COLLEGE OF PHARMACY
2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019 November 13-14, 2019
Conference Proceeding
Organized by: College of Pharmacy Hawler Medical University
Conference theme: “Medicines From Discovery to Optimal Use”
2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Table of Contents
Page Content
I Welcome Note
II Conference Committees
III - VII Scientific Program
VIII Poster Presentations
IX Scientific Articles
1-10 Heat shock protein 90 regulates neutrophil Macexpression and extravascular recruitment in acute pancreatitis related with lung injury
11-21 Changes of Endothelin-1 Receptors Activity in Induced-Hyperthyroid Isolated Rat Aorta
22-32 Formulation and Evaluation of Valsartan Oral Granule
33-41 Heavy Metals Estimation of Canned Food in Erbil City Markets
42-50 Serum vitamin D level discriminates between the primary and the associated clinical manifestations of fibromyalgia
51-59 Estimation of serum 25-hydroxy vitamin D and zinc levels in patients with acne vulgaris and their association with disease severity
60-65 Metastatic Breast Cancer to Stomach Mimicking Primary Gastric cancer
66-72 The Fate of Expired Medications in Kurdistan region of Iraq
73-80 Antimicrobial activity extracts from three species of fresh water algae and used against some pathogenic microorganisms and analysis of minerals in algal species
81-87 Rac 1 project against diabetes mellitus via attenuation of platelet chemokines
88-97 In silico investigation to select the most fitting mTOR inhibitors using AutoDock Vina Oral Granule
98-112 Simultaneous Spectrophotometric Determination of Resorcinol, Caffeic Acid and Ascorbic Acid Using Artificial Neural Network Technique
113-116 Pro-inflammatory cytokines “Tumor necrosis factor alpha (TNF-α) and Interleukin beta (IL-1 β)” levels in albino rats after co-administration of diclofenac sodium and dexamethasone
117-121 Description of onthophagus lucidus(sturm, 1800) (coleoptera: scarabaeidae) from Kurdistan region –Iraq 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Welcome Note
On behalf of the organizing committee, it is my pleasure to welcome you to attend the 2nd Hawler Pharmaceutical Sciences Conference, scheduled during November 13-14, 2019 at Dedeman Hotel- Erbil -Kurdistan. The conference was held under the kind patronage of the president of Hawler Medical University, Assist. Professor Dr. Dara O. Meran, I would like to refer to his pioneering role and valuable guidance to raise the level of scientific performance of the conference. The organizing committee expects pharmacy academicians, researchers and professionals from Kurdistan region and all other parts of Iraq to attend the conference to gain new knowledge and share their experiences with the upcoming scientists. This two days event will be dedicated to cover a wide spectrum of sessions related to drug discovery and development as well as pharmacy practice, pharmacy curriculum development and other related fields of pharmacy, and will feature keynote forum, presentations, panel discussions, poster session, and workshops. The 2nd Hawler Pharmaceutical Sciences Conference will provide a unique opportunity to gather all the sectors of pharmacy (i.e. academia, community, institutional, wholesale, manufacturing, consultant, etc.) to come together, debate and attempt to resolve issues that are common to all in pharmacy and to reflect on the recommendations that were made on the 1st conference of the College of Pharmacy/ Hawler Medical University 2017. We look forward to greeting you at the conference and wish you a pleasurable experience.
Assist. Professor Dr. Alaadin M Naqishbandi President of the Conference
I 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Executive Committee: Head : Assist. Prof. Dr. Alaadin Mohammed Naqishbandi Members: Assist. Prof. Dr. Shatha Raoof Assist. Prof. Dr. Safa Toma Akka Assist. Prof. Dr. Hemn Abdul Qader Assist. Prof. Dr. Ava Tahir Ismael
Scientific Committee: Head : Assist. Prof. Dr. Zahra Abdulqader Amin Members: Assist. Prof. Dr. Lazeeza Sattar Omer Assist. Prof. Dr. Abdulkadir Aziz Hassan Assist. Prof. Dr. Rafal Abdulrazaq Abdulhameed Assist. Prof. Dr. Nozad Rashid Hussein Assist. Prof. Dr. Huner Kamal Omer Assist. Prof. Dr. Aryan Rzgar Ganjo Assist. Prof. Dr. Muslih Abdulkarim Ibrahim Assist. Prof. Dr. Dana Mohamed Amin Dr. Suha Saeed Aziz Dr. Abdulrahman Jawdat Mohammed
Consultancy Committee Assist. Prof. Dr. Sanaa Gadbaan Hama Prof. Dr. Lamaan Janab AL- Hayaly Prof. Dr. Abbas Mohamed Faraj Assist. Prof. Dr. Bushra Ahmed Hamdi Assist. Prof. Dr. Ansam Naji Abood Assist. Prof. Dr. Aras Najmaddin hamad
Administrative and Finance Committee: Head : Assist. Prof. Dr. Hawre Mustafa Baker Members: Dara Abdulla Qader Rawaz Delzar Tawfeeq Tareq Fareeq Abdulla Shahin Hazim Mustafa Bestoon Qasim Saeed Aza Omer Qader Nada Yahya Ahmad
II 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Scientific Program
III 2ⁿ Hawler Pharmaceu cal Sciences Conference: HPSC 2019.
November 13-14, 2019 Tuesday, 12 November 2019, Opening Ceremony Welcome Speeches 18:00 - 20:00 Exhibi on Opening & Coffee Break Registra on desk: Opening hours (17:00 : 20:00) Day I: Wednesday, 13 November 2019 09:00 - 15:30 Poster Presenta on First Session 09:00 : 13:00 Hall A Chairpersons: Dr. Lamaan AL-Hayaley & Dr. Sanaa Ghadban
Keynote lecture: 09:00 - 09:30 Prof. Dr. Alaa Abdulhussein, Ne w Aspects in Pharmaceu cal science University of Baghdad
Keynote lecture: Dr. Asos M. Rasoul / Pharmacist's roles in 21st century 09:30 - 10:00 Chair of Komar Pharmacy
Neurohumoral effects of Neprilysin inhibitor alone and in combina on with Renin-Angiotensin-Aldosterone 10:00 - 10:15 Kawa Dizaye, Rojgar H. Ali system blockers in rats with experimentally induced heart failure
Heat shock protein 90 regulates neutrophil Mac- 10:15 - 10:30 Mohammed Merza expression and extravascular recruitment in acute pancrea s related with lung injury 10:30 - 11:00 Coffee Break Closing Ceremony Panel Session: Kurdistan Syndicate of Pharmacists: Obstacle in the way of good Pharmacy prac cing in 11:00 - 13:00 Dr. Ammer S. Chalaby, Kurdistan with the possible solu on Dr. Adnan B. Qadir
13:00 - 14:00 Lunch Break Second Session 14:00 - 15:00 Hall A Chairpersons: Dr. Ansam Naji & Dr. Abdulrahman Jawdat Chiman M Jawdat, and Ismail Changes of Endothelin-1 Receptors Ac vity in Induced- 14:00 - 14:15 M. Maulood Hyperthyroid Isolated Rat Aorta Anjam H. Abdalla, Anoosh 14:15 - 14:30 Formula on and Evalua on of Valsartan Oral Granule Haqupian, Hewa Abdulla) Hemn K. Qadir, Heavy Metals Es ma on of Canned Food in Erbil City 14:30 - 14:45 Hemn A. Qader and Tariq F. Markets Abdullah
Talar A.M. Mohammad and Serum vitamin D level discriminates between the 14:45 - 15:00 Marwan S.M. Al-Nimer primary and the associated clinical manifesta ons of fibromyalgia
Sara Tharwat and Marwan The status of cardiometabolic risk factors that predict 15:00 - 15:15 AlNimer the future cardiovascular e vents in the breast cancer survivors in Kurdistan-Iraq
IV 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Asmaa Awni Haydar, Nazanin Sadq Ismail, Isra Mohammed Taha Alsaadi, Marwa Miqdad Es ma on of serum 25-hydroxy vitamin D and zinc levels Tahir, Azgar Nash Hussen, 15:15 - 15:30 in pa ents with acne vulgaris and their associa on with Balen Nariman Tahir, Mustafa disease severity Wrya Ikram, Aveen Nozad, Lana Yousif Mutalib, Bashdar M. Hussen
First Session 11:00 - 13:00 Hall B Chairpersons: Dr. Abdulqadir Naqishbandi & Dr. Rafal Abdulrazaq
Workshop: Pharmacy Curriculum Development (Applica on of 11:00 - 13:00 Dr. Salah Ismail Yahya / Bologna process) Koya University
13:00 - 14:00 Lunch Break Second Session 14:00 - 15:30 Hall B Chairpersons: Dr. Bushra Hamdi & Dr. Raad Kaskus Metasta c Breast Cancer to Stomach Mimicking Primary Ava Tahir 14:00 - 14:15 Gastric cancer
Zalina Zahari, Chee Siong Lee, Muslih Abdulkarim Ibrahim, Comparison of Pain Tolerance between Opioid Dependent Nurfadhlina Musa, Mohd 14:15 - 14:30 Pa ents on Methadone Maintenance Therapy (MMT) and Azhar Mohd Yasin, Yeong Yeh Opioid Naive Individuals Lee, Soo Choon Tan, Nasir Mohamad and Rusli Ismail
U lity of biomarkers for differen a ng between diabe c Muhanad Salah Mawlood 14:30 - 14:45 re nopathy and diabe c with no re nopathy
Hewa A. Hamadameen, Anjam H. Abdullah, Anoosh 14:45 - 15:00 The Fate of Expired Medica ons in Kurdistan region of Iraq Bashir Hakub, Mohamad O. Qader
Mahmud S. Yaseen and Time-dependent Effect of Candesartan on Blood Pressure 15:00 - 15:15 Muslih A. Ibrahim and Serum Potassium Level in Kurdish Hypertensive Pa ents
Frequency of biofilm produc on gene bla exo S by 15:15 - 15:30 Bashdar M. Hussen Pseudomonas aeruginosa in clinical samples
END OF DAY I
V 2ⁿ Hawler Pharmaceu cal Sciences Conference: HPSC 2019.
Day II: Thursday, 14 November 2019
09:00 - 15:30 Tuesday, 12P Novosterember Presen 2019,ta on Opening Ceremony First Session 09:00 - 13:00 Hall A Chairpersons: Dr. Shahla Jihad & Dr. Badraddin Kareem Keynote lecture: Asst. Prof. 09:00 - 09:30 Dr. Ibrahim Majeed, Clinical Pharmacy Program in Iraq DaUnivy I: erWsityednesda of Baghdady, 13 November 2019 Evalua on the Effects of Vitamin D3 as Add on Treatment 09:30 - 09:45 Alan Riyadh Mohammed in Asthma c Pa ents in Erbil Governorate/Iraq.
Younis Sadeq Smael, Pentoxifylline significantly reduces inflammatory 09:45 - 10:00 Suha Saed Aziz, biomarkers in knee Osteoarthri c pa ents: an open- Diar Abdullah AbuBaker label clinical interven onal study
Balsam Q. Saeed, Hussain Effect of different medicinal plant extracts on 10:00 - 10:15 Fadhil Hassan, Hssain Ismail Glutamic Oxaloace c Transaminase (GOT), Glutamic Arteen and Ahmed Omar Pyruvic Transaminase (GPT) enzyme ac vi es in Adrees Leishmania tropica promas gotes
An microbial Ac vity Extracts from Three Species of 10:15 - 10:30 Kwestan Sdiq Fresh Water Algae and Used Against Some Pathogenic Microorganisms and Analysis of Minerals in algal species 10:30 - 11:00 Coffee Break Panel Session:KMCA: Dr. Salam Mohammed Jawad, 11:00 - 13:00 Dr. Shaima Wahbe Abdulla, Role of quality control in pharmaceu cal op miza on Dr. Rawsht ClosingAhmad Abdulla, Ceremon y Dr. Essa AbdulRahman Esaa
13:00 - 14:00 Lunch Break Second Session 14:00 - 15:30 Hall A Chairpersons: Dr. Farhad Hamad & Dr. Idrees Bakir RAC 1 protect against Diabetes Mellitus via a enua on 14:00 - 14:15 Rundik Hwaiz of platelet chemokines Bashdar M. Hussen, Molecular detec on of bla ctx-M gene among Sahar M. Zaki, 14:15 - 14:30 Pseudomonas aeruginosa strains isolated from different Fa ma A.Ali, clinical samples in Erbil City. Rozhan M.Ismael Me ormin ameliorates diabetes mellitus in Kurds 14:30 - 14:45 Muslih A. Ibrahim pa ents by a enua on of serum cor sol and copper levels
Azilsartan as “Add-On” Treatment with Methotrexate Naza M. Ali Mahmood 14:45 - 15:00 Improves the Disease Ac vity of Rheumatoid Arthri s
In silico inves ga on to select the most fi ng mTOR 15:00 - 15:15 Hazem Abbas Al-Bustany inhibitors using AutoDock Vina
Salar A. Ahmed, Sazgar A. Hameed, Bashdar M. Prevalence of G20210A muta on in prothrombin gene 15:15 - 15:30 Hussen, Theodora Choli- among pa ents suffering from ischemic stroke in Hawler Papadopoulou city VI 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
First Session 11:00 - 13:00 Hall B Chairpersons: Dr. Kazhal Mahmoud & Dr. Hiwa Omer Workshop: 11:00 - 13:00 Dr. Nazar P. Shabila Wri ng Research Proposals and Grant Applica on Dr. Abubakir Majeed Saleh
13:00 - 14:00 Lunch Break Second Session 14:00 - 15:15 Hall B Chairpersons: Dr. Dana Hamadamin & Dr. Mohammed Merza Hemn A. Qader, Nabil A. Simultaneous Spectrophotometric Determina on of 14:00 - 14:15 Fakhri, Sarmad B. Dikran, Resorcinol, Caffeic Acid and Ascorbic Acid Using Ar ficial Idrees B. Qader Neural Network Technique
Pro-inflammatory cytokines “Tumor necrosis factor alpha (TNF-α) and Interleukin beta (IL-1 β)” levels in albino rats 14:15 - 14:30 Zahra A. Amin a er co-administra on of diclofenac sodium and dexamethasone.
Abbas M. Faraj, Nabeel A. New record of Onthophagus lucidus (Sturm, 1800) 14:30 - 14:45 Mawlood, Noor N. Polse (Coleoptera: Scarabaeidae) from Kurdistan Region –Iraq
Development of a β-arres n recruitment assay for the 14:45 - 15:00 Moein Mustafa characteriza on of na ve and mutant adenine receptors and their ligands
Assessment of Inhibin B , An -mullerian hormone and pro Shahin Hazim 15:00 - 15:15 –inflammatory cytokines among infer le males in Erbil
Closing Ceremony
END OF DAY I
VII 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Poster Presentations
Hevin Salar Yassin, Shatha Rouf Moustafa Association of Dyslipidemia with type two Diabetes Mellitus
Sayran H. Haji, Salah T. Jalal, Sharmin A. Omer, Molecular detection of SHV-Type ESBL in E. coli Ahang H. Mawlood and K. pneumoniae and their antimicrobial resistance profile
Sakar B. Smail, Aryan R. Ganjo Bacterial agents and antibiotic resistance pattern of infections that occurred among patients in Erbil city
Jwan M. Ahmed, Dr. Andrew Ingham Using Phase Solubility Analysis for the Determination of the Ionised and the Unionised Species' Solubility and Investigating the Effect of pH on the Complexation between Quinine Sulphate and Ibuprofen
Sheila Mufeed Nuraddin Antibacterial Effects Of Punica granatum Peel Extract
Shokhan Osman and Zahra A. Amin Vitamin K Accelerates Wound Healing Process On Rat Skin Achieved By Common Wound Dressing Agents In Erbil City/ Kurdistan
Suren Azad and Hemn A. Qader Pros and Cons of Fluoride, Chlorhexidine and Triclosan in Oral Care Products
Sazan M. Talat and Suha S. Shangula Obesity and Depression: an entwined nature? Exploring the link between obesity and depression in medical students in Erbil city
Marwan Qassim, Zahra A. Amin Gastro-protective effects of Hypericum perforatum peels extract on ethanol induced stomach ulcer in rats
Baraa Nazar, Aryan R. Ganjo The development of antimicrobial resistance in Escherichia coli in patients from hospitals in Erbil-city
Haidar Hawar, Shatha Rouf Moustafa Association of Creatinine and Urea as prognostic markers in type two Diabetes Mellitus patients with Renal Function Impairment
VIII 2nd Hawler Pharmaceutical Sciences Conference: HPSC 2019
Scientific Articles
IX The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Heat shock protein 90 regulates neutrophil Mac-1 expression and extravascular recruitment in acute pancreatitis related with lung injury
Mohammed Yousif Merza1* Abstract Background and objective: Severe acute pancreatitis (AP) is associated with infiltration of leukocytes and tissue necrosis but the cellular signaling mechanisms controlling organ damage in the pancreas remain elusive. Heat shock protein 90 (HSP 90) is a potent regulator of specific cellular processes effectuating pro-inflammatory activities. Herein, we examined the role of HSP90 signaling in acute pancreatitis. Methods: Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct in C57BL/6 mice. Animals were treated with radicicol (60 mg/kg) prior to induction of pancreatitis. Results: Administration of radicicol significantly decreased the taurocholate-induced increase in serum amylase, pancreatic neutrophil infiltration, acinar cell necrosis and edema formation in the pancreas related with lung injury. In addition, inhibition of heat shock protein 90 reduced the MPO levels in the pancreas and lung in response to taurocholate challenge. However, treatment with radicicol had no effect on taurocholate-provoked formation of macrophage inflammatory protein-2 (MIP-2) in the pancreas. Interestingly, Heat shock protein 90 inhibition abolished neutrophil expression of Mac-1 in mice with pancreatitis. Moreover, radicicol markedly decreased MIP-2-induced Mac-1 upregulation in isolated neutrophils in vitro, suggesting a direct role of heat shock protein 90 in regulating Mac-1 expression in neutrophils. Finally, inhibition of heat shock protein had no direct effect on secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro. Conclusion: These results demonstrate that heat shock protein 90 signaling plays a significant role in acute pancreatitis by regulating neutrophil infiltration and tissue injury via expression of Mac-1 on neutrophils. Thus, our findings not only elucidate signaling mechanisms in pancreatitis or lung injury but also suggest that heat shock protein might constitute a novel target in the management of severe AP related with lung injury. Keywords: Amylase, Chemokines, Inflammation, Leukocytes, and Pancreas.
Introduction trypsinogen activation, inflammation and Acute pancreatitis (AP) presents with impaired microvascular perfusion are a wide range of disease severity ranging integrated components in the from simple and transient pain to local pathophysiology of pancreatitis.2 Knowing and systemic complications.1 Due to the that activation of trypsinogen appears to limited understanding of the underlying be an early and transient process, pathophysiology, management of patients inflammation in the pancreas persists with severe AP poses a major challenge longer and might be a more rational target to clinicians, which is largely limited to for treatment.2 Leukocyte accumulation is supportive therapies. Today, there is no a hallmark of inflammation and several effective method to predict the severity and reports have shown a key role of outcome of AP. The literature suggests that leukocytes in the development of AP.3
1 Department of Clinical Analysis, College Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 1 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The extravasation process of leukocytes administration of radicicol. comprises multiple sequential steps Methods mediated by specific adhesion molecules, such as P-selectin, Mac-1 and LFA-1.4 Animals Tissue navigation of leukocytes is All experiments were conducted using orchestrated by secreted chemokines.5 C57BL/6 male mice, weighting 20-25 g (6 CXC chemokines, such as macrophage to 8 weeks) purchased from Taconic. The inflammatory protein-2 (MIP-2), stimulate animals were maintained under a 12 h extravascular recruitment of neutrophils. light/ dark cycle in a climate at 22ºC and CXCR2 is the high affinity receptor on fed water and standard chow ad libitum. murine neutrophils for MIP-2 and KC and Mice were anesthetized by intraperitoneal it has been shown that CXCR2 is critical (i.p) administration of 75 mg/kg of ketamine in supporting neutrophil infiltration in the hydrochloride (Hoffman-La Roche, Basel, pancreas.6 Although, the role of specific Switzerland) and 25 mg/kg of xylazine adhesion molecules and chemoattractants (Janssen Pharmaceutics, Beerse, Belgium) in leukocyte infiltration in the pancreas is in 200 µl saline. Analgesia was obtained by relatively well known, the understanding subcutaneous injection of buprenorphine of the signaling pathways coordinating hydrochloride 0.1 mg/kg (Schering-Plough pro-inflammatory actions in AP is limited. Corporation, New Jersey, USA). Trauma and infection trigger multiple Animal model of acute pancreatitis signaling cascades that converge on Anaesthetized mice underwent mildline specific transcription factors controlling laparotomy and the second part of gene expression of pro-inflammatory duodenum and papilla of vater were substances. This signal transmission is identified. Traction sutures (7–0 prolene) predominately regulated by intracellular were placed one cm from the papilla. kinases phosphorylating down-stream Parallel to the papilla of vater, a small targets.2 Targeting Hsp90 protects against puncture was made through the duodenal intestinal inflammation and leakage and wall with a 23 G needle. A non-radiopaque might be a useful strategy to ameliorate polyethylene catheter (ID 0.28 mm) intestinal failure in polymicrobial connected to a micro infusion pump sepsis.inflammatio.7 In several disease (CMA/100, Carnegie Medicin, Stockholm, models, including pancreatitis, heat shock Sweden) was inserted through the proteins (HSPs) are chaperone proteins punctured hole in the duodenum and that protect living cells against one mm into the common bile duct. The injury‐inducing stimuli. However, common hepatic duct was identified at dysregulated expression of HSPs has been the liver hilum and clamped with observed in various disease conditions a neurobulldog clamp. Infusions of 10 µl of including cancer. Inhibition of HSP 5% sodium taurocholate (Sigma, St. Louis, especially HSP 70 expression in pancreatic MO, USA) for 10 min were retrogradly cancer cells leads to caspase dependent infused into the pancreatic duct. After apoptotic cell death and it has been completion, the catheter was withdrawn shown as a novel therapeutic modality and the common hepatic duct clamp was for pancreatic cancer.8 Based on these removed. The duodenal puncture was considerations, we hypothesized that heat closed with a purse-string suture (7–0 shock protein signaling might play a role in monofilament). The traction sutures were severe AP. We used an experimental removed and the abdomen was closed in model of severe AP with lung injury in two layers. Animals were allowed to wake mice based on retrograde infusion of up and were given free access to food taurocholate in the pancreatic duct and and water. Radicicol (60 mg/kg, Sigma) or interfered with HSP 90 activity by vehicle (phosphate-buffered saline [PBS])
2 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) was administered i.p. prior to bile duct previously described.18 All pellets cannulation. This dose and scheme of were mixed with 1 ml of 0.5% administration of the HSP90 inhibitor was hexadecyltrimethylammonium bromide. based on a previous investigation.2 Animals Next, the sample was frozen for 24 h and exposed to taurocholate were pretreated then thawed, sonicated for 90 s, put in with vehicle (n = 8) or radicicol (n = 8). a water bath 60°C for 2 h, after which Control mice undergoing laparotomy and the MPO activity of the supernatant sodium chloride infusion into the pancreatic was measured. The enzyme activity was duct were pretreated with vehicle (Sham n determined spectrophotometrically as the = 8). In separate experiments were mice MPO-catalysed change in absorbance in exposed to taurocholate treated with the redox reaction of H2O2 (450 nm, with vehicle (n = 8) or radicicol (n = 8) 2 h after a reference filter 540 nm, 25°C). Values induction of pancreatitis. All animals were are expressed as MPO units·per g tissue. killed 24 h after pancreatitis induction and Tissue preparation assessed for all parameters included in this Pancreas samples were fixed in 4% study. Blood was collected from the tail formaldehyde phosphate buffer overnight vein for systemic leukocyte differential and then dehydrated and paraffin counts. Blood samples were also embedded. Six micrometre sections were collected from the inferior vena cava for stained (haematoxylin and eosin) and determination of serum amylase levels. examined by light microscopy. The severity Pancreatic tissue was removed and kept in of pancreatitis was evaluated in a blinded two pieces; one piece was snap frozen in manner by use of a pre-existing scoring liquid nitrogen for biochemical analysis of system including oedema, acinar cell myeloperoxidase (MPO) and MIP-2 and the necrosis, haemorrhage and neutrophil other piece was fixed in formalin for later infiltrate on a 0 (absent) to four (extensive) histological analysis. Lung tissue was also scale as previously described in detail.2 harvested for MPO measurements. MIP-2 levels Amylase measurements MIP-2 levels in serum and pancreas were Amylase was quantified in blood with determined in stored supernatants from a commercially available assay homogenized pancreatic tissues. MIP-2 (Reflotron®, Roche Diagnostics GmbH, levels were assessed using double- Mannheim, Germany). antibody Quantikine enzyme linked Systemic Leukocyte counts immunosorbent assay kits (R & D Systems Tail vein blood was mixed with Europe, Abingdon, UK) using recombinant Turks solution (0.2 mg gentian violet in murine MIP-2 as standard. The minimal 1 ml glacial acetic acid, 6.25% v/v) detectable protein concentration is less in a 1:20 dilution. Leucocytes were than 0.5 pg/ml. identified as monomorphonuclear and Flow cytometry assay polymorphonuclear cells in a Burker Blood was collected (1:10 acid citrate chamber. dextrose) from vehicle and pancreatitis MPO activity mice. To block Fcg III/II receptors and All frozen pancreatic and lung tissues were reduce non-specific labeling samples were pre-weighed and homogenized in 1 ml incubated with an anti-CD16/CD32 for mixture (4:1) of PBS and aprotinin 5 min. Then samples were stained with 10 000 KIE·ml−1 (Trasylol®, Bayer a PE-conjugated anti-Gr-1 (clone RB6- HealthCare AG, Leverkusen, Germany) for 8C5, eBioscience, San Diego, CA, USA) 1 min. The homogenate samples were antibody. Erythrocytes were lysed and centrifuged (15339×g, 10 min) and the the cells were fixed. Cells were recovered supernatant was stored at −20°C and following centrifugation before being the pellet was used for MPO assay as analyzed with a FACSCalibur flow
3 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) cytometer (Becton Dickinson, Mountain performed by using non-parametrical tests View, CA, USA). A viable gate was used to (Mann–Whitney). P <0.05 was considered exclude dead and fragmented cells. After significant and n represents the number of gating the neutrophil population based on animals. forward and side scatter characteristics, Results Mac-1 expression was determined on cells positive for Gr-1, which is a neutrophil Heat shock protein 90 controls tissue marker. damage in pancreatitis Trypsinogen activation in isolated acinar In order to examine the role of heat shock cells protein 90 in severe AP, serum amylase Pancreatic acini cells were prepared levels were first examined as an indicator by collagenase digestion and gentle of tissue damage. We observed that shearing as described previously.2 Cells retrograde infusion of taurocholate in the were suspended in HEPES-Ringer buffer pancreatic duct increased serum amylase (pH 7.4) saturated with O2 and passed levels by 5-fold (Figure 1, P <0.05 vs. through a 150 µm cell strainer (Partec, Sham, n = 8). Treatment with the heat England). Isolated acinar cells (1 × 107 shock protein inhibitor radicicol decreased cells per well) were preincubated with taurocholate-induced serum amylase vehicle or radicicol (200 µM, 30 min) and levels from 410 ± 0.61 µKat/l to 208 ± stimulated with 100 nM cerulein (37°C, 30 0,37 µKat/l, corresponding to a 50.7% min) in duplicate. The buffer was then reduction (Figure 1, P <0.05 vs. vehicle + discarded and the cells were washed taurocholate, n = 8). twice with buffer (pH 6.5) containing 250 mM sucrose, 5 mM 3-(morpholino) propanesulphonic acid (MOPS) and 1 mM MgSO4. The cells were next homogenized in cold (4°C), MOPS buffer using a potter Elvejham-type glass homogenizer. The resulting homogenate was centrifuged (56×g, 5 min), and the supernatant was used for assay. Trypsin activity was measured flourometrically using Boc-Glu- Ala-Arg-MCA as substrate as described previously.21 For this purpose, a 200 µl aliquot of the acinar cell homogenate was added to a cuvette containing assay buffer (50 mM Tris, 150 mM NaCl, 1 mM CaCl2 and 0.1% BSA, pH 8.0). The reaction was Figure 1: blood amylase (µKat/l) in sham initiated by the addition of substrate, and and taurocholate-exposed mice pretreated the fluorescence emitted at 440 nm in with PBS or the HSP 90 inhibitor radicicol response to excitation at 380 nm was (60 mg/kg). Blood samples were obtained monitored. Trypsin levels (pg/ml) were 24 h after pancreatitis induction. Data calculated using a standard curve represent means ± SEM and n = 8. generated by assaying purified trypsin. # P < 0.05 versus sham and *P < 0.05 Viability of the pancreatic acinar cells was versus PBS + taurocholate. higher than 95% as determined by trypan blue dye exclusion. Analysis of data Data are presented as mean values ± SEM. Statistical evaluations were
4 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Examination of tissue morphology revealed by 66.6 % and edema by 52 % in the that control mice had a normal pancreatic pancreas (Figure 2 b and c, P < 0.05 vs. microarchitecture (Figure 2, n = 8), vehicle + taurocholate, n = 8). Moreover, whereas taurocholate lead to significant radicicol decreased the number of destruction of the pancreatic tissue extravascular leukocytes by 57.1 % in structure characterized by acinar cell pancreatitis mice (Figure 2d, P < 0.05 vs. necrosis, edema formation and neutrophil vehicle + taurocholate, n = 8). Challenge accumulation (Figure 2, n = 8). We found with taurocholate increased the number of that inhibition of heat shock protein 90 circulating MNLs and PMNLs, suggesting protected against taurocholate-provoked on-going systemic activation (Table 1). tissue destruction (Figure 2, n = 8). Inhibition of heat shock protein 90 reversed For example, it was observed that changes in leukocyte differential counts in administration of radicicol reduced the circulation towards the levels in control taurocholate-evoked acinar cell necrosis animals (Table 1).
Table 1: Systemic leukocyte differential counts. PMNL MNL Total Sham 1.3 ± 0.3 9.4 ± 0.3 10.7 ± 0.6 PBS + Pancreatitis 0.9 ± 0.2# 6.2 ± 0.2# 7.1 ± 0.4# Radicicol + Pancreatitis 1.4 ± 0.1 11.3 ± 0.3 12.5 ± 0.4 blood was collected from sham, saline control, taurocholate-treated animals receiving PBS or the heat shock protein inhibitor radicicol (60 mg/kg). Cells were identified as monomorphonuclear leukocytes (MNL) and polymorphonuclear leukocytes (PMNL). Data represents mean ± SEM, 106 cells/ml and n = 8. #P < 0.05 versus sham and *P < 0.05 versus PBS + taurocholate.
Figure 2: HSP 90 regulates tissue damage in acute pancreatitis. (A) Acinar cell necrosis (B) edema formation (C) hemorrhage and (D) extravascular leukocytes in sham and taurocholate-exposed mice pretreated with PBS or the HSP 90 inhibitor radicicol (60 mg/ kg). (E) Representative hematoxylin & eosin sections of the pancreas. Samples were harvested 24 h after pancreatitis induction. Bars represent 100 µm. Data represent means ± SEM and n = 8. #P < 0.05 versus sham and *P < 0.05 versus PBS + taurocholate. 5 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Heat shock proteine regulates As part of a systemic inflammatory neutrophil infiltration in pancreatitis response in severe AP, activated Tissue levels of MPO were used as an neutrophils accumulate in the pulmonary indicator of neutrophil infiltration. We found microvasculature. Indeed, it was observed that challenge with taurocholate increased that challenge with taurocholate markedly pancreatic MPO activity of by 10-fold enhanced the MPO activity in the lung. (Figure 3a, P <0.05 vs. Sham, n = 8). Heat shock protein 90 inhibition Inhibition of heat shock protein 90 inhibition reduced MPO levels in the lung by reduced taurocholate-provoked pancreatic more than 30.7% in mice challenged levels of MPO by 27 % (Figure 3a, with taurocholate (Figure 3b, P <0.05 vs. P <0.05 vs. vehicle + taurocholate, n = 8). vehicle + taurocholate, n = 8).
Figure 3: HSP 90 controls taurocholate-induced neutrophil accumulation. MPO levels in the (A) pancreas and (B) lung in sham and taurocholate-exposed mice pretreated with PBS or the HSP 90 inhibitor (60 mg/kg). Samples were harvested 24 h after pancreatitis induction. Data represent means ± SEM and n = 8. #P < 0.05 versus sham and *P < 0.05 versus PBS + taurocholate.
6 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) In addition, we observed that challenge experimental model. Inhibition of heat with taurocholate markedly increased shock protein 90 signaling markedly MIP-2 levels in the pancreas from reduced neutrophil expression of Mac-1 0.08 ± 0.04 to 8.8 ± 0.05 pg/mg (Figure 4, in pancreatitis (Figure 5a, P < 0.05 vs. P < 0.05 vs. Sham, n = 8). Administration vehicle + taurocholate, n = 8). In order of radicicol had no significant effect on MIP to test whether this inhibitory effect of -2 levels in the inflamed pancreas (Figure 4 radicicol on neutrophil expression of Mac-1 P > 0.05 vs. vehicle + taurocholate, n = 8). was indirect or direct, we stimulated Moreover, we noted that Mac-1 expression isolated neutrophil with MIP-2 in vitro and was increased on the surface of found that radicicol markedly decreased neutrophils in mice with pancreatitis MIP-2-induced upregulation of Mac-1 on (Figure 5a, P <0.05 vs. Sham, n = 8), isolated neutrophils (Figure 5b, P < 0.05 indicating systemic activation in this
Figure 4: Chemokine formation in the pancreas. Pancreatic levels of MIP-2 were determined in sham and taurocholate-exposed mice pretreated with PBS or the HSP 90 inhibitor (60 mg/kg). Samples were harvested 24 h after pancreatitis induction. Data represent means ± SEM and n = 8. #P < 0.05 versus sham and *P < 0.05 versus PBS + taurocholate.
Figure 5: HSP 90 regulates Mac-1 expression on neutrophils. Mac-1 expression (A+B) on neutrophils in PBS and radicicol (60 mg/kg) treated animals 24 h after induction of pancreatitis and (C+D) on isolated neutrophils incubated with MIP-2 (5 µg/ml) and PBS or radicicol (100 µM). Fluorescence intensity is shown on the x-axis and cell counts on the y-axis. Histograms are representative of 8 samples. Data represent means ± SEM and n = 8. #P < 0.05 versus sham and *P < 0.05 versus PBS + taurocholate.
7 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Trypsinogen activation in acinar cells in shock protein 90 is involved in the surface vitro upregulation of Mac-1 on neutrophils. We nest asked whether heat shock protein Inhibition of heat shock protein 90 activity 90 may regulate trypsinogen activation in not only decreases neutrophil infiltration in pancreatic acinar cells in vitro. For this the pancreas but also attenuates acinar purpose, we isolated acinar cells from cell necrosis and serum amylase levels the pancreas of mice and incubated the in AP. In addition, we found that cells with cerulein. It was found that heat shock protein 90 inhibition abolished cerulein stimulation increased trypsinogen accumulation of neutrophils in the lung, activation by more than 30-fold compared suggesting that heat shock protein to unstimulated cells (Figure 7, P < 0.05 90 controls both local and systemic vs. control, n = 5). However, preincubation inflammation in severe AP. Heat shock of the acinar cells with radicicol had no protein 90 activity has been reported effect on secretagogue-induced activation to control pro-inflammatory actions in of trypsinogen (Figure 6, P < 0.05 vs. experimental models of multiple sclerosis vehicle + cerulein, n = 5). and sepsis.7 Herein, we found that heat shock protein 90 inhibition with a specific Discussion heat shock protein 90 inhibitor (Radicicol) Signaling cascades controlling pro- significantly reduced tissue injury in inflammatory pathways in pancreatitis are severe AP. For example, administration of incompletely understood. Our present radicicol decreased taurocholate-provoked study demonstrates for the first time increase in blood amylase and acinar that heat shock protein 90 is an important cell necrosis suggesting that heat shock regulator of the pathophysiology in severe protein 90 activity regulates a significant AP. These results demonstrate that heat part of the tissue damage in severe AP.
Figure 6. Acinar cell activation of trypsinogen was measured in negative control cells and cerulein-exposed acinar cell homogenate pretreated with PBS or radicicol (200 mM). Activation of trypsinogen activation was quantified by measuring enzymatic activity of trypsin fluorometrically by using Boc-Gln-Ala-Arg-MCA as the substrate as described in detail in Materials and Methods. Trypsin levels (pg/ml) were calculated using a standard curve generated by assaying purified trypsin. Data represent means ± SEM and n = 8. #P < 0.05 versus PBS control. 8 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) These data constitutes the first evidence in Notably, treatment with radicicol greatly the literature that inhibition of the heat decreased neutrophil expression of Mac-1, shock protein 90 signaling pathway suggesting that heat shock protein protects against severe AP. In this context, regulates Mac-1 expression on neutrophils it is interesting to note that HSP, which are in AP. We next asked whether this mainly used to regulate severe levels in inhibitory effect of radicicol might be patients with cardiovascular diseases, have indirect and related to formation of CXC been reported to attenuate experimental chemokines, such as MIP-2, which is pancreatitis.8 It is widely held that a particularly potent activator of neutrophil infiltration is a prominent feature neutrophils.9 It was therefore of great in pancreatitis.9 For example, neutrophil interest to examine formation of MIP-2 in depletion has repeatable been reported to the pancreas in this study. It was found ameliorate tissue damage in AP.9 In the that taurocholate provoked a significant present study, we found that taurocholate increase in MIP-2 levels in the pancreas. challenge markedly enhanced MPO However, inhibition of heat shock protein activity and the number of extravascular 90 had no effect on taurocholate-induced neutrophils in the pancreas. Administration production of MIP-2 in the pancreas. We of radicicol significantly reduced MPO next asked whether radicicol might directly levels and the number of extravascular inhibit Mac-1 upregulation on activated neutrophils in the pancreas, indicating neutrophils. Indeed, we found that radicicol that heat shock protein 90 activity is abolished MIP-2-induced upregulation an important regulator of neutrophil of Mac-1 on the surface of isolated recruitment in the inflamed pancreas. neutrophils, suggesting that heat shock Considering the critical role of neutrophils protein 90 directly regulate Mac-1 in the pathophysiology of pancreatitis.2 expression in neutrophils. Trypsinogen it might be forwarded that the inhibitory activation is generally considered as effect of radicicol on neutrophil responses a central feature in the pathophysiology of might explain the protective effect of AP. 1 A recent study showed that GTpase radicicol in AP. In addition, systemic signaling regulates trypsinogen activation complications of severe AP include in acinar cells.5 However, we observed that pulmonary accumulation of neutrophils.10 Inhibition of heat shock protein 90 activity We found that lung levels of MPO had no effect on secretagogoue-induced clearly increased in response to activation of trypsin in isolated acinar cells taurocholate challenge. Interestingly, in vitro. The precise role of HSP in AP radicicol decreased pulmonary MPO needs to be addressed in future studies. activity, suggesting that heat shock protein Nonetheless, these findings suggest that 90 also regulates systemic activation and the protective effects of HSP90 are infiltration of neutrophils in the lung in downstream of trypsin activation in severe AP. Numerous studies have shown pancreatitis. This notion is in line with the that specific adhesion molecules control concept in the present study suggesting the extravasation process of leukocytes.2 that a dominant role of fHSP in AP is Although the detailed role of certain related to inhibition of Mac-1 expression adhesion molecules in facilitating leukocyte and neutrophil accumulation in the accumulation in the pancreas is relatively pancreas and lung. In conclusion, these unclear, several reports have documented findings demonstrate that HSP 90 signaling that shown that Mac-1 is a dominating regulates tissue damage in severe AP. molecule in mediating tissue infiltration of Our results show that inhibition of HSP 90 neutrophils.5 Herein, it was observed that attenuates neutrophil expression of Mac-1 Mac-1 expression on neutrophils increased and infiltration in the pancreas and the in response to taurocholate administration. lung, suggesting that HSP 90 controls both
9 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) local and systemic inflammation in 8. Boudesco C, Cause S, Jego G, Garrido C. Hsp70: pancreatitis. Thus, these data not only A Cancer Target Inside and Outside the Cell. Methods Mol Biol 2018;1709:371–96. delineate a key signaling mechanism in 9. Merza M, Hartman H, Rahman M, Hwaiz R, AP but also indicate that targeting HSP Zhang E, Renström E, et al. Neutrophil might be an effective way to ameliorate to extracellular traps induce trypsin activation, pathological inflammation in severe AP. inflammation, and tissue damage in mice with severe acute pancreatitis 2015 Gastroenterology; Conclusion 149(7):1920–31. 10. Abdulla A, Awla D, Thorlacius H, Regnér S. Role These results demonstrate that heat shock of neutrophils in the activation of trypsinogen in protein 90 signaling plays a significant severe acute pancreatitis 2012 JLB; 90(5):975– role in acute pancreatitis by regulating 82. neutrophil infiltration and tissue injury via expression of Mac-1 on neutrophils. Thus, our findings not only elucidate signaling mechanisms in pancreatitis or lung injury but also suggest that heat shock protein might constitute a novel target in the man- agement of severe AP related with lung injury. Conflict of interest The author declares no competing interests. References 1. Merza M, Awla D, Hwaiz R, Rahman M, Appelros S, Abdulla A, et al. Farnesyltransferase regulates neutrophil recruitment and tissue damage in acute pancreatitis Pancreas 2014; 43(3):427–35. 2. Merza M. Isoprenylation and NET formation in acute pancreatitis. PhD. Thesis book, Lund University 2015. 3. Merza M, Wetterholm E, Zhang S, Regner S, Thorlacius H. Inhibition of geranylgeranyltransferase attenuates neutrophil accumulation and tissue injury in severe acute pancreatitis. JLB 2013; 94(3):493–502. 4. Madhi R, Rahman M, Taha D, Linders J, Merza M, Wang Y, et al. Platelet IP6K1 regulates neutrophil extracellular trap-microparticle complex formation in acute pancreatitis. JCI insight 2019. 5. Wetterholm E, Linders J, Merza M, Regner S, Thorlacius H. Platelet-derived CXCL4 regulates neutrophil infiltration and tissue damage in severe acute pancreatitis. Translational Research 2015; 176:105–18. 6. Merza M, Rahman M, Zhang S, Hwaiz R, Regner S, Schmidtchen A, et al. Human thrombin -derived host defense peptides inhibit neutrophil recruitment and tissue injury in severe acute pancreatitis AJPGLP 307(9):914–21. 7. Zhao Y, Huang ZJ, Rahman M, Luo Q, Thorlacius H. Radicicol, an Hsp90 inhibitor, inhibits intestinal inflammation and leakage in abdominal sepsis. J Surg Res 2013; 182(2):312–8.
