Journal of Genetic Engineering and Biotechnology (2012) 10, 213–219

Academy of Scientific Research & Technology and

National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology
www.elsevier.com/locate/jgeb

ARTICLE

Role of hyaluronidase inhibitors in the neutralization

of toxicity of Egyptian horned viper Cerastes cerastes
venom
A.F. Wahby a, El-Sayed M.E. Mahdy b, Hatem A. EL-mezayen b,
Walaa H. Salama a, Nihal M. Ebrahim a, Azza M. Abdel-Aty a,*, Afaf S. Fahmy a

a
Department of Molecular Biology, National Research Center, Dokki-Cairo, Egypt
b
Department of Chemistry, Faculty of Science, Helwan University, Egypt

Received 1 August 2012; revised 13 September 2012; accepted 3 October 2012

Available online 11 November 2012

KEYWORDS Abstract Hyaluronidase ‘‘venom spreading factor’’ is a common component of snake venoms and
Cerastes cerastes venom; indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite
Hyaluronidase activity; site/systemic collapse of the envenomated victim. The present study was performed to assess the
Rosmarinus officinalis; benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc).
Inhibition; The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluron-
Hemorrhage; idase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the
Survival time most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhib-
itory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previ-
ously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is
efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also
neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract mark-
edly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h
in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min),
respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neu-
tralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a
therapeutic value in complementary snakebite therapy.
ª 2012 Academy of Scientific Research & Technology. Production and hosting by Elsevier B.V.
All rights reserved.

