Characterization and optimization of lyophilization and storage conditions of Leech saliva extract from the tropical leech Hirudinaria manillensis

Abdualrahman Mohammed Abdualkader*1, Abbas Mohammad Ghawi2, Mohamed Alaama1, Mohamed Awang1 and Ahmed Merzouk3

1) Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

2) Basic Medical Science Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

3) Biopep Solutions Inc., Vancouver, BC Canada

*Corresponding author: e-mail: armak1983@gmail.com

[Abstract]

The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract (LSE) was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized (solid state) and non-lyophilized (liquid state) were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20°C as the optimum temperature. Samples stored at ambient temperature and +4°C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light.

Keywords: Antithrombin, leech, lyophilization, protein, storage conditions.

JOURNAL

http://www.pjps.pk/wp-content/uploads/pdfs/CD-PJPS-26-3-13/Paper-14.pdf

Published 22-04-2015

Anticancer Effects of Medical Malaysian Leech Saliva Extract (LSE)

Ahmed Merzouk1*, Abbas Mohammad Ghawi2, Abdualrahman M. Abdualkader3, Abubakar Danjuma Abdullahi2 and Mohamed Alaama3

1) Biopep Solutions Inc.,Vancouver,BC Canada

2) Basic Medical Science Department,Faculty of Pharmacy,International Islamic University Malaysia,Kuantan, Pahang,Malaysia

3) Pharmaceutical Chemistry Department,Faculty of Pharmacy,International Islamic University Malaysia,Kuantan,Pahang,Malaysia

*Corresponding Author :Ahmed Merzouk (E-mail: ahmed.merzouk@gmail.com)

[Abstract]

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 IC50 of 119.844 µg/ml compared with IC50 values of two reference standard drugs Irinotecan (5.81 µg/ml) and Carboplatin(18.754 µg/ml). In a combination regimen, LSE reduced the IC50 of Carboplatin & Irinotecan by 65% & 11.5% respectively. Carboplatin reduced the IC50 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.

Keywords Cancer; Carboplatin; Cytotoxic; Irinotecan; Leech

JOURNAL

http://www.omicsonline.org/anticancer-effects-of-medical-malaysian-leech-saliva-extract-lse-2153-2435.S15-001.php?aid=5370

Published 22-04-2015

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

Abdualkader Abdualrahman Mohammed1*, Ghawi Abbas Mohammad2, Alaama Mohamed1, Awang Mohamed1 , Merzouk Ahmed3

1) Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia

2) Basic Medical Science Department, Faculty of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia

3) Biopep Solutions Inc., 235-11590 Cambie Road, Richmond, BC V6X 3Z5, Canada

[Research funding] This project was supported by the grant (No. EDW B 10-0393) from Research-Management Centre, International Islamic University Malaysia.

[*Corresponding author] Abdualkader, Abdualrahman Mohammed: E-mail: armak1983@gmail.com

[ABSTRACT]

The 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.

[KEY WORDS] Alloxan; Diabetes mellitus; Insulin; Leech; Hirudinaria manillensis

JOURNAL

http://www.biopeps.com/wp-content/uploads/2014/03/In-vivo-anti-hyperglycemic-activity-of-saliva-extract-from-the-tropical-leech-Hirudinaria-manillensis.pdf

Published 22-04-2015

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

Abbas Mohammad Ghawi1 *, Abdualrahman M. Abdualkader2 , Ahmed Merzouk3 and Mohamed Alaama2

1) Basic Medical Science Department, Faculty of Pharmacy, International Islamic University, Malaysia, Kuantan, Pahang, Malaysia

2) Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

3) Biopep Solutions Inc., Vancouver, BC, Canada

*Corresponding author: Abbas Mohammad Ghawi, E-mail: azawiabbas@yahoo.com

[ABSTRACT]

Antioxidants from natural sources have been arisen as prophylactic and therapeutical agents in many lifethreatening 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 IC50 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.

Keywords: Antioxidants; DPPH; Free radical; leech; leech saliva

JOURNAL

http://www.omicsonline.org/free-radical-scavenging-activity-of-the-medicinal-malaysian-leech-saliva-extract-hirudinaria-manillensis-jbb.S14-001.pdf

Published 11-05-2015

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

Abbas Mohammad Ghawi1*, Abdualrahman M Abdualkader2, Ahmed Merzouk3 and Mohamed Alaama2

1)Basic Medical Science Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

2)Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

3) BIOPEP SOLUTIONS INC., Vancouver, BC Canada

[ABSTRACT]

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.