1010 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Changes of Endothelin-1 Receptors Activity in Induced-Hyperthyroid Isolated Rat Aorta
Chiman M Jawdat1 Ismail M. Maulood2* Abstract Background and objective: Endothelin has an excessive diverse set of actions that affect homeostatic mechanisms throughout the body. The current study was designed to show the effects of induced-hyperthyroid on the ET-1 receptor and their antagonists were investigated. Methods: The study was carried out with two group of female rats: control and induced-hyperthyroidism. Induced-hyperthyroidism in rats occurred by administration of L-thyroxin for four weeks. The results confirmed the induction of hyperthyroidism because L-thyroxine-treated rats associated with increased serum levels T3 and T4. Results: In an in vitro study, the dose-response curve (DRC) of ET-1 were found in isolated intact and denuded aortic rings of both control and induced-hyperthyroid groups. On the other hand, denudation of aortic rings shifted the DRC of ET-1 to the left and the pD2 increased non-significantly in control group. The pD2 significantly increased in induced-hyperthyroid aortic rings and shifted the DRC of ET-1 to the left. Furthermore, pre -incubation with selective endothelin-A (ETA) receptor antagonist (BQ-123) shifted the DRC of ET-1 to the right in both groups and significantly reduced pD2 in control aortic rings. Selective endothelin B (ETB) receptor antagonist (BQ-788) pre-incubation significantly raised the pD2 value in both groups and shifted the DRC to the left in induced-hyperthyroid aortic rings, while it decreased Emax in control and increased in induced-hyperthyroid aortic rings. Conclusion: In induced-hyperthyroid rats BQ-788, but not BQ-123 enhanced contractile response to ET-1 Pre-incubation of BQ-123 did not lead to change the contractile response of denuded rings in control rats, but pre-incubation of this antagonist in hyperthyroid denuded aortic rings led to inhibition of contractile response to ET-1. Keywords: Endothelin-1; ET A and ETB receptors; Hyperthyroidism; Isolated Rat Aorta.
Introduction normotensive subjects.3 Arterial VSMCs Endothelin-1, a 21-amino-acid long peptide express both ETA and ETB receptors.4 isolated from the cultured endothelial cells, Endothelin A receptors are expressed it has long-lasting vasoconstriction activity on SMCs that contract on activation. In in most vascular smooth muscle cells contrast, ETB receptors are expressed (VSMCs).1 ET-1 has two main receptors, both on smooth muscle cells to evoke ETA and ETB; ETA receptors are contractions and endothelial cells to induce expressed on VSMCs and ETB receptors NO and EDHF-dependent relaxation.5 on both endothelial and.2 The ETA Depending on a type of cell, type of tissue antagonist BQ-123 also improved the or physiological situations, ETA and impairment of vasodilation in hypertensive ETB receptors can possess synergetic or patients, whereas BQ-788, the ETB opposing effects.6 Vascular endothelial antagonist, exerted a vasoconstrictor effect cells appear to express ETB receptors on forearm resistance arteries in primarily, and the activation of these ETB 1 Department of Biology, Faculty of Science, University of Soran, Erbil, Iraq. 2 Department of Biology, College of Science, Salahaddin University, Erbil, Iraq. * Correspondence: [email protected] 111 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) receptors increases intercellular [Ca+2], temperature (12 hrs. light: 12 hrs. dark resulting in increased NO production and photoperiod and 22 ± 4 oC). vasodilation.7 Endothelin has an excessive Induction of hyperthyroidism diverse set of actions that affect Animals were induced thyrotoxicosis by homeostatic mechanisms throughout the administration of L-thyroxin (0.4 mg/100g body. ET-1 is a potent and long-lasting food) (BERLIN-CHEMIE AG –Berlin, vasoconstrictor both in vivo and in-vitro.8 It Germany) for four weeks. Hyperthyroidism has a greater role in the maintenance of confirmed by determination of serum T3 vascular tone and modulation.9 Endothelins and T4. Both hormones significantly also promote growth and proliferation of elevated as compared to the control rats. VSMCs, an effect that appears to be ETA Animal ethics receptor-mediated and involves activation The care of animals has been approved of mitogen-activated protein (MAPK)s and by the animal research committee in perhaps the transactivation of epidermal Salahaddin University-Erbil. The study growth factor receptor.10 Endothelial protocol has been endorsed by the ethics dysfunction is a result of an imbalance committee of our department. between vasoconstriction and relaxing Estimation of serum T3 and T4 factors, procoagulant and anticoagulant Triiodothyronine, thyroxine were substances, as well as among pro- determined by a fully automated immune inflammatory mediators. Detection of ED analyzer (Cobas e 411 Roche Diagnostics, based on the assessment of circulating HITACHI, Japan) based on the ECL markers of endothelial function; ET-1.11,12 technology. In thyroid carcinoma, the overexpression of Preparation of aortic rings ET-1 and ETA receptor observed these Female albino rats were injected with suggest a mitogenic role of ET-1 that heparin (1500units /kg body weight) to could be countered theoretically via ETA avoid blood clotting and possible damage receptor antagonists. Also, in thyroiditis of aortic endothelium and anesthetized overexpression of ET-1 and ETA receptor with the injection of ketamine (40 mg/kg) supports the ET-1 role in the inflammatory and xylazine (10 mg/kg) intraperitoneal process.13 However, in hyperthyroidism, (Struck et al., 2011). The animal abdomen vascular tones will change, but according wall opened until chest cavity, after that to our knowledge, the exact mechanism for excess fat tissues removed to obtain the such changes is not fully understood. appropriate length of rings and isolate it Therefore, the present study aimed to smoothly without being stretching and investigate the role of ET-1 receptors in any mechanical force to get intact aorta. In induced-hyperthyroid rat aortic constriction experiments on denuded endothelial cells, -induced by ET-1. the isolated aorta gently rubbed by cotton many times to get denuded aorta. The Methods aorta transferred to a petri-dish filled with Animals and housing cold Krebs-Henseleit solution aerated with Female albino rats of about weighting about 95% oxygen in 37Co. Krebs solution 200-300gm used in the present study. Ani- is a physiological buffered solution that mals were housed and bred in the animal contains (NaCl 0.118, NaHCO3 0.025, house belong to Biology Department, Col- MgSO4 0.0012, KCl 0.0047, KH2PO4 lege of Science; Salahaddin University – 0.0012, CaCl2 0.0025, C6H12O6 0.011 Erbil. The animals kept in plastic cages and EDTA 0.001) in mole/L with pH 7.4. (4-5 rats/cage) bedded with wood chips These solution maintains the viability of and given a standard rat diet and tap the cell, provide the suitable conditioner water ad libitum. The animals housed in medium for it and has a sensitive pH standard laboratory conditions of light and for that reason should be used freshly.
122 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The aortic rings then cut into 4-segments Experimental design each with 2-2.5mm length approximately. The in vitro experiment was done using The confirmation to ensure the denudation, isolated thoracic aorta pre-incubated with the aortic rings was contracted by (KCl ET-1 receptor antagonists. DRC was found 60mM) then relaxed by ACh, if the muscle for ET-1 (Bachem AG Company, Germany) not relaxed, then the aorta denuded. in intact and denuded aortic rings of Isometric tension recording in isolated both control and Induced-hyperthyroid vessel rats. Also, the experiment carried out to The aortic rings held up by two stainless investigate the roles of ETA and ETB steel clamps (Tissue clamp, Model Le receptors in aortic smooth muscle tone in 0140, Panlab Harvard Apparatus, USA). both control and induced hyperthyroid rats. One of the clamps connect to a hook at Here EC50 was calculated for ET-1 dose the bottom of the organ bath jacket, response curve in the absence and and the other was connected to the force presence of selective ETA (BQ-123) transducer through a thread to record (Bachem AG Company, Germany) and vibrations from the rings to transduce it selective ETB receptor antagonists (BQ- by the amplifier (Quad bridge amplifier 788) (BachemAGCompany, Germany). Powerlab 8/35) (Fig 3.4). Isometric tension Furthermore, these ET-1 receptor detected by the transducer which is antagonists also applied to denuded aortic connected to the amplifier when the muscle smooth muscle tissues. of the ring contracts or relax, and the data The following groups included: recorded by data acquisition software Group 1: Dose - response curve of ET-1 (LabChart 7.1). Then the aortic rings (Control) immersed in Krebs solution contained in a Aortic rings of control and induced 10 ml organ chamber. Krebs solution was hyperthyroid rats were allowed to maintained at pH 7.4 and continuously equilibrate for 60-90 min at 2g tension. aerated with carbogen in about 95% The dose-response curve and the EC50 oxygen 5% carbon dioxide at 37oC obtained for ET -1 (10-10 - 10-7M) by its (LE 13206 Thermostat, Panlab Harvard cumulative addition with time interval 8 Apparatus, USA). The aortic rings were minutes to organ bath in both intact tensed to a stable basal strain of 2gm (n=5 control, n=8 TIH) and denuded aortic before left to equilibrate for an hr. rings (n=8 control, n=7 TIH). To remove any cellular metabolites, Group 2: BQ-123 (ETA Receptor Krebs solution was replaced every 15-20 antagonist) minutes intervals in the bath chamber. The aortic rings of control and induced After stabilization, the viability test was hyperthyroidism pre-incubated with performed to know whether the aorta is still selective ETA Receptor antagonist (BQ- alive or not by pre-contraction with KCl 123, 0.3µM) for 20 minutes. DRC and 60 mM (EC50, Dose response curve EC50 obtained for ET-1 (10-10 - 10-7M) by previously obtained in the laboratory). its cumulative addition with time interval In order to reach stability of the aorta, 8 minutes to organ bath in both intact it washed many times and the experimental (n=6 control, n=8TIH) and denuded aortic substances introduced into bath chambers rings (n=8 control, n=8 TIH). according to the protocols. The DRC for ET Group 3: BQ-788 (ETB Receptor -1 (10-10 - 10-7M) obtained in the absence antagonist) of incubation also, the aorta incubated with The aortic rings of control (n=8) and chemicals for 20 minutes then ET -1 (10-10 induced hyperthyroidism (n=5) pre- - 10-7M) DRC received in time interval of 8 incubated with selective ETB Receptor minutes. antagonist (BQ-788, 1µM ) for 20 minutes then DRC and EC50 obtained for ET-1 133 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) (10-10 - 10-7M) by its cumulative addition achieved by a post hoc Bonferroni’s with time interval 8 minutes to organ bath in multiple-comparison test. The data intact aortic rings. considered to be significantly different Data analysis when P <0.05. ET-1 induced contractions expressed as a percentage, and for each dose–response Results curve the maximum effect (Emax) and the The present results show that ET-1 concentration of ET-1 which produced half induced- aortic ring contraction (ET-1, of Emax (EC50) were calculated. Maximum 10-10-10-7 M) for each control and effects expressed as mean ± S.E.M. induced-hyperthyroid rats are shown in and EC50 as the geometric mean with the Table 1 , Figure 1. The pD2 value of its confidence limits (95%) for repeated concentration-response curve for ET-1 was experiments. Statistical comparisons of non-significantly lower in aortic rings in Emax and −log EC50 (pD2) values of the induced-hyperthyroid rats than in aortic dose–response curves for ET-1 obtained rings of control rats. with the different treatments in the arteries
Table 1: Maximum efficacy (Emax) and pD2 values of dose–response curves for the effects of ET-1 receptor antagonists on response to ET-1 in control and induced-hyperthyroidism in rats isolated intact and denuded aortic rings. Control Induced –Hyperthyroidism
Emax pD2 Emax pD2 Control Intact 115.1 ± 12.65 7.499 ± 0.208 100.0± 21.51 7.007 ± 0.500 BQ-123 Intact 103.1 ± 4.526 6.125 ± 0.436 100.0 ± 20.84 6.639 ± 0.763
BQ-788 Intact 88.94 ± 10.96 7.517 ± 0.190 143.7 ± 24.68 7.669 ± 0.264
Control denuded 100.0 ± 20.92 8.220 ± 0.276 100.0 ± 14.02 8.260 ± 0.187 BQ-123 denuded 100.0 ± 11.49 7.868 ± 0.217 100.0 ± 2.544 7.544 ± 0.082
Values are means ± S.E. Emax = maximum efficacy , pD2 = -log EC50.
Figure 1: Dose -response contractile curve for ET-1 in intact aortic rings of control and thyroxine-induced hyperthyroidism (TIH) (A). pD2 value for ET-1 in both groups (B), A Typical chart view traces (C). Values represented as mean ± S.E. 144 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The contractile potency of ET-1 and significantly (P <0.001) increased pD2 their DRC, did not change in endothelium- values of the concentration-response denuded aortic rings of thyroxine-induced curves for ET-1 from (7.007 ± 0.500 hyperthyroidism comparing to control rats to 8.260 ± 0.187). It means that the as shown in the Table 1 and Figure 2. contractile potency of ET-1 increased Furthermore, as seen in the Table 1, significantly. Endothelial denudation shifted Figure 3 and 4 mechanical removal of the DRC of ET-1 to the left versus intact endothelial cells in both groups, non- endothelium, and it has a significant significantly increased the pD2 value of difference with endothelium-intact DRC of ET-1 in control aortic rings. While, ET-1 at the dose(10-8M).
Figure 2: Dose -response contractile curve for ET-1 in denuded aortic rings of control and thyroxine-induced hyperthyroid rats (TIH) (A). pD2 value for ET-1 in both groups (B), A Typical chart view traces (C). Values represented as mean ± S.E.
Figure 3: Dose -response contractile curve for ET-1 in intact and denuded aortic rings of control rats (A). pD2 value for ET-1 in intact and denuded aortic rings (B), A Typical chart view traces (C) Values represented as mean ± S.E.
155 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Our data presented that incubation of minutes significantly increased the pD2 control intact aortic rings with ETA value in comparison with DRC for an receptor antagonist (BQ-123, 0.3 µM) for ET-1in absence of antagonists of control 20 min decreased the contractility of ET-1 rings (7.499 ± 0.208 control versus BQ-788 produced vasoconstriction with rightward 7.517 ± 0.190). It also decreased Emax displacements of the ET-1 DRC and significantly from 115.1 ± 12.65 of control caused a significant reduction of the pD2 to 88.94 ± 10.96 of BQ-788. Moreover, value (7.499 ± 0.208 control versus BQ- there is a significance P <0.05 difference 123 6.125 ± 0.436). Also, it is significantly of pD2 value between the DRC of ET-1 pre different at doses (10-8M and 5×10-8M -incubated with BQ-123 and DRC of ET-1 versus control). While, pre-treatment of pre-incubated with BQ-788, Table 1 and control intact aortic rings with the ETB Figure 5. receptor antagonist (BQ-788, 1µM) for 20
Figure 4: Dose -response contractile curve for ET-1 in intact and denuded aortic rings of thyroxine-induced hyperthyroid rats (TIH) (A). The pD2 value for ET-1 (B). A Typical chart view traces (C). The star sign shows the comparison with Intact-TIH, *P <0.05, ***P <0.001. Values represented as mean ± S.E.
Figure 5: Dose -response contractile curve for ET-1 in intact aortic rings with absence (Control) and presence of ETA receptor antagonist (BQ 123, 0.3 µM) and BQ-788, 1µM) in rats with control (A). The pD2 value for ET-1 (B). A Typical chart view traces (C). The star sign show the comparison with control: *P <0.05, **P <0.01, ***P <0.001 while hash sign show comparison with BQ-788, # #P <0.01.Values represented as mean ± S.E. 166 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Pre-incubation of induced-hyperthyroid different with DRC for ET-1in intact aortic intact aortic rings with ETA receptor rings of TIH at doses (5×10-8M and 10-7 antagonist (BQ-123, 0.3 µM) for 20 min non M). Despite these, there is a significant -significantly affected the ET-1 induced difference observed between intact aortic contraction in a concentration-dependent rings pre-incubated with BQ-123 (0.3µM) manner decreased pD2 and without the and intact aortic rings pre-incubated with change in Emax. In contrast, pre-treatment BQ-788 (1µM) of pD2 value from 6.639 ± of intact aortic rings in induced- 0.763 of TIH-BQ-123 to 7.669 ± 0.264 TIH- hyperthyroid rats with the ETB receptor BQ-788. The Emax significantly increased antagonist (BQ-788, 1µM) for 20 minutes from 100.0 ± 20.84 of TIH-BQ-123 to 143.7 led to a significant change in the ET-1 ± 24.68 of TIH-BQ-123.Besides, the DRC induced contraction. It also shifted the of ET-1 pre-incubated with BQ-123 curve to the left and increased pD2 value (0.3µM) significantly different at doses (10- p<0.001 from 7.007 ± 0.500 of TIH to 7.669 8M, 5×10-8M, and10-7M) in comparison ± 0.264 of TIH-BQ-788. As well as Emax with TIH-BQ-788 as shown in the Table 1 from 100.0 ± 21.51 of TIH to 143.7 ± 24.68 and Figure 6. of TIH-BQ-788. Also, it is significantly
Figure 6): Dose -response contractile curve for ET-1 in intact aortic rings with absence (Control) and presence of ETA receptor antagonist (BQ 123, 0.3 µM) and BQ-788, 1µM) in thyroxine-induced hyperthyroidV (TIH) rats (A). The pD2 value for ET-1 (B). A Typical chart view traces (C). The star sign show the comparison with TIH, *P <0.05, **P <0.01, ***P <0.00 while hash sign show comparison with TIH-BQ-788, # P <0.05, # #P <0.01, # # #P <0.001. Values represented as mean ± S.E.
177 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Pre-incubation of denuded aortic rings with augmented the contractile potency of ET-1, ETA receptor antagonist (BQ-123, 0.3 µM) significantly decreases ET-1 pD2 value for 20 minutes in control slightly shifted from 8.260 ± 0.187 of TIH-denuded to the DRC for ET-1 to the right and non- 7.544 ± 0.082 of TIH-BQ123-denuded. significantly slightly reduced pD2 value as Furthermore, it was significantly different at seen in the Table 1 and Figure 7. Contrary, doses (10-8M and 5×10-8M) in comparison BQ-123 pre-incubated in denuded aortic with TIH-denuded as shown in the Table 1 rings of induced-hyperthyroid group and Figure 8.
Figure 7: Dose-response contractile curve for ET-1 in denuded aortic rings with absence (Control) and presence of ETA receptor antagonist (BQ 123, 0.3 µM) in rats with control (A). The pD2 value for ET-1 (B). A Typical chart view traces (C). Values represented as mean ± S.E.
Figure 8: Dose-response contractile curve for ET-1 in denuded aortic rings with absence (Control) and presence of ETA receptor antagonist (BQ 123, 0.3 µM) in rats with thyroxine-induced hyperthyroidism (TIH) (A). The pD2 value for ET-1 (B). A Typical chart view traces (C). The star sign show the comparison with TIH: *P <0.05, ***P <0.001. Values represented as mean ± S.E.
188 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Discussion ET-1 DRC in the absence of BQ-123. He et The results obtained from the in vitro al. (2007) recorded that the ETA antagonist experiment revealed that l-thyroxine BQ-123 induced a higher relaxation than inducing hyperthyroidism did not the BQ-788 did in the ET-1-precontracted significantly alter the dose-response curve internal mammary artery.18 Marrachelli of ET-1 and the pD2 value of ET-1with their et al. (2006) reported that BQ-123 Emax slightly decreased without being significantly inhibited the contractile the significant change. The similar results response to ET-1 of renal arteries from obtained by the.14 In the VSMCs, both ETA control rabbits.19 Inversely, pre-incubation and ETB receptors may couple to PLC via of endothelium-denuded control aortic a GTP-binding protein. Activation of PLC rings with BQ-123 did not significantly causes phosphatidyl inositol hydrolysis, change the DRC of ET-1 with their pD2 rapid formation of IP3, and accumulation and Emax value when compared to of DAG. IP3 stimulates the release denuded aortic rings in the absence of of Ca2+ from intracellular stores, BQ-123. These findings indicate that including endoplasmic reticulum.15 Our endothelium has a great role in relaxation. data presented that endothelial denudation In contrast, the current result showed that of aortic rings of control rats shifted in induced-hyperthyroid rats pre-incubation the dose response curve of ET-1 to of aortic rings with BQ-123 did not make the left and increased pD2 value non- any significant change in dose-response significantly. It confirms that ET-1 makes curve induced by ET-1 compared to ET-1 a vasocontraction via ETA receptor present DRC in the absence of BQ-123 incubation. on SMCs.16 Meanwhile, ET-1 induce Interestingly, endothelial denudation in contraction is endothelium independent. induced-hyperthyroid rats pre-incubation Tirapelli et al. (2005) reported that of aortic rings with BQ-123 lead to endothelium denudation leads to an a significant decrease in contractile enhancement in the Emax values with potency of ET-1 induced contraction no differences in the pD2 value when without the change in Emax value as well compared to endothelium-intact carotid as the decrease in pD2 value. Accordingly, rings.17 Moreover, in the induced- the main reason that caused this result hyperthyroid group, the endothelium may be due to ETA receptor expression denudation of aortic rings significantly up-regulated on SMCs of induced shifted the DRC of ET-1 to the left, and hyperthyroid rat aorta or due to ED caused the potency of ET-1 increased without by hyperthyroidism. In contrast, Donckier changing the Emax compared to the intact et al. (2003) reported that ET-1 and ETA aortic rings. Hyperthyroidism has a great receptor overexpressed in thyroid papillary effect on endothelium as previously carcinoma and Hashimoto's thyroiditis.13 described; it causes ED. This finding may Furthermore, pre-incubation of aortic rings be due some changes happened to of control rats with ETB receptor antagonist ETA receptor on SMCs and may be (BQ-788) significantly increased the pD2 of overexpressed in hyperthyroid rats ET-1it means increases ET-1 potency Incubation of control rat aortic rings with while it is significantly declined the Emax BQ-123 significantly inhibited the ET-1 in comparison with DRC of ET-1 in the induced contraction in a concentration- absence of BQ-788. Previously it has been dependent manner and produced shown that pretreatment of mesenteric a rightward displacement of ET-1 dose arteries with the BQ-788 significantly response curve. Meanwhile, the pD2 value increased the potency of ET-1 in the Wistar significantly decreased it means that the group but led to the non-significant change ET-1 potency decreased and the Emax inGoto-kaki Zaki diabetic group.20 The pD2 slightly reduced compared to aortic rings value of ET-1 of aortic rings incubated with 9 19 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) BQ-788 significantly increased compared and Et A/Et B receptors, But not in calcium to aortic rings incubated with BQ-123, while handling mechanisms, In deoxycorticosterone Emax slightly decreased. In the present acetate-salt hypertension. Brazilian journal of medical and biological research 2002; 35:1061– study, pre-incubation of aortic rings of 8. induced-hyperthyroid rats with BQ-788 5. Thorin E, Lucas M, Cernacek P, Dupuis J. Role of shifted the DRC of ET-1 to the left. Despite, ETA receptors in the regulation of vascular it is significantly increased the contractile reactivity in rats with congestive heart failure. American journal of physiology-heart and potency of ET-1 with the significant circulatory physiology 2000; 279:844–51. increase in Emax of ET-1 in comparison 6. De Mey J G, Compeer M G, Lemkens P, Meens M with ET-1 DRC in the absence of BQ-788. J. ET A-Receptor antagonists or allosteric This result may be due to inhibition of modulators? trends in pharmacological sciences vasodilation effect of ETB receptor via 2011; 32:345–51. 7. Ohuchi T, Kuwaki T, Ling G Y, Dewit D, Ju K H, a release of NO, prostacyclin and ATP- Onodera M, Cao W H, Yanagisawa M, 21 gated K+ channels . On the other hand, Kumada, M. Elevation of blood pressure by the Emax and pD2 of ET-1 incubated genetic and pharmacological disruption of with BQ-788 of induced-hyperthyroid rats the ETB receptor in mice. American Journal of Physiology-Regulatory, Integrative and significantly higher than Emax and pD2 comparative physiology 1999; 276:1071–7. value of ET-1 incubated with BQ-123. 8. Ruffolo Jr, R. R. Endothelin receptors: From the So, the possible reason for the present gene to the human 1995; Crc Press. results may be due to down-regulation of 9. Scanlon V C, Sanders T. Essentials of anatomy ETB receptor in aortic rings of induced and physiology 2014; Fa Davis. 10. Kedzierski R M, Yanagisawa M. Endothelin hyperthyroidism. system: The double-edged sword in health and disease. Annual review of pharmacology and Conclusion toxicology 2001; 41:851–76. In induced-hyperthyroid rats BQ-788, but 11. Tamer I, Sargin M, Sargin H, Seker M, not BQ-123 enhanced contractile response Babalik E, Tekce M, et al. The evaluation of left ventricular hypertrophy in hypertensive patients to ET-1Pre-incubation of BQ-123 did not with Subclinical hyperthyroidism. Endocr J 2005; lead to change the contractile response of 52. denuded rings in control rats, but pre- 12. Canaris G J, Manowitz N R, Mayor G, Ridgway E incubation of this antagonist in hyperthyroid C. The Colorado Thyroid Disease Prevalence Study. Arch Intern Med 2000; 160:526–34. denuded aortic rings led to inhibition of 13. Donckier J E, Michel L, Van Beneden R, contractile response to ET-1. These new Delos M, Havaux X. Increased expression of findings taken together returned to ETA endothelin‐1 and its mitogenic receptor ETA receptor activity located smooth muscle in human papillary thyroid carcinoma. Clinical aortic rings in hyperthyroidism. Endocrinology 2003; 59:354–60. 14. Mcallister R M, Luther K L, Pfeifer P C. Thyroid References status and response to endothelin-1 in rat arterial vessels. American Journal of Physiology- 1. Yanagisawa M., Kurihara H., Kimura S, Tomobe Endocrinology and Metabolism 2000; 279:252–8. Y, Kobayashi M, Mitsui Y, et al. A novel potent 15. Katzung G, Bourne H, Von Zastrow M, Holford vasoconstrictor peptide produced by vascular N, Correia M, Berkowitz B. Basic principles of endothelial cells. Nature 1988; 332:411–5. pharmacology. Basic and clinical pharmacology, 2. Vignon-Zellweger N, Heiden S, Miyauchi T, Emoto 12th edition 2010; New York: Mcgraw-Hill N. Endothelin and endothelin receptors in the Medical. renal and cardiovascular systems. Life Sciences 16. Barrett K E, Ghishan F K, Merchant J L, Said H 2012; 91:490–500. M, Wood J D, Johnson L R. Physiology of the 3. Cardillo C, Kilcoyne C M, Waclawiw M, Cannon R gastrointestinal tract, elsevier science 2006. O, 3Rd & Panza J A. Role of Endothelin In 17. Tirapelli C R, Casolari D A, Yogi A, The Increased Vascular Tone Of Patients With Montezano A C, Tostes R C, Legros E, et al. Essential Hypertension. Hypertension 1999; Functional characterization and expression of 33:753–8. endothelin receptors in rat carotid artery: 4. David F, Montezano A, Rebouças N, Nigro D, Involvement of nitric oxide, A vasodilator Fortes Z, Carvalho M, Tostes R. Gender prostanoid and the opening of K+ channels differences in vascular expression of endothelin in ETB‐induced relaxation. British journal of
1020 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) pharmacology 2005; 146:903–12 18. He GW, Liu MH, Yang Q, Furnary A, Yim AP. Role of endothelin-1 receptor antagonists in vasoconstriction mediated by endothelin and other vasoconstrictors in human internal mammary artery. The annals of thoracic surgery 2007; 84:1522–7. 19. Marrachelli VG, Miranda FJ, Alabadí, JA, Lloréns S, Alborch E. Contribution of endothelin receptors and cyclooxygenase-derivatives to the altered response of the rabbit renal artery to endothelin-1 in diabetes. European journal of pharmacology 2006; 534:178–86. 20. Matsumoto T, Ishida K, Nakayama N, Kobayashi T, Kamata K. Involvement of no and MEK/ERK Pathway In Enhancement Of Endothelin- 1-Induced Mesenteric Artery Contraction In Later-Stage Type 2 Diabetic Goto-Kakizaki Rat. American Journal Of Physiology-Heart And Circulatory Physiology 2009; 296:1388–97. 21. Yuan J X J, Garcia J G N, Hales C A, Rich S, Archer S L, West J B. Textbook Of Pulmonary Vascular Disease 2011; Springer Us.
1121 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Formulation and Evaluation of Valsartan
Oral Granule
Anoosh Bashir Hagop1* Anjam Hama Abdalla1 Hewa Abdulla Hamadameen1 Abstract Background and objective: Oral dosage form is a popular route of drug delivery and the most common types are tablets and capsules because of their ease administration, patient acceptance and stability of formulation, while administration of such dosage forms have drawback such as difficulty in swallowing in pediatric and geriatric patients. Oral granules dosage form as sachet or dry syrup of valsartan is an alternative dosage form to be used. Methods: Wet granulation method was used to prepare two different formulations of valsartan granules, novel formula (F1) and conventional formula (F2). The two formulas prepared by the same method with different technique of mixing. Where F1 was prepared by dissolving the drug in limited quantity of ethanol and PVP in D.W separately then both solutions were mixed and added to the remaining dry ingredients slowly to form a granules after sieving and drying process in the oven. While in the case of F2 all dry ingredients including the drug were mixed together except the PVP which dissolved separately in D.W then the prepared solution was added slowly and the granules were formed after sieving and drying the prepared mixture in oven. Results: The study revealed that F1 has accepted value of dissolution profile where within 30 minutes about 96.84% of drug was released from F1 which is within the acceptable limit, while for F2 only 64.60% of the drug was released within the same time. This differences in the release profile was statistically significant (P <0.05) between F1 and F2, but a non- significant difference (P >0.05) was seen between F2 and conventional valsartan tablet using diovan as a reference. Conclusion: The study concluded that formulation of F1 dry granule can be used as an alternative method to prepare dry granules and to be used by pediatrics and geriatrics patients instead of tablets and capsules. While F2 needs improvement in solubility before using in the preparation of poorly water soluble drugs like valsartan which is within class II BCS (low solubility and high permeability). Keyword: Valsartan; Sachet; Wet granulation method; Oral granule.
Introduction other classes of antihypertensive drug.2 About one third of hypertensive patients will Valsartan is effective in treatment of reached goal blood pressures of <140/90 pediatric, adolescents and the elderly mm Hg with monotherapy, regardless of patients with mild to moderate the medication used.1 Valsartan is an hypertension. 3 There are several classes angiotensin II-receptor antagonist that has of pharmaceutical dosage forms, an oral been available overseas for several years route is the most accepted route of which can be use to decrease blood administration. Tablets and capsules are pressure by the same ratio such as unsuitable for given large doses of other angiotensin II-receptor antagonists. medicine, since single high dose is difficult This class of antihypertive drug have same to swallow, or needed the administration of blood pressure lowering effects to those of several pills or capsules at the sometime,
1 Department of Pharmaceutics, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 221 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) it is not accepted by patients. The chewing 1.2. Required characteristics of oral process need teeth, taste cannot be granules for reconstitution8,9 masked completely and lack of control - Powder blend must be a uniform mixture release probability chewable tablets are of the suitable concentration of each not ideal with pediatric and geriatric ingredient and during reconstitution. patients. Hence it is essential to develop -The powder blend must disperse rapidly a reconstitutable granule dosage form.4 and completely in the aqueous vehicle. Oral granules are solid dosage form that - Final product must have an acceptable can be reconstituted by the addition of appearance, odor and taste. water to administer by oral route. Mostly 1.3. Rapidly dispersed excipients on antibiotics, some moisture sensitive and reconstitution9 pediatric drugs are available in the form of - Granule disintegrant: It results in dry granules and prepared as dry powder prevention of the particles aggregation. mixtures or granules that are intended to - Granule binder: It helps to reduce the be suspended in water or some other settling of particles in suspensions and vehicle before oral administration. Oral also used as a stabilizer such as high granules can offer many positive points like molecular weight povidone. remaining of the chemical stability of the - Suspending agents: It should be easily active compounds until reconstitution at dispersed during reconstitution. Some of the time of use and can be easily given to the suspending agents that are children of different ages by adjusting the recommended for use are acacia, volume to swallow.5,6 Hence, the aim of this carboxymethylcellulose sodium and study is to prepare a new dosage form of xanthan gum. valsartan as oral granules and sachet by - Sweeteners: Sweeteners can mask the wet granulation method to be used as unfavorable taste and enhance patient alternative dosage form that can occupy acceptance in the pediatric population the place of the conventional valsartan that uses this product. It is a significant tablets and also to study different component of granules for reconstitution parameters affecting dissolution behaviour such as sucrose, mannitol, dextrose and of the prepared formulas (F1 and F2) aspartame. then comparing it with that of valsartan - Wetting agents: Many drugs are conventional tablet (Diovan® 80mg tablet) hydrophobic; they repel water and are not as a reference tablet. easily wetted. The appropriate wetting 1.1. Advantages of oral granules for oral agent must be selected for optimum use6, 7 dispersion of the drug at the lowest - Accurate single dosing as the dose is effective concentrations such as packed in single dose sachets making polysorbate 80 and sodium lauryl sulfate. the formulation easy to carry and - Other excipients: The other excipients enhanced convenience of single dosage include buffers, preservatives, flavors and regimen. colors.10 - Drug dose is relatively independent 1.4. Method of preparation of dry of any physical factors like temperature, mixture11 sedimentation rate and liquid flow - Direct mixing method: it is the easiest properties. way to manufacture tablets and granules. - Colored, flavored, sweetened formulation The great advantages of this method are is advantageous for administration to the the low manufacturing cost, conventional pediatric population. equipment, commonly available excipients - Stable on storage and when and a limited number of processing steps reconstituted with an ingestible liquid for involved in direct compression. administration.
232 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) - Dry Granulation (Slugging) method: A tiny amount of valsartan powder was Dry granulation also referred to as introduced in to a small glass capillary tube precompression or double compression is 10-15 cm long, which is closed at one end, a size enlargement process designed to attaching this to the stem of a thermometer improve the flow and compression centred in a melting point apparatus. characteristics of powders that would The sample was heated slowly, while the otherwise be unsuitable for compression. sample was carefully observed. The The process involves compaction of temperature at which melting begins and powder particles into large pieces or complete was recorded as a melting compacts which are subsequently broken point.12 down into granules to produce granules 2.4. Determination of calibration curve that can be further processed into dosage of valsartan forms. 2.4.1.Preparation of standard stock - Wet Granulation (wet massing) solution13 method: is a process of size enlargement Standard drug solution of valsartan in which fine powder particles are was prepared by dissolving 10mg pure agglomerated or brought together into valsartan in methanol and distilled water larger, strong and relatively permanent then transferred into 100ml volumetric flask structure called granules using a suitable to obtain 100µg/ml of stock solution from non-toxic granulating fluid such as water or which desired concentrations of solutions ethanol. Powder mixing, in conjunction with were prepared. the cohesive properties of the granulating 2.4.2. Determination of calibration curve agent, enables the formation of granules. of valsartan14 The characteristics and performance of the Standard stock solution was suitably final product, greatly depends on the extent diluted with distilled water to obtain to which the powder particles interact with concentrations ranging from 1-10 µg/ml. each other to form aggregates (granules). Absorbance of these solutions was Therefore wet granulation technique uses measured at 255nm (λmax of valsartan) the same preparatory and finishing steps of using UV-spectrophotometer, calibration direct compression and dry granulation (dry curve was obtained by plotting graph screening and mixing); with additional between concentration and absorbance. steps of wet massing, wet screening and 2.5. Drug–excipient compatibility drying. study Fourier Transforms Infrared Spectroscopy (FT-IR) Studies: Methods The FT-IR (Jasco, FT/ IR- 4600)) was used 2.1.Materials for the IR analyses in the frequency range Valsartan was obtained as a gift powder between 4000 and 400 cm-1 and at 1 cm-1 from Awamedica Pharmaceutical resolution. The instrument was calibrated Company, Erbil/ Iraq, polyvinyl pyrollidone, by using polystyrene film. Pure drug and lactose, orange flavour, sorbitol and excipients were selected separately.15 The distilled water were also used. IR peaks of pure valsartan were analyzed 2.2.Instruments used in the study and compared with the peaks of the The instruments used in the study include obtained formulations (F1 and F2). sensitive balance, melting point apparatus, 2.6. Formulation of valsartan sachet pH meter, UV-spectrophotometer, FTIR, using wet granulation method: distillator, dissolution apparatus type II Wet granulation method was used to paddle, magnetic stirrer and shaker. prepare two different formulations of 2.3.Melting point determination valsartan sachet named novel formula (F1) The melting point of valsartan was and conventional formula (F2) as shown in measured by using capillary tube method. Table1. Both formulas were prepared by
243 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) the same ingredients and method, the only 2.7.3. Dissolution test: differences between them was in the The release of valsartan sachet was technique of mixing. For F1 which is the determined using USP dissolution testing novel formula prepared by dissolving apparatus type II (paddle method). The the drug and polyvinyl pyrollidone (PVP) dissolution test was performed using 900 separately in small quantities of ethanol ml of phosphate buffer solution (pH 6.8) at and distilled water (D.W), respectively, then 37 ± 0.1°C and 50 rpm. A sample (5 ml) these liquid mixtures were mixed together of the solution was withdrawn from the and finally added to the previously dissolution apparatus at different time thoroughly mixed dry ingredients. While for intervals and the samples were replaced F2 which is the conventional formula, only with fresh dissolution medium. The the PVP was dissolved in small quantity samples were filtered through a 0.45μ of distilled water then the formed liquid membrane filter and diluted to suitable mixture was added to the remaining concentration with buffer solution (pH 6.8). previously mixed dry ingredients. After Absorbance of these solutions was preparation of both formulas, all ingredients measured at λmax 255 nm using a UV- were passed through mesh # 60 and dried visible spectrophotometer. Cumulative in the oven for 30 minutes to get a granules percentage drug release was calculated and form a sachet. using regression equation obtained from 2.7.Post preparation evaluation a standard curve.16 parameters of valsartan sachet 2.7.4. Comparison with marketed 2.7.1. Weight variation test: product (diovan): Ten sachets were taken and their weight The release characteristics of valsartan was determined individually and collectively sachet (F1 and F2) were compared with on a digital weighing balance. The average available marketing product of valsartan weight of one sachet was determined from tablet (diovan)R the collective weight.16 2.8. Statistical Analysis 2.7.2. Weight content uniformity: All experiments were carried out in Six sachets of 300 mg equivalent weight of triplicate. The values were represented valsartan dry granules was accurately as mean ± standard deviation. The weighed and transferred into a 100 ml differences in release profile of new volumetric flask containing (20ml ethanol developed formula (F1) and marketed and 80ml buffer solution). The solution conventional tablet were tested for in the volumetric flask was shaked for significance by using independent two 10 minutes using magnetic stirrer and samples t-test with the aid of the statistical shaker then filtered, diluted suitably and package for the social sciences (version drug content was analyzed using UV- 18) program. Differences were considered Visible spectrophotometer (analytiKjena, statistically significant when (P <0.05). SPECORD 40) at λmax 255 nm.16
Table 1: Formulation of valsartan oral granule (sachet).