* Corresponding author.
E-mail address: azzasdm@hotmail.com (A.M. Abdel-Aty). 1. Introduction
Peer review under National Research Center, Egypt.
Snake’s envenomation is a serious socio-medical problem,
especially in the places where snakes are abundant, with an
estimated 1.8–2.5 million incidences per year, a mortality level
Production and hosting by Elsevier
of 100,000–125,000 persons annually and more than 100,000
1687-157X ª 2012 Academy of Scientific Research & Technology. Production and hosting by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jgeb.2012.10.001
214
individuals suffering from severe complications, which may
end in amputation of the attacked limb [22,36]. In Egypt,
approximately 1000–10,000 envenomings per year with about
11–100 persons are estimated to die annually [22].
The Egyptian desert horned viper (genus Cerastes) is the
best known, most distinctive and most abundant venomous
snakes of the great deserts of North Africa and the Middle
East. This viper is popular among snake-keepers and thus
encountering snakebite is not rare outside the area of its ori-
ginal distribution [11,43]. Envenomings by Cerastes viper in-
flict prominent local tissue damage, which disrupts the
extracellular matrix and basement membrane surrounding
blood vessels and capillaries that are essential for rapid dis-
semination of target-specific toxins. Therefore, locally acting
factors, especially hyaluronidases, hemorrhagic metallopro-
teinases and myotoxins (enzymatic/non-enzymatic) have been
received more attention [2,16]. These spreading factors are
facilitating the diffusion of toxins into the circulation leading
to systemic effects, which include spontaneous hemorrhage–
cerebral hemorrhage being the most serious manifestation
defibrinogenation, disseminated intravascular coagulation,
and cardiovascular shock secondary to hypovolaemia, vaso-
dilation, and direct effects on the myocardium, acute renal
failure and acute respiratory distress syndrome [48]. It is
likely that inhibition of these enzymes will not only minimize
local tissue damage such as hemorrhage, edema, necrosis and
inflammation in site of the bite, but will also retard the dis-
tribution of lethal toxins; hence their importance in the man-
agement of snakebite.
Although the available antivenom neutralizes the active
venom components, stopping further damage but do not re-
verse the damage already done [26–28,52]. It is important
to search for different venom inhibitors, either synthetic or
natural, which would complement the action of antivenom,
particularly in relation to the neutralization of local tissue
damage. Hence, hyaluronidase is an important target for
minimizing envenomation-associated morbidity, so the inhibi-
tion of hyaluronidase not only prevents local tissue destruc-
tion but also retards the diffusion of toxins into the blood,
resulting in delay in time to death in severe cases of envenom-
ation [13,14,53].
Compounds such as proteins, glycosaminoglycans, polysac-
charides, flavonoids, alkaloids, antioxidants, anti-inflamma-
tory agents, and synthetic organic have been investigated as
hyaluronidase inhibitors. [12–14,23,25,29,33,35,40,44,53]. In
addition, aqueous, methanolic or ethanolic extracts prepared
to different parts of numerous plant species have been investi-
gated as hyaluronidase inhibitors [9,15,18,30,38,39,47]. In pre-
viously study, novel hyaluronidase CcHaseII (33 kDa) of the
most dangerous horned viper Cerastes cerastes was purified
and characterized in a set of biochemical assays. CcHaseII en-
hanced one hundred percent of hemorrhagic activity of the po-
tent purified hemorrhagic SVMP of corresponding venom and
the antiserum prepared against it showed highly cross-reactiv-
ity with many of the Egyptian snake venom proteins [51]. This
study aims to screen the aqueous extracts of different common
medicinal herbs for their inhibitory effect on the hyaluronidase
activity of Cc venom. Select the most potent inhibitor among
the tested extracts as a natural inhibitor and studies its role
for inhibition of hemorrhagic activity, edema inducing activity
and lethality of the Cc venom.
A.F. Wahby et al.
2. Materials and methods
2.1. Collection of medicinal plants
Plants names and their families: (Cuminum cyminum, Foenicu-
lum vulgare, Pimpinella anisum; Apiaceae), (Ceratonia siliqua,
Glycyrrhiza glabra, Tamarindus indica; Fabaceae), (Mentha
peprinta, Ocimum basilicum, Origaum majorana, Rosmarinus
officinalis, Thymus monglicus; Lamiaceae), (Hibiscus sabda-
riffa; Malvaceae; Sesamum inddicum; Pedaliaceae), (Hordeum
vulgare; Poaceae), (Nigella sativa; Ranunculaceae), and (Cur-
cuma longa, Zingiber officiale; Zingiberaceae). All plants for
the present study were obtained from Agricultural Research
Center, Dokki, Egypt.
2.2. Extraction of plants
Air-dried plant material collected, washed thoroughly with
water, shade-dried and grounded into coarse powder. Five
grams of the powdered shade dried plant material was soaked
in 50 ml distilled water (w/v) overnight at room temperature
with constant stirring. The obtained extract was filtered using
Whatman filter paper No. 1, the residue obtained was re-
suspended in freshly distilled water (1:2 ratios). The process
was repeated three times. Then, crude extracts were centri-
fuged at 3000 rpm for 10 min, and were stored in tightly stop-
per bottles at 4 C for further study.
2.3. Animals
Male Swiss albino mice weighting 20 ± 1 g were used for
lethality test. Albino and Swiss rabbits weighting (1, 1.5 kg)
were used for hemorrhagic test and antiserum preparation,
respectively. The animals were obtained from the laboratory
animal (National Research Centre). They housed in stainless
steel cages at room temperature (28–32 C) under a light peri-
od of 16–18 h daily and fed on standard commercial feed. The
experiments were carried out in the same lab in accordance
with the institutional guidelines for animal care and use.
2.4. Snake venom and chemicals
Venom of C. cerastes (Cc) was milked from adults collected
from the field. The venom was lyophilized and stored at
20 C. Samples were thawed and centrifuged before use.
CcHaseII, hyaluronidase enzyme previously purified and char-
acterized from Cc venom [51] Rooster comb hyaluronic acid
(HA) and cetyltrimethylammonium bromide (CTAB) were ob-
tained from Sigma. All other chemicals and reagents were of
analytical grade. The buffers were prepared according to
Gomorie [17] and Blanchard [6] and the final pH was checked
