ANTIMICROBIAL SCREENING AND ANTIINFLAMMATORY ACTIVITIES OF METHANOL EXTRACT OF CISSUSARALIOIDES

ABSTRACT

The aim of this study was to investigate the antimicrobial effect of Cissusaralioides extracts and their anti-inflammatory activity using experimental rat models. Phytochemical analyses of  Cissusaralioides   showed   the  presence   of  flavonoids,   saponins,   tannins,   alkaloids, terpenoids, steroids and glycosides. Antimicrobial activity of C. aralioides was investigated against  some gram negative  bacteria  such as  Escherichia  coli, Pseudomonas  aeruginosa, Klebsiellaaerogenes   and  gram  positive   bacteria   such  as  Staphyllococcus   aureus  and Streptococcus  pneumonia  using  agar  well  diffusion  method.  Anti-inflammatory  activities were  tested   on  egg   albumin-induced   rat  paw  oedema,   acetic   acid-induced   vascular permeability  in  rats,  agar-induced  leukocyte  migration  in  rats  and  heat  and  hypotonic solution-induced  haemolysis  of human  red  blood  cell membrane.Cissusaralioides  extract administered orally up to the dose of 5000 mg/kg caused no deaths after 24 hours indicating that the lethal dose of Cissusaralioides  is > 5000 mg/kg. Acetone, methanol  and aqueous extracts displayed various degrees of antibacterial activity but the methanol extract showed higher activity against the bacteria examined. The extract  significantly (p< 0.05) inhibited egg albumin-induced  oedema  in rats  treated  with  the  extract  (100-400  mg/kg)  and  also significantly (p< 0.05) reduced exudate volume and vascular permeability induced by acetic acid  in rats. The  extract  significantly  (p<  0.05) stabilized  human  erythrocyte  membrane subjected to heat and hypotonic-induced  lysis in the treated groups (100-800  µg/ml). This study  has  shown  that  the  acetone,  aqueous  and  methanol  extracts  of  Cissusaralioides possessed antimicrobial properties, and the methanol extract had anti-inflammatory activity.

CHAPTER ONE

INTRODUCTION

Inflammation  is  the  response  of  tissue  to  injury  (infection,  trauma  and  hypersensitivity), characterized  in the acute phase (microscopically)  by increased blood  flow (vasodilation) and vascular  permeability  along  with  the  accumulation  of  fluid,  leukocytes,  and  inflammatory mediators such as cytokines. Macroscopically, it is characterized by redness, swelling, heat, pain and loss of function and these cause  considerable pain and discomfort and most often require treatment (Anosikeet al., 2012).At the onset of an inflammation, the cells undergo activation and release inflammatory  mediators.  These mediators include histamine,  serotonin,  slow reacting substances of anaphylaxis (SRS-A), prostaglandins and some plasma enzyme systems such as the   complement system, the clotting system, the fibrinolytic system and the kinin system(Perianayagamet   al.,  2006).  These  mediator  molecules  work  collectively  to   cause increased vasodilation and permeability of blood vessels. Thus, leading to increased blood flow, exudation  of  plasma  proteins  and  fluids,  and  migration  of  leukocytes,  mainly  neutrophils, outside the blood vessels into the injured tissues.  However, several medicinal plants are used in the treatment of disorders arising from inappropriate deployment of the inflammatory mediators, among them isCissusaralioides.This is even more compelling with the realization that currently available anti-inflammatory agents such as steroids and non-steroidal anti-inflammatory drugs are fraught with several drawbacks  or limitations to their use. Increased  incidence of stroke, atherosclerosis, cancer, gastric disorder and coronary heart related diseases has been attributed to prolonged  use of  synthetic inhibitors of cyclooxygenase,  the non-steroidal  anti-inflammatory drugs such as indomethacin, largely due to the implication of prostanoids in these pathological conditions  (Grosser  et  al.,  2006).  Specific  inhibitors  of  the  cyclooxygenase  isoenzymes- cyclooxygenase-1   and  cyclooxygenase-2   are  associated  with  gastrointestinal  and   vascular systems toxicities  respectively (Joseph et al., 2005).Recently,  these traditional  medicines are receiving  more  scientific  support  which  helps  in  not  only  authenticating  the  use  of  these medicines  for  treatment  but  also  understanding  the  mechanism  of  action  of  these  drugs. Cissusaralioides  found in Asia and Africa shows antidiabetic,  antimicrobial  as well as anti- inflammatory properties (Maxwellet al., 2015). Cissusaralioides as an anti-inflammatory agent may reduce the formation of pro-inflammatory cytokines that stimulate bone resorption, thereby reducing bone loss and also act as an estrogen receptor agonist. Available experimental evidence showed that the  extracts of the leaf caused moderate anti-diabetic activity (Igoliet al., 2012).

