Toxicological Evaluation of Medicinal Plants. Chrysophyllum Albidum Seed Cotyledon

Table of Content

INTRODUCTION AND LITERATURE REVIEW

TRADITIONAL MEDICINE
Traditional Medicine in Nigeria
Advantages of Traditional Herbal Medicine
Limitations of Traditional Herbal Medicine
Safety Evaluation of Traditional Herbal Medicine

TOXICOLOGICAL SIGNIFICANCE OF PLANT SECONDARY METABOLITES

TOXICOLOGICAL STUDIES AND HERBAL MEDICINE
Overview of Pre-Clinical Toxicity Studies
Cell-Based Cytotoxicity Tests
Single Dose (Acute) Toxicity Studies
Repeated Dose Toxicity Tests
Reproductive Toxicity and Genotoxicity Studies
Carcinogenicity Studies
Local Tolerance Studies
Toxicokinetic Studies
Relevance of Recovery Studies in Toxicity Studies

PATHOLOGICAL ASSESSMENT IN PRE-CLINICAL TOXICITY STUDIES
Clinical Pathology
Anatomical Pathology

RELEVANCE OF ANIMAL MODELS IN TOXICOLOGY

RECOGNITION OF THE ADVERSE AND NON-ADVERSE EFFECT IN TOXICITY STUDIES

TOXIC SUBSTANCES
Types of Phytotoxicity

CHRYSOPHYLLUM ALBIDUM (CA)
Nomenclature
Botanical Description of Chrysophyllum albidum
Ethnomedicinal Significance of Chrysophyllum albidum
Pharmacological Profile of Chrysophyllum albidum

FINAL REMARKS

Reference

INTRODUCTION AND LITERATURE REVIEW

TRADITIONAL MEDICINE

Traditional medicine (TM) also known as ‘Complementary and Alternative’ medicine is an indigenous form of medicine (WHO, 2005a; Alves and Rosa, 2007). TM is an essential aspect of the rich cultural heritage that has survived through many generations and includes the indigenous way of preventing, diagnosing and managing diseases. Among the widely known and globally practiced forms of TM are: Ayurveda, a form of traditional medicine in India (Morgan, 2002); Unani, an indigenous form of medicine originating from Greece (Sofowora, 2012); Tibetan medicine, a derivative of methods from both Ayurvedic medicine and the Chinese traditional medicine (Li, 2000); Neo - Western Herbalism, encompassing the European and American herbal medicine (Elvin-Lewis, 2001; WHO, 2002); Traditional Chinese Medicine, which accounts for around 40% of all health care delivery in China (Wu, 2005); and African Traditional Medicine, accounting for up to 80% of the African health care needs (Gurib-Fakim, 2006). These underscore the global relevance and increasing recognition of traditional medicine as a veritable tool in addressing Man’s health needs.

In a bid to accommodate the worldwide diversity of cultures and their indigenous mode of traditional medicine practices, the World Health Organization (WHO) defines traditional medicine as: “the sum total of all the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures whether explicable or not, used in maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness” (WHO, 2005b). In addition, Medicinal plants are defined as herbal preparations produced by subjecting plant materials to extraction, fractionation, purification, concentration or other physical or biological processes which may be produced for immediate consumption or as a basis for herbal products (WHO, 2001).

Man’s knowledge of medicinal plants and traditional system of medicine dates back to 1500 BC from the Eberus Papyrus in Egypt. Such knowledge may have been acquired through instinct, experiences or careful observations of the effects of effects of such plants on domestic animals, and subsequently passed from generation to generation through tutelage or other anecdotal forms of communication (Sofowora, 2012).

Traditional medicine in Africa has grown considerably, having approximately 60,000 of the world’s higher plant species (Dzoyem et al., 2013). Its ease of accessibility and affordability has made it “the most economical and available system of health care and highly favoured by a large number of the African population in rural and semi-urban areas” (Kasilo et al., 2010; Kamsu-Foguem and Foguem, 2014). Despite its popularity, information bordering on African traditional medicine is still largely insufficient when compared to its contemporaries around the world (Ndhlala et al., 2009; Egharevba et al., 2015a, b) due to several challenges. One of such is the quality control of herbal medicine, an issue of global importance that is indispensable for the advancement of the herbal medicine system (Sen et al., 2011). Zhang et al. (2012) pointed out that issues on quality control of herbal medicines involve internal factors arising from the drug and external factors in clinical use. Another important challenge is the issue of adverse effects caused by herbal medicines. Kamsu-foguem and Foguem (2014) noted that from the huge patronage of herbal medicine in Africa countries, it is most likely that many adverse drugs reactions will go unnoticed and unrecorded, either as a result of patients failing to report cases of adverse effect to health services, or non-availability of pharmacovigilance analysis. In spite of this, a few African countries notably South Africa, Nigeria, and Cameroon have subsequently introduced herbal / traditional medicine as part of their pharmacovigilance systems (Fokunang et al., 2011).

Traditional Medicine in Nigeria

Nigeria abounds in its huge biodiversity of flora especially medicinal plants used in the treatment of many tropical diseases. This curative property has been attributed to the presence of certain phytochemicals present in these tropical medicinal plants (Okwu and Okwu, 2004; Onwuliri, 2004).

In Nigeria, the use of herbal medicine singly or in combination with orthodox medicine for management of various ailments is a frequent practice (Ezuruike and Prieto, 2014). Several studies have reported effective use of herbal medicine in Nigeria for the management of diseases including those of adults with various forms of chronic illness (Amira and Okubadejo, 2007; Ogbera et al., 2010); on pregnant women (Fakeye et al., 2009); children with chronic illness (Oshikoya et al., 2008). Also, increasing patronage of herbal preparations has been reported for, among other purposes - the treatment of malaria and hypertension (Oreagba et al., 2011). Such increasing patronage may be attributed to but not limited to the perceived safety of herbal formulation when compared to orthodox medicine (Amira and Okubadejo, 2007; Fakeye et al., 2009), mostly due to their natural origin, efficacy and perceived lack of adverse effect (Oreagba et al., 2011). Typical example of some Nigerian Medicinal Plants with their folkloric uses are: Rauwolfia vomitoria (used in hypertension, stroke, insomnia and convulsion) (Amole et al., 2009); Citrus parasidi seed (treatment of urinary tract infections) (Oyelami et al., 2005); Carica papaya L. (treatment of intestinal parasitosis) (Okeniyi et al., 2007); Garcinia kola (treatment of osteoarthritis) (Adegbehingbe et al., 2008); Pygeum africanum (prostatitis, aphrodisiac, Laxative) (Kim et al., 2012); Securidac longepedunculata (epilepsy, wounds, venereal diseases, dysmenorrhea) (Luyckx, 2012); Agathosma betulina (Diuretic and urinary tract antiseptic, arthritis, cellulite, cystitis, kidney infections) (Moolla and Viljoen, 2008).

