WOUND HEALING EFFECT OF METHANOL EXTRACT AND FRACTIONS OF NEWBOULDIA LAEVIS ROOT-BARK ON 5-FLUOROURACIL INDUCED LEUKOPENIA IN RATS

ABSTRACT

This study ascertained the wound healing potential of Newbouldia laevis root-bark in experimentally induced leukopenia in rats using excision wound model. 5-Flourouracil (12 mg/kg.b.w) was administered intraperitoneally for three (3) consecutive days after which a maintenance dose of (6 mg/kg.b.w) was administered every four (4) days. The methanol extract, ethyl acetate and methanol fractions of Newbouldia laevis root-bark (0.2 and 0.4 % w/w) were formulated into ointment and applied topically on the wounds of the rats once daily for 18 days. The percentage yields of the methanol extract, ethyl acetate and methanol fractions   were   5.95%,   0.87%   and   1.77%   respectively.   Quantitative   phytochemical constituents of the methanol extract revealed the presence of terpenoids (6.10 ± 0.14), flavonoids (5.07 ± 0.25), cardiac glycosides (4.71 ± 0.41), saponins (4.71 ± 0.32), alkaloids (4.03 ± 0.19), tannins (2.53 ± 0.45) and steroids (1.3 ± 0.11). Acute toxicity test showed that the methanol extract was not toxic up to 5000 mg/kg body weight. The induction with 5- fluorouracil  significantly  (p  <  0.05)  decreased  the  total  WBC  count  of  all  the  groups, compared to those of the normal control. During post wound healing days (3, 6, 9, 12, 15 and

18), there were non-significant (p > 0.05) decrease in wound area on day 3 compared to day 0 in all the treatment groups. On day 6, there was significant (p < 0.05) decrease in wound areas compared to day 0 in all the treatment groups, with corresponding increase in the percentage wound  contraction  and  the  untreated  group  2  showing  the  lowest  percentage  wound contraction (07.32%) and (group 9 0.4% w/w MFO methanol fraction ointment) with percentage wound contraction of (21.19%) and wound contraction significantly (p < 0.05) increase across the groups in all the days. Day 18 showed the highest percentage wound contraction with group 9 having (98.41%) compared to the untreated group 2 (51.59%) and the lowest wound healing area was also observed on the 18th day, hence percentage wound contraction was shown to be time dependent. Malondiadehyde concentration (MDA) was significantly (p < 0.05) higher in the untreated group, compared to the normal control. However, all the other treatment groups showed non-significant (p > 0.05) difference in their MDA  values.    SOD  activity  significantly  decreased  (p  <  0.05)  in  the  untreated  group compared to the normal control with the exception of group 4 that showed non-significant (p > 0.05) difference. CAT activity of the untreated group was significantly (p < 0.05) lower when compared to the normal control. There were non-significant (p > 0.05) difference in all the treatment groups compared to the normal control group. GSH and Vitamins C concentrations of the untreated group were significantly (p < 0.05) lower compared to the control and significantly (p < 0.05) lower in all the treatment groups when compared to the normal control of Vit. C values. Hydroxylproline showed significant (p < 0.05) increase on day 11 and 18 across the group compared to group 2 while hexosamine showed significant (p < 0.05) increase on day 11 and significant (p < 0.05) decrease on day 18 in all the group compared to group 2. These results demonstrated that the extract and fractions of Newbouldia laevis root-bark contain wound healing potentials as shown in the antioxidant activities, MDA results and in the stabilization of the connective tissue parameters with the methanol fraction showing higher efficacy.

CHAPTER ONE

INTRODUCTION

Plants  have long  been  the source of important  products  with  nutritional  and  therapeutic properties. Natural products, specifically those derived from plants, have been the major source of traditional medicine system all over the world. Human beings have been aware of medicinal plants possibly since 3,000 BC (Sofowara, 1982). More than 80% of the total population of the world depends on plant-based drugs in other to satisfy their primary health care need (WHO, 1993). Almost every indigenous culture uses medicinal plants in one way or the other for treatment of ailments throughout the world. This is due to the availability, low cost and minimal side effect of plants derived drugs. Plant cells fundamentally are chemical factories and many rich supply of therapeutically useful constituents (Rankin, 2009). It has been realized that a number of important modern pharmaceuticals have been derived from or are plants used by indigenous people (Balick and Cox, 1996). A number of modern drugs like aspirin, atropine, ephedrine, digoxin, morphine, quinine, reserpine and tubocurarine are examples of drugs, which were originally discovered through observations of traditional cure methods of indigenous  peoples (Gilani, 2005). Traditional medicine work in a way that depends on a typical methodology, however, the use of plants as medicine has being an ancient practice common to all societies especially the African society. These practices continue to exist in the developing nations. It is on this basis that researchers keep on working on medicinal plants in order to produce or develop the best medicines for physiological or therapeutic uses (Usman and Osuji, 2007). However, plants are used in traditional medicine for the treatment of various diseases or ailments including wound healing.