Flavonoids and some other compounds isolated from Cissusaralioides inhibit the release of β- hexosaminidase  in  rat  basophilic  leukemia  cells  (Xu  et  al.,  2009).  Flavonoids  have  anti- inflammatory (Yamamoto and Gaynor, 2011), antimicrobial (Cushnine and Lamb, 2005; 2011), anticancer and anti-diarrheal properties (Schuieret al., 2005).Terpenoids have been well studied for their pharmacological activities and are known to have anti-inflammatory properties and are used as anti-cancer drugs and they target the phospholipase, cyclooxygenase and lipoxygenase (Bracaet al., 2010).

1.1       Cissusaralioides:

Fig. 1: Cissusaralioides

Cissusaralioides is a natural climber only found from the countries of Arabia through Eastern and Western Africa.

1.1.1    Description of Cissusaralioides

Cissusaralioides  is  a  lofty  climber,  woody  at  the  base  with  stout  green  succulent  stems constricted at the nodes and sometimes sub-succulent leaves. Flowers are greenish or whitish, comparatively large and horizontal. The fruit is 2½ cm long, mostly red in colour. The whole plant is covered with irritating hairs and leaves contain an acid and slightly acrid red sap. They are commonly found in deciduous forests and fringing jungle across the region from Senegal to Northern  and  Southern  Nigeria.  Cissusaralioides   is  found  commonly  in  Tropical  Africa especially Cameroon (common name- kindamine) and Nigeria (Igbo name- eririagwo) (Burkill,

2000).

1.1.2    Taxonomic Classification of Cissusaralioides

Taxonomy classification of Cissusaralioides

Kingdom:                   Plantae

Division:                     Magnoliophyta (Flowering plants) Class:                         Magnoliopsida (Dicotyledons) Order:                         Vitales

Family:                       Vitaceae (grape fruit family) Genus:                        Cissus

Species:                      aralioides                   (Burkill, 2000). Common name:          Guinea Bissau

Nsukka name:            Eriri-agwo

1.1.3    Origin and Geographic Distributionof Cissusaralioides

Cissusaralioidesoriginated    from   Congo   extending   from   Arabia   through   Eastern   Africa

Southwards to Mozambique and the Transvaal (Wild et al., 1963).

1.1.4    Edible Uses of Cissusaralioides

Cissusaralioides is a climbing or prostate shrub found throughout Africa, Egypt and the Arabian Peninsula and is used as a vegetable. It has minor economic importance as a  medicinal plant (Balogun  and Fetuga,  1986). The stem  is sold as a food condiment  in  local markets  in the Eastern and Northern parts of Nigeria.

1.1.5Medicinal Uses of Cissusaralioides

In Nigeria folkloric medicine, the leaves are used for treatment of cuts, wounds, internal and external  microbial  infections  and  swellings.  It  is  also  used  for  the  treatment  of  arthritis, rheumatism, dropsy, gout swelling, oedema, analgesic, pulmonary troubles. (Burkill, 2000). In Cameroon  traditional  medicine,  Cissusaralioides  leaves  and  roots are  used  as antimicrobial agents against microorganisms of the gastrointestinal and urogenital tracts (Assobet al., 2011). In Gabon, the grinded leaves of Cissusaralioides mixed with sugar cane juice are used to combat gonorrhea. The liane (freed from the leaves) is used in Congo for its analgesic and antiseptic attributes  to  relieve  cough,  abdominal  and  kidney  problems  (Burkill,  2000).  Phytochemical analyses  of  Cissusaralioides  showed  that  it  contains  alkaloids,  tannins,  saponins,  terpenes, flavonoids and cardiac glycosides (Borokini and Omotayo, 2012).

