review article chinese medicinal leech: ethnopharmacology...

Review ArticleChinese Medicinal Leech Ethnopharmacology Phytochemistryand Pharmacological Activities

Han Dong1 Ji-Xiang Ren2 Jing-Jing Wang345 Li-Shuai Ding345 Jian-Jun Zhao2

Song-Yan Liu1 and Hui-Min Gao34

1Department of Neurology China-Japan Union Hospital Jilin University Changchun 130033 China2The Affiliated Hospital to Changchun University of Chinese Medicine Changchun 130021 China3Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing 100700 China4National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicine Beijing 100700 China5College of Pharmacy Henan University of Traditional Chinese Medicine Zhengzhou 450008 China

Correspondence should be addressed to Song-Yan Liu yan1966163com and Hui-Min Gao huimin gao126com

Received 5 January 2016 Accepted 22 March 2016

Academic Editor Hyunsu Bae

Copyright copy 2016 Han Dong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hirudo (Shuizhi in Chinese) is an important Chinese medicine which possesses many therapeutic properties for the treatment ofthe cerebral hemorrhage and other thrombosis-related diseases The phytochemical investigation gave more than 51 compoundsincluding pteridines phosphatidylcholines glycosphingolipids and sterols as well as some bioactive peptides from the Shuizhiderived from three animal species recorded in the current Chinese Pharmacopoeia The pharmacological studies on the Shuizhihave revealed various activities such as anticoagulation antithrombosis antiatherosclerosis antiplatelet aggregation antitumorand anti-inflammatory as well as hemorheology improvement and protective effects against cerebral ischemia-reperfusion injuryHowever some important issues based on the traditional uses of Shuizhi are still not clear The aim of the present review is toprovide comprehensive knowledge on the ethnopharmacology phytochemistry and pharmacological activities of Shuizhi It willprovide a potential guidance in exploring main active compounds of Shuizhi and interpreting the actionmechanism for the furtherresearch

1 Introduction

Cardiovascular diseases (CDs) are the leading cause of deathworldwide [1] They resulted in over 173 million deaths(315) in 2013 [2] The pathological thrombus formation isresponsible for most of CDs such as myocardial infarctionand strokes Compared with the synthetic drugs TraditionalChinese Medicines (TCMs) have been evidenced to beactive in the prevention and treatment of thrombosis-relateddiseases with few side effects Hirudo a representative ofanimal-sourced TCMs also known as Chinese medicinalleech (Shuizhi in Chinese) is featured on promoting bloodcirculation and removing blood stasis It was firstly recordedin the Shen-Nong-Ben-Cao-Jing and had been widely used forthe treatment of cardiovascular diseases and other chronicdiseases [3] More than 300 prescriptions containing Shuizhihave been used in the clinical practice for 2000 years [4] and

there are hundreds of manufacturers making single Shuizhior its formula preparations in China [5] Since 1963 Shuizhihas been listed in the Chinese Pharmacopoeia [6]

Hirudotherapy was once popular in Europe especiallyduring the 1825 and 1850 owing to the excluded effecton treating various human diseases [7] For several yearsthere has been renewed interest in the use of medicinalleeches especially for reducing blood coagulation relievingvenous pressure from pooling blood and treating congestivecomplications after plastic and reconstructive surgery [8 9]Widespread recognition and acceptance of leech usagewithinthe medical community came in 2004 when the US Food andDrug Administration officially approved the marketing andsale of the medicinal leech as a medical device for relievingvenous congestion [10 11] 650 species of leeches are found allover the world andHirudo medicinalis is a kind of leechmed-ically used in the western countries [9] However according

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7895935 11 pageshttpdxdoiorg10115520167895935

2 Evidence-Based Complementary and Alternative Medicine

to the description in the Chinese traditional literatures onlyseveral species with the features of small body living inwaterand blood-sucking are possible sources of Shuizhi such asHnipponiaWhitman (HN)H pulchra Song Poecilobdella nan-jingensis sp Nov P manillensis (Lesson) and P hubeiensisYang [12]Three species includingWhitmania pigraWhitman(WP) H nipponica Whitman and W acranulata Whitman(WA) are recorded in the current Chinese Pharmacopoeiaas the source of Shuizhi [13] Among them WP is the mostcommonly available from Chinese commercial leech marketBased on the potent anticoagulant effect of Shuizhi manyinvestigations on phytochemistry and biological activitieshave been reported The present review summarized theethnopharmacology phytochemistry and pharmacologicalproperties of Shuizhi concerning three legally listed Chinesespecies which can provide some evidences for the interpre-tation of its effective constituents and action mechanism

2 Ethnopharmacology

The earliest record of Shuizhi appeared in the first classicbook on Chinese Materia Medica Shen-Nong-Ben-Cao-Jingas a downgrade drug The debate on the animal origins ofShuizhi has been continued in the follow-up ChinesemedicalclassicsMing-Yi-Bie-Lu (206 BCndash8AD)Ben-Cao-Jing-Ji-Zhu(480ndash498 AD) Xin-Xiu-Ben-Cao (657ndash659 AD) Shu-Ben-Cao (935ndash960 AD) Ben-Cao-Tu-Jing (1020ndash1101 AD) Ben-Cao-Meng-Quan (1565 AD) and Ben-Cao-Gang-Mu (1590AD) As an important TCM characterized by eliminatingblood stasis and stimulating menstruation discharge theChinese Pharmacopoeia has officially accepted it since 1963for the treatment of amenorrhea induced by blood stasismasses in the abdomen apoplexy and hemiplegic paralysisas well as traumatic injuries

Shuizhi is often used in combination with other TCMingredients to treat ailments resulting from blood stasisBased on the textual literatures more than 300 prescrip-tions containing Shuizhi have been described in the clinicalpractice including the different dosage forms such as pilldecoction powder sublimed preparation and plaster Thefamous ldquoDi-Dang decoction or pillrdquo and ldquoDa-Huang-Zhe-Chongpillrdquo arewell employed bymedical sage ZhangZhong-jing living in the Eastern Han Dynasty (25ndash220 AD) In thementioned prescriptions there are up to 290 TCMs beingcompatible with Shuizhi among which the top ten drugs areTabanus (Mengchong) Persicae Semen (Taoren) Rhei Radixet Rhizoma (Dahuang) Angelicae Sinensis Radix (Danggui)Cinnamomi Cortex (Rougui) Moutan Cortex (Mudanpi)Achyranthis Bidentatae Radix (Niuxi) Ginseng Radix et Rhi-zoma (Renshen) Natrii Sulfas (Mangxiao) and ToxicodendriResina (Ganqi) (Table S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167895935) The herbalcouple of Hirudo (Shuizhi) and Tabanus (Mengchong) is themost common unit in the prescription compositions

According to recorded formula compositions and clin-ical evidences most of these prescriptions have been sub-jected to modern drug research and development As aresult to date there are hundreds of patented prepara-tions officially approved to the medical market in China

containing single Shuizhi or its compatibility form withother herbs (Table S2) [5 13 14] The representatives offrequently prescribed preparations are Nao-Xue-Kang Huo-Xue-Tong-Mai Capsule and Tong-Xin-Luo Capsule Theformer two are derived from single Shuizhi and the latter isprepared from eleven ingredients Ginseng Radix et Rhizoma(Renshen) Hirudo (Shuizhi) Scorpio (Quanxie) PaeoniaeRadix Rubra (Chishao) Cicadae Periostracum (Chantui)Eupolyphaga (Tubiechong) Scolopendra (Wugong) San-tali Albi Lignum (Tanxiang) Dalbergiae Odoriferae Lignum(Jiangxiang) Olibanum (Ruxiang processed with vinegar)Ziziphi Spinosae Semen (Suanzaoren fried) and BorneolumSyntheticum (Bingpian)

3 Chemical Constituents

The enormous information gathered from the ethnopharma-cological applications of Shuizhi and its preparations needsthe global investigation of the chemical constituents Thesestudies have led to the isolation of macromolecule substancessuch as protein and polypeptide and small molecules includ-ing pteridines phosphatidylcholines glycosphingolipids andsterols

