CHAPTER ONEINTRODUCTION1.1 Background StudySexual interaction is the physical manifestation of our emotional need for acceptance, our need for affirmation and our need for life. Sexual function is an important component of quality of life and subjective well-being in humans (Kandeel, 2007). In recent times, it is believed that because of altered lifestyle, stressful living conditions, diverse pollutants, certain prescription and several nonprescription drugs, dietary toxins and certain nutritional deficiencies sexual life is negatively affected (Kenneth, 2001).Sexual problems are widespread and adversely affect mood and interpersonal functioning. The main problems are related to sexual desire and male erectile dysfunction (Shin et al.
, 2010). Male sexual dysfunction affects not only sexual relationships, but also the overall quality of life and includes erectile dysfunction, ejaculation dysfunction, hypogonadism and represents a serious public health problem (Pare, 2014).A variety of synthetic medications (Sildenafil, Alprostadil, Cyproheptadine, Buspirone) are reported to be used in the treatment and management of male sexual dysfunction, but they are associated with some serious side effects (severe allergic reactions, memory loss, painful or prolonged erection, seizure, severe or persistent dizziness, loss of hearing, sudden decrease or loss of vision, insomnia etc.), are not readily available and are expensive (Boyarsky and Hirschfeld, 2000). Therefore, the search for natural supplement from medicinal plants is being intensified probably because of fewer side effects, availability and affordability. These medicines are expensive, not easily available and accessible by people in rural areas and have some serious side effects (Adbillahi and Van Staden, 2012).
In ancient times, people used external agents in the form of food, drinks and self?made preparation to maintain or enhance the sexual power. These external substances possessed pharmacological and psychological action to fortify the sexual or reproductive system. The allopathic drugs used for sexual dysfunction are believed to produce side effects and affect other physiological processes and, ultimately, general health (Vitezic and Pelcic, 2002).
In developing countries on the contrary, the inability to afford modern medical healthcare has forced patients to seek traditional medical attention. In these countries, many plants extract are traditionally used to improve sexual performances (Kamtchouing et al., 2002; Carro-Juarez et al., 2004). The gradual shift to herbal therapy with its attendant increasing acceptance, even among the elites, make the herbal practitioners lay claims to having the cure to a myriad of ailments, including male fertility, irrespective of the etiology of such disease (Anthony et. al, 2006).
The search for aphrodisiac of natural plant of herbal origin has been increased as opposed to synthetic compounds which are known to cause severe unwanted side effects (Wani et al, 2011).Melicope ptelefolia locally known as “tenggek burung,” is one of the most common medicinal herbs that are widely distributed in many areas of Peninsular Malaysia and also in several other Asian countries (David, 1995). Apart from being one of the most popular traditional fresh vegetables among the Malays of Malaysian community, different parts of Melicope ptelefolia has been used traditionally for centuries as natural remedy for fever, emmenagogue, stomach ache, and rheumatism as well as treatment of wounds and itches (Perry and Metzger, 1980). This herb is known to prevent premature ejaculation by lowering blood pressure.
However, these usages are not substantiated by any written document (Abas et al., 2006). Hence, this study is being proposed to simultaneously identify Melicope ptelifolia towards its aphrodisiac potential.1.2 Problem StatementMale sexual dysfunction, that is the repeated inability to achieve normal sexual intercourse, includes various forms such as premature ejaculation, retrograded, retarded or inhibited ejaculation, erectile dysfunction, arousal difficulties (reduced libido), compulsive sexual behaviour, orgasmic disorder and failure of detumescence.
Male sexual dysfunction is on the rise worldwide because of ageing population and other increasing aetiological factors (Yakubu, Akanji and Oladiji, 2007). Erectile dysfunction’ (ED) or (male) impotence is a sexual dysfunction characterised by the inability to develop or maintain an erection of the penis (Monga, 1999; Malviya, Jain, and Vyas, 2013; Malviya, Malviya, and Jain, 2013). The causes of erectile dysfunction may be physiological or psychological (Bosch et al.
, 1991). Penile erection is a complex process involving psychogenic and hormonal input, and a neurovascular nonadrenergic, noncholinergic mechanism. Nitric oxide (NO) is believed to be the main vasoactive nonadrenergic, noncholinergic neurotransmitter and chemical mediator of penile erection. Impaired no bioactivity is a major pathogenic mechanism of erectile dysfunction (Burnett, 2006; Kaminetsky, 2008).In Malaysia, it is reported 13% of men have untreatable sterility, 11% have treatable conditions and 76% have disorders of sperm production (Concept Fertility Centre, 2006). A large number of plants have been tested throughout the world for the possible fertility properties (Bhatia et al.
, 2010). In Malaysia, information should be provided to enable the nation to understand and be knowledgeable consumers besides appreciating our local herbs. Melicope ptelifolia is one of the alternative herbal resources which have a great potential to be marketed worldwide as it can help to prevent premature ejaculation and as an aphrodisiac potential for its blood pressure lowering effects. Although the Melicope ptelifolia appears to have some traditional use to increase fertility, globally there is no substantial pharmacological report on the possible aphrodisiac potential effect of this plant up to this date (Sulaiman et al., 2010).Based on these facts, there is a need to monitor medicinal plants presence in Malaysia towards its potential health benefit.
Hence, this study is being proposed to simultaneously identify Melicope ptelifolia towards its aphrodisiac potential.1.3 Research JustificationMalaysia has a great potential to develop its abundant natural resources to increase the market based on herbal products. This is due to Malaysia is endowed with tropical rainforests which are rich in medicinal and aromatic plants which can be utilized as medicines, food, cosmetics by Malays, Indian, Chinese and aborigine’s communities. There are about 1, 200 medicinal plants in Malaysia (Aman, 2006).
Most of them are reported of having potential pharmaceutical values and only a small number of these species have been utilized as active ingredients in cosmetics, fragrances and health care products. With advancement in knowledge, expertise, research and development (R&D), growth of the herbal and biotechnology sector in Malaysia is expected to become alternative medicines and supplements to enhance human’s health and practices in the future (Karim et al., 2011). Such an example, different parts of Melicope ptelefolia has been used traditionally for centuries as natural remedy for fever, emmenagogue, stomachache, and rheumatism as well as treatment of wounds and itches (Sulaiman et al.
, 2010). In addition, there have been many other usages of the herb, for example, to prevent premature ejaculation, as an aphrodisiac, and for its blood pressure lowering effects. However, many of these so-called usages are not substantiated by any written documents (Corner, 1952).Therefore, this study will simultaneously identify the effects of Melicope ptelifolia towards its aphrodisiac potential. Upon completion of the study Melicope ptelifolia on aphrodisiac potential and its effective dose, a study of toxicity will be done to finally answer the burning question on the effect of consuming Melicope ptelifolia in a long term. Such a study has not been reported previously.
1.5 OBJECTIVES1.5.1 General Objective To study the effect Melicope ptelifolia on aphrodisiac potential, male sexual behaviours, penile erection index, sperm parameters, testosterone level, anabolic effects and histology of testes.
1.5.2 Specific ObjectivesTo determine and to compare aphrodisiac potential, male sexual behaviours, penile erection index, sperm parameters, testosterone level, anabolic effects and histology of testes between control negative, control positive and Melicope ptelifolia treated groups for 4 weeks’ treatment.To study the acute toxicity of aqueous extract of Melicope ptelifolia on male Sprague-Dawley (SD) rats.1.6 Null HypothesisHO1: There are no significant differences in mean of: a) Aphrodisiac potentialb) Male sexual behaviours c) Sperm parametersd) Testosterone level e) Anabolic effects f) Histology of Testesbetween control negative, control positive and C.
betacea treated groups after 10 weeks’ post-obesity induction and 7 weeks’ post-treatment. HO2: There are no significant differences in mean of toxicity between control negative, control positive and Melicope ptelifolia treated groups for 4 weeks’ treatment.CHAPTER 2LITERATURE REVIEW2.1 Infertility2.
1.1 DefinitionInfertility has become one of the serious problems among mankind in the whole world. However, there are no consistent records for global prevalence of infertility (Mascarenhas et al., 2012). Infertility can be defined as the inability of couples who are sexually active and are not taking contraceptives to achieve pregnancy within one year (WHO, 2000).
This problem has affected 15% of couples who do not practice unprotected intercourse ( Sharlip et al., 2002). Approximately 48.
5 million couples whom have unprotected intercourse had been identified as intertile (Marttinez et al., 2012). However, infertilifty in men was reported to be atleast 30 million worldwide with the highest rate recorded in Africa and Eastern Europe (Agarwal et al., 2015).Generally, according to Sharlip et al.
, (2002), it was stated that approximately 50% of infertility cases are due to women, and about 20-30% of cases are due to men’s problem. The remaining 20-30% are due to combination of both female and male factors. Referring to the National Center for Health Statistics, the total numbers of impaired fecundity increased by about 2.7 million in women, which is from 4.56 million in 1982 to 7.26 million in 2002, then fell slighlty to 6.
71 million in 2006-2010 (Chandra et al., 2006). Moreover, the fertility rate in men younger than the age of 30 years has also decreased worldwide by 15% (Minino, 2007). A study that had been carried out in Finland found that there is a temporal reduction in semen quality in the general population over a period of 8 years from 1998 to 2006 (Jorgesen et al., 2011).
2.1.2 Sexual dysfunctionSexual dysfunction or sexual disorders has been defined by The World Health Organization, (WHO) as “the various ways in which an individual is unable to participate in a sexual relationship as he or she would wish.” According to Lewis et al.
(2010), sexual dysfunctions are conditions when the individuals are lost or have no feeling of sexual interest or desire, sexual thoughts or fantasies, and a lack of responsive desire, besides having less or no motivations to attempt to become sexually aroused. Sexual function basically involves four main components which are libido, erection, ejaculation and orgasm. Sexual dysfunction occurs when these component(s) are interfered. Male sexual dysfunction has been categorized according to the incident happening in the sexual response cycle into desire disorder, arousal (erectile dysfunction) disorder, or orgasm (premature or delayed ejaculation, or anorgasmia) disorder, although they might overlap with each other (Rösing et al.
, 2009). In addition, sexual dysfunctions are also usually classified based on their cause that is whether they are organic- or psychogenic-caused dysfunctions although they might be influenced from one to another. As defined on Merriam-Webster online dictionary, organic is (1) not using artificial chemicals, or (2) of, relating to, or obtained from living things. Meanwhile, psychogenic is defined as originating in the mind or in mental or emotional conflict. These terms are more commonly used to describe the etiology of erectile dysfunctions after further diagnostic studies.
Organic erectile dysfunction is erectile dysfunction caused by physical problems. In contrast, psychogenic erectile dysfunction is dysfunction caused by anxiety, guilt, depression, or other conflicts related with sexual issues.2.1.3 Erectile dysfunctionErectile dysfunction (ED) is the inability experienced consistently or concurrently by men in obtaining or maintaining sufficient penile erection for sexual activity. It is usually diagnosed after 3 months of the persistent symptoms except in the circumstances of ED caused by trauma or surgery (Lewis et al.
