Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

Contents lists available at ScienceDirect

Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com/locate/prmcm

Review

Development of Leech extract as a therapeutic agent: A

chronological review
Mohamed Alaama a, b, *, Omer Kucuk c, Birdal Bilir c, Ahmed Merzouk d,
Abbas Mohammad Ghawi e, Mukerrem Betul Yerer b, f, Mohamed Alaa Ahmado g, b,
Abdualrahman Mohammed Abdualkader h, A.B.M. Helaluddin i
a
Biopep Medikal İlaç Sanayi ve Ticaret, Kayseri, Türkiye
b
Drug Application and Research Center (ERFARMA), Erciyes University, Kayseri, Türkiye
c
Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
d
Biopep Solutions Inc., Vancouver, BC Canada
e
Department of Basic Medical Science, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia
f
Department of Pharmacology, Faculty of Pharmacy, Erciyes University, Kayseri, Türkiye
g
Department of Medical Biology, School of Medicine, Erciyes University, Kayseri, Türkiye
h
Faculté de Pharmacie, Université de Montréal, Montréal, Québec, Canada
i
Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia

A R T I C L E I N F O A B S T R A C T

Key words: Introduction: Leech extract contains many identified bioactive substances which have a variety of biological ef­
Leech fects. Leech extract was discovered in the late 19th century and since then many pharmaceutical products have
Leech extract been produced using leech extract for different ailments, but many have been withdrawn. Ongoing studies focus
Leech saliva extract
on developing health authority compliant pharmaceuticals to be used as modern medicine.
Hirudotherapy
Methodology: The online databases including Google Scholar, Scopus, PubMed, and Web of Science, were
searched using different keywords: "Leech", "Leech extract", "Leech salivary gland extract" and "Leech saliva
extract". The purpose of this review was to discuss the development of leech extract as a therapeutic agent,
including the evolution of extraction techniques, and the successful manufacturing of leech extract-based
pharmaceuticals.
Results: Leech extract has been successfully developed as a therapeutic agent. Some of the developed leech
extract-based pharmaceutics were withdrawn and some are still in the market. The extraction methods played a
vital role in the quality and efficacy of leech extract-based pharmaceutics and ultimately on their sustainability in
the market. Also, the full characterization of leech extract components is a key factor in the development of leech
extract as a therapeutic agent.
Discussion: This review provides a comprehensive historical perspective on the development of leech extract
therapy, exploring its various stages of development and the key scientific and medical advances that have
shaped its current state. The discussion also delves into potential future applications.
Conclusion: Leech extract is an invaluable source of bioactive substances that can be utilized for the treatment of
mild and life-threatening medical disorders. This review will encourage other scientists to continue their research
on leech extract, especially in the areas of formulation and marketing.

1. Introduction habitats include freshwater, estuarine, and marine aquatic ecosystems,

as well as moist terrestrial ecosystems [2].
Leeches are segmented worms belonging to the Phylum Annelida, Some leeches are carnivorous; while others are sanguivorous feeding
with about 700–1000 species of leeches all over the world [1]. Leeches on the blood of diverse animals including amphibians, birds, reptiles,
are found all over the earth, except terrestrial Antarctica, and their fish and mammals. Blood feeding leeches, like other hematophagous

* Corresponding authors
E-mail address: hass83pharm@gmail.com (M. Alaama).

https://doi.org/10.1016/j.prmcm.2023.100355
Received 25 September 2023; Received in revised form 29 December 2023; Accepted 31 December 2023
Available online 1 January 2024
2667-1425/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

animals, possess active materials in their saliva to facilitate the feeding adverse effects and complications. Patient non-compliance, bleeding,
process. Because of these materials and their therapeutic effects, leeches the need for blood transfusion, local or systemic infection, allergic re­
have been used for medical purposes for centuries, mainly for blood­ actions and inflammation are the most reported complications of live
letting which was considered the key factor for its usefulness in medical leech therapy [7].
treatment. Many leech species are considered medicinal and used for The theory of leech therapy underwent a significant change with the
medical purposes such as Hirudo medicinalis, Hirudo verbena and Hirudo discovery of anticoagulants in the leech head extract by Dr. John H.
orientalis [3], Fig. 1: Haycraft in the late 19th century. Haycraft’s discovery challenged the
The first documented evidence of the use of leeches in medicine goes traditional belief that the benefit of leech therapy was exclusively due to
back to Greek physicians, who described leech therapy for the treatment bloodletting. Instead, he suggested that the real therapeutic benefit was
of various diseases. They believed that most diseases could be treated by due to the substances injected into the patient’s body by the leech [8].
bloodletting, thus they have used many methods to achieve that, This discovery led to a deeper understanding of the mechanism of action
including leeching. It was reported that Greek physicians had used of leech therapy and paved the way for further research into the ther­
leeches for the treatment of arthritis pain, gout, ocular paralysis, acute apeutic benefits of leech extract and its active compounds. Haycraft’s
liver disease, abdominal diseases associated with dyspepsia as well as for contribution was an important turning point in the history of leech
the treatment of satyriasis [4]. With the rise of Arabic medicine during therapy and had a lasting impact on the field [9]. The discovery of an­
the medieval period, leech therapy was well described and documented. ticoagulants in leech extract opened the door for a new era of leech
Arab physicians utilized leeches for the treatment of skin diseases, alo­ therapy and encouraged researchers and physicians to explore the use of
pecia, joint diseases, after surgery and for pain relief. The medicinal leech extract as a therapeutic agent. This helped to overcome some of
leeches were well described and differentiated from the non-medicinal the limitations and potential risks associated with live leech therapy,
leeches by Avicenna and Ibn Al-Quf. In Europe, during the 17th and such as the risk of infection, bleeding, and allergic reactions. Over the
18th centuries, leech therapy was used for almost all diseases [5]. years, numerous studies have been conducted to identify the active
In the early 20th century, the use of leech for medicinal purposes compounds in leech extract and to develop safe and effective medicinal
declined due to the discovery of chemical drugs and the development of products based on leech extract [10–12].
specific theoretical bases of disease and treatment. However, in the late Various terms were used to describe this extract, such as leech extract
20th century, leech therapy witnessed resurgence in popularity, [13], leech head extract [14], leech salivary glands extract [11], and
particularly in plastic surgery and microsurgery. The United States leech saliva extract [15]. The use of leech extract as a therapeutic agent
Federal Drug Administration (FDA) approved the use of the medicinal has a long history and has evolved through several eras of development.
leech, Hirudo medicinalis, in plastic surgery in 2004 as a medical device, The first era, which took place between 1884 and 1930, witnessed the
leading to an increased use of leech in medical practice [6]. discovery of leech extract and the manufacturing and marketing of
Leech therapy, like any other therapy, may be associated with commercial leech extract injections, which were primarily used to treat
thrombosis and other circulatory disorders. However, the popularity of
leech extract declined with the advent of modern pharmaceutical
products, and it eventually disappeared from medical practice.
The second era, which lasted from 1930 to 1980, was characterized
by a total lack of research and development in the field of leech extract
therapy. During this period, leech extract was completely forgotten as a
potential therapeutic agent.
The third era began in the 1980s with the reintroduction of leech
extract, and specifically leech saliva extract, as a potential therapeutic
agent. This period has been marked by a renewed interest in the use of
leech extract for medical purposes, including the treatment of skin
grafts, venous insufficiency, and other conditions.
In this review, the development of leech extract as a therapeutic
agent, the evolution of extraction techniques, and the successfully
manufactured leech extract-based pharmaceuticals are discussed.