31 Proteins and Peptides

311 Hirudin and Recombinant Products As the represen-tative of active proteins and peptides from Shuizhi hirudinhas been considered as the most potent natural inhibitorof thrombin for a long time Hirudin is an active peptideisolated from the saliva of H medicinalis by Marquanrdtof Germany in 1950 and it is composed of 65 amino acidswith themolecularweight of approximately 71 kDa includinga compact N-terminal domain containing three S-S bondsand a C-terminal domain that is disordered in uncomplexedhirudin [15] Natural hirudin presents inminuscule amountsand it is not enough to obtain hirudin as an antithromboticagent for considerable clinical use Therefore recombinanttechniques are used to produce homogeneous preparationsof hirudin In a successful case the recombinant RGD-hirudin containing a recognizable sequence of Arg-Gly-Asp(RGD) is a bifunctional molecule according to the structureof wild-type hirudin variant 2 These structural changesimprove the hydrophobicity of the protein and allow therecombinant RGD-hirudin to interact more effectively withthe fibrinogen recognition exosite of thrombin resultingin a specific activity of 12000ATUmg [16] Owing to thesignificant anticoagulant effects [17 18] recombinant hirudinhas been approved into the market for the treatment ofthrombosis-related diseases

Hirudin naturally occurs in the fresh saliva of medicinalleeches and it is almost not detected in the processed Shuizhisamples Moreover hirudin is easily degraded when orallyadministrated by the pepsin secreted from gastric chief cellsHowever according to the traditional uses Shuizhi wascommonly decocted in hot water for 1-2 h and orally adminis-trated in the clinical practice resulting in the good anticoag-ulant effect [19] The evidences from the comparative studyof different extracting methods also suggested the extracts

Evidence-Based Complementary and Alternative Medicine 3

produced by water-boiling and alcohol-precipitating showedthe strongest anticoagulant activity and good antithromboticeffect [20] These clues led to more investigations for discov-ering other potent substances from Shuizhi

312 Other Peptides Recent studies reported novel proteinsor peptides with anticoagulant activity from Shuizhi relatedanimal origin species NLP-1 (New Leech Protein-1) a low-abundant protein (Mw 13800Da) was obtained from HN onthe basis of biomimetic ligand library screening and one-steppurification method [21] Recently three peptides were alsoidentified fromdried body ofHNwith theirmolecular weightseparately 14998 15988 and 15956Da by ultra-performanceliquid chromatography coupled with electrospray ionizationquadrupole time-of-flight mass spectrometric detector [22]Using the same methods an oligopeptide with a much lowermolecular weight of 19971 Da whitide was obtained fromdried WP and it might be an oral anticoagulant for its hotand trypsin stability [23] A novel anticoagulant peptidewhitmanin was isolated and purified from the dried bodyof WP by anion-exchange chromatography on SephadexDEAE A-50 gel permeation chromatography on SephadexG-25 and Sephadex LH-20 columns and reversed phase highperformance liquid chromatography successively [24 25]

32 Small Molecule Constituents Apart from the macro-molecules small molecule constituents are considered aspotent active substances including pteridines phosphatidyl-cholines glycosphingolipids sterols and fat acids

321 Pteridines Two new heterocyclics hirudonucleodisul-fide A 1 and hirudonucleodisulfide B 2 were isolated fromthe dried material of WP displaying the moderate antianoxicactivity with EC

50values of 2701 plusmn 223 120583gmL and 1954 plusmn

153 120583gmL respectively [26] The same authorsrsquo continualinvestigation on WP reports the isolation and structuralelucidation of a new pteridinedione 3 [27] From the samespecies three new pteridine derivatives 4ndash6 and a new120572-pyrone glycoside 7 with novel structural features werepublicated recently [28 29] Interestingly the phytochemicalstudy onHN another legally listed species also afforded threenew pteridines hirudinoidines A 8 B 9 and C 10 [30] whichsuggested both species shared the same compound type Thestructures of compounds 1ndash10 are presented in Figure 1

322 Phosphatidylcholines and Glycosphingolipids Ninelysoglycerophospholipids 11ndash19 were isolated from thedried body of HN [31] Among them two (15 and 18) arelysophosphatidylcholines and the other seven are lyso-platelet-activating factors Eight new glycosphingolipids20ndash27 along with four known ones 28ndash31 were alsoobtained from this species including six zwitterionicmonogalactosylceramides carrying a choline phosphategroup 20-21 and 28ndash31 and six neutral trigalactosylceram-ides 22ndash27 [32 33] The structures of compounds 11ndash31 arepresented in Figure 2

323 Other Components Eight known compounds havebeen reported for the first time from HN collected

from Jiangsu province China including campesterol32 hexadecyl ether of glycerol 33 (2S3S4E)-45-didehydro-octadecasphingosine pentacosanoic amide 34 1-O-(120573-D-gal-actopyranosyloxy)-substituted-2-(docosenoylamino)octa-dec-4-en-3-ol 35 succinic acid 36 hypoxanthine 37propylamine 38 and L-isoleucine 39 [34] In addition tothe compounds 32 33 and 36ndash39 other 12 first-reportedconstituents are obtained from commercial samples of HNincluding cholesterol 40 nicotinic acid 41 uracil 42 uridine43 inosine 44 xanthine 45 phenylalanine 46 adenosine47 proline 48 valine 49 glycerin 50 and palmitic acid 51[35] Fatty acid methyl esters and sterols were identified byGC-MS in the anticoagulant extract of WP [36]

4 Qualitative and Quantitative Analyses

Since the protein and amino acids are the major componentsof Shuizhi the amino acids targeted analysis on Shuizhi isone of the main aspects in the quality control A reversedphase high performance liquid chromatography-evaporativelight scattering detectionmethodwas developed for the directdetermination of fourteen underivatized amino acids in thesamples ofWP including serine histidine glycine threoninearginine alanine tyrosine tryptophan methionine valinephenylalanine isoleucine leucine and lysine [37] Comparedwith direct determinationmethodwithout derivatization theindirect methods with derivatization of amino acid (pre- orpost-column derivatization) were commonly used for thetotal amino acids and free amino acids in the dried Shuizhisamples [38 39] For the small bionic zymolysis peptidesLorry method recorded in the Chinese Pharmacopoeia wasmore preferable than biuretmethod and ninhydrin colorime-try method [40 41] Dot blotting was also employed forthe determination of hirudinrsquos hydrolysates in the processedShuizhi samples with rat antibody of anti-hirudin as the firstantibody resulting in the concentration of 29651 165479558 and 29805 120583gg [42] The new anticoagulant peptidewhitmanin has the average concentration of 0074 in thedifferent batches of commercial samples fromWP [43]

Nucleosides have been proven as the bioactive com-pounds involved in the multiple biological activities suchas antiplatelet aggregation and antiarrhythmic and anti-seizure effects [44] Several quantitative studies have beenreported concerning the recently reported small moleculecompounds as marker constituents for the quality evaluationof Shuizhi samples including uracil hypoxanthine xanthineuridine hirudonucleodisulfide C hirudonucleodisulfide Aand hirudonucleodisulfide B [45ndash50] The content of uracilhypoxanthine xanthine uridine hirudonucleodisulfide Chirudonucleodisulfide A and hirudonucleodisulfide B in thedifferent batches of samples varies over the range of 0063ndash017 090ndash148 0052ndash023 0051ndash017 018ndash028015ndash029 and 012ndash021 respectively The total amountsare 212ndash241 [48] The content of hirudonucleodisulfide Cwas the highest and that of hirudonucleodisulfide B was thelowest In addition the total amounts of three compoundsdepended on the species and cultivating areas [49]

In terms of the ongoing debate on the undefined effectivesubstances of Shuizhi the quality control using the chemical


HN

NH

N

N

SO

O

O

OH

S

HN

NH

N

N

SO

O

OHS

OH

HN

NH

N

N

SO

O S O

N

N N

N

SO

O

O

HN

S

COOH

1 2 3

N

N N

N

SO

O

COOH

SO

HN

N N

N

O

O

O

OO

O

HO

O

OH

HO

HO

HO

Whitmanine B 5 6

N

N N

N

SO

O SO

67

88a9

9a123 4 4a

55a HN

N N

N

SO

O S

CH3

O

HN

NH

N

N

SO

O SO

Hirudinoidine C 10

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3CH3

CH3

CH3

CH3

CH3

H3C

H3C

H3C

H3C

H3C

H2N

(CH2)5-

Hirudinoidine B 9Hirudinoidine A 8Whitmanoside A 7

Whitmanine A 4

Figure 1 Structures of the pteridine derivatives isolated from Shuizhi

markers displayed certain limitation Therefore the bioassayhas been paidmore attention to Apart from the items includ-ing description TLC identification tests (water content totalash ash insoluble in hydrochloric acid and pH value) andethanol soluble extractive the antithrombin titrationmethodfor the quantitative assay was legally recorded in the currentquality standard of Shuizhi [13] It contains not less than160U of antithrombin per g for HN and 30U for WP andWA However this quantitative method is not so objectiveand it could lead to poor accuracy of the test results Manymethods based on the biological activity were attemptedto improve the quality control strategy of Shuizhi suchas fibrinogen-thrombin time (Fibg-TT) [51] and activatedpartial thromboplastin time (APTT) [52ndash54]

