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INTERNATIONAL JOURNAL OF ADVANCES IN

PHARMACY, BIOLOGY AND CHEMISTRY

Research Article

Pharmacological Research Progress and Prospects of especially on Ion Channels

Guike Li 1, Zizhong Yang 1, Chenggui Zhang 2, Chenggong Li 1, Yuanyuan Zheng 2, Xiumei Wu 1*, Zhibin Yang 2, and Yu Zhao 1,2*.

1 Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University,

Dali 671000, Yunnan Province, China.

2 National-local Joint Engineering Research Center of Entomoceutics, Dali 671000, Yunnan

Province, China.

ABSTRACT Spider toxins are huge combinatorial libraries composed of natural peptides and proteins with high medicinal values and different medicinal properties, including a variety of pharmacological effects like , anti-bacterial, insect repellent, anti-tumor, etc. Various toxins acting on different ion channels play different pharmacological efficacies. Along with the in-depth studies of spider toxins in recent years, this biotoxin family is expected to be more widely used in new drug discovery as well as in the treatment of various refractory diseases. Following worldwide research progress in this area, China has also made a unique and significant development in the past decades. This report gave a general review of the above-mentioned researches and the development outlook of this family.

Keywords: Biotoxin, cardiovascular diseases, ion channels, natural active components, new drug R&D, pharmacological activities, spider .

INTRODUCTION The spider, Araneae, is the biggest group except for in defense or hunting 2. Spider venom is composed of insects in terrestrial . The Araneae is a large diverse components, whose bioactive effects are also order of Arachnida with about 3935 genera 44906 various. The venom can be used for local ischemia, or described all over the world 1, while in China used as neuroprotective agents, pesticides, and the estimate is no less than 3,000 species. Many analgesic drugs. Traditional Chinese Medicine discharge venom from their cheliceral claws emphasizes a widely applied remedy called "fight

171 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 poison with poison". Based on this conception and composed of peptides and proteins, including historically practical experiences, the medicinal polypeptide , cytotoxins, bradykinin spiders were recorded as "to cure all furuncle analogs, antimicrobial peptides, enzymes swollen, suppurative osteomyelitis corrosive sores, (sphingomyelinase, hyaluronidase, phospholipase, polypus and sarcoma" in Chen Zangqi's "Chinese isomerase) and agglutination active peptides. It also Materia Medica" published at Tang dynasty. As a contains low molecular weight substances such as natural toxin, spider toxins hold rich prospects of polyamines, nucleotides, amino acids, monoamines, medical application 3. and inorganic salts 8. Spider venom contains so many ingredients making it possess a huge role in the TYPE AND COMPOSITION OF SPIDER pharmacological efficacies, thus providing a broad TOXINS AND THEIR PHARMACOLOGICAL prospect to take advantage of these biotoxins for the EFFECTS development of new drugs. Type and Composition of Spider Toxins Currently, nearly 500 spider toxins have been In accordance with the role and functions, the spider structurally identified, less than 1% of the speculated toxins could be divided into three categories such as all spider toxins. Scrutiny on the identified toxin nerve poisons, poison necrosis, and mixed poison, components, it could be generally divided into three while the most important of which is 4,5. categories according to molecular weight difference 9. Neurotoxins can be further differentiated into The first class indicated small molecules less than polypeptides and polyamines, and in accordance with 1000 Daltons. This category includes inorganic the difference in molecular size, the peptidic toxins ions/salts (Ca2+, Na+, K+, Mg2+, Cl-, etc.), organic could be subsequently divided into high molecular acids (e.g., citric acid, lactic acid, weight proteins and low molecular weight peptide dihydro-phenylacetic acid), glucose, amino acids, toxins. The high molecular weight toxin protein could biogenic (e.g., histamine, spermine, be represented by such as the α- (α-LTX) spermidine, putrescine, etc.), and neurotransmitters isolated from the venom of Latrodectus (glutamate, aspartic acid, epinephrine, dopamine, tredecimguttatus, α-latroerustatoxin (α-LCT) and five GABAN-methyl-3,4-dihydrocarbylphenylethylamine kinds latroinsectotoxins (LIT) (α-ε) 4,5, whose ), etc.. Vassiklevski implied that, such small molecular weight is 131 KDa, 120 KDa, 120KDa, molecules play a role not only as neurotransmitters 140 KDa, 120 KDa, 110 KDa and 110 KDa, and neuromodulators in an insect, but also as an respectively 6. Polyamines neurotoxins could be antifeedant since they cause pain. Furthermore, these represented by Americas funnel web spider small molecules in spider toxins can also increase (Agelenpsis aperta Gertsch) venom toxin, vascular permeability 10. Category 2 means protein α-agatoxins family. There typical necrosis poisonous components possessing a molecular weight of 10 toxin such as brown recluse spider especially KDa or more, mainly indicates the aforementioned Loxosceles spp. can produce severe local skin polymer toxins and proteases. The third category necrosis 7. The mixed spider toxins, could be indicated the peptidic toxins with their molecular exampled by spider toxins and Clubiona weights between 1000 Dalton and 10 KDa, these spider toxins, which exhibit neurotoxic poisons and substances is most abundant in the spider toxin necrotic features. components, and occupies an important position on According to difference of chemical composition, the spider toxins' bio-actions. Neurotoxins is an spider toxins contain a variety of constituents mainly important part of these substances. As of 2009,

172 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 statistics results shows that among all of the peptidic of the total category. toxins have been identified, 174 contains three Among various types of small molecules in the spider disulfide bonds, 128 containing four disulfide bonds, venom, the most thoroughly studied part is 53 containing five pairs of disulfide bonds, containing polyamines, especially the arylpolyamines. This 24 owns six pairs of disulfide bonds, 5 comprising article lists some typical spider polyamines for seven pairs of disulfide bonds or more. In addition, reference (Fig. 1). Table 1 lists some spider toxins only 16 peptidic toxins possess odd number of reported to possess medicinal activities 11-61. cysteine residues, which represent a small proportion

Table 1. Comparison of some important spider toxins possessing medicinal activities.