Formula name Valsartan PVP Orange flavour Sorbitol Lactose Total (mg)
F1 40 2.5 5 20 232.5 300
F2 40 2.5 5 20 232.5 300
254 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Results 18 λ max at 255 nm, as shown in Figure 1. 3.1.Melting point determination 3.3.Determination of calibration curve of The measured melting point of valsartan valsartan was 115-120°C which is within the reported Calibration curve data and calibration value of 116°C ;17 this indicates the rational curve parameters for valsartan in methanol purity of drug powder. with distilled water analyzed by UV- 3.2. Determination of λ max (maximum spectrophotometer at 255 nm demonstrate absorbance) that calibration curve was linear in the Valsqartan solution was scanned by concentration range from 1 to 10μg/ml. UV-spectrophotometer at a wave length of The correlation coefficient was found to be 200-400 nm in 1 cm cell. The spectrum (0.999) and the calibration plot is illustrated reveals that valsartan shows a well defined in Figure 2.
Figure 1: UV-Scan of valsartan in methanol with phosphate buffer (pH 6.8).
Figure 2: Calibration curve of valsartan in methanol with phosphate buffer (pH 6.8).
265 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 3.4.Drug–excipient compatibility study taken for the characterization studies Fourier Transformation Infra-red (FTIR) as shown in figures (3), (4) and (5), analysis respectively. IR spectrum of pure valsartan This study was done by FTIR to powder showed characteristic peaks at evaluate the compatibility of valsartan with 2963.09 cm-1 due to C=N stretching, at pharmaceutical excipients of common use 1731.76 cm-1due to carboxylate stretching. as diluents and binder. The infrared (IR) The spectra also showed bands at 1731.76 spectra of pure valsartan drug powder and cm-1 due to C=O bending, at 1105.01 cm-1 the prepared formulas (F1 and F2) were due to C-N bonding. 19
Figure 3: FTIR spectrum of pure valsartan powder.
Figure 4: FTIR spectrum of F1.
Figure 5: FTIR spectrum of F2.
276 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 3.5. Post preparation evaluation results are expressed as a mean ± S.D parameters of valsartan sachet (n=6). 3.5.1.Weightvariatioen test: 3.5.3.Dissolution test of F1 and F2 in Weight variation of the prepared valsartan comparison with diovan: sachet was determined and listed in The dissolution test of the novel (F1) and Table 2. The result show that there was conventional (F2) formulas of the prepared a non-significant differences (P >0.05) valsartan sachet as well as valsartan between F1 and F2 and the results are conventional tablet (Diovan® 80 mg tablet) expressed as a mean ± S.D (n=10). as a reference was done according to 3.5.2.Weight content uniformity: USP, using 900ml of phosphate buffer The calculated percentage of the weight solution (pH 6.8) as a dissolution medium content uniformity of the prepared formulas at 37°C with constant stirring speed of 50 F1 and F2 with diovan are listed in rpm for 45 minutes and the results listed Table 3. Where there was a non-significant in Table 4 and are expressed as a mean ± differences (P >0.05) between the prepared S.D (n=6). valsartan sachet and diovan, also the
Table 2: Weight variation test of F1 and F2 with diovan.
Formula name Weight variation (mg)
F1 299.4±4.86
F2 296.9±4.39
Diovan 80.9±6.54
Table 3: Weight content uniformity of F1 and F2 formulations with diovan.
Formula name Weight uniformity (mg)
F1 96.06±1.96
F2 95.93±3.31
Diovan 95.45±1.43
287 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Figure 6 revealed the comparison of the 75% of the drug content.12 The results release profile of the prepared valsartan indicate that within 30 minutes about sachet (F1 and F2) with that of the 96.84% and 78.24% of drug was released valsartan conventional tablet using from F1 and diovan respectively which is diovanas a reference. Depending on the within the acceptable limit, while for F2 USP pharmacopeia for the solid dosage only 64.60% of the drug was released forms 30 minutes is needed to release within the same time.
Table 4: Dissolution profile of F1 and F2 with diovan.
Percentage of release Percentage of release Percentage of release Time (min.) of F1 (%) of F2 (%) of diovan (%)
10 55.51 35.7 40.19
20 77.18 50.77 60.97
30 96.84 64.6 78.24
45 86.09 37.55 68.95
Figure 6: Dissolution profile of F1 and F2 with diovan.
298 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
References that there was a non-significant differences Pre-preparation evaluation parameters (P >0.05) between F1 and F2 with diovan. of pure valsartan powder: Dissolution test of F1 and F2 in The rational purity of pure valsartan powder comparison with diovan: indicated by the result obtained from the The rate and extent of dissolution of the measurement of melting point with the active ingredient from any dosage form measurement of the maximum absorbance often determines the rate and extent of and the preparation of the calibration curve. absorption of the drug.20 In case of Post preparation evaluation parameters valsartan which has low water solubility, of valsartan sachet: dissolution may be the rate- limiting step in Post preparation evaluation parameters the process of drug absorption; therefore were done for the prepared formulas F1 drugs with low water solubility have been and F2 with the conventional tablet of shown to be unpredictably and slowly valsartan using diovan as a reference. absorbed compared with drugs of higher Drug–excipient compatibility study: solubility and a better oral formulation Fourier Transformation Infra-red (FTIR) like valsartan sachet (F1 and F2) can analysis be developed by increasing the water The comparison of the IR spectrum of F1 solubility of such drugs. Based on the USP and F2 with that of pure drug showed all pharmacopeia for the solid dosage forms the characteristic peaks of pure valsartan 30 minutes is needed to release 75% of and revealed that there is no appreciable the drug content.12 The results indicate that change in the positions of characteristic within 30 minutes about 96.84% and absorption bands of groups and bonds. 78.24% of drug was released from F1 and The spectra of these, even though slightly diovan respectively which is within the differ in appearance but no change is acceptable limit, while for F2 only 64.60% observed in the positions of the bands in of the drug is released within the same the spectra. This clearly suggests that the time. Also the results showed that after 10, drug remains in the same form even in 20, and 45 minutes the percentage of its formulations indicating that there is no drug release from F1 was 55.51%, 77.18% interaction between the drug and excipients and 86.09% respectively and for diovan used for the preparation of valsartan was 40.19%, 60.97%, and 68.96% sachet. respectively. Whereas the percentage of Weight variatioen test: drug release from F2 for the same The results of weight variation test time was 35.70%, 50.77% and 57.85% indicated that both formulas (F1 and F2) respectively. So, this indicates that there were within pharmacopoeial limits (not was a significant differences (P <0.05) in more than two tablets differ from the the dissolution of the novel formula (F1) average by no more than 10% and no which was faster than the conventional tablets differ by more than 20%),19 the formula (F2) of the prepared valsartan result indicate that there was a non- sachet, while a non- significant differences significant differences (P >0.05) between (P >0.05) was obtained between F1 and F1 and F2. the conventional valsartan reference Weight content uniformity: tablets (Diovan® 80 mg tablet). This The drug content uniformity of the prepared significant improvement in the dissolution valsartan sachet (F1 and F2) complies of the prepared valsartan sachet can with USP criteria; it was found that no be related to that F1 does not sachet from ten sachets lies out of the need disintegration process because is label claim,19 which indicates a uniform a granule dosage form and it is known that distribution and a proper dose of the drug decreasing of the disintegration time leads in the sachet. Therefore the results indicate to increase in dissolution rate,21 because 30 9 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) faster disintegration delivers a fine of Universal Pharmacy and Bio Sciences 2014; suspension of drug particles resulting in 3(2):93–111. 7. Bardeskar C, Geeverghese R. Reconstitutable a higher surface area and faster 22 Oral Suspensions (Dry Syrups): An Overview. dissolution. While the same improvement World Journal of Pharmaceutical Research. not observed with F2 due to the 2014; 4(3):462–84. problem of solubility of valsartan which 8. Damor SR, Jethara SI, Patel MS, Patel MR. A is within the class II drugs depend in Review On Dual Release Oral Reconstitutable Suspension. World Journal of Pharmaceutical on biopharmaceutical classification system Research. 2015; 4(3):592–613. (BCS). Therefore F2 needs improvement in 9. Bhandare P, Yadav A. A Review on “Dry Syrups its solubility before preparation as a sachet ForPaediatrics”. Int J Curr Pharm Res 2015; or any other dosage form. 9(1):25–31. 10. Pavane M, Shirsat M, Dhobale A, Joshi D, Conclusion Dhembre G, Ingale P. Formulation, Development and Evaluation of Oral Reconstitutable Dry 5.Conclusion Syrup. Indo American Journal of Pharmaceutical Valsartan oral granule as sachet or dry Sciences 2018; 5(1):483–91. granules can be used as alternative 11. Tsue S, Abe S, Shimotori T, Sugisawa K. Evaluation of Direct Compression Tablet dosage form The prepared novel formula prepared by a new Super fine Powder of (HPC) F1 provided a significant improvement in Hydroxypropyl Cellulose. Nihongi Laboretory, the drug release profile in comparison with Nippon Soda Co., Ltd., Japan 2011. conventional formula F2 and the marketed 12. USP-30 NF-25 (2007), United State Pharmacopoeial Convention. Electronic Version. conventional valsartan tablet. The overall 13. Kishanta Kumar Pradhan and U. S. Mishra. results indicates the possibility of using the Development and validation of stability indicating novel formula F1 in the preparation of RP-HPLC method for the determination valsartan oral granules, as a new dosage of valsartan. Int. J. of Pharmacy and form for the oral administration with fast pharmaceutical sciences 2015; 7(8): 57–61. 14. Nataraj KS, Ramakrishnama Charya SV, dissolution time, that can occupy the place Swathi Goud E, Saigeethika S, Ramanjineyulu of the conventional valsartan tablets. K. Simple quantitative method development and validation of valsartan in pureform and Competing interests pharmaceutical dosage forms byuv – The authors declare that they have no spectroscopy. International Journal of Pharmacy and Biological Sciences. IJPBS 2011; 1(2). competing interests. 15. Usha Sri B, Indira Muzib Y, DVRIN Bhikshapathi, Sravani R. Enhancement of solubility and oral References bioavailability of poorly soluble drug valsartan by 1. Tripathi KD. Essentials of Medical Pharmacology novel solid self emulsifying drug delivery system. 2005; 5 th Edition: 476–554. Int J of drug delivery 2015; 7:13–26. 2. Alfred Goodman Gilman, Theodore W. Rall, Alan 16. Bhabani Shankar Nayak, Sruti Ranjan S. Nies, Palmer Taylor. The Pharmacological Mishra, Harekrishna Roy. Valsartan Fast Basis of Therapeutics, Eighth Edition, Volume II Dissolving Tablets: Formulation and In vitro 50–64. Characterization. Journal of Chemical and 3. Flesch G, Muller P, Lloyd P. Absolute Pharmaceutical Research 2018; 10(3):182–9. bioavailability and pharmacokinetics of valsartan, 17. Clarke’s Analysis of Drugs and Poisons (2011). an angiotensin II receptor antagonist, in man. Eur Electrical edition, pharmaceutcal press. J Clin Pharmacol 1997; 52(2):115–120. 18. Khan F, Lohiya RT, Umekar MJ. Development 4. Mali RR, Goel V, Gupta S. Novel study in of UV Spectrophotometric method for the sustained release drug delivery system: simultaneous estimation of Meloxicam and A Review. Int J Pharm Med Res 2015; 3(2):204– Paracetamol in tablet by simultaneous Equation, 15. Absorbance ratio and Absorbance Correction 5. Kundu S, Patil AV, Srinivasan G, Borkar N. method. Int J Chem Tech Res 2010; 2(3):1586– Controlled Release Suspension: A Review. 91. International Journal of Pharmaceutical 19. Balamuralidhara V, Sreenivas SA, Gangadhara Innovations 2011; 2(4):1–18. HV, Pramodkumar TM. Investigation on the 6. Patel M, Patel KR, Patel MR, Patel NM. Effect of Different Disintegrants on the Formulation and Evaluation of Microemulsion Orodispersible Tablets of rabeprazole. Asian J Based Gel of Ketoconazole International Journal Sci Res 2009; 4:12–9.
1031 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 20. El-badry M, Fathy M. Enhancement of dissolution and permeation rates of meloxicam by formation of its freeze dried solid dispersion in polyvinyl pyrrolidine K-30. Drug DevInd Pharm 2006. P. 141–2. 21. Takeuchi H, Tanimura S, Nagira S, Yamamoto H, Kawashina Y. Tableting of Solid Dispersion Particles Consisiting of Indomathacin and Porous Silica Particles. Chem Pharm Bull 2005; 53 (5):487–91. 22. Malikarjuna SC, Prasad DV, Gupta VR. Development of fast dispersible aceclofenac tablets: Effect of functionality of super disintegrants. Ind J pharm Sci 2008; 70(2):180–5.
1132 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Investigation Evaluation of some heavy metals in different canned food samples in Erbil markets
Hemn K. Qadir1* Hemn A. Qader 1 Tariq F. Abdullah1 Abstract Background and objective: Metal pollution of waterways directly affects human health and can impact the food chain. The purpose of this study was to analyze and quantify some heavy metals in fifty-six of different canned food samples (Luncheon meat, powder milk, beans, peas, tomato paste, beverage, black olive, coffee powder, and cheese) that available in Erbil markets. Methods: Concentrations (μg/g) of some heavy metals were determined using atomic absorption spectroscopic technique. Results: The average (mean) range of Iron (Fe) was between 5.31-63.64 (μg/g), whereas zinc (Zn) range is 0.484 (μg/g) to 13.81 (μg/g). And Manganese (Mn) was an average range between 0.64-8 (μg/g). Although mercury was found with 0.03 (μg/g) in one milk sample, however for other heavy metals cobalt (Co), tin (Sn), cadmium (Cd), and lead (Pb), in canned samples were not found. Conclusion: Based on the WHO-FAO Guidelines for the Evaluation of Heavy metal in Food, there is no health risk associated with (iron, zinc, manganese) concentration in the samples analyzed. But in all samples are less than the level content of the same types in natural freshly. Some descriptive statistical analysis with one way ANOVA was performed on the obtained results. Keywords; Canned food; Heavy metals; Atomic absorption spectroscopy.
Introduction cobalt (Co), lead (Pb), and Tin (Sn), The definition of ‘‘heavy metals seems very These elements may be imported into the simple on a first glance: heavy metal is environment like fruits and vegetable by a metal having high specific weight’’.1 various anthropogenic activities, such Heavy metals are present in various as fertilizer, mining use, metal-based environmental partitions (soil, sediments, pesticides, and a broad range of current atmosphere, water, and living organisms) industrial actions, which release metals and these forms are controlled by physical into the surrounding environment and and chemical conditions as well as by foods. 4 Heavy metals are probable biological processes.2 Heavy metals are environmental contaminants with the rationally exciting elements, are present in effectiveness of causing human health mutable concentrations in all environment trouble if present to excess in the food system in the earth.3 In all the worldwide we eat. They are toxic effects even at care, the problem of heavy metal addition very small concentration.5 The eating of has been studied one of the most worrying contaminated food breed uptake of factors creating in the late 19th and recent toxic elements by humans, can involve 20th centuries. a little metals, such as physiological boost and central nervous mercury (Hg), zinc (Zn), arsenic (As), system activity, disturb the function of
1 Department of Pharmaceutical Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected]
331 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) organs like kidneys and change the heavy metals such as, arsenic (As), blood composition, liver, and lung.6 mercury (Hg), copper (Cu), lead (Pb) and The corruption of canned foods is the cadmium (Cd). Over the years as reported occurrence of any other changes desirable in several literatures (Ray, 1994; in the characteristics of the food whether Oehlenschlager, 2002; Damek-Poprawa the impact of this corruption in the form of et al., 2003; Yargholi et al., 2008).12 the box from the outside or did not appear A research study of lead (Pb) contents because the normal shape of the box is to of fresh and canned foods in the United be concave on both ends resulting from the Kingdom UK in 1984 shown high level of unloading which occurs within the effect of lead concentration.13,14 Some metals such the primary heating process and if there is as zinc, iron, and copper are vital to life a convexity at the end of the coop or both, and play energetic roles in the working of this usually indicates the occurrence of critical enzyme systems. However zinc is extortion.7,8 The use of convective heat an important requirement for a human means the elimination of all microscopic healthy body, too much can be hurtful organisms (cells and spores), with damage and extreme absorption of Zinc can also to the enzymes in the food, resulting in suppress copper and iron absorption the process of self-degradation of the food eating or consumptions of 140– 450 mg tissue. It was found that the use of heat for of (Zn) for each day have been related long periods of time ensures access to with such chronic effects as low (Cu) food, free of germs, but the process grade, improved iron role, reduced levels includes some negative effects.9 The idea of high-density lipoproteins (HDL) and of food damages is of great importance in reduced immune function. The free zinc ion the resolution to fight hunger, increase pay is also a great acid treated as Lewis acid and better food security in the international up to the point of being sever and harsh. lowliest countries. Food losses have an Stomach acid contains hydrochloric acid effect on food safety for poor persons, (HCl), in which salt of zinc and metallic zinc on food safety and quality, on economic dissolves readily to give corrosive zinc 15-17 development and on the environment. The chloride(ZnCl2). Then cobalt and its perfect reasons of food losses differ during compounds are classified by International the world and are extremely dependent (IARC) as possibly risk cancer-causing to on the unique conditions and the local humans.18 Tuna and Sardine fishes can situation in a given country.10 The danger contain a high amount of rare and heavy associated with the contact to heavy metals metals like Mercury (Hg) and cobalt (Co) like mercury, Tin, Cadmium, and Lead, concentrate which can be a human health present in meat and meat products is a call worry, science it a main canned seafood for concern for both human health as well and can be spent by human directly, as food safety because of their toxic remember that toxic elements can be very nature. Then, regulatory bodies such as the dangerous even at low level concentration (FAO), (WHO), and (USEPA), are famous when taken over long time.19 Evaluation of in from other countries in the world have tin in canned food come to be very important set of maximum limits for heavy metals in as it deals scientific information about foodstuffs.11 Heavy metal in canned food. the industrial pollution process and be Canned foods contain a different range of responsible for helping to increase canned metal elements such as sodium (Na), food safety.20 Processing or packaging is potassium (K), (Ca) calcium, iron (Fe), another way of entrance of metal and (Zn) zinc and (Cu) copper. However, heavy metals.13 Manganese it does not a large number of them are harmful to originate as an alone element in animals, plants and human even at low environment and nature; it is frequently concentration. This is typically true of found in minerals in combination with
342 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) iron.21 Also, manganese is a mineral Methods element that is both nutritionally vital and Sample collection possibly has a toxic effect.22 Manganese A total of (56) different types of canned (Mn) plays a significant and important role food samples (milk powder, cheese, in a number of physiologic processes as luncheon meat, beans, black olive, coffee a basic of manifold enzymes and an powder, and beverages) from the local activator of other enzymes.23 Rich main markets were randomly collected from sources of manganese contained in nuts, retail outlets in Erbil city, during sample whole grains, teas, and leafy vegetables. collection, the (products’ retail prices were Although teas are rich sources of recorded. Other information was also manganese, the tannins present in tea recorded, such as the Batch number, may reasonably decrease the absorption country of manufacturer’s, as well as of manganese.24 Fresh food may be product and expire date information on contaminated by little amounts of metal nutritional composition, and packages were from brief effect, twenty particularly also noted. Complete information was eminent in vegetables such as onions, shown in Table 1. pepper, lettuce and tomatoes which, in addition, may be contaminated during irrigation agriculture.25,26 Aim and objective Table 1: types of food used in the study The aim of this study was to evaluate and there information. amount a heavy metals, (iron, mercury, cobalt, tin, cadmium, manganese, zinc, and lead) respectively in some canned food transported from other countries and to proffer data that may be used as the support for protective measures. This was resolved to be managed through the following objectives: -To determine the metal contents of canned food in Erbil city major markets by using inductive Shimadzu AA-7000, Japan polarized Zeeman Atomic Absorption Spectrophotometer. -To compare the levels of the metals in canned food, (Milk powder, Cheese, Tuna fish,, Luncheon meat, Beans, Black olive, Tomato paste, tea leaf, o Coffee powder, Beverage, and Jam) with the global certified value. -To determine the risk associated with the consumption of these products. Limitations of the study The study was limited to these brands of canned food due to their possibility in all the selected markets from the different geographical zones of the capital city (Erbil). Also, the sampling sites per zone were limited due to financial constraint as at the time of the study.
353 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Reagents solution was filtered into a 25.0 mL The concentrated acids Perchloric acid, volumetric flask; the volume was complete Nitric acid, and Sulfuric acid (Merck to the mark by de-ionized water. Millipore, Germany) were used of analytical 7. Each extract solution of canned food grade. Stock standard solution (500 ppm) sample was analyzed by Shimadzu AA- of mercury, cadmium, lead, iron, cobalt, tin 7000, polarized Zeeman Atomic Absorption and zinc (Hg), (Cd), (Pb), (Fe), (Co), Spectrophotometer. (Sn), and (Zn) respectively were used in 8. Blank solution was prepared by using qualifying their calibration curve after little the same procedures. serial dilutions. Standard of Ammonium Instrumentation dihydrogen phosphate (NH4H2PO4) solution The Shimadzu AA-7000, Japan was prepared by dissolving anhydrous it in polarized Zeeman Atomic Absorption 500 ml de-ionized water and stored in dark Spectrophotometer was used in this study. brown container glass bottle. Two modes of atomization, graphite Quality control furnace AAS (GFAAS) and flame AAS All samples three times were analyzed (FAAS) were used. in order to reliably provide the GMP (The Statistical analysis of notes data results are based on three repetitions of the The Microsoft® Excel 2010 software experiment for each canned product) of the program and statistical software package manufacturers; we ensured that the canned IBM SPSS 23.0 was used for statistical samples that had difference batch numbers analysis to calculate descriptive statistics on their packages were sampled at three (i.e. the frequency in count and different sellers. However, they were percentage, sum, average, median and carefully gained from different retailers who interquartile range) with bar chart graphs. handle it at different times. With one way ANOVA was performed. Sample preparation and acid digestion method Results and discussion In this study, sample solutions were The concentrations of heavy metals(metals prepared as recommended by Tuzen and Iron(Fe), Mercury(Hg), Cobalt(Co), Tin(Sn), Soylak28 and Voegborlo et al29 as in the Cadmium(Cd), Manganese(Mn), Zinc(Zn), following arrangement of steps: and Lead(Pb) ) in 66 different brands of 1. Every canned food sample was canned food are analyzed, In addition, homogenized carefully using a food results are compared between different blender with stainless steel cutters. brands and types of canned food. So the 2. A weight of 10.0 g of every uniform food result discusses more closely as follows: sample was placed into a borosilicate Iron (Fe) evaporating dish and dried in an oven at 70 It has been reported that the average °C for 24 h. (mean), maximum and minimum level of 3. A weight of 1.0 g dry weight sample was concentration by microgram per gram placed into a borosilicate evaporating dish, (μg.g-1) for determination of Iron in all (56) then 10.0 mL of 70% (v/v) HNO3 and 70% samples are shown in Table 2, revealed (v/v) HClO4 was added with a ratio of from the results, Fe has the highest (7:3 v/v). concentration in 10 (17.85%) samples of 4. The solution in a dish was heated till black olives by mean (average)(63.64 μg.g dryness. -1) and milk powder (61.45 μg.g-1) among 5. Few drops of 0.15% (w/v) NH4H2PO4 the samples were recorded. While the were added to each sample as a matrix concentration is in the lowest level of iron modifier. noted in 18(32.14%) samples cheese (5.31 6. After cooling, an aliquot of 5.0 mL of μg.g-1), and coffee powder (7.36 μg.g-1) 0.1% (v/v) HNO3 was added, then the respectively, the detailed comparison
364 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) among the canned samples are shown in Iron in all samples are less than the level Figure 1. it also important to mention that content of the same types in natural the concentration of iron a respectively with freshly samples according to WHO-FAO increase of storage period due internal Guidelines for the Evaluation of Heavy erosion.30 Also, the percent content of metal in Food.31
Table 2: Fe residues (μg.g-1) in canned food samples.
Sample No. of Average(mean Minimum Maximum Samples μg.gm -1)
Milk powder 5 61.45 37.401 79.5
Cheese 5 5.31 1.28 9.25
Luncheon meat 9 15.37 9.64 31.97
Black olive 5 63.64 18.25 152.01
Tomato paste 5 26.26 9.38 32.07
Coffee powder 3 7.36 7.25 7. 5
Beverage 12 12.51 1.17 19.73
Peas and bean 12 14.42 4.82 20.49
Figure 1: comparison of iron in all kind of canned samples.
375 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Zinc (Zn) whereas the other 5 (8.9 %) samples had The results show in Table 3 that zinc maximum level for Zn mean concentration occurrence in canned samples, Among the (13.81) μg.gm -1 seen in milk powder. All 56 canned samples, 32 (57.14 %)(had details are simplified shows in Figure 2. concentrations were negligible in samples It also important to the discussion that the beverage, coffee powder, black olive, and concentration of zinc(Zn) fluctuates in peas canned with the content mean (0.484, same types of all canned samples sources 0.683, 1.36, 2.67) (μg.gm -1) respectively. and are very less than the minimum level The other19 (28.78 %) samples of tomato content of the same kinds in natural freshly paste, cheese, and luncheon meat had sample according to WHO-FAO Guidelines Zn concentration increased gradually with for the Evaluation of Heavy metal in a mean (3.14, 7.17, and 8.88) μg.gm -1, Food.31
Table 3: Zn residues (μg.gm -1) in canned samples. Sample No. of Average(mean) Minimum Maximum Samples μg.gm -1
Milk powder 5 13.81 4.89 37.23
Cheese 5 7.17 1.89 13.86
Luncheon meat 9 8.88 5.29 13.45
Black olive 5 1.36 0.65 2.21
Tomato paste 5 3.14 1.72 4.22
Coffee powder 3 0.683 0.65 0.71
Beverage 12 0.484 0.244 1.14
Peas and bean 12 2.67 0.55 5.79
Figure 2: comparison of zinc in all kind of canned samples.
386 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Manganese (Mn) sequentially, but in 31(55.35%) evaluate In this study, Manganese (Mn) canned food the lowermost close concentration samples of different brands were evaluated understood mean value in milk powder and display in Table 4. The content by (0.64 μg.gm -1 ), black olive (0.9 μg.gm - μg.gm -1 is varying from sample brand 1), beverage (2.13 μg.gm -1 ), and to another as illustrated in Figure 3. The luncheon meat (2.38 μg.gm -1). The concentration decrease dramatically from concentration of manganese, such as iron coffee powder mean (8μg.gm -1) to milk and zinc, compared with the quantitative powder mean(0.64 μg.gm -1) , therefore as concentrations in the fresh form is shown 25 (44.64%) samples of tomato significantly lower according to WHO-FAO paste, cheese, peas with beans, and coffee guidelines for the evaluation of heavy powder are include the highest level metal in Food.31 content (4.43, 6.711, 7.27, and 8) μg. gm -1
Table 4: Mn residues (μg.gm -1) in canned food samples. Sample No. of Average(mean) Minimum Maximum Samples μg.gm -1
Milk powder 5 0.64 0.52 0.70
Cheese 5 6.711 0.224 30.85
Luncheon meat 9 2.38 1.88 3.175
Black olive 5 0.900 0.5 1.5
Tomato paste 5 4.43 3.56 4.86
Coffee powder 3 8.00 7.8 8.25
Beverage 12 2.13 0.1 13.4
Peas and bean 12 7.27 0.285 66.2
Figure 3: comparison of manganese in all kind of canned samples.
397 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Mercury (Hg) a statistically significant difference in Mercury was found in one sample milk, so time to complete the problem between for evaluating the milk powder seen that the group that Fe and the Zn metal the level concentration (0.03 μg.gm -1 ) is (P = 0.028), as well as between the much more greater than the maximum limit Fe metal and Mn metals (P = 0.014). of mercury by WHO-FAO Guidelines for However, there were no differences the Evaluation of Heavy metal (0.001 between the groups that took the Zn and μg.gm -1). Clearly, this type of sample milk Mn metals (P = 0.949). So shortly for powder is contaminated and should not be all samples the average percent of Fe, Zn, used or treated because of its danger to and Mn. Shown in Figure 4. Other Heavy human health, especially the health of metals. The presented study proves that all children and infants.31 In order to test canned samples hade concentration of the hypothesis that the level of heavy heavy metals cobalt (Co), tin (Sn), metals (Fe, Zn, Mn) had an effect on cadmium (Cd), and lead (Pb) as presented different types of canned food confidence, in table 3, under detection limit and did not a between-groups ANOVA was performed. found by our study dominate that harmless Prior to conducting the ANOVA, the result and safe for the metals above according shows the output of the ANOVA analysis the guideline level well-known by EU the test statistic is the F value of 9.59. and FAO/WHO Expert Board on Food Using an α of 0.05, we have F 0.05; 2, 24 = Additives .31 3.403. Since the test statistic is much larger than the critical value (F = 5.699), we reject Conclusion and recommendations the null hypothesis of equal population In this study project the overall aim was to means and conclude that there is obtain more knowledge about the level of a (statistically) significant difference among concentration of heavy metals (Fe, Hg, the population means. The p-value for Zn, Co, Cd, Sn, Mn, and Pb) present or 5.699 is 0.009, so the test statistic is suspected of being present in different significant at that level. There is a canned food samples.The results of this statistically significant difference in the study concluded that for all the canned mean length. This is great to know, but samples the concentration of Co, Cd, Sn for multiple comparisons testing the and Pb was not detected (under detection differences between means results the limit) while in this study, the heavy Tukey post hoc test had been done we elements Fe, Zn, Mn were detected in all can see from the table below that there is types of canned foods. Also the research
Figure 4: Comparison of average mean in percent of Fe, Zn, and Mn in all canned food samples.
408 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) showed that the ratio of these elements Arabian Journal of Chemistry 2017; 10(7):906– is altered in all types of samples on 13. a different scale and has a lower value 9. Fiamegos Y, Vahcic M, Emteborg H, Snell J, Raber G, Corderio F et al. Determination of compared with different types of fresh toxic trace elements in canned vegetables: The condition depend upon the internationally importance of sample preparation. TrAC Trends level content EU and FAO/WHO. We in Analytical Chemistry 2016; 85(B):57–66. recommend reducing the use of or keep 10. Rawat S. food spoilage microorganisms and their prevention. Asian Journal of plant Science and away from eating and using canned food, Research 2015; 5(4):47–56. especially for child and infants. We also 11. Zahrana D A, Hendy B. Heavy metals and trace recommend that the government adjust elements composition in certain meat and meat and evaluate the basic criteria for the products sold in Egyptian markets. Int J Sci: quality control laboratory and measurement Basic Appl Res 2015; 20(1):282–93. 12. Yakubu A D, Stephen E A, Hamza A, Usman S of heavy metals canned foods and the M , Eneyi C. Level of heavy metals in fresh and serious interest in this matter. This study canned foods consumed in North Central Nigeria. recommends more research and more Scholarly Journal of Agricultural Science 2013; accurate studies. Details the content of 3(6):210–13. 13. Elinder C G. Zinc. In: Friberg L, Nordberg G F, heavy metals in various types of Vouk V B. Handbook on the toxicology of metals. canned foods more broadly. Using new 2nd edition. Elsevier, Amsterdam, New York; technologies and modern equipment and 1986. P. 78–117. finding quick and useful methods for quality 14. Adetutu O A, Muhammad D F, Aminuddeen H A. control the heavy and rare metals in Seasonal Evaluation of Mineral Elements, Heavy Metals, Essential Amino Acids, Proximate canned foods. Compositions and Pesticides in Goat Milk. World Journal of Analytical Chemistry 2015; 3(1):1–9. References 15. Kiri H J, Samuel Y G, Samson I W and Joseph 1.Klaus J A. What are ‘‘heavy metals’’ in Plant A. Levels of Zinc, Iron and Lead in Canned Fish Sciences. Acta Physiol Plant 2010; 32(10):615–9. Sold in Jos, Nigeria. Advances in Research 2016; 2. Mortatti J and Probst H, Jean L. Characteristics of 7(4):1–6. heavy metals and their evaluation in suspended 16. Hooper P L, Visconti L, Garry P J, Johnson G E. sediments from Piracicaba river basin (São Zinc lowers high-density lipoprotein-cholesterol Paulo,Brazil). Revista Brasileira de Geociências levels. J Am Med Assoc 1980; 244:1960–1. 2010; 40(3):375–9. 17. Muyssen B T, Karel A C, Schamphelaere D E, 3. Ilyin I, Travnikov O, Ass W, Ugerud H. Heavy Colin R J. Mechanisms of chronic waterborne Zn metals: transboundary pollution of the toxicity in Daphnia magna. Aquatic Toxicology environment. EMEP Status 2003. MSC-E 2006; 77:393–401. Report; 2(6):40–3. 18. Petra K,Ingrid M, Boris P, Venčeslav P. 4. Azevedo R A. What is new in the research on Determination of trace cobalt concentrations cadmium induced stress in plants. Food and in human serum by adsorptive stripping Energy Security 2012; 1(2):133–40. voltammetry. Journal of Trace Elements in 5. Salama A K, Radwan A M. Heavy metals (Cd, Pb) Medicine and Biology 2003; 17(3):153–8 and trace elements (Cu, Zn) contents in some 19. Zarie M, Mollaie A, Eskandari M H, Pakfetrat S. foodstuffs from the Egyptian market Emir. J Agric Histamine and heavy metals content of canned Sci 2005; 17(1):34–2. tuna fish. Global Veterinaria 2010; 5(5):259–63 6. Rajeev B, Vicente M G. Practical Food Safety: 20. Manzoori J L, Amjadi M, Abolhasani D. Contemporary Issues and Future Directions. Spectrofluorimetric determination of tin in canned 1st Edition. USA: Wiley & Sons; 2014. P. 125–8. foods. J Hazard Mater 2006; 137(3):1631–5. 7. Hajeb P, Sloth J J, Shakibazadeh S H, Mahyudin 21. De Bièvre P. Atomic weights and isotopic NA, Afsah H L. Toxic elements in food: abundances of the elements: a future concern for Occurrence, binding, and reduction approaches. the analytical chemist. Fresenius' Zeitschrift für Comprehensive Reviews in Food .Science and analytische Chemie 1973; 264(5):365–71. Food Safety 2014; 13(4):457–72. 22. Keen C L, Ensunsa J L, Watson M H. Nutritional 8. Hana R A, Hope K, Emmanuel A, Tsdale MA, aspects of manganese from experimental ThorntonbC M, BabyakbS F. Determination of studies. Neurotoxicology 1999; 20(2-3):213–23. macro, essential trace elements, toxic heavy 23. Nielsen F H. Ultratrace minerals. In: Shils M, metal concentrations, crude oil extracts and Olson J A, Shike M, Ross AC, eds. Modern ash composition from Saudi Arabian fruits Nutrition in Health and Disease. 9th ed. and vegetables having medicinal values. Baltimore: Williams & Wilkins; 1999; 283–303.
399 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 24. Kies C. Bioavailability of manganese. In: Klimis-Tavantzis DL, Manganese in health and disease. 1st ed. Boca Raton: CRC Press, Inc; 1994; 39–58. 25. Awode U A, Uzairu A, Balarabe M L, Harrison G F, Okunola O J. Assesment of Peppers and Soils for some Heavy metals from Irrigated Farmlands on the banks of River Challawa, Nigeria Pakistan Journal of Nutrition 2008; 7(2):244–8. 26. Abdullahi M S, Uzairu A, Harrison G F, Balarabe M L, Okunola O J. Comparative study of tomatoes and onions from irrigated farmlands on the banks of river Challawa, Kano, Nigeria. Interanational Journal of Environmental Sciences 2008; 2(1):65–70.
1041 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Serum vitamin D level discriminates between the primary and the associated clinical manifestations of fibromyalgia patients: A preliminary report
Talar Ahmad Merza Mohammad1* Marwan Salih Mohammad Al-Nimer1 Abstract Backgrounds and objective: Recent research focus of the association of low serum vitamin D level with the fibromyalgia. This study aimed to prove that serum vitamin D level served as a discriminator of the chronic pain and the associated clinical signs and symptoms. Methods: This study conducted in the Rizgary Teaching Hospital through 2019, on the 37 patients presented with chronic widespread pain suggestive fibromyalgia. Data related to the characteristics, anthropometric measurements, cardiometabolic risk factors, and serum vitamin D level were collected. The number of tender points, the scores of the revised fibromyalgia impact questionnaire (FIQR), fatigue severity scale (FSS), insomnia, and Hamilton's scale for depression were assessed as outcome measures of the study. A serum vitamin D of < 3ng/ml is used as a cutoff value to discriminate the outcome measures. Results: Evidence of cardiometabolic risk factors was observed in the term of overweight, high systolic and diastolic blood pressure and abnormally high level of serum triglyceride. The mean ± SD of the scores of tender points, total FIQR, FSS, insomnia, and depression were 15.1±2.1, 158.6±31.4, 6.3±0.7, 20.1±3.9, 26.2±5.2, respectively. The area under the curve of the tender points, fatigue, insomnia and depression was higher than 0.5. Conclusion: serum vitamin D level (<3ng/ml) significantly discriminates the score of the tender points and the symptoms domain of the revised fibromyalgia impact questionnaire. Moreover, severe vitamin D deficiency plays a role in the associated clinical manifestations, including fatigue, insomnia and depression that commonly observed in fibromyalgia. Keywords: Fibromyalgia; Vitamin D; Fatigue; Insomnia; Depression.