2. Methodology

A comprehensive literature search was conducted using the key­

words ″Leech extract″, ″Leech saliva extract″, ″Leech salivary gland
extract″ in the subject area of pharmacology and pharmaceutical tech­
nology. The data were collected from scientific publications and data­
bases, including Scopus, PubMed, Web of Sciences, and Google Scholar.
The English, German, and French literature between 1884 and 2022
were included in the search. The main criteria for including any article
in this study was that it contained information about the preparation,
composition, development or formulation of leech extract as a thera­
peutic agent.
A total of 316 articles were reviewed and only 106 articles were
included in this review according to the main criteria.

3. Results

Leech extract had been discovered in 1883 by John Haycraft, and it

Fig. 1. some medicinal leech species, A: Hirudo verbena, B: Hirudo medicinalis, was studied thoroughly in terms of pharmacological effects and chem­
C: Hirudo orientalis. ical characteristics. In the meantime, many experiments were performed

2
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

to prepare a leech extract-based pharmaceutics to replace the hir­ especially as an anticoagulant for blood transfusions. Later, as more was
udotherapy and utilize its therapeutic effect without exposing the pa­ learned about the biological properties of leech extract, researchers
tients to live leech therapy complications. The results showed that the began to explore its potential use in other areas, such as the treatment of
journey of developing leech extract as a therapeutic agent has witnessed eclampsia and as an anticancer agent. On the other hand, many studies
three major steps as follow: were conducted to prove the safety or toxicity of leech extract.
The mechanism of anticoagulation activity of leech saliva extract
3.1. The discovery of “Leech extract” was also studied, and the antithrombin and antiplatelet aggregation
effects of leech extract were described by [14]. In 1914, leech extract
The old use of leeching and leech therapy depended on the theory of was used for blood transfusion at a dose of 7 mg/500 ml of blood and
four humours when bloodletting was believed to help restoring the showed no toxic reaction except for some chills and febrile reactions
balance between body fluids, which was ruined by the disease [16]. [21]. However, other research showed toxicity of some hirudin prepa­
Haycraft had noticed that, unlike normal wounds, the bleeding from the rations, which was explained by the lack of hirudin standardization and
wounds induced by leeches’ bites continued for at least one hour after the impossibility of sterilization of hirudin by heating, which destroyed
leech detached without coagulation. He reported that surgeons knew the anticoagulation effect of hirudin [22]. Leech extract was also used
that the blood remained in a liquid form inside leech’s body for a long for the treatment of eclampsia, a condition characterized with seizures
time. His-theory was that the leeches secrete ferment-containing juice in pregnant women who develop high blood pressure and damage to
which prevents coagulation of blood within their bodies and enough organs such as the liver and kidneys. The use of leech extract in the
remaining around the edges of the wound to prevent, for some time, the treatment of eclampsia was based on the belief that leech saliva could
outflowing blood from clotting. To prove his theory, he incubated rabbit help to regulate blood flow and reduce the risk of blood clots, which
blood with either 6% saline or a crude extract of leech intestines or leech could be a complication of preeclampsia [23].
heads in saline. The leech head extract prevented the blood from The first report of the use of leech extract as an anticancer agent goes
coagulation for 24 h. In this way, he demonstrated that the leech head back to 1913 [24]. While testing different materials on carcinoma in
contains a substance that inhibits blood coagulation. For in-vivo studies, mice, they reported that leech extract not only inhibited the growth of
Haycraft developed an extraction method by soaking the anterior part of tumor but also caused some regression of tumor. However, subsequent
leeches in absolute alcohol for three days, then evaporating the alcohol experiments showed that cancer cells eventually became resistant to the
off, grinding the leech with broken glass, extracting with water and effects of hirudin, and the potential for long-term effectiveness as an
filtering. Dogs and rabbits were injected with this newly obtained leech anticancer agent was limited [25]. Additionally, there were concerns
extract and the withdrawn blood showed prolonged coagulation time about the toxicity of hirudin, as seen in experiments with Guinea pigs
and the leech extract was proven to be eliminated by the kidney [8]. In [26].
the following years, researchers conducted many studies to understand Haas in 1924 reported the use of leech extract for hemodialysis and
the nature of leech extract and its action on blood [17]. applied it on humans after a series of experiments on animals. The first
To obtain a better yield and higher efficacy, the extraction method experiment on human subjects aimed to confirm the non-toxicity of
was modified by Bock, who cut the leeches’ heads, rubbed them with dry hirudin and the safety of the procedure. The hemodialysis lasted for 15
sand, mixed them with 0.7% sodium chloride and shook the mixture for min with satisfactory results [27]. At the same time, Necheles used
2 h. Then centrifuged the mixture, collected the supernatant and labeled another commercial hirudin, prepared by Passek and Wolf Hamburg, for
it as non-diluted leech extract. The remaining billets in centrifuge tubes the dialysis in animals and he claimed that this preparation was less
were washed with sodium chloride and centrifuged again to obtain a toxic [28]. Yet, the following experiments were not encouraging because
diluted leech extract [18]. of the toxicity of different hirudin preparations, and at the same time
Later, Franz reported a more developed method for the extraction of heparin was discovered and replaced hirudin in hemodialysis [29].
leech extract. While conducting his study, he described for the first time Along with the development of leech extract as a therapeutic agent,
the leech salivary glands and he believed that they were the source of the many scientists and physicians suggested the utilization of leech bite to
bioactive materials. He had also discovered and proved that the active deliver the active materials to patients rather than using impure and
material was in the head of a leech. The extraction method depended on non-standardized extract thereof. Termier was the first to suggest and
Prof. Jakobi’s studies, who named the full extract “Hirudin” for the first implement this method and called it “hirudinization of blood”. The
time [19]. The extraction procedure was applied on the full leeches’ method was successfully applied for the treatment of pulmonary em­
heads or dissected leeches’ heads, where only the pharyngeal rings are bolism [30]. This method resembles the traditional hirudotherapy (leech
taken and used for the extraction. The heads were rubbed with sand for therapy) in applying live leeches but differs in the mechanism (hirudi­
20 min then the thymolized saline was added and extracted for 1 h at nization of blood), which was completely understood at that time.
37◦ –38 ◦ C with stirring and then centrifuged to separate the Unfortunately, this revival of medical leeches as therapeutic agents
non-dissolved parts. The extract was heated to 60 ◦ C for 2 h and then stopped completely when their supply from natural sources in South­
centrifuged. After that, it was exposed to chloroform vapors for 3 days, eastern Europe was interrupted during World War II. Moreover, new
centrifuged again and finally dried in a desiccator over sulfuric acid. The powerful but much less expensive natural (heparin) and synthetic oral
yield was 8 − 9 mg extract per head which corresponded to 12.5%. The (coumarin) anticoagulants became available and soon proved to be
study was extended to discover the time needed for leeches to recover useful in clinical settings. Thus, the chance of developing hirudin as a
their active materials after feeding, and the time needed was 14 days. therapeutic agent had declined, just because of the natural limitations in
The study also reported a suitable method to stabilize the leech extract supply and the existence of competitor drugs which did not have these
by drying the extract and keeping it away from moisture and air or by limitations [31].
keeping it in thymol-sterile glass tubes. This product was then marketed To summarize the first era of developing leech extract as a thera­
as a medicine for the first time by (Sachsse and Co., Leipzig, Germany) peutic agent, it is obvious that researchers and physicians succeeded in
[20]. There was likely growing interest in the therapeutic potential of producing an injectable leech extract which was used clinically. Com­
leech extract during this period, and companies were actively working mercial leech extract (hirudin) was produced and marketed by Sachsse
to commercialize and market leech extract-based medicines. Overall, the and Co. Leipzig, Germany, and Passek and Wolff of Hamburg. Regardless
development of this extraction method and the subsequent commer­ of the reports about the toxicity and incapability of standardization of
cialization of leech extract-based medicines likely represented a signif­ some hirudin preparations at that time, many papers stated that leech
icant milestone in the history of leech extract therapy. extract was used clinically for the treatment of some diseases and ulti­
The studies on the use of leech extract in the clinic had accelerated mately injected into human patients. The reported toxicity of hirudin