In spite of these benefits of traditional herbal medicine, cases of adverse effects to some plant based herbal preparations has been reported either when used singly (Oshikoya et al., 2007) or concurrently with conventional orthodox medicine (Langlois-Klassen et al., 2007). More so, it is becoming common knowledge that the indiscriminate or non-regulated use of several herbal medicines in Nigeria and the rest of the world may put the health of their users at risk of toxicity (Kloucek et al., 2005; Nnorom et al., 2006). To address some of these concerns, in Nigeria, advanced investigations has been introduced to effectively explore the opportunities for the integration of herbal medicine into the national health system with applicable regulations (Awodele et al., 2011; Awodele et al., 2012).

Advantages of Traditional Herbal Medicine

Developing countries all over the world are faced with preventable or curable diseases (e.g. malaria) which unfortunately, are a major killer disease in this regions as a result of the poor economic conditions and lack of simple healthcare. Thus, many individuals in these countries largely rely on local traditional herbal remedies as first-line treatment of some of these tropical diseases (Willcox and Bodeker, 2004; Willcox et al., 2011). It is therefore important that the advantages of traditional medicine be viewed against the background of socio-economic status of developing countries, the magnitude of their health problem, and the few resources available (Sofowora, 1993). Generally, the advantages of traditional medicine include (a) cost-effectiveness (Pal and Shukla, 2003); (2) accessibility (WHO, 2002); (3) wider cultural acceptability among the population (Pal and Shukla, 2003); and as source of raw material for drug development, for example, reserpine from Rauwolfia species (Amole et al., 2009).

Limitations of Traditional Herbal Medicine

Despite the invaluable contributions of traditional herbal products especially in the developing countries for the treatment of various diseases such as malaria, diabetes, mental disorders, cancer, among others (Okigbo et al., 2009), the available literature points to some drawbacks which are briefly discussed below.

- Lack of Reproducibility in Bioactivity : A major constraint in plant based drug discovery is the lack of reproducibility of activity, attributable to such factors as time of plant collection, location, variation in the methods used for extraction and biological activity determination among others (Cordell, 2000). Adequate standardization, regulation of the production process, and full control of growing conditions, resulting in cost-effective and quality-controlled production, thereby improving the herbal medicines (Goldman, 2001; Ahmad et al., 2006).
- Herb – Drug Interactions: it has been reported that when medications are used together, they can interact in the body, causing changes in the way the herbs and/or the drug works. Such changes are referred to as herb–drug interactions (Ahmad et al., 2006). These interactions have been further described as either Pharmacodynamic or Pharmacokinetic. Various studies have reported cases of alteration in the pharmacokinetic profile of drugs concomitantly administered with herbal preparations (Fugh-Berman, 2000; Kane and Lipsky, 2000). St John’s wort, for example, induces the cytochrome P450 isoenzyme, CYP 3A4 and intestinal P-glycoproteins, accelerating the metabolic degradation of many drugs including cyclosporin, antiretroviral agents, digoxin, and warfarin (Moore et al., 2000). Moreover, the consequence of these herb – drug interactions may lead to potentiation of drug effect or antagonism of drug absorption or metabolism, thus resulting in unwanted side effects (Blumenthal, 2000), antagonism in therapy (Elvin-Lewis, 2001) among others.
- Adulteration and Contamination of Herbal Preparations: Available evidences have revealed cases of herbal medicine preparations being adulterated with heavy metals (Nnorom et al., 2006; Obi et al., 2006), orthodox medicines (Floren and Fitter, 1999) or contaminated with microbes (Adeleye et al., 2005). The high price of adulteration and contamination in the use of extracts from plants is particularly distressing considering the fact that these problems often go undetected unless linked with the outbreak of an epidemic (Thomson et al., 2000). For example, Veno-occlusive disease is associated with ingestion of plants containing pyrrolidizine alkaloids, which can be life threatening (Ahmad et al., 2006).
- Adverse Effects Associated with Herbal Medicines: Despite the potential usefulness of herbal products, there are evidences of adverse effects associated with their use, though often under-reported (Foster, 2000). For instance, some medicinal plants of African origin have been reported to be toxic including: Catharanthus roseus (medullary aplasia, leucopoenia, ataxia, convulsion) (Morel and Talbot, 2010); Amaranthus dubius (Hypotension, skin irritations) (Halberstein, 2012); Callilepis laureola (Chronic renal disease, hyperkalemia, convulsions, and liver failure) (Kamsu-foguem and Foguem, 2014). It is also important to emphasize that it is more difficult to recognize adverse effects that develop chronically (e.g., hypokalemia from anthranoid laxatives); those that occur infrequently, or are readily ascribed to an underlying disease (e.g., hepatitis from the bile-duct remedy celandine) (Benninger et al., 1999). Also, as with all forms of self-treatment, there is the potential risk of adverse effect in the illicit use of herbal medicine (De Smet, 2002).

Safety Evaluation of Traditional Herbal Medicine

According to Wooley (2008), the term “safety” is a rather complex concept. Arguably, it is fairly easy to determine for instance, what dose of a chemical is toxic but more difficult to predict safety. This is essentially because even at low dose of a substance, sensitive individuals may develop adverse responses. In spite of this, it is a widely accepted principle that to predict safety, it is necessary to demonstrate the toxic dose and mechanism of toxicity.

Fundamentally, one of the most crucial aspect in drug discovery and development process is the assessment of the toxicological properties of a potential medicinal agent. Toxicology is an aspect of pharmacology that deals with the adverse effects of bioactive substances on living organisms (Anisuzzaman et al., 2001). Currently, the global drug market is experiencing a dramatic increase in sales of herbal products owing to their alleged efficacy but unfortunately, knowledge base on their potential toxicity is rather scarce (Cupp, 2000). More so, as reported by Boullata and Nacen (2000) and Ernst (2007), commonly consumed food / food supplements (e.g. rye, oats, pulses, red kidney beans) originally regarded as safe are now known to contain toxic or anti-nutritional factors like phytates, cyanogenic glycosides, thiocyanates, alkaloids and lectins.

