Leech Saliva Extract Research Articles

The Investigation of Effects of Medicinal Leech Saliva Extract on the Breast Fibroblast Cell Line In Vitro: An Experimental Study

The Investigation of Effects of Medicinal Leech Saliva Extract on the Breast Fibroblast Cell Line In Vitro: An Experimental Study

Nanoencapsulation of Hirudo medicinalis proteins in liposomes as a nanocarrier for inhibiting angiogenesis through targeting VEGFA in the Breast cancer cell line (MCF-7).

Introduction: Breast cancer is the most serious cause of women’s death throughout the world. Using nanocarrier vehicles to the exact site of cancer upgrades the therapeutic efficiency of the drugs. Capsulation of active proteins in the vesicular liposomes’ hydrophilic core is essential to develop a therapeutic protein carrier system. We aimed to encapsulate the medicinal leech saliva extract (LSE) and assess the inhibition of angiogenesis of breast cancer cells by targeting vascular endothelial growth factor A (VEGFA). Methods: In this research, enhanced formulation of liposomal protein was determined by zeta potential analysis, droplet size, drug release assay, and transmission electron microscopy (TEM). Furthermore, a cytotoxicity assay of liposomal LSE was performed to determine the cytotoxic activity of components. For assessing the expression of VEGFA, P53, and hypoxia-inducible factor subunit alpha (HIF1a) genes, Real-Time PCR was applied. Results: Nano liposome was chosen as an enhanced formulation due to its much smaller size (46.23 nm). Liposomal LSE had more practical actions on the MCF-7 cells. As noticed by DAPI staining, apoptosis was extensively greater in treated MCF-7 cells. Wound healing assay demonstrated that MCF-7 cells could not sustain growth at the presence of liposomal LSE and expression of the VEGFA gene was declined in treated cells. Downregulation of VEGFA was evaluated with western blotting technique. Conclusion: It can be concluded that our investigation of the tests confirmed the fact that nano liposomal LSE is a novel promising formulation for anticancer drugs and can significantly improve the penetration of protein drugs to cancer cells.

A study on antioxidative and antimicrobial activities of saliva extract of Indonesian local leeches

Antioxidative and antimicrobial activities of saliva extract of Indonesian leeches were assessed. Leeches were obtained from Situgede Bogor, Sawangan Depok, and Sijunjung Regency, West Sumatra. A completely randomized design with three treatments and five replicates was used. Treatments consisted of three places of leech origins. Measurements were taken on the amount, total protein content, IC50 value, zone of inhibition to bacterial growth, and minimum inhibitory concentration of crude leech saliva extract (CLSE). Results showed that the amount of CLSE obtained was 3.696-4.633g/g BW and total protein content of CLSE was 48.303-64.742 μg/mg. The IC50 value was 47-108 μg/mg while that of ascorbic acid was 3.312 μg/mg. Antimicrobial activity was found to the growth of S. aureus but not to the growth of E. coli with a minimum inhibitory concentration at 2.472-4.528 μg/ml.

Effectiveness of topical gel of medical leech (Hirudo medicinalis) saliva extract on patients with knee osteoarthritis: A randomized clinical trial

Leech saliva extract (LSE) in the liposome-based gel was used as a supplementary treatment to relief the signs and symptoms of osteoarthritis (OA).The saliva of medical leech was extracted and nano liposomes were used to formulate the supplement to enhance skin absorption. A clinical trial was designed to evaluate the therapeutic effects of LSE liposomal gel. Lenquesne and VAS questionnaires were used as indexes of this supplement therapy efficacy for 30 days.Questionnaires analysis showed that after one-month administration of LSE liposomal gel, patients' pain was relieved approximately up to 50%; also, due to reduction in joint inflammation and stiffness, the range of motion was increased and the patients' quality of life was enhanced (p < 0.001).LSE nano scaled liposomal gel as an innovative supplement therapy in OA patients, makes desirable therapeutic approach, which seems to make a significant impact on patient's quality of life and self-care capability.

Abstract 107: Mechanism of anticancer activity of BPS-001 (lyophilized leech saliva extract)