3
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

could have been due to allergy and shock, which could also be partly reconstructive surgery continued to gain popularity and was further
explained by the presence of endotoxin in leech extract. The lack of optimized during the 1990s. This led to the FDA recognizing and
standardizability can be overcome now by the utilization of suitable approving the use of medicinal leeches as a medical device for the
analytical methods for leech extract. The adverse event of bleeding treatment of venous congestion in graft tissue in plastic surgery [6].
could now be addressed by adjusting the dose and blood transfusion. Alongside with the comeback of leech therapy, leech salivary gland
extract (SGE) had also gained more attention after the discovery of its
3.2. The discovery of leech extract constituents as pure compounds unique composition and the possibility of employing the leech SGE for
the treatment of complicated diseases such as lung tumor [11,78]. This
The second era showed a decline in the use of leech extract as a development helped leech extract to revive and find its way back to
therapeutic agent, while the traditional use of live leeches or leech modern pharmacies as commercial products. Since 1983, many studies
therapy continued without any support or approval from international have reported promising and successful leech extract-based pharma­
health bodies. The studies on leech extract were limited to the isolation ceutical formulations, especially in the Chinese market. The extraction
and purification of pure compounds and testing their activities on method has also been modified and optimized to obtain the full leech
different diseases. The first isolated pure compound was the leech hy­ saliva components without killing the leeches. Therefore, the following
aluronidase [32], followed by the pure compound “hirudin” [33], text will discuss the “leech extract in Chinese medicine” and the “leech
bdellins [34], eglins [35], decorsin [36], and many other active com­ saliva extract” as different products according to their extraction
pounds. The biological activities of these isolated compounds were methods and their application and their commercial products.
studied and they can be summarized in Table 1.
Recombinant hirudin was produced [73], which opened the door to 3.3.1. Leech extract in chinese medicine
produce injectable drugs such as lepirudin, desirudin, and bivalirudin. Leech therapy has been used in Chinese traditional medicine for
Lepirudin was marketed under the brand name Refludan (Bayer) as an thousands of years and the leech (Shuizhi) has been listed in Chinese
anticoagulant in patients with heparin-induced thrombocytopenia (HIT) pharmacopeia and identified as the full body dried powder of Whitmania
and thromboembolic disease to prevent further thromboembolic com­ pigra, Hirudo nipponica and Whitmania acranulata leech species. Leech
plications for the patients who are not able to use heparin [74]. Desir­ extract can be found as oral, topical, or injected medicine in the Chinese
udin was approved by the FDA under the brand name Iprivask as a direct market.
thrombin inhibitor. Iprivask is indicated for the prophylaxis of deep vein
thrombosis, which may lead to pulmonary embolism and in patients 3.3.1.1. Oral leech extract in Chinese traditional medicine. Oral leech
undergoing elective hip replacement surgery [75]. Bivalirudin was extract is prepared as tablets or capsules, containing dried leech powder
approved by the FDA under the brand name Angiomax and is indicated only or combined with other herbal or animal extracts. Many approved
for use as an anticoagulant in patients with unstable angina undergoing medicines can be found in the Chinese Pharmacopeia and the Chinese
percutaneous transluminal coronary angioplasty (PTCA) [76]. market such as:

1. Zhixiong (capsules) has been used clinically for the treatment of

3.3. The return of natural whole leech extract as a better therapeutic
acute and chronic cerebrovascular diseases due to its contents’
agent
abilities to improve the blood circulation, decrease blood coagula­
tion, activate meridians and relieve stasi [79].
Although leech therapy was not so popular during the first half of the
2. Shenyuandan (capsules) has been used for the treatment of coronary
20th century, it started to have a great comeback beginning in the 1960s
heart disease and angina pectoris. It protects against myocardial
when physicians noticed its remarkable benefits in relieving venous
ischemia/reperfusion injury, ameliorates oxygen-free radical injury
congestion of skin transplants [77]. The use of leech therapy in plastic
in ischemic myocardium, and improves the antioxidant ability of
myocardial tissue. It attenuates atherosclerosis by promoting auto­
Table 1
phagy, probably through regulating genomic DNA methylation and
Isolated chemical compounds from different leech species.
Atg13 promoter demethylation [80]. Clinically, Shenyuandan’s ef­
Leech species Isolated compounds ficacy and safety in reducing the incidence of peri‑procedural
Hirudo medicinalis Hirudin [33], Destabilase [37], Bdellin B [38], Calin [39], myocardial injury among patients with unstable angina who un­
Factor Xa inhibitor, Bdellastasin [40], Hirustatin, derwent elective percutaneous coronary intervention were tested
Leech-derived tryptase inhibitor (LDTI) [41], Hyaloronedase
using a prospective, randomized, double-blinded, placebo-controlled
[32], Eglins [35], Apyrase [10], Collagenase [10], Leech
carboxy peptidase inhibitor [42], Lipase (triglyceridase),
trial. The results showed a significant decrease in the number of
Cholesterol-esterase [43], Steroids, Histamine, and peri‑procedural myocardial injury in the Shenyuandan group
Serotonin [43]. compared to the placebo group [81].
Hirudinaria Bufrudin [44], HM1, HM2 [45], Hirullin P6, Hirullin P18 3. Naoxintong (capsules) has been reported to have multiple protective
manillensis [46], Manillase [47], Gelin [48].
effects on cardiovascular diseases including coronary artery disease,
Hirudo nipponia Leech granulin [49], Guamerin [50], Piguamerin [51]
Theromyzon Theromin [52], Therostasin [53], Cytin [54], Therin [55], coronary microembolization, myocardial infarction, acute coronary
tessulatum Tessulin [56], Theromacin and Theromyzin [57], Renin-like syndrome, ischemic stroke, and ischemia-reperfusion injury [82].
enzyme [58] 4. Tongxinluo (capsules) has been used for coronary heart disease,
Haementeria Leech antiplatelet protein (LAPP) [59], Antistasin [60] unstable angina pectoris, and acute stroke, in addition to its ability to
officinalis
Haementaria Hementin [61], Tridegin [62], Ghilantin [63], Saratin [64],
improve serum lipid levels [83].
ghilianii 5. Dahuang Zhechong (pills) has been reported to improve pulmonary
Haementeria Hementerin [65], Lefaxin [66], function, quality of life, and exercise capacity of silicosis patients
depressa [84], reduce serum biomarkers of liver fibrosis in patients with
Haementarea lutzi Hementerin [67]
chronic hepatitis B [85] and suppress colorectal cancer liver metas­
pintu
Haemadipsa Haemadin [68], tasis via ameliorating exosomal CCL2 primed pre-metastatic niche
sylvestris [86].
Macrobdella decora Decorsin [69]
Placobdella ornate Ornatins [70]
Leech in general 100++ unidentified proteins [71,72]

