EVALUATION OF ANTIINFLAMMATORY, ANTIPYRETIC AND ANALGESIC PROPERTIES OF VITEX SIMPLICIFOLIA LEAF EXTRACTS

ABSTRACT

Natural products are sources of bioactive compounds and have potential for developing some novel therapeutic agent, due to their enormous chemical diversity. These diverse natural products form the basis of many traditional medicine systems throughout the world, due to their accessibility,   efficacy   and   minimal   side   effects.   The   present   study   evaluated   the antiinflammatory, antipyretic and analgesic properties of Vitex simplicifolia leaf. The extraction of 464.67 g of Vitex simplicifolia leaves gave an exact yield of 82.26 g and percentage yield of

17.7% using 99.5% ethanol. Subsequent partitioning using 60 g of the crude ethanol extract gave percentage yields of 4.47% for n-hexane, 2.77% for ethylacetate and 9.93% for 10% ethanol. The phytochemical  composition of crude extract of Vitex simplicifolia included tannins, cardiac glycosides, anthraquinones, hydrogen cyanides, terpenoids, steroids, phenols, alkaloids, flavonoids and saponins.  The acute toxicity study of the extract showed that the leaf extract was safe even at the higest dose of 5000 mg/kg body weight. The 10% ethanol fraction (100 mg/kg and 200 mg/kg b.w.) significantly (p < 0.05) inhibited the paw oedema formation induced by egg albumin at the earlier stage of inflammation (0.5 – 2 h), but the inhibition was non significant (p < 0.05) for later stages of inflammation (3 – 5 hr) when compared to that of the control. The rats treated with 400 mg/kg b.w. significantly (p < 0.05) inhibited paw oedema at 0.5hr, but the inhibition was non significant (p < 0.05) from 1hr -5hr when compared to that of the control. All the doses  of the 10%  ethanol  fraction  of the  extract  produced  a dose  and  time dependent inhibition of paw oedema when compared with the control. The standard anti-inflammatory drug diclofenac sodium (10 mg/kg b. w.) had a similar trend. Similarly, varying doses of 10% ethanol fraction of the extract (0.1 mg/ml – 0.5 mg/ml) significantly (p < 0.05) inhibited phospholipase A2  activity in a concentration-related manner when compared to the control. The extract had higher inhibition percentage when compared to prednisolone of same concentration. The 10% ethanol fraction (100 – 400 mg/kg b. w.) significantly inhibited nociceptive response in dose dependent manner. The highest analgesic activity (71.84%) was obtained with extract dose of 400 mg/kg b.w., while that of the standard drug diclofenac sodium (10 mg/kg b. w.) was 38.83%. The 10% ethanol fraction of the extract (0.1 mg/ml – 0.25 mg/ml) significantly (p < 0.05) inhibited hypotonicity-induced haemolysis of human red blood cell more than indomethacin a standard, anti-inflammatory drug. The n-hexane fraction (100, 200 and 400 mg/kg b. w.) significantly (p < 0.05) inhibited yeast-induced pyrexia in rats in both dose and time dependent manner when compared to that of the control. The results of the study show that 10% ethanol fraction of the extract inhibited significantly (p < 0.05) inflammatory activity induced by egg albumin as well as nociceptive response induced by acetic acid when compared with the control. From the results, it could be inferred that the n-hexane fraction of the extract inhibited yeast- induced  pyrexia. The results  suggest  that  the  inhibition  of phospholipase A2   is  one of the mechanisms of the anti-inflammatory effect of the plant. The investigation provides empirical evidence for the use of Vitex simplicifolia leaf extract in folkloric treatment of inflammatory, analgesic and pyretic disorders.

CHAPTER ONE

INTRODUCTION

Natural product is a source for bioactive compounds and has potential for developing some novel therapeutic agent (Deepa and Rebuke, 2014). Over the last decade there has been a growing interest in drugs of plant origin and such drugs formed an important class for disease control (Kumar et al., 2013). Herbs are staging a comeback and herbal ‘renaissance’ is happening all over the globe. The herbal products today symbolize safety in contrast to the synthetics that are regarded as unsafe to human and environment (Kumar et al., 2013). This is because unlike modern  drugs  which  are single active components  that  target  one specific pathway,  herbal medicines work in an orchestral manner, as the chemical compounds they contain act synergistically  on  targeted  elements  of  a  complex  cellular  pathway  (Kumar  et  al.,  2013). Nigeria’s biodiversity is rich in medicinal plants. The World Health Organization (WHO) reported that 70─90% of the world’s population relies chiefly on traditional medicine and a major part of the traditional therapies involve the use of plant extracts or their active constituents to treat diseases including inflammatory related ones.