by pH meter (Hanna, pH 211 Microprocessor pH meter).
2.5. Immunization of rabbits and preparation of CcHaseII
antiserum
According to a low dose and low volume immunization proto-
col assessed by Chotwiwatthanakun et al. [10] with slight mod-
ification. Two male Swiss rabbits (1.5 kg) were injected
intramuscularly with dose containing 10 lg of CcHaseII dis-
solved in 0.5 ml isotonic saline. The primer dose was emulsified
Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom
with 0.5 ml of complete Freund’s adjuvant (CFA), whereas the
first and second booster doses were emulsified with 0.5 ml of
incomplete Freund’s adjuvant (IFA). Blood samples of the
individual rabbits were collected at one-week intervals, left
overnight at 4 C, centrifuged at 3000 rpm for 10 min to sepa-
rate the antisera. Sera samples before immunization were used
for control purposes.
2.6. ELISA procedure
ELISA was performed according to the method of Maria et al.
[32] Briefly, ELISA plates were incubated overnight at 4 C
with 0.1 ml of 0.5 lg Cc venom dissolved in 50 mM sodium
carbonate; pH 9.6. After blocking and washing, 100 ll/well
of each serial dilution from the non-immune and the immune
rabbit sera dissolved in washing buffer were incubated at
37 C for 1 h. The plates were washed and incubated with
100 ll/well of anti-rabbit IgG (whole molecule) peroxidase
conjugate, diluted (1:5000) for 1 h at 37 C. Then the wells
were washed and 100 ll of substrate buffer (0.33 mg OPD/ml
dissolved in citrate buffer, pH 5.2 containing 0.04% hydrogen
peroxide) were added. The reaction was stopped after 20 min
by the addition of 20 ll of a 1:20 dilution of sulfuric acid,
and the absorbance values were determined at 490 nm with
the ELISA reader photometer (Titrec Multiscan, Flow Labo-
ratories). All the values were recorded in duplicate, and the
mean results were obtained. The dilution that gives 0.5 at
490 nm (OD) was taken as the endpoint for calculation of
the ELISA titer in ELISA units (EU), calculated as reciprocal
of the dilution at the end.
2.7. In vitro inhibition of the hyaluronidase activity of Cc venom
Hyaluronidase enzyme activity was determined turbidimetri-
cally by the method of Pukrittayakamee et al. [41]. For inhibi-
tion studies, the Cc crude venom (10 lg) and purified CcHaseII
(1.75 lg) were pre-incubated separately with various watery
plant extracts for 15 min at 37 C. Then, the mixture was
added to 50 lg of hyaluronic acid in 0.2 M sodium acetate buf-
fer, pH 5.5 containing 0.15 M NaCl at 37 C for 15 min prior
addition of 1 ml of stopping reagent (2.5% CTAB in 2%
NaOH). The absorbance was monitored at 400 nm. The ve-
nom and reaction mixture without plant extract or anti-CcHa-
seII were referred as control or 100% activity.
2.8. Inhibition of Cc venom spreading property
Hemorrhagic activity was determined according to the method
of Kondo et al. [24]. The back of an albino rabbit was shaved
and after overnight incubation, samples were injected dorsally
and intradermally in 100 ll of saline. Sample 1, one unit (1U)
of hemorrhagic CcHTI (1.9 lg) previously purified and charac-
terized from Cc venom alone [50]. Sample 2, 1U of
CcHTI+1U CcHaseII, sample 3, 1U CcHTI+1U CcHaseII
individually pre-incubated for 5 min at 37 C with 50 ll of
Ro extract. Sample 4, 5 and 6, CcHaseII (1U), Ro extract
(50 ll), and saline (100 ll) alone serve as controls, respectively.
The next day, the skin was rapidly removed, fixed on a glass
plate and the diameter of each hemorrhagic spot was measured
and compared with the control. Results were expressed as
mean hemorrhagic diameters (mm) ±SEM.
215
2.9. Edema-inducing activity
The edema inducing activity assayed according to the method
of Vishwanath et al. [49] with slight modifications. Groups of 5
male Swiss mice (20 ± 1 g)were injected individually in the
right footpads with two-times the minimum edema dose
(2MED) of Cc venom (8 lg) in 100 ll saline, and the left foot-
pads received equal volume of saline as controls. Inhibition
studies were performed by preincubtation of 2MED of Cc with
Ro extract at 1:0,1:10, 1:20, 1:30, 1:40, 1:50 ratios at 37 C for
15 min before injection. The mice were anesthetized by diethyl
ether inhalation; the legs were removed at the ankle joint after
1 h increase in weight due to edema was calculated as edema
ratio, which equals the weight of edematous leg · 100/weight
of control leg. The MED defined as the amount of venom re-
quired to cause an edema ratio of 120%.
2.10. Lethal potency and neutralization of lethal toxicity of Cc
venom
LD50 was determined according to the method of Meier and
Theakston [34]. Five groups of male Swiss albino mice
(n = 5) 20 ± 1 g were injected intraperitonially (i.p.) with Cc
venom in 0.3 ml of saline with the doses ranging from 2 to
64 lg, and control group has received only saline. Survival
time of each animal in each set was recorded for 24 h. LD50
was calculated as the venom dose that killed 50% of animals.
For neutralization studies, the effects of the plant extract (Ro)
and prepared anti-CcHaseII on the lethal potency of Cc stud-
ied by six groups of mice (n = 5). Group I received 2LD50 of
venom alone. Group II and group III each divided to five sub-
groups (a–e), in which either anti-CcHaseII or Ro-extract in-
jected i.p. immediately after 2LD50 venom injection, with
ratios (w/w) of 1:20, 1:40, 1:80, 1:160 and 1:320 in a–e, respec-
tively. Groups IV, V and VI received anti-CcHaseII, Ro-ex-
tract and saline alone, respectively, as control groups. The
mice were constantly observed for symptoms/signs of toxicity,
and the survival time was recorded.
2.11. Protein determination
Protein concentrations were determined according to the
method of Bradford [8], using bovine serum albumin as a stan-
dard protein.
2.12. Statistical analysis
Each experiment was repeated thrice and the data in this study
are presented as mean ± SEM.
3. Results and discussion
Edema, hemorrhage and necrosis associated with extensive tis-
sue damage are the characteristic symptoms of Cc envenoma-
tion. In addition, the envenomation systemically affects
hemostasis resulting in bleeding disorders [43,48]. In view of
the limitations of antivenom therapy, information in ancient
books recommending the use of many plants against snakebite
is highly inspiring. Degradation of hyaluronan by venom hyal-
uronidase during natural envenomation increases the easy dif-
fusion of venom toxins, which otherwise would have diffused
216 A.F. Wahby et al.