, 2010). ED is the major sexual arousal disorder in men that is highly prevalent and is age-related. The prevalence of ED worldwide was between 2% in men younger than 40 years to 86% in men 80 years or older (Prins et al., 2002). Meanwhile, based on the systematic review done by Park et al. (2011) the prevalence of ED in Asia from January 2000 to September 2010 ranges from 2% to 88%.
In Malaysia, based on the cross-sectional study carried out by Rahman et al. (2011), it was reported that the prevalence of ED was 69.5%. This prevalence increased with age such that they were 49.
7%, 66.5%, 92.8% and 93.9% of men in their 40s, 50s, 60s and 70s respectively (Rahman et al., 2011).
Aside from age, the other risk factors of ED are sedentary lifestyle, obesity, alcohol consumption and smoking. It has been found that active lifestyle, ideal weight, medium alcohol intake and to be smoking-free can lower the risk (Bacon et al., 2010). Furthermore, the underlying diseases such as lower urinary tract symptoms of benign prostatic hyperplasia, cardiovascular disease, diabetes mellitus, psychologic/ psychiatric conditions, cancer, hypertension and stroke are also associated with ED. Lewis et al.
(2010) suggested that there are common pathways between ED and other vascular diseases as the conditions such as vascular smooth muscle and endothelial dysfunction happened in many ED cases. Besides, there is also evidence of ED being associated with medication.2.
1.4 Treatments for EDTreatments available for ED can be targeted for two main factors – modifying the underlying risk factors or giving direct treatment to the patients. Lifestyle change, diet, stop smoking or medical treatment can help correct the underlying factors that lead to ED, as well as controlling drug use for medication-associated ED and hormone replacement therapy for individuals with abnormal hormone production.
Direct treatment can be local therapy such as getting injection therapies, psychotherapy, undergo surgical procedure or by using the most common prescribed treatment which is of phodphodiesterase-5 (PDE5) inhibitors.2.2 Male Reproductive System2.2.1 DefinitionThe male reproductive system is made up of several organs that are important for gamete production and reproduction purposes including scrotum, testes (testicles), epididymis, vas deferens, penis, urethra and the accessory sex glands that consist of seminal vesicles, prostate gland and bulbourethral glands (Marieb and Hoehn, 2007). The scrotum is the pouch-like muscular sac that encases and protects the testes, which extends from the body cavity between the upper thighs, behind the penis.
The subcutaneous muscle layer of the scrotum is made up of dartos muscle that continues internally to make up the scrotal septum. The septum divides the scrotum into two sides, each contains one testis. Two cremaster muscles that descend from the internal oblique muscle of the abdominal wall cover each testis like a muscular net.
During cold weather, both muscles contract simultaneously, ascending the testes closer to the body thus decreasing the surface area of the scrotum to retain heat and vice versa happens when the temperature is higher. This ensures the testes to be in optimal temperature to aid in sperm production (Marieb and Hoehn, 2007). The testes that are located in the scrotum are the location of sperm and hormone production in male.
They are surrounded by two layers of connective tissues which are tunica vaginalis and tunica albuginea. Tunica vaginalis is a serous membrane that covers the testis. Tunica albuginea that lies beneath the tunica vaginalis is a tough, fibrous connective tissue layer that also covers the testes and forms the septa inside the testes, dividing it into lobules. Sperm develop inside the seminiferous tubules, within the lobules. Adjacent to the seminiferous tubules are Leydig cells or interstitial cells that secretes testosterone hormone (Marieb and Hoehn, 2007). Epididymis is a coiled tube attached to the testis that becomes a transit site where sperm continues to mature and be transported.
Then, the sperm will be carried from the epididymis to the ejaculatory duct through vas deferens (Marieb and Hoehn, 2007). The penis is the male copulation organ that consists of three parts which are the root, body (shaft) and glans penis. The shaft of the penis surrounds urethra, the channel that transports semen and urine. The penis is made up of three column-like sinuses of erectile tissues. The two larger lateral columns, corpora cavernosa that occur side-by side makes up the bulk of the penis. Meanwhile, the third sinus which is the corpus spongiosum surrounds the penile urethra and can be felt as a raised ridge during erection (Marieb and Hoehn, 2007).
The seminal vesicle joins with vas deferens to form ejaculatory duct. Together with the prostate, they produce fluids that contribute to almost 60% of the semen volume. Fructose content in the seminal vesicle fluid becomes the source of ATP production by the sperm. Bulbourethral gland release fluid that lubricates the urethra and helps clean urine residues from the penile urethra (Marieb and Hoehn, 2007).2.2.
2 Hypothalamic-pituitary-gonadal axisTestosterone hormone is necessary for male reproductive functions. It is produced by the Leydig cells in the testes. Not only it is secreted into the interstitial spaces of the testis, testosterone hormone is also secreted into the systemic circulation. The regulation of testosterone production in male body involved the interplay between the endocrine system and the reproductive system through the interaction known as the hypothalamic-pituitary-gonadal axis.
The regulation begins from the pulsatile release of a hormone called gonadotropin-releasing hormone (GnRH) in the hypothalamus. On the anterior pituitary gland, GnRH binds to its receptor, the gonadotropin-releasing hormone receptor (GnRHR), stimulating the anterior pituitary gland to release two gonadotrophins which are luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These two hormones play a major role for reproductive function in both men and women. In men, LH binds to LH receptors on Leydig cells in the testes and stimulates the production of testosterone. Meanwhile, FSH binds to the Sertoli cells within the seminiferous tubules and stimulates the Sertoli cells to release androgen- binding protein (ABP) and inhibin.
ABP binds with testosterone, thus keeping its level high inside the seminiferous tubules. The synthesis of the LH and FSH is a negative feedback loop system. When the concentration of inhibin reach its threshold, the hormone inhibin signals the anterior pituitary to stop the release of FSH. On the other hand, testosterone negatively feeds back the hypothalamus and pituitary to inhibit further release of GnRH, LH and FSH.
2.2.3 Phosphodiesterase type 5 (PDE5) inhibitorsKnown as the first-line of treatment for ED, PDE5 inhibitors are oral therapies that have become the drug of choice due to their less invasive property and broad efficacy when given to patients despite the different underlying causes of ED. PDE5 inhibitors can improve erectile function by increasing penile cyclic guanosine monophosphate (cGMP), resulting in relaxation of smooth-muscle cells (McVary et al., 2007).
Four most potent PDE5 inhibitors approved by European Medicine Agency (EMA) to treat ED are sildenafil, tadalafil, vardenafil and avanafil. Sildenafil was the first one launched, which was in the year 1998, and it is available in doses of 25, 50 and 100 mg. After the launching of sildenafil into the market, other PDE5 inhibitors started to evolve with them having different efficacies and onset. The introduction of PDE5 inhibitors clearly has become the major breakthrough in ED treatment as the drugs were proven to improve erectile function and is safe. However, there is always two sides of a thing.
No drug is 100% safe. Despite their effectiveness, PDE5 inhibitors are also reported to have adverse effects on patients. The common adverse event that comes with the use of various PDE5 inhibitors are dizziness, headache, flushing, abnormal colour vision, eye redness, nasal congestion, rhinitis, dyspepsia, back pain, myalgia, urinary tract infection, infection, diarrhoea, dizziness, rash, back pain, flu-like syndrome, and arthralgia (Mukherjee and Shivakumar, 2007). There has also been a report on the use of sildenafil citrate causing myocardial infarction, ventricular arrhythmia, cerebrovascular hemorrhage, transient ischaemic heart attack and hypertension. Sildenafil also caused some disturbing effects such as seizure and anxiety, persistent erection, priapism and haematuria (Krenzelok, 2000; Pryor, 2000; Christiansen et al., 2000).
Mukherjee and Shivakumar (2007) also reported a first case of sudden sensorineural hearing loss of a patient following 15 days when he started taking sildenafil. Khan et al. (2011) later then reported a total of 53 cases of hearing loss associated with ingestion of sildenafil. 2.3 Animal model 2.
3.1 Animal model for ED studiesAt present, many of the in vivo and in vitro research on animals provide knowledge of physiology of erection. Gajbhiye et al. (2015) had reviewed several animal models of erectile dysfunction frequently used by researchers and classified them into three- nerve stimulation models, models based on sexual behavior in conscious animals, and models of male ED based on specific predisposing conditions.
The use of sexual behavior model is not new as Agmo (1997) stated assessment of male rat sexual behavior to evaluate drug action is gaining more interest in the behavioral pharmacology field even in the earlier years. The sexual behavior animal model can be used for mating tests, which include introducing a hormonally induced ovariectomized female rat into a single observation chamber in which the tested male was placed. The number of mounts, intromissions, and ejaculation are recorded and scored. Non-contact erection tests that divide male and female at opposite sides of the observational chamber can also be carried out to observe the penile erection of the male rats in the presence of female but without direct contact.
Another two erection tests that can be done are compound-induced erections and reflexive erection tests. Both carried out in the absence of female rat, compound-induced erection test identifies the penile erection after injecting drugs or test substances, meanwhile reflexive erection test involves observing the penile reflexes while the animals are restrained on their back (Gajbhiye et al., 2015).
In addition, copulation studies that use representative parameter of erectile function such as intromission ratio can also be carried out by using the sexual behavior animal model.2.3.2 Sexual behaviors of male ratsMale rat usually begins the sexual meeting with the female by exploring the face and anogenital region of the female. The male will mount the female from its rear, gives a few quick shallows thrusts, and gives deeper thrusts, inserting its penis into the vagina for 200-300 seconds once the female’s vagina is detected. Then, it will rapidly spring back and grooms its penis.
Ejaculation usually follows between 1 to 2 minutes apart after the male experiences 7 to 10 intromissions. It can be differentiated based on the longer and deeper thrust observed and a much slower withdrawing, with a post-ejaculation posture. The male rat will groom itself and then rests after the ejaculation, in a period called as post-ejaculatory interval (PEI) that usually lasts 6 to 10 minutes before it continues mating (Hull and Dominguez, 2007). Male rats acquire copulatory ability between 45 and 75 days of age.
However, it declines as the rat ages and the rat loses the ability to ejaculate. It was found that the condition may be due to the decline in estrogen receptor but not androgen receptor (Roselli, Thornton and Chambers, 1991). 2.3.3 Hormonal activation of male rat mating behaviorsTestosterone is the principal steroid hormone that control and regulates male sexual behavior in vertebrate species. Testosterone hormone which is secreted by Leydig cells in male testis is metabolized into estradiol (E2) by aromatization or reduced to dihydrotestosterone (DHT) by 5?-reduction (Hull and Dominguez, 2007).