Inflammation is the response of living tissue to injury. It involves a well-organized cascade of fluidic and cellular changes. It can be acute or chronic (Ferrero-Miliani et al., 2007). Chronic inflammation  is  associated  with  many  diseases  of  advanced  age  such  as  heart  attacks, Alzheimer’s disease and cancer (Coussens and Werb, 2002; Libby et al., 2002). It has both beneficial and detrimental effects locally and systemically. Fundamentally it is a response that protects the body with its ultimate goal as to get rid of noxious chemicals, but sometimes maybe potentially harmful and needs pharmacological treatment to control its symptoms (Kumar et al.,

2004). Inflammation is normally treated with the use of both steroidal and non-steroidal anti inflammatory drug. Long term uses of NSAIDs causes side effects including gastric ulceration and renal toxicity (Payne, 2000; Ezekwesili et al., 2011) since they concurrently inhibit both isoforms of cyclooxygenase (COX). The development of NSAIDs which are selective COX inhibitors still have side effects as reports have connected these drugs with an increased risk of heart attack and stroke (Salmon, 2006; Nelson and Cox, 2008). Studies on their safety profiles shows that non of them is completely safe (Rang et al., 2003). There is therefore, a need for potent anti-inflammatory drugs with fewer side effects. This has prompted the research into plants used in folk medicine to treat inflammation.

The plant Vitex simplicifolia has traditionally been used to treat malaria, stomach ache, as well as joint pains and fever among the locals of Umumba Ndiagu in Ezeagu Local Government Area of Enugu State. Young twigs are used as tooth picks in Nigeria. Vitex Simplicifolia belongs to the family labiatae, Verbenaceae. The plant is a small tree or shrub with dense, pale indumentum and mauve flowers. In Savanna, it grows to a height of approximately 8 m. There is little or no scientific reason for the use of the plant especially as an anti inflammatory agent. This work therefore, seeks to find the scientific basis for the use of the plant as an anti inflammatory agent.

1.3 Description  Vitex simplicifolia

Vitex Simplicifolia belongs to the family labiatae, Verbenaceae. The plant is a small tree or shrub with dense, pale indumentum and mauve flowers; in Savanna. It grows to a height of approximately 8 m and is commonly found in savanna (Salim and Dikko, 2016). Vitex simplicifolia Oliv is very aromatic. The vitex genus family consists of about 250 species of shrubs and trees it is widely cultivated in warm temperate and subtropical regions (Bodade et al.,

2012). The plants in the vitex family have different uses which include:

1.1.1 The plant (Vitex simplicifolia)

Source: Personal picture

1.1.2 Taxonomical classification of Vitex simplicifolia

Family: Labiatae, Verbenaceae Specie: Vitex simplicifolia Synonyms: Vitex madiensis Oliv. Sub specie. Madiensis

1.1.3 Uses of the Plant

Many researches have been embarked on the study to identify the possibility of using plant components to solve human health problems (Kanife et al., 2012). This include health related problem like cancers as well as environmental pollution for example using maize cob to remove heavy metals in environmental waste water

Vitex  trifolia  var.  simplicifolia  is  basically  a  sea  side  shrub  from  the  family lamiaceae  or verbenaceae. The vitex genus family consists of about 250 species of shrubs and trees it is widely cultivated in warm temperate and subtropical regions (Bodade et al., 2012). The plants in the vitex family have different uses which include their use as food as well as their ethnomedicinal uses

1.1.4.1 As Food

Vitex simplicifolia is used to enhance the aroma of food, the plant was used to prepare traditional dessert among Siamese communities in kelanthan called “khanom Bai kunthi”. The ingredients were rice flour, salt and extract of Vitex trifolia var simplicifolia leaf. Extracts from leaves of Vitex trifolia var simplicifolia will give colour, flavour and fragrance to the dessert (Philip et al.,