4
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

6. Maixuekang (capsules) consists of leech Whitmania pigra/Hirudo in leech body extract and that could reduce the possibility of allergy and
nipponica extract only and has been prescribed for acute coronary shock which may result from whole leech body extract injection [10].
syndrome [87]. LSE can be sterilized, depyrogenated, standardized and formulated as a
topical or parenteral drug. Up to date, there are no approved commercial
3.3.1.2. Parenteral leech extract in Chinese medicine. The Chinese products of LSE, but the literature contains many proven active products
injectable leech extract Shuxuetong was prepared from the whole leech which could be marketed soon.
body and earthworm using different techniques. The leeches (Hirudo LSE was prepared as a topical liposomal gel for the treatment of knee
nipponica Whitman) and earthworms (Pheretima aspergillum) were osteoarthritis and the effect was assessed clinically [100]. A clinical trial
cleaned and sterilized by soaking them in normal saline repeatedly. was designed to evaluate the effect of adding LSE liposomal gel treat­
Then they were grinded and homogenized, and then exposed to freeze- ment to the physiotherapy of patients with knee osteoarthritis. The re­
thaw cycles with a freezing temperature of − 15 C. The mixture was sults showed that the group receiving the combined treatment had
centrifuged, and the supernatant was separated. The supernatant was significant pain reduction and quality of life improvement compared
ultrafiltered using a cut-off membrane of 6000 Dalton and the filtrate with the group receiving only physiotherapy [100].
was obtained, and the Shuxuetong was ready for medical use. Shux­ Another LSE product is produced using Asian leech (Hirudinaria
uetong was prepared by mixing leech extract and earthworm extract in manillensis) and prepared for the treatment of different types of cancer,
the percentages of 33%− 84% and 16%− 67%, respectively, as injectable especially prostate cancer. LSE effects were evaluated in vitro and in vivo,
solutions 1–20 ml, infusion 50–200 ml/ bottle, or freeze-dried powder but no clinical study has been performed yet [12]. LSE IC50 was deter­
for injection [88]. mined in vitro in five prostate cancer cell lines using MTS cell viability
Shuxuetong injection has been included in the Chinese Pharmaco­ assay. In vivo efficacy of LSE was determined in Lymph Node Carcinoma
poeia and had total sales of over $93 million in 2017 in China. Based on of the Prostate (LNCaP) and 22RV-1 in nude mice xenograft models. LSE
the source of the medicine, the main active ingredients were believed to was injected subcutaneously at a dose of 5 mg/kg once a week for 4
be hirudin and lumbrokinase, so it is extensively prescribed as an anti­ weeks and docetaxel was used as a positive control. The results showed
coagulant and fibrinolysis in acute cerebral infarction, acute phase of that LSE has significantly reduced the tumor size and improved the PSA
ischemic stroke and other coagulation-related diseases [89]. Later, a and other biological markers. LSE anticancer effect was similar to
study showed that hirudin is not detected in Shuxuetong, while the main docetaxel effect with no side effects [101]. The studies revealed that LSE
ingredients are free amino acids, peptides, monosaccharides, poly­ inhibited Proteinase Activated Receptor 1 (PAR-1) which is responsible
saccharides, nucleic acids and other components. The extraction method for hormones like cellular signals in different cancer cells including
could be the reason behind the absence of hirudin which is broken down those of the breast and prostate. Moreover, in LNCaP cells the Androgen
into smaller polypeptides during the preparation of Shuxuetong [90]. Receptor (AR) activation and the Prostate Specific Antigen (PSA)
Regardless of the presence or the absence of hirudin, Shuxuetong expression were significantly reduced after treatment with LSE. In­
showed a high anticoagulation effect making it a suitable candidate for hibitions of phosphoinositide-3-kinase (PI3K) and Phospholipase C,
the treatment of cerebral infarction. Animal studies have shown that Gamma 2 (PLCG2) were also observed. It has been found that LSE
Shuxuetong can effectively improve spinal cord injury due to its active modulates a multitude of pathways which makes it a very promising
materials’ ability to enhance spinal cord blood flow [91]. In addition to candidate for the treatment of cancer with an important advantage of
improving blood flow to the injured area, Shuxuetong facilitates low toxicity and reduced potential for the development of drug resis­
angiogenesis during wound healing following traumatic brain injury in tance [102].
rats. The formation of new capillary blood vessels was enhanced after
the treatment with Shuxuetong which promoted the endothelial cell 4. Discussion
proliferation via the vascular endothelial growth factor/vascular endo­
thelial growth factor receptor-2 pathway [88]. Leech extract has been found to be effective for the treatment of
Clinically, Shuxuetong injection was used in combination with other many diseases due to its unique composition. Its extraction method has
chemical drugs for the treatment of ischemic stroke [92]. gone through huge development starting with the whole-body extract
through the extraction of leeches’ heads and salivary glands to the
3.3.2. Leech saliva extract (LSE) extraction of leech saliva without killing the leeches. Many leeches
In leech farms, leeches are fed on blood using many techniques, such extract-based pharmaceuticals have been produced and marketed
as feeding on living frogs and fish, or on animals’ blood through sieve, or (Table 2).
artificial membranes. To study the effect of some chemicals found in The success of leech extract-based pharmaceuticals and the devel­
blood on the feeding response of leeches, Galun and Kindler developed a opment of new extraction, filtration and formulation techniques along
method to feed leeches through artificial membrane consisting of silastic with the proven benefits of leech therapy enhance the scientific com­
membrane [93]. This method was further developed and used for the munity’s confidence in the possibility of obtaining a leech extract-based
extraction of diluted leech saliva extract [10]. Leeches were fed on a product which is in compliance with modern health authorities’ rules
pre-heated phagostimulatory solution consisting of NaCl 0.015 M and and regulations. However, apart from medicines marketed in China,
Arginine 0.001 M through a sausage membrane. While feeding, leeches there are no FDA-approved leech extract-based pharmaceuticals yet. The
secrete saliva into the solution. Once the feeding is completed, the published data shows promising products with enhanced pharmaco­
diluted saliva is obtained by squeezing leeches from the posterior sucker logical effects and a wide safety margin comparing to existed chemical
towards the anterior sucker. This method was further optimized to ease drugs.
the extraction process as leeches show some resistance against A study was conducted using the propensity score method to analyze
squeezing. Leeches can be stressed to regurgitate the saliva by adding the effect of Maixuekang capsule on the treatment outcome of coronary
salt or wood ash [94], ethanol [95] and ice [96]. The LSE extracted using heart disease. The study team examined the electronic medical record
this method showed remarkable biological activity such as anticoagu­ database of 22 large-scale tertiary hospitals in China identifying patients
lant [10], antidiabetic [97], antibacterial [98] and anticancer [99]. with coronary heart disease taking or not taking Maixuekang capsule in
Many attempts have been reported to prepare a pharmaceutical combination with other chemical drugs. After matching, there were
formulation using LSE which is extracted using this method [95]. Re­ 2464 cases in the Maixuekang and non-Maxiuekang groups. The results
searchers claim that LSE is the suitable candidate as it contains the active showed that the Maixuekang group had better recovery and improve­
materials only without any leech body components which were present ment compared to the non-Maixuekang group [87].
Clinically, the effects of Shuxuetong in adjuvant treatment of

5
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

Table 2
Leech extract-based pharmaceuticals.
Leech extract-based product Route of Claimed usage Year of Used for
administration marketing