Wounds are bodily injuries that result in an opening or break of the skin. The skin forms a protective hitch against the environment. Wound healing is a process by which a damaged tissue is restored as closely as possible to its ordinary state, it is a very complex, multifactor sequence of events involving several cellular and biochemical processes in order to regenerate and reconstruct the disrupted anatomical continuity and functional status of the skin (Mahato and Pal, 1986). Wound healing involves continual cell-cell and cell-matrix interactions that are divided into four phases: (1) Haemostasis phase which occurs immediately after injury, (2) Inflammatory phase: (0-3 days), (3) Proliferative phase: (3-12 days) and (4) Maturation and Remodeling phase (3-6months). These four phases overlap each other (Gantwerker and Hom, 2011, Sun et al., 2014; Kurahashi and Fujii, 2015). The inflammation stage which begins   with   haemostasis   occurs   immediately   after   injury.   It   is   characterized   with

vasoconstriction that enables homeostasis and release of inflammation mediators. This phase also involves coagulation, which controls excessive blood loss from the damaged vessels. The proliferative phase is characterized by granulation tissue proliferation formed mainly by fibroblast and the angiogenesis process. The remodelling stage is also characterized by reformulations and improvements in the mechanisms of collagen fibre that leads to increase in tensile strength (Bodeker and Hughes, 1996). Wound contraction is the process of mobilizing healthy  skin  surrounding  the  wound  to  cover  the  denude  area.  It  is  determined  by  the reparative abilities of the tissue, type and extent of damage and overall state of the health of the tissue. The granulation tissue of the wound is mainly composed of fibroblast, collagen, edema, and small new blood vessel (Nayak et al., 2007).

5-Flourouracil is a chemotherapeutic drug. It is a cytostatic anti-tumour drug that possesses immunosuppressive  activities   (Wang   et   al.,   2002).   When   administered   alone  or  as combination therapy with surgery and radiation, it may have detrimental effects on the rapidly dividing tissues of healing wounds (Payne et al., 2008) thereby causing delay in wound healing. Delayed healing processes are observed in animals under certain conditions, such as advanced age, diabetes and immunosuppression (Kurahashi and Fujii, 2015). Wound leads to oxidative stress (Hamidi and Ghourchian, 2015). Nowadays, emphasis on healthy living based on antioxidant ingestion and the implication of oxidative stress molecules/free radicals on certain diseased conditions including delayed wound healing (Rasik and Shukla, 2000), has led to the screening of plants with high anti-oxidative properties and potent wound healing drugs with fewer side effects for the management of wound healing in cancer patients. The healing process can be physically observed by assessing the rate of wound contraction. An example of such plant, which is a rich source of active phytoconstituents that can provide medicinal  benefits  against  several  aliments  or  diseases  including  wound  healing,  is Newbouldia laevis.

Newbouldia laevis is a plant commonly found in tropical Africa (Kaey, 1989). It grows from Guinea Savannahs to dense forests (Arbonnier, 2004). It is a boundary tree.  Some parts of Nigeria commonly regard this tree as the “tree of fertility” or the “tree of life”.  Scientifically, Newbouldia laevis has been reported to have medicinal value which includes anti- inflammatory; the flowers are known for their anti-inflammatory activities (Tanko et al.,

2008), wound healing properties, antioxidant, anti-microbial, anti-fungal, and analgestic (Aladesanmi et al., 1998; Chukwujeku et al., 2005; Kuete et al., 2007; Akerele et al., 2011) activities. A number of naphthoquinones

present in the root of Newbouldia laevis have been reported to have wound healing potentials, anti-bacterial and anti-fungal activities. The biochemical and physiological importance of naphthoquinones are the K vitamins. Vitamin K, aids blood coagulation and is necessary for the formation of prothrombin; deficiency of this vitamin in the body, causes haemorrhage and prolongs blood clotting thereby leading to poor wound healing (Dorsett‐Martin, 2004). Many herbal plants play very important roles in wound healing, plants have the tendency of being more potent healers of wound such that they promote the repair mechanisms in the natural way (Nithya and Baskar, 2011). Research in this regard, is ensuring the use of medicinal plants around us in an attempt to find new compounds of therapeutic interest (Akinpelu et al.,

2009).