Spider species Toxin’s name Amino acid sequence Pharmaco- Molecular weight (Da) logical activity

Latrodectus α-Latrotoxin EGEDLTLEEKAEICSELELQQKYVDIASNII Ca2+ 131.5K tredecimguttatus (α-LTX) 5 GDLSSLPIVGKIAGTIAAAAMTATHVASGR LDIEQTLLGCSDLPFDQIKEVLENRFNEIDR KLDSHSAALEEITKLVEKSISVVEKTRKQM NKRFDEVMKSIQDAKVSPIISKINNFARYFD TEKERIRGLKLNDYILKLEEPNGILLHFKES RTPTDDSLQAPLFSIIEEGYAVPKSIDDELAF KVLYALLYGTQTYVSVMFFLLEQYSFLAN HYYEKG

α- EMSRADQCKLLAYTAVGYETVGNVAADIA Effective against 120K Latroinsectoto SIEGANLVAAPVAAGGHLGKGLTDAAMIA insects xin MDCSSIPFEEIKEILNKEFKEMGRKLDKNT

5 EALEHVSKLVSKTLSTVEKIRVEMREGFKL (α-LIT) VIETIENIATKEIVFDINKIVQYFNNERENIN SRQKEEFVAKLQEPAPGNFLLYLRNSRTSE SGTLYSLLFRIIDQELAIPNNAGDNNAIQAL YALFYGTETFISIMFYLVKQYSYLAEYHYQ KG

δ-Latroin-secto DEEDGEMTLEERQAQCKAIEYSNSVFGMI Effective against 110K toxin (δ-LIT) 5 ADVANDIGSIPVIGEVVGIVTAPIAIVSHITS insects AGLDIASTALDCDDIPFDEIKEILEERFNEID RKLDKNTAALEEVSKLVSKTFVTVEKTRN EMNENFKLVLETIESKEIKSIVFKINDFKKF FEKERQRIKGLPKDRYVAKLLEQKGILGSL KEVREPSGNSLSSALNELLDKNNNYAIPKV VDDNKAFQALYALFYGTQTYAAVMEFLLE QHSYLADYYYQKG

Agelenpsis aperta ω-Aga-IA 11-13 AKALPPGSVCDGNESDCKCYGKWHKCRC Acting on 7.5K PWKWHFTGEGPCTCEKGMKHTCITKLHCP voltage -gated NKAEWGLDW calcium channels (L-type in DRG)

ω-Aga-IIA 12-14 GCIEIGGDCDGYQEKSYCQCCRNNGFCS Acting on 10K voltage -gated calcium channels (N-type in chick synaptosomes)

ω-Aga-IIIA SCIDIGGDCDGEKDDCQCCRRNGYCSCYS Acting on 8.5K 11,12,14 LFGYLKSGCKCVVGTSAEFQGICRRKARQ voltage -gated CYNSDPDKCESHNKPKRR calcium channels (L-,P/Q-,R-, N-type in rat brain synaptosomes)

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ω-Aga-IVA KKKCIAKDYGRCKWGGTPCCRGRGCICSI Acting on 5220.39 12,14,15 MGTNCECKPRLIMEGLGLA voltage -gated calcium channels (P/Q-type and P-type currents in cerebellar Purkinje neurons)

ω-Aga-IVB 15 EDNCIAEDYGKCTWGGTKCCRGRPCRCS P-type VCCC 5287.13 MIGYNCECTPRLIMEGLSFA blocker

μ-Aga-I 16 ECVPENCHCRDWYDECCEGFYCSCRQPPK Induces 4264 CICRNNN-NH2 repetitive firing

16 of action μ-Aga-II ECATKNKRCADWAGPWCCDGLYCSCRSY potennials in 4137 PGCMCRPSS ventrolateral 16 muscles of μ-Aga-III ADCVGDGQRCADWAGPYCCSGYYCSCRS 4188 MPYCRCRSDS-NH2 Musca domestica

μ-Aga-IV 16 ACVGENQQCADWAGPHCCDGYYCTCRYF 4199 PKCICRNNN-NH2

μ-Aga-V 16 ACVGENKQCADWAGPHCCDGYYCTCRYF 4199 PKCICRNNN-NH2

μ-Aga-VI 16 DCVGESQQCADWAGPHCCDGYYCTCRYF 4159 PKCICVNNN

Hadronyche versuta ω-ACTX-Hv2a LLACLFGNGRCSSNRDCCELTPVCKRGSC VSCC in 4478 17-19 VSSGPGLVGGILGGIL neurons of honeybee (Insect voltage-gate calcium)

ω-ACTX-Hv1a SPTCIPSGQPCPYNENCCSQSCTFKENENG Acting on 4050 18 NTVKRCD voltage -gated calcium channels (VSCC in abdominal ganglia neurons of cockroach)

δ-Atracotoxin- CAKKRNWCGKTEDCCCPMKCVYAWYNE Blocks 4847.2 Hv1 QGSCQSTISALWKKC inactivation of (δ-ACTX-Hv1, Na+ currents in Versutoxin) 20 DRG (Rat dorsal root ganglion) (Fig. 3) Janus-faced AICTGADRPCAACCPCCPGTSCKAESNGV Mediated 3772.31 atracotoxin-Hv SYCRKDEP blockage of 1c (J-ACTX) K+/Ca2+ channels 21,22 (Inserticidal toxin)

Atrax robustus δ-Atracotoxin- CAKKRNWCGKNEDCCCPMKCIYAWYNQ Blocks 4854 Arl QGSCQTTITGLFKKC inactivation of (δ-ACTX-Arl, Na+ currents in Robustoxin) DRG 11,20

Thrixopelma pruriens Protoxin-I ECRYWLGGCSAGQTCCKHLVCSRRHGWC Nav1.2,1.5,1.7,1. 3987.55 (ProTx-I) 23 VWDGTFS 8 Protoxin-II YCQKWMWTCDSERKCCEGMVCRLWCKK Nav1.2,1.5,1.7,1. 3826.64 (ProTx-II) 23 KLW 8 Ornithoctonus HWTX-I 15,24 ACKGVFDACTPGKNECCPNRVCSDKHKW N-type calcium 3764.48 huwena CKWKL channel blocker HWTX-II 15,25 LFECSFSCEI/QEKEGDKPCKKKKCKGKWK Insecticidal toxin 8484.00 CKFNMCVKV 174 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688