Abstract Recent studies, investigating the composition and therapeutic potential of leech saliva have identified many peptides and proteins with multiple therapeutic properties including anti-thrombotic, antimicrobial and anti-metastatic. In vitro anti-cancer effects were shown in breast, prostate and lung cells lines. In vivo anticancer activity of the extract was shown in multiple breast and prostate cancer xenografts studied by our lab. In our previous studies, BPS-001 was non-toxic and demonstrated various activities including pro-apoptotic properties, inhibition of cellular adhesion, angiogenesis and downregulation of AR expression. To investigate the affected pathways, mechanistic studies were carried out using in vivo and in vitro treated samples. Protein expression in samples was determined by Western blotting of in vitro and in vivo samples. AR expression was measured using an eGFP reporter assay in LNCaP cells, with PSA also measured in the media using cobas e 411 analyzer. BPS-001 inhibited Proteinase Activated Receptor 1 (PAR-1) which is responsible for hormone like cellular signals in different cancer cells including those of the breast and prostate. Moreover, in LNCaP cells AR activation and PSA expression were significantly inhibited after treatment with BPS-001. Inhibition of PI3K and PLCG2 were also observed. It has been found that BPS-001 modulates a plethora of pathways which makes it a very promising candidate for treatment of cancer with an important advantage of low toxicity and potential resistance. Citation Format: Amr Ammar, Emma Guns, Omer Kucuk, Abdualrahman Abdualkader, Mohamed Alaama, A.B.M. Helal Uddin, Abbas Ghawi, Mohamed Hassona. Mechanism of anticancer activity of BPS-001 (lyophilized leech saliva extract) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 107. doi:10.1158/1538-7445.AM2017-107

Evaluation of the Anti-Glucotoxic Effects of Leech Saliva Extract (LSE) on Mouse and Human Pancreatic Islets

Evaluation of the Anti-Glucotoxic Effects of Leech Saliva Extract (LSE) on Mouse and Human Pancreatic Islets

In vivo assessment of the therapeutic effects of lyophilized leech saliva extract from (Huridinaria manillensis) on LNCaP tumor xenograft model in nude mice.

271 Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. A large number of peptides and proteins have been identified and characterized in leech saliva extract (LSE), including anti-thrombotic agents, cancer metastasis inhibitors and anti-microbials Currently, leech therapy is established as an important tool in microsurgery and reconstructive operations having demonstrated superior clinical outcomes for the optimal salvage of grafted tissues. Methods: In the current study, we have determined the in vivo efficacy of LSE from (Huridinaria manillensis) on castration resistant LNCaP xenograft mouse model. Mice were divided into three groups of six, mice were subcutaneously injected with either LSE (5 mg/kg), docetaxel (10 mg/kg), or vehicle once a week. PSA and tumor volume were measured weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and immunohistochemical (IHC) analysis. Results: There was a significant decrease in the tumor volume and PSA with either docetaxel (10mg/kg) or LSE (5 mg/kg) treated groups compared to the control. While there was no significant difference between the anti-tumor activity of docetaxel (10mg/kg) and LSE (5 mg/kg). IHC showed significant increase in caspase-3 and significant decrease in Ki-67 and PCNA expression in the LSE treated mice compared to the control group. Interestingly, transcriptome analysis of tumor samples showed that LSE modulated cytokine production, monocyte adhesion, steroidogenesis, and P38 MAPK signaling pathways. Conclusions: LSE has significant anti-tumor activity in LNCaP tumor xenograft models with no apparent side effects. This can be attributed, at least partly, to its inhibition of cellular proliferation, induction of apoptosis, modulation of immunity and steroidogenesis.

In vivo anti-hyperglycemic activity of saliva extract from the tropical leech Hirudinaria manillensis

AbstractThe anticoagulant effect of leech saliva was traditionally employed in the treatment of diabetes mellitus complications such as peripheral vascular complications. This study was carried out to examine the effect of leech saliva extract (LSE) on blood glucose levels in alloxan-induced diabetic rats. First, LSE was collected from leeches which were fed on a phagostimulatory solution. Second, total protein concentration was estimated using the Bradford assay. Third, diabetic rats were injected subcutaneously (sc) with LSE at doses of 500 and 1 000 μg·kg−1 body weight (bw). Other diabetic rats were injected sc with insulin at doses of 10 and 20 U·kg−1 bw. Another group was injected simultaneously with LSE (250 μg·kg−1 bw) and insulin (10 U·kg−1 bw). Fasting blood glucose (FBG) concentrations were monitored during a study period of eight hours at regular intervals. Findings showed that both doses of LSE resulted in a significant and gradual decrease in FBG starting from 10%-18% downfall after two hours of injection reaching the maximal reduction activity of 58% after eight hours. Remarkably, LSE was sufficient to bring the rats to a near norm-glycemic state. The high dose of insulin induced a severe hypoglycemic condition after 2-4 h of injection. The lower dose was able to decline FBG for 2-6 h in rats which became diabetic again after 8 h. On the other hand, the concurrent injection of low doses of LSE and insulin produced a hypoglycemic effect with all rats showing normal FBG levels. Taken together, these findings indicated that the subcutaneous injection of LSE of the medicinal Malaysian leech was able to provide better glycemic control compared with insulin. Moreover, the synergism between LSE and insulin suggests that LSE could be utilized as an adjuvant medication in order to reduce insulin dosage or to achieve better control of blood glucose.