1.1 Description of Newbouldia laevis

Newbouldia laevis is an angiosperm, a member of the Bignoniaceae family (Kaey, 1989). Bignoniaceae is  a  family  of flowering  plants which  belongs  to  the order Lamiales  and is commonly known as the bignonias (Heywood et al., 2007). The largest tribe in the family is called Bignonieae, which are mostly trees or lianas, sometimes shrubs and subshrubs or herbs. The   family   has   a   nearly cosmopolitan   distribution,   but   is   habitually tropical,   with uncommon species native to  the temperate  zones  (Eberhard  et  al.,  2004).  According  to different researchers, the number of species in the family is about 810 (David, 2008) or about

860 (Eberhard el al., 2004). The last monograph of the entire family was published in 2004 (Eberhard el al., 2004). In that work, 104 genera were discovered, since that time, molecular phylogenetic studies have greatly clarified relationships within the family and the number of accepted genera is now between 80 and 85 (De Lucia et al., 2006).

Newbouldia   laevis   is   a   fast-growing   evergreen   shrub   or   small   tree   with height   of approximately 7–8 m high in the West Africa and up to 20m in Nigeria. The bole can be up to

90 cm in diameter, but is usually less (Burkill, 1985). It is erected vertically with ascending branches.  The plant has  shiny dark green leaves and bears large showy terminal purple flowers. It is easily recognized by its short branches, coarsely toothed leaflets, purple and white flowers (Ejele et al., 2012). It is usually cultivated as an ornamental tree where it is valued for its flowers and is easily propagated by cutting. It is a familiar live-fence and boundary tree throughout its distribution. It is a very popular plant in the African continent and is highly valuable due to its numerous immense benefits to human race. The wood is pale brown, durable, equally textured and hard. It tends to remain alive for a long time even after

cutting it. This makes it viable for usage as posts, woodworks, yam stakes, house posts and bridges. It has different symbols and meanings to different villages and countries for example: The Igbo part of Nigeria refers to Newbouldia laevis (ogilisi) tree as a sacred or symbolic tree, usually planted in small groves in front of a chief’s house.

1.2 Common Names of Newbouldia laevis

Newbouldia laevis has different names by which it is known by different African Counties, for example, in Ghana sesemasa, Togo lifui, Senegal gimgid, Mali kinkin, Guinea canhom, Gambia killihi, Sierra Leone sherbro. In Nigeria, it is commonly known as, ogirisi or ogilisi in Igbo, akoko in Yoruba and aduruku in Hausa (Hutchinson and Dalziel, 1963), in Tiv kontor, Ibibio Itomo while in Edo ikhimi.

Figure 1: Diagrammatic representation of Newbouldia laevis plant

Figure 2: Dried Newbouldia laevis root-bark

1.3 Taxonomic Classification of Newbouldia laevis

Kingdom:                              Plantae

Sub kingdom:                       Viridaeplantae Phylum:                                 Tracheophyta Class:                                     Magnoliopsida Order:                                    Lamiales Family:                                  Bignoniaceae Genus:                                   Newbouldia

Species:                                  Newbouldia laevis

Source:  (Gbile and Adesina, 1986; Anaduaka et al., 2014)

1.4 Geographical Distribution, Climate and Soil type of Newbouldia laevis

Newbouldia laevis is a native to tropical Africa and grows from Guinea Savannahs to dense forests (Arbonnier, 2004) and west tropical Africa such as Senegal to Cameroon and Gabon. It is a sun-loving plant, which grows best in well-drained fertile soil (Barwick, 2004). N. laevis is fast- growing and drought-tolerant specie. The species blooms during the warmer months of the year and has an upright growth habit and not taking up much room, but also not providing much shade. It is also found in regenerating and dry forests (Huxley, 1992). One thing remarkable about this plant is that it hardly dies hence it is used to indicate boundary marks among the Igbo people of South Eastern Nigeria (Ejele et al., 2012), and also to mark graves.