Huwentoxin-I ECLEIFKACNPSNDQCCKSSKLVCSRKTRW Inhibit the 4113.83 V (HWTX-IV) CKYQI neuronal 26 tetredotoxinsensi tive (TTX-S) voltage-gated N+ channel HWTX-V 27 H2-ECRWYLGGCSQDGDCCKHLQCHSNYE Insecticidal toxin 4111.4 WCVW DGT-COOH HWTX-X 13 KCLPPGKPCYGATQKIPCCGVCSHNKCT Inhibition of rat 2937.62 dorsal root ganglion high threshold N- type calcium currents SHLP-I GCLGDKCDYNNGCCSGYVCSRTWKWCV Erythrocyte 3540.05 (SHL-I) 28,29 LAGPW HWTX-XVI 30 CIGGGVPCAGAAPACCSGLVCLLPTLHGIT N-type calcium 4437.4 TLSTTCTLL channels HWTX-XI 31 IDTCRLPSDRGRCKASFERWYFNGRTCAKF Trypsin 6166.2 IYGGCGGNGNKFPTQEACMKRCAKA inhibitor , Kv1.1 channel blockers (double-knot GDCAKEGEVCSWGKKCCDLDNFYCPMEF Capsaicin toxin, DkTx) 32 IPHCKKYKPYVPVTTNCAKEGEVCGWGS receptor (TRPV1 KCCHGLDCPLAFIPYCEKYR ion channel) Grammostola Hanatoxin 1 ECRYLFGGCKTTSDCCKHLGCKFRDKYCA Kv2.1 4114.73 spatulata (HaTx1) 33 WDFTFS Hanatoxin2 ECRYLFGGCKTTADCCKHLGCKFRDKYCA K+ channel 4102 (HaTx2) 15,34 WDFTFS Grammostola rosea Grammostola YCQKWMWTCDEERKCCEGLVCRLWCKRl Stretch-activated 3934.77 mechanotoxin2 lNM channels (MSC, (GsMTx2) 35 SAC) in Adult rat astrocytes Grammostola GCLEFWWKCNPNDDKCCRPKLKCSKLFK Stretch-activated 4095.90 mechanotoxin4 LCNFSF channels (MSC, (GsMTx4) 35 SAC) Chilobrachys JZTX-I 23,36 ACGQFWWKCGEGKPPCCANFACKIGLYLC Kv2.1,Kv4.1, 3675.4 jingzhao IWSP Kv4.2 JZTX-II 15 GCGTMWSPCSTEKPCCDNFSCQPAIKWCI E- type calcium 3726.35 WSP channel blockers JZTX-III 15,36 DGECGGFWWKCGRGKPPCCKGYACSKT Kv2.1, Nav1.5 3918.46 WGWCAVEAP JZTX-IV 37 ECTKFLGGCSEDSECCPHLGCKDVLYYCA Inhibits current 3774.88 WDGTF and slows the inactivation of JZTX-V 36 YCQKWMWTCDSKRACCEGLRCKLWCRKI Kv2.1, Kv4.1, 3605.73 I Kv4.2

38,39 + JZTX-VIII NH2-LFECSFSCDIKKNGKPCKGSGEKKCSG Block Ca 4329.37 GWRCKMNFCVKV-COOH channe1 in DRG cells JZTX-XI 40,41 ECRKMFGGCSVDSDCCAHLGCKPTLKYC Kv2.1, Kv4.1, 3726.38 AWDGTF Kv4.2 JZTX-XII 41,42 YCQKWMWTCDSERKCCEGYVCELWCKY Kv4.1 3665.4 NL

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JZTX-XIII 36,41 ECRWLFGGCEKDSDCCEHLGCRRAKPSW Kv2.1, Kv4.1, 4122.5 CGWDFTV Kv4.2

43 Acanthoscurria Gomesin ZCRRLCYKQRCVTYCRGR-NH2 Affect bacteria, 2270.4 gomesiana fungi, yeasts and eukaryotic parasites Lycosa singariensis Lycocitin 1 44 GKLQAFLAKMKEIAAQTL-NH2 Inhibit growth of 2034.20

44 Gram-positive Lycocitin 2 GRLQAFLAKMKEIAAQTL-NH2 and 2340.28 Gram-negative (bacteria and fungi at micromolar concentrations LSTX-A1 45 KECIPKHHECTSNKHGCCRGNFFKYKCQC Erythrocytes 7335.33 TTVVTQDGEQTERCFCGTPPHHKAAELVV GFGKKIFG Anti-tumor Psalmopoeus Psalmopeotoxi ACGILHDNCVYVPAQNPCCRGLQCRYGKC Erythrocytes 3615.60 cambridgei n I (PcFK1) 17 LVQV Anti malaria Psalmopeotoxi RCLPAGKTCVRGPMRVPCCGSCSQNKCT Erythrocytes 2948.30 n II (PcFK2) 17 Anti malaria Lycosa chilea Psalmotoxin1 EDCIPKWKGCVNRHGDCCEGLECWKRRR Acid-sensitive 4689.45

46 SFEVCVPKTPKT ion channel (PcTx1) (ASIC1a) (Fig. 3) Diguetia canities DTX9.2 47,48 AKDGDVEGPAGCKKYDVECDSGECCQKQ The 6371 voltage-depende YLWYKWRPLDCRCLKSGFFSSKCVCRDV nt sodium channels of insect nerve membrane. Selenocosmia Hainantoxin-I ECKGFGKSCVPGKNECCSGYACNSRDKW Kv2.1, Kv4.2 3607.22 hainana (HNTX-I) 49 CKVLL (Fig. 2) Hainantoxin-II GCKGFGDSCTPGKNECCPNYACSSKHKW Kv4.2, Kv4.3 3607 I (HNTX-III) CKVYL 23,50

Hainantoxin-I ECLGFGKGCNPSNDQCCKSSNLVCSRKHR Nav TTX-S 3987.59 V (HNTX-IV) WCKYEIX 51

Hainantoxin-V ECLGFGKGCNPSNDQCCKSANLVCSRKHR Nav TTX-S 3972.57 (HNTX-V) 51 WCKYEI Phoneutria PhTxl 15,52,53 AELTSCFPVGHECDGDASNCNCCGDDVYC Na+ channel 8600 nigriventer GCGWGRWNCKCKVADQSYAYGICKDKVN blocker C PhTx2-1 13,15,54 ATCAGQDKPCKETCDCCGERGECVCALSY Na+ channel 5838.8 EGKYRCICRQGNFLIAWHKLASCKK blocker PhTx2-5 13,54 ATCAGQDQTCKVTCDCCGERGECVCGGP Na+ blocker 5116.6 CICRQGNFLIAAYKLASCKCK PhTx2-6 13,54 ATCAGQDQPCKETCDCCGERGECVCGGP Na+ channel 5291.3 CICRQGYFWIAWYKLANCKK blocker PhTx2-9 13,54 SFCIPFKPCKSDENCCKKFKCKTTGIVKLC Na+ channel 3742.1 RW blocker PhTx3-1 55,56 AECAAVYERCGKGYKRCCEERPCKCNIV K+ channel 4582.93 MDNCTCKKFISE