1.2   Medicinal Plants

1.2.1    History of Medicinal Plant

Medicinal plants have been used as native treatment for numerous human diseases for thousands of years and in many parts of the world and can be alternatives since their reputed efficacies have been experienced and passed on from one generation to another (Akinyemiet al., 2005; Jachak and Saklani, 2007).

In rural areas of developing countries, they continue to be used as the primary source of medicine (Chitmeet al., 2003). About 80% of the people in developing countries use traditional medicines for their health care (Kim, 2005). It is estimated that there are 250,000 to 500,000 species of plants on earth (Borris, 1996). Relatively small percentages (1 to 10%) of these are used as foods by both humans and other animals. It is possible that even more are used for medicinal purposes (Moerman, 1996). Hippocrates (in the  late fifth century B.C.) mentioned

300 to 400 medicinal plants (Schultes, 1978). In the first century A.D., Dioscorides wrote De MateriaMedica,   a  medicinal  plant  catalogue  which  became  the  prototype  for   modern pharmacopoeias.

The  Bible  offers  descriptions  of  approximately  30  healing  plants  (Cowan,  1999). Indeed,  frankincense  and  myrrh  probably  enjoyed  their  status  of great  worth  due  to  their medicinal properties. They were reported to have antiseptic properties and were even employed as  mouthwash.   Thus  the  mainstream  medicine  is  increasingly  receptive  to  the  use  of antimicrobial  and  other  drugs  derived  from  plants,  as  traditional  antibiotics  (products  of microorganisms or their synthesized derivatives)  become ineffective and as new, particularly viral diseases remain intractable to this type  of drug. Another driving factor for the renewed interest in plant antimicrobials  in the  past 20 years has been the rapid rate of plant species extinction (Lewis and Elvin-Lewis, 1995).

1.2.2     The Phytochemicals in Medicinal Plants

Plants  have almost  a limitless  ability to synthesize  aromatic  substances,  most of which  are phenols  or their  oxygen-substituted  derivatives  (Lahlou,  2004).  Most  of  the  derivatives  are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total (Schultes,  1978). In many cases,  these  substances  serve as plant defence  mechanisms  against predation  by  microorganisms,  insects,  and herbivores.  Some of them, such as terpenoids, give plants their odours; others (quinones and tannins) are responsible for plant pigments. Many compounds areresponsible for plant flavour (the terpenoid capsaicin from chili peppers), and some of the same herbs and spices used by humans to season food yield useful medicinal  compounds. Such secondary metabolites like tannins, terpenoids, flavonoids, alkaloids,  saponins,  reducing  sugars  and  sterols  have been found  to have anti-diarrhoeal  or antimicrobial activity (Yu et al., 2000; Al-Rehailyet al., 2001).

1.3   Antimicrobial Agents

Man is in constant contact with a large number of different bacteria which temporarily or permanently inhabit his body creating temporary or permanent community. Relations which are thus  established   are  dangerous  and  very  complex,  from  those  positive  to   those  whose consequences for man are extremely negative. Very often, both on and in man’s body, bacteria which have the ability to cause an infection are present. This ability of pathogenic bacteria is reflected in possession of certain pathogenicity factors. A set of factors which enable successful invasion and damage of the host are: toxins, surface structures and enzymes. Between the host and the pathogen,  very complex relations are  established  whose outcome depends  on host’s characteristics as well as on pathogen’s characteristics (Bartizilaet al., 1992).

Infections  caused  by bacteria  can  be  prevented,  managed  and  treated  through  anti- bacterial group of compounds known as antibiotics. Antibiotics are natural,  semi-synthetic  or synthetic compounds that kill or inhibit the growth of bacteria. When bacteria are exposed to an antibiotic, they respond doubly:

(i)        They are sensitive to what caused the inhibition of their growth, division and death or

(ii)        ii) They can remain unaffected or resistant (Okorie, 2005).

The resistance of bacteria to antibiotics can be natural (intrinsic) or acquired.  Natural resistance is achieved by spontaneous gene mutation. The acquired resistance  occurs after the contact  of bacteria  with an antibiotic  and  as a result  of adaptation  of a  species  to  adverse

environmental conditions. In such population, an antibiotic as a selective agent, acts on sensitive organism,  while  resistant  organism  survive  and  become  dominant.  Bacteria  gain  antibiotic resistance due to three reasons which are:

(i)        Modification of active site of the target site resulting in reduction in the efficiency of binding of the drug

(ii)       Direct  destruction  or  modification  of  the  antibiotic  by  enzymes  produced  by  the organism (iii) Efflux of antibiotic from the cell (Sheldon, 2005).