depends on the abundance of specific antibody against the

Table 1 ELISA titers and in vitro neutralization of crude Cc
principle lethal toxin of the venom also on its binding affinity
hyaluronidase activity by anti-CcHaseII.
for the toxin [19,45].
Weeks ELISA titer % Neutralization of
hyaluronidase activity 3.2. Inhibition of Cc hyaluronidase activity by plant extracts
1 1:100 43
2 1:50 33 An important point of this work was the manner in which the
3 1:40 21 neutralizing effects of plant extracts against snake venoms
4 1:4600 44 evaluated. Neutralization studies could be performed by
5 1:1600 24 adopting two basic strategies: (1) Pre-incubation of venom
6 1:1000 20
and antivenom prior to testing and (2) Injecting venom fol-
7 1:5500 67
8 1:4500 48
lowed by anti-venom or plant extract separately to experimen-
9 1:4000 25 tal mice [15,21]. In case of screening the active plant extracts
against venom, we have used pre-incubation method. In Ta-
ble 2, the initial screening studies involving aqueous extracts
of different parts (leaf, seed and flower) of 17 commonly
slowly. Thus, the inhibition of hyaluronidase may result in a medicinal herbs belonging to different plant families against
reduction in the spread of toxins [14]. Cc hyaluronidase activity at (venom: extract) ratio of 1:10
(w/w), only the aqueous extract of R. officinalis (Ro) com-
3.1. Detection of antibodies and immunoreactivity of anti- pletely inhibited the hyaluronidase activity of Cc venom. In
CcHaseII with Cc venom addition, the aqueous extracts of Mentha peprinta < Ocimum
basilicum < Origanum majorana < R. officinalis belong to the
The anti-CcHaseII prepared in rabbit showed different levels same Lamiaceae family inhibited the hyaluronidase activity of
of immunoreactivity with whole venom in indirect ELISA at Cc venom to a varied extent. Lamiaceae family consists mainly
a week interval after injection. The ELISA titer of anti-CcHa- of herbs containing essential oil, but also shrubs, trees and lia-
seII against Cc venom increased significantly across weeks nas are represented. It made up of 236 genera and 7173 species
intervals, and showed the highest titer of 1:5500 in the 7th that are abundant in pharmacopoeias throughout the world
week (Table 1) which is less than the titer of the prepared [4,5]. The herbal remedy of Lamiaceae species most often used
anti-NNH1 antiserum against NN venom (1:8000) [13]. The against snakebite is a decoction or a poultice of primarily
pre-immune rabbit serum did not reveal any titer. In addition, leaves, but all parts are used either orally or externally [3,36].
the in vitro neutralization of hyaluronidase activity of the Cc
venom by using the anti-CcHaseII showed the maximum of 3.3. Dose dependent inhibition of CcHaseII and Cc venom
67% at the 7th week. A good correlation, r = 0.65, was found hyaluronidase activity
between the in vitro hyaluronidase neutralization values and
the ELISA titers of anti-CcHaseII against the corresponding Different concentrations of aqueous Ro-extract were pre-incu-
venom at 1st, 4th and 7th weeks, suggesting that the antiserum bated with Cc venom to inhibit its hyaluronidase activity. Inhi-
in these periods more avid because more antigenic epitopes bition of 50% of the hyaluronidase activity (IC50) and the
were involved. A set of high correlations was reported for 100% (complete inhibition) were achieved at a ratio of 1:1.2
the neutralization of specific venom activities with ELISA ti- and 1:8 (w/w) of Cc venom: Ro extract respectively, (Fig. 1).
ters [1,42]. The success of toxin neutralization by antivenom In addition, IC50 and 100% inhibition were achieved at a ratio