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PhTx3-2 55-57 ACAGLYKKCGKGASPCCEDRPCKCDLAM Ca2+ channel 3540.84 GNCICK PhTx3-3 56 GCANAYKSCNGPHTCCWGYNGYKKACIC Ca2+ channel 6300.00 SGXNWK PhTx3-4 55,56,58 SCINVGDFCDGKKDCCQCDRDNAFCSCSV Ca2+ channel 8449.60 IFGYKTNCRCE PhTx3-5 55,56 GCIGRNESCKFDRHGCCWPWSCSCWHKE Ca2+ channel 5063.6 GQPESDVW PhTx3-6 55,56 ACIPRGEICTDDCECCGCDNQCYCPPGSSL Ca2+ channel 6044.39 GIFKCSCAHANK YFCNRKKEKCKKA PhTx4 (6-1) 59 CGDINAACKEDCDCCGYTTACDCYWSKS Blocks 5244.6 CKCREAAIVIYTAPKKKLTC inactivation of insect Na+ currents

Cupiennius salei CSTX-1 53,60 SCIPKHEECTNDKHNCCRKGLFKLKCQCS Ca2+ channels 8352.6 TFDDESGQPTERCACGRPMGHQAIETGLNI FRGLFKGKKKNKKTK Scodra griseipes SGTX1 61 TCRYLFGGCKTTADCCKHLACRSDGKYCA K+ 3776.32 WDGTF Heteropoda Heteropodatoxi DCGTIWHYCGTDQSECCEGWKCSRQLCK Kv4.2 3910.57 venatoria n1 (HpTxl) 49 YVIDW HpTx2 49 DDCGKLFSGCDTNADCCEGYVCRLWCK Kv4.2 3412.72 LCW HpTx3 49 ECGTLFSGCSTH Kv4.2 3599.38 ADCCEGFICKLWCRYERTW gigas SNX-482 61 GVDKAGCRYMFGGCSVNDDCCPRLGCHS Cav E class (act 4495.06 LFSYCAWDLTFSD on R-type and L-type Ca2+ channels)

Pharmacological Effects Analgesic Effect Many biotoxins isolated from spider venom shows in right ankle arthritis of rats. The results showed that analgesic effects. Since most of the traditionally HWTX-I can significantly improve the pain response effective analgesic drugs containing side effects with in rats with unilateral adjuvant arthritis. The authors addiction, which makes spider toxins have certain supposed that by inhibiting the expression of advantages in terms of new type of analgesic. inflammatory cytokines in the spinal cord pain in rats -I (HWTX-I), a spider peptide toxin model, by reducing its activation of primary afferent isolated from venom of Selenocosmia huwena Wang neurons, HWTX-I can improve the excitement valve, 62 63,64 (Fig. 2) allows transmission of pain to be suppressed thus reducing inflammatory pain . Chen et al. and thus exhibited analgesic effect. As a natural constructed a pain model by rapid injection of peptidic neurotoxin 24, HWTX-I (Fig. 3) exhibited as formalin into the submucosa of sigmoid colon in the N-type calcium channel blocker on presynaptic Sprague Dawley rats, and proved that after membrane, which has important implications in subarachnoid administration of HWTX-I and terms of analgesic against sports injury. Tao et al. 62 huwentoxin-IV (HWTX-IV, Fig. 3), the rats with performed solitary inflammatory pain model acute inflammatory visceral pain showed experiments by using of complete Freund's adjuvant dose-dependent inhibitory effect. HXTX-1 and

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amino acid linker

O NH2 H H H H N N N N N NH2 General aromatic H part Polyamine backbone O primary amino acid structure CONH 2 or guanidine group OH

O O O O HO HO N HO N H H

OH O H H H N N N NH2 N JSTX-1 H O HO CONH2 CONH2 OH O O H H H N N N NH2 N N N JSTX-2 H H H O O HO NH2 OH O O H H N N NH2 N N N JSTX-3 H H H O HO CONH2

OH O O NH2 H H H N N N NH2 N N JSTX-4 H H O O NH HO CONH2 H2N

OH O NH2 H H H H N N N N N NH2 NPTX-594 H O O HO CONH2 OH O NH2 H H H N N N NH2 N N N NSTX-3 H H H O O HN HO CONH2

OH O O NH H N Argiopine N N N N N NH2 H H H H H O NH2 HO CONH2

OH O O Me O NH2 H H H H N N N N NH2 Clavamine N N N N H H H H O O O NH HO CONH2 OH O O H N NH2 N N N Spidamine H H H O HO CONH2

O O H N NH2 N N N Joramine H H H O HO CONH2

Figure 1 Chemical structures of acylpolyamines from spider .

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HXTX-IV show a stronger action than morphine venom-induced inflammatory pain model, hydrochloride, and with a longer duration. Both postoperative pain model, hot plate test, thermal drift exhibit similar antinociceptive effects when experimental and scotch experiments using both comparing with ω- MVIIA (SNX-111) 63,64. epidural and intramuscular route of administration. It In addition to , Jingzhao toxins is reported that Zeneca Inc. declared a protein (JZTXs), a series of polypeptide toxins purified from obtained from Chile tarantula also exhibit effective the venom of Chilobrachys jingzhao, also exhibits analgesic efficacy that can be used to relieve severe irreplaceable analgesic activity. Zeng et al. 65 proved pain. This protein may play a similar role in pain JZTX-V possesses certain analgesic effect via series inhibition with heroin formulations, but the of experiments such as formalin pain model, bee mechanism is completely different from heroin 14.

Figure 1. Chemical structures of acylpolyamines from spider venoms. (Continued)

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Latrodectus mactans Fabricius Nephila clavata L. Koch (Black widow spider) (Bang Luo Xin Fu)

Selenocosmia huwena Wang Selenocosmia hainana Liang (Hu Wen Bu Niao Zhu) (Hai Nan Bu Niao Zhu)

Figure 2. Biological morphology of selected spiders with reported pharmacological efficacies.

67 Antibacterial Effect a synergistic antibacterial effect . Gomesin, Spider venom contains many antimicrobial peptides, obtained from the crude venom of Tarantula which are poisonous natural ingredients in the spider (Acanthoscurria gomesiana), can effectively inhibit venoms. These bioactive peptides have inhibitory both the growth of bacteria and the formation of effect against Bacillus pumilus, Bacillus subtilis and hyphae of fungus, meanwhile, this peptide can also Escherichia coli 66. From Huwena crude venom two affect the survival of Leishmania parasites 43 The kinds of peptidic toxins, HWTX-I and HWTX-II, other two antibacterial peptides, Lycocition 1, was isolated, both could inhibit the growth of lycocition 2, isolated from venom of Burrowing Gram-negative bacteria, Gram-positive bacteria and tarantula also shown to have a significant inhibitory brewer's yeast, and these two peptides toxin showed effect of E. coli 44,68.