Principally, from safety standpoint, there are three (3) groups of herbs, namely (1) those that contain near therapeutic concentrations of poisonous constituents, such as Atropa belladonna, Aconitum spp, Digitalis spp (Mcrae, 1996); (2) those with very powerful actions, often causing nausea or vomiting but are perfectly safe if used under appropriate conditions, such as Lobelia and Eonymus spp.; and (3) those that exhibit specific kinds of toxicity, such as the hepatotoxicity of pyrrolizidine alkaloid-containing plants (Symphytum) (Philomena, 2011).

TOXICOLOGICAL SIGNIFICANCE OF PLANT SECONDARY METABOLITES

Plants produce an amazing diversity of biochemicals, whose structures have and are currently being elucidated (De Luca and St Pierre, 2000). Of these wide array of plant-based metabolites are ‘ primary ’ and ‘ secondary ’ metabolites (Pichersky and Gang, 2000). The ability for plants to synthesize toxic chemicals (in form of secondary metabolites) (Chandra et al., 2012) serve to ward off pathogens and herbivores; and for allelopathic activities among other roles (Siemens et al., 2002). Based on their role in plant defense mechanism, these metabolites can be categorized into constitutive substances, also called prohibitins or phytoanticipins (acting as first line of chemical defense) and induced metabolites (formed in response to infection, involving de novo enzyme synthesis) also known as phytoalexins (Dixon, 2001). These plant biochemicals may gain assess into the body via inhalation, ingestion or by skin contact. In addition, these plant biochemicals have also been shown to be distributed either throughout the whole plant or concentrated in one or more parts of the plant including the flower, bud, leaf, stem, and root (Chandra et al., 2012). The secondary metabolites, sub-classes and mechanisms of action are highlighted in Table 1.

Table 1.1: Mechanisms of action of plant toxins

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Table 1.1(continued): Mechanisms of action of plant toxins

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Source: William and Carl (2001); Tomris (2002).

TOXICOLOGICAL STUDIES AND HERBAL MEDICINE

The indiscriminate use of botanical preparations together with increasing reports of adverse effects have necessitated the need to demonstrate the safety of herbal products and to globally harmonize standards of toxicity testing methods that can be used for toxicological characterization of herbal medicine (Chan and Fu, 2007; Verma et al., 2012). Principally, two (2) important factors may be put into consideration in evaluating the safety of any herbal drug. These are - the nature / significance of the adverse effect and the exposure level where the effect is observed (Obidike and Salawu, 2013).

Overview of Pre-Clinical Toxicity Studies

Toxicological screening of potential drugs is an indispensable aspect of the drug development process and it is required by regulatory agencies to support various stages of drug development and registration (Jacobson-Kram, 2003). Essentially, some of the objectives of the pre-clinical toxicological screening include (a) to identify doses that can cause toxicity (including dose-response relationships); (b) to determine appropriate starting dose and to aid dose escalation strategy for the first human trials, as well as the maximum human dose, among others (Scientific Working Group (SWG), 2002; Pandher et al., 2012). In addition, the pre-clinical toxicity testing is important in the estimation of “No Observed Adverse Effect Level” (NOAEL) which is needed to initiate the clinical evaluation of investigational products (Parasuraman, 2011).

A battery of tests have been recommended for conducting non-clinical safety evaluation of pharmaceuticals to include single and repeated dose toxicity tests, reproduction toxicity tests, genotoxicity tests, pharmacokinetic (ADME) tests, local tolerance tests and for drugs that have special cause for concern or intended for a long duration of use, an assessment of carcinogenic potential, according to the guidelines of Organization for Economic, Cooperation and Development (OECD) (2001).

Cell-Based Cytotoxicity Tests

Cytotoxicity assays (CTAs) are used to predict potential toxicity, using cultured cells which may be normal or transformed cells (Obidike and Salawu, 2013). These tests usually involves short term exposure of cultured cells to test substances, to detect how basal or specialized cell functions may be affected by the substance being examined, prior to performing safety studies in whole organisms. The assessment parameters for cytotoxic effects include inhibition of cell proliferation, cell viability markers (metabolic and membrane), morphologic and intracellular differentiation markers (O’Brien and Haskings, 2006). Alternatively, Brine shrimp assay could be used to screen medicinal plants for cytotoxicity (Gadir, 2012). This assay has already been used for detection of fungal toxin, plant extract toxicity, heavy metals, pesticides and cytotoxicity testing of dental materials (Sam, 2010). In addition, in vitro cytotoxicity tests could also be employed as adjuncts to the alternative animal tests (Fixed Dose Procedure, the Acute Toxic Class Method and the Up and Down Procedure) recommended by OECD (2001), which will be highly invaluable in improving dose level selection and in the determination of sample size of the experimental study (Botham, 2004).

Single Dose (Acute) Toxicity Studies

Acute toxicity is the ability of a substance to cause detrimental effect within a relatively short period of time (usually defined in terms of minutes, hours or days, rarely longer than 14 days) after a single oral administration (Senin, 2006). Acute toxicity was also referred to as lethality toxicity in a report by Onwusonye et al. (2014) and was used to define the concept of median lethal dose (LD50), an amount of the investigational substance that can be expected to cause death in half (i.e. 50 %) of a group of animal species (such as rats and mice), when administered through a particular route of administration. Data from acute toxicity studies should provide information about possible dose levels for first applications to humans, and give indications as to the possible effects to be expected with overdosing (accidental or intentional) (SWG, 2002). Furthermore, according to the United Nation report (2011), substances can be allocated to one of five toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to numeric cut-off criteria (Table 2). Acute toxicity could also be expressed as lethal concentration (LC50) (a measure of chemical toxicity resulting from inhalation) values or as Acute Toxicity Estimates (LD50).

Table 1.2: Acute toxicity estimate (LD50) values (United Nation, 2011)

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Note: ppmV = parts per million per volume (ppmV)

In conducting acute toxicity studies for drug development, it is recommended that two (2) different mammalian species be used, often by two different routes namely the clinical route and a parenteral route, usually intravenous (IV) with an exception of when the clinical route is IV, in which case, only the intravenous route is tested. In addition, following the administration of the compound, the test requires a 14-day period to observe and evaluate the clinical signs of toxicity (e.g. behavioural changes, decreased body weight) while monitoring the duration, and reversibility of the toxic effect (OECD, 2001; Chapman and Robinson, 2007).