Table 2 Inhibition of hyaluronidase activity of Cc venom by various aqueous plants extracts at a venom: extract ratio of 1:10 in vitro.
Plant extracts Plant family Plant parts Inhibition %
None – – 0
Cuminum cyminum Apiaceae Seed 15
Foeniculum vulgare Apiaceae Seed 0
Pimpinella anisum Apiaceae Seed 32
Ceratonia siliqua Fabaceae Seed 9
Glycyrrhiza glabra Fabaceae Whole 0
Tamarindus indica Fabaceae Seed 30
Mentha peprinta Lamiaceae Leaf 69
Ocimum basilicum Lamiaceae Leaf and flower 78
Origanum majorana Lamiaceae Leaf 89
Rosmarinus officinalis Lamiaceae Leaf 100
Thymus mongolicus Lamiaceae Leaf 10
Hibiscus sabdariffa Malvaceae Flower 37
Sesamum indicum Pedaliaceae Seed 1
Hordeum vulgare Poaceae Seed 3
Nigella sativa Ranunculaceae Seed 4
Curcuma longa Zingiberaceae Rhizome 4
Zingiber officinale Zingiberaceae Seed 8
Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom 217

Cc+Ro-Extract Table 3 Effect of hyaluronidase inhibition using Ro extract

100
CcHaseII+Ro-Extract
on hemorrhagic activity of CcHT1 + CcHaseII mixture.
Hyaluronidase activity %

80 Number Samples Hemorrhagic

injected (w/w) spot (mm)
60
(IC50) a 1U of CcHT1 (1.9 lg) alone 10 ± 1
b 1U of CcHT1 + CcHaseII (1U) 22 ± 2
40
c 1U of CcHT1 + CcHaseII 0
(1U) + Ro extract (50 ll)
20
d Ro extract alone (50 ll) 0
e CcHaseII alone (1U) 0
0 f Saline alone (100 ll) 0
1:0 1:2 1:4 1:8 1:16 1:32 1:64
Venom : Extract (w/w) Values represent mean ± SEM (n = 2).

Figure 1 Inhibition of hyaluronidase activity of Cc (10 lg) and

the purified CcHaseII (1.75 lg) were preincubated separately with
various watery RO extract for 15 min at 37 C. The mixture was
added to 50 lg of hyaluronic acid in 0.2 M sodium acetate buffer, 150
pH 5.5 containing 0.15 M NaCl at 37 C ,then hyaluronidase
activity was carried out as described in Section 2. The values
120
represent mean ± SEM (n = 3).