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Toxicity A549 cells, hepatomacells BEL-7402, human Spider venom secreted by the venom gland can enter gastric cancer cell BGC-823, and Hela cells 74,78. In into the human body through the sting wound of the addition, Selenocosmia huwena spider toxin has a skin. Spider venom contains the spider toxin significant inhibitory effect on the proliferation of hemolysin, neurotoxins and tissue toxins. glioma cell line U251 72. Neurotoxins may stimulate the central nervous system, peripheral nerves, and autonomic nervous Fibrinolysis system causing clinical symptoms such as headache, The process of the decomposed liquefaction of fibrin abdominal pain, muscle pain, dizziness, salivation, formation in the blood coagulation is called weakness, convulsions, increased heart rate, coma, fibrinolysis. The researchers isolated a new etc.. Tissue toxins may cause tissue necrosis, skin polypeptide from Chilobrachys guangxiensis spider rash, induce chest tightness caused by myocardial venom which can play a pronounced inhibitory effect necrosis, palpitation, arrhythmia, etc. 69. If human on platelet formation, and have a good anti- clotting beings are bitten by the spider, it occur local skin effect, which make it a novel human platelet irritation, swelling, oozing around, getting the aggregation inhibitor 79. occurrence of necrosis, as well as the formation of ulcers. In severe cases, the bite can cause systemic Nerve-muscle junctions and Neuroprotection + + 2+ toxic reactions, vomiting, high fever, convulsions Spider venom may act on the Na , K , Ca channels, and pulmonary edema embolism 70. Furthermore, some act as activator, causing massive release of some cases will demonstrate hemolytic anemia, neurotransmitters, some play a role of blockers, disseminated intravascular coagulation, and renal blocking the ion channel currents thereby inhibiting failure 71. These toxic effects of spider toxins are neurotransmitters' delivery. Studies have shown that detrimental to human, but it showed cytotoxicity spider toxin can compete with Ach for the Ach against tumor cells, which can effectively inhibit the receptor subunits on endplate membrane, thereby proliferation of various cancer cells. For example, a block nerve impulse transmission, and subsequent cytolytic peptide LSTX-A1, newly isolated from cause the loss of muscle contractility. HWTX-I is a burrowing tarantula venom in Xinjiang province strong irreversible inhibitor to mammalian 24 exhibit good anti-tumor effect, which can inhibit the neuromuscular transmission . With their proliferation of HeLa cells 45. The spider venoms subarachnoid medication it has a neuroprotective secreted by Selenocosmia huwena (Fig. 2), effect on rat hippocampus in a global cerebral Haplopelma hainanum, Chilobrachy jingzhao, ischemia-reperfusion injury rat model. The Macrothele raveni also inhibit tumor cell activity mechanism was supposed to be that the HWTX-I 72-74. The four crude spider venoms may significantly restrain the dead signal transduction (Fas, FasL, inhibit the growth of human gastric adenocarcinoma FADD) apoptosis pathway of hippocampal neurons 80 81 cells BGC-823 and human hepatocellular carcinoma of rats . Mao et al. utilize global ischemic injury BEL-7402 cells 75,76. Feng et al. 77 found that the model of rat to observe the mophological changes by crude toxin of Macrothele raveni inhibits both the Nissel-staining of hippocampus CA1 pyramid proliferation of human esophageal cancer TE-1 cell neurons. Combined with subarachnoid catheter, they lines and the VEGF expression, while inducing measured SOD, CAT activity and the concentration apoptosis. Chilobrachys guangxiensis, the crude of MDA of the rat brain tissue after Nissl-staining the spider venom and its constituents were cytotoxic on hippocampal pyramidal cells. The results further

181 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 proved that HWTX-I displayed a significant of JZTX, and proved that JZTX has neuroprotective protective effect on rat brain cells induced by global effect in cerebral ischemia-reperfusion injury, whose cerebral ischemia-reperfusion injury rat model. Mi et mechanism might be related to the improvement of al. 82 also utilize cerebral ischemia-reperfusion injury antioxidant capacity and the down-regulation of model of mice to observe the neuroprotective effect COX-2 expressions after cerebral ischemia.

Huwentoxin-IV (4113.83 Da) Huwentoxin-XI (6166.20 Da) Minassian NA et al., J. Biol. Chem, 2013; Kuan Peng K, et al., Acta Biochim. Biophy. Sin, 288: 22707-20. 2006, 38: 457-66.

HWTX-I (3764.48 Da) HNTX-I (3607.22 Da) Qu Y et al, J. Protein Chem, 1997, 16: 565-74. Li D et al, FEBS Lett, 2003, 555: 616-2.

δ-Atracotoxin-Hv1 (4862.73 Da) PcTx1 (4689.45 Da) Fletcher JI et al, Structure, 1997, 5: 1525-35. Saez NJ et al, Mol. Pharmacol, 2011, 80: 796-808. Figure 3. Typical 3D structures of the reported spider venoms. means Beta strand; means turn; means 3/10 alpha helix.