1991).  The  factor  that  determines  the  colour  is  the  plant  pigments  include  chlorophyll, xantophyll, carotene, flavone, flavonol and anthocyanin. Chlorophyll can be destroyed after certain temperature. However, as the chlorophyll is destroyed, the other pigments such as carotenoid and anthocyanin are expressed. Anthocyanins are oxidant flavonoids which improve human health condition. Besides, antioxidant supplementation can block NF-kB (nuclear factor kappa-light-drain enhancer of activated B cells) inhibits cancer, wound healing and antibacterial property

1.1.3.2 Ethnomedicinal use

Vitex simplicifolia Oliv is used as internal and external remedies to treat disease such as dermatitis, migrains fever, aches, amoebiasis, sore teeth, and infant tetanus (Salim and Dikko,

2016). Ethno-botanical investigations have revealed that the plant is also used in the treatment of skin infections and wound healing. The healing process is an immune response that begins after injury and takes place in three stages: vascular and inflammatory stage, the phase of tissue repair and phase of maturation. A drug having simultaneously the potential antioxidant and antimicrobial activities may be a good therapeutic agent to accelerate cicatrisation and wound healing (Philip et al., 1991; Heike et al., 1999).

Vitex simplicifolia Oliv is very aromatic. Aroma therapy is now considered to be another alternative way in healing people, and therapeutic values of aromatic plants lie in their volatile constituents such as monoterpenoids, sesquiterpenoids and phenolic compounds that produce a definite physiological action on the human body (Chopra et al., 1956). Vitex simplicifolia Oliv also has antitrypanasomial and antinflammatory activities (Houghton et al., 2005).

The bark of Vitex simplicifolia is used in medicines for treatment of dropsy, swellings, oedema, gout, oral treatments, pain-killers; skin, mucosae, fruit-pulp and young twig is used as food. In agri-horticulture it is used as shade-trees across Africa. Young twigs are used as tooth-sticks in Nigeria; bark decoction is used as a lotion in Ivory Coast on edemas, skin infections, and for toothache (Burkill, 2000).

1.1.4 Other Plants in the Vitex Family

Researches into the uses of plants belonging to the vitex family have also revealed that other members of the family have verifiable medicinal importance as documented by some research journal.  Examples include;

   Vitex rotundifolia

Vitex rotundifolia was historically used to surpress sexual desire in women and for similar reasons become a culinary spice in monastries hence the common name Monk’s pepper. Some of the  active  chemical  compounds  have  been  linked  to  female  hormone  balance,  female reproductive organs, menopause, actions on the pituitary glands, and treatment for acne. In Korea, it has been used for the rehabilitation and land scaping in sea board areas (Ono et al.,

2008).

   Vitex negundo

Vitex negundo Linn, commonly known as five-leave chaste tree or Monk’s pepper is used as medicine fairly throughout the greater part of India and found mostly at warmer zones and ascending to an alttitude of 1500 m in outer Western Himalayas (Ganpaty and Vidyadhar, 2005). The plant is a large aromatic shrub or sometimes a smaller slender tree with quadrangular densely whitish tomentose branchlets up to 4.5-5.5m in height. Bark thin, yellowish grey, leaves

3-5 forliolate; leaflets lanceolate; terminal leaflets 5-10×1.6-2.3cm, lateral one smaller, all nearly glabrous. Upper surface of the leaves are green and the lower surface are silvery in colour. Flower, bluish purple, roots are cylindrical (Ono et al., 2008).

1.2 Inflammation

Inflammation is generally described as the response of living tissue to injury. Inflammation is a complex body protective reaction to eliminate or limit the spread of an injury (Purnima et al.,

2010). It involves a well-organized cascade of fluidic and cellular changes elicited by numerous stimuli that include infectious agents, ischemia, antigen-antibody interaction and thermal or physical  injury (Burke  et  al.  2006;  Hunskaar  1987).  Inflammation  has  both  beneficial  and detrimental effects locally and systemically. Some form of inflammatory response is seen in virtually all living organisms, but the higher life forms have the unique ability to use the blood vascular system to transport and deposit fluid and cells in the extra-vascular space (Purnima et al., 2010).

The mechanism of action through which the body responds to inflammation is common to all not minding the triggering factor (Ferrero-Miliani et al., 2007). Inflammatory mechanism involves a cascade of biochemical events comprising of the local vascular system and the immune system (Da Silveira e Sá et al., 2013). It also involves the production of factors that could cause damage to tissues when not properly regulated. Thus, genes that play effector roles in inflammatory responses are actively repressed under normal conditions and are only induced when cells senses danger.