The   evolution   of   antibacterial   resistance   in   human   pathogenic   and   commensal microorganisms is the result of the interaction between antibiotic exposure and the transmission of resistance within and between individuals. It is especially interesting in the phenomenon of horizontally gene transfer, extra-chromosomal  DNA material,  so-called plasmids, often carry genes of resistance and can transfer information within and between the individuals of the same or related bacterial species, thus also spreading the resistance.

Having in mind the current progress of resistance spreading and resilience of larger and larger number of bacteria to traditional antibiotics as well as a way of transmitting the gene of resistance,  above  all via plasmids,  one can conclude  that the ability of obtaining  bacterium resistance  to antibiotics  represents  a very dynamic  and unpredictable  phenomenon.  For that reason,  bacterial  resistance  to  antibiotics  represents  a  major  health  problem.  Solving  this problem and search for new sources of antimicrobial agents is a worldwide challenge and the aim of many scientific researches, academic institutions and pharmaceutical companies is testing biologically active compounds of plant origin (Cowan, 1999).

1.3.1    History of Antimicrobial Agents

The first anti-microbial  agent was Salvarsan,  a remedy for syphilis that was synthesized  by Ehrlich in 1910. In 1935, sulfonamides were developed by Domagk and other researchers. These drugs were synthetic compounds and had limitations in terms of safety and efficacy.  In 1928, Fleming  discovered  penicillin.  He  found  that  the  growth  of  Staphyllococcus  aureus  was inhibited in a zone surrounding a contaminated blue mold (a fungus fromPenicilliumgenus)  in culture dishes, leading to the finding that a microorganism would produce substances that would inhibit the growth of other microorganisms (Cushnie and Lamb, 2011). Penicillin was originally effective  against  gram-positive  organisms  such  as  S.  aureus  which  produces  the  penicillin hydrolyzing  enzyme  penicillinase  and later  methicillin  was developed.  The drugs have been

developed   to  achieve  better  pharmacodynamics   including  the  absorption  of  oral   drugs, concentration in the blood and distribution to the inflammatory focus.

Antimicrobial  agents that are associated with serious side effects have been replaced  by other  safer  drugs.  Quinolone  antimicrobials  represent  an  example  of  drugs  with  improved pharmacodynamics and safety. Nalidixic acid, the first drug of this class, was active only against gram-negative bacteria. S. aureus is the resistant bacterium that rapidly acquired resistance to sulfonamides when they were in use. However, in 1961, methicillin-resistant S. aureus (MRSA) was isolated  in the UK. MRSA acquires resistance  to most β-lactam  antibiotics,  through its acquisition of the penicillin-binding protein (PBP)2 gene. PBP2 is an enzyme involved in cell wall synthesis that has low binding affinity for β-lactam antibiotics. Although P. aeruginosa are intrinsically  resistant  to  many  antimicrobial  agents,  the  emergence  of P.  aeruginosa  strains resistant to all the classes of antimicrobials, i.e., carbapenems, quinolones and amino glycosides is a recent concern(Chambers,  1997). This has spurred our interest to undertake antimicrobial screening especially against P. aeruginosa.

1.4   Biochemistry of Inflammation

Inflammation is a complex reaction of the body in response to cellular injury that is marked by tissue swelling, capillary dilation, anti-histamine activity, redness, heat and pain. It serves as a mechanism initiating the elimination of noxious agents of damaged tissue by intercepting and destroying  invading  microorganism  (Anosikeet  al., 2012b).At  the onset  of inflammation,  the cells undergo activation and release inflammatory mediators. These mediators include histamine, serotonin, slow reacting substances of anaphylaxis (SRS-A),  prostaglandins  and some plasma enzymes such as the complement system, the clotting  system, the fibrinolytic system and the kinnin system (Perianayagamet al., 2006). These mediator molecules work collectively to cause

increased   vasodilation   and   permeability   of   blood   vessels.   The   damaged   cell   signals inflammatory response releasing NF Kappa B, the key regulator to our inflammatory response system. This results in the expression of several pro-inflammatory proteins such as COX-2 and iNOS that cause pain, fever, swelling and heat in an affected area. Simultaneously, a series of pro-inflammatory cytokines such as IL-2, TNF-α and  interferon-γ are released (Cushnine and Lamb,  2005).Following  inflammation,  injured  tissue  is  usually  replaced  by  new  cells  and extracellular materials, with undamaging surrounding cells proliferating and migrating to fill the void, although some tissues especially surface epithelium can grow back efficiently (Ferrero et al., 2007).The basic components of inflammatory response are:

1.4.1    Increased Vascular Permeability

The endothelial lining of capillaries   becomes leakier, allowing more fluid (blood plasma)  to exude into  the connective  tissue spaces.  There  is normally a balance  between  fluid  leaving vascular  spaces  and  fluid  re-entering  the system.   Inflammation  shifts  this  balance,  causing accumulation of interstitial fluid. The fluid build-up, which follows this permeability change, is called oedema and is visible as puffiness or swelling (Albertset al., 2002).

1.4.2    Emigration of Leukocytes

Vasodilation  and  increased  vascular  perfusion  are  designed  to  prepare  the  way  for   the inflammatory  infiltrate  to  enter  the  inflamed   tissue.A  combination   of   vasodilation  with thickening of the blood (due to fluid leaking out of the vessels) causes a slowing of flow rate, which encourages leukocytes to stick to the sides of the vessels.  This is called “margination” or “pavementing”  (the  white  blood  cells  gather  along  the  endothelium,  like  bricks  paving  a road).From  here the  leukocytes  crawl  between  the  endothelial  cells and  enter  the  inflamed connective tissue.  Increased metabolic activity associated with leukocyte activity also generates heat, contributing to local warmth (Pancer and Cooper, 2006).

1.4.3    Inflammatory Infiltrate

The inflammatory or leukocytic infiltrate consists of white blood cells which leave the blood and enter  (infiltrate)  the inflamed  connective  tissue.  Cells  of the  inflammatory  infiltrate  include neutrophils, lymphocytes and monocytes.  Immigration of these cells into  peripheral tissues is one of the principal purposes for inflammation,  bringing to a site of  injury the immune cells which combat infection and clean up damaged tissue (Moreau et al., 2001).

1.4.3.1 Neutrophils

Neutrophilic  leukocytes are the first white blood cells to enter the tissue during  acute inflammation.   Neutrophils  are anti-bacterial  cells which lyse (break down) bacterial  cells by releasing lysosomal enzymes (Langermanset al., 1994).  Neutrophils are fairly uniform in size with  a diameter  between  12  and  15  micrometers  and  are  polymorphonuclear.  The  nucleus consists of two  to five  lobes joined  together  by hair  like filaments.  Neutrophils  move with amoeboid  motion.  They extend  their  long  projection  called  pseudopodium  into  which  their granules flow; this action is followed by contraction of filaments based in the cytoplasm, which draws the nucleus and rear of the cell forward. In this way neutrophils rapidly advance along a surface. The bone marrow of a normal adult produces about 100 billion neutrophils  daily.  It takes about one week to form a mature neutrophil from a precursor cell in the marrow; yet, once in the blood, the mature cells live only a few hours or perhaps a little longer after migrating to the tissues  (James  and  Michael,  1996).  To  guard  against  rapid  depletion  of the short-lived neutrophils (for example, during infection), the bone marrow holds a large number of them in reserve to be mobilized in response to inflammation or infection.

Within the body, the neutrophils migrate to areas of infection or tissue injury. The force of  attraction  that  determines  the  direction  in  which  neutrophils  will  move  is  known  as chemotaxis and is attributed to substances liberated at sites of tissue damage. Of the 100 billion neutrophils circulating outside the bone marrow, half are in the tissues and  half in the blood vessels; of those in the blood vessels,  half are within the mainstreams  of rapidly circulating blood and the other half move slowly along the inner walls of the blood vessels (marginal pool), ready to enter tissues on receiving a chemotactic signal from them (Agerberth and Gudmunsson, 2000).