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The Role of Sensory Organs contains a variety of peptidic toxins which has been feeling to external stimuli may not only identified to be active on insects resistance 86. All of related to stimulus intensity and manner, but also have the spider venom have toxic effects on insects, the a close relationship to some of its in vivo physiological median lethal dose LD50 value is rather low, which conditions. The body needs to maintain a stable allows them killing some forest pests and agricultural internal environment, in order to support bodily pests quickly. Compared with conventional pesticides, functions produce a corresponding sense of the the proteinic property of the spider venom allow them appropriate stimulation and make a normal reaction. A to be easily and completely denatured into the soil stable pH value is the necessary factor for body to after decomposition, thus avoid of polluting the natural produce and transport of taste, the sense of pain, and environment legacy. This leads people to realize the the perception properly. The acidity of the extracellular favorable advantage of spider toxins in agricultural fluid is not only directly related to the pain, but also utilizations, as evidenced by the Institute of Biology at fluctuate the perception and taste transduction with the the University of Queensland that spider venom pH changes in the brain. The changes of pH values in peptides forms potentially extractable pesticides 87. the brain is affected by acid-sensing ion channels Moreover, introduction of the key insecticidal peptide (ASICs), the tarantula toxin psalmotoxin, PcTx gene into crops may enhance their resistances to pests extracted from the South American tarantula and plant diseases, which has potentially important Psalmopoeus cambridgei and Lycosa chilea can block applications in the biological control of agroforestry. ASIC channels. Thus changing acidity of the The representative two polypeptidic pesticides are extracellular fluid in the brain, therefore influences the Tx4(6-1) isolated from the Brazilian spider Phoneutria generation and transmission of pain, taste and nigriventer Keys, and DTX9.2 obtained from the 83,84 perception . Diguetidae spider Diguetia canities, both can cause rapid paralysis of insects, causing hyperexcitability of The Role of Cardiovascular and Cerebrovascular flies' sensory nerves and neuromuscular, as well as Action The peptide GSMtX-4 purified from Lycosa chilea induce depolarization of the huge neuronal cell 59,88 venom can effectively prevent permeability of sodium membrane of cockroach . Furthermore, Gomesin, a ions, potassium, and calcium ion via the specific polypeptic antimicrobial toxin isolated from the channels located in the heart cells 85. These ion tarantula spider Acanthoscurria gomesiana can inhibit channels are often considered to be a special biological the in vitro growth of intraerythrocytic forms of 43,89 ontogeny source of atrial fibrillation. Atrial fibrillation Plasmodium falciparum . So did two other is a common arrhythmia disease, when the onset of the polypeptidic toxins Psalmopeotoxin I (PcFK1) and disease, the heart beats rather irregularly, which may Psalmopeotoxin II (PcFK2) exhibit, which were cause blood clots and stroke. GSMtX-4 can effectively purified from the Psalmopoeus cambridgei and suppress and shorten the time of atrial fibrillation, possesses inhibitory efficacy against Plasmodium 17 therefore reduces the risk of blood clots and prevent falciparum . Moreover, the spider toxin ω-ACTX the effects of heart disease. Another studies indicated HV2 separated from Hadronyche versuta is also active that JZTX-I owns strong cardiac pharmacological on the insects but invalid to mammalian. This effects. entomological calcium channel blocker may provide a natural resource for the development of new kinds of Insect Resistants and Antigen Activity insecticides 19. Another study found that certain spider Insects are the main prey of spiders, the spider venoms

183 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 venom also has the characteristic of antigen. Therefore neuronal sodium channel blocker, but does not affect several appropriate antitoxic serums have been the action potential of neural stem. Its target receptors prepared according to this feature, such as antitoxic may be presynaptic membrane sodium channels. serum against Loxosceles reclusa and antitoxic serum HNTX-IV (Fig. 3) is a toxic blocker on TTX- sensitive against L. reclusa. sodium channel pore sites-1 of nerve cells, while HNTX-V displayed the similar effect on sodium Other Biological Effects channels exactly as , and Selenocosmia huwena lectin-I (SHL-I), a lectin μ- 50,51. Two spider polypeptidic toxins, isolated from the crude venom of Selenocosmia Jingzhaotoxin-1 and Jingzhaotoxin-2, isolated from huwena, which is currently the peptidic lectin venom of the Chinese tarantula Chilobrachys jingzhao, 28 possessing the smallest molecular weight . Liu et al. both exhibit myocardial inhibition upon Nav1.5 identified that two peptides, m-HWTX-III and SHL-II, channel current, which can be used as optional tool which was purified from the crude venom of a reagents to distinguish Nav1.5 and Nav1.8 and Nav1.9 poisonous spider, have the same haemagglutination channels 23,91. Luo et al. 92 proposed Jingzhaotoxin-V 29 activity to each other , which can stimulate β cells can completely suppress the Tetrodotoxin-insensitive and lead to insulin release. Furthermore, α-LTX, sodium channel current expressed by the rat dorsal isolated from black widow spider venom also have this root ganglion cells, and has a good analgesic effect. 90 effect . ProTx-I and ProTx-II are isolated from the venom of pruriens, both can reversely THE PHARMACOLOGICAL ACTION inhibit the tetrodotoxin-resistent sodium channel MECHANISM OF SPIDER VENOMS 93 Nav1.8 . Derived from the venom of the Australian Ion Channels funnel web spider three toxins δ-ACTX-Arla, Sodium Channel δ-ACTX-Hvl, δ-ACTX-Arlb could delay the Sodium channels play an important role in the TTX-sensitive sodium channel inactivation 94. From generation and regulation of sense of pain, while Brazil Phoneutria nigriventer venom researchers different types of sodium channels demonstrate isolated Txl, which is a reversible toxin inhibitor of the different actions during the generation of various types mammalian recombinant Nav1.2 channel current 95, of pain. Many pharmacological analgesic owns the while Tx2-6 also exhibit efficacy upon enhancement of same mechanism of inhibiting the neuronal sodium male erectile function 96. channel, blocking the conduction pathway of excitement, thereby reducing the sense of pain. Xiao Calcium Channel Yu-cheng 50 observed the effects of venoms from Jingzhaotoxin-I (JZTX-I) is an inhibitor toxin causing 23 Selenocosmia huwena Wang, S. jainana Liang, and inactivation of calcium channel . Jingzhaotoxin-III Macrothele raveni on tetrodotoxin-sensitive (TTX-S) (JZTX-III) is a voltage-gated sodium channel inhibitor 15 voltage-gated sodium channels (VGSCs) and , while Jingzhaotoxin-VIII (JZTX-VIII) also showed delay-rectified potassium channels using to be a calcium ion channel inhibitor by patch clamp 38,39 undifferentiated NG108-15 cells. It was found that all experiment . In addition, indicated by patch clamp 97 of the crude spider venoms exhibited dose-dependent techniques, Peng et al. found Huwentoxin-I inhibitions against TTX-S sodium currents, but have (HWTX-I) can selectively inhibit the N-type calcium no significant effect on outward delay-rectified channel but showed rather weak effect on L-type potassium currents. The crude venom has the role of calcium channel in prostaglandin E1 differentiated