1.3 Signs and Characteristics of inflammation

The four principal effects of inflammation (rubor, tumor, calor and dolor) were described nearly

2,000 years ago by the Roman Aulus Cornelius Celsus, more commonly known as Celsus Silva. Later, another cardinal sign known as function laesa was added by Rudolf Virchow in 1858 to the list of features described in Celsus’ written work. The five cardinal signs which characterize inflammation include redness (rubor), swelling (tumor), heat (calor), pain (dolor) and loss of function (function laesa)

1.3.1 Redness (rubor)

An acutely inflamed tissue appears red, due to dilatation of small blood vessels within the damaged area (hyperemia) (Stankov, 2012).

1.3.2 Swelling (tumor)

Swelling results from edema, the accumulation of fluid in the extravascular space as part of the inflammatory fluid exudate, and to a much lesser extent, from the physical mass of the inflammatory cells migrating into the area (Stankov, 2012).

1.3.3 Heat (calor)

Increase in temperature is readily detected in the skin. It is due to increased blood flow (hyperemia) through the region, resulting in vascular dilation and the delivery of warm blood to the area (Stankov, 2012).

1.3.4 Pain (dolor)

Pain results partly from the stretching and distortion of tissues due to inflammatory edema and, in part from some of the chemical mediators of acute inflammation, especially bradykinin and some of the prostaglandins (Klos et al., 2009).

1.3.5 Loss of` Function (functio laesa)

Loss of function is a well known consequence of inflammation, it is manifested as inhibition of movement of an inflamed area by pain, either consciously or by reflexes, while severe swelling may   physically   immobilize   the   affected   area 

2018). Inflammation also has the following as its characteristics;

1.   The inflammatory process is redundant and complex. This makes it a challenging subject to study.  Many mediators of inflammation have the same functions and many mediators have multiple functions. Also, the same mediator may have different effects on different tissues.

2.   The process is continuous over a period of time. Peracute, acute, subacute, and chronic are terms used to describe different stages of inflammation.

3.   Inflammation  is  caused  by a  stimulus  and  removal  of the stimulus  should  result  in

abatement of inflammation. If it doesn’t get fixed in the acute period, it becomes chronic.

4.   Blood is the primary delivery system for inflammatory components.

5.   Inflammation is on a continuum with the healing process.

1.4 Causes of Inflammation

Inflammation is caused by a number of reactions triggered by the immune system in response to a physical injury or an infection. Among the many causes of inflammation are microbial infection, hypersensitive reaction, physical agents, irritant and corrosive chemicals,

1.5 Mediators of inflammation

Biochemical mediators released during inflammation intensify and propagate the inflammatory response. These mediators are soluble, diffusible molecules that can act locally and systemically. Inflammatory mediators are substances triggered by inflammatory stimuli. They are derived from inflammatory cells, or released as plasma proteins (Vishal et al., 2014). Mediators derived from plasma include complement and complement-derived peptides, kinins and C-reactive proteins (CRP). While cell-derived mediators include vasoactive amines (serotonine and histamine), interleukins, arachydonic acid derivatives (prostaglandins) and platelet activating factors (Vishal et al., 2014).

1.5.1 Plasma Derived Mediators

Plasma derived madiators are mostly proteins that circulate in the plasma in their inactive form (precursors). These inactive proteins undergo proteolytic cleavage to become active. Mediators found in plasma include; complement, clotting, fibrinolytic and kinin systems (Vishal et al.,

2014).

1.5.1.1 The Complement System

The complement system consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect. The complement system plays the role of generating biologically active products from various pathways of complement activation which include: classical, lectin, alternative, properdin and thrombin pathways (Neher et al., 2011). Complement activation (fixation) leads to lysis of cells. Once activated the complements engages in opsonization of pathogen, recruits other inflammatory cells and kills pathogens. The activation of one protein enzymatically cleaves and activates the next protein in the cascade. Complement can be activated via three different pathways as shown in the figure below, which can each cause the activation of C3, cleaving it into a large fragment, C3b, that acts as an opsonin, and a small fragment C3a (anaphylatoxin) that promotes inflammation. Activated C3 can trigger the lytic pathway, which can damage the plasma membranes of cells and some bacteria. C5a, produced by

this process, attracts macrophages and neutrophils and also activates mast cells (Neher et al.,

2011).