Introduction a co-adjuvant nutraceutical in FM therapy.2 Fibromyalgia is a chronic disease Vitamin D supplementation of 50000UI characterized by widespread pain weekly for 8 weeks improved the scores associated with sleep disturbances, fatigue, of the questionnaires that related to the and depressive symptoms. Plenty of fibromyalgia, sleep, pain, but not the research deals with the role of vitamin D in quality of life as assessed by short the fibromyalgia. In one meta-analysis Form-36 in FM patients with low vitamin D included 12- eligible studies with 851 levels of insufficient category.3 Moreover, fibromyalgia patients and 862 controls, the vitamin D supplementation of 50000 UI serum level of vitamin D in the fibromyalgia once weekly for 12 weeks non-significantly group was lower than the corresponding reduced the number of tender points in FM level of the controls with a standard patients with insufficient category of mean difference -0.56.1 FM patients are serum vitamin D.4 Interestingly, vitamin D usually responded to the vitamin D supplementation improves non-significantly supplementation which can consider as the score of widespread pain which is not
1 Department of Pharmacology and Toxicology, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 421 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) related to the increasing the serum level of Rheumatology criteria of which confirmed vitamin D.5 Another study found that there by the specialist in Rheumatology, with is a appositive correlation between the a negative laboratory investigations of serum levels of vitamin D with the Berg rheumatic profile (including C-reactive Balance Scale which comprise of 14 tasks protein, rheumatoid factor above upper related to simple balance, and non- limit of erythrocyte sedimentation rate, significantly correlated with visual rheumatoid factor, antinuclear antibody, analogue scale for pain in FM.6 Therefore, and anticyclic citrullinated peptide). The the effectiveness of the vitamin D exclusion criteria are patients with chronic supplementation on the pain or other FM rheumatic illnesses, including rheumatoid symptoms is still not clear.7 A published arthritis, systemic lupus erythematous, Observational Research Design included polymyalgia rheumatica, chronic fatigue 50,834 patients with pain-related syndrome, diabetes mellitus, hypertension, conditions, it has been found that vitamin neurological and psychiatric disorders, deficiency was observed in articular, hematological disorders, renal or liver muscular, and widespread pain.8 The diseases, pregnancy and patients with rationale of this study is the low serum a history of using non-steroidal or steroidal vitamin D of severe deficiency category in anti-inflammatory drugs within one month FM was not thoroughly investigated. This of inclusion in the study. The consultant study aimed to investigate the FM patients rheumatologists working in the hospitals, in with severely deficient of vitamin D in Erbil, clinically evaluated each patient and attempting to derive a cutoff value of serum whether they met the inclusion criteria or vitamin D that can discriminate the primary not. Then each patient was interviewed and secondary outcome measures in newly by the researchers and each item of the diagnosed FM patients. research questionnaire was explained to them by their native language. The Methods researcher under supervision of the This study took place between January consultant rheumatologists also carried out and May 2019 at the Department of the following measurements. Fibromyalgia 10 Pharmacology and Toxicology, Pharmacy Impact Questionnaire Revised (FIQR). College at Hawler Medical University in the Hamilton’s Depression Rating Scale for 11 Erbil, Iraq. The study was approved by depression (21-items). Fatigue Severity 12 the Hawler Medical University Institutional Scale (FSS). and the Insomnia Severity 13 Scientific Committee. The selected patients index (ISI). The ISI is a questionnaire of were those who attended the Rizgary seven-items and the items are recorded Teaching Hospital in Erbil. A total number on a five-point scale with 0=no issue and of 37 (6 males and 31 females) patients 4= serious. A sleeping disorder was were enrolled into the study. The diagnostic characterized as a complete score of the criteria that approved by the American SIS ≥ 10. The FSS is questionnaire of College of Rheumatology (ACR 2010) a nine-item and the items are recorded were used to confirm the diagnosis of on a seven- point scale with 1 = strongly fibromyalgia.9 With respect to patient disagree and 7= strongly agree. The mean privacy and medical ethics, all participants value of all results has been determined. have been reported that their data will be The researchers evaluated the pain that m used for goals of the study, and each eets the following requirements: spread in participant signed a consent form. The all four body quadrants plus axial pain, and inclusion criteria are patients with the signs at least 11 out of 18 predefined tender poin and symptoms that suggest a diagnosis of ts that were caused by a peak pressure ofa 2 fibromyalgia (length of at least three pproximately 4 kg / cm using algometry. months), and met the American College of Measurements of blood pressure in the 432 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) seated position were registered using (E411, Roche, Germany), and the 25(OH) a mercury digital sphygmomanometer. D values were categorized as Normal The height (in meter) and body weight (>30ng/ml), Insufficiency (20-29.9ng/ml), (in kilogram) were evaluated. The and Deficiency (<20ng/ml). A serum circumference of the waist (in centimeter) vitamin D of < 3ng/ml is used as a cutoff was measured by the measuring tape at value to discriminate the outcome the umbilical point. The Body mass index measures. has been calculated. Triglyceride-Glucose Statistical analysis index was calculated as a formula: Statistical analyses were conducted using Ln (triglyceride [mg/dL]×glucose [mg/dL]/2). SPSS version (20) for windows. The Under sterile conditions, (5 mL) of the research variables were analyzed using blood samples were obtainedfrom each descriptive statistics such as a mean± participant who was ata minimum ten hours standard deviation, percentage, and fasting. Part of the blood sample was number. Receiving operating characteristic added into disposable tubes, kept at room analysis test was applied to discriminate temperature, and then centrifuged for 10 the primary and secondary outcome minutes at 3,000 r.p.m to separate the sera measures by calculating the area under the for determination of the rheumatic profile curve and 95% confidence intervals. panel (anticyclic citrullinated peptide, qualitative C-reactive protein, rheumatoid Results factor, and antinuclear antibodies), Table 1 shows that the proportion of the serum glucose and lipid profile (total male-to-the female is 1:5.2 with a mean cholesterol, total triglyceride, high density age 38.2 (range: 20-56) years. 24.3% of lipoprotein-cholesterol). The other part of participants were current smokers and the sera was used to determine the 75.7% of the patients were habitants in the serum levels of 25-hydroxyvitamin D of urban area. 34 out of 37 (91.9%) patients patients using the Hormone Analyzer had a serum level of vitamin D <20ng/ml).
Table 1: Characteristics of the patients. Variables Results Sex (Male: Female) 6: 31 Age (year) 38.2±10.2 Smoking (No.) Current 9 (24.3) Previous (ex-smoker) 1 (2.7) Marital status Single 4 (10.8) Married 27 (73.0) Widow 5 (13.5) Divorced 1 (2.7) Residency Rural 9 (24.3) Urban 28 (75.7) Vitamin D status Normal (>30ng/ml) 2 (5.4) Insufficiency (20-29.9ng/ml) 1 (2.7) Deficiency (<20ng/ml) 34 (91.9) The results are expressed as number (%) and mean ±SD. 443 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Cardio-metabolic risk factors were detected of the FM patients. The mean ± SD value in the participants of this study. The means of pulse pressure-to-systolic blood of anthropometric measurements showed pressure that indicates the arterial stiffness that the participants were in the category of is 0.348±0.056 (Table 2). The fasting the overweight (25-29.99kg/m2) and had a serum lipid profile and glucose showed waist-to-height ratio exceeded the upper that 27.0% (10 out of 37 patients) have cutoff value of 0.5 (Table 2). High systolic hypertriglyceridemia and the TYG index is and diastolic blood pressures were 8.49±0.62 (Table 2). detected in 32.4% and 29.7%, respectively
Table 2: Cardio metabolic risk factors. Metabolic syndrome components Results
Body mass index (kg/m2) 29.4±7.0 Waist circumference (cm) 88.4±15.0 Waist-to height ratio 0.564±0.095
Blood pressure (mmHg) Systolic 125.6±18.0 >140 12 (32.4) Diastolic 81.4±10.4 >90 11 (29.7) Mean 96.1±12.3 Arterial stiffness 0.348±0.056
Fasting serum lipid profile (mg/dl) Triglyceride 117.2±66.7 >150 mg/dl 10 (27.0) Total Cholesterol 165.8±36.6 High density lipoprotein-cholesterol 48.5±12.7 Non-high density lipoprotein-cholesterol 117.3±40.5
Fasting serum glucose (mg/dl) 96.6±14.6 Triglyceride-Glucose index 8.49±0.62
The results are expressed as number (%) and mean± SD. Two of patients have serum TG > 500 excluded from mean
454 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Tables 3 showed the scores of the primary index, and Hamilton's scale for depression and secondary outcome measures that are 15.1, 6.3, 20.1, and 26.2 respectively. assessed by several questionnaires. The The mean total score of the FIQR is 158.6 means of the tender points, the scores of (Table 3). the fatigue severity scale, insomnia severity
Table 3: Scoring of the subjective symptoms assessed by revised fibromyalgia impact questionnaire, fatigue severity symptom, insomnia scale, depression (using the Hamilton's rating scale), and objective signs assessed by tender points. Determinants Results
Duration of symptoms (weeks) 13.1±14.1
Domain of FIQR Function 66.5±14.7 Global impact 16.8±3.6 Symptoms 75.3±18.1 Total 158.6±31.4
Symptoms-related FIQR Pain 8.2±1.8 Energy 7.7±2.1 Stiffness 7.4±2.5 Sleep 8.9±1.4 Depression 7.6±2.6 Memory problem 7.9±2.2 Anxiety 8.2±1.9 Tenderness to touch 8.1±2.0 Problems balance 7.1±2.7 Sensitivity to loud noises, bright lights, odors 7.3±2.5 and cold
Tender points 15.1±2.1
Fatigue severity scale 6.3±0.7
Insomnia severity index 20.1±3.9
The Hamilton's scale for depression 26.2±5.2
The results are expressed as mean± SD.
465 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Figure 1 shows that the area under the 0.550 (95%C.I. 0.357-0.742) and 0.535 curve of the tender points and the v( 95%C.I. 0.346-0.725) at a cutoff value of symptoms of FM that detected by FIQR serum vitamin D of <3ng/ml). as the primary outcome measures are
Test Result Variable(s) Area 95% Confidence Interval Under the curve Lower Bound Upper Bound Tender points 0.550 0.357 0.742 Function 0.421 0.233 0.609 Global 0.370 0.188 0.552 Symptoms 0.535 0.346 0.725 FIQR-total 0.477 0.283 0.670
Figure 1: The area under the curve of the primary outcome measures of fibromyalgia taking the level of serum vitamin D < 3ng/ml (n=18) as a discriminator.
476 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Figure 2 shows that the area of the under insomnia, and depression has exceeded the curve of each of the secondary the 0.5 at a cutoff value of serum vitamin D outcome measures including fatigue, of <3ng/ml).
Test Result Variable(s) Area 95% Confidence Interval
Lower Bound Upper Bound Fatigue severity scale 0.626 0.440 0.812
Insomnia 0.542 0.352 0.733
Depression 0.529 0.340 0.719
Figure 2: The area under the curve of the secondary outcome measures of fibromyalgia taking the level of serum vitamin D < 3ng/ml as a discriminator.
487 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Discussion fatigue syndrome.17 The status of vitamin D did not play a role in the sleep The results of this study show that a cutoff disturbances or insomnia in conditions not value of serum vitamin D of <3ng/ml is related to the fibromyalgia.20 In one a helpful discriminator of primary outcome meta-analysis study, including 9397 and secondary outcome measures. The recruited from nine studies (non- characteristic of patients, including the randomized, non-controlled), vitamin D predominance of the female gender, living deficiency (< 20ng/ml) was associated with in urban areas and current smoking, can a higher risk of sleep disorder.21 Further influence the serum level of vitamin D. study showed that vitamin D insufficiency Vitamin D deficiency is more common in is associated with short sleep.22 The odd female than male and this gender-based ratio of vitamin D deficiency in patients effect shared the other determinants diagnosed with depression is 1.22, taking that cause vitamin D deficiency in FM 14 in consideration the geographic latitude patients. Another study found that vitamin that strongly played a role in the status D deficiency were similar in both sexes, but of vitamin D deficiency.23 Therefore, low its prevalence is increased in patients with serum vitamin Dis a useful discriminator of cardio metabolic risk factors, including the secondary outcomes of fibromyalgia. high body mass index, high blood pressure, The strength of this study is the receiving abnormal lipid profile and impaired glucose operating characteristic is used for analysis level as some of these factors reported in 15 and not the simple correlation test which our study. Heavy smokers are more likely previous studies showed conflicting results. to have a low serum vitamin D deficiency, One of the most important limitations of the and in our study about quarter of patients study is small sample size. The sensitivity were currently smokers which may share 16 and specificity were not determined with low serum vitamin D in FM patients. because healthy people haven't severe The areas under the curves of the number deficiency of vitamin D. of the ender points and the subjective symptoms that assessed by FIQR are Conclusion exceeded the value of 0.5 taking a cutoff We conclude that a severe vitamin value of serum vitamin D <3ng/ml, which deficiency is common in FM patients and indicating that low serum vitamin D can a cutoff value of <3ng/ml can discriminate discriminate the widespread pain and the the primary and secondary outcome associated symptoms from other disorders measures of fibromyalgia. like chronic fatigue syndrome.17 Previous studies did not show correlation between References the clinical symptoms of fibromyalgia and 1. Makrani AH, Afshari M, Ghajar M, Forooghi Z, serum vitamin d by using used simple Moosazadeh M. Vitamin D and fibromyalgia: correlation and multiple logistic regression a meta-analysis. Korean J Pain 2017; 30(4):250– 18 7. analysis tests. In one study carried on 2. Martins YA, Cardinali CAEF, Ravanelli MI, Saudi patients, the mean serum level Brunaldi K. Is hypovitaminosis D associated with of vitamin D in FM patients was 4.76 fibromyalgia? A systematic review. Nutr Rev ng/ml and , the serum vitamin D level 2019; pii: nuz033. significantly and inversely correlated with 3. Mirzaei A, Zabihiyeganeh M, Jahed SA, Khiabani 19 E, Nojomi M, Ghaffari S. Effects of vitamin D the widespread pain index. Moreover, optimization on quality of life of patients with low serum vitamin D can discriminate fibromyalgia: A randomized controlled trial. Med J the secondary outcome measures of Islam Repub Iran 2018; 32:29. fibromyalgia including fatigue, insomnia, 4. de Carvalho JF, da Rocha Araújo FAG, da Mota LMA, Aires RB, de Araujo RP. Vitamin D and depression. Previous studies Supplementation Seems to Improve Fibromyalgia demonstrated the low serum vitamin D did Symptoms: Preliminary Results.Isr Med Assoc J not contribute to the fatigue in the chronic 2018; 20(6):379–81. 49 8 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
5. Yong WC, Sanguankeo A, Upala S. Effect of et al. Serum Vitamin D Status in Iranian vitamin D supplementation in chronic widespread Fibromyalgia Patients: according to the Symptom pain: a systematic review and meta-analysis. Clin Severity and Illness Invalidation. Korean J Pain Rheumatol 2017; 36(12):2825–33. 2016; 29(3):172–8. 6. Kasapoğlu Aksoy M, Altan L, Ökmen Metin B. 19. Abokrysha NT. Vitamin D deficiency in women The relationship between balance and vitamin 25 with fibromyalgia in Saudi Arabia.Pain Med 2012; (OH)D in fibromyalgia patients.Mod Rheumatol 13(3):452–8. 2017; 27(5):868–74. 20. Niu J, Sahni S, Liao S, Tucker KL, 7. Ellis SD, Kelly ST, Shurlock JH, Hepburn ALN. Dawson-Hughes B, Gao X. Association between The role of vitamin D testing and replacement in Sleep Duration, Insomnia Symptoms and Bone fibromyalgia: a systematic literature review.BMC Mineral Density in Older Boston Puerto Rican Rheumatol 2018; 2:28. Adults. PLoS One 2015; 10(7):e013234. 8. Wu Z, Malihi Z, Stewart AW, Lawes CM, 21. Gao Q, Kou T, Zhuang B, Ren Y, Dong X, Scragg R. The association between vitamin D Wang Q. The Association between Vitamin D concentration and pain: a systematic review Deficiency and Sleep Disorders: A Systematic and meta-analysis.Public Health Nutr 2018; Review and Meta-Analysis. Nutrients 2018; 21(11):2022–37. 10(10). pii:E1395. 9. Wolfe F, Clauw DJ, Fitzcharles MA, Goldenberg 22. Gong QH, Li SX, Li H, Chen Q, Li XY, DL, Katz RS, Mease P et al. The American Xu GZ. 25-Hydroxyvitamin D Status and Its College of Rheumatology preliminary diagnostic Association with Sleep Duration in Chinese criteria for fibromyalgia and measurement of Schoolchildren. Nutrients 2018; 10(8). pii: E1013. symptom severity. Arthritis Care Res 2010; 62(5): 23. Schaad KA, Bukhari AS, Brooks DI, Kocher JD, 600–10. Barringer ND. The relationship between vitamin 10. Bennett RM, Friend R, Jones KD, Ward R, D status and depression in a tactical athlete Han BK, Ross RL. The revised fibromyalgia population. J Int Soc Sports Nutr 2019; 16(1):40. impact questionnaire (FIQR): validation and psychometric properties. Arthritis Res Ther 2009; 11(4):120. 11. Hamilton M. A rating scale for depression. J Neurol Neurosurg Psychiatry 1960; 23:56–62. 12. Learmonth YC, Dlugonski D, Pilutti LA, Sandroff BM, Klaren R, Motl RW. Psychometric properties of the fatigue severity scale and the modified fatigue impact scale. J Neurol Sci 2013; 331(1-2): 102–7. 13. Morin CM, Belleville G, Belanger L, Ivers H. The Insomnia Severity Index: psychometric indi- cators to determine insomnia cases and evaluate treatment response. Sleep 2011; 34(5):601–8. 14. Clemente MG, Argiolas D, Blue ME, Argiolas L, Bitti A, Saderi L, et al. Family-related factors may affect serum vitamin D levels. Acta Paediatr 2019; 1–2. 15. Al Zarooni AAR, Al Marzouqi FI, Al Darmaki SH, Prinsloo EAM, Nagelkerke N. Prevalence of vitamin D deficiency and associated comorbidities among Abu Dhabi Emirates population. BMC Res Notes 2019; 12(1):503. 16. Islam S, Sarkar NK, Mujahid AA, Bennoor KS, Hossain SS, Attar MM, et al. Association of Serum Vitamin D (25OHD) Level with Acute Exacerbation of Chronic Obstructive Pulmonary Disease. Mymensingh Med J 2019; 28(2):441–8. 17. Earl KE, Sakellariou GK, Sinclair M, Fenech M, Croden F, Owens DJ, et al. Vitamin D status in chronic fatigue syndrome/myalgic encephalomyelitis: a cohort study from the North-West of England. BMJ Open 2017; 7(11):e015296. 18. Maafi AA, Ghavidel-Parsa B, Haghdoost A, Aarabi Y, Hajiabbasi A, Shenavar Masooleh I,
509 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Estimation of serum 25-hydroxy vitamin D and zinc levels in patients with acne vulgaris and their association with disease severity
Asmaa Awni Haydar1* Nazanin Sadq Ismail2 Isra Mohammed Taha Alsaadi2 Marwa Miqdad Tahir1 Azgar Nash Hussen1 Balen Nariman Tahir1 Mustafa Wrya Ikram1 Aveen Nozad Adham3 Lana Yousif Mutalib3 Bashdar M. Hussen3 Abstract Backgrounds and objective: Acne vulgaris is a common inflammatory skin disease. Vitamin D3 plays an important role in the immune system. Zinc also is an essential element for human which plays a vital role in the immune system and their deficient subjects may experience increased susceptibility to a variety of pathogens. Our study was done to determine the effect of vitamin D and zinc level deficiency on the development of acne. Methods: This study was performed at Hawler Medical University/Medical Research Center and DNA laboratory, Erbil, Iraq, from August 2018 to September 2019.Thirty three samples were collected from acne patients of both genders participated in the study and twenty five samples were collected from subjects (with supplement treatment) as control group. Serum levels of 25(OH) D and zinc were determined by mini VIDAS® from bioMérieux. Results: There were statistical significant differences in age (20.52year versus 35.52 year, respectively, and body mass index between Group I (Control) and Group II (Patients) (23.55 kg/m2 versus 26.74 kg/m2), respectively. A significant high value of vitamin D levels (P ≤0.001) and non-significant high frequency of zinc levels in acne vulgaris patients when compared with patients treated with supplements (P ≤ 0.248). Conclusion: We found that vitamin D deficiency was more frequent in patients with acne, which was inversely related with disease severity. A further study with a larger sample size is needed to confirm our results because of the small number of patients in the supplementation study and the natural fluctuation of acne. Keywords: Acne vulgaris, Serum 25(OH) vitamin D, Zinc.
Introduction especially adult women with acne, is Acne vulgaris is a chronic form of skin elevated. The higher prevalence of adult disease that involves sebaceous follicles acne in the female population prompted us which is a common basis of face, chest and to investigate whether other variables back. Androgenic changes in serum sexual could present significant differences hormones which stimulate of sebum between gender.3 Adult female acne production by the adrenal glands of both affects around 40% of women and the males as well as females. The disease prevalence of the disease in adult women commonly occurs during with adolescents, is rising. Adult female acne is considered however would also children also would to be different from adolescent acne both be affected.1, 2 Acne is one of the most in terms of its clinical presentation and common skin disorders worldwide and pathogenesis.4 A combination of increased occurs primarily at puberty with a sebum formation and abnormal hyper prevalence of almost 95%. Although it is proliferation of keratinocytes results in the principally a disorder of adolescence, formation of a small microscopic lesion the prevalence of adult patients with acne, known as microcomedo.5 In addition, 1 Department of Pharmacology, College of Pharmacy, Hawler Medical University, Erbil, Iraq. 2 Ministry of Health, Erbil, Iraq. 3 Department of Pharmacognosy, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 511 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Propionibacterium acnes (P. acnes) the (VDR).13 However, it is confirmed that main predisposing factor in acne vulgaris the vitamin D via its anti-apoptotic effect which is triggers cytokine activation by protects the epidermal melanin and Toll-like receptors, which means that the melanocyte through a control over the innate immune system is also important activation, proliferation, migration of for acne development.6 Many conditions melanocytes and melanogenesis by which exacerbate the disease have been modulating T cell activation. The exact suggested including diet, menstruation, mechanism of these effects is not fully sweating, personal stress, ultraviolet understood. Vitamin D probably exerts radiation, application of pomades and melanocyte physiology through occupation. Use of medications like lithium, melanogenic cytokines (such as endothelin steroids, and anticonvulsants, exposure to -3) and regulatory factors of melanocyte excess sunlight, use of occlusive wear like life and maturation process.14 Zinc is shoulder pads, headbands backpacks, and a trace element found in the structure of underwire brassieres, endocrine disorders many metalloenzymes that play a role in like polycystic ovarian syndroms.7 Food important functions such as protein with a high glycemic index is rapidly synthesis, DNA and RNA replication and absorbed, increases serum glucose levels cell division. Zinc is therefore required for and stimulates increased glucose- growth and development.15 Also zinc plays dependent insulin signaling.8 Apart from a vital role in the development of metabolic the named consequences, depression and syndrome, taking part in the regulation eating complaints are the most prominent of cytokine expression, suppressing problems, which expose patients who are inflammation, and is also required to the risk of mental and dietary factors of activate antioxidant enzymes that severe and resistant disease. Concerning scavenge reactive oxygen species, dietary factors, studies have been decreasing oxidative stress. Zinc is found conducted to show the role of zinc in to stimulate production of IL-2 and induces treatment of drug-resistant cases of a shift from Th2 to Th1 response. It has depression. Also these kinds of also been illustrated to decrease the performances have done for vitamin D and serum levels of TNF-ߙ and inhibit the its effects on acne vulgaris.9 Vitamin D TNF- ߙ induced apoptosis of peripheral as an antioxidant agent would have blood mononuclear cells that helps in positive impression on the disease.10,11 controlling the disease activity and At the basal layer of epidermis, the reactional states.16 Zinc also have a role in cholesterol is converted to pro- vitamin D3 the correct functioning of lipid and glucose (7-dehydrocholesterol), which undergoes metabolism, regulating and forming the a photochemical transformation due to expression of insulin.17 It has also been ultraviolet B radiation (UVB) into the demonstrated to possess anti-androgenic intermediate compound pre- vitamin D3.12 properties as it causes modulation of Cholecalciferol synthesized in the skin is 5 ߙ-reductase type 1 and 2 activity.18,19 transferred to a circulatory system. The first This study headed to compare serum activation is made in hepatic cells into levels of 25(OH) vitamin D between 25-hydroxychole- calciferol – calcidiol [25 cases of acne vulgaris and age- plus (OH)D], which is the main form of vitamin sex-matched of supplemental control group circulating in the blood. The second stage to find any relationship between the vitamin occurs in the kidney cells with production and the occurrence of the disease besides of 1(OH)25-di- hydroxycholecalciferol – comparing the two groups in terms of calcitriol [1,25(OH)D], which is the main vitamin and zinc level deficiency active form of the vitamin and a specific conditions. The aims of the present study ligand for a nuclear vitamin D receptor to determine the effect of vitamin D and
522 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) zinc level deficiency on the development of Statistical Analysis acne in comparison with treatment group. The statistical result were computed by SPSS version 24 where many statistical Methods tests were used to answer the objectives of A prospective case-control study of the study. For example, the Chi-sqaure test acne vulgaris was performed at Hawler was conducted to discover the association Medical University, College of Pharmacy between the categorical variables and in cooperation with acne vulgaris patients P values were also discussed given in who referred to dermatology departments Table (3-1), also for changes in both and clinics at Awat Center and DNA measurements for all categorical variables laboratory/Erbil/Iraq. The patients were were considered and tested using Kruskal recruited from the Dermatology department Tests in case of more than two groups at Awat consultant center from April to followed by Man-Whitney U test for November 2019. Eligible patients of both multiple comparison as mentioned in gender whatever age with a history of Table 2. Acne vulgaris. Exclusion criteria prohibited enrollment of patients and controls who Results were receiving therapeutic interventions The current study was the first, to our such as systemic hormonal therapy, knowledge, in the evaluation of serum pregnant women, severe illness, infants, concentration of “25(OH) vitamin D and elderly, a vitamin D and zinc supplements zinc levels in patients with acne vulgaris (group I). History and examination were which compared cases and controls in this carried out severity of acne was recorded regard. The mean age of the of acne using the Global Acne Grading System patients was 20.52 ± 6.15 years and in (GAGS). This system divides the face, control those treated with supplements chest and back into six areas (forehead, was (35.52± 6.14).There were statistical left cheek, right cheek, nose, chin and significant differences between group I chest and back) and assigns a factor to (case) and group II (control with each area on the basis of severity. A total supplements) in gender male (M): female number of 33 patients with acne vulgaris (F) ratio of ratio of (3:30 versus 18:7) and 25 as control (with supplements 5000 accordingly. As for prevalence, adult acne IU vitamin D, Zinc gluconate 50 mg; is more prevalent in women than in men, daily oral dose for 3 months). Information while in adolescents, the prevalence is on the whole population was recorded quite similar in both sexes. Our results are including their gender and age group. based on the participant's own perception Anthropometric measurements including of the presence or absence of acne rather the height (m), weight (kg) and the than a clinical evaluation. Acne continues calculated body mass index kg/m2 were to be a common skin problem past determined. A venous blood sample drew the teenaged years, with women being from each patient’s serum test tube (for affected at higher rates than men in all age determination of vitamin D and zinc levels), groups 20 years or older, although in sex, and plain test tube to separate the serum BMI, and sun exposure hours in a day, by centrifugation at 3000 rpm for 15 there was significant difference when minutes to determine the inflammatory comparing between two groups (P ≤0.01). markers, they were determined by using However, the prevalence of 25(OH)D enzyme linked immune sorbent assay was significantly lower in patients with technology according to the instruction of severe acne compared to supplements the manufacturer of mini VIDAS® from administrated group (11.34 vs 35.47; bioMérieux. P ≤0.001). Statistical comparison of acne and control groups revealed that patients 533 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) with acne had no significantly lower 25(OH)D serum levels (13.88± 9.27 in serum zinc levels (P = 0.248) (Table 1). To female, 35.61± 47.70 in male). Although understand the vitamin D and levels of zinc obesity and decreased sun exposure using status associated with acne patients, we sunscreen are known to be associated investigated the factors the mean age of with low 25(OH)D levels, they were not the patients who were under 20 years old associated with vitamin D and zinc levels were defined as having adolescence acne deficiency in this study (P ≥0.05). However, and those over 20 years old as having we demonstrate that skin types are not adult acne, most cases of adult acne are associated with zinc and vitamin D levels of persistent acne in a population of which has non-significantly difference randomly selected individuals with similar according to severe acne patients. demographic characteristics, the levels of Furthermore, we considered family history zinc mean according to age of patients and onset of acne in male and female with acne lower than 20 years was adults. Furthermore, we saw that no (83.51±28.35) while in those over 20 years significant difference was found in mean old the mean was (78.82± 15.07) while in of family history according to both zinc 25(OH)D was (11.51±9.16) in adolescence and vitamin D levels in acne patients acne and (25.98±35.72) in adult acne (P ≤0.001). Furthermore, no significant which revealed that patients with acne had difference was found in mean GAGS score highly significantly differences (P ≤ 0.001). of patients who had exposed to sunlight Statistically variance occur when compared less than two hours per day and patients zinc levels according to gender, mean were with sun-exposure time longer than two (77.14 in female and 85.58 in male) with hours per day (Table 2).
Table 1: Baseline demographic and clinical characteristics of patients with acne and controls. Cases I Control II P-values
Age (Years) (M ± SD) 20.52 ± 6.15 35.52 ± 6.14 ≤ 0.001**
BMI (M± SD) 23.55 ± 4.68 26.74 ± 3.25 0.012
Gender (F:M) (n/%) 30 (90.9%): 3 7(28%): 18 ≤ 0.001** (9.1%) (72%) Sun Exposure hours in a day 1.32 ± 1.35 1.70 ± 1.80 0.315 (M ± SD)
Vitamin D level (M± SD) 11.34 ± 7.75 35.47 ± 43.16 ≤ 0.001**
Zinc Levels (M± SD) 73.68 ± 25.07 83.75 ± 11.64 0.248
The results were expressed as number and mean ± SD. P value was calculated by using Chi-square test for category data and independent two sample- two tailed t-test for continuous data.
544 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The threshold of 25(OH)D deficiency in this randomly assigned in comparison with study was defined as deficient ≤12 ng/ml, control group (n=25) which treated with Adequate≥ 20ng/ml and Inadequate 12_20 vitamin D supplements for 3 months ng/ml of vitamin D, according to the resulted in a statistically highly significant guideline set by the Food and Nutrition increase in 25(OH) levels (P ≤ 0.001) and Board of the institute of Medicine.24 In produced a clinical improvement compared total we determined whether vitamin D to control group. The acne patients deficiency, 15% inadequate levels, 79% in showing zinc level’s deficiency where given the severe acne patients were 25 (OH)-D zinc supplement for 3 months where put deficient, whereas only 6% adequate levels in control group, the two group didn’t of vitamin D in acne patients (Figure 1). significantly differ by any demographic or We also assessed the efficacy of vitamin D clinical factor (Figure 2) deficiency in acne patients (n=33) were
Table 2: Results of vitamin D and Zinc levels according to influencing factors.
Vitamin D Zinc P-values Age ≤20 11.51 ± 9.16 83.51 ± 28.35 0.001 ≥20 25.98 ± 35.72 78.82 ± 15.07 Gender F 13.88 ± 9.27 77.14 ± 21.07 ≤0.001 M 35.61±47.70 85.58 ± 16.28 Skin Type Dry 19.08 ± 7.53 84.2 ± 15.73 Oily 18.09 ± 12.77 77.89 ± 22.6 0.869 Normal 34.82 ± 64.82 83.24 ± 13.24 BMI Normal or Less (<23) 15.25 ± 10.26 75.71 ± 22.21 0.068 Overweight or obese (≥23) 24.34 ± 36.13 82.05 ± 18.61 Family History No 26.84 ± 36.67 82.88 ± 14.58 ≤0.001 Yes 11.27 ± 6.29 74.68 ± 27.18 Sun Exposure < 2hr sun exposure 23.42 ± 34.85 75.17 ± 18.17 0.695 ≥ 2hr sun exposure 15.92 ± 8.75 97.58 ± 14.80 25(OH)D values are presented as mean ± SD. P values are for comparison of vitamin D with Zinc levels. Age, Gender **<0.001
Figure 1: Percentages of patients with Figure 2: Clinical effects of vitamin D and different vitamin D levels. zinc level in patients with acne in comparison with supplemented control group. 555 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Discussion production of Vitamin D in the skin.24 The Acne vulgaris is a chronic inflammatory current study has evaluated the serum disease of the pilosebaceous follicles, concentration of “25(OH) vitamin D” in mainly identified by comedones and patients with acne vulgaris (11.34) which inflammatory lesions such as pustules, significantly increased in control group papules, nodules, cysts, and scars. 20 This (35.47) (shown in Table 1).The mechanism study was carried out in order to determine of vitamin D functions in some medical the levels of vitamin D and zinc levels conditions has been focused by several in acne vulgaris in comparison with researches. Youssef et al (25) as well as those treated with their supplementation other investigators explained an antibiotic therapies on the blood levels. The current effect for vitamin D and its derivatives study showed that female was more which help infection prevention too. Zinc, frequent in acne vulgaris, included 33 alone or as an adjuvant, has been found patients with acne vulgaris (30 females useful in many dermatological infections and 3 males) and 25 controls; treated with owing to its modulating actions on supplements (7 females and 18 males). macrophage and neutrophil functions, This was based on the results of previous natural killer cell/phagocytic activity, and reports which showed the importance of various inflammatory cytokines.16 In our supplementation therapy which improved study zinc levels presented in Table 2 the state of acne.21,22 However, little is were no significant difference between known about the effects of the 25(OH)D acne patients and therapeutic regimen and zinc levels supplementation on the as (73.68) and (83.75), respectively at acne vulgaris therapy. In the current study P = 0.248. The basis of zinc utilization there was statically significant difference in depends on anti-inflammatory activity. mean age between patients with acne Cytokine production and antioxidant (20.52) and control with supplementation activity are not well known. It can be (35.52). An association between age and used in acne treatment based on its acne development is unclear, but current anti-inflammatory effects. Zinc levels are research suggests that patients with acne evaluated to better understand acne were younger than controls. Several large pathogenesis and different results are studies have reported a prevalence of yielded.26 Since patients with severe acne adolescent acne ranging from 81 to 95 % in included in the study, there is a strength in young men and 79 to 82 % in young our results to document the relationship women.2 Our study found there was no between the serums zinc levels and the statistically significant association in BMI severity of acne (Figure 2). Acne vulgaris between patients with acne (23.55 kg/m2) can appear at any age but it is more and control (26.74 kg/m2) (Table.1). It has frequent during adolescence, acne vulgaris also been reported that body fat content is more common in males than in females, is inversely related to serum 25D however in adulthood, acne vulgaris is concentration, and that this associations is more common in women than in men. stronger than those between 25D and BMI Adolescent acne usually begins with the and body weight.23 Also found there was no onset of puberty, when the gonads begin statistically significant association in Sun to produce and release more androgens.27 Exposure hours in a day in acne patients As seen in (Table 2) the adolescent acne, (1.32) and control group (1.70). The main males were predominant. In persistent reason for its deficiency is the change of adult acne, males and females were lifestyle, i.e. increase in sunscreen use and equally affected, while in late-onset environmental factors such as reduced adult acne, females were predominant. presence sunlight, which is required for Hypovitaminosis D is well-documented in ultraviolet (UV) B rays to induce the those who have had bariatric or gastric 56 6 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) bypass procedures, in which a severity was influenced the most by malabsorptive state is deliberately mother’s acne history. In (Table 2), no induced,28,29 but there is no evidence that significant difference was found in mean obesity itself results in reduced absorption GAGS score of patients who had exposed of dietary vitamin D. Studies done by to sunlight less than two hours per day and vanlint30 showing no effect of vitamin D patients with sun-exposure time longer supplementation on weight included than two hours per day (Table 2). Although participants who were vitamin D replete, solar exposure is described by several and may thus have shown that giving patients as beneficial in controlling acne, supplemental vitamin D to those who are it is not known which wave length is replete has no additional effect (Table 2). responsible for this improvement: UV, Mechanism to discern body weight and visible light, or both. Provitamin D (7- Vitamin D relationship, which include: dehydrocholesterol, 7-DHC) is converted to Vitamin D Receptor (VDR) polymorphism previtamin D in the skin by exposure to shown in transgenic mice and its UVB radiation. The previtamin D is then overexpression in adipocytes that led to isomerized by body heat to form vitamin fatty acid β-oxidation, lipolysis and reduced D3. Vitamin D3 is then transported by the energy metabolism; increased parathyroid blood to the liver, where it is converted to hormone levels in Vitamin D deficiency that 25-hydroxyvitamin D (25(OH)D.36 Exposure can increase adiposity by influx of calcium to sunlight is having the major role in into adipocytes promoting lipogenesis; providing sufficient amount of Vitamin D. Vitamin D as “essential factor” in leptin Everyone should sit in the sun at least for depletion which may contribute to 15 min daily without applying sunscreen so increased appetite and obesity in Vitamin D that UV B rays reach to the skin and deficient conditions; and outdoor activity, Vitamin D production will be there.37 food intake and exercise which can also People who spend their time indoors or influence Vitamin D levels as confounding wear clothes that cover a large portion of factors.31 To understand the vitamin D the skin will have more chances of Vitamin status associated with acne patients, D deficiency due to lack of sunlight we investigated the factors that influence exposure. Vitamin D is also known by vitamin D deficiency as well as zinc levels some authorities as a preventive factor in acne vulgaris patients. In this study, against cancers 38,39and skin inflammatory noticed the oily type skin have lower levels diseases like psoriasis, etc. In addition to of vitamin D and zinc (18.09), (77.89), mediating the beneficial dermatologic respectively. It is also noteworthy that skin effects of light.40 In the randomized type determines a person’s effectiveness in sample collection of 33 acne patients producing vitamin D.32 Serum levels of 25 with vitamin D deficiency, oral vitamin D (OH)D in a broad spectrum of different skin supplementation in group II of 25 sample diseases and allergic conditions. Although produced a significant improvement in clear definitions of vitamin D insufficiency acne inflammation (Figure 2). The and deficiency are still lacking, according to observed anti-inflammatory effects of vita- the present study we see the highest need min D have several biological mechanisms. to consider vitamin D supplementation.33 The expression of inflammatory Our results suggested that family history of biomarkers, such as interleukin (IL)-6, IL-8, acne was significantly more frequent in and matrix metallo- proteinase 9, is subjects with acne (Table 2). Moreover, Di reduced by treatment with vitamin D in Landroet al34 suggested that acne vulgaris cultured sebocytes.41 Our vitamin D is associated with a family history of acne supplementation trial had a few potential in first degree relatives, mother and father. limitations, such as the use of a low dose Ghodsi35 noticed that acne prevalence and and short duration of treatment because
577 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) the risk of hypercalcemia occurs in up to 4. Dreno B. Treatment of adult female acne: a new 4% of the population in association with challenge. JEADV 2015; 29(5):14–9. malignancy, primary hyperparathyroidism, 5. Golnick H, Cunliffe W, Berson D. Management of acne: a report from a Global Alliance to Improve ingestion of excessive calcium and/or Outcomes in Acne. J Am Acad Dermatol 2003; 42 vitamin D. Our study revealed statistically 49:1–38. no significantly lower serum zinc levels in 6. Tanghetti EA. The role of inflammation in the patients with acne, as compared with the pathology of acne. J Clin Aesthet Dermatol 2013; 6(9):27– 35. control group. We believe that the zinc 7. Mohiuddin AK. A Comprehensive Review of Acne deficiency among the university students Vulgaris. J Clin Pharm 2019; 1(1):17–45. participating in this study may be related to 8. Romańska-Gocka K, Woźniak M, Kaczmarek- inadequate and imbalanced nutrition. The Skamira E, Zegarska B. The possible role of diet probable association between zinc and in the pathogenesis of adult female acne. Postepy Dermatol Alergol 2016; 33: 416–20. acne may be due to the anti-inflammatory 9. Toossi P, Azizian Z, Yavari H, Fakhim TH, Sadat action of zinc together with its effects Amini S, and Enamzade R. Serum 25-hydroxy on androgen metabolism and vitamin A vitamin D levels in patients with acne vulgaris metabolism(43). Further studies are required and its association with disease severity. Clin Cases Miner Bone Metab 2015; 12(3):238–42. to evaluate the potential effect of zinc 10. FedirkoV, Bostick RM, Long Q, Flanders WD, gluconate on the acne patients (Figure 2). McCullough ML, Sidel- nikov E, Daniel CR, In conclusion, we found that vitamin D Rutherford RE, Shaukat A. Effects of deficiency was more frequent in patients supplemental vitamin D and calcium on oxidative with acne, which was inversely correlated DNA damage marker in normal colorectal mucosa: a randomized clinical trial. Cancer with disease severity, indicating a potential Epidemiol Bio- markers Prev 2010; 19:280–91. role of vitamin D and zinc deficiency in 11. Hamden K, Carreau S, Jamoussi K, Miladi S, acne pathogenesis. A further study with a Lajmi S, Aloulou D, Ayadi F, Elfeki A. 1Alpha, 25 larger sample size is needed to confirm our dihydroxyvitamin D3: therapeutic and preventive effects against oxidative stress, hepatic, results because of the small number of pancreatic and renal injury in alloxan-induced patients in the supplementation study and diabetes in rats. Nutr Sci Vitaminol 2009; 55:215 the natural fluctuation of acne. Evaluation –22. of the tissue level of vitamin D in patients 12. Bilewicz-Stebel1 M, Beata Bergler-Czop B, with acne will also require a further study Sierant K, Wcisło-Dziadecka D. Vitamin D in dermatology. Post N Med 2018; XXXI(1A):55–60. to reveal direct evidence of the effect of 13. Gupta MA, Gupta AK. The psychological vitamin D on acne. comorbidity in acne. Clin Dermatol 2001; 19:360– 3. Conclusion 14. Birlea SA, Costin GE, Norris DA: Cellular We found that vitamin D deficiency was and molecular mechanisms involved in the action of vitamin D analogs targeting vitiligo more frequent in patients with acne, which depigmentation. Curr Drug Targets 2008; 9:345– was inversely related with disease severity. 59. A further study with a larger sample 15. Kaymak Y, Adisen E, Erhan M, Celik B, Gurer size is needed to confirm our results MA. Zinc Levels in Patients with Acne Vulgaris. J Turk Acad Dermatol 2007;1(3):71302a. because of the small number of patients in 16. Gupta M, Mahajan VK, Mehta KS, and Chauhan the supplementation study and the natural PS. Zinc Therapy in Dermatology: A Review. fluctuation of acne. Dermatology Research and Practice 2014; 2014: 1-11. References 17. Olechnowicz J, Tinkov A, Skalny A and 1. Bolognia JL. Textbook of dermatology. Elsevier. Suliburska J. Zinc status is associated with 3th edition 2012; (1):545–59. inflammation, oxidative stress, lipid, and glucose 2. Burns T. Rook’s textbook of dermatology. metabolism. J Physiol Sci 2018; 68(1):19–31. Blackwell science. 8th edition 2013; 4. 18. Nitzan YB and Cohen AD. Zinc in skin pathology 3. Skroza N, Tolino E, Mambrin A, Zuber S, Balduzzi and care. J of Dermatological Treatment 2006; V, Marchesiello A. Adult Acne Versus Adolescent 17(4):205–10. Acne: A Retrospective Study of 1,167 Patients. 19. Brocard A, Knol A, Khammari A, and Dr´eno B. J Clin Aesthet Dermatol 2018; 11(1):21–25. Hidradenitissuppurativa and zinc: a new 588 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) therapeutic approach—a pilot study. school pupils: a community-based study. J Invest Dermatology 1007; 214(4):325–7. Dermatol2009;129:2136–41. 20. Shen Y, Wang T, Zhou C. Prevalence of acne 35. .Di Landro A, Cazzaniga S, Parazzini F. vulgaris in chinese adolescents and adults. Acta Family history, body mass index, selected dietary Derm Venereol 2012; 92:40–4. factors, menstrual history, and risk of moderate 21. Kramer C, Seltmann H, Seifert M, Tilgen W, to severe acne in adolescents and young adults. Zouboulis CC, Reichrath J. Characterization of J Am Acad Dermatol 2012; 67:1129–35. the vitamin D endocrine system in human 36. Ola Engelsen. The Relationship between sebocytes in vitro. J Steroid Biochem Mol Biol. Ultraviolet Radiation Exposure and Vitamin D 2009;113:9–16. Status. Nutrients 2010; 2:482–95. 22. Lim S-K, Ha J-M, Lee Y-H, Lee Y, Seo Y-J, 37. Wadhwa Sh, Shikha D, Sharma1, Mehta M, Kim C-D, et al. (2016) Comparison of Vitamin D Thakur D, Mahajan S, Singh SK, Satija S. Levels in Patients with and without Acne: Vitamin D deficiency, skin, and sunshine: A Case- Control Study Combined with a A review. International J of Green Pharmacy Randomized Controlled Trial. PLoS ONE 2016; 2018(Suppl); 12(2):S345. 11(8):e0161162. 38. Marashian SM, Farnia P, SeyfSh, Anoosheh S, 23. Arunabh S, Pollack S, Yeh J; Aloia J F. Body fat Velayati AA. Evaluat- ing the role of vitamin D content and 25-hydroxyvitamin D levels in healthy receptor polymorphisms on susceptibility to women. J Clin Endocrinol Metab 2003; 88:157– tuberculosis among Iranian patients: a case- 61. control study. Tüberkülozve Toraks Dergisi 2010; 24. Holick MF. Vitamin D deficiency. N Engl J Med 58(2):147–53. 2007; 357:266–81. 39. Holick MF. Vitamin D: a millenium perspective. 25. Youssef DA, Miller CW, El-Abbassi AM, Cutchins J Cell Biochem 2003; 88:296–307. DC, Cutchins C, Grant WB, Peiris AN. Antimicro- 40. Reichrath J, Lehmann B, Carlberg C, Varani J, bial implications of vitamin D. Dermatoendocrinol Zouboulis CC. Vitamins as hormones. Horm 2011 Oct; 3(4):220–9. Metab Res 2007 Feb; 39(2):71–84. 26. Ozuguz P, Kacar SD, Ekiz O, Takci Z, Balta I, 41. Lee WJ, Choi YH, Sohn MY, Lee SJ, Kim do W. and Kalkan G. Evaluation of serum vitamins A Expression of Inflammatory Biomarkers from and E and zinc levels according to the severity of Cultured Sebocytes was Influenced by Treatment acne vulgaris. Cutan Ocul Toxicol 2014; 1–4. with Vitamin D. Indian J Dermatol 2013; 58:327. 27. Raza K, Talwar V, Setia A, Katare O. Acne: 42. Tebben PJ, Singh RJ, and Kumar R. Vitamin D- An understanding of the disease and its impact Mediated Hypercalcemia: Mechanisms, on life. International J of drug development & Diagnosis, and Treatment. Endocr Rev 2016; research 2012; 4(2):14–20. 37(5):521–47. 28. Cusack C, Danby C, Fallon JC, Ho WL, Murray 43. Igic PG, Lee E, Harper W, Roach KW. Toxic B, Brady J et al. Photoprotective behavior effects associated with consumption of zinc. and sun- screen use: impact on vitamin D Mayo Clin Proc 2002; 77:713–6. levels in cutaneous lupus erythematosus. Photodermatol Photoimmu- nol Photomed 2008; 24:260–7 29. Hewitt S, Søvik T, Aasheim E, Kristinsson J; Jahnsen J, Birketvedt G, et al. Secondary hyperparathyroidism, vitamin D sufficiency, and serum calcium 5 years after gastric bypass and duodenal switch. Obes Surg 2013, 23, 384–90. 30. Vanlint S. Vitamin D and obesity. Nutrients 2013; 5:949–56. 31. Mehmood ZH and Papandreou D. An Updated Mini Review of Vitamin D and Obesity: Adipo- genesis and Inflammation State. J of Medical Sciences2016; 4(3):526-532. 32. Mostafa WZ and Hegazy RA. Vitamin D and the skin: Focus on a complex relationship: A re- view .J of Advanced Research 2015; 6: 793–804 33. Amon U, Baier L, Yaguboglu R, Ennis M, Holick MF, and Amona J. Serum 25-hydroxyvitamin D levels in patients with skin diseases including psoriasis, infections, and atopic dermatitis. Dermatoendocrinol 2018; 10(1):e1442159. 34. Ghodsi SZ, Orawa H, Zouboulis CC. Prevalence, severity, and severity risk factors of acne in high
599 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Metastatic Breast Cancer to Stomach Mimicking Primary Gastric cancer
Ava T. Ismael1* Jalal A. Jalal2 Sarhang Hussein3 Nazar M.T. Jawhar4 Muhammad Yhya Shafeek5 Rafal Abdulrazaq1 Abstract Although breast cancer is quite common, metastases to the stomach are rare.. Unlike infiltrative ductal carcinoma, invasive lobular carcinoma (ILC) has a high tendency to metastasize to the stomach. The present study reports a case of a 61-year-old female who had undergone a modified radical mastectomy of the right breast for ILC eleven years before and now presented at the clinic seeking treatment for epigastric discomfort, from sour regurgitation and belching that had persisted for three months. Gastroscopy revealed a constricting obstructing narrowed lesion in the stomach, biopsy revealed a poorly differentiated carcinoma of single cell type with areas showing Indian file arrangement; the lesion was multifocal in the stomach at which the mucosa was intact. Surgery was performed because of obstructing nature of the tumor. The diagnosis of metastatic lobular carcinoma from the breast was further established using histological and immunohistochemical markers for gross cystic disease fluid protein-15, cytokeratin (CK)7 and CK20, ER, PR. Aim: Few cases of breast cancer metastasizing to the stomach have been reported, particularly those that have been confirmed using gastroscopy. This case highlights the importance of considering gastric metastasis of breast cancer as a differential diagnosis in patients who present with a gastric lesion and a history of breast cancer and to raise awareness of the condition. Keywords: breast cancer; infiltrative lobular carcinoma; metastatic gastric cancer; histopathology; immunohistochemistry.