1.1, 1:4(w/w) of the purified CcHaseII: Ro extract, respectively
(Fig. 1). In previously study, the prepared anti-CcHaseII inhib-
ited in vitro the hyaluronidase activity of purified CcHaseII
and crude Cc venom in a dose-dependent manner. The
(IC50) and 100% inhibition were achieved at a ratio 1: 4 and
1:32 (w/w) of crude Cc venom: anti-CcHaseII, respectively.
In addition, IC50 and 100% inhibition were achieved at a ratio
1: 1.2 and 1:16 (w/w) of the purified CcHaseII: anti-CcHaseII,
respectively [51]. Both anti-NNH1 and aristolochic acid inhib-
ited the hyaluronidase activity of purified hyaluronidase,
(NNH1) and of whole Naja naja venom in a dose-dependent
manner [13]. Furthermore, the methanolic extracts of Vitis
vinifera and Andrographis paniculata have shown 100% and
59% of hyaluronidase inhibition of Vipera russeli and N. naja
venoms at venom: extract ratio of 1:2 and 1:20, respectively
[18,30].
3.4. Inhibition of spreading property of CcHaseII
In rabbit co-injected with the purified hemorrhagic CcHTI and
CcHaseII, hemorrhagic activity was approximately twice that
of rabbit injected with hemorrhagic CcHTI alone 10 ± 1,
22 ± 2 mm, respectively. Rabbit co-injected with hemorrhagic
CcHTI and CcHaseII that had been pre-incubated with Ro ex-
Edema Ratio %
90
60
30
1:0 1:10 1:20 1:30 1:40 1:50 1:60
Venom: extract (w:w)
Figure 3 Inhibition of the edema inducing activity of Cc venom.
The 2 MED of crude venom (8 lg) was preincubated with different
concentrations of Ro extract in a total volume of 100 ll saline for
15 min at 37 C and injected into the right footpads of mice. The
mice were anesthetized and scarified after 1 h and the edema ratio
was calculated. The values represent mean ± SEM (n = 5).
tract completely neutralized the diameter of hemorrhagic spot
(Table 3, Fig. 2). Therefore, Ro. not only neutralized the
CcHaseII hyaluronidase activity but also neutralized the
CcHTI hemorrhagic activity. It suggests that Ro. may contain
an endogenous inhibitor of venom-induced hemorrhage might
chelate the metal ion or interact with a specific domain to
inactivate the hemorrhagic protease, and that could have led
to inhibition of the hemorrage. Similarly, the aqueous extract
of Hibiscus aethiopicus totally inhibited the hemorrhagic

Figure 2 Inhibition of spreading property of purified CcHaseII on hemorrhagic activity according to Kondo et al. (1960). The spots were
developed in the skin of rabbits with doses of (a) 1U (1.9 lg) of CcHT1, (b) 1U of CcHT1 + (1U) CcHaseII, (C) 1U of CcHT1 + (1U)
CcHaseII pre-incubated with Ro extract (50 ll) (d,e,f). CcHaseII (1U), Ro extract (50 ll), and Saline (100 ll) servers as controls.
218 A.F. Wahby et al.