Essentially, in view of recently updated international policies bordering on animal welfare; in 2002, OECD deleted the test guideline, TG 401 on grounds of its lethality endpoint and large number of animals required and later introduced three OECD guidelines for conducting oral acute toxicity tests based on non – lethality endpoints namely TG 420 (fixed dose procedure), TG 423 (acute toxic class method) and TG 425 (up and down procedure) (OECD, 2008). Meanwhile, it is noteworthy that in all methods specified by OECD, amidst all aspect of the guidelines, it was recommended that all test animals (including those that die during the test or are euthanized for animal welfare reasons) be subjected to both gross necropsy and microscopic examination of organs. This is however in contrast to the guidelines of the Society of Toxicologic Pathology (STP) which argues against the need to evaluate organ weights of animals (dead or euthanized) in the course of the studies as the differences in nutritional status, exsanguination, tissue congestion and edema, and the absence of matched concurrent control data could in themselves be confounding variables that may affect the interpretation of organ weights (Sellers et al., 2007). Table 3 highlights the principles in the alternative methods for estimating acute oral toxicity as specified by OECD while Table 4 shows some typical signs of toxicity and the corresponding body systems affected.

Table 1.3: The principles of the three alternative methods (Botham, 2003; OECD, 2008)

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Table 1.4: Typical signs of toxicity and related body systems (Lu and Kacew, 2003)

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Repeated Dose Toxicity Tests

In principle, the aim of repeated dose toxicity is to characterize the toxicological profile of the test substance following repeated administration. Consequently, it is intended for garnering information on toxic effects (including potential for persistence or reversibility) of the test substance; identifying potential target organs by evaluating parameters such as hematology, clinical chemistry, and histopathology, amongst other endpoints (Setzer and Kimmel, 2003; Katsumi et al., 2010). Broadly, these tests may be categorized into short-term (which involves repeated administrations of the test substance, usually on a daily or five times per week basis, over a period of about 10 % of the life-span of the experimental animal) and long-term (which involves repeated administrations over the entire life span of the test animals or at least a major fraction of it) (Lu and Kacew, 2003; Ballantyne et al., 2009; Parasuraman, 2011). Literature search revealed the use of various terms namely: ‘ sub-acute’, ‘sub chronic’, ‘chronic toxicity studies’ to depict various forms of repeated dose toxicity studies. Although, a report by Obidike and Salawu, (2013) depicted the sub-acute and sub-chronic toxicity (on the basis of duration of exposure) as 28 - 30 days and 60 – 90 days respectively, there are still conflicting views on the duration of the repeated dose toxicity studies among toxicologists.

Reproductive Toxicity and Genotoxicity Studies

Reproductive toxicity study aims at characterizing the toxicological profile of the substance with respect to effects on fertility and early embryonic development; embryo-foetal development; pre – and postnatal development (including maternal function) (SWG, 2002; Parasuraman, 2011; United Nation, 2011).

Genotoxicity testing is basically intended to assess the potential of a test material to induce genetic damage whether in the form of gene mutations, chromosome changes, and alterations in the DNA sequence (Oliveira et al., 2010). Several studies have been recommended for conducting mutagenicity / genotoxicity studies including: mammalian in vivo micronucleus test (OECD, 2014a, b, c, d)

Carcinogenicity Studies

Carcinogenicity studies are aimed at providing information on the possible health hazards likely to arise from repeated exposure of a test substance for a period lasting up to the entire lifespan of the species used (SWG, 2002; OECD, 2009b). Although other animal species can be used, majority of this sort of studies are carried out in rodent species over a greater portion of an animal's lifespan.

Principally, the specific objectives of carcinogenicity studies (United States Environmental Protection Agency, USEPA, 2005); Boobis et al., 2006; OECD, 2009b) include:

- identification of the carcinogenic properties of a chemical.
- identification of target organ(s) of carcinogenicity;
- identification of the time of appearance of neoplasms;
- characterization of the tumour dose-response relationship;
- identification of a no-observed-adverse-effect level establishment of a Benchmark Dose (BMD);
- extrapolation of carcinogenic effects to low dose human exposure levels.

Consequently, as expressed by Parasuraman (2011), during and after exposure to test substances, the experimental animals are evaluated for signs of toxicity and development of tumors. If these evaluations are negative, the test may be terminated after 18 months in the case of mice and hamsters and after 24 months with rats. However, if the animals are healthy, clinical and morphological pathology examination is performed after the 12 months and the 18 months respectively, and the study terminated.

Local Tolerance Studies

The objective of these studies is to ascertain whether medicinal products are tolerated at sites in the body which may come into contact with the product as a result of its administration in clinical use (European Medicinal Evaluation Agency (EMEA), 2001). An example of this sort of studies include the Ocular/Skin irritancy test (also known as Draize test) (Obidike and Salawu, 2013).

Toxicokinetic Studies

Toxicokinetic analysis may be conducted in vivo or via in vitro cell lines (Payan et al., 2003; Zepnik et al., 2003). Toxicokinetic (TK) studies serves as adjunct in toxicology studies to improve understanding of the mechanism of toxicity of the investigational substance and also to demonstrate systemic exposure of the test animals to the investigational substance by revealing the circulating moieties (parent substance/metabolites). TK studies may also provide useful information for determining dose levels for toxicity studies (linear vs. non-linear kinetics), route of administration effects, bioavailability, and issues related to study design. Certain types of TK data can be used in physiologically based toxicokinetic (PBTK) model development (OECD, 2010).

With respect to herbal medicine, toxicokinetics deals with the prediction of toxicity on the basis of the pharmacokinetic disposition of an herb (or purified xenobiotics derived from the herbal product), as a result of the influence of genetics or potential herb - drug interactions (Maurer, 2012). Some herbs, notably St. John's Wort (Hypericum perforatum), ginkgo (Ginkgo biloba), ginseng (Panax ginseng), kava (Piper methysticum) and garlic (Allium sativum) have been reported to cause significant alterations in the pharmacokinetic profile of certain co-administered drugs modulated by cytochrome P450 enzyme system (Wienkers and Heath, 2005; Izzo and Ernst, 2009).