Introduction infiltration of the uterus, peritoneum, Although breast cancer is quite common, retroperitoneum, meninges, intestine, metastases to the stomach are rare.1,2 and stomach,5 autopsies of patients The most frequent sites of breast cancer with metastatic lobular cancer showed metastasis are the local and distant lymph a 6–18% incidence of gastrointestinal (GI) nodes, brain, lung, liver and bone, metastases 6 suggesting that the GI lumen with metastasis to the stomach being is a relatively common eventual site of relatively rare. An Italian single-institution progressive metastases. Linitisplastica of retrospective review of 980 breast cancer the stomach is a potential long-term cases demonstrated a 0.3% incidence of sequela of metastatic breast cancer. It is gastric metastases.1 When metastasis to essential that the clinician be aware of this Stomach occurs, lobular carcinoma is by pathologicentity when evaluating women, far the most common breast cancer with or without a history of breast cancer, histological type, with an estimated because the treatment for metastatic prevalence of 853 to 97%.4 However, breast cancer presenting as linitisplastica autopsy series have reported metastatic is dramatically different from that 1 Department of Clinical Analysis, College of Pharmacy, Hawler Medical University, Erbil, Iraq. 2 Department of Pathology, College of Medicine, Hawler Medical University, Erbil, Iraq. 3 Department of General Surgery, College of Medicine, Hawler Medical University, Erbil, Iraq. 4 Department of Pathology, College of Medicine, Ninevah University, Ninevah , Iraq. 5 Consultant in Gastroenterology and Hepatology, Ministry of Health, Iben Sena Hospital, Ninevah , Iraq. * Correspondence: [email protected] 601 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) implemented to treatprimary gastric cancer adjuvant chemotherapy AC (Adriamycine presenting as linitisplastica.7 60 mg/m2 and Cyclophosphomide 600mg/ m2Q 3 Weeks’ time 4, NSABP B-15 Case Report regime) with fourth cycle completed in A 61-year-old female presented to the 9/1/2007. She received Paclitaxel (Taxol Department of General Surgery at Rezgary 80 mg/m2 weekly for three weeks for total Teaching Hospital of Erbil- Kurdistan of four complete cycles, she completed her region/Iraq seeking treatment for epigastric fourth cycle in 5/5/2007, and then referred discomfort from sour regurgitation and to adjuvant chemotherapy. belching that had been persisted for three Diagnosis months. The patient had no history of bleeding, tarry stools or associated Gastroscopy was done twice to the patient hemorrhagic symptoms. The patient had with interval of three weeks that revealed previously undergone a modified radical multiple small nodules in lesser curve of mastectomy for ILC of the right breast in the stomach (Figure 1 A, B & Figure 4). May 2007, with metastasis to seven axiliary Multiple endoscopic biopsies were lymph nodes out of twelve with no extra performed. Histopathological diagnosis nodal infiltrate (T2N2MX).Her mastectomy based on hematoxylin and eosin (H&E) sample immunohistochemical profile (IHC) staining slides revealed scattered poorly was positive for estrogen (ERs) and differentiated cells in the lamina propria progesterone receptors (PRs) and it was and submucosa with focal surface questionably negative for Her 2 neu (border ulceration, the cells were arranged in line +2). Work up for distant metastatic indian file pattern suggesting metastatic disease was negative, and her complete rather than primary diffuse gastric hematology and oncology profile were carcinoma of signet ring and single cell unremarkable. She was treated with patterns (Figure 2).
A B
Figure 1 (A & B): Gastroscopy revealing multiple small lesions in the body of the stomach.
612 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Chest, abdomen and pelvic CT with IV (CK) 7+ which was positive, CK20 which contrast revealed normal gastric wall was negative. Based on the medical thickness and no mass was detected history of the patient and the findings of (Figure 3). Moreover, because of limitation IHC and gastroscopy, suggestion of breast of tumor cells in both gastroscopic cancer metastasis to the stomach was biopsies, a wide histochemical pannel made. was not performed apart from cytokeratin
Figure 2: Histopathology revealed intact mucosa with infiltration of single cells in the lamina propria. (H&E, X100).
Figure 3: CT scan of abdomen revealed no mass.
Figure 4: Repeating endoscopy and biopsy. 623 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The patient undergone total gastrectomy, adenocarcinoma predominantly involving with Roux-en-Y esophagojejunostomy the muscularispropria and serosal fat reconstruction, partial colectomy (Figure 6). All five perigastric lymph nodes (transverse colon) which was indicated due were tumor free. The stricture area in to the presence of colonic strictures with transverse colon showed hemorrhagic dissection of omental fat, were sequentially infarction and it was tumor free. The performed as a part of palliative surgery omental fat showed congestion only and it duo to obstruction. The patient was was tumor free too. Immunohistochemical discharged home on postoperative day stains revealed CK7(+), CK20 (-), ER (+), 9 after tolerating oral diet (Figure 5). PR (+), and E-cadherin (faintly+),GCDFP Final histopathological analysis of (gross cystic disease fluid protein-15) the stomach, demonstrated diffuse feaures that favor a breast primary . infiltration with a poorly differentiated
Figure 5: Gastrectomy specimen representing a constricting infiltrating mass.
Figure 6: Final histological section of the gastrectomy specimen, showing single cells with indianfile arrangement (H&E X100).
634 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Discussion a specific tissue marker of breast 19 infiltrative lobular carcinoma is the second carcinomas. The recommended most common type of breast cancer, treatment of gastric metastases from it accounts for 6–14% of breast cancer breast cancer is typically a systemic with distinctive biological behavior,8,9 in treatment. Surgical intervention should be comparison with infiltrative duct carcinoma reserved for palliation or certain cases of solitary resectable gastrointestinal tract (IDC), ILC occurs in older age group, 20 mostly postmenopausal with larger metastases. The present case represents tumor size, low grade, ER-positive and a typical metastatic breast cancer to the less lympho-vascular invasion9,10 and stomach, which was detected using associated with better overall and disease gastroscopy eleven years after the surgical free survival9 Local and distant lymph removal of ILC. It is essential to use nodes, brain, lung, liver and bone are the GCDFP-15 and CK7/20 immunostaining most common sites for metastasis while for the biopsy tissue in order to identify metastasis to the stomach is relatively breast cancer metastases in the stomach. rare.9 Although studies have reported that, When a patient has a history of ILC, unlike IDC, ILC has a higher tendency endoscopic examinations should be to metastasize to the stomach, ovaries, performed carefully. Moreover, physicians meninges, pleura, skin, peritoneum, should provide the clinical history of duodenum and colon.11,12 This can be the patient to the endoscopist and the explained by the presence of discohesive endoscopist should provide sufficient small cells, a phenotypic trait that information to the pathologist in order to characterizes ILC and loss of E-cadherin, obtain an accurate diagnosis of breast which is observed in the majority of cancer metastasis to the stomach and ILC, may lead to changes in cell-cell improve the patient’s quality of life. adhesion and advantages for growth and Conclusion distant metastasis.13 Symptoms of gastric metastasis are non-specific, includes metastatic breast carcinoma to the epigastric pain, hemorrhage, anorexia, stomach is a rare event which can be dysphasia and vomiting.14 Although many misdiagnosed as primary gastric cancer. methods are used to confirm the diagnosis A diagnostic approach should include of gastric metastases, sometimes it is clinical suspicion, repeated endoscopy, difficult. The identification of gastric correct histpathological examination and metastases using endoscopy is also hard. finally immunohistochemistry is quite Endoscopic findings, includes observation helpful in confirming the diagnosis. of benign appearing lesions or11,15 diffusely References infiltrative lesions.14 Histology confirms ~90% of gastric metastatic lesions16 and 1. Ambroggi M, Stroppa EM, Mordenti P, Biasini C, Zangrandi A, Michieletti E, Belloni E, Cavanna L. immunohistochemical confirmation can aid Metastatic breast cancer to the gastrointestinal for the definite diagnosis of metastases. tract: report of five cases and review of the Since ER and PR positive expression are literature. Int J Breast Cancer 2012; 439023. not seen in gastric tumors so they are 2. Ciulla A, Castronovo G, Tomasello G, Maiorana useful for diagnosing breast cancer AM, Russo L, Daniele E, Genova EDG. Gastric 17 metastases originating from occult breast lobular metastases to the stomach but if the carcinoma: diagnostic and therapeutic problems. primary lesion was negative for ER and World J Surg Oncol 2008; 6:78. PR, then other markers should be 3. Hussain T, Elahi B, McManus P, Mahapatra T, performed such as CK20 and CK7 which and Kneeshaw P J. Gastric obstruction 18 secondary to metastatic breast cancer: also aids in the diagnosis. Also GCDFP- a case report and literature review. J Med Case 15 isn't found in benign and malignant Rep 2012; 6:232. lesions of the stomach, thus GCDFP-15 is 64 5 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 4. Hara F, Kiyoto S, Takabatake D, Takashima S, stomach: clinical and endoscopic features. World Aogi K, Ohsumi Sh, Teramoto N, et al. J Gastroenterol 2006; 12:7326–8. Metastatic breast cancer to the stomach 17. vanVelthuysen ML, Taal BG, van der Hoeven resembling early gastric cancer. Case Rep Oncol JJ, Peterse JL. Expression of oestrogen receptor 2010; 3:142–7. and loss of E-cadherin are diagnostic for 5. Harris M, Howell A, Chrissohou M, et al. gastric metastasis of breast carcinoma. A comparison of the metastatic pattern of Histopathology 2005; 46:153–7. infiltrating lobular carcinoma and infiltrating duct 18. Tot T. Cytokeratins 20 and 7 as biomarkers: carcinoma of the breast. Br J Cancer 1984; 50:23 usefulness in discriminating primary from –30. metastatic adenocarcinoma. Eur J Cancer 2002; 6. Ghirarduzzi A, Sivelli R, Martella E, Arrangoiz R, 38:758–63. Papavasiliou P, Dushkin H, et al. Gastric 19. Wick MR, Lillemoe TJ, Copland GT, Swanson metastasis from breast carcinoma: report of PE, Manivel JC, Kiang DT. Gross cystic disease three cases, diagnostic-therapeutic critical close fluid protein-15 as a marker for breast cancer: examination and literature review. Ann Ital Chir Immunohistochemical analysis of 690 human 2010; 81:141–6. neoplasms and comparison with alpha- 7. Lisa A. Whitty, David L. Crawford, Jay H. lactalbumin. Hum Pathol 1989; 20:281–7. Woodland, Jitendra C. Patel, Bryce Nattier, 20. Pectasides D, Psyrri A, Pliarchopoulou K, Charles R. Thomas, Jr. Metastatic breast cancer Floros T, Papaxoinis G, Skondra M, et al. Gastric presenting as linitisplastica of the stomach metastases originating from breast cancer: Gastric Cancer 2005; 8:193–7. report of 8 cases and review of the literature. 8. Arpino G, Bardou VJ, Clark GM, Elledge RM. Anticancer Res 2009; 29:4759–63. Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome. Breast Cancer Res 2004; 6:149–56. 9. Pestalozzi BC, Zahrieh D, Mallon E, Gusterson BA, Price KN, Gelber RD, Holmberg SB, et al. Distinct clinical and prognostic features of infiltrating lobular carcinoma of the breast: combined results of 15 international Breast Cancer Study Group clinical trials. J Clin Oncol 2008; 26:3006–14. 10. Cristofanilli M, Gonzalez-Angulo A, Sneige N, Kau SW, Broglio K, Theriault RL, Valero V, Buzdar AU, et al. Invasive lobular carcinoma classic type: response to primary chemotherapy and survival outcomes. J Clin Oncol 2005; 23:41– 8. 11. Almubarak MM, Laé M, Cacheux W, Arpino G, Bardou VJ, Clark GM, Elledge RM. Gastric metastasis of breast cancer: a single centre retrospective study. Dig Liver Dis 2011; 43:823– 7. 12. Abid A, Moffa C, Monga DK. Breast cancer metastasis to the GI tract may mimic primary gastric cancer. J Clin Oncol 2013; 31:106–7. 13. Ferlicot S, Vincent-Salomon A, Médioni J, Genin P, Rosty C, Sigal-Zafrani B, Fréneaux P, et al. Wide metastatic spreading in infiltrating lobular carcinoma of the breast. Eur J Cancer 2004; 40:336–41. 14. Taal BG, Peterse H, Boot H. Clinical presentation, endoscopic features, and treatment of gastric metastases from breast carcinoma. Cancer 2000; 89:2214–21. 15. Malhotra A, Guturu P, Basim MS, Raju GS. A rare case of breast cancer metastasis presenting as linitisplastica of the stomach and colon (with videos) Gastrointest Endosc 2009; 70:552–3. 16. De Palma GD, Masone S, Rega M, Simeoli I, Donisi M, Addeo P, et al. Metastatic tumors to the
656 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Investigation The fate of unused or expired medicines in Erbil governorate
Hewa A. Hamadameen1* Anjam H. Abdullah1 Anoosh Bashir Hagop1 Mohamad O. Qader1 Abstract Background and objective: Currently there is no clear guidance for the methods to dispose expired and unused medications in Kurdistan region of Iraq. Medications disposed improperly may contaminate surface water, ground water and drinking water. The best method which is available for medication waste is incineration. The aim of this study is to evaluate the background information and also determine current practices carried out by healthcare professionals and especially pharmacists with regards to unused medication disposal. Methods: A total of 200 healthcare professionals which includes pharmacists, nurse and others who work in community and hospital pharmacies completed a survey about practices of medication dispose and beliefs. Results: Returning unwanted medications to pharmaceutical companies and stores is the preferred way of medication disposal (78.1%); the major concern about current medication practices is water supply and environmental contamination (46.2 %). Managing pharmacy storage and amount of medication is the major route to limit medication waste product (77.2 %). Conclusion: Pharmacists consider medication waste minimizing activities are very crucial, but there is no a clear guideline for proper medication disposal. Keywords: Expired medications; Waste products; Pharmacist; Unused medicines.
Introduction these levels pose a chronic threat to Medications dispensed to patients might human or animal health.8 Many researches not be completed because of side effect, have also undertaken on the levels of intolerance, changing the dose and pharmaceuticals in waste water and their discontinuation of the medication or possible environmental consequences.9-14 medications expired that the patient will However there is not enough data behind have it after taking the medicine. Therefore the reason or factors behind having it is very common for patients to have such huge amount of unused or expired unused medications which are not used medications. Clearly a major factor is the or it is expired and they need scientific human excretion of medicines and their recommendations to be disposes properly. metabolites into sewage15 but another There have been many recent significant factor is the human through reports of pharmaceutical compounds and which unused medicines is disposed. their metabolites in the environment.1-4 Pharmaceuticals may enter the Pharmaceutical compounds have been environment through improper ways of detected in surface5 and drinking water,6 disposal of the pharmaceutical products and even though the concentrations into the toilet or sink.16.17 Also, the steps or detected are of the order of nanograms methods which are used to dispose the to micrograms per liter7 there remains expired or unused medicines might not be uncertainty about whether compounds at a good method to remove all the disposed 1 Department of Pharmaceutics Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected]
661 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) pharmaceutical products; consequently it medications which should be flushed might have a negative effect on the water down the toilet, 30 but for all the other ways and environment.18-20 If unwanted medications and in the absence of pharmaceuticals are disposed of in the a take-back program, one of the best garbage, they eventually end up in landfills method to dispose of unwanted or expired with the potential to be discharged medications in the form of solid dosage into the environment as leachate.21 form is to grind them and mix them with cat Expired, unused, split and contaminated litter and put them in a sealed container. pharmaceutical products, vaccines that are This method of disposing will decrease no longer required are considered as the poisoning chance, on the other hand Unwanted Medications (UMs) and need to the consequences or results of land be disposed appropriately.22 Unwanted fill disposal is not clear so far on the medications should never be dispensed environment.31 In addition, there are other and should always consider as methods for disposing which include pharmaceutical waste.23 There are different ‘reverse distribution network’ or drug sources for unwanted or expired take-back programs which guarantee medications which is not only includes a safer avenue for disposing of households, but also it does include pharmaceutical from homes. The aim of hospitals and healthcare organizations this study is to evaluate the background which is about 3% of total waste information and also determine current products.24 In 1999, the WHO released practices carried out by healthcare the first guidance which is a global and professionals and especially pharmacists comprehensive document on how to with regards to unused medication manage waste products safely, and in disposal. which it addresses regulatory framework, planning issues, waste minimization Methods and recycling, handling, storage and A cross sectional study (survey) was transportation, treatment and disposal performed in Erbil city about disposal options, and training of workforce.22 This of unused medications from February document explains the factors which 2019 to March 2019, Participants were contributes to proper waste management assured that free of risks in this study which includes awareness on the risks of and information obtained would be bad or inappropriate management of waste considered anonymous and confidential. products, financial issues, lack of training A consent form, explaining the research and having a bad system to manage all of purposes, attached to each questionnaire these. The use of pharmaceutical products was read. A total of 200 people is on the rise, with an estimated increase including pharmacists, nurses, doctors from 2 billion to 3.9 billion prescription assistant pharmacists and other numbers yearly between 1999 and 2009 healthcare professionals who work in the in the United states of America alone.25 pharmaceutical field participated in the Additionally, having a high percentage of survey, the participants were either visited wasting unused medicines is due to lack of by the author at their work sites and invited patient compliance to the medicine which to participate, or replied to the online results in stopping to take the product survey tool via google form of the which is prescribed by doctor.26-28 In order questionnaire with access to the link of to decrease the bad impact of wasting the survey. Also, items which are used expired or unwanted medicines to the to increase awareness on and the environment, then we should determine the consequences on environment were used cause and the reasons behind this. The in order to spread the idea of protect our FDA has listed 26 drugs as ‘dangerous’ environment before devastating it such as
672 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) recycling.33,34 The questionnaire which is professionals) aged 20-41 years. The available in appendix, divided into individual pharmacists who are doing some three sections: Background information, kind of activities in order to decrease awareness which is connected to wasting of medicines are classified as environmental hazards Practice of dispensing, prescribing a left over stage. disposing of unused/unwanted medications As it is explained in table 1; few questions from pharmacies 3. were asked the participants, and the Data management and statistical answers are put as number and analysis percentage for every question. In Data were recorded on a specially prescribing stage; in order to decrease the designed questionnaire, collected and medication waste product in prescribing entered in the computer and then analyzed stage, prescribers can manage the amount using appropriate data system which is of medications which are prescribed. Microsoft Excel worksheet (Excel 2010). Approximately 40 participants explained The results were compared between the that, the doctors can manage or tailor the responders regarding different variables, product amount based on the medicines with a statistical significance level of <0.05. feature such as cost of the product, age of The results were presented as rates, ratio, the patient and expected healing time. frequencies, percentages in tables and Additionally, in pharmacy related activities figures. the role of pharmacist on this can be done through adjusting the amount of prescribed Results medications, sometimes the patients might Two hundred participants replied to have more than the amount they need, so questionnaire (80.7% pharmacists, 9.6% the pharmacist can reduce the amount nurses, 4.6% assistant pharmacists, which consequently results in less drug 3.6% doctors and 2% other healthcare expiration or unused medicines, and this is
Table 1: Waste-reducing activities that can be done by pharmacists and the number of activities which are done to minimize medication waste product.
Activities N = 200 No. (%) Prescribing stage Prescribers tailor prescription amount 40 (20)
Dispensing stage Pharmacists adjust prescribed amounts 132 (66) Dispense opened medication package 114 (57) Advice patients how to store medications 146 (73) Advice patients how to get rid of unused medications 59 (29.5) check expiratory date on medications 180 (90) Manage medication amounts in stock Limiting storage amounts 154 (77) Exchange medications with other pharmacies 97 (48.5) Discuss the quantity needed for symptom improvement with the patient 126 (63) Leftover stage Collect unused medications 104 (52)
683 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) one of the activities by pharmacist. One and taking the required amount, example of such an activity is; when pharmacist also helping the patient on how a pharmacist notices that a physician has to manage the waste in case of expiration prescribed more medicines than they or not using the medicine. Lastly, in needed, they inform the patient and reduce leftover stage; approximately 52% of the dispensed amount. Concerning internal participants said that the amount of unused waste management at the pharmacy, medications is collected in the pharmacy pharmacists informed that they manage the for safe disposal. Majority of the amount of pharmaceutics which are kept in participants preferred returning of expired stock. For example, 48.5% reported that medications to pharmaceutical companies they exchange medications that are rarely which was 78.10% of the participants as it used or that are close to the expiry date to is shown in Figure 1. On the other hand, prevent disposal. Stock management was only 5.60% of participants preferred to most frequently reported activity; this was flush down expired medicines to the successfully performed in some pharmacy toilet. Unfortunately, 18.40% of participants for their patients. After talking to the patient preferred to throw away expired medicines about the amount of medications which to garbage which is very dangerous for the is prescribed for, also activities which are environment. Concerning about medication related to patient were reported which disposal was another question; majority of includes medication management and the participants (46.20%) were concerned storage by the patient. This includes about disposing used medications checking the medication for patient and improperly will affect environment and talking about the medicines they take and contaminate water as well as it is shown in reducing the extra quantity or taking the Figure 2. While, 44.60% of the participants required quantity. Furthermore, in addition answered yes for this questions that the to giving instruction to the patient by the expired medication which is thrown away pharmacist about the use of the medication to the garbage will be used by others.
Figure 1: Preferred ways responded by Figure 2: Major concerns about current the participants to dispose unused medication disposal practices. medications.
694 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Discussion and unnecessary waste. Patients receiving medications for more than 30 days, Collection of expired or unwanted they are mostly waste a part of those medicines is very crucial in order to prevent medications.45Additionally, pharmacists the harm on environment and provide can use a good way to decrease waste a safety zone for human living in that place. of medicines through providing a trail of In every country and especially developed medicines to the patient and if they countries, pharmacy and pharmacies have continue on it, then they can take the an important role to dispose unused rest of it. Paterson et al. showed that medications properly. According to our a split-fill supply could reduce the cost survey questionaries’ pharmacist showed of medication waste. Whars’ more, their enthusiasm to and the positive impact dispensing medications through increasing of collecting or disposing unwanted the frequency and giving in different medicines under a good system where batches on the same day, or either at government can implemented in hospital, it might reduce the medication everywhere of the country. Also, majority of waste rate. However, implanting this new the participants confirmed the harmfulness strategy might not be easy as it need more of throwing medicine to the environment. 31, 32 cost to the government through providing One of the common practices of this bigger amount of medicines with cheaper system is awareness and using the price is required Pharmacists should to disposal system by every individual of evaluate the patient individually when it society because the drug is used by every comes to increase the frequency of the population rather than only health care therapy because it does not save the cost professionals. Self-medications tendency all time. But, it might decrease pollution of increases in case of unused medications environment. This is the first study that and may result in pediatric intoxication, shows an overview of activities taken by while expired medications place human community pharmacists in Erbil city. For and environmental health in danger due 35-38 this study, several limitations could be to inappropriate disposal practices. identified. Most importantly is the role of National or local drug take-back programs pharmacists which are involved in the implemented in different countries to tackle study, however many other healthcare these unfavorable outcomes gained professionals work in the pharmacies. acceptance by the public with important 39, 29,40-43 Hence, it is assumed that the missing achievements. This study suggests answers would not have altered the that activities which are related to findings. pharmacy are easier and can be implemented much better compare to Conclusion others. Additionally, the activities which This study demonstrates that pharmacists focus on reducing or prevention of waste have developed many activities to reduce products are another important activity medication waste in all stages of the which can be one of the reliable and pharmaceutical supply chain. However, promising activities. A recent and current not all potential activities to decrease evidence and real example of waste medication waste have been implemented management is waste reducing activity in daily practice. Gaps exist in practices where fewer amounts of medications are of medicine disposal. The government is prescribed for the expensive one. Limiting responsible to provide safe and cost the medication amount supplied for a first effective medicine disposal program. Also, time to a two-week period, followed by an updated system is required for our 30 days for a repeat prescription,44 may community in order to collect the expired decrease the risk of unused medications or unused medications in order to dispose
705 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) them safely which do not harm the 13. Parolini M, Binelli A, Cogni D, Riva C, environment, in addition to doing media Provini A. An in vitro biomarker approach for the campaign activities. evaluation of the ecotoxocity of non-steroidal anti -inflammatory drugs (NSAIDs). Toxicol in Vitro 2009; 23:935–42. References 14. Rosal R, Rodea-Palomares I, Boltes K, 1. Kasprzyk-Hordern B, Dinsdale RM, Guwy AJ. The Fernandez-Pinas F, Leganes F, Gonzalo S, et al. occurrence of pharmaceuticals, personal care Ecotoxicity assessment of lipid regulators in products, endocrine disruptors and illicit drugs in water and biologically treated wastewater using surface water in South Wales, UK. Water Res three aquatic organisms. Environ Sci Pollut Res 2008; 42(13):3498–518. 2010; 17:135–44. 2. Snyder SA. Occurrence, treatment, and 15. Heberer T, Reddersen K, Mechlinski A. From toxicological relevance of EDCs and municipal sewage to drinking water: fate and pharmaceuticals in water. Ozone Sci Eng 2008; removal of pharmaceutical residues in the 30:65–9. aquatic environment in urban areas. Water Sci 3. Kummerer K. The presence of pharmaceuticals in Technol 2002; 46:81–8. the environment due to human use present 16. Ruhoy IS, Daughton CG. Types and quantities knowledge and future challenges. J Environ of leftover drugs entering the environment via Manage 2009; 90:2354–66. disposal to sewage — revealed by coroner 4. Snyder SA, Benotti MJ. Endocrine disruptors records. Sci Total Environ 2007; 388:137–48. and pharmaceuticals: implications for water 17. Comeau F, Surette C, Brun GL, Losier R. sustainability. Water Sci Technol 2010; 61:145– The occurrence of acidic drugs and caffeine in 54. sewage effluents and receiving waters from three 5. Bartlett-Hunt SL, Snow DD, Damon T, Shockley J, coastal watersheds in Atlantic Canada. Sci Total Hoagland K. The occurrence of illicitand Environ 2008; 396:132–46. therapeutic pharmaceuticals in wastewater 18. Nakada N, Tanishima T, Shinohara H, Kiri K, effluent and surface waters in Nebraska. Environ Takada H. Pharmaceutical chemicals and Pollut 2009; 157:786–91. endocrine disrupters in municipal wastewater in 6. Benotti MJ, Trenholm RA, Vanderford BJ, Holady Tokyo and their removal during activated sludge JC, Stanford BD, Snyder SA. Pharmaceuticals treatment. Water Res 2006; 40:3297–303. and endocrine disrupting compounds in U.S. 19. Kim S, Aga DS. Potential ecological and drinking water. Environ Sci Technol 2009; 43:597 human health impacts of antibiotics and –603. antibiotic-resistant bacteria from wastewater 7. Moldovan Z, Schmutzer G, Tusa F, Calin R, treatment plants. J Toxicol Environ Health Part B Alder AC. An overview of pharmaceuticals and 2007; 10:559–73. personal care products contamination along the 20. ZoritaS, Martensson L, Mathiasson L. river Somes watershed, Romania. J Environ Occurrence and removal of pharmaceuticals Monit 2007; 9(9):986–93. in a municipal sewage treatment system in the 8. Zuccato E, Castiglioni S, Fanelli R, Reitano G, south of Sweden. Sci Total Environ 2009; Bagnati R, Chiabrando C, et al. Pharmaceuticals 407:2760–70. in the environment in Italy: causes, occurrence, 21. Barnes KK, Christensen SC, Kolpin DW, effects and control. Environ Sci Pollut Res 2006; Focazio MJ, Furlong ET, Zaugg SD, et al. 13:15–21. Pharmaceuticals and other organic waste 9. Pomati F, Castiglioni S, Zuccato E, Fanelli R, water contaminants within a leachate plume Vigetti D, Rossetti C, et al. Effects of a complex downgradient of a municipal landfill. Ground mixture of therapeutic drugs at environmental Water Monit Rem 2004; 24:119–26. levels on human embryonic cells. Environ Sci 22. WHO. Waste from health-care activities. Technol 2006; 40:2442–7. F a c t s h e e t 2 5 3 . W o r l d H e a l t h 10. Lienert J, Gudel K, Escher BI. Screening method Organization;2011.Available:http://www.who.int/ for ecotoxicological hazard assessment of 42 mediacentre/factsheets/fs253/en/index.html pharmaceuticals considering human metabolism (accessed 29.11.11). and excretory routes. Environ Sci Technol 2007; 23. Kaiser Family Foundation, 2010. Prescription 41:4471–8. Drug Trends. Available http://www.kff. Org/ 11. Schmitt-Jansen M, Bartels P, Adler N, rxdrugs/upload/3057-08.pdf. (Accessed July 11 Altenburger R. Phytotoxicity assessment of 2010). diclofenac and its transformation products. Anal 24. Lindberg MJ, Andersen SE, Christensen Bioanal Chem 2007; 387:1389–96. HR, Kampmann JP. Compliance to drug 12. Li ZH, Randak T. Residual pharmaceutically prescriptions. Ugeskr Laeger 2008; 170:1912–6. active compounds (PhACs) in aquatic 25. Ekedahl ABE. Reasons why medicines are environment — status, toxicity and kinetics: returned to Swedish pharmacies unused. Pharm a review. Vet Med 2009; 54:295–314. World Sci 2006; 28:352–8.
716 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 26. Mitka M. FDA: flush certain unused medications. 40. Maeng DD, Ann L, Wright EA. Patient J Am Med Assoc 2009; 302:2082. characteristics and healthcare utilization patterns 27. Tong AYC, Peake B M, Braund R. Disposal associated with unused medications among practices for unused medications around the medicare patients. Res SocAdm Pharm 2016; world. Environ Int 2011; 7(1):292–8. 13:1090–4. 28. Glassmeyer ST, Hinchey EK, Boehme SE, 41. Bekker CL, Van Den Bemt B J F, Egberts ACG, Daughton CG, Ruhoy IS, Conerly O, et al. Bouvy M L, Gardarsdottir H. Patient and Disposal practices for unwanted residential medication factors associated with preventable medications in the United States. Environ Int medication waste and possibilities for 2009; 35(3):566–72. redispensing. Int J Clin Pharm 2018; 40:704–11. 29. Guagnano GA. Altruism and market-like 42. Paterson J M, Anderson GM. Trial prescriptions behavior: an analysis of willingness to pay for to reduce drug wastage: Results from Canadian recycled paper products. Popul Environ 2001; programs and a community demonstration 22(4):425–38. project. Am J Manag Care 2002; 8:151–8. 30. Centers for Disease Control and Prevention 43. Fasola G, Aprile G, Marini L, Follador A, Mansutti (CDC). Nonfatal, unintentional medication M, Miscoria M. Drug waste minimization and cost exposures among young children–United States, -containment in Medical Oncology: two-year 2001–2003. MMWR Morb Mortal Wkly Rep 2006; results of a feasibility study. BMC Health Serv 55(1):1–5. Res 2008; (1)8–70. 31. De Bolle L, Mehuys E, Adriaens E, Remon JP, 44. Tilson V, Dobson G, Haas CE, Tilson D. Van Bortel L, Christiaens T. Home medication Mathematical modeling to reduce waste of cabinets and self-medication: a source of compounded sterile products in hospital potential health threats. Ann Pharmacother. 2008; pharmacies. Hosp Pharm 2014; 49:616–27. 42(4):572–9. 45. Millar J, McNamee P, Heaney D, Selvaraj S, 32. Pharmaceutical Waste Reduction in the NHS. Bond C, Lindsay S, et al. Does a system of Report, Version 1, June 2015 [cited 2017 April 6]. instalment dispensing for newly prescribed Available from: https://www.england.nhs.uk/wp medicines save NHS costs? Results from content/uploads/2015/06/pharmaceutical-waste a feasibility study. Fam Pract 2009; 26:163–8. reduction.pdf. 33. Kusturica MP, Tomas A, Sabo A. Disposal of unused drugs: knowledge and behavior among people around the world. Rev Environ Contam Toxicol 2017; 240:71–104. 34. Coma A, Modamio P, Lastra C F, Bouvy M L, Mariño E L. Returned medicines in community pharmacies of Barcelona, Spain. Pharm World Sci 2008; 30(3):272–7. 35. Akici A, Aydin V, Kiroglu A. Assessment of the association between drug disposal practices and drug use and storage behaviors. Saudi Pharm J 2018; 26(1):7–13. 36. Perry LA, Shinn BW, Stanovich J. Quantification of ongoing communitybased medication take-back program. J Am Pharm Assoc 2014; 54:275–9. 37. Persson M, Sabelström E, Gunnarsson B. Handling of unused prescription drugs: knowledge, behaviour and attitude among Swedish people. Environ Int 2009; 35(5):771–4. 38. Thach AV, Brown CM, Pope N. Consumer perceptions about a community pharmacy-based medication takes back program. J Environ Manage 2013; 127:23–7. 39. Ministry of Health Welfare and Sports. Arrangement: Preventing Waste by Limited Prescribing. Available online: https:// www.rijksoverheid.nl/ministeries/ministerie-van- volksgezondheid-welzijn-ensport/ nieuws/2016/11/29/afspraak-verspilling- voorkomen-doorkorter-voorschrijven(accessed on 12 December 2016).
