ABSTRACT
Conventional chemotherapy has always taken a prominent place in dealing with diseased states; but owing to their various side-effects, natural products that play similar therapeutic roles have become the thrust of much research work. Hence, this study sought to investigate the possible reasons for the use of honey in combating the human male reproductive challenges. It also investigated the probable cause of colicky pains following ingestion of honey. Phytochemistry showed that the honey had high concentrations of simple reducing sugars, saponins, terpenoids and potassium. Flavonoids, glycosides, resins, proteins, steroids, calcium and vitamin C were in moderate concentrations; alkaloids, acidic compounds and magnesium were in low concentrations while tannins were not detected. A total of twelve (12) male rabbits of between nine and eleven months old with average body weight (b.wt.) of 1.0 ± 0.02 kg and mice (12) of about nine months old and average body weight of 27.1 ± 0.16 g were used. The rabbits were divided into four groups of three each. Group 1 served as control, group 2 rabbits were treated with 2.5 ml/kg b.wt of honey, group 3 rabbits were treated with 5.0ml/kg b.wt of honey, and group 4 rabbits with
7.5 ml/kg b.wt of honey. Treatment lasted for 14 days. The control group received only normal saline and normal rat feed. The activities of smooth muscles of prostate and jejunum of rabbits fed raw honey was compared with those of prostate and jejunum smooth muscles of rabbits that were not given honey. Administration of honey brought about enhanced muscle activity in the jejunum smooth muscle of the experimental rabbits compared with the control. On the administration of acetylcholine (2 µg and 4 µg, w/v) to the muscle segment in student organ bath, the activity was amplified significantly (p<0.05) in group 2 compared with that of the control group. The mid and high doses of honey, 5.0 and 7.5 ml/kg b.wt produced increased cholinergic-elicited contractions compared with the contraction in the control muscle but the low dose, 2.5 ml/kg b.wt produced more appreciable (p<0.05) modified smooth muscle activity. With a threshold dose of adrenaline (20 µg, w/v) in the organ bath, the low honey dose of 2.5 ml/kg b.wt elicited significant (p<0.05) reproducible increase in the adrenergic-induced smooth muscle contraction of the prostate muscle compared with the control. Compared with the control group, the modified smooth muscle contraction also increased significantly (p<0.05) with the mid and high doses, 5.0 and 7.5 ml/kg b.wt in groups 3 and 4 respectively. The serum concentrations of some electrolytes implicated in smooth muscle activity were determined. There was a non-significant (p>0.05) reduction in serum concentrations of zinc and magnesium ions in the test groups relative to the control group. Ca2+ concentration in the serum increased non-significantly (p>0.05) in the test groups when compared with the control group. Serum [K+] increased significantly (p<0.05) in the test group 1 relative to the control group. The variations in the other test groups compared with the control group were not significant (p>0.05). The superoxide dismutase, prostatic specific antigen, prostatic acid phosphatase and high-density lipoprotein cholesterol were non-significantly (p>0.05) reduced in groups 2, 3 and 4 compared with group 1. There were non-significant variations in the levels of low- density lipoprotein cholesterol, and triacylglycerol in groups 2, 3 and 4 compared with group 1. The findings show that consumption of raw honey in relatively small quantities can modify the behaviour of the smooth muscles, leading to enhanced contractile responses. At low doses, the honey brought about stimulatory effect on the smooth muscles of the jejunum and the prostate of the rabbit. At higher doses, however the honey had inhibitory effect on the smooth muscle. The results show that honey facilitates luminal flow of the prostate fluid. The activation and amplification of the auto-rhythmic activity of jejunum may safely be responsible for the frequent and painful gut movements following honey intake.