Hirudin Sachsse and Co., Injection Anticoagulant [20] 1904–1925 Blood transfusion [21], Eclampsia [23], hemodialysis [27]
Leipzig, Germany
Hirudin Passek and Wolf Injection Anticoagulant [28] 1920–1925 hemodialysis [28]
Hamburg
Piyavit Oral Anticoagulant [103] 1993–1995 acquired heart valvular disease [103]
Shuizhi and other oral Oral Anticoagulant, antiplatelet 1960s- today Acute and chronic cerebrovascular diseases [79], atherosclerosis [80],
Chinese formulations aggregation [104] ischemic stroke [82], acute coronary syndrome [87].
Shuxuetong Injection Anticoagulant [105] 1990s-today Ischemic stroke [92]
Recombinant hirudin derivatives
Lepirudin Injection Anticoagulant [74] 1997 heparin-induced thrombocytopenia [74]
Desirudin Injection Thrombin inhibitor [75] 2003 Antithrombin in elective hip replacement surgery [75]
Bivalirudin Injection Anticoagulant [76] 2000 unstable angina [76]

ischemic stroke on the degree of nerve injury, lipid metabolism and Writing – review & editing. Ahmed Merzouk: Writing – review &
blood coagulation function were studied. The study included 74 patients editing. Abbas Mohammad Ghawi: Writing – review & editing.
who were divided into two groups, where the control group received Mukerrem Betul Yerer: Writing – review & editing. Mohamed Alaa
regular chemical treatments, while the Shuxuetong group received Ahmado: Writing – review & editing. Abdualrahman Mohammed
Shuxuetong injections in addition to the regular treatments for three Abdualkader: Writing – review & editing. A.B.M. Helaluddin: Writing
months. The chemical drugs included 100 mg aspirin enteric-coated – review & editing.
tablets, 75 mg sulfate clopidogrel tablets, 20 mg atorvastatin and 30 mg
edaravone injections with 250 mL of normal saline. After one and three Declaration of competing interest
months of treatment, the degree of nerve injury, lipid metabolism and
blood coagulation function indices were evaluated. The results showed The authors declare that they have no known competing financial
that Shuxuetong had significantly alleviated the injury of nerve func­ interests or personal relationships that could have appeared to influence
tion, improved blood lipid metabolism and coagulation function in pa­ the work reported in this paper.
tients treated of ischemic stroke [92].
The wound healing properties of leech extract cream were compared Acknowledgments
to phenytoin cream, and the results showed that leech cream and
phenytoin cream could promote wound regeneration by accelerating the The authors expressed their gratitude to staff of Department of
re-epithelialization process and initial angiogenesis [106]. The anti­ Pharmaceutical Chemistry, Faculty of Pharmacy, International Islamic
tumor properties of LSE and docetaxel in prostate cancer were assessed. University Malaysia and staff of the Drug Application and Research
The LSE has significant in vitro and in vivo anti-tumor activity with no Center, Erciyes University for their technical assistance.
apparent side effects compared to the chemical drug docetaxel [101].
References
5. Conclusion
[1] M.E. Siddall, Phylogeny of the leech family Erpobdellidae (Hirudinida:
oligochaeta), Invertebr. Syst. 16 (1) (2002) 1–6.
The complexity of some diseases, such as cancer and diabetes, or the [2] A.J. Phillips, F.R. Govedich, W.E. Moser, Leeches in the extreme: morphological,
complications associated with aging which affect the whole body re­ physiological, and behavioral adaptations to inhospitable habitats, Internat. J.
quires, patients to receive many medications at the same time increased Parasitol.: Parasit. Wildlife 12 (2020) 318–325.
[3] S. Utevsky, N. Kovalenko, K. Doroshenko, L. Petrauskienė, V. Klymenko,
the need for combined drugs. However, the increased number of medi­ Chromosome numbers for three species of medicinal leeches (Hirudo spp, Syst.
cations taken by the patients at the same time led to severe and serious Parasitol. 74 (2009) 95–102.
side effects which left some important organs at risk such as the liver. [4] M.R. Montinari, S. Minelli, From ancient leech to direct thrombin inhibitors and
beyond: new from old, Biomed. Pharmacother. 149 (2022) 112878.
Therefore, the health community confirms the need for a medication [5] I.A. Younis Munshi, H. Rafique, Z. Ahmad, Leeching in the history-a review, Pak.
which can work on different sides with high efficacy and less side effects. J. Biol. Sci. 11 (13) (2008) 1650–1653.
An increasing number of scientific reports suggest the health benefits of [6] C. Rados, Beyond bloodletting: FDA gives leeches a medical makeover, FDA
Consum 38 (5) (2004) 9. -9.
leech therapy. Centuries of use in diverse populations attesting to its [7] M. Pourrahimi, M. Abdi, R. Ghods, Complications of leech therapy, Avicenna J.
relative safety warrant further investigation of leech saliva extract as a Phytomed. 10 (3) (2020) 222.
pharmaceutical agent. Leech saliva extract could be a treatment of [8] J.B. Haycraft, On the action of a secretion obtained from the medicinal leech on
the coagulation of the blood, Proc. R. Soc. Lond. 36 (228–231) (1883) 478–487.
choice for complex diseases due to the multitude of ingredients it con­
[9] S. Lemke, A. Vilcinskas, European medicinal leeches—new roles in modern
tains, which are combined naturally and demonstrate diverse biological medicine, Biomedicines 8 (5) (2020) 99.
activities. However, more research is needed to elucidate the full [10] M. Rigbi, H. Levy, F. Iraqi, M. Teitelbaum, M. Orevi, A. Alajoutsijärvi,
chemical composition and mechanism of action of each compound that A. Horovitz, R. Galun, The saliva of the medicinal leech Hirudo medicinalis–I.
Biochemical characterization of the high molecular weight fraction, Comp.
is present in leech saliva extract. Biochem. Physiol., B 87 (3) (1987) 567–573.
[11] G.J. Gasic, A. Iwakawa, T.B. Gasic, E.D. Viner, L. Milas, Leech salivary gland
extract from Haementeria officinalis, a potent inhibitor of cyclophosphamide-and
Financial support
radiation-induced artificial metastasis enhancement, Cancer Res 44 (12_Part_1)
(1984) 5670–5676.
This work received no financial support. [12] M. Alaama, M. AlNajjar, A. Abdualkader, A. Mohammad, A.J.I.E.J. Merzouk,
Isolation and analytical characterization of local Malaysian leech saliva, IIUM
Engineer. J. 12 (4) (2011).
CRediT authorship contribution statement [13] C.H. Wang, S. Pandey, K. Sivalingam, M.A. Shibu, W.-W. Kuo, V.P. Viswanadha,
Y.-C. Lin, S.-C. Liao, C.-Y. Huang, Leech extract: a candidate cardioprotective
Mohamed Alaama: Conceptualization, Data curation, Formal against hypertension-induced cardiac hypertrophy and fibrosis,
J. Ethnopharmacol. 264 (2021) 113346.
analysis, Investigation, Methodology, Project administration, Writing – [14] R.I. Lee, B. Vincent, A study of the effect of anaphylaxis and leech extract on the
original draft. Omer Kucuk: Writing – review & editing. Birdal Bilir: coagulation of the blood, J. Med. Res. 32 (3) (1915) 445.