5 Pharmacological Activities

As an important TCM to control cerebral hemorrhageand other thrombosis-related diseases Shuizhi showedvarious pharmacological effects including anticoagulationantithrombosis antiatherosclerosis antiplatelet aggregation

antitumor and anti-inflammatory as well as hemorheol-ogy improvement and protective effects against cerebralischemia-reperfusion injury Of all the activities describedfor Shuizhi the anticoagulation and antithrombosis are themost widely studied subject to date In terms of traditionaluses we provide a general overview of main bioactivity ofShuizhi extract powder ormicropowderThe progress on thepharmacological activity of hirudin is beyond the scope ofthis paper

51 Effect on the Hematological System511 Anticoagulation Numerous studies have evidenced thatthe anticoagulation is the main pharmaceutical effect ofShuizhi [55] However the action intensity of anticoagulationis affected by many factors such as origin species [56ndash58]harvesting and postharvesting process [59ndash61] and prepara-tion methods of tested samples [62 63] The Shuizhi samplesfrom blood-sucking HN displayed stronger anticoagulationactivity than those from snail or clam-feeding WP andmoreover the fresh samples are more active than the dried


C HHO O

P O

R R R R R

11 12 13 14

15 16 17 18 19

O PO

O

OOH

OHHO (Ceramide)

RONH

OH

4 8 11 22RO

NH

OH

20 21

RONH

OH

4 18RO

NH

OH

4 18

28 29

RONH

OH

4 18RO

NH

OH

417

30 31

O

OH

OHHO

OO

HO

OHHO

OO

OH

OHHO O

NH

OH

22

CH2OR

CH2O CH2 CH2 N+

CH3

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

-(CH2)15CH3 -(CH2)17CH3 -(CH2)13CH3 -(CH2)8(CH2)5CH3

-CO(CH2)14CH3 -(CH2)14CH3 -(CH2)14CH(CH3)2 -CO(CH2)16CH3 -(CH2)16CH3

H3C N+ CH2

CH2

CH2

Ominus

24998400

O=C

O=C

O=C

O=C

O=C

O=C

O=C

CH2OH

CH2

CH2

17998400

16998400

18998400

24998400

Ominus

Figure 2 Continued


RONH

OH

4 18RO

NH

OH

4 19

23 24

RONH

OH

417 RO

NH

OH

4 17

25 26

O

OH

OHHO

OO

HO

OHHOO

OH

OHHO O

NH

OH

4 18O

27

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3O=C

O=C O=C

O=C

O=C

CH2OH

CH2

CH2

16998400

16998400

16998400

2499840022

998400

Figure 2 Structures of phosphatidylcholines and glycosphingolipids

ones [56] The extracts obtained by different extract solventsand methods showed various anticoagulant activities Pro-thrombin time (PT) thrombin time (TT) and APTT wereemployed as evaluation markers for the discovery of activefractions of Shuizhi and as a result ethyl acetate fractionremarkably prolonged PT TT and APTT [62] Nonheatingleech extract could prolong bleeding time (BT) and clottingtime (CT) in the model mice and PT and APTT in the modelrats and decrease the activity of coagulation factor II andplatelet aggregation in the model rats [64] Nonheating leechextract produced stronger anticoagulant effect on the micewith blood hypercoagulable state than water-decocted leechextract [65] Altogether the different species habitat harvesttime extraction methods and drying process would resultin the different quality of Shuizhi with variable anticoagulantactivity

Most of published literatures report that the anticoagu-lation mechanism of Shuizhi is related to hirudin Howeverhirudin level is very low in the processed Shuizhi sampleseven almost not detected The increasing evidence indicatesthat other small molecules may be attributed to the wholeanticoagulation effect Therefore more efforts are to be madefor understanding its action mechanism

512 Antithrombosis Effect The antithrombosis effect sharespart of the endogenous or exogenous signal pathways withanticoagulation Besides anticoagulation Shuizhi showedpotent antithrombosis effect The ethanol extract fromShuizhi significantly inhibited the thrombosis induced bycollagen-adrenaline in the mice and thrombosis in artery-vein bypass in the rats This effect may be associated withthe enhancement of erythrocyte and thrombocytemembrane

fluidity [66 67] The water-soluble extract of Shuizhi signifi-cantly activated tissue factor pathway inhibitors and inhibitedoverexpression of tissue factors in the HUVECs induced bythrombin [68]

513 Effect onHemorheology Shuizhiwater-decocted extractcan decrease blood viscosity and plasma viscosity of rats orhuman and shorten the electrophoresis time of erythrocyte[69] The active extracts of Shuizhi can significantly improvehemorheological parameters of the acute blood stasis rats[70] Shuizhi micropowder can improve blood rheology ofrats with acute blood stasis and possess obvious effect ofpromoting blood circulation and removing blood stasis Itsaction mechanism might be related to the content decreaseof plasma von Willebrand factor and plasminogen activatorinhibitor-1 [71]

514 Antiatherosclerosis Effect Atherosclerosis is a mainevent and fundament of many cardiovascular diseases andit has now been considered as a chronic inflammatorydisease Shuizhi extract from WP can obviously attenuatethe area of atherosclerosis lesion in ApoEminusminus mice in adose-dependent manner and this effect is mainly a result ofreduced invasion ofmonocyte in arterywalls by blockingNF-120581B translocation [72] Shuizhi powder may be involved in thedecrease of plasma total cholesterol level and inflammatoryfactor further inhibition on the proliferation of smoothmuscle cells in the atherosclerotic lesions in apolipoprotein Edeficient mice [73] Shuizhi extract at the dosage of 3 15 and75mgkg decreased significantly the content of plasma totalcholesterol triglyceride low density lipoprotein-cholesteroland nitric oxide and activities of serum NOS and iNOS and


increased activities of serum cNOS in hyperlipidemia rats[74] An experiment carried out in blood stasis syndromerabbits further confirmed that Shuizhi could regulate lipidmetabolism which is related to increasing LDL-R and ApoEmRNA expression [75] Liu and Sui have drawn similarconclusion that Shuizhi superfine powder played a role inantagonizing atherosclerosis by regulating level of serumlipid [76] Tongxinluo capsule a compound preparationcontaining Shuizhi as a main ingredient has similar effectsas simvastatin in lowering serum lipid levels inhibitingplaque inflammation and preventing vulnerable plaquesfrom rupture and may provide an alternative therapy foratherosclerosis [77] Dahuang Zhechong pill a famous andclassical Chinese herbal prescription containing Shuizhisignificantly inhibited the proliferation of vascular smoothmuscle cells (VSMCs) in vivo and in vitro The inhibitoryeffect is partially attributed to depressing PDGF expressionin VSMCs retarding the cell cycle and promoting apoptosisofVSMCs [78] Reversion of vascular endothelial dysfunctionis another important mechanism of antiatherosclerotic effectof Shuizhi [79]

52 Protective Effect against Cerebral Ischemia-ReperfusionInjury As an important ingredient in the clinical pre-scriptions for cerebral hemorrhage anticoagulation cannotfully explain the efficacy of Shuizhi The cerebral ischemia-reperfusion injury was characterized by the injury apoptosisand necrosis of nerve cells The Shuizhi extract obtainedby water decoction and ethanol precipitation significantlyprotected cerebral cells from apoptosis in middle cerebralartery ischemiareperfusion rats by reducing the apoptoticrate of cerebral cells [80] and regulation of Bcl-2 and Bax pro-tein [81] Shuizhi micropowder also displayed the protectiveeffect on cerebral ischemia-reperfusion injury by increasingSOD activity and decreasing MDA and NO contents inthe serum or cerebral tissues as well as reducing the pro-duction of inflammatory factors such as intercellular adhe-sion molecule-1 (ICAM-1) vascular cell adhesion molecule-1 (VCAM-1) and platelet-derived growth factor (PDGF)[82ndash84] Similar mechanism was shown that Sheng-Nao-Kang decoction (SNK) including Shuizhi as one of fifteenTCM ingredients a modified traditional Chinese medicinefor the treatment of acute and chronic cerebrovascularrelated diseases demonstrated a strong and ameliorativeeffect on cerebral IR damage in rats due to the propertiesof antiapoptosis and antioxidation as well as regulation ofiNOS and TNOS [85] In addition the effect of Shuizhi oncerebral hemorrhagemight be associatedwith the productionimprovement of capillary cell and glial cells in rats [86]Under the guidance of ldquoNao suirdquo theory proposed by RenJixue National Master of TCM Poxue Huayu and TianjingBusui decoction containing Shuizhi in combination withother seven TCMs was used for cerebral hemorrhage Itsaction mechanism is through upregulating the expression ofbrain-derived neurotrophic factor [87]