CHAPTER ONE
INTRODUCTION
Internal organs such as intestines, urinary bladder, stomach, blood vessels and uterus have smooth muscles. These muscles are described as “smooth” because they are not striated as the skeletal and cardiac muscles. Smooth muscles are innervated by autonomic nervous system (Elenkov et al., 2000; Webb, 2003). The muscles develop different types of contractile responses namely, phasic and tonic, depending on the prevailing changes in load or length. The very complex prostate gland contains secretory elements and tubualveolar glands that are lined with smooth muscles. While the glandular elements help in secreting most constituents of the seminal fluids, the muscular elements help in ejaculation and micturition (White et al., 2013). Smooth muscles maintain a consistent continuum from the mid gut to the anal portion of the alimentary canal (Sanders, 2008). Smooth muscles have a unique capacity to contract rhythmically for a great stretch of time. The length of the small intestines is about
6.9m. It has three portions: duodenum, jejunum and ileum. The entire length of the small intestine undergoes spontaneous motility in a wave-like pattern under the control of pacemaker cells. The GIT is replete with nerve fibres (Prins, 2011). Vagal and spinal primary afferent nerves innervate the GIT viscera. The GIT possesses intrinsic enteric nervous system which contains intrinsic primary neurons. The neurons are specifically concerned with the gut motility and peristalsis (Akbar et al.,
2009). The afferent nerves are sensitive to chemical, mechanical and thermal stimuli. Contraction of the smooth muscles and secretions are, however, regulated by the efferent nerve fibres. The fibres also control inflammatory and immune processes in the intestine (Gershon, 2005; Furness, 2007). Since these smooth muscles are richly innervated and the nerves are sensitive to different stimuli, it follows that certain food substances that man ingests can elicit various muscular responses. The responses could have scores of implications for the health of the organism concerned.
The ability to contract rhythmically makes it possible for the gastrointestinal tract (GIT) organs to do various jobs. These jobs include the movement, storing and mixing of the contents of the lumen with secretions of the GIT. The mixing facilitates proper breakdown of food. Peristalsis is a mechanism mediated in the gut by the smooth muscles. This mechanism moves food substances in their different stages of digestion through the gut for the optimal absorption of various small food molecules
across portions of the GIT. The removal of undigested food mass by the GIT is still a function of the contractile activities of the smooth muscles in the walls of the tract (Walker et al., 2008). Other roles include the release of urine by the urinary system, regulation of anal opening, regulation of air movement through the lungs, foetal expulsion, ejaculation of semen, regulation of blood flow in blood vessels, release of bile by the gall bladder and churning of food by the stomach walls among many other functions. Each of these roles is critical to the life of man and other organisms of similar musculature. Anything that can preserve the natural physiology of smooth muscles should be sought.
Honey is a viscous brown natural liquid produced by bees from nectars of flowers. There are many evidence-based health benefits ascribed to honey in recent years. This liquid is often consumed as food by man (Erejuwa et al., 2012). Some people take it alone while some others take it in a mixture with pap, tea or beverage drink. Honey is also taken as medicine. This application could arise from its phytochemical composition. The therapeutic applications of honey benefit so much from the phenolic compounds, minerals and vitamins found in it. The use of honey is becoming increasingly popular for human health promotion. Some of the phenolic compounds in honey include: galangin, acacetin, quercetin, phenylethyl ester, kaempferol and caffeic acid. These phenolics have promising uses in the treatment of cardiovascular diseases (Khalil and Sulaiman, 2010). Ascorbic acid, riboflavin, niacin and panthothenic acid are some of the vitamins usually found in honey. Minerals such as zinc, calcium, copper, phosphorus, potassium, iron, manganese and magnesium are also found in honey (Bogdanov et al., 2008; Ajibola et al., 2012).
The purpose of this study is to determine the effect of raw honey on the smooth muscles of rabbits. This was informed by the folkloric emetic effect of honey after ingestion, disturbing facilitated intestinal and bowel movement as well as the use of raw honey as base medium for traditional therapy for male sterility.
1.2 Muscles
Muscle is a tissue that has the ability to contract. It develops from the mesoderm of animal germ cells (Krause, 2005). There are three types of muscles namely: skeletal, cardiac and smooth. Each of these muscles has characteristic structure, contractile
features, and regulatory mechanisms. Muscles are generally known for force generation which usually brings about motion (Booth and Wyman, 2008). Muscles bring about the movement of body parts or the entire body of organisms. The muscles are mainly powered by fat and carbohydrate oxidation (Nelson and Cox, 2000). These metabolic processes generate adenosine triphosphate (ATP) molecules for powering the contractile apparatus. There are over six hundred muscles in a human being each of which is served by nerves. The nerves connect each of the muscles to the brain and the spinal cord for co-ordination (Lateva et al., 2002). Human physiological needs demand that muscles accomplish different functions and as such, the body has been endowed by nature with three types of muscles.