Keywords: Amidolytic; Antithrombin; leeches; leech saliva; Thrombin time.

[JOURNAL]

https://www.researchgate.net/file.PostFileLoader.html?id=51555ca5d2fd64ec6b000001&assetKey=AS:272106950987800@1441886760963

Published 31-10-2016

Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer.

Mohamed Hessein, Amr Ammar, Mei Chin, Abdulrahman Abdualkader, Mohamed Alaama, Ahmed Merzouk, Abul Bashar Helaluddin, Abbas Ghawi, Omer Kucuk and Emma Guns

Vancouver Prostate Centre, UBC, Vancouver, BC, Canada; International Islamic University Malaysia, Kuantan, Pahang, Malaysia; The Winship Cancer Institute of Emory University, Atlanta, GA

[ABSTRACT]

Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. Leech saliva contains a large number of peptides and proteins, which have anti-thrombotic, antimicrobial, antitumor and anti-metastatic activities. Currently, leech therapy has an established role as an important tool in microsurgery, reconstructive surgery and salvage of grafted tissues. Methods: LSE IC50 was determined in-vitro in five prostate cancer cell lines using MTS cell viability assay. In-vivoefficacy of LSE was determined in LNCaP and 22RV-1 in nude mice xenograft models. Castrated tumor bearing mice were divided into four groups of 6-8 each. Mice were subcutaneously injected with either LSE (5 mg/kg) once a week, LSE (5 mg/kg) twice a week, docetaxel (10 mg/kg) or vehicle twice a week for four weeks. PSA, tumor volume, and weight were measured periodically. Results: In-vitro, LSE induced cell death in a panel of five prostate cancer cell lines. In vivo studies showed that LSE once and twice weekly regimens both caused significant decrease in PSA and tumor volume compared to control with no significant difference from docetaxel treated group. Interestingly, once weekly treatment was associated with significant weight gain (due to good dietary intake) at several time points. Immunohistochemical staining (IHC) showed significant decrease in P21and Ki-67 expression in the LSE treated mice compared to the control group in the LNCaP model. Active caspase-3 was increased at once and twice weekly doses in both models confirming the pro-apoptotic properties of LSE.. Transcriptome analysis of tumor samples showed that LSE had significant immunomodulatory, anti-inflammatory, along with significant effects on cell-cell adhesion, induction of glutathione transferase, inhibition of certain growth factors, and modulating several transcription factors. Conclusions: LSE has significant in vitro and in vivo anti-tumor activity with no apparent side effects. This can be attributed, at least partly, to its ability to inhibit cellular proliferation, induce apoptosis and arrest cell cycle through its modulation of immunity, cell-cell adhesion, and inflammation.

[JOURNAL]

http://hwmaint.meeting.ascopubs.org/cgi/content/abstract/33/15_suppl/e16120

[POSTER]

http://www.biopeps.com/biopep-wp/wp-content/uploads/ACCR-Poster.pdf

Published 31-10-2016

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

Amr Ammar, Mohamed Hessein, Emma Guns, Mei Chin, Abul Bashar Helaluddin, Abdulrahman Abdualkader, Mohamed Alaama, Ahmed Merzouk, Abbas Ghawi and Omer Kucuk

Vancouver Prostate Centre, UBC, Vancouver, BC, Canada; International Islamic University Malaysia, Kuantan, Pahang, Malaysia; The Winship Cancer Institute of Emory University, Atlanta, GA

[ABSTRACT]

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.

[JOURNAL]

http://hwmaint.meeting.ascopubs.org/cgi/content/abstract/33/7_suppl/271

Published 31-10-2016

Starvation Time and Successive Collection Effects on Leeches Saliva Collection Quantity and Proteins Quality and Quantity in Wet Season

Mohamed Alaama1, ABM. Helaluddin*1 & Abdualrahman Mohammed Abdualkader1, Abbas Mohammad2, Ahmed Merzouk3 and Mohamed Bin Awang4

1)Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy International Islamic University Malaysia (IIUM) 25200 Kuantan, Pahang Malaysia

2) Department of Basic Medical Science, Kulliyyah of Pharmacy International Islamic University Malaysia (IIUM) 25200 Kuantan, Pahang Malaysia

3) Biopep Solutions Inc. Vancouver BC Canada

4) Faculty of Pharmacy Cyberjaya University College of Medical Sciences (CUCMS) 63000 Cyberjaya, Selangor Malaysia

*Corresponding author; email: mohdhelal@hotmail.com

[ABSTRACT]