Relevance of Recovery Studies in Toxicity Studies

In pre-clinical toxicity studies, routine procedures such as clinical and morphological pathology are conducted to examine the laboratory animal(s) immediately after completion of the dosing phase. However, some studies include cohorts of animals that undergo a dosing phase followed by a non-dosing phase of a specified duration, termed a recovery phase or reversibility phase. This non dosing phase is designed to understand whether toxicities observed at the end of the dosing phase are partially or completely reversible (Pandher et al., 2012). Other reasons for the inclusion of recovery groups in toxicity studies include: (a) to assess whether effects observed at the end of the dosing phase are persistent; (b) to determine whether the test article has the potential to produce delayed toxicity after dosing has ended; (c) to allow for detection of antidrug antibodies, which may not be detectable in the presence of the test article, depending on the assay used; and (d) for the purpose of complying with recommendations of regulatory authorities (e.g., addition of requirements for alternative first-in-human [FIH] approaches) (International Conference for Harmonization (ICH), 2009, 2010, 2011).

Reversibility studies are also a key part of the ‘weight-of-evidence’ approach in the interpretation of toxicological data. It could also be used for assessing if there should be cause for concern inter alia an effect. For example, a change that is readily and completely reversible on cessation of treatment is considered an indication of a lower level of concern. Hence, it follows that knowledge of whether or not an effect is reversible may influence significantly the overall interpretation and differentiation of adverse from non-adverse effects (Lewis et al., 2002; Pandher et al., 2012).

Conversely, while inclusion of a recovery phase in non-clinical toxicity studies seem imperative for interpretation; concerns have arisen about what should constitute the most judicious use of recovery groups (Pandher et al., 2012). Such concerns include: (i) the responsible use of animals in research, as the addition of recovery groups generally leads to increased animal use; (ii) elongation of study period which in turn increases the study cost and (iii) increased work with the generation of both an interim report (without recovery data) and full final report (Pandher et al., 2012).

PATHOLOGICAL ASSESSMENT IN PRE-CLINICAL TOXICITY STUDIES

Most routinely, toxicological screenings require a pathological assessment of potentially detrimental effect of the investigational substance, hence, the relationship between the two (2) fields. Pathology as a field of science can be categorized into two major subspecialties namely – Clinical pathology and Anatomical pathology.

Clinical Pathology

Clinical pathology can be subdivided into clinical hematology, clinical biochemistry, clinical microbiology and molecular diagnostics (Kemal, 2014). For the purpose of this study, the implications of hematological and biochemical parameters are discussed.

Basically, the hematopoietic system is an important index of physiological and pathological status in man and animals (Adeneye et al., 2006). Studies have revealed the susceptibility of the hematopoietic system as reflected in the derangement of certain blood parameters after the ingestion of toxic plants (Adedapo et al., 2007; Adewoye et al., 2012). Some of the hematopoietic parameters that are routinely studied for clinical pathology in toxicity studies by researchers include: packed cell volume (PCV), hemoglobin, white blood cell count, differential leukocyte count, coagulation time determination, bleeding time, red blood cell indices and platelets count (Venkatesh et al., 2014).

Conversely, clinical chemistry (also known as chemical pathology and clinical biochemistry) is concerned with analysis of body fluid (Kemal, 2014). Serum biochemical parameters can provide important and useful information in assessing not only the extent and severity of organ damage, but also the type of damage (Ramaiah, 2007). Certain biochemical tests routinely employed in preclinical toxicity studies including liver function tests, renal function tests, urinalysis amongst others, to assess the integrity of this vital internal organs (Aniagu et al., 2004; Arneson and Brickell, 2007; Rebar, 2010). In addition, complete urinalysis includes observation of color and clarity, urine specific gravity (measured by refractometry), urine pH, bilirubin, glucose, occult blood, ketones and screening for proteinuria (Robertson and Seguin, 2006). Urine sediment should be examined for red blood cells, white blood cells, epithelial cells, casts, organisms and crystals (Robertson and Seguin, 2006).

More importantly, it is worthwhile to note that in interpreting results of clinical pathology particularly, in biochemical profiling, certain pitfalls are likely to arise. Some of which include the fact that (1) normal animals may have occasional abnormal test results, (2) ill animals can have normal test results, and (3) abnormalities in one organ system may cause abnormal results in a test that is run to assess the status of a second organ system (Rebar, 2010).

Anatomical Pathology

In addition to clinical pathology, the morphological evaluation of the disease pattern is essential for a holistic interpretation of toxicity studies. Common histopathological manifestations of chemical induced organ damage include: adaptive response such as atrophy (decrease in cell size), hypertrophy (increase in cell size), hyperplasia (Increase in the number of cells in an organ or tissue) and metaplasia (replacement of one cell type by another); cell death (necrosis and apoptosis); inflammation; and neoplasm (FDA Working Group, 2000; Mitchell and Cortan, 2004a, b). In the assessment of morphological tissue changes, the OECD guideline TG 407 has pointed out that marked changes in organ weights of the various tissues should generally be accompanied by significant morphologic (gross and microscopic) changes in the same tissues (OECD, 2008).

In addition, as an index for toxicity, organ weight has been regarded as one of the most sensitive indicators in the evaluation of the toxic effect of the investigational substance as significant differences between treated and untreated (control) animals may occur in the absence of morphological changes in toxicological experiments (Bailey et al., 2004; Nirogi et al., 2014). In fact, it has been asserted in previous studies that animals cannot survive after loss of more than 10 % of the initial body weight (Raza et al., 2002; Obici et al., 2008). Additionally, in a report by Sellers et al., (2007), it was pointed out that a relationship exist between organ weight and body weight of the animal. This association was attributed to the biological variation of the animal. Therefore, it was suggested that normalization of organ weights to body weight (in case of large inter-animal variability) could help reduce variations due to body weight differences. Alternatively, organ-to-brain weight ratios could also be useful, as test materials that alter body weight generally do not alter brain weight (Wilson et al., 2000) making organ-to-brain weight ratios useful in cases where significant increase or decrease in body weight could impact organ-to-body weight ratios (Nirogi et al., 2014). More so, a report by Bailey et al., (2004) suggested that in evaluating organ toxicity, organ-to- brain weight ratios may be more appropriate in evaluating toxicity in the ovary and adrenal glands while organ-to- body weight ratios may be best suited for evaluation of liver and thyroid gland weights.