1.5 Health Benefits of Newbouldia laevis

Newbouldia  laevis  plants  are  used  as  traditional  remedies  in  the  treatment  of  different ailments among many tribes in West Africa (Akinpelu et al., 2009). A decoction of the leave is taken by women to treat fertility problems. Traditional healers in Nigeria use it to facilitate labour. The decoction of the root is laxative and it is used to treat constipation.  Various parts of Newbouldia laevis are used for pain and several inflammatory conditions. Among the Igbo’s (South Eastern Nigeria) and parts of the Midwestern Nigeria, this plant is used for the treatment of septic wounds and eye problems (Akerele et al., 2011). The leaves, stem and flower have been used for wound dressing; febrifuge and for the treatment of stomach ache

(Iwu, 2000).  The leaves  are  used  among  the  Igbos  also  for the  treatment  of dysentery, conjunctivitis, ear ache, cough and hernia. The plant is used to stop vaginal bleeding in threatened abortion (Ejele et al., 2012). It is used in traditional medicine of Togo for the treatment of sickle cell disease, (Joppa et al., 2008). Specifically, the stem bark mixed with clay and red pepper has been reported to be effective against pneumonia, fever, cold, cough and different illness like bone lesions (Yapici et al, 2009). It can also be used for the treatment of diseases such as malaria, tooth aches, sexually transmitted diseases (STDs) and diarrhoea (Eyong et al., 2005). The root alone is used as round worm vermifuge (Kuete et al., 2007). The leaves and roots mixed together and boiled are used to treat fever and convulsion. The stem bark is used in the treatment of impotence, infertility and various skin infections. The roots and leaves are also used for the treatment of elephantiasis, migraines and epileptic seizure (Odunbaku and Amusa, 2012). The bark and twigs are used to treat haemorrhoids, women pelvic pain, peptic ulcer disease, earache, skin ulcer, constipation and epilepsy (Akerele et al., 2011).

1.6 Pharmacological Effects of Newbouldia laevis

Ethnobotanicals are important for pharmacological research and drug development, not only when plant constituents are used directly as therapeutic mediators, but also as preliminary materials for the synthesis of drugs or as prototypes for pharmacologically active compounds (Li  and  Vederas,  2009).  Studies  over  the  years  have  proven  the  value  of  extracts  of Newbouldia laevis in herbal medicine; some of these studies are as follows:

1.6.1 Analgesic and Anti-inflammatory Effect of Newbouldia laevis

Tanko et al. (2008) investigation has shown the analgesic and anti-inflammatory effect of N. laevis flower in rodents. The study carried out by Ainooson et al. (2009) on the effect of a hydro-alcoholic  extract  of  N.  leavis  stem  bark  in  formalin-induced  pain,  a  model  of neuropathic pain, revealed that N. laevis extract contains central and peripheral analgesic properties. The ethanol flowers were shown to exert anti-nociceptive and anti-inflammatory effects in laboratory animals at different doses Tanko et al. (2008). Olajide et al. (1997) reported that the methanol stem bark extract of this plant also exhibited anti-inflammatory activity on oedema induced in rats. The work of Ahmadiani, et al. (1998); Ahmadiani et al. (2000) were supported by other workers who found that flavonoids and tannins which are present in N. laevis were found to have antinociceptive and anti-inflammatory activities

1.6.2 Anti-oxidant Effect of Newbouldia laevis

Phytochemicals are naturally occurring compounds and are believed to be active in contesting and preventing diseases due to their antioxidant effect (Halliwell et al., 1992; Ejele et al.,

2012). Researchers in recent scientific advancements have developed interest in plants as a result of their medicinal properties, due to their potent antioxidant properties and profitable viability (Geethalakshmi et al., 2013 and Koneru et al. (2011). Anaduaka, et al. (2014) observed that the ethanol extracts of N. laevis, stem and leaves possessed anti-oxidant effect produced by the increased activities of superoxide dismutase, catalase and glutathione levels in diabetic rats. The ethanol leaf extract of N. laevis is a potential source of antioxidants and free radical scavengers as a result of important metabolites which may be linked to its ethno- medicinal use as studied by Habu and Ibeh (2015).

1.6.3 Anti- bacterial and Anti-microbial Effect of Newbouldia laevis

The study of Kuete et al. (2007) reported the antimicrobial activity of methanol extract of N. laevis root bark on some bacterial isolates, while the leaf extract also exhibited antimicrobial activity  Usman  and  Osuji  (2007).  Antimicrobial  activities  of  N.  laevis  have  also  being reported by Ejele et al. (2012). Akerele et al. (2011) studied the phytochemical and antibacterial evaluations of the stem bark of N. laevis against isolates from infected wounds and eyes.

1.6.4 Cardioprotective Effect of Newbouldia laevis

Agbafor et al. (2015) findings on cardioprotective effect of leaf and root extracts of N. laevis against carbon tetrachloride induced-cardiotoxicity in albino rats, shows an indication of possible cardioprotective potential of N. laevis root and leaf extracts which may be partly responsible for their efficacy against cardiovascular diseases.