Aromatization of testosterone takes place in the hypothalamus and limbic system. The “aromatization hypothesis” suggested that estradiol is the major hormone that involves in activating the sexual behavior of male rats but the behavior is not fully maintained by it (Hull, Wood and McKenna, 2006). Androgens influence the motivation and performance in male rats and it is also sufficient to maintain ex copula genital reflexes although it is ineffective (Hull and Dominguez, 2007). 2.4 AphrodisiacsAphrodisiacs are any substances that can enhance sexual desire or sexual performance when consumed.
It is a term that originates from the Greek word Aphrodite, which refers to the Greek goddess of love and beauty. The subject regarding aphrodisiacs has been gaining more interests as the citation based on a simple search with the word aphrodisiac itself on Medline gives out more than 146 results between the year 2000 to 2009 as compared to only 86 articles between 1990 to 1999 (Shamloul, 2010). The practices of using natural aphrodisiacs is not a new thing as the search for treatment to improve the sexual function especially in male can be dated back as early as 3000 to 4000 years back. For instance, the poem Samhita of Sushruta from Hindu population that describes several remedies and treatment be it the ones which is highly nutritious or even mixing heavy metal oxide with musk, amber and spices (Hollister, 1975). There are several characterizations that can be used in order to classify these aphrodisiacs. From the mechanism of action, aphrodisiacs can be categorized into substances that increase libido, potency or sexual pleasure (Sandroni, 2001). Besides, Shamloul (2010) suggested that aphrodisiacs to be categorized into two which are natural aphrodisiacs and non-natural aphrodisiacs based on their sources, and then further classify natural aphrodisiacs based on whether they are from plant or non- plant sources.2.5 Toxicity study2.5.1 DefinitionToxicity means the extent to which the substance can harm humans or animals. It can be measured by its effects on the target organism, organ, tissue or cells. The toxic effects of a substance on animal physiology can range from minor changes such as reduced weight gain, small physiological alteration or change in the levels of circulating hormones, to severe effects in organ functional loss leading to death. Intermediate levels of toxicity may cause pain and suffering (Home office, 2004). The types of toxicity tests which are routinely performed by pharmaceutical manufactures in the investigation of a new drug involve acute, sub-acute and chronic toxicity. Acute toxicity is involved in estimation of LD50 the dose which has proved to be lethal (causing death) to 50% of the tested group of animals. Determination of acute oral toxicity is usually an initial screening step in the assessment and evaluation of the toxic characteristics of all compounds. (Akhila et al., 2007).As per the OECD guidelines, in order to establish the safety and efficiency of a new drug, toxicological studies are very essential in animals like mice, rat, pig, dog, rabbit, monkey etc. under various conditions of drug. Toxicological studies help to make decision whether a new drug should be adopted for clinical use or not. OECD 401, 423 ; 425 does not allow the use of drug clinically without its clinical trial as well as toxicity studies. Depending on the duration of drug exposure to animal’s toxicological, studies may be three types such as acute, sub-acute and chronic toxicological studies (Jayacitra et al., 2014).In order to support an application for a clinical trial or for the registration of a new drug, it is necessary to satisfy legislation that requires that certain data should be produced from a variety of toxicological investigations that show the safety profile of the compound to which humans may be exposed. Therefore, in the majority of cases of evaluation of the toxicity of most substances, rodents and non-human primates are first used in preclinical animal safety studies before further studies are done in humans. These animals are mainly used because of their biological similarity to humans that allows them to be regarded as the suitable metabolic models for humans in a broad range of investigations (Loomis and Hayes, 1996; Pascoe, 1983).2.5.2 Acute ToxicityAcute toxicity is produced after administration of a single dose or multiple doses in a period not exceeding 24 hours, up to a limit of 2000 mg/k g. Objective of acute toxicity studies is to identify a dose causing major adverse effects and an estimation of the minimum dose causing lethality (Robinsonet al., 2007). As per Jayacitra et.al., 2014, Acute toxicity refers to the adverse effects that occur on first exposure to a single dose of a substance. Separate tests are needed to detect the effects of contact with the skin and eye (corrosion, irritancy and sensitisation; topical or local toxicity)In acute toxicity studies, single dose of drug is given in large quantity to determine immediate toxic effect. Acute toxicity studies are commonly used to determine LD50 of drug or chemicals and natural products. (Abrar et.al., 2013).2.5.3 Toxicity Aspects of Use of Herbal Products There is an on-going world-wide “green” revolution which is mainly premised on the belief that herbal remedies are safer and less damaging to the human body than synthetic drugs (Williamson et al., 1996). Many writers claim that it is assumed that “all things natural are good” (Gaillard and Pepin, 1999) and, generally, the extensive traditional use of herbal products is not assumed to be based on a comprehensive well documented logic, but rather on empirical wisdom accumulated over many years, often arrived at through trial and error and transmitted orally from generation to generation. This traditional methodology has enabled those herbal medicines producing obvious signs of toxicity to be recognized and their use avoided. However, the premise that “traditional use of a plant for perhaps many hundreds of years establishes its safety does not necessarily hold true” (Ojewole, 2004). The subtler and subacute forms of toxicity, that can lead to chronic carcinogenicity, mutagenicity, and hepatotoxicity, may well have been overlooked by previous generations and it is these types of toxicity that are of most concern when assessing the safety of herbal remedies (Williamson et al., 1996; Tomlinson and Akerele, 1998)2.6 Melicope ptelefolia 2.6.1 DefinitionMelicope is a genus of shrubs, or small trees of the Rutaceae family. There are about 230 species that have been recorded and distributed in the world ranging from Madagascar to India, South China, throughout Malesia, Polynesia, the Hawaiian Islands, Australia and New Zealand (Soepadrno and Wong, 1995). There are eight species in the Malay Peninsula ( Burkill, 1966), and about fourteen species are found in Sabah and Sarawak, i-e; Melicope triphylla, M. jugosa, M sororia, M bonwickii, M denhii, M. latifolia, M clamensiae, M. subunifoliolata, M. confme, M. glabra, M. lunu-ankeda, M accedens, M hookeri and M. incana (Soepadmo and Wong, 1995).Melicope is the largest genus of Rutaceae with about 235 species and it accounts for one third of the diverse species of the family together with Zanthoxylum (Kubitzki et al., 2011). It is largely distributed in areas ranging from India throughout Southeast Asia, Malesia, Australasia and many Pacific Islands, reaching Hawaii in the Northeast and the Marquesas Islands in the Southeast (Appelhans, Wen and Wagner, 2014). Melicope ptelefolia (M. ptelefolia) also locally known as “tenggek burung, pauh-pauh, cabang tiga, and tapak itik”, and in Javanese it is popular as “sampang” while Siamese called it as Uam, or Sam Ngam (Shoji et al., 1989). MP grows wildly in open area, shrub edge, paddy field and turf areas of land and also grows well in peat and sulphate acid soil. Birds are main spreader of this plant. This plant is easy to grow and does not need intensive care.2.6.2 Characteristics of M.ptelefoliaThe Rutaceace family usually has twigs and branches sometimes armed with spines or thorns. The leaves are in trifoliate which means there are three leaves on each stalk. Besides that, it comes with long stalks and the leaves are dotted with oil-glands that appear as dark green spots when the leaf is held to the light (Jones, 1995). They can be easily recognized from the aromatic or lime-like smell from the broken twigs, fruits or crushed leaves. Specifically, in terms of its physical characteristics, M.ptelefolia has trifoliate, green, thick, broad leaves and has small white and greenish flowers. M.ptelefolia also has a slightly bitter taste, crunchy young leaves, pungent, and lemon-lime aroma. People love to eat this leaves raw as ulam (Karim et al., 2011).2.6.3 Therapeutic properties of M.ptelefoliaM.ptelefolia is believed to be high in nutritional and medicinal value. According to Van et al. (1998), the leaves and twigs part of MP are used for treatment of itches, wound infections, trauma, abscess, eczema, dermatitis and hemorrhoids whereas its roots and bark parts are served as an appetizer, digestive and emmenagogue. Besides, there are also reports of other uses of this species as an antipyretic, anti-inflammatory and analgesic. The almost ripe fruit of this herb are used in Korean folk medicine as an analgesic, antiemetic, astringent and also as a hypertensive agent (Yuk et al., 1981). In Chinese medicine, dried and unripe fruit was recommended for the treatment of abdominal pain, diarrhea and also headache (Shoji et al., 1989). Melicope ptelefolia has also been reported to have antibacterial (Manadhar et al., 1985) and fungicidal activities (Kumar et al., 1990). Aman (2006) reported that this herb is used as medicine to treat high blood pressure, reduces fatigue, improve blood circulation and relieve body stamina. It also acts as an aphrodisiac especially for men. Besides, this herb also contains a lot of antioxidants which is good for cancer. This herb is also used to treat high blood pressure, reduce fatigue, improve blood circulation and stamina as well as an aphrodisiac for men (Aman, 2006).In previous studies, ethanol extracts of plants are reported to have antinociceptive activities (Sulaiman et al., 2010) and pharmacologically, this plant also consists several other medicinal properties including anti-inflammatory (Shaari et al., 2011), antimicrobial and cytotoxic properties (Abas et al., 2006; Afendi et al., 2015). This plant is also rich in anti-oxidant (Abas et al., 2006) which plays a main role in increase the production of steroidal hormones.2.6.4 Phytochemical composition of M. ptelefoliaThere have been some previous reports regarding the compounds isolated from M. ptelefolia such as 2,2-dimethyl-2H-1-benzopyans (Kamperdick et al., 1997), N-methylflindersine, melicobisquinolinone A and melicobisquinolinone B (Kamperdick et al., 1999). Some other isolated compound also includes kokusagine, 5 –methoxymaculine (Abas et al., 2010), and several polyphenol including 2,4,6-trihydoxy-3-geranylacetophenone (tHGA) and 2,4,6-trihydroxy-3-prenylacetophnone (Seema et al., 2013; Abas et al., 2010). Abas et al. (2006) also stated that the antioxidant activity of M. ptelefolia might have been caused by the presence of several derivatives of isoquinoline (alkaloid), found by McCormick et al., (1996) and benzopyran, including those with a phenolic moiety, found by Kamperdick et al (1997). Phytochemical studies of the plant reveals that it contains kokusaginine, ?