53 Antitumor Effect Shuizhi is the representative of TCMsderived from animal insect sources with the propertiesof eliminating blood stasis softening hard masses and

dissolving lumps The single Shuizhi or its formula prepa-rations in combination with other TCM ingredients werecommonly used to treat various cancers in the clinical prac-tice including esophagus cancer gastric carcinoma intestinalcancer hysterocarcinoma and breast cancer The increasingevidence indicated that Shuizhi could inhibit the proliferationof human HepG2 [88 89] and leukemic HL-60 cells [90]The action mechanism could be involved in the inducedcell differentiation and apoptosis [88 90 91] and cell cyclearrest in the G1 phase [92] as well as participation inDNA demethylation by inhibiting the expression of DNAmethyltransfer [89 93] Downregulating the expression ofMDR1 and upregulating caspase 3 are considered to be pos-sible mechanism of Shuizhi extract inducing apoptosis andenhancing the chemotherapeutic sensitivity of 5-fluorouraciland adriamycin in the Hep2 cell [94] In addition a recentinvestigation suggested that Shuizhi could inhibit tumorangiogenesis via improving tumor hypoxia microenviron-ment which was partly attributed to decreasing the expres-sion of mRNA and protein level of hypoxia inducible factor-1120572 (HIF-1120572) and degrade the mRNA expression of vascularendothelial growth factor (VEGF) downstream gene of HIF-1120572 [95 96]

54 Anti-Inflammatory Effect Shuizhi has displayed antico-agulant antithrombin and hypolipidemic effect as well asprotective effect against cerebral ischemia-reperfusion injuryThese effects could be attributed to its anti-inflammation anddetumescence Earlier reports indicated that Shuizhi extractfromWP at a dose of 2 g crude materialkg reduced abdomi-nal capillary permeability in themice and relieved croton oil-induced mouse ear edema and carrageenan-induced rat pawedema and subcutaneous embedding of filter paper inducedgranulation tissue hyperplasia in themice [97]Theprocessedproducts showed a stronger anti-inflammatory trend thanthat of crude materials [98]

6 Concluding Remarks andFuture Perspectives

As a valuable traditional medicine the research on chemi-cal compositions pharmacological actions and therapeuticproperties of Shuizhi has been ongoing in China and upto now great progress has been made however its activesubstances and action mechanisms remain unclear

61 Influence of Origin Species on the Chemical Differenceand Therapeutic Properties of Shuizhi Of all three specieslegally described in the Chinese Pharmacopoeia WP is therichest resource and it has becomemainstream in theChinesecommercial Shuizhi market However compared with blood-sucking HN WP showed weaker anticoagulation effect andmoreover it is not in accordance with the description ofmedicinal leech in the Chinese traditional textures [12]For the multiorigin TCMs each of different species dis-played various pharmacological intensity which is bound tobring to the effectiveness difference in the clinical practiceTherefore the comprehensively systematic comparison ofthe chemistry and pharmacology of three legally recorded


species is necessary for the consistency of clinical efficacy ofcommercially available Shuizhi

62 The Debate on the Active Substances Is Ongoing andMuch Effort Is to Be Made Most studies regarded hirudinand its homologue peptides as active substances responsiblefor its anticoagulation activity however in our opinionthe following points support that more components areattributed to the whole effect of Shuizhi Hirudin is presentin the fresh saliva of HN and it is easily destroyed at hightemperature or by the pepsin It is active in the directinjection form Traditionally Shuizhi was commonly used inthe processed forms (calcining in the hot talcumpowder) anddecocted in the hot water before being orally administratedTherefore based on the ancient tradition it is not enoughto take hirudin as active substances for Shuizhi samples Thestudies on the active substances of Shuizhi and their actionmechanism are to be continued employing the advancedextraction purification and analytical technologies includ-ing proteomics for macromolecules and phytochemistry formicromolecules as well as the integration of metabonomicsnetwork pharmacology and chemoinformatics

63TheArt and Science of Processing for Shuizhi in the ClinicalPractice According to traditional Chinese medicine theorypaozhi processing transforms raw drugs into ldquodecoctionpiecesrdquo thus instilling them with the desired properties fortheir medical application including improved flavor anddetoxification or alteration of their therapeutic efficacy [99]Based on the ancient tradition it is necessary for Shuizhi tobe processed before prescribing by calcining or frying withor without adjuvants In the current Chinese Pharmacopoeiathe only calcined Shuizhi in the hot talcum powder isrecorded The studies indicated that processing at the hightemperature could decrease the anticoagulation activity ofShuizhi or improve its anti-inflammatory activity [100] Howto obtain the targeted therapeutic features by processingis very important which is associated with the differentfactors such as tested samples processing parameters andbioassay markers More investigations are to be done forunderstanding the scientific importance of processing ofShuizhi for the clinical prescription and drug preparationsand further standardization of the processing techniques

Generally speaking the present review summarized thetraditional uses and phytochemistry and pharmacologicalproperties of Shuizhi Some important unanswered questionsand directions for future research are outlined which arousesus to take more efforts to assure the safety and efficacy ofShuizhi in the clinical practice

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Hui-Min Gao designed the framework of the paper HanDong Ji-XiangRen Jing-JingWang andLi-ShuaiDingwrotethe paper All authors including Jian-Jun Zhao and Song-Yan

Liu and Hui-Min Gao discussed and commented on thepaper All authors reviewed and approved the final paper

Acknowledgments

This work was supported by the Fundamental ResearchFunds for theCentral PublicWelfare Research Institutes (nosQZPT004 ZZ070820 and ZXKT15015) The authors thankProfessor Zhi-MinWang for correcting the English grammarand spelling of the paper

References

[1] S Mendis P Puska and B Norrving Global Atlas on Car-diovascular Disease Prevention and Control World HealthOrganization inCollaborationwith theWorldHeart Federationand the World Stroke Organization 1st edition 2011

[2] GBD 2013 Mortality and Causes of Death CollaboratorsldquoGlobal regional and national age-sex specific all-cause andcause-specific mortality for 240 causes of death 1990ndash2013a systematic analysis for the Global Burden of Disease Study2013rdquoThe Lancet vol 385 no 9963 pp 117ndash171 2015

[3] Y M Liu J Zhang F Shi and A Liu ldquoAdvance on thechemical constituents of hirudordquo China Journal of InformationonTraditional ChineseMedicine vol 18 no 12 pp 108ndash109 2011

[4] httpcoworkcintcmcomengineoutlinepage=1ampchannelid=37595ampsearchword=CBAEF2CEampispage=yes

[5] httpdbyaozhcomchufang[6] Chinese Pharmacopoeia CommissionChinese Pharmacopoeia

vol 1 Peoplersquos Medical Publishing House Beijing China 1963[7] L Petrauskiene O Utevska and S Utevsky ldquoCan different

species of medicinal leeches (Hirudo spp) interbreedrdquo Inver-tebrate Biology vol 128 no 4 pp 324ndash331 2009

[8] A P Singh ldquoMedicinal leech therapy (Hirudotherapy) a briefoverviewrdquoComplementaryTherapies in Clinical Practice vol 16no 4 pp 213ndash215 2010

[9] N Sobczak and M Kantyka ldquoHirudotherapy in veterinarymedicinerdquoAnnals of parasitology vol 60 no 2 pp 89ndash92 2014

[10] A J Phillips andM E Siddall ldquoPoly-paraphyly of Hirudinidaemany lineages ofmedicinal leechesrdquo BMCEvolutionary Biologyvol 9 no 40 article 246 2009

[11] C Rados Beyond Bloodletting FDA Gives Leeches a MedicalMakeover FDA Consumer Magazine 2004 httpwwwfdagov

[12] W Zhang R-X Zhang J Li F Liang and Z-Z QianldquoSpecies study on Chinese medicine leech and discussion onits resource sustainable utilizationrdquo China Journal of ChineseMateria Medica vol 38 no 6 pp 914ndash918 2013

[13] Chinese Pharmacopoeia Commission Chinese Pharmacopoeiavol 1 China Medical Science Press Beijing China 2015

[14] Drug Standards of Ministry of Health of Peoplersquos Republic ofChina China Medical Science and Technology Press BeijingChina 1997