1.6.5 Hepatoprotective Activity of Newbouldia laevis

The study of Hassan et al. (2015) shows the hepatoprotective activity of N. laevis stem bark. Investigation conducted on effects of leaf and root extracts of N. laevis on hepatic and renal systems in Albino Rat, which shows that doses of 200 and 400 mg/kg deionized water extracts of N. laevis leaf extract may be hepatoprotective Agbafor and Ezeali (2015).

1.6.6 Antihyperglycemic Activity of Newbouldia laevis

Osigwe et al., (2015) researched on the antihyperglycemic Studies of the leaf extract and active fractions of N. laevis, findings shows that the leaf extract and fractions of N. laevis possesses antihyperglycemic activities and can be the base for the traditional use of N. laevis in management of diabetes mellitus.

1.6.7 Anti-malaria Activity of Newbouldia laevis

Currently,  leaf  and  root  extracts  of N.  laevis have  been  shown  to  possess  Anti-malaria. Gbeassor et al. (2006) and Eyong et al. (2006). The phytochemical screening and in vitro antiplasmodial  activity  of  the  ethanol  extracts  as  well  as  the  fractions  from  N.  laevis buttressed the use of this plant for the treatment of malaria associated symptoms in traditional setting Andrew et al. (2016).

1.6.8 Wound Healing Activity of Newbouldia laevis

The  investigation  of  Omokpo  et  al.  (2012)  on  the  comparative  study  on  the  effects  of methanol extract of N. leavis stem bark and honey on ulcerated soft tissue injury of wistar rat, suggested that  N. laevis extract can be recommended for the treatment of soft tissue injuries. Only few work have being done to show the efficacy of N. leavis, leaves, stem and roots on wound healing, hence this research.

1.7.0 Phytochemicals

Phytochemicals  are  chemical  compounds  formed  during  the  plants  normal  metabolic processes, these chemicals are often referred to as “secondary metabolities” of which there are several classes including alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1973; Okwu, 2001). These can act as agents to prevent unwanted side effects of the main active substances or to assist in the assimilation of the main substances. Phytochemicals are present in a variety of plants utilized as important components of both human and animal diets. These include fruits, seeds, herbs and vegetables. Herbs and spices are accessible sources for obtaining natural antioxidants (Okwu, and Josiah, 2006).

In contrast to synthetic pharmaceuticals based upon single chemicals, many medicinal and aromatic plants exert their beneficial effects through the additive or synergistic action of several chemical compounds acting at single or multiple target sites associated with a physiological  process.  These  synergistic  pharmacological  effects  can  be  beneficial  by

eliminating the difficult side effects associated with the predominance of a single xenobiotic compound in the body (Tyler et al, 1999). The synergistic interactions that underlie the effectiveness of a number of Phytomedicines have been extensively documented (Kaufman,

1999). Most of these phytochemical constituents are potent bioactive compounds found in medicinal plant parts which are precursors for the synthesis of useful drugs (Soforawa, 1993).

1.7.1 Classification of Phytochemical Constituents

Phytochemical constituents are classified as primary or secondary constituents, depending on their role in plant metabolism. Primary constituents include the common sugars, amino acids, proteins, purines and pyrimidines of nucleic acids, chlorophylls and so on. Secondary constituents are the remaining plant chemicals such as alkaloids, terpenes, flavonoids, lignans, plant steroids, curcumines, saponins, phenolics, flavonoids and glucosides (Hahn, 1998).

1.7.1.1 Flavonoids

Flavonoids consist of a large group of polyphenolic compounds having a  benzo-��-pyrone structure and are ubiquitously present in plants. They are synthesized  by phenyl-propanoid pathway (Shashank and Abhay, 2013). Flavonoids constitute one of  the most ubiquitous groups of plant phenolics. They have been reported to exert  multiple  biological property including antimicrobial, cytotoxicity, anti-inflammatory as  well as anti-tumor activities but the best-described property of almost every group of  flavonoids is their capacity to act as potent antioxidants which can protect the human body from free radicals and reactive oxygen species (Mamta et al., 2013). The position of  hydroxyl groups and other features in the chemical  structure  of  flavonoids  are   important   for  their  antioxidant  and  free  radical scavenging activities. Flavonoids  constitute a wide range of substances that play important role in protecting biological  systems against the harmful effects of oxidative processes on macromolecules, such as carbohydrates, proteins, lipids and DNA (Atmani et al., 2009).

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