Furthermore, a number of histopathological examinations have been recommended in conducting toxicological experiments. These include tissues from the high dose and control groups; all tissues from animals dying or killed during the study; all tissues showing macroscopic abnormalities; target tissues, or tissues which showed treatment-related changes in the high dose group, from all animals in all other dose groups; in the case of paired organs, e.g., kidney, adrenal, both organs should be examined (Crissman et al., 2004; OECD, 2009c).

RELEVANCE OF ANIMAL MODELS IN TOXICOLOGY

Michael et al. (2001) reported that in the process of drug discovery and development, part of the ultimate goals of toxicological assessments include the use of animal models to characterize toxicity in short and long term pre - clinical studies; identifying the conditions under which toxicity occurs, and evaluating the extent to which the data from animal studies warrant extrapolation to humans. Essentially, rats and mice are the most frequently used rodent species while dogs are the most widely used non-rodent species, with occasional use of miniature pigs and non-human primates. More so, it has also been pointed out that in search of knowledge on normal human functions and mechanisms which underlie dysfunction in normal body physiology, rodents are often the most preferred animal species of choice because of factors including: similarity between pharmacodynamic parameters of both man and rodents; availability, low cost, ease of breeding, and an extensive literature database to enable comparisons to present findings (Lu and Kacew, 2003).

Fundamentally imperative in the non – clinical toxicological aspect of drug development is concern over the issue of laboratory animal welfare (Society of Toxicology (SOT), 2006). In a bid to satisfy conditions of appropriate use of animals in biomedical science, the Russell and Burch’s principles (1959) of the ‘3Rs’ is usually employed. The Three Rs are: Replacement (replacing whole animal models with non-whole animal models); Reduction (reducing the number of animals required to a minimum), and Refinement (minimizing harms to animals in both husbandry and experimental procedures). The principle of Three Rs is widely accepted in animal research communities and is typically used by ethics committees to guide their review of animal care and use protocols (Stephens et al., 2001; Glaxo Smith Kline 2001; Pfizer 2002; Eli Lilly 2004).

RECOGNITION OF THE ADVERSE AND NON-ADVERSE EFFECT IN TOXICITY STUDIES

Fundamentally, in view of reports of adverse effects (historical or contemporary) from the use of pharmaceutical products like in the case of the infamous thalidomide tragedy of 1956 (van Meer, 2013), it has become quite imperative to clarify certain issues as it relates to the safe use of pharmaceuticals and also whether animal models are as predictive and appropriate in detecting possible adverse effects associated with use of such pharmaceuticals (Lewis et al., 2002). Hence, the call for a pragmatic, standard approaches in recognizing toxicity, especially in pre-clinical toxicity studies. Basically, with respect to data extrapolation of animal studies to humans, one perpetual challenge has been about how to interpret data from toxicity studies on animal models as either treatment related or non-treatment related. Hence, it was suggested by Lewis et al. (2002) that in discriminating between the adverse and the non-adverse effects, consideration is dependent on the following: whether the effect is an adaptive response; whether it is transient; the magnitude of the effect; its association with effects in other related endpoints; whether it is a precursor to a more significant effect; whether it has an effect on the overall function of the organism; whether it is a specific effect on an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. More importantly, it has been emphasized in several studies that the identification of the “No Observed Effect Level (NOEL)” is one of the most important quantitative outputs of toxicity considered relevant to human health risk assessment (Hayes, 2001; FDA, 2002). Furthermore, the concept of NOEL is considered to be dependent on the design of the study, indication of the drug, expected pharmacology, and spectrum of off-target effects (Dorato and Engelhardt, 2005).

In addition, it has been asserted that among factors relevant in predicting adverse effects for instance, identification of toxic effects in animals is generally expected to follow a dose – response pattern in relation to incidence and severity, thus allowing the determination of dose levels where substantial, pertinent or relevant effects may or may not occur (Ballantyne et al., 2009). In fact, it is seen that opinions on where harmful and non-harmful effects are likely to occur may change as additional information related to the test substance is generated. For example; a liver enzyme elevation in a short-term non-clinical study without a histopathology correlate, will be noted but may not be considered to be harmful (Dorato and Engelhardt, 2005). Meanwhile, data from longer-term studies, indicating a progression of effect and the occurrence of histological alteration in the liver, provides a new interpretation of the importance and/or relevance of the response and the duration of exposure associated with the first indications of an unwanted response (Calabrese and Baldwin, 2003a, b; Dorato and Engelhardt, 2005).

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Figure 1.1: Classifying toxicological profile as adverse or non-adverse (Lewis et al., 2002)

A number of determinants have been presented in the diagram below (Figure 1) to aid the determination of treatment and non-treatment related effects.

TOXIC SUBSTANCES

Toxic substances come in a variety of forms from a number of different sources. Those that come from natural sources are commonly called toxins, whereas those produced by human activities are called toxicants. These toxic substances may be classified according to several criteria such as (a) Chemical form (e.g. heavy metals, polycyclic aromatic hydrocarbons) (b) Physical form (dusts, vapors, or lipid-soluble liquids) (c) Source (plant material, combustion by-products) (d) Target organs (neurotoxins, hepatotoxicants) (e) Biochemical effects (alkylating agent, sulfhydryl inhibitor) and (f) Poisoning potential (extremely toxic, very toxic, slightly toxic) (Eaton and Klaassen, 2001; Manahan, 2003).

Types of Phytotoxicity

Chemically, plant toxins are low molecular weight compounds such as alkaloids, terpenoids, saponins among other secondary metabolites, used by plants to adapt to environmental stress like attack from microbes and herbivores (Wink, 2008a; Wink and van Wyk, 2008). These phytochemicals may be additive or even synergistic in their overall properties and activities (Wink 2008b). More so, it is important to note that part of the risk posed by phytotoxins is that some of these phytochemicals synthesized by plants against herbivorous insects for instance, also end up being harmful to humans. This phytotoxicity has been attributed to the highly conserved biological similarities shared between both taxa as seen in pathways involving protein, nucleic acid, carbohydrate and lipid metabolism (Kawashima et al., 2007). In addition, there are several biochemicals in humans such as signaling molecules, neuropeptides, hormones and neurotransmitters whose functions can be mimicked or antagonized by phytochemicals like alkaloids, flavonoids, terpenoids and saponins (Daniels et al., 2008; Ismail et al., 2008; Nassel and Winther, 2010).