727 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Antimicrobial activity extracts from three species of fresh water algae and used against some pathogenic microorganisms and analysis of minerals in algal species
Kwestan Hassan Sdiq1* Trifa Kamal Jalal Farkha2 Awara Khdir Smail3 Abstract Background and objective: Algal organism are affluent funds for pharmaceuticals on the basses of presence some primary , secondary metabolic content and The distinctive Chemical compounds which have the biological activity such as toxicity, antibacterial, antifungal, antiviral, antitumor and other specific activities. the algae species antimicrobial activities have trivial effective side and have precious therapeutic potential rather than the synthetic antimicrobials objective of the study was to determine antimicrobial activity and mineral analysis of crude extract of three fresh water algal species which isolated from Dukan lake water stations then comparison their inhibition activity against 2 types of pathogenic bacteria and one type of fungus species with negative (organic solvent) and positive control (antibiotic and antifungal). Three fresh water algal species (Chlorella vulgaris), (Chara vulgaris) and (Spirogyra pratensis). Methods: The algal species were cultivated was extracted by using 3 different organic solvents (Ethanol, Ethylacetate and Chloroform) then used to produced their inhibitory effect against 2 bacterial pathogen (Escherichiacoli) gram negative and (Staphylococcus aureus) gram positive and one fungus species (Aspergillusniger) by using agar well diffu- sion methods. Results: The obtained results elevated that the ethanol and ethyl acetate extract of all algal species in this study showed antimicrobial activity against all organisms tested except Staphylococcus aureus which was resistant against the ethanol extract of Chlorellavulgaris while the chloroform extract of Chara vulgaris and Spirogyrapratensis having the antibacterial activity only against the gram negative bacteria (Escherichia coli). In the end of this study analysis for three species of freshwater algae, shows that contain these minerals (Na, K, Ca, Mg, Ni, Fe, Zn, Mn, Cu, Pb, Al, Cd, Cr, and Co) by using Flame atomic absorption spectrophotometer. Conclusion: These results of algae species in these study give an indication of the presence promising antimicrobial compounds. Keywords: Antimicrobial activity; Algal species; Mineral analysis.
Introduction organisms are rich source of novel and Algae are a large and diverse group of biologically active primary and secondary organisms from which a wide range of metabolites and also They contain more secondary metabolites have been isolated. than sixty trace elements including A number of these compounds possess minerals, proteins, iodine, bromine and biological activity such as toxicity, many bioactive substances.2 To date, antibacterial, antifungal, antiviral, antitumor many chemically unique compounds of and other specific activities. Many bioactive fresh water origin with various biological and pharmacologically active substances activities have been isolated and some of have been isolated fromalgae.1 Algal them are under investigation while some
1 Department of Medical Microbiology, College of Health Sciences, Hawler Medical University, Erbil, Iraq. 2 Department of Biology, Faculty of Science and Science Education, School of Science, Sulaimani University, Iraq. 3 Department of Biology, Faculty of Science and Health, School of Science, Koya University, Erbil, Iraq. * Correspondence: [email protected] 731 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) are being used to develop new intervals, this method was repeated till pharmaceutical industry.3,4,5,6,7 The cell a uni-algal culture or cultures have been extracts and active constituents have been gained.15 shown to have antibacterial activity in Cultivation of the purified algae: vitro against Gram positive and Gram A small part of uni-algal culture which was negative bacteria. In addition, a wide range microscopically confirmed as uni-algal of results of in vitro antibacterial and culture was transferred into media nutrient antifungal activities of extracts of fresh solutions within a 250 ml sterile conical water algae have been reported.8 flask to get appropriate growth and The existence of bioactive compounds incubated for 2-3 weeks according to in algae is to be expected due to method of Jawad.16 Then the cultivated occurrence of these organisms in aquatic algae was centrifuged by centrifuge natural communities, where an inhibitory 10000rpm for 15 minute the algae pellet interaction occurred between producers was collected and dried by oven at 40 ̊C and competitors within the same habitat. and the dried algae was preserved in These metabolites may be synthesized plastic bottle in refrigerator .In order to under stress conditions and low growth sustain the viability of the uni-algal growth, rate.9The use of antimicrobial drugs against these cultures were renewed every two infectious diseases has certain limitations weeks by sub culturing into another media due to changing patterns of resistance in nutrient solution.17 pathogens and side-effects they produce. Collection of macro algae: These limitations demand for improved Algal blooms samples were collected from pharmacokinetic properties, which water of studied area. The blooming of continued research for new antimicrobial Algal biomass was handpicked, samples compounds for the development of drugs.10 were cleaned of epiphytes, extra generous matter and necrotic particles are removed. Methods , and placed in clean plastic containers Isolation and identification of algal with their original water and brought to the species: laboratory, they identified as Spirogyra and Three algal species (Chlorellavulgaries, Chara, Then the samples were rinsed with Charavulgaris and Spirogyrapratensis) distilled water and shaded dried then cut were isolated from dukan lake have been into small pieces and powdered in a mixer identified by using those key of grinder and dried at 40 ̊C in an oven for 2-3 7 identification and references.11,12,13 days till the dry weight was constant. Culturing of algae: Preparation of crude extract of algae: Uni-algal culture was obtained culturing The samples of algae were dried at room algae, the surface of each media plates temperature under shade and powdered. was inoculated with 1 ml of sampled water, The powder was dissolved with three the inoculum distributed with a sterile different organic solvent (ethanol, ethyl spreader or streaking using a sterile loop. acetate and chloroform) (1/10w/v) and The inoculated plates were incubated in soaked overnight. The solvent extracts a controlled temperature and illumination, were centrifuged at 2000 rpm for 15 min (White fluorescent tubes).14 In the present (sigma). The supernatant which contained study 1000 lux for each 10 plates, polyphenols was recovered. The pellet was temperature is 25±3 and the photo period dissolved twice in same solvent extract time of 16 light and 8 dark time were used. (1/10 w/v). The supernatants were filtered Aggregated colonies were observed on through a watt man filter paper and the surface of plates. Part from these concentrated by using Rotary evaporator colonies was stroke on other plates. Each (40-45˚C). The dried extract was dissolved subculture was examined at different in the same organic solvent and stored at 742 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 4°C before analyzing.18 after 24 hr. Assays were run in triplicate. Antimicrobial activity: After incubation the inhibition zones around All organisms used for antimicrobial activity the wells as an evidence of antibacterial obtained from Media Diagnostic Center, in activity were measured underside and Kurdistan region in Erbil, crude extract expressed in millimeter.9, 17, 25 was used for antimicrobial activity.19 The Anti-fungal assay: crude solvent extract were tested against Antifungal activity was evaluated by agar the gram positive bacterial species; well diffusion method. For antifungal Staphylococcus aureus ''ATCC:25923'' and activity 20 ml of Potato dextrose agar were the gram negative bacterial species; poured in petri dishes after solidify and Escherichia coli ''ATCC:25922'' and one then inoculated with 0.1 ml of 5 days fungus Aspergillus niger''ATCC:16404''. glucose peptone broth culture of test fungi The inoculated plates were incubated for and yeast. The agar plates inoculated with 24 h at 37°C for bacteria and inoculated for the test microorganisms were incubated for 3 days at 30°C for fungi.17,20 The turbidity 1h before placing extract, following this of the suspension of the activated broth wells of 25 µl of crude extract of algae which used for culturing compare with were applied on agar medium. After 0.5 McFarland standard solutions (1.5x10 incubation all plates were observed for 8x10 cell /ml) then 1ml of bacterial zones of growth inhibition, and the suspension was added to 9ml of sterile diameters of these zones were measured normal saline after that added to a Petri in millimeters. All tests were performed on dish agar. 1ml of the prepared bacterial sterile conditions in duplicate and repeated suspension was added to the Petri three times. Itraconazole was used as dish.21Three plates were tested per target positive control, and a solvent extracts as microorganism. The plates were left 2 h at negative control.9, 26 4 ◦C then incubated at 37 ◦C for 24 h and Mineral analysis: examined for zones of inhibition around Samples for mineral analysis were thedisc.22 dissolved in dilutes nitric acid and Antibacterial assay: analyzed through flame atomic -For antibacterial activity the bioactivities absorption spectrophotometer, the of algae were tested by agar well procedures described by.27 diffusion method with the extract of algae species.23,24 Results -For inoculum preparation and assay of an- Antimicrobial activity: tibacterial activity, Mueller Hinton Agar was Three algal species were tested for used. antimicrobial activity against two At pH 7.4±0.2 after autoclaving at 121°C pathogenic bacteria and one fungus for 15 min. poured in petri dishes, species (Table 1), the degree of microbial inoculated with 0.1ml of a 24 hr. broth activity was varied with references to culture of test bacteria. Four wells of 6 mm algal species, type of solvent used for each were made onto the medium extraction, and the type of microorganism. using a cork borer. Petri dishes left for Antimicrobial activity of crud extracts were 15 minutes until bacteria were absorbed to evaluated using agar well diffusion method medium, then extracts (50 µl) were poured in comparison to the reference drug into wells. The agar plates were incubated antibiotic (Erythromycin) for bacterial at 37 ̊C for 24 hr. and the inhibition zones strains and antifungal (Itroconazol) for measured with a ruler and compared with fungus .The ethanol extract of Chlorella the negative control well (well containing vulgaris showed antimicrobial activity only the respective solvent) and against E. coli and Aspergillusniger which erythromycin (15μg) as positive control their inhibition zone was 13.5mm and
753 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 11.9mm in diameter respectively, while antibacterial activity against both gram Staphylococcus aureus, was resistant positive and gram negative bacterial against chlorella vulgaris. Ethanol extract of strains which was 12.5mm and 10.5 mm, Chara vulgaris and Spirogyra pratensis respectively. Chloroform extract of showed antimicrobial activity for all Chlorella vulgaris was failed to inhabit the microorganisms which used in this growth of any microorganisms used during study .The highest inhibition zone of was the study. While the chloroform extract of recorded by Chara vulgaris crud extract Chara vulgaris and Spirogyra pratensis against Staphylococcus aureus with the inhibited the growth of E. coli which was inhibition zone of 17.3mm in diameter and 10.3mm and 11mm, respectively. From the the minimum inhibition zone was recorded result in Table1 It was clear that Chara by Spirogyra pratensis which was 7.0mm vulgaris extracted by ethanol showed in diameter against Staphylococcus a highest activity against Aspergillusniger aureus. The ethyl acetate extract of which was reached 18.5 mm in diameter. Chlorella vulgaris and Chara vulgaris The results of positive control for showed antimicrobial activity against all erythromycin were 8.7 mm for gram microorganisms which used in the study positive bacteria and 9.7 mm for gram and the highest inhibition zone was negative bacteria. The result for recorded against the gram positive bacteria Itraconazole was recorded 19.0mm in which were 14.3mm and 16.0 mm in diameter of inhibition zone against diameter respectively, While the ethyl Aspergillusniger . acetate extract of Spirogyra showed
Table 1: Antimicrobial activity of three different extract against. Algae +Controls Inhibition zone (mm) of Microrganisms Staph.aureusE.coliAsp.niger
Chlorella culgarisethanol 0.00 13.5 11.9 Ethyle acetate 14.3 13 10.5 Chloroforn 0.00 0.00 0.00 Chara vulgaris ethanol 17.3 16.5 18.5 Ethyle acetate 16 13.5 17 Chloroforn 0.00 11 0.00 Spirogyra pratensisethanol 7 13.5 10 Ethyle acetate 12.5 10.5 12 Chloroforn 0.00 10.3 0.00 Erythromycin and Itraconazol 8.7 9.7 19
( Positive control) Negative control ethanol 0.00 0.00 0.00 Ethyle acetate 0.00 0.00 0.00 Chloroforn 0.00 0.00 0.00
764 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Mineral analysis: mineral analysis of all algal species, In Table 2, we summarize our results of analysis by diluted nitric acid
Table 2: Mineral analysis of algae µg/gm.
Minerals Chlorella Chara Spirogyra
Sodium (Na) 500 380 270
Potassium (K) 560 180 105
Calcium(Ca) 600 85 73
Cobalt (Co) 1.84 1.20 0.72
Chromium(Cr) 20.3 6.20 3.41
Copper (Cu) 3.20 1.25 2.00
Iron(Fe) 1625 521 422
Magnesium(Mg) 5241 1896 1284
Zink( Zn) 80.5 60.24 48.35
Manganese (Mn) 18.65 65.2 22.3 18.65
Cadmium (Cd) 1.31 0.54 0.21
Aluminum (Al) 210.2 55.4 20.1
Nickel ( Ni) 1.27 1.32 0.95
Lead (Pb ) 2.31 0.99 0.84
From the results appear that contain high amount (5241 µg/gm) Mg in Chlorella, followed by (1625 µg/gm) Fe in Chlorella, followed by (600 µg/gm) Ca in Chlorella, while minimum amount (0.21 µg/gm) Cd in Spirogyra.
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Discussion While chloroform extract of Chlorella Chlorella vulgaris extracted by ethanol vulgaris was failed to show growth showed moderate antibacterial activity inhibition against any microorganisms against the gram negative E. coli. While which used during the study. Low ethyl acetate extract of Chlorellavulgaris, antimicrobial activity was detected in the showed antimicrobial activity against all chloroform extracts. This probably was microorganisms which were used in this because of polar nature of the active study. The chloroform extract of Chlorella components. It revealed that the chance of vulgaris was failed to show any bioactivities producing antimicrobial activity is higher in ethanol and ethyl acetate extracts than against all microorganisms used during the 38 study. Several researchers proved that chloroform. Indicated that there are many the fresh water algae have anti-microbial factors affecting the algae extraction. activity.17,23,28,29,30,31,32,33 The ethanol extract Among them, extraction temperature, the of Chlorella vulgaris showed the concentration and extraction time are antibacterial activity against E. coli which key factors, intraspecific variability in the have the inhibition zone was 13.5mm production of secondary metabolites in diameter this result was similar to occasionally related to seasonal variations, the results recorded by which was differences in the extraction protocols used 18mm and 20.5mm, respectively.34,35 While to recover the active metabolites as well as Staphylococcus aureus, was resistant this differences in the assay methods may be result was agreed to the result obtained by causes the variation between our results (Al-Wathnani) using the mixture of three and those from other studies. solvents methanol:acetone:diethylether as Conclusion 5:2:1 v/v. and disagree with the result of ( Vishnu and Sumathi, Abdin and Antimicrobial activity was carried out Hala).4,23,34Aspergillusniger inhibition zone against two bacterial pathogens was reached 11,9mm in diameter this ( Escherichiacoli ) gram negative bacteria result similar to that investigated by and (staphylococcus aureus) gram positive (Ghasmeni, Abdin and Hala).4,35 Ethanol bacteria and one fungus species and ethyl acetate extracts of Spirogyra (Aspergillusniger). The obtained results pratensis showed the antibacterial activity elevated that the ethanol and ethyl acetate against both gram positive and extract of all species in this study showed gram negative bacterial strains, inhibition antimicrobial activity against all organisms zones recorded was12.5mmand 10.5 mm tested except Staphylococcus aureus respectively, and the antifungal activity which was resistant against the ethanol was 12mm in diameter, agreement to the extract of Chlorellavulgaris while the result of those references.35,36 Generally chloroform extract of Chara vulgaris and gram negative bacteria was resistant Spirogyrapratensis having the antibacterial against antibiotic compared with gram activity only against the gram negative positive bacteria because of their more bacteria (Escherichia coli). These results of complex multi layered cell wall structure, algae species in these study give an which makes it more difficult for the indication of the presence promising active compound to penetrate.37 Results of antimicrobial compounds. Further Spirogyra extract by chloroform revealed phytochemical studies are needed to that the spirogyra produce antibacterial elucidate the components responsible for agent against the gram-negative bacteria antimicrobial activity of these extracts (E. coli) inhibition zone recorded was against human pathogen. 10.3mm in diameter; this result was agreed with the result of (Hemavani, et al.,).19
786 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) References 15. Littler MM, Littler MM, Stein-Taylor JR, Littler DS, editors. Handbook of phycological methods: 1. Rout S and Kumar A. A review on the potentiality volume 4: ecological field methods: macroalgae. of marine seaweeds as a medicinal source. World Cambridge University Press 1985. journal of pharmacy and pharmaceutical sciences 16. Jawad AM. Interaction between cyanobacteria 2015; 4(10). and other micro-organisms. Ph.D. Thesis. 2. Asthana RK, Tripathi MK, Srivastava A, Liverpool University. England 1982. Singh, AP, Singh SP, Nath G. Isolation and 17. Abdo SM, Hetta MH, Samhan FA, Din RE, identification of a new antibacterial entity from Ali GH. Phytochemical and antibacterial study of the antarcticcyanobacterium Nostoc CCC 537. five freshwater algal species. Asian journal of J Appl Phycol 2009; 21:81–8. plant sciences 2012; 11(3):109. 3. Choudhary S, Sree A, Mukherjee SC, Patnaik P, 18. Cho SH, Kang SE, Cho JY, Kim AR, Park SM, Bapuji M. In vitro antibacterial activity of extracts Hong YK, Ahn DH. The antioxidant properties of selected marine algae and mangroves against of brown seaweed (Sargassumsiliquastrum) fish pathogens. Asian Fish Sri 2005; 18:285–94. extracts. Journal of medicinal food 2007; 4. Abedin RMA, Hala MT. Antibacterial and 10(3):479–85. antifungal activity of cyanobacteria and green 19. Hemavani C, Thippeswamy B. Evaluation of microalgae. Evaluation of medium components antimicrobial property of Spirogyra species. by Placketi-Burman design for antimicrobial International Multidisciplinary Research Journal. activity of. Spirulinaplatensis. Glob J Biotechnal 2012; 2(2). Biochem 2008; 3:22–31. 20. Zakaria NA, Ibrahim D, Sulaiman SF, Supardy 5. Desbois AP, Lebi T, Yan L, Smith VJ. Isolation NA. Journal of Chemical and Pharmaceutical and structuralcharacterisation of two antibacterial Research. J Chem 2011; 3(3):182–91. free fatty acids from the marine diatom, 21. Medina-Jaritz NB, Perez-Solis DR, Ruiloba de Phaeodactylumtricornutum. Appl Microbiol Leon SL, Olvera-Ramírez R. Antimicrobial Biotechnol 2008; 81:755–64. activity of aqueous and methanolic extracts from 6. Kamble SM, Chavan AM. Antibacterial activity of Arthrospira maxima. Science against microbial some fresh water algae. J Exp Sci 2010; 1(2):5– pathogens: communicating current research and 6. technological advances. A. Méndez-Vilas (Ed.). 7. Elsie BH, DhanaRajan MS. Evaluation of 2011:1267–71. antimicrobial activity and phytochemical 22. Al-Wathnani H, Ara I, Tahmaz RR, Al-Dayel TH, screening of Gelidiumacerosa. Journal of Bakir MA. Bioactivity of natural compounds Pharmaceutical Sciences and Research 2010; isolated from cyanobacteria and green algae 2(11):704. against human pathogenic bacteria and yeast. 8. Naik Ansari A, Hemavani C, Thippeswamy B. Journal of Medicinal Plants Research. 2012; Evaluation of antimicrobial property of Spirogyra 6(18):3425–33. species. Int multidisciplinary Res J 2012; 2:13– 23. Bauer AW, Kirby WM, Sherris JC, Turck M. 15. Antibiotic susceptibility testing by a standardized 9. Patil KJ, Patil VA, Mahajan SR, Mahajan RT. single disk method. American journal of clinical Bio-activity of algae belonging to Bhusawal pathology 1966 ; 45(4):493–6. region, Maharashtra. Current Botany 2011 Feb 24. TAŞKIN E, Taşkin E, Öztürk M. Inhibitor 24. Activities of some seaweeds from the Aegean 10. Al-Haj NA, Mashan NI, Shamsudin MN, Coast of Turkey. Journal of Applied Biological Mohamad H, Vairappan CS, Sekawi Z. Sciences 2011; 1(1):11–5. Antibacterial activity in marine algae 25. Karabay‐Yavasoglu NU, Sukatar A, Ozdemir G, Eucheumadenticulatum against Staphylococcus Horzum Z. Antimicrobial activity of volatile aureus and Streptococcus pyogenes. Research components and various extracts of the red Journal of Biological Sciences 2009; 4(4):519–24. alga Janiarubens. Phytotherapy Research: An 11. Prescott GW. Algae of the western great lakes International Journal Devoted to Pharmacological area, Otto Kaetz Science publishers. W German and Toxicological Evaluation of Natural Product 1982. Derivatives 2007; 21(2):153–6. 12. Komárek JT, Anagnostidis K. Cyanoprokaryota. I 26. Adrian WJ. A comparison of a wet pressure -Chroococcales, II-Oscillatoriales, III-Nostocales. digestion method with other commonly used Stigonematales, Stuttgart 1989. wet and dry-ashing methods. Analyst 1973; 13. Krammer K. Bacillariophyceae 2. Teil: Bacillari- 98(1164):213–6. aceae, Epithemiaceae, Surirellaceae. Sub- 27. Pradhan AK, Pradhan N, Sukla LB, Panda PK, wasserflora von Mitteleuropa. 1988. Mishra BK. Inhibition of pathogenic bacterial 14. Hreeb KK, AL-Asadi MS, Talal AA. Organic biofilm by biosurfactant produced by manure (horses stool) affects the green alga Lysinibacillusfusiformis S9. Bioprocess and Chlorella vulgaris Beijernick growth. Journal of biosystems engineering. 2014; 37(2):139– Misan Researches 2007; 3(6):15–28. 49.
797 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 28. Bhattacharya D, Price DC, Chan CX, Qiu H, Rose N, Ball S, Weber AP, Arias MC, Henrissat B, Coutinho PM, Krishnan A. Genome of the red alga Porphyridiumpurpureum. Nature communications 2013; 4:1941. 29. Dhanalakshmi M. Phytochemistry And Antibacterial Activity Of Chlorosarcinopsis Species. International Journal of Scientific & Technology Research 2013; 2(10):315–21. 30. Khalid MN, Shameel M, Ahmad VU, Shahzad S, Leghari SM. Bioactivity and phycochemistry of Gloeotrichiaraciborskii (Cyanophycota) from Sindh. International Journal of Phycology and Phycochemistry (Pakistan) 2010. 31. Ghazala B, Shameel MU, Choudhary MI, Shahzad SA, Leghari SM. Phycochemistry and bioactivity of certain freshwater green algae of Sindh. Pakistan Journal of Botany 2004; 35(5):695–704. 32. Fadoul HE, Juntawong N. Antimicrobial Activity of Extracts from Aquatic Algae Isolated From Salt Soil and Fresh Water in Thailand. International Journal of Research Studies in Biosciences (IJRSB) 2004; 2:149–52. 33. Vishnu N, Sumathi R. Isolation of fresh water microalgae Chlorella sp and its antimicrobial activity on selected pathogens. Int. J. Adv. Res. Biol. Sci 2014; 1(3):36–43. 34. Abedin RM, Taha HM. Antibacterial and antifungal activity of cyanobacteria and green microalgae. Evaluation of medium components by Plackett-Burman design for antimicrobial activity of Spirulinaplatensis. Global Journal of Biotechnology and Biochemistry 2008; 3(1):22– 31. 35. Ghasemi Y, Yazdi MT, Shafiee A, Amini M, Shokravi S, Zarrini G. Parsiguine, a novel antimicrobial substance from Fischerellaambigua. Pharmaceutical biology 2004; 42(4-5):318–22. 36. Ghasemi Y, Moradian A, Mohagheghzadeh A, Shokravi S, Morowvat MH. Antifungal and antibacterial activity of the microalgae collected from paddy fields of Iran: characterization of antimicrobial activity of Chroococcusdispersus. J Biol Sci 2007; 7:904–10. 37. Ördög V, Stirk WA, Lenobel R, Bancířová M, Strnad M, Van Staden J, Szigeti J, Németh L. Screening microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites. Journal of applied phycology 2004; 16(4):309–14. 38. Goud MJ, Seshikala D, Charya MS. Antibacterial activity and biomolecular composition of certain fresh water microalgae collected from river Godavari (India). International Journal on Algae 2007; 9(4).
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Rac 1 project against diabetes mellitus via attenuation of platelet chemokines
Rundk Ahmad Hwaiz1* Abstract Background and Objective: Diabetes mellitus is a major risk factor for platelet activation. Inflammation and abnormal platelet function contribute to morbidity and mortality in diabetes mellitus. So far, the molecular mechanisms involved in the inflammation effects of diabetes are only partially known. Rac1 has been reported to regulate diverse functions in platelets; we hypothesized herein that Rac1 might regulate platelet release of CCL5 and CXCL4 in Diabetes Mellitus. Methods: We examined the effect of diabetes mellitus on platelet chemokines and Rac1 activation, a small G -protein involved in the activation of platelet. A total of 90 subjects were examined, which included 60 patients with type 2 diabetes and 30 non-diabetic controls. Serum concentrations platelet chemokines (CCL5 and CXCL4) were assayed using ELISA technique. Moreover platelet from C57BL/6 mice were pre treated with a specific Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6- methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride), and activated with 25 mmol/lglucose and examined for Rac1 activation by electrophoresis. Results: Our results showed that platelet chemokines of diabetic individuals and in platelet treated with high glucose is associated with an increased platelet chemokines and Rac1 activation respectively. More important, selective Rac1 inhibition by NSC23766 protected from increased platelet chemokines secretion induced by glucose. Conclusion: Our study demonstrates that Rac1 plays a crucial role in diabetes-induced inflammation, and it could be a target of novel therapeutic approaches to reduce inflammation in diabetes mellitus. Targeting inflammatory pathways could possibly be a component of the strategies to prevent and control diabetes and related complications. Keywords: Rac1; Platelet; Chemokines; Diabetes Mellitus.
Introduction fasting glucose group.3 Diabetic patients Diabetes mellitus is a serious common are more prone to increase platelet chronic, metabolic disease resulting from reactivity. High blood glucose contributes complex interactions between genetic to greater platelet reactivity by promoting and environmental factors, including glycation of platelet proteins.4 Insulin which risk factors such as sedentary lifestyle produced by beta cells of the pancreatic and obesity.1 Patients with diabetes islets has a crucial role in allowing glucose have a high risk of developing other in the blood to enter cells, providing health condition like macrovascular them with the energy to function. Diabetic and microvascular complications.2 patients usually have low insulin production Macrovascular and microvascular or insulin resistance.5 Insulin deficiency complications contribute to morbidity and and insulin resistance increase platelet mortality in diabetes mellitus. Previous reactivity. Insulin inhibits activation of study showed that mean platelet volume platelets. Previous studies have showed higher in diabetic group than in impaired that diabetic patients with chronic
1 Department of Clinical Biochemistry, College of Health Sciences, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected]
811 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) hyperglycemia have also high reactive Roche, Basel, Switzerland) and 25 mg oxygen species (ROS) production in xylazine (Janssen Pharmaceutica, Beerse, endothelium as a result of platelet Belgium) per kg body weight. A total of 90 activation. In addition the overproduction of subjects were examined, which included ROS during DM may occur directly through 60 patients with type 2 diabetes and glucose metabolism and auto oxidation 30 non-diabetic controls in (University or indirectly through the formation of Hospitals Malmö) has systematically proinflammatory cytokine Receptor for collected plasma and all consenting DM advanced glycation end products (RAGE). patients. Patients didn’t have drugs ROS playing an important role in the affecting platelet for previous 2 weeks. The development of vascular damage and general inclusion criteria for entering in the enhance the activation of other signaling study were: male and female, age 18–65 molecule like, NF-B and protein kinase years. The subjects who included in this C (PKC), leading to redox sensitive study were diagnosed DM according to the gene transicription.6 An important definition established by the International proinflammatory derivative such as CXCL4 Diabetes Federation. The criteria were and CCL5, present in the cytosol of confirmed in order to subject to be platelets, are produce from mRNA and diagnosed with DM should have the release up on activation of platelets. Upon following conditions: Overweight male or platelet activation, these proinflammatory female with waist circumference ≥ 94 cm, derivatives release by platelets. The and ≥ 80 cm, respectively; Fasting glucose activation of Platelet during CLP in mice level ≥ 100 mg/dL; and, Male HDL- leads to expression of CXCL4 and CCL5, cholesterol < 40 mg/dL, female HDL- which induces other immune cells to cholesterol < 50 mg/dL. The matched age aggregate and induce more inflammation control group inclusion criteria were: during sepsis.7,8 Rac1 is a small GTPase Healthy volunteers were recruited among protein which Rac-1, a small G protein, is personal and plasma donors of the center. an important signaling molecule plays key All of them were normolipidemic (fasting function in diabetes mellitus.9 Moreover, total cholesterol and triglycerides below the Rac1 controls platelet secretion of CXCL4 90th percentile of the population), and CCL5 in sepsis.7,8,10 Other studies normoglycemic (fasting glucose < 100 mg/ have identified an essential role Rac1 in dL), and non-obese (BMI < 30 kg/m2). lamellipodia formation, phospholipase Cγ 2 Plasma samples activation, granule secretion and clot Plasma samples were obtained following retraction in platelets.10 Thus, I asked the standard procedures: a 4 mL (EDTA) whether Rac1 might be involved in the blood sampling tube wasused for collection secretion of CXCL4 and CCL5 from of plasma from peripheral venous, (BD platelets in DM the present study. vacutainers, Plymouth, UK). The blood sampling tubes were centrifuged (1300 ×g, Methods 10 min at 4° C) within 1 hour. Plasma Animals was then isolated and immediately frozen The experiments were performed using in aliquots at −20°C. Samples were male C57Bl/6 mice (20-25 g) in accordance transferred to −80° C for long-term storage. with the legislation on the protection of ELISA animals and were approved by the Plasma CCL5 and CXL4 levels Regional Ethical Committee for Animal were successfully measured in 90 Experimentation at Lund University, individuals assessed by enzyme-linked Sweden. Animals were anesthetized by immunosorbent assay (ELISA) using the intraperitoneal (i.p.) administration of 75 Quantikine ELISA kit for Human CCL5/ mg ketamine hydrochloride (Hoffman-La RANTES (R&D Systems, Minneapolis,
822 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) USA, cat no. DRN00B) and Human CXCL4 the rest of the volume was used for the (R&D Systems, Minneapolis, USA, cat pull-down assay. Supernatant containing no. AF795) following manufacturer’s equal amount of proteins were then diluted instructions. Absorbance was measured at with 2 x SDS sample buffer and boiled 450 nm. Readings at 570 nm were for 5 min. Proteins were separated subtracted from these at 450 nm, to correct using SDS-PAGE (10–12% gel). After for plate imperfections. All samples were transferring to a nitrocellulose membrane assessed in duplicate. On each microplate, (Bio-Rad, Hercules, CA, USA), blots were a new standard curve was established blocked with Tris-buffered saline/Tween-20 by diluting a standard with known containing 3 % bovine serum albumin at concentration. Mean absorbance for the room temperature for 1 h, followed by duplicate wells was used to determine the incubation with an anti-Rac1 antibody chemokines concentration for each sample, (1:1000) at 4 °C overnight. Binding of the using a logistic curve-fitting algorithm. With antibody was detected using peroxidase- appropriate dilutions, all absorbance values conjugated anti-mouse antibody (1:100 were within the linear portion of the 000; Pierce Biotechnology) at room standard curve. Concentrations read from temperature for 2 h and developed by the standard curve were multiplied by the Immun-Star WesternC dilution factor. The results were expressed Chemiluminescence Kit (Bio Rad). Total as ng/mL. Rac1 was used as a loading control. Platelet isolation and Rac1 activity Statistics Blood was collected in 1-ml syringes Data were presented as mean containing 0.1 ml of acid–citrate–dextrose values±s.e.m. Statistical evaluations were anticoagulant, immediately diluted with performed by using nonparametric test equal volumes of modified Tyrode solution (Mann–Whitney). P˂0.05 was considered (1 mg/ml prostaglandin E1 and 0.1 U/ml significant and n represents the total apyrase), and centrifuged at 200 g for 5 number of mice in each group. Statistical min at room temperature. Platelet-rich analysis was performed by using plasma was collected and centrifuged at SigmaPlot 10.0 software (Systat Software, 800 g for 15 min at room temperature, and Chicago, IL, USA). pellets were resuspended in modified Results Tyrode solution. After being washed one more time at 10 000 g for 5 min, platelets NSC23766 Prevents High Glucose– were resuspended at a count of 0.5 x 108 Induced platelet activation by platelets per tube in Tyrode solution. attenuating Rac1 activation Platelets from wild-type C57BL/6 mice To evaluate the in vitro effects of were treated with 2 different glucose NSC23766 on glucose induced platelet concentrations, mimicking normoglycemia activation, isolated platelet from wild-type (5 mmol/L) or hyperglycemia (25 mmol/L) C57BL/6 mice were treated with 2 for 30 minutes, with and without NSC23766 different glucose concentrations, (100 mM) at 37 °C. After stimulation, cells mimicking normoglycemia (5 mmol/L) or were immediately fixedby the addition of hyperglycemia (25 mmol/L). Isolated 0.5% paraformaldehyde, samples were platelet exposed to 25 mmol/ L of glucose centrifuged at 10 000 g for 10 min at 4° for 30 minutes showed a significant Rac1 C.Cells were resuspended in 1000 µl lysis activation compared with platelet with 5 buffer on ice, and then centrifuged at mmol/L of glucose (Figure 1A, B). These 16 000 g for 15 min; 10 µl from each data confirm the detrimental effects of high supernatant was removed to measure the glucose levels on activation of Rac1 in protein content using Pierce BCA Protein platelets. Interestingly, pretreatment with Assay Reagent (Pierce Biotechnology) and Rac1 inhibitor, NSC23766 (100 mM), was
833 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) able to protect from platelet activation induced by high glucose (Figure 1A, B).
A
B
Figure1: Rac1 activity in platelet lysate. A) Rac1-GTP was determined by Western blotting by use of GST-PAK pull-down beads, 30min after induction of glucose. B) Band intensities were quantified in isolated platelets by densitometry and normalized to total Rac1. Western blots are representative of 3 independent experiments. Isolated platelets were treated with the Rac1 inhibitor NSC23766 (100mM) and induced by different dose of glucose (NG=5mmol/l) or (HG=25mmol/l), B) n=3, P ˂0.05.
844 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Increased circulating CCL5 and CXCL4 this study I foundthat the stimulation of in Diabetes Mellitus isolated platelets by high concentration of Plasma levels of CCL5 in control group glucose induce the activation of Rac1 and were low but detectable (Figure 2A, interesitingly this increse is abolished by n = 90). Hyperglycemia significantly NSC23766. This indicate the importance of enhanced plasma levels of CCL5 from 0.6 platelet and Rac1 in molecular mechanism ± 1.1 ng/mL up to 135.0 ± 8.2 ng/mL of DM. Morover the platelet chemokines (Figure 2A, P <0.05 versus Ctrl, n =90). CCL5 and CXCL4 are found in high In addition, I observed that the plasma level concentration of diabetic subjects, of CXCL4 was increased in DM compared demonstraiting the molecular mechanism with control group from 0.9 ± 0.2ng/mL up of platelet in regulating DM. CCL5 and to 125± 7.2 (Figure 2B, P <0.05 versus Ctrl, CXCL4 are most abundant chemokines in n = 90). Thus, DM significantly induced platelets.7,8 Several studies have shown plasma levels of CCL5 (Figure 2 A, B). the higher level of soluble inflammatory markers, including interleukin-6 (IL-6), Discussion tumor necrosis factor-α (TNFα), CXCL16, The present findings indicate an important and high-sensitivity C-reactive protein function of Rac1-mediated activation of (CRP) in patients with metabolic syndrome platelet and secretion of CXCL4 from compared to the healthy subjects.12,13,14 In platelets in DM. These results point to an addition, metabolic syndrome has been important role of platelets in DM and related to an increased number of several implicate that inhibition of Rac1 signaling immune cells like leukocyte, monocyte and/or CXCL4 function might be useful and platelet activation. 12,13,14 Interestingly, strategies to ameliorate DM. Platelets are I found that the level of CXL4 and CCL5 in not only critical in wound healing and plasma from diabetic patients are higher thrombosis but also exert numerous than in normal subjects (Figure 2A, B). pro-inflammatory functions in the host Previous studies showed the importance of response to bacterial invasion.11 For Rac1 in regulation of platelet chemokines example, data have shown that platelets secretion.7,8,10 Morover, it is well regulate numerous aspects of leukocyte established that high glucose level in the responses to severe infections.7,8,10 blood promote the production of reactive Previous studies have showed the oxygen species (ROS) on endothelial lining importance of platelet in DM however, in of the vessels.15,16 ROS is an important
Figure 2: Activated platelets secret chemokines in diabetic patients. ELISA was used to quantify the levels of CCL5 and B) CXCL 4 in the diabetic patient’s plasma. Data represent mean ± SEM and n = 90 (Ctrl=30, Diabetic=60). *P ˂0.05 vs. control.
855 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) signaling molecules which produced by Mean platelet volume in type 2 diabetes mellitus. NADPH, play a crucial role in the J Lab Physicians 2012; 4(1):5–9. 4. Kumari Shilp, R. M. Potekar. A Study of Platelet progression of inflammation and vascular 17 Indices in Type 2 Diabetes Mellitus Patients., damage in DM. Rac1, a small G protein, is Indian J Hematol Blood Transfus 2018; 34(1):115 an important signaling molecule integrating –20. intracellular transduction pathways toward 5. Balakumar P, Maung-U K, Jagadeesh G. NADPH oxidase activation.18 In this study Prevalence and prevention of cardiovascular disease and diabetes mellitus. Pharmacol Res I determined the role of Rac1 in platelet 2016;113(Pt A):600–9. activation. Hence, Rac-1 activation, could 6. Daniela Cosentino-Gomes, Nathália Rocco- be a crucial mechanism for the Machado, José Roberto Meyer-Fernandes. Cell hyperglycemia-evoked platelet activation, Signaling through Protein Kinase C Oxidation and Activation. Int J Mol Sci 2012; 13(9):10697– chemokines secretion and vascular 721. oxidative stress in DM. In this study 7. Hwaiz R, Rahman M, Zhang E, Thorlacius H. I demonstrated the role of Rac1 in platelet Platelet secretion of CXCL4 is Rac1-dependent activation as well as the hyper expression and regulates neutrophil infiltration and tissue of the chemokines CXCL4 and CCL5 as damage in septic lung damage. Br J Pharmacol 2015; 172(22):5347–59. a consequences of platelet activation. 8. Hwaiz R, Rahman M, Syk I, Zhang E, Thorlacius Taking together, Rac1 regulate H. Rac1-dependent secretion of platelet-derived chemokines secretion by activated platelet CCL5 regulates neutrophil recruitment via in DM, Rac1 inhibotor, thus, might be activation of alveolar macrophages in septic lung injury. J Leukoc Biol 2015; 97(5):975–84. a therapeutic agent to Control DM via 9. Schiattarella GG, Carrizzo A, Ilardi F, Damato A, attenuation of chemokines which Ambrosio M, Madonna M, et al. Rac1 Modulates aresignaling molecules in exaggerationg Endothelial Function and Platelet Aggregation in inflammation. However, I need to do more Diabetes Mellitus. J Am Heart Assoc 2018; 7(8). invivo experiment in order to strengthen my 10. Hwaiz R, Rahman M, Zhang E, Thorlacius H. 2014. Rac1 regulates platelet shedding of CD40L hypothesis. in abdominal sepsis. Lab Invest. Sep;94(9):1054 –63. Conclusion 11. Wang Y, Hwaiz R, Luo L, Braun OÖ, Norström In this study I demonstrated the role of E, Thorlacius H. Rac1 regulates bacterial Rac1 in platelet activation as well as the toxin-induced thrombin generation. Inflamm Res 2018; 65(5):405–13. hyper expression of the chemokines 12. Mankowska A, Pollak J, Sypniewska G. CXCL4 and CCL5 as a consequences of Association of C-Reactive Protein and Other platelet activation. Taking together, Rac1 Markers of Inflammation with Risk of regulate chemokines secretion by activated Complications in Diabetic Subjects. EJIFCC platelet in DM, Rac1 inhibotor, thus, might 2006; 17(1):8–11. 13. Marques P, Collado A, Martinez-Hervás S, be a therapeutic agent to Control DM via Domingo E, Benito E, Piqueras L, et al. Systemic attenuation of chemokines which are Inflammation in Metabolic Syndrome: Increased signaling molecules in exaggerating Platelet and Leukocyte Activation, and Key inflammation. However, I need to do more Role of CX3CL1/CX3CR1 and CCL2/CCR2 Axes in Arterial Platelet-Proinflammatory Monocyte invivo experiment in order to strengthen my Adhesion. J Clin Med 2019; 8(5). hypothesis. 14. Yao L, Herlea-Pana O, Heuser-Baker J, Chen Y1, Barlic-Dicen J. Roles of the chemokine References system in development of obesity, insulin 1. Liu C, Foti K, Grams ME, Shin JI, Selvin E. Trends resistance, and cardiovascular disease. J in Self-reported Prediabetes and Metformin Use Immunol Res 2014; 181450. in the USA: NHANES 2005-2014. J Gen Intern 15. Volpe CMO, Villar-Delfino PH, Dos Anjos Med 2019. PMF, Nogueira-Machado JA. Cellular death, 2. Michael J. Fowler, MD. Microvascular and reactive oxygen species (ROS) and diabetic Macrovascular Complications of Diabetes. complications. Cell Death Dis 2018; 9(2):119. Clinical Diabetes 2008; 26(2):77–82. 16. Kaneto H, Katakami N, Matsuhisa M, 3. Kodiatte TA, Manikyam UK, Rao SB, Jagadish Matsuoka TA. Role of reactive oxygen species TM, Reddy M, Lingaiah HK, Lakshmaiah V. in the progression of type 2 diabetes and
866 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) atherosclerosis. Mediators Inflamm 2010; 453892. 17. Henríquez-Olguin C, Knudsen JR, Raun SH1, Li Z, Dalbram E, Treebak JT, et al. Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise. Nat Commun 2019; 10(1):4623. 18. Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res 2006; 98(4):453–62.