[15] M A Corral-Rodrıguez S Macedo-Ribeiro P J BarbosaPereira and P Fuentes-Prior ldquoLeech-derived thrombininhibitors from structures to mechanisms to clinical applica-tionsrdquo Journal of Medicinal Chemistry vol 53 no 10 pp3847ndash3861 2010

[16] Y N Huang Y L Zhang B Zhao et al ldquoStructural basis ofRGD-hirudin binding to thrombin tyr3 and five C-terminal


residues are crucial for inhibiting thrombin activityrdquo BMCStructural Biology vol 14 article 62 2014

[17] G Nowak and K Schror ldquoHirudinmdashthe long and stony wayfrom an anticoagulant peptide in the saliva of medicinalleech to a recombinant drug and beyondmdasha historical piecerdquoThrombosis and Haemostasis vol 98 no 1 pp 116ndash119 2007

[18] A Greinacher and T E Warkentin ldquoThe direct thrombininhibitor hirudinrdquo Thrombosis and Haemostasis vol 99 no 5pp 819ndash829 2008

[19] X F Liu Y J Yang L J Wu and J Xu ldquoResearch progressand prospect on pharmacognosy and chemical composition ofHirudordquo Global Traditional Chinese Medicine vol 5 no 8 pp637ndash640 2012

[20] J X Ding X C Ou Q H Zhang L Zhang and Z L LiuldquoComparison among 5 different extractingmethods ofWhitma-nia pigrawhitmanrdquo Chinese Journal of Integrative Medicine vol14 no 3 pp 165ndash134 1994

[21] Y L Gui D X Dong and R X Li ldquoBiomimetic affinitypurification and identification of a new anticoagulant proteinfromHirudo nipponiardquoChinese Journal of Biochemical Pharma-ceutics vol 29 no 3 pp 145ndash148 2008

[22] L Xiao J Nie D P Li and K L Chen ldquoPeptides fromtwo sanguinovorous leeches analyzed by ultra-performanceliquid chromatography coupled with electrospray ionizationquadrupole time-of-flight mass spectrometric detectorrdquo Phar-macognosy Magazine vol 11 no 41 pp 32ndash37 2015

[23] X B Zheng J Li ZW Chen YM Liu and K L Chen ldquoPurifi-cation and characterization of an anticoagulant oligopeptidefrom Whitmania pigra Whitmanrdquo Pharmacognosy Magazinevol 11 no 43 pp 444ndash448 2015

[24] S Zhong Z Cui N Sakura D Wang J L Li and Y Zhai ldquoArapid method for isolation and purification of an anticoagulantfromWhitmania pigrardquoBiomedical Chromatography vol 21 no5 pp 439ndash445 2007

[25] S Zhong D P Yang and Z Cui ldquoStudies on anticoagulantconstituents in the dried Whitmania pigrardquo China Journal ofChinese Materia Medica vol 33 no 23 pp 2781ndash2784 2008

[26] Y-F Zheng X-F Huang and G-P Peng ldquoStructures of twonovel heterocyclics fromWhitmania pigrardquo Planta Medica vol74 no 5 pp 562ndash564 2008

[27] Y F Zheng C Y Li G P Peng and H Y Li ldquoA new pteridine-dione fromWhitmania pigrardquo Chinese Chemical Letters vol 21no 5 pp 603ndash605 2010

[28] T Li G-C Wang C-H Wang and W-C Ye ldquoThree newpteridines from the LeechWhitmania pigrardquo Chemistry Lettersvol 42 no 9 pp 983ndash985 2013

[29] T Li G-C Wang X-J Huang and W-C Ye ldquoWhitmanosideA A new 120572-pyrone glycoside from the leechWhitmania pigrardquoHeterocycles vol 87 no 7 pp 1537ndash1543 2013

[30] Y-B Li W-H Huang and Y Xiang ldquoThree new pteridineshirudinoidines A-C from Hirudo nipponica Whitmanrdquo Hel-vetica Chimica Acta vol 91 no 2 pp 303ndash307 2008

[31] N Noda R Tanaka M Nishi S Inoue and K MiyaharaldquoIsolation and characterization of seven lyso platelet-activatingfactors and two lyso phosphatidylcholines from the crudedrug lsquoSuitetsursquo (the Leech Hirudo nipponica)rdquo Chemical andPharmaceutical Bulletin vol 41 no 8 pp 1366ndash1368 1993

[32] N Noda R Tanaka K Tsujino M Miura K Miyahara andJ Hayakawa ldquoTwo amphoteric galactocerebrosides possessinga tri-unsaturated long-chain base from the leech (Hirudonipponica)rdquo Chemical and Pharmaceutical Bulletin vol 43 no4 pp 567ndash570 1995

[33] N Noda R Tanaka K Miyahara and T Sukamoto ldquoSixtrigalactosylceramides from the leech (Hirudo nipponica)rdquoChemical and Pharmaceutical Bulletin vol 44 no 5 pp 895ndash899 1996

[34] Y B Li Y Xiang W H Huang and N Yao ldquoChemicalconstituents from Hirudo nipponicaWhitmanrdquo Strait Pharma-ceutical Journal vol 21 no 5 pp 75ndash77 2009

[35] W G Jing J Fu Y M Liu and A Liu ldquoChemical constituentsof HirudordquoChinese Journal of Experimental Traditional MedicalFormulae vol 20 no 19 pp 120ndash123 2014

[36] R Q Huang C H Luo J N Peng et al ldquoThe active consituentsof small molecules in W pigra by GC-MS analysisrdquo ChineseTraditional Herbal Drugs vol 34 no 9 pp 789ndash790 2003

[37] D Yan G Li X-H Xiao X-P Dong and Z-L Li ldquoDirectdetermination of fourteen underivatized amino acids fromWhitmania pigra by using liquid chromatography-evaporativelight scattering detectionrdquo Journal of Chromatography A vol1138 no 1-2 pp 301ndash304 2007

[38] XHYuan XD Yang C TWang and S LWang ldquoDetermina-tion of animo acids in the extract of Hirudo by HPLCrdquo ChineseJournal of Pharmaceuticals vol 38 no 8 pp 590ndash592 2007

[39] Y Liu Y Xie Y Yi et al ldquoDetermination of animo acids inShuizhi and Dilong samples by the pre-column derivatizationandHPLCrdquoHunan Journal of Traditional ChineseMedicine vol28 no 3 pp 138ndash139 2012

[40] X W Qiu P M Yang L Dai and G F Liu ldquoMethod fordetermination of small bionic zymolysis peptides of LeechesrdquoChinese Journal of Experimental Traditional Medical Formulaevol 17 no 8 pp 60ndash62 2011

[41] X W Qiu P M Yang L Dai and G F Liu ldquoDeterminationof small bionic zymolysis peptides of leeches by Folin-phendmethodrdquo Medical Information vol 24 no 5 pp 2557ndash25582011

[42] H-WWang and J-Z Tian ldquoRapid determination of contents ofhirudinrsquos hydrolysates in processed leech by dot blottingrdquoChinaJournal of ChineseMateriaMedica vol 33 no 19 pp 2193ndash21952008

[43] S Zhong D P Yang and Z Cui ldquoDetermination of whitmaninin the samples ofWhitmania pigra by RP-HPLCrdquo in Proceedingsof the 9th Conference on the Latest Advance Carried Out byYoung Pharmaceutical Researchers pp 414ndash416 Beijing China2008

[44] J Wang Z-G Huang H Cao et al ldquoScreening of anti-plateletaggregation agents from Panax notoginseng using humanplatelet extraction and HPLCndashDADndashESI-MSMSrdquo Journal ofSeparation Science vol 31 no 6-7 pp 1173ndash1180 2008

[45] X H Yuan L H Tong and H T Zhao ldquoDetermination ofhypoxanthine in the leech by HPLCrdquo Journal of MudanjiangMedical University vol 27 no 1 p 68 2006

[46] L F Liu R L Jin and G J Xu ldquoThe comparison of contentsof uracil xanthine and hypoxanthine in five species of leechrdquoJournal of Chinese Medicinal Materials vol 22 no 1 pp 8ndash101999

[47] Y Wan P M Yang L Dai and G F Liu ldquoDetermination ofhypoxanthine and uridine in leech by HPLCrdquo Chinese Journalof Experimental Traditional Medical Formulae vol 17 no 13 pp74ndash76 2011

[48] W Z Liu J W Fan Y F Li et al ldquoSimultaneous determinationof seven components in the dried Whitmania pigra by HPLCrdquoChinese Journal of Pharmaceutical Analysis vol 34 no 8 pp1417ndash1421 2014