- sitosterol, p-0-geranylcoumaric acid, 3-geranyl-2,4,6-trihydroxyacetophenone, benzopyranone, 4′,5-dihydroxy-3,3′,7-trimethoxyflavone, scoparone and 2,4,6- trihydroxy-3-geranylacetophenone (tHGa), n-octadecanyl palmitate, palmitic acid, 3,5,3′-trihydroxy-8,4′-dimethoxy-7-(3-methylbut-2-enyloxy) flavone, daucosterol, salylic acid, and kaempferol-3-O-alpha-D-arabinpyranoside (Shaari et al., 2006; Xie et al., 2011). M. ptelefolia also contains glycosides where seven of them were new diglycosidic constituents named pteleifosides A-G (Zhang et al., 2012). It was also reported to contain alkaloid, flavonoid, benzopyrans and acetophenones.On the other hand, phytochemical analysis of this plant showed the presence of three new compounds, identified as melicoester, melicopeprenoate and p-O-geranyl-7″-acetoxycoumaric acid by Shaari et al. (2011). The compounds were isolated along with 21 other known compounds mainly lupeol, oleanolic acid, gensteinn, 4-stigmasten-3-one, 3-beta-hydoxystigma-5-en-7-one, cis-phytylpalmitate, dodecane, dodecan-1-ol, ceryl alcohol, hentriacontanoic acid, eicosane, n-amyl alcohol, caprylic alcohol, octatriacontane, nonatracontane, hexatriencontan-1-ol, methyloctacosanoate, beta-sitosterol, and beta-sitosterol glucoside. Glucoside present in this plant is secondary metabolites that help in the secretion of steroidal hormones and enhances sexual activity.Yakubu and Afloyan (2009) stated the androgenic and vasodilatory effect of saponins and alkaloids. Glycosides are molecules consisting of a glycone (sugar) and aglycone (non-sugar group) group. The classification of pteleifosides are not mentioned. However, glycosides such as the one from Fenugreek (Trigonella foenum-graecum) seeds shows significant effect on the reproductive system (Wankhedea et al., 2016). CHAPTER 3METHODOLOGY3.1 Experimental study design3.1.1 Plant preparation and aqueous extraction of Melicope ptelefoliaFresh young leaves of M. ptelefolia were purchased from a local supplier market (Pasar Borong Selangor) in the Seri Kembangan city of Selangor state, Peninsular Malaysia in compliance with Good Manufacturing Practice (GMP) standard. M. ptelefolia was further identified by a botanist from the Department Institute of Bioscience (IBS), Universiti Putra Malaysia. The voucher herbarium specimen was deposited at the Mini Herbarium of the Department Institute of Bioscience (IBS), Universiti Putra Malaysia (Vouncher no. SK 2934/15). Approximately 4 kg of the whole plant, including the stems and leaves, were collected. To make sure there were no more sand, debris and pesticides, the leaves were washed thoroughly using distilled water upon separation from the stems. Sample Extraction was carried out according to the method described by Zakaria et al. (2011) with slight modifications. In order to obtain the MPAE, 250 g of the freshly collected leaves of M. ptelefolia were dried in an oven at 50°C overnight. The dried plant was then grounded to a fine powder using a grinder and was stored at -20°C until used. Next, the dried plant powder was extracted by boiling it in distilled water (1.5 L) for 30 minutes. The extract was then filtered with Whatman No. 1 filter paper and the resulting filtrate was froze-dried. The froze-dried material was reconstituted separately in distilled water to give the required doses for each experiment. Resulting water extract was then filtered through a tea strainer. Boiling of the powder in 1.5 L distilled water was repeated and pooled. The filtrate will then centrifuge in 50 mL batches at 2000 rpm for 10 min in a centrifuge (Eppendorf-5810 R). The resulting supernatant was then filtered through filter paper Whatman (No. 1) placed on a funnel. The filtrate was dried in a freeze dryer and stored at 4°C in an amber glass vials until use (Raidah & Mahanem, 2015).3.1.2 Experimental animalsSprague Dawley rats strain of both sexes (male and female) were used for the experiment. The rats were 7-8 weeks of age and weighed between 200-250 g. They were kept at the animal house facility in the Faculty of Medicine and Health Sciences of Universiti Putra Malaysia. The animals were housed in groups of three, separately for each sex and kept at room temperature with 12 h light-dark cycle starting at 6.00 am and 6.00 pm. Upon arrival, the animals were allowed for acclimatization for 14 days, with provided standard pellet and water ad libitum prior to experimentation. All the animal experimentations were carried out after prior approval from the Institutional Animal Care and Use Committee (IACUC) of Universiti Putra Malaysia, Serdang, Selangor.3.1.3 Preparation of test samples The doses of 100 mg/kg, 200 mg/kg and 300 mg/kg of MPAE were prepared by suspending the respective amount of crude dried extract obtained in distilled water and were administered orally to the male rats using metal canula. Hormonal solutions used for the experimentation were suspended in arachis oil, making doses 0.125 mg kg-1 estradiol benzoate and 1.25 mg kg -1 of progesterone.3.1.4 Ovariectomy The surgical procedure was carried out based on the method modified from Khajuria, Razdan and Mahapatra, (2012). Before the surgery was carried out, the weight of each animal were measured using a digital balance. Then, the animals were anesthetized under ketamine/xylazine anesthesia, intraperitoneally. The anesthetized animal was placed on its dorsal surface before the fur on its abdomen was removed by using an electronic animal hair trimmer. The area for surgery was thoroughly cleaned with 70% ethanol, followed by prepping with iodine. A small peritoneal incision of about 1.0-1.5 cm was made transversely with a surgical scalpel blade no. 11, right on the middle part of the abdomen. After the peritoneal cavity was accessed, the adipose tissue was pulled away until the right uterine tube and ovary was exposed. Ligation was done at the distal uterine horn before complete removal of the ovary. The same procedure was then repeated for the left uterine tube and left ovary. The uterine horn was carefully inserted back into the peritoneal cavity after the removal of the ovaries. The two layers of the wound which consist of muscle and skin were sutured with one absorbable suture (Ethicon chromic sutures 3/0, Johnson & Johnson Ltd.) and the skin were sutured with one non-absorbable suture (Ethicon mersilk sutures-3/0, Johnson & Johnson Ltd). Iodine was applied to the skin for disinfection for 3 respective days. Meloxicam (0.3 mg/kg) were given to the rats for 5 respective days postoperatively for pain relief.3.1.5 Treatments The experiment was designed with four experimentation groups whereby the sexually naïve Sprague Dawley male rats were randomly divided into four groups of six animals each (refer Appendix B). Group I was the control group administered vehicle only. Meanwhile, group II, III, and IV were treatment groups receiving 100 mg/kg, 200mg/kg and 500 mg/kg MPAE respectively. The animals were treated continuously for 28 days (4 weeks) and were subjected to sexual behavior analysis on day 63 of treatment. Female animals of the same strain were prepared for experimentation by first being ovariectomized under ketamine/xylazine anesthesia and brought into estrous by sequential subcutaneous injections of 0.125 mg kg-1 estradiol benzoate and 1.25 mg kg -1 of progesterone, 48 and 4 hours before the copulatory studies, respectively. Then they were used as a stimulus for evaluation of sexual behavior. 3.1.6 Cage-side observations The rats were observed individually and special attention was given to the treatment groups. The cage-side observations for their physiological and behavioural changes and mortality pattern assessments were performed daily throughout the study period. The cage-side observations included the evaluation of the following: changes in skin, fur, and eyes; respiratory effects; autonomic effects, including salivation, diarrhea, and urination; central nervous system effects, including tremors and convulsions (Kumarnsit et al., 2006; Demma et al., 2006; Mukinda and Syce, 2007; Obici et al., 2008).3.2 Toxicity Healthy male Sprague Dawley rats aged between 8 and 12 weeks were starved for 3- 4 h and subjected to acute toxicity studies as per Organization of Economic Co-operation and Development (OECD) guidelines No: 423. They were divided into 4 groups of 6 animals each and kept singly in separate cages during the experiment. Group 1 represented the control group, which received 10 ml/kg of distilled water orally. Groups 2- 4 received suspension of different extract Melicope ptelifolia aqueous extract orally at the doses of 1000, 2000 and 5000 mg/kg once. The rats were observed continuously for 2 hours for behavioural, neurological and autonomic profile, and for the next 24 hours for any lethality or death. The rats were weighed and visual observations for mortality, behavioral pattern (Salivation, fur, lethargy, and sleep), changes in physical appearance, injury, pain and signs of illness were conducted once daily during that period.3.3 Anabolic effectsThe body weights of rats were recorded on the first day and once every 7 days by using a table top electronic balance for 28 consecutive days prior to the administration of the extract. On day 28 of treatment, the liver, kidney and testes of each group were carefully removed after sacrifice and their relative organ weights were recorded. The relative organ weight (ROW) for each organ was calculated using the following formula; Relative organ weight = (Absolute organ weight (g)/body weight of rat on the day of sacrifice (g) x 100% (Rajeh et al., 2012). Relative Organ Weight =Absoluteorganweight(g)/Bodyweightofratonthedayofsacrifice(g)×1003.4 Male Sexual Behaviour test.On day 7, 14, 21 and 28, 2 hours after oral administration, male sexual behaviours analysis were carried out according to Sumaia et.al., 2015. 