The complement system plays a critical role in inflammation and defence against some bacterial infections. Complement may also be activated during reactions against incompatible blood transfusions, and during the damaging immune responses that accompany autoimmune disease. Deficiencies of individual complement components or inhibitors of the system can lead to a variety of diseases

1.5.1.1.1 Classical Pathway

This pathway involves complement components C1, C2 and C4. The pathway is triggered by antibody-antigen complexes binding to C1, which itself has three subcomponents C1q, C1r and C1s. The pathway forms a C3 convertase, C4b2a, which splits C3 into two fragments; the large fragment, C3b, can covalently attach to the surface of microbial pathogens and opsonise them; the small fragment, C3a, activates mast cells, causing the release of vasoactive mediators such as histamine (Neher et al., 2011).

1.5.1.1.2 Alternative Pathway

This pathway involves various factors, B, D, H and I, which interact with each other, and with C3b,  to  form  a  C3  convertase,  C3bBb,  that  can  activate  more  C3,  hence  the  pathway  is sometimes called ‘the amplification loop’. Activation of the loop is promoted in the presence of bacterial  and  fungal  cell  walls,  but  is  inhibited  by  molecules  on  the  surface  of  normal mammalian cells (Neher et al., 2011).

1.5.1.1.3 Mannose-Binding Lectin Pathway

This pathway is activated by the binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface. This in turn activates the MBL-associated serine proteases, MASP-1 and MASP-2, which activate C4 and C2, to form the C3 convertase, C4b2a.

1.5.1.1.4 Lytic Pathway

This pathway is initiated by the splitting of C5, and attachment of C5b to a target. C6, C7, C8 and C9 unite with C5b, and this membrane-attack complex (MAC), when inserted into the outer membrane of some bacteria, can contribute to their death by lysis. Red cells which have antibody

bound to the cell surface can also activate the classical and lytic pathways, and become susceptible to lysis (Neher et al., 2011).

1.5.1.2 The Clotting System

Normal function and control of the blood-clotting system ensures the maintenance of circulation in higher animal. However, over activity of this system results in the formation of unwanted blood  clot  leading to  the blockage of  critical  blood  vessels.  Blood  coagulation  mechanism involves activation, aggregation and adhesion of platelets, along with the deposition and maturation of fibrin. The clotting cascade occurs through two separate pathways that interact with each other they include, the intrinsic and the extrinsic pathway (Neher et al., 2011).

1.5.1.2.1 Extrinsic Pathway

The extrinsic pathway is activated by external trauma that causes blood to escape from the vascular system.  This pathway is quicker than the intrinsic pathway.  It involves factor VII. Here a cell membrane protein called tissue factor (TF), present on the outside of all human cells with the exception of red blood cells and endothelium, binds with a plasma protein, Factor VII (FVII) converting FVII to the active FVIIa. The TF/FVIIa complex initiates the clotting cascade. TF/FVIIa complex reacts with plasma proenzymes factor IX and factor X converting them to active enzymes factor IXa and factor Xa. Small amounts of factor Xa react with prothrombin (FII)  in  the  presence  of  cofactor  factor  Va  (FV  is  released  from  platelets).  This  produces thrombin (FIIa). Thrombin catalyzes fibrin formation from fibrinogen.

When the body has made a small amount of fibrin, a substance known as Tissue Factor Pathway Inhibitor (TFPI) is released. This inhibitor binds to the TF:FVIIa/FXa complex, preventing further formation of factor FXa. It is thought that TFPI is released to protect against overreation of the coagulation system. At this point, the intrinsic pathway is activated (Neher et al., 2011).

1.5.1.2.2 Intrinsic Pathway

The small amount of factor IIa (thrombin) formed from the initial pathway activates the inactive pro-cofactor factor VIII to factor VIIIa. Factor VIIIa forms a complex with activated factor IXa. Factor VIIIa/factor IXa complex is responsible for the continuous formation of thrombin, which in turn cleaves fibrinogen into fibrin. This step is crucial for the formation of a durable secondary hemostatic plug. The fibrin form interract with aggregated platelets to form thrombus (Klos et al., 2009)

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