[49] Y F Zheng J M Chen and G P Peng ldquoContent determinationof three pteridine active ingredients in leech by RP-HPLCrdquoJournal of Nanjing TCM University vol 27 no 6 pp 573ndash5742011

[50] P Chen W Li Q Li et al ldquoIdentification and quantificationof nucleosides and nucleobases in Geosaurus and Leech byhydrophilic-interaction chromatographyrdquo Talanta vol 85 no3 pp 1634ndash1641 2011

[51] B Su Z B Wang Y D Guo et al ldquoResearch on the method offibrinogen-thrombin time by coagulometer for quality controlof Hirudordquo Chinese Journal of Pharmaceutical Analysis vol 34no 10 pp 1802ndash1806 2014

[52] C C Song Study on the quality control methods of Hirudo basedon the bioassaymdashAPTTmethod [MS thesis] Beijing Universityof Traditional Chinese Medicine Beijing China 2014

[53] J J Jin Z B Wang B Su C C Song Y D Guo and Y CHu ldquoResearch of anticoagulation effect and establishement ofreaction system of bioactivity assay of Hirudo in vitrordquo ChinesePharmaceutical Journal vol 49 no 22 pp 1997ndash2001 2014

[54] L Xiao X-L Xu K-Y He and K-L Chen ldquoAssay of antico-agulation activity of leeches by using biological standardizationmethodrdquoChinese Pharmaceutical Journal vol 50 no 3 pp 258ndash262 2015

[55] J Q Liang N S Wang and S Q Mi ldquoThe effects of antico-agulation and anti-thrombosis of hirudo and the relationshipbetween the activity and origin species as well as extractmethodrdquo Chinese Journal of Integrative Medical Cardio-CerebroVascular Disease vol 7 no 9 pp 1096ndash1098 2009

[56] H Z Zhang S M Nie L P Zhang Y Zou and Y L GuildquoComparison of anticoagulation of hirudo samples collected theleech species with different eating habitsrdquo Journal of ChineseMedicinal Materials vol 21 no 12 pp 599ndash600 1998

[57] W Li F L Liao X J Yin and J Peng ldquoExperimental study onanti-platelet aggregation and anti-coagulation of seven speciesof leecchesrdquo Pharmacology and Clinics of Chinese MateriaMedica vol 13 no 5 pp 32ndash34 1997

[58] X C Ou Q H Zhang J X Ding Z Liu L Zhang and T YangldquoStudies of the anticoagulant activity of 4 kinds of leechesrdquoNatural Product Research andDevelopment vol 8 no 2 pp 54ndash56 1996

[59] H Z Shi Q S Guo S S Lu andH Li ldquoQualities ofWhitmaniapigra in different-month-age and best harvest timerdquo ChinaJournal of Chinese Materia Medica vol 34 no 23 pp 3060ndash3063 2009

[60] F Liu Q S Guo H Z Shi TWang and S S Lu ldquoComparationstudy on quality of different population of wild and breedingWhitmania pigrardquoChina Journal of ChineseMateriaMedica vol35 pp 1280ndash1283 2010

[61] W H Lv F J Qiu and Z M Wang ldquoPreliminary studies onprocess and ultramicro-pulverization on pharmacodynamics ofleechrdquo China Journal of Chinese Materia Medica vol 26 no 4pp 241ndash244 2001

[62] Y L Li R Q Huang and X D Sun ldquoExperimental studieson anticoagulant activity of different extract of leech in vitrordquoJournal of Chinese Medicinal Materials vol 27 no 2 pp 123ndash125 2004

[63] Z B Li S H Zhao Y R Wang Z Y Wang and Y J ZhangldquoComparison of leech extracts in anticoagulant and fibrinolyticactivities and the composition of its enzymolysis productsrdquoChinese Traditional Patent Medicine vol 33 no 1 pp 42ndash452011

[64] Q Z Zhang J T Liu X L Shi and J L Li ldquoEffect ofnon-heating leech extract on coagulation system and plateletaggregation in the animal model with blood hypercoagulablestaterdquo Chinese Journal of Experimental Traditional MedicalFormulae vol 12 no 5 pp 47ndash49 2006

[65] X L Shi J T Liu X K Li W Gou C H Liu and C HDeng ldquoEffects of anticoagulation and antithrombosis of non-heating leech extractrdquo Traditional Chinese Drug Research andPharmacology vol 15 no 2 pp 95ndash97 2004

[66] Y Z Tan P Xu X Y Zhang and S Y Zhao ldquoResearch onanti-thrombosis effect of ethanol extract from beheaded leech(EEBL)rdquo China Journal of Chinese Materia Medica vol 24 no10 pp 622ndash640 1999

[67] X G Wu G Y Xiao and Z Han ldquoResearch on the anti-thrombus function and prospects of alcohol refined materialfromHirudordquo Journal ofMudanjiangMedicalUniversity vol 30no 2 pp 35ndash37 2009

[68] L H Liu Q TanM F Lu X Q Zhang Z D Lei and Y HuangldquoEffects of leech extract on the release of TFPI and expressionof TF in the HUVECs induced by thrombinrdquo Chinese Journalof Integrative Medical Cardio-Cerebro Vascular Disease vol 12no 5 pp 594ndash595 2014

[69] J J Xu J M Huang and X H Chen ldquoEffects of leech water-decocted extract on platelet aggregation and hemorheologyin rats and human volunteersrdquo Pharmacology and Clinics ofChinese Materia Medica vol 4 no 4 pp 36ndash39 1988

[70] H Zheng S X Fang X M Chen and R-B Wei ldquoEffect of theactive fraction of the leech on hemorheology of the acute bloodstasis model ratsrdquo China Journal of Hospital Pharmacy vol 24no 10 pp 587ndash588 2004

[71] Z Y Sui J Liu and X J Li ldquoEffect of hirudo micropowderon hemorheology in rats with acute blood stasisrdquo HenanTraditional Chinese Medicine vol 33 no 3 pp 353ndash355 2013

[72] Y Wang X Zhao Y-S Wang S-L Song H Liang and A-G Ji ldquoAn extract from medical leech improve the function ofendothelial cells in vitro and attenuates atherosclerosis in ApoEnull mice by reducing macrophages in the lesionsrdquo Biochemicaland Biophysical Research Communications vol 455 no 1-2 pp119ndash125 2014

[73] G W Chen Y Pan L Shang and H Yu ldquoEffects of leechon atherosclerotic lesion in apolipoprotein E deficient MicerdquoMedical Journal ofWuhanUniversity vol 34 no 3 pp 344ndash3472013

[74] H T Wang C Z Li S L Xiao and C Z Hong ldquoEffectsof extract of hirudo on blood lipid metabolism nitric oxideand nitric-oxide synthase in experimental hyperlipidemia ratsrdquoModern Medicine Journal of China vol 10 no 5 pp 25ndash262008

[75] H Y Yang X D Zhang K Y Liu Z H Du and X J Bai ldquoEffectof hirudo on blood metabolism and its related gene expressionin blood stasis syndrome rabbitsrdquo Chinese Journal of ModernApplied Pharmacy vol 30 no 9 pp 959ndash963 2013

[76] J Liu and Z Y Sui ldquoEffects of leech superfine powder on thelevel of serum lipid in the rabbit of atherosclerosis modelrdquoJournal of Liaoning University of Traditional Chinese Medicinevol 17 no 6 pp 26ndash28 2015

[77] W Q Chen L Zhong L Zhang et al ldquoChinese medicinetongxinluo significantly lowers serum lipid levels and stabilizesvulnerable plaques in a rabbit modelrdquo Journal of Ethnopharma-cology vol 124 no 1 pp 103ndash110 2009

[78] Y-H Zhang J-T Liu B-Y Wen and N Liu ldquoMechanismsof inhibiting proliferation of vascular smooth muscle cells by


serum of rats treated with Dahuang Zhechong pillrdquo Journal ofEthnopharmacology vol 124 no 1 pp 125ndash129 2009

[79] X Y Liu F W Li L S Zhang H Liu and J H Pan ldquoEffectsof hirudo on vascular endothelial dysfunction of experimentalatherosclerosis in rabbitsrdquo Chinese Journal of Basic Medicine inTraditional Chinese Medicine vol 4 no 3 pp 15ndash17 1998

[80] C L Cao C S Ma W L Ma and T Z Yang ldquoAnti-apoptoticeffect of traditional Chinese medicine hirudo on cerebral cellsafter occlusionreperfusionrdquo Journal of Hebei Medical Univer-sity vol 21 no 4 pp 193ndash195 2000