Essentially, for the plant defense compounds (secondary metabolites) to be effective, they must be able to interfere with molecular targets at the different organizational levels of the animals’ life (Wink, 2009). Several studies have highlighted some of the biochemical mode of action of these secondary metabolites to include:

(a) Neurotoxins: E.g. ion channel modulators as in the case of Na+ - K+ ATPase inhibition by cardiac glycoside (Wink, 2003). They could act as mimetics of endogenous neurotransmitters such as the biogenic amines, either as agonists or antagonists (Alberts et al., 2008).
(b) Inhibitors of cellular respiration: β-glucosidase and nitrilase are released upon contusion of plants and break down of cellular compartment. These enzymes hydrolyse the cyanogenic glucosides leading to the release of toxic HCN, which interferes with the mitochondrial respiratory chain by binding to iron ions of the terminal cytochrome oxidase) (Mutschler et al., 2008).
(c) Cytotoxins : a typical example is saponins, which are stored as inactive bidesmosidic saponins in plant vacuoles, which upon contusion and decompartmentation are converted into the membrane-active monodesmosidic saponins, which are amphiphilic with detergent properties (Wink and van Wyk, 2008). In addition, they could interfere as inhibitors of ribosomal protein biosynthesis as in the action of amanitins from Amanita phalloides (Alberts et al., 2008) and also cytoskeleton poisons such as colchicine, podophyllotoxin, vinblastine (Wink, 2007).
(d) Alkylating and intercalating DNA toxins: some secondary metabolites such as β-carboline alkaloids, anthraquinones or furanocoumarins, are known to attack DNA and RNA by either intercalation or alkylation. These agents stabilise DNA, thus inhibiting DNA replication (Wink, 2000).
(e) Toxins of skin and mucosal tissues: examples of these toxins are the diterpenes present in members of the Euphorbiaceae and Thymelaeaceae family. These diterpenes classified as phorbol esters can stimulate protein kinase C, cause severe and painful inflammation, with ulcers and blister formation, upon contact with the skin, mucosal tissues or the eye) (Alberts et al., 2008; Mutschler et al., 2008).

In addition, toxic effects could also be described as idiosyncratic in nature (Levine, 1978); reversible or irreversible (Eaton and Klaassen, 2001); immediate or delayed (Hatch et al., 1998); local or systemic (Eaton and Klaassen, 2001). Table 5 shows the classification of toxic effects according to the organs affected.

Table 1.5: Toxic effects by plants (on basis of organ affected) (Norton, 2001)

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Table 1.5 (contd.): Toxic effects by plants (on basis of organ affected) (Norton, 2001)

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CHRYSOPHYLLUM ALBIDUM (CA)

Nomenclature

Chrysophyllum albidium (also known as White Star Apple) belongs to the family, Sapotaceae (Hutchinson and Dalziel, 1963). Morphologically, the tree plant has been reported to be closely related to the African star apple (Chrysophyllum africanum) which is also a common variety in West Africa (Adepoju et al., 2013). In South-western Nigeria, the tree plant is called “agbalumo” and popularly referred to as “udara” (South-eastern Nigeria); agwaluma (Hausa) (Egharevba et al., 2015) . It is a dominant canopy tree of lowland mixed rain forest widely distributed throughout the tropical Central, East and West Africa regions (Madubuike and Ogbonnaya, 2003; Orwal et al., 2009; Germplasm Resources Information Network [GRIN], 2013). The fruit of C. albidum is seasonal. In Nigeria for instance, it is found in urban and rural centres in the period from December to April (Amusa, 2003).

Botanical Description of Chrysophyllum albidum

The genus Chrysophyllum belongs to the family Sapotaceae and order Ericales which consists of a large biodiversity of medicinal plants. Previous studies estimated the world distribution of plant species in the Sapotaceae family at about 70 genera and 800 species (Gill, 1988; Keay, 1989), however, a more recent estimate have put the world distribution at 53 genera and 1,100 species (Pennington, 2004a, b). In West Africa alone, twenty-three (23) genera and more than 300 species have been documented. These genera include; Omphalocarpum, Ituridendron, Manikara, Mimusops, Kanton, Breviea, Dehpdora, Chrysophyllum, Pachystela, among others (Hutchinson and Dalziel, 1963; Inyama et al., 2011). Fifteen genera have been recorded in Nigeria among which is Chrysophyllum (Keay, 1989). It should also be noted that of all the Nigerian species of Chrysophyllum (including albidum, perpulchrum, cainito, welwitschii, delvoyi, pruniforme, giganteum, subnudum) which are characterized by their towering height, C. welwitschii has been identified as being a woody climbing shrub (Jessup and Short, 2011).

The genus Chrysophyllum (Greek) means “golden-leaf” which depicts the colour of the hairs of some species (although a few others are silvery – white in colour) while the species albidum, refers to the white or silvery-grey undersurface of mature leaves, easily seen when looking up into the tree’s canopy (Orwal et al., 2009). The species has been described to have a set of structural features (Orwal et al., 2009; Emmanuel and Francis, 2010; Inyama et al., 2011; Jessup and Short, 2011), which includes:

- a small to medium buttressed tree species, reaching up to 25-37 m in height;
- dehiscent fruits, which are almost spherical, slightly pointed at the tip, about 3.2 cm in diameter, greenish-grey when immature, turning orange-red, yellow-brown or yellow, sometimes with speckles;
- seeds (1-1.5 x 2 cm) is composed of a seed coat which appears bony - hard, shiny, dark brown in colour and an inner white coloured cotyledon. Seed count of about 1 – 6 in number; laterally compressed; prominent hilum scar; large embryo);
- leaf is simple, dark green above, pale tawny below when young but silver-white below when mature; oblong-elliptic in shape, 12-30 cm long, 3.8-10 cm broad; apex shortly acuminate, base cuneate; primary lateral nerves widely spaced, 9-14 on each side of the midrib; secondary lateral nerves indistinct or invisible;
- flowers shortly pedicellate, in dense clusters in the leaf axils or from above the scars of fallen leaves; calyx 5 - lobed, 3 mm long, rusty pubescent outside, creamy white, the lobes equaling the tube in length; bisexual or unisexual;
- The bark is thin, pale grey-brown or pale brownish-green, while the pale brown slash exudes copious white gummy latex, a characteristic of the Sapotaceae family.