The salivary gland secretion of the haematophagous animals, leeches, has attracted the attention of therapists since the extreme old ages due to its wide range of medical properties. Thus, many researches have been done to develop and optimize new methods to collect leech saliva with high quality and quantity. In the present study, we aimed to evaluate the effects of starvation period and repeated collection on the quality and quantity of leech saliva extract LSE and its contents of proteins during the rainy season. Protein recovery in the LSE was also studied after first collection. It was found that leeches are able to produce protein-containing saliva whenever fed during the whole study period of 18 weeks with varied protein concentrations. The results showed that the highest protein concentrations (105-91 ?g/mL) were produced after 12-15 weeks of starvation. The results of successive collection showed that leeches are able to produce proteins and peptides whenever they suck the solution after first collection with some varies in the concentrations. The concentrations varied between 0 and 72% of the initial concentration. Gel electrophoresis results showed absence for some bands when the concentrations are too low. Also the results showed that leeches are able to recover about 42% of their initial proteins concentration within four weeks of starvation after first feeding. The gel electrophoresis results showed the closeness between the first and second collections. To conclude, all test factors (starvation period, successive collection and recovery test) were shown to have an important impact on protein concentration of leech saliva and therefore its medicinal affectivity. The mentioned results are reported for the first time and they open the gate for further studies.

Keywords: Leeches; saliva; starvation; time; quality; quantity; wet season

[JOURNAL]

http://www.ukm.my/jsm/pdf_files/SM-PDF-43-11-2014/07 Mohamed Alaama.pdf

Published 31-10-2016

Isolation and Characterization of Antithrombin Peptides from the Saliva of Malaysian Leeches

Mohamed Alaama1*, ABM Helal Uddin1*, Abbas Mohammad Ghawi2 , Ahmed Merzouk3, Abdualrahman Mohammed Abdualkader¹, and Mohamed bin Awang4

¹)Department of Pharmaceutical Chemistry, 2)Department of Basic Medical Science, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Malaysia,

3) Biopep Solutions Inc, Vancouver, BC, Canada,

4) Faculty of Pharmacy, CUCMS, Cyberjaya, Selangor.

*For correspondence: Email: hass83pharm@yahoo.com, abmhelal@iium.edu.my

[ABSTRACT]

Purpose: To isolate and characterize the antithrombin compounds of Malaysian leeches’ saliva collection (LSC) for use as anticoagulant proteins and peptides. Method: Reversed phase - high performance liquid chromatography (RP-HPLC) was used to isolate all proteins from LSC. All isolated proteins were tested for anticoagulant activity by applying two tests, thrombin time test and the inhibition of the amidolytic activity of thrombin on the chromogenic substrate (S-2238). The molecular weights of isolated active compounds were identified using tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). Results: More than 40 proteins were isolated from LSC using RP-HPLC. Two compounds (protein 1 and protein 2) were found to be active as they increased thrombin time by 30.26 and 36.75%, respectively and inhibited the amidolytic activity of thrombin. as evaluated by measuring the conversion of the chromogenic substrate (S-2238). Decrease in the conversion of the substrate (S-2238) by 31.10 and 41.61%, respectively was observed. The molecular weights of the proteins (6.289 and 14.255 kDa, respectively) had no similarity with other leech species. Conclusion: Two antithrombin proteins with anticoagulant properties were isolated from the saliva of Malaysian leeches using one-step isolation procedure. The proteins did not show any similarity to previous isolated proteins from leech.

Keywords: Leech, Saliva, Antithrombin, Anticoagulant, Peptides, Molecular weight

[JOURNAL]

http://www.ajol.info/index.php/tjpr/article/view/103808

Published 31-10-2016

SOME BIOLOGICAL ACTIVITIES OF MALAYSIAN LEECH SALIVA EXTRACT

ABDUALRAHMAN M.ABDUALKADER*1, AHMED MERZOUK1, ABBAS MOHAMMED GHAWI2 AND MOHAMMED ALAAMA1

1) Pharmaceutical Chemistry Department,

2) Basic Medical Science Department, Kulliyyah of Pharmacy, International Islamic University Malaysia , Jalan Istana 25200, Kuantan, Pahang, Malaysia.

*Corresponding author: armak1983@gmail.com

[ABSTRACT]

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 43.205 ?g/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 78.253 ?g/ml), S.aureus (MIC 78.253 ?g/ml) and E .coli (MIC 121.256 ?g/ml).

KEYWORDS: leech; anticoagulant; thrombin time; antibacterial activity

[JOURNAL]

http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/156/196

Published 01-11-2016