2 hours prior to the experiment, male rats (n=6) were weighed before being transferred to a dark room. Then, they were placed into individual cages and were left for at least 1 hour before the commencement of sexual behaviour study. Female rats used as mating stimuli were exposed to heat with a single intramuscular injection of 0.125 mg kg-1 estradiol benzoate and 1.25 mg kg -1 of progesterone, 48 and 4 h before testing respectively. Each male rat was placed in the observation chamber (42 cm x 25 cm x 14 cm) and was allowed for 5 minutes’ acclimatization with the chamber environment before the analysis began. A sexually receptive female rat prepared for the experimentation was then introduced silently from one side of the chamber as stimulus. The male rat should be expected to reach receptive female within 3 minutes. The time that the male started to mount the receptive female was considered as positive sexual behaviour. Male which failed to respond within 3 minutes after being placed in the cage should be considered as having negative sexual behaviour. The time taken for the male rat’s intromission activity was recorded. Male rats that could not have an intromission within the first 15 minutes fail the mating test. The following male sexual parameters were recorded during the observation period:Mount latency (ML): the time from the introduction of the female to the occurrence of the first mount. Intromission latency (IL): time for first intromission after introduction of the female in the cage. Intromission frequency (IF): number of intromissions observed during the observation period from of introduction of female until ejaculation. Intromission ratio (IR): determined by dividing the number of intromissions by the sum of number of mounts and number of intromissions (Agmo, 1997). Post ejaculatory interval (PEI): Calculated as time from ejaculation until next intromission.3.5 Penile erection index (PI)On day 7, 14, 21 and 28, Penile Erection (PE) were determined 30 minutes after all the male rats in the group (n=30; 6 per group) were given their respective treatments, using the method reported by Benassi- Benelli, Ferrari and Pellegrini-Quarantotti (1979). For each group, all 6 rats were placed inside an observation cage at a time and continuously observed for a period of 30 minutes. The movement of the rats when the rat bent down to lick their erected penis was recorded as PE. Penile Erection Index (PI) will be determined as follows: Penile Erection Index (PI) = Percentage (%) of rats showing at least one episode of PE × Mean number of PE 3.6 Sperm AnalysisSperm Analysis from the cauda epididymis was determined by using the method described by WHO, 2000 and was carried out for this test. On day 29, the control and treated group were sacrificed under ketamine/xylazine anesthesia and dissected to obtain cauda epididymis. The sperm sample preparation was then used for the following analysis. Sperm CollectionThe caudal part of epididymis was isolated from each rat. The epididymis was minced, excited and incubated in a pre-warmed petri dish with 10ml of normal saline (0.9% sodium chloride) at 37°C. The spermatozoa were allowed to disperse into the normal saline. The suspension was gently shaken to homogenize and was analyzed under a microscope.3.6.1 Sperm Count Dilution ratio of 1:20 was used to determine the sperm count by diluting 50?L of the sperm from the semen with 950 ?L of diluents (10% formalin in saline). An amount of 10 ?L from the diluted solution was transferred into the haemocytometer. The fixed sperms were counted and evaluated under light microscope (OLYMPUS 3X31) at 400x magnification. Sperm count = Total number of sperm in 5 square x 50000 x Dilution factor3.6.2 Sperm MotilityAn amount of 40 ?L of ss placed on the slide and was covered with a cover slip for motility analysis and was observed under light microscope (400xmagnification). Sperm motility was classified into progressive, non-progressive and non-motile movement. The spermatozoa with progressive motility were those with linear forward movements; non-progressive motility refers to circular or local movements, and immobile sperm were spermatozoa without movements. The total sperms in 10 different views were counted and the percentage of motility were recorded.3.6.3 Sperm ViabilitySperm viability was assessed by staining 40 ?L of sperm suspension with 10 ?L of Eosin-Nigrosin stain on a glass slide. The smear was observed under a light microscope (OLYMPUS CXCI) at 400x magnification. Live sperm remained unstained while those that showed pink or red coloration were classified as dead. Approximately 200 sperm were counted from the smear and percentage of live sperms was recorded.3.6.4 Sperm MorphologyThe slides prepared to evaluate sperm viability was used to observe the morphology of the sperm. Sperm morphology was assessed under the light microscope using the x100 objective lens. The morphological features of spermatozoa were classified according the abnormality of head, neck, tail and multiple abnormalities.3.7 Testosterone LevelSerum Testosterone Level was determined by using the method described by Raidah and Mahanem, 2015. On day 29, the control and treated group were sacrificed under ketamine/xylazine anesthesia. Blood samples from all rats were collected from cardiac puncture. Serums were separated from blood samples by centrifugation at 2500 rpm for 10 minutes and it was stored at -20°C until used. The blood was then sent to Clinipath (M) Sdn. Bhd. for analysis.3.8 Histology of TestesThe testes were fixed in a Bouin’s solution overnight, washed in 0.9% NaCl, dehydrated through graded concentration of ethanol series, cleared in toluene and embedded in paraffin wax. Tissues were sectioned at 5 ?m thicknesses and stained with Hematoxylin and Eosin (H;E) stains. The slides of testicular spermatogenesis were observed and evaluated qualitatively under light microscope. Histopathology studies were done at the Histopathology Laboratory, FMHS, UPM. Following fixation, the organs were trimmed into smaller pieces at 5 mm thickness and were placed into plastic cassettes before being processed using a standard overnight method in an Automated Tissue Processor (Leica TP1020, Germany) where the machine carried out the dehydration, clearing, and impregnating processes.After processing, the tissue samples were then embedded in molten paraffin with appropriate molds with Leica EG 1160 (Leica Microsystems, Germany). The blocks were trimmed at 16 ?m thickness and sectioned into a thickness of 0.4 ?m using a rotary microtome. (Leica RM2135, Germany). The thin sections of the tissue samples were placed in a water bath (Leica H1210, Germany) at 35°C to 37°C, and were then fished and mounted onto glass slides using a slide warmer. The slides were stained with Haematoxylin and Eosin (H;E) stain, by using Autostainer (Tubesha et al., 2013). This was then followed by mounting of p-xylene-bis-pyridinium bromide and observed under a light microscope in a random order and without knowledge of animal or group. At least ten fields from each slide of each group were examined to evaluate the histological changes. The renal injury was based on degeneration of Bowman space and glomeruli, degeneration of proximal and distal tubules, vascular congestion and interstitial edema. Meanwhile, the criteria for liver injury were vacuolization of hepatocytes and pyknotic hepatocyte nuclei, number of Kupffer cells and enlargement of sinusoids.3.9 Statistical analysis Statistical analysis was performed using SPSS version 22. All data were expressed as the mean ± standard error of mean (S.E.M). The mean differences between the control and treatment groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc comparison test. A mean difference was considered statistically significant. when p < 0.05CHAPTER 4RESULTS AND DISCUSSION4.1 ToxicityNo mortality and changes in the behavioural, neurological and autonomic profile were observed in treated groups of the rats up to highest dose of 5000 mg/kg body weight. Hence, one tenth of treated dose was selected for present investigation. It is concluded the LD 50 is more than 5000 mg/kg of Melicope ptelifolia aqueous extract. 4.2 Anabolic Effects4.2.1 Body Weight AnalysisFigure 4.2(a) shows the pattern of mean bodyweight changes of rats supplemented with distilled water throughout 28 days. Figure 4.2(b), Figure 4.2(c) and Figure 4.2(d) shows the pattern of mean bodyweight changes of rats supplemented with MPAE at dosage of 100 mg/kg, 200 mg/kg and 500 mg/kg throughout 28 days. Whereas Figure 4.2(e) shows the pattern of mean bodyweight changes of rats supplemented with 4.5 mg/kg sildenafil citrate throughout 28 days. After receiving their respective treatments for 4 weeks, there were no significant weight differences (p<0.05) observed between control negative group and treatment groups. left10776040060960424434000Figure 4.2(a) : Changes in body weight of rats supplemented with distilled water throughout 28 days.Figure 4.2(b) : Changes in body weight of rats supplemented with 100 mg/kg of MPAE throughout 28 days.252095414808500252095100952700Figure 4.2(c) : Changes in body weight of rats supplemented with 200 mg/kg of MPAE throughout 28 days. Figure 4.2(d) : Changes in body weight of rats supplemented with 500 mg/kg of MPAE throughout 28 days.156210106410800 Figure 4.2(e): Changes in body weight of rats supplemented with 4.5 mg/kg sildenafil citrate throughout 28 days. 4.2.2 Organ weight-to-body weight ratio.Similar results obtained for the relative ratios of organ-to-body weight of livers, kidneys, and testes of the rats are presented in Table 4.6.2. There were no significant changes (P<0.05) noted in relative organ weight between the control and all treatment groups (Table 4.6.2).Organ NegativeControl 100 mg/kgMPAE 200 mg/kgMPAE 500 mg/kgMPAE PositiveControlLiver 4.08 ± 0.04 4.04 ± 0.19 4.03 ± 0.15 4.09 ± 0.14 4.23 ± 0.05Kidney 0.47 ± 0.01 0.50 ± 0.03 0.49 ± 0.03 0.49 ± 0.02 0.51 ± 0.02Testis 0.04 ± 0.01 0.67 ± 0.01 0.68 ± 0.01 0.66 ± 0.01 0.65 ± 0.01-1397031661100Table 4.2 : Organ weight-to-body weight ratio of rats treated orally with distilled water, and Melicope ptelefolia extract (values are mean ± SEM of six rats).In general, increases or decreases in the body weights of animals can be used as an indicator of adverse effects of drugs and chemicals (Teo et al., 2002). However, Harizal et al. (2010) reported rather than to the toxic effects of drugs or chemicals, body fat accumulation are more closely related to increases in the body weights of animals. According to Teo et al. (2002), there will be a slight reduction in body weight gain and internal organ weights after some exposure to potentially toxic substances. Following exposure to drugs and chemical toxic substances, the reduction in body weight exceeding 10% of initial body weight and internal organ weights is considered as an indicator and sensitive index of toxicity (Teo et al., 2002; Rajeh et al., 2012). Rhiouani et al. (2008) suggested that reductions in the body weights of animals in toxicity studies may be associated with normal physiological adaptation responses to the plant extracts or compounds, which lead to low appetite and, hence, lower caloric intake by the animal. Reducing their food intake, which may lead to reductions in their body weights also might be effect from the stress in animals after being administered high doses of plant extract (Harris et al., 1998). In the present toxicity study, all rats at each dosage group continued to gain weight throughout the 28-day toxicological study and no female rats treated with MPAE groups lost more than 10% of the body weight from that of the control groups. In addition, the anabolic study for selected internal organ weight is an important index of physiological status in animals. The function of organ whether suffered any treatment related injury or not is diagnosed by the fundamental relative internal organ weight. In drug metabolism context, the liver and kidney are the primary organs that will be affected by metabolic reactions in the presence of toxicants (Jothy et al., 2011; Vaghasiya et al., 2011). The present study found that the relative organ weights-to-body weight ratio in all the MPAE-treated groups did not show any changes from the control groups, signifying that the MPAE exhibited no grossly toxic effect from the treatment.4.3 Male Sexual Behaviour test4.3.1 Ejaculation latency (ML)0210618100After 4 weeks of treatment, all groups resulted in a longer time of ejaculation latency from week 1 to week 4. Negative control showed the longest ejaculation latency meanwhile high dose group of 500 mg/kg showed the shortest ejaculation latency time (Table 4.3.1)Figure 4.3.1 Effect of MPAE on Ejaculation Latency (EL) of the male ratsThe longest time taken for ejaculation latency was by the negative control group which were 1336.17 ± 28.06 (s) for week 1, 1364.83 ± 25.8 (s) for week 2, 1392.17 ± 19.73 (s) for week 3 and 1437.17 ± 23.68 (s) for week 4.The shortest time taken for ejaculation latency was by the high dosage group of 500 mg/kg which were 867.00 ± 25.74 (s) for week 1, 886.17 ± 26.05 (s) for week 2, 915.33 ± 25.77 (s) for week 3 and 945.83 ± 23.42 for week 4.SPSS data showed the low dosage group of 100mg/kg had a significant difference in week 1 compared to the negative control group. Medium dosage group of 200 mg/kg and high dosage group of 500 mg/kg showed significant difference in week 1, week 2, week 3 and week 4 compared to the negative control group. These results suggest that MPAE really does show the effect in increasing the ejaculation latency05506720Organ Week 1 Week 2 Week 3 Week 4Negative Control 1336.17 ± 28.06 1364.83 ± 25.81 1392.17 ± 19.73 1437.17 ± 23.68100 mg/kg *1225.67 ± 24.14 1257.83 ± 22.26 1308.50 ± 22.61 1372.33 ± 14.75200 mg/kg *1151.33 ± 17.65 *1167.67 ± 17.06 *1190.00 ± 15.47 *1222.33 ± 16.73500 mg/kg * 867.00 ± 25.74 *886.17 ± 26.05 *915.33 ± 25.77 * 945.83 ± 23.42Positive Control * 899.83 ± 5.15 *910.50 ± 4.08 *929.17 ± 4.87 * 948.83 ± 3.8300Organ Week 1 Week 2 Week 3 Week 4Negative Control 1336.17 ± 28.06 1364.83 ± 25.81 1392.17 ± 19.73 1437.17 ± 23.68100 mg/kg *1225.67 ± 24.14 1257.83 ± 22.26 1308.50 ± 22.61 1372.33 ± 14.75200 mg/kg *1151.33 ± 17.65 *1167.67 ± 17.06 *1190.00 ± 15.47 *1222.33 ± 16.73500 mg/kg * 867.00 ± 25.74 *886.17 ± 26.05 *915.33 ± 25.77 * 945.83 ± 23.42Positive Control * 899.83 ± 5.15 *910.50 ± 4.08 *929.17 ± 4.87 * 948.83 ± 3.83Table 4.3.1: Effect of MPAE on Ejaculation Latency (ML) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.4.3.2 Intromission latency (IL) 1514475253746000After 4 weeks of treatment, all groups resulted in a shorter time of intromission latency from week 1 to week 4. Negative control showed the longest ejaculation latency meanwhile positive control showed the shortest ejaculation latency time.Figure 4.3.2 Effect of MPAE on Intromission (IL) of the male ratsThe longest time taken for intromission latency was by the negative control group which were 778.50 ± 46.26 (s) for week 1, 700.50 ± 41.57 (s) for week 2, 153.00 ± 10.50 (s) for week 3 and 261.17 ± 32.44 (s) for week 4.The shortest time taken for ejaculation latency was by the positive control group which were 299.17 ± 6.25 (s) for week 1, 190.17 ± 15.21 (s) for week 2, 915.33 ± 25.77 (s) for week 3 and 84.50 ± 11.99 for week 4.left4261485Organ W1 W2 W3 W4Negative Control 778.50 ± 46.26 700.50 ± 41.57 500.00 ± 29.13 261.17 ± 32.44100 mg/kg * 565.17 ± 23.83 * 399.17 ± 35.02 * 286.83 ± 9.97 255.50 ± 15.36200 mg/kg * 534.50 ± 17.34 * 340.17 ± 15.12 * 286.17 ± 5.03 233.67 ± 12.34500 mg/kg *15.50 ± 18.15 * 264.83 ± 8.44 * 158.33 ± 4.36 * 110.67 ± 10.21Positive Control * 299.17 ± 6.25 *190.17 ± 15.21 * 153.00 ± 10.50 * 84.50 ± 11.990Organ W1 W2 W3 W4Negative Control 778.50 ± 46.26 700.50 ± 41.57 500.00 ± 29.13 261.17 ± 32.44100 mg/kg * 565.17 ± 23.83 * 399.17 ± 35.02 * 286.83 ± 9.97 255.50 ± 15.36200 mg/kg * 534.50 ± 17.34 * 340.17 ± 15.12 * 286.17 ± 5.03 233.67 ± 12.34500 mg/kg *15.50 ± 18.15 * 264.83 ± 8.44 * 158.33 ± 4.36 * 110.67 ± 10.21Positive Control * 299.17 ± 6.25 *190.17 ± 15.21 * 153.00 ± 10.50 * 84.50 ± 11.99SPSS data showed that the low dosage group of 100mg/kg and medium dosage group of 200 mg/kg showed a significant difference in week 1, week 2 and week 3 compared to the negative control group. The high dosage group of 500 mg/kg and positive control showed significant difference in week 1, week 2, week 3 and week 4 compared to negative control group.Table 4.3.2 : Effect of MPAE on Intromission Latency (IL) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.054.3.3 Mount latency (ML) 13982702447053After 4 weeks of treatment, all groups resulted in a shorter time of mount latency from week 1 to week 4. Negative control showed the longest mount latency meanwhile positive control showed the shortest ejaculation latency time.Figure 4.3.3 Effect of MPAE on Mount Latency (ML) of the male ratsThe longest time taken for mount was by the negative control group which were 736.17 ± 35.48 (s) for week 1, 670.67 ± 40.04 (s) for week 2, 471.50 ± 24.89 (s) for week 3 and 246.67 ± 33.45 (s) for week 4.The shortest time taken for ejaculation latency was by the positive control group which were 280.67 ± 6.45 (s) for week 1, 168.00 ± 17.08 (s) for week 2, 119.17 ± 11.87 (s) for week 3 and 71.33 ± 10.56 for week 4.-254002893695Organ W1 W2 W3 W4Negative Control 736.17 ± 35.48 670.67 ± 40.04 471.50 ± 24.89 246.67 ± 33.45100 mg/kg * 540.00 ± 28.98 * 377.17 ± 30.28 *276.50 ± 9.73 236.83 ± 17.58200 mg/kg * 518.50 ± 17.79 * 323.00 ± 16.02 *270.67 ± 5.52 222.83 ± 11.89500 mg/kg * 344.50 ± 17.28 * 238.67 ± 10.45 *133.83 ± 7.83 * 101.00 ± 9.78Positive Control * 280.67 ± 6.45 * 168.00 ± 17.08 * 119.17 ± 11.87 * 71.33 ± 10.560Organ W1 W2 W3 W4Negative Control 736.17 ± 35.48 670.67 ± 40.04 471.50 ± 24.89 246.67 ± 33.45100 mg/kg * 540.00 ± 28.98 * 377.17 ± 30.28 *276.50 ± 9.73 236.83 ± 17.58200 mg/kg * 518.50 ± 17.79 * 323.00 ± 16.02 *270.67 ± 5.52 222.83 ± 11.89500 mg/kg * 344.50 ± 17.28 * 238.67 ± 10.45 *133.83 ± 7.83 * 101.00 ± 9.78Positive Control * 280.67 ± 6.45 * 168.00 ± 17.08 * 119.17 ± 11.87 * 71.33 ± 10.56SPSS data showed that the low dosage group of 100mg/kg and medium dosage group of 200 mg/kg showed a significant difference in week 1, week 2 and week 3 compared to the negative control group. High dosage group of 500 mg/kg and positive control showed significant difference in week 1, week 2, week 3 and week 4 compared to the negative control group.Table 4.3.3: Effect of MPAE on Mount Latency (ML) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.4.3.4 Intromission frequency (IF)1460310268860900After 4 weeks of treatment, all groups resulted in a longer time of intromission frequency from week 1 to week 4. The negative control showed the longest mount latency meanwhile positive control showed the shortest ejaculation latency time.Figure 4.3.4 Effect of MPAE on Mount Latency (ML) of the male rats.The longest time taken for mount was by the negative control group which were 11.00 ± 1.41 (s) for week 1, 12.83 ± 1.47 (s) for week 2, 471.50 ± 24.89 (s) for week 3 and 246.67 ± 33.45 (s) for week 4.The shortest time taken for ejaculation latency was by the positive control group which were 280.67 ± 6.45 (s) for week 1, 168.00 ± 17.08 (s) for week 2, 16.33 ± 2.03 (s) for week 3 and 20.67 ± 2.99for week 4.SPSS data showed that the high dosage group of 500mg/kg showed a significant difference in week 2 at 20.00 ± 1.81 (s) compared to the negative control group. Positive control showed significant difference in week 2, week 3 and week 4 compared to the negative control group.left3848100Organ W1 W2 W3 W4Negative Control 11.00 ± 1.41 12.83 ± 1.47 16.33 ± 2.03 20.67 ± 2.99100 mg/kg 12.83 ± 0.60 16.17 ± 0.83 20.17 ± 1.51 24.50 ± 2.40200 mg/kg 13.17 ± 1.19 17.00 ± 1.53 20.67 ± 2.16 24.00 ± 2.73500 mg/kg 15.50 ± 0.96 * 20.00 ± 1.81 23.67 ± 2.32 29.67 ± 2.26Positive Control 15.33 ± 1.33 * 21.67 ± 2.08 * 28.67 ± 2.39 * 34.17 ± 2.320Organ W1 W2 W3 W4Negative Control 11.00 ± 1.41 12.83 ± 1.47 16.33 ± 2.03 20.67 ± 2.99100 mg/kg 12.83 ± 0.60 16.17 ± 0.83 20.17 ± 1.51 24.50 ± 2.40200 mg/kg 13.17 ± 1.19 17.00 ± 1.53 20.67 ± 2.16 24.00 ± 2.73500 mg/kg 15.50 ± 0.96 * 20.00 ± 1.81 23.67 ± 2.32 29.67 ± 2.26Positive Control 15.33 ± 1.33 * 21.67 ± 2.08 * 28.67 ± 2.39 * 34.17 ± 2.32Table 4.3.4: Effect of MPAE on Intromission Frequency (IF) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.054.3.5 Post-ejaculatory interval (PEI) 1460206271549700After 4 weeks of treatment, all groups resulted in a shorter time of post-ejaculatory interval from week 1 to week 4. Negative control showed the longest mount latency meanwhile positive control showed the shortest ejaculation latency time. Figure 4.3.5: Effect of MPAE on Post-Ejaculatory Interval (PEI) of the male rats.The longest time taken for post-ejaculatory interval was by the negative control group which were 349.67 ± 4.33 (s) for week 1, 339.83 ± 3.62 (s) for week 2, 328.00 ± 6.11 (s) for week 3 and 314.00 ± 6.22 (s) for week 4.The shortest time taken for ejaculation latency was by the positive control group which were 139.17 ± 3.18 (s) for week 1, 131.33 ± 3.38 (s) for week 2, 124.67 ± 2.95 (s) for week 3 and 120.67 ± 3.16 for week 4.-412753765493Organ W1 W2 W3 W4Negative Control 349.67 ± 4.33 339.83 ± 3.62 328.00 ± 6.11 314.00 ± 6.22100 mg/kg * 308.50 ± 7.50 * 300.33 ± 6.94 * 293.33 ± 6.56 *283.17 ± 6.12200 mg/kg * 272.50 ± 2.92 * 269.67 ± 2.47 *266.17 ± 2.87 *258.67 ± 2.38500 mg/kg * 154.67 ± 2.92 * 145.83 ± 2.27 *141.50 ± 2.47 *137.50 ± 2.09Positive Control * 139.17 ± 3.18 * 131.33 ± 3.38 *124.67 ± 2.95 *120.67 ± 3.160Organ W1 W2 W3 W4Negative Control 349.67 ± 4.33 339.83 ± 3.62 328.00 ± 6.11 314.00 ± 6.22100 mg/kg * 308.50 ± 7.50 * 300.33 ± 6.94 * 293.33 ± 6.56 *283.17 ± 6.12200 mg/kg * 272.50 ± 2.92 * 269.67 ± 2.47 *266.17 ± 2.87 *258.67 ± 2.38500 mg/kg * 154.67 ± 2.92 * 145.83 ± 2.27 *141.50 ± 2.47 *137.50 ± 2.09Positive Control * 139.17 ± 3.18 * 131.33 ± 3.38 *124.67 ± 2.95 *120.67 ± 3.16SPSS data showed all treated groups low dosage group of 100mg/kg and medium dosage group of 200 mg/kg, high dosage group of 500 mg/kg and positive cotrol showed a significant difference on week 1, week 2, week 3 and week 4 compared to negative control group.Table 4.3.5: Effect of MPAE on Post-Ejaculatory Interval (PEI) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.From this study, mount latency (ML) and intromission latency (IL) are two useful parameters to indicate the sexual motivation of the male rats. High or increased sexual motivation can be evaluated based on the decreased latencies of males before they show certain sexual behaviors such as mount and intromission (Hamson et al., 2009). Outcome of this study showed MPAE’s success to decrease both mount and intromission latencies, indicating that it did contribute to the sexual motivation of the male rats. In addition, there was also a significant improvement in intromission frequency (IF) for 500 mg/kg in week 2, which was the indicator for mating. According to Tajuddin et al. (2004), intromission frequency is an important parameter that shows the sexual vigour, libido and potency of the rats. It can be affected by the level of brain monoamines. Sexually active rats treated with reserpine and agomelatine showed low intromission frequency (Dewsbury and Davis, 1970; Canpolat et al., 2016) as they deplete the level of brain dopamine. Post-ejaculatory interval (PEI) is important in measuring the rats’ copulatory ability. PEI indicates potency, libido and time taken to recover from exhaustion after the first series of mating (Tajuddin et al., 2004). Male sexual behaviour is regulated in many different brain