1.2 Smooth Muscles
Smooth muscle does not have striations peculiar to the cardiac and skeletal muscles; hence, the smooth muscle is described as smooth (Sheerwood et al., 2012). The contractile function of smooth muscle is under involuntary control. Sheets of smooth muscle cells are found in the walls of several organs and tubes in the body such as the blood vessels, stomach, intestines, bladder, respiratory tract, uterus, penile and clitoral cavernosal sinuses (Booth and Wyman, 2008). Smooth muscle cells are of various lengths in different organs, from 20 µm in small blood vessels to 500 µm and 600 µm in the pregnant uterus. The shape of each smooth muscle cell is like a spindle, with a centrally positioned nucleus (Krause, 2005). These muscles in the blood vessels regulate blood flow through and to vital organs. Smooth muscle in the digestive and urinary systems, forms rings called sphincters which regulate the movement of materials along internal passageways (Saladin, 2003). When smooth muscles occur in bundles, layers, or sheets, they play many important roles in the body. Those found around blood vessels regulate the flow of blood to the superficial dermis of the skin while smooth muscles of the arrector pili elevate hairs (Standring, 2008; Burgdorf et al., 2009; Wang et al., 2010; Morioka et al., 2011). In the heart circulatory system, the smooth muscle encircling the blood vessels helps in regulating blood distribution and pressure of blood flow. The diameters of the airways and the resistance to airflow are modified by the contraction and relaxation of the smooth muscle. Smooth muscle extends throughout the layers of the digestive tract for peristaltic movement of materials in the lumen. The ejection of bile into the GIT from the gall bladder is carried out by the smooth muscle wall. The rate of blood filtration in the kidneys is
altered by the activities of the smooth muscle tissue in the walls of the blood vessels. The smooth muscle in the walls of the ureters transports urine to the urinary bladder and the contractile function of the smooth muscle in the bladder forces urine out of the body via the urethra (Saladin, 2003; Fox, 2010). In the males, smooth muscle layers in the reproductive system help to move sperm along the reproductive tubules. The muscle also causes release of secretions from accessory glands into the reproductive system. Layers of smooth muscle facilitate the movement of female gametes along the reproductive tract in females. The contractile function of the
uterine smooth muscle expels foetus from the uterus at child delivery (Fox, 2010).
The smooth muscle bundles at hair follicles contract to make the hairs erect for homeostasis. The muscle bundles in the eye ball contract and relax to enable the lens make adjustments for accommodations (David, 2001). The contractile state of smooth muscle is under the control of autocrine and paracrine agents, hormones and other local chemical signals (Webb, 2003). Mechanically, smooth muscle can be described as phasic, or fast contracting and tonic, that is, slow contracting. Phasic smooth muscle has the unique features of relative rapid rates of activation and relaxation forces. It also has a great actomyosin ATPase activity. The tonic smooth muscle, however, has relatively slow rates of activation and relaxation forces. Its actomyosin ATPase activity is slow (Rhee and Brozovich, 2000). Changes in load and length cause smooth muscle cells to respond with phasic and tonic contractions. Cross-bridge cycling between actin and myosin generates force following the initiation of
contraction by calcium ions (Ca2+) (Spudich, 2001; Andersson and Arner, 2004). To
sustain force generation, Rho A/Rho kinase pathway inhibits dephosphorylation of light chain by myosin phosphatase following a Ca2+ sensitization of contractile proteins. The muscle relaxes when Ca2+ is withdrawn from the cytosol and when the myosin phosphatase activity is stimulated (Chitaley et al., 2001).
Smooth muscle can occur as single unit or multi-unit in various organs. Rhythmic contractions are myogenic in single muscles, this means that they can contract regularly without input from a motor neuron. They are neurogenic in the multi-unit smooth muscles. The contraction of the multi-unit smooth muscle must be initiated by a motor neuron. While single unit smooth muscle contracts in response to rapid stretch, multi-unit smooth muscle does not contract as such (Sheerwood, 2010). Single unit smooth muscle is characterized by possession of many gap junctions which enable the entire muscle to contract at once. The multi-unit smooth muscles have fewer gap junctions. The multi-unit smooth muscle cells are so richly innervated that individual cell can contract independently (Fox, 2010). Pacemaker cells in the single smooth muscles bring about spontaneous rhythmic contractions. Nerve activity and hormones can affect the contractile activity of the single unit smooth muscles. Single unit smooth muscles are found in certain parts of the body, namely: intestinal tract, bladder, uterus and blood vessels. The multi-unit smooth muscles on the other hand, are found in lungs, erectile tissues of hair follicle, and arteries. Multi-unit smooth muscles have motor units which determine the response of the smooth muscles. Other factors that influence their response are frequency of discharge in the fibres and the relative amount of excitatory and inhibitory input (Sheerwood, 2012).