Anticancer Effects of Medical Malaysian Leech Saliva Extract (LSE)

Leech saliva contains biologically active compounds that are mainly proteins & peptides. Small cell lung cancer (SCLC) is a form of cell lung carcinoma. In this study a modified and smooth extraction method of saliva was used without leech scarification. Trying to find out the biological activity of medical Malaysian Leech Saliva Extract as cytotoxic in vitro, the SW 1271 cell line was grown and maintained in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum at 37 ° C in normal atmospheric air. Serial dilutions of LSE were added to the cell line SW 1271 media for testing the cytotoxic activities.Result revealed that the LSE has a cytotoxic activity against small cell lung cancer(SW 1271 cell line) with IC 50 of 119.844 µg/ml compared with IC 50 values of two reference standard drugs Irinotecan (5.81 µg/ml) and Carboplatin(18.754 µg/ml). In a combination regimen, LSE reduced the IC 50 of Carboplatin & Irinotecan by 65% & 11.5% respectively. Carboplatin reduced the IC 50 of LSE by 4.6%, while Irinotecan reduced it by 57%. This results provides a promising novel agent for treatment of small cell lung cancer (SCLC) at least in vitro, more researches are needed.

Free Radical Scavenging Activity of the Medicinal Malaysian Leech Saliva Extract, Hirudinaria manillensis

Antioxidants from natural sources have been arisen as prophylactic and therapeutical agents in many life-threatening disorders such as cancer and cardiovascular diseases. Leech therapy has been used since the extreme old ages in a variety of abnormalities. The current study was executed to examine the antioxidant activity of the salivary gland secretion of the medicinal Malaysian leech using DPPH free radical scavenging activity method. Leech saliva extract (LSE) was collected from starved leeches after feeding them on the phagostimulatory solution of 0.001M arginine in 0.15 M sodium chloride. Total protein concentration was 78.753 ± 2.406 μg/ml. A series of different dilutions of LSE were mixed with DPPH in a methanolic medium and the changes in absorbance were measured at 516nm. Results showed that LSE expressed a free radical scavenging activity with IC 50 of 7.282 μg/ml compared with 5.803 μg/ml of L-ascorbic acid as a positive control. Therefore, this study revealed that the proteomic contents of LSE are promising natural antioxidants.

Season Variation and Starvation Period Influence on the Antithrombotic Activity of Leech Saliva Extract From the Medicinal Malaysian Leech, Hirudinaria Manillensis

Leech therapy has been practiced for a wide range of therapeutical purposes since the extreme old ages. Nowadays, leech application in plastic and microsurgery has been considered as a promising tool. In Malaysia, traditional physicians have used the medicinal leeches as an effective remedy for bloodletting and many body disorders. Leech saliva extract (LSE) was collected after feeding leeches on the phagostimulatory solution through parafilm membrane. The total protein concentration was estimated using Bradford assay. The antithrombin activity was evaluated using the amidolytic assay of the synthetic substrate S-2238 and thrombin time assay in vitro. It was found that LSE could inhibit thrombin-medicated hydrolysis of the substrate. The extract effectively prolonged thrombin time of the citrated plasma in a linear dose-dependent manner. It was found that the extract collected during the dry season was more biologically active than those collected during the rainy season. Likewise, results revealed that the longer the starvation period, the lower the antithrombin activity. For effective utilization of leech therapy or leech products, we recommend to be used during the dry season and after a starvation period not more than 16 weeks.

Some Biological Activities of Malaysian Leech Saliva Extract

Leeches were fed on the phagostimulatory solution through parafilm membrane. The satiated leeches were forced to regurgitate the solution by soaking them in an ice-container. The anticoagulant activity was ascertained using thrombin time assay (TT). The result revealed that the saliva concentration which increases TT by 100% (IC100) is 43.205µg/ml plasma. The antimicrobial activity of the saliva was tested against several bacterial spp. (E.coli, P.aeruginosa, B.cereus, Sal.typhi and S.aureus) and fungi spp. (C.albicans and C.neoformans). It was found that saliva has an inhibition activity against Sal.typhi (minimal inhibitory concentration MIC 78.253µg/ml), S.aureus (MIC 78.253µg/ml) and E.coli (MIC 121.256µg/ml).ABSTRAK: Pacat-pacat diberi makan larutan phagostimulatory menerusi membran parafilem. Pacat-pacat yang kekenyangan itu dipaksa memuntahkan larutan tersebut dengan direndam di dalam bekas berisi ais. Aktiviti antigumpal ditentukan menggunakan cerakin masa trombin (TT). Keputusan menunjukkan kepekatan air liur pacat menyebabkan pertambahan TT sebanyak 100% (IC100) iaitu 43.205µg/ml plasma. Aktiviti antimikrob air liur telah diuji dengan pelbagai jenis bakteria (E.coli, P.aeruginosa, B.cereus, Sal.typhi dan S.aureus) dan pelbagai jenis kulat (C.albicans and C.neoformans). Didapati air liur menghasilkan aktiviti perencatan terhadap Sal.typhi (kepekatan perencat minima (Minimal inhibitory concentration - MIC) 78.253µg/ml), S.aureus (MIC 78.253µg/ml) dan E.coli (MIC 121.256µg/ml).