[81] M B Lin X Huang and J Zhang ldquoEffects of the antagonismof leech extract on the apoptosis of primary cultured neonaterat cerebral cortical neurons caused by hypoxiardquo West ChinaJournal of Pharmaceutical Science vol 23 no 5 pp 543ndash5452008

[82] K M Li J B Wu Z Y Sui and A J Wang ldquoEffects ofhirudo micropowder on ICAM VCAM and PDGF in rats withcerebral ischemia-reperfusion injuryrdquo Traditional Chinese DrugResearch and Clinical Pharmacology vol 20 no 2 pp 136ndash1372009

[83] J B Wu K M Li Z Y Sui and A J Wang ldquoThe effect ofhirudo micro-powder on the levels of SOD MDA and NO inthe blood serum and brain homogenate of the rats with cerebralischemia-reperfusion injuryrdquo Shandong Journal of TraditionalChinese Medicine vol 27 no 4 pp 265ndash267 2008

[84] K M Li J B Wu Z Y Sui and A J Wang ldquoEffects of Hirudomicropowder on the rats with cerebral ischemia-reperfusioninjuryrdquo Pharmacology and Clinics of Chinese Materia Medicavol 27 no 4 pp 56ndash58 2011

[85] L Chen Y Zhao T Zhang et al ldquoProtective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusioninjury in ratsrdquo Journal of Ethnopharmacology vol 151 no 1 pp228ndash236 2014

[86] G H Chen Q Wu S J Yin X Z Wang Z W Chen and XH Chu ldquoThe effect of hirudo on cerebral hemorrhage in ratsrdquoChinese Journal of Critical Care Medicine vol 10 no 6 pp 329ndash333 1998

[87] J X Ren X Y Zhou JWang J J Zhao and P G Zhang ldquoPoxueHuayu and Tianjing Busui decoction for cerebral hemorrhage(upregulation of neurotrophic factor expression) upregula-tion of neurotrophic factor expressionrdquo Neural RegenerationResearch vol 8 no 22 pp 2039ndash2049 2013

[88] Y L Guo X F Tian and Z Xiao ldquoStudy on inhibition effects offreeze-thawing leech extract onHepG2 cellsrdquoChinese Journal ofInformation on Traditional Chinese Medicine vol 16 no 8 pp30ndash31 2009

[89] X F Tian J Sun Y Fang C R Wu Q Zhou and X HLiao ldquoStudy of leech extract on DNA demethylation in HepG2rdquoJournal of Traditional Chinese Medical University of Hunan vol31 no 9 pp 8ndash11 2011

[90] Y S Xiao F S Liao Z D Zhao A R Zuo andD C Zhu ldquoStudyon inhibitory functions of leech extract on HL60 cells in vitroexperimentrdquo Journal of Jiangxi University of Traditional ChineseMedicine vol 25 no 4 pp 63ndash66 2013

[91] Y S Xiao J H Hou F S Liao and D C Zhu ldquoThe effectof hirudo extract on the proliferation and differentiation ofleukaemia HL-60 cellsrdquo Jiangxi Journal of Traditional ChineseMedicine no 9 pp 32ndash34 2014

[92] Y S Xiao J H Hou Q Li and JMWang ldquoThe effect of hirudoextract on the proliferation and cell cycles of leukaemia HL-60cellsrdquo Jiangxi Journal of Traditional Chinese Medicine no 6 pp31ndash33 2014

[93] Y Ji L Li and M H Wu ldquoResearch progress in the studies onantitumormechanisms of leechrdquoChinese Journal of Informationon Traditional Chinese Medicine vol 22 no 3 pp 131ndash133 2015

[94] X D Huang Y L Guo L Z Huang Q Zhou and X F TianldquoStudy on the effects and mechanism of hirudo extract on thesensitivity of chemotherapeutic drugs and apoptosis inducingin humanhepatomaHepG2 cellsrdquoChinese Journal of TraditionalChinese Medicine and Pharmacy vol 30 no 6 pp 2094ndash20962015

[95] Q L Wu J L Guo H D Lu and Q Z Kong ldquoImpacts of leechhirudo and blister beetle mylabris on the angiogenesis of chickchorioallantonic membrancerdquoWorld Journal of Integrative Tra-ditional and Western Medicine vol 5 no 10 pp 855ndash857 2010

[96] X J Li H D Lu W Q Chen Z Lei Y Hu and Q Z KongldquoEffects of hirudo on tumor angiogenesis and its mechanismsrdquoCancer Research on Prevention and Treatment vol 40 no 1 pp46ndash50 2013

[97] Y H Xie S W Wang and Q Guo ldquoExperimental study onanti-inflammatory function of Hirudordquo Journal of the FourthMilitary Medical University vol 17 no 6 pp 431ndash433 1996

[98] W H Lv Z G Du S Q Gong Z M Wang and Q WangldquoA pharmacological study on the different processed samples ofWhitmania pigrardquo Journal of Chinese Medicinal Materials vol17 no 12 pp 21ndash24 1994

[99] H Sheridan B Kopp L Krenn D A Guo and J SendkerldquoTraditional Chinese herbalmedicine preparation invoking thebutterfly effectrdquo Science vol 350 no 6263 pp S64ndashS66 2015

[100] L Xiao and K L Chen ldquoReview and analysis of processingtechnology and active ingredients of medicinal leechrdquo ChinaPharmacist vol 17 no 10 pp 1760ndash1762 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


to the description in the Chinese traditional literatures onlyseveral species with the features of small body living inwaterand blood-sucking are possible sources of Shuizhi such asHnipponiaWhitman (HN)H pulchra Song Poecilobdella nan-jingensis sp Nov P manillensis (Lesson) and P hubeiensisYang [12]Three species includingWhitmania pigraWhitman(WP) H nipponica Whitman and W acranulata Whitman(WA) are recorded in the current Chinese Pharmacopoeiaas the source of Shuizhi [13] Among them WP is the mostcommonly available from Chinese commercial leech marketBased on the potent anticoagulant effect of Shuizhi manyinvestigations on phytochemistry and biological activitieshave been reported The present review summarized theethnopharmacology phytochemistry and pharmacologicalproperties of Shuizhi concerning three legally listed Chinesespecies which can provide some evidences for the interpre-tation of its effective constituents and action mechanism

2 Ethnopharmacology

The earliest record of Shuizhi appeared in the first classicbook on Chinese Materia Medica Shen-Nong-Ben-Cao-Jingas a downgrade drug The debate on the animal origins ofShuizhi has been continued in the follow-up ChinesemedicalclassicsMing-Yi-Bie-Lu (206 BCndash8AD)Ben-Cao-Jing-Ji-Zhu(480ndash498 AD) Xin-Xiu-Ben-Cao (657ndash659 AD) Shu-Ben-Cao (935ndash960 AD) Ben-Cao-Tu-Jing (1020ndash1101 AD) Ben-Cao-Meng-Quan (1565 AD) and Ben-Cao-Gang-Mu (1590AD) As an important TCM characterized by eliminatingblood stasis and stimulating menstruation discharge theChinese Pharmacopoeia has officially accepted it since 1963for the treatment of amenorrhea induced by blood stasismasses in the abdomen apoplexy and hemiplegic paralysisas well as traumatic injuries

Shuizhi is often used in combination with other TCMingredients to treat ailments resulting from blood stasisBased on the textual literatures more than 300 prescrip-tions containing Shuizhi have been described in the clinicalpractice including the different dosage forms such as pilldecoction powder sublimed preparation and plaster Thefamous ldquoDi-Dang decoction or pillrdquo and ldquoDa-Huang-Zhe-Chongpillrdquo arewell employed bymedical sage ZhangZhong-jing living in the Eastern Han Dynasty (25ndash220 AD) In thementioned prescriptions there are up to 290 TCMs beingcompatible with Shuizhi among which the top ten drugs areTabanus (Mengchong) Persicae Semen (Taoren) Rhei Radixet Rhizoma (Dahuang) Angelicae Sinensis Radix (Danggui)Cinnamomi Cortex (Rougui) Moutan Cortex (Mudanpi)Achyranthis Bidentatae Radix (Niuxi) Ginseng Radix et Rhi-zoma (Renshen) Natrii Sulfas (Mangxiao) and ToxicodendriResina (Ganqi) (Table S1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520167895935) The herbalcouple of Hirudo (Shuizhi) and Tabanus (Mengchong) is themost common unit in the prescription compositions