1.3 Innervations of Some Smooth Muscles
The innervations of the smooth muscles are from the autonomic nervous system (ANS). The nerves originate from diverse sources. The trachea and the GIT for example, have plexuses of intrinsic nerves which are tantamount to independent nervous system that control the smooth muscle activities in these organs. There are sensory and motor neurons as well as intermediate neurons in these plexuses. The intrinsic innervations are vital for human life. This is because should there be a damage or surgery on the central nervous system (CNS); the activities of the smooth muscle in these areas are not hampered. Smooth muscles generally, however, receive some inputs from the CNS in a way described as ‘extrinsic innervations’ (Philips and Powley, 2007). These inputs for most smooth muscles could be opposing from the sympathetic and parasympathetic arms of the ANS (Fox, 2010). The intrinsic nervous system does the major work in systems where it occurs along with the extrinsic nervous system. There is no specialized connection between the nerve fibre and the muscle cell (Alpers et al., 2011).
The smooth muscle cells and nerve fibres are very closely situated. The nerve fibres release neurotransmitters such as acetylcholine from the varicosities in the fibres. The neurotransmitter covers a long distance but it can bind to any nearby smooth muscles (Burnstock, 2007). In the digestive system, sympathetic and parasympathetic nerve fibre divisions innervate the system. The parasympathetic control takes pre-eminence (Alpers et al., 2011). Smooth muscle contractions and relaxations bring about movements in the GIT. The GIT has an outer longitudinal smooth muscle layer, an inner circular smooth muscle layer, and submucosal smooth muscle layer. Each of these layers is innervated with circular and longitudinal fibres. These fibres cause the movement of the villi of the mucosa. Mucosal epithelium lines the inner surface of the tract. The outer layer consists of serosa that is a continuum with the mesentery. The mesentery contains blood vessels, lymphatic vessels and nerve fibres. In the brain stem is located the CNS centres that regulate the digestive functions. The sensory taste fibres from olfactory, tactile and gustatory receptors terminate on the cell bodies of the vagal motor and salivary nuclei. The state of the gut and its luminal content are communicated to the central autonomic system via the numerous afferent and sensory vagal nerve fibres. The parasympathetic outflow and motor centres of the brain stem are both influenced by the higher cortical and olfactory centres. While the sympathetic system has a resultant inhibitory effect on digestive activities, secretion and motility, the parasympathetic system has stimulatory effect (Johnson, 2003). The effect of the sympathetic system is a secondary function of vasoconstriction which deceases blood flow in the digestive tract. The GIT, especially the transverse colon, is innervated by the vagus nerve. The nerves include the efferent and afferent fibres. The extremities of the GIT possess parasympathetic nerves from the pelvic plexus. The stimulation of local neurons of the intrinsic, enteric nervous system in the wall of the gut results in increased digestive activities. This stimulation is done by the parasympathetic fibres (Prins, 2011). The two sets of nerve plexi in the intrinsic, enteric nervous system namely: submucosal Meisssner and myenteric Auerbach plexi affect the gut differently. While submucosal Meissner plexus regulates the digestive glands, myenteric Auerbach plexus regulates gut motility. Motor neurons in the myenteric plexus makes acetylcholine as well as substance P available (Brodal, 2010). The acetylcholine when bound to muscarinic receptors causes smooth muscles cells to contract (Thorp, 2008). The relaxation of these smooth muscle cells is brought about by vasoactive intestinal peptide (VIP) and nitric oxide (NO) released by inhibitory
motor neurons. Food causes some tension in the gut wall to which stretch receptors respond. These stretch receptors have connections with sensory neurons. Mucosal chemoreceptors also have connections with the sensory neurons. The chemoreceptors detect the presence of various chemical substances in the lumen of the gut. Short effector neurons promote digestive gland secretions and also induce the contraction of smooth muscles. The enormous neuronal connections make up the intrinsic, enteric nervous system that stems the brain influence on digestive functions. The systems of neurons and their supporting cells in the walls of the GIT, along with pancreas and gall bladder constitute enteric nervous system (ENS). This ENS is often described as the little brain (Webb, 2003). Gastrointestinal tract functions such as motility, secretions and immune system and blood flow are regulated by the ENS. The CNS plays its role on the GIT by sending messages through the two components of the extrinsic ANS: parasympathetic and sympathetic nervous system; hence it modulates the GIT function. The CNS does not carry out total control on the GIT function (Serio et al., 2011). The GIT does the job of moving ingested materials as well as secreting digestive juices from the mouth to the anus. The totality of the gut movement and nonpropulsive contractions of the gut are known as motility. This motility is under the modulation of adrenergic mechanisms (Orlando, 2003). The sphincters in the GIT have adrenergic alpha1 receptors (α1-receptors). The stimulation of these α1-receptors leads to their contraction (Mills et al., 2008).