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Figure 1.2: Fruit of Chrysophyllum albidum (source: the author)

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Figure 1.3: Seeds of Chrysophyllum albidum (source: the author)

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Figure 1.4: The white cotyledons of Chrysophyllum albidum (after separation from brown seed kernel) (source: the author)

Ethnomedicinal Significance of Chrysophyllum albidum

The plant parts of Chrysophyllum albidum are used for diverse ethnomedicinal purposes as outlined in the Table 1.6.

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Table 1.6: Ethnomedicinal Uses of Chrysophyllum albidum (Houessou et al., 2012)

Pharmacological Profile of Chrysophyllum albidum

Published reports have also revealed a plethora of pharmacological / biological activities in the different morphology of Chrysophyllum albidum that could be exploited for drug development. Some of the previously explored pharmacological activity are outlined in Table 7 .

According to Egharevba et al. (2015), phytochemical screening of Chrysophyllum albidum cotyledon from crude sample to methanol extract reveals the presence of saponin, alkaloid, tannin, flavonoid, sterol and anthraquinone. However, these phytoconstituents in the cotyledon were observed to be absent in non – polar medium (hexane and ethylacetate) except for presence of sterol. The methanol extract meanwhile showed presence of these phytochemicals (same as crude sample), with the exception of anthraquinone.

Table 1.7: Biological properties of part(s) of Chrysophyllum albidum

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Several studies have been conducted to demonstrate the role of plant secondary metabolites in man (Schafer and Wink, 2009; Mazid et al., 2011). In Chrysophyllum albidum for instance, the phytochemicals found in the methanol extract of the cotyledons of Chrysophyllum albidum have proven to be highly important for their biological activities including

(i) Flavonoids are important in preventing oxidative damage in cells and have been shown to also have strong anti-cancer and anti-allergic activities (Cushine and Lamb, 2005). The phytochemicals in the seed of Chrysophyllum albidum (CA) have also been proven to have good anti-inflammatory and anti-microbial activities and have been suggested to play role in its ethnomedicinal use for wound healing (Lotito et al., 2006).
(ii) Alkaloids: One of the most common biological properties of alkaloids is their toxicity against foreign organisms (Egharevba et al., 2015). The methanol extract of the seed has been reported to contain β – carboline alkaloids like eleagnine (1, 2, 3, 4 -tetrahydro-1-methyl-β- carboline), tetrahydro-2-methylharman (1, 2, 3, 4- tetrahydro-1, 2-dimethyl-β-carboline) and skatole (3- methylindole) (Idowu et al., 2003, 2006). Alkaloids belonging to β-carboline group possess antimicrobial, anti-HIV and anti-parasitic activities (Bouayad et al., 2011). Other pharmacological activities of the β-carboline alkaloids has been attributed to their interaction with receptors / enzymes like benzodiazepine, opiates, imidazole, dopamine, serotonin and monoamine oxidase (Herraiz et al., 2010; Farzin et al., 2011). The antinociceptive activity of the cotyledon of CA has also been reported and has been attributed to eleagnine (Idowu et al., 2003). On the other hand, skatole has been implicated to be responsible in causing pulmonary edema in several animal species such as goats, sheep, rats, and some strains of mice (Egharevba et al., 2015). The pulmonary edema was attributed to be as a result of skatole’s selective activity on ‘club cells’, which are the major site of cytochrome P450 enzymes in lungs. These enzymes converts skatole to a reactive intermediate, 3-methyleneindolenine, which damages cells by forming protein adducts. In contrast, Idowu et al., (2003) maintained that although the cotyledon of Chrysophyllum albidum contains pharmacologically active b -carboline alkaloids, it is not known to be cytotoxic.

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Source: Structure of one of several bioactive compounds in Chrysophyllum albidum

(iii) Tannins: The cotyledons of CA have been reported to possess high tannin content (Egharevba et al., 2015). According to Enzo (2007), these tannins could be responsible for the anti-diarrheal activity of the seed. Tannins also act as iron depriver, interacting with specific proteins and enzymes in microbial cells through hydrogen bonding of the polar regions and hydrophobic interactions of non-polar regions, forming tannin-protein complexes (McRae and Kennedy, 2011). More so, it has been reported that herbs that possess tannins are astringent in nature and are beneficial in the treatment of intestinal disorder such as diarrhea and dysentery (Okoli and Okere, 2010), hence the antimicrobial activity of cotyledon of CA. Also, tannins exhibit remarkable activity in cancer treatment (Li et al., 2003).
(iv) Saponins: Saponins are associated with numerous pharmacological properties (Estrada et al., 2000). These phytochemicals are important for their roles as dietary supplements, expectorant and anti-inflammatory agent (Marjan and Hossein, 2008). In spite of reports on the role of the seeds of Chrysophyllum albidum as dietary supplement, the study conducted by Egharevba et al. (2015) revealed that as a result of low total ash value (0.45 %) (suggesting a low amount of inorganic substances) in the seed, it might not be a good source of mineral elements as dietary supplement.

In addition, studies have also been conducted on ethanol extract of seed cotyledon of CA where the cotyledon of CA has been reported to possess anti-hyperglycemic potential (Olorunnisola et al., 2008), thus, providing alternative medicine in the treatment of diabetes mellitus. In Western Nigeria, the ointments of the seed cotyledons are reported to be used in the treatment of vaginal and dermatological infections (Egunyomi et al., 2005; Adebayo et al., 2011). Furthermore, studies on nutritive value of the cotyledon of CA revealed that the seed flour of CA is rich in mineral components including potassium (5100.00 mg/kg); magnesium (2100.00 mg/kg), calcium (1960.00 mg/kg); sodium (210.00 mg/kg); iron (47.20 mg/kg); manganese (24.20 mg/kg), copper (12.90 mg/kg) and zinc (6.70 mg/kg) (Ajayi and Ifedi, 2015).

FINAL REMARKS

The extracts of the seed cotyledon of Chrysophyllum albidum have been shown to possess an array of potentials including anti-microbial, anti-inflammatory, anti-oxidant, anti-diabetic, hypolipidemic and fertility related activities, thus providing scientific evidence for its ethnomedicinal uses and indicating that the seeds are new drug candidate. Despite the plethora of pharmacological potentials inherent in this plant, particularly the cotyledon, the toxicological profile remains largely unexplored, hence the need for further studies.

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