Suan
Mei using the venom of A. b. ussuriensis,

and the other named Defibrinogenase using
the venom of D. acutus also appeared inclinical application, but their efficiency andside reactions differed from each other.

Venom Capsules, made by
Xin-LeDistrict Hospital using theShanghai
crude venom of A. b. brevicaudus fortreatment of cancers has a magicallyinhibitory effect on the growth of malignant
tumour cells. These observations aroused
787 Snake

our interest to understand the differences ofAgkistrodon venom properties.

The components and properties ofvenoms from these species are strikinglydifferent1981).

from each other (Zhao etal.,

From the standpoint of venoms thegeneral designation of Chinese Agkistrodon
as only one species, A. halys Pallas,should not be accepted. Recently in China,

and enzymatic

activities

of arginine

esterase, proteolytic and fibrinolytic
enzymes to evaluate the quality of selectedpit-viper venoms.

Methods

Agkistrodon venoms have been used tomake medicines to cure thrombotic diseaseand cancers.

In thispaper we determined the toxicity (LD50),

Snake Venoms

SVATE (Snake Venom Anti-thrombotic Enzymes) was first preparedfrom the venom of A. shedaoensis fromSnake Island in Dalian, and was effective inSince there was acuring thrombosis.
shortage of A. shedaoensis venom, thevenom of A. b. brevicaudus from Zhejiang
in eastern China was also used.

the product of A.

Howevershedaoensis seemed+This publication was previously published inChinese by Chen et al. (1990).

1992 by Asiatic Herpetological Research

Agkistrodonb.

brevicaudus werepurchased from the Shanghai Experimental
Animal Supply Station. Agkistrodon b.
ussuriensis, A. shedaoensis, and A.
strauchii were kindly provided byProfessor Ermi Zhao of the Chengduof Biology. Agkistrodon
intermedius was kindly provided by Mr.
Jinbao Yu from Xinjiang Institute ofChemistry. Deinagkistrodon acutus werepurchased from Jindezeng Snake Institute,InstituteJiangxi.

Asiatic Herpetological Research

February 1992

ml of 15%and mixed it
thoroughly, filtering after 30 min. TheWe
filtrate was measured at 280 nm.

minutes

Chemical Reagents

weaddedtrichloroacetic

BAEE (N-benzoyl arginine ethyl ester
products of th
Biochemicalof
Dongfeng Factory
Humanfibrinogen and thrombin
Reagents.
oftheare products
Shanghai Institute ofandcasein is a productProducts
Biologicalof
ChemicaltheofReagents,Factory
Shanghai. All other chemical reagents arehydrochloride),areanalytical grade.

Toxicity (LD 50)

and then diluted the venom solution to 1-5
Swiss mice of 18-20
concentrations.
ofbody weight, were divided intograms
five groups, with six individuals each.
Mice were injected intra-abdominally with
0.4 ml of venom solution. After injection
observations were made within 48 hours.

The death rate (LD50) was then calculated.

venom.
Fibrinolytic enzyme (Deogny et al.,dissolved 0.5 grams of human1975).
in
10 ml of pH 7.75 barbiturate
fibrinogenbuffer and transfered 5 ml of the solution
3.

—Weof two petri dishes (8We subsequently added

put 10 \i\ of venom solution; incubated at37°C for 18 hours. One unit of activity isequal to 1
activity ismm 2 of lysis on the plate. The
denoted by units = mm 2/mg ofResults—M

pH 8.0 trisdissolving BAEE in 0.05
HC1 buffer solution. Three ml of BAEE
solution was inserted into a cuvette and 0.1
ml of snake venom solution was added.
Spectrophotometric measurements weremade at 253 nm and 25°C. The unit ofcalculated according to theTformula, U/mg =Ai
A2/O.OO3 x
The last value is at the linear part of theactivity

isTWA.

curve, where A2 is the initial value, T is theis the weight of venom
time tested, andWin miligrams.

—Proteolytic enzyme (Rick, 1963).
One gram of casein was dissolved in 100
Tris-HCl pH 7.8 buffer in a
ml of 0.05
bath and the undissolved
waterboilingtransfered 2 ml
materials were filtered.
of filtrate to a test tube and incubated it in a2.

MWe

37° C water bath.

1venom.

Arginine esterase (United States
Pharmacopea, 1980). BAEE solutionmoles) was prepared by(0.8mmcmml ofthrombin (about 4 units) and mixed it
thoroughly. Then we immediately added 5
ml of 2% agar solution (below 60°C) andmixed it again to get a uniform plate. Wells
were punched in the plate into which wasdiameter).

Enzymatic Activities
1.

15

acid

calculated the tyrosine released from thestandard curve of tyrosine. The unit ofactivity is denoted by jig of Tyr/15 x mg of

into each

Toxicity (LD50) was assayed (Litchfieldand Wilcoxon, 1949). We dissolved 3-5
mg of snake venom in physiological saline,

Vol. 4, p. 59

Then two ml of venom

solution (2 mg / ml) was added. After 15

The venom toxicity of A. intermedins istimes that oftenacutus(Table 1). The
Deinagkistrodon
venoms of A. b. brevicaudus and A. b.
ussuriensis are the next strongest.
Neurotoxins were isolated from these
thestrongest,venoms.

A

presynaptic neurotoxin,Agkistrodotoxin, has been purified fromthe venom of A. b. brevicaudus (Chen et1981) and its amino acid sequence also
has been determined (Kondo, 1989).
al.,presynaptic neurotoxins werepurified from the venom of A. intermedius.
Their LD 50 are 38, 49 and 49 ^ig/kg ofmice respectively, higher than that ofAgkistrodotoxin which has a LD50 of 55
Hg/kg (Zhang and Hsu, 1985a). A fraction
from column chromatography of the venomof A. b. ussuriensis has been confirmed tobe neurotoxic (Zhang and Hsu, 1985b).
The venoms of A. shedaoensis and D.
acutus are non-neurotoxic. Therefore thepotency of toxicity appears to be related toThreethe neurotoxin content.

Asiatic Herpetological ResearchVol. 4, p. 60

TABLE 1
Snake venom.

February 1992

Comparison of toxicity and enzymatic activities of Agkistrodon and Deinagkistrodon venoms.

February 1992

Asiatic Herpetological ResearchVol. 4, p. 61

ZHANG, J., AND K. HSU.

835 pp.

1985b. [Presynaptic
toxins from Agkistrodon intermedius venom].

XU, X., C. WANG, J. LIU, AND Z. LU.

1981.

Purification and characterization of hemorrhagic

components from Agkistrodon aculus (hundredpace snake venom). Toxicon 19:633-644.

Acta Herpetologica Sinica 4:291-295.

(InChinese).

[A comparison

ZHAO, E., G. WU, X. WU, Y. CHEN, M. JIANG,J. ZHANG, AND K. HSU. 1981. [A comparisonof snake venom electrophoretogram ofof neurotoxin components in the venoms ofActa
Chinese Agkistrodonspecies].
Herpetologica Sinica 4:287-290. (In Chinese).

Agkistrodon species of China and their value insnake taxonomy].
Acta Zoologica Sinica
27:211-217. (In Chinese).

ZHANG, J., AND K. HSU.

1985a.