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NG108-15 cells. Its mechanism of action was proved current, whilst HaTx1 also has a weak inhibitory effect to be through inhibition of presynaptic upon Kv4.2 channel 33. The first purified toxin SGTX1 neurotransmitter's release and thereby blocking nerve isolated from Scodra griseipes exhibited more than conduction. The site of action was proved to be on 40% and reversible blocking the fast transient N-type calcium channels 98. ω-AgaIIIA is a peptide potassium channel and delayed rectified purified from the crude ω-Agatoxin of North American potassium channel in cerebellar granule cells 104. These funnel web spider Agelenopsis aperta. ω-AgaIIIA can toxins, together with Heteropodatoxin1-3 (HpTxl-3), not only block the L-type and N-type calcium channel, isolated from the spider venom of Heteropoda but also act on the high voltage-activated calcium venatoria are all Kv4 channel inhibitors 49. channels (P/Q-type and R-type) 99. A cluster of spider toxin ω-ACTX-Hv1, isolated from Hadronyche Regulatory Effect of Insulin Secretion versuta, is composed of six members, which can α-LTX is a proteinic neurotoxin extracted from the reversibly inhibit insect calcium channel currents, but black widow spider (Latrodectusm actans) venom exhibit no effect on calcium channels in mammals 100. which can induce exocytosis of neuroendocrine cells. The half-maximal inhibitory concentration of α-LTX stimulates β cells and causes the release of Huwentoxin-XVI (HWTX-XVI) in rat dorsal root insulin. There are two key mechanisms of α-LTX 105 ganglion neurons N-type calcium channel is 60 promoting insulin release , one is perforated effect, nmol/L, which convey HWTX-XVI a highly selective since α-LTX embedding the cell membrane and form a 2+ blocker on the N-type calcium channels with a low permeable ion channels of Ca , causing influx of 2+ toxicity and reversibility, can effectively inhibit extracellular Ca triggers exocytosis. The other mammalian N-type calcium channels 30. Brazilian mechanism is transmembrane signal transduction. "armed" spider venom toxins of Phoneutria α-LTX binding to specific receptors in the membrane nigriventer Keys contains another group of calcium surface to achieve a transmembrane signal channel blockers, three kinds of components Txl, Tx2, transduction, thus promote the secretory vesicles' 90 and Tx3 were obtained from this venom. Tx3 type aggregation, maturation, and membrane fusion . neurotoxin contains six peptidic toxins (Tx3-1~Tx3-6), Acting on Other Channels intracerebroventricular injection of Tx3-1~Tx3-6 can Zeng et al. disclosed that HNTX-XXI can continually lead to different types and degrees of paralysis, all are activate the transient receptor potential vanilloid-1 acting on calcium channels 54,56,58. From Araneae (TRPV1). Thereby the influx of extracellular Ca2+ cell spider Cupiennius salei another toxin CSTX-1 was was constantly kept in an excitement state, thus no isolated, which can block the L-type high threshold longer sensitivity to external stimuli can take place, calcium channels on glutamate synaptosomes 101. which led to analgesic anti-inflammatory effects 106. Furthermore, the double-knot toxin (DkTx) purified Potassium Channels Huwentoxin-XI (HWTX-XI, Fig 3) isolated from from the venom of tarantula Ornithoctonus huwena Ornithoctonus huwenna belongs to subfamily of also acts on TRPV1, kept in an ongoing liberalization 32 Kunitz-type toxin (KTT), which act simultaneously as state . Moreover, GsMTx2 and GsMTx4, a class of a serine protease inhibitor, as well as a potassium peptidic toxins isolated from tarantulas (Grammostola channel blocker 102,103. Hanatoxins (including HaTx1 spatulata) act on the mechanically sensitive ion 35 and HaTx2) isolated from the venom of tarantulas channels . Grammostola spatulata can block the Kv2.1 channel

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THE CLINICAL APPLICATION OF SPIDER infarct size. All these support its future medical VENOM TOXINS development. Moreover, two drugs developed by In recent years, the investigations of Chinese anti-aging research center with spider venom ion-channel-targeted becomes a new hot "Brain Pill" and "Zeng Wei Pill-I" has spot. Ion channels can direct regulate the neuronal achieved good results in the treatment of cerebral excitability, participate the release of the pain media, vascular diseases and tumors 111. In 2006, Zhang which directly affect the generation and transmission performed the investigation of preparing the crude of pain signals. Therefore, these ion channel drugs tend spider toxins of Selenocosmia huwenna and to have a potent analgesic effect, and have no tolerance Chilobrachys jingzhao to be lyophilized powder and dependence. The common utilization of the spider injections as analgesic 112, while the single toxin toxins can be divided into two categories. One is used Jingzhao toxin-V (JZTX-V) was also proved to be directly as a drug or as a precursor during molecule useful to prepare analgesic for the patients such as drug design, while another is the direct medicinal use cancer, AIDS, intraoperative and postoperative pain, as a research tool reagent upon various ion channels. rheumatism and rheumatoid arthritis, sciatic nerve pain Partial spider venom toxins can block the opening of and trigeminal neuropathic pain 113. In addition, VGSCS, stop the sodium current, thereby chock the Brazilian researchers described the spider venom toxin nervous hyperexcitability, which led to their function Tx2-6 from Phoneutria nigriventer can promote as analgesic drugs. The above-mentioned HWTX-I, relaxation and penile erection due to NO increases, the HWTX-V, HWTX-IV, Jingzhaotoxin-III (JZTX-III), main factor provoke an erection. It can be new drugs HNTX-III, and HNTX-IV all performed as this or combined with other existing drugs used for mechanism. HWTX-I, HWTX-I, in addition to its recovery of erectile dysfunction such as hypertension analgesic activity, it has no addictive side-effect, which individual's erectile function 96. Moreover, the make this drug does not need dose increase in the recombinant spider protein toxin PnTx2-6-α and post-treatment period, and its duration of analgesia PnTx2-6-β can also be used to treat male erectile was significantly longer than that of morphine. In dysfunction. In a number of hospitals in Brazil, addition to the analgesic effect, spider toxins' Argentina and Israel, doctors have conducted years of formulation can also effectively withdraw from drug clinical application, with spider toxin treatment of dependence 107. Another medicinal promising spider male ED 114. American Neurex Corporation identified venom toxin is trypsin inhibitor HWTX-XI, whose SNX-482 isolated from an African spider venom has inhibitory activity is 30 times stronger than bovine been demonstrated as an R-type calcium channel pancreatic trypsin inhibitor. The scientist has selective inhibitor which can be used in the treatment successfully expressed recombinant toxin rHWTX-XI of neurological disorders and severe anxiety disorders. in Saccharomyces cerevisiae with the same activity Modern investigation indicated that the biological with the wild toxin 108. The researcher already peptidic toxin may bind either with the receptors on developed it into a pre-clinical stage against the target cell membrane, or with specific proteins' pancreatitis 95,109. In addition, the crude toxin of domain of the ion channels, inducing activation or Chilobrachys guangxiensis also showed good modification of the receptors or ion channels, and medicinal prospects in the treatment and prevention of thereby generating symptoms such as shock, stroke and/or nerve damage of neurons 110. The numbness, pain and even death 61. Therefore, they can experimental results of the venom demonstrated serve as good tools to study membrane receptors and significant anti-coagulation, scavenging free radicals, channel structures as well as their functionalities. For alleviate the symptoms of ischemic stroke, and reduce