6
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

[15] M. Alaama, A.B.M. Helaluddin, A. Mohammad, A. Merzouk, A.M. Abdualkader, [47] M. Kordowicz, D. Gussow, U. Hofmann, T. Pacuszka, A. Gardas, Hyaluronidase
M. Awang, Starvation time and successive collection effects on leeches saliva from the Hirudinaria manillensis isolation, purification and recombinant method
collection quantity and proteins quality and quantity in wet season, Sains Malays. of production, Google Patents, 2006.
43 (11) (2014) 1693–1697. [48] A. Electricwala, N.A.E. Von Sicard, R.T. Sawyer, T. Atkinson, Biochemical
[16] B. Okka, Hirudotherapy from past to present, Eur. J. Basic Med. Sci. 3 (3) (2013) characterisation of a pancreatic elastase inhibitor from the Leech Hirudin Aria
61–65. Manillensis, J. Enzy. Inhib. 6 (4) (1993) 293–302.
[17] W.L. Dickinson, Note on “leech-extract” and its action on blood, J. Physiol. 11 [49] S.J. Hong, K.W. Kang, Purification of granulin-like polypeptide from the blood-
(Suppl) (1890) 566. sucking leech, Hirudo nipponia, Protein Expr. Purif. 16 (2) (1999) 340–346.
[18] J. Bock, Untersuchungen über die Wirkung verschiedener Gifte auf das isolirte [50] H.I. Jung, S.I. Kim, K.-S. Ha, C.O. Joe, K.W. Kang, Isolation and characterization
Sängethierherz, Archiv. für experiment. Pathol. Pharmakol. 41 (2) (1898) of guamerin, a new human leukocyte elastase inhibitor from Hirudo nipponia,
158–178. J. Biol. Chem. 270 (23) (1995) 13879–13884.
[19] C. Jacoby, Über Hirudin, Dtsch. Med. Wochenschr. 30 (1904) 786–787. [51] D.R. Kim, K.W. Kang, Amino acid sequence of piguamerin, an antistasin-type
[20] F. Franz, Ueber den die Blutgerinnung auf hebenden Bestandtheil des protease inhibitor from the blood sucking leech Hirudo nipponia, Eur. J.
medicinischen Blutegels, Archiv. für experimen. Pathol. Pharmakol. 49 (4) (1903) Biochem. 254 (3) (1998) 692–697.
342–366. [52] M. Salzet, V. Chopin, J.-l. Baert, I. Matias, J. Malecha, Theromin, a novel leech
[21] H.S. Satterlee, R.S. Hooker, The use of herudin in the transfusion of blood, J. Am. thrombin inhibitor, J. Biol. Chem. 275 (40) (2000) 30774–30780.
Med. Assoc. 62 (23) (1914) 1781–1783. [53] V. Chopin, M. Salzet, J.-l. Baert, F. Vandenbulcke, P.-E. Sautiere, J.-P. Kerckaert,
[22] E.K. Marshall, The toxicity of certain hirudin preparations, J. Pharmacol. Exp. J. Malecha, Therostasin, a novel clotting factor Xa inhibitor from the
Ther. 7 (1) (1915) 157–168. rhynchobdellid leech, Theromyzon tessulatum, J. Biol. Chem. 275 (42) (2000)
[23] A. Wallace, The suppression of the convulsion in eclampSIA, Lancet North Am. 32701–32707.
Ed. 180 (1912) 1574–1576. [54] V. Chopin, T.V. Bilfinger, G.B. Stefano, I. Matias, M. Salzet, Amino-acid-sequence
[24] L. Loeb, M.S. Fleisher, Intravenous injections of various substances in animal determination and biological activity of cytin, a naturally occurring specific
cancer, J. Am. Med. Assoc. 60 (24) (1913) 1857–1858. chymotrypsin inhibitor from the Leech Theromyzon tessulatum, Eur. J. Biochem.
[25] M.S. Fleisher, M. Vera, L. Loeb, Immunization against the action of substances 249 (3) (1997) 733–738.
inhibiting tumor growth, J. Exp. Med. 20 (5) (1914) 522–541. [55] V. Chopin, I. Matias, G.B. Stefano, M. Salzet, Amino acid sequence determination
[26] M.S. Fleisher, L. Loeb, The effect of the intravenous injection of substances and biological activity of therin, a naturally occuring specific trypsin inhibitor
affecting tumor growth on the cyclic changes in the ovaries and on placentomata, from the leech Theromyzon tessulatum, Eur. J. Biochem. 254 (3) (1998)
J. Exp. Med. 20 (2) (1914) 180–190. 565–570.
[27] G. Haas, Versuche der blutauswaschung am lebenden mit hilfe der dialyse, Klin. [56] V. Chopin, G.B. Stefano, M. Salzet, Amino-acid-sequence determination and
Wochenschr. 4 (1) (1925) 13–14. biological activity of tessulin, a naturally occurring trypsin-chymotrypsin
[28] H. Necheles, Über dialysieren des strömenden Blutes am Lebenden, Klin, inhibitor isolated from the leech Theromyzon tessulatum, Eur. J. Biochem. 258
Wochenschr 2 (27) (1923) 1257. -1257. (2) (1998) 662–668.
[29] G. Haas, über Blutwaschung, Klin. Wochenschr. 7 (29) (1928) 1356–1362. [57] A. Tasiemski, F. Vandenbulcke, G. Mitta, J. Lemoine, C. Lefebvre, P.-E. Sautiere,
[30] A. Ochsner, H. Mahorner, The use of leeches in the treatment of phlebitis and the M. Salzet, Molecular characterization of two novel antibacterial peptides
prevention of pulmonary embolism, Ann. Surg. 98 (1933) 408. inducible upon bacterial challenge in an annelid, the leech Theromyzon
[31] G. Nowak, K. Schrör, Hirudin–the long and stony way from an anticoagulant tessulatum, J. Biol. Chem. 279 (30) (2004) 30973–30982.
peptide in the saliva of medicinal leech to a recombinant drug and beyond, [58] V. Laurent, M. Salzet, Isolation of a renin-like enzyme from the leech Theromyzon
Thromb. Haemost. 98 (07) (2007) 116–119. tessulatum, Peptides 16 (8) (1995) 1351–1358.
[32] A. Claude, Spreading properties of leech extracts and the formation of lymph, [59] T.M. Connolly, J.W. Jacobs, C. Condra, An inhibitor of collagen-stimulated
J. Exp. Med. 66 (3) (1937) 353. platelet activation from the salivary glands of the Haementeria officinalis leech. I.
[33] F. Markwardt, Hirudin, Blut 4 (3) (1958) 161–170. Identification, isolation, and characterization, J. Biol. Chem. 267 (10) (1992)
[34] H. Fritz, K. Krejci, Trypsin-plasmin inhibitors (Bdellins) from leeches. Methods 6893–6898.
Enzymol, Elsevier, 1976, pp. 797–806. [60] G.P. Tuszynski, T.B. Gasic, G.J. Gasic, Isolation and characterization of antistasin.
[35] U. SeemÜLler, M. Meier, K. Ohlsson, H.-P. MÜLler, H. Fritz, Isolation and An inhibitor of metastasis and coagulation, J. Biol. Chem. 262 (20) (1987)