Neutrophils are actively phagocytic; they engulf bacteria and other microorganisms and microscopic  particles.  The  granules  of  the  neutrophils  are  microscopic  packets  of  potent enzymes capable of digesting many types of cellular materials. When bacterium is engulfed by a neutrophil, it is encased in a vacuole lined by the invaginated membrane. The granules discharge their contents  into  the vacuole  containing  the organism.  As this  occurs, the granules  of the neutrophil  are  depleted  (degranulation).  A  metabolic  process  within  the  granules  produces hydrogen peroxide and a highly active form of oxygen (superoxide), which destroy the ingested bacteria. Final digestion of the invading organism is accomplished by enzymes.

Neutrophils constitute 40 to 70% of total WBCs; they are the first line of defense against infection. Mature neutrophils have a half-life of about 2 to 3 days (Albertset al., 2002). During acute inflammatory response (e.g., infection), neutrophils are drawn by chemotactic factors and alerted  by  the  expression  of  adhesion  molecules  on  blood  vessel  endothelium,  leave  the circulation   and   enter   tissues.   Their   purpose   is  to   phagocytose   and   digest   pathogens. Microorganisms   are  killed  when  phagocytosis   generates   lytic  enzymes  and  reactive  O2 compounds (e.g., superoxide, hypochlorous acid) and triggers release of granule contents (e.g., defensins,  proteases,  bactericidal  permeability-increasing  protein,  lactoferrin,  and lysozymes). DNA and histones are also released, and they, with granule contents such as elastase, generate fibers  in the  surrounding  tissues;  the  fibers  may  facilitate  killing  by trapping  bacteria  and

focusing  enzyme  activity.  Severe  inflammation  may  increase  the  numbers  of neutrophils  in blood, resulting in neutrophilia (Langermanset al., 1994).

1.4.3.2 Lymphocytes

Lymphocytes are the cells responsible for the body’s ability to distinguish and react to an almost infinite number of different foreign substances, including those of which microbes are composed (Moreau et al., 2001). Lymphocytes are mainly a dormant population, awaiting the appropriate signals to be stirred to action.  Lymphocytesaccumulate  somewhat later during the inflammatory process.   Their presence  in large numbers indicates the continuing presence of antigen and thus may suggest an established infection (Boyton and Openshaw, 2000).

Lymphocytes produce the multitude of diverse antibody molecules (one specific type of antibody per lymphocyte)  which provide  the mechanism  for chemical recognition of  foreign materials  (distinguishing  between  self  and  non-self)  and  also  for  mediating  and  regulating immune responses.  Lymphocytes  travel in the blood, but they routinely leave  capillaries and wander through connective tissue.  Therefore, lymphocytes may be normally encountered at any time in any location.  They even enter epithelial tissue, crawling between the epithelial cells. They re-enter  circulation  via  lymphatic  system  channels  (hence  their  name)  (Yenuguet  al., 2003).  Lymph channels drain into lymph nodes, where dense aggregations of lymphocytes form lymph  nodules.   Each  lymph  nodule  has  a  “germinal  center”,  where  activated  lymphocytes proliferate.  Lymph nodules with proliferating lymphocytes also characterize the tonsils and the appendix and may be encountered in other sites as well (James and Michael, 1996).

Recent  research  suggests  that  some  types  of  lymphocytes  are  compartmentalized  to particular tissues or body regions. Lymphocytes are small cells, 7-9 micrometer in diameter in blood smears, and are the second most common white blood cell type (about 30% of the WBCs). They have a round heterochromatic  (deeply staining) nucleus surrounded by a relatively thin rim of cytoplasm.   Lymphocytes  are most easily  recognized in histological sections as small “naked” nuclei (the cytoplasm is usually  inconspicuous)  which occur here and there in most tissues  and especially  commonly near  mucous membranes.   Lymphocytes  are found densely packed in lymphoid  tissue-spleen  and lymph nodes. Plasma cells are lymphocytes  which are specialized for mass production and secretion of circulating antibodies.  Plasma cells have more extensive   cytoplasm   filled   with  rough  endoplasmic   reticulum   (for  synthesizing   protein, specifically antibody molecules).  This cytoplasm is distinctly basophilic, a consequence of the large numbers of ribosomes associated with the rough ER, and typically forms a lopsided bulge on one side of the nucleus.   The heterochromatin  of plasma  cells is typically  clumped  in a characteristic  “spoke-wheel”  arrangement  which also aids plasma cell  recognition (Agerberth and Gudmunsson, 2000).

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