areas. However, these different areas were found to be regulated by the level of dopamine. MPOA lesions were found to be associated with copulation but not sexual motivation (Hull and Dominguez, 2007). Instead, lesions of the basolateral amygdala (blAMY) is the one responsible for reduced sexual motivation (Becker, 2009). On the other hand, lesions on VTA and nucleus accumbens (NAc) increased PEIs and decreased noncontact erections but did not affect copulation (Hull, Wood and McKenna, 2006). Among these areas, MPOA is the major brain area that is responsible for male sexual behaviour. According to Hull and Dominguez (2007), the level of dopamine in MPOA is increased before and during copulation and NO is the major factor that stimulates the release of dopamine in MPOA. Sexual behaviour in male rat needs both estrogen and testosterone for its function. There are two components of sexual behaviour which are sexual motivation (appetitive) and sexual ability (consummatory) (Becker, 2009). As proposed in “aromatization hypothesis”, sexual behaviour in male rats is primarily activated by estrogen but androgen also plays some contribution towards motivation and performance (Hull and Dominguez, 2007). Therefore, it is suggested that administration of MPAE is able to increase the level of the androgens in male rats that are sufficient to induce penile erection.The result might also be influenced by the factor of time when the experiment was carried out. The male rats in the study were kept at normal light-dark cycle and were tested during the light cycle between time 14:00 to 17:00. Many other studies (Yakubu and Afloyan, 2009; Das et al., 2016; Suresh et al., 2009) carried out the behaviour tests in the evening or during the dark phase between the time 19:00 to 22:00. However, there was also a study that house the animals in a normal 12-hour light-dark cycle and tested the sexual behaviour during the light phase (Chauhan et al., 2007). Fantie et al. (1984) demonstrated that lighting condition could affect copulatory behaviour in rats although Agmo (1997) contrastingly reported that there would be no differences between the sexual behaviour carried out under dim or bright light.4.4 Penile Erection Index-4283248392800Following 4 weeks of treatment, all treated groups showed a positive result of post- ejaculatory penile Erection IndexFigure 4.4 Effect of MPAE on Penile Erection Index (PI) of the male ratsLowest PI recorded was by the negative control group at 11.81 ± 5.12 whereas the highest PI was by the positive control group at 84.72 ± 17.44.SPSS data showed a significant difference of high dosage group of 500 mg/kg at 68.75 ± 15.65 as compared to negative control. The positive control group also showed a significant difference as compared to the negative control group. Yet, there were no significant difference (p>0.05) found between other treatment groups compared to 63501949672Groups PINegative Control 11.81 ± 5.12100 mg/kg 27.78 ± 10.64200 mg/kg 43.75 ± 16.13500 mg/kg *68.75 ± 15.65Positive Control *84.72 ± 17.4400Groups PINegative Control 11.81 ± 5.12100 mg/kg 27.78 ± 10.64200 mg/kg 43.75 ± 16.13500 mg/kg *68.75 ± 15.65Positive Control *84.72 ± 17.44negative control.Table 4.4: Effect of MPAE on Penile Erection index (PI) of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.The findings from this experiment showed significant effect on PI for 500 mg/kg and Positive control which could possibly involve NO-cGMP pathway mechanism, but not on other tested parameters. As stipulated, the involvement of NO from penile erection mechanism should have increased the level of brain dopamine, thus leading to significant changes on the other sexual behaviour parameters. Penile erection occurs as a result of consequent relaxation of the arterial and trabecular smooth muscle that causes vasodilation of the penile arteries, thus increasing the blood flow to the penis (Corbin, 2004). The significant effect of MPAE on PI might be contributed from its alkaloid, daucosterol and glycosidic constituents known as ptelefosides (Zhang et al., 2012). Daucosterol is a steroid saponin that is sitosterol attached to a ?-D-glucopyranosyl residue at position 3 via a glycosidic linkage. Yakubu and Afloyan (2009) mentioned the androgenic and vasodilatory effect of saponins and alkaloids. Glycosides are molecules consisting of a glycone (sugar) and aglycone (non-sugar group) group.4.5 Sperm Parameters4.5.1 Sperm countsGroups Description Sperm Count (x10 7 cell/ml)I Distilled Water 7.55 ± 0.12II 100 mg/kg 7.75 ± 0.11III *200 mg/kg 8.17 ± 0.10IV *500 mg/kg 8.37 ± 0.10V *SildenafilCitrate8.91 ± 0.08Table 1.0 shows the sperm count for group I, II, III, IV and V of the Sprague-Dawley male rats after consuming certain doses of MPAE (for treated group), distilled water (for negative control group) and sildenafil citrate (for positive control group). Group IV (at 8.91 ± 0.08) displayed the highest mean sperm count followed by Group IV (8.37 ± 0.10), Group III (8.17 ± 0.10), Group II (7.75 ± 0.11) and the lowest, Group I (7.55 ± 0.12). The increased in sperm count indicates the effect of MPAE on testicular spermatogenesis. Testiscular histology studies are underway to verify this conclusion.Table 4.5.1: Effect of MPAE on Sperm count of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.left1017850Figure 4.5.1 Effect of MPAE on Sperm Count of the male rats4.5.2 Sperm MotilitySperm Motility revealed that male rats from treated and non-treated groups had non-significant differences. The increase in grade sperm motility with MPAE suggests that MPAE affects sperm maturation process in epididymis. The body weight of the control and treated animal did not show significant changes throughout the course of the investigation (data not shown).Groups Description Progressive Non-Progressive ImmotileI Distilled Water 0.49 ± 0.01 0.15 ± 0.01 0.36 ± 0.01II 100 mg/kg 0.51 ± 0.03 0.11 ± 0.00 0.38 ± 0.03III 200 mg/kg 0.52 ± 0.02 0.10 ± 0.02 0.38 ± 0.04IV 500 mg/kg 0.50 ± 0.01 0.10 ± 0.02 0.40 ± 0.02V Sildenafil Citrate 0.52 ± 0.02 0.09 ± 0.02 0.39 ± 0.03Table 4.5.2: Effect of MPAE on Sperm Motility of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.0513500103883660Figure 4.5.2 Effect of MPAE on Sperm Motility of the male rats.4.5.3 Sperm ViabilitySperm viability was also increased with MPAE. For example, at lower dose Group 1 (distilled water) the viability was 0.78% ± 0.01, Group II (100 mg/kg), the viability was 0.80% ± 0.01, Group III (200 mg/kg), Group IV (500 mg/kg) and Group IV (sildenafil citrate), the viability was 0.83% ± 0.01. Group IV (at 8.91 ± 0.08) displayed the highest mean sperm count followed by Group IV (8.37 ± 0.10), Group III (8.17 ± 0.10), Group II (7.75 ± 0.11) and lowest by Group I (7.55 ± 0.12)Groups Description Sperm Viability (%)I Distilled Water 0.78 ± 0.01II 100 mg/kg *0.80 ± 0.01III *200 mg/kg *0.83 ± 0.00IV *500 mg/kg *0.83 ± 0.01V *SildenafilCitrate*0.83 ± 0.01-164465595376000Table 4.5.3: Effect of MPAE on Sperm viability of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.Figure 4.5.3 Effect of MPAE on Sperm Viability of the male rats.4.5.4 Sperm MorphologySimilarly, to the sperm count parameter, results in the sperm morphology showed significant increase for Group II, III and IV. Group IV (at 0.93 ± 0.01) displayed the highest Normal Sperm Morphology (%) (x10 7 cell/ml) followed by Group V (0.92 ± 0.01), Group III (0.89 ± 0.00), Group II (0.85 ± 0.01) and lowest by Group I (0.80 ± 0.01). The result indicated that MPAE treatment was able to maintain normal morphology of sperm as any sperm deformities were associated with functional deficiencies. This caused reduced motility and fertilization ability. Besides that, morphological sperm parameters were important for fertilization step meanwhile DNA integrity were crucial as it was related with the establishment and continuation of pregnancy (Thomlison et al., 2001). Apart from that, sperm morphology is the most informative semen measurement for discriminating between fertile and infertile male.Groups Description Normal Sperm Morphology (%) (x10 7 cell/ml)I Distilled Water 0.80 ± 0.01II 100 mg/kg 0.85 ± 0.01III *200 mg/kg 0.89 ± 0.00IV *500 mg/kg 0.93 ± 0.01V *SildenafilCitrate0.92 ± 0.01Table 4.5.4: Effect of MPAE on Sperm morphology of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p<0.05.014033500Figure 4.5.4 Effect of MPAE on Sperm Morphology of the male rats.This study was conducted to assess the effect of MPAE on sperm characteristic in adult male Sprague Dawley rats for 28 days by comparing different dosages of MPAE (100 mg/kg, 200 mg/kg and 500 mg/kg) and distilled water as negative control.Some andrological parameter was used including sperm count, viability, motility and morphology in order to monitor the fertility of rats. The mean sperm count in rats treated with different dosage (100 mg/kg, 200 mg/kg and 500 mg/kg) showed significant difference when compared to negative control group. One possibility for this effect is due to increase in hormones such as FSH, LH or testosterone which tend to increase sperm count (Jana et al., 2006). LH and FSH are essential for quantitatively normal spermatogenesis in pubertal rats (Kulin and Reither, 1973). The increase of LH and FSH are consequent rising in testosterone production, which may therefore be held responsible for spermatogenesis process in rat treated with MPAE. Moreover, MP exhibit anti-oxidant properties (Abas et al., 2006). Trans et. al., 2005 reported that antioxidants could increase sperm output in healthy rats. Generally, anti-oxidant reduces cell wide oxidative damage, support redox balance within leydig cells, release leydig cells from oxidative inhibition of testosterone synthesis and the rate of testosterone secretion (Glade et al., 2015). Therefore, by reducing oxidative stress, it can safely increase the testosterone status.In sperm count, the methods such as counting chambers, computed assisted sperm analysis system (CASA) and automated system such as IVOS HTMIDENT; HTM integrated Visual Optical System semen analyzer were commonly used (Krause and Viethen., 1999; Strade., 1996; Clement et al., 1995). However, among these methods, the use of counting chambers was problematic because of variation (Choi et al., 2008). Computer-Aided Sperm Analysis (CASA) systems are the evolution of multiple photomicrography exposure and video-micrography techniques for spermatozoa track, using computer equipped with Imaging software. A CASA system refers to the physical equipment used to visualize and digitalize static and dynamic sperm images and to the methods used to process and analyze them (Boyer et.al., 1989).Sperm motility is an important parameter to evaluate sperm quality and fertilizing potential (Mnagelsdorf et al., 2003). Furthermore, it was clear that spermatozoa which were highly motile will have a greater opportunity of fertilization. This present study showed that the total mean percentage of motile sperms was significant for group IV and V compared to the negative control. A correlation between mitochondrial activities and motile sperm has been shown by using flow cytometry (Auger et al., 1993). Hung et al., (2008) demonstrated that ATP from mitochondrial sources did contribute to sperm motility in rhesus monkey sperm. Moreover, the