characterisation of a low molecular weight inhibitor (of chymotrypsin and human 9718–9723.
granulocytic elastase and cathepsin G) from leeches, Hoppe Seylers Z Physiol. [61] S.M. Malinconico, J.B. Katz, A.Z. Budzynski, Fibrinogen degradation by
Chem. 358 (1977) 1105–1107. hementin, a fibrinogenolytic anticoagulant from the salivary glands of the leech
[36] P.A. Orlandi, F.W. Klotz, J.D. Haynes, J. Cell, T.J. Hadley, Structure of the RGD Haementeria ghilianii, J. Lab. Clin. Med. 104 (5) (1984) 842–854.
Protein Decorsin: conserved Motif and Distinct Function in Leech Proteins That, [62] S. Finney, L. Seale, R.T. Sawyer, R.B. Wallis, Tridegin, a new peptidic inhibitor of
Chem. (Easton) 17 (1978) 4756. factor XIIIa, from the blood-sucking leech Haementeria ghilianii, Biochem. J. 324
[37] I.P. Baskova, G.I. Nikonov, Destabilase: an enzyme of medicinal leech salivary (3) (1997) 797–805.
gland secretion hydrolyzes the isopeptide bonds in stabilized fibrin, Biokhimiia [63] R. Brankamp, G. Manley, D. Biankenship, T. Bowlin, A.D. Cardin, Studies on the
50 (3) (1985) 424–431. anticoagulant, antimetastatic and heparin-binding properties of ghilanten-related
[38] E. Fink, H. Rehm, C. Gippner, W. Bode, M. Eulitz, W. Machleidt, H. Fritz, The inhibitors, Blood Coagul. Fibrinol. 2 (1991) 161–166.
primary structure of bdellin B-3 from the leech Hirudo medicinalis. Bdellin B-3 is [64] C.S. Barnes, B. Krafft, M. Frech, U.R. Hofmann, A. Papendieck, U. Dahlems,
a compact proteinase inhibitor of a “non-classical” Kazal type. It is present in the G. Gellissen, M.F. Hoylaerts, Production and characterization of saratin, an
leech in a high molecular mass form, Biol. Chem. Hoppe Seyler 367 (1986) inhibitor of von Willebrand factor-dependent platelet adhesion to collagen,
1235–1242. Semin. Thromb. Hemost. (2001) 337–348.
[39] R. Munro, C.P. Jones, R.T. Sawyer, Calin–a platelet adhesion inhibitor from the [65] A.M. Chudzinski-Tavassi, E.M.A. Kelen, A.P. de Paula Rosa, S. Loyau, C.A.
saliva of the medicinal leech, Blood Coagul. Fibrinol. 2 (1991) 179–184. M. Sampaio, C. Bon, E. Anglés-Cano, Fibrino (geno) lytic properties of purified
[40] M. Moser, E. Auerswald, R. Mentele, C. Eckerskorn, H. Fritz, E. Fink, Bdellastasin, hementerin, a metalloproteinase from the leech Haementeria depressa, Thromb.
a serine protease inhibitor of the antistasin family from the medical leech (Hirudo Haemost. 80 (07) (1998) 155–160.
medicinalis), 253(1) (1998) 212–220. [66] F. Faria, E.M.A. Kelen, C.A.M. Sampaio, C. Bon, N. Duval, A.M. Chudzinski-
[41] C.P. Sommerhoff, C. Söllner, R. Mentele, G.P. Piechottka, E.A. Auerswald, Tavassi, A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech,
H. Fritz, A Kazal-type inhibitor of human mast cell tryptase: isolation from the Thromb. Haemost. 82 (11) (1999) 1469–1473.
medical leech Hirudo medicinalis, characterization, and sequence analysis, Biol. [67] E.M.A. Kelen, G. Rosenfeld, Fibrinogenolytic substance (Hementerin) of Brazilian
Chem. Hoppe Seyler 375 (10) (1994) 685–694. blood-sucking leeches (Haementeria lutzi Pinto 1920), Pathophysiol. Haemost.
[42] D. Reverter, J. Vendrell, F. Canals, J. Horstmann, F.X. Avilés, H. Fritz, C. Thromb. 4 (1) (1975) 51–64.
P. Sommerhoff, A carboxypeptidase inhibitor from the medical leech Hirudo [68] K.-H. Strube, B. Kröger, S. Bialojan, M. Otte, J. Dodt, Isolation, sequence analysis,
medicinalis: isolation, sequence analysis, cDNA cloning, recombinant expression, and cloning of haemadin. An anticoagulant peptide from the Indian leech, J. Biol.
and characterization, J. Biol. Chem. 273 (49) (1998) 32927–32933. Chem. 268 (12) (1993) 8590–8595.
[43] I.P. Baskova, Z. Ferner, A.S. Balkina, S.A. Kozin, O.V. Kharitonova, L.L. Zavalova, [69] J.L. Seymour, W.J. Henzel, B. Nevins, J.T. Stults, R.A. Lazarus, Decorsin. A potent
V.G. Zgoda, Steroids, histamine, and serotonin in the medicinal leech salivary glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech
gland secretion, Biochem. (Moscow) Suppl. Series B: Biomed. Chem. 2 (3) (2008) Macrobdella decora, J. Biol. Chem. 265 (17) (1990) 10143–10147.
215–225. [70] P. Mazur, W.J. Henzel, J.L. Seymour, R.A. Lazarus, Ornatins: potent glycoprotein
[44] A. Electricwala, R.T. Sawyer, C.P. Jones, T. Atkinson, Isolation of thrombin IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella
inhibitor from the leech Hirudinaria manillensis, Blood Coag. Fibrinol. 2 (1991) ornata, Eur. J. Biochem. 202 (3) (1991) 1073–1082.
83–89. [71] V.V. Babenko, O.V. Podgorny, V.A. Manuvera, A.S. Kasianov, A.I. Manolov, E.
[45] E. Scacheri, G. Nitti, B. Valsasina, G. Orsini, C. Visco, M. Ferrera, R.T. Sawyer, N. Grafskaia, D.A. Shirokov, A.S. Kurdyumov, D.V. Vinogradov, A.S. Nikitina, S.
P. Sarmientos, Novel hirudin variants from the leech Hirudinaria manillensis: I. Kovalchuk, N.A. Anikanov, I.O. Butenko, O.V. Pobeguts, D.S. Matyushkina, D.
amino acid sequence, cDNA cloning and genomic organization, Eur. J. Biochem. V. Rakitina, E.S. Kostryukova, V.G. Zgoda, I.P. Baskova, V.M. Trukhan, M.
214 (1) (1993) 295–304. S. Gelfand, V.M. Govorun, H.B. Schiöth, V.N. Lazarev, Draft genome sequences of
[46] V. Steiner, R. Knecht, K.O. Börnsen, E. Gassmann, S.R. Stone, F. Raschdorf, J. Hirudo medicinalis and salivary transcriptome of three closely related medicinal
M. Schlaeppi, R. Maschler, Primary structure and function of novel O- leeches, Bmc Genom. 21 (1) (2020) 331.
glycosylated hirudins from the leech Hirudinaria manillensis, Biochemistry 31 (8) [72] I.P. Baskova, E.S. Kostrjukova, M.A. Vlasova, O.V. Kharitonova, S.A. Levitskiy, L.
(1992) 2294–2298. L. Zavalova, S.A. Moshkovskii, V.N. Lazarev, Proteins and peptides of the salivary