According to Pennefather et al., (2000), prostatic stroma has much noradrenergic innervations. The stimulation of the noradrenergic nerves produces contractions of prostate smooth muscles. Guanethine and α1-adrenoceptor antagonists inhibit the contractile activity of the smooth muscle. The antagonists likely act at the α1L- adrenoceptor. These actions underscore the clinical use of α1 adrenoceptor antagonists in treating benign prostatic hyperplasia (BPH). Prostatic stroma and epithelium are innervated by acetylcholinesterase-positive nerves. Nerve-mediated contractions of the muscle of stroma of rat, guinea-pig and rabbit are reduced by atropine. Guinea- pig, dog and rat have been found with M1, M2 and M3 muscarinic receptors respectively which are implicated in the contraction of the prostatic stroma (Nguyen et al., 2013). Smooth muscle tone, prostate innervation and pelvic perfusion are regulated by nitric oxide (NO). The NO is derived from nitric oxide synthase (NOS). The NOS is an enzyme that synthesizes NO from L-arginine in terminals of non- adrenergic/non-cholinergic nerves and endothelium (Bond et al., 2013).
The innervation of the prostate is responsible for the control of ejaculation and micturition. The individualization of the many nerves and intramural ganglia of the prostate is very difficult. The capsule and caudal prostate especially have nerve fibres with diameters beyond 95mm. The urethra in turn has fibres greater than 30mm in diameters. The complex innervation has some implications in prostate cancer. The nerve path is the route of intracapsular invasion of considerable number of cases of prostate cancer. Cancer may spread to striated sphincter when it develops in the prostate apex. The spread may be along nerves to the bladder neck when the cancer growth is towards the supramontanal urethra (Hale et al., 2013).
1.4 Receptors on Some Smooth Muscles
Smooth muscles of human and rabbit prostate glands possess adrenergic and cholinergic receptors. In the respiratory tract, there is a significant distribution of the human beta2-adrenoceptor (β2-adrenoceptor). The β2-adrenoceptor is a member of 7- transmembrane group of receptors. Cyclic adenosine monophosphate and protein kinase affect intracellular signalling. The signalling cascade occurs when they activate β2-adrenoceptor (Johnson, 2006).
Fig. 3: Stimulation of adrenergic receptors
Alpha1-receptors relative to beta2-receptors are associated with the smooth muscle of the blood vessels. Epinephrine has a relatively higher affinity for beta2-receptors than the alpha1-receptors. Vasodilation is produced following the activation of the beta2- receptors while vasoconstriction results from the activation of the alpha1-receptors. Hence, the relative amount of epinephrine and the affinity of alpha1-receptors and beta2-receptors for epinephrine determine its effect (Budhiraja, 2009). When the concentration of epinephrine is low, epinephrine selectively stimulates beta2-receptors and this brings about muscle relaxation. However, when the concentration reaches a threshold that can bind to the alpha1-receptors, there will be constriction (Talwar and Srivastava, 2006).
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EFFECT OF HONEY ON JEJUNUM AND PROSTATE SMOOTH MUSCLES OF RABBITS
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