study also found a close and positive relationship between sperm motility and mitochondrial enzyme-specific activities, suggesting that more specific mitochondrial dysfunction could be the underlying cause of idiopathic asthenozoospermia (Ruiz-Pesini et al., 1998). However, there was argument about whether ATP produced by the mitochondria can be effectively delivered from the mitochondria supply to entire flagellum (Nevo and Rikmenspoel, 1970; Adam and Wei, 1975; Du et al., 1994).As expected such measurement have, in general, led to variable results between laboratories or observers and the value of such measurement of motility in predicting fertility was still questionable due to the subjectivity of the technique. Subjective estimation of the sperm quality was affected by various causes; particularly variation in the observer’s experience, the endpoints that are chosen and how these measures are interpreted (Rurangwa et al., 2004).Sperm motility was one of the most important factors in determining the ability to produce viable sperms (Oyeyemi et al., 2000). True viability of spermatozoa was eventually defined by their capacity to move and fertilize an egg (Rurangwa et al., 2004). In this study, the viability of the sperm had been investigated by using Eosin-Nigrosin staining where the stain was based on the integrity of the sperm membrane. The ratio of the in vitro dead sperm cells was observed due to Eosin penetrating and staining the dead autolysing sperm cells while viable sperm repel the stain. In motility analysis, there were sperms that were graded with immotile as no movement, observed under the microscope. But, in certain condition, the immotile sperm were still viable. Therefore, in cases of low motility, viability analysis was carried out in order to confirm the status of sperm viability. MPAE was able to preserve the sperm viability as MPAE shows significant changes when compared to normal (negative control) for group IV. This might be due to Melicope ptelifolia having the potential in increasing the secretion of testosterone. Testosterone hormone is the principal of male reproductive hormones and play an important role in sperm quality (Parhizkar et al., 2013). Still, the mechanism on how sperm viability was maintained is still unknown (Smith and Nothnick, 1997).The percentage of normal sperm morphology show significant increase for Group III, IV and V. The result indicated that MPAE treatment was able to maintain normal morphology of sperm as any sperm deformities was associated with functional deficiencies. This caused reduced motility and fertilization ability. Besides that, morphological sperm parameters were important for fertilization step meanwhile DNA integrity were crucial as it was related with the establishment and continuation of pregnancy (Thomlison et al., 2001). Apart from that, sperm morphology is the most informative semen measurement for discriminating between fertile and infertile male. This present study concludes that the treatment with Melicope ptelifolia aqueous extract did produce a clear change in the sperm quality.4.6 Testosterone LevelGroups Description Testosterone Level (ng/mL) I Distilled Water 2.7 ± 0.23II 100 mg/kg 4.10 ± 0.22III 200 mg/kg 6.68 ± 0.38IV *500 mg/kg 11.02 ± 0.66V *Sildenafil Citrate 19.80 ± 2.02Table 1.4 shows the oral administration of MPAE (100 mg/kg, 200 mg/kg and 500 mg/kg) increased the testosterone level. At 500 mg/kg, it showed a significant increase compared to the control group. The significantly increased level of testosterone in 500 mg/kg group compared to control showed that testosterone has correlate with the increase of libido and sperm quality. The receptive female is believed to affect the activation of the hypothalamic-pituitary-testicular complex (HPTC) in the male which is indicated by increased serum testosterone and luteinizing hormone levels (Bartke and Dalterio, 1975).Table 4.6: Effect of MPAE on Testosterone level of the male rats. Each column represents the mean ± S.E.M. of 6 rats. * significantly different at p;0.0513648300260Figure 4.6 Effect of MPAE on Testosterone Level of the male rats.The significant increased level of testosterone in 500 mg/kg group compared to control showed that testosterone has correlate with the increase of libido and sperm quality. The receptive female is believed to affect the activation of the hypothalamic-pituitary-testicular complex (HPTC) in the male which is indicated by the increased serum testosterone and luteinizing hormone levels (Bartke and Dalterio, 1975). The excessive reactive oxygen species (ROS) generated by cell metabolism may suppress the ability of sperm function leading to infertility. ROS is physiologically generated during mitochondrial respiration in normal cell metabolism (de-Lamirande et al., 1997). The level of ROS reflects the highly specific lipid composition of sperm membrane cells as the main substrate for lipid peroxidation. At the low level of lipid peroxidation, where the ROS is low, the motility and functional ability of sperm cells to interact with zona pellucida will increase. However, the pathological lipid peroxidation of sperm membrane due to the high level of ROS will undergo unbalance oxidative stress in the testes (Aitken et al., 1989; Aitken and Roman, 2008). The presence of the intra and extracellular antioxidants of enzymatic and non-enzymatic system however will scavenge free radicals as self-protection mechanism (Alvarez et al., 1987). Intake of superoxide dismutase supplement was leading to the progressive sperm motility and improved the acrosome reaction (Griveau and Le Lannou, 1997). In this study, the administration of MPAE has succeeded in increasing the antioxidant enzyme activity of superoxide dismutase (SOD) and catalase (CAT) suggesting the MPAE has anti-oxidative effect that contribute to male fertility.4.7 Histology of TestesPhotomicrograph of rat testis in Group IV shows active germinal epithelium with successive stages of spermatogenesis and normal connective tissue. Lumen of seminifeous tubules were filled with spermatozoa. The high number of spermatogenic cells contributed to the high sperm count. Photomicrograph of rat testis in V shows normal appearance of spermatogenesis and normal intertubular spaces with connective tissue. Lumen of seminifeous tubules has spermatozoa as control group. While the treated groups II, III, IV and V appeared more than normal histological characteristics (a high density) of spermatozoa as compared with group I.05038033004735076274268347749202730146A00AFigure 4.7(a) Photomicrograph of rat testis in Group I (Distilled water) showing degeneration of spermatogenic elements with appearance of vacuoles. Loosened intertubular spaces increased4807225206053748613522073872BB47487962074877B00Bleft122500400 Figure 4.7(b) Photomicrograph of rat testis in Group II (100mg/kg MPAE) showing less degeneration of spermatogenic elements with appearance of vacuoles.48613992231921CC4814599222564580010518829000Figure 4.7(c) Photomicrograph of rat testis in Group III (200 mg/kg of MPAE) showing lesser degeneration of spermatogenic elements with appearance of vacuoles.4982807227394450560572272987D00Dcenter152654000Figure 4.7(d)Photomicrograph of rat testis in Group IV (500 mg/kg of MPAE) showing normal features with successive stages of spermatogenesis and normal Intertubular spaces with connective tissues.51101202105698E00Ecenter528031900Figure 4.7(e)Photomicrograph of rat testis in Group V (Sildenafil Citrate) showing normal features with less successive stages of spermatogenesis and normal intertubular spaces with connective tissues.The results showed that the spermatogenic cell counts increased in the seminiferous tubules of group IV compared to group I. MPAE may play an active role in steroidogenesis and spermatogenesis, leading to an increase in the production of testosterone in the testis and enhancement of germ cell proliferation, which in turn has increased spermatozoa production (Chan et al., 2009). In the testis, one may suggest that the quassinoid Eurycomanone (Burkill, 1966) has passed through the blood-testis barrier and acted on the Leydig cells to initiate testosterone production. The resultant testosterone may subsequently bind to the androgen binding protein (ABP) produced by Sertoli cells and then initiate the development of spermatozoa from the spermatogonial germ cells in the seminiferous tubules (Rmmerts, 1992.CHAPTER 5CONCLUSION AND RECOMMENDATION OF WORK5.1 ConclusionIt is well known now that herbs have been tested as an alternative medicine to treat different types of human disease including erectile dysfunction. This present study was undertaken to find possible contribution and benefits of Melicope ptelifolia towards erectile dysfunction and related diseases. After the rats received their respective dose for 4 weeks, there was no death recorded for acute toxicity test. Also, there was no significant weight differences (p;0.05) observed between control negative group and treatment groups. Interestingly, there was a significant increment of ejaculation latency and intromission latency (p;0.05) detected in the MPAE treated group when compared to the control negative group. Positive reduction could also be seen in the intromission latency, mount latency and post-ejaculatory interval in the MPAE treated group when compared to control negative group. MPAE has also showed a positive result of post-ejaculatory penile Erection Index. There was a significant difference (p;0.05) detected in the MPAE treated group at high dose 500 mg/kg when compared with the control negative group. MPAE also successfully showed a significant difference in sperm counts , sperm viability and sperm morphology of MPAE treated groups compared to the control negative group. However, no significant difference was recorded for the MPAE treated group compared to the control negative group for sperm motility. This present study concludes that the treatment with Melicope ptelifolia aqueous extract did produce a clear change in the sperm quality.Testosterone level for MPAE treated groups showed a significant difference compared to the control negative group. The significant increased level of testosterone in 500 mg/kg group compared to the control showed that testosterone has correlate with the increase of libido and sperm quality. As the testes section of the MPAE treated group was observed, the testes appeared normal. Overall, the present study provides scientific data that Melicope ptelifolia have helped in potential erectile dysfunction. No mortality was found during the 28 days after the oral administration of a single oral dose of the extract. LD50 for MPAE exceeded 5000 mg/kg. Therefore, consumption of Melicope ptelifolia is a one-step action towards the prevention of erectile dysfunction. 5.2 Recommendation of workMelicope ptelifolia contained various bioactive constituents which may act to increase fertility effect. Yet, these specific bioactive compound are not investigated in this present study. It can be anticipated that a fertility boost constituent and most appropriate level of intake in Melicope ptelifolia will be determined in the near future for both the general population and populations at risk of developing particular diseases.