186 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 example, the double-knot toxin is a satisfactory tool external diseases, (5) sting by centipede, wasp, and used to investigate the conformation and 116. functionalities of TRPV1 32. As a reagent to be used in Seen in this light, although the spider has not been the ion channel tools, the toxins from the American listed as one of traditional Chinese "the five poisonous Dipluridae spider, ω-agatoxin IV A, was chosen to creatures" (the five poisonous creatures, scorpion, identify calcium channels due to its capability of viper, centipede, house , and toad), but the selectively inhibiting P/Q-type calcium channels. virulence of its pharmacological sense is significantly Jingzhaotoxin-III possesses high affinity and higher than several other poisonous animals, such as selectivity on voltage-gated cardiac sodium channel , centipedes, Wasps and so on. According to subtypes, therefore make it promising to be a powerful theory of traditional Chinese medicine, "fight fire with research tool for cardiac sodium channel 9. As a novel fire (counteract one toxin/poison with another)", peptide from spider venom which can influence spiders are indeed worth of further in-depth study. In TTX-S sodium channel, HNTX-IV also owns great the period of construction of the National-local Joint significance to rich the toxicology types of spider Engineering Research Center of Entomoceutics” at toxins. Meanwhile, HNTX-IV provides an important Dali University, this center has collected more than tool agent to elucidate the relationships concerning 36,000 head spiders from the Yunnan-Guizhou Plateau structures and functionalities of the sodium channels area, thus forming the largest Germplasm Bank spider 50. in Southwest China. From the more than 1,200 identified species of spider, more than 50 new species DISCUSSION AND OUTLOOK have been already found 117-120. There are more than Chinese traditional medicine recorded medicinal use of 200 kinds of new species by estimation whichneed spider began in 739 years AD in "Herbal Supplements through identification before publication. The (Ben Cao Shi Yi)" (Tang dynasty). In Ming Dynasty "National-local Joint Engineering Research Center of (AD 1578) the "Compendium of Materia Medica (Ben Entomoceutics" (NJERCE, Yunnan, China) continuing Cao Gang Mu)" written by Li Shi-zhen also recorded a to establish cooperation between various disciplines of number of prescription medicine of spiders. The above pharmaceutical spider R&D with domestic and foreign description of the spider species of precious books are institutions, and has developed and performed a series mainly distributed in five families with eight species, of pharmacological screenings with these spiders which are Latouchia cornuta Song et Qiu and extracts against cardiovascular and cerebrovascular Latouchia davidi Simon (Ctenizidae), Uroctea diseases, cancer, endocrine diseases, analgesic, fungal compactilis Koch and Uroctea lesserti Schenkel, infections, and viral diseases. Numerous exciting Araneus ventricosus L. Koch and Argiope bruennichii findings have been achieved from these spider extracts Scopoli (Araneidae), Agelena labyrinthica Clerck or single components, which is hopeful for the (Agelenidae), and Menemerus confusus Boes. et Str. development of new drugs and are undergoing further (Salticidae) 115. According to entry in the record of the experiments 121-123. spider in the 1977 book "Chinese Dictionary", this medicinal animal has been mainly used in the civil CONFLICTS OF INTEREST clinical at the following applications: (1) foxy hernia, The authors confirm that this article contents has no (2) stroke deviated facial paralysis, (3) chronic conflict of interest. This work was supported by infantile convulsion, children with lockjaw, infantile IRTSTYN (2010-ZY-011), the P-MOST Programme malnutrition, (4) Furuncle swollen, scrofula, skin and for Yunnan Innovative Research Team, Key Projects

187 www.ijapbc.com IJAPBC – Vol. 4(1), Jan - Mar, 2015 ISSN: 2277 - 4688 of Science and Technology Development Plan of Hebei Araneae; Hebei Science and Technology Yunnan Province 2012, 2011 Collaborative Innovation Publishing House: Wuhan, China, 2001. Center of Yunnan Province, and Special Funds for the 3. Chen ZQ, Shang ZJ. Ben Cao Shi Yi Ji Shi Development of Strategic Emerging Industries Project (Series Release of Supplement to Materia in Yunnan Province 2013. Medica); Anhui Science and Technology Publishing House: Hefei, China, 2002; pp ACKNOWLEDGEMENTS 262-263. We thank Prof. Dr. Joachim Stoeckigt and Dr. Zhang 4. Grishin E. Polypeptide neurotoxins from spider Jingxin (Sinofi Co. USA) for the helpful discussions. venoms. Eur. J. Biochem, 1999; 264(2): 276-280. ABBREVIATIONS 5. Grishin EV. Black widow spider toxins: the Ach = Acetylcholine present and the future. Toxicon, 1998; 36(11): AIDS = Acquired immunodeficiency 1693-1701. syndrome 6. Yan YZ. Purification and characterization of ASICs = Acid-sensing ion channels toxins from the eggs of tredecimguttatus. PhD CAT = Catalase Thesis, Hunan Normal University: Hunan, COX-2 = Cyclooxygenase-2 May 2012. ED = Erectile dysfunction 7. David L, Swanson MD, Richard S, Vetter MS. FADD = Fas-associated death domain, Bites of brown recluse spiders and suspected Fas-associating protein with a novel death domain necrotic arachnidism. New Eng. J. Med, 2005; Fas = Factor associated suicide, The 352(7): 700-707. apoptosis-associated factors 8. Escoubas P, Diochot S, Corzo G. Structure and FasL = Fas Ligand pharmacology of spider venom neurotoxins. GABA = gamma-aminobutyric acid Biochimie, 2000; 82(9): 893-907. KDa = Killo dalton 9. Chen JJ. Molecular diversities and functions of LD50 = Median lethal dose the venoms from the tarantula Chilobrachys MDA = Malondialdehyde jingzhao. PhD Thesis, Hunan Normal NO = nitrogen monoxide University: Hunan, November 2008. TTX-S = Tetrodotoxin sensitive 10. Vassilevski AA, Kozlov SA, Grishin EV. TRPV1 = Transient receptor potential 1 channel Molecular diversity of spider venom. VEGF = Vascular endothelial growth factor Biochemistry (Moscow), 2009; 74(13): VGSCs = Voltage-gated sodium channels 1505-1534. SOD = Superoxide dismutase 11. Brown MR, Sheumack DD, Tyler MI, Howden REFERENCES MEH. Amino acid sequence of versutoxin, a 1. Platnick NI. 2014. The world spider catalog, lethal neurotoxin from the venom of the version 15. American Museum of Natural funnel-web spider Atrax versutus. Biochem J, History, online at 1988; 250: 401-405. http://research.amnh.org/entomology/spiders/c 12. Sheumack DD, Claassens R, Whiteley NM, atalog/index.html. (Accessed on 24 June, Howden MEH. Complete amino acid sequence 2014) of a new type of lethal neurotoxin from the 2. Song DX, Zhu MS, Chen J. The Fauna of venom of the funnel-web spider Atrax robustus.

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