According to recorded formula compositions and clin-ical evidences most of these prescriptions have been sub-jected to modern drug research and development As aresult to date there are hundreds of patented prepara-tions officially approved to the medical market in China

containing single Shuizhi or its compatibility form withother herbs (Table S2) [5 13 14] The representatives offrequently prescribed preparations are Nao-Xue-Kang Huo-Xue-Tong-Mai Capsule and Tong-Xin-Luo Capsule Theformer two are derived from single Shuizhi and the latter isprepared from eleven ingredients Ginseng Radix et Rhizoma(Renshen) Hirudo (Shuizhi) Scorpio (Quanxie) PaeoniaeRadix Rubra (Chishao) Cicadae Periostracum (Chantui)Eupolyphaga (Tubiechong) Scolopendra (Wugong) San-tali Albi Lignum (Tanxiang) Dalbergiae Odoriferae Lignum(Jiangxiang) Olibanum (Ruxiang processed with vinegar)Ziziphi Spinosae Semen (Suanzaoren fried) and BorneolumSyntheticum (Bingpian)

3 Chemical Constituents

The enormous information gathered from the ethnopharma-cological applications of Shuizhi and its preparations needsthe global investigation of the chemical constituents Thesestudies have led to the isolation of macromolecule substancessuch as protein and polypeptide and small molecules includ-ing pteridines phosphatidylcholines glycosphingolipids andsterols

31 Proteins and Peptides

311 Hirudin and Recombinant Products As the represen-tative of active proteins and peptides from Shuizhi hirudinhas been considered as the most potent natural inhibitorof thrombin for a long time Hirudin is an active peptideisolated from the saliva of H medicinalis by Marquanrdtof Germany in 1950 and it is composed of 65 amino acidswith themolecularweight of approximately 71 kDa includinga compact N-terminal domain containing three S-S bondsand a C-terminal domain that is disordered in uncomplexedhirudin [15] Natural hirudin presents inminuscule amountsand it is not enough to obtain hirudin as an antithromboticagent for considerable clinical use Therefore recombinanttechniques are used to produce homogeneous preparationsof hirudin In a successful case the recombinant RGD-hirudin containing a recognizable sequence of Arg-Gly-Asp(RGD) is a bifunctional molecule according to the structureof wild-type hirudin variant 2 These structural changesimprove the hydrophobicity of the protein and allow therecombinant RGD-hirudin to interact more effectively withthe fibrinogen recognition exosite of thrombin resultingin a specific activity of 12000ATUmg [16] Owing to thesignificant anticoagulant effects [17 18] recombinant hirudinhas been approved into the market for the treatment ofthrombosis-related diseases

Hirudin naturally occurs in the fresh saliva of medicinalleeches and it is almost not detected in the processed Shuizhisamples Moreover hirudin is easily degraded when orallyadministrated by the pepsin secreted from gastric chief cellsHowever according to the traditional uses Shuizhi wascommonly decocted in hot water for 1-2 h and orally adminis-trated in the clinical practice resulting in the good anticoag-ulant effect [19] The evidences from the comparative studyof different extracting methods also suggested the extracts
















HN

NH

N

N

SO

O

O

OH

S

HN

NH

N

N

SO

O

OHS

OH

HN

NH

N

N

SO

O S O

N

N N

N

SO

O

O

HN

S

COOH

1 2 3

N

N N

N

SO

O

COOH

SO

HN

N N

N

O

O

O

OO

O

HO

O

OH

HO

HO

HO

Whitmanine B 5 6

N

N N

N

SO

O SO

67

88a9

9a123 4 4a

55a HN

N N

N

SO

O S

CH3

O

HN

NH

N

N

SO

O SO

Hirudinoidine C 10

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3CH3

CH3

CH3

CH3

CH3

H3C

H3C

H3C

H3C

H3C

H2N

(CH2)5-


Whitmanine A 4








C HHO O

P O

R R R R R

11 12 13 14

15 16 17 18 19

O PO

O

OOH

OHHO (Ceramide)

RONH

OH

4 8 11 22RO

NH

OH

20 21

RONH

OH

4 18RO

NH

OH

4 18

28 29

RONH

OH

4 18RO

NH

OH

417

30 31

O

OH

OHHO

OO

HO

OHHO

OO

OH

OHHO O

NH

OH

22

CH2OR

CH2O CH2 CH2 N+

CH3

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3



H3C N+ CH2

CH2

CH2

Ominus

24998400

O=C

O=C

O=C

O=C

O=C

O=C

O=C

CH2OH

CH2

CH2

17998400

16998400

18998400

24998400

Ominus

Figure 2 Continued


RONH

OH

4 18RO

NH

OH

4 19

23 24

RONH

OH

417 RO

NH

OH

4 17

25 26

O

OH

OHHO

OO

HO

OHHOO

OH

OHHO O

NH

OH

4 18O

27

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3O=C

O=C O=C

O=C

O=C

CH2OH

CH2

CH2

16998400

16998400

16998400

2499840022

998400






















Competing Interests





Acknowledgments


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































HN

NH

N

N

SO

O

O

OH

S

HN

NH

N

N

SO

O

OHS

OH

HN

NH

N

N

SO

O S O

N

N N

N

SO

O

O

HN

S

COOH

1 2 3

N

N N

N

SO

O

COOH

SO

HN

N N

N

O

O

O

OO

O

HO

O

OH

HO

HO

HO

Whitmanine B 5 6

N

N N

N

SO

O SO

67

88a9

9a123 4 4a

55a HN

N N

N

SO

O S

CH3

O

HN

NH

N

N

SO

O SO

Hirudinoidine C 10

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3CH3

CH3

CH3

CH3

CH3

H3C

H3C

H3C

H3C

H3C

H2N

(CH2)5-


Whitmanine A 4








C HHO O

P O

R R R R R

11 12 13 14

15 16 17 18 19

O PO

O

OOH

OHHO (Ceramide)

RONH

OH

4 8 11 22RO

NH

OH

20 21

RONH

OH

4 18RO

NH

OH

4 18

28 29

RONH

OH

4 18RO

NH

OH

417

30 31

O

OH

OHHO

OO

HO

OHHO

OO

OH

OHHO O

NH

OH

22

CH2OR

CH2O CH2 CH2 N+

CH3

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3



H3C N+ CH2

CH2

CH2

Ominus

24998400

O=C

O=C

O=C

O=C

O=C

O=C

O=C

CH2OH

CH2

CH2

17998400

16998400

18998400

24998400

Ominus

Figure 2 Continued


RONH

OH

4 18RO

NH

OH

4 19

23 24

RONH

OH

417 RO

NH

OH

4 17

25 26

O

OH

OHHO

OO

HO

OHHOO

OH

OHHO O

NH

OH

4 18O

27

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3O=C

O=C O=C

O=C

O=C

CH2OH

CH2

CH2

16998400

16998400

16998400

2499840022

998400






















Competing Interests





Acknowledgments


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C HHO O

P O

R R R R R

11 12 13 14

15 16 17 18 19

O PO

O

OOH

OHHO (Ceramide)

RONH

OH

4 8 11 22RO

NH

OH

20 21

RONH

OH

4 18RO

NH

OH

4 18

28 29

RONH

OH

4 18RO

NH

OH

417

30 31

O

OH

OHHO

OO

HO

OHHO

OO

OH

OHHO O

NH

OH

22

CH2OR

CH2O CH2 CH2 N+

CH3

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3



H3C N+ CH2

CH2

CH2

Ominus

24998400

O=C

O=C

O=C

O=C

O=C

O=C

O=C

CH2OH

CH2

CH2

17998400

16998400

18998400

24998400

Ominus

Figure 2 Continued


RONH

OH

4 18RO

NH

OH

4 19

23 24

RONH

OH

417 RO

NH

OH

4 17

25 26

O

OH

OHHO

OO

HO

OHHOO

OH

OHHO O

NH

OH

4 18O

27

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3O=C

O=C O=C

O=C

O=C

CH2OH

CH2

CH2

16998400

16998400

16998400

2499840022

998400






















Competing Interests





Acknowledgments


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















RONH

OH

4 18RO

NH

OH

4 19

23 24

RONH

OH

417 RO

NH

OH

4 17

25 26

O

OH

OHHO

OO

HO

OHHOO

OH

OHHO O

NH

OH

4 18O

27

CH3

CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

CH3O=C

O=C O=C

O=C

O=C

CH2OH

CH2

CH2

16998400

16998400

16998400

2499840022

998400






















Competing Interests





Acknowledgments


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Competing Interests





Acknowledgments


References















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article chinese medicinal leech: ethnopharmacology...

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