877 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
In silico investigation to select the most fitting mTOR inhibitors using AutoDock Vina Oral Granule
Hazem Abbas Al-Bustany1* Abstract Background and objective: Mammalian/Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase protein associated with PI3K/Akt/mTOR pathway, which has many roles in different cellular functions such as cell survival, growth, proliferation, metabolism, apoptosis and autophagy. mTOR pathway considered as one of the most regularly activated pathways in human cancers. mTOR originate from two complexes, mTORC1 and mTORC2. mTORC1 is a master regulator of cellular growth and metabolism; which has generate the development of small molecule known as inhibitors that target various sites in the pathway. The function of mTORC2 is less well understood, but is activated by growth factors and is important for cell survival, proliferation, and cytoskeleton organization Methods: The present study, the binding pose and molecular interactions of the most fitting mTOR inhibitors among commercially available ones have been investigated on mTOR protein kinase using AutoDock Vina software. Results: The molecular docking analysis showed that there are different affinities toward mTOR protein, Torin 2 (5), Torin 1 (4), and XL 388 (3) have gaves -10.3, -10.1, and -9.5 kcal/mol (lowest energy) respectively. They formed hydrogen bond and hydrophobic interactions. These interaction results were great when compared with mTOR ligand (phosphothiophosphoric acid-adenylate ester) which gave -7.8 kcal/mol. According to their specific binding and potency, it can be recommended that compound no. 5, 4, and 3 are most fitting mTOR inhibitors.In the future, it is important to depend on Molecular Dynamic Simulations to reach information related to dynamic behaviors and stability of the generated complexes. In vitro and in vivostudies are required to find the role and impact of mTOR inhibitors on living cells. Keywords: mTOR inhibitor; mTOR; cell signaling; AutoDock Vina; Docking software.
Introduction translation. mTORC2 consists of: TOR, Mammalian (or mechanistic) target of Rictor (Rapamycin insensitive protein), rapamycin (mTOR) is a conserved protein mLST8, and Sin1. The main function of kinase that exists in simple microorganisms Rictor, with corporation with Sin, is to like yeasts and in more sophisticated living stabilize mTOC2 integrity. mTORC2 act as being like human. mTOR found to play the upstream target of Akt, and with PDK1 critical role in health and disease.1,2,3 can fully phosphorylate Akt (the upstream mTOR is found in two complexes known target for many vital cellular process), and as mTORC1 and mTOR2.4,5,6 mTORC1 found to be involved in regulation of consists of the following proteins: TOR, cytoskeleton and specifically in actin Raptor (Rapamycin sensitive protein), and polymerization. Both mTOR complexes are mLST8 (or GbL). Raptor main function is negatively regulated by an endogenous to recruit mTORC1 substrates, p70S6K inhibitor known as Deptor.7, 8, 9, 10 mTOR and 4EBP1 that are involved in protein found to be involved in the emergence of
1 Department of Basic Science, College of Medicine, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 881 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) metabolic disorders, immune disease, of human mechanistic target of rapamycin aging, and cancer.8,12,13 There are growing (mTOR)26 downloaded from the RCSB evidences about the involvement of mTOR Protein Data Bank (PDB) using available in development of cancer, angiogenesis, (PDB-ID: 4JSP) (http://www.rcsb.org).27 and metastasis either through mutations Discovery Studio 4.1 (http://accerys.com)28 that activate oncogenes or others and Molecular Graphics Laboratory (MGL) deactivating tumor suppress genes, and Tools 1.5.6 (http://mgltools.scripps.edu)29 these mutations causing activation of used to prepare mTOR protein for mTOR even in the absence of stimulating molecular docking and saved in (.pdbqt) signals like growth factors.14 mTOR file format (MGLTools, 2017) after adding dysregulation (either over or down polar hydrogens,also the Mg+2 recharged regulation) have been reported in many by editing the .pdbqt file manually using types of cancer. Thus mTOR is considered notepad. Among the commercially as a valuable target in treating different available inhibitors, a total of 21 mTOR types of cancer including, but not restricted inhibitors (Table 1) were investigated in to, the followings: lung cancer, pancreatic this study. The 2D structures (Figure 2) cancer, hepatic cancer, gastric cancer, of all inhibitors gained from: https:// breast cancer, and colorectal pubchem.ncbi.nlm.nih.gov/,ChemDraw Pro cancer.15,16,17,18,19,20,21 For targeting mTOR 12.0 (www.cambridgesoft.com),30 and different types of inhibitors have been Discovery Studio 4.1 (http:// identified or created like: Rapamycin, accerys.com), 28 used to convert 2D to 3D Everolimus, Torin1, AZD 3147, and many structures then saved in (.pdb) file format, others.22,23,24,25 In this study we have and viewed using UCSF Chimera Ver. investigated the most fitting mTOR inhibitor 1.10.1 program (http://www.cgl.ucsf.edu/ (s) among available ones to target mTOR chimera/),31 and convert to (.pdbqt) file protein kinase in the most specific and format which required for AutoDock Vina effective way via using AutoDock Vina using Open Babel graphical user interface software. (GUI) (http://openbabel.org/).32 Methods Preparation of mTOR protein and inhibitors: The crystal structure complex
Figure 1: (a) Diagram showing domain structure, (b) components and known substrates of mTOR protein kinase.11 892 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Table 1: List of mTOR inhibitors.
Product Name Cat. No. CAS Number: Description
Everolimus 6188/10 159351-69-6 mTOR inhibitor; Immunosuppressant23 eCF 309 5955/10 2001571-40-8 Potent mTOR inhibitor33 Potent and selective mTOR inhibitor; XL 388 4893/10 1251156-08-7 antitumor34
Torin 1 4247/10 1222998-36-8 Potent and selective mTOR inhibitor24
Torin 2 4248/10 1223001-51-1 Potent and selective mTOR inhibitor23 KU 0063794 3725/10 938440-64-3 Selective mTOR inhibitor35 Temsirolimus 5264/10 162635-04-3 mTOR inhibitor; antitumor23 WYE 687 4282/10 1062161-90-3 Potent and selective mTOR inhibitor36 dihydrochloride Rapamycin 1292/1 53123-88-9 mTOR inhibitor; immunosuppressant22
PP 242 4257/5 1092351-67-1 Dual mTORC1/mTORC2 inhibitor37
Potent and selective dual PI 3-K/mTOR PF 05212384 4823/10 1197160-78-3 inhibitor38 Potent and selective dual mTORC1 and 2 AZD 3147 5615/10 1101810-02-9 inhibitor; orally bioavailable25
Serine/threonine protein kinase 16 (STK16) STK16-IN-1 6036/10 1223001-53-3 inhibitor; also inhibits PI 3Kδ, PI 3Kγ and mTOR kinases39
Potent PI 3-kinase inhibitor; also inhibits DNA ETP 45658 4702/10 1198357-79-7 -PK and mTOR40 Inhibitor of PI 3-kinase, mTOR and DNA- PI 103 hydrochloride 2930/1 371935-79-4 PK41
Compound 401 3271/10 168425-64-7 Selective DNA-PK and mTOR inhibitor42
Naturally occuring flavonoid and antioxidant; Fisetin 5016/50 528-48-3 neuroprotective43
(R)-2- Hydroxyglutaric acid 6124/50 103404-90-6 KDM4A inhibitor; also NF-κB activator44 disodium salt
LY 294002 Prototypical PI 3-kinase inhibitor; also inhibits 1130/5 934389-88-5 hydrochloride other kinases45 STAT3 inhibitor; also inhibits mTORC1 Niclosamide 4079/50 50-65-7 signaling46 Dual kinase inhibitor; inhibits PI 3-K family PP 121 3894/5 1092788-83-4 kinases47
903 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
mTOR Protein (4JSP) Phosphor thiophosphoric mTOR (4JSP) Ligand 2D Everolimus (1) and ligand in active site acid-adenylate ester, mTOR (4JSP) Ligand 3D
eCF 309 (2) XL 388 (3) Torin 1 (4) Torin 2 (5)
KU 0063794 (6) Temsirolimus (7) WYE 687 Rapamycin (9) dihydrochloride (8)
PP 242 (10) PF 05212384 (11) AZD 3147 (12) STK16-IN-1 (13)
ETP 45658 (14) PI 103 hydrochloride (15) Compound 401 (16) Fisetin (17)
(R)-2-Hydroxyglutaric acid LY 294002 hydrochloride Niclosamide (20) PP 121 (21) disodium salt (18) (19) Figure 2: Diagrams showing 2D structures of studied mTOR inhibitors.
914 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) AutoDock Vina separately well docked to mTOR protein by The mTOR active site for docking the which six runs were performed for every inhibitors viewed, and line cube that compound. involved manipulating a colored box (x, y, z), 26 × 26 × 26 Å established Results which is flexible and the volume can After making a successful docking be changed as required in the active protocol, mTOR inhibitors were docked into site, but simultaneously increasing its active site of mTOR protein. The docked volume will cause exponential increase in conformations with the lowest energy computational time, with center grid boxes (highest affinities) were carefully chosen to -23.008, -29.906 and -55.202 used as evaluate the mode of binding. Our results x, y and z, respectively to include the showed that studied inhibitors have inhibitor binding site with grid point spacing different affinities toward mTOR protein of 1.0 Å. After specifying the coordinates (as shown in Table 2), and Torin 2 (5), written in a text document (configuration Torin 1 (4), and XL 388 (3) have gave the file);receptor name, ligand name, grid box, following interaction results, respectively: center, and the exhaustiveness of the -10.3, -10.1, and -9.5kcal/mol (lowest search which set to 8 the most exhaustive energy). These interaction results were search, which was required to feed the very good when compared them with AutoDock Vina 1.1.2.48 AutoDock Vina was mTOR ligand (phosphothiophosphoric acid run on the Windows 7 operating system -adenylate ester) which gives -7.8 kcal/mol. with 4 CPUs and studied inhibitors
Table 2: Average docking scores for mTOR inhibitors docked into mTOR protein. Product Name Docking Scores (kcal/mol) lowest energy 1st 2nd 3rd 4th 5th 6th mTOR Inhibitor Average Run Run Run Run Run Run Phosphothiophosphoric acid-adenylate ester -7.7 -8.0 -7.8 -7.8 -7.8 -7.8 -7.8 Everolimus -5.7 -5.7 -5.7 -5.7 -5.7 -5.7 -5.7 eCF 309 -7.4 -7.4 -7.4 -7.3 -7.4 -7.4 -7.4 XL 388 -9.5 -9.5 -9.5 -9.5 -9.5 -9.5 -9.5 Torin 1 -10.1 -10.1 -10.1 -10.1 -10.1 -10.1 -10.1 Torin 2 -10.3 -10.3 -10.3 -10.3 -10.3 -10.3 -10.3 KU 0063794 -8.4 -8.4 -8.4 -8.4 -8.4 -8.4 -8.4 Temsirolimus -7.8 -8.2 -7.8 -7.8 -7.8 -7.8 -7.9 WYE 687 dihydrochloride -8.9 -8.9 -8.9 -8.9 -8.8 -8.8 -8.9 Rapamycin -8.3 -8.3 -8.0 -8.3 -8.0 8.3- -8.2 PP 242 -7.5 -7.4 -7.8 -7.1 -7.4 -7.4 -7.4 PF 05212384 -8.2 -8.7 -8.3 -8.2 -8.2 -8.3 -8.3 AZD 3147 -8.2 -8.3 -8.1 -8.1 -8.2 -8.1 -8.2 STK16-IN-1 -8.7 -8.7 -8.7 -8.7 -8.7 -8.7 -8.7 ETP 45658 -7.8 -7.8 -7.8 -7.8 -7.8 -7.8 -7.8 PI 103 hydrochloride -8.4 -8.4 -8.4 -8.4 -8.4 -8.4 -8.4 Compound 401 -8.7 -8.7 -8.7 -8.7 -8.7 -8.7 -8.7 Fisetin -8.3 -8.3 -8.3 -8.3 -8.3 -8.3 -8.3 (R)-2-Hydroxyglutaric acid disodium salt -4.3 -4.6 -4.3 -4.3 -4.2 -4.6 -4.4 LY 294002 hydrochloride -9.3 -9.3 -9.3 -9.2 -9.3 -9.3 -9.3 Niclosamide -7.8 -7.8 -7.8 -7.8 -7.8 -7.8 -7.8 PP 121 -7.9 -7.9 -7.9 -7.9 -7.9 -8.1 -7.9
925 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Torin 2 (5) inhibitor (Figure 3) were well with ILE2356. Torin 1 (4) inhibitor (Figure 4) docked into mTOR protein which showed which showed second highest affinity highest affinity formed: hydrogen bond, formed: hydrogen bond, electrostatic and halogen, and hydrophobic interactions, and hydrophobic interactions. Hydrogen bond conquered the affinity of the most favorable interactions were with residues: HIS2247, binding pockets as well. Hydrogen bond ARG2251, an electrostatic interaction with interactions were with LYS2187, VAL2240, ASP2244, a hydrophobic interaction - Pi-Pi THR2245 as hydrogen acceptor, a halogen Stacked with TRP2239, Alkyl with ILE2163, with SER2342, and hydrophobic interactions Pi-Alkyl with LEU2185,CYS2243, ALA2248, Pi-Pi Stacked with LEU2185, TRP2239, MET2345, ILE2356. ASP2244, a Pi-Sulfur with MET2345, a Pi-Alkyl
(a)
(c) (b) Figure 3: Inhibitor Torin 2 (5) docked to mTOR protein, (a) showing the protein as ribbons and the inhibitor in active site (b) 2D of the inhibitor and (c) showing interacted residues of protein with inhibitor.
(a)
(c)
(b) Figure 4: Inhibitor Torin 1 (4) docked to mTOR protein, (a) showing the protein as ribbons and the inhibitor in active site (b) 2D of the inhibitor and (c) showing interacted residues of protein with inhibitor. 936 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The XL 388 (3) inhibitor (Figure 5) selective mTOR inhibitor among the which showed third highest affinity studied ones (Table 2 & Figure 3). Torin 1 formed: hydrogen bond, and hydrophobic (4) inhibitor showed the second highest interactions. Hydrogen bond were with docking score which was -10.1 kcal/mol, residues GLN2167, LYS2187, a hydrophobic thus it found to be the second best interaction –alkyl- withILE2163, PRO2169, selective inhibitor for mTOR among the LEU2185, MET2345, a Pi-Alkyl with PRO2169, studied ones (Table 2 & Figure 4).Finally, ILE2237, TRP2239, ILE2356, and Pi-sulfur with the third highest docking scores generated MET2345. from XL 388 (3) inhibitor and mTOR protein interaction was -9.5 kcal/mol, thus Discussion it found to be the third best selective Docking studies involved the interactions of inhibitor for mTOR among the studied ones examined 21 inhibitors with mTOR active (Table 2 & Figure 5). site residues. In these interactions the proper orientation and conformation fitting Conclusion between mTOR binding site and the Compound Torin 2 (5) is the best formed protein-ligand complex were the candidate for targeting mTOR protein most significant requirements. Due that, among the studied mTOR inhibitors the docked results with the lowest energy (recommended and commercially available (highest affinities) were selected to ones). Interaction studies also confirmed investigate the mode of binding and that the inhibitors interacted with mTOR used as criteria to understand the best by building hydrogen bonds, halogen and conformation output among the studied hydrophobic interactions with binding mTOR inhibitors. The best ranked inhibitor pocket residues, which predicted to take in this regard was Torin 2 (5) that gave place in mTOR active site. In the future, the lowest energy requirement to fulfill it is important to depend on Molecular a successful docking, which was -10.3 kcal/ Dynamic Simulations to reach information mol and due to that it found to be the best related to dynamic behaviors and stability
(a)
(b) (c)
Figure 5: Inhibitor XL 388 (3) docked to mTOR protein, (a) showing the protein as ribbons and the inhibitor in active site (b) 2D of the inhibitor and (c) showing interacted residues of protein with inhibitor.
947 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) of the generated complexes. In vitro and 11. Chiang, Gary G., and Robert T. Abraham. in vivostudies are required to research, and "Targeting the Mtor Signaling Network in Cancer." Trends in Molecular Medicine 13, no. proteomic profiling are required to find the 10 (2007):433–42. role and impact of mTOR inhibitors on 12. Thomson, Angus W., Hēth R. Turnquist, and living cells and their outcome within living Giorgio Raimondi. "Immunoregulatory Functions system. of Mtor Inhibition." Nature Reviews Immunology 9 (05/01/online 2009):324. References 13. Lamming, Dudley W, and David M Sabatini. "A Central Role for Mtor in Lipid Homeostasis." 1. Heitman, Joseph, N. Rao Movva, and Michael N. Cell Metabolism 18, no. 4 (2013):465–69. Hall. "Targets for Cell Cycle Arrest by the 14. Populo, H., J. M. Lopes, and P. Soares. 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958 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Production." [In eng]. Stem Cell Reports 6, no. 5 Cell-Permeable Mtor Inhibitor." Med Chem Comm (May 10 2016):679–91. 7, no. 3 (2016):471–77. 23. Galluzzi, L., J. M. Bravo-San Pedro, B. Levine, D. 34. Takeuchi, C. S., B. G. Kim, C. M. Blazey, S. R. Green, and G. Kroemer. "Pharmacological Ma, H. W. Johnson, N. K. Anand, A. Arcalas, Modulation of Autophagy: Therapeutic Potential et al. "Discovery of a Novel Class of Highly and Persisting Obstacles." [In eng]. Nat Rev Drug Potent, Selective, Atp-Competitive, and Orally Discov 16, no. 7 (Jul 2017):487–511. Bioavailable Inhibitors of the Mammalian Target 24. Peterson, T. R., S. S. Sengupta, T. E. Harris, of Rapamycin (Mtor)." [In eng]. J Med Chem 56, A. E. Carmack, S. A. Kang, E. Balderas, D. A. no. 6 (Mar 28 2013):2218–34. Guertin, et al. "Mtor Complex 1 Regulates Lipin 1 35. 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Dietary Antioxidant, Delphinidin, Ameliorates 37. Zhao, L., P. Yue, F. R. Khuri, and S. Y. Sun. Psoriatic Features in Vitro and in an Imiquimod- "Mtor Complex 2 Is Involved in Regulation of Induced Psoriasis-Like Disease in Mice." [In eng]. Cbl-Dependent C-Flip Degradation and Antioxid Redox Signal 26, no. 2 (Jan 10 2017): Sensitivity of Trail-Induced Apoptosis." [In eng]. 49–69. Cancer Res 73, no. 6 (Mar 15 2013):1946–57. 27. Yang, H., D. G. Rudge, J. D. Koos, B. 38. Mallon, R., L. R. Feldberg, J. Lucas, I. Vaidialingam, H. J. Yang, and N. P. Pavletich. Chaudhary, C. Dehnhardt, E. D. Santos, Z. "Mtor Kinase Structure, Mechanism and Chen, et al. "Antitumor Efficacy of Pki-587, Regulation." [In eng]. Nature 497, no. 7448 (May a Highly Potent Dual Pi3k/Mtor Kinase 9 2013):217–23. Inhibitor." [In eng]. Clin Cancer Res 17, no. 10 28. Houde, A. L. S., A. D. Schulze, K. H. Kaukinen, (May 15 2011): 3193-203. J. Strohm, D. A. Patterson, T. D. Beacham, A. P. 39. Liu, F., J. Wang, X. Yang, B. 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969 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 44. Carbonneau, M., M. Gagne L, M. E. Lalonde, M. A. Germain, A. Motorina, M. C. Guiot, B. Secco, et al. "The Oncometabolite 2-Hydroxyglutarate Activates the Mtor Signalling Pathway." [In eng]. Nat Commun 7 (Sep 14 2016):12700. 45. Bain, J., L. Plater, M. Elliott, N. Shpiro, C. J. Hastie, H. McLauchlan, I. Klevernic, et al. "The Selectivity of Protein Kinase Inhibitors: A Further Update." [In eng]. Biochem J 408, no. 3 (Dec 15 2007):297–315. 46. Jin, Y., Z. Lu, K. Ding, J. Li, X. Du, C. Chen, X. Sun, et al. "Antineoplastic Mechanisms of Niclosamide in Acute Myelogenous Leukemia Stem Cells: Inactivation of the Nf-Kappab Pathway and Generation of Reactive Oxygen Species." [In eng]. Cancer Res 70, no. 6 (Mar 15 2010):2516–27. 47. Apsel, B., J. A. Blair, B. Gonzalez, T. M. Nazif, M. E. Feldman, B. Aizenstein, R. Hoffman, et al. "Targeted Polypharmacology: Discovery of Dual Inhibitors of Tyrosine and Phosphoinositide Kinases." [In eng]. Nat Chem Biol 4, no. 11 (Nov 2008):691–9. 48. Trott, Oleg, and Arthur J. Olson. "Autodock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading." Journal of computational chemistry 31, no. 2 (2010):455–61.
1097 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Simultaneous Spectrophotometric Determination of Resorcinol, Caffeic Acid and Ascorbic Acid Using Artificial Neural Network Technique
Hemn A. Qader1* Nabil A. Fakhri2 Sarmad B. Dikran3 Idrees B. Qader1 Abstract Background and objective: Severely overlapped absorption spectra of compounds cannot be analyzed with the aid of normal UV/Visible spectrophotometric techniques. Spectrophotometric assisted chemometric techniques are powerful tools to analyze overlapped absorption spectra of multicomponent mixture. Chemometric artificial neural network (ANN) and spectrophotometric technique were applied for simultaneous determination of ternary mixtures of resorcinol, caffeic acid and ascorbic acid without any prior separation steps. Methods: In this method an ANN consisting of three layers of input, hidden and output layers (multiple layer type), 30 nodes in the input layer corresponding the data of the absorption spectra of calibration samples in the training set, 8 nodes in the hidden layer, 3 nodes in the output layer corresponding the concentration of resorcinol, caffeic acid and ascorbic acid, respectively, was trained and successfully applied for simultaneous determination of resorcinol, caffeic acid and ascorbic acid. Results: The absorption spectra of these components (resorcinol, caffeic acid and ascorbic acid) severely overlapped in UV spectral range. By optimizing the network architecture of the ANN system, the predication accuracy was enhanced. The validation of the model was made basing upon the calculation of Error% of the prediction of each one of resorcinol, caffeic acid and ascorbic acid, respectively. The value of predicted residual error sum of squares (PRESS), mean squared error (MSE) and root mean squared error (RMSE) for the prediction of the validation set are within the acceptable range. Conclusion: The proposed trained spectrophotometric assisted ANN-chemometric technique was successfully applied for simultaneous determination of resorcinol, caffeic acid and ascorbic acid in samples of Salvadora persica L. without any preliminary chemical separation and good recovery. Keywords Salvadora persica L.; resorcinol; caffeic acid; ascorbic acid; chemometry and artificial neural network.
Introduction benefits of siwak in maintaining the oral Miswak is a chewing stick that derives hygiene.1 The WHO recommends and from Arak tree (Salvadora persica L.) which encourages the use of chewing sticks of is used as cleaning teeth. It is used in Salvadora persica L. as an effective oral different part of Africa, Asia especially in hygiene procedure in areas where its use Middle East, South of America, India, is traditional.2 The main use of Salvadora Pakistan and mostly of Muslim community. persica L. is as a tool for teeth, tongue and This custom was adopted and Islamized by gum cleaning and has also been used to Prophet Muhammad (peace and blessings treat toothache. Pharmacological studies of Allah be upon him/PBUH), because indicated that Salvadora persica L. there are several haddith mentioning the plant possess anti-microbial, antifungal, 1 Department of Pharmaceutical Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Iraq. 2 Department of Chemistry, College of Education, Salahaddin University, Erbil, Iraq. 3 Department of Chemistry, College of Education-Ibn-Haitham, Baghdad, Iraq. * Correspondence: [email protected] 981 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) anti-plaque, aphrodisiac, alexiteric, been used as an important raw material for analgesic, anti-inflammatory, anti-pyretic, chemical industries such as: in the fields hypoglycemic activities besides astringent of rubber, plastics and organic synthesis and detergent effects, diuretic and better industries, wood adhesives, fire retardants stomachic activities. It has great medicinal and UV stabilizer, and also as a solvent. It use in the treatment of nose troubles, piles, is also employed in cosmetic products as scabies, leucoderma, scurvy, gonorrhea, a disinfectant and exfoliating agent and boils and to treat hook worm, vesicle catar- therapeutically in the treatment of human rah, venereal diseases, in rheumatism, acne. It is derived from various resins and cough and asthma, to lower cholesterol tannins but most commonly by fusing plasma levels, spleen troubles, liver sodium hydroxide with meta-benzene- diseases, swollen joints, inflamed glands, disulfuric acid.8, 9 Caffeic acid (CA) (3,4- reestablishment of the components of dihydroxycinnamic acid), (Figure 1b), is gastric mucosa, and as a laxative. The found in a wide variety of foods including plant in various forms is applied to treat vegetables, fruits, tea, coffee, and wine. female sterility, as an antidote to allsorts of CA elicits several interesting and various poison and has anti-scorbutic properties. biological responses, such as antibacterial, Leaves and flowers also used for skin anti-fungal, anti-inflammatory, antiviral, diseases, kidney stones, constipation and anticancer, antioxidant, antimutagenic, and anthelmentic. Also the plant has been anti-diabetic activities. Due to these incorporated into commercially available biological activities, caffeic acid could toothpaste.3-5 The phytochemical studies of be a good lead compound for new Salvadora persica L.have found that it drug development.10 Ascorbic acid (AA) contains almost nineteen active ingredients (vitamin C), (Figure 1c), is a water-soluble in different parts of the plant. The three antioxidant, an unstable, easily oxidized main components which are essential for acid and can be destroyed by oxygen, oral care are chloride, calcium oxalate alkali and high temperature.11 It is a six- and fluoride. Other chemical contents of carbon lactone that is synthesized from Salvadora persica L. including vitamin C, glucose in the liver of most mammalian saponins along with tannins (tannic species, but not by humans. Consequently, acid), salvadourea, resins, alkaloids, when humans do not ingest vitamin C in trimethylamine, silica, gallic acid, benzyl- their diets, a deficiency state occurs with a isothiocyanate aromatic oils (essential oil), wide spectrum of clinical manifestations. high content of minerals in the root, Thus, humans must ingest vitamin C elemental sulfur (S8 a monoclinic form) in to survive.12 Ascorbic acid occurs in root, sodium bicarbonate, poly phenols, different concentrations in a variety of flavonoids and sterols.6, 7 The stem extract natural samples. It is added to several of Salvadora persica L. which identified and pharmaceutical products as an essential determined by RP-HPLC technique, found ingredient, a stabilizer for vitamin B to contain; caffeic acid (5.82%), rutin complex (common ingredient of multivita- trihydrate (2%), trans-cinnamic acid min preparations), food additives and phar- (1.58%), gallic acid (1.53%), resorcinol maceuticals, and as an anti-oxidant it is (1.33%), chlorogenic acid (1.14%), commonly added to foods, juices and quercetin dihydrate (0.67%), naphtho- beverages. Consequent upon its desirable resorcinol (0.48%), catechine hydrate effects, it is widely used in the treatment (0.24%), p-coumaric acid (0.11%), and of certain diseases such as scurvy, 3,4-dihydroxyphenylacetic acid (0.01%).3 anemia, haemorrhagic disorders etc. It is Resorcinol (R) (1,3-dihydroxybenzene), considered essential for the development (Figure 1a), has two hydroxyl groups in the and regeneration of muscles, bones, teeth meta position of the benzene ring, it has and skin. Also it has been identified as
992 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) a radical scavenger in vivo.13, 14 component problems. Recently, it has been proposed that ANN can be used to Methods solve regression problems by acting as Chemometrics is defined as the use of nonparametric calibration methods that statistical and mathematical techniques have the ability to learn from a set of to analyze chemical data, which are examples without requiring any knowledge transformed into information used for of the model type and to generalize this decision making.15 Chemometric is a field knowledge to new situations. ANN has which is related with various disciplines. the outstanding power for modeling both It is used as a guide to the chemist in linearity and nonlinearity systems and has extraction of maximum chemical shown better prospects as a calibration information from complex observations, model than PLS and PCR methods thus chemometric deals with chemical data in nonlinearity systems.17, 18 ANN is a and how to obtain information from it.16 mathematical model, of which composition Multivariate analysis is synonymous with is inspired by the structure of the human the term chemometric. It is a method which brain18, the sole requirement for an takes into consideration many variables analytical system to be accurately defined acting together. The method is fast by an ANN is the use of a large enough and efficient in determination as well number of calibration samples. The as extraction of information. Multivariate primary element of an ANN is the neuron. analysis, as the name suggests, involves Arranged in input and output layers looking simultaneously at the relationship sandwiching one or more "hidden" between multiple data values, rather than processing layers, neurons act as weighted at individual ones.16 There are various transfer functions and can have single or chemometric or multivariate methods. All multiple inputs.19 These methods (artificial these methods commonly share the basic intelligence, especially ANNs) are effective principle in multivariate analysis. They are in spectrophotometric analysis because used to resolve the problem in analyzing the simultaneous analysis of several multi component mixture by allowing rapid spectral intensities can greatly improve and simultaneous determination of each the precision and applicability.20 In the component in the mixture. The accuracy spectrophotometric analysis, the input and precision is achieved without prior parameters are the absorbance of the separation of the components. This implies spectra or at specified wavelengths. The that these methods are time and cost parameters are connected to neurons in effective.16 Artificial neural network (ANN) the hidden layer. The number of hidden is a powerful chemometric method and is layers and the number of neurons in each one of the most broadly used mathematical layer is flexible and is determined by the algorithms for regression problems. ANN examination of errors in the results in the has been often applied to simultaneous output layer in terms of concentrations.21 determination of analytes in multi The aim of this study is to develop a rapid,
OH OH HOHO OH OOH OH H
OO OH HO OH OO
. (a) (b) (c) Figure 1: Chemical structure of (a) R (b) CA (c) AA.
1003 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) non-destructive, environment friendly and 240 nm/min scan speed, and 1.0 nm slit efficient ANN-spectrophotometric method width) against D.W. as a reagent blank for the simultaneous determination of R, in the range of 200 – 350 nm on the CA and AA in Hibiscus sabdariffa L. double-beam UV/Vis spectrophotometer samples. and the data of the absorption spectra Experimental were collected. Apparatus UV-spectrum of CA A Perkin Elmer Precisely Lambda Different aliquots containing CA were taken 25 double-beam scanning UV/Vis from the stock solution and transferred to spectrophotometer equipped with 10-mm a series of 25 mL volumetric flasks in order path length quartz cell and computer with to cover the concentration range of (25.0 – Pentium IV and 512 MB RAM was used for 500.0) μg in a final volume of 25 mL. recording normal absorption spectra and The solutions were scanned (0.5 nm data collecting the data of the absorption intervals, 240 nm/min scan speed, and 1.0 spectra for each one of R, CA, AA and their nm slit width) against D.W. as a reagent mixture solutions. blank in the range of 200 – 350 nm on the Software double-beam UV/Vis spectrophotometer. Portable JMP 8.0 program established with The data of the absorption spectra were a Pentium IV computer having 512 MB collected. RAM (Windows XP operating system) was UV-spectrum of AA used for processing the data in the An aliquot of standard solutions containing chemometric ANN model for simultaneous AA in the concentration range 75.0 – 875.0 determination of R, CA and AA. μg were transferred into 25 mL volumetric Reagents and solutions flasks, then dilution was made to the mark All chemicals used were of analytical with D.W. and a portion of the solution was reagent grade, all of the solutions must be placed in 1-cm quartz cell and scanned prepared freshly. (0.5 nm data intervals, 240 nm/min scan Resorcinol solution (250 μg/mL) speed, and 1.0 nm slit width) against (Scharlau): D.W. as a reagent blank in the range 0.025 g of the compound was dissolved of 200 – 350 nm on the double-beam and diluted to 100 mL with D.W. UV/Vis spectrophotometer. The data of the Caffeic acid solution (250 μg/mL) absorption spectra were collected. (Sigma-Aldrich): Training set of R, CA, AA mixtures 0.025 g of the compound was dissolved A training set were prepared by with mild heating and diluted to 100 mL transferring different aliquots of the with D.W. standard stock solutions of R, CA and AA Ascorbic acid solution (250 μg/mL) to a series of 25 mL volumetric flasks and (Scharlau): 0.025 g of the compound was diluting to the mark with D.W in order to dissolved and diluted to 100 mL with D.W obtain a series of mixtures of calibration in a volumetric flask and placed in a cold samples as summarized in Table 1. The place. Only fresh solutions were used for absorbance of each one of the solutions the measurements. does not exceed 2.0. Recommended procedures Chemometric-ANN analysis of R, CA UV-spectrum of R and AA Different volumes of the stock solution The calibration sample solutions in the of R were diluted with D.W. to obtain training set of R, CA and AA mixtures the working standard solutions in the were scanned on the double beam UV/Vis concentration range of 50.0 – 2500.0 μg spectrophotometer against D.W. The in a final volume of 25 mL, then the recording condition was 1.0 nm data solutions scanned (0.5 nm data intervals, intervals, 240 nm/min scan speed and
1014 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 1.0 nm slit width. Then the data of the layers (multiple layer type), 30 nodes in the absorption spectra were collected. The input layer corresponding the data of the matrix of the absorbance and concentration absorption spectra of calibration samples was obtained from the transpose of the in the training set, 8 nodes in the collected data (converting from columns hidden layer, 3 nodes in the output layer wise to rows wise). The design of the corresponding the concentration of R, network in the ANN model constructed by CA and AA, respectively, and maximum the portable JMP 8.0 software as follows: iterations number of 300. three layers of input, hidden and output
Table 1: Calibration samples of R, CA and AA mixtures. CA R AA Mixtures (μg/mL) (μg/mL) (μg/mL) 1.0 2.0 10.0 (1:2:10) 1.0 2.0 35.0 (1:2:35) 1.0 2.0 30.0 (1:2:30) 1.0 5.0 20.0 (1:5:20) 1.0 10.0 5.0 (1:10:5) 1.0 20.0 5.0 (1:20:5) 1.0 50.0 3.0 (1:50:3) 1.0 75.0 3.0 (1:75:3) 1.0 100.0 3.0 (1:100:3) 3.0 2.0 20.0 (3:2:20) 3.0 5.0 5.0 (3:5:5) 3.0 10.0 10.0 (3:10:10) 3.0 20.0 5.0 (3:20:5) 3.0 50.0 5.0 (3:50:5) 3.0 75.0 3.0 (3:75:3) 3.0 100.0 3.0 (3:100:3) 5.0 2.0 20.0 (5:2:20) 5.0 5.0 10.0 (5:5:10) 5.0 10.0 10.0 (5:10:10) 5.0 20.0 5.0 (5:20:5) 5.0 50.0 5.0 (5:50:5) 5.0 75.0 3.0 (5:75:3) 10.0 2.0 10.0 (10:2:10) 10.0 5.0 5.0 (10:5:5) 10.0 10.0 10.0 (10:10:10) 10.0 20.0 5.0 (10:20:5) 10.0 50.0 3.0 (10:50:3) 10.0 75.0 3.0 (10:75:3) 20.0 2.0 3.0 (20:2:3) 20.0 2.0 5.0 (20:2:5)
1025 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Preparation of Salvadora persica L. Results and Discussion sample It can be seen from Figure 2 that the Dry stems of Salvadora persica L. (normal spectra of these compounds R, CA and and flavored type-Al madinah siwak) AA overlapped seriously and quantitative imported from Saudi Arabia were obtained. estimations cannot be carried out Extraction was made with a mixture of successfully by direct UV absorption acetone:water (80:20, v/v). The extract was spectrophotometry without separation prepared by placing amount of small steps. Consequently, chemometric ANN particle plant material in a household method was applied to quantitatively blender to obtain 4.0 g powder of the resolve the overlapped UV spectra of these stem samples, then 40 mL extracting compounds in their mixtures. agent solvent was added to the powder ANN model for R, CA and AA samples and allowing the mixtures to The training set of 30 calibration samples stand overnight at room temperature, the for the application of ANN model supernatants were filtered with Whatman corresponding to the rule of 10 (the rule of filter paper No. 42, and dried/evaporated 10 is better still, if 10 times the number of on a heater with gentle heating. They were as there are components is used, can be stored at 4°C until analysi.22 The stored able to create a solid calibration for samples were re-dissolved in D.W. and typical applications23) were prepared and phenolic compounds of R, CA and summarized in Table 1 to be used for AA were immediately analyzed with training the ANN model for R, CA and AA). the aid of standard addition method by The compositions of the mixtures of the recommended procedures for the calibration samples for application of ANN determination of R, CA and AA with model tried to be representative as much derivative spectrophotometry, PLS and as possible over the linear ranges of each ANN chemometric techniques.
Figure 2: normal absorption spectrum of R (a), CA (b), AA (c) and their mixture (d).
1036 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) one of R (2 – 100) μg/mL, CA (1 – 20) μg/ of R, CA and AA, respectively, with mL and AA (3 – 35) μg/mL, which maximum iteration number of 200. The previously determined to obey Beer’s law value of RMSE of the training set was used for each one of the compounds under as an index to obtain the best design of the study. The absorption spectra of all network during the training process of the calibration sample solutions in the training proposed network. The lowest value for set were recorded and collected with RMSE of the training set data was selected scanning condition of data interval of 1 nm, as optimum case for the parameters under 240 nm/min scan speed and 1 nm slit study. The number of neurons in the width in the range of 200 – 350 nm. hidden layer was studied via introducing The transpose of the absorbance and a new node to the hidden layer in the concentration data matrix (rows wise network and recording RMSE value and matrix) was obtained and processed by fixing the number of iteration in 200 a Pentium IV computer having 512 MB of as maximum iterations. The results at RAM (Windows XP operating system). The Table 2 indicate that minimum RMSE is ANN module was carried out with portable observed if the network includes 8 nodes in JMP 8.0 software. Initial structure of the the hidden layer. Another factor which af- network in this work was included three fects the performance of the ANN model is layers of nodes or neurons (multiple layer the maximum iteration number. In order to type); the input layer with 30 neurons (the obtain optimum number of iterations the data of the absorption spectra of training value of the iteration was varied from 50 – set), one hidden layer with one active 5000 and basing upon RMSE value the neuron and output layer with 3 active best iterations number can be obtained neurons representing to the concentration when number of iteration is 300.
Table 2: Effect number of nodes in the hidden layer of ANN of ternary system.