7
M. Alaama et al. Pharmacological Research - Modern Chinese Medicine 10 (2024) 100355

gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. [89] Z.Y. Sun, F.J. Wang, H. Guo, L. Chen, L.J. Chai, R.L. Li, L.M. Hu, H. Wang, S.
orientalis, Biochemistry (Moscow) 73 (2008) 315–320. X. Wang, Shuxuetong injection protects cerebral microvascular endothelial cells
[73] M. Courtney, G. Loison, Y. Lemoine, N. Riehl-Bellon, E. Degryse, S.W. Brown, J. against oxygen-glucose deprivation reperfusion, Neural. Regen. Res. 14 (5)
P. Cazenave, G. Defreyn, D. Delebassee, A. Bernat, Production and evaluation of (2019) 783–793.
recombinant hirudin, Semin. Thromb. Hemost. 15 (1989) 288–292. [90] Z. Yu, X. Liu, Y. Xing, X. Wang, X. Wang, Y. Huang, L. Han, G. Pan, Identification
[74] T.E. Warkentin, Management of heparin-induced thrombocytopenia: a critical and quantification of characteristic peptides (Oligopeptides) in Shuxuetong (SXT)
comparison of lepirudin and argatroban, Thromb. Res. 110 (2) (2003) 73–82. injection by LC–MS/MS, Chromatographia 85 (12) (2022) 1029–1039.
[75] T.J. Graetz, B.R. Tellor, J.R. Smith, M.S. Avidan, Desirudin: a review of the [91] L.-Y. Jia, A.-H. Yao, F. Kuang, Y.-K. Zhang, X.-F. Shen, G. Ju, Beneficial effect of
pharmacology and clinical application for the prevention of deep vein the traditional Chinese drug Shu-Xue-Tong on recovery of spinal cord injury in
thrombosis, Expert Rev. Cardiovasc. Ther. 9 (9) (2011) 1101–1109. the rat, Evid. Based Complem. Alternat. Med. 2011 (2011) 862197.
[76] T.E. Warkentin, A. Greinacher, A. Koster, Bivalirudin, Thromb. Haemost. 99 (11) [92] W. Zhang, Effects of Shuxuetong injection applied in acute ischemic stroke,
(2008) 830–839. J. Acute Disease 5 (6) (2016) 507–511.
[77] M. Deganc, F. Zdravic, Venous congestion of flaps treated by application of [93] R. Galun, S.H. Kindler, Chemical specificity of the feeding response in Hirudo
leeches, Br. J. Plast. Surg. 13 (1960) 187–192. medicinalis (L.), Comp. Biochem. Physiol. 17 (1) (1966) 69–74.
[78] G.J. Gasic, E.D. Viner, A.Z. Budzynski, G.P. Gasic, Inhibition of lung tumor [94] B.R. West, L.S. Nichter, D.E. Halpern, Emergent reuse leech therapy: a better
colonization by leech salivary gland extracts from Haementeria ghilianii, Cancer method, Plast. Reconstr. Surg. 93 (5) (1994) 1095–1098.
Res 43 (4) (1983) 1633–1636. [95] A. Shakouri, N. Adljouy, J. Abdolalizadeh, Anti-cancer activity of liposomal
[79] J. Zhou, Z. Song, M. Han, B. Yu, G. Lv, N. Han, Z. Liu, J. Yin, Evaluation of the medical leech saliva extract (LSE), in: Proceedings of the 3rd World Congress on
antithrombotic activity of Zhi-Xiong Capsules, a traditional Chinese medicinal Recent Advances in Nanotechnology (RAN’18), April, Budapest, Hungary, 2018.
formula, via the pathway of anti-coagulation, anti-platelet activation and anti- [96] M. Alaama, A.M. Abdualkader, A.M. Ghawi, A. Merzouk, R.S. Khalid, A.B.
fibrinolysis, Biomed. Pharmacother. 97 (2018) 1622–1631. M. Helaluddin, Assessment of trace heavy metals contamination in the tissues and
[80] M. Zhou, P. Ren, Y. Zhang, S. Li, M. Li, P. Li, J. Shang, W. Liu, H. Liu, Shen-Yuan- saliva of the medicinal Leech Hirudinaria manillensis, Turk. J. Fisher. Aquatic Sci
Dan capsule attenuates atherosclerosis and foam cell formation by enhancing 21 (5) (2021) 225–231.
autophagy and inhibiting the PI3K/Akt/mTORC1 signaling pathway, Front. [97] A.A. Mohammed, G.A. Mohammad, A. Mohamed, A. Mohamed, M. Ahmed, In
Pharmacol. 10 (2019) 603. vivo anti-hyperglycemic activity of saliva extract from the tropical leech
[81] X. Li, Y. Zhang, H.-X. Liu, J.-J. Shang, Q. Zhou, A.-Y. Li, X.-L. Lai, W.-L. Xing, S.- Hirudinaria manillensis, Chin. J. Nat. Med 11 (5) (2013) 488–493.
H. Jia, A randomized, placebo-controlled, double-blind trial to evaluate efficacy [98] B. Malik, D.A. Astuti, D.J.F. Arief, M. Rahminiwati, A Study On Antioxidative and
and safety of Shen-Yuan-Dan capsules, a traditional Chinese medicine, for Antimicrobial Activities of Saliva Extract of Indonesian local Leeches, IOP
treatment of peri-procedure myocardial injury following percutaneous coronary Publishing, 2019 012061.
intervention, Complement. Ther. Med. 69 (2022) 102841. [99] A. Merzouk, A.M. Ghawi, A.M. Abdualkader, A. Abdullahi, M. Alaama,
[82] J. Han, H. Tan, Y. Duan, Y. Chen, Y. Zhu, B. Zhao, Y. Wang, X. Yang, The Anticancer effects of medical Malaysian leech saliva extract (LSE), Pharm. Anal.
cardioprotective properties and the involved mechanisms of NaoXinTong Acta S 15 (2012) 2–6.
Capsule, Pharmacol. Res. 141 (2019) 409–417. [100] A. Shakouri, N. Adljouy, S. Balkani, M. Mohamadi, H. Hamishehkar,
[83] J. Lv, S. Liu, S. Guo, J. Gao, Q. Song, X. Cui, Tongxinluo capsule as J. Abdolalizadeh, S.Kazem Shakouri, Effectiveness of topical gel of medical leech
supplementation and cardiovascular endpoint events in patients with coronary (Hirudo medicinalis) saliva extract on patients with knee osteoarthritis: a
heart disease: a systematic review and meta-analysis of randomized, double- randomized clinical trial, Complement. Ther. Clin. Pract. 31 (2018) 352–359.
blind, placebo-controlled trials, J. Ethnopharmacol. 289 (2022) 115033. [101] A.E. Ammar, M.H. Hassona, G.R. Meckling, L.G. Chan, M.Y. Chin,
[84] W.-Y.-N. Tang, J.-T. Liang, J. Wu, L. Liu, M.-Z. Lu, X.-Y. He, L.-J. Wu, H.-Y. Jiang, A. Abdualkader, M. Alaama, A. Merzouk, A. Helaluddin, A. Ghawi, O. Kucuk, E.
F. Wang, X. Meng, S.-P. Li, Efficacy and safety of Dahuang Zhechong pill in S. Guns, Assessment of the antitumor activity of leech (huridinaria manillensis)
silicosis: a randomized controlled trial, Evid. Based Complement. Alternat. Med. saliva extract in prostate cancer, Cancer Res (2015) 75.
2021 (2021) 4354054. [102] A. Ammar, E. Guns, O. Kucuk, A. Abdualkader, M. Alaama, A.H. Uddin, A. Ghawi,
[85] F. Wei, Y. Lang, D. Gong, Y. Fan, Effect of Dahuang zhechong formula on liver M.J.C.R. Hassona, Mechanism of anticancer activity of BPS-001 (lyophilized leech
fibrosis in patients with chronic hepatitis B: a meta-analysis, Complement. Ther. saliva extract), 77(13_Supplement) (2017) 107–107.
Med. 23 (1) (2015) 129–138. [103] I.P. Baskova, O.M. Aguejouf, F. Azougagh-Oualane, L.L. Zavalova, A.V. Basanova,
[86] C. Chen, X. Yao, Y. Xu, Q. Zhang, H. Wang, L. Zhao, G. Wen, Y. Liu, L. Jing, C. Doutremepuich, Arterial antithrombotic effect of piyavit, the novel
X. Sun, Dahuang Zhechong Pill suppresses colorectal cancer liver metastasis via pharmacological preparation from the medicinal leech, and of its components,
ameliorating exosomal CCL2 primed pre-metastatic niche, J. Ethnopharmacol. prostanoids and enzyme destabilase, Thromb. Res. 77 (6) (1995) 483–492.
238 (2019) 111878. [104] J. Qiu, W. Lingna, H. Jinghong, Z. Yongqing, Oral administration of leeches
[87] R. Chen, G. Gai, W. Zhang, Y. Zhuang, Y. Xie, Application of propensity score (Shuizhi): a review of the mechanisms of action on antiplatelet aggregation,
method to analyze the effect of Maixuekang capsule on the treatment outcome of J. Ethnopharmacol. 232 (2019) 103–109.
coronary heart disease, J. Chinese Phy. (2020) 365–368. [105] Y. Su, A. Jing, Y. Wang, Clinical research of Huangqi Injection combined with
[88] X. Jin, G. Shen, F. Gao, X. Zheng, X. Xu, F. Shen, G. Li, J. Gong, L. Wen, X. Yang, Shuxuetong Injection therapy on aged acute cerebral infarction, Chinese
X. Bie, Traditional Chinese drug ShuXueTong facilitates angiogenesis during Traditional Patent Medicine (1992).
wound healing following traumatic brain injury, J. Ethnopharmacol. 117 (3) [106] L. Amani, N. Motamed, M. Mirabzadeh Ardakani, M. Dehghan Shasaltaneh, M.
(2008) 473–477. Malek, F. Shamsa, E. Fatemi, M. Amin, Semi-solid product of medicinal Leech
enhances wound healing in rats, 16(4) (2021) e113910.