edema ratio % due to 2 MED of Cc crude venom is neutralized

Table 4 Effect of anti-CcHaseII and Ro extract on the
from 150% to 110% by the prepared anti-CcHaseII at 1:100
survival time of mice injected with 2LD50 of the Cc venom.
ratio [51].
Groups Samples injected (w/w) Survival time (min)
I Cc venom (2LD50) 15 ± 5 3.6. Effect of anti-CcHaseII and Ro extract on survival time of
IIa Cc venom + anti-CcHaseII (1:20) 25 ± 5 mice injected with Cc venom
IIb Cc venom + anti-CcHaseII (1:40) 45 ± 8
IIc Cc venom + anti-CcHaseII (1:80) 55 ± 4 Herbal compounds that possess snake venom neutralization
IId Cc venom + anti-CcHaseII (1:160) 60 ± 3 properties in experimental animal usually apply the protocol
IIe Cc venom + anti-CcHaseII (1:320) 75 ± 4 of venom followed by herbal compound because this technique
IIIa Cc venom + Ro extract (1:20) 56 ± 2
is similar to clinical conditions; therefore, we have used this
IIIb Cc venom + Ro extract (1:40) 97 ± 7
IIIc Cc venom + Ro extract (1:80) 125 ± 3
protocol. In Table 4, when mice were injected (i.p.) with Cc ve-
IIId Cc venom + Ro extract (1:160) 243 ± 8 nom (2LD50) followed by anti-CcHaseII (Group II) or the
IIIe Cc venom + Ro extract (1:320) 439 ± 7 aqueous Ro extract (Group III), the survival time of each
Iv Anti-CcHaseII (0:320) All alive group was increased compared to mice injected with (2LD50)
V Ro extract (0:320) All alive of Cc venom alone (Group I). Both the anti-CcHaseII and
VI Saline All alive Ro extract showed a dose-dependent effect, and the survival
Values are mean ± SEM (n = 5). time increased from 15 ± 5 min (Group I) to a maximum of
75 ± 4 min at venom: anti-CcHaseII ratio of 1:320 (w/w), we
found a little effect on survival time during additional increas-
activity of Echis ocellatus venom [20]. In addition, the metha- ing in the anti-CcHaseII concentrations. However, the Ro ex-
nolic extract of V. vinifera effectively neutralized the hemor- tract increased the survival time from 15 ± 5 min to a
rhagic activity of Vipera russilli venom at the venom: extract maximum of 439 ± 7 min at venom: Ro extract ratio of
ratio of 1:25 [30]. However, the prepared anti-CcHaseII at 1:320 (w/w). Hence, the Ro extract retard the time death up
(50, 100 ll) decreased the diameter of hemorrhagic spots in- to 7 h. however, mice injected with anti-CcHaseII (Group
duced by mixture of 1U of hemorrhagic CcHTI + 1U of IV), Ro extract (Group V) alone, and saline were all alive in
CcHaseII from 22.2 to 10, 8 mm, respectively, [51]. No hemor- the experiment time. The internal hemorrhage induced by Cc
rhage was observed in rabbit injected with CcHaseII, Ro ex- venom was less prominent in the presence of Ro extract
tract, or saline alone (Table 3, Fig. 2). (Fig. 4). Similarly, the methanol extract of A. paniculata has
shown a significant inhibition on neurotoxic symptoms caused
3.5. Neutralization of Cc edema-inducing activity by the N. naja venom and prolonged the survival time of mice
(22 ± 2 g) to 14.44 ± 0.55 h at the venom: extract ratio of
The edema-forming activity was assessed for Cc, and Ro ex- 1:500 [18]. Similar results were observed by different plant ex-
tract was found to be effective in the neutralization of edema tracts and bioactive components [7,31,37,46].
induced by venom (Fig. 3). The edema ratio due to 2 MED
is neutralized from 150 ± 5% to 115 ± 2% by Ro-extract at 4. Conclusion
1:50 (w/w) ratio, suggesting the inhibition of inflammatory
reactions, most likely phospholipase A2 activity of the venom. Hence, inhibitors of hyaluronidase are of great interest as they
Grape seeds extract completely abolished the edema induced could stop or delay the diffusion of toxins after the natural
by the Daboia russelli venom dose-dependently and at the ve- envenomation. The natural herbal remedies are showing prom-
nom extract ratio of 1:20 the edema ratio due to 6MED of ising expectations against snakebite. The aqueous rosemary ex-
170 ± 2% neutralized to 117 ± 3.1% [30]. In addition, the tract is cheap, easily available, stable at room temperature and

Figure 4 The effect of Ro extract on internal hemorrhage induced by Cc venom. Mice injected with: Cc alone (A), Cc followed with anti-
CcHaseII (B), Cc followed with Ro extract (c) saline alone as control (D), respectively.
Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom
could strongly neutralize many local effects of Cc venom. We
have not found any studies done on its effect in inhibiting the
spreading property of the venom of either the selected Cc viper
or any snake species. Therefore, the application of the aqueous
rosemary extract might be useful as first aid treatment for vic-
tims of snakebites, complete action of antivenom. In addition,
application of such inhibitors at the site of a bite may minimize
mortality and morbidity while transporting the patient to re-
ceive anti-venom therapy.
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