Nodes RMSE R2 number R CA AA R CA AA
1 15.351293 5.1782916 3.6805661 0.6878 0.2320 0.8073
2 6.5140593 1.3607091 1.1191642 0.8850 0.9350 0.8665
3 0.9265785 0.3031899 0.4373885 0.9990 0.9969 0.9975
4 0.8909437 0.2383830 0.3561930 0.9993 0.9982 0.9984
5 0.7187220 0.1800638 0.2788245 0.9994 0.9990 0.9991
6 0.6244522 0.1773154 0.2262686 0.9996 0.9993 0.9995
7 0.5720926 0.1577883 0.2134032 0.9997 0.9996 0.9996
8 0.4953578 0.0957219 0.1916801 0.9998 0.9997 0.9997
9 0.5252320 0.0988669 0.2017735 0.9998 0.9996 0.9996
10 0.5375663 0.1006392 0.2079205 0.9997 0.9996 0.9996
12 0.5441662 0.1079005 0.2110053 0.9997 0.9997 0.9997
1047 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The network of the ANN model for R, CA concentration of R, CA, and AA and their and AA was constructed under optimum predicted concentrations as shown in conditions as shown in Figure 3. Under Figure 4, 5 and 6, respectively. The the optimum design of the network for model quality of the proposed ANN model the proposed ANN model for ternary was expressed with R2 = 0.99972 and system three calibration curves from the SSE of 0.0241628405. training set were obtained between the
Figure 3: Network of the ANN model with 30 neurons in input layer, 8 nodes (H1 – H8) in hidden layer and 3 nodes (column 152, 153 and 154) in the output layer which represent concentration of R, CA and AA, respectively. 1058 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Figure 4: Calibration curve of prediction of R in the training set with ANN model.
Figure 5: Calibration curve of prediction of CA in the training set with ANN model.
Figure 6: Calibration curve of prediction of AA in the training set with ANN model.
1069 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) ANN analysis of the validation set of R, Samples held in reserve this way are CA and AA known as validation samples and pair of It is highly desirable to assemble an absorbance and concentration matrices additional data set containing independent holding these data is called a validation measurements on samples that are set. 24 In order to evaluate the performance independent from the samples used to of the proposed ANN model 34 different create the training set. This data set is not mixture solutions as a validation set or used to develop the calibration. Instead, it testing set were prepared and the ratio of is held in reserve so that it can be used to the mixture solutions are summarized in evaluate the calibration’s performance. Table 3.
Table 3: Validation samples of validation set of R, CA and AA mixtures. CA R AA Mixtures (μg/mL) (μg/mL) (μg/mL) 2.0 3.0 7.0 (2:3:7) 2.0 3.0 25.0 (2:3:25) 2.0 15.0 4.0 (2:15:4) 2.0 15.0 7.0 (2:15:7) 2.0 30.0 5.0 (2:30:5) 7.0 3.0 4.0 (7:3:4) 7.0 3.0 25.0 (7:3:25) 7.0 15.0 4.0 (7:15:4) 7.0 30.0 3.0 (7:30:3) 7.0 30.0 4.0 (7:30:4) 3.0 25.0 5.0 (3:25:5) 7.0 7.0 7.0 (7:7:7) 2.0 40.0 4.0 (2:40:4) 4.0 4.0 4.0 (4:4:4) 1.0 3.0 3.0 (1:3:3) 3.0 3.0 3.0 (3:3:3) 5.0 3.0 3.0 (5:3:3) 10.0 3.0 3.0 (10:3:3) 15.0 3.0 3.0 (15:3:3) 20.0 3.0 3.0 (20:3:3) 3.0 2.0 3.0 (3:2:3) 3.0 5.0 3.0 (3:5:3) 3.0 10.0 3.0 (3:10:3) 3.0 20.0 3.0 (3:20:3) 3.0 50.0 3.0 (3:50:3) 3.0 75.0 3.0 (3:75:3) 3.0 100.0 3.0 (3:100:3) 5.0 5.0 3.0 (5:5:3) 5.0 5.0 5.0 (5:5:5) 5.0 5.0 15.0 (5:5:15) 5.0 5.0 20.0 (5:5:20) 5.0 5.0 30.0 (5:5:30) 5.0 5.0 35.0 (5:5:35) 3.0 2.0 35.0 (3:2:35)
10710 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) The validation of the model was made set, the results are tabulated in basing upon the calculation of Error% of Table 4, 5, 6 and 7. Thus, with the aid the prediction of each one of R, CA and of ANN-chemometric technique mixtures of AA, respectively, and some statistical R, CA and AA were predicted successfully parameters such as: PRESS, MSE and without any separation steps. RMSE for the prediction of the validation
Table 4: Prediction and Error % of validation set of R with ANN model. R Predicted concentration Error % (μg/mL) (μg/mL) 3.0 2.975596 - 0.81 3.0 3.043632 + 1.45 15.0 15.21151 + 1.41 15.0 15.19611 + 1.31 30.0 29.88829 - 0.37 3.0 3.058031 + 1.93 3.0 3.1151 + 3.84 15.0 15.16171 + 1.08 30.0 29.88678 - 0.38 30.0 30.45119 + 1.50 25.0 24.41069 - 2.36 7.0 6.930671 - 0.99 40.0 40.59991 + 1.50 4.0 4.011965 + 0.30 3.0 3.138967 + 4.63 3.0 3.039117 + 1.30 3.0 2.95073 - 1.64 3.0 3.078745 + 2.62 3.0 3.08780 + 2.93 3.0 3.041577 + 1.39 2.0 2.038262 + 1.91 5.0 5.059017 + 1.18 10.0 10.36797 + 3.68 20.0 19.74553 - 1.27 50.0 49.71028 - 0.58 75.0 75.58726 + 0.78 100.0 99.70710 - 0.30 5.0 5.050843 + 1.02 5.0 5.080887 + 1.62 5.0 5.089668 + 1.79 5.0 5.210455 + 4.21 5.0 4.910179 - 1.80 5.0 5.069853 + 1.40 2.0 2.027090 + 1.35
10811 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Table 5: Prediction and Error % of validation set of CA with ANN model.
CA Predicted concentration Error% (μg/mL) (μg/mL)
2.0 2.018515 + 0.93 2.0 2.055212 + 2.76 2.0 1.96047 - 1.98 2.0 2.026625 + 1.33 2.0 2.013999 + 0.70 7.0 6.803898 - 2.80 7.0 6.835147 - 2.36 7.0 6.875921 - 1.77 7.0 7.113586 + 1.63 7.0 7.185876 + 2.66 3.0 2.948542 - 1.72 7.0 7.000492 + 0.007 2.0 1.957696 - 2.12 4.0 3.860565 - 3.49 1.0 1.011639 + 1.16 3.0 3.054324 + 1.81 5.0 4.875355 - 2.49 10.0 9.848121 - 1.52 15.0 15.41319 + 2.75 20.0 19.82333 - 0.88 3.0 3.059664 + 1.99 3.0 2.879087 - 4.03 3.0 2.935812 - 2.14 3.0 2.925373 - 2.49 3.0 2.887533 - 3.75 3.0 2.84600 - 5.13 3.0 2.904795 - 3.17 5.0 4.942231 - 1.16 5.0 4.762617 - 4.75 5.0 4.915734 - 1.69 5.0 4.969708 - 0.61 5.0 5.118633 + 2.37 5.0 5.011599 + 0.23 3.0 2.967241 - 1.09
10912 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Table 6: Prediction and Error % of validation set of AA with ANN model. AA Predicted concentration Error % (μg/mL) (μg/mL) 7.0 6.989404 - 0.15 25.0 25.70707 + 2.83 4.0 3.969857 - 0.75 7.0 7.180548 + 2.58 5.0 5.058359 + 1.17 4.0 3.947259 - 1.32 25.0 25.61963 + 2.48 4.0 4.137041 + 3.43 3.0 2.890074 - 3.66 4.0 3.981552 - 0.46 5.0 5.134536 + 2.69 7.0 7.202378 + 2.89 4.0 4.128909 + 3.22 4.0 3.889528 - 2.76 3.0 3.113209 + 3.77 3.0 3.11082 + 3.69 3.0 3.09867 + 3.29 3.0 3.063483 + 2.12 3.0 3.087844 + 2.93 3.0 3.097443 + 3.25 3.0 3.088401 + 2.95 3.0 3.082675 + 2.76 3.0 2.940589 - 1.98 3.0 3.136663 + 4.56 3.0 3.146541 + 4.88 3.0 2.921768 - 2.61 3.0 2.919161 - 2.69 3.0 3.138510 + 4.62 5.0 4.885275 - 2.29 15.0 15.41834 + 2.79 20.0 20.74377 + 3.72 30.0 30.64189 + 2.14 35.0 35.43126 + 1.23 35.0 35.67778 + 1.94
Table 7: Model quality of validation set of ternary system with ANN model. Values Parameter R CA AA PRESS 1.903219 0.583057 2.987794 RMSEP 0.236595 0.130953 0.296439 MSEP 0.055977 0.017149 0.08786
11013 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Interferences study References The selectivity of the proposed ANN model 1. Mahanani ES, Samuel SV. MISWAK (Salvadora for simultaneous determination of R, CA persica) as a Cleansing Teeth. Mutiara Medika: and AA was checked by studying the Jurnal Kedokteran dan Kesehatan 2016; 7(1):38 effects of different compounds, 1500 μg/mL –42. 2. Sharma V, Ramawat KG. Salinity-induced of (citric acid, scopoletine, rutine, modulation of growth and antioxidant activity quercetine and catechine HCl), 1000 μg/mL in the callus cultures of miswak (Salvadora of (chlorogenic acid and ferulic acid) and persica). 3 Biotech 2013; 3(1):11–7. 250 μg/mL of (caffeine, gibberlic acid and 3. Noumi E, Snoussi M, Trabelsi N, Ksouri R, m-coumaric acid) on the determination of Hamdani G, Bouslama L, et al. Antioxidant activities and RP-HPLC identification of 5.0 μg/mL of R, CA and AA. Results polyphenols in the ethyl acetate extract of indicated that the compounds under study Tunisian Juglans regia L treated barks. Journal did not interfere in the proposed methods. of Medicinal Plants Research 2012; 6(8):1468– Application of the method 75. 4. Ahmad H, Ahamed N, Dar JM, Mohammad UJ. The proposed ANN model was successfully Ethnobotany, Pharmacology and Chemistry of applied for simultaneous determination of Salvadora persica L. A Review. Research in R, CA and AA in stem samples of Plant Biology 2012; 2(1). Salvadora persica L. (normal and flavoured 5. Tiwari S, Sarkar B, Dubey G, Jain A. type-Al madinah siwak) with the aid of Comparative evaluation of in vitro free radical scavenging activity of different extract of standard addition method. Also, recovery Salvadora persica L. Asian J Pharm Life Sci study was performed by standard addition 2011; 1:133–6. method. Table 8 shows the results. 6. Nordin FN. A review on the sunnah of miswak (Salvadora Persica) and its potentiality to Conclusion improve oral health. Revelation and science 2012; 2(01). The proposed chemometric ANN model 7. Al Sadhan ReI, Almas K. Miswak (chewing stick): is rapid and successfully applied for the a cultural and scientific heritage. Saudi Dent J simultaneous determination of ternary 1999; 11(2):80–8. 8. Guedes SF, Mendes B, Leitão AL. Resorcinol mixtures of R, CA and AA in their mixture. degradation by a Penicillium chrysogenum strain The proposed method is rapid, non- under osmotic stress: mono and binary substrate destructive, economy and environment matrices with phenol. Biodegradation 2011; friendly (it does not require expensive 22(2):409–19. solvents and reagents, do not use toxic and 9. Keyvanfard M. A New Inhibition Kinetic Spectrophotometric Method for the ozone depleting organic solvents and Determination of Resorcinol. Journal of polluting reagents). Chemistry 2010; 7(3):727–32.
Table 8: Simultaneous determination of R, CA and AA in Salvadora persica L. samples with ANN model.
Amount found with proposed Recovery % PLS method (mg/g) Salvadora persica L. samples R CA AA R CA AA
Normal 9.194 25.09 8.660 93.04 96.22 93.41
Almadinah (flavonoid) 9.072 24.813 8.582 94.66 95.97 94.98
11114 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) 10. Wang L, Hsu K, Hsu F, Lin S. Simultaneous RP-HPLC identification of polyphenols in the determination of caffeic acid, ferulic acid acetone 80 extract of Salvadora persica. African and isoferulic acid in rabbit plasma by high Journal of Pharmacy and Pharmacology 2011; performance liquid chromatography. Journal of 5(7):966–71. Food and Drug Analysis 2008; 16(1):34. 23. Kramer R. Chemometric techniques for 11. Iqbal K, Khan A, Khattak M. Biological quantitative analysis: CRC Press 1998. significance of ascorbic acid (vitamin C) in human 24. Lomillo MA, Renedo OD, Martınez MA. health-a review. Pakistan Journal of Nutrition Resolution of ternary mixtures of rifampicin, 2004; 3(1):5–13. isoniazid and pyrazinamide by differential pulse 12. Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, polarography and partial least squares method. Lee J-H, et al. Vitamin C as an antioxidant: Analytica chimica acta 2001; 449(1-2):167–77. evaluation of its role in disease prevention. Journal of the American college of Nutrition 2003; 22(1):18–35. 13. Revanasiddappa H, Veena M. Sensitive spectrophotometric methods for the determination of ascorbic acid. Journal of Chemistry 2008; 5(1):10–5. 14. Vishnikin AB, Svinarenko TY, Sklenářová H, Solich P, Bazel YR, Andruch V. 11-Molybdobismuthophosphate—A new reagent for the determination of ascorbic acid in batch and sequential injection systems. Talanta 2010; 80(5):1838–45. 15. Slutsky B. Chemometrics: A Practical Guide By Kenneth R. Beebe, Randy J. Pell, and Mary Beth Seasholtz. Wiley-Interscience Series on Laboratory Automation. John Wiley & Sons: New York, 1998. xi+ 348 pp. ISBN 0-471-12451-6. $69.95. Journal of Chemical Information and Computer Sciences 1998; 38(6):1254–. 16. Kassa M. Chemometrics Assisted UV- Spectrophotometric Determination of Topical Binary Mixtures Containing Benzoicacid, Salicylic Acid or Resorcinol: Addis Ababa University 2008. 17. Ni Y, Xia Z, Kokot S. A kinetic spectrophotometric method for simultaneous determination of phenol and its three derivatives with the aid of artificial neural network. Journal of hazardous materials 2011; 192(2):722–9. 18. Gao L, Ren S. Prediction of nitrophenol-type compounds using chemometrics and spectropho- tometry. Analytical biochemistry 2010; 405(2):184 –91. 19. Bahram M, Afkhami A. Recent applications of kinetic methods in multi-component analysis. Journal of the Iranian Chemical Society. 2008; 5(3):352–66. 20. Hasani M, Moloudi M. Application of principal component-artificial neural network models for simultaneous determination of phenolic compounds by a kinetic spectrophotometric method. Journal of hazardous materials 2008; 157(1):161–9. 21. Dumancas GG, Muriuki M, Purdie N, Reilly L, editors. Simultaneous spectrophotometric and chemometric determination of oleic, linoleic, and linolenic fatty acids in vegetable oils. Proceedings of the World Congress on Engineering 2011. 22. Noumi E, Hajlaoui H, Trabelsi N, Ksouri R, Bakhrouf A, Snoussi M. Antioxidant activities and
11215 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Pro-inflammatory cytokines “Tumor necrosis factor alpha (TNF-α) and Interleukin beta (IL-1 β)” levels in albino rats after co-administration of diclofenac sodium and dexamethasone
Zahra Abdulqader Amin1* Abstract Background and objective: Dexamethasone is a corticosteroid that prevents the release of substances in the body that cause inflammation; it is used to treat many different inflammatory conditions such as allergic disorders and skin conditions, while diclofenac sodium (Voltaren) is a nonsteroidal anti-inflammatory drug (NSAID) works by reducing substances in the body that cause pain and inflammation. It is used to treat mild to moderate pain, or signs and symptoms of osteoarthritis or rheumatoid arthritis.Indeed, Tumor necrosis factor alpha (TNF-α)and Inter leukin-1 beta (IL-1β) are two powerful mediators of inflammation primarily produced by activated monocytes or macrophages. The aim of this study was to investigate the side effects of co-administration of diclofenac and dexamethasone on albino rats by measuring the proinflammatory cytokinesTNF-α and IL-1 βlevels in rat’s blood. Methods: Forty male Albino rats, weighing 170-200 g, were used in this study, divided into 4 groups, each group containing 10 animals. Group I rats were intramuscularly injected with physiologic saline (4ml/kg) daily, Group II rats were intramuscularly injected with dexamethasone (1.6 mg/kg) daily. Group III rats intramuscularly injected with Diclofenac Sodium (30 mg/kg) daily and Group IV rats received a daily dose of both dexamethasone (1.6 mg/kg) and Diclofenac Sodium (30mg/kg). After one month all the rats were sacrificed then blood was collected, serum separated and used for the cytokines analysis. Results: Our findings demonstrate that rats treated with each of diclofenacsodium and dexamethasone has shown to inhibit the release of both TNF-α and IL-1β ( 43.1±1.1, 47.5±1.6 pg/ml)(28±2.5, 28.8±0.4 pg/ml) respectively in comparison to the control group (55.8±0.5,42.3±1.1pg/ml). However, the co-administration of them significantly increased the production of the two cytokines (69±12.9, 37±0.6 pg/ml). Conclusion: A possible inhibition role of the Diclofenac sodium and Dexamethasone on each other have been observed that may recommend more detailed analysis research to state the exact mechanism of action. Keyword: Diclofenac sodium; Dexamethasone; TNFα; IL-1β.
Introduction of pathological pain 1. IL-1β is a small (17.5 Cytokines are small secreted proteins kDa) neutral proinflammatory cytokine released by cells have a specific effect belonging to the IL-1 gene family. IL-1β is on the interactions and communications the prototypical multifunctional cytokine, between cells. Proinflammatory cytokines having the ability to induce the expression are produced predominantly by activated of other proinflammatory mediators, and macrophages and are involved in the is central to setting in motion the host’s up-regulation of inflammatory reactions. inflammatory and immune responses. There is abundant evidence that certain pro IL-1β has a range of biological effects that -inflammatory cytokines such as IL-1β, reflect the expression of the IL-1 receptor 1 IL-6, and TNF-α are involved in the process (IL-1R1) on target cell types. While TNFα
1 Department of Pharmacognosy, College of Pharmacy, Hawler Medical University, Erbil, Iraq. * Correspondence: [email protected] 1131 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) belongs to a superfamily of ligand/receptor Laboratory animals. Rats were divided into proteins called the tumor necrosis 4 groups, each group containing 10 factor/tumor necrosis factor receptor animals. Group I rats were intramuscularly superfamily proteins. TNF is an important injected with physiologic saline (4ml/kg) proinflammatory cytokine for both daily, Group II rats were intramuscularly inflammatory and immune processes, injected with dexamethasone (1.6 mg/kg) as well as in the generation of pain. daily. Group III rats intramuscularly injected TNF receptors are either constitutively with Diclofenac Sodium (30 mg/kg) daily expressed (TNFR1, p55-R) or inducible and Group IV rats received a daily dose (TNFR2, p75-R) under inflammatory/injury of both dexamethasone (1.6 mg/kg) and conditions.2 Dexamethasone is Diclofenac Sodium (30mg/kg). After one a corticosteroid indicated for allergic month all the rats were sacrificed then states dermatologic diseases, endocrine blood was collected, serum separated disorders, gastrointestinal diseases, and used for the cytokines analysis. hematologic disorders, neoplastic Rat IL-1 β and TNF α Platinum ELISA kits diseases, nervous system, ophthalmic (affymetrix-eBioscience, Vienna, Austria) diseases, renal diseases, respiratory were used following the manufacturer’s diseases, and rheumatic disorders.3 instructions. Moreover, diclofenac sodium is Statistical analysis: a nonsteroidal anti-inflammatory drug All values are reported as Mean ± S.E.M. (NSAID) used to treat pain and and the statistical significance of differ- inflammatory diseases. It is taken by mouth ences among groups were assessed using or applied to the skin. Improvements in one-way ANOVA. LSD test was used to pain typically occur within half an hour and test the significance. A value of P ≤0.05 last for as much as eight hours. It is was considered significant. used commonly to treat mild to moderate postoperative or post-traumatic pain, in Results particular when inflammation is also Our findings demonstrate that rats treated 4 present. The interaction between the with each of diclofenac sodium and mechanisms of action of non-steroidal dexamethasone alone has shown to inhibit anti-inflammatory drugs (NSAIDS) and the release of both TNF-α and IL-1β steroids suggest that co-therapy may (43.1±1.1, 47.5±1.6 pg/ml) (28±2.5, provide beneficial inflammatory and pain 28.8±0.4 pg/ml) respectively in comparison relief in the absence of side effects. The to the control group (55.8±0.5, 42.3±1.1pg/ aim of the study was to compare the effect ml). However, the co-administration of of co-administered dexamethasone and them significantly increased the production diclofenac sodium with dexamethasone of the two cytokines (69±12.9, 37±0.6 pg/ and diclofenac sodium alone on the levels ml), As shown in Table 1 and Figures 1 of the proinflammatory cytokines IL-β1 and and 2. TNFα. Methods Forty male Albino rats, weighing 170-200 grams, were used in this study from the animal house unit of the College of Medicine /Hawler Medical University. Animals were handled and received humane care depending on the ethical principles of the National Institutes of Health’s Guide for the Care and Use of
1142 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Table 1: The changes of cytokines level in different experimental groups. TNF(pg/ml) IL-1(pg/ml) Group I 55.8±0.5 42.3±1.1 Group II 43.1±1.1a 28±2.5a,b Group III 47.5±1.6a 28.8±0.4a,b Group IV 69±12.9 37±0.6b Data are presented as Mean ± SEM. Group I (Physiologic saline), Group II (Dexamethasone), Group III (Diclofenac Sodium) and Group IV (Dexamethasone and Diclofenac Sodium). Significance was set when P ≤0.05. (a when compared to Group IV and b when compared to Group I).
(A) (B) Figure 1: (A) The effect of dexamethasone and diclofenac sodium alone and their co-administration on Rat’s blood TNFβ level.Group I (Physiologic saline), Group II (Dexamethasone), Group III (Diclofenac Sodium) and Group IV (Dexamethasone and Diclofenac Sodium). (B) The standard curve of the work showing the accuracy.
(A) (B) Figure 2: (A) The effect of dexamethasone and diclofenac sodium alone and their co-administration on Rat’s blood IL-1β level. Group I (Physiologic saline), Group II (Dexamethasone), Group III (Diclofenac Sodium) and Group IV (Dexamethasone and Diclofenac Sodium). (B) The standard curve of the work showing the accuracy. 1153 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Discussion more detailed analysis research to state Conventional chronic and acute treatments the exact mechanism of action is for osteoarthritis (OA) many diseases recommended. are done recently by oral NSAIDs References (such as diclofenac) and intra-articular 1. Zhang J-M, An J. Cytokines, inflammation and injected glucocorticosteroids (such as pain. International anesthesiology clinics 2007; dexamethasone). Steroids affect the 45(2):27. inflammatory response in multiple ways, 2. Clark AK, Old EA, Malcangio M. Neuropathic one of which is the inhibition of arachidonic pain and cytokines: current perspectives. Journal of pain research 2013; 6:803. acid synthesis, NSAIDs decrease 3. De Gans J, Van de Beek D. Dexamethasone in inflammation by inhibition the activity adults with bacterial meningitis. New England of cyclo-oxygenase, thus decreasing Journal of Medicine 2002; 347(20):1549-56. the production of prostaglandins from 4. Özgüney IS, Karasulu HY, Kantarci G, Sözer S, arachidonic acid. Diclofenac indirectly Güneri T, Ertan G. Transdermal delivery of diclofenac sodium through rat skin from various modulates also the lipoxygenase pathway formulations. AAPS pharmscitech 2006; 7(4):39– in the arachidonic acid cascade. This dual 45. mechanism of action of diclofenac may 5. Laurell C, Zetterström C. Effects of make it comparable in anti-inflammatory dexamethasone, diclofenac, or placebo on the 5 inflammatory response after cataract surgery. activity to the corticosteroids. Diclofenac British Journal of Ophthalmology 2002; sodium is well known to decrease the 86(12):1380. inflammatory cytokines TNFα and IL-1β in 6. Vieira V, Glassmann D, Marafon P, Pereira P, many conditions6 on the other hand, Gomez R, Coitinho AS. Effect of diclofenac the treatment with dexamethasone alone, sodium on seizures and inflammatory profile induced by kindling seizure model. Epilepsy decreased Tumor Necrosis Factor research 2016; 127:107–13. α and Interleukin 1-β levels in experimental 7. Roach BL, Kelmendi-Doko A, Balutis EC, Marra animals as well.7 Another study revealed KG, Ateshian GA, Hung CT. Dexamethasone that treatment with both Diclofenac and release from within engineered cartilage as a chondroprotective strategy against interleukin-1α. Dexamethasone showed high efficiency Tissue Engineering Part A 2016; 22(7-8):621–32. and performance in local osteoarthritis 8. Elron-Gross I, Glucksam Y, Margalit R. treatment.8 Also the enhanced effects Liposomal dexamethasone–diclofenac of co-administered dexamethasone and combinations for local osteoarthritis treatment. diclofenac K on short-term post-operative International Journal of Pharmaceutics 2009; 376(1-2):84–91. pain and swelling, compared to diclofenac 9. Bamgbose BO, Akinwande JA, Adeyemo WL, 9 potassium alone has been reported. In Ladeinde AL, Arotiba GT, Ogunlewe MO. this study our findings showed that the Effects of co-administered dexamethasone and co-administration of Diclofenac sodium and diclofenac potassium on pain, swelling and trismus following third molar surgery. Head & Dexamethasone increased the production Face Medicine 2005; 1(1):11. of the TNFα and IL-1β cytokines. These 10. Assali M, Shawahna R, Shareef M, results are in agreement to another study Alhimony I-A. Dexamethasone-diclofenac loaded which proofed that Diclofenac sodium and polylactide nanoparticles: Preparation, release Dexamethasone exhibited a synergistic and anti-inflammatory activity. European Journal of Pharmaceutical Sciences 2018; 122:179–84. effect in resuming the TNFα level near to the normal in comparison to each drug alone.10 Conclusion The combination of non-steroidal anti-inflammatory drugs (NSAIDs) with glucocorticoids provides a synergistic anti-inflammatory in rat’s blood. However,
116324 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
Description of onthophagus lucidus (sturm, 1800) (coleoptera: scarabaeidae)
from Kurdistan region –Iraq
Abbas M. Faraj1* Nabeel A. Mawlood2 Noor N. Polse2 Abstract Background and objective: Dung beetles, family Scarabaeidae are magnificient group of insects noted for both their physical beauty, and ecologically significant role in parasite suppression and agricultural management. These insects feed on feces in their larval and adult forms. The study was carried out to conduct a comprehensive survey of different dung beetles locations of Kurdistan Region-Iraq to collect the samples and describe the new record of dung beetles, Onthophagus lucidus (Sturm, 1800) in Kurdistan Region-Iraq. Methods: The samples were collected from the corpses of sheep and goats in many locations of Kurdistan Region-Iraq. Dissecting, preparing and photographing the important taxonomic parts by using digital and comprised microscope. Results: Body medium to small-sized species, 6.1–9.4mm in length. Head rounded, black, surface with a dense and simple punctuation. Anterior and posterior margins of labrum are straight. Mandible irregular shaped, apical oval, single denticles, 4th segment of maxillary palps elongate, oval , bare 3.1 times as long as 3rd segment. Apical margin of labium slightly concave, posterior margin V-inverted shaped, nine segmented, the club (7-9) segments are capitated. Abdomen is brown with seven visible sternites. Pygidium oval, generally exposed weakly convex, without groove or depressions. Aeadegus moderately sclerotized in dorsal view, apical part of the parameres slightly curved downward, external margin straight to very feebly curved. Conclusion: In highlighting of this article, we conclude: New record of Onthophagus lucidus (Sturm, 1800) is described for the first time in Kurdistan Region–Iraq, with detailed description of this species, with new characters which are not previously mentioned by other researchers. Keywords: Coleoptera; Scarabaeidae; first record; Onthophagus (Palaeonphagus) lucidus; Kurdistan Region-Iraq.
Introduction play a remarkable role in agriculture and Dung beetles, family Scarabaeidae are tropical forests by burying and consuming a magnificent group of insects noted for dung, they improve nutrient recycling both their physical beauty and ecologically and soil structure.3 Dung beetles, family significant role in parasite suppression and Scarabaeidae are detritivores insects that agricultural management. These insects feed on decomposing plant materials, feed on feces in both their larval and adult carrion. Some live in the nests or burrows forms and are classified into one of three of vertebrates or in the nests of ants or groups based on the way they procure termites. A few feed on fungi. Many feed fecal resources to their young. Dung on plant materials such as grasses, foliage, beetles live in many habitats, including fruits, and flowers, and some of these desert, grasslands and savannas desert, are serious pests of lawns, golf greens, or grasslands and savannas.1 Farmlands, and various agricultural crops.4 Dung beetles native and planted forests.2 Dung beetles have been further shown to improve soil 1 College of Pharmacy, Hawler Medical University, Erbil, Iraq. 2 College of Agriculture, University of Salahaddin, Erbil, Iraq. * Correspondence: [email protected] 1171 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) conditions and plant growth on rehabilitated camera). The measured proportions of coal mines in South Africa.5 Interactions body parts are given in points of an between dung beetles and helminthes eye piece linear micrometer in a binocular are with both positive and negative microscope. The body length was consequences for successful transmission measured from the apex of the clypeus to of parasites.6 Some species of dung the apex of the elytra, and the body width beetles act as biological control agents was measured at the base of the pronotum for gastrointestinal Parasites of livestock, (with the specimen in dorsal view). The Ostertagia ostertagi.7 Many species of dung species confirmed by Dr. Guid Sabatinelli beetles are major intermediate host of specialist of Scarabaeidae in Natural the dog esophageal worm Spirocerca lupi.8 History Museum of Geneva, Switzerland. Dung beetles also play an important role Characters selected for identification in the transmission of some helminthes are those generally easily observed, to human and cattle.9 The genus with depending on male genitalia. The Onthophagus is a hyper diverse genus specimens were deposited in the within the subfamily Scarabaeinae. insect museum at the Department of According to the most recent catalogue plant protection, College of Agriculture, of the American Onthophagini,10 and Salahaddin University, Erbil Iraq. considering the new species described over the last decade.11,12,13,14,15 The genus Results and Discussion Onthophagus Latreille, currently comprises Description 174 species and subspecies. In Iraq; Body (Figure 1a): Medium to small-sized (16) indicated eight species of the family in species 6.1 – 9.4 mm in length. Color. five genera. (17) mentioned one species. Head and pronotum black, elytra reddish- (18) recorded three species and (19) brown- black. recorded one species. The aim of this Head (Figure 1b): Rounded, black, surface study is to conduct a comprehensive of with a dense and simple punctuation. survey of different dung beetles locations Clypeus distinctly punctated, elongated of Kurdistan Region – Iraq to collect the forward, triangular shaped, genal and samples and describe the new record clypeal margins continuous, barely of dung beetles, Onthophagus lucidus sinuated at theclypeo-genal junction. (Sturm) in Kurdistan Region–Iraq with Fronto-clypeal region without transversal photographing the important parts carina, frontal horns straight and parallel. especially the male genitalia. Methods The present paper is based on 14 specimens which collected from sheep's wool in different localities of Erbil and Sulimani Governorate, Kurdistan region– Iraq during March-August of 2018, using hand picking. The specimens were placed in boiling water for 10-15 minutes to soften a b their parts. The mouthparts and abdomen were separated and cleared in a hot solution of 10% KOH for 24 hours. Then Figure 1: Onthophagus lucidus (Sturm, the parts were studied under immersion in 1800) distilled water.20, 21Photographs of habitus a. Head,15X and important parts were captured through b. body,10X a digital camera (Ucmas series microscope
1182 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) Labrum (Figure 2a) rectangular, slightly downward and obtuse at the apex, the sclerotized, anterior and posterior margins outer once 3 times as long as the inner, straight, surface sparsely dark brown very protarsal five segmented tubular segments, short setose. Mandible (Figure 2b) Low- 2nd segment 2 times as long as the 1st, high sclerotized, irregular shaped, apical 2nd-4th segments nearly same length, 5th oval, single denticle, molar area with mass segment 2.8 times as long as the 4th. Fore of fine pale yellow setae. Maxilla (Figure claws long, weak and slightly curved. 2c) brown-dark brown, cardo triangular, Middle legs similar to the fore legs except, sparsely very long dark yellow setose, the coxa nearly conical, outer margin of stipes triangular, densely very long, dark tibia with row of small spines, apical part yellow setoe,galea triangular densely long with row of small spines and two long yellow setose, lacinia triangular bare.1st-3rd spurs, 1st tarsal segment 2.2 times as long segments of maxillary palps cup shaped, as the 2nd, 2nd segment 1.8 times as long 3rd segment 1.1 as long as 2nd segment, as the 3rd, each segment with 3-4 yellow 4th segment elongate oval , bare 3.1 times short spines. Hind legs similar to the as long as 3rd segment. Labium (Figure 2d) middle legs except, the coxae is bot darkyellow, mentum nearly square shaped, shaped. Elytra (Figure 2g)nearly triangular, apical margin slightly concave, posterior yellow, slightly swollen, striae very shallow, margin V-inverted shaped, surface with interstriae flat to weakly convex at the base very long dark yellow setose. Labial palps and apex margins dark brown, surface brown, 1st segment short, oval, 2and 3rd very finely microreticulated, interstrial segment cup shaped, 3rd segment 1.2 punctuation shallow and simple, punctures times as long as the 2nd, apical part of 3rd sparse and devoid of conspicuous segment with small process. Antenna brownish setae, lateral interstriae (VI–VII) (Figure 2e) brown- dark brown, length 1.0– with deeper punctures. Prosternum surface 1.4 mm, consist of nine segments, 1st black finely microreticulated and with long segment is the longest, 5 times as long setae at middle. Mesosternal surface black as 2nd segment, 2nd segment globular, with coarse punctures, and short setae. 3rd-6th segments cup shaped, the club (7-9) Metasternum with fine and densely segments are capitate, 7 and 8 segments punctures. cup shaped, 7th segment slightly longer Abdomen than the 8th, 9th segment oval. The Brown, with seven visible sternites segments sparsely short yellow setose. (Figure 2h),1st-4th abdominal sternites Thorax nearly same length, 5th abdominal sternite Pronotum dark brown, hump-like slightly 1.1 times as long as 6th,posterior margin convex, lateral margins moderately curved, of 5th and 6th sternites concave, 2nd -6th anterior angles triangular, posterior margin abdominal sternites transverse, Pygidium of pronotum striate, prontal surface densely oval, generally exposed weakly convex, punctate, most of the punctures with without groove or depressions, anterior a central pit, anterior angles with fine margin of each sternites with a row of punctures, posteromedia region with punctures with a central pit. obsolete punctures. Anterior angles of Male genitalia pronotum with a weak and rounded Aeadegus 1.1-1.5mm long, moderately tubercle. Legs dark brown, surface with sclerotized, in dorsal view (Figure 2i), yellow setose. Fore coxae (Figure 2f) apical part of the parameres slightly curved cylinderical, fore trochanter small and downward, external margin straight to very triangular, fore femure nearly cylindrical, feebly curve. In lateral view (Figure 2j) expanded at the middle, outer edge of distal-inferior area with sinuate near the protibia with fourstout teeth, apical part of apex, lateral margin straight and strongly protibiae with two spurs weakly curved angled on the posterior side.
1193 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan)
a b c d 0.5 mm
e f g 0.5 mm
h i j 1 mm 0.5 mm
Figure 2: Onthophagus lucidus (Sturm, 1800) a. Labrum b. Mandible c. Maxilla d. Labium e. Antennae f. Fore leg g. Elytra h. Abdominal sternites i. Aedeagus (Dorsral view) j. Aedeagus(Lateral view)
1204 The Second Scientific Conference for The College of Pharmacy/Hawler Medical University, (13th - 14th of November 2019, Dedeman Hotel - Erbil, Kurdistan) References 13. Genier F, Howden HF. Onthophagus fragosus n. sp. a second endemic species of Onthophagus 1. França FM, Korasaki V, Louzada J, Vaz-de-Mello, Latreille from Cuba (Coleoptera: Scarabaeidae: FZ. First report on dung beetles in Scarabaeinae). Zootaxa 2014; 3795(5):597–9. intra-Amazonian savannahs in Roraima, 14. Arriaga-Jimenez A, Moctezuma V, Rossini M, Brazil. Biota Neotropica 2016; 16(1). Zunino M, Halffter G. A new species of 2. Beiroz W, Slade EM, Barlow J, Silveira JM, Onthophagus (Scarabaeoidea: Scarabaeinae) Louzada J, Sayer E. Dung beetle community from the Mexican transition zone, with remarks dynamics in undisturbed tropical forests: on its relationships and distribution. Zootaxa implications for ecological evaluations of land-use 2016; 4072:135–43. change. Insect Conservation and Diversity 2017; 15. Moctezuma V, Rossini M, Zunino M, Halffter G. 10(1):94–106. A contribution to the knowledge of the mountain 3. Brown J, Scholtz CH, Janeau JL, entomofauna of Mexico with a description of Grellier S, Podwojewski P. Dung beetles two new species of Onthophagus Latreille, (Coleoptera: Scarabaeidae) can improve soil 1802 (Coleoptera: Scarabaeidae: Scarabaeinae). hydrological properties. Applied Soil Ecology ZooKeys 2016; 572:23–50. 2010; 46:9–16. 16. Derwesh AI. Direct. Gen. Agri. Res. Proj. 4. Triplehorn CA, Johnson NF. Borror and Delon,g th Baghdad. Bull 1965; 121–3. introduction to study of insects. 7 ed. Brooks / 17. El-Haidari H, Fattah YM, Sultan JA. Cole, Cengage Learning Australia 2005. P. 411. Contribution to the fauna of Iraq. Bulletin 1972; 5. Badenhorst J, Dabrowski J, Scholtz C H, 18(4):1–19. Truter W F. Dung beetle activity improves 18. Abdul-Rassoul MS. Checklist of Iraq natural herbaceous plant growth and soil properties on history museum insect collection, Nat Hist Res confinements simulating reclaimed mined land Cent Iraq Publ 1976; (30). in South Africa . Applied Soil Ecology 2018; 19. Al-Ali AS. Phytophagous and entomophagous 132:53–59. insects and mites of Iraqi. Nat. l Hist. Res. 6. Nichols E, Gomez A. Dung beetles and fecal Center publ 1977. P. 33–142. helminth transmission: patterns, mechanisms and 20. Lane RP, Crosskey RW. Medical insects questions 2014; 141(5):614–23. and arachnids. The Natural History Museum. 7. Fincher G T. Dung beetles as biological control Chapman and Hall, London 1993. agents for gastrointestinal parasites of livestock. 21. Mawlood NA, Hamad MI, Abdullah YM. A new J. Parasitol 1973; 59(2):396–9. record of glaphyrid scarab beetles, Eulasia vitatta 8. Du Toit CA, Holter P, Lutermann H, Scholtz CH. (Fabricius, 1775) (Coleoptera, Glaphyridae) from Role of dung beetle feeding mechanisms in Erbil Kurdistan region-Iraq. ZJPAS 2016; 28(3): limiting the suitability of species as hosts for the 1–4. nematode Spirocerca lupi, Medical &Veterinary Entomology 2012; 26:455–7. 9. Mowlavi G, Mikaeili E, Mobedi I, Kia E, Masoomi L, Vatandoost H. A Survey of dung beetles infected with larval nematodes with particular note on Copris lunaris Beetles as a vector for Gongylonema sp. in Iran. Korean J. Parasitol 2009; 47(1):13–7. 10. Pulido-Herrera LA, Zunino M. Catalogo preliminar de los Onthophagini de America (Coleoptera: Scarabaeinae). In: Zunino M, Melic A, editors. Escarabajos, diversidad y conservación biológica. Ensayos en homenaje a Gonzalo Halffter. Vol. 7. Zaragoza: Monografía Tercer Milenio, Sociedad Entomologica Aragonesa 2007; P. 93–129. 11. Kohlmann B, Solis A. New species and revalidations of scarab beetles (Coleoptera:Geotrupidae: Athyreini and Coleoptera: Scarabaeidae: Scarabaeinae) from Costa Rica and Panama. Zootaxa 2012; 3193:28 –52. 12. Delgado L, Curoe DJ. Panamanian Onthophagus (Coleoptera: Scarabaeidae): description of a new species, and a revised key to the species. Fla Entomol 2014; 97(1):61–7.
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